404 not found 3 niluferaamin.cdr page 1 page 2 page 3 page 4 issn 1978-3477, eissn 2087-8575 volume 11, number 1, march 2017 cloning of synthetic lipase gene from rhizomucor miehei with original signal peptide in pichia pastoris effect of tempeh supplementation on the profiles of human intestinal immune system and gut microbiota studies for iaa (indole-3-acetic acid) production by isolates h6 with nitric acid mutation analysis of human immune response against salivary glands protein extract of anopheles sundaicus. l in malaria endemic area construction and expression of single recombinant peptide surfactant for enhanced oil recovery (eor) application martha eka cahyani, is helianti, niknik nurhayati, and abinawanto stephanie, nine kirana ratih, susan soka, and antonius suwanto rahayu fitriani wangsa putrie, tiwit widowati, sylvia jr lekatompessy, and harmastini sukiman mohammad mirza nuryady, sugeng setyo utomo, yunita armiyanti, sri mumpuni wahyu widjajati, and kartika senjarini cut 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171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference 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available with a minimum order of 50 copies. guide for author microbiol indones formulir berlangganan (subscription form) microbiology indonesia nama (name) institusi (institution) alamat pengiriman (address): phone/fax/email : pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com formulir berlangganan (subscription form) microbiology indonesia nama (name) institusi (institution) alamat pengiriman (address) phone/fax/email pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) ) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com guide for author 145-148 (shirly kumala)..pmd infectious disease is one of the main health problems in the developing countries including indonesia. according to household health survey 1997, the main cause of death are as follows: 28.1% are caused by infectious and parasitic diseases, 18.9% are due to vascular diseases, and 15.7% are caused by respiratory diseases (depkes 1997). this evidence demonstrates that the prevalence of infectious disease in indonesia is still high, and to solve this problem new antiinfection drugs that are potent and affordable must be available especially for those in the low to middle economic society classes. this encourages research to find new natural low cost medicinal sources with potent anti-microbial activity. the microbes existing in plants are called endophytic microbes. these microbes spent part or their entire lifetime in the living tissue of the host plant without causing any harm (petrini et al. 1992). microbes such as fungi, yeast, and bacteria associated with the host plant assist in the metabolism of the host plant, and produce secondary metabolites with potent medicinal activity such as anti-tumor, anti-bacterial, and anti-fungal compounds as decomposing enzymes, and also plant growth hormones (petrini et al. 1992; strobel et al. 1996; strobel 2002). mengkudu is a tropical plant that has been used to treat a variety of diseases since thousands of years ago. studies showed that parts of the mengkudu plant (morinda citrifolia) such as the fruit, leaf, root, and bark apparently have a therapeutic effect. some of these effects are the lowering of high blood pressure. improving body resistance, pain relief, anti-tumor and anti-cancer, anti-inflammatory and antibacterial activity. microbiology indonesia, december 2007, p 145-148 volume 1, number 3 issn 1978-3477 short communication isolation and screening of endophytic microbes from morinda citrifolia and their ability to produce anti-microbial substances shirly kumala* and endro budi siswanto faculty of pharmacy, universitas pancasila, jalan srengseng sawah, jagakarsa, jakarta 12460, indonesia assaying for, and isolation and screening of endophytic microbes from the morinda citrifolia plant and their ability to produce anti-microbial substances was carried out. endophytic microbes are microorganisms that live asymptomatically within the living tissue of host plants. microorganisms such as bacteria, fungi, and yeast can be associated with the host and produce secondary metabolites. these secondary metabolites may be enzymes and other bioactive substances with medicinal activity such as anti-arthritic, anti-cancer, and anti-microbial compounds. the aims of these experiments was to investigate the ability of endophytic microbes isolated from m. citrifolia to produce secondary metabolites which can act as anti-microbial agents. a direct seed inoculating technique was used by planting the plant sample onto the surface of nutrient agar and potato-dextrose agar. assessment of their ability to produce anti-microbial substances was conducted by growing the endophyte isolates in muller hinton broth for bacterial isolates, and in potato-dextrose yeast broth for fungal isolates. the agar diffusion method using paper disk was applied to assay the anti-microbial activity of each substance. the results of the endophyte isolation in these experiments gave five bacterial isolates and eleven fungal isolates. all of the bacterial isolates showed a broad antimicrobial spectrum while ten of the fungal isolate demonstrated broad anti-microbial activity and four out of the ten fungal isolates had activity towards candida albicans. key words: anti-microbial, endophytic microbe, morinda citrifolia (mengkudu), secondary metabolite _____________________________________________ ________________________ * corresponding author, phone: +62-21-6902104, fax: +62-21-7864727, e-mail: fskumala@yahoo.com up to now there have been few studies, which focus on the medicinal activity of endophytic microbes as antimicrobial substances. the objective of this study is to identify the endophytic microbes in the mengkudu plant and their capacity to produce secondary metabolites that have anti-microbial effect. the twig of the mengkudu plant which was used in this study had been identified at the herbarium bogoriense, balitbang botani, puslitbang biologi lipi, bogor (the bogor herbarium, botanical research and development agency). the test microbes used in this assay were staphylococcus aureus, escherichia coli, bacillus subtilis, salmonella typhimurium, and candida albicans. these microorganisms were derivates of atcc and were obtained from laboratory of microbiology, medical faculty, university of indonesia. twigs from the second and the third branches of the mengkudu plant (diameter of 0.3-0.6 cm) were selected for use in this study. leaves were removed and the twigs were cut into lengths of 2 cm and washed with under running water (tap water) for about 10 min. the sections of the twigs were sterilized by soaking in 75% v/v ethanol for 1 min in the laminar air flow cabinet (gelman sciences, australia), and then in a 5.3% w/v solution of naocl for 5 min. following the sterilization step, samples were air-dried on sterile tissue paper for 1 min. after drying, they were placed on a sterile glass slide and cut longitudinally with a scalpel into two equal parts. one part was placed on an nutrient agar (na) medium that had been treated with nystatin (0.01% w/v) as an antifungal agent and the other part was on potato dextrose agar that had been treated with chloramphenicol (0.005% w/v) as antibiotic. each sample was positioned longitudinally such that the surface of the cut was placed in contact with the medium. samples were then placed in an incubator for 5-7 days at 27-29 °c (bacon 1988). 146 short communication microbiol indones second branching first branching second branching second branching second branching natgpc 5a natgpc 3b natgpc 2a natgpc 1b natgpc 1c cocco bacil cocco bacil cocco bacil cocco bacil cocco bacil gram negative gram positive gram positive gram positive gram negative table 1 data of endophytic bacteria part of plant isolate code shape gram stain first branching first branching second brancing second brancing first branching second brancing first branching second brancing first branching first branching second brancing pdapgpc 5a1-1 pdapgpc 4a1-a2 pdatgpc 3a2 pdatgpc 3a pdapgpc 1c pdatgpc 5a pdapgpc 1d pdapgpc 4-c2 pdatgpc 2 pdapgpc 4a1 pdatgpc 5a1-2 white undulate, reverse white, aerial concentric green to black velvety, reverse black brown to withish,, reverse white, aerial concentric white hyphae, reverse white, with aerial concentric black at center, reverse white milk white undulate, reverse white, aerial concentric white milk grey velvety, edge of colony entire, reverse white black velvety, reverse grey yellow, reverse white milk, aerial concentric white, reverse white white edge of colony undulate, reverse white table 2 macroscopic morphology of endophytic fungi part of plant isolate code morphology and color of colony on pda medium at 27-29 °c bacterial colony identification was through observation and was carried out based on the following criteria: color, surface, the margin of the colony as well as gram staining. fungal colony identification was through observation based on the color of colonies and color contrast. sample that matched with this observation criteria were considered to be the same isolate while those that did not match with the criteria were considered as a different isolate. furthermore, individual colonies with different morphology were separated and considered as individual isolates. observation of the morphology was again conducted after 5-7 days of incubation and each isolate was transferred into two different types of culture media (stock and working culture). each stock culture was kept in paraffin lig, while each working culture was kept in slant agar. the 24 h bacterial sample in the na slant was suspended by adding 5 ml of muller hinton broth (mhb). the opacity of bacteria is made equal to the opacity of 0.5 mcfarland standard. then, 1 ml of bacterial suspension was put into a 50 ml erlenmeyer flask containing 9 ml of mhb medium. the fermentation process was done using a shaking incubator for 48 h with a speed of 120 rpm. the secondary metabolites, as product of fermentation, were centrifuged with a speed of 3 000 rpm for 20 min. the supernatant was used for antimicrobial assay (rosana et al. 2001). a section of the fungal isolate that had sporulated (aged 7-10 days) was taken out using a sharp forceps, (dimension of 1 x 1 cm) and was placed into 10 ml liquid fermentation medium of potato dextrose yeast extract broth in a 50 ml erlenmeyer flask. the fermentation process was conducted by using a shaker for 7 days with a speed of 170 rpm. the product of fermentation was centrifuged with a speed of 3 000 rpm for 20 min, and the supernatant was used for antimicrobial assay (rosana et al. 2001). the assay was performed using a paper disk for the fungi and using stainless cylindrical tubes for the bacteria (kumala et al. 2006). the gram positive test bacterial were s. aureus and b. subtilis, the gram negative test bacterial were e. coli and s. typhimurium, and the yeast test microbe was c. albicans. nutrient agar was used as the medium for s. aureus, b. subtilis, e. coli, and s. typhimurium bacterial test and pda was used as the medium for c. albicans yeast test microbe. disks were dipped into the suspension until it was saturated, and then placed on the agar medium that had been inoculated with test microbes. the test microbe was adjusted with 0.5 mcfarland standard. for the bacteria, 100 ml of suspension of the product of fermentation was put into the stainless cylinder that had been placed on the agar medium that had been inoculated with the test microbe. then, it was incubated at a temperature of 37 °c and assayed for two days. assay was by observation which was carried out to determine a clear zone around the disk and stainless cylindrical tubes. the endophytic bacteria isolated from the piece of twig were grown in na medium that had been added with nystatin as an anti-fungal agent. three gram positive endophytic bacteria and two gram negative endophytic bacteria were obtained (table 1). endophytic fungi isolated from the piece of twig were grown in pda medium that had been added with chloramphenicol as an anti-bacterial agent to grow the fungi. eleven endophytic fungi were obtained (table 2). in this study, we screen endophytic microbes that can produce secondary metabolite that has potential antimicrobial substances. the in vitro testing showed that the secondary metabolite of the endophytic microbes produced a zone of inhibition of the test microbes. of the 5 bacteria, all of them (100%) had activity against the test bacteria (s. aureus, e. coli, s. typhimurium, b. subtilis) and on the yeast (c. albicans). the diameter of the zone of inhibition and the illustration of the zone of inhibition can be seen in table 3 and fig 1. table 4 shows the inhibition zone of the endophytic fungi. this study is a preliminary study to find a source of antimicrobial compounds from endophytic microbes. the isolates obtained were fungal and bacterial endophytic microbes. from the isolations carried out, we obtained five endophytic bacterial isolates and eleven endophytic fungal isolates. of the five endophytic bacterial isolates obtained, all of them had the same cell shape, that is rod-shaped, in which 3 isolates were gram positive and 2 isolates were gram negative. identification of endophytic bacterial and fungal isolates from the mengkudu plant was only carried out based on observed morphology and grouping by gram staining. these results showed that endophytic microbes living in the mengkudu plant contained more gram positive than gram negative. the products of bacterial fermentation were tested for anti-microbial activity. the test microbes used were volume 1, 2007 microbiol indones 147 table 3 data for anti-microbial assay of endophytic bacteria using a 1.0 cm cylindrical tube diameter of zone inhibition (cm) staphylococcus aureus bacillus subtilis escherichia coli salmonella typhimurium candida albicans isolate code natgpc 5a napgpc 3b natgpc 2a natgpc 1b natgpc 1c ++ + + + + + + + + + + + + + + + + + + + + + + + + +: < 1.2, ++: > 1.2. fig 1 anti-microbial assay of endophytic fungi towards staphylococcus aureus. èno. 9 shows inhibition zone. pdapg pc 5a1-1 pdapg pc 4-a2 pdatg pc 3a2 pdatg pc 3a pdapg pc 1c pdatg pc 5a pdapg pc 1d pdapg pc 4-c2 pdatg pc 2 pda pg pc 4a1 pdatg pc 5a1-2 + + ++ + + + ++ ++++ + + + + + + + + + ++ + + + + ++ + + + + +++ + + + + + + ++ + ++ +++ + + ++ ++ +++ + table 4 data for anti-microbial assay of endophytic fungi using 0.6 cm paper disk diameter of zone inhibition (cm) staphylococcus aureus bacillus subtilis escherichia coli salmonella typhimurium candida albicans isolate code -: no inhibition, +: < 0.80, ++: > 0.80 < 1.00, +++: > 1.00 < 1.20, ++++: > 1.20. s. aureus, b. subtilis, e. coli, and one yeast c. albicans. the results of the anti-microbial activity test showed that the five endophytic bacterial isolates of the mengkudu plant produced anti-microbial substances. this was demonstrated by the formation of a clear zone surrounding the paper disk, indicating that there was a zone of inhibition of growth of the test microbe. of the five test microbes, s. aureus was the most sensitive to the anti-microbial substances produced by the endophytic bacteria. shaking was used in this assay as kumala et al. (2005) stated that isolated fungal or bacterial endophytes produced more secondary metabolites than if still fermentation methods were used. the product of fungal fermentation in the pdy medium showed that ten endophytic fungal isolates from the mengkudu produced anti-microbial substances. this was demonstrated by the formation of a clear zone surrounding the cylindrical tube, indicating that there was a zone of inhibition of the growth of the test microbe. the four types of test bacteria used, which were s. aureus, b. subtilis, e. coli, s. typhimurium, and the test yeast used, which was c. albicans, were sensitive to the anti-microbial compounds produced by the endophytic fungi. of the five test microbes, s. aureus was the most sensitive to the anti-microbial substance produced by the endophytic fungi. there was one endophytic fungal isolate that was not capable of producing anti-microbial substances. this showed that no zone inhibition of growth of the test microbes. using cylindrical tubes not paper disks, the bacteria isolate needs to obtain a clear zone. for the smalles quantities of anti-microbial agent it is easier to assay using cylindrical tubes rather than paper disks (kumala et al. 2006). the result of this anti-microbial assay showed that the anti-microbial substances produced by the endophytic bacteria and fungi had a broad spectrum because it was capable of inhibiting the growth of gram positive bacteria, gram negative bacteria, and a yeast. furthermore, the number of isolated fungi which were obtained in this study were more than for the bacteria endophytes. these results agree with a previous investigation (kumala et al. 2006). acknowledgements we would like to thank the microbiology laboratory of the medical faculty of the universitas indonesia for the test microbes used in this study. references bacon cw. 1988. procedure for isolating the endophytes from fall rescue and screening isolates for ergot alkaloid. appl environ microbiol 54:2615-2618. [depkes] departemen kesehatan. 1997. survei kesehatan rumah tangga. jakarta: badan penelitian dan pengembangan kesehatan, depkes. kumala s, mangunwardoyo w, budiarti p. 2005. fermentasi diam dan goyang isolat kapang endofit dari brucea javanica l. merr dan uji aktivitas anti-mikroba. j ilmu kefarmasian indones 3:121 5 . 148 kumala et al. microbiol indones kumala s, syarmalina, handayani ar. 2006. isolasi mikroba endofit ranting tanaman johar (cassia siamea lamk) serta uji aktivitas anti-mikroba substansi bioaktif mikroba endofit. j ilmu kefarmasian indonesia 4:15-24. petrini o, sieber tn, toti l, viret o. 1992. ecology, metabolite production, and substrate utilization in endophytic fungi. nat toxin 1:185-196. rosana y, ibrahim f, susilo j, sukara e. 2001. isolasi dan seleksi jamur endofit dari tanaman belimbing wuluh (averhoa blith linn) yang menghasilkan bahan anti-mikotik. j mikol ked indones 2:134-139. strobel ga. 2002. microbial gifts from rain forest: symposium contribution. can j plant pathol 24:14-20. strobel ga, hess wm, ford e, sidhu rs, yang x. 1996. taxol from fungal endophytic and the issue of biodiversity. j indust microbiol 17:417-423. 7.mi690-d barrie *corresponding author; phone: +44-01248382358; e-mail: d.b.johnson@bangor.ac.uk “biomining” is generic term to describe the application of living organisms to extract and recover metals from mineral ores and waste materials. since its inception as a crude technology (“dump leaching”) for treating “run of mine” rocks and boulders that contained too little copper to be processed by conventional processing, engineering options used in biomining have become increasingly refined and diverse. currently, microbiological processing is used to extract both base metals (copper, and to lesser extents nickel and zinc) and precious metals (mostly gold) from ores and mineral concentrates in heaps and stirred-tank bioreactors, as well as in dumps. recent developments include the demonstration, at pilot-scale, of indrect leaching of zinc sulfide concentrates, in which the biological step (regeneration of ferric iron) is carried out independently of abiotic mineral oxidation, and using microbiologically-mediated reductive dissolution of ferric iron minerals to liberate nickel from lateritic ores. keywords: biomining, microbiological process “biomining” adalah istilah umum yang digunakan untuk menggambarkan penggunaan organisme hidup untuk mengekstrak dan memisahkan logam dari bijih mineral dan limbah. biomining pada awalnya dikenal sebagai teknologi untuk mengolah bahan tambang berupa batu-batuan yang memiliki kandungan tembaga yang terlalu sedikit yang tidak mampu diolah menggunakan prosedur konvensional, tetapi penerapan teknologi ini secara perlahan meningkat dan meluas. saat ini, aplikasi mikroorganisme dalam pertambangan digunakan untuk mengekstraksi baik logam dasar (tembaga dan logam yang memiliki nilai yang lebih rendah seperti nikel dan zink) maupun logam mulia (terutama emas) dari bijih logam dan konsentrat mineral yang diolah dalam tangki bioreaktor. pengembangan teknologi ini telah diterapkan dalam skala besar untuk memurnikan konsentrat zink sulfida, dimana dalam tahapannya regenerasi besi ferri dilakukan tanpa menggunakan senyawa abiotik pengoksidasi mineral, tetapi pelarutan reduktif mineral besi ferri secara mikrobiologi sehingga nikel dapat dimurnikan dari bijih tambang. keywords: biomining, proses mikrobiologi evolution of biomining as a niche technology for mineral processing the idea that microorganisms could be used to extract metals from sulfidic ore arose from the discovery in the late 1940's of a bacterium that was able to generate ferric iron from ferrous in acidic liquors, a n d s u b s e q u e n t d e m o n s t r a t i o n s t h a t t h i s microorganism could grow autotrophically on pyrite and other sulfide minerals, causing their dissolution. thiobacillus ferrooxidans, as it was known at the time (it was renamed acidithiobacillus (at) ferrooxidans in 2000) has been one of the most widely studied of all bacteria outside of those of medical importance. while other acidophilic bacteria and archaea can also degrade sulfide minerals in pure or mixed cultures, the early work with at. ferrooxidans remains an important foundation stone for the entire biomining industry. the first recognized application of biomining was by the kennecott copper corporation in the 1950's who obtained a patent for using bacteria to extract copper from their waste rock dumps and developed operations at the bingham canyon mine in utah and later at the chino mine in new mexico (fig 1). since then, mine operations using dump bioleaching of low-grade copper ore have been established world-wide. gradation from dump to heap leaching (described below) began with the use of “thin layer” engineering of mounds (crushed ore, stacked 2.5 to 6 m in height) at the lo aguirre mine, and later demonstration of the benefits of forced aeration of heaps at the quebrada blanca mine (both copper mines, located in chile; brierley 2008). heap bioreactors used to recover metals other than copper have been established in nevada (since 1999; pretreatment of refractory gold ore) and finland (since 2008; bioleaching of a polymetallic black schist at the talvivaara mine, extracting nickel, zinc, and copper). stirred tank bioreactors processing sulfidic ores have review biomining: an established and dynamic biotechnology david barrie johnson college of natural sciences, bangor university, bangor ll57 2uw, united kingdom available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.4.7issn 1978-3477, eissn 2087-8575 vol 6, no 4, december 2012, p 189-193 mines to extract copper from low-grade “run of mine” ores that contain too little of the metal (typically < 0.5% by weight) to merit conventional processing (grinding, concentrating and smelting). heap leaching (fig 2) is a more refined approach for bio-processing low-grade mineral ores, concentrates heap leaching, which entrails agglomeration of the concentrate onto a coarse substrate, has been investigated. the main differences between heap and dump leaching systems are: (i) ores chrushed to facilitate more rapid breakdown of sulfide minerals and, where fine-grain particles are produced, these may be agglomerated with sulfuric acid; (ii) the heaps themselves are generally smaller in height (typically 610 m) than are dumps (though their base areas may exceed those of dump operations) and several successive heaps (or “lifts”) may be stacked upon each other as the operation develops; (iii) heaps are often aerated from beneath as well as irrigated from above, to provide the primary autotrophic mineral-oxidizing microorganisms with both oxygen and carbon dioxide; (iv) heaps are constructed on pads that are lined with impermeable synthetic liners (generally high density polyethylene on which drain pipes, used to collect and convey pls, are placed); (v) inoculation ponds, in which mineraldegrading bacteria and archaea can be grown to high cell densities and then introduced either in the irrigation liquor or else during the mineral agglomeration process, may be used in heap operations. the higher capital cost expenditure results in more rapid (heaps typically operate for about a year) and more extensive (up to 90%) been operating since 1986. most of these are used to oxidize arsenopyrite and pyrite that are present in refractory gold ores, thereby liberating otherwise occluded fine particles of the precious metal. the first plant to be commissioned, at the fairview mine in barberton, south africa, is still in operation today. its design capacity (55 t/day) has far been exceeded by more recent plants established in western australia (wiluna; 158 t/d), china (jinfeng; 790 t/d), ghana (ashanti; 960 t/d) and elsewhere (brierley 2008). one of the more recent stirred tank bioleaching systems to have been commissioned (at kokpatas, uzbekistan) is designed to process over 160,000 t of refractory gold concentrate each day. engineering designs and operational parameters currently, there are two major engineering variants for biomining-irrigated systems and stirred tanks. in the first of these, low grade sulfidic rocks (ranging from large boulders to ground and agglomerated ore) are piled into large dumps or heaps, which are then irrigated with an acidic liquor to stimulate the activities of microorganisms that either occur naturally with the ore or else are inoculated into the heaps. the percolating liquor becomes enriched with metals (and sulfate) released from the sulfide minerals and also other minerals (referred to as gangue minerals) that are unstable in acidic solutions. this “pregnant leach solution” (pls) is collected from outflow channels from the base of the dump or heap, and the target metal(s) extracted. dump leaching was the first practice of biomining to be adopted, and is still operated at many fig 1 dump leaching of “run-of-mine” ore at the kennecott chino mine, new mexico. acid irrigation impermeable membrane pregnant leach solution air blowers metal extraction than is achieved in dump operations. again, the main metal that is recovered in heap biomining operations is copper, though a polymetallic black schist ore (where the major metal in terms of its economic value is nickel) is currently processed via heap leaching in finland, and heap leaching of a refractory gold ore was operated for over a decade in a site in nevada (brierley 2007). bioprocessing of sulfidic mineral concentrates in fig 2 schematic of a heap leaching operation. 190 johnson microbiol indones stirred tanks that are continually aerated and maintained at prescribed temperatures and ph values constitute the most efficient biomining systems currently in operation, in terms of both speed and efficiency of the operation. however, the greater costs involved in constructing and maintaining the vast 3 bioreactors used (each up to 1,350 m -the largest reactors used for any biological-based operation) means that these have been operated as full-scale commercial systems for recovering gold rather than base metals, with the single exception of a bioleaching plant in uganda where cobalt is the target metal. most commercial stirred tank biomining systems operate at ph of about 1.5 and temperatures between 40 and 45 °c. since the oxidative dissolution of sulfide minerals is an exothermic reaction, cooling rather than heating of the tanks is required, constituting (along with stirring of the dense mineral slurries -20% pulp density is typical) a significant part of the running costs of any single operation. mechanisms of oxidative dissolution of minerals by microorganisms the mechanism(s) by which microorganisms catalyze the oxidative dissolution of sulfide minerals has been the subject of considerable debate for many years. minerals, such as pyrite, are stable in environments that are totally dry or from which oxygen is excluded. in biomining operations, where solution ph is generally < 2.5, soluble ferric iron is the main oxidant of sulfide minerals, as illustrated (for pyrite) in equation [1]: 3+ 2+ 2+ fes + 6fe + 3h o 7fe + s o + 6h [1]2 2 2 3 ferric iron is consumed in this reaction and its, regeneration is primarily mediated (except in very high temperature liquors) by iron-oxidizing bacteria and archaea, in an oxygen-requiring reaction [2]: 2+ + 3+ fe + 0.25o + h fe + 0.5h o [2]2 2 sulfide minerals can be divided into those that are a c i d s o l u b l e ( e . g . s p h a l e r i t e , c h a l c o p y r i t e , arsenopyrite and galena) and others that are not susceptible to proton attack (e.g. pyrite, molybdenite and tungstenite (ws ). in acidic liquors, the latter are 2 oxidized by soluble ferric iron, and a total of six successive one-electron oxidation steps are required to break the sulfur-metal bonds. since the initial sulfur product released from the degrading mineral is thiosulfate, this form of oxidative sulfide mineral dissolution has been described as the “thiosulfate mechanism” (rohwerder et al. 2003). thiosulfate is unstable in acidic liquors (more so when ferric iron is present) and oxidizes via tetrathionate and other sulfur oxy-anions, ultimately to sulfate. the microbiology of biomining operations microorganisms that are involved in biomining operations have been described in a number of reviews (e.g. schippers 2007); rawlings and johnson 2007b. acidophiles that can have either a direct or indirect process in the dissolution of sulfide minerals can be categorized as primary, secondary and tertiary microorganisms. primary bacteria and archaea are those chemo-autotrophs that can catalyze ferrous iron oxidation, and thereby initiate mineral oxidation; secondary microorganisms are sulfur-oxidizers that generate the acidity required for the acidophilic consortia, while tertiary acidophiles are those that degrade organic compounds excreted from living bacteria (most of which in biomining operations are autotrophic) or lysed from dead cells, and thereby help maintain suitable conditions for the more “organic sensitive” lithotrophs, such as leptospirillum spp.. in reality, there is considerable overlap between these categories. for example, some acidithiobacillus spp. (at. ferrooxidans and at. ferrivorans) can oxidize both ferrous iron and reduced sulfur as can all sulfobacillus spp., and some heterotrophic acidophiles (e.g. ferrimicrobium acidiphilum and ferroplasma spp.) can also oxidize iron as can some mixotrophic acidophiles (e.g. acidimicrobium ferrooxidans). primary producers in stirred tank operations are exclusively chemolithoautotrophic bacteria. another important characteristic that can be use to differentiate acidophilic bacteria and archaea is their response to temperature. a pragmatic and much-used distinction has been made between (extreme) thermophiles, that have temperature optima above 60 °c, moderate thermophiles (40-60 °c) and mesophiles (2040 °c). archaea account for almost all of the first group, most moderate acidophiles are gram-positive bacteria, and gram-negative bacteria make up the majority of mesophilic species. although oxidative dissolution of simple and complex sulfide ores and concentrates may be mediated by pure cultures of iron-oxidizing acidophiles, as has often been described in laboratory studies, axenic cultures are never found in actual volume 6, 2012 microbiol indones 191 acidophiles contribute to the robustness and efficiency of the consortia by removing material that might otherwise inhibit the primary mineral-degraders. in recent years, the identities of acidophiles involved in pilot-scale and full-scale biomining operations have been revealed by the application of biomolecular techniques, while advances in techniques for isolating and cultivating physiologically and phylogenetically diverse acidophiles in the laboratory have facilitated understanding of the nature and importance of microbial interactions in biomining environments. indirect leaching of metal sulfide concentrates one alternative engineering design option for biomining that has been demonstrated successfully at the pilot-scale is “indirect leaching”. in this, the biological regeneration of ferric iron is separated from the oxidation of the target mineral by ferric iron (which is an abiotic reaction). in this way, it is possible to optimize conditions for the different reactions, e.g. the abiotic reaction is generally faster at elevated temperatures and does not require oxygen, in contrast to the biological process (fig 3). another advantage with this approach is that the sulfide moiety in the mineral can be oxidized to elemental sulfur, rather than sulfate. bioprocessing of oxidized metal ores by reductive dissolution at present, all biomining operations use sulfide ores, and metals are recovered by a process of oxidative dissolution of metal-bearing minerals. some metal ores, however, are already oxidized (such as nickel laterites, biomining operations. consortia of microorganisms with synergistic (and sometimes complimentary) metabolic physiologies, have been identified in all commercial-scale systems that have been examined. laboratory studies using defined populations of acidophiles have demonstrated that mixed cultures are more robust and are also frequently superior to pure cultures in terms of leaching kinetics. all biomining operations necessarily operate as open and non-sterile systems. however, because of the prevailing conditions, only living organisms that are able to survive in environments that are both highly acidic and that contain greatly elevated concentrations of toxic metals and other dissolved solutes are active in biomining operations, which restricts indigenous life forms almost exclusively to some “extremophilic” bacteria and archaea. metal extraction occurs due to the oxidative dissolution of sulfide minerals in which the metals either occur (e.g. in the case of copper) or else are associated (e.g. in the case of gold) with sulfides, and the primary prokaryotes involved are autotrophic iron-oxidizers that continually regenerate the main oxidizing agent (ferric iron) that initiates mineral dissolution. however, microbial consortia rather than individual species are involved in mineral dissolution in both heaps and stirred-tanks. autotrophic sulfuroxidizers generate sulfuric acid from reduced sulfur species that originate from the degraded sulfide minerals and, by so-doing, maintain the extremely low ph environment that promote both the activities of the iron-oxidizers and the effectiveness of ferric iron as an oxidizing agent. a third group of prokaryotic acidophiles that degrade organic carbon compounds that derive from active and dead carbon-fixing mineral leaching reactor (anoxic: 80-90 °c) zns bacteria pls 2+ 2+ zn & fe zn stripping 3+ fe 2+ fe 3+ fe regeneration reactor (aerated; ~45 °c) fig 3 schematic of the “indirect” bioleaching of zinc sulfide concentrate. 192 johnson microbiol indones which represent about 70% of nickel ores in the lithosphere) and therefore such an approach cannot be used to process them. a novel technique has, however, recently been devised and demonstrated for extracting nickel from lateritic ores using specialised bacteria (fig 4; hallberg et al. 2011). in this, ground ore is mixed with elemental sulfur and inoculated with the acidophilic iron-reducing bacterium, at. ferrooxidans. when oxygen is depleted, the bacteria couple the oxidation of sulfur to the reduction of ferric iron present in goethite, which is the most abundant mineral in the ore and with which much of the nickel is intimately associated. the goethite is disrupted and ultimately destroyed in this process (known as “reductive mineral dissolution”), and the nickel is solubilised. since the process operates in an acidic liquor (ph ~ 1.8) the nickel remains in solution, which facilitates its downstream recovery by ionexchange, solvent extraction/electrowinning or sulfide precipitation. this new approach to biomining demonstrates further the potential for using biological systems in “green” technologies relating to the mining industry. acknowledgment the author is grateful to the royal society (u.k.) for the provision of an industrial fellowship. references brierley cl. 2008. how will biomining be applied in future? t nonferr metal soc. 18(6):1302-1310. doi: 10.1016/s1003-6326(09)60002-9. brierley ja. 2007. a perspective on developments in biohydrometallurgy. hydrometallurgy 94(1-4):2-7. doi: 10.1016/j.hydromet.2008.05.014. hallberg kb, grail bm , plessis c.du, db johnson. 2011. reductive dissolution of ferric iron minerals: a new approach for bioprocessing nickel laterites. miner eng. 24(7): 620-624. doi: 10.1016/j.mineng.2010.09.005 . johnson db. 2010. the biogeochemistry of biomining. in: barton l, mandl m, editors. geomicrobiology: molecular and environmental perspective. dordrecht: springer. p 401-426. doi: 10.1007/978-90-481-92045_19. rawlings de, johnson db. 2007a. biomining. springerverlag, heidelberg. doi: 10.1007/978-3-540-34911-2. rawlings de, johnson db. 2007b. the microbiology of biomining: development and optimization of mineraloxidizing microbial consortia. microbiology 153(2): 315-324. doi: 10.1099/mic.0.2006/001206-0. rohwerder t, gehrke t, kinzler k, sand w. 2003. bioleaching review part a: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. appl microbiol biotechnol. 63(3): 239-248. doi: 10.1007/s00253-003-1448-7. schippers a. 2007. microorganisms involved in bioleaching and nucleic acid-based molecular methods for their identification and quantification. in: donati er, sand w, editors. microbial processing of metal sulfides. dordrecht, the netherlands, springer. 3-33. doi: 10.1007/1-4020-5589-7_1. reductive dissolution 2+ 2+ fe , ni , oh fig 4 extracting from lateritic ores using specialised bacteria.nickel volume 6, 2012 microbiol indones 193 5.mi712-gunnar sandstorm available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.3.5issn 1978-3477, eissn 2087-8575 vol 7, no 3, september 2013, p 124-128 *corresponding author; phone/fax: +46-8-524-832-79, email: gunnar.sandstrom@ki.se aloe sinkatana is a clumping rosettes plant belonging to family xanthorrhoeaceae, subfamily asphodeloideae, genus aloe. it is found in eastern of sudan in red sea mountains mainly in arkawit area. the plant is up to 60 cm tall and 60-90 cm in diameter (reynolds 1957). anthraquinone glycosides; is an aromatic organic compound with the formula c h o with several isomers, each of 14 8 2 them is known as a quinone derivative. three different solvents such as water, ethanol and acetone were used to extract the bioactive compounds from the leaves of a. vera to screen the antimicrobial activity of selected human pathogens by agar diffusion method. the the active component in aloe vera is which antifungal activity of a. vera was analysed against aspergillus flavus and aspergillus niger. the maximum antifungal activity was observed in acetone extracts when compared with other extracts. a. vera plant extract with acetone can be used as antimicrobial agents (arunkumar and muthuselvam 2009). the comparative antimicrobial activities of the gel and leaf of a. vera were tested against staphylococcus aureus, pseudomonas aeruginosa, trichophytonmenta graphytes, t. schoeleinii, microsporium canis, and candida albicans (pandey and mishra 2010). ethanol was used for the extraction of the leaf after obtaining the gel from it. antimicrobial effect was measured by the appearance of zones of inhibition (pandey and mishra 2010; du plessis and hamman 2013). antimicrobial susceptibility test showed that both the gel and the leaf inhibited the growth of s. aureus. aloe sinkatana is a plant belonging to the family xanthorrhoeaceae, subfamily asphodeloideae, genus aloe. it is cultivated in the red sea mountainseastern of the sudan. in the present study, the extract of a. sinkatana leaves was screened for its antibacterial and antifungal activity. the phytochemical screening of a. sinkatana extracts was carried out using thin layer chromatography (tlc) technique. four extracts of a. sinkatana were prepared using chloroform, ethanol, methanol, and water. antibacterial activity of the extract was performed following the cup-plate agar diffusion method. also, the antifungal activity of the extract was tested. the result showed that the extracts of a. sinkatana leaves revealed antimicrobial activities greater than the commercial antifungal (nystatin) which can be used for treatment of candidiasis and ketoconazole that is used for treatment of fungal infection. the antimicrobial activity might be due to specific plant compounds, which was found to be more effective than commercial antifungal compounds. however the commercial antibiotic used for treatment of bacterial infection, displayed better antimicrobial activity than the a. sinkatana extracts. in conclusion a. sinkatana extract can be a useful treatment against fungal infections. key words: antibacterial, antifungal aloe sinkatana adalah tanaman yang termasuk keluarga xanthorrhoeaceae, subfamili asphodeloideae, genus aloe . tanaman ini dibudidayakan di red sea mountainseastern, sudan . dalam penelitian ini, dilakukan skrining aktivitas antibakteri dan antijamur dari ekstrak daun a. sinkatana. skrining secara fitokimia dari ekstrak a. sinkatana dilakukan dengan menggunakan teknik kromatografi lapis tipis ( klt ). enam jenis ekstrak a. sinkatana dipersiapkan dalam kloroform, etanol, metanol, dan air. aktivitas anti bakteri dari ekstrak dilakukan mengikuti metode cup-plate agar diffusion. demikian pula dilakukan uji aktivitas antijamur telah dilakukan. hasil penelitian menunjukkan bahwa ekstrak dari a. sinkatana mengungkapkan kegiatan antimikroba lebih besar daripada anti jamur komersial, nistatin, yang digunakan untuk pengobatan candidiasis, dan ketoconazolethat yang digunakan untuk pengobatan infeksi jamur. aktivitas anti mikroba mungkin disebabkan karena senyawa tertentu, yang lebih efektif daripada senyawa anti jamur komersial. akan tetapi, antibiotik komersial untuk pengobatan infeksi bakteri, mempunyai aktivitas anti mikroba lebih baik daripada ekstrak a. sinkatana. sebagai kesimpulan ekstrak a. sinkatana berpotensi sebagai obat infeksi jamur. kata kunci: aloe sinkatana, anti bakteri, anti jamur aloe sinkatana, antimicrobial activity of aloe sinkatana 1 2 abubaker ali , elham abdelbasit suleiman , 3 3 amir saeed , and gunnar sandström * 1 university of medical sciences and technology, faculty of medical laboratory sciences, khartoum 12810, sudan; 2 veterinary research institute department of mycology, khartoum 12810, sudan; 3 karolinska institute, department of laboratory medicine, division of clinical microbiology, karolinska university hospital, huddinge, se-14186 stockholm, sweden only the gel inhibited the growth of t. mentagrophytes while the leaf possesses inhibitory effects on both p. aeruginosa and c. albicans. the results of this stimulate the use of both a. vera gel and leaf (o et al. 2005; pandey and mishra 2010). the aim of this study is to test the antimicrobial activities of the a. sinkatana. there is no work done on a. sinkatana so far and accordingly, this is the first report on investigation on its antimicrobial activity. materials and methods plant material. a. sinkatana plant was obtained from faculty of pharmacy collection at the university of medical science and technology, khartoum, sudan. preparation of the crude extracts. coarsely powdered a. sinkatana leaves (80 g) were extracted for twenty hours with chloroform in soxhlet apparatus. the chloroform extract was filtered and evaporated under reduced pressure. the extracted leaves were airdried, re-packed in soxhlet till exhaustively extracted with methanol. the methanolic extract was filtered and evaporated under reduced pressure. the residue of chloroform was re-dissolved in a mixture of petroleum ether and methanol in the ratio of (1:2 v/v) and the methanol extract was re-dissolved in methanol. the final product was kept in a refrigerator till used. simultaneously, water extract was prepared by adding 10 ml of boiled distilled water to a sample of 10 g of the coarsely powdered plant materials in a beaker, with occasional shaking for four hours and the final volume was adjusted to 10 ml with boiled distilled water at a temperature of 25 °c. the aqueous extract was filtered and the precipitate was washed in distilled water and the filtrate was used immediately (almagboul, 1992) . phytochemical screening of a. sinkatana extracts. the phytochemical screening was carried out according to qualitative methods described. six extracts have been obtained such as chloroform extract, methanol extract, and aqueous extract. the powdered dried samples of the leaves of a. sinkatana were separately screened for the following constituents: carbohydrates and/or glycosides, tannins, flavonoids, saponins, alkaloids and/or nitrogenous bases, anthraquinons, unsaturated sterols and/or triterpenes, and coumarins using water and organic solvents according to the required material. preparation of the test organisms. one ml of a 24 h broth culture of the test organism was aseptically distributed onto nutrient agar slopes and incubated at 37 °c for 24 h. the bacterial were harvested and washed in sterile normal saline, and suspended in a small volume of normal saline to produce a suspension containing 8 9 about 10 -10 colony forming units per ml. the suspension was stored in a refrigerator at 4 °c till used. the average number of viable organism per ml of the stock suspension was determined by means of the surface viable counting technique (miles and amyes, 1996). serial dilutions of the stock suspension were made in sterile normal saline, and dropping pipettes to the surface of dried nutrient agar plates transferred 0.02 ml of the appropriate dilutions. the plates were allowed to stand for 2 h at room temperature, following incubation at 37 °c for 24 h. after incubation the number of colonies in each drop was counted. the average number of colonies per drop (0.02 ml) was multiplied by 50 and by the dilution factor to give the viable count of stock suspension, expressed as the number of colony forming units per ml of suspension. each time a fresh stock suspension was prepared. antibacterial activity of the extract. the cupplate agar diffusion method (kavanagh, 1972), was adopted with some minor modification to assess the antibacterial activity of the prepared extracts. one ml of 8 9 the standardized bacterial stock suspension (10 -10 colony forming units per ml) was thoroughly mixed with 100 ml of sterile molten muller-hinton agar, which was maintained at 45 °c. 20 ml aliquots of the inoculated muller and hinton agar were distributed into sterile petri dishes. the agar was left to set. on each of these plates, four cups (10 mm in diameter) were cut using a sterile cork borer and agar discs were removed. the cups were filled with 0.1 ml sample of each of the extract using standard fine pipette adjustable volume digital pipette, and allowed to diffuse at room temperature for two hours. the plates were incubated in the upright position, at 37 °c for 18 h. three replicates were made for each extract against each of the tested organisms. simultaneously, positive control was inoculated using respective solvents. after incubation, the diameters of the resultant growth inhibition zones were measured. the average mean values were tabulated. to determine whether the plant extracts were bacteriostatic or bactericidal subcultures were made from within the zones of inhibition onto muller and hinton agar and incubated at 37 °c for 24 h. then the plates were examined for bacterial growth. growth of the organism indicates the bacteriostatic activity of extract and no growth indicates its bactericidal effect. antifungal activity of the extracts. the fungal tested organism a. flavus and a. niger were spread over the sabouraud’s dextrose agar plates after the volume 7, 2013 microbiol indones 125 microbial, four cups (10 mm in diameter) were cut using a sterile cork borer and agar discs were removed. the cups were filled with 0.1 ml samples of each of the extract, and allowed to diffuse at room temperature for 2 h. the plates were incubated in the upright position, at 30 for 72 h. three replicates were made out for each extract against each of the tested organisms. simultaneously, positive control was involved by adding respective solvents instead of the extracts. after incubation, the diameters of the resultant growth inhibition zones were measured in mm, the average mean values were tabulated. for other fungal tested organisms, one ml of the extract was thoroughly mixed with 100 ml of sterile molten sabouraud’s dextrose agar which was maintained at 45 °c. twenty ml aliquots of the inoculated sabouraud’s dextrose agar were distributed into sterile petri-dishes. the agar plates were left to set; the test organisms were inoculated in the center of plate. positive control was inoculated in the same manner as above. antimicrobial susceptibility-test susceptibility test procedure. plates with mueller and hinton agar were prepared according to a method previously described (bauer and driesen 1966). for rapidly growing aerobic organisms, 3-4 similar colonies from pure cultures as inoculum were selected and transferred into 5 ml of tryptone soya broth. incubation was performed at 35 °c for 2-8 h until moderate turbidity developed. a sterile non-toxic cotton swab on a wooden applicator was dipped into the standarized °c inoculum and the soaked swab was firmly rotated against the upper inside wall of the tube angle between each streaking. the inoculum was allowed to dry for 515 min with lid in place. using aseptic technique the discs were applied. the discs with centers at least 24 mm apart were deposited. for penicillin and cephalosporin, the discs were deposited with centers 30 mm apart. the plates were incubated immediately at 37 and examined after 14-19 h. zones showing complete inhibition was measured (bauer and driesen 1966) . result the antimicrobial activity against s. arues, e. coli, and p. aerginosa. the antimicrobial activity of different antibiotica and a. sinkatana in different solvent systems against s. arues, e. coli, and p. aerginosa was estimated and revealed inhibition zones (table 1). in comparison virtually no difference could be seen between antibiotica tested and extracts from a. sinkatana tested. the antimicrobial activity of different antibiotica and a. sinkatana in different solvent systems against c. albicans, a. niger, a. flavus, t. mentaegraphytes, and phialophorarichardsiae revealed inhibition zones (table 2). all dermatophytes tested revealed sensitivity to all extracts. furthermore, no growth was observed on sabouraoud dextrose agar after a. sinkatana extracts treatment. thus, the extracts of a. sinkatana tested against dermatophytes included in experiments °c table 1 effect of aloe sinkatanain in different solvent systems and antibiotic against against staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa s. aureus p. aeruginosa e.coli solvent system solvent inhibition zone (mm) chloroform 19 18 17 methanol 22 20 20 water 0 14 14 antibiotic co-trimexazole 30 32 gentamycin 20 19 21 ciprofloxacin 35 35 38 amoxycillin 30 ceftriaxone 26 30 cephalothin 34 ampicillin 34 amikacin 30 imipenem 32 126 ali et al. microbiol indones flavonoids, tannins, saponins, carbohydrates, steroids, and reduced sugars. these results are similar to previous results (arunkumar and muthuselvam 2009) showing that a. vera contains tannin, saponin, and flavonoids and there are findings reported a. vera to have monoand polysaccharides, tannins, sterols, organicacids, enzymes, saponins, vitamins, and minerals (rodriguez et al. 2010; nejatzadeh-barandozi 2013). thus, this shows that a. sinkatana and a. vera share almost the same constituents. antibacterial activity of a. sinkatana against s. aureus, p. aeruginosa, and e. coli revealed antibacterial activity in the methanol extract compared to the other extracts. among the three bacterial organisms tested the maximum growth suppression was observed with s. aureus (22 mm) compared with e. coli and p. aeruginosa. this finding harmonise to a previous finding, (arunkumar and muthuselvam 2009) in which it was shown that a. vera leafs inhibited growth of s. aureus, s. pyogen, p. aeruginosa, and e. coli. this might be due to the anthraquinones compound which disclosed to have an antimicrobial activity (ernst 2000). according to available literature there is no work done on antimicrobial activity of a. sinkatana and performed show antimicrobial activity. two fungi were tested on the antimicrobial activity of a. sinkatana, t. verrucosum, and m. canis were tested in different solvent system of a. sinkatana and revealed growth inhibition (table 3). clearly the effect of a. sinkatana extracts against t. verrucosum was indisputable since all extracts rendered no growth detectable. m. cains on the other hand showed growth after treatment of one extract which was chloroform. all dermatophytes tested revealed high sensitivity to all extracts. no growth was observed when culturing them on sabouraoud’s dextrose agar. discussion it can safely be presumed that the major part of a traditional medicine involves the use of plants and their derived active principles, although their use is not always verified by the scientific means, very little and scattered investigations have been carried out searching for plants with antimicrobial activity. in the present study the phytochemical screening of a. sinkatana showed that the plant contains same ingredients as in a. vera such as anthracene, alkaloids, table 2 inhibition zones of aloe sinkatana against candida albicans, aspergillus niger, flavus, tricophyton mentagrophytes, and phialophorarichardsiae in different solvent systems aspergillus table 3 inhibition of growth by aloe sinkatana against microsporum cains, and tricophyton verrucosum in different solvent c. albicans a. niger a. flavus t. mentaegraphytes p. richardsiae solvent system solvent inhibition zone (mm) chloroform 23 18 20 22 20 methanol 22 20 21 18 15 water 19 0 0 0 0 antifungal agents nystatin 17 ketoconazole 100 mg 27 25 - ketoconazole 50 mg 24 20 - ketoconazole 25 mg 18 18 - fungi growth inhibition solvent system tricophyton verrucosum chloroform methanol water no growth no growth no growth no growth microsporum canis growth growth volume 7, 2013 microbiol indones 127 bauer km, driesen d. 1966. [cerebral aneurysm and seminoma as a combined malformation. report on 2 clinical cases]. med welt. 46:2492-2494. du plessis l.h, hamman j h. 2013. in vitro evaluation of the cytotoxic and apoptogenic properties of aloe whole leaf and gel materials. drug chem toxicol. doi:10.3109/0148 0545.2013.83435. ernst e. 2000. adverse effects of herbal drugs in dermatology. br j dermatol. 143(5):923-929. doi:10.1046/j.13652133.2000.03822.x. farnsworth nr, akerele o, bingel as, soejarto dd, guo z. 1985. medicinal plants in therapy. bull world health organ. 63(6):965-981. miles rs, amyes sb. 1996. laboratory control of antimicrobial therapy. in practical medical microbiology, new york, churchill livingstone. nejatzadeh-barandozi f. 2013. antibacterial activities and antioxidant capacity of aloe vera. org med chem lett. 3(1):5. doi:10.1186/2191-2858-3-5. newall ca, anderson la, phillipson jd. 1996. herbal medicines: a guide for health-care professionals. london, england: pharmaceutical press. agarry oo, olaleye mt, bello-michael co. 2005. comparative antimicrobial activities of aloe vera gel and leaf. afr j biotechnol. 4(12):1413-1414. pandey r, mishra a. 2010. antibacterial activities of aloe barbadensis to clinically isolated bacterial pathogens. appl biochem biotechnol. 160(5):1356-1361. doi:10.1 007/s12010-009-8577-0. reynolds. 1957. classification of aloe sinkatana. j african botany. 23-29. rodriguez er, martin jd, romero cd. 2010. aloe vera as a functional ingredient in foods. crit rev food sci nutr. 50(4):305-326. doi:10.1080/10408390802544454. sharma d, lavania aa, sharma a. 2009. in vitro comparative screening of antibacterial and antifungal activities of some commo plants and weeds axtracts. asian j exp sci. 23:169-172. van gorkom ba, de vries eg, karrenbeld a, kleibeuker j h. 1999. review article: anthranoid laxatives and their potential carcinogenic effects. aliment pharmacol ther. 13(4):443-452. doi:10.1046/j.1365-2036.1999.0 0468.x. accordingly this is the first report on such work. antifungal activity of a. sinkatana against c. albicans, a. flavus, a. niger, microsporum canis, trichophyton mentagraphytes, t. verrcosum spp., and phialophorarichardsiae could be estimated and the revealed maximum antifungal activity with ethyle acetate extract was observed against a. niger (28 mm). this finding is similar to previous findings, where it has been shown that, the maximum antifungal activity of a. vera was observed in a. flavus (15 mm) (arunkumar and muthuselvam 2009). moreover, a. sinkatana extracts have shown to inhibit the growth of fungi that cause tinea since complete inhibition of growth of m. canis and t. verrucosum was observed. in the present study, the extracts of a. sinkatana leaves revealed antimicrobial properties greater than commercial antifungal agent (nystatin) used for treatment of c. albicans and ketoconazole used for treatment of a. niger and a. flavus, in conclusion present study revealed the presence of secondary metabolites in the leaves of a. sinkatana. it was further shown that the plant extracts may be used for the treatment of fungal infections such as ringworm and aspergillosis. the results lend credence to the folkloric use of this plant in treating microbial infection and shows that a. sinkatana could be exploited for new potent antimicrobial agents especially antifungal agents. this becomes more relevant as current antimicrobial agents in use are fast loosing effectiveness due to emergence of resistant microorganisms. reference almagboul az. 1992. antimicrobial and phytochemical investigation of vernonia and other sudanese medicinal plants. phd, university of khartoum. arunkumar s, muthuselvam m. 2009. analysis of phytochemical constituents and antimicrobial activities of aloe vera l. against clinical pathogens. world j agr sci. 5(5):572-576. 128 ali et al. microbiol indones 03 putrie.cdr vol.11, no.1, march 2017, p 18-22 doi: 10.5454/mi.11.1.3 studies for iaa (indole-3-acetic acid) production by isolates h6 with nitric acid mutation rahayu fitriani wangsa putrie*, tiwit widowati, sylvia jr lekatompessy, and harmastini sukiman plant symbiotic microbes laboratory, research center for biotechnology, indonesian institute of sciences (lipi) jalan raya bogor 46 cibinong 16911, indonesia nitric acid mutations are known could be used for strain improvement. this research aimed to study indole3-acetic acid (iaa) production by nitric acid mutan were compared with wild type. mutation were conducted with some different treatment time such as 0, 30, 60, 90, and 120 min subsequently it were measured for iaa production. isolate h6 as wild type isolates were also molecularly identified. the wild strain exhibited 53.83 µg -1 -1 -1 ml of iaa while the nitric acid mutan within a range 77.39 µg ml to 95.70 µg ml . isolates h6.60 exhibited -1 the highest iaa production which 39.87 µg ml higher were compared with wild-type. based on 16s rrna gene analysis, isolate h6 had similarity with lysobacter sp. es2-22. key words: iaa production, mutation, nitric acid mutasi asam nitrat diketahui dapat digunakan untuk perbaikan galur. tujuan dari penelitian ini adalah untuk mempelajari produksi indole-3-asam asetat (iaa) yang dihasilkan oleh mutan asam nitrat dibandingkan dengan tipe liarnya. mutasi dilakukan dengan beberapa perbedaan perlakuan waktu yaitu 0, 30, 60, 90, dan 120 menit kemudian dilakukan pengukuran produksi iaa. isolat h6 sebagai isolat tipe liar juga diidentifikasi secara -1 molekuler. tipe liar dapat menghasilkan 53,83 µg ml iaa, sedangkan mutan asam nitrat menghasilkan iaa -1 -1 dalam kisaran 77,39 µg ml sampai 95,70 µg ml . isolat h6.60 menghasilkan produksi iaa tertinggi yaitu -1 39,87 µg ml lebih tinggi dibandingkan dengan tipe liar. berdasarkan identifikasi gen 16s rrna isolat h6 mempunyai kemiripan dengan lysobacter sp. es2-22. kata kunci: asam nitrat, mutasi, produksi iaa microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-5663232 ext. 8765, fax:+62-21-5602575; email: raha009@lipi.go.id regulation and nutritional balance, resistance induction for against plant pathogens, solubilizing nutrients for easy uptake by plants also synergistic and antagonistic interactions with microorganisms within the rhizosphere and beyond in bulk soil, which indirectly boosts plant growth rate (vejan et al. 2016). production of indole-3-acetic acid (iaa) hormone are widespread in a major property of rhizosphere bacteria that could stimulate and facilitate the plant growth (glick and pattern 1996; damam et al. 2016). iaa function is to promote the growth by several ways in plant. iaa are known promote growth of auxiliary bud and bud formation, help in the apical dominance, stimulate lateral and adventitious root development and growth. besides development, iaa were also play crucial role in leaf and flower abscission (kundan et al. 2015). genus of pgpr could produce iaa by several pathway. biosynthesis of iaa which are used by single bacterial strain sometimes containing more than one pathway (patten and glick 1996). the most widely pathway of iaa biosynthetic in bacteria is indole-3pyruvate (ipa) and indole-3-acetamide (iam) anabolic pathways. those pathways used tryptophan as plant growth promoting rhizobacteria (pgpr) are a group of microorganisms which inhabiting around or on the root surface. they are known for plant growth promotion and development,both directly or indirectly by producing and secreting of various regulatory chemicals in the vicinity of rhizosphere (ahemad and kibret 2014). pgpr could be used as inoculants of biofertilizers in agriculture (daman et al. 2016). the genera of pgpr include azoarcus, azobacter, azorhizobium, azospirillum, azotobacter, bacillus, beijerinckia, burkholderia, chryseobacterium, frankia, gluconacetobacter, herbaspirillum, mycobacterium, paenibacillus, phyllobacterium, p s e u d o m o n a s , r h i z o b i u m , s i n o r h i z o b i u m , sphingomonas, and streptomyces (vejan et al. 2016). in addition, genus acetobacter, enterobcter, actinobacteria, brevibacillus, lysinibacillus, terribacillus and jeotgalibacillus also included pgpr (kundan et al. 2015). the mechanisms of pgpr include hormonal volume 11, 2017 microbiol indones 19 precursor. ipa pathway mainly conducted by plant growth promoting bacteria (pgpb), whereas the iam pathway is present in phytopathogenic bacteria (vegaceledon et al. 2016). rhizobium sp. could produce iaa through the iam and ipya pathway (spaepen et al. 2007). the expression level of iaa are depending on biosynthesis pathway, location of genes involved, either on chromosomal or plasmid dna, their regulatory sequences, and the presence of enzymes that could convert it to more active, free iaa into an inactive also conjugated form. (patten and glick 1996). pseudomonas spp. were isolated from the rhizosphere of soybean plants in cirebon, west java has been known have ability to produce the iaa hormone iaa within a range 0.33 ppm to 23.04 ppm in medium culture were added of tryptophan. those isolates could promote the growth of seeds in vitro assay (wahyudi, astuti and giyanto 2011). isolates of bacillus sp. were derived from soybean rhizosphere in cirebon, west java also known had ability to produce iaa with -1 different concentrations within a range 0.81 mg ml to -1 15.16 mg ml (wahyudi et al. 2011). level of iaa concentration is regulated in plants. bacterial rhizosphere have been known could modulate that iaa levels (vega-celedon et al. 2016). iaa hormone which produced by bacteria are known an efficient biofertilizer inoculants to promote the plant growth (damam et al. 2016). therefore required a technique to improve a potential bacterial strain that had ability for iaa production. that one way were conducted by mutation. mutations are known as change in the nucleotide sequence of the genetic material, which could result in amino acid sequence modification of the protein encoded by the gene (tanja van mourik 2013). nitric acid mutations are known could be used for strain improvement. production of lasparaginase from a marine fungus beauveriabassiana ss18/14 which were obtained from nitric acid mutants had higher asparaginase production. the wild strain -1 produced 6.32 iu ml of l-asparaginase activity while -1 nitrous acid mutant uvf4-n-2 exhibited 10.44 iu ml enzyme activity. the strain improvement programme could increased l-asparaginase activity 1.65 times if compared to the wild strain (kamalakumari et al. 2015). therefore, this research aimed to improve of iaa production by h6 isolate with nitric acid mutation. materials and methods nitric acid mutation. the suspension of parent strain was prepared by using acetate buffer ph 7.5. a total of 1 ml of culture suspension was centrifuged subsequently the pellets were subjected to nitrous acid (0.1m sodium nitrite in buffer of phosphate) treatment with different time such as 0, 30, 60, 90 and 120 min by incubating the mixture at 30 °c. after incubation, the suspension was centrifuged at 10.000 rpm. the pellets were washed twice with phosphate buffer (ph 7.0) and suspended in phosphate buffer. samples were diluted -1 and plated on to nutrient agar (na) (23 g l ) medium. the plates were incubated at 28 °c for 24 h. (kamalakumari et al 2015). each of colony are grown subsequently calculated for number of colonies. test of iaa production. mutant isolates of each treatment and wild-type were inoculated into 10 ml nb medium supplemented with 0.2 mm tryptophan and incubated for 24 h on a shaker. a total of 2 ml bacterial culture mutant and wild-type h6 centrifuged at 10.000 g for 10 min the temperature of 4 ºc. iaa production was evaluated by the colorimetric method as described by gordon and weber (1951). the supernatant was taken and inserted into a test tube susequently added 2 ml of salkowsky reagent (150 ml concentrated h so , 250 ml of distilled water, and 2 4 7.5 ml of 0.5 m fecl ). reagent was mixture with 3 supernatant and incubated at room temperature in the dark condition for 30 min. furthermore, an absorbance was measured using a spectrophotometer at a wavelength of 520 nm. absorbance results are used to calculate the concentration of iaa with the equation obtained from the iaa standard curve. identification based on 16s rrna gene analysis and phylogenetic tree. isolates endophytic h6 derived from root nodule of green beans which were planted in wonosari, central java. h6 isolates were molecularly identified based on 16s rrna gene. one colony of isolates was taken with a sterile toothpick then inserted into eppendorf tube containing 100 ml dh o subsequently it were vortex. a total of 2 1 ml suspension was used for the amplification with polymerase chain reaction (pcr) technique. pcr (tc 5000 techne) of 16s rrna gene by using universal primer 520 f (5'-gtgccagcagccgcgg-3') and 920 r (5'-gtcaattcctttgagttt-3'). a total volume 50 µl of pcr were contains 1 µl dna template, 2 µl of primer for each forward and reverse, 25 µl of 2x kapa taq ready mix (kapa biosystem) and ddh o 20 µl. amplification was performed for 30 2 cycles that include initial denaturation stage at a temperature 96 ºc for 5 min, denaturation at a temperature 96 ºc for 30 s, annealing a temperature 55 ºc for 30 s, extension at a temperature 72 ºc for 1 min, final extension at a temperature 72 ºc for 7 min. the success of the pcr were checked by electrophoresis for 30 min with a voltage of 100v 500ma (bio-rad mupid-exu-75577 advance japan). amplicon dna were purified and sequenced with two directions. sequence analysis were conducted by comparing the sequences with genbank database using blastn in the national center for biotechnology information (http://www.ncbi.nlm.nch.gov) to determine similarity of the sequence homology. sequence of 16s rrna gene were also analyzed for their phylogenetic tree using mega5 and clustalw software with neighboor joining method. results the result showed that the colonies number of survivals from each treatment time with nitric acid mutation. the number of colonies decreased in the period of exposure 0-90 min afterwards the number of colonies increased in the time span to be exposed for 120 min (table 1). colonies are grown then measured for iaa production. the wild strain exhibited 53.83 µg -1 ml of iaa while the nitric acid mutan within a range -1 -1. 77.39 µg ml to 95.70 µg ml isolates h6.60 exhibited the higgest iaa production which 39.87 µg -1 ml higher were compared with wild-type (table 2). h6 isolate without exsposure to mutagen or parent isolates were analyzed and compared for homology of 16s rrna gene sequence. in genbank using blastn program. based on 16s rrna sequence analysis, isolate h6 as much as 92% sequence similarity belonged to lysobacter sp. es2-22 with accession n u m b e r k j 8 7 8 6 0 4 . 1 . f u r t h e r m o r e , t h r o u g h phylogenetic tree known phylogenetic relationship with other pgpr isolates which sequence of base are known (fig 1). discussion the number of mutant colonies are grown in the time tratment of 0-90 min exposure had inversely proportion to the treatment time. more longer of exposure time, mutans growth are slightly, except in the treatment time of 120 min. it could happen due to differences in exposure times of nitric acid as mutation agent. nitric acid is a very strong oxidizing agent. reaction with nitric acid could produce nitric oxide (n o) that is identified by frazier and hage as a 2 reproductive toxin (young 2002). it toxin cause the death of the organism or mutation. in the treatment time of 120 min more colonies were grown. those colonies might be had the high mutation probability more than other treatment time exposure previously. the nitrosating agent are formed from nitric oxide autoxidation, it could cause dna damage or deamination of dna bases. the damage are followed with nitrosation of primary amine functions. the result of deamination are the formation of xanthine and uracil within g:c base pairs caulfield, wishnok and tannenbaum 1998). deamination are known occur in dna, rna, and their precursors via a hydrolytic and a nitrosative reaction. the generated deaminate products had mutagenic potential because of their structure similarity to natural bases, which in turn leads to fault nucleotide base pairing and disruption of cellular metabolism. incorporation of deaminate precursors into the nucleic acid strand occured during nucleotide synthesis by dna and rna polymerases or base modification by dna and or rna editing enzymes (kuraoka 2015). mutations treatment with nitric acid for 60 min produced mutants with the highest iaa production if compared with wild type. based on the result, it proved that strain improvement with nitric acid as classical strain improvement became one of effective way. classical strain improvement such as uv and x-ray irradiation or mutagenic chemicals such as nitrous acid, formic acid or hydrazine has been known for their history of success more than 50 years ago. bacillus subtilis out 8103 which are mutated with classical methode by nitrosoguanidine (ntg) resulted mutan arghx4, arghx6, arghx7 and arghx13 that had shown high arginine production titers in the range of 1.2 mg -1 -1 ml up to 1.65 mg ml . out of the four colonies, arghx 7 produced the highest levels of arginine until -1 1.62 mg ml (rao et al. 2012). based on analysis of 16s rrna gene, h6 isolates were closely related to the species lysobacter sp. es222. lysobacter are used in this study proved could produce iaa hormone. their potency which could produce iaa hormone supporting their role as plant growth promoter. other studies showed that lysobacter also had another potency as plant growth promoting rhizobacteria (pgpr) beside of produce iaa. lysobacter enzymogenes are known could produce antibiotics and some enzyme activities which made it became attractive candidates for use in biological control of plant diseases and of nematodes (hayward et al. 2009). l. capsici tm5405 and l. enzymogenes tm2502 had proved for their ability as consistent disease suppression of a broad range two 20 putrie et al. microbiol indones volume 11, 2017 microbiol indones 21 fig 1 phylogenetic tree based on 16s rrna gene of h6 isolate compared with 16s rrna gene of other plant growth promotion species. scale showed that distance evolution on the branch length, while the numbers on the branches indicate bootstrap values​​. table 1 effect of nitric acid for number of colonies after exposure exposure time (min) -1 number of colonies (ml ) 0 7 8.10 30 7 5.10 60 7 2.10 90 5 4.10 120 6 10.10 table 2 iaa production of isolate mutan h6 mutan h6 -1 iaa production (µg ml ) h6.0 93.89 h6.30 89.99 h6.60 95.70 h6.90 77.39 h6.120 84.24 wild type 53.83 important soilborne diseases in worldwide such as root rot diseases caused by rhizoctonia solani ag-8 and caused by gaeumannomyces graminis var. tritici with p r o d u c e e x t r a c e l l u l a r m e t a b o l i t e , s u c h a s sidherophores and protease. lysobacter sp. strains also have broad antifungal and antibacterial activities a g a i n t s p y t h i u m u l t i m u m , c o l l e t o t r i c h u m gloeosporioides, fusarium oxysporum, botrytis cinerea, r. solani, botryosphaeria dothidea, and bacillus subtilis which produced xanthobaccins a, b and c (wang et al. 2014). therefore, their phylogenetic relationship are known through the phylogenetic tree. h6 isolates showed the fit results between blastn with the phylogenetic tree. bootstrap value of 100% on the phylogenetic tree directly justify of blastn results showing that isolates h6 included in lysobacter sp. es2-22. lysobacter isolates have closely genetic relationship with pgpr group that has been previously known. this is indicated by bootstrap values on each branch of the phylogenetic tree. acknowledgment authors thank and appreciate to all staff of plant symbiotic microbes laboratory for the supports are given to carry out this research. references ahemad m, kibret m. 2014. mechanisms and applications of plant growth promoting rhizobacteria: current perspective. j king saud university. 20 p [on line]. http://dx.doi.org/10.1016/j.jksus.2013.05.001. caulfield jl, wishnok js, tannenbaum sr. 1998. nitric oxide-induced deamination of cytosine and guanine in deoxynucleosides and oligonucleotides. j biol chem 273(21): . 12689–12695. damam m, kaloori k, gaddam b, kausar r. 2016. plant growth promoting substances (phytohormones) produced by rhizobacterial strains isolated from the rhizosphere of medicinal plants. int j pharm sci rev res. 37(1): 130-136. gordon sa, weber rp. 1951. colorimetric estimation of indole acetic acid. plant physiol 26: 192-195. hayward ac, fegan n, fegan m, g.r. stirling gr. 2009. stenotrophomonas and lysobacter: ubiquitous plantassociated gamma-proteobacteria of developing significance in applied microbiology. j appl microbiol 108(3): 756-770.issn 1364-5072. kamalakumari pv, sankar gg, prabhakar. 2015. strain improvement studies for the production of lasparaginase by beauveria bassiana ss18/41. int j pharm sci rev res 31(2):173-176. issn 0976-044x. kundan r, pant g , jadon n, agrawal pk. 2015. plant growth promoting rhizobacteria: mechanism and current 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tropicales 37:33-39. vejan p, abdullah r, khadiran t, ismail s, boyce an. 2016. role of plant growth promoting rhizobacteria in agricultural sustainability-a review. molecules 21(573): 1-17. doi:10.3390/molecules21050573. wahyudi at, astuti ri, giyanto. 2011. screening of pseudomonas sp. isolated from rhizosphere of soybean plant as plant growth promoter and biocontrol agent. am j agri biol sci., 6 (1): 134-141. issn 1557-4989. wahyudi at, astuti rp, widyawati a, meryandini a, nawangsih aa. 2011. characterization of bacillus sp. strains isolated from rhizosphere of soybean plants for their use as potential plant growth for promoting rhizobacteria. j microbiol antimicrob. 3(2):34-40. issn 2141-2308. wang x, mavrodi dv, ke l, mavrodi ov, yang m, thomashow ls, zheng n, weller dm, zhang j. 2014. biocontrol and plant growth-promoting activity of rhizobacteria from chinese fields with contaminated soils. microbial biotech 8:404-418. young ja. 2002. chemical laboratory information profile. j chem ed. 79(12):1413. 22 putrie et al. microbiol indones page 1 page 2 page 3 page 4 page 5 1.mi698-ratna setyaningsih available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.2.1issn 1978-3477, eissn 2087-8575 vol 7, no 2, june 2013, p 45-50 *corresponding author; phone/fax: +62-271-663375, email: ratnasetya@yahoo.co.id denitrification is defined as the dissimilatory reduction of nitrate (no ), or nitrite (no ) to nitrous 3 2 oxide (n o) or nitrogen gas (n ) (hayatsu . 2008). 2 2 denitrification is part of the bioenergetic apparatus of bacterial cell. the complete denitrification leads to n 2 formation while the incomplete process produces n o 2 (zumft 1997). nitrous oxide is one of the principal greenhouse gases. concentration of n o in the 2 atmosphere increased from 270 in pre-industrial era to 319 ppb in 2005. the primary driver of n o increase is 2 microbial production in expanding and fertilized agricultural land (forster and ramaswamy 2007). differences in soil microbial community composition affect n o emission (holtan-hartwig 2000).2 the species ochrobactrum anthropi was described et al first by holmes et al. (1988) as the sole species of ochrobactrum. since then, some other species of ochrobactrum had been discovered (kämpfer et al. 2007). o. anthropi is a gram negative, rod shaped, motile, and obligately aerobic bacteria (holmes et al. 1988). o. anthropi are widely distributed in diverse habitat including human body (holmes et al. 1988), activated sludge taken from reclaimed land (song et al. 2002), rice field water (reche and fiuza 2005), and wastewater treatment plant (zuo et al. 2008). some o. anthropi strains have been identified as denitrifier (sung et al. 2002; doi et al. 2009). nevertheless, n o 2 reduction activities of these strains have not been investigated. setyaningsih et al. (2010) reported that o. anthropi bl1 and bln1 could grow on n o as a sole electron 2 acceptor. in 5 days, bl1 and bln1 reduced n o up to 2 nitrous oxide (n o) is one of the principal greenhouse gases. differences in soil microbial community 2 composition affect n o emission. ochrobactrum anthropi bl1 and bln1 isolated from rice field in tangerang, 2 banten, indonesia can grow on and reduce n o. this study investigated the patterns of n o reduction activity 2 2 and growth of o. anthropi bl1 and bln1 on denitrification media and also examined the ability of bln1 strain to reduce n o in flooded rice soil. nitrous oxide reduction activity and growth of strains bl1 and bln1 occurred 2 simultaneously, indicating that the bacteria used n o for growth. bl1 and bln1 showed the same specific 2 growth rate, but the n o reduction rate of bln1 was higher than that of bl1. increase of the n o concentration in 2 2 the surface water of flooded soil without bln1 isolate six hours after the addition of no was significantly 3 greater than the surface water from soil that had been inoculated with the isolate. key words: n o, ochrobactrum anthropi, reduction, rice field2 dinitrogen oksida (n o) merupakan salah satu gas rumah kaca utama. perbedaan komposisi komunitas 2 mikrobia dalam tanah mempengaruhi emisi n o. ochrobactrum anthropi bl1 dan bln1 yang diisolasi dari 2 sawah di daerah tangerang, banten, indonesia dapat tumbuh dengan mereduksi n o. penelitian ini mengkaji 2 pola pertumbuhan dan aktivitas reduksi n o dari o. anthropi bl1 dan bln1 dalam media denitrifikasi serta 2 kemampuan galur bln1 mereduksi n o di tanah sawah tergenang. aktivitas reduksi n o dan pertumbuhan 2 2 galur bl1 dan bln1 berlangsung serentak, menunjukkan bahwa bakteri menggunakan n o untuk tumbuh. nilai 2 kecepatan pertumbuhan spesifik antara bl1 dengan bln1 sama, sedangkan kecepatan reduksi n o galur bln1 2 lebih tinggi dari pada bl1. peningkatan konsentrasi n o dalam air permukaan tanah tergenang setelah enam jam 2 penambahan no di tanah tanpa isolat bln1 lebih besar secara signifikan dari pada di tanah yang ditambah 3 isolat. kata kunci: n o, ochrobactrum anthropi, reduksi, sawah2 nitrous oxide reduction activity of denitrifying ochrobactrum anthropi isolated from rice field 1 2 3 ratna setyaningsih *, iman rusmana , prihasto setyanto , 2 and antonius suwanto 1 department of biology, faculty of mathematics and natural sciences, universitas sebelas maret, jalan ir sutami 36a kentingan, surakarta 57126, indonesia; 2 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 3 balai penelitian lingkungan pertanian, jalan jakenan-jaken km 05, jakenan, pati 59182, indonesia -1 4.09 and 3.91 μmol ml cultures respectively. to confirm and further characterize n o reduction 2 capacity of the isolates, this study investigated the patterns of n o reduction activity and growth of o. 2 anthropi bl1 and bln1 on denitrification media. this study also examined the ability of bln1 strain to reduce n o in flooded rice soil. n o reducing 2 2 denitrifier maybe able to be used as n o emission 2 controller in rice field. materials and methods bacterial strains and media. the bacterial strains used in this study were isolated from rice field in tangerang, banten, indonesia and have been characterized previously (setyaningsih et al. 2010). experiments were conducted using denitrification -1 media containing ch coona·3h o 2.72 g l , 3 2 -1 -1 -1 k hpo 0.80 g l , kh po 0.30 g l , nh cl 0.40 g l , 2 4 2 4 4 -1 mgso ·7h o 0.40 g l (barford et al. 1999), and yeast 4 2 -1 extract 3 g l . soil. soil samples were obtained from the top 20 cm of a paddy field. the soil was air-dried, broken up, sieved (2 mm) and thoroughly mixed. the particle size distribution was 51 % clay, 34 % silt, and 15 % sand. nitrous oxide reduction activity and bacterial growth patterns. n o reduction activity and bacterial 2 growth patterns on denitrification media were observed using n o as a sole terminal electron acceptor. 2 experiments were carried out in anaerobic condition with 10.1 ml sterile media in the 20.1 ml test tubes with rubber stoppers. media were flushed with n for 10 2 minutes. n o was injected into each test tube. n o 2 2 concentration dissolved in the media at the beginning of the experiment was 12.75 mm. inoculum was prepared by anaerobic growth in media supplemented with -1 nano (1.275 g l ). cells from exponential phase 3 inoculum culture were twice washed by centrifugation in sterile distilled water and resuspended in fresh media without no . after inoculation, cultures were incubated 3 at 30 °c with rolling. periodically, n o were sampled 2 from the headspace of test tubes by gas-tight syringes and measured by gas chromatography with a shimadzu 14a ecd gas chromatograph equipped with q porapak column. carrier gases were methane (5%) and argon (95%) and the operating temperatures were as follows; detector 340 °c, column 35 °c, injector 200 °c. growth of bacteria was monitored based on the culture’s turbidity using nephelometer eel unigalvo ds29. n o concentration in solution was calculated with 2 the formula: y = α*x (solution volume/headspace 46 setyaningsih et al. microbiol indones volume), where y was amount of the dissolved n o 2 (µmol) in a closed system, α was solubility of n o 2 -1 expressed as dissolved n o (ml n o ml h o), and x 2 2 2 was amount of n o in the flask headspace (µmol) 2 (carter 1993). the specific growth rate was calculated with the formula μ = (∆lnn)/∆t, where n was the cells number per milliliter and t was the corresponding time (hours) (el hassan et al. 1985). reduction rate of n o 2 (v ) was calculated by the analogous equation v = red red δs/δt where s was dissolved n o concentration.2 n o reduction in rice soil. n o reduction in rice 2 2 soil experiment was carried out using rice soil flooded with distilled water in close system. two hundreds and twenty grams of soil was filled into the 603 ml bottle with rubber stopper. soil thickness in the bottle was 6 cm. soil was flushed with n for 15 min to minimize the 2 residual no and incubated for 2 d. flushing the soil 3 with n was repeated to remove the n o that might 2 2 have been produced from residual no . distilled water 3 was added up to 2 cm above the soil surface. the flooded soil was incubated for 7 d with the bottle cap opened to mimic the rice field condition. the soil ph was measured. 0.6 mmol no was added as nano . o. 3 3 anthropi bln1 inoculum was added as suspension in 8 sterile distilled water (5 x 10 cfu). after no and 3 inoculum addition, the bottle was closed with rubber stopper. control was prepared without bacterial inoculum. the headspace gas was sampled every 3 h to determine n o concentration. after gas sampling from 2 the headspace, bottle cap was opened. surface water sample for dissolved n o analysis was taken using 2 syringe and transferred into test tube containing 100 μl of formaldehyde (38% (v/v)) and capped with rubber stopper. water samples in the test tubes were shaken vigorously and n o concentration in the headspace 2 was measured using shimadzu 14a ecd gas chromatograph with q porapak column. carrier gas used was n and the operating temperatures were as 2 follows; detector 350 °c, column 60 °c, and injector 150 °c. the emission value was calculated by subtracting the n o concentration in the headspace at 2 the certain time by the initial n o concentration .2 results nitrous oxide reduction activity and bacterial growth. fig 1 reported the n o reduction activity and 2 growth of bl1 and bln1 bacterial strains. the slopes of curves indicate the specific growth rate (μ) and n o 2 reduction rate (v ) (table 1). bl1 and bln1 strains red showed the same µ. however bln1's v was higher red than bl1’s. the μ:v ratio of strains bl1 and bln1 red were 0.65 and 0.45, respectively. this ratio expresses the efficiency of each bacterial strain in utilizing n o 2 as electron acceptor. nitrous oxide emission from rice soil. the amount of n o in the headspace of bottle filled with 2 rice soil at the beginning of experiment was 6.86 nmol. there was the fluctuation of the amount of n o in the 2 headspace during 9 h observation (fig 2). nevertheless, the n o emission did not show any 2 significant difference between soil incubated with bln1 strain or without bacterial strain after 3, 6, and 9 h of incubation (table 2). the concentration of n o in 2 the surface water at the beginning of the treatment was -1 0.70 nmol l . the concentration of n o in the surface 2 water increased sharply for 6 h following the addition of no in soil without isolate. however, the 3 concentration started to go down after 6 h of incubation (fig 3). increase of the concentration of n o in the 2 surface water of flooded soil after incubation was significantly greater for soil without bln1 isolate than soil with the addition of isolate (table 3). discussion nitrous oxide reduction activity and growth of volume 7, 2013 microbiol indones 47 b ac te ri al g ro w th -1 (l n o f ce ll s n u m b er m l ) 0 2 4 6 8 10 19 20 21 22 23 24 0 3 6 9 12 15 18 n it ro u s o x id e -1 re d u ce d ( μ m o l m l ) time (h) bl1 0 2 4 6 8 10 19 20 21 22 23 24 0 3 6 9 12 15 18 time (h) bln1 b ac te ri al g ro w th -1 (l n o f ce ll s n u m b er m l ) n it ro u s o x id e -1 re d u ce d ( μ m o l m l ) table 1 nitrous oxide reduction rate (v ) and specific growth rate (μ) of strains bl1 and bln1red strain v (red -1 -1 μmol ml h ) ± se -1 µ (h ) ± se bl1 0.26 ± 0.03 0.17 ± 0.02 bln1 0.43 ±0.03 0.17 ±0.03 fig 1 nitrous oxide reduction activity and bacterial growth of strains bl1 and bln1. bars represent standard error, calculated from three experiments. :bacterial growth; :nitrous oxide reduced. se: standard error. reduction. the concentration of n o in the surface 2 water of flooded soil decreased after six h of incubation since n o went through further reduction to become 2 n . in spite of the increasing n o concentration in the 2 2 surface water of flooded soil without isolate at the sixth hour, n o released into the headspace did not go 2 through any significant rise, then. according to heincke and kaupenjohan (1999), the speed of n o 2 dissolving in the water and escaping into the atmosphere depended, among others, on the turbulence and the speed of water current. if there was a current with sufficient aeration, then the dissolved n o would 2 go in to the atmosphere in several min. if n o stayed in 2 the ground water sufficiently long then there would be sufficient time for n o to be reduced to n . 2 2 bln1 strain is expected to maintain its high ability to reduce n o in the environment among the bacterial 2 community. bln1 strain could use no , no , and o 3 2 2 (setyaningsih 2011) in addition to n o as the terminal 2 electron acceptor so that its sustainibility in the environment could be supported. according to martin strains bl1 and bln1 occurred simultaneously, indicating that the bacteria used n o for growth. o. 2 anthropi bl1 and bln1 had high ability to grow on and reduce n o. they grew well for 15 h on n o. 2 2 snyder et al. (1987) found that pseudomonas aeruginosa pao1 and p1 lost the ability to grow on n o after 1-3 h due to the loss of n o uptake activity. 2 2 p. aeruginosa p2 also exhibited a loss of n o uptake 2 activity but the loss was not as extensive as strains pao1 and p1. p2 could still grew at slower rate until n o became exhausted after 8 h. synthesis and 2 destruction of enzyme could control n o reduction 2 activity in denitrification bacteria. bacterial strains having the ability to reactivate or synthesize new enzyme could grow longer on n o.2 the increase of n o in the surface water for 6 h 2 following the addition of 0.6 mmol of no indicated an 3 no reduction activity of denitrifying bacteria in the 3 flooded soil. the reduction of no could be done by 3 the native denitrifiers and the inoculants. nevertheless addition of the inoculants increased the activity of n o 2 microbiol indones48 setyaningsih et al. table 2 emission of n o with and without bln1 inoculation following the addition of 0.6 mmol of no2 3 0 2 4 6 8 10 12 0 3 6 9 a m o u n t o f n it ro u s o x id e in th e h ea d sp ac e (n m o l) time (h) fig 2 amount of n o in the headspace without and with bln1 inoculation following the addition of 0.6 mmol of no bars 2 3 . represent standard error. :without isolate; :with isolate. time (h) -2 without isolate (nmol m ) -2 with isolate (nmol m ) 3 6 9 0.30 a 0.12 a 0.20 a -0.11 a 0.29 a 0.28 a all of the numbers followed by the same alphabet indicates no significant difference in the analysis of variance with α=0.05. bacterium ochrobactrum anthropi yd50.2 tolerates high levels of reactive nitrogen oxide. appl environ microbiol. 75(16):5186-5194. doi:10.1128/aem.0060409. el hassan ga, zablotowicz rm, focht dd. 1985. kinetics of denitrifying growth by fast-growing cowpea rhizobia. appl environ microbiol. 49(3): 517-521. forster p, ramaswamy v. 2007. change in atmospheric constituents and radiative forcing. in: solomon, s et al., editor. climate change 2007: the physical science basis. contribution of working group i to the fourth assesment report of the intergovernmental panel on climate change. cambridge: cambridge university press. p 130-217. hayatsu m, tago k, saito m. 2008. various players in the nitrogen cycle: diversity and functions of the microorganisms involved in nitrification and denitrification. soil sci plant nutr. 54(1):33-45. doi:10.1111/j.1747-0765.2007.00195.x heincke m, kaupenjohann m. 1999. effect of solution on the dynamics of n o emissions: a review. nutr cycl 2 agroecosyst. 55(2):133-157. doi:10.1023/a:10098420 11599. holmes b, popoff m, kiredjian m, kersters k. 1988. ochrobactrum anthropi gen. nov. from human clinical volume 7, 2013 microbiol indones 49 et al. (1988) the population of denitrifying bacteria had persistent and stable characteristics. in conclusion, our results seems to indicate that the bln1 strain could be used to reduce the emission of n o from the rice fields.2 acknowledgment this research was supported by bpps scholarship grant from dikti to the first author. thanks also due to db nedwell, emeritus professor of department of biological sciences, university of essex, colchester, united kingdom for his great support and advice. references barford cc, montoya jp, altabet ma, mitchell r. 1999. steady-state nitrogen isotope effects of n and n o 2 2 production in paracoccus denitrificans. appl environ microbiol. 65(3): 989-994. carter mr, editor. 1993. soil sampling methods of analysis. boca raton: crc press llc lewis publisher. p 354. doi y, takaya n, takizawa, n. 2009. novel denitrifying 0 5 10 15 20 25 30 35 40 45 0 3 6 9 c o n ce n tr at io n o f n it ro u s o x id e -1 in t h e su rf ac e w at er ( n m o l l ) time (h) fig 3 concentration of n o in the suface water without and with bln1 inoculation following the addition of 0.6 mmol of no2 3 bars represent standard error. :without isolate; :with isolate.. table 3 increase of the concentration of n o in the surface water with and without bln1 inoculation following the addition of 2 0.6 mmol no3 3 6 9 6.07 a 31.12 b 7.59 a 11.53 a 12.94 a 5.95 a time (h) -2without isolate (nmol m ) -2 with isolate (nmol m ) all of the numbers followed by the same alphabet indicates no significant difference in the analysis of variance followed by duncan's multiple range test with α=0.05. setyaningsih r, rusmana i, setyanto p, suwanto a. 2010. physiological characterization and molecular identification of denitrifying bacteria possesing high nitrous oxide reduction activity isolated from rice soils. microbiol indones. 4(2):75-79. snyder sw, bazylinski da, hollocher tc. 1987. loss of n o reductase activity as an explanation for poor 2 growth of pseudomonas aeruginosa on n o. appl 2 environ microbiol. 53(9):2045-2049. song sh, yeom sh, choi ss, yoo yj. 2002. effect of aeration on denitrification by ochrobactrum anthropi sy509. biotechnol bioprocess eng. 7(6):352-356. sung dw, song sh, kim jh, yoo yj. 2002. effects of electron donors on nitrate removal by nitrate and nitrite reductases. biotechnol bioprocess eng. 7(2):112-116. zumft wg. 1997. cell biology and molecular basis of denitrification. microbiol mol biol rev. 61(4):533616. zuo y, xing d, regan jm, logan be. 2008. isolation of the exoelectrogenic bacterium ochrobactrum anthropi yz-1 by using u-tube microbial fuel cell. appl environ microbiol. 74(10):3130-3137. doi:10.1128/aem.0273 2-07. doi: 10.5454/mi.4.2.5. specimen and previously known as group vd. int j syst bacteriol. 38(4):406-416. holtan-hartwig l, dörsch p, bakken lr. 2000. comparison of denitrifying communities in organic soils: kinetics of no and n o reduction. soil biol biochem. 32(6):833-3 2 843. kampfër p, citron dm, goldstein ejc, scholz hc. 2007. difficulty in the identification and differentiation of clinically relevant ochrobactrum species. j med microbiol. 56(11): 1571-1573. doi:10.1099/jmm.0.473 50-0. martin k, parsons ll, murray re, smith ms. 1988. dynamics of soil denitrifier populations: relationships between enzyme activity, most-probable-number counts and actual n gas loss. appl environ microbiol. 54(11):2711-2716. reche mhlr, fiuza lm. 2005. bacterial diversity in ricefield water in rio grande do sul. braz j microbiol. 36(3):253-257. doi:10.1590/s1517-838220050003000 09. setyaningsih r. 2011. karakterisasi dan uji aktivitas bakteri denitrifikasi pereduksi dinitrogen oksida (n o) yang 2 diisolasi dari tanah sawah [characterization and nitrous oxide (n o) reduction activity of denitrifying bacteria 2 isolated from rice field] [dissertation]. bogor (id): institut pertanian bogor. microbiol indones50 setyaningsih et al. 6 466 diah us silmi rich dedi... isolation and characterization of simian retrovirus type d from and in indonesiamacaca fascicularis m. nemestrina diah iskandriati , uus saepuloh , silmi mariya , richard f grant , dedy duryadi solihin , dondin sajuthi , joko pamungkas 1 1 1 2 3 1,4 1,4 * simian type d retroviruses (srvs) are one of the causative agents of simian acquired immunodeficiency syndrome (aids) in asian macaques. in the past, srv isolates from macaques had only been identified at the us primate centers, outside the country of origin and after the animals had been introduced into a new environment. in this study, we report the first isolation, cultivation and molecular characterization of the type d simian retrovirus naturally infecting wild caught macaques in their natural habitats in the country of origin, in this case, indonesia. when peripheral blood mononuclear cells (pbmc) from (mf) and (mn) were co-cultured with raji human b-cell line, syncytia were observed microscopically and confirmed by immunofluoresence assay using antibody to srv-2. immunoblot analysis of purified mf-et1006 from cell culture supernatants demonstrated that the viral core and envelope proteins reacted with rabbit anti-srv. sequence analysis of mf isolates in the viral envelope region revealed high homology to srv-2 (94-96%). on the other hand, the homologies in the envelope region of mn isolates were less than 80% to srv-1, srv-2, srv-3 and mf isolates. this study suggests that the isolate from mn may be different from any other published srv isolates.. key words: type d simian retrovirus, , and 1 2 3 4 primate research center, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; virology and molecular biology, shin nippon biomedical laboratories (snbl) usa, everett, washington, usa; department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; faculty of veterinary medicine, institut pertanian bogor, darmaga campus , bogor 16680, indonesia macaca fascicularis m. nemestrina macaca fascicularis macaca nemestrina for many years, the retroviridae family has been studied primarily because of the ability of these viruses to transform mammalian cells and cause naturally occurring tumors in many animal species. it was recognized that this virus family could also cause non-oncogenic disease, including immunosuppresive disorders such as aids (gardner 1988). among family retroviridae, the lentivirus (simian immunodeficiency virus, siv) and exogenous type d retroviruses (simian retrovirus, srvs) have been identified as the etiologic agents of infectious immunodeficiency diseases in several macaque species that have some clinical similarities to human aids (lerche 1995). srv infection is endemic and for the first time was reported in sp. populations at some primate research centers in the united states in the early 1980s (daniel . 1984; gardner . 1988; pamungkas . 1991). until 1988, five serotypes of srv had been isolated and identified from macaques (srv-1 to srv-5). however, from these five serotypes, only four (srv-1, srv-2, srv-3 and srv-4) had been completely sequenced and reported to genbank (daniel 1984; marx 1984; gardner 1988; li . 2000). srvs have emerged as significant pathogens in captive macaques, although infection appears to be sporadically prevalent in asian macaques at breeding facilities and these species are probably the natural hosts of srv, but prevalence of infection in feral macaques remains undetermined (daniel 1984; gardner 1988; pamungkas . 1991). serological studies of and in indonesia show variable prevalence of srv-2 leading to the assumption that the animals have been infected with srv (iskandriati 1998a; iskandriati 1998b; warren 1998). et al. et al. macaca et al et al et al et al. et al. et al. et al et al. et al. et al m. fascicularis, m. nemestrina, pongo pygmeus et al. et al. et al. srv isolates, found to date from macaques at primate centers out of the animals' original country, have been obtained after the animals were introduced to the new environment. according to grant (1995), the isolates obtained over many years from the washington primate center have a very high homology level, which shows that these srv isolates probably came from the same source or a small number of source animals. however, the characteristics of virus growth and genetic sequence variability from wild type isolates have not been described yet. until today there has been no report on findings of wild isolate in its natural population. since srv infection has negative impact on the management of macaques breeding facility, it is very important that we be able to identify and characterize new srv isolates in the animals' original population. the objective of this research was to identify srv from and in indonesia using virus isolation technique, and to identify and characterize provirus dna from those isolates. the resulting amino acid sequences obtained were then compared with those from other type d retroviruses to describe their genetic relationship . fifty-three blood samples from lampung and palembang, indonesia and 76 blood samples from in palembang were obtained from several breeding facilities in indonesia. and with a total number of 129 were placed in individual cages during the screening process. blood samples were taken within the first week of the animals' arrival at the breeding facilities. this was carried out to get accurate data about the animals' condition. if the animals were infected by srv, it can confirm that the m. fascicularis m. nemestrina m. nemestrina m. fascicularis m. nemestrina . materials and methods sample collection and processing *corresponding author, phone: +62-251-8347519, fax. +62-251-8310033, e-mail: atie@indo.net.id issn 1978-3477 vol 4, no 3, dec 2010, p 132-136 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.6 volume 4, 2010 microbiol indones 133 infection occurred not at the breeding facilities, but in their places of origin. the peripheral blood mononuclear cells (pbmcs) were recovered from samples on a ficoll gradient (organon teknika, usa) as described in hunt (1987). the cell suspensions were washed in phosphate-buffered saline (pbs) and aliquoted. the cells were further used for virus isolation and polymerase chain reaction (pcr) of proviral dna. . raji cells (atcc ccl-86) were cultured in rpmi 1640 supplemented with 10% fetal bovine serum, 100 iu ml penicillin and 100 ug ml streptomycin and used for isolation. the cells were subcultured every four days for the duration of three weeks for syncytium induction as described previously (rosenblum 2000). to prove that syncytia formed on infected raji cells were caused by srv, immunofluores cence assay was carried out using rabbit anti-srv serum and detected by a -labeled goat anti-rabbit immunoglobulin antibody. . the supernatant from infected raji cells was inoculated into a549 cells (atcc ccl-185). virus was harvested from culture supernatant using ultrafiltration and ultracentrifugation techniques and purified into chromatography column using sepharose-cl4b as described by grant (1995b). immunoblotting was modified from harlow and lane (1988). purified virus was electrophoresed on a 7.5-17.5% gradient sds-page and transferred onto a nitrocellulose membrane. individual cut strips were incubated with various rabbit anti-srv polyclonal sera. bound antibodies were detected by an alkaline phosphatase-labeled goat anti-rabbit immuno-globulin antibody (sigma, usa) with bcip-nbt (5-bromo-4-chloro-3-indolylphosphate toluidiniumnitroblue tetrazolium) substrates (sigma, usa). . infected raji cells were fixated on object slide using methanol and cold acetone with 1:1 ratio for 5 min. then the object slides were washed with pbs and dried in room temperature (22 ). cell blood smear was then incubated with rabbit anti-srv serum and washed with pbs. flourescein isothiocyanate-conjugated anti-rabbit immunoglobulin g was added to cell blood smear, incubated for 45-60 min and re-washed with pbs. then, object slide was given counter staining evans blue and expelled with mounting medium pbsglycerol 90% before closing it with glass lid. cell blood smear was observed by using fluorescent microscope to see color luminescent from cytoplasm. . total dna was extracted using dna purifying kit (qiagen, usa). approximately 1800 bp fragment from this dna was amplified with a primer set consisting of an upstream primer ' 5 c a c c t c t y t r c t yaya g a g c t g a 3 ' a n d a downstream primer 5'-gaaactgcgcctgtct-3' located in the gene (grant 2004, personal communication). pcr amplification were performed by following protocol: denaturation for 2 min at 92 c, amplification for 10 cycles consisting of 10 sec at 92 c, 30 sec at 60 c and 2 min at 68 c continued with amplification for 20 cycles consisting of 15 virus isolation virus purification and immunoblotting immunofluorescence technique (immunofluorescence assay, ifa) dna extraction and pcr amplification et al. fitc et al. env -1 -1 o o o o °c sec at 92 c, 30 sec at 60 c and 2 min at 68 c, with time prolongation of 20 sec for each cycle and ended with an extension at 68 c temperature for 7 min. the pcr amplified dna fragments were cloned into the ta topo vector (invitrogen, san diego, california) and sequenced. . sequencing of nucleotides was carried out in automated dna sequencer abi-prism model 310 version 3.7 (applied biosystem, foster city, california) using m13 primers. alignment of the sequences was done with clustalw (embl-ebi). the phylogenetic analyses for protein were done by the neighbor-joining (nj) method using mega package software version 4.0 (kumar 2008). distances were estimated by kimura's two parameter method with one thousand bootstrap replicates. genebank accession numbers of the comparison sequences were m11841 ( ), m16605 ( ), m12349 ( ), d10032 and af142988 (baboon endogenous virus, type c retrovirus), and af284693 ( type d endogenous retrovirus) and endogenus retrovirus of (grant, data not yet published) out of 129 pbmcs samples, there were only four samples (mf-et1006, mn-5055, mn-5281, and mn-5378) showing retrovirus infection, as shown by the appearance of multinucleated giant cells (syncytial formation) on day 7 after inoculation, a typical cytopathic effect (cpe) resulted from the fusion of infected cells. fig 1b shows the appearance of multinucleated giant cells on raji cells inoculated with pbmcs from et1006 compared with normal uninoculated raji cells (fig 1a); while fig 1c shows smaller size syncytia on raji cells inoculated with pbmcs from 5378. the other two (5055 and 5281) pbmcs produced syncytia with similar size as shown by 5378. lack of retrovirus infection was shown by the absence of syncytial formation on the remaining 125 pbmcs samples after co-cultured with raji cells. from the four samples showing syncytial formation on raji cells, et1006 gave the most obvious cpe positive result with larger size of giant cells, while isolates from the gave smaller syncytial formation on raji cells. ifa was carried out to confirm that the syncytia or multinucleated giant cells formed on raji cells were caused by srv infection, not by other retroviruses. foamy virus, a member of the retroviridae family from the genus of spumavirus also shows the appearance of syncytial formation when co-cultured with peripheral blood mononuclear cells . the positive ifa result was indicated by the greenish fluoresceinated color under a fluorescence microscope resulted from the reaction of antigen/ viral protein bond with anti-srv-2 antibodies produced in rabbit (rabbit anti srv-2 serum) which later reacted with anti-rabbit immunoglobulin g conjugated to fluorescein isothiocyanate (fitc-anti-rabbit igg conjugate) from the four samples showing syncytial formation on raji cells, et1006 gave the most obvious fluorescein iluminescence (fig 2b) indi o o o o dna sequencing and analysis et al. srv-1 srv-2 mpmv/srv-3 trichosurus vulpecula macaca m. fascicularis m. nemestrina m. nemestrina m. nemesterina m. fascicularis m. nemestrina in vitro . m. fascicularis . results 134 skandriatii et al. microbiol indones m. nemestrina cating a strong positive result, while isolates from the gave weak positive result (fig 2c). unino culated normal raji cells were used as negative control. (fig 2a). due to the limited syncitial formation shown by all mn isolates on raji cells, further multiplication on a549 cells could only be carried out on mf-et1006 sample. to see the relationship between antigens et1006 and reference srv-2, immunoblotting analysis was carried out by using antibody panel of some reference simian retroviruses as samples. fig 3a shows the results of immunoblotting from et1006 antigen reacted with polyclonal antibody of srv-1, srv-2, srv-3, srv-5, retrovirus type c from baboon, srv-pc and smrv. as a standard for comparison, immuno-blotting of srv-2 antigen was carried out (fig 3b). most of proteins which are typical property of srv-2 such as protein (gp70 and gp22) and protein (p45, p27, p14 and p10) were seen in the mf-et1006 strips reacted with antibodies to srv-1, srv-2 and srv-3 with different gp70 intensity levels. lack of gp22 protein was seen when the mf-et1006 strip was reacted with antibody to srv-5, while reaction to p27 protein was seen in all samples. the same immunoblotting analysis on et1006 antigen showed cross reactivity in all polyclonal antibodies to simian retroviruses as shown by srv-2 antigen. however, gp70 env gag-pol env fig 1 co-culture of monkey pbmcs with raji cells: a, uninoculated raji cells; b, raji cells inoculated with pbmcs from et1006; c, raji cells inoculated with pbmcs from 5378. macaca fascicularis m. nemestrina a b c a b c fig 2 ifa of raji inoculated with monkey pbmcs: a, uninoculated raji cells; b, raji cells inoculated with pbmcs from et1006 c, raji cells inoculated with pbmcs from 5378. macaca fascicularis ; m. nemestrina protein of et-1006 showed a double band instead of one thick band. a fragment of 500 bp from gp70 gene of srv-2 was amplified in 18 isolates. however, there was no amplification of 600 bp fragment from gp70 gene of srv-1 and srv-3 in any of the samples tested. the results of pcr analyses of the region gp70 and the whole region are shown in fig 4. phylogenetic tree (fig 5) shows that mf-et1006 isolate is very close to srv-2 with amino acid homology level 95-96%. on the other hand, isolate obtained from mn has lower homology (less than 80%), to either mf-et1006 isolate or other srv isolates (srv-1, to srv-3). both indonesian isolates have no similarities to either primate or non-primate endogenous virus the appearance of syncytial formation on raji cells inoculated with pbmcs from et1006 and from 5378, 5055 and 5281 samples shows the illustration of cytopathic effect (cpe) as an indication that the viruses replicated in raji cells. the difference of the syncytial formation on raji cells caused by and isolates is thought to be caused by serotype difference of both viruses that probably have different receptor on the target cells. isolate replicates very well in raji cells that were originally b lymphocytes. due to the limitation of cell lines availability in the laboratory, virus isolation was not carried out on t lymphocytes cell lines, so it is unknown whether mn isolate would be able to replicate better in t lymphocytes. the illustration of ifa results from the and mn isolates showed different degree of reactivity to srv-2 antibody. -et1006 gave a very strong positive reaction, indicating that mf-et1006 virus proteins had a high specificity or homology to bind with its antibody. it was also supported by immunoblotting result on the mfet1006 virus protein strip reacted with some antibodies to simian retroviruses. the illustration of mf-et1006 protein env env env env m. fascicularis m. nemestrina m. fascicularis m. nemestrina m. fascicularis m. fascicularis m. fascicularis . discussion profile reacting with its antibody was very identical with protein profile illustration of srv-2. this phenomenon supports an assumption that the mf-et1006 isolate has a closer genetic relationship with srv-2 than the mn isolate. relatively high genome variation is found at gene (grant . 1995a; li . 2000; nandi . 2003) in srv. some retrovirus isolates including hiv, mulv, felv, srv1, srv-2 and srv-3 show that sequence variation on the surface unit or n-terminal domain of gene is higher than env et al et al et al env a b fig 3 results of et1006 immunoblotting: a, and srv-2; b, with rabbit polyclonal antibody against many srvs. 1, antibody against srv-1; 2, antibody against srv-2; 3, antibody against srv-3; 4, antibody against srv-5; 5; antibody against srv pc; 6, antibody against type c retrovirus; 7, antibody against smrv; 8, negative control; 9, conjugate control. volume 4, 2010 microbiol indones 135 fig 4 results of provirus dna amplification against srv-2 measuring 500 base pairs. m is a marker measuring 1000 base pairs; 1, reagent control; 2, mn 5281; 3, mn 5378; 4, mn 5505; 5, mf et1006; 6, positive control srv-2 from mf; 7, reagent control. srvmf-et1006 srv-2 m126467 srv-2 m16605 srvmn-5278 srv-1 srv-3 d-servpc c-servpc d-servtv 0.05 fig 5 phylogenetic tree based on amino-acid sequences using kimura two-parameter and the neighbor-joining method. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7m 12000 5000 2000 2650 1000 850 650 500 400 300 200 kd 70 45 27 22 20 14 10 kd 70 45 27 22 20 14 10 that of the other area in its genome. the surface unit of this is binding the target of antibody resulted from adoptive host immune cells in the frame of neutralizing virus. to deceive the immune system attack of host, virus changes by mutation mechanism especially on the envelope surface, resulting high variation in this area (nandi . 2003). some aspects may be influenced by gene change, among others are host, pathogenesis and antigenic characteristics. the phylogenetic tree shows that mf-et1006 isolate has a very close relatedness with srv-2 with homology level reaching 96%. in contrast, the isolate from mn has much lower homology (less than 80%) to either mf-et1006 isolate or other srv isolates (srv-1 to srv-3). based on this and some other information showing the differences in both microscopic cytopathic effects and ifa illustration, we conclude that the isolate from is indicated to be a new serotype of type d retrovirus, which is quite different from the other published srvs his work was supported by competitive research grant from directorate of higher education, ministry of national education, republic of indonesia, and washington national primate research center, university of washington, seattle, usa envelope et al env m. nemestrina . t . acknowledgements references daniel md, king nw, letvin nl, hunt rd, seghal pk, desrosiers rc. 1984. a new type d retrovirus isolated from macaques with an immunodeficiency syndrome. science 223:602-5. gardner mb, luciw p, lerche n, marx p. 1988. nonhuman primate retrovirus isolates and aids. adv vet sci comp med 32:171-90. grant rf, malinak cj, wu, h, sabo a, tsai cc. 1995a. pcr amplification and dna sequencing of srv-2 from archieved tumor tissue. virus res 36:187-200. grant rf, windsor sk, malinak cj, bartz cr, sabo a, benveniste re, tsai c. 1995b. characterization of infectious type d retrovirus from baboon. virology 207:292-6. harlow e, lane d. 1988. immunobloting. : antibodies: a laboratory manual. new york: cold spring harbor. hunt sv. 1987. preparation of lymphocytes and accessory cells. : klaus ggb, editor. lymphocytes: a practical approach. oxford: irl pr. iskandriati d, pamungkas j, suparto ih, grant r, agy mb, morton wr, sajuthi d. 1998a. evidence for retroviruses infection in captive orangutans ( ) returned to indonesia. j med primatol 27:173-80. iskandriati d, pamungkas j, surya m, mariya s, budiarsa in, sajuthi d. 1998b. prevalence of simian retrovirus type d serotype 2 (srv-2) antibodies on and in indonesia. j primatol indones 2:5-8. kumar s, tamura k, nei m. 2008. mega: molecular evolutionary genetics analyses, version 4.0. pennsylvania: pennsylvania state university, university park. lerche nw, heneine w, kaplan je, spira t, yee jl, khabbaz rf. 1995. an expanded search for human infection with simian type d retrovirus. aids res hum retroviruses 11:527-9. li b, axthelm mk, machida ca. 2000. simian retrovirus serogroup 5: partial sequence and viral rna distribution in an infected rhesus macaque. virus genes 21:241-8. marx pa, maul dh, osborne kg. 1984. simian aids: isolation of type d retrovirus and disease transmission. science 223:1083-6. nandi js, tikute sa, chhangani ak, potdar va, tiwari-mishra m, ashtekar ra, kumari j, walimbe a, mohnot sm. 2003. natural infection by simian retrovirus-6 (srv-6) in hanuman langurs ( ) from two different geographical regions of india. virology 311:192-201. pamungkas j, joeniman b, watanabe ra, kuller l, schmidt a, thouless me, morton wr. 1991. simian aids retrovirus (srv) antibodies on cynomolgus macaques ( ) from indonesia. j ilmu pert indones 1:52-6. rosenblum ll, weiss ra, mcclure mo. 2000. virus load and sequence variation in simian retrovirus type 2 infection. j virol 74:3449-54. warren ks, niphuis h, heriyanto, verschoor ej, swan ra, heeney jl. 1998. seroprevalence of specific viral infections in confiscated orangutans ( ). j med primatol 27:33-7. in in pongo pygmeus macaca fascicularis macaca nemestrina gag-prt semnopithecus entellus macaca fascicularis pongo pygmaeus 136 skandriatii et al. microbiol indones 05 wibowo.cdr a conventional pulping process involve application of chlorine in bleaching process of the pulp. the used of chlorine in the bleaching process not only affects the waste water released into the environment but also the final paper products. indonesia is one of the top ten pulp producer countries using chemical pulp production method (popp et al. 2011). chemical processes in the pulp and paper industries are generally done at high temperature and ph, especially in the kraft pulping process, the process of sulfate and chlorine bleaching process. recently, demand of elemental chlorine free (ecf) or totally chlorine free (tcf) in the world market of paper product has dramatically increased in the last decade. in order to fulfill the market demand, utilization of biotechnology might can be done by the application biobleaching (bajpai et al. 1999). biobleaching is the process of bleaching the pulp using xylanase (buchert et al. 1994; da silva et al. 1994). the use of xylanase can give a good impact on the environment which replaces or reduces the amount of chlorine used in the bleaching process of pulp. enzymes are biocatalysts that can speed up a chemical reaction without changing its structure. it is also widely used in industry as an alternative to the use of chemicals that pollute the environment. application of thermoalkalophilic xylanase in pulp and paper process has been studied extensively since the last two decades (buchert et al. 1994; kulkarni et al 1996; zheng et al. 2000; oakley et al. 2002; ibarra et al. 2010; paes et al. 2012). xylanase is an extracellular enzyme that can hydrolyze xylan (hemicellulose) into short chain xylooligosaccharide (kulkarni et al.1999). according to kulkarni et al. (1999) in the process of bleaching paper (pulp) requires a temperature of 80 °c with a ph vol.10, no.3, september 2016, p 112-117 doi: 10.5454/mi.10.3.5 application of response surface method in optimization of medium composition for xylanase production by bacillus halodurans cm1 in submerged fermentation 1 2 3 2* sara gustia wibowo , is helianti , ani suryani , and budiasih wahyuntari 1 post graduate program, biotechnology, institut pertanian bogor, jalan raya dramaga, bogor 16680, indonesia; 2 center for technology of bioindustry, agency for the assessment and aplication of technology (bppt), laptiab 1, puspiptek#611, serpong, south tangerang 15314, indonesia; 3 department of agroindustrial technology, faculty of agricultural engineering and technology, institut pertanian bogor, jalan raya dramaga, bogor 16680, indonesia a two level factorial design was performed to optimize xylanase production by alkalothermophilic bacillus halodurans cm1 using response surface method. the variables involved in this experiment were carbon (x ), 1 nitrogen source (x ) concentration, and ph (x ), corn cob and fish powder were used as carbon and nitrogen 2 3 source respectively. statistical analysis of the experimental results in the range studied, only carbon source gave significant effect on xylanase production. a second-order model was proposed to represent the enzyme activity as a function of xylan concentration (x ) and ph (x ). the optimum corn cobs concentration was 4.37% (w/v), 1 3 and the fish powder (with high p content) concentration was 1.75% (w/v) and ph 9. these conditions were tested and validated experimentally since the maximum growth rate achieved with these parameters, and the highest -1 xylanase activity observed was 522 u ml after 24 h fermentation. key words: bacillus halodurans cm1, production optimization, rsm, xylanase disain faktorial dua tingkat digunakan untuk optimasi produksi enzim xylanase alkalitermofilik dari bacillus halodurans cm1 dengan metode statistik respons permukaan. variabel yang terlibat dalam penelitian ini adalah konsentrasi sumber xilan tongkol jagung (x ), konsentrasi tepung ikan dengan kandungan fosfor (p) (x ), dan ph 1 2 (x ). analisis statistik hasil ccd menunjukkan bahwa, dalam kisaran yang diamati, konsentrasi tepung ikan 3 tidak memberi pengaruh yang signifikan terhadap produksi xilanase. sebuah model orde kedua diusulkan untuk mewakili aktivitas enzim sebagai fungsi konsentrasi xilan (x ) dan ph (x ). konsentrasi xilan tongkol jagung 1 3 dan tepung ikan p optimum masing-masing adalah 4,37% (b/v) dan 1,75% (b/v) pada ph 9. kondisi ini diuji dan divalidasi secara eksperimental karena laju pertumbuhan maksimum dicapai dengan parameter ini. hasil -1 pengamatan aktivitas xilanase terbaik adalah 522 u ml setelah fermentasi berlangsung 24 jam. kata kunci: bacillus halodurans cm1, optimasi produksi, rsm, xylanase microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone/fax: +62-21-7560536; email: budiasih.solichin@gmail.com volume 10, 2016 microbiol indones 113 range of 6.0-8.0. therefore, the xylanase needed is the alkalothermophilic one. bacillus halodurans cm1 is a xylanase-producing bacterium and has been isolated by ulfah et al. (2011) of hot springs cimanggu, west java. b. halodurans cm1 is a gram-positive bacterium that produces extracellular xylanase in submerged fermentation system. however, the optimum conditions for the enzymes production include ph, source concentration of carbon and nitrogen has not been known yet. based on preliminary experiment, b. halodurans cm1 produced xylanase in the xylan containing medium (ulfah et al. 2011). therefore, the production of xylanase used xylan carbon source. this study would be planned to be continued through to the industrial scale, so inducer xylan must be substituted with a carbon source (xylan) from xylan-containing agricultural waste (corn cobs and sugarcane bagasse), as well as the source of nitrogen substitution with fish powder. experiment optimization of xylanase production conditions implemented using response surface optimization techniques methodology (rsm). the aims of this study was to obtain the optimum condition of ph, carbon, and nitrogen source concentration for b. halondurans cm1 xylanase production using response surface method. materials and methods microorganism. bacillus halodurans cm1 (culture collection of laboratory of bioindustry, bppt), isolated from cimanggu hot pond, west java was used in this study. stock cultures were maintained on culture medium, which was used for the submerged culture, refreshed at 55 °c in modified luria broth medium containing 0.5 % (w/v) bactopeptone, 0.25 % (w/v) yeast extract, 0.25 % (w/v) sodium chloride, and 1 % (w/v) beech wood xylan. cultivation medium. the medium composition used in the experiment was modification of medium according to mamo et al. (2006). in 100 ml medium consisted of 0.5 g corn cobs xylan, 0.5 g fish powder rich in p, 0.29 g kh po , 1 g na co , 0.2 g naci, 0.01 g 2 4 2 3 mgso , 0.01 g caci . for the optimization 4 2 experiments, the cultivation medium was composed of different concentration of corn cob, fish powder, and ph according to the experimental design. cultivation. the optimization experiments were carried out in 500 ml erlenmeyer flasks containing 100 ml of cultivation medium. shake flasks were seeded with inoculum, at initial concentration of 2.2 x 8 10 cells ml, which were incubated for 24 h at 50 °c 120 rpm. enzyme assays. xylanase activity was determined by dinitrosalicylic acid (dns) method, according to the miller (1959) for quantifying reducing sugar. 50 µl of crude enzyme extract at appropriate dilutions in tris-base buffer was mix with 450 µl of 0,5 % (w/v) beechwood xylan in 50 mm of buffer at the indicated ph 9. the mixture was incubated for 5 min in the thermomixer at the optimum temperature for enzyme activity of 70 °c. subsequently, 750 µl dns reagent (1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulfite, and 1% sodium hydroxide, 20% (w/v) potassium sodium tartrate) was added to stop the reaction. then the mixture was boiled at 100 °c for 5 min, and kept at room temperature. afterward 250 µl of distilled water was added. reducing sugar released during incubation was measured as xylose equivalents at 540 nm. as blank we used the same mixture as in the above sample; however, enzymes were added following addition of dns into the reaction mixture. one unit (u) of enzyme activity is defined as the amount of enzyme releasing 1 µmol of reducing sugar equivalent per minute under the assay conditions. 3 experimental design. a 2 central composite design (ccd) was performed in order to determine the optimal conditions for producing xylanase by b. halodurans cm1. the dependent variable selected for -1 this study was the enzyme activity, expressed in u ml , and the independent variables chosen were corn cob concentration, fish powder (rich in p) concentration, and medium ph. the range and the levels of these variables are given in table 1. statistical analysis. statistics, software design expert 7 version 7.0.0, stat-ease corporation minneapolis, usa, was used for regression and graphical analysis. results based on earlier studies the media selected was modified medium with corn cob was selected as a carbon source and fish powder was selected as nitrogen source. the variables involved in this study were corn cob concentration (x ), fish powder concentration (x ), 1 2 and ph (x ). the following experiment was to 3 determine which factors affected the enzyme production. a two level factorial design was defined to determine factors that affected the enzyme production (data not shown). the experimental results showed that the model of corn cob concentration, fish powder concentration and ph value was significant so was the curvature f value. in this work, these variables were statistically optimized with the help of central composite design (ccd) using response surface method (oh et al. 1995; wang et al. 2004). the experimental design and the results are shown in table -1 2. the highest xylanase activity (522.512 u ml ) was observed at run number 8, where the factors corn cob concentration and fish powder concentration was 4.37% (b/v) and (1.75% (b/v), respectively. the medium ph was 9.0 and the incubation time was 24 h. this activity was 15.7 fold higher than that of observed at run number 11, where the related factors were used at highest levels for x , and lowest for x and x . table 3 2 1 3 shows the regression analysis for this experiment, presenting the estimates and hypothesis test to the coefficients of regression. at the 5% probability level the linier and quadratic coefficient of x (corn cob 1 concentration), and the coefficient of the interaction x x (corn cob concentration and ph) were found to be 1 3 significant. the mathematical model representing the xylanase activity (ŷ) in the experimental region studied can be expressed by eq.(1) ŷ = -18.89893 + 2.07786x + 4.33193x – 0.19490 1 3 2 x x – 0.22459 x (1)1 3 3 the independent variables, corn cobs, ph and interaction between corn cobs and ph had a significant effect on xylanase production (p > 0.05) (table 3). the statistical significance of a second-order model equation was evaluated by the f-test analysis of variance (anova). the computed f-value (19.61) indicated that the model was significant at high confidence level. the probability p-value was very low independent variables symbol levels -1 0 1 xylan x1 0.63 1.31 2.00 fish powder p x2 0.54 0.99 1.44 ph x3 8 9 10 1 table 1 values of independent variables a different levels of the ccd run number coded levels xylanase activity (u ml) x1 x2 x3 observed predicted 1 0 0 0 345.18 505.01 2 0 0 0 97.089 90.2609 3 1 1 -1 196.26 266.453 4 1 -1 1 138.47 168.974 5 -1 -1 -1 42.496 51.88 6 -1 1 1 103.85 112.534 7 0 0 0 197.37 212.716 8 0 0 0 522.51 521.328 9 -1 -1 1 99.26 112.534 10 1 1 1 243.19 169.974 11 -1 1 -1 33.19 51.88 12 1 -1 -1 272.19 266.453 13 0 0 0 276.38 212.71 14 0 -1682 0 153.85 212.71 15 0 0 -1.682 254.61 212.71 16 -1682 0 0 67.49 43.09 17 0 0 0 198.58 212.71 18 1.682 0 0 50.34 56.74 19 0 0 1.682 234.75 212.71 20 0 1.682 0 187.32 212.71 1 table 2 experimental design and result of the ccd 114 wibowo et al. microbiol indones *statistically significant at 99% of confidence level **statistically significant at 95% of confidence level table 3 results of regression analysis of the ccd (p=<0.0001) reflecting the significance of the model (liu et al. 2003). the model show lack of fit not significant and presented a fairly high determination 2 coefficient (r = 0.80), explaining 80% of the variability in the response and indicates correlation between the experimental observed and predicted values (table 4). in general, a regression model having 2 r value higher than 0.9 considered to have a very high correlation (haaland 1989). the value of the 2 determination coefficient (r = 80%) was also high enough to indicate the significance of the model. the response surface described by the model equation (ŷ) to estimate xylanase activity over independent variables xylan concentration (x ) and ph 1 (x ) is shown in fig 1. that illustrated the relationship 3 between the response and experimental data. there is a rather broad plateau region over which the enzyme activity changes relatively litle when xylan concentration is varied. as observed, the fish powder p concentration in the range studied (0.23-1.75% (b/v)), did not affect xylanase production therefore the fish powder concentration was maintained at 1.75 % (b/v). the validation steps of the models using solution from regression model (eq. 1). based on the model obtained, the optimal working conditions were defined to attain high xylanase activity minimizing the xylan concentration and cultivation time. thus, the point assigned as optimum corresponded to 4.37% (b/v) of corn cob concentration and 1.75% (b/v) of fish powder p concentration, ph 9.0 and 24 h cultivation time. under this condition, the model predicted a xylanase -1 activity of 521.238 u ml was slightly different with -1 the predicted value of 519.5-523.1 u ml . as a result, the models developed were considered to be accurate and reliable for predicting the production of xylanase by alkalophilic b. halodurans cm1. discussion although previous research regarding alkalo thermophilic xylanase production has been reported, little information on the optimization of its production is available (heck et al. 2005). some of these studies with bacteria have not been reported using statistical designs (brodel et al. 1990; subramanian et al. 1998). in this paper, experimental method of 2 level factorial design, and rsm design demonstrated that the ccd and regression analysis methods were effective to find the optimized corn cobs, fish powder concentration and ph for the production of alkalo-thermostable xylanase from alkalophilic b. halodurans cm1. the bacterium a newly isolated strain growing on some economical and term coefficient standard error p-value intercept 2.33 0.067 0.0060 x1 0.22 0.045 0.0011* x1x1 -0.0049 0.044 0.2939 x2 0.053 0.045 0.2679 x2x2 0.037 0.044 0.4178 x3 0.033 0.045 0.4811 x3x3 -0.23 0.044 0.0008* x1x2 0.024 0.058 0.6938 x1x3 -0.13 0.058 0.0518** x2x3 0.064 0.058 0.3079 1 source ss df ms f-ratio p-value model 1.55 3 0.52 19.61 <0.0001 residual 0.37 14 0.026 lack of fit 0.35 11 0.032 6.44 0.0759 total (corr.) 1.93 19 1 2 r = 0.80;ss, sum of squares; df, degrees of freedom; ms,mean square table 4 analysis of variance (anova) for the model regression representing xylanase activity volume 10, 2016 microbiol indones 115 abundant substrate as corn cobs replaces the expensive carbon source-xylan and fish powder replaces the expensive nitrogen source-pepton. using the rsm analysis it was found that maximum production xylanase activity was obtained at 4.37% (w/v) corncobs (w/v), 1.75% (w/v) fish powder p , ph 9.0 after 24 h incubation the activity was increased nearly twice compared to the original medium. according to lin et al. (2007) experiment alkalophilic bacillus sp under high ph conditions around 9.0-10.0 the cell growth and the production of xylanase were comparatively desirable, it corresponds to b. halodurans optimum ph 9.0. statistical method for optimizing fermentation process could overcome the limitations of classic empirical methods and was proved to be a powerful tool to optimize the production of alkalothermostable xylanase from alkaliphilic b. halodurans cm1. in this work, rsm model was proposed to study the combined effects of culture media composition. under optimal composition, the predicted production of xylanase after 24 h incubation -1 was 52 u ml . optimization study of xylanase production by bacillus circulans d1 using rsm by bocchini et al. (2002), showed that optimum xylanase activity of 19 -1 ml was gained at the optimum xylan concentration -1 and cultivation time of 5 g l and 48 h respectively. in our work the xylanase production was much higher and the highest xylanse activity was reached in a shorter period of time than that of bocchini et al. (2002) report. response surface methods were also used to optimizing a culture medium with regards to xylanase production by a schizophyllum commune strain (haltrich et al. 1993), the optimum concentration of -1 -1 -1 73.4 g l avicel, 55.5 g l yeast extract, and 1.38 g l nh no were determined by a central composite 4 3 -1 design. the highest xylanase production (5.74 u ml ), resulted in an increase of 330% compared initial medium and was reached within 11 days fermentation. the cultural conditions for thermostable xylanase production of thermomyces lanuginosus was also studied by purkarthofer et al. (1993), an experiment using ccd was performed to optimize the medium component concentrations. when the fungus was -1 grown in the optimized medium 31.2 g l corn cob, -1 -1 30.2 g l yeast extract, and 5.0 g l kh po , the 2 4 highest xylanase production observed within 7 days -1 was 2.70 u ml . validation experiments were also carried out to verify the availability and the accuracy of the models, and the result showed that the predicted values were well agreed with the experimental values. it strongly suggested that the production of alkalo-thermostable xylanase by wild-strain alkaliphilic b. halodurans cm1 could improve in a large-scale fermentation process. the result of this study also provides some useful information for other extremozymes fermentation processes. these facts are important in making the whole process economically more feasible, in view of the high cost of pure xylan and the difficulty of its extraction from agricultural waste material. the alkalophilic ph of the fermentation medium 9.0 and the fig 1 response surface described by the model ŷ, which represents xylanase activity (u ml) as a function of xylan concentration and ph. 116 wibowo et al. microbiol indones high temperature used 50 °c also reduced the chances of contamination by opportunist microorganisms. references bajpai p. 1999.application of enzymes in the pulp and paper industry. biotechnol prog. 15:147-157. bocchini da, alves-prado hf, baida lc, roberto ic, gomes e, da-silva r. 2002. optimization of xylanase production by bacillus circulans d1 in submerged fermentation using response surface methodology. process biochem. 38:727–31. brodel b, samain e, debeire p. 1990. regulation and optimization of xylanase production in clostridium thermolacticum. biotechnol lett. 12(1):65-70. buchert j, tenkanen m, kantelinen a, viikari l. 1994. application of xylanases in the pulp and paper industry. bioresour technol. 50:65-72. chen c, chen jl, lin ty. 1997. purification and characterization of a xylanase from trichoderma longibrachiatum for xylooligosaccharide production. enzyme microbiol technol. 21:91-6. da silva r, yim dk, park yk. 1994. application of thermostable xylanases from humicola sp for pulp improvement. j ferment bioeng. 77(1):109-11. gupta s, bhusnan b, hoondal gs. 1999. enhanced production of xylanase from staphylococcus sp. sg-13 using amino acids. world j microbiol biotechnol. 15:511-512. haaland, p.d. (ed.), 1989. separating signals from the noise. in: experimental design in biotechnology. marcel dekker, new york, p.61-83. haltrich d, preiss m, steiner w. 1993. optimization of a culture medium for increased xylanase production by a wild strain of schizophyllum commune. enzyme microbiol technol. 15:854-60. heck jx, de barros soares lh, ayub maz (2005) optimization of xylanase and mannanase production by bacillus circulans strain bl53 on solid-state cultivation. enzyme microbiol technol. 37:417–423. katapodis p, christakopoulou v, chistakopoulos p. 2006. optimization of xylanase production by sporotrichum thermophile using corn cobs and response surface methodology. eng life sci. 2006. 6(4): 410-415. doi: 10.1002/elsc.200520134. kubata bk, suzuki t, horitsu h, kawal k,takamizawa k. 1994. purification and characterization of aeromonas caviae me-1 xylanases v, which produces exclusively xylobiose from xylan. appl environ microbiol. 60: 531-535. kulkarni, n., a. shendye & m. rao. 1999. molecular and biotechnological aspects of xylanases. fems microbiol rev. 23: 411-456. lin, s.,dou w,xu h, li h, xu z, ma y.2007. optimization of medium composition for the production of alkaline β-mannanase by alkaliphilic bacillus sp. n16-5 using response surface methodology. appl microbiol biotechnol. 75:1015-1022. doi 10.1007/s00253-0070907-y. liu jz, weng lp, zhang ql, xu h, ji ln. 2003. optimization of glucose oxidase production by aspergillus niger in a benchtop bioreactor using response surface methodology. world j microbiol biotechnol. 19:317–323. mamo g, rajni hk, mattiason b. 2006. a thermostable alkaline active endo-β1-4 xylanase from bacillus halodurans s7: purification and characterization. enzyme microbiol technol. 39(7):1492. miller gl. 1959. use of dinitrosalycylic acid as reagent for the determination of reducing sugars. anal chem. 31(3):208-218. doi:10.10.1021/ac60147a030. oh s, rheem s, sim j, kim s, back y. 1995. optimizing conditionsfor the growth of lactobacillus casei yit9018 in tyrptone-yeast extract-glucose medium by using response surface methodology. appl environ microbiol. 61:3809–3814. purkarthofer h, sinner m, steiner w. 1993. cellulase-free x y l a n a s e f r o m t h e r m o m y c e s l a n u g i n o s u s : optimization of production in submerged and solidstate culture. enzyme microbiol technol. 15:677-82. subramaniyan s, prema p. 2002. biotechnology of microbial xilanases: enzymology, molecular biology and application. critical rev biotechnol. 22(1):33-46. ulfah m, helianti i , wahyuntari b, nurhayati n. 2011. characterization of a new thermoalkalophilic xylanaseproducing bacterial strain isolated from cimanggu hot spring,west java, indonesia . j microbiol indonesia. 5(3): 139-143. doi: 10.5454/mi.5.3.7. viikari l, kantelinen a, sundquistj, linko m. 1994. xylanases in bleaching: from an idea to the industry. fems microbiol rev. 13: 335-350. wang yx, lv fx, lu zx. 2004. optimization of cultivation medium clitocybe sp.as5.112 for the extracellular polysaccharide production and mycelial growth by response surface methodology. j nanjing agriculture university. 27:89–94. volume 10, 2016 microbiol indones 117 page 1 page 2 page 3 page 4 page 5 page 6 01 munaeni.cdr vol.11, no.3, september 2017, p 75-80 doi: 10.5454/mi.11.3.1 in vitro phytochemical and inhibitory potential tests of buton forest onion extract (eleutherine palmifolia) on vibrio harveyi 1,2* 1 2 2 waode munaeni , arman pariakan , munti yuhana , mia setiawati , 1 and la ode baytul abidin 1 department of aquaculture, faculty of fisheries and marine science, universitas halu oleo, jalan kampus hijau bumi tridharma, kendari, sulawesi tengah, indonesia; 2 department of aquaculture, faculty of fisheries and marine science, institut pertanian bogor, jalan dramaga, bogor, indonesia. the objectives of this study were to analyze phytochemical content of buton forest onion extract and to test the inhibitory potential of buton onion extract on the growth of vibrio harveyi bacteria at different doses. test parameter included: (1) phytochemical test through the method of color visualization, (2) inhibitory potential test using two methods namely agar diffusion and co-culture. treatment of dose consisted of positive control/k+ -1 (chloramphenicol 30 mg ml ), negative control/k(sterile aquadest) and treatment of extract included a (20 mg -1 -1 -1 -1 ml ), b (40 mg ml ), c (60 mg ml ), d (80 mg ml ). qualitatively, result of phytochemical test showed that buton forest onion extract contained flavonoid, tannin, saponin, quinone, steroid and triterpenoid compounds. result of inhibitory potential test indicated that treatment d exhibited the highest inhibitory potential, while the minimum inhibitory potential was found in treatment a. the best co-culture test result was also found in treatment d. the extract of buton forest onion in this study the showed its capability in hibiting the growth of v. harveyi key words: eleutherine palmifolia, inhibitory potential, phytochemical, vibrio harveyi tujuan penelitian ini adalah menganalisa kandungan fitokimia dari ekstrak bawang hutan buton dan menguji daya hambat ekstrak bawang hutan buton terhadap pertumbuhan bakteri vibrio harveyi dengan dosis berbeda. parameter uji meliputi: (1) uji fitokimia dengan metode visualisasi warna, (2) uji daya hambat menggunakan dua metode yaitu metode diffusi agar dan metode kultur bersama. perlakuan dosis terdiri dari kontrol positif/k+ -1 -1 (chloramphenicol 30 mg ml ), kontrol negatif/k(aquades steril) dan perlakuan ekstrak meliputi a (20 mg ml ), -1 -1 -1 b (40 mg ml ), c (60 mg ml ), d (80 mg ml ). hasil uji fitokimia secara kualitatif menunjukkan bahwa ekstrak bawang hutan buton mengandung senyawa flavonoid, tanin, saponin, quinon, steroid dan triterpenoid. uji daya hambat terbesar terdapat pada perlakuan d, sedangkan daya hambat minimum terdapat pada perlakuan a. perlakuan terbaik dari uji kultur bersama juga ditemukan pada perlakuan d. ekstrak bawang hutan buton pada penelitian ini dapat menghambat pertumbuhan v. harveyi. kata kunci: eleutherine palmifolia, daya hambat, fitokimia, vibrio harveyi microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82188196428; email: unalaspan@yahoo.co.id diseases caused by microorganism in aquaculture may lead to serious loss, while the use of antibiotic for disease treatment causes undesired side effect (li et al. 2006) such as bacterial resistance on antibiotic. the use of natural compound originated from harmless plants has potential in fish culture as an alternative of antibiotic use (van hai et al. 2015). medicinal plant is considered to be able to show potential effect on the growth and survival and also has antimicrobial properties on aquatic organism (immanuel et al. 2004; citarasu 2010). phytochemical compound in medicinal plants can be used as chemotherapy in aquaculture (chang 2000; sivaram et al. 2004). phytochemical compound from several medicinal plants contained phenolic, polyphenol, alkaloid, quinone, terpenoid, lectin, and polypeptide compounds (harikrishnan et al. 2011). in addition, medicinal plant vibrio harveyi is a bacterium that causes vibriosis disease or luminescent disease in aquatic organism (phuoc et al. 2009). v. harveyi is a saprophytic and opportunistic bacterium, living in fish culture environment in normal condition and becomes pathogen in worsening environmental and host condition (garrity et al. 2004). vibriosis disease occurs in shrimp including in larvae stadia up to adult shrimp (soto-rodriguez et al. 2012, munaeni et al. 2014), is acute and infectious. shrimp attacked with vibriosis shows several signs such as brownish and skin damage, reddish tail and swimming legs, necrosis, black lymphoid organ, brown gills, brownish muscle, empty intestine, and weak movement (cano-gomez et al. 2009). is rich in various nutrients (chang 2000). the use of medicinal plants has advantages namely cheap, easy to prepare, effective with less side effect during treatment (jian and wu 2003; 2004), not dangerous and does not create environmental problems (citarasu 2010). the use of alcohol solvent resulted higher efficiency in extracting secondary bioactive metabolites compared with water extraction method (bulfon et al. 2015). the aim of this study were to analyze phytochemical content of buton forest onion extract and to test the inhibitory potential of buton onion extract on the growth of vibrio harveyi bacteria at different doses. materials and methods time and place. this study was conducted during march-may 2017 in testing laboratory of fisheries and marine science faculty, universitas halu oleo and laboratory of fish health, aquaculture department, fisheries and marine science faculty and laboratory of biopharmaca, institut pertanian bogor. preparation of powder and ethanol extraction. the bulbs of buton forest onion that had flowered or already reached 3-4 months old were cleaned and further thinly sliced and dried in the oven for 48 h at o temperature of 60 c. later, the slices of dry onion were grinded using blender and separated using sieve until flour/powder was produced. buton forest onion powder was further extracted using ethanol solvent of 96% at ratio of 1:4 (w/v). later, maceration process was performed at room temperature for 24 h using magnetic stirrer. the product of first maceration was filtered and further remacerated for two times. the products of maceration were collected together and filtered using whatman filter paper no 41, then treated to generate more concentrated product using evaporator vacuum at o temperature of 40 c. the yield of dry buton forest onion extract obtained was calculated using the equation below : observational parameter. phytochemical test was done using color visualization method and inhibitory potential test was conducted using two methods, namely agar diffusion and co-culture. phytochemical test. phytochemical test was performed through color visualization method by the standard method of department of health of republic of indonesia (1995), which included tests of flavonoid, alkaloid, tannin, saponin, quinone, steroid, and triterpenoid. inhibitory potential test through agar diffusion method. inhibitory potential test through agar diffusion method was performed to observe the zone of inhibition around the paper disk. buton forest onion extract were made into various concentrations, -1 -1 -1 those were 20 mg ml , 40 mg ml , 60 mg ml , 80 mg -1 ml and negative control (k-) using sterile aquadest, -1 and positive control (k+) using 30 mg ml antibiotic chloramphenicol (bernofarm). pure culture of v. harveyi amounted to one innoculation loop was taken and cultured in 25 ml liquid swc (sea water complete: 5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 750 ml sea water, 250 ml aquadest) media and further incubated in waterbath o shaker at temperature of 37 c for 24 h. suspension of bacteria was moved into eppendorf of 2 ml and serial 8 dilution was performed until bacterial density of 10 -1 cfu ml was obtained. approximately 50 µl of bacterial suspension was spread on tcbs (thiosulphate citrate bile-salt sucrose) agar media. sterile paper disk was dipped into the stock of buton forest onion extract and further put on tcbs media in which the bacteria has been spread. each treatment of dose was given mark with replication of four times. furthermore, treatments o were incubated for 24 h at temperature of 37 c. after being incubated, measurement of the diameter of the zone of inhibition was done. inhibitory potential test through co-culture method. in vitro antibacterial test of buton forest onion extract on v. harveyi was also performed through coculture method. buton forest onion extract at different doses were put into a test tube of 90 µl, while for control, pbs (phosphate buffer saline: 8 g nacl, 0.2 g kh2po4, 1.5 g na2hpo4, 0.2 g kcl, 1000 ml aquadest) was filled into the sterile tube. each tube was filled with suspension of v. harveyi of 10 µl. further, it was homogenized and incubated for 24 h at o temperature of 37 c. later, it was serially diluted and spread on tcbs media and incubated for 24 h at o temperature of 37 c. moreover, the number of colony grew in each treatment was calculated. data analysis. research data were analyzed descriptively and statistical test was done using microsoft excel 2010, anova (analysis of variance) test, if the result was significantly different, post hoc test of duncan was performed using the program of spss (statistical program software system) version 16. 76 munaeni et al. microbiol indones volume 11, 2017 microbiol indones 77 results fresh buton forest onion bulbs was processed into the powder or flour of buton forest onion (fig 1a) and further was extracted using 96% ethanol thus resulted in buton forest onion extract (fig 1b) with yield value of -1 10.11% (v v ). result of phytochemical test (table 1) showed that the extract of buton forest onion bulbs positively contained flavonoid, tannin, saponin, quinone, steroid and triterpenoid compound, yet it was negative or did not contain alkaloid compound using the reagent of dragendorff, meyer's and wagner. result of inhibitory potential test through agar diffusion method using tcbs media (fig 2a) and swc media (fig 2b) indicated that higher dose of treatment led to larger clear zone. it can be found in the result of statistical test (fig 3) that the treatment of buton forest onion extract generated the largest zone of inhibition in treatment d which was not significantly different (p>0.05) from treatment k+ (positive control using -1 chloramphenicol of 30 mg ml ) and significantly different (p<0.05) from other treatments.treatment a was not significantly different (p>0.05) from treatment k-, yet it was significantly different (p<0.05) from other treatments. treatment b was significantly different (p<0.05) from all treatments, so was treatment c. result of inhibitory potential test through coculture method (fig 4) also indicated the same finding as agar diffusion result that higher dose will result in decreasing density of bacteria. treatment d (dose of 80 -1 mg ml ) was significantly different (p<0.05) from other treatments. in this treatment, v. harveyi colonies -1 were not found (0 log 10 cfu ml ) after incubation for 24 h. discussion the yield of buton forest onion extract produced in -1 this study amounted to 10.11 % (v v ). this yield value was higher than the result of study conducted by febrinda (2014) by macerating fresh bulb using water and ethanol solvent which resulted in yield of 8.11% and 6.47%. the result of phytochemical test was also different from the research finding of febrinda (2014) where buton forest onion extract obtained from -1 fig 1 powder (a) and extract of buton forest onion with yield of 10.11 % (v v ) (b). (a) (b) fig 2 inhibitory potential test of v. harveyi through agar diffusion method using media of tcbs (a) and swc (b) -1 with treatments: k+ (positive control using chloramphenicol of 30 mg ml ), k(negative control using -1 -1 -1 -1 aquadest), a (20 mg ml ), b (40 mg ml ), c (60 mg ml ), d (80 mg ml ). (a) (b) kalimantan, positively contained alkaloid compound, both extracts were produced using water and ethanol solvent. the difference of this phytochemical content was expected due to the difference of the type of soil where the bulbs of buton forest onion extract used in this study grew, that was buton forest of south east sulawesi. furthermore, the use of solvent also affected the compound composition of extraction product. it was explained by stanojević et al. (2009) that the total phenol and flavonoid content in hieracium pilosella 78 munaeni et al. microbiol indones fig 3 diameter of zone of inhibition in v. harveyi through agar diffusion method. different letters over each treatment bar (mean ± sd) indicated significant difference (duncan; p < 0.05). k+ (positive control using -1 -1 -1 chloramphenicol of 30 mg ml ), k(negative control using aquadest), a (mg ml ), b (40 mg ml ), c -1 -1 (60 mg ml ), d (80 mg ml ). fig 4 total of v. harveyi resulted from inhibitory potential test through co-culture method. different letters over each treatment bar (mean ± sd) indicated significant difference (duncan; p < 0.05). k+ (positive control -1 -1 using chloramphenicol of 30 mg ml ), k(negative control using aquadest), a (20 mg ml ), b (40 mg -1 -1 -1 ml ), c (60 mg ml ), d (80 mg ml ). table 1 result of phytochemical test of buton forest onion extract parameter reagent result flavonoid positive wagner negative alkaloid dragendorff negative meyer’s negative tannin positive saponin positive quinone positive steroid positive triterpenoid positive extract was higher by using ethanol solvent compared with water and methanol maceration. according to febrinda (2014), buton forest onion extract produced using ethanol solvent contained higher phytochemical compound, total phenol, and total flavonoid than that of water maceration. this study showed that higher dose of buton forest onion extract resulted in larger clear zone obtained from agar diffusion method or less bacterial density through co-culture method. result of this research was in line with the research finding of pandhi and panda (2015) that the use of e. bulbosa extract with solvent of ethyl acetate, chloroform, butanol, ethanol, and -1 -1 aqueous at dose of 30 mg ml (w v ) was able in inhibiting gram negative bacteria (escherichia coli, pseudomonas aeruginosa, pseudomonas fluorescens, shigella boydii, shigella dysentriae, shigella flexneri, salmonella typhimurium, shigella sonnei, vibrio alginolyticus, vibrio cholerae) and gram positive bacteria (bacillus brevis, bacillus subtilis, bacillus licheniformis, staphylococcus aureus, staphylococcus epidermidis). the use of extract from medicinal plants was able to penetrate the outer membrane of bacteria, disrupt cellular and metabolism function, and led to the loss of cell content that eventually resulted in death (kang et al. 2011). these naturally occuring phenols and phenolic compounds of medicinal plants may accumulate in bacterial membrane which led to energy depletion (conner 1993), inhibited bacterial cell wall synthesis (marcucci et al. 2001). extract to inhibit v. harveyi was due to the existence of flavonoid, tannin, saponin, quinone, steroid, and triterpenoid compounds which were expected to be able to inhibit metabolism and caused v. harveyi. cell bacteria to lyse since polyphenolic, flavonoid, terpenoid, and otthe ability of buton forest o n i o n h e r v o l a t i l e c o m p o u n d s h a v e s t r o n g antimicrobial properties (gulluce et al. 2007; tassou et al. 2000). lin and tang (2007) mentioned that phenolic and flavonoid content in fruits and vegetables functioned as immunomodulator and was able to kill microorganism. extract of medicinal plants rich of phenolic compound contained high level of antimicrobial activity with the ability to inhibit the growth of various gram-negative and gram positive bacteria (silici et al. 2010; negi 2012). phenolic compound may cause bacterial cell wall to lyse, disrupt cytoplasmic membrane, and lead to leakage in cellular structure of bacteria which later resulted in cell death (negi, 2012). according to kim et al. (1995), medicinal plant compound was able to hinder the permeability of cell membrane and caused changes in cell structure and function, cell damage until cell death. in conclusion, the use of buton forest onion extract resulted in the death of v. harveyi cell. it was shown by the result of inhibitory potential test through co-culture method in this study, in which the colony of v. harveyi -1 -1 was 0 cfu ml at dose treatment of d (80 mg ml ). -1 whereas, in dose treatment of b and c (60 mg ml and -1 40 mg ml ), there was still v. harveyi some colonies, yet it surpressed the v. harveyi growth significantly higher compared with control treatment. acknowledgment the research is financially supported by pekerti (penelitian kerja sama antar perguruan tinggi) scheme from ministry of research, technology, and higher education, strategic research halu oleo university to arman pariakan with contract number : 065/sprh/lt/drpm/iv/2017. references bulfon c, volpatti d, galeotti m. 2015. current research on the use of plant-derived products in farmed fish. aquacult res. 46:513-551. chang j. 2000. medicinal herbs: drugs or dietary supplements? biochem pharmacol. 59:211-219. citarasu t. 2010. herbal biomedicines: a new opportunity for aquaculture industry. aquacult int. 18:403-414. conner de. 1993. naturally occurring compounds. in antimicobials in foods, davidson pt and branen al (eds.). marecl dekker , new york, pp. 441-468. 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pilosella l. extracts. sensors. 9:5702-5714. doi:10.3390/s90705702. tassou c, koutsoumanis k, nychas gje. 2000. inhibition of salmonella enteritidis and staphylococcus aureus in nutrient broth by mint essential oil. food res int. 33:273–280. van hai n. 2015. the use of medicinal plants as immunostimulants in aquaculture: a review. aquaculture. doi: 0.1016/j.aquaculture.2015.03.014. 80 munaeni et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 06 probiotic administration in coping of various disease caused by pathogenic bacteria that resistant to antibiotic nowadays has become a trend (mileti et al. 2009). probiotics are living microorganism which if given in adequate manner will produce many advantages for the health of the host (kaboosi 2011). probiotic bacteria are generally used and can be isolated from lactobacillus groups (romeo et al. 2010). vol.8, no.4, december 2014, p 183-190 doi: 10.5454/mi.8.4.6 effect of lactic acid filtrate and bacteriocins of lactobacillus acidophillus on phagocytosis activity of macrophages cell againts enteropathogenic escherichia coli (epec) 1* 1 2 iis herawati , diki hilmi , and prima nanda fauziah 1 medical technology department, sekolah tinggi ilmu kesehatan jenderal achmad yani , jalan terusan jenderal sudirman, cimahi 40533, indonesia; 2 department of biology, school of life sciences and technology, institut teknologi bandung, jalan ganesha 10 bandung 40132, indonesia immunity development known as one of effective ways in avoiding infection. antibacterial agent product isolated from lactobacillus acidophillus has been reported can activate t lymphocyte as part of adaptive immunity. this experimental study aimed at investigation of lactic acid and bacteriocins filtrate from l. acidophillus in modulating phagocytosis activity of human macrophages infected by enteropathogenic escherichia coli (epec). each of human macrophages culture was supplemented with lactic acid and -1 bacteriocins filtrate at concentration of 3.125, 6.25, and 12.5 µg ml as well as control without filtrate addition and incubated for 24 h. macrophages culture was then infected with epec for 30 minutes and was microscopically observed after being stained by giemsa. percentage of phagocytosis activity was gained from active macrophages in 100 observed cells. macrophages cultures supplemented with bacteriocins filtrate showed augmented phagocytosis activity while cultures supplemented with lactic acid filtrate showed decreased phagocytosis activity. anova analysis showed significant difference in phagocytosis activity of macrophage cultures supplemented with lactic acid (p=0.038) and bacteriocins (p=0.016 and 0.023). tukey hsd analysis for phagocytosis activity of macrophage cultures supplemented by bacteriocins, each group of treatment showed significant difference againts control. in conclusion, lactic acid from l. acidophillus has no effect in modulation of macrophages phagocytosis activity while bacteriocins can improve phagocytic activity. bacteriocins from l. acidophillus then can be suggested to have a role as immunomodulator. key words: bacteriocins, enteropathogenic e. coli (epec), lactic acid, lactobacillus acidophillus, phagocytosis peningkatan imunitas merupakan salah satu cara yang efektif dalam menghindari penyakit infeksi. produk antibakteri yang diisolasi dari lactobacillus acidophillus telah dilaporkan dapat mengaktifkan limfosit t yang berperan dalam imunitas adaptif. penelitian eksperimental ini bertujuan untuk menguji kemampuan filtrat asam laktat dan bacteriosin dari l. acidophillus dalam memodulasi aktivitas fagositosis dari makrofag manusia yang terinfeksi oleh enteropathogenic escherichia coli (epec). setiap kultur makrofag manusia diberi filtrat asam -1 laktat dan bacteriosin dengan berbagai konsentrasi, yaitu 3,125, 6,25, dan 12,5 µg ml , serta kontrol tanpa penambahan filtrat dan kemudian diinkubasi selama 24 jam. kultur makrofag yang telah diinkubasi kemudian diinfeksi oleh epec selama 30 menit dan diamati secara mikroskopis setelah diwarnai oleh giemsa. persentase aktivitas fagositosis diperoleh dengan menghitung jumlah makrofag yang aktif dalam 100 makrofag. kultur makrofag yang diberikan filtrat bacteriosin menunjukkan peningkatan aktivitas fagositosis, sementara kultur makrofag yang diberikan filtrat asam laktat menunjukkan penurunan aktivitas fagositosis. analisis anova menunjukkan perbedaan yang signifikan pada aktivitas fagositosis makrofag yang diberikan filtrat asam laktat (p = 0,038) dan bacteriosin (p = 0,016 dan 0,023). analisis tukey hsd untuk aktivitas fagositosis pada kultur makrofag yang diberikan filtrat bakteriosin, masing-masing kelompok perlakuan menunjukkan perbedaan yang signifikan terhadap kelompok kontrol. dari penelitian ini dapat disimpulkan bahwa asam laktat dari l.acidophillus tidak berpengaruh dalam modulasi aktivitas fagositosis makrofag sementara bacteriosin dapat meningkatkan aktivitas fagositosis makrofag. bacteriosin l. acidophillus kemungkinan dapat berperan sebagai imunomodulator. kata kunci: asam laktat, bakteriosin, e.coli enteropatogenik (epec), fagositosis, lactobacillus acidophillus *corresponding author; phone: +62-22-6631622, fax: +6222-6631624 ; email: iis.herawati73@yahoo.com lactobacillus acidophillus is one of probiotic bacteria from lactobacillus group. bacteriocins is a peptide synthesized by bacteria, including probiotic bacteria. antibacterial agent namely lactic acid and bacteriocins from l. acidophillus are known to have inhibitory effect to pathogenic bacterial growth and have an effect to improve immune system through lymphocyte activation (kim et al. 2009; kaboosi 2011; khazaie et al. 2012). this inhibitory effect by antibacterial agent involves its activity in lowering ph circumstance that influence the viability of pathogenic bacteria. in addition, the filtrate has been reported as an immunomodulator (bourhis et al.2009). immunomodulator is a substance or drug that recover the function of defect or hampered immune system, while immunostimulator is one of immunomodulator which improve the function of immune system (borchers et al. 2009; patil et al. 2011; khazaie et al. 2012). macrophages are cells of innate immune system that can be found in most of many organs. macrophages play a role as phagocyte as well as an antigen precenting cell (apc) that initiate adaptive immune response by activating t lymphocyte. macrophages can be activated by toll-like receptor (tlr) signals after recognize many kinds of pathogen associated molecular patterns (pamps) such as component of pathogenic bacteria and virus, fungi, and many others (taylor et al. 2010; fabriek et al. 2011). infectious disease is one of global basic problem need to be overcomed comprehensively. beside antibiotic treatment, natural substance administration which improve immune system must be considered as a part of controlling and eradication of infectious disease (sivick et al. 2010; fengyi et al. 2011). infectious disease caused by bacteria commonly occured in gastrointestinal tract. escherichia coli is one of causative agent in human gastrointestinal tract infections. enteropatoghenic e. coli (epec) is an important causative agent of children diarhhea in developing countries. epec infections mostly observed as diarrhea with manifestations life fever, blood, and convulsions (rodrı´guez-ban˜o et al. 2010). in this work, we conducted investigations about lactic acid filtrate and bacteriosins effect as immunostimulator on macrophages phagosytosis activity especially againts epec. adminstration of lactic acid filtrate and bacteriosins from l. acidophillus is expected to have a role in avoiding and handling epec infections in the future. materials and methods bacteria strain. the cultures used were lactobacillus acidophillus cps1 from the isolation of whole milk of lembang, and enteropathogenic escherichia coli (epec) bacteria culture, from the collection of microbiology laboratorium, medical technology department, sekolah tinggi ilmu kesehatan jenderal achmad yani. l. acidophillus and epec was grown in the man rogosa sharpe (mrs) agar (oxoid cm0361 b) supplemented by 0.5% caco and mcconkey agar (mca) (oxoid 3 cm0007) media respectively at a temperature of 37 °c for 24 h. production of lactic acid filtrate of l. acidophillus. bacterial filtrate was obtained by centrifugation of l.acidophillus culture that had been active in the man rogosa sharpe (mrs) broth at 6000 rpm at 4 °c for 15 min to separate the cells from the filtrate. filtrate supernatant was taken and put into a sterile tube. filtrate was then exposed to uv light at a distance of 40 cm for 40 min (moghaddam et al. 2006; fauziah et al. 2013), this treatment can distinguish bacteriosin from lactic acid filtrate according to uv sensitive characterisitic of bacteriocins. this filtrate was qualitatively confirmed by uffelman's method (salkowski 2009) and diluted with sterile aquadest to -1 gain lactic acid filtrate stock equal to 1000 µg ml (v/v). every stock then diluted for the second time so we got the concentration of each microtube -1 consecutively 25, 12.5, and 6.25 µg ml . production of bacteriocins filtrate of l. acidophillus. bacterial filtrate was obtained by centrifuging l. acidophillus bacteria that had been active in the man rogosa sharpe (mrs) broth at 6000 rpm at 4 °c for 15 min to separate the cells from the filtrate. filtrate supernatant was taken and put into a sterile tube. it was neutralized with naoh, and the filtrate was sterilized with 0.22 μm millipore filter (moghaddam et al. 2006; fauziah et al. 2013). this filtrate was qualitatively confirmed by visual zones of inhibition on lawns of l. lactis (ulrich and hughes 2001), and was then diluted with sterile aquadest to -1 gain bacteriocins filtrate stock equal to 1000 µg ml (v/v). every stock was then diluted for the second time so we got the concentration of each microtube -1 consecutively 25, 12.5, and 6.25 µg ml . preparation of peripheral blood mononuclear cell (pbmc) and macrophages culture. pbmc isolated from whole blood of healthy individual 184 herawati et al. microbiol indones (confirmed by protein electrophoresis analysis as uninfected subject at the time of investigations). pbmc isolation was performed according to bagiada and linawati (2009) briefly, leucoytes were separated by centrifugation of whole blood at 1500 rpm for 15 min to form three layers consist of red blood cells at the bottom, buffy coat containing leucocytes, and plasma as the upper layer. the buffy coat was then transferred into falcon tube containing hank's balanced salt solution (hbss) (1:1). this mixture was transferred into another falcon tube containing histopaque (1:1) and followed by centrigugation at 1500 rpm for 30 min. isolated pbmc transferred into new falcon tube and washed by hbss (1:1). macrophages culture was performed according to herawati et al. (2013) briefly, two hundreds microliter of isolated pbmc was dispensed into each well of multidish 24 wells which contains coverslip, and incubated for two hours at 37 o c and 5% co . culture supernatants from every well 2 were discarded and washed by hbss followed by giving 500 µl of complete rpmi (roswell park memorial institute), incubation was then continued to o 7-10 d at 37 c and 5% co .2 macrophages phagocytosis activity examination. macrophages phagocytosis activity examination againts epec was performed in duplicate according to chairul et al. (2009) and herawati et al. (2013). briefly, 500 µl of lactic acid filtrate or bacteriosins from various concentration dispensed into each well of culture containing mature macrophages followed by adding rpmi to get the final indicated concentration of lactic acid filtrate or bacteriosin in -1 each well 12.5, 6.25, and 3.125 µg ml , including control without filtrate addition. after incubation for 24 h, culture medium from each well were discarded and washed by hbss followed by addition of 500 µl of pbs and epec suspension into each well including control. the plate was then incubated for 30 min, washed twice by pbs, fixed with absolute methanol for 1 min and stained by giemsa for 10 min. every well was then gently washed by tap water and the coverslip was taken from each well and dried before observation under light microspcope using objective lens 100x with immersion oil. phagocytosis activity measurement was determined by the number of actively phagocytosing macrophages in one hundred macrophage cells. preparation of epec bacteria was started by inoculation of epec in mca, after incubation for 24 h epec inoculum was then suspended in 5 ml sterile phosphat buffer saline (pbs), and measured by nephelometer of mcfarland until its turbidity in 6 proportion to 0.5 mcfarland (150 x 10 ). results to confirm that the subject of investigations did not encounter any infections, we examine protein electrophoresis of subject blood before used as the source of macrophage culture. the value of albumin, alpha 1 and 2, beta 1 and 2, was at normal range of healthy individual confirmed the subject was not encounter any infections despite gamma globulin level describe increased subject antibody level (fig 1a). pbmc isolation from blood subject described that centrifugation of whole blood formed three layers consist of red blood cells at the bottom, buffy coat containing leucocytes, and plasma as the upper layer. the isolated pbmc (arrow) used as a source for macrophages culture (fig 1b). macrophages maturation from monocyte achieved after 7 d incubation and typical features of the cells was recognized by its attachment characteristic when obeserved on inverted microscope (fig 1c). phagocytosis activity of mature macrophages againts epec was seen after giemsa staining and actively phagocytosing macrophages can be recognized by epec's present inside the cell when observed on light microscope (fig 1d). measurement of macrophages phagocytosis activity againts epec supplemented by lactic acid filtrate of l. acidophillus was described. the highest activity level of -1 macrophages occured at concentration 3.25 µg ml with 72% active macrophages (fig 2). lowest activity -1 occured at concentration 12.5 µg ml with 62% active macrophages. anova analysis shows significant difference between control and treatment with p<0.05 (p=0.027), as control cells activity are better than the treatment. low phagocytosis activity of treated cells due to more lactic acid supplemented more acid ph circumstance, result in inappropriate optimal condiditon for macrophages phagocytosis. based on this evidence we suggest that lactic acid as an excretion product of probiotic l. acidophilus has no potential role as an immunostimulan. the highest activity level of macrophages supplemented by bacteriocins occured at concentration -1 6,25µg ml with 93% active macrophages (fig 3). in addition the lowest activity occured at control cells with 71% active macrophages. anova analysis shows significant difference between control and treatment with p<0.05 (p=0.014). tukey hsd test on table 1 volume 8, 2014 microbiol indones 185 a 186 herawati et al. microbiol indones b c volume 8, 2014 microbiol indones 187 fig 1(a) serum protein electrophoresis from whole blood of healthy individual before isolation of pbmc. albumin, alpha and beta globulin was at normal level despite gamma globulin level increased. this figure indicates that the subject did not encounter any infections. (b) isolation of peripheral blood mononuclear cell (pbmc) from whole blood of the subject. blood divided into three layers, red blood cell at the bottom, buffy coat in the middle and plasma at the upper layer. macrophages was grown from pbmc area in buffy coat (arrow). (c) macrophages maturation from monocyte. mature macrophages recognized from their attach features on the coverslip while being observed on inverted microscope. (d) phagocytosis activity of macrophages againts epec after being supplemented with 12,5 ug/ml of bacteriosin of l. acidophillus while being observed on light microscope 1000x. active macrophage can be distinguished by its epec engulfing-pseudopodia. d shows significant difference between control and treated cells with concentration of -1 bacteriocins 6.25 and 12.5 µg ml . discussion delphine et al. (2009) reported that l. crispatus can modulate gen expression of tlr-2 dan tlr-4.tlr-4 has been known as receptor o n p h a g o c y t i c c e l l s r e c o g n i z i n g lippopolysachharides (lps) on the wall of gram negative bacteria include epec. because l. acidophilusis one of probiotic bacteria, increased phagocytosis activity of macrophage culture supplemented by bacteriosin from l. acidophilus suggested due to up regulation of t l r 4 g e n e x p r e s s i o n . t l r 4 s i g n a l transduction initiated through lps binding by lps-binding protein (lbp). the bound lps to lbp continue to be bound by cluster of differentiation 14 (cd14). this lps-cd14 complex will be recognized by tlr-4 through md2 (modulation 2). md2 required for triggering signal induction of tlr-4. tlr-4 signal transduction can activate my88 (myeloid differentiation primary response gene 88) d e p e n d e n t p a t h w a y a n d d a n m y d 8 8 independent pathway. these activations will lead to activate nuclear factor kappa-b (nf-kb) which mediate gene expression of proinflammatory cytokine interleukin-1 (il-1) and expression of interferon-type gene (lu et al. 2008). karlsson (2012) also reported that probiotic bacteria can modify both innate and adaptive immune response dependent on strain-type of probiotic bacteria. l.rhamnosus gr-1 is one of probiotic can activate human macrophage nfkb. nf-kb known as transcription factor play important role in initial immune response againts pathogen. cytokine production as response to antigen determined by nf-kb translocation into nucleus. the role of probiotic bacteria on improving nf-kb activation made it a basis for improvement of pro-inflammatory c y t o k i n e p r o d u c t i o n ( tu m o r n e c r o s i s factor/tnf) that also has been reported. a study of l.casei administration to mice fig 3 phagocytosis activity of macrophages againts epec from culture supplemented with bacteriosin of l. -1 acidophillus. increase activity are significant at 6.25 and 12.5 g ml compared to control group with p value = 0.016 and 0.023, respectively. fig 2 phagocytosis activity of macrophages againts epec from culture supplemented with lactic acid filtrate of -1 l. acidophillus. decrease activity are significant at 12.5 g ml compared to control group with p value = 0.038. 188 herawati et al. microbiol indones -1 concentration of lactic acid (g ml ) -1 concentration of bacteriocin (g ml ) challenged by salmonella reported that tnf production increased compared to control group with no l.casei administration. nevertheless production of cytokin has not improved for all of pro-inflammatory cytokine, such as il-6 and il-8 that showed decrease level as of more investigations about the expression of various cytokine by human macrophage supplemented by bacteriosin from l.acidophillus need to be elucidated due to improvement of phagocytosis activity againts epec in this study. in addition probiotic bacteria had been reported can modulate adaptive immune response especially limfocyte b function observed from increased antibody level of mice administered by l.casei and produce better protective antibody titer againts salmonella infection. other investigation reported that l.rhamnosus gg administration reduce antibody ige production by mice so that bacteriosin administration effect on adaptive immune response especially cytokine production need to be revealed through more investigations (delphine et al. 2009; taylor et al. 2010; karlsson 2012). bacteriocins from l. acidophilus was able to improve phagocytosis activity of macrophage, while lactic acid had no ability to improve macrophage phagocytosis activity. bacteriocins from l. acidophilus can be suggested play a role as an imunostimulator, therefore require more investigations especially its effect on production of various cytokines. acknowledgment this research was supported by the directorate general of higher education, department of national education (hibah dosen pemula) 2013, contract number: 1789/k4/kl/2013, date 26 august 2013. we would also like to thank the lembaga penelitian dan pengujian terpadu (lppt), universitas gadjah mada, yogyakarta for the aid in providing research materials. references bagiada m, linawati m. 2009. pengaruh propolis terhadap sekresi interleukin-12 pada supernatan kultur makrofag dari penderita tuberkulosis paru yang diinfeksi mycobacterium tuberculosis. 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email: nrachmania@ipb.ac.id or melon aphid or cotton aphid was one of the leaf miners that attack many kinds of plants, including chili pepper. chili peppers attacked by have wrinkled leaf, obstructed shoot, and damaged leaf tissue caused by the chili veinal mottle virus (cvmv) carried by adult aphids. chili pepper yields may decrease up to 60% due to the attack of this virus (wagiman . 2001). is the most effective vector for viruses; aside of cvmv, the aphid also takes part as a vector for (garzo 2003). insectiside have been used in high frequency to control pest, but the limitation in chemical control is increasing due to a rapid development of insectiside resistance and also the accumulation of chemical residue is difficult to degrade. therefore, biological control has been chosen as an alternative to reduce chemical pesticides usage. root colonizing bacteria (rhizobacteria) that exert beneficial effects on plant development via direct or indirect mechanisms have been defined as plant growth-promoting rhizobacteria (pgpr). the pgpr may become a promising biocontrol agent for plant diseases. large scale application of pgpr to crop as inoculants will substantially reduce the use of chemical fertilizers and pestisides, which often pollute the environment (bloemberg and lugtenberg 2001). chitin is the most abundant polymer after cellulose. it is a common constituent of algae and fungal cell walls, insects, and nematodes. insect's exoskeleton or cuticle is composed of chitin. the cuticle is divided into three layers, namely the cover layer, epicuticle, and procuticle (merzendorfer and zimoch 2003; schwarz and moussian 2007). chitinases (ec 3.2.1.14) hydrolyse the glycosidic -1,4 bonds in chitin to its oligomer or monomer and are found in a broad range of aphis gossypii the aphids et al a. gossypii cucumber mosaic virus et al. organisms, including bacteria, fungi, and higher plants, and play different roles in their origin (lonhienne . 2001; funkhouser and aronson 2007). the presence of chitinolytic bacteria in the crop rhizosphere soils is highly beneficial as they are able to suppress plant pathogenic fungi near the root zone and provide sustainable plant protection against root diseases. saikia . (2005) reported the presence of pgpr sp. that produced chitinase and -1,3-glucanase and showed ability in inhibiting the growth of . previous research by mubarik (2010) showed that the chitinase of sp. strain i.5 and i.21 showed their ability to degrade exoskeleton chitin of the whitefly . the objectives of this research are to screen potential pgpr from pepper rhizosphere that produce extracellular chitinase and to use the enzyme for controlling leaf miner such as . twenty five bacteria isolates from the rhizosphere of chili pepper (damayanti 2007) were collected at microbiology laboratory of biology department, faculty of mathematics and natural sciences, institut pertanian bogor (ipb) and ipb culture collection. were obtained from suffering plants around ipb. twenty five pgpr from the rhizosphere of chili pepper were grown on nutrient agar and incubated for 48 h at 37°c, ph 7.0. each isolate was reinoculated to chitin agar (1% colloidal chitin, 0.1% mgso .7h o, 0.02% k hpo , 0.1% yeast extract, and 1.5% agar) and incubated for 48 h at 37°c, ph 7.0. the isolates were screened based on chitinolytic index, defined as the ratio of clear zone to colony diameter on nutrient agar. the identification of bacterial isolate was carried et al et al pseudomonas fusarium oxysporum et al. bacillus bemisia tabaci a. gossypii et al. a. gossypii materials and methods bacteria and test-objects. bacterial growth media and chitinolytic bacteria selection. 4 2 42 issn 1978-3477 vol 4, no 3, dec 2010, p 103-107 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.1 out based on morphological structure, gram staining, endospore staining, and catalase test. a number of 10 cells ml of selected isolate was inoculated to production media consisting of 0.3% colloidal chitin, 0.1% mgso .7h o, 0.02% k hpo , and 0.1% yeast extract. the culture was incubated on a dry shaker at 37°c and 100 rpm. cell cultures were collected every 12 h until 96 h and their density were measured spectrophotometerically at 600 nm. the cultures were then centrifuged at 8400 x g for 10 min to obtain the crude extract of extracellular chitinase enzyme in supernatant. chitinase activity was measured as described by spindler (1997). a volume of 450 l enzyme crude extract was added to 900 l 0.3% colloidal chitin and 450 l 0.1 m phosphate buffer. the mixture was incubated at 55ºc for 30 min. enzyme reaction was stopped at 100ºc for 10 min. after centrifugation at 8400 x g for 5 min, 600 l filtrate was added to 1.5 ml distilled water and 3 ml schales reagent, and the mixture was boiled at 100ºc for 10 min. enzyme activity was determined by measuring absorbance at 420 nm. one unit of enzyme activity was defined as the amount of enzyme which released 1 mol reducing sugar (n-acetyl glucosamine) per min. protein content was determined using as describe by bradford (1976). bovine serum albumin was used as a protein standard. enzyme characterization was done to define optimum ph and temperature of chitinase activity. chitinase activity was measured at ph ranging from 4 to 10 using 0.1 m citrate buffer (ph 4.0-6.0), 0.1 m phosphate buffer (ph 7.0-8.0), and 0.1 m glycine-naoh buffer (ph 9.0-10.0). the effect of temperature on chitinase activity was measured at temperature ranging from 25-60ºc using 5ºc interval at optimum ph obtained from previous experiment. the data were based on duplicate experiments. live aphids were selected based on their shape and size. aphids with the same size were assumed as aphids of the same age and chitin thickness. the test were done by dropping 20 μl cells supernatant and 20 μl enzyme crude extract to each aphid at room temperature. chitin hydrolysis was observed every 3 h until 24 h by comparing the thickness of chitin layer on each aphid (mubarik . 2010). treated aphid were first fixed with 70% ethanol for 24 h and then serially dehydrated for 10 min with 80, 95, and 100% ethanol. clove oil was dropped onto the aphids for 15 min and cleaned with xylol. each aphid was placed on the object glass, then covered and tightened with synthetic resin entellan (a particularly rapid embedding agent). chitin thickness was observed with light microscope at 40 times magnification. a total of 25 pgpr isolates were obtained from the rhizosphere of chili pepper. they formed a clear zone when grown on chitin agar. it showed that they all produced extracellular chitinases that could hydrolyze bacterial growth curve and enzyme production. c h i t i n a s e a c t i v i t y a n d p r o t e i n q u a n t i t y measurement. chitinase characterization. chitinase test to . results chitinolytic index. 8 -1 4 2 2 4 � � � � � aphis gossypii et al chitin in the media. among the 25 chitinolytic bacteria, only 3 isolates produced 0.5 cm clearing zone, i.e. i.5, i.21, and ii.14. interestingly, the isolates also had both proteolytic and cellulolytic activities (table 1). chitinase activity and characterization of i.5 and i.21 had been reported by mubarik (2010). therefore in this research only characterization of ii.14 chitinase activity were described, and the isolate is a gram-positive, endospore forming, rod shaped bacterium with catalase positive reaction. based on the physical characteristics, the isolates are most likely belonging to sp. (holt 1994). the isolate was grown in liquid chitin media at ph 7.0 and 37ºc. the growth rate of isolate ii.14 ascended from 0-24 h incubation and was relatively stable for 24-84 h, and descended afterwards (fig 1). the chitinase produced was detected since the 24 h and the highest chitinase activity were found at the 48 h, after 60 h the activity was not detected (fig 1). the activity of the isolate was measured at 55ºc ph 7.0. among the different temperature tested, maximum chitinase activity was observed at 55 c being 0.115 unit per mg protein. there were no chitinase activity lower than 30ºc and between 30-50ºc (fig 2). among different ph values tested, at ph 7.0 the enzyme showed maximum activity of 0.1 unit per mg protein. there was no chitinase activity at phs lower than 5.0 and higher than 8.0 (fig 3). et al. bacillus et al. growth curve and chitinase activity of ii.14 isolate. characterization of chitinase from ii.14 isolate. th th o isolate colony diameter (cm) clear zone diameter (cm) chitinolytic index other hydrolytic index proteolytic index cellulolytic index i.1 0.88 0,90 0.02 0.16 i.2 0.70 0.70 0.00 0.06 i.3 0.80 0.90 0.13 0.12 0.15 i.4 0.85 0.90 0.06 i.5 0.60 1.20 1.00 0.51 0.10 i.8 0.76 0.80 0.05 i.14 0.96 1.00 0.04 i.15 0.66 0.71 0.08 0.38 i.21 0.70 1.30 0.86 0.82 0.18 i.25 0.52 0.69 0.33 1.08 0.08 i.26 0.87 0.97 0.11 0.13 i.28 0.59 0.65 0.10 1.00 i.33 0.57 0.60 0.05 i.34 0.81 0.92 0.14 0.63 0.25 ii.7 0.62 0.65 0.05 ii.8 0.94 1.00 0.06 ii.10 0.84 0.92 0.10 ii.11 0.59 0.65 0.10 ii.12 0.75 0.77 0.03 ii.13 0.76 0.79 0.04 0.05 ii.14 0.60 1.05 0.75 0.28 0.40 ii.15 1.00 1.10 0.10 ii.16 0.65 0.67 0.03 ii.17 0.57 0.58 0.02 ii.34 0.90 0.99 0.10 table 1 chitinolytic index of rhizobacteria isolates no hydrolytic activity 104 nurdebyandaru et al. microbiol indones aphid chitin hydrolysis. discussion chitinases from i.5, i.21, and ii.14 isolates were used to observe their activities on the aphids' exoskeleton. microscopic observation showed that the aphid exoskeleton were thinner in the presence of enzymes. this was even observed at 3 h after treatments. treatment with the free cell supernatant made the exoskeleton thinner 12 h (i.21 and ii.14) and 9 h (i.5) after treatment. the thickness of aphids' exoskeleton after treatments with chitinase or the cell culture were significantly different compared to the control (fig 4). shanmugaiah . (2008) reported that among the 39 chitinolytic bacteria isolated from rice rhizosphere, only 11 isolates produced clearing zone over 0.5 cm on the chitin colloidal agar. chitinase produced by showed the highest clear zone of 1.1 cm around the colony in chitin colloidal agar. in this research, among the 25 chitinolytic bacteria, only 3 isolates produced clearing zone over 1.05 cm with chitinolytic index over 0.5, i.e. i.5, i.21, and ii.14. all isolates most likely belong to sp. (holt 1994). chitinase production was reported in different species of such as (sabry 1992), (huang 2005), (chen . 2004), (waldeck 2006), (sabry 1992), (sakai 1994), (wang 2006), sub sp. (vega 2006), and sub sp. (driss 2005). previous reports have shown that pgpr sp. strain i.5 and i.21 demonstrated their ability to degrade exoskeleton chitin of whitefly (mubarik 2010). however, this is the first report on the production of chitinase by sp. strain ii.14 and its application to degrade exoskeleton of aphid cell culture and chitinase of 1.5 and 1.21 isolates were used as comparison. ii.14 isolate secreted chitinase that were induced by the presence of substrates such as colloidal chitin. the highest specific activity was observed at the stationary phase, when the cell density began to decrease after 48 h incubation. from this time on, the activity started to decrease and no activity was detected after 72 h until the end of observation et al b. laterosporus bacillus et al. bacillus b. amyloliquefaciens b. cereus et al. b. circulans et al b. licheniformis et al. b. megaterium b. stearothermophilus et al. b. subtilis et al. b. thuringiensis aizawai et al. b. thuringiensis kurstaki et al. bacillus bemisia tabaci et al. bacillus a. gossypii. fig 1 growth and chitinase activity of ii.14 isolate on production media enriched with colloidal chitin. log of cell number; specific chitinase activity. fig 2 effect of temperature on chitinase activity of ii.14 isolate. fig 3 effect of ph on chitinase activity of ii.14 isolate. fig 4 aphids without treatments or control (a), cell culture treatment (b-d), enzyme treatment (e-g). cell culture treatment of i.5 isolate at 9 h after treatment (b), i.21 isolate at 12 h (c), ii.14 isolate at 12 (d). enzyme treatment isolated fromi.5 at 3 h after treatment (e), i.21 at 3 h (f), ii.14 at 3 h (g). microscope observation was conducted with 40 times magnification. isolate isolate isolate c e ll s a m o u n t lo g a ri th m 8.4 8.3 8.2 8.1 8.0 7.9 7.8 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 0 12 24 36 48 60 72 84 96 time (h) x temperature ( c) o 25 30 35 40 45 50 55 60 0.12 0.10 0.08 0.06 0.04 0.02 0 4 5 6 7 8 9 10 0.12 0.01 0.08 0.06 0.04 0.02 0 ph x x x x xx x x x a b c d e f g volume 4, 2010 microbiol indones 105 (96 h). it was estimated that after 72 h, the bacterial culture were already at the end of stationary phase and in the beginning of death phase. the enzymes were no longer produced and toxic metabolites, such as antibiotic, were detected in stationary phase. chitinase activity was measured using the method of spindler (1997). this method defines the amount of n-acetyl glucosamine formed following hydrolysis, which is then mixed with schales reagent [0.05% k fe (cn)6 and 5.3% na co ] and measured with spectrophotometer. low chitinase activity shows that there are a high amount of unhydrolyzed chitins and little amount of released n-acetyl glucosamine. chitinase of ii.14 had the highest activity at 55ºc. chitinase activity of i.21 and i.5 were also measured at 55ºc (mubarik 2010). chitinase of ii.14 also showed activity at 30ºc and 50ºc, but not as high as at 55ºc. there was no chitinase activity at 30-50ºc. we assumed there were more than one chitinase molecules in the crude enzymes that had optimum activity at 30 and 55ºc. chitinases have been isolated from a variety of bacteria, including spp. and some of them were reported to produce multiple forms of chitinases with different molecular masses (sanmugaiah . 2008). enzyme activity was also affected by ph. chitinase of isolate ii.14 showed activity at ph 5.0-8.0. the highest activity was observed at ph 7.0. bacterial chitinase generally shows optimum activity at low ph (ueda and arai 1992; evvyernie . 2000; natsir 2000), but some also reported optimum activity at neutral ph (wang and chang 1997; purwani 2004). chitin hydrolization test against showed that cell cultures and chitinase crude extracts of isolate i.5 and i.21 were able to hydrolyse the insect’s exoskeleton (mubarik 2010). this research analysed the ability of cell culture and chitinases of i.5, i.21, and ii.14 isolate to degrade the exoskeleton of leaf miner . damage level was caused by the treatment were observed at every 3 h for 24 h. crude extracts worked better than the cell cultures in degrading chitin. microscopic observation showed that aphids cuticle were hydrolyzed at 3 h after treatment. cell culture treatment showed that the hydrolysis started after 12 h (i.21 and ii.14) and after 9 h (i.5) after treatment. it showed that chitinase were faster than the cell cultures in hydrolyzing the cuticles. in cell cultures treatment, more time is needed by the cells to produce chitinase, while the chitinase treatment made it possible to directly hydrolyse the chitin in exoskeleton. dark exoskeletons were degraded and became more transparent, therefore objects with higher transparency showed a higher level of hydrolyzed chitin. chitinases-encoding gene may be cloned into high economy plant. expression of the chitinase genes on tomato plants decreased the growth of colorado potato beetle (lawrence and novak 2006). koga (2005) reported that transgenic strawberry plants carrying chitinase gene were resistant to pathogenic fungi. chitinase genes transformation on papaya increased its resistancy to red spiders (mccafferty . 2006). however, the general attitude of indonesians toward transgenic plants is still not positive (abbas 2003). i.5, i.21, and ii.14 isolates showed their ability to degrade chitin of . chitin degradation with enzyme 3 3 2 3 et al. bacillus et al et al bemisia tabaci et al. a. gossypii et al a. gossypii treatment was better than the cell culture treatment. chitinase produced by the three selected isolates were potential to be improved and used as a biocontrol agent for insect such as aphid application of chitinase may be done by spraying the enzyme directly to the plants. leaves and fruits of chitinase-sprayed-strawberry showed no presence of any insects or pathogenic fungi (koga 2005). combination between chitinase and -toxin of were found more effective in killing pest insects (patil . 2000). spreading chitin surrounding the plant is also an alternative, as this will induce chitinolytic rhizosphere bacteria to secrete chitinase (metcalfe . 2002). a. gossypii. b. thuringiensis et al et al σ references abbas n. 2003. controversion around the food product as a result of genetically modified organism. analis 4:41-7. bloemberg gv, lugtenberg bjj. 2001. molecular basis of plant growth promotion and biocontrol by rhizobacteria. curr opin plant biol 4:343-50. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. anal biochem 72:248-54. chen cy, wang yh, huang cj. 2004. enhancement of the antifungal activity of f29-3 by the chitinase encoded by gene. can j microbiol 50:451-4. damayanti ta, pardede h, mubarik nr. 2007. utilization of rootcolonizing bacteria to protect hot-pepper against hayati j biosci 14: 105-109. driss f, kallassy-awad m, zouari n, jaoua s. 2005. molecular characterization of a novel chitinase from subsp. . j appl microbiol 99:945-53. evvyernie d, yamazaki s, morimoto k, karita s, kimura t, sakka k, ohmiya k 2000. identification and characterization of m-21, a chitinolitic, mesophilic, and hydrogen producing bacterium. biosci bioeng 89:596-601. funkhouser jd, aronson nn. 2007. chitinase family gh18: evolutionary insights from the genomic history of a diverse protein family. bmc evol biol 7:96-111. garzo ei, diaz b, duque m, fereres a. 2003. settlement rate of (hemiptera, aphididae) and transmissionefficiency of in melons protected with kaolin-particle films. span j agric res 4:65-71. holt jg, krieg nr, sneath pha, staley jt, williams st. 1994. bergey's manual of determinative bacteriology 9 ed baltimore: williams & wilkins. huang cj, wang tk, chung sc, chen cy. 2005. identification of an antifungal chitinase from a potential biocontrol agent, 28-9. j biochem mol biol 38:82-8. koga d. 2005. application of chitinase in agriculture. j met mater miner 15:33-6. lawrence sd, novak ng. 2006. expression of poplar chitinase in tomato leads to inhibition of development in colorado potato beetle. biotechnol lett 28:593-9. lonhienne t. 2001. cloning, sequences, and characterization of two chitinase genes from the antarctic sp. strain tad20: isolation and partial characterization of the enzymes. j bacteriol 183:1773-9. mccafferty hr, moore ph, zhu yj. 2006. improved tolerance to carmine spider mite by the expression of chitinase transgene. trans res 15:337-47. merzendorfer h, zimoch l. 2003. chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. j exp biol 206:4393-412. metcalfe ac, krsek m, gooday gw, prosser j, wellington emh. 2002. molecular analysis of a bacterial chitinolytic community in an upland pasture. appl environ microbiol 68:5042-50. bacillus subtilis bacillus circulans chia tobacco mosaic tobamovirus. bacillus thuringiensis kurstaki . clostridium paraputrificum aphis gossypii cucumber mosaic virus . . bacillus cereus arthrobacter carica papaya manduca sexta th 106 nurdebyandaru et al. microbiol indones mubarik nr, mahagiani i, anindyaputri a, santoso s, rusmana i. 2010. chitinolytic bacteria isolated from chili rhizosphere : chitinase characterization and its application as biocontrol for whitefly ( genn.). am j agr biol sci 5:430-5. natsir h, chandra d, rukayadi y, suhartono mt, hwang jk, pyun yr. 2002. biochemical characteristics of chitinase enzyme from sp. of kamojang crater, indonesia. j biochem mol biol biophys 6:279-82. patil rs, ghormade v, desphande mv. 2000. chitinolytic enzymes: an exploration. enzyme microb technol 26:473-83. purwani ey, suhartono mt, rukayadi y, hwang jk, pyun yr. 2004. characteristics of thermostable chitinase enzymes from the indonesian sp. 13.26. enzyme microb technol 35:147-53. sabry sa. 1992. microbial degradation of shrimp-shell waste. j basic microbiol 32:107-11. saikia r, singh bp, kumar r, arora dk. 2005. detection of pathogenesisrelated proteins-chitinase and -1,3-glucanase in induced chickpea. curr sci 89:659-66. sakai k, narihara m, kasama y, wakayama m, moriguchi m. 1994. purification and characterization of thermostable beta-nacetylhexosaminidase of ch-4 isolated from chitin-containing compost. appl environ microbiol 60:2911-5. schwarz h, moussian b. 2007. electron microscopic and genetic dissection of arthropod cuticle differentiation. in: mendez-villas, diaz j, editors. modern research and educational topics in microscopy tuebingen: formatex. p 316-25. bemisia tabaci bacillus bacillus b bacillus stearothermophilus . volume 4, 2010 microbiol indones 107 shanmugaiah v, mathivanan n, balasubramanian n, manoharan pt. 2008. optimization of cultural conditions for production of chitinase by mml2270 isolated from rice rhizosphere soil. af j biotechnol 15:2562-8. spindler kd. 1997. chitinase and chitosanase assays. in: muzarelli raa, mg peter, editors. chitin handbook. grottamare: alda tecnografica. p 229-35. ueda m, arai m. 1992. purification and some properties of chitinase from sp. no. 10s-24. biosci biotech biochem 56:460-4. vega lm de la, barboza-corona je, aguilar-uscanga mg, ramirez-lepe m. 2006. purification and characterization of an exochitinase from subsp. and its action against phytopathogenic fungi. can j microbiol 52:651-7. wagiman fx, hussein my, muhamad r, sajap as, ismail a. 2001. age structure of aphidophagous fabricius and glover. hayati 8:1-4. waldeck j, daum g, bisping b, meinhardt f. 2006. isolation and molecular characterization of chitinase deficient strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. appl environ microbiol 72:7879-85. wang sl, chang wt. 1997. purification and characterization of two bifunctional chitinases/lysozymes extracellularly produce by k-187 in a shrimp and crab shellpowder medium. appl environ microbiol 63:380-6. wang sl, lin ty, yen yh, liao hf, chen yj. 2006. bioconversion of shellfish chitin wastes for the production of w-118 chitinase. carbohydr res 341:2507-15. bacillus laterosporous aeromonas bacillus thuringiensis aizawai menochilus sexmaculatus aphis gossypii bacillus licheniformis pseudomonas aeruginosa bacillus subtilis 3.mi691-charis amarantini available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.1.3issn 1978-3477, eissn 2087-8587 vol 7, no 1, march 2013, p 17-23 *corresponding author; phone: +62-274-563929, fax: +62274-513235, e-mail: charis@ukdw.ac.id molecular techniques have become increasingly popular and potentially useful tool for the classification and identification of bacterial strains in most bacterial genera. one of which is 16s rdna gene sequence analysis. the 16s rdna gene is highly conserved within a species and among species of the same genus, and therefore it can be used as a reference for the speciation of bacteria (woo et al. 2000). this method plays an important role in the identification of bacterial pathogens, which is useful not only for diagnosis, but also for phylogenetic classification. most salmonella strains are identified serologically as having o (somatic) and h (flagellar) antigens. there are, however, often serological cross-reaction between strains that phylogenetic classification is important to distinguish them from one another (woo et al. 2001). currently there are three known species in the salmonella genus , salmonella enterica, salmonella bongori, and salmonella subterranea (shelobolina et al. 2004). salmonella enterica comprises of six subspecies. they are s. enterica subsp. enterica (subsp. i), s. enterica subsp. salamae (subsp. ii), s. enterica subsp. arizonae (iiia subsp.), s. enterica subsp. diarizonae (subsp. iiia), s. enterica subsp. houtenae (subsp. iv), and s. enterica subsp. indica (subsp. vi). salmonella bongori was originally classified as s. enterica subspecies v. the majority of s. enterica subsp. enterica (subsp. i) cause most infections in humans and warm-blooded animals (truper 2005; tajbakhsh et al. 2011). salmonella typhi, the agent of typhoid fever, is included in subspecies i: s. enterica subsp. enterica serotype typhi (s. enterica serovar typhi; salmonella a total of thirteen isolates representative of salmonella typhi from different geographical locations in sumba island, east nusa tenggara, indonesia were identified by 16s rdna gene sequences. bacterial dna tm extraction was prepared by using a purelink genomic dna kit. the bacterial dna and control were amplified using the specific primers for s. typhi. these 16s rdna gene sequence data were aligned with the corresponding available s. typhi sequence and the reference organisms from the family enterobacteriaceae from ncbi database by using the clustal x software. phylogenetic trees were generated using the phylip software package and the matrix of nucleotide similarity and nucleotide difference were generated by using the phydit software. the results from the 16s rdna analysis showed that the degree of similarity within these strains ranged from 99.13-100%. the percentage of sequence similarity between s. typhi strains was very high (>99 %). molecular phylogenetic analysis showed that all of the isolates formed a new center of diversity with s. typhi t atcc 19430 as a reference strain. based on these results, all of the tested strains belonged to species of s. typhi t suggested by their relatedness with the type strain of s. typhi atcc 19430 . key words: 16s rdna, phylogenetic analysis, salmonella typhi, typing sebanyak 13 isolat salmonella typhi yang mewakili berbagai wilayah geografis di pulau sumba, nusa tenggara timur, indonesia diidentifikasi berdasarkan sekuen gen 16s rdna. dna bakteri diekstraksi sesuai tm dengan petunjuk dari purelink genomic dna kit. dna bakteri dan kontrol diamplifikasi menggunakan primer spesifik untuk s. typhi. urutan basa sekuen gen 16s rdna dianalisis bersama dengan sekuen 16s rdna s. typhi dan anggota famili enterobacteriaceae yang diperoleh dari database ncbi menggunakan program clustalx. pohon filogeni dikonstruksi dengan menggunakan program phylip sedangkan matriks similaritas dan perbedaan nukleotida dianalisis dengan menggunakan program phydit. hasil analisis sekuen 16s rdna menunjukkan bahwa kemiripan di antara isolat s. typhi berkisar antara 99,13-100%. persentase kemiripan sekuen di antara isolat s. typhi sangat tinggi (>99%). analisis filogeni menunjukkan bahwa semua isolat t membentuk pusat keanekaragaman baru dengan isolat standard s. typhi atcc 19430 . berdasarkan hasil tersebut, dapat disimpulkan bahwa semua isolat yang diuji tergolong dalam spesies s. typhi yang ditunjukkan t dari kedekatan hubungan kekerabatan dengan isolat standard s. typhi atcc 19430 . kata kunci: 16s rdna, analisis filogenetik, salmonella typhi, typing 16s rdna typing of salmonella typhi strains from different geographical locations in sumba island east, nusa tenggara, indonesia charis amarantini* and tri yahya budiarso biology department, universitas kristen duta wacana, jalan dr wahidin sudirohusodo 5-19, yogyakarta 55224, indonesia typhi). typhoid fever is most prevalent in tropical areas, including indonesia (moehario 2009). in sumba island, east nusa tenggara especially in soutwest sumba materials and methods extraction of bacterial dna, pcr amplification, and dna sequencing. bacterial dna was extracted in accordance with the protocol's instructions tm using a purelink genomic dna mini kit (invitrogen k1820-00). the bacterial dna and control were st amplified with 25 pmol primers (1 base custom oligos tm tm fbco) and pcr supermix (illustra pure taq tm ready-to-go pcr beads. pcr amplification for the 16s rdna sequences showed bands of 428 bp, 484 bp, and 483 bp. these fragments were amplified using district over 197 cases or 725 infection/100.000 inhabitants were recorded in the database of the karitas hospital in 2006 (amarantini et al. 2009). this number was higher than the average infection cases in rural areas in indonesia, and it was nearly the same as the average cases in urban areas (810/100.000) according to world health organization (who) in 2003. these numbers suggested that the area was a good niche for fast growing microorganisms. analysis of various s. typhi strains using numerical systematic method showed that there were a big diversities in the use of the carbon sources by the s. typhi isolates in these region (amarantini et al. 2009). in addition to obtaining accurate data and strong discriminative ability to distinguish the strains, this study was aimed to identify and unravel the diversities of s. typhi isolates from typhoid fever patients using molecular phylogenetic approach based on 16s rdna gene sequences. bacterial strains. thirteen isolates used in this research were isolated from the blood cultures of typhoid patients in sumba island. they came from different geographical regions in karitas hospital in weetabula, a private clinic in elopada subdistrict in southwest sumba district, and lendemoripa hospital in waikabubak in west sumba district. specimen collection methods were described in the journal article published previously (amarantini et al. 2009). these isolates were identified using microbiological standard methods. all cultures were screened in chromocult coliform agar (cca). typical colonies appear white and transparents due to the lack of ßgalactosidase and ß-glucoronidase enzymes. these colonies were confirmed using triple sugar iron agar (tsia) (who 2003). all cultures were grown at 30 °c for 24 h on brain heart infusion (bhi) agar before used. 18 amarantini and budiarso microbiol indones primer r1 fr, r3 fr, and r5 fr respectively. the pcr product was gel purified with a qiaquick pcr purification kit (qiagen, hilden, germany). the purified pcr product was sequenced with abi prism 3100-avant genetic analyzer in accordance with the manufacture's instructions (applied biosystems, usa) using pcr primers. analysis and alignment of 16s rdna nucleotide sequences. the 16s rdna nucleotide sequences were analyzed, edited and assembled with finch tv 1.4.0 and dna baser sequence analysis software. complete assembled sequences were aligned with the corresponding s. typhi sequences retrieved from the ncbi database with custal x software (thompson et al. 1997). results the primers used to amplify these fragments are shown in table 1 (massi et al. 2005). the pcr mixtures were amplified for 40 cycles at 94 °c for 1 minute, 55 °c for 1 min, and 72 °c for 2 min, with a final extension at 72 °c for 10 min in automated applied biosystems geneamp pcr system 2400. an aliquot of 5 µl of each amplified product was electrophoresed in 3.0% (w/v) agarose gel, tm with a 100 bp dna ladder (gene ruler ). the pcr product was gel purified with a qiaquick pcr purification kit (qiagen, hilden, germany). the purified pcr product was sequenced with abi prism 3100-avant genetic analyzer according to the manufacturer's instructions (applied biosystems, usa) using the same primers as used in pcr. construction of phylogenetic tree. based on 16s rdna nucleotide sequences, a phylogenetic tree was constructed with phylip software package (felsenstein 1993) with neighbor-joining algorithm (saito and nei 1987). the evolutionary distance matrix of the neighbor-joining method was generated according to the description from jukes and cantor (1969). the phylogenetic distances were obtained by adding only the values of the horizontal components. eventually, the matrix of the nucleotide similarity and difference was generated with phydit software (chun 1999). thirteen isolates used in this research came from different locations; seven isolates from east wewewa, three isolates from kodi, two isolates from north wewewa, and one isolate from waikabubak. these isolates were selected to represent their geographical origins. they're mapped according to the infected patient's place of residence based on global positioning system (fig 1). fig 1 distribution of typhoid fever patients and 13 strains representation from different geograophic location in sumba island, east nusa tenggara. tnumber z47544 (s. typhi atcc 19430 ), af029227 (s. bongori br 1859), x80724.1 (escherichia coli atcc 25922), m59291 (citrobacter freundii atcc29935), m59160 (serratia marcescens), m59149 (erwinia carotovora atcc 15713), x75275 (yersinia ruckeri atcc 29473), x75279 (yersinia intermedia er-3854), m59155 (hafnia alvei atcc 13337), x82248 (photobacter luminescens dsm 3368), x82251 (xenorhabdus nematophilus dsm 3370), x07652 (proteus vulgaris ifam 1731), and m59159 (plesiomonas shigelloides) the phylogenetic analysis showed 13 representative strains of s. typhi originated from different locations in sumba island, east nusa tenggara, which were then divided into three clades. the first clade consisted of seven strains. one isolate (hb01) originated from wailabubu, north kodi subdistrict and six isolates were obtained from east wewewa subdistrict, which were hb05 (isolated from wanowitu), hb09 (isolated from hb07 (isolated from weerambo, east wewewa), hb06 (isolated from ombawawi north wewewa), hb03 (isolated from watubero wailabubur north kodi), and hb04 (isolated from pakamutu, the third clade consisted of two strains. they were hb08 (isolated from palekki/mahaloko in north t wewewa subdistrict) and s. typhi atcc 19430 , which was the reference strain. the 16s rdna nucleotide similarity values (%) and the number of nucleotide differences among 13 s. typhi isolates from infected patients in different geographical locations in sumba island and the t reference strain s. typhi atcc 19430 are shown in table 1 and 2. the sequences of the 13 strains and s. t typhi atcc 19430 showed >99% similarity. it was also evident that strain hb10 was identical with the strain hb09, and the strain hb11 was identical with the strains hb09 and hb10 (table 2). the two other strains (hb03 and hb04) were also identicals (table 3). bondokodi kodi). katikutana kodi kodi bangedo north kodi waikabubak lamboya laura loli mamboro tana righu umbu ratu nggay west umbu ratu nggay wanokaka west wawewa south wawewa east wawewa 119'00' 119'10' 119'20' 119'30' 119'40' 119'50' 9 '2 0 ' 9 '3 0 ' 9 '4 0 ' 9 '5 0 ' 119'00' 119'10' 119'20' 119'30' 119'40' 119'50' 9 '2 0 ' 9 '3 0 ' 9 '4 0 ' 9 '5 0 ' strain representation patient residence phylogenetic analysis results of 13 s. typhi isolates based on 16s rdna gene sequences are shown in fig 2. the 16s rdna gene sequences of these isolates were initially compared to those of the enterobacteriaceae family reported in the gene bank with the accesion omba rade), hb 10 (isolated from weedindi), hb11 (isolated from durru lodo), hb12 (isolated from kongge), and hb13 (isolated from elopada). the second clade consisted of five strains, which were hb02 (isolated from kampung sawah waikabubak), volume 7, 2013 microbiol indones 19 table 2 16s rdna similarity values (%) and the number of nucleotide differences between seven strains and the reference t strains of s.typhi atcc 19430 within the first clade 20 amarantini and budiarso microbiol indones primer sequence nucleotide position r1f 5' agtttgatcctggctcag 3'* 3-20 (ac: z47544) r1r 5' agtactttacaacccgaagg 3'* 411-430 (ac: z47544) r3f 5' aagtactttcagcgggga 3'* 424-441 (ac: z47544) r3r 5' ttgagttttaaccttgcgg 3'* 898-916 (ac: z47544) r5f 5' aactcaaatgaattgacgg 3'* 901-919 (ac: z47544) r5r 5' aggcccgggaacgtattcac 3'* 1364-1383 (ac: z47544) table 1 the primers used for pcr amplification of 16s rdna gene sequence of salmonella typhi hb09 hb10 hb11 hb12 hb01 hb13 hb05 s. typhi atcc t 19430 s. bongori br1859 hb09 --0/1383 0/1381 7/1382 4/1383 1/1382 1/1381 5/1381 33/1377 hb10 100.00 -- 0/1381 7/1381 3/1382 1/1382 1/1381 5/1381 33/1377 hb11 100.00 100.00 -- 7/1381 3/1381 1/1381 1/1381 5/1381 33/1377 hb12 99.49 99.49 99.49 -- 11/1382 8/1381 8/1381 12/1381 40/1377 hb01 99.71 99.78 99.78 99.20 --4/1382 4/1381 8/1381 36/1377 hb13 99.93 99.93 99.93 99.42 99.71 2/1381 6/1381 34/1377 hb05 99.93 99.93 99.93 99.42 99.71 99.86 --4/1381 32/1377 99.64 99.64 99.64 99.13 99.42 99.57 99.71 -- 36/1497 s. bongori br1859 97.60 97.60 97.60 97.10 97.39 97.53 97.68 97.60 -- s. typhi atcc t 19430 hb03 hb04 hb06 hb02 hb07 hb08 s. typhi atcc t 19430 s. bongori br1859 hb03 -- 0/1384 5/1381 5/1381 6/1381 4/1381 7/1381 35/1377 hb04 100.00 -- 5/1381 5/1381 6/1381 4/1381 7/1381 35/1377 hb06 99.64 99.64 -- 4/1381 5/1381 3/1381 6/1381 34/1377 hb02 99.64 99.64 99.71 -- 1/1382 3/1381 6/1381 34/1377 hb07 99.57 99.57 99.64 99.93 -- 4/1381 7/1381 35/1377 hb08 99.71 99.71 99.78 99.78 99.71 -- 3/1381 33/1377 99.49 99.49 99.57 99.57 99.49 99.78 -- 36/1497 97.46 97.46 97.53 97.53 97.46 97.60 97.60 -- s. typhi atcc t 19430 s. bongori br1859 table 3 16s rrna similarity values (%) and the number of nucleotide differences between six strains and the reference strains t of s.typhi atcc 19430 within the two and the third clades ac: genbank accession no. *reference: massi et al. 2005 0.01 t reference strain s. typhi atcc 19430 . it was also shown that strains within the species have diverse 16s rdna gene sequences. in fact, all of the strains fall into three clades. these clades demostrated that genetic diversity of the tested strains could be unraveled using phylogenetic tree based on 16s rdna sequences. it is shown in phylogenetic tree that each clade is made up of strains from different geographical areas in the sumba island especially in southwest sumba district. the six tested strains within the first clade volume 7, 2013 microbiol indones 21 discussion entatives of the genus salmonella clearly showed that all of the tested strains form a new center t of diversity with s. typhi atcc 19430 (fig 2). this result directly proved that all of the isolates belong to s. typhi species because of their relationships with the phylogenetic analysis based on comparison of 16s rdna nucleotide sequences of the 13 strains with the corresponding nucleotide sequence of available repres fig 2 neighbour-joining phylogeny tree constructed on the basis of 16s rdna gene sequences showing relationship amongst the thirteen representatives of s. typhi isolates from the different locations in sumba island, east nusa tenggara. the arrow indicates estimated root position of the three as determined using plesiomonas shigelloides (m59159) as an outgroup. bar, 1 substitution per 100 nucleotides. indicated that these strains are indigenous from the southwest sumba district. acknowledgments this research was supported by the directorate general of higher education, department of national education (hibah bersaing) 2012, contract no: 560.7/k5/kl/2012 date 10-02-2012. a special gratitude is given to karitas hospital in weetabula in southwest sumba district and lende moripa hospital in waikabubak in west sumba district east nusa tenggara for their assistance in collecting the samples. i should also thank sr. sili bouka adm-the director of karitas hospital, dr. loeta lapoe moekoe-the director of lende moripa hospital and all doctors of karitas hospital for their assistance during the research. references amarantini c, sembiring l, kushadiwijaya h, asmara w. 2009. seleksi bakteri salmonella typhi dari kultur darah penderita demam tifoid [selection of samonella typhi bacteria from the blood culture of typhoid patients]. in: wijaya a, darmawan d, tuti r, atmanto t, nurohman s, editors. revitalisasi mipa dan pendidikan mipa dalam rangka penguatan kapasitas kelembagaan dan profesionalisme menuju world class university. proceeding of national seminar on research, education, and applied of mathematics and natural sciences; 2009 may 16. yogyakarta(id): p b13-b20. amarantini c, sembiring l, kushadiwijaya h, asmara w. 2009. isolasi, karakterisasi dan pengelompokan strain salmonella typhi asal kabupaten sumba barat daya nusa tenggara timur berdasarkan sifat-sifat fenotip [isolation, characterization and grouping of salmonella typhi strains in the southwest sumba regency east nusa tenggara based on phenotypic characteristics. berkala penelitian hayati 14(2):191-196. baumler aj, tsolis rm, ficht ta, adams lg. 1998. evolution of host adaptation in salmonella enterica. infect immun. 66(10):4579-4587. christensen h, nordentoft s, olsen je. 1998. phylogenetic relationship of salmonella based on rrna sequences. int j syst bacteriol. 48(2):605-610. doi:10.1099/0020771348-2-605. chun j. 1999. phylogenetic editor (phydit). windows version. drancourt m, bollet c, carlioz a, martelin r, gayral jp, raoult d. 2000. 16s ribosomal dna sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. j clin microbiol. 38(10): 3623-3630. felsenstein j. 1993. phylogeny inference package version 3,5c. departement of genetics. seattle (usa): university of washington. were derived from east wewewa subdistrict, whereas the other isolates were derived from north kodi subdistrict. the second clade consisted of two strains originally from kodi, one strain derived from waikabubak, and the two strains derived from east wewewa and north wewewa. based on the coordinate position of the infected patient's residences (fig 1), we learned how the typhoid fever spreads among the inhabitants. most of the patients lived in east wewewa subdistrict. it appears that the strains were distributed in finger pattern to nearby subdistrict such as north wewewa, south wewewa, west wewewa, and then goes further out to the kodi bangedo, kodi, and north kodi. therefore, these results indirectly showed that there were inter geographical distribution of the strains due to migration of people in this area. identification to the species level requires that the tested strains 16s rdna sequence has 99% similarity with the sequence of the reference strain in genbank (drancourt et al. 2000). it is shown in this study that the 16s rdna sequence of these 13 isolates had >99% similarity with the sequence of the closest strain in genbank. thus these isolates were identified as s. typhi. in terms of nucleotide similarity and nucleotide differences (table 1), it was shown that the strain hb10 was identical with hb09, and the strain hb11 was identical with hb09 and hb10. however, this result did not entirely coherent with its phylogenetic relatedness based on phylogenetic tree. it is apparent in fig 2 that the closest strain was found to be the strain hb12. two tested strains, hb03, and hb 04 (table 2), were fully congruent in their similarity values, nucleotide differences and phylogenetic analysis. it is especially interesting that the strain hb 08 from palekki/mahaloko north wewewa t was found to be closely related to s.typhi atcc 19430 . based on analysis of housekeeping and rrna genes, it has been understood that the genus salmonella contains two lineages that had diverged considerably from each other during evolution. these lineages represent two distinct species, s. enterica and s. bongori (baumler et al. 1998). christensen et al. (1998) also demostrated that the analysis of 16s rdna sequences separated s. bongori from s. enterica, and these two species from the complex of e. coli and shigella species. this corresponds to the topology of this phylogeny tree (fig 2). in summary, all the isolates were identified as s. typhi species because of their association with the type t strain of s. typhi atcc 19430 . the fact that the 13 isolates belonging to s. typhi species formed a new center of diversity within the 16s rdna gene tree microbiol indones22 amarantini and budiarso thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids res. 25(24):4876-4882. doi:10.1093/nar/25.24.4876. truper hg. 2005. the type species of the genus salmonella lignieres 1900 is salmonella enterica (ex kauffmann and edwards 1952) le minor and popoff 1987, with the t type strain lt2 , and conservation of the epithet enterica in salmonella enterica over all earlier epithets that may be applied to this species. opinion 80. judicial commission of the international committee on systematics of prokaryotes. ijsem. 55(1):519-520. doi:10.1099/ijs.0.63579-0. who 2003. background document: the diagnosis, treatment and prevention of typhoid fever. communicable disease surveillance and response vaccines and biologicals. p 1-30; 103-120. woo pcy, leung pkl, leung kw, yuen ky. 2000. identification by 16s ribosomal rna gene sequencing of an enterobacteriaceae species from a bone marrow transplant recipient. j clin pathol. 53(4):211-215. doi:10.1136/mp.53.4.211. woo pcy, fung amy, wong ssy, tsoi hw, yuen ky. 2001. isolation and characterization of a salmonella enterica serotype typhi variant and its clinical and public health implications. j clin microbiol. 39(3):1190-1194. doi:10.1128/jcm.39.3.1190-1194.2001. jukes th, cantor cr. 1969. evolution of protein molecules. in: munro hn, editors. mammalian protein metabolism. new york: academic press. 13:2l-l32. shelobolina es, sullivan sa, o'nell kr, nevin kp, lovley dr. 2004. isolation, characterization, and u(vi)-reducing potential of a facultatively anaerobic, acid-resistant bacterium from low-ph, nitrateand u(vi)-contaminated subsurface sediment and description of salmonella subterranea sp. nov. appl environ microbiol. 70(5):2959-2965. doi:10.1128/aem.70.5.29592965.2004. massi mn, shirakawa t, gotoh a, hatta m, kawabata m. 2005. identification and sequencing of salmonella enterica serotype typhi isolates obtained from patients with perforation and non-perforation typhoid fever. southeast asian j trop med publ health. 36(1):118122. moehario lm. 2009. the molecular epidemiology of salmonella typhi across indonesia reveals bacterial migration. j infect dev ctries. 3(8):579-584. doi:10.3855/jidc.548. saitou n, nei m. 1987. the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol biol evol. 4(4):406-425. tajbakhsh m, nayer bn, motavaze k, kharaziha p, chiani m, zali mr, klena jd. 2011. phylogenetic relationship of salmonella enterica strains in tehran iran using 16s rrna and gyrb gene sequences. j infect dev ctries. 5(6): 465-472. doi:10.3855/jidc.1504. volume 7, 2013 microbiol indones 23 02 stephanie.cdr vol.11, no.1, march 2017, p 11-17 doi: 10.5454/mi.11.1.2 effect of tempeh supplementation on the profiles of human intestinal immune system and gut microbiota 1 1 1 1,2* stephanie , nine kirana ratih , susan soka , and antonius suwanto 1 faculty of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia; 2 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, dramaga campus, bogor 16680, indonesia tempeh is a traditional fermented soybean product from indonesia. although tempeh is consumed as daily menu in indonesia, its nutrigenomic study employing human has not been reported yet. on the other hand, our study in mice showed that tempeh could enhance immune system, especially by increasing secretory immunoglobulin a production in ileum and colon. tempeh was also found to be potential in modulating composition of gut microbiota. therefore, the objective of this study was to analyze the impact of tempeh supplementation on the profiles of human intestinal immune system and gut microbiota analysis. a total of 16 participants, comprising of each 8 healthy females and males, aged between 20 and 23 were recruited to this study. the volunteers consumed 200 ml milk from day 1-8 followed by consumption of 100 grams steamed tempeh each day from day 9-24. fecal samples taken on day 9 and 25, were analyzed with half sandwich elisa for iga enumeration. in addition, fecal samples collected on day 0, 9, and 25, were analyzed for total akkermansia muciniphila employing quantitative real time pcr. the result of this study suggested that tempeh supplementation might act as paraprobiotic and slimming agents since tempeh enhanced production of iga and increased population of a. muciniphila in human intestinal tracts. key words: akkermansia muciniphila, elisa, iga, rt-pcr, tempeh tempe merupakan produk fermentasi kedelai asal indonesia. meskipun tempe dikonsumsi sebagai menu sehari-hari di indonesia, studi nutrigenomik tempe dengan subjek penelitian manusia belum pernah dilaporkan sebelumnya. studi nutrigenomik tempe dengan tikus sebagai hewan model menunjukkan bahwa tempe dapat meningkatkan sistem imun, yaitu dengan meningkatkan produksi immunoglobulin a sekretori di usus halus dan usus besar. tempe juga memiliki potensi dalam memodulasi komposisi mikrobiota usus. tujuan dari penelitian ini yaitu menganalisa efek suplementasi tempe terhadap sistem imun usus manusia dan menganalisa perubahan komposisi mikrobiota usus. sebanyak 16 responden, yang terdiri dari 8 wanita dan 8 pria sehat, berumur 20-23 tahun berpartisipasi dalam penelitian ini. responden mengkonsumsi 200 ml susu dari hari ke 1-8, dan 100 gram tempe dari hari ke 9-24 setiap harinya. sampel feses diambil pada hari ke 9 dan ke 25 untuk perhitungan iga dengan elisa, sedangkan sempel feses yang diambil pada hari ke 0, 9, dan 25 dianalisis untuk perhitungan akkermansia muciniphila dengan quantitative real time pcr. hasil penelitian ini menunjukkan bahwa suplementasi tempe dapat berperan sebagai agen paraprobiotik dan penurun berat badan karena tempe mampu meningkatkan produksi iga dan meningkatkan jumlah a. muciniphila dalam saluran pencernaan manusia. kata kunci: akkermansia muciniphila, elisa, iga, rt-pcr, tempe microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mioline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-5703306 ext. 449, fax:+62-21-5719060; email: asuwanto@indo.net.id composition. previous study had shown that tempeh has beneficial effects for immune system and cardiovascular health (babu et al. 2009). tempeh nutrigenomics studies were mostly conducted in mice and rats as animal models and have not been reported yet to involve human as research object (utama et al. 2013). previous study showed positive impact of tempeh consumption, that tempeh supplementation increased ileum immunoglobulin a (iga) gene and protein expressions in sprague dawley rats (soka et al. 2015 and soka et al. 2015). furthermore, nurrahman et al. (2011) reported that tempeh consumption in salmonella typhimuriuminduced rats had enhancement of iga concentration tempeh is a traditional fermented soybean product from indonesia. badan standarisasi indonesia (bsn) also known as standardization body of indonesia (2012) reported that 81 000 tempeh producers produce 2.4 tons tempeh every year in indonesia. tempeh is formed as the result of microorganisms' work, such as rhizopus spp. and lactic acid bacteria. as the result of fermentation product, tempeh became easily digested. since tempeh also has higher amino acids compared to raw soybean, tempeh could play an important role in intestinal cells proliferation and affect gut microbiota and accelerated the rats' resilience. immune system exploration is commonly detected by analyzing gut microbiota in the host's digestive 11 system. human gut microbiota contains at least 10 bacterial cells per ml intestinal fluid. this microorganism's consortium could act as the source of energy and nutrition for intestinal metabolism. intestinal stability is also guaranteed by the presence of probiotics, which is enhanced by the presence of prebiotics (flint et al. 2010). in this study, tempeh's ability as prebiotic agent will be tested and one of probiotics bacteria, such as akkermansia muciniphila would be enumerated. therefore, the objective of this study was to analyze the impact of tempeh supplementation on human's immune profiles, specifically by enumerating iga level and a. muciniphila in fecal samples. materials and methods human study. this study was reviewed and approved by the ethics committee of research and community service center in atma jaya catholic university of indonesia for employing human as research subject. research was done by following all institutional guidelines. a total of 8 healthy females and 8 healthy males aged 20-23 years volunteered for this study. woman volunteers were neither pregnant nor breast-feeding. the volunteers were not smoking, did not consume any antibiotics at least one month before the intervention, and did not suffer lactose intolerance. the volunteers were required to be discipline in consuming given tempeh regularly and willing not to consume any kinds of prebiotics and other tempeh during the intervention. the intervention was designed for 24 days. at day 0-8, the volunteers consumed 200 ml ultra-heat temperature (uht) milk per day. at day 9-24, the volunteers consumed 100 grams of 10-minutessteamed tempeh emp. fecal specimen collection was done in day 0, 9, and 25. fecal specimen for iga analysis was stored in -80 °c and fecal specimens for gut microbiota analysis were stored in -20 °c for further analysis. fecal immunoglobulin a extraction. the method was adapted from peters et al. (2004). one gram of fecal specimen was diluted in 10 ml extraction buffer containing 0.01 m phosphate buffer saline (pbs) ph 7.4 containing 0.5% w/v tween-20, and 0.05% w/v sodium azide. the fecal specimen was homogenized by mechanical homogenization with a vortex mixer. the fecal suspension was centrifuged at 1500 × g for 20 min at 5 °c. two milliliters of supernatant was transferred to sterile tubes containing 20 µl protease inhibitor cocktail (sigma-aldrich, st louis, missouri, usa), which were prepared by following the manufacturer's instruction. the mixture was homogenized using vortex mixer and centrifuged at 10000 × g for 10 min. the supernatant was transferred to sterile tubes and stored in -20 °c. immunoglobulin a analysis. ninety-six well ® polystyrene plates (nunc maxisorp , wiesbaden, germany) were coated with human colostrum iga (sigma-aldirch, st. louis, mo, usa) and fecal iga samples, diluted in pbs (1:10) and incubated for 18 h at 4 °c. the next day, the plates were washed with pbs tween for 4 times. the remaining protein sites were blocked with blotto, consisting of 5% skim milk in pbs tween for 1 h at 37 °c. the plates were washed then added with rabbit anti-human iga labeled with horseradish peroxidase (sigma-aldirch, st. louis, mo, usa) diluted in blotto (1:10000) and incubated for 1 h at 37 °c. the plates were washed and added with 3,3',5,5'-tetramethylbenzidine as a substrate diluted in phosphate citrate buffer. the colorimetric reaction was stopped by adding 2 n sulfuric acid. the absorbance was measured with elisa reader (bio-rad, san fransisco, ca, usa) at 450 nm. data were standardized into the iga standard curve, and iga -1 concentration was expressed in ng ml fecal samples. total fecal bacterial dna extraction. bacterial dna quantification method was adapted from soka et al. ® (2014). the extraction was done using qiaamp dna stool mini kit (qiagen, hilden, germany) by following the manufacture's instruction. the modification was done by adding glass bead in sample homogenization step. standard curve construction and bacterial ® specific enumeration. pgem t plasmid containing 16s rdna of a. muciniphila was obtained from pt nutrifood indonesia (jakarta, indonesia) which was adapted from from prawira (2014). the plasmids were transformed by employing escherichia coli top 10 using cacl and mgcl method. plasmid extraction was 2 2 ® done using wizard plus sv minipreps dna purification system (promega, san luis obispo, ca, usa) by following the manufacture's instruction. plasmid concentration serially diluted for dna template in standard curve construction with a 4 10 minimum five standard concentration between 10 -10 dna copies per reaction. pcr primers and amplification for a. muciniphila was conducted and reported previously (derrien 2007). 12 stephanie et al. microbiol indones a. muciniphila from bacterial dna and diluted plasmid were quantified with iq5 multicolor real time pcr detection system (bio-rad, san fransisco, ca, ® usa). a reaction was consisted of 10 µl kapa sybr tm fast master mix 2x bio-rad icycler , 1 µl 10 rmol -1 µl of each specific primers, 1 µl dna template (100 -1 ng ml ), and 7 µl nuclease free water, with total volume 20 µl. condition applied for this analysis consisted of 1 cycle of 94 °c for 5 min and 40 cycles of 94 °c for 20 s, 50 °c for 20 s, and 72 °c for 50 s. every diluted plasmid samples fecal dna samples were duplicated with the same pcr reaction. statistical analysis. significant difference between before and after intervention were analyzed using paired parametric t-test (p<0.05). data was analyzed using graphpad prism version 6.0 (graphpad software inc, la jolla, ca, usa). presented data is the mean of personal data from each volunteer ± standard error mean (sem). results from a total of 8 female and 8 male participants, 3 male and 3 female participants were excluded due to their low physical fitness and less fulfillment of tempeh consumption during the intervention. fecal samples were stored for 6 weeks in -80 °c before iga extraction process. the data shows that both male and female shows enhancement of iga production (fig 1) and the enhancement of a. muciniphila population in fecal samples (fig 2). higher secretion of male iga was seen bigger in day 25 -1 -1 compared to day 9, i.e. 2573 ng ml vs 2098 ng ml , -1 respectively, while in female i.e. 2421 ng ml vs 2376 -1 ng ml , respectively. when the data is combined between male and female, the same pattern also occurred that higher iga secretion was seen on day 25 -1 -1 compare to day 9, i.e. 2497 ng ml vs 2237 ng ml . the number of a. muciniphila also increased after day 9 and day 25 compared to the beginning of experiment, day 0. from sampling on day 0, 9, and 25, male iga enumeration was the highest after tempeh intervention on day 25, while on female the highest number of a. muciniphila was on day 9, before tempeh intervention. if male and female data were combined, the result showed the enhancement of a. muciniphila population kept increasing and was the highest after tempeh supplementation on day 25. fig 1 iga level in fecal samples in male participants only (a), female participants only (b), and the combination between male and female participants (c). day 9 represents the day after milk intervention and day 25 -1 represents the day after tempeh intervention. values are in ng ml fecal iga samples ±sem (n=5 for each male and female). lg a c o n c en tr a ti o n ( n g /m l ) d ay 9 d ay 2 5 0 1000 2000 3000 4000 5000 male participants onlya lg a c o n c en tr a ti o n ( n g /m l ) d ay 9 d ay 2 5 0 1000 2000 3000 4000 female participants onlyb lg a c o n c en tr a ti o n ( n g /m l ) d ay 9 d ay 2 5 0 1000 2000 3000 4000 male and female participantsc microbiol indones 13volume 11, 2017 discussion all participants were uniformed with uht milk consumption. in general, uht milk, which was taken from cow, and tempeh, which was made from soybean contain different protein profile. these facts brought the expectation of discrete results on before and after tempeh intervention. on the other hand, tempeh is rich of various microorganisms that form a distinctive flavor of tempeh. the variations may occur due to differences of tempeh raw material and procession steps from the producers. tempeh empang (emp), which is made in empang, bogor was used in this study as it was previously explored before (soka et al. 2014, soka et al. 2015). emp is produced by 6 main steps, which are soybean cooking, soaking, de-hulling, washing, mixing with inoculum, and incubation until tempeh was ready for harvest in 2-3 days. metagenomics study showed that emp tempeh had higher number of bacterial cells, compared to wjb tempeh, which was produced in warung jambu, bogor. emp provides more antigens compare to wjb tempeh. acetobacter indonesiensis, klebsiella pneumoniae, bacillus subtilis, and flavobacterium sp. were dominant bacteria found in emp tempeh, while klebsiella sp. and pseudomonas putida were the dominant bacteria in wjb tempeh (barus et al. 2008). a study by ayu et al. (2014) showed that klebsiella pneumoniae from indonesian tempeh were genetically different from pathogenic isolates. a study by soka et al. (2014) suggested that indonesian tempeh might modulate the composition of gut microbiota toward a healthier gut. in addition, soka et al. (2015) also reported that indonesian raw and cooked tempeh might stimulate iga secretion, and also both viable and non-viable fig 2 akkermansia muciniphila level in fecal samples in male participants only (a), female participants only (b), and the combination between male and female participants (c). day 0 represents the day before any intervention, day 9 represents the day after milk intervention, and day 25 represents the day after tempeh intervention. values are in log copy number of 16s rdna gene in fecal samples ±sem (n=5 for each male and female). one asterisk represents significant difference between two days of intervention. a ă▄ś t ăʼnċ╜ľ╜♫ă■ċℓ h ■▄ŧ l o g c o p y n u m b e r d ay 0 d ay 9 d ay 2 5 0 2 4 6 8 10 a cś▓ă▄ś t ăʼnċ╜ľ╜♫ă■ċℓ h ■▄ŧ l o g c o p y n u m b er d ay 0 d ay 9 d ay 2 5 0 2 4 6 8 10 * b [ o g c o p y n u m b er d ay 0 d ay 9 d ay 2 5 0 2 4 6 8 10 * c male and female participants male participants only female participants only 14 stephanie et al. microbiol indones male and female participants microorganisms might be stimulating iga gene expression in sprague dawley rats. secretory immunoglobulin a (siga) is an antibody which presence in mammals' digestive tract, including human. siga act as a first defense barrier for protecting the intestine from pathogenic bacteria and toxins. siga production against specific antigens is likely influenced by the presence of antigen-presenting cells, such as dendritic cells. the performance of this antibody is influenced by the activation of t cells and b cells (mantis et al. 2011). siga also has an important role in the activation of various non-inflammatory pathways and control intestinal microbiota balance (corthesy 2010). the production of siga is affected by the presence of antigens in the intestinal tract. siga is the form of two dimeric iga that is connected thru j chain. this dimeric structure prevents siga from proteolytic enzyme activity, so then siga works optimally to protect mucosal immune system (woof and russel 2011). in this study, monomeric iga level was measured. iga would not present in membrane-bound form and would not be attached to j-chain. therefore, iga should be easier to be quantified to see the reflection on intestinal immune profile. since emp tempeh was previously steamed for 10 minutes, the microorganisms in tempeh became inactive in our bodies. although the microorganisms were made to be inactive, intestinal immune cells could recognize the dead cells as antigens and lead to iga stimulation in the form of intestinal immune system defense. this concept is also known as paraprobiotics effect (taverniti and guglielmetti 2011). milk supplementation was done as comparison to tempeh supplementation. for the first 8 days, the volunteers consumed 200 ml milk, which could act as p r e b i o t i c a g e n t d u e t o t h e p r e s e n c e o f galactooligosaccharides (patel and goyal 2012). since the volunteers were not allowed to consume any probiotics during the intervention, it might lead to iga reduction. iga production is affected by the presence of probiotics bacteria (anandharaj et al. 2014). in our findings, milk supplementation could increase probiotic bacteria and led to iga production, which might explain that iga concentration on day 9, as the baseline was pretty high and close to iga concentration after tempeh intervention. tempeh intervention was done for the next 16 days and the concept of paraprobiotics was proven. our findings showed that tempeh intervention slightly increased iga production compared to milk intervention on day 9. the result also showed that tempeh could act as prebiotics and paraprobiotics agents for human body because tempeh contains reducing sugars and significant amount of dead cells. akkermansia muciniphila is a gram negative bacteria belonging to verrumicrobia phylum. a. muciniphila colonizes in cecum, mucin's biggest production site in digestive tract. this bacterium has the ability to degrade mucin, a glycoprotein which composed of serine and threonine peptides and linked by oand nglycosidic bond. mucin has four main oligosaccharides, which are n-acetyl glucosamine, nacetyl galactosamine, galactose, and fucose. mucin degradation employs several enzymes, such as glycosidase and sulphatase that disrupt oligosaccharide bonds. the presence of mucin provides advantage for a. muciniphila, which acted as alternative energy source for intestinal metabolism. a. muciniphila regulates immune system, cell proliferation, cell adhesion, and cell apoptosis and mucosal gene expression (derrien et al. 2004; derrien et al. 2011). previous study also reported that a. muciniphila dominates 3-5% gut microbial communities of healthy people. a. muciniphila also shows to have positive correlation with obesity and type 2 diabetes reductions s i n c e e n d o c a n a b i n o i d c o m p o n e n t c o n t r o l s inflammation, intestinal balance, and intestinal peptides secretion. moreover, the presence of a. muciniphila also increases 2-oleoglyserol level, which stimulates the secretion of glucagon-like-peptide from l cells (everard et al. 2012). both milk and tempeh were tested to act as prebiotic agents since milk contains galactooligosaccharides and tempeh contains various peptides and oligosaccharides. the result showed that tempeh supplementation increased the number of a. muciniphila significantly on combined male and female participants, compared to day 0, or before intervention. the increase of a. muciniphila population in this study suggested that consuming tempeh could be promising for weight loss and to reduce diabetes type-2 syndrome. to conclude, our findings suggested that tempeh supplementation might modulate human intestinal immune system by increasing iga production. in addition, tempeh consumption could increase population of a. muciniphila. volume 11, 2017 microbiol indones 15 acknowledgment this research was supported by dana dipa ipb (code: 2013.089.521219). references anandharaj m, sivansankari b, rani rp. 2014. effects of p r o b i o t i c s , p r o b i o t i c s , d a n s y n b i o t i c s o n hypercholesterolemia: a review. chin j biol. 2014:1-7. doi: 10.1155/2014/572754. ayu e, suwanto a, barus t. 2014. klebsiella pneumoniae from indonesian tempeh were genetically different from that of pathogenic isolates. microbiol indones. 8:9-15. doi: 10.5454/mi.8.1.2. babu pd, bhakyaraj r, vidhyalakshmi r. 2009. a low cost nutritious food “tempeh”a review. world j dairy food sci. 4:22-27. badan standardisasi nasional. 2012. tempe: persembahan indonesia untuk dunia (tempeh: indonesian tribute for the world) badan standardisasi nasional press, jakarta, indonesia. barus t, suwanto a, wahyudi at, wijaya h. 2008. role of b a c t e r i a i n t e m p e b i t t e r t a s t e f o r m a t i o n : microbiological and molecular biological analysis based on 16s rrna gene. microbiol indones. 2:17-21. doi: 10.5454/mi.2.1.4. clemente jc, ursell lk, parfrey lw, knight r. 2012. the impact of the gut microbiota on human health: an integrative view. cell. 148:1258-1270. doi: 10.1016/j.cell.2012.01.035. corthesy b. 2010. role of secretory immunoglobulin a and secretory component in the protection of mucosal surfaces. future microbiol. 5:817-829. doi: 10.2217/fmb.10.39. derrien m, vab baarleen p, hooived g, norin e, müller m, devos vm. 2011. modulation of mucosal immune response, tolerance, and proliferation in mice colonized by the mucin-degrader akkermansia muciniphila. front microbiol. 2:1-14. doi: 10.3389/fmicb. 2011.00166. derrien m, vaighan ee, plugge cm, de vos wm. 2004 akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. int j syst evol. microbiol. 54:1469-1476. doi: 0.1099/ijs.0.02873-0. derrien m. mucin utilization and host interactions of the novel intestinal microbe akkermansia muciniphila. 2007. [dissertation] wageningen (nl): wageningen university. everard a, belzer c, geurts l, ouwerkwerk jp, druart c, bindels lb, guiot y, derrien m, muccioli gg, delzenne nm, devos wm, cani pd. 2012. cross-talk between akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. pnas. 110:9066-9071. doi :10.1073/pnas.1219451110. ferreira cs. 2005. refractive index matching applied to fecal smear clearing. 2005. rev inst med trop s paulo. 47:347-350. doi: 10.1590/s0036-46652005000600007. flint hj, o'toole pw, walker aw. 2010. special issue: the human intestinal microbiota. microbiology. 156: 32033204. doi: 10.1099/mic.0.045443-0. kabeerdoss j, devi rs, mary rr, vidy dr, mechenro j, mahendri nv, pugazhendhi s, ramakrishna bs. 2011. effect of yoghurt containing bifidobacterium lactis ® b b 1 2 o n f a e c a l e x c r e t i o n o f s e c r e t o r y immunoglobulin a and human beta-defensin 2 in healthy adult volunteers. nutr j 10:138. doi: 10.1186/1475-2891-10-138. mantis nj, rol n, corthesy b. 2011. secretory iga's complex roles in immunity and mucosal homeostasis in the gut. mucosal immunol. 4:603-611. doi: 10.1038/mi.2011.41. nurrahman, astuti m, suparmo, marsetyawan, soesatyo hne. 2011. the effect of black soybean tempe and it's ethanol extract on lymphocyte proliferation and iga secretion in salmonella thyphimurium induced rat. afr j food sci. 5: 775-779. doi: 10.5897/ajfs11.128. patel s, goyal a. 2012. the current trends and future perspectives of prebiotics research: a review. 3 biotech 10 p [on line]. doi: 10.1007/s13205-012-0044-x. peters ir, calvert el, hall ej, day mj. 2004. measurement of immunoglobulin concentrations in feces of healthy dogs. clin diagn lab immunol. 11:841-848. doi: 10.1128/cdli.11.5.841-848.2004. prawira ma. preliminary study on human gut microbiota profiles in type 2 diabetes mellitus patients. 2014. [thesis] jakarta (id): atma jaya catholic university of indonesia. soka s, suwanto a, rusmana i, sajuthi d, iskandriati d, jessica k. 2015. analysis of intestinal mucosal immunoglobulin a in sprague dawley rats supplemented with tempeh. hayati j. biosci. 22:4852. doi: 10.4308/hjb.22.1.48. soka s, suwanto a, sajuthi d, rusmana i. 2014. impact of tempeh supplementation on gut microbiota composition in sprague-dawley rats. res j microbiol. 9:189-198. doi: 10.3923/jm.2014.189.198. soka s, suwanto a, sajuthi d, rusmana i. 2015. impact of tempeh supplementation on mucosal immunoglobulin a in sprague-dawley rats. food sci biotechnol 24:1481-1486. doi: 10.1007/s10068-015-0191-z. taverniti v, guglielmetti s. 2011. the immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics:proposal of paraprobiotic concepts). genes nutr 6:261-274. doi: 10.1007/ 16 stephanie et al. microbiol indones s12263-011-0218-x. utama z, okazaki y, tomotake h, kato n. 2013. tempe consumption modulates fecal secondary bile acids, mucins, immunoglobulin a, enzyme activities, fecal microflora, and organic acids in rats. plant foods hum nutr. 68:177-183. doi: 10.1007/s11130-013-0357-x. woof jm, russel mw. 2011. structure and function relationships in iga. mucosal immunol. 4:590-597. doi: 10.1038/mi.2011.39. volume 11, 2017 microbiol indones 17 page 1 page 2 page 3 page 4 page 5 page 6 page 7 2.mi-regina available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.4.2issn 1978-3477, eissn 2087-8575 vol 10, no 4, december 2016, p 125-130 *corresponding author; phone: +62-21-8765066, email: reginawanti@unpad.ac.id indonesia has 76,022,000 ha potential dry land from overall 148,000,000 ha of cultivation land. entisols is a soil order in which vegetable production has been carried out. in indonesia, entisols constitute up to 14,540,000 ha (balai penelitian tanah 2006). low amount of nutrient in entisols may lead to the limited crop production. inorganic fertilizer has been used for years to supply the nutrient to the crops to improve the yield. however, excess amount of inorganic fertilizer will reduce the soil fertility and it is hazardous to the environment (gruhn et al. 2000). in sustainable agricultural system, part of the inorganic fertilizer can be replaced by plant growth promoting rhizobcteria (pgpr). instead of using merely inorganic fertilizer, a combination of both fertilizer and pgpr should be used. one of the well known pgpr is aerobic non symbiotic nitrogen fixer azotobacter sp., which also produces phytohormones (tripathi et al.2015). azotobacter sp. release the phytohormones such as indole acetic acid (iaa) to induce cell development (kumar et al. 2014). nowadays, the ability of leafy vegetables contributes to the inflation rate in ambon city due to low productivity in rainy season. some vegetables are imported from other islands while importantvegetables such as local petsai (brassica chinensis l.) and chili (capsicum annum l.) are cultivated in low nitrogen soil, entisols. lack of nitrogen could be overcome by using inorganic fertilizeras well as biofertilzer. the soil can be inoculated with rhizobacteria, such as azotobacter, to increase the nitrogen uptake and improve the quality of vegetables. this research was conducted to isolate and select azotobacter from rhizosphere of vegetables and to examine the effect of azotobacter inoculation on chili-seedling growth and nitrogen uptake by using bioassay method. azotobacter sp. was isolated in nitrogen-free ashby’s media. the bioassay was held in the green house with randomized block design experiment, which examined the combination of isolates and population of azotobacter sp. on chili. two best isolates which was selected based on ph, nitrogen content and cell viability were s2a10 (from petsai’s rhizosphere) and c2a9 (from chili’s rhizosphere). bioassay showed that azotobacter inoculation followed by reduced npk fertilizer doses had no effect on transplant dry weight and nitrogen uptake. all azotobacter 8 -1 inoculation except 10 cfu ml s2a10 maintain soil nitrogen although azotobacter population in soil was slightly reduced. this showed that azotobacter sp. potentially reduce the use of inorganic biofertilizer. keywords: azotobacter, chili, entisols, nitrogen fixation komoditas sayuran di kota ambon adalah salah satu penyumbang inflasi. berbagai jenis sayuran masih diimpor dari luar pulau padahal kota ambon memiliki sawi (brassica chinensis l.) dan cabai (capsicum annuum l.) lokal yang telah dibudidayakan di tanah entisols dan kekurangan nitrogen. untuk memperkuat produksi benih kedua sayuran lokal tersebut, selain pupuk anorganik, inokulasi rizobakteri azotobacter dapat meningkatkan serapan unsur hara nitrogen dan meningkatkan kualitas tanaman. penelitian ini dilakukan untuk mengisolasi dan menseleksi isolat azotobacter dari rizosfer kedua komoditas dan menguji kapasitasnya untuk meningkatkan pertumbuhan dan serapan n bibit tanaman cabai (capsicum annuum l.). bakteri azotobacter sp. diisolasi dengan metode pengayaan dan dilanjutkan dengan metode gores pada media selektif ashby bebas n. uji hayati di rumah kaca dirancang dalam rancangan acak kelompok yang menguji kombinasi isolat dan kepadatan inokulan cair azotobacter sp. berdasarkan n tersedia, kepadatan sel dan ph kultur, maka dua isolat terbaik adalah s2a10 dari rizosfer sawi dan c2a9 dari rizosfer cabai. uji hayati memperlihatkan bahwa inokulasi azotobacter disertai penuruan dosis pupuk npk tidak mempengaruhi berat kering dan serapan nitrogen cabai. 8 -1 seluruh perlakukan inokulasi azotobacter kecuali 10 cfu ml s2a10 menjaga kadar nitrogen tanah meskipun populasi azotobacterdi tanah menurun. percobaan ini memberikan gambaran bahwa azotobacter sp. berpotensi menurunkan penggunaan pupuk anorganik. kata kunci: azotobacter, cabai, entisol, fiksasi nitrogen selection and bioassay of azotobacter sp. isolates to improve growth of chili (capsicum annum l.) on entisols in ambon 1 1 reginawanti hindersah *, priyanka , 2 2 wilhelmina rumahlewang , and a marthin kalay 1 soil science department, faculty of agricuture universitas padjadjaran, jalan raya km 21 jatinangor, sumedang, bandung 45363, indonesia; 2 plant diseases department, faculty of agriculture, universitas pattimura, jalan ir. m. putuhena, azotobacter to produce exopolysaccharide (eps) has been well documented. the eps is extracellular structure that protects nitrogenases in the nitrogen fixation process (sabra et al. 2000). azotobacter has been found in the rhizosphere of various plants.tarigan et al. (2013) has isolated the azotobacter from soybean (glycine max l.) rizosphere. widiastuti et al. (2010) has also isolated azotobacter from the rhizosphere of palm(elaeis guineensis), coffee (coffea arabica), rubber tree (hevea brasiliensis), cashew tree (anacardium occidentale), and corn (zea mays). in attempt to develop local azotobacter biofertilizer to support leafy vegetable production in ambon city, indigenous isolates were needed. indigenous azotobacter biofertilizer is expected to increase vegetable yield as well as its quality. this study has been carried out to isolate and select azotobacter from the rhizosphere of chili (capsicum annuum) and petsai (brassica chinensis) and to examine the effect of azotobacter inoculation on chili-seedling growth and nitrogen uptake by using bioassay method. materials and methods this experiment was conducted from september 2015 to march 2016 at the faculty of agriculture, universitas padjadjaran. azotobacter was isolated from petsai’s rhizosphere grown in entisols in desa waiheru, kecamatan teluk baguala, ambon and chili rhizosphere grown in the same soil order in desa hative besar, kecamatan teluk ambon, ambon. bioassay was performed in entisols from negeri hative besar, kecamatan teluk ambon, kota ambon (ph 5.8; 1.67% organic carbon, 0.12% total nitrogen, -1 9.08 mg 100 g available p, 7.86 ppm total p, 0.44 cmol -1 -1 kg available k, and 35.01 mg 100 g total k) to grow chili unpad-ck5. azotobacter isolation. isolation of azotobater has been carried out using nitrogen free ashby’s media (kh po 0.2 g; mgso .7h o 0.2 g; mannitol 10 g; 2 4 4 2 nacl 0.2 g; caso .2h o 0.1 g; caco 5 g per liter 4 2 3 distilled water). one g of rhizosphere were poured into 50 ml autoclaved nitrogen free ashby’s broth and incubated for five days at 30 °c until the pellicle was appeared. one loop of pellicle was streaked on ashby’s agar plate and incubated for 2 d at 30 °c. single colony were transferred to slantsof the same medium. isolates were subcultured several times to finally get five pure cultures, which were maintained in slant agar at room temperature. the azotobacter isolates were characterized based on the colony morphology, gram and 126 hindersah et al. microbiol indones capsule staining. isolates selection. all isolates were cultured in 100 ml ashb’s broth in 200 ml glass bottle placedon 115 rpm gyratory at room temperature (25-27 °c) for three days. at the end of the incubation, ashby’s broth acidity was determined and the available nitrogen was analyzed by kjeldhal method after 9 000 rpm centrifugation at 4 °c. the number of cells was counted by using haemocytometer under light microscope. two best isolates were selected based on rank. bioassay on chili seedling. the final stage of the experiment was plant inoculation with different azotobacter isolates and determination of the best inoculum cell density using chili (c. annuum l.) as the indicator plant. this bioassay was set up in randomized block design consisting of five treatment combinations: control (without azotobacter sp. inoculation) + 100% of npk fertilizer; 6 -1 azotobacter sp.s2a10 inoculation (10 cfu ml ) + 75% of npk fertilizer; 8 -1 azotobacter sp.s2a10 inoculation (10 cfu ml ) + 75% of npk fertilizer; 6 -1 azotobacter sp.c2a9 inoculation (10 cfu ml ) + 75% of npk fertilizer; 8 -1 azotobacter sp.c2a9 inoculation (10 cfu ml ) + 75% of npk fertilizer. each treatment was performed five times. before experiment, azotobacter population in bulk soil was counted by using dilution plate method in free-n ashby medium. the initial population of indigenous azotobacter has been used as a reference to determine the effect of treatments on azotobacter population in the rhizosphere of chili. chili seeds were soaked in azotobacter liquid inoculum and 1% gum arabic (m:v) for 30 sec. single seed was planted in growth media containing100 g of entisols and 100 g cow manure in black polybag; and maintained in green house for 21 d. the dose of npk -1 fertilizer for early growth of chili was 200 kg ha , which is applied 7 d after planting. dry weight of 21 d old transplants, units of azotobacter in ashby’s plate agar, and the amount of available nitrogen in soil were analyzed using analysis of variance (f test) and duncan multiple range test, to determine if f test were significant. the count of azotobacter population and the amount of available nitrogen in untreated soil were also analyzed and later used as a reference to determine the effects of azotobacter and npk treatments to the soil result indigenous azotobacter isolates. based on morphological observation and gram staining, five isolates were identified as azotobacter sp. azotobacter had been cultivated in free nitrogen ashby’s broth (ph 7) for three days and at the end of incubation, we found + that available n (nh and no ), cell density and broth 4 3 acidity differed from one isolate to another (table 1). the observation results, however, showed that there was no great acidity difference between these liquid cultures. the acidity ranged between 7.22 and 7.79. the 6 populations of azotobacter also varied between 5 × 10 -1 7 -1 cfu ml and 43 ×10 cfu ml . the highest number of cells by direct counting using haemocytometer was shown by s2a10 inoculum. regardless of the isolates, + nh content was always lower than no content, 4 3 indicating that nitrification had taken place. bioassay on chili plant. statistical analysis showed that the biomass, indicated by dry weight, of control plants without azotobacter were not significantly different from that of the other plants which received the treatment (table 2). however, inoculation of azotobacter with reduced dosage of npk significantly affected either the population of the azotobacter in the rhizosphere or the soil nitrogen content. the population of azotobacter in bulk soil before experiment was 1.83 × 10 cfu ml . the population was increased after inoculation of azotobacter. rhizosphere containing the highest azotobacter 6 -1 population (1.6 × 10 cfu ml ) were found in control 8 -1 plant. plant inoculated with 10 cfu ml c2a9 in combination with 75% of npk gave similar 6 -1 azotobacter population count (1.51 × 10 cfu ml ) with control.total nitrogen in entisols untreated soil was 0.12%, which is categorized as low according to the indonesian soil research institute (2009). all treatments enhanced the total nitrogen in soil up to 0.48%. the amount of total available nitrogen in control soil with npk 100% was equal with that of soil 6 -1 inoculated with 10 cfu ml azotobacter sp. s2a10 + 8 -1 75% npk as well as 106 cfu ml and 10 cfu ml azotobacter sp. c2a9 + npk 75%. the lowest total 8 -1 nitrogen was shown by plant treated with 10 cfu ml azotobacter sp. s2a10 + npk 75%. due to limited transplant biomass, we combined all five replicates prior to analyzing n uptake (table 3). without analytical statistics, the results showed that n uptake of control plant was the lowest. seedling inoculation followed by lower level of inorganic fertilization enhanced n uptake. 5 -1 volume 10, 2016 microbiol indones 127 table1. available nitrogen, azotobacter cell density and acidity of liquid culture after 3 d incubation + isolates were selected based on four aspects, i.e.no and nh concentrations, cell 3 4 density, and acidity. considering these parameters (table 1), two isolates, s2a10 from petsai rhizosphere and c2a9 from chili rhizosphere, were selected. tabel 2 effect of azotobacter and npk fertilizer on plant biomass, azotobacter population, and soil nitrogen of chili transplant note: the same letter (a, b or c) in a column indicates that the correponding values were not significantly differentaccording to the duncan multiple range test (p>5%). isolates + nh (ppm) 4 no (ppm) 3 cell density 7 -1 (10 cfu ml ) acidity c2a8 15.6 364.5 0.5 7.71 c2a9 20.0 508.7 12.1 7.47 s 1a 26.2 423.9 22.1 7.22 s 2a10 22.4 288.2 43.0 7.79 s2a9 10.2 457.8 15.0 7.72 combination dry weight (g) population of azotobacter 6 -1 (10 cfu gram ) soil total nitrogen (%) control (without azotobacter) + npk 100% 0.02 1,60 c 0.44 b 6 -1 azotobacter sp. s2a10, 10 cfu ml + npk 75% 0.04 0,98 b 0.39 b 8 -1 azotobacter sp. s2a10, 10 cfu ml + npk 75% 0.03 1,10 b 0.31 a 6 -1 azotobacter sp. c2a9, 10 cfu ml + npk 75% 0.02 0,33 a 0.48 b 8 -1 azotobacter sp. c2a9, 10 cfu ml + npk 75% 0.02 1,51 c 0.42 b microbiol indones128 hindersah et al. + to available nitrogen n-nh and n-no . both forms 4 3 of ionic nitrogen are detectable in nitrogen-free ashby’s broth following azotobacter application. the presence of these ionic nitrogens after incubation proves the capability of nitrogen fixation (hoffman et al. 2014). the results of the bioassay involving inoculation of chili seedling with azotobacter sp. indicated that the pgpr can potentially reduce inorganic fertilizer dosage. the combination of azotobacter (azotobacter sp. c2a9, 6 -1 8 -1 10 cfu ml or 10 cfu ml ) with reduced dose of inorganic fertilizer (npk 75%) produced equal amount of biomass as the plant treated with 100% inorganic fertilizer (without azotobacter). similar experiment by jarak et al. (2010) showed that chili plants inoculated with azotobacter produced the same average biomass (0.03 g) as the plant without azotobacter inoculation. the number of azotobacter cells in rizosphere indicates the capacity of the plant roots to provide nutrient for bacterial growth. it also indicates the survival rate of azotobacter, which will be used as biofertilizer. all treatments increased the population of azotobacter in soil, including the control treatment, compared to population before experiment. indigenous azotobacter from either liquid inoculum or crude exopolysaccharide is able to proliferate in rhizosphere by using plant-derived small organic molecules which is excreted from roots as carbon aand nitrogen sources (ahemad and kibret, 2014). the composition and population density of azotobacter in rhizosphere should be equivalent to the condition in either soil or plant. in this experiment, mixture of entisols and cow manure (1:1; v:v) were shown to support plant growth and root exudates, serving as carbon and other nutrition sources for heterotrophic azotobacter. 6 -1 except soil with 10 cfu ml azotobacter sp. c2a9 and npk 75%, the amount of total nitrogen inoculated soil with npk 75% was similar to that of control which indicated that azotobacter fixed nitrogen in low discussion azotobacter obtained from the rhizosphere of vegetables could be formulated as biofertilizer to enhance the quality of vegetable production while reducing the use of npk fertilizer. the first step of this experiment was carried out to screen the isolates based on ph, the population density of the isolates, and the available nitrogen produced, then, the selected bacteria were used in bioassay. the acidity of inoculum is one of the quality parameters in standardized biofertilizer, since each microbe lives and proliferates within specific ph level. according to the government regulation no 70, the indonesian ministry of agriculture (peraturan menteri pertanian no. 70/2011) in indonesia the value ranges between 5.0-8.0. the ph of the liquid culture of all isolates after three days fermentation were slightly around the range of 6.257.44, which was optimum for the growth of azotobacter species (becking 2006). azotobacter sp. is empirically known for its ability to fix nitrogen. slight increase of ph inhibits the azotobacter proliferation in the inoculum, since alkaline condition clearly reduces nitrogen fixation. acidic growth environment influences the azotobacter population density, and, generally, azotobacter is uncommonly found in the lower ph. previous research showed that all soils within ph range 7.07-8.56 contained azotobacter (mazinani et al. 2012).based on the measurement of the selective parameters (no3 + and nh content, cell density, and acidity) as presented 4 in table 1, two isolates, s2a10 from petsai’s rhizosphere and c2a9 from chili's rhizosphere, were selected. the population density of the isolates represents their ability to adapt and to perform their activity in soil (schmidt et al. 2014). azotobacter has an instinctive ability to fix dinitrogen to nh catalyzed by 3 nitrogenase. once nh is formed, it will be transformed 3 combination n uptake (mg/plant)* 1,02 1,44 1,52 1,62 2,56 control (without azotobacter) + npk 100% 6 -1 azotobacter sp. s2a10, 10 cfu ml + npk 75% 8 -1 azotobacter sp. s2a10, 10 cfu ml + npk 75% 6 -1 azotobacter sp. c2a9, 10 cfu ml + npk 75% 8 -1 azotobacter sp. c2a9, 10 cfu ml + npk 75% tabel 3. nitrogen uptake by chili at 21 d after transplanting *data from two nitrogen analysis (duplo). volume 10, 2016 microbiol indones 129 education indonesia. we thank neni rostini to provide chili unpad-ck5 seeds. references ahemad m, kibret m. 2014. mechanisms and applications of plant growth promoting rhizobacteria: current perspective. journal of king saud university-sciene. 26(1):1-20. doi:10.1016/j.jksus.2013.05.001. balai penelitian tanah. 2006. luas lahan kering yang sesuai untuk pertanian (dry land area for cultivation). online: http://balittanah.litbang.pertanian.go.id/ th accessed at july 30 2015. balai penelitian tanah.2009. analisis tanah, tanaman, air dan pupuk (soil, plant, water, and fertilizer analysis). online: http://balittanah.litbang.pertanian.go.id/ind/ th accessed atjuly 30 2015. becking jh. 2006. the family azotobacteraceae. prokaryotes 6:759-783. cejudo fj, and a. paneque. 1987. correlation between nitrate uptake rate and nitrate inhibition of nitrogenase activity in azotobacter chroococcum. fems microbiology letters. 43(1):5-7. doi:10.1007/0-387-30746-x_26. gruhn p, goletti f, yudelman m. 2000. integrated nutrient management, soil fertility, and sustainable agriculture: current issues and future challenges. intl food policy res institute. isbn 0-89629-638-5. hamilton iii ew, frank da. 2001. can plants stimulate soil microbes and their own nutrient supply? evidence from a grazing tolerant grass. ecology 82(9):2397-2402. doi:10.1890/0012-9658(2001)082[2397:cpssma]2.0. co;2. harmens h, stirling cm, marshall c, farrar jf. 2000. is partitioning of dry weight and leaf area with in dactylis glomerata affected by n and co enrichment?. annals 2 of botany. 86(4): 833-839.doi:10.1006/anbo.2000.1243. hindersah r, sulaksana a, herdiyantoro, d. 2014. perubahan kadar n tersedia dan populasi azotobacter di rizosfer sorgum (sorghum bicolor l.) yang ditanam di dua ordo tanah dengan inokulasi azotobacter sp. (n concentration change and population of azotobacter in sorghum rhizosphere on two kind of soil order) agrologia. 3(1):10-17. halbleib cm, ludden pw. 2000. regulation of biological nitrogen fixation. the journal of nutrition 130(5):1081 -1084. hoffman bm, lukoyanov d, yang zy, dean dr, seefeldt lc. 2014. mechanism of nitrogen fixation by nitrogenase: the next stage. chemical reviews. 114(8):4041-4062. doi:10.1021/cr400641x. jarak mn, duric ss. dordevic bd. 2010. benefits of inoculation with azotobacter in the growth and production of tomato and peppers. proceedings for natural sciences matica srpska. 119:71-76. doi:10.2298/zmspn1019071j. kubat j, klie j, pova d. 2003. the dry matter yield, nitrogen uptake, and the efficacy of nitrogen fertilization in long-term field experiment. plant soil environ. 49(8):337-345. nitrogen soil. azotobacter inoculation potentially decreased the use of npk fertilizer to 25%. untreated soil contained 0.12% total nitrogen, low nitrogen give benefit to nitrogen fixation process since nitrogenase system is repress when high rate nitrate or nitrite taken up by the azotobacter’s cells (cejudo and peneque 1987) and cellular level of fixed nitrogen is sufficiently high (halbleib and ludden, 2000). the increase of available nitrogen was also reported by hindersah et al. (2014) in sorghum inoculated with indigenous azotobacter in bioassay experiment using inceptisol and entisols soil orders. plants stimulate microbial growth by secreting root exudate that functions as a source of nutrient (hamilton and frank 2001). this mechanism might have something in common with the relation between s2a10 isolate, which had been isolated from petsai rhizosphere, and chili plant. different hosts might have negative effect to the azotobacter nitrogenase activity (swain and abhijita 2013). seedling inoculation followed by lower level of inorganic fertilization enhanced n upatake. this finding indicated that local indigenous azotobacter was able to increase n uptake by chili plant but did not yet play a significant role in dry matter enhancement. it is generally assumed that n uptake correlates with partitioning of dry matter but based on our bioassay, increased n uptake did not significantly improve the dry matter content. however, in long term, mixed agricultural input in certain commodities increased n uptake,which, in turn, is correlated with the yield (kubat et al. 2003). two azotobacter isolates, s2a10 and c2a9, which were isolated from petsai’s and chili’s rhizosphere, respectively,were selected based on their ph, cell density, and available nitrogen. bioassay showed that azotobacter inoculation followed by reduced npk fertilizer doses had no effect on transplant dry weight and nitrogen uptake. all azotobacter inoculation 8 -1 except 10 cfu ml s2a10 maintain soil nitrogen although azotobacter population in soil was slightly reduced. acknowledgment the authors convey their gratitude to rafael osok as chief of research institution of universitas pattimura, indonesia, for giving the opportunity to undertake the research on pekerti scheme of universitas pattimura. the research was funded by the directorate of higher education, department of microbiol indones130 hindersah et al. swain h, abhijita, s. 2013. nitrogen fixation and its improvement through genetic engineering. j global biosci. 2(5):98-112. tarigan rs, elimasni d. 2013. seleksi bakteri penambat nitrogen dan penghasil hormon iaa (indole acetic acid) dari rizosfer tanah perkebunan kedelai (glycine max l.) (nitrogen fixer and iaa producing bacteria bacteria selection from soybean (glycine max l.) rhizosphere). saintia biologi. 1(2):42-48. tripathi s, barua s, chakrabarti k. 2015. site specific bioinoculants for sustainable agriculture in coastal saline soil. halophiles 6:209-234. doi:10.1007/978-3319-14595-2_8. widiastuti h, siswanto, suharyanto. 2010. karakterisasi dan seleksi beberapa isolat azotobacter sp. untuk meningkatkan perkecambahan benih dan pertumbuhan tanaman (characterization and selection of azotobacter sp to enhance the seedling and plant growth). buletin plasma nutfah.16(2):160-167. doi:10.21082/blpn.v16n2.2010.p160-167. kumar a, kumar k, kumar p, maurya r, prasad s, singh sk. 2014. production of indole acetic acid by azotobacter strains associated with mungbean. plant archive.14(1):41-42. mazinani z, aminafshar m, asgharzadeh a, chamani m. 2012. effect of azotobacter population on physicochemical characteristics of some soil samples in iran. annals of biological research. 3(7):3120-3125. peraturan menteri pertanian no 70 tahun 2011 mengenai pupuk organik, pupuk hayati dan pembenah tanah. (indonesian ministry of agriculture regulation no. 70 in 2011 about organic fertilizer, biofertilizer and soil amendment) sabra a, zeng p, lonsdorf h, deckwer wd. 2000. effect of oxygen on formation and structure of azotobacter vinelandii alginate and its role in producing nitrogenase. appl environ microbiol. 66(9):40374044. doi:10.1128/aem.66.9.4037-4044.2000. schmidt sk, king aj, meier cl, bowman wd, farrer ec , suding k n, nemergut dr. 2014. plant-microbe interactions at multiple scales across a high-elevation landscape. plant ecol div. 8(5-6):703-712. doi:10.1080/ 17550874.2014.917737. 1: 125 2: 126 3: 127 4: 128 5: 129 6: 130 9.mi717-elizabeth situmorang available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.9issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 198-204 *corresponding author; phone/fax: +62-21-50338899/+6221-50389999 , email: situmorang_elizabeth@yahoo.com endophytes refer to a group of fungi that reside asymptomatically inside the living plant tissues as reported by hyde and soytong (2008). huang et al. (2008) reported that recent surveys of various host plants have demonstrated that fungal endophytes are ubiquitous in plant species . globally, there are at least one million species of endophytic fungi as reported by ganley et al. (2004), which represent an important genetic resource for biotechnology. endophytes have been recognized as potential sources of novel natural products for agricultural, industrial, and pharmaceutical uses, especially those secondary metabolites produced by fungal endophytes colonizing medicinal plants as reported by mitchell et al. (2008). according to shiomi et al., (2006), endophytic fungi are able to penetrate and become systemically disseminated in the host plant, actively colonize the apoplast, conducting vessels and occasionally the intracellular spaces. this colonization presents an ecological niche, similar to that occupied by plant pathogens therefore endophytic fungi are more able to inhibit plant pathogens by antibiosis mechanism, competition for nutrients and spaces for proliferation, and induce plant resistance. zaiton (2006) studied the isolation and characterization of microbial endophytes from oil palm roots in endophytic fungi defined as fungi that colonize internal plant tissues without causing visible damage to their host plant. as they are internal colonisers, therefore more able to compete within the vascular systems with capacity to arrest the spread of pathogens such as ganoderma boninense causal agent of basal stem rot (bsr) disease in oil palm. endophytic microbes acted against plant pathogen by antibiosis mechanism, nutrient and space competition, and induce plant pathogen resistance by producing metabolites. the objective of the present study was to identify endophytic fungi from oil palm roots in g. boninense endemic area padang halaban estate, north sumatera, based on morphological and histological character. at each site, five random palms were sampled. seventy five endophytic fungi had been isolated and selected from bsr symptomless palm root. identification of fungal endophytes were carried out by observing the reproductive structures (sexual and asexual) under a light-field microscope with camera attachment. seventy five isolates were classified to eight genera, consisting of trichoderma (20), fusarium (10), aspergillus (5), penicillium (5), gliocladium (4), phoma (4), alternaria (4), and curvularia (3). twenty others were unidentified due to sterile mycelia. key words: basal stem rot, elaeis guineensis, endemic area, oil palm, root endophytic-fungi fungi endofit hidup dan berkoloni dalam jaringan tanaman namun tanpa menunjukkan gejala perusakan pada tanaman inang. sebagai mikroba pengkoloni internal, akan lebih memudahkan dalam bersaing dalam system vascular dengan patogen seperti ganoderma boninense penyebab busuk pangkal batang tanaman kelapa sawit. mikroba endofitik bekerja melawan patogen tanaman melalui mekanisme antibiosis, persaingan ruang dan nutrisi untuk pertumbuhannya, dan menginduksi ketahanan tanaman melalui metabolit yang dihasilkan. tujuan dari penelitian ini adalah mengidentifikasi fungi dari akar tanaman kelapa sawit asal kebun endemik g.boninense di sumatera utara berdasarkan karakteristik morfologi dan histologi. lima sampel diambil secara acak dari masing-masing lokasi. sebanyak 75 isolat diisolasi dari akar tanaman kelapa sawit sehat yang tumbuh di kebun endemik. identifikasi morfologis dan mikroskopis fungi endofit dilakukan dengan mengamati struktur reproduksi seksual maupun aseksual menggunakan kamera terhubung dengan mikroskop. isolat diidentifikasi ke dalam 8 genera yaitu trichoderma (20), fusarium (10), aspergillus (5), penicillium (5), gliocladium (4), phoma (4), alternaria (4), dan curvularia (3). isolat lainnya tidak teridentifikasi secara morfologis maupun histologis, karena tidak berspora. kata kunci: area endemik, elaeis guineensis, busuk pangkal, fungi endofit akar, tanaman kelapa sawit short communication morphology and histology identification of fungal endophytes from oil palm roots in ganoderma boninense endemic area elizabeth caroline situmorang, andriessa prameswara, hana christine sinthya, nurita toruan-mathius, and tony liwang microbiome technology laboratory, plant production and biotechnology division, pt smart tbk, sinarmas land plaza tower 2, 28th fl. jalan mh thamrin no. 51, kav 22, gondangdia, jakarta pusat,10350, indonesia malaysia. the results showed that the fungal isolates were mainly from the genera fusarium and trichoderma. fusarium is most ubiquitous and has been isolated from many host plants and constitutes 72.70 per cent of the isolated fungi. detection of fungal endophytes was carried out by observation of histological oil palm root sections under light microscope (100× magnification). fungal hyphae were stained by toluidine blue have been observed intra and inter cellularly in roots from symptomless palms, especially in the epidermal cells running parallel into the schlerenchyma cells and the cortex. mitrousia (2012) stated that some rhizospheric fungi are pathogens to plants such as phoma. but it is still to be confirmed if it also the case of endophytes fungi. nur amin et al. (2014) reported that endophytes gliocladium, fusarium, aspergillus, and others have been isolated from cocoa and supposed to be beneficial for plant. mathan et al. (2013) reported antimicrobial metabolites could be produced by fungi such as aspergillus sp. basal stem rot (bsr) disease caused by ganoderma species is the most destructive disease in oil palm. the use of conventional chemical fungicide has not given much green light, as the disease is known systemic. so far, cultural practices, combined to some extent with biological control, have been considered as the best approach for controlling the bsr. recently, study on endophytic fungi as biological control agents in suppressing plant disease has gained much attention in pathological research. some of endophytic bacteria and fungi might have potentiality to control g. boninense since they showed ability to inhibit g. boninense in vitro as reported by wicaksono et al. (2011). the study of sharaf et al. (2013) also reported that chitinase produced by trichoderma sp. could be used as antifungal agent. the objective of this current study was to identify as much as possible specific endophytic fungi living in bsr symptomless oil palm plant, for the first stage by morphological and histological characterizations. this study was conducted to obtain potential endophytic fungi against g. boninense in view of formulating indigenous biofungicide. root sampling and isolation of fungal endophytes. oil palm roots were obtained from padang halaban estate pt smart, tbk in north sumatera, indonesia. the age of palms was 28 years with bsr (basal stem rot) symptomless at symptom areas. the roots were sampled from three sites selected for bsr infection. at each sites, five random palms were sampled with the roots diameter 0.5 cm, taken about 1.0 m away from their bases at 25-30 cm depth. root samples from each palm were rinsed under running tap water for 20 min to remove any adhering soil from their surface, then surface sterilized using 5.25% of sodium hypochlorite. after dipping in 50, 70, and 90% of ethanol, samples were rinsed with sterilized distilled water following procedure described by arnold et al. (2003). root samples were then dried on a sterilized filter paper. one centimeter from the ends of each section was discarded. the cuticle from the middle centimeter was removed and was cut into two sub-sections of 0.5 cm each. the sub-sections were, in turn, split longitudinally into four before transferring to the pda media for culturing. purification of fungal endophytic isolates. fungal endophytes which showed emergence at cutting sample then were selected to be purified. colonies were cut and transferred aseptically into new cultures media. colonies which were pure then selected and transferred in slant agar and petri dish containing pda media. colonies were incubated at 28+2 °c. pure fungal isolates were then observed macroscopically and microscopically for identification. morphological and histological identification of fungal endophytes. fungal endophytes were selected for further characterization and identification based on barnett and hunter (1998). morphological observation was followed every 24 h during 30 d and identification of fungal isolates was done according to colony or hyphal morphology of the fungal culture, surface and reverse colony color, and colony texture. histological identification of fungal endophytes was carried out by observing the characteristics of the spores or conidia, and reproductive structures (sexual and asexual) under a light-field microscope with camera attachment (model nikon eclipse-50i, japan). growth of fungal endophytes. only a few of pure cultures of fungi started to grow out of the surface of sterilized root sections after 24 h of incubation on pda at 28+2 °c, with 1.1 to 2.6 cm of diameter. almost all of them started growing after 48 h with diameter range from 0.4 to 5.3 cm. some were fast growing and others were even very slow growing fungi, which started to grow after 168 h (2.0 and 9.0 cm of diameter respectively). seventy-five endophytic fungi isolates were obtained from palm roots. according to morphology and histology characters, the isolates of endophytic fungi belonged to genera trichoderma (20), fusarium (10), aspergillus (5), penicillium (5), volume 7, 2013 microbiol indones 199 gliocladium (4), phoma (4), alternaria (4), and curvularia (3). twenty others could not be identified due to sterile mycelia. trichoderma sp. trichoderma sp. were typically fast growing observed on pda at 28 °c. colonies were transparent to white at beginning on agar media, which was developed 24 h after incubation, with diameter of 1.5-2.0 cm, and becoming compact in time. the surface of colony color was white and scattered greenish patches become visible as the conidia were formed starting from 72 h and concentric rings were slowly formed in time. on the reverse side, the color was pale to yellowish. a yellow pigment seemed clearly to be secreted into the agar (fig 1a). taxonomy of trichoderma were based largely on histological character such as conidial form, size, color and ornamentation, branching pattern with short side branches, short inflated phialides and the formation of sterile, and fertile hyphal elongation from conidiophores. microscopic of trichoderma showed the presence of septate hyaline hyphae of around 200 µm. phialides were hyaline, branched, and flask-shaped, inflated at the base, represented in cluster, and attached to the conidiophores at right angles. conidiophores were also hyaline and branched. conidia were unicellular, ellipsoidal form, green in color, smooth walled, with an average diameter of 5 µm, and were grouped in sticky heads at the tips of the phialides (fig 1b). the genus trichoderma (ascomycetes, hyprocreales) contains species that are of vast economic importance owing to their production of antibiotics, industrial enzymes, and ability to act as biological control agents (bca) against plant pathogens since 1920s. some trichoderma strains have been developed and widely applied in agricultural practices as bio-control agents against plants’ fungal diseases. the various mechanisms include antibiosis, parasitism, inducing host-plant resistance, and competition. strains producing enzymes such as chitinase and glucanase have been selected as criteria for bca. fusarium sp. morphological observation of fusarium sp. showed that these cultures were fast growing starting to grow after 24 h, and produced woolly and cottony spreading colonies. from the front, the color of the colony were observed creamy white. from the reverse side, it was almost colorless. some species also produce distinctly different conidia in the aerial mycelium (referred to as microconidia). aerial mycelium represented the growth of hyphae above the agar surface, often forming a convex shape, with a cottony or somewhat ropey texture (fig 2a). species of fusarium typically produce both macroand microconidia from slender phialides. macroconidia were hyaline, twoto three-celled, comma shaped, mostly with an elongated apical cell and pedicellate basal cell. microconidia were formed from 1to 2celled, hyaline and ovoid, straight or curved. chlamydospores were present in this fusarium culture. according to summerel et al. (2003), fusarium were characterized by the production of slimy, hyaline, canoe-shaped conidia (known as macroconidia) that in most species are produced in fruiting-structures called sporodochia. some fusarium species produce morphologically distinct conidia in the aerial mycelium, which are usually smaller and have fewer septa than the macroconidia produced in sporodochia. therefore, they are called microconidia (fig 2b) showing that macroconidia was a type c, straight. microconidia were represented in comma shaped or ellipsoidal. chlamydospores were produced singly or in pairs. monophialide is phialide that produces conidia from one opening only. f. oxysporum-non pathogenic have been developed as bio-control agents against plant pathogen by antibiosis and mycoparasitism mechanism (rubini et al. 2005). aspergillus sp. aspergillus sp. characteristics which are essential in genera identification are the growth rate, colony color and thermo-tolerance. texture of colonies varies from downy to powdery. the colonies were fast growing, with average diameter of 1.5 cm at 24 h and 6.0-9.0 cm at 168 h on pda at 28 °c. surface colony color was black with powdery aspect, and the reverse was mostly pale yellow (fig 3a). the only thermo-tolerant aspergillus which can grow at temperature range of 20 °c to 50 °c is a. fumigatus. histological microscopic of aspergillus showed that the hyphae of these genera were septate and hyaline. conidiophores arised from the basal foot cell found at the supporting hyphae and terminate in a vesicle at the tip. vesicle is typically for the genus aspergillus. phialides were flask-shaped attached to the vesicle via a supporting cell, metula. conidia were attached over the phialides forming radial chains (fig 3b). conidia were round with 1 µm of diameter in average. penicillium sp. penicillium sp. is comparable to aspergillus. the genus penicillium falls into the order eurotiales. in this order, organisms produce asci within cleistothecia. penicillium is often reffered to as deuteromycetes, or fungi imperfecti. the name penicillium comes from the word “brush”; this refers to 200 situmorang et al. microbiol indones histological microscopic showed that hyphae of this genera were septate and hyaline to brown (fig 6b). conidia were hyaline, oval shaped, unicellular and each conidium usually had two oil droplets inside. chlamydospores were brown and appear in long chains. alternaria sp. alternaria sp. grew rapidly and the colony size reached a diameter of 3 to 9 cm after incubation at 28 °c for 7 d on potato dextrose agar. the colony was flat, downy to woolly and was covered by grayish, short, aerial hyphae in time. the surface was grayish white at the beginning, which later darkened and became greenish black or olive brown with a light border (fig 7a). the reverse side was typically brown to black due to pigment production. alternaria spp. had septate, brown hyphae. conidiophores were also septate and brown in color, occasionally producing a zigzag appearance. they beared simple or branched large conidia (7-10 × 23-34 µm) which have both transverse and longitudinal septations. these conidia were observed singly or in acropetal chains and may produce germ tubes (fig 7b). they were ovoid to obclavate, darkly pigmented, muriform, and smooth or roughened. the end of the conidium which were the nearest the conidiophore was round while it tapered towards the apex. this gave the typical beak or clublike appearance of the conidia. curvularia sp. curvularia sp. produced rapidly growing, woolly colonies on potato dextrose agar at 28 °c. from the front, the color of the colony was white to pinkish gray initially and turned to olive brown as the colony matured. from the reverse, it was dark brown to black (fig 8a). curvularia hyphae were septates brown, of which brown conidiophores, and conidia were visualized. conidiophores were simple or branched and bent at the points where the conidia originated. this bending pattern was called sympodial geniculate growth. the conidia (8-14 × 21-35 µm), which were also called the poroconidia, were straight or pyriform, brown, multiseptate, and had dark basal protuberant hila. the septa were transverse and divided each conidium into multiple cells. the central cell was typically darker and enlarged compared to the end cells in the conidium. the central septum may also appear darker than the others. the swelling of the central cell usually gave the conidium a curved appearance (fig 8b). the number of the septa in the conidia, the shape of the conidia (straight or curved), the color of the conidia (dark vs pale brown), existence of dark median septum, and the prominence of geniculate growth pattern were the appearance of spores in penicillium. bancerz et. al. (2005) found that this species was one of the best lipase producers among other fungi they studied in the arctic tundra. penicillium has high enzymatic activity and has the ability to produce alpha-amylase. most fungi contain secondary metabolites. these were used to produce antibiotics such as penicillin. certain components of fungal genetic structure that create these secondary metabolites are common across not just species, but across orders (carlile et al. 2001). penicillium is known as filamentous fungi. the colonies of penicillium were rapid growing, flat, filamentous, and velvety in texture. the colonies were initially white and became pinkish in time. the plate reverse was yellowish and pale (fig 4a). they had branched conidiospores. conidia were observed round and unicellular. penicillium culture seemed to have small hyphae. glucans are reported common in the cell walls of penicillium species. penicillium hyphae were septate hyaline (1.5 to 5 µm in diameter), simple or branched conidiophores, metulae, phialides, and conidia were observed. metulae were secondary branches that were formed on conidiophores. the metulae carried the flask-shaped phialides. the organization of the phialides at the tips of the conidiophores was very typical. they form brush-like clusters, which referred to as “penicilli”. the conidia (2.5-5 µm in diameter) were rounded, unicellular, and visualized as unbranching chains at the tips of the phialides (fig 4b). gliocladium sp. gliocladium sp. is most close related to penicillium and paecilomyces. colonies on potato dextrose agar were white initially, but typically become green, granular, poorly demarcated, and grew across the entire plate, resembling a “green lawn” (fig 5a). phialides were branched and tapered at tips. spherical conidia were gathered at tip of phialides in a tight,ball-shaped cluster and were often somewhat larger than trichoderma (fig 5b). conidiophores had penicillate branches and conidial masses collecting in a mucilaginous droplet. spores were smooth-walled. small spines, which were microscopically observable, distribute sparsely on the main stem of conidiophores. phoma sp. phoma sp. is a cosmopolitan, dematiaceous filamentous fungus that inhabits the soil and plant material. colonies growth rate was rapid, starting from 48 h, texture powdery to velvety, spreading and frequently submerged in the pda medium. surface colony color was initially white becoming olive gray, sometimes with a tint of pink while reverse color was purple to yellow diffusible pigment. volume 7, 2013 microbiol indones 201 fig 1 culture of trichoderma growing on potato dextrose agar. the white areas did not contain spores, while the green areas were covered with dense masses of spores (conidia) (a). trichoderma was repeatedly branched conidiophores, irregularly verticillate, bearing clusters of divergent, irregularly bent, flask-shaped phialides (b). conidia/spores (c). fig 2 culture of fusarium growing on potato dextrose agar (a). microconidia, macroconidia (2 4 septates), septate hyphae, and chlamydospores were present (arrows showed in b). fig 3 culture of aspergillus growing on potato dextrose agar. the spores come in black color (a); microscopic histology of aspergillus. phialides attached to the vesicle via a supporting cells called metula (arrow showed in b). conidia/spores (c). a b c a b c a b c fig 4 culture of penicillium growing on potato dextrose agar. colonies texture are very powdery (a); conidia, phialides and metula are present. phialides are attached to the vesicle via a supporting cells called metula (b). a b c 202 situmorang et al. microbiol indones fig 5 culture of gliocladium growing on potato dextrose agar (a); microscopic structure of phialides with branches and tapered at tips (arrows showed in b). a b fig 8 culture of curvularia growing on potato dextrose agar (a); conidia are brown and multiseptate (b). a b fig 6 culture of phoma growing on potato dextrose agar. septate hyaline hyphae and hyaline conidia were present (b). fig 7 culture of alternaria growing on potato dextrose agar (a); conidia darkly pigmented with germ tubes (b). a b a b c volume 7, 2013 microbiol indones 203 ananas ananassoides in the bolivian amazon. fungal divers. 31:3743. mitrousia g, huang y, hall a, fitt bdl. 2012. severity of phoma leaf spotting and stem canker on brassica napus cultivars with rlm7 resistance against leptosphaeria maculans in the uk”. aspects of applied biology, 117: 217-222. nur amin salam m, junaid m, asman, baco ms. 2014.isolation and identification of endophytes fungi from cocoa plant resistante vsd m.05 and cocoa plant susceptible vsd m.01 in south sulawesi. int’l j curr microbiol appl sci. 3(2):459-467. rubini mr, silva-ribeiro rt, pomella awv, maki cs, araujo wl, dos santos dr, azedevo jl. 2005. diversity of endophytic fungal community of cacao (theobroma cacao l.) and biological control of crinipellis perniciosa, causal agent of witches broom disease. int j bio sci. 1:24-33. doi:10.7150/ijbs.1.24. sharaf ef, el-sarrany, aeq, el-deeb m. 2012. biorecycling of shrimp shell by trichoderma viride for production of antifungal chitinase. afr j microbiol res. 6(21):45384545. shiomi hf, harllen sas, itamar sdm, flavia vn, wagner b. 2006. bioprospecting endophytic bacteria for biological control of coffee leaf rust. j sci agri. 63(1):32-39. summerel ba, salleh b, leslie jf. 2003. a utilitarian approach to fusarium identification. plant dis. 87(2):117-128. doi:10.1094/pdis.2003.87.2.117. wicaksono wa, buana rf, situmorang ec. 2011. endophyte microbes from oil palm (elaeis guineensis) tissues and its potential as a biocontrol for ganoderma boninense in vitro. in: suharsono, ehara h, minarsih h, wiryawan kg, miftahuddin, yunus m, ermayanti tm, widyastuti u, mardatin nf, editors. improving th food, energy and environment with better crops. the 7 acsa conference; 2011 september 27-30. bogor (id). bogor: rcbb. p360-365. zaiton s, sariah m, zainal abidin ma. 2006. isolation and characterisation of microbial endophytes from oil palm roots: implication as biocontrol agents against ganoderma. the planter, kuala lumpur. 82(966):587597. the major microscopic features that help in differentiation of curvularia spp. among each other. for instance, the conidia of c.lunata have 3 septa and 4 cells, while those of c. geniculata mostly have 4 septa and 5 cells. references arnold ae, meija cl, kyllo d, rojas ie, maynard z, robbins n, herre ae. 2003. fungal endophytes limit pathogen damage in a tropical tree. the national academy of sciences of the usa. 15659-15654. bancerz r, ginalska g, fiedurek j, gromada a. 2005. cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus penicillium chrysogenum. j ind microbiol biot. 32(6):253-260. doi:10.1007/s10295-005-0235-0. barnett hl, hunter bb. 1998. illustrated genera of th imperfect fungi. 4 ed. usa: prentice-hall, inc. carlile mj, watkinson sc, gooday gw. 2001. the fungi. second edition. san diego, california: academic press. ganley rj, brunsfeld sj, newcombe g. 2004. a community of unknown, endophytic fungi in western white pine. proc of natl acad sci u s a. 101(27):10107-12. doi:10.1073/pnas.0401513101. huang wy, cai yz, hyde kd, corke h, sun m. 2008. biodiversity of endophytic fungi associated with 29 traditional chinese medicinal plants. fungal divers. 33:61-75. hyde kd, soytong k. 2008. the fungal endophyte. fungal divers. 33:163-173. jinantana j, sariah m. 1997. antagonistic effect of malaysian isolates of trichoderma harzianum and gliocladium virens on sclerotium rolfsii. pertanika: j tropagri sci. 20(1):35-41. mathan s, subramanian v, nagamony s. 2013. optimization and antimicrobial metabolite production from endophytic fungi aspergillus terreus kc 582297. eur j exp biol. 3(4):138-144. mitchell am, strobel ga, hess wm, vargas pn, ezra d. 2008. muscodor crispans, a novel endophyte from 204 situmorang et al. microbiol indones 1: 198 2: 199 3: 200 4: 201 5: 202 6: 203 7: 204 8.mi701-raden haryo bimo setiarto available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.8issn 1978-3477, eissn 2087-8587 vol 7, no 4, november 2013, p 192-197 *corresponding author; phone/fax: +62-81-327025330/+6221-87907612 , email: haryobimo42@yahoo.com rice straw is a by-product of rice production in indonesia. it becomes a problem because it contains lignocellulose compound that is very difficult to decompose (arantes et al. 2012). on the other hand, it can potentially be converted into organic fertilizer. during harvesting months, rice straw is produced in huge quantity and cannot be optimally utilized. in the fact, rice straw can be used to increase soil organic nutrient by composting process. long term utilization of rice straw compost can recover soil fertility because it contains lignocelullolytic microbes as bio-remedy agents (chang et al. 2012). rice straw compost contains phosphate (6.86 ppm), carbon (35.83%), water (35.83%), nitrogen (1,57%), phosphor (0.02%), iron (4.04 ppm), and zinc (0.09 ppm). based on this composition, each ton rice straw compost contains soil nutrition equivalent to 41.2 kg urea and 4.5 kg sp36 (liu et al. 2012). it can substitute more than half of chemical fertilizer stock. lignocelullolytic enzymes such as laccase, cellulase, and xylanase can be produced by lignocelullolytic fungi isolated from spent mushroom substrate that had been used to grow champignon (agaricus) (dixon and webb 1979). the lignocellulolytic fungi have a main role of converting bio-mass of rice straw into compost. so, they can degrade and use lignin, cellulose, and hemicelluloses respectively as carbon source and hydrogen source for the main problem in soil conservation is the lack of carbon source from organic material. rice straw from spent mushroom substrate (sms) can be used as organic fertilizer to supply organic carbon for soil. it can also improve soil structure and increase macro-elements and micro-elements required by plants. this research focused on analyzing lignocellulolytic activity and indole acetic acid (iaa) concentration produced by 14 fungal strains isolated from rice straw that had previously been used as substrate for champignon (agaricus sp.). four strains were isolated (jpf 2, jpf 5, jpf 13, and jpf 14) and then characterized. jpf 14 isolate had the highest -1 laccase activity (1.767 u ml ) and produced the highest concentration of indole acetic acid (iaa) hormone (6.78 -1 -1 mg ml ). jpf 13 isolate had the highest amylase activity (0.746 u ml ). jpf 2 isolate had the highest cellulase -1 -1 activity (0.502 u ml ). jpf 5 isolate had the highest xylanase activity (0.560 u ml ). based on their ability to grow on certain ph and temperature, isolate jpf 2 was classified as mesophylic-acidophylic microbe, isolates jpf 5 and jpf 14 were psycrophylic-acidophylic microbes, and isolate jpf 13 was psycrophylic-alkalophylic microbe. key words: champignon, indole acetic acid hormone, lignocellulolytic fungi, rice straw, spent mushroom substrate jerami padi merupakan salah satu substrat yang kaya lignoselulosa dan memiliki potensi untuk dapat diolah menjadi pupuk organik. penambahan kompos jerami akan menambah kandungan bahan organik tanah, sehingga dapat mengembalikan kesuburan tanah. tujuan penelitian ini adalah seleksi dan karakterisasi jamur pendegradasi lignoselulosa dan penghasil hormon asam indol asetat (iaa) yang diisolasi dari jerami padi limbah budidaya jamur champignon (spent mushroom substrate). hasil yang diperoleh menunjukkan bahwa dari 4 isolat jamur lignoselulolitik terpilih (jpf 2, jpf 5, jpf 13, dan jpf 14), isolat jpf 14 memiliki aktivitas enzim -1 lakase tertinggi yaitu sebesar 1.767 u ml dan mampu menghasilkan hormon iaa (asam indol asetat) dengan -1 kadar tertinggi yaitu 6.78 mg ml . isolat jamur jpf 13 memiliki aktivitas enzim amilase tertinggi dengan nilai -1 -1 0.746 u ml . isolat jamur jpf 2 memiliki aktivitas enzim selulase tertinggi yaitu sebesar 0.502 u ml . isolat -1 jamur jpf 5 memiliki aktivitas enzim xilanase tertinggi dengan nilai 0.560 u ml . selain itu berdasarkan pertumbuhan pada suhu dan ph tertentu, isolat jamur jpf 2 dapat dikelompokkan sebagai mikroba mesofilikasidofilik, isolat jamur jpf 5 dan jpf 14 dapat dikelompokkan sebagai mikroba psikrofilik-asidofilik, isolat jamur jpf 13 dapat dikelompokkan sebagai mikroba psikrofilik-alkalofilik. kata kunci: hormon asam indol asetat, jamur champignon, jamur lignoselulolitik, limbah jerami padi fungal short communication the lignocellulolytic activity and ability to produce indole acetic acid hormone of fungal inoculant isolated from spent mushroom (agaricus sp.) substrate raden haryo bimo setiarto* and iwan saskiawan microbiology division, research center for biology lipi jalan raya jakarta-bogor km 46, cibinong science center, bogor 16911, indonesia growth (dewi 2002). cellulase is an enzyme that hydrolyzes β-1,4 glucoside chain on cellulose and derivatives into glucose. it has a multi-enzyme system consisting of endoglucanase (ec.3.2.1.4), selobiohydrolase (ec.3.2 .1.91), and β-glucosidase (ec.3.2.1.21) (ahamed 2008; bhat 1997). xylanase is an enzyme group which can hydrolyze polymer xylan into xylose. it can be classified into β-xilosidase, exoxylanase, and endoxylanase. exoxylanase can cut the chain of xylan polymer at the end of reduction, so it can produce xylose as the main product as well as other short chain oligosaccharides (da silva et al. 2005, goddess 2002, linko et al. 1984). laccase (benzendiol: oxygen oxidoreductase, ec 1.10.3.2) is an extracellular enzyme that uses oxygen to carry out oxidation reaction in aromatic and non aromatic compound. it belongs to oxidase enzyme class which requires metal ion. the laccase enzyme requires oxygen and produces water as one of the side products. the main substrate of laccase is lignin and oxidation reaction will not produce hydrogen peroxide compound (couto et al. 2006; couto et al. 2007; kirk and farrel 1997; kruus 2000). lignin peroxidase, and manganese peroxidase are two ligninase enzymes which can degrade lignin in addition to the laccase enzyme (jeffries 1994, kruus 2000). fungi isolated from spent champignon substrate not only can produce lignocellulolytic enzyme, but also indole acetic acid (iaa) hormone to induce and increase plant growth (aryantha et al. 2002). iaa is an endogenic auxin hormone that is available in plant body. it is a regulatory growth hormone that was first found as the main signal for plant growth (ahmad et al. 2005). it is formed in the root and crops meristem tissue. moreover, it can push elongation cell in coleoptil and plant internode. elongation cells especially exist in vertical turn near the amplification cell. the iaa hormone also has a role in activating cell component, producing cell wall, and reorganizing into matrix whole cell (bric et al. 1991, levean et al. 2004). the iaa hormone produced by fungi will be absorbed by plant, that the plant grow faster and bigger (arshad and frakenburg 1991). based on previous research, so this study will concern to selecting fungi isolates from spent champignon substrate which can produce high concentration iaa hormone and high activity of lignocellulolytic enzyme. moreover, this research focused on producing organic fertilizer starter inoculant made of lignocellulolytic fungi isolated from spent champignon substrate which can produce iaa hormone. the starter inoculant was then used to produce compost from rice straw as spent mushroom substrate. concentration of iaa hormone and activity of laccase, cellulase, and xylanase enzymes from fungal isolates were used as the parameters for selection and characterization process. fungi isolated from spent champignon substrate were grown on potato dextrose agar (pda) medium. pda medium contained 400 g potato extract boiled in 1 l of aquadest, 7 g bacto agarose, and 20 g dextrose. next, the fungal isolates were grown in 50 ml potato dextrosa broth (pdb) medium (37 °c, 72 h, 120 rpm -1 min ) to analyze the lignocellulolytic activity. extraction process was done using buffer phosphate 0.2 m (ph 7) with ratio of culture : buffer phosphate (1:2) (v/v). then, it was centrifuged using kubota high speed refrigerated centrifuge model 6500, capacity (6 x 500 ml), and dimension (500 x 740 x 940 mm) with parameter (20 000 x g, 10 min, 0-4 °c). crude lignocellulolytic enzyme and iaa hormone were isolated on supernatant fraction. next, it was filtered using whatman filter paper grade 41 (diameter porous 20-25 µm) to get crude extracts of laccase, cellulase, xylanase enzymes, and iaa hormone. assay of laccase enzyme activity was conducted based on previous report (bourbonais and paice, 1990). three hundred seventy five µl sample of crude extract containing laccase enzyme was mixed with 375 µl (1.8 mm) abts (2,2-azinobis-3-ethylbenzthiazoline-6sulfonate) in sodium acetic buffer (ph 4.5). the mixture was then incubated in water bath at 37 °c for 10 min. enzymatic reaction was stopped using 250 µl 1 % (b/v) sds (sodium dodecyl sulphate). then, it was measured by using spectrophotometer uv-vis (λ=420 nm). one international unit of laccase enzyme activity was defined as the total laccase enzyme that can oxidize 1 µmol abts each minute. assay of cellulase and xylanase enzyme activity was conducted based on previous report (miller 1959). one hundred twenty five (125) μl sample of crude extract containing cellulase enzyme was mixed with 0.5 % (b/v) cmc (carboxy methyl celullose) liquid substrate in 0.05 m acetic acid buffer ph 5. then, it was incubated in water bath at 37 °c for 10 min. later, 500 μl dns (3,5-dinitro salisilic acid) was added. then, the mixture was heated in water bath (100 °c, 5 min) to stop enzymatic reaction. before measurement using spectrophotometer uv-vis (λ= 540 nm), 5 ml aquadest was added to dilute th sample. one international unit of cellulase enzyme activity was defined as the total μmol product of glucose as the volume 7, 2013 microbiol indones 193 results of cellulase enzyme hydrolysis each minute. for xylanase, 150 μl crude xylanase extract was mixed with 0.5% (b/v) suspension of beechwood xylan in 0.05 m acetic acid buffer ph 5. then, the mixture was incubated in water bath at 37 °c for 10 min. later, 200 μl dns (3,5dinitro salisilic acid) was added. then, the mixture was heated in water bath (100 °c, 5 min) to stop enzymatic reaction. two ml aquadest was added to dilute the sample before being measured with spectrophotometer uv-vis (λ=540 nm). one international unit of xylanase activity was defined as the total μmol xylose produced from hydrolysis by xylanase enzyme each minute. assay of indole acetic acid (iaa) hormone was conducted based on previous report (gordon and webber 1950). zero point five ml crude extract of iaa hormone was mixed with 1 ml salkowski reagent (0.5 m fecl .6h o in h so ). the mixture was then 3 2 2 4 incubated in dark room for 30 min until color is formed. then, the absorbance was measured using spectrophotometer uv-vis (λ=530 nm). concentrationiaa hormone was calculated by linear regression analysis using iaa standard curve. the standard curve was made using varying concentration of iaa from 0.005 -1 mg ml to 0.050 mg ml . in general, all 14 fungal isolates showed low cellulase and xylanase activities. nevertheless, their -1 laccase activities and concentrations of iaa hormone differ from one another. fungal isolate jpf 2 had the -1 highest cellulase activity (0.502 u ml ) (table 1). this results was higher than cellulase activity expressed by -1 aspergillus sp. (0.04 u ml ), fusarium sp. (0.05 u -1 -1 ml ), aspergillus flavus (0.34 u ml ), and -1 botryotrichum sp. (0.0098 u ml ) (table 1) as reported by kerem et al. (1992). however, it was still lower than the celllulase activity showed by -1 trichoderma reesei (1.66 u ml ) and aspergillus -1 niger (1.69 u ml ) (table 1) grown on rice straw medium (ahamed and vermette 2008). fungal isolate jpf 5 showed the highest xylanase -1 activity (0.560 u ml ). it was still lower than that of aspergillus niger grown on rice straw medium, of -1 which the activity was 15.33 u ml (da silva et al. 2005). the highest laccase activity was shown by -1 fungal isolate jpf 14 (1.767 u ml ), followed by -1 fungal isolate jpf 4 (1.691 u ml ), of which the activity was still higher than that of omphalina (1.162 -1 u ml ) isolated from empty bunch of palms (kruus 2000). nevertheless, the activities shown by the fungal isolates were lower than that of marasmius sp. (4.560 -1 -1 u.ml ) and pleurotus ostreatus (4.394 u ml ), which were isolated from industrial pulp paper (couto and toca herera 2007; kirk and farrel 1997). fungal isolate jpf 14 not only showed the highest fungal isolate cellulase activity -1 (u ml ) xylanase activity -1 (u ml ) laccase activity -1 (u ml ) iaa hormone concentration -1 (mg ml ) jpf 1 0.340 0.412 0.988 5.34 jpf 2 0.502 0.476 0.654 4.23 jpf 3 0.344 0.464 1.467 5.17 jpf 4 0.380 0.493 1.691 5.48 jpf 5 0.423 0.560 0.756 5.20 jpf 6 0.353 0.458 0.935 4.00 jpf 7 0.315 0.431 0.965 3.91 jpf 8 0.438 0.527 0.969 5.64 jpf 9 0.387 0.533 1.113 5.53 jpf10 0.302 0.453 1.603 3.98 jpf 11 0.385 0.508 0.403 4.09 jpf 12 0.473 0.541 0.889 6.11 jpf 13 0.243 0.479 1.281 4.84 jpf 14 0.371 0.482 1.767 6.78 table 1 cellulase, xylanase, laccase activities and iaa hormone concentration expressed by 14 fungal isolated from spent champignon (agaricus sp.) substrate microbiol indones194 setiarto and saskiawan pda (potato dextrose agar) medium and incubated at room temperature for 5 d. to make starter inoculant f0, fungal isolates were transferred from pda medium -1 into bean sprout extract-sugar (60 g l ) medium and incubated at room temperature for 7 d. then, the starter inoculant f0 was transferred again into sterile cassava dregs (onggok) to make starter inoculant f1 and incubated for 14 d. after incubation, it was dried and mixed again with sterile cassava dregs with ratio (1:20) (w/w). then, it was incubated for another 14 d to make starter inoculant f2, which will be used to inoculate rice straw (spent champignon substrate) to produce organic fertilizer. population density of the starter inoculant f2 was estimated by using tpc (total plate count) method (table 2). the population density of inoculant f2 made from isolates jpf 2 and jpf 13 was 5 -1 5 5.6 x 10 cfu ml , fungi isolate jpf 5 was 4.6 x 10 -1 5 -1 cfu ml , and jpf 14 was 2 x 10 cfu ml . tpc 3 method was performed by serial dilution factors 10 , 4 5 6 10 , 10 , and 10 . tpc (total plate count) method indicated that the total microbial population on organic fertilizer starter 5 -1 inoculant f2 was between 2 x 10 cfu ml and 5.6 x 5 -1 10 cfu ml . microbes growing on starter inoculant f2 of organic fertilizer was dominated by fungi isolate. so, it can be assumed that there is no contamination on the production process of organic fertilizer starter inoculant. lignocellulolytic fungi was applied as starter to produce compost organic fertilizer from rice straw (chang et al. 2012). laccase, cellulase, and xylanase enzymes are very important in composting to produce organic fertilizer from rice straw. laccase enzyme helps to degrade lignin from rice straw. cellulase and xylanase enzyme have an important role main role in hydrolyzing cellulose and hemicelluloses, respectively, to become glucose and xylose (liu et al. 2012). glucose and xylose are sources of carbon and hydrogen for fungi, which are essential nutrition source to induce plant growth. in addition, there is also another important reason why fungal is used as starter for organic fertilizer, that is the fungal ability to produce endogenous auxin hormone such as indol acetic acid (iaa) hormone (ahmad et al. 2005). combination of lignocellulolytic enzyme and iaa hormone will produce organic fertilizer that is nutrition rich and containing high concentration of growth hormone regulator, which is necessary to increase the soil fertility and can accelerate the growth of roots, branches, bars, and leaves (isikhuemhen and mikiashvilli 2012). laccase activity, but also produced the highest -1 concentration of iaa hormone (6.78 mg ml ) in comparison to the other fungal isolates. the second best iaa producer is jpf 12 with iaa hormone -1 concentration 6.11 mg ml . all 14 fungal isolates isolated from spent champignon substrate produced with high concentrations of iaa hormone between -1 -1 3.91 mg ml and 6.78 mg ml . this was much higher than the amount produced by rhyzobium sp. (14.40 μg -1 ml ), which formed symbiosis with plant root, and -1 pseudomonas putida (1.1225 μg ml ) (levean and lindow 2004; ahmad et al. 2005). fungal isolates having high activity of lignocellulolytic enzyme and iaa hormone were characterized by measuring the biomass dry weight at varying temperature and ph.m cultivation was performed on -1 shaker incubator (72 h, 100 rpm min ) with ph variation from 4.0 to 8.0 (with interval ph 1.0) and temperature variation between 20 °c and 60 °c (with interval temperature 10 °c). after incubation, the fungal biomass was separated from medium by centrifugation (20 000 x g, 10 min, 0-4 °c) using kubota high speed refrigerated centrifuge model 6500, capacity (6 x 500 ml), dimension (500 x 740 x 940 mm) followed by filtration using whatman filter paper grade 41 before being weighed. the optimum growth temperature and ph of the lignocellulolytic fungi is defined as the condition giving the best dry weight. characterization of optimum growth ph and temperature of the selected lignocelullolytic fungi (isolates jpf 2, jpf 5, jpf 13, and jpf 14) was conducted by measuring the cellular dry weight (fig 1,2). cultivation 4 strains of lignocellulolytic fungi was -1 performed on shaker incubator (72 h, 100 rpm min ) with ph variation from 4.0 to 8.0 (with interval ph 1.0) and temperature variation between 20 °c and 60 °c (with interval temperature 10 °c). fungi isolate jpf 2 grew optimum at temperature of 30 °c with weight of biomass cell 1.3985 g and ph 5 with weight of biomass cell 1.0887 g. then, fungi isolate jpf 5 grew optimum at temperature of 20 °c with weight of biomass cell 1.3584 g and ph 5 with weight of biomass cell 1.5375 g. meanwhile, fungi isolate jpf 13 grew optimum at temperature of 30 °c with weight of biomass cell 1.1986 g and ph 8 with weight of biomass cell 1.9778 g. later, fungi isolate jpf 14 grew optimum at temperature of 20 °c with weight of biomass cell 1.2978 g and ph 6 with weight of biomass cell 0.9113 g. for production of lignocelullolytic fungi starter inoculant for organic fertilizer, the experiments was conducted as follow. fungal isolates were cultivated on volume 7, 2013 microbiol indones 195 references ahamed ap, vermette. 2008. culture-based strategies to enhance cellulase enzyme production from trichoderma reesei rut-c30 in bioreactor culture conditions. biochem eng j. 40(3):399-407. doi:10.1016 /j.bej.2007.11.030. ahmad f, ahmad l, khan ms. 2005. indole acetic acid production by the indigenous isolates of azotobacter and pseudomonas in the presence and absence of acknowledgments author extends full gratitude for all researchers and technicians at microbial biochemistry laboratory research center for biology lipi, who had supported the ongoing of this project. this research activity was funded by project dipa pn v 2012 microbiology division, research center for biology, indonesian institutes of sciences. 2.5 2 1.5 1 0.5 0 w ei g h t b io m as s fu n g i ce ll ( g r) 0 2 4 6 8 10 ph variation w ei g h t b io m as s fu n g i ce ll ( g r) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10 20 30 40 50 60 70 temperature variation (°c) fig 2 characterization for optimum temperature for growth of fungi isolates jpf 2 ( ), jpf5 ( ), jpf 13 ( ), and jpf 14 ( ). fig 1 characterization for optimum ph for growth of fungi isolates jpf 2 ( ), jpf5 ( ), jpf 13 ( ), and jpf 14 ( ). fungi isolates -1 results of tpc (cfu ml ) average tpc results -1 (cfu ml ) 1 2 3 jpf 2 5 7 x 10 4 x 10 5 6 x 10 5 5.6 x 10 5 jpf 5 3 x 10 5 6 x 10 5 5 x 10 5 4.6 x 10 5 jpf 13 5 x 10 5 8 x 10 5 4 x 10 5 5.6 x 10 5 jpf 14 2 x 10 5 3 x 10 5 1 x 10 5 2 x 10 5 table 2 population density of organic fertilizer starter inoculant f2 calculated based on tpc method microbiol indones196 setiarto and saskiawan dixon m, webb ec. 1979. enzyme. third edition. new york: academic press. gordon sa and weber rp. 1997. colorimetric estimation of indole acetic acid. plant physiol. 26:192-195. doi:10.1104/pp.26.1.192. hanafiah ka. 2005. biologi tanah: ekologi dan mikrobiologi tanah. 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[principle of biochemistry part 1]. alih bahasa maggy t. jakarta: erlangga. levean jh, lindow se. 2004. utilization of plant hormone indole acetic acid for growth by pseudomonas putida strain 1290. american society for microbiology. 1(5):2365-2370. linko m, vilkari l, suiko mi. 1984. hydrolysis of xylan and fermentation of xylose to ethanol. biotechnol adv. 2(2):233-252. doi:10.1016/0734-9750(84)90007-7. liu d, zhang r, yang x, wu h, xu d, tang z, shen q. 2012. thermostable cellulase production of aspergillus fumigatus z5 under solid-state fermentation and its application in degradation of agricultural wastes. international biodet biodegrad. 65(5):717-725. doi:10.1016/j.ibiod.2011.04.005. miller gl. 1959. use of dinitrosalicylic acid reagent of determination of reducing sugar. anal chem. 31(3):246248. doi:10.1021/ac60147a030. tryptofan. turk j biot.29:29-34. alexopolous cj, mims sw, and blackwell m. 1996. introductory mycology, 4th ed. new york: john wiley and sons, inc. arantes v, milagres amf, filley tr, goodell b. 2012. lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic fenton-based reactions. j ind microbiol biotechnol. 38(4):541-555. doi:10.1007/s10295-010-0798-2. aryantha in, lestari dp, pangesti npd. 2002. mikrobia penghasil fitohormon. [microbes producer phytohormone]. bandung: institut teknologi bandung. arshad m and frankerburger wt. 1991. microbial production of plant hormones. plant soil. 133(2):1-8. doi:10.1007/bf00011893. bhat mk, bhat s. 1997. cellulose degrading enzymes and their potential industrial applications. biotechnol adv. 15(3-4):583-620. doi:10.1016/s0734-9750(97)00006-2. bourbonnais r, paice mg, reid di, lanthier p, yaguchi m. 1995. lignin oxidation by laccase isozymes from trametes versicolor and role of the mediator 2,2’azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. appl environ microbiol. 61:1876-1880. bourbonnais r dan paice mg. 1990. demethylation and delignification of kraft pulp by trametes versicolor laccase in the presence of abts. applied microbial technol. 36(6):823-827. doi:10.1007/bf00172202. bric jm, bostock rm, silverstone se. 1991. rapid in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane. appl environ microbiol. 57(2):535-538. chang aj, fan j, wen x. 2012. screening of fungi capable of highly selective degradation of lignin in rice straw. int biodeter biodegr. 72: 26-30. doi:10.1016/j.ibiod.2012. 04.013. couto sr, toca herrera jl. 2006. industrial and biotechnological applications of laccases: a review. biotechnol adv. 24(5):500 -513. doi:10.1016/j.biotechadv.2006.04.003. couto sr, toca hererra jl. 2007. laccase production at reactor scale by filamentous fungi. biotechnol adv. 25(6):558-569. doi:10.1016/j.biotechadv.2007.07.002. da silva r, lago es, merheb cw, machione mm, park yk, gomes e. 2005. production of xylanase and cmcase on solid state fermentation in different residues by thermoascus auranticus miehe. braz j microbiol. 36(3):235-241. doi:10.1590/s1517-838220050003000 06. deacon jw. 1997. modern micology. new york:blackwell science. dewi. 2002. hidrolisis limbah hasil pertanian secara enzimatik. [hydrolysis agricultural product waste using enzyme]. akta agrosia. 5(2):67 -71. volume 7, 2013 microbiol indones 197 1: 192 2: 193 3: 194 4: 195 5: 196 6: 197 yunianto et al25042012 issn 1978-3477, eissn 2087-8575 vol 6, no 1, march 2012, p 23-29 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.4 isolation and identification of endophytic fungi from srikaya plants (annona squamosa) having potential secondary metabolites as anti-breast cancer activity 1, 3 2 2 prasetyawan yunianto *, syofi rosmalawati , indra rachmawati , 3 1 wahyudi priyono suwarso , and wahono sumaryono 1 center for pharmaceutical and medical technology, bppt, th bppt building ii 15 floor, jalan mh thamrin 8, jakarta 10340, indonesia 2 biotechnology research center, bppt , puspiptek building 630, serpong, tangerang selatan 15314, indonesia 3 department of chemistry, faculty of mathematics and natural science, universitas indonesia, depok 16424, indonesia annonaceous acetogenin was extracted from annona squamosa (srikaya) seeds. it has cytotoxic activity against cancer cells and lower toxicity compared to other cancer drugs. endophyte from annonaceae is expected to have similar extracted metabolites to the host, thus increasing the economic value. this research is a preliminary study to obtain active compounds with potential as anti-cancer agents from endophytic fungi of srikaya plants. four endophytic fungal strains were isolated from srikaya plants (annona squamosa) and identified based on 28s rdna sequence. the isolates are sky ii.3.1, sky i.1.2, sky ii.3.2, and sky iii.3.1, and have similarity with fusarium sp. vega760, fusarium sp. nrrl 22354 nrrl223, nectria rigidiuscula, and fusarium sp. bol35, respectively. the identified isolates were fermented in liquid media for three weeks. the liquid and mycelium were extracted using ethyl acetate. whole extract of each fermented isolate was partitioned and evaporated to obtain ethyl acetate extract. cytotoxicity assay of ethyl acetate extract was carried out at level 100 ppm by methyl thyazole tetrazolium (mtt) viability test towards mcf-7 (breast cancer cell). the result indicated that each ethyl acetate extract could inhibit the viability of cell mcf-7 with 11.34 %, 99.78 %, 91.48 %, and 96.84 %, for sky ii.3.1, sky i.1.2, sky ii.3.2, and sky iii.3.1 respectively. based on the results of cytotoxicity assay on mcf-7 breast cancer cells, endophytic fungi isolates sky i.1.2, sky ii.3.2, and sky iii.3.1 are potential as sources of anti-breast cancer compounds. key words: 28s rdna, annona squamosa, endophytic fungi, mtt viability test senyawa acetogenin yang diekstrak dari biji srikaya menunjukkan potensi sitotoksik terhadap sel kanker, dan toksisitasnya lebih rendah dibandingkan dengan obat kanker lainnya. endofit dari srikaya diharapkan memiliki metabolit yang sama dengan senyawa hasil ekstraksi dari inangnya, sehingga lebih ekonomis daripada mengekstraksi dari bijinya. penelitian ini merupakan studi pendahuluan untuk mendapatkan senyawa aktif yang mempunyai potensi sebagai anti kanker yang berasal dari metabolit sekunder jamur endofit tanaman srikaya. empat galur jamur endofit telah berhasil diisolasi dari tanaman srikaya (annona squamosa) dan diidentifikasi secara genetik menggunakan metode 28s rdna. hasil identifikasi menunjukkan bahwa isolat jamur sky ii.3.1, sky i.1.2, sky ii.3.2, dan sky iii.3.1 mempunyai kemiripan tinggi dengan masing-masing fusarium sp. vega760, fusarium sp. nrrl 22354 nrrl223, nectria rigidiuscula, dan fusarium sp. bol35. selanjutnya empat isolat jamur tersebut difermentasi dalam media cair selama tiga minggu, dan dilakukan ekstraksi baik terhadap media cair maupun miseliumnya dengan menggunakan etil asetat. hasil ekstraksi dari masing-masing isolat, dipekatkan dan dipisahkan lebih lanjut, sehingga dihasilkan ekstrak etil asetat. uji sitotoksisitas ekstrak etil asetat dilakukan masing-masing dengan konsetrasi 100 ppm mengunakan metode methyl thyazole tetrazolium secara viabilitas pada sel kanker payudara mcf-7. hasilnya menunjukkan bahwa ekstrak etil asetat dari masing-masing isolat secara berurutan dapat menghambat pertumbuhan sel mcf-7 sebesar 11,34%, 99,78%, 91,48%, dan 96,84%. berdasarkan seleksi dengan uji sitotoksisitas terhadap sel mcf7, maka yang layak untuk dilakukan fermentesi lebih lanjut untuk mendapatkan senyawa anti kanker payudara adalah isolat jamur sky i.1.2, sky ii.3.2, dan sky iii.3.1. kata kunci : 28s rdna, annona squamosa, jamur endofit, uji mtt *corresponding author; phone: +62-21-7560536, fax: +62-21-7566922; e-mail: pyunianto@gmail.com of co-evolution or genetic transfer between the host plant and the endophytes, which is highly dependent on environmental conditions where the host lives (tan and zou 2001). bioactive natural compounds produced by endophytes have promising potential applicability in medicine, agriculture, and industry (joseph and priya 2011). million dollars have been generated after the first endophytic microbes are bacteria or fungi that live in plant tissue for a certain time period, forming colonies without harming its host. many higher plants may contain several endophytic microbes capable of producing secondary metabolites. it is allegedly a result 24 yiunianto et al. microbiol indones discovery of taxol (paclitaxel) as an anti-cancer drug that endophytic fungi isolated from the plant taxus brevifolia nutt (wani et al. 1971). currently, several other endophytic species have been isolated from various taxus species such as taxus baccata, t. cuspidata sieb. the compounds also show efficacy as anti-tumor (strobel and daisy 2003). srikaya also called sugar apple or sweetsop (annona squamosa) one of the known species of annona fruits. based on ethnobotanical experience, part of the sugar apple in the form of roots, leaves, seeds, and bark have been utilized as traditional medicine for efficacious anti-inflammatory, antidepressant, astringent, laxative to treat intestinal worms cases (anthelmintic), abortivum, insecticide, and tonic. this plant contains alkaloids, generally of asporfin type (anonaine) and bisbenziltetrahydroisoquinoline (reticuline). the seeds contains derivatives of polyketide bistetrahydrofuran; acetogenin (squamostatin c, d, anonaine, annonacine a, anonin 1, iv, vi, viii, ix, xvi, squarnostatin a, bulatacin, bulatacinon, squamon, neoanonin b, neo desacetylluraricin, neo reticulacin a, squamosten a, asmicin, squamocin, annonacine, anonastatin, and neoanonin) (wu 2007; hai et al. 2003). according to landolt et al.(1995) annonaceae acetogenin is a new compound and natural material having potential activity as antitumor, pesticide, and bioactive that can act as a barrier to electron transport in mitochondria (mclaughin and david 2001). chih and yang (2008) found a new compound that have a cytotoxic potential against cancer cells. it is a derivative of mono-tetrahydrofuran (thf), an annonaceae type acetogenin extracted from sugar apple seeds. research concerning annonaceous acetogenin as anticancer drug is still being carried out in the united states pharmaceutical industry. according to hollingworth et al. (1994), up-john corporation has conducted in vivo testing of annonaceous acetogenin compounds and the results showed that they were active against mice l-1210 leukemia. therefore, annonaceous acetogenin has a greater potential than taxol due to its low toxicity. cancer's prevalence tends to increase from time to time. according to an estimation by globocan iarc, in 2002 the breast cancer incidence was 26 per 100 000 women and cervical cancer was 16 per 100 000 women (karrosi et al. 2009). thus new drugs from natural resources such as plants or their endophytic microbes are still required. endophytes from annonaceae is predicted to contain similar metabolites as those extracted from the host, so it is expected that the extraction from the endophytes is more economical than extraction from the seeds of the host fruit. this research is a preliminary study to obtain active compounds potential as anticancer from endophytic fungi of sugar apple plants. we screened endophytic fungi from sugar apple plants as potential anti-cancer based on the observation of viability of breast cancer cells (mcf-7) using in vitro assay with mtt method. the potential fungi were isolated and identified using 28s rdna. materials and methods isolation of endophytic fungi. twigs of sugar apple plants from tangerang, banten, indonesia, were washed with distilled water, cut into 1-2 cm. the surface was sterilized by three times immersion, first in -1 ethanol 70% v v for 1 min, second in 5.25% naocl solution for 5 min and the last, in 70% ethanol solution -1 v v for 0.5 min. the outer skin is discarded with a sterile knife and placed on an agar medium in a petri dish containing dried host plant leaves and antibiotics. the media composition was 15 g agar, 15 g dry powder of the host plant leaves, 0.2 g chloramphenicol, and up to 1 l distilled water (rusman 2006). incubation was carried out at room temperature for 1-3 weeks depending on the fungal growth rate. the fungal colony then moved to a new media with the composition of 15 g agar, 15 g malt extract, and up to 1 l of distilled water, and adjusted to ph 7.4 to 7.8. liquid fermentation of isolated fungi. a single isolate of endophytic fungus (the macroscopic and microscopic) is used as mother culture for cultivation. this isolate was incubated for 3 to 7 days before being used as inoculums of the starter for the subsequent cultivation or fermentation. liquid fermentation was carried out in 150 ml of wickerham medium (1 l of distilled water consisted of 3 g yeast extract, 3 g malt extract, 5 g peptone, 20 g glucose monohydrate, with ph 7.2 to 7.4) in 300 ml erlenmeyer flask of the culture surface at room temperature, between 14-21 d (ebel et al. 2006), with -1 amount of starter about 5% v v . ethyl acetate extracts preparation. a 250 ml ethyl acetate was added into each fungal culture in an erlenmeyer flask and the mixture was kept overnight to ensure that the fungal cells died. the mixture was then sonicated three times for 15 minutes (for cell destruction) and then filtered using büchner vacuum. volume 6, 2012 microbiol indones 25 the ethyl acetate phase was then separated from water phase (medium) using separation funnel. to remove the remaining salts and other polar constituents, the ethyl acetate phase was washed with water two times. just after evaporation, the ethyl acetate extracts was diluted in 90% methanol and extracted with n-hexane to remove fatty acids and other non-polar constituents. after that the methanol phase was evaporated. at the same time, the water phase (medium) was extracted with water-saturated nbutanol to collect the polar constituents. dna isolation. fungal dna isolation and ® purification was performed using prepman ultra s a m p l e p r e p a r a t i o n r e a g e n f r o m a p p l i e d biosystems®. the edge of filamentous fungus colony from a culture plate was suspended in 100 µl of prepman ultra sample preparation reagent in microcentifuge tube. the sample was heated in heat o block for 10 min at 100 c and then cooled at room temperature for 2 min. after centrifugation at 15 000 x g for 2 min, supernatant was ready for pcr. pcr amplication and dna sequencing. the dna isolated from pure fungal isolates was then used as a template for pcr amplification of the 28s ribosomal gene using the universal fungal primers nl1 and nl4 (o'donnell 1993; ainslie and cameron 2007). tm the pcr was carried out using takara la taq from tm takara bio inc. contains takara la taq dna polymerase, pcr buffer (with mgcl ) and dntps. 2 primer nl1 (5'-gcatatcaataagcggaggaaa ag-3') and nl 4 (5'-ggtccgtgtttcaagacggtm -1 3') were mixed with takara la taq and 200 ng µl dna template. the pcr reaction was the performed using the o following program: initial denaturation for 2 min at 98 c, followed by 30 cycles of amplification (denaturation for o o 20 s at 98 c, annealing for 20 s at 52 c, and extension for o o 2 min at 72 c) and final extension of 4 min at 72 c. the pcr products were gel electrophoresed in 1% agarosegel-electrophoresis for 25 min in tae buffer. the agarose gel was then stained using 1% sybr safe (invitrogen). a 600 bp stained dna fragment was then excised from the agarose gel and then purified using geneaid gel extraction kit. the amplified fungal dna was then submitted for sequencing using genetic analyzer 3130. the sequencing cycle was performed as follows: initial o denaturation at 96 c for 3 min, 25 reaction cycles o o (denaturation for 10 s at 96 c, annealing for 5 s at 50 c, o and extension for 4 minutes at 60 c). nl1 and nl4 primers were used for sense and antisense sequencing, respectively. then, dna sequence allignment was performed using clustaix . the phylogenetic tree was constructed by comparing the base sequences to the data in genebank with a help of blast-algorithmus and visualized using tree view 1.6.6. cytotoxicity test in vitro with methyl thyazole tetrazolium (mtt) assay. the ethyl acetate extract fraction was tested in vitro for its cytotoxicity against cancer cells using mcf-7 cell line (breast cancer cell line) using mtt assay based on the protocol from cancer chemoprevention research center, universitas gajahmada. the mtt assay is commonly used to evaluate cell survival, based on the ability of viable cells to convert mtt, a soluble tetrazolium salt [3-(4,5-dimethylthyazole-2-yl)-2,5 diphenyl-tetrazolium bromide], into an insoluble formazan precipitate, which is quantified by spectrophotometry. mcf-7 cell was treated by each ethyl acetate extract with concentration 100 ppm (µg -1 ml ) in 96-well tissue culture dishes and incubated -1 with mtt (5 mg ml ) for 4 h. mcf-7 cells not treated by the extract was used as a control . the cells were then solubilized in 100 µl of dmso. the amount of mtt dye reduction was calculated based on its absorbance at 570 nm. cell viability in treated cells was expressed as the amount of dye reduction relative to that of untreated control cells. the wells which contained only medium and 10 ml of mtt were used as blanks for the plate reader. results isolation of endophytic fungi. four endophytic fungi were isolated from srikaya plants (annona squamosa l.). isolations of the fungi were performed based on observation on macrocospic and microscopic level. the fungal isolates show different morphological characteristic (table 1 and fig 1). pcr amplification and identification of endophytic fungus. the 28s rdna regions were amplified using the universal fungal primers nl1 and nl4 and isolated dna as templates by for. as table 1 the morphology of endophytic fungi isolated from the twigs of sugar apple on agar plate with media pda isolat code co lor and form o f colonies on agar p late sky ii.3.2 redish, smooth sky iii.3.1 white, coarse sky i.1.2 whiteyellowish, sky ii.3.1 white, smooth isolat code identification highest similarity (%) sky i.1.2 fusarium sp nrrl 22354 nrrl223 98 sky iii.3.1 fusarium sp bol35 100 sky ii.3.2 nectria rigidiuscula 100 sky ii.3.1 fusarium sp vega760 96 26 yiunianto et al. microbiol indones expected, all pcr products showed only a single visible band about 600 bp on agarose gel electrophoresis (fig 2). identification of the endophytic fungi have been conducted from d1-d2 region sequence of 28s rdna. phylogenetic relationships were inferred based on the sequences of the d1-d2 regions in 28s rdna of four fungal isolates. the phylogenetic analysis showed relationshipswith 31 strains cited from genbank (fig 4). phylogenetic tree indicated that the four fungal isolates consist of three groups, the genera nectria, clyndrocladium, and fusarium with similarity 96-100 %. cytotoxicity assay in vitro with mtt method on breast cancer cells mcf-7. cytotoxicity assay of ethyl acetate extract was carried out at level 100 ppm by mtt viability test towards mcf-7 (breast cancer cell). fig 3 showed that the ethyl acetate extract from secondary metabolites of sky i,1.2, sky ii.3.2 and sky iii.3.1 (except sky ii.3.1) could inhibit more than 50 % growth of mcf-7 cell at extract -1 concentration 100 ppm (µg ml ) in comparison to the untreated control cells (negative control). discussion based on morphological differences, four fungal isolates were obtained in this study and identified using their 28s rdna sequence (table 2). in this study, we sequenced about 600 nucleotides from the 5' end of the large subunit rdnas from each isolated fungus and used phylogenetic analyses of these sequences to evaluate species assignments to the various genera. was used and developed by o'donnell (1996) to classify fusarium or fungi. this method is widely used to determine other fungi, like the identification of ascomycetous yeasts by kurtzman (1997), yeast species by lachance (2003) and black yeast by ainsle (2007). in the phylogentic relationship analysis, we compared the rdna sequences with the sequences determination of the d1-d2 regions from 28s rdna iii.3.1 ii.3.2 i.1.2 ii.3.1 fig 1 morphology isolates of endophytic fungi from twig sugar apple (annona squamosa). 700 bp 500 bp 1 2 3 4 5 fig 2 pcr amplification of sequence dna by nl1 and nl4 primer (lane1: ladder, lane 2,3,4,5: sky i.1.2, sky ii.3.2, sky ii.3.1 and sky iii.3.1.) table 2 identification of isolates fungus based on their 28s rdna. isolated the endophytic fungi of anonna squamosa growing in one location (tangerang, indonesia). the ethyl acetate fraction extracts, instead of the butanol extracts as previously conducted by kumala et al. (2007), were screened for their cytotoxicity. the use of ethyl acetate extracts was intended to facilitate further isolation of single compound compared to the butanol extract, which is more polar. sreening of fungal isolates efficacy as anti-cancer was performed in vitro by cytotoxicity assay using mtt method. the mtt test was performed to measure the abilty of extracts at a concentration of 100 ppm (µg -1 ml ) to inhibit the growth of mcf-7 cancer cells. according to meyer et al. (1982), if an extract has an -1 lc less than 1000 ppm (µg ml ), it is considered 50 active. although swanson and pezzuto (1990) claimed that a compound is considered active, only when it has lc less than 20 ppm. in this study, the mtt test was 50 carried out at concentrations 100 ppm. the isolated fungi was considered potential as anti cancer, if treatment with the ethyl acetate extract showed percentage viability of mcf-7 cells less than 50 %, whichmeans that the extract can inhibit growth of mcf-7 cell more than 50 % at concentration extract on -1 100 ppm (µg ml ). screening of endophytic fungi as anti-cancer was conducted using extracts of secondary metabolites such as conducted by kumala et al. (2007) and karrosi et al. (2009). meanwhile, xiao lin et al. (2010) performed screening based on the sequence of bases from endophytic fungi that have a homologous sequence with primer that produces polyketide synthase (pks). the active anti-cancer compounds from the host are polyketides that are extracted from the seed of sugar ofseven fungal genera of (acremonium, phomopsis, colletotrichum, clyndrocladium, acremonium, nectria and fusarium) available from the gene bank. the results showed that two isolates belongs to the genus fusarium, and isolate sky ii. 3.2, which was identified as nectria, showed some genetical similarities with the species nectria rigidiuscula (fig 4). sky ii.3.1, although having some similarities with fusarium sp vega760, in the phylogenetic analysis was classified as group 5, genus clyndrocladium. these results are similar to research conducted by roberta et al (2006) who isolated endophytic fungi from leaves, stems and roots of 110 sweetsop and 90 soursop plants from pernambuco-brazil, and were identified as acremonium (10.34%), aspergillus (3.45%), chaetomium (3.45%), colletotrichum (10.34%), cylindrocladium (13.8%), fusarium (31.03%), glomerella (3.45%), nigrospora (6.9%), penicillium (6.9 %), and phomopsis (10.34%). meanwhile, xiao lin et al. (2010) had isolated 131 strains of endophytic fungi from the plant annona squamosa during different seasons in china. phylogenetic analysis showed that these fungi were distributed in 10 orders and 19 genera. they were mostly in the orders of diapothales and hypocreales. this confirmed that the endophytes of sugar apple plants are mainly from diapothales and hypocreales orders, in this phylogenetic study the four fungi isolated were of the order hypocreales. the different distribution patterns of fungal isolates into different orders between brazil and indonesia on one side and china on the other hand, confirmed that endophytes highly dependent on environmental conditions where the host lives (strobel 2003). in this study, we only microbiol indones 27volume 6, 2012 negative control sky i.1.2 sky ii.3.1 sky ii.3.2 sky iii.3.1 ce ll v ia b il it y % 120 100 80 60 40 30 0 -20 fig 3 effect of each ethyl acetate extract (100 ppm) on cytotoxicity in breast cancer cell mcf-7 as determined by the mtt cell viability assay. 28 yiunianto et al. microbiol indones apple fruit, so that endophytic fungi are expected to have the pks gene that can produce the same polyketide compounds as produced by their host plant. however, according to tan and zou (2001), anti-cancer compounds from endophytic fungi is not only polyketide, but could also be alkaloids, terpenoids, or phenol. this is relevant with the results of investigation by lu et al. (2008), who managed to isolate a tenmembered lactone, namely phomolide c, l-methoxy-8hydroxy-9.10-anthraquinone, 1.8-dihydroxy-anthraquinine-9, 10 (1.8. dha), cytospo-rone c and altiloxin a from phomopsis sp.b27, an endophytic fungal strain of annona squamosa. based on the results of cytotoxicity assays on mcf7 breast cancer cells (fig 3), endophytic fungal isolates of sky i.1.2, ii.3.2 and iii.3.1 are potential as producers of anti-cancer by fermentation. the potential compounds had been isolated using chromatography technique. to further continue this study, we plan to perform cytotoxicity assays of the isolated compounds fig 4 phylogenetic tree of endophytic fungal isolates from twigs annona squamosa. 0.05 volume 6, 2012 microbiol indones 29 on mcf-7 and cho (normal cell) cell lines, in also comparison to the commercially available anti-cancer drugs (cisplatin or doxo) as positive control. if the lc50 of the isolated compounds to mcf-7 and cho cell line is not significant compared to the positive control, then, they are potential as anti-cancer agents. references ainslie ef, cameron rc. 2007. symbiotic complexity: discovery of a fifth symbiont in the attine ant -microbe symbiosis. biol lett. 3(5): 501-504. doi:10.1098/rsbl. 2007.0253. chih cl, yang cl. 2008. mono-tetrahydrofuran annonaceous acetogenins from annona squamosa as cytotoxic agents and calcium ion chelators. j nat prod. doi:10.1021/ np0704957. ebel r. 2006. secondary metabolites from marine-derived fungi. in: proksch p, müller weg, editors. frontiers in marine biotechnology. norwich: horizon bioscience pr. p 73-144. guang xz, chen ry, zhang yc, yu d q. 2000. calamistrin e, the first annonaceous acetogenin with double bond in aliphatic chain from genus uvaria. chin chem lett . 11( 4): 341-342. hai h x, xiao y w, wang j d, liu , yang rz. 2003. a new cytotoxic acetogenin from the seeds of annona squamosa. chin chem lett. 14(6):588-590. hollingworth rm, ahammadsahib ki, gadelhak g, and mclaughlin jl. 1994. new inhibitors of complex i of the mitochondrial electron transport chain with activity as pesticides. biochem soc trans. 22(1):230-233. joseph b, priya rm. 2011. bioactive compounds from endophytes and their potential in pharmaceutical effect: a review. am j biochem mol biol. (8):291-309. doi: 10.3923/ajbnb.2011.291.309. karrosi at, poniah s, linar z, udin. 2009. anti breast cancer activity of ethyl acetat extract of fermentation broth employing endophitic taxus sumatrana isolates. j kimia terapan indonesia. 11(1):22-25. kumala s, utji r, pratiwi s, kardono lbs. 2007. isolation of endophytic fungi from brucea javanica l.(merr.) and cytotoxic evaluation of their n-butanol extract from fermentation broth. pak j biol sci. 9: 825-832. doi: 10.3923/jm.2007.625.631. kurtzman cp, robnet cj. 1997. identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 59 end of the large-ubunit (26s) ribosomal dna gene. j clin microbiol. 35(5):1216– 1223. lachance ma, daniel hm, meyer w, prasad, gautam sp, boundy-mills k. 2003. the d1/d2 domain of the largesubunit rdna of the yeast species clavispora lusitaniae is unusually polymorphic. fems yeast res. 4:253-258. doi:10.1016/s1567-1356(03)00113-2. landolt jl, ahammadsahib ki, hollingworth rm. 1995. determination of structure-activity relationships of annonaceous acetogenins by inhibition of oxygen uptake in rat liver mitochondria. chem biol interact. 98(1):1-13. lu ch, xiao l, shen ym. 2008. one new ten-membered lactone from phomopsis sp． b27， an endophytic fungus of annona squamosa． chin j nat med. 6: 0391-0394. doi: 10.3724/sp.j.1009.2008.00391. mclaughlin jl, david ch, inventors; purdue research foundation, assignee. 2001 june 5. selectively cytotoxic acetogenin compounds. united states patent. us 6,242,483. meyer bn, ferrigni nr, pulman je, jacobson, nicholas de, mclaughlin. 1982. brine shrimp: a convenient general bioassay for active plant constituents. plant med. 45:31-34. o'donnell k. 1996. progress towards a phylogenetic classification of fusarium. sydowia 48(1): 57-70. roberta los, jaqueline sl, barbosa e, cavalcante. 2006. fungos endofíticos em annona spp.: isolamento, caracterizaçãoenzimática e promoção do crescimento em mudas de pinha (annona squamosa l.) [endophytic fungi of annona spp.: isolation, enzymatic characterization of isolates and plant growth promotion in annona squamosa l. seedlings]. acta bot bras. 20(3): 649-655. doi:10.1590/s0102-330620060003 00015. rusman y. 2006. isolation of secondary metabolites from sponge-assosiated and plant-derived endophtic fungi. gottingen: cuvillier verlag gottingen. strobel ga, daisy b. 2003. bioprospecting for microbial endophytes and their natural products. microbiol mol biology rev. 67(4):491-502. doi : 10.1128/mmbr.67. 4.491-502.2003. swanson sm, pezzuto jm. 1990. bioscreening technique for cytotoxic potential and ability to inhibit macromolecule biosynthesis. in thompson eb, editor. drug bioscreening; drug evaluation techniques in pharmacology. new york: wch publishers inc. 273297. wani mc, taylor hl, aal me, goggon p, mcphail at. 1971. plant antitumor-agent iv,the isolation and structure of taxol, a novel antileukemic and anti-tumor. j am chem soc. 9:2325 – 2327. wu yc, inventor; advpharma, inc, assignee. 2007 may 29. cytotoxic annonaceous acetogenins from annona muricata. united states patent. us 7,223,792 b2. tan rx, zou wx. 2001. endophytes: a rich source of functional metabolites. nat prod rep. 18 (4): 448-459. doi : 10.1039/b100918o. xiao ly, huang j, zheng zh, su wj, qian xm, shen ym. 2010. endophytes from the pharma-ceutical plant, annona squamosa: isolation, bioactivity, identification and diversity of its polyketide synthase gene. fungal divers. 41 (1): 41-51. doi: 10.1007/s13225-010-0017-5. 1: 23 2: 24 3: 25 4: 26 5: 27 6: 28 7: 29 1.mi718-neti yuliana available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.8.1.1issn 1978-3477, eissn 2087-8575 vol 8, no 1, maret 2014, p 1-8 *corresponding author; phone/fax: +67-721-781498, email: yuliana_thp@unila.ac.id sweet potato flour is generally produced conventionally by drying of sliced peeled tubers followed by grinding and sieving to pass different mesh size. this process has disadvantages such as limited use in application, primarily in food system, and low whiteness index. sweet potato flour has viscosity profile, rehydration, and solubility that are less favorable than those of wheat flour. in addition native flour did not show any thermo pasting behavior when subjecting to amylograph brabender (yuliana and nurdjanah 2013). the native sweet potatoes flour has low viscosity that makes it just useful for the food that require lower viscosity (aprianita et al. 2009) in this research we tried to improve or modify sweet potato flour properties trough spontaneous fermentation of the fresh tuber, so that the flour is whiter and has broader application in various food products. previous studies showed lactic fermentation has been implemented in some flour and starch production, such as in the production of sweet potato starch (deng et al. 2013), sour cassava flour (putri 2011), cocoyam flour (oke and bolarinwa 2012) and rice flour (lu et al. 2005). mutwiri (2007) and kasim (2012) worked on naturally fermented sweet potato flour that was prepared by drying a fresh sweet potato pulp before fermenta sausages filling (mutwiri 2007). in african countries, lactic acid fermentation has been practiced to improve nutritional value and taste. for examples, amylolytic lab are mainly distributed in directly nigerian fermented meals (sanni et al. 2002), and traditional african fermented sorghum (yousif et al. 2010). lactic acid bacteria as dominant organisms in food fermentations will convert free sugars to lactic acid, produce amylolytic enzymes that degrade starch granules and hydrolyze the short chain amilose and amylopectin in amorphous region of starch granules. the degradation of starch granule by lactic acid during fermentation could change the porosity and surface et al. native sweet potato flour is usually has low whiteness index and limited application to food systems due to its inherent functional properties. therefore, it needs modification process to improve this property. in this study, sweet potatoes cubes were lactic spontaneously fermented for 120 h before being processed to flour to modify its properties. selected physico-chemical properties of flour were then determined and compared with the control (without fermentation). the results showed that lactic acid fermentation significantly caused more changes on flour properties. the lactic acid fermentation caused an alteration in the starch granules as evident by scanning electron microscopy. when compared to the control flour, spontaneous fermented flour had lower solubility, higher swelling power, and paste viscosity. the results suggested lactic spontaneous fermentation within 120 h period of time could provide a greater extent of flour modification. key words: modified sweet potato flour, spontaneous lactic acid fermentation tepung ubi jalar biasanya mempunyai indeks putih yang rendah dan sifat fungsional alami yang menyebabkan keterbatasan aplikasi dalam sistem pangan. dengan demikian tepung ubi jalar perlu dimodifikasi untuk memperbaiki sifat-sifat fungsionalnya. pada penelitian ini, potongan ubi jalar difermentasi laktat secara spontan selama 120 hari sebelum diproses menjadi tepung untuk memperbaiki sifat fungsionalnya. sifat fisiko kimia terpilih kemudian diukur dan dibandingkan dengan tepung hasil perlakuan yang tidak difermentasi (kontrol). hasil penelitian menunjukkan bahwa fermentasi asam laktat secara nyata menyebabkan perubahan sifat-sifat tepung. fermentasi menyebabkan perubahan granula pati seperti yang ditunjukkan pada gambar hasil scanning electron microscopy. jika dibandingkan dengan kontrol, tepung hasil fermentasi mempunyai kelarutan yang lebih rendah, kemampuan pembengkakan granula, dan viskositas pasta yang lebih tinggi. hasil ini mengindikasikan bahwa fermentasi secara spontan selama 120 jam dapat menghasilkan proses yang berdampak besar terhadap modifikasi tepung. kata-kata kunci: fermentasi asam laktat spontan, tepung ubi jalar termodifikasi effect of spontaneous lactic acid fermentation on physico-chemical properties of sweet potato flour neti yuliana*, siti nurdjanah, ribut sugiharto, and deary amethy department of agricultural product technology (thp), agriculture faculty, universitas lampung, jalan sumantri brojonegoro no 1, bandar lampung 35145, indonesia area of the granule and would modify the properties of flour. information about these physicochemical properties is important to determine the usability of fermented sweet potatoes flour on various food products. beside amylolitic enzymes, lactic acid bacteria also produce proteolitic enzymes as well as organic acids which degrade protein and inactivate polyphenol oxidase in sweet potatoes. lower protein content, inactivation of polyphenol oxidase together with low free sugar content as a result of the conversion of free sugar to lactic acid in fermented sweet potatoes flour may reduce the non enzymatic browning during flour drying. thus, it will result in increasing whiteness of sweet potatoes flour. materials and methods sweet potato fermentation. white-fleshed sweet potato samples were obtained from pasar gintung, a local market at bandar lampung, province of lampung-indonesia. the sweet potatoes were washed, 3 peeled, and cut into cubes (1x1x1 cm ). the potato cubes (40 g) were put into a 150 ml-fermenting container, then it was made up to 150 ml volume with the 3% brine solution. the fermenting containers containing the samples were pasteurized using a microwave oven (sharp) at high level setting for 10 min to reach temperature around 72-73 °c, and then they were left at room temperature to cool. the fermentation was conducted at room temperature (30 °c) for 120 h to let them reach completely fermentation. this was indicated by drop in ph to 4.0 and unique acid aroma of fermented sweet potatoes cube as described in previously author (yuliana et al. 2010). microbial growth during fermentation was evaluated by enumerating total of lactic acid bacteria (lab) using total plate count method. appropriate dilutions were placed on duplicate plates of mrs medium (oxoid) with 0.1% (w/v) caco . the cultures were incubated at 3 30 °c for 48 h. lab was identified by the presence of clear zones around the colonies; the experiment was designed in three replicates. the control (non fermented flour) was prepared by drying fresh cubes of sweet potatoes followed by milling and sieving. sweet potatoes flour production. after fermentation has been completed, the cubes were washed with running tap water until the water runs clear and then drained. the fermented cubes of sweet potatoes were dried in an oven at 65 °c overnight until the moisture content reaches approximately 12%. these dry 2 yuliana et al. microbiol indones fermented cubes were then ground into flour to pass through an 80 μm mesh sieve size. the same procedure was applied to dry cubes from fresh sweet potatoes without fermentation to make control sweet potatoes flour. the flour was packed in polythene bags and stored at ambient temperature of around 25±2 °c for further analysis. ph and ta. sweet potatoes flour samples (5 g) were weighed in triplicate into a beaker, mixed with 20 ml of distilled water. the resulting suspension stirred for 5 min and left to settle for 10 min. the ph of the water phase was measured using a calibrated ph meter (aacc, 2000). the ta was measured using titration method with phenolpthalein as indicator of end titration. morphology of sweet potatoes starch granules. the morphology of starch granules was evaluated by scanning electron microscope (jsm-6510lv sem). flour samples were suspended in 95% ethanol and mounted on circular aluminium stubs with doublesided sticky tape. the flour granules were evenly distributed on the surface of the tape, and the ethanol was allowed to evaporate. the samples were then coated with 12 nm gold, examined and photographed at an accelerating voltage of 15 kv with a magnification of 2000×. swelling power and solubility. swelling power and water solubility index (wsi) determinations were carried out in the temperature range 60-80 °c at 10 °c intervals. briefly, 0.5% flour suspensions were prepared in 15 ml tubes and heated in a water bath at 60, 70, and 80 °c for 30 min with constant agitation to avoid sedimentation. this was followed by centrifugation at 1000× g for 15 min at 20 °c. the sediment fraction was weighed and its mass related to the mass of dry starch was expressed as swelling power (w/w). the solubility was reported as the ratio of the weight of total soluble starch to the weight of dried sample. pasting properties of flour. the pasting properties of the flour were evaluated by using a micro visco amylo graph (brabender). flour suspensions (10%, w/w) were equilibrated at 30 °c for 1 min, -1 heated at 95 °c for 5.5 min, at a rate of 6 °c min , held at 95 °c for 20 min, cooled down to 50°c at a rate of 6 -1 °c min and finally held at 50 °c for 20 min. it was a programmed heating and cooling cycle. parameters recorded were pasting temperature (pt), peak viscosity (pv), minimum viscosity (mv), or trough viscosity (tv), final viscosity (fv), and peak time (ptime). breakdown viscosity (bv) was calculated as the difference between pv minus mv, while total setback viscosity (tsv) was determined as the fv minus mv. all determinations were performed in duplicate. flour whiteness. sweet potatoes flour whiteness was determined using a powder whiteness tester model c 100, kett electric laboratory. a 25 g sample of flour was weighted and put in the sample container. the top of container was then closed and container was inserted into the sample holder in the system. the values of whiteness were the average from three measurements. results ph and ta of flour. changes in ph and titratable acidity (ta) of flour are shown in table 1 and fig 1. fermentation was found to cause a gradual reduction in a ph from 6.49 to 4.5. concomitant with the drop in ph, there was a rise in ta of sp flours throughout the fermentation process from 0.02 % to 1.68 %. flour whiteness. fermentation improved the whiteness of sp flour (fig 2). fermented flour was observed to have higher whiteness index compared to that in control. solubility. sweet potato flour is difference in solubilitty. in general, fermented flour has lower solubility in spite of the temperature over 60 to 80 °c. swelling power. any significant change in swelling power of sp flour, as shown in fig 4. at 60 °c the swelling power of both treatments was very low, and this value increased when the heating reached 70 °c. the granules of control sweet potato flour swelled at a lower temperature (~73 °c) in comparison to those of spontaneous sweet potato flour, which swelled at ~75 °c. the swelling power of control sweet potatoes flours increased steadily with a temperature rise from 60 to 80 °c, as opposed to fermented sweet potato flour with a rapid change of swelling power in this temperature region. at 80 °c, the swelling power of the fermented sweet potato flour was greater than that of control flours. pasting properties. table 2 demonstrates the viscosity profile of different flour. the fermented flour samples had the higher maximum viscosity, break down and set back than those of control. however, the control had the higher peak time (10 min), and pasting temperature (93.87 °c) which may indicate different structural rigidity in comparison to fermented sweet potato flour. morphology of granule. fig 5 shows mild superficial corrosions on some starch granules by volume 7, 2013 microbiol indones 3 control spontaneous 8 7 6 5 4 3 2 1 0 p h 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 t a ( % ) control spontaneous fig 1 ph and titratable acidity (ta) of control (fresh sp) and spontaneous fermented sp flour. table 1 ph and titratable acidity (ta) at control (fresh sp) and spontaneously fermented sp flour treatment ta (%) ph control 0.02±0.005 6.49±0.63 spontaneous 1.68±0.16 4.5±0.61 microbiol indones4 yuliana et al. w h it en es s (% ) 0 10 20 30 40 50 60 70 80 90 control spontaneous fig 2 whiteness (%) of control and spontaneous fermented sp flour. 60 °c 70 °c 80 °c temperature s o lu b il it y ( g /g ) 25.00 20.00 15.00 10.00 5.00 0.00 60 °c 70 °c 80 °c temperature 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.0 s w el li n g p o w er ( g /g ) fig 3 solubility of control (fresh) and spontaneous fermented sp flour. : control and : fermented. fig 4 swelling power of control (fresh) and spontaneous fermented sp flour. : control and : fermented. volume 7, 2013 microbiol indones 5 during fermentation in this research was around 0.619 at day 1 to 7.803 (log 10 cfu) at day 5 (120 h). this could explain the apparent increase in lactic acid towards the end of fermentation accompanied by decrease in ph. fermentation was observed to improve the whiteness index of sp flour (fig 2). this can be ascribed to flour purification by spontaneous fermentation and the decrease of ash, protein and sugar content. it is generally accepted that the ash content, protein and free sugar are factor affecting the whiteness of flours such as those from cassava and rice (sobowale 2007; lu et al. 2005). during fermentation, bacteria would produce proteolityc which degraded protein in sweet potatoes, convert free sugar to lactic acid thus the content of protein and free sugar in fermented flour was lower than those at control. a higher protein and free sugar content in control flour may cause the non enzymatic browning during flour drying which result in darker color, thus fermentation. the spontaneous fermented samples were much smoother surface angle, etched and had very shallow pits but the control samples had no pits when examined using scanning electron microscopy. discussion fermentation was found to cause a gradual reduction in a ph. this result is in agreement with yuliana et al. (2013) and adebayo-oyetoro (2012) who reported that lactic acid fermentation causes a rapid drop in ph as in sweet potatoes cube and cassava tubes fermentation. spontaneous fermentation of carbohydrate-rich biomass such as sp, cassava, sorghum, and caper berries, is mainly lactic acid fermentation (yuliana et al. 2013; kakou et al. 2010; yousif et al. 2010; pulido et al. 2005). the ph of the fermented sp flour is lowered due to the production of organic acids by lactic acid bacteria. total lab grew et al. table 2 pasting properties of control and fermented sp flour a b fig 5 scanning electron micrographs of control and fermented sp flours (15 kv, 2000 ). the arrows show etches on the starch granules. a: control flour and b: spontaneous fermented flour . beginning of gelatinization 73.78 ±0.65 75.38±0.83 maximum viscosity (bu) 215.00±4.16 473.50±89.41 peak time 10 min 7.5 min temperature at max viscosity 93.87±0.69 84.30±2.63 breakdown (bu) 21.50±10.47 216.00±6.98 setback (bu) 62.25±5.50 156.50±20.14 pasting properties control fermented amorphous areas in the starch granule are relatively susceptible to hydrolytic agents such as various enzyme and acids . beside producing enzyme, lab also produced organic acids, mainly lactic acid during fermentation. the pasting behavior of the control and spontaneous sp flour was studied by observing changes in the viscosity of a flour system based on the rheological principals. during heating in water, starch of flour began to gelatinize as the granules became swollen and partially solubilized, contributing to a viscous starch paste. the control flour samples, had the higher peak time (table 2), which may indicate a greater structural rigidity in comparison to fermented sweet potato flour. this structural rigidity was also observed from the lower swelling power as discussed previously. the control sweet potato flour had higher (93.87 °c) pasting temperature than the fermented flour having the lower (84.30 °c). also control sweet potato flour had lower peak viscosity as opposed to the fermented sp flour (table 2). this observation might have been influenced by lower rigidity of starch granules in fermented sweet potato, which in turn caused instability and consequently disruption upon the heating and stirring treatment (leon et al. 2006). on the other hand, the higher peak viscosity of the spontaneous fermented flour compared to control flour samples could be due to the increase of granule size (fig 1a), which also led to higher swelling power (fig 2a) and subsequently higher viscosity. the high viscosity of fermented sp would make them very useful in food applications where high thickening power is required. however, the viscosity of this flour decreased substantially afterwards. this phenomenon is probably likely due to lower protein content and free leaching of amylose and amylopectin from the granules (leon et al. 2006). fermented flour also showed a retrogradation tendency, indicated by the rise of viscosity during cooling period as shown in its setback value. setback is a measure of recrystallization of gelatinizatied starch during cooling or measurement of retrogradation. the spontaneous fermented sp flour had setback value higher than control flour. a higher retrogradation tendency of fermented sp makes it suitable for use in jelly foods and noodle. a good quality of noodle is thought to result from the pasted starch that exhibit a high set back value on cooling (lii and chang 1981). breakdown viscosity measure of the vulnerability or susceptibility of the cooked starch to disintegration. reducing whiteness of flour. fermentation was found to make difference in solubility of sweet potato flour as shown in fig 3. this could be caused by starch structural differences, such as chain length distribution and granular size. belloperez et al. (2000) reported that the distributions of chain length in the starches cause differences in solubility, and tian et al. (1991) stated that granular size also affects solubility of the starches where the smaller the granule size, the higher the starch solubility. in this study it was revealed that based on sem analysis, the granular size of the fermented flour was bigger than that of control. fermentation caused any significant change in swelling power of sp flour. there was difference start in rapid swell beetween the granules of control sweet potato flour and those of spontaneous sweet potato flour. the starch granules start to swell rapidly only after the temperature reached the onset of the gelatinization temperature (jacguier et al. 2006). the onset gelatinization temperature of control sweet potatoes flour was 73.78±0.65 °c, and those of spontaneous sweet potatoes flour was 75.38±0.83 °c as determined by micro visco amylograph, corresponded to the start of the rapid increase of swelling power of these flours. there was also difference pattern in rapid change of swelling power between control and fermented sp flour. the swelling power of flour samples is often related to their protein and starch contents (woolfe 1992). during fermentation, bacteria would produce proteolytic enzymes which degrade protein in sweet potatoes, thus the content of protein in fermented flour was lower than those at control. a higher protein content in control flour may cause the starch granules to be embedded within a stiff protein matrix, which subsequently limits the access of the starch to water and restricts the swelling power. furthermore, during fermentation, lactic acid bacteria would produce amylase that hydrolyzes amylose thus reduce amylose content. lower amylose content would increase the swelling factor of starch (tester and morisson 1990). according to leach et al. (1959), the major factor that controls the swelling behavior of a starch is the strength and character of the micellar network within the granule. the control flour samples may have a greater structural rigidity in comparison to fermented sweet potato flour. lab is known to produce amylases, and when starch were treated with amylases there was an initial attack on the amorphous regions of the starch granule. french (1984) observed that intercrystalline microbiol indones6 yuliana et al. volume 7, 2013 microbiol indones 7 bello-pérez la, contreras-ramos sm, jimenez-aparicio a, paredes-lopez o. 2000. acetylation and characterization of banana (musa paradisiaca) starch. acta cientifica venezuela 51:143-149. deng f-m, mu t-m,zhang m, abegeunde ok. 2013. composition, structure and physicochemical properties of sweet potato starches isolated by sour liquid processing and centrifugation. starch/starke 65(12):162-171. leach hw, mccowen ld, schoch tj. 1959. structure of the starch granule. 1. swelling and solubility patterns of various starches. cereal chem. 36:534-544 lii c-y, chang s-m. 1981. characterization of red bean starch and its noodle quality. j food sci. 46(1):78-81. doi:10.1111/j.1365-2621.1981.tb14535.x. leon ae, barrera gn, perez gt, ribotta pd, rosell cm. 2006. effect of damaged starch levels on flour-thermal behaviour and bread staling. eur food res technol. 224(2):187-192. doi:10.1007/s00217-006-0297-x. lu z-h, li l-t, min w-h, wang f, tatsumi e. 2005.the effects of natural fermentation on the physical properties of rice flour and the rheological characteristics of rice noodles. int j food sci technol. 40(9):985-992. doi:10.1111/j.1365-2621.2005.01032.x. french, d. 1984. organization of starch granules in starch:chemistry and technology. eds rl whistler, ef paschal, and jn bemiller 2rd ed, academic press, london:184-247. jacguier jc, kaara, lyng jg, morgan dj, mckenna bm. 2006. influence of granule size on the flow behaviour of heated rice starch dispersions in excess water. carbohydrate polymers 66(4):425-434. doi:10.1016/j. carbpol.2006.03.029. kakou ca, guehi st, olo k, kouame fa, nevry rk koussemon cm. 2010. biochemical and microbial changes during traditional spontaneous lactic acid fermentation process using two varieties of cassava for production of a “alladjan” starter. int food res j. 17:563-573. kassim, sm. july, 2012. effect of fermentation period on functional and pasting properties of sweet potato flour “elubo”. food sci and technol, federal university of agriculture, abeokuta. mutwiri tw. 2007. textural characteristics of lactic fermented sweet potato and its performance as sausage. filler http://erepository.uonbi.ac.ke:8080/xmlui/handle /123456789/19104. oke mo, bolarinwa if. 2012. effect of fermentation on physicochemical properties and oxalate content of cocoyam (colocasia esculenta) flour, isrn agronomy volume 2012:1-4. pulido rp, omar nb, abriouel h, lo ṕez rl,canamero mm, gaĺvez a. 2005. microbiological study of lactic acid fermentation of caper berries by molecular and culture-dependent methods. applied and environ microbiol. 71(12):7872-7879 doi:10.1128/aem.71.12. 7872-7879.2005. putri wdr, haryadi dw, marseno, cahyanto mn. 2011. the higher the breakdown in viscosity, the lower the ability of the starch sample, to withstand heating and shear stress during cooking (adebowale et al. 2005). therefore fermented sp flour might be less able to withstand more heating and shear stress compared to control flour because of their higher breakdown value. other modified technique fermentation was needed to reduce the breakdown value of this flour. the stability of fermented flour against heat and mechanical treatment would also be useful in many other food applications. in regard to morphology of granule, there was presence of the pits and smoother surface angle in the starch granules seems to indicate some breakdown of the starch. we presume that they might be caused by the digestion of some starch by lactic acid bacteria. in addition, cell wall material on control flour still attached to the starch granules in control flour, while those has been released in fermented flour. the size of starch granules in fermented flour become bigger than those in control. fermentation may thus change the amorphous region of the starch granule, size of the granule as well as the chemical components and thereby modify both physical properties of sp flour and its rheological characteristics. based on the systematic analysis of the physical properties of sp flour and its rheological characteristics, it can be concluded that lactic spontaneous fermentation had great effect on starch crystalline and the amorphous region. the results revealed that fermentation may change the amorphous region of the starch granule, size of granules, as well as the chemical components and it may modify the physical and pasting properties of sp flour. therefore, fermented sp flour is easily swollen and soluble. further research work is needed to apply this fermented flour for making product such as noodle that is suitable from these change properties. references aacc, american association of cereal chemists. 2000. approved methods of the aacc. methods 02-52 adebayo-oyetoro ao, olatidoye op, ogundipe oo, balogun io, apara to. 2012. effect of local cassava fermentation methods on functional pasting and sensory properties of lafun. continental j agricl sci. 6(2):1-8. aprianita a, purwandari u, watson b, vasiljevic t. 2009. physico-chemical properties of flours and starches from selected commercial tubers available in australia. int food res j. 16:507-520 yousif nmk, huch m, schuster t, cho gs , dirar ha, holzapfel wh, franz map. 2010. diversity of lactic acid bacteria from hussuwa, a traditional african fermented sorghum food. food microbiol. 27(6):757768. doi:10.1016/j.fm.2010.03.012. yuliana n, nurdjanah s. 2013. development of sweet potatoes pickle as modified flour: effort to balance the need of wheat. research report. universitas lampung. yuliana n, nurdjanah s. 2009. sensory properties of spontaneously fermented purple sweet potato pickle at various salt concentration. jurnal teknologi dan industri hasil pertanian 14:120-128. yuliana n, nurdjanah s, ocatrini zh. 2010. some biochemical and total lactic acid bacteria changes during natural fermentatioan of the purple sweet potatoes. proceeding international seminar on holticulture to support food security: b209-b214. yuliana n, nurdjanah s, margareta m. 2013.the effect of a mixed-starter culture of lactic acid bacteria on the characteristics of pickled orange-fleshed sweet potato (ipomoea batatas l.). microbiol indones. 7(1):1-8. doi:10.5454/mi.7.1.1. effect of biodegradation by lactic acid bacteria on physical properties of cassava starch. int food res j. 18(3):1149-1154 sanni a, morlon-guyot j, guyot jp. 2002. new efficient amylase-producing strains of lactobacillus plantarum and l. fermentum isolated from different nigerian traditional fermented foods. int j food microbiol. 72(12):53-62. doi:10.1016/s0168-1605(01)00607-9. shimelise, meaza m, rakishit s. 2006. physicochemical properties, pasting behaviour and functional characteristics of flours and starches from improved bean (phaseolus vulgaris l) varieties grown in east african. cigr ejournals 8:1-18. sobowale ao, olurin to, oyewole ob. 2007. effect of lactic acid bacteria starter culture fermentation of cassava on chemical and sensory characteristics of fufu flour. afr j biotechnol. l 6(16):1954-1958. tester rf, morrison wr. 1990. swelling and gelatinization of cereal starches. i. effect of amylopectin, amylose and lipids. cereal chem. 67:551-559. tian sj, rickard je, blanshard jm. 1991. physicochemical properties of sweet potato starch. j sci food agri. 57(4):451-491. doi:10.1002/jsfa.2740570402. microbiol indones8 yuliana et al. 3 akhmaloka_350 (11-16) the effect of mutation at thr 295 of saccharomyces cerevisiae erf1 on suppression of nonsense codons and erf1 structur prima endang susilowati1,3, pingkan aditiawati2, fida madayanti1, and akhmaloka1* 1biochemistry research division, faculty of mathematics and natural sciences, 2school of life science and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia 3department of chemistry, faculty of mathematics and natural sciencies, universitas haluoleo, jalan anduonohu, kendari, indonesia the termination of translation in saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, erf1, and erf3. two regions in erf1, at position 281-305 and 411-415, were proposed to be involved on the interaction to erf3. in this study we have constructed and characterized erf1 mutants at position 295 from threonine to alanine and serine residues resulting in erf1(t295a) and erf1(t295s) respectively. the mutations did not affect the viability or temperature sensitivity of the cells. the stop codons readthrough of the mutants were analyzed in vivo using pgk-stop codon-lacz gene fusion and the results showed that the suppression of the mutants was increased in all of the codon terminations. the suppression of the uag codon was the high for both mutants, with a 7-fold increased for erf1(t295a) and a 9 fold increase for erf1(t295s). the suppressor activity of erf1(t295s) was higher compared to that of erf1(t295a), suggesting that the accuracy of translational termination in erf1(t295s) was lower than that of erf1(t295a). computer modeling analysis using swiss-prot and amber version 9.0 programs revealed that the overall structure of erf1 mutants has no significant difference with the wild type. however, substitution of threonine to serine on erf1(t295s) triggered a secondary structure change on the other motif of the c-terminal domain of erf1. this observation did not occur for on erf1(t295a). this suggests that the high stop codon suppression on erf1(t295s) is probably due to the slight modification of the structure of the cterminal motif. keywords: saccharomyces cerevisiae, erf1, mutation, nonsense codon suppression _____________________________________________ ________________________ *corresponding author, phone: +62-22-2515032, fax: +62-22-2502360, e-mail: loka@chem.itb.ac.id protein biosynthesis is carried out in three distinct steps: initiation, elongation, and termination. while the first two steps have been extensively studied, our understanding of the termination process has lagged behind. two classes of release factors mediate translation termination in the eukaryotic system, codon-specific rf’s, and codon-non specific rf’s (kisselev et al. 2003). eukaryotic release factor 1 (erf1), encoded by sup45 gene, is a class i release factor that recognizes any of the three stop codons, when they are located in the ribosomal a site (bertram et al. 2000). eukaryotic release factor 3 (erf3), encoded by sup35 gene, is a class ii release factor that facilitates stop codon recognition and stimulates the termination reaction in a gtp dependent manner (frolova et al. 1996; salas-marco and bedwell 2004). following stop codon recognition, erf1 also induces polypeptide chain release by activating the peptidyltransferase center of the ribosome. both erf1 and erf3 are essential for the viability of yeast cells and deletion of the c-terminal part of each protein separately, lead to lethality (inge-vechtomov et al. 2003). the crystal structure of human erf1 has been determined and found to be a protein is arranged in three domains. the n-terminal domain (domain1) has been proposed to be responsible for stop codon recognition (song et al. 2000). this proposal was supported by investigations using the mutational approach (bertram et al. 2000) and by crosslinking experiments (chavatte et al. 2001). the middle domain (domain 2) is responsible for peptidyl-tranferase hydrolytic activity and includes a ggq motif that has been highly conserved through evolution (frolova et al. 1999). mutations in ggq (g residues) are dominant-negative in vitro and lethal in vivo in s. cerevisiae cells (song et al. 2000). the domain 3 corresponds to the c-terminal part of erf1 that is necessary for the interaction with erf3 although there are some discrepancies in the precise location of the region of erf1 which interacts with erf3. progressive deletion of the c-terminal region of erf1, 6-19 amino acids in s. cerevisiae (eurwilaichitr et al. 1999) and 17 amino acids of schizosaccharomyces pombe (ito et al. 1998) resulted in a corresponding loss of erf3 binding affinity. in any case, the core erf3-binding region identified for homo sapiens erf1 (by yeast two-hybrid and deletion analysis), showed that two regions in each release factor are critical for mutual binding. these are position 281-305 and 411415 (gilry) of erf1, and position 478-530 and 628-637 of erf3 (merkulova et al. 1999). although deletion of residues within domain 3 of erf1 resulted in the loss of erf3 interaction, detail of the position and amino acid residues for this interaction remain unclear. preliminary studies using computer modeling analysis of the structure of erf1, especially of the above two regions, showed that tyrosine at position 410 (y410) and threonine at position 295 (t295) of erf1 are exposed to the molecular surface and are predicted to be involved in its interaction with erf3. mutation on tyrosine to serine at position of 410 in erf1 has been reported to decrease the binding affinity of the protein to erf3 protein (subandi 2002; akhmaloka et al. 2008). in order to further probe the role of t295 in yeast erf1 protein, we report here the effect of mutation t295 of yeast erf1 on the termination of protein biosynthesis. the nonsense codons read-through process has been used to assay the accuracy of the termination process. issn 1978-3477 volume 2, number 1, april 2008 p 11-16 12 susilowati et al. microbiol indones materials and methods microbial strains, plasmids, and growth conditions. saccharomyces cerevisiae strains used in this study are listed in table 1. all strains were suq5 and [psi-]. yeast cultures were grown in a standard-rich-medium, ypd: 1% (w/v) bacto peptone, 0.5% (w/v) yeast extract; and 2% (w/v) glucose or in minimal medium, sm: 0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose, supplemented with appropriate amino acids, at 30°c with gentle shaking. for the plasmid shuffling, the strains were grown on medium ynb-foa: 0.67 % (w/v) yeast nitrogen base, 2% (w/v) glucose, 0.1% (w/v) 5-fluorootic acid (5-foa), 20 μg ml-1 uracil. the escherichia coli strain used in this work was dh5α [supe44 lacu169 (q80lacz m15) hsdr17 reca1 enda1 gyra96 thi-1 rei a1] for the cloning experiments. the e. coli culture was grown in luria bertani medium lb: 1% (w/v) bacto tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) nacl, supplemented with relevant antibiotics for selection (e.g. 50 μg ml-1 ampicillin). plasmid pukc1901, a shuttle vector between e. coli and s. cerevisiae cells, carrying sup45-y410s gene was used for the construction of plasmid carrying sup45 mutants. the other plasmids, pukc815, 817, 818, and 819 were used in the nonsense codon read-through assay on s. cerevisiae. dna-manipulation and plasmid constructions. all dna manipulation and plasmid constructions were carried out according to standard protocols (sambrook et al. 1989). to generate plasmids pspes-t295s and pspes-t295a, a 1 400 bp bglii/hindiii fragment from plasmid pepes-t295s and a fragment from pepes-t295a containing the entire sup45 mutant genes, were cloned into bglii and bamhi sites of plasmid pukc1901 (a gift from m f tuite, university of kent at canterbury, uk), which is a shuttle vector of e. coli and s. cerevisiae. the recombinant plasmids were used to transform e. coli dh5α for propagating all plasmids. plasmid shuffling. the plasmid shuffling was carried out based on the standard method of akhmaloka (1991). the haploid le(803) (sup45::his3, ura3, sup45) was used in plasmid shuffling. the strain was transformed with (leu2 sup45) plasmids (pspes-t295s and pspes-t295a). the transformants were selected on media without uracil and leucine (-ura-leu) and then checked by replicating onto 5-foa medium to counter select against ura3 plasmids (kaiser et al. 1994). growth was also assayed using serial dilution overnight culture with od 600nm = 1.0. serially (10-fold) diluted yeast cell cultures were spotted on plates containing 5-foa to determine the ability of sup45 mutant alleles to support cell growth. the wild type yeast sup45 gene carried by the ura3 plasmid is eliminated since 5-foa is toxic to cells expressing the ura3 gene. βββββ-galactosidase assays. centromeric plasmids pukc815, 817, 818, and 819 (akhmaloka 1991; stansfield et al. 1995) were transformed into suppressor strains (le-t295s and let295a). the transformants were grown in plasmid-selective medium and 3 x 106 cells were inoculated into 5 ml ypd respectively. triplicate samples from cultures grown, to a density of od 600nm ~1.0, were treated essentially as described by coligan et al. (1995), with the following modifications. the cells were suspended in 500 µl z buffer, 10 µl 0.1% (w/ v) sds, and 20 µl chloroform. the samples were then vortexed for 15 s and equilibrated for 15 min at 30°c using a water bath. after adding 100 µl 4 mg ml-1 o-nitrophenyl-βd-galactosidase (onpg), samples were vortexed for 5 s and the reactions were carried out for 30 min at 30°c in a water bath before stopping the reaction by adding 500 µl 1.0 m sodium carbonate. after centrifugation at 5 000 x g, the optical density of supernatants were measured at both 420 and 550 nm wavelengths and the miller unit of each of the samples were calculated as (od 420 )-(od 550 x 1.75) (stansfield et al. 1995). the read-through for each of the three stop codons was calculated as being the percentage of lacz expression relative to the construct lacking a stop codon (pukc815) in the same medium. computer simulation. the initial three-dimensionalstructure of erf1 was constructed by homology protein structure modeling from swiss-prot using predict protein program (rost et al. 2004). the comparative modelling program used three steps: alignment of the amino acid sequence of yeast erf1 with the template of human erf1 (song et al. 2000) (pdb number 1dt9.pdb.), structure prediction based on primary sequences homology, and validation of the structure using what_if program in swissprot. the amber version 9.0 program package (case et al. 2006) was used for the molecular simulation. the structure was subjected to energy minimization calculation using the steepest descent method with 500 iterations followed by the conjugate gradient method with 4 500 iteration to be used as starting lowest energy structure. the energy minimized for the protein was then submitted to molecular dynamic simulation after equilibrating for about 100 ps at 350 k. temperature and pressure were maintained constant during the simulation. the trajectories and the coordinates of the proteins were saved every 2 fs for structural analysis. simulation image of the protein was generated using visual molecular dynamic (vmd) software (humphrey et al. 1996). the root mean square deviation (rsmd) was calculated for strain genotype source suq5, ade2-1, his3-11, 15, ura3-1 leu2-1, can1-100, [psi-], sup45::his3 suq5, ade2-1, his3-11, ura3-1, leu2-1 can1-100, [psi-], sup45::his3, [leu2-1-sup45] suq5, ade2-1, his3-11, 15, ura3-1, leu2-1, can1-100, [psi-], sup45::his3, [leu2-1-sup45-t295a] suq5, ade2-1, his3-11, 15, ura3-1, leu2-1, can1-100, [psi-], sup45::his3, [leu2-1-sup45-t295s] δle2(803) δle2(sup45) δle2(t295a) δle2(t295s) university of kent at canterbury, uk this study this study this study table 1 yeast strains used in this study volume 2, 2008 microbiol indones 13 the backbone atoms with reference to starting structure of time zero. results construction and characterization of yeast-strains le2(t295a) and le2(t295s). le2(t295a) and le2 (t295s) are haploid yeast strains that carry chromosomal sup45 disrupted by the his3 gene (sup45::his3) and sup45 gene in plasmids psepes-t295a and psepes-t295s respectively. the strains were constructed from le2(803), a haploid strain which carries sup45::his3 and sup45 genes in a cen-ura3-based plasmid. the le2(803) strain was transformed with leu2 based plasmid carrying sup45 or sup45 genes. the transformants were then subjected to plasmid shuffle analysis to verify whether strains containing sup45 could lose cell viability (fig 1). all transformants carrying sup45 alleles and sup45 (control) were able to grow in the presence of 5-foa, indicating that all tested mutations can replace the wild type of sup45 for the viability test (data not show). however, plasmid shuffle was less efficient for sup45 mutants than for the wild type sup45. the viable transformants on media containing 5-foa were characterized, including genotypes, temperature sensitivity, and the allosuppressor phenotypes. all of viable cells carrying sup45 and sup45 mutants could grow on media without adenine (-ade) or without -leu, however they were unable to grow on media without -ura (fig 2). the viability of the cells on media -ade showed that all transformants still possessed an allosuppressor phenotype. the viability of the transformants on media -leu and -ura was negative, showing that ura3 based plasmid had been replaced by leu2 based plasmids. the data confirmed that the plasmid shuffling had been successfully carried out. furthermore, none of the transformants were temperature sensitive mutants (data not shown). efficiency of le2(t295a) and le2(t295s) on stopcodon-suppression. stop codon read-through on le2(t295a) and le2(t295s) were measured based on the ability of the cells to terminate the translational process on the fusion gene between pgk-termination codon-lacz carried by centromeric (single copy), ura3 based plasmid (fig 3). the strains were transformed by each of four serial plasmids (pukc815, 817, 818, and 819). plasmid pukc817, 818, and 819 carry uaa, uag, and uga termination codons respectively, while plasmid pukc815 was used as the non-termination control. the efficiency of all stop codon read-through in le2(t295a) and le2(t295s) were decreased compared to that the wild type [le2(sup45)]. mutation on t295s showed a higher stop codon read-through compared to that t295a. the uag codon showed the highest read-through among the stop codons from the results comparing mutants with the wild type. the increasing read-through of the uag codon fig 1 strategy used for plasmid shuffling. (1) the mutagenized dna on plasmid carryinf leu2 marker introduced into the yeast cell, (2) transformants carrying double plasmids (ura3 and leu2 based plasmid) were grown on media containing 5-foa to replaced ura3 based plasmid. δle2 (803) ura3 ura3 leu2 leu2 leu2 ura3 sup45 sup45sup45 1 2 tr an sf o rm at io n f o a s el ec ti on δle2 -sup45 fig 2 the viability of mutant yeast strains on variation media. (-ade) minimum media without adenine, (-leu) minum media without leucine, (-ura) minimum media without uracil. δle2(t295s) -ade -leu -ura δle2(t295a) -ade -ura-leu fig 3 plasmid used for suppression assay. pukc815, a ura3 based plasmid carrying pgk-lacz gene fusion without codon termination in between; pukc817, 818, and 819 are ura3 based plasmids carrying pgk-stop codon-lacz gene fusion taa, tag, and tga on oligonucleotides respectively; ( ), pukc 817: gat cta taa gct ttg gat, pukc 818: gat cta tag gct tta aag gat, pukc 819: gat cta tga gct tta aag gat. ri (1)p (1 120) c (26) b (925) bg (9 200) ri (3 926) b (4 126) rv (5 650) pukc 815 ura3 cen 4 ars 1 pgk-5’ lacz bla was over 7 fold for le2(t295a) and 9 fold for le2(t295s) (fig 4). structural modelling of erf1(t295a) and erf1(t295s). structural modelling of erf1 mutants and the wild type were performed using the amber 9 program followed by visualization using vmd. based on the rmsd value, the overall structure of erf1(t295a) and erf1(t295s) showed no significant difference compared to the wild type. however, detailed analysis on the secondary structure of erf1 mutants showed that erf1(t295s) contained secondary structure changes which had not occurred on the erf1(t295a) (table 2). the structure of the amino acid residues change on erf1(t295s) was not in the same motif with the position of mutation (fig 4). discussion domain 3 of erf1 is considered to be responsible for the interaction with erf3 protein (ito et al. 1998; ebihara and nakamura 1999; frolova et al. 2000). serial deletion of this region has been reported to lose on its interaction (eurwilaichitr et al. 1999), however there was no information concerning the effect of amino acid substitution in this region. deletion of an amino acid usually causes an overall change of structure of a protein, while substitution of amino acid does not affect the overall structure. in order to probe the amino acids which are responsible for the function of the protein, the last strategy is preferable. we have constructed and characterized sup45 mutants which have a mutation in codon no 295 for threonine to alanine or serine. threonine at position 295 in yeast erf1 was predicted to be involved on the interaction with erf3 (subandi 2002). threonine at 295 is also one of the amino acids that are critical for mutual binding (merkulova et al. 1999). structural modeling of yeast erf1 showed that the amino acid exposed to the surface of the protein is the one which has the requirement of amino acid residues to interact with other molecules. sup45 is an essential gene in haploid s. cerevisiae cell (inge-vechtomov et al. 2003). disruption of the gene causes lethality in haploid yeast cells. in order to assay mutation in the gene in vivo, the mutant gene was introduced into le(803) which carries disrupted chromosomal sup45::his3 and sup45 gene in single copy ura3 based plasmid. the transformants containing double plasmids (ura3 and leu2 based plasmids) were examined for plasmid shuffle procedure (chabelskaya et al. 2007) by growing the cells on a medium containing 5-foa. the wild type yeast sup45 gene carried on the ura3 plasmid is eliminated because 5-foa is toxic to cells expressing the ura3 gene (boeke et al. 1984). mutation of sup45-t295a and sup45-t295s showed an allosuppressor phenotype (fig 2). this suggested that mutation of threonine at position 295 was not essential for cell growth but caused a defect in the functioning of the mutant protein. le2(803) carries ade2 and suq5 in addition to sup45::his3 and sup45 cen ura3 based plasmids. suq5 is a weak trna suppressor (cox 1977). this trna suppressor does not suppress the nonsense codon on the ade2 mutant with a [psi-] genetic background (ingevechtomov et al. 1988). however, mutation on the sup45 gene can enhance the activity of suq5 (stansfield et al. 1996) to suppress the nonsense mutation on the ade2 gene with the result that the cells showed viable on the medium without supplemented by adenine (fig 2). the viability of transformants on medium without adenine showed that the mutation enhanced the activity of suq5 and thus displayed an allosuppressor phenotype. mutation on sup45-t295a and sup45-t295s enhanced all of the stop codon suppressions (fig 5). however, the quantitative values were variable depending on the type of stop codons and the mutations. the uaa codon showed the most read-through in all of the strains examined, including the wild type. this was not surprising since all strains carried the suq5 genetic background. suq5 is a codon specific trna suppressor for the uaa codon (stansfield et al. 1995). for the uag and uga codons, the suppression of the wild type strain was very low, or insignificant (fig 5). however, for the mutant strains, the suppression of uag and uga was significantly increased. the uag codon was the most leaky in both mutants. this is to be unexpected since erf1 is reported to recognize all of stop codons in the same manner (frolova et al. 1994; zhouravleva et al. 1995; frolova et al. 1996). termination of translation in eukaryotic system that involves interaction between erf1 and erf3 is a complex phenomenon. detail mechanism of the process is still unclear yet. erf1 was reported to be phosphorylated by ck2 protein but the product did not directly affect translation termination (kallmeyer et al. 2006). urakov et al. (2001) demonstrated that ittip could modulate the efficiency of translation termination in yeast. meanwhile, a recent report showed that erf1 protein participates not only in the termination of translation but also in mrna degradation and the initiation of translation via interaction with other proteins (chabelskaya et al. 2007). in order to further characterize the causes of an elevation of suppression in sup45-t295a and sup45-t295s, the three dimensional structure of erf1(t295a) and erf1(t295s) were table 2 root mean square deviation (rmsd) value and the secondary structure change on some amino acid residues. the rmsd value was calculated using the erf1 wild type as control secondary stuctures of amino acid residues amino acid no. 343-350 amino acid no. 351-354 rmsd erf1-sup45 erf1-t295a erf1-t295s 0 0.0455 0.0251 turn turn α-helix turn turn coil δle2 -sup45 δle2[t295s]δle2[t295a] s up pr es si on l ev el s (% ) 60 50 40 30 20 10 0 fig 4 codon termination read-through of yeast strains carrying sup45 and sup45 mutants genes. 1, uaa; 2, uag; 3, uga. 1 2 3 1 2 3 1 2 3 14 susilowati et al. microbiol indones volume 2, 2008 microbiol indones 15 analyzed using molecular dynamic simulation. the results showed that there was no significant change on the overall structure of erf1 mutants. however, detail analysis of erf1(t295s) showed that secondary structure of a few amino acids changed, from turn to α-helix form (table 2). the structural modification of erf1(t295s) had not directly affect on the structure of the t295 motif but it did trigger structural modification of the neighboring motif (fig 4). this modification might affect the interaction of erf1(t295s) with erf3 and thus increasing the suppression of codon terminations. acknowledgement we would like to thank m f tuite, university of kent, uk, for plasmid and yeast cell gifts. this research was supported by a fundamental research grant from the directorate for higher education, department of national education, indonesia to akhmaloka, and a bpps scholarship to prima endang susilowati. references akhmaloka. 1991. a molecular genetic analysis of the allosuppressor gene sal4 in saccharomyces cerevisiae. [dissertation]. kent: university of kent. akhmaloka, susilowati pe, subandi, madayanti f. 2008. mutation at tyrosine in amrly (girly like) motif of yeast erf1 on nonsense codons suppression and binding affinty to erf3. int j biol sci 4:879 5 . bertram g, bell ha, ritchie dw, fullerton g, stansfield i. 2000. termination eukaryote translation: domain 1 of release factor erf1 function in stop codon recognition. rna 6:1236-1247. boeke jd, lacroute f, fink gr. 1984. a positive selection for mutants lacking orotidine-5’-phospate decarboxylase activity in yeast: 5-flouro-orotic acid resistence. mol genet 197:345-346. case da, darden ta, cheatham te, simmerling cl, wang j, duke re, luo r, merz km, pearlma da, crowley m, walker rc, wang w, wang b, hayik s, roitberg a, seabra g, wong kf, paesani f, wu x, brozel s, tsui v, gohlke h, yang l, tan c, mongan j, homak v, cui g, beroza p, mathews dh, schafmeister c, ross ws, kollman pa. 2006. amber 9. san francisco: university of california. chabelskaya s, gryzina v, moskalenko s, le goff c, zhouravleva g. 2007. inactivation of nmd increases viability of sup45 nonsense mutants in saccharomyces cerevisiae. bmc mol biol 8:71. fig 5 three dimensional structure of domain 3 erf1 based on molecular dynamic simulation: a, wild type of erf1; b, erf1-t295a; and c, erf1t295s. areas of conformational change shown within dotted circles. a b c thr-295 tyr-410 ala-295 tyr-410 ser-295 tyr-410 chavatte l, frolova ly, kisselev l, favre a. 2001. the polypeptide chain release factor erf1 specifically contacts the s(4)uga stop codon located in the a site of eukaryotic ribosomes. eur j biochem 268:2896-2904. coligan j, dunn bm, ploegh hl, speicher dw, wingfield pt. (eds). 1995. current protocols in protein science. new york: wiley. cox bs. 1977. allosuppressors in yeast. genet res 30:187-205. ebihara k, nakamura y. 1999. c-terminal interaction of translational release factor erf1 and erf3 of fission yeast: g-domain uncoupled binding and the role of conserved amino acid. rna 5:739-750. eurwilaichitr l, graves fm, stansfield i, tuite mf. 1999. the cterminus of erf1 defines a functionally important domain for translation termination in saccharomyces cerevisiae . mol microbiol 32:485-496. frolova ly, le gx, rasmussen hh, cheperegin s, drugeon g, kress m, arman i, haenni al, celis je, philippe m, kisselev l. 1994. a highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. nature 372:701703. frolova ly, le gx, zhouravleva g, davydova e, philippe m, kisselev l. 1996. eukaryotic polypeptide chain release factor erf3 is an erf1 and ribosom-dependent guanosine triphosphatase. rna 2:234-341. frolova ly, merkulova ti, kisselev ll. 2000. translation termination in eukaryotes: polypeptide release factor erf1 is composed of functionally and structurally distinct domains. rna 6:381-390. frolova ly, tsivkovskii ry, sivolobova gf, oparina ny, serpinsky oi, blinov vm, tatkov si, kisselev ll. 1999. mutations in the highly conserved ggq motif of class 1 polypeptide release factor abolish ability of human erf1 to trigger peptidyl-trna hydrolysis. rna 5:1014-1020. humphrey w, dalke a, schulten k. 1996. vmd. j mol graphics 14:33-38. inge-vechtomov sg, tikhodeev on, karpova ts. 1988. selective system for obtaining recessive ribosomal suppressors in yeast saccharomyces cerevisiae. genetika 24:1159-1165. inge-vechtomov sg, zhouravleva g, philippe m. 2003. eukaryotic release factor (erfs) history [review]. biol cell 95:195-209. ito k, uno m, nakamura y. 1998. single amino acid substitution in prokaryotic polypeptide release factor 2 permits it to terminate translation at all three termination codons. proc natl acad sci usa 95:8165-8169. kaiser c, michaelis s, mitchell a. 1994. cold spring harbor laboratories: methods in yeast genetics. new york: cold spring harbor laboratory pr. kallmeyer ak, keeling km, bedwell dm. 2006. eukayotic release factor 1 phosphorylation by ck2 protein kinase is dynamic but has little effect on the efficiency of translation termination in saccharomyces cerevisiae. eukaryotic cell 5:1378-1387. kisselev l, ehrebreg m, frolova l. 2003. termination of translation: interplay of mrna, rrnas, and release factor. embo j 22:175182. merkulova ti, frolova ly, lazar m, camonis j, kisselev ll. 1999. c-terminal domains of human translation termination factors erf1 and erf3 mediated their in vivo interaction. febs lett 443:41-47. rost b, yachdav g, liu j. 2004. the predict protein server. nucleic acids res 32:321-326. salas-marco j, bedwell dm. 2004. gtp hydrolysis by erf3 facilitates stop codon decoding during eukaryotic translation termination. mol cell biol 24:7769-7778. sambrook j, fritsch ef, maniatis t. 1989. molecular cloning: a laboratory manual. new york: cold spring harbor pr. song h, mugnier p, webb hm, evans dr, tuite mf, hemmings ba, barford d. 2000. the crystal structure of human eukaryotic release factor erf1-mechanism of stop codon recognition and peptidyl-trna hydrolysis. cell 100:311-321. stansfield i, akhmaloka, tuite mf. 1995. a mutant allele of the sup45 (sal4 ) gene of saccharomyces cerevisiae shows temperature dependent allosuppressor and omnipotent suppressor phenotypes. curr gennet 27:417-426. stansfield i, eurwilaichitr l, akhmaloka, tuite mf. 1996. depletion in the levels of the release factor erf1 cause a reduction in the efficiency of translation termination in yeast. mol microbiol 20:1135-1143. subandi. 2002. pengaruh mutasi y410s erf1 pada interaksi erf1erf3 saccharomyces cerevisiae. [dissertation]. bandung: institut teknologi bandung. urakov vn, valouev ia, lewitin ei, paushkin sv, kosorukov vs, kushnirov vv, smirnov vn, ter-aanesyan md. 2001. ittip, a novel protein inhibiting translation termination in saccharomyces cerevisiae. bmc mol biol 2:9. zhouravleva g, frolova ly, le gx, le guellec r, inge-vechtomov s, kisselev l, philippe m. 1995. termination of translation in eukaryotes in governed by two interacting polypeptide chain release factors, erf1, and erf3. embo j 14:4065-4072. 16 susilowati et al. microbiol indones sopandi24042012 issn 1978-3477, eissn 2087-8575 vol 6, no 1, march 2012, p 35-41 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.6 sub-acute toxicity of pigment derived from penicillium resticulosum in mice 1 tatang sopandi * and wardah 1 department of biology, faculty of mathematics and natural science. universitas pgri adi buana surabaya, jalan dukuh menanggal xii, surabaya 60234, indonesia; 2 department of food technology, faculty of food industry, universitas17 agustus 1945 surabaya, jalan semolowaru 45, surabaya 60234, indonesia 2 pigments derived from penicillium have different toxicities depending on the pigment components. this study was intended to evaluate the sub-acute toxicity of oral exposure of balb/c mice to penicillium resticulosum pigment. a total of 50 healthy adult male and female mice were divided into 5 treatment groups and different -1 doses of pigment (0, 125, 250, 500 and 1000 mg kg body weight) were orally administered. oral feeding of -1 pigment with doses 125 to 1000 mg kg body weight daily to adult mice did not cause mortality nor any clinical abnormalities. there were no significant differences in body, liver and kidney weights, nor liver and kidney -1 functions of mice when pigment was given orally with intake doses of 125 to 1000 mg kg body weight daily for 28 d in comparison to mice without pigment intake (control groups). there was a slight difference in liver -1 histopathology of mice exposed to 500 and 1000 mg kg body weight of pigment for 28 d in comparison to mice control groups, although there were no differences in kidney histopathology. thus, we can conclude that the -1 pigment of p. resticulosum can be cathegorized as low toxic pigment and well tolerated at dose below 500 mg kg body weight daily for 28 d. key words: mice, penicillium, pigment, toxicity pigmen yang berasal dari kapang penicillium diketahui mempunyai toksisitas yang berbeda tergantung pada komponen yang dihasilkan. penelitian ini bertujuan untuk mengevaluasi toksisitas sub-akut secara oral pigmen dari penicillium resticulosum pada mencit balb/c. sebanyak 50 ekor mencit jantan dan betina dewasa sehat dibagi menjadi 5 kelompok perlakuan masing-masing 5, dan pada masing-masing kelompok diberi pigmen -1 secara oral dengan dosis yang berbeda yaitu diberi 0, 125, 250, 500, dan 1000 mg kg bobot badan per hari selama -1 28 hari. mencit yang diberi pigmen 125-1000 mg kg bobot badan tidak menunjukkan adanya kematian dan -1 kelainan klinis. fungsi hati dan ginjal mencit yang diberi asupan pigmen 125-1000 mg kg bobot badan setiap hari selama 28 hari tidak berbeda signifikan dibandingkan dengan mencit yang tidak diberi asupan pigmen (control). terdapat sedikit perubahan profil histologi hati mencit yang diberi asupan pigmen dengan dosis 500 -1 dan 1000 mg kg bobot badan setiap hari selama 28 hari dibandingkan profil histologi hati mencit yang tidak diberi asupan pigmen namun tidak terdapat perbedaan profil histology ginjal mencit pada semua dosis asupan pigmen dibandingkan dengan kontrol. dapat disimpulkan bahwa pigmen dari p. resticulosum termasuk kategori -1 toksik rendah dan aman dikonsumsi di bawah dosis 500 mg kg bobot badan setiap hari selama 28 hari kata kunci : mencit, penicillium, pigmen, toksisitas the use of synthetic pigments as dyes has an important role in the food industry (himri et al. 2011). however, since consumption of food containing certain synthetic pigment is known to cause health problems (duran et al. 2002; babitha et al. 2006), public attention towards the development of pigment from materials of natural origin has been increasing (aberoumand 2011). as manufacturers demand rises for naturally-derived ingredients, particularly in food applications, naturally derived colorants appear set to overtake synthetic colorants in market value (mapari et al. 2009). it is predicted that naturally derived pigment will have a bright future with a predicted annual growth rate of 5-10% while synthetic dye color are forecasted to grow at a lower rate of between 3 and 5% (downham and collins 2000). there have been searches to find new sources of natural pigments from plants and animals to replace the synthetic pigment. microorganisms have lately been receiving more attention as sources of natural pigment (mapari et al. 2005). production of pigments by fermentation has a number advantages; possibly easier extraction, higher yields, no lack of raw materials, no seasonal and agroclimate variations, and can be produced in a short period of time (mortensen 2006; aberounmand 2011; poorniammal et al. 2011). microorganisms are known to produce various types of polyketide pigments, such as carotenoids, fenazine, acilphenol, pyrone, sclertiorine, and anthraquinone. only carotenoid pigments and polyketide are known to be toxic (poorniammal et al. 2011). meanwhile, various species of fungi found in nature have reported but only a few have been explored *corresponding author, phone: +62-81231681744, fax: +62-31-5042804, e-mail: tatang-sopandi@yahoo.co.id 36 sopandi and wardah microbiol indones for the purpose of production of food dyes (mapari et al. 2006). production of pigment by the genus penicillium is more efficient and profitable than the production of pigment by bacteria and yeasts. penicillium can secrete enzymes and pigments out of the cell. the secreted pigment is relatively stable, thus easily purified. penicillium can also grow on a variety of lignocellulosic materials. in previous studies, a penicillium indigenous pigment producer was obtained from soil of baluran national park indonesia and tentatively identified as penicillium resticulosum based on morphological characteristics. however, the toxicity of pigment from this fungus is not yet known. this study aims to evaluate the sub-acute toxicity of pigment produced by p. resticulosum in mice. materials and methods fermentation and pigment production. penicillium sp. was obtained from the laboratory of microbiology collection, faculty of mathematics and natural sciences, the universitas of pgri adi buana surabaya indonesia. pigment production by p. resticulosum performed on liquid fermentation using a formulation media consisting of 5 mg kh po , 0.2 g 2 4 mgso . h o, 3.0 g nano , 4.0 g of nh no , 3.0 g 4 2 3 4 3 yeast extract, and 30.0 g carboxymethylcellulose (cmc) in 1000 ml of hydrolysed corn cob medium with initial ph of 5.5. medium was placed in erlenmeyer flask, homogenized and sterilized in an o autoclave at temperatures 121 c for 15 min, then cooled to room temperature. all flasks were inoculated 6 with 10 ml of the spore suspension containing 10 cells -1 ml conidia of p. resticulosum (estimated based on haemocytometer count). fermentation was carried out o in batch condition at 28-29 c, 50% relative humidity with 60 rpm agitation for 12 days under dark condition. after incubated, whole of fermented matter was mixed with 200 ml distilled water and filtered through whatmann no. 1. filter followed by centrifugation at 3000 x g (hettich eba 8s germany) for 15 min to separate pigment from spore and other material. one liter ethyl acetate was added to an equal volume of supernatant pigment and the mixture was adjusted to ph 3.0 with 2 n hcl with vigorous hand mixing and then let to stand for 15 min until two layers were formed. the ethyl acetate layer (upper layer) was obtained by pipette and evaporated using a rotary o vacuum (heidolph vv2011, germany) at 50 c. the pigment was collected and dried to constant weight at o 50 c for 48 h. experiments on animals and pigment administration. a total of 50 healthy male and female balb/c mice aged 3 months, weighing 30-35 g were obtained from pusvetma surabaya and used for 28days sub-acute toxicity evaluation. the mice were acclimatized for 14 days and assigned to five groups each consisting of five males and five females. the mice were housed in polypropylene cages (5 mice in one cage) on soft chip bedding, changed twice per week in a controlled room with a 12 hours light-dark cycle o and temperature 26 ± 2 c with relative humidity of 55%. they were kept with free access to water and dry commercial pellets feeding. pigment p. resticulosum was fed by oral doses 0 (control), 125, 250, 500, and -1 1000 mg kg body weight daily in nacmc (sodium carboxymethylcellulose) daily for 28 d. prior to dosing, animals were fasted overnight. animals were observed thoroughly for onset of any immediate toxic signs and also during observation period of one week. general behaviors observed for the first 1 h, 24 h, and one week of test pigment administration were motor activity, tremors, convulsions, straub reaction, aggressiveness, pilo-erection, loss of lighting reflex, sedation, muscle relaxation, hypnosis, analgesia, ptosis, lacrimation, diarrhea and skin colour. at the end of the study (28 d), all animals were measured for the following variables; b o d y, l i v e r a n d k i d n e y w e i g h t , a s p a r t a t e aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), lactate dehydrogenase (ldh), blood urea nitrogen (bun), and liver and kidney histopathology. blood sampling. blood sampling was done by the cardiac puncture after the mice were anaesthetized by chloroform. blood samples from each mouse were collected in tubes containing edta. blood samples were stored for 1 hour and centrifuged in the cold at 2500 x g for 10 min. serum was separated and placed in sterile plastic vials stored at -20 °c until use. biochemical studies. activity of serum aspartate aminotransferase (ast) and alanine aminotransferase (alt) were determined by kinetic method (teco diagnostics). blood urea nitrogen (bun) was determined by enzymatic colorimetric method (chronolab cat. no. 101-0248). alkaline phosphatase (alp) was determined by kinetic method (diachem ltd cat. no. 48263). lactate dehydrogenase (ldh) was determined by modification of scandinavian comitee on enzymes (chemhouse cat. no. 065-0150). histophatological studies. after 28 d pigment treatment, mice were anesthetized with chloroform and volume 6, 2012 microbiol indones 37 sacrificed. liver and kidneys were removed from abdominal cavity and weighed. liver and renal samples of the control and treated mice were fixed in 10% phosphate buffered formalin for 24 h and then the samples were dehydrated and embedded in paraffin. sections of 5 µm were done and stained with hematoxylin and eosin stains for histopathological studies under a bright field microscope with a magnification 400 times. a semi-quantitative analysis was done to assess the extent of the histopathological changes (wang et al. 2000). statistical analysis. the statistical significance of the differences between control and experimental groups was evaluated by student's t-test using statistical package for the social sciences 13.0 (spss 13.0) software. results mortality, clinical signs, body and organ weights. there was no difference found in the mortality and clinical abnormalities in mice treated p. resticulosum pigment in comparison to control group (table 1). however, we found one aggressive male -1 mouse in the group that was given a dose 1000 mg kg body weight p. resticulosum pigment daily. absolute body, liver, and kidney weight in both male and female mice fed p. resticulosum pigment did not differ significantly (p>0.05) in comparison to control group mice. there were no significant (p>0.05) difference in the ratios of liver to body weight and kidney to body weight in comparison to control group mice (table 2). biochemical studies. there was no significant difference (p>0.05) in the level of ast, alt, alp, ldh and bun in both male and female mice (table 3). histopathological liver and kidney. there were no specific abnormalities of liver histopathology profile in the mice treated with p. resticulosum pigment -1 (125 and 250 mg kg body weight daily) for 28 d in comparison to that of the control group (fig 1). very mild sinusoidal congestion was found around the vascular central vein as a response to chloroform anesthesia. the lobular and sinusoids appeared normal and no congestion of sinusoids was seen at liver of mice treated with doses 0 (fig 1a), 125 (fig 1b), and 250 -1 (fig 1c) mg kg body weight p. resticulosum pigment, but fat degeneration and necrosis in some lobus were found in the liver of mice treated with 500 (fig 1d) and -1 1000 (fig 1e) mg kg body weight p. resticulosum pigment daily for 28 d. observations (fig 2) of the pigment effects on kidney histology of mice showed that there was no found abnormal glomerulus, tubular necrosis, and atrophy in the kidneys of mice treated dose 0 (fig 2a), 125 (fig 2b), 250 (fig 2c), 500 (fig 2d), and 1000 -1 (fig 2e) mg kg body weight p. resticulosum pigment for 28 d. clinical sign p. resticulosum pigment dose (mg kg -1 bod y weight daily) 0 (con trol) 125 250 500 1000 m f m f m f m f m f mortality 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 motor activity 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 tremors 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 convulsion s 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 straub reaction 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 aggressiveness 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 0/5 pilo-erection 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 loss of lightin g reflex 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 sedation 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 muscle relaxation 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 hypn osis 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 analgesia 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 ptosis 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 lacrimation 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 diarrh ea 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 skin color 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 table 1 mortality and clinical signs of balb mice /c treated with oral administration of p. resticulosum pigment for one week m: male of five measurements; f: female of five measurements values represent the mean ± sd of five measurements, p<0.05 indicates significant difference from controls. table 2 effect of oral administration of p. resticulosum pigment on body, liver, and kidney weight of mice 38 sopandi and wardah parameters p. resticulosum p igment dose (mg kg -1 body w eight daily) 0 (con trol) 125 250 500 1000 ast (ul-1 ) male 76.0 2±0.75 76.04±1.11 76.70±1.27 77.01±1.27 77.84±0.72 female 83.6 2±1.33 84.19±0.88 84.74±1.50 85.86±1.38 85.96±2.08 alt (ul -1 ) male 75.1 5±0.81 74.97±0.52 75.80±1.13 75.73±0.92 76.40±1.09 female 74.6 3±1.31 75.18±0.97 75.52±1.68 75.54±0.76 76.35±118 alp (ul-1 ) male 38.3 0±1.80 38.81±2.35 40.72±1.31 40.16±0.95 40.42±0.69 female 34.7 0±1.45 34.81±2.19 36.52±1.03 36.56±0.95 37.02±1.23 ld h (ul -1 ) male 1288.47±6 7.62 1342 .59±5 4.31 1348.74±30.23 1351.77±45.82 1367.54±28.80 famale 1199.51±7 2.58 1249 .64±5 5.64 1269.86±39.88 1280.03±36.71 1263.79±47.33 bun (mg dl-1) male 16.2 6±3.32 15.94±3.35 16.06±3.60 17.94±3.01 15.90±3.07 female 16.9 0±2.38 18.28±1.12 16.68±2.40 18.10±1.26 18.14±1.55 table 3 effect of p. resticulosum pigment administration on biochemical parameters of mice microbiol indones discussion evaluation of oral toxicity using 28 d toxicity tests generally has been conducted in sub-acute toxicity study which is the fundamental test to determine the level of product safety(arts et al., 2004: wang et al. 2007; poorniammal et al. 2011 ), including the safety of food coloring pigments produced by microorganisms p. resticulosum pigment dose (mg kg -1 body weigh t daily) 0 (control) 125 250 500 1000 absolute body weigh t (g) prior treatment n.d. male 29.18±2.36 31.64±3.63 30.98±0.97 30.42±2.36 27.86±3.63 female 26.20±1.30 27.20±1.92 27.40±1.14 26.01±1.58 26.80±3.63 on day 28 male 29.38±2.32 31.78±3.63 31.10±0.83 30.66±2.31 28.46±3.22 female 26.40±1.34 27.46±1.84 27.66±1.12 26.32±1.51 27.06±0.83 liver male 1.37±0.054 1.4 1±0.084 1.40±0.021 1.40±0.058 1.35±0.075 female 1.30±0.019 1.3 3±0.054 1.32±0.045 1.29±0.022 1.28±0.027 kidney male 0.51±0.046 0.5 2±0.059 0.51±0.016 0.51±0.027 0.51±0.066 female 0.44±0.005 0.4 7±0.014 0.44±0.010 0.44±0.014 044±0.007 relative (% g animal -1 ) liver male 4.72±0.22 4.49±0.25 4.53±0.12 4.61±0.18 4.89±0.40 female 4.97±0.19 4.88±0.18 4.83±0.09 4.97±0.23 4.79±0.09 kidney male 1.74±0.04 1.64±0.02 1.65±0.09 1.66±0.06 1.71±0.02 female 1.75±0.13 1.70±0.07 1.70±0.08 1.77±0.12 1.67±0.02 values represent the mean ± sd of five measurements, p<0.05 indicates significant difference from controls. n.d.: not detected volume 6, 2012 microbiol indones 39 (kumari et al. 2009). in this study, during the administration period, no death occurred in animals from any group. there were no abnormal signs in the groups. pigments from penicillium have varied toxicity depending on the comonents of the pigment. pigment omponent such as atrovenetin and norherqueinone from p. antrovenetum (raistrick et al. 1958), antrovenetin and herquinones from p. herquei (robinson et al. 1992), anthraquinone from p. citrinum (duran et al. 2002), phoenicin from p. antrosanguineum, xanthoepocin from p. brevicompactum, anthraquinone derivatives arpink red from p. oxalicum (mapari et al. 2005), mitosrubin, mitosrubinol and purpurogenone from p. purpurogenum (mapari et al. 2006) have been known as non-toxic pigment. on the other hand, toxic pigment components include citrinine from p. citrinum (duran et al. 2002), and acid-secalonic d from p. oxalicum (mapari et al. 2005). toxicity of pigment produced by p. resticulosum has not been reported by previous investigators. shridhar et al. (2009) reported that the culture filtrate of p. resticulosum did not contain mycotoxins and 1.5 ml of culture filtrate of p. resticulosum only causes the death of mice after administration of the filtrate for 45 d. body and organ weights and ratio of organ with body weight are indicators of organ damages or abnormalities due to provision of treatment (poorniammal et al. 2011). this study indicates that the administration of p. resticulosum pigment for 28 d produced no effect on the body weight gain and abnormalities in liver and kidney of mice. shridhar et al. (2009) reported that although the administration of 1a 1b 1c 1d 1e fig 1 liver section (magnification 400 x) of mice treated with orally administered various doses of p. resticulosum pigment: 0 -1 (a), 125 (b), 250 (c), 500 (d), and 1000 (e) mg kg body weight daily for 28 d. vena centralis (cv) marked with black arrows, hepatocyte cell (hc) marked with red arrow, and sinusoids (ss) marked with white arrows. fig 2 kidney section (magnification 400 x) of mice treated with orally administered various doses p. resticulosum pigment: 0 -1 (a), 125 (b), 250 (c), 500 (d) and 1000 (e) mg kg body weight per day for 28 days. glomeruli (gb) marked with black arrows, tubules (tb) marked red arrows, and proximal tubules (ptb) marked with white arrows. 2a 2b 2c 2d 2e microbiol indones 1.5 ml of culture filtrate of p. resticulosum gave no effect on the body weight of mice, the body weight decreased gradually until the provision of 45 d. activities of ast, alt, alp, and ldh are indicators of liver damage. our study demonstrated that the daily intake of p. resticulosum pigment for 28 day exhibited no significant effect on ast, alt, alp, and ldh when compared to the both male and female control mouse. the culture filtrate of p. resticulosum with a dose of 1.5 ml per day for 45 d can increase the concentration of ast and alt enzymes, indicating liver damage (shridhar et al. 2009). fumaryl-dlalanine (fumaromono-dl-alanine) is a metabolic product of p. resticulosum that might have caused altered permeability and/ cell necrosis, leaked of the enzymes into the bloodstream (birkinshaw et al 1942). consumption of synthetic or natural food colorant after 30 days of treatment increased serum creatinine and urea in rats (helal et al. 2000; himri et al. 2011). blood urea nitrogen is an indicator of kidney damage as a result of enzymatic hydrolysis of urea by urease to ammonia (zafar et al. 2010). our study showed upon the administration p. resticulosum pigment in mice for 28 days there were no differences in bun levels in both male and female mice compared to mice control groups. loss of the structure and abnormal function of cells are the results fat metabolism disorder in cells, inducing degeneration. cells undergoing fatty degeneration was characterized by the accumulation of metabolic products such as molecules of fat, protein and glycogen in abnormal amounts. fatty degeneration of the cells indicates the presence of biochemical disturbances caused by abnormal metabolism. toxic chemicals that are microscopically visible as grains of fat accumulated in the liver lobes, especially the perilobular tissue. the present study revealed that mouse consuming high dose -1 of p. resticulosum pigment (500 and 1000 mg kg body weight) daily for 28 days exhibited slight existence of fatty degeneration and necrosis in some lobus. kidney proximal tubule is the most vulnerable to damage from toxic substances. in the proximal tubule occur the process of absorption and secretion of various substances. if there is absorption of toxic materials in the tubular epithelium, it would interfere with the metabolism and absorption. in addition, levels of cytochrome p-450 in the proximal tubule increased to detoxify toxic substances. in the present study, microscopic observations of kidneys histopathologic preparations found no abnormalities in glomeruli, tubules, proximal tubules and capillaries between the tubules. in conclusion, the 28 d sub-acute toxicity evaluation indicates that pigments produced by p. resticulosum can be categorized as low toxicity pigments. provision of p. resticulosum pigment up to a -1 dose of 500 mg kg body weight daily for 28 days had no effect on body weight, organ weights, and activities of ast, alt, alp, ldh enzymes and bun. however, mouse taking p. resticulosum pigment above 500 mg -1 kg body weight daily showed fatty degeneration and mild necrosis of liver cells indicating that the consumption of pigment from p. resticulosum was still -1 safe up to doses below 500 mg kg body weight daily for 28 d. acknowledgment the authors would like thank to dp2m higher education for financial support of the research through research grants for fundamental research year 2011. references aberoumand a. 2011. a review on edible pigments properties and sources as natural biocolorants in foodstuff and food industry. world j dairy food sci. 6(1):71-78. arts jhe, muijser h, appel mj, kuper cf, bessems jgm, woutersen ra. 2004. sub-acute (28-day) toxicity of furfural in fischer 344 rats: a comparison of oral and inhalation route. food chem toxicol. 42(9):1389-1399. doi:10.1016/j.fct.2004.03.014. babitha s, soccol cr, pandey a. 2006. jackfruit seeda novel substrate for the production of monascus pigments through solid-state fermentation. food technol biotechnol. 44(4):465-471. downham l, collins p. 2000. colouring our foods in the last and nex millenium. int j food sci technol. 35:5-22. birkinshaw jh, raistrick h, smith g. 1942. studies in the 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edelstein cl. 2010. sirolimus attenuates disease progression in an orthologous mouse model of human autosomal dominant polycystic kidney disease. kidney int. 78(2): 754-761. doi:10.1038/ki.2010.250. 1: 35 2: 36 3: 37 4: 38 5: 39 6: 40 7: 41 6.mi724-diana nurani available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.6issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 177-185 *corresponding author; phone/fax: +62-21-3169513/+6221-3169510, email: diana.nurani@yahoo.co.id tropical peat consists of lignin, hemicellulose, cellulose, and protein. lignin biodegradation in peat soil produces several organic acids. the organic acids resulted in lignin biodegradation process are from the groups of aliphatic and aromatic compounds. aliphatic compounds found in peat soil are originated from carboxylic acid derivatives such as acetic, formic, propionic, and butyric acids, while the aromatic compounds are from phenolic acid derivatives such as vanillic, p-hydroxybenzoic, p-coumaric, ferulic, and syringic acids (alexander 1977; stevenson 1994; hartley et al. 1984). phenolic acid generally has negative effects to plant nutrient absorption and growth (tadano et al. 1992). phenolic acid, with a range of concentration between 4-30 mm, showed toxic effects in several some organic acids derived from lignin degradation in peat soil have significant role in lowering ph of peat soil and have phytotoxic effect for plant. a series of experiments has been conducted to decrease the amount of organic acids in peat soil by microbial treatment. the aim of the study was to characterize the growth of selected fungus using organic acids extracted from peat soil as a sole carbon source.the fungus strain mgs-2 was grown in a liquid medium containing various concentrations of organic acids extracted from peat soil, nitrogen and salt at initial ph of 3.8. the cell growth, ph of medium and the content of organic acids were analyzed. the identification of fungus mgs-2 was based on the 28s rdna.the ability of hypocrea sp. mgs-2 to degrade toxic organic acid derived from peat soil has never been reported. the organic acid and nitrogen optimum for the growth were 33.1 mn-naoh and 2 g (nh ) so per liter medium, respectively. the interaction between carbon 4 2 4 and nitrogen source was found to be significantly influenced by the increment of ph medium, however this interaction did not effect to the cell growth and reduction of organic acids. the carbon source affected significantly to the cell growth and acid metabolism by fungus strain mgs-2 the fungus could not grow well in the medium without yeast exract, but grew well in the limitation of nh so , suggested that yeast extract was 2 4 metabolized as nitrogen source. the optimum degradation of organic acids extracted from peat soil by mgs-2 was obtained at ph 3.8. these result suggested that the cell mass production of mgs-2 can be performed in optimal defined medium utilizing organic acid derived from peat soil. molecular identification revealed that the mgs-2 was closed to hypocrea sp. key words: acid tolerant-fungus, peat soil, organic acid degrading-fungus beberapa asam organik yang berasal dari degradasi lignin dalam tanah gambut memiliki peran penting dalam menurunkan ph tanah dan toksisitas terhadap tanaman. serangkaian riset telah dilakukan untuk mengurangi jumlah asam organik dalam tanah gambut dengan perlakuan mikroba. tujuan dari riset ini adalah untuk mengkarakterisasi pertumbuhan mikroba terpilih menggunakan asam organik yang diekstraksi diekstraksi dari tanah gambut sebagai sumber karbon tunggal. strain jamur mgs-2 ditumbuhkan dalam medium cair yang mengandung berbagai konsentrasi asam organik, nitrogen dan garam pada ph awal 3,8. pertumbuhan sel, ph dan kandungan asam organik dianalisis. identifikasi jamur mgs-2 didasarkan pada 28s rdna. kemampuan hypocrea sp.strain mgs-2 dalam menurunkan asam organik beracun yang berasal dalam tanah gambut belum pernah dilaporkan. asam organik dan nitrogen yang optimal untuk pertumbuhan adalah 33,1 mn-naoh dan 2 g (nh ) so per liter media, masing-masing. interaksi antara sumber karbon dan nitrogen berpengaruh terhadap 4 2 4 peningkatan ph, namun interaksi ini tidak berpengaruh terhadap pertumbuhan sel dan pengurangan asam organik. sumber karbon terpengaruh secara nyata terhadap pertumbuhan sel dan metabolisme asam oleh strain mgs-2. strain hypocrea sp.mgs-2 tidak bisa tumbuh dengan baik di media tanpa ekstrak ragi, tetapi tumbuh baik dengan pemberian nh so secara terbatas. degradasi optimum asam organik oleh strain hypocrea 2 4 sp.mgs-2 berlangsung pada ph awal ph 3,8. hasil ini menunjukkan bahwa produksi sel mgs-2 dapat dilakukan dengan medium minimal menggunakan asam organik dari tanah gambut. identifikasi molekuler mengungkapkan bahwa mgs-2 adalah hypocrea sp. kata kunci: asam organik, jamur toleran asam, tanah gambuthypocrea sp. strain mgs-2, growth characteristic of fungus strain mgs-2 in a defined medium containing organic acids derived from peat soil diana nurani* and koesnandar laboratory of industrial technology development for agro and biomedical center for bioindustrial technology, agency for the assessment and application of technology (bppt) th bppt buiding ii 15 floor, jalan mh thamrin no 8, jakarta 10340, indonesia species of plant (guenzi et al. 1966). phenolic acids such as ferulic, p-coumaric, and p-hydroxybenzoic acids are considered more harmful compared to carboxylic acids, because of its greater toxicity. other than harming plants, organic acid found in peat soil also causes low ph. several microbes in soil have the ability to quickly decompose organic acids. studies have been conducted to develop the utilization of organic acids as carbon source for microbes (mccarty et al. 1986). phenolic acid, which was chosen as one of the organic acid that can be utilized by the microbes in soil and rhizosphere, has been studied by adding one type of -1 phenolic acid with a concentration of ≥ 0,25 µmol g of soil. the addition of p-hydroxybenzoic acid in soil showed that microbes were able to utilize the acid as their single source of carbon (mohamed et al. 2009). several genera of bacteria are able to degrade phenolic acid and use it as a carbon source, one of them is azotobacter (juarez et al. 2005), while from the group of actinomycetes, streptomyces sannanensis can grow optimally by using ferulic acid with a concentration of 5 mm as its carbon source (ghosh et al. 2007). beside bacteria and actinomycetes, there are also several fungi that are able to utilize phenolic acid, such as the white rot fungus schyzophyllum commune, pycnoporus cinnabarinus, and trametes sp., using coumaric and ferulic acids as their single carbon source (sachan et al. 2010). according to ghosh et al. (2006), the fungi paecilomyces variotii grows optimally by using ferulic acid as its carbon source with a concentration of 10 mm. we have been conducting a series study to decrease the amount of phytotoxic organic acid in peat soil. the aim of the current study was to characterize the growth of selected fungus using organic acid extracted from peat soil as a sole carbon source. materials and methods extraction of organic acid and liquid medium. organic acids were extracted from peat soil using a modified method from maciak and harms (1986). ten grams of soil was added by 100 ml of 2 m naoh and then agitated on a shaker for 3 h at room temperature. the solution was separated from the solid fraction by centrifugation for 20 min at 8 000 rpm (8770 x g). h so solution was added gradually to the 2 4 liquid fraction while being mixed, until precipitation was fully formed. liquid fraction was separated by centrifugation for 20 min at 8 770 x g and then used as 178 nurani et al. microbiol indones carbon source in this study. fungi and liquid medium. a medium containing organic acid at low ph (3.8) was used to isolate a potent fungal-degrading organic acid of peat soil. the growth characterization of mgs-2 was done using modified czapek's media (labeda, 1990), with the following -1 composition (g l ) : 0.5 g of mgso .7h o; 3 g of 4 2 (nh ) so ; 1 g of kh po ; 0.5 g of yeast extract; and 4 2 4 2 4 33.1 mn-naoh organic acid, with the ph medium 3.8. analysis of total acid and phenolic acid. total acid was quantified by titration method (aoac, 19190). two ml of sample was added to a 250 ml erlenmeyer. the liquid was diluted with aquades until it reached 20 ml of volume (dilution factor = 10) and was added several drops of phenolphthalein indicator. the solution was titrated with 0.1 n naoh until a pink color was formed. the concentration of phenolic acid derivatives was detected by hplc. ten ml of sample or medium was acidified to ph 2.5 with 1 m hcl. the solution was then diluted by adding 10 ml of methanol (ch o) : water (h o) : acetic acid (c h o ) solution. 4 2 2 4 2 the solution was then used in hplc to determine the phenolic acid concentration. the types of phenolic acid which constitute the sample was determined by partition separation system using a reversed-phase column (bondapak c18 column, 3.9 x 300 mm) and a uv detector with d2 light at 240 nm wavelength. fungal growth characterization. growth characterization was determined by optimizing carbon and nitrogen source. the concentrations of organic acid used as carbon source (c1, c2, c3, and c4) are -1 33.1; 66.2; 99.3; 132.4 mn-naoh l of medium, while the concentrations of (nh ) so as nitrogen 4 2 4 -1 source (n1, n2, n3, n4) are 1, 2, 3, 4 g l of medium. 10 ml of mgs-2 culture was inoculated into 100 ml medium (with composition as stated in table 1) and incubated at room temperature for 48 hours with agitation at 120 rpm. observation was conducted by measuring total acid, ph, cell growth (dry cell mass). the study was done with two repetitions for each treatment. 28s rdna-based identification of mgs-2. the 28s rdna of the mgs-2 were amplified by pcr using a pair of specific primers, nl1(5’-gcatatcaataag cggaggaaaag-3’) and nl4 (5’-ggtccgtgttt caagacgg-3’) (park et al. 2002). the pcr product was analyzed using 1% agarose gel electrophoresis. after the dna band was confirmed, to obtain nucleotide sequences of 28s rdna gene, the pcr product was purified and directly sequenced using both primers by an automated dna sequencer volume 7, 2013 microbiol indones 179 (applied biosystem 3130). the sequences were then analyzed for identity using ncbi blast (http://www.ncbi.nlm.nih.gov) with 28s rdna sequences available at the ncbi database. multiple sequence alignment of mgs-2 sequence and its blast result sequences was carried out using clustal x software (http://www.clustal.org/clustal2/). phylogenetic tree was then constructed by distance method in phylip software (http://evolution.genetics. washington.edu/phylip.html). statistical analysis. data resulted from this study was analyzed as a factorial experiment with completely randomized design (2 factors). every treatment was repeated two times. significant difference between treatments was carried out by duncan analysis. results growth characterization of mgs-2. mgs-2 reached its optimum growth when cultured in media with a concentration of organic acid of 33.1 mn-1 naoh, and a concentration of nitrogen source of 2 g l . the ph of the medium increased to 5.43 at optimum growth (table 2, fig 1), followed by a decrease in organic acid as much as 4.8 mn-naoh during 48 hours of fermentation (fig 2). the results of variety analysis towards ph showed that the interaction of c and n significantly influenced the ph at 5% level (p < 0.05 → 0.00 < 0.05). this result suggested that the c and n source was strongly needed by strain mgs-2 in degrading organic acid. the duncan test also indicated that the combination factor -1 of c1n2 (33.1 mn-naoh of carbon source and 2 g l of nitrogen source) significantly produced a different ph compared to other combination factors of treatment. the results of variety analysis towards cell growth showed that interaction of c and n did not significantly affects the cell growth at 5% level (p < 0.05 → 0.00 < 0.05). a single factor analysis showed that only c factor significantly influenced the cell growth at 5% level (p < 0.05 → 0.00 < 0.05). the duncan test towards the c factor also showed that there were two treatment groups produced a significant influence to the cell growth. the duncan test also indicated that carbon source was strongly required for the growth of strain mgs-2. in total acid analysis, the interaction of c and n did not affects the total acid reduction at 5% level (p < 0.05 → 0.00 < 0.05). however, in a single factor analysis, c factor significantly influenced the total acid reduction at 5% level (p < 0.05 → 0.00 < 0.05), and the n factor also significantly influenced the total acid reduction at 5% level (p < 0.05 → 0.00 < 0.05). the duncan test towards the c factor showed that two groups of treatment exhibited different effects to the cell growth. the duncan test towards the n factor also showed a similar result to the cell growth. the analysis of variance and duncan test indicated that each carbon and nitrogen source is strongly required by strain mgs-2 during degradation of organic acid. optimization of nitrogen. strain mgs-2 was unable to grow in medium without yeast extract. in this medium, the ph increase to 4.39 at 36 h incubation. in medium without ammonium sulphate, the strain mgs2 was still capable to grow, and the ph medium was found increasing to 5.79 at 36 h incubation (fig 4). optimization of ph. effect of initial ph value on strain mgs-2 growth was investigated by treatment of initial ph variation in medium with optimum c and n concentration range from 3.8-6.5. the result showed higher initial ph gave slower rate of organic acid degradation (fig 5). it can be concluded that strain mgs-2 is tolerant towards low ph and showed highest carbon source = organic acid -1 (mn-naoh l medium) -1 nitrogen source = (nh ) so (g l medium)4 2 4 n1=1 n2=2 n3=3 n4=4 c1=33.1 c1n1 c1n2 c1n3 c1n4 c2=66.2 c2n1 c2 n2 c2n3 c2n4 c3=99.3 c3n1 c3n2 c3n3 c3n4 c4=132.4 c4n1 c4n2 c4n3 c4n4 table 1 carbon source and nitrogen source variation in growth medium *) other compositions of the growth media include mgso4.7h2o; kh2po4 and yeast extract. microbiol indones180 nurani et al. nitrogen -1 source (g l ) 33,1 66,2 99,3 132,4 1 ef5,34 bc5,2 bcd5,22 e5,32 2 g 5,43 cd5,25 b5,18 bcd5,22 3 fg 5,39 bcd5,22 b5,19 a5,08 4 d5,26 ef5,34 bc5,2 a5,12 carbon source (mn-naoh) table 2 final culture ph of strain mgs-2 with c & n source variations after 48 hours of incubation fig 1 ph value of strain mgs-2 growth media with carbon and nitrogen source variations. fig 2 decreased total acid consumed by strain mgs-2 in growth media with carbon and nitrogen source variations n1 n1 n1 n1 n2 n2 n2 n2 n3 n3 n3 n3 n4 n4 n4 n4 carbon and nitrogen source variations 4.9 5 5.1 5.2 5.3 5.4 5.5 5.34 5.2 5.22 5.32 5.43 5.25 5.18 5.22 5.39 5.22 5.19 5.08 5.26 5.34 5.2 5.12 p h c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 carbon and nitrogen source variations c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 n1 n1 n1 n1 n2 n2 n2 n2 n3 n3 n3 n3 n4 n4 n4 n4 d ec re as ed t o ta l ac id ( m n -n ao h ) 0 1 2 3 4 5 6 2.9 0.9 1.8 2.1 4.8 2.6 1.9 2 3.4 0.8 0.7 0.5 3.6 3.4 1.8 1.6 volume 7, 2013 microbiol indones 181 degradation activity of organic acid as a sole carbonat ph 3.8. growth of mgs-2 in the optimum medium. the rise of ph in the medium until 12 h incubation showed that strain mgs-2 was not only capable in consuming phenolic acid (p-hydroxybenzoic acid), but also capable to utilize non-phenolic organic acids for their growth (fig 6 and fig 8). non-phenolic organic acids consumption possibly started from the initial growth phase until the maximum growth phase (6-12 h incubation). the increment of culture ph was also followed by reduction in total acid, started from 6 to 12 h incubation (fig 6). during the period of incubation, the cell growth was inclined, and then declined after the 12 h (fig 7). depleting of organic acid in the medium possibly caused lysis in the fungal cells. the phydroxybenzoic acid was even already consumed during the early growth phase. in addition,the phenolic acid (p-hydroxybenzoic acid) was also possibly already degraded during the early growth phase (6-12 h incubation), because it was not detected in the medium during that incubation period (fig 8). molecular identification. the blast result of the 28s rdna sequence of strain mgs2 showed 100% fig 3 cell growth of strain mgs-2 in growth media with carbon and nitrogen source variations. -1 c el l g ro w th ( g l ) 0 20 40 60 80 100 120 140 160 180 4.65 32.78 12.35 117.47 8.9 36.72 89.59 7.8 18.57 42.8 8.28 155.09 32.56 29.84 75.05 carbon and nitrogen source variations c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 n1 n1 n1 n1 n2 n2 n2 n2 n3 n3 n3 n3 n4 n4 n4 n4 6.5 6 5.5 5 4.5 4 3.5 3 p h 0 12 24 36 48 60 72 incubation time (h) fig 4 yeast extract effects on the growth of strain mgs-2. : carbon and nitrogen optimal, : non (nh ) so , : non 4 2 4 yeast, and : non non (nh ) so and yeast.4 2 4 soil as a sole carbon source has been isolated and characterized. carbon and nitrogen are essential nutrition required by microbes for their growth and metabolism. gao et al. (2007) stated that carbon and nitrogen demand varies in different strains of fungi depending on each strains growth characteristic. strain mgs-2 can grow optimally in medium containing 2 g -1 l of ammonium sulphate. the same concentration of ammonium sulphate was also reported capable to increase the growth of phytophthora capsici (perveen, 2010). hypocrea sp. strain mgs-2 was also optimally similarity and query coverage to several species of trichoderma/hypocrea. the highest similarity is hypocrea sp. (gu048594.1). the phylogenic tree (fig 9) indicated that strain mgs-2 showed a close phylogenetic relationship to hypocrea sp. (gu048594. 1) genus hypocrea is a telemorph state (sexual) of trichoderma. discussion a fungus utilizing organic acid extracted from peat microbiol indones182 nurani et al. fig 5 influences of initial ph on the growth of strain mgs-2. : initiated ph, : final ph, and :∆ ph. fig 6 ph and total acid value during the growth of strain mgs-2 in optimum medium for 48 h. :ph and :acid total. 3.8 4.5 5 5.5 6 6.5 variation of initial ph 8 7 6 5 4 3 2 1 0 p h 5.85 3.8 2.05 4.5 6.18 1.68 5 6.32 1.32 5.5 6.55 1.05 6 6.64 0.64 6.5 6.9 0.4 p h 3.00 4.00 5.00 6.00 7.00 8.00 0 6 12 18 24 30 36 42 48 14 12 10 8 6 4 2 0 a ci d t o ta l (m n -n ao h ) incubation time (h) volume 7, 2013 microbiol indones 183 fig 7 cell growth of strain mgs-2 in optimum medium during 48 h incubation. fig 8 phenolic acid consumption by strain mgs-2 in optimum medium. fig 9 phylogenetic analysis of strain mgs-2 based on 28s rdna analysis. the mgs-2 strain is identified as hyprocrea sp (circled). the other strain are h.sch.chta: hypocrea schweinitzii (gu048594.1), h.jec.28s : hypocrea jecorina (af127154.1), h.sch.lsu1: hypocrea schweinitzii lsu,(ay281095.1), h.jec.lsu: hypocrea schweinitzii lsu(ay281097.1), h.jec.2444: hypocrea jecorina 24449 lsu (hm466681.1, t.longibra: trichoderma longibrachiatum atcc 18648 lsu (hm466682.1), h.sch.icmp: hypocrea schweinitzii icmp1694 lsu (ay283549.1), h.sch.lsu2: hypocrea schweinitzii lsu (af279395.1), h.jecorina : hypocrea jecorina, tri.sp.: trichoderma sp. (kj372238.1). 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 -1 d ry w ei g h t ce ll ( g l ) 0 6 12 18 24 30 36 42 48 incubation time (h) 0 2 4 6 8 10 12 14 16 18 p h en o li c ac id ( p p m ) 0 12 24 36 48 incubation time (h) h.sch.chta h.jec.28s h.sch.lsu1 h.jec.lsu h.jec.2444 t.longibra h.sch.icmp h.sch.lsu2 h.jecorina 3128sl tri.sp. grown at 33.1 mm-naoh. several species of microbes have the ability to degrade phenolic acid and utilize it as their carbon source, these include azotobacter, streptomyces sannanensis, schyzophyllum commune, pycnoporus cinnabarinus, trametes sp., paecilomyces variotii (juarez et al. 2005; ghosh et al. 2006, 2007; sachan et al. 2010). the studies on microbial degradation of hydroxybenzoic acids provide a wealth of knowledge on the metabolism of this compound (chatterjee et al. 1987; suemori et al. 1995). during the growth of hypocrea sp. strain mgs-2, the availability of yeast extract was proven as essential factor. yeast extract possibly contains vitamins or certain amino acids which are important for the growth of hypocrea sp. strain mgs-2. other studies, using clostridium thermoaceticum grown in mineral medium, showed that only nicotinic acid derived from yeast extract essentially needed in the formation of + nad during its growth (koesnandar et al. 1990). the requirement of yeast as a growth factor by hypocrea sp. is an interesting phenomenon, because fungi usually require a simple medium. it is well known that bacteria require a more complex medium requirements. the ability hypocrea sp. strain mgs-2 to utilized organic acids as sole carbon sources derived from peat soil, suggested that the increment of culture ph was due to the deletion of organics acid in the culture. the ability of hypocrea sp. to degrade toxic organic acid has never been reported. this phenomenon is advantages because some organic acids derived from lignocellulose in peat soil is toxic to plant to grow (guenzi and mccalla, 1966). mendonca et al. (2004) reported that fusarium flocciferum was able to degrade phenolic acid, while according to sachan el al. (2010), strain schizophyllum commune was able to convert coumaric acid into p-hydroxybenzoic acid. streptomyces sannanensis was also reported for its ability to degrade ferulic acid (ghosh et al. 2007). among the group of bacteria, azotobacter chroococcum was able to degrade p-hydroxybenzoic acid in 24 hours of incubation (juarez et al. 2005). bacillus sp. and pseudomonas sp. produce phydroxybenzoic acid hydroxylase which is used in degrading p-hydroxybenzoic acid into protocatechuic acid (karegoudar et al. 2000). since hydroxybenzoic acid are the most important intermediate compound in the microbial degradative pathways of various aromatic compound (karegoudar et al. 2000), this is might be the reason that hydroxybenzoic acid was metabolized by hypocrea sp. strain mgs-2 very fast (fig 8), followed by utilization of other organic acids derived from peat soil (fig 8). thus capability hypocrea sp. strain mgs-2 to metabolize organic acid is important to release toxic compound and decreased soil acidity. previously we reported that a mixture of undefined microbes was able to improve ph and other chemicals properties of peat soil (nurani, et al. 2007), suggested that the effectivity of hypocrea sp. strain mgs-2 to improve peat soil characteristics is required. the present result showed the ability of hypocrea sp. mgs-2 to metabolize organic acid derived from peat soil in a defined medium. the application the hypocrea sp. mgs-2 to eliminate phytotoxic and improve peat soil characteristics as well as increase plant pruduction is under investigation. acknowledgment we would like to thank andreas prof. dr. ir. dwi santosa, ms. of institut pertanian bogor for his valuable discussion. we like to thank ahmad fauzi, reni giarni, ruzka radwamina, and farah of the agency for the assessment and application of technology for their support on technical matters references alexander m. 1977. introduction to soil microbiology. john willey & sons new york chatterjee dk, bourquin aw . 1987. metabolism of aromatic compounds by caulobacter crescents. j bacteriol. 169(5):1993-1996. ghosh s, sachan a, a mitra. 2006. formation of vanillic acid from ferulic acid by paecilomyces variotii mtcc 6581. curr sci. 90(6):825-829. ghosh s, sachan a, sen sk , mitra a . 2007. microbial transformation of ferulic acid to vanillic acid by streptomyces sannanensis mtcc 6637. j ind m i c r o b i o l b i o t e c h n o l . 3 4 ( 2 ) : 1 3 1 1 3 8 . doi:10.1007/s10295-006-0177-1. guenzi wd, mccalla tm. 1966. phenolic acids in oats, wheat, sorghum, and corn residues and their p h y t o t o x i c i t y. j a g r o n . 5 3 ( 3 ) : 3 0 3 3 0 4 . doi:10.2134/agronj1966.0002196200580003001 7x. hartley rd, whitehead, dc. 1984. phenolic acids in soil and their influence of plant growth and soil microbial processes. dalam vaughan, d. dan r.e. malcolm. soil organic matter and biological activity. martinus nijhoff/dr. w. junk publisher. lancaster. juarez b, martinez tmv, gonzalez lj. 2005. growth of azotobacter chroococcum in chemically defined media containing p-hydroxybenzoic hcid microbiol indones184 nurani et al. an d p r o to catech u ic acid . c h emo s p h er e. 59(9):1361-1365. doi:10.1016/j.chemosphere.20 04.11.037. karegoudar tb, kim ck. 2000. microbial degradation of monohydroxybenzoic acids. j microbiol. 38(2):53-61. koesnandar n. nishio sn. 1990. stimulation by cysteine on growth of clostridium thermoaceticum in minimal medium. appl microb biotechnol. 32(6):711-714. doi:10.1007/bf00164746. labeda dp. 1990. isolation of biotechnological organisms from nature. mcgraw-hill publishing company. new york. li g, sun mh , liu xz, yong cs. 2007. effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi. myco res.111(1):87-92. doi:10.1016/j.mycres.2006.07.019. maciak f, h harms. 1986. the effect of agricultural utilization on the composition and yield of phenolic acids in low peat soils. plant and soil 94(2):171-178. doi:10.1007/bf02374341. mccarty gw, bremner jm. 1986. effects of phenolic compounds on nitrification in soil. soil sci soc am j. 50(4):920-922. doi:10.2136/sssaj1986.036 15995005000040018x. mendonca ea. martins ama. 2004. biodegradation of natural phenolic compounds as single and mixed substrates by fusarium flocciferum. j biotechnol. 7(1):30-37. mohamed man, sebai tnm, hartmann a. 2009. effect of co-enrichment, soybean rhizosphere and p-hydroxybenzoic acid, on microbial metabolic diversity and p-hba degradation. j agric biol sci. 5(4):301-309. nurani d, s, h, g, koesnandar. 2007. increase in ph of peat soil by microbial treatment. international symposium and workshop on tropical peatland. yogyakarta, 27-31 august 2007. park hs, kim gy, nam bh, lee sj, lee jd. 2002. the determination of the partial 28s ribosomal dna sequences and rapid detection of phellinus linteus and related species. mycobiology 30(2):82-87. doi:10.4489/myco.2002.30.2.082. perveen r. 2010. effect of nitrogen and carbon from different organic supplements on pathogenic potential of phytophthora capsici, in the collar rot of chillies. eur j sci res. 43(1):107-112. sachan a, gosh s, mitra a. 2010. transforming pcoumaric acid into p-hydroxybenzoic acid by the mycelial culture of a white rot fungus schizophyllum commune. afr j microbiol res. 4(4):267-273. stevenson fj. 1994. humus chemistry : genesis, composition, reaction. john wiley & son. new york. suemori a, nakajima k, kurane r, nakamura y. 1995. o-, mand p-hydroxybenzoate degradative pathways in rhodococcus erythropolis. fems microbiol lett. 125(1):31-36. doi:10.1111/j.15746968.1995.tb07331.x. tadano t, yonebayashi k, saito n. 1992. effect of phenolic acids on the growth and occurrence of sterility in crop plants. pp: 358-369. in: k. kyuma, p. vijarnsorn, and a. zakaria (eds). coastal lowland ecosystems in southern thailand and malaysia. showado-printing co. skyoku. kyoto. parmiyatni purwanta angkoso volume 7, 2013 microbiol indones 185 1: 177 2: 178 3: 179 4: 180 5: 181 6: 182 7: 183 8: 184 9: 185 04 ambarsari.cdr performance optimization of microbes from shrimp pond sediment by adding em4 in nitrification process for the treatment of wastewater containing high ammonia concentration 1 2 hanies ambarsari * muhammad rahmadi harahap and 1 institute for water and waste treatment technology (btpal), agency for assessment and application of technology (bppt), gedung 820 geostech kawasan puspiptek serpong, tangerang selatan 15314, banten, indonesia; 2 department of biology, faculty of life science, universitas surya, tangerang, indonesia. in liquid wastes, especially domestic wastewater, many organic substances are disposed causing water quality degradation, one of them is ammonia. liquid wastes containing ammonia can be treated using an activated sludge system. one of the active sludge that can be used is shrimp pond sediment. this experiment investigated the performance of microbes in shrimp pond sediments with the addition of em4 in the nitrification process for the treatment of wastewater with high ammonia concentration in a 8 l batch reactor capacity. the results showed that the addition of shrimp pond sediment as the active sludge could remove high ammonia level almost completely and there was known interaction between time and variation of shrimp pond quantity (p value <0,05) to the decreasing of ammonia level. efficiency of decreasing the concentration of ammonia up to 100% th could be reached on the 15 day in each treatment. the addition of em4 could shorten the period of the ammonia decreasing level by 50%. key words: activated sludge, ammonia, em4, nitrification, shrimp pond sediment dalam limbah cair, terutama air limbah rumah tangga, banyak zat organik dibuang menyebabkan degradasi kualitas air, salah satunya adalah amonia. limbah cair yang mengandung amonia dapat diolah dengan menggunakan sistem lumpur aktif. salah satu lumpur aktif yang bisa digunakan adalah sedimen tambak udang. percobaan ini meneliti kinerja mikroba pada sedimen tambak udang dengan penambahan em4 dalam proses nitrifikasi untuk pengolahan limbah cair dengan konsentrasi amonia tinggi dalam kapasitas reaktor batch 8 l. hasil penelitian menunjukkan bahwa penambahan sedimen tambak udang sebagai lumpur aktif dapat menghilangkan kadar amonia tinggi hampir seluruhnya dan diketahui adanya interaksi antara waktu dan variasi jumlah tambak udang (p value <0,05) terhadap penurunan kadar amonia. efisiensi penurunan konsentrasi amonia hingga 100% dapat dicapai pada hari ke 15 pada setiap perlakuan. penambahan em4 dapat mempersingkat lama penurunan tingkat amonia hingga 50%. kata kunci: ammonia, em 4, lumpur aktif, nitrifikasi, sedimen tambak udang vol.11, no.3, september 2017, p 94-102 doi: 10.5454/mi.11.3.4 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21 , 75791377 ext. 4089 fax:+62-21; email: 75791381 hanies.ambarsari@bppt.go.id harm health. nitrate and nitrite with too high concentration can cause diarrhea, gastrointentinal disorders, until death if not given help (soemirat 1994). wastewater containing ammonia can be processed using activated sludge system for the ammonia nitrification process. the active mud utilizes biological waste treatment in the presence of microorganisms decomposing organic substances within the sludge. the consortium of microorganisms in shrimp pond sediments is known to have the ability to break down ammonia such as nitrosomonas sp. in the process of nitrification. sediments of shrimp ponds are formed from the remains of shrimp feed and filth that settles on the bottom of the pond (burford et al. 1998). microbes in shrimp pond sediments are expected to have the ability to decompose organic substances ie the ammonia content in water. however, it is necessary to add microbiological fertilizer in the form of effective in liquid wastes, especially domestic wastewater, many organic substances are mixed and cause a decrease in water quality, one of them ammonia. in general, ammonia already exists in waters derived from urine and fish feces, but in a small concentration and can decompose naturally. according to the regulation of the minister of environment number 5 year 2014, the standard of ammonia levels for businesses or -1 activities that do not have a fixed standard is 5 mg l . the presence of too high ammonia concentrations can be detected from a very strong odor (plog et al. 1996). with high concentrations of ammonia the water can be toxic to fish, humans and also can disrupt the ecosystem in the waters. ammonia in the nitrification process will turn into nitrate and nitrite which can also microorganisms 4 (em4) as a media optimization step to the microorganism consortium of shrimp pond sediment. em4 is a useful mixture of microbial cultures that function to reduce pollutant parameters and increase nutrient ammonia (fitria et al. 2008). thus in this study several experiments were conducted to determine the performance of microbes in shrimp pond sediments with the addition of em4 for water treatment containing high concentration of ammonia so that the results expected from this research can be beneficial to the community. materials and methods time, place, and research variables. this research started from february 2017 to september 2017, located in microbiology laboratory, wastewater treatment and water treatment center (btpal), agency for assessment and application of technology (bppt) serpong, tangerang selatan, banten. the variables in this study are as follows: independent variables that are value variations in this study was the composition of sediment shrimp ponds; dependent variable which is variable of research center point that were ph as well as the concentrations of cod, ammonia, nitrite and nitrate; control variables which are controlled and constant variables were aerator, em4 concentration, mixture volume in the reactor. sediment take. the preliminary treatment in this research was sediment sampling in shrimp ponds of high school of fisheries (sekolah tinggi perikanan or stp), serang, banten. the selected shrimp farm was a pond that had been at least one month after seed stocking, so that the sediment had been collected and was expected to have many microbial decomposition of ammonia in it. sediments were taken at the center of the pond, where the sediments conical in the middle. sediment samples were taken and placed in an ammonia container. further water containing ammonia was made by adding the parent ammonia to the aquades -1 to an ammonia concentration of 100 mg l . i n i t i a l tr e a t m e n t o f e m 4 . e f f e c t i v e microorganism 4 (em4) needed to be activated before being mixed into domestic wastewater as a starter, by mixing em4 and aquades. 1:20 comparison was required for em4 and aquades, then the mixture was fermented at room temperature for 5-7 d. it is necessary to activate the microorganisms contained in em4 from dormant conditions, so that the active microorganisms return and can work optimally when mixing with ammonia solution (higa and widiana 1994). reactor design. the reactor made was a reactor that can support the working system of the batch reactor and was assisted by the aerator. the batch reactor was a simple reactor system with the working principle of inserting reactants (tambak sediments) into the reactor, then left for several days to be tested for quality within a given time range. the reactor was simply designed that of gallon measuring 12 liters, then a small hose was connected to the aerator and affixed at the bottom of the gallon. the small hose formed a circle and was given a hole every 8 cm. in the diameter of the circle was installed with a connecting hose and given a hole in the middle to drain the air from the aerator. a pvc pipe with a diameter of 1.75 cm and a height of 8 cm was given at the center of the gallon. at the bottom of the pvc pipe many small hollows were made up to 2 cm. the perforated hose and pvc pipe in the center were aimed to drain the air in all directions so that aeration is evenly distributed in each section (figure 1). research procedure. ammonia solution with -1 concentration of 100 mg l was fed into gallons vertically along with diluted em4 and shrimp pond sediments with the following variations based on the results from our preliminary study: -1 a. control (k0): ammonia solution (100 mg l ) 8000 ml, fig 1 interior design of a nitrification reactor. volume 11, 2017 microbiol indones 95 -1 b. control 1 (k1): ammonia solution (100 mg l ) 7600 ml + 400 ml em4, -1 c. control 2 (k2): ammonia solution (100 mg l ) 7500 ml + 500 ml sediment of shrimp ponds, -1 d. treatment 1 (p1): ammonia solution (100 mg l ) 7500 ml + 400 ml em4 + 100 ml sediment of shrimp ponds, -1 e. treatment 2 (p2): ammonia solution (100 mg l ) 7300 ml + 400 ml em4 + 300 ml sediment of shrimp ponds, -1 f. treatment 3 (p3): ammonia solution (100 mg l ) 7100 ml + 400 ml em4 + 500 ml sediment of shrimp ponds, -1 g. treatment 4 (p4): ammonia solution (100 mg l ) 6900 ml + 400 ml em4 + 700 shrimp pond sediment. all treatment variations were incubated at 25 °c for 18 days and treated with mechanical aerators during the study. the parameters tested were cod, ammonia (nh ), nitrite (no ), nitrate (no ) and ph (apha 3 2 3 standard method 2012). the parameters were tested on days 0, 3, 6, 9, 12, 15, and 18. all treatments were duplicated so that there were 8 experimental units and 6 controls. characterization and identification of microbes. after testing was completed, the sediment samples were taken to characterize the bacteria contained therein. samples were taken from the reactor to be cultured into nutrient agar (na) medium using o spread plate technique then incubated at 30 c for 24 h. after forming several colonies, each colony was subcultured into selective media ie steiner's medium (nitrosomonas medium) and autotrophic nitrobacter medium (nitrobacter medium). the petri dish was then o closed tightly and incubated for 24 h at 30 c. bacteria grown in selective media were then tested for catalase and gram staining before being observed under a light microscope to be identified. the catalase test was done by taking the culture of the sample by ose aseptically and then the culture was scraped on the object glass. microbial cultures spilled with 1-2 drops of hydrogen peroxide (h o ) were then observed. the presence of 2 2 bubbles formed indicates positive catalase bacteria, if there are no bubbles including the negative catalase bacteria. gram staining was also performed to morphologically characterize bacteria with the aid of a light microscope and determine the gram positive or gram-negative species of the bacterial isolate. data analysis. the data of ammonia observation were analyzed by using analysis of variance or anova (analysis of variance) method of completely random design with 4 repetitions followed by benferroni test with 5% confidence level using minitab 16. while the observation data of nitrite, nitrate and cod were processed with microsoft excel 2013 qualitatively. all data is then presented in the form of a line graph using microsoft excel 2013. microbes acclimatization. acclimatization process in this research was done in such a way with experiment design and addition of aerator at each reactor. the shrimp pond sediment that had been taken was put into the reactor for the acclimation process. the sediment composition was used in accordance with the experimental design of 500 ml for the control reactor 2 (k2), 150 ml for the treatment reactor 1 (p1), 300 ml for the treatment reactor 2 (p2), 500 ml for the treatment reactor 3 (p3), and 700 ml for treatment reactor 4 (p4). the 5% activated 5% effective microorganisms (em4) were also incorporated into the 400 ml reactor for each of the experimental reactors, -1 while the ammonia water at concentrations of 30 mg l was added to the reactor until each the reactor had a total volume of 8 liters. acclimatization aimed to select and adapt microorganisms in shrimp pond sediments so that they were more accustomed to higher concentrations of ammonia compounds. this process was performed for ten days and at the end of the process it was known that ammonia had decreased its -1 -1 concentration perfectly from 30 mg l to 0 mg l . treatment of ammonia high water in batch reactors. after the acclimatization of the shrimp pond sediment was done and showed that the ammonia content could be derived, then the operation of the test reactor was started with the addition of ammonia -1 solution 100 mg l up to the total volume of the reactor to 8 l. results characteristics of shrimp ponds in stp serang can be seen in table 1. the condition of the sediment showed a more acidic figure with its ph of 6.7 when compared with the shrimp pond water with its ph of 7.8. decreased ammonia concentrations in batch reactors. based on the result of absorbance determination of ammonia standard solution a linear regression was got y = 0.0049 + 0.9358x, with r2 = 0.9995. the results of the efficiency of decreasing the ammonia concentration in the batch reactor for eighteen days can be seen from figure 2. based on the graph in figure 2 it can be explained qualitatively that the zero control (k0) containing only the ammonia -1 solution of 101.92 mg l and the control one (k1) 96 ambarsari et al. microbiol indones -1 containing an ammonia solution of 87.13 mg l with the addition of 400 ml em4 5% showed an efficiency in ammonia concentration decrease of only 10.66% and 25.87%, respectively, hence the ammonia th -1 concentration on the 18 day became 91.05 mg l and -1 64.59 mg l , respectively. it shows that k0 and k1 reactors did not have good ammonia decreasing abilities. at reactor k2 containing an ammonia solution -1 of 97.25 mg l with addition of 500 ml of shrimp pond sediment could be seen to be able to decrease of ammonia concentration up to 99% starting at day 14 th hence the concentration of ammonia on the 18 day had -1 become 0.063 mg l . while at reactor p1, p2, p3 and p4 showed that the ammonia concentration decreased almost 100% so that the ammonia concentration on the th -1 -1 -1 18 day became 0.04 mg l , 0.12 mg l , 0.11 mg l , -1 and 0.14 mg l , respectively. thus, k2, p1, p2, p3 and p4 reactors have positive results in lowering ammonia concentrations. result data in p1, p2, p3 and p4 test reactor were analyzed by anova variety to know the real difference in each treatment. the result of variance analysis showed that variation of volume and time had significant effect (p<0,05). there was a real interaction fig 2 curve of ammonia decrease efficiency. the above efficiency values were calculated using the following equation: description: h = value on day 1, h = value on day n0 n (irmanto and suyata 2009) source description: * = sni 01-7246-2006 ** = ministry of environment of the republic of indonesia regulation no. 5/2014 volume 11, 2017 microbiol indones 97 table 1. results of shrimp pond quality analysis between the two factors so that the analysis test was continued with bonferroni test (cleophas and zwinderman 2011). the average of ammonia concentration on volume variation with time can be seen in figure 3. the line graph in figure 3 shows the depletion of ammonia concentration in reactor p1, p2, p3 and p4 along with the length of operation of the reactor. on day 3 and day 6 the p3 reactor showed a marked difference in the decrease of ammonia concentration between the test reactors. this showed that the p3 reactor had the ability to lower the ammonia concentration faster than the other reactors. the ability of this p3 reactor with shrimp pond sediment volume to be added as much as 500 ml could be assumed as the best composition in the 8 l capacity test reactor, while for 3 day operation time on each test reactor showed a rd significant decrease among other days. thus the 3 day was suggested to be the most optimum time for the bacteria in the reactor started to decrease the concentration of ammonia. concentrations of nitrite and nitrate in the batch reactors. from the nitrite test using the standard method of bppt ip no. 5 (2012), nitrite values were obtained as described in figure 3. from figure 3 it could ben seen that the nitrite concentration increased on day 4 and decreased thereafter until day 16. according to the nitrification process, nitrite is converted to nitrate. therefore in this study nitrate concentration measurement was also conducted. the results of nitrate concentration measurement using standard method from lab bppt ip no. 6 (2012) were shown in figure 4. the graph in figure 4 showed that during 16 days time the new nitrate concentration th increased after the 4th day. the 16 day nitrate concentration in p1, p2, p3, and p4 was each 101.25 -1 -1 -1 -1 mg l , 75.25 mg l , 79.05 mg l , and 82.3 mg l , respectively. the concentrations of the four reactors were close to the ammonia concentration at the start of the operation. thus, it can be assumed that ammonia had been converted to nitrate. characterization of nitrifying microbes in the batch reactor. sampling for isolation was taken from the p3 reactor because the reactor showed a decrease in the ammonia concentration faster than the other reactors. prior to the sampling of the p3 reactor, the -1 reactor was recharged with 50 mg l ammonia as the feeding for nitrosomonas sp., nitrobacter or th nitrospira sp. bacteria to be active again. after the 5 day of feeding, the sample was taken for inoculation with the general medium of nutrient agar media (na) by the spread plate method on two petri dishes. from the isolation results in two na media petri dishes three different colonies were visible visually. subsequently each colony was transferred to in two petri dishes of bacterial nitrification selective media such as stanier's medium for nitrosomonas sp. bacteria and nitrobacterial autotrophic medium for nitrobacter sp. (atlas 2010). there are three different colonies in na media that grow in stainer's medium (nitrosomonas medium), but there was no colony at all in the autotrophic nitrobacter medium for nitrobacter sp.. to ensure that the bacteria nitrobacter sp. present or not in the medium, the sample was recovered from within the p3 reactor to be isolated directly to the selective medium of nitrobacter autotrophic medium with an incubation time of 48 h. apparently the results showed that bacteria did not grow on the autotrophic selective nitrobacterial medium. furthermore, to ensure bacteria that grow on stainer's media selective media is indeed the bacterium nitrosomonas sp., it was then morphologically observed under a microscope with gram staining and activity test by catalase test on the three colonies formed. while the results of gram staining seen in figure 5 showed that the three colonies were gram negative by showing the red color in the microscope. the results of the observations can be seen in table 2 and table 3. the three colonies showed positive results in the catalase test by generating air bubbles after the addition of hydrogen peroxide (h o ). 2 2 it shows that the three colonies could produce catalase enzymes that could break down h o into oxygen and 2 2 water. these three colonies were then labeled as nitrosomonas sp1., nitrosomonas sp2., and nitrosomonas sp3. discussion shrimp pond characterization. the shrimp farms used in the study were eighty days after seed stocking. during the period of eighty days the sediment formed was quite a lot and it was expected that the ammonia degradation microbes had been in sufficient quantities. according to suwoyo et al (2016), as time increases after seed stocking there will be an increase in sediment formation which is the accumulation of shrimp feces, dead organisms (either shrimp or plankton), shrimp feed residues containing organic ingredients, high, and mud particles carried by the flow of water supply from the sea. sediment collection from shrimp ponds at stp serang was done at the center of 98 ambarsari et al. microbiol indones fig 4 curve of nitrate concentration. fig 3 curve of nitrite concentration. fig 5 identification using light microscope with 100x enlargement after gram staining (a) nitrosomonas sp3., (b) nitrosomonas sp2., and (c) nitrosomonas sp1. (a) (b) (c) volume 11, 2017 microbiol indones 99 the pond due to the shrimp pond sunken form towards the middle and also to avoid the different sediment composition when the taking was done at different points. high concentrations of nitrate in the shrimp ponds of stp serang can stimulate the growth of natural food for shrimp such as clekap, plankton and moss (sawyer et al. 2008). sediment ph values were known to be more acidic than the ph value of shrimp pond water the longer the age of the pond, then the sediment deposition was more increased due to the decomposition process from the remaining shrimp feed (suwoyo et al. 2016). decreased ammonia concentrations on batch reactors. the systems in the k0 and k1 reactors were not able to reduce the ammonia concentration well because the reactors were intended as the negative controls. the k0 reactor containing only ammonia solution with the aid of the aerator had no ability in reducing ammonia concentration since no bacteria culture was added. whereas the k1 reactor added with em4 as a biofertilizer also had not been seen to have the ability to reduce ammonia concentration well. this could be possible because the nitrogen element in the form of ammonia in the test reactor was too high so that in the absence of cultures of ammonia oxidizing bacteria had caused the bacteria in em4 could not work to break down the substrate in the reactor well by themselves. bacteria in em4 will work well as biofertilizer if the nutrients in the substrate are well met which can break down the substrate optimally (javaid and bajwa 2010). each treatment reactor similarly contained shrimp pond sediment so that it could be seen that there were ammonia decomposing bacteria in the sediment. according to burford et al. (1998), in shrimp pond sediments there are bacteria capable of processing the nutrients accumulated in the sediment. shrimp pond sediments contain bacteria that can recycle nitrogen especially using the nitrification process to convert ammonia (nh ) to nitrite (no ) by the aid of 3 2 nitrosomonas sp bacteria. and nitrobacter sp. or nitrospira sp. in converting nitrites to nitrates (no ) 3 under aerobic conditions, while the conversion of nitrate to nitrogen was done by denitrifying bacteria in anaerobic conditions in the sediment. the addition of em4 to reactor p1, p2, p3 and p4 showed the time difference of ammonia concentration decreasing. similarly, if compared between reactor treatments with k2 reactor which was not added em4, the rate difference of ammonia concentration decrease would be seen and indicated on the gradient difference from each line in graph of figure 2. it showed that addition of em4 also had an effect to improve microbial performance in shrimp pond sediments to decompose ammonia in the reactor, which could accelerate the decrease of ammonia concentration. this was clearly evident from the comparison of the graph line of k2 (control 2 which is only filled with 500 g of shrimp pond sediments without additional em4) with the graph line of p3 (treatment 3 filled with 500 g of shrimp pond sediment with an additional 400 ml of em4 solution). at the p3 treatment the efficiency of ammonia concentration decreased by more than 90% on day 6, while on the same day in the new k2 the efficiency of ammonia concentration decreased by table 2 morphological characterization of the microbes in the batch reactor table 3 catalase test on the microbes in the batch reactor 100 ambarsari et al. microbiol indones colony observation nitrosomonas sp1. nitrosomonas sp2. nitrosomonas sp3. morphology colour white yellowish yellowish white shape round irregular irregular margin filamented undulate lobate elevation flat flat umbonate under light microscope gram staining negative negative negative cell form rod (streptobacillus) rod (bacillus) rod (diplobacillus) 1 colony observation nitrosomonas sp1. nitrosomonas sp2. nitrosomonas sp3. catalase test positive positive positive 1 about 65% and the efficiency of the decrease was almost 99% on day 14 ( figure 2). our results were in accordance with the results indicated in previous studies using em4 for the treatment of wastewater from various sources (jasmiati et al. 2010; munawaroh et al. 2013; pitriani 2015). concentrations of nitrite and nitrate in batch reactors. based on the nitrogen cycle of the nitrification process, the decreased ammonia concentration is converted to nitrites by the bacterium nitrosomonas sp. and then nitrite is converted to nitrate by the bacterium nitrobacter sp. or nitrospira sp. (atlas and bartha 1993; widiyanto 2005). the increase th of nitrite concentration on the 4 day was followed by its decrease thereafter until the end of the study was possible because the bacteria nitrosomonas sp. in the reactor began to break down the ammonia into nitrites th so that nitrites increased on the 4 day, while the decline that occurs was possible because after that day the nitrites had been converted to nitrate by bacteria nitrobacter sp. or nitrospira sp. in the shrimp pond sediments. according to prosser (2005), the decomposition of ammonia into nitrites under aerobic conditions performed by nitrosomonas sp. experienced the following reaction: + nh + o + 2h + 2e ------> nh oh + h o 2 2 2 2 + nh oh + h o ------> no + 5h + 4e2 2 2 ammonia is oxidized to hydroxylamine by oxygen in endergonic reactions, subsequently hydroxulamine is oxidized to nitrite by the aid of oxygen in water by exergonic reactions. th new nitrate concentrations increased after the 4 day showing that the nitrate was formed only after nitrite was formed. according to prosser (2005), nitrites oxidized to nitrates in aerobic conditions by nitrobacter sp. or nitrospira sp. by catalyzing nitrite with oxidoreductase as the following reaction: + no + h o ------> no + 2h + 2e2 2 3 the nitrate concentration that continued to increase until day 16 showed that in the reactor there were no denitrifying bacteria that convert nitrates to nitrogen in the form of gas. denitrification is a process that requires anaerobic conditions. while the reactor was aerated with the aerator hence the nitrate in the reactor was high because there was no anaerobic condition to perform the process of denitirification. the nitrate concentration on the last day of the study within each of the four reactors was close to the concentration of ammonia at the beginning of the operation. it could then be assumed that ammonia had almost all converted to nitrate. characterization of nitrifying microbes in the batch reactor. it was known that the nitrate test showed the presence of nitrate compound in the reactor meaning that there was the nitrification process in converting nitrite to nitrate which should be done by nitrobacter sp. bacteria. however after isolation, nitrobacter sp. did not grow in the selective media of autotrophic nitrobacterial media. this was possible because the bacteria nitrobacter sp. which was in the reactor could not be cultured (unculturable). according to koops and roser (2001), some nitrifying bacteria can not be cultured on the media due to their prolonged development or the bacteria lose nutrients to the other bacteria in the media. molecular techniques are hence required to ensure the presence of these unculturable bacteria. acknowledgment this research was jointly funded by insinas 2017 program managed by indonesian ministry of research, technology and higher education (kemenristekdikti), btpal–bppt and surya university. high appreciation and sincere thank are given to all members in laboratory of environmental microbiology and laboratory of chemical analysis btpal-bppt for their supports during the research. references th atlas, rm. 2010. handbook of microbiological media. 4 ed. crc press:florida, usa. atlas rm, bartha r. 1998. microbial ecology: fundamentals and applications. benjamin/cummings publishing company: usa. apha. 2012. standard methods for the examination of nd water and wastewater. 22 edition, american public health association, american water works association, water environment federation: usa. bppt ip no. 5. 2012. procedure for nitrite analysis. serpong, tangerang selatan. bppt ip no. 6. 2012. procedure for nitrate analysis. serpong, tangerang selatan. burford ma, peterson el, baiano jcf, preston np. 1998. bacteria in shrimp pond sediments: their role in mineralizing nutrients and some suggested sampling strategies. aquacult res. 29 (11): 843–849. doi: 10.1111/j.1365-2109.1998.tb01110.x. cleophas tj, zwinderman ah. 2011. bonferroni t-test. in: statistical analysis of clinical data on a pocket volume 11, 2017 microbiol indones 101 calculator. springer: dordrecht. doi: 10.1007/978-94007-1211-9_15. fitria y, ibrahim, bustami, desniar. 2008. liquid organic fertilizer production from fishery industrial wastewater using acetic acid and em4 (effective microorganism 4). j water resources-akuatik 2, 1. higa t, wididana. 1994. effective microorganism technology. jakarta: forestry department officers cooperation. javaid a, bajwa r. 2010. field evaluation of effective microorganisms (em) application for growth, nodulation, and nutrition of mungbean. tubitak res article: 443-452. doi: 10.3906/tar-1001-599. jasmiati et al. 2010. bioremediaton of tofu industrial liquid waste using effective microorganism (em4). j env sci. 52, 2(4). republic of indonesia ministry of environment regulation no. 5 year 2014. wastewater quality standards. republic of indonesia state news. koops hs, roser ap. 2001. distribution and ecophysiology of the nitrifying bacteria emphasizing cultured species. fems microbiol ecol. 37: 1-9. doi: 10.1016/s01686496(01)00137-4. munawaroh et al. 2013. allowance parameter environmental pollutans in wastewater industry know using effective microorganism 4 (em4) and utilization. j of nat inst of tech. (itenas), 2 (1):12-21. pitriani, natsir mf, daud a. 2015. the effectiveness of em4 addition into biofilter to reduce of bod, cod and mpn coliform of hospital wastewater. int j of pharm tech res. 8 (4): 702-708. plog ba, niland j, quinland pj. 1996. fundamentals of th industrial hygene (4 ed). illinois: national safety council. prosser ji. 2005. nitrification. elsevier: university of aberdeen, aberdeen, uk. soemirat sj. 1994. kesehatan lingkungan [environmental health]. yogyakarta: gadjah mada university press. suwoyo hs, undu mc, makmur. 2014. sedimentation rate and sediment characterization of litopenaeus vannamei shrimp super intensive pond. proceeding of aquaculture technology innovation forum. brackish water aquaculture research and development body of south sulawesi: 327-339. widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): bogor agriculture institute. 102 ambarsari et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 yusra (characterization of an... currently the use of harmful chemicals as a preservative of food like tofu, noodles, meatballs, chicken meat, and fish is prohibited. we must search an alternatives to the particular safe food and fish preservatives and for consumption. bacteriocins from lactic acid bacteria (lab), especially their antibacterial activities, have attracted much attention and have been the subject of intensive investigation (mataragas . 2002). the limited existence of data regarding bacteriocins from spp. makes this genus an interesting object to investigate, since it produces diverse array of antimicrobial peptides representing several different basic chemical structures (adetunji and olaoye 2011) the production of bacteriocins or bacteriocin-like substances has already been described for some spp., such as , , et al bacillus bacillus bacillus substilis b. cereus b. . bacillus cereus escherichia coli staphylococcus aureus salmonella thypi bacillus subtilis listeria monocytogenes bacillus cereus staphylococcus aureus bacillus cereus staphylococcus aureus ss28 isolated from budu, a fermented fish product from west sumatra, produced antimicrobial compound that had broad spectrum of inhibition against five microorganisms ( , , , , and ). the aims of this research are characterization of ss28 antimicrobial activity and observation of its effect to the cellular morphology of with electron microscope. antimicrobial compound produced by ss28 was stable at ph range between 2 and 11 and to heating at 121 c for 15 min. maximum antimicrobial activity was expressed at ph 2-3 and 70 °c for 45 min. the activity remained after 15 min exposure to uv light. the main changes observed under sem and tem were the alteration of structural cell membrane 48 h after exposure to the antimicrobial compound from bacillus cereus ss28 o . key words: kata kunci: antimicrobial bacteriocin, ss28, budu, characterization, west sumatera ss28 diisolasi dari ikan budu, produk fermentasi ikan yang berasal dari sumatera barat yang dapat menghasilkan komponen antimikroba bakteriosin dengan spektrum yang luas dan dapat menghambat lima bakteri ( dan ). tujuan dari penelitian ini adalah melakukan karakterisasi komponen antimikroba dari bakteri ss28 dan mempelajari pengaruhnya terhadap morfologi sel dari bakteri . komponen antimikroba yang dihasilkan oleh bakteri ss28 stabil pada perlakuan ph 211 dan pemanasan suhu 121 c selama 15 menit. aktivitas antimikrobial yang paling tinggi terdapat pada ph 2-3, suhu 70 c selama 45 menit.bakteriosin masih stabil setelah terpapar di bawah sinar uv selama15 menit.dengan menggunakan sem dan tem terlihat perubahan struktur membran sel bakteri setelah terpapar selama 48 jam oleh komponen antimikroba dari ss28 antimikroba bakteriosin, ss28, budu, karakterisasi, sumatera barat bacillus cereus bacillus cereus escherichia coli, staphylococcus aureus, salmonella thypi, bacillus subtilis listeria monocytogenes bacillus cereus staphylococcus aureus bacillus cereus staphylococcus aureus bacillus cereus bacillus cereus o o . characterization of antimicrobial bacteriocin produced by ss28 isolates from budu, a traditionally fermented fish product of west sumater bacillus cereus a yusra *, fauzan azima , novelina , periadnadi 1 1 1 2 and 1 2 departement of agricultural processing technology, faculty of agricultural technology departement of biology, faculty of matematics and natural sciences, universitas andalas, padang, 25163, indonesia stearothemophilus bacillus et al et al listeria monocytogenes streptococcus pyogenes et al b. megaterium b. amyloliquefaciens et al bacillus . et al et al bacillus bacillus cereus and other spp. (zheng 1999; cherif . 2001; stein . 2002). some strains produce bacteriocin with broad spectrum of activity including important pathogens such as and (cherif . 2001). some produced well characterized bacteriocins, such as lichenin and megacin produced by . bacteriocin had also been isolated from (lisboa . 2006). a number of general physicochemical properties has been studied to provide information about the composition and structure of bacteriocins. various studies stated that bacteriocins produced by sp showed resistance to heat treatment and tolerance to ph, as described by sharma . (2009), and khalil . (2009) about the effects of ph, heating and exposure to uv light towards sp mtcc 43 bacteriocins. ss28 isolated from budu showed very high antimicrobial activity against all tested issn 1978-3477, eissn 2087-8575 vol 8, no 1, maret 2014, p 24-32 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.8.1.4 *corresponding author; phone/fax: , email: +62-751-7051678/+62751-55475 yusra@bunghatta.ac.id strains ( , , , and ), with range of inhibition zone 14-35 mm (yusra 2013). budu is a fermented fish product from west sumatera, mainly originated from the coastal areas, such a pariaman, tiku and pasaman. normally made from bigger size marine fish such as spanish mackerel ( sp.) and leatherskin ( sp.), locally, knowns as ikan tenggiri and ikan talang (yusra 2012). however, studies related to the antibacterial characteristics of these organisms have been limited and not fully exploited. therefore, the purpose of this research were to characterize the antimicrobial compounds isolated from ss28 and to observe its effect to cell morphology with electron microscopy (sem and tem) . materials used in this study were isolated from ss28. the indicator strains used in this work were provided by the laboratory of clinical microbiology research, faculty of medicine and microbiology, universitas indonesia, and laboratory microbiology, department of food science and technology, faculty of agricultural technology, institut pertanian bogor. they include both gram negative and gram positive strains ( , , , , and ) . the strain ss28 provided by yusra . (2013) was maintained at -4 c and as frozen stock cultures in equal volumes of 10% glycerol. ss28 was grown in mrs broth, , , , and were grown in nutrient broth (nb). the cultures were grown at 37 c for 24 h in mrs broth or nb medium. ss28 was grown in the mrs broth media as much as 200 ml, incubated at 37 c for 30 h. one ml culture sample was taken hourly and put in a test tube. changes in the optical density of the cultures were recorded at 600 nm wavelength (olivera 2004). . ss28 was cultivated in 250 ml erlemeyer flask escherichia coli staphylococcus aureus salmonella thypi bacillus subtilis listeria monocytogenes et al. budu scomberomorus chorinemus bacillus cereus staphylococcus aureus bacillus cereus e. coli s. aureus s.thypi b. subtilis l. monocytogenes . b.cereus et al b. cereus e. coli s. aureus s. thypi b. subtilis l. monocytogenes b. cereus et al. bacillus cereus . materials and methods bacterial strains bacterial cultures growth and production of bacteriocin by ss28 in a mrsb at 37 c. production of crude bacteriocin o o o b. cereus o containing 100 ml of mrs broth and incubated for 48 h at 37 c. supernatants were harvested by centrifugation o at 6000 g for 10 min at 4 c. the ph of the cell free supernatant was adjusted to 6.5 using 1 m naoh solution to prevent the inhibitory effect of organic acids. the supernatants were then filtered using 0.22 μm membrane filter (millipore). the filtrates were used for the characterization of bacteriocin. o a n t i m i c r o b i a l a c t i v i t y o f e x t r a c t e d bacteriocin characterization of bacteriocin effect of ph on antimicrobial activity effect of temperature on antimicrobial activity effect of uv light on antimicrobial activity scanning electron microscopy. . agar well diffusion and paper disc methods were used to study antimicrobial activity of the extracted bacteriocin. in the agar well diffusion assay 0.1 ml culture of the tested microorganisms ( and ) were spread on sterile nutrient agar. twenty l placed in each well and the plates were aerobically incubated at 37 °c for 24 hrs. . supernatant from culture was diluted with deionized water. the diluted supernatant was then divided into several parts, each of which was adjusted to different ph levels between 2 to 11 using sterile 10 mm/l naoh or 10 mm/l hci solution. the solutions were then heated at 100 °c for 30 min, before the ph was adjusted to 6.5 with sterile dh o and assayed for its activity (nofisulastri . 2006). the antimicrobial activity were determined by paper disc assay. . supernatant of ss28 was exposed to various heat treatments: 40, 55, 70, 85, 100, and 121 ºc. aliquot volumes of each fraction were then removed after 0, 30, 60, or 90 min and assayed for bacteriocin (ogunbarwo . 2003). . ten ml supernatant of ss28 was placed in a sterile petri dish and exposed to short wave uv light (wavelength 340 nm, 220-240 v, 50 hz) situated at a distance of 30 cm from petri dishes. time of exposure to uv light is 30 minutes after which the bacteriocin activity was estimated by the papper disc method (ogunbarwo . 2003). culture that has been exposed to bacteriocin from ss28 at 37 c for 48 h were examined by sem to visualize any morphological change occuring in the cell following exposure to bacteriocins and pressure. the cell suspensions were fixed with 3% gluteraldehyde in na-cacodylate buffer (100 mm, ph 7.1). then the cells were pelleted and washed to e. coli, staphylococcus aureus, salmonella thypi, b. subtilis, listeria monocytogenes b. cereus ss28 et al b. cereus et al b. cereus et al s. aureus b. cereus μ extracted bacteriocin preparation (cbp) was 2 o volume 7, 2013 microbiol indones 25 remove gluteraldehyde before resuspended in the same buffer. a drop of each suspension was transferred to a poly-l-lysine-treated silicon wafer chips that were kept for 30 min in a hydrated chamber to let the cells adhere. the attached cells were post fixed by immersing the chips in 1% osmium tetroxide (oso ) in cacodylate buffer for 30 min, then rinsed in the same buffer and dehydrated in ethanol in ascending concentrations (%): 50, 70, 95 (2x) and 100 (2x), for 10 min each. the chips were mounted on aluminum stubs and coated with gold-palladium in a sputter coater (emitech k550, ashford, kent, england). the chips were viewed at 3 kv accelerating voltage in a hitachi s-4000 field emission scanning electron microscope (jem-jeol jsm-5310lv type) and secondary electron image of cells for topography contrast were collected at several magnifications (bolshakova . 2004). the cell suspensions that has been exposed to bacteriocin from ss28 at 37 c for 48 h were harvested by centrifugation and washed twice with 0.1 m phosphate buffer (ph 7.3). the cells were fixed with 2.5% (v/v) glutaraldehyde, 2.0% (v/v) formaldehyde in 0.12 m phosphate buffer for 10 days and then postfixed in 2% (w/v) osmium tetroxide in the same buffer for 45 min. the samples were dehydrated in a graded acetone series (30-100%) and embedded in araldite-durcupan for 72 h at 60 °c. thin sections 4 et al s. aureus b.cereus transmission electron microscopy. o (microtome upc-20, leica) were mounted on grids, covered with collodion film and poststained with 2% uranyl acetate in reynold's lead citrate. its preparation were observed with transmission electron microscope tipe jeol-1010 (bozzola and russel 1999, with modification). the growth and bacteriocin production of is slight increase of cell dry weight was observed for 28 h of fermentation. during log phase (6 22 hours fermentation), medium ph decreased rapidly. it occured concurrently with the increase of the cell dry weigt. the data indicated that the alteration of the medium ph was inversely proportional with growth of ss28. the effect of ph on bacteriocin activity was studied. it was observed that bacteriocin produced by ss28 was stable between ph 2-11 (fig 2). the inhibitory activity towards the test isolates was heat stable (fig 3). the antimicrobial activity remained constant after heating at 121 ºc for 15 minutes. the activity was highest when being heated at 70 ºc for 45 min. results growth of ss28 and the production of bacteriocin in mrsb at 37 c. effect of ph on antimicrobial activity. effect of temperature on antimicrobial activity. b. cereus o b. cereus ss28 b. cereus b. cereus fig 1 the growth curve of ss28 isolate on mrs broth mediumbacillus cereus 26 et al.yusra microbiol indones 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 5 10 15 20 25 30 a b so rb a n (n m ) time (h) fig 2 effect of ph on the activitiy of antimictobial compound from ss28 determined based on the size of the inhibition zone (mm) bacillus cereus . fig 3 effect of temperature on activity of antimictobial compound from ss28, determined based on the size of the inhibition zone (mm) bacillus cereus . effect of uv light on antimicrobial activity. scanning electron microscopy. bacteriocin produced by the test isolates was tested for their sensitivity (loss of activity) to uv light exposure. the antimicrobial activity was lost or unstable after exposure to uv-light for 15 and 30 min (fig 4). sem has been widely used in microbiology to study the surface structure of biomaterials and to measure cell attachment and changes in morphology of bacteria. the sem-generated photomicrograph of pathogen after treatment with antimicrobial compound from ss28 is presented in figure 5. s. aureus b. cereus ph 0 5 10 15 20 25 30 2 3 4 5 6 7 8 9 10 11 in h ib it io n z o n e s iz e (m m ) escherichia coli staphylococcus aureus salmonella thypi bacillus subtilis listeria monocytogenes 0 5 10 15 20 25 30 35 40 50 70 85 100 121 in h ib it io n z o n e si z e (m m ) temperature (°c) e. coli s. aureus s. thypi b. subtilis l. monocytogenes volume 7, 2013 microbiol indones 27 fig 5 scanning electron microscopy of cells control (a) (bajpal . 2009) and after treatment with antimicrobial compound of ss28 (b). staphylococcus aureus et al bacillus cereus bacteria 0 5 10 15 20 in h ib it io n z o n e si z e (m m ) uv, 15 menit uv, 30 menit fig 4 effect of uv light on activity of antimictobial compound from ss28, determined based on the size of the inhibition zone (mm) bacillus cereus . a b transmission electron microscopy. the effect of antimicrobial compound from ss28 on bacterial cells was studied using as a representative of gram-positive cells. morphological investigations were performed using 48-h culture treated with antimicrobial compound from ss28 l). control has exhibited typical coccus morphology of (fig 6). untreated b. cereus s. aureus s. aureus b. cereus s. aureus (20 μg/m s.aureus s. aureus cells) shows a typically structured nucleus of and a perfect cell wall (fig 6a). after 48 hours of exposure to the antimicrobial compound, a slight alteration can be observed in the cell cytoplasm (fig 6b), the cells exhibited notable alteration in cell cytoplasm. bacterial cells completely collapsed 48 h after treatment with the antimicrobial compound (fig 6). 28 et al.yusra microbiol indones discussion bacteriocin activity remained stable up to 24 h fermentation, then the activity started to drop after 28 h fermentation. koroleva (1991) stated that most of the metabolism products resulted in the log phase were in the form of lactic acids, which causes the decrase in ph of the medium. this acidic condition will eventually inhibit growth of the respective bacteria (negative feed back effect). bacteriocin is extracellular secondary metabolite. the increase in the amount of biomass produced in the bacterial culture caused the increase in the amount of the bacteriocin produced. after reaching the stationary phase, the amount started to decrese (boe 1996). synthesis of bacteriocin by lab occurred during the exponential growth phase, usually following the protein synthesis (schnell 1998). torkar and matijasic (2003) who did research on the characterization of bacteriocins produced by from milk and other dairy products, found that the production of bacteriocins entered the stationary phase after 10-16 hours of incubation. the research by naclerio . (1993) on the production and activity of bacteriocins cerein present in the stationary phase also demonstrated similar result. the highest antibacterial activity was exhibited at ph range 2 to 3, while inactivation occurred between ph 9 to 11. khalil . (2009) showed that bacteriocins produced by 22 has activity antimicrobial against at ph range 2-8. naclerio . (1993) who studied the antimicrobial activity of bacteriocins cerein from found that the compound's activity was et al., b. cereus et al from b. cereus et al b. megaterium s. thypimurium et al b. cereus, fig 6 transmission electron microscopy of cells control (a) (santhana ., (2007) and after treatment with antimicrobial compound of ss28 (b). staphylococcus aureus et al bacillus cereus stable between ph 3-12. growth temperature plays an important role and is often correlated with bacteriocin production (todorov and dicks 2006). similar to the results of alam . (2011), who stated that bacteriocin of bs15 retained activity up to 80 c for 30 min, other bacteriocin produced by ssp. diacetilactis was reported to maintain its activity even after boiling for up to 60 min. on the other hand, lactacin f was reported to completely lose the activity when treated at 50 c for 30 min (kojic . 1991; kim . 2005). cleveland . (2001) suggested several potential advantages of bacteriocins to serve as biopreservatives, namely: a) the material is not toxic and susceptible to degradation by proteolytic enzymes because it is a protein compound, b) the material does not harm the intestinal microflora because it is easily digested by gastrointestinal enzymes, c) the material can reduce the chemicals as a food preservative, d) flexibility of use, and e) stability towards sufficiently broad range of ph and temperature that it is resistant to treatment processes involving acids and bases, as well as hot and cold conditions. antimicrobial activity of ss28 was the highest against with inhibition zone diameter 20 mm, after 15 minutes exposure, which decreased to 10 mm after 30 minutes. species and other gram negative bacteria were sensitive to nisin and other bacteriocins after exposure to treatments that change the permeability barrier properties of the outer membrane (stevens . 1991). khalil . (2009) 19 bacteriocin was stable after 15 min exposure to uv light and was completely destroyed after 90 min. et al b. subtilis l. lactis et al et al et al b. cereus s. thypi, s. et al et al b. megaterium o o a b volume 7, 2013 microbiol indones 29 bajpai vk, al-reza sm, choi uk, lee jh, kang sc. 2009. chemical composition, antibacterial and antioxidant activities of leaf essential oil and extracts of miki ex hu. food and chem toxicol. 47(8):1876-1883. doi:10.1016/j.fct.2009.04.043. bhunia ak, johnson mc, ray b. 1987. direct detection of an antimicrobial peptide of in s o d i u m d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l electrophoresis. j ind microbiol. 2(5):319-322. doi:10.1007/bf01569434. bizani d, brandelli a. 2002. characterization of a bacteriocin produced by a newly isolated sp. strain 8a. j appl microbiol. 93(3):512-519. doi:10.1046/j.1365-2672.2002.01720.x. boe, young j. 1996. evaluation of optimum for production for bacteriocin from sp. jb42 isolation from kimichi. j microbiol biotechnol. 6(1):63-67. bol’shakova av, golutvin ia, nasikan ns, yaminskii v. 2004. determination of mechanical characteristics of surface of block copolymers by atomic force microscopy techniques. polymer scie ser a. 46(9):926-932. bozzola jj, russell ld. 1999. ultramicrotomy e , 2nd edition. sudbury, massachusetts, jones & bartlett. brotz h, bierbaum g, leopold k, reynolds pe, sahl hg. 1998. the lantibiotic mesarcidin inhibits peptidoglycan synthesis by targeting lipid ii. antimicrob agents chemother. 42(1):154-160. cherif a, quazri h, daffonchio d, cherif h, siama bk, hassen a, japua s and boudabous a. 2001. thurin 7: a novel bacteriocin produced by bmg 1.7, a new strain isolated from soil. lett appl microbiol. 32(4):2432-2247. doi:10.1046/j.1472765x.2001.00898.x. cleveland j, monteville tj, nes if, chikindas mi. 2001. bacteriocins: safe, natural antimicrobials for food preservation. int j food microbiol. 71(1):1-20. doi:10.1016/s0168-1605(01)00560-8. dalmau m, maier e, mulet n, vinas m, benz r. 2002. bacterial membrane injuries induced by lactacin f and nisin. int microbiol j. 5(2):73-80. doi:10.1007/s10123002-0063-2. diop mb, dauphin rd, tine e, ngom a, thonart dj, philippe t. 2007. bacteriocin producers from traditional food products. biotechnol agron soc environ. 11(4):275-281. hartmann m, berditsch m, hawecker j, ardakani mf, gerthsen d, ulrich as. 2010. damage of the bacterial cell envelope by antimicrobial peptides gramicidin s and pgla as levealed by transmission and scanning electron microscopy. antimicrob agents chemother. 54(8): 3132-3142. doi:10.1128/aac. 00124-10. jack rw, wan j, gordon j, harmark k, davidson be, hillier aj, wettenhall reh, hickey mw, coventry mj. 1996. characterisation of the chemical and antimicrobial properties of piscicolin 126, a bacteriocin produced by jg 126. j appl environ microbiol. 62(8):2897-2903. metasequioa glyptostroboides pediococcus acidilactici bacillus lactobacillus lectron microscopy: principles and techniques for biologists bacillus thuringenesis carnobacterium piscicola the effect of antimicrobial compound from supernatant ss28 from wall and cell membrane was investigated. it could be associated with the damage in the cell wall and cell membrane and subsequent lysis and reduction. immediately after treatment, 80% of the cell's surface appeared rough, which is quite different from the normal cells. in a previous study with , which has an inducible autolytic enzyme, bacteriocin treatment, pressurization or their combination did not only produce cell death and cell lysis, but also triggered the autolytic enzyme, which, by hydrolyzing the wall, disintegrated the cells (bhunia . 1987; kalchayanand . 2002). electron microscopy showed cell lysis after treatment with antimicrobial compound of ss28. the cell damage caused by antimicrobial compound resembles that observed with a crude bacteriocin treatment (ocana . 1999). bizani . (2005) tried to truestigate the effect of cerein 8a against spore. an approximately 4-5 log reduction was observed when spores were plated in pca containing 800 au ml . as cerein 8a concentration increased to 1600 au ml , complete inhibition of colony development was observed. when spores were treated with cerein 8a in bhi broth before plating, similar results were observed. the bactericidal effect of the antimicrobial compound from ss28 apparently works by disrupting the membrane function of target organisms. to conclude antimicrobial bacteriocin from was stable over a broad range of ph (between ph 2 to 11) and to heat-treatment at 121 c for 15 min. the antimicrobial activity was the highest at being heated at 70 c for 45 min and for 15 min of exposure to uv light. the main changes observed under sem and tem analyses were structural disorganization of the cellular membrane 48 h after exposure to the antimicrobial compound of ss28. b. cereus s. aureus layconostoc mesenteroides et al et 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biopreservation of fresh-cut salads using bacteriocinogenic lactic acid bacteria isolated from commercial produce.m.sc (thesis). collected in the aafc, ns and dalhousie university, halifax. canada. sharma n, kapoor g, gautam n, neopaney b. 2009. characterization of a partially purified bacteriocin of sp mtcc 43 isolated from rhizosphere of radish ( ) and its application as a potential food biopreservative. j sci industrial res. 68(10):881-886. stein t, brochert s, conrad b, feesche j, hofemeister b and hofemeister j. 2002. two different lantibiotic-like peptides originate from the ericin gene cluster of a1/3. j bacteriol. 184(6):1703-1711. doi:10.1128/jb.184.6.1703-1711.2002. stevens ka, sheldon bw, klapes na, klaenhammer tr. 1991. nisin treatment for inactivation of species and other gram-negative bacteria. j appl environ microbiol. 57(12):3613-3615. sumner ss, wallner-pendleton ea, froning gw, stetson le. 1995. inhibition of on agar medium and poultry skin by ultraviolet energy. j food prot. 59(3):319-321. todorov sd and dicks lmt. 2006. screening of bacteriocin-producing lactic acid bacteria from boza, a traditional cereal beverage from bulgaria. comparison of bacteriocins. proc biochemi. 41(1):11-19. doi:10.1016/j.procbio.2005.01.026. torkar kg, matijasic bb. 2003. partial characterisation of bacteriocins produced by isolates from milk and milk products. food technol biotechnol. 41(2):121-129. yang r, johnson mc, ray b. 1992. novel method to extract large amounts of bacteriocins from lactic lactic bacteria. j appl environ microbiol. 58(10):3355-3359. yusra. 2012. isolation and identification of lactic acid bacteria from budu, fish fermented product spanish mackerel ). fundamentals grant final report. dp2m higher education. bung hatta university, padang. bacillus cereus bacillus raphanus sativus bacillus subtilis salmonella salmonella typhimurium bacillus cereus (scomberomorus guttatus volume 7, 2013 microbiol indones 31 zheng gs. 1999. isolation, partial purification and characterization of a bacteriocin produced by a newly isolated strain. lett appl microbiol. 28(5):363-367. doi:10.1046/j.1365-2672.1999. 00545.x. bacillus subtilis yusra, azima f, novelina, periadnadi. 2013. antimicrobial activity of lactic acid bacteria isolated from of west sumatra to food biopreservatives. pakistan j of nutr. 12(7):628-635. doi:10.3923/pjn. 2013.628. 635. budu 32 et al.yusra microbiol indones 1. mi-lisa pratama.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.11.2.1issn 1978-3477, eissn 2087-8575 vol 11, no 2, june 2017, p 35-45 *corresponding author; phone: +62-21-7560536, email: solichin.budiasih@gmail.com advanced technology in oil palm based industries might change triglyceride’s double bonds into single bond which is more stable than that of hydrogenation technique. the technique eliminates double bond in oil/fat by adding h to change unsaturated oil to 2 saturated one. the aim of the technique is to obtain saturated oil which has specific characteristic in flavour and texture by modifying solid fat content (sfc) and melting point (mp). the high temperature used in hydrogenation process tends to change cis form of double bonds in unsaturated fat into trans form. partial hydrogenation in double bond produces translipid acid. several studies reported that trans form adversely affects health, i.e. increasing the risk of coronary heart disease (mozaffian et al. 2006). to decrease the negative impact, the lipid can be modified using interesterification reaction. interesterification reaction can be carried out in two method, i.e. chemical interesterification (cie) and enzymatic interesterification (eie) (rodriguez et al. 2009). chemical interesterification usually uses sodium methoxide or sodium ethoxide as catalyst while the enzymatic one uses lipase (amir et al. 2012). lipase (triacylglycerol hidrolase) is biocatalyst with ability to catalyze lipid hydrolysis reaction into lipid acid and glycerol. lipase use in industrial’s enzymatic bioconversion lipase catalyses hydrolysis and esterification of lipids. the aim of this study was to obtain lipase producing indigenous fungi, to identify the selected fungi, to study optimum temperature and ph of the enzyme activity, as well as the enzyme ability in interesterification reaction. the isolates used in the experiment were isolated from tempeh, oncom and bppt laboratory culture collection. the results showed that three fungal isolate observed were positive produced lipase after qualitative assay using rhodamine b, olive oil, and pva. the morphology and molecular identification of the isolates, revealed that r isolate was aspergillus niger, o isolate was neurospora sp. and t isolate was rhizopus oligosporus. upon quantitative assay from determination of the media and time production, potato dextro broth (pdb) with 2% olive oil after 48 h fermentation showed the highest specific activity of the enzymes. lipase produced from three isolates have the optimum at ph 4, temperatures at 40-45 °c, and stable in interesterification reaction (55 °c) for 30-40 min. hplc analysis after interesterification enzymatic reaction in mixture palm kernel olein (pkoo) and palm stearin (pos) showed that the composition of triglycerides (tag) did not change as compared to the commercial lipase (lypozyme tl1m). key words : enzymes, fungi, interesterification, lipase lipase mengkatalisis hidrolisa dan esterifikasi lipid. tujuan dari penelitian ini adalah mendapatkan lipase yang diproduksi oleh jamur asli indonesia, mengidentifikasi jamur yang terpilih, mempelajari suhu optimum dan ph aktivitas enzim, serta untuk mengetahui kemampuan enzim dalam reaksi interesterifikasi. isolat jamur yang digunakan dalam percobaan diisolasi dari tempe, oncom, dan koleksi kultur laboratorium bppt. hasil penelitian menunjukkan bahwa tiga isolat jamur yang diamati positif menghasilkan lipase setelah uji kualitatif menggunakan rhodamine b, minyak zaitun, dan pva. identifikasi morfologi isolat tersebut, menunjukkan bahwa isolat r adalah aspergillus niger, isolat o adalah neurospora sp., dan isolat adalah rhizopus oligosporus. pada uji kuantitatif dari penentuan media dan produksi waktu, kaldu dekstro kentang (pdb) dengan minyak zaitun 2% setelah 48 jam fermentasi menunjukkan aktivitas enzim tertinggi. lipase yang dihasilkan dari tiga isolat memiliki optimum pada ph 4, suhu 40-45 °c dan stabil dalam reaksi interesterifikasi (55 °c) selama 30-40 menit. analisis hplc setelah reaksi interesterifikasi enzimatik pada campuran palm kernel olein (pkoo) dan palm stearin (pos) menunjukkan bahwa komposisi trigliserida (tag) tidak berubah dibandingkan dengan lipase komersial (lypozyme tl1m). kata kunci : enzim, interesterifikasi, jamur, lipase isolation, characterization, and production of lipase from indigenous fungi for enzymatic interesterification process 1 2 3 2 lisa pratama , is helianti , ani suryani , and budiasih wahyuntari * 1 post graduate program, biotechnology, institut pertanian bogor, jalan raya dramaga, bogor 16680, indonesia; 2 center for technology of bioindustry, agency for the assessment and aplication of technology (bppt), laptiab 1, puspiptek 611, serpong, south tangerang 15314, indonesia; 3 department of agroindustrial technology, faculty of agricultural engineering and technology, institut pertanian bogor, jalan raya dramaga, bogor 16680, indonesia process, however, faces obstacles, i.e. the availability and the high price of imported lipase because lipase producers are only a few in the world (suzuki et al.1988; panji et al. 2008). the dependence of several food industries in indonesia to use only imported lipase encourages enzyme experts or researchers to isolate lipase from indigenous microorganisms. in microorganisms, lipase is produced by bacteria, yeast, and fungi (faloni et al. 2006). the use of microbial lipase is considered cost-effective. several lipase-producing bacteria are bacillus sp., pseudomonas sp., burkholderia sp., and staphylococcus sp.; lipaseproducing fungi are rhizhopus sp., aspergillus sp., geothricum sp., mucor sp., thricoderma reseei, fusarium sp., and rhzhomucor sp.(treichel et al. 2010); while lipase-producing yeasts are candida rugosa (crl) and c. antarctica (cal) (bussamara et al. 2010). fungi are potential lipase producing agents (rajesh et al. 2010) and the best lipase producer (ming et al. 1998). several indigenous fungal species were studied to explore which fungal species capable on producing lipase. indigenous fungi capable on producing lipase in high activity are necessary to carry out enzymatic interesterification technique to modify fat of margarine raw material. lipase from rhizopus sp. has been applied in interesterification of amazonian patauá oil and palm stearin better than that of commercial thermomyces lanuginosus lipase (speranza et al. 2015). this study aimed to select indigenous lipase producing fungi from several sources, to identify the selected isolates, to study the enzyme properties and determine the capability on enzymatic interesterification in the mixture of stearin and olein. material and methods isolation, screening of lipolytic fungus, and morphological identification. lipase-producing fungi were isolated from soya-tempeh and oncom. the screening of lipase-producing fungi was using -1 petridish with potato dextrose agar (pda) in 39 g l concentration. in addition, pure fungal isolate from bppt-cc (badan pengkajian dan penerapan teknologi-culture collection) was also used.the morphological identification of macroscopic and microscopic fungal isolates was carried out following gandjar et al. (1999) and watanabe (1937). identification of the fungal isolates based on molecular marker. genomic dna was extracted from the fungal isolates (r, o, and t) using purelink® 36 pratama et al. microbiol indones genomic dna kits for purification of genomic dna (invitrogen, usa). pcr of 28s rrna region was conducted using nl4 5’-ggtccgtgtttcaag acgg-3’ and nl1 5’-gcatatcaataagcggagg aaaag-3’ as a primer pair ( , with the o’donnell 1992) following cycles condition: 30 cycles of 94 °c 1 min, 55 °c 35 s, 72 °c 2 min, and followed by extension at 72 °c 10 min. the pcr product then purified and st sequenced in 1 base sequencing service (singapore). sequencing results were compared to the data base from the ncbi website (http://www.ncbi.nlm.nih.gov) with the basic local alignment search tool (blast). nucleotide sequence alignment and phylogenetic tree construction were conducted using mega 5.2 software. construction of a phylogenetic tree was conducted using the neighbour joining method. qualitative and quantitative enzyme assay, protein content, and specific activity. qualitative of lipolytic activity on solid media can be determined by using olive oil rhodamine method (modifed from -1 kouker and jaeger 1987) comprising of 39 g l pda, 0.01% rhodamin, 1.5% (w/v) pva, and 4% (v/v) olive oil substrate. there are other analysis using modified method from silva (2005) and titration method (yang et al. 2006). silva’s modified method (2005): 3 mg pnpp was dissolved in 1 ml 2-propanol (solution a) and mixed with 10 mg stabiliser and 40 mg triton x-100 to dissolution in 9 ml 50 mm buffer (solution b). 0.1 ml enzyme sample mixed with 0.9 ml substrate solution and incubated at 40 °c for 30 min. the appearance of the sample can be monitored by reading the absorbance at 410 nm. titration method (yang et al. 2006): substrate contained 25% olive oil and 1.5% pva in water, 1 ml crude extract enzyme, 5 ml substrate, and 4 ml 0.05 m ph 7 phospat buffer was incubated at 37 °c for 20 min shaker incubator and added 5 ml methanol (ethanol 100%) for stop the reaction. the liberated fatty acids were titrated with 0.05 m naoh. all experiments were carried out in triplicate for the calculation of the mean value. enzyme protein concentration was determined following bradford’s method (1976) using bovine serum albumin (bsa) standard solution. one unit enzyme activity was defined as the amount of enzyme that generated 1.0 mmol of fatty acid from a triglyceride in one min or the amount of enzyme that released 1 mmol of pnitrophenol from pnpl in one minute. media selection and production period of enzyme. cultivation media selected were (1) pdb and -1 olive oil media consisted of 24 g l pdb and 2%(v/v) olive oil; (2) pdb and crude palm oil (cpo) media -1 consisted of 24 gl pdb and 2%(v/v) crude palm oil. effect of ph, temperature, and stability enzyme. to evaluate the effect of reaction temperature and ph on lipase activity, the lipase activity was assayed at various ph 4,5 (citric acid buffer), 6,7 (phospat buffer), and 8 (tris-hcl buffer) and also at various temperature (25, 30, 35, 40, 45, 50, and 55 °c). to determine the lipase stability assay, the lipase was conducted at interesterification reaction temperature of 55 °c up to 3 h. the analysis by sampling per 15 min after incubation for 1 h, 2 h, and 3 h. enzyme production, concentration, and freeze drying. ten percent spore suspension was put into 1000 ml sterile production media in erlenmayer 2000 ml to incubated using shaking incubator at 30 °c for 150 rpm. after 48 h, the mycelia of the fungi were filtered using whatman paper to separate from their cells. the result of filtration then centrifuged at 8000 rpm and 4 °c for 20 min to obtain crude extract enzyme. the enzyme was concentrated using polyetylene glycone (peg) 20000 with dialysis tubing cellulose and acetonein various concentrations, i.e. 20, 40, and 60% (v/v) as comparators. after that, the concentrated enzyme was freeze dried with the addition of 0.5% maltodextrin. interesterification reaction. material mixture followed zainal and yusoff (1999) consisted of palm kernel olein (pkoo): palm stearin(pos) in 75:25 (w/w) ratios. ten percent enzyme (w/w) put in the melted pkoo-pos mixture. interesterification reaction was conducted at 150 rpm and 55 °c (the result of enzyme stability) for 24 h. to stop the reaction, the sample mix was filtered through whatman paper. analysed of oils and interesterified products using high performance liquid chromatography (hplc) were determined by the rapid method of aocs official method ce 5b-89. the column used was rp c-18 (250 × 4 mm i.d.), with particle size of 5-µm (merck, darmstadt, germany). detector used was refractive index and aseton and asetonitril with ratio of 75:25 v/v) was used as mobile -1 phase. the elution rate was 1 ml min . standard used was mixture of tri acyl glycerole (tag) that consisted of gliseryl tridecanoate (ccc), glyseryl tridodecanoate (lalala), glyseryl trimyristate (mmm), glyseryl trioctanoate (cacaca), dan glyseryl tripalmitin (ppp). individual peaks was identified by comparing qualitative of retention times with those of pure tag standards, negative control, palm stearin, palm kernel oils and the fat blend after interseterification, also the result of interesterification of commercial lipase (lypozyme tl1m). result fungal isolation, lipase qualitative activity assay and morphological identification. fungal isolation from several sources after 2-3 d of incubation resulted in 3 pure fungal groups, i.e. isolate r from ferment (bppt-cc), isolate t from tempeh, and isolate o from oncom. the result of activity assay of lipase crude extract from the three fungal isolates under uv irradiation on rhodamine b and olive oil media indicated that the three isolates positively produced lipase on the media assay. lipid acid products released by enzyme from hydrolysis process were responsible for colour development around the colonies under uv irradiation (kouker and jaeger 1987). the three isolates were capable of producing orange fluorescence (table 1) and forming clear zone on media and isolate o produced the highest fluorescence intensity (fig 1). microscopic and macroscopic identification of the three isolates were matched with their respective fungal morphology guidance books which resulted in isolate r, t and o belonged to genus aspergillus, mucor, and neurospora respectively (fig 2). based on blast and phylogenetic tree analyses, the fungal isolates r, o, and t belong to aspergillus niger, nerospora sp. and rhizopus oligosporus species, respectively (fig 3). volume 11, 2017 microbiol indones 37 table 1 the diameter of clear zone and the intesity of crude enzyme fluorescence of the three isolates under uv rays on rhodamin olive oil agar (roa) media modified from koeker and jaeger (1987) information: [+] low intensity, [++] moderate intensity, [+++] high intensity, and [++++] very high intensity. variables isolate r t o diameter of zona (cm) 0.2 0.3 0.3 fluorescence + +++ ++++ microbiol indones38 pratama et al. fungi produce lipase during their early growth period. however, the decrease in lipase produced by the three fungi was not followed with the decrease in biomass. media selection and production period. the result of media selection and production period in fig 5 indicated that media having the highest enzyme specific activity was pdb and olive oil media with optimum activity period occurred at 48 h. the highest lipase production by isolate r, t, and o was 43.963, -1 41.096, and 80.847 u mg , respectively. pdb and crude palm oil media produce the highest enzyme specific activity at 24 h, i.e. ranged from 20.918 to -1 25.868 u mg . enzyme specific activity of the three these fungal species are confirmed as gras for food product application. enzyme activity. fungal growth of the three isolates experienced both increase and decrease during 6 days fermentation (fig 4). on pdb and 2% olive oil media, enzyme specific activity of the three fungi reached the highest on day 2 of fermentation (isolate -1 -1 r88.96 u mg , isolate t 84.53 u mg , and isolate o -1 96.42 u mg ) before decreasing afterwards while on pdb and 2% crude palm oil occurred on day 1 of -1 fermentation (isolate r 24.43 u mg , isolate t 25.86 u -1 -1 mg , and isolate o 20.92 u mg ) before also decreasing afterwards. this indicates that the three fig 1 qualitative activity of lipase from three fungal isolates under uv on roa media, modified from koeker and jaeger (1987), after 2 days incubation at room temperature, a) isolate r from bppt-cc, b) isolate t from tempeh, and c) isolate o from oncom. fig 2 the microscopic identification result of morphological characteristics of 2 days-pure fungal isolates using microscope, a) isolate r, b) isolate t, and c) isolate o. a b c a b c volume 11, 2017 microbiol indones 39 gelasinospora tetrasperma atcc 96230 neurospora intermedia 28s large subunit ribosomal rna gene partial sequence uncultured fungus 5b e2h5615 neurospora crassa or74a neurospora pannonica trtc51327 neurospora crassa 28s o sordaria fimicola 28s gelasinospora tetrasperma aftol-id 1287 sordaria lappae dl05 sordaria tomento-alba 0.5 aspergillus niger sf-6095 aspergillus niger ifm 54309 aspergillus niger bk 01 r aspergillus niger nja-1 aspergillus awamori vtcc:f099 aspergillus niger kaml02 aspergillus niger ra402 aspergillus niger 18s aspergillus niger uwfp 696 aspergillus awamori vtcc:f-296 0.0005 rhizopus oligosporus cbs 338.62 rhizopus oligosporus 28s t rhizopus azygosporus cbs 357.93 rhizopus rhizopodiformis ifm 46417 rhizopus microsporus fsu 753 rhizopus azygosporus cbs 357.92 rhizopus chinensis cbs 631.82 uncultured fungusa1a10711 rhizopus microsporus cbs 699.68 rhizopus microsporus fsu 5256 0.001 fig 3 phylogenetic tree analyses of 28s rrna gene sequences of fungal isolates: o (a), r (b), and t (c). construction of a phylogenetic tree was conducted using the neighbour joining method. a b c fig 4 comparison of enzyme specific activity of isolate r ( ), isolate t( ) , and isolate o ( ) during 6 days fermentation at room temperature, [a] pdb and 2% olive oil media, and [b] pdb and 2% crude palm oil media. fig 5 enzyme specific activity of [a] isolate r, [b] isolate t, and [c] isolate o on pdb and olive oilmedia ( ), pdb and crude palm oil ( ), and blain ( ). 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 -1 s p ec if ic a ct iv it y ( u m g ) day a 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 -1 s p ec if ic a ct iv it y ( u m g ) day b 10 20 30 40 50 60 70 80 90 24 48 72 96 s p ec if ic a ct iv it y -1 (u m g ) time (h) a 0 10 20 30 40 50 60 70 80 90 24 48 72 96 s p ec if ic a ct iv it y -1 (u m g ) time (h) b 0 10 20 30 40 50 60 70 80 90 24 48 72 96 s p ec if ic a ct iv it y -1 (u m g ) time (h) c microbiol indones40 pratama et al. lipase incubation at reaction temperature of 55 °c failed to maintain enzyme catalytic activity hence since minute 15 to 180 the enzyme's activity gradually decreased (fig 7). lipase enzyme activity of isolate r -1 -1 decreased from 0.368 u ml to 0.193 u ml , isolate t -1 -1 from 0. 318 u ml to 0.024 u ml , and isolate o from -1 -1 0.424 u ml to 0.031 u ml . the result indicated that the longer the enzyme incubated at 55 °c, the lower the enzyme stability. the result of enzyme stabilty test was used as determinator of interesterification reaction period in the mixture of margarine material consisting of palm kernel olein (pkoo) and palm stearin (pos). enzyme concentration using polyethylene glycol (peg). enzyme concentration aimed to increase lipase activity before being used for enzymatic interesterification reaction. the result of comparison of enzyme specific activity before and after 10 times concentration (v/v) using peg indicated that peg was capable of increasing enzyme specific activity 2.9-5.1 times of before concentration (fig 8). isolates on blain media, however, reached only 4.729-1 47.771 u mg with 48 h as optimum production period. the effect of ph and temperature on enzyme’s activity and stability. based on the result of determining interesterification reaction temperature using enzyme stability test at 50 °c and 60 °c and compared to the enzyme activity at optimum temperature (40 °c), the activity of lipase at 50 °c decreased 26.67% in isolate r, 27.2% in isolate t and o 22.63% in isolate o. where as at 60 c, the enzyme specific activity of isolate r decreased 64.44%, isolate t 57%, and isolate o 32.85% (fig 6). such decrease was considered not too significant and therefore the closest temperature (55 °c) was used for interesterification reaction. this is due to interesterification process in the mixture of margarine material consisting of palm kernel olein (pkoo) and palm stearin (pos) requires a rather high reaction temperature. temperature brings about significant effects, not only for enzyme activity but also enzyme stability. fig 6 enzyme activity of isolate r ( ), isolate t ( ), and isolate o ( ) on several ph conditions using method of titration and silva. (a) at 50 °c and (b) at 60 °c. 0 1 2 3 4 5 6 7 8 9 10 4 5 6 7 8 -1 s p ec if ic a ct iv it y ( u m l ) a ph 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 4 5 6 7 8 -1 s p ec if ic a ct iv it y ( u m l ) ph b volume 11, 2017 microbiol indones 41 fig 8 enzyme specific activity of the three isolates before ( ) and after concentration ( ) using polyethylene glycole (peg). analysis result before and after enzymatic interesterification reaction using indigenous lipase of raw material formula consisting of palm kernel olein (pkoo) and palm stearin (pos), it was known that the result remained the same, or in another word there was no significant change in tag composition while in commercial lipase, the tag composition changed (fig 9). the highest increase (5.1 times) belonged to isolate t where the activity in isolate r increased 3.1 times and in isolate o 2.9 times. interesterification reaction. the quality of margarine is usually can be determined based on the characteristic of raw materials and tag composition (pandiangan 2008). based on the profil data of hplc fig 7 enzyme specific activity of isolate r ( ), isolate t ( ), and isolate o ( ) in several temperature conditions using method of titration (a) and silva (b). 0 1 2 3 4 5 6 7 8 9 10 25 30 35 40 45 -1 s p ec if ic a ct iv it y ( u m l )a temperature (°c) temperature (°c) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 25 30 35 40 45 -1 s p ec if ic a ct iv it y ( u m l ) b 10 20 30 40 50 60 70 80 90 100 110 -1 s p ec if ic a ct iv it y ( u m l ) r t o isolate 0 microbiol indones42 pratama et al. et al. 2003), while pva functions as emulsifier that can help stabilize oil and water soluble (ota and yamada 1966). test result indicated that the three isolates were capable of producing orange fluorescence and forming clear zone on the media. according to hou and johnston (1992), fluorescence zone formed on selection media with cpo as lipid source also indicated that the isolate was capable of producing lipase which break triacylglycerol (tag) in cpo into dag, mag, or ffa, and with rhodamin b forming fluorescence compounds.specific lipase hydrolyses ester bond at 1,3 position, producing fatty acid, monoacylglycerole, and diacylglycerole (suharyanto et al. 2011). enzyme activity indicated the enzyme ability to catalyze the change of a substrate into product in unit. a unit of lipase activity was defined as the amount of lipase capable of releasing 1 μmol fatty acid per ml discussion lipase was used as biocatalyst on enzymatic interesterification reaction in the making of margarine raw material. the test of lipase activity of the three fungi was qualitatively carried out following the modification of koeker and jaeger’s method (1987). this method could only confirm the lipase was produced but not for quantifying the enzyme activity. media used was selective media with rhodamin b and olive oil, and polyvinyl alcohol (pva) as modification. rhodamin b functions as the absorber of orange illuminating zone when lipase-producing fungi was placed under uv rays. rhodamin b forms dimer complex with monoacylglycerol (mag), diacylglycerol (dag), and free fatty acid (ffa) (koeker and jaeger 1987). olive oil functions as lipid source (gupta fig 9 high performance liquid chromatography (hplc) analysis result. triglyceride mix standart; specification peak: [1]glyceryl trioctanate; [2]glyceryl tridecanoate; [3]glyceryl tridodecanoate; [4] glyceryl trimyristate; [5]glycerol tripalmitate. volume 11, 2017 microbiol indones 43 interesterification reaction using high performance liquid chromatography (hplc) showed that tag composition showed that there was not any change before and after the reaction using indigenous lipase and raw material consisting of palm kernel olein (pkoo) and palm stearin (pos), as compared to commercial lipase. this is allegedly because the activity of indigenous lipase was still lower than that of commercial one. interesterification reaction requires lipase with high activity and thermostable characteristics under high temperature. therefore before using lipase in interesterification reaction, optimization of enzyme and production media is necessary to obtain lipase with high productivity and that can be used for high temperature interesterification reaction. acknowledgment the authors would like to thank agency for the assessment and application of technology technology (bppt), serpong for financial support and facilitated in this research. the work was also partially funded by insinas research grant of ministry of research and higher education through consortium scheme. references amir rm, shabbir ma, khan mr, hussain s. 2012. interesterification of fats and oils (review). j food sci. 22(3):143-153. bradford mm. 1976. a rapid and sensitive method for the quantitation of microorganisms quantities of protein in utilizing the principle of protein dye binding. anal biochem. 72(1):248�254. doi:10.1016/00032697(76)9 0527-3. bussamara r, fuentefria am, oliveira ed, broetto l, simcikova m, valente p, schrank a, vainstein. 2010. isolation of a lipase secreting yeast for enzyme production in a pilot plant scale batch fermentation. biores technol. 101(1):268-275. doi:10.1016/j.biortech. 2008.10.063. crueger w, crueger a. 1984. biotechnology: a textbook of industrial microbiology. brock td, editor. madison (us): science tech. faloni g, armas jc, mendoza cd, hernandez jlm. 2006. production of extracelluler lipase from aspergillus niger by solidstate fermentation. biotech. 44(2):235240. gandjar i, samson ra, van k, vermeulen t, oetari a, santoso i. 1999. pengenalan kapang tropik umum [introduction of general tropical fungus]. jakarta (id): yayasan obor indonesia. -1 -1 min , written in u ml . media selection and enzyme production period were carried out based on enzyme activity curve at hour 24 to 96 on various production media and inducers. a good fungal fermentation media to produce lipase contains carbon source such as fructose, nitrogen source such as peptone, and oil added such as palm oil and olive oil as inducer (sharma et al. 2001). inducer concentration highly affects enzyme activity. too much oil as inducer in production media leads to low lipase activity due to low oxygen transfer into medium (lima et al. 2003). pramitasari et al. (2012) used 2% olive oils inducer to produce lipase and suharyanto et al. (2011) used 2% crude palm oil (cpo) to produce 1.3 glyceride specific lipase from rhizopus oryzae tp-2. media selection and production period based on the comparison of enzyme specific activity (fig 11) indicates that the optimum activity period of pdb and olive oil media was at 48 h. in margarine raw material selection and making, pos can be mixed with pkoo which has shorter lipid chain to make the mixture better functional property such spread ability at room temperature (noor et al. 2002). interesterification using pkoo and palm oil stearin poswill likely have margarine free of trans fatty acid if the lipase produced by the three fungal isolates capable of performing interesterification reaction. palm stearin (pos) is solid fraction from oil palm husks which has physical characteristic of easily solidified at room temperature. therefore, interesterification reaction using the mixture of margarine material consisting of palm kernel olein (pkoo) and palm stearin (pos) requires high temperature, i.e. 60-70 °c. zainal and yusoff (1999) carried out interesterification reaction using commercial lipase from rhizomucor miehei (lipozyme im 60) with the ratio of pos and pkoo of 30:70 at 60 °c. enzyme is protein sensitive to high temperature as denaturation can easily occurred along with temperature increase. denatured protein adversely affects enzyme activity, and eventually decrease enzyme concentration and reaction rate. the optimum temperature of lipase produced from fungi isolated from tempeh, oncom, and bppt-cc was 40 °c and then decreased along with the increasing incubation temperature. the decrease in lipase activity at 50 °c was 22.63%-27.2% while at 60 °c 32.85%-64.44%; such decrease is considered not highly significant. however, because of the low lipase activity of the three isolates compared to commercial lipase, it eventually decreased the effectiveness of lipase activity. qualitative analysis based on profile data from microbiol indones44 pratama et al. 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40(1):321326. doi:10.1016/j.procbio.2004.01.005. speranza p, ribeiro ap. 2015. lipase catalyzed interesterification of amazonian patauá oil and palm stearin for preparation of specific-structured oils. j food sci technol. 52(12):8268-75. doi:10.1007/s13197-0151943-8. suharyanto, tri p, urip p. 2011. optimasi produksi diasilgliserol dari crude palm oil menggunakan lipase spesifik 1,3-gliserida dari rhizopus oryzae tp-2 [optimization of diacylglycerol production of crude palm oil using 1,3-specific lipase from rhizopus oryzae glycerides tp-2]. menara perkebunan. 79(1):23-29. suzuki y, mushiga y, yamane t, shimizu s.1988. mass production of lipase by fed-batch culture of pseudomonas fluorescens. appl microbial biotechnol. 27(1):417-422. doi:10.1007/bf00451606. treiche lh, oleivera d, mazzuti ma, luccio md, oleivera jv. 2010. a review on microbial lipases production. food bioproc technol. 3(1):182-196. doi:10.100 7/s11947-009-0202-2. yang jg, wang yh, yang b, mainda g, guo y. 2006. degumming of vegetable oil by a new microbial lipase. food technol biotechnol. 44(1):101-104. watanabe t. 1937. pictorial atlas of soil and seed fungi, morphologies of cultured fungi and key to species. london (gb):crc press. zainal z, yusoff msa.1999. enzymatic interesterification of palm stearin and palm kernel olein. jaocs. 76(9):1003-1008. gupta r, rathi p, gupta n, bradoo s. 2003. review lipase assays for conventional and molecular screening. biotechnol appl biochem. 37(1):63-71. doi:10.1042/b a20020059. hou ct, johnston tm. 1992. screening of lipase activities with culture from agricultural research services culture collection. jaocs. 69(1):1088-1097. doi;10.1007/bf0 2541042. illanes a, editor. 2008. enzime biocatalysis. de valpara´ıso: springer science, business media b.v. doi:10.1007/97 8-1-4020-8361-7. kitts d. 1996. toxiciti and safety of fats and oils. bailey’s industrial oil and fat products. 1(1):215-280. kouker g, jaeger ke. 1987. specific and sensitive plate assay for bacterial lipases. appl environ microbiol. 3(1):211-213. lima vmg, krieger n, sarquis mim, mitchell da, ramos lp, and fontana jd. 2003. effect of nitrogen and carbon sources on lipase production by penicillium aurantiogriseum. food technol biotechnol. 41(2):105110. ming lo, ghazali hm, let cc. 1998. effect of enzymatic transesterification on the fluidity of palm stearin palm kernel olein mixtures. food chem. 63(2):155-159. doi:10.1016/s0308-8146(98)00046-6. mozaffarian d, katan mb, ascherio a, stampfer mj, willet wc, 2006, trans fatty acids and cardiovascular disease (rev), n engl j med. 354(15):1601-1613. doi:10.1056/ nejmra054035. murni sw, kholisoh sd, tanti dl, petrissia em. 2011. produksi, karakterisasi, dan isolasi lipase dari aspergillus niger [production, characterization, and isolation of lipase from aspergillus niger]. in: murni sw, kholisoh sd, tanti dl, petrissia em, editor. pengembangan teknologi kimia untuk pengolahan sumber daya alam indonesia. indonesian congress and seminar on tenik chemistry. 2011 feb 22;. yogyakarta (id): upn. p 1-7. noor hmd, sundram k, siew wl, aminah a, mamot s. 2002. tag composition and solid fat content of palm oil, sunflower oil, and palm kernel olein blends before and after chemical interesterification. jaocs. 79 (11):1137-1144. doi:10.1007/s11746-002-0617-0. ota y, yamada k. 1996. lipase from candida paralipolytica: part 1 anionic surfactant as the essential activator in the systems emulsified by polyvinyl alcohol. agr biol chem. 30(1):351-358. panji t, suharyanto, arini n. 2008. lipase spesifik 1,3gliserida dari fungi lokal untuk biokonversi cpo menjadi diasilgliserol [1,3 specific lipase-glycerides of local fungi for the bioconversion of oil into diacylglycerol]. men perkeb. 76(1):11-22. volume 11, 2017 microbiol indones 45 1: 35 2: 36 3: 37 4: 38 5: 39 6: 40 7: 41 8: 42 9: 43 10: 44 11: 45 2 tresnalisrinaagustin.cdr page 1 page 2 page 3 5.mi-mahmud available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.4.5issn 1978-3477, eissn 2087-8575 vol 10, no 4, december 2016, p 149-152 *corresponding author; phone: +62-11116178/+62-2518624907, email: kcl_51@yahoo.co.id since the industrial revolution in the middle of the 19th century, average female life expectancy has increased in western societies from 45 years to 80 years, which corresponds to an increase of 2.3 years per decade (oppen et al.2002). the increase in average life span observed in all developed countries is accompanied by an incremental burden of age associated diseases. elderly are among vulnerable groups due to weakening immune system. the aging process is related to changes of bmi (body mass index). aging is also associated with chronic, low-grade inflammatory activity. systemic chronic inflammation has been found to be related to mortality risk from all causes. agerelated diseases such as alzheimer, atherosclerosis, diabetes mellitus, sarcopenia, and osteoporosis are initiated or worsened by systemic inflammation, (westendorp 2012). catfish’ flour has been proven to be able to improve the nutritional status of undernourished five years old children (kusharto et al. 2008; adi 2010). catfish flour is produced, while catfish oil as by product. catfish’ oil contains essential fatty acids, i.e., 22.65%, 17.79%, 1.21% of oleic acid (c18:1), linoleic acid (c18:2) (omega 6), and linolenic (c18:3) (omega 3) respectively (srimiati 2011).various studies revealed that the essential fatty acid has anti-inflamatory and antithe aim of the research was to study the effect of provision of catfish’ flour, oil and probiotic faecium is-27526 on body weight and crp (c-reactive protein) of aged female cynomolgus monkey (macaca fascicularis). nine aged female cynolmolgus monkeys were randomly divided into three groups. the age is determined by dentition, with body weight in a range of 2 4 kg. animals were placed in individual cages in the position where they can interact audiovisually. feed composition consists of sugar, egg, soy flour, wheat flour, sweet potatoes flour, butter, egg yolk flour, catfish’ flour and oil, and microencapsulated probiotic e. faecium is27526 and administered for 90 days effective. evaluation of body weight and crp were conducted. the study showed that there is no significant effect of experimental diets on body weight in each group. probiotic tends to suppress the body weight gain where the body weight of cynomolgus fed with probiotic diet has more stable than others. the body weight of cynomolgus in probiotic diet is shown more stable than others. there is no effect of experimental diets on crp which is marked by negative result of crp test. probiotic e. faecium is-27526 is potential for body weight homeostasis regulation to reduce the risk of overweight and obesity. key words : body weight, clarias gariepinus, crp, enterococcus faecium is-27526, macaca fascicularis tujuan dari penelian adalah untuk mempelajari pengaruh pakan fungsional berbasis tepung, minyak ikan lele dan probiotik enterococcus faecium is-27526 terhadap berat badan, dan crp (c-reactive protein) pada monyet ekor panjang (macaca fascicularis) betina usia tua. sembilan ekor monyet ekor panjang dibagi dalam tiga kelompok secara acak. usia ditentukan melalui pergigian (dentisi), dengan berat badan antara 2-4 kg. hewan coba ditempatkan pada kandang individu dengan posisi masih dapat berinteraksi secara audiovisual satu dengan yang lain. pakan yang diberikan terdiri atas gula, telur, tepung kedelai, tepung terigu, tepung ubi jalar, butter, tepung kuning telur, tepung ikan lele, minyak ikan lele, dan probiotik e. faecium is-27526. pengukuran berat badan (bb) dan crp dilakukan setiap bulan. tidak ada pengaruh signifikan pemberian pakan terhadap berat badan hewan coba pada setiap kelompok. namun, probiotik memiliki kecenderungan menekan peningkatan berat badan. tidak ada pengaruh pakan terhadap crp ditandai dengan hasil negatif pada uji crp. probiotik e. faecium is-27526 berpotensi dalam regulasi homeostasis berat badan untuk menurunkan risiko kelebihan berat badan dan obesitas. kata kunci: berat badan, clarias gariepinus, crp, macaca fascicularis, probiotik enterococcus faecium is-27526 enterococcus effect of catfish’ (clarias gariepinus) flour and oil with probiotic enterococcus faecium is-27526 based functional feed provision on body weight and c-reactive protein (crp) of aged atherogenic female cynomolgus monkey 1 2 mahmud aditya rifqi , clara m kusharto *, 3 2 ingrid s surono , and sri anna marliyati 1 department of nutrition and health, universitas airlangga, jalan mulyosari, surabaya 60286, indonesia; 2 department of community nutrition, institut pertanian bogor, kampus ipb dramaga, bogor 16680, indonesia; 3 department of food technology, universitas bina nusantara, jalan jalur sutera barat kav. 21, alam sutera, tangerang 15143, indonesia atherosclerosis effects (demonty et al. 2006). enterococcus faecium is-27526 was isolated from dadih fermented buffalo milk in west sumatera. based on in vitro and in vivo studies, e. faecium is-27526 has been proven to have the ability of adherence to the intestinal mucosa (collado et al. 2007). novel probiotic e. faecium is-27526 has significant positive effects on humoral immune response, salivary siga, in underweight preschool children, and on body weight gain of pre-school children (surono et al. 2011). administration of microencapsulated e.faecium is27526 in the pasta cream has been proven to increase the body weight and fecal lactic acid bacteria of mice (harianti 2009). this study was conducted to the aged female atherogenic cynomolgus monkey (macaca fascicularis) to evaluate the effect of. functional feed of catfish’ flour, oil and probiotic e. faecium is-27526 based on body weight and crp (c-reactive protein) materials and methods strain and probiotic preparation. e. faecium is27526 was isolated from dadih fermented milk and was identified by 16s rrna gene sequencing as e. faecium (genbank accession no. efo68251) (surono et al. 2011). the probiotic culture was cultivated in mrs broth in a 10 l fermentor for 22 h at 37 °c under aerobic condition, harvested by centrifugation (3200 × g, 4 °c, 20 min), washed twice with phosphatebuffered saline (pbs, ph 7.0) and frozen. purity and viability of probiotic was tested on mrs agar plates before and during the study period. experimental animals. nine aged female cynomolgus monkeys (m. fascicularis) or cm of 2-4 kg quarantined in the center of primate study of the institut pertanian bogor. animals were adapted in the individual cages in the position where they can interact audiovisually each other. diets were given for 120 -1 calori kg of body weight per day, while the drink was 150 rifqi et al. microbiol indones supplied ad libitum. the ethical clearance had been obtained from animal care and use committee (acuc) of pt bimana indomedical number p.01-13ir. experimental design. this preliminary study was conducted in randomized factorial (raf) design. the trial required oral consumption of intervention diet over a total period of 90 d. the diet formula is based on previous research of kusharto et al. (2012). the diets provision to animals were divided into three groups (n=3). the groups namely a1 standard diet (410 cal -1 100 g , fat 18.76%), a2 standard diet + probiotic e. 8 -1 -1 faecium is-27526 (10 cfu g ), (425 cal 100 g and fat of 19.84%), a3) standard of diet + probiotic e. faecium 8 -1 -1 is-27526 (10 cfu g ) + fish oil (7 ml 100 g ) (energy -1 435 cal 100 g , fat 21.86%). egg yolk flour was added as atherogenic diet (4% of diet). the subjects were conditioned to abstain from other probiotic and prebiotic during intervention. compliance of intake were monitored and assured by veterinarian. body weight and crp were collected at baseline and monthly during 3 months intervention. analysis of samples. body weight weighing-scale was conducted. the crp test was aglutination test. the reagent/test kit was produced by pt. rajawali nusindo, indonesia. analysis of data. comparison of body weight and lipid profile within and between group were tested by analysis of variance (anova). further test the difference between the group was analyzed using duncan multi range range test (dmrt) with 5% significance level. results subject. nine apparently healthy monkeys (m. fascicularis) were included in the research and no adverse effect was found. percentage of feed consumption is shown in table 1. the percentage of animals diet consumptions more groups body weight average (kg) consumption average (gram) consumption percentation (%) a1 3.41 ± 0.29 90.91 ± 0.31 90.91 a2 3.28 ± 1.22 85.75 ± 0.36 85.75 a3 3.30 ± 0.58 89.18 ± 0.44 89.18 note: (a1) standard diet, (a2) standard diet+probiotics, (a3) standard diet+ probiotics + catfish oil table 1 percentage of feed consumption 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 ∆ b o d y w ei g h t (k g ) month 1* month 2* month 3* a1 a2 a3 0.36 0.41 0.47 0.20 0.21 0.18 0.18 0.26 0.29 than 85%, not significant difference exist within the groups. body weight. the variety of diets affect of animal body weight. measurement of body weight was performed during the intervention. the percentage of body weight is shown in fig 1. based on anova, the difference of body weight not significant (p>0.05) between groups. all diets tend to increase the animal body weight during intervention. body weight in probiotic diet more stable than others. crp (c-reactive protein). crp is an important component of the immune system, collection of protein that is made by body when a major infection or trauma. crp can be an indicator of inflammation in the body. many studies have found that crp as marker of inflammatory process are elevated among individuals at high risk for future heart disease. inflammation is important in all phases of heart disease, including the early initiation of atherosclerotic plaques within the arteries, as well as the acute rupturing of these plaques that results in heart attack and, all too often, sudden death (ridker 2003). table 2 shows the negative results of crp during intervention. this result indicates no inflammation as an indicator of atherosclerosis in all interventions which are characterized with negative results. discussion factors that influencing the animals’ consumption are palatability, form and type of diet (bennet et al. 1996). in adaptation period the animal consumes the monkey chow which is common feed of monkeys. monkey chow diet didn’t significantly affect cm body weight and lipid profile. while, high fat diet with the addition of eggyolk flour is able to raise the palatability of feed and blood cholesterol of cm (astuti et al. 2010; pelletier 1996). measurement of body weight during intervention is required as the indicator of health of animals (fortman et al. 2002). the growth of animal depends on the nutrition content of diet. eggyolk flour is added to improve the palatability affecting the body weight (astuti et al. 2010). oktarina (2009) revealed that yolkflour (fat content ±19.62%) tended to cause more obesity compared with monkey chow (fat 5.55%). it is due to higher fat compared with protein and carbohydrate. probiotic tends to supress the body weight gain. nugraha (2013) revealed that administration of probiotic e. faecium is-27526 increased fecal lactic acid volume 10, 2016 microbiol indones 151 table 2 evaluation of crp of during intervensionmacaca fascicularis fig 1 alteration of macaca fascicularis body weight month* treatment n 1 2 3 4 standard (a1) 3 ( ) ( ) ( ) ( ) standard + probiotics (a2) 3 ( ) ( ) ( ) ( ) standard + probiotics + fish oil(a3) 3 ( ) ( ) ( ) ( ) *note: (1) month 0 (baseline data), (2) month 1, (3) month 2, (4) month 4 microbiol indones152 rifqi et al. primates in biomedical reseach: biology and management. new york (us): academic pr. collado mc, surono is, meriluoto j, salminen s. 2007. potential probiotic characteristics of lactobacillus and enterococcus strains isolated from tradisional dadih fermented milk against pathogen intestinal colonization. j food protection. 70(3):700-705. doi:10.4315/0362028x-70.3.700. demonty i, chan y, pelled d, jones p. 2006. fish-oil esters of plant sterol improve the lipid profile of dyslipidemic subjects more than do fish-oil or sunflower oil esters of plant sterols. am j clin nutr. 84(6):1534-42. fortman jd, hewett ta, bennett 8t. 2002. the laboratary non-human primate. florida (us): crc pr. harianti r. 2009. the effect of high protein biscuit added with probiotic cream administration on the profile of fecal microbiota and body weight of rats [thesis]. bogor (id): bogor agricultural university. kusharto c, marliyati s, surono, dainy n. 2012. functional clarias biscuit riched protein, mineral and fiber, added fish oil as nutritious and emergency food for elderly. bogor (id): institut pertanian bogor. nugraha es. 2013. effect of probiotic enterococcus faecium is-27526, and catfish (clarias gariepinus) oil in functional biscuit enriched with catfish flour and sweet potato (ipomea batatas) flour on fecal microbiota of aged female macaca fascicularis [thesis]. bogor(id): bogor agricultural university. oktarina r. 2009. the study of high energy feed source on the formation of obese monkeys [thesis]. bogor (id): bogor agricultural university. oeppen j, vaupel jw. demography. broken limits to life expectancy. science 296(5570):1029-31. doi:10.1126/ science.1069675. pelletier x, thouvenot p, belbraouet s. 1996. effect of egg consumption in healthy volunteers: influence of yolk, white or whole-egg on gastric emptying and on glycemic and hormonal responses. ann nutr metab. 40(2):109-115. doi: 10.1159/000177903. ridker pm. 2003. c-reactive protein, a simple test to help predict risk of heart attack and stroke. american hearth association. doi: 10.1161/01.ctr.0000093318.57779. 67. rifqi ma. 2014. effect of catfish (clarias gariepinus) flour and oil with probiotic enterococcus faecium is27526 based functional feed provision on body weight, lipid profile and c-reactive protein (crp) of aged female cynomolgus monkey (macaca fascicularis) [thesis]. bogor (id): bogor agricultural university. srimiati m. 2011. refining of catfish oil by product catfish flouring as alternative of source omega 6 fatty acid [thesis]. bogor(id): bogor agricultural university. surono is, koestomo pf, novitasari n, zakaria fr, yulianasari, koesnandar. 2011. novel probiotic enterococcus faecium is-27536 supplementation increased total salivary siga level and bodyweight of pre-school children: a pilot study. elsevier ltd. 17(6):496-500. bacteria and decreased fecal coliform bacteria of macaca significantly (p<0,05). probiotic causes the balance of microflora in gastrointestinal tract, that supressing the pathogenic bacterial growth. lactobacillus and enterococcus strain significantly reduced pathogen adhesion to mucus (collado et al. 2007) the healthy intestinal tract optimizes the absorption and causes the beneficial to health (harianti 2009). probiotic e. faecium is-27526 is potential for body weight homeostasis regulation to reduce the risk of over weight and obesity supported by significantly lowering ldl and total cholesterol (rifqi 2014). inflammation is measured by crp in this research. the result shows the negative result (-) in all treatment groups. crp is a powerful predictor of risk, particularly when combined with cholesterol evaluation. a persistently elevated crp level is indicative of the risk of heart disease and of the accelerated atherosclerosis that affects individuals with diabetes and others metabolic syndrome, the older adult who are over weight/obese are more likely than those who are not obese to the symptoms of metabolic syndrome. overweight as a major underlying factor contributing to atherosclerotic cardiovascular disease, including abnormal lipid profile. probiotic e. faecium is-27526 is potential of body weight homeostasis regulation to reduce the risk of over weight and obesity. acknowledgment this research was supported by the grant from competitive research grant of directorate general of higher education. secial special thanks delivered to director of pt. carmelitha lestari, primate research centre, department of microbiology, atmajaya university and microbiology of seafast centre, lppm-ipb, pt ultra jaya milk co. for their support and advices. references adi ac. 2010. efficacy of biscuit enriched with the catfish protein flour (clarias gariepinus), soy protein isolate and enterococcus faecium is-27526 microencapsulated probiotic as a supplementary food on underweight underfive children (2-5 years old) [dissertation]. bogor (id): bogor agricultural university. astuti d, mansjoer, sajuthi. 2010. blood lipid profile of cynomolgus monkey (macaca fascicularis) induced by high fat diet. j primatologi indonesia. 7(1): 16-20. bennet bt, abee cr, henrickson r. 1996. non human 1: 149 2: 150 3: 151 4: 152 5.mi685-junianto available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.2.5issn 1978-3477, eissn 2087-8575 vol 7, no 2, june 2013, p 75-83 *corresponding author; 21-7566922, e-mail: solichin.budiasih@gmail.com phone: +62-21-7560536, fax: +62chitin is a homopolymer of n-acetyl-d-glucosamine residues linked by β-1, 4 bonds. chitin and its derivatives have many applications in food, pharmaceutical, photography, cosmetic, paper, and textile industries (dutta et al. 2004). chitin can be found in various animals and plants and the crustacean is the most important chitin source for commercial use. in crustacean waste chitin is associated with protein, minerals, especially calcium carbonate, lipids, and pigments (mahmoud et al. 2007). consequently, to extract chitin from crustacean waste will involve removal of the protein (deproteination), minerals (demineralization), lipids, and pigments. conventional chitin extraction used harsh chemicals that caused environmental pollution, since concentrated hydrochloric acid was used for demineralization and sodium chitin extraction from shrimp shells involves two processing steps that are demineralization followed by deproteination process. lactobacillus acidophilus fncc 116 and bacillus licheniformis f11.1 were used in demineralization and deproteination respectively. the overall objectives of this experiment were to determine fermentation systems which resulted in the highest mineral and protein removal. the demineralization experiments consisted of three different batch fermentation designs: batch fermentation (a ); subsequent batch m fermentation 1, in which 100% medium was replaced with fresh medium after 24 h fermentation (b ); and m subsequent batch fermentation 2, in which 50% medium was replaced with the same amount of fresh medium after 24 h fermentation (c ). the demineralization was conducted at 30±2 °c, 50 rpm for 60 h. the deproteination m experiments consisted of 3 different batch fermentation designs: batch fermentation 1, inoculum was added once at the beginning of the fermentation (a ); batch fermentation 2, inoculum was added twice, at the beginning and p after 24 h fermentation (b ); and subsequent batch fermentation, 100% medium was replaced with fresh medium p after 24 h fermentation (c ). the deproteination was carried out at 55 °c, ph 7.8-8.0, aeration 2.3 vvm and p agitation 275 rpm for 96 h. the experimental results showed that in the demineralization process, fermentation design b gave the highest ash removal. ash removed in the fermentation design a , b , and c was 97.19, 99.69, m m m m and 97.69% respectively. the protein removed in the fermentation design a , b , and c was 94.42, 94.51, and p p p 95.37% respectively. key words: demineralization, deproteination, microbiological chitin extraction, shrimp shells ekstraksi kitin dari kulit udang terdiri atas dua tahap proses yaitu proses demineralisasi dilanjutkan dengan deproteinasi. lactobacillus acidophilus fncc 116 dan bacillus licheniformis f11.1 masing-masing digunakan untuk proses demineralisasi dan deproteinasi. tujuan penelitian ini adalah untuk menentukan sistem fermentasi yang dapat menghilangkan mineral dan protein dari kulit udang terbanyak. percobaan demineralisasi terdiri atas 3 rancangan fermentasi sistem tumpak yang berbeda: fermentasi sistem tumpak (a ); fermentasi sistem tumpak m berurutan 1, di mana 100% medium diganti dengan medium segar sesudah fermentasi 24 jam (b ); dan m fermentasi sistem tumpak berurutan 2, di mana 50% medium diganti dengan medium segar dalam jumlah yang sama sesudah fermentasi 24 jam (c ). proses demineralisasi dilakukan pada 30±2°c, 50 rpm selama 60 jam. m percobaan deproteinasi terdiri atas 3 rancangan proses yang berbeda: fermentasi sistem tumpak (a ), inokulum p ditambahkan sekali pada awal fermentasi; fermentasi sistem tumpak 2 (b ), inokulum ditambahkan 2 kali, pada p awal dan setelah fermentasi 24 jam; fermentasi sistem tumpak berurutan: 100% medium diganti dengan medium segar setelah 24 jam (c ). proses deproteinasi dilakukan pada 55 °c, ph 7,8-8,0, aerasi 2,3 vvm dan agitasi 275 p rpm selama 96 jam. hasil percobaan menunjukkan bahwa dalam proses demineralisasi, rancangan fermentasi bm dapat menghilangkan kadar abu terbanyak. kadar abu yang dihilangkan dalam rancangan fermentasi a , b , dan m m c masing-masing 97,19%, 99,69 %, dan 97,69%. pada proses deproteinasi, protein yang dihilangkan dari m rancangan fermentasi a , b , dan c masing-masing 94,42%, 94,51%, dan 95,37%. p p p kata kunci: demineralisasi, deproteinasi, ekstraksi kitin secara mikrobiologi, kulit udang selection of methods for microbiological extraction of chitin from shrimp shells 1 2 2 junianto , budiasih wahyuntari *, and siswa setyahadi 1 faculty of fishery and marine sciences, universitas padjajaran, jalan raya jatinangor km 21, sumedang 45363, indonesia; 2 center for bioindustrial technology, gedung 611, laboratoria pengembangan teknologi agro-biomedika, badan pengkajian dan penerapan teknologi, kawasan pusat penelitian ilmu pengetahuan dan teknologi, tangerang selatan 15314, indonesia hydroxide for deproteination. some studies have been done to reduce or even to eliminate the environmental problems (healy et al. 2003; beaney et al. 2005; rao and stevens 2005; jung et al.2006; and prameela et al.2010). these studies include deproteination of the crustacean shells using proteolytic enzyme or microorganisms. beaney et al. (2005) showed that the functional properties of the bioprocessed chitin were similar to those of chemically extracted chitin. microbiological demineralization and deproteination have been done by healy et al. (2003), rao and stevens (2005), jung et al. (2006), and prameela et al. (2010). based on our previous study, demineralization prior to deproteination in chitin extraction resulted in better ash and protein removal (wahyuntari et al. 2011). the objective of trying different demineralization experiments was to find the optimum lactic acid production in order to get maximum ash removal. parameters observed in this experiment were growth of lactobacillus acidophilus fncc 116, glucose consumed by the bacteria during fermentation, lactic acid produced, and ph course during fermentation. three different deproteination experiments were done to optimize protease production for hydrolyzing protein in the shrimp shells. the parameters studied were bacterial growth of bacillus licheniformis f11.1, protease produced and protein content of the protease supernatant (cell free fermented broth). material and methods shrimp shells and microorganisms. headless shrimp shells of penaeus vannamei were obtained from frozen shrimp processing company “pt wirontono baru” north jakarta, indonesia. the shells were washed and disintegrated into 5-10 mm size, kept in -20 °c before using it for experiments. l. acidophilus fncc 116, a lactic acid producing bacterium was used for demineralization process. the bacterium was obtained from food nutrition culture collection of faculty of agicultural technology, universitas gadjah mada, yogyakarta, indonesia. the stock cultures in 10% glycerol and 10% skimmed milk were stored in deep freezer at -80 °c. b. licheniformis f.11-1 used for deproteination process of shrimp shells was isolated from shrimp shell waste of pt lauraindo, a frozen shrimp processing company, palembang, sumatera, indonesia. the bacterium was isolated and identified by waldeck et al (2006) and molecularly modified by hoffman et al (2010). the stock culture was kept in 10% glycerol and 10% skimmed milk at -80 . demineralization process. completely randomized design was used in experiment of shrimp shell demineralization. the demineralization experiments consisted of 3 different batch fermentation designs: batch fermentation (a ); subsequent batch fermentationm 1 (b ), 100% medium was replaced with fresh medium m after 24 h fermentation (the fermented medium was centrifuged to separate the cells and shrimp shells waste, the supernatant was discarded and replaced with fresh medium); subsequent batch fermentation 2 (c ), in which 50% medium was replaced with the m same amount of fresh medium after 24 h fermentation. the demineralization was conducted in 2 l fermentor (contained 1 l medium) at 30±2 °c, 50 rpm for 60 h. refreshing frozen stock culture was conducted by transferring 1 ml stock culture into 9 ml of sterile de man rogosa sharpe (mrs) broth and incubated at 37 ºc for 24 h. to prepare a starter inoculum, 10 ml refreshed culture was transferred into 90 ml mrs broth in 250 ml erlenmeyer flask and incubated at 37 °c until its optical density reached 0.85 at wavelength 9 of 600 nm with cell concentration about 1x10 cfu -1 ml . based on previous study optimum demineralization condition was at ambient temperature (30 ± 2 °c) and 50 rpm agitation (junianto et al. 2009). three hundred grams frozen shrimp shell waste (69.5% moisture) were added with 900 ml liquid media and 100 ml starter inoculums. in 100 ml medium contained 6 g glucose, and 0.05 g yeast extract, ph was adjusted to ph 7. the fermentation was done at 37 °c and 50 rpm agitation for 48 h. each experiment was repeated twice. when demineralization process was completed, the shells were separated from the broth and washed with running water until the washed water became neutral (ph 7) and drained. the demineralized shells then were kept in -20 °c for the following process. deproteination process. completely randomized design was used in the deproteination experiment of demineralized shrimp shells. the deproteination experiments consisted of 3 different batch fermentation designs: batch fermentation 1 (a ), inoculumn was p added once at the beginning of the fermentation; batch fermentation 2 (b ), inoculum was added twice, at the p the beginning and after 24 h fermentation; subsequent batch fermentation (c ), 100% medium was replaced p with fresh medium after 24 h fermentation (the fermented medium was centrifuged to separate the cells and shrimp shells waste, the supernatant was discarded and replaced with fresh medium). the frozen stock °c 76 junianto et al. microbiol indones culture of b. licheniformis f11.1 was refreshed in luria broth media. the culture was incubated in shaker incubator at 55 °c, 180 rpm for 6 h or until the optical density of the culture reached 0.9, which based on 9 previous experiment the cell density was equal to 1 × 10 -1 cfu ml (junianto et al. 2009). each 100 ml fermentation medium contained 0.5 g kh po , 0.5 g 2 4 nacl, 0.5 g yeast extract, 0.05 g mgso , and 0.1 g 4 cacl . two hundreds ml inoculum was added into 300 2 g of shrimp shells in 800 ml medium. the fermentation was carried out at 55 °c, 2.5 vvm aeration, 275 rpm agitation for 60 h and the ph was maintained at the range of 7.8-8.2. samples for analytical assays were taken every 6 h. after the deproteination process completed, the shells were separated from the broth, washed, drained, and kept at 20 °c for the following process. all fermentations were conducted in custom made fermentor consisted of 2 l glass cylinder jar equipped with jake and kunkel rod agitator for agitation, a compressor connected to lkb-bromma, flow meter for aeration and heated water in water bath that was being circulated using coil into the jar for temperature control. the shrimp shells were not sterilized prior to any fermentation. analytical procedures. demineralization process of the shrimp shells (before, during, and after fermentation), moisture, ash, and insoluble protein content of the shrimp shells were observed, whereas the parameters observed of the fermented broth in demineralization process were bacterial density, glucose, lactic acid, and ph. parameters observed during deproteination process were bacterial density and protease production in the fermented broth, as well as protein content of the shrimp shells. moisture content was determined by heating samples at 110 °c in “kett” infrared moisterure meter model f-1a (tokyo, japan). ash content was determined after combustion of 5 g dried sample in a crucible at 600 °c for 4 h in muffle furnace (aoac 1984). insoluble protein content of the shrimp shells and fermented solid sample was solubilized using 1m naoh. seven poin five (7.5) ml of 1m naoh was added to 0.5 g sample and then incubated for 24 h. the protein content of the supernatant was measured according to lowry et al. (1951) method using bovine serum albumin fraction iv (sigma) as a standard. glucose and lactic acid content was analyzed using hplc (merck-hitachi), aminex column hpx-87h (300mm × 7.8mm), at 65 °c (l-5025-column thermostat), isocratic mobile phase of 0,005 n h so with 2 4 -1 flow rate of 0.6 ml min (l-6200a-pump, merck hitachi), differential refractometer detector ri-71 (merck). glucose standard used 1% glucose (sigmaaldrich) and lactic acid standard was 10% l-lactic acid (oxoid). protease activity in fermented broth was assayed using azocasein as a substrate according to the method described by waldeck et al. (2006). one unit was defined as the amount of enzyme releasing 1 mol azocasein per min under reaction conditions. the density of bacterial growth in fermentation broth was assayed after serial dilution by counting colony -1 forming unit (cfu ml ) on mrs agar plate after incubation at 37 °c, 24 h for l. acidophilus fncc116 and on luria bertani agar plate after incubation at 55 °c, 24 h for b. licheniformis f11.1. decrease of the ash and protein content in shrimp shells were analyzed statistically using f test and duncan’s multiple range tests at 95% confidence. experimental results of other parameters were descriptively analyzed in the graphic forms. chitin concentration of the demineralized and deproteinized shrimp shells was calculated based on equation 1, according to mizani and aminlari (2007). chitin (%) = chitin nitrogen ×14.25 total nitrogen of the demineralized and deproteinized shrimp shells (chitin) was analysed using kjeldhal method (aoac 1984). results demineralization. bacterial growth in batch fermentation a reached the maximum growth (1.96 × m 9 -1 10 cfu ml ) after 24 h fermentation, and then started decreasing afterward until the end of observation (2.53 × 8 10 cfu after 60 h) (fig 1). maximum bacterial growth in fermentation b was also reached after 24 h (1.81 × m 9 10 cfu). eighteen h after replacing the medium or 42 h from the beginning of fermentation, the cell amount 9 reached 2.07 × 10 cfu, and then the cell amount 8 decreased to 5.78 × 10 cfu at the end of fermentation (60 h observation) (fig 1). in the second subsequent 9 batch c , the maximum cell growth reached 1.99 × 10m cfu after 24 h. after replacing 50% medium with the fresh one, the cells reached maximum amount of 1.55 × 9 10 cfu after 12 h or after 36 h since the beginning of fermentation, then the cell amount decreased to 5.68 × 8 10 cfu at the end of observation (fig 1). initial glucose concentration was 6% (w/v), and after 24 h fermentation the remaining concentration of the (1) volume 7, 2013 microbiol indones 77 78 junianto et al. microbiol indones fermentation b and replacement of medium in c , the p p cell amount started increasing. the cell amount in b p started declining 6 h after addition of inoculum and in c declined gadually for the following 12 h and p decreased fast for the last 6 h of observation (fig 6). maximum production of protease during deproteination in a , b and c was reached after 48, 72, p p p and 72 h incubation respectively. the highest enzyme -1 activity was 16.51 u ml in b , the second was 16.06 u p -1 -1 ml in a , and the lowest was 11.63 u ml in c (fig p p 7). protein concentration of the shrimp shells in fermentation system a , b , and c 5.58, 5.49, and m m m 4.63% respectively which represented 94.42, 94.51, and 95.37% protein removed. aproximate analysis of chitin produced by combination of demineralization bm and deproteination a showed as follows: 0.84% ash, p 1.46% protein, and 97.85% chitin (% dry base). discusion based on our previous report, in the microbiological chitin extraction from shrimp shells, demineralization prior to deproteination gave a better chitin yield (wahyuntari et al. 2011), therefore in this experiment, demineralization was carried out before deproteination process. demineralization. fermentation system of l. acidophilus fncc 116 used for lactic acid production in this experiment was batch and modification of fed batch. batch, fed batch, or modification of both systems are common fermentation system used in industries (hsu and wu 2002). the efficiency of lactic glucose of all fermentation design was about 1% (w/v) (fig 2). in fermentation b and c , the medium was m m replaced after 24 h with a 100% and 50% fresh medium, thus there was glucose addition. the rapid decrease of glucose concentration in the first 6 to 24 h afterward (fig 2) corresponded to the increase of bacterial cells (fig 1). after 48 h fermentation, glucose in the medium was continuously consumed until the end of the observation, where less than 2% glucose remained. lactic acid produced in all fermentation designs after the first 24 h of fermentation was about the same which was 2.17, 2.30, and 2.26%, repectively. after that, lactic acid concentration in batch fermentation am did not increase. however, the lactic acid concentration in fermentation b and c increased after fresh medium m m addition. the highest lactic concentration was produced in b (4.19%) and in c (3.42%) (fig 3). m m the ph value of the fermentation broth depended on the lactic acid concentration. the lowest ph was achieved in fermentation b (ph 3.88) and the highest m ph in a (ph 4.61) (fig 4). the ash concentration in the m shell in all treatments decreased rapidly for the first 24 h, and then the ash removal was slowing down until the end of observation. the ash concentration of the shells in a , b , and c was 0.55, 0.06, and 0.45% which m m m represented 97.19, 99.69, and 97.69% ash removed respectively (fig 5). deproteination. maximum bacterial growth of all fermentation systems was reached after 12 h incubation, and then started declining for the following 12 h, in fermentation a continuously declining until p the end of observation. after addition of inoculum in fig 1 growth of lactobacillus acidophilus fncc 116 in different fermentation systems. a ( ): batch fermentation; b ( ): m m subsequent batch fermentation, in which 100% medium was replaced with fresh medium after 24 h fermentation; c ( ): m subsequent batch fermentation, in which 50% medium was replaced with the same amount of fresh medium after 24 h fermentation. r£ 7 10 8 10 9 10 10 10 0 12 24 36 48 60 -1 c el l a m o u n t (c f u m l ) fermentation time (h) fig 2 glucose consumption during demineralization of shrimp shells by lactobacillus acidophilus fncc 116 in different fermentation systems. a ( ): batch fermentation; b ( ): subsequent batch fermentation, 100% medium was replaced m m with fresh medium after 24 h fermentation; c ( ): subsequent batch fermentation, 50% medium was replaced with the m same amount of fresh medium after 24 h fermentation. fig 3 lactic acid production during demineralization of shrimp shells by lactobacillus acidophilus fncc 116 in different fermentation systems. a ( ): batch fermentation; b ( ): subsequent batch fermentation, 100% medium was replaced m m with fresh medium after 24 h fermentation; c ( ): subsequent batch fermentation, 50% medium was replaced with the m same amount of fresh medium after 24 h fermentation. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0 12 24 36 48 60 l ac ti c ac id ( ) g r/ 1 0 0 m l fermentation time (h) 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 0 12 24 36 48 60 p h fermentation time (h) fig 4 ph of fermented broth during demineralization of shrimp shells by lactobacillus acidophilus fncc 116 in different fermentation systems. a ( ): batch fermentation; b ( ): subsequent batch fermentation, 100% medium was replaced m m with fresh medium after 24 h fermentation; c ( ): subsequent batch fermentation, 50% medium was replaced with the m same amount of fresh medium after 24 h fermentation. r£ r£ r£ volume 7, 2013 microbiol indones 79 0 1 2 3 4 5 6 7 0 12 24 36 48 60 g lu co se ( g r/ 1 0 0 m l ) fermentation time (hr) 80 junianto et al. microbiol indones fig 5 ash content in shrimp shells during demineralization of shrimp shells by lactobacillus acidophilus fncc 116 in different fermentation systems. a ( ): batch fermentation; b ( ): subsequent batch fermentation, 100% medium was m m replaced with fresh medium after 24 h fermentation; c ( ): subsequent batch fermentation, 50% medium was replaced m with the same amount of fresh medium after 24 h fermentation. fig 6 growth of bacillus licheniformis f.11.1 during deproteination of shrimp shells. a ( ): batch fermentation 1, inoculum p was added once at the beginning of the fermentaion; b ( ): batch fermentation 2, inoculum was added twice, at the p beginning and after 24 h fermentation; c ( ): subsequent batch fermentation, 100% medium was replaced with fresh p medium after 24 h fermentation. fig 7 protease production during deproteination of shrimp shells using bacillus licheniformis f11.1. a ( ): batch fermentation p 1, inoculum was added once at the beginning of the fermentation; b ( ): batch fermentation 2, inoculum was added twice, p at the beginning and after 24 h fermentation; c ( ): subsequent batch fermentation, 100% medium was replaced with p fresh medium after 24 h fermentation. 0 5 10 15 20 25 0 12 24 36 48 60 a sh c o n te n t (% ) fermentation time (h) 5 10 6 10 7 10 8 10 9 10 10 10 0 12 24 36 48 60 72 84 96 -1 b ac te ri al c el l am o u n t (c f u m l ) fermentation time (hr) 0 4 8 12 16 20 0 12 24 36 48 60 72 84 96 -1 p ro te as e ac ti v it y ( u m l ) fermentation time (h) r£ r £ r £ volume 7, 2013 microbiol indones 81 batch a followed by stationary phase until 36 h m incubation. when 100% medium was replaced with the fresh one in b , the cell undergone exponential phase m again from 24 h to 36 h incubation. however, the maximum cell amount between a and b was not m m much different. when the medium was only replaced 50% (c ), the cells did not grow as much as in b . in the m m exponential phase, the cells consumed most of the nutrients in the medium and in this experiment, the change of glucose concentration in the medium was observed. in the first 24 h, glucose concentration declined very rapidly in all fermentation system from 6% (w/v) and down to < 1% (w/v). after replacement of the medium, the glucose concentration returned to the original concentration in b since the medium was m 100% replaced with the fresh one, whereas in c the m medium was only replaced 50%, the glucose concentration became 3.16% (w/v) (fig 2). after 48 h of incubation until the end of observation (60 h) the glucose concentration barely changed (< 0.2%). glucose consumed by the bacteria (fig 2) was associated with lactic acid production (fig 3). lactic acid concentration increased rapidly up to 2.19-2.3% in the first 24 h in all fermentation systems which were related with the decrease of glucose concentration (fig 2) and ph (fig 4) in the medium. the data show that l. acidhophilus fncc 116 converted some of glucose to lactic acid. maximum concentration of lactic acid reached in a was 2.19% (w/v) and it did not change m until the end of observation (60 h). among the three fermentation systems, the highest lactic acid was in the system with 100% medium replacement (b ). the m highest lactic acid concentration was 4.19% (w/v) 12 h acid fermentation depends on some factors such as inoculum density, glucose concentration, and initial ph, during fermentation as well as fermentation time (rao et al. 2000). l. acidophilus is a homofermentative lactic acid bacterium which produces lactic acid as a major product from glucose (jafarei and ebrahimi 2011). based on experimental data, the bacterial exponential growth was associated with the glucose consumed and the lactic acid produced. in this demineralization experiment, initial ph and glucose concentration in the medium used in all batch process was ph 7 and 6%, respectively, with cell density of 8 -1 bacterial starter 8.5 × 10 cfu ml . the effect of glucose concentration in the medium on lactic acid production was observed by applying three different fermentation schemes (fermentation a , b , and c ). m m m the aim of applying three different fermentations of lactic acid bacteria was to find the optimum lactic acid production. since the lactic acid produced would react with calcium carbonate in chitin fraction of the shrimp shells to form calcium lactate which precipitate and can be removed by washing (rao and stevens 2005). therefore, the more lactic acid produced, as expected the more calcium carbonate in the shrimp shells could be removed. our proximate analysis showed that calcium content of our sample was 39.21% of the ash content. the calcium removed was observed by analyzing the ash content of the shells, thus the more calcium removed the less ash content of the shells. experimental data showed that exponential growth phase of l. acidophilus fncc116 lasted for 6 h for all batch system (a , b , and c ) (fig 1). after the first 6 h m m m of fermentation, the bacteria grew exponentialy in fig 8 protein content of shrimp shells during deproteination using bacillus licheniformis f11.1. a ( ): batch fermentation 1, p inoculumn was added once at the beginning of the fermentaion; b ( ): batch fermentation 2, inoculumn was added twice, p at the beginning and after 24 h fermentation; c ( ): subsequent batch fermentation, 100% medium was replaced with p fresh medium after 24 h fermentation. r 0 5 10 15 20 25 30 0 24 48 72 96 p ro te in c o n te n t (% ) fermentation time (h) £ based on the experimental data, demineralization process of shrimp shells suggested was the fermentation b with 36 h incubation, which was m subsequent batch with 100% medium replacement after 24 h. the ash content in b after 36 h incubation m was 0.08% ± 0.00% (dry base) or 99.61% ± 0.01% ash removed from the shrimp shells. the results of this demineralization process was better than previous study done by healy et al (2003), rao and stevens (2005), daum et al. (2007), and prameela et al. (2012). healy et al (2003) used a mixture of bacteria (l. plantarum, l. salivarius, streptococcus faecium, and pediococcus acidilacti) to demineralize and deproteinize prawn (nephros norvegicus) at 30 °c for 7 d, and the final ash removed from the shells was 87.9%. rao and stevens (2005), used l. plantarum 541 for demineralization and deproteination, 63% ash 66% protein were removed. daum et al (2007) used shells of penaeus sp and lactobacillus sp for demineralization process at 37 °c for 3 d, with 8% glucose added, 10% 8 -1 inoculum (10 cells ml ), resulted in 89.6% ash removed. prameela et al (2012) used l. plantarum nrrl b-14768 with 15% glucose added, the fermentation was done at 37 °c for 72 h resulting 76% ash, and 89% protein removed. deproteination. deproteination of demineralized shrimp shells was done by cultivating protease producing bacterium b. licheniformis f11.1. three different fermentation systems were applied to optimize protease production for hydrolyzing the protein which associated with the shrimps shells. in the system a , after 12 h incubation, the bacterial p cells amount declined until the end of observation (96 h). in system b the cells amount decreased from 12 h to p 24 h, then after addition of medium the cell amount increased again until 36 h incubation, then decreased again until the end of observation (96 h). in system c , p after 12 h incubation, the cell amount decreased until 24 h of incubation. after addition of inculum after 24 h, the cells amount increased for 12 h (36 h incubation) then started decreasing until the end of incubation (96 h). among the three fermentation systems, the least cell amount declining rate was in c . p the highest protease production in a (16.06 ±0.03 p -1 u ml ) was reached after 48 h incubation, in b was p -1 after 72 h (16.51±0.01 u ml ) and in c was after 72 h p -1 (11.63±0.04 u ml ). addition of inoculum after 24 h (b ) only slightly increased the protease production but p delayed the maximum production from 48 h in a to 72 p h in b , whilst replacement of medium (c ) even p p delayed and decreased the protease production. after the medium replacement and the concentration remained at that value (4.2% w/v) until the end of observation (60 h) (fig 3). in fermentation system c , m where only 50% of the medium was replaced, the maximum lactic acid concentration was only 3.44% (w/v) (fig 3). these data indicate that glucose added into the medium was mostly converted to lactic acid. prameela et al (2010) study on chitin extraction using l. plantarum (atcc 8014 and nrrl b-14768), l. pentosus (atcc 8041), and l. fermentum (nrrl b 1840) showed that the more glucose added the more lactic acid produced. the lactic acid produced affected the ph of the medium as (fig 4). the initial ph of all system was 6.97-6.98. during the first 24 h, the ph of the medium decreased drastically to 4.53, 4.65, and 4.71 in a , b , and c , respectively. after 30 h, the m m m lowest ph was in b (ph 3.88). in a the ph was 4.61. m m the ph value stayed the same during 60 h observation. however, in c after 30 h the ph of the medium was m 4.35 and slightly decreased to 4.15 after 60 h observation ph value reached in fermentation b ph m 3.88±0.00 during 60 h incubation with total glucose added 12%, this ph was lower than that in prameela study (prameela et al. 2010), were ph 4.173±0.01 was reached using l. plantarum nrrl b-14768 after 72 h fermentation with 15% initial glucose added. jung et al (2005) reported that the ph value (ph 3.52±0.15) of fermentation using l. paracasei kctc-3074 for 3 d (72 h) was slightly lower than our study (ph 3.88±0.00). the glucose added in jung’s study was 10% (jung et al. 2005). bacterial growth, conversion of glucose to lactic acid and change of ph medium affected ash content of the shrimp shells (fig 5). for the first 24 h of fermentation, ash content declined rapidly from initial concentration of 19.26-19.61% (w/v) down to 2.93.18% (w/v) with reduction rate of the ash concentration in the range of 0.034-0.035% (w/v) per hour. the reduction rate of the ash content in the shrimp shells was slowing down afterward until the end of observation (60 h) in the range of 0.022-0.027% (w/v) per hour. the highest ash reduction of the shrimp shells was in b (99.69%), followed by c (97.69%) and the m m lowest was in a (97.17%). therefore, the lowest ash m content of the shrimp shells after 60 h incubation was in fermentation b (0.06 ± 0.01% dry base) followed by m c (0.45 ± 0.02% dry base) and a (0.55 ± 0.06% dry m m base). based on duncan multiple range test, in demineralization process b , there was no significant m difference in reduction rate of ash content in the shrimp shells after 24, 36, 42, 48, 54, and 60 h incubation. 82 junianto et al. microbiol indones appl environ microbiol. 76(24):8211-8221. hsu yl, wu wt. 2002. a novel approach for scaling up a fermentation system. biochem eng j. 11(2-3):123-130. doi:10.1016/s1369-703x(02)00016-5. jafarei p, ebrahimi mt. 2011. lactobacillus acidophilus cell structure and application. african j microbiol res. 5:4033-4042. jung wj, jo gh, kuk jh, kim ky. park rd. 2005. extraction of chitin from red crab shell waste by cofermentation with lactobacillus paracasei sbsp tolerans kctc-3074 and serratia marcescens fs-3. appl microbiol biotechnol.71(2):234-237. doi 10.1007/s00253-005-0126-3. junianto, mangunwidjaja d, suprihatin, mulyorini, wahyuntari b. 2009. pengaruh tingkat aerasi dan kecepatan agitasi terhadap tingkat hidrolisis protein kulit udang pada tahapan ekstraksi kitin secara biologis [effect of aeration and agitation rate on protein hydrolysis of shrimp shells in biological chitin extraction]. j bionatura. 11(2):107-117. lowry oh, rosenbrough nj, farr al, randall rj. 1951. protein measured with folin phenol reagent. j biochem. 193:265-275. mizani a, aminlari bm. 2007. a new process deproteination of chitin from shrimp head waste. proceedings of european congress of chemical engineering; 2007 september 16-20. copenhagen (dk). p 1-8. mahmoud ns, ghay ae, arab f. 2007. unconventional approach for demineralization of deproteinized crustacean shells for chitin production. am j biochem biotech. 3(1):1-9. doi:10.3844/ajbbsp.2007.1.9. prameela k, mohan cm, hemalatha kpj. 2010. extraction of pharmaceutically important chitin and carotenoid from shrimp biowaste by microbial fermentation method. j pharmacy res. 3:2393-2395. rao ms, munoz j, stevens wf. 2000. critical factors in chitin production by fermentation of shrimp biowaste. appl microbiol biotechnol. 54(6):808-813. doi:10.100 7/s002530000449. rao ms, stevens wf. 2005. chitin production by lactobacillus fermentation of shrimp biowaste in a drum bioreactor and its chemical conversion to chitosan. j chem technol biotechnol. 80(9):10801087. doi:10.1002/jctb.1286. wahyuntari b, junianto, setyahadi s. 2011. process design of microbiological chitin extraction. microbiol indones. 6(1):36-45. doi:10.5454/mi.5.1.7. waldeck j, daum g, bisping b, meinhardt f. 2006. isolation and molecular characterization of chitinase-deficient bacillus licheniformis strains capable of deproteiniation of shrimp shell waste to obtain highly viscous chitin. appl environ microbiol. 72(12):7879-85. doi:10.1128/ aem.00938-06. protease produced by the bacterium would hydrolyze protein in the shrimp shells in the fermentation mixture. during the first 12 h incubation, the protein content in the shrimp shells was reduced rapidly from 26% to 13% (±50% protein removed). at the end of observation in a ; b , and c 94.42%, 94.51 p p p %, and 94.85 % was removed respectively (fig 8). some studies on microbiological demineralization of shrimp shells were also reported. daum et al (2003) showed that fermentation using lactobacillus sp for 48 h could reduce 95% protein of shrimp (penaeus sp) shells. prameela et al (2010) reported that demineralization of penaeus monodon shells for 72 h using l. plantaraum nrrl b-14768 removed 76% ash, while rao and steven (2005) reported that one step deproteination and demineralization of shrimp shells using l. plantarum 541 could removed 83% protein and 88% ash. therefore, considering the operational cost, the fermentation design b (subsequent batch fermentation)m in which 100% medium was replaced with fresh medium after 24 h fermentation, followed by deproteination system a (batch fermentation) was p suggested. a system was the easiest and cheapest p compared to the other ones. system a needed neither p addition of inoculum nor medium. references th aoac. 1984. official methods of analysis. 15 edition. association of official analytical chemistry, inc. virginia: arlington. beany p, lizardi-mendoza j, healy m. 2005. comparison of chitins produced by chemical and bioprocessing methods. j chem technol biotechnol. 80(2):145-150. doi:10.1002/jctb.1164. daum g, stöber h, veltrup k, meinhardt f, bisping b. 2007. biotechnological process for chitin recovery out of shrimp waste. j biotechnol. 131(1):s188. doi:10.1016/j. jbiotec.2007.07.334. dutta pk, dutta j, tripathi vs. 2004. chitin and chitosan: properties and applications. j sci industrial res. 63:2031. healy m, geen a, and healy a. 2003. bioprocessing of marine crustacean shell waste. acta biotechnol. 23 (23):151260. doi: 10.1002/abio.200390023. hoffman k, daum g, köster m, kulicke wm, meyerrammes h, bisping b, meinhardt f. 2010. genetic improvement of bacillus licheniformis strains for efficient deproteinization of shrimp shells and production of high-molecular-mass chitin and chitosan. volume 7, 2013 microbiol indones 83 1: 75 2: 76 3: 77 4: 78 5: 79 6: 80 7: 81 8: 82 9: 83 vol.1 , no. , 202 , p -6 2 december 2 15 23 doi: 10.5454/mi.1 . .6 2 15-23 multidrug resistance and extensively drug-resistance in staphylococcus aureus, staphylococcus epidermidis, staphylococcus haemolyticusand cliff clarence haliman , dimas seto prasetyo , conny r tjampakasari , 1 2 2 and t mirawati sudiro*jahjani 2 1 program of clinical microbiology residency, faculty of medicine, universitas indonesia, jakarta 10320, indonesia; 2 department of clinical microbiology, faculty of medicine, universitas indonesia dr. cipto mangunkusumo hospital, jakarta 10320, indonesia antimicrobial resistance in bacteria has become a leading global public health issue. hasstaphylococcus sp. an efficient mechanism to deal with antimicrobial agents that make them hard to treat in hospital-acquired and community-acquired infections. this study was conducted due to limited data about multidrug resistance and extensively drug resistance in . in indonesia. this study was a descriptive retrospective studystaphylococcus sp using a cross-sectional design to get the prevalence and antimicrobial susceptibility of ,s. haemolyticus s. aureus, and the data w secondary data extracted from whonet 2022 software. this study's data weres. epidermidis. ere from bacteria from samples sent to ukk lmk fkui, jakarta from 2017 to 2021 for routine diagnostic. in this study, we found that the prevalence of methicillin-resistant was 24.9%, methicillin-resistants. aureus s. epidermidis s. haemolyticus s. aureuswas 65,5%, and methicillin-resistant was 86.8%. the prevalence of mdr is less than and respectively. mdr consistently above 85%s. epidermidis s. haemolyticus, s. haemolyticus was each year, while was above 50% and was below 50%. xdr staphylococcus was onlys. epidermidis s. aureus found in and , i.e. three and seven xdr isolates ofs. aureus s. haemolyticus s. aureus and s. haemolyticus respectively during 2017-2021. although we could not find any pan-resistant isolates from all samples, we found methicillin-resistant and isolates that were also resistant to vancomycin and linezolid.s. aureus s. haemolyticus s. haemolyticus s. epidermidis coagulase-negative staphylococcusdan were an important species that can't be neglected due to the high percentage of mdr and the discoveries of xdr in so that they have thes. haemolyticus potential to disseminate resistance plasmids to the more virulent bacteria. therefore we need to control the use of antimicrobial agent to prevent this resistance. key words: indonesia, jakarta, mdr, ,methicillin resistant staphylococcus aureus, staphylococcus epidermidis staphylococcus haemolyticus,, xdr resistensi antimikroba adalah salah satu masalah kesehatan utama di dunia. . memilikistaphylococcus sp mekanisme yang efisien dalam mengatasi antimikroba sehingga menyebabkan sulitnya pengobatan infeksi baik di maupun penelitian ini dilakukan karena keterbatasanhospital acquired infection community acquired infection. data mengenai prevalensi multidrug resistance (mdr) dan extensively drug resistance (xdr) staphylococcus sp., di indonesia. penelitian ini menggunakan data sekunder yang diambil dari perangkat lunak whonet 2022 dan merupakan penelitian deskriptif retrospektif dengan pendekatan potong lintang untuk mengetahui prevalensi dan pola kepekaan antimikroba dari danstaphylococcus haemolyticus, staphylococcus aureus staphylococcus epidermidis. sampel yang dianalisis merupakan sampel yang dikirim ke ukk lmk fkui, jakarta pada tahun 2017 sampai dengan 2021 untuk diagnosis rutin. dari hasil penelitian ini ditemukan prevalensi methicillin resistant s.aureus methicillin resistant s.epidermidis methicillin resistantadalah 24,9%, adalah 65,5% dan s.haemolyticus s.aureus s.epidermidisadalah 86,8%. prevalensi yang merupakan mdr lebih sedikit daripada dan yaitu berturut-turut konsisten diatas 85% tiap tahun, konsisten di atas 50% dan konsisten dis.haemolyticus, bawah 50%. staphylococcus yang merupakan xdr, hanya ditemukan pada dan , yaitus.aureus s.haemolyticus berturut turut pada sebanyak tiga isolat dan pada sebanyak tujuh isolat selama tahuns.aureus s.haemolyticus 2017-2021. walaupun dari keseluruhan sampel, tidak ditemukan pan-resistensi, ditemukan dans.aureus s.haemolyticus s.haemolyticus s.resisten metisilin yang juga resisten terhadap vankomisin dan linezolid. dan epidermidis coagulase negative staphylococcusmerupakan yang perlu diperhatikan, karena tingginya persentase mdr dan ditenukannya xdr pada , sehingga berpotensi dapat mendiseminasikans.haemolyticus plasmid resistensi kepada organisme yang lebih virulen sehingga diperlukan adanya pengendalian penggunaan antimikroba untuk mencegah penyebaran resistensi tersebut. kata kunci: indonesia, jakarta, mdr, ,methicillin resistant staphylococcus aureus, staphylococcus epidermidis staphylococcus haemolyticus,, xdr microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 * c o r r e s p o n d i n g a u t h o r : p h o n e ;: + 6 2 e m a i l :mrwtsoediro@gmail.com leading global public health issue due to its ineffective treatment of hospital acquired or community-acquired infections. (magiorakos 2012; patel 2011)et al. et al. antimicrobial resistance in bacteria has become a staphylococcus sp. has an efficient mechanism to deal with antimicrobial agents that make them hard to treat, especially in life-threatening diseases. (almanaa et al. 2 0 2 0 ; k a u r a n d c h a t e , 2 0 1 5 ) a m o n g a l l staphylococcus species, staphylococcus aureus, staphylococcus epidermidis, staphylococcusand haemolyticus are the most common species in nosocomial infection. (de giusti 1999; dzenet al. et al. 2005; graham 2000; heilmann 2019; kimet al. et al. and jang, 2017; takeuchi 2005; suhartonoet al. et al. 2019) is the major pathogenstaphylococcus aureus species for humans. (riedel 2019, p.205)et al. staphylococcus haemolyticus inhabits human skin as commensal species, at first not considered a pathogen, now it is the second most common species after staphylococcus epidermidis among the coagulasenegative staphylococcus group that can be isolated in nosocomial infection.(de giusti 1999; dzenet al. et al. 2005; graham 2000; heilmann 2019; kimet al. et al. and jang, 2017; takeuchi 2005)et al. staphylococcus haemolyticus has genomic flexibility that facilitates the survival mechanism to the antimicrobial agents and can be disseminated to other species in the staphylococcus group. (kim 2012; takeuchi 2005)et al. et al. . in the previous study in aceh, staphylococcus haemolyticus infection prevalence in hospital is 32 2%,. and among the isolate, 96 6% is. methicillin-resistant staphylococcus haemolyticus. et al.(suhartono 2019) while in a global study of multi drugs resistance to staphylococcus haemolyticus that involved eight countries, the prevalence is 77 7%, where multi drugs. resistance is defined as resistance to at least three antimicrobial agents. (cavanagh 2014; czekajet al. et al. 2015). due to limited data about multidrug resistance and extensively drug resistance in .staphylococcus sp especially in indonesia. this study aims to determine the prevalence of multidrug resistance and extensively drug resistance in , ands. haemolyticus s. epidermidis, s. aureus from secondary data obtained from clinical isolates during routine diagnostic in ukk lmk fkui, jakarta. materials and methods s t a p h y l o c o c c u s s p . i d e n t i f i c a t i o n a n d antimicrobial susceptibility were obtained from secondary data extracted from whonet 2022 software. this study was a descriptive retrospective study using a cross-sectional design to get the prevalence and antimicrobial susceptibility of s. haemolyticus s. aureus, s. epidermidis., and this study's data were from bacteria from samples sent to ukk lmk fkui, jakarta from 2017 to 2021 for routine diagnostic. this study has been approved by the ethics committee of the faculty of medicine, universitas indonesia – cipto mangunkusumo h o s p i t a l . ( n o : k e t1 2 7 2 / u n 2 . f 1 / e t i k /ppm.00.02/ 2022). clinical samples were inoculated to blood agar plates, and the plates were incubated for 24 hours in 37 c before being identified by gram o staining. blood samples were first inoculated into bactec blood culture system and positive samples ® were spread onto blood agar plates as above. identification and drug susceptibility tests were generated using vitek 2 gp and vitek 2 ast-gp ® ® 67 (biomerieux, france) and converted using baclink software (whonet, boston). then the data of species and drug sensitivity were extracted and presented in frequency percentage using whonet 2022 software (whonet, boston). gnu pspp 1.6.2 (free software foundation, boston) and microsoft excel software was used to calculate the percentage that was not calculated in whonet and make the graph. t h e p h e n o t y p e o f m e t h i c i l l i n r e s i s t a n t staphylococcus was detected by susceptibility to cefoxitin. (magiorakos 2012; riedelet al. et al. 2019,p.215) multidrug resistance (mdr) was determined by antimicrobial susceptibility test results when the isolate has non-susceptibility to ≥1 antimicrobial agent in ≥3 antimicrobial categories phenotype or has methicillin resistant phenotype (magiorakos 2012). while extensively druget al. resistance (xdr) was determined by antimicrobial susceptibility test results when the isolate has nonsusceptibility to ≥1 antimicrobial agent in all but ≤ 2 antimicrobial categories phenotype (magiorakos et al. 2012). a bacteria is determined as pandrug resistant (pdr) when the antimicrobial susceptibility test showed non-susceptibility to all antimicrobial categories (magiorakos 2012).et al. results a total of 1099 samples were used to analyse the prevalence and sample characteristics of ,s. aureus s. haemolyticus, s. epidermidis,and which can be seen in table 1 and table 2. from all the data mentioned before, a total of 168 other than ands. aureus, s. epidermidis, s. haemolyticus were excluded from antimicrobial susceptibility 16 haliman et al. microbiol indones volume 1 , 2026 2 microbiol indones 17 table characterization of sp. according to the specimen type of origin in 2017-2 staphylococcus isolates 2021 table 1 sp. in 2017-2021number of isolatesstaphylococcus organism 2017 n (%) 2018 n (%) 2019 n (%) 2020 n (%) 2021 n (%) total s. aureus 151(53.0) 74(32.5%) 69(27.0%) 42(29.2%) 53(28.5%) 389 s. epidermidis 51(17.9%) 55(24.1%) 70(27.3%) 37(25.7%) 42(22.6%) 255 s. haemolyticus 45(15.8%) 72(31.6%) 80(31.3%) 26(18.1%) 64(34.4%) 287 other staphylococcus 38(13.3%)a 27(11.8%)a 37(14.5%)b 39(27.1%)c 27(14.5%)d 168 a s. capitis, s. cohnii, s. hominis, s. lentus, s. ludgunensis, s. pseudintermedius, s. saprophyticus, s. sciuri, s. warneri, s. xylosus. b s. capitis, s. cohnii, s. hominis, s. lentus, s. ludgunensis, s. saprophyticus, s. sciuri, s. warneri c s. capitis, s. cohnii, s. hominis, s. lentus, s. pseudintermedius, s. saprophyticus, s. sciuri, s. simulans, s. warneri, s. xylosus. d s. capitis, s. cohnii, s. hominis, s. lentus, s. ludgunensis, s. pseudintermedius, s. saprophyticus, s. sciuri, s. warneri, s. xylosus specimen species s. aureus (n (%)) s. epidermidis (n (%)) s. haemolyticus (n (%)) other staphylococcus* (n (%)) abdominal fluid 1(0.3%) 1(0.4%) 2(0.7%) 2(1.2%) abscess and pus 88(22.6%) 27(10.6% 11(3.8%) 11(6.5%) blood 34(8.7%) 71(27.8%) 20(7.0%) 55(32.7%) brain and csf 4(1.6%) 2(1.2%) bronchoalveolar lavage and bronchial washing 5(1.3%) 7(2.7%) 2(0.7%) 1(0.6%) cervix 1(0.4%) 3(1.0%) cornea and eye 3(0.8%) 3(1.2%) 4(1.4%) 5(3.0%) external urethra 1(0.3%) 3(1.2%) 8(2.8%) nose 47(12.1%) 12(4.7%) 4(1.4%) 9(5.4%) semen 2(0.8%) 19(6.6%) 7(4.2%) skin swab 7(1.8%) 23(9.0%) 4(1.4%) 5(3.0%) sputum 57(14.7%) 43(16.9%) 36(12.5%) 3(1.8%) throat and trachea 67(17.2%) 3(1.2%) 2(0.7%) 8(4.8%) tissue 30(7.7%) 17(6.7%) 9(3.1%) 8(4.8%) urine 16(4.1%) 19(7.5%) 145(50.5%) 38(22.6%) vagina 3(0.8%) 5(2.0%) 8(2.8%) wound and ulcer 10(2.6%) 2(0.8%) 4(1.4%) 1(0.6%) others** 20(5.1%) 12(4.7%) 6(2.1%) 13(7.7%) total 389 255 287 168 * other :staphylococcus s. capitis, s. cohnii, s. hominis, s. lentus, s. ludgunensis, s. pseudintermedius, s. saprophyticus, s. sciuri, s. simulans, s. warneri, s. xylosus. ** others: specimen less than 1% (aspirate, bile, bone, central venous catheter, ear, joint fluid, kidney, leg swab, liver, male genital, mediastinum, mouth, no data, placenta, pleural fluid, rectal, sinus, prosthesis) 18 haliman et al. microbiol indones analysis to specify the antimicrobial susceptibility test only in three species. therefore, 926 samples were used to analyse the antimicrobial susceptibility profile shown in table 3, table 4, and table 5. in total, methicillin-resistant s. aureus (mrsa) prevalence was 24 9%, methicillin-resistant s.. epidermidis methicillin-(mrse) was 65 5% and. resistant s. haemolyticus(mrsh) was 86 8%.. from the antimicrobial susceptibility test, we found the prevalence of xdr in ands. aureus, s. haemolyticus were 0 77% and 2 43% respectively. while we could not. . found xdr in . mdr and xdrs. epidermidis phenotypes were found in ands. aureus, s. epidermidis, s. haemolyticus were shown in table 6 and fig 1 shows the percentage of ethicillin-resistant phenotype asm . shown in fig 2, ancomycin-resistant were found inv s. aureus, s. epidermidis, s. haemolyticusand in methicillin-resistant. in this study, we found that among the methicillinresistant phenotype, four isolates on weres. aureus vancomycin and linezolid resistant, and ten s. haemolyticus isolates were vancomycin and linezolid resistant. while in , we did not find ans. epidermidis isolate resistant to vancomycin and linezolid in the methicillin-resistant phenotype. discussion in this study, we found that s. aureus, s. haemolyticus, s. epidermidisand , respectively, were the most common species that can be isolated from clinical specimens during routine diagnostic tests. s. aureus s. epidermidis s. haemolyticus, , and are important hospital-acquired infection causative pathogens, especially in patients using a venous catheter and medical devices in the intensive care unit.(cerca 2007; daniel 2014; horanet al. et al. et al. 2008; klingenberg 2007)et al. . in this study, was commonly found ins. aureus abscesses and pus, oropharynx and tracheal swab, and sputum. was commonly found in blood,s. epidermidis sputum, abscesses, and pus. at the same time, s. haemolyticus was commonly found in urine, sputum, and blood. and coagulase-negatives. aureus staphylococcus are bacteria that colonize human skin, nails, and nares. hence, they can invade to form pus in the tissue if there is a disruption of human barriers, such as damage to the skin layer, hair follicle trauma, and using medical devices.(do carmo ferreira 2011;et al. lowy, 1998; schuenck 2008) whileet al. s. haemolyticus is associated with infection in the urinary tract. (gunn and davis, 1988; hovelius 1984;et al. john and o'dell, 1978; lozano 2015; ruppet al. et al. s.1992). from a study conducted in aceh, haemolyticus was predominantly found in the intensive care unit. (suhartono 2019) in anotheret al. study conducted in nepal, wass. aureus predominantly found from pus in the intensive care unit, while was predominantly found ons. epidermidis catheter tips in the intensive care unit. (shrestha et al.2018) in this study, we could not determine whether the staphylococcus that we isolated were from the intensive care/ hospital ward or outpatient due to lack of data. from the result of antimicrobials. aureus susceptibility that has been shown before, we found that the prevalence of methicillin-resistant s. aureus (mrsa) was below 40% each year. the highest prevalence was discovered in 2018 and decreased in 2019 and 2021. this prevalence is slightly lower compared to studies conducted in afghanistan (naimi et al. et al.2021) and pakistan (ullah 2016) but similar to the study conducted in china (wang 2021).et al. from the antimicrobial susceptibility profile, we found in this study that 90% of is susceptible tos. aureus nitrofurantoin, rifampicin, vancomycin, linezolid, and q u i n u p r i s t i n / d a l f o p r i s t i n , t i g e c y c l i n e , a n d trimethoprim-sulfamethoxazole. while below 90% of s. aureus is susceptible to ciprofloxacin, clindamycin, e r y t h r o m y c i n , g e n t a m y c i n , l e v o f l o x a c i n , moxifloxacin, tetracycline, cefoxitin, oxacillin, and penicillin g. penicillin g was the least susceptible antimicrobial agent to compared to others. aureus. studies mentioned before, the resistance to penicillin g of is similar to studies conducted ins. aureus afghanistan, pakistan, and china, but slightly different from other antimicrobial agents such as in afghanistan. they found that is relatively resistant tos. aureus erythromycin and ciprofloxacin, in china, they discovered that is relatively resistant tos. aureus erythromycin and clindamycin and in pakistan, they found that is relatively resistant tos. aureus erythromycin.(naimi 2021; ullah 2016;et al. et al. wang 2021)et al. . from the result of antimicrobials. epidermidis susceptibility that has been shown before, we found that the prevalence of methicillin-resistant s. epidermidis was relatively high. this result is similar to the study conducted in tianjin, china (xu 2020),et al. which found a high prevalence of methicillin-resistant s. epidermidis. the result we found was slightly lower than their study result, but, in our discovery, s. volume 1 , 2026 2 microbiol indones 19 table percentage of antimicrobial sensitivity3 s. aureus * breakpoints according to eucast version 5.0 table percentage of antimicrobial sensitivity4 s. epidermidis antibiotic 2017 (n=51) 2018 (n=55) 2019 (n=70) 2020 (n=37) 2021 (n=42) total (n=255) cefoxitin 23.5% 41.8% 52.9% 16.2% 23.8% 34.5% oxacillin 23.5% 41.8% 52.9% 16.2% 23.8% 34.5% penicillin g 2.0% 7.3% 37.1% 5.4% 2.4% 13.3% tetracycline 52.9% 76.4% 91.4% 78.4% 90.5% 78.4% erythromycin 15.7% 40.0% 54.3% 48.6% 26.2% 38.0% clindamycin 11.8% 43.6% 50% 43.2% 28.6% 36.5% ciprofloxacin 33.3% 69.1% 61.4% 45.9% 28.6% 49.8% moxifloxacin 35.3% 69.1% 61.4% 45.9% 28.6% 50.2% levofloxacin 33.3% 69.1% 61.4% 48.6% 28.6% 50.2% trimethoprim/sulfamethoxazole 31.4% 65.5% 68.6% 40.5% 38.1% 51.4% gentamycin 39.2% 80.0% 67.1% 70.3% 47.6% 61.6% nitrofurantoin 100% 98.2% 98.6% 100% 100% 99.2% vancomycin 86.3% 83.6% 98.6% 97.3% 97.6% 92.5% rifampicin 86.3% 90.9% 77.1% 83.8% 54.8% 79.2% tigecycline* 98.0% 98.2% 98.6% 100% 97.6% 98.4% quinupristin/dalfopristin* 100% 98.2% 100% 100% 100% 99.6% linezolid 100% 98.2% 98.6% 100% 100% 99.2% antibiotic 2017 (n=151) 2018 (n=74) 2019 (n=69) 2020 (n=42) 2021 (n=53) total (n=384) cefoxitin 90.7% 60.8% 65.2% 61.9% 73.6% 75.1% oxacillin 90.7% 60.8% 65.2% 61.9% 73.6% 75.1% penicillin g 9.9% 5.4% 21.7% 7.1% 20.8% 12.3% tetracycline 54.3% 73.0% 59.4% 81.0% 75.5% 64.5% erythromycin 84.1% 77.0% 72.5% 66.7% 71.7% 77.1% clindamycin 79.5% 79.7% 73.9% 66.7% 69.8% 75.8% ciprofloxacin 94.7% 78.4% 82.6% 78.6% 83% 86.1% moxifloxacin 94.7% 79.7% 81.2% 78.6% 83% 86.1% levofloxacin 94.7% 79.7% 82.6% 78.6% 83% 86.4% trimethoprim/sulfamethoxazole 98.7% 90.5% 91.3% 83.3% 90.6% 93.1% gentamycin 93.4% 82.4% 82.6% 88.1% 90.6% 88.4% nitrofurantoin 100% 97.3% 95.7% 100%% 100% 98.7% vancomycin 87.4% 87.8% 92.8% 85.7% 100% 90% rifampicin 98% 90.5% 87% 95.2% 94.3% 93.8% tigecycline* 100% 97.3% 100% 97.6% 100% 99.2% quinupristin/dalfopristin* 100% 98.6% 95.7% 100% 100% 99.0% linezolid 100% 97.3% 95.7% 100% 100% 98.7% * breakpoints according to eucast version 5.0 epidermidis isolates were widely resistant to more than one antimicrobial agent. we found that the sensitivity of to penicillin g was below 10%,s. epidermidis except in 2019,which is similar to several studies conducted in tianjin, china(xu 2020), shanghai,et al. china(du 2013), and scotland (zalewskaet al. et al. 2021) where they have found more than 90% of s. epidermidis are resistant to penicillin g. in 2019, penicillin g sensitivity was slightly higher than usual, maybe this finding due to fewer methicillin-resistant phenotype that could be isolated from clinical specimens that year. from the result of antimicrobials. haemolyticus susceptibility that has been shown before, we found 20 haliman et al. microbiol indones table percentage of antimicrobial sensitivity5 s. haemolyticus table sp mdr and xdr percentage from 2017-20216 staphylococcus antibiotic 2017 (n=45) 2018 (n=72) 2019 (n=80) 2020 (n=26) 2021 (n=64) total (n=287) cefoxitin 17.8% 12.5% 12.5% 3.8% 15.6% 13,2% oxacillin 17.8% 12.5% 12.5% 3.8% 15.6% 13,2% penicillin g 6.7% 4.2% 1.3% 3.8% 6.3% 4,2% tetracycline 64.4% 61.1% 65% 53.8% 60.9% 62,0% erythromycin 20.0% 19.4% 40% 23.1% 21.9% 26,1% clindamycin 15.6% 13.9% 25% 19.2% 17.2% 18,5% ciprofloxacin 42.2% 33.3% 52.5% 42.3% 26.6% 39,4% moxifloxacin 44.4% 37.5% 50% 42.3% 29.7% 40,8% levofloxacin 44.4% 36.1% 52.5% 38.5% 28.1% 40,4% trimethoprim/sulfamethoxazole 66.7% 61.1% 66.3% 50% 73.4% 65,2% gentamycin 62.2% 63.9% 73.8% 61.5% 51.6% 63,4% nitrofurantoin 97.8% 94.4% 100% 100% 98.4% 97,9% vancomycin 91.1% 83.3% 93.8% 76.9% 96,9% 89,9% rifampicin 75.6% 69.4% 76.3% 69.2% 71.9% 72,8% tigecycline* 88.9% 87.5% 86.3% 80.8% 84.4% 86,1% quinupristin/dalfopristin* 93.3% 83.3% 93.8% 88.5% 98.4% 91,6% linezolid 93.3% 90.3% 97.5% 96.2% 98.4% 95,1% * breakpoints according to eucast version 5.0 s. aureus s. epidermidis s. haemolyticus mdr xdr mdr xdr mdr xdr 2017 19.2%(29/151) 0% 84.3%(43/51) 0% 86.7%(39/45) 4.4%(2/45) 2018 44.6%(33/74) 1.4%(1/74) 60%(33/55) 0% 94.4%(68/72) 4.2%(3/72) 2019 44.9%(31/69) 2.9%(2/69) 50%(35/70) 0% 88.8%(71/80) 1.3%(1/80) 2020 40.5%(17/42) 0% 86.5%(32/37) 0% 100%(26/26) 0% 2021 41.5%(22/53) 0% 78.6%(33/42) 0% 90.6%(58/64) 1.6%(1/64) fig 1 percentage of methicillin resistant sp. in 2017 to 2021staphylococcus . that the prevalence of methicillin-resistant s. haemolyticus was over 80% each year. this result is similar to a study conducted in aceh (suhartono et al. 2019) and brazil (barros 2012), where 96 6 andet al. . 88% of from clinical specimens,s. haemolyticus respectively, were methicillin-resistant. besides, we found that is widely resistant tos. haemolyticus antimicrobial agents such as erythromycin, c l i n d a m y c i n , c i p r o f l o x a c i n , l e v o f l o x a c i n , moxifloxacin, oxacillin, and penicillin g. this result similar with a study conducted in aceh, indonesia (suhartono 2019) and review from several studieset al. that conducted in poland (czekaj 2015), theyet al. found that many are multidrugs. haemolyticus resistant. another important finding in this study is that we found ancomycin resistan in eachv t s. haemolyticus year, and the highest prevalence was in 2020. this result differs from a study conducted in brazil (barros et al. s. haemolyticus2012), where all isolates were susceptible to vancomycin. this finding needs further attention, since the drug of choice in methicillinresistant staphylococcal infections is vancomycin, and the drug of choice in vancomycin-resistant staphylococcal infections is linezolid. (choo and chambers, 2016; loomba 2010) this study foundet al. isolates resistant to vancomycin and linezolid in the methicillin-resistant phenotype. mainly we found them in and , but we could nots. haemolyticus s. aureus find them in .s. epidermidis we found that ands. epidermidis s. haemolyticus were more resistant to antimicrobial agents than s. aureus s.. this was proven by the mdr percentage in aureus s. epidermidis s.being lower than in and haemolyticus, respectively. interestingly, from our study, the prevalence of mdr wass. haemolyticus consistent above 85% each year, while s. epidermidis was s. aureusabove 50% and was below 50% each year. besides, we found that the xdr phenotype only can be found in and althoughs. aureus s. haemolyticus, the prevalence of xdr is higher. wes. haemolyticus discovered that only ten isolates have xdr phenotype where seven isolates of the xdr phenotype were s. haemolyticus, s. aureusand three isolates were . in this study, we could not find the pdr phenotype. in conclusion, ands. haemolyticus s. epidermidis were important coagulase-negative staphylococcus species that can't be neglected, although in earlier times, they were not considered a pathogen species, due to their high prevalence in clinical isolate. besides, s. haemolyticus are resistant to many antimicrobial agents in a high percentage. thus, we should worry about their potential ability to disseminate the plasmid to virulent species. 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an important role in lignocellulose degradation. many research found that streptomyces have cellulolytic and ligninolytic enzymes that are sufficient to degrade lignocellulosic materials. however, a minimum lignocellulosic material condition that can efficiently be degraded by sp. hasstreptomyces not been fully understood. in this research, three pretreatment conditions (physical, alkaline-hydrothermal, and hydrogen-peroxide chemical treatments) of sugarcane bagasse were used as lignocellulosic material to be further degraded by sp. s2. lignocellulose component measurement concluded that raw (physicallystreptomyces treated only) bagasse wasn't efficiently degraded by sp s2. hydrogen-peroxide was effective instreptomyces reducing both syringic and guaiacyl lignin. meanwhile, alkaline-hydrothermal pretreatment was very effective in lowering syringic lignin. this study suggests that hydrogen-peroxide pretreatment can be used in any type of lignocellulosic material, which can be further degraded by sp. s2. on the other hand, alkaline-streptomyces hydrothermal pretreatment is best suited to degrade lignocellulosic material with a high percentage of syringic lignin. key words: alkaline-hydrothermal treatment, hydrogen peroxide treatment, lignocellulose, sp.streptomyces s2, sugarcane bagasse genus berperan penting dalam degradasi lignoselulosa. penelitian terdahulu telahstreptomyces membuktikan bahwa memiliki enzim selulolitik dan lignolitik yang dapat mendegradasi bahanstreptomyces lignoselulosa. namun hingga saat ini belum diketahui kondisi awal bahan lignoselulosa yang sesuai agar dapat didegradasi secara efisien oleh . pada penelitian ini, bagas tebu sebagai sumber lignoselulosastreptomyces mendapatkan tiga macam perlakuan pendahuluan (perlakuan fisik, perlakuan alkali-hidrotermal dan perlakuan kimiawi hidrogen peroksida), dan kemudian diikuti dengan degradasi oleh sp. s2. berdasarkanstreptomyces hasil analisis komponen lignoselulosa memperlihatkan bahwa perlakuan fisik melalui pengecilan ukuran belum dapat mendegradasi secara efisien. perlakuan hidrogen-peroksida secara efektif dapat menurunkan komponen syringil dan guaiacyl lignin, sementara perlakuan alkalin-hidrotermal hanya efektif menurunkan komponen syringic lignin. kajian ini memperlihatkan pengaruh perlakuan hidrogen peroksida dapat digunakan untuk berbagai bahan lignoselulosa, kemudian dilanjutkan dengan degradasi oleh sp. s2; sedangkanstreptomyces perlakuan alkali-hidrotermal dapat mendegradasi bahan lignoselulosa dengan persentase syringic lignin yang tinggi. kata kunci: bagas tebu, degradasi lignoselulosa, perlakuan alkali-hidrotermal, perlakuan hidrogen peroksida, streptomyces sp. s2 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 251-8621974;/fax: +62email: titi-cs@apps.ipb.ac.id country. in 2017, about 150 million tonnes of dried rice grain, 30.000 thousands tonnes of corn and 2000 thousands of sugarcane were produced in indonesia (kementerian pertanian republik indonesia 2018). bioethanol and bioplastics can be produced from sugarcane bagasse, since sugarcane bagasse has high performance index and low environmental effect (sari et al. 2021). however, 17.52%˗23.37% of lignin in sugarcane bagasse makes utilization of its cellulose to simple sugar hampered (maryana 2014; pinet al. et cellulose fiber with hemicellulose fiber and lignin together form lignocellulose materials. in recent years, bioethanol, biogas, bioplastics, and simple sugar were derived from lignocellulose materials (azeredo et al et al..2015; machado and ferraz 2017; fan 2018; sari .2021). indonesia has significant potential toet al use lignocellulose material since it is an agricultural stanislaus aditya agung , rismawati , dede heri yuli yanto biotechnology study program, graduate school, ipb university, j l kamper, dramaga, bogor 16680, indonesia biology department, faculty of mathematics and natural sciences, ipb university, kampus dramaga bogor 16680; a an ; department of agroindustrial technology, faculty of agricultural technology, ipb university, kampus ipb darmaga bogor 16680. biotechnology research centreipb university, jalan kamper, dramaga, bogor 16680, indonesia; research center for biomaterials, national research and innovation agency (brin). cibinong science center, jalan raya bogor km. 46 cibinong, bogor 16911 indonesia; lenovo typewritten text 2 lenovo typewritten text 1 lenovo typewritten text 3 lenovo typewritten text anja meryandini , titi candra sunarti lenovo typewritten text 2,4 lenovo typewritten text 5* lenovo typewritten text 2 lenovo typewritten text 3 al.2019). high crystallinity and high polymeryzation degree of sugarcane bagasse cellulose also builds sugarcane bagasse strength as lignocellulosic material. thus, the accessible cellulose area becomes small (lee et al. 2014). pretreatment and/or bleaching can be used to reduce lignin content. the bleaching process has been used in the paper industry, reduce lignin content and brighten lignocellulose color. usually the bleaching process use chemicals such as h o , naclo , or clo , which can2 2 2 2 contain chlorine or not. pretreatment focuses are to reducing lignin content and reducing the size of lignocellulose particles. some pretreatments types are physical (extrusion, ultrasonic radiation, milling), physicochemical (supercritical co ammonia fiber2, explosion). chemical pretreatment utilizes alkaline or acid solution in high concentration or dilute, ionic liquid, deep-eutectic-solvent (des) or organosolv. biological pretreatment uses whole organisms (bacteria or fungi) or their enzymes to degrade lignin (baruah et al. et al. et al.2018; septevani 2018; singh 2019). streptomyces sp. is gram-positive bacteria that live in soil and extreme environments such as volcanoes, deep sea and extremely cold places like the arctic. most streptomyces sp. produce secondary metabolites, such as anti-cancer and antibacterial, also can degrade substrate in dead plants (chater 2016; sivalingam et al streptomyces, xanthomonas,.2019). alongside pseudomonas flavobacteria,and are major lignocellulose degraders on the first three days of degradation (ma . 2020). ability toet al streptomyces degrade plant biomass is influenced by the high number of binding sites called cebr. enzymes associated with plant biomass deconstruction such as endoglucanase, glycoside hydrolase, β-glucosidase, cellobiohydrolase, xylanase, mannanase, chitinase and lpmo (lytic polysaccharide monooxygenase) is determined by the amount of the cebr sites (book 2016).et al. ligninolytic enzymes such as lignin-peroxidase (lip), low laccase (lac), dye decoloring enzyme (dyp), and aryl alcohol oxidase (aao) activities can be found in ligninolytic , but without manganese-streptomyces peroxidase (mnp) enzyme activities (riyadi et al. 2020). streptomyces sp. is potentially used to degrade lignocellulose material, since it has cellulolytic and ligninolytic enzyme activities. however, streptomyces sp degradation ability on different lignocellulose conditions hasn't been fully understood. biological degradation was done using sp s2 thatstreptomyces isolated from oil palm plantation in jambi, indonesia. streptomyces sp s2 degradation ability had been tested on three sugarcane conditions (control (milled only), alkaline-hydrothermal, hydrogen-peroxide). alkalinehydrothermal and hydrogen-peroxide pretreatment are widely used to reduce lignin content. both pretreatment can make bagasse easier to degrade by spstreptomyces s2, compared to milled treated-only bagasse as control. each sugarcane bagasse condition was evaluated its crude fiber content, lignocellulose component, crystallinity using x-ray diffraction before and after degraded by sp s2. the bagasse chemicalstreptomyces contents were analyzed using fourier transform infrared, and lastly its 3d structure was scanned using scanning electron microscope (sem). amount of reducing sugar, total sugar and polymerization degree were also measured before and after enzymatic degradation using sp s2.streptomyces materials and methods sugarcane bagasse preparation. total of 40 kg wet weight of sugarcane bagasse from sugarcane juice seller in bogor, was cut, washed until clean, then sundried for 3 days, or accelerated using oven until its water content was lower than 9%. all dried bagasse was then size reduced using wiley mill, filtered through 40 mesh sieve. bagasse that had passed through the sieve was used for further treatment. alkaline-hydrothermal pretreatment. each 100 g prepared bagasse was put into 2l erlenmeyer and treated with 1.5 l 1m naoh (solid to liquid ratio 1:1.5) . erlenmeyer was closed using rubber bands and plastic, before being heated in an autoclave at 121 c for o 30 minutes (lemoes 2018 modified temperatureet al. used). the alkaline-autoclaved pretreated bagasse was washed using aquadest until the neutralized ph solution was same or near as the aquadest used (ph 5.5). washed sugarcane bagasse was filtered using filter paper and then dried using 60˗80 c oven. o hydrogen peroxide pretreatment. every 100 g sieved bagasse (using 40 mesh sieve) was treated with 1% (w/v) trisodium citrate dihydrate, 6% (w/v) h o ,2 2 1% naoh (w/v) and 92% (w/v) aquadest with total solution of 1l in 2l erlenmeyer. erlenmeyer was closed using holed-plastics for gas release that formed during the pretreatment and tightened using rubber bands. peroxide treatment was done in a waterbath at 60 c for 90 minutes (yan . 2019). peroxide o et al pretreated bagasse was further washed using aquadest until the solution ph was near or same as the aquadest used (ph 5.5). bagasse that had been washed then 14 agung et al. microbiol indones volume 1 2026, 2 microbiol indones 15 filtered before dried on a 60-80 c oven. o application using sp. s2streptomyces culture preparation. streptomyces sp s2 strain is indigenous to indonesia and found to have lytic polyssacharide monooxygenase (lpmo) activities that help to open cellulose bonds (utarti 2020).et al. streptomyces sp.s2 was inoculated into isp 4 agar medium, incubated for 5 days in room temperature. degradation using sp. s2.streptomyces prepared culture of sp. s2 then moved using a 10streptomyces mm diameter corkborer into 200 ml isp 4 liquid medium (ph=6.0) then incubated for 14 days in 100 rpm and 27 c shaker. after the incubation, the liquid o medium containing sp. s2 was pouredstreptomyces into 500 ml erlenmeyer containing 15 g of previously sterilized (autoclaved 125 c, 40 minutes) sugarcane o bagasse from previous pretreatment (control, alkalinehydrothermal treated, peroxide treated). degradation of treated bagasse using was taken 4streptomyces days. on the 4 days of bagasse degradation, bagasse th was filtered through filter paper before being put into the freezer, to stop enzymatic degradation. each pretreatment (control, alkaline-hydrothermal, h y d r o g e n p e r o x i d e ) w a s t r e a t e d d u p l o f o r streptomyces sp. s2 pretreatment. enzymatic degradation using spstreptomyces s2 crude enzyme. one 10 mm corkborer of prepared streptomyces sp. s2 was moved into 30 ml isp-4 liquid medium, for enzyme production. optimum amount of streptomyces sp. s2 crude enzyme was added to control, alkaline pretreated and peroxide pretreated bagasse in different ratios (1:2; 1:3; 1:4). enzymatic degradation was done at 120 rpm and 27 c shaker, o incubated for four days. measurement of total sugar, reducing sugar and observation of fiber was done after four days of incubation. measurement of total sugar after enzyme incubation was done using miller (1959) dns method, meanwhile, total sugar was done using dubois (1956) phenolic sulfate method.et al. observation of bagasse fiber was done by observing fiber under the microscope. degree of polymerization was based on total sugar divided by reducing sugar. fiber characterization .crude fiber measurement crude fiber was measured using aoac method (1998). crude fiber content calculated based on the formula: neutral detergent fiber. both neutral detergent fiber and acid detergent fiber were measured using crude fiber: lignocellulose weight sample weight x 100% (1) aoac (1998) method. each 1 gram sample (a) and 100 ml neutral detergent solution was added into a 600 ml beaker. the sample and solution were heated until they boiled. after 60 minutes of boiling time, the sample was extracted and filtered through a fritted disc (b). residues were washed using acetone and water, then further dried using an oven at 105 c until stable o weight (c), then calculated using the formula: acid detergent fiber and hemicellulose. each gram of sample (a2) and 100 ml acid detergent solution was added into a 600 ml beaker, then heated until boiled. the sample was then extracted after 60 minutes of boiling time, filtered through a fritted disc (b2). residues were washed using water and acetone, then further dried using 105 c oven until stable weight (c2). o each acid detergent fiber and hemicellulose calculated using the formula: cellulose content measurement. sample from acid detergent fiber measurement (c2) was soaked in 72% h so for 3 hours. residues that had been formed2 4 then was washed using hot water and acetone. remaining sample was dried using a 105 c oven until o the stable weight was achieved (d). cellulose content was calculated using the formula: lignin content. sample from cellulose content measurement (d) was dried in a furnace at 600 c, until o the stable weight was achieved (e). lignin content was calculated using the formula: crystallinity index assay. crystallinity index assay was done using xrd shimadzu maxima 7000 (japan) in the research center of biomaterials, national research and innovation agency (brin), cibinong, west java, indonesia. the sample was analyzed between 10 to 80 on continous scanning at 2 o o o speed per minute, with cu radiation (1. 506 å). background noise was separated from the selected xrd pattern, and then the x-ray pattern was corrected ndf(%)= c-b a x 100% (2) adf(%)= c2-b2 a2 x 100% (3) hemicellulose(%)= ndf (%) adf(%) (4) cellulose(%)= c2-b a2 x 100% (5) lignin(%)= d-e a2 x 100% (6) and normalized using a computer. crystallinity index was calculated using the formula: (i / (i + i )) x 100%,cr cr a (7) where i is crystalline area meanwhile i is amorphcr a area. functional group analysis. fourier-transform analysis was done using spectrum two, perkin elmer (usa) on absorbance between 400–4000 cm and -1 spectrum area between 4 cm and 16 scans. wave -1 intensity was based on the percent of transmittance, which was calculated and observed later. m o r p h o l o g i c a l c h a r a c t e r i s t i c . e a c h pretreatment sample was weighed around one gram (without repetition) and dehydrated using gradual ethanol (ethanol concentration: 50%, 70%, 80%, 90% and last 100%) before being coated with gold. each gold-coated bagasse sample was observed on 200, 500 and 1000 times magnification using electron microscope sem jeol jsm-it200 (japan) at 3 kilovolt. a n a l y t i c a l s t a t i s t i c s . c r u d e f i b e r a n d lignocellulose were duplo repeated, data were expressed as mean ± standard deviation. data were further processed using anova (analysis of variance) along with a t-test assuming equal variances with α=0.05 and α= 0.01, respectively. the amount of reducing sugar, total sugar and polymerization degree was duplo repeated, expressed as mean ± standard deviation and processed using anova. results lignocellulosic component of sugarcane bagasse. around 31.90% total dry weight of raw bagasse was crude fiber. crude fiber of control bagasse was ineffectively degraded by sp s2,streptomyces concluded from not statistically significant crude fiber between control and control. crude fiberstreptomyces of alkaline and peroxide pretreated bagasse was increased by 19.96% and 10.95%, respectively. naohstreptomyces bagasse crude fiber was increased by 5%, which was not statistically significant to alkalinehydrothermal pretreated bagasse. sp s2streptomyces can significantly decrease hydrogen-peroxide pretreated bagasse crude fiber by 4.6% (table 1). raw sugarcane bagasse used in this research contains 46.65% cellulose, 31.83% and 9.90% lignin. control bagasse cellulose content was increased by 2.51% after degradation; meanwhile,streptomyces hemicellulose and lignin were slightly increased by 0.45% and 0.88%, respectively. both alkalinehydrothermal and hydrogen peroxide pretreatment reduces hemicellulose to 12.46% and 21.38%, respectively. on the other hand, the cellulose content of alkaline-hydrothermal and hydrogen peroxide treated bagasse was increased by 31.56% and 4.99%, respectively. however, lignin content was increased significantly by 6.34% after peroxide pretreatment, while lignin content decreased by 4.08% after alkaline pretreatment. streptomyces degradation to alkaline pretreated bagasse reduces cellulose significantly by 4.86% from 78.21% to 73.35%. on the other hand, streptomyces degradation only slightly increases both hemicellulose and lignin content of alkaline pretreated bagasse. cellulose content was increased by 1.52% to 53.16% and hemicellulose content was increased significantly by 3.87% to 25.25%, when peroxide bagasse was further degraded using sp s2.streptomyces streptomyces degradation reduces the lignin of peroxide bagasse significantly by 6.48% to 9.76% compared to peroxide treatment only, meanwhile not significant compared to control bagasse (table 1). crystallinity index analysis. raw bagasse cellulose consists of 54% cellulose distributed unevenly ( amorph), while 45.34% was distributed in a well-ordered crystalline area. alkaline-hydrothermal pretreatment caused a wider amorph area and loosened cellulose bonds, reducing crystallinity to 35.44%. crystalline area after peroxide pretreatment was increased by 5%, which can also mean cellulose purity had increased. streptomyces sp s2 enzyme was more efficiently digest cellulose crystalline area in peroxide bagasse. this was indicated from higher crystalline area reduction (11% crystalline area reduction) in peroxide bagasse compared to only 9% crystalline area reduction in control bagasse, after both bagasse was degraded by . the crystallinity ofstreptomyces alkaline pretreated bagasse remained stable after being degraded by only slightly increased bystreptomyces, 1.51% (fig 1 ). functional group analysis. compared to raw bagasse, both alkaline-hydrothermal and hydrogenperoxide pretreatment can weaken bonds between 3500-3000 cm . degradation increase -1 streptomyces peak number 3300 cm of control and alkaline -1 pretreated bagasse. on the other, streptomyces decrease the intensity of peak number 3300 of peroxide pretreated bagasse. band on 1726 cm was found on -1 raw bagasse; this bond was weakened after all treatment. raw bagasse has bonds between 1300-1200 cm , -1 16 agung et al. microbiol indones volume 1 2026, 2 microbiol indones 17 with a higher 1300 bond than the 1200 cm . the peak -1 on 1300 cm-1 was reduced after the alkalinehydrothermal treatment, but the peak on 1200 cm -1 wasn't. on the other hand, peroxide pretreatment had reduced both 1300-1200 cm bonds. although the drop -1 on 1300 cm was not as high as after alkaline -1 pretreatment. degradation of peroxidestreptomyces bagasse can further weaken 1300 cm and 1200 cm -1 -1 (fig 2). both alkaline and peroxide treatment weaken bands between 1100-900 cm compared to control and -1 controlbagasse.streptomyces streptomyces degradation had increased the peak on 1160 cm of -1 previously alkaline pretreated bagasse; meanwhile, the same peak number of peroxide pretreated bagasse was decreased. a slight decrease of peak number 832 cm -1 was only found in peroxidebagasse (figstreptomyces 2). morphological characteristic. raw sugarcane bagasse has been shown to have neatly and regularly bundled fiber, without difference in depth (fig 3a). a l k a l i n e h y d r o t h e r m a l p r e t r e a t m e n t c r e a t e s multilayer folding (consists of several bundles of fiber) and big holes on the outer layer of bagasse (fig 3b). shallow holes were scattered around the outer layer of peroxide pretreated bagasse. peroxide bagasse also has multilayer bundles with rough edges and differences in depth (fig 3c). controlbagasse shownstreptomyces to have some folding and deep holes from streptomyces activities were scattered around its outer layer (red arrows, fig 3d) . degradationstreptomyces to alkaline pretreated bagasse seems to remove lignin from the outer layer, leaving a smooth layer of bagasse and deep holes scattered around its surface (red arrows, fig 3e). degradation creates deep holesstreptomyces on peroxide pretreated bagasse (red arrows, fig 3f); most of the bagasse outer layer was also completely removed, exposing another layer of cellulose and hemicellulose. enzymatic degradation using spstreptomyces s2. control sugarcane bagasse had the highest drop in polymerization degree (from 37 to 11) alongside the highest drop in total sugar, resulting in the highest reducing sugar amount on the highest enzyme concentration ratio (1:4). the significant drop in polymerization degree and total sugar, with a significant increase in reducing sugar of control, only happened in 1:4 enzyme ratio. meanwhile enzyme ratio 1:3 wasn't change anything of control bagasse compared to the lowest (1:2) enzyme ratio. alkaline pretreatment bagasse had the lowest polymerization degree at the lowest enzyme ratio (24 at 1:2 ratio ), had a significant drop (to 13) as enzyme concentration i n c r e a s e d t o 1 : 4 . t h e s i g n i f i c a n t d r o p i n polymerization degree of alkaline bagasse only happened on the highest enzyme concentration (1:4) compared to the lowest enzyme concentration to previous alkaline bagasse and control (1:2). meanwhile drop in polymerization degree of alkaline bagasse (1:4 enzyme ratio) wasn't significant compared to when using 1:3 enzyme ratio. the lowest polymerization degree and most significant drop were achieved in peroxide pretreatment bagasse (8) on 1:4 bagasse to enzyme concentration ratio, decreased by 22 points from 30 on 1:2 enzyme concentration and 3 points from 1:3 enzyme concentration (table 2). the highest hydrolysis efficiency was achieved by peroxide pretreatment bagasse on 1:4 enzyme concentration (highest percentage of reducing sugar from its total sugar amount), compared to raw bagasse and alkaline pretreated bagasse. alkaline pretreated bagasse had the lowest hydrolysis efficiency (lowest reducing sugar produced from its total sugar amount); the significant drop in total sugar of alkaline bagasse only resulted in a slight increase of its total sugar. discussion cellulose, hemicelulose, lignin and some amount of β-glucan, dextrin, pectin, mucilages, inulin, also oligosaccharides/ oligofructose are form crude fiber (slavin 2009). alkaline pretreated bagasseet al. increased cellulose content and reduced lignin was similar to srivastava (2017). meanwhile,et al. peroxide pretreated bagasse lignin content was increased, which was inconsistent compared to yan et al. (2019) findings. increased lignin content might be because sugarcane bagasse has high (around 130 kj/mol) lignin enthalpy, which makes sugarcane has high lignin thermal stability. higher thermal stability makes hydrogen-peroxide harder to extract bagasse lignin compared to other lignocellulose with lower thermal stability, within the same temperature (chen et al. et al.2016; watkins 2015). another possible explanation is lignin was moved to the surface of bagasse because of the acid nature of peroxide pretreatment. lignin on the bagasse surface was easier to be accessed by bacteria, resulting in significant lignin content drop in peroxide bagasse when further treated using (table 1). a wider specificstreptomyces surface area accessible to bacteria, caused by increased crystallinity, can be further digested by bacteria. these findings were consistent and similar to xu (2017)et al. 18 agung et al. microbiol indones table 1 crude fiber and lignocellulose component of sugarcane bagasse treatment crude fiber (%) hemicellulose (%) cellulose (%) lignin (%) control 31.90 ± 0.50 31.83 ± 0.74 46.65 ± 0.66 9.90 ± 0.55 alkaline-hydrotermal (naoh) 51.86 ± 1.09b 12.46 ± 1.09b 78.21 ± 0.42b 5.82 ± 1.00a hydrogen-peroxide (h2o2) 42.85 ± 0.93b 21.38 ± 0.34b 51.64 ± 0.59a 16.24 ± 0.08b control-streptomyces 31.25 ± 1.03 32.28 ± 0.25 44.14 ± 0.91 10.78 ± 0.45 naoh-streptomyces 55.15 ± 0.67b 13.20 ± 0.21b 73.35 ± 0.21bb 6.54 ± 0.31a h2o2-streptomyces 38.25 ± 0.40ba 25.25 ± 0.97ba 53.16 ± 0.08b 9.76 ± 0.18ns-b hemicellulose percentage was based on neutral detergent fiber (ndf) substracted by acid detergent fiber (adf); data were not shown. (a,b) the first superscript letter show statistical significance compared to control only, (a,b) the second superscript shows statistical significance from its previous treatment (example, control from controletc.)streptomyces, (ns) not statistically significant α=0.05 (a) statistically significant based on p (t< t) two-tail value on two-sample t-test assuming equal variance, α=0.05 (b) α=0.01 fig xrd graph used to measure the crystalline area1 . fig fourier transform infrared graph2 . fig 3 scanning electron microscope (sem) photograph (a). control (b). alkaline-hydrothermal (c). hydrogen. peroxide (d). control(e). naoh(f). h o -streptomyces streptomyces streptomyces2 2 . all photographs shown are at 1000x magnification. table 2 measurement of reducing sugar and total sugar using different enzyme concentrations (1:2, 1:3, 1:4) also polymerization degree of sugarcane bagasse after four days of enzymatic degradation treatment enzyme concentration ratio reducing sugar (mg ml-1) total sugar (mg ml-1) polymerization degree control (1:2) 0.21 ± 0.02 7.64 ± 0.05 37.13 ± 3.22 (1:3) 0.27 ± 0.02ns 7.41 ± 0.13ns 27.81 ± 1.71ns (1:4) 0.39 ± 0.01b-a 4.24 ± 0.25b-b 10.98 ± 0.98b-b alkaline-hydrotermal (naoh) (1:2) 0.19 ± 0.01ns 4.57 ± 0.07b 24.17 ± 1.44a (1:3) 0.23 ± 0.03ns-ns 4.07 ± 0.06b-a 17.95 ± 2.33a-ns (1:4) 0.26 ± 0.02ns-ns-ns 3.52 ± 0.06b-b-a 13.89 ± 1.39a-a-ns hydrogen-peroxide (h2o2) (1:2) 0.11 ± 0.01a 3.36 ± 0.08b 30.91 ± 2.55ns (1:3) 0.26 ± 0.02ns-a 2.89 ± 0.15b-ns 11.36 ± 0.52b-b (1:4) 0.32 ± 0.01a-b-ns 2.67 ± 0.04b-b-ns 8.34 ± 0.32b-b-a volume 1 2026, 2 microbiol indones 19 research that use to degradecupriavidus basilensis acid pretreated rice straw. hydrogen bonds in cellulose were cut after both alkaline and peroxide pretreatment, indicated from lower peaks between 3500-3000 cm , compared to -1 control. increase on peak number 3300 cm in control-1 streptomyces streptomyces,and naohexcept peroxideindicates bonds betweenstreptomyces. cellulose were stronger in controlandstreptomyces naohbagasse, despite the lowerstreptomyces cellulose content than its previous treatment. meanwhile cellulose bonds were weaker in peroxidestreptomyces bagasse compared to peroxide bagasse. raw bagasse contains ester, indicated from an available peak of 1726 cm (nandiyanto .2019). -1 et al all pretreatment weakens these ester bonds. raw sugarcane bagasse has higher s (syringyl) compared to (g) guaiacyl lignin, indicated from higher bands around 1300 cm compared to around 1200 cm -1 -1 (watkins . 2015). miyamoto . (2018) researchet al et al also found that most lignin in sugarcane bagasse is syringil lignin. alkaline pretreatment was only effective to reduce syringil lignin on peak 1300 cm . -1 meanwhile wasn't effective in reducing guaiacyl lignin, indicated from unchanged 1200 cm peak. -1 syringil lignin and guaiacyl bonds remained unchanged when alkaline bagasse was degraded by streptomyces sp s2. peroxide pretreatment weakens both lower peaks around 1300-1200 cm , indicating -1 that peroxide treatment cut both syringil and guaiacyl lignin. degradation further weakens bothstreptomyces syringil and guaiacyl lignin, indicated from lower 1300-1200 cm (fig 2). this explains the drop in lignin -1 content after peroxide bagasse degraded by streptomyces sp s2, although the final lignin content was similar to control (table 1). hemicellulose identifier bands are stacked with lignin and cellulose identifier bands, between 1200800 cm (gogna and goacher 2018). raw bagasse has -1 hemicellulose that was highly bonded to lignin, indicated from low bands around 1100-900. this result was similar to rice straw in zulyadi (2016)et al. research. alkaline and peroxide pretreatment was reduce hemicellulose content, correspond to weakened bands between 1100-900 cm . alkaline bagasse -1 h e m i c e l l u l o s e c o n t e n t w a s ' t c h a n g e d a f t e r streptomyces degradation, indicated from an unchanged peak on 1160 cm . however, -1 streptomyces was weakening bonds on 1160 cm of peroxide -1 pretreated bagasse. this indicates some of the hemicellulose bonds of peroxide bagasse were cut by streptomyces sp. or remains bonded to lignin, despite an increase in the percentage of hemicellulose (table 1). sugarcane bagasse hemicellulose contains βglycosidic bonds, indicated from a present band on 897 cm (rashid 2020) a slight decrease of peak -1 et al. number 832 cm was only found in the peroxide-1 streptomyces streptomycesbagasse. this indicates sp. s2 can decrease p-hydroxyphenyl only after sugarcane is treated with hydrogen-peroxide (porterobarahona et al. 2019), (fig 2). qi research (2018) show similar folding in 4%et al. alkaline (naoh) pretreated wheat straw. streptomyces sp. s2 was likely to reduce the lignin from the bagasse surface by creating holes and digging out the lignin. this was similar to xu (2017) research that usedet al cupriavidus basilensis to degrade rice straw. the amount of reducing sugar released from degraded sugarcane bagasse was consistent with the crystallinity index value (cri, fig 1). this was consistent with li (2016) findings, that higheret al. crystallinity index before saccharification process will increase hexoses sugar that released. the amount of sugar released somehow seems not related to the increase in cellulose percentage after alkaline or peroxide pretreatment (table 1). another factor that might affect in decreased hydrolysis efficiency of alkaline pretreated bagasse was higher temperature used (121 c) in alkaline pretreatment than peroxide o (60 c), despite shorter time (30 minutes of alkaline vs o 90 minutes of peroxide pretreatment). higher temperature used in alkaline pretreatment makes oligomers such as cellobiose was decomposed faster than glucose yield rate (mohan . 2015). alkalineet al pretreatment alone produce higher amount of acetic acid, compared to alkaline-peroxide pretreatment. higher acetic acid inhibits reducing sugar production (li 2016). these result suggest hydrogen-et al. peroxide pretreatment using lower temperature within optimum time can be applicable in fermentation process within pilot plant or industrial settings, since produce higher reducing sugar amount. in conclusion, hydrogen-peroxide pretreated bagasse was the best substrate to degraded by streptomyces sp s2. hydrogen-peroxide can weaken both syringil and guaiacyl lignin in bagasse. which causes lower lignin content when peroxide bagasse is further degraded by sp s2.streptomyces streptomyces sp also increase cellulose and hemicellulose content of peroxide bagasse. these factors affect hydrogenperoxide bagasse to have the lowest polymerization degree and most effectively hydrolyzed when treated 20 agung et al. microbiol indones with sp s2.streptomyces competing interest the authors declare that they have no competing interest. references aoac. 1998. official methods of analysis, 16th edition, 4th revision. gaithersburg: aoac international. azeredo hmc, kontou-vrettou charis, moates gk, wellner n, cross k, pereira phf, waldron kw. 2015. wheat straw hemicellulose films as affected by citric acid. food hydrocoll. 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jiamin w, feng y, caiyun t, qiongtao h. 2019. effect of h o bleaching treatment on the properties of2 2 finished transparent wood. polymers 11(776): 1-13. zulyadi nh, saleh sh, sarijo sh. 2016. fractionation of hemicellulose from rice straw by alkaline extraction and ethanol precipitation. malays j anal sci. 20 (2): 329-334. 22 agung et al. microbiol indones 4.mi696-arif nurkanto available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.1.4issn 1978-3477, eissn 2087-8575 vol 7, no 1, march 2013, p 24-36 *corresponding author; phone: +62-21-8765066, fax: +6221-8765062, e-mail: arif.nurkanto@lipi.go.id diseases caused by microbial infection have been increasing progressively in the past decades (anand et al. 2008; who 2011). some of the reasons behind the increasing number of cases of the diseases are the growing number of new pathogenic microbes and the development of resistance in infectious microorganism. the confounding fact is instantly followed by the ever growing need for drugs. however, the number and variety of drugs available in the market are barely adequate for the new emerging diseases and resistant pathogen (phoebe et al. 2001; donaldio et al. 2010; pozzi et al. 2011). thus, it is crucial to search for potential new antimicrobial compounds to cope with the problems. this research was aimed to obtain general antimicrobial compounds which were focused on natural products. these natural products can be isolated from plants, animals, and microorganisms (newman et al. 2000; dewick 2002; newman and cragg 2004). microorganisms, especially actinomycetes, are potential sources of natural products. actinomycetes are distributed in soil and leaf litter, and produce many important bioactive compounds with commercial values, including antibiotics (takizawa et al. 1993). streptomyces is the largest group of actinomycetes with potential biological activities and antibiotic (omura et al. 2001; bentley et al. 2002). antimicrobial screening of two hundred organic the objective of this research was to investigate bacterial response after treatment with active metabolite produced by streptomyces sp. isolated from batanta island. minimum inhibitory concentration (mic) values of four clinically tested bacteria (escherichia coli, bacillus subtilis, staphylococcus aureus, and micrococcus luteus) were successfully determined in this research using microdilution method. leakages of nucleic acids and proteins from the tested microbes were detected using uv/vis spectrophotometry method at 260 and 280 nm. uracil leakage was analyzed using hplc. morphological changes of the bacterial cells were observed using scanning electron microscope (sem). a streptomyces isolate bl225 was identified based on the 16s rrna gene sequence (1500 bp). when tested agains microbes, the mics values of this compound were between 16-64 µg -1 ml . the results indicated leakages of protein, nucleic acid and uracil from e. coli and b. subtilis cells after treatment with pure metabolite isolated from bl225. treatment using metabolite from bl225 also caused morphological changes and damages of the target bacterial cell. bl225 had been identified as a strain that has closed relation to streptomyces badius (98.9%). key words : batanta island, nucleic acid, protein, sem, streptomyces, uracil leak tujuan penelitian ini adalah untuk mengetahui respon bakteri setelah pemaparan dengan metabolit aktif dari streptomyces sp. yang diisolasi dari pulau batanta. nilai konsentrasi hambat minimum (khm) terhadap empat isolat klinik (escherichia coli, bacillus subtilis, staphylococcus aureus, dan micrococcus luteus) telah berhasil ditentukan dalam penelitian ini dengan menggunakan metode mikrodilusi. kebocoran asam nukleat dan protein dari bakteri uji dideteksi dengan menggunakan metode spektrofotometri uv/vis pada panjang gelombang 260 dan 280 nm. kebocoran urasil dianalisa menggunakan hplc. perubahan morfologi sel bakteri uji diamati dengan menggunakan mikroskop elektron payar (sem). isolat streptomyces sp. bl225 telah diidentifikasi berdasarkan sekuen gen 16s rrna (1500 bp). nilai mic dari senyawa aktif produksi streptomyces sp. bl225 -1 terhadap bakteri uji berkisar antara 16-64 µg ml . hasil penelitian mengindikasikan adanya kebocoran protein, asam nukleat, dan urasil pada sel bakteri e. coli dan b. subtilis setelah perlakuan dengan metabolit murni bl225. perlakuan dengan metabolit bl225 juga menyebabkan perubahan morfologi dan kerusakan pada sel bakteri. isolat bl225 teridentifikasi sebagai strain yang mempunyai kekerabatan tinggi dengan streptomyces badius (98,9%). kata kunci : asam nukleat, kebocoran urasil, pulau batanta, protein, sem, streptomyces bacterial response after exposure with pure metabolite produced by streptomyces sp. bl225 isolated from batanta island's leaf litter 1 1 2 arif nurkanto *, andria agusta , wellyzar sjamsuridzal , 1 and heddy julistiono 1 research center for biology, indonesia institute of sciences (lipi), jalan raya bogor km 46, cibinong 16911, west java, indonesia; 2 department of biology, faculty mathematics and natural science, universitas indonesia, depok 16424, indonesia volume 7, 2013 microbiol indones 25 and water solvent extracts derived from raja ampat's actinomycetes showed that 43% isolates had antimicrobial activities against bacteria and yeast (nurkanto et al. 2012). isolate capable of producing bioactive compound with the highest antibacterial activities was selected and used in this research. the major bioactive compound from actinomycetes bl225, the selected isolate, was isolated from broth fermentation. pure bioactive compound was used to study bacterial responses of both gram negative and gram positive bacteria. bacterial responses to exposure towards various antibiotic substances are relatively diverse . one of the factors influencing these responses is the mechanism of action of the antibacterial compounds. through study of bacterial cell reaction to antibiotic exposure, the mechanism of action of the specific substance against targeted bacteria could be revealed (kohanski et al. 2010). some of the relatively easily observed cell responses are excreted cell products or components such as protein, nucleic acid, and ions. changes in cell morphology are also important parameter in studying cell response (harold and thomas 1996). the objectives of this research are to determinate mic value on some clinical bacterial and to study the mechanism of the action of newly discovered antibacterial compound. the mechanism of action was studied by observing protein and nucleic acid leakages and their effects to cell morphology. taxonomical status of the isolated actinomycetes was also studied in this research. material and methods sources of isolate. actinomycetes used as metabolite sources were isolated from litter sample in batanta island, raja ampat, west papua using sds-ye method (hayakawa and nanomora 1987). we had previously isolated more than one hundred actinomycetes (unpublished result). our previous study focusing on the screening of antimicrobial activity from metabolites produced by these isolates (nurkanto et al. 2012) had demonstrated that isolate bl225 had the highest antimicrobial activity, and thus selected to produce active compound. production of active substance by fermentation. actinomycetes isolate bl225 was inoculated into a 5000 ml flask containing 2000 ml of actino medium no. 1 (daigo, japan) (with composition per liter: 5 g polypeptone, 3 g yeast extract, at ph 7.2). the flask was incubated at 28 °c for 7 d in shaker incubator. extraction and purification bioactive compound. broth culture was extracted using ethyl acetate and methanol (4:1) solvent. these organic solvents were mixed thoroughly by shaking and left to stand for 1 h. the two layers, the organic and water layers, were separated. the organic layer was concentrated by evaporation under vacuum. dry extract of supernatant and biomass were purified using column chromatography (merck, 60-120 mesh). dichloromethane and methanol 20:1 (v/v) mixture was used as eluting solvent. two milliliters of crude extract was added on silica gel column surface and the extract was adsorbed on top of the silica gel. eight fractions were collected and tested for their antimicrobial activities (usha et al. 2010). the purification of the active fraction from the column was confirmed using tlc gf 254 (merck) and hplc (shimadzu rf-10axl). column ascentis ® c18 (supelco, usa) 5 µm 4.6 x 150 mm for hplc was used in this reserach. the volume injected was 100 µl per injection under conditions of average pressure of 1.350 psi in 254 nm wavelength, and the flow rate was -1 1 ml min where the mobile phase was methanol : water (8:2) and time period was 25 min. determination of minimum inhibitory concentration (mic). calculation of mic value from pure extract produced by streptomyces sp. bl225 against four bacterial srain was performed by micro dilution method (rahman et al. 2005). the pure extract was tested against escherichia coli (lipimc 186 = nbrc 3301), bacillus subtilis (lipimc 187 = nbrc 3134 = atcc 6633), micrococcus luteus (lipimc 76=nbrc 13867=atcc 10240), and staphylococcus aureus (lipimc 114 = nbrc 12732 = atcc 6538p = dsm 1790). compounds (bl225 metabolite and chloramphenicol) were diluted in 50% dmso and filtered with 0.22 µm cellulose membrane. compounds were prepared one step higher or two times concentration than the final dilution range required, to compensate for the additional of an equal volume of inoculum. after added with microbial inoculum, the compounds' final concentrations were 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 -1 7 -1 µg ml . bacterial test suspensions (1-5 x 10 cell ml ) were inoculated into microplate before incubation at 37 °c for 12 h. after the incubation period had elapsed microbes should have grown in all wells of the antibiotic free control plate. mic were defined as the lowest concentration of compound at which there is no visible growth of the organism. cell leakage test. cell leakage test was performed on two bacterial species, e. coli and b. subtilis, as representation of gram negative and gram positive bacteria. methods used based on miksusanti et 26 nurkanto et al. microbiol indones al. (2008) with some modification. cell leakage test was conducted using several approaches, analyses of protein and nucleic acid leakages, uracil leakage, and cell morphology observation using scanning electron microscope. these analyses were performed simultaneously. the first step in cell leakaege test is preparation of experimental microoraganisms. microbes were cultivated in mueller hinton media (difco) for 24 h at 37 °c with shaking. 0.5 ml of 0.1% tween 80 was added to about 10 ml cell suspension, and then mixed. the mixture was then centrifuged at 3500 rpm speed for 20 min in cold temperature (4 °c). supernatant was discarded and the pellet was washed twice with phosphate buffer (ph 7). pure extract was then added at concentrations 1mic and 2mic to the cell suspension in phosphate buffer. only filtered 50% dmso solution (without compound) was added to the negative control. chloramphenicol solution was used as comparison control. samples were then incubated in shaker incubator for 24 h at 37 °c. the suspension was centrifuged for 15 min. afterward, supernatant and pellet were separated. this was then followed by analyses of protein, nucleic acid, and uracil leakages. cell pellet was observed using sem. protein and nucleic acid leakages were analysed by measuring absorbance using uv/vis spectrophotometer at 260 and 280 nm wavelength. uracil leakage was detected using hplc. hplc (shimadzu, japan) analyses were performed using acetonitrile: aquabidest (1:9) as solvent. one ml samples was injected. the column used was puresil 5m c18 4.6 x 150 mm. the -1 flow rate used was 1 ml min and column pressure was 1350 psi with 254 nm wavelength. sem preparation. bacteria was treated accordingly with bl225 compound at suband supramics (1 mic and 2 mic) for 12 h at 37 °c in phosphate buffer. untreated controls were also prepared in this experiment. after being fixed for 1 h with 2% glutaraldehydeand washed with cocodylate buffer ph 7.2, the bacteria was then post-fixed with 1% oso . 4 samples were dehydrated with graded ethanol series (30, 50, 70, 80, 90, and 100%), with 10 min incubation after treatment with each ethanol concentration. bacterial cells were observed using sem (joel, jsm5310lv). electron images were taken at low electron energies (20 kv). actinomycetes identification. actinomycete isolate was identified based on its 16s rrna gene sequence. the isolate was cultured in 5 ml yeast starch -1 broth medium (yeast extracts 2 g l , soluble starch 15 g -1 l ph 7.2) (miyadoh 2001). pellet was collected during log phase growth. chromosomal dna was isolated using pitcher et al. (1989) method. twenty to 100 ng genomic dna was used as a templete for polymerase c h a i n r e a c t i o n ( p c r ) a m p l i f i c a t i o n o f a n approximately 1500 base segment. the pcr primers used were 20f (5’-gattttgatcctggctcag-3’) and 1500r (5’-gttaccttgttacgactt-3’) (suriyachadkun et al. 2010). pcr product was purified using hiraishi et al. (1995) method. the purified pcr product was sequenced using an abi 3130 genetic analyzer with bigdye terminator version 3.1 sequencing method. for completed sequence result, we used 6 primers. the sequence of the primers were 520f (5’-gtgccagcagccgcgg-3’), 920r (5’ccgtcaattcatttgagttt-3’), 520r (5’a c c g c g g c t g c t g g c 3 ’ ) , 9 2 0 f ( 5 ’ aaactcaaatgaattgacgg-3’), 20f (5’ gattttgatcctggctcag-3’) and 1500r (5'gttaccttgttacgactt-3’) (yukphan et al. 2004; suriyachadkun et al. 2010; techaoei et al. 2011). full sequence analysis was conducted using reference strains from the ribosomal database project-ii (http://www.rdp.cme.msu.edu), which were chosen based on high similarity rank with the strains in this study. approximately 1400 bases were included in the phylogenetic analysis, which was performed with the clustal x version 1.83 and nj plot computer program (thomson et al. 1997; felsenstein 1985). results pure compound derived from streptomyces sp. bl225 was investigated and checked (fig 1) and was used in our experiments. the compound's structure is still currently being investigated using lcms and nmr. the chemical structure and elucidation study of this active compound will be published separately. in this article, we focused on the evaluation of the pure compound to determine mics and cell leakages. examination of minimum inhibitor concentration value. mic values of bl 225 pure extract differed from one tested microbe to another (between 16-64 µg -1 ml ). compared to chloramphenicol, mic values of the extract were relatively higher (table 1). nucleic acid and protein leak analyses. generally, the microbes treated with the pure extract showed considerable changes. the level of nucleic acid leakage of the tested microbes increased after treatment with 1 and 2 mic. this level of leakage was higher compared to the negative and positive controls. the pattern of protein leakage level was similar to the increased uracil leakage relative to the negative control. uracil leakage in b. subtilis when treated with extract at 1 mic and 2 mic, and chloramphenicol were 2.274 ppm, 2.784 ppm, and 3.152 ppm respectively (table 2). those values were higher compared to the negative control, which showed uracil leakage of only 1.483 ppm. similar result occurred in e. coli, where uracil leakage was higher in treated cell than in cell without treatment (negative control cell). in e. coli uracil leakage in bacterial cells treated with 1 and 2 mic, and with chloramphenicol were 8.169, 17.784, and 18.158 ppm respectively (table 2). uracil leakage in b. subtilis negative control was 0.092 ppm. cell morphology observation using sem. sem observation on e. coli showed diverse cells' morphology (fig 7). the negative control cells seemed to be all intact. the positive control cells showed significant changes in morphology. the cells were clearly damaged and rounder in form. treatment with 1 mic and 2 mic extracts also changed the cells' morphology. a big hole was observed in each cell (fig 7c and 7d). treatment with 2 mic had apparently damaged the cells causing the cells' death. b. subtilis negative control showed intact cells with rod shape (fig 8). there were no changes observed in the cell morphology. treatment with chloramphenicol, as positive control, did not seem to disrupt the cells or change the cells' morphology. however, treatment with one of nucleic acid leakage. treatment with pure extract produced higher protein leakage than the negative control, but still lower than the positive control (chloramphenicol treated cell) (fig 2 and 3). the leakage profiles of e. coli and b. subtilis showed similar pattern although the values were different. in e. coli, the nucleic acid leakage was -1 highest at 2 mic, which was 38.01 µg ml . the negative control showed the lowest nucleic acid -1 leakage with 16.56 µg ml . chloramphenicol treated cells showed the highest protein leakage with 995.85 -1 µg ml . the lowest protein leakage was observed in -1 the negative control with 278.14 µg ml . b. subtilis showed the highest nucleic acid leakage level at 2 mic -1 with 10.08 µg ml and the lowest in the negative -1 control cell with 3.92 µg ml . the highest protein leakage was in observed in chloramphenicol treated -1 cell with 282.16 µg ml , and the lowest was in -1 negative control cell with 97.44 µg ml . analyses of uracil leakage. to observe uracil leakage, hplc grade uracil standard (sigma) was used at concentration 0.31-5 ppm (fig 4). uracil standard was detected at retention time about 2.7 mins. hplc analyses indicated several peaks with different retention time. uracil leakage was detected on every treatment (control cell, treatment with 1 and 2 mic, and treatment with cholramphenicol) (fig 5 and 6). generally, all treatments of e. coli and b. subtilis 1500 1250 1000 750 500 250 0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min mv fig 1 pure active compound produced by bl225 isolate after purification by column chromatography. this compound was detected by hplc using methanol: water (8:2) as mobile phase and time period was 25 min. 64 16 64 16 16 16 16 8 bacterial tested metabolite -1 (µg ml ) chloramphenicol -1 (µg ml ) e. coli m. luteus s. aureus b. subtillis table 1 mic values of pure metabolite produced by bl225 isolate against some bacteria, incubation temperature 37 °c for 12 h volume 7, 2013 microbiol indones 27 fig 2 the leakages of nucleic acid and protein (µg ml ) in e. coli after treatment with metabolite from bl225 isolate, incubated at 37 °c for 12 h. control: without compound. values represent the mean ± standard deviation (sd) of three measurements. a,b,c,d: means within a selected graph are significantly difference based on lsd-test at p ≤ 0.05. -1 28 nurkanto et al. microbiol indones fig 3 the leakages of nucleic acid and protein (µg ml ) in b. subtilis after treatment , incubated at 37 °c for 12 h. control : without compound. values represent the mean ± of three measurements. a,b,c,d: means within a selected graph are significantly difference based on lsd-test at p ≤ 0.05. -1 with metabolite from bl225 isolate standard deviation (sd) 45 40 35 30 25 20 15 10 5 0 -1 ce ll l ea k ag e (μ g m l ) control 1 mic 2mic chloramphenicol -1 64 μg ml nucleic acid leakage from e.coli 16.56 ± 1.05 26.2 ± 2.71 38.01 ± 2.98 26.02 ± 1.78 c b a b protein leakage from e.coli control 1 mic 2mic -1 c e ll l e a k a g e ( μ g m l ) 1200 1000 800 600 400 200 0 278.14 ± 12.98 379.49 ± 34.76 443.86 ± 32.67 995.85 ± 54.66 a b c d chloramphenicol -1 64 μg ml nucleic acid leakage from b. subtillis 12 10 8 6 4 2 0 -1 c el l le ak ag e (μ g m l ) control 1 mic 2mic c b c a 3.92 ± 0.42 8.48± 0.66 10.08 ± 0.33 8.31 ± 0.78 chloramphenicol -1 64 μg ml 350 300 -1 c el l le ak ag e (μ g m l ) 250 200 150 100 50 0 control 1 mic 2mic 282.16 ± 25.66 d c b a188.5 ± 17.34 132.77 ± 9.56 97.44 ± 10.23 protein leakage from b. subtillis chloramphenicol -1 64 μg ml fig 4 uracil standard 5 ppm (a), 2.5 ppm (b), 1.25 ppm (c), 0.63 (d), and 0.31 ppm (e) on hplc analysis. 30 25 20 15 10 5 0 mau a mau 15.0 12.5 10.0 7.5 5.0 2.5 0.0 b 7.5 5.0 2.5 0.0 mau 3 2 1 0 1.5 1.0 0.5 0.0 -0.5 mau mau 0.0 2.5 5.0 7.5 10.0 12.5 min c d e volume 7, 2013 microbiol indones 29 taxonomical status of actinomycetes bl225. bl225 isolate was identified based on 16s rrna gene sequence. after contig assembly from sequences pure extract at 1 and 2 mic had caused morphological changes, indicating cell damage. there seemed to be some holes formed in the cell wall (fig 8c and 8d). fig 5 hplc analyses of uracil leakage on e. coli after treatment with metabolite from bl225 isolate. a: cell control; b: 1 mic; c: 2 mic; and d: chloramphenicol. 2.0 1.5 1.0 0.5 0.0 12.5 10.0 7.5 5.0 2.5 0.0 20 15 10 5 0 20 15 10 5 0 a b c d 0.0 2.5 5.0 7.5 10.0 12.5 min mau mau mau mau 30 nurkanto et al. microbiol indones to s. badius (t) nrrl b-2567 and streptomyces parvus (t) nrrl b-1455t with 98.9 % and 98.7 % homology, respectively. phylogenetic analysis (fig 9) derived from 6 primers, we obtained 1391 bases of nucleotide for complete sequence analysis of this gene. blast result showed that bl225 had high similarity 2 1 0 -1 2.0 1.5 1.0 0.5 0.0 -0.5 4 3 2 1 0 3 2 1 0 a b c d 0.0 2.5 5.0 7.5 10.0 12.5 min mau mau mau mau fig 6 hplc analyses of uracil leakage on b. subtilis . a: cell control; b: 1mic; c : 2mic; and d : chloramphenicol. after treatment with metabolite from bl225 isolate volume 7, 2013 microbiol indones 31 discussion the mic values were used for standard activity of the active compound. mics are defined as the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. higher the mic value indicates weaker demonstrated that bl225 was monophyletic with s. badius and s. parvus with bootstrap value more than 900. the 16s rrna sequence was submitted in ncbi gene bank with accession number kf146316. the isolate was also deposited in indoesia culture collection (inacc) at research center for biology, lipi, indonesia. e. coli microbes treatment control uracil concentration (ppm) d 0.092 ± 0.002 1 mic 2 mic c 8.169 ± 0.026 b 17.784 ± 0.054 chloramphenicol a 18.158 ± 0.055 b. subtilis control 1 mic 2 mic chloramphenicol c 1.483 ± 0.006 b 2.274 ± 0.009 b 2.784 ± 0.100 a 3.152 ± 0.011 table 2 uracil concentration in medium after treatment, incubated at 37 °c for 12 h. fig 7 morphology of e. coli using sem observation (a: control; b: cloramphenicol; c: 1 mic; d: 2 mic), incubated at 37 °c for 12 h. holes are indicated by white arrows. a b c d control: without compound; a,b,c,d: values are significantly different 32 nurkanto et al. microbiol indones fig 8 morphology of b. subtilis using sem observation (a: control; b: cloramphenicol; c:1 mic; d : 2 mic), incubated at 37 °c for 12 h. holes are indicated by white arrows. fig 9 phylogenetic tree analysis of bl225 based on 16s rdna sequence and relationship to other streptomyces constructed by nj plot software with 1000 times bootstrap. the scale bar, the mean number of nucleotide substitutions per site. a b c d volume 7, 2013 microbiol indones 33 by metabolite compound activities which was given. at 2mic treatment, some cells were completely disrupted. cell contents were ruptured. these processes indicated cell death was the result of the extract treatment. this statement is supported by the recorded protein, nucleic acid, and uracil leakage data. nora et al. (2001) and ronald and chopra (1986) had previously mentioned that cellular mambrane rupture caused the loss of free + amino acid, protein, nucleic acid, uracil, and k ion and release cytoplasmic protein from bacteria. our result indicated similar nucleic acid, protein, uracil profiles and morphological changes after treatment. active compound could attach to cell wall or cell membrane or insert in bacterial cells. cells metabolism could be disturbed. cell walls leaked and intracellular material were released. protein and nucleic acid are intracellular materials, whereas uracil is a component of rna. the higher the activityof the compound or extract used for treatment, the more intracellular material (nucleic acid, protein and uracil) were released out of the cell. cellular leakage could be investigated using morphological observation. sem observation clearly show us morphological differences between treated and untreated cells. holes formation in extract treated cells will affect the cells. these holes will disturb cellular metabolism and inhibit or even halt cell proliferation and growth. our result clearly demonstrated that pure extract produced by isolate bl225 caused protein, nucleic acid, and uracil leakages and disrupted the cell. however, specific mechanisms of this disruption was unclear. the results of identification and taxonomical studyindicated that bl225 belongs to s. badius because of its close homology. the closest species, s. badius, have antibiotic and antifungal activities (debananda et al. 2011). however, up to now, commercial drugs derived from this species had never been discovered. study on s. badius was limited only to antagonistic test on microbial pathogens. acknowledgments this research was funded by dipa from the research center for biology, indonesian institute of sciences, ministry of research and technology, and directorate general of higher education, indonesia. we would like to thank achirul nditasari for pre review and dian alfian (puslit biologi lipi) for sequencing preparation. antimicrobial activity, whereas lower mic value indicates stronger antimicrobial activity. the presence of proteins in the media indicated cell damage. pure extract at 1 and 2 mic, as well as chloramphenicol treatment caused secretion of high concentration of protein. the higher the extracellular concentration, the higher the level of cell damage. nucleic acid was also detected in the medium. pure extract-treatedand chloramphenicol-treated (positive control) cells showed higher extracellular concentration of nucleic acid compared to cells without treatment (negative control cells). nucleic acid extracellular concentration also indicated the level of cell damage. uracil is part of rna material. if high amount of uracil were secreted, translation process would be disturbed and thus lead to the failure or inhibition of protein synthesis. although mic value of chloramphenicol was relatively lower compared to the pure extract, uracil leakage observed after treatment with the pure extract was higher compared to chloramphenicol treatment. it indicated that the bl225 pure extract was more effective in causing uracil leakage than chloramphenicol. it was widely known that chloramphenicol is a bacteriostatic antibiotic. chloramphenicol stops bacterial growth by inhibiting protein synthesis. the antibiotic prevents protein chain elongation by inhibiting the peptidyl transferase activity on the bacterial ribosome. chloramphenicol binds to 23s rrna of the 50s ribosomal subunit, preventing peptide bond formation (jardetzky 1963; wolfe and hahn 1965; hahn et al. 1955). our results showed that cell leakages still occured in untreated cell, both e. coli and b. subtilis. however, cell leakages in untreated cell was less than treated cell. these phenomenon might be caused by some factors. cells would be dead after a certain period of time because of nutrient deficiency. in our research, we cultivated cell for 12 h in buffer with no nutrient sources. some cells might die naturally at this condition. dead cells could release intracellular material such as protein, nucleic acid, and uracil. these materials were detected in our research. sem observation on e coli showed changes in morphology of the cell after treatment with the extract. those cells underwent shrinkage, elongation, and produced protusions on the cell wall. according to miksusanti (2008), protusion forming was caused by the inability of peptidoglycan to sustain intracellular pressure as the effect of given metabolite compound. cytoplasm and membrane cell were leaking. biosynthesis of cell wall also did not occur or disturbed 34 nurkanto et al. microbiol indones references anand pa, kunnumakkara b, sundaram c, harikumar kb, tharakan st, oiki sl, sung b, anggarwai bb. 2008. cancer is a preventable disease that requires major lifestyle changes. j pharma res. 25(9):2097-2116. doi:10.1007/s11095-008-9661-9. bentley sd, chater kf, cerdeno-tarraga a-m, 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prathumpai w, santiarwarn d, leelapornpisid p. 2011. screening characterization and stability of biosurfactant produced by pseudomonas aeruginosa scmu106 isolated from soil in northern volume 7, 2013 microbiol indones 35 wolfe ad, hahn fe. 1965. mode of action of chloramphenicol ix. effect of chloramphenicol upon a ribosomal amino acid polymerization system and its binding to bacterial ribosome. biochim biophys acta. 95(1):146-155. doi:10.1016/0005-2787(65)90219-4. world health organization. 2011. global health observatory metadata. http://apps.who.int/athena/. yukphan p, potacharoen w, tanasupawat s, tanticharoen m, yamada y. 2004. asaia krungthepensis sp. nov., an acetic acid bacterium in the α-proteobacteria. int j syst evol microbiol. 54(2):313-316. doi: 10.1099/ijs.0.02734-0. thailand. asian j biol sci. 4(4):340-351. doi:10.3923/ ajbs.2011.340.351. thompson jd, gibson tj, plewniak f, higgins dg. 1997. clustal x windows interface: flexible strategies for multiple sequence aligment aided by quality analysis tools. j nucleic acids res. 25(24):4876-4882. doi:10.1093/nar/25.24.4876. usha r, ananthaselvi p, venil ck, palaniswamy n. 2010. antimicrobial and antiangeogenesis activity of streptomyces parvulus kuap106 from mangrove soil. eur j biol sci. 2(4):77-83. 36 nurkanto et al. microbiol indones 2.mi707-aris tri wahyudi available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.3.2issn 1978-3477, eissn 2087-8575 vol 7, no 3, september 2013, p 94-104 *corresponding author; phone/fax: 0251-8622833 , email: aristri2011@gmail.com rhizospheres are containing microorganisms that could increase plant growth. these bacteria are call plant growth promoting rhizobacteria (pgpr) that have a significant effect for plant growth, directly and indirectly. pgpr have been capability in producing indole acetic acid (iaa) hormone, exopolysaccharide (eps), acc-deaminase, as phosphate solubilizing, and biocontrol agents of pathogenic fungi (dey et al. 2004; kaci et al. 2005; husen et al. 2011). iaa hormones is one of the plant growth hormone that plays a role in several aspects of plant growth, including cell division and elongation, differentiation, tropisms, apical dominance, aging, abscission, and flowering (zhao et al. 2001). drought can affect microorganisms and plant growth since water becomes a limiting factor for plants rhizobacteria have been known for their capability as plant growth promoter through some mechanisms, directly and indirectly. the purpose of this research to screen rhizobacteria of bacillus spp. and pseudomonas spp. for their drought tolerance as plant growth promoter of maize (zea mays). screening of rhizobacteria as growth promoter of 47 isolates of bacillus sp. cr and 34 isolates of pseudomonas sp. crb resulted 24 and 9 isolates were able to stimulate the growth of maize sprouts, respectively. further screening of those growth promoter of the rhizobacterial isolates to drought tolerance resulted 7 isolates of bacillus sp. cr and 6 isolates of pseudomonas sp. crb that were able to grow on medium with osmotic pressure -1 and -2 mpa, respectively. potential rhizobacterial isolates of growth promoter and drought tolerance were tested for antagonist mechanisms which aims to determine ability to live together in one carrier medium if to be made formulation. both non antagonist rhizobacterial isolates were evaluated for their potential in producing exopolysaccharide -1 (eps) revealing that crb 19 and cr 90 exhibited the highest activity of eps production up to 0.346 mg ml on -1 medium with -2.0 mpa and 0.107 mg ml on medium with -0.73 mpa, respectively. based on 16s rrna sequence analysis, it revealed crb 19 and cr 90 had highest similarities to pseudomonas aeruginosa strain b2 and brevibacillus brevis b33, respectively. those growth promoter and drought tolerant of bacillus sp. cr and pseudomonas sp. crb had potency to be developed as inoculants in dry land agriculture. key words: bacillus sp. cr, drought tolerant, growth promoter, pseudomonas sp. crb, rhizobacteria rizobakteria diketahui memiliki kemampuan sebagai pemacu tumbuh tanaman melalui beberapa mekanisme, baik secara langsung maupun tidak langsung. penelitian ini bertujuan untuk menyeleksi rizobakteria bacillus spp. dan pseudomonas spp. toleran kekeringan sebagai pemacu pertumbuhan tanaman jagung (zea mays). penapisan rizobakteria pemacu tumbuh dari 47 isolat bacillus sp. cr dan 34 isolat pseudomonas sp. crb mendapatkan masing-masing 24 dan 9 isolat yang secara signifikan memacu pertumbuhan. isolat pemacu tumbuh selanjutnya diseleksi terhadap toleran kekeringan, lalu didapatkan 7 isolat bacillus sp. cr dan 6 isolat pseudomonas sp. crb yang dinyatakan toleran kekeringan. masing-masing isolat mampu tumbuh pada media dengan tekanan osmotik -1 dan -2 mpa. isolat potensial pemacu tumbuh toleran kekeringan diuji sifat antagonisnya untuk mengetahui kemampuannya hidup bersama pada satu media pembawa jika dibuat formula. isolat yang tidak saling antagonis selanjutnya dievaluasi potensinya dalam menghasilkan eksopolisakarida. -1 crb 19 dan cr 90 masing-masing menghasilkan eksopolisakarida tertinggi hingga 0.346 mg ml pada medium -1 -2.0 mpa dan 0.107 mg ml pada medium -0.73 mpa. berdasarkan sekuens gen 16s rrna, crb 19 memiliki kemiripan tertinggi dengan pseudomonas aeruginosa strain b2 dan cr 90 dengan brevibacillus brevis b33. bacillus sp. cr dan pseudomonas sp. crb pemacu tumbuh dan toleran kekeringan tersebut berpotensi untuk dikembangkan sebagai inokulan di lahan kering. kata kunci : bacillus sp. cr, pemacu tumbuh, pseudomonas sp. crb, rizobakteria, toleran kekeringan screening of rhizobacteria for plant growth promotion and their tolerance to drought stress 1 1 rahayu fitriani wangsa putrie , aris tri wahyudi *, abdjad asih 2 3 nawangsih , and edi husen 1 department of biology, faculty of mathematics and natural sciences, jalan agatis, institut pertanian bogor, kampus dramaga bogor 16680, indonesia; 2 department of plant protections, faculty of agriculture, institut pertanian bogor, jalan agatis kampus dramaga bogor 16680, indonesia; 3 soil research institute, bbsdlp, jalan tentara pelajar, bogor 16114, indonesia and microorganisms to survive. one way drought adaptation of microorganisms are secretion of exopolysaccharide (eps) in higher amounts. eps are structural component of extracellular matrix in biofilms are synthesized by cells to respond physiological stress in the environment (marvasi et al. 2010). eps were produced as response biotic and abiotic stress factors to adaptation in extreme environments. the main function is to assist the protection against environmental stresses. microorganisms such as agrobacterium sp., alcaligenes faecalis, xanthomonas campestris, bacillus sp., zygomonas mobilis, leuconostoc, pseudomonas sp., acetobacter xylinum, and several other genera of microorganisms are known to produce eps (donot et al. 2011). eps production by rhizobium sullae kygt207 strain was isolated from dry land in south algeria (gassi touil) and was known to contribute of water absorption and nutrients by roots through modification of physical properties of triticum durum rhizosphere. in vivo test which inoculated kygt207 strain on wheat plants was also known to give significant influence as the plant growth promotion. eps in sandy soil can protect plants from stress, lack of water and contributes to the formation of soil aggregates (kaci et al. 2005). previously we have isolated two groups of pgpr, bacillus sp. cr and pseudomonas sp. crb from rhizosphere of soybean plant of cirebon field area west java (wahyudi et al. 2011 a.b). the use of drought tolerance of pgpr in plant growth promotion is an effective and environmentally friendly step. objective of this study was to screen pgpr pseudomonas sp. crb and bacillus sp. cr for plant growth promoting of maize under drought stress condition. materials and methods screening of pseudomonas sp. and bacillus sp. as growth promoter of maize. initial stage was seed surface sterilization (somasegaran and hoben 1985). sterilized seeds were then soaked in distilled water for 24 h to speed up the germination process. after 24 h, seeds were placed in petri dishes covered by filter paper that was previously moistened with distilled water for 24 h. fourty seven isolates of bacillus sp. cr and 34 isolates of pseudomonas sp. crb were cultured on nutrient -1 -1 broth (nb) (yeast extract 2 g l , peptone 5 g l , nacl 5 -1 -1 g l ) and king’s b medium (peptone 20 g l , k hpo 2 4 -1 -1 -1 1.5 g l , mgso 1.5 g l , and glycerol 15 ml l ) shaked 4 at 120 rpm for 24 h at room temperature, respectively. seeds that have been germinated with 2-3 mm long of primary roots were transferred to a petri dish that contains 1% water agar medium and each seed was inoculated with 100 µl culture of bacillus sp. cr or 9 pseudomonas sp. crb with a cell density of 10 cells -1 ml . seeds with medium without inoculum were used as a control. seeds that had been inoculated with bacteria were incubated for 7 d at room temperature in dark conditions. growth parameters measured were length of stem, root length, and number of lateral roots (dey et al. 2004). data obtained from this experiments were statistically analyzed by one-way analysis of variance (anova) using sas 9.1 software and followed by duncan’s test (dmrt) at 5% level. screening of drought tolerance. bacillus sp. cr and pseudomonas sp. crb were cultured in nutrient broth (nb) medium. two hundred µl preculture of each isolates was inoculated in 20 ml of medium containing polyethylene glycol 6000 (peg 6000) with concentrations of 0, -0.73, -1, -1.5, -2, and -2.5 mpa. concentration of osmotic pressure arrangements according to peg 6000 calculated by michel and kauffman (1973). incubation of medium that had been inoculated with bacterial isolate was performed at room temperature for 24 h by shaking at 120 rpm. optical density (od) of each bacterial culture was measured using spectrophptometer at a wavelength of 570 nm. medium without bacterial inoculation was used as a control. bacteria that were able to grow minimum at -0.73 mpa with od 0.4 catagorized as drought tolerant (sandhya et al. 2009). antagonism test. the purpose of this test was to determine capability of bacillus sp. cr and pseudomonas sp. crb to live together in one medium with no competition each other if to be made formulated. each isolates was grown in lb medium -1 -1 (tryptone 10 g l , nacl 10 g l , and yeast extract 5 g l 1 8 ) at 120 rpm for 24 h to reach concentration of 10 cells -1 ml . antagonism test was done by the following ways: 10 ml of la medium was inoculated with 100 µl targets culture, then poured into a sterile petri dish. a total of 1 ml test culture on lb medium with cell 8 -1 density of 10 cells ml was centrifuged 10 min at 10 000 rpm to obtain the supernatant futher used to antagonism test. sterile paper discs were given for each supernatant test culture then placed on the dish containing la medium that had been inoculated with the targets culture for further incubated. each isolate cr and crb were used as the test culture and targets culture. antagonism was shown by a clear zone around the paper disc. test of exopolysaccharide (eps) production. volume 7, 2013 microbiol indones 95 isolates of bacillus sp. cr and pseudomonas sp. crb were grown in nb medium added by peg 6000 to induction of drought stress. culture incubation was performed in an incubator shaker at room temperature for 72 h, furthermore the cultures were centrifuged to separate the supernatant and pellet. each supernatant was mixed with 3 ml of cold absolute alcohol and incubated overnight at 4 °c. eps was obtained by centrifugation for 15 min at 10 000 rpm. quantity of total carbohydrate content which settles on the eps was measured using the method described by dubois et al (1956). optical density of each eps sample was measured using spectrophotometer at a wavelength of 490 nm. phospate solubilization test. test of phosphate solubilization was performed by standard methods. pseudomonas sp. crb or bacillus sp. cr was grown by streaking on pikovskaya agar plate medium -1 containing tricalcium phosphate (glucose 10 g l , -1 -1 -1 nacl 0.2 g l , kcl 0.2 g l , mgso .7h o 0.1 g l , 4 2 -1 -1 mnso .2h o 2.5 mg l , feso .7h o 2.5 mg l , yeast 4 2 4 2 -1 -1 extract g l , (nh ) so 1 gl-1, and ca (po ) 5 g l ). 4 2 4 3 4 2 plates were incubated at room temperature for 72 h. clear zone formed around the colony indicated the bacteria solubilized phosphate. the clear zone was formed by these bacteria were measured to determine phosphate solubilization index (psi). molecular identification based on 16s rrna sequence. bacterial genomes were extracted by the cetyl trimetyl ammonium bromide (ctab) method. 16s rrna gene was amplified by pcr with 63f primers (5’-caggcctaacacatgcaagtc-3’) and 1387r (5’-gggcggwtggtacaaggc-3’) (marchesi et al. 1998) in total volume of 50 µl. the pcr volume contains of 1 µl of genomic dna template; 1 µl of primer for each forward and reverse, 25 µl of 2x phusion master mix (thermo scientific phusion high fidelity), 1 µl of dmso, and ddh o to a 2 volume of 50 µl. amplification was performed for 30 cycles that include stages of initial denaturation at a temperature of 94 °c for 2 min, denaturation at a temperature of 92 °c for 30 sec, annealing at a temperature of 55 °c for 30 sec, extension at 72 °c for 1 min, and final extension at 72 °c temperature for 7 min. dna of pcr products were purified and sequenced and analyzed by comparing the sequences with genbank database using the blastn program to determine similarity. the dna sequences were also analyzed their phylogenetic tree using mega5 and clustalw software. results results of this study, 24 out of 47 isolates of bacillus sp. cr and 9 out of 34 isolates of pseudomonas sp. crb screened for growth promotion revealed that they had capability in promoting growth of maize sprouts. bacillus could increase the growth of shoot length, root length, and number of lateral root of maize sprouts. pseudomonas could increase the growth of root length and stem length of maize sprouts. this results also revealed that bacillus sp. cr 36 was able to increase all parameters of the growth of maize, significantly (table 1,2). all of the rhizobacteria selected as plant growth promoting were screened for their drought tolerance. seven isolates of bacillus sp. cr and six isolates of pseudomonas sp. crb were classified as drought tolerant, they were able to grow in medium with -1.0 mpa for bacillus sp. cr, and -2.0 mpa for pseudomonas sp. crb (table 3 and 4). rhizobacteria pseudomonas sp. crb, and bacillus sp. cr which could grow in the highest osmotic presssure were subjected to antagonism test to know their capability to survive in one medium. results of this experiment are performed in table 5 and table 6. moreover, the potent isolate of rhizobacteria, crb and cr were examined for their eps production. isolate crb 19 exhibited the highest activity of eps production to -1 0.346 mg ml in nb medium with -2.0 mpa of osmotic pressure. average of eps produced by pseudomonas sp. crb was higher than bacillus sp. cr. bacillus sp. cr produced eps within a range of -1 0.050-0.107 mg ml on medium with -0.73 mpa of -1 osmotic pressure and 0.072-0.096 mg ml on medium with -1 mpa of osmotic pressure (fig 1a). pseudomonas sp. crb was able to produce eps -1 within a range of 0.062-0.196 mg ml on medium with -0.73 mpa of osmotic pressure and within a range -1 0.055-0.197 mg ml on medium with -1 mpa of osmotic pressure (fig 1b). majority, bacillus sp. cr and pseudomonas sp. crb have been known their ability as phospate solubilizing on pikovskaya agar medium. pseudomonas sp. crb 10 was known as the highest in phospate solubilizing. phosphate solubilizing index of pseudomonas sp. crb and bacillus sp. cr within a range of 0.18-0.67. all those isolates of rhizobacteria were able to solubilyze phosphate solubilizing index clear zone diameter colony colony diameter = 96 putrie et al. microbiol indones pseudomonas sp. crb and bacillus sp. cr classified as growth promoter of maize and drought tolerant, were phosphate, except cr 67, cr 90, and crb 4. futhermore, eight potential isolates of rhizobacteria treatment shoot length (cm) root length (cm) number of lateral root cr 61 15.5 a 13.8 n 14.0 lmnopqr cr 39 15.0 ab 15.0 lnm 24.4 c cr 6 14.6 abc 16.8 ijkl 24.8 c cr 59 14.5 abcd 19.7 defgh 12.6 pqr cr 32 14.3 abcd 17.4 hijk 14.8 klmnopq cr 69 14.2 abcd 25.2 b 20.2 de cr 42 13.9 abcde 15.1 lnm 11.0 r cr 12 13.7 abcdef 22.5 c 19.6 defg cr 36 13.5 abcdefg 25.5 b 28.0 ab cr 3 13.3 abcdefgh 25.7 b 12.2 pqr cr 51 13.1 abcdefgh 21.1 cdef 19.4 defgh cr 81 12.9 bcdefghij 20.8 cdef 13.6 mnopqr cr 79 12.8 bcdefghij 29.6 a 26.0 bc cr 30 12.5 bcdefghijk 17.7 hijk 13.4 nopqr cr 83 12.4 bcdefghijk 28.8 a 17.8 efghijk cr 22 12.4 bcdefghijk 22.3 c 13.8 mnopqr cr 86 12.3 bcdefghijk 14.4 mn 29.8 a cr 31 12.2 cdefghijk 25.2 b 21.4 d cr 74 12.2 cdefghijk 22.5 c 16.4 ghijklmn cr 54 12.1 cdefghijk 18.4 ghij 16.2 hijklmn cr 2 12.1 cdefghijk 25.1 b 19.4 defgh cr 56 12.1 cdefghijk 18.8 fghi 13.2 nopqr cr 66 12.0 cdefghijk 21.7 cd 16.2 hijklmn cr 25 12.0 cdefghijk 21.9 cd 15.2 jklmnop cr 90 11.9 cdefghijk 25.5 b 17.6 efghijk cr 21 11.9 cdefghijk 21.8 cd 24.4 c cr 88 11.8 defghijk 16.2 jklm 17.2 efghijkl cr 34 11.8 defghijk 17.0 ijkl 26.6 bc cr 15 11.7 defghijk 19.1 efghi 11.8 qr cr 29 11.4 efghijk 20.8 cdef 19.0 defghi cr 26 11.4 efghijk 22.2 cd 15.0 jklmnopq cr 17 11.2 efghijk 20.7 cdefg 12.6 pqr cr 64 11.1 fghijk 21.5 cd 18.2 efghij cr 50 11.0 fghijk 15.7 klmn 12.8 opqr cr 27 10.9 ghijk 25. 1 b 11.2 r cr 13 10.7 ghijk 14.2 nm 20.0 def cr 91 10.7 ghijk 22.7 c 14.0 lmnopqr cr 23 10.7 ghijk 17.0 ijkl 18.0 efghij cr 75 10.7 ghijk 21.4 cde 18.6 defghi cr 55 10.6 hijk 22.2 c 16.0 ijklmno cr 8 10.6 hijk 25.2 b 12.6 pqr cr 67 10.4 ijk 22.1 cd 24.6 c control 10.3 ijk 20.2 cdefg 17.4 efghijk cr 38 10.2 jk 18.8 fghi 19.0 defghi cr 24 10.2 jk 22.2 cd 14.0 lmnopqr cr 46 10.1 jk 25.0 b 24.6 c cr 33 10.0 k 27.9 a 13.6 mnopqr cr 47 9.70 k 21.7 cd 16.8 fghijklm table 1 effect of bacillus sp. isolates in some growth parameters of maize numbers within a column followed by the same letter are not significantly different at 5% level by dmrt (α=0.05). bold indicated growth promoter isolates compared with control. volume 7, 2013 microbiol indones 97 discussion some studies suggest that bacillus sp. and molecularly indentified based on 16s rrna gene and sequence (table 7 and 8). phylogenetic tree analysis using neighboor joining method (fig 2 a,b). treatment root length (cm) shoot length (cm) treatment root length (cm) shoot length (cm) group 1 group 4 control 10.8 abc 17.5 abc control 6.6 abc 12.7 abcd crb 42 9.6 abc 12.1 abc crb 64 9.2 cdef 18.9 d crb 55 10.1 abc 13.1 abc crb 85 6.1 ab 12.8 abcd crb 107 11.7 abc 15.4 abc crb 71 10.2 ef 18.8 d crb 88 11.4 abc 19.4 abc crb 3 9.3 cdef 18.0 cd group 2 crb 4 10.6 f 15.0 abcd control 10.9 ab 10.1 a crb 11 2 8.9 cdef 19.1 d crb 34 12.4 abc 16.2 b crb 90 8.7 bcdef 13.8 abcd crb 111 11.9 ab 14.4 ab crb 25 7.5 abcde 11.2 ab crb 19 14.2 bcd 16.9 b crb 104 5.7 a 10.4 a crb 24 18.7 d 15.6 b crb 76 8.0 abcdef 13.9 abcd crb 115 7.9 a 13.1 ab crb 113 8.7 bcdef 13.4 abcd crb 47 17.8 cd 14.7 b crb 96 7.6 abcde 12.9 abcd crb 77 9.3 ab 10.1 a crb 33 8.7 bcdef 12.0 abc crb 58 10.4 ab 12.6 ab crb 36 8.1 abcdef 15.7 abcd crb 10 12.3 abc 16.9 b crb 69 8.8 bcdef 17.4 bcd group 3 crb 63 8.2 abcdef 16.9 bcd control 11.3 a 10.2 a crb 40 9.1 cdef 17.9 cd crb 23 14.0 a 16.6 b crb 92 7.1 abcd 18.6 cd crb 98 13.0 a 16.5 b crb 100 9.6 a 9.8 a table 2 effect of pseudomonas sp. isolates in some growth parameters of maize table 3 optical density values of bacillus sp. isolates in osmotic pressure of 0, -0.73, -1, -1.5 at λ 570 nmand mpa numbers within a column followed by the same letter are not significantly different at 5% level by. dmrt (α = 0.05). bold indicated growth promoter isolates compared with control. bold of figures and letters indicated drought tolerant isolates. mpa: mega pascal isolates code osmotic pressure 0 mpa -0.73 mpa -1 mpa -1.5 mpa cr 33 1.514 0.504 0.400 0.071 cr 61 1.273 0.400 0.290 0.000 cr 69 1.011 0.681 0.271 0.000 cr 36 1.425 0.471 0.412 0.064 cr 83 1.073 0.758 0.464 0.017 cr 39 1.118 0.763 0.483 0.056 cr 51 1.317 0.496 0.138 0.000 cr 46 1.056 0.547 0.476 0.022 cr 67 0.95 0.503 0.432 0.033 cr 31 1.069 0.698 0.329 0.000 cr 90 1.063 0.682 0.559 0.028 cr 32 1.11 0.763 0.326 0.000 98 putrie et al. microbiol indones might be caused by their iaa concentration is too high, for example isolate cr 55 producing iaa up to 44.66 ppm (wahyudi et al. 2011a). the highest of iaa concentration was known to stimulate the growth of maize using biological fertilizer within a range 54.55 ppm in leaves and 22.68 ppm in roots, respectively. leaf tissue contains a higher iaa concentration rather than root tissue (wibowo 2008). high levels of iaa hormone would promote formation of ethylene and pseudomonas sp. have been capability to increase plant height by producing iaa hormones (patten and glick 2002; leveau and lindow 2005; wahyudi et al. 2011a,b). rhizobacteria as growth promoter in this research previously also had been tested in producing iaa hormone within a range 2.82-22.79 ppm (wahyudi et al. 2011a,b). other isolates, 23 strains of bacillus and 25 strains of pseudomonas did not have ability as growth promotion of maize sprouts. this isolates code osmotic pressure 0 mpa 0.73 mpa 1 mpa 1.5 mpa 2 mpa 2.5 mpa crb 4 1.742 1.362 1.254 0.966 0.792 0.490 crb 10 1.084 0.773 0.727 0.843 0.725 0.173 crb 19 1.789 1.058 1.017 1.086 0.872 0.563 crb 23 1.719 1.654 1.331 0.506 0.431 0.000 crb 47 1.709 1.249 1.151 0.771 0.630 0.000 crb 98 1.713 1.092 1.042 1.015 0.831 0.664 table 4 od values of pseudomonas sp. isolates in osmotic pressure of 0 mpa to -2.5 mpa at λ 570 nm table 5 inhibition zone formation by bacillus sp. (cr) as target bacteria against pseudomonas sp. (crb) as test bacteria table 6 inhibition zone formation by pseudomonas sp. (crb) as target bacteria against bacillus sp. (cr) as test bacteria od value ≥ 0.4 indicated drought tolerant isolates. mpa: mega pascal target isolates test isolates crb 98 crb 23 crb 10 crb 47 crb 4 crb 19 cr 33 cr 39 + + cr 83 + cr 67 cr 90 + + cr 36 + + + + + cr 46 target isolates test isolates cr 33 cr 39 cr 83 cr 67 cr 90 cr 36 cr 46 crb 98 + + + + crb 23 + + crb 10 + crb 47 + crb 4 + crb 19 + + volume 7, 2013 microbiol indones 99 cr : bacillus sp. (+) : no clear zone crb : pseudomonas sp. ( ) : no clear zone-information : cr : bacillus sp. (+) : no clear zone crb : pseudomonas sp. ( ) : no clear zone-fig 1 eps concentration produced by bacillus sp. cr (a) and pseudomonas sp. crb (b) at different osmotic pressure. table 7 identification of 16s rrna gene sequence homology of isolates of bacillus sp. cr with sequences available in genbank using blastn program isolates species most related sequence similarity length (bp) query cover e-value accession number cr 46 bacillus isabeliae strain cvs-8 83% 846/1018 82% 0.00 nr 042619.1 cr 67 brevibacillus brevis strain nbrc 15304 100% 659/659 100% 0.00 nr 041524.1 cr 83 bacillus cereus atcc 14579 strain atcc 14579 100% 566/566 100% 0.00 nr 074540.1 cr 90 brevibacillus brevis strain bb33 97% 1200/1243 92% 0.00 jf772474.1 osmotic presure (mpa) e p s c o n c e n tr a ti o n -1 (m g m l ) b 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 -0.73 -1 -1.5 -2 -2.5 crb 98 crb 23 crb 10 crb 47 crb 4 crb 19 e p s c o n c e n tr a ti o n -1 (m g m l ) 100 putrie et al. microbiol indones were the most sensitive organ to fluctuations of iaa levels and its response of increase exogenous iaa widespread in the number of primary root elongation, stop the growth. bacteria pgpr had ability in producing iaa and insert that into pool auxin hormone of plant. plant roots isolates species most related sequence similarity length (bp) query cover e-value accession number crb 10 pseudomonas aeruginosa strain l1 99% 434/440 94% 0.00 jx292018.1 crb 19 pseudomonas aeruginosa strain b2 91 % 966/1062 98 % 0.00 jq900536.1 crb 23 pseudomonas fragi strain atcc 4973 84% 657/780 83% 0.00 nr 024946.1 crb 98 pseudomonas sp. cl 3.1 97% 1297/1334 99% 0.00 fm173664.1 table 8 identification of 16s rrna gene sequence homology isolates of pseudomonas sp. crb with sequences available in genbank using blastn program fig 2 phylogenetic tree based on 16s rrna gene of bacillus sp. (a) and pseudomonas sp. (b) compared with 16s rrna gene of other species. scale showed that distance evolution on the branch length, while the numbers on the branches indicate bootstrap values. nr 027552.1| bacillus subtilis subsp. subtilis strain dsm 10 nr 074977.1| bacillus pumilus safr-032 strain safr-032 nr 074923.1| bacillus licheniformis dsm 13 atcc 14580 strain atcc 14580 dsm 13 cr 83 nr 074540.1| bacillus cereus atcc 14579 strain atcc 14579 nr 042619.1 bacillus isabeliae strain : cvs-8 cr 67 nr 041524.1 brevibacillus brevis strain nbrc 15304 nr 040979.1| brevibacillus formosus strain dsm 9885 jf772474.1| brevibacillus brevis strain bb33 cr 46 cr 90 100 93 87 58 99 99 92 0.1 a nr 024946.1| pseudomonas fragi strain atcc 4973 nr 044974.1| pseudomonas chlororaphis subsp. chlororaphis strain dsm 50083t nr 041715.1| pseudomonas stutzeri atcc 17588 lmg 11199 strain atcc 17588 crb 19 jq900536.1| pseudomonas aeruginosa strain b2 jx292018.1| pseudomonas aeruginosa strain l1 eu833948.1| pseudomonas putida strain fwc30 fm173667.1| pseudomonas sp. cl3.5 isolate cl3.59 crb 98 fm173664.1| pseudomonas sp. cl3.1 isolate cl3.13 crb 10 crb 23 93 77 93 52 100 82 78 74 88 0.1 b volume 7, 2013 microbiol indones 101 of eps linearly with stress experienced by cells as a form of physiological adaptation so that cells could survive. therefore, the capability in producing eps by bacterial cells used as drought tolerance criteria for bacteria (sandhya et al. 2009). production of eps would increased by cells during stress experienced as a form of physiological adaptation so that cells could survive. eps were produced to protect bacterial cells from drought, heavy metals or other environmental stresses, including host immune response and to produce a biofilm which could increase survival cell in specific ecological niches (ozturk and aslim 2010). quantity and composition of eps vary greatly depending on genus and species of bacteria, in some cases dependent on environmental conditions for growth. in addition, the carbon sources in medium was functioning as a component of cell formation, source of energy required for synthesis and eps excretion (santi 2011). b. subtilis could produce levan exopolysaccharide -1 within a range 0.32-86.3 g l using sucrose as substrate. a total of 16 genes of operon eps (yvekyvff) involved in the biosynthesis, modification and expenditure. recently, two genes epsg (yveq) and epsh (yver) had been identified that might be involved in the biosynthesis. epsg encodes a protein that might be involved in eps polymerization and epsh encodes a glycosyltransferase (marvasi et al. 2010). species of pseudomonas has been known for their capability producing eps in alginate type. approximately 24 genes in p. aeruginosa were identified. cluster consisted of 12 structural genes (algd, alg8, alg44, algk, alge, algg, algx, algl, algl, algj, algf, and algae) were clustered in a single operon approximately 3.96 mb. only algc genes located on separate chromosomes. those gene encodes for phosphomannomutase involved in rhamnolipid and lipopolysaccharide biosynthesis. operon was containing all the genes that encode proteins involved in the biosynthesis of alginate (algd and algae for precursor synthesis, algl, algj, and algf for acetylation, algg to epimerisasi, algl for degradation) (hay et al. 2010). the potent isolates crb 19 and cr 90 exhibited the highest activity of eps secretion and based on 16s rrna analysis revealed crb 19 and cr 90 belonged to p. aeruginosa strain b2 and brevibacillus brevis b33, respectively other studies showed that those group was known as promoting growth in plants. p. aeruginosa fp6 isolated from rhizosphere had been known for their capability as phosphate solubilizing, producer of iaa hormone, ammonia, sidherophore, and cells wall . lateral root formation and adventitious roots until termination of growth on the plant (leveau and lindow 2005). patten and glick (2002) also stated that the high of exogenous iaa would increase ethylene production which would effect to inhibition of roots growth. other mechanism that had capable to support as plant promoting growth by cr and crb are their ability in phosphate solubilizing which one of the essential nutrients for plants. phosphate in soils were present in bound form. phosphate bound with 3+ 3+ 2+ aluminium (al ), iron (fe ), calcium (ca ), and 2+ magnesium (mg ). thus, only it a small fraction which could be absorbed by plants (trivedi and pandey, 2007), pgpr had been ability to solubilyze bound phosphate so that could be absorbed by plants. screening of drought tolerant aimed to determine of isolates which could survive on drought condition so their can survive when applied in the field. the addition of peg 6000 on medium which bound water molecule were simulated as drought stress so that could reduce of potential water value. consequently, reduction of potential water value by the high of peg concentration on medium would cause decreasing survival of bacterial population. optical density values linearly related to water potential contained on medium. reduction of water potential values would decrease number of bacterial population. therefore, peg 6000 which soluble in water used to reduce of water potential value. water potential associated with level of drought would affect to capability survive of bacteria. isolates that had cappability to grow on medium with a certain of potential water value and od value ≥ 0.4 classified as drought tolerant isolates (alikhani and mohamadi 2010). majority, the result of antagonism test for plant promoting growth and drought tolerant isolates showed the same result for isolate cr and crb were used as the test culture and targets culture, respectively. formation of clearing zone around paper disc occured by production of certain compounds by bacteria test that inhibited growth of bacterial target or while back of test. isolates with different results founded in isolates cr 33 which wasn’t antagonis to crb 98 when cr 33 was used as target isolates and isolate cr 83 wasn’t antagonis to crb 19 when cr 83 as a target. this result might be caused by differences substance when isolates were used as targets and test isolates. when isolate used as targets, the substances were intact cells while tested back were used supernatant. tolerances to the drought stress were characterized by production of eps. this indicates that production 102 putrie et al. microbiol indones biotechnol. 85(6):752-759. husen e, wahyudi at, suwanto a, giyanto. 2011. growth enhancement and disease reduction of soybean by 1aminocyclopropane-1-carboxylate deaminase-producing pseudomonas. am j appl sci. 8(11):1073-1080. kaci y, heyraud a, barakat m, heulin t. 2005. isolation and identification of an eps-producing rhizobium strain from arid soil (algeria) : characterization of its eps and the effect of inoculation on wheat rhizosphere soil structure. res microbiol. 156(4):522-531. leveau jhj, lindow se. 2005. utilization of the plant hormone indole-3-acetic acid for growth by pseudomonas putida strain 1290. appl environ microbiol. 71(5):2365-2371. doi :10.1128/aem.71.5.2 365-2371.2005. marvasi m, visscher pt, martinez lc. 2010. exopolymeric substances (eps) from bacillus subtilis : polymers and genes encoding their synthesis. fems microbiol lett. 313(1):1-9. doi:10.1111/j.1574-6968.2010.02085.x. michel be, kauffman mr. 1973. the osmotic potential of polyethylene glycol 6000. plant physiol. 51(5):914916. marchesi jr, sato t, weightman aj, martin ta, fry jc, et al. 1998. design and evaluation of useful bacterium specific pcr primers that amplify genes coding for bacterial 16s-rrna. appl environ microbiol. 64(2):795-799. ozturk s, aslim b. 2010. modification of exopolysaccharide composition and production by three cyanobacterial isolates under salt stress. environ sci pollut res. 17(3):595-602. patten cl, glick br. 2002. role of pseudomonas putida indoleacetic acid in development of the host plant root system. appl environ microbiol. 68(8):3795-3801. doi:10.1128/aem.68.8.3795-3801.2002. trivedi p, pandey a. 2007. low temperature phosphate solubilization and plant growth promotion by psychrotrophic bacteria, isolated from indian himalayan region. microbiol. 2(5):454-461. sandhya, ali skz, minakshi g, gopal r, venkateswarlu. 2009. alleviation of drought stress effects in sunflower seedlings by the exopolysaccharides producing pseudomonas putida strain gap-p45. biol fertil soils. 46(1):17-26. santi lp. 2011. the role of exopolysaccharide producing bacteria in aggregation of a sandy soil texture [dissertation]. bogor (id) : institut pertanian bogor. somasegaran p, hoben hj. 1985. methods in legumerhizobium technology. hawaii:department of agronomy and soil science, university of hawaii. wahyudi at, astuti rp, widyawati a, meryandini a, nawangsih aa. 2011a. characterization of bacillus sp. strains isolated from rhizosphere of soybean plants for their use as potential plant growth for promoting rhizobacteria. j microbiol antimicrob. 3(2):34-40. wahyudi at, astuti ri, giyanto. 2011b. screening of pseudomonas sp. isolated from rhizosphere of soybean doi:10.1002/jctb.2372. doi:10.1002/ jctb.2372. doi:10.1007/s00374-009-0401-z. degrading enzyme such as cellulase, chitinase, and protease. inoculation of cowpea (vigna unguiculata) seeds by those bacteria given significantly effect (p<0.05) for enhanced seed germination (92%), seedling vigor index, plant height, and also fresh and dry weight in compared with control (bhakthavatchalu et al. 2013). b. brevis strain ipc11 also known could increase seed germination, producing phenylalanine ammonia lyase enzyme and reduced cancer disease in tomato (girish and umesha 2005). finally, the potent isolates of bacillus sp. cr and pseudomonas sp. crb have capability to live in drought condition and could be developed as inoculants in dry land agriculture when it put in medium carrier as biofertilizer. acknowledgments the research was supported by i-mhere b2c. institut pertanian bogor (ipb) and a grant from the collaborative research project of kkp3n, department of agriculture, indonesia to atw. therefore, authors thank and appreciate for all the supports given to carry out this research. references alikhani ha, mohamadi l. 2010. assesing tolerance of rhizobial lentil symbiosis isolates to salinity and th drought in dry land farming condition. 19 world congress of soil science, soil solutions for changing world. australia, 1-6 august 2010. bhakthavatchalu s, shivakumar s, sullia sb. 2013. characterization of multiple plant growth promotion traits of pseudomonas aeruginosa fp6, a potential stress tolerant biocontrol agent. ann biol res. 4(2):214-223. dey, pal kk, bhatt dm, chauhan sm. 2004. growth promotion and yield enhancement of peanut (arachis hypogaea l.) by application of plant growth promoting rhizobacteria. microbiol res. 159(4):371-394. doi: 10.1016/j.micres.2004.08.004. donot, fontana, baccoua, 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cultivated crops in response to the application of biofertilizer [thesis]. bogor (id) : institut pertanian bogor. 104 putrie et al. microbiol indones guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can be used as long as there is consent from its authors. all pages, including the pages with figures and tables at the end of the paper, must be numbered consecutively. research paper may occupy up to 4500 words, some figures and tables; or 15 pages maximum. reviews, written as continuous 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carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 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microbiology.indonesia@gmail.com formulir berlangganan (subscription form) microbiology indonesia nama (name) institusi (institution) alamat pengiriman (address) phone/fax/email pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) ) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com guide for author (14) 1.mi692-neti yuliana available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.1.1issn 1978-3477, eissn 2087-8575 vol 7, no 1, march 2013, p 1-8 *corresponding author; phone/fax: +62-721-781498, email: yuliana_thp@unila.ac.id orange fleshed sweet potatoes have important nutritional values because of high concentration of β carotene that functions as provitamin a. the carotenoid content of orange-fleshed sweet potatoes has been reported around 18 mg/100 g fresh weight (panda et al. 2007). this potential character of orangefleshed sweet potatoes should be responded by many food manufacturers to produce a healthy food. an alternative to diversify the sweet potato utilization is to use it as a raw material for pickle processing. most pickled vegetables are processed through spontaneous fermentation without inoculum addition. for this reason, the result of the process is often very unpredictable due to the diversities of the initial microflora. this has caused inconsistency in the quality of the pickle, off-flavoring, and the growth of different types of microorganisms. fresh vegetables may contain various wild microflora, dominated by non lactic acid bacteria responsible for spoilage (fleming 1982). an alternative to control the fermentation process is by adding lactic acid bacteria (lab) cultures during pickle processing. lab could rapidly produce lactate and other organic acids, and accumulate them in the raw materials. furthermore, they can also produce various metabolites that contribute to the development of some desirable characteristics such as visual appearance, texture, shelf-life, acceptability, and safety (holzapfel 1997; holzapfel 2002; leroy and vuyst 2004; dalie et al. 2010 ). some researchers have shown potential benefits of using starter cultures in fermentation, for example in the productions of low-salt sauerkraut, sauerkraut, in this study, fermentation process was carried out on orange-fleshed sweet potato cubes to produce sweet o potato pickle using a mixed culture of lactobacillus plantarum and leuconostoc mesenteroides at 30 c over 12 d period. spontaneous fermentation was also performed as a control. samples were withdrawn at various time intervals for analyses of reducing sugar content, total number of lactic acid and non-lactic acid bacteria, lactic acid concentration, ph, and sensory attributes. the results showed that using a mixed culture of l. plantarum and l. mesenteroides could greatly reduce contamination of non-lactic acid bacteria, retaining low amount of reducing sugar, rapidly producing lactic acid and consequently decreasing ph value of the pickle, as well as giving better sensory score. after 12 d of fermentation, sample of pickle inoculated with mixed culture showed -1 the following characters: total lactic acid content 0.5%, total lactic acid bacteria 8.46 log cfu ml , total non-10 -1 -1 lactic acid bacteria 1 log cfu ml , total reducing sugar 0.84 g l , and hedonic 10 sensory score for both taste and aroma 4 (like) in a scale of 5. these results indicated the potential ability of the mixed culture of lactic acid bacteria to improve the quality of the pickle fermented spontaneously. -1 -1 -1 0,84 g l tekstur 64,92 mm 50 g s , -1 -1 texture 64.92 mm 50 g s , key words: fermentation, lactic acid bacteria, mixed-starter cultures, pickle, sweet potato pada penelitian ini, potongan ubi jalar kuning difermentasi untuk memproduksi pikel menggunakan kultur o campuran lactobacillus plantarum dan leuconostoc mesenteroides pada 30 c selama 12 hari. fermentasi spontan juga dilakukan sebagai kontrol. parameter yang diamati meliputi gula reduksi akhir, total bakteri asam laktat (bal), total non-bal, konsentrasi asam laktat, ph, tekstur, dan sensori. hasil penelitian menunjukkan bahwa pengunaan kultur campuran l. plantarum dan l. mesenteroides dapat menekan pertumbuhan kontaminan non-bal, menyisakan sejumlah kecil gula reduksi, menghasilkan asam laktat dengan cepat sehingga menurunkan ph secara cepat, serta menghasilkan sensori yang lebih baik. karakteristik pikel setelah 12 -1 hari fermentasi adalah sebagai berikut: total asam laktat 0,5%, total bal 8,46 log cfu ml , total non-bal 1 10 -1 log cfu ml , total gula pereduksi sisa , dan nilai sensori untuk rasa dan 10 aroma masing masing adalah 4 (suka) dari 5 skala, untuk pikel dengan perlakuan kultur campuran. hasil ini menunjukkan bahwa penggunaan kultur campuran bal pada fermentasi pikel ubi jalar dapat memperbaiki kualitas pikel yang difermentasi secara spontan. kata kunci: bakteri asam laktat, fermentasi, kultur campuran pikel, ubi jalar the effect of a mixed-starter culture of lactic acid bacteria on the characteristics of pickled orange-fleshed sweet potato l.) (ipomoea batatas neti yuliana*, siti nurdjanah, and mika margareta department of agricultural product technology (thp), agriculture faculty, universitas lampung, jalan sumantri brojonegoro no 1, bandar lampung 35145, indonesia sauerkraut juice, and sausages (tolonen et al. 2002; wiander and ryhanen 2005; johanningsmeier et al. 2007; rai et al. 2006; hu et al. 2007). materials and methods preparation of starter culture. pure cultures of l. plantarum fncc 0123 and l. mesenteroides fncc 0023 were purchased from food and nutrition inter university center (pau) universitas gadjah mada. the starter was prepared by inoculating one loop of pure cultures of l. mesenteroides fncc 0023 and l. plantarum fncc 0123 into tubes containing 10 ml of sterile mrs broth and incubated at 37 °c for 24 h. brine solution preparation. brine solution was prepared by mixing 15 g nacl with 2.5 g sucrose, and then dissolved in 250 ml distilled water. the brine solution were heated using microwave oven (sharp) at high level setting for 6 min to achieve 60 °c , and then left for 7 min in a container containing warm water (3540 °c). this solution was distributed into 150 ml of fermenting containers. research on the production of pickled sweet potatoes has not been done as intensively as on pickled cucumber. several groups have reported the fermentation of pickled sweet potatoes without inoculation with a mixed culture (panda et al. 2007; panda et al. 2009; sivakumar et al. 2010; yuliana and nurdjanah 2009; and yuliana et al. 2010). so far there is no report mentioning the effect of inoculation with bacterial cultures commonly used for pickle processing, such as leuconostoc mesenteroides and lactobacillus plantarum, on the characteristics of pickled sweet potatoes. in this study, we compared the effects of adding l. mesenteroides and l. plantarum cultures on pickled sweet potatoes to pickle that had been processed spontaneously (without culture addition), to determine whether the addition of these starter cultures might have given positive effects on the fermentation. one ml of these cultures was then sub cultured twice (using pour plate and streak plate methods) in mrs agar at 37 °c for 24 h. single colony was picked up and inoculated twice into a centrifuge tube containing 10 ml mrs broth and incubated for 24 h 37 °c for the first and 48 h 37 °c for the second incubation. finally, the broth of each culture of l. plantarum and l. mesenteroides were combined and harvested by centrifugation (iec centra cl2 centrifuge) at 349 x g for 5 min. the amount of cells in this preparation was 6 -1 approximately 2 x 10 cfu ml . 2 yuliana et al. microbiol indones sweet potato pickle processing. orange-fleshed sweet potato samples were obtained from pasar gintung, a local market at bandar lampung, province of lampung-indonesia. the sweet potatoes were 3 washed, peeled, and cut into cubes (1x1x1 cm ). the potato cubes (40 g) were put into a 150 ml-fermenting container, then it was made up to 150 ml volume with the brine solution. the fermenting containers containing the samples were pasteurized using a microwave oven (sharp) at high level setting for 10 min to reach temperature around 72-73 °c, and then they were left at room temperature to cool. each sample was inoculated with the mixed culture of l. 6 plantarum and l. mesenteroides at level of 2 x10 cfu -1 ml . in addition, fermenting container without inoculum addition was also prepared as a control. the fermentation was conducted at room temperature (30 °c) for 12 d. the experiment was designed in four replicates. the starch content was determined enzymatically at the beginning and the final day of fermentation. the sample was homogenized, gelatinized, and subjected to αamylase and amyloglucosidase (sigma-aldrich) digestion for 20 min at 55 °c then the enzyme activity was stopped by adding few drops of tri chloroacetic acid (tca) and centrifuged at 1398 x g (slightly modified from nurdjanah 2008). the glucose in the supernatant was quantified using phenol-sulfuric acid method (dubois et al. 1956). 30 °c for 48 h. lab was identified by the presence of clear zones around the colonies; otherwise they were identified as non-lactic acid bacteria. evaluation of the characteristics of the pickled sweet potatoes. evaluation on the physicochemical properties was performed on lactic acid by titration method (aoac 1980), reducing sugar using nelsonsomogyi method (somogyi 1952), starch, and texture. total starch content was calculated by multiplying the result by 0.90 (aoac 1980). texture was determined according to nurdjanah et al. (2007) using penetrometer (precision, scientific petroleum instruments) and reported as the depth of the sweet potato tissue penetrated by the cone probe when applied -1 -1 with 50 g loading force per second (mm 50 g s ). microbial growth during fermentation was evaluated by counting the total lactic acid bacteria (lab) and non-lactic acid bacteria (non-lab) using total plate count method. appropriate dilutions were placed on duplicate plates of mrs agar medium (oxoid) containing 0.1% (w/v) caco . the cultures 3 were then incubated at the sensory evaluation was conducted at day 12 of fermentation according to method by meilgaard et al. (2007) and performed by a sensory panel composed of the increase of cell numbers (represented in cfu -1 ml ) produced during 12 d fermentation with prior inoculation of l. plantarum and l. mesenteroides mixed culture is shown in fig 1a-1b. during 12 d of fermentation, the populations of these strains increased approximately 5 log cycles, while the number of lab in control increased only approximately 2 log cycles. the total number of lab in the spontaneous fermentation was lower than those in batch with mixed 18 panelists. a five-point hedonic scale, varying from dislike very much (score 1) to like very much (score 5) was used. each of 18 panelists evaluated all samples in randomized order of presentation and evaluated the pickle in relation to taste and aroma using the score sheet for hedonic testing. results inoculum throughout the 12 d of fermentation. the lactic acid was already observed at early stage of fermentation and rapidly increased to an amount of 0.3% (v/v) on day three of fermentation in batch with mixed inoculum. however, inspontaneous fermentation, rapid increase was observed on day six of fermentation (fig 2a). lactic acid content increased linearly with fermentation time from 0.03 to 0.27% and 0.007 to 0.52% at rate 0.06 and 0.11% for 12 d in spontaneous and mixed inoculum batch, respectively. the final lactic acid content of the pickle inoculated with mixed culture and spontaneous fermentation was 0.52 and 0.27%, respectively. the reducing sugar increased quadratically with fermentation time from 0.46 to 0.71 and 0.44 to 0.84 g -1 l in both spontaneous and mixed inoculum pickle, in contrast to the patterns observed on reducing sugar and starch utilization, during 12 d of fermentation the texture of the pickles seemed to alter respectively, during 12 d fermentation (fig 2b). volume 7, 2013 microbiol indones 3 fig 1 total lab (a) and non-lab (b) population during 12 d fermentation. -1 t o ta l l a b ( l o g c f u m l ) 1 0 10.0 9.0 8.0 7.0 6.0 4.0 3.0 1.0 0.0 5.0 2.0 0 3 6 9 12 fermentation lenght (d) a -1 t o ta l n o n -l a b ( l o g c f u m l ) 1 0 6.0 4.0 3.0 1.0 0.0 5.0 2.0 -1.0 0 3 6 9 12 fermentation lenght (d) b spontaneous mixed culture spontaneous mixed culture -1 and spontaneous fermentation ranged 50-60 mm 50 g -1 s , indicating that the texture of the pickle cube was slightly softer. the sensory evaluation showed that panelists prefered the pickle that had been inoculated withmixed culture. the scores for taste and aroma of the pickle inoculated with mixed culture were 3.9 and 4.2 (liked), while scores of those with spontaneous fermentation in similar pattern, whether inoculated with mixed culture or not (fig 3). the firmness of the pickled vegetable decreased quadratically with fermentation time, as indicated by the increasing depth the probe could penetrate the tissue, that is from 43.16 to 59.31 in -1 spontaneous fermentation and 43.71 to 64.92 mm 50 g -1 s in pickled that had been inoculated with mixed culture.the final texture of pickle with mixed culture microbiol indones4 yuliana et al. fig 2 total lactic acid (a) and reducing sugar (b) production during 12 d fermentation. table 1 the starch content of fresh and pickled sweet potato fresh sweet potato* 29.11±1.11 spontaneous 29.33±1.07 25.43±0.09 13.30 inocultaed with mixed culture 26.84±0.31 21.06±1.91 21.57 treatments starch content (%) efficiency (%)before fermentation after fermentation 0.70 0.60 0.40 0.50 0.30 0.20 0.10 0.00 t o ta l la ct ic a ci d ( % ) 0 3 6 9 12 fermentation lenght (d) 0.70 0.80 0.40 0.60 0.30 0.20 0.10 0.00 -1 r ed u ci n g s u g ar ( g l ) 1.00 0.90 0.50 0 3 6 9 12 fermentation lenght (d) a b spontaneous mixed culture spontaneous mixed culture *moisture content of fresh sweet potato was 81% fermentation was higher than those in the batch that was inoculated with mixed cultures (fig 1 b). the high population of lab at the initial stage of fermentation had an important role in inhibiting growth of contaminants even after fermentation had started (hu et al. 2007; rai et al. 2006). the different lab population densities could affect the amount of lactic acid produced. it has been found that the faster the production of lactic acid, the faster the ph decreased (tolonen et al. 2002; wiander and ryhanen 2005), thus the faster the completion of in order to control the fermentation, lab starter cultures have been used in the fermentations of sauerkraut, olives, cucumber, sausages, and other fermented products (fleming et al. 2001; hu et al. 2007; rai et al. 2006; zdolec et al. 2008). volume 7, 2013 microbiol indones 5 were 3.2 and 3.3 (neither liked nor disliked). the total lab, total non-lab, lactic acid content, and reducing sugar during fermentation, as well as the sensory of final product. throughout the 12 d of fermentation the total population of lab in the spontaneous fermentation was lower than those in batch inoculated with mixed culture. the higher population of lab in the pickle that had been inoculated with mixed culture greatly reduced the total contaminating non-lab. consequently, the population of total non-lab in the batch with spontaneous discussion the addition of lab culture significantly affected fig 3 change of texture during 12 d fermentation. fig 4 hedonic sensory score at day 12 fermentation of pickle with spontaneus fermentation and mixed culture inoculation. score 1= dislike very much, 2= dislike , 3 = moderate (neither like nor dislike), 4 =like, 5 =like very much. 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 s e n so re s c o re spontaneous mixed culture taste aroma 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 -1 -1 t ex tu re ( m m 5 0 g s ec ) 0 3 6 9 12 fermentation lenght (day) spontaneous mixed culture the addition of lab caused a rapid acid accumulation in the raw material, with the production of lactic and several organic acids as well as other various metabolites. furthermore, the presence of lab culture could suppress the total non-lab contaminants contributing to off-flavor of the pickle. consequently, a inoculating the pickle with mixed lab culture helped to develop good sensory properties on the aroma and the tastes. the benefits of lab culture addition have been reported by some authors. the mixed cultures containing l. mesenteroides and l. plantarum gave sauerkraut and sauerkraut juice the most desirable sensory characteristics (wiander and ryhanen 2005). no significant off-flavor was observed on salted cabbage inoculated with l. mesenteroides at the end of the fermentation, sauerkraut which had been inoculated with this strain was more uniformly fermented in comparisios to the one produced bys spontaneous fermentations (johanningsmeier et al. 2007). in conclusion, the results indicated the positive effect of a mixed culture of l. plantarum and l. mesenteroides on the safety and sensory quality (aroma, taste, and texture) of pickled orange-fleshed sweet potato. the addition of these lab cultures significantly suppressed the total of non-lab as contaminant that could contribute to the off-flavor of the pickle. this finding supported the previous similar studies on fruit and vegetable fermentation. references ampe f, brauma a. 1995. origin of enzymes involved in detoxification and root softening during cassava retting. world j microbiol biotechnol. 11(2):178-182. doi:10.1007/bf00704644. aoac. 1980. official methods of analysis of the association of official analytical chemist. arlington, virginia. binner s, jardine w, renard c, jarvis m. 2000. cell wall modifications during cooking of potatoes and sweet potatoes. j sci food and agric. 80(2):216-218. doi:10.1002/(sici)1097-0010(20000115)80:2<216::aidjsfa507>3.0.co;2-6. dalie dkd, deschamps am, richard-forget f. 2010. lactic acid bacteria potential for control of mould growth and mycotoxins a review. j food control. 21(4):370-380. doi:10.1016/j.foodcont.2009.07.011. dubois m, gilles ka, hamilton jk, rebers pa, smith f. 1956. colorimetric method for determination of sugars and related substances. anal chem. 28(3):350-356. doi:10.1021/ac60111a017. duvetter t, sila dn, van buggenhout s, jolie r, van loey a, hendrickx m. 2009. pectins in processed : fermentation. reducing sugar remained relatively lower in the culture inoculated batch than that in spontaneous batch throughout the fermentation. this was probably due to a faster growth rate of starter culture. lab produced amylase enzyme which rapidly degraded starch to simple sugars thatcould then be utilized for lactic acid production (petrova et al. 2013). therefore, rapid decrease of reducing sugar in substrate (fig 2b) was observed in the lab culture inoculated batch. the degradation of starch was indicated by the decrease of starch content in the pickled sweet potatoes after fermentation (table 1). amylase enzyme was reported to present naturally in sweet potatoes (takahata et al. 1995; hagenimana et al. 1994; krishnan et al. 2010) and produced by lab as well (reddy et al. 2008; petrova et al. 2013 ). this enzyme was able to rapidly degrade starch to a simple sugar such as glucose. after day 12, the texture of the culture inoculated and spontaneously fermented pickled potatoes were both slightly soft. it has been documented that the texture is influenced by the main components of plant cell wall polysaccharides, such as cellulose, pectin, and matrix polysaccharides that are composed of interconnected hemicelluloses bound to cellulose microfibrils (imam et al. 2005). this structural components give the cell wall its rigidity. it is assumed that enzymes produced by lab and non-lab during fermentation along with the enzymes present in the sweet potatoes were to be the major contributors to this cell wall degradation and caused the disruption of the texture of the sweet potatoes pickle. lactic acid bacteria and other microbes are able to produce depolymerizing enzymes that act on cell-wall components (marin-rodriguez et al. 2000; mokemiabeka et al. 2011) and on starch (petrova et al. 2013), thus destroying the integrity of the plant tissues. enzymatic tissue softening caused by microbial agents has been reported in pickled cucumber (maruvada and mcfeeters 2009) and other fruits and vegetables containing pectin (duvetter et al. 2009). however, the depolymerizing enzymes contributing to the softening of the pickle might have come from the sweet potatoes. it has been found that pectin esterase, pectin methyl esterase (pme), α amilase were natural enzymes present in sweet potatoes (sunmola and bukoye 2011; binner et al. 2000; hagenimana et al.1994; ikemiya and deobald 1966). however, the activity of pme could be inhibited by rapid ph reduction (turk 1989). this might be the rational reason why the slight softening was observed in both batch fermentations. and β microbiol indones6 yuliana et al. mokemiabeka s, dhellot j, kobawila sc, diakabana p, ntietie-loukombo rn, nyanga-koumou ag, louembe d. 2011. softening and mineral content of cassava (manihot esculenta crantz) leaves during the fermentation to produce ntoba mbodi. adv j food sci and technol. 3(6):418-423. nurdjanah s, susilawati, sabatini mr. 2007. prediksi kadar pati ubi kayu (manihot esculenta) pada berbagai umur panen menggunakan penetrometer. 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degradation-pathways and prevention. florida:crc press. p 563-597. johanningsmeier s, mcfeeters rf, fleming hp, thompson rl. 2007. effects of leuconostoc mesenteroides starter culture on fermentation of cabbage with reduced salt concentrations. j food sci. 72(5):m166-m172. doi:10.1111/j.1750-3841.2007.00372.x. leroy f, vuyst ld. 2004. lactic acid bacteria as functional starter cultures for the food fermentation industry. trends food sci tech. krishnan jg, padmaja g, moorthy sn, suja g, sajeev ms. 2010. effect of pre-soaking treatments on the nutritional profile and browning index of sweet potato and yam flours. innov food sci emerg technol. 11(2): 387-393. doi:10.1016/j.ifset.2010.01.010. marin-rodriguez mc, orchard j, seymour gb. 2002. pectate lyases, cell wall degradation and fruit softening. fruit development and ripening special issue. j exp bot. 53(377):2115-2119. doi:10.1093/jxb/erf089. maruvada r, mcfeeters rf. 2009. evaluation of enzymatic and non-enzymatic softening in low salt cucumber fermentations. int j food sci technol. 44(6): 1108–1117. doi: 10.1111/j.1365-2621.2009.01925.x meilgaard d, civile gv, carr bt. 2007. sensory evaluation th techniques (4 edition). florida:crc press. volume 7, 2013 microbiol indones 7 2003.09.004. 15(2):67-78. doi:10.1016/j.tifs. konsentrasi garam. [sensory properties of spontaneously fermented purple sweet potato pickle at several levels of salt concentrations]. j teknologi & industri hasil pertanian. 14(2):119-126. zdolec n, hadziosmanovic m, kozacinski l, cvrtila z, filipovic i, skrivanko m, leskovar k. 2008. microbial and physicochemical succession in fermented sausages produced with bacteriocinogenic culture of lactobacillus sakei and semi-purified bacteriocin mesenterocin. meat sci. 80(2):480-487. doi:10.1016/j.meatsci.2008.01.012. sauerkraut juice by using starters in combination with mineral salt with a low nacl content. eur food res technol. 220(2):191-195. doi:10.1007/s00217-0041080-5 yuliana n, nurdjanah s, oktarini zh. 2010. some biochemical and total lactic acid bacteria changes during natural fermentation of the purple sweet potatoes (ipomea batatas l) pickle. work paper. international seminar on horticulture to support food security, june 2010. bandar lampung (id): p b209b214. yuliana n, nurdjanah s. 2009. sensori pikel ubi jalar ungu yang difermentasi spontan pada beberapa tingkat microbiol indones8 yuliana et al. 5 roga.cdr page 1 page 2 page 3 404 not found issn 1978-3477, eissn 2087-8575 volume 10, number 3, september 2016 enhancing the removal of highly concentrated co through 2 synergism between microalgae consortium and nutrient ratio in photobioreactor screening of antibiofilm activity from marine bacteria against pathogenic bacteria cloning and heterologous expression of extracellular plantaricin f produced by lactobacillus plantarum s34 isolated from “bekasam” in lactococcus lactis molecular identification of endospore-forming rhizobacteria from organic cabbage farm potential as biocontrol against phytopathogen xanthomonas campestris application of response surface method in optimization of medium composition for xylanase production by bacillus halodurans cm1 in submerged fermentation astri rinanti and kania dewi alianda budhiriani rossati camesi, agustina lukito, diana elizabeth waturangi, and hwang jae kwan apon zaenal mustopa, hidayah murtiyaningsih, fatimah, and suharsono maya fitriana ilul fahmi, endang kusdiyantini, agung suprihadi, dyah wulandari, and anto budiharjo sara gustia wibowo, is helianti, ani suryani, and budiasih wahyuntari 79 87 95 107 112 issn 1978-3477, eissn 2087-8575 volume 10, number 3, september 2016 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000, 200 000,-5 institutional rate (institution or library) (idr) 240 000, 400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; 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dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi new-september-cover depan 0916 (2) 1.mi-dessy natalia available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.4.1issn 1978-3477, eissn 2087-8575 vol 10, no 4, december 2016, p 119-124 *corresponding author; phone: +62-22-2502103, email:dessy@chem.itb.ac.id hepatitis b virus infection can cause a chronic liver disease. it is estimated that 240 million people are chronically infected with hepatitis b and more than 786 000 people die every year due to complication of hepatitis b, including cirrhosis and liver cancer (world health organization c2015). in indonesia, there are about 28 million people infected with hepatitis b and 14 million people are potentially becoming chronic with 10% progress into hepatocellular carcinoma (kementerian kesehatan republik indonesia 2013). the most effective approach to control and prevent the spreading of hepatitis b is by vaccination (who 2010). in indonesia, hepatitis b vaccine has been integrated into national immunization programmes since 1997 (kementerian kesehatan republik indonesia 2013). therefore, in order to fulfill the need of hepatitis b antigen, here we report the production of local recombinant small hepatitis b surface antigen (shbsag) using hansenula polymorpha expression system. h. polymorpha expression system offers more advantages than non-methylotrophic yeast expression system, such as saccharomyces cerevisiae. h. polymorpha expression system has a strong and tightly-regulated alcohol oxidase (aox) inducible promoter, and a high-frequency of non-homologous recombination (kang and gelissen 2005). moreover, h. polymorpha does not hyperglycosylate protein, is able to grow in simple medium, and has thermotolerant property (kang and gelissen 2005; reinders et al. 1999). recombinant small hepatitis b surface antigen (shbsag) is used as a vaccine component to prevent hepatitis b virus infection. as an attempt to produce local recombinant shbsag, a pcr-amplified dna fragment encoding indonesia shbsag which belongs to b genotype and adw2 subtype was cloned into hansenula polymorpha expression vector phipx4 by using recombination method. the resulted phipx4-shbsag was integrated into the alcohol oxidase locus of h. polymorpha ncyc495 genome and the shbsag expression was regulated under the control of h. polymorpha aox promoter. h. polymorpha ncyc495 carrying the shbsag coding sequence was grown in mineral medium and methanol 0.5% (v/v) was added to induce the expression of recombinant shbsag. the expression of shbsag was detected by hbsag diagnostic kit test, elisa, and western blot analysis. key words: aox promoter, hansenula polymorpha, hepatitis b, shbsag antigen permukaan virus hepatitis b berukuran kecil (shbsag) rekombinan digunakan sebagai komponen vaksin untuk mencegah infeksi virus hepatitis b. sebagai upaya untuk memproduksi shbsag rekombinan lokal, fragmen dna amplifikasi pcr yang mengkode shbsag indonesia yang tergolong ke dalam genotipe b dan subtipe adw2 diklon ke dalam vektor ekspresi hansenula polymorpha phipx4 dengan menggunakan metode rekombinasi. plasmid rekombinan phipx4-shbsag terintegrasi ke dalam lokus alkohol oksidase (aox) dari genom h. polymorpha ncyc495 dan ekspresi shbsag diregulasi di bawah kendali promotor aox h. polymorpha. h. polymorpha ncyc495 yang membawa urutan kode shbsag ditumbuhkan di dalam medium mineral dan metanol 0,5% (v/v) ditambahkan untuk menginduksi ekspresi shbsag rekombinan. ekspresi shbsag dideteksi melalui uji kit diagnostik hbsag, elisa, dan western blot. kata kunci: promotor aox, hansenula polymorpha, hepatitis b, shbsag cloning and expression of small hepatitis b surface antigen (shbsag) in hansenula polymorpha 1 1 1 christian heryakusuma , fernita puspasari , ihsanawati , ernawati arifin 2,3 3 4 giri-rachman , marselina irasonia tan , ekaputra ramadhani , 4 1,3 neni nurainy , and dessy natalia * 1 biochemistry research group, faculty of mathematics and natural sciences, institut teknologi bandung, bandung 40132, indonesia; 2 genetics and molecular biotechnology research group, school of life sciences and technology, institut teknologi bandung, bandung 40132, indonesia; 3 physiology, animal development, and biomedical sciences research group, 4 school of life sciences and technology, institut teknologi bandung, bandung 40132, indonesia; 5 pt bio farma (persero), bandung 40161, indonesia this paper described cloning of shbsag coding sequence into h. polymorpha expression vector phipx4 and expression of the shbsag in h. polymorpha ncyc495. the shbsag expression was analysed with hbsag diagnostic kit test, elisa, and western blot. materials and methods construction of phipx4-shbsag. the shbsag coding sequence of hepatitis b virus was amplified by pcr method using ppiczα-a-hbsag (nurfitriani 2012) as a template and a set of primers sf (ctaaagtacaaaaacaagcttatggagaac acgcatcagg) and sr (gatcgatcctctagag tcgacttaaatgtatacccaaagac). the amplified shbsag dna fragment and phipx4 previously digested with hindiii/sali were ® homologously recombined according to cloneez pcr cloning kit procedure (genscript, usa). the resulted recombinant plasmid was designated as phipx4-shbsag. the nucleotide sequence of shbsag in phipx4-shbsag was determined by dideoxy-chain termination method (macrogen, korea). transformation of h. polymorpha ncyc495 and recombinant shbsag expression. h. polymorpha ncyc495 leu1.1 was transformed with recombinant plasmid phipx4-shbsag, which had been linearized with scai, according to the method described by faber et al. (faber et al. 1994). h. polymorpha ncyc495 transformants were grown on ynd medium, which composed of 0.67% (w/v) yeast nitrogen base without amino acids, 1% (w/v) dextrose, and 1.6% (w/v) bacto agar. pcr colony was performed using primers sf (ctaaagtacaaaaa caagcttatggagaacacgcatcagg) and sr (gatcgatcc tctagagtcgacttaaatgtata cccaagac) to verify plasmid integration into the h. polymorpha ncyc495 genome. the transformants were precultured in 5 ml mineral medium (van dijken et al. 1976) containing 0.25% (w/v) glucose and incubated with shaking at 200 rpm for 18 h at 37 °c. the culture was then centrifuged at 2,900 g and the whole pellet cell were inoculated in 50 ml fresh mineral medium containing 0.5% (v/v) methanol as the sole carbon source. the culture was incubated with shaking at 200 rpm, 37 °c. to induce recombinant shbsag expression, 0.5% (v/v) methanol was added to the culture medium every 22 h for 66 h. the yeast cells were harvested by centrifugation at 120 heryakusuma et al. microbiol indones 2900 g, 4 °c, and the cell pellet was resuspended in lysis buffer which contained 10 mm potassium phosphate ph 8, 500 mm nacl, 5 mm edta, 8% (v/v) glycerol, 1% (v/v) triton x-100, and 1% (v/v) -1 leupeptin 2.5 μg ml . g, the cells were lysed by manual grinding in liquid nitrogen. the protein was collected by centrifugation at 7200 g, 4 °c for 10 min. hbsag diagnostic kit test. recombinant shbsag was analysed with hbsag diagnostic kit test, uji hepatitis bsag (pakar biomedika indonesia, indonesia). the test was conducted using 50 μl protein samples. elisa. recombinant shbsag was examined with commercial elisa kit, murex hbsag version 3 (diasorin, italy). elisa was performed using 1 μg protein samples and 75 μl of elisa negative and positive control. all protein samples and controls were analysed in duplicate. the negative and positive controls were provided by the kit manufacturer. the negative control was a normal human serum diluted in a buffer containing protein of bovine origin and 0.05% (w/v) bronidox® preservative, while the positive control was an inactivated human serum diluted in a buffer containing protein of bovine origin and 0.05% (w/v) bronidox® preservative. the elisa threshold value was calculated according to the instruction of elisa kit manufacturer by adding a value of 0.050 to the a of 450 negative control provided in the elisa kit. western blot. 10 μg protein samples were first separated on a 12% sds-polyacrylamide gel electrophoresis. the proteins were then transferred onto nitrocellulose membrane and then blocked with roti block (carl roth, germany) for 2 h at room temperature. the shbsag protein was detected using monoclonal anti-hbsag (virostat, usa), goat antimouse antibody-alkaline phosphatase conjugate (biorad, usa), and visualized with nbt/bcip. purified recombinant shbsag, produced in-house by pt. bio farma (persero), was used as a positive control. results integration of linearized phipx4-shbsag into h. polymorpha ncyc495 genome. the shbsag coding sequence was subcloned into h. polymorpha expression vector phipx4 which has saccharomyces cerevisiae leu2 gene under its endogenous promoter. the resulted recombinant plasmid phipx4-shbsag (fig 1) was verified by restriction enzyme analysis using hindiii and sali (fig 2a). the digested phipx4shbsag gave two dna fragments with the size of 7.0 kb and 0.7 kb which represented the phipx4 vector and shbsag insert, respectively. the recombinant plasmid phipx4-shbsag was linearized in the promoter aox region to allow its integration in the aox locus of h. polymorpha ncyc495 genome which enabled the transformants to grow on minimal medium without leucine. pcr colony of h. polymorpha ncyc495 transformants gave a 0.7 kb dna fragment which confirmed the presence of shbsag coding sequence in the h. polymorpha ncyc495 genome (fig 2b). identification of recombinant shbsag. crude protein extracts from two h. polymorpha ncyc495 transformants (designated as s1 and s2) were evaluated using commercial hbsag diagnostic kit. the s1 and s2 crude protein extracts gave positive interaction with hbsag antibody, whereas the interaction was negative for crude protein extract from h. polymorpha ncyc495 (table 1). this result indicated that crude protein extracts of s1 and s2 contained recombinant shbsag. further identification of the expression of recombinant shbsag in s1 and s2 was conducted using elisa. the crude protein extracts from s1 and s2 gave the value of a that was higher than elisa 450 threshold value (fig 3a). in contrast, the crude protein extract from h. polymorpha ncyc495 had lower a 450 value than that of the threshold value (fig 3a). this elisa result suggested the presence of recombinant shbsag in s1 and s2. western blot analysis was performed to demonstrate volume 10, 2016 microbiol indones 121 1 2 3bp 8000 6000 750 a 1 2 3bp 750 b fig 1 phipx4-shbsag recombinant plasmid. the plasmid map was generated using snapgene® viewer 3.0.1. fig 2 restriction enzyme analysis of phipx4-shbsag recombinant plasmid with its uncut control (a) and colony pcr of h. polymorpha ncyc495-phipx4-shbsag (b). (a) lane 1, dna ladder; 2, phipx4-shbsag digested with hindiii and sali; 3, uncut phipx4-shbsag. (b) lane 1, dna ladder; 2-3, positive transformant. microbiol indones122 heryakusuma et al. subtype. to determine the immunogenicity of “a” determinant of recombinant shbsag, the shbsag sequence in the research was also examined for possibility of immune-escape mutant. the immuneescape mutant of hbv was known to have some rare substitution in amino acids residues in the “a” determinant region, such as g145r (purdy et al. 2007), q129r, and g145a (koyanagi et al. 2000). interestingly, one study also found a rare amino acid substitution y161s located outside the “a” determinant region was an immune-escape mutant (jinata et al. 2012). based on multiple amino acid sequence alignment in the “a” determinant region of shbsag, the sequence used in this research was not identified as an immune-escape mutant (fig 4). the recombinant plasmid phipx4-shbsag was linearized in the aox promoter region to facilitate its integration in the aox locus of h. polymorpha ncyc495 (saraya et al. 2012). several papers have reported that the integration of shbsag in the h. polymorpha genome by the use of autonomously replicating sequence (hars1) allowed higher integration frequency (diminsky et al. 1997; heijtink the expression of shbsag in h. polymorpha ncyc495 phipx4-shbsag. a protein band at molecular weight of 19.8 kda appeared in the crude protein extracts from s1 and s2, as well as in the purified shbsag (fig 3b). as expected, there was no protein band detected in the h. polymorpha ncyc495 crude protein. taken together, this result confirmed that s1 and s2 produced recombinant shbsag. the negative control of elisa is a normal human serum, while the positive control is an inactivated human serum. the positive control of western blot is purified recombinant shbsag from pt. bio farma (persero). discussion the indonesia shbsag sequence used in this work was derived from local clinical isolates of hasan sadikin hospital bandung in which the virus has b genotype and adw2 subtype (suhandono et al. 2007). amino acid sequence alignment of the “a” determinant region of several shbsags showed some amino acid variations due to differences in virus genotype and sample ncyc495 s1 s2 interaction – + + 3.20 2.80 2.40 2.00 1.60 a 4 5 0 0.40 0.00 ncyc495 s1 s2 + 19.80 kda ncyc 495 s1 s2 + b a theshold 0.301.903.120.23 3.06 table 1. hbsag diagnostic kit test of crude protein extracts fig 3 elisa (a) and western blot analysis (b) of crude protein extracts from h. polymorpha ncyc495-phipx4-shbsag (s1 and s2) and h. polymorpha ncyc495. all elisa value bar represent an average value of two repeated assay. volume 10, 2016 microbiol indones 123 dunker ak, kenyon aj. 1976. mobility of sodium dodecyl sulphate-protein complexes. biochem j. 153(2):191197. doi:10.1042/bj1530191. faber kn, haima p, harder w, veenhuis m, ab g. 1994. highly-efficient electrotransformation of the yeast hansenula polymorpha. curr genet. 25(4): 305-310. doi:10.1007/bf00351482. gasteiger e, gattiker a, hoogland c, ivanyi i, appel rd, bairoch a. 2003. expasy: the proteomics server for indepth protein knowledge and analysis. nucleic acids res. 31(13): 3784-3788. doi:10.1093/nar/gkg563. heijtink ra, van bergen p, melber k, janowicz za, osterhaus adme. 2002. hepatitis b surface antigen (hbsag) derived from yeast cells (hansenula polymorpha) used to establish an influence of antigenic subtype (adw2, adr, ayw3) in measuring the immune response after vaccination. vaccine. 20(1718):2191-2196. doi:10.1016/s0264-410x(02)00145-7. jinata c, giri-rachman ea, retnoningrum ds. 2012. molecular analysis of immune-escape mutants of hepatitis b virus from local clinical samples. microbiol indones. 6(1): 9-14. doi:10.5454/mi.6.1.2. kang ha, gellissen g. 2005. hansenula polymorpha. in: gelissen g, editor. production of recombinant proteins: novel microbial and eukaryotic expression systems. weinheim (de): wiley-vch verlag gmbh & co. kgaa. p. 111-142. kementerian kesehatan republik indonesia. c2013. jakarta (id): pusat data dan informasi kementerian kesehatan ri; [accessed 2015 feb 15, 2016 jan 12]. http://www.depkes.go.id/folder/view/01/structure-pub likasi-pusdatin-info-datin.html. koyanagi t, sakai h, sugimoto r, enjoji m, koto k, iwamoto h, kumazawa t, mukaide m, nawata h, nakamuta m. 2000. analysis of hbs antigen negative variant of hepatitis b virus: unique substitutions, 129 145 glu to asp and gly to ala in the surface antigen gene. med sci monit. 6(6):1165-1169. nurfitriani. 2012. construction of recombinant vector ppiczα-a-hbsag for expression of hepatitis b surface antigen (hbsag) extracellular in pichia pastoris [thesis]. bandung (id): institut teknologi bandung. ottone s, nguyen x, bazin j, berard c, jimenez s, letourneur o. 2007. expression of hepatitis b surface antigen major subtypes in pichia pastoris and purification for in vitro diagnosis. protein expres purif. 6(2):177-188. doi:10.1016/j.pep.2007.07.008. purdy ma. 2007. hepatitis b virus s gene escape mutants. asian j transfus sci. 1(2):62-70. doi:10.4103/0973et al. 2002; bian et al. 2009). the expression of shbsag in h. polymorpha ncyc495 transformants (s1 and s2) has been verified by diagnostic kit test and elisa. further analysis using western blot showed that the recombinant shbsag appeared as a protein band at 19.8 kda, which is similar with the purified shbsag used as a control (fig 3b). the predicted molecular weight of shbsag calculated using expasy (gasteiger et al. 2003) was 25.3 kda. this molecular weight difference could be due to incomplete reduction of disulfide bonds in recombinant shbsag (ottone et al. 2007). the presence of disulfide bonds will introduce a more compact shape of the proteins (dunker and kenyon 1976), hence faster migration rates of recombinant shbsag would be expected (rath et al. 2009). taken together, the h. polymorpha capable of expressing indonesian shbsag developed in this work is an alternative source of recombinant hbsag vaccine production. acknowledgment we thank ida van der klei from university of groningen for providing hansenula polymorpha strain and expression vector. this research was partially funded by ministry of research, technology and higher education for the national hepatitis b consortium. references bian g, cheng y, wang z, hu y, zhang x, wu m, chen z, shi b, sun s, shen y, chen ej, yao x, wen y, yuan z. 2009. whole recombinant hansenula polymorpha expressing hepatitis b virus surface antigen (yeasthbsag) induces potent hbsag-specific th1 and th2 immune responses. vaccine. 28(1):187-194. doi:10.10 16/j.vaccine.2009.09.101. diminsky d, schirmbeck r, reimann j, barenholz y. 1997. comparison between hepatitis b surface antigen (hbsag) particles derived from mammalian cells (cho) and yeast cells (hansenula polymorpha): composition, structure and immunogenicity. vaccine. 15(6/7): 637-647. doi:10.1016/s0264-410x(96)002393. fig 4 multiple amino acid sequence alignment in the “a” determinant region of shbsag. microbiol indones124 heryakusuma et al. saraya r, krikken am, kiel jakw, baerends rjs, veenhuis m, van der klei ij. 2012. novel genetic tools for hansenula polymorpha. fems yeast res. 12(3):271278. doi:10.1111/j.1567-1364.2011.00772.x. van dijken jp, otto r, harder w. 1976. growth of hansenula polymorpha in a methanol-limited chemostat:physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. arch microbiol. 111(1):137-144. doi:10.1007/bf00446560. world health organization. c2015. 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6.mi711-nanik rahmani available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.3.6issn 1978-3477, eissn 2087-8575 vol 7, no 3, september 2013, p 129-136 *corresponding author; phone/fax: 021-8765066/0218765062, email: rahmani_btk@yahoo.com starch is the most abundant storage polysaccharide in cereal and legume grains, many roots, and in tubers (vander maarel et al. 2002; belitz et al. 2004); the polysaccharide consists of the two glucose polymers amylose and amylopectin. the former is a linear α-(1-4) linked glucose chain with a plant-specific degree of polymerization of 200-6000; the latter consists of short linear α-(1-4) linked chains with α-(1-6) linked side chains (vander maarel et al. 2002). starch is hydrolyzed into smaller oligossaccharides by α-amylase, wich is one of the most important commercial enzyme processes (souza and pérolade 2010). oligosaccharides (os) have been commercialised since the 1980s as low-calorie bulking agents. the functional food concept was first introduced in japan. in 1991, several oligosaccharides were classified as “foods for specified health use” (foshu) in japan. recent research findings across the globe, have led to the inclusion of non-digestible oligosaccharides (ndos) under functional food. currently, there exist a 27 billion usd market for functional foods and experts forecast its 8.5-20% growth. the global market for functional foods was valued at 73.5 billion usd in 2005, whereas by 2013, it is expected to reach a value of 90.5 billion usd (patel and goyal 2011). in general, various kinds of oligosaccharides can be produced from starch as the raw material, such as maltooligosaccharides (maltose, maltotriose, maltotetraose, maltopentaose, and maltoheptaose), isomaltooligosaccharides (isomaltose, panose, and isomaltotriose), cyclodextrins (cds) (α-cd, β-cd, γ-cd, hp β-cd, and branched cds), maltitol, gentiooligosaccharides, trehalose, and nigerose. maltooligosaccharides are by definition glucose oligomeres consisting of 2 to 10 glucopyranosyl residues linked via α-1-4 bonds high quality maltooligosaccharides were produced from indigenous indonesian black potato starch by making use of an amylase from brevibacterium sp. optimal production was achieved at 2.5% (w/v) substrate concentration, an enzyme-substrate ratio of 1:5 (w/v) and hydrolysis time of 4 h. under such conditions the yield of reducing sugars was 14 240 ppm with a polymerization degree of 16. thin layer chromatography (tlc) revealed the formation of glucose, maltose, and maltotriose with rf values of 0.60, 0.52, and 0.37, respectively. hplc analysis of freeze-dried samples disclosed rf values of 0.60, 0.50, 0.37, and 0.12. maltooligosaccharide profile analysis both using tlc and hplc showed that the enzymatically hydrolyzed samples contained glucose, maltose, and maltotriose. thus, black potato starch can be randomly converted into simple sugars and maltooligosaccharides applying by amylolytic enzymes from the marine microbe brevibacterium sp. key words: black potatoes, brevibacterium sp., maltooligosaccharides maltooligosakarida dengan kualitas baik diproduksi dari pati kentang hitam asli indonesia menggunakan enzim amylase dari brevibacterium sp. kondisi terbaik hidrolisis pati kentang hitam telah diperoleh yaitu pada konsentrasi substrat pati kentang hitam 2.5% (b/v), perbandingan enzim-substrat 1:5 (b/v) serta waktu hidrolisis 4 jam. gula pereduksi yang dihasilkan pada kondisi tersebut sebesar 14 240 ppm dengan derajat polimerisasi 16. hasil analisis maltooligosakarida menggunakan kromatografi lapis tipis (klt) menunjukkan jenis maltooligosakarida yang terbentuk adalah glukosa, maltosa, dan maltotriosa dengan nilai rf berturut-turut 0.60, 0.52, dan 0.37. spot kromatogram hplc sampel hasil freeze-drying memiliki nilai rf 0.60, 0.50, 0.37, dan 0.12. analisis profil maltooligosakarida dengan klt dan hplc menunjukkan bahwa hidrolisat mengandung glukosa, maltosa, dan maltotriosa. munculnya gula-gula tersebut menunjukkan bahwa pati kentang hitam dapat terdegradasi menjadi gula-gula sederhana dan maltooligosakarida secara acak dengan menggunakan enzim amilase dari mikroba laut brevibacterium sp. kata kunci: brevibacterium sp., kentang hitam, maltooligosakarida production of maltooligosaccharides from black potato (coleus tuberosus) starch by α-amylase from a marine bacterium (brevibacterium sp.) 1 2 2 1 1 nanik rahmani *, rohanah , sukarno , ade andriani , and yopi 1 biocatalyst and fermentation laboratory, research center for biotechnology, indonesian institute of sciences, cibinong, indonesia; 2 department of food science and technology, faculty of agricultural engineering and technology, institut pertanian bogor, kampus darmaga, bogor 16820, indonesia (nakakui 2005). maltooligosaccharides are produced from starch commercially by the action of debranching enzymes such as the pullulanase (ec 3.2.1.41) or isoamylase (ec 3.2.1.68) and controlled hydrolysis by various αamylases (ec 3.2.1.1). alpha-amylases are enzymes that catalyses the hydrolysis of internal-1,4-glycosidic linkages in starch in low molecular weight products, such glucose, maltose, and maltotriose units (souza and pérolade 2010). depending on the organism from which they are produced the latter display diverse reaction specificities facilitating the production of syrups rich in maltooligosaccharides of different chain lengths (crittenden and playne 1996). in recent years there is a steadily growing interest to use maltooligosaccharides in the food industries: as biopreservatives, functional food, and as important components in a great number of other nutritional products (barreteau et al. 2006). they have been used as sweeteners, anti-hygroscopic or truncating agents or humectants (lee et al. 2003). various kinds of maltooligosaccharides-containing syrups (maltose ~ maltopentaose) having low sweetness impart resistance to retrogradation of starch gels and prevent sucrose crystallization; their rather low browning tendency is due to the improved heat stability. accordingly, they are useful for enhancing intrinsic properties of various foods, and can be applied as powdering materials, dry milk saccharides, in liquid diets, and for increasing viscosity in refreshing drinks (nakakuki 2005). maltooligosaccharides produced by α-amylases can reduce retrogradation which is of practical importance for bakery products (smits et al. 2003). amylases producing specific maltooligosaccharides have been reported from a number of microorganisms, such as bacillus circulans and b. subtilis. the maltopentaose-generating amylase of b. licheniformis is used for the production of maltotriose, maltotetraose, and maltopentaose syrups. research reagarding maltooligosaccharides produced by the amylase from a marine bacterium is relatively scarce. in this research maltooligosaccharides were produced by brevibacterium sp. isolated from pari island. previously, we have demonstrated that the bacterium is capable of secreting a starch hydrolizing enzyme withan activity of 2.5 u -1 ml which can potentially be used for maltooligosaccharides production (rahmani et al. 2011). the most popular tubers in indonesia comprise cassava and sweet potatoes but others, such as the black potatoes are locally planted as well. however, indonesian black potatoes are only rarely exploited and 130 rahmani et al. microbiol indones they are cultivated exclusively in rather small scale in java, bali, and the madura islands (heyne 1987). the use of indigenous indonesian black potato tubers as processed foodstuff is still rather limited due to the fact that enzymatic treatment of black potato starch is not thoroughly explored in indonesia. in this study we determined the optimal conditions for the enzymatic hydrolysis of starch from coleus tuberosus by enzymes from brevibacterium sp. and analyze the profiles of the generated maltooligosaccharide by thin layer chromatography (tlc) and high performance liquid chromatography (hplc). materials and methods extraction of black potato starch and physicochemical analysis. black potato tubers used in this study were provided by the cell and tissue culture laboratory, research center for biology, lipi, cibinong, bogor (fig 1a). the used variety 3.2 resulted from a tissue culture of a plant from the sangian area, east java. the starch was extracted through the stages of the process, such as stripping, washing, grater, extraction, filtration, precipitation, drying, and sieving (fig 1b). fresh black potato tuber were peeled and washed by using manually to clean tubers from soil and the other dirt. the tuber be shredded using grater machine and then starch extracted by added water with a ratio of material and water was 3.5 : 1. furthermore, the filtering was done to separate the starch from the residue. residue obtained from the screening process again extracted 5 times with the same ratio of the water addition and precipitation on night. after precipitation, the supernatant were removed until the only remaining part of the wet starch deposition. furthermore, drying starch obtained using the sun. starches continue were crushed with mortar and then continue to the sifting process to obtain a uniform particle size using a filter pore size of 50 mesh. finally, starch obtained from the varieties 3.2 were weighed and subsequently analyzed physico-chemically: moisture, protein, lipid, and ash contents of the isolated samples were determined using approved methods (aoac 1984). the amylose content was determined by the iodine blue complex method of sowbhagya and bhattacharya (1979) using a solution of 0.2% iodine in 2% potassium iodide. scanning electron microscopy (sem) analysis. the purity of the isolated starch was additionally checked by scanning electron microscopy (sem) according to the method described by tharanathan and ramadas bhat (1988). microorganism. amylase production was carried out using the brevibacterium sp. from the marine bacterium collection of the biocatalyst and fermentation laboratory, research center for biotechnology, lipi , cibinong bogor. crude enzyme production. production of the amylase was carried out by submerged fermentation. -1 the medium consisted of 38 g l artificial sea water (asw), 2% commercial starch (merck, darmstadt, -1 -1 germany), 1.5% agar, 1 g l yeast extract, and 5 g l peptone, ph 8. media were sterilized at 121°c for 15 min. fermentation was performed for 4 d at 150 rpm, 30 °c (stuart orbital incubator s1500, staffordshire, united kingdom). the crude amylase enzyme preparation was obtained as the culture supernatant by centrifugation (6 764 ×g, 15 min, 4 °c). subsequently, the supernatant was analyzed for the enzymatic activity at ph 6.6 in phosphate buffer (0.02 m) at 30 °c (rahmani et al. 2011). crude extracts amylase assay. the amylase activity was assayed according to bernfeld (1955) by incubating 0.5 ml of the enzyme solution with 0.5 ml of a starch solution (0.5% w/v) (merck, darmstadt, germany) prepared in phosphate buffer ph 6.6 (0.02 m) at 30 °c for 30 min. the reaction was stopped by immersing the test tubes in boiling water for 20 min and subsequent cooling on ice. color formation was measured in a spectrophotometer at λ 540 nm (hitachi, u-3900h, tokyo japan). one unit is defined as the production of 1 mm maltooligosaccharides per min under the above conditions. enzymatic hydrolysis conditions of black potato starch varieties 3.2. enzymatic hydrolysis was carried out under various conditions, such as diverse substrate concentrations (w/v) 1, 2.5, and 5%, enzymesubstrate ratio (v/v) 1:10, 1:5, 1:2, and 1:1, and the reaction time (hours from 1, 2, 4, 6, and 8). reactions were carried out in 100 ml erlenmeyer flasks containing 20 ml of reaction mixtures in a rotary shaker (stuart orbital incubator s1500, staffordshire, united kingdom) at room temperature. samples were taken at regular intervals (after 1, 2, 4, 6, and 8 h); reactions were stopped by heating the samples in boiling water. chemical analysis of maltooligosaccharides. product hydrolysis was analyzed by calculating the total sugar content, reducing sugars and the degree of polymerization by tlc and hplc. analysis of the total sugar content was performed by applying the phenol-sulfuric acid method with modifications described by dubois et al. (1956). reducing sugars were determined by the dns method (miller 1959). the degree of polymerization was calculated based on the ratio between total and reducing sugar. thin layer chromatography (tlc) of maltooligosaccharides products was carried out by the ascending method (three time development) on silica gel 60f plates 254 (merck art20-20cm, darmstadt, germany). all samples were applied in equal quantities (1 µl) and then resolved by two runs with a solvent mixture of nbutanol/aceticacid/water (12:6:6, by volume). spots were visualized by spraying the sugar color (0.5 g αdiphenylamine, 25 ml acetone, 2.5 ml phosphate acid, and 0.5 ml aniline) and subsequent heating at 100 °c for 15 min. maltooligosaccharide products were freeze dry and analyzed by high performance liquid chromatography (hplc) (lee et al. 2003; kandra et al. 2002) using the agilent system (agilent technology 1290 infinity, united state). the column used was zorbax sil column (silica) coated with 3-amino propilsilen and the mobile phase was acetonitrile and distilled water in a ratio of 75:25 (v/v). the temperature -1 was kept at 30 °c with a flow rate of 1.4 ml min and a sample volume of 20 µl. the effluent from the column was monitored with a refractive index detector (rid). results optimization of enzymatic hydrolysis conditions of black potato starch varieties 3.2. the yield of starch from black potato was 18.73 % on a grain dry matter basis. the relatively low yield could be attributed to losses occurring during the repeated washing needed for the starch extraction process. the moisture content of the isolated starch was 10.13%. the lipid and protein contents of the starch were 0.81% and 0.51%, respectively, indicating that the isolated starch was quite pure (table 1). indeed, the purity of the isolated starch was confirmed by sem micrographs at 2500x magnifications showing integrity of the starch granules (fig 2). the amylopectin content of the black potato starch was 67.69%, which agrees with the observed maltooligosaccharide production. maltooligosacharides formation by enzymatic hydrolysis. production of maltooligosaccharides was carried out by making use of the amylase from brevibacterium sp. that was isolated from pari island. optimization of hydrolysis conditions for maltoligosaccharides from starch is a promising method. to determine the most suitable conditions, enzyme reactions were carried out using the same amount of -1 enzyme (2.5 u ml ) in various substrate concentrations ranging from 1, 2.5 to 5% as starch concentrations volume 7, 2013 microbiol indones 131 microbiol indones132 rahmani et al. fig 2 scanning electron micrographs of the starch granules of variety 3.2 at 2500 times magnifications. table 1 yield and chemical composition of isolated black potato starch varieties 3.2 yield (%) moisture protein lipid ash total carbohydrates (%) viscosity (cp) amylose content (%) amylopectin content (%) 18.73 10.34 0.81 0.50 0.44 83.87 14.2 32.31 67.69 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 2 4 6 8 r ed u ci n g s u g ar co n ce n tr at io n ( p p m ) time (h) fig 3 analysis of reducing sugar content of black potato starch varieties 3.2 were hydrolyzed by amylase enzyme of brevibacterium sp. 10 ml reaction volume consisting of starch and enzyme substrate with a ratio of 1:1 with enzyme -1 activity 2.5 u ml . the reaction consists of 3 variations of black potato starch concentration were 1% ( ), 2.5% ( ), -1 and 5% ( ) in phosphate buffer ph 6.6, the volume of 1 ml enzyme (2.5 u ml ), 30 °c. fig 1 (a) black potato tubers of the tissue culture coleus tuberosus variety 3.2 and (b) the respective starch powder. a b 8 h at the substrate concentration of 1, 2.5, and 5%, reducing sugars were 490, 11 435, and 15 570 ppm, respectively. though the amount of reducing sugars were highest at 5% starch, determination the of the optimal enzyme substrate ratio by tlc was performed at 2.5% starch (fig 4) as maltooligosaccharides were volume 7, 2013 microbiol indones 133 exceeding 5% resulted in jelly like solutions. the formation of reducing sugars were assayed to monitor the hydrolysis of starch by the amylase; with respect to the starch concentration different amounts of reducing sugar were produced (fig 3). the reducing sugar concentration increased from 1 to 5%, for example after a 1 2 3 4 5 6 1 2 3 4 5 6 g m1 m3 m5 b 1 2 3 4 5 6 g m1 m3 m5 c fig 4 thin layer chromatography analysis of the black potato (coleus tuberosus) starch hydrolyzed by brevibacterium sp. amylase on the substrate concentration 1% (a), 2.5% (b), and 5% (c). lane 1, control; lane 2, 1 h; lane 3, 2 h; lane 3, 4 h; lane 4, 6 h, and lane 5, 8 h. lane 7 (g),standard glucose; lane 8 (m1) standard maltose, lane 9 (m3) standard maltotriose, and lane 10 (m5) standard maltopentaose. fig 5 the results of reducing sugar analysis on a variety of enzyme-substrate ratio (v/v) 1:5 ( ) , 1:2 ( ), 1:1 ( ), and 1:10 -1 ( ) at a concentration of 2.5% substrate, phosphate buffer ph 6.6, the volume of 1ml enzyme (2.5u ml ), 30 °c. 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 1 2 3 4 5 6 7 8 r e d u c in g s u g a r c o n c e n tr a ti o n ( p p m ) time (h) higher quantity of maltotriose (aiyer 2005; yang and liu 2004 ), but there was also evidence of maltotetraose product dominating amylase (murakami et al. 2008 ) and jana et al. (2013) described that potato starch hydrolysis by thermophilic α-amylase from b. megaterium vumb109 produced higher quantity of maltopentaose than maltotriose. patel and arum (2011) described that thin layer chromatography (tlc) can reveal the degree of polymerization of oligosaccharides. from the products separated by tlc it can be inferred readily produced at this concentration and the spots at 2.5% resulted in an increasingly clear separation compared to 5% (fig 4). there are three kinds of maltooligosaccharides produced: maltose, maltotriose, and a maltooligosaccharides mix, which is dominated by maltose and maltotriose (fig 6). discussion generally α-amylase on starch hydrolysis yielded microbiol indones134 rahmani et al. enzyme: substrate ratio hydrolysis time (hours to) dp 1:5 1 22 2 16 4 9 6 10 8 9 1:2 1 18 2 17 4 15 6 15 8 14 1:1 1 14 2 13 4 11 6 12 8 11 fig 6 elution profile of maltooligosaccharides (freeze dry) from hydrolysis the black potato (coleus tuberosus) starch by amylase from brevibacterium sp. chromatographic conditions:column (zorbaxsil(silica) coated with 3-1 aminopropilsilen); eluent (75:25 acetonitrile:water); flow rate (1.4 ml min ); detector (refractive index/rid). table 2 degree of polymerization (dp) analysis with a variety of enzyme-substrate ratios (v/v) (1:5, 1:2, and 1:1) at a -1 concentration of 2.5% substrate, phosphate buffer ph 6.6, the volume of 1 ml enzyme solution (2.5 u ml ), 30 °c 10 145, and maltotriose 16 746. hplc analysis of samples resulted in simple sugars such as the glucose monosaccharide, disaccharides such as maltose, and oligosaccharides such as maltotriose. the type of both, simpleand oligosaccharides determined in both, hplc and tlc analyses are similar. jana et al. (2013) describe that the potato starch which is normally resistant to enzymatic hydrolysis. but, the results of degradation black potato starch, indegeus indonesia into simple sugars and oligosaccharides can efficiently be performed using amylolytic enzymes from the marine microbe brevibacterium sp. the end products of α-amylase action are oligosaccharides with varying length with an-configuration and-limit dextrins (vander et al. 2002), which constitute a mixture of maltose, maltotriose, and branched oligosaccharides of 6-8 glucose units that contain both-1,4 and 1,6 linkages (souza and pérolade 2010). this research suitable with other marine bacteria amylase. starch hydrolysis by chromohalobacter sp. tvsp 101 amylase formed maltotetraose, maltotriose, maltose, and glucose as end products (prakash et al. 2009). chakraborty et al. 2011 has been reported that major starch hydrolysis by halophilic saccharopolyspora sp. a9 were glucose, maltose, and maltotriose as major products. kumar and khare (2012) have reported total 72 % soluble starch hydrolysis was achieved in 4 hours by marinobacter sp. emb8 amylase with the major products were maltotetraose, maltotriose, and maltose by tlc and hplc analysis. references aiyer pv. 2005. amylases and their applications. afr j biotechnol. 4:1525-1529. aoac. 1984. official methods of analysis, 14th edn. arlington, va: association of official analytical chemists.inc. washington d.c. barreteau h, cédric d, philippe m. 2006.production of oligosaccharides as promising new food additive generation. food technol biotechnol. 44(3):323-333. belitz, hd, grosch w, schieberle p. 2004. food chemistry, rd 3 edn. heidelberg: springer. doi:10.1007/978-3-66207279-0. bernfeld p. 1955. amylase αand β. meth enzymol. 1:149158. crittenden rg and playnemj.1996. production, properties and applications of food-grade oligosaccharides. trends in food science and technology. 7(11):353361. doi:10.1016/s0924-2244(96)10038-8. dubois, gilles ka, hamilton jk, rebers pa, smith f. 1956. colorimetric method for determination of sugar and related substance. j anal chem. 28(3): 350-356. . . volume 7, 2013 microbiol indones 135 that the hydrolysis is due to an endo-type in which random starch hydrolysis produced oligosaccharides with dp≥2. the substrate:enzyme 1:10 has the trend to gradually increase the amount of reducing sugars (fig 5). at the substrate:enzyme ratio of 1:1; 1:2, and 1:5 the reducing sugar production increased up to 4 h and then decreased for 6 h, subsequently it raised slowly and constantly up-to 8 h but with different degree of polymerization (table 2). the utilization of very high enzyme dosages caused suboptimal hydrolysis, as low amounts of reducing sugars were produced, as for the ratios of 1:1 and 1:2. the enzyme substrate ratio of 1:5 was chosen as the most effective for further analysis. similarly, the time for maltooligosaccharides production was determined from the results obtained by investigating enzyme substrate ratios (fig 5). hence, a reaction time 4 h were chosen because in this time (substrate concentration 2.5%; enzyme substrate 1:5 ratio) production of reducing sugars were at 14 240 ppm. maltooligosaccharides profile analysis by hplc. patel and arum (2011) described that to understand the relations between physicochemical properties and the functionality of oligosaccharides, it is important to characterize their structure. structural analysis requires determination of monosaccharides, their sequence, type of linkages, branching, and anomeric configuration. the oligosaccharides can be isolated using high performance liquid chromatography (hplc) (patel and arum 2011). for the determination of oligosaccharide profiles using hplc, polar degassed solvents (75% acetonitrile and 25% distilled water) were used according to eliasson (2006). separation techniques involved liquid-liquid partition chromatography with retention mechanisms followed by normalphase with a polar stationary phase and a 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2002. present status and future of functional oligosaccharides development in japan. j pure appl chem. 74(7):1245-1251. doi:10.1351/pac2002740712 45. microbiol indones136 rahmani et al. 7 476 siti chandra reka selly... identification and selection of entomopathogenic fungi as biocontrol agents for from south sumatra aphis gossypii siti herlinda , chandra irsan, reka mayasari, selly septariani* aphid, is a vector of curly virus disease. the damage of chili due to its feeding is only 35% and it can achieved 100% if the damage caused by the aphid as a vector. the objectives of this research were to explore, to isolate, to identify, and to select entomopathogenic fungi as biocontrol agents for . the fungi were explored using insect bait in soil and collected infected insects from south sumatra, indonesia. then, the fungi were isolated and identified, and finally the bioefficacy tests were done using 1 x 10 conidia ml against the third instar of the explorations found 25 isolates of enthomopathogenic fungi consisting 10 isolates of and 15 isolates of selection of the fungi isolates on the aphid nymphs showed that isolate bpm isolated from caused the highest mortality rate (80.80%), while the lowest (47.20%) was caused by the isolate bagtb isolated from the shortest time needed to produce 50% mortality (lethal time ) was 2.54 days (isolate of ). the longest time (3.66 days) was produced by isolate of . key words: , , fungi and department of plant pests and diseases, faculty of agriculture, universitas sriwijaya, jalan raya palembang-prabumulih, km 32, ogan ilir, inderalaya 30662, south sumatra, indonesia aphis gossypii a. gossypii a. gossypii. beauveria bassiana metarhizium anisopliae. pseudoplusia chalcites a. gossypii. chrysodeixis chalcites from muarasiban tenebrio molitor from tanjung raja aphis gosypii beauveria, metarhizium 6 -1 50 t . he most important pest for chili is which is the vector of curly virus disease aphid, is a vector of curly virus disease. the damage of chili due to its feeding is only 35% and it can achieved 100% if the damage caused by the aphid as a vector (fuller . 1999). is a carrier for 76 viruses attacking various host plants (satar . 1999) and has been reported resistant to various insecticides (wang 2002). it is crucial to identify an alternative control that is relatively safer for both agricultural product and environmental health. biological control is the main component of integrated pest management (ipm) that is a safer pest control than other control methods (lopes . 2009). biological control is needed for green consumers in the world who prefered pesticide free agricultural products. this can be achieved by controlled biologically of the vector insects using natural enemies, such as entomopathogen. and are common soil-borne entomopathogenic fungi that occur worldwide. cause a disease known as the white muscardine disease because infected insects covered with a layer of white mold (alves 2002; klinger 2006) and the green muscardine disease for (santiago 2001). both fungi attack the immature and adult stages of several insect orders, such as hemiptera (liu . 2002) and diptera (moraga . 2006). in order to develop successful biological control, a basic research is needed to find entomopathogenic fungi which are most pathogenic againts the vector, therefore, the objectives of this research were to explore, to isolate, to identify, and to select entomopathogenic fungi as biocontrol agents for aphis gossypii, aphis gossypii et al a. gossypii et al et al. et al beauveria bassiana metarhizium anisopliae b. bassiana et al. et al. m. anisopliae et al. et al et al a. gossypii. a. gossypii materials and methods exploration of entomopathogenic fungi. isolation and identification of entomopathogenic fungi. entomopathogenic fungi exploration was done by using two methods to obtain many species or strains of the fungi. the first method by collecting aphid nymphs and adults, larvae of lepidoptera, hemiptera, and other insect ordo that were sick or dead due to fungus infection (herlinda 2008). the infected insects that showed symptoms of dry body and the presence of conidia and fungal conidia, white or green body of the larvae were isolated or purified. surveys to explore the fungi were carried out five times on each location. then, the fungus-infected insects were isolated in the laboratory at a cabinet of laminar air flow that had been sterilized with 70% alcohol. the second method of fungus exploration was to use insect as bait following method of hashim and azwana (2003). the insect used was third instar of (hongkong caterpillar) that had been newly molting. soil used to trap the fungi was taken by purposive sampling from forests in south sumatra. the soil sample was taken by digging at a depth of 50-10 cm, brought to the laboratory as much as 400 g, and then it was put into a plastic tray (13 x 13 x 10 cm ). the hongkong caterpillars were immersed 0.5 cm deep in the soil, and 20 larvae of the caterpillars were put in bottom of the tray. this treatment was repeated 20 times. then, the tray was covered with a piece of black cloth that had been moistened. three days later, the infected caterpillars were examined and isolated in the laboratory. isolation of entomopathogenic fungi used methods of herlinda (2006). the fungus-infected insects and caterpillars were sterilized with 1% sodium hypochlorite or 70% alcohol for three minutes. then insects were rinsed with sterile water three times, and dried on top of sterile filter paper. then, they were placed in a petri dish (diameter 9 cm) containing moist sterile paper and incubated to stimulate et al. tenebrio molitor et al. 3 *corresponding author, phone +62-711-580663, fax: +62-711-580276, email: sherlinda_hpt_fp@unsri.ac.id issn 1978-3477 vol 4, no 3, dec 2010, p 137-142 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.7 conidial germination. fungi were isolated, cultured on saborroud dextrose agar (sda) medium, and incubated for seven days at 25-27 c and relative humidity 80-85%. then, a pure culture fungus was identified by using reference of toledo (2010). the isolate was grown on slants of sda medium supplemented with chitin from the small mole cricket, kept in 1.5 x 13 cm glass test tubes, and incubated for 7 days. the fungal spores were harvested from the slant culture, and each 1 g medium of the isolate was separately suspended in 9 ml water. conidial density was calculated after analysis of a 1 ml sample of the suspension in a haemocytometer. conidial germination as a measure of viability was obtained by spreading 1x10 propagules in 100 μl on glucose yeast agar (gya) medium. the suspension was incubated at room temperature for 24 h. to calculate conidial viability per unit volume, total counts estimated with the haemacytometer were multiplied by the percentage of germination. after culturing isolates of the fungi, was also cultured on the chili to get the available colony that would be used to entomopathogenic fungi selection. the selection was done using method of herlinda (2008). conidia l fungal suspension (density of 1x10 conidia ml ) on the third instar of . in this experiment, ° et al. a. gossypii et al. a. gossypii 6 6 -1 selection of entomopathogenic fungi as biocontrol agents. of the fungal isolates were used by dripping topically 10 μ each isolate (table 1) was inoculated on 25 newly moltingthird instar of and repeated five times. nymphs that had been exposed to the fungus conidia were subsequently maintained in plastic cylinders (diameter 9 cm and height 30 cm) covered with cloth. in the cylinders, there was a pot (diameter 9 cm) of chili growth. every 6 h during the nymph stage, the number of dead nymph were recorded, while the number of nymphs growing to be adults were also recorded daily until all the nymphs became adult. the difference of mortality data and adult percent emerging were analyzed using analysis of variance, with the tukey test. time of nymph death was analyzed to determine the lt using probit analysis and also calculated by using sas-stat program two species of entomopathogenic fungi from south sumatra were identified, and consisted of 10 isolates, whereas were 15 isolates (table 1) the fungi could be found in lowland and highland areas of south sumatra, however they were more found in lowland areas than in highlands. exploration method by dipping bait insecs in the soil was more effective than collecting infected insects from the field. isolates more often found during survey than isolates. a. gossypii b. bassiana m. anisopliae. b. bassiana m. anisopliae . m. anisopliae b. bassiana data analysis. isolates of entomopathogenic fungi. 50 . results table 1 entomopathogenic fungi isolates collected from south sumatra isolate codes source host insects origins exploration methods lowland areas bagtb aphis gossypii talang buruk collection of infected insects bnlptr nilaparvata lugens pantura collection of infected insects btmso tenebrio molitor soak bait insects in soil btmtb tenebrio molitor talang buruk bait insects in soil maagin aphis gossypii inderalaya collection of infected insects malain leptocorisa acuta inderalaya collection of infected insects magin aphis gossypii inderalaya collection of infected insects mlaptr leptocorisa acuta pantura collection of infected insects mtmbb tenebrio molitor bukit besar bait insects in soil mtmin tenebrio molitor inderalaya bait insects in soil mtmgb tenebrio molitor gelumbang bait insects in soil mtmkt tenebrio molitor kenten bait insects in soil mtmtk tenebrio molitor talang kelapa bait insects in soil mtmtr tenebrio molitor tanjung raja bait insects in soil highland areas blepd lipaphis erysimi pagardin collection of infected insects bpm chrysodeixis chalcites muarasiban collection of infected insects bpcpd1 chrysodeixis chalcites pagardin collection of infected insects bpcpd2 chrysodeixis chalcites pagardin collection of infected insects btmpd tenebrio molitor pagardin bait insects in soil bccc chrysodeixis chalcites curup collection of infected insects magpd aphis gossypii pagardin collection of infected insects mtmbk tenebrio molitor bedeng kresek bait insects in soil mtmjr tenebrio molitor jarai bait insects in soil mtmkj tenebrio molitor kerinjing bait insects in soil mtmms tenebrio molitor muarasiban bait insects in soil isolate code using first letter b refers to and m refers tobeauveria bassiana metarhizium anisopliae. 138 herlinda et al. microbiol indones isolate codes conidial density (x 10 6 conidia ml -1 ) range mean±se beauveria bassiana bagtb 36.60-37.40 37.00 ±0.40 ef bccc 21.55-22.23 21.87 ±0.34 abc blepd 43.30-45.15 43.98 ±1.02 f bnlptr 20.60-43.35 21.53 ±0.54 abc bpm 37.15-59.60 45.39 ±12.36 f bpcpd1 24.60 -27.52 26.25 ±1.49 cd bpcpd2 20.60-22.83 21.61 ±1.13 abc btmpd 42.17-43.65 42.73±0.80 f btmso 17.23-21.23 19.85±2.27 ab btmtb 20.40 -24.52 22.81±2.15 abc metarhizium anisopliae maagin 19.08-20.25 19.63±0.59 a magin 37.40-38.00 37.78±0.33 ef magpd 43.95-44.85 44.46±0.46 f malain 19.37-19.45 19.38±0.04 a mlaptr 17.88-20.90 19.74±1.63 a mtmbb 18.75-20.45 19.36±0.95 a mtmbk 38.05-39.20 38.72±0.59 ef mtmgb 29.50-34.37 31.98±2.44 f mtmin 35.70-38.47 38.96±3.53 ef mtmjr 36.87-38.70 37.62±0.96 ef mtmkj 42.45-44.70 44.06±1.40 f mtmkt 27.65-23.35 25.05±0.32 bc mtmms 42.87-43.42 43.10±0.29 f mtmtk 18.00-18.63 18.36±2.29 a mtmtr 38.37-44.27 41.98±3.16 f data in the same column followed by the same letter showed they were not significantly different (hsd test, p 0.05). and isolates had very dense conidia, but the trend of conidial density of isolates was significantly higher compared to isolates (table 2). the highest conidial density of entomopathogenic fungi reached 45.39x10 conidia ml that was found on isolates coded bpcm, the lowest found on mtmtk isolate of (18.36x10 conidia ml ). the highest conidial viability of entomopathogenic fungi reached 47.50% with a 41.87% average viability that was found in isolates coded by bpcm (table 3). the lowest conidial viability averaged of 11.90% found on isolates coded by mtmbb. the conidial viability of isolates tended significantly higher than those of isolates. twenty five isolates were selected to determine the most virulent isolate nymphs. the results showed that all and isolates were pathogenic to the nymphs of causing mortalities between 42.40 and 80.80% (table 4). the most virulent isolate was bpm of isolate causing an average of 80.80% mortality, and the two least ones were bagtb of isolate and mlaptr of isolate. mortality caused conidial density and viability of entomopathogenic fungi. virulence isolates of entomopathogenic fungi. b. bassiana m. anisopliae b. bassiana m. anisopliae b. bassiana m. anisopliae b. bassiana m. anisopliae b. bassiana m. anisopliae againts a. gossypii b. bassiana m. anisopliae a. gossypii b. bassiana b. bassiana m. anisopliae a. gossypii 6 -1 6 -1 isolate codes percentage of conidial viability (%) range mean±se beauveria bassiana bagtb 14.30-24.20 20.67±5.52 abcd bccc 19.89-33.29 25.20±7.12 abcdef blepd 20.00-28.10 25.20±4.51 abcdef bnlptr 14.80-20.30 16.80±3.04 abc bpm 38.10-47.50 41.87±4.97 f bpcpd1 22.72-34.78 28.37±6.07 abcdef bpcpd2 14.83-31.37 21.50±8.72 abcde btmpd 12.50-19.40 16.01±3.45 abc btmso 29.03-36.40 36.58±3.79 def btmtb 26.30-40.90 31.5 ±8.16 bcdef metarhizium anisopliae maagin 5.49-21.37 13.15±7.95 ab magin 10.50-15.15 13.15±2.39 ab magpd 15.40-23.50 18.83±4.19 abcd malain 9.69-18.28 14.80±4.52 abc mlaptr 15.70-21.53 16.80±2.92 abcd mtmbb 8.71-14.47 11.9±2.93 a mtmbk 10.80-17.80 14.87±3.64 abc mtmgb 25.00-29.80 28.13±2.72 abcdef mtmin 20.70-34.60 26.09±7.46 abcdef mtmjr 14.30-24.20 20.67±5.52 abcd mtmkj 20.00-27.02 23.61±3.51 abcdef mtmkt 28.00-43.50 32.21±7.89 cdef mtmms 20.00-27.60 21.00±6.16 abcd mtmtk 15.68-26.71 21.70±5.58 abcde mtmtr 31.43-53.30 39.91±11.7 ef table 3. conidial viability of and m isolates beauveria bassiana etarhizium anisopliae data in the same column followed by the same letter showed they were not significantly different (hsd test, p 0.05). volume 4, 2010 microbiol indones 139 beauveria bassiana etarhizium anisopliae table 2. conidial density of and m isolates by bpm isolate was significantly different from bagtb and mlaptr isolates. bpcm isolate was isolated from in highland areas, bagtb isolate from , and mlaptr isolate from both of the bagtb mlaptr isolates were from lowland areas. the level of mortality caused by treatment tended to be higher than those of treatment. all and isolates were able to infect nymphs and almost had low value of lethal time median (lt ). the time median when death occured differed among isolates and it varied between 2.54 and 3.66 days. (table 5). the result indicated that bpm isolate of had the lowest lt value (2.54 days) againts nymphs, while mtmtr isolate of was the highest one (3.66 days) in this study, exploration methods that were able to find entomopathogenic fungi were deeping insect bait in the soil and collecting the infected insects from the fields. preliminary survey following method of feng (2007) had tried to find the fungal conidia of entomopathogen from air using sticky cards but they were unable to be found. the fungi were easier to be found from the soil compared to infected insects from the fields. herlinda . (2008) found that the entomopathogenic fungi obtained from infected chrysodeixis chalcites aphis gossypii leptocorisa acuta. and a. gossypii b. bassiana m. anisopliae b. bassiana m. anisopliae a. gossypii b. bassiana a. gossypii m. anisopliae et al. et al 50 50 . discussion isolate codes nymph mortality (%) range mean ± se beauveria bassiana bagtb 32-60 42.40±13.44 a bccc 36-64 47.20±12.13 ab blepd 24-72 52.80±17.29 ab bniptr 32-64 46.40±13.44 ab bpm 72-92 80.80± 7.69 b bpcpd 52-84 67.20±14.25 ab bpcpd2 32-64 47.20±13.38 ab btmpd 36-52 44.80± 7.15 ab btmso 32-76 53.60±21.46 ab btmtb 52-80 54.40±16.63 ab metarhizium anisopliae maagin 44-60 53.60± 6.69 ab magin 20-92 47.20±29.03 ab magpd 32-68 48.00±16.73 ab malain 36-68 49.60±13.73 ab mlaptr 32-64 42.40±17.57 a mtmbb 32-52 44.80± 7.69 ab mtmbk 52-60 53.60± 8.29 ab mtmgb 40-80 54.40 ±15.3 ab mtmin 36-64 47.20±11.09 ab mtmjr 40-80 57.60±10.43 ab mtmkj 28-88 46.40±23.93 ab mtmkt 40-80 56.80±14.80 ab mtmms 32-68 52.00±12.96 ab mtmtk 36-68 50.40±15.12 ab mtmtr 36-56 44.00± 7.48 ab table 4 mortality of nymph exposed to conidia of and at a concentration of 10 conidia ml aphis gossypii beauveria bassiana metarhizium anisopliae 6 -1 data in the same column followed by the same letter showed they were not significantly different (hsd test, p 0.05). 50table 5 lethal time median (lt ) of nymph exposed to conidia of and at a concentration of 1 x 10 conidia ml aphis gossypii beauveria bassiana metharizium anisopliae 6 -1 isolate codes mean lt50 (days) 95% confidence limit regression equation lower upper beauveria bassiana bagtb 3.18 2.97 3.50 y= 0.176+0.066x bccc 3.02 2.73 3.53 y= 0.155 +0.061x blepd 2.91 2.69 3.23 y= 3.070+1.060x bnlptr 3.06 2.83 3.39 y= 0.167+ 0.065x bpm 2.54 2.42 2.67 y= 0.194+ 0.077x bpcpd1 2.80 2.67 2.96 y= 0.194 +0.075x bpcpd2 3.02 2.72 3.56 y= 0.194 +0.075x btmpd 3.08 2.85 3.44 y= 2.980 +0.097x btmso 3.10 2.86 3.46 y= 0.185 +0.078x btmtb 3.08 2.84 3.43 y= 0.157 +0.062x metarhizium anisopliae maagin 2.81 2.56 3.21 y= 0.142 + 0.058x magin 3.03 2.78 3.42 y= 2.890 + 0.950x magpd 3.02 2.75 3.47 y= 0.188 + 0.079x malain 2.89 2.57 3.48 y= 0.144 + 0.058x malaptr 3.11 2.80 3.71 y= 0.151 + 0.060x mtmbb 3.09 2.81 3.55 y= 0.155 + 0.062x mtmbk 2.95 2.75 3.25 y= 0.166 + 0.060x mtmgb 3.03 2.89 3.20 y= 3.060 + 1.010x mtmin 3.14 3.10 3.89 y= 0.127 + 0.047x mtmjr 2.87 2.63 3.26 y= 3.050 + 1.060x mtmkj 3.20 2.93 3.39 y= 0.179 + 0.069x mtmkt 3.23 2.98 3.60 y= 0.135 + 0.049x mtmms 3.07 2.92 3.27 y= 0.910 + 0.073x mtmtk 2.91 2.62 3.39 y= 2.820 + 0.970x mtmtr 3.66 3.36 4.13 y= 0.155 + 0.540x insects tended to be more difficult to be isolated. the other from the infected insects often were contaminated by air fungi. hashim and azwana (2003) reported that the conidia in the soil tended to be more persistent, they could be easily trapped using insect bait. fuxa and richter (2004) found that soils with high clay content improved persistence of the fungal conidia. thus, future research using clay will be required to formulate the fungus conidia to increase efficacy as biological control. condial density observed were densed, and the highest conidial density of entomopathogenic fungi reached 45.39x10 conidia ml that was found on isolates coded by bpcm. soundarapandian and chandra (2007) stated that conidial density were determined by mass production media and temperature of the incubated room. liquid media tended to produce more conidia than those of solid ones. the optimum temperature and ideal ph for the mass production of was found to be 25-30°c and 7, respectively. few germinated conidia were observed at 24 h of incubation that only reached 47.50%. the fungal isolates used in this study generally had low viability, it could be caused by shorter time (24 h) used in incubation of conidial suspension. conidia were considered germinated if germtube lengths were two times in diameter of the propagules or if with conspicuous swelling (toledo . 2010). they reported that germinated conidia in vitro for and at 72 hours could be 95.50% and 100%, respectively bidochka . (2000) stated that 6 -1 b. bassiana m. anisopliae et al b. bassiana m. anisopliae . et al 140 herlinda et al. microbiol indones conidial viability was determined by temperature. the optimum temperature needed for entomopathogenic fungal conidia to germinate was 22-27 c with optimum humidity above 90%, and at under 86% humidity, the virulence would decrease continuously. twenty five isolates of and found in this reasearch were almost pathogenic againts nymphs. the most virulent isolate was bpm of isolate causing an average of 80.80% mortality. bpcm isolate was isolated from that was unrelated to no relationship between pathogenicity and the origin of the isolates was observed. bagtb of isolate isolated from had the lowest pathogenicity and caused only 42.40% mortality of . liu . (2002) also found that virulent isolates of or could be originally isolated from related and unrelated hosts. the ability of the bpcm isolate to produce the highest mortality rates might be caused either by their genetic characteristics, or by their conidial viability. aregger (1992) stated that conidial viability factor might be the factor affecting virulence. rate of loss of conidial viability of varies among the strain. decline of conidial viability of this fungus correlated with decline of host mortality due to its infection. and isolates needed just 2.54 days and 2.81 days, respectively to kill . it takes shorter time than they killed other species of host, such as planthopper (toledo 2010). thompson and brandenburg (2005) reported that death caused by the fungi usually occured more than 48 h after attachment of conidia to the insect cuticle. toledo (2010) found that germ tubes on host cuticular surface began to be found at 24 and 48 h, and they were observed penetrating directly through the host cuticle in regions near the hairs of the second antennal segment and on the laterosternites of abdomen. after 72 h, long and errant germ tubes were detected on the cuticular surface. fuxa and richter (2004) stated that hyphae from conidia entered the host's body with the help of enzymes or mechanical pressure. in the end, the host was covered all over with propagules and the soft parts of the body were penetrated so hyphal growth could be observed outside the host insect's body. external hyphal growth would produce conidia which spread spores into the environment upon reaching maturity, then infect other healthy insects. we concluded that and were able to kill nymphs. twenty five isolates of and found were almost pathogenic againts them. the most virulent isolate was bpm of isolate causing an average of 80.80% mortality. dead insect hosts infected by showed the same symptoms as those infected by , except for the color of the hyphae which was greenish white we would like to thank riyanto and cheppy wati for their assistance in the surveys. financial support of this o b. bassiana m. anisopliae a. gossypii b. bassiana chrysodeixis chalcites a. gossypii. b. bassiana a. gossypii a. gossypii et al b. bassiana m. anisopliae b. bassiana b. bassiana m. anisopliae a. gossypii peregrinus maidis et al. et al. m. anisopliae b. bassiana m. anisopliae a. gossypii b. bassiana m. anisopliae b. bassiana m. anisopliae b. bassiana . acknowledgements volume 4, 2010 microbiol indones 141 research was provided by the project of incentive research fund, ministry of research and technology, republic of indonesia, budget year 2010 with contract number: 106/ rd-df/d.psiptn/insentif/ppk/1/2010, 15 january 2010. references alves sb, rossi ls, lopes rb, tamai ma, pereira rm. 2002. yeast phase on agar medium and its pathogenicity against (lepidoptera: cerambidae) and (acari: tetranychidae). j invertebr pathol 81:70-7. aregger e. 1992. conidia production of the fungus on barley 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[online]. http://www.insectsicence.org/10.35. [sep 2010]. beauveria bassiana diatraea saccharalis tetranychus urticae beauveria brongniartii aphis gossypii beauveria bassiana beauveria bassiana cosmopolites sordidus leptocorisa oratorius beauveria bassiana plutella xylostella beauveria bassiana leptinotarsa decemlineata beauveria bassiana, metarhizium anisopliae lygus lineolaris beauveria bassiana metarhizium anisopliae ceratitis capitata metarhizium anasopliae locusta migratoria manilensis aphis gossypii metarhizium anisopliae beauveria bassiana. beauveria bassiana metarhizium anisopliae peregrinus maidis o , wang ky, liu tx, yu ch, jiang xy, yi mq. 2002. resistance of (homoptera: aphididae) to fenvalerate and imidacloprid and aphis gossypii activities of detoxification enzymes on cotton and cucumber. j econ entomol 95:407-13. 142 herlinda et al. microbiol indones 4 455 usamah.cdr a comparison of serological and bacteriological methods for detection of in experimentally-infected chickens mycloplasma gallisepticum usamah afiff department of infectious diseases and veterinary public health, faculty of veterinary medicine, institut pertanian bogor, darmaga campus, bogor 16680, indonesia. phone: +62251-8310185, fax: +62251-8629466, email: uafiff@yahoo.co.uk an indirect enzyme-linked immunosorbent assay (elisa) was developed to detect antibodies to . three antigens were used in this experiment. antigen 1 was prepared from whole cell of , antigen 2 was a sodium dodecyl sulfatesolubilized preparation from whole cells, and antigen 3 was prepared by sonication of the whole cell antigen. the assay was then used to detect (anti)antibodies in experimentally-infected chickens compared with serum-plate-agglutination (spa), haemagglutination-inhibition (hi) tests, and tracheal culture. data obtained in this experiment showed that there was a correlation between seropositivity and rate of isolation of . elisa was found to be less sensitive, but more specific than spa, and more sensitive than the hi test. the whole cell antigen gave the highest optical densities but was less specific than the other two antigens. the elisa using all three antigens successfully identified the -infected chickens uniformly and positively through 14-35 days post infection, and correctly identified the control group as negative through the 35 day experimental period. the elisa obviously has a place in the serodiagnosis of avian mycoplasma. improved-specificity and -sensitivity of the antigen is desirable. key words: serology, detection, infected chickens. mycoplasma gallisepticum m. gallisepticum m. gallisepticum m. gallisepticum m. gallisepticum m. gallisepticum mycoplasma gallisepticum, the mycoplasmas are the tiniest and simplest prokaryotic cells capable of self replication. the genus is composed of over 100 species of small prokaryotes (consisting of a genome containing 600-1800 kb). the mycoplasmas are separated from the eubacteria in the class mollicutes (“soft skin”) which consist of the single order mycoplasmatales. this order contains six genera ( , and ) with generic distinctions stand mainly on differences in morphology, genome size and some nutritional requirements (weisburg . 1989). recently advance techniques have been applied for classification and analysis of the genetic relationships amongst strains of such as 16s-rdna-based technique/amplified-rdna restriction analysis (stakenborg . 2005), amplified fragment length polymorphism technique (hong . 2005), random amplified polymorphic dna (rapd), and pulsed-field gel electrophoresis (pfge) (charlton . 1999; mettifogo . 2006). avian mycoplasmosis can be caused by several species of including s, and . is the most important pathogen in poultry (nascimento . 2005). infection is also known as a chronic respiratory disease (crd) of chickens (soeripto 2009). infection can cause significant economic losses from decreased egg production, reduced feed efficiency, and decreased growth in chicken flocks and other avian species. infection with has a wide diversity of clinical manifestation, but even in the absence of apparent clinical signs, the economic impact may be significant (levisohn and kleven 2000). infections are transmitted both horizontally and vertically and it remains in the flock constantly as a subclinical form (bencina . 1988; mycoplasma a c h e l o p l a s m a , a n a e ro p l a s m a , a s t e ro l e p l a s m a , mycoplasma, spiroplasma ureaplasma et al mycoplasmas et al et al et al et al mycoplasma mycoplasma gallisepticum, m. synoviae, m. meleagridi m. iowae m. gallisepticum et al m. gallisepticum m. gallisepticum m. gallisepticum m. gallisepticum et al feberwee . 2005b). infection can be diagnosed by clinical manifestation, serology assay, and cultural and biochemical characteristic (soeripto 2009). cultivation techniques used for mycoplasmas are laborious, expensive, and time-consuming, and therefore far from a routine procedure. problems experienced with culture include overgrowth by faster growing species (amin and jordan 1978). the objectives of this study were to compare the isolated from detection of antibodies from experimentally-infected (ei) chickens with and to set up an elisa test to detect mycoplasma antibodies in serum which would be of practical to use for routine diagnosis in field samples. twelve specific-pathogen-free (spf) white leghorn chickens at 5 weeks of age were determined to be free of and by tracheal-culture (timms 1967) and serology (avakian 1988) prior to experimental infection. birds were divided into two groups and caged separately in an isolation house and treated as follows. each of four birds in first group received 0.2 ml intratracheally, 1 drop (approximately 50 l) by eye-drop, and 1 drop intranasally, of 24 h broth culture of strain s6. two uninoculated birds were placed in group 1 as contact to infected birds. group 2 consisted of six uninoculated chickens. the inoculum was titrated by 10-fold serial dilution in eaton´s broth just after the chickens were inoculated and was found to contain 2.3 x 10 cfu ml . chickens in group 1 were bled and tracheal-cultured at weekly intervals starting on day 0 and continuing until the end of the trial on day 35. chickens in group 2 were bled and tracheal-cultured at the beginning of the trial (day 0) and at the end of the trial (day 35). et al m. gallisepticum mycoplasma mycoplasmas m. gallisepticum m. gallisepticum m. synoviae et al. m. gallisepticum materials and methods experimental design. μ 8 -1 issn 1978-3477 vol 4, no 3, dec 2010, p 119-126 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.4 viable count. growth inhibition test. epi-immunofluorescent (eif) test technique. a n t i g e n p re p a r a t i o n . these were performed by the method described by miles and misra (1938) with some modifications. serial tenfold dilution of the cultures were prepared in eaton´s broth, and five 0.02 ml drops were immediately transferred onto eaton's agar from each dilution. plates were incubated at 37°c in a moist air tight candle jar for one week before colonies were counted. for the isolation of mycoplasma, the method described by timms (1967) was adopted with some modification. tracheal swabs were inoculated into eaton´s broth and serial dilutions were made to dilute out possible contamination. the culture was incubated at 37°c in a candle jar for primary isolation. the culture was checked every 24 h to detect colour changes in the medium. changes of colour might indicate growth of the organism. in the case of suspected contamination, filtering was used to remove the contaminant. after incubation for 4 days, the broth cultures were plated onto eaton´s agar and passed onto a second broth, and were then incubated in incubator with a moist 5% co atmosphere at 37°c for 4 days and examined every day. the identification of the isolates was performed by growth-inhibition tests and the epiimmunofluorescent technique. this test was performed by using eaton´s plates. plates were divided into two, in which each side was seeded by the running-drop technique. after plates were dried, paper disks which have been saturated with specific antisera to were placed on top. then, they were incubated in incubator with a moist 5% co -atmosphere at 37°c for 2-3 days and were then examined for size of zones of growth inhibition. plates were seeded by the running-drop technique which consists of undiluted and 1 in 100 dilutions of the test sample being dropped onto the same plate. the plates were incubated at 37°c. agar blocks with mycoplasma colonies were cut and placed colony side upwards on a slide. for each sample two blocks of agar were cut. one block was treated with specific non-inactivated rabbit antiserum to in a dilution of 1:20. the other was treated with antiserum from a mycoplasma of bovine origin, and the block incubated at room temperature for 30 min in a moist chamber. after incubation, the blocks were tipped into a 10 ml tube containing approximately 7 ml of pbs, ph 7.2-7.4. the tubes were rotated slowly for 10 min and then the pbs was decanted, replaced by fresh pbs, and the tubes were again rotated for 10 min. the pbs was gently decanted and the block tipped out onto a slide and placed colony-side upwards. each block was flooded with anti-rabbit igg antiserum conjugated with fitc in a dilution of 1:10. the blocks were again incubated for 30 min at room temperature in a moist chamber. after incubation the blocks were washed twice as before, using rotation. the blocks were examined under a fluorescence microscope. to p r e p a r e a n t i g e n , approximately 10 ml of eaton's was inoculated with this volume was used to inoculate 100 ml 2 2 mycoplasma m. gallisepticum m. gallisepticum m. gallisepticum. of eaton's broth which was incubated for 24 h at 37 c. each 100 ml aliquot was then used to inoculate 1000 ml of eaton's broth which was incubated for 24 h in air at 37 c. the culture was then stirred gently and continuously. three different antigenic treatments were performed, i.e. wholecell antigen, sonicatedand sds-solubilized antigens. the whole cell was harvested by centrifugation at 12 000 x g for 30 min at 4 c. it was resuspended in pbs (ph 7.2) and recentrifuged three times. the final pellet was resuspended in sterile pbs to give a final concentration of 5 mg protein ml and stored at -70 c in a small volume (aliquot). this was used as whole cell antigen. three ml of whole cell antigen were sonicated for 10 min, 30 sec on and 30 sec off. this was used as sonicated antigen. for the preparation of sds-solubilized antigen, the method described by talkington (1984) was adopted. whole-cell-antigen was diluted to give a final concentration of 1 mg protein ml and incubated with 1% sds (1 mg sds mg protein) for 90 min at 37 c, and centrifuged at 20 000 g for 30 min at 4 c. the supernatant was then removed and stored at 4 c and used within 2 wk. the protein concentration was determined by the bca protein assay (pierce laboratories, rockford, usa) as directed by the manufacturer. the substrate was prepared by dissolving 5 mg of p-nitrophenyl phosphate (sigma, 1 tablet) in 5 ml freshly made substrate buffer. for the elisa, the method described by talkington (1984) was adopted. the antigen was diluted in coating buffer and 100 l of the mixture was dispensed into each well (nunc-immuno microtitre plate, polysorp f16, inter med-denmark) except for wells in rows a and b. the plates were incubated for 2 h at 37°c. after incubation the plates were washed 5 times in washing solution using a commercial washer (dynatech). washing was done twice and in between washes the plates were filled with washing solution for 1 min. one hundred micro litre aliquot of diluted serum was placed onto the test microtitre plate and incubated at 37°c for 30 min. after incubation the plates were washed as in step 2 and 100 l of conjugate was then added. the plates were incubated for 30 min at 37°c. after incubation the plates were then washed as in step 2, 100 l of substrate was then added. the plates were incubated at 37°c for 30 min and read on an elisa reader (dynatech mr500) at approximately 410 nm. before the addition of conjugate, 100 l of monoclonal antibody to igm was placed onto the test microtitre plate and incubated at 37°c for 30 min. this was conducted for detection of immunoglobulin m (igm). as baseline for elisa, values indicating at which point a result was positive or negative were established using 60 sera from chickens known to be mycoplasma-free. both antigen and sera were diluted 1:400 and the conjugate 1:2000. the test was carried out as described earlier. the baseline value was then interpreted as the average of these 60 values plus 4 standard deviation values above their mean. a checkerboard titration was carried out to determine the working dilutions of antiserum o o o -1 o -1 -1 o o o determination of protein concentration. the indirect elisa procedure. m. gallisepticum et al. m. gallisepticumet al. μ μ μ μ 120 afiff microbiol indones and antigens. two-fold serial dilutions of the antiserum and antigen were made in the diluting medium. the conjugate was diluted 1:2000. the test was carried out as the same procedure as before. these were done using antisera to different mycoplasma. the antisera were first treated so as to remove the anti-medium component in order to minimize cross-reactions. rabbit anti-sera against , and were used. one-way crossreactivity was done using antisera obtained from experimentally (ei) and contact-infected (ci) chickens tested with whole cell antigen in spa test, hi test, and elisa. the rabbit sera were diluted 1:10 in eaton's broth and incubated for 30 min at 37 c. they were then centrifuged once at 20 000 x g and the floating layer of fat was removed. both sera (treated and untreated) and antigen was diluted 1:200 and the test was carried out as described earlier. these were done using antisera obtained from ei chickens at 35 days. two-fold dilution of the antiserum from 1:100 to 1:55 200 were made. antigen was diluted 1:400 and the conjugate was diluted 1:2000. for the hi test the method described by timms (1967) was adopted. using pbs, 200 l aliquot of serial two-fold antigen dilution were prepared in a who agglutination plate. to each dilution 200 l of pbs and 1% chicken red blood cells were added, giving a total of 600 l in each well. an rbc control consisting of 400 l pbs and 200 l of 1% chicken red blood cells was placed in the last well. the plate was shaken to ensure thorough mixing and readings were made after 50 min at room temperature. using wells a an initial 1:5 dilution was prepared from each test serum (i.e. 25 l of serum + 100 l pbs). 25 l of this dilution was transferred to wells h and g. 25 l of pbs was placed in wells g to b and serial two-fold dilutions were prepared, discarding the final 25 l from well b at the end of the titration. fifty micro litre of the 1:5 serum dilution was discarded from well a, leaving 25 l for provision of a serum control. then, 25 l of 4ha antigen was added to wells h to b. in place of antigen, 25 l of pbs was added to well a. twenty five micro litre of 1% chicken rbc's was added to wells h to a, thus bringing the total volume in each well to 75 l. controls treatments included were: test serum control, known positive and negative control sera, antigen control and rbc control. in order to ensure freedom from “non-specific” agglutination, 25 l of 1:5 serum dilution, pbs and 1% rbc were placed at the end of each titration (i.e. in well a), and it was used as test serum control. known positive and negative control sera were tested in the same manner. antigen control: at the end of each assay (row 12), the ha activity of the antigen was checked, using the same technique as previously described for “antigen preparation”, but with 25 l volumes in place of 200 l. the last well of the plate (a12) was used to check that the rbc's button specificity tests. treatment of sera to minimize cross-reactions. sensitivity tests. haemagglutination inhibition (hi) test. haemagglutination inhibition (hi) procedure. m. gallisepticum, m. synoviae m. meleagridis m. synoviae o μ μ μ μ μ μ μ μ μ μ μ down clearly without signs of haemolysis or non-specific agglutination (25 l of 1% rbc's and 50 l of pbs), and this was used as rbc control. the plate was shaken lightly to ensure thorough mixing of the well contents and read after 50 min at room temperature, or when the antigen titration was reading exactly 4ha. interpretation of the hi method: hi titres of 5 and above were considered positive. for serum plate agglutination the method described by timms (1967) was adopted. stained antigen was allowed to warm up to room temperature and shaken well before use. twenty five litre of serum and stained antigen were placed on ceramic tiles and were mixed well with a clean glass or plastic rod. known positive and negative sera were included and tested in the same manner. ceramic tiles were rocked gently for 2 min. at weekly intervals, starting at day 0 and continuing until day 28, all chickens were examined for the presence of species by tracheal culture. at the end of the trials, all chickens were killed and their air-sacs, trachea and lungs were collected for mycoplasma isolation. isolated myco-plasma were identified to the species level by epiimmunofluorescent technique and growth inhibition test, and only the species used for inoculation was recovered. for determination of the optimum antigen and serum dilution, known positive and negative chicken sera to at various dilutions were titrated against various dilutions of antigen using checker-board design procedure. antigen was tested at 1:100, 1:200, 1:400, and 1:800 dilutions against 1:100, 1:200, 1:400 serum dilutions, and alkaline phosphatase conjugate at 1:2000 dilutions was used. fig 1a, 1b, and 1c showed the optical density values obtained in checker-board titration of whole cell, sonicated-, and sds-solubilized antigen, respectively from which the working dilution of antigen and serum were calculated. the optimum antigen and serum dilution for whole cell antigen was 1:400 (fig 1a). the optimum antigen and serum dilution for sds-solubilized and sonicated antigen was 1:400 for the antigen and 1:200 for serum (fig 1b and 1c). however when these dilutions were applied with sera obtained from ei chickens they gave a high background. therefore some adjustment was necessary, resulting in the choice of a 1:400 dilution for both antigen and serum. baseline values, indicating at which point a result was positive or negative, were established using 60 negative chicken sera known to be mycoplasma-free (spf chickens of mycoplasmosis). the result showed the mean value of absorbance was 0.102± 0.038. the baseline value was fixed at absorbance+ 4sd (equal to 0.255) for 30 min substrate reaction time at 410 nm. in the ei chickens, the spa test showed no activity at 0 days post infection. by 7-35 days post infection, 100% of the samples were positive to . in the ci μ μ serum plate agglutination. results mycoplasma mycoplasma m. gallisepticum m. gallisepticum m. gallisepticum � volume 4, 2010 microbiol indones 121 chickens the spa test showed no activity at 0-14 days postinfection and by 21-35 days post-infection, 100% of the samples were positive. in the ei chickens the hi test had no activity until 7 days post-infection and 100% were positive at 14 through 35 days post-infection. in the ci chickens the hi test showed no activity at 21 days post-infection, and 100% were positive at 28 to 35 days post-infection. in the ei chickens, 75% of the samples were positive at 7 days post-infection with elisa using whole cell, sdssolubilized and sonicated antigen, and 100% were positive by 14 days post-infection (table 1). at 7-35 days post infection was recovered from all ei chickens, and at day 21 post infection was recovered from 1 of 2 ci chickens, and by days 21-35 days post infection was recovered from all ci chickens. thus, in comparing serological and bacteriological methods, the spa test and tracheal-culture were the most sensitive, and the hi test the least sensitive assay. elisa was less sensitive than the spa test and tracheal-culture, but more sensitive than the hi test (table 1). fig 2 shows absorption (a 410 nm) values of whole cell, sds-solubilized and sonicated antigen of in m. gallisepticum m. gallisepticum m. gallisepticum m gallisepticum 0 0.5 1 1.5 2 2.5 100 200 400 800 ag dilution (fold) a (4 10 n m ) 0 0.5 1 1.5 2 2.5 100 200 400 800 ag dilution (fold) a (4 10 n m ) 0 0.5 1 1.5 2 2.5 100 200 400 800 ag dilution (fold) a (4 10 n m ) a b c fig 1 determination of optimum antigen (ag) dilution of at conjugate dilution 1:2000: a, whole cell antigen; b, sonicated antigen; c, sds-solubilized antigen. ◊,100 fold diluted + sera; ♦, 100 fold diluted sera; δ, 200 fold diluted + sera; ▲, 200 fold diluted sera; □, 400 fold diluted + sera; ■, 400 fold diluted sera; +sera, known positive chicken sera to ; -sera, known negative spf chicken sera to mycoplasma gallisepticum m. gallisepticum m. gallisepticum. table 1 comparison of spa test, hi test, elisa, and tracheal-culture for detection of infection in experimentally-infected chickens (ei) and contact-infected chickens (ci) mycoplasma gallasepticum days post infect ion spa test hi test elisa a ei ( positive sample/tested sample ) ci ei ci ei ci 0 0/4 0/2 0/4 0/2 0/4 0/2 7 4/4 0/2 0/4 0/2 3/4 0/2 24 4/4 0/2 4/4 0/2 4/4 0/2 21 4/4 2/2 4/4 0/2 4/4 2/2 28 4/4 2/2 4/4 2/2 4/4 2/2 35 4/4 2/2 4/4 2/2 4/4 2/2 elisa b elisa c trachealculture ei ci ei ci ei ci 0/1 0/2 0/4 0/2 0/4 0/2 0/2 3/4 0/2 4/4 0/2 4/4 0/2 4/4 0/2 4/4 0/2 4/4 2/2 4/4 2/2 4/4 2/2 4/4 2/2 4/4 2/2 4/4 2/2 4/4 2/2 4/4 2/2 4/4 2/2 days post infection 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 7 14 21 28 35 a (4 1 0 n m ) fig 2 development of immunoglobulin g (igg) antibodies in experimentally-infected (ei) and contact-infected (ci) chickens with strain s6 detected by elisa. ◊, ei whole cell antigen; ♦, ci whole cell antigen; δ, ei sds-solubilized antigen; ▲, ci sds-solubilized antigen; □, ei sonicated antigen; ■, ci sonicated antigen. mycoplasma gallisepticum 3/4 122 afiff microbiol indones detecting antiantibodies in ei chickens and ci chickens. it was observed that the whole cell antigen gave the highest absorbance value and sds-solubilized antigen gave the lowest. the sensitivity between elisa and the hi test using sera obtained at 35 days post infection was compared (table 2). serum was diluted starting 1:100 and using 2-fold dilution to 1:51,200. it was observed that the elisa was more sensitive than the hi test. absorbance values of cross-reaction test between whole cell, sds-solubilized, and sonicated antigens with treated and untreated and antisera prepared in rabbits were compared (table 3). whole cell antigen gave highest optical densities both with treated and untreated antisera to . sds-solubilized and sonicated antigen gave high absorbance with untreated antisera, but gave low absorbance with treated antisera. thus sds-solubilized and sonicated antigens were more specific than whole cell antigen. table 4 compares the one-way cross-reactivity of the spa test, hi test and elisa, using a antiserum obtained from ei and ci chickens. the spa test had the highest incidence of cross-reactivity, by 28 days post infection, all the samples were reacting non-specifically in the agglutination test. the hi test showed no cross-reaction. elisa using sera obtained form ei chickens at 14 and 21 days post-infection showed a high incidence of crossreaction, but by day 35 post-infection there was no indication of cross-reactivity by elisa. elisa using sera from ci chickens showed no cross-reaction. therefore elisa was more specific than spa, but less specific than the hi test. the development of immunoglobulin g (igg) in ei and ci chickens detected by elisa using different preparations of antigen showed that the whole cell antigen gave a higher result than sds-solubilized and sonicated antigen. all m gallisepticum m. synoviae m. meleagridis m. meleagridis m. gallisepticum table 2 sensitivity of the elisa hi test (hit) with sera from chickens experimentally-infected (ei) with strain s6 and with sera from contact-infected (ci) chickens obtained at 35 days post infection. and mycoplasma gallisepticum serum dilution hit elisa a elisa b elisa c ei ci ei ci ei ci ei ci (positive sample/tested sample) 100 4/4 0/2 4/4 2/2 4/4 2/2 4/4 2/2 200 4/4 0/2 4/4 2/2 4/4 2/2 4/4 2/2 400 4/4 0/2 4/4 2/2 4/4 2/2 4/4 2/2 800 0/4 0/2 4/4 2/2 4/4 2/2 4/4 2/2 1600 0/4 0/2 4/4 0/2 4/4 0/2 4/4 1/2 3200 0/4 0/2 4/4 0/2 4/4 0/2 4/4 0/2 6400 0/4 0/2 4/4 0/2 3/4 0/2 4/4 0/2 12800 0/4 0/2 4/4 0/2 0/4 0/2 ¼ 0/2 25600 0/4 0/2 0/4 0/2 0/4 0/2 0/4 0/2 51200 0/4 0/2 0/4 0/2 0/4 0/2 0/4 0/2 antigen dilution 1:400 and conjugate dilution 1:2000. ei, chicken were inoculated intratracheally, intranasally, and by eyedrop with approximately 2.3 x 10 colony forming units per ml of 24 h broth culture strain s6. ci, uninoculated chicken placed in the same cage with inoculated chickens. positive sample/tested sample, number of positive samples/ number of samples tested. for hi tests positives values 5 hi titre; for elisa, positive values absorbance at (410 nm) 0.255. a, tested with whole cell antigen; b, tested with sds-solubilized antigen; c, tested with sonicated antigen. 8 m. gallasepticum > > table 3 absorbance values of cross-reaction test obtained in elisa using untreated and treated rabbit anti-sera to and with antigens mycoplasma gallisepticum, m. synoviae, m. meleagridis m. gallisepticum antigen antisera mg ms mm untreated treated untreated treated untreated treated wc 1.500 0.973 0.651 0.267 0.343 0.032 ss 1.362 0.762 0.290 0.158 0.294 0.030 s 0.974 0.597 0.326 0.236 0.113 0.027 for elisa untreated: antigen and serum dilution of 1:400 dilution was used; for elisa treated: treated sera and antigen were diluted 1:200, rabbit anti, , and sera were used. mg, ms, mm, wc, whole cell antigen; ss, sds-solubilized antigen; s, sonicated antigen. m. gallisepticum m. synoviae m. meleagridis m. gallisepticum; m. synoviae; m. meleagridis; table 4 comparison of serum plate agglutination (spa), haemagglutination inhibition (hi) test, and elisa : cross-reactivity of antisera obtained from experimentally infected (ei) chickens and contact-infected (ci) chickens with antigens mycoplasma gallisepticum mycoplasma synoviae ei, chicken were inoculated intratracheally, intranasally, and by eyedrop with approximately 2.3 x 10 colony forming units per ml of 24 h broth culture strain s6. ci, uninoculated chicken placed in the same cage with inoculated chickens. positive sample/tested sample, number of positive samples/ number of samples tested. for hi tests positives values 5 hi titre; for elisa positive values absorbance at (410 nm) 0.255. 8 m.gallasepticum > > day post infection spa test hi test elisa ( positive sample /tested sample ) ci ei ci eiei ci 0 0/4 0/2 0/4 0/2 0/4 0/2 7 2/4 0/2 0/4 0/2 0/4 0/2 14 2/4 0/2 0/4 0/2 0/4 0/2 21 3/4 1/2 0/4 0/2 0/2 28 4/4 2/2 0/4 0/2 0/2 35 4/4 2/2 0/4 0/2 0/4 0/2 3/4 3/4 volume 4, 2010 microbiol indones 123 antigen was first detected the igg by day 7 post-infection in ei chickens, and by day 21 in ci chickens (table 5). the development of immunoglobulin m (igm) in ei and ci chickens detected by elisa of whole-cell, sdssolubilized, and sonicated antigen were compared. the wholecell antigen gave the highest result. in ei chickens igm titres increased from day 7 to day 21 post-infection and decreased from day 28 to day 35 post-infection. in ei chickens igm titres increased from day 7 to day 21 post-infection and decreased from day 28 to day 35 post-infection (fig 3). serum plate agglutination, elisa and tracheal-culture can detect infection in ei chickens by day 7 post-infection, and by day 21 in ci chickens. data obtained in these experiments showed there is correlation between seropositivity and the rate of isolation of thus serum-plate-agglutination and elisa were effective in identifying infection in ei and ci chickens. these data suggest that serum plate agglutination and elisa can identify culture positive chickens, but the rate of discussion m. gallisepticum m. gallisepticum. m. gallisepticum table 5 development of immunoglobulin g (igg) antibodies in experimentally infected (ei) chickens and contact-infected (ci) chickens, detected by elisa days post infection elisa a elisa b elisa c ei ci ei ci ei ci 0 0.104 0.082 0.106 0.070 0.075 0.070 7 0.386 0.088 0.355 0.080 0.381 0.073 14 1.356 0.221 0.734 0.103 0.981 0.111 21 1.473 0.485 0.783 0.363 1.143 0.355 28 1.690 0.879 1.176 0.567 1.433 0.596 35 1.731 1.045 1.414 0.841 1.631 0.925 ei, chicken were inoculated intratracheally, intranasally and by eyedrop with approximately 2.3 x 10 colony forming units per ml of 24 h broth culture strain s6. ci, uninoculated chicken placed in the same cage with inoculated chickens. elisa : mean absorbance (a ), obtained in elisa, using whole cell antigen. elisa : mean absorbance (a ), obtained in elisa, using sds-solubilized antigen. elisa : mean absorbance (a ), obtained in elisa, using sonicated antigen. 8 a b c mycoplasma gallasepticum 410 nm 410 nm 410 nm isolation of from field material does not always correlate with seropositivity (salami . 1992). these authors reported that mycoplasma could not be isolated from several seropositive birds; in addition fritz (1992) reported that only a small number of and was isolated from serologically-positive wild turkeys. feberwee (2005a) found in their study that a certain level of false positive results can be expected in any serological test. isolation of avian mycoplasma from field material is influenced by several factors. according to amin and jordan (1978) these factors can be classified into two main groups. first, those associated with the organs and tissues of the host, such as the duration of infection, intercurrent infection with other avian pathogens, the presence of competing flora, especially fast growing mycoplasma, transfer and treatment of tissue. second, those associated with the provision of growth requirements, such as the components, form and ph of the medium, temperature and humidity of incubation. the serum-plate agglutination and haemagglutination tests have been used for many years to diagnose infection. the non-specific reaction which occurs with the serum plate agglutination test makes it necessary to resort to the haemagglutination-inhibition test to confirm a diagnosis (patten 1984). however the haemagglutination inhibition test lacks sensitivity, especially in early stages of infection, and certain strains of appear incapable of eliciting a response which can be detected by the haemagglutination test (sahy and olsen 1981). thus a more sensitive and specific assay is needed. the elisa is a sensitive test which could overcome some deficiencies of the other tests. however, one of the main disadvantages has been the appearance of crossreactivity between and (talkington . 1984) and false positive reactions with negative sera (jordan and mustafa 1983). three different preparations of antigen, whole cell, sdssolubilized, and sonicated antigen were used in these experiment. both sds-solubilized and sonicated antigen gave lower absorbance than the whole cell antigen. it can only be assumed that sonication breaks down the protein to peptides that are not recognized by the antibody and also sonication generated heat that can denature the protein causing changes in its conformation. to determine whether an absorbance value was positive or negative, fixed baseline values were calculated using wholecell antigen as a standard, a threshold absorbance value of mean ± 4sd (0.255) were chosen. this caused a loss of sensitivity but was necessary in order to maintain specificity. the performance of elisa was assessed by comparison with the spa and hi test. when compared with the hi test, elisa proved sensitive. elisa successfully confirmed 75% of -infected chickens as positive by day 7 post infection, and by days 14 to 35 post infection elisa successfully confirmed 100% of infected chickens. in the comparison, hi test showed no activity until day 7 post-infection and by days 14 to 35, the hi test gave positive results with all infected chickens. this m. gallisepticum et al et al. m. gallisepticum m. synoviae et al. m. gallisepticum et al. m. gallisepticum m. gallisepticum m. synoviae et al m. gallisepticum m. gallisepticum 124 afiff microbiol indones 0 0.1 0.2 0.3 0.4 0.5 0.6 0 7 14 21 28 35 days post infection a (4 1 0 n m ) fig 3 development of immunoglobulin m (igm) antibodies in experimentally-infected (ei) and contact-infected (ci) chickens with strain s6 detected by elisa. ◊, ei whole cell antigen; ♦, ci whole cell antigen; δ, ei sds-solubilized antigen; ▲, ci sds-solubilized antigen; □, ei sonicated antigen; ■, ci sonicated antigen. mycoplasma gallisepticum may be because the hi test detected antibody in the 7s igg class, which normally does not appear until approximately 2 weeks after primary exposure (roberts and olesiuk 1967). although it was less sensitive than elisa, the hi test was much more specific. when compared with the spa test, elisa was much more specific, but less sensitive. however, because the spa test measures igm antibodies and elisa primarily measure igg antibodies, although light-chain cross-reactivity is possible. it is therefore difficult to effectively compare the sensitivities of these assays. many of the false positive reactions in the elisa have been correlated prior to inoculation of poultry with commercial fowl coryza ( ) bacteria, also inactivated infectious bursal disease virus vaccine produce strong systemic antibody responses to components of mammalian sera (avakian and kleven 1990). it is known that serum components from the growth medium become associated with the cell during growth (thorn and boughton 1980). it is suggested that these associated medium components contribute significantly to false positives in serology. timms and cullen (1974) suggested that the presence of rheumatoid factor in chicken sera was a cause of false positive reactions, and ansari (1982) suggested a common mycoplasma antigen exists between and because of the extreme sensitivity of elisa. in these experiments sds-solubilized antigen was more specific than sonicatedand whole cellantigen, it may be because solubilization with sds causes a removal of common antigen (higgins and whithear 1985). avakian and kleven (1990) suggested that purified antigen would be necessary to develop more specific elisa. serology tests are particularly helpful in screening poultry flock. it is important to know the mycoplasma infection status of poultry flock, as this will influence decisions related to use of antibiotics, vaccination programme and biosecurity planning. immunological responses of can be followed by quantification of igg and/or igm concentrations. this technique is nowadays widely applied for diseases control in mycoplasmosis. the concentrations of igg and/or igm are be measured prior to and post challenges. the effectiveness of mg vaccines are compared between serological responses of vaccinated groups to control animals are given by gatesa . (2008). haemophilus paragallinarum m. gallisepticum in vitro m. gallisepticum et al. m. gallisepticum m. synoviae m. gallisepticum-infected chickens, et al references amin mm, jordan ftw. 1978. a comparative study of some cultural methods in the isolation of avian mycoplasma from field material. avian pathol 7:455-70. ansari aa, taylor rf, chang ts. 1982. application of enzyme-linked immunosorbent assay for detecting antibody to infection in poultry. avian dis 27:21-35. avakian ap, kleven sh. 1990. the humoral immune response of chickens to and studied by immunoblotting. vet microbiol 24:155-69. avakian ap, kleven sh, glisson jr. 1988. evaluation of the specificity and sensitivity of two commercial enzyme-linked immunosorbent asay kits the serum plate agglutination test and the hemagglutination-inhibition test for antibodies formed in response to . avian dis 32:262-72. m. gallisepticum mycoplasma gallisepticum mycoplasma synoviae mycoplasma gallisepticum bencina d, tadina t, dorrer d. 1988. natural infection of ducks with and and myocplasma egg transmission. avian pathol 17:441-9. charlton br, bickford aa, chin rp, walker rl. 1999. randomly amplified polymorphic dna (rapd) analysis of isolates from turkeys from the central valley of california. j vet diagn invest 11:40815. feberwee a, mekkes dr, de wit jj, hartman eg, pijpers a. 2005a. comparison of culture, pcr, and different serologic tests for detection of and infections. avian dis 49:260-8. feberwee a, mekkes dr, klinkenberg d, vernooij j, gielken a, stegeman j. 2005b. an experimental model to quantify horizontal transmission of . avian pathol 34:355-61. fritz ba, thomas cb, yuill tm. 1992. serological and microbial survey of in wild turkeys from six western states. j wildlife dis 28:10-20. gatesa ae, frascaa s, nyaokeb a, gortona ts, silbart lk, geary sj. 2008. comparative assessment of a metabolically attenuated mutant as a live vaccine for the prevention of avian respiratory mycoplasmosis. vaccine 26:2010-9. higgins pa, whithear kg. 1985. detection and differentiation of and antibodies in chicken serum using enzyme-linked immunosorbent assay. avian dis 30:160-8. mycoplasma species. j clin microbiol 43:90912. jordan ftw, mustafa a. 1983. preliminary studies on the elisa in examining chicken turkey and quail sera for antibodies to avian mycoplasma. yale j biol med 56:854. levisohn s, kleven sh. 2000. avian mycoplasmosis ( ). rev sci tech 19:425-42. miles aa, misra ss. 1938. the estimation of the bacterial power of the blood. j hyg 38:732-48. mettifogo e, buzinhani m, buim mr, piantino ferreira aj, kleven sh, timenetsky j. 2006. molecular characterization of mg isolates using rapd and pfge isolated from chickens in brazil. j vet med b 53:445-50. nascimento er, pereira vla, nascimento mgf, barreto ml. 2005. avian mycoplasmosis update. rev bras cienc avic 7:1-9. patten be, higgins pa, whithear kg. 1984. a urease-elisa for detection of mycoplasma infection in poultry. aust vet j 61:151-5. roberts dh, olesiuk om. 1967. serological studies with avian dis 11:104-9. sahy sp, olsen no. 1981. characterisation of an isolate of mycoplasma wvu 907 which possesses common antigens to . avian dis 25 4:943-53. salami jo, addo p, umoh ju, adegboye ds. 1992. chicken mycoplasmosis: a review with special reference to and vet bull 62:511-20. soeripto, 2009. [chronic respiratory disease (crd) in chicken] [in indonesian]. wartazoa 3:134-42. stakenborg t, vicca j, butaye p, maes d, baere td, verhelst r, peeters j, de kruif a, haesebrouck f, vaneechoutte m. 2005. evaluation of amplified rdna restriction analysis (ardra) for the identification of species. bmc infect dis 5:46, doi: 10.1186/14712334-5-46. talkington fd, kleven sh, brown j. 1984. an enzyme-linked immunosorbent assay for the detection of antibodies to in experimentally infected chickens. avian dis 29:53-70. thorns cj, boughton e. 1980. studies on the effect of growth medium composition on the antigenicity of j hyg 84:29-36. timms lm. 1967. isolation and identification of avian mycoplasma. j med lab technol 24:78-89. mycoplasma synoviae mycoplasma gallisepticum mycoplasma gallisepticum mycoplasma gallisepticum mycoplasma synoviae mycoplasma gallisepticum mycoplasma gallisepticum meleagridis galloparo mycoplasma gallisepticum m. gallisepticum m. synoviae mycoplasma gallisepticum m. synoviae. m. gallisepticum m. gallisepticum m. synoviae. mycoplasma mycoplasma gallisepticum mycoplasma bovis. hong y, garcı´a m, levisohn s, lysnyansky i, leiting v, savelkoul phm, kleven sh. 2005. evaluation of amplified fragment length polymorphism for differentiation of avian volume 4, 2010 microbiol indones 125 weisburg wg, tully jg, rose dl, petzel jp, oyaizu h, yang d, mandelco l, sechrest j, lawrence tg, van etten j, maniloff l, woese cr. 1989. a pylogenetic analysis of the mycoplasmas: basis for their classification. j bacteriol 171:6455-67. 126 afiff microbiol indones timms lm, cullen ga. 1974. detection of infection in chickens and its differentiation from infection. british vet j 130:75-84. mycoplasma synoviae mycoplasma gallisepticum 05 prethrombin-2 (pt2) is a thrombin precursor that structurally has one glycosylation site and four disulphide bonds. as one of the fibrin glue components, thrombin is able to stick and to cover the wound. so, thrombin can be potentially applied in replacing suture technic (spotnitz and prabhu 2005). eventhough the fibrin glue has widespread applications, commercial fibrin glue has several disadvantages, i.e. allergic effect; pathogenic contamination, since the use of any plasma-derived product in the surgical setting carries a potential risk of viral transmission; and expensive protein therapeutics since must be imported (spotnitz 2001; enus et al. 2011). generally, glycosylated protein therapeutics are produced from mammalian cells. chinese hamster ovary (cho) is the most commonly used mammalian vol.8, no.4, december 2014, p 177-182 doi: 10.5454/mi.8.4.5 codon optimization and chaperone assisted solubilization of recombinant human prethrombin-2 expressed in escherichia coli 1,2 1 saronom silaban , iman permana maksum , 1 3 4 1 shabarni ghaffar , khomaini hasan , sutarya enus , toto subroto , 1* and soetijoso soemitro 1 biochemistry laboratory, department of chemistry, universitas padjadjaran, bandung, indonesia; 2 department of chemistry, universitas negeri medan, medan, indonesia; 3 bioscience and biotechnology research center, institut teknologi bandung, indonesia; 4 national eye hospital, rumah sakit mata cicendo, bandung, indonesia; prethrombin-2 (pt2) is a thrombin precursor, which plays a role in the conversion of fibrinogen into fibrin during blood clotting process. previous study reported that the expression of human prethrombin-2 (rhpt2) in escherichia coli formed inclusion bodies. the aim of this study was to establish a strategy to express a soluble rhpt2 in e. coli. this study was aimed to design and optimize the codon of human prethrombin-2 gene as well as to optimize the expression analyses condition using four strains of e. coli. the codon adaptation index (cai) of the unoptimized hpt2 gene was 0.336, with 56.8% gc content. after optimization, the cai of optimized hpt2 became 1.000 with 53.1% gc content. the optimized gene was successfully cloned into ptwin1 expression vector. expression analysis indicated that only e. coli arcticexpress strain could successfully express a soluble rhpt2 protein, with only small part of rhpt2 being expressed in insoluble form. however, the rest of the e. coli strains used in the experiments failed to express the rhpt2 in soluble form. we are deducing that the success in achieving soluble expression was not only due to the availability of chaperones cpn60/cpn10 in psychrophilic environment, which played a crucial role in the protein folding in e. coli arcticexpress strain, but also due to the codon optimization of hpt2 gene. key words: chaperons, codon optimization, e. coli, inclusion bodies, prethrombin-2 pretrombin-2 (pt2) adalah prekursor trombin yang berperan dalam mengubah fibrinogen menjadi fibrin selama proses pembekuan darah. penelitian sebelumnya melaporkan bahwa ekspresi pretrombin-2 manusia rekombinan (rhpt2) dalam inang escherichia coli selalu menghasilkan badan inklusi. tujuan penelitian ini adalah mencari strategi dalam mengekspresikan rhpt2 dalam bentuk terlarut dalam inang e. coli. desain eksperimen meliputi perancangan dan optimasi kodon pretrombin-2 manusia (hpt2) serta mengoptimasikan ekspresi rhpt2 terlarutdengan menggunakan empat galur e. coli. gen hpt2 menunjukkan codon adaptation index (cai) sebesar 0,336 dengan presentase gc 56,8%, sementara hasil optimasi menunjukkan berturut-turut sebesar 1,000 dan 53,1%. selanjutnya, gen hpt2 berhasil diklon ke dalam vektor ekspresi ptwin1. uji ekspresi protein rhpt2 terhadap empat galur inang e. coli menunjukkan bahwa galur arcticexpress mampu mengekspresikan protein rekombinan dalam bentuk terlarut walapun sejumlah protein juga terekspresi dalam bentuk badan inklusi. sementara galur lain menunjukkan hasil ekspresi protein rekombinan selalu dalam bentuk badan inklusi. selain preferensi kodon hpt2 yang sesuai dengan e. coli, peran chaperon cpn60/cpn10 dalam lingkungan psikrofilik yang terdapat pada e. coli arcticexpress memiliki peran yang sangat krusial dalam membantu pelipatan rhpt2, sehingga dapat diekspresikan dalam bentuk terlarut. kata kunci: badan inklusi, chaperon, e. coli, optimasi kodon, pretrombin-2 *corresponding author; phone: +62-818423339, fax: +6222-4218292 ; email: s_soemitro@unpad.ac.id cells due to safety consideration (septisetyani 2014). the first registered recombinant human thrombin in the united states was produced from its precursor, prethrombin-1, by using cho as a host (bishop et al. 2006). clinical study revealed that this product had homeostatic effect and was highly tolerance toward proteolytic digestion (burnouf 2011). however, this expression host is very expensive and has low economic value. escherichia coli remains the system of first-choice for expressing proteins, as it is cheap, easy to be handled, and has short-life cycle (cabrita et al. 2006). additionally, the genome of e. coli well studied, so it can be easily to be manipulated. however, inability of e. coli to express proteins with high molecular weight, disulphide bond rich, or posttranslational modification become a bottleneck . application of e. coli as a host on pathogenic free-thrombin production from prethrombin-2 had been reported. prethrombin-2 had previously been expressed in e. coli, however only small amount of thrombin was active due to the formation of inclusion bodies (soejima. et al 2001; freydell et al. 2007). in this study, several strategies were implemented to prevent the expression of rhpt2 as inclusion bodies in e. coli. we used codon optimization, host optimization and chaperone co-expression to express rhpt2 in soluble form. so, we established the expression system of soluble rhpt2 in e. coli. materials and methods strains, chemicals, vector, and medium. transformation and cloning was performed using e. coli top10f' (invitrogen, usa). e. coli er2566 expression host was purchased from new englands (new englands, biolabs, usa). e. coli bl21(de3), bl21(de3)_rosetta and bl21(de3)_arcticexpress were kind gifts from dr. jiri damborsky (masaryk university, brno, czech republic). the hosts were cultivated in luria bertani medium, (1% tryptone, 0.5% yeast extract, and 1% sodium chloride) supplemented by appropriate antibiotics (tetracycline -1 -1 100 µg ml or ampicillin 100 µg ml ). luria bertani medium with addition of 2% agar was used as solid medium. all restriction enzymes, t4 dna ligase, and ptwin1 expression vector were purchased from new englands (new england biolabs, usa). prethrombin2 gene was commercially synthesized by geneart (life technologies, jerman). isolation kit (roche applied science, usa) and gene extraction kit (geneaid, taiwan) were purchased commercially. isopropyl-β-d-thiogalactoside (iptg) dan βmercaptoethanol (βme) were from sigma aldrich (sigma aldrich, usa). polyacrilamide and commasie brilliant blue were from biorad (biorad, richmond, usa). construction and codon optimization of hpt2. the hpt2 synthetic gene was designed to be cloned into ptwin1 expression vector, using two restriction sites at 5' and 3' ends, xhoi and bamhi, respectively (fig 1). we extracted the sequence database at genbank with accession number: nm_000506.3: tatseyqtffnprtfgsgeadcglrplfekksle dkterellesyidgrivegsdaeigmspwqvml frkspqellcgaslisdrwvltaahcllyppwd knftendllvrigkhsrtryerniekismlekiy ihprynwrenldrdialmklkkpvafsdyihpv clpdretaasllqagykgrvtgwgnlketwt anvgkgqpsvlqvvnlpiverpvckdstririt dnmfcagykpdegkrgdacegdsggpfvmks pfnnrwyqmgivswgegcdrdgkygfythvf rlkkwiqkvidqfge (308 amino acids). afterwards, the sequence was optimized by optimizer free software (http://gnomes.urv.es/ optimizer; puigbo et al. 2007) based on codon usage database (http://www.kazusa.or.jp/codon/) and e. coli codon was used as codon preference. subsequently, optimized codon was analyzed by graphical codon usage analyzer (gcua) (http://gcua.schoedl.de/; mclnerney 1998). 178 silaban et al. microbiol indones fig 1 construction of ptwin1-hpt2. hpt and ptwin1 were cut using restriction enzymes xhoi and bamhi to obtain hpt2 and ptwin1 fragments. hpt2 fragment was ligated to ptwin1 using t4 dna ligase. cloning. general molecular biology experiment was performed according to sambrook and russel (2001). pma-t-hpt2 was cut by xhoi and bamhi. in parallel, ptwin1 was also cut by same restriction enzymes. the restriction fragments were characterized using 1% agarose gel electrophoresis. then, hpt2 and ptwin1 fragments were extracted from the agarose. the extracted hpt2 was ligated into ptwin1 by t4 dna ligase resulting ptwin1-hpt2. the cloned gene was verified by dna sequencing (macrogene, korea). expression analyses and characterization. hpt2 expression was tested in four different e.coli hosts, i.e. bl21(de3) arcticexpress, er2566, bl21(de3), and bl21(de3) rosetta. the protein expression was induced by iptg. single transformant o colony was grown overnight at 37 c in 5 ml lb medium supplemented with the appropriate antibiotics. one ml of overnight culture was then transferred into 100 ml of lb medium supplemented with appropriate antibiotic. subsequently, the culture was incubated for 3-4 h with shaking at 150 rpm until its optical density at 600 nm (od ) reached 0.5. 600 before induction, one ml culture was taken and labelled as 'before induction' sample. the culture was then induced with 0.1 mm of iptg. after induction, er2566, bl21(de3) and bl21(de3) rosetta strains o continued to be incubated at 30 c for 4 h, while the bl21(de3) arcticexpress were incubated at 12 °c overnight. one ml was taken and labelled as 'after induction' sample. the cell was harvested by o sentrifugation at 4 c, 7500 g. then, the cell was resuspended with 2 ml of 50 mm glycine buffer ph 7.5 and sonicated for 15 min. forty ml of the clear supernatant was kept and labelled as 'crude extract'. 50 l of 8 m urea was added into pellet and then the o sample was boiled for 15 min at 100 c. the sample was labelled as 'insoluble fraction'. the labelled samples were analysed by 12% sds page according to laemmli (1970). results codon optimization. codon sequence of hpt2 was analysed using optimizer. the result showed that gc percentage was 56.8% with cai 0.336. analysis of the codons by graphical codon usage analyzer (gcua) using e. coli codon preference indicated that 10 amino acids were encoded by non-preference codon. after codon optimization, gc percentage of hpt2 was 53.1% with cai of 1.000. cloning. the cloning strategy was depicted in fig 1. the hpt2 gene was cut from plasmid pma-t-hpt2 using xhoi and bamhi. in parallel, plasmid ptwin1 was cut with the same restriction enzymes. the agarose gel electrophoresis showed that hpt2 fragment was successfully cut from pma-t. two bands with molecular weight approximately 939 bp and 2.374 bp were indicated as hpt2 and pma-t, respectively (fig 2). ptwin1 digest also produced two bands with molecular weight 6.536 bp and 839 bp, corresponding to ptwin1 dan mcs (multi cloning site), respectively (fig 2). furthermore, the hpt2 and ptwin1 bands were extracted and purified from the gel. agarose gel electrophoresis proved that hpt2 and ptwin1 were successfully extracted and purified (fig 3). two bands appeared with molecular weight 939 bp and 6.536 bp indicating hpt2 and ptwin1. volume 8, 2014 microbiol indones 179 table 1 pattern of rhpt2 expression in e. coli then, the hpt2 fragment was ligated into ptwin1 vector (figure 1) using t4 dna ligase. to confirm the ligation process, the sequence ptwin1-hpt2 was verified by dna sequencing. the nucleotide alignment revealed that hpt2 was successfully inserted into ptwin1. protein expression analyses. rhpt2 expression was tested in four different e. coli strains (table 1). the rhpt2 was designed as fusion protein. the total molecular weight of fusion protein was 63.36 kda. the protein expression was analyzed by 12.5% sdspage. the results showed that e. coli bl21(de3) arcticexpress was able to express soluble cbd-inteinrhpt2 fusion protein (fig 4a). in contrast, er2566 (fig 4b), bl21(de3) (fig 4c) and bl21(de3) rosetta (fig 4d) failed to express rhpt2-fusion protein in soluble form, instead, these strains expressed the protein target inclusion bodies. discussion several parameters may affect the protein expression and solubilisation efficiency, for instance e. coli strain cbd-intein sspdnab-rhpt2 soluble insoluble er2566 + bl21(de3) arcticexpress +++++ ++ bl21(de3) +++ bl21(de3) rosetta + +++ fig 2 restriction analysis of pma-t-hpt2 and ptwin1 vector by using 1% agarose gel electrophoresis. line 1: pma-t-hpt2 was cut with xhoi and bamhi; line 2: ptwin1 vector was cut with the same restriction enzymes; m: 1 kb dna marker. fig 3 extraction analysis of hpt2 and ptwin1 by using 1% agarose gel electrophoresis. line 1: hpt2 extracted from agarose gel; line 2: ptwin1 extracted from agarose gel; m: 1 kb dna marker. fig 4 sds-page analysis of protein expression of cbd-intein-rhpt2 in 4 strains of e. coli. (a) bl21 (de3) arcticexpress, (b) er2566, (c)bl21(de3) and (d) bl21(de3) rosetta. line 1: cells before induction; line 2: cells after induction; line 3: soluble protein; line 4: insoluble protein; m: molecular weight marker. 180 silaban et al. microbiol indones ���� 3000 1000 1 2 m ptwin1 [6536 bp] pma-t [2374 bp] hpt2 [939 bp] mcs [839 bp] bp ���� 1000 1 2 m hpt2 [939 bp] ptwin1 [6536 bp] bp codon sequence, choice of host, chaperonins, or environment. the compatibility between expression system and host is important to express a soluble protein recombinant. in order to achieve that goal, we used a commercial ptwin1 vector. this vector has two advantages: first is as expression vector and second, the vector carried a highly specific purification tag, chitin-binding domain with self cleavableintein system. these advantages will be used for mass production and purification of rhpt2. the ptwin1 vector which carries t7 promoter, which has been used to express large quantity of soluble recombinant protein (studier et al. 1991; smith et al. 2012). differences between the host codon preference and the original hpt2 codon created a problem in the expression of heterologous protein. the hpt2 contains rare codons for e. coli, thus inefficient and/or misstranslation of the gene might occur (merkl 2003; gustafsson et al. 2004). rare codons for e. coli, such as agg or aga encoding arginine, is common in human gene (rinehart 2005). formation of inclusion bodies in e. coli is a major problem in the expression of recombinant protein (freydell et al. 2007). although it may also offer an advantage, that it is easily purified, as long as the protein can latter be refolded (singh and panda 2005). however, refolding involves multistep processes and the recovery of correctly folded and active enzyme are still uncertain (soejima et al. 2001). the first step in this study was codon optimization of hpt2 gene according to the codon preference of e. coli (gustafsson et al. 2004). rarely used codons were replaced with high frequency codons (xiong et al. 2008). every gene in the genome has numeric number called codon adaptation index (cai). the cai can be used predict the expression of heterologous protein. (carbone et al. 2003). the optimized codon was then chemically synthesized. this technology is so much more effective and efficient than gene isolation and, also, can prevent from disease transmission (hughes et al. 2011). it is well-known that e. coli is the most widely used o and suitable host of heterologous proteins at 21-49 c, o with an optimum at abou 37 c (ferrer et al. 2003), but when high expression of protein is needed, the e. coli's capability to accurately express recombinant proteins decreased, and inclusion bodies can be formed. low temperature has been proposed to improve protein solubility, but slower growth and low synthesis rates may downgrade protein yields (sorensen and mortensen 2005). to e l i m i n a t e t h i s o b s t a c l e , e . c o l i bl21(de3)_arcticexpress, an engineered e. coli strain capable of growing at low temperature and expressing the psychrophilic bacterium oleispira antarctica chaperones cpn60/10, was employed. these chaperones display high refolding activities and govern growth of e. coli at low temperatures (ferrer et al. 2004). as expected, expression analyses demonstrated that e. coli bl21(de3)_arcticexpress could produce hpt2 in soluble form. it can be concluded that the synergy among optimized-codon usage, e. coli bl21(de3)_ arcticexpress as a host, and psychrophilic environment, could be used to successfully produce a soluble form of recombinant hpt2. acknowledgment this work is supported by grant “penelitian unggulan strategis nasional (pusnas)” from directorate of higher education, ministry of national education, indonesia. references bishop pd, lewis kb, schultz j, walker km. 2006. comparison of recombinant human thrombin and plasma-derived human α-thrombin. semin thromb hemost. 32:086-097. doi: 10.1055/s-2006-939558. burnouf t. 2011. recombinant plasma proteins. vox sang. 100:60-83. doi:10.1111/j.1423-0410.2010. 01384.x. cabrita ld, dai w, bottomley sp. 2006. a family of e. coli expression vectors for 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4 page 5 page 6 2.mi669-chusnul hanim available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.2.2issn 1978-3477, eissn 2087-8575 vol 7, no 2, june 2013, p 51-58 *corresponding author; phone/fax: +62-274-889477, email : c.hanim@ugm.ac.id β-1,4-linked xylopyranose is the principal component of plant cell wall hemicellulose. the heteropolymer xylan represents the second most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid (dodd et al. 2009). hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. microbes use enzymes to hydrolyze xylan into its constituent sugars (dodd and cann 2009). a wide variety of microorganisms, including sea and soil bacteria, rumen bacteria, fungi, sea algae, and protozoa, as well as insect and cereals (sunna and antranikian 1997), are known to produce xylan-degrading enzymes (gessesse 1998). most of the bacteria and fungi secrete extracellular xylanases that hydrolyze hemicellulosic materials to liberate xylose as the end product, allowing the organisms to grow on this study was conducted to obtain xylanolytic mutants that have higher xylanase activity than their wildtype counterparts. a mutant with the best xylanolytic activity was selected and identified based on its 16s rrna sequence. its optimum growth condition was also characterized and its phylogenetic relations to other xylanolytic bacteria were analyzed. wild type xylanolytic alkalophlic bacteria were grown in medium containing xylan as a substrate. mutation was performed using ethidium bromide (etbr) or ethyl methanesulfonate (ems) at -1 concentrations 50, 100, and 150 mg ml and times of exposure 30, 60, 90, and 120 min for each treatment. twenty two mutants were obtained from etbr and 24 mutants from ems mutageneses. the mutants were analyzed for their capability to secrete xylanase into xylan medium containing xylose or glucose or glycerol. growth optimizations of the mutant were done in media with ph range 6-11 and temperature range 30 to 60 °c. -1 mutant number 19, which was obtained by treatment using 50 mg ml ems for 120 min, had the highest -1 xylanase activity (15.057 u g ). this activity was obtained at optimum growth conditions: ph 9.5 and temperature 55 °c. chromosomal dna of this mutant was extracted and amplified by pcr using 16s rrna gene specific primers. the amplified fragments were sequenced by dideoxynucleotide chain terminator method. the phylogenetic analysis based on 16s rrna gene sequence showed that mutant 19 was closed to an anaerobic xylanase producing bacteria. key words: ethidium bromide, ethyl methanesulfonate, mutagenesis, xylanolytic bacteria penelitian ini bertujuan untuk memperoleh mutan bakteri xilanolitik yang mempunyai aktivitas xilanase lebih tinggi dari aslinya, dan mengetahui kondisi optimum pertumbuhan mutan, serta mengidentifikasinya menggunakan 16s rrna untuk menjelaskan kekerabatan dengan bakteri penghasil xilanase lain. bakteri penghasil enzim xilanolitik alkalofilik dari hasil penelitian terdahulu ditumbuhkan dalam medium xilan. -1 mutasi dilakukan menggunakan etbr atau ems pada konsentrasi 50, 100, dan 150 mg ml , dengan lama pemaparan 30, 60, 90, dan 120 menit setiap perlakuan mutagen. mutan yang diperoleh ditumbuhkan dalam medium xilan yang mengandung xilosa atau glukosa atau gliserol, kemudian dianalisis aktivitas xilanasenya. optimalisasi pertumbuhan mutan dilakukan pada ph medium 6 sampai 11 dan suhu 30 sampai 60 °c di dalam medium yang mengandung xilan sebagai substrat. dari hasil penelitian diperoleh 22 mutant hasil mutasi menggunakan etbr dan 24 mutan dari mutasi dengan ems. mutan dengan kode 19 yang merupakan hasil -1 mutasi menggunakan 50 mg ml ems selama 120 menit menghasilkan aktivitas xilanase tertinggi yaitu -1 15,057 u g . mutan tersebut tumbuh pada ph optimum 9,5 dan suhu optimum 55 °c yang menghasilkan aktivitas xilanase tertinggi. dna kromosom mutan diekstraksi dan diamplifikasi menggunakan pcr, kemudian dilakukan sekuensing gen 16s rrna menggunakan metode dideoxynucleotide chain terminator. berdasarkan analisis pohon filogenetik 16s rrna diketahui bahwa mutan kode 19 termasuk dalam kerabat bakteri anaerob penghasil xilanase. kata kunci: bakteri xilanolitik,etidium bromida, etil metansulfonat, mutagenesis mutagenic improvement of xylanase production from xylanolytic bacteria and its phylogenetic analysis 1 1 2 chusnul hanim *, lies mira yusiati , muhammad nur cahyanto , 1 and ali wibowo 1 faculty of animal science, universitas gadjah mada , jalan fauna 3,yogyakarta 55281, indonesia; 2 faculty of agricultural technology, universitas gadjah mada, jalan sosio yustisia, bulaksumur, yogyakarta 55281, indonesia xylan. many xylanase producers are ruminal microorganisms, possibly due to the high hemicellulose content in the dietary of ruminant animals (kulkarni et al. 1999). two principally important enzymes involved in the recycling of hemicellulosic material are endo1,4-β–xylanases (ec 3.2.1.8), which cleave the xylan backbone, and β-d-xylosidases (ec 3.2.1.37), which cleave xylose monomers from the nonreducing end of xylo-oligosaccharides (dodd et al. 2009). in many cases the synthesis of these enzymes is induced by xylan and repressed by xylose. mutagenesis can degrade operator gen so that the enzymes are synthesized when repressors are presence (rajoka et al. 1997). in the past few years further studies on induction and secretion of xylanases had been performed to develop xylanase producers with possible commercial applications. xylanase production by various bacteria and fungi has been shown to be inducible. however, rare examples of constitutive xylanase expression have also been reported. in general, induction of xylanase production is a complex phenomenon and the level of response to individual inducer varies with the organisms. an inducer for producing maximum xylanase activity in one species may be the inhibitor of the activity in another species. the substrate derivatives and the enzymatic end products may often play a key positive role in the induction of xylanases. they can also act as the end-product inhibitors, possibly at much higher concentrations (kulkarni et al. 1999). ethidium bromide (etbr) (2,7-diamino-9phenylphenanthridium-1-ethyl bromide) has been used for various biological purposes. etbr induces petite mutations in yeast. it has been used to eliminate penicillinase plasmids and resistance transfer factors from bacterial cells. it is an inhibitor of nucleic acid biosynthesis and functions as mutagen in bacterial cells. etbr binds to nucleic acids in vitro and is a commonly used intercalating agent (bishop and brown 1973). intercalating agents induce dna damages which may cause either killing of cells or induction of mutation and cancer. most of such damages are subjected to common cellular dna repair mechanisms, such as excision repair and post-replication repair (kataoka et al. 1983). in this study we utilized etbr and ems as mutagen to enhance xylanase activity produced by xylanolytic bacteria. we have tried to optimize several growth parameters of the mutants, including medium ph and temperature. in addition, we studied the phylogenetic relationship between the best mutant and other xylanolytic bacteria. material and methods bacteria and culture conditions. the wild-type xylanolytic bacteria were isolated from crabs (eriocheir sinensis) from previous study as reported by hanim (2003). ethidium bromide (etbr) from wako and ethyl methane sulfonate (ems) from alfa aesar were used as mutagens. xylanolytic bacteria were grown in enrichment medium and recultured in growth medium with xylan as substrate. incubation was done at 35 °c, ph 11 for 7 d in anaerobic condition (hanim 2003). the enrichment medium contained 1 g (nh ) so , 0.5 g 4 2 4 mgso .7h o, 1 g k hpo , 2 g caco , 10 g oat spelt 4 2 2 4 3 xylan, in 1 l water, 10 ml nacl 1%, and 0.1% resazurin (skinner 1971). the growth medium contained 1 g (nh ) so , 0.1 g mgso .7h o, 7g nacl, 4 2 4 4 2 2gk hpo , 1 g yeast extract, in 1 l water, 0.1% 2 4 resazurin, 2% oat spelt xylan, and 0.5% cystein hcl (skinner 1971). mutagenesis and screening of mutants. the cultures were grown in defined medium with xylan as substrate, centrifuged (14 000 × g, 15 min, 5 °c), and exposed to different challenging doses of etbr -1 (concentration 50, 100, and 150 mg ml ) (chand et al. 2005), and ems (concentration 50, 100, and 150 mg -1 ml ) (rakariyatham et al. 2006), and incubated for 30, 60, 90, 120 min (chand et al. 2005). cells surviving these doses were grown in agar medium containing 1% oat spelt xylan in the presence of (2% w/w from oat spelt xylan) glucose, glycerol, or xylose. two days old colonies were picked and restreaked. larger colonies were picked for mutant selection. the clear zone that produced on xylan+xylose medium was larger than those in presence of glucose or xylose. the larger clear zones around the colonies indicated the higher production of xylan-degrading enzymes. the isolated colonies were subsequently replica-plated on xylan+xylose agar plate. each colony was studied for production of xylanase in vivo by measuring the diameter of the clear zone as visualized with congo red spread on the agar plates (gupta et al. 2000). optimization of xylanase production. optimization of xylanase production of the best mutant was performed at ph range 6 to 11, and temperature range 30 to 60 °c on xylan+xylose substrate. the incubation was performed at anaerobic condition for 5 d. the composition of growth medium was described above. culture medium was centrifuged at 14 000 × g for 15 min at 5 °c. the supernatant was carefully separated 52 hanim et al. microbiol indones from the pellet and measured for xylanase activity. we measured the protein concentration during the growth. enzyme assays. xylanase activity was determined by measuring the increase of reducing sugar concentration formed by enzymatic hydrolysis of soluble oat spelt xylan. enzyme sample (0.2 ml) was mixed with 0.2 ml of 4% (w/v) soluble oat spelt xylan and 0.4 ml 50 mm sodium acetate buffer (ph 6). the mixture was then incubated at 50 °c for 20 min (ruiz-arribas et al. 1995). the reducing sugar generated was quantified by nelson somogyi method (plummer 1978) using d-xylose as standard. one unit of enzyme activity was defined as the amount of enzyme that catalyzed the liberation of 1 µmol of reducing sugar per min. protein determination. protein content was determined by lowry method as described by plummer (1978). five ml of alkaline copper sulphate reagent was added to 2 ml extracellular protein sample. the mixture was then left to stand for 10 min at room temperature, followed by addition of 0.5 ml folin-ciocalteu phenol reagent, standing for 30 min at room temperature, and measuring the absorbance at 750 nm using bovine serum albumin as the standard. dna extraction and pcr conditions. bacterial dna was extracted using versatile quick-prep method for genome of gram-positive bacteria (pospiech dan neumann 1995 modified). the dna extraction was prepared with lysozyme and proteinase k, and then purified using microclean kit. 16s rdna from mutants were amplified by pcr with the 16s rdna degenerate primer f8 [5’agagtttgatc(a/c)tggctc-3’] and universal reverse primers r1492 [5’-gntaccttgttacgac tt-3’] (bera-maillet et al. 2004). pcr was done using mega mix blue kit. for pcr amplification, 30 cycles were conducted in an automated thermocycler. the following parameters were used; denaturation step for 30 s (2 min for the first cycle) at 94 °c, annealing step for 2 min at 52 °c, and extension step for 45 s at 72 °c. phylogenetic analysis. the purified dna was sequenced with an automatic sequencer. the 16s rrna nucleotide sequences were aligned using clustal_x (thompson et al. 1997). all sequence data retrieved from genbank database were used for comparative purposes. the alignments were analyzed using phylip program (felsenstein 1993), followed by analysis of the output file using neighbor program. similarity and difference of the nucleotide sequences were analyzed by phydit program (chun 1999). results screening for xylan-degrading activities. the mutation using etbr and ems showed different effects on the viability and xylanase activity of xylanolytic bacteria. etbr inhibited the growth of xylanolytic bacteria at much lower concentrations than ems. several bacterial colonies were still viable upon subculturing after 120 min exposure to the chemicals. there were 22 mutants surviving after exposure to etbr, while 24 mutants were found in ems. fourty six colonies were screened by substrate overlay method for their ability to degrade oat spelt xylan. twenty six mutants, that is 21 ems (87.5%) and 5 etbr mutants (23.81%), showed clearing zones around the colonies, thus indicating production of xylan-degrading enzymes. twenty six other mutants failed to produce clearing zone. mutant code 19, which was exposed to -1 50 mg ml ems for 120 min, was re-cultured in medium with xylan plus xylose as substrates. the mutant had the highest specific xylanase activity -1 (15.057 u g ). previous study showed that xylanase -1 activity of the wild-type was 9.317 u g . therefore, there was an increase (61.61%) of xylanase activity of mutant code 19 compared to its wild-type. effect of ph and temperature on xylanase production. mutant 19’s optimum incubation time for optimum xylanase activity and microbial protein concentration was 5 d (data is not shown). it was shorter than the wild-type's (7 d). the results (fig 1) clearly demonstrated that medium ph influence xylanase production of this mutant. fig 2 showed that temperature affected xylanase production. the optimum temperature was 55 °c, which produced the maximum xylanase activity. however, enzyme activity decreased sharply after incubation at lower or higher than the optimum temperature. phylogenetic analyses. phylogenetic relationships of mutant 19 to other xylanolytic bacteria available in the genbank (xylanibacterium ulmi lmg 21721 [accesion number nr 029089], xylanibacterium ulmi xil08 [ay273185], ruminobacter amylophilus dsm 43089 [y15992], xylanibacter oryzae kb3 [ab078826], ruminococcus albus atcc 27210 [l76598], ruminococcus flavefaciens atcc 19208 [l76603], blautia hansenii dsm 20583 [m59114], s. caprinus strain acm3969 [y10868], streptococcus gallolyticus acm 3611 [x94337], s. alactolyticus atcc 43077 [af201899], s. caballi strain 151 volume 7, 2013 microbiol indones 53 obtained could be used to subdivide the isolates into three groups (fig 3). the first group included strains of x. ulmi, x. oryzae, blautia hansenii, and several other strains from different geographic areas. the second and the third group contained streptococcus. mutant 19 had more than 82% similarity to b. hansenii dsm 2058, r. albus atcc 27210, and r. flavefaciens atcc 19208 (table 1). [ef364098], s. dysgalactiae subsp. equisimilis strain cip 105120 [dq232540], s. ictaluri strain 707-05 [dq462421], and s. gallinaceus ccug42692t [aj307888]), were analyzed. similarity value and number of nucleotide differences derived from analysis of 16s rrna sequences between mutant 19 and the other xylanase-producing strains are shown (table 1). in spite of their overall similarity, the sequence fig 1 effect of ph on the extracellular xylanase activity produced by xylanolytic mutant 19 grown anaerobically on xylan medium containing 2% xylose, at 35°c for 5d. xylanase activity was measured in the culture supernatant, and values are means of duplicated measurements. each point is the mean of standard deviation (sd) (indicated by error bar) from two different experiments. fig 2 effect of temperature on the activity of extracellular xylanase of xylanolytic mutant 19 grown on xylan medium containing 2% xylose at anaerobic condition, ph 9.5 for 5d. xylanase activity was measured in the culture supernatant and values are means of duplicated measurements. bars represent standard deviation (sd) for two different cultures. 54 hanim et al. microbiol indones 0 5 10 15 20 25 30 35 40 6 -1 x y la n as e sp ec if ic a ct iv it y ( u g ) 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 ph 0 10 20 30 40 50 60 -1 x y la n as e sp ec if ic a ct iv it y ( u g ) 30 35 40 45 50 55 60 temperature (°c) volume 7, 2013 microbiol indones 55 t ab le 1 s im il ar it y v al u e (% ) an d n u m b er o f n u cl eo ti d e d if fe re n ce s d er iv ed f ro m a n al y si s o f 1 6 s r r n a s eq u en ce s b et w ee n m u ta n t 1 9 a n d t h e o th er x y la n as e p ro d u ci n g s tr ai n s x . u lm i l m g 2 1 7 2 1 x . u lm i x il 0 8 r . a m yl o p h il u s d s m 4 3 0 8 9 s . ca p ri n u s st ra in a c m 3 9 6 9 s . g a ll o ly ti cu s a c m 3 6 1 1 s . a la ct o ly ti cu s a t c c 4 3 0 7 7 s . d ys g a la ct ia e su b sp . eq u is im il is st ra in c ip 1 0 5 1 2 0 s . ic ta lu ri st ra in 7 0 7 -0 5 s . ca b a ll i st ra in 1 5 1 s . g a ll in a ce u s 1 9 r . a lb u s a t c c 2 7 2 1 0 r . fl a ve fa ci en s a t c c 1 9 2 0 8 b . h a n se n ii d s m 2 0 5 8 3 x . o ry za e k b 3 x . u lm i l m g 2 1 7 2 1 -0 /1 5 1 3 3 2 6 /1 4 9 7 2 8 4 /1 2 3 2 3 4 2 /1 4 4 5 3 0 1 /1 3 9 2 3 1 6 /1 4 4 2 3 3 3 /1 4 2 6 3 1 7 /1 3 8 0 3 1 8 /1 4 5 7 3 2 1 /1 4 0 9 2 9 8 /1 3 8 8 3 0 4 /1 3 6 3 2 9 8 /1 3 8 0 3 5 7 /1 3 1 0 x . u lm i x il 0 8 a y 2 7 3 1 8 5 1 0 0 .0 0 -3 2 6 /1 4 9 7 2 8 4 /1 2 3 2 3 4 2 /1 4 4 5 3 0 1 /1 3 9 2 3 1 6 /1 4 4 2 3 3 3 /1 4 2 6 3 1 7 /1 3 8 0 3 1 8 /1 4 5 7 3 2 1 /1 4 0 9 2 9 8 /1 3 8 8 3 0 4 /1 3 6 3 2 9 8 /1 3 8 0 3 5 7 /1 3 1 0 r . a m yl o p h il u s d s m 4 3 0 8 9 7 8 .2 2 7 8 .2 2 -3 1 8 /1 2 5 7 3 7 7 /1 4 6 9 3 4 0 /1 4 1 7 3 7 1 /1 4 6 7 3 7 3 /1 4 5 1 3 6 0 /1 4 0 5 3 7 3 /1 4 8 2 3 3 5 /1 4 0 6 3 0 1 /1 3 9 1 3 0 4 /1 3 6 6 3 2 2 /1 3 9 0 3 8 2 /1 3 1 6 s . ca p ri n u s st ra in a c m 3 9 6 9 7 6 .9 5 7 6 .9 5 7 4 .7 0 -2 2 /1 2 6 6 3 9 /1 2 7 7 7 6 /1 2 7 7 7 7 /1 2 7 7 6 1 /1 2 5 6 7 5 /1 2 7 5 2 2 5 /1 2 3 4 2 6 3 /1 2 3 1 2 5 2 /1 2 2 8 2 6 1 /1 2 1 2 3 3 1 /1 2 0 8 s . g a ll o ly ti cu s a c m 3 6 1 1 7 6 .3 3 7 6 .3 3 7 4 .3 4 9 8 .2 6 - 6 2 /1 4 2 6 9 8 /1 4 6 7 9 6 /1 4 5 9 8 1 /1 4 1 1 1 0 1 /1 4 7 5 2 6 7 /1 4 0 6 2 8 6 /1 3 8 6 2 7 2 /1 3 6 5 2 8 8 /1 3 7 7 3 6 4 /1 3 1 2 s . a la ct o ly ti cu s a t c c 4 3 0 7 7 7 8 .3 8 7 8 .3 8 7 6 .0 1 9 6 .9 5 9 5 .6 5 -7 8 /1 4 3 7 7 8 /1 4 2 8 5 9 /1 3 9 6 6 8 /1 4 2 6 2 4 5 /1 3 7 8 2 7 2 /1 3 9 0 2 6 2 /1 3 7 2 2 7 6 /1 3 6 7 3 6 2 /1 3 2 3 s . d ys g a la ct ia e su b sp . eq u is im il is st ra in c ip 1 0 5 1 2 0 7 8 .0 9 7 8 .0 9 7 4 .7 1 9 4 .0 5 9 3 .3 2 9 4 .5 7 - 4 5 /1 4 6 6 7 7 /1 4 2 2 7 8 /1 4 6 4 2 4 8 /1 4 1 5 2 6 1 /1 3 9 8 2 6 1 /1 3 7 4 2 9 1 /1 3 9 4 3 6 6 /1 3 2 3 s . ic ta lu ri st ra in 7 0 7 -0 5 7 6 .6 5 7 6 .6 5 7 4 .2 9 9 3 .9 7 9 3 .4 2 9 4 .5 4 9 6 .9 3 -6 5 /1 4 2 2 8 2 /1 4 6 9 2 5 2 /1 4 1 5 2 7 2 /1 3 8 3 2 6 8 /1 3 7 4 2 9 0 /1 3 7 3 3 6 5 /1 3 2 3 s . ca b a ll i st ra in 1 5 1 e f 3 6 4 0 9 8 7 7 .0 3 7 7 .0 3 7 4 .3 8 9 5 .1 4 9 4 .2 6 9 5 .7 7 9 4 .5 9 9 5 .4 3 -7 3 /1 4 2 2 2 5 2 /1 3 8 1 2 7 5 /1 3 5 5 2 6 3 /1 3 5 3 2 8 7 /1 3 4 8 3 5 6 /1 3 2 5 s . g a ll in a ce u s 7 8 .1 7 7 8 .1 7 7 4 .8 3 9 4 .1 2 9 3 .1 5 9 5 .2 3 9 4 .6 7 9 4 .4 2 9 4 .8 7 -2 5 5 /1 4 1 6 2 6 4 /1 3 8 4 2 6 0 /1 3 7 5 2 7 7 /1 3 7 3 3 6 0 /1 3 2 4 7 7 .2 2 7 7 .2 2 7 6 .1 7 8 1 .7 7 8 1 .0 1 8 2 .2 2 8 2 .4 7 8 2 .1 9 8 1 .7 5 8 1 .9 9 -2 4 5 /1 3 7 0 2 4 1 /1 3 6 8 2 3 5 /1 3 5 8 3 5 2 /1 3 0 6 r . a lb u s a t c c 7 8 .5 3 7 8 .5 3 7 8 .3 6 7 8 .6 4 7 9 .3 7 8 0 .4 3 8 1 .3 3 8 0 .3 3 7 9 .7 0 8 0 .9 2 8 2 .1 2 -1 1 6 /1 3 7 9 2 2 7 /1 3 7 0 3 5 0 /1 3 0 0 1 9 r . fl a ve fa ci en s a t c c 1 9 2 0 8 7 7 .7 0 7 7 .7 0 7 7 .7 5 7 9 .4 8 8 0 .0 7 8 0 .9 0 8 1 .0 0 8 0 .4 9 8 0 .5 6 8 1 .0 9 8 2 .3 8 9 1 .5 9 - 2 1 9 /1 3 4 7 3 6 0 /1 2 9 5 b . h a n se n ii d s m 2 0 5 8 3 7 8 .4 1 7 8 .4 1 7 6 .8 3 7 8 .4 7 7 9 .0 8 7 9 .8 1 7 9 .1 2 7 8 .8 8 7 8 .7 1 7 9 .8 3 8 2 .7 0 8 3 .4 3 8 3 .7 4 - 3 4 1 /1 2 7 2 x . o ry za e k b 3 7 2 .7 5 7 2 .7 5 7 0 .9 7 7 2 .6 0 7 2 .2 6 7 2 .6 4 7 2 .3 4 7 2 .4 1 7 3 .1 3 7 2 .8 1 7 3 .0 5 7 3 .0 8 7 2 .2 0 7 3 .1 9 - discussion etbr mutation was more lethal than ems. etbr inhibited bacterial growth at much lower concentrations than ems. thus, much fewer resistant xylanase producing bacteria were obtained by etbr mutagenesis. although etbr is known to cause cell death, it is not known whether the lethality is due to mutagenicity or to some other toxic effects. many bacteria are resitant to dna-intercalating compounds (lee et al. 1996). tomchick and mandel (1964) reported that the growth -4 of escherichia coli was partially inhibited by 1.2 x 10 m etbr, however, during growth in the presence of ethidium, rna and protein contents were relatively unaffected. etbr binds to nucleic acids in vitro and is commonly used as intercalating agent (bishop and brown 1973). alkylating agents induce dna damages, which may cause either cell death or induction of mutation and cancer (kataoka et al.1983). the use of different mutagenic agents including ultraviolet (uv), x-rays, gamma radiation, ethyl methane sulfonate (ems), n-methyl-n-nitro-n-nitrosoguanidine (ntg) and mustards demonstrated improvement for cellulase production (sangkharak et al. 2012). lee et al (1985) reported a mutant of strain atcc 824 was obtained by selection on larch xylan agar medium after exposure of the wild-type strain to 1% (vol/vol) ethyl methanesulfonate. the xylanase activity of this mutant, xyn 1, was about two fold higher when grown on oat spelt xylan at ph 6.0 than when grown on larch xylan at ph 5.2. in this study, we resulted xylanase activity of mutant code 19 was higher than the wild-type. the maximum -1 xylanase activity was detected at ph 9.5 (27.09 u g ), which was lower than the wild-type's optimum ph. the wild-type's optimum ph and temperature to produce maximum xylanase activity were 11 and 35 °c, respectively (hanim 2002). in order to determine the distribution of xylanase xylanibacterium ulmi lmg 21721 (nr 029089) xylanibacterium ulmi xil08 (ay273185) ruminobacter amylophilus dsm 43089 (y15992) xylanibacter oryzae kb3 (ab078826) ruminococcus albus atcc 27210 (l76598) ruminococcus flavefaciens atcc 19208 (l76603) blautia hansenii dsm 20583 (m59114) 19 (xylanolytic bacteria) streptococcus caprinus strain acm3969 (y10868) streptococcus gallolyticus acm 3611 (x94337) streptococcus alactolyticus atcc 43077 (af201899) streptococcus caballi strain 151 (ef364098) streptococcus dysgalactiae subsp. equisimilis strain cip 105120 (dq232540) streptococcus ictaluri strain 707-05 (dq462421) streptococcus gallinaceus ccug42692t (aj307888) 0.1 fig 3 phylogenetic tree constructed by 16s rrna gene sequences analysis of mutant 19 and other related xylan-degrading bacteria, with streptococcus gallinaceus ccug42692t (aj307888) as the out-group. scale bar shows 1 substitution per 10 nucleotides. 56 hanim et al. microbiol indones chun j. 1999. phylogenetic editor (phydit). windows version. dodd d, cann ik. 2009. enzymatic deconstruction of xylan for biofuel production. glob change biol bioenergy. 1:2-17.doi:10.1111/j.1757-1707.2009.01004.x. dodd d, kocherginskaya sa, spies ma, beery ke, abbas ca, mackie ri, cann iko. 2009. biochemical analysis of a β-d-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in prevotella ruminicola 23. j bacteriol. 191 (10): 33283338. doi:10.1128/jb.01628-08. felsenstein j. 2002. phylip (phylogeny inference package), department of genome sciences. university of washington, seattle, usa. gessesse a. 1998. purification and properties of two thermostable alkaline xylanases from an alkaliphilic bacillus sp. appl environ microbiol. 64(9):3533-3535. gupta n, reddy vs, maiti s, ghosh a. 2000. cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic bacillus sp. strain ng-27. a p p l e n v i r o n m i c r o b i o l . 6 6 ( 6 ) : 2 6 3 1 2 6 3 5 . doi:10.1128/aem.66.6.2631-2635.2000. hanim c. 2002. characterization and stability analysis of xylanase from xylanolytic bacteria [thesis]. yogyakarta (id): biotechnology study program inter university centre, universitas gadjah mada.. hanim c. 2003. optimalisasi pertumbuhan bakteri xilanolitik alkalofilik dari ketam (eriocheir sinensis) [optimalization of growth of xylanolytic alkalophylic bacteria from crabs (eriocheir sinensis)]. buletin peternakan universitas gadjah mada 27(4):168-176. kataoka h, yamamoto y, sekiguchi m. 1983. a new gene (alkb) of escherichia coli that controls sensitivity to methyl methane sulfonate. j bacteriol. 153(3):13011307. kulkarni n, shendye a, rao m. 1999. molecular and biotechnological aspects of xylanases. fems microbiol rev. 23:411-456. doi:10.1111/j.1574-6976.1999.tb0040 7.x. lee lf, huang yj, chen cw. 1996. two classes of ethidiurn-bromide-resistant mutants of streptomyces lividans 66. microbiology 142(4):1041-1047. doi:10.1 099/00221287-142-4-1041. lee sf, forsberg cw, gibbins ln. 1985. xylanolytic activity of clostridium acetobutylicum. appl environ microbiol. 50(4):1068-1076. plummer dt. 1978. an introduction to practical rd biochemistry. 3 ed. tata mc graw hill publ. co. ltd. p 156-157. pospiecch a, neumann b. 1995. a versatile quick-prep of genomic dna from gram-positive bacteria. trends genet. 11(6):217-218. doi:10.1016/s0168-9525(00)89 052-6. rajoka mi, bashir a, malik ka. 1997. mutagenesis of cellulomonas biazotea for enhanced production of xylanases. bioresour technol. 62(3):99-108. doi:10.101 6/s0960-8524(97)00116-8. new delhi: during growth on xylan at different temperature and ph value, the xylanase activity was measured. it was observed previously that the production of xylanase by wild-type bacteria was affected by ph and temperature of medium (hanim 2003). hence, it was important to determine whether the production of xylanase of mutant was also influenced by the ph and temperature of medium, since this could affect bacterial growth in medium containing xylan as a substrate. the xylanolytic mutant showed maximal xylanase activity at alkaline ph (9.5). this optimum ph was 1.5 point lower than its wild-type (11). the optimum temperature was 55 °c. under these optimum conditions the enzyme showed maximum activity after 5 d incubation. kulkarni et al. (1999) reported that the optimum temperature for endoxylanase from bacterial and fungal sources varies between 40 and 60 °c. it was also reported by kulkarni et al. (1999) that d-xylanases from different organisms are usually stable over a wide ph range (3-10) and show optimum ph in the range of 4-7, however, certain alkaliphilic bacilli are known which have ph optima for growth and enzyme production at 9-10. the result of phylogenetic analysis showed that the 16s rrna sequences of mutant 19 had more than 82% similarity with the sequence of the closest strains b. hansenii dsm 2058, r. albus atcc 27210, and r. flavefacie atcc 19208. based on the similarity, the mutant was included in the first group including xylanase and cellulose-producing anaerobic bacteria, but this similarity was still less than 99%, thus the mutant cannot be classified in the same genera. acknowledgments we thank the laboratory of nutritional biochemistry for supporting our research. this work was supported by research grants from the ministry of education and culture of indonesia. references bera-maillet c, ribot y, forano e. 2004. fiber-degrading of different strains of the genus fibrobacter. appl environ microbiol. 70(4):2172-2179. doi:10.1128/ae m.70.4.2172-2179.2004. bishop pe, brown, l. r. 1973. ethidium bromide-resistant mutant of bacillus subtilis. j bacteriol. 115(3):10771083. chand p, aruna a, maqsood am, rao lv. 2005. novel mutation method for increased cellulose production. j app microbiol. 98(2):318-323. doi:10.1111/j.13652672.2004.02453.x. volume 7, 2013 microbiol indones 57 skinner fa. 1971. isolation of soil clostridia. in: isolation of anaerobes. the society for applied bacteriology technical series no. 5. london: academic press. sunna a, antranikian g. 1997. xylanolitic enzymes from fungi and bacteria. in: stewart gg, russell i, editors. crit rev biotech. 17(1):39-67. doi:10.3109/073885597 09146606. thompson jd, gibson tj,plewniak f, jeanmougin f, higgins dg. 1997. the c lustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucl acids res. 25(24):4876-4882. doi:10.1093/nar/25.24.4876. rakariyatham n, burt-indr b, niamsup h, shank l. 2006. improvement of myrosinase activity of aspergillus sp. nr4617 by chemical mutagenesis. j biotech. 9(4): 379385 on line. doi:10.2225/vol9-issue4-fulltext-13. ruiz-arribas a, fernandaz-abalos jm, sanchez p, garda al, santamaria ri. 1995. overproduction, purification, and biochemical characterization of a xylanase (xys 1) from streptomyces halstedii jm 8. appl environ microbiol. 61(6):2414 2419. sangkharak k, vangsirikul p, janthachat s. 2012. strain improvement and optimization for enhanced production of cellulase in cellulomonas sp. tsu-03. african j microbiol res. 6(5):1079-1084. doi:10.5897/ajmr11. 1550. 58 hanim et al. microbiol indones 1: 51 2: 52 3: 53 4: 54 5: 55 6: 56 7: 57 8: 58 8.mi684-dewi zilda available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.4.8issn 1978-3477, eissn 2087-8575 vol 6, no 4, december 2012, p 194-200 *corresponding author; phone: e-mail: seswitazilda@yahoo.com +62-21-53650157, proteolytic enzymes do not only play important roles in cellular metabolic processes but have also gained considerable interest for industrial applications. proteases account for nearly 60 % of the total annual turnover of the world enzyme market (chen et al. 2004; chu 2007). they are widely applied in detergent, protein modification, leather, meat, brewing, photographic, dairy, membrane cleansing, and waste treatment industries (kumar et al. 2002; chu 2007). the advantages of using thermostable enzymes in industry include; the capability to use elevated temperatures, thus facilitating faster reactions, increasing the solubility of nongaseous reactants/products and reducing the incidence of microbial contamination by mesophilic organisms (sokkheo et al. 2000; chen et al. o 2004). thermostable proteases are active above 60-70 c and withstand organic solvents, detergents, low and high ph values and other denaturing agents (covan et al. 1985; covan 1997; gupta and khare 2006). these properties are of particular interest for industrial processes. as the substrate of the proteases is unfolded at elevated temperatures, thermostable enzymes have-due to their characteristics-higher specific activities. they also appear to be rather useful in the synthesis of high molecular weight peptides due to their resistance against organic solvents, when processes are performed in media with low water content. (sellek and chaudhuri 1998; bruins et al. 2001; synowiecki 2008). hot springs are promising sources for direct isolation of thermostable enzymes producers. microorganisms in a hot spring are not only adapted to the elevated temperature but also to the prevalent ph and the presence of certain chemical compounds. due to the growing market and the potential applications of proteases, there is an increasing interest in the isolation of new bacterial species as proteolytic short communication hot springs represent one of the most promising sources for the isolation of thermostable enzyme producers. the microorganisms living in a hot spring not only have to withstand elevated temperatures but also extreme environmental ph and certain chemical compounds that are often toxic to other microbes. a bacterial strain denoted as brevibacillus thermoruber lii has been isolated from padang cermin hot spring, lampung, indonesia. optimization of the conditions for protease production by this strain revealed that the isolate produced o a thermostable protease optimally at temperature and ph ranges 45-55 c and 6-7, respectively, with keratin as substrate. the strain’s keratinolytic activity was shown by the ability to degrade untreated chicken feathers after 24 h incubation in liquid medium. key words: brevibacillus thermoruber, hot spring, thermostable protease sumber air panas merupakan salah satu sumber yang paling menjanjikan untuk isolasi penghasil enzim tahan panas. mikroorganisme yang hidup di sumber air panas tidak hanya tahan terhadap suhu tinggi tapi juga terhadap ph lingkungan yang ekstrim dan senyawa kimia yang seringkali bersifat racun bagi mikroorganisme lain. isolat brevibacillus thermoruber lii telah diisolasi dari sumber air panas padang cermin, lampung, indonesia. optimasi kondisi untuk menghasilkan protease oleh isolat tersebut menunjukkan bahwa isolat ini menghasilkan protease tahan panas secara optimal pada suhu dan ph masing-masing 45-55 °c dan 6-7 dengan keratin sebagai substrat. aktivitas keratinolitik strain ini ditunjukkan dengan kemampuannya mendegradasi bulu ayam tanpa perlakuan setelah inkubasi 24 jam pada medium cair. kata kunci: brevibacillus thermoruber, protease tahan panas, sumber air panas optimization of culture conditions to produce thermostable keratinolytic protease of brevibacillus thermoruber lii isolated from the padang cermin hot spring, lampung, indonesia 1 2 2 2 dewi seswita zilda * , eni harmayani , jaka widada , widya asmara , hari 1 1 1 eko irianto , gintung patantis , and yusro nuri fawzya 1 research and development center for marine and fishery product processing and biotechnology (kkp), jalan ks tubun petamburan vi, jakarta 10260, indonesia; 2 biotechnology study program, universitas gadjah mada, jalan teknika utara, barek yogyakarta 55281, indonesia enzyme producers with novel properties for industrial applications. therefore, rarely exploited regions and niche habitats are likely to yield such strains. there are many hot springs spread in lampung province which is connected to anak krakatau mountain, the active volcano located in sunda strait. one of those is hot spring which is an unnamed and unexplored spring located in padang cermin. there are many such springs in lampung, hence no reports on exploration of thermophile in this area are exist. gene encoding lon protease in b. thermoruber wr-249. lon proteases are atp-dependent serine peptidases belonging to the merops peptidase family s16 (lon protease family, clan sf). b. thermoruber had also been reported for b. thermoruber lii was isolated from the hot spring at padang cermin, lampung after extensive screening on minimal synthetic medium (msm plate agar containing 0.1% nacl, 0,1% k hpo , 0.1 % 2 4 nacl, 0.01% mgso .7h o, and 0.05 % yeast extract 4 2 supplemented with 1% skim milk). the purified cultures were preserved in 40% glycerol and stored at 70 °c. preliminary investigations revealed that out of three proteolytic isolates (clear zone around colony), strain lii produced a thermostable protease with highest activity when fermented at 50 °c in liquid medium after 22 h incubation. the protease was stable up to 100 min at 75 °c. for proper identification of lii by 16s rrna analysis, the dna of the bacterium was extracted using the tianamp dna kit. the 16s rdna (1.47 kbp) fragment was amplified by pcr using a pair of universal primers 16s rdna-27f and 16s rdna1492r (gee et al. 2003) and 1 µl chromosomal dna, 2µl b. thermoruber was reported at first as a new species of bacillus by manachini et al (1988) and showed protease producing capacity at optimum temperature 45 °c and optimum ph 9. wang et al. (2012) reported about a brevibacillus species that produced thermostable protease with slightly different characters to the one produced by strain lii (optimum temperature 75 °c and optimum ph 9). another experiment on this bacteria was reported by lee et al (2004). lee's group reported the presence of a its ability to degrade fibroin which is one of the substrates of protease (suzuki et al. 2009). all reports on protease produced by b. thermoruber showed that thermostable protease produced by lii had unique characters regarding optimum temperature and stability against thermal. 50 μl of the reaction mixture containing primer mix, 22 µl ultrapure water, 25 µl 2x tag pcr master mix (tiangen biotech, china). denaturation at 95 °c for 5 min, extension at 72 °c for 90 sec and denaturation at 95 °c for 60 sec were performed, followed by a final extension at 72 °c for 10 min. the nucleotide sequence of the amplified fragment was identified on a 3730 dna sequencer (applied biosystems, ca, usa) and subjected to a homology search against a dna database using the fasta program (http://fasta.ddbj.nig.ac.jp/). the nucleotide sequence was deposited at genbank with ab757752.1. the phylogenetic tree constructed on the basis of the sequence and presented in fig 1 identifies the strain as a representative of the b. thermoruber species. the protease activity was determined using a modified takami method (1989). the mixture containing 0.25 ml 1% casein in 0.025 m tris-cl buffer, ph 7 was incubated with 0.25 ml of the enzyme for 10 minutes. the reaction was stopped by adding 0.5 ml 0.4 m tca. the mixture was centrifuged at tm @ 10.000 rpm (beckman coulter micfuge centrifuge f 241.5p) for 10 min. supernatant (0.5 ml) was mixed with 2.5 ml 0.4 m na co and 0.25 ml folin-2 3 ciocalteu's phenol solution and incubated for 30 minutes at room temperature. the absorbance of the solutions were read against the sample blanks at 660 @ tm nm using spectronic 20 genesys . (spectronic instrument inc, rochester, ny). tyrosin standard -1 solution, in the range of 0-1000 mg l was prepared in triplicate to obtain a standard curve. one unit (u) of protease was defined as the amount of enzyme that could produce 1 μmol of tyrosine in one min under the defined assay conditions. zymogram analysis was carried out to check the finger print of the thermostable protease produced by strain lii based on the effect of incubation condition and substrate supplement. the samples, without prior heating, were loaded on a 10 % acrylamid gel supplemented with 1% gelatin for 1.5-2 h. before staining, the gel was immersed in 0.025 m tris-cl buffer ph 9 at the optimum temperature 75 °c for 30 min. the protease band appeared as clear zone surrounded by dark blue color of the gel. t m (gymorax amerex instrument inc.usa) temperatures 45 after 30 cycles of annealing at 55 °c for 30 sec, accession number the optimum temperature for enzyme production was investigated in 15 ml culture medium by supplementing 1.5 % skim milk in msm. the culture was incubated in a shaking incubator (150 rpm) at , 50, 55, and 60 °c. one loop-full of volume 6, 2012 microbiol indones 195 24 h culture from plate agar was inoculated into starter medium which had the same composition with working medium and incubated at temperature ; 1 % casein, 1 % collagen, 1 % skim milk, and 0.5 % keratin. strain lii is a gram positive, short rod with terminal or subterminal oval endospore. and grows optimally at temperatures range 45-55 °c. 16s rrna gene sequencing showed that lii belongs to b. thermoruber with 98% homolog. b. thermoruber lii produced the highest activity of previously mentioned. after 24 h incubation, 5% of the starter culture was inoculated into 15 ml working medium. the cell free supernatant was measured for the activity after 22 h incubation. the ph optimation of the b. thermoruber lii culture medium to produce the thermostable protease was carried out at ph 4, 5, 6, 7, 8, 9, and 10. for determination of the optimum substrate, four kinds of proteins were used keratin was prepared by washing chicken feathers with tap water followed by sun drying. subsequently, the dried feathers were boiled in 0.0125 m naoh for 20 min, neutralized with hcl and then again sun dried. the above procedure was repeated twice before the samples were ground and used as the substrate. thermostable protease when cultivated at 50 °c. high activity was found at 45 and 55 °c and although not too high, the activity of thermostable protease was still detected when cultivated at 60 °c (fig 2a) some reports showed the different temperatures for enzyme production, for examples bacillus subtilis strain 38's optimum production temperature was 47°c ( 2002), bacillus sp. mtg 30°c (gauda, 2006) and bacilus sp smta-2 60°c ( frankea et al. (1986) revealed on their study that the link between enzyme synthesis and energy metabolisms in bacteria was controlled by temperature as well secretion of extracellular enzyme. the temperature possibly changed the physical properties of the cell membrane for enzyme secretion (rahman et al. 2005). the activity of thermostable ( 2002), nevertheless, chantawannakula et al. nascimento & martins 2004). protease was detected over a broad ph range with the optimum ph at 6 (fig 2b). the result showed that lii preferred acidic to basic medium to produce the protease. nevertheless, up to ph 10 the activity was still detected. the optimum ph of medium for protease production was different from those of b. subtilis, of which the optimum ph was 6.5 chantawannakula et al. and bacillus sp strain fig 1 phylogenetic tree of strain lii based on 16s rrna gene sequences. genbank accesion numbers are given after the spesies name. bar, 10 substitution per 100 nucleotides. brevibacillus reuszeri jx999618.1 brevibacillus parabrevis kc172029.1 brevibacillus brevis jx286699.1 brevibacillus formosus nr_040979.1 brevibacillus nitrificans ab507254.2 brevibacillus choshinensis nr_040980 brevibacillus laterosporus jx155768.1 brevibacillus ginsengisoli nr_041376.1 brevibacillus nitrificans ab507254.2 brevibacillus invocatus nr_041836.1 brevibacillus thermoruber nr_026514.1 brevibacillus thermoruber ab112722.1 brevibacillus sp. fj490610.1 brevibacillus thermoruber ab362290.1 lii ab757752.1 brevibacillus borstelensi jx517230.1 10% 196 zilda et al. microbiol indones 1000 2000 3000 4000 5000 6000 0 s p ec if ic a ct iv it y ( u ) -1 m g 4 5 6 7 8 9 10 medium ph s p ec if ic a ct iv it y ( u ) -1 m g 1000 2000 3000 4000 5000 6000 7000 8000 0 temperature (°c) 45 50 55 60 fig 2 effect of temperature, ph, and substrate supplementation of culture medium on protease production by thermoruber lii. (a) effect of incubation temperature on protease production; (b) effect of medium production ph on protease production; (c) effect of substrate supplementation in culture medium on protease production. bacillus b a -1 s p e c if ic a c ti v it y ( u m g ) 1000 2000 3000 4000 5000 6000 7000 8000 0 substrates control skim milk casein colagen keratin c volume 6, 2012 microbiol indones 197 fig 3 effect of substrate supplementation in culture medium on protease finger print of brevibacillus thermoruber lii. (a) control, (b) skim milk, (c) casein, (d) colagen, (e) keratin, (m) marker. fig 4 scanning electron micrographs of feather. (a) before incubation with brevibacillus thermoruber lii culture; (b) after 24 h incubation with b. thermoruber lii culture. a b thermostable protease with high activity in liquid medium supplemented with 0.1% keratin showed that it had keratinolytic activity. the untreated chicken feather showed the damage after incubating with b. thermoruber in liquid medium as shown on fig 4. keratinases are a class of proteolytic enzymes which have capability degrading keratin to soluble form. several feather-degrading bacteria have been reported as keratinase producers that mostly belong to the genera streptomyces and bacillus (marcedo et al. 2005; kim et al. 2001: bressolier et al. 1999; chitte et al. 1999). among gram-positive bacteria, novel feather-degrading isolates have been identified as arthrobacter sp. (lucas et al. 2003), microbacterium sp. (thys et al. 2004), and kocuria rosea (bernal et al. 2006). unfortunately, the properties of thermostable keratinases have been reported only in a few cases. a m o n g t h e m i s k e r a t i n a s e i s o l a t e d f r o m smi-2 nascimento and martins 2004), which produced protease optimally at ph 8. ph of culture medium affected many enzymatic processes and transport of various component across the cell membrane, as reported by moon and parulekar (1991). the result showed that b. thermoruber lii produced protease optimally in medium supplemented with 0.1% feather keratin (fig 2c). the activity in medium with collagen and casein supplementation showed lower activity than control. the temperature and ph gave no effect on the type of thermostable protease produced by b. thermoruber lii as showed on acrylamid gel supplemented by 2 % gelatin (figure were not presented). however, the zymogram analysis showed that b. thermoruber lii produced more than one protease when the medium was supplemented with 1 % sikim milk as well as 1 % casein. the ability of b. thermoruber lii to produce ( 66.0 kd 97.0kd 20.1 kd 14.4 kd 45.0 kd 30.0 kd a b c d e ml 198 zilda et al. microbiol indones fervidobacterium islandicum aw-1, which had been characterized (nam et al. 2002). this experiment showed that protease produced by b. thermoruber lii had different character to those produced by other strain of b. thermoruber. there are no known reports about thermostable protease produced by b. thermoruber, which has keratinolytic activity since this paper submitted. further experiment to purify and characterize the enzyme is currently on work. references bernal c, cairó j, coello n. 2006a. purification and characterization of a novel exocellular keratinase from kocuria rosea. enz microb tech. 38(1-2): 49-54. doi: 10.1016/j.enzmictec.2005.02.021. bressolier p, letourneau f, urdaci m, verneuil b. 1999. purification and characterization of a keratinolytic serine proteinase from streptomyces albidoflavus. app environ microbiol. 65(5): 2570-2576. bruins me, janssen aem, boom rm. 2001. thermozymes and their applications. appl biochem biotechnol. 90(2): 155-186. doi: 10.1385/abab:90:2:155. chantawannakula p, oncharoena a, klanbuta k, chukeatiroteb e, lumyong s. 2002. characterization of proteases of bacillus subtilis strain 38 isolated from traditionally fermented soybean in northern thailand. sci asia. 28: 241-245. doi: 10.2306/scienceasia15131874.2002.28.241. chen xg, stabnikova o, tay jh., wang j.y., tay s.t.l. 2004. thermoactive extracellular proteases of geobacillus caldoproteolyticus, sp. nov., from sewage sludge. extremophiles 8(6): 489-498. doi: 10.1007/s00792-0040412-5. chitte rr, nalawade vk, dey s. 1999. keratinolytic activity from the broth of a feather-degrading thermophilic streptomyces thermoviolaceus strain sd8. lett app microbiol. 28(2): 131-136. doi: 10.1046/j.1365-2672.1999.00484.x. chu wh. 2007. optimization of extracellular alkaline protease production from species of bacillus. j ind microbial biotechnol. 34(3):241-245. doi: 10.1007/s10295-0060192-2. covan d, daniel r, morgan h. 1985. thermophilic proteases: properties and applications. trends biotechnol. 3(3):6872. doi:10.1016/0167-7799(85)90080-0. covan da. 1997. thermophilic proteins: stability and function in aqueous and organic solvents. comp biochem physiol., part a: mol integr physiol. 118(3): 429-438. doi: 10.1016/s0300-9629(97)00004-2. frankena j, koningstein, gm, verseveld hw, stouthamer ah. 1986. effect of different limitations in chemostat cultures on growth and production of exocellular protease by bacillus licheniformis. appl microbiol biotechnol. 24(2): 106-112. gauda mk. 2006. optimization and purification of alkaline proteases produced by marine bacillus sp. mig newly isolated from eastern harbour of alexandria. pol j microbiol. 55(2): 119-126. gee je, sacchi ct, glass mb, de bk, weyant rs, levett pn, whitney am, hoffmaster ar, popovic v. 2003. use of 16s rrna gene sequencing for rapid identification and differentiation of burkholderia pseudomallei and b. mallei. j clint microbiol. 41(10): 4647-4654. doi: 10.1128/jcm.41.10.4647-4654.2003. gupta a, khare sk. 2006. a protease stable in organic solvents from solvent tolerant strain pseudomonas aeruginosa. bioresour technol. 97(15): 1788-1793. doi: 10.1016/j.biortech.2005.09.006. kim jm, lim wj, suh hj. (2001). feather-degrading bacillus species from poultry waste. proc biochem. 37(3): 287-291. doi: 10.1016/s0032-9592(01)00206-0. kumar, d., h. gajju and t.c. bhalla, 2002. production of a thermostable protease by bacillus sp. apr-4. asian j microbiol biotechnol env sci. 4(4): 533-540. lee ayl, tsay s, chen my, wu sh. 2004. identification of a gene encoding lon protease from brevibacillus thermoruber wr-249 and biochemical characterization of its thermostable recombinant enzyme. eur j biochem. 271 (4): 834-844. doi: 10.1111/j.14321033.2004.03988.x. lucas fs, broennimann o, febbraro i, heeb p. 2003. high diversity among feather-degrading bacteria from a dry meadow soil. microbial ecology. 45(3):282-290. 10.1007/s00248-002-2032-x. manachini pl, fortina mg, carlo p. 1988. thermostable alkaline protease produced by bacillus thermoruber-a new species of bacillus. app microbiol biotech. 28(45): 409-413. doi: 10.1007/bf00268205. macedo aj, silva wob, gava r, driemeier d, henriques jap, termignoni c. 2005. novel keratinase from bacillus subtilis s14 showing remarkable dehairing capabilities. appl environ microbiol. 71(1): 594-596. doi: 10.1128/aem.71.1.594-596.2005. moon sh, parulekar sj. 1991. aparametric study of protease production in batch andfed-batch cultures of bacillus firmus. biotechnol bioeng. 37(5):467-483. doi: 10.1002/bit.260370509. nam gw, lee dw, lee hs, lee nj, kim b j, choe ea. 2002, native feather degradation by fervidobacterium islandicum aw-1, a newly isolating keratinaseproducing thermophilic anaerobe. arc microbiol. 178(6): 538-547. doi: 10.1007/s00203-002-0489-0. nascimento wca, martins mll. 2004. production and properties of an extracellular protease from thermophilic bacillus sp. braz j microbiol. 35 (1-2): 91-96. doi: 10.1590/s1517-83822004000100015. rahman rnzr, geok lp, basri m, salleh ab. 2005. physical factors affecting the production of organic solvent-tolerant protease by pseudomonas aeruginosa strain k. bioresource technol. 96 (4): 429-436. doi: 10.1016/j.biortech.2004.06.012. sellek ga, chaudhuri jb. 1998. biocatalysis in organic media using enzymes from extremophiles. enz microb technol. 25 (6): 471-482. doi: 10.1016/s0141-0229(99)00075-7. volume 6, 2012 microbiol indones 199 sookkheo b, sinchaikul s, phutrakul s, chen st. 2000. purification and characterization of the highly thermostable proteases from bacillus stearothermophilus tls33. protein expres purif. 20(2): 142-151. doi: 10.1006/prep.2000.1282. suzuki y, matsui h, tsujimoto y, watanabe k. 2009. enzymatic degradation of fibroin fiber by a fibroinolytic enzyme of brevibacillus thermoruber yas-1. j of biosci bioeng. 108(3):211-215. doi: 10.1016/j.jbiosc.2009.04.005. synowiecki j. 2008. thermostable enzymes in food processing, in recent research developments in food biotechnology. enzymes as additives or processing aids. in: porta r, pierro p. di, mariniello l, editor. kerala: research signpost. p 29-54. takami h, akiba t, horikoshi k. 1989. production of extremely thermostable alkaline protease from bacillus sp. no. ah-101. app microbiol biotech. 30(2): 120 124. doi: 10.1007/bf00263997. thys rcs, lucas fs, riffel a, heeb p, brandelli a. 2004. characterization of a protease of a feather-degrading microbacterium species. lett appl microbiol. 39 (2): 181-186. doi: 10.1111/j.1472-765x.2004.01558.x. wang s, lin x, huang x, zheng l, zilda ds. 2012. screening and characterization of the alkaline protease isolated from pli-1, a strain of brevibacillus sp. collected from indonesia’s hot springs. j oc univ china. 11(2): 213-218. doi: 10.1007/s11802-012-1845-6. 200 zilda et al. microbiol indones 10 adi.cdr page 1 page 2 page 3 1 susan soka anastasia.cdr page 1 page 2 page 3 issn 1978-3477, eissn 2087-8575 volume 10, number 4, december 2016 i n d o n e s i a accredited at level “a” until februari 2019 no.040/p/2014 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 diana e waturangi, 2020 endang purwantini, 2020 friedhelm meinhardt, 2016 managing editors is helianti, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 netty widyastuti sigit, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan 15314, indonesia phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology. published in march, june, september, and december subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 150 000,200 000,institutional rate (institution or library) (idr) 240 000,400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi astutiati nurhasanah, 2020 kartini kramadibrata, 2020 maggy thenawijaya suhartono, 2016 maria inge lusida, 2016 neung tiaamroeng, 2020 norio kurosawa, 2020 raija laiho, 2016 wellyzar sjamsuridzal, 2020 yuan kun lee, 2020 yaya rukayadi, 2020 available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 vol 10, no 4, december 2017 acknowledgment volume 10 siti khodijah chaerun, institut teknologi bandung, triwibowo yuwono, universitas gadjah mada, indonesia valan arasu, king saud university, saudi arabia verawat, biotec national center for genetic engineering and biotechnology, thailand wahono sumaryono, badan pengkajian dan penerapan teknologi, indonesia wo ro h a s t u t i s a t y a n t i n i , universitas airlangga, indonesia wangsa tirta ismaya, dexa laboratories of biomolecular sciences, indonesia wellyzar sjamsuridzal, universitas indonesia, indonesia yaya rukayadi, universiti putra malaysia, malaysia yusra, universitas bung hatta, indonesia abu bakar salleh, universiti putra malaysia, malaysia achmad dinoto, lembaga ilmu p e n g e t a h u a n i n d o n e s i a , indonesia alucia anita artarini, institut teknologi bandung, indonesia amarila malik, universitas indonesia, indonesia antonius suwanto, institut pertanian bogor, indonesia astutiati nurhasanah, badan pengkajian dan penerapan teknologi, indonesia budiasih wahyuntari, badan pengkajian dan penerapan teknologi, indonesia brett neilan, university of newcastle, australia catur riani, institut teknologi bandung, indonesia d e b b i e s r e t n o n i n g r u m , institut teknologi bandung, indonesia dominngus male, universitas patimura, indonesia ekowati chasanah, department o f m a r i n e a ff a i r s a n d fisheries, indonesia ekachai chukeatirote, mae fah luang university,thailand eny ida riyanti, ministry of agriculture, indonesiaelfahmi, institut teknologi bandung, indonesia e r n a w a t i a r i f i n g i r i rachman, institut teknologi bandung, indonesia erni martani, universitas gadjah mada, indonesia fernita puspasari, institut teknologi bandung, indonesia hideki takahashi, tohoku university, japan hiroaki okamoto, jichi medical university, japan i made sudiana, lembaga ilmu pengetahuan indonesia, indonesia iman rusmana, institut pertanian bogor, indonesia is helianti, badan pengkajian dan penerapan teknologi, indonesia jignesh patel, university of texas medical branch at galveston, usa kartini kramadibrata, lembaga ilmu pengetahuan k h o m a i n i h a s a n , i n s t i t u t teknologi bandung, indonesia mahyudin abdul rachman, badan pengkajian dan penerapan teknologi, indonesia maria inge lusida, universitas airlangga, indonesia maria magdalena coman, università di camerino, italia maelita ramdani moeis, institut teknologi bandung, indonesia misri gozan, universitas indonesia, indonesia moritz müller, swinburne university of technology, malaysia n venkatesh, st. joseph’s college of engineering, omr chennai india nagendra p. shah, the university of hong kong, hong kong neung tiaamroeng, suranaree university of technology, thailand n i k n i k n u r h a y a t i , b a d a n pengkajian dan penerapan teknologi, indonesia oslan jumadi, universitas negri makasar, indonesia puspita lisdiyanti, lembaga ilmu pengetahuan indonesia, indonesia prasetyawan yunianto, badan pengkajian dan penerapan teknologi, indonesia priyo wahyudi, badan pengkajian dan penerapan teknologi, indonesia sabar pambudi, badan pengkajian dan penerapan teknologi, indonesia sartaj alam syed, the university of agriculture peshawar, pakistan sathianeson satheesh, king abdulaziz university, saudi arabia silva abraham, badan pengkajian dan penerapan teknologi, indonesia sinosh skariyachan, dayananda sagar institutions, india siswa setyahadi, badan pengkajian dan penerapan teknologi a.front cover 10(4)-december 3.mi720-muhammad sahlan available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.3issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 152-158 *corresponding author; phone/fax: +62-21-7863516/+6221-7863515 , email: sahlan@che.ui.ac.id apoptin is chicken anemia virus (cav) protein which contains 121 kinds of amino acid and molecular weight about 14 kda (noteborn et al. 1991; maddika et al. 2006). apoptin was reported can induces apoptosis in human transformed or tumor cells but not in normal cells (dannen et al. 1997). this specificity and selectivity properties may be the reason to develop apoptin production in larger scale. the common method that used by any researchers to develop production of this protein is by recombination. one of the major obstacles in apoptin recombinant production is to produce protein in native form. recombinant protein which is expressed in escherichia coli is often in an inclusion body (insoluble aggregate) which cause the protein can not form a proper tertiary structure and will be inactive (ahsan et al. 2005). besides, the use of isopropyl β-d1 thiogalacto pyranoside (iptg) as inducer in e. coli expression system would also inhibits the development of apoptin production in larger scale because it is not economical. recently, we reported that he succeded to produce recombinant apoptin in native form with bacillus subtilis 168 as cell host which has been transformed by gateway system the success of recombinan apoptin production in native form in the previous results open the way to develop this anticancer protein production to the larger scale. we optimized cultivation process of recombinant bacillus subtilis 168 harbouring poxgw12his8arg with apoptin gene in a stirred tank fermentor and shake flasks. the parameters to optimize cultivation process are xylose-inducer concentration, agitation speed, and aeration rate. the xylose-inducer concentration variations are carried out in a shake flasks with 100 ml volume broth, while the agitation speed and aeration rate variation is carried out in a stirred tank fermentor with 3 l volume broth. the xylose concentration is varied between 0-5% w/v, while agitation speed and aeration rate are varied between 150-1 250 rpm and 0.5-1.5 nl min respectively. the best condition in this cultivation is 1% w/v of xylose, 250 rpm of -1 agitation speed and 1,5 nl min of aeration rate giving the specific growth rate value for each parameter of 0.628 -1 -1 -1 h ; 0.630 h ; and 0.747 h respectively. the recombinant apoptin were purified by ni-nta column using akta system. however, the results showed that the optimum condition of producing recombinant apoptin was 1% w/v -1 of xylose, 250 rpm of agitation speed, and 0.5 nl min of aeration. key words: aeration, agitation, apoptin, optimization, xylose keberhasilan produksi apoptin rekombinan dalam bentuk native pada penelitian sebelumnya membuka jalan untuk mengembangkan produksi protein antikanker ini ke skala yang lebih besar. di dalam studi ini, kami melakukan optimasi kultivasi bakteri rekombinan apoptin dalam stirred tank fermentor dengan bakteri bacillus subtilis 168 rekombinan apoptin. parameter yang dioptimasi adalah konsentrasi induksi xylose, kecepatan agitasi dan laju aerasi. variasi konsentrasi induksi xylose dilakukan dalam shake flasks dengan volume kultur 100 ml dengan konsentrasi 0-5% b/v sedangkan variasi kecepatan agitasi dan laju aerasi dilakukan dalam stirred tank fermentor dengan volume kultur 3 l dengan kecepatan dan laju masing-masing adalah 150-250 rpm dan 0,5-1,5 -1 nl min . hasil yang didapat adalah pertumbuhan bakteri optimum dicapai pada konsentrasi xylose 1% b/v, -1 kecepatan agitasi 250 rpm, dan laju aerasi 1,5 nl min dengan nilai laju pertumbuhan spesifik bakteri untuk -1 -1 -1 masing-masing variasi adalah 0,628 h ; 0,630 h ; dan 0,747 h . apoptin rekombinan dimurnikan dengan menggunakan kolom ni-nta pada sistem akta. namun, hasilnya menunjukkan bahwa kondisi optimum untuk memproduksi apoptin rekombinan dicapai pada konsentrasi xylose 1% b/v, kecepatan agitasi 250 rpm, dan -1 laju aerasi 0,5 nl min . kata kunci: aerasi, agitasi, apoptin, optimasi, xylose cultivation process optimization of bacillus subtilis harbouring apoptin gene recombinant 1 1 1 muhamad sahlan *, yuki desiandini , muhammad iqbal , 2 3 4 nunuk widhyastuti , amarila malik , and siswa setyahadi 1 chemical engineering department, faculty of engineering, universitas indonesia, depok 16424, indonesia; 2 research center for biology, indonesian institute of sciences (lipi), cibinong 16911, indonesia; 3 facultry of pharmacy, universitas indonesia, depok 16424, indonesia; 4 center for bioindustrial technology, agency for assesment and aplication of technology (bppt), jalan mh thamrin 8, jakarta 10340, indonesia (japan) with poxgw12his8arg plasmid and use xylose as the inducer. this method is more realistic to be applied in the larger scale because it needs a cheaper inducer compared to iptg (sahlan et al. 2013). before doing the apoptin production in large scale, optimization studies are needed to determine the optimum operating conditions. in this study several parameters of recombinant bacteria cultivation are considered, which are agitation speed, aeration rate, and xylose concentration. optimization of agitation speed and aeration rate are carried out in a stirred tank fermentor (eyela) with 3 l volume broth while the optimization of xlose concentration is carried out in shake flasks with 100 ml volume broth. this study will show how these parameters affect the growth profile, the specific growth rate value of recombinant bacteria and production of the protein. material and methods microorganisms. bacteria used in this study is bacillus subtilis 168 has been transformed by gateway system (funded by jsps young researcher invitation program, naist, japan) with poxgw12his8arg plasmid for apoptin expression. cultivation medium. cultivation mediun used in -1 this study is luria bertani (lb) which contains (l ) 10 g of bacto tryptone (becton, dickinson & company), 10 g of nacl (merck), and 5 g of yeast extract (becton, dickinson & company). these three components are dissolved in aquadest and ph was adjusted to 7. this solution was then autoclaved at 121 °c for 15 min. the medium was then cooled down to ambient temperature and tetracycline (darya varia) is added as -1 antibiotics until its concentration reach 10 µg ml in the medium. cultivation in stirred tank fermentor for agitation seed and aeration rate optimization. optimization of agitation speed and aeration rate starts with inculating 10% v/v of preculture to 3 l of -1 sterilized medium that has contained 5 µg ml of o tetracycline at normal ph and 37 c. agitation speeds are varied between 150-250 rpm and aeration rates are -1 varied between 0.5-1.5 nl min . when od reaches 600 0.4, tetracycline was added again until its -1 concentration was 10 µg ml in medium. cultivation was then continued until od value reaches 0.6-0.8, 600 xylose (sigma-aldrich) is added in this step with 1% w/v concentration. the culture was cultivated again until stationary phase is reached. cultivation in shake flasks for xylose concentration optimization. lb medium which has -1 contained 10 µg ml of tetracycline is prepared in 18 flasks and volume of medium in each flask is adjusted so the xylose concentration in medium reach 0%, 1%, 2%, 3%, 4%, and 5% in 100 ml medium (each concentration is done in 3 repetitions). one mililitre of inoculums that has been dissolved in sterilized aquadest is added to each flasks. these flasks was then incubated in shaker incubator at 37 °c and 120 rpm until od value reach 0.6-0.8. next step is xylose 600 addition with the varied concentration and the culture was incubated again for 2 h and the growth profile will be evaluated. purification of recombinant apoptin. the recombinants apoptin were purified by akta system using ni-nta column (histrap ff) with buffer a (100 mm tris hcl ph 7.4, 100 mm nacl, and 20 mm imidazole) as binding and washing buffer and buffer b (100 mm tris hcl ph 7.4, 100 mm nacl, and 500 mm imidazole) as elution buffer. the purified recombinant apoptin was confirmed by 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (sds-page). the concentration proteis analyzed by bradford method with bovine serum albumin (bsa) as standard. results optimization of agitation speed in stirred tank fermentor. optimization of agitation speed was carried out in stirred tank fermentor with 3l of volume broth at constant temperature and aeration rate which -1 are 37 °c and 0,5 nl min respectively and three variations of agitation speed (150, 200, and 250 rpm). the growth profile on fig 1 shows that bacteria grows faster in higher agitation speed. it can be seen on the highest speed, the lag phase is shorter and the specific growth rate value is the highest (fig 2). optimization of aeration rate in stirred tank fermentor. optimization of aeration rate was also carried out in stirred tank fermentor with 3l volume broth at constant temperature and agitation speed which are 37 °c and 250 rpm respectively, with 3 -1 variation of aeration rates (0.5, 1, and 1.5 nl min ). based on specific growth rate value (µ) for each aeration rates (fig 4), the highest rates gives the highest µ which means the bacteria grows faster in higher aeration rate. the aeration rate that gives the best -1 bacterial growth in this study is 1.5 nl min . optimation of xylose concentration as inducer. optimization of xylose concentration is aimed to volume 7, 2013 microbiol indones 153 determine if increasing xylose concentration will affect the cell growth or not since xylose is not the secondary nutrition but as inducer to apoptin expression. this optimization was carried out in shake flasks with 100 ml of volume broth and the xylose concentrations are varied between 0-5% w/v. the growth profile on fig 5 shows that there is no significant effect in increasing xylose concentration to the bacterial growth. based on specific growth rate calculation (fig 6), the highest µ value is reached at 1% xylose concentration and decrease as the xylose concentration increase. the apoptin recombinan expression. the cells were collected and purified by liquid chromatography akta system using histrap ff column. the cell was lysed by sonication process, centrifugation and applied to the column. unbound protein was washed by binding buffer, after unbound protein was removed, the recombinant apoptin was eluted gradiently by the elution buffer. the apoptin was eluted in conductivity -1 about 17-22 ms cm (fig. 7). the fraction of protein signal peak was confirmed by sds-page. the results fig 1 bacterial growth profile in each agitation speed. : 150 rpm, : 200 rpm, and : 250 rpm. -1 µ ( h ) 0.7 0.6 0.5 0.4 150 200 250 agitation speed (rpm) 0.570 0.630 0.538 fig 2 specific growth rate value on each agitation speed. o d v al u e 6 0 0 2.5 2 1.5 1 0.5 0 0 2 4 6 8 10 12 time (h) 154 sahlan et al. microbiol indones oxygen more captured in the behind of agitator blades and is dispersed into medium. the oxygen is mixture with medium and contact with cell surface. the agitation also acts to homogenous ph and temperature in the medium. the cell growth fast when the agitation speed is increased (ducros et al. 2009). the agitation speed that gives the best bacterial growth in this study is 250 rpm. the interesting part that seen on fig 1 is there are tendency to form diauxic growth pattern at 150 and 200 rpm growth profile but not at 250 rpm. it shows that the agitation speed may be affect the bacterial growth pattern on substrate consumption because there is more than one carbon source in this cultivation (lb and xylose) so this phenomenon may be happened, but still need further study to prove this showed that the molecular weight of apoptin about 15 kda (fig 8). the amount of protein produced in each cultivation process was analysed by bradford method. the results showed in table 1. the results showed that the optimum cultivation process for production the recombinant apoptin for agitation speed, aeration rate, and xylose concentration for induction which are 250 -1 rpm, 0.5 nl min , and 1% w/v respectively. discussions the process of agitation in the aerobic cell culture is important to capture oxygen and disperse it into medium. the results are appropriate to the theory that state when the impeller speed increase, air containing fig 3 bacterial growth profile in each aeration rate. : -1 -1 -1 0.5 nl min , : 1 nl min , and : 1.5 nl min . -1 aeration rate (nl min ) 0.5 1 1.5 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -1 µ ( h ) 0.63 0.593 0.747 fig 4 specific growth rate value on each aeration rate. o d 6 0 0 v al u e 0 0.5 1 1.5 2 2.5 0 2 4 6 8 time (h) volume 7, 2013volume 7, 2013 microbiol indones 155 fig 5 bacterial growth profile in each xylose concentration. : 0%, : 1%, : 2%, : 3%, : 4%, and : 5% xylose concentration. -1 µ ( h ) 0.6 0.5 0% 1% 2% 3% 4% 5% xylose concentration (w/v) 0.613 0.628 0.622 0.601 0.603 0.602 fig 6 specific growth rate value on each xylose concentration. tabel 1 the apoptin productionrecombinant no. cultivation condition weight of the apoptin recombinant (mg) agitation (rpm) -1aeration (nl min ) 1. 150 0.5 0.025 2. 200 0.5 0.012 3. 250 0.5 0.152 4 . 250 1.5 0.149 o d 6 0 0 1.400 1.200 1.000 0.800 0.600 0.400 0.200 0.000 0 1 2 3 4 5 time (h) 156 sahlan et al. microbiol indones crude sample 24 23 22 21 20 19 18 marker 97 kda 66 kda 45 kda 30 kda 20.1 kda 14.4 kda fig 7 chromatogram of the purification step the recombinant apoptin in ni-nta column. (blue) uv detection at 280 nm, (red) conductivity, and (green) gradient program. fig 8 sds page of the recombinant apoptin. the molecular weight of recombinan apoptin was about 15 kda. volume 7, 2013 microbiol indones 157 danen-van oorschot a a a m, fischer d, grimbergen j, klein b, zhuang s m, falkenburg j h f, backendorf c, quax p h a, van der eb a j, noterborn m h m. 1997. apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. proc natl acad sci usa. 94(11):5843-5847. doi:10.1073/pnas.94.11.5843. doran p m. 1995. bioprocess engineering principles. california: academic press limited. ducros e, ferrari m, pelegrino m, raspanti c, bogni c. 2009. effect of aeration and agitation on the protease production by staphylococcus aureus mutant rc128 in a stirred tank bioreactor. bioprocess biosyst eng. 32(1):143-148. doi:10.1007/s00449-008-0233-5. maddika s, mendoza f, hauff k, zamzow c, paranjothy t, los m. 2006. cancer-selective therapy of the future: apoptin and its mechanism of action. cancer biol ther. 5(1):10-19. doi:10.4161/cbt.5.1.2400. noteborn m, de boer g, van roozelaar d, karreman c, kranenburg, vos j, et al. (1991). characterization of cloned chicken anemia virus dna that contains all elements for the infectious replication cycle. j virol. 65 (6):3131-3139. sahlan m, savitri i k, prasetyo a a, onuma c, ishikawa s, malik a, ogasawara n. 2013. efficient expression of recombinant soluble apoptin in escherichia coli and bacillus subtilis. int j chem environ biol sci. 1(1): 2320-4087. possibility. similar with agitation effect, the aeration rates also increased the dissolved oxygen level in the medium so the metabolism activity that also increased and the cell growth goes faster. the xylose is not directly influence cell growth, it is an inducer molecules for protein expression and not affect cell growth. it shown that apoptin is not toxic protein for b. subtillis. the fig 5 showed phenomenon of cell growth inhibition after supplemented by xylose. this inhibition phenomenon may be happened due to catabolite repression during cultivation. as for metabolites that may be formed are still being obstacle to be analyzed since the medium contains complex compounds. this phenomena is also related to the expression of the recombinant protein. in fast cell growth rate the recombinant protein is not well expressed. the results showed that the optimum cell growth rate was different with the recombinant protein production. references ahsan n, aoki h. 2005. over expression in escherichia coli and functional reconstitution of anchovy trypsinogen form the bacterial inclusion body. mol biotechnol. 30 (3):193-205. doi:10.1385/mb:30:3:193. 158 sahlan et al. microbiol indones 1: 152 2: 153 3: 154 4: 155 5: 156 6: 157 7: 158 05 sari layout baru.cdr vol.11, no.1, march 2017, p 28-34 doi: 10.5454/mi.11.1.5 construction and expression of single recombinant peptide surfactant for enhanced oil recovery (eor) application 1 1 2 1 cut nanda sari , usman , riesa kw rohmat , leni herlina , ken sawitri 1 1 2 2 suliandari , onie kristiawan , dwiyantari , tati kristianti , 2* and sony suhandono 1 ppptmgb lemigas, cipulir, kebayoran lama, jakarta 12330, indonesia; 2 school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia. surfactant is generally synthetic chemical, which is effective and reliable. however, the chemicals usually cannot be degraded easily in the environment and could cause damage to the environment. the other possible alternative to produce surfactant is using genetic engineering in order to produce peptide based surfactant. in this research, peptide surfactant was produced using a gene construct which was created using overlapped polymerase chain reaction method (oe-pcr). sds-page analysis showed that single surfactant peptide construction can be expressed by induction of iptg 1 mm and after at least twice sonication. this research proved that both two constructions have been successfully expressed by producing peptide in expected size (approximately 15 kda). key words: enhanced oil recovery, overlapped pcr method, surfactant peptide surfaktan digunakan untuk meningkatkan perolehan minyak bumi (enhanced oil recovery). surfaktan hasil sintesis ini bersifat cepat dan efektif, tetapi banyak menimbulkan masalah lingkungan. alternatif lain yang bisa digunakan untuk menghasilkan surfaktan dengan rekayasa genetika untuk produksi rekombinan peptida bersifat surfaktan dalam bakteri. pada penelitian ini dilakukan konstruksi genetik untuk menghasilkan surfaktan peptida dengan menggunakan metode overlapped-pcr (oe-pcr). hasil analisis sds-page menunjukkan konstruksi surfaktan peptida dapat diekspresikan dengan cara diinduksi iptg 1 mm dan setelah dua kali pemecahan sel. penelitian ini membuktikan bahwa konstruksi genetik berhasil mengekspresikan peptida pada ukuran yang sesuai (sekitar 15 kda). kata kunci: enhanced oil recovery, metode overlapped-pcr, peptida surfaktan microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-22-2511575, fax:+6222-2534107; email: sony@sith.itb.ac.id (salter et al. 2011). this compound causes damage in hydrodynamics open water and cause co exchange 2 effectivity that will harm the life at sea. beside that, oleyl alcohol is also irritant to human especially skin and eyes. one of eco-friendly surfactant may be produced using genetic engineering, a biosurfactant. biosurfactant production using natural microbial is usually called microbial enhanced oil recovery (meor). meor is the technology using microbial and the metabolic way for oil recovery (bachmann et al. 2014). oil recovery using microbial that is developed into biosurfactant has become advanced as seen from the numerous researches that studied and generated about biosurfactant (desai and banat 1997; van hamme et al. 2003; sandrea and sandrea 2007; maneerat 2005; dwyer et al. 2014; pacheco et al. 2010). meanwhile, biosurfactant has also weakness, the quality is hard to be standardized. biosurfactant need to be developed in recombinant in order to generate in large scale with assured quality. peptide based biosurfactant in this research has been designed by lemigas. this peptide sequence is then called as oil recovery from production wells still cannot fulfill the world oil demands (bachmann et al. 2014). oil is non-renewable resource, as the world demand of crude oil increased, the oil industry find difficulties to discover new oil resources. the industry should more focus on mature or even abandoned wells (brown and vadie 2000). primary oil recovery requires high cost with the result 20% 50% from original oil in place (ooip) (sandrea and sandrea 2007). therefore, the better oil recovery method with less production cost is needed. one of the existing technologies is using surfactant in order to mixed oil and water. the emulsion between oil and water results oil recovery and may increase crude oil recovery up to 30% 200%. however, the existing surfactant is not eco-friendly, because it is difficult to degrade in nature. one of the non ecofriendly surfactant is oleyl alcohol (ch (ch2) -3 7 ch=ch-(ch ) oh) which is produced synthetically 2 8 supel. earlier the peptide was synthesized chemically, which was very expensive. in order to make supel production cheaper, genetic engineering will be used in this research. peptide sequence was translated into dna sequence with consideration of optimum codon usage for escherichia coli, then the dna is made synthetically and constructed into plasmid pet 32-b+, which is then called as ss (single supel) construct. peptide will be produced by e. coli cell naturally and the result will be analyzed using poly acrilamid gel electrophoresis (page). materials and methods optimization of supel codon usage .this research will use e. coli strain bl21 (de3), therefore codon optimisation for translation in e. coli is needed for efficient production of the peptide. this codon usage optimation was made by codon adaptation indexed (cai) with reference of highly expressed gene (maloy et al. 1996). the optimise codon usage of the supel presented in table 1. single supel construction design. the dna sequenced was ordered to be synthesised chemically at genescript. the cloned gene was inserted with tobacco etch virus (tev) protease peptide recognition side. this has been made by inserting dna sequence into a primer that was designed to amplified supel. a pair of primer is designed to have (30 bp) sequence that complements each other at tip point 3' (tev-f primer and tev-r primer). as a result the supel fragment contain dna sequence encode tev protease peptide recognition side. the cloned dna inculding the protease recognition site was construted into pet32b+ using overlapped pcr. specific primer pair which is used for amplification [tev + surfactant's composer nucleotide] is: tev-f primer and tev-r primer. pcr profile of tev amplification and surfactant's coding nucleotide is started with pre-denaturation 95 °c for 3 min, cycle 1 is 15 times (95 °c for 30 s, 55 °c for 30 s, 72 °c for 1 min), cycle 2 is 10 times (95 °c for 30 s, annealing temperature touchdown 50 55 °c for 30 s, 72 °c for 1 min), cycle 3 is 15 times (95 °c for 30 s, 50 °c for 30 s, 72 °c for 1 min), additional elongation temperature 72 °c for 7 min and 4 °c to deactivate the enzyme. the further pcr product becomes template for a pair of primer that overlapped pcr. figure 1 shows pcr design of tev construction and nucleotide. primer was designed having tm = 60-65 °c at part that overlapped with pet32b vector. the overlapped primer with pet-32b vector has 25-35 bp in length. primer for pcr overlap extension has 15-20 bp in length at part that overlapped with template [tev + nucleotide surfactant]. the pcr product was digested with dpni enzyme which will destroy the template (pet-32b+ vector). this enzyme will degrade wild type of pet-32b+ vector because this vector endogenously methylated. the addition result of dpni enzyme will only leave the pcr overlap product and can be transformed into e.coli strain bl21 (de3). supel insert confirmation with pcr (polymerase chain reaction), restriction enzyme and sequencing. putative recombinant plasmid is amplified using tevpep_f forward primer sequence and tev-pep_r amino acid codon usage % emergence amino acid codon usage % emergence m atg 100 l ctg 55 d gac 41 e gaa 70 f ttc 49 n aac 61 s tct 19 i atc 46 s tct 19 l ctg 55 m atg 100 d gac 41 a gct 19 k aaa 76 k aaa 76 a gct 19 l ctg 55 r cgt 42 c tgc 57 n aac 61 h cac 48 s tct 19 t acc 43 stop taa 62 table 1 codon usage for single peptide supel at e.coli volume 11, 2017 microbiol indones 29 30 sari et al. microbiol indones reserve primer with using green taq ready mix. sequencing is done in macrogen inc., south korea with two ways, using universal primer t7 promoter and t7 terminator. sequencing result is then analyzed and aligned using some softwares such as bioedit and snapgene™v.1.1.3. expression test for single supel construction. single supel construction result using pcr overlap is then transformed and made glycerol stock. from that glycerol stock, solid lb (luria bertani) is streaked into bowl and made into liquid culture. overnight culture result 1 ml is put into erlenmeyer 100 ml of lb. the culture is incubated into incubator 37 °c with shaker 180 rpm for 2-4 h to obtain od 0.5-0.7. after od 600 600 reach that number, 5 ml from culture is separated as non-inducted culture and then the other 95 ml is inducted using 1 mm iptg (final concentration). culture is then incubated for 4 hours measured by od 600 using pattern: v x od = 1 to uniform sds-page 600 sample amount. the further culture is pelleted at the speed of 14000 rpm for 10 min. the pelet was sonicated once and twice. the further sonicated cell result was then analysed by sds-page is done using 15% or 20% tris-glisin gel. results single supel construction. pcr amplicon to synthetize self supel peptide template using a pair of primer that has 30bp overlapped pcr, was obtained with the size around 120 bp (fig 2). this pcr product is then become template to make overlapped pcr using mega primer. pcr product with the further mega primer is used to overlap pcr using expression vector pet-32b as a template. through this overlap pcr of tev + supel + codon stop will insert between trx*tag and his*tag sequence of pet32b+, producing construction map as shown in fig 3. based on the map above, supel peptide is above signal strength control of transcription and translation t7 bacteriophage, where the expression is induced by the availability of t7 rna polymerase in cell and induced with iptg at final concentration 1 mm (novagen 2003). the tev sequnce presence is useful for target protein purification. the sequence existence confirmation result of tev + supel + codon stop is validated using pcr with specific primer to amplify tev and ss construction coding sequence, that is fig 1 primer design for pcr overlap extension. forward primer has total length of 56 bp that consists of 30 bp that overlaps with trx tag from pet-32b (green) and 26 bp that overlaps with tev sequence and nucleotide the composer of surfactant (blue and orange). reverse primer has total length of 51 bp that consists of 28 bp and overlaps with mcs part from pet-32b (green), 3 bp codon stop (red), and 20 bp with nucleotide the composer surfactant (orange). volume 11, 2017 microbiol indones 31 fig 3 single supel construction map. amp_f and amp_r. pcr result shows the size under ladder tape 250 bp, has been successfully amplified because the size is supposed to be 81 bp (fig 4). expression analysis of recombinant single supel peptide (ss). expression from supel peptide was tested with sds-page analysis. supernatant of culture was used for sds-page analysis (fig 5). ss construction expressed supel peptide protein at the size under ladder 15 kda. this result is according to online calculation result of the combination of tev protein molecule weight, 1x supel, and codon stop which is obtained ̴ 14.9 kilo dalton. this result indicates that supel protein has been expressed correctly. protein expression occured after 2 to 3 times sonication. this result can be assumed that 1x supel protein is expressed intracellular. discussion amino acid series at this supel causes peptide has two polar of different side chain, that is hydrophobic and hydrophylic. modelling using online software innovagen http://pepcalc.com/, around 67% of amino acid is hydrophobic and the other 33% is hydrophylic (fig 6). this indicates that supel peptide can bind with non-polar compound like oil. the condition of both characteristic in this supel peptide causes supel peptide is amphipathic when in liquid, which is the hydrophylic is inside the water while the hydrophobic will be on the water surface. this supel peptide is designed to form helix structure. the helix structure is designed in order that peptide has surfactant characteristic, depleted in water, and stable fig 2 pcr electrophoregram of supel; 1 and 2 of pcr samples, m. dna 1 kb ladder. at high temperature and high salinity (dwyer et al. 2014). peptide surfactant must have the characteristic that similar to surfactant which can be used in crystalization, that is peptide surfactant must be designed to have hydrophylic residual and 3 – 6 hydrophobic residual at the tail (yeh et al. 2005; kiley et al. 2005; matsumoto et al. 2009). the tail of supel fig 5 sds-page of ss construction supernatant sample; a. lysed cells of bl21 de3 with no plasmid, b. pet non-induction, c. pet induction, d. recombinant non-induction, e. ladder, f. recombinant 1 induction non sonication, g. recombinant induction and sonication, h. recombinant induction and two times sonication, i. recombinant induction and three times. fig 6 simulation of hydropathy in silico using innovagen (http://pepcal.com/). 32 sari et al. microbiol indones fig 4 (a) side attachment of prime ramp_f and amp_r in sequence; (b) electrophoregram of existence confirmation result of tev sequence and 1x supel peptide sequence. a b peptide is designed to have 3 amino acid residual of hydrophobic, that is arginine (r), lysine (k) and aspargarine (n). prediction of protein structure of 1x supel peptide that fused with thioredoxine and tev is analyzed in silico with bioinformatics program. this structure shows the presence of trx protein that forms alphahelix and tev and also 1x supel peptide forms linear structure (fig 7). simulation of fusion protein structure between thioredoxine, tev and single peptide supel shows that in fused condition, single peptide supel forms linear structure so it is assumed that peptide hasn't had surfactant characteristic yet. that's why, the separation of single peptide supel from thioredoxine using tev protease needs to be made before surfactant characteristic test. in conclusion, construct of surfactant supel sequences (ss) has been successfully conducted into pet-32b expression vector using overlapping pcr methods. constructs of ss can express supel peptide at sizes below 15 kda ladder by sds-page analysis. this is consistent with the result of online program calculation which show molecular weight of combination of tev protein, 1x supel, and stop codon at the size of ~ 14.9 kilo dalton references bachmann rt, johnson ac, edyvean rg. 2014. biotechnology in petroleum industry: an overview. int biodeterior biodegradation. 86:225-237. doi: 10.1016/ j.ibiod.2013.09.011. brown lr and vadie aa. 2002. slowing production decline and extending the economy life of an oil field: new meor technology. spe reservoir eval eng. 5(1): 3341. doi: 10.2118/75355-pa. desai, j. d., banat, i. m. 1997. microbial production of surfactants and their commercial potential. microbiol mol biol rev. 61(1): 47-64. dwyer md, brech m, yu l, middelberg, anton pj. 2014. intensified expression and purification of a recombinant biosurfactant protein. chem eng sci. 105: 12-21. doi: 10.1016/j.ces.2013.10.024. kiley p, zhao x, bruce bd, baldo m, zhang s. 2005. selfassembling peptide detergents stabilize isolated photosy i on a dry surface for an extended time. plos biol. 3:1181–1186. doi: 10.1371/journal.pbio.0030230. maloy s, stewart v, taylor r. 1996. genetic analysis of pathogenic bacteria. new york: cold spring harbor laboratory press. maneerat, suppasil. 2005. production of biosurfactants u s i n g s u b s t r a t e s f r o m r e n e w a b l e r e s o u r c e s . slongklanakarin j sci technol. 27 (3): 675-683. matsumoto k, koutsopoulos s, vaughn m, bruce bd, zhang s. 2009. designer lipid-like peptide surfactants stabilize functional photosy i membrane complex in solution. j phys chem. 115:75–83. doi: 10.1021/jp8021425. novagen, 2003. pet sy manual. us: novagen. pacheco gj, ciapina, elisa mp, gomes, edelvio de b, pereira junior, nei. 2010. biosurfactant production by rhodococcus erythropolis and its application to oil removal. braz j microbiol. 41: 685-693. doi: 10.1590/ s1517-83822010000300019 salter me, upstoll-goddard rc, nihtingale pd, archer sd, blomquist b, ho dt, huebert b, schlosser p, yang m. 2011. impact of an artificial release on air-sea gas fluxes during deep ocean gas exchange experiment. j geophys res. 116: 1-6. doi: 10.1029/2011jc007023. sandrea i and sandrea r. 2007. global oil reserves-recovery factors leave vast target for eor technologies. oil gas j. 1-8. van hamme jd, singh a, ward op. 2003. recent advances in petroleum microbiology. microbiol mol biol rev. 67 volume 11, 2017 microbiol indones 33 fig 7 1x peptide that still fused with trx and tev. blue protein shows thioredoxine while the orange shows tev and peptide surfactant. (4): 503-549. doi: 10.1128/mmbr.67.4.503-549.2003. yeh ji, du s, tordajada a, paulo j, zhang s. 2005. peptergent: peptide detergents that improve stability and functionality of a membrane protein glycerol-3phosphate dehydrogenase. biochemistry. 44:1691216919. doi: 10.1021/bi051357o. 34 sari et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 2 475 characterization (yoko)... characterization of moderately thermophilic bacteria isolated from saline hot spring in japan yoko kawasaki, madoka aoki, yoriyasu makino, hiroyuki sakai, yuki tsuboi, junko ueda, kazuhiko sonoda, keiko watanabe, shuichi yamamoto, norio kurosawa*and department of environmental engineering for symbiosis, faculty of engineering, soka university, 1-236 tangi-cho, hachioji, tokyo 192-8577, japan bacillus aeolius bacillus alveayuensis bacillus b. aeolius b. alveayuensis bacillus bacillus aeolius bacillus alveayuensis bacillus b. aeolius b. alveayuensis bacillus twelve strains of moderately thermophilic bacteria were isolated from a saline hot spring (55 °c, ph 8.3) located in odaito, hokkaido, japan. all strains showed over 99% sequence similarities in the 16s rrna gene. however, only eight strains grew well at 55 °c and demonstrated detectable activities. the representative strains, bek6 and bek11, were chosen from individual phenotypic groups and were further characterized. the nucleotide sequence of the almost full-length 16s rrna genes of the representative strains showed 97.1% and 96.6% similarity with and , respectively, indicating that the strains belonged to a novel species of the genus . the cells of both strains were gram-positive and showed catalase and oxidase activities. the optimal growth temperature and ph of strains bek6 and bek11 was approximately 50 °c and 7-8, respectively. these strains grew in a medium containing 10% nacl, in contrast to and that grew in a medium containing maximum of 4-5% nacl. the strain bek11 showed relatively strong protease and amylase activities in the presence of 3% nacl, suggesting the potential industrial uses of these enzymes under saline conditions. key words: thermophile, hot spring, novel species, dua belas galur bakteri termofilik telah diisolasi dari sumber air panas berkadar garam tinggi (55 °c, ph 8.3) yang terletak di odaito, hokkaido, jepang. semua galur bakteri menunjukkan lebih dari 99% persamaan sekuens dalam gen rrna 16s. hanya delapan galur bakteri yang dapat tumbuh dengan baik pada suhu 55 °c dan mempunyai aktivitas -galaktosidase. dari kelompok fenotipe individu bek6 dan bek11 dipilih sebagai perwakilan. urutan nukleotida 16s rrna secara parsial dari bek6 dan bek11 menunjukkan masing-masing 97.1% dan 96.6% kesamaan dengan dan , menunjukkan bahwa galur tersebut adalah spesies baru dari genus . kedua bakteri adalah gram-positif dan mempunyai aktivitas katalase dan oksidase. suhu pertumbuhan optimal galur bek6 dan bek11 ialah sekitar 50 °c dan ph 7-8. berbeda dengan dan yang hanya dapat tumbuh dalam medium yang mengandung nacl maksimum 4-5%, kedua galur bakteri tersebut dapat tumbuh dalam medium yang mengandung nacl 10%. galur bek11 menunjukkan aktivitas protease dan amilase yang yang relatif tinggi dalam kondisi nacl 3%, sehingga menjanjikan potensinya sebagai produsen enzim industri. kata kunci: termofil, sumber air panas, spesies baru, β-galactosidase β thermophiles are microbes that live in geothermal hot springs, hydrothermal vents, and artificially hot environments such as fermented compost and show optimal growth above 45-50 °c (lebedinsky . 2007). these microbes are roughly classified as moderately thermophilic and extremely thermophilic. the former have an optimal growth temperature ranging from 45-50 °c to approximately 65 °c; microbes belonging to both classes may have the potential for industrial application. moreover, thermophiles and their enzymes are interesting subjects for research in fundamental microbiology, evolutionary biology, biochemistry, and protein engineering. the biodiversity of thermophiles has been well studied until date using cultivation-dependent and independent methods; however, the existence of numerous uncultured thermophilic species in hot environments is indicated. during a recent attempt to et al identify novel thermophiles, we isolated twelve strains of moderately thermophilic bacteria from a saline hot spring in hokkaido, japan. preliminary experiments for the taxonomic classification of the isolated strains based on the 16s rrna gene (16s rdna) sequences indicated that the strains were closely related to each other and could be classified as a novel species of the genus , which consists of many species including the biotechnologically valuable strains (helianti . 2010). in this paper, we describe the physiological properties, chemotaxonomic characteristics, and phylogeny of the newly isolated strains and analyze their enzymatic activities. a pumped, hot, spring water sample was obtained from the odaito hot spring in hokkaido, japan, with the permission of the pumping facility bacillus et al bacillus materials and methods sampling, isolation, and cultivation of microbes. *corresponding author, phone: +81-42-6918175, fax: +81-42-6918175, e-mail: kurosawa@soka.ac.jp issn 1978-3477, eissn 2087-8575 vol 5, no 2, june 2011, p 56-60 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.2.2 owner. the temperature and ph of the water were approximately 55 °c and 8.3, respectively. the sample was plated onto modified brock's basal salts (kurosawa 1998) supplemented with 0.1% (w/v) yeast extract and artificial sea salts (1% w/v) at ph 8.3, solidified by 0.7% (w/v) gelzan (sigma), and incubated at 50 °c for two days. colonies were picked up and purified by repeated single-colony isolation. for further characterization of the isolates, a “routine medium,” in which artificial sea salts and gelzan were replaced with nacl and 1.5% agar, respectively, was used. the cultures were maintained at 50 °c and ph 8.0 for routine cultivation. the representative strains (bek6 and bek11) were subjected to standard tests including gram staining and tests for motility, catalase, and oxidase activities according to standard method (aygani and arikan 2007). anaerobic growth was examined by an anaeropack jar (mitsubishi gas chemical), and cell morphology was examined by a phase contrast microscope (axioskop 40; carl zeiss). to determine the temperature and ph optima, the strains were cultivated in the “routine medium” and various ph values were used to determine the ph dependence of growth. in addition, the nacl tolerance of growth was examined. all cells were grown aerobically in standing cultures of loosely-capped glass tubes. growth was monitored by measuring the optical density (od ) of the culture; utilization of lactose, maltose, glucose, dsorbitol, d-galactose, melibiose, d-mannose, dfructose, d-cellobiose, sucrose, d-xylose, trehalose, and citrate (1.0 g l of each carbohydrate) was tested. fatty acids of the cells in the exponential growth phase were hydrolyzed and methylated by tetramethylammonium hydroxide at 315 °c. the resultant fatty acid methyl esters were then analyzed using gc/ms (6850 network gc system and 5975cvl-msd with a triple-axis detector; agilent). various enzymatic activities were analyzed by an api zym kit (biomerieux) according to the manufacturer's instructions except for the incubation temperature, which was adjusted to 45 °c. the protease, amylase, chitinase, cellulase, and xylanase activities of the strains were examined using solidified media (plates) containing individual substrates and detected by a clear zone (halo) appearing around the colonies. the 16s rdna of the strains was amplified by pcr using the bacterial u n i v e r s a l p r i m e r s b 2 7 f ( f o r w a r d ; 5 ′ agagtttgatcmtggctcag, positions 8-27 based on numbering) and u1492r (reverse; 5′-ggytaccttgttacgactt, positions et al. escherichia coli morphology and phenotypic characteristics. phylogenetic analysis. 600 -1 1510-1492) (kurosawa . 2010). the following thermal cycle was used 30 times: 94 °c for 30 s, 60 °c for 30 s, and 72 °c for 1.5 min. dna sequencing was performed by the dideoxynucleotide chain termination method. the 16s rdna sequences of strains bek6 and bek11 were compared with the available 16s rdna sequences in the genbank dna database using blastn (http://www.ncbi.nlm.nih.gov/blast/; altschul 1990). seventeen 16s rdna sequences of related species belonging to the family were aligned using clustalw (thompson 1994); all sites with gaps in any of the sequences and pcr primers were eliminated from the alignment. phylogenetic trees with bootstrap sampling were reconstructed by the neighbor-joining method (saitou and nei 1987) using the genetyx-win software (genetyx). the almost full-length 16s rdna sequences of strains bek6 and bek11 were deposited in the dna database with the accession numbers ab607229 and ab607228, respectively. we isolated twelve moderately thermophilic bacterial strains designated bek3, bek5, bek6, bek7, bek9, bek10, bek11, bek12, bek13, bek14, bek15, and bek16 from a saline hot spring in hokkaido, japan. preliminary analysis of the partial 16s rdna sequences (approximately 500 bp) of these isolates indicated that they were closely related to each other and belonged to the genus . however, the analysis also indicated that strains bek6, 7, and 10 showed no or very low activity compared with other strains such as bek3, 5, 9, 11, 12, 13, 14, 15, 16. moreover, the negative strains, particularly bek6, showed poor growth at 55 °c compared with the other strains. strain bek11 showed the highest cell density at 55 °c. therefore, we chose strains bek6 and bek11 as representatives of the individual phenotypes for further characterization (table 1). strains bek6 and bek11 were obligate aerobes and gram-positive motile rods. the colonies of both strains were matt white, circular, and convex (average, 0.5-1.0 mm) with slightly undulate margins. the lengths of bek6 and bek11 cells were 0.6 × 2-3 × 3(fig 1). in the stationary growth phase, both strains formed endospores. in addition, they showed catalase and oxidase activities. nitrate reduction and indole production by these strains were not detected. optimal growth temperatures of strains bek6 and bek11 were approximately 45 °c and 50 °c, respectively. both strains appeared to be slightly alkaliphilic and grew at ph 7-10. the strains et al et al. bacillaceae et al. bacillus results β-galactosidase se β-galactosidase μm and 0.6 7 μm, respectively volume 5, 2011 microbiol indones 57 were relatively halotolerant and grew in a medium containing 10% (w/v) nacl. the major fatty acid identified was c , which constituted approximately 50% of the total fatty acid content; no unsaturated fatty acid was found in either strain (table 2). phylogenetic analysis indicated that strains bek6 and bek11 were most closely related to (gugliandolo 2003) and (bae 2005; fig 2). the 16s rdna sequences of the isolated strains showed 97.1% and 96.6% similarity with those of and , respectively. we used the api-zyme kit to analyze the enzymatic activities of strains bek6 and bek11; both strains showed alkaline phosphatase and esterase activities. as mentioned earlier, the 16 b. aeolius et al. b. alveayuensis et al. b. aeolius b. alveayuensis β-galactosidase 58 kawasaki et al. microbiol indones activity was detected only in strain bek11. protease and amylase activities were detected by the plate tests performed at 45 °c and 1% nacl, and the strain bek11 produced larger halos on both substrates. activities of chitinase, cellulose, and xylanase were not detected. subsequently, the protease and amylase activities of strain bek11 were examined at higher temperatures and nacl concentrations; these activities were observed at 50 °c in the presence of 3% nacl. the utilization of carbohydrates also differed between strains bek6 and bek11. strain bek6 utilized maltose, glucose, fructose, cellobiose, sucrose, and trehalose, while strain bek11 was able to use galactose and melibiose, in addition to those used by strain bek11. bek6 bek11 bacillus aeolius b. alveayuensis colony color white white nd cream cell form rods rods rods rods cell width×length (μm) 0.6×2-3 0.6×3-7 0.5×2.0 0.5-1.5×2.5-5 motility + + + + spore forming + + + + aerobic growth + + + + anaerobic growth growth temperature range ( o c) 25-55 25-55 37-65 40-65 optimal growth temperature ( o c) 45 50 55 55 growth ph range 7.0-10.0 6.0-10.0 7.0-9.0 6.5-9.0 optimal growth ph 7.0-8.0 7.0-8.0 8.0 7.0-7.5 nacl range for growth (%) 0-13 0-11 0.5-5.0 0-4.0 optimal nacl concentration (%) 2-3 2-3 2 3 catalase + + + oxidase + + + nitrate reduction indole production gram reaction + + + + b. aeolius, b. aeolius et al. b. alveayuensis, b. alveayuensis et al.4-1 (gugliandolo 2003); tm1 (bae 2005); nd, not determined. t t properties fig 1 phase contrast micrographs of the strains. a, bek6 and b, bek11 10 μm a b 10 μm table 1 phenotypic characteristics of the strain bek6, bek11 and relative species. volume 5, 2011 microbiol indones 59 bek6 bek11 “b. aeo” 1 b. alv 2 10:0 1.7 12:0 4.4 13:0 2.2 14:0 2.3 2.2 iso-14:0 0.6 15:0 2.0 3.5 iso-15:0 8.5 15.5 anteiso-15:0 2.5 3.5 16:0 51.5 49.6 33.2 11.1 iso-16:0 12.5 10.1 anteiso-16:0 7.3 8.8 16:1ω9 0.9 16:1ω13t 1.4 17:0 9.6 8.7 4.9 0.7 iso-17:0 8.2 10.5 11.1 15.4 anteiso-17:0 10.1 11.1 cyclohexan-17:0 1.5 18:0 14.8 11.1 12.8 4.8 anteiso-18:0 8.6 8.0 18:1ω9 3.0 19:0 1.6 table 2 fatty acid composition of the strain bek6, bek11 and relative species. 1 t 2 t b. aeolius et al. b. alveayuensis et al.4-1 (gugliandolo 2003), tm1 (bae 2003). discussion phylogenetic analysis using 16s rdna sequences indicated that strains bek6 and bek11 were affiliated with the genus . as mentioned in the results section, the phylogenetically closest species was . however, strains dsm 15084 and cip 107628, which have been deposited as the type strains of , are not representative of the original type bacillus b. aeolius b. aeolius strain, strain 4-1. attempts to obtain a subculture of the original strain from the depositor were in vain because the strain is no longer available in the depositor's laboratory (pukall . 2008). according to rule 18c of the international code of nomenclature of bacteria (lapage . 1992), pukall . 2008 proposed that the judicial commission of the international committee on systematics of prokaryotes places the name (gugliandolo 2003) on the list of rejected specific and subspecific epithets in names of species and subspecies of bacteria, if a suitable replacement for the type strain or a neotype is be found within two years of publication of their request. although has not been placed on the rejected list until date, the closest available strain (to strains bek6 and bek11) is possibly strain tm1 (bae . 2005). the similarity of 16s rdna sequences between the representative strains and was 96.6%, suggesting that strains bek6 and bek11 differ from . as shown in tables 1 and 2, many phenotypic differences between the isolates and or . in particular, strains bek6 and bek11 were more tolerant to alkaline and saline conditions than strains of the most closely related species, . phenotypic and phylogenetic evidence suggested that the newly isolated strains were novel species of the genus . moreover, we examined the secreted hydrolyzing enzymatic activities of strains bek6 and bek11 and detected relatively strong protease and amylase activities from strain bek11 at 50 °c in the presence of 3% nacl, indicating that these et al et al et al b. aeolius et al. b. aeolius b. alveayuensis et al b. alveayuensis b. alveayuensis b. aeolius b. alveayuensis were also identified b. alveayuensis bacillus t fatty acid composition fig 2 nj-tree for the strain bek6, bek11 and relative species. the bootstrap values (1000 sampling) above 50% are shown at nodes. 60 kawasaki et al. microbiol indones enzymes can be used at relatively high temperatures and saline environments. further enzymatic characterization and chemotaxonomic analysis for the validation of the isolates as a novel species are now in progress. references altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j mol biol. 215(3):403-410. aygani a, arikan b. 2007. an overview on bacterial motility detection. int j agri biol. 9(1):193-196. bae ss, lee jh, kim sj. 2005. sp. nov., a thermophilic bacterium isolated from deep-sea sediments of the ayu trough. int j syst evol microbiol. 55(3):1211-1215. doi: 10.1099/ijs.0.63424-0. gugliandolo c, maugeri tl, caccamo d, stackebrandt e. 2003. sp. nov. a novel thermophilic, halophilic marine species from eolian islands (italy). syst appl microbiol. 26(2):172176. doi:10.1078/072320203322346001. helianti i, nurhayati n, ulfar m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated aq1 in . j biomed biotechnol. 12 p [on line]. doi:10.1155/2010/980567. kurosawa n, itoh yh, iwai t, sugai a, uda i, kimura n, horiuchi t, itoh t. 1998. gen. nov., sp. nov., a novel bacillus alveayuensis bacillus aeolius bacillus bacillus subtilis escherichia coli sulfurisphaera ohwakuensis extremely thermophilic acidophile of the order sulfolobales. int j syst bacteriol. 48(2):451456. kurosawa n, sato s, kawarabayasi y, imura s, naganuma t. 2010. archaeal and bacterial community structures in the anoxic sediment of antarctic meromictic lake nurume-ike. polar sci. 4(2):421-429. doi:10.1016/j.polar.2010.04.002. lapage sp, sneath pha, lessel ef, skerman vbd, seeliger hpr, clark wa. (editors) 1992. international code of nomenclature of bacteria (1990 revision). bacteriological code. washington, dc: american society for microbiology. lebedinsky av, chernyh na, bonch-osmolovskaya ea. 2007. phylogenetic systematics of microorganisms inhabiting thermal environments. biochemistry (mosc). 72(12):1299-1312. doi: 10.1134/ s0006297907120048. pukall r, schumann p, clermont d, bizet c. 2008. dsm 15084 (=cip 107628 ) is a strain of . int j syst evol microbiol. 58(5):1268-1270. doi: 10.1099/ijs.0.2008/ 001388-0. saitou n, nei m. 1987. the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol biol evol. 4(4):406425. thompson jd, higgins dg, gibson tj. 1994. clustal_w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. nucleic acids res. 22(22):4673-4680. bacillus aeolius bacillus licheniformis t t 04 nuryady.cdr vol.11, no.1, march 2017, p 23-27 doi: 10.5454/mi.11.1.4 analysis of human immune response against salivary glands protein extract of anopheles sundaicus. l in malaria endemic area 1 1 2 mohammad mirza nuryady , sugeng setyo utomo , yunita armiyanti , 1 1* sri mumpuni wahyu widjajati , and kartika senjarini 1 department of biology, mathematic and natural science faculty, universitas jember, jalan kalimantan 37, kampus tegal boto, jember 68121, indonesia; 2 faculty of medicine, universitas jember, jalan kalimantan 37, kampus tegal boto, jember 68121, indonesia malaria is an infectious disease caused by plasmodium, which is transmitted by anopheles mosquitoes as vectors. malaria transmission begins when an infected mosquito takes blood meal from healthy human. mosquitoes will release parasite and components of saliva into the host's body. saliva contains components (proteins) that affect the host's hemostasis and immune response, such as vasomodulator and immunomodulators. imunomudulator could act as immunosuppressive factors that can suppress nonspecific immune system of the host and modulate the change of t helper 1 (th1) toward t helper 2 (th2) response, which is advantageous for malaria parasite to infect human host. this research wanted to evaluate human immune respons in endemic area against salivary gland protein extract (sgpe) from its major malaria vector i.e. anopheles sundaicus (an. sundaicus). analysis of human immune response was conducted quantitatively by elisa (enzyme-linked immunosorbent assay) towards igg from human sera samples after cross reacted with sgpe. the results showed that exposures to an. sundaicus were able to induce high levels of igg. igg anti salivary proteins of an. sundaicus is higher than the levels of igg anti salivary proteins of ae. aegypti. furthermore, the age group 11-40 years with the highest bites probability, had the highest igg levels compared to other age groups. key words: anopheles sundaicus, igg, malaria, salivary malaria merupakan penyakit infeksi disebabkan oleh plasmodium, yang ditransmisikan oleh vektor nyamuk anopheles. transmisi malaria diawali ketika nyamuk yang terinfeksi melakukan blood feeding ke manusia sehat. selama blood feeding, nyamuk juga akan melepaskan parasit bersamaan dengan komponen saliva ke tubuh inang manusia, saliva mengandung kompoten (protein) yang dapat mempengaruhi hemostasis dan respon imun inang seperti vasomodulator and immunomodulators. imunomudulator dapat bersifat sebagai faktor yang immunosuppressive sehingga dapat menekan sistem imun non spesifik serta memodulasi perubahan respon imun spesifik t helper 1 (th1) ke arah t helper 2. hal ini sangat menguntungkan parasit sehingga memudahkan infeksinya ke dalam tubuh manusia. penelitian ini ingin menguji respon imun manusia yang hidup di daerah endemik terhadap ekstrak protein kelenjar saliva (sgpe) dari vektor dominan di daerah tersebut yaitu anopheles sundaicus (an. sundaicus). analisis respon imun dilakukan secara kuantitatif dengan elisa (enzyme-linked immunosorbent assay) dengan mengamati titer igg dari sampel sera penduduk terhadap sgpe. hasil penelitian menunjukkan bahwa paparan berulang dari an. sundaicus mampu memicu meningkatnya titer igg. konsentrai igg terhadap sgpe an. sundaicus lebih tinggi dibandingkan dengan igg terhadap sgpe ae. aegypti. kelompok usia 11-40 tahun yang memiliki kemungkinan terpapar gigitan nyamuk lebih tinggi, menunjukkan titer igg yang tertinggi dibandingkan kelompok-kelompok usia lainnya. kata kunci: anopheles sundaicus, igg, kelenjar saliva, malaria microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-331, fax:+62-330225 331; email: senjarini@unej.ac.id330225 modulator. vasomodulatory factors can manipulate the host's vasomodulator mechanism by preventing vasoconstriction, platelet activation and aggregation, and blood coagulation. immunomodulator factor can supress the host's nonspecific immune system that modulates the changing response from t helper 1 (th1) to t helper 2 (th2) (titus et al. 2006). the changing of immune system can be seen through the decrease of ifn-g production and the increase of interleukin-4 (il4). il4 will influence the proliferation of cell b, which, in turn, will raise humoral antibody. because malaria is an infectious disease which is caused by plasmodium and spread by anopheles mosquito as the vector. the dissemination of the disease starts when a mosquito that carries plasmodium takes blood meal from healthy human. the mosquito will release saliva components to the host's body. mosquito's saliva contains components that can influence host's homeostasis including vasomodulator and immunothe role of salivary proteins are mediating easier phatogen transmission, the increasing of humoral antibody against salivary protein will also affect the transmission of phatogen which means exposure against mosquitoe's bite will increase host immune resistancy against its transmitted phatogen (donovan et al. 2007). bites from plasmodium infected mosquito can spread malaria. on the other hand, reexposure to sterile mosquito can cause protection, cause the salivary component give the sensitisation effect. in a study conducted by (morris et al. 2001) injection with low dose saliva component can increase the transmission of pathogen, while injection with high dose can give protection. it means that people who live in the endemic area and often get exposure to vector's bite will have protection to pathogen. recurring exposure causes the changing of immune response like in the normal condition (th1 response), which are the activation of macrophage and production of nitric oxide (no) so that it will be effective to kill parasite (donovan et al. 2007). because of the increasing production of antibody against salivary antigen (igg) increase by repeated exposure, it can mediate to block the infection, therefore people living in endemic area who often get more exposure to vector's bite tend to have more protection from the infection. (fontaine et al. 1995). the measurement of anti-salivary protein antibody (igg) can be used as biomarker of the exposure to anopheles mosquito. in indonesia, especially in endemic area of bangsring village, banyuwangi, there has not been any analysis of immune response towards anopheles saliva protein in the population. analysis of host's immune response towards salivary gland protein of an. sundaicus is an important step to determine if human exposure to mosquito bite is related to the production of specific antibody. if such a relationship can be proven, this antibody might be used as a biomarker of the exposure. materials and methods landing collection, identification and rearing an. sundaicus. anopheles mosquito was reared in mosquito cage at the zoology laboratory, biology department, faculty of mathematic and natural science, universitas jember. rearing process started with collecting (landing collection) mosquitoes from their habitat in bangsring village at wongsorejo, banyuwangi. anopheles larva was collected from lagoons near the coast, while adult anopheles mosquitoes were gathered from around livestock pens near people's house. the anopheles mosquitoes identified in the laboratorium based on the book of determination key of insect. the rearing started from adult mosquitoes that were kept at room temperature o (25-28 c) and given 10% sucrose solution diet, and periodically were given the body of wistar rat as the source of blood. the mosquitoes were given this diet since the first day. humidity was kept by wrapping the mosquito cage with wet fabric. extracting an. sundaicus salivary gland protein. salivary gland was isolated by micro dissection, with the addition of lysis buffer (1:1 ratio). then, the sample was homogenized, sonicated for 30 min by using water sonicator, centrifuged with 12690 o rpm for 15 min at 4 c. then, supernatant was taken and o kept at 80 c. salivary protein was concentrated using o epi membrane centrifuged at 10000 rpm at 4 c for 30 s. o the concentrated protein was kept at 80 c until further use. preparing blood serum. serum samples of the were taken from healthy people's blood at endemic area in bangsring, banyuwangi. the volunteers were grouped based on their age, children (<10 year old), adult (11-40 year old), and old (>40 year old). blood sample was taken from the branchial vein in the upper arm. three ml blood was taken and placed in vacutainer without heparin. then, it was kept for 15 to 45 min. after that, upper transparent layer was taken o and centrifuged at 27 c, 3200 rpm for 10 min. the serum from the supernatant was then kept at the o temperature of -80 c. indirect elisa (enzyme linked immuno-1 sorbent assay). the plate was coated with 5 µg ml -1 (50 µl well ) an. sundaicus sgpe, which has been diluted with 0.1 m natrium bicarbonate buffer (ph 9.6). o coating was performed overnight at 4 c. then, the plate was washed with 250µl pbs-t (ph 7,4). the plate was blocked with 200µl blocking buffer (pbs-t ; o 1% bovine serum albumine) for 2 h at 37 c. serum -1 was diluted with 1:25 ratio (50 µlwell ) and incubated o at 37 c for 1 h. then, 50µl horse radish peroxidase (hrp)-conjugated rabbit anti human igg (1:5.000) o was added and incubated for 1 h at 37 c. after that, 50 µl tetra methyl benzidine substrate was added and incubated for 10 min at room temperature. then 50µl 1m h so4 was added to stop the reaction. the level of 2 igg was determined using elisa reader set at 450nm. the control well also applied with the same methods, but without adding serum into the well, it substitutes by blocking buffer. 24 nuryady et al. microbiol indones data analysis. the data obtained in this research were analyzed using softwares graphpad prism 5.0 and spss, with one way anova (p<0.05) and duncan's (p<0.05) tests. results salivary gland and salivary gland protein extract from an. sundaicus. there are 800 pairs of female an. sundaicus' salivary gland were isolated by micro dissection technique (bruce 1980). female anopheles only had one pair of salivary gland which was located in each side of esophagus at the anterior thorax (wright 1969). one salivary gland consisted of three lobes, two lateral lobes and one medial lobe. salivary duct connects medial lobe and salivary pump, which is located near hypopharynx. lateral lobes are divided into proximal, intermediate, and distal area (dhar and kumar 2003). an. sundaicus salivary gland can be seen in figure 1. the total amount of sgpe -1 extracted from an. sundaicus was 4.2 mg ml . human igg level towards sgpe from an. sundaicus. this research used two different mosquito salivary glands protein extracts (sgpes), which were an. sundaicus sgpe and aedes (ae.) aegypti. the result of igg measurement can be seen in figure 2. the comparison between two antigens was done to determine the level of igg introductory to sgpe. figure 2 shows higher od score was observed on detection of igg against an. sundaicus sgpe than ae. aegypti sgpe. discussion in this research, there were 2000 anopheles mosquitoes were collected from the field in 2014. the species of anopheles mosquitoes which found based on the result of landing collection consisst of: an. annularis, an. vagus, an. subpictus, an. idenfinitus, an. barbirostris, and an. sundaicus. the most dominant species collected was an. sundaicus. this result was consistent with the previously published results (lyimo and takken 2008), which stated thatat least seven species of anopheles had been found in the bangsring village, in which the most dominant was an. sundaicus. sgpe consists of many different proteins, some of which can be conserved into genus and even family level (fontaine et al. 2011). the high respond of igg anti sgpe from an. sundaicus in figure 2 is due to the place where the serum was taken was endemic area of malaria with high population of an. sundaicus. it is in line with previous research (shinta et al. 2003) which stated that bangsring village in wongsorejo; banyuwangi was endemic of malaria, in which an. sundaicus was the primary vector. the level of ae. aegypti sgpe reactive igg was also high. most probably, this was because ae. aegypti is ubiquitousin indonesia, as shown by the high case of dengue fever all over the country(depkes ri 2004). the high level of ae. aegypti sgpe recognising igg might also be because whole protein extract was used. tthere is high chance that plenty of homology occur in the salivary proteins ofmosquitos in the cullicidae family, to which group both ae. aegypti and an. sundaicus belong to (fontaine et al. 2011). in this research, the grouping was based on age. there was no level of igg or the igg level was zero in the control group. there was no antigen in the control group so that antibody within the serum could not attach to its specific antigen. however, in the neonates group fig 1 salivary gland of female anopheles. note: pl: proximal lobe, dl: distal lobe, ml: medial lobe. a: anopheles sp. salivary gland. (jariyapan et al. 2007). b: the result of an. sundaicus salivary gland isolation (lw scientific microscope, 400 x magnifications, optilab camera). volume 11, 2017 microbiol indones 25 showed low od score. it was assumed that there was anti salivary protein in the serum from the mother. the group of 11-40 year old showed the highest level of igg compared to the other groups. the high od score in group of 11-40 year old might due to two reasons which were the influence of age which could influence the activation of t memory cell and influence the production of specific antibody (lyimo et al. 2008). at the active age group (11 to 40) the immune response was more mature so that the igg level was high while the group of >40 year old the level of antibody production had decreased. on the other hand, in the group of <10 year old the immune system is still developing (bratawidjaja and rengganis 2014). the second possibility was supported by the result of the questionnaire which had been gathered before the blood sampling. most of people in bangsring village who were 11 to 40 years old had activities outside their home at night. this was also related to the behavior of an. sundaicus which was more active at night (nocturnal) (cdc 2010) and the species of an. sundaicus was found more outside than inside of house (mardiana et al. 2003). the measurement of igg based on age group can be seen in figure 3. a person who is often exposed to mosquito bite will have higher anti salivary protein igg level than a person who is rarely exposed to mosquito bite. the result of this research was consistent with the previous research, stating that people living in malaria endemic area who were often exposed to anopheles saliva would express higher immunity level by producing antibody in the form of anti salivary protein igg (waitayakul et al. 2006). the data from questionnaire states that volunteers younger than 10 years old spent most of their night at home. they used anti-vectorial such as insecticide netting, body lotion, and mosquito coil, fig 2 graph of igg level based on salivary protein antigen (ag). there is significant differences between each group (anova p=0.000 < 0.05), and the higher salivary anti protein igg level is an. sundaicus antigen (duncan p=0.000 < 0.05). fig 3 the result of igg measurement based on age group. the five experimental groups show significant difference (anova p=0.000 < 0.05). then, the highest level of anti salivary protein was in the group of 11-40 years old (duncan p=0.00 < 0.05). 26 nuryady et al. microbiol indones which could prevent them from the mosquito bites. from our data, therefore, it is indicated that there is correlation between antibody response against sgpe with exposure to an. sundaicus. high level of antisgpe antibody against an. sundaicus in sera samples from human living in endemic area can be used as a potential source of indicators of exposures to an. sundaicus. even in comparison to anti-sgpe antibody against aedes aegypti, which is also in indonesia, our results demonstrated that the level of antibody against an. sundaicus was still higher. serum analysis also supported the hypothesis that anti sgpeantibody level increased with the probability of exposure to mosquito bites. acknowledgement we are very grateful to ministry of research, technology, and higher education especially” hibah pascasarjana” for providing financial support for this study. references bratawidjaja kg, rengganis i. 2014. immunologi dasar [basic immunology] edisi ke-11(cetakan ke-2). jakarta: badan penerbit fakultas kedokteran universitas indonesia. bruce-chwatt lj. 1980. essential malariology. london: william heinemann medical books ltd. cdc. 2010. anopheles mosquitoes http://www.centers for . disease control and prevention (cdc)/anopheles (diakses tanggal 27 februari 2014). departemen kesehatan republik indonesia. 2004. profil kesehatan indonesia [profile of health in indonesia]. jakarta: departemen kesehatan republik indonesia. dhar r, kumar n. 2003. role of mosquito salivary gland. curr sci. 85(9):1308-1313. donovan jm, messmore as, scrafford da, sacks dl, kamhawi s, mcdowell ma. 2007. unifected mosquito bites confer protection against infection with malaria parasites. j infect immun.75:2523-2530. doi: 10.1128/iai.01928-06. fontaine a, pascal a, orlandi-paradines e, diouf i, remoue f, pages f, fusai t, rogier c, almeras l. 2011. relationship between exposure to vector bites and antibody responses to mosquito salivary glands extracts. j of immun markers expo. 6(12):1-10. doi: 10.1371/journal.pone.0029107. lyimo e, takken w. 2008. effects of adult body size on fecundity and the pre-gravis rate of anopheles gambiae females in tanzania. j med vet entomol. 7 : 328-332. doi: 10.1111/j.1365-2915.1993.tb00700.x. mardiana w, wigati w, suwaryono t. 2003. aktivitas menggigit anopheles sundaicus di kecamatan wongsorejo, kabupaten banyuwangi, jawa timur. [biting activities of anopheles sundaicus at wongsorejo district, banyuwangi, east java]. j media litbang kesehatan. 13(2):26-30. morris rv, shoemaker cb, david jr, lanzaro gc, titus rg. (2001). sandfly maxadilan exacerbates infection with leishmania major and vaccinating against it protects against l. major infection. j immunol. 167:5226-5230. doi: 10.4049/jimmunol.167.9.5226. shinta ss, supratman s, mardiana. 2003. komposisi spesies dan dominasi nyamuk anopheles di daerah pantai banyuwangi, jawa timur. [species composition and anopheles mosquitos domination at the coastal area of banyuwangi, east java]. j media litbang kesehatan. 8(3):1-8. ti t u s r g , b i s h o p j v, m e j i a j s . 2 0 0 6 . t h e immunomodulatory factors of arthropod saliva and the potential for these factors to serve as vaccine targets to prevent pathogen transmission. parasite immunol. 28:131-141. doi: 10.1111/j.1365-3024.2006.00807.x. waitayakul a, somsri s, sattabongkot j, looareesuwan s, cui l, udomsangpetch r. 2006. natural human humoral response to salivary gland proteins of anopheles mosquitoes in thailand. acta trop. 98:66-73. wright ka. 1969. the anatomy of salivary glands of anopheles stephensi liston. can j zool. 47:579-587. doi: 10.1139/z69-101. volume 11, 2017 microbiol indones 27 page 1 page 2 page 3 page 4 page 5 5 471 dono.cdr the life cycle of on synchytrium pogostemonis pogostemon cablin dono wahyuno indonesia medicinal and aromatic crops research institute, departemen pertanian indonesia, jalan tentara pelajar no. 3, bogor 16111, indonesia phone: +62-251-8321879, fax: +62-251-8327010, email: dwahyuno@yahoo.ca synchytrium pogostemonis s. pogostemonis. s. pogostemonis synchytrium, pogostemon cablin became a serious disease of patchouli cultivation in indonesia, since it spread widely in many patchouli producing areas in indonesia. the fungus caused warts on leaves, petioles and young stems of infected patchouli. the infected plant developed rosette habit, lost its vigour, was susceptible to drought period and finally died. few information regarding the eco-biology and life cycle of the fungus were available the present research aimed at describing the life cycle of the diseased patchouli was obtained through artificial inoculation. mass inoculation was carried out by placing the healthy patchouli seedling close to diseased patchouli as source of inoculum and was watered regularly using the top head sprinkler . the infected leaves were observed both under disecting and light compound microscopes, and the existing fungal structure were recorded, described and measured. it was observed that is a long-cycle type fungus, the sexual reproduction was initiated by zoospores, followed subsequently by development of resting-structure spores, vesicles, sori, and sporangial formation. key words: , life cycle patchouli ( ) is a patchouli-oil pro ducing plant species, which is an important source of income for many farmers in indonesia. recently pest and diseases has become a serious constraint in patchouli cultivation, including viruses and nematodes. in the last three years, a disease named (wart in local language) has spread widely in patchouli production centres in indonesia. the warts appeared on leaves, petioles, young stems, and young shoots (fig 1a). the disease was believed to be caused by virus or micoplasm-like organisms as indicated by their typical symptom appearances, such as leave curl and rosetting (sitepu and asman 1991; mustika and asman 2004). however, detailed observation on disea sed plants collected from various patchouli plantations in indonesia, the resting spore of the biotrophic fungus was noticed consistenly from the samples (wahyuno 2007). artificial inoculation experiment indicated that the presence of water film on soil particles was favourable for fungus spreading and infection development (fig 1b). based on the existing morphological characteris tics of the fungus, it is identified as (wahyuno and sukamto 2010) the same species was also reported occuring in india (dayal 1997) and its world wide distribution was still limited to south asia and south east asia (thronton 2002), where patchouli plants have been widely cultivated. up to now there is still limited information regarding the life cycle of karling (1954 1964) in his compilation of world wide did not mention the occurence of ; and there were various life cycle types in (karling 1964; webster 1988). the objective of the present study is to find out the life cycle of with the final objective of controlling the disease in the future. pogostemon cablin ralstonia solanacearum, budok synchytrium et al. synchytrium pogostemonis . s. pogostemonis. , synchytrium s. pogostemonis synchytrium s. pogostemonis materials and methods the diseased patchouli plants studied were obtained and propagated by artificial inoculation. the diseased patchouli plants collected from bogor (syn-tro-01) and kuningan in west java (syn-tro-03) were used as source of inoculums. the inoculation was conducted by placing infected leaves, petioles and young stems around the bases stems of 4-node young patchouli cuttings planted in polybags (wahyuno and sukamto 2010). the inoculated patchouli cuttings were incubated in the green house for about one month until the warts appeared on stems, petioles or leaves. during this period, watering the soil was carried out to maintain high humidity to create a suitable condition for the infection of the zoospores of . mass inoculation was carried out to obtain sufficient amounts of diseased patchouli cuttings by placing the healthy patchouli cuttings around those infected cuttings, obtained from previous artificial inoculation over head watering by sprinkle spraying was carried out daily to enhance the occurrence of mass infection of patchouli cuttings. the infected leaves were characterized by the presence of warts on its either abaxail or adaxial surfaces. prior to the observation under compound microscope, the leaf tissues were softened, using the modified method of rao and pavgi (1986) by adding a soaking treatment in sodium hyphoclorite solution before the leaf tissues were soaked in sterilized water the infected leaves were cut into pieces of about 1 cm each, which were then soaked in a 1% sodium hypochlorite solution for one minute, rinsed with sterilized water and finally incubated in sterilized water in a botle for two days. under a dissecting microscope slide preparation was made from softened pieces of leaves and the slides were then observed under a compound microscope. the existing structures of were recorded, measured and characterized. the existing fungal structures were categorized and named according to the system proposed by karling (1964). s. pogostemonis , . synchytrium 2 issn 1978-3477 vol 4, no 3, dec 2010, p 127-131 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.5 128 wahyuno microbiol indones results warts appeared on the surface of stems and newly developed shoots as well as leaves on stems arising from below the soil surface. the wart formation tended to develop more toward the younger upper part of the plant than toward the older base of the stem. thus the warts on the lower sections of the stems were older than those on the upper parts. the old warts were characterized by the presence of yellow resting spores inside which could be easily observed by using a hand lens. observations, with a light microscope, of cells around the warts revealed that the cells contained granulle-like structures inside (fig 1c). such structures were not observed in uninfected leaves. the fungal thalli were not presemt in the cells of infected plant leaf tissues. fig 1 symptoms and resting spore developmental stages of . (a) advanced symptom, with warts on young stems, leaves and petioles, (b) warts on artificially inoculated stems, (c) summer spores (sporangia) inside the infected plant cells ( ), (d) an imature resting spore, (e) an advanced stage of imature resting spore, (f) a mature resting spore, (g) a vesicle is emerging from a resting spore (resting spore functions as prosorus), (h) cell division forming sporangia within a vesicle which functioned as a sorus, (i) broken sorus liberating the sporangia, leaving the resting spore empty (j) individual sporangium of a sorus emerged from a resting spore, and (k) an empty resting spore with conical releasing pore as seen from top view. s pogostemonis volume 4, 2010 microbiol indones 129 synchytrium further observations on the infected plants showed that resting spores (winter spores) of were noted in warty leaf tissue. the yellow to light orange or light brown resting spores with thick cell wall were generally recognizable inside enllarged plant cells. the immature resting spores were ellipsoid, oblong to globose, and the nucleus could be identified inside the spore as hyaline spot (fig 1d, 1e), while but mature resting spore were ge nerally oblong, light orange to dark brown, having rough surfaces, with diameter range of 70.0-(106.7)-150.0 µm (fig 1f). the number of resting spores within a single wart varies from 1 to 11, depending on the size of the warts. vesicle-like structures could be identified in some resting spores, whose size was about the same as that of the resting spores (fig 1g). each vesicle contained 20 35, yellow-orange sporangia. in some other vesicles, the s yellow-orange sporangia were hyaline, thin-walled oblong to globose or irregular, with size range of 25.0(29.7)-35.0 x 17.5-(24.9)-30.0 µm (fig 1h, 1i and 1j). the empty resting spores were also noted and some of them had conical releasing pores (fig 1k). in the observed samples, the true fungal thalli were not noted in this stage. discussion present microscopic observations indicated that the summer sporangia (the asexual reproductive sporangia) did exist in nature. the summer sporangia were present as granulle-like structures inside of the infected leaves. it was not easy to fnd and identify the summer sporangia in the warts, even the samples were soaked in distilled water prior to observations under a light microscope. fig 2 the life cycle of . (1) zoospores, (2) encysting and ingression of the zoospore into a healthy plant cell, (3) summer sporangia in an infected plant cell, (4) sporangium liberates zoospores, (5) mating of two different type of zoospores, (6) encysting and ingression of the mating zoospores into healthy plant's cell, (7) young resting spore inside a plant cell, (8) a mature resting spore, (9) vesicle formation, (10) the vesicle transforms into a sorus with initial formation of sporangia, (11) further stage of sorus development, (12) a sorus breaks and releases the sporangia, (13) single sporangium and the life cycle repeated by liberating the zoospores. synchytrium pogostemonis s. pogostemonis synchytrium desmodii s. pogostemonis synchytrium synchytrium et al. s. pogostemonis p. cablin s. pogostemonis mycrosynchytrium. synchytrium mycrosynchytrium, mesochytrium, synchytrium, exosynchytrium, pycnochytrium, the microscopic observation sugested that the asexual cycle of initiated by liberation of zoospores from summer sporangia.and the liberated zoospores swam to the surrounding cells. they. were then encysting on the plant cells and eventually they infected the plant cells (fig 2.1, 2.2, and 2.3). in the subsequent stage the summer sporangia were formed within the plant cells. the mature summer sporangia released the zoospores into adjacent cells or on to plant surfaces in searching for other healthy plant cells, and the asexual cycle was repeated (fig 2.4). the zoospore dispersal through water movement or water film seemed more prevalent than dispersal by wind. artificial inoculation experiment on patchouli seedlings with water as a planting medium was successfully conducted in a green house (wahyuno and sukamto 2010). it sugested that summer spores of were naturally dispersed by water movement, water splash or movement within cells of wartz but not by wind as dry spores (price 1987). the present study indicated also that had a macrocylic life cycle. the presence of orange to light brown and thick walled spores inside the warts was a clear evidence of the existence of the resting spores. the resting spore is an indicator of the sexual reproduction occurring in nature. the resting spores are the product of pairing of two different types of zoospores (fig 2.5), encysting and adhering on plant cells prior to infecting (fig 2.6), deve loping young resting spores (fig 2.7) and formation of mature resting spore (fig 2.8). a resting spore germinates by producing a vesicle-like structure without rupturing the resting spore cell wall, but by emerging through the conical structure which is a part of the resting spore, as the releasing pore (fig 2.9). in this stage the resting spore functions as prosorus. the vesicle-like structure acts as a sorus where initial formation of a sporangial structure takes place (fig 2.10). further cleavage continues to take place in the sori, leading to the advanced stage of sporangial formation (fig 2.11). in the advanced stage, the sorus tear its thin cell wall, and release the sporangia into soils or plant surfaces (fig 2.12 and 2.13), followed by liberating the zoospores from the sporangia, and the sexual reproduction cycle is repeated. the result of present observation differs from that reported by dayal (1997), who shows that the vesicle-like structures emerge from sori, instead of resting spores. karling (1964) points out that the presence of conical pore of empty sheath is an indicator of the presence of prosorial stage in the life cycle of . the resting spore of the product of mating of two different types of zoospore (karling 1964; garcia massini 2007), especially when the environment is unfavorable (hoffman 2008). based on the current observation, it is inferred that infecting belongs to the long lifecycle type, and this phenomenon is reported for the first time in the present study. therefore, suppose belongs to member of the subgenus karling (1964) proposed to consist of six subgenera, i.e. and s s woroninella mycrosynchytrium s. pogostemonis s. endobioticum s. endobioticum mesochytrium p. cablin synchytrium psophocarpi et al. s. pogostemonis p. cablin s. pogostemonis s. pogostemonis s. pogostemonis . is characterized by its long life-cycle, the presence of prosori, sori, sporangia zoospores and resting spore stages, as well as the nature of its resting spore that do not function as sporangia (karling 1964). the life-cycle of differs from infecting potatoes. at the sexual reproductive cycle stage, the resting spore of transforms its function to become the sporangium without passing through either prosori nor sorial stage ( sub genus) (karling 1964). in the warts appeared at the stem bases and young shoots emerging from the below soil surface three to four weeks after artificial inoculation (wahyuno and sukamto 2010); and one to weeks if the inoculation were carried out on leaves (sukamto and wahyuno 2008). on winged bean, warts appeared one week after leaves were inoculated with (karami 2009). lange and olson (1981) mentioned that the warts were seen developing at the base of the potato plants three to four weeks after artificial inoculation. garcia massini (2007) pointed out that the grouping into holocarpic and eucarpic is based on the conversion of thallus into reproductive structures. since the thalli were not present in the infected patchouli, it is suggested to include infecting in the holocarpic type. the presence of prosorial and sorial stages prior to the sporangium formation is a clear indication that the has a long lifecycle. it implies the existence of a more complex process in the epidemic development of in the field, the strategy and approach of controlling might differ from those applied for potatoes, especially in exercising all efforts to erradicate the sources of inoculum in the soil and infected plant debris. references dayal m. 2007. indian chytridomycetes. m.d. new delhi: publications pvt. hoffman y, aflalo c, zarka a, gutman j, james ty, boussiba s. 2008. isolation and characterization of a novel chytrid species (phylum blastocladiomycota), parasitic on the green alga mycol res 112:70-81. karami a, ahmad zam, sijam k. 2009. morphological characteristics and pathogenicity of (rac.) gäumann associated with false rust in winged bean. am j appl sci 6: 1876-9. karling j. 1954. host reaction, host-parasite relationship, hosts and taxonomic criteria in . mycologia 46:292-313. karling j. 1964. . new york: academic pr.. lange l, olson lw. 1981. development of the resting sporangia of the causal agent of potato wart disease. protoplasma 106:83-95. garcia-massini jl. 2007. a possible endoparasitic chytridiomycetes fungus from the permian of antarctica. palaeontol electron 10:1-14. mustika i, asman a. 2004. [controlling insect pests and diseases of patchouli in indonesia ] [in indonesian].. perkembangan teknol tan rempah obat 16:40-6. price tv. 1987. liberation and germination of sporangia. trans br mycol soc 89:333-40. rao nnr, pavgi ms. 1986. acid-temperature-shock treatment as a method for inducing resting spore germination in some tropical synchytria. curr sci 55:196-7. haematococcus. synchytrium psophocarpi synchytrium synchytrium synchytrium endobioticum, synchytrium desmodii 130 wahyuno microbiol indones sitepu d, asman a. 1991. [research on diseases of patchouli in nad]. [in indonesian]. laporan kerja sama pupuk iskandar muda dan balittro. 22 p. sukamto, wahyuno d. 2008. inokulasi jamur sp. pada tanaman nilam. seminar nasional pengendalian terpadu opt jahe dan nilam. baliitro; 2008 nov 4; bogor, indonesia. p147-52. thronton h. 2002. bio-geography. //synchytrium bio-geography [29 oct 2010]. synchytrium synchytrium http://bama.ua.edu/ ~nsfpeet/biogeography wahyuno d. sukamto. 2010. [the resistance of and against ] (in indonesian). j penel tan industri 16:91-7. wahyuno d, sukamto, manohara d, kusnanta m.a, c. sumardiyono c, hartono s. 2007. a potential threat of patchouli in indonesia. proceeding international seminar on essential oil; 2007 nov 7-9; jakarta: indonesian essential oil council. p 92-9. webster j. 1988. introduction to fungi, 2 ed. cambridge: cambridge univ pr. . pogostemon cablin pogostemon heyneanus synchytrium pogostemonis . synchytrium nd volume 4, 2010 microbiol indones 131 4.mi721-praptiwi available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.4issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 159-166 *corresponding author; phone/fax: +62-21-8765066/ 21-8765062, email: bislunatin@yahoo.com +62endophytic microbes are microorganisms that live inside the plant tissues without causing negative effects to residential plant (petrini 1991). endophytic microbes, particularly endophytic fungi in healthy plant tissues are known to produce broad spectrum and highly diversed bioactive secondary metabolites (zhang et al. 2006). various bioactive compounds have been reported to be isolated from endophytic microbes associated with various plant species, for examples anticancer compound, taxol, which is produced by fungus taxomyces andreanae (stierli et al. 1993), leusinostatin a and leusinostatin a α-o-glucoside produced by endophytic fungus acremonium sp. tbp-5 (strobel et al. 1997; strobel et al. 1997) and antibiotic sitosporon d and e produced by endophytic fungi cytospora sp. cr 200 (brady et al. 2000) and many other examples. an important metabolite from endophytic fungus diaporthe sp. gnbp-10 isolated from gambier plant uncaria gambier roxb.: rubiaceae (ilyas et al. 2009) that possesses strong activity against pathogenic bacteria escherichia coli and micrococcus luteus was isolated and characterized in this current study. the metabolite was identified as (+)-1,1’-bislunatin (fig 1), which is a dimeric form of lunatin, an anthraquinone produced from curvularia lunata (jadulco et al. 2002). prior to this present study the compound was isolated from endophytic fungus diaporthe sp. f associated with a tea plant camellia sinensis (l.) kuntze and reported to produce cytotoxicity against kb cells with ic 3.5 µg (agusta et al. 2006). to the best of our 50 knowledge, there is no antibacterial activity of (+)1,1’-bislunatin reported yet. in this paper we deal with the antibacterial activity of (+)-1,1’-bislunatin against several bacteria and its effect on the cell morphology of the tested bacteria. materials and methods fungus cultivation. the endophytic fungus diaporthe sp. gnbp-10 isolated from young stem of gambier plant (uncaria gambier roxb. : rubiaceae) was cultivated on potato dextrose agar (pda) (25 x 200 ml) (becton, dickinson and company), and -1 ml endophytic fungi has been known as a source of biologically active compunds with a broad sprectrum of activities. one of the endophytic fungi isolated from young stem of gambier plant (uncaria gambier roxb.:rubiaceae), diaporthe sp. gnbp-10, produced (+)-1,1’-bislunatin when cultivated on potato dextrose agar (pda). (+)-1,1’-bislunatin showed moderate antibacterial activity against 7 bacteria (bacillus subtilis, staphylococcus aureus, escherichia coli, micrococcus luteus, shigella flexneri, proteus vulgaris, and p. -1 mirabilis) with mic value in the range of 32-64 µg ml . the wall disruption and morphological changes in the cells affected by exposure of (+)-1,1’-bislunatin are also discussed in this article. key words: (+)1,1’-bislunatin, antibacterial, diaporthe sp. gnbp-10, effect on bacterial cells, endophytic fungus, uncaria gambier roxb. fungi endofitik telah diketahui sebagai pengahasil senyawa biologi aktif yang memiliki spektrum aktivitas yang luas. salah satu fungi endofitik diisolasi dari akar tanaman gambir yang masih muda (uncaria gambier roxb.:rubiaceae), diaporthe sp. gnbp-10 menghasilkan (+)-1,1’-bislunatin ketika ditumbuhkan pada media potato dextrose agar (pda). (+)-1,1’-bislunatin menunjukkan aktivitas aktivitas antibakterial yang moderat terhadap 7 spesies bakteri patogen (bacillus subtilis, staphylococcus aureus, escherichia coli, micrococcus luteus, shigella flexneri, proteus vulgaris, dan p. mirabilis) dengan nilai penghambatan minimum antara 32-64 -1 µg ml . pemberian (+)-1,1’-bislunatin terhadap sel bakteri menimbulkan kerusakan dinding sel dan perubahan morfologi sel bakteri. kata kunci: (+)1,1’-bislunatin, antibakterial, diaporthe sp. gnbp-10, efek terhadap sel bakteri, fungi endofitik, uncaria gambier roxb. antibacterial activity of bisanthraquinone (+)-1,1’-bislunatin praptiwi, yuliasri jamal, ahmad fathoni, arif nurkanto, and andria agusta* research center for biology, indonesian institute of sciences jalan raya bogor 46, cibinong, indonesia incubated at room temperature for one month. after the incubation fungal biomass including the medium, were cut into small pieces and soaked overnight with ethyl acetate before being filtered. the filtrates were evaporated using rotary evaporator to afford 1.21 g of dark red crude extract. isolation and identification of (+)-1,1’bislunatin. the crude extract (1.21 g) was then separated through sephadex lh-20 (300 ml, amersham biosciences) column chromatography and eluted with methanol. eluate from column chromatography was analyzed tlc silica gel plate ( m e r c k , t y p e 6 0 , f ) u s i n g m i x t u r e o f 2 5 4 tetrahydrofuran-chloroform-water (6:4:0.5) as solvent. the eluate that has similar spot was combined into 1 fraction. analysis of purity level and identification of (+)-1,1’-bislunatin were performed through high performance liquid chromatography (hplc) (shimadzu, cbm-20a). the hplc analysis was performed using capcellpak c18 column (5 µm, 25 cm x 4,6 mm, shiseido) with solvent gradient 1 70 % acetonitrile (v/v in water) at 40 °c. the flow rate was -1 set at 1 ml min and uv detector at 254 nm. compound 1 13 identity was analyzed by h-nmr (500 mhz) and cnmr (125 mhz) (joel eca-50, japan) using dmso solvent and compared with the published d6 data of (+)-1,1’-bislunatin previously isolated from diaporthe sp. f (agusta et al. 2006). antibacterial activity assay and determination of minimum inhibitory concentration (mic). bacillus subtilis lipimc 0073, staphylococcus aureus lipimc 0114, eschericia coli lipimc 0186, micrococcus luteus lipimc 0076, shigella flexneri (clinical isolate), proteus vulgaris (clinical isolate), and p. mirabilis (clinical isolate) had been used in this study. the antibacterial activity of (+)-1,1’-bislunatin were assayed by paper disc (diam. 6 mm, whatman) method. ten microliters of (+)-1,1’-bislunatin (1 mg -1 ml in acetone) was put onto paper disc and dried for 10 min, before the discs were placed on muller hinton medium that had previously been inoculated with the bacteria to be tested. then incubation was performed at 37 °c for 24 h. ten microliters acetone was put on paper disc as negative control. the antibacterial activity of the substance was characterized by the formation of clear zone around the paper disc. minimum inhibitory concentration (mic) was determined by broth microdilution method that has been previously validated (pfaller et al. 2002). microbial isolate was grown at 35 °c in muller hinton broth (becton, dickinson and company). the 160 praptiwi et al. microbiol indones population density used for antimicrobial testing was 1 5 × 10 cfu . (+)-1,1’-bislunatin was disolved in dimethyl sulfoxide (dmso, phyto technology laboratory). the stock solution was prepared through double concentration in yma medium (becton, dickinson and company). the concentrations of (+)1,1’-bislunatin used for mic test ranged between 128.0 -1 to 0.25 µg ml . the mic value of (+)-1,1’-bislunatin that inhibited the bacterial growth was determine by -1 adding 15 µl (0.5 mg ml ) of p-iodonitrotetrazolium violet indicators (int, sigma-aldrich) into each well. each measurement was done in triplicate. chloramphenicol and erythromycin were used as positive controls. analysis of (+)-1,1’-bislunatin influence on bacterial cells. the mechanism of bacterial growth inhibition was determined based on the protein and nucleic acid contents in the culture media. escherichia coli and b. subtilis were cultured in muller hinton broth medium at room temperature. after 48 h, (+)1,1’-bislunatin (in dmso) was added into culture medium then incubated again for 24 h. ten ml bacteria suspension was centrifuged at 4 °c, 3 500 rpm for 15 min, and the filtrate was discarded. pellet was washed with phosphate buffer ph 7.4 twice, then resuspended with 9 ml phosphate buffer and 1 ml of (+)-1,1’bislunatin at 1 mic concentration followed by incubation in shaker incubator at 100 rpm, and 37 °c for 18 24 h. the suspension was centrifuged again for 15 min, at 3 500 rpm. supernatant was filtered, and its absorbance was measured using spectrophotometer uv-vis shimadzu at 260 nm and 280 nm (castillo et al. 2006). 2+ + ca and k ions that leaked out from cell membrane due to (+)-1,1’-bislunatin exposure was detected by atomic absorption spectrophotometer (aas, perkin elmer). supernatant was prepared as for the analysis of protein and nucleic acid (castillo et al. 2006). e. coli suspension which has been previously treated with (+)-1,1’-bislunatin was centrifuged for 5 min at 5 000 rpm. supernatant was discarded and pellet then was fixed with 2% glutaraldehyde for 2 h and centrifuged again. cocodylate buffer 0.2 m ph 7.2 was added to the pellet and left for 20 min, then 1% osmium tetraoxide was added into the cocodylate buffer and left in refrigerator for an hour before being centrifuged again. this pellet was then suspended in 70, 80, and 96% alcohol, for 10 min each respectively. then, the pellet was finally suspended in butanol. one loopful of suspension was placed on a cover glass glued to an -1 ml aluminium stub and frozen. then it was freeze-dried for 1 h. the dried cell suspension on the cover glass was then vacuum coated (6 to 7 pa) with gold for 20 min and analysed with scanning electrone microscope (sem) jeol 6300. results cultivation of the fungi, isolation and characterization of (+)-1,1’-bislunatin. after one month incubation culture media (including the fruiting bodies) were isolated with ethylacetate and dried at low pressure to obtain the dark red extract. separation of 1.21 g crude extract using sephadex-lh20 column chromatography produced six fractions ie. fraction 1 (f1: 0,219 g), fraction 2 (f2: 0,0358 g), fractions 3 (f3: 0,235 g), fraction 4 (f4: 0,1168 g), fractions 5 (f5: 0,0019 g), and fraction 6 (f6: 0,3611 g) respectively. the target metabolite (+)-1,1’bislunatin appeared as a single spot on f6. further analysis by cochromatography technique showed 99.94 % purity of the obtained (+)-1,1’-bislunatin based on hplc chromatogram peak area (fig 4). finally, the chemical structure of (+)-1,1’-bislunatin was confirmed by 1 13 comparing its chemical shift values in the hand cnmr spectra as shown on fig 5 and 6 and tabel 1. antibacterial activity of (+)-1,1’-bislunatin. in the preliminary disc difussion assay, the isolated metabolite (+)-1,1’-bislunatin showed antibacterial activities against e. coli and s. aureus. further investigation implementing dilution method also showed a moderate antibacterial activities against some pathogenic bacteria with mic values ranging -1 from 32 to 64 µg ml . interestingly, (+)-1,1’-1 bislunatin (mic 64 µg ml ) showed conspicuously stronger activity against clinical isolate proteus mirabilis. it was even more obvious than the widely known commercial antibiotics chloramphenicol (mic -1 113.78 µg ml ) and erythromicin (mic 113.78 µg ml 1 ). on the other hand, antibacterial activity of (+)-1,1’-1 bislunatin (mic 64 µg ml ) was conspicuously -1 stronger than erythromycin (mic 113.78 µg ml ) but -1 equally strong with chloramphenicol (mic 64 µg ml ) against clinical isolate of p. vulgaris. inhibition mechanism of bacterial growth. the 2+ + leakages of protein, nucleic acid, ca , dan k were the parameters observed in the bacterial cells membrane breakdown when exposed to (+)-1,1’-bislunatin. the result showed that, when exposed to (+)-1,1’bislunatin, the concentration of protein, and nucleic acid in e.coli and b.subtilis culture media significantly increased compared to the control treatment (fig 2). volume 7, 2013 microbiol indones 161 table 1 13cand 1h-nmr data in dmso-d6 for (+)-1,1’-bislunatin isolated from the culture of diaporthe sp. gnbp-10 and diaporthe sp. f atom (+)-1,1’-bislunatin from diaporthe sp. gnbp-10 (+)-1,1’-bislunatin from diaporthe sp. f* 1,1’ 124.0 123.7 2,2’ 164.2 6.70, 2h,s 164.0 6.74, 2h,s 3,3’ 107.4 107.3 4,4’ 163.7 163.6 4a,4a’ 108.6 108.6 6.73,2h,d,j=2.1 hz 5,5’ 164.2 6.78, 2h,d,j=2.1 hz 164.1 6,6’ 106.7 6.96,2h,d,j=2.1 hz 106.5 6.96,2h,d,j=2.1 hz 7,7’ 165.6 165.5 8,8’ 106.9 106.9 8a,8a’ 135.0 134.9 9,9’ 181.6 181.4 9a,9a’ 130.9 130.9 10,10’ 188.5 188.5 10a’10a 109.4 3.82,6h,s 109.2 3.81,6h,s 7,7’-och 3 56.2 56.0 oh 12.28, 12.82, 2x2h,s 12.24, 12.82, 2x2h,s 13c-nmr 1h-nmr 1h-nmr13c-nmr *agusta et al. 2006 1,1’-bislunatin was greater than any of those standard antibiotics. this indicates that (+)-1,1’ bislunatin possesses weaker antibacterial activity compared to any of commercial erythromycin-based antibiotics. in order to determine the mechanism of (+)1,1’ bislunatin inhibition of e.coli and b.subtilis growth, 2+ + protein, nucleic acid, ca and k leakage from cells were observed the protein leakage was observed at the wavelength of 280 nm, while the nucleic acid leakage was observed at 260 nm (castillo et al. 2006). there were increasing concentration of protein and nucleic acid in e.coli and b.subtilis culture media of when exposed to (+)1,1’bislunatin. this indicates the presence of protein and nucleic acid transfer from cytosol in the bacteria cells into culture medium. this is in conformity with the increase of the contents of 2+ + metal ions ca and k in same medium (fig 2). the result of this study is in accordance with (castillo et al. 2006) that the potassium leakage as the first sign of membrane damage in microorganisms, a process that leads to the increase presence of protein, nucleic acid, 2+ + and ions ca and k in the culture medium transferred there from the cytosol cell due to the breaking down of bacteria cell walls. lavlinesia (2007) also noted that . observation of e. coli and b. subtilis cell morphology when exposed to (+)-1,1’-bislunatin indicated abnormalities. treatment e.coli (fig 3b) and b.subtilis (fig 3d) with of 64 µg/ml (+)-1,1’-bislunatin caused uneven cell surface. discussion the isolation and characterization of (+)1,1’ bislunatin was done by sephadex lh-20 column chromatography. in order to validate the purity of (+)1,1’-bislunatin, the f6 was analysed using hplc by co-chromatography technique using (+)-1,1’bislunatin as the internal standard. the hplc chromatogram indicated that the only single peak in f6 was identical with the (+)-1,1’-bislunatin standard, and had 99.94% purity. the chemical structure of f6 1 was elucidated by 1d-nmr analisys. both the h13 nmr and c-nmr spectra of the purified f6 showed identical chemical shift value with (+)-1,1’-bislunatin isolated from the culture of diaporthe sp. f (agusta et al. 2006). based on the above data, it was clear that the metabolite in f6 was (+)-1,1’-bislunatin. the isolated (+)-1,1’-bislunatin was tested for antibacterial activity against several bacterial isolates including clinical isolate p. mirabilis. p. mirabilis is a species of gram-negative bacteria that causes cystitis, acute pyelonephritis,urinary stones production (mobley research laboratory 2013). usually, gram negative bacteria are less sensitive to antibiotic substances due to their cell membrane structure that is obviously different from gram positive bacteria. nevertheless, the mic value of (+)-1,1’-bislunatin against p. mirabilis was still lower than that of erythromycin. the result of the present study showed that generally, the mic value of bacteria exposed to (+) microbiol indones162 praptiwi et al. table 2 mic values of (+)-1,1’-bislunatin against tested bacteria o o o o oh ho och3 ho ho oh oh och3 fig 1 molecular structure of (+)-1,1’-bislunatin. p. mirabilis (clinical isolate) microbial isolates -1 mic values (ug ml ) chloramphenicol (+)-1,1’-bislunatin erythromycin b. subtilis lipimc0073* 64 8 0.03 s. aureus lipimc0114 64 16 0.06 e. coli lipimc0186 64 16 32 m. luteus lipimc0076 64 16 16 s. flexneri (clinical isolate) 32 1 4 p. vulgaris (clinical isolate) 64 64 113.78 64 113.78 113.78 *lipimc: lembaga ilmu pengetahuan indonesia collection. 2+ + fig 2 the quantities of (ca , k ), protein and nucleic acid leakages from bacterial cells after being exposed by (+)-1,1’bislunatin on 1 mic values. volume 7, 2013 microbiol indones 163 negative control b. subtilis, 1 mic negative control e.coli, 1 mic a b so rb an ce 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 protein leakage negative control b. subtilis, 1 mic negative control e.coli, 1 mic 0.3 0.25 0.2 0.1 c o n ce n tr at io n ( p p m ) ++ ca ion leakage 0.15 0.05 0 c o n ce n tr at io n ( p p m ) nucleic acid leakage negative control b. subtilis, 1 mic negative control e.coli, 1 mic 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 c o n ce n tr at io n ( p p m ) negative control b. subtilis, 1 mic negative control e.coli, 1 mic + k ion leakage50 49 48 47 46 45 44 43 42 41 + protein, nucleic acids, and k were indicators of the disruption in the bacterial membranes. this indicated the damage in the bacterial cells. the excessive cytoplasm loss was due to membrane leakage. it also supports the previous results of henie et al. (2009) which stated that the release of bacterial microbiol indones164 praptiwi et al. fig 3 sem micrograph of the cell morphology of e. coli without (a) and after being treated (+)-1,1’-bislunatin (b). the same treatment in bacillus subtilis cells without (c) and after being treated (+)-1,1’-bislunatin (d). the arrows indicate the deformed shrinking cells. fig 4 hplc chromatogram of purified (+)-1,1’-bislunatin. a b dc 0.0 5.0 10.0 15.0 20.0 25.0 min 100 200 300 mv detector a:254nm 0 1 fig 5 h nmr spectrum of purified (+)-1,1’-bislunatin. volume 7, 2013 microbiol indones 165 evidently exposure to (+)-1,1’-bislunatin caused an obvious change in the permeability of bacterial cell membrane and degeneration of cell membrane. according to kvam et al. (2012) the cell poration and lysis are believed to be indicators of gradual cellular protein and nucleic acid leakage from the cell. the observation on cellular morphology showed that the bacteria cells deflated after treatment with (+)1,1’-bislunatin, causing losses of cell wall function as protector or barrier between organs and cellular fluid. as a consequence the cell loss most of its contents due to the destruction of the bacterial cell wall or necrosis and the cellular fluid migrate irresistibly into the culture medium. this is in accordance with our results in which the concentration of proteins and nucleic 2+ + acids, ca and k increased in the culture medium of the cells after being treated with (+)-1,1’-bislunatin. to conlclude (+)-1,1’-bislunatin isolated from endophytic fungus diaporthe sp. gnbp-10, obtained from young stem of gambier plant (uncaria gambier roxb.: rubiaceae) showed moderate antibiotic activity against several bacterial isolates. exposure of (+)1,1’ bislunatin caused disruption of the cell wall and changes in the cell morphologies of b. subtilis and e. coli cells. acknowledgment this work was supported by program riset kompetitif of indonesian institute of sciences 20112012. the authors would also like to express their gratitude to ary p. keim to kindly proofread this manuscript. references agusta a, ohashi k, shibuya h. 2006. bisanthraquinone metabolites produced by the endophytic fungus diaporthe sp. chem. pharm bull. 54(4):579-582. doi:10.1248/cpb.54.579. annon. 2002. national committee for clinical laboratory standards. brady sf, singh mp, janso je, clardy j. 2000. guanacastepene, a fungal-derived diterpene antibiotic with a new carbon skeleton. j am chem soc. 122(9):2116-2117. doi:10.1021/ja993835m. castillo ja, clapes p, infante mr, comas j, manresa a. 2006. comparative study dihydrochloride and chlorhexidine dihydrochloride against staphylococcus aureus and escherichia coli. j antimicrob chemother. 57(4):691-8. doi:10.1093/jac/dkl012. henie efp, zaiton h, suhaila m. 2009. bacterial membrane disruption in food pathogens by psidium guajava leaf extracts. int food res j. 16:297 -311. ilyas m, kanti a, jamal y, hertina and agusta a. 2009. biodiversity of endophytic fungi associate with uncaria gambier roxb. (rubiaceae) from west sumatra. j biol diversity. 10(1):23-28. jadulco r, brauer g, erada ra, ebel e, ray v, sudarsono, proksch p. 2002. new metabolites from sponge derived fungi curvularia lunata and cladorsorium herbarum. j nat prod. 65(5):730-733. doi:10.1021/np010390i. 13 fig 6 c nmr spectrum of purified (+)-1,1’-bislunatin. microbiol indones166 praptiwi et al. kvam e, davis b, mondello f, garner al 2012. nonthermal atmospheric plasma rapidly disinfects multidrugresistant microbes by inducing cell surface damage. antimicrob agents chemother. 56(4):2028-2036. doi:10.1128/aac.05642-11. lavlinesia. 2007. kajian pola dan mekanisme aktivasi bakteri oleh ekstrak etil asetat biji atung (purinarium glaberium hassk.) [dissertation]. bogor(id): institut pertanian bogor. mobley research laboratory. 2013. proteus mirabilis. available at : http://www.umich.edu/~hltmlab/research/ mirabillis/infection.htm (accesed : 27 -03-2013). petrini o. 1991. fungal endophytes of tree leaves. in: nj fokkema, j van den heuvel, editors. microbial ecology of the leaves. cambridge: cambridge university press. p 185-187. doi:10.1007/978-1-4612-3168-4_9. stierle a, strobel g, stierli d. 1993. taxol and taxane production by taxomyces andreanae, an endophytic fungus of pacific yew (taxus brevifolia). science 260(5105): 214-216. doi:10.1007/978-1-4612-31684_9. strobel ga, torczynski r, bollon a. 1997. acremonium sp. a leucinostatin a producing endophyte of european yew (taxus baccata). plant science 128(1):97-108. doi:10.1016/s0168-9452(97)00131-3. strobel ga, hess wm. 1997. glucosylation of the peptide leucinostatin a,produced by an endophytic fungus of european yew, may protect the host from leucinostatin toxicity. chemistry and biology 4(7):529-536. doi:10.1016/s1074-5521(97)90325-2. zhang hw, song yc, tan rx. 2006. biology and chemistry of endophytes. nat prod rep. 23(5):753-771. doi:10.10 39/b609472b. 1: 159 2: 160 3: 161 4: 162 5: 163 6: 164 7: 165 8: 166 chandra et al19042012 issn 1978-3477, eissn 2087-8575 vol 6, no 1, march 2012, p 9-14 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.2 only causing chronic infection, this virus can also induce liver cirrhosis and hepatocellular carcinoma formation. as an early stage in preventing hbv infection, small hepatitis b surface antigen (shbsag) is used as a major component of hepatitis b vaccine (mast et al. 2005; clements et al. 2010; chan et al. 2011). furthermore, to control hbv infection in postexposure stage, chemotherapeutic agents such as molecular analysis of immune-escape mutants of hepatitis b virus from local clinical samples 1 1 chandra jinata , ernawati arifin giri-rachman , and 2 debbie soefie retnoningrum * 1 school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, west java, indonesia; 2 school of pharmacy, institut teknologi bandung, jalan ganesha 10, bandung 40132, west java, indonesia small hepatitis b surface antigen (shbsag) is used as a component of hepatitis b vaccine. even though this vaccine is known to be effective in preventing hepatitis b disease, natural mutation may induce hepatitis b virus (hbv) to form immune-escape mutant. this mutant is not only capable of infecting hepatitis b-vaccinated people, but also causing commercial diagnostic assay failure. immune-escape mutant is generally detected from amino acid change at major hydrophilic region (mhr) of shbsag while the change occurred outside the region may also lead to immune-escape mutant formation. this research was aimed to investigate the presence of hbv immune-escape mutants in local clinical samples in indonesia. shbsag gene of seventeen hbv samples from local patients were amplified by polymerase chain reactions then subjected to two-directional sequencing. the dna sequences later were analyzed by bioinformatics programs. fifteen out of seventeen samples were genotype b and subtype adw2, while the other two were genotype c and subtype adrq+. among fifteen genotype b samples, twelve of them were not immune-escape mutants, two were immune-escape mutants that have been previously reported (gln129arg and met133leu), and one was a mutant outside mhr that has not been previously reported as an immune-escape mutant (tyr161ser). both samples of genotype c group were not immune-escape mutants. as conclusion, by investigating seventeen local clinical hbv samples, it was known that two of seventeen samples were confirmed as immune-escape mutants and one of seventeen samples was a mutant outside mhr. key words: hepatitis b virus, immune-escape mutant, major hydrophilic region, small hepatitis b surface antigen antigen permukaan berukuran kecil dari virus hepatitis b (shbsag) digunakan sebagai salah satu komponen vaksin hepatitis b. meskipun vaksin ini diketahui efektif dalam mencegah penyakit hepatitis b, namun mutasi alami pada virus hepatitis b (hbv) dapat menginduksi terbentuknya mutan lolos imun. mutan ini selain dapat menginfeksi orang-orang yang sudah divaksinasi hepatitis b sebelumnya, juga mengakibatkan kegagalan deteksi oleh kit diagnostik komersial. mutan lolos imun biasanya dideteksi dari perubahan asam amino pada major hydrophilic region (mhr) dari shbsag, meskipun perubahan asam amino di luar daerah ini juga dapat mengakibatkan pembentukan mutan lolos imun. penelitian ini bertujuan untuk mempelajari keberadaan mutan lolos imun hbv pada sampel klinis lokal di indonesia. gen shbsag pada tujuh-belas sampel hbv dari pasien lokal diamplifikasi dengan reaksi polimerisasi berantai kemudian disekuensing pada dua arah pembacaan. sekuens dna yang didapat kemudian dianalisis dengan program bioinformatik. lima-belas dari tujuh-belas sampel termasuk genotipe b dan subtipe adw2, sementara dua lainnya termasuk genotipe c dan subtipe adrq+. pada lima-belas sampel genotipe b, dua-belas di antaranya bukan termasuk mutan lolos imun, dua termasuk mutan lolos imun yang sudah dilaporkan sebelumnya (gln129arg dan met133leu), dan satu termasuk mutan di luar mhr yang belum dilaporkan sebelumnya sebagai mutan lolos imun (tyr161ser). kedua sampel dari genotipe c bukan termasuk mutan lolos imun. sebagai kesimpulan, berdasarkan hasil investigasi tujuhbelas sampel klinis lokal hbv, diketahui bahwa dua dari tujuh-belas sampel termasuk mutan lolos imun dan satu dari tujuh-belas sampel termasuk mutan di luar mhr. kata kunci: antigen permukaan berukuran kecil dari virus hepatitis b (shbsag), major hydrophilic region, mutan lolos imun, virus hepatitis b hepatitis b virus (hbv) is one of the most common infectious disease causing agents in the world. this virus was estimated to infect one third of total world population and approximately around 20% of them are infected chronically (chan et al. 2011). not *corresponding author, phone/ fax: +62222504852, e-mail: retnoningrum@indo.net.id 10 jinata et al. microbiol indones materials and methods pcr and dna sequencing. seventeen hbv dna samples were isolated at pramita diagnostic laboratory, jakarta, indonesia. the shbsag gene was amplified by pcr using primers designed by kusumawardhani (2009) for forward sequence and utari (personal communication 2011) for reverse sequence. the nucleotide sequences of the primers were as follows: f:5’-gaattcatggagaacatcg catcagg-3’ and r:5’-ggtcaccttaaatgtata cccaaagac-3’. the pcr mixture contained 1 µl genomic dna template, final concentration of: 1 x taq buffer, 2 µm mgcl , 0.2 mm dntps, 0.2 u taq dna 2 polymerase, and 0.4 µm of each primer per 25 µl reaction. the pcr program consisted of preo denaturation at 94 c for 2 min, followed by 30 cycles at o o o 94 c for 30 sec, 55 c for 45 sec, and 72 c for 1 min. o the last step was at 72 c for 7 min. as positive control, shbsag gene-containing-plasmid bamafsv from zainuddin (2008) was used. pcr results were visualized by electrophoresis on 1.2% (w/v) agarose gel in 1 x tae, followed by gel immersion in diluted -1 ethidium bromide (0.5 µg ml ). positive result was based on the presence of dna band of 681 bp on the agarose gel. specific dna band later was purified by gel or pcr dna fragments extraction kit (geneaid) then subjected to two-directional direct sequencing at st 1 base company, singapore. sequence analyses. raw sequences from direct sequencing were confirmed by contig analysis and blast-n (http://www.ncbi.nlm.mih.gov/blast). genotyping was based on the similarities to dna s e q u e n c e r e f e r e n c e s i n g e n b a n k n c b i (http://www.ncbi.nlm.nih.gov). each sample sequence was aligned to a conserved sequence based on alignment of shbsag coding genes deposited in the genbank ncbi database. mutation and amino acid change analyses were performed by bioedit program (http://www.mbio.ncsu.edu/bioedit) and later each of mutant shbsag amino acid sequence was analyzed further on b cell epitopes prediction program utilizing emini surface accessibility prediction, kolaskar and tongaonkar antigenicity, parker hydrophilicity prediction, and bepipred linier epitope prediction tools program (http://tools.immuneepitope.org). results pcr, dna sequencing, and sequence analyses. amplified dnas from pcr migrated interferon- and nucleoside or nucleotide analogs are used in combinations with different effectiveness and results (mast et al. 2005; glebe 2007). even though there are strategies in controlling hbv in preand post-exposure stages, hbv is still one of the most difficult infectious agents to defeat since the virus is variable due to the intrinsic activity of hbv dna polymerase that results in high rate of natural mutations. the high variability of hbv is shown by the coexistence of different viral population in various proportions. moreover, different environmental conditions also determine variant compositions of the population that occurs during hbv infection. hbv immune-escape mutant is the result of natural mutation that occurred on the small hepatitis b surface antigen coding gene and its existence is also supported by selection pressure of both hepatitis b vaccine (vaccineinduced escape mutant) and nucleoside or nucleotide analogs which affect not only hbv polymerase gene structure directly, but also its overlapped gene-the surface antigen gene-structure indirectly (pawlotsky 2005; cuestas et al. 2006; valsamakis 2007; clements et al. 2010). hbv immune-escape mutant is a variant of hbv that cannot be recognized by neutralizing antibody because its antigenicity profile has changed due to the mutation (pawlotsky 2005; glebe 2007). this mutant is related to clinical cases such as occult hepatitis b infections, reactivation of hepatitis b, and diagnostic assay failure (purdy 2007). immuneescape mutant issue becomes more and more urgent since its variability has been increasing with time and associated with the development of hepatocellular carcinoma (carman 1997; purdy 2007; salpini et al. 2011). about 3% of total hbv infections was hbv immune-escape mutant cases in china (he et al. 2001). generally, mutation on the major hydrophilic region (mhr) of shbsag coding gene, especially on antigenic (“a”) determinant or epitope, causes the i n d u c t i o n o f h b v i m m u n e e s c a p e m u t a n t generation. moreover, amino acid change outside mhr also affects epitope conformation of shbsag (weinberger et al. 2000; purdy 2007). this research was aimed to investigate the presence of hbv immune-escape mutants in local clinical samples in indonesia. in this study, we observed the presence of hbv immune-escape mutants from local patients in indonesia and analyzed the amino acid change and its effect to the physical profile of shbsag by bioinformatics prediction tools. volume 6, 2012 microbiol indones 11 fig 1 electrophoregram result from seventeen amplified dna samples. the dnas were migrated around 680 bp, indicating the presence of shbsag genes (681 bp). notes: l: dna ladder 1 kbp; number listed above is sample number; : negative control. around 680 bp-long of dna ladder of 1 kbp in the agarose gel electrophoresis, indicating the presence of 681 bp-long shbsag genes (fig 1). after the dnas were sequenced, partially confirmed dna sequences from each sample were obtained (genbank ncbi accession number: jq646056-646072). based on high similarity score (99%) to dna sequence references in genbank ncbi, fifteen out of seventeen samples were found to be genotype b, while the other two belonged to genotype c. using hbv subtype determination algorithm (purdy 2007), all genotype b samples were known to be subtype adw2 and all genotype c samples belonged to subtype adrq+. twenty-seven hbv full genome dna sequence references from genbank ncbi were used to obtain a conserved shbsag coding gene from genotype b and c. utilizing the conserved sequences, mutation and amino acid change analyses were performed. among fifteen samples from genotype b pool, twelve of them were not immune-escape mutants, two of them were immune-escape mutants (accession number jq646059 and jq646069) and which have previously been reported (met133leu and gln129arg, respectively), and one was both a mutant with amino acid change outside its mhr (accession number jq646056) and has never been reported as an immune-escape mutant (tyr161ser). b cell epitopes prediction program analysis. shbsag amino acid sequences from wild-type and 3 mutants were analyzed by the four tool programs. table 1 showed that compared to the wild-type, amino acid change from each mutant also caused different amino acid index specifically to the designated amino acid. fig 2 showed different antigenic propensity between wild-type and mutants in amino acid 101-161 of shbsag based on kolaskar and tongaonkar antigenicity prediction tool, while fig 3 represented the different antigenic score between wild-type and mutants in amino acid 101-161 of shbsag based on bepipred linier epitope prediction tool. discussion based on this research, hbv genotype b was found more dominant (88.2%) than the genotype c (11.8%) in this study. this result was supported by thedja et al. (2011) who found that hbv genotype b is more common in indonesia, especially in the western and southern part. as genotype of hbv is correlated to its infection severity, hbv genotype c is known to be more progressive in enhancing clinical phenotype to form hepatocellular carcinoma (zumbika et al. 2005; aljarallah 2006). assuming that all patients had the same genetic background, this result suggests that probability of hepatocellular carcinoma formation that caused by hbv infection on indonesian patients may be lower than in the region where hbv genotype c is dominant. as for hbv subtype, in this research hbv genotype b subtype adw2 was found highly predominant and two samples of hbv genotype c belonged to subtype adrq+. this result was supported esap: emini surface accessibility prediction; kta: kolaskar and tongaonkar antigenicity prediction; php: parker hydrophilicity prediction; blep: bepipred linier epitope prediction. table 1 index changes of b cell epitopes from three mutants showed amino acid change causes different index between wild-type and mutant l 1 8 9 11 15 19 26 28 29 30 36 37 40 42 43 59 65 + 2000 bp 1500 bp 1000 bp 750 bp 500 bp 250 bp mutant esap kta php blep gln129arg 2,511 à 2,819 0.978 à 0.958 4,686 à 4,429 1,376 à 1,204 met133leu 1,666 à 1,395 0.970 à 1,030 1,800 à 1,086 0.438 à 0.290 tyr161ser 2,306 à 1,984 1,034 à 1,013 -2,100 à -0.900 -0.959 à 0.599 1 microbiol indones12 jinata et al. fig 2 kolaskar and tongaonkar antigenicity prediction results from mutant tyr161ser (a), gln129arg (b), and met133leu (c). mutant tyr161ser and gln129arg showed minor difference of antigenic propensity in amino acid 158-161 and 127-128 of shbsag respectively compared to wild-type, while mutant met133leu gave major difference of antigenic propensity in amino acid 129-136 of shbsag compared to wild-type. fig 3 bepipred linier epitope prediction results from mutant tyr161ser (a), gln129arg (b), and met133leu (c). mutant tyr161ser had different antigenicity score only in amino acid 161 of shbsag compared to wildtype, while mutant gln129arg showed major antigenicity score difference on amino acid 123-132 of shbsag compared to wild-type and met133leu gave minor antigenicity score difference on amino acid 128-131 of shbsag compared to wild-type. by lusida et al. (2008) who represented that in indonesia hbv genotype b subtype adw2 has been known to be predominant in sumatera, java, the southern part of borneo, bali, lombok, ternate, and morotai while hbv genotype c subtype adrq+ was found mostly in papua. three mutants that were found in this research-two of them were known as immune-escape mutants-were from hbv genotype b. based on the multiple reference sequence alignment of shbsag coding sequence from hbv genotype b and c (data not shown), it was known that genetic variability was more common present on the genotype b. this result may suggest that hbv genotype b tends to have higher probability to generate mutant than genotype c. mutant tyr161ser had been reported before by sánchez et al. (2002) and cuestas et al. (2006) from a mexican and argentinian patient but was not confirmed as an immune-escape mutant-in contrast to a confirmed-immune-escape mutant tyr161leu (salpini, et al. 2011)-and had not been reported previously from indonesian patient. amino acid replacement from tyrosine (uncharged polar amino acid) to leucine (non-polar amino acid) changes antigenicity profile of mutant tyr161leu found in shbsag that causes immune-escape mutant case. serine, which replaces tyrosine at tyr161ser, is in the same amino acid group with tyrosine. based on this amino acid characteristic, it is assumed that tyr161ser yqgmlpvcplipgssttstgpcktcttpaqgtsmfpsccctkptdgnctcipipsswafaky 100 110 120 130 140 150 160 s wild-type tyr161ser 1400 1200 1000 800 600 400 200 0 a n ti g en ic p ro p en si ty a wild-type tyr161ser yqgmlpvcplipgssttstgpcktcttpaqgtsmfpsccctkptdgnctcipipsswafaky 100 110 120 130 140 150 160 r wild-type gln129arg wild-type gln129arg 1400 1200 1000 800 600 400 200 0 a n ti g en ic p ro p en si ty b yqgmlpvcplipgssttstgpcktcttpaqgtsmfpsccctkptdgnctcipipsswafaky 100 110 120 130 140 150 160 l wild-type met133leu wild-type met133leu 1400 1200 1000 800 600 400 200 0 a n ti g en ic p ro p en si ty c yqgmlpvcplipgssttstgpcktcttpaqgtsmfpsccctkptdgnctcipipsswafaky 100 110 120 130 140 150 160 s wi l d t y p e ty r 1 6 1 s e r s co re 2500 2000 1500 1000 500 0 -500 -1000 -1500 a wild-type tyr161ser yqgmlpvcplipgssttstgpcktcttpaqgtsmfpsccctkptdgnctcipipsswafaky 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 r wi l d t y p e g l n 1 2 9 a r g wild-type gln129args co re 2500 2000 1500 1000 500 0 -500 -1000 -1500 b s co re 2500 2000 1500 1000 500 0 -500 -1000 -1500 c yqgmlpvcplipgssttstgpcktcttpaqgtsmfpsccctkptdgnctcipipsswafaky 100 110 120 130 140 150 160 wild-type met133leu wild-type met133leu l volume 6, 2012 microbiol indones 13 may not be an immune-escape mutant, even though further clinical investigation needs to be done. based on the b cell epitopes prediction program, from surface accessibility, antigenicity, hydrophilicity, and linier epitope perspectives, all mutants gave different indices compared to wild-type (table 1). this difference overall might significantly affect shbsag antigenicity (fig 2) and changes its epitope conformation (fig 3). since three-dimensional structure of shbsag is still unknown, using prediction tools to determine antigenicity profile change as the effect of amino acid replacements is merely speculative and specific research regarding to antigenicity profile change and clinical immune-escape mutant impact need to be done (cuestas et al. 2006). however at the bepipred linier epitope prediction result in fig 3b, where mutant gln129arg showed major antigenicity score difference on amino acid 123-132 of shbsag compared to wild-type, this change indicated that mutant’s epitope structure may have changed so that binding capacities of this mutant to several monoclonal antibodies were much lower (chiou et al. 1997). in brief, this study showed that immune-escape mutant from hbv genotype b, was present in local clinical samples in indonesia. the amino acid replacement occurred on shbsag may change its antigenicity profile. further study about mutant tyr161ser should be done regarding to references aljarallah, bm. 2006. hepatitis b genotyping and its clinical implications. the saudi j of gastroenterol. 12(3): 146148. carman, wf. 1997. the clinical significance of surface antigen variants of hepatitis b virus. j viral hepat. (4):s11-20. chan hl-y, jia jd. 2011. chronic hepatitis b in asia-new insights from the past decade. j gastroenterol hepatol. 26(s1):131-137. doi:10.1111/j.1440-1746.2010. 06544.x. chiou h-l, lee t-s, kuo j, mau y-c, ho m-s. 1997. altered antigenicity of 'a' determinant variants of hepatitis b virus. j gen virol. 78:2639-2645. clements cj, coghlan b, creati m, locarnini s, tedder rs, torresi j. 2010. global control of hepatitis b virus: does treatment-induced antigenic change affect immunization? bull world health organ. 88:66-73. doi:10.2471/blt.08.065722. cuestas ml, mathet vl, ruiz v, minassian ml, rivero c, sala a, corach d, alessio a, pozzati m, frider b, in vitro study of shbsag–anti-shbs antibody interactions, since it may also be another immune-escape mutant variant. oubiňa jr. 2006. unusual naturally occurring humoral and cellular mutated epitopes of hepatitis b virus in a chronically infected argentine patient with anti-hbs antibodies. j clin microbiol. 44(6):2191-2198. doi:10.1128/ jcm.00057-06. glebe d. 2007. pathogenesis of hepatitis b virus infection. world j gastroenterol. 13(1):82-90. he c, nomura f, itoga s, isobe k, nakai t. 2001. prevalence of vaccine-induced escape mutants of hepatitis b virus in the adult population in china: a prospective study in 176 restaurant employees. j gastroenterol hepatol. 16 (12):1373-1377. kusumawardhani s. 2009. kloning dan ekspresi hbsag isolat indonesia secara intraselular pada pichia pastoris km71 [thesis]. bandung (id): institut teknologi bandung. a comprehensive immunization strategy to eliminate transmission of hepatitis b virus infection in the united states. mmwr 54 (rr-16). pawlotsky j-m. 2005. the concept of hepatitis b virus mutant escape. j clin virol. 34:s125-s129. purdy ma. 2007. hepatitis b virus s gene escape mutants. asian j transf sci. 1(2):62-70. salpini r, cento v, scopelliti f, bertoli a, gori c, micheli v, gubertini g, sanctis gmd, sarrecchia c, andreoni m, svicher v, perno cf. 2011. prevalence of drugresistance hbv strains is relatively low in drugexperienced patients and nearly absent in drug-naïve patients [presentation]. cyprus: european workshop on hiv and hepatitis. sánchez lv, maldonado m, bastidas-ramīrez be, norder h, panduro a. 2002. genotypes and s-gene variability of mexican hepatitis b virus strains. j med virol. 68:24-32. doi: 10.1002/jmv.10166. thedja md, muljono dh, nurainy n, sukowati chc, verhoef j, marzuki s. 2011. ethnogeographical structure of hepatitis b virus genotype distribution in indonesia and discovery of a new subgenotype, b9. arch virol. 155:855-868. doi:10.1007/s00705-0110926-y. valsamakis a. 2007. molecular testing in the diagnosis and management of chronic hepatitis b. clin microbiol rev. 20(3):426-439. weinberger km, bauer t, bohm s, jilg w. 2000. high genetic variability of the group-specific a-determinant of hepatitis b virus surface antigen (hbsag) and the corresponding fragment of the viral polymerase in chronic virus carrier lacking detectable hbsag in serum. j gen virol. (81):1165-1174. zainuddin im. 2008. kloning dan konstruksi hbsag ke dalam vektor ekspresi tumbuhan dengan promoter terinduksi luka [thesis]. bandung (id): institut teknologi bandung. lusida mi, nugrahaputra ve, soetjipto, handajani r, nagano-fujii m, sasayama m, utsumi t, hotta h. 2008. novel subgenotypes of hepatitis b virus genotypes c and d in papua, indonesia. j clin microbiol. 46(7):2160-2166. mast ee, margolis hs, fiore ae, brink ew, goldstein st, wang sa, moyer la, bell bp, alter mj. 2005. microbiol indones14 jinata et al. zumbika e, ruan b, xu ch, ni q, hou w, chen z, liu kz. 2005. hbv genotype characterization and distribution in patients with hbv-related liver diseases in zhejiang province, pr china: possible association of coinfection with disease prevalence and severity. hepatobiliary pancreat dis int. 4(4):535-543. 1: 9 2: 10 3: 11 4: 12 5: 13 6: 14 2 433 i made kartika.cdr bioenergetic analysis of flag tagged-subunit 8 of mitochondrial atp synthasesaccharomyces cerevisiae i made artika department of biochemistry, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor16680, indonesia phone/fax.+62-251-8423267, e-mail: imart171@yahoo.com the majority of cellular energy in the form of adenosine triphosphate (atp) is synthesized by the f f -atp synthase. the yeast mitochondrial f f -atp synthase is a multisubunit complex that contains at least 17 different subunits grouped into f and f sectors. subunit 8 of yeast mitochondrial atp synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial gene. subunit 8 has three distinct domains; an n-terminal domain, a central hydrophobic domain and a c-terminal domain. flag tag addition to the c-terminus of subunit 8 and its variants has facilitated elucidation of subunit 8's membrane topology. in order to analyze its detailed structure and function, a set of strains expressing flag tagged-subunit 8 and its variants were subjected to bioenergetic analysis at cellular and mitochondrial levels. results obtained showed that the hydrophobic character of the central hydrophobic domain of subunit 8 is essential for functional coupling between f and f sectors, hence for mitochondrial atp synthase function. key words: bioenergetics, atp synthase, mitochondria, yeast 1 0 1 0 1 0 1 0 atp8 the majority of cellular energy in the form of adenosine triphosphate (atp) is synthesized by the f f -atp synthase. power for atp synthesis is derived from an electrochemical proton gradient generated by an electron transport chain in an energy transducing membrane. this force drives rotation of membranous (f ) motor components. rotation of the components of the f motor is transmitted to the eccentric shaft subunit to elicit conformational changes in the catalytic site f , leading to atp synthesis (van ballmos . 2009). in some organisms, the atp synthase also works in the reverse direction by hydrolyzing atp and generating an electroc hemical proton gradient across a membrane to support locomotion or nutrient uptake (hong and pedersen 2008). atp synthase is an exceptionally complicated protein complex. the yeast mitochondrial atp synthase is composed of at least 17 subunits grouped into two sectors, a membrane-extrinsic sector (f ) and a membrane-embedded sector (f ). the two sectors are linked by protein stalks. the f sector is comprised of five subunits , , , , and , and is coupled to proton flux through the f sector. the f sector spans the membrane and is composed of subunits b, oscp, d, e, f, g, h, i/j, k which are encoded by nuclear genes, and subunits 6, 8, and 9, which are encoded by mitochondrial genes (stephens . 2003). in the inner mitochondrial membrane the atp synthase complex can form a dimer (fronzes . 2006). the subunit 8 of yeast mitochondrial atp synthase is essential for oxidative phosphorylation (macreadie . 1983). it has three distinct domains; an n-terminal domain, a central hydrophobic domain (chd) and a c-terminal domain (devenish . 1992). the topology of subunit 8, which is determined by introduced unique cysteine residues, indicates that its n-terminus is located in the intermembrane extrinsic to the lipid bilayer, indicating the existence of a 21 1 0 0 0 1 1 0 1 0 0 et al et al et al et al et al space of mitochondria whereas the c-terminus is located within the mitochondrial matrix (stephens . 2000). further analysis employing cysteine-scanning mutagenesis showed that the first 14 and the last 13 amino acids were et al amino acid transmembrane spanning region (stephens . 2003). it is essential for subunit 8 to maintain transmembrane topology for functioning (artika 2009). as a mitochondrially encoded protein, subunit 8 is transcribed and translated entirely within the organelle. subunit 8 is not present in prokaryotes which means that prokaryotic atp synthase can naturally function without the presence of subunit 8 (artika 2007). the immediate question therefore is to resolve the detailed structure and roles of this subunit in the enzyme complex. several lines of evidence suggested that subunit 8 is part of the stator stalk in the yeast mitochondrial atp synthase. no amino acid of subunit 8 directly participates in either atp synthesis/hydrolysis or proton pumping, suggesting that subunit 8 is a structural component of the mitochondrial atp synthase complex (stephens . 2003). in order to elucidate its detailed structure, function, and membrane topology, an allotropic expression system for subunit 8 has been developed (gearing . 1985). this system has been applied to study various aspects of subunit 8 molecular biology and has also been successfully used to express flag tagged-subunit 8 (artika 2006) and flag tagged-subunit 8 variants (artika 2007). the main purpose of the flag tag addition to subunit 8 was to enable detection of subunit 8 protein by means of an anti-flag tag monoclonal antibody. the flag tag system has been found to be useful in the studies of abundance, cellular location, posttranslational modifications, protein-protein interactions, and purification of particular tagged-protein (kolodziej and young 1991). the practicability of the flag tag system relies on the specificity of the binding of the anti-flag antibody to the flag fusion proteins (schafer and braun 1995). the small size of the flag epitope is proposed as having a minimal effect on protein conformation thereby reducing the possibility of disrupting the function of any tagged-protein. flag tagged-subunit 8 and its variants have been employed to study structure, function, and topology of this subunit et al et al et al (artika 2007, 2009). the incorporation of flag epitope issn 1978-3477 vol 4, no 3, dec 2010, p 108-112 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.2 volume 4, 2010 microbiol indones 109 . strain m31 [atp8, mit 6, 1] a collection strain of the department of biochemistry and molecular biology, monash university, has previously been described (nagley 1988). strains ftc2, df66, df68, and df71 have been described (artika 2007). strain ftc2 is strain m31 expressing flag-taggedsubunit 8 gene fused with a mitochondrial signal peptide. strains df66 is strain m31 expressing flag-taggedsubunit 8 variant (g16d, f17d) gene fused with a mitochondrial signal peptide. strain df68 is strain m31 expressing flag-tagged-subunit 8 variant (l23d, l24d) gene fused with a mitochondrial signal peptide. strain df71 is strain m31 expressing flag-tagged-subunit 8 variant (q29r, f30r) gene fused with a mitochondrial signal peptide. . isolation of mitochondria for bioenergetic analysis was carried out as described by law . (1995). unless otherwise stated, centrifugation steps were carried out using sorvall ss34 rotor. yeast cells were grown in 2 l flasks containing 450 ml salt medium [1% (w/v) yeast extract, 0.12% (w/v) (nh ) so , 0.1% (w/v) kh so , 0.01% (w/v) cacl , 0.0005% (w/v) fecl , 0.07% (w/v) mgcl , 0.05% (w/v) nacl] supplemented with 2% (v/v) ethanol until early midlogarithmic phase. after harvesting the cells by centrifugation using sorvall rc3 rotor (4 500 rpm; 5 min), the cells were washed twice with ice cold distilled water containing 0.1% (w/v) galactose. the cells were then resuspended in buffer 1 (0.1 m tris-hydrochloric acid, 0.5 m 2-mercaptoethanol, ph 9.3) and incubated for 10 min at 30 c. about 20 ml buffer 1 g dry weight of cell was used. in order to remove the 2-mercaptoethanol, which may inhibit the enzymatic digestion step, the pellet was washed three times by resuspending the pellet in ice cold buffer 2 (10 mm tris-hydrochloric acid, 0.5 m kcl, ph 7.0) followed by centrifugation. about 50 ml buffer 2 was used per g cell. the cell walls were then digested by incubating the pellet in buffer 3 (10 mm citrate, 10 mm disodium phosphate, 1.35 m sorbitol, 1 mm egta, ph 5.8) containing 1.0 mg.ml of zymolyase 20t at 30 c. the status of the digestion process was followed by measuring the optical density (od) of the suspension. the digestion was stopped when the optical density of the suspension reduced to 80%. following centrifugation (10 000 rpm; 10 min) the pellet was washed twice by resuspending the pellet in ice cold buffer 4 (10 mm tris-maleate, 0.75 m mannitol, 0.4 m sorbitol, 2 mm egta, 0.1% (w/v) bovine serum albumin, ph 6.8) at 4 c followed by centrifugation (10 000 rpm; 10 min). the pellet was then resuspended in buffer 5 (10 mm tris-maleate, 0.6 m mannitol, 2 mm egta, 0.2 % (w/v) bovine serum albumin, materials and methods materials. isolation of mitochondria saccharomyces cerevisiae his ade et al. et al saccharomyces 4 2 4 2 4 2 3 2 650nm ° ° ° −1 −1 tag on the wild-type subunit 8 has been shown not to cause functional impairment on mitochondrial atp synthase (artika 2010). in the present study, strains expressing flag tagged-subunit 8 variants were subjected to cellular and mitochondrial respiration assays to analyze detailed subunit 8's structure and function 0.5 mm phosphate, ph 6.8). about 10 ml buffer 5 was used per g dry cell weight. to release mitochondria from the spheroplast, the suspension was homogenized at high speed for 6 x 5 sec. the suspension was diluted 4 times with buffer 5 followed by low speed centrifugation at 2000 rpm for 20 min. the supernatant was decanted and centrifuged at high speed (12 000 rpm; 10 min) to recover the mitochondria. the mitochondrial pellet was washed twice by firstly resuspending the pellet in buffer 6 (10 mm tris-maleate, 0.6 m mannitol, 2 mm egta, 0.5 mm phosphate, ph 6.8) followed by low speed centrifugation as before. secondly, the supernatant was decanted and centrifuged at high speed (12 500 rpm; 10 min). finally the mitochondria were resuspended in a small volume of buffer 6 prior to determination of protein concentration. . cellular bioenergetic analysis was carried out by measuring the whole cell respiration as described by law . (1995). yeast cells were grown at 23 c on salt medium [1% (w/v) yeast extract, 0.12% (w/v) (nh ) so , 0.1% (w/v) kh so , 0.01% (w/v) cacl , 0.0005% (w/v) fecl , 0.07% (w/v) mgcl , 0.05% (w/v) nacl] plus 2% (v/v) ethanol, until they reached their mid-logarithmic growth phase. the cells were then harvested by centrifugation, washed twice in distilled water and then resuspended at od = 30. the cellular respiration rate was determined by measuring the oxygen consumption rate using the clarke electrode (gilson). the oxygraph was calibrated so that the amount of oxygen in o -saturated distilled water in the chamber represented 100% and in the presence of sodium dithionate in the chamber represented 0% oxygen concentration. to the chamber was then added 300 l respiration buffer ( salt medium without ethanol). following the addition of 1.0 l of absolute ethanol, 10 l of cell suspension was added to the chamber. the rate of oxygen consumption, j (nmol o min mg dry-cell-weight ) was measured. the oxygen consumption rate measured in this state represents the physiological functioning of the cells. to measure the respiration rate in the absence of oxidative phosphorylation, j (nmol o min mg dry-cell-weight ), triethyltin (tet) was added to the chamber to a final concentration of 80 m. from these measurements, the respiratory control ratio (rcr), j /j , was calculated. to determine the uncoupled (maximal) respiratory rate, carbonylcyanide -chlorophenylhydrazone (cccp) was added to the oxygraph chamber to a final concentration of 5 mm. the maximal respiration rate, j ( nmol o min mg dry-cell-weight ) was then measured. from these measurements, the maximum respiratory capacity (mrc), j /j , was calculated. . the mitochondrial respiration rate was measured in the same oxygraph chamber used for determination of the cellular respiration rate. to the chamber 300 l of respiratory substrate containing 100 g available protein of mitochondria was added. the state 4 respiration [state 4(a)] (nmol o min mg mitochondrial protein ) was then measured. in order to induce state 3 cellular respiration assay mitochondrial respiration assay et al saccharomyces saccharomyces m ° 4 2 4 2 4 2 3 2 550nm 2 0 0 0 0 0 0 0 −1 −1 tet −1 −1 tet cccp −1 −1 cccp tet −1 −1 strains mitochondrail respiration parameters state 4 (b) uncoupled rate mrc (nmol o min −1 mg mitochondrial protein −1 ) (uncoupled rate/state 4b) ftc2 109 ± 5 314 ± 13 2.9 ± 0.1 df66 106 ± 9 372 ± 19 3.5 ± 0.1 df68 65 ± 3 194 ± 3 3.0 ± 0.2 strains mitochondrail respiration parameters state 4(a) state 3 rcr (nmol o min −1 mg mitochondrial protein −1 ) (state 3/state 4a) ftc2 130 ± 9 304 ± 23 2.3 ± 0.1 df66 111 ± 7 342 ± 20 2.2 ± 0.1 df68 80 ± 13 120 ± 3 1.5 ± 0.2 strains cellular respiration parameters j0 j0 tet j0 cccp rcr (j0/j0 tet ) mrc (j0 cccp /j0 tet ) ftc2 21.7 ± 0.1 10.3 ± 0.7 27.9 ± 3.1 2.1 ± 0.1 2.7 ± 0.2 df66 17.2 ± 2.5 10.1 ± 1.8 30.4 ± 1.6 1.7 ± 0.1 3.1 ± 0.5 df68 13.9 ± 0.9 8.4 ± 0.9 26.6 ± 2.1 1.7 ± 0.1 3.2 ± 0.6 df71 23.3 ± 2.1 10.9 ± 1.9 37.8 ± 2.5 2.2 ± 0.2 3.4 ± 0.5 (nmol o min −1 mg dry-cell-weight −1 ) 110 artika microbiol indones respiration, adp was added to a final concentration of 1.0 mm. the state 3 respiration (nmol o min mg mitochondrial protein ) was then measured. from this measurement, the respiratory control ratio (rcr) was calculated. to measure the maximal respiratory capacity, the chamber was washed several times with distilled water. a fresh portion of 300 l respiratory buffer containing 100 g available protein of mitochondria was then added. the state 4 [state 4(b)] respiration (nmol o min mg mitochondrial protein ) was measured as before and then cccp was added to a final concentration of 13.3 m and the maximal respiration rate (nmol o min mg mitochondrial protein ) was measured. from these measurements the maximal respiratory capacity (mrc) was calculated. the cellular respiration parameters of strains ftc2, df66, df68 and df71 are shown in table 1. these data show the followings. the rate of oxygen consumption, j , of df66 and df71 were similar while j of df68 was significantly reduced compared to that of ftc2. the j of the flag tagged-subunit 8 variants were not different from that of the wild-type version strain ftc2. the j of strains df66 and df68 was similar to that of the ftc2, while the j of strain df71 was found to be higher. the rcr of df66 and df68 were lower compared to that of the wild-type ftc2. the rcr of strain df71 was similar to that of the ftc2. the mrc of the variants were similar to that of the ftc2. the j and rcr of df68 were significantly lower compared to that of the ftc2. the mitochondrial respiration parameters of strains ftc2, df66, and df68 are shown in table 2. these data show the followings. the state 3 respiration rate of strain df68 was significantly reduced. similarly, state 3 respiration of strain df66 was reduced compared to that of ftc2. while state 4 respiration rate of df68 was reduced, the corresponding value of df66 was not altered compared to that of the wild-type ftc2. the rcr of df68 was reduced while that of df66 was maintained. the maximal respiration rate of df68 was lower compared to that of the wild-type, while that of df66 was found to be higher. −1 −1 −1 −1 −1 −1 tet cccp cccp results cellular respiration parameters of strains expressing wild-type and flag tagged-subunit 8 variants. mitochondrial respiration parameters of strains expressing wild-type and flag tagged-subunit 8 variants. 0 0 0 0 0 0 higher maximal respiration rate observed in strain df66 resulted in the higher mrc of this strain. by contrast, the mrc of strain df68 was not altered. the present study mainly concerns the bioenergetic analysis of flag tagged-subunit 8 variants in order to verify the bioenergetic consequences of adjacent charged residues within the central hydrophobic domain (chd) in combination with the presence of charged hexapeptide flag tag at the c-terminus. since all of the strains tested bear flag epitope tag, it was therefore concluded that the aberrant functional performances displayed by the variants are likely mainly caused by the introduction of the charged residues within the chd. these functional consequences may well be related to the fact that the presence of the charged residues within the chd drastically reduces the hydrophobicity of the transmembrane region. in addition, the presence of flag epitope tag on the wild-type subunit 8 has been shown not to cause functional impairment on mitochondrial atp synthase (artika 2010). the bioenergetic parameters of flag tagged-subunit 8 variants were analyzed at both cellular and mitochondrial discussion table 1 cellular respiration parameters of strains ftc2, df66, df68, and d 71f table 2 mitochondrial respiration parameters of strains ftc2, df66, and df68 state 4(a): state 4 respiration (absence of phosphorylation) prior to addition of adp to induce state 3 respiration; state 3: a state of respiration at maximal phosphorylation rate; rcr: respiratory control ratio, is defined as state 3 respiration over state 4(a) respiration. state 4(b): state 4 respiration (absence of phosphorylation) prior to addition of cccp to induced uncoupled respiration; uncoupled respiration is a state in which the respiratory chain is not kinetically controlled by the electrochemical proton gradient and therefore is dependent only on the activity of the respiratory chain; mrc: maximal respiratory control, is defined as uncoupled respiration over state 4(b) respiration. a b volume 4, 2010 microbiol indones 111 levels. measuring bioenergetic parameters at the cellular level has been found to be a convenient preliminary test of mitochondrial performance. in addition, this procedure also allows the assessment of the bioenergetic characteristics of mutant strains having functional mitochondrial atp synthase deficiencies from which good mitochondria may be difficult to isolate in sufficient amount, thus precluding bioenergetic studies on isolated mitochondria. bioenergetic measurements made at cellular level also exclude functional alterations caused by artifacts during mitochondrial preparation (law . 1995). the data of cellular bioenergetic analysis of strains ftc2, df66, df68, and df71 (table 1) showed that the rate of oxygen consumption, j , of df66 and df71 were similar while j of df68 was significantly reduced compared to that of ftc2. j is defined as the physiological respiration rate, intermediate between state 3 and state 4 respiration. as the respiration rate reflects the activity of the mitochondrial atp synthase, these data suggest that the functional coupling between the f and f sectors in df68 is reduced as a consequence of the presence of adjacent negatively charged aspartate residues in the middle of the chd. generally these results are in agreement with the previously reported basic growth characteristics of the flag tagged-subunit 8 variants (artika 2007) in that strain df68 displayed the slowest growth rate. the generation times of ftc2, df66, df68, and df71 were 6.9, 8.2, 11.1, and 7.2 hour respectively (artika 2007). the generation time reflects the performance of the corresponding subunit 8 in the enzyme complex. the respiration rate of the flag tagged-subunit 8 variants was also measured in the present of tet. the oxygen consumption rate (j ) under these conditions reflects the permeability of the inner mitochondrial membrane. j is defined as the inhibited rate or the apparent state 4 rate of respiration. the j of the flag taggedsubunit 8 were not different from that of the wild-type strain ftc2. this indicates that the physical integrity of the inner mitochondrial membrane is maintained in the variants strains. in the presence of protonophore, cccp, the respiration rate (j ) reflects the maximal respiratory capacity of the mitochondria. the j of strains df66 and df68 was similar to that of the wild-type, while the j of strain df71 was found to be higher. the reason for high j of df71 is unclear. the rcr df68 was lower compared to that of the wild-type ftc2. this was mainly due to lower absolute values of respiration rate (j ) of the df68 compared to that of ftc2. the rcr of strains df66 and df71 was similar to that of the ftc2 indicating that the activity of the mitochondrial atp synthase of these variants is maintained. the mrc of the variants were similar to that of the wildtype. to this end, the most profound functional defect was observed in the strain df68 carrying adjacent negatively charged residues in the middle of chd. the j and rcr of df68 were significantly lower compared to that of the et al 0 0 0 0 1 0 0 0 0 0 0 0 0 0 tet tet tet cccp cccp cccp cccp ftc2. this indicates that the efficiency of the utilization of the proton motive force for f activity is reduced in this strain. data from measurement of the gross cellular respiration rate suggested that the activity of the mitochondrial atp synthase in strains df66 and df68 is reduced while that of df71 is maintained. to confirm these findings, respiration measurements were also carried out on isolated mitochondria of df66 and df68. to obtain intact (well coupled) mitochondria for bioenergetic analysis, mitochondria were prepared from strains df66 and df68 according to the procedure described by law . (1995). in this procedure, the mitochondria are released from the cells by enzymatic digestion of the cell walls rather that using mechanical force to break cells open. strain df66 expresses variant having aspartate substitutions at residue positions 16 and 17. data from analysis at the mitochondrial level (table 2) showed that state 3 respiration of df66 is lower than that of ftc2. the rcr of df66, however, was similar to that of ftc2. these results indicate that there is possible functional impairment conferred by the strain df66 due to in efficient coupling between the f and the f sectors. the mrc of strain df66 is well maintained. this result indicates that the introduction of double aspartate residues at position 16 and 17 does not have major consequences on the respiratory chain. consistent with the data obtained from the cellular respiration assays, it was observed that state 3 respiration rate of strain df68 was significantly reduced. state 4 respiration, rcr, and uncoupled respiration rate of df68 were also reduced compared to that of the wild-type ftc2 (table 2). since the addition of the flag tag on the wildtype subunit 8 does not cause functional impairment on mitochondrial atp synthase, it is therefore concluded that the aberrant functional performances displayed by df68 are likely mainly caused by the introduction of the charged residues in the middle the central hydrophobic domain (chd). these functional consequences may well be related to the fact that the presence of the charged residues in the middle the chd drastically reduces the hydrophobicity of the transmembrane region. significant bioenergetic defects observed on strain df68 having double aspartate substitutions in the middle of the chd indicates that the functional coupling between f and f sector of df68 is compromised. the respiration rate (j ) is reduced by around 33%. this indicates that significant reduction of mitochondrial atp synthase has occurred due to the introduction of asp23, 24. these results indicate that the position of the charged residues within the chd is critical whether they are functionally defective or not. these data led to a conclusion that the hydrophobic character of the central of the chd is important for the function of the mitochondrial atp synthase. the two aspartate residues in the chd may reduce the proton flow through the f channel by diverting protons to go in different directions in the membrane. another possibility is that the charged residues alter protein-protein interaction of the coupling subunits between f and f sectors. 1 1 0 0 1 0 0 1 0 et al 112 artika microbiol indones acknowledgements references i am grateful to ausaid for sponsoring me during the course of this study. i would like to thank rodney j devenish and phillip nagley of the department of biochemistry and molecular biology, monash university, victoria, australia, for guidance and provision of facilities. artika im. 2006. allotopic expression of a gene encoding flag taggedsubunit 8 of yeast mitochondrial atp synthase. hayati j biosci 13: 36-8. artika im. 2007. structural and functional analysis of flag taggedsubunit 8 of yeast mitochondrial atp synthase. microbiol indones 1:33-6. artika im. 2009. membrane topology of subunit 8 of yeast mitochondrial atp synthase. microbiol indones 3:37-41. artika im. 2010. bioenergetic consequences of flag tag addition to the c-terminus of subunit 8 of yeast mitochondrial atp synthase. hayati j biosci 17:151-4. devenish rj, papakonstantinou t, galanis m, law rhp, linnane aw, nagley p. 1992. structure/function analysis of yeast mitochondrial atp synthase subunit 8. ann nyacad sci 671:403-14. fronzes r, weimann t, vaillier j, velours j, brethes d. 2006. the peripheral stalk participates in the yeast atp synthase dimerization independently of e and g subunits. biochemistry 45:6715-23. gearing dp, mcmullen gl, nagley p. 1985. chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus. biochem int 10:907-15. saccharomyces cerevisiae saccharomyces cerevisiae saccharomyces cerevisiae hong s, pedersen pl. 2008. atp synthase and the actions of inhibitors utilized to study its roles in human health, disease, and other scientific areas. microbiol mol biol rev 72:590-641. kolodziej pa, young ra. 1991. epitope tagging and protein surveillance. meth enzymol 194:508-19. law rhp, manon s, devenish rj, nagley p. 1995. atp synthase from . meth enzymol 260:133-63. macreadie ig, novitski ce, maxwell rj, john u, ooi b, mcmullen g, lukins hb, linnane aw, nagley p. 1983. biogenesis of mitochondria: the mitochondrial gene ( ) coding for mitochondrial atpase subunit 8 in . nucl acids res 11:4435-51. nagley p, farrell lb, gearing dp, nero d, meltzer s, devenish rj. 1988. assembly of functional proton-translocating atpase complex in yeast mitochondria with cytoplasmically synthesised subunit 8, a polypeptide normally encoded within the organelle. proc natl acad sci usa 85:2091-5. schafer k, braun t. 1995. monoclonal anti-flag antibodies react with a new isoform of rat mg dependent protein phosphatase . biochem biophys res commun 207:708-14. stephens an, khan ma, roucou x, nagley p, devenish rj. 2003. the molecular neighborhood of subunit 8 of yeast mitochondrial f f -atp synthase probed by cysteine scanning mutagenesis and chemical modification. j biol chem 278:17867-75. stephens an, roucou x, artika im, devenish rj, nagley p. 2000. topology and proximity relationships of yeast mitochondrial atp synthase subunit 8 determined by unique introduced cysteine residues. eur j biochem 267:6443-51. van ballmoos c, wiedenmann a, dimroth p. 2009. essentials for atp synthesis by f f -atp synthase. ann rev biochem 78:649-72. saccharomyces cerevisiae aap1 saccharomyces cerevisiae 2+ 1 0 1 0 2.mi671-ruth chrisnasari available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.1.2issn 1978-3477, eissn 2087-8575 vol 7, no 1, march 2013, p 9-16 *corresponding author; phone: , e-mail: +6231-2981399, fax: +6231-2981278 ruth_c@staff.ubaya.ac.id because of the recent increase in the gas price and interest in environmental issues, the demand of ethanol as substitute of gasoline has been rapidly increased (bai et al. 2008). bioethanol can be produced from many sources of biomass, thus access to raw material is virtually unlimited. the most common raw material is starch and sugar based material because it is easier to convert to ethanol than lignocellulose material (dumitriu, 1998; gray et al. 2006). the hydrolysis of starch to monomeric sugars can be achieved in many different ways, including chemical hydrolysis by acid or alkaline and enzymatic hydrolysis. nowadays, enzymatic hydrolysis is the most widely selected because it gives higher specific product, requires mild reaction conditions, and does not generate side products that inhibit microorganisms. in the last decade, comparison and selection of hydrolysis and fermentation process to obtain higher ethanol yield has gained more interest (söderström et al. 2005; ollson et al. 2006; ohgren et al. 2007; tu et al. 2009; drissen et al. 2009). there are two configurations for hydrolysis and fermentation processes, namely separate hydrolysis and fermentation (shf) and simultaneous saccharification and fermentation (ssf). when enzymatic hydrolysis and fermentation are performed sequentially, it is referred to shf. however, the two process steps are conducted simultaneously, it is referred to ssf. ssf results in a lower capital cost, reduced risk of contamination and higher ethanol yields than shf (wyman et al. 1992; stenberg et al. 2000). however, when adding biomass solids directly to the bioreactor makes cell recirculation very difficult. in addition, ssf requires compatible fermentation and saccharification reaction conditions, including ph, temperature, and two different process configurations, simultaneous saccharification and fermentation (ssf), and separate hydrolysis and fermentation (shf), were compared for ethanol production from shorgum bicolor grain. optimization modeling for glucoamylase and zymomonas mobilis concentration in both of ssf and shf were carried out to obtain optimal concentration of ethanol production. the optimum condition was achieved using 0.021% (v/v) of glucoamylase, and 30.19% (v/v) of z. mobilis for shf. in contrast, the optimum condition for ssf was 0.021% (v/v) of glucoamylase and 17.51% (v/v) of z. mobilis. the model predicted shf processing to be superior. the superiority of shf over ssf was confirmed experimentally, the result showed ethanol yield of -1 shf was 134.80 g l and ethanol yield of ssf was 115.66 after 72 h incubation time. a high similarity was observed between the predicted and experimental results, demonstrating the accuracy of the model. key words: ethanol, shorgum bicolor, separate hydrolysis and fermentation, simultaneous saccharification and fermentation dua konfigurasi proses yang berbeda, sakarifikasi dan fermentasi simultan (ssf) serta hidrolisis dan fermentasi terpisah (shf) dibandingkan pada proses produksi etanol dari shorgum bicolor. optimasi pemodelan pada konsentrasi glukoamilase dan zymomonas mobilis dengan konfigurasi ssf dan shf dilakukan untuk mendapatkan konsentrasi etanol yang optimal. kondisi optimum dicapai pada konsentrasi glukoamilase 0,021% (v/v) dan z. mobilis 30,19% (v/v) pada kofigurasi shf. namun, kondisi optimum untuk ssf dicapai pada glukoamilase 0,021% (v/v) dan z. mobilis 17,51% (v/v). prediksi model menunjukkan konfigurasi shf lebih unggul. keunggulan shf dibanding ssf telah dikonfirmasi secara eksperimental, hasilnya menunjukkan etanol -1 -1 hasil shf adalah 134,80 g l , dan etanol hasil ssf adalah 115,66 g l setelah 72 jam waktu inkubasi. kesamaan yang tinggi antara hasil prediksi dan eksperimental menunjukkan keakuratan model. kata kunci: etanol, shorgum bicolor, sakarifikasi dan fermentasi simultan, hidrolisis dan fermentasi terpisah -1 g l optimization modeling of ethanol production from shorgum bicolor grain: comparison between separate hydrolysis fermentation and simultaneous saccharification fermentation ruth chrisnasari*, damiati hartini susetyo, adrian pratama sugianto, and tjandra pantjajani department of biology, faculty of biotechnology, universitas surabaya, jalan raya kalirungkut, surabaya 60292, indonesia optimum substrate concentration (ballesteros et al. 2004). one problem associated with ssf are the different optimal conditions for saccharification and fermentation, this means that the conditions chosen for ssf are not optimal for both the microorganism and the enzymes. on the other hand, in the shf process the optimal condition of enzymatic hydrolysis and fermentation can be performed (gupta et al. 2009). however, shf leads to high production of sugars, which is problematic if the enzyme is end product inhibited. ssf may thus exhibit lower inhibition of the enzymes because glucose released by enzyme is simultaneously fermented by the microorganism (prashant et al. 1999). in the present study, shorgum bicolor, a grain having high sugar accumulation, was used as raw material for ethanol production. s. bicolor has potential as a raw material for fuel-grade ethanol production due to its rapid growth rate and early maturity, greater water use efficiency, limited fertilizer requirement, high yield production, and wide adoptability (reddy et al. 2005; prasad et al. 2007; antonopoulou et al. 2008). although ssf and shf have been investigated extensively, there are still no comparison ssf and shf modeling to achieve the optimal operating conditions for ethanol production from s. bicolor. optimization modeling using response surface methodology (rsm) has been reported to be an efficient method for identifying the effect of individual variables and for seeking the optimum conditions for a multivariable system (ambati and ayyanna 2001; ratnam et al. 2005; bandaru et al. 2006). this method has superiority compare to other methods due to rapidity, limited number of required experiments, and accuracy for describing the optimal conditions. in this present study, shf and ssf were compared for ethanol production from s. bicolor and optimized by rsm. the rsm approach was adopted to locate optimum level of glucoamylase and zymomonas mobilis concentration for ethanol production, since these parameters play a key role in the enhancement of ethanol yield (bandaru et al. 2006; chrisnasari et al. 2011). materials and methods bacterial strain. z. mobilis zm4 (nrrl b14234) obtained from ars culture collection national center for agricultural utilization research, peoria il, usa, was used throughout the research. e n z y m e . t h e r m o s t a b l e a l p h a a m y l a s e (liquozyme®sc ds, novozymes) originated from 10 chrisnasari et al. microbiol indones bacillus licheniformis and glucoamylase (dextrozyme® ga, novozymes) from aspergillus niger, were employed for starch hydrolysis. the enzymatic activities of these enzymes as defined by novozymes were 240 -1 knu-s g and 270 agu , respectively. cultivation medium and conditions. z. mobilis -1 was maintained on medium containing (g l ): glucose, 100; yeast extract, 10; kh po , 1; (nh ) so , 1; 2 4 4 2 4 mgso .7h o, 0.5. cells were cultured at 35 °c and ph 4 2 of 5.5 with 24 h incubation time. raw material pretreatment. fermentation medium was made by gelatinized s. bicolor flour (30% w/v) at 100 °c for 15 min. gelatinized slurry maintained under temperature of 85 °c and ph of 5.5, then added -1 with 20 μl l of alpha amylase and incubated for 2 h during liquefaction process. the liquefied starch then cooled down and adjusted according to the next step condition. separate hydrolysis and fermentation. saccharification was done by keeping the liquefied shorgum starch at 55 °c and a ph of 4.3, followed by addition of glucoamylase at vary concentrations and incubated for 48 h. saccharified starch was adjusted into ph of 5.5 and centrifuged at 4000 rpm for 15 min. supernatant was taken for fermentation process. optimization studies of ethanol production by z. mobilis were carried out in 250 ml flasks and fermentation conditions were maintained at temperature of 30 °c and shake of 200 rpm for 72 h. the inoculums was derived from z. mobilis cells that grown overnight which od at 560 nm was maintained at 1.5 for both shf and ssf configuration and for each replication. simultaneous saccharification and fermentation. the saccharification and fermentation of liquefied starch was carried out simultaneously in one flask. the liquefied medium was added with vary concentration of glucoamylase and inoculated with vary concentration of z. mobilis suspension, followed by incubation process which maintained at 37 °c and 200 rpm for 72 h. analytical methods. the concentration of reducing sugars was determined by dinitrosalicylic (dns) colorimetric assay using glucose as sugar standard (miller 1959). the concentration of starch was determined according to hall et al. (2000). the amount of ethanol was measured by gas chromatography (hewlett packard, hp c1540a). cell concentration was measured by spectroscopy at 560 nm (od ).560 experimental design and optimization. the aim of this study was to find the optimum levels of glucoamylase and inoculum concentrations for ethanol -1 g volume 7, 2013 microbiol indones 11 production from s. bicolor. central composite experimental design rsm (ccd, box and wilson, 1951) was used in the optimization of ethanol production. glucoamylase concentration (x , % v/v) 1 and inoculum (x , % v/v) were chosen as independent 2 -1 variables and ethanol concentration (yi, g l ) was used as output variable. for statistical calculations the variables xi were coded as xi according to equation (1). where, x is the dimensionless value of an i independent variable, xi is the real value of an independent variable, xi; is the real value of the independent variable at the center point and ∆xj is step change. table 1 shows independent variable that used 2 in this experimental plan. a 2 -factorial ccd, with four axial points (α =√2) and five replications at the center points (n =5) leading to a total number of 13 0 experiments was employed in table 1 and 2. the second degree polynomials (equation (2)) were calculated with the statistical package (stat-ease inc, minneapolis, mn, usa) to estimate the response of the dependent variable: where yi is the predicted response, x , x , are 1 2 independent variables, b , is the offset term, b , b , are 0 1 2 linear effects, b ,b are squared effects and b is 11 22 12 interaction terms. results the two factors that most influence the enzymatic starch hydrolysis and fermentative production of ethanol are glucoamylase concentration and inoculum concentration. rsm was applied to find the optimal condition for each of these. the vicinity of the optimum condition for ethanol production was estimated through steepest ascent step at the preliminary study by varying concentrations of glucoamylase and z. mobilis inoculum in both shf a n d s s f c o n f i g u r a t i o n . t h e g l u c o a m y l a s e concentration was varied from 0.005-0.04 % (v/v) and the inoculum was varied from 10-40 % (v/v). the three points closest to the optimum range were used as selected values for the rsm experiment. the preliminary results showed that each fermentation configuration (shf and ssf) led to a different optimal range for glucoamylase and inoculum concentrations. both of shf and ssf showed optimal sugar concentration at 0.02% (v/v) of glucoamylase concentration (fig 1a) therefore the optimum range of glucoamylase for shf was 0.015-0.025 % (v/v). in other hand, there was insignificant difference of sugar concentration between 0.02 and 0,025 % (v/v) of glucoamylase concentration in ssf configuration, consequently the optimal range was expanded to 0.010-0.030 % (v/v). in contrast, the optimal inoculums range for shf configuration was 25-35 % (v/v) and for ssf configuration was 10-25 % (v/v) (fig 1b). these points then were used as the lower factorial point (-1 of coded factor), center point (0 of coded factor), and upper factorial point (1 of coded factor) in ccd matrix design as listed in table 1. using ccd, a total number of 13 experiments with different combinations of glucoamylase and inoculum were performed (table 2). the response was taken at the maximum ethanol production which was observed at 72 h incubation time. the estimation model analysis was done using sequential model sum of squares, lack of fit tests and model summary statistics. the result showed that suggestion model was quadratic which means relationship between variables and response followed a quadratic function. the following second order polynomial equation was found to represent the ethanol production adequately for shf and ssf configuration: equation for shf configuration: equation for ssf configuration: the experimental data as shown at table 2 were statistically analyzed using the fischer's statistical test for analysis of variance (anova) and the results are shown in table 3. the anova of the quadratic regression model indicated that the model for shf and ssf were highly significant where the prob >f-value of the model was less than 0.0500. the coefficient estimate and the corresponding prob >f-values demonstrated that glucoamylase concentration had a significant effect on ethanol production in shf configuration but not in ssf configuration. however, inoculum concentrations were observed to have an insignificant effect on ethanol production in both ssf and shf configuration. the analysis also showed that there were insignificant interactions between glucoamylase and inoculums concentration for both xi-xi ∆ xj i = 1,2,3,....kxi = (1) 2 2 yi=b +b x +b x +b x +b x +b x x +0 1 1 2 2 11 1 22 2 12 1 2 (2) y=-39.30774+6029.10590 x +7.54986 x +6.99500 1 2 2 2 x x -1.52150e+005 x -0.12739 x1 2 1 2 (3) y=-89.32138+9446.26618 x +11.83360 x +21.04800 1 2 2 2 x x -2.30690e+005 x -0.35069 x1 2 1 2 (4) 12 chrisnasari et al. microbiol indones ssf and shf. in addition, there were significant effects of quadratic function for ssf and shf configuration respectively. the coefficient determination of shf configuration, 2 2 r is 0.9785 and for ssf configuration, r is 0.9278 which implies that most of the sample variation in the ethanol yield was attributed to the independent variables. adequate precision measures the signal to noise ratio. adequate precision ratio of 18.228 for shf configuration and 9.837 for ssf configuration indicate an adequate signal, so that these models can be used to navigate the design space. the predicted optimum levels glucoamylase and inoculums were obtained by applying the regression analysis to the equation (3) and (4). the predicted and experimental ethanol production at the optimum levels of glucoamylase and inoculums also determined by using equation (3) and (4). fig 2 represent the response fig 1 the effect of glucoamylases concentration to the amount of sugar released and the effect of inoculum concentration to the amount of ethanol produced at 72 h incubation time in (a) shf configuration, (b) ssf configuration. table 1 independent variable in the experimental plan variable actual value of each coded level -1.414 -1 0 1 1.414 shf: 0.0129 22.9 0.015 25.0 0.020 30.0 0.025 35.0 0.0271 37.1 ssf: glucoamylase concentrations (% v/v), x 1 inoculum (% v/v), x 2 concentration 0.0059 6.9 0.010 10.0 0.020 17.5 0.030 25.0 0.0341 28.1 glucoamylase concentrations (% v/v), x 1 0.04 30 0 0.01 0.02 0.03 0.04 glucoamylase (% v/v) -1 r ed u ci n g s u g ar ( g l ) 240 220 200 180 160 140 120 100 240 220 200 180 160 140 120 100 -1 r ed u ci n g s u g ar ( g l ) 0 0.01 0.02 0.03 glucoamylase (% v/v) 140 135 130 125 120 115 110 105 -1 e th an o l (g l ) 100 0 10 20 30 50 inoculum (%) 40 140 135 130 125 120 115 110 105 -1 e th an o l (g l ) 100 0 5 10 15 25 inoculum (%) 20 a b inoculum concentration (% v/v), x2 volume 7, 2013 microbiol indones 13 shf ssf glucoamylase (% v/v) inoculum (% v/v) actual ethanol -1 (g l ) predicted -1 ethanol (g l ) glucoamylase (% v/v) inoculum (% v/v) actual -1 (g l ) ethanol predicted -1 ethanol (g l ) 0.015 25.0 129.38 128.65 0.010 10.0 67.82 67.44 0.025 25.0 130.31 129.83 0.030 10.0 77.51 76.03 0.015 35.0 128.73 128.76 0.010 25.0 58.37 63.99 0.025 35.0 130.36 130.64 0.030 25.0 74.38 78.89 0.020 30.0 135.86 136.46 0.020 17.5 116.65 114.38 0.020 30.0 135.77 136.46 0.020 17.5 100.58 114.38 0.020 30.0 136.60 136.46 0.020 17.5 120.78 114.38 0.020 30.0 137.38 136.46 0.020 17.5 109.66 114.38 0.020 30.0 136.69 136.46 0.020 17.5 124.24 114.38 0.0129 30.0 127.37 127.77 0.0059 17.5 62.80 59.94 0.0271 30.0 129.88 129.93 0.0341 17.5 77.83 76.55 0.020 22.9 128.99 129.76 0.020 6.9 72.97 75.14 0.020 37.1 130.73 130.42 0.020 28.1 81.03 74.72 table 2 experimental and the predicted value of ethanol yield table 3 analysis of variance (anova) for the response surface quadratic model source shf ssf sum of square df mean square f-value p-value prob>f sum of square df mean square f-value p-value prob>f model 157.04 31.41 <0.0001 5965.76 5 1193.15 17.98 0.0007 x1 0.0177 1 275.67 4.15 0.0809 x2 0.3799 1 0.18 0.00265 0.9604 x x1 2 0.6334 1 9.97 0.15 0.7099 2 x1 100.65 100.65 204.43 <0.0001 3702.11 1 3702.11 55.78 0.0001 2 x2 170.55 143.30 <0.0001 2707.05 1 2707.05 40.78 0.0004 residual 7 66.37 lack of fit 63.79 9.51 0.88 0.25 1.29 0.3916 3 36.18 0.41 0.7573 pure error 4.68 0.43 0.12 70.55 3.45 1.70 1.75 4.68 0.43 0.12 0.49 0.57 0.44 275.67 0.18 9.97 464.56 108.53 356.03 4 89.01 corrected total 160.49 5 1 1 1 1 1 7 3 4 12 6430.32 12 variables optimum level -1 optimum ethanol yield (g l ) experimental predicted shf: glucoamylase concentrations (% v/v), x 1 inoculum concentration (% v/v), x2 0.021 30.19 134.80 136.50 ssf: glucoamylase concentrations (% v/v), x 1 inoculum concentration (% v/v), x 2 0.021 17.51 115.66 114.76 table 4 the predicted and experimental ethanol yield at the optimum levels of glucoamylase and inoculums predicted. in other hand, the optimum condition for ssf configuration can be achieved at the glucoamylase concentration of 0.021% (v/v) and inoculum concentration of 17.51% (v/v) which gives the -1 maximum ethanol production of 114.76 g l . either experimental or predicted ethanol production at the surface and contour plots for the optimization of inoculums vs. glucoamylase on ethanol production. the optimum condition of shf configuration for highest ethanol production can be achieved at the glucoamylase concentration of 0.021% (v/v) and 30.19 % (v/v) of inoculums. at these optimum conditions -1 maximum ethanol production of 136.50 g l was microbiol indones14 chrisnasari et al. fig 2 response surface and contour plot of glucoamylase vs. inoculums on ethanol production: (a) shf configuration, (b) ssf configuration. 35.00 128.5 130.725 132.95 135.175 137.4 e th an o l 0.015 0.018 0.020 0.022 0.025 25.00 27.50 30.00 32.50 a: glucoamylase b: inoculum a 25.00 58 74.75 91.5 108.25 125 e th an o l b a: glucoamylase b: inoculum 0.0100 0.0150 0.0200 0.0250 0.0300 10.00 13.75 17.50 21.25 25.00 production from palmyra jaggery using response surface method. world j microbiol biotechnol. 17(4): 331-335. doi:10.1023/a:1016613322396. antonopoulou g, gavala hn, skiadas iv, angelopoulos k, lyberatos g. 2008. biofuels generation from sweet sorghum: fermentative hydrogen production and anaerobic digestion of the remaining biomass. bioresour technol. 99(1):110-119. doi:10.1016/j.biortech.2006.11.048. anuradha r, suresh ak, venkatesh kv. 1999. simultaneous saccharification and fermentation of starch to lactic acid. bai fw, anderson wa, young mm. 2008. ethanol fermentation technologies from sugar and starch feedstock. biotechnol adv. 26(1):89-105. doi:10.1016/s0032-9592(99)00080-1. bioresour technol. process biochem. 35(3-4):367-375. doi:10.1016/s00329592(99)00080-1. ballesteros m, oliva jm, negro mj, manzanares p, ballesteros i. 2004. ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (ssf) with kluyveromyces marxianus cect 10875. process biochem. 39(12):1843-1848. doi:10.1016/j.procbio.2003.09.011. bandaru vvr, somalanka sr, mendu dr, madicherla nr, chityala a. 2006. optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of zymomonas mobilis using response surface methodology. enzyme microbiol technol. 38(12):209-214. doi:10.1016/j.enzmictec.2005.06.002. box gep, wilson kb. 195. on the experimental attainment of optimum conditions. j r stat soc series b stat methodol. 13(1):1-45. box gep, hunter js, hunterwg. 2005. statistic for experimenters: design, innovation and discovery. new jersey: johnwilley & sons. p 450-453. chrisnasari r, wardani ak, murdiyatmo u. 2011. optimization of ethanol production from palmyra sap by zymomonas mobilis using response surface method microbiol indones. 5(2):61-67. doi:10.5454/mi.5.2.3. drissen ret, maas rhw, tramper j, beeftink hh. 2009. modelling ethanol production from cellulose: separate hydrolysis and fermentation versus simultaneous saccharification and fermentation. biocatal biotransfor. 27(1):27-35. doi:10.1080/10242420802564358. dumitriu s. 1998. polysaccharides. new york: markker deccer inc. gray ka, zhao l, emptage m. 2006. bioethanol. curr opin chem biol. 10(2):141-146. doi:10.1016/j.cbpa.2006.02.035. gupta r, sharma kk, kuhad rc. 2009. separate hydrolysis and fermentation (shf) of prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by saccharomyces cerevisiae and pichia stipitis-ncim 3498. 100(3):1214-1220. doi:10.1016/j.biortech.2008.08.033. hall mb, ennings jp, lewis jba, robertson jb. 2000. evaluation of starch analysis methods for feed samples. j sci food agric. 81(1):17-21. doi:10.1002/10970010(20010101)81:1<17::aid-jsfa758>3.0.co;2-b. optimum condition for shf and ssf configuration were also determined (table 4). the modeling result predicted shf processing to be superior to ssf. the superiority of shf over ssf was confirmed experimentally, the result showed ethanol yield of shf -1 was 134.80 g l and ethanol yield of ssf was 115.66 g -1 l after 72 h incubation time. discussions in the present study, we demonstrated the production of high ethanol concentrations from s. bicolor using the shf and ssf configurations. the results demonstrated that glucoamylase and inoculum concentrations influenced to the enzymatic starch hydrolysis and fermentative production of ethanol. increasing glucoamylase concentration positively influenced ethanol production in shf configuration, as the liquefied starch in sorghum gets converted to glucose in the presence of glucoamylase enzyme. the increasing of glucoamylase concentration has lead to increasing of rate conversion of liquefied starch to glucose which resulted increasing of glucose concentration in medium. as in shf hydrolysis and fermentation are separate, the best reaction conditions for each can be independently selected (gupta et al. 2009; ohgren et al. 2007). however, for ssf one set of conditions has to be selected. this usually means selecting a lower than optimal temperature for the enzyme to accommodate the microorganism (anuradha et al. 1999). this problem recommends the superiority of ethanol production of shf over than ssf configuration. previous investigation on ethanol production had to compare the ssf and shf configuration at the same reaction condition in order to be able to compare the performance for both systems (zhang et al. 2011). the previous result showed that ssf was superior to shf due to the comparison was done at the same reaction condition. in this study, shf and ssf were optimized before comparing ethanol yields for shf and ssf. optimal conditions were determined using rsm for both experimental efficiency and accuracy which may lead to the different reaction conditions for each system. at their each optimum condition, shf was found to be superior to ssf on the basis of final ethanol yield. references ambati p, ayyanna c. 2001. optimizing medium constituents and fermentation conditions for citric acid volume 7, 2013 microbiol indones 15 alternate raw material for bio-ethanol and bioenergy. icrisat 1(1):1-8. söderström j, galbe m, zacchi g. 2005. separate versus simultaneous saccharification and fermentation of two step steam pretreated softwood for ethanol production. j wo o d c h e m te c h n o l . 2 5 ( 3 ) : 1 8 7 2 0 2 . doi:10.1080/02773810500191807. stenberg k, bollok m, reczey k, galbe m, zacchi g. 2000. effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steampretreated softwood for ethanol production. biotechnol bioeng. 68(2):204-210. doi:10.1002/(sici)10970290(20000420)68:2<204::aid-bit9>3.0.co;2-4. tu m, zhang x, paice m, mcfarlane p, saddler jn. 2009. effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine. biotechnol progr. 25(4):1122-1129. doi:10.1002/btpr.198. wyman ce, spindler dd, grohmann k. 1992. simultaneous saccharification and fermentation of several lignocellulosic feedstocks to fuel ethanol. biomass bioenerg. 3(5):301-307. doi:10.1016/09619534(92)90001-7. zang l, zhao h, gan m, jin y, gao x, chen q, guan j, wang z. 2011. application of simultaneous saccharification and fermentation (ssf) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales. . 102(6): 4573-4579. doi:10.1016/j.biortech.2010.12.115. bioresour technol miller gl. 1959. use of dns reagent for determination of reducing sugars. anal chem. 31(3):426-428. doi:10.1021/ac60147a030. ohgren k, bura r, lesnicki g, saddler j, zacchi g. 2007. a comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover. process biochem. 42(5):834-839. doi:10.1016/j.procbio.2007.02.003. olsson l, soerensen hr, dam bp, christensen h, krogh km, meyer as. 2006. separate and simultaneous enzymatic hydrolysis and fermentation of wheat hemicellulose with recombinant xylose utilizing saccharomyces cerevisiae. appl biochem biotech. 129 (1-3):117-129. doi:10.1385/abab:129:1:117. prasad s, singh a, jain n, joshi hc. 2007. ethanol production from sweet sorghum syrup for utilization as automotive fuel in india. energy fuel 21(4):2415-2420. doi:10.1021/ef060328z. prashant v, iyer, lee yy. 1999. simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid in a two-zone fermentor-extractor system. appl biochem biotechnol. 77(79):409-418. ratnam bvv, rao ss, rao dm, rao nm, ayyanna c. 2005. optimization of medium constituents and fermentation conditions for the production of ethanol from palmyra jaggery using response surface methodology. world j microbiol biotechnol. 21(4):399-404. doi:10.1007/s11274004-2461-4. reddy bvs, ramesh s, reddy ps, ramaiah b, salimath pm, kachapur r. 2005. sweet sorghum-a potential microbiol indones16 chrisnasari et al. 1.mi714-leong sui sien available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.1issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 137-143 *corresponding author; phone/fax: +60-82-581-388, email: leongsuisien87@gmail.com abuse of antibiotic use is becoming a major concern of today’s society. it is also the major cause of antibiotic resistance in clinical practice. since the discovery of penicillin in the 1940s, the emergence of antibiotic resistance has been highlighted. according to dorsch (2007), the excessive use of antibacterials in agriculture and clinical therapy has led to the phenomenon of antibiotic resistance. antibiotic resistance decreases our ability in treating infections and diseases, affecting the proper infection control, and prevention strategies (dorsch, 2007). antibiotic resistant bacteria have also widely spread in the environment. transmission of resistant gene in these pathogenic bacteria may cause health problem to human. according to varaldo (2002), streptococcus pyogenes has become resistant to erythromycin and able to enter human respiratory cells. staphylococcus aureus has also been found to be resistant to a variety of antibiotics (akano et al. 2009). thus, the studies of there is a growing concern on the occurrence of antimicrobial resistance. development of multiple antibiotic resistant bacteria has overtaken new drug development and threatened the patients with untreatable infections. this study was conducted to isolate and characterize the antibiotic resistant bacteria from swiftlet farm houses located in various places including kota samarahan, semarang, saratok, betong, sarikei, sibu, sepinang, maludam, miri, and kuching in sarawak, malaysia. five feces samples were collected randomly from each site. one gram of the feces sample was diluted in 9 ml of 0.85% normal saline solution. the diluted sample was plated on trypticase soy agar plates and incubated at 37±1 °c for 24 h. a total of 500 bacteria isolates were then identified using 16s rrna analysis method. disc diffusion method was then used to confirm the resistant phenotypes of these isolates. the results showed that the means of the bacterial colony count were significantly -1 different (p<0.05) from one another, with the highest log cfu g (9.22±0.72) found in kota samarahan and the 10 -1 lowest log cfu g (6.03±0.62) in betong. besides, the isolated bacteria were identified as 96% gram positive 10 bacteria and 4% gram negative bacteria. the isolated bacteria were highly resistant to penicillin g (36.80±23.87%), ampicillin (28.60±17.13%), and rifampicin (16.90±13.70%). thus, swiftlet feces are good reservoir for a range of antibiotic resistant bacteria which may pose a potential health hazard to human. key words: antibiotic resistance, bacteria, isolation, swiftlet feces dewasa ini, tingkat kekhawatiran pada munculnya resistensi terhadap antimikroba semakin meningkat. laju kemunculan bakteri dengan resistensi ganda terhadap antibiotik lebih tinggi dibandingkan dengan penemuan obat baru, sehingga membahayakan pasien-pasien dengan infeksi yang tidak tertangani. penelitian ini ditujukan untuk mengisolasi bakteri yang resisten terhadap antibiotik dari peternakan walet (swiftlet) yang tersebar di kota samarahan, semarang, saratok, betong, sarikei, sibu, sepinang, maludam, miri, dan kuching di sarawak, malaysia dan mengkarakterisasinya. lima sampel feses diambil secara acak dari masing-masing situs. satu gram sampel fees didilusi dalam 9 ml 0.85% larutan salin normal. sample terdilusi kemudian di sebar pada media agar tripticase soy dan diinkubasi pada 37±1 °c selama 24 jam. lima ratus isolat bakteria kemudian diidentifikasi berdasarkan urutan 16s rrna. resistensi dibuktikan menggunakan metode cakram difusi (disk diffusion method). hasil penelitian ini menunjukkan bahwa nilai rata-rata penghitungan koloni bakteri berbeda secara -1 signifikan (p<0.05) antara isolat satu dengan lainnya. nilai log cfu g tertinggi ditemukan di kota samarahan 10 (9.22±0.72) sementara nilai terendah ditemukan di betong (6.03±0.62). identifikasi juga menunjukkan bahwa 96% isolat adalah bakteri gram positif sementara hanya 4% bakteri gram negatif. bakteri terisolasi menunjukkan resistensi tinggi terhadap penisilin g (36.80±23.87%), ampisilin (28.60±17.13%), dan rifampisin (16.90±13.70%). maka dapat disimpulkan bahwa feses walet merupakan sumber bakteri yang resisten antibiotik, sehingga boleh jadi berpotensi berbahaya terhadap manusia. kata kuci: bakteri,feses walet, isolasi, resistensi antibiotik isolation and characterization of antibiotic resistant bacteria from swiftlet feces in swiftlet farm houses in sarawak, malaysia 1 1 2 leong sui sien *, samuel lihan , ling teck yee , 1 3 chia hwa chuan , and lim chan koon 1 2 department of molecular biology; department of chemistry; 3 department of zoology, faculty of resource science and technology, universiti malaysia sarawak, kota samarahan 94300, sarawak, malaysia antibiotic resistance are as vital as providing optimum treatment for patients (dorsch 2007) and predicting the emerging of resistant pathogens (allen et al. 2010). swiftlet feces are usually used as organic fertilizer in agricultural industry because it is rich in nutrient, water, nitrogen, phosphorus, potassium, and minerals (nyakundi and mwangi 2011). most bacteria are able to grow in the swiftlet feces. organic fertilizers containing these antibiotic resistant bacteria may contaminate vegetables. improper washing of the contaminated vegetables may also spoil our foods in the kitchen, causing harmful diseases to human. intensive and extensive abuse of antibiotics usage have caused the formation of an antibiotic resistant genes pool in the environment. thus, there is a growing public health concern on the microbiological risk of antibiotic resistance of these bacteria in the swiftlet feces to human health. therefore, this study was conducted to identify and characterize the antibiotic resistant bacteria present in the swiftlet farm houses. materials and methods locations of the study areas. swiftlet houses were situated at kota samarahan, semarang, saratok, betong, sarikei, sibu, sepinang, maludam, miri, and kuching in sarawak, malaysia. sample collection. sampling and sample processing procedures for the isolation of bacteria from the swiftlet feces were carried out as described by nyakundi and mwangi (2011). five samples were collected randomly from the floor of the sampling sites in each swiftlet house. the samples were collected using the spatula and then transferred into sterile bag for storage and then further analysed in the laboratory. sample was diluted by mixing 1 g of dropping in 9 ml of sterile 0.85% saline solutio\n. the diluted sample was plated on trypticase soy agar (scharlau, spain) plates and incubated at 37±1 °c for 24 h. the number of -1 bacterial colony forming unit (cfu g ) was calculated according to nyakundi and mwangi (2011) as the number of colony per plate multiplied by the dilution factor. five to 10 colonies were randomly picked for further identification. dna extraction. the bacterial dna was extracted by boiling method described by maria et al. (2008) with minor modification. the bacterial cultures were prepared by growing the bacteria in luria broth (scharlau, spain) at 37 ± 1°c for 24 h. one thousand and 500 µl of overnight bacterial culture was transferred into 1.5 ml microcentrifuge tube and centrifuged at 10 138 sien et al. microbiol indones 000 rpm for 5 min. the supernatant was then discarded. five hundred microliters of sterile distilled water was added and vortexed to resuspend the cell pellet. the content of the microcentrifuge tubes were then boiled for 10 min together with the tubes and then immediately placed on ice for 5 min. after 5 min, the microcentrifuge tubes were centrifuged at 10 000 rpm for 10 min and the supernatant was collected. dna sequencing polymerase chain reaction analysis. the isolated bacteria were genotypically characterized using 16s rrna identification method with minor modification (woo et al. 2000; kumar et al. 2009). primers 27f (5’-agagtttgatc ctggctca g-3’) and 519r (5’-gwattaccgcgg ckgctg-3’) were used. pcr was performed in reaction mixtures containing 20 µl of dna, 1.0 µl of each 20 pmol primers (first base), 3 µl of 10 mm deoxynucleoside triphosphates mix (promega), 6.0 µl of 25 mm mgcl (promega), 10.0 µl of 5x buffer 2 solution (promega), 8.0µl of distilled water, and 1µl of taq polymerase (promega). pcr was performed for 35 cycles with initial denaturation at 95 °c for 10 min, denaturation at 94 °c for 30 sec, annealing at 55 °c for 1 min, primer extension 72 °c for 1.5 min, and final extension at 72 °c for 10 min. the pcr products were visualized by ethidium bromide staining after agarose gel electrophoresis for 30 min at 90 volt. sequencing analysis. dna purification was carried out using qiagen sequencing purifying kit (qiaquick gel extraction kit, usa). purified dna product was sent for dna sequencing at first base laboratories sdn. bhd. in selangor, malaysia. all the dna sequences were compared using blast program to determine the closest matches of the identities. disc diffusion method. antibiotic susceptibility testing was conducted using disc diffusion method according to the method described by national committee for clinical laboratory standards (nccls) (2012) and zulkifli et al. (2009). antibiotic discs (oxoid, england) used for testing represents agents historically or currently used in clinical practice. escherichia coli atcc 25922 was used as positive control. among the antibiotic discs used in this testing were chloramphenicol (30 µg), ampicillin (10 µg), tetracycline (30 µg), streptomycin (10 µg), gentamycin (10 µg), erythromycin (15 µg), cephalothin (30 µg), nitrofurantoin (300 µg), tobramycin (10 µg), rifampicin (5µg), kanamycin (30 g), sulphamethoxazole/trimethoprim (1.25/ 23.75 µg), amikacin (30 µg), imipenem (10 µg), ceftriaxone (30 µg), penicillin g (10 u), doxycycline (30 µg), ceftazidime (30 µg), norflaxacin (10 µg), vancomycin (30 µg), piperacillin (100 µg), ciprofloxacin (5 µg), and nalidixic acid (30 µg). the medium used in this study was muellerhinton agar (mha). a sterile cotton swab was dipped into a bacterial suspension with turbidity 0.5 mcfarland and used to inoculate the surface of the mha plate evenly, using different bacteria for each plate. the plate was allowed to dry for 5 min. a pair of sterile forceps was used to pick up the antibiotic disc and placed on the surface of the mha agar plate. the plate was incubated at 37±1 °c for 24 h. the diameter of inhibition zone was measured and the results were recorded as sensitive (s) or resistance (r) base on who drug information and nccls (yah et al. 2007). statistical analysis. statistical analysis of the data was conducted using spss statistics version 21.0 program. besides, all the data were analyzed for analysis of variance (anova). all differences between the means were compared using turkey multiple range test after a significant f-test at p<0.05. results bacterial colony count. table 1 showed that the -1 means of the bacterial colony counts (log cfu g ) 10 were significantly different (f=8.265, p<0.05) from -1 one sampling site to another the highest log cfu g10 of 9.22±0.72 was found in kota samarahan and -1 the lowest log cfu g of 6.03±0.62 in betong. the 10 mean bacterial colony counts collected from kota samarahan, maludam, miri, kuching, and sibu were significantly higher than those from other sampling sites such as saratok, betong, semarang, sepinang, and sarikei. identification of bacteria in swiftlet feces. a total of 500 bacterial isolates were obtained from kota samarahan, saratok, betong, maludam, miri, kuching, semarang, sepinang, sarikei, and sibu. the results showed that a total of 480 (96%) bacterial isolates were identified as gram positive, while only 20 (4%) were identified as gram negative bacteria. the distribution of the bacterial isolates in the swiftlet feces from the different sampling sites is shown in fig 1. although all these bacterial isolates were found in all the sites, staphylococcus sp. was the most highly prevalent bacteria, followed by bacillus sp., lysinibacillus sp., enterococcus harae, sporosarcina sp., e. coli, paenibacillus sp., pseudomonas aeruginosa, leucobac-ter iarius, and dermacoccus sp. antibiotic susceptibility test. a total of 500 bacterial isolates were identified and disc diffusion method was then used to confirm the resistant phenotypes of these isolates. the percentages of resistance to 23 types of antibiotics among bacteria isolated from kota samarahan, saratok, betong, maludam, miri, kuching, semarang, sepinang, sarikei, and sibu was shown in tables 2. the highest frequencies of antibiotic resistance were detected against penicillin g (36.80±23.87%), followed by ampicillin (28.60±17.13%), and rifampicin (16.90±13. volume 7, 2013 microbiol indones 139 site -1 mean of bacterial colony counts (log cfu g )10 kota samarahan saratok betong maludam miri kuching semarang sepinang sarikei sibu total 9.22 ± 0.72 a* 6.23 ± 0.93 b 6.03 ± 0.62 b 7.47 ± 0.49 abcd 8.76 ± 0.14 ad 8.38 ± 0.16 acd 7.14 ± 0.50 bcd 6.64 ± 0.83 bc 6.95 ± 1.68 bc 7.52 ± 1.01 abcd 7.43 ± 1.26 table 1 the means of colony counts of bacteria isolated from kota samarahan, saratok, betong, maludam, miri, kuching, semarang, sepinang, sarikei, and sibu in sarawak, malaysia bacteria were capable to grow both aerobically and anaerobically. besides, kota samarahan is situated at the coastal area which has sufficiently nutrient-rich water considering the shallow coastal depth waterlevel and the abundance of sunlight in this are, thus more conducive for bacterial growth. the present bacterial count was higher than the findings reported by nyakundi and mwangi (2011). in their report, the 6 total bacterial viable count was between 2.4 x 10 and 6 -1 7.8 x 10 cfu g from marabou stock (leptoptilos crumeniferus) droppings. the results of this microbiological examination on the bacterial distribution in the samples of swiftlet feces randomly collected at kota samarahan, semarang, saratok, betong, sarikei, sibu, sepinang, maludam, miri, and kuching in sarawak, malaysia (fig 1) revealed that most of these bacterial isolates were gram positive bacteria. staphylococcus sp. was the most prevalent, followed by bacillus sp., lysinibacillus sp. b4, e. harae, sporosarcina sp., e. coli, paenibacillus sp., p. aeruginosa, l. iarius, and dermacoccus sp. staphylococcus sp. and bacillus sp. were highly prevalent bacteria mainly because they are facultative anaerobic bacteria capable of growing both aerobically 70%). the bacteria isolated from different sites showed different levels of resistance toward the antibiotics (table 2). the bacteria have developed resistance to at least one antibiotic. the analysis of variance test showed that the levels of the resistance toward 23 tested antibiotics were significantly different (f=6.780, p<0.05) from one another. the mean resistance levels toward penicillin g and ampicillin were significantly higher (p <0.05) than the other tested antibiotics such as chloramphenicol, tetracycline, streptomycin, gentamycin, erythromycin, cephalothin, nitrofurantoin, tobramycin, rifampicin, kanamycin, sulphamethoxazole/trimethoprim, amikacin, imipenem, ceftriaxone, doxycycline, ceftazidime, norflaxacin, vancomycin, piperacillin, ciprofloxacin, and nalidixic acid. discussion bacterial colony count was performed to determine the total number of microorganisms present in the collected swiftlet feces samples. the log of mean 10 bacterial colony count per g was the highest (9.22±0.72) in samples collected from kota samarahan (table 1), mostly because these facultative anaerobic microbiol indones140 sien et al. fig 1 swiftlet feces bacterial distributions (%) in kota samarahan, semarang, saratok, betong, sarikei, sibu, sepinang, maludam, miri, and kuching in sarawak, malaysia. 0 20 40 60 80 100 120 d is tr ib u ti o n o f b ac te ri a (% ) sampling sites leucobacter iarius strain 40 proteus sp. pseudomonas aeruginosa strain 123 dermacoccus sp. 103 paenibacillus sp. gh-134 sporosarcina sp. enterococcus harae strain ss33b escherichia coli staphylococcus sp. lysinibacillus sp. b4 bacillus sp. se m ar an g k ot a sa m ar ah an sa ra to k b et on g sa rik ei si bu se pi na ng m al ud am m iri k uc hi ng volume 7, 2013 microbiol indones 141` a n ti b io ti c r e si st a n c e r a te ( % ) m e a n k o ta s a m a ra h a n (n = 5 0 ) s a ra to k (n = 5 0 ) b e to n g (n = 5 0 ) m a lu d a m (n = 5 0 ) m ir i (n = 5 0 ) k u c h in g (n = 5 0 ) s e m a ra n g (n = 5 0 ) s e p in a n g (n = 5 0 ) s a ri k e i (n = 5 0 ) s ib u (n = 5 0 ) p e n ic il li n a m p ic il li n 4 2 6 2 3 8 1 0 2 8 4 2 8 2 0 1 6 2 0 2 8 .6 0 ± 1 7 .1 3 a b * p e n ic il li n g 4 0 6 6 7 2 1 4 4 6 6 4 1 2 2 0 1 6 1 8 3 6 .8 0 ± 2 3 .8 7 a p ip e ra c il li n 4 8 0 0 0 0 4 0 0 0 1 .6 0 ± 2 .8 0 c c e p h a lo sp o r in c e ft ri a x o n e 4 2 8 0 4 0 2 2 0 6 0 6 .4 0 ± 1 2 .8 2 c c e ft a z id im e 3 8 1 6 1 2 6 0 2 4 2 1 8 1 8 2 1 5 .1 1 ± 1 5 .5 0 b c c e p h a lo th in 4 4 2 0 4 4 1 8 4 1 6 1 2 4 0 1 2 .6 0 ± 1 3 .0 7 b c p h e n ic o l te tr a c y c li n e c h lo ra m p h e n ic o l 0 8 0 0 1 8 0 2 0 0 0 2 .8 0 ± 5 .9 0 c q u in o lo n e s n o rf lo x a c in 4 1 6 0 0 0 0 0 0 0 0 3 .4 5 ± 6 .8 2 c n a li d ix ic a c id 4 8 0 0 0 0 0 0 0 0 1 .2 0 ± 2 .7 0 c f lu ro q u in o lo n e c ip ro fl o x a c in 4 1 6 4 0 4 2 0 0 0 0 3 .0 0 ± 4 .9 2 c m a c ro li d e e ry th ro m y c in 0 3 0 0 2 4 1 0 2 0 8 0 5 .6 0 ± 9 .2 8 c a m in o g ly c o si d e g e n ta m ic in 5 4 2 0 4 0 0 0 2 2 0 1 8 0 1 1 .8 0 ± 1 7 .2 2 b c k a n a m y c in 2 8 0 0 0 2 2 0 0 0 0 3 .2 0 ± 7 .0 7 c a m ik a c in 0 8 0 0 0 0 8 0 0 0 1 .6 0 ± 3 .3 7 c t o b ra m y c in 0 8 0 0 0 2 2 0 0 0 0 3 .0 0 ± 7 .1 3 c c a r b a p e n e m im ip e n e m 0 8 0 0 0 0 0 0 0 0 0 .8 0 ± 2 .5 3 c n it ro fu r a n n it ro fu ra n to in 0 1 6 0 0 1 8 2 2 2 0 0 0 5 .8 0 ± 9 .0 2 c a n sa m y c in r if a m p ic in 4 4 2 8 4 6 2 4 2 6 2 0 9 6 2 1 6 .9 0 ± 1 3 .7 0 b c s tr e p to m y c in 4 8 0 0 0 0 1 0 0 0 0 2 .2 0 ± 3 .8 2 c t ab le 2 r es is ta n ce t o 2 3 t y p es o f an ti b io ti cs a m o n g b ac te ri a is o la te d f ro m s w if tl et f ec es c o ll ec te d a t k o ta s am ar ah an , s ar at o k , b et o n g , m al u d am , m ir i, k u ch in g , s em ar an g , s ep in an g , s ar ik ei , a n d s ib u in s ar aw ak , m al ay si a * m ea n s w it h t h e sa m e su p er sc ri p ts a re n o t si g n if ic an tl y d if fe re n t at 5 % l ev el . in conclusion, the mean of bacteria colony count -1 (log cfu g ) was the highest in kota samarahan and 10 the lowest in betong. there were more gram positive than gram negative bacteria isolated from the swiftlet feces in sarawak, malaysia. staphylococcus sp. and bacillus sp. were the most highly prevalent bacteria in the swiftlet farm houses. the isolated bacteria showed high resistance to penicillin g and ampicillin when tested against 23 types of antibiotics commonly used in current medical practice. if the antibiotic resistant bacteria cannot be controlled or killed by antibiotics, it might become a serious threat to public health. acknowledgmet this research was funded partially by fundamental research grant scheme under the grant no. frgs/01(1 6)/745/2010(31). references akano so, daini oa, ojo mo, smith si, akinside ka. 2009. comparative analysis of antibiotic resistance and r-plasmid of staphylococcus aureus isolates from human and dog samples. afri j clin exp microbiol. 10(3):136-143. allen hk, donato j, wang hh, cloud-hansen ka, davies j, handelsman j. 2010. call of the wild: antibiotic resistance genes in natural environments. nat rev microbiol. 8:251-259. doi:10.1038/nrmicro2312. bozdogan bu, esel d, whitener c, browne fa, appelbaum pc. 2003. antibacterial susceptibility of a vancomycin-resistant staphylococcus aureus strain isolated at hershey medical center. j antimicrob chemoth. 52(5):864. doi:10.1093/jac/dkg457. chih my, ming fl, chung hl, yi th, chia th, ming ll. 2009. characterization of antimicrobial resistance patterns and integrons in human fecal escherichia coli in taiwan. jpn j infect. 62(3):177-181. cirz rt, chin jk, andes dr, de crecy-lagard v, craig wa, romesberg fe. 2005. inhibition of mutation and combating the evolution of antibiotic resistance. plos biol. 3(6): e176. doi:10.1371/journal.pbio.0030176. clements ld, miller bs, streips un. 2002. comparative growth analysis of the facultative anaerobes bacillus subtilis, bacillus licheniformis, and escherichia coli. us national library of medicine national institute of health. 25(2):284-286. dorsch mr. 2007. rapid detection of bacterial antibiotic resistance: preliminary evaluation of pcr assays targeting tetracycline resistance genes. human protection and performance division 2007. enne vi, delsol aa, roe jm, bennett pm. 2004. rifampicin resistance and its fitness cost in enterococcus faecium. j antimicrob chemoth. 53(2):203-207. and anaerobically (clements et al. 2002). besides, bacillus sp. was able to produce endospores that can stay dormant under stressful environment for a long period of time (wayne et al. 2000), thus able to survive even in dry swiftlet feces. contamination of bird feed in the feces sample could also be the reason for the occurrence of staphylococcus sp. according to kocijan et al. (2009), staphylococcus sp. was ubiquitous and commonly found in bird feed. according to simpson (2002), it is normal to find e. coli in swiftlet feces samples because almost all wild birds have e. coli as their normal flora. this study showed that e. coli found in the swiftlet feces samples was normal because almost all wild birds contain e. coli (simpson, 2002). nyakundi and mwangi (2011) and literak et al. (2007) had isolated e. coli from the marabou stock (leptoptilos crumeniferus) droppings and rooks droppings samples. the results, as shown in table 2, have revealed that most of the bacteria were resistant to penicillin g and ampicillin antibiotics. according to li and nikadio (2009), bacteria exhibit an enzymatic deactivation mechanism by producing β-lactamases. in my study, it was shown that staphylococcus sp. was the most highly prevalent bacteria occurring in all the swiftlet houses. bozdogan et al. (2003) reported that staphylococcus sp. had higher antibiotic resistance tendency toward penicillin, methicillin, tetracycline, and erythromycin. besides, cirz et al. (2005) showed the similar result showing that enterococcus sp. had higher resistance towards rifampicin. acquired resistance such as mutation of genes encoding the bacterial protein (cirz et al. 2005; enne et al. 2003) occur and caused antibiotic resistance in most bacteria. cirz et al. (2005) also mentioned that the occurrence of vancomycin resistant enterococcus was high and a lot of researcher had shown the data as early as 1987. pseudomonas sp. was another highly prevalent pathogen and antibiotic resistance bacteria (poole 2004). pseudomonas sp. exhibits multiple antibiotic resistance mechanisms by developing multidrug efflux pumps encoded by antibiotic resistant genes and bacterial cellular envelopes with lower permeability to antibiotics (poole 2004). most of the e. coli were highly resistant to piperacillin and streptomycin (wittwer et al. 2005). streptomycin has been the first-line antimicrobial and widely used as growth promoters in human and agricultural field in taiwan (chih et al. 2009). similar result was shown by chih et al. (2009) stated that extensive usage of streptomycin antibiotics caused resistance in e. coli. microbiol indones142 sien et al. volume 7, 2013 microbiol indones 143 simpson vr. 2002. wild animals as reservoirs of infectious diseases in the uk. vet j. 163(2):128-146. doi:10.1053/ tvjl.2001.0662. varaldo p. 2002. antimicrobial resistance and susceptibility testing: an evergreen topic. h, henry jm, peter s. 2000. resistance of bacillus endospores to extreme terrestrial and extraterrestrial environments. microbiol mol biol. 64(3):548-572. doi:10.1128/mmbr.64.3.548572.2000. wayne ln, nobuo m, gerda h, henry jm, peter s. 2000. resistance of bacillus endospore to extreme terrestrial and extraterrestrial environment. microbiol mol biol rev. 64(3):548-572. doi:10.1128/mmbr.64.3.548572.2000. wittwer m, keller t, wassenaar tm, stephen r, howald d, regula g, bissig-choisat b. 2005. genetic diversity and antibiotic resistance patterns in campylobacter population isolated from poultry farm in switzerland. appl environ microbiol. 71(6):2840-2847. doi:10.1128 /aem.71.6.2840-2847.2005. woo pcy, leung pkl, leung kw, yuen ky. 2000. identification by 16s ribosomal rna gene sequencing of an enterobacteriaceae species from a bone marrow transplant recipient. molecular pathology. 53(4):211215. doi:10.1136/mp.53.4.211. yah cs, chineye hu, eghafona no. 2007. multi-antibioticsresistance plasmid profile of enteric pathogens in pediatric patients from nigeria. biokemistri. 19(1):3542. zulkifli y, alitheen nb, son r, raha ar, samuel l. 2009. random amplified polymorphic dna-pcr and eric pcr analysis on vibrio parahaemolyticus isolated from cockles in padang, indonesia. int food res j. 16:141-150. doi:10.1093/jac/dkh044. kocijan ie, prukner-radovcic, beck r, galov a, marinculic a, susic g. 2009. microflora and internal parasites of the digestive tract of eurasian friffon vultures (gyps fulvulus) in croatia. eur j wildl res. 55(1):71-74. doi:10.1007/s10344-008-0209-4. kumar p, ramakritinan cm, kumaraguru ak. 2009. 16s rrna based identification of aeromonas sp. kumar by constructing phylogenetic tree and identification of regulatory elements from the harmful red tide bloom, gulf of mannar. int j oceans oceanogr. 3(2):29-35. li x, nikadio h. 2009. efflux-mediated drug resistance in bacteria. an update. drug 69(12):1555-1623. doi:10.21 65/11317030-000000000-00000. literak i, vanko r, dolejska m, cizek a, karpyskova r. 2007. antibiotic resistant escherichia coli and salmonella in russian rooks (corvus frugilegus) intering in czech republic. lett appl microbial. 45(6):616-621. doi:10.1111/j.1472-765x.2007.02236.x. maria iqo, juan ddc, manuel m, maria jb, pilar m. 2008. preparation of bacterial dna template by boiling and effect of immunoglobulin g as an inhibitor in realtime pcr for serum samples from patient s with brucellosis. american society for microbiology. 15(2):293-296. national committee for laboratory standards (nccls). 2012. performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. in approved standard m100-s22. volume 32(3).villanova, pa: united states. nyakundi wo and mwangi w. 2011. isolation and characterisation of pathogenic bacteria and fungi from leptoptilos crumeriferu (marabau strok) dropping. j appl sci tech environ sanit. 1(1):93-103. poole k. 2004. efflux-mediated multiresistance in gramnegative bacteria. clin microbiol infect. 10(1):12-26. doi:10.1111/j.1469-0691.2004.00763.x. 1: 137 2: 138 3: 139 4: 140 5: 141 6: 142 7: 143 8 483 juniastuti etal.cdr analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia juniastuti , eduardus bimo aksono , takako utsumi , yoshihiko yano , soetjipto , yoshitake hayashi , hak hotta , fedik abdul rantam , hernomo ontoseno kusumobroto , maria inge lusida 1,4 3 4,5 5 2,4 5 5 5 1,4 * mutations of precore (a1896) and core promoter (t1762/a1764) of hepatitis b virus can reduce hbeag production. these mutations are frequently found in the late hbeag seroconversion. however, it has been a controversy about the role played by precore and core promoter mutations in determining outcome of chronic hepatitis b. in the present study, the variability of precore and core promoter of hepatitis b virus were analyzed using pcr amplification and sequencing, according to the outcome (viral load and hbeag/anti-hbe) in chronic hepatitis b patients in surabaya. the study groups included 5 patients with uncomplicated chronic hepatitis b and 10 patients with chronic hepatitis b and liver cirrhosis in dr. soetomo hospital, surabaya. the control group included 6 blood donors obtained from indonesia red cross, surabaya. all groups were hbsag positive. precore mutation a1896 was predominant in all groups (60%-67% of each), together with precore variant t1858. as reported, precore variant t1858 is a prerequisite for precore a1896 and characteristic for viral genotype. nevertheless, core promoter mutations t1762/a1764 were predominant only in lc patients (60%). all of these mutations were found mostly after hbeag seroconversion (anti-hbe+). of most samples with anti-hbe+, precore mutation was related with low viral load (<10 copies/ml), but core promoter mutations with high viral load ( 10 copies/ml). precore mutation a1896 was predominant in all groups, but core promoter mutations t1762/a1764 were only predominant in lc patients. the precore mutation alone is possible not critical to indicate a poor outcome, the core promoter mutations must be considered also. key words: hepatitis b virus, precore mutations, core promoter mutations, chronic hepatitis b 6 and 1 2 3 4 5 6 department of microbiology, school of medicine, universitas airlangga, jalan mayjen prof dr moestopo 47, surabaya 60131, indonesia; department of biochemistry, school of medicine, universitas airlangga, jalan mayjen prof dr moestopo 47, surabaya 60131, indonesia; institute of tropical disease, universitas airlangga, jalan mulyorejo, surabaya 60115, indonesia; indonesia-japan collaborative research center for emerging and re-emerging infectious diseases, institute of tropical disease, universitas airlangga, jalan mulyorejo, surabaya 60115, indonesia; center for infectious diseases, kobe university graduate school of medicine, 7-5-1 kusunoki-cho, kobe, hyogo, 650-0017, japan; department of internal medicine, dr. soetomo general hospital, jalan mayjen prof dr moestopo, surabaya 60131, indonesia 5 5 > chronic hepatitis b virus (hbv) infection is a major health problem worldwide, especially in indonesia which belongs to the moderate-to-high hepatitis b endemic region (sastrosoewignjo . 1991; khan 2004). hbv belongs to the and replicates its dna genome via a reverse transcription step. the high spontaneous error rate of the viral reverse transcriptase is responsible for viral genome evolution during the course of infection under the antiviral pressure of the host immune response or specific therapy (summers and mason 1982). hbv infection is associated with various courses of liver diseases ranging from asymptomatic carrier state to fulminant hepatitis. recently, the clinical importance of hbv genome variability has been discovered. however the results of recent molecular studies are a matter of controversy, and these issues have yet to be resolved (davidson 2005). in the natural course of chronic hbv infection, the early seroconversion from hbeag to anti-hbe usually indicates a favorable outcome, because it is usually associated with the cessation of viral replication and non-progressive liver disease (chen 1993; chu 2000). in contrast, the late seroconversion of hbeag after multiple bouts of reactivation and remission may accelerate the progression of chronic hepatitis to liver cirrhosis and, thus have a poor clinical outcome (liaw 1987; perrillo 2001). in clinical practice, the most frequently encountered variant form of et al hepadnaviridae et al. et al. chronic hbv infections is hbeag-negative chronic hepatitis b associated with the replication of precore region mutants that terminate precore protein and hbeag expression (carman 1989; rizetto and ciancio 2008). the most frequently detected precore mutation is nucleotide transition at the codon 28 (a1896) which converts into a tag stop codon (okamoto 1990). evidence has been proposed that the pattern of precore mutation is restricted by the secondary structure requirements of the epsilon encapsidation signals which are essential for its function and therefore by hbv genotypes. the presence of this mutation is dependent upon the nucleotide (c/t) of the precore at position 1858 (codon 15) (lok 1994). besides, the core promoter region also plays an important role in the viral life cycle, concerning the production of the precore mrna and pregenomic rna. hbv carrying the most common core promoter mutations, t1762 and a1764 displayed reduced levels of hbeag synthesis and were associated with enhanced viral replication (buckwold 1996). previous reports have suggested that hbeag mutations are associated with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (rodriguez-frias 1995). however, conflicting evidence suggests that hbeag mutations are present in hbv carriers and in those individuals with mild forms of hbv infection or without liver disease (davidson 2005). the aim of this study was to analyze the precore and core promoter mutations in patients with uncomplicated chronic hepatitis b, patients with chronic hepatitis b and liver et al. et al. et al. et al. et al. et al. *corresponding author: phone: 62-31-5030252 ext. 159, fax: +6231-5022472, e-mail: koeraisindewi@yahoo.co.id + issn 1978-3477 vol 4, no 3, dec 2010, p 143-148 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.8 cirrhosis, and blood donors as controls, according to the outcome (viral load and hbeag/anti-hbe status) three groups of individuals were included in this study. group 1 included 5 patients with uncomplicated chronic hepatitis b, i.e. patients who were hbsag positive more than 6 months without any clinical significance, confirmed by the medical records of the laboratory and clinical data. group 2 included 10 patients with chronic hepatitis b and liver cirrhosis, i.e. patients who were hbsag positive more than 6 months and had liver cirrhosis shown by the medical records of the laboratory and ultrasonography data. group 1 and 2 were obtained from dr. soetomo general hospital, surabaya, indonesia. group 3 as the control group included 6 blood donors, i.e. individuals who were hbsag positive and alt normal, obtained from blood transfusion unit – indonesian red cross in surabaya. exclusion criteria included hepatitis c infection, hiv infection and use of antiviral therapy. the descent of each individual was documented, both maternally and paternally. all sera from each individual were stored at -20 for further examinations. ethical clearance of this study was obtained from the ethics committee of the dr soetomo general hospital, in surabaya, indonesia. all participants signed an agreement to participate in this study. hbeag and anti-hbe serologic assays were performed by microparticle eia (abbott). quantitative determination of viral dna in serum was performed by cobas amplicor hbv monitor (roche). the dna of hbv was extracted from each serum sample using the high pure viral nucleic acid (roche) following the manufacturer's guidelines. the extracted dna was used as a template for the amplification of the respective gene regions. pcrs were performed with the high fidelity pcr enzyme mix (fermentas). the reactions contained 2.5 l high fidelity pcr buffer (with mgcl ), 2.5 l dntp with a concentration 2 mm, 0.25 l high fidelity pcr enzyme mix, 10 l dna, and 0.5 l of each primer with a concentration of 100 pmol l , in a total reaction volume of 25 l. the thermocycling condition included a 5-min denaturation step of 94 , followed by 40 cycles of 1 min at 94 , 1 min at 55 , and 2 min at 72 . the partial core promoter gene and the precore gene were amplified in the first round using hbc1 (5'-tta cat aag agg act ctt gg-3', nt 1650 to 1669) and hb9r (5'-gga tag aac tag cag gca t-3', nt 2654-2635) (sugauchi 2001). if the first round pcr was negative, the second round pcr was performed using primers hbc1 and hbc2 (5'-taa agc cca gta aag ttt cc-3', nt 2494 to 2475) (lusida . 2008). to shed more light on the association between hbv genotype and precore mutation, part of the s gene was amplified in the first round using primers p7 (5'-gtg gtg gac ttc tct caa ttt tc-3', nt 256 to 278) and p8 (5'. c c c c c materials and methods patients and controls. hbeag/anti-hbe serology and viral dna load. viral dna extraction, pcr amplification and sequencing. ° ° ° ° ° � � � � � � � 2 -1 et al. et al cgg taw aaa ggg act cam gat-3', nt 796 to 776). if the first round pcr was negative, the second round pcr was performed using primers hbs1 (5'-caa ggt atg ttg ccc gtt tg-3', nt 455 to 474) and hbs2 (5'-aaa gcc ctg cga acc act ga-3', nt 713 to 694) (lusida 2008). nucleotide sequences of the amplified fragments were determined using the big dye terminator v1.1 cycle sequencing kit with an abi prism 310 (applied biosystems, usa). all analyses of the sequences data were performed with genetyx-mac version 9 (software development co., ltd., tokyo, japan). after aligning the sequences obtained from this study and from international dna databases (ddbj/embl/genbank), the variability of the precore and core promoter sequences were analyzed. the hbv genotypes were determined based on the homologous percentage of >96% in the s gene sequences compared with hbv isolates from international dna databases (ddbj/embl/genbank) (magnius and norder 1995; arauz-ruiz . 1997) a total of 21 hbsag-positive sera were obtained from 6 women and 15 men (aged 22 – 68 years). they were people of java (85.7%), flores (4.8%), sulawesi (4.8%) and acehbatak (4.8%) living in surabaya. of these, 13 (61.9%) were hbeag-/anti-h be+, 6 (28.6%) were hbeag+/ anti-hbe-, and 2 (9.5%) were hbeag-/anti-h be-. the high hbv dna levels, 10 copies ml (13/21, 61.9%) were frequently found in patients with chronic hepatitis b and liver cirrhosis (7/10, 70%). the low hbv dna levels were detected in 38.1% of the total sera, mostly found in controls (3/6, 50%). hbv genotypes could be assigned to 15 hbv isolates. based on part of the s gene, all of these isolates were classified into hbv genotype b (fig 1). the most frequently detected mutation was g1896a (61.9%) in the precore region, and it was predominant in all groups (60-66.7% of each). the precore g1896a was the most prevalent in hbv isolates with genotype b (10/15, 66.7%) (table 1). besides, all isolates studied had precore variant t1858. the precore mutation, a1846t was detected in 33.3% isolates (7/21), mostly (5/7, 71.4%) associated with the virus carrying an a1896 or a1899. the other precore mutation g1899a was detected in 23.8% isolates (5/21), and some of these were combined with a1896 (3/5, 60%) (table 1, fig 1). in the core promoter region, a1762t and g1764a were detected in some isolates (38% and 33.3%, respectively), and these mutations were predominant in patients with chronic hepatitis b and liver cirrhosis (60% and 50%, respectively) but were few in the other groups. the other core promoter mutations, t1753c (amino acid t127) and t1754g (amino acid m127) were detected in 33.3% isolates (7/21), and found around 30% 40% in each group (table 1, fig 2). [a/t] [a/c] 5 -1 et al. et al sequences analyses for detection of precore and core promoter mutations and determination of hbv genotypes. . results > 144 juniastuti et al. microbiol indones 1814 1846 1858 1896 1899 fig 1 nucleotide sequences of the precore region of hbv isolates from patients with uncomplicated chronic hepatitis b (u), patients with chronic hepatitis b and liver cirrhosis (s) and controls (p). the two upper sequences were downloaded from international dna databases ((ddbj/embl/genbank) the hbv genotypes/subtypes are indicated following the accession number of the two sequences from international databases and the sample number of fifteen sequences obtained in this study. . table 1 variability of precore and core promoter in patients with uncomplicated chronic hepatitis b, patients with chronic hepatitis b and liver cirrhosis and controls 1742 1753 1754 1762 1764 1813 * ** * * * d00330 badw2 tgggggaggaggtt fig 2 nucleotide sequences of the partial core promoter region of hbv isolates from patients with uncomplicated chronic hepatitis b (u), patients with chronic hepatitis b and liver cirrhosis (s) and controls (p). the two upper sequences were downloaded from international dna databases (ddbj/embl/genbank). volume 4, 2010 microbiol indones 145 the most prevalent precore mutation, a1896 was frequently detected in hbeag-/anti-hbe+ status (77%). the core promoter mutations, t1762 and a1764 were also predominant in sera with hbag-/anti-hbe+ (75% and 85.7%, respectively). these precore and core promoter mutations were detected few in the other hbeag/anti-hbe status (0-25%) (table 2). of most sera with antihbe+, precore a1896 was mostly related with low hbv dna levels (60%). on the contrary, core promoter t1762 and a1764 were frequently related with high hbv dna levels (66.7%) (table 3) a nucleotide transition which converts into a tag stop codon mutation (a1896) of the precore region develops during viral replication and displacement of the wild type by mutant can take several years (okamoto 1990; akarka 1994). this mutation prevents the translation of the precore protein and completely abolishes the production of hbeag (kao 2002). although hbeag is not required for viral replication or infectivity, its exact function is not known (kramvis and kew 2005). hbeag is believed to be an important humoral and cellular immune target, this loss of hbeag production may represent escape mutation, thus contributing to viral persistence (carman 1993). detailed studies showed a significant link between precore mutation with remission of liver disease (chan 1999), but others found a high prevalence of precore mutation in patient with severe liver disease (chisari 1997; friedt 1999; kao 2000). the result of this study showed that the precore a1896 was predominant not only in patients with chronic hepatitis b and liver cirrhosis, but also in patients with uncomplicated chronic hepatitis b and controls (60-66.7% of each) (table 1). it has been considered that a contributing factor in precore mutation frequency is viral genotype (kramvis and kew 2005). the precore a1896 is found only in patients infected with hbv genotypes b, d, e, and in minority only of the genotypes c and f strains that bear a variant t at nt 1858 (hadziyannis 1995; kramvis and kew 2005). the precore a1896 and t1858 tighten the stem structure by making a t-a pair (lok 1994), although t1858 can also make a wobble pairing with g1896 (kao 2002). the hbv isolates with certain genotypes which had precore t1858 are more frequent in geographic regions such as asia and the mediterranean basin (rodriguez-frias 1995; lindh 1997) related with the ethnic origin of the individual carriers of the hbv (davidson 2005). as previous . discussion et al. et al. et al. et al. et al. et al. et al. et al. et al. studies (lusida 2003; mulyanto 2009), this study showed that hbv genotype b was predominant in patients with hbv infection (mostly javanese) in surabaya, and it agreed with the predominance of precore a1896 in all groups. however, the association between precore a1896 and other genotypes can not be explained in this study. considering clinical outcomes, this study supports some previous studies (zarski 1994), that precore a1896 alone may have no direct pathogenic role, especially in hbv isolates with some genotypes having precore t1858. the precore a1896 combined with other precore mutations was detected in some hbv isolates. as much as 7 isolates (33.3%) had an a to t mutation at nt 1846 in the precore region, mostly associated with the presence of 1896 or 1899 mutation (table 1, fig 1). the double precore mutations at nt 1896 and nt 1899 was detected in 3 isolates (14.3%) (fig 1). as proposed, precore a1896 and t1858 together with precore a1899 and t1855 could enhance the stability of the lower stem by replacing t-g pair with more stable t-a pair (lok 1994). besides, although the mutation at nt 1846 will destroy base pairing at nt 1896 or nt 1899, the combined mutation at 1896 or 1899 may stabilize stem loop (cho 1999). some of these combined mutations at nt 1846-nt 1896/1899 and at nt 1896-nt 1899 were found in sera with low hbv dna levels (60% and 66.6%, respectively) and with high hbv dna levels (40% and 33.3%, respectively) (data not shown). further studies are necessary to confirm the significance of these mutations on viral replication. mutations in the core promoter that is part of the x region can also prevent hbeag production by selectively down-regulating the transcription of the precore mrna. the most common mutations involved a to t change at nt 1762 (amino acid k130m) and g to a change at nt 1764 (amino acid v131i) (okamoto 1994). these mutations were mostly detected in patients with chronic hepatitis b and et al. et al. et al. et al. et al. et al. 146 juniastuti et al. microbiol indones table 2 mutations of precore and core promoter according to the hbeag/anti-hbe status liver cirrhosis (50-60%), and found few in other groups (16.7-20%) (table 1). utama (2009) also reported that the double mutations were only found in 1.96% blood donors in makasar, indonesia. as kramvis and kew reported (2005), the core promoter mutations, t1762 and a1764 were found to be significantly associated with more severe liver disease (liver cirrhosis with or without hepatocellular carcinoma). it has been proposed that core promoter, t1762/a1764 may enhance hbv virulence by increasing host immune response, increasing viral replication, or altering the coding region for the x (kidd-ljunggren 1995; buckwold 1996; li 1999; hunt 2000). by diminishing circulating hbeag, core promoter mutants may augment the host immune response to hbv-infected hepatocytes, hence increasing hepatocytes apoptosis and regeneration, which leads to liver injury (hunt 2000). interestingly, most hbv isolates with core promoter, t1762/a1764 were not accompanied with precore a1896 (5/8, 62.5%), especially in patients with chronic hepatitis b and liver cirrhosis (fig 1-2). it is still unclear the importance of the precore a1896 in pathogenesis of hbv with core promoter t1762/a1764. the other core promoter mutations, c1753 (amino acid t127) and g1754 (amino acid m127) were detected in 7 hbv isolates (33.3%) (table 1, fig 2). the t127 mutation was recently described in japanese patients with fulminant and chronic hepatitis (kreutz 2002), and it was shown that this mutation affecting the crucial domain for transactivation suppress the transcription of both precore and core mrna. in this study, the t127 mutation was detected around 0 – 20% in each group (table 1). it needs further investigation to confirm its role. the most frequently encountered variant form of chronic hbv infections is hbeag negative chronic hepatitis b due to replication of naturally occurring hbv variants with nucleotide substitutions in the precore and/or core promoter regions of the genome and represents a later phase of chronic hbv infection. hbv can mutate into an hbeag negative phenotype to evade the anti-hbe immune pressure that develops sooner or later in hosts. hbeag-negative mutants frequently become the predominant virus population in chronic hbsag carriers, possibly indicating a selection advantage against the wild type (okamoto 1990; okamoto 1994). the major mutations focused in this study, precore a1896 and core promoter t1762/a1764 were mostly found in sera with antihbe+ (table 2). of most sera with anti-hbe+, precore a1896 was related with low viral load (<10 copies ml ), but core promoter t1762/a1764 with high viral load ( 10 copies ml ) (table 3). lin (2007) also reported that patients with core promoter t1762/a1764 mutant had significantly higher serum hbv dna levels than those with core promoter a1762/g1764 wild type strain, regardless of precore 1896 status. it appears that the mutations of core promoter, t1762/a1764 were related with severe liver disease after hbeag seroconversion not as precore a1896, and it might not to worry about the high prevalence of precore a1896 in surabaya. et al. et al. et al. et al. et al. et al. et al. et al. et al. 5 -1 5 -1 > interestingly, two sera obtained from one patient with uncomplicated chronic hepatitis b (7rs) and one patient with chronic hepatitis b and liver cirrhosis (17rs), had precore mutation a1896 but hbeag did not disappear (data not shown). it might be due to presence of a mixed infection of the mutant and wild type viruses. the presence of wild type virus is needed by some of the mutants for infection of hepatocytes. it appears that hbv exists as a quasi species of wild type and mutant clones even in the hbeag positive phase (davidson 2005). the inno-lipa assay will be able to pick up the mixed infection (abbas 2006). a heterogenous virus population circulating in patients with chronic hbv infection must be considered to determine the outcome. overall, it could be concluded that precore mutation a1896 was predominant in all groups, but core promoter mutations t1762/a1764 were only predominant in patients with chronic hepatitis b and liver cirrhosis. the precore mutation alone is possibly not critical to indicate a poor outcome, core promoter mutations must be considered also. further studies with a larger population are needed to investigate which type of specific mutations or which combined mutations is associated with severe liver disease and thus might play a role in pathogenesis he authors are grateful to slamet riyadi, nur achmad tjipto, and nasronudin for their cooperations; also mochamad amin and koen poedijati for their technical assistance. we also deeply appreciate the patients at dr soetomo general hospital, surabaya and blood donors at the blood transfusion unit-indonesia red cross, surabaya, who had given their blood in this study. this study was supported by a grant from the directorate general of high education, department of national education, indonesia, and also by the program of founding research centers for emerging and reemerging infectious diseases, the ministry of education, culture, sports, science and technology (mext), japan et al. et al. . t . acknowledgements references abbas z, muzaffar r, siddiqui a, naqvi saa, rizvi sah. 2006. genetic variability in the precore and core promoter regions of hepatitis b virus strains in karachi. bmc gastroenterology 6:20, doi: . akarka us, greene s, lok asf. 1994. detection of precore hepatitis b virus mutants in asymptomatic hbsag-positive family members. hepatology 19:1366-70. arauz-ruiz p, norder h, visoná ka, magnius lo. 1997. molecular epidemiology of hepatitis b virus in central america reflected in the genetic variability of the small s gene. j infect dis 176:851-8. buckwold ve, xu zc, chen m, yen ts, ou jh. 1996. effects of a naturally occurring mutation in the hepatitis b virus basal core promoter on precore gene expression and viral replication. j virol 70: 5845-51. carman w, hadziyannis s, mcgarvey mj, jacyna m, karayiannis p, makris 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summers j, mason ws. 1982. replication of the genome of a hepatitis b-like virus by reverse transcription of an rna intermediate. cell 29:403-15. utama a, octavia ti, dhenni r, miskad ua, yusuf i, tai s. 2009. hepatitis b virus genotypes/subgenotypes in voluntary blood donors in makasar, south sulawesi, indonesia. virol j 6:128, doi:10.1186/1743-422x-6-128. zarski jp, marcellin p, cohard m, lutz jm, bouche c, rais a, 1994. comparison of anti-hbe-positive and hbe-antigen-positive chronic hepatitis b in france. j hepatol 20:636-40. et al. 148 juniastuti et al. microbiol indones 6.mi663-sri wilarso budi available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.4.6issn 1978-3477, eissn 2087-8575 vol 6, no 4, december 2012, p 180-188 *corresponding author; phone: +62-251-8626806, e-mail: wilarso62@yahoo.com arbuscular mycorrhizal fungi (amf) is one of the soil microorganisms composing the essential components of sustainable soil-plant system. these fungi provide numerous benefits to their host, including better phosphorus uptake (gossous and mohammad 2009), increased absorption of nitrogen (rotor and delima 2010), production of plant growth hormones (herrera-medina et al. 2007), defending root against soil borne diseases (bakhtiar et al. 2010) and increasing plant growth and productivity (duponnois et al. 2005). several types of microorganisms were reported to be associated with the rhizospheres of different host plants colonized by arbuscular mycorrhizal fungi (amf). these microbes were identified as n -fixing bacteria (secilia and 2 bagyaraj 1987), plant growth-promoting rhizobacteria (jargeat et al. 2004), phosphate solubilizing bacteria (cruz and ishii 2011), and antagonists of plant pathogens (budi et al. 1999a, bakhtiar et al. 2010). the interaction between amf with different types of mycorrhizosphere bacteria can influence their d e v e l o p m e n t a n d s y m b i o t i c e s t a b l i s h m e n t . hildebrandt et al. (2002) reported the increase of amf fungal growth when combined with bacteria. the am fungal colonization (mamatha et al. 2002), inhabitation of plant pathogen fungi ( ), and agriculture and forestry plant growth (barea et al. 2002) have been reported to increase by mycorrhizosphere bacteria inoculation. recently some bacteria were reported to be associated with amf like mycelium and surface sterilized spore. the bacterial genus paenibacillus was intimately associated with the mycelium of amf glomus intraradices (mansfeld-giese et al. 2002). the bacteria species janthinobacterium lividum maier et al. 2004 four isolates (bgi1, bgi2, bgi3, and bgi4) bacteria were isolated from surface sterilized arbuscular mycorrhizal fungi (amf) spores of gigaspora margarita (gm). based on 16s rdna analyses and phylogenetic tree, it was revealed that isolates bgi1, bgi3 and bgi4 belong to genus bacillus, whereas bgi2 was very close to bacillus megaterium eg 24. enzymatic activity test showed that all four isolates had cellulase and protease activities; while one isolate (bacillus sp. bgi4) also has pectinase activity in addition to the celulase and protease activities. dual inoculation of melia azedarch linn roots by b. megaterium bgi2 and amf spores g. margarita enhanced mycorrhizal root colonization by 58.3 %. combination of bacillus sp. bgi1 and g. margarita increased height, diameter, shoot biomass, and root biomass of m. azedarch by 353, 4.8, 4546, and 2810%, respectively, in comparison to the uninoculated control plant. key words: bacteria, arbuscular mycorrizal fungi, stimulation effects, root colonization, plant growth empat isolat bakteri (bgi1, bgi2, bgi3, dan bgi4) telah diisolasi dari spora fungi mikoriza arbuscula gigaspora margarita (gm) yang dsterilisasi permukaannya. berdasarkan pada analisis urutan gen 16s rdna dan pohon filogenetik, keempat isolat tersebut termasuk ke dalam genus bacillus sedangkan isolat bgi2 mempunyai kemiripan tinggi dengan bacillus megaterium eg 24. uji enzimatik menunjukkan bahwa keempat isolat menghasilkan enzim selulase dan protease. satu isolat bacillus sp. bgi4 menghasilkan enzim selulase, protease, dan pectinase. inokulasi tanaman melia azedarch linn dengan b. megaterium bgi2 dan spora g. margarita meningkatkan kolonisasi akar sebesar 58,3 %. kombinasi bacillus sp. bgi1 dan g. margarita meningkatkan pertumbuhan tinggi, diameter, biomass pucuk, dan biomass akar m. azedarch berturut-turut 353 %, 4,8%, 4546 %, dan 2810%, dibanding tanaman kontrol. kata kunci: bakteri, fungi mikoriza arbuskula, pengaruh rangsangan, kolonisasi akar, pertumbuhan tanaman bacteria associated with arbuscula mycorrhizal spores gigaspora margarita and their potential for stimulating root mycorrhizal colonization and neem (melia azedarach linn) seedling growth 1 2 3 sri wilarso budi *, yenni bakhtiar , and nunang lamaek may 1 department of silviculture faculty of forestry, institut pertanian bogor, campus ipb darmaga. bogor 16680, indonesia; 2 center of biotechnology, badan pengkajian dan penerapan teknologi, gedung 630 kawasan puspitek serpong tangerang selatan 15314, indonesia; 3 faculty of forestry, universitas cendrawasih, jalan camp wolker, jayapura 99358, papua, indonesia (kcigm01), and paenibacillus polymixa (kcigm04) have been reported to be associated with amf spore of gigaspora margarita (cruz et al. 2008). cruz and ishii (2011) reported that some bacteria bacillus sp (ktcigm01), bacillus thuringiensis (ktcigm02), and paenibacillus rhizospherae (ktcigm03) have been isolated from surface sterilized amf spore of gigaspora magrarita. the bacteria associated with amf spore of glomus geosporum and glomus constrictum have been reported by roesti et al. (2005). the fact that bacteria isolated from surface sterilized amf spores can increase phosphate solubilisation and suppression of pathogenic fungi had been reported by cruz et al. (2008). however, no data on the effects of associated bacteria isolated from surface sterilized amf spore on the growth of forest trees are available. the neem, melia azedarach linn. (meliaceae) are native trees of asia, including indonesia (orwa et al. 2009) and widely planted in west java as a community forest tree (yulianti et al. 2011). neem tree is one of the potential forest trees species for reforestation and medicinal plants (selvaraj and mosses 2011). this plant has a fairly coarse root system with very few root hairs, which are common characteristics of plants that are very responsive to arbuscular mycorrhizal symbiosis. the objectives of the present study were to isolate and identify bacteria from surface sterilized spores of amf g. margarita and their effects on mycorrhization and growth of neem seedling. materials and methods isolation of bacteria from the arbuscular mycorrhizal spores. the spores of amf g. margarita originated from the collection of silviculture laboratory, faculty of forestry, institut pertanian bogor (ipb) were sieved by wet sieving according to the method of gardeman and nicholson (1963). fifty spores were then surface sterilized by the method prescribed by budi et al. (1999b). the surfaced sterilized spores were then placed in petri dishes containing sterile pseudomonas agar and nutrient agar and then incubated in the dark at 30 °c. the bacteria were then isolated and purified. each single colony was then transferred to agar slants and stored at 4 °c until used. identification of bacterial isolates. isolates of bacteria were identified following the method described by budi et al. (1999a) and bakhtiar et al. (2010), by sequence analysis of the small ribosomal subunit (16s ribosomal dna) after pcr amplification with eurobacterial primers 765 r (5'-ctgtttgctccccacgtttc-3') and 1141 r (5'-gccttgcgctcgttgc-3') from alpha dna canada (bakhtiar et al. 2010). the dna contigs were assembled using atgc program that connects the 765 r and 1141 r primers. the sequences were compared with dna sequences available in genbank database of ncbi using blast programme. alignment of 16s rdna sequences were performed using clustalx. the phylogenetic tree was constructed by comparing 16s rdna sequences of four isolated bacteria with 16s rdna sequences from genebank dna database and then visualized using tree view 1.6.6. test of enzymatic activity of bacteria in vitro. the characterization of cellulalitic and pectinolytic activity was carried out according to the method of teather and wood (1982) whereas the proteolitic activity was characterized according to the method described by dunne et al. (1997). the experiment was performed in a completely randomized design in petri dishes in triplicates. dual inoculations of bacteria and arbuscular mycorrhizal spores on m. azedarach. the experiments were performed by factorial design with two different treatments of arbuscular mycorrhizal inoculations (inoculated and uninoculated), and five different types of isolate used as inoculums (uninoculated, and inoculated with isolate bgi1, bgi2, bgi3, and bgi4). the experiment was arranged in a completely randomized design in a polybag culture in five replicates. three week old m. azedarach seedlings with four leaves were transplanted into 500 ml polybags containing sterilized ultisol soil. the properties of soil medium were determined following the standard method used in soil laboratory, ipb. the soil medium was very low in phosphorus and other nutrients but had high aluminium concentration (table 1). the plant was either inoculated with surface sterilized amf spores g. margarita and bacterial isolates or not inoculated, as a control at transplanting. each inoculated plant received 50 surface sterilized 8 amf spores and 5 ml bacteria solution containing 10 -1 colony forming unit (cfu) ml of bacteria placed near the roots plant. plants were grown for twelve weeks in the green house and watered as needed. plants were harvested twelve weeks after transplanting and evaluated for height, diameters shoot, and root dry weight. amf root colonizations were evaluated after being cleaned with 2.5 % koh, and stained with 0.05 % trypan blue in acidic glycerol (koske and gemma 1989). percent amf roots colonizations were determined according the method of biermann and volume 6, 2012 microbiol indones 181 linderman (1981). all data were analyzed by analysis of variance procedure using spss software program. results isolation of spore associated bacteria. a total of four bacterial isolates were isolated and purified from surface sterilized spores of amf g. margarita. the morphological characteristic of each isolate bacterium such as color, shape, and colonies were used for separating the isolates. the bacterial isolates showed different morphological characteristic (table 2). identification of bacterial isolates. bacterial isolates were identified based on their 16s rdna analyses (table 2). phylogenetic analyses of 16s rdna showed correlation with 18 strains cited from genbank and all isolates belonged to the genera bacillus spp. (fig1). test of enzymatic activity of bacteria in vitro. enzymatic test activity showed that bacterial isolates bacillus sp. bgi1, bacillus megaterium bgi2, bacillus sp. bgi3, and bacillus sp. bgi4 produced zones of hydrolysis on carboxyl methyl cellulose (cmc) and protein medium. in addition, the bacterial isolate bacillus sp. bgi4 also produced zone of hydrolysis on pectine media tested as indicated by clear zones (halo) around the colonies (table 4). the medium containing pectin was weakly degraded (1 mm zone surrounding ph h o2 4.5 na (me/100 gram) 0.13 ph hcl 3.7 cec (me/100 gram) 23.38 c-organic (walkley & black) % 2.64 base saturation (%) 4.49 n-total (kjeldhal) % 0.24 al-dd (me/100 gram) 1132 c/n 11 h-dd (me/100 gram) 0.51 p (bray i) ppm 8.6 fe (me/100 gram) 4.08 p (hcl 25 %) ppm 206.6 cu (me/100 gram) 0.08 ca (me/100 gram) 0.47 zn (me/100 gram) 0.68 mg (me/100 gram) 0.28 mn (me/100 gram) 7.56 k (me/100 gram) 0.17 table 1 chemical properties of growing media used in this experiment table 2 species identification and characteristic morphology of isolates of bacteria isolated from arbuscula mycorrhizal spores of gigaspora margarita no. isolats code morphological characteristic of colony (colour, colony surface and shape) gram species name with the highest identity (%) 1 bgi1 irregular, lobate, raised, opaque. positive bacillus sp. bd-107 (99%) 2 bgi2 circular, entiere, convex, glossy, brown cream, oaque positive bacillus megaterium eg 24 (100%) 3 bgi3 circular, undulate, pulvinate, opaque positive bacillus subtilis df 61 (100%) 4 bgi4 irregular, undulate, cream, opaque positive bacillus flexus strain ksc_sf9c (100%) 182 budi et al. microbiol indones diameter, shoot and root dry weights of neem seedling (table 5). no significantly different effects were observed on any growth parameters when different bacterial isolates were applied on their own. all isolates tended to increase all growth parameters of neem seedling (table 5). dual inoculation of amf and bacteria significantly increased growth and development of neem seedlings in comparison to uninoculated plants. inoculation with amf alone gave the best growth and increased the height, diameter, shoot and root dry weights by 399, 5.4, 5304, and 4038 %, respectively. dual inoculation with amf and bacterial isolates produced lower results on all growth parameter than single inoculation with amf alone. however, these results were not significantly different the colony), whereas the media containing cmc and protein were very strongly degraded (> 2 mm zone surrounding the colony). effect of bacteria and arbuscular mycorrhizal fungi on root colonization and plant growth. after 12 weeks inoculation, significantly higher amf roots colonization was found in plants inoculated by bacterial isolate b. megaterium bgi2.there was an increase by 58.3 % in comparison to plants inoculated with arbuscular mycorrhiza alone (table 3). on the other hand, plants inoculated with bacterial isolates bacillus sp. bgi1, bacillus sp. bgi3, and bacillus sp. bgi4 only had the amf roots mycorrhization increased by 10, 55, and 0 %, respectively. amf inoculation significantly increased height, fig 1 phylogenetic tree of bacteria isolates from surface sterilized amf spores gigaspora margarita. genbank accession numbers are given after the strain name. bar, 1 substitution per 100 nucleotides. escherichia coli strain wab1892 bgi-1 bgi-3 bgi-4 b. flexus-uvba (kc431015) b. flexus-asu ( kc342254) b. flexus-dneb (kc178613) b. aryabhattai md03 (kc247687) b. megaterium yi (kc311785) b. megaterium hat1 (jn995603) b. megaterium vkk-4ht (jx852575) b. megateriumi eg24 (kc122688) b. megaterium q-36 (jx122812) b. megaterium atcc154151 (fj969753) b. megaterium kccm 11761 (fj969757) b. megaterium tzq1 (hq143625) b. megaterium pdd-31b-1 (hq256822) b. aryabhattai e3 (jf683656) bgi-2 b. aryabhattai 340 (jq860098) 0.01 volume 6, 2012 microbiol indones 183 had been detected in several member of the gigasporaceae. bianciotto et al. (2004) found intracellular bacteria in all fungal life cycle; spores, germ tube, extra, and intraradical hyphae, except arbuscula of gigaspora rosea. an average of about 20000 bacteria in one spore of g. margarita had been detected (bianciotto et al. 2003), and identified as a new taxon termed candidatus glomeribacter gigasporarum (jargeat et al. 2004). recently cruz et al. (2008) and cruz and ishii (2011) found three genera o f b a c t e r i a j a n t i b a c t e r i u m , b a c i l l u s , a n d paenibacillus from surface sterilized spores g. margarita. this research revealed that g. margarita, originally from tropical region, also harbored other bacteria in addition to those already reported. the morphological features of the bacteria including shape, and color confirmed the information provided by other researchers. based on the bacterial isolates' enzymatic activity tests, it was shown that all bacterial isolates produced different amount hydrolytic enzyme activities as indicated by clear zone (table 3). it was demonstrated that the hydrolytic enzyme played important roles in on combination using bacterias isolates bacillus sp. bgi1, and b. megaterium bgi2. on the other hand, dual inoculation of amf and bacillus sp. bgi1 increased the height, diameter, shoot and root dry weights of neem seedlings by 353, 4.9, 4512, and 2752 %, respectively. the values were increased by 335 %, 4.5, 3700, and 1847 %, respectively, when inoculation was performed in combination with b. megaterium bgi2. when plants were inoculated with bacterial isolates alone, their growth and development tended to increase in comparison to the uninoculated plants (table 5). the height was increased by 17, 11.1, 21. 6, and 14. 1 % when inoculated with bacillus sp. bgi1, bacillus megaterium bgi2, and bacillus sp. bgi3, and bacillus sp. bgi4, respectively. discussion the present study showed that surface sterilized amf spores of g. margarita, a member of gigasporaceae, harbor several different bacterial strains. based on phylogenetic correlation analyses, all isolated bacteria belong to bacillus sp. genera. previous research showed that endosymbiotic bacteria table 3 clear zone (mm) of celluase, protease and pectinase activity produced by bacterial isolat after 3 d incubation no strain’s name cellulase protease pectinase 1 bacillus sp. bgi1 4.8a 6.6a 0.0b 2 bacillus megaterium bgi2 2.0b 3.0c 0.0b 3 bacillus sp. bgi3 5.1a 5.2b 0.0b 4 bacillus sp. bgi4 5.0a 6.7a 1.0a table 4 effect of bacterial isolates on amf roots colonization of melia azedarach 12 weeks after inoculation mycorrhizae strains’s name roots colonization (%) no bacillus sp. gi1b 0.00 bacillus megaterium gi2b 0.00 bacillus sp. gi3b 0.00 bacillus sp. i4bg 0.00 yes 40.00 b bacillus sp. i1bg 44.00 b bacillus megaterium i2bg 63.33 a bacillus sp. i3bg 62.00 a bacillus sp. i4bg 39.33 b uninoculated *values in a column followed by the same letter do not differ significantly from each other *values in a column followed by the same letter do not differ significantly from each other 184 budi et al. microbiol indones in particular phosphorous. according to smith et al. (2003) in am symbiosis, plants receive all of their phosphorous via their fungal symbiosis, in consequence the symbiosis establishment is very critical and hydrolytic enzyme could facilitate hyphae of amf enter to the root. the role of the bacteria in the growth and development of arbuscular mycorrhizal fungi had been well documented (frey-klett and garbaye 2005). they play roles in promoting the establishment of symbiosis, stimulating mycelia extension, increasing root-fungus contacts and colonization and reducing the impact of adverse environmental condition on the mycelium of the mycorrhizal fungi. different mechanisms of bacterial effects on mycorrhizal development had been reported by several researchers. keller et al. (2006) found that secondary metabolites responsible for growth promotion of arbuscula mycorrhizal fungi were produced by bacteria. the production of hydrolytic enzymes by mycorrhizosphere bacteria that caused cell wall degradation of pathogenic fungi had been reported (budi et al. 2000). the evidence that arbuscular mycorrhizal fungi might enhance plant growth and development was well documented (johansson et al. 2004). this research demonstrated that neem seedling grown in unfertile soil (table 1) and inoculated by arbuscular mycoorhizal fungi g. margarita significantly facilitating hyphae to enter the epidermis roots in small amount (bonfante and perotto 1995). in this research, as shown in table 3, three bacterial isolates produced cellulolytic and proteolytic activities, while one isolate produced cellulolytic, proteolytic and pectinolytic activities at different amounts as indicated by the width of the clear zones. the bacterial isolate b. megaterium bgi2 produced cellulase and protease in smaller amount compared to other bacterial isolates and had greater mycorrhizal root colonization compared to other bacterial isolates (table 4). the present studies showed that bacteria isolated from surface sterilized amf spores of g. margarita produced hydrolytic enzymes and played important roles on facilitating amf symbiosis development on neem seedling. as shown in table 4, the degree of mycorrhizal root colonization also depended on the amount of enzymes produced. the enhancement of amf root colonization in the presence of non-spore-associated bacteria had been reported previously by other researchers (artursson 2005; duponnois and plenchette 2003) and we assume that those bacteria also produce hydrolytic enzyme activity. arbuscular mycorrhizal fungi are obligate symbionts that establish symbioses with plants in order to obtain carbon, enabling them to grow and complete their life cycle (harrison 2005). in turn, the fungus will assist the plant with the acquisition of mineral nutrient, mycorrhizae strain’s name height (cm) diameter (cm) shoot weight (g) dry root dry weight (g) no no bacteria 3.98 c 0.909 d 0.026 c 0.021 d bacillus sp. i1bg 4.66 c 0.911 cd 0.037 c 0.025 d bacillus megaterium gi2b 4.42 c 0.912 cd 0.031 c 0.026 d bacillus sp. gi3b 4.84 c 0.918 cd 0.039 c 0.033 d bacillus sp. gi4b 4.54 c 0.911 cd 0.031 c 0.027 d yes no bacteria 19.86 a 0.958 a 1.405 a 0.869 a bacillus sp. gi1b 18.02 a 0.953 a 1.199 ab 0.599 ab bacillus megaterium gi2b 17.30 ab 0.952 a 0.988 ab 0.409 bc bacillus sp. gi3b 13.88 b 0.942 b 0.706 c 0.395 c bacillus sp. gi4b 7.86 c 0.923 0.374 c 0.143 dc table 5 effect of bacteria and amf on growth and development of melia azedarach seedling 12 weeks after inoculation *values in a column followed by the same letter do not differ significantly from each other volume 6, 2012 microbiol indones 185 increased their height, diameter and shoot and root biomass growth in comparison to uninoculated control (table 5). increasing growth and development of azadirachta indica a. juss, a forest tree of the same family with m. azedarchta, inoculated by arbuscular mycorrhizal fungi, had been reported (phavaphutanon et al. 1996). the present study is in accordance with the above findings, showing that m. azedarach grown in unfertile soil and inoculated with arbuscular mycorrhiza increased their growth. phosphorous is one of the mineral nutrients essential for plant growth and development (shtark et al. 2010), although in this research the phosphorous absorption was not evaluated, it was demonstrated that am fungi improve phosphorous uptake from soils in particularly in soil with low p availability (olsson et al. 2006). although bacterial isolates enhanced plant growth and development, co-inoculation with arbuscular mycorrhizal fungi decreased their growth in comparison to plants inoculated with arbuscular mycorrhizal alone (table 5). this was probably due to the neem seedling was still in the early growth stage and the nutritional contribution from bacteria and arbuscular mycorrhizae was limited. correa et al. (2006) reported that ectomycorrhiza formation may have a detrimental rather than a beneficial effect on plant productivity during their establishment and early development stage. the effect depends on the amount of n available to the plant, on the nutritional status and on the age of the plant. the interaction between arbuscular mycorrhizal fungi and bacteria could positively enhance mycorrhizal development and function and plant growth (hameeda et al. 2007). other researcher found a negative effect by reduced spore germination and hyphae length, decreased root colonization, and a decline in the metabolic activity of the internal mycelium (gryndler et al. 1996) or no effect to mycorrhizal development (edwards et al. 1998). in this research three bacterial isolates i.e. bacillus sp. bgi1, b. megaterium bgi2, and bacillus sp. bgi3 positively enhance mycorrhizal root colonization and one isolate bacillus sp. bgi4 showed a neutral effect. it was reported that bacillus subtilis isolated from oil palm mycorrhizosphere can be used as biocontrol agent of ganoderma (bakhtiar et al. 2010). furthermore, it is necessary to investigate the possible combination of amf and bacillus sp. bgi3 most related to b. subtilis towards against ganoderma fungi. based on the findings of this study, it can be concluded that bacterial isolates bacillus sp. bgi1, b. megaterium bgi2 and bacillus sp. bgi3 isolated from surface sterilized amf spores g. margarita are potential for enhancing amf root colonization on neem seedling. all bacterial isolates enhanced neem seedling growth, but when combinations of amf and bacterial isolates were applied, it tended to decrease early growth of neem seedlings in the nursery. acknowledgments the authors wish to thank to prijanto pamungkas, head of silviculture laboratory who gave permission using facilities and chemical reagents for this reasearch. thank also goes to josepha for her technical assistant during experiment. references artursson v, finlay rd, jansson jk. 2006. interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth. environ m i c r o b i o l . 8 ( 1 ) : 1 1 0 . d o i : 1 0 . 1111 / j . 1 4 6 2 2920.2005.00942.x. bakhtiar y, yahya s, sumaryono w, sinaga ms, budi sw, and tajudin t. 2010. isolation and identification of mycorrhizosphere bacteria and their antagonistic effects towards ganoderma boniense in vitro. microbiol indones. 4 (2) : 96-102. b a r e a j m , a z c o n r , a z c o n a g u i l a r c . 2 0 0 2 . mycorrhizosphere interactions to improve plant fitness and soil quality. antonie van leeuwenhoek. 81: 343351.doi: 10.1023/a:1020588701325. bianciotto v, lumini e, bonfante p, vandamme p. 2003. 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av, delima pc. 2010. mycorrhizal association, n fertilization and biocide application on the efficacy of bio-n on corn (zea mays l.) growth and productivity. e-int j sci res. 2 (3): 267-290. selvaraj m, mosses m. 2011. efficacy of melia azedarach on the larvae of three mosquito species anopheles stephensi, culex quinquefasciatus, and aedes aegypti (diptera: culicidae). eur mosquito bull. 29: 116-121. secilia j, bagyaraj dj. 1987. bacteria and actinomycetes associated with pot cultures of vesiculararbuscular mycorrhizas. can j microbiol. 33(12): 1069-1073. doi: 10.1139/m87-187. shtark oy, borisov ay, zhukov va, provorov na, tikhonovich ia. 2010. intimate associations of beneficial soil microbes with host plants. in: dixon gr and tilston el, editors. soil microbiology and sustainable crop production. the netherlands: springer science+business media b.v. p 119-196. doi: 10.1007/978-90-481-9479-7_5. smith se, smith fa, jakobsen i. 2003. mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. plant physiol. 133(1): 16-20. theather rm, wood jp. 1982. use of congo red polysacharide interaction in enumeration and characterization of cellulolytic bacteria from the bovine rumen. appl environ microbiol. 43(4) : 777-780. yulianti, siregar iz, wijayanto n, darma igk t. 2011. genetic variation of melia azedarach in community forests of west java assessed by rapd. biodiversitas 12(2): 64-69. 188 budi et al. microbiol indones ernin (abundance).cdr indonesia has marginal land, such as dryland, swamp, swampy, tides and peat. however, they are not utilized optimally (renstra the ministry of agriculture 2009). based on wide area, ecological composition and abundance of culturable bacteria of four soil samples (ktr50ii, d50ii, g50ii, and a50ii) were analyzed. the soil samples were collected from maize rhizosphere that planted in dryland lombok island. each soil sample give different growth performance of maize in greenhouse experiment. this study was to investigate the relation of maize growth performance with culturable bacterial community of their rhizosphere and the effect of culture media on number of bacterial isolates recovery. the rhizosphere bacteria were cultured and isolated on commercial media (sea) and non commercial modification media (na n, na n-se and na nre). rhizosphere bacteria were obtained from four maize rhizophere soil samples. d50ii is the soil sample that caused the better growth performance to the maize, contrary to ktr50ii. d50ii has significantly highest number of culturable bacterial types, while significantly lowest on ktr50ii. in d50ii, at least 17 bacterial isolates contributed to better growth performance in maize and have relative abundance of dominant isolate not more than 35 34%. in comparing the rhizosphere bacteria recovered using different culture media, bacteria cultivated on sea have different growth characteristic compared with bacteria cultivated on na n, na n se and na n-re. six bacterial isolates showed antagonistic ability when grew on sea but not in all of three media. compared with commercial media, non commercial modification media can increase total isolates recovery about 70 6% culture media, dryland, maize, rhizosphere bacteria telah dilakukan kajian komposisi dan kelimpahan bakteri terkultur dari empat sampel tanah (ktr50ii, d50ii, g50ii, dan a50ii). sampel tanah dikoleksi dari rizosfer tanaman jagung yang tumbuh di lahan kering pulau lombok. pada penelitian skala rumah kaca diketahui bahwa setiap sampel tanah memberikan performa pertumbuhan berbeda pada tanaman jagung. penelitian ini bertujuan untuk mengetahui hubungan antara performa pertumbuhan tanaman jagung dengan komunitas bakteri terkultur penghuni daerah perakarannya serta pengaruh media kultur terhadap perolehan isolat bakteri. bakteri rizosfer di kultur dan diisolasi menggunakan media komersil (sea) dan media modifikasi non komersil (naln, naln-se and naln-re). sebanyak 34 isolat bakteri diperoleh dari keempat sampel tanah. d50ii diketahui memberikan performa pertumbuhan yang lebih baik pada tanaman jagung, sebaliknya ktr50ii memberikan performa pertumbuhan yang buruk. d50ii mempunyai jumlah jenis bakteri terkultur paling banyak dan sebaliknya pada ktr50ii. pada d50ii, sedikitnya 17 isolat bakteri terlibat dalam menghasilkan pertumbuhan yang lebih baik pada tanaman jagung, serta dengan jumlah kelimpahan relatif isolat dominan tidak lebih dari 35,34%. bila membandingkan bakteri rizosfer yang diperoleh dengan media kultur berbeda, terlihat bahwa bakteri yang terkultivasi pada sea mempunyai karakter pertumbuhan yang berbeda dibandingkan bakteri yang terkultivasi pada naln, naln-se and naln-re. enam isolat bakteri menunjukkan kemampuan antagonis saat tumbuh pada sea, namun kemampuan tersebut tidak muncul saat tumbuh pada tiga media lainnya. dengan media modifikasi non komersil dapat meningkatkan perolehan isolat sekitar 70,6% dibandingkan hanya menggunakan media komersial bakteri rizosfer, lahan kering, media kultur, tanaman jagung l l l thirty four strains . l l l . . key words: . kata kunci: abundance of culturable bacteria isolated from maize rhizosphere soil using four different culture media ernin hidayati , aris tri wahyudi *, antonius suwanto , rahayu widyastuti 1,3 1 1 2 and 1 2 3 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680, indonesia; department of soil science and land resources, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia; department of biology, faculty of mathematics and natural science, universitas mataram, west lombok 83125. indonesia potency, social and economic potency, dryland is appropriated to optimize. west nusa tenggara (ntb) is one of the provinces in indonesia with a relatively wide dryland. from the total area (about 1 673 476.307 ha in lombok and sumbawa islands), only about 626 034.60 ha that can be developed (bappeda ntb . . . issn 1978-3477, eissn 2087-8587 vol 8, no 1, maret 2014, p 33-40 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.8.1.5 *corresponding author; phone/fax: , email:+62-251-8622833 aristri2011@gmail.com 2003). now, the dryland is being actively used primarily for maize plantation soil microbes have a significant role in the soil ecosystem. so that they can be used as a selective indicator of soil and also as the main index to evaluate the soil quality and global diversity (zhang 2013). therefore, soil microbe are feasible for fundamental components that could be considered as one of the management strategies for sustainable agriculture in dryland. soil microbial communities can be influenced by many factors. several studies showed that rhizosphere bacterial communities of maize influenced by complex interaction, such as soil type (castellanos . 2009), plant growth stage (di cello . 1997), cultivar (dohrmann . 2013) and genotype (schmalenberger and tebbe 2003). although growing on the same soil type, same species and age of plant, sometimes individually of maize has a different growth performance. this case has not been ever studied yet, especially in dryland lombok island. according to li . (2014), rhizosphere bacteria have significant contribution to crop health, productivity and carbon sequestration. thus it is predicted that the difference of plant growth performances have relation to rhizosphere bacterial community of that plant. rhizosphere bacterial communities have been analyzed using cultivation technique and metagenome. although many molecular techniques used to study many aspects of environmenal microbe, but this technique has disadvantage, such as inavailability of culture isolates. as a result, it is impossible to study some aspects related to the life and function of the isolates. cultivation technique have significantly contributed to our understanding of living microbes (pham and kim 2012). until now, cultivation technique is fundamental tool to provide access to diverse characteristics and physiology of microbe (reid and buckley 2011). culture isolates obtained can be stored for further analysis and application. cultivation technique is also not expensive relatively. this method can be applied by many laboratories, including laboratories in developing countries. because of this technique has been done since long time ago, so that the results of this technique has been well documented that could be useful for comparison of metagenome. the study of microbial communities based on cultivation techniques highly depend on culture media. the problem is that soil as complex media for microbial growth in which they are interacting each other. many progress have been obtained from . et al. et al et al et al et al cultivation-based methods, especially related to modification of nutrient culture media, such as by reducing nutrient component and concentration of culture media (aagot 2001; connon and giovanni 2002; schoenborn . 2004), adding growth stimulant (nichols 2008; d'onofrio 2010), soil extract amended (hamaki 2005; o'neill 2009; george 2011) plant juice supplemented (stamer 1970; nour 2010) and adding exudates of artificial root and root exudates (baudoin 2003; kozdroj; louvel 2011 and van elsas 2000). to date, no single media that can bring satisfaction for microbial cultivation. because of the facts that lombok island dryland will be developed for plantation of agricultural crops, especially maize, it is important to explore many aspects of culturable bacterial community of the dryland related to maize plantation. to cover greater bacterial isolate, this study improved modification of culture media by supplementing soil extract and root extract of maize on low nutrient concentration media. the goal of this study was to explain the contribution of dryland culturable bacterial community to give good growth performance of maize. accordingly, this investigation examines the relation of maize growth performance with culturable bacterial community of their rhizosphere and the effect of culture media on number of bacterial isolates recovery four types of rhizosphere soil samples used in this study, namely ktr50ii, d50ii, g50ii, and a50ii. the soil samples were collected from rhizosphere of two months age of maize ( var bisi 2) that grew in dryland field located at lombok island, west nusa tenggara, indonesia (s 08º13'42.4'', e 116º21'24.4''). study of soil samples continued in greenhouse experiment on the growth of maize for 30 d. as a result, each soil sample application showed different vegetative growth performance of maize in greenhouse. d50ii gave better growth performance of maize, a50ii and ktr50ii gave worse growth performance, and g50ii gave medium growth performance. maize root mass were taken from the field and placed in plastic bags labeled. the rhizosphere soil samples were collected as described by phillips and fahey (2006). all soil samples were placed into sterile plastic bags labeled and stored in the refrigerator at 6-7 c. et al. et al et al. et al. et al. et al. et al. et al. et al. et al. et al. . zea mays . materials and methods soil sampling 0 34 hidayati et al. microbiol indones preparation of culture media cultivation and growth ability of rhizosphere soil bacteria . rhizosphere bacteria were cultivated using commercial culture media (soil extract agar) and non commercial modification culture media (naln, naln-se and naln-re). soil extract agar (sea, himedia, mumbai, india) consisted of 1 g l glucose, dipotassium phosphate 0.50 g l , 17.75 g l soil extract, and 15 g l agar. nutrient agar low nutrient (naln) consisted of 1% (0.08 g l ) nutrient broth (criterion, santa maria, ca) and 18 g l agar powder. nutrient agar low nutrient supplemented with soil extract (naln-se) consisted of naln and 50% soil extract. nutrient agar low nutrient supplemented with root extract of maize (naln-re) consisted of naln and 25% soil extract. on each medium, of antifungal nystatin was added. firstly, soil extract and root extract prepared. soil extract was taken from soil in lombok island dryland. one part of soil sample was mixed with two parts of sterile water ( ). soil slurry was sterilized for 1 h (o'neill 2009) and then allowed 24 h at room temperature. the supernatant was filtered with thick layer sterile cotton. the liquid of soil extract was stored at 6-7 c. the maize root extract was prepared from maize root mass ( var bisi 2). one part of the root mixed with two parts of sterile water ( ). root slurry was sterilized for 20 min and then allowed 24 h at room temperature. the supernatant was filtered with thick layer sterile cotton. the liquid of soil extract was stored at 6-7 c. ten grams of soil samples were suspended in 90 ml of sterile saline (0.85% nacl). the soil suspension was shaken on a rotary shaker for 15 min. from dilutions 10 , 10 , 10 , 10 , and 10 at 28 c for 7 days. during incubation, total colony forming unit (cfu), number of bacterial isolates, and cfu of each isolate were determined. cultural morphology, colony texture, colony pigmentation, and cell characteristics were recorded. the colonies were also streaked on each medium to verify their characteristic to avoid double identification. interesting isolates were identificated based on size of terminal restriction fragment (trfs) of 16s rdna were digested with the mspi restriction enzyme. purified bacterial isolates were stored in 20% glycerol at -20 c for use throughout the study. -1 -1 -1 -1 -1 -1 -1 0 0 -3 -4 -5 -6 -7 0 0 50 μg ml , 100 μl aliquots of suspensions were spreaded on the surface of all of four culture media and incubated w/v et al. zea mays w/v . antagonistic assay statistical analysis. bundance of bacteria isolated from maize rhizosphere soil . some previous studies suggested that bacteria in mix culture could inhibit the growth of other bacteria by producing antibacterial substance. in this study, the bacterial isolates were also examined for their antagonistic ability to inhibit other isolates. the suspected of antagonist isolates were streaked on the edge of agar plate of all culture media and incubated at 28 c. three days after incubation, tested isolates were streaked horizontally 5 mm in opposite to antagonist and further prolonged incubating for 2 weeks. inhibition activity of antagonists were determined as the positive ability when the colony of testing isolates was retarded. each treatment was made in 3 replicate. all result enumerations of isolates and colony were analyzed using annova 5% and then followed by using turkey method of minitab 16. . thirty-four bacteria isolates were cultivated from four maize rhizosphere soil (ktr50ii, d50ii, g50ii, and a50ii ) using four culture media (sea, naln, naln-se, and naln-re). the number and isolate types in each rhizosphere soil sample are showed in fig 1 a. number of isolate types were significantly higher in d50ii and significantly lowest in ktr50ii. at least 17 of the 34 isolates (50%) were found in d50ii. a further 12 of 34 isolates (35.29%) were found in a50ii, nine of the 34 isolates (26.47%) were found in g50ii, and five of the 34 isolates (14.70%) were found in ktr50ii. based on bacterial isolates distribution of each soil sample, 18 of 34 isolates (52.9%) were found in certain soil samples and the other found in more than one soil samples. based on colony forming unit (cfu), the number of cfu was significantly higher in g50ii (13.05x10 ) and then decreased from d50ii (5.07x10 ), a50ii (1.87x10 ), and ktr50ii (1.25x10 ) (fig 1 b). comparison of isolate abundance showed that each rhizosphere soil sample has different relative abundance pattern. d50ii has the most abundance isolates and then decreasing from a50ii, g50ii, and ktr50ii (fig 2). each soil sample was inhabited by different dominat isolate type. for examples, cdl 30 is the most abundant isolate found in ktr50ii, cdl 38 found abundance in d50ii, cdl 6 found abundance in g50ii, and cdl 33 found abundance in a50ii. relative 0 7 7 7 7 results a volume 7, 2013 microbiol indones 35 abundance of dominant isolate found in d50ii showed not more than 35.34%, while in the other soil samples more than 37.60%. cdl 6 was detected as a predominant isolate was found in all soil samples. cdl 6 is known to having 123 trf size digested with the mspi restriction enzyme. . fourteen of the 34 isolates were cultivated on naln-re, 12 of 34 isolates were ability of each culture media to cultivate the rhizosphere bacteria cultivated on naln, 11 of the 34 isolates were cultivated on naln-se, and 10 of the 34 isolates were cultivated on sea (fig 3). at least 5 isolate types shared cultivated on naln, nal-se and naln-re, 6 isolate types shared on naln-se, and naln-re, while 7 isolate types shared on naln, naln-se and nalnre. at least 29.4% of bacterial isolates were cultivated on sea only while the remaining (70.6%) were cultivated on naln, naln-se, and naln-re. a b fig 1 comparison of number and isolate type (a) and colony forming unit (b) of maize rhizosphere bacteria cultivated from maize rhizosphere soil samples cultivated using four culture media (sea, naln, naln-se, and naln-re). fig 2 estimation of relative abundance of maize rizosphere bacteria from lombok island dryland related to different growth performance of maize made by cultivation technique using four different culture media. 36 hidayati et al. microbiol indones growth differences of bacterial isolates on each culture medium. nearly all bacterial colonies developed on sea have different morphological appearances compared with naln, naln-se and naln-re (fig 4). the bacterial colonies were cultivated on sea developed during the first day of incubation. the characteristic of these colonies were as follows: fast growing, colony diameter average of 2.0 2.5 mm with clear differences among each colony morphology (fig 4 a). when the dilution higher than 10 , only dominated with growing of certain isolates. 5 the growth of these isolates usually followed by appearance the yellowish color surrounding the colonies. bacterial colonies were cultivated on naln, naln-re, and naln-se developed after 3 days of incubation. the characteristic of the colonies were as follows: slow growing, colony diameter average of 0.5-2.0 mm, and many of these are very thin and transparent (fig 4 b, c, d). cultivated bacteria from dilution higher than 10 not showed phenomena such as on sea. physiological differences were also found only in six isolates such as cdl 12, cdl 19 , cdl 25, 5 fig 3 comparison of number and isolate type isolated using four culture media (a) and number of isolate shared among culture media (b). 5 5 6 7 10 7 9 6 10 14 11 12 sea naln naln-re naln-se a b sea naln naln-se naln-re c d a b fig 4 appearance of colony development of maize rhizosphere bacteria on sea (a), naln (b), naln-se (c), and naln-re (d) at 10 dilution after 6 days of incubation. -3 volume 7, 2013 microbiol indones 37 cdl 26 , cdl 30, and cdl 32, where those isolates produced yellowish color around the colonies when grew on sea. this evidence did not happen when they grew on naln, naln-se, and naln-re. . antagonistic assay was done to six isolates (cdl 12, cdl 19, cdl 25, cdl 26, cdl 30, and cdl 32) that produced yellowish color when grew on sea. this assay showed that ability to produce yellowish color is related to antagonistic ability of the isolates in inhibiting the growth of other isolates (fig 5). the antagonistic ability only showed when the antagonist grew on sea (fig 5 a, b, c) and not on naln, nalnse and naln-re (fig 5 d, e, f). the antagonist inhibited the growth of certain isolates but not in other isolates. the cdl 30 and cdl 32 were detected as the higher relative abundance in ktr50ii. cdl 30 known to having 486 trf size and showed 98% score identity similar with (nr_114751), while cdl 32 known to having 485 trf size and showed 97% score identity similar with (nr_037000). in the other soil samples, relative abundance of the antagonist less than 15%. his study is the first recorded of the maize rhizosphere bacterial states in lombok island dryland. antagonistic ability in some bacterial isolates pseudomonas stutzeri pseudomonas pseudoalcaligenes discussion t fig 5 bacterial antagonist (in vertical streak) isolated from maize rhizosphere soil bacteria. antagonistic ability developed when grew on sea (a, b, c) but not on naln (d), naln-se (e), and naln-re (f). the results of this experiment showed that based on total number isolates and colony forming unit (cfu), d50ii has higher number of bacterial isolates compared with other soil samples. despite d50ii has lower number of cfu compared with g50ii, the number of cfu about 5.07x10 is more likely produced better growth. as for the g50ii, although that soil sample has higher of cfu compared with d50ii, less number of isolate likely it was not enough to produce better growth as d50ii. despite of a50ii has higher number of isolates but less number of cfu may caused poor growth compared with g50ii. in ktr50ii, lowest number of isolates and cfu may caused the worse growth. this result explained that the number of bacterial isolates more important to maize growth than total cfu. in addition, each kind of bacteria should be present in a sufficient number to be able to affect the growth of maize. the results indicated that the maize growth performance influenced by number and isolate type on the soil samples. although the number of isolate types in a50ii significantly higher than g50ii, but the community seemed to be dominated by only one type of isolate is cdl33, which the relative abundance about 81.99% (fig 2). in addition to number of isolate type and cfu, the relative abundance pattern that composed each soil type may contribute to differentiation of the growth performance of maize. it predicted that more abundant baceria with balance composition should give better effect to maize growth. 7 38 hidayati et al. microbiol indones more previous study showed many aspects that known to influence the microbial community (di cello 1997; schmalenberger and tebbe 2003; dohrmann 2013). in this study, when the influence of the abiotic factors and plant are reduced, it appeared that the rhizosphere bacterial community contributed to influence the growth of maize. it is assumed that abundance of the origin microbial status in dryland is the initial factor that influence the growth of maize. the results also showed that 52.9% of bacterial isolates were found in certain soil sample and the other found on more than one soil samples. the similarity of isolate type was found in the soil samples may it is because of the soil samples taken from the same soil type from the same location. while the variation of isolate type was found in the soil samples may it is because of each soil sample has different origin microbial diversity since they are in the land. furthermore, the differences of diversity which contributed to the differences of maize growth performance. by doing variation in composition of media such as low concentration of nutrients (naln), low concentration of nutrients supplemented by soil extract (naln-se) and supplemented by maize root extract (naln-re) increased the number of culturable bacteria obtained from the soil sample compared with commercial media only. it was suggested that the different nutrient composition is likely affected the number and type of bacterial cultivated on each media. in this experiment, rich nutrient composition of the sea may provide conditions that allow for certain groups of bacteria to grow rapidly and dominate each other, while the slow growing bacteria will eliminated with no space to grow. in contrast to the naln, nalnse and naln-re, poor nutrient composition provide conditions that allow the slow growing and dormant bacteria to grow. the similar results also found by other researcher (brozel and cloete 1992; joseph 2003). the soil extract and root extract were supplemented to naln-se and naln-re respectively, may also contribute as a selection factor of the types of rhizosphere soil bacteria. according to hamaki (2005), soil bacteria and actinomycetes can grow on soil extract agar media but not on conventional media. some experiments also showed that medium of soil extract amended was suitable for growth of fastidious soil bacteria (davis . 2005) and also reduce et al. et al. et al. et al. et al diversity and dominance of fast growing bacteria (kozdroj and van elsas 2000). according to tamaki (2005), bacteria in mix culture can produced antibacterial substances to inhibit the growth of other bacteria. dominance also contributed to the ability of organisms to produce extracellular antibacterial component (rao 2005). refer to tamaki (2005) and rao (2005), it is likely that the antagonistic ability of six isolates (cdl 12, cdl 19, cdl 25, cdl 26, cdl 30, and cdl 32) may caused by antibacterial substances that produced by the isolates. the antagonistic ability of these rhizosphere soil bacteria not only when they are in the form of mixed cultures but also explained the relationship between type of culture media and 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american academy of microbiology. washington, dc 20036. p 1-28. [renstra] 2009. strategic plan of the ministry of agriculture 2010-2014. schmalenberger a, tebbe cc. 2003. bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on pcr-amplified partial small subunit rrna genes in ecological studies. mol ecol. 12(1): 51-262. doi: 10.1046/j.1365-294x.2003. 01716.x. zhang x, ma l, gilliam fs, wanga q, li c. 2013. effects of raised-bed planting for enhanced summer maize yield on rhizosphere soil microbial functional groups and enzyme activity in henan province, china. field crops res. 130: 28-37. doi:10.1016/ j.fcr.2012.02.008 pseudoalteromonas tunicate. . 40 hidayati et al. microbiol indones 1.mi709-dyah noor available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.3.1issn 1978-3477, eissn 2087-8575 vol 7, no 3, september 2013, p 85-93 *corresponding author; phone/fax: 021-7563120/0217560208, email: bougenviella@yahoo.com escherichia coli is a frequent cause of lifethreatening bloodstream infections and other common infections, such as urinary tract infections. antibioticresistant e. coli are readily acquired via the diet (food and water), and other factors such as continuous drug usage, the in appropriate dose usage, and the natural factors of that microbe itself. the resistance results in increased mortality, morbidity, and health care cost. in indonesia resistant e. coli have emerged, whereas the rates of resistance to some antibiotics were significantly higher, especially to beta-lactam classes (lestari et al. 2008). beta-lactams remain the most widely utilized antibiotics owing to their comparatively high effectiveness, low cost, and minimal side effects. among the various classes of antibiotics, penicillin, and cephalosporins are the most frequently used agents in treatment of bacterial infection. the most common mechanism of resistance to this class of antibiotics is the ability of bacteria to produce beta-lactamases, enzymes that degrade or modify the antibiotic before it can reach the appropriate target site. these enzymes are very important for gram-negative bacteria, including e. coli, as they constitute a major defense mechanism against the emerge of antibiotic resistance has been an important issue all over the world, on the other hand, infectious diseases have been one of the highest causes of death causes in the world. therefore, the discovery of a new antimicrobial drug is very important, and the group of rare actinomycetes are really promising as the producer of new bioactive compounds, in this case antibiotics. in this study we screened and characterized the actinomycetes with antibacterial activity against escherichia coli atcc 35218 resistant beta-lactam antibiotics. a total of 96 strains collected in biotechnology microbial culture collection (biomcc), bppt, were screened for their antibacterial activities by the agar plug method. three strains, at-hh-64, at-hh-78, and at-hh-259, showed antibacterial activity. the selected strains were cultured on four different media, both solid and liquid media, e.g. isp2, isp4, micromonospora starch medium (ms), and bennet’s medium (bm), and we characterized their morphology and growth patterns. morphological characterization showed that all strains belonged to the genera micromonospora. the active strains were also identified based on 16s rrna partial sequence. blast search of the 16s rrna sequences of all tested strains with the sequences available in the ncbi data bank showed a maximum similarity 99% with micromonospora chersina. key words : antibacterial activity, characterization, micromonospora, screening munculnya resistensi antibiotik telah menjadi isu penting di seluruh dunia, di sisi lain, penyakit menular telah menjadi salah satu penyebab kematian tertinggi di dunia. oleh karena itu, penemuan obat antimikroba baru adalah sangat penting, dan kelompok aktinomiset langka benar-benar menjanjikan sebagai penghasil senyawa bioaktif baru, dalam hal ini antibiotik. dalam penelitian ini kami melakukan penapisan dan karakterisasi aktinomiset yang memiliki aktivitas antibakteri terhadap e. coli atcc 35218 resisten antibiotik beta-laktam. sebanyak 96 galur aktinomiset yang merupakan koleksi dari biotechnology microbial culture collection (biomcc), bppt, dilakukan penapisan aktivitas antibakteri terhadap e. coli atcc 35218 dengan metode agar plug. tiga galur menunjukkan aktivitas antibakteri, yaitu galur hh-64, hh-78, dan hh-259. galur dengan aktivitas antibakteri selanjutnya ditumbuhkan di dalam empat jenis media, baik padat maupun cair, yaitu isp2, isp4, micromonospora starch medium (ms), dan bennet's medium (bm), dan dilakukan karakterisasi morfologi dan pola pertumbuhannya. karakterisasi morfologi menunjukkan bahwa seluruh strain termasuk ke dalam kelompok genus micromonospora. pada galur aktif juga dilakukan identifikasi berdasarkan sekuen 16s rrna. hasil blast sekuen 16s rrna dibandingkan dengan data yang terdapat pada ncbi menunjukkan bahwa ketiga strain memiliki kedekatan tertinggi (99%) dengan micromonospora chersina. kata kunci: aktivitas antibakteri, karakterisasi, micromonospora, penapisan characterization of micromonospora spp. with activity against beta-lactam antibiotic-resistant escherichia coli atcc 35218 1,3 1,2 4 dyah noor hidayati *, yulin lestari , and bambang marwoto 1 department of biology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 2 biopharmaca research center, institut pertanian bogor, taman kencana campus, bogor 16151, indonesia; 3 center for biotechnology bppt, kawasan puspiptek-serpong, tangerang selatan 15314, indonesia; 4 center for pharmaceutical and medical technology bppt, kawasan puspiptek-serpong, tangerang selatan 15314, indonesia beta-lactam based drugs (wilke et al. 2005). on the other hand the antibiotic resistant has been an important issue all over the world, on the other side, the discovery of new antibiotics is slower than the rate of resistance. consequently, it is important to focus on screening programs of microorganisms, primarily of rare actinomycetes, for their production of antibiotics (donadio et al. 2002). actinomycetes are well known as producers of bioactive secondary metabolites which include antibiotics, anticancer agents, enzymes, and biocontrol agents. according to bérdy (2005), from 16 500 antimicrobial compounds isolated from microbes, 52.7% (8700 antimicrobial) were from actinomycetes, while the remaining 29.7 and 17.6% respectively were isolated from fungi and bacteria. of 8 700 antimicrobial compounds isolated from actinomycetes, 6 550 have been isolated from the genus streptomyces and the rest, 2 250 from rare actinomycetes. the discovery of a new antimicrobial compounds from rare actinomycetes have been reported by several researchers over the world (hopmann et al. 2002; beltrametti et al. 2006). micromonospora, which are a group of rare actinomycetes are widely distributed in nature, inhabiting variated environments such as coastal sediment (zhao et al. 2004), soil rhizosphere (qiu et al. 2008), peat swamp forest (thawai et al. 2005), and from root nodules of the leguminous plants (trujillo et al. 2007). micromonospora species are best known for synthesizing antibiotics, especially aminoglycosides including gentamicin and netamicin, oligosaccharide antibiotics, and enediyne (bérdy 2005). in this study, we utilized the microbes collected in the biotechnology microbial culture collection (biomcc), bppt, specifically actinomycetes, as the source for screening of antibacterial compounds against e.coli atcc 35218 resistant beta-lactam antibiotics, and we characterized the active strains. materials and methods actinomycetes isolates. the strains were collection of microbial biotechnology culture from the (biomcc)-bppt, isolated from soil using highheating method, cultured on isp2 agar medium, and maintained as frozen cell in 20% glycerol solution. screening of antibacterial activity. antibacterial activity was assayed by the agar plug method on mueller hinton agar (mha). the ninety-six actinomycete isolates were subcultured on yeast-extract maltextract agar medium (isp2) for 14-21 d, and the agar . . . 86 hidayati et al. microbiol indones containing the cells was cut off (about 7 mm in diameter) and placed on the assay plates inoculated 6 -1 with 10 cfu ml tested bacteria. after 24 h incubation at 36 ± 1 °c, the antibacterial activities were determined by clearing zone around the tested isolates (sharma et al. 2011). antibacterial activity patterns. the antibacterial activity pattern of micromonospora spp. was conducted using the agar plug method (as described above). the active isolates were cultured on isp2 agar medium and incubated at 28 °c for 15 d. every 3 d of interval incubation, the plates were picked up and assayed their antibacterial activities againts test bacteria. for the antibacterial activities were expressed as the diameter of the growth inhibition zone (mm) and measured using calipers (mitutoyo, japan). morphological and growth pattern characterization of active actinomycetes. the potent actinomycetes isolates were selected and characterized by morphological characteristics in four different agar media (yeast-extract malt-extract (isp2), inorganic salt-starch (isp4), micromonospora starch medium (ms), and bennett’s medium (bm)). the morphological methods consist of macroscopic and microscopic characterization. macroscopically, the actinomycete isolates were grown in four different media, and characterized their growth and colony color after 21 d of incubation. for microscopy, the plates from different media were observed for their mycelial structure, spores, and sporophore development directly under a microscope (olympus bx51, 1000× magnifications). the observed morphology of the isolates was compared with the actinomycetes morphology provided in bergey’s manual for the presumptive identification of the isolates (kawamoto 1989). for growth patterns characterization, a pure culture of the actinomycetes isolates was inoculated individually into the seed medium (isp2 broth). after 72 h of incubation, the seed culture at a rate of 10 % (v/v) was transferred into the four different fermentation media, using the same media as for morphological characterization (described before). the fermentation was carried out at 28 °c for 15 d under agitation at 220 rpm (performed using rotary shaker incubator, takasaki scientific instruments corp). after every 3 d of incubation, the flasks were harvested, and the amount of packed mycelial cells (pmv) was measured. culture broth was taken (10 ml) and centrifuged at 3 000 x g for 5 min (performed using kubota 7780) to separate cells from fine particles of the medium. the separated mycelia were resuspended in a saline solution (0.85 % sodium chloride) to give the original volumes using 15 ml centrifuge tubes. this step were replicated three times, then the mycelia was allowed to sediment for 20 min. the pmv was measured and expressed as % of cells to total volume (10 ml) (rho et al. 1991). molecular identification of active strains based on 16s rrna sequence. the dna from active actinomycetes was isolated using instagene matrix (biorad). the pure colony of the isolate was collected from fresh culture on isp2 agar medium, transfered to the microtubes consisting of 10 µl ddh o, added with 2 90 µl instagene matrix, and heated at 100 °c for 15 min. the mixed solution was centrifugated at 15 000 x g (tomy mx-301) for 3 min. the dna amplification was performed via polymerase chain reaction (pcr) by pcr thermal cycler (takara, japan). the dna template (8 µl) was added to the amplification mixture containing 1.6 µl ddh o, 1 µl dmso, 0.2 µl primer 2 178 f, 0.2 µl primer 179 r, and 9 µl master mix. after the initial denaturation for 2 min at 96 °c, 35 cycles of amplification were performed, 20 s at 90 °c denaturation, 20 s at 60 °c annealing and 1 min 72 °c elongation. these cycles were followed by a final 5 min elongation cycle at 72 °c. the pcr product purification was done using gel/pcr dna fragments extraction kit (df buffer, washing buffer, elution buffer, df column) and amplified by pcr using the mixture reaction from go taq green master mix (promega). the pcr product was directly sequenced using abi 3130 genetic analyzer, and 16s rrna sequences data from each isolate was compared to the database available at genbank by using the blast software (blastn) on the national center biotechnology information site (ncbi) (http://www.ncbi.nlm.nih.gov/). the 16s rrna sequences of the active strain were aligned using the clustal w2 program. phylogenetic and molecular evolutionary analyses were conducted using mega software version 5. the evolutionary distances were computed using the maximum composite likelihood method and were in units of the number of base substitutions per site (tamura et al. 2011). results antibacterial activity of micromonospora spp. from the ninety-six actinomycetes strains which were assayed, three strains showed inhibition activity against e.coli atcc 35218, they were strains hh-64, hh-78, and hh-259, with the inhibition diameters of 13.55, 13.88, and 14.71 mm, respectively (fig 1). characterization of antibacterial activity, based on the age of culture (fig 2) showed that the highest activities were at 9 d of culture. antibacterial activities started on the third day of culture, except for the strain hh-259, and continued to increase until the 9 d of culture. the antibacterial activities began to decline at 12 d of culture. overall, up to 15 d of culture, all of the strains still showed their antibacterial activities. morphological and growth patterns characteristic of micromonospora spp. micromonospora spp. showed varied growth patterns in different liquid media, indicated by the yielded biomass (% pmv). the strain hh-64 showed the best growth in isp4 broth, followed by the growth in the isp2, ms, and bm media. this pattern also occured for strain hh-259. the best growth medium for strain hh-78 was isp4, followed by isp2, bm, and ms (fig 3). macroscopically, morphological colony observations of three strains in the four different growth media varied. strain hh-64 grew abundantly in all media and sporulated more slowly on bennet’s medium. strain hh-78 grew abundantly and sporulated well in all media. strain hh259 grew abundantly on isp2 medium and moderately on three other media, and it sporulated slowly on ms and bm media. overall, the color of vegetative mycelium of the strains was colorless to light orangevolume 7, 2013 microbiol indones 87 fig 1 the antibacterial activities of micromonospora spp. against e. coli atcc 35218 (shown by clear zones around the tested strains). with 16s rrna gene sequences in the genbank database. the homology search by the blast program showed that all strains showed similarities to micromonospora chersina, with 16s rrna genesequence-similarity values of 99%. the results indicated that the closest related species for strain hh yellow, then turned to greenish-black or brownish-black after sporulation (fig 4). microscopic observation showed that all active strains belong to the genus micromonospora (fig 5). molecular identification of micromonospora spp. the partial sequences of each strain was compared microbiol indones88 hidayati et al. d ia m et er o f in h ib it io n ( m m ) 20 18 16 14 12 10 8 6 4 2 0 0 3 6 9 12 15 culture time (d) fig 2 antibacterial activity patterns of micromonospora spp. from isp2 agar culture performed using agar plug method. : hh-64; : hh-78; : hh-259. 12 10 8 6 4 2 0 0 3 6 9 12 15 culture time (d) 10 8 6 4 2 0 0 3 6 9 12 15 culture time (d) b c t h e am o u n t o f p ac k ed m y ce ll ia c el ls , p m v ( % ) t h e am o u n t o f p ac k ed m y ce ll ia c el ls , p m v ( % ) 12 10 8 6 4 2 0 0 3 6 9 12 15 culture time (d) a t h e am o u n t o f p ac k ed m y ce ll ia c el ls , p m v ( % ) fig 3 the growth patterns of micromonospora spp. in four various liquid media (isp2, isp4, micromonospora medium (ms), and bennet’s medium (bm). (a) isolate hh-64; (b) isolate hh-259; (c) isolate hh78. : isp2; : isp4; : ms; : bm. hh259 hh78 hh64 discussion from the results we obtained, 3 strains showed antibacterial activities, and the patterns of antibacterial activity based on the age of the cultures showed that until 15 d of culture they still have activities against the target bacteria. antibacterial activities are characterized by the formation of a clear zone around the actinomycetes colony being tested (fig 1), the wider the clear zone formed, the greater the antibacterial activity produced. this indicates that the strains are capable of secreting a volume 7, 2013 microbiol indones 89 64 was with m. chersina r-ac138 (fn649453), for strain hh-78 with m. chersina rtiii8 (eu274367), and for strain hh-259 was with m. chersina r-ac135 (fn649452), respectively. the phylogenetic dendogram was inferred using the neighbor-joining method and the results showed that two strains, hh-64 and hh259, were clustered together and were closely related with other m. chersina. the strain hh-78 was separated from the anothers and it was clustered together with m. olivasterospora, m. viridifaciens, and m. echinaurantiaca (fig 6). fig 4 micromonospora spp. colonies after 21 d incubation on four different agar media (a. isp2; b. isp4; c. micromospora medium; d. bennet’s medium). fig 5 microscopic structures of micromonospora spp. 21 d old on isp2 medium, the spherical spore indicated by the red arrow (1000 x magnification). the bacterial target we used in this study was e. coli atcc 35218, it is a beta-lactam resistant bacteria, a non-pathogenic that produces tem-1 betalactamase. this bacteria is used as a bacterial surrogate for ehec e. coli o157:h7 in microbial challenge studies (gurtler et al. 2010). in gram-negative substance that inhibits the growth of test bacteria, in this case e. coli atcc 35218. the bioactive compounds could be a beta-lactamase inhibitor or antibacterial compounds that cannot be hydrolyzed or are hydrolysed poorly by beta-lactamase activity produced by the target bacteria (babic et al. 2006). microbiol indones90 hidayati et al. 0.02 fig 6 the phylogenetic dendogram based on 16s rrna gene sequences of micromonospora spp. and other genera (root of bootstrap). the strains are shown in bold symbols. percentage bootstrap values based on 500 resampled data sets are shown at the nodes; bootstrap value lower than 40% were cut off. varied among the strains, which may due to their ability to assimilate the medium components variably. microbial growth and antibiotic production by actinomycetes and other spore forming strains in batch culture are mainly influenced by two factors. the first is the nature and concentrations of different medium composition such as carbon, nitrogen, phosphate, and metals sources. carbon sources that can readily serve as growth substrates, such as glucose, often repress antibiotic production but are excellent for growth. polysaccharides such as, starch, are often preferable in antibiotic yield. the second factor is cultivation conditions such as temperature, ph, incubation time, oxygen supply, type and concentration of inoculum. in shaking flask cultures, the important factors that influence the gas-mass transfer in the cultivation media are mainly the shape and volume of the cultivation vessel, medium volume, the degree of agitation, and the medium viscosity (el-enshasy et al. 2000; himabindu and jetty 2006). in order to maximize the fermentative production of secondary metabolites, in this case antibacterial compounds, it’s important to characterize the producing organism. characterization of the active strains showed that all strains belonged to the genus micromonospora and they closely related to m. chersina. microscopically, they were characterized by the presence of single spherical spores located terminally on short or long hyphal branches and sometimes developed into clusters,with no aerial mycelium being formed. m. chersina was first proposed by tomita et al. (1992), the vegetative mycelium of this strain is branched monopodially and forms single spores (spherical with short blunt spines) which are sessile or born on shortor long-monopodial sphorophores, and no aerial mycelium is formed. this strain is known as a dynemicin producer, an antitumor antibiotic, with broadspectrum activity. not only did it have activity against tumor cells, it also had activity against bacteria and fungi (konishi et al. 1991). the phylogenetic analysis (fig 6), showed that the strain hh-78 was separated from the two others and its closely related species, m.chersina. instead, it clustered together with m. olivasterospora, m. viridifaciens, and m. echinaurantiaca, this was probably due to their menaquinone concentration and fatty acid composition (tomita et al. 1992; koch et al.1996). besides streptomyces, micromonospora is one of the genera of actinomycetes that is known as world antibiotics producer and this genus has remained poorly studied compared to streptomyces. some new volume 7, 2013 microbiol indones 91 bacteria, beta-lactamases are the most important mechanism of resistance to beta-lactam antibiotics. beta-lactamases are bacterial enzymes that hydrolyse the beta-lactam ring and render the antibiotic inactive before it reaches the penicillin-binding-protein (pbp) target. there are two principal ways to overcome the beta-lactam resistance involving finding inhibitors or inactivators of beta-lactamases and finding a new betalactam antibiotic that demonstrates great affinity for the target pbp which is not hydrolized or hydrolized poorly, by beta-lactamases (babic et al. 2006). currently, there are 3 beta-lactamasei inhibitors used clinically: clavulanic acid, sulbactam, and tazobactam, which are combined with other antibiotics (miller et al.2001). antibacterial agents are one of the secondary metabolites produced by actinomycetes. secondary metabolites are produced as a response to the limited availability of nutrients, and are not essential for the growth of the producing cultures but serve diverse survival functions in nature. in contrast with secondary metabolites, the primary metabolites are associated with growth and maintenance of life, its concern with the release energy and the synthesis of important macromolecules such as sugars, protein, nucleic acids, and organic acids. the organism will be die if primary metabolism were stopped (demain and fang 1995). in actinomycetes, the production of secondary metabolites coincides with, or precedes, sporulation. its occurs when cultured on the agar medium. in the liquid culture, the secondary metabolites generally limited to the stationary phase, and its frequently assumed to the result of nutrient limitation. because both secondary metabolites production and sporulation occurs close to the beginning of the stationary phase, one may suspect that these two process are regulated by an overlapping mechanism. however, in some cases there is no close connection between the spore formation and antibiotic production, particulary, by non-sporulating organisms (bibb 2005; glazer and nikaido 2007). the growth pattern of active strains showed that the patterns varied in different media (fig 4). the best growth is demonstrated by the growth in isp4 medium. the medium has a more complex composition than other media we used, it contains soluble starch, ammonium sulphate, and potassium phosphate, as a source of carbon, nitrogen, and phosphate, respectively. in addition, the media also provides micro-elements as a sources of metals, such as ferrous iron and manganese. the growth patterns in this medium also 2002. targets and assays for discovering novel antibacterial agents. j biotechnol. 99(3):175-185. doi:10.1016/s0168-1656(02)00208-0. el-enshasy h, farid ma, el-sayed sa. 2006. influence of inoculum type and cultivation conditions on natamycin production by streptomyces natalensis. j basic microbiol. 40(5-6):333-342. doi:1521-4028(200012)4 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genetic engineering of micromonospora sp. tpma0041. j ind microbiol biotechnol. 39(11):1693-1701. doi:10.1007/s10295012-1173-2. sharma d, kaur t, chadha bs, manhas rj. 2011. antimicrobial activity of actinomycetes against multidrug resistant staphylococcus aureus, e. coli and various other pathogens. trop j pharm res. 10(6): 801808. doi:10.4314/tjpr.v10i6.14. tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics 1: 85 2: 86 3: 87 4: 88 5: 89 6: 90 7: 91 8: 92 9: 93 3.mi703-israwati harahap available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.3.3issn 1978-3477, eissn 2087-8575 vol 7, no 3, september 2013, p 105-112 *corresponding author; phone/fax: 021-8765066/0218765062, email: imanhidayat@yahoo.com . shorea spp. (dipterocarpaceae) dominantly inhabit rainforests in southeast asia, especially in malaysia and indonesia. in indonesia, these trees spread across the island of sumatra, bangka-belitung, kalimantan and several locations in java island (the ministry of forestry 2007). in natural ecosystems, shorea spp. posses an important role in maintaining the balance of the ecosystem because they produce abundant lignocellulosic rich substrates as a source of nutrients for the survival of microorganisms including saprobic fungi. the saprobic fungi are known as the major wood and leaf decomposing organisms which play an important role in the nutrient cycle of forest areas including shorea spp. plantations. during the decomposition process, the saprobic fungi degrade lignocellulosic materials into more simple compounds (osono 2007). hyde and taylor (2003) noted that there are many benefits from studying the diversity of saprobic fungi. these include novel agents with important medicinal properties (yang et al. 2011), and for other bioactive compounds (strobel 2003), and discoveries of new the community structure of sporulating fungi on decaying branch and leaf litters of shorea spp. were studied to reveal the common saprobic fungi. the study was mainly based on morphological observation. twenty-nine species of the sporulating fungi were found on shorea spp. litters at situ gede and bubulak forest area, bogor, west java. the fungi included seven species of ascomycetes (annulohypoxylon purpureonitens, diatrype chlorosarca, didymosphaeria epidermidis, lophiostoma sp., lophodermium sp., pemphidium sp., and valsa sp.) and 22 species of anamorphic taxa that consisted of 12 coelomycetes (coniella musaiaensis, coryneum betulinum, hendersoniopsis thelebola, lasiodiplodia theobromae, lasmeniella guaranitica, leptodothiorella sp., massariothea themedae, pestalotia guepinii, pestalotiopsis sp., pseudolachnea hispidula, septoriella sp., and unidentified species of coelomycetes) and 10 hyphomycetes (beltraniella portoricensis, cryptophialoidea fasciculata, hermatomyces spaerichus, kiliophora ubiensis, minimidochium setosum, monodisma fragilis, nodulisporium sp., stilbella fimetaria, virgatospora echinofibrosa, and unidentified hyphomycetes). the most common taxa occuring on decaying leaf litter were b. portoricensis and pemphidium sp., while those on decaying branch material were l. theobromae and c. fasciculata. the fungal community was subtrate specific. the community on decaying branch litter was more diverse than that on leaf litter. the c/n ratio of the substrate was closely related to the structure of the community. key words: community structure, frequency of occurrence, fungi, shorea spp. struktur komunitas cendawan berspora pada serasah ranting dan daun shorea spp. dipelajari untuk mengetahui cendawan saprob yang umum pada serasah tersebut. penelitian dilakukan berdasarkan pada pengamatan morfologi. sebanyak 29 spesies cendawan berspora ditemukan pada serasah shorea spp. yang dikoleksi dari areal hutan yang terletak di situ gede dan bubulak, bogor. jawa barat. cendawan terdiri dari 7 spesies askomiset (annulohypoxylon purpureonitens, diatrype chlorosarca, didymosphaeria epidermidis, lophiostoma sp., lophodermium sp., pemphidium sp., dan valsa sp.) dan 22 spesies cendawan anamorfik yang terdiri dari 12 soelomiset (coniella musaiaensis, coryneum betulinum, hendersoniopsis thelebola, lasiodiplodia theobromae, lasmeniella guaranitica, leptodothiorella sp., massariothea themedae, pestalotia guepinii, pestalotiopsis sp., pseudolachnea hispidula, septoriella sp., dan soelomiset yang tidak teridentifikasi) dan 10 hifomiset (beltraniella portoricensis, cryptophialoidea fasciculata, hermatomyces spaerichus, kiliophora ubiensis, minimidochium setosum, monodisma fragilis, nodulisporium sp., stilbella fimetaria, virgatospora echinofibrosa, dan hifomiset yang tidak teridentifikasi). spesies cendawan yang umum ditemukan pada serasah daun adalah b. portoricensis dan pemphidium sp., sedangkan pada serasah ranting adalah l. theobromae dan c. fasciculata. komunitas cendawan bersifat spesifik substrat. komunitas cendawan pada serasah ranting lebih beragam dibandingkan pada serasah daun. rasio c/n pada substrat mempengaruhi struktur komunitas cendawan tersebut. kata kunci: cendawan, frekuensi keberadaan, shorea spp, struktur komunitas community structure of sporulating fungi on decaying litters of shorea spp. 1 1 2 israwati harahap , gayuh rahayu , and iman hidayat * 1 department of biology, faculty of mathematics and natural sciences, kampus dramaga, institut pertanian bogor, bogor 16680. indonesia; 2 microbiology division, research center for biology, indonesian institutes of sciences, cibinong 16911, indonesia taxa (genera and species). ho et al. (2001) found ten new species and a new genus during their study on the saprobic fungi on submerged wood from streams. in addition, numerous new species have been discovered from palms (goh and hyde 1996; yanna et al. 1997; 1998; hidayat et al. 2006). there are many fungal community studies on plants that have been reported worldwide. however, a study based on the fungal community structure of decaying litter of shorea spp. has not widely been reported. few studies were reported from thailand (osono et al. 2009) and india (soni et al. 2011), and so far none from indonesia. therefore, research on the fungal community of shorea spp. was undertaken in order to provide information regarding the fungal community inhabiting shorea spp. litter. materials and methods collection of samples. samples of shorea spp. litter were collected from shorea plantation research area managed by the research and development centre for forest conservation and rehabilitation, located at the situ gede village and bubulak village, bogor, west java. samples were taken from 13 sampling sites which were designed plots (50 m x 50 m). the distance between each sampling site is 3 m. in total, 260 samples of shorea spp. (130 branch and 130 leaf samples) were collected. the size of each branch is 10 cm lengths, regardless the diameter. samples were placed in resealed plastic bags. after sealing, each bag was labeled as follows: name of the sample, collecting site, collector, and collection date. the environment parameters such as ph, light intensity, soil water content, water content of branch and leaf litter, the c/n ratio of branch and leaf litter were also measured. examination of materials. on returning to the laboratory, the materials were immediately incubated and examined periodically over one month. the materials were examined for saprobic ascomycetes, coelomycetes, and hyphomycetes. the materials were examined using an olympus szx7 dissecting microscope to determine the presence of the fungal fruiting structures, and an olympus cx41 to determine microscopic structures. for ascomycetes and coelomycetes, a sharp one sided razor blade or a pair of inox 5 fine forceps were used to carefully remove the top of the fruiting body. the specimens were rehydrated if the contents were dry or crystalline by using distilled water or potassium hydroxide (koh) 3% w/v before extraction. the 106 harahap et al. microbiol indones contents were placed in a drop of distilled water on a slide and covered with an 18 x 18 mm coverslip. hyphomycetes examination was prepared by using a pair of inox 5 fine forceps. water was used for all examinations, spore/conidia measurements, and most of the photographs/line drawings. shear’s solution was added to the slides for permanent fixation. the slides were heated to remove air bubbles in the shear’s solution and the edges of the coverslip sealed with two layers of clear nail varnish. the slides were labeled with the number of each specimen. detailed observations of morphological characters were carried out by means of an olympus cx41 light microscope using an oil immersion lens (1000×). single spore isolation of each new fungus encountered was as reported by choi et al. (1999) with modification. identification procedures. the following texts were consulted for basic identification such as hyde et al. (2000) and hyde and taylor (2003) for ascomycetes; nag raj (1993) and sutton (1980) for coelomycetes; and ellis (1971, 1976), carmichael et al. (1980) and seifert et al. (2011) for hyphomycetes. further identification of fungal specimens was by reference to the recent publications in various journals of mycology. the following fungal database websites were also used such as index fungorum (), mycobank (http://www. mycobank.org) and usda fungus-host database (http://nt.ars-grin.gov/fungaldatabases/fungus host/fungushost.cfm. data analysis. frequency of occurence (fo) of a taxon was calculated according to the following formula: the total number of species and the number of fungi per sample were recorded and calculated. based on the percentage occurrence of different species, grouping was done as follows: very frequent (>10%), frequent (>5%-10%), less frequent >1%<5%), and rare (<1%). shannon-wiener diversity (h’) and evenness indices (e) were calculated for each sampling point along with margalef’s species richness. calculations were carried out according to magurran (1988). the margallef index on diversity (d ) was calculated as mg follows: margallef index d = (s-1)/lnn ;mg d = margalef indexmg frequency of occurrence of taxon a = occurrence of taxon a total number of samples examined x 100% s = the number of species n = the total number of fungal occurrences shannon index (h’) = -σ pi. ln pi pi = the proportional abundance of th the i species= (ni/n) evenness (e) = h’ = shannon index, s = total species number the relationship between assemblage of the fungal community and different organ type of shorea spp. was also analysed using a simple correspondence analysis (ca). the analysis were performed using minitab 15 software. results community structure of sporulating fungi on decaying litters of shorea spp. examination of decaying branch and leaf litter of shorea spp., indicated that only 50% of the litter containing fungi. in those litter, 29 of the sporulating fungal taxa, comprising seven species of ascomycetes (representing 24.1% of all taxa), and 22 species of anamorphic fungi (representing 75.9% of all taxa) were found. the anamorphic fungi were composed of 12 coelomycetes (41.4%) and 10 hyphomycetes (34.5%) (fig 1). the number of fungal taxa found on decaying branch litter was higher than those on leaf litter (table 1). twenty-two species of fungi were recorded for the branch litter, and seven species were found in the leaf litter. beltraniella portoricensis and pemphidium sp. appeared to be the most common taxa inhabiting leaf litter with fo of 16.9 and 11.5% respectively (table 1). during the process of decaying branch litter, lasiodiplodia theobromae and cryptophialoidea fasciculata were determined to be the most frequent fungi found during this study. the remaining fungi . were found either less frequently or were rare based on their fo on branch or leaf litter (table 1). based on fungal groups, coelomycetes appeared as the most dominant group found on decaying branch litter with 11 species (total abundance 36), followed by hyphomycetes with 6 species (total abundance 28), and ascomycetes with 5 species (13 total abundance) (fig 2, fig 3). in the case of leaf litter, hyphomycetes were found to be the highest fungal group in species richness and abundance, with 4 species and score of 55, respectively. ascomycetes appeared as the second highest in species richness with 2 taxa recorded and score of 32, followed by coelomycetes taxa with 1 species and score of 1 (fig 2, fig 3). the shannon-wiener index was measured on thirteen sites in the sampling location (fig 4). the diversity of sporulating fungi on branch litter was higher than those on leaf litter. this data was also supported by the margalef index calculation (table 1). evenness was higher on the branch litter than on the leaf litter (table 1). this means that every fungal species in branch litter has a higher frequency of occurrence than for leaf litter. correspondence analysis (ca) of the fungal community on branch and leaf litter showed that taxa such as annulohypoxylon purpureonitens, diatrype c h l o ro s a rc a , d i d y m o s p h a e r i a e p i d e r m i d i s , lophiostoma sp., valsa sp., coniella musaiaensis, coryneum betulinum, hendersoniopsis thelebola, lasiodiplodia theobromae, lasmeniella guaranitica, leptodothiorella sp., massariothea themedae, pestalotia guepinii, pseudolachnea hispidula, septoriella sp., cryptophialoidea fasciculata, hermatomyces spaerichus, minimidochium setosum, monodisma fragilis, nodulisporium sp., virgatospora echinofibrosa, and coelomycetes sp.1 were common on branch litter. however, on leaf litter, taxa such as lophodermium sp., pemphidium sp., pestalotiopsis sp., beltraniella portoricensis., kiliophora ubiensis, stilbella fimetaria, and hyphomycetes sp. 1 were more common (fig 5). volume 7, 2013 microbiol indones 107 h ln s fig 1 comparison of fungal community on shorea spp. based on number of species. r i=1 al. 2009). the current study shows that species diversity (h') and species richness (d ) of the fungal community on mg the branch litter was higher than for leaf litter (table 1). the higher the c/n ratio and density of wood, rather than that of leaves best supported the growth of fungi on the wood (kodsueb et al. 2008). on palm, pinnoi et al. (2006) suspected that thicker cell walls may yield more nutrients, in particular cellulose and starch. these nutrients could sustain the growth most of the fungi. they found that the palmicolous fungi were more prevalent on palm petioles (53%) than on rachides discussion the present study is one amongst a few fungal community studies on branch and leaf litter of shorea spp., and it is the first study in indonesia. osono et al. (2009) and soni et al. (2011) studied the fungal community on leaf litter only. several studies of fungal community on other host plants, such as palmae, bamboo, mangrove, etc. have been carried out in several areas of the tropics (huhndorf and lodge 1997; hyde and alias 2000; hyde and sarma 2001; maria and sridhar 2003; hyde and taylor 2003; karamachand et taxa branch leaf total fo (%) ascomycetes annulohypoxylon purpureonitens (aap) 2 0 2 0.7 diatrype chlorosarca (adc) 5 0 5 1.9 didymosphaeria epidermidis (ade) 3 0 3 1.1 lophiostoma sp. (als) 2 0 2 0.7 lophodermium sp. (ald) 0 2 2 0.7 pemphidium sp.(apd) 0 30 30 11.5 valsa sp.(avs) 1 0 1 0. 3 anamorphic fungi coelomycetes coniella musaiaensis (ccm) 3 0 3 1.1 coryneum betulinum (ccb) 1 0 1 0.3 hendersoniopsis thelebola (cht) 1 0 1 0.3 lasiodiplodia theobromae (clt) 17 0 17 6.5 lasmeniella guaranitica (clg) 1 0 1 0.3 leptodothiorella sp. (cld) 1 0 1 0.3 massariothea themedae (cmt) 2 0 2 0.7 pestalotia guepinii (cpg) 2 0 2 0.7 pestalotiopsis sp. (cps) 0 1 1 0.3 pseudolachnea hispidula (cph) 1 0 1 0.3 septoriella sp.(cst) 6 0 6 2.3 coelomycetes sp. 1 (csp) 1 0 1 0.3 hyphomycetes beltraniella portoricensis (hbp) 0 44 44 16.9 table 1 frequency of occurence (fo) of sporulating fungi on branch and leaf litter of shorea spp. microbiol indones108 harahap et al. *fo = frequency of occurence volume 7, 2013 microbiol indones 109 12 10 8 6 4 2 0 ascomycetes coelomycetes hyphomycetes fungal group 5 2 1 6 4 11 s p ec ie s ri ch n es s 0 10 20 30 40 50 60 ascomycetes coelomycetes hyphomycetes fungal group 13 32 36 1 28 55 sampling locations s h an n o n -w ei n er i n d ex 1 2 3 4 5 6 7 8 9 10 11 12 13 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1.3 1.3 1 0.6 1.3 1.7 1.2 1 0.6 0.3 0.8 1.2 1 0.9 1 0.8 0.8 0.6 0.6 0 0 0 0 0 0 0 fig 2 distribution of all taxa recorded from of shorea spp. the graphics are presented based on qualitative data of species richness. : branch litter; :leaf litter. fig 3 distribution of all taxa recorded from of shorea spp. the graphics are presented based on quantitative data (total abundance). : branch litter; : leaf litter. fig 4 histogram of shannon-wiener diversity index (h’) showing the diversity of the fungal community on thirteen sites in the sampling location. : branch litter; : leaf litter. of each organ type. on the contrary, the fo of fungi in leaf litter was higher than that of branch litter. this probably relates to water content of substrates. the water content of leaf litter of shorea spp. was higher than that of branch litter (table 2). water has been recognized as an important factor in fungal growth, in particular during the germination process and the dispersal of fungal propagules. the water content is directly related to a high relative humidity required for spore germination, growth, and reproduction of fungi (yoder and wood 1973). fungi are well-recognized as organisms that utilized sugar due to sugar occupying a central position in fungal metabolism. hyde and sarma (2001) noted that other parameters to be considered as the factors affecting the frequency of occurrence of the fungal community on different habitats included ph, incubation time, ecological niches, availability of the substrate, quality of the substrate (old or young samples/ soft or hard tissue), nutrient availability, dod, cod, and bod, seasonality and succession, temperature (tropical or subtropical), and host specificity/ main host samples. different fungal communities occured on branch and leaf litters of shorea spp. indicating that substrates might play an important role in determining species composition by selectively stimulating or inhibiting the growth of specific fungi. different parts of the shorea spp. (i.e. leaf and branch) were found to support different fungi, pointing to the existence of a distinct ecological niche. this indicated that some fungi may preferentially develop on certain tissue types, as previously reported by hyde and alias (2000). (30%) and leaves (17%). hyde and sarma (2006) noted that branch thickness may also offer a better substrate for fungal colonization than that of leaves which were thinner. leaves contain mainly parenchymatous cells which are thin-walled, with chloroplasts and rich in starch, while rachides and petioles have more sclerenchyma associated with their vascular bundles. our analyses of fungal community structure, based on the artificial taxonomical groups, showed that anamorphic fungi were more dominant than teleomorphic fungi, and this result is in agreement with previous studies carried out by osono et al. (2009) and soni et al. (2011). osono et al. (2009) noted that hyphomycetes fungi were commonly found at the early stage of fungal community succession. however, the reasons for the higher dominance of anamorphic fungi over ascomycetes (teleomorphic fungi) are unknown. several fungal species commonly found on decayed litter of shorea spp. by osono et al. (2009) and soni et al. (2011), such as beltraniella, pestalotiopsis, and lophodermium were also found in the current study. shirouzu et al. (2009) noted that beltraniella species most commonly occured on newly fallen leaves and their fo usually decreased with decay. the distributions of the fungal community on branch and leaf litter were different (fig 5). it is apparent that several taxa only occured either on branch or leaf littter. distinct fungal community composition occurs on different organs of shorea spp. is indicated by the fact that fungi on shorea litter are substrate specific. there is limited information regarding the physical structure of each organ of shorea spp., therefore, it is difficult to determine the factors affecting the distinct community microbiol indones110 harahap et al. 1,00,50,0-0,5-1,0 1,0 0,5 0,0 -0,5 -1,0 component 1 c o m p o n en t 1 leaf bran ch hv e hsf hn s hmf hms hk u hh 1 hh s hcf hb p cst cp h cp s cpgcmt cld clg cltch t ccb ccm csp av s ap d ald als ad e ad c aap symmetric plot fig 5 correspondence analysis of fungal community on branch and leaf litter. taxa are shown by their acronym. in conclusion, there were different fungal community structures which occupied decaying branch and leaf litters of shorea spp. however, the factors that influenced the distinct fungal communities structures on branch and leaf litters were difficult to determine. therefore, future studies should take into consideration all factors effecting the frequency of occurence of fungi in shorea spp., in particular from the hosts point of view. references cai l, ji kf, hyde kd. 2006. variation between freshwater and terrestrial fungal communities on decaying bamboo culms. antonie van leeuwenhoek 89(2):293301. doi:10.1007/s10482-005-9030-1. carmichael jw, kendrick wb, conners ll, sigler l. 1980. genera of hyphomycetes. edmonton: the university of alberta press. choi yw, hyde kd, ho wwh. 1999. single spore isolation of fungi. fungal divers. 3:29-38. ellis mb. 1971. dematiaceous hyphomycetes. kew, surrey: cab international. ellis mb. 1976. more dematiaceous hyphomycetes. kew, surrey: cab international. goh tk, hyde kd. 1996. a new species of nectria from mauritia flexuosa (arecaceae) in ecuador and a key to nectria and allied genera on palms. mycoscience. 37(3):277-282. doi:10.1007/bf02461298. hidayat i, jeewon r, to-anun c, hyde kd. 2006. the genus oxydothis: new palmicolous taxa and phylogenetic relationships within the xylariales. fungal divers. 23:159-179. ho wh, hyde kd, hodgkiss ij, yanna. 2001. fungal communities on submerged wood from streams in brunai, hong kong, and malaysia. mycoln res. volume 7, 2013 microbiol indones 111 in relation to the fungal community on decayed plant substratum, the nature of the host substratum (chemical), minor components of the substratum, lignin/cellulose ratio, and nitrogen/carbohydrate ratio probably play important roles as selective factors determining which group of fungi colonize the substrate at different stages of decomposition. host plants may contain low to high amounts of compounds that are toxic or inhibit the growth of fungi e.g. phenols, while wood density of a substratum may also influence the ability of fungi to colonize (pinruan et al. 2007). based on phytochemical studies of shorea spp., the main secondary metabolites of this plant genus is a class of phenolic compounds, such as oligostilbenoid, flavonoids, phenyl propanoid, and phenolic acid derivates. rohaiza et al. (2011) have been isolated four oligostilbenes of (-)-e-viniferin, (-)-ampelopsin e, (-)hopeaphenol and shoreaphenol from the acetone extract of s. hopeifolia. oligostilbene compounds exhibit a variety of significant bioactivities, including anti-bacterial (nitta et al. 2002) and anti-fungal (kusuma and tachibana 2007). stilbene derivates are known to be abundantly distributed in the plants belonging to the dipterocarpaceae, vitaceae, leguminosae, and cyperaceae (ohguchi et al. 2003). therefore, the smaller number of sporulating fungi in this study, as compared to studies on another hosts such as bamboo (cai et al. 2006), palm (pinruan et al. 2007), mangrove plants (maria and sridhar 2003), were probably due to the phenolic compound contents in the shorea plants which inhibited fungal growth and thus protected plant tissues (including leaf and branch material) from the fungal colonization. parameters value ph of soil 6 light intensity 120 water content of soil 38.34% water content of 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measurement. london sydney: croom helm. p 11-37. doi:10.1007/978-94-015-7358-0. maria gl, sridhar kr. 2003. diversity of filamentous fungi on woody litter of five mangrove plant species from the southwest coast of india. fungal divers. 14:109-126. nag raj tr. 1993. coelomycetous anamorphs with appendage-bearing conidia. waterloo: mycologue publications. nitta t, arai t, takamatsu h, inatomi y, murata h, iinuma m, tanaka t, ito t, asai f, watabe k. 2002. antibacterial activity of extracts prepared from tropical and subtropical plants on methicillin-resistant staphylococcus aureus. j health sci. 48(3):273-276. doi:10.1248/jhs.48.273. ohguchi k, tanaka t, ito t, iinuma m, matsumoto k, akao y, nozawa y. 2003. inhibitory effects of resveratrol derivatives from dipterocarpaceae plants on tyrosinase. biosci biotechnol biochem. 67(7):1587-1589. doi:10.1271/bbb.67.1587. microbiol indones112 harahap et al. 1: 105 2: 106 3: 107 4: 108 5: 109 6: 110 7: 111 8: 112 3.mi-sarjiya anton.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.4.3issn 1978-3477, eissn 2087-8575 vol 10, no 4, december 2016, p 131-138 *corresponding author; phone: +62-81316499731, email: sarj.antonius@gmail.com the phytohormone auxins play a key role in plant growth and development as a regulator of numerous biological processes, such as cell division, elongation and differentiation to tropic responses. auxins are employed to induce rooting, callus formation, flowering and so on (teale et al. 2006). the action and interaction of some growth regulators like auxins regulate most of the physiological activities and growth in plants. aplication of liquid or solid organic fertilizer containing iaa producing bacteria could improve not only the growth of various vegetables and crops, but also increased the yield under green house and field trial (antonius & agustiyani 2011a; antonius and agustiyani 2011b; antonius et al. 2012; antonius et al. 2015). naturally occurring substances with indole nucleus possessing growth-promoting activity are referred to as auxins, chemically it is indole acetic acid (iaa). the auxins and cytokinins could be synthesized by microorganisms as well plants. the ability to synthesize phytohormone is widely found among plant-associated bacteria (spaepen et al. 2007). about 80% of the bacteria isolated from plant rhizosphere able to produce iaa. tryptophan is used by plants and bacteria as physiological precursors to produce iaa. the high content of tryptophan in an organism or bacterial growth media, normally following with high content of iaa. in order to produce iaa by using rhizobacteria, it is necessary to find correct natural resources as substrate that may contain amino acid as precursor for iaa indole-3-acetic acid (iaa) is the main member of the auxin family that controls many important physiological processes in plant. such beneficial iaa that produced by plant growth promoting rhizobacteria (pgpr), enhances plant growth and was believed to increase the access to more nutrients in the soil. the precursor for syntetizing iaa is tryptophan, and it was also found in the sprout or other sources of protein. the aim of this study was to investigate the best bacteria and growth medium supplemented with extract of bean sprout or fish meal as the sources of precursor for the iaa production. several bacterial isolates were screened for iaa production. iaa production was measured with high performance liquid chromatography. all of isolates were able to produce iaa and isolates ps1 was selected for the further assay by cultivating under fermentor system. sequencing of 16s rdna of ps1 isolate indicated as acinetobacter sp. the result showed that the highest iaa production during fermetation was 62,428 ppm found in under medium supplemented with mung bean sprout extracts grown in fermentor, after 24 hours incubation. key words: plant growth promoting rhizobacteria, indole acetic acid, mung bean sprout extract, acinetobacter sp. asam indole-3-asetat (aia) adalah bagian utama dari kelompok auksin yang mengontrol banyak proses fisiologis penting dalam tanaman. peran penting aia yang dihasilkan oleh rizobakteri pemacu pertumbuhan tanaman (rppt) meliputi meningkatkan pertumbuhan tanaman dan dipercaya semakin menngkatkan penyerapan nutrisi dalam tanah. prekursor untuk memproduksi iaa adalah triptofan, dan itu juga ditemukan dalam kecambah atau sumber protein lainnya. tujuan dari penelitian ini adalah untuk mendapatkan bakteri terbaik dan media pertumbuhan yang dilengkapi dengan ekstrak kecambah atau tepung ikan sebagai sumber prekursor untuk produksi iaa. beberapa isolat bakteri diseleksi untuk produksi iaa tertinggi. produksi iaa diukur dengan high performance liquid chromatography. semua isolat yangdigunakan mampu menghasilkan iaa dan isolat ps1 dipilih untuk pengujian lebih lanjut dengan menumbuhkannya dalam fermentor. analisa sekuen 16s rdna dari isolat ps1 diketahui tergolong sebagai acinetobacter sp. hasil penelitian menunjukkan bahwa produksi iaa tertinggi ps1 selama fermentasi adalah 62.428 ppm, dengan ditumbuhkan pada media yang mengandung ekstrak kecambah kacang hijau pada ferementor setelah inkubasi 24 jam. kata kunci: rizobakteri pemacu pertumbuhan tanaman (rppt), asam indole-3-asetat, ekstrak kecambah kacang hijau, acinetobacter sp. the use of sprout as precursor for the production of indole acetic acid by selected plant growth promoting rhizobacteria grown in the fermentor 1 2 1 sarjiya antonius *, rachel budisatria , and tirta kumala dewi 1 research center for biology lipi, jalan raya jakarta-bogor km 46, cibinong, bogor, jawa barat 16911, indonesia; 2 universitas pelita harapan, lippo village, jalan mh thamrin boulevard, tangerang, banten 15811, indonesia production. according to mubarak (2005), germination process also lowers antinutrition component and increase the content of amino acids. applying of mung bean sprouts extract and fish meal might be possible to get high containing tryptophan. according kay (1979) sprout contained high amino acid, tryptophan and serin. this paper discusses the results of screening of iaa producing rhizobacteria by applying mung bean sprout extract and fish meal as sources of precursor and its iaa production ability by selected best isolate with the suitable precursor under fermentor conditions. material and methods bacterial screening of iaa producing activity by using sprout extract or fish meal as precursor. seven bacterial isolates that were screened of iaa producing activity qualitatively in the laboratory of agricultural microbiology, research center for biology-lipi were used for this study. the medium used for growing the bacteria and iaa production was tryptic soy broth (tsb) 50% (half strength), with the following composition: 10 g of peptone 10 g, 2.5 g of nacl, 20 g of agar, and 1000 ml of distilled water. after the medium was sterilized, filter sterilized precursor ltryptophan 200 ppm was added to the medium (glickmann and deseaux 1995). one loop of the pure culture of iaa producing bacteria was inoculated into 25 ml tsb medium in 50 ml flask and incubated at 25 °c, 120 rpm for 72 h. each of bacteria culture at 0, 24, 7 -1 48, and 72 h with 0.6 optical density (10 cells ml ) was picked up 3 ml and centrifuged at 12 000 rpm for 15 min for measured the iaa concentration. to study the potency of mung bean sprout as precursor, instead of tryptophan, 50 % volume of media was suplemented the sprout extract or fish meal. dna extraction and amplification of 16s rdna. ps1 isolates was growth on the nutrient agar (na) medium and incubated for 48 h. the dna of colonies of bacteria was extracted by using ges method ( pitcher et al 1989). the dna amplification was performed using polymerase chain reaction ( pcr ). a total pcr volume used was 25 µl, containing 12.5 µl gotaq green® master mix (promega), 1 µl of 10 µm 27f (5’-agagtttgatcctggctcag3’), 1 µl of 10 µm 1492r (5’-tacggytaccttgtt acgactt-3’) ; and 10.5 µl nucleus free water and 1 µl dna template. the pcr protocol was as follows pre-denaturation at 95 °c for 2 min, followed by denaturation at 95 °c for 30 sec, annealing at 50 °c for 30 sec, extension at 72 °c for 1,5 min, final-extension 132 antonius et al. microbiol indones at 72°c for 10 min. the pcr cycle was used 30 cycles. the pcr product was verified by electrophoresis for 30 min and 100 v, on 1 % agarose in 1x tae buffer. formed band profiles were observed under the uv transilluminator. dna sequencing and phylogenetic analysis. the pcr product was further analyzed by dna st sequencing (1 base, singapore). the sequenced data were processed using bioedit programme. the homology of 16s rdna sequence were searched using blast software (blastn) on the national center biotechnology information site (ncbi) (http://www.n cbi.nlm.nih.gov/). constructions of phylogenetic tree was done using neighbor-joining tree method (njt) implemented in mega 5.05 software (tamura et al 2011). model of k2+g+i (kimura2-parameter and gamma distributed) was selected as the best-fit substitution model for the current analysis. strength of internal branches of the phylogenetic tree was tested with bootstrap analysis using 1000 replications. sprout extract preparation. a total of 1 kg of mung bean (vigna radiate) seed was prepared and germinated for ± 3 d to get out the young plants with a length of 4 cm sprouts. sprouts were then boiled in 1 l of distilled water for 30 min or until the half water volume left. the rest of sprout boiled water collected by filtered. the extract obtained was collected into the bottle and stored in the refrigerator. production of iaa pilot scale. the 20 l volume of fermentor (shanghai bailun-bio technology co., ltd) was filed with tsb media and sprout extract or fish meal (1:1) and on site sterilized. the ph of media was adjusted at 7 and under room temperature was inoculated with 1 l bacterial inoculant. the fermentor was suplied oxygen and adjusted the oxygen desolve near about 80 % with the rotor speed 100-150 rpm. the growth of bacteria and the iaa production were monitored by taking sample for measuring optical density and iaa concentration until late stationare phase. sample preparation and determination of iaa concentration. a 3 ml of clarified supernatant of mung bean sprout extract were adjusted to ph 2.8 with hcl and extracted three times using 3 ml ethyl acetate in each extraction. the extract was drying by rotary evaporator to eliminate the solvent. in parallel, the recovery took place with percentage of 98%. the indol compounds were dissolved in 1.0 ml methanol solution and analyzed by hplc. the resulting solution was injected into a hitachi hplc system consisting uv-vis detector, autosampler. chromatographic separation was performed on analytical column microsorb c18 (150 mm × 4.6 mm), eluted with methanol:acetic acid:water (30:1:70) with isocratic elution at a flow rate of 1.0 ml min-1 at room temperature. the iaa presence was detected at 280 nm wavelength for 15 min (mehnaz dan laravoits 2006). the iaa concentration was calculated using the peak area and the calibration curve between 0-10 ppm. each standard of calibration curve was injected by triplicates. each extract was run with duplicates. results screening of the pgpr that has the highest iaa producing activity quantitatively. first step screening of bacterial isolates for iaa producing activity is usually based on quality analysis. in this case, the information was only positive or negative regarding the bacterial isolate ability to produce iaa, it needed therefore to screened based on quantitative iaa producing activity in relation with the growth. seven different bacterial isolates (ps, es, rs, rl, ms, ps1, and ae) collection of agriculture microbiology laboratory-lipi that originated from various ecosystem and showed iaa producing activity qualitatively before, were chosen to use in the study. all isolates were selected based on their ability to produce iaa by measuring the activity quantitively. in order to know the correlation of growth phase of each isolate with the optimum iaa production, the growth curve of all isolate were monitored (fig 1). measurement of iaa by using hplc showed that growth medium of all the isolates contained substantial amount of iaa. spectrophotometrically monitoring of bacterial growth indicated that ps isolate showed the best growth by showing the highest optical density data. however the production of iaa by ps isolate was relatively low, with the maximum iaa production of about 18 ppm (fig 2). the other isolates with code es, rs, rl, ms, and ae produced various conctration of iaa in the growth media and ps1 isolate showed the highest production of iaa. the highest production of iaa by ps1 was about 140 ppm, acheived after 72 h incubation (fig 2). based on sequence analisis of 16s rdna of ps1 isolate indicated as acinetobacter sp. phylogenetic characterization of indole-3acetic acid producing bacteria. the homology search by the blast program showed that ps1 isolate closely related to acinetobacter sp. the phylogenetic tree was inferred using the neighbor-joining method and the result showed that ps1 and acinetobacter baylyi strain b2 (fig 3) were clustered together with 16s rdna sequence-similarity values of 99%. production of iaa by acinetobacter sp. with sprout extract and fish meal as precursor. it is well known that tryptophan is a good precursor for production of iaa by pgpr. it has been reported that production of iaa and ila (indole-3-lactic acid) increased with increasing concentrations of tryptophan -1 (1-100 μg ml ) by azospirillum brasilense sp13t and sr2, respectively (tien et al. 1979). the growth indicator of each acinetobacter sp. culture by plating method in tsb media suplemented with tryptophan, mung bean sprout extract or fish extract were comparable (data not shown), however volume 10, 2016 microbiol indones 133 number isolates code source of samples sampling locations 1 ps rhizosphere of healthy banana plant lampung, sumatra 2 es endophytes of diseased banana plant lampung, sumatra 3 rs rhizosphere of healthy banana plant lampung, sumatra 4 rl rhizosphere of healthy banana plant lampung, sumatra 5 ms rhizosphere from mix soil of banana plant lampung, sumatra 6 ps 1 rhizosphere of healthy banana plant lampung, sumatra 7 ae rhizosphere of edamame plant cibinong, west java table 1 source of isolates and sample locations microbiol indones134 antonius et al. reached maximum at early stationary phase (fig 6). the maximum production of iaa by acinetobacter sp under fermentor cultivation was obtained in the 24 h incubation of time (fig 6). discussion the results of quantitative iaa producing activity screening obviously showed that the diverse the production of iaa were much different especially in the media suplemented with 200 ppm of tryptophan, the iaa containing in the media was much higher about 150 ppm (fig 4). the growth of acinetobacter sp. culture in the fermentor was very good and after 12 h incubation started to aproaching in the stationare phase. in paralel with the growth of acinetobacter sp., the iaa production was started at the beginning growth and 0 24 48 72 4 3.5 3 2.5 2 1.5 1 0.5 0 incubation (h) o p ti ca l d en si ty ( o d ) ia a ( p p m ) incubation (h) 0 24 48 72 0 20 40 60 80 100 120 140 160 fig 1 growth curve of several isolates during screening for the highest production of iaa with tryptophan as precursor. the experiments were performed in triplicates. ps: , es: , rs: , rl: , ms: , ps1: , and ae: . fig 2 iaa production by several isolates during screening for the highest production of iaa with tryptophan as precursor. the experiments were performed in triplicates. ps: , es: , rs: , rl: , ms: , ps1: , and ae: . volume 10, 2016 microbiol indones 135 than the growth media supplemented with mung bean sprout extract or fish meal as natural source of tryptophan. sprout extract and fish meal contain high concentration of protein, it might contain high concentration of amino acid including tryptophan. therefore both source of amino acid were used in the experiments as precursor of iaa production by acinetobacter sp. basically, the hypothesis reaction was protein of raw material converted first to amino acid (tryptophan), then use as precursor for iaa production. it was possible that conversion of the mung bean sprout extract and fish extract to tryptophan was much lower than 200 ppm and consequently the production iaa were about 25 ppm. according to lee et al. (2004), in presence of higher concentration of tryptophan, the microbes release greater quantities of iaa and related compounds. the using of tryptophan in this work was as positive control. although comparing to tryptophan, applying mung bean sprout extract as precursor for the production of iaa was much lower, it was still higher than applying fish meal as precursor. it was great interest to produce iaa by acinetobacter sp. with applying mung bean sprout extract as precursor under pilot plant in the fermentor. antonius et al. (2012) reported that production of liquid organic biofertilizer with row material containing mung bean sprout could promote the higher concentration of iaa in the final product after 3 weeks relationship between the growth rate and the ability to produce iaa. it has been reported that iaa production by bacteria can vary among different species and strains, and it is also influenced by culture condition, growth stage and substrate availability (mutluru and konada 2007). the ideal selected bacterial isolate should be best on growth parameter and its iaa production activity. however, there was no isolate such best criteria and ps1 isolate was chosen to further study. this ps1 isolate showed the highest iaa production activity (140 ppm) compare to other isolates although maximum growth was not so high (about od λ 436: 1.5). the highest growth was ps isolate (about od λ 436:1.5) even though only showed relatively low iaa production (18 ppm). interestingly, both isolates ps1 and ps were originally from rhizosphere of healthy banana plant. the other isolate were endophytes or soil bacteria showed no special iaa producing activity nor maximum growth. sarwar and kremer (1992) reported that isolates from the rhizosphere are more efficient auxin producers than isolates from the bulk soil. based on phylogenetic analysis (fig 3) ps 1 isolate has closely relatedwith acinetobacter sp., it was probably due to their activity to producing iaa (kwon and song 2014). rokhbakhsh-zamin et al. (2011) found that acinetobacter sp. was isolated from rhizosphere has ability to produce iaa. the production of iaa with the supplement of 200 ppm of tryptophan was much higher (about 150 ppm) 0.05 aj276351 bacillus subtillis strain dsm10 jq771132 acinetobacter nectaris sap 763.2 jx402203 acinetobacter apis strain hyn18 aj295007 acinetobacter venetianus atcc 31012 x81666 acinetobacter radioresistens dsm6976 x81662 acinetobacter haemolyticus dsm6962 aj626712 acinetobacter beijerinckii luh 4759t x81660 acinetobacter baumannii dsm30007 hq180184 acinetobacter pittii lmg 1035 aj888983 acinetobacter calcoaceticus strain nccb 22016 eu290155 acinetobacter soli strain b1 af509820 acinetobacter baylyi strain b2 contig ps191 100 92100 53 53 59 100 0.05 aj276351 bacillus subtillis strain dsm10 jq771132 acinetobacter nectaris sap 763.2 jx402203 acinetobacter apis strain hyn18 aj295007 acinetobacter venetianus atcc 31012 x81666 acinetobacter radioresistens dsm6976 x81662 acinetobacter haemolyticus dsm6962 aj626712 acinetobacter beijerinckii luh 4759t x81660 acinetobacter baumannii dsm30007 hq180184 acinetobacter pittii lmg 1035 aj888983 acinetobacter calcoaceticus strain nccb 22016 eu290155 acinetobacter soli strain b1 af509820 acinetobacter baylyi strain b2 contig ps191 100 92100 53 53 59 100 fig 3 phylogenetic tree of ps1 isolate based on 16s rdna sequences of the using neighbor-joining tree method, model kimura2-parameter and gamma distributed with 1000 replications. microbiol indones136 antonius et al. incubation (h) -10 0 10 20 30 40 50 60 70 80 ia a ( p p m ) 0 20 40 60 80 100 120 140 160 0 12 24 36 48 60 incubation (h) 0.3 3.0 o d ( 4 3 6 n m ) 0 12 18 24 36 42 48 60 64 incubation (h) 10 20 30 40 50 60 70 0 ia a ( p p m ) fig 4 iaa production by acinetobacter sp. with tryptophan (black bar), mung bean sprout extract red bar) and fisch meal (green bar) as precursor. the experiments were performed in triplicates and the bars indicate standard deviation. : tryptophan, : mung bea sprout, and :fish meal. fig 5 growth curve of achinetobacter sp. during scale up production of iaa with mung bean sprout extract as precursor in the 20 l fermentor. fig 6 iaa production by by acinetobacter sp. during scale up production of iaa with mung bean sprout extract as precursor in the 20 l fermentor. the experiments were performed in triplicates and the bars indicate standard deviations. volume 10, 2016 microbiol indones 137 biofertizier containing plant growth promoting rhizobacteria on water melon growth and yield, and soil biochemical properties under filed experiment in malinau-north kalimantan. berk. penel. hayati. 16: 203-206. antonius s, laili n, imamuddin h, agustiyani n. 20012. development of sustainable agriculture: the role of beyonic-startmik lipi biofertilizer on yield improvement of various crops and conservation of soil biochemical properties of various ecosystem in indonesia. in abdulhadi r, tjahjono bse, waluyo eb, delinom rm, prijono sn, fizzanty t, lesmana t (eds). proceedings “mobilizing science toward green economy”, the 12 th sciences council os asia (sca) conference and international symposisum 10-12 july, 2012-bogor, indonsia.p.119-126. antonius s, rahmansyah m, agustiyani d. 2015. the use of microbial inoculants for compost enrichment on vegetables cultivation. berita biologi. 14:223-234. glickmann e. and dessaux y. 1995. a critical examination of the specifity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. appl enviro microbiol. 61(2):793-796. kay de. 1979. food legume. tropical product institute london kwon h and song h. 2014. interactions between indole-3acetic aczid producing acinetobacter sp. sw5 and growth of tomato plant. korean j microbiol. 50(4):302307. doi:10.7845/kjm.2014.4050. lee s, flores-encarnacion m, contreras-zentalla m, garcia-flores l, escamilla je, kennedy c. 2004. indole-3-acetic acid biosynthesis is deficient in gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes. j bacteriol. 186 (16):5384-5391. doi:10.1128/jb.186.16. 5384-5391.2004. liu y, chen l, zhang n, li z, zhang g, xu y, shen q , zhang r. 2016. plant-microbe communication enhances auxin biosynthesis by a root-associated bacterium, bacillus amyloliquefaciens sqr9. mol plant-microb interact. 29(4):324-330. doi:10.1094/mpmi-10-15-0239-r. mehnaz s, lazarovits g. 2007. inoculation effects of pseudomonas putida, gluconacetobacter azotocaptans, and azospirillum lipoferum on corn plant growth under greenhouse conditions. microbiol ecol. 51(3):326-335. doi:10.1007/s00248-006-9039-7. mubarak ae. 2005. nutritional composisition and antinutritional factors of mug bean seeds (phaseolus aureus) as affected by some home traditional processes. food chemistry 89(4):4. doi:10.1016/j.foodchem.2004. 01.007. mutluru s and konada v m. 2007. bioproduction of indole acetic acid by rhizobium strains isolated from root nodules of green manure crop, sesbania sesban (l.) merr. iranian j biotechnol. 5(3):178-182 pitcher dg, saunders na, owen rj. 1989. rapid extraction of bacterial genomic dna with guanidium thiocyanate. lett appl microbiol. 8(4):109-114. doi:10.1111/j.1472765x.1989.tb00262.x. fermentation. although the production of iaa was not so high, natural row material mung bean extract as precursor for the production of iaa was chosen for pilot iaa production under fermentor system. such natural raw material is not only cheap but also easily to be found in every market. the pilot scale of iaa production under fermentor system was obviously much higher compare to the erlenmeyer culturing system incubated in the rotary shaker. under this fermentor system, the solubility and homogeneity of the substrate and precursor were much better and also very good in the diffusion of oxygen, consequently the bacterial could growth and produce iaa much better. it was likely that optimization of medium composition and the fermentation condition needed. from this study, it is clear that seven qualitatively selected isolates of iaa producing bacteria has the ability to produce various amount of iaa in a tryptophan-supplemented medium. ps1 isolate show best iaa producing activity (about 150 ppm) in tryptophan-supplemented medium. sequencing of 16s rdna of ps1 isolate indicated as acinetobacter sp. natural sources mung bean sprout extract was able to be used as precursor for iaa production by acinetobacter sp. with concentration of the product about 30 ppm and 60 ppm under erlenmeyer shaking culture and fermentor pilot scale production, respectively. it is concluded that mung bean as precursor could be effective and potent for the precursor of iaa production quantitatively. this research information was the first evidence that mung bean sprouts extract could be used as precursor for the production of iaa by acinetobacter sp. acknowledgments we are thankful to our colleagues ms. achirul nditasari and ms. tri ratna sulistiyani who provided expertise that greatly assisted on identifying bacterial isolate. financial assistance to the corresponding author from dipa and biovillage programs, are gratefully acknowledged. references antonius s and agustyani d. 2011a. effects of biofertilizer containing microbial of p solubilizer and plant growth factor producer on cabbage (brassica oleraceae var. capitata) growth and soil enzymatic activities: a green house trial. berk. penel. hayati. 16:149-153 antonius s dan agustiyani d. 2011b. impact of organic microbiol indones138 antonius et al. tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics analysis using maximum likehood, evolutionary distance, and maximum parsimony methods. mol biol evol. 28(10):2731-2739. doi:10.1093/molbev/msr121. teale wd, paponov ia, palme k. 2006. auxin in action: signaling, transport and the control of plant growth and development. j mol cell biol. 7:847-859. tien, t.m., gaskins, h. and hubbell, d.h. 1979. plant growth substances produced by azospirillum brasilense and their effect on the growth of pearl millet (pennisetum americanum l.). appl environ microbiol. 37(5):1016-1024. rokhbakhsh-zamin, farokh, sachdev d, kazemi-pour n, engineer a, pardesi kr, zinjarde s, dhakephalkar pk, chopade ba. 2011. characterization of plant-growthpromoting traits of acinetobacter species isolated from rhizosphere of pennisetum glaucum. j microbiol biotechnol. 21(6):556-566. sarwar m, kremer, rj. 1992. determination of bacterially derived auxins using a microplate method. lett appl microbiol. 20(5):282-285. doi:10.1111/j.1472765x.1995.tb00446.x. spaepen s, vanderleyen j, remans r. 2007. indole-3-acetic acid in microbial and microorganism-plant signaling. fems mirobiol rev. 31(4):425-448. doi:10.1111/j.157 4-6976.2007.00072.x. 1: 131 2: 132 3: 133 4: 134 5: 135 6: 136 7: 137 8: 138 1.mi731-eveline ayu new available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.8.1.2issn 1978-3477, eissn 2087-8575 vol 8, no 1, maret 2014, p 9-15 *corresponding author; phone/fax: +62-21-5703306/5727615 ext. 450/+62-21-5719060, email: antoniussuwanto@gmail.com tempeh is an indigenous indonesian fermented food and has become an important part of the indonesian diet for hundreds years. tempeh is consumed in relatively large portion and can be found in a variety of types of cooking and processing methods. excellent protein quality has made tempeh become a meat substitute and becomes popular among vegetarians (liem et al.1977). the process of tempeh making in indonesia is still using conventional methods with uncontrolled condition (barus et al. 2008). during the fermentation process, not only fungi involved but also bacteria play important roles in the formation of flavor and nutrition. bacterial growth during tempeh production begins in the process of soybean soaking. during the fermentation process, the producers inadvertently adding bacteria so that the bacteria eventually become an inseparable part of tempeh, and even has an important role in determining the quality of tempeh (barus et al. 2008; seumahu et al. 2013). several studies have reported the presence of bacteria in tempeh as klebsiella pneumoniae and citrobacter freundii (keuth and bisping 1994), and also bacteria of the phylum proteobacteria and firmicutes (seumahu et al. 2012) which are reported as bacteria producing vitamin b in tempeh. other 12 contaminants such as brevibacterium epidermidis and micrococcus luteus known to play a role in the formation of antioxidants in tempeh (klus and barz 1995). furthermore, bacteria in tempeh is also known as one of the factors that play a role in the formation of a bitter taste in tempeh (barus et al. 2008). a diverse array of lactid acid bacteria and yeasts also play important role in indonesian tempeh production (efriwati et al. 2013). k. pneumoniae which is included in family enterobacteriaceae is also known both of causing tempeh is important traditional indonesian fermented food made​​ from soybeans employing rhizopus oligosporus or r. microsporus. during the process of tempeh production, some bacteria from the environment and tempeh starter become an integral part of tempeh, and even have important roles in determining the final quality of tempeh it self. several studies reported the presence of klebsiella pneumoniae in tempeh as one of vitamin b12 producing bacteria in tempeh. however, k. pneumoniae also known as opportunistic pathogens causing pneumonia and liver abscess in human. in this study, enterobacterial repetitive intergenic consensuspolymerase chain reaction (eric-pcr) was employed to determine genetic diversity of k. pneumoniae isolated from tempeh and compared them with medical isolates. the result indicated that isolates from tempeh were genetically distinct from those of medical isolates. key words: eric-pcr, tempeh tempe merupakan salah satu makanan utama tradisional indonesia yang terbuat dari kacang kedelai dengan fermentasi menggunakan cendawan rhizopus oligosporus atau r. microsporus. selama proses pengolahan, bakteri yang berasal dari lingkungan dan inokulum awal menjadi bagian yang tidak terpisahkan dari tempe, bahkan memiliki peranan yang penting dalam menentukan kualitas akhir pada tempe. beberapa penelitian telah melaporkan keberadaan klebsiella pneumoniae pada tempe sebagai salah satu bakteri penghasil vitamin b12 pada tempe. akan tetapi, k. pneumoniae juga dikenal sebagai patogen oportunis penyebab penyakit pneumonia dan abses hati pada manusia. pada penelitian ini, metode enterobacterial repetitive intergenic consensuspolymerase chain reaction (eric-pcr) digunakan untuk membandingkan keragaman genetik k. pneumoniae pada tempe dibandingkan dengan isolat medis. hasil penelitian ini menunjukkan bahwa secara genetik pneumoniae pada tempe berbeda dengan isolat medis. kata kunci: eric-pcr, tempe klebsiella pneumoniae, klebsiella pneumoniae, klebsiella pneumoniae from indonesian tempeh were genetically different from that of pathogenic isolates 1 1,2 1 eveline ayu , antonius suwanto *, and tati barus 1 department of biology, faculty of biotechnology, universitas katolik atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia; 2 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia pneumonia disease, an acute infection that attacks the alveoli (gori et al. 1996) and liver abscess in human (wang et al. 1998). therefore, the study of genetic diversity is important for the identification and characterization of bacterial pathogenicity (rademaker and de bruijn 1997). one of the many molecular techniques for the study of genetic diversity is enterobacterial repetitive intergenic consensus (eric)-pcr. eric sequences are short sequences, which is 126 bp long, with sequences that are conserved as internal repeat and as non-coding sequences (lupski et al. 1992). this technique is often used because it is simple, rapid, reproducible, and discriminative (olive et al. 1999) and has been successfully analyzed the diversity of different types of bacteria, such as mycobacterium tuberculosis (sechi et al. 1998) and vibrio parahaemolyticus (khan et al. 2002). ericpcr has also been used to analyzed genetic diversity of klebsiella spp. isolated from tempeh (barus et al. 2013), as well as to study genetic heterogeneity of many types of vibrio cholerae (waturangi et al. 2012). materials and methods this research was conducted in research laboratory, faculty of biotechnology, atma jaya catholic university of indonesia from may to december 2013. medical isolates of k. pneumoniae. four medical isolates ware used for comparison with k. pneumoniae from tempeh, i.e. k. pneumoniae atcc baa-2146, k. pneumoniae subsp. pneumoniae atcc 10031, k. pneumoniae atcc 35657, and one k. pneumoniae isolate originated from pneumonia patient, named as fk isolate, collection of department of microbiology, faculty of medicine, atma jaya catholic university. k. pneumoniae isolates from tempeh. tempeh emp and wjb was produced in bogor, west java, indonesia (barus et al. 2008). a 10 g of fresh tempeh was placed into 90 ml of sterile physiological salt 0.85% (w/v) nacl and homogenized in orbitar shaker (yih der) at a speed of 24 x g for one min. dilution was -1 -6 made ​​from 10 until 10 and a 100 µl from dilution of -4 -5 -6 10 , 10 , and 10 was spread on eosin methylene blue (emb) agar (oxoid) and incubated overnight at 37 °c. a single purple mucoid colony was typical character of k. pneumoniae. these colonies were further analyzed by cultivating them on simmons’ citrate agar (sca) (difco), and incubated at 37 °c for 24 h. tempeh sampling were conducted twice, i.e. august and october 2013. klebsiella sp. 135 isolated from tempeh 10 ayu et al. microbiol indones was used for control (maysella 2010). analysis of 16s rrna genes. suspected blue colonies on simmons’ citrate agar were further verified using sequencing of genes encoding 16s rrna. the whole cells from single colonies on plates were used directly in pcr reaction as described by rademaker and de bruijn in 1997. the 16s rrna gene was amplified employing a pcr machine (applied biosystems, 2720 thermal cycler) using primer 63f (5’caggcctaacacatgcaagtc3’) and 1387r (5’gggcggwt gtacaaggc -3’) (marchesi et al. 1998). total pcr volume was 50 µl containing 2 µl dna template, 25 µl gotaq green® master mix (promega), 2 µl primer forward dan -1 reverse (25 pmol µl ) dan 19 µl nuclease free water. the pcr protocol was as follows: initial denaturation at 94 °c for 5 min, denaturation at 92 °c for 30 s, annealing at 62 °c for 30 s, elongation at 72 °c for 30 s, and post extention at 72 °c for 7 min. the cycle was repeated for 30 times. a 5 µl of pcr amplification products were further verified by electrophoresis in 1% agarose (bioline) in 1x tae buffer for 60 min, 80 v. sequencing of pcr products were performed in macrogen inc., korea, and were analyzed employing a program seqtrace. sequencing results were compared to the database with the basic local alignment search tool (blast) program which is provided by national centre of biotechnology information (ncbi). genetic profiling of k. pneumoniae isolates. 13 bacterial isolates were identified as k. pneumoniae from emp tempeh and 10 isolates from wjb tempeh. a total pcr volume used was 25 µl containing 12.5 µl gotaq green® master mix (promega), 1 µl of 25 pmol eric1r (5’-atgtaagctcctggggattca c-3’), 1 µl of 25 pmol eric2f (5’-aagtaagtgac tggggtgagcg-3’)give references for the primers (versalovic et al. 1991), 9.5 µl nuclease free water, and 1 µl dna template which was obtained directly from the isolates using a sterile toothpick. the pcr protocol was as follows: initial denaturation at 95 °c for 7 min, denaturation at 95 °c for 30 s, annealing at 49 °c for 1 min, elongation at 65 °c for 8 min, and post extention at 65 °c for 16 min (applied biosystems, 2720 thermal cycler). the pcr cycle was used 30 times. a 5 µl of pcr products was verified by electrophoresis for 90 min and 70 v, on 1% agarose in 1x tae buffer. formed band profiles were observed under the uv transilluminator. formed band profiles were then compared as biner number and analyzed using freetree (hampl et al. 2001) and treeview to construct a phylogenetics tree (http://taxonomy.zoology.gla.ac.uk/ rod/treeview.html. results isolation of k. pneumoniae. a total of 58 bacterial isolates (table 1) were isolated from emp and wjb tempeh. the bacterial colonies were purple in the center of colony, mucoid, and rounded shape on emb medium. the colonies of these isolates changed from green to blue on simmons’citrate medium, which is specific character of klebsiella sp. fig 1a and 1b showed hypermucoviscocity of k. pneumoniae atcc35657 or fk colonies when touched with inoculating loop. however, colonies of k. pneumoniae from emp (fig 1c) and wjb tempeh (fig 1d) did not show hypermucoviscosity. this character showed significant phenotypic difference between tempeh and those of medical isolates. analysis of 16s rrna genes. a total of 18 isolates from the emp tempeh and 25 isolates from wjb tempeh were selected for further amplification of genes encoding 16s rrna. based on 16s rrna gene sequence alignments with the ncbi database, k. pneumoniae were identified in 13 isolates obtained from emp tempeh and 10 isolates from wjb tempeh (table 1). the other microorganisms were found from emp tempeh based on 16s alignment analysis were: klebsiella sp., rhizobium sp., and enterobacter sp. while bacterium sp., klebsiella sp., and cronobacter sakazakii were isolated from wjb tempeh. genetic profiling of k. pneumoniae isolates. a total 23 isolates which were identified as k. pneumoniae were further subjected to eric-pcr analysis in order to compare their genetic diversity from those of medical isolates. eric-pcr of 13 isolates of k. pneumoniae from emp tempeh showed a similar pattern (fig 2). the eric-pcr profiles of medical isolates showed a diverse patterns but distinctively different from tempeh isolates. this result indicated that k. pneumoniae presence in tempeh was not the same as pathogenic k. pneumoniae. similar result were also found when we analysed isolates from wjb tempeh (fig 3) which showed different ericpcr profiles from the medical isolates. although genetic profiles of k. pneumoniae isolates in wjb tempeh were more varied than emp tempeh, we found noi dentical profiles when compared to those of medical isolates. the resulting electrophoresis band profiles were further converted into binary data matrix. the results were used to construct a phylogenetic tree using freetree program and treeview. fig 4 showed the genetic relationship of k. pneumoniae isolated from emp tempeh. based on this analysis, the medical isolates clustered into a separate and distict group. tempeh isolates formed two groups, the first one contains isolates which have similar eric profiles, i.e., i emp16, i emp13, i emp9, i emp8, i emp5, i emp4, and i emp. the other group contains of i emp1, ii emp5, ii emp2, ii emp4, ii emp1, and ii emp3. isolates from wjb tempeh formed a separate group under one branch except one isolate, i wjb2 (fig 5). again, in this analysis, the medical isolates formed a separate group outside the branches which contained isolates from wjb. discussion k. pneumoniae presence in both of tempeh samples (emp and wjb) and consistantly exist from the first volume 8, 2014 microbiol indones 11 tempeh sample number of sampling number of isolates sequencing of 16s rrna emb agar sca dna sequence alignment (k. pneumoniae) emp i 18 18 10 8 ii 8 8 8 5 total of emp 26 26 18 13 wjb i 17 10 10 7 ii 15 15 15 3 total of wjb 32 25 25 10 grand total 58 51 43 23 table 1 klebsiela pneumoniae from fresh tempeh microbiol indones12 ayu et al. and second sampling of each of these tempeh samples. the presence of k. pneumoniae in tempeh have been reported before (keuth et al. 1994, barus et al. 2008). growth of k. pneumoniae on emb medium showed distinctive colony morphology, which were dark purple in the center, mucoid, and rounded shape. c d a b fig 1 the phenotype of k. pneumoniae atcc 35657 (a), fk isolate (b), k. pneumoniae from emp (c), and wjb tempeh (d). 700 bp fig 2 eric-pcr from k. pneumoniae from emp tempeh, ie. (m) molecular markers, (a) atcc 10031, (b) atcc 2014, (c) atcc 35657, (d) fk, (e) klebsiella sp. 135, (f) i emp1, (g) i emp3, (h) i emp4, (i) i emp5, (j) i emp8, (k) i emp9, (l) i emp13, (n) i emp16, (o) ii emp1, (p) ii emp2, (q) ii emp3, (r) ii emp4, and (s) ii emp5. volume 8, 2014 microbiol indones 13 700 bp fig 3 eric-pcr profiles of k. pneumoniae from wjb tempeh, ie. (m) molecular markers, (a) atcc 10031, (b) atcc 2014, (c) atcc 35657, (d) fk, (e) klebsiella sp. 135, (f) i wjb1, (g) i wjb2, (h) i wjb3, (i) i wjb4, (j) i wjb5, (k) i wjb6, (l) i wjb16, (n) ii wjb3, (o) ii wjb5, and (p) ii wjb8. medical isolate medical isolates group 2 group 3 medical isolates group 2 group 3 medical isolate fig 5 philogenetic tree generated from eric-pcr profiles of k. pneumoniae isolated from wjb tempeh. fig 4 philogenetic tree generated from eric-pcr profiles of k. pneumoniae isolated from emp tempeh. disease associated with k. pneumoniae in tempeh. on the other hand, their presence in tempeh might be beneficial due to their ability to synthesize vitamin b12 and their immunomodulatory effects in human. to conlude tempeh produced in indonesia naturally harbors k. pneumoniae with unique genomic profiles. k. pneumoniae from tempeh samples in this study showed that they were genetically different from isolates known to be pathogenic to human. acknowledgment this work was supported by dana dipa ipb (code : 2013.089.521219). we also acknowlegde the department of microbiology, faculty of medicine, universitas katolik atma jaya for the fk isolate. references barry t, colleran g, glennon m, dunican lk, gannon f. 1991. the 16s/23s ribosomal spacer region as a target for dna probes to identify eubacteria. pcr methods appl. 1:51-56. doi:10.1101/gr.1.1.51. barus t, hanjaya i, sadeli j, lay bw, suwanto a, yulandi a. 2013. genetic diversity of klebsiella spp. isolated from tempe based on enterobacterial repetitive intergenic consensus-polymerase chain reaction (eric-pcr). hayati j biosci. 20(4):171-176. doi:10.4308/hjb.20. 4.171. barus t, suwanto a, wahyudi at, wijaya h. 2008. role of b a c t e r i a i n t e m p e h b i t t e r t a s t e f o r m a t i o n : microbiological and molecular biological analysis based on 16s rrna gene. microbiol indones. 2(1):1721. doi:10.5454/mi.2.1.4. efriwati, suwanto a, rahayu g, nuraida l. 2013. population dynamics of yeasts 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colonies of medical isolates showed more sticky on solid media than those isolated from tempeh. mutation in maga is one of the factors that mutants lost the hypermucoviscosity phenotype and became susceptible tophagocytes and a virulent to mice (fang et al. 2004). 16s rrna gene sequences are the most common house keeping genetic marker used for identifying bacteria in the laboratory (janda et al. 2007). although 16s rrna gene (approximately 1 500 bp ) is large enough for bioinformatics purposes, but usually it is not reliable for intra-strain differentiation. for example, escherichia coli o104: h4 that is known pathogenic as well as isolate in recent out breaking germany (mellmann et al. 2011) were confirmed only as e. coli based on 16s dna sequence analysis. we confirmed colonies obtained from tempeh samples as k. penumoniae based on 16s dna sequence analysis before subjected them for ericpcr analysis to reveal intra-species genetic diversity (barus et al. 2013). in this study, eric-pcr method has been successfully employed to differentiate genetic profiles of k. pneumoniae isolated from tempeh and those of medical isolates. the eric-pcr profiles of k. pneumoniae from emp tempeh (fig2) and wjb tempeh (fig3) were different from those of medical isolates. keuth and bispingin 1994 reported that k. pneumoniae isolated from indonesian tempeh were negative for three known enterotoxins, i.e. shiga-like toxin sltiia, heat-labil enterotoxin ltih, and heatstable enterotoxin stih. therefore, intraspecies genetic diversity within k. pneumoniae might reflect different phenotypes which could make the pathogenic or non-pathogenic. the phylogenetic analysis showed two separate clusters representing k. pneumoniae from tempeh and the medical isolates. the phylogenetic trees generated from emp tempeh showed unambiguously that tempeh isolates were genetically different from pathogenic k. pneumoniae (fig4). that figure showed three main groups were formed from tempeh isolates, 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perils, and pitfalls. j clin microbiol. 45(9):27612784. doi:10.1128/jcm.01228-07. keuth s and bisping b. 1994. vitamin b production by 12 citrobacter freundii or klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by pcr. appl environ microbiol. 60(5):1495-1499. khan aa, mccarthy s, wang rf, cerniglia ce. 2002. characterization of united states outbreak isolates of vibrio parahaemolyticus using enterobacterial repetitive intergenic consensus (eric) pcr and development of a rapid pcr method for detection of o3:k6 isolates. fems microbiol. 206(2):209-214. doi:10.1111/j.1574-6968.2002.tb11011.x. klus k and barz w. 1995. formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempeh. archives microbiol. 164(6):428-434. doi:10.1007/bf02529741. lane dj, pace b, olsen gc, stahl da, sogin ml, pace nr. 1985. rapid determination of 16s rrna sequences for phylogenetic analyses. pnas 82(20):6955-6959. doi:10.1073/pnas.82.20.6955. liem ith, steinkraus kh, cronk tc. 1977. production of vitamin b in tempeh, a fermented soybean food. appl 12 environ microbiol. 34(6):773-776. lupski jr and weinstock gm. 1992. short, interspersed repetitive dna sequences in prokaryotic genomes. j bacteriol. 174(14):4525-4529. marchesi jr, sato t, weightman aj, martin ta, fry jc, hiom sj, wade wg. 1998. design and evaluation of useful bacterium-specific pcr primers that amplify genes coding for bacterial 16s rrna. appl environ microbiol. 64(2):795-799. maysella. 2010. isolation and genetic diversity analysis of klebsiella poneumoniae in tempe based on 16s rrna encoding gene [thesis]. jakarta (id): faculty of biotechnology, atma jaya catholic university of indonesia. meacham kj, zhang l, foxman b, bauer rj, marrs cf. 2003. evaluation of genotyping large numbers of escherichia coli isolates by enterobacterial repetitive intergenic consensus-pcr. j clin microbiol. 41(11):5224-5226. doi:10.1128/jcm.41.11.5224-5226.2003. mellmann a, harmsen d, cummings ca, zentz eb, leopold sr, rico a, prior k, szczepanowski r, ji y, 4.mi-trismillah available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.4.4issn 1978-3477, eissn 2087-8575 vol 10, no 4, december 2016, p 139-148 *corresponding author; phone:+62-8161435792, fax: 7566922, email: trismilah@bppt.go.id +62 lipases are triacylglycerol acyl-hydrolases (ec 3.1.1.3) that catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids (verma et al. 2012). the enzymes are capable of hydrolyzing the ester bonds of water-insoluble substrates at the interface between the substrate and water. it is well known that the reaction is reversible and the enzymes catalyze both ester synthesis and transesterification. since lipases catalyze a number of different reactions, they have been widely used in such industries as food, chemical, and pharmaceutical, detergent industries as well as in leather processing industry, and also for producing better quality products as part of an eco-friendly process (selvamohan et al. 2012; hanifi et al. 2013; francoiset al. 2014). lipases are ubiquitous enzymes lipase is a lipids hydrolyze enzyme which are widely used in various industries such as chemical, pharmaceutical, food industries, and detergents. bacillus licheniformis f11.4 is one of the bacteria with potential source of lipase. this study aimed to obtain optimum production of lipase from b. licheniformis f11.4 by optimizing the composition of media and ph values w​​ ith fish flour as a replacement for peptone and yeast extract based medium. selection of the significant factors used a 2-level factorial design. the upper limit and lower limit of the selected factors was optimized using central composite design (ccd) and the data analysis was performed using the response surface methodology (rsm). fermentation was carried out in erlenmeyer at initial ph 8 and a temperature of 37 °c, using a shaker incubator at 150 rpm. a fermentation system for lipases production is considered optimal when its desirability value closes to 1. by using numerical optimization, an optimal medium could be obtained, i.e. consisting of olive oil (oo): crude palm oil (cpo) 0.14 % (w/v) and fish flour 2% -1 (w/v), at ph 8 and 150 rpm, which produced lipase with enzyme activity of 1.563 u ml and protein level of 0.08 -1 mg ml . furthermore, the results are verified in the erlenmeyer, working volume of 50 ml, ph = 8, t = 37 °c, -1 agitation 150 rpm, t=18 hours, the activity of lipase and protein levels are 1.568 ± 0.014 u ml and 0.072 ± 0.006 -1 -1 mg ml respectively.the results showed that the optimum condition lipase activity was 1.568 u ml so that the the activity of only 75% compared to that before optimization. key words: bacillus licheniformis f11.4, central composite design, enzyme activity, protein content, response surface methodology lipase merupakan enzim penghidrolisis lipida yang banyak digunakan di berbagai industri seperti kimia, farmasi, makanan, maupun aditif detergen. bacillus licheniformis f11.4 adalah salah satu bakteri yang potensial sebagai sumber lipase. penelitian ini bertujuan untuk mendapatkan produksi lipase yang optimal dari b. licheniformis f11.4 melalui optimasi komposisi media dan nilai phdengan tepung ikan sebagai pengganti pepton dan yeast ekstrak dalam medium. pemilihan faktor-faktor yang berpengaruh secara signifikan menggunakan desain faktorial 2 level. batas atas dan batas bawah faktor-faktor yang terpilih selanjutnya dioptimasi menggunakan central composite design (ccd) dan analisis data dilakukan dengan menggunakan respone surface methodology (rsm). fermentasi dilakukan di dalam erlenmeyer ph awal 8 dan suhu 37 °c, menggunakan shaker inkubator 150 rpm. sistem fermentasi untuk produksi lipase dikatakan optimal jika nilai desirability mendekati 1. dengan optimasi numerik nilai desirability mendekati satu, komposisi media fermentasi terbaik adalah minyak olive (oo) : minyak sawit mentah (cpo) = 0,14 % (b/v); tepung ikan = 2% (b/v) dan nilai ph = 8, agitasi 150 rpm, aktivitas enzyme dan kadar protein yang akan diperoleh masing-1 -1 masing adalah 1,563 u ml , dan 0,08 mg ml . selanjutnya hasil tersebut diverifikasi di dalam erlenmeyer, volume kerja 50 ml, ph = 8, t = 37 °c, agitasi 150 rpm, t = 18 jam, memberikan nilai aktivitas lipase dan kadar -1 -1 protein masing-masing adalah 1,568 ± 0,014 u ml dan 0,072 ± 0,006 mg ml . hasil penelitian menunjukkan -1 bahwa pada kondisi optimum aktivitas lipase adalah 1,568 u ml sehingga aktivitas hanya 75% dibandingkan dengan kondisi sebelum optimasi. kata kunci: aktivitas enzim, bacillus licheniformis f11.4., central composite design, aktivitas enzim, kadar protein, response surface methodology optimization of lipases production bybacillus licheniformis f11.4 using response surface methodology 1 2 1 trismilah *, prih sarnianto , and edi wahjono 1 center for bioindustrial technology, agency for the assessment and application of technology (bppt), laptiab, bld.610-612, puspiptek, south tangerang , 15314, indonesia; 2 faculty of pharmacy, universitas pancasila, srengseng sawah, jagakarsa, jakarta 12640, indonesia synthesized by all biological system, including animals, plants, and microorganisms (bacteria, fungi, yeast, and actinomycetes). microbial lipases have gained special attention due to their ability towards both extreme temperature and ph, organic solvents as well as chemo-, region-, and enantio-selectivity (devi et al. 2012; verma et al. 2012; kumar et al. 2007). among lipases producing bacteria, pseudomonas aeruginosa, enterobacter aerogenes and some species of bacillus such as b. subtilis, b. pumilis, b. licheniformis, b. thermoleovorans, b. stearothermophilus, and b. sphaericus produce lipases suitable for biotechnologi cal applications. widely known as a multipurpose organism, b. licheniformis has gained popularity along with b. subtilis. b. licheniformis, which is commonly found in soil and on the feathers of ground dwelling birds (maria et al. 2013), produces extracellular lipases. lipases production by b. licheniformisis affected by many physicochemical (particularly ph and temperature) and nutritional factors (the nature and availability of carbon and nitrogen substrates as well as lipids). in bacterial fermentation, peptone is usually used as the main source of nitrogen with yeast extract as the source of growth factors, including vitamins.to increase the production of lipases, olive oil is often used both as inducer and carbon substrate (niyonzima et al. 2013). however, peptone, yeast extract, and olive oil are quite expensive and not very easy to obtain. in the present study, peptone and yeast extract were entirely substituted with fish flour as a source of nitrogen, then olive oil and crude palm oil (cpo) can be used as a carbon source or inducer in media production lipase (limpon et al, 2012). to determine the optimal substrate composition as well as fermentation condition (ph, temperature), response surface methodology (rsm) was used along with central composite design (ccd) technique. rsm is the most popular optimization method used in recent years. there are so many works based on the application of rsm in chemical and biochemical process (deniz et al. 2007). rsm experimental design is an efficient approach to determine which explanatory variables (e.g. medium componentand/or fermentation condition) have impacts to the response variables of interests (e.g. growth rate, protein level or productivity) (box et al. 2005), and there are several reports on application of rsm for the production of primary and secondary metabolites through microbial fermentation (devi et al. 2012; dash et al. 2002). ccd technique contains an imbedded factorial or fractional factorial design with “center points” that is 140 trismilah et al. microbiol indones augmented with a group of “star points”, allowing estimation of curvature (sematech 2010). this study aimed to obtain optimum production of lipase from b. licheniformis f11.4 through optimization of media composition and ph values ​​with fish flour as a replacement for peptone and yeast extract based on sharma media that has been modified (sharma et al. 2012). materials and methods materials. b. licheniformis f11.4 is a collection of the laboratory of bioindustrial technologylaptiab, bppt, serpong. fish flour from cv pratama hikmah karya, cimahi, bandung, indonesia; olive oil (oo) a trademark of le riche; crude palm oil (cpo) a product of pt sinar mas, indonesia; tryptone, agar, peptone, yeast extract, nacl from himedia laboratories pvt. ltd., mumbai, india. other ingredients and equipment are commonly used for bioprocess research. regeneration of the microorganisms. a colony of b. licheniformis f11.4 was transferred from the stock culture onto luria bertani agar and incubated at 37 °c or 50 °c, ph 7, for 16 h. propagation of the microorganisms. one colony of regenerated b. licheniformis f11.4 was innoculated into luria bertani (lb) liquid media and incubated at 37 °c in a shaking incubator (kuhner) with constant agitation at 150 rpm, for 8-16 h, up to optical density (od) of 0.6-0.8. the suspension of bacterial cells were then used as bacterial starter in the production of lipases. the lb liquid media were prepared by dissolving into ro water adjusting its ph to 7 by adding hcl 1m or naoh 1m, and sterilizing the mixture in autoclave for 20 min at 121 °c. production of lipase. the bacterial starters (100 ml) were innoculated into liquid medium for lipase production (900 ml), incubated at 37 °c with initial ph of 8 and agitation at 150 rpm, for 18 h. the number of 7 -1 bacteria were minimum 10 cells ml , determined prior to the incubation (hour zero). the fermentation medium used was described by sharma with some modification (sharma et al. 2012) consisted of 3% pepton ,1% yeast extract, 0.3% nano ,0.01% kh po , 3 2 4 1% olive oil (oo), and 1% cpo. in the modification, peptone and yeast extract were substituted with fish flour of equal protein content. the protein content was determined by using modified lowry’s method. in the present study, two kind of fish flours, one made of bony fish (p) and the other of more-fleshy fish (n), were compared. regarding the inducer, the proportion of oo and cpo was modified to 0.25% (w/v) and 0.75% (w/v), respectively, in accordance with the results of the preliminary study on oo and cpo requirementf o r lipase production by b. licheniformis f11.4. harvesting of lipase: the enzyme harvesting were carried out by separating the supernatant from biomass through centrifugation at 10 000 rpm for 15 min at 4 °c. the supernatant, termed as crude (extracellular) enzyme fraction, was assayed for its lipase activity. assay for lipase activity. the crude enzyme’s lipase activity was determined using spectrophotome tric method. as the substrate, 3 mg of paranitrophenyl palmitate (p-npp) was dissolved in 1 ml isopropanol and mixed with 10 mg of arabic gum and 40 mg of triton x-100 in 9 ml of 0.05m tris-hcl buffer solution of ph 8. into 0.9 ml of substrate, 0.1 ml crude enzyme was added and then incubated at 37 °c in a thermoshaker with constant agitation at 300 rpm, for 30 min. the absorbance of the incubated mixtures was measured at λ410 nm against blank. lipase activity was calculated using the following formula (silva et al. 2005): where c = concentration of p-npp (mg ml-1) , t = time (minute), v = volume of crude enzyme (m), and molecular mass of p-npp (mr p-npp) = 377.52. protein content of the crude enzyme was analyzed by spectrophotometric method using bradford reagent (bradford 1976). optimization using rsm. in the present study, optimization using rsm was performed in two stages. in the first stage, two-level factorial design, the proportion of oo and cpo (0.1-0.5% w/v) as inducers, fish flour (1.8-5.4%) as the main source of nitrogen, and ph (8-10) were treated as independent variables with lipase activity as the response variable. the fermenting media also contained 3% nano and 0.1% 3 kh po ; and b. licheniformis f11.4. innoculated were 2 4 incubated at 37 °c with constant agitation at 150 rpm. the data were analyzed with design expert (version 7.1.5, stat-ease inc., usa) software. media composition that gave the highest lipase activities were used to determine the upper limit and lower limit of independent variables for the second stage optimization experiment that used central composite design (ccd). 3 in the second stage, factorial design 2 which was expanded with 6 starting points and 6 center points were used, allowing 20 combinations of oo, cpo, and fish flour. for each combination, fermentation was carried out in a 125 ml erlenmeyer flask using 25 ml fermenting media of ph 8, containing 3% nano and3 0.1% kh po , at 37 °c and 150 rpm for 18 h. 2 4 numerical optimization of the results was performed using the design expert software.the ideal desirability value in numerical optimization is 1.0. the results of the numerical optimization that close to the ideal desirability value are verified in the laboratory with a minimum of 6 replicates. results optimization using response surface methodology. in the first stage, optimization of lipase production was conducted by using a two-level factorial design experiment toward the three affecting factors, i.e. oo:cpo was coded a, fish flour was coded b, and ph was coded c, with lipase activity as the variable response. the design expert software was used to generate the experimental designs, of which produced 12 experimental design models (table 1). the statistical significance of the models in lipase activities of crude enzymes produced were evaluated using analysis of variance (anova) and the results are presented in table 2. from the first stage of research the two-level factorial, used to specify an upper limit and a lower limit on the central composite design (ccd) as shown in table 3. based on the data in table 3 of research second stage using composite design center (ccd) with 3 factors a, b, and c and two response variables are the activity and protein levels and resulting 20 combination of treatments as shown in table 4. further data enzyme activity and protein levels of 20 combinations of research results incorporated into the program design expert 7.1.5. and the results of the analysis can be seen in fig 1 and fig 2. the results of anova for lipase activity response can be seen in table 5, and the interaction between the media composition and ph value on the response of lipase activity can be seen in fig 3a, fig 3b, and fig 3c. the test results of analysis of variance (anova) response protein levels can be seen in table 6, while the interaction between the media composition and ph value on the response of lipase activity can be seen in fig 4a, fig 4b, and fig 4c. the results of numerical calculation models media optimization and the ph value based on the central volume 10, 2016 microbiol indones 141 -1 lipase activity (u ml ) = c × 1000 mrpnpp × t × v microbiol indones142 trismilah et al. model is larger than f table (28.96> 4.87) and p-value = 0.0094 <α = 0.05 shows a model of a significant or unacceptable to see the estimated effect of each variable and interaction factor with the response. the 2 coefficient of determination (r ) 0.9854 shows a high correlation between the influence of factors on the response variable. point arc curve (curvature) also showed a significant result so that the model can be used to define the design of the central composite design of experiments process (central composite design/ccd) to determine the optimal media composition in the production of lipase b. licheniformis f11.4. of the two level factorial experiments obtained the highest lipase -1 activity of 1.198 u ml in the combined treatment of 0.1% oo:cpo; 1.8% fish flour and 8 ph value is then used to determine an upper limit and a lower limit on ccd (table 3). from the central composite design (ccd) by design expert 7.1.5 program provides 20 combined treatment (table 4). results of analysis of variance design expert 7.1.5 in table 5 and table 6 is the response to enzyme activity and protein levels indicate that a significant model analysis with p values ​​<5%, which means that the model can be used for process composite design (ccd) and response surface method (rsm) using design expert 7.1.5 program delivering provide desirability 0.764 with composition 0.1% (w/v) oo:cpo; v2.04% (w/v) fish flour and ph 8. desirability value approaching 1 (one) obtained by trial and error using design expert 7.1.5 program. from the trial and error were obtained twelve combination with desirability value = 0.921. discussion factors that influence the production of lipase b. licheniformis f11.4. were determined by two level factorial experimental design. response lipase b. licheniformis f11.4. was observed on hour to 8, 14, and 18. the result of the interaction between the concentration oo:cpo, the concentration of fish flour and ph values ​​were analyzed using analysis of variance (anova), selected to give the highest activity on the observation hours to 18. the analysis results in table 2 show that the model of interaction between the concentration oo:cpo, the concentration of fish flour and the ph value to yield significant results. from the analysis of variance, the value of f of the table 1. generated experimental design models for lipase production by b. licheniformis f11.4 run oo:cpo [a] fish flour [b] ph [c] -1 activity, u ml 1 0.50 5.40 10.00 0.969 2 0.50 5.40 8.00 0.511 3 0.10 1.80 8.00 1.198 4 0.30 3.60 9.00 0.695 5 0.50 1.80 10.00 0.463 6 0.50 1.80 8.00 1.150 7 0.30 3.60 9.00 0.691 8 0.10 1.80 10.00 0.618 9 0 .30 3.60 9.00 0.751 10 0.10 5.40 8.00 0.875 11 0.10 5.40 10.00 0.933 12 0.30 3.60 9.00 0.801 factors affecting lipase production by b. licheniformis f11.4: a = ratio of olive oil and cpo = oo/cpo, upper limit 0.5% (w/v), lower limit 0.1% (w/v) b = fish flour p, upper limit 5.4 % (w/v), lower limit 1.8% (w/v) c = ph, upper limit 10, lower limit 8 volume 10, 2016 microbiol indones 143 second equation (quadratic) was obtained from design expert 7.1.5 (equation 2), which explains the data response on protein content. the accuracy of the model can also be seen from a comparison of the actual value of research with model predictions. prediction model (predicted) expressed as a straight line and the actual results of the study (actual) expressed as a box. from fig 1 and fig 2 can be seen that the actual value (of the box) scattered approach the predicted values (​​ straight line), it indicates the optimization of lipase production. addition of test results lack of fit to the model can be seen that there are no inaccuracies models, it can be proved of value for lack of fit obtained p value 0.052 (not significant) for the response to the activity and the p value 0.2136 (not significant) for response to the protein content, meaning that the regression model is accepted. second equation (quadratic) was obtained from design expert 7.1.5 (equation 1), which explains the data response on enzyme activity. table 2. anova of the factors affecting lipase production by b. licheniformis f11.4 table 3 the upper limit and lower limit factors for central composite design (ccd) factor name low actual high actual a oo:cpo (% w/v) 0.05 0.18 b fish flour (% w/v) 0.8 3.1 c ph 7 9 y = + 2.197 8.314 a + 0.209 b 0.411 c + 3.512 ab + 0.827 ac 0.032 bc + 5.702 a2 + 8.122 b2 – 0.021 c2.................................................... (eq.2) y = 20.024 + 5.773a + 0.338 b +5.219 c + 0.148 ab + 0.189 ac + 0.075 bc – 25.056 a2 – 0.255 b2 – 0.337 c2................................................... (eq.1) – microbiol indones144 trismilah et al. -1 table 4 the data activity (u ml ) and protein content (mg ) of were combined treatments of oo:cpo, fish flour and the ph value, the result of the central composite design (ccd) by the program design expert 7.1.5. -1 ml fig 1 distribution of actual and predicted values in the lipase production activity respon. volume 10, 2016 microbiol indones 145 saddle (saddle point) so that the optimum point of the curve a little bit difficult to determine. the calculation numerical models of media optimization and the ph value is based on the ccd and the analysis of rsm provides desirability value 0.764. trial and error using design expert 7.1.5 program there are twelve combinations that provide value desirability 0.921. (w/v) (w/v) f -1 ml -1 ml (w/v) (w/v) f validation study was conducted in 125 ml erlenmeyer, initial ph 8 fermentation time 18 h with the addition 0.3% nano and 0.1% kh po with six 3 2 4 replications, providing value lipase activity 1.568 ± -1 0.014 u ml and 0.072 ± lipase protein content of -1 0.006 mg ml or the value of a specific activity is -1 21.210 u mg . from india reported by sharma et al. 2012, produce lipase from b. licheniformis mtcc10498, inducers tween 80 0.5% w/v, ph 7.5, a temperature of 55 °c, 150 rpm, fermentation time of 72 -1 h the enzyme activity of 2.1 u ml . from india was also reported by devi et al. 2012, that for the as for the lower limit and upper limit, 0.010,14 % oo:cpo; 0.8 to 2 % ish flour and ph 7.8 to8; value will be the activity of 1.563 u and the protein content of 0.08 mg with media compositions 0.14% oo:cpo; 2 % ish flour and ph 8. deviation is low both in response to the activity as well as on the response of the protein content is evidenced by the standard deviation of anova each is 0:21 and 0:06. a low standard deviation shows that the model has good accuracy or the model correspond (fit model). according gaspersz (1995) the model is said to be proper if the assumption residual meet the assumption of normal distribution. if the residual plot spread randomly around zero and likely to approach a straight line (linear line) so as to be normally distributed. anova test results in table 5 obtained determina 2 tion coefficient (r ) 0.8824 for the response to enzyme activity and in table 6 for a response to the protein 2 content obtained determination coefficient (r ) 0.7312. it shows that 88% correlation activity response variable and 73.12% variable response to the production of lipase protein levels are influenced by independent variables (oo:cpo; fish flour: ph value). the optimum area of independent variables (factors) that produces the maximum response can be seen from the threedimensional contour plot curves in fig 3a that the interactions between fish flour, the ph of the enzyme activity showed that the parabolic curve is obtained. in fig 3b; 3c and fig 4a; 4b; 4c curve obtained shaped fig 2 distribution of actual and predicted values in the lipase production protein content respon. microbiol indones146 trismilah et al. -1 specific enzyme activity of 32.2 u mg . the results of the optimization study b. licheniformis f11.4 lipase production gives the -1 enzyme activity 1568 ± 0014 u ml , lipase protein -1 content of 0.072 ± 0.006 mg ml and a specific enzyme -1 activity of 21.77 u mg . the optimum medium composition of fishflour 2% (w/v), olive oil 0.07% (w/v), cpo 0.5% (w/v), nano 0.3% (w/v), and 3 kh po 0.1% (w/v) in the working volume of 50 ml 2 4 production of lipase b. subtilis with inducers tween 80 and using the method rsm. media optimization -1 results with yeast extract 9.3636 g ml , cacl 0.8986 g -1 ml and incubation periods 1.813 d, gives the activity -1 -1 enzyme 16.627 u min ml . anahita et al (2011) from malaysia also reported that the research of production the lipase acinobacter sp. in submerged fermentations using rsm at the optimum condition at the fermentation time 24 h, t = 29 °c, ph = 6, the value of a table 5 anova lipase activity response of experimental design ccd fig 3 interaction oo: cpo, fishflour, ph on lipase activity response analysis results surface method (rsm) using the program design expert 7.1.5 volume 10, 2016 microbiol indones 147 assistance during the completion of this study and ptb-bpp technology, which has provided funding and laboratory facilities for this study. references anahita k, afshin e, boon kb, and oi ml.2011. production of a solvent, detergent, and thermotolerant lipase by a newly isolated acintobacter sp. in erlenmeyer with the operating conditions of ph 8, temperature 37 °c, agitation 150 rpm and fermentation time of 18 h. the increase in the activity of only 75% -1 compared to before the optimization ie 1,176 u ml . acknowledgments we thank tiara purnamasari tirtarasa for her table 6 anova response lipase protein content of experimental design ccd fig 3 interaction oo:cpo, fish flour, ph on lipase activity response analysis results surface method (rsm) using the program design expert 7.1.5 bacillus sp isolated from hotspring of arunachal pradesh, india. braz j microbiol. 43(1):30-42. doi:10.1590/s1517-83822012000100004 . maria g, asma a, afsheen a, rashida rz, nadir ns, and shah aulq.2013. isolation and characterization of different strains of bacillus licheniformisfor the production of commercially significant enzymes. pak j pharm sci. 26(4):691-697 691. niyonzima fn, more ss.2014. concomitant production of detergent compatible enzymes by bacillus flexus xju-1. braz j microbiol. 45(3):903-910. doi:10.1590/s151783822014000300020. prasanth kmp, valsa ak. 2007. optimization of culture media and cultural conditions for the production of extracellular lipase by bacillus coagulans. indian j biotechnol. 6(1):114-117. selvamohan t, ramadas v, sathya ta. 2012. international journal of modern engineering research (ijmer). 2(6):4231-4234. sematech. 2010. engineering statistic handbook. http://itl,nist.gov/div898/handbook/pri/section3/pri33 61.htm. sharma chander k, kanwar shamsher s.2012.purification of a novel thermophilic lipase from b. licheniformis mtcc-10498. isca. j biol sci. 1(3):43-48. silva wdb, mitidieri s, schrank a, vainstein mh. 2005. production and extraction ofan extracelluler lipase from the entomopathogenic fungus metharizumanisopliae. process biochemistry 40(1):321-326. doi:10.101 6/j.procbio.2004.01.005. verma n, thakur s, bhatt ak. 2012. microbial lipases: industrial applications and properties (a review). international research journal of biological sciences. international science congress association.issn 2278-3202. 1(8):88-92. submerged and solid-state fermentations. j biomed biotechnol. hindawi publishing corporation, article id 702179, 12 pages. doi:10.1155/2011/702179. bradford m. 1976.a rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. anal biochem.72(1-2):248-254. doi:10.1016/00032697(76)90527-3. box gep, hunter js, hunter wg . 2005.statistic for experiments : design, innovation and discovery. new jersey : john willey & sons. dash vv, panda t . 2002. optimization of microbiological parameters for enhanced griseofulvin production using response surface methodology. bioprocess engineering 22(1):45-49. doi:10.1007/pl00009099. deniz b, ismail h. boyacı. 2007. modeling and optimization i: usability of response surface methodology. journal of food engineering 78(3):836-845. doi:10.1016/j.jfoodeng.2005.11.024. devi a, devi kc, rajendiran r. 2012. optimization of lipase production using bacillus subtilis by respone surface methodology. world academy of science, engineering and technology. int j biological, veterinary, agricultural and food engineering. 6 (9):840-845. francois n. niyonzima and sunil s.more. 2013. screening and identification of a novel alkaline lipase producing bacterium. int j pharm bio sci. 4(2):1037-1045. gaspersz v. 1995. teknik analisis dalam penelitian percobaan. bandung (id): tarsito pr. hanifi b, mustafa d, mehmet k.2013.environmentally friendly detergent production. research in civil and environmental engineering (rcee).1(03):169-176. limpon b, minakshi b. 2012. optimization of extracellular thermophilic highly alkaline lipase from thermophilic microbiol indones148 trismilah et al. 1: 139 2: 140 3: 141 4: 142 5: 143 6: 144 7: 145 8: 146 9: 147 10: 148 4.mi693-rofiq sunaryanto black available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.2.4issn 1978-3477, eissn 2087-8575 vol 7, no 2, june 2013, p 68-74 *corresponding author; phone/fax: +62-21-7560208; email: rofiqsn@yahoo.com starch production from sago palm is common in the asia pacific region and south east asia (singhal et th al. 2008). at the end of 20 century, it was estimated that about 60 million tons of sago starch, derived from sago palms, were produced annually in south-east asia alone (wee et al. 2011). the yield of sago starch is higher (25 tons starch/ha/year) than other crops like cassava (2 tons) and maize (1 ton) (karim et al. 2008). due to its abundance, sago may be utilized for the production of fermentable sugars. this can be accomplished economically at large scale with the use of starch-saccharifying enzymes. adinarayan and suren (2005) have reported that hydrolysis using starchsaccharifying enzymes have replaced the conventional acidic hydrolysis method. these enzymes account for approximately a 15% share in the global enzyme market. products of starch hydrolysis such as maltodextrin, corn syrup, glucose syrup, and high glucose syrup have a wide application in the food, textile, brewing, ethanol, and pharmaceutical industries (soto et al. 2012). the sugars from sago starch can be used as feedstocks in fermentation industries, ethanol fermentation and also for the production of highfructose syrup (suraini 2002). an example of the enzymes that might be involved in the starch hydrolyitic process is glucoamylase (ec 3.2.1.3), which hydrolyzes starch into reducing sugars such as glucose and maltose. fermentation-derived ethanol can be produced from sugar, starch or lignocellulosic biomass. sugar and starch-based feedstocks are currently predominant at the industrial level and, so far, economically favourable (subashini et al. 2011). starch-based materials are currently most utilized for the ethanol production in north america and europe (kim and dale 2004). the hydrolysis of starch may be considered as a key step in the processing of starch-based feedstock for the bioethanol production (drapoch 2008). this step plays a role in effectively converting two major starch polymer components, amylose, a mostly linear α-d-(14)-glucan,and branched amylopectin,to fermentable sugars that could subsequently be converted to ethanol by yeasts or bacteria (woiciechowski et al. 2002). the a series of studies on the hydrolysis of sago starch for ethanol fermentation had been conducted. hydrolysis of sago starch was carried out using sulfuric acid 2.5% and amylase(s) enzymes. the concentrations of sago starch used in this experiment were 5, 10, 15, 20, and 30% (w/v). the highest hydrolysate containing reducing sugar was used as substrate for ethanol fermentation by saccharomyces cerevisiae fncc 3012. the results indicated that hydrolysis using 2.5% sulfuric acid for 120 min at 121 °c produced 6.6% (w/v) reducing sugar and hydrolysis using α-amylase and dextrozyme dx produced more reducing sugar 7% (w/v) and 17.1% (w/v), respectively. the fermentation of hydrolyzed sago starch by s. cerevisiae fncc 3012 produced ethanol 7.98% (v/v). key words: enzyme and acid hydrolysis, ethanol fermentation, saccharomyces cerevisiae, sago starch telah dilakukan penelitian hidrolisis pati sagu dan fermentasi hidrolisatnya menjadi etanol. hidrolisis pati sagu dilakukan dengan menggunakan asam sulfat encer 2,5% dan enzim amilase. hidrolisa pati sagu dilakukan pada konsentrasi 5, 10, 15, 20, and 30% (b/v). hidrolisat dengan gula pereduksi tertinggi digunakan sebagai substrat untuk fermentasi etanol dengan menggunakan strain saccharomyces cerevisiae fncc 3012. hidrolisis menggunakan asam sulfat 2,5% pada suhu 121 °c selama 120 menit menghasilkan gula pereduksi 6,6% (b/v) sedangkan hidrolisis dengan menggunakan α-amylase dan dextrozyme dx menghasilkan konsentrasi gula pereduksi tertinggi berturut-turut sebesar 7% (b/v) dan 17,1% (b/v). fermentasi hidrolisat pati sagu menggunakan isolat s. cerevisiae fncc 3012 menghasilkan etanol dengan konsentrasi sebesar 7,98% (v/v). kata kunci: fermentasi etanol, hidrolisa enzim dan asam, pati sagu, saccharomyces cerevisiae enzymatic and acid hydrolysis of sago starch for preparation of ethanol production 1 2 2 rofiq sunaryanto *, berti hariasih handayani , and ratu safitri 1 center of biotechnology bppt, kawasan puspiptek serpong, tangerang selatan 15314,banten, indonesia; 2 deparment of biology, faculty of mathematics and natural sciences, universitas padjadjaran, jalan raya bandungsumedang km 21 jatinangor, sumedang 45363, west java, indonesia volume 7, 2013 microbiol indones 69 most commonly used distiller's yeast cerevisiae is unable to hydrolyse starch. traditional production of ethanol from starch requires a three-stage process; liquefaction of starch by α-amylase, saccharification of liquefied starch by enzymes to sugars followed by fermentation using s. cerevisiae. amylolytic enzymes from bacteria and fungi are used for the saccharification of starch and this adds to the overall cost of the bioethanol production process (yetty et al. 2000). in this study, 2.5% sulfuric acid, α-amylase, and also dextrozyme dx (contaning glucoamylase and pullulanase) were used to hydrolyze sago starch into reducing sugars, which were then used for ethanol fermentation by s. cerevisiae fncc 3012. the purpose of this study was to determine the degree of sago starch hydrolysis by acidic and enzymatic methods and to determine the ethanol production profile using the hydrolyzed sago starch. material and methods preparation of acid-treated sago starch. sago starch of 5, 10, 15, 20, and 30 g were dissolved in 200 ml demineralized water. sulfuric acid 2.5% were added to adjust to ph range 1.0-2.0. the starch suspension was then refluxed at 121°c for 120 min. after cooling, naoh was added to the starch to neutralize the ph. the final volume was noted. during incubation, samples were taken every 20 min (at 0, 20, 40, 60, 80, and 120 min). the reducing sugars concentration was measured. preparation of enzyme-treated sago starch. there were two stages in enzymatic sago starch hydrolysis, liquefaction and saccharification. -1 liquefaction was performed using 1.5 μl g (enzyme -1 volume/weight of substrate) α-amylase (8 222 u ml ) from bacillus amyloliquefaciens a7595 (sigma) at 85 °c for 140 min and agitation 150 rpm. during incubation, samples were withdrawn every 20 min (at 0, 20, 40, 60, 80, 100, 120, and 140 min) and the reducing sugars concentration was measured. the product of the liquefaction process was then used as a substrate in saccharification. prior to saccharification, the substrate temperature was cooled down from 85 °c to 61°c. then, the ph of the substrate was set at about 4.3 using 2 n hcl solution. a beaker glass containing the substrate was placed in a shaking-waterbath for 15 min at 61°c, 150 rpm -1 agitation speed. then 0.7 μl g dextrozyme dx (24 -1 904 u ml ) (volume of enzyme/weigh of substrate) saccharomyces was added followed by further incubation for 60 h at the same condition. dextrozyme dx is the one of a novozymes product, that is a mixture of glucoamylase (produced by aspergillus niger) and pullulanase (produced by bacillus licheniformis). while incubating, samples were withdrawn every 12 h (at 12, 24, 36, 48, and 60 h). the reducing sugars concentration was measured. determination of -amylase activity. prior to the enzymatic reaction, a aliquot of the soluble starch solution (1.9 ml, 2% (w/v) in acetate buffer ph 5) was heated at 85 °c for 15 min in a shaking-waterbath. the enzymatic reaction was initiated by adding 0.1 ml of the α-amylase solution to the pre-heated substrate. the mixture was then incubated for 20 min at 85 °c and 150 rpm. the reaction was stopped by heating in boiling water for 5 min, followed by cooling on the room temperature. afterwards, the reduction sugar was analyzed using method specified by nelson and somogyi (1952). one unit of α-amylase activity was defined as the amount of α-amylase required to liberate 1 μmol of reduction sugar (maltose) from starch per minute at 85 °c, ph 5. determination of dextrozyme dx activity. prior to enzymatic reaction, a soluble starch solution (1.9 ml 2% (b/v)) in acetate buffer ph 5 was heated at 61°c for 15 min in a shaking-waterbath. the enzymatic reaction was initiated by adding 0.1 ml of the dextrozyme dx solution to the pre-heated substrate. the mixture was then incubated for 20 min at 61°c, 150 rpm. the reaction was stopped by heating in boiling water for 5 min, followed by cooling at room temperature. afterwards, the reducing sugar was analyzed using method specified by nelson and somogyi (1952). one unit of dextrozyme dx activity was defined as the amount of enzyme required to liberate 1 μmol of reduction sugar (glucose) from starch per minute at 61°c, ph 5. ethanol fermentation. hydrolyzed sago starch (saccharified) containing the highest concentration of reducing sugar is used as fermentation substrate. hydrolyzed sugar was filtered using standard filter paper. then, the concentration of the reducing sugars in the substrate was set between 14-18% (w/v) (putri and sukandar 2008). nutritions, in the form of 4% (w/v) ammonium sulfate [(nh ) so ] and 1% (w/v) peptone, 4 2 4 were added. afterwards, the ph was adjusted to 4.5-5.0 using 0.1 n hcl. fermentation substrates were sterilized in an autoclave for 15 min at 121°c, 1 atm. five ml culture of s. cerevisiae fncc 3012 5 containing 2 × 10 cells was used to inoculate 50 ml α 70 sunaryanto et al. microbiol indones fermentation substrate (9% (v/v) inoculum size). then, the culture was incubated while shaking for 48 h at 28 °c, 150 rpm. samples were obtained every 12 h (at 0, 12, 24, 36, and 48 h). analysis of ethanol concentration. ten ml of fermentation broth was centrifuged at 5000 rpm for 30 min and the supernatant was used to estimate the ethanol concentration. ethanol was analyzed by gas chromatography (gc-14a shimadzu) equipped with carbowax column at 150 °c, flame ion detector (fid) 200 °c and injector 200 °c. nitrogen was used as 3 -1 carrier gas at flow rate 30 cm min and the combustion gas was a mixture of hydrogen and air. analysis of reducing sugar. the reducing sugar concentration in the samples was estimated using method specified by nelson and somogyi (1952). twenty five ml of sample was withdrawn at regular intervals cooled to room temperature and then filtered through muslin cloth. the filtrate was centrifuged at 3000 rpm for 15 min. the clear supernatant of the hydrolysate was taken for determination of reducing sugar. sugar was extracted with 80% ethanol twice. the supernatant was evaporated by keeping it in water bath at 80 °c and the residue was dissolved by adding 10 ml water. aliquots (0.1 ml) of each sample was pipetted into test tubes and simultaneously 0.2, 0.4, 0.6, 0.8, and 1 ml of working standard were pipetted out into test tubes. volumes of samples and standards were made up to 2 ml with distilled water. one ml of alkaline copper tartarate reagent was added to each tube containing 1 ml of distilled water blank. the tubes were placed in boiling water for 10 min and after cooling 1 ml of arsenomolybdate reagent was added. finally, the volume was made up with distilled water to 10 ml and left for 10 min. absorbance was read at 620 nm using spectrophotometer and the values were plotted in a graph against concentration. reducing sugars present in the sample were calculated and expressed in terms of percentage. results effect of different sago starch concentrations on the saccharification using sulphuric acid at different time intervals. effect of different concentrations of sago starch on saccharification by 2.5% h so was tested at different time points with 20 2 4 min intervals. the maximum amount of reducing sugar observed at 2.5% h so showed that both 20% (w/v) 2 4 and 30% (w/v) sago starch produced the highest concentrarion of 6.6 % (w/v) reducing sugar (fig 1a). at sago starch concentration range 5%-20% (w/v) there was proportional correlation between starch concentration and reducing sugar, where higher starch concentration produced higher concentration of reducing sugar (fig 1a). this is reasonable since higher concentration of starch meant there were more contacts between molecules of starch and acid, so more reducing sugar is produced. but this did not occur at 30% (w/v) sago starch concentration. substrate viscosity might also affect the hydrolysis process. sago starch of 30% (w/v) seemed thicker and stickier than sago starch 20%, 15%, 10%, and 5% (w/v). however, the amount of reducing sugar produced from this acid hydrolysis was low. effect of different sago starch concentrations on liquefaction using α-amylase at different time intervals. prior to being used in the experiments, the volumetric activities of the α-amylase and dextrozyme dx were determined using soluble starch as substrate. the volumetric activies of the α-amylase and -1 -1 dextrozyme dx were 8 222 u ml and 24 904 u ml , respectively. effect of different sago starch concentrations on the liquefaction using α-amylase was tested at different time points with 20 min time intervals. the maximum amount of reducing sugar (7% (w/v)) was observed when 20% (w/v) sago starch and 140 min production time was used (fig 1b). this result was better than when other sago starch concentrions (30%, 15%, 10%, and 5% (w/v)) were used, attaining 6, 5.2, 3.8, and 1.3 % (w/v), respectively. higher substrate concentration would normally produce more product. however, in this case, 30% (w/v) sago starch produced less reducing sugar in comparison to 20% (w/v) sago starch. this might be related to the viscosity of the substrate solution. high viscosity will affect the substrate-enzyme interaction, thus reducing the amount of reaction product. effect of sago starch different concentrations on saccharification using dextrozyme dx at different time intervals. effect of different sago starch concentrations on saccharification using dextrozyme dx was tested at different time points with 10 h intervals. the maximum amount of reducing sugar was observed at 20% sago starch. the result was higher than when 30%, 15%, 10%, and 5% (w/v) sago starch were used (fig 1c). apart from having the best effect on the liquefaction process, 20% (w/v) sago starch also produced the highest concentration of reducing sugar (17.1% (w/v)). this was better than 30%, 15%, 10%, and 5% (w/v) volume 7, 2013 microbiol indones 71 -1 fig 1 profile of sago starch hydrolyses by 2.5% h so (a), liquefaction using α-amylase (enzyme activity 8 222 u ml ) (b), 2 4 -1 and saccharification using dextrozyme dx (enzyme activity 24 904 u ml ) (c), at different concentrations (5, 10, 15, 20, and 30% (w/v)) of sago starch. : starch 5%; : starch 10%; :starch 15%; : starch 20%; : starch 30%. 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 140 r ed u ci n g s u g ar ( % ) a time (min) 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 140 160 time (min) r ed u ci n g s u g ar ( % ) b 0 2 4 6 8 10 12 14 16 18 0 10 20 30 40 50 60 70 time (h) r ed u ci n g s u g ar ( % ) c 72 sunaryanto et al. microbiol indones by saccharification using 2.5% h so was 6.6% (w/v). 2 4 acid breaks down starch molecules at random and generates more reducing sugars. theoretically, hydrolysis of starch into glucose produces 60% glucose (woiciechowski et al. 2002). however, in this study the highest concentration of reducing sugar obtained was 6.6% (w/v). the major products of glucose decomposition that were identified as 50hydroxymethylfurfural (hmf), 1,6-anhydroglucose, levulinic acid, and formic acid (xiang et al. 2004). effect of different sago starch concentrations on liquefaction by α-amylases showed that 20% (w/v) sago starch produced the highest concentration of reducing sugar (7% (w/v)), followed by 30% (w/v) sago starch that produced 6 (w/v)% reducing sugar. αamylases (1,4-d-glucan glucanohydrolases) are endohydrolases which cleave 1,4-α-d-glucosidic bonds and can bypass but cannot hydrolyse 1,6-α-dglucosidic branchpoints. commercial enzymes used for the industrial hydrolysis of starch are produced by b. amyloliquefaciens. the principal requirement for liquefaction is to reduce the viscosity of the gelatinized starch to ease subsequent processing. at liquefaction process, “maltodextrin” is obtained which contains mainly different oligosaccharides and dextrins. maltodextrins are only slightly sweet and they usually sago starch, which produced only 16, 12, 8.2, and 4 % (w/v) reducing sugar, respectively (fig 1c). figure 1c showed that the saccharification using 20% (w/v) sago starch produced more reducing sugar than 30% (w/v) sago starch. similarly to liquefaction process, the high viscosity of sago starch reduced the possibility of substrate-enzyme interaction, thus the conversion yield was lower. in the subsequent study, saccharification using 20% (w/v) sago starch were used. the ethanol fermentation was conducted by s. cerevisiae fncc 3012 for 48 h at 28 °c, 150 rpm. the highest sugar consumption rate occurred in the logarithmic phase (fig 2). at the same time, cell growth was also high and ph decreased. ethanol production occurred simultaneously with cell growth. stationary phase was achieved after 36 h, where there was no more increase of cell number and the rate of ethanol production was constant. at the same time, sugar consumption declined. declining of sugar concentration had slowed down the cell growth and many cells started to lyse. maximum ethanol concentration (7.98% (w/v)) was attained after 36 h. discussion the maximum amount of reducing sugar obtained 0 2 4 6 8 10 12 14 16 18 0 10 20 30 40 50 60 time (h) e th an o l( % , v /v ), r ed u ci n g s u g ar ( % , w /v ), p h 0 20 40 60 80 100 120 6 c el l n u m b er × 1 0 fig 2 time course of ethanol fermentation using hydrolyzed sago starch by saccharomyces cerevisiae fncc 3012. :ethanol % (v/v); : ; : ph ; and : cell number. reduction sugar (%) (b/v) volume 7, 2013 microbiol indones 73 undergo further conversion. various manufacturers use different approaches to starch liquefaction using αamylases but the principles are the same. on the saccharification step using dextrozyme dx, 20% (w/v) sago starch produced the highest reducing sugar concentration (17.1% (w/v)). dextrozyme dx containing amyloglucosidase-pullulanase enzyme. a glucoamylase is used to further break down the maltodextrins. the glucoamylase can hydrolyze starch completely to glucose along with, a little maltose and isomaltose. a pullulanase is a de-branching enzyme that can also be used to aid saccharification. reducing sugars of various sizes are formed when starch is converted to fermentable sugars. normally, higher reducing sugar levels indicates higher proportion of smaller sugar molecules in the mixture, which are easier to ferment. in the ethanol fermentation, the reducing sugars concentration in the substrate was set between 14-18% (w/v). this concentration was selected according to previous results published by others (putri and sukandar (2008)). putri and sukandar (2008) had produced 8.6% (v/v) ethanol from s. cerevisiae fncc 3012 fermentation using 14% (w/v) hydrolyzed ganyong starch as carbon source. our study using the same isolate but instead, hydrolyzed sago starch, as carbon source produced 7.98% (w/v) ethanol. the ethanol formation occurred along with lag and exponential phase and showed maximum concentration after the growth reached stationary phase. this indicates that ethanol production was associated with cell growth. at the exponential phase, sugar consumption was highest and ph decreased sharply. decomposition of sugar releases proton and causing ph to decrease sharply. the highest ethanol concentration (7.98% (v/v)) was obtained after 48 h cultivation. however, after 36 h the productivity of ethanol had already slowed down and the cell number no longer increased. the low productivity of ethanol can be affected by several factors; first of all, the ability of isolates to produce ethanol (type of isolate), especially for wild type strain such as s. cerevisiae fncc 3012, which had never been tested as industrial strain, secondly, the presence of product (ethanol) inhibition. in the previous study, s. cerevisiae fncc 3012 had been used to produce ethanol (8.98% (v/v)) using nipah (nypa fruticans) sap as carbon source (trisasiwi et al. 2011). according amenaghawon et al. (2012) the inhibitive effect of ethanol can affect the specific growth rate, product yield and specific ethanol production rate during batch ethanol fermentation. solid state fermentation (ssf) method enables attainment of higher ethanol yields by removing endproduct inhibition like ethanol inhibition (lin and tanaka 2006). however, in this study we did not carry out the solid state fermentation for ethanol production. references adinarayaan k and suren s. 2005. response surface optimization of enzymatic hydrolysis of maize starch for higher glucose production. biochem eng j. 6(3):179190. doi:10.1208/pt060348. amenaghawon na, okieimen co, ogbeide s. 2012. kinetic modelling of ethanol inhibition during alcohol fermentation of corn stover using saccharomyces cerevisiae. inter j eng res appl. 2(4):798-803. drapoch. 2008. biofuel feed stock. new york: mcgraw-hill companies inc. p 69-78. karim aa, tie ap, manan dma, zaidul ism. 2008. starch from the sago (metroxylon sagu) palm tree; properties, prospects, and challenges as a new industrial source for food and other uses. rev food sci food safety. 7(3): 215-228. doi: 10.1111/j.1541-4337.2008.00042.x. kim s, dale be. 2004. global potential bioethanol production from wasted crops and crop residues. biomass bioenerg. 26(4):361-375. lin y, tanaka s. 2006. ethanol fermentation from biomass resources: current state and prospects. appl microbiol biotechnol. 69(6): 627-642. doi:10.1007/s00 253-005-0229-x. nelson n, somogyi m. 1952. a photometric adaptation for the somogyi method for the determination of glucose. j biol chem 153: 375-380. putri lse, sukandar d. 2008. starch conversion of ganyong (canna edulis ker.) to bioethanol using acid hydrolysis and fermentation. biodiversitas 9(2):112-116. ren q, huang y, ma h, wang f, gao j, xu j. 2013. convertion of glucose to 5-hydroxymethylfurfural catalyzed by metal halide in n,n-dimethylacetamide. bioresources 8(2):1563-1572. singhal r s, kennedy j f, gopalakrishnan sm, kaczmarek a, knill cj, akmar pf. 2008. industrial production, processing, and utilization of sago palm-derived products. carbohyd polym. 72(1):1-20. doi:10.1016/j. carbpol.2007.07.043. somda mk, savadogo a, ouattara cat, ouattara as, traore as. 2011. improvement of bioethanol production using amylasic properties from bacillus licheniformis and yeasts strains fermentation for biomass valorization. asian j biotechnol. 3(3):254261. soto jlm, garcia lm, gonzalez jv, nicanor ab, cruz lg. 2012. influence of starch source in the required hydrolysis time for the production of maltodextrins with different dextrose equivalent. afr j biotechnol. woiciechowski al, nitsche s, pandey a, soccol cr. 2002. acid and enzymatic hydrolysis to recover reducing sugars from cassava bagasse: an economic study. braz arch biol technol. 45(3):393-400. doi:10.1590/s151689132002000300018 . xiang q, yong y, lee, torget rb. 2004. kinetic of glucose decomposition during dilute acid hydrolysis of lignocellulosic biomass. app biochem biotechnol. 115(1-3):1127-1138. doi:10.1385/abab:115:1-3:1127. yetti m, saari n, hassan z, radu s. 2000. improvement in raw sago starch degrading enzyme production from acremonium sp. endophytic fungus using carbon and nitrogen sources. enzyme microbiol tech. 27(7):511515. doi:10.1016/s0141-0229(00)00243-x. 11(69):13428-13435.doi: 10.5897/ajb12.2257. subashini d, ejilane j, radha a, jayasri ma, suthindhiran k. 2011. ethanol production from sago waste using saccharomyces cerevisiae vits-m1. curr res j biol sci. 3(1):42-51. suraini aa. 2002. review: sago starch and its utilization. j biosci bioeng. 94: 526-529. trisasiwi w, asnani a, setyawati r. 2011. optimization of bacterial doses and incubation time on bio-ethanol fermentation of nipah (nypa fruticans) for biofuel energy. j life sci. 5(12):1022-1029. wee ll, annuar msm, ibrahim s. 2011. energetics of glucoamylase-catalyzed hydrolysis of commercial sago starch. asia pac j mol biol biotechnol. 19(4):117-120. 74 sunaryanto et al. microbiol indones 5.mi678-ekowati chasanah available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.1.5issn 1978-3477, eissn 2087-8575 vol 7, no 1, march 2013, p 37-44 *corresponding author; phone: 62-21-53650157, fax: +6221-53650158, email: ekowatichasanah@gmail.com aaptos sp., one of marine sponges that can be found abundantly in indonesian waters, has been reported to produce bioactive metabolite against tumor, pathogenic microbes, and herpes simplex virus type i (hsv-1) (coutinho et al. 2002). it was detected that the compounds produced were alkaloids, e.g aaptamin, aaptosin and isoaaptamine; the latter compound showed potent activity against human immunodeficiency virus type i (hiv-1) (gul et al. 2006). aaptos sp. was also been reported to produce sterol compounds (rachmat and muniarsih 2001), and other novel aaptamine alkaloids possesing various biological activities, including cytotoxic against murine lymphoma l5178y cell line, antiviral, antimicrobial, antifungal, antiparasitic, α-adrenergic antagonistic, radical scavenging, and antifouling activity (pham et al. 2013). sponge such as aaptos sp. is very rich in microorganism including bacteria associated within their body. about 40 % of sponge body is bacteria, and its role is very significant in the sponge metabolism (taylor et al. 2007). the sponge produces similar metabolites from their associated bacteria (radjasa et aaptos sp. is a marine sponge that could produce bioactive compounds such as aaptamin, aaptosin, and isoaaptamin which have activities as antitumor, antimicrobial, and antiviral. community of bacteria associated with the sponge might correlate with production of those bioactive compounds and be affected by water environment where the sponge grow. the presence of anthropogenic stressor such as pollutans might become a burden to the waters where the biota grown and could affect the microbial biodiversity in the sponge and its active metabolite produced. the objective of this research was to analyze bacterial community associated with aaptos sp. from rote island and seribu islands, using t-rflp method. the results showed that bacterial community associated with aaptos sp. from both sampling sites shared 40.81% similarity in which they were dominated by the same bacteria class of actinobacteria, flavobacteria, α-proteobacteria, δ-proteobacteria, and γ–proteobacteria. the bacteria collected from rote island were more highly distributed and diverse than those from seribu islands. a total of 23 classes of microorganism were identified in rote island waters, while in seribu islands was 14 classes of microorganism. the presence of actinobacteria and proteobacteria in aaptos sp., is allegedly involved in the production of secondary metabolites. key words: aaptos sp., microbial diversity, rote island, seribu islands, t-rflp aaptos sp. merupakan spons laut yang dapat memproduksi senyawa bioaktif seperti aaptamin, aaptosin, dan isoaaptamin yang memiliki aktivitas sebagai antitumor, antimikrobial, dan antiviral. komunitas bakteri yang berasosiasi dengan spons tersebut kemungkinan berkorelasi dengan produksi komponen bioaktif dan akan dipengaruhi oleh perairan dimana spons tumbuh. adanya stresor yang disebabkan oleh manusia seperti polutan akan menambah beban perairan ditempat biota tumbuh yang dapat mempengaruhi keragaman mikroba dalam spons dan metabolit aktif yang diproduksi. tujuan penelitian ini adalah menganalisa komunitas bakteri yang berasosiasi dengan aaptos sp. dari perairan pulau rote dan kepulauan seribu, menggunakan metoda t-rflp. hasil penelitian menunjukkan bahwa komunitas bakteri yang berasosiasi dengan aaptos sp. dari kedua daerah sampling memiliki kesamaan 40,81%, yang didominasi oleh kelas bakteri yang sama yaitu kelas actinobacteria, flavobacteria, α-proteobacteria, δ-proteobacteria, dan γ-proteobacteria. bakteri dari spons yang dikoleksi dari pulau rote lebih terdistribusi dan lebih beragam dibanding yang dari spons yang dikoleksi dari kepulauan seribu. secara total ada 23 kelas mikroorganisme sebagian besar bakteri yang diidentifikasi ada di spons dari pulau rote, sedangkan spons dari kepulauan seribu terdapat 14 kelas. keberadaan actinobacteria dan proteobacteria dalam aaptos sp., diduga berhubungan dengan produksi metabolit sekunder. kata kunci : aaptos sp., kepulauan seribu, keragaman bakteri, pulau rote, t-rflp analysis of bacterial community associated with aaptos sp. from rote and seribu islands 1 1 1 ekowati chasanah *, gintung patantis , ariyanti suhita dewi , endar 1 1 2 2 2 marraskuranto , hedi indra januar , stella , susan soka , and yogiara 1 research and development center for marine and fisheries product processing and biotechnology, jalan ks tubun, petamburan 6, jakarta, indonesia; 2 faculty of biotechnology, universitas katolik atmajaya, jalan jenderal sudirman 51, jakarta 12930, indonesia al. 2007), and there is a strong correlation between bacterial richness and secondary metabolites produced by the host (haygood et al.1999). since they are filter feeder, the sponge associated bacteria are highly affected by the water quality where the sponges grown. the presence of anthropogenic stressor such as pollutans which is more frequently occured nowadays, might become a burden to the waters and it could affect the bacterial diversity in the sponge and its active metabolite produced. aaptamine is a bioactive compound that was once becoming chemotaxonomic marker for sponges of the order hadromerida where aaptos sp. is one of this ordo's member. latest report showed that there are number of species in the ordo hadromerida producing clinically active compounds in association with microorganism including bacteria (thomas et al. 2010). our previous study on the screening of the bioctive compounds from sponges including aaptos sp from karimunjawa, bali, and lombok, yogyakarta, binuangen (banten) waters found that the aaptamine group compound from the sponge had relatively high yield (chasanah et al. 2007). aaptamine-like compounds have also been found in sponges of other genera such as xestospongia, suberites, hymeniacidon, and luffarriella (pham et al. 2013). the objective of this research was to analyze the bacterial community associated with aaptos sp. from two different locations, i.e. rote island and seribu islands.the two locations represent the less polluted and polluted waters due to human activities. material and methods samples collection. aaptos sp. samples were taken by hand using scuba diving equipment from territorial waters at rote island (batu termanu) th indonesia on may 10 , 2010 (geographic location s10º40.228'; e123º05.73) and seribu islands (penjaliran barat) on april 2010, at geographic o o location of s05 27.720'; e106 33.603', at the same depth of 5m. three samples of aaptos sp from each location were mixed for genomic dna isolation and analyzed to generate the bacterial community profile of the sampels from both locations. water (200 ml) around the aaptos sp. was collected in triplicates using nansen jar and analyzed on board. dissolved anorganic nitrogen (nitrate, nitrite, ammonia) and phosphate ions were analyzed by a colorimetric method using hach dr-890 colorimeter. salinities and ph were recorded using refractometer and ph meter. dissolved oxygen (do) and physical characteristics (water temperature, transparancy and water flow) of the waters was conducted in situ using do meter, thermometer, and secchi disk, respectively. samples pre-treatment. about 25 g of aaptos sp. sample was added into 25 ml of ctab extraction buffer, blended, and centrifuged at 200 x g for 2 min. supernatant was transferred into new microtube and centrifuged at 600 x g for 2 min, the supernatant was subsequently transferred into a microtube sterile and centrifuged at 10 000 x g for 5 min. genomic dna isolation. genome isolation was conducted following zhou et al. (1996) with a modification. pellets which were formed from the pretreatment were homogenized with 750 ctab extraction buffer and homogenized for 2 min at full speed. samples were then centrifuged at 14 000 x g for 30 sec, and supernatant were transferred into sterile microtubes, frozen at -70 °c, and thawed at 65 °c (this process was done in duplicate). afterward, samples were placed at room temperature, then, proteinase-k were added and incubated at 37 °c for 30 min, and were added with 150 μl 10% sarkosyl detergent and incubated at 65 °c for 30 min. samples were flipped every 10 min, centrifuged at 10 000 x g for 5 min, and the supernatants were transferred into sterile microtubes and added with equal volume of phenol:chloroform:isoamylalcohol (25:24:1), then shaked until well mixed. samples were then centrifuged at 14 000 x g for 5 min., the liquid phase were transferred into sterile microtubes, then equal volume of chloroform were added to the samples and shaked until well mixed. samples were centrifuged at 14 000 x g for 5 min and the liquid phase formed was transferred into sterile microtubes and added with 0.6 x volume of cold isopropanol and incubated at -20 °c for 20 min. subsequently the samples were centrifuged at 16 000 x g for 5 min. supernatant were discarded and the pellets formed were washed with 70% ethanol and then dried. pellets were homogenized with 50-100 μl tris-cl ph 8. genomic dna were visualized on 1% agarose in 1 x tae buffer, which were visualized with cyber-gold. dna genomes were purified using dna wizard genomic dna purification kit® (promega). 16s rrna gene amplification and t-rflp analyses.16s-rrna gene was amplified using bio-rad c-1000 with universal primers 27f-fam (5'-aga gtttgatcctggctcag3') which was labeled at the 5'-end with the phosphoramiditefluorochrome 5carboxyfluorescein (fam) and 1387r (5'-gggcgg wgtgtacaaggc-3') (marchesi et al. 1998). to μl 38 chasanah et al. microbiol indones perform gene encoding 16s rrna pcr, master mix was made with the following composition: 12.5 μlgotaq (promega, madison, wi, usa), 1 μl primer 27f and -1 1387r primers (25 pmol μl ), 9.5 μl ddh o, and 1 μl of the dna genome as template. pcr was performed at 95 °c for 5 min, 30 cycles of 95 °c for 30 sec, 55 °c for 30 sec, and 72 °c for 1 min, followed by 72 °c for 10 min. pcr products were visualized on 1% agarose gel in 1x tae buffer. the 16s rdna was then purified using qia quick pcr purification kit (qiagen, germany) according to the manufacturer's protocol. t-rflp analysis was conducted as follow: fluorescently labeled pcr products were single digested with 3 endonuclease enzymes namely bsh 1236i, rsai, and hpaii for 24 h at 37 °c. digestion was performed in a total volume of 20 μl containing 2 μl restriction enzymes, 2 μl of 10x restriction buffer, and 16 μl of pcr products. digestion products were purified using edta-naoac precipitation method (sambrook and russel 2001). pellet of the digested products were mixed with 12 μl deionized formamide and 0.5 μl of internal size standard (rox-500, applied biosystems, foster city, ca, usa). this mixture was denatured for 5 min at 95 °c and immediately chilled on ice before electrophoresis on an abi prism 310 genetic analyzer (applied biosystem, foster city, ca, usa) operated in genescan. after electrophoresis, the length of fluorescently labeled trfs was determined by comparison with internal standards by using genescan software (applied biosystems, foster city, ca, usa). fragment sizes were analyzed using fragsort ver.5.0 software (www.oardc.ohiostate.edu/trflpfragsort/index.php) with data base from microbial community analysis (shyu et al. 2007). to evaluate richness and evenness, diversity statistics were calculated from each population. population richness (s) was total number of distinct population. diversity index analysis was analyzed using shannon-wiener index (h), simpson's index (d), and evenness (e). the shannon-wiener diversity index was calculated from equation h= -σ(pi)(lnpi) and the simpson's index was calculated from equation d = 1((σ ni (ni-1) / n (n-1)). evenness value was calculated from equation e = h/h , h = ln s (allen et al. 2009).max max analysis of aaptamine and isoaaptamine. for analysis of aaptamine and isoaaptamine, triplicates of 5 g wet weight of frozen aaptos sp. were extracted using 10 ml of methanol (pa), and centrifuged to coagulate suspended solid. the supernatan were concentrated with nitrogen and freezed dried, and redissolved in methanol -1 providing concentration of 10 mg ml . concentrated 2 supernatants (5 μl) was injected into hplc system shimadzu 10-ad with pda detector and shimpack vp ods coloumn (2.0 mm x 150 mm). eluent used was 20% acetonitrite/h o (1% tfa) at a flow rate 0.2 ml 2 -1 min . concentration of aaptamine and isoaaptamine were determined according to dewi et al. (2012) as average of triplicate samples. result bacterial community profiles and its electropherograms observed from aaptos sp. from two sampling areas of rote island and seribu islands can be seen at fig 1 and fig 2. aaptos harvested from rote island contained more diverse bacteria than those of seribu islands as shown in table 1, using shannon-wiener index, i.e. the h value for rote and seribu islands was 5.53 and 4.78, respectively. this was supported by simpson's index, in which the d value of the bacterial population from aaptos sp. from rote island (0.894) was higher than that of seribu islands (0.884). based on the calculation of evenness value (e), the distribution of the bacteria population from aaptos sp. in rote island (0.653) was more highly distributed than that of seribu islands (0.676). the higher d values the higher the diversity, and the bigger e values the lower in variation distribution of bacterial community. there were 24 main classes and 1985 microorganisms of bacterial population identified along with uncultured microorganisms, uncultured organisms, uncultured bacteria, and unidentified organisms detected from aaptos sp. from seribu islands, while aaptos sp. from rote island which was considered richer had a total of 6128 microorganisms. sponges from both locations shared 40.81% similarity of bacterial community, and were dominated by the same classes, i.e. actinobacteria, bacilli, bacteroidia, deinococci, α-proteobacteria, δproteobacteria, and γ-proteobacteria. clostridia was the only class which was found in sponge sample from seribu islands but was not found in those from rote island. on the contrary, there were 10 classes that were found in sponge from rote island but were not found in those from seribu islands. water quality analysis was conducted in the sampling areas at the same time of sampling (table 2). analysis of the bioactive compounds, i.e. aaptamine and isoaaptamine, was only carried out with samples from seribu islands but it could not be conducted with those from rote island due to the technical problem of sample storage. the aaptos sp. from seribu islands contained 2.197% of aaptamine and 0.578% of volume 7, 2013 microbiol indones 39 compared to those of seribu islands. low water flow in seribu islands might cause no much change of the inhabitants (bacteria) in the aaptos including clostridia from this area. high value of phosphate in rote island could isoaaptamine (dried extract weight). discussions t-rflp method has been used in this study since 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% p re ce n ta g e o f d et ec te d m ic ro o rg an is m s clostridia thermomicroba thermodesulfobacteria fusobacteria cryptophyta thermotogae negativicutes nitrospirae synergistia deferribacteres cyanobacteria planctomycetacia cytophagia β-proteobacteria sphingobacteria chlorobia deinococci bacteroidia bacilli unidentified organism δ-proteobacteria γ-proteobacteria uncultured bacterium flavobacteria α-proteobacteria uncultured bacterium actinobacteria uncultured bacterium rote island seribu islands sampling location fig 1 percentage of detected microorganisms in aaptos sp. collected from rote island and seribu islands. this method was reported to be effective in determining bacterial communities in a range of environments, highly reproducible, rapid, and amenable to field-scale experiments (liu et al.1997; dunbar et al. 2000). the diversity of bacterial communities associated with aaptos sp. in both sampling sites showed that the bacterial community structures were different eventhough dominated by similar bacteria. based on the water quality (table 2), rote island waters showed higher water flow, indicating that the water in that location was more circulated, and this would affect the bacteria in the sponge as filter feeder organism. stronger water flow in rote island waters might lead to frequent water exchange that enters the sponge body, and this might be the reason why aaptos sp. from rote island waters contained more diverse micoorganisms indicate there was domestic contaminations from human activity such as sewage containing detergent or it might be an accumulation of phosphate ion from rock decay in the marine waters that might be affected by high water flow in the waters. this contamination might be indicated by the presence of phosphate degrading bacteria such as bacilli which was detected in more quantity in the sponge from rote island (fig 1). those bacteria could produce phophatase enzyme which plays a key role in mineralizing organic phosphate into inorganic phosphate. from the data, it can be seen that water quality of rote island was better than seribu islands waters. ammonia content of seribu islands waters depicted that the waters was highly polluted with domestic waste. the ph value of seribu islands waters which 40 chasanah et al. microbiol indones fig 2 electropherograms of bacterial community t-rflps generated from rdnas with a labeled reverse primer and 3 endonuclease digest namely bsh 1236i, rsai, and hpaii obtained from representative water samples from penjalinan barat (seribu islands) and batu termanu (rote island) waters. penjaliran baratbsh 1236i 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 100 200 300 400 500 600 batu termanubsh 1236i 100 200 300 400 500 600 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 penjaliran barat rsai 100 200 300 400 500 600 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 batu termanursai 100 200 300 400 500 600 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 penjaliran barathpai 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 100 200 300 400 500 600 batu termanuhpai 100 200 300 400 500 600 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 volume 7, 2013 microbiol indones 41 khan et al. 2010). study conducted by (2010) also reported that marine actinomycetes was very potential in producing the active metabolites. streptomyces has been for various therapeutic agents such as tetracycline as antibacteria, amphotericin as antifungal, and as immunosuppressant ( . another dominating class was proteobacteria (α, β, δ, and γ-proteobacteria) which was found more abundant in aaptos sp. from rote island, i.e. about 7% compared to 4% in aaptos sp. from seribu islands waters. li et al. (2006) reported that proteobacteria are common in marine environment and might be used as a biomarker on sponge host. proteobacteria have been suggested to have varied effects on sponge hosts such as nitrogen fixation. proteobacteria especially γproteobacteria has been reported to produce the most chemically diverse bioactive natural products such as agrochelin, a cytotoxic thiazole alkaloid from agrobacterium and b-90063, a dimericoxazolepyridone analog from blastobacter, which indicates the biomedical potential of this class of proteobacteria. in addition, an early investigation of the α-proteobacterium thalassospira sp. strain cnj328, resulted in the discovery of unique immunosuppressive peptides, thalassospiramides a and b (oh et al. 2007). suthindhiran and kanabiran used for commercial production of different compounds tacrolimus suthindhiran and kannabiran 2010) was below the ph required for corals growth (7.8-8.8) (jameson and kelty 2004) suggested that an acid contamination might be occured in the waters. low water flow in seribu islands waters also causes the pollutants stay longer in the waters, therefore it affects the living microorganisms and marine biota in the waters. previous work showed that water quality has affected the metabolite richness of the nephthea spp harvested from numbers of sampling point at alor waters (januar et al. 2012). aaptos sp. tissue from both locations were dominated by the same bacteria, but different in quantity. the dominant bacteria from both samples were actinobacteria, proteobacteria, and flavobacteria. class actinobacteria was very abundant in aaptos sp harvested from seribu islands i.e. 50% compared to 26% of rote island waters. seribu island was consider more polluted than rote island waters. on the other hand, class proteobacteria was found more likely higher in rote island waters compared to that of seribu islands waters. actinobacteria was reported to be the most morphologically diverse prokaryotes, and are widely distributed in both terrestrial and aquatic ecosystems (servin et al. 2008). one of the well studied actinobacteria species is streptomyces sp. metabolite compounds produced by streptomyces sp. was reported could exhibit cytotoxic activity against cancer cell lines (chasanah et al. 2009; table 1 the diversity index of bacterial community associated with aaptos sp. table 2 water quality of rote island and seribu islands rote island seribu islands hmax 5.533 4.787 h 3.611 3.235 d 0.894 0.884 e 0.653 0.676 water flow (m/sec) temperature (°c) salinity (‰) ph do phosphate (ppm) nitrate (ppm) nitrite (ppm) ammonia (ppm) rote island 0.23 31 33.33 8.1 6.83 0.533 0.050 0.002 0.033 seribu islands 0.09 32 31.00 7.7 6.73 0.017 0.067 0.000 0.100 42 chasanah et al. microbiol indones d: simpson’s index; e: evenness; h: shannon-wiener index; h : ln population richnessmax fisheries postharvest biotechnol special edition:1-7. chasanah e, januar hi, wright ad. 2007. activity completion report. public sector linkages program (pslp). abn 78 961 616 230. coutinho af, chanas b, souza tml, frugrulhetti icpp, epifanio ra. 2002. anti hsv-1 alkaloids from a feeding deterrent marine sponge of the genus aaptos. heterocycles 57(7):1256-1272. dewi as, hadi ta, januar hi, pratitis a, chasanah e. 2012. study on the effect of pollutants on the production of aaptamine and the cytotoxicity of crude extract from aaptos suberitoides. squalen 7(3):97-104. dunbar j, ticknor lo, kuske1 cr. 2000. assessment of microbial diversity in four southwestern united states soils by 16s rrna gene terminal restriction fragment analysis. appl environ microbiol. 66(7):2943-2950. gul w, hammond n, yousaf m, bowling j, schinazi r, wirtz, s, de castro ag, cuevas c, hamann m. 2006. modification at the c9 position of the marine natural product isoaaptamine and the impact on hiv-1, mycobacterial, and tumor cell activity. bioorg med chem. 14(24):8495-8505. doi:10.1016/j.bmc.2006.08.042. haygood mg, schmidt ew, davidson sk, faulkner dj. 1999. microbial symbionts of marine invetebrates: opportunities for microbial biotecnology. mol microbiol biotechnol. 1(1):33-43. jameson sc, kelty ra. 2004. a review of indikatorsfo land-based pollution stress on coral reefs, a background paper for joint epa/noaa/usga/doi workshop on assessing pollution stress on coral reefs; 2004, august 31-september 2, honolulu, hawaii (usa). januar hi, marraskuranto e, patantis g, chasanah e. 2012. lc-ms metabolomic analysis of environmental stressor impacts on the metabolite diversity in nephthea spp. chronicles of young scientists 3(1):5762. doi:10.4103/2229-5186.94319. kalinovskaya ni, ivanova ep, alexeeva yv, gorshkova nm, kuznetsova ta, dmitrenok as, nicolau dv. 2004. low molecular-weight, biologically active compounds from marine pseudoalteromonas species. curr microbiol. 48(6): 441-446. khan st, komaki h, motohashi k, kozone i, mukai a, takagi m, shin-ya k. 2010. streptomyces associated with a marine sponge haliclona sp.; biosynthetic genes for secondary metabolites and products. environ microbiol.13(2):391403. doi:10.1111/j.1462-2920.2010.02337.x. li zy, he lm, wu j, jiang q. 2006. bacterial community diversity associated with four marine sponges from the south china sea based on 16s rdna-dgge fingerprinting. j exp marine biol ecol. 329(1):75-85. doi:10.1016/j.jembe.2005.08.014. liu wt, marsh tl, cheng h, forney lj. 1997. characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16s rrna. appl environ microbiol. 63(11):4516-4522. marchesi jr, sato t, weightman aj, martinta, fry jc, hiom sj, wade wg. 1998. design and evaluation of kalinovskaya et al. (2004) reported that proteobacteria could produce low molecular-weight biological active compounds with antimicrobial and surface-active properties. another research by radjasa et al. (2007) reported that metabolites of proteobacteria which was isolated from aaptos sp. from panjang island, jepara, were active against multi drugs resistant strains of microorganism. all of the above studies indicated that there is correlation of sponge bioactive compounds with proteobacteria and/or actinobacteria, which were dominant microorganism in aaptos sp. used in this study. sponge from ordo suberitidae where aaptos sp. is one of the member was reported to produce therapeutic important bioactive compounds of microbial origin. alpha, β, γ, δ-proteobacteria, and actinobacteria has been reported to be possible contributors of pharmacologically relevant secondary metabolites of sponges, in addition to firmicutes, cyanobacteria, and fungi (thomas et al. 2010). in this study, the bioactive compounds, i.e. aaptamine and isoaaptamine have been extracted from the aaptos sp. tissue from seribu islands, but we failed to extract those from sample from rote island waters due to the failure of the sample storage. aaptamine in the aaptos sp. tissue harvested from seribu islands was 2.197% and isoaaptamine was 0.578% (dried extract weight). since the same compounds from rote samples could not be analyzed, we could not compare both samples. to conclude, the structure of bacterial community in the aaptos sp. harvested from seribu islands and rote island waters was affected by water quality, but in this study, we can not yet correlate the bacterial community with the bioactive content of the sponge, i.e. aaptamine and isoaaptamine. bacterial community associated with aaptos sp. from rote island which was less polluted and having higher water flow, was more diverse and highly distributed than that of seribu islands. however, they both shared 40.81% similarity of microorganism, from which they had the same dominant bacteria, i.e. actinobacteria and proteobacteria that are well known as bioactive producer. references allen b, kon m,yaneer by. 2009. a new phylogenetic diversity measure generalizing the shannon index and its application to phyllostomid bats. am nat. 174(2). doi:10.1086/600101. chasanah e, januar hi, irianto he, bourne d, liptrot c, wight a. 2009. screening of anticancer activity of fungi derived from indonesian marine sponge. j marine volume 7, 2013 microbiol indones 43 servin ja, herbold cw, skophammer rg, lake ja. 2008. evidence excluding the root of the tree of life from the a c t i n o b a c t e r i a . m o l b i o l e v o l . 2 5 ( 1 ) : 1 4 . doi:10.1093/molbev/msm249. suthindhiran k, kannabiran k. 2010. diversity and exploration of bioactive marine actinomycetes in the bay of bengal of the puducherry coast of india. ind j microbiol. 50(1):76-82. doi:10.1007/s12088-0100048-3. shyu c, soule t, bent sj, foster ja, forney lj. 2007. mica: a web-based tool for the aof microbial communities based on terminal-restriction fragment length polymorphisms of 16s and 18s rrna genes. j microb ecol. 53(4):562-570. doi:10.1007/s00248-0069106-0. taylor mw, radax r, steger d, wagner m. 2007. spongeassociated microorganisms: evolution, ecology and biotechnological potential. microbiol mol biol rev. 71(2):295-347. doi:10.1128/mmbr.00040-06. thomas tra, kavlekar dp, loka bharathi pa. 2010. marine drugs from sponge-microbe association-a review. mar drugs. 8(4):1417-1468. doi:10.3390/md8041417. zhou j, bruns ma, tiedje jm. 1996. dna recovery from soils of diverse composition. appl environ microbiol. 62(2):316-322. useful bacterium-specific pcr primers that amplify genes coding for bacterial 16s rna. appl environ microbiol. 64(2):795-799. oh dc, strangman wk, kauffman ca, jensenpr, fenical w. 2007.thalassospiramides a and b, immunosuppressive peptides from the marine bacterium thalassospira sp. org lett. 9(8):1525-1528. doi:10.1021/ol070294u. pham cd, hartmann r, müller weg, voogd n, lai d, proksch p. 2013. aaptamine derivatives from the indonesian sponge aaptossuberitoides. j nat prod. 76(1):103-106. doi:10.1021/np300794b. rachmat r, murniasih t. 2001. identifikasi senyawa sterol dari spons aaptos sp. asal spermonde [identification of sterol of aaptos sp. from spermonde]. prosiding seminar nasional iv kimia dalam pembangunan; hotel santika yogyakarta, 2001. 27-28 mar. jaringan kerjasama kimia indonesia. yogyakarta (id). p 89-92. radjasa ok, kencana ds, sabdono a, hutagalung ra, lestari es. 2007. antibacterial of marine bacteria associated with sponge aaptos sp. against multi drugs resistant (mdr) strains. j matematika sains. 12(4):147-152. sambrook j, rusell dw. 2001. molecular cloning: a rd laboratory manual. 3 edition. cold spring harbor (ny): cold spring harbor laboratory press. 44 chasanah et al. microbiol indones 3.mi699-uus saepuloh available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.2.3issn 1978-3477, eissn 2087-8575 vol 7, no 2, june 2013, p 59-67 *corresponding author; phone: +62-251-8320417, fax. +62251-8360712, e-mail: uussaepuloh@yahoo.com reverse transcription is an essential and critical step in the life cycle of all retroviruses replication (telesnitsky and goff 1997; sarafianos et al. 2009). reverse transcriptase (rt) is a multifunctional enzyme that catalyzes the formation of doublestranded deoxyribonucleic acid (dna) from singlestranded ribonucleic acid (rna) viral genomes (telesnitsky and goff 1997; hizi and herschhorn 2008). this enzyme is responsible for genome replication of viruses possessing rna and dna polymerase and rnase h activities (hizi and herschhorn 2008). rt has become an important enzyme in molecular biology, genetics and medicine for the synthesis of complementary dna (cdna) from messenger rna (mrna) (telesnitsky and goff 1997; herschhorn and hizi 2010). the application of this enzyme combining with pcr amplification technique (rt-pcr) has expanded our knowledge of numerous cellular regulatory mechanisms and has become a gold standard to facilitate the coding region of any gene of interest. the development of biotechnology has been relying on the availability of this efficient rts (herschhorn and hizi 2010). until nowadays, the reverse transcriptases of human immunodeficiency virus type-1 (hiv-1) and moloney murine leukemia virus (mmlv) have been extensively characterized. the rt of hiv-1, the virus causing acquired immunodeficiency syndrome (aids), is a prime target for the development of antiretroviral drug therapy of hiv-1/aids and the most studied enzyme in biomedical research (kohlstaedt et al.1992). meanwhile, the mmlv-rt is the most widely used in this study, we isolated simian betaretrovirus (srv-2) reverse transcriptase (rt) gene from infected indonesian cynomolgus monkey (macaca fascicularis). the gene was then cloned in escherichia coli expression system. the srv-2 rt gene is located between nucleotides 3284-4925 in the polyprotein (pol) region encodes 547 amino acids. analysis of expression using sds-page and western blot techniques showed a specific band of 64.9 kda, indicating srv-2 rt recombinant enzyme. purification of srv-2 rt recombinant -1 -1 enzyme produced 312 µg ml protein with 7.1 u µl enzyme activities. application of this recombinant enzyme in reverse transcription-pcr (rt-pcr) of β-globin and β-actin genes produced dna fragments of 206 and 350 bp, indicating amplification of β-globin and β-actin genes, respectively. therefore, the expressed srv-2 rt enzyme was proven to be functional, although the activity was low. key words: escherichia coli expression system, recombinant enzyme, reverse transcriptase, srv-2 pada penelitian ini, dilakukan isolasi gen reverse transcriptase (rt) simian betaretrovirus serotipe-2 (srv2) yang menginfeksi monyet ekor panjang (macaca fascicularis) asal indonesia. gen tersebut selanjutnya diklonakan ke dalam sistem ekspresi escherichia coli. gen rt srv-2 berada pada posisi nukleotida 3284-4925 di daerah polyprotein (pol) yang mengkodekan 547 asam amino. analisis hasil ekspresi menggunakan teknik sds-page dan western blot menunjukkan adanya pita protein spesifik berukuran 64,9 kda yang diperkirakan -1 sebagai enzim rekombinan rt srv-2. pemurnian enzim rekombinan rt srv-2 menghasilkan 312 µg ml -1 protein yang memiliki aktivitas enzim sebesar 7,1 u µl . aplikasi enzim rekombinan rt srv-2 pada teknik reverse transcription-pcr (rt-pcr) terhadap target gen β-globin dan β-aktin menghasilkan pita dna berukuran 206 dan 350 pb. dengan demikian, enzim rekombinan rt srv-2 terbukti berfungsi memiliki aktivitas enzim walaupun aktivitasnya masih rendah. kata kunci: enzim rekombinan, reverse transcriptase, sistem ekspresi escherichia coli , srv-2 serotype-2 cloning and expression of serotype-2 simian betaretrovirus reverse transcriptase gene isolated from indonesian cynomolgus monkey in escherichia coli 1 1 2 1 uus saepuloh *, diah iskandriati , fungkey hoetama , sela septima mariya , 3 1,4 1,4 dedy duryadi solihin , joko pamungkas , and dondin sajuthi 1 primate research center, institut pertanian bogor (pssp lppm ipb), jalan lodaya ii/5, bogor 16151, indonesia; 2 faculty of biotechnology, unika atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia; 3 department of biology, faculty of mathematics and natural science, institut pertanian bogor, kampus darmaga, bogor 16680, indonesia; 4 faculty of veterinary medicine, institut pertanian bogor, kampus darmaga, bogor 16680, indonesia enzyme for cdna synthesis and rna amplification due to its robust catalytic activity and high fidelity (kotewitcz et al.1988; taube et al. 1998). simian betaretroviruses (srvs) are the causative agents of simian acquired immunodeficiency syndrome (saids) in asian macaques with varying severity (marx et al. 1984; gardner et al. 1988; lerche 2010). regarding potentially active infection and immune abnormality affected by this virus, srvs is a pathogenic agent that should be eliminated in the macaca breeding colony (marx et al. 1984; morton et al. 2008). this srvs infection poses some problems for breeders providing a population of srv-free macaques, since macaques are frequently used as animal model in biomedical researches (lerche and osborn 2003). while srv is an important pathogen in asian macaques and causes a potential confounding variable in biomedical research, srv also provides a valuable viral model to compare with other retrovirals for a better understanding of aspects of retroviral-host interactions, infection mechanism, retroviral structure, antiretroviral, and vaccine development (montiel et al, 2010; lerche and osborn 2003). recently, srv-2 have been isolated and characterized from wild indonesian macaca fascicularis of which the sequence analysis of envelope region showed 96% homology to srv-2 (iskandriati et al. 2010). the srvs’ genome contains three major genes, 5’-gag-pol-env-3’. each gene plays a role in the production of viral proteins (marracci et al. 1995; marracci et al. 1999). the rt sequence is located within the pol coding domain of this retrovirus. the gene encoding rt can be isolated and cloned to produce rt recombinant enzyme. in this study, we report the expression of srv-2 rt gene isolated from indonesian m. fascicularis in escherichia coli, its purification, and its enzymatic activity measurement of the gene product. materials and methods isolation of srv-2 rt gene using pcr technique. dna was extracted from pbmcs (peripheral blood mononuclear cells ) of indonesian m. fascicularis infected srv-2 (archived sample of pssp lppm ipb) using qiamp blood dna minikit (qiagen, hilden, germany). the srv-2 rt gene was amplified using specific primers srv-2: rt3284u (5’cacccctgtttgggttgatc-3’) and srv-2 rt4925r (5’-ctagtgattaccttgagataaagg tcc-3’). the forward primer was designed with the addition of cacc nucleotides that would facilitate the cloning of the gene into the pentr/sd/topo entry vector. meanwhile, reverse complement of stop codon uag (cta) was added to the reverse primer for correct expression in n-terminal fusion pdest17 vector. pcr amplification was performed using pfx dna polymerase of thermococcus sp that has low error rate and proof reading activity. additional primers for sequencing were m13f (5’ -tgtaaaacgacggcca gt-3’), m13r (5’-ggtcatagctgtttcctg-3’), srv-2 rt 3751r (5’-gcccaagattttctgattgg -3’), srv-2 rt 3799f (5’-gattggcgaacaagttt tgc-3’), srv-2 rt 4247f (5’-tactggcctcttctg g-3’), and srv-2 rt 4604f (5’-ggcatagccgcata cacttt-3’). these primers were developed using primer3 program (http://primer3.wi.mit.edu/) based on srv-2 complete genome (genbank af126467.1). srv-2 rt gene cloning and expression. srv-2 rt gene was inserted to pentr/sd/topo according to procedure from invitrogen, usa and transformed to e.coli top10. transformants were selected on lb agar -1 plate containing 50 µg ml kanamycin antibiotic. the clone was then cultured in lb broth and incubated at 37 °c for 18 h. plasmid was extracted using qiaprep spin miniprep kit (qiagen, hilden, germany), then analyzed by pcr and sequencing techniques using m13 universal primer to prove the gene target was inserted correctly. srv-2 rt gene in pentr/sd/topo (invitrogen, california, usa) was then sub-cloned to pdest17 (invitrogen, california, usa) destination vector between nucleotide positions 147 and 1830, downstream of 6× his-tag gene and transformed to e. coli dh5α. analysis of recombination was performed by pcr and sequenced using t7 promotor forward primer and srv-2 rt4925r primer. pdest17 containing target gene was transformed to e.coli bl21ai using heat shock methods. the tranformant was then grown in 5 ml lb broth containing 100 μg -1 ml ampicillin until the od was 0.6-1.0. the culture 600 was used to inoculate fresh lb medium containing 100 -1 μg ml ampicillin, which was then let to grow until mid-log phase (od of ~0.4) prior to induction with 600 20% l-arabinose. purification and expression analysis of srv-2 rt recombinant enzyme. e. coli bl21ai expressing srv-2 rt recombinant protein was cultured overnight in 1 l of lb broth and then centrifuged at 10 000 × g for 10 min. pelleted cells were lyzed using native binding buffer containing 50 mm potassium phosphate ph 7.8, 400 mm nacl, 100 mm kcl, 10% glycerol, 0.5% triton x 100, and 10 mm imidazole, and added with 8 mg lysozyme. the affinity 60 saepuloh et al. microbiol indones 2+ chromatography with ni -nta chelating agent was used for further purification (qiagen, hilden, germany). recombinant enzyme concentration was determined using bicinchoninic acid (bca) assay (thermo scientific, illinois, usa). expression of srv-2 rt recombinant enzyme was detected by sdspage. meanwhile, western blot was performed to analyze the serological reaction using antibody against 6× histidine-tag (invitrogen, california, usa) to srv2 rt. srv-2 rt recombinant enzyme activity analysis. srv-2 rt enzyme was determined quantitatively using rt chemiluminescent assay (roche, penzberg, germany) based on the ability of rt enzyme to synthesize new dna from template/primer hybrid poly (a) × oligo (dt) . assay was conducted in reaction mixture containing 46 mm tris-hcl, 266 mm potassium phosphate, 27.5 mm magnesium chloride, 9.2 mm ddt, 10 mm dutp/dttp, and template/primer hybrid, at final ph of 7.8. the reaction mixture was incubated for 1 h at 37 °c. the amount of this synthesized dna is used as a parameter of rt enzyme activity following a sandwich elisa (enzyme-linked immunosorbent assay) protocol. digoxigeninand biotin-labeled nucleotides in an optimized ratio are incorporated into the same dna molecule, which is newly synthesized by the rt. biotin-labeled dna binds to the surface of microplate that have been precoated with streptavidin. an antibody to digoxigenin, conjugated to horseradish peroxidase (anti-dig-pod), binds to the digoxigenin-labeled dna. the chemiluminescent peroxidase substrate luminol/4iodophenol is added. peroxidase in the presence of hydrogen peroxide (h o ) catalyzes the oxidation of 2 2 luminol, resulting in reaction products that quantified using elisa reader at the absorbance 490 nm that directly correlated to the level of rt activity in the sample. the specific activity of dna polymerase were expressed in unit (u) that means as the amount of enzyme required for the incorporation of 1 nmol of labeled dntp in 10 min at 37 °c using poly (a)× oligo(dt) as template/primer hybrid. 15 meanwhile, this srv-2 rt rombinant enzyme was applied to reverse transcription-pcr (rt-pcr) technique to qualitatively analyze the enzyme’s ability in reverse transcribing the rna template to the cdna. the methods was referred to superscript iii reverse transcriptase (invitrogen, california, usa) and the cdna pcr amplification was performed to amplify 15 β-globin and β-actin as housekeeping genes. results srv-2 rt gene isolation and cloning. in this study, reverse transcriptase gene was isolated from srv-2 pol located at positions 3284-4925 specific band of about 1600 bp was shown in gel electrophoresis (fig 1), indicating the presence of the targeted gene. pcr analysis using m13 forward and reverse primers was performed to verify the srv-2 rt in pentr/sd/topo plasmid, resulted the specific amplicon of about 2000 bp (fig 2). the nucleotides sequence analysis was analyzed by blast program (http://blast.ncbi.nlm.nih.gov/), indicating the presence of the gene target, with 99% (1631 of 1641 nucleotides) similarities to srv-2 rt d2/rhe/or isolate (genbank af126467.1). the gene of interest was inserted to the vector in correct direction without any nucleotide shifts nor mutations in the reading frame. the insertion site was downstream of the ribosome binding site (rbs) and flanked by attl1 and attl2. it was proven that the target gene had been successfully inserted in the plasmid (fig 3). srv-2 rt recombination into pdest17 e. coli expression system. the srv-2 rt cloned in pentr/sd/topo entry plasmid had been subcloned to pdest17 destination vector by lr recombination reaction. pdest17 is a vector, which is n-terminally fused with 6× his tags for purification purposes. atg initiation codon lies upstream of the 6× his tags. analysis of recombination in the pdest17 vector by pcr amplification using the t7 promoter primer to srv-2 rt 4925r produced a specific fragment of of about 1800 bp (fig 4). nucleotide sequence analysis indicated that the srv-2 rt gene had been inserted properly, in frame within the vector. srv-2 rt enzyme expression analysis. sdspage analysis of srv-2 rt recombinant protein showed the presence of a band at molecular weight approximately 64.9 kda (fig 5). this result was confirmed by molecular weight calculation of 547 amino acids srv-2 rt and six histidines, using the software from website http://www.expasy.org/compute _pi/mw. srv-2 rt recombinant protein has been 2+ successfully purified using ni -nta spin column affinity chromatography. the purified expressed protein was eluted using imidazole, which binds nickle ion more tightly than 6xhis. in this research, purification method was performed under native conditions that the 6× his-srv-2 rt enzymes was in soluble condition. however, under this condition, the 2+ non-tagged proteins will also interact with ni -nta volume 7, 2013 microbiol indones 61 100 bp 600 bp 1500 bp m 1 2 1600 bp fig 1 srv-2 rt gene amplification using specific primer srv-2 rt 3284f and 4925r. m: 100 bp dna ladder (invitrogen); 1 2: 1600 bp pcr product of srv-2 rt. 1650 bp 100 0 bp 50 0 bp 500 0 bp 2000 bp a fig 2 (a) pcr amplification to pentr/sd/topo srv-2 rt plasmids using m13 f and m13 r primer. m: 1kb dna ladder (invitrogen); 1-2: 2000 bp amplicon contained srv-2 rt gene. (b) genetic map illustration of pentr/sd/topo-srv2 rt vector. b 62 saepuloh et al. microbiol indones from invitrogen, cdna of β-globin and β-actin using hela cell as mrna template (invitrogen). the pcr produced 206 bp and 350 bp fragments corresponding to β-globin and β-actin, respectively (fig 6). the result proved the functionality of the expressed srv-2 rt. discussions srv-2 rt gene was cloned in pentr/sd/topo entry vector before being subcloned by site specific recombination into pdest/17destination e.coli expression system. the recombination was mediated by lr clonase ii enzyme mix, a mixture of the bacteriophage λ integrase (int) and excisionase (xis), and the e. coli integration host factor (ihf) protein (shuman 1994; landy 1989; hunt 2005). this vector was designed to facilitate high-level, inducible expression of recombinant proteins in e. coli using the pet system. the system takes advantage of the high activity and specificity of the bacteriophage t7 rna polymerase to allow regulated expression of heterologous genes in e. coli from the t7 promoter resin at higher affinity than in denaturating condition. this was shown by the large number of proteins appearing in the binding buffer (fig 5a). the result of the bca assay showed that protein concentration was -1 2280 µg ml . this non-specific binding could have been reduced by adding low concentration of imidazole in the washing buffers. the concentration of purified rt-srv-2 eluted with acidic buffer containing high concentration (250 mm) of imidazole was 306 µg -1 ml protein (table 1). for further expression analysis, western blot was performed using anti-6xhis-tag antibody showed the presence of a specific band around 64.9 kda (fig 5b). analysis of srv-2 rt recombinant enzyme activities. purified srv-2 rt recombinant enzyme had the activity of 1420 unit in 200 μl elution buffer, therefore, the enzyme volumetric activity was 7.1 u -1 -1 µl and the enzyme specific activity was 22.76 u mg total protein (table 1). the purified srv-2 rt recombinant enzyme (3 µl of 21 u is the optimum reaction) was then applied in rt-pcr, to create in comparison to 1 µl of 200 u commercial mmlv-rt fig 3 nucleotide sequences of indonesian isolate’s srv-2 rt at nucleotide positions 3284-4925 inserted in the pentr/sd/topo vector. nucleotide sequences of vector and srv-2 rt gene were highlighted in yellow and grey, respectively. primer positions were indicated with red letters. m13 forward primer tgtaaaacgacggccagtcttaagctcgggccccaaataatgattttattttgactgatagtgacctgttcgttgca attl1 acaaattgatgagcaatgcttttttataatgccaactttgtacaaaaaagcaggctccgcggccgccttgtttaact rbs srv-2 rt3284f srv -2 rt gene ttaagaaggagcccttcacccctgtttgggttgatcaatggcccctaactcaagaaaaacttgctgctgcccaacagt tagtgcaagaacaattacaggcagggcatattatagaaagtaattctccctggaatacacctatatttgtcataaaaa agaagtctggtaaatggaggcttttgcaagatttaagggcggtaaatgccaccatggtattaatgggagctctccaac ctgggctgccctcaccagtggctattcctcagggatattttaaaatagtcattgatcttaaagattgtttttttacta tcccccttcagcccgttgatcaaaagcgatttgcttttagtctcccgtctaccaactttaaacaaccaataaaacgtt atcaatggaaagtgttgcctcaaggcatggccaatagtcctaccttgtgtcaaaaatatgtagctgctgctatagagc caatcagaaaatcttgggcacaaatgtacattatacactatatggatgacattctaatagcaggaaagattggcgaac aagttttgcagtgttttgctcaacttaaacaagcattgacaactactgggttacaaatagctccagaaaaggtacagc tacaagatccatatacctaccttgggtttcaacttaatggtcccaaaattaccaatcataaggcagttattcgtagag ataagttacaaacccttaatgattttcaaaaacttttgggagatatcaattggcttagaccctatttacacctcacta caggagatctaaaacctctttttgatatcctaaaaggggattctaatcctaactcacccagatttttatctgaagcag cccttacgtctcttaaaaaggtagaaacagctattgctgaacaatttgttacacaaatagattatacacagccattga cctttttaatttttaatactacactgacacctactggcctcttctggcaaaataatcctgtcatgtgggttcacttgc ctgcatcgccaaaaaaggtattgttcccctattatgatgctatagcagatcttattatccttggaagggacaacagta aaaaatattttggacttgaaccatctactattatacaaccctattctaaatctcaaatccattggttaatgcaaaaca cagaaacatggccaattgcctgcgcttcctatgcaggcaacattgataatcattatccacccaataaacttattcaat tttgcaaactccatgcagttgtttttccccgaatcattagtaaaactcctctagacaatgccttactggtattcactg atggatcctctaccggcatagccgcatacacttttgaaaagaccactgtcaaatttaaaacctcccatacgtcagccc aattagtagaattacaagccctaattgcagtgttatcggcctttcctcatcgggccctcaatatttatacagatagtg catacttagctcattctatacctctacttgagacagtatcgcaaatcaaacatatctcagacacagcaaaattatttt tacagtgccaacaactaatatgcaacaggtctatacccttttatttaggacatatcagggcccattcaggattaccag gacctttatctcaaggtaatcactagaagggtgggcgcgccgacccagctttcttgtacaaagttggcattataagaa srv-2 rt4925r agcattgcttatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgccatccagctgata attl2 tcccctatagtgagtcgtattacatggtcatagctgtttcctg m13 reverse primer volume 7, 2013 microbiol indones 63 comparison of nversus c-terminal positioning of his tag on several proteins expressed in bacteria and eukaryotes as well as analyses of the protein expression level and solubility indicated that, in most cases, the nterminal tags improved protein expression (busso et al. 2003; svensson et al. 2006). however, in this study we had only the soluble active srv-2 rt enzyme, while any attempts to recover enzymatically active preparation of the insoluble inclusion bodies had been unsuccessful. in the case of the gateway expression vector pdest-17, the n-terminal 6× his tag is followed by an additional 21 amino acids that arise from transcription through the gateway recombination and topoisomerase sites. (rosenberg et al. 1987). sds-page and western blot analyses indicated that the molecular weight of our srv-2 rt 6× his nterminal fusion protein was 64.9 kda. the 6× his tag is small enough that it does not affect the structure and functionality of the expressed protein. in addition the tag allows the protein to be purified under denaturing and native conditions, which is useful if refolding of insoluble proteins is to be attempted. however, nterminal 6× his tags does not only plays a role in protein recombinant purification but also seems to have stabilizing effect on the mrna structure in the translation initiation region (svensson et al. 2006). 1650 bp 1000 bp 500 bp 5000 bp a b 1800 bp fig 4 (a) pcr amplification to pdest17 srv-2 rt plasmids using t7 forward primer and srv-2 rt4148 reverse primer. m: 1 kb dna ladder (invitrogen); 1-2: 1800 bp amplicon containing srv-2 rt gene. (b) genetic map illustration of pentr/sd/topo-srv-2 rt vector. 64 saepuloh et al. microbiol indones kd 216 132 78 64.9 45.7 32.5 18,4 7,6 kd 210 125 101 64.9 56.2 35.8 29 21 6.9 m 1 2 3 4 m 1 2 3 4 6x his rt srv -2 a b 2+ fig 5 srv-2 rt recombinant enzyme expression analysis of preand postni -nta purification produced specific band at 64.9 kda. (a) 10% sds page stained with coomassie brilliant blue. m: broad range prestained protein marker (biorad); 1: pre-purified, 2: binding buffer, 3: washing buffer, 4: elution buffer. (b) western blot analysis using hrp conjugated anti-polypeptide histidine antibody. m: kaleidoskop™ prestained protein marker (biorad); 1: pre-purified; 2: elution buffer; 3: washing buffer; 4: binding buffer. 2+ table 1 protein concentration and activities of the ni -nta purified rt srv-2 fig 6 comparison of two steps rt pcr products of β-globin and β-actin house keeping genes using srv-2 rt enzyme and ssiii rt commercial enzyme (invitrogen). the pcr produced 206 bp and 350 bp bands representing the β-globin and β-actin, respectively. 1: 100 bp dna ladder (invitrogen); 1-4: repeated samples; 5: master mix control. 350 bp 206 bp 1500 bp 500 bp β-globin β-actin m 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 rt sstiii rt srv-2 rt sstiii rt srv-2 fraction of purification step protein concentration -1 (µg ml ) total protein (mg) enzyme concentration -1 (u µl ) pre-purified 2760 0.10 60 0.04 binding buffer 2280 0.15 90 0.07 washing buffer 578 0.24 144 0.42 elution buffer 312 1656 1368 346.8 62.4 7.1 1420 22.76 total enzyme activity (u) enzyme specific activity -1 (u mg protein) volume 7, 2013 microbiol indones 65 the protein concentration was determined by pierce bca protein assay kit (thermo scientific) and the enzyme activity was measured by reverse transcriptase assay chemiluminescent kit (roche). in conclusion, srv-2 rt gene was successfully isolated, cloned and expressed in e. coli. the expressed protein had also been purified and successfully used in rt-pcr. although the functionality of this recombinant srv-2 rt enzyme was proven indeed the activity was lower than commercial enzymes. therefore, it seems still necessary to optimize the expression of the recombinant srv-2 rt enzyme and to concentrate the product to increase the activity. in addition, it is also necessary to characterize the biochemical and kinetic properties of the enzyme, if we intend to further study the enzyme or to use it for commercial production. this preliminary result was for us very intriguing to explore this enzyme in depth in the next study. acknowledgments this study was supported financially by the directorate general of higher education (dikti), ministry of education and cultural, republic of indonesia through ipb competitive grant 2012 and dipa seameo biotrop 2012. references busso d, delagotte-busso b, moras d. 2005. construction of a set gateway-based destination vectors for highthroughput cloning and expression screening in escherichia coli. j anal biochem. 343(2):313-321. doi:10.1016/j.ab.2005.05.015. gardner mb, luciw p, lerche n, marx p. 1988. nonhuman primate retrovirus isolates and aids. adv vet sci comp med. 32:171-190. herschhorn a, hizi a. 2010. retroviral reverse transcriptase. cell mol life sci. 67(16):2717-2747. doi:10.1007/s00018-010-0346-2. hizi a, herschhorn a. 2008. retroviral reverse transcriptases (other than those of hiv-1 and murine leukemia virus): a comparison of their molecular and biochemical properties. virus res. 134(1-2):203-220. doi:10.1016/j.virusres.2007.12.008. hunt i. 2005. from gene to protein: a review of new and enabling technologies for multi parallel protein expression. protein expr purif. 40(1):1-22. doi:10.1016/j.pep.2004.10.018. iskandriati d, saepuloh u, mariya s, grant rf, solihin dd, sajuthi d, pamungkas j. 2010. kohlstaedt la, wang j, friedman jm, rice pa, steitz ta. 1992. crystal structure at 3.5 a resolution of hiv-1 reverse transcriptase complexed with an inhibitor. science 256(5065):1783-1790. doi:10.1126/science.13 77403. isolation and characterization of simian retrovirus type d from macaca fascicularis and m. nemestrina in indonesia. microbiol indones. 4(3):132-136. doi: 10.5454/mi.4.3.6. although we did not remove the additional amino acids, we were quite certain that the properties of the recombinant srv-2 rt are similar, if not identical, to those of the native rt enzyme. western blot analysis of this soluble n-terminal 6× his srv-2 rt using anti-6× his antibody indicated the presence of a specific band of 64.9 kda, confirming the presence of the tagged protein. three dimensional protein modelling demonstrated that, like other rts, srv-2 was a multi-functional enzyme possessing the rna-dependent dna polymerase (rddp), dna-dependent dna polymerase (dddp), and rnase h activities (saepuloh, submitted for publication). in this study, we only analyzed quantitatively the rddp catalytic activities based on oligo(dt) primers elongation on poly(ra)n templates 15 (table 1). as already found for most rts, among the synthetic template-primers suitable for the rddp activity, poly (ra)n (oligo(dt) is the most efficient 12-18 (taube et al. 1998). divalent cations are required for the catalytic activities of most dna polymerases, including 2+ the srv-2 rt, which used mg as an enzyme cofactor. srv-2 rt enzyme activity was also measured qualitatively by using the enzyme in a two-step reverse transcription pcr. the ability of this enzyme in reverse transcribing mrna template into cdna followed by pcr amplification using a pair specific primer designed for coding sequences gene, indicated that this srv-2 rt has the rddp and dddp activities. the volumetric activity of the purified srv-2 rt -1 recombinant enzyme was 7.1 u µl . the activity was lower compared to the commercial enzymes, for examples themmlv rt superscript iii (invitrogen) and avian myoblastosis virus (amv) rt (promega, wisconsin, usa), of which the activities were 200 u -1 -1 µl and 20-25 u µl , respectively. it is possible that 2+ the one step ni -nta chromatography was not enough to remove protein contaminants from the enzyme eluate, thus causing the low enzymatic activity. on the other hands, it is also possible that different groups of retrovirus indeed have different activities. if this is the case, then we will also have to bear in mind that the rt enzymes whose activities we were comparing came from different groups, mmlv is a gammaretrovirus, amv is an alpharetrovirus, and srv-2 is a betaretrovirus. it is known that although rts from the different groups of retrovirus share similar functional catalytic activities, they differ significantly in various parameters, such as structure and subunit compositions, molecular weights, catalytic properties, biochemical and biophysical characteristics and sensitivity to different inhibitors (herschhorn and hizi 2010). 66 saepuloh et al. microbiol indones 39(5):303-314. doi:10.1111/j.1600-0684.2010.00412.x. morton wr, agy mb, capuano sv, grant rf. 2008. specific pathogen free macaques: definition, history and current production. ilar j. 49(2):137-144. doi:10.1093/ilar.49.2.137. rosenberg ah, lade bn, chui ds, lin sw, dunn jj, studier fw. 1987. vectors for selective expression of cloned dnas by t7 rna polymerase. gene 56(1):125135. doi:10.1016/0378-1119(87)90165-x. sarafianos sg, marchand b, das k, himmel d, parniak ma, hughes sh, arnold e. 2009. structure and function of hiv-1 reverse transcriptase: molecular mechanisms of 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transcriptase lacking ribonuclease h activity. nucleic acid res. 16(1):265-277. doi:10.1093 /nar/16.1.265. landy, a. 1989. dynamic, structural, and regulatory aspect of site-specific recombination. annu rev biochem. 58:913-949. doi:10.1146/annurev.bi.58.070189.004405. lerche nw. 2010. simian retroviruses: infection and disease-implications for immunotoxicology research in primates. j immunotoxicol. 7(2):93-101. doi:10.3109/1 5476911003657406. lerche nw, osborn kg. 2003. simian retrovirus infections. potential confounding variables in primate toxicology studies. toxicol pathol. 31(suppl):103-111. marracci gh, avery na, shiigi sm, couch g, palmer h, pilcher ky, nichols h, hallick lm, axthelm mk, machida ca. 1999. molecular cloning and cellspecific growth characterization of polymorphic variants of type d serogroup 2 simian retroviruses. virology 261(1):43-58. doi:10.1006/viro.1999.9858. marracci gh, kelley rd, pilcher ky, crabtree l, shiigi sm, avery n, leo g, webb mc, hallick lm, axthelm mk. 1995. simian aids type d serogroup 2 retrovirus: isolation of an infectious molecular clone and sequence analyses of its envelope glycoprotein gene and 3’ long terminal repeat. j virol. 69(4):2621-2628. marx pa, maul dh, osborne kg. 1984. simian aids: isolation of type d retrovirus and disease transmission. science 223(4640):1083-1086. doi:10.1126/science.66 95196. montiel na. 2010. an updated review of simian betaretrovirus (srv) in macaque hosts. j med primatol. volume 7, 2013 microbiol indones 67 04 the increased demand for fossil fuel while it is running low has raised efforts to explore and utilize alternative energy sources. the use of feed stocks for biofuel production may cause problems in food security (actionaid 2013). cellulose is the most abundant biomass in nature and is not used as food source. the content of cellulose in plant reaches 3550% of plant dry weight (lynd et al. 2002). cellulases consist of three types of enzymes: (1) endoglucanase (eg, e.c.3.2.1.4), breaks internal bonds and opens the polysaccharide chains, (2) exoglucanase (e.c.3.2.1.9), breaks 2-4 units from the ends of the exposed chains produced by eg resulting in tetrasaccharides or disaccharides, such as cellobiose, (3) β-glucosidase (e.c.3.2.1.21), hydrolyses cellobiose into glucose. the production of cellulase is mainly regulated at the transcriptional level, by induction and catabolite repression (glucose). generally, there is a low level of constitutive expression of the cellulases. hydrolysis products of cellulose or their derivatives will induce expression of the cellulase genes (aro et al. 2005). simple sugars that are not hydrolysis products of cellulose can also induce cellulase expression. for example lactose can induce expression of cellobiohydrolase i gene from trichoderma reesei (messner and kubicek 1991) and eg genes from bacillus brevis (singh and kumar 1998) and bacillus sp. mtcc 10046 (sadhu et al. 2014). bacillus species have long been used for the production of various industrial enzymes such as vol.8, no.4, december 2014, p 170-176 doi: 10.5454/mi.8.4.4 cloning and expression of endoglucanase gene from thermophilic bacteria bacillus sp. rp1 1* 2 1 maelita ramdani moeis , dessy natalia , rahma widya ningrum , 1 and ari dwijayanti 1 school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia; 2 faculty of natural sciences and mathematics, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia an endoglucanase gene belonging to glycoside hydrolase family 5, had been isolated from bacillus sp. rp1 and cloned into escherichia coli. the cloned gene comprised the promoter, coding sequence, and terminator of the gene. this gene encoded a protein with 499 amino acid residues (predicted molecular weight=55.2 kda) with a typical bacillus signal peptide. the recombinant endoglucanase (eg) had optimum activity at ph 5.0 and 50 °c. the recombinant eg was expressed in the extracellular, intracellular, and periplasmic fractions with the highest total activity (60.15%) in the intracellular fraction, measured at three hours after isopropyl-β-d thiogalactopyranoside (iptg) induction. three hours after the addition of 1% carboxymethyl cellulose (cmc), there was a two-fold increase in intracellular eg specific activity compared to the uninduced cells. three hours after the addition of 1 mm iptg, 1% glucose, 1% galactose, or 1% cellobiose the intracellular eg specific activity decreased compared to the uninduced cells. key words: bacillus sp. rp1, native promoter, native signal peptide, recombinant endoglucanase gen endoglukanase yang termasuk famili 5 glikosida hidrolase telah diisolasi dari bacillus sp. rp1 dan diklon ke escherichia coli. gen yang telah diklon mencakup promotor, daerah pengkode, dan terminator dari gen. gen ini mengkode protein yang terdiri dari 499 asam amino (perkiraan berat molekul 55,2 kda) dengan peptida sinyal khas bacillus. endoglukanase (eg) rekombinan ini mempunyai aktivitas optimum pada ph 5.0 dan suhu 50 °c. eg rekombinan ini terdapat di dalam sel, di periplasma dan di luar sel, dengan aktivitas total terbanyak di dalam sel (60.15%), yang diukur tiga jam setelah induksi dengan isopropyl-β-d thiogalactopyranoside (iptg). tiga jam setelah penambahan 1% carboxymethyl cellulose (cmc) terjadi peningkatan aktivitas spesifik eg intraseluler, dua kali lebih tinggi dibandingkan sebelum diinduksi. tiga jam setelah penambahan 1 mm iptg, 1% glukosa, 1% galaktosa, atau 1% sellobiosa terjadi penurunan aktivitas spesifik eg intraseluler dibandingkan sebelum diinduksi. kata kunci: bacillus sp. rp1, endoglukanase rekombinan, peptida sinyal asli, promoter asli *corresponding author; phone: +62-22-2511575, fax: +6222-2534107 ; email: maelita@sith.itb.ac.id cellulase (sukumaran et al. 2005). cellulases belong to glycoside hydrolase (gh) enzymes and many bacterial egs belong to gh family 5. bacillus sp. rp1, a cellulolytic thermophilic bacterium, produced seven cmcases, six fpases, and four xylanases extracellularly (puspitasari and moeis 2008). in this study, we focused on its endoglucanase. the objectives of this study were to isolate one of its eg gene, determine the optimum ph and temperature of the recombinant enzyme, investigate its subcellular location, and determine the influence of the addition of specific inducers on gene expression through its native promoter. materials and methods bacterial strain and plasmids. bacillus sp. rp1 was isolated from a hot spring in cimanggu (west java, indonesia) (puspitasari and moeis 2008) and was used as source for genomic dna. escherichia coli strains used as host cells for cloning were dh5α and top10. ® the plasmid used for cloning was pgem -t easy (promega). dna isolation. a colony of bacillus sp. rp1 was inoculated into 3 ml of lb media and grown at 41 °c, 150 rpm for 16 h. dna was then isolated by wang method as described in doi and mcgloughlin (1992). the cells were incubated in lysis buffer (0.15 m nacl, -1 0.1m edta ph 8.0, 0.5 mg ml lysozyme), extracted with chloroform/isoamyl alcohol (24:1) and dna was precipitated with isopropanol. the dna pellet was washed with 70 % ethanol, centrifuged and dissolved in 200 μl water. cloning of the eg gene. pcr was performed using reagents from fermentas on a gene amp pcr system 2400 (applied biosystem).the primers used to amplify the partial eg gene were designed based on the consensus sequences of bacillus spp. eg genes from gh5 family as described by rachim (2008) (table 1). touched down pcr was performed: 94 °c, 3 min; 5 cycles of 94 °c for 30 s, 55 °c for 30 s, and 72 °c for 1 min; 10 cycles of 94 °c for 30 s, 55 to 45 °c at 1 °c interval for 30 s, and 72 °c for 1 min; 10 cycles of 94 °c for 30 s, 45 °c for 30 s, and 72 °c for 1 min.the pcr product was purified after electrophoresis using geneaid preparation purification kit, ligated to ® pgem -t easy (promega). transformation of e. coli dh5α (partial gene) was performed by the cacl heat 2 shock method and recombinant clones were selected by blue-white screening (sambrook and russell 2001). dna was sequenced by sanger method in macrogen inc., korea. to clone the complete gene, primers were designed based on the sequencing results of the partial eg gene, so that the pcr product would include the regulatory region and terminator. primers were designed based on conserved sequences from six bacillus subtilis strains (acc. no. ay044252.1, x04689.1, d01057.1, al009126.3, z29076.1, and x67044.1) and one bacillus sp. (acc. no. af045482.1). the sequence of the forward primer was gtttgcattcagttca gttgtc and reverse primer was tcagtatttcatc cacaacgca. the initial pcr condition was denaturation at 94 °c for 3 min followed by 25 cycles of denaturation at 94 °c for 30 s, annealing at 55.9 °c for 30 s, elongation at 72 °c for 2 min and final elongation at 72 °c for 7 min. the pcr product was gel purified and cloned into e. coli dh5α top 10. the cloning and sequencing methods used were the same as previously described. nucleotide sequence analysis. sequencing results were analyzed using programs bioedit (hall 1999), vecscreen (https://www.ncbi.nlm.nih.gov/ tools/vecscreen/), blast (altschul et al. 1990) (http://blast.ncbi.nlm.nih.gov/blast/). the in silico translated product of the eg gene was characterized using online programs from ncbi (www.ncbi.nlm. nih.gov), expasy proteomic server (gasteiger et al. 2003) (http://expasy.org/), and interproscan (zdobnov and apweiler 2001) (http://www.ebi.ac.uk/tools/ pfa/iprscan/) to know the domain and motif generated. e x p re s s i o n a n d r e c o m b i n a n t e n z y m e preparation. e. coli top10 expressing recombinant eg was inoculated into 10 ml of lb broth containing -1 100 µg ml of ampicillin at 37 °c for 16 h. after that, 0.1 ml of overnight culture was inoculated into 10 ml -1 of new lb broth containing100 µg ml ampicillin and grown at 37 °c until the optical density (od) reached 0.5 at 600 nm. then, appropriate inducers were added into the culture broth. the culture was incubated at 37 °c for 3 and 6 h after induction. the fractionation of extracellular, periplasmic, and intracellular eg were * r(a/g), y(c/t), h(a/c/t), s(c/g), m(a/c), w(a/t) primer sequences 5’-trtatgtcatyattgaytggca-3’ 5’-aygaaattgcraaygarcc-3’ 5’-ccccaytchstmacraawa-3’ forward f1 forward f2 reverse r table 1 primer sequences used to isolate partial endoglucanase gene (rachim 2008) volume 8, 2014 microbiol indones 171 carried out by the method of yamabhai et al. (2008). extracellular fraction was obtained from the supernatant by centrifugation. the periplasmic fraction was released by osmotic shock. the cells were resuspended in cold (4 °c) spheroplast buffer (100 mm tris–cl, ph 8.0, 0.5 mm edta, 0.5 mm sucrose, and -1 20 µg ml phenylmethylsulfonyl fluoride). after incubation for 5 min on ice, the bacterial cells were collected by centrifugation and resuspended in ice-cold sterile water supplemented with 1 mm mgcl and 2 incubated on ice for 5 min with frequent shaking. the supernatant was then collected by centrifugation as the periplasmic fraction. the precipitated cells were washed once with lysis buffer (50 mm tris–cl + 0.5 mm edta), resuspended in lysis buffer and sonicated (bransonic 32, 150 watt), for 30 s on and 30 s off repeatedly for 5 times at 4 °c. the cell debris was centrifuged and the supernatant was collected as the intracellular fraction. for e. coli top10 containing pgem-t easy without dna insertion, only intracellular fraction was taken. eg activity assay and total protein estimation. eg activity was measured by incubating 0.05 ml of enzyme solution with 0.15 ml of 1% (w/v) cmc, prepared in 50 mm phosphate citrate buffer (ph 5) for 1 h at 50 °c. the release of reducing sugar was measured by ferricyanide alkali method (walker and harmon 1996) using a calibration curve for d-glucose. one unit of eg activity was defined as the amount of enzyme which produced one µmol of reducing sugar (glucose equivalent) per minute under the assay condition. protein content in the supernatant was estimated by bradford method (bradford 1976) using bovine serum albumin (bsa) as standard. results characterization of the eg gene of bacillus sp. rp1. two clones containing the partial eg genes were obtained, named ega (406 bp) and egb (286 bp). pairwise alignment of the sequences of both clones using software bioedit showed that egb was part of ega. sequence analysis of the partial genes using blast n showed that ega and egb had high similarities with eg genes from bacillus subtilis strains, reaching 98% identity in nucleotide sequence. the complete eg gene was named egc (submitted to ncbi with acc. no. kj652953). using orf finder program, the coding sequence of egc was found to be 1497 nucleotides at position 651 – 2147, producing a protein consisting of 499 amino acids. the promoter sequence of egc was tacaat (pribnow box) and tagacg (-35 consensus sequence) and the shine dalgarno was aggagg (fig 1). two inverted repeats were found in the sequence which could be terminator sequences. one was downstream the egc coding sequence and the other was upstream the egc promoter, which could be the terminator of a gene located upstream the eg gene (fig 2). amino acid sequence analysis revealed that egc had high amino acid sequence homology (97-100%) with egs of b.subtilis strains. at the n terminus there was a signal peptide consisting of 29 amino acid residues (mkrsisifitcllitlltmggmias pasa). this was followed by a cellulase domain which had a motif for glycoside hydrolase family 5 (viyeianepn). on the c terminus there was a carbohydrate binding module (cbm3). subcellular location of recombinant enzyme in e. coli. the optimum eg activity in the crude extract was obtained at ph 5 and 50 °c (fig 3).the majority of the eg activity was in the intracellular fraction, both at 3 h (60.15%) and 6 h (66.58%) after iptg induction. three hours after iptg induction, eg mainly accumulated in the cytoplasm and some were secreted into the periplasm (1.92%) and out of the cell (37.93%). the total activity of eg in all fractions after six hours induction was higher than after three hours induction. six hours after induction, the activity in the periplasmic fraction increased to 6.56% and the extracellular fraction decreased to 26.86% compared to three hours after induction (data not shown). since most of the proteins were present in the cytoplasmic fraction, the effect of specific inducers on eg activities were analysed in this fraction. effect of the addition of specific inducers on eg expression. the specific activity of eg in non-induced cells after 6 h incubation (equivalent to 3 h after -1 induction) was 23.5 u mg and the activity after 9 h incubation (equivalent to 6 hours after induction) was -1 2.1 u mg . there were about two-fold increases in eg specific activities three hours after the additions of 1% cmc and 1% cmc+ 1 mm iptg compared to noninduced cells. at six hours after induction, the increases in eg specific activities after addition of 1% cmc, 1% cmc+ 1 mm iptg and 1 mm iptg were six-fold (13.1 -1 -1 -1 umg ), 12-fold (24.7 umg ) and 10-fold (20.3 mg ) -1 respectively compared to non-induced cells (2.1 u mg ). additions of 1% glucose, 1% galactose, 1% cellobiose and 1 mm iptg (specifically three hours after induction) gave eg activities that were either similar or lower than non-induced cells (fig 4). 172 moeis et al. microbiol indones fig 1 positions of promoter (-35, -10), transcription start site (tss), ribosome binding site (rbs), start codon (start), stop codon (stop) and terminator (repeat region) of the cloned endoglucanase gene (egc) of bacillus sp. rp1.zone. fig 2 full length endoglucanase gene of bacillus sp. rp1 with its regulatory elements that was inserted into pgem-t easy plasmid and cloned into e. coli top10. repeat regions = terminators; tss = transcription start site;rbs = ribosomal binding site. fig 3 effect of ph (a) and temperature (b) on the bacillus sp. rp1 recombinant eg activity a b volume 8, 2014 microbiol indones 173 discussion optimum ph and temperatures of bacillus sp. egs. the optimum ph and temperature of egc was the same as the crude extract of extracellular eg of bacillus sp. rp1, i.e. at ph 5 and 50 °c (puspitasari and moeis 2008). this is different from the optimum conditions of other bacillus egs. there was a wide range of optimum phs (4-10) and temperatures (40-65 °c) for egs in bacillus spp. the optimum conditions for eg activities were ph 4 and 40 °c for bacillus thuringiensis var. israelensis and bacillus thuringiensis var. thompsoni (lin et a., 2012), ph 5 and 60 °c for bacillus sp. m9 (bajaj et al. 2009), ph 7 and 50 °c for bacillus sp. (sadhu et al. 2014), ph 9 10 and 50 °c for bacillus sp. (nizamudeen and bajaj 2009), ph 6-8 and 65 °c for bacillus subtilis lh (zhao et al. 2012). secretion of egc. there was an increase in total activity of the cell compartments along with inducer exposure time. the signal peptide sequence of egc was a typical signal peptide for bacillus subtilis. this signal peptide consisted of hydrophilic amino acid such as lysine and arginine, followed by 18 hydrophobic amino acids (rich in leucine and isoleucine) and a short sequence before the cleavage site which contained several alanine residues (mackay 1986). typical bacillus signal peptide could be recognized by e. coli secretion machinery (yamabhai et al. 2008). this suggested that the mechanisms of protein secretion in gram-negative and gram-positive bacteria were highly conserved although there was a great difference in the structure of the cell wall and cell membrane (yamabhai et al. 2008; mergulhão et al. 2005). however, the e. coli protein secretion system could not secrete this recombinant protein through the inner and the outer membranes efficiently. this was shown in this experiment by the fact that most of the activities were in the intracellular fractions. the intracellular eg specific activity was lower at six hours compared to three hours after the addition of the inducers, except after the addition of iptg. iptg cannot be metabolized by the cell, eg synthesis remained relatively constant. on the other hand, all the other inducers could be metabolized by the cell. this could decrease the rate of eg synthesis lower than the rate of secretion. other bacillus spp. may have different secretion efficiency. secretion efficiency of recombinant enzyme depends on the inducer exposure time, the type of signal peptide, and the molecular mass of the enzyme (yamabhai et al., 2008). expression of the cellulase gene from b. subtilis h12 in e. coli jm109 were mainly in the periplasm (80%) and none were found in the medium (oh et al. 2008). on the other hand, eg of b. subtilis i15 that was cloned in e. coli bl21 was mostly extracellular (yang et al. 2010). basal eg activity. the promoter of egc was not identical to any of the constitutive promoters of e. coli (shimada et al. 2014), nor b. subtilis (radeck et al. 2013). however, eg activity was present in noninduced recombinant e. coli, suggesting low level of expression which was needed for the regulation of the cellulase synthesis (aro et al. 2005; lynd et al. 2002; sukumaran et al. 2005). the lb medium that was used could have contributed to the relatively high basal activity. in b. subtilis, basal activities from promoters was higher when the bacteria were grown in lb medium compared to mose medium (mops-based chemically defined medium with succinate and glutamate) (radeck et al. 2013). lb medium contains yeast extract and carbohydrates present in the yeast extract might have contributed to the low level of egc fig 4 specific activity of intracelullar eg with addition of specific inducers (non-induced, 1 mm iptg, 1 % cmc, 1 % cmc + 1 mm iptg, 1 % glucose, 1 % galactose, and 1 % cellobiose) at 3 hours (dark gray column) and 6 hours (light gray colum) after induction. the error bars are based on three experimental replicates. 174 moeis et al. microbiol indones expression. effect of the addition of inducers on eg activity. since there was a possibility of inefficient secretion and a decrease in inducer concentrations affecting the results at six hours after induction, analysis of the effect of inducer on the recombinant eg were performed at three hours after induction. iptg addition increased eg expression in bacillus sp. rp1 (unpublished data) but decreased it in recombinant e. coli. iptg is a synthetic molecule that is stable because it is not metabolized by the cell and can replace lactose as an inducer. no sequence similar to the lac operator in the regulatory region of egc was present, therefore a different regulatory protein would be involved. the molecular mechanisms of lactose induction of eg in bacillus spp. are still unknown. lactose had variable effects on eg expression of bacillus species. lactose induces eg gene expression in bacillus brevis (singh and kumar 1998) and bacillus sp. mtcc 10046 (sadhu et al. 2014), but had no effect on bacillus sp. x10-1-2 (yan et al. 2013). since iptg could not increase eg activity from egc promoter in e. coli, the regulatory protein involved in egc induction by iptg in bacillus sp. rp1 might be absent in e. coli. among all the inducers tested three hours after induction, only cmc showed eg activities greater than without induction. cmc is a soluble cellulose derivative that can induce cellulase synthesis in many fungal and bacterial species. cmc is a macromolecule that could not enter the cell. low level of eg synthesis would break down cmc to smaller molecules that could induce cellulase synthesis (aro et al. 2005). the addition of glucose decreased eg expression, however the eg activities after the additions of galactose and cellobiose were even lower. these sugars were usually known as cellulase inducers. glucose as the final product of cellulose hydrolysis is known as a carbon catabolite repressor that can inhibit the production of cellulase (sukumaran et al. 2005). on the other hand, chundakkadu (1998) stated that the addition of glucose can be used as an inducer in the production of cellulase. addition of galactose inhibited eg expression, however the mechanism is yet to be known. e. coli top10 used in this study had a mutation in galk, so it cannot metabolize galactose. because the galactose was not metabolized by cells, the cells remained inhibited six hours after induction. cellobiose was known to be an inducer for many cellulase genes. at high concentrations, cellobiose was known to inhibit cellulase production (kubicek and penttila 1998). cellobiose (two β-1,4-linked glucose unit) can be hydrolyzed by β-glucosidase into glucose molecules. β-glucosidase produced by the host cell (e. coli has bgl operon to produce β-glucosidase) will help in the formation of inducers for eg synthesis (aro et al. 2005) through the transglycosylation mechanism. βglucosidase has an ability to transglycosylate cellobiose to sophorose (two β-1,2-linked glucose unit), which would induce cellulase synthesis. the effect of cellobiose addition on the eg expression o c c u r s i n t h e b a l a n c e o f h y d r o l y s i s a n d transglycosylation mechanism (aro et al. 2005). sophorose is a known inducer for fungal cellulases, but there are no report on the effect of sophorose inducing bacillus cellulases. a full length eg gene from bacillus sp. rp1was cloned in e. coli. no eg gene with the same sequence of egc has been characterized before. this protein has potential to be used in industrial applications. references actionaid. 2013. fuelling hunger: new data reinforces why the uk must tackle damaging biofuels policies at the g 8 a n d t h e e u . 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2 research center for detection of pathogenic bacteria, lembaga penelitian dan pengabdian kepada masyarakat, universitas negeri jakarta, jl. rawamangun muka, jakarta timur 13220, indonesia; 3 center forensic laboratory of the criminal investigation, police of the republic of indonesia, cipambuan babakan madang, bogor, 1681, indonesia; 4 institute of bioproduct development, universiti teknologi malaysia (utm), skudai, johor bahru, malaysia; 5 school of chemical and energy engineering, faculty of engineering, universiti teknologi malaysia (utm), skudai, johor bahru, malaysia; 6 city of scientific research and technology applications, new burg al arab, alexandria, egypt. cases of food poisoning often occur due to food contamination caused by pathogenic bacteria. one of the pathogenic bacteria is which is found in seafood. thus, a fast, accurate and specificvibrio parahaemolyticus detection method is needed. the purpose of this study was to quickly detect bacteria invibrio parahaemolyticus seafood samples targeting the gene using real time pcr. in a previous study, gradient pcr was used totoxr optimize ideal annealing temperature ranges from 53-62°c and revealed that 58°c produced the best outcomes for the primer with a size of 171 base pairs. real-time pcr was utilized to amplify, specify, and test fortoxr sensitivity under the ideal conditions from the pcr gradient. the confirmation results show that the primer pairs could amplify of with the amount of concentration as much as 50 ng µl with cttoxr vibrio parahaemolyticus -1 10.69 and 10.32 and melting curve at temperature 82.18°c and 82.23°c. this primer pair can also distinguish nontarget bacteria with different ct and melting curve temperature. the sensitivity assay for this primer can amplify dna templates at concentration 0.0032 ng µl . shrimp samples that are contaminated artificially can still be -1 detected at ct 13.02 and ct 13.09. based on these results, it can be concluded that real time pcr with toxr primer can be applied to develop a detection kit for in seafood.vibrio parahaemolyticus key words: detection kit, foodborne diseases, real-time pcr, ,toxr vibrio parahaemolyticus kasus keracunan makanan sering terjadi karena kontaminasi makanan yang disebabkan oleh bakteri patogen. salah satu bakteri patogen adalah yang banyak ditemukan pada makanan laut. olehvibrio parahaemolyticus karena itu, diperlukan metode pendeteksian yang cepat, akurat dan spesifik. tujuan dari penelitian ini adalah untuk mendeteksi secara cepat bakteri pada sampel makanan laut yang menargetkan genvibrio parahaemolyticus toxr menggunakan real time pcr. dalam penelitian sebelumnya, pcr gradien digunakan untuk mengoptimalkan suhu annealing ideal antara 53-62°c dan mengungkapkan bahwa 58°c menghasilkan hasil terbaik untuk primer dengan ukuran 171 pasangan basa. pcr real-time digunakan untuk memperkuat,toxr menentukan, dan menguji sensitivitas di bawah kondisi ideal dari gradien pcr. hasil konfirmasi menunjukkan bahwa pasangan primer dapat mengamplifikasi dengan jumlah konsentrasitoxr vibrio parahaemolyticus sebanyak 50 ng µl dengan ct 10,69 dan 10,32 dan kurva leleh pada suhu 82,18°c dan 82,23° c. pasangan primer -1 ini juga dapat membedakan bakteri non-target dengan ct dan suhu kurva leleh yang berbeda. uji sensitivitas untuk primer ini dapat mengamplifikasi template dna pada konsentrasi 0,0032 ng µl . sampel udang yang tercemar -1 artifisial masih dapat dideteksi pada ct 13,02 dan ct 13,09. berdasarkan hasil tersebut, dapat disimpulkan bahwa real time pcr dengan primer dapat diterapkan untuk mengembangkan kit deteksi vibrio parahaemolyticustoxr pada makanan laut. kata kunci: keracunan pangan, kit deteksi, real time pcr, toxr, vibrio parahaemolyticus microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-81517249667; fax: +6221-4894909; e-mail: muktiningsih@unj.ac.id world. this disease comes from consuming contaminated food. everyone is at risk for foodborne disease. however, some people may be at greater risk of serious symptoms or even death (bari and ukuku 2016). 52.4% of the 208 instances of food poisoning foodborne disease is one of the public health challenges that causes death every year around the reported to dki jakarta in 2016 were attributable to seafood intake. (mabruroh and ciptaningtyas 2018). vibrio parahaemolyticus is one of the pathogenic bacteria that can cause foodborne diseases in seafood. in estuarine and marine environments around the world, vibrio parahaemolyticus is a naturally occurring part of the microbial community and is a major cause of bacterial sickness linked to seafood consumption. (newton 2012). a culture method is one of theet al. several approaches that have been proposed to identify v. parahaemolyticus ( it is requiredduan and su 2005). to design a new quick, sensitive, and targeted way to identify the because thevibrio parahaemolyticus culture method has a lengthy execution time and is difficult to define limits in samples at low concentrations (dorak 2006). bacterial identification at the lowest concentration is possible with real-time polymerase chain reaction. additionally, it only takes 2.5 hours, or, when combined with sample preparation, real-time pcr can be finished in about 5 hours (nurjayadi 2019).et al. a detection kit with thesalmonella typhi fimc gene, whose product length is 95 base pairs, was successfully developed in a prior study. (nurjayadi et al. 2017). the aim of this research is to create a precise, perceptive, and quick detection kit of vibrio parahaemolyticus toxrwith gene using real-time pcr. ideal annealing temperature gene has beentoxr optimized at 58°c with a size of 171 base pairs (nurjayadi 2022)et al. . based on the previous experience and literature reviewed, the detection method of s s as a rapid,vibrio parahaemolyticu sensitive, and specific detection kit is expected to be developed. materials and methods preparation of culture sample. vibrio parahaemolyticus atcc 17802 in kwik-stiktm (microbiologist, minnesota) was employed in this study. was further cultured inv. parahaemolyticus thiosulfate citrate bile sucrose (tcbs) agar (merck) at 37ºc for 24h. after incubation, one single colony was inoculated to tryptic soy broth (tsb) + 2.5% nacl and incubated in an incubation shaker (yihder lm-400d) at 37ºc for 24h and 150 rpm. for artificially contaminated food samples, vibrio parahaemolyticus inoculum was then diluted at 10 dilution with -1 -7 -10 nacl 0.85%. tcbs media (merck) was used to grow a one ml solution of 10 -10 dilutions using spread plate -5 -7 method and incubated at 37ºc for 24h to determine the number of colonies contaminated food samples. colonies result used for artificial contamination is a sample dilution that formed 30-300 colonies on a based on the fda bam's plate count standard (food and drug administration bacteriological analytical manual). cfu ml was then determined based on the -1 number of colonies. seafood artificially contaminated with vibrio parahaemolyticus. first, shrimp samples were boiled. the boiled samples were then ground until smooth in a sterile plastic. samples were put in a petri dish and exposed to uv light for a half-hour. after that, the shrimp sample was transferred to a sterile erlenmeyer flask and infected with a suspension of vibrio parahaemolyticus at a volume ratio of 8:2. the positive control used was in the form of non-dilute suspension of bacteria to be inoculated on shrimp samples, samples of uncontaminated food were used as a negative control. at a speed of 150 rpm, an orbital shaking incubator from the lm series (yihder co. ltd.) was used to incubate each sample for 24 hours at 37 c.º isolation of dna. dna isolation, 1.5 ml ofbefore the bacterial culture from thevibrio parahaemolyticus tsb + 2,5% nacl was centrifuged at 5000 x g for 10 minutes. thermo scientific genejet genomic dna purification kit (thermofisher, 2012) and geno plus genomic dna extraction miniprep system (viogene) were used to isolate dna. the concentration of dna isolation results was determined using nanodrop (nanovue plus), then confirmed by electrophoresis on 0.7% agarose gel (nzytech) and checked with an uv transilluminator (vilber lourmat). optimization annealing temperature of toxr primer pairs: synthesized primer pairs were then optimized using gradient pcr with temperature length of 53-62°c. a pcr cocktail contained of colorless master mix (nzytaq), genomev. parahaemolyticus as a dna template, nuclease free water (qiagen), toxr toxr-f (forward primer) and -r (reverse primer) with a total volume of single reaction is 25µl. optimization results are followed by 40 cycles in reaction conditions of 95ºc for 3 minutes (initial denaturation), 95ºc for 10 second (denaturation), annealing temperature 53-62ºc for 30 second, 72 ºc for 30 seconds (extension), and 72 ºc for 7 minutes (final extension) (smobio 2017). vi b r i o p a r a h a e m o l y t i c u s to x r g e n e confirmation assay. the confirmation assay of vibrio parahaemolyticus was run in a magnetic induction cycler qpcr (bio molecular system). a pcr cocktail contained of qpcr master mix with sybr green dye 10 krisdawati et al. microbiol indones (smobio), genome as a dnavibrio parahaemolyticus template, nuclease free water (qiagen), toxr-f and toxr-r with a total volume of single reaction is 20µl. to verify the fluorescence signals not contaminated by any contaminant; negative or non-template controls were also run. as a negative control, a pcr mixture does not contain any dna template. positive control is salmonella typhi fimcwith primers that have already study before (nurjayadi 2019). the pcr cycleet al. conditions are followed by 40 cycles in reaction conditions of 95ºc for 3 minutes (initial denaturation), 95ºc for 10 second (denaturation), annealing temperature 58 ºc for 30 second, 72ºc for 30 seconds (extension), and 72ºc for 7 minutes (final extension) (law 2015)et al. . specificity and sensitivity assay. the specificity assay of primer pairs -f and -r were testedtoxr toxr with non-target bacteria, such as, vibrio alginolyticus, cronobacter sakazakii, listeria monocytogenes, yersinia enterocolitica, salmonella typhi, klebsiella pneumoniae, staphylococcus aureus. meanwhile, for the sensitivity assay, culturevibrio parahaemolyticus stock was diluted to establish the real-time pcr primer pairs' limit of detection (lod). by graphing the ct (cycle threshold) value against the plasmid dna copy number, the standard curves for vibrio parahaemolyticus copy number were created. ( . the pcr cycle conditions weremirasoli 2011) described above. confirmation assay of seafood sample. amount of 10 milligram shrimp contaminated artificially by vibrio parahaemolyticus bacteria were used as dna template samples in pcr mixture; meanwhile, a positive control of this assay is genome dna of culture stock of bacteria nonvibrio parahaemolyticus dilute results, and a negative control are dna template from shrimp non-contaminated as samples. pcr mixture and cycle conditions were the same as described above. results cultivation of v. parahaemolyticus. bacteria on selective media thiosulfate citrate bile sucrose (tcbs) agar showed the formation of green colonies on the surface of tcbs media (fig 1). in bacterial suspension 10 achieve the fda bam (food and drug -6 administration bacteriological analytical manual)recommended colonies. there are reportedly 88 colonies of forming.v. parahaemolyticus qualitative and quantitative dna analysis. dna purity and concentration were measured using nanodrop (nanovue plus) with a result as described in table 1. bacteria wasvibrio parahaemolyticus successfully isolated, as shown in fig 2 based on the. electrophoresis analysis of dna isolates, vibrio parahaemolyticus-specific dna bands are visible in the upper section, higher than the 1 kb marker size. optimization of primer pairs annealingtoxr temperature. before real-time pcr assay, primer pairs were optimized in annealing temperature range 53ºc-62ºc. the results of gradient pcr were then visualized using 2% agarose gel electrophoresis. this optimization showed in fig 3, whereas the positive control is , which produced 151cronobacter sakazakii base pairs. amplification band of primer pairs istoxr at 171 bp, and the optimum annealing temperature result is at 58ºc. real-time pcr amplification. confirmation assay resulted from that and primer pairstoxr-f toxr-r can amplify dna at ct 10.32vibrio parahaemolyticus and 10.69, as shown in fig 4. the melting curve (fig 5) of was at 82.18ºc andvibrio parahaemolyticus 82.23ºc. non template control amplified ct 33.11 with the differences 23 cycles and different melt curve with low peak at 82.34 ºc. specificity sensitivity assay. vibrioand parahaemolyticus isolate was diluted as much as five times of dilution. this assay gave the results that vibrio parahaemolyticus dna template can still detect at a lower concentration of 0,0032 ng µl at (fig 6 -1 28.60 and table 2) standard curve has a regression value of r = 0,9969 and equation y = -4.194 x + 18.68.2 toxr primer pairs were tested with non-target bacteria as mentioned before and showed a good result as shown in fig 7. primer pairs can amplify to non-target bacteria volume 17, 2023 microbiol indones 11 table 1 design of cacao beans fermentation experiment sample concentration (ng µl-1) purity (a260/a280) v. parahaemolyticus dna isolated (lane 1 & 2) 50 1,92 v. parahaemolyticus dna isolated (lane 3 & 4) 47 1,85 with a difference range amount o dnaf 17-21 cycles. and non-target, bacteria have a different melting curve (fig 8 and table 3). confirmation assay of seafood sample. artificial contaminated shrimp samples and negative control non contaminated shrimp samples checked 12 krisdawati et al. microbiol indones fig 1 culture results on tcbs agar.vibrio parahaemolyticus fig 2 gel electrophoresis dna isolates results of . lane 1. dna marker 1 kb ladder. lanev. parahaemolyticus 2, 3, 4, 5. the genome of bacteria.vibrio parahaemolyticus fig 3 optimization of gene fragments with an annealing temperature range of 53-vibrio parahaemolyticus toxr 62°c (1) dna marker 100 basepair; (2) nfw+mm (negative control); (3) ntc; (4) positive control grxb cronobacter sakazakii; (5) dna fragment at temperature 53ºc; (6) dna fragment at temperature 54ºc; (7) dna fragment at temperature 55ºc; (8) dna fragment at temperature 56ºc; (9) dna fragment at temperature 57ºc; (10) dna fragment at temperature 58ºc; (11) dna fragment at temperature 59ºc; (12) dna fragment at temperature 60ºc; (13) dna fragment at temperature 61ºc; (14) dna fragment at temperature 62ºc; (15) dna ladder 100 bp. then compared with non-dilute positive controls (fig 9). primer pairs can amplify the shrimp sample attoxr the melt curve in fig 10,ct 13,02 and ct 13,09. showed that both samples have the similar melting temperature at 82.28ºc and 82.22ºc. discussion according tocultivation of v. parahaemolyticus. ( b i s h a 2 0 1 2 ) t h e r e s u l t i n ge t a l . vi b r i o parahaemolyticus colonies were in accordance with having a round size with a diameter of 2-3 mm and greenish colour on tcbs media. in the 10 dilution -6 there were 88 bacterial colonies, so that the number of bacterial cultures in one milliliter were obtained: 88 x 10 cfu ml . the results of this calculation will be5 -1 applied to determine the number of bacterial colonies formed in the confirmation test using seafood sample. qualitative and quantitative dna analysis. the requirements for the purity of a good dna isolate are 1.8-2.0. if it is less than 1.8, it indicates that there is still an impurity in the form of rna and if it is more than 2.0, it indicates that there are still contaminants in the form volume 17, 2023 microbiol indones 13 fig 4 amplification curve of a concentration 50 ng dna templated bacterial stock culture,v. parahaemolyticus positive control , and negative control ntc, nfw+mm.salmonella typhi fig 5 stock culture melting curve with 50 ng dna template.v. parahaemolyticus fig 3 the sensitivity test and amplification curves for the gene.toxr vibrio parahaemolyticus 14 krisdawati et al. microbiol indones of protein (wasdili and gartinah 2018). measurement of concentration and purity of pure culture isolates of vibrio parahaemolyticus dna showed good results. vibrio parahaemolyticusspecific dna bands are visible in the upper section, higher than the 1 kb marker size, indicating that the results are consistent with data of the genome size that is 3.288.558 bp (markino et al. 2003). optimization of primer pairs annealingtoxr temperature. an important factor in real-time pcr is optimization annealing temperature for primer pair design (green and sambrook 2019) annealing temperatures were set based on ± 5 ºc from andtoxr-f toxr-r primer pairs theoretical result which is ± 5 c0 from 58 ºc and also this melting curve can be calculated by inserting the amount of nucleotides content in the formula tm= 4(g+c) + 2(a+t) (innis 1997). the results of gradient pcr were then visualized using 2% fig 7 amplification curve for the gene's primer specificity.vibrio parahaemolyticus toxr table 2 the phytochemical component from ethanol extract of fermented cacao beans table 3 findings from an analysis of the gene primer specificitytoxr v. parahaemolyticus volume 17, 2023 microbiol indones 15 agarose gel electrophoresis. this optimization showed a good result as revealed in fig 2. amplification band of toxr primer pairs is at 171 bp, and the optimum annealing temperature result is at 58 ºc. the band has a generally constant band thickness when it is annealed at a temperature between 53-62 ºc. based on the brightest, most stable, and single band that appears, the primer's ideal annealing temperature can amplify the template in the pcr process between 58-62 degrees celsius. therefore, if used in conjunction with in silico analysis, 58 degrees celsius is the most ideal annealing temperature for the pcr procedure (nurjayadi et al. 2022). real-time pcr amplification. confirmation fig 9 the amplification curve in the confirmation evaluation of primer withtoxr vibrio parahaemolyticus shrimp samples. fig 10 the melting curve in the confirmation evaluation of primer with shrimptoxr vibrio parahaemolyticus samples. fig 8 melting curve of the primer specificity of the gene.vibrio parahaemolyticus toxr assay resulted from that and primer pairstoxr-f toxr-r can amplify . the fewervibrio parahaemolyticus number of cycles that required the fluorescent signal is first recorded as statistically significant above background and thus lower the ct value. the melting curve result shows that there is no mis-priming, which means that the primer just amplifies the target dna as indicated by the formation of one peak (biorad 2006). specificity sensitivity assay. the sensitivityand assay gave the results that vibrio parahaemolyticus dna template can still detect at a lower concentration of 0.0032 ng µl with lod 0.00396 cfu ml at ct -1 -1 28.60 (fig 6 and table 2). the standard curve can be determined by prolonging the amplification curve by the value of the x-axis, in which the value of the yaxis is the sample concentration. the curve's ct connection and concentration were made. standard curve has a regression value of r = 0,9969 and equation y = -4.1942 x + 18.68 or value regression, which has superb linearity (dorak 2006). primer pairs were tested with non-toxr target bacteria as mentioned before and showed a good result as shown in fig 7. primer pairs can amplify to non-target bacteria with a difference range amount of 17-21 cycles. which can be considered as a negative control as stated by that if the cycles between target and nontarget bacteria had a different range of 10 cycles, it considered that the primer pairs can amplify the nontarget bacteria . dna and non-(nurjayadi 2017)et al. target, bacteria have a different melting curve (fig 8 and table 3). so that this melting curve result can be a reference that non-target dna can be considered as a negative control. confirmation assay of seafood sample. plate count results show that 10 dilution samples have 88-6 colonies. this result is chosen since it followed fda bam recommendation. so, the non-dilute bacteria that is contaminated artificially in food sample is 88x105 cfu ml . artificial contaminated shrimp samples and -1 negative control non contaminated shrimp samples checked then compared with non-dilute positive control (fig 9). primer pairs can amplify the shrimptoxr sample at the melt curve in figct 13.02 and ct 13.09. 10, showed that both samples have the similar melting temperature compared to non-dilute at 82.28ºc and 82.22ºc. so, it can be stated that the concentration of vibrio parahaemolyticus contaminated in the shrimp sample is cfu ml . this result can be stated -1 2,04x10-1 that the method could be developed as a detection kit model to determine pathogenic bacteria as rapid, sensitive, and specific and show an accurate result. acknowledgement this research is funded by kemendikbudristek by the scheme pdupt under the agreement number ontracts 1/pg.02.00.pt/lppm/v/2022. additionally, we would like to thank pt sinergi indomitra pratama for providing the instruments for this study, the indonesian national police forensic laboratory center (puslabfor polri), sentul bogor indonesia, for supporting this study based on the mou between unj and puslabfor, and the entire salmonella team at the center of excellence for pathogenic bacteria detection (pui pendeteksi bakteri patogen) lppm unj for their hard work and contribution. our appreciation is also conveyed to our international partner, ibd-utm, whose support is essential to all collaborative efforts. references bari l, ukuku do. 2016. foodborne pathogen and food safety. in .crc press bio-rad. 2006. real-time pcr applications guide. bior a d l a b o r a t o r i e s , i n c , p . 1 1 . d o i : 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i, putri gi, declan jl, juliansyah da, azzahra m, maulana i, rahmawati an, kartika ir, kurniadewi f, sukmawati d, rahayu s, saamia v, saputro dao, wiranatha im, enshay he. 2022. the potential of the toxr gene as a detection tool for vibrio parahaemolyticus in seafood. in: smic 2022. nurjayadi m, pertiwi yp, islami n, azizah n, efrianti ur, saamia v, wiranatha im, nastassya l, el-enshasye ha. 2019. detection of the salmonella typhi bacteria in contaminated egg using real-time pcr to develop rapid detection of food poisoning bacteria. biocatalysis and agricultural biotechnology 20, . doi: 10.1016/j.bcab. 2019.101214. nurjayadi m, santoso i, kartika ir, kurniadewi f, saamia v, so han w, nurkhasanah d. 2017. isolation,fi ampli cation and characterization of foodbornefi pathogen disease bacteria gene for rapid kit test development. in: aip conference proceedings 1862 030077. doi: 10.1063/1.4991181. smobio. 2017. product information exceltaq series 2x q-pcr master mix (sybr, rox). retrieved from. www.smobio.com. thermofisher. 2012. dna isolation kit catalog number 761001d. retrieved from. www.thermofisher.com. wasdili faq, gartinah t. 2018. penentuan kualitas isolasi dna dengan metodesalmonella typhimurium spektrofotometri dan elektroforesis. prosiding pertemuan ilmiah nasional penelitian & pengabdian masyarakat (pinlitmas 1), 1(1), 578–583. volume 17, 2023 microbiol indones 17 4. mi-milani.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.11.2.4issn 1978-3477, eissn 2087-8575 vol 11, no 2, june 2017, p 62-68 *corresponding author; phone: +62-21-7560536 ext 7119, fax: +62-21-7566299. email: is.helianti@bppt.go.id lipases are abundant in the nature, including in plants, animals, and microorganisms. microbes are a major source of many enzymes including lipase for industrial application (vakhlu and kour 2006). lipases had more than 60% usage in the industries, such as detergent, food, and starch industry. most of the industries use recombinant enzymes for their product (cowan 1996). yeast has been used in many industrial enzymes, because it’s considered to be easily handled and has high cell-density than bacteria (cregg et al. 1993). gene cloning and protein expression of lipase gene require expression vectors and suitable host. synthetic thermomyces lanuginosus lipase in previous studies has been cloned and expressed in escherichia coli using puc57 and pet plasmid vector, and bacillus subtilis using vector pske194 (haniyya 2016). the result of studies showed that expression levels of lipase was low. because of that, it needed another alternative to increase expression of lipase gene. researchers are looking for alternative hosts for increasing the expression of lipase such as methylotrophic yeast pichia pastoris. lipase is one of the most important industrial enzymes, which is widely used in the preparation of food additives, cosmetics, and pharmaceutical industries. in the previous study, we have cloned synthetic thermomyces lanuginosus lipase gene into bacillus subtilis and escherichia coli and resulting low expression for enzyme activity. the aim of this research was to construct the t. lanuginosus lipase (tll) gene into pichia pastoris vector expression with tll original signal peptide. tll gene was amplified by pcr and contained original signal peptide and then inserted into ppiczα a between xhoi and xbai site, and transformed into competent cell e.coli dh5α. from the transformant, two of positive recombinants were analyzed by sequencing analysis. as the result, both of two recombinant have a positive target gene which has lipase gene. the correct plasmid was linearized and then was transformed in p. pastoris x-33 by electroporation method. thermomyces lanuginosus synthetic lipase gene successfully integrated into chromosome of p. pastoris x-33, which revealed by clear zones around the colony on yeast extract peptone dextrose tributyrin (ypd.tb) plate with zeocin. the t. lanuginosus lipase had an open reading frame of 942 bp encoding tll of 314 amino acids with theoretical molecular mass of 35 kda. the recombinant enzyme, t. lanuginosus lipase had optimal temperature at 80 °c and optimal ph at ph 8.0. key words: original signal peptide, pichia pastoris, ppiczα a, synthetic lipase gene, thermomyces lanuginosus lipase merupakan salah satu enzim industri yang penting, dan banyak digunakan dalam pembuatan zat aditif makanan, kosmetik, dan industri farmasi. pada penelitan sebelumnya, telah dilakukan kloning gen sintetik thermomyces lanuginosus lipase ke dalam bacillus subtilis dan eschericihia coli dan menghasilkan aktivitas enzim lipase yang rendah. penelitian ini bertujuan untuk mengkloning gen sintetik t. lanuginosus lipase (tll) ke dalam vektor ekspresi pichia pastoris menggunakan sinyal peptida alami ttl. gen tll yang mengandung sinyal peptida alami diamplifikasi dengan pcr, dan disisipkan ke dalam ppiczα a diantara situs xhoi dan xbai, kemudian ditransformasikan ke dalam sel kompeten e. coli dh5α. dari hasil transformasi dipilih dua rekombinan positif untuk dilakukan analisa sekuensing. hasil sekuensing, kedua rekombinan mengandung gen target lipase. plasmid yang telah dikonfirmasi kemudian dilinearisasi dan ditransformasikan ke dalam pichia pastoris x-33 dengan menggunakan metoda elektroporasi. gen t. lanuginosus lipase berhasil diintegrasi ke dalam kromosom p. pastoris x-33, yang ditunjukkan dengan terbentuknya zona bening pada media yeast extract peptone dextrose tributyrin (ypd.tb) agar yang mengandung zeocin. t. lanuginosus lipase memiliki daerah open reading frame (orf) 942 bp yang mengkode 314 asam amino dengan massa molekul teoritis 35 kda. enzim rekombinan t. lanuginosus memiliki suhu optimum 80˚c dan ph optimum 8.0. kata kunci: pichia pastoris, sinyal peptide alami, ppiczα a, gen sintetik lipase, thermomyces lanuginosus cloning of synthetic lipase gene from thermomyces lanuginosus into pichia pastoris with its original signal peptide 1 2 2 1 milani anggiani , is helianti *, niknik nurhayati , and abinawanto 1 department of biology, faculty of mathematics and natural science, universitas indonesia, depok 16424, indonesia 2 center of bioindustrial technology, agency of assessment and application of technology (bppt), building 611, laptiab-bppt, puspiptek area, tangerang selatan 15314, indonesia recombinant enzymes in eukaryotic systems are a good alternative to expression of enzymes extracellularly (yang et al. 2009). recombinant lipase enzyme can be expressed and secreted in the methylotrophic yeast p. pastoris. it can be used as the host to expression of lipase gene in high quantities (fang et al. 2014). one of the common strategies to increase the expression level of a specific gene is to screen and select a strong promoter. expression system p. pastoris can be used several promoter, including aox1, gap, fld1, pex8, and ypt1. the paox1-regulated systems are the most common (cregg et al. 2003). t. lanuginosus lipase (zheng et al. 2011), rhizopus chinensis lipase (yuet al. 2009), pseudomonas fluorescens (yang et al. 2009) were expressed under the alcohol oxidase promoter. many researchers used aox1 promoter to control expression of heterologous protein, because the promoter was able to control protein expression in large number with methanol as sole carbon source (cereghino and cregg 2000). pichia protein expression system can use a different signal peptide. researchers can clone a foreign gene in frame with sequence encoding either the native signal, the saccharomyces cereviceae α-factor signal peptide, or the p. pastoris acid phosphatase (pho1) signal. although several different secretion signal sequences, including the native secretion signal present on heterologous proteins, have been used successfully, results have been variable (cereghino and cregg 2000). in some case, many studies using α-factor signal peptide in pichia protein expression system including, rhizopus chinensis lipase yu et al. (2009); beauveria bassiana lipase vici et al.(2015); or rhizopus oryzae lipase (minning et al. 1998). this paper describes cloning of a synthetic lipase gene from t. lanuginosus into pichia vector expression with its original signal peptide and under promoter aoxi for expression of protein recombinant using p. pastoris strain x33 as a host. it also reports partial characterization of the recombinant lipase produced by the recombinant p. pastoris. materials and methods strains, plasmids, and media. e.coli dh5α was used for the cloning and propagation of plasmid. p. pastoris x-33 were used as hosts for lipase expression. plasmid pske 194 containing t. lanuginosus lipase gene (tll) is used as template for amplification of lipase gene. the vector ppiczα a was supplied from invitrogen. e.coli was grown at 37 °c in luria-bertani -1 (lb), or in lb medium containing 25 μg ml zeocin when used for selection. p. pastoris recombinant was grown in ypd plate or ypd tributyrinplate at 30 °c, in medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar, 2% -1 tributyrin, and 100 μg ml zeocin. p. pastoris recombinants were grown in buffered glycerolcomplex (bmgy)/buffered methanol-complex (bmmy) medium1% (w/v) yeast extract, 2% peptone for expression of lipase recombinant. recombinant dna techniques and construction of the plasmid ppiczα a-tll. standard recombinant dna methods were carried out according to the methods described in sambroock et al. (1989).the plasmid vector pske 194 containing the tll gene served as a template for polymerase chain reaction (pcr). the open reading form of the tll gene was amplified from this template dna using a pair of primers, sequence-natsp-tllip-xhoi-f1 (5’ gcatcct cgagaaaagagaggctgaagctatgaggagc tcccttgtgctgttc-3’) and his-tllip-xbai-r2 (5’-gcatctctagagcaagacatgtcccaattaa cccgaagtac-3’), by high fidelity phusion dna polymerase (thermo scientific). the restriction sites xhoi and xbai were incorporated into the forward and reverse primer sequence respectively. the pcr fragment was ligated into the respective sites of ppiczα a resulting in ppiczα a-tll.natsp under the control of the methanol inducible alcohol oxsidase 1 promoter (aox1). the mixture of ligation then was transformed -1 into e.coli dh5α in lb low salt containing 25 μg ml zeocin. transformants were analyzed by pcr with specific primer,and analyzed with restriction enzymes digestion (xhoi, xbai, and psti) and sequenced to confirm the correct frame for expression. dna-sequencing. for the sequence determination, samples of plasmid recombinant were sent to pt. genetika science for sequencing. furthermore, the tm result of sequencing was analyzed by snapgene 1.1.3 software. transformation of pichia pastoris. the recombinant plasmids ppiczα a-tll.natsp confirmed with sequencing method then was linearized with pmei, resolved on a 1% agarose gel, and purified. 6.5 μl linearized ppiczα a-tll.natsp and 100 μl p. pastoris competent cells x33 was incubated on ice for 30 min and electroporated into p. pastoris x33 competent cellswith gene pulser, biorad electroporator under 1,5 kv, 25 μf, 200 ω, using a 0.2 cm cuvette (invitrogen). one hundred μl of sample was spread on ypd plates -1 containing 100 μg ml zeocin and incubated for 2-3 d volume 11, 2017 microbiol indones 63 at 30 °c until colonies with appropriate size appeared. confirmation of recombinant lipase expression. after 2-3 d, all recombinants grown on ypd plate containing zeocin were transformed on ypd tributyrin plate. the colonies grew on ypd tributyrin plates have clear zones were picked up for further analyzes. all of these colonies were picked and screened by colony pcr, and the pcr products weredigested with enzyme restriction psti. several clones were obtained but only 2 were chosen for further use, respectively.the recombinant colonies then were analyzed by pcr with specific primer, and the pcr product were digested with restriction enzymes (xhoi, xbai, and psti) and sequenced to confirm the presence of tll gene. cultivation of recombinant p. pastoris producing t. lanuginosus lipase in shaking flask. colonies of recombinant p. pastoris grown on ypd plate containing zeocin, were picked and inoculated into a 125 ml erlenmeyer flask containing 25 ml of bmgy medium at 30 °c and 250 rpm for culture starter. after 16-18 h incubation, the optical density (od) of culture cells were measured by spectrophotometer, untill optical density reached od 0.2, the cells were 600 harvested (3800 rpm for 15 min), washed and resuspended in 200 ml bmgy medium. then cultivation continued until optical density reached od between 4.0 and 6.0, the cells were harvested (3800 rpm for 15 min), washed and resuspended in 50 ml bmmy medium. the cultures were grown at 30 °c and 250 rpm in 250 ml shake flasks for 120 h the cultures were supplemented daily with 3% (v/v) methanol, and sampled every 24 h. the sample was collected by separating the supernatant from the cells by centrifugation (4 °c, 3800 rpm for 15 min). sds-page analysis. the recombinant lipase in the supernatant was analyzed by sds-page, which was conducted using a 6% stacking gel and a 12% separating gel on a vertical mini gel apparatus (biorad, usa), as described by laemmli (1993). protein molecular weight marker was purchased from fermentas (burlington, canada). samples were mixed equally with 2× loading buffer and heated at 100 °c for 5 min before electrophoresis. proteins were stained with coomassie brilliant blue r-250 (bio-rad, usa). partial characterization of lipase. the experiment of the effect of temperature on lipase activity was conducted at different temperatures (4080 °c) for 20 min under ph 8.0. each measurement was performed three times under ph 8.0. the effect of ph on lipase activity was measured at different ph (510) using the following buffers na-citrate (ph 5), naphosphate (ph 6-8), tris/hcl (ph 8-9), dan glycine/naoh (ph 9-10). lipase activity assay. the lipase activity of supernatants was determined by the alkaline titration method, using olive oil as substrate. the reaction was conducted in a mixture of 5 mm tris-hcl (ph 8.0), 4 ml of emulsion of olive oil [25% (v/v) olive oil emulsified with 1.5% (w/v) polivinyl alcohol solution and 1 ml of proper dilute enzyme solution at 70 °c for 20 min in shaking incubator. finally, a 5 ml methanol absolute was added to terminate the reaction. the amount of liberated fatty acids was measured by titration with 50 mm naoh using phenolphthalein as an indicator. one unit (u) of lipase activity was defined as the amount of lipase necessary to liberate 1 μmol per min of fatty acids from the olive oil. result construction of recombinant plasmid ppicz α a containing t. lanuginosus lipase gene (tll). the fragment dna encoding recombinant lipase gene with natural signal peptide was amplified by pcr using the primer pair natsp-tllip-xho-f1/ his-tllip-xbar2 with pske194-tll as the template. the total size of tll was 942 bp. after digested with xhoi and xbai the fragment was inserted into the same restriction enzymes site of the vector ppiczα a (3568 bp after cutting). the ligation mixture then used to transform competence cells e. coli dh5α. transformants were grown in media low salt lb + zeocin that plasmids extracted and authenticated using restriction enzymes xhoi, xbai, and psti. positive results were confirmed using restriction enzymes (fig 1), restriction enzyme xhoi and xbai have one restriction site, the results seen in agarose gel that was one band in 4405 bp. meanwhile, restriction psti has two restriction site, as shown on agarose gel has two bands in 4137 bp and 268 bp. further sequencing to confirm the recombinant plasmid was conducted. the sequencing result show that the fragment dna was inserted correctly in frame with the alpha factor in the ppicz alpha vector. based on the sequence analyses the deduced amino acid were homologous to the lipases from t. lanuginosus lipase (lgy, 100% similarity, genbank accession no. eu022703.1), (mgy, 99% similarity, genbank accession no. eu370914.1) and (lip, 99% similarity, genbank accession no. af054513.1) (fig 2). sequence analyses confirmed that there was a start codon (methionine) in the 5’ fragment, six histidine 64 anggiani et al. microbiol indones in ypd tributyrin showed clear zones that was indicated expression of lipase activity (fig 4). sds-page electrophoresis. supernatant from shaking flask cultivication culture was running on 12% sds-page, and the target lipase recombinant migrated as a 35 kda band, as expected (fig 5). there were contaminating proteins present in the supernatant from shaking flask cultivation (fig 5). nevertheless, the molecular weight of protein contained in the range of 35 kda, which constantly appears in every addition of methanol per 24 h. partial characterization of recombinant lipase cultivated in shaking flask. to determine the optimal temperature and ph of the lipase recombinant, the enzymes were incubated at ph 8.0 for residue, and the stop codon.the predicted mature lipase consisted of 314 amino acid (fig 2) with a molecular weight of approximately 35 kda. transformation of p. pastoris plasmid ppiczα a.tll.natsp (clone iiib7) extracted and linearized with restriction enzyme pmei. then the results of linearized transformed into competent cells p. pastoris strain x33 using electroporation methods. colonies growth in ypd tributyrin medium, eight colonies of transformantsn p. pastoris x-33 picked up for screening positive recombinant. transformant was verified by pcr technique, in order to screen positive recombinant (fig 3). colonies showed positive results, subsequently grown on ypd tributyrin plates containing zeocin. all transformants x33 were grown fig 1 confirmation result of recombinant plasmid with restriction enzymes (xhoi, xbai, and psti). fig 2 dna sequence from thermomyces lanuginosus lipase (tll.natsp) and the deduced amino acid sequence tll.natsp. met r s s l v l f f v s a w t a l a s p i r r e v s q d l f n q f n l f a q y s a a a y c g k n n d a p a g t n i t c t g n a c p e v e k a d a t f l y s f e d s g v g d v t g f l a l d n t n k l i v l s f r g s r s i e n w i g n l n f d l k e i n d i c s g c r g h d g f t s s w r s v a d t l r q k v e d a v r e h p d y r v v f t g h s l g g a l a t v a g a d l r g n g y d i d v f s y g a p r v g n r a f a e f l t v q t g g t l y r i t h t n d i v p r l p p r e f g y s h s s p e y w i k s g t l v p v t r n d i v k i e g i d a t g g n n q p n i p d i p a h l w y f g l i g t c l a l e q k l i s e e d l n s a v d h h h h h ctcgagaaaagagaggctgaagctatgaggagctcccttgtgctgttctttgtctctgcgtggacggccttggccagtcc tattcgtcgagaggtctcgcaggatctgtttaaccagttcaatctctttgcacagtattctgcagccgcatactgcggaaaaa acaatgatgccccagctggtacaaacattacgtgcacgggaaatgcctgccccgaggtagagaaggcggatgcaacgtttct ctactcgtttgaagactctggagtgggcgatgtcaccggcttccttgctctcgacaacacgaacaaattgatcgtcctctcttt ccgtggctctcgttccatagagaactggatcgggaatcttaacttcgacttgaaagaaataaatgacatttgctccggctgca ggggacatgacggcttcacttcgtcctggaggtctgtagccgatacgttaaggcagaaggtggaggatgctgtgagggagca tcccgactatcgcgtggtgtttaccggacatagcttgggtggtgcattggcaactgttgccggagc agacctgcgtggaaatg ggtatgatatcgacgtgttttcatatggcgccccccgagtcggaaacagggcttttgcagaattcctgaccgtacagaccggc ggaacactctaccgcattacccacaccaatgatattgtccctagactcccgccgcgcgaattcggttacagccattctagccc agagtactggatcaaatctggaacccttgtccccgtcacccgaaacgatatcgtgaagatagaaggcatcgat gccaccggc ggcaataaccagcctaacattccggatatccctgcgcacctatg gtacttcgggttaattgggacatgtcttgctctaga 4.137 bp 268 bp 5.000 bp 4.000 bp 250 bp 500 bp 4.405 bp 5.000 bp 4.000 pb 4.137 bp 268 bp 4.405 bp 5.000 bp 4.000 bp ib.1 iiib.4 iiib.9 iiib.10m iiib.11 iiib.5 iiib.5 iiib.7 mm volume 11, 2017 microbiol indones 65 1000 pb 700 pb 916 pb -499 pb -268 pb -149 pb 75 bp 200 bp 300 bp 500 bp fig 3 the verification of colony pcr pichia pastoris x-33 recombinant by colony pcr (a) the restriction analysis of pcr colony product pichia pastoris x-33 with psti (b). fig 4. pichia pastoris x33 recombinant expressing lipase in ypd tb plate. fig 5 sds-page analysis of the recombinant expression lipase from pichia pastoris strain x33 (line 1-6: day 0-5; line 7: marker lmw). clear zone d.0 d.1 d.2 d.3 d.4 d.5 mr (kda) 97 66 45 30 20.1 14.4 66 anggiani et al. microbiol indones x-33 chromosome under the control of aox1 promoter. the p. pastoris expression system can be used several promoters, including aox1, gap, fld1, pex8, and ypt1. the aox1 promoter was used in several the study, zheng et al. (2011), yan et al. (2014), yu et al. (2009). under the control of aox1 promoter for expression protein using methanol as a major carbon source. in addition aox1 promoter, the use of multiple promoters were also used to increase the expression of heterologous proteins (fang et al. 2014). the use of the lipase gene original signal peptide 20 min at different temperatures (40-80 °c) or at 70 °c at different ph, respectively. it was found that the enzyme had an optimal temperature at 80 °c and an optimal ph at 8.0 (fig 6). discussion protein expression system is commonly used in cell-based, which is a package consisting of expression vector, cloned dna, and a host cell, so that the foreign gene can be expressed by the host cell and is produced 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 40 °c 50 °c 60 °c 70 °c 80 °c a ct iv it y o f l ip as e temperature a 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 5 6 7 8 9 10 a ct iv it y o f l ip as e ph b naturally rare, researchers generally used a signal peptide derived from the host expression system. in p. pastoris expression systems commonly used α factor signal peptide available on plasmid vector (zheng et al. 2011; yan et al. 2014; yu et al. 2009). in this study, the optimum temperature and ph on -1 lipase activity reached 7.25 ± 0.26 u ml and 7.25 ± -1 0.12 u ml . zheng et al. (2011) used the signal peptide from expression vector for expression of lipase, the in large quantities. for example, a commonly used host is bacteria (such as e. coli (li et al. 2014), yeast (such as s. cereviceae, p. pastoris (yuet al. 2009; minning et al. 1998; zheng et al. 2011; kademi et al. 2003). while commonly used vectors are viruses, plasmids, artificial chromosomes, and bacteriophage (such as lambda). synthetic t. lanuginosus lipase gene has been successfully constructed into plasmid ppiczαa using natural peptide signal and integrated into the p. pastoris fig 6 the optimum reaction temperature of expression lipase (a) the optimum reaction ph of expression lipase (buffers na/citrate (ph 5), na-phosphate (ph 6-8), tris/hcl (ph 8-9), dan glycine/naoh (ph 9-10)) (b). :na-citrate, : na-phospate, :tris-cl, and :glycine-naoh. volume 11, 2017 microbiol indones 67 recombinant bacillus subtilis db104 carrying pske194-lip plasmid [skripsi]. universitas indonesia. kademi a, lee b, houde, a. 2003. production of heterologous microbial lipases by yeast. indian j biotechnol. 2(3):346-355. laemmli uk. 1970. most commonly used discontinuous buffer system for sds electrophoresis. nature 227:680685. li xj, zheng rc, wu zm, ding x, zheng yg. 2014. thermophilic esterase from thermomyces lanuginosus: molecular cloning, functional expression and biochemical characterization. prot expr purifi. 101:17. doi:10.1016/j.pep.2014.05.006 minning s, dannert cs, schmid rd. 1998. functional expression of rhizopus oryzae lipase in pichia pastoris: high-level production and some properties. j biotechnol. 66(23):147-156. doi:10.1016/s01681656(98)00142-4. sambrook j, fritsch ef, maniatis t. 1989. molecular nd cloning: a laboratory manual (2 ed). new york:cold spring harbor laboratory. vakhlu j, kour a. 2006. yeast lipase: enzyme purification, b i o c h e m i c a l p r o p e r t i e s a n d g e n e c l o n i n g . electronic j biotechnol. 9(1):69-85. doi:10.2225/vol9issue 1-fulltext-9. vici ac, cruz af, facchini fda, carvalho cc, pereira mg, maldonado rf, ward rj, pessela bc, lorente gf, torres fag, jorge ja, polizeli mltm. 2015. beauveria bassiana lipase a expressed in komagataella (pichia) pastoris with potential for biodiesel catalysis. j front microbiol. 6(1083):1-12. doi:10.3389/ fmicb.2015.01083. yan j, zheng x, li s. 2014. a novel and robust recombinant pichia pastoris yeast whole cell biocatalyst with intracellular over expression of a thermomyces lanuginosus lipase: preparation, characterization and application in biodiesel production. j bior tech. 151:4348. doi:10.1016/j.biortech.2013.10.037. yang j, zhang b, and yan y. 2009. cloning and eexpression of pseudomonas fluorescens 26-2 lipase gene in pichia pastoris and characterizing for transesterification. 159(2):355-365. appl biochem biotechnol. doi:10.1007/s12010-008-8419-5. yu x-w, wang l-l, xu y. 2009. rhizopus chinensis lipase: gene cloning, expression in pichia pastoris and properties. j mol catalysis b enzymatic. 57:303-311. doi:10.1016/j.molcatb.2008.10.002. zheng y-y, guo x-h, song n-n, li d-c. 2011. thermophilic lipase from thermomyces lanuginosus: gene cloning, expression and characterization. j mol catalysis b enzymatic. 69(3-4):127-132. doi:10.1016/j. molcatb.2011.01.006. -1 activity of lipase reached 61 u ml . whereas, fang et al. (2014) inserted lipase gene into several expression vector loci to increase expression of lipase, the activity -1 of lipase reached 4350 u ml . methanol concentration used as a major source of carbon in heterologous protein expression varied. pichia strains that used affects the concentration of methanol induction. in this study, 2.5% methanol used for p. pastoris x-33. it is also found varies in others study, 1.2% (fang et al. 2014), 1% (zheng et al. 2011) methanol for induction in the host p. pastoris gs115, 1% methanol for induction in the host p. pastoris x-33 (yan et al. 2014). in conclusion, this study reported the cloning, expression, and partial characterization of recombinant t. lanuginosus lipase. t. lanuginosus synthetic gene lipase has been successfully integrated into chromosome of p. pastoris, which have clear zones in ypd. tba agar with zeocin. the optimum temperature of activity recombinant lipase at 80 °c and optimum ph at 8.0. acknowledgment this work was supported by the incentive research grant 2015-2016 from the indonesia ministry of research and technology, republic of indonesia through research consortium scheme. references cereghino lj, cregg jm. 1999. heterologous protein expression in the methylotrophic yeast pichia pastoris. fems microbiol rev. 24(1):45-66. doi:10.1111/j.15746976.2000.tb00532.x. cowan d. 1996. industrial enzyme technology. trends biotechnol. 14(6):177-178. doi:10.1016/01677799(69)300009-7. cregg jm, vedvick ts, raschke wc. 1993. recent advances in the expression of foreign genes in pichia pastoris. nat biotechnol.11:905-910. doi:10.1038/nbt0893-905. fang z, xu l, pan d, jiao l, liu z, yan y. 2014. enhanced production of thermomyces lanuginosus lipase in pichia pastoris via genetic and fermentation strategies. j ind microbial biotechnol. 41(10):1541-1551. doi:10.10 07/s10295-014-1491-7. haniyya. 2016. characterization of synthetic thermomyces lanuginosus lipase gene product expressed by 68 anggiani et al. microbiol indones 1: 62 2: 63 3: 64 4: 65 5: 66 6: 67 7: 68 modeling and optimising the growth of lasiodiplodia theobromae during gathotan fermentation umi purwandari, novia nava, darimiyya hidayatiand department of agroindustrial technology, universitas trunojoyo madura po box 2 kamal, madura, indonesia 69162 gathotan is fungal fermented cassava, and a raw material for a javanese snack called 'gathot'. this type of food is now hardly to find, and the process of making gathotan is relatively lack of process control, leads to failure in process. to make gathotan, peeled cassava tubers are left on the ground or roof for several weeks or months until they become black inside an important characteristic of gathotan. this work aims to improve gathotan fermentation by optimizing fermentation process. the effect of incubation temperature and time, inoculum level, soaking time, and drying, on the growth of the main gathotan fungus, in cassava lasiodiplodia theobromae, tubers was studied. experimental design was set up according to response surface methodology. five parameters measured were ph, titratable acidity, and fungal growth. results showed that incubation temperature affected ph in linear (p<0.01) and quadratic functions (p<0.05). titratable acidity was not affected by any treatment. fungal growth was significantly affected by incubation time (p<0.01) or inoculum level (p<0.05), and interaction of several factors: incubation time and incubation temperature (p<0.05) or drying time (p<0.01). optimization model indicated that incubation temperature at 34.5°c for 2.4 days, soaking for 26.4 hours, drying time of 3.7 hours at 40°c, and inoculum level of 2% resulted in maximum growth of in gathotanl. theobromae . key words: gathotan, , fermentationlasiodiplodia theobromae gathotan adalah singkong yang terfermentasi oleh jamur, dan merupakan bahan mentah jajan tradisional jawa yang bernama 'gathot'. makanan ini saat ini sulit ditemukan di pasaran, dan proses pembuatan gathotan umumnya tidak memiliki pengendalian proses yang cukup baik, sehingga kegagalan proses sering terjadi. gathotan dibuat dengan cara mengupas singkong, lalu membiarkannya di permukaan tanah atau di atas atap rumah beberapa minggu atau beberapa bulan sehingga menjadi berwarna hitam di dalamnya. warna hitam merupakan salah satu ciri penting gathotan. penelitian ini bertujuan mengoptimasi proses fermentasi gathotan. dikaji pengaruh suhu dan waktu inkubasi, kadar inokulum, dan waktu perendaman terhadap pertumbuhan lasiodiplodia theobromae, jamur utama pada gathotan, di singkong. rancangan percobaan disusun mengikuti rancangan pada metodologi permukaan tanggap. ada lima parameter yang diukur, yaitu ph, keasaman tertitrasi, dan pertumbuhan jamur. hasil menunjukkan bahwa suhu inkubasi mempengaruhi ph sesuai fungsi linear (p<0.01) dan kuadratik (p<0.05). keasaman tertitrasi tidak dipengaruhi oleh perlakuan. pertumbuhan jamur dipengaruhi oleh waktu inkubasi (p<0.01) atau kadar inokulum (p<0.05), atau interaksi beberapa faktor berikut: waktu dan suhu inkubasi (p<0.05) atau waktu inkubasi dan waktu pengeringan (p<0.01). model optimasi menunjukkan bahwa suhu inkubasi pada 34,5°c selama 2,4 hari, perendaman selama 26,4 jam, pengeringan selama 3,7 jam pada suhu 40°c, dan kadar inokulum 2% memberikan hasil pertumbuhan jamur l. theobromae maksimum. kata unci : k gathotan, , fermentasilasiodiplodia theobromae vol.8, no.3, september 2014, p 112-120 doi: 10.5454/mi.8.3.4 *corresponding author; phone: +62-31 3011146, fa : x +61 31 3011506 ; email: umipurwandari@yahoo.com gathotan is dried fermented cassava tuber, seemingly mostly found in central and east java, indonesia. fungal types and distribution in gathotan has been reported previously (purwandari 2000), consisting of spore forming hypomycetes on surface, and -a dematiaceous lasiodiplodia theobromae fungusthe dominant fungus grew inside the tuber. dominant spore-forming hypomycetes are rhizopus oryzae aspergillus flavusand . gathotan is characterized by black colour which comes mostly from mycelia of , since it is the only l. theobromae fungus having dark colour of hyphae and the fungus isolated mostly from the inside part of gathotan (purwandari 2000). pairing with either l. theobromae r. oryzae a. flavusor on three types of plate agar (dg18/dichloran glycerol agar, afpa/a. flavusparasiticus agar, and mea/malt extract agar) consistently shows dominant growth of l. theobromae and restricted growth of the other fungus (purwandari 2000). in the beginning of growth stage, is r. oryzae dominating over , but on the later stage l. theobromae of growth, grows overcompeted l. theobromae r. oryzae. gathot, the cooked gathotan, is characterised by its chewy texture. the chewy nature may relate to the mailto:umipurwandari@yahoo.com change in starch nature. such change maybe due to enzyme(s) or acid produced by fungus/fungi. l. theobromae is potentially a producer of a starch hydrolizing enzyme called glucoamylase (navaratman et al. 1996) to result in d-glucose and degraded starch fraction. one possibility of starch fraction is dextrin, a hydrocolloid. the chewy nature of gathot enables it to be used as raw material of gluten-free noodle (purwandari . et al 2014b), since elasticity is a very essential characteristic of noodle. moreover, gathotan flour has also been used as texturing agent of various gluten-free noodle made from some types of tubers or starchy fruit such as breadfruit (purwandari . 2014a). gathotan noodle et al has been identified for several health-relating benefits including hypoglycemic and antioxidant properties (purwandari . 2014c). the hypoglycemic effect of et al gathotan noodle may come from fermentation product of starch during gathotan making, while antioxidant activity may come from melanin produced by l. theobromae et al(purwandari . 2014c). the growth of in gathotan can raise l. theobromae health concern, since the fungus is a plant pathogen. it is an important plant pathogen, and highly prevalent in the tropical areas. factors affecting growth of the fungus have been reported with reference to tropical tuber (kihurani . 2008) or fruits (mortuza and ilag et al 1999; muniz . 2011; sivakumar . 2011; pawar et al et al 2012), such as temperature, water activity, and uv exposure (uduebo and madelin 1974). antagonistic studies of several microorganisms against l. theobromae has also been reported, which involves fungi (mortuza and ilag 1999; haggag and nofal 2006; fadahunsi . 2013), yeast (hashem and alamri et al 2009), or bacteria (fadahunsi . 2013; sajitha . et al et al 2014). so far, there is no study in the area of safety of gathot in relation to growth. however, l. theobromae there seems to be no report on incidence of gathot toxicity caused by the fungus. instead, one may start thinking of potential health benefits can be generated by the growth of in gathot, such as anti-l. theobromae cancer (pandi . 2010), anti-thrombotic et al (vasconcelos . 2013), and antioxidant (giese et al et al. 2015). the fungus also produces -d-glucan called  lasiodiplodin with several functional properties (vasconcelos 2013).et al. l. theobromaealthough growth of as plant pathogen has been extensively studied, its growth during gathotan fermentation does not seem to attract much attention. traditionally, gathotan making process is conducted without any addition of inoculum, thus allowing different types of soil fungi including those antagonistic to , grow during the process l. theobromae (purwandari 2000). this can lead to failure in the fermentation, or very little grows in the l. theobromae tuber, a common problem faced by gathotan makers. therefore, this work is dedicated to construct a model and to optimise the growth of during l. theobromae gathotan fermentation, using gathotan powder as inoculum. materials and methods raw material for gathotan making is materials. fresh cassava tuber. the tuber was purchased commercially from local market in kamal, madura. the tubers were intact without any sign of cut. it had white flesh, and diameter of base of tuber varied between 3 to 5 cm. typical commercial cassava is characterised by relatively low water content and optimum starch content, without fibrous part in the flesh. tuber was free from any defect caused either by biochemical reaction so called 'vascular streak', or signs of visible microbial growth. the second material was gathotan powder. gathotan chunk taken from previous batch was characterised by black colour covering more than 50% of inside part of tuber. the chunks were washed with running tap water, until washing water became clean, did not show discolouration of tuber. washed gathotan chunk was then dried in a cabinet dryer at 40 °c for one hour, after which it was ground with a mechanical grinder. the flour resulted from grinding was passed through 60 mesh sieve, and this flour was used as inoculum in the experiment. black part inside gathotan is the place for the growth of (purwandari 2000).l. theobromae cassava tubers preparation. tubers were peeled, and washed thoroughly with tap water three times to ensure they were free from dirt, and cut into about 10 cm long chunks. the chunks were drained well, and then treated according to experimental design. the next step was fermentation of gathotan. soaking the tubers in tap water, according to experimental design: 0, 12, 24, 36, and 48 hours. after soaking, tubers were again washed to remove soaking water from tubers. tubers were then dried in a cabinet dryer (40°c) for several hours according to experimental design: 1, 2, 4, and 6 hours. after that, tubers were mixed thoroughly with gathotan suspension (gathotan powder : distilled water, 1:2, w/w). level of inoculum used in the experiment was 2, volume 8, 2014 microbiol indones 113 3, 4, 5 and 6 % (w/w) of fresh cassava tubers. then 1 kg of tubers of each treatment combination were placed in a sterile 5 l plastic container. the plastic container was then incubated at different temperatures: 27, 30, 35, 37, and 40 °c, according to experimental design. incubation time was 2, 3, 4, 5, and 6 days. response surface methodology observation. experimental design was constructed according to response surface methodology, using five factors (soaking time, drying time, inoculum level, incubation time, and incubation temperature), with five levels for each factor. during fermentation, samples were taken for examination on ph, total acidity, and growth of l. theobromae. determination of ph of gathotan chunk was carried out using electronic ph-meter. gathotan chunk was first washed by spraying chunk with distilled water thoroughly to remove excess of soaking water. then, chunk was put in a blender, and was added with distilled water 5 times of weight of sample. it was then blended for around 3 minutes, until slurry was formed. the slurry was then passed through a number 40 whatman filter paper to collect filtrate. filtrate was examined for ph, using ph meter. the same slurry was examined for titratable acidity, by first added with 1 ml of 1% phenolphtalein, and then titrated with 1n naoh. titratable acidity was expressed as percentage of lactic acid. fungal growth examination was carried out by examining the presence of hyphae of l. theobromae inside tubers, using a light microscope connected with a camera (olympus cx31 led, japan) and a pc. mycelium of is septate and grows l. theobromae parallel to longitudinal rays (muniz . 2011). tuber et al chunk was dissected using a manual microtome without freezing the sample. the dissected piece was divided into three sections towards center. first section (zone a) was the outer zone division. part one-third near the surface of tuber. the middle section (zone b) was between one-third area from surface and one-third was from the center of tuber. zone c was one-third area from the center of tuber. statistical analysis was statistical calculation. performed on minitab 14 (minitab inc., pensylvania, ® usa) for response surface methodology, employing a quadratic equation. response was expressed as following equation: y = + x + x + x + x + x            1 2 3 4 5 2 2 2 2 2 + x + x + x + x + x + x x +           1 2 3 4 5 1 2             x x + x x + x x + x x + x x + 1 3 1 4 1 5 2 3 2 4 x x + x x + x x + x x + …. (1)2 5 3 4 3 5 4 5       where, y was response (ph, total acidity, or fungal mycelia), x was incubation temperature (°c), x was 1 2 drying time (hour), x was inoculum level (% w/w), x 3 4 was soaking time (hours), and x was incubation time 5 (days). results ph. result on ph of gathotan chunk throughout fermentation showed that ph was influenced significantly (p<0.01) only by linear and quadratic function of incubation temperature (table 1), and ph was not affected significantly (p 0.05) by other factors > such as drying time nor inoculum level, following the equation: ph = 31 043 + 0.0160 x 0.0396 x 0.0509 x  1 2 3 2 2 2 0.0002 x 0.0071 x + 1.3165 x 0.0075 x 4 5 1 2 2 2 0.9866 x + 0.0177 x 1.0465 x + x x + 3 4 5 1 2    x x + x x + 0.0185 x x - 1 3 1 4 1 5 x x + x x + 0.0625 x x + 0.0004 x x + 2 3 2 4 2 5 3 4 0.0625 x x 0.0625 x x …. (2)3 5 4 5 where, x was incubation temperature (°c), x was 1 2 drying time (hour), x was inoculum level (% w/w), x 3 4 was soaking time (hours), and x was incubation time 5 (days). high regression coefficient of incubation temperature (1.3165), fermentation time (-1.0465), and inoculum level (-0.9866) showed that the three factors affected ph of gathotan in a relatively considerable extent. the correlation between ph and incubation temperature was negative, indicating that ph was lo w e r e d by i nc r e a s in g te m pe r a t ur e du r in g fermentation. contrary, longer fermentation time or higher inoculum level reduced ph. the coefficient of determination for ph was 72.24% (data not shown), indicating relatively high sufficiency of model. incubation temperature between 35 to 40 °c resulted in final ph of 4.5 (fig 1). incubation at room temperature to 34 °c resulted in ph between 7 to 5. similarly, at high level of inoculum (6 %), low ph at 4.8 could be reached at incubation temperature of 32 °c (data not shown). although statistically not significant (p 0.05), lower drying time seemed to > reduce ph at high incubation temperature near 40 °c (data not shown). titratable acidity. in contrast to ph, titratable acidity was not affected significantly (p 0.05) by any > factor examined. regression model for titratable acidity was as follows: titratable acidity = 0.0009 x + 0.0006  1 2 x + 0.0032 x -0.00004 x + 0.0012 x 0.0776 x 2 3 4 5 1 2 2 2 2 114 p urwandari et al . microbiol indones 0.0233 x 0.0238 x 0.0002 x + 0.0852 x 2 3 4 5  x x x x 0.00001 x x 0.0026 x x + 1 2 1 3 1 4 1 5 0.0009 x x + x x + 0.0004 x x + 0.0002 2 3 2 4 2 5 x x + 0.0011 x x 0.0005 x x …. (3)3 4 3 5 4 5 where, x was incubation temperature (°c), x was 1 2 drying time (hour), x was inoculum level (% w/w), 3 4 5x was soaking time (hours), and x was incubation time (days). although all regression coefficients in equation 3 were small, those of incubation temperature (-0.0776) and fermentation time (0.0852) were relatively greater then the others. while higher incubation temperature table 1 ph of cassava chunk during fermentation of gathotan source ph titratable acidity fungal growth in zone a coefficient p coefficient p coefficient p constant 31.3043 0.004 -1.31990 0.152 -60.0810 0.141 incubation temperature -1.3165* 0.010 0.07765 0.092 1.5516 0.426 drying time 0.0075 0.994 0.02336 0.805 -6.4361 0.145 inoculum level -0.9866 0.335 -0.02380 0.811 9.8713* 0.043 soaking time 0.0177 0.824 -0.00020 0.979 -0.6726 0.075 incubation time -1,0465 0.308 0.08526 0.399 14.0059* 0.008 incubation temperature * incubation temperature 0.0160* 0.022 -0.00098 0.130 -0.0083 0.762 drying time * drying time -0.0396 0.518 0.00061 0.919 -0.3822 0.173 inoculum level * inoculum level -0.0509 0.410 0.00324 0.594 -0.1322 0.624 soaking time * soaking time -0.0002 0.635 -0.00004 0.349 0.0078* 0.001 incubation time * incubation time -0.0071 0.907 0.06124 0.838 -0.5072 0.079 incubation temperature * drying time 0.0047 0.836 -0.00091 0.689 0.4279* 0.001 incubation temperature * inoculum level 0.0352 0.142 -0.00023 0.920 -0.2163* 0.050 incubation temperature *soaking time 0.0003 0.888 -0.00001 0.964 0.0084 0.326 incubation temperature *incubation time 0.0185 0.424 -0.00264 0.257 -0.2644* 0.021 drying time*inoculum level -0.0644 0.439 0.00087 0.914 -0.3125 0.397 drying time*soaking time 0.0002 0.976 0.00019 0.783 -0.0156 0.607 drying time*incubation time 0.0550 0.507 0.00038 0.963 -1.1875* 0.006 inoculum level*soaking time -0.0004 0.951 0.00021 0.759 -0.0156 0.607 inoculum level*incubation time 0.0625 0.453 0.00113 0.890 0.0625 0.863 soaking time*incubation time -0.0042 0.541 0.00048 0.485 0.0365 0.243 *indicated significance level of 0.05 fig 1 the effect of incubation temperature and incubation time on ph of cassava chunk during gathotan fermentation volume 8, 2014 microbiol indones 115 resulted in reducing titratable acidity, longer incubation time led to the increase in titratable acidity. coefficient of determination for titratable acidity was 65.97% (data not shown), indicating that data fitted model relatively well. surface plot of incubation temperature against fermentation time indicated that high temperature from 36 to 40°c resulted in high titratable acidity (fig 2). similarly, high temperature (30 to 36°c) and longer incubation time also caused high concentration of titratable acidity. inoculum level at 3% also gave high titratable acidity (data not shown). fungal growth. fungal growth was expressed as the number of hyphae inside gathotan. l. theobromae hyphae of looked as relatively large l. theobromae septate hyaline hyphae that grew parallel to vascular tissue. every parallel hyphae of the fungus was counted as one hyphae. young hyphae of is l. theobromae hyaline, and becomes dark, grey, or black in later stage of growth (alasoadura 1970). data of the number of hyphae of growth inside cassava chunk l. theobromae during fermentation of gathotan was only meaningful when in zone a. the model for fungal colonization in zone b and c showed negative number of hyphae, thus possibly indicated unreliable model correlatively absence of fungus in the area. therefore, data on the number of hyphae in zone b and c were not analysed further. regression equation for mycelial growth in zone a was: number of hyphae = 0.0083 x  1 2 0.3822 x 0.1322 x + 0.0078 x 0.5072 x 2 3 4 5 2 2 2 2 1.5516 x 6.4361 x + 9.8713 x 0.6726 x + 1 2 3 4 14.0059 x + 0.4279 x x x x + 0.0084 x x 5 1 2 1 3 1 4 0.2644 x x 0.3125 x x 0.0156 x x 1.1875 1 5 2 3 2 4 x x 0.0156 x x + 0.0625 x x 0.0365 x x …. (4)2 5 3 4 3 5 4 5 where, x was incubation temperature (°c), x was 1 2 drying time (hour), x was inoculum level (% w/w), x 3 4 was soaking time (hours), and x was incubation time 5 (days). high regression coefficients were shown by fermentation time (14.0059) and inoculum level (9.8713), indicating significant role of the two factors in determining fungal growth, with positive correlation. coefficient of determination for the equation was 88.62%, reflecting good fitness of data to the regression. the number of hyphae in zone a was affected positively and significantly by inoculum level (p<0.05) and incubation time (p<0.01) (table 1). several interactions between factors showed significant effect were: quadratic function of soaking time (p<0.005), interaction between incubation temperature and drying time (p<0.005), incubation temperature and incubation time (p<0.05), and incubation time and drying time (p<0.01). no mycelium was detected during the first three days, 5 0 fig 2 the effect of incubation temperature and fermentation time on titratable acidity of cassava chunk during gathotan fermentation fig 3 growth of during gathotan fermentation as affected by incubation time and inoculum levellasiodiplodia theobromae fig 4 growth of during gathotan fermentation as affected by incubation time and incubation lasiodiplodia theobromae temperature cur high low0.93445 d optimal d = 0.66134 targ: 5.0 ph y = 4.9066 d = 0.94072 tar g: 0.150 total ac y = 0.1494 d = 1.0000 maximum zone a y = 5.0054 0.93445 desir ability composite 2.0 6.0 0.0 48.0 2.0 6.0 0.0 4.0 27.0 40.0 drying t inoculum soaking fermentatemper at [34.5214] [3.6768] [2.0] [26.4159] [2.3636] fig 4 optimization of conditions for growth during gathotan fermentationlasiodiplodia theobromae volume 8, 2014 microbiol indones 117 when inoculum level was below 4 %. however, when inoculum level was high enough (over 5 %), hyphae started colonizing cassava as soon as two days incubation (fig 3). maximum fungal growth was reached after four days at 6 % inoculum level, or six days at 4 % inoculum level. incubation temperature around 30 °c and fermentation time at 4 d or longer resulted in highest mycelium number (fig. 4). discussion the changes of ph during gathotan fermentation was not as fast as when it was grown in liquid cassava medium with added nitrogen source (navaratnam et al. 1996). in this liquid cassava medium, ph was lowered to reach ph 1.8 during 50 hours of fermentation, showing a significant acid production by l. theobromae when nitrogen source was available (navaratnam 1996). cassava is low in nitrogen et al. content, so that ph reduction in gathotan fermentation was not as rapid as that in the liquid medium. relatively slow ph reduction in gathotan fermentation seemed to be of benefit to the growth of , l. theobromae since the fungus grow well at relatively high ph (eng et al. 1998). high ph reduction rate would not allow sufficient colonization of cassava tuber by the fungus, resulting in failure of fermentation. previous report mentioned that mycelial production of l. theobromae was maximum at ph 8 to 10 (eng . 1998). the ph et al during gathotan fermentation did not seem to go lower than 4, thus allowed sufficient fungal growth inside tuber. assuming that chewy texture of gathot, the cooked gathotan, is caused by glucoamylase activity, ph during gathotan fermentation (4-6) seems to support activity of the enzyme (navaratnam . et al 1996), resulted in good and chewy gathot. titratable acidity indicates free proton neutralised during titration using a strong base solution. titratable acidity also includes buffering capacity of sample (rajkovic . 2007). not all of hydrogen ions can be et al detected during titration, due to probable influence of monovalent cation exchange (boulton 1980) and dissociation strength of acids. there was no significant difference of titratable acidity among our samples, although ph was different among gathotan samples. measurement of ph was reported to be more reliable method to determine acidity in wine (rajkovic et al. 2007). this may also apply to gathotan, where measurement of ph gives more meaningful information than titratable acidity. incubation temperature at around 30°c seemed to support growth of not only in gathotan l. theobromae fermentation, but also in various media. for example, in a liquid medium (eng . 1998), cassava-et al containing medium without any addition of nitrogen source (navaratnam . 1996), potato dextrose et al et alagar medium (kausar . 2009), and mango during postharvest (pawar 2012). it was also reported that light was important to support growth of the fungus (kausar . 2009). in our work, fermentation was et al conducted inside building with minimum light, so that growth of may not be at optimum level.l. theobromae g a t h o t a n f e r m e n t a t i o n w a s p r o n e t o contamination, as it is usually carried out in an open and unsterilized container. some fungi are antagonistic to , such as some species of l. theobromae trichoderma (mortuza and ilag 1999, haggag and nofal 2006, fadahundi 2013). one of antagonistic bacteria is et al. b. subtilis which grows during soaking of cassava (anyogu 2014). a species of yeast, et al. pichia anomala l. theobromaerestricts growth of (hashem and alamri 2009). thus, reducing contamination of those antagonistic microorganisms is essential, and it can be carried out by improving sanitation of process. lowering ph can inhibit bacterial contamination, but ph may not be allowed to be too low as it can inhibit growth of . therefore, it is important to l. theobromae ensure fermentation conditions to support dominant growth of over antagonistic l. theobromae microorganisms. we tried to determine optimum condition for gr o wth of d ur ing g at hot an l. th eo brom ae fermentation. using optimisation procedure, and by putting relative importance and weight of fungal colonization at 10, titratable acidity at 2 and ph at 2, we found that maximum fungal growth was achieved at incubation temperature of 34.5°c for 2.4 days, 3.7 hours drying at 40°c, inoculum level of 2%, and 26.4 hours soaking (fig 5). relative importance and weight is determined approximately to reflect distribution of desirability between lower or upper bound and the target value. the setting used in this optimization is evaluated by determining composite desirability value which is between 0 and 1. our setting resulted in composite desirability of 0.934, indicating that the setting gave satisfactory results for all response as a whole. we noticed that during duration of fermentation in this work, there was no black fungal coloration of tubers, suggesting that additional time to allow maturation is needed in order to achieve final products with black colour characteristics of gathotan. 118 p urwandari et al . microbiol indones acknowledgment this work is funded by directorate general of high degree education, ministry of education and culture, the government of indonesia, as research grant for “penelitian hibah bersaing” scheme in 2014. references alasoadura so. 1970. culture studies on botryodiplodia theobromae . pat. mycopathol mycol appl. 42(1-2):153-160 anyogu a, awamaria b, sutherland jp, ouoba lij. 2014. molecular characterisation and antimicrobial activity of bacteria associated with submerged lactic acid cassava fermentation. food control. 39:119-127. http://dx.doi.org/10.1016/j.foodcont.2013.11.005. boulton r. 1980. the relationships between total acidity, titratable acidity and ph in grape tissue. vitis 19:113-120. eng f, gutierrez-rojas m, favela-torres e. 1998. culture conditions for jasmonic acid and biomass production by in submerged botyrodiplodia theobromae fermentation. process biochem. 33(7):715-720. fadahunsi i, ayansina d, okunrotifa a. 2013. biocontrol of mushroom spoilage fungi and aflatoxin evaluation during storage. nat and sci. 11(7):7-13. giese ec, gascon j, anzelmo g, barbosa am, da cunha maa, dekker rfh. 2015. free-radical scavenging properties and antioxidant activities of botryosphaeran and some other ß-d-glucans. int j biol macromol 72:125-130. http://dx.doi.org/10.1016/j.ijbiomac. 2014.07.046. haggag wm, nofal ma. 2006. improving the biological control of on some botryodiplodia disease annona cultivars using single or multi-bioagents in egypt. biol control. 38:341-349.doi:10.1016/j.biocontrol.2006.02.010 hashem m, alamri s. 2009. the biocontrol of postharvest disease ( ) of guava botryodiplodia theobromae ( l.) by the application of yeast strains. psidium guajava post harve st biol a nd te chnol . 53: 123-130. doi:10.1016/j.postharvbio.2009.04.001. kausar p, chohan s, parveen r. 2009. physiological studies on and , the lasiodiplodia theobromae fusarium solani cause of shesham decline. mycopath. 7(1):35-38. kihurani aw, narla rd, shibairo s, imungi j, carey e. 2008. effect of soil ph on postharvest pathological deterioration of sweet potato storage roots. afr j hort sci 1:1-8. 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of guava fruit. curr botryodiplodia bot. 3(3):16. purwandari u, arifin s, tamam b, hidayati d 2014b. . glutenfree noodle made from gathotan (an indonesian fungal fermented cassava) flour: cooking quality, textural, and sensory propertis. int food res j. 21(4):1615-1621. purwandari u, khoiri a, muchlis m, noriandita b, zeni nf, lisdayana n, fauziyah, e. 2014a. textural, cooking quality, and sensory evaluation of gluten-free noodle maden from breadfruit, konjac, or pumpkin flour. int food res j. 21(4):1623-1627. purwandari u, tristiana gr, hidayati d. 2014c. gluten-free noodle made from gathotan flour: antioxidant activity and effect of consumption on blood glucose level. int food res j. 21(4):1629-1634. purwandari u. 2000. aflatoxin in gathotan in relation to fungal distribution. master thesis. royal melbourne institute of technology university. rajkovic mb, novakovic id, petrovic a. 2007. determination of titratable acidity in white wine. j agric sci. 52(2):169-184. sajitha kl, florence ejm, dev sa. 2014. screening of bact erial biocontrol against sapstain fungus ( pat.) of rubberwood lasiodiplodia theobromae ( muell. arg.). res in microbiol. hevea brasiliensis 165:541-548. http://dx.doi.org/10. 1016/j.resmic. 2014.07.002. sivakumar d, jiang y, yahia em. 2011. maintaining mango ( l.) fruit quality during the export mangifera indica chain. food res int. 44:1254-1263. doi:10.1016/ j.foodres.2010.11.022. uduebo ae, madelin mf. 1974. germination of conidia of botryodiplodia theobromae in relation to age and environment. trans br mycol soc. 63(1):33-34. volume 8, 2014 microbiol indones 119 http://dx.doi.org/10.1016/j.foodcont.2013.11.005. http://dx.doi.org/10.1016/j.ijbiomac. http://dx.doi.org/10. vasconcelos afd, dekker rfh, barbosa am, carbonero er, silveira jim, glauser b, pereira ms, da silva mlc. 2013. sulfonation and anticoagulant activity of fungal exocellular b-(16)-d-glucan (lasiodiplodan). carbohydr polym. 92:1908-1914. http://dx.doi.org/ 10.1016/j.carbpol.2012.10.034. 120 p urwandari et al . microbiol indones http://dx.doi.org/ vol.1 , no.1, march 202 , p -6 2 1 12 doi: 10.5454/mi.1 .1.6 1-12 lipase activity enhancement of kc4j mutant rom oil palm waste usingf response surface method nd partial characterizationa aris indriawan , trismilah , wibowo mangunwardoyo 1 2 1* , and dadang suhendar 2 1 department of biology, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia; 2 agency for the assessment and application of technology, laptiab, bld.610-612, puspiptek, south tangerang, 15314, indonesia. lipase has an important role in industry. the kc4j mutated isolates from oil palm waste had 100% similarity to strain ra204, a fungus known produce lipase. the study aimed to increase lipase activityaspergillus fumigatus of kc4j mutant through media optimization using response surface methodology (rsm) and partial characterization. the three variables of media composition (olive oil, soy flour, and ph were optimized using central composite design (ccd). the lipase characterization measured the influence of ph, temperature and metal ions. the ph tested on range 6 to 12, while the temperature variation tested on 30 to 70 °c. the metal ions tested were mg , ca , zn , mn , fe and k with concentrations of 1 mm and 10 mm. the production medium 2+ 2+ 2+ 2+ 2+ + containing 1.25% of olive oil, 3.5% of soy flour and 7.5 ph resulting 11.25 u/ml of lipase activity, which was higher than the previous media composition (10.00 u/ml). the results of ccd and quadratic analysis showed that the source of carbon, nitrogen and ph had an effect on lipase activity which showed r 0.93. the optimum lipase 2 activity produced at ph 6 and on 60 °c, and the lipase stable at ph 6-8 and on 30-70 °c. all metal ions tested were able to increase lipase activity with ca ion gave the highest result. 2+ key words: central composite design, kc4j mutant, ipase, il alm astel o p w lipase memiliki peran penting dalam industri. mutan kc4j hasil mutasi isolat dari limbah kelapa sawit memiliki similaritas 100% dengan spesies strain ra204 merupakan kapang yang dapataspergillus fumigatus menghasilkan lipase. produksi lipase dipengaruhi oleh beberapa faktor, seperti jenis mikroorganisme, sumber nutrisi dan faktor lingkungan. penelitian bertujuan meningkatkan aktivitas lipase mutan kc4j melalui optimasi media dengan menggunakan metodologi permukaan respon (rsm) dan karakterisasi parsial. tiga variabel komposisi media yang digunakan adalah minyak zaitun, tepung kedelai dan ph. ketiga variabel tersebut dioptimalkan dengan (ccd), karakterisasi lipase yang dilakukan adalah pengaruh ph,central composite design ​​suhu dan ion logam. kisaran ph yang diuji yaitu ph 6-12, sedangkan variasi suhu 30-70°c. ion logam yang diuji adalah mg , ca , zn , mn , fe dan k dengan konsentrasi 1 mm dan 10 mm. hasil penelitian menunjukkan 2+ 2+ 2+ 2+ 2+ + bahwa media produksi yang mengandung 1,25% minyak zaitun, 3,5% tepung kedelai dan ph 7,5 mampu menghasilkan aktivitas lipase 11,25 u/ml, yang lebih tinggi dari komposisi media sebelumnya (10,00 u/ml). hasil dan analisis kuadrat menunjukkan bahwa sumber karbon, nitrogen dan phcentral composite design berpengaruh terhadap aktivitas lipase yang menunjukkan r 0,93. aktivitas lipase optimum pada ph 6 dan suhu 2 60°c, stabil pada ph 6-8 dan suhu 30-70 °c. semua ion logam yang diuji dapat meningkatkan aktivitas relatif lipase. ion ca dapat meningkatkan aktivitas relatif tertinggi dibandingkan ion lainnya. 2+ kata kunci: central composite design, pase, utan kc4jlimbah kelapa sawit, li m microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ;: +62 8161435792 fax: +62-21-7566922 e-mail: trismilah@bppt.go.id; conventional biodiesel production using acid or alkaline catalysts still requires several processes for final product purification and wastewater treatment, so alternative catalytic agents are needed to overcome these problems (ghaly 2010). lipase is anet al. alternative biocatalyst agent that can be used for biodiesel production. in addition, the lipase is more environmentally friendly (kotogan 2014). someet al. fungi can produce lipase, namely ,aspergillus niger aspergillus fumigatus candida rugosa colletotrichum, , gloeosporioides mortierellaechinospaera penicillium, , restricum rhizopus miehei rhizopus stolonifer, and (prabakaran 2009; kotogan 2014; el-batalet al. et al. et lipase (triacylglycerol acylhydrolase, e.c. 3.1.1.3) is an enzyme that has great potential for commercial applications due to its broad selectivity and substrate specificity. lipases can catalyze various reactions such as the hydrolysis of fats and oils to glycerol and fatty acids. lipases can also catalyze esterification and transesterification reactions, so they can be used as catalyst for biodiesel production. (jaeger and eggert 2002; ghaly 2010, sharma 2011,et al. et al. prasad 2012, andualema and gessesse 2012).et al. al. 2014; el-batal et al.). types of microorganisms, nutrient sources and environmental factors influence lipase production (lima 2003; pinheiroet al. et al. 2008; ulker 2011; kotogan 2014).et al. et al. according to khausik (2010), lipase productionet al. increased by media optimization. the response surface method (rsm) is one of the methods that can be used. this method used to determine the optimum conditions using mathematical and statistical analysis. this method used to obtain the best solution from a combination of several variables that affect the production process. jia (2015) proved that rsmet al. can be used for optimization of lipase production. in addition, increasing lipase activity can also be done by optimizing ph and temperature and metal ions addition. metal ions function as cofactors for lipases which can make lipases more stable in binding to the substrate (palmer 1991; dandavate 2009; iqbal and rehmanet al. 2015). mutations have been carried out with the use of ultraviolet (uv) light on c kernel mold isolates from oil palm waste, malinping, lebak, banten, west java, indonesia. in figure 1, the result of phenotypic and phylogenetic approaches (28s rrna) kc4j isolate mutated by uv light has 100% similiarity with aspergillus fumigatus strain ra204. changes in the mean character that occur in the branches are indicated by the values ​​contained in the maximum likelihood phylogenetic tree, so that the kc4j mutant isolate (aris-28s) has the closest ancestor to strain ra204. the maximuma fumigatusspergillus likelihood phylogenetic tree compiled was based on dna changes (reece 2014; indriawanet al. et al. 2018). reported by indriawan 2018 that mutatedet al. kernel c isolates with uv light from oil palm waste whichwere fermented at room temperature (28-30 ºc) for 7 days in a 200 rpm rotary shaker able to produce lipase. the media used was fish meal as nitrogena source and olive oil as carbon source as well as an inducer (murni 2015). the kc4j mutant gave theet al. highest lipase activity and transesterification activity with 10 u / ml and 0.114 u / mg, respectively. therefore, it was expected that the kc4j mutant lipase can be used as catalyst in biodiesel production. this study aim to increase the activity of mutant kc4j lipase which is similar to strain ra204aspergillus fumigatus by optimizing the media using rsmand also performing partial characterization. partial characterization of kc4j mutant lipase included the influence of ph, temperature and metal ions. the ph tested was on 6 to 12 range, while the temperature variation was tested at 30 up to70 °c. the metal ions tested were mg , ca , zn , mn , fe , and k with 2+ 2+ 2+ 2+ 2+ + concentrations of 1 mm and 10 mm. reported by djafar et al. 2010, that the optimization of the production medium needs to be done to determine the composition of the media that can produce optimum lipase activity, and lipase characterization is needed to determine the characteristics of lipase when applied to the industrial sector. materials and methods microorganisms. this study used kc4j mutant, a collection of the bioindustrial technology laboratory, fig 1 position of kc4j mutant isolate (aris28s) shown on the phylogenetic tree based on the 28s rrna gene sequence using maximum likelihood (ml).the position of the substitution per nucleotide is shown in 0.1 bar. 2 indriawan et al. microbiol indones volume 1 , 2026 2 microbiol indones 3 laptiab-bppt, puspiptek, banten, which is similar to strain ra204. theaspergillus fumigatus mold culture was grown on slant potato dextrose agar (pda) medium that stored at 4 °c rejuvenated every 30 days. preparation of inoculum and liquid media. the kc4j mutant was grown in a pda slant test tube and incubated for 7 days at 28 °c. after incubation, 10 ml of sterile distilled water was put into the slant culture and scraped off using inoculation loop. the culture furthermore homogenized for 30 seconds and the spore suspension used to inoculate was adjusted to 10 spores/ml (el-batal 2015). lipase production 7 et al. was carried out in 50 ml of liquid production medium on250 ml erlenmeyer flasks based on the results of preliminary research (unpublished) which contain 3% of soy flour and 1% of olive oil on ph 7 which sterilized for 15 minutes at 121 °c. the spore suspension was inoculated into the flask with 10% (w / v) ratiofrom the total production medium. the fermentation process were conducted for 7 days on a rotary shaker at 200 rpm agitation in room temperature (28-30 °c) (maia et al. 2001; ellaiah 2004; pinheiro 2008; el-batalet al. et al. et al. 2015). experimental design and analysis experiments were carried out with rsmusing ccd experimental design. the design used three variables with two lipase activity as the measured response. each variable consists five level of codes. the variables used were carbon source (x ), nitrogen1 source (x ) and ph (x ), as shown in table 1. lipase2 3 production was carried out according to the variation in the combination determined by rsm. all data obtained were processed using rsm analysis. based on ccd experimental design using design expert v. 7.0.0. software (statease, usa), all the data obtained were plotted on a second order polynomial equation. furthermore, each coefficient of significance was seen based on the p and f values ​​in the anova. each parameter has a significant effect if the p value gets smaller and the f value gets bigger (demirel and kayan 2012). lipase activity assay. lipase activity assay can be done using li (2014) method withet al. modifications. the substrate was made with composition: 1.5% of polyvinyl alcohol (pva), 25% of olive oil, and distilled water, which were mixed until homogeneous. a total of 5 ml of the substrate was added with 4 ml of 0.05 m phosphate buffer solution, ph 6 and 1 ml of crude lipase extract.the mixture were incubated at 50 °c for 20 minutes in shaker 150 rpm).( after the incubation completed, 5 ml of methanol was added to stop the reaction. 2 drops of phenolphthalein (pp) indicator were added to the solution and then titrated with 0.05 m naoh. the titration was stopped when the color of the solution turned to and remained pink, and the titration volume was recorded. the blank solution was made in the same way as the sample, whilst methanol addition before incubation were to stop the lipase reaction. lipase characterization. lipase characterization was carried out by determining the optimum ph and temperature of the lipase, test the ph and temperature stability of the lipase, and verifythe effect of metal ions on lipase. optimum ph and temperature. determination of the optimum ph of lipase was carried out by testing lipase activity at ph 6, 7, 8, 9, 10, 11 and 1 using2 titration method (li 2014). several buffer solutionset al. used were: 0.05 m phosphate buffer solutions ph 6 and 7, 0.05 m tris-hcl buffer solutions ph 8 and 9, and 0.05 m glycine-naoh buffer solutions ph 10, 11, and 12. the lipase activity assay was incubated at 50 °c with 200 rpm agitation for 20 minutes. using li (2014)et al. method with modification, determination of lipase optimum carried out at the optimum ph with different temperature variation (30, 40, 50, 60 and 70 °c).s ph and temperature stability. lipase ph stability was carried out by incubating lipase in ph 6, 7, 8, 9, 10, 11 and 12 with one part of enzyme incubate in four parts of buffer solutions at optimum temperature. sampling was carried out at 0, 30, 60 and 90 minutes. also using li (2014) method with modification,et al. the effect of temperature on lipase stability was determined by incubating the lipase at 30, 40, 50, 60 and 70 °c at the optimum ph. sampling was carried out at 0, 30, 60 and 90 minutes. table 1 independent variables ange and level in lipase production optimization using rsm designr independent variable variable ranges and levels α -1 0 +1 + α x1 -1.6817 -1 0 +1 +1.6817 x2 -1.6817 -1 0 +1 +1.6817 x3 -1.6817 -1 0 +1 +1.6817 effect of metal ions. the effect of metal ions determined by testing the activity of lipases containing ions such as mn , zn , ca , k , mg and fe . the ion 2+ 2+ 2+ + + 2+2 concentration used are 1 mm and 10 mm (ghori et al. 2011; mokodongan 2013). results optimization of media using rsm. the media used for the production of lipase contained olive oil, soy flour and was adjusted to ph 7. the composition and conditions of the media were optimized with a ccd design and the design response measured was lipase activity. in this paper, only design respondto lipase activity are reported, as can be seen in table 2. the variables used were olive oil, soy flour and ph. rsm was used to examine the effect of each variable on the response. multiple regression analysis was used to analyze the data obtained and the second order polynomial equation is described from the regression analysis below: lipase activity response y = +10,34 + 0,051x +0,24x + 0,30x 0,040x x +1 2 3 1 2 0,12x x + 0,21x x – 0,11x – 0,028x + 3,523e-1 3 2 3 1 2 2 2 003x3 2 the y equation shows the responds to lipase activity; x , x and x show the independent variables of carbon1 2 3 source, nitrogen source and ph. data from ccd designthen analyzed by anova with design expert v.7.1.5 program. design expert program 7.1.5. used for model selection analysis based on the sequential model sum of square, model mismatch testing (lack of fit test) and model summary statistics. the results of model selection on the responds to lipase activity are shown on table 3, whilst the anova calculation on lipase activity shown in table 4. the three-dimensional image of the interaction between olive oil media composition, soy flour and ph on lipase activity was shown in figure 2.the actual and predictive values ​​of research for lipase activity was shown in figure 3. optimum ph and temperature. the lipase activity test was carried out at several variations of ph and temperature, in order to obtain the optimum ph and temperature of the lipase. the ph variation used was ph 6-12, while the temperature variation used was 3070 c. figure 4 shows that the highest lipase activity is o at ph 6 at 11.50 u/ml and decreases at ph 7-9. lipase activity stops at ph 10-12. figure 5 shows that lipase activity increases at 3060 °c and decreases in lipase activity at 70 °c. the highest lipase activity was at 60 ° c at 13.44 u/ml. ph and temperature stability. the effect of ph on the relative stability of lipase activity is presented in figure 6 and the effect of temperature in figure 7. the effect of ph on the stability of the relative activity of lipases was tested at ph 6-12.relative lipase activity at 0 minutes, lipase had no activity.the 30th minute of lipase relative activity increased until the 60th minute and decreased by the 90th minute at all tested phs.the highest relative lipase activity was at ph 6 min. 60, which was 129.08%.the relative activity of lipase tended to be stable at ph 6-8, but above ph 8, the relative activity of lipase was 0%.lipase stability at ph 6 showed a relative lipase activity pattern that increased until the 60th minute incubation time and decreased at the 90th minute.kc4j mutant lipase similar to strain ra204 hasaspergillus fumigatus optimal lipase activity at acidic ph, namely ph 6 and stable at ph 6-8. effect of metal ions. the effect of metal ions on the relative activity of lipases is presented in figure 8. all metal ions can increase the relative activity of lipases at concentrations of 1 mm and 10 mm and act as activators. the highest relative lipase activity was found in the addition of ca 1 mm ion concentration of 2+ 146.55%. discussion he choice ofoptimization of media using rsm. t model in table 3 was based on the description of the sum of squares of the sequence model (sequential model sum of square) which is suggested to use the quadratic model versus two interaction factors (2fi), because it has a p value of 0.0305 or p 0.305% (p <5%) . this shows that the probability of error from the model was less than 5%, so that the model has a significant impact on explaining the effect of experimental factors or variables on the response. in the selection of the model mismatch test (lack of fit test), it is suggested that a quadratic model with an f value of 0.52 and a p value of 0.75 indicates that the cascade model is not significant because the p value is 0.75. model summary statistics suggested a quadratic model for the optimization of media composition. the analysis of variance (anova) model was used to determine the effect of each variable and the interaction between experimental variables on lipase activity. the model with an f value of 15.60 shows that mistakes do not play an important role and errors are likely to occur by 0.01% (table 4). f value or 4 indriawan et al. microbiol indones table 2 central composite design and the responds to lipase activity run olive oil (%) soybean powder (%) ph lipase activity (u/ml) 1 1,50 3,00 7,00 9,94 2 0,75 3,50 6,50 10,13 3 1,25 3,50 6,50 9,81 4 0,75 3,50 7,50 9,94 5 1,00 3,00 7,00 10,44 6 1,00 3,00 7,00 10,19 7 1,00 3,00 7,00 10,50 8 0,75 3,50 7,50 10,81 9 0,75 2,50 6,50 9,81 10 1,00 3,00 6,00 9,75 11 1,00 4,00 7,00 10,63 12 1,00 3,00 7,00 10,19 13 1,25 2,50 6,50 9,94 14 1,00 3,00 7,00 10,5 15 0,50 3,00 7,00 9,81 16 1,00 3,00 8,00 10,88 17 1,25 3,50 7,50 11,25 18 1,25 2,50 7,50 10,25 19 1,00 3,00 7,00 10,13 20 1,00 2,00 7,00 9,75 table 3 analysis of model selection with the sequential model sum of squares, lack of fits test and model summary statistics on lipase activity responds sequential model sum of squares source sum of squares df mean square f value p-value prob > f mean vs total 2094,08 1 2094,08 12,72 0,0002 linear vs mean 2,41 3 0,80 3,87 0,0352 2fi vs linear 0,48 3 0,16 4,49 0,0305 suggested quadratic vs 2fi 0,31 3 0,10 0,53 0,7221 aliased cubic vs quadra 0,059 4 0,015 residual 0,17 6 0,028 total 2097,50 20 104,87 lack of fit tests source sum of squares df mean square f value p-value prob > f linear 0,86 11 0,078 2,62 0,1484 2fi 0,38 8 0,048 1,61 0,3109 quadratic 0,078 5 0,016 0,52 0,7523 suggested cubic 0,019 1 0,019 0,64 0,4591 aliased pure error 0,15 2 0,030 model summary statistics source std. dev. rsquared adjusted r-squared predicted r-squared press linear 0,25 0,7046 0,6492 0,5013 1,70 2fi 0,20 0,8440 0,7720 0,6669 1,14 quadratic 0,15 0,9335 0,8737 0,7678 0,79 suggested cubic 0,17 0,9508 0,8441 -0,2136 4,14 aliased volume 1 , 2026 2 microbiol indones 5 table 4 analysis of variance (anova) for response to lipase el summary statistics on lipase activity responds source sum of square df mean square f value p-value prob > f model 3,19 9 0,35 15,60 <0,0001 significant x1-olive oil 0,042 1 0,042 1,85 0,2035 x2-soybean powder 0,91 1 0,91 40,17 <0,0001 x3-ph 1,45 1 1,45 63,96 <0,0001 x1 x2 0,013 1 0,013 0,56 0,4700 x1 x3 0,11 1 0,11 4,87 0,0519 x2 x3 0,35 1 0,35 15,54 0,0028 x1 2 0,29 1 0,29 12,56 0,0053 x2 2 0,019 1 0,019 0,85 0,3779 x3 2 3,120e-004 1 3,120e-004 0,014 0,9090 residual 0,23 10 0,023 lack of fit 0,078 5 0,016 0,52 0,7523 not significant pure error 0,15 5 0,030 cor total 3,41 19 std. dev 0,15 r-squared 0,9335 mean 10,23 adj r-squared 0,8737 c.v. % 1,47 pred r-squared 0,7678 press 0,79 adeq r-squared 12,544 fig 2 the interaction of factor variables of olive oil, soybean flour and ph on the response to lipase activity. fig 3 distribution of research results and predictive value to lipase activity response. 6 indriawan et al. microbiol indones probability less than 0.05 indicates that the model components are significant, b and c are significant components. lack of fit f value of 0.52 indicates that the model is not significant to the response, there is a chance that the model is not suitable. insignificant model mismatch can be interpreted well and the selected model is correct. the interaction between media composition (olive oil, soy bean flour and ph) on lipase activity was shown in the form of a three-dimensional image. the highest desirability value of the research results was 1 with a media composition of 1.25% olive oil, 3.50% soybean flour, ph 7.00, 11.25 u/ml of lipase activity was obtained. the composition of media that has a desirability value of 1 has been verified in the laboratory. the verification results resulted: 11.50 u/ml of lipase activity, 3.02 mg/ml of protein content,0.95 grams of biomass, and 0.14 u/g of transesterification activity. rsm can be used for optimization of lipase production as reported by kaushik (2010) and jia (2015).one of rsmet al. et al. design are ccd, which maximizes both the precision and accuracy of the estimated extreme point of secondorder response surface for the constructed model parameters fixed values (coetzer 2011).researchet al. of variables or numerical factors of olive oil, soybean flour and ph gave the maximum effect to lipase activity response, because it had an r value of 93.35%.the 2 concentration of olive oil and soy flour are the main nutrients that regulate lipase biosynthesis. the highest concentration of these two nutrients can inhibit lipase synthesis, so that the appropriate concentration can increase the optimum lipase production. in addition, ph has an effect on the biosynthesis of the lipase. too low and high ph can inhibit lipase synthesis and is supported by research jayaprakash and ebener (2012). optimum ph and temperature. based on these results, it is known that lipase activity is influenced by ph. ph 6.00 is the optimum ph because it has the highest lipase activity. according to christakopoulus (1992), low or high ph can denature lipase thus decrease lipase activity. lipase is a protein, changes in ph can cause protein molecules to ionize, thus changing the three-dimensional structure of lipases. these structural changes can cause disruption of the catalytic function of lipases. in addition, low ph hydrolysis will occur in unstable peptide bonds, while at high ph there will be irreversible denaturation and partial damage (ghaima 2014). the optimum phet al. obtained in this study is the same as the optimum ph obtained by faloni (2006) onet al. aspergillus niger lipase, which is ph 6.00. this is also supported by the results of research by crueger and crueger (1993), other strains are also optimum at phaspergillus niger 6.00. based on the data obtained, it is known that lipase activity is influenced by temperature. according to septiningrum and moeis (2009), an increase in temperature will have a positive correlation with an increase in lipase activity before it reaches the optimum temperature, while at temperatures above the optimum, the lipase activity will decrease rapidly. the optimum temperature obtained in this study is quite high, namely 60 °c. the optimum temperature conditions increase the kinetic energy which accelerates the rotation and movement of the lipase molecules and the substrate, thus increasing the collision frequency which is the opportunity for both of them to react. if it is above the optimum temperature, the lipase activity will decrease due to changes in the tertiary structure of the protein in the lipase (gomes et al. et al.2006). according to daniel (2010) stated that high temperatures can reduce lipase activity due to denaturation of the protein structure. ph and temperature stability. the lipase characteristics are similar to the research of mhetras et al.(2009), reported that lipase ncimaspergillus niger 1207 was stable at alkaline ph (ph 8-11), but had optimum activity at acidic ph. according to colla .et al (2015), has ph stability at ph 3.5-aspergillus flavus 6.5 for 24 hours of incubation time with a residual activity of> 80%, while at ph 7-10 it has a residual activity of around 50%. ph can affect lipase stability by changing the electrostatic interaction of the lipase protein structure, which causes changes in amino acid ionization, secondary and tertiary structures of proteins (sharma 2002; rajakumara 2008).et al. et al. the effect of temperature on the relative stability of lipase activity was tested at 30-70 °c. the relative activity of lipase at 0 minutes was 0%. the 30th minute of lipase relative activity increased until the 60th minute and decreased by the 90th minute at all tested temperatures. the highest relative lipase activity was at 60 °c in the 60th minute, which was 130.68%. temperature 30-70 °c, the relative lipase activity tends to be stable. lipase stability at 60 °c indicated that the relative lipase activity pattern increased until the 60th minute incubation time. the 0th minute there is no lipase activity. lipases are stable at high temperatures (> 50 °c) probably due to the presence of polyamines in the protein structure. in addition, a high proportion of thermophilic amino acids, salt bridges and the number volume 1 , 2026 2 microbiol indones 7 fig 4 lipase activity (u ml) vs ph (6-12) kc4j mutant lipase similar to strain ra204./ aspergillus fumigatus fig 5 lipase activity (u / ml) vs temperature (30-70 c) kc4j mutant lipase similar to 0 aspergillus fumigatus strain ra204. fig effect of ph on the relative stability of lipase activity during 90 minutes of incubation6 . 8 indriawan et al. microbiol indones of hydrogen bonds can also affect the stability of lipases against temperature (bora and bora 2012). rhizopus oligosporus lipase can maintain residual activity of 80% at 25-30 °c and in rhizopus oligosporus mutant, residual activity of lipase can be 100% at 20-50 °c. increasing incubation temperature can cause lipase activity to be inhibited (iftikhar et al. 2011) and ncim 1207 is stable at 40aspergillus niger °c for 3 hours and at 50 ° c for 1 hour causing 52% loss of activity (mhetras 2009).et al. effect of metal ions. several metal ions are required for lipase to increase its activity metal ions at. certain concentrations can act as activators or inhibitors for lipases.in addition, metal ions also function as cofactors for lipases and make lipases more stable when binding to the substrate (palmer 1991). in general, ca ions play an important role in 2+ increasing lipase activity and these ions can also influence structural changes rather than the catalytic role of lipases (dandavate 2009). in addition,et al. these ions at a concentration of 1 mm can induce a conformational change in the lipase structure to become more stable, thus increasing lipase activity (iqbal and rehman 2015). according to glogauer (2011), the ionet al. concentration of 1 mm ca , cu and mn can increase 2+ 2+ 2+ the relative activity of lipase. the relative activity of rhizopus oryzae lipase can also be increased with the addition of ca and mg ions (10 mm) (dali 2+ 2+ et al. 2009). lipases produced by other rhizopus oligospores can also be increased by the addition of ca , mg and mn ions (5 mm) (kareem 2017). 2+ 2+ 2+ et al. shah and bhatt (2012) and wahyuni ​​(2016), reported that lipase activity can be increased by the addition of ions such as ca , mg , mn and fe . 2+ 2+ 2+ 2+ nin conclusio , the r value of 0.93 results from the 2 central composite design analysis and the quadratic model shows that carbon, nitrogen and ph sources have an effect on lipase activity. the composition of the medium for production of 1.25% olive oil, 3.5% soy fig 7 effect of temperature on the relative stability of lipase activity during 90 minutes of incubation. fig 8 effect of addition of 1 mm and 10 mm metal ions on the relative activity of lipase. volume 1 , 2026 2 microbiol indones 9 flour and a ph of 7.5 are the optimum conditions for lipase fermentation. the optimum lipase activity is at ph 6 and temperature 60 °c, relatively stable at ph 7-8 and temperature 30-60 °c. all of the ions tested could increase lipase activity and ca ions were the ones that 2+ could increase the highest lipase activity. references 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wahyuni g. 2016. pemurnian dan karakterisasi lipase dari yeast m2 sebagai biodeterjen. tesis. sekolah pascasarjana institut pertanian bogor, bogor: vii + 27 hlm. 12 indriawan et al. microbiol indones 2.mi719-achmad dinoto available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.2issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 144-151 *corresponding author; phone/fax: +62-21-8765066/+6221-8765062, email: achmaddinoto@yahoo.com porang (amorphophallus muelleri blume) is a robust herbacous plant commonly found in asia that produces the potential corms, large globulose depressed tubber. the corms contain carbohydrate in which mostly are in the form of mannan (kay 1973). depending on the cultivar, the glucomannan contents in the corms of a. konjac are 29 to 59% of total dry weight (chua et al. 2010). in several countries, the corms were eaten as vegetable and also used for industrial purposes such as the source of flour and the corms of amorphophallus muelleri blume contain a large amount of glucomannan, a kind of polysaccharide that are commonly consumed by people as gelly foods. in order to improve the beneficial properties of glucomannan, we previously have established the enzymatic process to produce the mannoligosaccharides from flour of glucomannan using microbial mannanase. the effects of mannoligosaccharides on the growth modulation of human intestinal microbiota were investigated in this study. a set of in vitro single batch culture experiment was conducted to study the effect of mannooligosaccharides on human-origin lactobacillus fermentum aa0014 and lactobacillus plantarum fu0811. a modified mrs medium containing 10% (w/v) sucrose, glucomannan, and mannoligosaccharide was used instead of glucose as -1 carbon source. the results showed the highest growth rate (0.13 h ) with both l. fermentum aa0014 and l. plantarum fu0811 in the presence of mannooligosaccharides. we confirmed this result by a similar in vitro experiment using human fecal samples of six healthy adults as innocula and analyzed the microbial population by fluorescence in situ hybridization (fish). lactobacilli were proliferated higher in the presence of mannoligosaccharide than other carbon sources, yielding the microbial proportion as much of 10.9% of total microbiota. overall, this study demonstrated the potential use of mannoligosaccharides synthesized from a. muelleri glucomannan as prebiotic candidate of modulating the beneficial human intestinal microbiota. key words: amorphophallus muelleri, growth modulation, human intestinal, mannoligosaccharides, microbiota, prebiotic umbi amorphophallus muelleri blume mengandung sejumlah tinggi senyawa glukomannan, yaitu sejenis polisakarida yang umum dikonsumsi oleh masyarakat sebagai produk pangan jeli. untuk meningkatkan manfaat glukomannan, penelitian sebelumnya telah dilakukan untuk mendapatkan produk mannooligosakarida hasil reaksi enzimatik tepung glukomannan menggunakan enzim mannanase. pada penelitian ini, mannooligosakarida hasil reaksi enzimatik dikaji pengaruhnya terhadap modulasi pertumbuhan mikrobiota saluran cerna manusia. percobaan kultur curah telah dilakukan untuk memahami efek stimulasi mannooligosakarida terhadap bakteri lactobacillus fermentum aa0014 and lactobacillus plantarum fu0811 asal manusia. medium mrs modifikasi yang mengandung 10% (b/v) sukrosa, glukomannan dan mannoligosakarida digunakan sebagai sumber karbon -1 menggantikan glukosa. hasil menunjukkan bahwa kecepatan pertumbuhan tertinggi (0.13 jam ) ditunjukkan oleh l. fermentum aa0014 and l. plantarum fu0811 pada substrat mannooligosakarida. konfirmasi dilakukan dengan percobaan in vitro serupa, namun menggunakan sampel feses manusia asal enam orang dewasa sehat sebagai inokulum mikrobiota usus. populasi mikrobiota dianalisis dengan fluorescence in situ hybridization (fish) dan hasil menunjukkan bahwa kelompok lactobacilli dapat digandakan populasinya lebih tinggi dengan substrat mannooligosakarida dibandingkan sumber karbon lainnya, yaitu mencapai 10.9% dari total mikrobiota. secara umum, penelitian ini menunjukkan potensi penggunaan mannooligosakarida yang disintesis dari glukomannan a. muelleri sebagai kandidat prebiotik yang berperan dalam memodulasi pertumbuhan mikrobiota saluran cerna manusia yang menguntungkan. kata kunci: amorphophallus muelleri, mannoligosakarida, manusia, modulasi pertumbuhan, mikrobiota saluran cerna, prebiotik in vitro modulation of human intestinal microbiota by mannoligosaccharides synthesized from amorphophallus muelleri glucomannan 1 2 3 achmad dinoto *, cory corazon watumlawar , and yopi 1 research center for biology, indonesian institute of sciences (lipi). jalan raya jakarta-bogor km 46, cibinong 16911, jawa barat, indonesia; 2 sekolah tinggi teknologi industri dan farmasi (sttif), jalan kumbang 23, bogor 16151, jawa barat, indonesia; 3 research center for biotechnology, indonesian institute of sciences (lipi), jalan raya jakarta-bogor km 46, cibinong 16911,jawa barat, indonesia mannose (kay 1973). the corms of a. konjac has been used as food and food additives in china and japan for more than 1000 years. in addition, the potential use of glucomannan derived from konjac tuber have been studied for the application in pharmaceuticals, cosmetics, coating materials, and emulsifier (alososande et al. 2009; chua et al. 2010; zhang et al. 2005). as food compounds, the glucomannan of a. konjac have been investigated previously for anti-obesity, antihyperglycemic and hypercholesterolemia, antiinflammatory, and laxative activities as well as prebiotic properties (chen et al. 2006; chua et al. 2010). glucomannan was considered to have diet therapy in primary prevention in high risk hypercholesterolemic children (martino et al. 2005). there is increasing interest in the development of mannan-based prebiotics. the mannanase-producing bacteria were applied to produce prebiotic mannooligosaccharides, using copra as raw materials (titapoka et al. 2008). basically glucomannan can be hydrolized to produce mannoligosaccharides. in previous study, we have succesfully established the processing methods for synthesizing the mannoligosaccharides from glucomannan by using the mannanase of actinomycetes isolates (yopi et al. unpublished data). oligosaccharides are important to stimulate efectively the beneficial microbiota in gastrointestinal tract, especially bifidobacteria in human and animal (dinoto et al. 2006a; dinoto et al. 2006b). lactobacilli was also increased in rat cecal after administering raffinose and encapsulated bifidobacteria (dinoto et al. 2006b). similary, modulation of beneficial microbes by fructooligosaccharides and galactooligosaccharides were also reported (rycroft et al. 2001). several oligosaccharides have been investigated intensively in human subject or using human feces as innoculum, including fructooligosaccharides, galactooligosaccharides, maltooligosaccharides, and xylooligosaccharides (rossi et al. 2005; rycroft et al. 2001; tuohy et al. 2005). the role of mannoligosaccharides in modulation of human intestinal microbiota is not completely understood. although some information is available on the effects of feeding mannoligosaccharides in the health status of human and animal, there is a limited information about the potential uses a. muelleri corms-based mannoligosaccharides for modulating gut microbial population. in this study, we investigated whether mannoligosaccharides influence the microbial population and metabolite profiles in the static batch culture of human intestinal microbiota. materials and methods strains and material preparation. bacterial strains used in this study are lactobacillus fermentum strain aa0014 and lactobacillus plantarum strain fu0811. those strains are human-origin and belonging to the collection of research center for biology, indonesian institute of sciences, indonesia. in this study we used glucomannan flour of a. muelleri (provided by pt. ambico, indonesia). mannoligosaccharides sample used in this study was synthesized by the reaction of glucomannan flour and mannanase enzyme of saccharopolyspora flava btcc id040555, which was kindly provided by the collection of the biotechnology culture collection, research center for biotechnology, indonesian institute of sciences. the liquid products of glucomannan and mannoligosaccharides used in the experiment were adjusted to final concentration of 10 g per liter medium. measurement of the growth rate. to determine the growth rate of representative probiotic candidate lactobacilli in several carbon sources, a seed culture for l. fermentum aa0014 and l. plantarum fu0811 growth experiments was grown overnight in mrs medium (de man et al. 1960). at an optical density at od = 0.8 of 600 seed culture was transferred to the same mrs-based medium, but containing one of the following -1 carbohydrates (10 g l ): glucose (merck), sucrose (merck), glucomannan, and mannoligosaccharides. bacterial growth was periodically monitored by determining the od of the culture broth. all the 600 cultures were incubated at 37 °c under anaerobic conditions using carbondioxide. specific growth rates -1 (µ) (in h ) were calculated during the logarithmic growth phase using the following equation: µ = (ln xt lnxt )/(t 2 1 2 t ), where xt and xt are the od values at times t and 1 2 1 600 2 t , respectively.1 static batch culture fermentations. the experiment was conducted as described by olanomartin et al. (2000). in autoclaved 50 ml serum bottles containing each substrate (0.5 g), aliquot of 45 ml autoclaved nutrient medium was added by filter sterilization to each bottle. the basal medium contained -1 (g l ): peptone water, 2; yeast extract, 2; nacl, 0.1; k hpo , 0.04; kh po , 0.04; mgso .7h o, 0.01; 2 4 2 4 4 2 cacl .6h o, 0.01; nahco , 2; hemin (dissolved in a 2 2 3 -1 few drops of 1 mol l naoh), 0.05; cysteine. hcl, 0.5; bile salts, 0.5; tween 80, 2 and 10 µl vitamin k . the 1 -1 medium was adjusted to neutral (ph 7.0). using 1 mol l hcl. before innoculation, the bottles containing medium were placed inside anaerobic jar (merck) with volume 7, 2013 microbiol indones 145 supplementation of anaerocult (merck) at 37 °c overnight to prereduce the media. a 10% (w/v) fecal slurry was prepared using fresh feces from six healthy donors (who had not taken antibiotics for 3 months -1 beforehand) and prereduced 0.1 mol l phosphate buffer (ph 7.0). fecal slurry was mixed in a sterilized tube for 2 min. in each serum bottle, 5 ml of the slurry were inoculated, mixed and capped. samples were removed from the fermenters at 24 h fermentation for enumeration of microbiota, and measurement of scfa. the fermentation experiments were performed in triplicate for each. fluorescence in situ hybridization analysis. the samples (100 µl) from the static batch culture at 24 h fermentation were removed and washed with phosphate buffered saline (pbs) (130 mm nacl, 10 mm sodium phosphate buffer; ph 7.2) at 9 000 ×g 4 °c for 2 min. the washed samples were fixed in 4% (w/v) paraformaldehyde in pbs (ph 7.2) for 24 h. fixed samples were washed once in pbs and stored in a known volume of 50% (v/v) ethanol-pbs at -20 °c until use. fish analysis was conducted as described by dinoto et al. (2006b). aliquots (3 µl) of fixed cells were applied to teflon printed glass slides (adcell; 12 wells; diameter, 5 mm; erie scientific company, portsmouth, n.h.), and air dried. the cells were then dehydrated with a series of solutions containing 50%, 80%, and 99.5% ethanol (3 min for each concentration). the cells fixed on the glass slides were hybridized by addition of 8 µl of hybridization buffer (0.9 m nacl, 0.01% sodium dodecyl sulfate, 20 mm tris-hcl, 20% deionized formamide; ph 7.2) with 1 -1 µl of cy3-labeled oligonucleotide probe (25 ng µl ; tsukuba oligo service co., ltd., tsukuba, japan). the 16s rrna-targeted oligonucleotide probes used for molecular analysis of lactobacillus spp. (lacb722, sg-lacb-0722-a-a-25, ycaccgctacacat grag ttccact) (sghir et al. 1999), bifidobacterium spp. (bif164m, s-g-bif-0164-b-a-18, catccggyatta ccaccc) (dinoto et al. 2006a), clostridium coccoides-eubacterium rectale group (erec482, s-*erec-0482-a-a-19, gcttcttagtca rgtaccg) (franks et al. 1998), and streptococcus spp. (strc493, s-*-strc-0493-a-a-19, gttagccgtccctttctg g) (franks et al. 1998). the slides were hybridized at 46 °c for 16 h in a moist chamber. after hybridization, the slides were rinsed with warm hybridization buffer at 48 °c and washed in prewarmed washing buffer (225 mm nacl, 0.01% sodium dodecyl sulfate, 20 mmtris-hcl; ph 7.2) for 20 min at 48 °c. the washed slides were stained with a dapi (4’,6-diamidino-2-phenylindole dihydrochloride n-hydrate) solution for 5 min at room temperature to stain the chromosomes as a control signal. the slides were washed with distilled water for 5 min at room temperature and air dried in the dark. the dried slides were mounted with vectashield (vector laboratories inc., burlingame, calif.) and examined with an nikon optihot-2 (nikon corporation, tokyo, japan) equipped with a nikon digital camera. the dapi and cy3 signals were captured in pairs of 10 random microscopic fields (about 500 cells per microscopic field). hybridization images were manually counted and were colorized when necessary using adobe photoshop 7.0 (adobe systems incorporated, san jose, calif.). specific signals from the probes were expressed as average percentages of the total cells visualized by dapi signals in the same microscopic field. short-chain fatty acid analysis and ph measurement. the measurement of short chain fatty acid was carried out by taking samples from the bottles of the static batch culture at 24 h fermentation. samples were centrifuged (13 000 ×g for 30 min) and filtered by 0.2 µm syringe to remove particulate material. ten microliter samples were then injected onto an hplc system (model water 1350t, biorad, uk). the column used in this analysis was an ion-exclusion aminex hpx-87h (bio-rad) which maintained at 35 °c with a column heater and the pressure of 2071 psi. the eluent, 0.008 n sulphuric acid in hplc-grade water was pumped through the column at a flow rate of 1.0 ml -1 min . data from the ri detector were integrated and by using calibration curves, acetate, propionate, succinate, and lactate were quantified in the samples. the ph of suspension was determenided by using iq120 tm minilab ph meter (hach company, loveland, co). statistical analysis. the data of total cell counts, microbial proportions, ph, and scfa concentrations for culture were analyzed statistically using spss software version 13.0 (spss, inc., chicago, il). bonferroni tests were performed for pair-wise multiple comparisons of the mean values for the glucose, sucrose, glucomannan, and mannoligosaccharides. results in this study, we determined the specific growth rate of selected l. plantarum fu0811 and l. fermentum aa0014 as representative human intestinal lactobacilli on the culture containing glucose, sucrose, glucomannan, and mannoligosaccharides as sole carbon source. under this experimental condition, both 146 dinoto et al. microbiol indones was monitored in glucomannan medium (3.2%) and sucrose medium (2.6%). the population of lactobacilli in glucose medium was only 0.7% of total microbiota. the highest population of bifidobacteria was found in mannoligosaccharides medium (3.3%) and we also confirmed that this group of bacteria was relatively low in sucrose and glucose media (lower than 0.1% of total microbiota). in contrast, the populations of eubacterium/clostridium showed a lower proportion in mannoligosaccharides medium than in sucrose medium or glucose medium (table 2). our data also showed no streptococci were detected in all tested carbon sources, indicating that this group of bacteria was absent or out of 7 detection limit in fish analysis (<10 cells). overall, this experimental system clearly demonstrated that the growth of human intestinal microbiota is much more stimulated by mannoligosaccharides. there was a change in the ph during fermentation of carbon sources by intestinal microbiota of human origin. we observed that the ph change at the static batch culture of human fecal slurry during 24 h fermentation with various carbon sources used in this study is about 2-3 (fig 2). in general, carbohydrate was rapidly metabolished by intestinal bacteria to produce organic acids. the occurences of nutrient metabolisms by intestinal microbiota could be simply recognized by the profile of short chain fatty acids (scfa). scfa are the products of microbial activities in the fermentations and only produced in appreciable levels in the presence of added carbohydrate. in this study, we observed that only lactate, acetate, and propionate were detected in all samples, whereas succinate was not detected. the l. plantarum and l. fermentum grew faster in the presence of mannoligosaccharides yielding the highest -1 specific growth rate as much of 0.13 h (table 1). we observed that the growth of those strains became slower when they were cultured in the glucomannan medium. interestingly, l. plantarum and l. fermentum have lower growth rates in the presence of glucose (0.05 and -1 0.04 h , respectively) as compared to sucrose medium -1 (0.09 and 0.10 h ), respectively (table 1). the cell morphology of intestinal microbiota in the batch culture inoculated with human fecal samples of six healthy adults were varied. based on fish analysis, we observed many cells were matched with oligonucleotide probe used in this study. the long-rod cells identified as lactobacilli under epifluorescence microscope were clearly distributed among other intestinal bacteria in batch culture (fig 1). since we did not monitored the population hour-by-hour, the 24 h observation could be used as representing impact of carbon source on human microbiota. the changes in selected bacterial populations with the carbon sources tested are presented in table 2. total cell counts of intestinal microbiota in -1 glucose medium was about 9.43 log cells ml 10 indicating that microbial cells in fecal slurry proliferated under experimental system. population of microbes before 6 h fermentation was observed under 7 log cells 10 -1 ml and it was unsufficient for analysis of fish. there is different cell number of microbiota when sucrose was used in this experiment (table 2). with all of the carbon source studied, a large significant increase in numbers of lactobacilli was observed in mannoligosaccharides medium (10.9%), whereas a relatively lower number table 1 the specific growth rate (µ) of human-origin lactobacillus fermentum aa0014 and lactobacillus plantarum fu0811 -1 specific growth rate (h ) ± sd* lactobacillus fermentum aa0014 glucose 0.04 ± 0.01 sucrose 0.10 ± 0.00 glucomannan 0.01 ± 0.01 mannoligosaccharides 0.13 ± 0.01 lactobacillus plantarum fu0811 glucose 0.05 ± 0.03 sucrose 0.09 ± 0.00 glucomannan 0.02 ± 0.02 mannoligosaccharides 0.13 ± 0.01 *sd, standard deviation volume 7, 2013 microbiol indones 147 scfa in medium was observed in mannoligosacchari-1 des medium (79.57 mmol l ) indicating that mannoligosaccharides was a substance in which intestinal microbiota prefered to use it as digestible substrate (table 3). discussion our data actually support the advantages of mannoligosaccharides as main compound in glucomannan hydrolysate on stimulating intestinal lactobacilli (table 1, table 2), in addition, without any significant changes in bifidobacteria and streptococci high level lactate were detected in sucrose medium and mannoligosaccharides medium, as much of 36.05 and -1 34.84 mmol l , respectively (table 3). in contrast, very low level of lactate was observed in glucomannan medium. the culture cultivated in the presence of glucose showed moderate level of lactate (24.06 mmol -1 l ). the highest concentration of acetate (36.19 mmol -1 l ) was observed in mannoligosaccharides medium. glucose medium tend to modulate the microbial community to produce slight acetate in medium. propionate was detected higher in mannoligosacchari-1 des and glucomannan media (8.54 and 9.58 mmol l ) than in other substrates. in general, the total detected dapi lacb722 fig 1 epifluorescence images of microbial cells (stained with dapi) and lactobacillus cells (hybridized with probe lacb722) in 24h static batch culture of human fecal slurry with mannoligosaccharides. table 2 microbial populations in the 24 h static batch culture fermentations with the various carbon sources population microbial population ± sd* -1 total microbiota (log10 cells.g ) lactobacillus (%) bifidobacterium (%) clostridium/eubacterium group (%) streptococcus (%) stain or probe glucose sucrose glucomannan mannoligosaccharides dapi b 9.43 ± 0.02 a 9.40 ± 0.03 c 9.47 ± 0.02 c 9.47 ± 0.03 lacb722 a 0.65 ± 0.59 ab 2.57 ± 0.67 b 3.18 ± 0.88 c 10.90 ± 3.65 bif164m a 0.89 ± 0.90 a 0.10 ± 0.22 b 2.46 ± 1.13 b 3.28 ± 1.17 erec482 b 21.14 ± 5.91 b 20.39 ± 5.56 ab 17.39 ± 4.55 a 13.93 ± 2.02 strc493 nd** nd nd nd *sd, standard deviation **nd, not detected 148 dinoto et al. microbiol indones increases in fecal total anaerobe counts (chen et al. 2005). similar phenomenon was observed where mannoligosaccharides significantly reduced fecal c. perfringens and escherichia coli counts (elamir et al. 2008). the effect of glucomannan hydrolysates added to the ultra-high temperature milk on the growth of lactic acid bacteria was evaluated (al-ghazzewi et al. 2007). the authors also reported that the glucomannan hydrolysates produced with either mannanase or cellulase enzymes were effective growth promoters (carbon sources) of lactic acid bacteria (al-ghazzewi and tester 2012). glucomannan is a kind of polysaccharide of the (table 2). we pressumed that mannoligosaccharides hydrolysate is likely preferred by lactobacilli rather than glucomannan itself in the term of simple utilization of oligosaccharides. in animal experiment, the increased population of lactobacilli in dog feces due to diet containing mannoligosaccharides was also reported (swanson et al. 2002). the effects of glucomannan and acid-hydrolyzed mannoligosaccharides of konjac on cecal microbiota in balb/c mice have been investigated where both glucomannan and mannoligosaccharides were capable of increasing bifidobacteria and oppositely decreasing clostridium perfringens in mice cecum. in addition, mannoligosaccharides caused table 3 scfa concentration in the 24 h static batch culture fermentations with the various carbon sources. fig 2 dicreases in ph value of the static batch cultures of human fecal slurry during 24 h fermentation with various carbon sources. ph decrease glucose sucrose glucomannan mannooligosacharides 0 1 2 3 4 5 a a b a -1 scfa concentration (mmol l ) ± sd* lactate acetate propionate succinate total glucose sucrose glucomannan mannoligosaccharides b 24.06 ± 3.98 b 36.05 ± 3.28 a 9.93 ± 2.80 b 34.84 ± 1.29 a 11.08 ± 0.72 a 17.63 ± 1.73 a 18.15 ± 2.30 a 24.47 ± 16.57 a 0.48 ± 0.08 a 0.09 ± 0.13 ab 9.58 ± 0.46 b 5.85 ± 3.79 nd** nd nd nd a 35.62 ± 3.34 a 53.76 ± 5.13 a 37.66 ± 0.95 a 65.17 ± 19.07 *sd, standard deviation **nd, not detected volume 7, 2013 microbiol indones 149 better protective effects on fecal water-induced dna damage as the prebiotic property than did the unhydrolysed konjac glucomannan (connoly et al. 2010). in conclusion, this study demostrated that mannoligosaccharides synthesized through enzymatic reaction of a. muelleri glucomannan have potential properties as prebiotic candidate. the further study could be conducted for clear characterization in order to improve the healthy condition of the host. acknowledgment this work was supported by the competitive research grant of indonesian institute of sciences. the authors wish to acknowledge the biotechnology culture collection (btcc) for providing actinomycetes isolate for producing mannanase. references al-ghazzewi fh, khanna s, tester rf, piggott j. 2007. the potential use of hydrolysed konjac glucomannan as a prebiotic. j sci food agric. 87(9):1758-1766. doi:10.1002/jsfa.2919. al-ghazzewi fh, tester rf. 2012. efficacy of cellulase and mannanase hydrolysates of konjac glucomannan to 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enzyme β-mannosidase that remove βmannose residues from β-1,4-linked mannoligosaccharides. specificaly, the degradation by β-glucosidase occurs only at a terminal glucose unit and stops at terminal oxidized residues or mannose units. the enzyme of β-mannanase and β-mannosidase are present in many microorganisms including bacteria harbored in human gut. in addition, the β-glucosidase genes are widespread among human gut bacteria (gill et al. 2006). a member of human gut lactic acid bacteria, l. gasseri, encoded twenty kinds of putative glycosyl hydrolases which mostly glucosidases and galactosidases with predicted substrate specificities for a diversity of diand trisaccharides. the β-glucosidase of this strain is specific and have no homolog among lactic acid bacteria (azcarate-peril et al. 2008). the substrate specifity was suggested to be important key in oligosaccharides utilization by lactobacilli. although several constraints of this study have included most detected microbes of intestinal samples are culturable and static culture condition may impact to tolerance against acidic condition where organic acid implicate to reduce the ph values, this study could explain the direct impact of prebiotic candidate to the growth selected individual strains (l. plantarum and l. fermentum). those strains were previously observed in separate study for reducing the cholesterol in medium after 36 h incubation (dinoto et al. unpublished data). thus, by understanding the suitable substrate, the development synbiotic product with mannoligosaccharides will be interesting aspect in term of functional foods. in mice experiment, konjac glucomannan and the products of partially-hydrolysed konjac glucomannan was compared. based on culture-dependent method, the total anaerobes and the populations of lactobacilli, and bifidobacteria were higher in partially-hydrolysed konjac glucomannan indicating the preference of those beneficial bacteria on specific digested product of glucomannan. 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6: 149 7: 150 8: 151 8 endang srimurni.pmd population density of wolbachia bacteria and the induction of the popcorn-effect in drosophila melanogaster endang srimurni kusmintarsih faculty of biology, universitas jenderal soedirman, jalan dr. soeparno no 63 grendeng, purwokerto 53122, indonesia phone: +62-281-638794, fax: 62-281-631700, e-mail: endang_sk@lycos.com drosophila melanogaster is known to harbour wolbachia bacteria that cause the early death of its adult host. the death occurs early as a result of cell rupture caused by the proliferation of wolbachia within it. however, the number of wolbachia causing the early death in d. melanogaster is unknown. in order to determine whether the number of wolbachia is related to the early death of its host, the quantity of wolbachia and nuclei were examined using the real-time pcr procedure. the results showed that an egg cell contains at least 2-3 bacterial cells. a 4 day old larva contains at least 45 cells. the number of wolbachia in adult flies of 1, 5, 8, and 12 days old were 422, 535, 964, and 610 per host cell, respectively. key words: wolbachia pipientis, real-time pcr, drosophila melanogaster _____________________________________________ issn 1978-3477 volume 2, number 2, august 2008 p 89 94 wolbachia are maternally inherited, intracellular, and obligate bacteria known to infect a wide range of arthropods. these bacteria were discovered by hertig and wolbach (1924) in the ovary of the mosquito culex pipiens. hertig (1936) formally named them as wolbachia pipientis in honour of his collaborator. recent surveys indicate that around 16-20% of all insect species may be infected with wolbachia (werren and jaenike 1995). wolbachia are also widespread in filarial nematodes. infections with wolbachia have been associated with host reproductive abnormalities such as cytoplasmic incompatibility, feminization, parthenogenesis, male killing (dying males caused by bacteria infectious in the ovaries of females) and the popcorn-effect. it is named popcorn-effect because when the bacteria multiply inside the fly’s cells they cause a sudden massive degeneration of host cells, which resemble the baking process of “popcorn” in a microwave (min and benzer 1997). the popcorn-effect of wolbachia has so far only been detected in one laboratory strain of drosophila melanogaster. it is characterised by a sudden onset of widespread degeneration in somatic tissue such as the brain, retina, and thoracic muscle of d. melanogaster. min and benzer (1997) and werren (1997) explain that death occurs early as a result of cell rupture caused by the proliferation of wolbachia within it. they note that wolbachia are present at a small population level during development through the embryonic, larval, and pupal stages. as soon as the adults emerge, the bacteria start to multiply rapidly and this leads to an abrupt death during the adult’s halfway lifespan. it might be possible to make use of the popcorn-effect induced by wolbachia to control insect vectors of important human diseases in which the pathogen or parasite is transmitted at the end of the insect’s lifespan after it has taken three to four blood meals. in the future, this may play a vital role in the prevention of arthropod-borne diseases. disease transmission depends on the older insect and wolbachia infection has been found to shorten the lifespan of insects. however, the cell population size of wolbachia to cause the popcorn-effect in d. melanogaster is unknown. up to recent days, there has been no in vitro system for culturing wolbachia within insect cells. the fastidious nature of wolbachia, together with the difficulty in counting bacterial numbers inside tissues, has made it difficult to assess wolbachia density within its host. this has severely impeded progress in the study of wolbachia. in recent years, more sophisticated methods have been developed in which amplified dna is quantified during the exponential phase of the pcr. these methods, some of which have been automated in real-time pcr, have eliminated much of the variation associated with end-point measurements. based on the above reasoning, real-time pcr was used to study wolbachia in order to determine whether the population density of wolbachia is related to the early death of its host. there are several steps required to work with real-time pcr. firstly, primers need to be designed to provide a product of less than 200 bp. the reason for using the short product is that a straight-line relationship of the starting amount versus threshold cycle needs to be obtained. a short sequence is needed to design these primers. therefore, we cloned and sequenced fragments of the wsp gene of wolbachia and the na+ pump á subunit of nuclei. these genes were used because they are present as single copies in the genome (lebovitz et al. 1989; bensaadi-merchermek et al. 1995; braig et al. 1998). in the present study, the cell population density of wolbachia which could induce the popcorn-effect in d. melanogaster was determined from eggs, larvae, and adult flies of 1, 5, 8, and 12 days old. this is based on previous observations that the survival of adult of d. melanogaster (w1118) infected with popcorn-effect inducing wolbachia was to a maximum of 13 days, while adult survivors of d. melanogaster (w1118) uninfected with popcorn-effect inducing wolbachia could survive up to 45 days at 29°c. mcgraw et al. (2002) reported in their research that after transinfection of the over-replicating wolbachia popcorn strain from d. melanogaster to d. simulans they showed that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. both 90 kusmintarsih microbiol indones densities and fitness costs associated with ovary infection rapidly decline in the generations after transinfection. the early death effect in d. simulans attenuated only slightly and was comparable to that induced in d. melanogaster. kondo et al. (2005) reported that wolbachia density was determined through a complex interaction between host genotype, symbiont genotype, and other factors. dutton and sinkins (2004) studied the strain-specific quantification of wolbachia density in aedes albopictus and effects of larval rearing conditions and showed that larval crowding always reduced adult size, but reduced the density of wolbachia strains relative to uncrowded conditions only if crowding was accompanied by restricted nutrient availability. crowded rearing conditions never resulted in strain segregation or in a reduction in the penetrance of cytoplasmic incompatibility (ci), however. the rate of maternal transmission and the penetrance of ci are the two most important variables that determine relative wolbachia population invasion dynamics, and both are considerably higher here than have been reported in the d. simulans model system. mouton et al. (2007) studied the interaction between three strains of wolbachia in two divergent homozygous lines of the wasp leptopilina heterotoma at two different temperatures and showed that wolbachia density varied between the two host genotypes at only one temperature, and the host genotype plays an important role in wolbachia density. they also highlighted its interaction with environmental conditions, making the evolution of local adaptations for the regulation of wolbachia density possible. materials and methods dna extraction from d. melanogaster. total genomic dna was extracted from 25 whole flies, 50 whole larvae, and 100 eggs. amplification of wsp and na+ pump á subunit genes, cloning, purification, and restriction of plasmids. primers used in the pcr were as follows: 610 bp for wolbachia primer, wsp 81f (5’ tggtccaataagtgatgaagaaac 3’) and wsp 691r (5’ aaaaat taaacgctactcca 3’); and 545 bp for nuclei primer, na+ pump á subunit s 1124 (5’ agcgtatggc(c/a)tc(a/g)aagaactg 3’), and a 1669 (5’ ag(c/t)tcc atgtaggcattgttga 3’). the conditions for the pcr were as follows: denaturation for 5 min at 95°c, followed by 35 cycle of denaturation at 95°c for 1 min, annealing at 55°c for 1 min and extension at 72°c for 2 min, and finally 4°c for an infinite amount of time. pcr products from d. melanogaster with bright bands were directly cloned into a ta cloning vector kit (invitrogen, usa) according to the manufacturer’s directions (topo ta cloning version l 01261 25-0184). plasmids were extracted from bacteria using the miniprep dna purification kit (qiagen, usa). in order to verify the presence of an insert, the plasmids were digested using ecor1 (promega, usa), and the products were run on 0.7% agarose gel. sequencing. dna of plasmids with an insert was sequenced using dye terminator cycle sequencing on ceq 2000 with quick start kit (qiagen, usa). primer design. primer select software provided by the dna star program was used for designing the primers. the value in determining a primer, set as the primary selection criterion of the software, was the length of amplicon (< 200 bp). the use of this software resulted in a series of best-fit suggestions for the primer set. the program checks for primer location, primer dimer, melting temperature (t m ), annealing temperature, and gc percentage value within primer sets. measurement of standard dna, of wolbachia bacteria and nuclei copy number using real-time pcr. the standards used were the wsp plasmid for wolbachia numbers and the sodium pump á subunit plasmid for nuclei numbers. these plasmids in real time pcr was calculated using pico green dsdna quantification reagent (molecular probe, netherlands). pico green is an ultrasensitive fluorescent nucleic acid stain for quantifying double-stranded dna (dsdna) in solution. pico green dsdna quantification reagent enables quantification of as little as 25 pgml-1 of dsdna (50 pg dsdna in a 2 ml assay volume) with a standard spectrofluorometer (vector). quantification of standard dna copy number. the standard dna was calculated based on the equation: copy number = standard dna sample x avogadro number mw quantification of wolbachia and nucleus copy number. wolbachia and nucleus amount (the number of wolbachia and nucleus) in unknown samples was calculated based on a single egg, larva, or adult fly. real-time pcr amplification efficiencies. real-time pcr efficiencies were calculated from the given slopes in the icycler (bio-rad, usa). the corresponding real-time pcr efficiencies (e) of one cycle in the exponential phase was calculated according to the equation exponential phase was calculated according to the equation : e = 10 (-1/slope) – 1. the new wolbachia and nucleus primers were used to amplify the genes. for icycler reaction, a master mix of reaction components was prepared as follows: 12.5 µl pcr master mix, 2x (promega), 0.5 µl forward primer (20 µm), 0.5 µl reverse primer (20 µm), 0.5 µl sybr green 1/dye (1:10 000 dilution), 5 µl of template was added to the standard or unknown (for negative control no template was added), 6 µl nuclease free water. finally, 25 µl aliquots of each mix were pipetted into rows of a 96-well thin-well pcr plate. the plate was covered with a piece of optically clear sealing tape and placed in the icycler iq detection system. pcr was carried out at 95°c for 2 min 18 sec; followed by 40 cycles of 15 sec at 95°c, 20 sec at 55°c, and 30 sec at 72°c. fluorescent data were collected during the 72°c step. three and four standards were set up (2 replicates of each) of wsp and na+ pump á subunit and unknowns were investigated [eggs, larvae, adult flies (day 1), adult flies (day 5), adult flies (day 8), and adult flies (day 12)]. statistical analysis. the density of wolbapphia and nuclei in d. melanogaster infected with wolbachia popcorneffect in eggs, larvae, and adult flies was analysed by using oneway anova. all of the data were transformed to logarithmic data before performing one-way anova. x avogadro numbercopy number = exponential statistical analysis. the density of wolbachia and nuclei in d. melanogaster infected with wolbachia popcorneffect in eggs, larvae, and adult flies was analysed by using one-way anova. all of the data were transformed to logarithmic data before performing one-way anova. volume 2, 2008 microbiol indones 91 results restriction of plasmids using ecor1. plasmids from two different amplifications were found to contain the expected inserts of approximately 610 bp for wsp and 545 bp for the na+ pump á subunit gene fragments. the gel electrophoresis of the restriction product is shown in fig 1. sequence from wolbachia surface protein (wsp) gene and sodium (na+) pump á subunit gene. the sequences reported here (table 1 and 2) showed similarity to genbank database (http://www.ncbi.nlm.nih.gov) accession no. 13173392 and 18487812 for wsp and na+ pump á subunit sequences. the sequences had identities of 98 and 97%, respectively. a standard curve was created and used to calibrate the pcr assay. the standard curve gives a correlation coefficient of 0.980 and slope of – 3.176 (fig 2). the efficiency was calculated using the equation : e = 10 (-1/slope) 1 = e = 10 (-1/-3.176) 1 and resulted in a high real-time pcr efficiency rate (100%). the correlation coefficient indicated how well the standards fit on the curve. given known starting amounts of the target nucleic acid, a standard curve can be constructed by plotting the log of the starting amount versus the threshold cycle. the efficiency was good when the value ranged 90% to 110%. table 2 sequence of na+ pump α subunit fragment 1 tttgtttggt ggagtttgaa ggggcacctc 31 gggcaatctt ctttattacg gcttggcgaa 61 tggttcatca catcgcccag agccaggttc 91 catgcacttg agcagagcag cctcggaggc 121 atctccactg acttctttct tgaggattgg 151 gacgccatct tggcctccct tgaactcggc 181 acggttacag agagtggcaa tgcgagagag 211 cgccttgaat ccagggctgg ttctatcgta 291 ttgaacaccc gactgatcct cagttgtgtc 241 ggcctcgatg atctgattat cgaaccacat 271 gtgggcgacc gtcattcggt tctgggtgag 301 ggtgccggtc ttatcggagc agatggtcga 331 tgtggagcca agggtctcca cggcctccag 361 attcttcacc agacagttct tcgataccat 391 acgctaaggg cgaattctgc agatatccat 421 cacactggcg gccgctcgag catgcatcta 451 gagggcccaa ttcgccctat agtgagtcgt 481 attac 1 gaatttttac ctcttttcac aaaagttgat 31 ggtattacct ataagaaaga caagagtgat 61 tacagtccat taaaaccatc ttttatagct 91 ggtggtggtg catttggtta caaaatggac 121 gacatcaggg ttgatgttga aggagtttat 151 tcatacctaa acaaaaatga tgttaaagat 181 gtaacatttg acccagcaaa tactattgca 211 gacagtgtaa cagcaatttc aggattagtg 241 aacgtgtatt acgatatagc aattgaagat 271 atgcctatca ctccatacat tggtgttggt 301 gttggtgcag cgtatattag cactcctttg 331 gaacccgctg tgaatgatca aaaaagtaaa 361 tttggttttg ctggtcaagt aaaagctggt 391 gttagttatg atgtaactcc agaagtcaaa 421 ctttatgctg gagctcgtta tttcggttct 451 tatggtgcta attttgatgg aaaaaaaaca 481 gatcctaaaa attcaaccgg acaggctgct 511 gatgcaggcg catac 526 table 1 sequence of wsp fragment fig 2 the standard curve of wolbachia surface protein: correlation coefficient of 0.980 and slope of -3.176. fig 1 restriction digest of plasmids containing wsp and na+ pump á subunit fragments. o: one kilobase ladder (promega, usa); a , lane 1: restriction enzyme treatment of wolbachia surface protein fragment; b, lane 1 and 2: restriction enzyme treatment of na +pump á subunit fragment. a b inserts of approximately 610 bp for wsp and 545 bp for the na+ pump á subunit gene fragments. the gel electrophoresis of the restriction product is shown in fig 1. sequence from wolbachia surface protein (wsp) gene and sodium (na+) pump á subunit gene. the sequences reported here (table 1 and 2) showed similarity to genbank database (http://www.ncbi.nlm.nih.gov) accession no. 13173392 and 18487812 for wsp and na+ pump á subunit sequences. the sequences had identities of 98% and 97%, respectively. a standard curve was created and used to calibrate the pcr assay. the standard curve gives a correlation coefficient of 0.980 and slope of – 3.176 (fig 2). the efficiency was calculated using the equation: e = 10 (-1/slope) 1 = e = 10 (-1/-3.176) 1 and resulted in a high real-time pcr efficiency rate (100%). the correlation coefficient indicated how well the standards fit on the curve. given known starting amounts of the target nucleic acid, a standard curve can be constructed by plotting the log of the starting amount versus the threshold cycle. the efficiency was good when the value ranged 90% to 110% 0 1 0 1 2 t h re s h o ld c y c le log starting quantity, copy number 92 kusmintarsih microbiol indones fig 3 showed that the melting temperature resulted in one peak of the product at 79°c. this indicated that the melting temperature of all dna templates, both standard and unknown, were the same. it also indicated that the amplified products were homogeneous and reassured that the correct product had been specifically amplified. the colours indicate different templates. copy number of wolbachia in drosophila melanogaster infected with popcorn-effect inducing wolbachia. fig 4 shows an increase in the number of wolbachia from eggs, larvae to adults at day 1, 5, 8, and 12. the analysis of bacterial cells (copy number) of wolbachia in eggs was significantly different with that of larvae day 4. the copy number of wolbachia in larvae at day 4 was also significantly different fig 3 melting curve of wolbachia surface protein product in real-time pcr. 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 -d ( r f u ) 5 0 5 2 5 4 5 6 5 8 6 0 6 2 6 4 6 6 7 0 8 0 9 6 9 86 8 7 2 7 4 7 6 8 2 8 4 8 6 8 8 9 0 9 2 9 4 temperature (oc) 100 fig 6 melting curve of ne product in real-time pcr. 5 0 -200 5 2 5 4 5 6 5 8 6 0 6 2 6 4 6 6 6 8 7 0 7 2 7 4 7 6 7 8 8 0 8 2 8 4 8 6 8 8 9 0 9 2 9 4 9 6 9 8 100 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 -d ( r f v ) temperature (oc) fig 5 standard curve of the ne primers in real-time pcr. correlation coefficient of 0.952 and slope of -2.685. fig 4 copy number of wolbachia in drosophila melanogaster infected with popcorn-effect inducing wolbachia. 5e+7 4e+7 2e+7 0 -1e+7 1e+7 3e+7 6e+7 7e+7 c o p y n u m b e r o f w o lb a c h ia egg larvae 4 adult 12 stages adult 1 adult 5 adult 8 log starting quantity, copy number t h re s h o ld c y c le volume 2, 2008 microbiol indones 93 (p< 0.001) with that of adults at day 1, 5, 8, and 12. how ever, there was no significant difference in the copy number of wolbachia found in adults (at day 1, 5, 8, and 12). the standard curve gave a correlation coefficient of 0.952 and slope of – 2.685 (fig 5). the efficiency was calculated using the equation mentioned above and resulted in a real-time pcr efficiency rate (135%). fig 6 showed that the melting temperature was obtained in one peak of the product at 86°c. this indicated that the melting temperature of all dna templates, both standard and unknown, were the same. it suggested that the amplified products might be homogeneous and the t m provided reassurance that the correct product had been specifically amplified. copy number of nuclei in drosophila melanogaster. fig 7 shows an increase in the number of nuclei from eggs, larvae to adults day 1, 5, 8, and 12. the copy number of nuclei in eggs was significantly different with the copy number of nuclei in adults day 8 and 12 and the number of nuclei in the larvae day 4 was significantly different with the number of nuclei in adults day 12. discussion the mean copy number of wolbachia present in egg and 4-day-old larva were 20 x 103 and 524 x 103, respectively. after the adults emerged, the bacteria reached 12 x 106, 18 x 106, 34 x 106, and 41 x 106 in adults aged 1, 5, 8, and 12 days, respectively. the copy numbers of wolbachia in the egg determined by confocal microscopy have been reported by boyle et al. (1993). they showed that there were 20 x 103 wolbachia in eggs, which is similar to the present findings. the number of wolbachia increase significantly from eggs, larvae, and adults. this was similar to the finding of min and benzer (1997), who reported that as soon as the adult flies emerge the bacteria started to multiply rapidly, causing the sudden death of their host. however, statistical analysis in our research showed that the copy number of wolbachia found in adults was not significantly at each day. the copy number of nuclei was 8 x 103, 18 x 103, 28 x 103, 33 x 103, 35 x 103, and 67 x 103 in eggs, larvae (day 4), adults (day 1), adults (day 5), adults (day 8), and adults (day 12), respectively. statistical analysis showed that there was a significant difference between eggs and the adults at day 8 or 12 and between larvae day 4 to adults day 12. it is assumed that some of cells in the adult fly at day 12 are apoptotic and therefore, slightly higher. cell division mainly occurred between immature stages and practically will not occur in adults other than in the reproductive tissue. the number of nuclei in eggs was 8 x 103 whilst the number of wolbachia in the egg was 20 x 103 and therefore, it is assumed that a cell contains at least 2-3 wolbachia bacteria cells. in larvae on day 4, the number of nuclei was 12 x 103, whilst the number of wolbachia was 52 x 103 and therefore, it can be said that a cell contains at least 45 wolbachia bacteria. furthermore, the number of wolbachia in a cell of adults at day 1, 5, 8, and 12 were 422, 535, 964, and 610, respectively. the differences observed in adult flies were statistically insignificant. the significant increase in the density of wolbachia from eggs to larvae and adult flies may be attributed to the increase in their total volume per cell and consequently in the population of the wolbachia population. the quantity/density of wolbachia causing cytoplasmic incompatibility (ci) in d. simulans was detected using competitive/quantitative pcr by sinkins et al. (1995). comparisons were initially made between three strains of d. simulans: dsr (d. simulans riverside), dsch (d. simulans coffs harbour), dsh (d. simulans hawaii), and between three strain of aedes albopictus (houston, koh samui, and mauritius). the result showed that wolbachia density in dsr was the highest: this strain shows the strongest expression of ci. wolbachia density in dsch was an intermediate between that of dsr and dsh (the relative densities being 100, 60, and 20, respectively). in this method, the number of wolbachia bacteria could not be measured absolutely, and therefore the exact number of wolbachia was unknown. recently, mcgraw et al. (2002) reported that the density of wolbachia in ovaries of d. simulans with riverside wolbachia and d. melanogaster popcorn-effect wolbachia was low and increased very little during the lifespan of the flies (7.37 and 2.7 copies per cell). in contrast, wolbachia density in ovaries of d. simulans with popcorn-effect wolbachia (resulted from artificial transfer of d. melanogaster popcorn-effect to d. simulans) rose rapidly (24.2 copies per cell). as a result, the number of wolbachia in d. melanogaster causing the popcorn-effect was higher than the number of wolbachia causing ci in l. triatellus or in s. furcifera (noda et al. 2001). this is reasonable since wolbachia causing ci did not destroy the cells. however, wolbachia in d. melanogaster cause the popcorn-effect in which cells rupture as a result of massive multiplication of bacteria within them. mouton et al. (2007) studied the interaction between three strains of wolbachia in two divergent homozygous lines of the wasp leptopilina heterotoma at two different temperatures, and showed that wolbachia density varied between the two host genotypes at only one temperature, and the host genotype played an important role in wolbachia density. they also highlighted its interaction with environmental conditions, making the evolution of local adaptations for the regulation of wolbachia density possible. acknowledgement this research was funded by the due project of the university of jenderal soedirman, purwokerto, indonesia. i would like to thank henk r. braig, school of biological sciences, university of wales, bangor, united kingdom for his guidance, advice, help, and for providing facilities. references bensaadi-merchermek n, salvado jc, cagnon c, karama s, mouches c. 1995. characterization of the unlinked 16s rdna and 23s-5s rrna operon of wolbachia pipientis, a prokaryotic parasite of insect gonads. gene 165:81-86. boyle l, o’neill sl, robertson hm, karr tm. 1993. interspecific and intraspecific horizontal transfer of wolbachia in drosophila. science 260:1796-1799. braig hr, zhou w, dobson sl, o’neill sl. 1998. cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont wolbachia pipientis. j bacteriol 180:2373-2378. dutton tj, sinkins sp. 2004. strain-specific quantification of wolbachia density in aedes albopictus and effects of larval rearing conditions. insect mol biol 13:317-322 hertig m. 1936. the rickettsia, wolbachia pipientis, and associated inclusions of the mosquito culex pipiens. parasitology 28:453490. hertig m, wolbach sb. 1924. studies on rickettsia-like micro-organisms in insects. j med res 44:329-374. kondo n, shimada m, fukatsu t. 2005. infection density of wolbachia endosymbiont affected by co-infection and host genotype. biol lett 22:488–491. lebovitz rm, takeyasu k, fambrough dm. 1989. sequences on genbank for drosophila melanogaster, leps, artemia, and fleas. embo 8:193-202. mcgraw ea, merritt dj, droller jn, o’neill sl. 2002. wolbachia density and virulence attenuation after transfer into a novel host. proc nat acad sci usa 99:2918-2923. min kt, benzer s. 1997. wolbachia, normally a symbiont of drosophila, can be virulent, causing degeneration and early death. proc nat acad sci usa 94:10792-10796. mouton l, henri h, charif d, boulétreau m, vavre f. 2007. interaction between host genotype and environmental conditions affects bacterial density in wolbachia symbiosis. biol lett 3:210-213. noda h, koizumi y, zhang g, deng k. 2001. infection density of wolbachia and incompatibility level in two planthopper species, laodelphax striatellus, and sogatella furcifera. insect biochem mol biol 31:727-737. sinkins sp, braig hr, o’neill sl. 1995. wolbachia pipientis: bacterial density and unidirectional cytoplasmic incompatibility between infected populations of aedes albopictus. exp parasitol 81:284291. werren jh. 1997. wolbachia run amok. proc nat acad sci usa 94:154-155. werren jh, jaenike j. 1995. wolbachia and cytoplasmic incompatibility in mycophagous drosophila and their relatives. heredity 75:320-326. 94 kusmintarsih microbiol indones 01 cahyani.cdr vol.11, no.1, march 2017, p 1-10 doi: 10.5454/mi.11.1.1 cloning of synthetic lipase gene from rhizomucor miehei with original signal peptide in pichia pastoris 1 2 2 1 martha eka cahyani , is helianti *, niknik nurhayati , and abinawanto 1 department of biology, faculty of mathematics and natural science, universitas indonesia, depok 16424, indonesia; 2 center of bioindustrial technology, agency for assessment and application of technology (bppt), building 611, laptiab-bppt, puspiptek area, tangerang selatan 15314, indonesia lipases (ec 3.1.1.3) are classified as hydrolases that hydrolyze lipids. these enzymes have potential application in biotechnology and industrial process. in previous study we have cloned the synthetic rhizomucor miehei lipase gene using the vector puc57 in escherichia coli dh5α, but only found the very low enzymes activity. this study aimed to clone synthetic rhizomucor miehei lipase gene into pichia pastoris expression plasmid for lipase expression with the original signal peptide. a dna fragment with the original signal peptide had been obtained by pcr, cut by xho i and xba i and then ligated into ppiczα a linearized with the same enzymes. the mixture of ligation reaction, then was transformed into escherichia coli dh5α. zeocin-resistant transformants were selected and contained plasmid was analyzed by restriction enzymes analyses and dna sequencing. as the result, a synthetic rhizomucor miehei lipase gene (rmlip) with the size of 1132 bp was successfully cloned to ppiczα a vector plasmid. the recombinant plasmid with the correct dna sequence was transformed into pichia pastoris x33. cultivation of recombinant p. pastoris was carried out with the addition of 1.5% methanol every day with appropriate aeration. the recombinant lipase produced by pichia pastoris x33 o containing rmlip in its chromosomal dna had optimal temperature and ph, 30 c and 9.0, respectively. key words: pichia pastoris, ppiczα a, rhizomucor miehei, synthetic lipase gene lipase (ec 3.1.1.3) diklasifikasikan sebagai enzim hidrolase yang menghidrolisis lipid. enzim ini memiliki potensi dalam berbagai aplikasi di bidang bioteknologi dan industri. dalam penelitian sebelumnya, kami telah mengklon gen lipase rhizomucor miehei sintetik menggunakan vektor puc57 dalam escherichia coli dh5α, dan mendapatkan aktivitas enzim yang sangat rendah. penelitian ini bertujuan untuk mengklon gen lipase rhizomucor miehei sintetik ke dalam plasmid ekspresi pichia pastoris untuk mengekspresikan lipase dengan menggunakan peptida sinyal asli. gen lipase sisipan diperoleh dengan pcr, dipotong dengan xho i dan xba i, kemudian diligasi ke dalam ppiczα a yang telah dilinearisasi dengan enzim yang sama. hasil ligasi kemudian ditransformasi ke dalam escherichia coli dh5α. transforman yang tahan terhadap zeocin dipilih dan kandungan plasmidnya dianalisis dengan enzim restriksi dan pengurutan dna. hasilnya diperoleh gen lipase rhizomucor miehei (rmlip)berukuran 1132 pb. plasmid rekombinan yang tepat kemudian ditransformasikan ke pichia pastoris x33. kultivasi dilakukan dengan penambahan 1.5% metanol setiap hari dengan aerasi yang sesuai. lipase rekombinan yang diproduksi oleh pichia pastoris x33 yang mengandung rmlip di dalam dna o kromosomnya mempunyai temperatur dan ph optimal masing-masing 30 c dan 9. kata kunci: gen lipase sintetik, pichia pastoris, ppiczα a, rhizomucor miehei microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-7560536 ext 7119, fax:+62-21-7566922; email: is.helianti@bppt.go.id vandenberghe 2003). lipase used commercially mainly come from microorganisms. microbial lipases chosen because it has a more stable properties such as selectivity and broad substrate specificity (dutra et al. 2008; griebeler et al. 2011). one of the microorganisms produces lipase is rhizomucor miehei (sarma et al. 2001). r. miehei is a mold that has lipase with high activity and good stability under diverse conditions (rodrigues and lafuente 2010). lipase of r. miehei has been used in in ester hydrolysis, ester synthesis, and transesterification reaction (huang et al. 2012). these lipase was able to successfully catalyze the synthesis of ethyl caproate in organic solvent displayed on the surface pichia pastoris (han et al. 2009), and also effective for the lipase (ec 3.1.1.3) is classified as hydrolase that hydrolyzes lipids. these enzymes are biocatalysts that can act on solid and liquid media (brunel et al. 2004). this character makes lipase potential in a variety of applications in biotechnology and industry, including pharmaceutical, pesticide, biosensors, biodiesel, detergents, and various food products (vakhlu and kour 2006). several techniques have been developed to obtain higher conversions, as highly specific enzymes for each application, improving the possibility of industrial applications for these biocatalysts (franken et al. 2008; soccol and transesterification of vegetable oils and other raw materials containing higher fatty acid alkyl ester into derivative (huang et al. 2014). since then, industry has continually searched for new components to improve the benefit of this development along with its costefficiency. center of bioindustrial technology, laptiab, bppt has conducted cloning of synthetic rhizomucor miehei lipase gene using the vector puc57 in escherichia coli dh5α. although the cloning process succeeded, but the enzyme activity obtained is very low. in this study we chose pichia pastoris as a host to replace e. coli. p. pastoris is a methylotrophic yeast that has been considered as an excellent host for the production of heterologous proteins (sreekrishna et al. 1997). p. pastoris has distinguished characteristics, yielding stable transformants of integration of foreign dna into chromosomal dna, have a high-density growth, have the ability to secrete high levels of foreign proteins (adrio and demain 2003), expressing heterologous protein can either be intracellular and extracellular (balamurugan et al. 2007). in addition, they have the simplicity of techniques needed for the molecular genetic manipulation and the ability to modify the eukaryotic protein after translation, such as glycosylation, disulfide bond formation and proteolytic processes (cereghino and cregg 2000). expression of any foreign gene in p. pastoris requires three basic steps, which is the insertion of the gene into an expression vector, introduction of the expression vectors into the genome of p. pastoris, and examination of potential expression strains for the foreign gene product (cereghino et al. 2000). therefore, this study aimed to clone r. miehei synthetic lipase gene in p. pastoris expression plasmid for lipase expression with the original signal peptide. materials and methods strains, plasmids, and media. e. coli dh5α (invitrogen) was used as the host for the recombinant plasmid cloning and propagation. p. pastoris x33 was used as the host for lipase expression. plasmid puc57 containing the dna of synthetic r. miehei lipase (rmlip) has been described elsewhere (hanniya 2015). the vector ppiczα a was supplied from o invitrogen. e. coli dh5α was grown at 37 c in luria bertani (lb) low salt agar medium containing 25 μg -1 ml zeocin for selection of clones transformed with the recombinant vector plasmid. p. pastoris x33 was o routinely grown in shaking flasks at 30 c, in a rich standard medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 100 mm potassium phosphate buffer, ph 6.0, 1.34% (w/v) yeast nitrogen base, 4 x 10 5 % (w/v) biotin, 1% (v/v) glycerol (buffered glycerolcomplex (bmgy)) before induction, or 1.5% (v/v) methanol (buffered methanol-complex (bmmy)) for induction. for maintaining cultures and plates, yeast extract peptone dextrose (ypd) medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose) -1 containing 50 μg ml zeocin was used, and for selection of transformants, yeast extract peptone dextrose sorbitol (ypds) medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, 1 m -1 sorbitol, 2% (w/v) agar) containing 50 μg ml zeocin and tributyrin agar (tba) medium (1% (w/v) yeast extract, 2% (w/v) special peptone, 2% (w/v) tributyrin, 2% (w/v) agar, 2% (w/v) dextrose) containing 50 μg -1 ml zeocin was used. construction of the vector ppiczα a-rmlip. construction of the vector ppiczα a-rmlip including cloning, transformation into e. coli and selection, were conducted as described by sambrook and russel (2001). the e. coli expression vector puc57 containing the synthetic rmlip gene served as a template for polymerase chain reaction (pcr). pcr w a s p e r f o r m e d u s i n g f o r w a r d p r i m e r ( 5 ' gcatcctcgagaaaagagaggctgaagctat ggtgctgaaacagcgcgc-3') and reverse primer (5'-gcatctctagagcggtgcacaggccggtg ttaatg-3'). the forward primer containing an xho isite (underlined) and the reverse primer containing an xba i-site (underlined) and the sequence that will detect 6xhis. the primers were ordered from integrated dna technologies (singapore). the pcr process is carried out by 30 cycles with predenaturation, denaturation, o o annealing, extantion, endextantion at 98 c, 30 s; 98 c, o o o 10 s; 69 c, 30 s; 72 c, 30 s; 72 c, 5 min, respectively. the obtained insert gene was digested with xho i and xba i. the restriction enzymes were purchased from fermentas (burlington, canada) and thermo scientific (waltham, usa), respectively. it was then ligated into ppiczα a linearized with the same enzymes. the mixture of ligation reaction, then was transformed into e. coli dh5α. zeocin-resistant transformants which grown on lb low salt media were selected and analyzed by plasmid digestion with bgl ii, xho i, and xba i. transformants showed the correct digestion pattern were sequenced to confirm the sequence. transformation into p. pastoris. plasmid with the correct dna sequence, were used for transformation into 2 cahyani et al. microbiol indones volume 11, 2017 microbiol indones 3 p. pastoris x33. the yeast was transformed with 1 μg of sac i-linearized ppiczα a-rmlip by the spheroplast method of cregg et al. (1985) with the following modifications. a fresh colony of p. pastoris x33 was inoculated into 10 ml of ypd and grown with shaking at o 250 rpm 30 c overnight. on the following day, 5 ml sample of the overnite culture was added to 45 ml of fresh ypd as starter od of 0.2 and grown cells under 600 the previous conditions until od reaches 1.4 to 1.6. the 600 cells were harvested by centrifugation at 6000 x g for 5 min and washed twice with 10 ml of ice-cold miliq h o 2 and then suspended in 8 ml of freshly prepared t buffer (10 mm tris-hcl ph 7.5, 0.1 m lithium acetate, 10 mm dtt, 0.6 m sorbitol) and incubated at 30 °c for 30 min. the spheroplasts were washed twice in 10 ml of ice-cold 1 m sorbitol and suspended in 400 μl of ice-cold 1 m sorbitol. one hundred μl of the spheroplasts were mixed with 1 μg of sac i-linearized ppiczα a-rmlip. then, the transformation performed by electroporation, which is applied electric pulse to the mixture (1.5 kv, 25 μf, 200 ω for gene pulser, bio-rad, usa) the mixture was added o by 1 ml of ice-cold 1 m sorbitol and incubated at 30 c without shaking for 1 h. the mixture then was added by o 1 ml of ypd and incubated at 30 c with shaking at 250 rpm for 1 h. one hundred μl of the mixture were -1 spreaded on tba medium plates containing 50 μg ml o zeocin and incubated at 30 c. transformants were o selected after 2-3 days at 30 c by plating cells on tba medium. lipase activity was detected by the appearance of clear zones around colonies. zeocin-resistant transformants which showed a clear zone were selected and analyzed by colony pcr and continued to pcr product digestion. cultivation. standard cultivation method was carried out according to method described in hu et al. (2013) and wang et al. (2016). a single positive colony o was cultivated in 100 ml of bmgy medium at 30 c overnite under constant agitation at 250 rpm, until an od = 2-5 was reached. then, these culture was 600 transfered into 900 ml of the bmgy medium for largescale cultivation until an od reached 5. the cells 600 were collected by centrifugation at 3800 rpm for 20 min, and resuspended in 200 ml of the bmmy medium. cultures were induced with absolute methanol to a final concentration of 1.5% every 24 h. the expression culture supernatants was harvested o every 24 h and stored at -4 c before analysis. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). the expression of lipase gene in the supernatant was analyzed by sdspage, which was conducted using a 12% polyacrylamide gel on a vertical mini gel apparatus (40 a, 200 v, 45 min, bio-rad, usa), as described by laemmli (1970). protein molecular weight marker was purchased from amersham (ge healthcare, uk). samples were mixed with 2× loading buffer, with a ratio 1:1. then, the mixture was boiled at 100 °c for 5 min before electrophoresis. proteins were stained with page blue staining solution (thermo scientific, waltham, usa). protein staining is done by polyacrylamide gel washing with miliq h o three 2 times, then incubated with page blue for 30 s in the o microwave and continued incubation at 24 c, 300 rpm, for 1 h. furthermore, it was rinsed and soaked by miliq h o overnight.2 characterization of recombinant lipase. the lipase properties of supernatants were determined by the alkali titration method, using olive oil as substrate, as described by fang et al. (2014) with the following modifications. the reaction was conducted in a mixture of 4 ml of 50 mm tris–hcl (ph 8.0), 5 ml of emulsion of olive oil (25% (v/v) olive oil emulsified with 1.5% (w/v) polyvinyl alcohol solution) and 1 ml of proper o dilute enzyme solution at incubation 50 c for 20 min in a shaker. finally, 5 ml of methanol was added to terminate the reaction. the amount of liberated fatty acids was measured by titration with 50 mm naoh using phenolphthalein as an indicator. one unit (u) of lipase activity was defined as the amount of lipase necessary to liberate 1 μmol per min of fatty acids from the olive oil. the optimal tempetarure was determined by examining the activity of the enzymes at tris-hcl buffer ph 8.0 in the various temperatures incubation o (30-80 c). the optimal ph was determined by measuring the enzymatic activity at the optimal temperature at ph range 5.0-10.0 (ph 5.0; ph 6.3-8.0; ph 8.0-9.0; ph 9.4-10.0 using na-citrate buffer 0.05 mm; na-phosphate 0.05 mm; tris-hcl 0.05 mm; glycine-naoh 0.05 mm, respectively). each sample was assayed in duplicate and the average value was determined. results construction of the recombinant plasmid ppiczα a-rmlip. a fragment dna encoding synthetic r. miehei lipase gene (rmlip) with the total size 1132 bp has been obtained by pcr. the vector ppiczα a is a p. pastoris expression vector. these vector size of 3593 bp. in order to the vector and rmlip can be ligated, the vector was also restricted at xho i and xba i sites. the total size ppiczα a-rmlip after fig 1 e. coli transformants verification. (a) analysis of transformants by plasmid digestion with bgl ii. (b) analysis of transformants by plasmid digestion with xho i. line 2: negative control. lines 3-8: transformants analyzed with enzime restriction, the sizes of 2992 bp and 1629 bp or 4629 bp, respectively. (c) analysis of transformants by plasmid digestion with xba i. lines 2-7: transformant analyzed with xba i, the sizes of 4629 bp. line 2: negative control. each transformant was screened in duplicate. ligated is 4621 bp. this ligated vector and inserted fragment was then transformed into e.coli. after transformation, there were four transformants grown on lb low salt agar medium. of the four transformants, only three transformants can be grown in lb low salt broth medium. three transformants cultures were verified by bgl ii, xho i, and xba i. xho i and xba i are the single cutting site located between the vector ppiczα a and rmlip, so that the dna size obtained by cutting was 4621 bp (fig 1a and 1b). while, bgl ii is the double cutting sites located at the vector ppiczα a and rmlip, so that the result was obtained by cutting the size of 2992 bp and 1629 bp (fig 1a). based on the verification by bgl ii, xho i, and xba i, showed that the three transformants are three positive colonies that carrying the vector ppiczα a-rmlip with a total size of 4621 bp. sequencing. sequence analysis was conducted to confirm the accuracy of the target gene sequence, the correct fusion of lipase gene to the vector, and the rate of mutation that occurred during the selection process. from dna sequencing result we found that the dna was in frame with alpha factor. the sequences were processed using online software www.genome.jp/ tools/clustalw/, later translated using software http://web.expasy.org/translate/ into the protein sequence of samples. based on the results of the sequencing, there are primers used (grey highlight), which is used as a marker of the original signal peptide, a marker of restriction enzymes xho i and xba i, as well as the marker his codon before the end translation process (fig 2a). sequence of synthetic r. miehei lipase is between the both primers. based on the protein sequence, there are amino acid of the original signal a b c 4 cahyani et al. microbiol indones volume 11, 2017 microbiol indones 5 peptide, amino acid of the propeptides, and amino acid of the mature enzyme, and obtained also size lipase protein produced is 32.9 kda (fig 2b). the results of the translation are then alignment with international protein sequence databases available on the website http://ncbi.nlm.nih.gov by selecting protein blast menu. alignment is performed to find the sample sequence similarity to sequences contained in the databases. protein blast results showed that the protein sequence ppiczαa-rmlip transformed into e. coli dh5α have similarities that are 100% identical to the protein lipase from rhizomucor miehei in international database (data not shown). transformation into p. pastoris x33 and selection of positive transformants. after transformation process of the correct sequence recombinant plasmid into p. pastoris, the recombinant colonies were grown. transformants expressing lipase activity, as indicated by the formation of a clear zone around colonies on tba medium. several transformants were picked for confirmation by colony pcr and continued plasmid digestion with bgl ii. the total size of rmlip after the colony pcr was 1132 bp and the total size of rmlip after the restriction by bgl ii was 680 bp and 452 bp. based on the confirmation by the colony pcr and bgl ii, showed that the transformants are positive colonies that carry the vector ppiczα a-rmlip (fig 3). sds-page electrophoresis. sds-page analysis showed that after induction by methanol, there was a band that appeared after induction with the measured molecular weight 32.9 kda (fig 4). characterization of lipase properties. temperature and ph are two important reaction parameters for enzymes. each enzyme has a distinct optimal temperature and ph levels. as well as recombinant lipase produced from recombinant p. pastoris x33 containing rmlip. the optimal reaction temperature and ph of this recombinant lipase o were 30 c and 9.0, respectively. discussion lipases has been successfully applied in various applications, such as in the food industry, organic synthesis, pharmaceutical (houde et al. 2004; diego et al. 2009) and in the field of biodiesel (huang et al. 2012; huang et al. 2014). the success is challenging us to keep looking for a better lipase expression with the aid of genetic engineering. this study aimed to clone r. miehei synthetic lipase gene into p. pastoris expression plasmid for lipase expression with the original signal peptide. in this study r. miehei synthetic lipase gene with signal peptide has been cloned in p. pastoris expression vector and transformed into p. pastoris x33. based on the analysis sequensing, rmlip has 363 amino acid residues, which consists of a 24 amino acid residues signal peptide, a 80 amino acids residues propeptida, and 269 amino acid residues of the mature enzyme. this is supported by boel et al. (1988), which said that, rmlip is synthesized as a precursor, which contained 70 amino acid residues propeptida before the 269 amino acid residues mature enzyme. differences in the number of amino acids propeptides affected by signal peptides are used. that is because propeptides are c-terminal domain of signal peptides containing site of the cutting, which will be cleaved off in the protein maturation steps (emanuelsson et al. 2007; wang et al. 2016). signal peptides and propeptides is a very important sequence in protein synthesis, because the functional region of the protein cannot form the native structure without the propeptide region (anderson et al. 1999). in addition, the protein molecular weight of lipase in ppiczα a-rmlip is 32.9 kda. this is reinforced by the statement wu et al. (1996) which says that the molecular weight of rmlip is 31.6 kda and the statement huge-jensen et al. (1989) states that, the molecular weight of rmlip is 32 kda. so it is suspected that the molecular weight of lipase rmlip is in the same range. temperature and ph is a very important parameter to determine the properties of the lipase expressed (wang et al. 2015). in this study, rmlip express lipase -1 with high levels at 30 °c, ie at 8.33 u ml ± 0.50. this is supported by the statement huang et al. (2014), having said that, the temperature tolerance for rmlip well expressed in p. pastoris x33 is at a temperature of o 0-40 c. for optimum ph, rmlip express lipase with high levels at ph 9.0 in tris-hcl buffer, ie at 8.44 u -1 ml ± 0.07. this is supported also by the statement huang et al. (2014) which says that, ph tolerant for rmlip in p. pastoris x.33 ie at ph 4.0—10.0. he et al. (2015) has been conducting research to increase the activity of lipase rmlip by combining the aox1 and gap promoter whose expression using the α factor signal peptide of the saccharomyces cerevisiae in p. pastoris gs115, lipase activity is generated is 140 u -1 ml . the result is higher of the control that only used -1 the aox1 and gap promoter, which is 25 u ml and -1 20 u ml , respectively. when compared with the control and the results of the such research, the lipase fig 2 analysis sequencing. (a) in the dna sequence, there are primers used (grey highlight), which is used as a marker of the original signal peptide, a marker of restriction enzymes xho i and xba i, as well as the marker his codon before the end translation process, sequence of synthetic rhizomucor miehei lipase is between the both primers. (b) in the protein sequence, there are amino acid of the original signal peptide, amino acid of the propeptides, and amino acid of the mature enzyme. these components are very important in protein expression. the protein sequence analysis by software http://web.expasy.org/ translate/ showed that the size of ppiczαa-rmlip lipase protein molecular weight is 32.9 kda. theoretical pi/mw: 5.03 / 32917.74 a b region of rhizomicor miehei lipase part of the a factor of ppiczaa region of signal peptide and propeptides 6 cahyani et al. microbiol indones activity generated in this study has not increased significantly. it may caused by several factors, the most striking is the incorporation of the promoter and signal peptide used. the combined use of the aox1 and gap promoters can be attributed to a positive effect on the expression level by increasing the copy number of the expression cassette. the transcription levels of the intracellular protein and secreted protein have been shown to increase greatly upon integration of multiple copies of the expression vector to an appropriate extent in p. pastoris (scorer et al. 1994; shen et al. 2012). signal peptides also affect the expression of recombinant lipase. there are three kinds of signal peptides that can be used directly in pichia pastoris to secrete and express a foreign gene that is the native signal peptides, the s. cerevisiae α factor prepropeptides (α factor signal peptide) and the p. pastoris acid phosphatase (pho1) signal. the original/native signal peptide is rarely used because there are still some weaknesses, one of which is described in the research brocca et al. (1998) regarding the effect of the original signal peptides on the expression and secretion of industrial lipase lip1 from candida rugosa. brocca et al. (1998) stated that the a b fig 3 pichia pastoris transformants confirmation. (a) rmlip confirmation result of transformation by colony pcr. line 1: generuler dna ladder 1 kb. line 2: negative control of pcr that used ddh o. line 3: 2 positive control of pcr that used extraction rmlip. line 4-8: rmlip result of transformation, the total size was 1132 bp. almost all the positive colonies, from five colonies, only 1 negative colony. (b) rmlip confirmation result of colony pcr by bgl ii. line 1: generuler dna ladder 1 kb. line 2: negative control of pcr that used ddh o. line 3: positive control of pcr that used extraction rmlip. line 4—7: 2 rmlip result of transformation, the total size was 680 bp and 452 bp. restriction results from four colonies, are all positive. fig 4 sds-page analysis of culture supernatans during the cultivation. the molecular weight of protein rmlip is 32.9 kda. line m: protein molecular weight marker. line 1-6: culture supernatant after 0, 24, 48, 72, 96, 120, and 144 h of cultivation. volume 11, 2017 microbiol indones 7 original signal peptide can secrete lipase lip 1, but its expression was hampered. there is an assumption that, in this study also experienced the same thing, signal peptide rmlip successfully constructed, but the expression of lipase inhibited. cereghino and cregg (2000) said that, although the original signal peptide and α factor prepropeptida (α factor signal peptide) from s. cerevisiae was adequate for foreign protein secretion and expression, but the highest levels of protein secretion was from clone with the full preprotein. this study obtained that synthetic rhizomucor miehei lipase gene with the size of 1132 bp was successfully cloned to ppiczα a vector with a total size of 4621 bp. dna sequencing analyses showed that the synthetic lipase gene ppiczα a-rmlip are 100% identical to the r. miehei lipase. the gene ppiczα armlip was successfully integrated into chromosome of p. pastoris x33. the enzymatic properties of ppiczαa-rmlip in p. pastoris x33 is to have the o optimal reaction temperature is 30 c and the optimal reaction ph is 9.0. acknowledgment this study was funded by national insentive for consortium research (insinas) 2015—2016 from ministry of research, technology, and higher education, republic indonesia. references adrio jl, demain al. 2003. fungal biotechnology. int. microbiol. 6: 191-199. doi: 10.1007/s10123-003-0133-0. anderson de, peters rj, wilk b, agard da. 1999. alphalytic protease precursor: characterization of a structured folding intermediate. biochemistry. 38: 4728-4735. doi: 10.1021/bi982165e. balamurugan v, reddy gr, suryanarayana vvs. 2007. pichia pastoris: a notable heterologous expression system for the production of foreign proteins-vaccines. ijbt. 6: 175-186. brunel l, neugnot v, landucci l, boze h, moulin g, bigey f, dubreucq e. 2004. high-level expression of candida parapsilosis lipase/acyltransferase in pichia pastoris. j biotechnol. 111: 41-50. boel e, hugejensen b, christensen m, thim l, fiil np. 1988. rhizomucor miehei triglyceride lipase is synthesized as a precursor. lipids. 23: 701-706. doi: 10.1007/bf02535672. brocca s, schmidt-dannert c, lotti m, alberghinaand l, schmid rd. 1998. design, total synthesis, and functional overexpression of the candida rugosa lip1 gene coding for a major industrial lipase. protein sci. 7: 1415-1422. cereghino jl, cregg jm. 2000. heterologous protein expression in the methylotrophic yeast pichia pastoris. fems microbiol. rev. 24: 45-66. cregg jm, barringer kj, hessler an, madden kr. 1985. pichia pastoris as a host system for transformations. fig 5 characterization of properties lipase. 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10.2225/vol9-issue1fulltext-9. wang z, pengmei lv, luo w, yuan z, he d. 2016. expression in pichia pastoris and characterization of rhizomucor miehei lipase containing a new propeptide region. j gen appl microbiol. 62: 25-30. doi: 10.2323/jgam.62.25. wu s, letchworth gj. 2004. high efficiency transformation by electroporation of pichia pastoris pretreated with lithium acetate and dithiothreitol. biotechniques. 36: 152-154. wu xy, jääskeläinen s., linko wy. 1996. purification and partial characterization of rhizomucor miehei lipase for ester synthesis. appl biochem biotechnol. 59:145-158. 10 cahyani et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 7 ari susilowati.cdr page 1 page 2 page 3 page 4 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 9, number 2, june 2015 expression and purification of phor sensor-domain histidine kinase of mycobacterium tuberculosis in escherichia coli genetic profiles of escherichia coli isolated from indonesian tempeh based on enterobacterial repetitive intergenic consensus-polymerase chain reaction (ericpcr) medium optimization for penicillin acylase (pac) production by recombinant containing pac gene from using response surface methodology bacillus megaterium ms941 bacillus thuringiensis bgsc bd1 inhibitory activity of lactobacillus plantarum u10 isolated from tempoyak (fermented durian) made in indonesia against salmonella typhi chemical constituens of an endophytic fungus aspergillus sp (sbd5) isolated from sambiloto (andrographis paniculata nees) cloning and expression of ha1 gene of h1n1 influenza virus 2009 pandemic (h1n1pdm09) indonesia strain in the pichia pastoris expression system for the development of influenza vaccine ernawati arifin giri-rachman, fenryco pratama, oktira roka aji, arum patriati, ihsanawati, edy giri rachman putra, and maelita ramdani moeis qurrota a'yun, antonius suwanto, and tati barus fentri paramitha putri, astutiati nurhasanah, niknik nurhayati, 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rukmi, m.kes.; jimmy hariantono, ph.d; ir. dwi kusuma indriani, mp; sugeng p sugiharto, m.sc.; rosdyana salim, a.md. kartini kramadibrata, 2016 koesnandar, 2016 maggy thenawijaya suhartono, 2016 maria inge lusida, 2016 neung tiaamroeng, 2016 norio kurosawa, 2016 raija laiho, 2016 ratih dewanti, 20 61 wellyzar sjamsuridzal, 2016 yuan kun lee, 2016 361-1025-1-pb (1) 2 diana_358 (8-10) isolation and identification of ice-nucleating-active bacteria from indonesian edible leafy plant poh-pohan (pilea glaberina) diana elizabeth waturangi1*, vicky meicy1, and antonius suwanto2 1faculty of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia, 2department of biology, faculty of science and mathematics, institut pertanian bogor, darmaga campus, bogor 16680, indonesia two ice-nucleating-active (ina) bacteria (isolates c and 6) were isolated from poh-pohan (pilea glaberina), an indonesian edible leafy plant (lalaban). the maximum nucleation temperature of aqueous suspensions of the two isolates is -5 °c. they were classified as a type ii ice nucleator. microscopic and morphological determination showed that these isolates had yellow pigmentation, rod shape, and were gram negative. biochemical analysis indicated that the isolates were exhibited catalase activity, but negative in oxidase and indole assays. dna sequencing of 16srrna gene of isolate a3 showed a 94% similarity to pseudomonas sp. while isolate a4 showed a 97% similarity to xanthomonas campestris. to our knowledge, this is the first report of ina bacteria isolated from a tropical edible leafy plant. keywords: ice-nucleating bacteria, indonesian edible leafy plant, pilea glaberina _____________________________________________ ________________________ *corresponding author, phone: +62-21-5703306 ext. 335, fax: +62-21-5719060, e-mail: diana.waturangi@atmajaya.ac.id leaf microbial communities of leaves are diverse and include many different genera of bacteria, filamentous fungi, yeasts, algae, and less frequently, protozoa and nematodes (andrews and harris 2000) bacteria are by far the most abundant inhabitants of the phyllosphere and ephiphytic bacterial populations differ sharply in size among and within plants of the same species (lindow et al. 1978). one of the bacterial components of leaf microbial communities are icenucleation-active (ina) bacteria (lindow and brandl 2003). these bacteria have been shown to incite frost damage to some plants (hirano and upper 2000). the bacteria commonly found on plants comprise strains of several species (such as pseudomonas syringae and erwinia herbicola) which produce a protein able to induce ice nuclei at temperatures as high as -2°c. leaf surface populations of these bacterial species limit supercooling in the plant parts on which they reside by initiating damaging ice formation at temperatures of -2 to -4°c. since plants do not have intrinsic ice nuclei active at these temperatures, these bacteria play an important role in initiating frost damage (lindow et al. 1978). while data of the presence of ina bacteria on the leaf surface are commonly found from subtropical areas, little is known about the presence of these bacteria in tropical areas. riupassa et al. (2005) investigated the abundance of phyllosphere bacteria in a number of indonesian edible leafy plants (lalaban) which could reach up to 3 x 108 cfu/g. in this study we are interested to focus on the isolation and identification of ice-nucleating active bacteria from an indonesian edible leafy plant poh-pohan (pilea glaberina). materials and methods sampling technique. isolations of ina bacteria were done from many edible leafy plants such as poh-pohan (p. glaberina), selada (lactuca sativa), kemangi (ocinum sanctum), and taoge kacang hijau (vigna radiata). samples were placed in sterile plastic bags and plated immediately upon return to the laboratory. samples of 2 grams of leaves were cut into pieces and washed with sterile distilled water 3x. samples were placed in sterile tubes containing 10 mm phosphate buffer (ph 8.0), vortexed for 2 minutes and then diluted to 100 fold. the suspensions were spread on king’s b medium agar and incubated at room temperature (28-30°c) for 2 days. detection of ina colonies by replica freezing assay. colony growth from the isolations were picked and prepared for ice-nucleation assay by the method of lindow et al. (1978). escherichia coli dh5α carrying pjl1703 was used as a positive control. ice nucleation activity was assessed with the drop-freezing assay. frequencies of the ice nucleation activity of the isolates were measured using the formula described by vali (1971). n(t) = (ln f)/v, where n (t) is the nucleation frequency at temperature t, f is the proportion of droplets unfrozen and v is the volume of individual droplets. biochemical tests. biochemical properties of these isolates were determined for indole, catalase, and oxidase assays. pcr amplification, cloning, and sequencing of 16s rdna. genomic dna was extracted and purified using the wizard genomic dna purification kit (promega, usa). the extracted dna was used as a template for pcr amplification of the 16s rrna gene using 63f (5’caggcctaacaca tgcaagtc-3’) primer and 1387r primer (5’-gggcggwgtg tacaaggc-3’) (marchesi et al. 1998). the reactions were performed in a volume of 25 µl containing 1 µl of dna template, 1 µl of each primer (25 pmol/µl) (research biolabs), 0.5 µl of dntp mix (reseearch biolabs) and 0.5 µl (2.5 u) of taq polymerase, 2.5 µl of 10 x buffer (new england biolabs, mass. usa). the total volume was adjusted to 25 µl with sterile doubledistilled water. the following cycles were applied for pcr: 94°c for 5 minutes then 25 cycles: 94°c for 30 seconds denaturation, 55°c for 30 seconds annealing and 72°c for 1 minutes elongation. after the amplification, an 8 μl of amplified dna was mixed with 3 µl of loading buffer and then loaded onto 1% (w/v) gel electrophoresis (mini-sub issn 1978-3477 volume 2, number 1, april 2008 p8-10 cell gt; biorad, richmond, ca) which was run for 60 minutes at 55 v prior to visualization using a uv transiluminator. the resulting amplicons were purified using a qiaquick pcr purification kit (qiagen, germany) and then cloned using the pgmt easy vector system i (promega, usa) and transformed into e. coli d h 5 á . e. coli transformants were selected on lb agar supplemented with x-gal (40 µg/ml). recombinant plasmids were isolated using plasmid wizard plus sv minipreps dna purification system (promega, usa). the cloned amplicons were sequenced (abi prism 3130x genetic analyser) on both strands using m13 reverse and forward primers. the sequences were compared to genbank sequences by using a standard nucleotide basic local alignment search tool (blast) search. results we recovered two isolates of bacteria (isolate a3 and a4) which showed ice-nucleation activity from poh-pohan (p. glaberina) (fig 1). the ice-nucleation-activity of isolate a4 is higher than isolate a3 with an ice-nucleation frequencies of 59.9/ml and 37.9/ml respectively (table 1). microscopic and morphological determination showed that these isolates had yellow pigmentation, rod shape and were gram negative. biochemical analysis indicated that the isolates were exhibited catalase activity, but negative in oxidase and indole assays. dna sequencing of 16srrna gene of isolate a3 showed a 94% similarity to pseudomonas sp while isolate a4 showed a 97% similarity to xanthomonas campestris. the dna sequences have been submitted to genbank with the accession number eu563218 for isolate a3 and eu531514 for isolate a4. discussion there are two isolates (a3 and a4) from poh-pohan, indonesian edible leafy plant, which were identified as icenucleation bacteria. these two isolates are categorized as type two ice-nucleation bacteria, having the maximum nucleation temperature of aqueous suspensions at -5°c. turner et al. (1990) classified ice nucleation proteins into three classes: class a which showed the ice nucleation activity between temperature -2°c to -5°c, class b within the temperature range of -5°c to -8°c, while class c is below -8°c. based on that system, these two isolates were categorized as class b. the presence of ina-bacteria on leaf surfaces could alter leaf habitats at subzero temperatures. frost-sensitive plants are injured when ice forms within plant tissues. without ice formation, such injury does not occur, and perhaps many ice-nucleation genes of ina-bacteria recovered from tropical leaf might not being expressed. this finding might raise question such as: what is the role of these bacteria on tropical leaves?; are these bacteria transmitted from subtropical habitats?; how is the distribution on plants in tropical areas? from the measurement of ice-nucleation frequency (table 1), isolate a3 have ice-nucleation activity of 37.9/ml, while isolate a4 is 59.91/ml. isolate a4 showed higher icenucleation activity than a3. identification of the isolates through pcr amplification, cloning and dna sequencing indicated that isolate a3 showed a 94% similarity to pseudomonas sp. while isolate a4 showed a 97% similarity to xanthomonas campestris. loper and lindow (1994) reported that the ice nucleation protein from pseudomonas sp. was classified as icec. while the ice nucleation protein from x. campestris was inax (turner et al. 1990; edwards et al. 1994). however, all of the publication data were derived from subtropical areas. to our knowledge, this is the first finding of ina bacteria from a tropical edible leafy plant. finding of pseudomonas sp. and x. campestris on p. glaberina was interesting and we need more studies on characterization of the activity of the protein and their role in their natural habitats. acknowledgements this research was supported by atma jaya research center. we also would like to give gratitude to steven lindow, university of california berkeley, usa for the scientific discussion and positive control of ina bacteria. references andrews jh, harris rf. 2000. the ecology and biogeography of microorganisms on plant surfaces. annu rev phytopathol 38:145180. edwards ar, ronald a, wichman ha, orser cs. 1994. unusual pattern of bacterial ice nucleation gene evolution. mol biol evol 11:911-920. hirano ss, upper cd. 2000. bacteria in the leaf ecosystem with emphasis on pseudomonas syringae a pathogen, ice nucleus, and epiphyte. mol microbiol biol rev 64:624-653. lindow se, arny dc, upper cd. 1978. distribution of ice nucleationactive bacteria on plants in nature. appl environ microbiol 36:831838. lindow se, brandl mt. 2003. microbiology of the phyllosphere. appl environ microbiol 69:1875-1883. loper je, lindow se. 1994. a biological sensor for iron available to bacteria in their habitats on plants surface. appl environ microbiol 60:1934-1941. fig 1 assay of ice nucleation activity. a, negative control; b, positive control e.coli dh5α carrying pjl1703; c, isolate a3; d, isolate a4. table 1 ice-nucleation activity of bacteria form poh-pohan (pilea glaberina) isolates f n(t) (-8 °c) a3 a4 17/20 19/20 37.9/ml 59.9/ml n(t) = ice-nucleation activity per ml. volume 2, 2008 microbiol indones 9 marchesi jr, sato t, weightman aj, martin ta, fry jc, hiom sj, wade wg. 1998. design and evaluation of useful bacteriumspecific pcr primers that amplify genes coding for bacterial 16s rrna. appl environ microbiol 64:795-799. riupassa pa, suwanto a, tjahjoleksono a. 2005. the abundance of phyllosphere bacteria isolated from some indonesian fresh leafy salad (lalaban). microbiol indonesia 10:96-98. turner ma, arellano f, kozloff lm. 1990. three separate classes of bacterial ice nucleation structures. j bacteriol 172:2521-2526. vali g. 1971. quantitative evaluation of experimental result on the heterogenous freezing nucleation of supercooled liquids. j atmos sci 28:402-409. 10 waturangi et al. microbiol indones 9 yasni short.pmd volume 3, number 3, december 2009 p 146 150 issn 1978-3477 short communication antimicrobial activity of black cumin extracts (nigella sativa) against food pathogenic and spoilage bacteria sedarnawati yasni1*, elvira syamsir1 and eva h direja2 1department of food science and technology, institut pertanian bogor, darmaga campus, po box 220, bogor 16002, indonesia 2flavor applicator, pt jerindo sari utama, jalan cempaka putih raya c-20, jakarta 10510, indonesia this study aimed to analyze the antimicrobial activity of black cumin (nigella sativa) extracts in inhibiting the growth of pathogenic and spoilage bacteria. black cumin was extracted by using steam distillation, single solvent extraction, and continuous solvent extraction. ethanol extract was the best extract in inhibiting the growth of bacteria while both aqueous and hexane extracts were less effective as antimicrobial agents. ethanol extract, essential oil, and ethyl acetate extract have a broad antimicrobial spectrum. the chemical composition of the essential oil was analyzed using a gc-ms technique. the major component of black cumin essential oil was para-cymene, followed by trans-anethole, alloaromadendrene, á-thujene, and thujyl alcohol along with many other components in minor amounts. the minimum inhibitory concentration (mic) value of ethanol extract in inhibiting the growth of salmonella typhimurium was 0.084% (w/w), of essential oil in inhibiting the growth of bacillus cereus was 1.72% (w/w), of ethyl acetate extract in inhibiting the growth of staphylococcus aureus was 1.88% (w/ w) and of methanol extract in inhibiting the growth of pseudomonas aeruginosa was 1.88% (w/w). keywords: antimicrobial activity, extract, nigella sativa, pathogenic bacteria, spoilage bacteria food and waterborne diseases are the leading causes of illness and death in developing countries. one strategy to reduce food-borne illness is by addition of antimicrobial food preservatives during the processing stage to inactivate or prevent the growth of microbes (thongson et al. 2004). since people awareness towards healthy living has changed their mind back to nature, this encourages many researches to pursue natural potensial including research on new antimicrobial compound from nature. black cumin (nigella sativa) is a herbaceous plant, whose seeds have been used for centuries to treat various ailments, including infectious diseases, and which posesses several medicinal properties (ali and blunden 2008; randhawa and al-ghamdi 2002). in middle east, black cumin seeds are used as traditional medicine to treat some health problems, and are also usually added to the traditional food, mixed with bread or honey to increase its flavor (al-saleh et al. 2006). in indonesia, black cumin seed locally known as jintan hitam is used to treat diarrhea and hypertension. diarrhea is one of the most frequent diseases that is caused by microbial contamination. thus, it is assumed that black cumin has antimicrobial activity, and the aim of this study is to analyze the antimicrobial activity of the crude extracts of black cumin seeds on several bacteria. sample material used in this research was black cumin seeds purchased from pasar tanah abang jakarta, and have been reidentified by herbarium bogoriensis in bogor. powdered sample of black cumin was obtained by milling the dried black cumin seeds with blender and filtered (20-40 mesh). the powdered sample was stored at a room *corresponding author. tel. +62251-8626725/8356889; fax+62251-8626725; email: sedarnawati@yahoo.com temperature in desiccator with plastic packaging until use. technical grade of hexane, ethyl acetate, methanol, pure analysis of dimethyl sulphoxide (dmso) and ethanol 96% were purchased from cv. panca pratama bogor. the bacteria were obtained from balivet culture collection (bcc), balai penelitian veteriner – institut pertanian bogor, such as staphylococus aureus bcc 1798, escherichia coli (bcc 207), salmonella typhimurium bcc 712, pseudomonas aeruginosa bcc 1993 and bacillus cereus bcc 2186. tested bacteria were prepared in 10 ml nutrient broth media, incubated aerobically at 37oc for 24 h. the testedbacteria culture (105 cfu ml-1) was used in the antimicrobial activity test of various extracts of black cumin. the extracts were diluted with dmso which has the ability to dilute polar and non-polar compounds, and has emulsifier and non-toxic properties. aqueous extract and ethanol extracts were obtained through single extraction by reflux method. the extraction process was conducted with the ratio of material to solvent 1:3 for 3 h at 100oc for distilled water and at 70oc for ethanol. at the end of extraction each extract was passed through whatman filter paper no.1. after filtration, the residue was re-extracted for 2 h using an additional volume of solvent with the same ratio of 1:3 and then filtered. the combined filtrate was concentrated by rotary evaporator at 40-45oc for ethanol extract. water is difficult to be vaporized so aqueous extract was not concentrated, but water content of aqueous extracts was analyzed using isotropic method (aoac 2005). essential oils were obtained by steam distillation which was conducted by balai tanaman rempah and obat (spice and medicine centre), bogor. the residue obtained from steam distillation was utilized as sample for continuous extraction. the chemical composition of the essential oils 06. tri asmira (sc genetic).cdr short communication genetic diversity of cucumber mosaic virus strain soybean from several areas tri asmira damayanti* and suryo wiyono department of plant protection, faculty of agriculture, institut pertanian bogor, jalan kamper, kampus ipb, darmaga bogor 16680. indonesia cucumber mosaic virus strain soybean (cmv-s) is one of economically important virus infecting soybean in indonesia. however, it is very few information related with the cmv-s indonesia isolates. thus, the aim of present work is to detect and identify cmv-s isolates from different origin. leaf samples collected from six different soybean fields in java and north sumatera. molecular detection was carried out by reverse transcription polymerase chain reaction (rt-pcr) using specific primer of the coat protein (cp) gene and dna sequencing. rt-pcr were successfully amplified the entire cp gene size ~657 bp from all samples and encoded 218 amino acids. analysis of nucleotide and amino acid sequences showed high homology among isolates ranging from 99.5% and 100%. however isolate from sindang laut either nucleotides or amino acids showed lower homology to that of five isolates ranging from 89.4-89.6% and 91.7%, respectively. the differences between cmv-s with other non legume strain found in six amino acid positions in the cp gene, suggesting the differences might related with host specificity. phylogenetic analysis of amino acid of six isolates to other cmv strain showed that the five cmv-s isolates were in the same cluster, while sindang laut isolate was in different cluster together with the non-legume cmv from india. it is indicating the present of genetic variability among cmv-s isolates in java. key words: cmv strain s, genetic variability, soybean cucumber mosaic virus strain soybean (cmv-s) adalah salah satu virus penting secara ekonomi yang menginfeksi kedelai di indonesia. namun, sangat sedikit sekali informasi terkait cmv-s isolate indonesia. oleh karena itu, tujuan penelitian ini untuk mendeteksi dan mengidentifikasi beberapa isolat cmv-s dari daerah yang berbeda. sampel daun dikoleksi dari enam pertanaman kedelai yang berbeda di jawa dan sumatera utara. deteksi molekuler dilakukan dengan reverse transcription polymerase chain reaction (rt-pcr) menggunakan sepasang primer spesifik gen protein selubung (coat protein) dan perunutan sekuen dna. rt-pcr berhasil mengamplifikasi seluruh gen cp yang berukuran ~657 pb dan mengkode 218 asam amino dari semua sampel. analisis sekuen nukleotida dan asam amino menunjukkan homologi yang tinggi antar isolat berkisar dari 99,5% dan 100%. namun homologi sekuen nukleotida dan asam amino isolat sindang laut menunjukkan homologi yang lebih rendah terhadap lima isolat lainnya berkisar 89,4-89,6% dan 91,7%. perbedaan cmv-s dengan strain non legum ditemukan dalam enam posisi asam amino dalam gen protein selubung. perbedaan ini diduga terkait spesifisitas inang. analisis filogenetik asam amino enam isolat cmv-s terhadap strain cmv lainnya menunjukkan lima isolat cmv-s berada dalam satu kluster, sedangkan isolat sindang laut berada dalam kluster yang berbeda bersama dengan cmv strain non legum dari india. hal ini menunjukkan ada variasi genetik pada isolat-isolat cmv-s di jawa. kata kunci : cmv strain s, kedelai, variasi genetik vol.9, no.1, march 2015, p 44-49 doi: 10.5454/mi.9.1.6 *corresponding author; phone : +62-251-8629364, fax: +62-251-8629362, e-mail : triasmiradamayanti@gmail.com cucumber mosaic virus (c m v) is one of cucumovirus genus member which well know has widely host range. the cmv genome is tripartite with ssrna consisting of rna1 and rna 2 which encoded protein 1a and 2a for replication, and rna 3 which play role as movement protein and coat protein (palukaitis and garcia-arenal 2003). there are several strain of cmv distributed worldwide. at least 15 strains of cmv whose nucleotide sequences have been determined were used for a complete phylogenetic analysis of the virus. cmv strain classified on 2 groups based on the similarities sequences such as group i (ia and ib) and group ii (roossinck 2002). cmv infecting soybean previously known as soybean stunt virus (ssv), later it known as cmv strain soybean (cmv-s) (hong et al. 2003). it is one of major virus infecting soybean in indonesia. infected young soybean will produce seed transmitted virus up to 74.1% (suseno et al. 1992). apparent symptom varied, depend on host variety, plant age when infection occur, and environment condition. the symptom on soybean is systemic mosaic occured at 710 days after mechanical inoculation. infected plants become stunted, leaf curling with mosaic, with fewer pod number, smaller seeds with brown mottling (hartman et al. 1999). recent field observation on 2013, the incidence of mosaic symptoms quite high in several soybean fields in java and identification of virus(es) associated with the mosaic symptoms is under progress (yf rahim 2014, personal communication). the study on biological characteristics of cmv-s (ssv) have been studied extensively, however there is no available sequence information of cmv soybean strain (hong et al. 2003; vo phan et al. 2014), especially for sequence information of indonesian isolates and its diversity still not available yet until present. the biological as well as molecular characteristic is an important to determine appropriate management strategies of viral disease. also, it will be usefull for breeder to produce tolerant or resistant cultivar against the virus. thus, the objective of the research was detect and identify the symptomatic leaves which may associated with cmv soybean strain (cmv-s) from several area in indonesia to obtain the gene information and its genetic variability. samples of soybean leaves showing mosaic symptom randomly were collected from various area such as samosir (north sumatera), bogor, cirebon (plumbon and sindang laut) (west java), magelang (central java), and jember (east java). total rna was extracted from 0.1 g leaves with simple direct tube (sdt) extraction method as described previously by suehiro et al. (2005) with minor modification in ratio of buffer and leaf samples. samples were grinded in buffer pbst 1x (phosphate buffer saline tween) with ratio 1:5 (w/v). plant sap as 50 μl was incubated in pcr tube for 15 min for total protein immobilization (plant and viruses). pcr tubes were washed 3-4 times by buffer pbst until no debris remain in the tube. total rna was extracted by providing 30 μl depc water containing 15u rnase inhibitor (ribolock, thermo), then pcr tubes heated at 95 ºc for 1 min and cooling down immediately on ice for at least 5 min. nucleic acid was detected by one-step rt-pcr (reverse transcription pcr). reagent composition used in one-step rt-pcr including; 2 μl of total rna, 2.5 μl of 10x buffer pcr, 1 μl of 10 mm dntp, 2 μl 25 mm mgcl , 1.25 μl 50 mm dtt, 1.25 μl of 10 μm 2 primer forward and reverse each, 0.05 μl of m-mulv -1 (200 uμl ) (revertaid, thermo), 0.1 μl of rnase -1 inhibitor (40 uμl ) (ribolock, thermo), 0.25 μl of -1 taq polymerase enzyme (5 uμl ) (hot start maxima, thermo) and nuclease-free water up to total volume 25 μl. premix one-step rt-pcr was mixed and was carried out on pcr machine gene amp 9700. pcr condition was applied as follows; rt reaction at 42 ºc for 60 min, 94 ºc for 2 min, continued by 1 cycle at 95 ºc for 3 min, 45 ºc for 1 min, 72 ºc for 1 min and followed by 35 cycles at 94 ºc for 1 min, 50 ºc for 1 min and 72 ºc for 1 min and final extension at 72 ºc for 10 min. rt-pcr was carried out by using specific primer for cmv cp gene (forward primer cmvcp-f 5'gcattctagatggacaaatctgaatc-3') and reverse primer (cmvcp-r 5'gcatggtacctc aaactgggagcac-3'). the rt-pcr product size is approximately 650 bp. the dna product was analyzed on 1% tbe (tris boric edta) agarose gel c o n t a i n i n g 0 . 5 μ g / μ l e t h i d i u m b r o m i d e . electrophoresis was conducted at 100 v for 20 min. the dna observed under uv transluminator and documented digitally. dna from rt-pcr was subjected to nucleotides sequencing at charoen pokphand tbk indonesia, jakarta. the dna sequences were analyzed by using clustal w (thompson et al., 1994) (bioedit version 7.05: http://www.mbio.ncsu.edu/bioedit/ bioedit.html) to obtain identity matrix of nucleotide and amino acid homology in compared to that of cmv deposited in the genbank. phylogenetic tree was constructed by using mega 6.0 software (tamura et al. 2013) with neighbor-joining method and pdistances model to estimated the distances, and bootstrap supported was estimated with 1 000 replicates. the c p gene of peanut stunt virus (ay775057) was used as out group. leaf samples collecting from several area of soybean fields showed symptom variations such as mild mosaic to severe mosaic, and leaf curling with mosaic (fig 1a-d). symptom of infected plants from sindang laut varied such as mild mosaic to yellow mosaic (data not shown). multiple virus infection and the differences of variety cultivated in each sampling area might contribute to the symptom variation in the fields. molecular detection by rt-pcr was amplified a ~650 base pair (bp) dna fragment from all six samples with specific primer for cmv cp gene (fig 2). it indicated that the symptomatic leaves collected from all area were infected by cmv. volume 9, 2015 microbiol indones 45 dna sequencing results successfully identified the full-length of cmv-s gene from all samples. the size of the cp gene is 657 nucleotides, and it is encodes 218 amino acids. analysis of nucleotide (nt) and amino acid (aa) sequences of the cmv-s cp gene of those i s o l a t e s ( i s o l a t e s b o g o rb g r, p l u m b o n p l b n , magelang-mgl, jember-jmbr, and samosir-smsr) showed high homology among isolates, ranging 99.5100% (nt) and 99-100% (aa) (table 1). however, isolate sindang laut (sdl) showed lower homology of either nucleotide (89.4-89.6%) or amino acid (91.7%) to that of five isolates, respectively (table 1). pair wise comparison of these sequences with corresponding nucleotide sequences in the genbank showed that the five isolates (bgr, plbn, mgl, jmbr, smsr) had homology ranging 88.8-91.7% (nt) and 90.3-92.6% (aa) with cmv non legume strain. however, c m v-s isolate sdl showed closely homology in nucleotides (91.9-94.5%) and amino acid (94.4-98.1%) to that of cmv non legume strain (table 2). it is indicating the genetic variability among cmvs isolates in the cp gene. comparison of amino acid sequences between five cmv-s isolates (bgr, plbn, mgl, jmbr, smsr) and sdl isolate showed the amino acid differences in 14 positions such as s12n, p25s, n31t, i32f, g34v, l53i, k65r, d98e, v142m, a144s, k180r, v192a, m196t and i200l (first symbols are amino acids of five isolates of cmv-s, last symbols are amino acids of cmv-s isolate sdl, number: the position of amino a b c d fig 1 variation of mosaic symptoms which caused by cmv-s infection or multiple infection of virus collected from the fields. ~650 bp 1 2 3 4 5 6 7 l fig 2 rt-pcr of cp gene of cmv-s isolates. l, dna ladder 1 kb plus (invitrogen), (1) positive control, (2) cmv-s bogor isolate, (3) plumbon isolate, (4) sindang laut isolate, (5) magelang isolate, (6) jember isolate, and (7) samosir isolate. 46 damayanti microbiol indones acids in cp gene). the differences of amino acid might related to the lower nucleotide homology of isolate sdl to that of other five isolates. comparison of amino acid sequences of cmv-s to other strain of cmv showed differ in 6 amino acids at position of l123v, s132a, t141a, e147a, i160v, and p171a. these might be related to strain differences and host specificity, however it needs further investigation. phylogenetic analysis of nucleotide sequences showed that all five isolates of cmv-s were in one cluster distantly related to those other cmv strains. however cmv-s sdl isolate more closely related to cmv ordinary strain from other countries (data not shown). similarly, phylogenetic based on amino acid sequences show that the five cmv-s isolates are located in one same cluster distantly related to other cmv ordinary strain, while sdl isolate separated far from others cmv-s isolates and closely to cmv infecting tomato from india (fig 3). cmv-s from various area of soybean fields in indonesia are distinct strain to cmv ordinary strain based on nucleotide and amino acid analysis of the cp gene except cmv-s sindang laut isolate. these results supported the previous report for cmv-s from japan (hong et al. 2003). cmv-s sindang laut isolate is locates in the same cluster to the cmv non legume strains. it is indicating the genetic variability of cmvs in java based on cp gene sequences. all indonesian isolates of cmv-s were a member of sub group ib (fig 3). the difference between c m v-s and c m v common/ordinary strain such cmv-y that, common strain could not infect soybean event locally. cmv-s can not systemically infect cucumber, whereas common strain of cmv can. similar strain of cmv-s table 1 homology of nucleotides and amino acids of cmv-s cp gene among isolates cmv-s isolates idn-bgr* idn-plbn idn-sdl idn-mgl idn-jmbr idn-smsr nt aa nt aa nt aa nt aa nt aa nt aa bogor 99.5 99.0 89.6 91.7 99.5 99.0 99.5 99.0 99.5 99.0 plumbon 89.4 91.7 100.0 100.0 100.0 100.0 100.0 100.0 s. laut 89.4 91.7 89.4 91.7 89.4 91.7 magelang 100.0 100.0 100.0 100.0 jember 100.0 100.0 samosir *cmv-s isolate from bgr-bogor, plbn-plumbon, sdl-sindang laut, jmbr-jember, smsr-samosir table 2 homology of nucleotides and amino acids of cmv-s cp with corresponding cmv in genbank cmv-s isolates idn-bgr idn plbn idn-s. laut idn-mgl idnjmbr idn-smsr nt aa nt aa nt aa nt aa nt aa nt aa california 91.6 92.6 91.4 92.6 93.6 98.1 91.4 92.6 91.4 92.6 91.4 92.6 india-luc 88.8 92.6 88.7 92.6 94.5 98.1 88.7 92.6 88.7 92.6 88.7 92.6 india-ama 91.7 90.3 91.6 90.3 93.7 97.2 91.6 90.3 91.6 90.3 91.6 90.3 italy 91.4 92.6 91.3 92.6 94.0 98.1 91.3 92.6 91.3 92.6 91.3 92.6 taiwan 90.8 90.3 90.7 90.3 91.9 94.4 90.7 90.3 90.7 90.3 90.7 90.3 thailand 89.6 90.8 89.4 90.8 93.9 98.1 89.4 90.8 89.4 90.8 89.4 90.8 *cmv-calfornia/tomato (af523340.1), cmv-india lucknow/tomato (ef153734.2),cmv-india/amaryllis (ef187825.1), cmv-italy/tomato (y16926.1), cmv-taiwan/yard long bean (d49496.1), cmv thailand/cucumber (aj810264.1) volume 9, 2015 microbiol indones 47 (cmv-209) from korea is only infects systemically on wild soybean and pea, but not infect the other legume species (vo phan et al. 2014). the ability to infect its host was related with the movement protein as host determinant of cmv (hong et al. 2007). there are 14 amino acids difference between five cmv-s isolates and sdl isolate on the cp gene, however the sdl isolate infects soybean systemically as well as other cmv-s isolates. it is support the previous report that host determinant of cmv rely on the movement protein rather than in the cp gene. the sdl isolate may different in several amino acid in the cp gene, but it may not contribute to its ability to infect soybean, suggesting the movement protein gene of sdl isolate may similar to those other cmv-s isolates. it needs further sequences analysis of the movement protein gene that is was not covered in these analysis. among 14 amino acids difference between five isolates of cmv-s and sdl isolate, one of it at position 25 is serine, while those other five isolates maintain amino acid proline at the position. the substitution of p25s on sdl isolate may imply a substantial structural change in protein. the amino acids at position 25, 76 and 214 are reported affect to transmission efficiency by aphid if substitution occurs at those positions (moury 2004). based on phylogenetic analysis it shown that cmv-s is cmv strain different to other cmv (nonlegume). ssv-in (ssv from indonesia) is one of divergent isolate compared to ssv isolate b, c, d and ae from hokkaido, japan, isolate from china and isolates wr from russia. ssv-in do not cause systemic infection on various wild type of soybean in japan. ssv-in is supposed to be adapted specifically to local indonesian soybean, which not influenced by gene flow from wild soybean (hong et al. 2003). the variability of the cp gene of cmv-s may leads to the differences of biological characteristics that need further investigation. cmv-113 cmv-tfn cmv-nt9 cmv-c7-2 cmv fny cmv-y cmv-tr15 cmv bb8 cmv-luc cmv s-sdl cmv m48 cmv s-bgr cmv s-jmbr cmv s-mgl cmv s-plbn cmv s-smsr cmv-ls cmv-q psv 100 96 100 100 93 85 100 72 99 ia ib ib ii fig 3 phylogeny of the cp gene amino acid of cmv-s isolates from several area in indonesia (idn). the cmv-s isolates are highlighted in grey. the tree were inferred by the neighbor-joining method using molecular evolutionary genetics analysis (mega) software version 6.0 based on the clustalw alignment sequences from indonesia, india, italy, taiwan, thailand and usa. the tree was rooted using peanut stunt virus (psv) as out groups. bootstrap values expressed as percentage of 1 000 replicates that greater than 70 are shown on the tree branches. 48 damayanti microbiol indones acknowledgement the research is conducted by financial support of competitive research grant of directorate general of higher education through dipa ipb with contract no 05/i3.24.4/spp/phb/2011 on march 28, 2011. references hartman gl, sinclair jb, rupe jc. 1999. compendium of th soybean disease. 4 ed. aps press. hong js, ohnishi s, masuta c, choi jk, ryu kh. 2007. infection of soybean by cucumber mosaic virus as determined by viral movement protein. arch virol. 152: 321-328. doi: 10.1007/s00705-006-0847-3. hong js, masuta c, nakano m, abe j, uyeda i. 2003. adaptation of cucumber mosaic virus soybean strain (ssvs) to cultivated and wild soybeans. theor appl genet. 107: 49-53. doi: 10.1007/s00122-003-1222-3. moury b. 2004. differential selection of genes of cucumber mosaic virus subgroups. mol biol evol. 21(8):16021611. doi:10.1093/molbev/msh164. palukaitis p, garcía-arenal f. 2003. cucumoviruses. adv virus res. 62:241–323. roossinck mj. 2002. evolutionary history of cucumber mosaic virus by deduced by phylogenetic analyses. j virol. 76:3382-3387. doi: 10.1128/jvi.76.7.33823387.2002. suehiro n, matsuda k, okuda s, natsuaki t. 2005. a simplified method for obtaining plant viral rna for rt-pcr. j virol methods. 12:67-73. doi:10.1016/ j.jviromet.2005.01.002. suseno r, suastika g, kusumah ym. 1992. studi beberapa aspek pengendalian virus terbawa benih kedelai yang ditularkan kutudaun, virus bantut kedelai (soybean stunt virus/ssv) dan virus mosaik kedelai (smv). laporan akhir penelitian pendukung pht dalam rangka pelaksanaan program nasional pengendalian hama terpadu. bogor; kerjasama proyek prasarana fisik bappenas dengan fakultas pertanian, institut pertanian bogor. tamura k, stecher g, peterson d, filipski a, kumar s. 2013. mega 6: molecular evolutionary genetics analysis version 6.0. mol biol evol. 30(12):2725-2729. doi: 10.1093/molbev/mst197. thompson jd, higgins dg, gibson tj. 1994. clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. nucleic acid res. 22: 4673-4680. vo phan ms, seo jk, choi hs, lee sh, kim kh. 2014. molecular and biological characterization of an isolate of cucumber mosaic virus from glycine soja by generating its infectious full-genome cdna clones. plant pathol j. 30(2):159-167.doi: 10.5423/ppj.oa. 02.2014.0014. volume 9, 2015 microbiol indones 49 page 1 page 2 page 3 page 4 page 5 page 6 jatmiko et al24042012 issn 1978-3477, eissn 2087-8575 vol 6, no 1, march 2012, p30-34 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.5 molecular identification of yeasts isolated from dadih by rflp-pcr and assessment on their ability in utilizing lactate 1 2 2 yoga dwi jatmiko *, miguel de barros lopes , and mary d barton 1 department of biology, faculty of sciences,universitas brawijaya, jalan veteran, malang, east java 65145, indonesia; 2 school of pharmacy and medical sciences, university of south australia, city east campus, adelaide, south australia 5000, australia *corresponding author, phone: +62-341-575840, fax : +62-341-575841. e-mail : jatmiko_yd@ub.ac.id. the existence of yeasts in a range of spontaneously fermented milk products has been investigated by several researchers (gadaga et al. 2000; abdelgadir et al. 2001; lore et al. 2005; kebede et al. 2007; zhang et al. 2008; rahman et al. 2009). as well as being potential spoilage microorganisms, the presence of indigenous yeasts in naturally fermented milk (nfm) products may contribute to the development of characteristic of texture and flavor (narvhus and gadaga 2003). the presence of indigenous yeasts in dadih has been noted but not well studied (hosono et al. 1989). yeasts were found in dadih as reported by some researchers which were endomyces lactis (hosono et al. 1989), candida tropicalis, geotrichum candidum (gandjar 1983; roositta et al. 2003), candida parapsilosis (gandjar 1983), and saccharomyces cerevisiae (roositta et al. 2003). yeasts in dadih generally have been identified traditionally based on phenotypic characteristics. this method is considered to be time consuming and fairly inaccurate (esteve-zarzoso et al. 1999; senses-ergul et al. 2006). molecular approach with reference to 5.8sits region has been successfully applied to identify yeast species from a range of fermented food products (abdelgadir et al. 2001; jeyaram et al. 2008; rahman et al. 2009). in this study, pcr amplification of the 5.8s-its region followed by restriction fragment length polymorphism (rflp) has been used to identify the yeast species present in dadih. to study the potential effect of these yeast on dadih, such as ability to utilize lactic acid -the major metabolite of lactic acid bacteria (lab)was evaluated. a wide variety of yeasts have been involved in traditional fermented foods, and potentially contributed to the development of product properties. the existence of indigenous yeasts in dadih, a traditionally fermented form of buffalo milk of west sumatera, has been reported but an accurate identification is still required to be conducted. this study was aimed to identify yeasts isolated from dadih using a molecular approach, and to evaluate their lactate utilization. a total of 51 isolates were characterized and identified as pichia jadinii and candida stellimalicola using pcr amplification of the 5.8s internal transcribed spacer region combined with restriction fragment length polymorphism (rflp) analyses and gene sequencing. the former species were the dominant one in this tested product. their ability to utilize lactate was demonstrated, indicating that they could modify the sensory characteristics of dadih, and hence interact with the indigenous lactic acid bacteria in dadih. the restriction profiles of the dadih yeasts can be used as a data base for rapid identification of yeasts in the future. further work is still needed to elucidate the dadih yeast ecology. key words: dadih, internal transcribed spacer (its), lactate, pcr, yeasts beragam jenis khamir telah terlibat dalam produk fermentasi pangan tradisional, dan khamir tersebut mampu mempengaruhi karakteristik dari produk tersebut. keberadaan khamir indigenus di dalam dadih, yaitu produk susu kerbau yang difermentasi secara tradisional dari sumatra barat, telah dilaporkan. tetapi identifikasi secara akurat masih diperlukan. penelitian ini bertujuan untuk mengidentifikasi khamir yang diisolasi dari dadih secara molekuler, dan mengevaluasi kemampuan khamir tersebut dalam menggunakan laktat. sejumlah 51 isolat khamir telah dikarakterisasi dan diidentifikasi sebagai pichia jadinii dan candida stellimalicola menggunakan amplifikasi pcr daerah 5.8s-its dan dilanjutkan dengan analisis restriction fragment length polymorphism (rflp) dan pembacaan deret dna dari gen. p. jadinii merupakan salah satu spesies khamir yang mendominasi pada produk dadih yang diamati. kedua jenis khamir tersebut juga mampu memanfaatkan laktat. hal ini menunjukkan bahwa khamir tersebut dapat memengaruhi karakteristik sensori dari dadih. selain itu, khamir dinilai mampu berinteraksi dengan bakteri asam laktat indigenus dalam dadih. pola restriksi hasil analisis rflp dari khamir tersebut dapat digunakan sebagai basis data untuk identifikasi khamir di penelitian mendatang. penelitian lebih lanjut masih diperlukan untuk mempelajari ekologi khamir di dalam produk dadih. kata kunci : dadih, internal transcribed spacer (its), khamir, laktat, pcr materials and methods isolates. fifty one of yeasts isolated from dadih by using de man rogosa sharpe (mrs) agar medium which were derived from previous research (jatmiko et al. 2010) were included in the study. a phenotypic characterization (shape of the cell and colony appearance) was carried out among these yeast isolates. media. yeast peptone dextrose (ypd) agar and broth supplemented with 2% (w/w) of d-glucose were used for cultivation of yeast isolates. minimal media containing yeast nitrogen base without amino acids (difco, bd diagnostics, usa) and 2% (v/v) of lactic acid (sigma-aldrich) was used for evaluation of utilization of lactate by yeasts. colony-pcr amplification. the oligonucleotide primers used in this experiment were its 1 (5'-tccgt aggtgaacctgcgg-3') and its 4 (5'-tcctccg cttattgatatgc-3'). colony-pcr was performed in this study where a small amount of yeast colony used directly as dna templates for the pcr amplification. a small amount of a single colony was taken from the solid media, and placed at the bottom of sterile pcr tubes using a sterile loop. subsequently, the colony in o the pcr tubes was heated at 100 c in a microwave oven (panasonic) for one minute. after this, the master mix reagents were dispensed into the tubes. the dna amplification of the 5.8s-its region was carried out in a mycycler thermal cycle (bio-rad laboratories, hercules, ca, usa) with following components: 5 µl of 5x pcr buffer, 1.25 µl of 50 mm mgcl , 2.5 µl of 2 2.5 mm dntps (2 mm each datp, dctp, dgtp and -1 dttp), 0.25 µl of taq polymerase (5 u µl ) and 1.5 µl of 5 µm each primer. to obtain a final volume of 25 µl, a quantity of sterile milli q water was added. the pcr o reaction was run with an initial denaturation at 94 c for o o 5 min, followed by 35 cycles of 94 c for 1 min, 56 c for o o 1 min and 72 c for 1 min, with a final extension at 72 c o for 5 min and a terminal hold at 4 c. amplified products were separated by electrophoresis in a 1.5% agarose gel -1 and stained with ethidium bromide (0.5 µg ml ). rflp analysis. after obtaining a good yield of product following electrophoresis, rflp analysis was performed for differentiation of 51 yeast isolates. a total volume of 10 µl was used for rflp analysis which comprised 9 µl of amplicons, 1 µl of buffer (composition dependent on the enzymes), and 0.2 µl of restriction enzymes (haeiii, hinfi, and ecori). the solution was incubated at the optimum temperature for the enzyme activity for at least 3 hours. following this incubation, 5 µl of the rflp product plus 2 µl of loading dye were dispensed into the wells of the agarose gel and then electrophoresed as mentioned in the previous section. since species identification of yeast can be determined by comparing the result of rflp patterns with the existing databases, sequencing of the its region product was also performed when restriction profiles of the reference strains are not yet available. purification of dna products. pcr products were purified for sequencing by using ultraclean pcr clean-up kit (mo bio laboratories inc, solena beach, ca, usa.) according to the manufacturer's instructions. dna sequencing analysis. sequencing of the dna products was performed using big dye terminator v3.1 cycle sequencing ready reactions (applied biosystem, foster city, ca, usa) at the dna sequencing facility, flinders medical centre, adelaide. the nucleotide sequences were analysed by using bioedit software (version 7.0.9.0). homology searches were performed using basic local alignment search tool (blast) at the national centre of biotechnology information website (http://www.ncbi. nlm.nih.gov/blast/). evaluation of lactate-utilizing yeasts. a number of yeast isolates were selected to represent the different strains obtained from the dadih samples. each selected isolate was revived from frozen stocks and subcultured o twice in ypd broth at 37 c in 5% co . following this 2 -1 incubation, 1% (v v ) of cultures was inoculated into -1 minimal medium containing 2% (v v ) of lactic acid, o and then incubated at 37 c in 5% co for 48 h. the 2 incubation condition was carried out without shaking in order to imitate the dadih fermentation condition. the cell count and ph were measured before and after incubation in duplicate. a spread plate method was used to determine the total yeast counts. the utilization of lactic acid was shown by subtracting between initial and final concentration of d/l-lactate measured using k-dlate kit (megazyme, ireland). the reagent preparation and calculation of the lactate were performed according to the manufacturer's instructions. minimal medium without cultures was used as a control. the isolates were also grown in the minimal medium without lactic acid. results molecular characterization and identification of dadih yeasts. a total of 51 yeast isolates were characterized and identified using pcr amplification volume 6, 2012 microbiol indones 31 of the 5.8s-its region combined with rflp and gene sequencing. the restriction analysis results were in consistent with cell and colony appearance, which differentiated the yeast isolates into two groups (table 1 and fig 1). the restriction enzymes of haeiii and hinfi successfully digested the amplicons and confirmed the presence of two different groups (species) indicates that the use of haeiii and hinfi to digest the its-pcr products could provide a reproducible result. based on dna sequencing, the two species were identified as pichia jadinii and candida stellimalicola, with the genbank accession number jq 927546 and jq 927545 respectively. the restriction profile of p. jadinii was also in agreement with the study conducted by villa-carvajal et al. (2006). interestingly, p. jadinii (20 isolates) was only found in dadih from gadut (district of agam). in dadih collected from sianok (district of agam), c. stellimalicola was the only identified yeast species in the two days fermentation while the three days fermentation yielded seven isolates of c. stellimalicola. at the same time, p. jadinii was dominant in both two and three days fermentation (six and 17 isolates, respectively). utilization of lactic acid by yeasts. during incubation in lactate medium, total lactate declined slightly in the presence of both yeasts (p. jadinii and c. stellimalicola). this was correlated with the increase in the ph values of the medium. all of the tested yeast strains were more active in utilizing d-lactate rather than l-lactate. interestingly, co-cultivation of the two yeasts did not increase use of lactate significantly (fig 2). discussion to our knowledge, this is the first report on the isolation of these two yeast species from dadih which means that the role of yeast in determining the flavor characteristics and sensory properties of dadih may have been overlooked. the morphological study of p. jadinii showed that the cell characters (shape of the cell) were in consistent with the literature, in that they were ovoid in shaped and large in size (3.5-4.5) x (7-13) µm (lodder 1970) . c. stellimalicola was first isolated from star apple (ma-fueng) in thailand (suzuki et al. 1994). in contrast with p. jadinii, the cellular morphology of c. stellimalicola is relatively smaller than that of p. jadinii. the cells are (1.8-4.0) x (1.8-4.2) µm, and ovoid in shape (suzuki et al. 1994). p. jadinii (anamorph of c. utilis) and c. stellimalicola was grouped in a similar group on the basis of 18s rrna gene sequence divergence (suzuki and nakase 2002). hence, based on the phylogenetic studies these yeast species are closely related. the presence of the two species of yeasts in dadih indicates that these species could reduce acidic properties of dadih. although unable to utilize lactose, these species' ability to use lactate may explain their presence in dadih (fig 2) (lodder 1970; suzuki et al. 1994) it should be noted that in our study the cultures . 500 bp 1,000 bp table 1 characterization and identification of dadih yeasts fig 1 internal transcribed spacer -pcr products and their restriction analysis with the endonuclease hinfi and haeiii lane m: 100 bp dna marker; lane 0: sterile milliq h o; lane 1: group ii (isolate s1.04), lane 2: 2 group i (isolate s2.31), lane 3: group ii (isolate g.20), lane 4: group i (isolate s2.29), lane 5-6: group ii (isolate g.19 and s2.03), lane 7: group i (isolate s2.33) (hinfi: lane 3-4; haeiii: lane 5-7). 0 m 1 2 3 4 5 6 7 g roup c ellm or ph ology n um be r of isola tes i t s -pc r (bp) r fl p ( bp) bl a s t ana lysis ( % ) ide ntif ic ation h ae iii h infi e cor i i s m all ovoid 8 575 390, 145 300, 275 575 98 c andida ste llim alicola ii l a r ge ovoid 43 520 520 300, 220 520 99 p ic hia jadinii 32 jatmiko et al. microbiol indones were incubated without shaking; as the two are obligate aerobes, it is predicted that if oxygenation was increased, growth and utilization of d/l lactate would be improved. lactic acid produced by lab can be assimilated further only by aerobic metabolic pathway (lowes et al. 2000). this experiment was designed to imitate dadih condition, which was semi-aerobic condition; as a result the lactate utilization in the medium was not increased significantly. their role in dadih requires further study. it is expected that these yeasts could live in the semi-aerobic condition as in the dadih fermentation process and may have adapted to the dadih environment by assimilating the lactic acid. the source of these yeasts also remains unknown and a further ecological study to assess microbiological condition of bamboo containers and the banana leaf covers, as well as the buffalo milk are interesting to study . to conclude, p. jadinii and c. stellimalicola were detected as non-lab cultures of tested dadih. their ability to utilize lactic acid was demonstrated, indicating that they could modify the sensory characteristics of dadih. a more detailed analysis using an appropriate yeast medium in place of mrs would be useful to further establish the dadih yeast ecology. in addition, the restriction profiles of the dadih yeasts can be used as a data base of the rapid identification of yeasts. acknowledgement this work was supported by australian development scholarships 2008-2010 from the australian agency for international development (ausaid). the authors wish to thank ingrid surono for the assistance during dadih sample collection. references abdelgadir ws, hamad sh, moller pl, jakobsen m. 2001. characterisation of the dominant microbiota of sudanese fermented milk rob. int dairy j. 11(1-2): 6370. doi: 10.1016/s0958-6946(01)00042-5. esteve-zarzoso b, belloch c, uruburu f, querol a. 1999. identification of yeasts by rflp 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115-121. doi: 10.2323/jgam.40.115. villa-carvajal m, querol a, belloch c. 2006. identification of species in the genus pichia by restriction of the internal transcribed spacers (its1 and its2) and the 5.8s ribosomal dna gene. anton leeuw. 90(2): 171181. doi: 10.1007/s10482-006-9071-0. zhang h, xu j, wang j, menghebilige, sun t, li h, guo m. 2008. a survey on chemical and microbiological composition of kurut, naturally fermented yak milk from qinghai in china. food control. 19(6): 578-586. doi: 10.1016/j.foodcont.2007.06.010. 34 jatmiko et al. microbiol indones 1: 30 2: 31 3: 32 4: 33 5: 34 7.mi727-kartika senjarini available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.7issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 186-191 *corresponding author; phone: +62-81358346388, email: kartika_senjarini@yahoo.com although malaria has been virtually eradicated from indonesia, it is currently recognized as a serious re-emerging threat to public health. anti-malarial drug resistances as well as vector resistance against insecticides are major public health problems hindering the control of malaria (e.g. yadouleton et al. 2010). dengue fever (df), caused by infection with dengue virus (denv), is not a new disease. today, dengue is considered as one of the most important arthropod-borne viral diseases in humans in terms of morbidity and mortality. however, life-threatening dengue cases are mostly occurred in west pacific and south east asia such as the philipines, thailand, and indonesia (gubler 1997). in indonesia, df case was first observed in 1968; since then, the incidence has been constantly increasing and the disease is now one of the principal causes of child lethality. the highest mosquito-borne diseases are rampant in most tropical regions of the world, especially rural, forested, and coastal areas such as indonesia. despite long-standing chemotherapeutic intercession and vector control programs, mosquito-borne diseases exact a heavy burden on human health in indonesia. two major public health problems transmitted by mosquito in indonesia are malaria and dengue haemorrhagic fever (dhf), causing millions of clinical episodes occurring annually. malaria is now recognized as a serious re-emerging threat to public health. dhf cases were first observed in 1968; since then, the incidence has been constantly increasing and the disease is now one of the principal causes of child lethality. it has been widely observed that saliva of mosquito that transmits the diseases contains several factors that could enhance pathogen infection. therefore, it should be possible to control pathogen transmission by vaccinating the host against the molecule(s) in saliva that potentiate the infection. however, specific component as a potential target for tbv in mosquito vectors of malaria & dengue, i.e. anopheles and aedes aegypti, has not been identified so far. this paper wanted to elaborate the potential role of salivary component from mosquitoes, particularly from indonesian vectors as molecular target for developing tbv against two major mosquito borne-diseases in indonesia i.e. malaria and dhf. key words: aedes, anopheles, immunomodulators, salivary gland, tbv penyakit yang disebarkan oleh nyamuk banyak ditemukan di kebanyakan daerah tropis di dunia khususnya daerah pedesaan, area sekitar hutan dan perairan pantai seperti yang banyak ditemukan di indonesia. walaupun berbagai usaha untuk menanggulangi penyakit tersebut telah lama dilakukan, baik dengan pengendalian penyakit dengan terapi kausatif (obat/bahan kimia) maupun pengendalian vector, penyakit diperantarai nyamuk ini masih merupakan masalah kesehatan utama di indonesia. malaria dan demam berdarah dengue (dbd) merupakan 2 masalah utama yang di transmisikan oleh nyamuk di indonesia. malaria masih menjadi masalah kesehatan yang serius karena pada dekade terakhir ini terjadi peningkatan prevalensi di beberapa daerah bahkan mulai muncul resistensi pada beberapa obat malaria (re-emerging). dbd pertama kali ditemukan pada 1968, sejak saat itu kasusnya selalu meningkat bahkan merupakan salah satu penyakit penyebab kematian utama pada anak usia sekolah. saliva nyamuk yang bertindak sebagai vektor, telah terbukti mengandung berbagai komponen yang mampu meningkatkan transmisi patogen yang dibawanya. oleh karena itu, transmisi patogen akan sangat mungkin untuk dihambat dengan mem”vaksinasi” inang dengan molekul “lawan” dari molekul yang meningkatkan proses transmisi tersebut sehingga dapat menghambat transmisinya (tbv). namun demikian, komponen spesifik sebagai target potensial untuk tbv dari vektor malaria dan dengue yaitu anopheles dan aedes aegypti sampai saat ini belum berhasil diidentifikasi. selama ini, telah dilakukan eksplorasi terhadap potensi saliva vektor dari berbagai vektor nyamuk yang ada di dunia tetapi belum ada laporan tentang hal ini dari indonesia. paper ini akan memberikan review tentang potensi saliva dari vektor nyamuk di indonesia sebagai kandidat target potensial untuk pengembangan tbv untuk 2 masalah utama kesehatan di indonesia yang disebarkan vektor tersebut yaitu malaria dan dbd. kata kunci: aedes, anopheles, imunomodulator, kelenjar saliva, tbv review potential use of mosquito’s salivary components as novel target for the development of transmission blocking vaccine (tbv) kartika senjarini* biology department-mathematics and natural sciences faculty, university of jember, jalan kalimantan 37, jember 68121, indonesia incidence of dengue occurred between 1998-2000 in surabaya, east java. the highest proportion of df related mortality occuring between 1999-2000 was associated with children aged 5-9 years (soegijanto 2004). dki jakarta, the capital city, was the province with the highest number of df cases, i.e 14,071 cases with case fatality rate (cfr) 0.42% in 2003. in 2005, df incidence increase up to 23,466 cases with cfr 0.34% (daniel 2008). despite the long-standing chemotherapeutic intercession and vector control programs, malaria and dengue fever outbreaks are always reported every year. this condition indicates that the quest for causative therapy is extraordinarily daunting. therefore, development of a vaccine could be a more efficient strategy to overcome the epidemic. in the last decade, a new approach in the development of vaccine for arthropode-borne diseases is by using the salivary components from vectors. this approach is developed based on a hyphothesis that the vector's saliva contains vasodilator and imunomodulator proteins (proposed by,e.g. sack and kamhawi 2001; titus et al. 2006). the vasodilatory factors play important role to help the vector during blood feeding. there are two hypotheses concerning the function of imunomodulatory factor in saliva of mosquitoes. many reports showed that salivary imunomodulators could enhance pathogen infection (e.g. donovan et al. 2007) (1). however, there is also evidence that saliva appeared to directly protect dendritic cells from infection in vitro (ader et al. 2004) (2). in relation to the first case, it should be possible to control pathogen transmission by vaccinating the host against the molecule(s) in saliva that potentiate the infection, thereby blocking the enhancing effects of saliva, thus preventing the pathogen from establishing infection in the host. in case of second condition, it could be used directly to protect host cells from infection of transmitted pathogens. these hypotheses led to closer examination of salivary factors, especially the imunomodulatory factors, as potential targets to control pathogen transmissions, i.e. transmission blocking vaccine (tbv) or also known as mosquito stage vaccine (ramirez et al. 2009). however, specific potential target for tbv in mosquitoes such as anopheles and ae (ae) aegypti has not been identified so far. therefore, exploration of mosquito’s salivary components of is an important step to find novel target for tbv development. this paper would like to elaborate the potential role of three potential malaria vectors in indonesia, a. aconitus, a. sundaicus and a. maculates, as well as a major dengue vector for, ae. aegypti as tbv target model . reduction of parasitemic rates by anopheles salivary components. the role of vector’s salivary components in mediating infection is a relatively new concept in term of arthropod borne-diseases. this is very important since mortality and morbidity caused by infectious diseases transmitted by blood sucking arthropods (haematophagous) have always been increasing in the last decade (gubler 1998). additionally, there are co-evolutionary functions between vectors’ and their transmitted pathogens’, for instance saliva plays active roles in homeostasis, inflammation and vertebrate host’s immune responses (ribeiro 1995). mosquito’s salivary components are immunogenic, that is, they induce strong immune response such as swelling and itching that accompanied mosquitoes bites (peng and simon 2004). the host’s immune response against mosquito’s saliva could reduce infectivity of transmitted pathogens (belkaid et al. 1998), therefore population living in leishmaniasis endemic sites showed natural resistance against leishmania parasites (davies & gavgani 1999). this has also been proven in leishmaniasis murine model. resistance against leishmania major was due to exposure of mouse model to uninfected sand flies. this process was associated with a strong delayed-type hypersensitivity response and with interferon-γ production at the site of parasite delivery (kamhawi et al. 2000). the hyphothetical mechanism of these processes was due to the natural immunity mediated by t-helper 1 (th1), which has protective properties and contains antibodies against sandflies saliva. mosquito bites have shown similar effects in animal models through cytokines systemic response in host (schneider et al. 2004). it may be that these imunomodulatory components has immunosupresive properties towards effector cells such as macrophage, t lymphocites, b lymphocites, natural killer cells, and granulocytes (andrade et al. 2005). another evidence showed that repeated exposure to bites from uninfected anopheles stephensi skewed the immune response towards th1 phenotype as indicated by increased levels of interleukin-12, gamma interferon, and inducible nitric oxide synthase, followed by reduction of parasitaemic rates in mouse model (donovan et al. 2007). other mechanisms related to this protection were very likely due to immunogenicity of salivary protein of anopheles. it has been proven with the salivary proteins from a. gambie (poinsignon et al. 2009), volume 7, 2013 microbiol indones 187 a 1 2 4 6 9 0 2 4 6 8 10 12 14 p ar as it em ic r at es ( % ) b 0 5 10 15 20 25 4 6 7 p ar as it em ic r at es ( % ) 0 5 10 15 20 2 3 4 5 6 p ar as it em ic r at es ( % ) c days after challenged with plasmodium fig 1 average rates of parasitaemia (%) in murine model: control (k), vaccinated with sge from pelet (p), and vaccinated with sge from supernatant (s) from a. maculatus (a), a. aconitus (b), dan a. sundaicus (c). k = control, p = pellet from salivary gland extract (sge) & s = supernatant from sge. x-axis represent the number of days after exposure to p. berghei (armiyanti et al. 2012; hanafy et al. 2012). : k, : p, and : s. 188 kartika microbiol indones components. mosquito bites have also been reported to influence immunity and potentiate viral disease in mouse models (e.g. limesan et al. 2003, schneider et al. 2006), possibly through the modulation of host systemic cytokine responses by the salivary component (schneider et al. 2004). this strategy may be important for the development of vaccines to combat mosquito-transmitted viral pathogens such as dengue fever. analyzing which protein portions of sg that can be recognized by human antibody would lead to the possibility to find sg’s protein with significant role in respond to dengue infection, especially those interacting with antibody of healthy individuals from endemic area. people living in endemic area who do not get the disease must have certain immune mechanisms conferring this protection. this is similar to natural resistance built by population living in endemic areas, as previously explained in the case of leishmaniasis. investigation of specific components important for immune response in relation to pathogen transmission started in our research group by using sg of ae. aegypti (oktarianti et al. 2013). as seen in fig 2, we were able to identify specific protein portions of aedes aegypti sg with molecular weight of ~ 56 and 31 kda, which were able to cross react only with sera of individuals from endemic area (individual response). these proteins did not seem to cross react with human sera who had never previously been exposed to mosquitoe bites. the ability of this protein a.arabiensis and a. funestus (e.g. lombardo et al. 2009; rizzo et al. 2011) as well as from a. barbirostris (jariyapan et al. 2012). our results showed that repeated exposures of salivary gland (sg) extract from 3 potential malaria vectors in indonesia i.e. a. aconitus, a. sundaicus and a. maculatus were able to reduce rates of parasitaemia in murine malaria model (fig 1.). the reduction of parasite burdens suggested the roles of host immune response in reducing the infectivity of transmitted pathogen. the data indicated that mosquito’s salivary components may serve as a nonspecific potentiator that induces a th1-biased environment known to be effective against malaria infection. therefore, addition of anopheles salivary components to antimalaria vaccines may be a suitable strategy to improve vaccine's efficacy. molecular characterization of salivary gland's components responsible for this process is needed to understand the roles of saliva in blocking transmission of malaria parasites. immunogenic proteins from sg of aedes aegypti. saliva of aedes aegypti could inhibit viral infection in dendritic cells (dc) in vitro (ader et al. 2004). pre-sensitization of dc with saliva could enhance the inhibition of infection. additionally, the same group also demonstrated that necrosis of dc was reduced after administration of aedes aegypti’s saliva. other effects of that application are the increase of il12 p70 and tnf-α production in cell culture. the data suggested the protective roles of aedes aegypti salivary fig 2 results of western blot analysis showed two specific proteins from sg ae. aegypti (31 & 56 kda) recognized by only human antibody from sera of people living in endemic area or sera of dengue patient. sera from people living in non endemic area were taken from japanese who had never traveled to tropical countries (oktarianti et al. 2013; 2014). volume 7, 2013 microbiol indones 189 investigated. acknowledgment we are very grateful for the financial supports in research on the development of tbv for malaria and dengue from risbin iptekdok depkes, l'oreal unesco fwis, department of higher education (dikti), ministrry of research and technology (insentif ristek) as well as lembaga penelitian universitas jember references ader db, celuzzi c, bisbing j, gilmore c, gunther v, peachman kk, rao m, bavir d, sun w, palmer dr. 2004. modulations of dengue virus infection of dendritic cell by aedes aegypti saliva. viral immunol. 17(2):252-256. doi:10.1089/0882824041310496. andrade bb, teixeira cr, barral a, barral-netto m. 2005. haematophagous arthropod saliva and host defense system: a tale of tear and blood. an acad bras cienc. 77(4):665-693. doi:10.1590/s0001-376520050004000 08. arcá b, lombardo f, de lara capurro guimarães m, della torre a, dimopoulos g, james aa, coluzzi, m. 1999. trapping cdnasencoding secreted proteins from the salivary glands of the malaria vector anopheles gambiae. proc natl acad sci usa. 96:1516-1521. doi:10.1073/pnas.96(4):1516-1521. armiyanti y, soraya i, vinny, senjarini k. 2012. efek penghambatan ekstrak kelenjar saliva nyamuk 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using combination of salivary components known to inhibit virus infection and human antibodies against salivary immunosupression factors would be an effective and efficient strategy to combat malaria, df and possibly also other mosquito borne-diseases in tropical countries such indonesia. conclusion and outlook to initiate eradication of two major mosquitoes borne-diseases in indonesia such as malaria and df, a comprehensive strategy is needed. chemical intercession and pathogen based vaccine development may not be sufficient to halt transmission. vectorbased strategy could be another alternative, because it targets not only an essential step in the transmission process, but also blocking the spread of pathogens, thus preventing rather than treating the illness. reduction in the rates of parasitaemia in mouse model vaccinated by salivary extract from a. aconitus and a. maculates indicated the potential role of vector’s saliva as a new vaccine target and novel strategy against malaria. many reports suggested that any measure that limits parasite densities will reduce the morbidity and mortality associated with malaria infection (mcerroy et al. 1994). the existence of specific proteins with mw 31 and 56 kda that may serve as important factors to confer resistance to dengue infection, suggesting their important role in human-virus-pathogen interaction. these are the first reports on exploration of salivary glands from indonesian mosquito’s vector. therefore, to further elucidate the role of host's salivary components in establishing and/or inhibiting infection, the predominant effectors mechanism in host immune response and molecular characterization of those specific sg’s protein should be further 190 kartika microbiol indones f. human igg response to a salivary peptide, gsg6-p1, as a new immuno-epidemiological tool for evaluating low-level exposure to anopheles bites. malar j. 8:198. doi:10.1186/1475-2875-8-198. ramirez jl, garver ls, domopoulos g. 2009. challenges 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junkum a, saeung a, et al. proteomic analysis of salivary glands of female anopheles barbirostris species a2 (diptera: culicidae) by two-dimensional gel electrophoresis and mass spectrometry. parasitol res. 111:1239-1249. kamhawi s, belkaid y, modi g, rowton e, sacks d. 2000. protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. science. 290:13511354. doi:10.1126/science.290(5495):1351-1354. king jg, kenneth d, vernick, julian fh. 2011. members of the salivary gland surface protein family (sgs) are major immunogenic components of mosquito saliva. jbc papers in press. 1-21. limesand kh, higgs s, pearson ld, beaty bj. 2003. effect of mosquito salivary gland treatment on vesicular stomatitis new jersey virus replication and interferon alpha/beta expression in vitro. j med entomol. 40(2):199-205. doi:10.1603/0022-2585-40.2.199. lombardo f, ronca r, rizzo c, mestres-simon m, lanfrancotti a, curra c, fiorentino g, bourgouin c, ribeiro jm, petrarca v, ponzi m, coluzzi m, arcà b. 2009. the anopheles gambiae salivary protein gsg6: an anopheline-specific protein with a blood-feeding role. insect biochem mol biol. 39(7):457-466. doi:10.1016/j.ibmb.2009.04.006. mcelroy pd, beier jc, oster cn, beadle c, sherwood ja, oloo aj, hoffman sl. 1994. predicting outcome in malaria: correlation between rate of exposure to infected mosquitoes and level of plasmodium falciparum parasitemia. am j trop med hyg. 51(5):523-532. nurdian y. 2004. identifikasi tempat-tempat perindukan dan kepadatan vektor demam berdarah dengue (dbd) pada beberapa lokasi di kota jember. jurnal biomedis, 2(1):13-22. oktarianti r, fatchiyah, aulani’am, senjarini k. 2013. individual human sera response against protein extracts from salivary gland of aedes aegypti. international conference on biological sciences. ugm. yogyakarta. oktarianti r, nguyen hh, czerkinsky c. senjarini k. human immune response of people living in endemic area dhf as indicator of immunogenic protein against protein salivary gland of aedes aegypti. 2014. 6th asean congress of tropical medicine and parasitology. malaysian society of parasitology and tropical medicine. kuala lumpur. poinsignon a, cornelie s, ba f, boulanger d, sow c, rossignol m, sokhna c, cisse b, simondon f, remoue volume 7, 2013 microbiol indones 191 1: 186 2: 187 3: 188 4: 189 5: 190 6: 191 5 no. 281 (happy).pmd protein patterns in arbuscular mycorrhizal roots and non-mycorrhizal roots of oil palm seedling happy widiastuti1∗, nampiah sukarno2, and latifah kosim darusman2 1balai penelitian bioteknologi perkebunan indonesia, jalan taman kencana no. 1, bogor 16151, indonesia 2faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia a comparison of the protein patterns in root extracts from none mycorrhizal and mycorrhizal oil palm roots has been made. the polypeptides were analyzed every three weeks up to 11 weeks. a factorial design of fungi species (no mycorrhizal, acaulospora tuberculata, gigaspora margarita) and with or without fertilizer was assessed. the result showed that specific polypeptides were detected in primary and secondary roots. in unfertilized oil palm root, a 60 kda polypeptide was detected while it was abcent in fertilized root. inoculation of a. tuberculata with the addition of fertilizer application yielded a specific 26.7 kda polypeptide in primary root on the 11th week after inoculation. a specific 64.2 kda polypeptide of g. margarita was detected in unfertilized secondary root also on the 11th week. key words: polypeptide, elaeis guineensis, acaulospora tuberculata, gigaspora margarita, fertilizer _____________________________________________ _________________ ∗ corresponding author, phone: +62-251-324048, fax: +62-251-328516, e-mail: happywidiastuti@yahoo.com arbuscular mycorrhizal obligate biothrop fungi that form mutualistic symbiosis with many of the important agricultural plants such as palm. the formation and function of arbuscular mycorrhiza (am) induced considerable morphological, physiological, biochemical, and molecular changes for both the host and the symbion (samra et al. 1997). these changes are presumably the result of complex sequence of interactions between the fungus and the plant’s roots. as in the microbe and plant interactions, regulation of these interactions requires a continuous exchange of signals between both partners, which when perceived through the corresponding receptors would induce a cascade of events, leading to changes in the expression of certain genes (benabdellah et al. 2000). pacovsky (1989) reported that during the early infection there are specific polypeptides. the results showed that the quantity and quality of plant protein shifted in response to the physiological changes resulting from infection by n 2 fixing bacteria or by endomycorrhizal fungi. moreover, samra et al. (1997) showed that there were 12 polypeptides in the mycorrhiza compatible to pea genotypes which were never observed in root extract from the mycorrhiza resistant mutant. the induction and accumulation of these polypeptides seem to be more correlated to the establishment of the functional symbiosis than to the recognition stage and apresorium formation. ferrol et al. (2002) showed that the expression of plasma membrane h+-atpase genes was regulated differentially in leaves and root both in wild type and mycorrhiza defective tomato plants. analysis of 2d-page gels revealed that am colonization induced changes in protein synthesis and the sequence of polypeptide showed a 75% identity with the nterminal sequence of the 69 kda catalytic subunit of the vacuolar type h+-atpase of plants (benabdellah et al. 2000). interaction between host and fungi in am fungi symbiosis with oil palm has never been studied extensively. understanding the mechanism of interaction is important and necessary in order to get more benefit from the symbiosis mechanism. therefore, it is a necessity to see the changes and gene expression involved in the infection process of the symbion. the ability to quantify protein involved in specific mycorrhizal symbiosis could be used to examine the changes in roots which were colonized with am fungi. the effect of am fungi symbiosis for the growth and nutrient absorption of oil palm has been reported before. however, the physiology of oil palm, especially the root’s polypeptides during the early infection, was never been reported. understanding the molecular and genetic basis of the plant-fungus interaction in a functional am symbiotic system is required for the identification of gene and it’s gene products involved in this process. in this paper we used sds page to study the effect of am colonization upon the oil palm polypeptide pattern. two compatible am fungi species were used in this work. materials and methods biological material and experimental design. plant material used were germinated oil palm of dura x pisifera from the indonesian oil palm research institute. acidic soil from cikopomayak, west java was used as planting medium (sterilized 3 x 8 hours intermitted overnight at 105 oc). spores and infected root fragments were used as inoculant raised on p. phaseoloides plants grown in a glasshouse. the experimental design used was a factorial complete blocked randomized design. two treatments assessed were am fungi species (acaulospora tuberculata and gigaspora margarita) and fertilizer (with and without fertilizer). these were used as the samples treatment. the dose of am fungi inoculant followed the report widiastuti et al. (2002), while the uninoculated treatment fertilizet as dose was recommended by lubis (1992). growth conditions. oil palms seeds were germinated in sterilized sand until they were 3-months-old. at the same time, the preparation of fresh inoculant was done by inoculating pueraria phaseoloides with am fungi. p. phaseoloides was grown in 60 cm x 60 cm sized polybags microbiology indonesia, april 2007, p 11-14 volume 1, number 1 issn 1978-3477 (10 kg absolute dried soil) without a hole containing sterilized acidic soil from cikopomayak. the oil palm was inoculated with am fungi by transferring to polybag. the plants were watered with pre-boiled water. plant harvesting was conducted at 3, 5, 8, and 11 weeks after inoculation. roots were washed and observed for the am infection, and analyzed for the polypeptide. a part of a root was sampled to determine the percentage of fungal colonization (koske and gemma 1989). the remaining root material was frozen at -20 oc before extraction for protein analysis. harvesting procedure and sampling. three plants from each treatment were taken and the roots were washed carefully with tap water and weighed for shoot and root of the plant. roots were pooled to make the root samples. primary and secondary roots were separately analyzed. protein extraction. freshly harvested oil palm roots were carefully cleansed in deionized water, weighed, and ground into fine powder in liquid nitrogen. subsequently the proteins were extracted. the protein content was determined by the bradford assay (bradford 1976) and bovine serum albumin (bsa) was used as the standard (samra et al. 1997). gel electrophoresis and staining. sodium-dodecylsulfate-polyacrilamide-gelelectrophoresis (sds page) was performed as described laemmli (1970). as much as 30 um protein was injected into the gel electrophoresis. gels were stained using the silver staining method (oakley et al. 1980). molecular weight of proteins were determined by using a commercial molecular weight marker kit (bio-red, munich germany). results development of am fungal infection. observation showed that the am fungi was absent in the uninoculated treatment while it was present in the inoculated plant. in the unfertilized treatment, am fungi infection was smaller compared to fertilized oil palm seedlings both in a. tuberculata and g. margarita (figure 1). in addition, there were differences of infection pattern between a. tuberculata and g. margarita. a. tuberculata infection was slower compared to g. margarita (widiastuti 2004). in addition, the results showed that am fungi infection in secondary root was higher compared to those in primary root (figure 1 and 2). generally, the enhancement of am fungi infection in secondary roots was higher after three weeks inoculation, while in primary roots the higher infection was found five weeks after inoculation, especially in g. margarita. however, the results showed that fertilizer enhanced am fungi infection. polypeptide pattern. the polypeptide pattern in inoculated root was slightly different compared to the uninoculated one. the same result was found between the fertilized and unfertilized oil palm roots. fertilizer also affected the root polypeptide pattern of inoculated seedling as well as uninoculated seedling. in general, polypeptide patterns of oil palm root in 3, 5, 8, and 11th week after inoculation were slightly different between treatment both in primary and secondary roots. however, there were only some polypeptides which appeared to be specific. sds page analysis of primary root in 11th 1 6 1 4 1 2 1 0 8 6 4 2 0 a m f u n g i c o lo n iz a ti o n ( % ) 0 3 5 8 11 weeks after inoculation a. tuberculata without fertilizer, 3 0 2 5 2 0 1 5 1 0 5 0 a m f u n g i c o lo n iz a ti o n ( % ) 0 3 5 8 11 weeks after inoculation weeks after inoculation is shown on figure 3. a polypeptide with a moleculer weight of 60 kda was present in unfertilized plant roots, but this polypeptide was not detected in fertilized plants (table 1). in addition, in the primary root of oil palm root inoculated with a. tuberculata and fertilized has a 26.7 kda polypeptide. this polypeptide was not found in plant root inoculated with fertilizer for a. tuberculata. this result showed that probably the polypeptide was specific for a. tuberculata colonies in fertilized roots (table 1). the observation of secondary roots on the 8th week after inoculation showed that there was a 60 kda polypeptide found in uninoculated oil palm root given no fertilizer (figure 4, table 2). the polypeptide was not found in the plant treated with fertilizer. however, there were no differences the polypeptide pattern of secondary and primary roots between inoculation with a. tuberculata and g. margarita. the same result was found in treatment with fertilizer in the two inoculated plants. sds page analysis of secondary oil palm root at the 11th week after inoculation showed that there were no differences in polypeptide pattern of the uninoculated root between fertilized and none-fertilized plants. a 35 kda polypeptide was present in palm root inoculated with a. tuberculata that was treated with fertilizer while it is absent in the plant that were not trated with fertilizer. in addition, a 64.2 kda polypeptide present in secondary roots that were 12 widiastuti et al. microbiol indones a. tuberculata with( g. margarita without fertilizer, g. margaritafertilizer, with fertilizer). a. tuberculata with( g. margarita without fertilizer, g. margaritafertilizer, with fertilizer). figure 1 am fungi colonization in primary root of oil palm. figure 2 am fungi colonization in secondary root of oil palm. a. tuberculata without fertilizer, data obtained, was conclude that infection of am fungi first took place in secondary roots followed by infection in primary roots. comparasion of polypeptide pattern between primary and secondary roots showed that a 60 kda polypeptide was detected from primary roots of uninoculated plants without treatment with fertilizer at week 11. however, it does not in the plant that were treated with fertilizer. similar results were found in observations at week 8 after inoculation with either a. tuberculata or g. margarita in which the 60 kda polypeptide was absent. the results suggested that there was a dynamic of pattern of polypeptide synthesis within oil palm roots. we suggest that the polypeptide was expressed in secondary roots in the first infection and followed by expression in primary roots. that’s the infection process was took place in secondary roots first, followed by infection in primary roots. sds-page analysis of primary roots harvested at the 11th week after inoculation with g. margarita that was not treated with fertilizer showed a specific polypeptide (64.2 kda). this polypeptide was also detected in secondary roots inoculated with g. margarita at week 11. this result is interesting since the same size of polypeptide was detected in two different root types within the same period. the result suggested that the response of oil palm to g. margarita inoculation both in primary and secondary root was typically the same. detection of similar polypeptide in both primary and secondary root showed that both root types have similar protein expression. pacovsky (1989) showed that in soybean inoculated with glomus fasciculatum has specific protein with the size of 16, 17, 18, 22, and 30 kda. samra et al. (1997) revealed that there were 12 polypeptides expressed in compatible peas. five polypeptides were detected in the early stage of symbiosis, kda 1 2 3 4 5 6 7 kda 2 0 0 14.5 21.5 3 1 4 5 6 6 97.4 1 1 6 6 0 26.7 figure 3 silver stained sds page of primary palm root extracts 11 week after inoculation. lane 1: marker, 2: uninoculated without fertilizer, 3: uninoculated with fertilizer, 4: inoculated with a. tuberculata without fertilizer, 5: inoculated with a. tuberculata with fertilizer, 6: inoculated with g. margarita without fertilizer, 7: inoculated with g. margarita with fertilizer. kda 1 2 3 4 5 6 7 kda 2 0 0 1 1 6 97.4 6 6 4 5 3 1 21.5 14.5 6 0 figure 4 silver stained sds page of secondary palm root extracts 8 week after inoculation. lane 1: marker, 2: uninoculated without fertilizer, 3: uninoculated with fertilizer, 4: inoculated with a. tuberculata without fertilizer, 5: inoculated with a. tuberculata with fertilizer, 6: inoculated g. margarita without fertilizer, 7: inoculated g. margarita with fertilizer. inoculated with g. margarita although the plant was treated with fertilizer. discussion infection of am fungi in secondary roots was higher compared to primary root (figure 1 and 2). the same result has been reported by widiastuti et al. (2003). based on the table 3 specific polypeptide developed at 11 week of secondary oil palm root size of specific polypeptide (kda) t r e a t m e n t a. tuberculata with fertilizer g. margarita without fertilizer 3 5 64.2 6 0 6 0 6 0 26.7 table 1 specific polypeptide developed at 11 week of primary oil palm root size of specific polypeptide (kda) t r e a t m e n t control: uninoculated without fertilization a. tuberculata without fertilizer g. margarita without fertilizer a. tuberculata with fertilizer table 2 specific polypeptide developed at eight weeks of secondary oil palm root size of specific polypeptide (kda) t r e a t m e n t control: uninoculated without fertilizer a. tuberculata without fertilizer g. margarita without fertilizer 6 0 6 0 6 0 3 5 kda 1 2 3 4 5 6 7 kda 2 0 0 1 1 6 97.4 6 6 4 5 3 1 21.5 14.5 64.2 figure 5 silver stained sds page of secondary palm root extracts at 11th week after inoculation. lane 1: marker, 2: uninoculated without fertilizer, 3: uninoculated with fertilizer, 4: inoculated with a. tuberculata without fertilizer, 5: inoculated with a. tuberculata with fertilizer, 6: inoculated with g. margarita without fertilizer, 7: inoculated with g. margarita with fertilizer. volume 1, 2007 microbiol indones 13 while others were observed later. it was thought that the induction and accumulation of these polypeptides seem to be more correlated to the establishment of the functional symbiosis rather than recognition stage and appresorium formation. benabdellah et al. (2000) showed that a specific polypeptide with a size of 69 kda in the plasma membrane of tomato root extract inoculated with am fungi. however, the role of the specific polypeptide detected in this research is not known. in addition, the origin of the polypeptide detected in this research is not known. it could be of fungal or plant. however, it was correlated with chitinase analysis and formation of am fungi organs especially the arbuscle (widiastuti 2004) it could be that the polypeptide detected in this research correlated with the final stage of recognition of symbion or early functional symbiosis. however, there was a different size of polypeptide detected in this study compared with the previously study. in this study the polypeptide was detected over a longer time (11 weeks) compared to the previously study. it seems likely that the different period of the appearance of specific polypeptide was correlated with the process of am fungi infection. however, the differences of am fungi species and host plant species suggested different characteristics of polypeptide and also the time of their detection. this research also showed that the expression of several polypeptides could be detected in same period. these are shown in figure 1, 2, and 3 both in fertilized and unfertilized oil palm and inoculated and uninoculated palm. but, from our study it is shown that the selected polypeptide was detected only in a certain period. this result in the same result as the result of garcia-garrido and ocampo (2002). in general, it has been shown that am fungi penetration inside roots and their development in roots involved a series of morphological and physiology differentiation in both the plant and am fungi. comparision of polypeptide pattern isolated from mycorrhizal and non-mycorrhizal oil palm root showed that in all fractions am colonization induced three major changes in oil palm root gene expression: up regulation and down regulation of some constitutive popypeptides already present in oil palm root and induction of some new polypeptides. this result was also shown in tomato as reported by benabdellah et al. (2000). however, at this point in time we do not know the role of the differentially expressed polypeptides. in addition, detection of polypeptide using sds-page in one dimension is not very sensitive. furthermore, the polypeptide induced in the early infection was very low and differentially expressed. acknowledgment financial support for this study was provided by indonesian agency for agriculture research and development, ministry of agriculture, republic indonesia through the apbn project. the authors wished to thank neneng and agustiningtyas wulandari for their excellent technical assistance. references benabdellah k, azcon-aguilar c, ferrol n. 2000. alteration in the plasma membrane polypeptide pattern of tomato roots (lycopersicon esculentum) during the development of arbuscular mycorrhiza. j exp bot 51:747-754. bradford mm. 1976. a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principal of protein dye binding. anal biochem 72:248-254. ferrol n, pozo mj, antelo m, azcon-aguilar c. 2002. arbuscular mycorrhizal symbiosis regulates plasma membrane h+atp ase gene expression in tomato plants. j exp bot 53:1683-1687. garcia-garrido jm, ocampo ja. 2002. regulation of the plant defence response in arbuscular mycorrhizal symbiosis. j exp bot 53:13771386. koske re, gemma jn. 1989. a modified procedure for staining roots to detect va mycorrhizas. mycol res 92:486-505. laemmli uk. 1970. cleavage of structural proteins during the assembly of the head of bacteriphage t4. nature 227:680-689. lubis au. 1992. kelapa sawit (elaeis guineensis jacq) di indonesia. pusat penelitian perkebunan marihat bandar kuala. pematang siantar. oakley br, kirsch dr, morris nr. 1980. a simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. anal biochem 105:361-363. pacovsky rs. 1989. carbohydrate, protein and amino acid status of glycine-glomus-bradyrhizobium symbioses. physiol plant 75:346-354. samra a, dumas-gaudot e, gianinazzi s. 1997. detection of symbiosisrelated polypeptides during the early stages of the establishment of arbuscular mycorrhiza between glomus mosseae and pisum sativum roots. new phytol 135:711-722. widiastuti h. 2004. biologi interaksi cendawan mikoriza arbuskula kelapa sawit pada tanah masam sebagai dasar pengembangan teknologi aplikasi dini [thesis]. bogor: institut pertanian bogor. widiastuti h et al. 2002. optimasi simbiosis cendawan mikoriza arbuskula acaulospora tuberculata dan gigaspora margarita pada bibit kelapa sawit. menara perkebunan 70:49-56. widiastuti h et al. 2003. arsitektur akar bibit kelapa sawit yang diinokulasi beberapa cendawan mikoriza arbuskula. menara perkebunan 71:26-39. 14 widiastuti et al. microbiol indones editorial board.cdr issn 1978-3477, eissn 2087-8575 volume 9, number 2, june 2015 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron koesnandar, 2016 chief editor debbie s retnoningrum, 2016 editorial board members antonius suwanto, 2016 brett neilan, 2016 dessy natalia, 2016 e awati arifin giri-rachman, 2016 rn friedhelm meinhardt, 201 6 john acton, 2016 managing editor is helianti, 201 , 20166 astutiati nurhasanah electronic editor iman rusmana, 2016 is helianti, 2016 business manager diana nurani, 2016 netty widyastuti sigit, 2016 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000, 200 000,-5 institutional rate (institution or library) (idr) 240 000, 400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print general executive board of indonesian society for microbiology 20 -20113 7 advisory board: prof. dr. pratiwi sudarmono, ph.d, sp.mk; dr. ir. listyani wijayanti; dr. roy a. sparringa, m.app.sc.; prof. dr. amin soebandrio, ph.d, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, m.sc.; dr. ir. koesnandar, m.eng.; prof. fedik president vice president i:: a. rantam, ph.d; dr. mardiastuti hw, m.sc.; diana nurani, m.si.; drs. nuki b. vice president general secretaries vice general secretaries ii: : : nugroho, m.si.; dr. niknik nurhayati; dr. debbie s. retnoningrum; dr. is helianti; prof. dr. witono treasurer scientific and publication committee: : basuki, m.sc.; prof. dra. netty widyastuti, m.si.; dr. ir. nur hidayat, mp; prof. dr. ir. eni harmayani m.sc.; dr. astutiati nurhasanah; siti zulaeha, s.si.; , certification committee: dr. siswa setyahadi; dr. ir. agustian; dra. dini riyandini, m.si. dr. ir. maman turjaman, de dr. andriansjah; dr. ir. trismilah, ; ; m.si.; dr. purwati, sppd, ph.d. prof. dr. ir. endang s. rahayu, ms; sri harjanti suhardai, ph.d; dr. puspita organization advancement and networking: lisdiyanti; dr. retno indrawati, drg., m.si.; alit pangestu, stp; dra. harmastuti sukiman, m.agr.; dra. mg. promo :tion and advocation committee isworo rukmi, m.kes.; jimmy hariantono, ph.d; ir. dwi kusuma indriani, mp; sugeng p sugiharto, m.sc.; rosdyana salim, a.md. kartini kramadibrata, 2016 koesnandar, 2016 maggy thenawijaya suhartono, 2016 maria inge lusida, 2016 neung tiaamroeng, 2016 norio kurosawa, 2016 raija laiho, 2016 ratih dewanti, 20 61 wellyzar sjamsuridzal, 2016 yuan kun lee, 2016 page 1 06 lg.cdr h1n1 influenza a viruses in pandemic 2009 are from mexico, south of america (xu et al. 2010). this virus firstly emerged in early april 2009 and spread rapidly around the world in june (belser et al. 2011; strengell et al. 2011). world health nation (who) assed the impact of this virus was moderate because the majority of infected patients experienced mild influenza like illness, but sporadic death found in individual with poor pre-existing immunity to h1n1 influenza virus. according to who, 18.000 cases of death caused by h1n1 in the world. (xu et al. 2010). h1n1 influenza a viruses belong to the orthomyxoviruses family (setiawan 2008; athmaram et al. 2011). this virus is an enveloped virus with eight segmented negative sense rna genome surrounded by a helical symmetry shell. influenza a virus genome encode eleven viral proteins which has a specific functions. hemagglutinin (ha), neuramidase (na), matrix m1 and m2 are known to have effects on the morphology of influenza virus particles. internal to the m1 matrix are found the nuclear export protein (nep; also called nonstructural protein 2, ns2) and the ribonucleoprotein (rnp) complex, which consists of the viral rna segments coated with nucleoprotein (np) and the heterotrimeric rna-dependent rna polymerase, composed of two “polymerase basic” and one “polymerase acidic” subunits (pb1, pb2, and pa) ( and palese 2008).bouvier h1n1 influenza a was a novel influenza virus which derived it gene from human, classical swine, and avian influenza (nelli et al. 2010). currently, available vol.9, no.2, june 2015, p 89-96 cloning and expression of ha1 gene of h1n1 influenza virus 2009 pandemic (h1n1pdm09) indonesia strain in the pichia pastoris expression system for the development of influenza vaccine 1 2 2,3 2,3 asri sulfianti , andi yasmon , budiman bela , and fera ibrahim* 1 biomedical science program, faculty of medicine, universitas indonesia, jalan salemba raya no. 6 jakarta, indonesia; 2 departement of microbiology, faculty of medicine, universitas indonesia, jalan salemba raya no. 6 jakarta, indonesia; 3 research center of virology and cancer health service pathobiology, universitas indonesia, jalan salemba raya no. 6 jakarta, indonesia among influenza viral proteins, hemagglutinin 1 (ha1) is the target for neutralizing antibodies which inhibit virus binding to receptor of target cells. this protein is widely developed as subunit recombinant vaccine. in this research, we expressed ha1 protein recombinant from dki271/2011 indonesian strain in pichia pastoris. the identity of this protein was confirmed by western blotting using anti-his tag and mouse specific antibody ha h1n1pdm09. the use of yeast p. pastoris as an alternative strategy to solve the problems which commonly found in influenza vaccine productions. expression protein in e. coli has been known to have many problems, while mammalian and insect cells requires special skills and relatively high cost. the analysis of ha1 gene sequences showed no mutation in epitope region which recognized by t dan b cells. further, this recombinant protein can be used as vaccine candidate in influenza vaccine development. key words: h1n1pdm09, hemagglutinin 1, influenza virus, pichia pastoris, vaccine dari sejumlah protein virus influenza, hemagglutinin 1 (ha1) adalah target antibodi penetralisasi yang menghambat pelekatan virus pada reseptor sel target. protein ini banyak dikembangkan sebagai vaksin subunit rekombinan. pada penelitian ini, telah diekspresikan protein rekombinan ha1 dari strain indonesia dki271/2011 pada pichia pastoris. identitas protein telah dikonfirmasi dengan western blotting menggunakan anti-his tag dan antibodi spesifik ha h1n1pdm09 tikus. penggunaan p. pastoris berperan sebagai strategi alternatif yang dapat mengatasai masalah umum pada produksi vaksin influenza. ekspresi protein di e. coli diketahui memiliki banyak masalah, sementara sel mamalia dan insekta membutuhkan teknik khusus dan relatif mahal. analisis sekuens gen ha1 menunjukkan tidak adanya mutasi di daerah epitope yang dikenali oleh sel t dan b. selanjutnya, protein rekombinan ini dapat digunakan sebagai vaksin kandidat dalam pengembangan vaksin influenza. kata kunci: h1n1pdm09, hemagglutinin 1, pichia pastoris, vaksin, virus influenza *corresponding author; phone: +62-85885266350; email: asrisulfianti88@gmail.com doi: 10.5454/mi.9.2.6issn 1978-3477, eissn 2087-8575 available online at http://jurnal.permi.or.id/index.php/mionline microbiology indonesia control measures for this virus are by antiviral and vaccine. however, because of drugs resistance and difficulties in threatening patient with severe, make antiviral is become not effective in controlling h1n1 virus in population (zu et al. 2012). so vaccine is another alternative tools to solve this problem (maikel et al. 2012). it is because not only can prevent the spread of viruses, vaccine also can mitigate the severity of illness and the impact of the disease. unfortunately, current available vaccine was produced in chicken embryo and cell culture which has a risk of allergic reaction, time consuming, and relative high cost. it has been a challenging task for pandemic influenza because of the insufficient time in preventing wide scale morbidity or mortality (athmaram et al. 2011). an approach to overcome the problem is development of vaccine based on genetic engineering. among viral proteins, ha1 is the target for neutralizing antibodies which inhibit virus binding to receptor of target cells. previously, we reported no substitution on 7 amino acid of ha1 gene of viruses isolated in 2009 (yasmon et al. 2012). recently, in 2013 we also reported the phylogenetic tree of virus isolated from 2009-2011 (sulfianti, 2013). based on this studies, all of viral strain showed conserved region of ha 1 gene particularly on 7 amino acid 2009 that are playing an important role in viral attachment to the host receptor. previous studies on bacterially-expressed ha protein of h5n1 avian influenza virus (aiv) reported an absence in it post translational modification such as glycosylation. this newly synthesized ha protein also showed are not likely to fold properly or trimerize like native ha molecule, which change the conformational epitope used to be recognized by immune responses. commonly, e. coli expressed recombinant protein is also characterized as inclusion bodies and requires careful optimization of the refolding condition. this may have an impact on the loss of recombinant protein and an increased costs of manufacture (sahdev et al. 2007; khow et al. 2012). other protein expression system that used in ha1 production is by mammalian and insect cells. but because the systems are still under development and clinical trial, these two systems are rarely used (athmaram et al. 2011). recently, pichia pastoris yeast become a new ideal organism to express viral antigens. this yeast has many advantages as eukaryotic expression (eg: protein processing, folding and posttranslational modification) and are technically faster, easier, and cheaper than another eukaryotic expression systems, such as mammalian and baculovirus (krainer et al. 2007). pichia pastoris can also express protein as secreted protein. this protein comprises the vast majority of the total protein in the medium, and serves as the first step in purification of the protein (athmaram et al. 2011). as one of the strategy to control pandemic influenza virus in indonesia, in this research we have demonstrated the cloning and expression of ha1 from ha1 dki271/2011 indonesian strain in pichia pastoris. the purpose of this research is to produce ha1 subunit recombinant protein which used as a subunit vaccine candidate in the development of influenza vaccine in indonesia. materials and methods viral ha1 gene. the ha1 gene of h1n1pdm09 strain a/jakarta/271/2011(h1n1pandemic2009) was amplified from ppiczα-a-ha1-02 recombinant vector. it was obtained from the department of microbiology, faculty of medicine, universitas indonesia. cloning of ha1 gene into ppiczα-a. the ha1 gene was re-amplified from ppiczα-a-ha1-02 following standard pcr conditions using a forward and reverse primer consisting of ecori and sfii restriction sites at their 5' end, respectively. ppiczα-aha1-02 was a recombinant vector contains tree fused genes, namely ha1, ma2, and ns1 of h1n1pdm09. the probability of mutation during amplification of ha1 gene was minimalized by used pfu high fidelity dna polymerase which has good accurate as dna polymerase. the amplicon was then digested by ecori and sfii, and cloned into ppiczα-a in e.coli. the bacterial clones containing new recombinant vector were verified by restriction digestion (ecori dan noti) and dna sequencing. this new recombinant vector was named by ppiczα-a-ha1 (h1n1pdm09). pichia pastoris transformation and selection of transformants. the recombinant vector (ppiczα-aha1 (h1n1pdm09)) was linearised by bstxi, and integrated at 5'aox1 locus on the p. pastoris gs115 genome (invitrogen) by electroporation (25 µf, 200 ω, 1500 v) (biorad, ca). this yeast was then plated onto yeast extract peptone dextrose (ypd) agar plates containing 100 µg/ml of zeocin (invitrogen, usa). the yeast transformants were subcultured on ypd broth. gene integration was verified by yeast genomic dna pcr with specific primers (5'swflseqf2 5'a gctcagtgtcatcatttgaa-3' and swflseq-r2 90 sulfianti et al. microbiol indones 5'-ccggctctactagtgtccag-3'). selection of putative transformants and differentiation a copy number of integrated were did by streaked this yeast transformants on the replica ypd agar plates containing -1 of 500 µg ml zeocin. the incubation was performed at 30 °c for two days. expression of rha1 in p. pastoris. the positive yeast transformants were inoculated into 2 ml of ypd -1 broth + ampicillin 50 µg ml and grown at 30 °c for 48 h at 250 rpm. addition of ampicillin to inhibit bacterial contamination during the protein expression. after 48 h, the cells were harvested by centrifugation at 5000 rpm for 5 m. the cells were washed three times with distillated water and moved to 10 ml buffered with 100 mm potassium phosphate medium ph 8,2 (bmmy). the cultures were induced with 1% methanol (v/v) for every 24 h until 120 h. after 120 h, the cells were harvested by centrifugation at 5000 rpm for 5 m in room temperature. the pellet contained cells were stored in -80 °c, and the supernatant contained soluble protein in medium were analyzed on 12% sds-page. western blot. the expressed of ha1 protein was confirmed by western blotting using anti-his detector tm nickel-hrp (kpl) and mouse anti ha1 polyclonal antibodies. 20x concentration of supernatant protein by peg (polyethylene glycol) was run on sds gel and transferred onto a nitrocellulose membrane. the membrane was blocked with blocking buffer containing bsa 1% and tbst 1x (kpl) at 4 °c for 1618 h. the membrane was then incubated with a 1:8000 tm anti his (his detector nickel-hrp) or 1:8000 mouse anti ha1 polyclonal antibodies at room temperature for 1 h. the membrane was washed twice with washing buffer containing tbst 1x for 5 min. membrane was then incubated in tmb membrane substrate at room temperature for 5-15 min. for membrane that detected by mouse anti ha1 polyclonal antibodies, it was incubated with hrp anti mouse igg as secondary antibody at room temperature for 30 min after washing twice. the reaction was terminated by adding sterile distillated water after protein band was detected in the membrane. results ha1 amplicon was showed as an expected single dna band corresponding to 1.076 bp (fig 1). the dna fragment consisted of ha1 gene (1.071 bp) and extension sequences (25 bp) at 5 and 3' end for ecori dan sfii sites. the amplicon was digested with ecori dan sfii and cloned into similary digested ppiczα-a vector (invitrogen). the clones which brings r e c o m b i n a n t p l a s m i d ( p p i c z α a h a 1 (h1n1pdm09)) were confirmed by restriction digestion with ecori dan and noti. it released a 3.593 bp ppiczα-a for vector and 1.129 bp for rha1 (fig 2). based on dna sequencing, this recombinant plasmid showed an inserted ha1 gene which had a correct inframe with vector (data not shown). electroporation p.pastoris with recombinant plasmid yielded ≥ 300 transformant per 2 µg of dna used. selection of this transformants resulted in 44 colonies showing resistance to ypd plate with 500 µg -1 ml zeocin. genomic dna pcr was performed by using specific primers resulted in amplification of 384 bp of ha1 gene (detection of ha1 gene). of 44 selected transformants, 23 clonal cells showed positive results (fig 3). the dna from non-recombinants did not show any amplification. unfortunately, variations in the amplicon band intensities of different clones cannot be attributed to difference in the transgene copy number. it happened because there was no standardization in dna templates which were used in pcr reaction. of 23 clones positive integration, only one clone (number 41) showed better expression of the ha1 recombinant protein. the ha1 recombinant was detected as a single band on sds-page with approximately 40 kda (fig 4). confirmation by western blotting using ha1 h1n1 polyclonal antibody from mice serum specific h1n1 and anti-his tag also showed a protein band with size of approximately 40 kda. the weight of the protein was smaller than the amino acid sequence prediction which yielded 48 kda. discussion pichia pastoris expression system in this research has been successfully expressed ha1 protein as soluble protein in supernatant culture. this protein comprises the vast majority of the total protein in the medium, and serves as the first step in purification of the protein (athmaram et al. 2011). the identity of this protein was confirmed by western blotting using antihis tag and mouse specific antibody ha h1n1pdm09. based on these detections, ha1 recombinant showed as a protein band with lower than 40 kda. this anti-tag recognized the marker of poly his tag on the downstream of recombinant ha1 protein, and the mouse specific antibody ha h1n1pdm09 detect the recombinant ha1 protein in supernatant culture. the size of these recombinant proteins was smaller volume 9, 2015 microbiol indones 91 fig 2 restriction analysis of recombinant ppiczα-a-ha1 (h1n1pdm09) using ecori and noti. lane 1-10: digestion product of dna recombinant plasmid. lane wt: digestion product of wild type dna ppiczα-a vector. lane ha1: amplification product of ha1 gene. lane m: dna ladder. bp: base pair. fig 1 amplification of ha1 gene from ppiczα-a-ha1-02 by pcr. lane 1: pcr with ppiczα-a-ha1-02 as dna template, lane 2: pcr negative control, lane m: dna ladder. bp: base pair. 92 sulfianti et al. microbiol indones fig 3 amplification ha1 gene from genomic dna of 44 yeast transformants by pcr. lane 1-44: amplification product of genomic dna pcr from pichia pastoris that transformed by ppiczα-a-ha1 (h1n1pdm09), clones no. 1 to 44. lane k+: amplification product of dna recombinant plasmid (ppiczα-a-ha1 (h1n1pdm09). lane k-: amplification product of dna genomic yeast which transformed by ppiczα wild type (negative control). lane m 100bp: dna ladder. bp: base pair. volume 9, 2015 microbiol indones 93 than amino acid sequence prediction, 48 kda. flint et al. (2000) described that it might happened because during the secretion of the recombinant ha1 protein, there was a process of cutting in the protein's alpha signal factor sequence. the cutting process resulted in a smaller protein size. the alpha signal factor cutting involves a signal peptidase enzyme and it happens during translocation in the reticulum endoplasm. the ha1 protein was only found in one clone (number 41). it was suggested that there had been a multicopy integration between ha1 gene and pichia genome in that particular clone. the multicopy integration caused the protein to be expressed and secreted more to the medium culture (athmaram et al. 2012). the recombinant protein was not expressed in the other clones. the first possible reason is spontaneous mutation in the aox1 locus or in the alpha signal factor sequence. spontaneous mutation in the aox1 locus of the recombinant plasmids prevented the process of homolog recombination between the plasmid and the yeast genome. therefore, the ha1 gene cannot integrate to the yeast genome and resulted in no protein expression (athmaram et al. 2012). the second reason is mutation in the alpha signal factor by means of pcr process. this mutation causes a failure to cut and bring the recombinant protein into the reticulum endoplasm. it prevents the protein in entering the reticulum endoplasm and therefore cannot be secreted through normal secretory pathway (murugan et al. 2013). the third reason is ha1 protein was retained in the reticulum endoplasm as inclusion bodies. this accumulation of this recombinant ha1 protein in the er limits the yield of ha1 secreted into the extracellular environment (athmaram et al. 2012). analysis of ha1 gene sequence showed variation that cause 4 amino acid differences to ha1 from influenza strain isolated in mexico 2009. two of these amino acids were suggested in changing the structural protein because it changed the hydrophobicity of the protein's amino acids (data not shown). comparison of amino acid sequences between ha1 recombinant, ha1 from mexico 2009, and several other ha1 sequences which were isolated in 2010 and 2011 (from jakarta, surabaya, china, india, thailand, and vietnam) showed no mutation on epitopes for b and t cells (data not shown); therefore, the recombinant ha1 protein will induce immunity responses that are crossfig 4 detection of ha1 expression secreted from recombinant p. pastoris by western blot using anti-his tag (a) and mouse specific antibody ha h1n1pdm09 (b). lane 41: yeast clone no. 41, showed ha1 recombinant protein band ha1 approximately 40 kda, lane k: yeast was transformed by ppiczα wild type (negative tm control), lane m (fig 4a): protein ladder (see blue plus2 ), lane m (fig 4b): protein ladder (chromatein prestained protein ladder). kda: kilo dalton. a b 94 sulfianti et al. microbiol indones reacted with other viral strains. with this ability, the subunit vaccine derived from this recombinant ha1 has a potential to be in charge in neutralization of influenza virus h1n1 pandemic 2009 that circulated in future. the recombinant ha1, which was secreted as an extra-cellular protein, also gives several advantages, such as easier and more economical method of isolation. it can increase effectivity of vaccine production, which in turn can prevent mortality and morbidity of the h1n1 influenza patients (murugan et al. 2013). acknowledgment national the authors express their gratitude to strategic research consortium as a funding research. references athmaram tn, saraswat s, santhosh sr, singh ak, suryanarayana ws, priya r, gopalan n, parida m, rao pv, vijayaraghavan r. 2011. yeast expressed recombinant hemagglutinin protein of novel h1n1 elicits neutralizing antibodies in rabbits and mice. virol j. 8(524):1-13. doi: 10.1186/1743-422x-8-524. athmaram tn, saraswat s, singh ak, rao mk, gopalan n, suryanarayana vv, rao pv. 2012. influence of copy number on the expression levels of pandemic influenza haemagglutinin recombinant protein in metyhlotrophic yeast pichia pastoris. virus genes. 45(3):440-51. doi: 10.1007/s11262-012-0809-7. belser ja, jayaraman a, raman r, pappas c, zeng h, cox nj, katz jm, sasisekharan r, tumpey tm. 2011. effect of d222g mutation in the hemagglutinin protein on receptor binding, pathogenesis and transmissibility of the 2009 pandemic h1n1 influenza virus. plos one. 6(9):1-7. doi: 10.1371/journal.pone.0025091. bouvier nm, palese p. 2008. the biology of influenza viruses. vaccine. 26(4):d49-53. flint j, enquist lw, racaniello vr, skalka am. 2003. p r i n c i p l e s o f v i r o l o g y : m o l e c u l a r b i o l o g y, nd pathogenesis, and control of animal virus, 2 edition, asm press, washington. khow o, suntrarachun s. 2012. strategies for production of active eukaryotic proteins in bacterial expression system. asian pacific j trop biomed. 2(2):159-162. doi: 10.1016/s2221-1691(11)60213-x. krainer fw, dietzsch c, hajek t, herwig c, spadiut o, glieder a. 2012. recombinant protein expression in pichia pastoris strain with engineered methanol utilization pathway. microb cell fact. 11(22):1-14. doi: 10.1186/1475-2859-11-22. lakdawala ss, lamirande ew, suguitan jr al, wang w, santos cp, vogel l. matsuoka y, lindsey wg, jin h, subbarao k. 2011. eurasian-origin gene segments contribute to the transmissibility, aerosol release, and morphology of the 2009 pandemic h1n1 influenza v i r u s . p l o s p a t h o g e n e s . 7 ( 1 2 ) : 1 1 4 . d o i : 10.1371/journal.ppat.1002443. murugan s, ponsekaran s, kannivel l, mangamoori ln, chandran d, villuppanoor alwar s, chakravarty c, lal sk. 2013. recombinant haemagglutinin protein of highly pathogenic avian influenza a (h5n1) virus expressed in pichia pastoris elicits a neutralizing antibody response in mice. j virol meth. 187(1):20-25. doi: 10.1016/j.jviromet. 2012.07.026. nelli rk, kuchipudi sv, white ga, perez bb, dunham sp, chang k. 2010. comparative distribution of human and avian type sialic acid influenza receptors in the pig. bmc vet res. 6(4):1-9. doi:10.1186/1746-6148-6-4. sahdev s, khattar sk, saini ks. 2008. production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. mol cell biochem. 307(1-2):249-264. setiawan im. 2008. vaksin virus influenza. majalah kedokteran indonesia. 58(12):517-524. strengell m, ikonen n, ziegler t, julkunen i. 2011. minor changes in the hemagglutinin of influenza a (h1n1) 2009 virus alter its antigenic properties. plos one. 6(10):1-13. doi: 10.1371/journal.pone.0025848. sulfianti a. pengklonaan dan ekspresi gen ha1 virus influenza h1n1 pandemi 2009 (h1n1pdm09) galur indonesia pada sistem ekspresi pichia pastoris untuk pengembangan kandidat vaksin influenza. jakarta: universitas indonesia; 2013. van der velden mv, aichinger g, pöllabauer em , löwbaselli a, fritsch s, benamara k, kistner o, müller m, zeitlinger m, kollaritsch h, vesikari t, ehrlich hj, barrett pn. 2012. cell culture (vero cell) derived whole-virus non-adjuvanted h5n1 influenza vaccine induces long-lasting cross-reactive memory immune response: homologous or heterologous booster response following two dose or single dose priming. vaccine. 30(43):6127-35. doi:10.1016/j.vaccine.2012. 07.077. xu l, bao l, lv q, deng w, ma y, li f, zhan l, zhu h, ma c, qin c. a single-amino-acid substitution in the ha protein changes the replication and pathogenicity of the 2009 pandemic a (h1n1) influenza viruses in vitro and in vivo. virol j. 7(325):1-7. doi:10.1186/1743-422x-7325. yasmon a, yulianti m, vivi s, beti ed, budiman b, fera i. 2012. five unique amino acide residues of hemagglutinin (ha) proteins of swine influenza a (h1n1) detected in 2009 in jakarta, indonesia. microbiol indones. 6(2):69-76. doi: 10.5454/mi.6.2.3. volume 9, 2015 microbiol indones 93 zu m, yang f, zhou w, liu a, du g, zheng l. 2012. in vitro anti-influenza virus and anti-inflammatory activities of the aflavin derivates. antiviral res. 94(3):217-224. doi: 10.1016/j.antiviral.2012.04.001. 96 elfita et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 3 457 ridawati.cdr genetic diversity of osmophilic yeasts isolated from indonesian foods with high concentration of sugar ridawati , betty sri laksmi jenie , ita djuwita , wellyzar sjamsuridzal 1 1 2 3* isolation of osmophilic yeasts from a total of 70 samples consisting of jam, sweet condensed milk, honey, sweet soy sauce, and palm sugar was conducted. sixty-eight osmophilic yeasts were isolated from strawberry jam, pineapple jam, and honey from south sumatera. no yeast was obtained from condensed milk, honey from sumbawa, sweet soy sauce, and palm sugar. sequence analysis based on the its region showed that isolates were identified as five species belong to two genera, and . those isolates were distributed in 5 species, , and was the predominant species in south sumatera honey, while group was predominant species in jams. those species were reported as osmophilic yeasts. in both jams and honey we found and , whilst was found only in pineapple jam. phylogenetic analysis based on sequence of its region showed that most of the osmophilic yeasts (67 out of 68 isolates) were located in the phylum and only one isolate nn38 from pineapple jam was located in the phylum . key words: genetic diversity, osmophilic yeasts, food with high sugar concentration and 1 2 3 department of food science and technology, faculty of agricultural technology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; department of anatomy physiology and pharmacology, faculty of veterinary medicine, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; department of biology, faculty of mathematics and natural sciences, universitas indonesia, ui campus, depok 16424, indonesia candida sterigmatomyces c. metapsilosis, c. etchellsii c. parapsilosis, c. orthopsilosis, s. halophilus. c. etchellsii c. parapsilosis c. parapsilosis c. metapsilosis c. orthopsilosis ascomycota sterigmatomyces halophilus basidiomycota *corresponding author and recent address, department of food and nutrition, faculty of engineering, universitas negeri jakarta, jalan rawamangun muka, jakarta 13220, indonesia; phone: +62-21-4715094; fax: +62-21-4715094; email: ridawatis@yahoo.co.id spoilage of food products by microorganisms results in the waste of valuable resources and causes large financial losses for the food industry that will inevitably affect the consumer. high sugar products are targets for spoilage by yeast. various kinds of traditional high sugar foods in indonesia such as jam, sweet condensed milk, sweet soy sauce and honey have the potential to be spoiled by yeast. some yeasts have the ability to grow or cause spoilage in foods, especially products with low ph, generally 5.5 or lower, and other products by the presence of sugars as carbon sources. the adverse media inflicted by the low ph, low oxygen levels, and high sugar concentration of these products prevents the growth of most organisms. however, these hurdles do not inhibit the growth of many spoilage yeasts, especially osmophilic yeasts. the reported food and beverage spoilage yeasts include a relatively large number of species representing both ascomycetes and basidiomycetes as has been published. many yeasts were frequently described as being osmophilic, suggesting a habitat restricted to a high solute (in this case, sugar) environment. the a of typical concentrated syrups (more than 50% soluble solids) ranges from 0.82-0.94. depending upon the solute and its concentration (salt, sugar, or glycerol), some yeasts are able to grow at relatively low temperatures. many osmophilic yeasts had been identified as spoilage agents in fruit concentrates and juices (arias 2002; fitzgerald 2004) and syrup (ancasi 2006). osmophilic yeasts can create a spoilage problem in food storage, because they are able to grow under conditions of high salt or sugar content. aside from their ability to grow in the restrictive habitat of high osmotic pressure, many yeasts w et al. et al. et al. are also extraordinarily resistant to common preservatives such as sulphur dioxide, sorbic acid, benzoic acid and acetic acid (warth 1985). yeasts can become adapted to preservatives by preexposure to low levels of these materials. many studies regarding these microorganisms have been carried out and indicate a desire to understand the diversity of yeasts in high sugar foods to prevent food spoilage. morphologically and physiologically, these osmophilic yeasts were found to be extremely similar to one another, with species differentiation being based mainly on the high sugar content of media and the ability to form ascospores or pseudomycelia. in order to understand the species interrelationships of these yeasts better, a phylogenetic study based on its sequence of 18s rdna was carried out upon all strains isolated in this work. it is important to understand the characteristics of the isolated yeasts. molecular comparisons have provided an understanding of yeast phylogeny that was not possible from analyses of morphology and physiology. gene-sequence determinations have also provided a rapid, accurate means for identification of individual strains to the species level. sequencing of species-diagnostic genes represents the most accurate means for isolate identification, and several rapid identification methods using molecular probes based on these sequences have been developed and are becoming available to food microbiology laboratories. over the last decade, different polymerase chain reaction (pcr) techniques and nuclear sequence analysis have been used for identifying many yeasts for the study of phylogeny. the its region of ribosomal dna had been used for this purpose. the use of 5.8s-its sequence is the best tool for rapid and accurate identification of yeast. some studies have reported that the its1 and its2 regions had higher degrees of variability and therefore higher differentiating power than d1/d2 region for closely related species (tavanti 2005;et al. issn 1978-3477 vol 4, no 3, dec 2010, p 113-118 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.4.3.3 kocsube 2007). partial sequencing of its region is convenient for the rapid characterization of yeast. using sequences available in the genbank database, sequences obtained could be used to compare the similarities of gene fragments among the currently recognized yeast species. in view of these issues, the objectives of the present study were (i) to obtain isolates of osmophilic yeasts from high sugar food products, (ii) to identify their genetic diversity and to estimate the taxonomic boundaries of the osmophilic species using the its rdna sequence information, and (iii) to reveal the phylogenetic relationship between osmophilic yeast isolated from many kinds of foods with high concentration of sugar in indonesia. a total of 70 samples of local high sugar food products (jams, sweet condensed milk, honey, sweet soy sauce, and palm sugar) were collected from different markets in jakarta, bogor, sumbawa, and south sumatera. osmophilic yeasts were isolated from these samples. a procedure for isolation and preparation of osmophilic yeast was the use of potato dextrose broth (pdb) and potato dextrose agar (pda) (oxoid) enriched with various concentrations (10-60% w/v) of sucrose were conducted. subsequently, the colonies which formed were isolated and purification by the quadrant streak method. total dna was extracted from osmophilic yeasts by boiling method according to sjamsuridzal and oetari (2003). the dna extract was amplified by pcr technique using its4 and its5 primers (white 1990). pcr reactions were prepared in 25 µl total volume containing 9 µl dna template; 0.5 pmol of each its4 and its5 primer; pcr buffer (10 mm tris-cl ph 9.0, 50 mm kcl); 200 µm (each) datp, dgtp, dctp, and dttp; 1.5 mm mgcl ; 2.5 unit puretaq dna polymerase (ge healthcare kit). the amplification procedure of the rdna its region was carried out using a published method (white 1990). the amplification profile consisted of 40 cycles of denaturation at 95°c for 15 sec, primer annealing at 58°c for 30 sec, and primer extension at 68°c for 1 min; followed by post extension at 68°c for 10 min. pcr products were analyzed on 2% (w/v) agarose gel, stained with ethidium bromide, and visualized under uv light using the chemidoc gel system (biorad). dna for sequencing was amplified using a 9600 perkin-elmer cetus thermal cycler apparatus as described by white (1990) with reverse primer (its5). dna sequencing was performed on abi prism 310 dna sequencer with protocols supplied by the manufacturer. the sequencing results were compared with dna sequences from genbank database at national center of biotechnology information (ncbi) using the basic local alignment search tool (blast) program for screening sequence similarity (altschul . 1997). sequence alignments were performed by clustal x1.83 program. et al. et al. et al. et al. et al materials and methods isolation of osmophilic yeasts. dna extraction and pcr analysis. partial sequencing of its region and phylogenetic analysis. 2 ascomycota basidiomycota basidiomycota ascomycota candida parapsilosis candida sterigmatomyces c. metapsilosis , c. etchelsii c. parapsilosis , c. orthopsilosis , s. halophilus . c. etchellsii c. parapsilosis c. parapsilosis c. metapsilosis c. orthopsilosis the phylogenetic tree was constructed using the neighborjoining (nj) method (saitou and nei 1987). the isolates could not grow in media supplemented with 10, 20, and 30% (w/v) sucrose, whereas most of the isolates showed a higher cell number at 50% (w/v) sucrose, and some growth was found in the presence of 40% (w/v) sucrose and even in the presence of 60% (w/v) sucrose. the result showed that osmophilic yeast strains grew better on pda enriched with 50% (w/v) sucrose than pda alone. colony size of the yeast grown in the media with lower concentration of sugar were smaller than in the media with a high sugar concentration (results not shown). a feature of the yeast strains isolated from the high sugar foods were ascomycetous characteristics. these yeasts are osmophilic and could frequently grow under extreme conditions. sixty eight osmophilic yeasts could be isolated from strawberry jam (a 0.84), pineapple jam (a 0.84), and honey from south sumatera (a 0.72). colonies were white to tannish white, creamy, shiny, smooth and produced a faintly acidic aromatic odor. no yeast was obtained from sweet condensed milk (a 0.84), sweet soy sauce (a 0.68), honey from sumbawa (a 0.67), and palm sugar samples (a 0.63) (data not shown). amplification of the its1-5.8s-its2 regions from the sixty eight osmophilic yeasts generated pcr products ranging in size from 400 to 500 bp. the profiles of bands representing the osmophilic yeasts were shown in fig 1. one isolate nn38) appeared to have a different profile from the others, so this isolate may be a different species. phylogenetic trees of rdna sequences were constructed based on the distance matrix methods as stated in the methodology. sixty-seven of the sequences clustered in the phylum , however one isolate formed a different branch far away from the others and clustered within the phylum (fig 2). thirteen yeast strains, representing four -lineage and nine -lineage and their accession numbers obtained from genbank are listed in table 1. comparative analysis of the its region data revealed that the fifty-two strains of group were highly related to one another, exhibiting sequence identity values that ranged from 99 to 100% (data not shown). sequence analysis based on its region showed that 68 isolates were identified as five species belonging to two genera and . those isolates were distributed in 5 species, i.e. (23) (15), (25) (4) and (1) was the predominant species in south sumatera honey and the group was the predominant species in strawberry jam and pineapple jam. in both jams and honey, and were found, while was found only in pineapple jam (data not shown). results isolation of osmophilic yeasts. analysis of the its regions and phylogenetic analysis. w w w w w w w ( 114 ridawati et al. microbiol indones fig 1 gel electrophoresis images showing pcr products of yeast its region rdna (about 400-500 bp in size). m, marker; nn14, nn23, nn26, nn27, nn38, nn39, nn31, isolate from pineapple jam. fig 2 dendrogram showing the phylogenetic relationship of isolates from jam and honey with related yeast species based on its region. bootstrap values, expressed as percentages of 1000 replications, are given at branch points (only values >50% are shown). candida parapsilosis and c. etchellsii ascomycota m n 14n nn23 nn26 nn37 nn38 nn39 nn31 500 bp 400 bp from fruit jam from fruit jam from fruit jam from fruit jam from honey basidiomycota volume 4, 2010 microbiol indones 115 discussion osmophilic yeasts have been isolated from indonesian foods with high sugar concentration sugar and water activity between 0.72-0.84. typically, the shelf-life of these products could be extended by pasteurization, evaporation, and/or the addition of chemical preservatives such as acid or benzoic. however, these preservation methods do not inhibit the growth of many spoilage-causing yeasts, especially osmophilic yeasts. a total of 68 isolates could be obtained from various samples. the results showed that osmophilic yeast strains grew better on pda enriched with 50% (w/v) sucrose than pda alone. water activity refers to the availability of water in foods or beverages and represents the amount of water that was available for microorganisms growth. a majority of food spoilage yeasts had been isolated from (foods with 65% (w/v) sucrose or 15% (w/v) nacl or foods with a 0.87) and could survive on a 0.80-0.85 as reported by beuchat (1983). in most cases, yeast contamination in these products occurred on the farm and they were therefore present in the fresh products. no yeast was obtained from sweet condensed milk (scm), even though these samples had a water activity value of 0.84. aside of water activity, the main factor causing a lack of yeast obtained from scm was the processing of these products. in scm manufacturing, heat-treated milk containing sugar is taken to the evaporator, where it is concentrated by heating the mixture 93.58 c for 3 min depending on the desired outcome of viscosity, bacteriological quality, and other physico-chemical properties, then evaporated, cooled, and tinned or bottled under hygienic conditions (beuchat 1983; ali and randal 2002). the method for preserving scm using aseptic technology means contaminant osmophilic yeast could not survive in these products. food with low water activity w w o w w w sweet soy sauce (a 0.68), honey from sumbawa (a 0.67) and palm sugar (a 0.63) have low water activity. no yeasts were obtained from these products. some unique foods like sweet soy sauce appear to be a high moisture product, but because salt, sugars, or other ingredients bind the water content, their water activities become quite low. the presence of sugar in such a high concentration will inhibit the growth of yeast contaminants. from sequencing results, a total of 68 strains-sequencedata were aligned together with known sequences of several species from the genbank database. a phylogenetic tree was constructed using the nj method as stated in the methodology, using as an outgroup. all species of and were closely related and formed a single cluster (fig 2). the species of this cluster were isolated from various sources, including honey, strawberry jam, and pineapple jam. they are widely distributed in nature and are common spoilage yeast in the food industry. tavanti . (2005) and andrew . (2009) had reported that all isolates within each group ( ) had similar d1/d2 sequences of the large (26s) ribosomal rna gene, and the 5.8s rdna sequences of isolates from all three groups were 100% identical. only minor differences were observed in sequences among the three groups, and considerable dissimilarities were found in the its1 sequences. from honey and strawberry jam were in a cluster with , (fig 2). this cluster was an assemblage of phylogenetically diverse species with large interspecific divergences. other yeasts used in the phylogenetic analysis were isolated from various sources, flowers and pollen nectar, wine, and grapes musk (table 1). many of these species are associated with insects, specifically bees, bumblebees and leaf-cutter bees, and many have been reported as the causative agent of spoilage of sugary foods, such as fruit juices and concentrates. many of the yeasts related to group have been implicated in the spoilage of foods, particularly sugary, lowfoods. spoilage of high sugar commodities has been reported and caused fruit juice and soft drink spoilage, . had been found in concentrated juice and . had been found in condensed milk as reported by stratford (2002). spoilage by . was relatively uncommon and it is considered to be an opportunist spoilage yeast. and . are spoilage yeasts in fruit juice concentrates and tomato sauce (deak and beuchat 1993). and have been isolated from pollen-nectar and bees. (acc gm620527) had been used as fermenting yeast for xylitol production (erisema . 2006). among the less frequently encountered species, had been isolated from fermented cocoa beans (daniel . 2009). ana . (2003) have isolated a new novel species from honey, pollen bee and nectar. the sequence of the d1/d2 domains of the large-subunit rdna showed that this novel species belongs to the cluster, and was most closely related to . . s. halophilus c. parapsilosis, c. orthopsilosis, c. metapsilosis et al et al c. parapsilosis, c. metapsilosis, c. orthopsilosis c. parapsilosis its2 candida etchellsii c. versatilis c. zemplinina, c. stellata, c. cellae c. parapsilosis a candida davenportii c. parapsilosis c bombicola c lactis condensii et al. c lactis condensi candida stellata c magnoliae candida riodocensis c. cellae candida parapsilosis et al c. orthopsilosis et al et al starmerella c. etchellsii w species name accesion number source candida versatillis ab196240 miso* c. zemplinina ay160762 wine c. stellata ay160766 grape musk c. bombi ab013576 bumblebee c. cellae ay861673 pollen nectar c. etchellsii ab196223 miso*` c. parapsilosis dq668350 marine environment c. metapsilosis ef190228 marine environment c. orthopsilosis eu557371 blood sample sporobolomyces sp. ay313072 leaf surfaces rhodotorula acuta ab038054 marine sterigmatomyces elviae ay373390 seawater s. halophilus af444556 marine and air table 1 species name, source, and genbank accession numbers of yeasts included in the phylogenetic analysis * a thick fermented paste made of cooked soybeans, salt, and often rice or barley, and used especially in making soups and sauces. 116 ridawati et al. microbiol indones candida parapsilosis, c. metapsilosis, c. orthopsilosis c. parapsilosis candida c. parapsilosis, c.metapsilosis, c orthopsilosis, c. etchellsii. candida c. parapsilosis, c. metapsilosis, c. orthopsilosis c. parapsilosis c. parapsilosis c. parapsilosis . et al et al et al. et al. et al. et al. . and strains collectively formed a distinct lineage which, apart from displaying a loose association with the group, showed little phylogenetic affinity to any other species examined (fig 2). kurtzman and robnett (1998) observed that strains showing greater than 1% difference in the its region were usually different species, whereas strains with zero to three nucleotide differences were either conspecific or sister species. alignment of contiguous yeast sequences demonstrated that both singlenucleotide differences and short lengths of sequence diversity due to insertions or deletions existed in the its regions among the species. a sequence similarity of 99-100% exists among . and the genus was the largest in the number of species of the yeast genera and was present in almost every environment. yeasts of this genus are abundantly distributed in nature. this genus is distributed across the ascomycetous yeast domain, overlapping with other genera according to phylogenetic analysis using ribosomal genes (kurtzman and robnett 1998). strains collectively form a distinct lineage which apart from displaying a loose association with group, showed little phylogenetic affinity to any other of the species examined. the sixty-seven isolates could be divided into the group and the nongroup, with the exception of one isolate which was clustered on a separate branch from all of data obtained in this study, we propose that indonesian high-sugar foods contain a lot of osmophilic yeasts. results of this analysis also indicated that most of the isolates belonged to the same clade as the isolates identified in the previous research such as isolates from miso (suezawa . 2006), pollen and flower (sjamsuridzal . 2010), nectar (matthias 2004), bumblebee (soon 2003; michael 2004), grape musk (belinda 2008), the air (cristina 2006), seawater (tekolo 2010) it is suggested that the existence of osmophilic yeasts diversity in products and manufacturing environment needs further investigation. such knowledge will aid the development of control strategies for food spoilage. we offer our regards and blessings to the department of biology, faculty of mathematics and natural sciences, university of indonesia 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a. 2003. rapid preparation of fungal and bacterial genomic dna for pcr. hayati 10:122-4. sjamsuridzal w, oetari a. kanti a, saraswati r, nakashima c, widyastuti y, katsuhiko a. 2010. ecological and taxonomical perspective of yeast in indonesia. microbiol indones 4:60-8. stratford m, bond cj, james sa, roberts in, steels h. 2002. sp. nov., a potential softdrinks spoilage yeast isolated from a wasp. int j syst evol microbiol 52:1369-75. soon gh, kyung sb, michel h, andreas t, marc al. 2003. sp. nov., a yeast associated with flowers and bumblebees. int j syst evol microbiol 53:367-72. suezawa y, kimura i, inoue m, gohda n, suzuki m. 2006. identification and typing of miso and soy sauce fermentation yeasts, and , based on sequence analyses of the d1/d2 domain of the 26s ribosomal rna gene, and the region of its1, 5.8s ribosomal rna gene and its2. biosci biotechnol biochem 70:348-54. tavanti a, davidson ad, neil arg, martin cjm, odds fc. 2005 and spp. nov. to replace groups ii and iii. j clin microbiol 43:284-92. candida parapsilosis candida metapsilosis candida orthopsilosis candida parapsilosis sensu lato candida zemplinina candida stellata. candida davenportii candida kunwiensis candida etchellsii c. versatilis candida orthopsilosis candida metapsilosis candida parapsilosis . volume 4, 2010 microbiol indones 117 tekolo om, jean m, alfred b, bernard ap. 2010. the osmotic stress tolerance of basidiomycetous yeasts. fems yeast res 10:482-91. warth ad. 1985. resistance of yeast spesies to benzoic and sorbic acid and to sulfur dioxide. j food prot 48:564-9. white tj, burns t, lee s, taylor j. 1990. amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis ma, gelfand dh, sninsky jj, white tj, editors. pcr protocols a guide to methods and applications. san diego: academic pr. p 315-22. : 118 ridawati et al. microbiol indones 04 fahmi.cdr cabbage has an important nutritional value as a source of vitamin a and c, protein, lipid, carbohydrates and fiber needed by human body (sastrosiswojo et al. 2005). production of cabbage in indonesia had been steadily increasing from 1 323 702 ton/ha in 2008 to 1 432 318 ton/ha in 2012 (directorate general of horticulture 2013). however, cabbage production is hampered by diseases and pests that reduce the quality and quantity of the cabbage. one of the major diseases of cabbage in indonesia and other asian countries is black rot which is caused by xanthomonas campestris. the leaves of cabbage attacked by x. campestris shows black spots and in short time the plant will die simultaneously (semangun 1989).the use of pesticides has been done to eradicate the problems of disease in plants, as it is considered effective and fast. however, increasing use of chemicals causes several negative effects, e.g. development of pathogen resistance to the applied agents as well as other environmental impacts (compant et al. 2005). the harmful effects of chemical pesticides has forced the search for environmentally friendly biocontrol to eliminate the diseases in plants. extensive research has demonstrated that plant growth promoting rhizobacteria (pgpr) could have an important role in agriculture and horticulture in improving crop productivity. while diverse microbes may contribute to the biological control of plant pathogens, most research and development efforts have focused on isolates of t h r e e g e n e r a , b a c i l l u s , tr i c h o d e r m a , a n d vol.10, no.3, september 2016, p 107-111 doi: 10.5454/mi.10.3.4 molecular identification of endospore-forming rhizobacteria from organic cabbage farm potential as biocontrol against phytopathogen xanthomonas campestris 1 1 1 maya fitriana ilul fahmi , endang kusdiyantini , agung suprihadi , dyah 2 1,2* wulandari , and anto budiharjo 1 biology department, faculty of sciences and mathematics,universitas diponegoro, jalan prof. soedharto sh, tembalang – semarang 50275, indonesia; 2 bacteriology laboratory, central laboratory of research and service, universitas diponegoro, jalan prof. soedharto sh, tembalang – semarang 50275, indonesia rhizobacteria are rhizosphere competent bacteria that colonize and proliferate in all the ecological niches found on the plant roots at all stages of plant growth, in the presence of a competing microflora. these bacteria are potential as biological control agent to inhibit the growth of phytopathogen. the aim of this study was to isolate endospore-forming rhizobacteria from cabbage farm and determine its ability as biocontrol against xanthomonas campestri, a pathogen causing black rot on cabbage. the methods used consisted of isolation, antibacterial activity test, biochemical characterization and molecular identification. fourteen isolates of endospore-forming rhizobacteria were obtained from cabbage farming. isolate k.9 had the highest ability to inhibit the growth of x. campestri. based on molecular characterization by sequence analyses of 16s rrna, isolate k9 had 97% homology with bacillus cereus strains bf15. key words: bacillus cereus, biocontrol, endospore-forming rhizobacteria, xanthomonas campestris rhizobakteri merupakan bakteri yang hidup disekitar perakaran tanaman, yang mampu mengkoloni dan berkembang disemua tahap pertumbuhan akar tanaman dan berkompetisi dengan bakteri lainnya. bakteri ini berpotensi sebagai agen biokontrol untuk menghambat pertumbuhan pathogen tanaman. penelitian ini bertujuan mengisolasi rhizobakteri pembentuk endospora dari tanaman kubis serta menguji kemampuan isolat tersebut untuk menghambat pertumbuhan patogen xanthomonas campestris penyebab penyakit busuk hitam pada kubis. metode yang digunakan meliputi isolasi bakteri, karakterisasi isolat bakteri secara morfologi, uji antibakteri, identifikasi secara molekuler dengan 16s rrna, dan uji biokimia. hasil isolasi diperoleh empat belas isolat rhizobakteri dan terdapat empat isolat yang memiliki potensi sebagai agen bakterisida terhadap patogen x. campestris. isolat k.9 memiliki daya hambat terbesar terhadap x. campestris yaitu 12,6 mm. identifikasi secara molekuler berdasar analisis 16s rrna menunjukkan isolate k.9 mempunyai homolog 97% dengan bacillus cereus bf15. kata kunci: bacillus cereus, biokontrol, rhizobacteria pembentuk endospora, xanthomonas campestris microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-24-76918147, fax: +6224-76918148; email: anto.budiharjo@fulbrightmail.org pseudomonas (mcspadden gardener and driks 2004). rhizobacteria are rhizosphere competent bacteria that colonize and proliferate in all the ecological niches found on the plant roots at all stages of plant growth, in the presence of competing microflora (antoun and kloepper 2001). endospore-forming rhizobacteria is one of the successful biocontrol agent commercially produced and used against plantpathogens. ryu et al. (2005) elucidated the benefit of endospore-forming rhizobacteria that is more stable under unfavorable condition. in addition, endospore makes the rhizobacteria easier to be applied and commercialized as biopesticide with long duration of storage. the advantage of endospore-forming rhizobacteria as biopesticide led to research towards the finding of new isolates potential as biocontrol agents. the aims of this study were to isolate endospore-forming rhizobacteria from organic cabbage farm, determine its ability against x. campestri and identify based on molecular methods. materials and methods isolation of endospore-forming rhizobacteria. endospore-forming rhizobacteria were isolated from organic cabbage farm in banyubiru, semarang, central java. isolation method was modified from ryu et al. (2005). five grams rhizosphere soil was placed in 100 ml flask containing of 45 ml sterile distilled water, o and then shaken and heat-treated at 80 c for 30 m. afterwards, the suspension was 10-fold serially diluted in sterile aquadest and plated in tryptic soy agar (tsa) medium. after 24-48 h of incubation at room t e m p e r a t u r e , s i n g l e c o l o n y i s o l a t e s w e r e characterized. bacteria was selected based on the morphological characteristics including color, form, elevation, and margin of the colony. the pure cultures were kept in slant agar. antibacterial activity test. x. campestri was obtained from laboratory of pest and plant disease brawijaya university. isolate of endospore-forming rhizobacteria and x. campestri were grown in 5 ml o tryptic soy broth medium overnight at 30 c on rotary shaker at 120 rpm. one ml of the x. campestri was then used to inoculate 100 ml tsa (40 °c), and evenly shaken. fifteen ml of the inoculated tsa was poured into petri dish and cooled down, and 5 paper discs were placed on the dish. fifteen µl of endospore-forming rhizobacteria isolate was dropped on the paper disc and the dish was incubated at 30 °c for 48 h. the antibacterial test was determined by the formation of clear zone. dna extraction. dna extraction was done by chelex method (walsh et al. 2013). three loops overnight culture of endospore-forming bacteria were placed in a tube containing 100 µl ddh o. 1 ml 0,5 % 2 saponin was added. then, it was incubated overnight at 4 °c before being centrifuged for 10 m at 12.000 rpm. the supernatant was removed, and 100 µl ddh o and 2 50 µl chelex 20% was added to the pellet. the mixture was then boiled for 10 m with vortexing every 5 m, and centrifuged. the dna would be in the supernatant and kept at 4 °c. dna amplification. dna amplification was performed using the following program: pre denaturation at 95 °c for 3 m, denaturation at 94 °c for 1 m, annealing at 55 °c for 1 m, extention at 72 °c for 1 m, and the final extention at 72 °c for 7 m. primers used were 27f (5'-agagtttgatcmtggctcag-3') and 1492r (5'-ggytaccttgttacgactt-3') (osborne et al. 2005). the pcr mixture contained 3 µl dna template, 1.5 µl of each primer, 25 µl kapa 2gfast kits, and 19 µl ddh o. visualization of the pcr 2 product was done using gel electrophoresis with 1 % agarose concentration run at 100v for 30 m. the band of pcr product was seen on gel documentation. sequences analyze. sequencing of pcr product was done at pt. genetika science, indonesia. the sequence was analyzed by base alignment using basic local alignment search tool (blast) to establish the percentage of base pair similarity with reference isolates from the genbank. phylogenetic tree. phylogenetic tree was constructed using mega 5 software. phylogenetic tree was constructed using neighbour-joining tree and bootstrap method. results seventeen endospore-forming rhizobacteria has been isolated from the rhizosphere of cabbage grown on organic farming. each isolate was characterized based on colony characteristic, morphology of the cell and gram stain. table 1 shows the characteristic of colony and cell of the isolates of endospore-forming rhizobacteria. all of the isolates were then tested their antagonistic activity against x. campestris, a causative agent of black rot disease. out of seventeen isolates tested, two isolates demonstrated their antagonistic activity showed by inhibition zone toward x. campestris. amongst those two isolates, isolate k-9 showed the highest antagonistic activity (table 2), 108 fahmi et al. microbiol indones hence, will be preceded or further molecular identification. dna extraction of k-9 isolate was done using chelex method. the dna obtained was then used for molecular identification based on 16s rrna. figure 2 shows visualization of the amplified 16s rrna gene of k-9 isolate on 1% agarose gel. the pcr product of 16s rrna gene was sequenced and obtaining sequence of 1360 bp length. the sequence of k-9 was analyzed using basic local alignment search tool (blast) program. the result showed that the sequence had 97% homology with bacillus cereus strain bf15. phylogenetic tree of the k-9 isolate showing appropriate affiliation is depicted in fig 3. discussion finding potential rhizobacteria that have the ability to eliminate plant pathogens is a great interest for the farmers and agricultural industries. the consumers demand agricultural products containing less pesticide residues. therefore, the discovery of potent biocontrol agents against pythopathogens is highly needed. one of the sources of biocontrol agents is rhizobacteria, e s p e c i a l l y e n d o s p o r e f o r m i n g b a c t e r i a . endopsoreforming bacteria have advantages in term of application and formulation compared to non endospore-forming bacteria (ryu et al. 2005). slepecky and hemphill (2006) stated that heating the sample at 80 °c for 30 m was enough to isolate endospore-forming bacteria, as vegetative cells of bacteria generally die at heat treatment of 60-70 °c. the highest ability of isolate k-9 in suppressing the growth of phytopathogen x. campestri amongst other isolates showed that this isolate has unique secondary metabolites. mcspadden gardener, 2004 stated that many strains of endospore-forming bacillus and pseudomonas showed ability to suppress pests and pathogens or otherwise promote plant growth. allabouvette et al, 2006 supported the idea that antibiosis is a mechanism used by biocontrols, like bacillus spp., trichoderma spp., pseudomonas spp., and streptomyces spp., due to the production of antibiotic, bacteriocin, degradation of cell wall enzyme, and other volatile compounds. based on 16s rrna molecular identification, the volume 10, 2016 microbiol indones 109 fig 1 antagonistic activity of endospore-forming rhizobacteria isolates against x. campestris. fig 2 visualization of amplified 16s rrna gene from k-9 isolate in 1% agarose gel using low molecular mass ladder (1 = k-9, m = marker). 1.500 bp 2.000 bp 1.200 bp 750 bp 110 fahmi et al. microbiol indones table 1 colony and cell morphology of endospore-forming rhizobacteria from rhizosphere of cabbage fig 3 phylogenetic tree of isolate k-9. isolate colony characteristic cell form gram color form margin elevation k-1 ivory irregular lobate flat rod positive k-2 cream circular entire raised rod positive k-3 white filamentous filiform flat rod positive k-4 ivory circular serrate raised rod positive k-5 ivory circular entire raised rod positive k-6 cream rhizoid filiform flat rod positive k-7 transparent circular entire flat rod positive k-8 white circular undulate flat rod positive k-9 cream circular entire raised rod positive k-10 orange circular entire raised rod positive k-11 yellow circular entire raised rod positive k-12 ivory irregular undulate flat rod positive k-13 ivory circular serrate raised rod positive k-14 ivory circular entire flat rod positive k-15 white circular undulate flat rod positive k-16 yellow circular entire raised rod positive k-17 yellow circular entire raised rod positive table 2 inhibition zone produced by endospore-forming rhizobacteria isolates against x. campestris isolate inhibition zone (mm) k-9 12.6 k-12 2.05 volume 10, 2016 microbiol indones 111 k-9 isolate was bacillus cereus strain bf15. the 16s rrna gene was used for molecular identification because this gene is present and conserved in all bacteria (janda and abbott 2007). phylogenetic tree analysis of the k-9 isolate compared to other a n t i b i o t i c p r o d u c i n g r h i z o b a c t e r i a , s u c h a s paenibacillus and bacillus, shows that k-9 isolate lies in the same clade with b. subtilis and b. azotoformans (fig 3). this means that k-9 isolae is more closely related to these bacteria rather that to the genus paenibacillus, which is in different clade. foldes et al. (2000) explained that many members of genus bacillus produced various antibacterial compounds. the compounds include the lipopeptide antibiotics iturin a and surfactin, which could suppress damping-off in tomato, and zwittermicin a, which si correlated to suppression of damping-off in alfalfa. hassi et al 2012, showed that bacillus produces variety of antibiotics able to suppress the growth of gram positive and gram negative bacteria. however, b. cereus is well known for its pathogenicity in animals and human (zhang et al. 2016). hence, it will limit the application as biocontrol in agricultural systems. in conclusion, endospore-forming rhizobacteria isolated from cabbage farm is potential as antibacterial compound against phytopathogen x. campestris. based on 16s rrna molecular identification, the isolate is bacillus cereus strain bf15. references allabouvette c, olivain c, steinberg c. 2006. biological control of plant diseases: the european situation. europ j plant pathol. 114: 329-341. doi: 10.1007/s10658-0050233-0. antoun h, kloepper jw. 2001. plant growth-promoting rhizobacteria (pgpr), in: encyclopedia of genetics, brenner, s. and miller, j.h., eds., academic press, ny.1447-1480. bps. 2013. laporan bulanan data sosial ekonomi. sensus pertanian edisi 40: 1-116. compant s, duffy b, nowak j, clement c, barka ea. 2005. use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. appl environ microbiol. 71(9): 49514959. doi: 10.1128/aem.71.9.4951-4959.2005. földes t, bánhegyi i, herpai z, varga l, szigeti j. 2000. isolation of bacillus strain from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms. j app microbiol. 89: 840-846. doi: 10.1046/j.1365-2672.2000.01184.x. hassi m, david s, haggoud a, guendouzi se, ouarti ae, souda si, iraqui m. 2012. in vitro and intracellular antimycobacterial activity of a bacillus pumilus strain. afr j microbiol res. 6 (10): 2299-2304. doi: 10.5897/ajmr11.1021. janda jm, abbott sl. 2007. 16s rrna gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. j clin microbiol. 45 (39): 2761-2764. doi: 10.1128/jcm.01228-07. mcspadden gardener bb. 2004. ecology of bacillus and paenibacillus spp. in agricultural systems. phytopathology 94:1252-1258. doi: 10.1094/phyto. 2004.94.11.1252. mcspadden gardener bb, driks a. 2004. overview of the nature and application of biocontrol microbes: bacillus spp. phytopathology 94:1244 doi: 10.1094/phyto. 2004.94.11.1244. musapa m, kumwenda t, mkulama m, chishimba s, noris de, thuma pe, mharakurwa s. 2013. a simple chelex protocol for dna extraction from anopheles spp. j vis exp. 71: 1-7. doi: 10.3791/3281. osborne ca, galic m, sangwan p, jansen ph. 2005. pcr generated artifact from 16s rrna gene-specific primers. fems microbiol lett. 248: 183-187. doi: 10.1016/j.femsle.2005.05.043. priest f, austin b. 1993. modern bacterial taxonomy. chapman and hall, london. ryu cm, kim jw, choi oh, park sy, park sh, park cs. 2005. nature of root-associated paenibacillus polymyxa from field-grown winter barley in korea. j microbiol biotechnol. 15 (5):984-991. sastrosiswojo s, uhan st, sutarya r. 2005. penerapan teknologi pht pada tanaman kubis. bandung: balai penelitian tanaman sayuran. semangun h. 1989. penyakit-penyakit tanaman hortikultura di indonesia. gadjah mada university press. yogyakarta. slepecky ra, hemphill he. 2006. the genus bacillusnonmedical. prokaryotes. 4: 530-562. doi:10.1007/0387-30744-3_16. walsh ps, metzger da, higuchi r. 2013. chelex 100 as a medium for simple extraction of dna for pcr-based typing from forensic material. biotechniques. 54 (3): 134-139. doi: 10.2144/000114018. zhang z, feng l, xu h, liu c, shah np, wei h. 2016. detection of viable enterotoxin-producing bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex pcr. j dairy sci. 99 (2): 1047-1055 doi: 10.3168/jds.2015-10147. page 1 page 2 page 3 page 4 page 5 vol.17, no.1, march 2023, p 1-8 doi: 10.5454/mi.17.1.1 8effect of cocoa bean fermentation using lactic acid bacteria and yeast starters on flavonoid formation and antioxidant activity anja meryandini irvan anwar , titi candra sunarti 1,2*, 3 and 4 1 department of biology, bogor agricultural university, jl. raya dramaga, bogor, jawa barat, 16680, indonesia; 2 biotech center, bogor agricultural university, jl. kamper, bogor jawa barat, indonesia; 3 biotechnology study program, graduate school, jl. raya dramaga, bogor, jawa barat, 16680, indonesia; 4 department of agroindustrial technology, jl. raya dramaga, bogor, jawa barat, 16680, indonesia. this study investigates the effect of fermentation using lactic acid bacteria and yeast as starters on the formation of flavonoid compounds and the antioxidant activity of cacao beans. the fermentation process were divided into 4 groups: f1: spontaneous fermentation, f2: fermentation using lactic acid bacteria (lab), f3: fermentation using yeast and f4: fermentation using lab and yeast. the extraction process was done using ethanol. flavonoid content was analysis using spectrophotometer assay. the antioxidant activity was analyzed by 1,1-difenil-2-pikrilhidrazil (dpph) method. all ethanol extract samples of fermented cacao beans contained alkaloids, polyphenols, flavonoids, and tannins. the flavonoid compounds from ethanol extract of cacao beans in f1 is 4.35 ± 0.20 mg l , f2 (5.64 ± 0.05), f3 (5.37 ± 0.17), and f4 (5.99 ± 0.23 mg l ). the antioxidant activity of -1 -1 cacao bean fermentation extracts using starter were increase compared to the spontaneous fermentation extract (f1). the antioxidant activity in f2 increased to 46.45 ± 2.00%, f3 (49.05 ± 0.58%), and f4 (50.33 ± 0.43%), while the antioxidant activity of f1 was 42.31 ± 0.66%. ic50 value as the ability of the extract to reduce 50% dpph radical on the ethanol extract of cacao beans from spontaneous fermentation (f1) was 141.67 mg l . the -1 ic50 value of the fermented cacao bean extract with the addition of starter was obtained at f2 at 109.30 mg l , f3 -1 (97.51), and f4 is 88.15 mg l . -1 antioxidant, dpph, ethanol extract, starterkey words: penelitian ini mengkaji pengaruh fermentasi menggunakan bakteri asam laktat dan khamir sebagai starter terhadap pembentukan senyawa flavonoid dan aktivitas antioksidan biji kakao. proses fermentasi dibagi menjadi 4 kelompok: f1: fermentasi spontan, f2: fermentasi menggunakan bakteri asam laktat (bal), f3: fermentasi menggunakan khamir dan f4: fermentasi menggunakan bal dan khamir. proses ekstraksi dilakukan dengan menggunakan etanol. kandungan flavonoid dianalisis menggunakan spektrofotometer. aktivitas antioksidan dianalisis dengan metode 1,1-difenil-2-pikrilhidrazil (dpph). semua sampel ekstrak etanol biji kakao fermentasi mengandung alkaloid, polifenol, flavonoid, dan tanin. senyawa flavonoid ekstrak etanol biji kakao pada f1 adalah 4,35 ± 0,20 mg l , f2 (5,64 ± 0,05), f3 (5,37 ± 0,17), dan f4 (5,99 ± 0,23 mg l ). aktivitas antioksidan -1 -1 ekstrak biji kakao fermentasi menggunakan starter meningkat dibandingkan dengan ekstrak fermentasi spontan (f1). aktivitas antioksidan pada f2 meningkat menjadi 46,45 ± 2,00%, f3 (49,05 ± 0,58%), dan f4 (50,33 ± 0,43%), sedangkan aktivitas antioksidan f1 adalah 42,31 ± 0,66%. nilai ic50 sebagai kemampuan ekstrak untuk mereduksi radikal dpph 50% pada ekstrak etanol biji kakao hasil fermentasi spontan (f1) adalah 141,67 mg l . -1 nilai ic50 ekstrak biji kakao fermentasi dengan penambahan starter diperoleh pada f2 sebesar 109,30 mg l , f3 -1 (97,51), dan f4 sebesar 88,15 mg l . -1 kata kunci: antioksidan, dpph, ekstrak etanol, starter microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ;: +62 fax: +62--251-8622833 251-8622833; ameryandini@apps.ipb.ac.ide-mail: fermentation is characterized by a succession of microbial activity from three groups of microorganisms, namely yeast, lactic acid bacteria (lab) and acetic acid bacteria (aab), which produce good cocoa beans (de vuyst and wecks 2016). cocoa beans can be naturally fermented, but the fermentation process should not be excessive because it can lead to the lowly quality aroma and taste of cacao beans. these problems can be controlled by using microbes as a starter in the fermentation process. lactic acid bacteria and yeasts are microbes that are widely used in the fermentation process. the addition of chocolates are produced from cocoa beans powder. as the 3rd largest country in cocoa production after ivory coast and ghana, indonesia plays a significant role in cocoa powder production (bps 2020). to obtain a good quality chocolate powder, fermentation of cocoa beans is one of the processes which can bring the right good aroma (chocolate flavor) due to chemical changes during fermentation. the chocolate flavor is a chemical change caused by microbial enzymatic activity. cocoa bacteria lactobacillus plantarum, acetobacter aceti and yeast ( ) in thesaccharomyces cerevisiae fermentation of cacao beans made the processing time shorter (sandhya 2016).et al. the application of cocoa powder in food and beverage products in the community is increasing, but the knowledge about its efficacy in health is still very limited. cocoa beans contain secondary metabolite compounds that have good antioxidant activity (brito et al. 2016). the antioxidant activity in cacao beans is due to the compound of polyphenols. polyphenol content will decrease along with the length of fermentation time (goya . 2022). given the importance of theet al chemical processes that occur during the fermentation of cacao beans and the importance of flavonoid compounds known to have antioxidant activity, so processing of cacao beans should be able to maintain the flavonoid content. one that can be done to maintain or increase the flavonoid content is the fermentation process with the addition of a starter. previous research has isolated lactic acid bacteria and yeasts from cacao beans. the isolates have characters as a starter in the cacao fermentation process which is resistant to acid conditions, high temperatures, and high ethanol content and are able to produce high acid. this study aims to analyze the effect of lactic acid bacteria and yeast as a starter in cocoa beans fermentation. materials and methods lactic acidfermentation of cacao beans. bacteria ( sp. h 2.34) and yeast (isolatelactobacillus ip4) used in this experiment were from spontaneously fermented cacao beans in parungkuda sukabumi west java. the fermentation was conducted in 1000 ml erlenmeyer, each containing 500 grams of cocoa beans and inoculated with 106 cfu microbes/g cocoa beans, according to meryandini (2019). cocoa beanset al. used in this research are forastero from central java coffee and cacao research center of central java. the design experiment is shown in table 1. ethanol extraction from cocoa beans. the fermented cocoa beans were washed with running water to remove the remaining pulp or impurities attached to the cocoa beans, then dried and ground. the supernatant was evaporated to obtain ethanol extract from fermented cocoa beans. the powder was sieved using a sieve (60 mesh), and 100 g of cocoa bean powder into a glass container with 300 ml of ethanol as solvent. samples were incubated for 3x24 hours; every 1x24 hours the solvent was replaced with new ethanol. phytochemical component of ethanol cacao bean extract. a. alkaloid content was determined using reagent dragendorff (raaman 2006). formation of orange color indicates presence of alkaloid. b. polyphenol content was determined using fecl3 5% (raaman 2006). formation of green blackish color indicates presence of polyphenol. c. flavonoid content was determined using concentrated hcl solution and mg powder (rusita and suhartono 2016). formation of red color indicates presence of flavonoid. d. tannin content was determined using nacl 10% and gelatin 2% (tiwari . 2011). whiteet al precipitation was formed, indicating presence of tannin. quercetinstandard curves of quercetin. compounds in ten series of concentrations of 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 mg l was used for -1 standard. each quercetin concentration was taken at 0.5 ml and mixed with 2 ml of distilled water and 0.15 ml of 5% nano2, allowed to stand for 5 minutes. a total of 0.15 ml of alcl3 10% was added to the solution, then allowed to stand for 5 minutes. the solution was reacted with 2 ml 4% naoh, diluted with distilled water to a volume of 5 ml, and the absorbance was measured by uv-vis spectrophotometer at λ 510 nm. quantification of flavonoid compound. the amount of flavonoid content was carried out by spectrophotometric methods using various standard concentrations of quercetin ( ). asmadaan . 2011et al much as 0.5 ml extract (100 mg l ) was mixed with 2 -1 2 meryandini et al. microbiol indones table 1 design of cacao beans fermentation experiment treatment duration of fermentation (hour) specific treatment f1 5 × 24 spontaneous fermentation f2 5 × 24 fermented by lab culture f3 5 × 24 fermented by yeast culture f4 5 × 24 fermented by combination of lab and yeast ml of distilled water and 0.15 ml nano 5% then2 incubated for 5 min. after incubation, 0.15 ml alcl3 10% was added to the solution and incubated for 5 minutes. the solution was mixed with 2 ml of 4% naoh, diluted with distilled water to a volume of 5 ml and measured by spectrophotometry at λ 510 nm. the results were expressed as mg of quercetin equivalents per g of dry cocoa bean in comparison with the standard curve, which was developed under the same conditions. analysis antioxidant activity by 1,1-difenil-of 2-pikrilhidrazil dpph) method.( measuring antioxidant activity was according to rahmi et al. (2016). ethanol extract of fermented cacao beans was prepared in 100 mg ml . three ml of each sample was -1 added to 2 ml dpph 0.1 mm, incubated at 37°c for 30 min, and measured with spectrophotometry at λ 517 nm. the control solution was used 3 ml of methanol in 2 ml dpph 0.1 mm. the percentage of inhibition is calculated based on the formula below: dpph scavenging activity (%) = [abs control – abs sample / abs control] × 100% (nishaa 2012). theet al. measuring for each extract was repeated 5 times. determination of ic50 value was done by ethanol extract of cacao beans prepared in 5 series concentrations: 50, 100, 150, 200, and 250 mg l . -1 three ml samples were mixed with 2 ml dpph 0.1 mm and incubated at 37°c for 30 minutes. the scavenging activity was measured spectrophotometrically by the decrease in absorbance at 517nm. quercetin compound was used as a control with concentrations of 10, 15, 20, 25, and 30 mg l . -1 data a .nalysis data were tested statistically by diversity analysis of variances (anova). if there was a significant difference, then proceed with tukey methods range test with a significant level of 95% using (sas)software statistical analysis system windows 9. results and discussion cacao beans fermentation. various aspects can view the success of a cacao bean fermentation process, one of which is the dry cacao bean split test. the split test is intended to determine the success rate of bean fermentation with color changes (fig 1). good fermented cacao beans produce dark brown color and hollow bean texture. the highest fermented cacao beans produced by fermentation were with the addition of a combination of lactic and yeast acid bacteria with 83% of fermented beans, followed by addition of lactic acid bacteria (69%), addition of yeast (55%), and without any addition, only 15%. the fermentation process will impact chemical compounds such as secondary metabolites found in cacao beans (meryandini . 2019).et al characteristic ethanol extract of cacao beans. yield, water content and residual solvents from ethanol extract of cacao beans. obtained yield from the ethanol extract of cacao beans is shown in table 2. the yield extracts from spontaneous fig 1 cocoa beans.(a) ; (b) ; (c)fermented unfermented nonfermented volume 17, 2023 microbiol indones 3 a b c treatment the yield extract (%) the water content and residual solvent (%) f1 3.92±0.09 b 7.67±0.12 f2 4.62±0.13 a 7.13±0.23 f3 3.99±0.12 b 7.27±0.31 f4 4.39±0.16 a 7.20±0.35 table 2 the yield of extracts, water content and residual solvents of ethanol extract of fermented cacao beans different numbers in the table have significant differences of p < 0.05. value are means ± sd (n = 3) fermentation (f1) was 3.92 ± 0.09%. adding a single starter or starter combination of lactic acid bacteria and yeast increased the yield value of the ethanol extract of cacao beans compared to f1. the f2 increased to 4.62 ± 0.13% or with a percentage of 15.2%, f3 (1.8%) and f4 10.7%. utami (2016) obtain 15 % polyphenolet al. from cocoa bean shell fermented 120 hours, using aceton:water (70:30). water content and residual solvents from the ethanol extract of cacao beans are presented in table 2. the water content and residual solvents from the ethanol extract of cacao beans from spontaneous fermentation were 7.67%. water content and residual solvents with the addition of a starter contained water and residual solvents which were not significantly different from the extracts of spontaneous fermentation (p > 0.137), where the percentage of water content and residual solvent at f2 are 7.13%, f3 (7.27%), and f4 (7.20%). the increase in the yield value of the extract (f2, f3, and f4) indicates that it is not affected by the water content or the remaining solvents contained in the extract but can occur due to the fermentation process carried out. increasing the yield of ethanol extract of cacao beans during fermentation with the addition of starter (f2, f3, and f4) occurs due to the stretching of carbon bonds between the constituents of cacao bean cells which causes the components of the covalently bonded compounds to the cell wall to be quickly released. the presence of lactic acid bacteria and yeast in the fermentation contributes to converting compounds in simplicia cacao beans, especially compounds with high molecular weight. so that when the extraction process takes place, it is suspected that the binding of compounds causes more yield extract becomes high. the ability of ethanol solvents to bind polar and nonpolar compounds is to withdraw compounds in cacao beans, such as fat, especially fat, with a short chain chemical structure because it is polar. the fat content in cacao beans is generally around 54% (afoakwa 2010). research by ristanti (2016)et al. reported that the fat content of cacao beans from 12 regions in south sulawesi was the highest at 38.21%, and the lowest was 20.73%. the high amount of fat in cacao beans causes fat extracted by ethanol solvents. so the extract yield was obtained in f1, f2, f3, and f4 containing secondary metabolites and other chemical compounds such as fat. phytochemical components ethanol extract of cacao beans. the increased quality of fermented cocoa beans using lactic acid bacteria and yeast as a starter is caused by the formation of compounds that play a role in taste and aroma precursors of cocoa beans. phytochemical component analysis was carried out to determine the presence of a phytochemical component in the tested extract. qualitative determination can be seen from changes in color or formation of foam or sediment if the extract or sample is reacted with certain chemicals. the test results of phytochemical components of ethanol extract of cacao beans from various fermentation treatments are presented in table 3. phytochemical components of ethanol extract of fermented cacao beans with a single starter or starter combination of lactic and yeast acid bacteria include alkaloids, polyphenols, flavonoids, and tannins. the alkaloid content of the cacao bean extract resulting from spontaneous fermentation (f1) contains moderate-intensity alkaloids. the results of f1 are directly proportional to the ethanol extract of fermented cacao beans with lactic acid bacteria (f2), which also contain moderate-intensity alkaloids. samples of f3 and f4 contain alkaloids with strong intensity, which is thought to have increased alkaloids in the extract. alkaloid compounds in cacao beans is theobromine which causes a bitter taste (sandhya et al. 2016). during fermentation occurs, theobromine will be degraded (aromolaran and motilola 2018; balc´azar-zumaeta 2023.)et al. polyphenols are secondary metabolites with the highest content in cacao beans that play a role in producing good taste and aroma in cacao beans (afoakwa 2012). one of the polyphenolet al. compounds in cacao beans is the flavonoid class. the content of polyphenols and flavonoids in f1 (ethanol extract of cacao beans from spontaneous fermentation) has moderate intensity. this can be due to the oxidation of polyphenols by polyphenol oxidase enzymes, diffusion to cotyledons than to the skin layer, polymerization, especially flavonoid compounds, and the formation of protein complexes. polyphenols and flavonoids in f2, f3, and f4 have strong intensity, meaning that there is an increase in the content of polyphenols and flavonoids in the extract compared to f1. the strong intensity of the sample can be due to the production of secondary metabolite produced by lactic acid bacteria and yeast at the stationary phase and affect the content of polyphenol or flavonoid compounds. the content of tannin compounds in extracts fermented with the addition of starter (f2, f3, f4) has the same content intensity as in f1 (ethanol extract of 4 meryandini et al. microbiol indones cacao beans resulting in spontaneous fermentation). these results indicate that the fermentation process with or without adding lactic acid and yeast acid starter to the tannin content did not change significantly. it is suspected that tannin compounds in cacao beans do not have a role in the formation of flavor or aroma in cacao beans. the content of flavonoid compounds ethanol extract of cacao beans. flavonoids are a phytochemical component with the highest range of cacao beans polyphenols group (chin 2013).et al. flavonoid compounds play a role in forming the aroma and taste of cacao beans. they are known to have high antioxidant activity, so quantitative analysis of the content of flavonoids is carried out. the flavonoid content of cacao beans ethanol extract was determined by using the quercetin standard. the flavonoid content of the ethanol extract of cacao beans from fermentation is presented in table 4. the content of flavonoid compounds from 100 mg l of ethanol extract of cacao beans on samples of -1 spontaneous fermentation extract (f1) contained flavonoid compounds of 4.35 ± 0.20 mg l . the -1 flavonoid compounds' content from fermentation extracts with the addition of a single starter or combination starter proved to increase compared to f1. f2 increased by a percentage of 22.9%, f3 (18.9%), and f4 (27.4%). improvement to the quality of cocoa beans by the addition of lactic acid bacteria dan yeast in the fermentation process is corelated to the high flavonoid compounds in the cacao beans ethanol extract. in the fermentation process, polyphenol compounds undergo oxidation by polyphenol oxidase enzymes and play a role in changing the color of beans (hoa . 2018,et al fang 2020).et al. increasing of flavonoid compounds after fermentation can be due to the breakdown of the storage cells. unfermented cocoa beans contain 12–18% polyphenols of the dry weight of beans on average. these compounds are stored in polyphenolic cells in unfermented beans. sugars from pulp are converted to acids during fermentation. these acids move into the beans and lower their ph, which leads to breakdown of storage cells (bariši´c . 2019et al ). increasing the content of flavonoid compounds can also be caused by plant cultivars used. the type of cacao used in the study was forestero cacao. research by hii . (2009) and towaha (2014) explains that theet al content of polyphenol compounds from cacao types forastero > trinitario > criollo are presented in table 5. the country of origin of cacao beans can also affect the chemical content in cacao beans. a country's climate difference causes cacao beans to contain different secondary metabolites. the results of the study by othman (2007) reported that the highestet al. polyphenol content of ethanol extract from cacao beans was produced by malaysia > indonesia (sulawesi) > ghana > ivory coast. antioxidant activity ethanol extract of cacao beans. antioxidant activity test in ethanol extract of cacao beans was carried out using 1,1-diphenyl-2pikrilhydrazyl (dpph) method. dpph is a stable free radical. parameters used to show the results of antioxidant activity by dpph method are % inhibition of dpph free radicals. the results of determination of dpph % inhibition of ethanol extract of cacao beans are presented in table 6. antioxidant activity (% inhibition) dpph shows that in the concentration of 100 mg l the ethanol -1 extract of cacao beans is capable of inhibiting various dpph free radicals. the antioxidant activity of spontaneous fermentation extract (f ) was 42.31 ±1 0.66%, f (46.45 ± 2.00%), f (49.05 ± 0.58%), and f42 3 at 50.33 ± 0.43%. antioxidant activity of ethanol extract of cacao beans fermented with the addition of a single starter or combination starter (f , f , and f )2 3 4 increased when compared with the ethanol extract of cacao beans from spontaneous fermentation (f ).1 antioxidant activity in f increased by 8.9%, f2 3 (13.7%), and f (15.9%). dordevic (2010),4 et al. explained that the increase in antioxidant activity in the fermentation process can be due to degradation of the cell wall so that it frees or induces bioactive components from a material. in general antioxidant activity is directly proportional to total flavonoids. this is because flavonoids' ability to capture free radicals is closely related to the presence of hydroxyl groups (tosun et al. 2009). the correlation between flavonoid content and antioxidant activity in general illustrates the effect of flavonoid compounds on antioxidant activity produced by extract samples. antioxidant activity of ethanol extract. the ic50 value is the concentration of the extract compound or an ingredient needed to reduce a free radical given as much as 50%. the smaller the ic50 value produced, the greater the ability of compounds, extracts, or ingredients to counteract free radicals. the results of % inhibition of dpph radicals were f1, f2, f3, and f4, which occurred at extract concentrations of 250, 200, 150, 100, and 50 mg l -1 volume 17, 2023 microbiol indones 5 6 meryandini et al. microbiol indones (fig 1). this shows that the higher the concentration of ethanol extract in cacao beans, the higher the ability to reduce dpph free radicals. these results are consistent with emelda (2015) study which reported that antioxidant activity (% inhibition) will increase with increasing extract concentration, where at a concentration of 5 mg l extract antioxidant activity -1 was obtained at 32.49%, at a concentration of 10 mg l -1 (53.11%), 25 mg l (83%), 50 mg l (91%), 100 mg l -1 -1 -1 (94%), and 200 mg l concentration of antioxidant -1 activity by 93%. the ic50 results of cacao bean ethanol extract is presented in table 7. it is stated that the ethanol extract from fermentation with the addition of starter has a table 5 the content of polyphenols based on cultivars and the country of origin of cacao beans country cultivar total polyphenol content (mg g-1) pantai gading forastero 81.5 columbia forastero 81.4 venezuela trinitario 64.3 peru criollo 50.0 dominika criollo 40.0 malaysia forastero 71.4 indonesia forastero 82.3 resources: hii 2009; tahowa 2014.et al. table 6 antioxidant activity (% inhibition) of dpph from 100 mg l ethanol extract of fermented cacao -1 beans treatment antioxidant activity (% inhibition) ethanol extract of fermented cacao beans (%) f1 42.31±0.66 c f2 46.45±2.00 b f3 49.05±0.58 ab f4 50.33±0.43 a different numbers in the table have significant differences of p < 0.05. value are means ± sd (n = 3) table 3 the phytochemical component from ethanol extract of fermented cacao beans type of testing phytochemical content f1 f2 f3 f4 alkaloid + + + + + + polifenol + + + + + + + flavonoid + + + + + + + tanin + + + + ++ = positive with a strong intensity, + = positive with a moderate intensity table 4 the flavonoid compounds from cacao beans at a concentration of 100 mg l of ethanol extract -1 treatment the flavonoid compounds from ethanol extract of cacao beans (mg l-1) f1 4.35±0.20 c f2 5.64±0.05ab f3 5.37±0.17b f4 5.99±0.23a different numbers in the table has significant differences of p < 0.05. value are means ± sd (n = 3) volume 17, 2023 microbiol indones 7 lower ic50 value compared to f1, which means the ability to extract by f2, f3, and f4 to capture dpph free radicals is higher because it requires extracts with a lower concentration, where f2 is 109.30 mg l , f3 -1 (97.51 mg l ), f4 (88.15 mg l ). in contrast, f1 can -1 -1 capture 50% dpph free radicals with a concentration of 141.67 mg l . -1 ic50 value of all cacao bean ethanol extract from spontaneous fermentation extract (f1) and from fermentation results with the addition of lactic acid and yeast acid starter (f2, f3, and f4) compared to ic50 from quercetin compound (comparator), then the ability of ethanol extract cacao beans to inhibit 50% dpph radicals lower than quercetin compounds. quercetin reduced 50% of dpph free radicals using only quercetin concentrations of 20.33 mg l . the high -1 ability of quercetin as an antioxidant because quercetin is known to have an oh group in its chemical structure that functions to capture dpph radicals by donating h atoms so that radical dpph becomes nonradical (salamah and widyasari 2015). in conclusion, cacao bean fermentation process with or without the addition of lactic acid and yeast acid starter is proven to contain flavonoid compounds. the ethanol extract of cacao beans with the addition of a single or combination starter has a high content of flavonoids compared to cacao bean extract resulting from spontaneous fermentation. the increase of flavonoid compounds from ethanol extract of cacao beans fermented with the combination of starter also increased the antioxidant activity to be higher than the ethanol extract of cacao beans from spontaneous fermentation. acknowledgement extended our thanks to directorate of research and community service directorate general of rasearch reinforcement and development, ministry of research, technology and higher education of the republic of indonesia in accordance with the letter of assignment of the implementation of research program number: 011/sp2h/lt/iv/2017. 20 april 2017 and addendum of contract number: 011/sp2h/ lt/drpm /vii /2017. august 21, 2017 for financial research. references afoakwa 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microbiol indones 01 lg.cdr phop/phor histidine kinase two-componentsystem in mycobacterium tuberculosis has been studied as potential target for new antitubercular drugs. the system is not found in mammals and the m. tuberculosis phop/phor amino acid sequence shows little homology with other prokaryotic proteins (suwanto 2012). these findings implied that phop/phor have low non-targetting potential, either on humans or other organisms, a required characteristic vol.9, no.2, june 2015, p 51-57 doi: 10.5454/mi.9.2.1 expression and purification of phor sensor-domain histidine kinase of mycobacterium tuberculosis in escherichia coli 1 + 1+ 1 ernawati arifin giri-rachman * , fenryco pratama , oktira roka aji , arum 3 2 3 1 patriati , ihsanawati , edy giri rachman putra , maelita ramdani moeis and 1 department of biotechnology, school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung, west java 40132, indonesia; 2 department of chemistry, faculty of mathematics and life sciences, institut teknologi bandung, jalan ganesha 10, bandung, west java 40132, indonesia; 3 center for science and technology of advanced materials, national nuclear energy agency (batan), gedung 43 kawasan puspiptek serpong, serpong, banten 15314, indonesia globally, tuberculosis (tb) remains a leading cause of death. the emergence of multidrug-resistant strains (mdr-tb) and extensively drug-resistant strains (xdr-tb) has fuelled the discovery for novel drugs and drug targets for its successful and better treatment. one of the potential candidates for drug target is phor sensory protein histidine kinase, a part of the two component system (tcs) phop/phor in mycobacterium tuberculosis (mtb). this protein system was known for its role on regulating hundred of mtb virulence factors, from genes for cell wall and lipid synthesis to genes for adaptation in human leukocyte and hypoxia response. previous studies have successfully characterized, isolated, and cloned theputative sensory domain of phor protein gene into prset vector expression system. in this study, escherichia coli was transformed with prset-sensphor and o cultivated at 37 c under iptg induction to express phor sensor-domain protein. most of the proteins were overexpressed in the form of inclusion bodies. subsequent protein purification in ni-nta system under refolding condition on urea gradient was performed to isolate phor sensor-domain protein in soluble form. arginine was supplemented in purified protein solution to prevent aggregation during long term storage. while highly purified protein was acquired, small angle x-ray scattering (saxs) analysis was conducted to obtain 3dimensional (3d) protein structures in solution. key words: multi-drug resistance, rational drug design, tuberculosis, two-component signal transduction system of histidine kinase secara global, tuberkulosis (tb) masih merupakan salah satu penyakit infeksi penyebab kematian tertinggi. semakin maraknya kemunculan kasus tb akibat infeksi oleh strain multidrug-resistant (mdr-tb) dan extensively drug-resistant (xdr-tb) telah mendorong dilakukannya pengembangan obat dan target obat baru untuk pengobatan yang lebih efektif dan aman. salah satu kandidat target obat baru yang cukup menjanjikan adalah protein sensor phor histidine kinase, anggota dari protein pensinyalan sistem dua komponen (tcs) phop/phor di mycobacterium tuberculosis (mtb). sistem phop-phor dikenal berperan dalam regulasi ratusan faktor virulensi dari mtb, mulai dari gen untuk sintesis dinding sel dan lipid hingga gen untuk adaptasi di leukosit manusia dan respon hipoksia. penelitian sebelumnya telah berhasil mengkarakterisasi, mengisolasi dan mengklon bagian dna yang merupakan domain sensor dari protein phor kedalam sistem vektor ekspresi prset. pada penelitian ini, escherichia coli ditransformasi dengan vektor prset-sensphor dan dikultivasi o pada temperatur 37 c dibawah induksi iptg untuk mengekspresikan domain sensor protein phor. sebagian besar protein diekspresikan dalam bentuk badan inklusi. purifikasi protein menggunakan sistem ni-nta dalam kondisi refolding di gradien urea dilakukan untuk mengisolasi domain sensor protein phor dalam bentuk terlarut. larutan protein disuplementasikan dengan arginin untuk mencegah pembentukan aggregat protein hasil refolding dan purifikasi selama proses penyimpanan jangka panjang. ketika domain sensor protein phor dengan kemurnian yang tinggi telah diperoleh, analisis small angle x-ray scattering(saxs) dilakukan untuk mempelajari struktur 3-dimensi protein tersebut di dalam larutan. kata kunci: desain obat secara rasional, kebal aneka obat, sistem transduksi sinyal dua komponen histidine kinase, tuberkulosis *corresponding author; phone: +62-81809418805, fax: +62-22-2534107 ; email: erna@sith.itb.ac.id + these authors contributed equally in this work. available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia for new drug target. previous studies also demonstrated that interrupting phop/phor two-component-system drastically decreased m. tuberculosis virulence in vivo (walters et al. 2006, gonzalo-asensio et al. 2008). phop/phor two-component-system also induces the expression of 114 virulence-related genes (pathak et al. 2011), including those involved in lipoarabinomanan (a cell wall component) metabolism and m. tuberculosis resistance inside macrophage (walters et al. 2006, gonzalo-asensio et al. 2008). inhibition of phor sensor-domain will block the first step in the signal transduction pathway and shut down the expression of the virulence-related genes. since many genes are affected by the inhibition, anti-tubercular drug which attack the phor sensor-domain should be more effective in treating m. tuberculosis infection and have shorter duration of treatment than existing drugs. previous studies by suwanto et al (2012) have successfully characterized, isolated, and cloned phor sensor-domain coding sequence from m. tuberculosis strain h37rv into prset expression vector. in this study, we over expressed recombinant phor sensordomain protein in e. coli bl21 (de3) and conducted subsequent purification process to isolate the protein. protein in pure condition is required to be proceed for structural study. the resulting phor sensor-domain 3d structure could be used for further characterization of protein function (shumilin et al. 2012). moreover, structural information will provide basis for in silico screening of new anti-tubercular drug using rational drug design, primarily from natural chemical products found in indonesia. instead of determining the 3d structure of protein crystal, solution scattering, a new promising method for studying conformational changes of protein in physiological conditions has been established (svergun et al. 2003, jacqueset al. 2010) and was proposed in this work. overall 3d structure of protein in low-resolution can be provided from the small-angle scattering (x-ray, saxs or neutron, sans) of protein solution as an important complementary method of protein crystallography due to the limitation in preparing the protein single crystal. an isotropic scattering function i(q) which is proportional to the averaged of over all structure orientations from a single protein molecule has to be attained in order to reconstruct the protein structure from solution. this constraint is only achieved from a monodisperse system as non-interacting of identical particles (scatterers) in dilute solutions. materials and methods cloning summary and overexpression of recombinant phor sensor-domain protein. previous study by suwanto et al (2012) has amplified dna segment encoding sensor-domain of phor protein (genbank, nc_000962.3, np_215272.1) from mycobacterium tuberculosis h37rv genome using pcr technique with forward (5'-gaattcgtcacct cgatgttgcagca-3') and reverse (5'-ccatggtt agacgtcggccagatcaatg-3') primers. the amplified dna then was cloned primarily into pgemt easy vector before transfered into prset expresion vector. the expressed protein will be observed around 17 kda according to in silico predicted product (mr​g​s​h​h​h​h​h​h​g​m​a​s​m​t​g​g​q​q​m​g​r​d​l​y​d​d​d​ d​k​d​r​w​g​s​ef​v​t​s​m​l​q​h​r​l​t​s​r​i​d​r​v​l​l​e​e​a​q​i​ w​a​q​i​t​l​p​l​a​p​d​py​p​g​h​n​p​d​r​p​p​s​r​f​y​v​r​v​i​s​p​d​ g​q​s​y​t​a​l​n​d​n​t​a​i​p​av​p​a​n​n​d​v​g​r​h​p​t​t​l​p​s​i​g​ g​s​k​t​l​w​r​a​v​s​v​r​a​s​d​g​y​lt​t​v​a​i​d​l​a​d​v​). prset-sensphor vector was transformed into e. coli bl21(de3) using heat shock method (sambrook and russel, 2001). a single transformant colony was -1 grown in lb medium containing 0.1 mg ml o ampicillin at 37 c and agitated at 200 rpm until the optical density at 600 nm reached 0.4-0.8. the overexpression was induced with 1 mm iptg for 4-6 h. o cells were harvested by centrifugation at 2000 g, 4 c, for 30 min. protein purification. the cell pellet from overexpression cultures was resuspended in lysis buffer containing 20 mm tris-hcl ph 8.0, 1 mm pmsf, disrupted by sonication, and then centrifuged at o 12,000 g, 4 c, for 10 min to separate soluble proteins (supernatant) from insoluble proteins and cell debris (pellet). the pellet was washed twice with triton x100 -urea (2% triton x-100, 2m urea, 0.5 m nacl, 20 mm tris-hcl ph 8.0) and once with tris-hcl 20 mm ph 8.0 followed by sonication and centrifugation at o 12,000 g, 4 c, for 10 min. the pellet containing inclusion bodies was dissolved in binding buffer containing 8 m urea, 0.5 m nacl, 5 mm imidazole, 2.5 mm β-mercaptoethanol, and 20 mm tris-hcl ph 8.0 stirred for 30-60 min, and then centrifuged at 12000 g, o 4 c, for 15 min. the resulting supernatant was filtered using millipore membrane of 0.22 μm. the filtrate was prepared for purification. protein purification was carried out using nickel ni-nta-agarose system on 50 ml of econo column (biorad) in the refolding state at flow rate of 0.5-1 ml -1 o min , 25 c. the column was equilibrated with solution 52 giri-rachman et al. microbiol indones containing 0,5 m nacl, 5 mm imidazole, 8 m urea, 2.5 mm β-mercaptoethanol,20 mm tris-hcl ph 8.0 (ge life sciences). the protein was refolded using a urea linear gradient of 8-0 m and then eluted with 0.5 m nacl, 250 mm imidazole, 2.5 mm β-mercaptoethanol, 20 mm tris-hcl ph 8 added with 200 mm arginine to prevent precipitation formation during storage. results from protein overexpression and purification were profiled by sds-page (fig 1). to confirm the identity of recombinant phor sensor-domain protein, slices of acrylamide gel containing a desired band were sent to center for mass spectrometry & proteomics university of minnesota for lc-ms analysis. small angle x-ray scattering (saxs) for determining protein structure in solution. the purified protein was dialyzed using servapor membrane (molecular weight cut off /mwco = 12-14 kda, serva) with protein sample:dialysis buffer volume ratio of 1:22 to dispose imidazole and βmerchaptoetanol and to reduce the nacl concentration in the purified sample to 200 mm. the sample was c o n c e n t r a t e d u s i n g n a n o s e p u l t r a f i l t r a t o r (mwco/molecular weight cut-off = 10 kda, pall). after filtration by millipore membrane of 0,22 µm, acquired protein in final concentration of 1.5 – 5 mg -1 ml then was subjected for saxs of protein in solution. sample equilibration was conducted prior to data collection by dialysing protein sample in its solution buffer. both of equilibrated protein sample and the background solution from its dialysed buffer were exposed to x-ray beam for 10 h and collected at o every hour, at 4 c. the collected scattering data of protein samples was subtracted by the background solution data in obtaining corrected scattering data of proteins. the corrected 2-dimensional scattering data was then radially averaged to obtain the scattering intensity i(q) as a function of the momentum transfer q -1 in å using data reduction software of bruker nanostar. results overexpression of recombinant phor sensordomain protein. since prset containing t7 promoter, iptg induction was conducted to increase the expression of the recombinant protein in e. coli bl21 (de3). sds page result showed that recombinant phor sensor-domain protein was overexpressed and found dominantly in pellet fraction of cell lysate to supernatant fraction (fig 1). ubsequent process to isolate and characterize inclusion bodies protein from pellet fraction confirmed that the estimated molecular weight of 17 kda protein existed in this form (fig 1). protein purification. phor sensor-domain protein was constructed in prset vector which contains hexahistidine as fusion tag. this tag facilitates rapid purification of recombinant protein by immobilized metal ion affinity chromatography (imac). purification was performed alongside the refolding process and highly purified protein was obtained from the single ni-nta column running (fig 2a). post-purification observation showed that the purified protein was unstable and aggregated after o storage at -80 c (fig 2b). arginine in final concentration of 0.2 m should be added into the protein solution immediately after elution to prevent further aggregation. lc-ms analysis was conducted to determine the peptide sequences of ~17 kda band of purified protein. several chemical modifications on the protein residues (fig 3) were performed prior to trypsin digestion to increase peptide fragments generation and readibility. all spectra of detected fragments were searched againts sequence reference of recombinant phor sensor-domain using peptide de novo sequence software peak 7.5 (ma et al. 2003). the result then confirmed the identity of recombinant phor sensor domain protein with 79% sequence coverage (fig 3). small angle x-ray scattering for determining protein structure in solution. the scattering intensity i(q) vs. momentum transfer q of recombinant phor sensor-domain protein in two different -1 -1 concentrations, (a) 3.9 mg ml (b) 1.56 mg ml was showed (fig 4). the scattering intensity still increased in low q-range indicating that this protein has a naturally propensity to aggregate under the buffer conditions used in the preparation whereas the protein solution was diluted two times. this aggregated form was also confirmed using dynamic light scattering (dls) measurements which dominant peak referred to particle with size 11 nm and molecular weight 1052 kda, significantly different with 3 nm and 17 kda of phor sensor-domain actual features. discussion the presence of recombinant phor sensor-domain in the inclusion bodies confirmed in silico analysis using protein-solubilization prediction software from university of oklahoma. the software had developed volume 9, 2015 microbiol indones 53 fig 1 sds page of phor sensor-domain recombinant protein expressed in e. coli. lane m shows molecular mass marker (in kda). lane p contains pellet-cell lysate; lane s contains supernatant-cell lysate; lane incbod the inclusion bodies. sample was run on 20% acrylamide and visualized using coomassie brilliant blue staining. 54 giri-rachman et al. microbiol indones on basis of logistic regression model with interaction of parameters that accurately predicted 96% insoluble protein from large set of data base (diaz et al. 2010). results from the analysis estimated that phor sensordomain protein was expressed as insoluble protein inside e. coli. the intrinsic factor of protein might affect the inclusion bodies formation of phor sensor domain in e.coli rather than overexpression factor since significant amount of protein was acquired in insoluble fraction (fig 1) (diaz et al. 2010). on different approach, the higher numbers of β-sheet structure than α helices in a pdc fold-like characteristic prediction (geneious) of phor sensor domain (suwanto 2012) was considered to be responsible for the insolubility character of this protein (vilasi et al. 2006). protein purification and refolding was performed to obtain phor sensor-domain protein in pure and native form. refolding protein is an elaborate process and achieving correct folded protein is sometimes tricky. in other cases, expressing the sensor domain of histidine kinase in particular vector that contain soluble promoting tag such as gst and mbp is preferable (cheung et al. 2012; tan et al. 2014). nevertheless, inclusion bodies production has several advantages for isolating and purifying particular protein such as high fig 2 sds-page of purified recombinant phor sensor-domain protein purificationin 20% acrylamide (a). lane m shows molecular mass marker (in kda); lane e the elution fraction. the gel was stained with coomassie brilliant blue. precipitation of purified protein was observed in the sample without additives (b). (kda) kda kda kda kda kda volume 9, 2015 microbiol indones 55 number of protein production, resistance from cellular protease, and shorter purifying step. the point that is stated later proven well in this experiment while recombinant phor sensor-domain protein could be purified merely in single column process using ni-nta agarose with high level of protein purity acquired. the heterogenity of protein sample might be decreased significantly during isolation of inclusion bodies step including removal of common co-purified contaminant of e.coli protein in nickle column (bolanos-garcia and thomas 2006), simplifying the subsequent process of protein purification to separate protein target from trace amount of other proteins. importantly, the lack of cysteine in primary sequence of phor sensor-domain protein trimming the elaborate process of protein refolding by finding out the suitable condition for arrangement of correct disulfide bridge. nevertheless, limitation was found in futher analysis of phor sensordomain recovery after refolding process due to the protein absence in enzymatic activity and lack of in te n s it y ( % ) fig 3 lc-ms analysis of purified recombinant phor sensor-domain protein. (a) shown is 155 amino acid sequences of expressed phor sensor-domain. the peptide sequence coverages by spectral analysis are bold and highlighted (79%). colorized boxes indicate the chemical modifications on particular residues of recombinant phor sensor-domain. list of chemical modifications is displayed at the right side. (b) the ms/ms spectrum of peptide gmasmtggqqmgr (m/z 656,16), which is the fragment of recombinant protein sensor-domain (amino acid residues 11-23) digested by trypsin, is shown as a representative spectra. -1 fig 4 phor sensor-domain was analyzed using saxs, in two different concentrations, (a) 3.9 mg ml and (b) -1 o 1.56 mg ml . analyzes was at 4 c. 56 giri-rachman et al. microbiol indones information about the protein ligand. post-purification observation showed that the purified and refolded protein was precipitated after o storage in -80 c. supplementation with additive such as arginine significantly reduced progression of protein aggregates. arginine might effectively prevent protein aggregation by several ways. ho et al. (2003) showed that addition of arginine in the refolding solution of lysozimes could shift the viral coefficient value into positive and accordingly increased repulsive interactions between proteins. in another mechanism, tsumoto et al. (2004) proposed that interaction of arginine with exposed aromatic amino acid in protein could increased its solubility that led to repress protein aggregation.it was later strongly indicated by das et al. (2007) experiment that cluster of arginine in aqueous solution displaying methyl groups that might interact with hydrophobic surface in protein and then prevented the aggregation. purified protein was confirmed accurate to recombinant phor sensor domain since mass spectral analysis shown high peptide coverage score (≥ 50%) and two and more unique peptides were identified(60 unique peptides) (link and labaer 2009). crystallization trial demands on high concentration of protein target. several attempts to produce final -1 solution of phor sensor-domain protein at ≥ 10 mg ml led to sample aggregation (data not shown). alternative method for analysing protein structure in lower concentration, such saxs, was preferable since it was able to give useful signal at concentration of protein -1 sample 2.5 mg ml (14 kda of molecular-weight) and -1 even at 0.5 mg ml (66 kda of molecular weight) (skou et al. 2014). it is not possible to have confidence in the structural models derived in obtaining useful biostructural information from small-angle scattering data if the samples aggregated. it should be monodisperse, identical particles, and the data are measured to low enough q to reliably characterize their largest dimensions as the plot will have a discernible flat region in the lowest q regime (independenton q) – dashed lines. moreover, it confirmed that subaggregate particles was still continually developed in the sample even after protein filtration and buffer supplementation with arginine. the highly sensitive technique was required to detect protein homogeneity preceding to saxs analysis or other related methods for protein structural study. nonetheless, from those initial saxs experimental works, it can be concluded that the sample preparation protocols for solution scattering experiment, experimental setup, data reduction and analysis are well established for protein solution. in conclusion, inclusion bodies of phor sensordomain protein was produced in e.coli by expressing prset-sensor phor system under cultivation o temperature of 37 c and iptg induction. while purification and refolding process dominantly isolated the protein in soluble form, protein homogeneity still an issue before proceeding the protein onto structural study. some improvements in protein production are required for providing a pure monodisperse protein which has a high stability.we propose to optimize this protein expression on alternative condition, host or vector system to obtain the soluble protein at the first time and to prevent elaborate process of protein refolding and storage optimization. acknowledgment this work was supported by the general directorate of higher education (dikti), indonesian ministry of education and culture through 2012 dikti decentralization research grant and 2014 itb research and innovation grant to e.a.g.r. the authors acknowledge robert knott, anna sokolova and agata rekas of australian nuclear science and technology organisation (ansto) for a benefit discussion and for providing the beam time. the saxs experiment was conducted under the batan-ansto joint research project on the biophysical study of virus like particles for targeted drug and vaccine development, (pi : e. g. r. putra). references ma b, zhang k, hendrie c, liang c, li m, doherty-kirby a, lajoie g. 2003. peaks: powerful software for peptide de novo sequencing by tandem mass spectometry. rapid commun mass spectrom. 17(20): 2337-2342. doi: 10.1002/rcm.1196. bolanos-garcia vm, davies or. 2006. structural analysis and classification of native proteins from e. coli commonly co-purified by immobilised metal affinity chromatography. biochim biophys acta. 1760(9): 1304–1313. doi: 10.1016/j.bbagen.2006.03.027. cheung j, le-khac m, hendrickson w. 2009. crystal structure of a histidine kinase sensor domain with similarity to periplasmic binding proteins. proteins 77(1):235–141. doi: 10.1002/prot.22485. das u, hariprasad g, ethayathulla as, manral p, das tk, pasha s, mann a, ganguli m, verma ak, bhat r, volume 9, 2015 microbiol indones 57 chandrayan sk, ahmed s, sharma s, kaur p, singh tp, srinivasan a. 2007. inhibition of protein aggregation: supramolecular assemblies of arginine hold the key. plos one 2(11): e1176. doi: 10.1371/journal.pone.0001176. diaz a, tomba e, lennarson r, richard r, bagajewicz mj, harrison rg. 2010. prediction of protein solubility in escherichia coli using logistic regression. biotechnol bioeng 105(2):374–383. doi: 10.1002/bit.22537. gasteiger e, hoogland c, gattiker a, duvaud s, wilkins mr, appel rd, bairoch a. 2005. protein identification and analysis tools on the expasy server. 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of arginine in protein refolding, solubilization, and purification. biotechnol prog. 20(5):1301–1308. doi: 10.1021/bp0498793. vilasi s, dosi r, iannuzzi c, malmo c, parente a, irace g, sirangelo i. 2006. kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences. febs lett. 580(6): 1681–1684. doi:10.1016/j.febslet.2006.02.018. walters sb, dubnau e, kolesnikova i, laval f, daffe m, smith i. 2006. the mycobacterium tuberculosis phopr two-component system regulates genes essential for virulence and complex lipid biosynthesis. mol microbiol. 60(2): 312–330. doi:10.1111/j.1365-2958.2006.05102.x. page 1 page 2 page 3 page 4 page 5 page 6 page 7 02 yudono.cdr vol.11, no.3, september 2017, p 81-88 doi: 10.5454/mi.11.3.2 oil recovery test using bio surfactant of halo tolerant bacteria brevundimonas diminuta and bhurkholderia glumae at variation of nacl salt concentrations 1* 2 3 bambang yudono , muhammad said , sri pertiwi estuningsih , 1 and aulia karima 1 2 3 department of chemistry; department of chemical engineering; department of biology, universitas sriwijaya, jalan raya palembang-prabumulih. km 32 inderalaya, south sumatera 30662. oil recovery test has been done by using crude biosurfactant from brevundimonas diminuta and bhurkholderia glumae -the indigenous halo tolerant bacteriawith the variation of nacl salt concentration 0; 1.5; 3; 4.5; 6; and 7.5%. oil recovery test was obtained by determining % tph (total petroleum hydrocarbon). the sample concentration was 28.19% tph. it was extracted by using biosurfactant of b. diminuta and b. glumae bacteria, the optimal salinity conditions were at 3, 4.5% salt concentrations with the value oil recovery as much as 50.41 and 69.97%, respectively. oil components which extraction by biosurfactant were analyzed by using gcms (gas chromatography-mass spectrophotometry). from the result of gc-ms analysis, it could be concluded that bacteria b. diminuta could dissolve hydrocarbon compounds short chain carbon atom at fraction <c –c 10 14 and long chain carbon atom at fraction >c , <c , c -c , and c -c ; whereas b. glumae could dissolve 22 10 11 14 15 17 hydrocarbon compounds short chain carbon atom at fraction <c –c and long chain carbon atom at fraction >c 10 14 22 according to the retention time. key words: bhurkholderia glumae, brevundimonas diminuta, crude biosurfactant, indigenous halo tolerant bacteria, oil recovery uji coba rekoveri minyak telah dilakukan dengan menggunakan biosurfaktan ekstrak kasar dari brevundimonas diminuta dan bhurkholderia glumae yang merupakan bakteri halo toleran asli indonesia dengan konsentrasi garam nacl 0; 1.5; 3; 4.5; 6; dan 7,5%. uji perolehan minyak diperoleh dengan cara menentukan % tph (total petroleum hydrocarbon). konsentrasi sampel adalah 28,19% tph. sampel diekstraksi dengan menggunakan biosurfaktan bakteri b. diminuta dan b. glumae, kondisi salinitas yang optimal berada pada konsentrasi garam 3 dan 4,5% dengan nilai perolehan minyak masing-masing sebanyak 50,41 dan 69,97%. komponen minyak yang diekstraksi dengan biosurfaktan dianalisis dengan menggunakan gc-ms (gas chromatography-mass spectrophotometry). hasil analisis gc-ms dapat disimpulkan bahwa bakteri b. diminuta dapat melarutkan senyawa hidrokarbon rantai pendek atom karbon pada fraksi <c –c dan atom 10 14 karbon rantai panjang pada fraksi >c , <c , c -c , dan c -c ; sedangkan b. glumae bisa melarutkan senyawa 22 10 11 14 15 17 hidrokarbon atom karbon rantai pendek pada fraksi <c –c dan atom karbon rantai panjang pada fraksi >c 10 14 22 sesuai waktu retensi. kata kunci: bakteri halo toleran asli indonesia, bhurkholderia glumae, biosurfaktan ekstrak kasar, brevundimonas diminuta, perolehan minyak microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8153821994, fax:+62711-580056; email: yudonob@hotmail.com provide the thrust to the oil to flow to the surface (lazar et al. 2007; shibulal et al. 2014). in order to increase cumulative oil production this acquisition should be increased as much as possible. the way is by applying the eor (enhanced oil recovery) method to the reservoir. the principle is to use external power to the reservoir that the power can help lifting the oil to flow to the surface. the eor is chemical surfactant and one of the eor techniques developed now is by utilizing microbes known as microbial enhanced oil recovery (meor). the meor method is more favorable than the eor method because this method can extract petroleum more efficient and environmentally friendly. the world's petroleum demand will increase from year to year along with the rapid development of global economy. therefore the oil industry players must continue to work to meet these demands. but the problem is that oil production is limited by the value of recovery factor (rf) that is a ratio showing the amount of oil that can be produced on the surface. this value will limit the amount of oil that can be produced by the primary recovery mechanism. the oil production is very dependent on the characteristics of reservoir and fluid and the type of driving mechanism that helps potential of microorganisms to degrade heavy crude oil to reduce viscosity is considered highly effective in meor (lazar et al. 2007 the basic ; enas 2007). principle of meor is the utilization of microbial secondary metabolite products to help increase the remaining oil gain or that is still trapped in the reservoir. bacterial decomposition by meor can include organic, inorganic acids, gases, water and bio surfactants. bio surfactant is one of the products produced by microbes and plays an important role in oil recovery because it reduces surface tension between two-phase fluids, thereby increasing oil mobilization .(sea and dhail 2013; chandankere et al. 2013) the ability of bacteria to produce bio surfactants is related to the ability of bacteria to use hydrocarbons as their substrate. microorganisms with large bio surfactant production generally have a great ability also in decomposing hydrocarbons. types of growth substrate, bacteria type, source of nutrition, and environment are the main factor of the researcher's attention in optimizing bio surfactant production. salt is an important environmental factor for bacterial growth and development. if the salt content of the environment is not compatible with microbial enzyme activity, bacteria can not be metabolized properly so it does not grow optimally (sarafin et al. 2014; shin et al. 2001; garcia-blanco et al. 2007). bhurkholderia glumae and brevundimonas diminuta are potential bacteria as biosurfactant and degradation producer (yudono 2010; yudono et al. et al. 2011). the presence or absence of crude biosurfactants that have been produced from bacteria with a density of 7 -1 ± 10 cells ml was tested using hemolysis tests (dhail 2012). potential bacteria in producing biosurfactant, were chosen based on the size of the inhibit zone/clear zone formed of colony of bacteria on blood agar. both bacteria is known to have the ability to produce a good biosurfactant because it has a clear zone that is 25.96 mm. a clear zone formed over 20 mm indicates that the bacteria tested have good potential for producing biosurfactants (yudono and estuningsih 2013). the growth of both of these bacteria depends on the concentration of nacl so it is potentially also possible in producing optimal bioproducts at certain salt levels (shin 2001). each bacterium has resistance to et al. different salts while oil wells in indonesia mostly have salinity characteristics (up to 40 000 ppm) (almeida et al. et al. 2004; hao 2008). for certain concentrations of nacl also causes a decrease in water-surface tension by surfactants, this is because the chemical bonds that make up nacl are ionic bonds capable of affecting hydrophobic and hydrophilic groups in lowering interphase surface tension (mnif and ghribi 2015; rufino et al. 2014). both bacteria have good tolerance to salinity concentration up to 7.5%. this research will be conducted in variation of salt content of 0, 1.5, 3, 4.5, 6, and 7.5% in biosurfactant for recovery in petroleum. the resulting biosurfactant was tested with a sludge sample, then the oil recovery was calculated using tph calculation (total petroleum hydrocarbon). to see the components of the degraded hydrocarbon compounds, a gc (gas chromatography) analysis of sludge produced the best percentage recovery (at the best salt level) of each bacterium (ibrahim 2013). et al. materials and methods materials. the brevundimonas diminuta and bhurkholderia glumae bacteria were isolated from oil field babat toman village, sludge oil (obtained from babat toman village, musi banyuasin, south sumatera). molasses was taken from sugar waste of pt cinta manis, tanjung raja, ogan ilir, south sumatra). maintainance of culture. a total of 1 ose of each bacterium b. diminuta and b. glumae were inoculated to solid medium na with zig zag movement. the bacteria that have been inserted into the na medium are incubated in the incubator for 24 h. after incubation bacteria are ready to use. medium preparation. zobell media was prepared by dissolving 5 g of peptone, 1 g of yeast extract, 0.012 g of k hpo , and 0.01 g of feso in aquadest with a 2 4 4 volume of 1000 ml solution. the mixture is boiled over the hotplate and homogenized with a magnetic stirrer. after boiling the mixture was sterilized with autoclave at 121 °c for 15 min (sunitha et al. 2007; behlugil 2002). bacterial starter. b. diminuta and b. glumae culture cultures were taken 5 ose, then subcultured into each erlenmeyer flask containing 100 ml of medium zobell, then aerated for 24 h, and after that it stopped. one hundred ml of medium was added to the mixture until the total volume is 200 ml and the mixture is reaerated until the shortest generation time of b. diminuta and b. glumae for 12 h. production of crude biosurfactants. the production process of crude biosurfactant is done by mixing bacterial starter, medium zobell, and 15% molasses concentration for b. diminuta bacteria and 20% for b. glumae bacteria with a total volume 82 yudono et al. microbiol indones volume 11, 2017 microbiol indones 83 composition of 200 ml. the mixture was incubated based on the shortest generation time of each bacteria ie 12 h. biosurfactant preparation with variation of salt level concentrations. the process of making saltcontaining biosurfactant solution is done by mixing crude biosurfactant from b. diminuta bacteria and variation of nacl 0, 1.5, 3, 4.5, 6, and 7.5%. the same treatment was performed using b. diminuta and b. glumae bacteria. crude biosurfactant application on sludge for oil recovery. a total of 25 g sludge (obtained from babat toman village, musi banyu asin, south sumatra) (b/v), were incorporated into each biosurfactant with variations of 0, 1.5, 3, 4.5, 6, and 7.5% (w/v against biosurfactant) to a total volume of 200 ml. the mixture is then aerated for 10 d. the mixture is then filtered with filter paper. the residue from the filtration result is then calculated by the final percentage of tph. tph measurement. two hundreds fifty ml boiling water with a socket-shake extract to be used was dried in the oven, then cooled in the desiccator. a total of 10 g sludge was inserted using a filter paper of the appropriate size. the filter paper containing the sludge sample was then inserted into the socket tube, then the top of the socket tube was connected to the condenser and the bottom was connected to a boiling flask containing n-hexane solvent with the volume wetting the entire filter paper. the sludge sample then was extracted until the solvent descends back into the boiling flask until it is clear. gc analysis. gc analysis was performed on sludge which yields the largest % oil recovery in each bacteria by analyzing gc sludge chromatogram before and after treatment at 0-7.5% salt. the type of column used is tg-5ms with a column length of 30 m and a diameter of 0.25 mm (yudono 1994). the samples were taken and injected to a thermo scientific gc tool with a pre-program temperature of 40 °c maintained for 4 min, the temperature was raised 5 °c every 1 min until the temperature reached 300 °c. data analysis. the data to be obtained from this research are; the initial sludge tph measurement data to determine the percentage of hydrocarbon compounds present in sludge prior to treatment and final tph data after the treatment of salinity variation and then compared with the initial tph values t​​ o see the biosurfactant ability to dissolve the hydrocarbon compound on sludge. the data obtained from the experiment was processed by using anova (analysis of variance) analysis to test the difference of salt and oil recovery. results total petroleum hydrocarbons (tph). tph were measured to determine the percentage of hydrocarbon compounds present in petroleum. measurements of total petroleum hydrocarbons (tph) in oil-contaminated sludge were used as samples and parameters to determine the bio surfactant's ability to reduce further tph values. initial tph measurements on sludge samples were performed by solvent extraction using n-hexane and using sochletation method. the initial tph data generated from the measurement of 28.19%. the samples were treated with the biosurfactants of both bacteria that had varied nacl salt by 0, 1.5, 3, 4.5, 6, and 7.5%. the results are characterized by reduced oil sludge levels after treatment. in this study, the biodegradation process is known from the calculation of heavy tph oil sludge from residual weight (oil residual) sludge weight, then compared with the initial weight added by gravimetric method, the results were presented in table 1. based on table 1, the final% tph value after biosurfactant treatment with variation of salt concentration of nacl has different tph value, respectively. as result, the salt concentration can affect the work of biosurfakan in ddissolving sludge. result of analysis of petroleum hydrocarbon compound on sludge using gc-ms. the following table 1 the average percentage of tph extracted from 28.19% tph samples %tph(average) salinity nacl (%) b. diminuta b. glumae 0 1.5 3 4.5 6 7.5 8.3283 10.8486 14.1992 7.7921 9.3863 8.3641 6.7539 9.8157 7.2273 19.7209 14.2114 7.7767 84 yudono et al. microbiol indones chromatograms show the initial components of sludge (before the treatment of variation of nacl salt content), sludge chromatogram with control, chromatogram sludge which having optimal nacl content yielding the largest petroleum recovery, and a histogram of oil abundance in each bacterium. from the data in table 1 and described in figure 2 showed that the two bacteria produced different biosurfactant yields at each salt concentration, in which the b. glumae. bacteria produced a more optimal biosurfactant compared with b. diminuta bacteria. data analysis were done by using data analysis with anova method, it shows that f value count on b. diminuta and b.a glumae bacteria bigger than f critical value, where f arithmetic for b. diminuta bacteria has value of f count 59, 24098 while bacteria b. glumae has a value of f arithmetic 87, 5864. the value of least significant different test value is higher for each bacteria, where it can be concluded that any difference in salt content of nacl gives a very significant oil recovery. result of petroleum recovery test by using control. petroleum recovery test using aquades is used as a control to be applied to a sludge sample that aims to determine the biosurfactant's ability to dissolve petroleum. the result of oil recovery at control that is equal to 4.7321%. the results of the recovery using the aquades will then be analyzed using gc to see components of the hydrocarbon compound after treatment, and used as control. after adding biosurfactant further decreases the peak area of ​​the chromatogram (fig 1). the peak areas decrease in chromatogram are caused by the breakdown of hydrocarbons into simple compounds. this is largely determined by the type of bacteria, where each bacteria has different capabilities in degrading the petroleum sludge. from the gc-ms data obtained, soluble and insoluble hydrocarbon analyzes that are left on the residue are shown (fig 3) and the histograms of percent abundance of each bacteria are presented (fig 4). the percent difference of abundance by subtracting percent abundance after addition of crude biosurfactant (at) from b. diminuta bacteria minus percent of peak abundance before addition of crude biosurfactant (a ) 0 (fig 3). a positive reduction results indicate that the hydrocarbon compound dissolves in the biosurfactant. an increased histogram shows that short-chain hydrocarbon compounds dissolve in biosurfactant, so that at the top of the chromatogram there is a missing carbon chain and breaks down into short chains. this histogram explains that the biosurfactant of the b. diminuta based on its retention time is capable of dissolving short c chain hydrocarbons, i.e., the <c -c 10 14 atomic chain at 40-140 °c and dissolving the long c chain hydrocarbons ie the atomic chain >c at a 22 o temperature of 265-290 c but the biosurfactants of the b. diminuta bacteria are not able to dissolve the long c chain hydrocarbons c -c and c -c at the 15 17 18 21 o temperature of 140-265 c. this histogram shows that the treatment with biosurfactant from b. glumae bacteria based on retention time is not capable of dissolving long chain c hydrocarbon compounds ie c -c , c c , and > c at 15 17 18 21 22 o a temperature of 140-290 c so that the component remains as residue. furthermore, biosurfactant can dissolve the hydrocarbon chain compound of short c atom, i.e., the atomic chain <c , c -c , and c -c at 10 11 14 15 17 o temperature 40-140 c (fig 4). discussion the optimal nacl concentration was 3% for brahmanimonas diminuta and 4.5% for bhurkholderia glumae bacteria. the higher levels of nacl salt that affect will result in different percentages of petroleum recovery, where salinity is one of the natural environmental factors present in the sludge sample area (shin et al. 2001; garcia-blanco et al. 2007). this indicates that b. glumae is an effective bacteria for oil recovery because the resulting biosurfactant can increase the solubility of hydrophobic compounds so as to increase and accelerate the rate of degradation by microbes. appropriate salt content will lead to a faster biodegradation process from bacterial culture. similarly, the type of bacteria itself will affect changes in salt levels obtained from bacterial metabolism activities. and vice versa if the level of salt is not suitable then the activity of bacteria will decrease that will affect the total of bacterial cells (shin et al. 2001). it is also revealed by charlena (2010) that salt conditions affect the growth of bacteria in degrading petroleum hydrocarbons. optimum salt conditions will enhance the species' ability to degrade hydrocarbons. gc-ms analysis is only performed for the treatment of the largest petroleum recovery results using biosurfactant with optimal saline nacl content obtained from each bacterium. in addition, gc analysis was conducted on sludge with treatment using aquades that served as a control to see the working ratio between biosurfactant and aquadest. objective gc-ms analysis volume 11, 2017 microbiol indones 85 fig 1 early tph chromatogram prior to the treatment of crude biosurfactant (a), final tph chromatogram after treatment using crude bosurfactant with variation of 3% salt on brevundimonas diminuta bacteria (b), final tph chromatogram after treatment using crude bosurfactant with variation in salt content 4.5% on the bhurkholderia glumae bacteria (c). (a) (b) (c) can provide information on the amount of the constituent components of petroleum before extraction and after the extraction using crude biosurfactant with variation of salt content (ibrahim et al. 2013; jovančićević et al. 2004). early tph chromatogram prior to the treatment of crude biosurfactant in obtaining the petroleum constituent component before treatment showed the presence of 16 peaks of the petroleum compound based on a total retention time of 56 min (fig 1a), whereas final tph chromatogram after treatment using crude bosurfactant with a variation of 4.5% salt on b. diminuta bacteria showed the presence of 15 petroleum compound peaks based on a total retention time of 56 min (fig 1b). tph chromatogram after treatment using crude bosurfactant with a 3% salt content variation in 86 yudono et al. microbiol indones fig 2 graph of petroleum recovery results after addition of salt content of nacl in brevundimonas diminuta and bhurkholderia glumae bacteria. fig 3 histogram changes of dissolved hydrocarbons before and after addition of crude biosurfactants from brevundimonas diminuta bacteria. fig 4 dissolved hydrocarbon histogram before and after addition of crude biosurfactant from bhurkholderia glumae bacteria. table 2 chain fraction c based on its temperature the identified chain c fraction temperature range <100 c 100-150 c 150-200 c 200-250 c 250-300 c (yudono 2014) <c10 c -c11 14 c -c15 17 c -c18 21 >c22 the b. glumae bacteria showed 21 peaks of the petroleum compound based on a total retention time of 56 min (fig 1c). it can be concluded if viewed from the peak amount after the treatment compared before the treatment of the addition of the peak amount that appears on the chromatogram. to see the degradability of hydrocarbon compounds can be seen from the change in the concentration of the initial hydrocarbon compound before treatment and the end after treatment. concentration changes can be analyzed using gas chromatography in the form of peak areas. of these differences may indicate a change in peak areas at baseline and after addition of biosurfactant (penet et al. 2006). if the chromatogram (a) is compared with (b) it can be seen that there is a decrease in ​​the peak area detected at retention time 25-50 min. then at 5-25 min retention periods small peaks indicate the presence of newly undetectable compounds. the increase is considered to be the result of degradation of a high molecular weight compound which then dissolves into a low molecular weight so that it appears at an earlier mooring time, resulting in a decrease in viscosity. this decrease in viscosity causes oil mobility to increase (li et al. 2002) (hao et al. 2008). while in figure (b) is a chromatogram of petroleum component of b. diminuta bacteria with the best salt concentration of 4.5% nacl compared with chromatogram of petroleum constituent components before treatment (a) there is a decrease of peak area areas at retention time 25-55 min, the decline in area is suspected because the bacteria can directly use and degrade the hydrocarbon compound into a simple compound. while at 5-25 min retention time new peaks appear degradation of c length to c short with the addition of wide peak area (penet et al. 2004). the soluble component in the biosurfactant and the residual component can be explained through the histogram of the abundance change. based on the histogram, the x axis is the temperature calculated from the retention time and the y-axis is the change of abundance. the histogram was derived from obtaining initial chromatogram data of the compound and after treatment of variation of salt nacl content in each bacteria, by comparing initial abundance before treatment and after variation of salt content of nacl based on retention time. changes in abundance are indicated by δa =% at-% ao if the resulting data is a positive number so it indicates the hydrocarbon chain component dissolved in biosurfactant, and if it is a negative number it indicates that the hydrocarbon chain component becomes residue. the carbon chain fraction contained in the chromatogram can be identified based on the program temperature methodology (yudono et al. 2010). the carbon chain fractions can be grouped according to the temperature range of the program as shown in table 2. the chromatogram ratio of petroleum and recovery chromatograms with biosurfactants from each bacterium has a peak change. changes in the peaks of chromatograms can occur due to the degredation process by bacteria. the degredation results may indicate a missing, emerging peak, larger peak height, or peak height reduction. missing peaks show that the hydrocarbon chains demineralized into co dan h o 2 2 (bharali et al. 2011). acknowledgment thanks to directorate general research and higher education of indonesia for supporting and funding this research, and university of sriwijaya which had given the facilities to do this research. references almeida f, 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sp. 2013. kinetika degradasi limbah minyak bumi menggunakan sinergi bakteri konsorsium (microccoccus sp, pseudomonas pseudomallei, pseudomonas pseudoalcaligenes dan bacillus sp) dan rumput eleusine indica (l.) gaertn [kinetics of petroleum-contaminated soil biodegraded by using bacterial consortium (microccoccus sp, p s e u d o m o n a s p s e u d o m a l l e i , p s e u d o m o n a s pseudoalcaligenes and bacillus sp) and eleusine indica (l.) gaertn].prosiding semirata fmipa universitas lampung 2013: 55-60. 88 yudono et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 404 not found 03 lg.cdr penicillin g (pen-g) was the most widely used antibiotics in the world market (approximately 19 %) (parmar et al. 2000). pen-g inhibits the enzyme transpeptidase involved in peptidoglycan synthesis, so that bacterial cell wall formation does not occur vol.9, no.2, june 2015, p 65-72 doi: 10.5454/mi.9.2.3 medium optimization for penicillin acylase (pac) production by recombinant bacillus megaterium ms941 bacillus thuringiensis containing pac gene from bgsc bd1 using response surface methodology 1 2 2 fentri paramitha putri , astutiati nurhasanah , niknik nurhayati ,* 2 3 is helianti , and khaswar syamsu 1 biotechnology major, graduate school, institut pertanian bogor, kampus ipb darmaga, bogor 16680, indonesia; 2 laboratory for the development of agroindustrial and biomedical technology bppt, gedung 611, kawasan puspiptek serpong, tangerang selatan, indonesia; 3 department of agroindustrial technology, faculty of agricultural technology institut pertanian bogor, kampus ipb darmaga, bogor 16680, indonesia penicillin g acylase (pac) hydrolyses of the amide bond of benzylpenicillin (pen-g) releasing paa and 6apa, key intermediate in the production of various semisynthetic penicillins. in this study, we optimised the pac production medium by rsm using two variables (xylose as inducer and cacl as divalent cations) to obtain the 2 optimum pac specific activity from bacillus megaterium btpacbd1. for this purpose, combinations of five different xylose concentrations (0.13 – 0.87 %) and five different cacl concentrations (0.64 – 4.36 mm) were 2 analysed, in a total of 22 experiments. ccd used for the analysis showed that in shake flask cultivations, xylose and cacl gave significant effects on pac volumetric activity and 2 the quadratic model was in good agreement 2 with the experimental results (r = 0.86 (p-value < 0.0001)). 130.669 ± 50.241 the maximum specific activity ( -1 units mg protein ) respectively, and was reached when xylose and cacl concentrations were 0.49% and 2.4 mm, 2 medium ph was around 7. under such conditions, the activity of pac and protein concentration achieved were -1 -1 1.318 ± 0.406 units ml and 0.01 ± 0.01 mg ml . the shake flask validation experiments demonstrated that with such medium composition the volumetric activity, protein concentration and specific activity achieved were -1 -1 -1 1.294 ± 0.171 units ml , 0.0102 ± 0.0003 mg ml and 125.91 ± 13.31 units mg , respectively . when the optimum medium composition was applied in 10 l bioreactor, the optimum volumetric activity (2.0687 ± 0.0820 -1 -1 units ml ) and protein concentration (0.0078 ± 0.0008 mg ml ) were achieved 48 h after the start of the -1 cultivation. however, the optimum pac specific acivity (1260.52 ± 27.57 units mg protein ) was achieved 18 h after the start of the cultivation. key words: bacillus megaterium btpacbd1, ccd, cacl , penicillin acylase, xylose2 penisilin asilase (pac) menghidrolisis ikatan amida dari benzilpenisilin (pen-g) dan menghasilkan paa dan 6-apa, bahan dasar produksi bermacam antibiotik penisilin semisintetis. pada penelitian ini, kami mengoptimasi media untuk produksi pac menggunakan rsm menggunakan dua variabel (xilosa sebagai induser dan cacl sebagai kation divalent) untuk mendapatkan aktivitas spesifik pac maksimal dari b. 2 megaterium btpacbd1. untuk mencapai tujuan ini, kombinasi dari lima konsentrasi xilosa (0,13 – 0,87%) dan lima konsentrasi cacl (0,64 – 4,36 mm) yang berbeda telah dianalisa sebanyak 22 percobaan. ccd digunakan 2 untuk menganalisa pada kultivasi skala labu erlenmeyer yang menunjukkan bahwa xilosa dan cacl 2 memberikan pengaruh yang nyata terhadap aktivitas pac dan model kuadratik dipilih berdasarkan hasil 2 -1 percobaan dengan (r =0,86 (nilai p < 0,0001)). aktivitas spesifik pac tertinggi (130,669 ± 50,241 unit mg ) didapatkan pada konsentrasi xilosa dan cacl masing-masing sebesar 0,49% dan 2,40 mm pada ph medium 2 -1 sekitar 7. pada kondisi ini, aktivitas pac dan kadar protein yang dihasilkan adalah 1,318 ± 0,406 unit ml dan -1 0,01 ± 0,01 mg ml . verifikasi hasil komposisi media optimal pada labu erlenmeyer dengan komposisi media hasil optimasi dapat menghasilkan aktivitas, kadar protein dan aktivitas spesifik masing-masing sebesar 1,294 ± -1 -1 -1 0,171 unit ml , 0,0102 ± 0,0003 mg ml dan 125,91 ± 13,31 unit mg . komposisi media optimal diaplikasikan -1 pada skala bioreaktor 10 l, aktivitas tertinggi (2,0687 ± 0,0820 unit ml ) dan kadar protein tertinggi (0,0078 ± -1 0,0008 mg ml ) dihasilkan pada waktu kultivasi 48 jam. sedangkan aktivitas spesifik tertinggi (1260,52± 27,57 -1 unit mg protein ). dihasilkan pada waktu kultivasi 18 jam. kata kunci: bacillus megaterium btpacbd1, cacl , ccd, penisilin asilase, xilosa2 *corresponding author; phone: +62-21-7560536 ext: 7119/ 7118, fax: +62-21-3169510; email: anpratomo@yahoo.com available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia (madigan et al. 2003). overuse of pen-g led to a bacterial pathogen resistance due to the production of β-lactamase which hydrolyzes β-lactam ring of penicillin rendering it inactive (navarro et al. 2004). pen-g's sensitivity to β–lactamase, its limited activity to gram positive bacteria and possibility to cause allergy raised problems of using this antibiotic (madigan et al. 2005). in industry, penicillin g acylase (penicillin amidohydrolase ec 3.5.1.11/ pac) catalyzes the conversion of benzyl penicillin (pen-g) via hydrolysis of the amide bond in the benzyl penicillin side chain, r e l e a s i n g p h e n y l a c e t i c a c i d ( pa a ) a n d 6 aminopenicillanic acid (6-apa). 6-apa is the key intermediate in the production of various semisynthetic penicillins (methicillin, ampicillin and amoxicillin) and the β-lactamase inhibitor (clavulanic acid) (wilson 1982). several bacterial isolates are known to produce pac, for examples escherichia coli (cole 1969), alcaligenes faecalis (verhaert et al. 1997), arthrobacter viscosus (ohashi et al. 1989), bacillus megaterium (chiang and bennett 1967), b. thuringiensis bgsc huazhongensis serovar strain 4cc1, 4bd1, 4aj1 and 4aw1 (nurhayati 2009). however, those that have been used commercially to produce pac enzymes are only e.coli and b. megaterium (vandamme and voets 1974). the production of pac can be improved by genetic engineering and/or bioprocess engineering. our study focused on the production of pac from recombinant b. megaterium. b. megaterium was chosen as the production host due to its relatively low extracellular protease activity (wittchen and meinhart 1995) and its capability to secrete pac directly into the medium, facilitating its purification and consequently decreasing the downstream processing and final production costs since the pac could be harvested simply by cold centrifugation (yang et al. 2006; hammond 1978). this is of course a great improvement in comparison to e. coli expression system, which retains the expressed protein intracellularly. the other disadvantage of e. coli was the presence of lipopolysaccharide (lps) as a component of the outer membrane. lps is endotoxin and harmful to humans. the strain used in this study contained a recombinant plasmid pmmbtpacbd1. the plasmid contained pac gene isolated from the genomic dna of b. thuringiensis bgsc bd1. the gene, btpac-bd1, was inserted into pmm1525 plasmid, by removing the lipase signal peptide (splipa) from the plasmid. the btpac-bd1 gene was predicted to have its own signal peptide that would lead the secretion of recombinant proteins out of the cell (nurhayati 2009). according to yang et al. (2006), b. megaterium yybm1 produced the highest pac with the addition of 0.5 % xylose and 2.5 mm cacl in medium. in contrast 2 to yang et al. (2006) who used the conventional methodology, this study used response surface methodology (rsm) via the design-expert software for the medium optimization, thus improving the accuracy and minimizing errors (box and draper 2007). in this project, in order to increase pac production, we tried to optimize the medium by addition of two variables, inducer and divalent cation, based on the previous research (yang et al. 2006). this study aimed to determine the optimum medium composition by xylose and cacl addition, try 2 pac production using bioreactors at its optimum conditions, and determine the best harvesting time. materials and methods microorganism. the strain used in this study (b. megaterium btpacbd1) was from the culturecollection of centre for bioindustrial technology, bppt, indonesia. stock cultures were maintained in -1 75% (v v ) glycerol. seed medium composition. luria broth (lb) agar contained 0.5% nacl, 1% tryptone, 0.5% yeast extract and 0.75% agar. lb liquid contained 0.5% nacl, 1% tryptone and 0.5% yeast extract. initial ph was adjusted to 7. media was autoclaved at 121°c for 15 min. lb tetracycline (lb-tet) medium was made by -1 adding tetracyclin (12.5 μg ml ) to the medium as a selection pressure. production medium composition. the minimal medium contained 50 mm mops (ph 7.0), 5 mm tricine (ph 7.0), 520 μm mgcl .6h o, 276 μm k so , 2 2 2 4 50 μm feso .7h o, 2.5 mm cacl , 100 μm 4 2 2 mncl .4h2o, 50 mm nacl, 10 mm kcl, 37.4 mm 2 -1 nh cl, 1.32 mm k hpo , 0.4 % (w v ) glucose, 1 ml 4 2 4 -1 -1 l trace element solution, and 1 ml l vitamin solution. the initial ph was adjusted to 7 and the medium was autoclaved at 121°c for 15 min. -1 antibiotic tetracyclin (12.5 μg ml ) was added to the medium as a selection pressure. the trace element solution contained 3.708 mg (nh4) 6mo7o .4h o, 24.73 mg h bo , 7.137 mg 24 2 3 3 cocl , 2.497 mg cuso , 15.832 mg mncl , and 2.875 2 4 2 -1 mg znso l . the vitamin solution consisted of 6 mg 4 66 putri et al. microbiol indones biotin, 20 mg niacin amid, 20 mg p-amino benzoate, 10 mg ca-panthotenate, 100 mg pyridoxal/hcl, 20 mg folacid, 50 mg riboflavin, 50 mg dl-6,8-thioctic acid -1 and 10 mg thiamine dichloride l . the minimal medium was supplemented with concentration of amino acid solution (1x). the stock solution (10x) was prepared separately according to the optimum solubility of amino acids in water as: 10 mg alanine, 10 mg arginine, 1 mg aspartic acid, 1 mg cysteine, 40 mg glycine, 4 mg isoleucine, 2 mg leucine, 10 mg lysine, 5 mg methionine, 5 mg proline, 5 mg serine, 5 mg threonine, 1.6 mg glutamic acid, 8 mg valine, 4 mg -1 histidine, 3 mg asparagines and 3 mg glutamine l (yang et al. 2006). all chemicals used for enzyme activity and protein assays were analytical grade. inoculum preparation and shake flask experiments. seed culture was prepared by inoculating a single colony from a 24 h lb-tet plate into 20 ml of lb-tet liquid medium (seed medium), ° followed by incubation at 37 c, 180 rpm for 6 h until od was 0.6 –0.8. after that, inoculum was prepared 578nm by inoculating 10 ml of seed culture to 90 ml of seed ° medium at 37 c, 180 rpm for 3 h until od was 0.6 – 578nm 0.8. batch cultivation was carried out as reported by ° rajendhran et al. (2003) with 180 rpm agitation, 28 c for 48 h in 500 ml erlenmeyer flasks containing 90 ml of cultivation medium. each flask was then inoculated with 10 ml of inoculum. e x p e r i m e n t a l d e s i g n . r e s p o n s u r f a c e methodology (rsm) with two variables, i.e. xylose as inducer and cacl as divalent cations, were used to 2 obtain the maximum and specific activity of pac produced by bacillus megaterium btpacbd1. for this purpose, five different xylose concentrations (ranging from 0.13% to 0.87%) and five different cacl 2 concentrations (ranging from 0.64 mm to 4.36 mm) (table 1) were analysed in combinations in a total of 22 experiments (table 2). design expert software version 9.0 was used for the experimental design and statistical analysis of the experimental data. batch cultivation in 10 l bioreactor. the batch cultivation was carried out in a 10 l biostat bioreactor equipped with three six-bladed disc impeller and oxygen under the following conditions: medium -1 volume 5 l, inoculation volume 10% (v v ), 28°c, 1.0 vvm aeration, 150 agitation and ph medium was 7. the ph of the medium was controlled by addition of sterile naoh and hcl. vegetable oil was also added as an antifoaming agent. to determine the best harvesting time, b. megaterium btpacbd1 was cultivated for 60 h and the volumetric activities were measured every 6 h. samples of the culture were withdrawn at 0, 6, 12, 18, 24, 30, 36, 42, 48, 54 and 60 h after the inoculation. samples of the culture were harvested every 6 h and the cultivations stopped at the best time from the result of the best harvesting time's experiment. assay of pac volumetric activity. culture were ° centrifuged at 4 c and 6000 rpm for 15 min (hitachi cr216, rotor r10a2) as reported by rajendhran et al. (2003). the supernatant was used as the source for the enzyme analysis. the pac activity in the clear ° supernatant was assayed at 50 c using penicillin g as substrate. one unit of pac activity was defined as the amount of enzyme required to release 1 μmol of 6-apa -1 min under the assay condition as reported by balasingham et al. (1972). protein concentration. protein assays were performed using bovine serum albumin as protein standard as reported by bradford (1976). specific activity. specific activity of pac (units -1 -1 mg ) was expressed as units mg of protein concentration or ratio from pac volumetric activity -1 -1 (units ml ) and protein concentration (mg ml ). results response surface methodology. the strategy used to attain the goal of this work was to explore the experimental space around the previously selected medium compositions. an experimental design was implemented in order to better evaluate the interactions between the variables. the ccd was chosen as the one that allows the fitting of several mathematical models from the data obtained. response 1: pac volumetric activity. the analysis of variance (anova) of the response surface model showed that when b. megaterium strain btpacbd1 was cultivated in shake flasks, xylose and cacl combination had significant effects on pac 2 volumetric activity. the quadratic model and two factors factorial design could explain the data achieved significantly (p-value < 0.0001) at 95% confidence level. the lack of fit test showed that only the quadratic model gave the incompatibility (p-value > 0.1142 and 2 r = 0.86). the anova of the quadratic response surface model demonstrated that the response surface quadratic model could explain the data (p-value < 0.0001). the variables that affected the model at 99% confidence level was indicated by p-value < 0.01. the application of the quadratic response model showed an volume 9, 2015 microbiol indones 67 note: -1: lower limit; 0: the midpoint; 1: the upper limit; α: starting point empirical relationship between pac activity and the test variables. the relationship could be expressed in the following equation: 2 2 y= 1.132 – 0.324 x – 0.129 x – 0.257 x *x1 2 1 2 -1 where, y is pac volumetric activity (units ml ), x is 1 xylose concentration (%) and x is cacl concentration 2 2 (mm). the response surface and contour plot of xylose and calcium ions combinations on pac volumetric activity was presented in fig 1a. response 2: protein concentration. the anova by treatment of the response surface model combinations showed that when b. megaterium strain btpacbd1 was cultivated in shake flasks, xylose and cacl did not have significant effects on protein 2 concentration (p-value > 0.0191). response 3: pac specific activity. the anova of the response surface model showed that when b. megaterium strain btpacbd1 was cultivated in shake flasks, xylose and cacl had significant effects on pac 2 specific activity. the quadratic model and two factors factorial design could explain the data from this results significantly (p-value < 0.0001) at 95% confidence level. the lack of fit test showed that only the quadratic model gave the incompatibility (p-value > 0.2688 and 2 r = 0.79). the anova of the quadratic response surface model demonsrated that the quadratic model could explain the data (p-value < 0.0001). the variables that gave the effects on the model at 99% confidence level was indicated by p-value <0.01. the application of the quadratic response model showed an empirical relationship between pac activity and the test variables. the relationship could be expressed in table 1 independent variable in the experimental plan table 2 the ccd matrix employed for two independent variables 68 putri et al. microbiol indones variable coded level coded -1.861 -1 0 1 1.861 xylose cacl2 x1 0.13 0.3 0.5 0.7 0.87 x2 0.64 1.5 2.5 3.5 4.36 experiment number x1 x2 1 1 -1 2 1 1 3 -1 1 4 -1 -1 5 -1 -1 6 0 0 7 0 0 8 -1 1 9 1 -1 10 1 1 11 1 -1 12 -1 1 13 -1 -1 14 1 1 15 0 0 16 -a 0 17 0 0 18 0 -a 19 a 0 20 0 a 21 0 0 22 0 0 the following equation: 2 2 y= 11.191 – 3.368 x – 1.519 x – 2.025 x *x1 2 1 2 -1 where, y is pac volumetric activity (units ml ), x is 1 xylose concentration (%) and x is cacl concentration 2 2 (mm). the response surface and contour plot of xylose and calcium ions combinations on pac specific activity was presented in fig 1b. the maximum specific activity was reached when xylose and cacl 2 concentrations were 0.49% and 2.4 mm, respectively, and medium ph was around 7. under such conditions, the volumetric activity of pac achieved was 1.318 ± -1 0.406 units ml with protein concentration 0.0101 ± -1 0.01 mg ml and specific activity 130.669 ± 50.241 -1 units mg . the validation experiment in shake flask demonstrated that with such medium composition the volumetric activity achieved was 1.294 ± 0.171 units -1 -1 ml , protein concentration 0.0102 ± 0.0003 mg ml -1 and specific activity 125.91 ± 13.309 units mg protein . batch cultivation in 10 l bioreactor. the optimum medium composition was applied in 10 l bioreactor. the growth of b. megaterium btpacbd1 and the volumetric activities were measured every 6 h for 60 h (fig 2). the exponential (log) growth phase occured between 0 – 30 h of cultivation. according to yang et al. (2006), the lag phase of the b. megaterium was 1 – 5 h after the start of cultivation. the adaptation (lag) phase might not have been detected in our data because the observation period was longer than the adaptation phase. b. megaterium btpacbd1 underwent the stationary phase between 30 – 42 h before subsequently entered the death phase. fig 2 also showed that pac secretion started in the exponential phase and reached -1 maximum (2163 units ml ) in the stationary phase (48 h), before eventually declining. since we aimed to obtain the highest possible enzyme activity on harvesting, then 48 h was selected as the best time point to harvest the enzyme, hence used in subsequent experiments as the final time point. the growth of b. megaterium btpacbd1, pac volumetric activity, protein concentration and pac specific activity during cultivation are presented in fig 3. as seen in fig 3 and fig 3a, the bacterial growth and pac volumetric activity showed the same pattern as previously observed (fig 2). at 48 h, the maximum pac -1 volumetric activity was 2.069 ± 0.0820 units ml which reached in the s meaning that 1 ml of pac enzyme harvested could degrade 2.0687 ± 0.0820 µmol ° pen-g to 6-apa per minute at 50 c. the pac volumetric activity was relatively constant between 18 – 42 h. fig 3b showed that the protein concentration started to increase in the exponential phase and continued to increase until the cultivation was terminated at 48 h fig 1 response surface and contour plot of xylose and calcium ions combinations on pac volumetric activity (a) response surface and contour plot of xylose and calcium ions combinations on pac specific activity (b). volume 9, 2015 microbiol indones 69 a b when the protein concentration was 0.0078 ± 0.0008 -1 mg ml . fig 3c showed that the pac specific activity was maximum during the exponential phase (18 h). the highest pac specific activity achieved was 1260.521 ± -1 27.5711 units (mgprotein) . discussion design experts software version 9.0 (stat ease fig 2 bacillus megaterium cell density ( ) and pac volumetric activity ( ) in optimum medium at 28°c, 1.0 vvm aeration and 150 rpm agitation for 60 hours cultivations. a b c fig 3 bacillus megaterium cell density ( ) and pac volumetric activity ( ) (a) protein concentration ( ) (b) and pac specific activity ( ) (c) in optimum medium at 28°c, 1.0 vvm aeration and 150 rpm agitation. each data point represents the average of three experiments, each experiment was performed in at duplicate/ triplicate. 70 putri et al. microbiol indones 7 -1 c el l d en si ty ( 1 0 c el ls m l ) -1 v o lu m et ri c a ct iv it y ( u n it s m l ) 7 -1 c el l d en si ty ( 1 0 c el ls m l ) 7 -1 c el l d en si ty ( 1 0 c el ls m l ) 7 -1 c el l d en si ty ( 1 0 c el ls m l ) -1 v o lu m et ri c a ct iv it y ( u m l ) -1 p ro te in c o n ce n tr at io n ( m g m l ) -1 s p ec if ic a ct iv it y ( u m g ) inc. usa) was used to determine the optimum xylose and cacl concentrations. as clearly and thoroughly 2 presented above, the results indicated that xylose and c a c l c o n c e n t r a t i o n s a ff e c t e d t h e p r o t e i n 2 concentration and the activity of pac enzyme s y n t h e s i z e d b y b . m e g a t e r i u m c o n t a i n i n g pmmbtpacbd1 plasmid that had the strong xyla promoter. gene btpac-bd1 was cloned so that its expression was directed by xyla promoter. so, when xylose was added to the medium, the promoter started trancription (mobitec gmbh 2012). the influence of calcium ions on the activity of b. megaterium pac was also tested. previous research showed that the calcium ions was a cofactor for membrane transport and important factor influencing the protein folding and maturation (ignatova et al. 2005; kasche et al. 2005). 2+ ca is a divalent cationic positive modulator, influencing the conformation of the catalytic enzymes, thus facilitating the enzyme-substrate interaction and enhancing the enzyme catalytic activity (susanti 2003). although the pac activity started to increase during the exponential phase (12 – 18h), the activity remained relatively constant between 18 – 42h (fig 2, 3a). however, when bacteria entered the death phase, the pac activity seemed to have increased rather significantly. presumably, the enzyme that was previously accumulated in the cell was released via cell lysis into the cultivation medium directly, without secretory process. this result might indicate that the protein was not secreted as expected. as the vector signal peptide gene had been removed, protein secretion had relied entirely on the host's secretion machinery. however, there is possibility that the host's secretion machinery does not suit that of the expressed protein, rendering the retention of the protein inside the cell, hence the stagnant activity during the exponential phase followed by the sudden increase of activity during the death phase optimization of the medium composition by yang et al. (2006) who used conventional methodology achieved volumetric activity of 1600 -1 units l . our experiment using rsm achieved higher -1 volumetric activity (2069 ± 82 units l ). however, this result was still lower than that of acevedo and -1 cooney (1973), who achieved 3000 units l and -1 panbangred et al. (2000), who achieved 9000 units l . differences in the results might be caused by the differences in the origin of the pac gene, the plasmid construct, the medium composition, the cultivation conditions and pac assays. in our study, the protein concentration was determined using crude extract enzyme without any purification step, so the measured protein concentration might not only contain pac but also other impurities accumulating during cultivation. this might have been the main cause of the significant reduction of the pac specific activity. when the cells started to lyse during death phase, releasing the intracellular proteins, the amount of impurities might increase significantly. this study was performed using b. megaterium ms941, which had been engineered by eliminating the extracellular proteases. so, when the cells were intact, extracellularly expressed protein can be protected from protease degradation. however, when the bacteria entered the death phase, the bacterial cells lysed releasing numerous proteins that might include various intracellular proteases into cultivation medium, causing hydrolysis of pac, further reducing the enzyme's specific activity. previous study by leonita (2013), who used the same strain as the one used in our study but did the optimization using conventional methodology, reported that the pac specific activity achieved was -1 1018.83 units mg , 36 h after the start of the cultivation. however, leonita (2013) used different medium with different composition so the result might not be comparable. in conclusion, it is clear that the methodology used for optimization of medium compositions, particularly the concentration of xylose and cacl , affect the pac-2 like activity. as clearly and thoroughly presented above, optimum medium composition as defined by the rsm aided optimization increased the pac-like activity produced. this is a better methodology for the optimization of pac production from b. megaterium btpacbd1 than the conventional methodology. however, further research must be performed to optimize the cultivation conditions, such as ph, agitation, aeration and temperature on a pilot scale in order to achieve a better pac volumetric activity and specific activity. references [mobitec] molecular biologische technologie gmbh. 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7 page 8 7 diana rev.pmd volume 3, number 3, december 2009 p 133 138 issn 1978-3477 rapid detection of virulence genes in vibrio cholerae from edible ice in jakarta diana e. waturangi* and marisa fransisca faculty of biotechnology, atma jaya catholic university, jalan jenderal sudirman 51, jakarta 12930, indonesia vibrio cholerae is a bacteria that lives naturally in an aquatic environment. it causes a waterborne disease which is called cholera. infection of waterborne disease occurs via the fecal-oral route, mostly through drinking water. as we know, ice is made from city water sources and it is commonly used in beverages. most of publications about v.cholerae come from clinical samples, while little is known about the presence of these bacteria in potable water, especially in ice. in this study, we isolated v. cholerae from ice in jakarta and continued with detection of the virulence genes. we recovered v. cholerae from ice samples and then continued with detection of virulence genes including toxr, ctxa, ompu, tcpa, ace, zot using multiplex pcr. the results indicated that all of the samples were non-toxigenic strains, but were classified as pathogenic strains because they have at least one of the virulence genes present. the presence of pathogenic v. cholerae in edible ice needs to be emphasized since they have some of the virulence factors and also the class 1 integron. keywords: vibrio cholerae, ice, multiplex pcr, virulence genes, vibrio cholerae is primarily an inhabitant of the aquatic environment and water plays an important role in the transmission and epidemiology of cholera. infection of waterborne disease happens via fecal-oral route, mostly through drinking water. the majority of beverages in jakarta are provided with ice cubes, made from local water sources. since indonesia lacks a public-health inspection program for monitoring ice quality, we suspect that ice may be a major source of water borne diseases, especially bacterial enteropathogens including v. cholerae. most publications on v. cholerae come from clinical samples while little is known about the presence of these bacteria in local water, especially in the form of ice. the pathogenicity of v. cholerae depends on combination properties of its virulence genes. there are at least six virulence genes in v. cholerae, the regulator factor toxr, cholera toxin enzymatic subunit a (ctxa), toxincoregulated pilus (tcpa), outer membrane protein (ompu), accessory cholera toxin (ace) and zonula occludens toxin (zot). toxr regulated genes are divided into four general classes, namely cholera toxin (ctx) genes, toxin coregulated pilus (tcp) genes, accessory colonization factor (acf) genes and toxr-activated genes (tag) of unknown function (peterson and mekalanos 1988; matson et al. 2007). toxin co-regulated pilus (tcp) has the function of being an essential colonization factor (taylor et al. 1987). the a and b subunits of cholera toxin are encoded by the ctx operon. it is now known that the ctx genes lie within the genome of a lysogenic filamentous phage (ctxö). ompu protein has the potential role in the adhesion of v. cholerae to mammalian cells. the accessory cholera enterotoxin (ace) has recently been identified as a third toxin of v. cholerae which alters ion transport and causes fluid accumulation. zonula occludens toxin (zot) has potential function to increase the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction (baudry et al. 1992). here we report the isolation of v. cholerae from edible ice in jakarta and the detection the presence of virulence genes using multiplex pcr. materials and methods sample collection. ice cubes were collected from five different regions of jakarta. each region of jakarta was represented by three ice suppliers. we also collected ice cubes from street vendors and restaurants around jakarta. ice cubes were collected and melted at room temperature. aliquots of samples were combined with alkaline peptone water (apw) (oxoid) with a ratio 1:1. incubations were undertaken for 24 hours at 37ºc. isolation of v. cholerae. enriched samples in apw were diluted by adding 0.85% of nacl (w/v). diluted samples were spread onto thiosulfate-citrate-bile-sucrose (tcbs) (oxoid) agar plates and incubated at 37ºc for 18 to 24 hours. yellow single colonies which grown on tcbs plates were randomly selected for pcr detection. each sample was chosen from 10-15 single colonies. the selected colonies were grown overnight at 37ºc. preparation of pcr template. a 5 ml aliquot of luria broth (lb) was inoculated with the selected colony to obtain template dna for multiplex pcr. incubation was done for 24 hours at 37ºc with shaking at 100 rpm. to prepare template dna, 1.5 ml of the culture was centrifuged, resuspended in sterile distilled water, and boiled for 10 min. multiplex pcr assay. a master mix was prepared for all of the samples into one reaction tube. the master mix for each sample contains 25 µl of go-taq master mix kit (promega), 10.5 µl of ddh 2 o and 12 µl of primers. we used six pairs of primer for detection of six virulence genes of v. *corresponding author, phone: +62-21-5703306 ext 449, fax:+62-21-5719060, e-mail: diana.waturangi@atmajaya.ac.id microbiol indones134 waturangi and fransisca cholerae, consisting of toxr, ctxa, ompu, tcpa, ace, zot 25 pmol.ul-1 for each primer (eurogentec). the sequences of the primers are listed in table 1. isolates from a previous study were used as a positive control. the multiplex pcr reaction was carried out as described by singh et al. (2002). pcr products were separated by 1.8% w/v agarose gel (promega) in 0.5x tbe buffer. the gel was visualized using gel-doc (biorad). serological assay. the v. cholera strains which were found to possess one or another of the virulence genes sought were examined for agglutination by the somatic-oantigen sero-grouping by using polyvalent-o and monovalent-ogawa and inaba serum (biofarma, bandung, indonesia). some of the isolates were confirmed as virulent through biochemical assays by using microbacttm gnb 12a + 12b (oxoid). antimicrobial resistance assays. positive colonies were inoculated on brain-heart-infusion-agar (bhia) (oxoid) plates using cotton swabs and seven antimicrobial disc (oxoid) was put on the surface of an agar plate (streptomycin 10 µg, tetracycline 30 µg, erythromycin 15 µg, ciprofloxacin 5 µg, kanamycin 30 µg, sulphamethaxole-trimethoprim 25 µg, ampycilin 10 µg). each disc must be pressed down to ensure complete contact with the agar surface. plates were incubated overnight at 37ºc. the zone of inhibition was measured by reduced diameter of the clear zone with in the diameter of the disc. the results were analyzed according to the 2003 nccls standard. results from 15 ice samples, we selected 330 isolates to be detected for the presence of virulence genes and the class 1 integron. each sample was alternated by 15 until 20 isolates. all of the presumptive colonies of v. cholerae were analyzed by multiplex pcr assay (fig 1). the results show that there are 92.06% isolates which were positive for toxr, 15.87% which were positive for ompu, 7.94% which were positive for zot and tcp, 87.3% which were positive for the class 1 integron. five isolates showed negative for toxr detection, but these isolates were positive for tcpa gene detection (table 2). none of the isolates were positive for ctx or ace, indicating that all of the isolates were non-toxigenic strains because they having at least one virulence gene. we used 7 antibiotics (streptomycin 10 µg, tetracycline 30 µg, erythromycin 15 µg, ciprofloxacin 5 µg, kanamycin 30 µg, sulphamethaxole-trimethoprim 25 µg, ampycilin 10 µg) to showed the occurrence of antibiotic resistance gene. all of the isolates have at least one antibiotic resistant gene (table 3). there are 53.97% that resistant to ampycilin, 44.44% that resistant to sulphamethaxole-trimethoprim, 58.73% that resistant to erythromycin, 66.67% that resistant to streptomycin, 50.79% that resistant to tetracycline, 25.40% that resistant to ciprofloxacin, and 66.67% that resistant to kanamycin. result of serological assay showed only eight isolates were belonging to o1 serogroup, 1 isolate is inabao1 and 7 isolates are ogawa-o1. therefore most of the isolates belong to the non-o1 serogroup (table 2). discussion multiplex pcr. the presence of toxr protein is important for the regulation of other virulence genes in pathogenic v. cholerae (bina et al. 2003; stonehouse et al. 2008). usually the presence of toxr gene is important for the function of other virulence genes, since it play as a table 1 primer sequence used in these study microbiol indones 135volume 3, 2009 regulator for other genes (matson et al. 2007). one of the possibilities is horizontal gene transfer from pathogenic strains to non-pathogenic strains, and the transfer is not included the toxr gene (covacci et al. 1997). our five isolates were also negative in ctxa detection. this is also contrary to the current assumption that most cholera toxin (ct)positive isolates are also positive for tcp. none of the detected isolates showed a positive result for ctxa and ace which indicates that all of the isolates do not have the genetic potential to produce ct. ompu has a potential role in the adhesion of v. cholerae to mammalian cells. five isolates showed positive result in zot gene detection. the presence of these protein could increase the permeability of the small intestinal mucosa by affecting the structure of the intracellular tight junction (baudry et al. 1992). although there are a lot of isolates which do not have any virulence genes detected in this study except for the toxr gene, it does not indicate that these bacteria could not evoke diarrhea. because there are some cases of mild diarrhea that are caused by non-pathogenic v. cholerae by an unknown mechanism (ramamurthy et al. 1993), and because there are some virulence genes that have not been detected in the present study. these nonpathogenic strains may be pathogenic strains, referenced by the current hypothesis that some pathogenic bacteria have evolved from non-pathogenic strains of the same species via horizontal transfer of virulence genes (covacci et al. 1997). serological and antimicrobial resistance assay. our results cannot indicate that the non-o1 serogroup bacteria are non-pathogenic strains. because the non-o1 strains have been reported to be involved in the emergence of a newer variant of v.cholerae, o139 strains could occur in epidemic and pandemic forms (bik et al. 1995). also, a lot of isolates in the present study contain many virulence genes. there are several isolates showed resistance to almost all of the antibiotic resistance genes. this finding need to aware since the virulence properties of the isolates and their relation with diarrheal disease. acknowledgement this research was funded by indonesia toray science foundation (itsf) research grant 2007. references baudry b, fasano a, ketley j, kaper jb. 1992. cloning of a gene (zot) encoding a new toxin produced by vibrio cholerae. infect immun 60:428-34. bik em, bunschoten ae, gouw rd, mooi fr. 1995. genesis of the novel epidemic vibrio cholerae o139 strains: evidence for horizontal transfer of genes involved in polysaccharide synthesis. embo j 14:209–16. bina j, zhu j, dziejman m, faruque s, calderwood s, mekalanos j. 2003. toxr regulon of vibrio cholerae and its expression in vibrios shed by cholera patients. proc natl aca sci 100:2801-6. covacci a, falkow s, berg de, rappuoli r. 1997. did the inheritance of a pathogenicity island modify the virulence of helicobacter pyroli. microbiology 5:205-8. fields pl, popovic t, wachsmuth k, olsvik o. 1992. use of polymerase chain reaction for detection of toxigenic vibrio cholerae o1 strains from the latin american cholera epidemic. j clin microbiol 30:2118–21. matson js, withey jh, dirita vj. 2007. regulatory networks controlling vibrio cholerae virulence gene expression. infect immun 75:5542-9. peterson km, mekalanos jj. 1988. characterization of the vibrio cholerae toxr regulon: identification of novel genes involved in intestinal colonization. infect immun 56:2822-9. ramamurthy t, bag pk, pal a, bhattacharya sk, shimada t, takeda t, karasawa t, kurasono h, takeda y, nair gb. 1993. virulence patterns of vibrio cholerae non-o1 strains isolated from hospitalized patients with acute diarrhea in calcuta, india. j med microbiol 39:310-7. rivera ing, jongsik c, anwar h, brad rs, rita rc. 2001. genotype associated with virulence in environmental isolates of vibrio cholerae. appl environ microbiol 67:2421–9. shi l, miyoshi si, hiura m, tomochika ki, shimada t, shinoda s. 1998. detection of genes encoding cholera toxin (cholera toxin), zonula occludens toxin (zot), accessory cholera enterotoxin (ace) and heat stable enterotoxin (st) in vibrio mimicus clinical strains. microbiol immunol 42:823–8. fig 1 results of multiplex pcr and pcr of class 1 integron detection (left) m, marker 100 bp (new england biolabs); negative control; 1, 3, 5, 6, isolates ja, mh, wl, jg toxr and ompu positive; 2 and 4, isolates jc, le toxr positive. (centre) m, marker 100bp (new england biolabs); -, negative control; 1, 2, 3, 4, 5, 6, isolates jm, jh, bb, ca, pc, mc toxr positive. (right) 1, marker 1 kb ladder (fermentas); 2, positive control (waturangi et al. 2003); 3, negative control; 4 and 5, class 1 integron with the amplicon size 750 bp; lane 6, 7, 8, class 1 integron with the amplicon size 500 bp. 869 bp 779 bp 2000 bp 750 bp 500 bp table 2 results of multiplex pcr and serological assay microbiol indones136 waturangi and fransisca region sample amp sxt e s te cip k west jakarta jc s s i r r r i ja s r r r s i r je s s s i s s s jt r r r r r i r jf s s r r r r i la s s i r s s r lb s i i r s s r lc s s i r s s r jd s s r r r r i jg s s r s s r r gh r r i s r r r gi s s r s s s s le r r r r r i i lk s s r i r s s jb s s r r r i r lh r r r i r r s jm r s i r s s s jh r r r i s s r lm s s s i r s r central jakarta mg r r r r i s s og r r r r r i i oj r r r r i s r ob r s s s i i r od r i r r r i r oe r r r r r i r nb r r r r i i s nj r r r r r s r ok r r r r r i i qf s i i i r s r mh r r r r s s s pc s i s r s s r mc r r r i i i r qh r i s r r i r xf s s s i i r r xg s s i i i r s xa s s s r r r r vb r r r r r i i vl r r s s s s r wk r r r r r i r wd r r r r i r r wc r r r r r i r we r r r r r i r wf r r r r r i i wl r r r r r r i vi s s s r i s r north jakarta df r r r r i s r dk s i i r s s r db s s i r s s r da s s i r s s r dc s i s r s s r de s i i r s s r south jakarta bb r i r r r i s be r r r r r i r ba r i i s s s r eg s i r s r i r ek r i i s s r i eg r r s s s i r eh r r r s s s i ec s i r r r i r east jakarta ca s s i i r r r cl r r r i r r r cb s i r r r r r ce s s r r r r r microbiol indones 137volume 3, 2009 table 3 results of class 1 integron detection and antibiotic resistance assay amp, ampycilin; sxt, sulphamethaxole-trimethoprim; e, erythromycin; s, streptomycin; te, tetracycline; cip, ciprofloxacin; k, kanamycin, r = resistant; i, intermediate; s, sensitive; v, present. singh dv, isac sr, colwell rr. 2002. development of a hexaplex pcr assay for rapid detection of virulence and regulatory genes in vibrio cholerae and vibrio mimicus. j clin microbiol 40:43214 . stonehouse e, kovacikova g, taylor rk, skorupski k. 2008. integration host factor positively regulates virulence gene expression in vibrio cholerae. bacteriology 190:4736-48. microbiol indones138 waturangi and fransisca taylor rk, miller vl, furlong db, mekalanos jj. 1987. use of phoa gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. proc natl acad sci 84:2833–7. waturangi de, suwanto a, schwarz s, erdelen w . 2003. identification of class 1 integron-associated gene cassettes in escherichia coli isolated from varanus spp. in indonesia. j antimicrob chemo 51:175-8. 01 wong.cdr endophytic fungi have attracted the interest of scientist and pharmaceutical companies, especially since the discovery of the world's first billion-dollar anticancer drug, paclitexel (taxol) from pestalotiopsis microspora, which is an endophytic fungus living within the himalayan yew tree, taxus wallichiana (maheswari 2006). endophytic fungi spend their entire life cycle within their host plant tissue without showing any apparent symptom (strobel et al. 1998). they live symbiotically with the host plants, which provide them profuse nutriment and restful habitation for the survival (firakova et al. 2007), while they protect their host plants from external biotic and abiotic stresses (rodriguez et al. 2009). for instance, the endophytic fungi, curvularia protuberate can be found on all of the non-embryonic tissues of the geothermal plant dichanthelium lanuginosum. neither the fungus nor vol.9, no.4, december 2015, p 141-149 doi: 10.5454/mi.9.4.1 isolation, identification and screening of antimicrobial properties of the marine-derived endophytic fungi from marine brown seaweed 1 2 1 3 changi wong *, peter proksch , lee tung tan , samuel lihan , 3 1 aazani mujahid , and moritz müller 1 faculty of engineering, computing and science, swinburne university of technology sarawak, kuching 93350, malaysia; 2 institut für pharmazeutische biologie und biotechnologie, universitätstraβe 1, düsseldorf 40225, germany; 3 faculty of resource science and technology, universiti malaysia sarawak, kota samarahan 93400, sarawak, malaysia marine seaweeds are known to produce valuable medicinal compounds such as antioxidants and anticoagulants, and have been reported to display antimicrobial activity against gram positive and gram negative bacteria. several studies have identified so-called endophytic fungi living inside their hosts as the source of active compounds. in this study marine brown seaweed, padina sp., was studied with regards to their endophytic fungi to assess if they are the source of the reported antimicrobial activity. twenty fungal isolates were isolated from padina sp. collected off talang-talang island, sarawak, malaysia. all isolates were screened for their antimicrobial properties and 11 out of 20 isolates displayed positive results. dna was successfully extracted for five isolates and sequence analysis grouped all of them with other endophytic fungi. “fungus 2” seems to be related to a so far uncultured endophytic fungus. “fungus 19” showed the most promising antimicrobial properties and was chosen for further agar well assay and cytotoxicity testing. its ethyl-acetate extract showed positive results in the agar well assay and also a cytotoxic effect on artemia nauplii. the extract was screened using hplc and showed a compound similar to a known anti-cancer compound, dihydromyricetin, which is also an anti-intoxicant, anti-inflammatory and anti-oxidative agent which may be responsible for the observed antimicrobial activity. key words: antimicrobial properties, endophytic fungi, marine brown seaweed, padina sp. rumput laut laut yang dikenal menghasilkan senyawa obat berharga seperti antioksidan dan antikoagulan, dan telah dilaporkan mempunyai aktivitas antimikroba terhadap bakteri gram positif dan gram negatif. beberapa studi telah melaporkan bahwa jamur endofit yang hidup di dalam rumput laut sebagai inang adalah sumber senyawa aktif. tujuan penelitian ini adalah mengevaluasi, apakah rumput laut coklat padina sp. dengan jamur endofitnya merupakan sumber dari aktivitas anti mikroba yang dilaporkan tersebut. dua puluh isolat jamur diisolasi dari padina sp. dikumpulkan dari pulau talang-talang, sarawak, malaysia. hasil skrining anti mikroba menunjukkan, bahwa 11 dari 20 isolat menunjukkan hasil positif. genom dna dari lima isolat berhasil diekstraksi dan dianalisa deret itsnya dan dianalisa dengan kelompok jamur endofit lainnya. "jamur 2" diduga terkait dengan jamur endofit yang tidak dapat dikultur (unculturable). "jamur 19" menunjukkan sifat antimikroba yang paling menjanjikan dan terpilih untuk agar well assay baik dan pengujian sitotoksisitas lebih lanjut. ekstrak etil asetat dari islat ini menunjukkan hasil positif dalam agar well assay dan sitotoksiksitas terhadap artemia nauplii. hasil skrining ekstrak tersebut dengan menggunakan hplc menunjukkan senyawa yang mirip dengan senyawa anti kanker, dihydromyricetin, yang juga merupakan anti-intoxicant, anti inflamasi, dan juga agen anti oksidan yang mungkin berkaitan dengan aktivitas anti mikroba. kata kunci: anti mikroba, jamur endofit, rumput laut coklat, padina sp. *corresponding author; phone: +60-168716911; email: cgwong@swinburne.edu.my available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia 142 wong et al. microbiol indones o the plant can tolerate temperatures above 38 c when they are grown non-symbiotically, but when grown symbiotically, they are able to tolerate temperatures up o to 65 c (márquez et al. 2006). in another study, a nonpathogenic mutant of an endophytic fungus, colletotrichum magna (path-1) was found living asymptomatically within watermelon (citrullus lanatus) and discovered to be involved in disease protection against virulent pathogens such as colletotrichum orbiculare (redman et al. 1999). marine seaweeds are known to produce valuable compounds such as antibiotics, natural antioxidants, anticoagulants, and anticancer compounds (ganesan et al. 2007; wijesinghe et al. 2012). they also contain high amounts of carotenoids, dietary fibres, proteins, essential fatty acids, vitamins and minerals (foon et al. 2013). besides that, rajasulochana and team (2009) have discovered that marine brown seaweed (padina sp.) extracts possess antimicrobial properties, and were able to inhibit the growth of bacillus megaterium, staphylococcus aureus and escherichia coli. no studies have assessed the possibility of endophytic fungi in brown seaweed as the potential origin of their active compounds and the aim of this study is to look at the endophytic fungi living inside padina sp. and assess them for their antimicrobial and anti-cancer potential. materials and methods endophyic fungi isolation and purification. endophytic fungi were isolated from padina sp., collected off talang-talang island, sarawak, malaysia. 2 the sample was cut into small pieces of 1 cm in size and then surface-sterilized by immersion in 70% ethanol for 5-15 seconds. after that, the sample was immersed in sterile artificial seawater (twice) to stop the sterilization. the sample was then dried using a sterile cotton cloth and placed on a yeast extract glucose chloramphenicol agar (ygca) plate. one millilitre of the artificial seawater that was used to clean the sample was taken out and poured on another ycga plate as negative control. the agar dish was then sealed with parafilm, labelled and incubated at 25 °c for 3 days. after 3 days, growth of hyphae was observed and those hyphae were isolated using sterile plastic straws and placed on fresh ycga dishes. this step was repeated until pure fungal colonies were obtained. purified fungi were cultured on potato dextrose agar (pda) and incubated at 25 °c. morphological identification. the identification of the isolated fungi was based on the description of colonies and morphological structures, and types of conidiophore described in descriptions of medical fungi by ellis et al. (2007). molecular identification. pure fungal isolates were cultured on potato dextrose agar (pda) for 5 days and a small amount of mycelia transferred into sterile 100 μl distilled water in 1.5 ml microcentrifuge tubes by using a sterile toothpick. after that, the mixture was vortexed thoroughly for 1 min and centrifuged at 10,000 rpm for 3 min. the supernatant was discarded and 100 μl of lysis solution (te buffer – 10 mm tris-hcl, 1 mm edta, at ph 8) added to the microcentrifuge tube. the mixture was incubated at 85 °c for 20 min and centrifuged for 5 min at 5,000 rpm. two (2) µl of the supernatant were used for the polymerase chain reaction. the rest of the crude extract was stored at -20 °c until further usage. a master mix was prepared which contained 20 μl -1 of 10x pcr buffer, 6 μl of dntp mixture (10 mmol l each), 8 μl of deioned formamide, 4 μl of mgcl (25 2 -1 mmol l ), 8 μl of each primer (its1 {5'tccgtaggtgaacctgcgg-3'} and its4 {5'-1 tcctccgcttattgatatgc-3'}; 10 mmol l ), 2 -1 μl of taq dna polymerase (5 u μl ) and 144 μl of sterile distilled water in a total volume of 200 μl. twenty (20) microliter of the master mix were transferred into a sterile 0.3 ml pcr tube together with 1 μl of the genomic dna. the polymerase chain reaction (pcr) consisted of an initial denaturing step of 5 min at 94 °c followed by 35 cycles (50 s at 94 °c, 50 s at 54 °c, and 50 s at 72 °c), followed by a final extension step at 72 °c for 10 min. the pcr products were resolved by electrophoresis through 1% agarose gels in tae and visualized by staining with ethidium bromide for 10 min and destaining for 15 min. the pcr products were then purified and sent for sequencing. the sequences obtained were analyzed against the ncbi (usa) database (zhang et al. 2000) and a phylogenetic tree was constructed from genetic distance and bootstrap values calculated using mega6 (see fig 1; tamura et al. 2013). the genus of the remaining 22 fungal isolates was identified using morphological characteristics and an overview is presented in table 1. fermentation. a single cylindrical block (agar plug) of 1 week old fungal cultures was inoculated into 200 ml of potato dextrose broth (pdb) and incubated o for 2 months at 25 c, static conditions. after 2 months volume 9, 2015 microbiol indones 143 of incubation, 200 ml of ethyl acetate were added and the mixture shaken overnight using a rotary shaker. the fungal biomass was filtered and crude extracts dried inside a fume hood. dried extracts were analyzed using a high performance liquid chromatography (hplc) system (dionex p580) coupled to pda detector (uvd340s). routine detection was carried out at 235, 254, 280, and 340 nm. the stationary phase was eurospher-10 c18 (knauer, germany), the mobile phase consisted of meoh and 0.02% h po in h o at 3 4 2 the following elution gradient: 0 min, 10% meoh; 5 min, 10% meoh; 35 min, 100% meoh; 45 min, 100% meoh. preliminary antimicrobial screening (agar diffusion assay). all 20 purified fungal samples were cultured on potatoes dextrose agar (pda) for 1 week before the agar diffusion assay was carried out. three different test organisms were used in the assay, a gram positive bacterium, bacillus subtilis, a gram negative bacterium, escherichia coli, and yeast, saccharomyces cerevisiae. the overnight broth cultures were swapped onto a fresh pda plate using a sterile cotton swap. a sterile plastic straw was used to transfer small segments of the purified fungal cultures onto the swapped pda plate. triplicates were prepared for each test organism, plates incubated at 37 °c for 24 h and the results recorded (see table 2). the purified “fungus 19”, which displayed the highest activity during the agar diffusion assay, was subjected to two more tests to assess his antimicrobial activity (see below). cross streaking assay. “fungus 19” was grown on pda for 4 days at 25 °c. five different types of test organisms were used in this test; a gram positive bacterium, bacillus subtilis, a gram negative bacterium, escherichia coli, two yeasts, saccharomyces cerevisiae and candida albicans, as well as a fungus, aspergillus niger. the overnight broth cultures were streaked on a pda plate containing “fungus 19” in the middle of the plate. the plate was then incubated at 25 °c for 3 days and results recorded (see table 3). agar well assay. thirty gram of agar from 15 days old “fungus 19” were transferred to a 250 ml sterile conical flask, 150 ml ethyl acetate added and shaken for 24 h. the mixture was filtered and 75 ml of the crude extract transferred into a 100 ml beaker to air dry inside a fume hood. the weight of the beaker with and without the crude extract was measured and recorded in order to obtain the weight of the dried extract which was utilized for the agar well assay and cytotoxicity testing. three different types of test organisms were used in the agar well assay, a gram positive bacterium, bacillus subtilis, a gram negative bacterium, escherichia coli, and a yeast, saccharomyces cerevisiae. overnight broth cultures were swapped onto a fresh pda plate using a sterile cotton swap. a sterile plastic straw was used to create 3 well on the plate and different volumes of the extract (10, 20, and 30 µl) were loaded into the wells using a micropippette. triplicates were prepared for each test organism. positive control experiments were carried out using chloramphanicol (1000 ppm) for bacillus subtilis and escherichia coli, and decon solution for saccharomyces cerevisiae; ethyl acetate as negative solvent control. the plates were incubated at 37 °c and the zones of inhibition were measured and recorded (see table 4). cytotoxicity testing (brine shrimp assay). national cancer institute (nci. usa) demonstrated that the cytotoxicity testing by using artemia nauplii has a significant correlation to the growth inhibition of human solid tumour cell lines in vivo (silva et al. 2006). serial dilutions of the dried extract were carried out and solutions of 1000, 100, 10, 1 and 0.1 ppm prepared. ten artemia nauplii (a. nauplii) (carballo et al. 2002) were drawn through a micropippette and placed in a well containing 50 µl of artificial seawater. fifty microliter (50 µl) of the different solutions (see above) were added to the wells to test for concentrations of 500, 50, 5, 0.5, and 0.05 ppm. triplicates were prepared for each concentration as well as a control experiment containing 50 µl of artificial seawater with two drops of dmso and ten a. nauplii. the wells were kept for 21 h at room temperature and number of alive a. nauplii recorded (see table 5). results in total of 20 fungal isolates were isolated from one marine brown seaweed (padina sp.) sample. fifteen of the isolates were identified based on their morphology (table 1) and five of the isolates were furthermore identified using molecular methods (fig 1). “fungus 1”, “fungus 12” and “fungus 3” were grouped with aspergillus aculeatus strain hsz15 [genbank accession number kj605160; 100% similarity], fungal endophyte sp. g81 [genbank accession number hm537058; 99% similarity] and cladosporium tenuissimum isolate hsw-17 [genbank accession number kj475817; 100% table 1 genus of 15 fungal isolates that were identified using morphological characteristics isolate (fungus) naming based on fungus morphology 4 18 5 19 6 11 9 14 8 13 10 15 20 16 17 botrydiplodia sp. botrydiplodia sp. botrydiplodia sp. penicillium sp. penicillium sp. botrydiplodia sp. botrydiplodia sp. similar to isolate 2 similar to isolate 2 penicillium sp. penicillium sp. botrydiplodia sp. penicillium sp. fusarium sp. botrydiplodia sp. fig 1 internal transcribed spacer (its) gene-based phylogenetic tree representing amplified fungal sequences. the phylogenetic tree was generated with distance methods, and sequence distances were estimated with the neighbour-joining method. bootstrap values ≥50 are shown and the scale bar represents a difference of 0.05 substitution per site. accession numbers for the reference sequences are indicated. similarity]. while “fungus 2” and “fungus 7” grouped with didymellaceae sp. c1-18 [genbank accession number jq717314; 100% similarity] and a so far uncultured endophytic fungus clone nr1-11 [genbank accession number jq717314; 95% similarity] (fig 1). among all 20 isolates, 45% of the fungi showed no inhibition on the test organisms, 25% showed inhibition of gram positive bacteria, 10% showed inhibition of gram negative bacteria, and another 5% showed inhibition of both gram negative and gram positive bacteria but no inhibition on yeast (table 2). our isolates seem to be able to inhibit gram positive 144 wong et al. microbiol indones fungus 12 aspergillus aculeatus strain hsz15 [kj605160] fungus 1 fungal endophyte sp. g81 [hm537058] fungus 3 cladosporium tenuissimum isolate hsw-17 [kj475817] fungus 2 uncultured endophytic fungus clone nr1-11 [fj882006] coniothyrium palmicola strain cbs 161.37 [jx681086] fungus 7 didymellaceae sp. c1-18 [jq717314] 100 100 99 91 100 100 96 0.05 fungus 12 table 2 screening of antimicrobial properties of the endophytic fungi isolated from brown seaweed, padina sp. from talang-talang island located in kuching. fungus 19 is highlighted in italics because it inhibited all test microorganisms. + indicates inhibition, indicates no inhibition. samples bacillus subtilis fungus 4 fungus 6 fungus 8 fungus 16 fungus 11 fungus 19 fungus 10 fungus 18 fungus 14 surface sterilized escherichia coli saccharomyces cerevisiae + + + + + + + + + + + + bacteria more than gram negative bacteria (15% more) which might be due to the composition of the cell wall of the negative bacteria which is more rigid and complex (hugo 1998). 'fungus 19' showed to be the most promising fungal strain, which is able to inhibit all the testing microorganism including yeast (table 2) and was therefore targeted for secondary screening, by using cross streaking assay and agar well assay. due to the observed wide activity, a fungus was further added as test organism for the secondary screening; aspergillus niger a fungal species that can cause chronic bilateral otomycosis (mishra et al. 2004). saccharomyces cerevisiae aspergillus niger test microorganism bacillus subtilis escherichia coli ++ + inhibition +++ +++ table 3 secondary screening of antimicrobial properties (cross streaking assay) of “fungus 19” against four different type of microorganisms. + indicate relative inhibition strength. 10 positive control (chloramphenicol) volume (μl) (1000 ppm) negative control (ethyl acetate) 30 20 0.63 (+/0.058) 2.8 zone of inhibition (cm) escherichia coli 1.07 (+/0.058) 0.87 (+/0.058) 0.67 (+/0.115) 3.1 bacillus subtilis 0.87 (+/0.208) 0.80 (+/0.100) table 4 overview of inhibition zones (in cm) for the crude extract of “fungus 19” tested against escherichia coli and bacillus subtilis. volume 9, 2015 microbiol indones 145 146 wong et al. microbiol indones “fungus 19” was also further assessed for its anticancer potency by a brine shrimp assay. the secondary screening (cross streaking) confirmed the antimicrobial properties of “fungus 19” as it inhibited 4 test organisms, gram positive bacterium, bacillus subtilis, gram negative bacterium, escherichia coli, a yeast, saccharomyces cerevisiae, and a fungus, aspergillus niger. it showed the biggest inhibition on both gram positive and negative bacteria, followed by yeast, and the least inhibition on the fungus aspergillus niger (see table 3). the compounds produced by the “fungus 19” were extracted by using ethyl acetatethe most efficient solvent for extracting extracting polar as well as nonpolar secondary metabolites from fungal cultures (jantamas and dusanee 2010). the crude extract of “fungus 19” exhibited antimicrobial properties against escherichia coli (inhibition zones of 0.63 – 1.07 cm, table 4) and bacillus subtilis (inhibition zones of 0.67 – 0.87 cm, table 4). it is noteworthy that all inhibition zones were smaller than the positive control, chloramphenicol. the crude extract of “fungus 19” did exhibit cytotoxicity against artemia nauplii (table 5). hplc analyses were carried out on crude extracts of all species showing activity and revealed many compounds with no known match in the database of the table 5 screening of cytotoxicity level of the crude extract produced by “fungus 19”. different concentrations (0.5 to 500 ppm), negative and positive controls are shown and mortality (in %) was calculated. the status “active”, “inactive” and “dead” indicates the mobility of the artemia nauplii which are high, low and immobile, respectively. a all dead 1 dead 9 active all active all active 5 active 5 inactive 1 dead 9 active all active b 2 inactive all active all active all active 1 active 5 inactive 4 dead all active 1 dead 9 active c 3 inactive all active all active 1 dead 9 active 5 active 3 inactive 2 dead 1 dead 9 active all active average 2 inactive all active all active all active 4 active 4 inactive 2 dead all active all active mortality 80% 0% 0% 0% 20% 0% 0% well concentration 500 ppm 0.05 ppm 5 ppm dmso + artificial seawater (negative control) 50 ppm original water from the artemia nauplii tank (negative control) 0.5 ppm fig 2 hplc chromatogram of compound from “fungus 4” and “fungus 7” that had a similar structure to the flavonoid dihydromyricetin heinrich heine universitaet (hhu). while “fungus 19” did show the most promising activity in the bioassays, the hplc spectra did not reveal any compound of interest. a possible reason may be that methanol was used as solvent for the hplc and the active compound of “fungus 19” was not soluble in methanol. further analyses are needed to clarify the origin of its observed activity. the spectra of “fungus 4” and “fungus 7”, however, did contain a compound with a structure related to dihydromyricetin, a flavonoid (fig 2), which has been shown to possess anti-intoxicant, anti-inflammatory, anti-oxidant, and anti-cancer activities (nazemiyeh et al. 2008; shen et al. 2012). discussion twenty (20) fungal strains were isolated from brown seaweed (padina sp.) samples, representing more than 7 genus of fungi. phylogenetic analyses of 5 isolates revealed that all of them are related to other endophytic fungi, confirming the cleanliness and proficiency of our isolation process. “fungus 2” was grouped with an uncultured endophytic fungus and could be a new species as many fungi remain to be discovered by mycologists (blackwell 2011). “fungus 7” grouped with known plant endophytes, didymellaceae sp. (fig 1). members of the cladosporium genus are known to produce antimicrobial agent such as cladospolide and isoclodospolide (zhang et al. 2001; dai et al. 2006). however, the “fungus 3” that was grouped with cladosporium tenuissimum, did not display antimicrobial properties. it could be because of the different plant hosts thus results in different production of secondary metabolites. this can be further supported by the studies done by zhang team (2001) and dai team (2006), both research groups isolated a cladosporium sp., one from marine sponge and the other one from maytenus hooker, both of the cladosporium sp. results in producing different type of bioactive compounds. fungus 1 was grouped with aspergillus aculeatus (figure 1) which is known to produce useful multipolysaccharide degrading enzymes such as βglucosidase, endoglucanase, β-xylosidase, and xylanase, and is a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry (suwannarangsee et al. 2014). we did not assess enzymatic properties in our study, however “fungus 1” could be analysed further in the future. a note of caution has to add though as the same fungal species can display varying activity in different host plants as explained above for “fungus 3”. only 1 out of 20 fungi, “fungus 19”, related to penicillium sp., displayed antimicrobial activity against yeast and both types of bacteria and therefore, this particular fungus was suspected to produce potential antimicrobial agents and has thus been chosen for further testing, such as cross streaking, agar well assay and cytotoxicity testing. penicillium sp. are known to produce a wide range of valuable bioactive compounds such as antibacterial and antifouling polyketides (bao et al. 2013), cytotoxic funicone (chen et al. 2014) and antifungal antibiotics (omura et al. 1988). in a recent study by subramani et al. (2013), a penicillium sp. isolated from a marine fijian sponge melophlus sp. exhibited both antibacterial against multi-drug resistant pathogens and cyotoxicity activity against brine shrimp larvae. in our study, the penicillium sp. related “fungus 19” ethyl-acetate extract showed to have positive results on both agar well assay and cytotoxicity testing. besides that, the cross streaking assay showed the inhibition of the yeast and fungus. therefore, this particular fungus can be suspected to produce some bioactive compounds that are cytotoxic and have wide antimicrobial properties or produce a variety of compounds at a sufficient level to inhibit several different test organisms. one of the compounds produced by “fungus 4” (related to botrydiplodia sp.) is related to an anticancer compound, dihydromyricetin, which is also an anti-intoxicant, anti-inflammatory and anti-oxidative agent (nazemiyeh et al. 2008; shen et al. 2012). zhang et al. (2014) have demonstrated that dihydromyricetin possesses antitumor activity against liver cancer cells without being cytotoxic to immortalized normal liver cells. “fungus 4” was however not tested for any of these activities as it only showed antimicrobial activity against b. subtilis during the initial screening (table 2). other botrydiplodia species have been reported to produce a different anti-cancer compound, fungal taxol, which is able to significantly suppress the 7, 12 dimethyl benz(a)anthracene (dmba)-induced breast cancer in sprague dawley rats (pandia et al. 2010). future studies should be carried out on “fungus 4” as it seems to produce a compound with a high potential for use in pharmaceutical industries. in conclusion, padina sp. was found to be a rich source of endophytic fungi and several of the isolates found in this study showed a promising range of volume 9, 2015 microbiol indones 147 148 wong et al. microbiol indones activities. the extracts produced by the isolates yielded some potentially valuable compounds as well as many unknown compound and are worth further investigations. acknowledgment we would like to express our great appreciation to prof. lee nyanti from universiti malaysia sarawak for providing the seaweed sample, and the lab technicians and my colleagues from swinburne university of technology sarawak. references bao j, sun yl, zhang xy, han z, gao hc, he f, qian py, qi sh. 2013. antifouling and antibacterial polyketides from marine gorgonian coral-associated fungus penicillium sp. scsgaf 0023. j antibiot (tokyo). 66(4): 219-223. doi: 10.1038/ja.2012.110. blackwell m. 2011. the fungi:1, 2, 3…5.1 million species? am j bot. 98(3): 426-438. doi: 10.3732/ajb.1000298. carballo jl, hernández-inda zl, pérez p, garcía-grávalos md. 2002. a comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. bmc biotechnol. 2: 17. doi: 10.1186/14726750-2-17. chen mj, fu yw, zhou qy. 2014. penifupyrone, a new 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macrolide antibiotic produced by cladosporium sp. ft-0012. j antibiot. 54(8): 635-641. zhang q, liu j, liu b, xia j, chen n, chen x, cao y, zhang c, lu c, li m, zhu r. 2014. dihydromyricetin promotes hepatocellular carcinoma regression via a p53 activation-dependent mechanism. scientific reports. doi:10.1038/srep04628. zhang z, schwartz s, wagner l, miller w. 2000. a greedy algorithm for aligning dna sequences. j comput biol. 7: 203-214. doi:10.1089/10665270050081478. volume 9, 2015 microbiol indones 149 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 02 lg.cdr liver cancer is one of the most common cancers worldwide with high mortality rate (ferlay et al. 2013) and it occurs mainly in the form of hepatocellular carcinoma (hcc). approximately 80% of the hcc cases are due to chronic hepatitis b virus (hbv) or hepatitis c virus (hcv) infection (el-serag 2012). hbv belongs to the hepadnaviridae family of virus and is the causative agent of acute and chronic liver diseases. while the acute hbv infection permits full recovery, the chronic hbv infection lasts for more than 6 months, defined by persistence of hbsag (chang 2007). increased risk of hcc development is observed in patients having chronic hbv infection and it can occur in absence of liver cirrhosis (chemin and zoulim 2009; el-serag 2012). hbv is classified into ten genotypes, i.e. genotype a – h, with genotype b and c predominantly exist in indonesia (sunbul 2014). the hbv genotype c is known to cause severe liver disease, cirrhosis, and hcc as compared to the hbv genotype b. however, hbv genotype b correlates with hcc incidence without cirrhosis (el-serag 2012). several mechanisms have been proposed to link hbv infection vol.10, no.1, march 2016, p 9-14 doi: 10.5454/mi.10.1.2 detection of hepatitis b virus x gene mutation from local clinical samples anita artarini*, hanary geby jessica, raden rini kartikasari, catur riani, and debbie soefie retnoningrum pharmaceutical biotechnology laboratory, school of pharmacy, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia hepatitis b virus (hbv) belongs to the hepadnaviridae family and it infects hepatocytes, which is the most common cell in liver. hbv infection is distinguished into acute and chronic infection based on the duration of infection. chronic infection of hbv exists more than six months and it can develop into liver cirrhosis and hepatocellular carcinoma (hcc). the development of chronic hbv infection is affected by viral particle load, genotype and subgenotype, as well as its association with hbv x protein (hbx). several mutations of the hbx gene are known to be associated with hcc. the aim of this study is to determine the genotype and subgenotype based on hbx gene, and to detect its mutation from 44 local clinical samples. the hbx gene was amplified using nested pcr, which produced two fragments with size of 469 and 395 bp. the obtained hbx gene sequences were aligned with hbx gene sequences from database to determine the genotype, subgenotype, and amino acid substitution. the analysis showed that patients were infected mainly by hbv subgenotype b3, which are common in indonesia. in this study, we found the presence of known hcc-related hbx mutant, i.e. v5l, a47t, i127t and k130m/v131i, as well as new hbx mutant, i.e. g22s and a85t. the presence of hbx t118n mutant was detected at the highest percentage and occurred from samples with high hbv dna titer. detection of hbx gene mutations might be necessary to predict the manifestation of liver disease, as well as development of hcc. key words: hepatitis b virus, indonesia, x gene mutation virus hepatitis b (vhb) tergolong dalam famili hepadnaviridae dan dapat menginfeksi sel hepatosit manusia. infeksi vhb dapat terjadi secara akut dan kronis berdasarkan lamanya infeksi. infeksi kronis terjadi selama lebih dari enam bulan dan dapat berkembang menjadi sirosis hati dan karsinoma hati. perkembangan infeksi vhb kronis menjadi sirosis dan karsinoma hati dipengaruhi oleh jumah virus yang menginfeksi, genotipe dan subgenotipe vhb, dan hubungannya dengan fungsi protein x vhb (hbx). mutasi pada gen pengkode hbx diketahui berasosiasi dengan status karsinoma hati. tujuan penelitian ini adalah menentukan genotipe dan subgenotipe vhb dan mendeteksi mutasi gen pengkode hbx dari 44 sampel lokal. gen pengkode hbx diamplifikasi menggunakan metode nested pcr yang menghasilkan 2 produk pcr berukuran 469 dan 395 bp. urutan nukleotida gen pengkode hbx disejajarkan dengan urutan gen pengkode hbx di database untuk penentuan genotipe, subgenotipe, dan substitusi asam amino hbx. hasil analisis menunjukkan keberadaan genotipe b dan subgenotipe b3 terdapat secara dominan. hasil analisis urutan gen pengkode hbx menunjukkan substitusi asam amino hbx yang telah diketahui berasosiasi dengan karsinoma hati, yaitu v5l, a47t, i127t dan k130m/v131i. hasil analisis juga menunjukkan substitusi asam amino baru, yaitu g22s and a85t. keberadaan hbx t118n mutan juga dideteksi pada penelitian ini dengan presentase tertinggi dan terdeteksi dari sampel dengan titer dna vhb tinggi. deteksi mutasi pada gen pengkode hbx dapat digunakan lebih lanjut sebagai prediksi manifestasi klinik infeksi hati oleh vhb dan kaitannya dengan perkembangan karsinoma hati. kata kunci: indonesia, mutasi gen x, virus hepatitis b microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone/fax: +62-22-2504842; email: anita@fa.itb.ac.id and hcc development, i.e. viral dna integration, the truncated form of hbsag and the x protein of hbv (hbx) (di bisceglie 2009). integration of viral dna to the host genome appears to happen at random and it was found in 80% of hbv infected patient with hcc. the mechanism of truncated hbsag in causing hcc remains unclear, whereas the hbx protein may contribute to hcc development in many ways. the hbx protein is known to act as a transcriptional activator of many cellular gene, activate cell survival signaling pathway, interact with cellular protein which has oncogenic potential and cause epigenetic change (di bisceglie 2009; kew 2011; shlomai et al. 2014). the hbx protein can modulate signaling transduction pathways, i.e. ras, raf, mapk, nf-ƙb and jak-stat, which then leads to stimulation of hepatocyte proliferation. the hbx protein also plays a role in apoptosis process and has proand anti-apoptotic effects (kew 2011). several hbx mutant proteins have been reported to be correlated with hcc incidence, i.e. v5m/l, p38s, h94y, i127t/n, and k130m/v131i (cho et al. 2011; jang et al. 2012; kim 2014). it has been reported that the k130m/v131i hbx mutation was present in 51% of hcc tissue (liu et al. 2014), whereas the t118n mutant was present in 50% and 30% of chronic hepatitis (ch) and hcc samples, respectively (utama et al. 2009). another study also reported the existence of t118n mutant in one out of ten samples from manado (fatimawali et al. 2014). the c-terminally truncated hbx was observed in 30-40% hcc patients, more frequently in tumor samples (liu et al. 2008; 2014; sze et al. 2013). a number of studies have also explored the contribution of hbx mutants to d e v e l o p m e n t o f h e p a t o c a r c i n o g e n e s i s . t h e k130m/v131i hbx mutant protein was shown to upregulate hif-1α expression and transactivation activity as compared to the wild-type hbx (lee et al. 2014; liu et al. 2014), as well as differentially influencing cell proliferation (lin et al. 2005; iyer and groopman 2011). in this study, we analyzed the hbx gene sequences from 44 local clinical samples. we also determined the genotype, subgenotype and the hbx gene mutation of the samples. the hbx gene was amplified using nested pcr and the sequence of hbx gene was analyzed in comparison to the existing sequence in the database. the amino acid substitution of hbx protein was compared to the known hbx mutant, especially the hcc-related hbx mutant, as well as determination of new hbx mutant. materials and methods pcr and dna sequencing. hbv dna samples were isolated at pramita diagnostic laboratory jakarta, indonesia, including the determination of hbv dna titer in the samples. the hbx gene was amplified using nested pcr. primer pairs were designed according to the sequence of hbv genome in the genbank database. primers used for the first round of pcr were designed to recognize the region outside the hbx gene at nucleotide 1233 and 1940 of hbv genome. for the second round of pcr, additional primers were designed at position 1685 and 1545 of the hbv genome. the primers used for the pcr reactions are as follows: hbvf1_1233_for (5'-catgcgtgg rca/gccttgtg-3'), hbvf1_1685_rev (5'gcctcaaggtcggtcgtt-3'), hbvf2_1545_for ( 5 ' c t c c c c g t c t g t g c c t t c 3 ' ) a n d hbvf2_1940_rev (5'-cagaaggcaaaaaa gagactaactc-3'). the pcr mixture for the first round of pcr contained 100 ng of hbv dna, 0.4 µm of each primer hbvf1_1233_for and hbvf2_ 1940_rev, 1x dreamtaq green master mix (thermo scientific) and pcr-grade water. the amplification was performed at 94 °c for 5 min, followed by 25 cycles of 94 °c for 30 s, 55 °c for 1 min and 72 °c for 1 min, with final elongation at 72 °c for 10 min. at the second round of pcr, the amplification of hbx fragment 1 (hbx_f1) used hbvf1_1233_for and hbvf1_1685_rev primer pair, while the the amplification of hbx fragment 2 (hbx_f2) used hbvf2_1545_for and hbvf2_1940_rev primer pair. the second round of pcr used 1 µl of 1/25 diluted pcr product from the first round of pcr as dna template. the pcr product from the second round of pcr was analyzed using 1.5% agarose gel electrophoresis. positive results were shown by existence of 469 bp of hbx_f1 and 395 bp of hbx_f2. specific dna bands of hbx_f1 and hbx_f2 from 44 samples were then purified using gel or pcr dna fragments extraction kit (geneaid). the samples were subjected to dna sequencing at macrogen, korea. confirmation of the nucleotide changes were performed by re-sequencing of the pcr product. sequence analyses. raw sequences from dna sequencing was confirmed using blast analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi) and the sequences of hbx_f1 and hbx_f2 were then assembled. determination of genotype and subgenotype was based on the similarity to the sequences deposited in the ncbi database. analysis of 10 artarini et al. microbiol indones amino acid substitution was performed by translating the dna sequence using expasy-translate tool (http://web.expasy.org/translate/) and then aligning the protein sequences with several hbx protein sequences from the database using multalin program (http://multalin.toulouse.inra.fr/multalin/). results to determine the nucleotide sequence, the hbx gene was amplified using nested pcr. the first round of pcr yielded 707 bp of pcr product which was undetectable in agarose gel electrophoresis. the pcr product from the first round pcr was then used as dna template for the second round of pcr. in the second round of pcr, hbx gene was amplified into 2 fragments, hbx_f1 and hbx_f2. the result from hbx_f1 amplification from several clinical samples showed band with experimental size of 463 bp, indicating presence of hbx_f1 with theoretical size of 469 bp (fig 1). meanwhile, the amplification of hbx_f2 showed band with size of 403 bp, which was in accordance to hbx_f2 theoretical size of 395 bp (fig 2). to analyze the nucleotide sequence of hbx gene, purified pcr product was analyzed by direct sequencing. the dna sequences were then assembled and the complete or partial sequences of hbx gene was analyzed (genbank ncbi accession number kx429616-59). determination of the genotype and subgenotype of hbv was performed by blast analysis based on the similarity of the hbx gene to the ncbi database. the result showed that genotype b, c and d were detected in 77.3%, 20.5% and 2.3%, respectively. analysis of subgenotype showed that b2, b3, c1 and c2 subgenotypes were detected in 18.2%, 59.1%, 15.9% and 4.5%, respectively (table 1). from the analyzed samples, genotype b and subgenotype b3 occured at the highest percentage. to analyze the hbx amino acid substitution, dna sequence results were translated and aligned with hbx protein sequences from the database with same genotype or subgenotype. some known hcc-related hbx gene mutation as well as new hbx mutation which occured in two or more samples were listed in table 2. the result showed the presence of known hcc-related hbx mutant, i.e. v5l (2.3%), a47t (6.8%), i127t (4.5%) and k130m/v131i (13.6%) (table 2). presence of hbx r87w was observed at 4.5% (table 2) and this mutant has been observed previously from hbv genotype f in chronic carrier patients (león et al. 2005). we also observed the presence of hbx v45l, g22s and a85t mutants in 11.4%, 9.1% and 4.5%, respectively. the hbx v45l was detected from samples with genotype b2, which was in accordance to the previous report (fatimawali et al. 2014). the presence of hbx g22s and a85t mutants was never been reported before. in this study, the hbx t118n mutant occurred at highest percentage (15.9%), while the hcc-related volume 10, 2016 microbiol indones 11 fig 1 electrophoregram result of hbx_f1 pcr product. amplification of hbx gene was performed by nested pcr from several clinical samples (27, 29, 30, 32, 33) to obtain hbx_f1 fragment. the pcr product was analyzed by 1.5% agarose gel electrophoresis. (m) : 1kb dna marker, (-): pcr negative control. fig 2 electrophoregram result of hbx_f2 pcr product. amplification of hbx gene was performed by nested pcr from several clinical samples (29, 30, 32, 33) to obtain hbx_f2 fragment. the pcr product was analyzed by 1.5% agarose gel electrophoresis. (m) : 1kb dna marker, (-): pcr negative control. 12 artarini et al. microbiol indones table 1 hbv genotype and subgenotype based on hbx sequence genotype subgenotype n (%) b c d b2 c1 c2 b3 8 (18.2) 7 (15.9) 2 (4.5) 26 (59.1) 1 (2.3) amino acid substitution n (%) -1sample (hbv dna titer in iu ml ) v5l* 1 (2.3) 8 43 (10 ) g22s 4 (9.1) 8 7 7 8 18 (10 ), 26 (10 ), 28 (10 ), 34 (10 ) v45l 5 (11.4) 7 6 6 5 5 3 (10 ), 17 (10 ), 19 (10 ), 22 (10 ), 25 (10 ) a47t* 3 (6.8) 7 8 7 26 (10 ), 29 (10 ), 31 (10 ) a85t 2 (4.5) 8 7 41 (10 ), 42 (10 ) r87w 2 (4.5) 7 6 5 (10 ), 15 (10 ) t118n 7 (15.9) 8 6 7 6 8 8 7 1 (10 ), 7 (10 ), 9 (10 ), 12 (10 ), 29 (10 ), 33 (10 ), 42 (10 ) *known hcc-related hbx mutation table 2 frequencies of some hbx mutations i127t* 2 (4.5) 6 8 7 (10 ), 43 (10 ) k130m/v131i* 6 (13.6) 6 7 6 6 7 8 2 (10 ), 4 (10 ), 7 (10 ), 15 (10 ), 40 (10 ), 43 (10 ) k130m/v131i mutant occurred at 13.6% (table 2). to date, the existence of hbx t118n was observed only from study using samples collected in indonesia (utama et al. 2009; fatimawali et al. 2014) and its direct correlation to hcc incidence was not investigated so far. the presence of hbx t118n mutant was observed from samples having high hbv 6 -1 dna titer (≥10 iu ml ), similarly to the known hccrelated hbx k130m/v131i mutant (table 2). discussion detection of hbv genotypes, subgenotypes and hbx gene mutations have been studied intensively to find association with hcc development. therefore, the studies may contribute in understanding clinical implication of diverse hbv genotypes and hbx gene mutation, as well as in finding better management of hbv infected patients. a meta-analyses study performed based on published data from 1950 to 2012 showed that hbv genotype c correlates with higher risk of hcc as compared to other hbv genotypes. association of hbv genotype b and c to hcc was found at 12% and 25%, respectively (wong et al. 2013). the hbx gene mutation may as well associates with hcc incidence, escpecially mutation that occurs in the hbx transactivation domain, protein binding site and immune epitopes (li et al. 2015). in this study, amplification of the hbx gene was performed using nested pcr which resulted 2 hbx fragments in the final pcr product. using this approach, amplification and hbx gene sequencing was successful for all 44 samples and for samples with 6 -1 hbv dna titer as low as 10 iu ml . previous attempt using conventional pcr showed successful amplification only for 12 out of 20 samples and successful nucleotide sequence analysis only from 5 out of 12 amplified samples. the amplification of hbx gene using conventional pcr was also only successful 8 -1 for samples with hbv dna titer of 10 iu ml (gunawan 2012). based on this research, hbv genotype b was found at higher percentage in comparison to genotype c and d. this is in accordance to previous report that genotype b is the most abundant hbv genotype found in indonesia, followed by genotype c (utama et al. 2009; sunbul 2014). it is previously shown that genotype b is found more abundantly in west area of volume 10, 2016 microbiol indones 13 indonesia while genotype c is predominant in east indonesia (thedja et al. 2011). the samples analyzed in this study were obtained predominantly from sumatra and java islands, thus more genotype b was shown. meanwhile, another study performed with samples from sulawesi island showed that genotype c was found at higher percentage (fatimawali et al. 2014). this study also showed that subgenotype b3 was the predominant hbv subgenotype. this is in accordance to the previous report that subgenotype b3 are the most abundant hbv subgenotype found in sumatra and java islands (thedja et al. 2011). in this study, analysis of hbx gene sequence showed the existence of known hcc-related mutants, such as v5l, a47t, i127t and k130m/v131i, with hbx k130m/v131i occurred at higher percentage as compared to the other hcc-related mutants. meanwhile, existence of hbx mutant t118n was detected at the highest percentage, 15.9%. in previous report, hbx t118n was found in 50% of ch patients and in 31% of hcc patients. meanwhile, the hbx k130m/v131i was found in 37% of hcc patients (utama et al. 2009). the percentage of hbx k130m/ v131i and hbx t118n detected in this study were rather low because it was detected from all hbvinfected patients and the clinical status of the patients was unknown. to date, there were no reports investigating the correlation of hbx t118n mutant to development of hcc, while there have been many r e p o r t s s t u d y i n g m e c h a n i s m o n h o w h b x k130m/v131i contributes to hcc in vitro. as the hbx t118n was found merely in samples with high hbv dna titer and detected as abundant as hbx k130m/v131i in indonesia, it might be necessary to study its correlation to hcc development. in conclusion, our study highlight the existence of known hcc-related hbx mutants as well as other previously uncharacterized hbx mutants. our observation of 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has never been reported previously. in this study, the aim is to identify whether bacteria play a role in the formation of bitter tastes in tempe. sensory tests were carried out in order to determine the scores of bitter-taste-intensity in tempe. the sensory test on emp, wjb, clr, drg, and mlb tempe shows that emp tempe has the highest score (2.3) and wjb has the lowest (1.3). it is revealed that the processing method has no impact on the formation of the bitter taste in tempe. plating analysis, showed that emp soaking water contained a higher number of enterobacteria group bacteria, approximately 103-104 cfu ml-1 and spore-forming bacteria groups, 102 cfu ml-1, compared to wjb. similarly, other bacteria groups in fresh emp tempe was 102 cfu g-1 higher than those in fresh wjb tempe. based on sequencing the16s rrna gene, the dominant bacteria on pca media in emp tempe are acetobacter indonesiensis, klebsiella pneumoniae, bacillus subtilis, and flavobacterium sp. on the other hand those in wjb tempe were klebsiella sp., brevundimonas sp., bacillus sp., pseudomonas putida, and acinetobacter sp. bacillus, a group of proteolytic bacteria was found 105 cfu m-1 higher in the soaking water of emp compared to wjb. nevertheless, the types and numbers of fungi were not significantly different between tempe types. accordingly, it is concluded that the difference in the number and the types of bacteria involved in the tempe production process leads to the difference in the bitter taste intensity in both emp and wjb tempe. key words: tempe, taste, bacteria community analysis _____________________________________________ ________________________ *corresponding author, phone/fax: +62-251-8315107, e-mail: asuwanto@indo.net.id tempe is a traditional food in indonesia wich is made through a fermentation process, mainly using rhizopus oligosporus. there are many different kinds of tempe depending on the raw material, but the most famous one is tempe with soybean as its raw material. the kind of tempe studied in this research is the soybean tempe, which in this research is defined as tempe. other than r. oligosporus, the presence of bacteria is very important in tempe production, as some of them play a role in improving the quality of tempe. for example, citrobacter freundii and klebsiella pneumoniae can increase the content of vitamin b 12 (keuth and bisping 1994), and micrococcus or arthrobacter play a role in the formation of isoflavones (klus et al. 1993). microorganisms have been reported to play a role in flavor formation in fermented foods (hagedorn and kaphammer 1994). for example, bitter taste in cheese is caused by protease activity of lactococcus lactis subsp. lactis (broadbent et al. 2002), the diversity of lactic acid bacteria influences wine quality (rodas et al. 2003), and staphylococcus xylosus affected the aroma of sausages (stahnke1994). the flavor of tempe is variable, but it is inconsistent and sometimes unpleasant flavors wich are not favoured by consumers. the flavor in food products is a very important factor that influences consumer’s decision in their choices of food product. one of the undesirable flavors is bitterness. bitterness is a main characteristic of fresh tempe. the intensity of bitterness in tempe can differ among producers even though they use the same raw material and inoculum (hartoyo 1994). although there is relatively the same fondness of the taste of tempe, bitterness is not one that is favored by consumers. tempe is locally produced through several kinds of processing methods, generally using uncontrolled conditions. the factors causing different intensities of bitterness in tempe have not been reported so far. therefore, the aim the study is to identify whether bacteria play a role in the formation of such bitter tastes in tempe. materials and methods bitterness sensory test. the examples of tempe were obtained from five producers in the bogor area. those five producers are clr, drg, mlb, emp, and wjb. all five kinds of tempe were produced using the same type of soybean as raw material. sensory test were conducted by panelists who had been selected after they went through several tests as follows: sixty students from the department science of food technology, bogor agricultural university, were made available as panelists. sensory sensitivity of the candidates was measured in three stages. the first stage was a matching test. five basic tastes were selected: sour (0.04% citric acid), sweet (0.7% sucrose), bitter (0.04% caffeine), salty (0.2% sodium chloride), and umami (1% monosodium glutamate). the second step was a triangle test for bitterness (0.02 and 0.04% caffeine), and the third stage was a bitterness ranking test (0.025, 0.04, 0.06, and 0.08% caffeine). the criterion used to select panelists from every stage was those giving 80% correct answers. the selection process resulted in 13 panelists who were then trained for the bitterness test. caffeine concentrations of 0.025 and 0.05% were used as the standard in the bitterness sensory test on tempe samples employing the rating test (meilgaard et al. 1999). issn 1978-3477 volume 2, number 1, april 2008 p 17-21 18 barus et al. microbiol indones fresh tempe 12 cm x 2 cm x 2 cm size were steamed for 15 min and then cut into 4cm x 2cm x 2cm pieces before being served to panelists. panelists were asked to determine the bitterness of tempe by comparing it to the standards provided. bitterness intensity scores were listed between 0-5 (score 0 for the lowest bitter taste intensity and score 5 for the highest). the data were then analyzed using analysis of variance and tested further with honestly significance difference (hsd). from these bitterness sensory tests, “non bitter” tempe (with the lowest intensity) and “bitter” tempe (with the highest intensity) were selected. tempe production. tempe was made using original tempe inoculum from pt aneka fermentasi industri (afi), bandung, indonesia, which is usually used by the wjb producer and also in the mixed tempe culture used by the emp producer. tempe was processed employing the wjb method (fig 1) and the products were then subjected to the bitterness sensory test. fungal analysis. fungal analysis was conducted on “non-bitter” and “bitter” fresh tempe. the medium used was potato dextrose agar (pda) (difco) with the addition of 0.3 g.l-1 (w/v) ampicillin and 0.3 g.l-1 (w/v). a quantity of 25 g of each tempe sample was added to 225 ml 0.85% (w/v) nacl, crushed and mixed to make a suspension prior to serial dilution. a 100 μl aliquot of suspension was spread on pda medium using the pour-plate method with two replications. three-day-old fungal colonies were morphologically observed under the microscope to determine the types of fungi present. bacterial analysis. bacterial analysis was conducted on “non-bitter” and “bitter” fresh tempe as well as the soaking water taken from each tempe production process. the soaking water was sampled in three stages at 1, 7, and 14 h after the soybean soaking. a quantity of 25 g of each tempe sample was added to 225 ml 0.85% (w/v) nacl and then crushed using a stomacher for 1.5 min. a 25 ml aliquot of the soaking water was diluted with 225 ml 0.85% (w/v) nacl and then a serial dilution was made for each sample. from each dilution, a 100 μl aliquot of suspension was spread on plate count agar media /pca (oxoid) to datermine the total cell number of mesophilic aerobic bacteria and spore-forming bacteria, and on eosin methylene blue agar/emb (difco) to reveal the total cell number of enterobacteria. spore-forming bacterial number was obtained by heating the sample to 85°c for 15 min. each test was done in replicate. colonies were counted and observed for their morphological characteristics. the bacteria that grew on pca, and the spore-forming bacteria, were tested for protease production using pca-media enriched with 2% skimed milk. test bacteria were spattered on the media and then incubated overnight at room temperature. positive testing for protease-producing bacteria was carried out by directly observing the clear zones which formed around the colonies. identification based on 16s rrna gene sequence. two types of dominant bacterial colonies on pca media were identified through their 16s rrna gene sequence. a 1.5 ml aliquot of bacterial suspension wich had been grown for one night in pca liquid media was centrifuged for 3 min at 10 000 x g (sorvall® pico, usa). the result an pellet was mixed with 200 μl 0.85% (w/v) nacl and the genomic dna was extracted according to the procedure of a genomic dna purification kit (fermentas, vilnius, lithuania). the 16s rrna gene was amplified employing a pcr machine (perkin elmer, gene amplification system 2400) using 63f primer (5’caggcctaacacatgcaagtc) and 1387 r (5’gggcggwt gtacaaggc -3’) (marchesi et al. 1998). the reaction conditions (50 μl) were as follows: 36μl ddh 2 o, 5 μl 10 x polymerase buffer, 1 μl dntps, 1μl taq dna polymerase (finnzymes, espoo, findland), 1μl of 63f primer (5pmol/μl), 1μl of 1387r primer (5pmol/μl), and 3μl of sample dna. the mixture was incubated in the pcr machine (perkin elmer, gene amp system 2400). the pcr protocol was as follows: initial denaturation at 94°c for 5 min, denaturation at 92°c for 30 s, annealing at 62°c for 30 s, elongation at 72°c for 30 s, and post pcr at 72°c for 7 min. the pcr cycle was used 25 times. the pcr results were observed employing electrophoresis using 0.8% (w/v) agarose gel. a dna band of 1.3 kb was cut and then purified using the wizard® sv gel and clean-up system (promega). purified dna was eluted with 35μl nuclease-free water. dna sequencing was carried out on an automatic dna sequencer abi prism 2400 (perkin elmer, usa). resulting sequences were compared with dna sequences available in the database of the european bioinformatics institute (ebi) using blastx 2.0 set to its default parameters. results bitterness sensory test. bitterness intensity scores of emp, drg, mlb, clr, and wjb tempe were different. the test results of hsd at 5% level showed that bitterness intensity fig 1 comparison of emp, drg, mlb, clr, and wjb tempe manufacturing methods. emp, drg, mlb, clr tempe wjb tempe cooking of soybean soaking dehulling washing mixing with inoculum incubation tempe cooking of soybean dehulling washing soaking cooking mixing with inoculum incubation tempe volume 2, 2008 microbiol indones 19 scores of the samples were categorized into three groups i.e. the highest scores emp (2.3) and drg (2.2), moderate scores mlb (1.7) and clr (1.6) and the lowest score wjb (1.3). scores followed by the same letter indicated that they were insignificantly different scores. based on this bitterness sensory test, emp that has the highest bitterness intensity score (2.3) was selected as representative of “bitter” tempe and wjb (1.3) as representative of “non-bitter” tempe. types of fungi. fungi that grew on tempe emp were r. oligosporus and mucor sp., each with a fungal abundance of 3.4 x 105 cfu g-1 and 3.5 x 103 cfu g-1 respectively. fungi that grew on fresh tempe wjb were r. oligosporus, mucor sp., and geotrichum sp. with a fungal abundance of 3.8 x 105 cfu g-1, 4.8 x 103 ccfu g-1, and <3.0 x 101 cfu g-1 respectively. bacterial abundance. the number of enterobacteria, spore-forming bacteria and proteolytic bacteria was found to be higher in soaking water and fresh tempe emp compared to that of wjb (table 1). plating analysis showed that emp soaking water contained a higher number of enterobacteria, approximately 103-104 cfu ml -1, and spore-forming bacteria groups, 102 cfu ml-1, compared to wjb. similarly, the number of other bacteria groups in fresh emp tempe was 102 cfu g-1 higher than those in fresh wjb tempe. based on morphological characteristics (table 2), the bacteria that grew dominantly on pca media from tempe emp had morphological characteristics of type a, b, f, and i, while tempe wjb had type b, d, g, h, and i. type i bacteria found in both tempe types has proteolytic activity. enterobacteria found in emp were dominated by bacteria with green metallic color (fecal) and other non-fecal bacteria, that were similar to the enterobacteria found in wjb. bacteria identification. two types of dominant bacterial colonies on pca media were identified through their 16s rrna gene sequence. the strategy used to determine the bacteria was carried out based on their 16s rrna gene sequence. dominant bacteria on tempe emp (table 2) w e r e a c e t o b a c t e r i n d o n e s i e n s i s , k l e b s i e l l a pneumoniae, bacillus subtilis, and flavobacterium sp., while dominants on tempe wjb were klebsiella sp., brevundimonas sp., pseudomonas putida, bacillus sp., and acinetobacter sp. bacillus, a group of proteolytic bacteria, was found in high abundance in the soaking water of emp than wjb. discussion fermentation is one of the technologies that have been used for a long time in food processing. one of the indonesian traditional foods that is processed through fermentation is tempe. in tempe processing, soybean, microbes, and technology blend into one to produce tempe which is superior in characteristics compared to raw soybean. based on the bitterness sensory test, tempe clr, drg, mlb, emp, and wjb have different bitterness intensity scores (fig 2). bitter taste was found in every sample but with different intensity. this was not caused by a 3 2 1 0s co re o f b it te r ta st e in te n si ty e m p drg mlb clr wjb types of tempe fig 2 the scores of bitter taste intensity in a number of tempe types. table 1 bacterial abundance in soak water 1,7, and 14 hours and fresh emp and wjb tempe. kind of bacteria spore-forming bacteria are proteolitic. 3.1 x 106 7.7 x 107 3.3 x 108 1.1 x 108 3.4 x 102 3.3 x 103 5.3 x 103 3.2 x 102 8.7 x 106 4.4 x 107 3.7 x 108 9.4 x 108 7 h1 h 14 h fresh tempe 6.1 x 102 5.3 x 104 6.0 x 105 4.2 x 105 <3.0 x 101 <3.0 x 101 <3.0 x 101 <3.0 x 101 7.1 x 106 1.3 x 107 7.6 x 108 1.8 x 106 (cfu ml-1) emp tempe enterobacteria spore-forming bacteria total bacteria (others) wjb tempe enterobacteria spore-forming bacteria total bacteria (others) table 2 the type of bacteria dominant in soak water and fresh tempe emp and wjb grown on pca medium sample dominant colonies cfu ml-1 cfu g-1 identities similarity (%) emp tempeh soak water 1 soak water 2 soak water 3 fresh tempeh wjb tempeh soak water 1 soak water 2 soak water 3 fresh tempeh type a type i type b type i type b type i type f type b type b type i type b type g type b type g type d type h 5.5 x 106 3.1 x 106 3.7 x 107 6.6 x 106 3.7 x 108 4.7 x 105 5.9 x 108 3.5 x 108 7.1 x 106 3.3 x 101 1.3 x 107 3.5 x 103 7.6 x 108 3.0 x 105 1.7 x 106 5.4 x 103 acetobacter i n d o n e s i e n s i s bacillus subtilis.* klebsiella pneumoniae bacillus subtilis* klebsiella pneumoniae flavobacterium sp. klebsiella pneumoniae klebsiella sp. bacillus sp.* klebsiella sp. brevundimonas sp. pseudomonas putida acinetobacter sp. 100 99 99 99 99 99 99 99 99 99 99 99 99 type a: globular, smooth periphery, mucous, curved, yellowish white, small; b: globular, smooth periphery, mucous, curved, white, small; d: globular, smooth periphery, mucous, curved, white, big; f: globular, not smooth periphery, mucous, curved, brownish white, big; g: globular, not smooth periphery, mucous, curved, white, medium; h: globular, smooth periphery, mucous, emerge, yellowish white, small; i: not uniform and spread out, mucous, plate shaped, white, big; -: not identified; *: poteolitic bacteria. 20 barus et al. microbiol indones soybean factor, because the same type of soybean was used in producing the tempe i.e. gcu, usa soybean no. 1. sensory test results of the five kinds of tempe showed that the strongest bitterness intensity was found in emp with the weakest in wjb (fig 2). the emp and wjb tempe were made by a different processing method. however, this research indicated that the difference in processing did not cause a significant differences in tempe bitterness intensity. this is supported by the results of laboratory tests, where tempe processing was conducted by using with emp and wjb methods of processing (fig 1). in this experiment, tempe was made with the same kinds of soybean, inoculum, and sterile water, but with different processing methods. the result of the sensory test of both tempe is shows that the bitterness scores were not significantly different, being 1.1 and 1.2. tempe emp and drg were produced employing the same method as tempe mlb and clr (fig 1). however they had significantly different intensity scores (fig 2). this result confirmed the result of hartoyo (1994) which stated that the bitterness intensity of tempe could differ among producers eventhough they used the same raw material and processing method. sensory test results of the five kinds of tempe showed that the strongest bitterness intensity was found in emp and the weakest in wjb (fig 2). the inoculum used to produce both tempe types came from the same source, pt. afi, bandung, indonesia. according to their fungal characteristics, the types of fungi in emp and wjb were found to be similar both in quantity and types. these similarities can occur because both manufactures used the same source of inoculum. the difference in bitterness intensities of emp and wjb could not come from the fungi because the sensory test results for both tempe products inoculated by the fungal inoculum from emp and wjb did not show significantly different intensity scores (1.1 and 1.2 respectively). the number of bacterial cells for emp was generally higher than for wjb. dominant bacteria found in soaking water and fresh tempe of emp were acetobacter indonesiensis, klebsiella pneumoniae, bacillus subtilis and flavobacterium sp. in wjb the bacteria were klebsiella sp. and pseudomonas putida (similarity 99%). other than the number of bacteria, bacterial diversity in the food ecosystem is a very important factor to determine food quality because different microbes have different functions and these are absolutely important in establishing the conditions of the environment during the fermentation process (scahwan 1998; ampe et al. 2001; randazzo et al. 2002). in food processing which includes a fermentation process, flavor is greatly influenced by the types of microorganisms involved. the total cell population of bacillus sp. found in emp soaking water was higher than wjb (table 2). bacillus sp. has proteolytic activity. according to omafuvbe et al. (2002), protease from different bacillus sp. produce a different free amino acid content of soy-daddawa. a different free amino acid content cause the different sensory attributes soy-daddawa (nigerian-fermented soyfood). soybean, as the raw material of tempe, contains around 40% protein from its dry weight. the protein consists of 11s glycinin and α, β, γ conglycinin (liu 1997). hydrolysis of soybean 11s glycinin by trypsin results in bitter peptides that have molecular weights of 2.4 to 3.5 kda (kim et al. 2003). furthermore myong et al. (2004) reported that enzymatic hydrolysis of soybean protein resulted in a bitter taste because of the formation of peptides with molecular weights around 2 to 4 kda. bitter taste in tempe can be caused by many factors, but result of this research indicated that the presence of bacteria involved during processing might be one factor that influences flavor formation, particularly the bitterness of tempe. this presumption is strengthened because the type of fungi present, methods used, and the raw material used did not affect bitterness intensity differences. therefore, bacterial analyses which do not depend on culturable bacteria are needed to further support this observation i.e. it is the bacteria present during tempe processing which affects flavor formation. references ampe f, sirvent a, zakhia n. 2001. dynamics of the microb i a l community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rrna hybridization. int j food microbiol 65:455 4 . broadbent jr, barnes m, brennand c, strickland m, houck k, johnson me, steele jl. 2002. contribution of lactococcus lactis cell envelope proteinase specificity to peptide accumulation and bitterness in reduced-fat cheddar cheese. appl environ microbiol 6 8 : 1 7 7 8 1 7 8 5 . hagedorn s, kaphammer b. 1994. microbial biocatalysis in the generation of flavor and fragrance chemicals. ann rev microbiol 48:773-800. hartoyo lk. 1994. usaha mengurangi rasa pahit pada tepung tempe dari bahan mentah tempe kedelai produksi beberapa pengrajin tempe di bogor [thesis]. bogor: institut pertanian bogor. keuth s, bisping b. 1994. vitamin b 12 production by citrobacter freundii or klebsiella pneumoniae during tempe fermentation and proof of enterotoxin absence by pcr. j appl environ microbiol 60:1495-1499. kim mr, kawamura y, lee ch. 2003. isolation and identification of bitter peptides of tryptic hydrolysate of soybean 11s glycinin by reverse-phase high-performance liquid chromatography. j food sci 68:2416-2422. klus k, borger-papendorf g, barz w. 1993. formation of 6,7,4 tryhidroxyisoflavone (factor 2) from soybean seed isoflavone by bacteria isolated from tempe. j phytochem 34:979-981. liu k. 1997. soybeans: chemistry, technology, and utilization. ney york: international thomson publ. marchesi jr, sato t, weightman aj, martin ta, fry jc, hiom sj, wade wg. 1998. design and evaluation of useful bacteriumspecific pcr primers that amplify genes coding for bacterial 16s r r n a . a p p l e n v i r o n m i c r o b i o l 6 4 : 7 9 5 7 9 9 . meilgaard m, civille gv, carr bt. 1999. sensory evaluation techniques. 3th ed. boca raton: crc pr. myong jc, unklesbay n, hsieh fh, clarke ad. 2004. hydrophobicity of bitter peptides from soy protein hydrolysates. j agric food chem 52:5895-5901. omafuvbe bo, abiose sh, shonukan oo. 2002. fermentation of soybean (glycine max) for soy-daddawa production by starter cultures of bacillus. j food microbiol 19:561-566. r a n d a z z o c l , to r i a n i s , a k k e r m a n s d a d l , d e vo s w m , va u g h a n e e . 2 0 0 2 . d i v e r s i t y, d y n a m i c s , a n d a c t i v i t y o f b a c t e r i a l c o m m u n i t i e s d u r i n g p r o d u c t i o n o f a n a r t i s a n a l sisilian cheese as evaluated by 16s rrna analysis. appl environ microbiol 68:18821892. r o d a s a m , f e r r e r s , p a r d o i . 2 0 0 3 . 1 6 s a r d r a : a t o o l f o r identification of lactic acid bacteria isolated from grape must and wine. j sys appl microbiol 26:412-422. schawan rf. 1998. cocoa fermentation conducted with a defined microbial cocktail inoculum. appl environ microbiol 64:14771483. stahnke lh. 1994. aroma component from dried sausages fermented with stapylococcus xylosus. j meat sci 38:39-53. volume 2, 2008 microbiol indones 21 1 sri hendrastuti_327 (1-7) identity and sequence diversity of begomovirus associated with yellow leaf curl disease of tomato in indonesia tri joko santoso1, sri hendrastuti hidayat2*, ati sri duriat3, muhammad herman1, and sudarsono4 1indonesian center for agricultural biotechnology and genetic resources research and development, jalan tentara pelajar no 3a, bogor 16111, indonesia 2department of plant protection, 4 department of agronomy and horticulture, institut pertanian bogor, darmaga campus, bogor 16680, indonesia 3indonesian vegetable research institute, jalan tangkuban perahu no 517, po box 8413, lembang 40391, indonesia infection of tomato by begomovirus is known to cause serious disease and yield losses. samples of tomato plants showing typical symptoms of begomovirus infection were collected from eight locations in java and sumatra. amplification of a putative av1 gene was performed using av1 specific primers for geminivirus, total nucleic acid isolated from tomato samples exhibiting leaf curl disease as the template, and the pcr technique. direct sequencing of pcr product was carried out, followed by nucleotide and predicted amino acid sequence analysis using the blast program. positive results were obtained, the pcr amplification proved that diseased tomato samples collected from eight locations in java and sumatra were infected with begomovirus. when nucleic acid and amino acid sequences of the eight isolates were compared to other begomovirus’s sequences present in the genbank it was found that the isolates determined in this research were indonesian isolates of ayvv. further phylogenetic analysis of eight begomovirus isolates identified in this study indicated they belonged into two different clades. results of this research also suggest that the existence of begomovirus genetic diversity in various regions in indonesia needs further investigation. moreover, the prevalence of distinct begomovirus species or isolates also need investigation. keywords: begomovirus, sequence analysis, tomato leaf curl virus _____________________________________________ ________________________ *corresponding author, phone: +62-251-8629363, fax: +62-251-8629362, e-mail: rihendrastutihidayat@gmail.com the family geminiviridae is one of the largest group of plant viruses. the morphology of geminivirus particles is unique having a twin shape and a small size (h” 30 x 20 nm). they are characterized by a circular, single stranded, dna genome which replicates in the host cell nucleus and is encapsulated in twin incomplete icosahedral particles. the family geminiviridae is divided into four genera, i.e., mastrevirus, curtovirus, topocuvirus, and begomovirus, based on the viral genome structure, host range and type of insect vector (van rogenmortel et al. 2000). mastreviruses and curtoviruses have a monopartite genome and are transmitted by various leafhopper species, but infect monocotyledonous and dicotyledonous plants, respectively. the genus topocuvirus is made up of the tomato pseudocurly-top virus, which has a monopartite genome and are transmitted by treehopper species, and which infects dicotyledonous plants. members of the genus begomovirus have monopartite (one ~2.9 kb dna) or bipartite (two ~2.6 kb dnas referred to as “dna-a” and “dna-b”) genome, and are transmitted by whiteflies (e.g. bemisia tabaci gennadius) and infect dicotyledonous plants (harrison 1985). begomoviruses are considered to be emerging plant viruses, due to their increasing incidence and the severity of the diseases which they cause in a number of economically important crops, mostly in tropical and subtropical regions in the world (polston and anderson 1997). in indonesia, begomoviruses are currently a spreading threat for cultivated tomatoes in some tomato production areas and causing substantial yield losses. these viruses have also been reported to infect some other plants such as chilli pepper (capsicum annuum), ageratum (ageratum conyzoides), and tobacco (nicotiana benthamiana) (sudiono et al. 2001). partial characterization of the genomic sequence of the indonesian tomato-leaf-curl virus (tolcidv) was first reported in 1999 (ddbj, accession number af189018). similar characterization was performed for six begomoviruses infecting tomato plants from bandung, west java (tobadi5, tobadii-20, tobadii-23, tobadiii-1); purwokerto, central java (topur-6); and magelang, central java (tomag-2) (sukamto et al. 2005). meanwhile, the complete nucleotide sequence identification has been reported for the tolcidv from java (kon et al. 2006). in this paper, we report sequence analysis of the coat protein gene isolated from eight begomovirus isolates infecting tomato plants collected from different locations in java and sumatra. it is important to understand that the genetic diversity of begomoviruses infecting tomato plants provides basic information for developing disease control strategies. materials and methods sample collection. tomato plants showing typical symptoms of begomovirus infection (yellow mosaic, leaf curling, and stunting) were collected from several tomato producing areas (8 districts, 5 provinces) in indonesia (table 1). samples were placed in plastic bags or bottles and carried to the laboratory for dna extraction and to a screenhouse for virus isolation and propagation in host plant. dna extraction and polymerase chain reaction (pcr) analysis. total dna was extracted from tomato leaves leaf according to doyle and doyle (1999) with slight issn 1978-3477 volume 2, number 1, april 2008 p 1-7 modification. leaf tissue was ground in a sterile mortar in 1.0 ml of extraction buffer. the extraction buffer used for the initial homogenization contained 100 mm tris ph 8.0, 1.4 m nacl, 20 mm edta ph 8.0, and 0.2% (v/v) βmercaptoethanol. the extraction buffer was autoclaved and 2% polyvinylpyrolidone (pvp) and 2% ctab were added immediately before use. immediately after grinding, 500 μl aliquotes were transferred to a 1.5 ml microfuge tube and incubated for 15 min at 65°c with occasional mixing to avoid aggregation of the homogenate. to the extract was added 500 μl of chloroform: isoamylalcohol (24:1.0) and the mixture was vortexed thoroughly. each tube was then centrifuged for 15 min at 10 000 x g. the debris-free supernatant was then transferred to a new tube and proteins precipitated by adding 2.5 x volume of absolute ethanol and washed twice with 70% ethanol (v/v). the pellet was dried and resuspended in 100 μl of sterile distilled water. this dna extract was stored at -20°c for further use. the coat protein gene was amplified by the pcr technique using two oligonucleotide specific primers for the geminivirus coat protein gene that were provided by dr. sylvia green from the asian vegetable research development centre (avrdc-taiwan), i.e the cpprotein-v1(5’-taattctagatgtcgagcga cccgccga-3’) and the cpprotein-c1(5’-ggccga attcttaattttgaacagaatca-3’). pcr reactions were prepared in 25 μl total volume, containing 10 x buffer (100 mm tris-hcl, 500 mm kcl, ph 8.3), mgcl 2 (75 mm), dntp mix (4 mm), 10 μm of each primer, 1 unit of taq dna polymerase, and 2 μl of the dna template. the amplication profile consisted of 30 cycles of denaturation at 94°c for 1 min, primer annealing at 55°c for 1 min and primer extension at 72°c for 2 min, and followed by postextension at 72°c for 5 min. pcr products were analysed on agarose gels (1%) and stained with ethidium bromide and visualized under uv light using the chemidoc gel system (biorad). direct sequencing of pcr products. products of the pcr were first visualized in agarose gels (1%) to estimate their concentration and to confirm their purity. further purification of pcr products used exoisap digestion [exonuclease i enzyme and shrimp alkaline phosphatase/ sap (biorad)] to remove the excess primers and dntps. the purified pcr products were then diluted, and mixed with a single primer (either forward or reverse primer). each sequencing reaction was prepared using a dtcs kit (beckman coulter) in a 20 μl volume containing 1.5 μm of either forward or reverse primer and 50 ng of template dna. the reaction profile consisted of 45 cycles of denaturation at 96°c for 20 sec, primer annealing at 50°c for 20 sec, and primer extension at 60°c for 4 min. reactions were analyzed in an ceq 800 analyzer (beckman coulter). determination of virus identity. database searches for the selected begomoviruses species were carried out using the national center of biotechnology information basic local alignment search tool or ncbi blast(http:// www.ncbi.nlm.nih.gov/blast) (altschul et al. 1990). the identity of the virus was determined based on the highest percentage value of the av1 gene nucleic acid and amino acid sequence among the evaluated isolates and available sequences in the genbank dna database. the sequences were aligned using clustalw (thompson et al. 1994) while phylogenetic analysis was conducted using online tool facilities available at http://www.genebee.msu.su/clustal/ advanced.html. the distance matrices were calculated using the kimura two-parameters model (kimura 1980). results of the analysis were used to construct phylogenetic tree and the robustness of the internal branches of the tree was tested by bootstrap analysis using 1 000 replicates. results the detection of begomovirus infection using specific primers for av1 gene specific primers resulted in a single dna fragment of approximately 780 bp (fig 1) for most of the tomato plants collected from the eight locations which showed typical begomovirus symptoms (fig 2). since the oligonucleotide primers used for pcr were specific for amplifying coat protein (av1) gene of begomovirus, results of this research suggested the presence of begomovirus in all of the tomato plants investigated. direct sequencing of pcr products generated sequences of the putative av1 gene ranging from 529-707 bp (table 1). the determined nucleotide sequences was submitted to the genbank database. homology among begomovirus isolates was shown when alignment was obtained for predicted amino acid sequence of partial av1 gene of eight begomovirus isolates identified in this research and other isolates available in the genbank dna database (fig 3). comparison of nucleotide and predicted amino sequences of putative av1 gene of the eight isolates with available av1 gene sequences in the genbank revealed that the eight isolates had homologies above 90% with ageratum yellow vein virus table 1 isolate identity, observed symptoms on collected tomato samples, location of collected samples, and number of determined nucleic acid and predicted amino acid sequences based on the polymerase chain reaction amplified putative av1 gene isolate identity observed symptoms on collected tomato sample location of collected sample size of sequences nucleotide (bp) amino acid (residues)* tolc-blt tolc-mlg tolc-srg tolc-mgl tolc-byl tolc-klu tolc-bgr tolc-btg leaf curling and stunting yellowing, severe upward leaf curling, and stunting leaf curling, stunting, and mosaic leaf curling, stunting, and smaller leaflet leaf curling and stunting severe upward leaf curling, yellowing, and stunting severe leaf curling, cupping, smaller leaf, and stunting leaf curling and stunting blitar, east java malang, east java sragen, central java magelang, central java boyolali, central java kaliurang, d.i. yogyakarta bogor, west java brastagi, north sumatra 580 529 685 707 702 605 666 706 193 176 227 235 233 201 221 234 *predicted based on the determined nucleotide sequences. 2 santoso et al. microbiol indones isolate from singapore (ayvv-genbank acc. no. x74516) (tan et al. 1995). the homology was less than 90% with a pepper leaf curl virus isolate from malaysia (peplcv-malaf414287) (shih et al. 1998) or cassava mosaic virus isolate from south africa (casmv-sa-aj575560) (table 2). distance matrices based on the av1 gene amino acid sequences of suspected begomovirus isolates examined in this research, ayvv, sclv-jpn, peplcv-mal, tolcv-jv, tolcv-jva, tolcv-mal, and sa-casmv, supported previous findings that the suspected begomoviruses were indeed isolates of ayvv since their distances (table 3) were generally less than 10%. on the other hand, the distances were generally more than 10% if av1 gene of the suspected begomovirus isolates from indonesia were compared with that of either peplcv, tolcv-jv or tolcv-jva, and more than 20% when compared with that of casmv-sa. these results indicated that the suspected begomovirus isolates from indonesia were not isolates of peplcv, tolcv-jv, tolcv-jva or casmv. the tolcv-jv and tolcv-jva were two other begomovirus isolates from indonesia that had previously been reported (kon et al. 2006). phylogenetic analysis was carried out based on the predicted amino acid sequences of the putative av1 gene determined in this research and those of other begomoviruses available in the genbank (fig 4). the eight begomovirus isolates determined in this research were all clustered in a similar clade with ayvv from singapore (ayvv) and taiwan (ayvv-tw). however, their sequences were quite diverse based on the arm-length of the phyllogenetic tree. results of this analysis also indicated that three begomovirus isolates from indonesia identified in previous study (tolcv-jv, tolcv-jva, and tylcv-lbg) did not belong to the same clade as the isolates identified in the present research. these three isolates were more closely related to peplcv-mal than to the eight isolates identified in the more recent research. fig 2 tomato plants exhibited various leaf-curl symptoms. subsequent experiment indicated they were infected by begomoviruses. a, leaf curling, smaller leaflet, and stunting symptoms on tomato plant from bogor, west java; b, leaf curling and mosaic symptoms on tomato plant from sragen, central java; c, severe upward leaf curling and yellowing symptoms on tomato plant from kaliurang, di yogyakarta; and d, leaf curling symptom on tomato plant from blitar, east java. 1 2 3 4 fig 1 agarose gel electropherogram of pcr amplified dna fragments of putative av1. the dna fragments were amplified by pcr using av1 specific primers and total nucleic acid of diseased tomato sample from (1) malang, east java; (2) blitar, east java; (3) sragen, central java; (4) magelang, central java; (5) boyolali, central java; (6) kaliurang, d.i. yogyakarta; (7) bogor, west java; and (8) brastagi, north sumatera; m, 1 kb plus (invitrogen) dna marker. av1 gene 1 000-bp 850 650 432 61 5 87 m volume 2, 2008 microbiol indones 3 tolc-btg vlvtnkrrtwtnrpmyrkprlyrmyrtpdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolc-srg vlvtnkrrtwtnrpmyrkprlyrmyrtpdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolc-bgr vlvtnkrrtwtnrpmyrkprlyrmyrtpdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolc-mlg vlvtnkrrtwtnrpmyrkprlyrmyrspdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolc-blt vlvtnkrrtwtnrpmyrkprlyrmyrspdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolc-klu vlvtnkrrtwtnrpmyrkprlyrmyrspdvpkgcegpckvqsyeqrhdishvgkvccvtd tolc-byl vfvtnkrrtwtnrpmyrkpsmysmyrspdvpkgcegpcnvqsyeqrhdishvgkvlcvsd tolc-mgl vlvtnkrrtwtnrpmyrkprlyrmyrspdvpkgcegpckvqsyeqrhdishvgkvlcvsd ayvv sclv-jpn vlvtnkrrtwtnrpmyrkprmyrmyrspdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolcv-mal vlvtnkrrawtqrpmyrkprmyrmyrspdvpkgcegpckvqsyeqrhdishvgkvlcvsd tolcv-jva vlvtnkrrtwtnrpmyrkprlyrmyrspdvpkgcegpckvqsfesrhdvshvgkvccitd tolcv-jv vlvtnkrrtwtnrpmyrkprmyrmyrspdvpkgcegpckvqsfesrhdvshvgkvccitd peplcv-mal vlvtnkrrswanrpmnrkpriyrmykspdvprgcegpckvqsyeqrhdvahvgkvicvsd casmv-sa vqgtnkrrswtlrpmyrkprmyrmyrspdvprgcegpckvqsyeqrddvkhtgavrcvsd * **:**:*: *** *** :* **::****:******:***:*.*.*: *.* * *::* tolc-btg vtrgnglthrvgkrfcvksvyvlgkvwmdeniktknhtntvmfylvrdrrpygt-amdfg tolc-srg vtrgnglthrvgkrfcvksvyvlgkvwmdgdiktknhtntvmfylvrdrrpygt-aldfg tolc-bgr vtrgnglthrvgkrfcvksvyvlgkiwmdeniktknhtntvmfylvrdrrpygt-amdfg tolc-mlg vtrgnglthrvgkrfcvksvyvlgkiwmdeniktknhtntvmfylvrdrrpygt-amdfr tolc-blt vtrgnglthrvgkrfcvksvymlgkiwmdeniktknhtntvmfhlvrdrrpygs-amdfg tolc-klu vtrgnglthrmgkrfcvksvyvlgkiwmdeniktknhtntvmfylvrdrrpygs-amdfg tolc-byl vtrgnglthrvgkrfcvrsvyvlgkiwmdeniktknhtntvmfylvrdrrpygs-amdfg tolc-mgl vtrgnglthrvgkrfcvksvyvlgkiwmdeniktknhtntvmfylvrdrrpygs-amdfg ayvv sclv-jpn vtrgnglthrvgkrfcvksvyvlgkiwmdeniktknhtntvmfylvrdrrpfgt-amdfg tolcv-mal vtrgngfthrvgkrfcvksiyvlgkiwmdeniktknhtntvmfflvrdrrpfgt-amdfg tolcv-jva vtrglglthrtgkrfcvksvyimgkvwmdeniktknhtntvmfflvrdrrpyss-pqdfg tolcv-jv vtrglglthrtgkrfcvksvyimgkvwmdeniktknhtntvmfflvrdrrpyss-pqdfg peplcv-mal vtrgnglthrvgkrfciksvyvlgkiwmdeniktknhtntvmfflvrdrrpfgt-pqdfg casmv-sa vtrgsgithrvgkrfcvksiyvlgkiwmdenikkqnhtnqvmfflvrdrrpygtspmdfg **** *:*** *****::*:*::**:*** :**.:**** ***.*******:.: . ** tolc-btg qvfnmydnepstatikndlrdryqvlrkftstvtggqyackeqamv tolc-srg qvfnmydnepstatikndlrdryqvlrkfsstvtggqyackrqawv tolc-bgr qvfnmydnepstatikndlrdryqvlrkytstvtggqyackeqalv tolc-mlg qvfnmydnepstatikndlrdryqvlrkftstvtggqyackeqawv tolc-blt qvfnmydnepstatikndlrdryqvlrkftstvtggqyaakeqasv tolc-klu qvftmydnepstatikndlrdryqgvrklsstvtggqyagkgqasv tolc-byl qvfnmydnepstatikndlrdryqvlrkftstvtggqyaskeqalv tolc-mgl qvynmydnepstatikndlrdryqvlrkftstvtggqyaskeqalv ayvv sclv-jpn qvfnmydnepstatikndlrdryqvlrkftstvtggqyackeqalv tolcv-mal qvfnmydnepstatvkndmrdryqvlrkftatvtggqyaskeqalv tolcv-jva qvfnmydnepstatvkndmrdrfqvlrkfsstvtggqyackeqalv tolcv-jv qvfnmydnepstatvkndmrdrfqvlrkftstvtggqyackeqslv peplcv-mal qvfnmydnepstatvkndnrdrfqvlrrfqatvtggqyaskeqaiv casmv-sa qvfnmfdnepstatikndlrdrfqvlrkfhatvvggpsgmkeqali **:.*:********:*** ***:* :*: :**.** . * *: : fig 3 alignment of partial amino acid sequences predicted from determine nucleotide sequences of av1 gene of eight begomovirus isolates determined in this research and seven begomovirus isolates available from genebank dna database. ayvv-ageratum yellow vein virus (x74516), sclv-jpn-soybean crinkle leaf virus-japan (ab050781), peplcv-mal-pepper leaf curl virus-malaysia (af414287), tolcv-jv-tomato leaf curl virus-java (nc-005031), tolcv-jva-tomato leaf curl virus-java [ageratum] (ab162141), tolcv-mal-tomato leaf curl virus-malaysia (nc-004648), and casmv-sa-south african cassava mosaic virus (aj575560) were obtained from genebank database at http://www.ncbi.nlm.nih.gov/blast/blast.cgi. table 2 percentages of nucleotide (nt) and amino acid (aa) sequence identities of av1 gene among suspected begomovirus isolates determined in this research and three begomoviruses available in the genebank database isolate identity nt (%) aa (%) nt (%) aa (%) nt (%) aa (%) tolc-bgr tolc-blt tolc-btg tolc-byl tolc-klu tolc-mgl tolc-mlg tolc-srg 95 93 95 92 93 95 94 94 97 95 96 93 90 96 96 93 81 81 81 80 82 81 81 82 85 85 85 83 81 86 86 81 78 89 87 86 84 86 79 82 77 77 79 78 75 80 75 76 ayvv-ageratum yellow vein virus ( x74516), peplcv-mal-pepper leaf curl virus-malaysia (af414287), and casmv-sa-south african cassava mosaic virus (aj575560) were obtained from genebank database at http://www.ncbi.nlm.nih.gov/blast/blast.cgi. ayvv peplcv-mal casmv-sa 4 santoso et al. microbiol indones table 3 distance matrices (%) based on predicted av1 gene amino acid sequences of suspected begomovirus isolates determined in this research, ageratum yellow vein virus (ayvv), soybean crinkle leaf virus (sclv), pepper leaf curl virus (peplcv), tomato leaf curl virus (tolcv), and cassava mosaic virus (casmv) ayvv-ageratum yellow vein virus (x74516), sclv-jpn-soybean crinkle leaf virus-japan (ab050781), peplcv-mal-pepper leaf curl virus-malaysia (af414287), tolcv-jv-tomato leaf curl virus-java (nc-005031), tolcv-jva-tomato leaf curl virus-java [ageratum] (ab162141), tolcv-maltomato leaf curl virus-malaysia (nc-004648), and casmv-sa-south african cassava mosaic virus (aj575560) were obtained from genbank database at http://www.ncbi.nlm.nih.gov/blast/blast.cgi. isolate tolc-blt tolc-mlg tolc-srg tolc-mgl tolc-byl tolc-klu tolc-bgr tolc-btg 3.7 7.7 3.1 6.3 7.7 4.4 4.4 5.0 4.4 16.2 14.0 14.7 9.0 22.5 5.0 3.1 6.3 8.3 2.5 2.5 3.7 2.5 15.5 14.7 15.5 8.3 23.3 7.0 10.4 10.4 5.0 3.7 6.3 6.3 18.5 15.5 17.7 12.5 27.6 4.4 7.7 3.1 3.7 3.1 3.1 15.5 14.0 14.7 7.7 21.7 11.1 6.3 7.0 6.3 5.0 18.5 17.7 17.0 9.7 23.3 8.3 9.0 9.7 9.0 20.9 15.5 17.7 14.7 27.6 1.8 2.5 2.5 16.2 14.7 15.5 8.3 23.3 3.1 3.1 16.2 14.0 14.7 9.0 24.2 tolc-blt tolc-mlg tolc-srg tolc-mgl tolc-byl tolc-klu tolc-bgr tolc-btg ayvv sclv-jpn peplcv-mal tolcv-jv tolcv-jva tolcv-mal casmv-sa fig 4 phylogenetic relationship based on predicted av1 gene amino acid sequences of suspected begomovirus isolates determined in this research, and other begomoviruses available in the genebank dna database. the av1 gene for ayvv-ageratum yellow vein virus (x74516), ayvv-tw-ageratum yellow vein taiwan virus (nc-004627), ayvv-ch-ageratum yellow vein china virus-[g68] (aj849916), sclv-soybean crinkle leaf virus (ab050781), slcv-jpn-soybean crinkle leaf virus-[japan] (ab050781), slcv-thai-soybean crinkle leaf virus-[thailand] (ef064788), tolcv-jva-tomato leaf curl java virus-[ageratum] (ab162141), tylcv-lbg-tomato yellow leaf curl indonesia virus-[lembang] (af189018), tolcvjv-tomato leaf curl java virus (nc-005031), tolcv-bang-tomato leaf curl bangladesh virus (af188481), tolcv-lao-tomato leaf curl laos virus (af195782), tolcv-mal-tomato leaf curl malaysia virus (nc-004648), tolcv-vt-tomato leaf curl vietnam virus (nc-004153), tolcv-ch-tomato leaf curl china virus (tolcv-ch), peplcv-mal-pepper leaf curl virus-[malaysia] (af414287), stlcv-stachytarpheta leaf curl virus (aj810157), casmv-sa-south african cassava mosaic virus (aj575560), blcv-mdgr-bean leaf curl madagascar virus (am701757), and wmchstv-watermelon chlorotic stunt virus (nc-003708) isolates, respectively, were obtained from genebank dna database, available at http://www.ncbi.nlm.nih.gov/blast/ blast.cgi. tolcv-ch peplcv-mal tolcv-vt tolcv-tw casmv-sa wmchstv blcv-mdgr tolcv-bang tylcv-lbg alcv tolcv-jva tolcv-jv tolcv-lao tolcv-mal syvv-jpn sclv tolc-btg tolc-srg tolc-bgr tolc-mlg tolc-blt tolc-klu tolc-byl tolc-mgl ayvv ayvv-tw ayvv-ch sclv-thai stlcv 0 0.06 0.18 0.200.02 0.04 0.08 0.10 0.12 0.14 0.16 volume 2, 2008 microbiol indones 5 discussion a high incidence of leaf curl disease in tomato plants in indonesia has been observed in the last 5 years and it has become a major problem in tomatoes growing in areas across the country (hidayat et al. 2006). association of begomiviruses with tomato leaf curl disease has been reported mainly from west java and central java (sudiono et al. 2001; aidawati et al. 2005). detailed analyses of the molecular properties and biological activities of begomoviruses from tomato plants with leaf curl in java has been described recently (sukamto et al. 2005; kon et al. 2006). in this paper, we reported the detection, sequencing, and phylogenetic analysis of several isolates of tomato begomoviruses collected from different locations in java and sumatra, indonesia. we conducted analysis of the genetic diversity based on coat-protein gene sequence after direct sequencing of pcr products. direct sequencing of pcr products, after the pcr parameters were optimimized, has an advantage compared with other strategies, i.e. it is extremely efficient for the analysis of a large number of sequences in a short period of time. previously it has been known that several begomoviruses are associated with tomato leaf curl disease in java, indonesia. based on sequence comparisons and phylogenetic analysis, the viruses were divided into several groups. it is an interesting fact that all begomoviruses asociated with tomato leaf curl disease in java formed separate groups from those of other tomato infecting begomoviruses. according to sukamto et al. (2005) and kon et al. (2006), tomato begomoviruses from java had a closest relationship with ayvv. similarly, begomovirus isolates identified in this research showed high sequence identities with that of ayvv, sclv, and also tolcv-mal. the av1 gene predicted amino acid sequences of the identified isolates exhibited distances of less than 10% against that of the three begomoviruses, indicating they were isolates of the same virus species. therefore, it was suggested that the identified begomovirus isolates in this study might be indonesian isolates of ayvv or sclv. based on av1 gene sequences analysis in this study, previously identified tomato begomoviruses from indonesia, tolcv-jv, tolcv-jva, and tylcv-lbg, had close relationships with peplcv and casmv. it was not the case for the eight identified begomovirus isolates in this study since their predicted amino acid sequence identities and their distances were either more than 90% and less than 10% (against peplcv) or more than 80% and less than 20% (against casmv), respectively. although the eight begomovirus isolates identified in this study exhibited more than 90% of the av1 gene amino acid sequence identities and less than 10% of the distances, results of phylogenetic analysis indicated they belonged into two different clades. such results indicated the their av1 gene might have originated from the same progenitor sequences but separated a different way because of accumulated mutations. another possible explanation might be through recombination. differences in accumulated mutations might not be the answer since the occurence of begomovirus-associated-tomato-diseases in indonesia is very recent. therefore, recombination might be the possible cause of such differentiation. more studies would be required before such a possibility could be decided. kitamura et al. (2004) has proposed that recombination is a very frequent event and widespread phenomenon among geminiviruses. such recombination might occur at both species and genera levels. it was also suggested that the process of genome recombination within geminiviruses contributed significantly to the evolution of geminiviruses. based on the analysis above, it is suggested that the existence of begomovirus genetic diversity in various regions in indonesia needs further investigation. moreover, the prevalence of distinct begomovirus species or isolates should also be investigated. such knowledge will aid the development of control strategies for viruses and support the development of begomovirus resistant tomato cultivars through plant breeding. acknowledgement part of this research was funded by usaid-agricultural biotechnology support project ii (usaid-absp ii), entitled: “development of multiple virus resistance of tomato”, task order no. 18. tjs was supported by a scholarship from department of agriculture and usaidabsp ii to pursue a phd (s3) at the agronomy study program, graduate school of bogor agricultural university, bogor, indonesia. the authors acknowledge hajrial aswidinnoor as member of phd advisory committee for tjs. references aidawati n, hidayat sh, suseno r, hidayat p, sujiprihati s. 2005. identifikasi geminivirus yang menginfeksi tomat berdasarkan pada teknik polymerase chain reaction-restriction fragment length polymorphism. j mikrobiol indones 10:29-32. altschul sf, gish w, miller w, myers ew, lipinan dj. 1990. basic local alignment search tool. j mol biol 215:403-410. briddon rw, robertson i, markham pg, stanley j. 2004. occurrence of south african cassava mosaic virus (sacmv) in zimbabwe. plant pathol 53(2):233-233. doyle jj, doyle jl. 1999. isolation of plant dna from fresh tissue. focus 12:13-15. harrison bd. 1985. advances in geminivirus research. ann rev phytopathol 23:55-82. hidayat sh, chatchawankanpanich o, rusli e, aidawati n. 2006. begomovirus associated with pepper yellow leaf curl disease in west java, indonesia. j microbiol indones 11:87-90. kimura m. 1980. a simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. j mol evol 16:111-120. kitamura k, murayama a, ikegami m. 2004. evidence for recombination among isolates of tobacco leaf curl japan virus and honeysuckle yellow vein mosaic virus. arch virol 149:1221-1229. kon t, hidayat sh, hase s, takahashi h, ikegami m. 2006. the natural occurrence of two distinct begomovirus associated with dna and a recombinant dna in a tomato plant. phytopathology 96:517-525. polston je, anderson pk. 1997. the emergence of whitefly-transmitted geminiviruses in tomato in the western hemisphere. plant dis 81:1358-1369. shih sl, roff mmn, nakhla mk, maxwell dp, green sk. 1998. a new geminivirus associated with a leaf curl disease of tomato in malaysia. j zhiwu baohuxue hui huikan 40:435-435. sudiono, hidayat sh, suseno r, sosromarsono s. 2001. molecular detection and host range study of tomato-infecting begomovirus. in: proceeding of indonesian phytopathology soc seminar. bogor, aug 22-24, 2001. p 208-217. 6 santoso et al. microbiol indones sukamto, kon t, hidayat sh, ito k, hase s, takahashi h, ikegami m. 2005. begomovirus associated with leaf curl disease of tomato in java, indonesia. j phytopathol 153:562-566. tan ph, wong sm, wu m, bedford id, saunders k, stanley j. 1995. genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed. j gen virol 76:2915-2922. thompson jd, higgins dg, gibson tj. 1994. clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. nuc ac res 22:4673-4680. van rogenmortel mhv, fauquet cm, bishop dhl, carstens e, estes mk, lemon sm, maniloff j, mayo ma, mcgeoch dj, pringle cr, wickner rb. 2000. virus taxonomy. 7th report of the international committee on taxonomy of viruses. san diego: academic pr. volume 2, 2008 microbiol indones 7 mi-ahmad wibisana available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.9.3.4issn 1978-3477, eissn 2087-8575 vol 9, no 3, september 2015, p 120-128 *corresponding author; phone: +62-21, fax: 21-75603120, email: awibisana2000@gmail.com 7560208 +62biosurfactants are microbial metabolites that work as surface active agents produced by some microorganisms, including bacteria, yeast and fungi. interests in biosurfactants are growing due to their broad possible applications such as in the pharmaceutical, cosmetic, agricultural and food industries, environmental protection and crude oil drilling (banat et al. 2010; meena et al. 2015). their important characteristics such as biological activities, emulsifying, foaming, soaping and dispersing acquire them for multiple applications. based on their chemical structures, biosurfactants are divided into five major classes, i.e. lipopeptides, glycolipids, phospholipids, neutral lipids, and polymeric compounds. one of the most important families of lipopeptide class is surfactin. surfactin is a cyclic molecules consisting of a fatty acid of variable length (hydrophobic moiety) linked to a short surfactin is a lipopeptide biosurfactant that show potential biomedical application due to its activities such as antiviral, antibacterial, antifungi, anticancer, and antimycoplasma. bacillus amyloliquefaciens md4-12, isolated from oil-contaminated soil, produced promising yield of surfactin in mckeen medium. the production of surfactin was influenced by many fermentation process parameters such as carbon, nitrogen, minerals and also environmental conditions such as ph and agitation. therefore, to obtain high yield of surfactin by b. amyloliquefaciens md4-12, optimization of process production was conducted in shake flask fermentation using response surface methodology. mckeen medium composition was used as basal medium. screening of the best carbon and nitrogen source were selected in preliminary experiments followed by selection of the influencing significant parameters on surfactin production using plackett-burman design. selected parameters were optimized by central composite design and for the data analysis was used response surface methodology. the -1 result showed that the optimum medium composition contained (g l ) 45.0 glucose, 6.33 urea, 1.0 monosodium glutamate, 1.85 mgso .7h o, 0.4 kcl, 0.5 k hpo , and 0.5 ml trace elements. the surfactin yield at optimal 4 2 2 4 -1 condition was 1.25 g l , increased 2.4 times compared to condition prior to optimization. key words: lipopeptide, optimization, response surface methodology, surfactin surfaktin adalah senyawa biosurfaktan lipopeptida yang sangat potensial untuk aplikasi di bidang biomedis karena mempunyai berbagai aktifitas biologi seperti antivirus, antibakteri, antijamur, antikanker, dan antimikoplasma. bacillus amyloliquefaciens md4-12, yang diisolasi dari tanah yang tercemar minyak menunjukkan kemampuan yang tinggi dalam menghasilkan surfaktin menggunakan medium dasar mckeen. produksi surfaktin secara fermentasi dipengaruhi oleh berbagai faktor seperti sumber karbon, sumber nitrogen, mineral serta kondisi lingkungan seperti ph dan agitasi. dengan menggunakan medium mckeen sebagai medium dasar, maka pada penelitian ini dilakukan optimasi produksi surfaktin menggunakan metode permukaan respon. pada percobaan awal dilakukan pemilihan sumber karbon dan nitrogen terbaik, dan selanjutnya diikuti dengan pemilihan faktor-faktor yang berpengaruh secara signifikan untuk produksi surfaktin menggunakan rancangan plackett-burman. faktor-faktor yang terpilih selanjutnya dioptimasi dengan menggunakan central composite design dan analisis data dilakukan dengan menggunakan metode permukaan respon. hasil penelitian -1 menunjukkan bahwa komposisi medium yang optimal terdiriatas (g l ) glukosa 45,0; urea 6,33; monosodium glutamat1,0; mgso .7h o 1,85; kcl 0,4,; k hpo 0,5 dan mikroelemen 0.5 ml. pada kondisi optimum 4 2 2 4 -1 perolehan surfaktin adalah 1,25 g l , meningkat 2,4 kali dibandingkan dengan kondisi sebelum optimasi. kata kunci: lipopeptida, metode permukaan respon, optimasi, surfaktin optimization of surfactin production by bacillus amyloliquefaciens md4-12 using response surface methodology 1 2,3 4 ahmad wibisana *, wahono sumaryono , tjahjani mirawati sudiro , and 4 pratiwi pudjilestari sudarmono 1 biotech center-badan pengkajian dan penerapan teknologi, gedung 630, kawasan puspiptek serpong, tangerang selatan, indonesia; 2 center of pharmaceutical and medical technology, badan pengkajian dan penerapan teknologi, gedung laptiab kawasan puspiptek serpong tangerang selatan, indonesia; 3 faculty of pharmacy universitas pancasila, jalan srengseng sawah jagakarsa jakarta selatan, 12640, indonesia; 4 department of microbiology, faculty of medicine, universitas indonesia/cipto mangunkusumo general hospital, jakarta, jalan pegangsaan timur 16 jakarta, 10320, indonesia peptide chain (hydrophilic moiety) of seven amino acids. it has ability to reduce surface tension of water -1 -1 from 72 mn m to 27 mn m (cooper et al. 1981). several biological activities have been reported for biomedical application, such as antiviral, antibacterial, antifungi, antitumor, antiinflammatory, antiadhesive, antimycoplasma and thrombolytic activity (huang et al. 2006; sabate et al.2013; liu et al. 2012; kim et al. 2007; zhang et al. 2015; zeraik et al. 2010, lim et al. 2005; meena et al. 2015). a large variety of bacillus has been reported to produce surfactin, nevertheless microbial exploration is still needed to obtain high productivity strain. bacillus amyloliquefaciens md4-12 isolated from oilcontaminated soil showed promising ability to produce surfactin. according to previous studies, the surfactin production was influenced by several nutritional factors including the nature and concentration of carbon and nitrogen substrate, the concentration of metal ion such as mg, fe, zn, and mn in the medium and environmental conditions such as temperature, ph, agitation and aeration (sen 1997; yi et al. 2013; sen et al. 1997; al-ajlani et al. 2007; abdel-mawgoud et al. 2008; amani et al. 2013; singh et al. 2014a). to achieve high yield of surfactin production by b. amyloliquefaciens md4-12, optimization of nutrient supply and environmental condition is very important. statistical technique, including combination of plackett-burman design (pbd), central composite design (ccd) and response surface methodology (rsm) are the most widely method used for optimization of biological processes. the plackettburman experimental design is a two-level factorial design, used to determine the critical physicochemical parameters. interaction effects among the variables are not considered (mnif et al. 2012). to screen n variables using pbd, n+1 experiments are needed. the significant variables obtained by pbd can be further optimized using ccd and (rsm). rsm has been used extensively in biological process optimization due to its efficiency and accuracy for building of a model, evaluating the effects of factors and predicting optimum conditions. the objective of the present study was to optimize of surfactin production by b. amyloliquefaciens md4-12 using response surface methodology. materials and methods microorganisms. a surfactin-producing microorganism has been isolated from oil-contaminated soil taken from palembang, indonesia. furthermore, it was identified as b. amyloliquefaciens md4-12 by morphological, biochemical, and 16s rrna sequence analysis. inoculum and culture conditions. the strain was stored lyophilized and working stock cultures were maintained on nutrient agar slant and stored at 4 °c. for surfactin production, two-stage inoculum were prepared as following: one isolated colony from fresh culture grown onto nutrient agar medium and incubated for 24 h, was dispensed in 5 ml of nutrient broth medium and incubated overnight at 37 °c. the culture (4 ml) was used to inoculate 100 ml nutrient broth medium in 500 ml erlenmeyer flasks and incubated in a rotatory shaker at 200 rpm and 37 °c (±0.5) for 16 h. at this condition, absorbance around 3 measured spectrophotometrically at 600 nm was reached. for fermentation, 2 ml of culture was inoculated to 250 ml erlenmeyer flask containing 50 ml of production medium. modified mckeen medium composition was -1 used as the production medium consist of (g l ) 20.0 glucose, 2.0 monosodium glutamate, 3.0 yeast extract, 1.0 mgso .7h o, 1.0 kcl with addition of 1 ml trace 4 2 element solution (in 100 ml deionized water : 0.64 g mnso .4h o, 0.16 g cuso .5h o, and 0.015 g 4 2 4 2 feso .7h o). ph was adjusted at 7.0. fermentation 4 2 was conducted for 28 h. experimental design and data analysis. preliminary experiments were conducted to screen carbon and nitrogen source. the effect of different carbon sources was evaluated by substitution of glucose in mckeen medium composition with sucrose, fructose, maltose, dextrose, mannitol, sorbitol, galactose, xylose, and starch. the best carbon source generated the highest surfactin production was used for further study. furthermore, the effect of the different nitrogen source addition was studied similarly by substitution of yeast extract with ammonium nitrate, ammonium sulphate, sodium nitrate, soy flour, peptone, casein hydrolysate, and urea. the next step was to screen the significant factors affecting surfactin production by b. amyloliquefaciens md4-12 using the pbd, as noted in table 1. all components in the production medium (seven factors) and some environmental conditions (two-factor) were used to determine the factors that influence significantly. this experiment required 12 runs as presented in table 2. the factors with confidence levels above 95% were considered as significant effect on surfactin production. the selected significant influence factor obtained volume 9, 2015 microbiol indones 121 from pbd experiments was optimized with ccd and rsm as presented in table 4. thirty experiments consisted of sixteen factorial points, eight star points and six center points were conducted to optimize four significant factors. for each factor was studied at three level concentration (low, middle and high levels) coded with (-1, 0, +1). all experiments were conducted triplicate and the final result was expressed as the average value. statistical analysis. design expert (version 7.0, stat-ease inc., usa) software was used to generate the experimental designs and data analysis. the quality of polynomial model equation was evaluated statistically using analysis of variance (anova) to determine the 2 2 coefficient of determination, r and adjusted r . the statistical significance of the model and the regression coefficient were determined using the f-test. surfactin analysis. the gravimetric method as described by liu et al. (2012) was used for determination of surfactin concentration with minor modification. the broth culture was centrifuged at 10 000 rpm for 10 min to obtain the cell free supernatant (cfs). an aliquot of the cfs was acidified to ph 2.0 using 6 n hcl, left overnight at 4 °c, and then centrifuged at 10 000 rpm for 20 min. the resulting supernatant was discarded and the remaining pellet was collected, and then extracted three times with methanol. the dry pellet was obtained by removal of methanol using a rotary vacuum evaporator. the net weight of pellet was determined and calculated as crude surfactin. structural characterization of the crude surfactin by mass spectrophotometry revealed that it contained surfactin as the major component (data not shown). results selection of carbon source and nitrogen source. among the carbon sources used in this experiment, the use of glucose yielded highest -1 surfactin production, i.e. 0.320 g l . furthermore, from the experiments result to screen the nitrogen source showed that the use of urea yielded the highest -1 surfactin production, i.e. 0.436 g l . therefore, glucose and urea were used as carbon and nitrogen source for the next study. plackett-burman experimental. the experiments for selection of nine critical factors affected surfactin production by b. amyloliquefaciens md4-12 namely medium composition (seven factors) and environment conditions (initial medium ph and fermentation temperature) were conducted using pbd. the experimental design and results were presented in table 2. the anova of the experiments (table 3) showed that the p-value (prob>f) of the model was 0.0402, implied that the model was significant. the magnitude of each factor affecting on surfactin production was indicated by p-value. among the factors screened, the concentration of glucose, urea, mgso .7h o, and kcl showed 4 2 significantly effecting on surfactin production with pvalues, 0.0438, 0.0100, 0.0221, and 0.0262 with the coefficient of the model 0.056, 0.12, -0.080, and 0.074 respectively. the other factors have p-value (prob>f) > 0.05 that means no significantly affecting on surfactin production. optimization using response surface methodology. optimization of four significant factors on surfactin production was conducted using rsm and the results were presented in table 4. the other factors were kept at their low level. the experiments result was modeled with a second-order polynomial equation to explain the effect of each factor on surfactin production, as follows: y = 1.18 0.034 x 4.758e-003 x + 0.069 x 0.080 1 2 3 x + 0.057 x x + 0.018 x x 0.11 x x 4 1 2 1 3 1 4 2.814e-003 x x + 0.047 x x + 8.887e-005 x 2 3 2 4 3 2 2 2 2 x 0.041 x 0.077 x 0.065 x + 0.023 x4 1 2 3 4 where y was the estimated surfactin production and x , x x and x were coded value for glucose, urea, 1 2, 3 4 mgso .7h o and kcl concentration, respectively. the 4 2 results of the second-order response surface model in the form of analysis of variance (anova) are given in table 5. it can be seen that two linear (x and x ) and 3 4 three quadratic (x , x , and x ) terms were significant, 1 2 3 with the p-values being very small (p<0.05). the quadratic effect of x was not significant (p>0.05). the 4 significant interaction of variables was shown by x .x ; 1 2 x .x , and x .x terms (p<0.05). the other interaction 1 4 2 4 of factors was insignificantly effected on surfactin production. by considering the only significant factors, the model for surfactin production could be written as following equation: y = +1.18 0.069 x 0.080 x + 0.057 x x 0.11 3 4 1 2 2 2 x x + 0.047 x x 0.041 x 0.077 x 1 4 2 4 1 2 2 0.065 x3 according to the equation, the yield of surfactin could be estimated as a linear effect of magnesium sulphate and potassium chloride concentration and squared effect of glucose, urea and magnesium 122 wibisana et al. microbiol indones variable code unit level -1 +1 carbon source a -1 g l 15 45 nitrogen source b 1 9 msg c 1 4 mgso .7h o 4 2 d 0.5 2 k hpo 2 4 e 0.5 2.5 kcl f 0.3 1 trace element g ml 0.5 2 ph h 6 8 temperature i °c 25 35 -1 g l -1 g l -1 g l -1 g l -1 g l std variables surfactin a b c d e f g h i -1 g l ml °c 1 45 9 1 2 2.5 1 0.5 6 25 0.366 2 15 9 4 0.5 2.5 1 2 6 25 0.413 3 45 1 4 2 0.5 1 2 8 25 0.133 4 15 9 1 2 2.5 0.3 2 8 35 0.048 5 15 1 4 0.5 2.5 1 0.5 8 35 0.207 6 15 1 1 2 0.5 1 2 6 35 0.116 7 45 1 1 0.5 2.5 0.3 2 8 25 0.085 8 45 9 1 0.5 0.5 1 0.5 8 35 0.572 9 45 9 4 0.5 0.5 0.3 2 6 35 0.508 10 15 9 4 2 0.5 0.3 0.5 8 25 0.180 11 45 1 4 2 2.5 0.3 0.5 6 35 0.039 12 15 1 1 0.5 0.5 0.3 0.5 6 25 0.064 -1 g l -1 g l -1 g l -1 g l -1 g l -1 g l table 1 plackett-burman design for screening of significant parameters on surfactin production by bacillus amyloliquefaciens md4-12 table 2 the plackett-burman design and the experimental results for the selection of important parameters on the surfactin production volume 9, 2015 microbiol indones 123 sulphate concentration. anova of the model for surfactin production showed that the regression model was highly significant and the lack of fit was insignificant (table 2 5). the high determination coefficient (r ) showed the goodness of fit of the second order model, and it implied that only 9.58% the total variations were not explained by the model. the high adjusted determination 2 coefficient (adj r ) was also satisfactory to confirm the significance of the model. a lower value of the coefficient of variation (cv) indicates that experiments were precise and reliable (box et al. 1978). the signal to noise ratio (adequate precision) for the model was higher than 4, indicating a good fit. visualization of the interaction between the response and experimental levels of each variable and the type of interactions between test variables in order to deduce the optimum conditions provided by the 3d response surface. fig 1 depicted the 3d response surface showing the interactive effect of the significant variable interaction. the other insignificant variables, i.e. concentration of monosodium glutamate, k hpo , 2 4 -1 and trace element were kept at a minimum level (1 g l , -1 -1 0.5 g l , and 0.5 ml l , respectively) and initial ph of medium and temperature were 7.0 and 30 °c, respectively. validation of the optimized condition. based on the model, the optimized medium composition consists -1 of (g l ): 45.00 glucose, 6.33 urea, 1 monosodium glutamate, 1.85 mgso .7h o, 0.40 kcl, 0.5 k hpo , 4 2 2 4 and 0.5 ml trace element. the model predicts the -1 maximum yield of surfactin as 1.35 g l . under optimized condition, validation experiments were conducted in triplicates. the result of experiments -1 showed that the yield of average surfactin was 1.25 g l , suggesting that experimental and predicted values of surfactin yield were in a good agreement. the usage of optimized medium determined by response surface methodology for surfactin production could increase 2.4-fold over the yield in non-optimized medium. discussion b. amyloliquefaciens md4-12 is a potential strain for surfactin production. the selection of potential strain is necessary for future mutation or bioengineering studies. the significant influence factors on surfactin production by b. amyloliquefaciens md4-12 consist of glucose, urea, mgso .7h o and kcl selected by pbd 4 2 experiments. b. amyloliquefaciens md4-12 can use source coeficient model sum of squares df mean square f value p-value prob > f model 0.39 9 0.043 24.26 0.0402* glucose 0.056 0.038 1 0.038 21.32 0.0438* urea 0.17 1 0.17 98.32 0.0100* yeast extract 0.019 4.376e-003 1 4.376e-003 2.48 0.2563 mgso .7h o 4 2 -0.080 0.077 1 0.077 43.83 0.0221* k hpo 2 4 -0.035 0.014 1 0.014 8.15 0.1039 kcl 0.074 0.065 1 0.065 36.72 0.0262* trace element -0.011 1.355e-003 1 1.355e-003 0.77 0.4737 initial ph -0.024 6.651e-003 1 6.651e-003 3.76 0.1920 temperature 0.021 5.243e-003 1 5.243e-003 2.97 0.2272 table 3 anova of the factors affecting on surfactin production using plackett-burman design *) significant at the level > 95.0% (for p-value < 0.05). 124 wibisana et al. microbiol indones table 4 central composite design and the experimental results for optimization of important parameters on surfactin production std x :glucose 1 -1 (g l ) x :urea 2 x :mgso .7h o 3 4 2 x :kcl 4 surfactin 1 30.00 5.00 0.70 0.40 1.088 2 45.00 5.00 0.70 0.40 1.261 3 30.00 9.00 0.70 0.40 0.922 4 45.00 9.00 0.70 0.40 1.064 5 30.00 5.00 2.00 0.40 1.170 6 45.00 5.00 2.00 0.40 1.313 7 30.00 9.00 2.00 0.40 1.033 8 45.00 9.00 2.00 0.40 1.175 9 30.00 5.00 0.70 1.00 1.190 10 45.00 5.00 0.70 1.00 0.556 11 30.00 9.00 0.70 1.00 1.000 12 45.00 9.00 0.70 1.00 0.868 13 30.00 5.00 2.00 1.00 1.180 14 45.00 5.00 2.00 1.00 0.790 15 30.00 9.00 2.00 1.00 1.103 16 45.00 9.00 2.00 1.00 0.988 17 22.50 7.00 1.35 0.70 0.953 18 52.50 7.00 1.35 0.70 1.020 19 37.50 3.00 1.35 0.70 0.783 20 37.50 11.00 1.35 0.70 0.864 21 37.50 7.00 0.05 0.70 0.630 22 37.50 7.00 2.65 0.70 1.008 23 37.50 7.00 1.35 0.10 1.388 24 37.50 7.00 1.35 1.30 1.061 25 37.50 7.00 1.35 0.70 1.169 26 37.50 7.00 1.35 0.70 1.079 27 37.50 7.00 1.35 0.70 1.159 -1 (g l ) -1 (g l ) -1 (g l ) -1 (g l ) volume 9, 2015 microbiol indones 125 mawgoud et al. 2008; ghribi et al. 2011). in this study, -1 the usage of glucose concentration higher than 40 g l still showed increasing of surfactin production and at -1 glucose concentration 45 g l reached maximum surfactin yield. although in this study the concentration of glucose required higher but keep in mind that surfactin produced was also higher than previous reported. the nature of nitrogen source also plays an important role in the production of surface-active several carbon sources for surfactin production. however, the nature and concentration of carbon source influenced on surfactin yield (abdel-mawgoud et al. 2008; yi et al. 2013; singh et al. 2014b). the best carbon source for surfactin production by b. amyloliquefaciens md4-12 is glucose since this carbon source is easy to assimilate. the concentration of glucose for maximum surfactin production was -1 reported at 40 g l and at glucose concentration higher -1 than 40 g l , the surfactin yield decreased (abdel source coeficient estimates sum of squares df mean square f value p-value prob > f model 0.91 14 0.065 10.11 < 0.0001* intercept 1.18 x -glucose 1 -0.0337 0.027 1 0.027 4.26 0.0567 x -urea 2 -0.0048 5.433e-004 1 5.433e-004 0.085 0.7747 x -mgso .7h o 3 4 2 0.06930 0.12 1 0.12 18.02 0.0007* x -kcl 4 -0.0798 0.15 1 0.15 23.89 0.0002* x .x 1 2 0.05693 0.052 1 0.052 8.11 0.0122* x .x 1 3 0.01790 5.127e-003 1 5.127e-003 0.80 0.3847 x .x1 4 -0.1136 0.21 1 0.21 32.29 < 0.0001* x .x 2 3 -0.0028 1.267e-004 1 1.267e-004 0.020 0.8899 x .x 2 4 0.04682 0.035 1 0.035 5.48 0.0334* x .x 3 4 8.89e -15 1.264e-007 1 1.264e-007 1.976e-05 0.9965 2 x 1 -0.0412 0.046 1 0.046 7.26 0.0166* 2 x2 -0.0767 0.16 1 0.16 25.23 0.0002* 2 x 3 -0.0647 0.11 1 0.11 17.97 0.0007* 2 x 4 0.0225 0.014 1 0.014 2.17 0.1610 residual 0.096 15 6.396e-003 lack of fit 0.082 10 8.195e-003 2.93 0.1236 pure error 0.014 5 2.798e-003 table 5 anova for respon surface with quadratic regression model 2 2 *) significant at the level > 95.0% (for p value < 0.05); r = 0.9042; adj r = 0.8148; coeficient variation (c.v. %) = 7.63; signal to noise ratio (adequate precision) = 14.605 126 wibisana et al. microbiol indones optimum condition of mgso .7h o and kcl were 1.85 4 2 -1 and 0.4 g l , respectively, which is equal to 2+ + concentration of mg and k of 7.5 and 5.3 mm, respectively. mgso4 concentration influenced cell growth as well as the surfactin production, while kcl was reported to relate with the permeation pressure by offering a buffer environment in cooperation with kh po for cell growth (yi et al. 2013). maintaining the 2 4 environmental condition is important to support biomass growth and surfactin production as well as inhibit byproduct formation in the fermentation process. compounds by microorganisms (singh et al. 2014b). in this study the use of urea showed highest surfactin production. the optimum urea concentration was 6.33 -1 g l . the adequate c/n ratio is an important factor for efficiently surfactin synthesis. the other significant factors effecting surfactin production in this study were mgso .7h o and kcl. 4 2 these results correspond to those reported in literature (wei et al. 2007). under optimal condition of the metal 2+ + ion mg and k (2.4 and 10 mm, respectively), surfactin yield was improved significantly. in this study the s u rf a c ti n x : urea2 x : kcl4 1.2075 1.065 0.9225 0.78 5.00 6.00 7.00 8.00 9.00 0.40 0.55 0.70 0.85 1.00 s u rf a c ti n 1.35 1.255 1.16 1.065 0.97 30.00 33.75 37.50 41.25 45.00 5.00 6.00 7.00 8.00 9.00 x : urea2 x : glucose1 1.35 a s u rf a c ti n x : glucose1 x : kcl4 1.35 1.2525 1.155 1.0575 0.96 30.00 33.75 37.50 41.25 45.00 0.40 0.55 0.70 0.85 1.00 b c fig 1 3d respon surface plot for effect combination of (a) glucose and urea (b) glucose and kcl and (c) urea and kcl on surfactin production. volume 9, 2015 microbiol indones 127 doi:10.1016/j/febslet.207.01.059. lim jh, park bk, kim ms, hwang mh, rhee mh, park sc, yun hi. 2005. the anti-thrombotic activity of surfactins. j vet sci. 6(4):353-5. liu x, ren b, gao h, liu m, dai h, song f, yu z, wang s, hu j, kokare cr, zhang l. 2012. optimization for the production of surfactin with a new ynergisticantifungal activity. plos one7(5). doi:10.1371/journal.pone.003 4430. meena kr, kanwar ss. 2015. lipopeptides as the antifungal and antibacterial agents: applications in food afety and therapeutics. biomed res int. doi:10.1155/2015/473050. mnif i, chaabouni-ellouze s, ghribi d. 2012. optimization of the nutritional parameters for enhanced production of b. subtilis spb1 biosurfactant in submerged culture using response surface methodology. biotechnol res int. doi:10.1155/2012/795430 sabate dc, audisio mc. 2013. inhibitory activity of surfactin, produced by different bacillus subtilis subsp. subtilis strains, against listeria monocytogenes sensitive and bacteriocin-resistant strains. microbiol res. 168(3):125-29. doi:10.1016/j.micres.2012.11.004. sen r. 1997. response surface optimization of the critical media components for the production of surfactin. j chem tech biotechnol. 68(3):263-70. doi:10.1002/(si ci)1097-4660(199703)68:3<263::aid-jctb631>3. 0.co;2-8. sen r, swaminathan t. 1997. application of response surface methodology to evaluate the optimum environmental conditions for the enhanced production of surfactin. appl microbiol biotechnol. 47(4):358-63. doi:10.1007/s002530050940. singh ak, rautela r, cameotra ss. 2014a. substrate dependent in vitro antifungal activity of bacillus sp. strain ar2. microb cell fact. 13:67. doi:10.1186/14752859-13-67. wei yh, lai cc, chang js. 2007 using taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by bacillus subtilis atcc 21332. proc biochem. 42(1):40-5. doi:10.1016/j.procbio.2006.07.025. yi l, zhang g, zhu z, wang x, ran w, shen q. 2013. optimization of medium composition for lipopeptide production from bacillus subtilis n7 using response surface methodology. korean j microbiol biotechnol. 41(1):52-9.doi:10.4014/kjmb.1207.07020. zeraik ae, nitschke m. 2010. biosurfactants as agents to reduce adhesion of pathogenic bacteria to polystyrene surfaces: effect of temperature and hydrophobicity. curr microbiol. 61(6):554-9. doi:10.1007/s00284010-9652-z. zhang y, liu c, dong b, ma x, hou l, cao x, wang c. 2015. anti-inflammatory activity and mechanism of surfactin in lipopolysaccharide-activated macrophages. inflammation 38(2):756-64. doi:10.1007/s10753-0149986-y. in this study, after optimization of significant factors by rsm the surfactin yield could be markedly enhanced (2.4-fold). using glucose, urea, mgso .7h o 4 2 and kcl at optimal concentration and keeping other factors at their minimum value, the yield of surfactin -1 reached 1.25 g l , which closely corresponds to the -1 model estimates (1.35 g l ). the surfactin yields obtained in this study was higher than some of reported yields in literature and also have shorter fermentation cycle (sen 1997; abdel-mawgoud et al. 2008; alajlani et al. 2007). references abdel-mawgoud am, aboulwafa mm,hassouna nah. 2008. optimization of surfactin production by bacillus subtilis isolate bs5. appl biochem biotechnol. 150(5):305-25. doi:10.1007/s12010-008-8155-x. al-ajlani mm, sheikh ma, ahmad z,hasnain s. 2007. production of surfactin from bacillus subtilis mz-7 grown on pharmamedia commercial medium. microb cell fact. 6(17). doi:10.1186/1475-2859-6-17. amani h,haghighi m,keshtkar mj. 2013. production and optimization of microbial surfactin by bacillus subtilis for ex situ enhanced oil recovery. petroleum scitechnol. 31(12):1249-58. doi: 10.1080/10916466.2010.542416. banat im, franzetti a, gandolfi i, bestetti g, martinotti mg, fracchia l, smyth tj, marchant r. 2010. microbial biosurfactants production, applications and future potential. appl microb biotechnol. 87(2):42744. box gep, hunter wg, hunter js. 1978. statistics for experimenters. new york: john wiley and sons 291334. cooper dg, macdonald cr, duff sjb, kosaric n. 1981. enhanced production of surfactin from bacillus subtilis by continuous product removal and metal cation additions. appl environ microbiol. 42(3):408-12. ghribi d, ellouze-chaabouni s. 2011. enhancement of bacillus subtilis lipopeptide biosurfactants production through optimization of medium composition and adequate control of aeration. biotechnol res int. doi:10.4061/2011/653654. huang x, lu z, zhao h, bie x, lü f, yang s. 2006. antiviral activity of antimicrobial lipopeptide from bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, newcastle disease virus and infectious bursal disease virus in vitro. int j peptide res therapeut. 12(14):373-377. doi:10.1007/s10989-0069041-4. kim sy, kim jy, kim sh, bae hj, yi h, yoon sh, koo bs, kwon m, cho jy, lee ce, hong s. 2007.surfactin from bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression. febs let. 581(5):865-71. 128 wibisana et al. microbiol indones 1: 120 2: 121 3: 122 4: 123 5: 124 6: 125 7: 126 8: 127 9: 128 5.mi730-hanna artuti available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.4.5issn 1978-3477, eissn 2087-8575 vol 7, no 4, november 2013, p 167-176 *corresponding author; phone/fax: +62-251-8626178, email: hanna.artuti@gmail.com numerous soils have been polluted by mercury (hg) as a result of anthropogenic activities, such as amalgamation of gold in gold-mining and the agricultural soils. hg is a toxic chemical which is not degraded because it is an element (us epa 2009). management of the areas, which have been exposed to either intense or diffuse hg pollution, has therefore become a major environmental concern. strategies oriented toward the use of plants and microbes, or both in combinations within the plant rhizosphere, have been proposed in the recent years as an effective cleanup technology in removing or stabilizing heavy metals in polluted soils. among these microbes, arbuscular mycorrhizal a river-sand culture experiment was conducted to investigate whether arbuscular mycorrhizal (am) colonization influenced mercury (hg) and nutrients accumulation, and whether am fungus (amf) gigaspora margarita enhance host plant sengon buto (enterolobium cyclocarpum) tolerance to hg. hg was applied as hgcl at different levels (375 and 750 µm) and added to the full strength of hoagland's solution, then applied to 2 seedlings in river-sands as growth media according to treatments. the non-mycorrhizal and mycorhizal e. cyclocarpum roots took up hg, but its translocation to the leaves was inhibited. am inoculation decreased significantly hg content of roots seedlings by 70.5% from non-am inoculation seedlings. mycorrhizae enhanced significantly ca and mg uptake in shoot by 1.29and 1.27-fold higher than non-mycorrhizal seedlings, but not enhanced significantly p uptake. based on the roots dry weight, the tolerance index of non-mycorrhizal or mycorrhizal seedlings treated with 750 µm hg supply was > 50%. it indicated that the seedlings can tolerate up to 750 µm hg added. considering the possible differences in amf response to hg in polluted soil from the field, it is not yet clear if gi. margarita could be applied for phytoremediation of hg in contaminated sites. therefore, more work needs to be done using amf isolates to reveal the possible application in the management of hg contaminated soils. key words: arbuscular mycorrhizal fungus, enterolobium cyclocarpum, gigaspora margarita, mercury percobaan untuk meneliti apakah kolonisasi mikoriza arbuskula (ma) mempengaruhi akumulasi merkuri (hg) dan unsur hara, dan apakah fungi ma (fma) gigaspora margarita meningkatkan toleransi tanaman inang sengon buto (enterolobium cyclocarpum) terhadap hg telah dilakukan. perlakuan hg diberikan dengan konsentrasi berbeda (375 dan 750 μm hgcl ) yang ditambahkan ke dalam larutan hara hoagland full strength, 2 kemudian diberikan pada semai di media tumbuh pasir sungai sesuai perlakuan. akar e. cyclocarpum tidak bermikoriza dan bermikoriza menyerap hg, tetapi translokasinya ke daun dihambat. inokulasi ma nyata menurunkan kadar hg akar semai 75% dari semai yang tidak diinokulasi ma. mikoriza nyata meningkatkan serapana ca dan mg di tajuk 1.29 dan 1.27 kali lebih tinggi dari semai tidak bermikoriza, tetapi tidak nyata meningkatkan serapan p. berdasarkan bobot kering akar, indeks toleransi semai tidak bermikoriza dan bermikoriza yang diberi perlakuan hg 750 µm lebih dari 50%. hal ini mengindikasikan bahwa semai dapat toleran hg hingga 750 µm. pertimbangan pada kemungkinan perbedaan respons fma terhadap hg di tanah tercemari dari lapangan, masih belum jelas apakah gi. margarita dapat diaplikasikan untuk fitoremediasi lahan terkontaminasi hg. oleh karena itu, penelitian lebih lanjut dengan memanfaatkan isolat-isolat fma diperlukan untuk mengembangkan kemungkinan aplikasinya dalam pengelolaan tanah-tanah terkontaminasi hg. kata kunci: enterolobium cyclocarpum, fungi mikoriza arbuskula, gigaspora margarita, merkuri the role of arbuscular mycorrhizal fungus (gigaspora margarita) on mercury and nutrients accumulation by enterolobium cyclocarpum seedlings 1,2 3 4 hanna artuti ekamawanti , yadi setiadi *, didy sopandie , 5 and dwi andreas santosa 1 tropical silviculture major, post-graduate school, institut pertanian bogor, jalan lingkar akademik kampus ipb darmaga, bogor 16680, indonesia; 2 faculty of forestry, universitas tanjungpura, jalan imam bonjol, pontianak, indonesia; 3 laboratory of forest biotechnology, research center for biological resources and biotechnology, institut pertanian bogor, jalan lingkar akademik kampus ipb darmaga, bogor 16880, indonesia; 4 department of agronomy and horticulture, institut pertanian bogor, jalan meranti, kampus ipb darmaga, bogor 16680, indonesia; 5 laboratory of biotechnology soil and environment, department of soil and land resources, institut pertanian bogor, jalan meranti, kampus ipb darmaga, bogor 16680, indonesia fungi (amf) are particular interest due to their unique position at the soil/root interface and their recognized role in element transport and immobilization (smith and read 2008). amf can tolerate a wide range of metal concentrations in soils (gonzález-guerrero et al. 2008), affect the accumulation of metals such as cu, cd, zn, and as by plants and enhance the tolerance of host plants to these metals exposure in soil (gonzálezchávez et al. 2004; janoušková et al. 2006; marques et al. 2006). however, few studies have addressed the interaction between amf and hg in growth medium or soil-plant system. therefore, the potential of amf has not yet been fully explored with respect to its hg phytoremediation. the first report about the effects of am inoculation on hg behavior in soil-plant system is done by yu et al. (2010) and it has been shown that hg uptake was lower by mycorrhizal roots of maize than by non-mycorrhizal roots. hg accumulation in several plants has been studied, such as white clover (greger et al. 2005), alfalfa (ortega-villasante et al. 2005), lentil, chickpea (rodríguez et al. 2007) and common vetch (sierra et al. 2008). nevertheless, very few studies have been conducted on the hg tolerance in tropical trees, such as sengon buto (enterolobium cyclocarpum). this species is one of the largest trees in the dry forest formation and a nitrogen-fixing species (world agroforestry centre 2013), therefore it could be a soil improver. if this tree species is proposed for phytoremediation on hg-polluted soil, plant hg uptake and resistance response should be evaluated under controlled conditions, prior to field establishment a screen to identify suitable candidate species. the interaction between amf, tree species (such as e. cyclocarpum) and hg was the subject of this study because of the possibility of the beneficial effect of mycorrhizae in improving the resistance of plants against hg toxicity. the study was carried out to determine the hg and nutrient accumulation in e. cyclocarpum seedlings inoculated with amf gigaspora margarita (collection of laboratory of forest biotechnology, research center for biological resources and biotechnology, bogor agricultural university) grown in artificial hg-polluted river sands media in pot experiment. such knowledge would help clarify the potential of tree species e. cyclocarpum and amf as phytoremediation agents of hg-polluted soils. materials and methods plant culture in river sands at different rates of hg. e. cyclocarpum seeds were surface-sterilized by 168 ekamawanti et al. microbiol indones immersing the seeds in 0.05% naocl for 60 sec and then rinsing them three times with distilled water (dh o). to break dormancy, the seeds were soaked in 2 boiling water for 5 min and then in dh o overnight. 2 the seeds were germinated on zeolite media. after formation of completed leaves, a seedling was transplanted on to zeolite media in polybag (15 cm x 25 cm). mycorrhizal seedlings were obtained by inoculating the seedlings with gi. margarita inoculums (laboratory of forest biotechnology, research center for biological resources and biotechnology, bogor agricultural university collection) as first inoculation first inoculation of amf inoculum when they were germinated and transplanted on zeolite. each nine-months old seedlings of non-mycorrhizal and mycorrhizal was grown in a pvc pot (10 cm in diameter and 30 cm in height), containing 5 kg coarse river sands (1-2 mm) as substrate and maintained in screen house. gi. margarita inoculum (25 g) was inoculated to mycorhizal seedlings for second inoculation and sterilized mycorrhizal inoculum (25 g) and its filtrate (10 ml each pot) was applied to non-mycorrhizal seedlings. all seedlings were maintained by adding the nutrient solution for 8 weeks. the composition of the -1 nutrient solution was as follows (mmol l ) (zornoza et al. 2010): ca(no ) 1.5; kno 4.0; kh po 1.5 and 3 2 3 2 4 -1 mgso 1.0. micronutrients were supplied (µmol l ): 4 fe-eddha 36; mnso .h o 33; znso .7h o 1.6; 4 2 4 2 cuso .5h o 1.6; h bo 46, and (nh ) mo o .4h o 4 2 3 3 4 6 7 24 2 0.1. the ph of nutrient solution ranged for 5.5 to 6.0. a factorial experiment with completely randomized design with three replicates for each treatment was used. hg treatments were 0, 375 and 750 µm, supplied as hgcl salt and applied to e. cyclocarpum seedlings, 2 either with or without am inoculation treatments. these concentration of hg treatments were threshold concentration of hg toxicity for e. cyclocarpum seedlings based on the result of previous research. the experiment was carried out in screen house of forest microbiology laboratory, agency for the research of forest rehabilitation and conservation, bogor, under the following conditions: 28 °c-33 °c temperature and 80%-95% humidity. harvesting. plants were harvested after 30 d to hg exposure and divided into roots, stems and leaves. all plant material was thoroughly washed with tap water followed by a subsequent rinse in 20 mm edta solution for 1 min and then in deionized water for 2 min. total fresh weight of each tissue was determined, then a representative plants were oven-dried to constant weight at 70 °c for 3 d prior to preparation for measuring dry weights and nutrients analysis, and at 50 °c prior to preparation for hg analysis. water content was determined based on the difference of fresh weight and dry weight. the rest of the material plants was frozen in liquid n and stored at -20 °c before 2 malondialdehyde (mda) concentration analyzing. analytical determination. dried plant sample was acid digested using 25 ml of deionized water, 3.5 ml of concentrated hno and 0.5 ml of hclo , added 3 4 to 0.5 g dry weight (dw) in a test tube. the digested solution was analyzed for hg concentration using inductively coupled plasma-optical emission spectrometry (icp-oes, spectro genesis) excitation, and calibration was performed by using standard hg solution (han et al. 2006).total hg and nutrient (p, ca, and mg) concentration were determined by icp-oes. lipid peroxidation in plant tissues was based on an estimate of malondialdehyde (mda) concentration, as described by heath and packer (ortega-villasante et al. 2005). am colonization was determined following brundrett et al. (1996). calculation. some variables were calculated to study the resistance to mercury stress of e. cyclocarpum seedlings (moreno-jiménez et al. 2007): where [hg] was hg concentration, and dw was the roots or leaves dry weight. nutrients (p, ca and mg) uptake and translocation were calculated as (wang and greger 2004): where dw was the entire-plant dry weight. tolerance index (ti) of mycorrhizal and nonmycorrhizal seedlings to hg treatments was determined based on roots dry weight, as (rabie 2005): water content (wc) of leaves, stems or roots was calculated as: where fw was fresh weight. statistical analyzes. a statistical analyzes for means comparison was carried out using anova and least significant difference (lsd) test using statistical software costat 6.400. results mercury accumulation. hg accumulation in e. cyclocarpum seedlings was affected significantly by hg treatment. hg concentration in roots of e. cyclocarpum seedlings was 8to 16-fold and hg content in roots was 3to 6-fold higher than the control (without hg) (table 1). however, at the 750 µm of hg, hg accumulation in roots was decreased significantly by 49.6% of hg concentration and 57.2% of hg content when compared to hg accumulation in roots of seedlings treated with 375 µm of hg. hg accumulation in roots without hg treatment shows that there was hg cross contamination in the air. table 1 shows that almost there are not hg accumulation in leaves of e. cyclocarpum. on the other hand, am inoculation decreased hg content of roots seedlings by 70.5% from non-am inoculation seedlings (data not shown). nutrients accumulation and distribution. the highest hg concentration had stronger effects regarding nutrients translocation than regarding nutrients uptake, especially in mycorrhizal seedlings (table 2). the supply of 750 µm hg caused reducing in p and mg translocation by 53% and 47%, respectively, lower than the control (without hg supply) in mycorrhizal seedlings. p uptake also followed a similar response to highest hg rate by 29% lower than the control in seedlings inoculated with amf. in contrast, nutrients (p, ca, and mg) uptake and translocation were not difference in non-mycorrhizal seedlings treated with the hg supply. moreover, nutrients uptake and translocation did not suffer decreases in seedlings at the lower hg supply (375 µm), either in non-mycorrhizal or mycorrhizal seedlings. table 2 also showed that am inoculation had negative effects regarding nutrients translocation, but not regarding nutrients uptake in seedlings treated with the hg supply. the am inoculation caused decreases significantly in p and mg translocation to leaves by 56% and 44%, respectively, lower than in nonmycorrhizal seedlings treated with 750 µm hg apply. in contrast, p and mg uptake showed no difference effects of between control (no am inoculation) and am inoculation in seedlings at 375 µm hg supply. this volume 7, 2013 microbiol indones 169 -1 hg content in roots (ng roots ) -1 and leaves (ng leaves ) = [hg] × g dw -1 nutrient uptake (nmol g dw) -1 nmol plant g dw = -1 nutrient translocation (nmol hg g dw) -1 nmol leaves g dw = ti (%) dw of roots at hg treated dw roots at non hg treated of the same treatments = × 100 wc (%) (fw-dw) of leaves, stems, or roots fw of leaves, stems, or roots = × 100 supply (table 4). dry weight of leaves, stems and roots decreased in the range 38-54%, 54-67%, and 43-54%, respectively. it indicated that there was reduction of plant growth caused by hg supply up to 750 µm during 30 d of hg exposure. on the other hand, biomass of seedling treated with am inoculation was decreased significantly (table 5). in stems and roots, e. cyclocarpum seedlings treated with am inoculation showed 63% and 68% decrease in the biomass respectively, but a sharp decrease (72%) was occurred in leaves. regarding water content (table 6), the effect of am inoculation depended on hg treatment. at control (without hg supply), leaves and stems water content in tendency was also found regarding ca uptake and translocation. on the other hand, am inoculation increased p uptake, significantly, and tended to increase ca and mg uptake and also nutrients translocation in seedlings treated without hg supply. in addition, am inoculation as a single factor treatment also enhanced ca and mg uptake in seedlings, significantly, by 1.29and 1.27-fold higher than nonmycorrhizal seedlings, respectively (table 3). e. cyclocarpum seedlings biomass and mda concentration. the negative effect of hg in plants was reflected in biomass (dry weight), being significantly (p < 0.05) reduced in non-mycorrhizal and mycorrhizal seedlings, when cultivated with hg microbiol indones170 ekamawanti et al. hg (µm) -1 hg concentration (ng g dw) hg content (ng) roots leaves roots leaves 0 38.2 ± 26.2 a 1.8 ± 3.0 363.5 ± 467.4 a 9.6 ± 14.7 375 611.0 ± 124.3 c n.d 2,066.5 ± 1,759.0 c n.d 750 302.8 ±154.8 b n.d 1,183.0 ± 991.1 b n.d hg (µm) am inoculation without with with out with -1-----p uptake (µmol g dw) ------------p translocation (µmol g-1 dw) ------- 0 109.8 ± 23.3 a(b) 164.7 ± 14.8 a(a) 19.3 ± 7.3 a(a) 29.2 ± 8.3 a(a) 375 127.0 ± 8.1 a(a) 162.5 ± 21.5 a(a) 23.4 ± 6.0 a(a) 28.5 ± 16.1 a(a) 750 142.1 ±36.7 a(a) 116.2 ±21.2 b(a) 30.2 ± 0.7 a(a) 13.3 ± 2.5 b(b) -1-------ca uptake (µmol g dw) ------- -1 -------ca translocation (µmol g dw) ------ 0 60.0 ± 3.3 a(a) 84.3 ± 14.3 a(a) 65.1 ± 21.8 a(a) 93.4 ± 30.5 a(a) 375 47.7 ± 2.6 a(a) 60.9 ± 12.2 a(a) 54.1 ± 10.3 a(a) 61.7 ± 18.9 a(a) 750 50.0 ± 3.2 a(a) 58.7 ± 3.6 a(a) 68.6 ±15.0 a(a) 42.6 ± 11.6 a(a) -1-------mg uptake (µmol g dw) ------- -1 ------mg translocation (µmol g dw) ------ 0 23.3 ± 2.3 a(a) 34.5 ± 8.6 a(a) 67.9 ± 19.7 a(a) 102.4 ± 32.1 a(a) 375 24.4 ± 1.3 a(a) 31.5 ± 2.2 a(a) 75.5 ± 15.5 a(a) 88.6 ± 31.1 a(a) 750 23.3 ±6.4 a(a) 24.6 ± 7.3 a(a) 83.6 ± 3.8 a(a) 46.9 ± 8.3 b(b) table 1 hg concentration and hg content in roots and leaves of e. cyclocarpum seedlings grown for 30 d in river sands with treatment of different hg concentration table 2 nutrients uptake and translocation in e. cyclocarpum seedlings grown for 30 d in river sands with treatments of different hg concentrations and am inoculation significant differences among hg treatments are indicated by different letter (mean ± sd, n = 6; lsd’s test, p < 0.05). dw: dry weight. significant differences among hg treatments are indicated by small different letter and among am inoculation treatments with different capital letters (mean ± sd, n = 3; lsd’s test, p < 0.05). dw: dry weight. volume 7, 2013 microbiol indones 171 mycorrhizal seedlings were increased by 11% each. even though am inoculation did enhance leaves water content, it increased stems water content significantly by 27% when the seedlings were exposed to 750 µm hg. moreover, mycorrhizal inoculation enhanced roots water content, 17% higher than non-mycorrhizal, in seedlings treated with or without hg treatments (data not shown). hg exposure for 30 d increased mda concentration in roots of e. cyclocarpum seedlings, significantly (fig 1). mda concentration of roots increased when the seedlings were grown in hg-treated river-sand media, either with or without am inoculation. am inoculation caused the highest increase in mda concentration by 485% in the roots of seedlings treated with 750 µm hg, when compared to the control (without am inoculation). on the other hand, 375 µm and 750 µm hg supply resulted in the increased mda concentration in leaves by 14% and 97%, respectively (data not shown). tolerance index of e. cyclocarpum seedlings grown for 30 d in river sands treated with 375 µm hg was < 50% but the tolerance index was > 54% when the seedlings were treated with 750 µm (table 7). roots of non-inoculated seedlings remained nonmycorrhizal while roots of inoculated ones were colonized by amf (table 7). the low amf colonization in seedlings treated with hg indicated that gi. margarita colonization was reduced in the presence of hg in substrate. discussion the results obtained during this research have shown that amf gi. margarita decreased hg content in root seedlings. we predicted that decreasing hg content in roots is one of hg tolerance mechanisms in mycorrhizal symbiosis. this result was in line with yu et al. (2010), amf glomus mosseae decreased significantly hg concentration in maize roots when hg -1 was applied at the rates of 2.0 dan 4.0 mg kg . all interactions of hg with soil/growth media, roots, and amf influence its uptake by plants, but this presume need to be investigated in further research. on the other hand, hg accumulation in roots treated with 750 µm of hg supply decreased significantly by 49.6% of hg concentration and 57.2% of hg content when compared to hg accumulation in roots of seedlings treated with 375 µm of hg. it shows that at the highest hg treatment, hg accumulation in root seedlings was limited. this result was in contrast with esteban et al. (2008) result, which found the pattern of long-term (over 28 d) hg accumulation in shoots and roots white lupin (lupinus albus l.) can be dissected into linear and hyperbolic (saturable) components, when they were grown in 5 and 10 µm hg supply in hydrophonics. the result of experiment also showed that a main part of hg accumulated by e. cyclocarpum am inoculation p uptake -1 (µmol g dw) ca uptake -1 (µmol g dw) mg uptake -1 (µmol g dw) without 126.3 ± 26.2 a 52.6 ± 6.3 b 23.7 ± 3.5 b with 147.8 ± 29.1 a 68.0 ± 15.6 a 30.2 ± 7.2 a hg (µm) dry weight (g) roots stems leaves 0 7.0 ± 4.8 a 11.1 ± 5.6 a 3.7 ± 2.2 a 375 3.2 ± 2.5 b 3.7 ± 2.7 b 1.7 ± 1.6 b 750 4.0 ± 2.9 b 5.1 ± 4.4 b 2.3 ± 2.1 b table 3 nutrients uptake in e. cyclocarpum seedlings grown for 30 d in river sands with treatment of mycorrhizal table 4 leaves, stems and roots dry weight of e. cyclocarpum seedlings grown for 30 d in river sands with treatments of different hg concentrations significant differences among hg treatments are indicated by different letter (mean ± sd, n = 9; lsd’s test, p < 0.05). dw: dry weight. significant differences among hg treatments are indicated by different letter (mean ± sd, n = 6; lsd’s test, p < 0.05). dw: dry weight. growth and nutrient uptake (smith et al. 2008). however, am symbiosis decreased nutrients (p and mg) translocation in seedlings treated with the highest hg supply (750 µm). we predict that disturbances of water fluxes altered p and mg distribution due to high concentration hg in growth substrate and also due to the existence of p and mg competition between plant and amf gi. margarita in a limited space of pot experiment. although a contribution of concentration effects after hg supply cannot be ruled out, the increase of some nutrients could also be a strategy to avoid toxicity in plants (moreno-jiménez et al. 2007). in other case, the increase of nitrogen by nitrogen supply prevents oxidative stress in roots (carrasco-gil et al. 2012). hg can induce toxicity symptoms in plants such as inhibition of plant growth or disturbances on water and nutrient uptake (patra at al. 2000). mercury accumulated in plants evokes severe phytotoxicity and impairs numerous metabolic processes including nutrient uptake, water status, and photosynthesis (chen et al. 2012). the inhibition of plant growth might be the first symptom of hg stress (cho et al. 2000). it was showed that hg decreased significantly dry weight of seedlings was in the roots. there was no translocation of hg to the aerial part (leaves) even though the hg concentration in the growth media was higher. it indicates that e. cyclocarpum is not an hg accumulator, but an hg excluder. this result in line with some studies which have indicated that only a very small amount of hg is translocated to plant shoot after root uptake and hg in leaf mainly comes from the uptake of air hg (ericksen et al. 2004; greger et al. 2005; fay et al. 2007; chen et al. 2009). even though it was not an hg accumulator, the potential of e. cyclocarpumas an hg phytoremediation agent could be explored because the roots accumulated hg. plants that accumulate hg in the roots convert the pollutant into less available forms, thus they could be used to prevent hg leaching and moving to other place. in contrast, arbuscular mycorrhizal symbiosis tend to enhance nutrients (p, ca, and mg) uptake and translocation, especially in seedlings treated without hg supply and 375 µm hg supply (table 2). nevertheless the enhancement was not significant but as a single factor, am inoculation enhanced p, ca and mg uptake, significantly (table 3). it is well documented that am symbiosis can increase plant microbiol indones172 ekamawanti et al. am inoculation dry weight (g) roots stems leaves without 7.2 ± 3.7 a 9.6 ± 5.1 a 3. 9 ± 1.9 a with 2.3 ± 1.4 b 3.6 ± 3.6b 1.1 ± 0.9 b hg (µm) am inoculation without with without with without with water content (%) --------------roots ------------- -------------stems -------------- ------------leaves ------------- 0 56.1 ± 9.3 a(a) 73.3 ± 3.3 a(a) 61.3 ± 2.8 b(b) 68.0 a ± 2.1 a(a) 68.3 ± 2.9 b(b) 75.8 ± 0.3 a(a) 375 66.3 ± 4.0 a(a) 74.9 ± 7.2 a(a) 67.1 ± 2.8 a(a) 66.7 a ± 2.6 a(a) 73.3 ± 1.2 a(a) 72.3 ± 4.0 a(a) 750 69.2 ± 9.8 a(a) 76.3 ± 3.3 a(a) 61.1 ± 1.7 b(b) 77.6 a ± 9.9 a(a) 69.5 ± 0.5 ab(a) 68.5 ± 2.3 b(a) table 5 leaves, stems and roots dry weight of e. cyclocarpum seedlings grown for 30 d in river sands with treatments of am inoculation table 6 roots, stems, and leaves water content of e. cyclocarpum seedlings grown for 30 d in river sands with treatments of different hg concentrations and am inoculation significant differences among hg treatments are indicated by different letter (mean ± sd, n = 9; lsd’s test, p < 0.05) significant differences among hg treatments are indicated by small different letter and among am inoculation treatments with different capital letters (mean ± sd, n = 3; lsd’s test, p < 0.05) volume 7, 2013 microbiol indones 173 of roots, stems and leaves significantly (table 5), even though am inoculation increased p, ca, and mg uptake (table 3). we predicted that increase in p, ca, and mg uptake might be not followed by increase in other macro and micro nutrients. therefore, increase in p, ca and mg uptake was not followed by increase in plant dry weight. additionally, growth depressions can be explained with respect to carbon (c) demand and p supply by the amf, where c drain to amf was not roots, stems, and leaves of e. cyclocarpum seedlings during 30 d exposure to hg supply (table 4). the reduction of plant growth caused by hg also been shown for rumex induratus and marrubium vulgare (moreno-jiménez et al. 2007). generally, the formed mycorrhizal symbiosis significantly improved plant growth performance, such as plant height, stem diameter, shoot, root or total dry weight. in contrast, as a single factor of am inoculation decreased dry weight a (a) b (a) b (a) a (a) b (a) c (b) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 375 750 m d a c o n ce n tr at io n ( n m o l g -1 r o o t f w ) hg treatment (µm) without am with am fig 1 mda concentration in roots of e. cyclocarpum seedlings with different hg concentration (mean ± sd, n = 3). significant differences among hg treatments are indicated by small different letter and among am inoculation treatments with different capital letters (lsd’s test, p<0.05). table 7 tolerance index and am colonization of e. cyclocarpum seedlings grown for 30 d in river sands with treatments of different hg concentrations and am inoculation hg (µm) am inoculation without with without with -----------tolerance index* (%) ----------- -----am colonization (%) ---- 0 100 100 0 65.9 375 46.3 45.6 0 47.2 750 54.2 66.9 0 40.8 remark: * the tolerance index was based on roots dry weight imbalance to p supply from amf (grace et al. 2009). kiers et al. (2011) stated that plants can detect, discriminate, and reward the best fungal symbionts with more carbohydrates and in turn, their fungal symbionts enforce cooperation by increasing nutrient transfer only to those roots providing more carbohydrates. it has been addressed that hg can disturbed water fluxes, therefore, it can alter nutrients distribution which depends on water movement within the xylem (moreno-jiménez et al. 2007). considering now water content, in each organ of e. cyclocarpum seedlings did not suffer reduction in water content during hg exposure. it indicated that water movement within the seedlings was not disturbed by hg apply. therefore, nutrients distribution (p, ca, and mg) in e. cyclocarpum seedlings were not inhibited, especially when the seedlings exposed to hg up to 375 µm concentration. this research only analyzed p, ca, and mg concentration, not other nutrients. therefore, we cannot state if other nutrients translocation is affected. the role of am inoculation to increase the water content of roots might be as an indirect strategy of seedlings to overcome hg-induced water stress when the roots contacted hg directly. it has been known, extraradical hyphae of amf can also increase water uptake in addition to nutrients uptake (smith et al. 2008). mda is one of organic compounds has been used as a biomarker, which can be useful in the early diagnosis of metal toxicity (prasad 2003). in our research, am inoculation enhanced mda concentration in roots, significantly, higher than non-mycorrhizal seedlings when exposed to the highest hg (750 µm) treatment. it suggested some degree of oxidative stress, probably due to the high hg concentration in the roots without any increase of the antioxidant (moreno-jiménez et al. 2007) in roots of mycorrhizal seedlings. in contrast, mda concentration in roots of mycorrhizal seedlings was not different with roots of non-mycorrhizal seedlings when exposed to the lower hg (375 µm) supply. increases of mda concentration by hg exposure have been observed in some previous results research (cho et al. 2000; ortega-villasante et al. 2005; moreno-jiménez et al. 2006; esteban et al. 2008). however, it has not clear yet how its mechanism, especially in mycorrhizal plants when they were exposed to hg and there is no information available, therefore further investigation is still needed. in other case, inoculation with amf caused reduction in mda content in comparison to salinized plants, indicating lower oxidative damage in the colonized plants (latef et al. 2011). in our research, there was no effect of am inoculation on mda concentration in leaves but only hg supply as a single factor resulted in the increased of mda concentration in leaves, nevertheless, there was no hg accumulation in leaves. it was probably due to indirect effect of hg accumulation in roots. this presumption has to be proved in further study. roots of inoculated plants were colonized by amf, while non-inoculated controls remained nonmycorrhizal (table 7). however, the percentage of am colonization was reduced in the presence of hg. this result indicated that the concentration of hg supply in the substrate was harmful to amf gi. margarita. this finding is in line with rabie (2005) who reported that sensitivity of am symbionts to heavy metal polluted soil expressed as a reduction in root colonization. in contrast, addition of hg in soil did not significantly influence maize root colonization rate (yu et al. 2010). another interesting result showed at table 7, the presence of amf could increase the metal tolerance index of e. cyclocarpum seedlings compared with nonmycorrhizal seedlings when they treated with 750 µm hg. this result emphasizes that amf could be potentially effective in protecting seedlings exposed to high levels of hg concentration. the amf ability to alleviate heavy metals stress of plants grown in heavy metal polluted soil was previously proved by rufyikiri et al. (2000); hildebrandt et al. (2007). in contrast, tolerance index of mycorrhizal or non-mycorrhizal seedlings grown for 30 d in river sands treated with 375 µm hg was lower than seedlings treated with 750 µm (table 7). it might be related to hg accumulation in roots treated with 375 µm hg was higher than in roots treated with 750 µm hg (table 1) then resulted in the decreased of roots dry weight. in conclusion, this study provides further evidence for the protective effects of amf gi. margarita on e. cyclocarpum against hg contamination, i.e. non significant effect on hg accumulation in roots, a tendency to increase nutrient uptake and translocation when seedlings treated with hg supply up to 375µm, the sharp decrease of dry biomass in seedlings, no different effect on water content in seedlings treated with hg supply and no increase in mda concentration in seedlings treated with 375 µm. it suggested that the role of symbiosis amf gi. margarita and e. cyclocarpum seedlings can tolerate up to 375 µm hg supply. even 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(an) = 1: 1: 1; ch: sc = 1: 1; dan ch: an = 1: 1. variasi komposisi nutrien yang digunakan adalah carbon (c) : nitrogen (n): phospor (p) = 100: 10: 1, c: n: p = 100: 50: 1, dan c: n: p = 100: 25: 1. sumber c, n, dan p masing-masing adalah co (anorganik), kno , dan kh po . 2 3 2 4 studi ini membuktikan bahwa sinergisme ditentukan oleh kemampuan jenis mikroalga dalam memanfaatkan sumber karbon anorganik. tanpa kehadiran ankistrodesmus sp., sinergisme antara scenedesmus obliquus dan chlorella sp. menunjukkan efisiensi pemanfaatan co dua kali lebih tinggi dibandingkan dengan sinergisme 2 antara ankistrodesmus sp. dan chlorella sp. rasio nutrien c: n: p sebesar 100: 50: 1 dapat meningkatkan efisiensi pemanfaatan co hingga 50% lebih tinggi dibandingkan rasio 100: 10: 1.2 kata kunci: konsorsium terbangun, pemanfaatan co , penyisihan co , rasio nutrien, sinergisme2 2 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-5663232 ext. 8765, fax:+62-21-5602575; email: astririnanti@trisakti.ac.id reduce excess amount of co emitted into the 2 atmosphere. due to the ability of microalgae to capture co during the process of photosynthesis, the algae are 2 n o w b e i n g d e v e l o p e d i n t o b i o l o g i c a l o r biotechnological carbon capture and storage (ccs) to reduce carbon emissions that would otherwise be the global warming issue, mainly caused by carbon dioxide (co ) as the major contributor of 2 greenhouse effects, has triggered various efforts to released into the atmosphere. the carbon capture process works based on the carbon requirement for the process of photosynthesis. microalgae consortium is a potential producer that requires co continuously to 2 perform photosynthesis reaction. compared to various terrestrial plants, microalgae are generally considered photosynthetically more efficient. chojnacka and marquez-rocha (2004) described that there are various types of microalgae based on their metabolism, i.e. autotrophs, heterotrophs, mixotrophs, photoheterotrophs, and able to change its metabolism as a form of adaptation or response to changing environmental conditions. autotroph microalgae in natural environment, that use inorganic co from the air and 2 the sun as the main energy source, could change into mixotroph that utilize organic compounds and inorganic co for growth in close system 2 photobioreactor. beside that, autotroph microalgae able to change into heterotroph as well when only organic co2 that naturally available to be transformed become another organic matters. it means the microalgae can change its metabolism as a response to changing environmental conditions. microalgae can utilize inorganic co from three different sources, i.e. 2 fixing co directly from the atmosphere, taking up co 2 2 from industrial waste gas, and taking up co from 2 dissolved carbonate (wang et al. 2008). microalgae growth is strongly influenced by environmental conditions and the availability of nutrients. carbon ©, nitrogen (n) and phosphorus (p) are macronutrients much needed by microalgae for making proteins. to achieve their optimum growth, source selection and concentration of nutrients must be adapted to the characteristics of strains of microalgae (westerhoff et al. 2010). many types of microalgae can utilize carbonate ions in the form of na co and 2 3 nahco as sources of c. other research shows slow 3 algal growth in medium without n (burkhard et al. 1999). therefore we need extra n to increase the rate of cell proliferation. n sources commonly used for the cultivation of microalgae is in the form of nitrate, ammonia, urea, or a combination of them. nitrogen can be obtained from kno , nano , or nh cl (cuaresma 3 3 4 et al. 2010). phosphorus is also the base material forming nucleic acids, enzymes, and vitamins that can be obtained from kh po , nah po , or ca po , 2 4 2 4 3 4 whereas elemental sulfur can be obtained from nh so , or cuso . the metabolism of these elements; 4 4 4 c, n, and p, are related to one another. phosphate (p) plays a central role in cellular energy transfer. low concentration has an impact on the balance in c and n assimilation. potassium (k) plays a role in the metabolism of carbohydrates and also a cofactor for several coenzymes. potassium can be obtained from kcl, kno , or kh po . iron (fe) plays a role in the 3 2 4 formation of chlorophyll and is an essential component in oxidation. this element can be obtained from fecl , 3 feso , or fecah o (graham and wilcox 2000). 4 5 7 elements si and ca are all ingredients of cell walls or shells. micro nutrients are also needed to perform various functions in the growth of microalgae. mn and zn are necessary for photosynthesis. mo, bo, co are required for the metabolism of nitrogen, while mn, b, cu are required to perform other metabolic functions (westerhoff et al. 2010). the aim of this study was determining the composition of microalgae consortium and the optimum nutrient ratios to obtain higher co removal 2 efficiency. materials and methods microalgae consortium and artificial growth medium. the consortium of green microalgae consisting of chlorella sp., scenedesmus sp., and ankistrodesmus sp. was originally isolated from the bojong soang wastewater treatment plant, bandung, indonesia. the microalga was screened, and then a potential candidate was selected as microbial carbon capture and storage (mccs) agent (rinanti et al. 2013). the microalgal cells were cultured in phm (phovasoli haematococcus media) artificial medium, which was prepared as follows (per litre): 1.0 g kno ,3 0.2 g kh po , 0.2 g mgso .7h o, 0.1 g fe stock 2 4 4 2 consisting of (per litre) 189 g edta and 24.4 g fecl .6h o. all nutrients were dissolved in distilled 3 2 water containing (per litre) 0.1 ml trace element made of (per 500 ml) 2.05 mg zncl , 30.5 mg h bo , 2.55 2 3 3 mg cacl .6h o, 3.0 mg cuso .5h o, 2.05 mg 2 2 4 2 mncl .4h o and 19 mg (nh ) .mo .o .4h o 2 2 4 6 7 2 4 2 (provasoli and pintner 1959). cultivation of microalgae in vertical column photobioreactor. the vertical photobioreactor used in this experiment was made ​​of glass with a capacity of 10 l containing 8 l phm growth medium and an initial 6 -1 cell density of 10 cell.ml . co was injected from the 2 bottom of the column to allow gas mixing with the medium. sparger was attached at the bottom of the photobioreactor to convert the gas into small bubbles. air was bubbled at the bottom. sparging with microbbuble allows thorough mixing, co mass 2 transfer and also removes o produced during photo-2 80 rinanti et al. microbiol indones synthesis. it was a strategy to provide good overall mixing, sufficient supply of co , and efficient removal 2 of o . pure co gas at 10% (v/v) was flowed from the 2 2 bottom of vertical column photobioreactor -1 continuously with optimum flow rate 5 l.min . a growth medium containing artificial nutrients (phm) -1 was flowed continuously at a flow rate 7l.day and the detention time of 3.8 days. four fluorescent lamps were positioned outside the photo-bioreactor to obtain light intensity of 4000 lux, set for 16 hours light exposure and 8 hours dark with operating temperature of 30 °c maintained during the study (rinanti et al. 2014). three compositional variations of microalgae consortium were used. they are as follows; ch : sc : an = 1: 1: 1; ch : sc = 1: 1; and ch : an = 1: 1, where ch, sc, and an were chlorella sp., scenedesmus obliquus, and ankistrodesmus sp., respectively. the following variations of nutrient composition were used; c: n: p = 100: 10: 1, c: n: p = 100: 50: 1 and c: n: p = 100: 25: 1. the c, n, and p sources were co 2 (inorganic), kno , and kh po , respectively.3 2 4 measurement of biomass dryweight. to measure the biomass dryweight, the microalgal culture must be dried by evaporating the liquid in the culture. prior to the evaporation, culture was centrifuged in 100ml tubes at 3,500 rpm for 10 minutes (weldy and huesemann 2007). supernatant was then removed from the tube and the cell pellet was kept. the pellet was then placed in a petri dish that had previously been weighed (x). the sample-containing petri dishes were then kept in the oven set at 105 °c overnight until constant weight (y) was reached. then, the sample was left in a desiccator for 30 minutes to evaporate any remaining liquid before re-weighed. biomass (dry weight) was calculated according to the formula from torzillo et al. (1991): dry weight (x; mg) = y (mg) x (mg). -1 specific growth rate (μ; d ) was calculated as follows: (1) measurement of co concentration and 2 determination of co removal efficiency. the co 2 2 concentration in the influent gas and effluent gas was measured by portable combination gas detector riken model rx-515. efficiency of co removal can 2 be calculated by the following formula: (2) carbon uptake rate. a formula (co h n p ) 0.48 1.83 0.11 0.01 suggested by grobbelaar (2004) was used to make an expected estimate of the dry biomass yield and carbon uptake rate was determined by using the following equation: carbon uptake rate = c x p (3) where c is the carbon content of the dried cell (g carbon.g -1 -1 -1 biomass ), p is the productivity (g biomass.l d ). results of elemental analysis in our study showed that the carbon content in the mix culture was 67.56%. co utilization efficiency. co utilization 2 2 efficiency was determined by using the following equation (ryu et al. 2009): (4) where c is the carbon content of the dried cell (g -1 carbon.g biomass ), p is the productivity (g biomass.l 1 -1 .h ), 44 and 12 are the molecular weights of carbon dioxide and carbon, respectively, and v is the rate of co supplied to the microalgal culture medium (g 2 -1 -1 co .l .h ).2 results species variation of microalgae consortium. the three species of microalgae composing the consortium used in this study, chlorella sp., scenedesmus obliquus, and ankistrodesmus sp., seemed to have different physiological characteristics. as mentioned by chojnacka and marquez-rocha (2004), every species of microalgae carries specific physiological properties. although these three species are capable of living synergistically by utilizing highly concentrated inorganic carbon source (10% v/v) together, synergism between chlorella sp. and scenedesmus obliquus showed better growth rate compared to the synergism between chlorella sp. and ankistrodesmus sp. (fig 1, fig 2, and table 1). determination of the optimum nutrient ratios. previous studies reported about algal cell vitality reduction due to the deficiency of various nutrients. this is related to the loss of the cell's ability to build functional structures associated with the limited amount of nutrients. hence, the following attempt to improve the co removal efficiency was done by adding 2 different concentrations of nitrogen to the growth medium (artificial phm). the co removal efficiency 2 increased in accordance to the increment of nitrogen addition. nutrient ratio c : n : p of 100 : 50 : 1 was found to be the optimum ratio and was very sufficient for the volume 10, 2016 microbiol indones 81 % co utilization2 efficiency carbon content ´ p ´ mco2 aeration rate of co2 100%˟ = m = 1 x dx dt % co removal2 efficiency influent of co effluent of co2 2 influent of co2 100%˟ = 82 rinanti et al. microbiol indones fig 2 profile of biomass dry weight (g/l) as a function of different species composition in microalgae consortium. (ch = chlorella sp.; sc = scenedesmus obliquus; an = ankistrodesmus sp.). fig 1 profile of co removal efficiency (%) as a function of different species composition in microalgae 2 consortium. (ch = chlorella sp., sc = scenedesmus obliquus, an = ankistrodesmus sp.). fig 3 profile of biomass dry weight (g/l) as a function of different species composition in microalgae consortium. (ch = chlorella sp.; sc = scenedesmus obliquus; an = ankistrodesmus sp.). volume 10, 2016 microbiol indones 83 growth of chlorella sp and scenedesmus obliquus consortium, consequently the highest co removal 2 efficiency of 87.63% was obtained when this ratio was applied (fig 3 and table 1). increased dry weight of the microalgae biomass occured in all tested nutrient ratio variations. in general, the higher the concentration of nitrogen, the higher the dry weight of the biomass produced. nutrient ratio c : n : p of 100 : 50 : 1 yielded an average -1 biomass dry weight of 8.86 (gl ) at steady state condition (fig 4). all variations of nutrient ratio reached steady state starting from day 7 until the end of the study. discussion species variation of microalgae consortium. synergism of chlorella sp. and scenedesmus obliquus gave the highest co removal efficiency (84.33%) and 2 -1 biomass dry weight (8.1 g.l ) compared to the other synergism. likewise, the synergism of these two green microalgae gave a higher co utilization efficiency and 2 carbon uptake rate than the synergism of other microalgae species in this study. extremely high co concentration can cause 2 physiological and metabolic changes consisting of stomatal density reduction, low availability of rubisco enzyme and chlorophyll, also low photorespiration -1 fig 4 profile of biomass dry weight (gl ) as a function of various nutrients composition. (c = carbon, n = nitrogen, p = phosphor). *) ch = chlorella sp.; sc = scenedesmus obliquus; an = ankistrodesmus sp.) **) c = co2 (inorganic), n = kno3, p = kh2po4 table 1 the effects of species variation in microalgae consortium and nutrient ratios towards co removal and utilization 2 efficiency, biomass dry weight and carbon uptake rate. 84 rinanti et al. microbiol indones (papazi et al. 2008). the microalgae species that is able to thrive in a highly concentrated co environment may 2 still have the genetic ability to photosynthesize (papazi et al. 2008). the species that can adopt transitional state in supporting the photosystem is able to grow well at high co concentration (miyachi et al. 2003). 2 microalgae also perform molecular mechanism in response to high co concentration by changing the 2 photosynthesis mode through carbon concentrating mechanism. saturated or half-saturated co 2 concentration can change the characteristics of cellular photosynthesis, for example, the affinity of co2 reached 0.5% in chlorella kessleri (papazi et al. 2008). composition of microalgae species making up the consortium found in this study indicated that the highest co utilization efficiency occurred in cultures 2 containing equal amount of chlorella sp. and scenedesmus obliquus, without ankistrodesmus sp.. in the next stage, we tried to find the optimum nutrient ratio to obtain higher co utilization efficiency by 2 culture containing equal amount of chlorella sp and scenedesmus obliquus. determination of the optimum nutrient ratios. a proper and sufficient nutrient ratio remarkably affects the co removal efficiency. nonetheless, it was 2 not significantly different from nutrient ratio with c : n : p = 100 : 25 : 1, which produced 86.03% co removal 2 efficiency. considering these results, adding higher nitrogen concentration were not expected to increase the co removal efficiency, and hence was not studied.2 microscopical observation showed that the size of chlorella cells tends to be bigger in the culture with high concentration of co gas (10% v/v). therefore, 2 despite the relatively lower cell density in 10% (v/v) co concentration, a higher biomass dry weight was 2 achieved (fig 4). it is easily understandable because with adequate supply of carbon, microalgae have greater opportunities to thrive than with limited carbon supply. this result is similar to the studies carried out by yoo et al. (2010) and chiu et al. (2008), where pure 10% co2 was also streamed into the reactor. thus, an optimum co concentration of 10% is needed to gain 2 high productivity of microalgae biomass. zhu et al. (2010) explained that the right composition of nutrients is one of the main factors affecting microalgal biomass productivity, in addition to light intensity, temperature, and co mass transfer 2 into the liquid (ono and j. cuello 2006). the efficiency of co utilization improves in 2 accordance to the escalation of nitrogen source concentration in the growth medium. the highest co 2 utilization efficiency of 5.22% occured in the culture with c : n : p ratio of 100 : 50 : 1. chojnacka and marquez-rocha (2004) described that microalgae could perform various types of metabolism, i.e. a u t o t r o p h , h e t e r o t r o p h , m i x o t r o p h , a n d photoheterotroph, and able to perform metabolic alteration as an adaptation measure or response to shifting environmental conditions. during this study, it seemed that the microalgae consortium comprising of chlorella sp and scenedesmus obliquus was mixotroph in conducting photosynthesis as the main energy source, yet it could maximize the utilization of nitrogen, organic compounds, and inorganic co for its 2 growth. adequate nutrient availability for microalgae is a precondition for optimum photosynthesis. nutrient deficiency will cause interferences with metabolism and production discrepancy in intermediary phase of photosynthesis. however, the correct ratio of carbon, nitrogen, and phosporus as the main nutrients also immensely affects the metabolism, for instance affecting carbon uptake rate. in this study, carbon uptake rate was defined as the absorption rate of co 2 dissolved in the phm growth medium by microalgae consortium. when the photosynthesis took place, pure co gas flow of 10% at 5 l/min flow rate was the 2 primary source of inorganic carbon consumed by microalgae to generate energy. the rate of co uptake 2 by microalgae cells was allegedly stimulated by an increase of co content in the media, so that the culture 2 without co addition absorbed less co , escalating the 2 2 culture ph, yet had never exceeded value of 8. stepan et al. (2002) stated that media ph ranging from 7.0–8.0 is good enough for laboratory microalgae culture. accordingly, the media ph was maintained at 7 during the study. microalgae commonly use nitrate as the primary nitrogen source. shi et al. (2007), bich et al. (1999), and qing-xue et al. (2010) explained that nitrogen compounds are strongly influenced by dissolved oxygen concentration in the water. nitrogen turns into ammonia (nh ) at low-level dissolved 3 oxygen and into nitrate (no ) at high-level dissolved 3 oxygen. nonetheless, when the environmental conditions are not favorable, then ammonia or urea can serve as nitrogen source (sassano 2007; soletto 2005). the lack of nitrogen content will result in the limited production of proteins needed for new cells (westerhoff et al. 2010). this study proved that in addition to the specific characteristics of microalgal cells' physiology, synergy between the types making up the consortium also volume 10, 2016 microbiol indones 85 determine the ability to utilize inorganic carbon source. chlorella sp have the highest synergy to interact together well with the scenedesmus obliquus or ankistrodesmus sp. without the presence of ankistrodesmus sp, synergism between scenedesmus obliquus and chlorella sp. provides twice higher co2 utilization efficiency than the synergism between ankistrodesmus sp and chlorella sp without the presence of scenedesmus obliquus. increased nitrogen proved to increase the growth of chlorella sp and scenedesmus obliquus as a consortium in line with the increase of co2 removal efficiency, co2 utilization efficiency and carbon uptake rate. the availability of macro nutrients, inorganic carbon, nitrogen, and phosphorus, in the appropriate ratio (c: n: p of 100: 50: 1) may increase co utilization efficiency 50% higher than when the c: 2 n: p ratio was 100: 10 : 1. however, the efficiency was not significantly different from when the c: n: p was 100: 25: 1. acknowledgment we thank dikti's (general directorate of higher education indonesia) decentralization program 2015 for funding some parts of this research. references anjos m, bruno d. fernandes, antónio a. vicente, josé a. teixeira, giuliano dragone. 2013 optimization of co 2 bio-mitigation by chlorella vulgaris. bioresour technol. 139: 149–154. http://dx.doi.org/10.1016/ j.biortech.2013.04.032. burkhard s, i. zondervan, u. riebesell. 1999. effect of co 2 concentration on c : n : p ratio in marine phytoplankton : a species comparisons. limnol ocean. 44 (3): 683 690. chiu sy, kao cy, tsai mt, ong sc, chen ch, lin cs. 2009. lipid accumulation and co utilization of 2 nannochloropsis oculata in response to co aeration. 2 bioresour technol. 100: 833-838. doi: 10.1016/ j.biortech.2008.06.061. chojnacka k, marquez-rocha fj. 2004. kinetic stoichiometric relationships of the energy and carbon m e t a b o l i s m i n t h e c u l t u r e o f m i c r o a l g a e . biotechnology 3(1): 21–34. doi: 10.3923/biotech. 2004.21.34. cuaresma m, casal c, forjan e, vilchez c. 2010. productivity and selective accumulation of carotenoids of the novel extremophile microalga chlamydomonas acidophila grown with different carbon sources in batch systems. j ind microbiol biotechnol. 38(1):167177. doi: 10.1007/s10295-010-0841-3. graham le, graham jm, wilcox lw. 2009. algae, 2nd ed., san francisco: pearson education, inc. grobbelaar ju. 1989. do light/dark cycles of medium frequency enhance phytoplankton productivity. j appl phycol. 1(4): 333-340. doi: 10.1007/bf00003470. kong qx, li l, martinez b, chen p, ruan r. 2010. culture of microalgae chlamydomonas reinhardtii in wastewater for biomass feedstock production. appl biochem biotechnol journal 160:9–18. doi: 10.1007/s12010-009-8670-4. miyachi s, iwasaki i, shiraiwa y. 2003. historical perspective on microalgal and cyanobacterial acclimation to low-and extremely high-co conditions. 2 photosynth. res. 77: 139-153. doi: 10.1023/ a:1025817616865. ono e, cuello jl. 2009. mini review : selection of optimal microalgae species for co sequestration. departemen 2 of agricultural and biosystem engineering. usa papazi a, makridis p, divanach p, kotzabasis k. 2008. bioenergetic changes in the microalgal photosynthetic apparatus by extremely high co concentrations induce 2 an intense biomass production. physiol. plant. 132: 338-349. doi: 10.1111/j.1399-3054.2007.01015.x. provasoli l, pintner ij. 1960. artificial media for freshwater algae: problems and suggestions. in: the ecology of algae. tryon, c.a. and hartman, r.t. (ed.). special publication no. 2. pymatuning laboratory of field biol., university pittsburgh, usa. rinanti a, kardena e, astuti di, dewi k. 2013. screening of potential photosynthetic microalgae from wastewater treatment plant for carbon dioxide capture and storage (ccs) agent. asian transactions sci technol j. 03(1): 1-8. rinanti a, kardena e, astuti di, dewi k. 2014. improvement of carbon dioxide removal through artificial light intensity and temperature by constructed green microalgae consortium in a vertical bubble column photobioreactor. malaysian j microbiol. 10 (1): 29-37. http://dx.doi.org/10.21161/mjm.56713. sassano cen, gioielli la, almeida ka, sato s, perego p, converti a, and carvalho jcm. 2007. cultivation of arthrospira (spirulina) by continuous process using ammonium chloride as nitrogen source. biomas b i o e n e r g . 3 1 : 5 9 3 5 9 8 . d o i : 1 0 . 1 0 1 6 / j.biombioe.2007.04.001. shi j, podola b, melkonian m. 2007. removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: an experimental study. j appl phycol. 19(5): 417-423. doi: 10.1007/s10811006-9148-1. 86 rinanti et al. microbiol indones stepan dj, shockey re, moe ta, dorn r. 2002. subtask, 2.3 carbon dioxide sequestration using microalgae systems. energy and environmental research center. university of north dakota. torzillo g, sacchi a, materassi r. 1991. temperature as an important factor affecting productivity and night biomass loss in arthrospira (spirulina) platensis grown outdoors in tubular phtobioreactors. bioresour technol. 38: 95-100. wang b, li y, wu n, lan cq. 2008. co bio-mitigation 2 using microalgae. appl microbiol biotechnol. 79(5): 707-718. doi: 10.1007/s00253-008-1518-y. wang j, wang j, zhou w, yang w, ruan r. 2016. application of nitrogen sufficiency conversion strategy for microalgae-based ammonium-rich wastewater treatment. environment technol j. (37) 20. http://dx.doi.org/10.1080/09593330.2016.1158744. weldy cs, huesemann m. 2007. lipib production by dunaliella salina in batch culture: effects of nitrogen limitation and light intensity. us department of energy. j undergraduate res. 7(1): 115–122. westerhoff p, hu q, esparza-soto m, vermaas w. 2010. growth parameters of microalgae tolerant to high levels of carbon dioxide in batch and continuous-flow photobioreactors. environment technol j. 31: 523-532. doi: 10.1080/09593330903552078. yoo c, jun sy, lee jy, ahn cy, oh hm. 2010. selection of microalgae for lipid production under high levels carbon dioxide. bioresour technol. 101(2010): 571574. doi: 10.1016/j.biortech.2009.03.030. zhu xg, long sp, ort dr. 2010. improving photosynthetic efficiency for greater yield. annu. rev. plant biol.61: 235-261. doi: 10.1146/annurev-arplant-042809-112206. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 2. aponzaenal revisi.cdr diversity of lactic acid bacteria isolated from indonesian traditional fermented foods apon zaenal mustopa fatimah 1* 2 and 1 research center for biotechnology, indonesian institute of sciences (lipi), cibinong bogor, indonesia; 2 indonesian center for agricultural biotechnology and genetic resources research and development (icabiograd), cimanggu, bogor 16111, indonesia the diversity of lactic acid bacteria was evaluated from indonesian fermented foods such as dadih (buffalo fermented milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous rice). lactic acid bacteria were enumerated using selective media and characterized based on a genotypic methods such as repetitive bacterial dna element (reppcr) and rapd-pcr, as well as 16s rrna gene sequencing of representative strains. fourty-six colonies had successfully been isolated from indonesian fermented foods. the great majority of these colonies originated from dadih (43.48%), tempoyak (39.13%), bekasam (13.04%), and tape (4,3%). the 46 isolates were characterized based on a genotypic methods such as rapd and rep-pcr as well as 16s rrna gene sequencing of representative strains. the rep-pcr result yielded seven clusters (i-vii) at a similarity level of 75-88% rapd-pcr used lb2 primer, m13 primer and primer a, b, c. the rapd result using lb2 primer yielded eight clusters (i-viii) at a similarity level of 82-91%. identification using 16s rrna showed that the majority strains were closed to lactobacillus plantarum, lactobacillus fermentum pediococcus pentosaceus, and strains. key words: 16s rrna, indonesian fermented foods, rapd, rep-pcr keragaman bakteri asam laktat telah di evaluasi dari pangan fermentasi tradisional indonesia seperti dadih (susu kerbau fermentasi), tempoyak (durian fermentasi), bekasam (daging fermentasi) dan tempe ketan. bakteri asam laktat diseleksi dengan menggunakan media selektif dan dikarakterisasi secara genotip menggunakan rep-pcr dan rapd pcr serta gen 16srrna. sebanyak 46 koloni bakteri asam laktat diisolasi dari pangan fermentasi dengan komposisi dadih (43,48%), tempoyak (39,13%), bekasam (13,04%) dan tape (4,3%). karakterisasi secara genotip dari 46 isolat dengan rep-pcr menghasilkan 7 kelompok dengan kesamaan 7588%, sedangkan rapd-pcr dengan primer lb2, m13, dan primer a,b,c terdapat 8 kelompok dengan kesamaan 82-91%. identifikasi menggunakan 16s rrna menunjukan bahwa isolate-isolat tersebut termasuk kedalam strain , and strainslactobacillus plantarum, lactobacillus fermentum pediococcus pentosaceus . kata unci :k 16s rrna, pangan fermentasi indonesia, rapd, rep-pcr vol.8, no.2, june 2014, p 48-57 doi: 10.5454/mi.8.2.2 *corresponding author; phone: +62-21-8754587; fax: +6221-8754588, email: azmustopa@yahoo.com lactic acid bacteria (lab) are of considerable economic significance because of their widespread use in industrial food fermentation processes (sudhamani et al. 2007). certain lab are also used as probiotics added to confer health benefits to consumers or to improve animal production. the lactic acid bacteria species are economically very important to the food fermentation industry (korhonen 2010). indonesian fermented foods, such as dadih (buffalo fermented milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous rice), have been consumed for centuries, but there is little investigation has been conducted to asses the diversity of lab in indonesian fermented foods. lab have complex nutritional requirements because of their limited biosynthetic capabilities. most lab strains must obtain essential components, such as carbohydrates, amino acids, peptides, fatty acid esters and vitamins, from their habitats. indonesia fermented foods should be a suitable environment for lab, since it contains plenty of protein and sugar units from the decomposed vegetables. moreover, because the environment in indonesia fermented foods differs from that in other fermented materials, it should be possible to collect lab strains with unique characteristics, unlike those of the strains found in ordinary fermented materials such as fermented milk or vegetables. molecular approaches for lab systematic studies include pulse field gel electrophoresis (pfge) (ventura and zink 2002), random amplified polymorphic dna (rapd) analysis (franciosi l.et a 2009; chao . 2013;), pcr-denaturing gradient gelet al electrophoresis pcr-dgge (ercolini . 2001; liuet al et al. et al2012), pcr-rflp (yu . 2011) and dna sequencing (liu 2012; sulistiani . 2014),et al. et al which have been extensively applied for the intraspecific identification and for genotyping lab isolated from several fermented foods as well as from human gastrointestinal tract (mc cartney 2002). for lab, the rapd is a method of choice for molecular typing. the profiles obtained can be stored in a computerized database, thus allowing rapid identification of unknown isolates (berthier and ehrlich 1999; corroler 1998). alternatively, pcret ai. amplification of repetitive bacterial dna elements (rep-pcr) has been recognized as a simple pcr-based technique with the following characteristics: (i) a high discriminatory power, (ii) low cost, (iii) suitable for a high-throughput of strains, and (iv) considered to be a reliable tool for classifying and typing a wide range of gram-negative and several gram-positive bacteria (gever . 2001; adimpong . 2012) our aim iset al et al . to investigate the diversity of the predominant lab present in fermented foods using rapd, rep pcr, and 16s ribosomal rna sequences. materials and methods samples ofsource and maintenance of culture. lab were isolated independently from dadih (buffalo fermented milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous). samples were serially diluted in saline solution and plated onto mrs agar (oxoid, england). the plates were incubated at 37 °c for 2 d a total of. 120 samples of lab isolated independently from dadih (fermented from fresh raw buffalo milk in bamboo tubes capped with banana leaves), tempoyak (durian ( ) meat was mixed with smallduria zibethinus amount of salt (2.5%) and placed in a sealed container. fermentation takes about 7 d), bekasam (meat is mixed with 10-20% salt (w/v) and grind roasted rice, then fermented (in sealed container) for 14 d), and tape ketan (glutinous rice is sleamed followed by inoculation with ragi tape, then fermentated about 1-2 d. this product is acid-alcoholic in taste) were used in this study. all lab were maintained by subculturing in de man rogosa and sharpe (mrs) broth (oxoid, england) supplemented with 0.02% (w/v) sodium azide, using 1% inoculum and overnight of incubation at 37 c; between transfer cultures were stored at 4 c. o o dna extraction. lab isolates were cultured in mrs broth (ph 7.0) for 1 d. bacterial cells were collected by centrifugation at 6000 rpm for 10 min. the genomic dna was extracted as previously described, with modification (zhu 1993). the pellet waset al. resuspended with te buffer (10 mm tris-hcl ph 8, 1 mm edta), 40 μ of lysozyme (60 mg ml ).l -1 incubated at 37 ºc for 60 min and 200 μl 10% sodium dodecyl sulfate, 100 μl 5 m nacl, 80 μl 10% ctab was added. warmed at 68 °c for 30 min and added an equal amount of chloroform. centrifugation was conducted at 13000 rpm for 10 min. the supernatant was harvested and an equal amount of ethanol was added. the mixture was shaken again and then centrifuged at 13,000 rpm for 10 min. after being air-dried, the dna was dissolved in te buffer (10 mm tris-hcl, 1 mm edta, ph 8.0) and the concentration was adjusted to 10 μg ml rnase were -1 stored at -20 °c until use. rapd-pcr and rep-pcr genomic fingerprinting. for both rapd-pcr and rep-pcr fingerprinting, total genomic dna from all isolates, as well as various reference strains, was isolated according to the methods of zhu . (1993) with modification.et al rapd-pcr reactions were done for each strain, each employing a different primer. the primers used were primer m13 (50-gag ggt ggc ggt tct-30) (huey and hall 1989) lb2 (5'-ggt gac gc-3') (ben 2 omar . 2000), primer a (5' ccg cag cca a 3'),et al primer b (5'aacgcg caa c 3'), and primer c (5' gcggaaata g 3') (chao . 2008).et al rapd was performed using methods and amplification conditions as described by chao .et al (2008). it lwas performed in 20 μ of a mixture containing 10 mm tris-hcl (ph 8.3), 50 mm kcl, 2.5 mm mgcl2, 1.6 μm of primer, 200 μm of each dntp, 0.96 u taq polymerase and 50 ng of genomic dna from the lab isolates. the cycling program consisted of 1 cycle of 94 °c for 2 min; 6 cycles of 94 °c for 30 s, 36 °c for 1 min, and 72 °c for 90 s; 30 cycles of 94 °c for 20 s, 36 °c for 30 s, and 72 °c for 90 s; and finally 1 cycle of 72 °c for 3 min. rep-pcr was performed using the primer gtg5 (5'-gtg gtg gtg gtg gtg-3') and methods as previously described by gevers . (2001). the cycling program consisted ofet al 1 cycle of 95 °c for 7 min; 30 cycles of 95 °c for 1 min, 55 °c for 1 min, and 65 °c for 8 min; and finally 1 cycle of 65 °c for 16 min. pcr products were separated by electrophoresis on 1.8% (w/v) agarose gel using 1 x tbe buffer. the gels were stained in ethidium bromide solution and photographed on a uv transilluminator. the rapd and the rep-pcr fingerprints obtained with both primers were analysed separately as a single data set by calculating the average matrix from the two separate similarity matrices for primer fingerprint sets volume 8, 2014 microbiol indones 49 to obtain a single dendrogram. it was coded in binary form 1 or 0, respectively. statistical analysis software ntsyspc 2.11p (exeter software, setauket, usa) was used for clustering. pcr amplification for 16s rrna. for 16s rrna sequencing, primers 8f (5'-aga gtt tga tca tgg ctc ag-3'; positions 8 to 27 bp) and 15r (5'aaggag gtg atc caa ccg ca-3'; positions 1541 to 1522 bp) were used to amplify the full length of bacterial 16s rrna fragment (cho .et al 2008). each 25 μ pcr mixture contained 10 mm trisl -hcl (ph 8.3), 50 mm kcl, 1.5 mm mgcl2, 200 μm of each dntp, 400 nm of each primer, 1 u of taq polymerase, and 10 ng of the dna template. the pcr conditions were 96 °c for 5 min; 35 cycles consisting of 96 °c for 1 min, 55 °c for 3 min, and 72 °c for 1 min; and 72 °c for 7 min. the pcr products were subjected to gel electrophoresis in 1% agarose gel, followed by ethidium bromide staining. dna sequencing and phylogenetic analysis. the dna sequencing was performed in macrogen, south korea. similarity searches with sequences were performed by online blast analysis in ncbi. for phylogenetic analysis, sequences were aligned by using the clustal x software (thompson .et al 1997) and the phylogenetic tree was constructed by the neighbor-joining method (saitou and nei 1987). results isolation of lactic acid bacteria. mean total of bacteria concentrations enumerated on selective media (mrs) agar were varied ranging from 1.0 x 10 to 9.0 x 7 10 cfu/ml. the latter samples were collected from 8 dadih (buffalo fermented milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous). several colony morphologies could be observed on most of the agar plates. colonies showing different characteristics (colour, shape, etc.) were collected (46 colonies) and plated again on the same agar medium for purification and preliminary identification. rapd and rep-pcr genomic finger printing. a taxonomical approach was utilised in this investigation to identify the predominant lab associated with indonesia fermented foods. pcrbased identification techniques (rapd-pcr and reppcr) and 16s rrna gene sequencing of representative strains showed that the majority of predominant isolates from indonesian tradisional food consisted of lactobacillus pediococcusand . a b c 50 mustopa et al. microbiol indones fig 1 dna profiling of lab using primer a) primer a b) primer (gtg) and c) primer lb on 2% agarose initially tested for5 2 their ability to type a subset of 18 lab. lane 1: 100 bp dna ladder; lane 2-11 lab isolated from dadih; lane 12-17 lab isolated from bekasam. * primer a, the random primer with 50% to 70% g+c content cho . 2008; (gtg) primer, theet al 5were designated by single oligonucleotide primer with repetitive gtg (gevers . 2000); primer lb , rapd-pcr fingerprinting (ben omaret al 2 et al. 2000) fig 2 dendrogram generated after cluster analysis of the digitized (gtg)5-pcr fingerprints of the lab isolated from indonesia traditional fermented foods. the majority of lab classified into four major groups (called g1, gii, giii and gvi). gi contained 12 isolates from dadih and gii contained 18 isolates from tempoyak. the isolates from bekasam clustered into group vi. volume 8, 2014 microbiol indones 51 for the evaluation of the rapd-pcr and rep-pcr fingerprinting technique, five single oligonucleotide primers for rapd (m13, lb2 and primer a, b, c) and single primer for rep pcr (gtg) were initially tested5 for their ability to type a subset of 46 lab. the 46 strains were submitted to rep pcr analysis using (gtg)5 primer. the rep-pcr result yielded seven clusters (i-vii) at a similarity level of 75-88%. the majority of lab could be classified into four major groups (called g1, gii, giii and gvi) (fig 2). group 1 (gi) contained 12 isolates, majority of isolates isolated from dadih and group 2 (gii) contained 18 isolates, majotity of isolates were from tempoyak. the majority of isolates from bekasam clustered into group vi. four groups were composed of only four isolates (giii) or one isolate (giv, gv, gvii)). the rep-pcr result confirmed the rapd-pcr using lb2 primer, m13 primer and primer a, b, c. the rapd using lb2 primer result yielded eight clusters (i-viii) at a similarity level of 82-91%; m13 primer yielded six cluster at similarity level 67-78% and a, b, c primer yielded ten cluster at similarity level 77-85%. majority of isolates emerged from rapd analysis using lb2 and m13 as primer pair could be classified into four major groups (gi, gii, giii and gii) (fig 3, 4). rapd analysis using a, b and c primers was done in separate reactions. for each strain, the three rapd patterns were merged for computations. the majority of strains could be classified into three major groups (fig 5). the bekasam isolates s12, s14 and s34, which were clustered together in group vi using rep-pcr analysis with primer (gtg)5, all clustered together with the type strain using rep-pcrl. plantarum fingerprinting, indicating that on the basis of these genotypic typing methods the strains can be characterised as . the dadih isolatesl. plantarum (dh1, dh2, and ds11 clustered together with) lactobacillus fermentum subgroup ia strain. the tempoyak isolate (u8, u11, az2 and az4) clustered together with type strain. thelactobacillus fermentum rep-pcr indicated there were isolates clustered together into subgroup iia strain. the isolate u10, az6, az8, az9, az10, az11, az23 and az23 clustered together with typelactobacillus plantarum strain. the rep-pcr indicated there were isolates clustered together into subgroup iib strain (fig 2). identification of the lactic acid bacteria. the partial 16s rrna gene sequences (1490 bp) of all the strains were determined. then, the sequences were compared with related bacteria in genbank and sequence similarities were determined using the blast program.the result confirmed that 12 isolates belong to 2 genera ( and ), 2lactobacillus pediococcus species groups, and 4 species: group,l. plantarum l. fermentum, pediococcus pentosaceus, pediococcus acidilactici (table 2). isolates s12, s14, s31 and s34 isolated from bekasam fermented meat showed similarity to they grouped on thel. plantarum. phylogenetic tree together with the corresponding type strain. fig 3 dendrogram generated after cluster analysis of the digitized rapd lb2 primer of the lab isolated from indonesia traditional fermented foods. majority of isolates classified into four major groups (gi, gii, giv and gv). gi contained 10 isolates from tempoyak and gii contained 7 isolates from dadih. 52 mustopa et al. microbiol indones volume 8, 2014 microbiol indones 53 fig 4 dendrogram generated after cluster analysis of the digitized rapd m13 primer of the lab isolated from indonesia traditional fermented foods. majority of isolates classified into four major groups (gi, gii, giii and giv). gi contained 12 isolates from tempoyak and gii contained 6 isolates from dadih. the bekasam strains clustered with the lactobacillus plantarum type strain. the almost complete 16s rrna gene sequence of some of these strains (s12, s14 and s34) was determined and showed high homology (98-99%) to that of the .l. plantarum the complete 16s rrna sequence of dh1 strain was high homology (98%) to . the tempoyakl. fermentum isolate 16s rrna gene sequencing showed that one of these strains (u11) could also be identified as l.plantarum (99 % similarity in 16s rrna gene sequence) the almost complete 16s rrna gene. sequence of strain u10 showed high homology (99%) with type strain.lactobacillus plantarum . fasta analysis ofphylogenetic relationships the 16s rrna gene sequence of strain s34 (a continuous stretch of 1561 bp) revealed that lactobacillus plantarum were the closest relatives (with 99% sequence similarity). the phylogenetic tree of the genus lactobacillus consisted of two separate clades (fig 6). the clade containing strain s12, t8, s34, dh7, s23, ds13, dh1, u11, t3 and s14 also included lactobacillus plantarum lactobacilluswcsf1 and plantarum plantarum lactobacillussubsp stiii, fermentum lactobacillus fermentummtcc 8711 and bcs36, kt3ce27pediococcus pentosaceus , pediococcus acidilactici ul5. the second clade comprised isolate u10 and s31. 54 mustopa et al. microbiol indones fig 5 dendrogram generated after cluster analysis of the digitized rapd a, b, c primer of the lab isolated from indonesia traditional fermented foods. majority of isolates classified into three major groups (gi, giii, and gix). gi contained 10 isolates from tempoyak. table 1 screening of lab isolated from indonesian fermented foods fermented foods sources location total lab isolated code of isolate dadih fermented buffalo milk padang, west sumatera 4 dh solok, west s umatera 16 ds tempoyak durian meat palembang, south sumatera 4 u musi banyuasin, south sumatera 14 az bekasam fermented meat way kanan, lampung, sumatera 6 s tape ketan glutinous rice kuningan, west java 2 t total 120 volume 8, 2014 microbiol indones 55 table 2 the lactic acid bacteria from indonesian tradisional fermented foods no isolat 1 s12 2 s14 3 s23 4 s31 5 s34 6 t3 7 t8 8 dh1 9 u10 10 u11 11 ds13 12 dh7 e spesies identity accession number lactobacillus plantarum 99% jn560843.1 lactobacillus plantarum 97% acgz01000098.1 pediococcus acidilactici 100% fj844982.1 lactobacillus plantarum 98 % acgz01000098.1 lactobacillus plantarum 99% al935263.2 lactobacillus plantarum 99% al935263.2 lactobacillus plantarum 100% al935263.2 lactobacillus fermentum 98% gu213430.1 lactobacillus plantarum 99% al935263.2 lactobacillus fermentum 99% fj462686.1 pediococcus pentosaceus 100% ab481102.1 pediococcus acidilactici 97% ef059987.1 fig 6 phylogenetic tree based on 16s rrna sequence analysis, showing the phylogenetic placement of strains isolated indonesian fermented food. the tree was constructed by the neighbor-joining method. 56 mustopa et al. microbiol indones discussion lactic acid bacteria biodiversity was evaluated from indonesian fermented foods such as dadih (buffalo fermented milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous rice). forty-six of lab were isolated from indonesian fermented foods and were phylogenetically characterized based on their diversity. the greatest majority of these active colonies was originated from dadih (43.48%), tempoyak (39.13%), bekasam (13.04%), and tape (4.35). predominant strains was well characterised based on a genotypic methods such as rapd and rep-pcr as well as 16s rrna gene sequencing of representative strains. identification using 16s rrna showed that the majority of strains were lactobacillus plantarum, l a c t o b a c i l l u s f e r m e n t u m , p e d i o c o c c u sa n d pentosaceus strains. a study conducted by leisner (2001), a totalet al. of 38 strains of lab were selected for comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their whole cell protein patterns. these strains were also examined for their carbohydrate fermentation patterns by use of api 50 ch. isolates belonging to the lactobacillus plantarum group were shown to be the predominant members of the lab flora. in addition, isolates belonging to the l a c t o b a c i l l u s b re v i s l e u c o n o s t o cg r o u p , mesenteroides lactobacillus mali lactobacillus, , fermentum, lactobacillusand an unidentified sp. were also observed. lab isolated from tempoyak was made in indonesia and malaysia. it was expected that strains of lab and other microorganisms varied depending on the place where the product was prepared. the identified two isolates from tempoyak that showedsimilarity to lactobacillus plantarum for u10 and lactobacillus fermentum, l.similarity to plantarum, l. brevis, l. mali, l. fermentum for u11 were also found in tempoyak from malaysia (issa 2000; leisner 2001), while wirawati (2002) andet al. ekowati (1998) had isolated l. plantarum, l. casei, l. corynebacterium l. fermentum, l. caseiand , respectively, from tempoyak in indonesia. leisner et al. lactobacillus, l.(2002) reported the new species of durianis sp., isolated from malaysian tempoyak. other lab presented in tempoyak from malaysia was leuconostoc mesenteroides et al.(leisner 2001) the identified three isolates from dadih originated from west sumatera showed that dh1 had similarity to l. fermentum, dh7 had similarity to pediococcus acidilactici and ds13 had similarity to pediococcus pentosaceus. surono and nurani (2001) reported lactobacillus sp lactococcus leuconostoc, and sp.sp., were dominant in dadih from bukit tinggi and padang p a n j a n g , we s t s u m a t e r a . l e u c o n o s t o c paramesenteroides was the dominant strain of lactic acid bacteria encountered in dadih originated from bukit tinggi (hosono . 1989). among tten lacticet al strains from bukit tinggi-originated dadih previously identified by the api 50ch system (api products, bio merieux, marcy l'etoile, france), (torshizi .et al 2008), 5 strains were re-identified by pcr as lactobacillus plantarum strains is-10506 and is20506; strains is-27526, is-enterococcus faecium 23427, and is-16183 showed potential probiotic properties with good survival rate at low ph value and in the presence of lysozyme, and short lag time in the presence of 0.3% oxgal (surono 2003). acknowledgment this work was financially supported by competitive lipi program 2013. we thank dwianty putri meitasari and muslihatun baroya for technical support. references adimpong db, nielsen ds, sørensen ki, derkx pmf, jespersen l. 2012. genotypic characterization and safety assessment of lactic acid bacteria from indigenous african fermented food products. bmc microbiology. 12:75. ben omar n, ampe f, raimbault m, guyot jp, tailliez p. 2000. molecular diversity of lactic acid bacteria from cassava sour starch. syst appl microbiol. 23:285290, doi: 10.1016/s0723-2020(00)80016-8. berthier e, ehrlich sd. 1999. genetic diversity within lactobacillus sakei lactobacillus curvatusand and design of pcr primers for its detection using randomly amplified polymorphic dna. int j syst bacteriol. 49: 997-1007. chao sh, tomii y, watanabe k, tsai yc. 2008. diversity of lactic acid bacteria in fermented brines used to make stinky tofu. int j food microbiol. 123:134-141. doi:10.1016/j.ijfoodmicro.2007.12.010 chao sh, huang hy, 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[ms thesis]. bogor (id): institut pertanian bogor. yu j, wang wh, menghe blg, jiri mt, wang hm , liu wj , bao qh , lu q , zhang jc , wang f, xu hy, sun ts, zhan hp. 2011. diversity of lactic acid bacteria associated with traditional fermented dairy products in mongolia. j dairy sci. 94 :3229-3241. doi: 10.3168/ jds.2010-3727. zhu h, qu, zhu lh. 1993. isolation of genomic dnas from plants, fungi and bacteria using benzyl chloride. nucl acids res. 21:5279-5280. volume 8, 2014 microbiol indones 57 3.mi-rachel budhisatria.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.11.2.3issn 1978-3477, eissn 2087-8575 vol 11, no 2, june 2017, p 55-61 *corresponding author; phone: +62-81932000311, email: rbudhisatria@gmail.com nutraceuticals and functional foods have become important tools for consumers to manage their health and well-being. many food components, especially food oligosaccharides and polysaccharides (including dietary fiber), have been shown to exhibit prebiotic activity. however, not all dietary carbohydrates are prebiotics. clear criteria of prebiotics have been established. these criteria are 1) resistance to gastric acidity, degree of hydrolysis by mammalian enzymes, and degree of gastrointestinal absorption; 2) degree of fermentation by intestinal microflora, and 3) selective stimulation on the growth and/or activity of the intestinal bacteria that contribute to health and wellin vitro and in vivo prebiotic activities of purified oligosaccharides derived from various local bananas (musa sp.): tanduk, uli, raja sereh, and cavendish banana is a good source of prebiotic, and in indonesia it is consumed as staple food. the aims of this research were to evaluate the activity of purified oligosaccharides (pos) as prebiotic from various different local bananas (musa sp.): tanduk (t), uli (u), raja sereh (rs), and cavendish (c); and to investigate their capacity in promoting the growth of lactobacillus sp., in vivo. in vitro investigation including purification of oligosaccharides from various different local bananas were by 80% ethanol extraction. subsequently, absolute ethanol was reconstituted before precipitation/centrifugation for glucose removal. water was also removed by freeze drying. pos from the four bananas were analyzed by thin layer chromatography (tlc). prebiotic activity of pos was investigated by measurement of prebiotic activity score (pas). in vivo investigation was conducted as followed, balb/c mice were grouped into 6 groups with different prebiotics supplementation: negative control (4 mice, standard feed), positive control (6 mice, 15 mg of inulin ), and samples (5 mice, -1 150 mg of t, u, rs, or c banana g day ) for 40 days. following 40 days after treatment, fecal viable counts of -1 lactobacillus sp. and enterobacteriaceae of balb/c mice was measured (cfu g ) and analysed. pas value revealed a positive correlation between the oligosaccharides from bananas and lactobacillus paracasei, with pas value for t (0.05), rs (0.15), u (0.33), and c (0.77). overall data suggest that fecal viable counts of lactobacillus sp. increased after 25 days administration of u, rs, and c banana when compared to controls. contrastingly, the fecal viable counts of enterobacteriaceae decreased after 40 days administration of u, rs, and c banana compared to the control. different types of local bananas demonstrate diverse prebiotic activities, u and c promote lactobacillus sp. growth and reduce enterobacteriaceae count. pas value of u and c suggest potential prebiotic activity, whereas t and rs do not. key words: banana, lactic acid bacteria, oligosaccharides, pas, prebiotics pisang merupakan sumber prebiotik, dan di indonesia pisang dikonsumsi sebagai makanan pokok. tujuan penelitian ini untuk mengevaluasi aktivitas dari oligosakarida yang sudah dipurifikasi (pos) sebagai prebiotik dari berbagai macam pisang lokal (musa sp.): tanduk (t), uli (u), raja sereh (rs), dan cavendish (c); dan untuk mengetahui kapasitas pisang dalam memicu pertumbuhan lactobacillus sp., in vivo. percobaan in vitro dilakukan sebagai berikut: oligosakarida dari berbagai macam pisang lokal yang berbeda dipurifikasi dengan 80% etanol. selanjutnya, etanol absolut ditambahkan sebelum presipitasi/sentrifugasi untuk menghilangkan glukosa. air dihilangkan dengan metode freeze drying. pos dari empat pisang dianalisis dengan thin layer chromatography (tlc). aktivitas prebiotik pos diukur dari hasil prebiotic activity score (pas). sedangkan percobaan in vivo sebagai berikut: mencit balb/c dibagi menjadi 6 kelompok, dengan pemberian suplemen prebiotik yang berbeda: kontrol negatif (4 mencit, pakan biasa), kontrol positif (6 mencit, 15 mg inulin/g/hari), -1 -1 dan sampel (5 mencit, 150 mg pisang t, u, rs, dan c g hari ) selama 40 hari. setelah 40 hari perlakuan, dihitung -1 jumlah lactobacillus sp. dan enterobacteriaceae dari sampel feses mencit dalam satuan cfu g dan dianalisa. nilai pas menunjukkan korelasi positif antara oligosakarida dari pisang dan lactobacillus paracasei, dengan nilai pas t (0,05), rs (0,15), u (0,33), dan c (0,77). secara keseluruhan data dari perhitungan sampel feses untuk lactobacillus sp. meningkat setelah 25 hari pemberian pisang u, rs, dan c dibandingkan dengan kontrol. sebaliknya, hasil perhitungan sampel feses untuk enterobacteriaceae menurun setelah 40 hari pemberian pisang u, rs, dan c dibandingan dengan kontrol. perbedaan jenis pisang lokal menunjukkan aktivitas prebiotik yang berbeda, u dan c memicu pertumbuhan lactobacillus sp. dan menurunkan perhitungan enterobacteriaceae. berdasarkan nilai pas dari u dan c diduga berpotensi memiliki aktivitas prebiotik, dimana t dan rs tidak. kata kunci: bakteri asam laktat, oligosakarida, pas, pisang, prebiotik -1 -1 g day -1 rachel budhisatria*, rosaria, lucy jap, and tan tjie jan biology department, universitas pelita harapan, jalan m.h thamrin boulevard, tangerang, 15811, indonesia being of host organism (thammarutwasik et al. 2009; gibson et al. 2004). prebiotic was first defined by roberfroid and gibson (1995), indigestible food ingredients that favorably affect the host by selectively promote growth and/or activity of beneficial bacteria in the colon, and thus improving the host health. indigestible oligosaccharides, particularly fructooligosaccharides, are prebiotics. intake of prebiotics can significantly modulate the colonic microbiota by increasing the number and activities of specific bacteria, such as lactobacilli and bifidobacteria and inhibit the growth or activities of pathogenic bacteria such as enterobacteriaceae causing change in the microbiome. prebiotics are known to indirectly promote physiological support of the gastrointestinal tract’s. (gibson and wang 1994; roberfroid 2007). acccording to kahlon and smith (2007) and gibson and wang (1994), banana is a source of prebiotics. indonesia is the leading banana producer in asia, producing more than 50 % of asia’s supply of bananas. indonesia has approximately 200 different kinds of local bananas (prabawati et al. 2009). this variety possesses the potential for development into prebiotics that can contribute to human health. however, studies of prebiotics in local bananas have been limited (kahlon and smith 2007; gibson and wang 1994b). herein, prebiotic activity of purified oligosaccharides from various local bananas: tanduk, uli, raja sereh, and cavendish were studied. tests were conducted to evaluate their capability to promote the growth of lactobacillus paracasei through pas value, which is a method to determine the ability of a given prebiotics to better promote the growth of probiotic microorganism relatively better than to other pathogenic microorganisms (huebner et al. 2007); and in vivo study were carried out on balb/c mice by feeding them with different types of local bananas. prebiotic activities were determined through their ability to promote the growth of lactobacillus sp. by measuring increase of the bacterial count in feces. material and methods samples and subjects. approximately 1.5 2 kg local bananas: tanduk (t), uli (u), raja sereh (rs), and cavendish (c) were used for oligosaccharides extraction, purification, and in vitro analysis. approximately 3 4 kg of each fresh local bananas (t, u, rs, and c), and a total of 30 male, twenty weeks old balb/c mice were used in the present in vivo study. all animal study were performed followed the 56 budhisatria et al. microbiol indones guidelines for the housing of mice in scientific institutions, animal welfare unit, new south wales department of industry (fawcett 2012). oligosaccharides extraction and glucose removal from samples. oligosaccharides extraction was performed according to method previously described by livingston (1990). eight hundred forty gram of each type of bananas (t, u, rs, and c) were blended with 1.1 l of 80% ethanol. blended banana ethanol mixtures were filtered with a cotton filter to remove the coarse particles. all collected filtrate were then heated at 70 °c for 15 min to inactivate potential oligosaccharide degrading enzymes, such as invertase. the resulting thick banana pulps were blended twice for about 5 min with 100 ml of distilled water and then agitated at 200 rpm at room temperature for 1 h, to homogenized. all extracts were then centrifuge at 900 g, for 20 min at 10 °c. supernatant were then collected and evaporated to remove residual ethanol. the thick extracts were mixed with absolute ethanol (1:1 v/v) and stored at -2 °c for 24 h. the extracts were further centrifuged at 900 g for 15 min at room temperature. glucose fraction in the supernatant was discarded. absolute ethanol was re-added into the remaining pellet, mixed and centrifuged at 900 g for 15 min at room temperature and the supernatant were decanted as above. the step was repeated for thirty times until glucose was not detected anymore through thin layer chromatography (tlc) with n-butanol/1-propanol/acetic acid/water (3:1:1:1) as eluent. after glucose removal, a thick milky suspension containing pure oligosaccharides was then subjected to freeze drying. dried oligosaccharide samples were then weighed. pure oligosaccharides were verified by tlc with 90% formic acid as eluent. prebiotic activity score (pas) from purified banana oligosaccharides. pas was performed according to the procedure as described by huebner et al. (2007). pas is defined as follow: whereby, p is defined as probiotics (lactobacillus paracasei/lp), e is defined as enteric bacteria (e. coli/ ec), p is total plate count of lp culture, and ec culture 24 in prebiotic added medium after 24 h incubation, respectively, p is total plate count of lp culture, and ec 0 culture in prebiotic added medium before 24 hours incubation, respectively, g is total plate count of lp 24 culture, and ec culture in glucose added medium after 24 h incubation, respectively, g is total plate count of 0 -pas = p p24 0 g g24 0 p p24 0 g g24 0 lp culture, and ec culture in glucose added medium before 24 h incubation, respectively. in the formula above, the probiotic lactobacillus paracasei (lp) was cultured in 3 ml modified de manrogosa sharpe broth without carbon source, agitated at 100 rpm at 37 °c, microaerophilic for 24 h. no prebiotic was added to negative control. 1% of inulin was added to positive control. 1% of purified oligosaccharide from banana t, u, rs, or c was added as tested samples. escherichia coli (ec) was cultured in 3 ml m9 minimal medium broth without carbon source, agitated at 100 rpm at 37 °c for 24 h to serve as basal media colony plate count. no prebiotic was added to negative control. 1 % of inulin was added to positive control. 1% of purified oligosaccharide from banana t, u, rs, or c was added as tested samples. additional control plate containing 1 % glucose on basal media was also prepared. the above pas plate count was performed as follow. a 50 μl of serial dilluted samples were spread on mrs agar for l. paracasei count, and on macconkey agar for e. coli count. serial dilution was performed to obtain countable cfu plate count (30 -1 300 cfu plate ). all plates were incubated at 37 °c for 24 h under microaerophilic condition for l. paracasei count and aerobic condition for e. coli count. colony -1 count in each plate was expressed in log cfu ml . quantitative analysis of lactobacillus sp. and enterobacteriaceae in mice feces treated with bananas. balb/c mice were grouped into negative control (4 mice, standard feed), positive control (6 -1 -1 mice, 15 mg of inulin g day ), and samples (5 mice, -1 -1 150 mg of t, u, rs, or c banana g day ). all supplemented prebiotics were measured in dry weight. fecal samples were collected from each mice at day 0, 17, 20, 25, and 40. for bacteria count, samples were diluted in physiological salt solution (1:9 v/v) (hartemink and rombouts 1999), and then 50 μl of each diluted feces was spread on rogosa agar for lactobacillus sp. and macconkey agar for enterobacteriaceae, at 37 °c for 24 48 h (adami and cavazzoni 1996). colony count in each plate (cfu -1 -1 ml ) was converted into log cfu g sample. the -1 difference percentage log cfu g relative to day 0 (d0) was calculated. results purified oligosaccharides (pos) from banana. the purification of banana from glucose after serial ethanol suspension and centrifugation produce excellent result (fig 1). the inclusion of glucose in oligosaccharide samples will interfere pas value because glucose will be used as carbon source by both bacteria, l. paracasei and e. coli (date et al. 2014; kunova 2011). the appearance of extracted pos from each bananas were different in color and texture (fig 2). in vitro analysis prebiotic activity. to ensure glucose was completely eliminated for the tlc analyses, oligosaccharides purification was conducted. each banana pos was further used to determine pas -1 value, as shown in (tabel 1) in log cfu ml . cavendish (c) banana display highest pas value of 0.77, followed by uli (u) banana sample with pas value of 0.33, raja sereh (rs) with pas value of 0.15 and lastly tanduk (t) with pas value of 0.15. the pas value of uli (u) is comparable to inulin pas value (0.33). negative control shows a negative pas value (-0.11). effect of feeding bananas on lactobacillus sp. growth in feces. balb/c mice were used to study the effect of banana feeding on lactobacillus sp. growth in gastrointestinal tract. treated mice were given t, u, -1 -1 rs, or c at 150 mg of banana g day for 40 d. as positive control inulin was given to animal feed at 15 -1 -1 mg of inulin g day for 40 d. feces of mice were collected as described in the methods. uli (u), raja sereh (rs), and cavendish (c) banana fed on mice result in a higher of lactobacillus sp. count than negative control. in contrast inulin gave an inconsistant result throughout time fed. tanduk (t) banana did not support the growth of lactobacillus sp. but enterobacteriaceae. other nutrient factors might play a part in promoting lactobacillus sp. growth. fig 4 showed that t and rs banana promote enterobacteriaceae growth, whereas u and c, and inulin as positive control did not promote growth. result displayed on day 40 reveal more disperse result on the effect of -1 different banana sample to the percentage log cfu g relative to d0. pas value of u and c indicates that these bananas promoted the growth of lactobacillus sp. and reduced the growth of enterobacteriaceae during in vivo study. discussion purified oligosaccharides from each different banana yield different mass after purification. according to livingston (1990), centrifugation and precipitation process at 900 g should successfully eliminate monosaccharide (glucose) and other simple volume 11, 2017 microbiol indones 57 microbiol indones58 budhisatria et al. a c b d a b c d e f fig 1 tlc result showing glucose elimination after repeated extraction. glucose detection was used n butanol: propanol: acetic acid: water with ratio 3: 1: 1: 1 as eluent. then, was dried and added with visualization solution and was dried again in oven until spots appeared. a: inulin standard (0,02 mg); b: glucose standard (0.02 mg); c: extract from t (10 µl); d: extract from u (10 µl); e: extract from rs (10 µl); and f: extract from c (10 µl). fig 2 purified oligosaccharides (pos) from banana samples. purifified oligosaccharides after freeze drying a: oligosaccharides from t; b: oligosaccharides from u, c: oligosaccharides from rs, and d: oligosaccharidess from c. fig 3 tlc result showing oligosaccharides detection after glucose elimination. oligosaccharides detection from each various local bananas. oligosaccharides detection was used 90 % of formic acid as eluent. then, was dried and added with visualization solution and was dried again in oven until spots appeared. a: inulin standard (0,02 mg); b: glucose standard (0,02 mg); c: t oligosaccharides (20 µl); d: u oligosaccharides (20 µl); e: rs oligosaccharides (20 µl); f: c oligosaccharides (20 µl); g: maltose standard (0.02 mg); and h: raffinose standard (0.02 mg). volume 11, 2017 microbiol indones 59 v t0 t24 t0 t24 t0 t24 t0 t24 1 inulin 9.59 5058.25 22.49 8669.62 5.98 8.70 6.35 8.94 2 negative control 13.00 128.82 26.18 831.76 6.11 7.11 6.42 7.92 3 glucose 17.30 1527.57 14.45 3767.04 6.24 8.18 6.16 8.58 4 t 89.81 3950.63 58.43 4786.30 6.95 8.60 6.77 8.68 5 u 28.71 3908.41 38.90 2766.94 6.46 8.59 6.59 8.44 6 rs 78.70 4813.93 57.28 4004.05 6.90 8.68 6.76 8.60 7 c 30.48 3265.88 35.48 164.44 6.48 8.51 6.55 7.22 lp ec 2.72 2.59 1.00 1.50 1.95 2.42 1.64 1.91 2.13 1.85 1.79 1.84 2.03 0.67 lp ec 1.40 1.07 0.51 0.62 0.84 0.79 1.10 0.77 0.92 0.76 1.04 0.28 0.33 -0.11 0.05 0.33 0.15 0.77 rgr iv samplesno pas value iii i ii -1 ∆ log cfu ml log -1 cfu ml-1 5cfu ml (×10 ) eclpeclp table 1 average results of pas value from t, u, rs, and c fig 4 progression of log cfu percentage of lactobacillus sp. per gram of fresh feces at day 20 (d20), day 25 (d25), and day 40 (d40) relative to day 0 (d0) in balb/c mice fed by various local bananas. : negative control, : inulin, : tanduk, : uli, : raja sereh, and : cavendish. fig 5 progression of log cfu percentage of enterobacteriaceae per gram of fresh feces at day 20 (d20), day 25 (d25), and day 40 (d40) relative to day 0 (d0) in balb/c mice fed by various local bananas. : negative control, : inulin, : tanduk, : uli, : raja sereh, and : cavendish. d0 d20 d25 d40 20 15 10 5 0 -5 % p ro g re ss io n l o g -1 c f u g o f l a ct o b a ci ll u s sp . fr o m d ay 0 day 15 10 5 0 -5 -10 -1 % p ro g re ss io n l o g c f u g o f e n te ro b a ct er ia ce a e sp . fr o m d ay 0 d0 d25 d40d20 day 5 this table shows results average pas value from inulin, negative control, t, u, rs, and c. columns i are cfu ml (x10 ); -1 columns ii are log from columns i; columns iii are are the difference (or δlog cfu ml ) from column ii, namely t -t for lp 24 0 -1 and ec, respectively.columns iv are relative growth ratio (rgr) obtained from column iii, namely δlog cfu ml from -1 inulin, t, u, rs, and c for lp and ec, which divided by glucose δlog cfu ml for lp and ec, respectively. the last column v is pas values which obtained from rgr (columns iv) lp-ec. -1 microbiol indones60 budhisatria et al. microbiota of the feces reflects that of the distal colon. many diseases, such as colon cancer, diverticular disease, ulcerative colitis, and inflammatory bowel disease originates from this organ (macfarlane et al. 1992). modulation in the microbiota of distal colon, such as higher count of lactobacillus sp. and a lower count of enterobacteriaceae, was able to prevent the onset of those diseases (roberfroid et al. 2010). cfu of lactobacillus sp. and enterobacteriaceae in the feces were quantified in selective media for minimizing the growth of contaminant microorganisms. lactobacillus sp. was quantified in rogosa medium which has been proven to be selective against lactobacillus sp. according to gram staining, catalase and endospora assays. meanwhile, macconkey medium was used to quantify enterobacteriaceae and its selectivity against this group of bacteria has been tested with gram staining method. this study was done to prove the possible positive health effect of fresh bananas on gastrointestinal tract by measuring the increasing number of lactobacillus in feces in comparison to enterobacteriaceae. in feces of the treated balb/c mice fed with the same bananas, the results showed that all bananas but t banana were able to promote lactobacillus sp. growth, while t and rs, but not u or c, were able to promote growth enterobacteriaceae. the contradicting result in vivo and in vitro could arise from different sample treatment. glucose is not omitted in in vivo assay which could result in glucose interference towards colon bacteria yield. it is recommended that glucose is removed from all samples before administration to animal model. however, attention of the limitation on technique, that result in significant sample loss, used for purification of pos from banana sample used in this paper need to be consider. acknowledgment the publication of this paper was supported by grant from lembaga penelitian dan pengabdian kepada masyarakat universitas pelita harapan (lppm uph). we are greateful for the collaboration with laboratorium biologi dasar (b202) and laboratorium biologi lanjutan (b407) universitas pelita harapan for providing us laboratory instruments. references adami a and cavazzoni v. 1996. a standard procedure for assessing the faecal microflora of swine. annali di glucose that have low degree of polymerization range (dp 3 5) from the extract which. (roberfroid 2007; slavin 2013). thus, considerable amount of oligosaccharides prebiotics were expected to be lost during purification. this is accountable to the minimum pas value measurement. banana pos from t, u, rs, and c were detected on tlc by using 90% of formic acid as eluent (fig 3) which possesses very high polarity or strong acid. this suggest that pos with high dp might be undetected on the tlc performed (holmes and o’brien 1979; zhang et al. 2007). eluent of 100% acetic acid, and another of 1-butanol: formic acid: aquades, 4: 8: 1, did not produce good result. appearances of purified pos from each banana were different. rs purified pos obtained as white color granule or powder. similar appearance was described by oliveira et al (2011) with agglomerate texture and difficult to ravel due to the strong glycosidic bond between the polisaccharides. t, u, and c purified pos appeared as a mixture of white and dark color granule and powder (oliveira et al. 2011). the positive values of pas for inulin, t, u, rs, and c, signifies that prebiotics or purified pos bananas are able to promote the growth of l. paracasei relatively better than e. coli. according to rubel et al. (2014), the rgr value >1 means that tested samples (t, u, rs, and c), prebiotics/oligosaccharides addition as growth media could support the growth of probiotics such as lactobacillus sp than using glucose as growth media (rubel et al. 2014). data reflected in table 1 showed average results of pas from in vitro analysis prebiotic activity. rgr value of prebiotic inulin and purified pos of u and c are >1, while t and rs are < 1 which indicates that t and rs could not promote l. paracasei growth well. as many types of oligosaccharides are present in fruits and plants, t and rs may have different oligosaccharide content than u and c (roberfroid 2007). other species of lactobacillus should be studied to examine its capability to metabolize the oligoaccharides. c has much higher pas value of 0.77 compare to inulin as control positive 0.33 (tabel 1) which calls for further investigation. the pas values showed that only two purified banana oligosaccharides, u and c, were capable of promoting the growth of l. paracasei with rgr >1, while the other two purified banana oligosaccharides t and rs were inconclusive, rgr <1. in vivo experiment was analyzed by using balb/c mice to study the effect of banana feeding on lactobacillus sp. growth in gastrointestinal tract. the volume 11, 2017 microbiol indones 61 macfarlane gt, gibson gr, cummings, jh. 1992. comparison of fermentation reactions in different regions of the human colon. j appl bacteriol. 72(1):5764. doi:10.1111/j.1365-2672.1992.tb04882.x. oliveira ajb, goncalves rac, chierrito tpc, santos mm, souza lm, gorin paj, sassaki g, lacomini m. 2011. structure and degree of polymerization of fructooligosaccharides present in roots and leaves of stevia rebaudiana (bert.) bertoni. food chem. 129(2): 305-311. doi:10.1016/j.foodchem.2011.04.057. prabawati s, suyanti, setyabudi, d. a. 2009. teknologi pasca panen dan pengolahan buah pisang. bogor: balai besar penelitian dan pengembangan pasca panen pertanian. roberfroid m. 2007. prebiotics: the concept revisited. j nutr. 137(3 suppl 2): 830s-837s. roberfroid m, gibson gr, hoyles l, mccartney al, rastall r, rowland i, wolvers d, watzl b, szajewska h, stahl b, guarner f, respondek f, whelan k, coxam v, davicco mj, léotoing l, wittrant y, delzenne nm, cani pd, neyrinck am, meheust a. 2010. prebiotic effects: metabolic and health benefits. br j nutr. 104 (suppl. 2), s1e63. rubel ia, pérez ee, genovese db, manrique gd. 2014. in vitro prebiotic activity of inulin-rich carbohydrates extracted from jerusalem artichoke (helianthus tuberosus l.) tubers at different storage times by lactobacillus paracasei. food res int. 62:59-65. doi:10.1016/j.foodres.2014.02.024. slavin j. 2013. fiber and prebiotics: mechanisms and health benefits. j nutr. 5(4):1417-1435. doi:10.3 390/nu5041417. thammarutwasik p, hongpattarakere t, chantachum s, kongkarn k, itharat a, reanmongkol w, tewtrakul s, ooraikul b. 2009. prebiotics-a review. songklanakarin j sci technol. 31(4):401-408. zhang z, xie j, zhang f, linhardt, r. j. 2007. thin layer chromatography for the analysis of glycosaminoglycan oligosaccharides. anal biochem. 371(1):118-120. doi:10.1016/j.ab.2007.07.003. microbiologia e enzimologia. 46:1-9. archibold hk. 1940. fructosans in the monocotyledons a review. new phytol. 39(2):185-219. doi:10.1111/j.14698137.1940.tb07132.x. date y, nakanishi y, fukuda s, nuijima y, kato t, umehara m, ohno h, kikuchi, j. 2014. in vitro evaluation method for screening of candidate prebiotic foods. food chem. 152: 251-260. doi:10.1016/j.foodchem.2013.11.126. fawcett a. 2012. guidilines for the housing of mice in scientific instutions. retrieved from: http://www.animalethics.org.au/__data/assets/pdf_file/0004/249898/ guideline-22-mouse-housing.pdf (4 november 2016). gibson gr and roberfroid mb. 1995. dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. j nutr. 125(6):1401-1412. gibson gr and wang x. 1994. regulatory effects of bifidobacteria on the growth of other colonic bacteria. j appl bacteriol. 77:412-420. doi:10.1111/j.13652672.1994.tb03443.x. gibson gr, probert hm, van loo jae, roberfroid mb. 2004. dietary modulation of the human colonic microbiota: updating the concept of prebiotics. nutr res rev. 17(2): 259-275. doi:10.1079/nrr200479. hartemink r and rombouts fm. 1999. comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples. j microbiol methods. 36(3):181-192. doi:10.1016/s0167-7012(99) 00031-7. holmes ew and o’brien js. 1979. separation of glycoprotein-derived oligosaccharides by thin-layer chromatography. anal biochem. 93:167-170. doi:10.1016/s0003-2697(79)80131-1. huebner j, wehling rl and hutkins rw. 2007. functional activity of commercial prebiotics. int dairy j. 17(7):770-775. doi:10.1016/j.idairyj.2006.10.006 . kahlon ts and smith ge. 2007. in vitro binding of bile acids by bananas, peaches, pineapple, grapes, pears, apricots and nectarines. food chem. 101(3):1046-1051. doi:10.1016/j.foodchem.2006.02.059. kunova g, rada v, lisova i, rockova s, vlkova, e. 2011. in vitro fermentability of prebiotic oligosaccharides by lactobacilli. czech j food sci. 29:s49-s54. livingston dp. 1990. fructan precipitation from a water/ethanol extract of oats and barley. plant physiol. 92(3):767-769. doi:10.1104/pp.92.3.767. 1: 55 2: 56 3: 57 4: 58 5: 59 6: 60 7: 61 05 lg.cdr women are three times more likely to get urinary tract infection (uti) than men, due to women's shorter urethra, sexual activity, pregnancy, and hormonal changes that occur very quickly (mitchell et al. 2002). hormonal changes of both estrogen and progesterone during the menstrual cycle affect urodynamic. estrogen deficiency can increase the resistance of the urethra as it can cause an increase in the threshold of the bladder or can boost the sensitivity of the receptors -adenoreseptor on the urethral muscle (robinson et al. 2013). estrogen deficiency also vol.10, no.1, march 2016, p 30-37 doi: 10.5454/mi.10.1.5 comparison of microbial pattern causing urinary tract infection in female outand hospitalized patients in jakarta 1 2 3 yeva rosana *, dwiana ocviyanti , and syadza rhizky putri akhmad 1 department of microbiology, faculty of medicine, universitas indonesia, jalan pegangsaan timur 16, jakarta 10320, indonesia; 2 department of obstetrics and gynecology, faculty of medicine, universitas indonesia jalan salemba raya 6, jakarta 10430, indonesia; 3 faculty of medicine, universitas indonesia, jalan salemba raya 6, jakarta pusat 10430, indonesia urinary tract infection (uti) is an infection in any part of the urinary system. women are 3 times more likely to have uti than men. the uti accounts for 15% infection cases in outpatients and 24% cases in hospitalized patients. although the most common cause of uti is certain bacteria, but it was not easy to choose the appropriate antimicrobial therapy. strategy for choosing empiric antimicrobial treatments for uti in female outand hospitalized patients should be based on the pattern of the causative organisms. the aim of this study was to understand the microbial pattern causing uti in female outand hospitalized patients in jakarta. the uti -1 causative microorganisms were obtained from urine culture containing 100,000 cfu ml . twenty nine microorganisms were found as the causative agents of uti in 317 pregnant women who came to six community health centres (puskesmas) in jakarta: makassar; pulogadung, cakung, pasar rebo, duren sawit and kramat jati for antenatal care. twenty nine microorganisms were isolated from 114 urine samples of female hospitalized patients who were diagnosed of uti. the samples were obtained from the microbiology laboratory clinic of fkui-rscm. the most common microorganisms causing uti in female outand hospitalized patients were gram negative bacteria. in female outpatients, klebsiella sp was the most common causative bacteria (31%), followed by escherichia coli (24.1%). in female hospitalized patients, escherichia coli was the most common causative bacteria (30%), followed by candida sp (24.1%) and klebsiella pneumonia (6.8%). there was more variation in the pattern of uti causative organisms in hospitalized female patients in comparison to that of the outpatients. candida sp. was only found in hospitalized uti patients but not in outpatients. key words: causative agent, female, inpatient, outpatient, uti infeksi saluran kemih (isk) adalah infeksi yang terjadi pada berbagai bagian saluran kemih. wanita memiliki risiko 3 kali lebih besar mengalami isk dibandingkan pria. kejadian isk mencapai 15% pada pasien rawat jalan dan 24% pada pasien rawat inap di rumah sakit. walaupun etiologi isk yang paling umum adalah bakteri tertentu, tetapi tidak mudah untuk memilih antimikroba yang tepat untuk terapi. strategi untuk memilih antimikroba empiris untuk isk pada pasien wanita rawat jalan dan rawat inap harus didasarkan pada pola mikroorganisme dari etiologi. tujuan penelitian ini adalah untuk melihat pola mikroorganisme penyebab isk pada pasien wanita rawat jalan dan rawat inap di jakarta. pola mikrooganisme penyebab isk didapatkan melalui -1 metode kultur urin, dengan jumlah koloni 100.000 cfu ml urin. sejumlah 29 bakteri didapatkan sebagai mikroorganisme penyebab isk pada 317 wanita hamil yang melakukan pemeriksaan kehamilan di 6 puskesmas di jakarta: puskesmas makassar, pulogadung, cakung, pasar rebo, duren sawit, dan kramat jati. sebanyak 29 bakteri didapatkan dari 114 sampel urin wanita rawat inap yang didiagnosis sebagai isk. sampel urin diperiksakan di laboratorium mikrobiologi klinik fkui-rscm. pola mikroorganisme penyebab terbanyak isk pada rawat jalan dan rawat inap adalah mikroorganisme gram negatif. pada rawat jalan klebsiella pneumoniae sebagai penyebab terbanyak isk (31%), diikuti escherichia coli (24,1%). pada rawat inap escherichia coli menjadi penyebab terbanyak isk (30%), diikuti oleh candida sp (24,1%) dan klebsiella pneumoniae (6,8%). pola mikroorganisme isk pada rawat inap lebih bervariasi dibandingkan rawat jalan. candida sp hanya ditemukan pada isk rawat inap, tetapi tidak pada rawat jalan. kata kunci: isk, penyebab infeksi, rawat jalan, rawat inap, wanita microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author; phone: +62-21-3160492; email: yeva.rosana@ui.ac.id disturbed the roles of the hormone in stimulating the proliferation of lactobacillus in the vaginal epithelium, reducing ph and avoiding vaginal colonization by enterobacteriaceae, which are the main pathogens of the urinary tract (raz r 2011). these conditions play an important role in the development of bacteriuria. around 35% of women aged between 20-50 years had experienced a uti in their lifetime (samirah et al. 2006). most women aged about 24 years old (reproductive age) have experienced a uti at least once in their life time. the high incidence of uti during the reproductive age might be related to the high sexual activity. therefore, the incidence of uti in sexually active women are generally higher than non sexually active women. other risk factors for uti include pregnancy, diabetes, obstruction of the urinary tract, older men with enlarged prostate, and other factors that disturb the physiology of the urinary tract (mitchell et al. 2002). uti acquired in hospitals are often associated with long term catheter use. data from research conducted on healthcare-associated infections (hais) in indonesia reported that the rate of hospital-acquired uti was between 0.9 to 3.5% (duerink et al. 2006). common bacteria causing urinary tract infection (uti) include escherichia coli, klebsiella p n e u m o n i a e , p ro t e u s s p , e n t e ro b a c t e r s p , streptococcus sp, and staphylococcus saprophyticus. in asymptomatic urinary tract infection, the number of significant bacteria to support uti diagnosis is -1 100,000 ml urine. appropriate therapy should be given as early as possible to prevent progression of the infection, such as development of pyelonephritis due to ascending spread or sepsis due to hematogenous spread. rational antimicrobial treatment should be based on the pattern of the causative microbes. selection of antimicrobial therapy in outpatient will be different from hospitalized patients, because the pattern of the causative microbes is also different. increased risk factors and exposure to antibiotics in hospitalized patients, can lead to more varied pattern of the causative microbes, that it is necessary to understand the difference of the causative microbial pattern in female outand hospitalized patient. materials and methods the pattern of uti causative microbes in female outpatients. urine was obtained in 2015 from female outpatients in six community health centers in jakarta, including makassar, pulogadung, cakung, pasar rebo, duren sawit, and kramat jati community health centers. the subjects were pregnant women who get antenatal care in the six community health centers. the urine was cultured at the clinical microbiology laboratory, faculty of medicine, universitas indonesia. urine samples used in this study were midstream urine from women with no risk factor. before culturing, urine sample was mixed well. urine was taken using sterile calibrated loop with 0.001 ml size and streaked on blood and mcconkey agar plates, followed by incubation at 35-37 °c, for 18-24 h. only urine samples containing microbes at ³100,000 cfu -1 ml urine were used for bacterial identification. the pattern of uti causative microbes in female hospitalized patients. the information of causative microbial pattern was obtained as secondary data from the clinical microbiology laboratory, faculty of medicine, universitas indonesia. culture of midstream urine from female patients hospitalized in several hospitals in jakarta were analyzed. only urine -1 samples containing microbes at ³100,000 cfu ml of urine used for bacterial identification. identification of uropathogen. identification of the uti-causing microorganisms from both clinical specimens were done using vitek 2 compact system® (biomérieux). colonies of a pure culture were taken and placed in a test tube containing 3.0 ml sterile saline (0.45%-0.50% nacl, ph 4.5-7.0) to make a suspension. suspension turbidity was adjusted to 0.5 8 -1 mcfarland, which was comparable to 1.5x10 cfu ml . test tube containing the microorganism suspension was placed into a special rack (cassette) and identification card was placed in the neighboring slot while inserting the transfer tube into the corresponding suspension tube. rack or cassette containing test tubes and the identification card was inserted into the machine. the filled cassette was placed manually into a vacuum chamber station. after the vacuum was applied and air was re-introduced into the station, the microorganism suspension was forced through the transfer tube into micro-channels that fill all the test wells. the cassette was incubated at 35.5±1.0 °c. each card was removed from the carousel incubator once every 15 min, transported to the optical system for reaction readings, and then returned to the incubator until the next reading. the identification results will appear on the monitor screen after 3-7 h. the databases of the vitek 2 identification machine were constructed with large strain sets of well-characterized volume 10, 2016 microbiol indones 31 microorganisms tested under various culture conditions. each of the composite values was compared to the others to determine if the data are sufficiently unique or close to one or more of the other database taxa. if a unique identification pattern was not recognized, a list of possible organisms was given, or the strain was determined to be outside the scope of the database. an unknown biopattern was compared to the database of reactions for each taxon, and a numerical probability calculation was performed. various qualitative levels of identification are assigned based on the numerical probability calculation. bacterial identification system used two types of vitek card, gn card for identification of gramnegative bacilli and gp card for identification of grampositive (primarily cocci). the yst card was used for identification of yeast-like microorganisms. the data in this study was analyzed by spss 20.0 for windows, using chi-square method. results the pattern of uti causative microbes in female outpatients. there were 27 of 317 urine samples from the female outpatients contained bacteria -1 at ³100,000 cfu ml . two of the samples indicated double infection (the presence of two bacteria in an i n d i v i d u a l ) . t h e r e f o r e , i t w a s f o u n d 2 9 microorganisms pattern in this study (table 1). gram-negative bacteria was the most common cause of uti found in female outpatient (found in 22 cases, (75.9%), while only 7 cases (24.1%) was caused by gram-positive bacteria found. klebsiella pneumoniae, a gram-negative bacteria, was found as the most common cause of uti in female outpatient in this study (found in 31% cases), while escherichia coli, which is also gram-negative, was found as the second most common cause of uti in female outpatients (24.1%). distribution pattern of the causative microbes by age group can be seen in table 2. in the age group 16-20 years, four species of bacteria were found, klebsiella pneumoniae, enterococcus cloacae, staphylococcus hemolyticus, and alcaligenes faecalis. however, these four microbes were found at equal frequency. in the age groups 21-30 and 31-40 years, escherichia coli was the most common uti-causing microbe. distribution pattern of the causative microbes by trimester of pregnancy can be seen in table 3. in the first trimester of pregnancy, three species of bacteria were found, acinetobacter baumannii, streptococcus agalactiae, and streptococcus viridans. there was no specific pattern in the frequency of occurence of the three uti-causing microorganisms in the first semester of pregnancy. in the second trimester, the most commonly found causative agent was escherichia coli, whereas in the third trimester of pregnancy, escherichia coli and klebsiella pneumoniae were the two most common causative bacteria found. the pattern of uti causative microbes in female hospitalized patients. of the 114 urine samples from female hospitalized patients, 29 of them showed significant positive culture results (containing -1 microbes at ³100,000 cfu ml of urine). the most common microbial cause of uti in hospitalized females were gram-negative bacteria (found in 55.2% cases), followed by fungi (24.1% cases), and grampositive bacteria (20.6% cases). among gramnegative bacteria, escherichia coli was the most common cause of infection (56.3% cases). however, all gram-positive bacteria were found at equal frequency. candida sp. was the only fungus found as a causative agent. comparison of the uti causative microbes in f e m a l e o u t a n d h o s p i t a l i z e d p a t i e n t s . microorganisms pattern of gram-negative as the most common cause of uti in female outpatient and hospitalized showed the same results of chi-square analysis (table 5). there were 75.9% uti in outpatients and 55.2% in hospitalized patients caused by gram-negative bacteria. in this study, klebsiella pneumoniae, a gramnegative bacteria, was found as the most common cause of uti in outpatients (31%) and the second most common cause in hospitalized patients (6.8%). escherichia coli is the most common uti causative bacteria in women. in this study, however, e.coli was found as the most common cause of uti in hospitalized patients (31%), while in outpatient e. coli was the second most common cause (24.1%). gram-positive microorganisms caused 24.1% uti in outpatients and the 48.3% in hospitalized patients. candida caused 24.1% (7/29) uti in inpatient, however, no fungus was found in outpatient (table 7). discussion the pattern of uti causative microbes in female outpatients. gram-negative bacteria was the most common cause of uti in female outpatients in this study. the enterobacteriaceae found as the main pathogen in the urinary tract might have come from the 32 rosana et al. microbiol indones table 1 the pattern of uti-causing microbes found in female outpatients gram bacteria number of infected patients percentage negative klebsiella pneumoniae 31% escherichia coli 24.1% alcaligenes faecalis ssp faecalis 3.4% enterobacter cloacae ssp cloacae 3.4% pseudomonas stutzeri 3.4% stenotrophomonas maltophilia 3.4% positive streptococcus viridans, alpha-hem 6.9% leuconostoc mesenteroides ss. cremoris 3.4% staphylococcus aureus ss. aureus 3.4% acinetobacter baumannii 6.9% streptococcus agalactiae 3.4% streptococcus sanguinis 3.4% total 100% staphylococcus haemolyticus 9 7 1 1 1 1 2 1 1 2 1 1 29 1 3.4% table 2 uti-causing microbes in female outpatient distributed by age age (in years) microbes 16-20 (n=4) 25% (1) 25% (1) 25% (1) 21-30 (n=10) 20% (2) 31-40 (n=6) 50% (3) no data (n=9) 66.7% (6) 25% (1) klebsiella pneumonia enterococcus cloacae alicaligenes faecalis escherichia coli escherichia coli klebsiella pneumonia staphylococcus hemolyticus percentage (number of infected patients) table 3 uti-causing microbes in female outpatients distributed by trimester of pregnancy trimester pregnancy microbes i (n=3) 33.3% (1) 33.3% (1) 50% (3) iii (n=12) 25% (3) 25% (3) no data (n=8) 75% (6) 33.3% (1) acinetobacter baumanii streptococcus agalacte escherichia coli escherichia coli klebsiella pneumonia klebsiella pneumonia streptococcus viridan percentage (number of infected patients) ii (n=6) volume 10, 2016 microbiol indones 33 34 rosana et al. microbiol indones table 5 comparison of gram-negative and non-gram-negative caused uti in female outand hospitalized patients pgram negative 0.09722 16 38 outpatients hospitalized patients total bacteria pattern table 4 the composition of uti-causing microorganisms found in female hospitalized patients gram bacteria number of infected patients percentage negative escherichia coli 31% klebsiella pneumonia 6.8% enterococcus faecalis 3.4% pseudomonas aeruginosa 3.4% stenotrophomonas maltophilia 3.4% proteus mirabilis 3.4% positive aerococcus urinae 3.4% leuconostoc mesentroides ss cremoris 3.4% actinomyces odontolyticus 3.4% alcaligenes faecalis (odorans) 3.4% staphylococcus haemolyticus 3.4% streptococcus sanguinis 3.4% streptococcus agalactiae 9 2 1 1 1 1 1 1 1 1 1 1 1 3.4% fungal candida sp. 24.1% total 100% 7 29 75.9 55.2 65.5 7 13 20 24.1 44.8 34.5 non gram negative n % n % intestinal microbiota. instead of e. coli, this study found that klebsiella pneumoniae, which is also a member of enterobacteriaceae, to be the most common cause of uti in female outpatients. this result was similar to a study reported by rajaratnam et al. in india (2013), showing that the most common bacteria causing uti in female outpatients was klebsiella pneumoniae (50%) and e. coli (14.2%). klebsiella pneumoniae is a gram negative bacteria, one of normal microorganisms in human's intestine. morphology of this bacteria is rod-shape, non-motile, and lactose fermenting. k. pneumoniae is a facultative anaerobe, therefore it is able to grow either with or without free oxygene. capsule as a virulence factor of this bacteria that act as physical barrier to overcome the host's immune response. this capsule surround this bacteria also protects the cell from desiccation. although found as normal microorganism, k. pneumoniae can progress into bacterial infections, including urinary tract infections. in the age groups 21-30 and 31-40 years, the most frequently found uti-causing microorganism in female outpatient in this study was escherichia coli. this bacteria is an enteric bacteria residing in the periurethral introitus of the vagina and can migrate up to the bladder through urethra. the lack of hygiene in the female reproductive tract and sexual activity can lead to the migration. e.coli infection in women can also come from the rectum, this is due to the close proximity between the rectum and urethral meatus. escherichia coli was also the most common uti causative microbe in the second and third trimesters of volume 10, 2016 microbiol indones 35 pregnancy. although uti in pregnant women often asymptomatic but it can develop to pyelonephritis. risk of life-threatening illness such as perinatal and neonatal morbidity can increase because of pyelonephritis. therefore, all pregnant women should be screened for bacteriuria and subsequently treated with appropriate antibiotics. the pattern of uti causative microbes in female hospitalized patients. the most common microorganisms causing uti in hospitalized females were gram-negative bacteria (55.2%), of which, escherichia coli consituted 56.3%. this result is similar to the study by alkhyat et al. in yaman (2013), where it was reported that the most common bacteria causing uti in hospitalized females was escherichia coli (46.7%). the emerging resistance of escherichia coli to several antibiotics was a challenge for uti treatment of hospitalized patients. evidence-based prevention guidelines is strongly recommended to reduce the morbidity and prevent the dissemination of drugresistant gram-negative microorganisms in hospital. the most effective management should be followed by removal of the risk factor such as urinary catheter. this study showed that 20.6% of the uti cases found in hospitalized females were caused by grampositive bacteria. this is similar to research conducted by beyene et al. (2011), who reported that 19.1% of uti cases in hospitalized females were caused by gram-positive bacteria. although gram-positive bacteria are fairly uncommon uti causative agents, investigating the efficacy of treatment is very important to reduce morbidity caused by uti in hospitalized patients. the predisposing factors in the urinary tract, such as obstruction, indwelling catheters, surgery and chronic debilitating diseases should be removed. fungi was the second most common uti causing microorganism in hospitalized females in this study (24.2%). this is supported by research conducted by wilson et al. (2004), who reported that the frequency of candida-caused uti in hospitalized patients ranged between 9.4-15.8%. the use of broad-spectrum antibiotics in hospitalized patients could cause an imbalance of the normal flora of the body. the imbalance condition could cause overgrowth of candida as part of normal flora in gastrointestinal tract that can lead to opportunistic uti in hospitalized patients. comparison of the uti causative microbes in female outand hospitalized patients. gramnegatives were found to be the most common uticausing microbes in female outand hospitalized patients with frequency of occurence 75.9% and 55.2%, respectively. this result was slightly different from angami et al. (2015), reported that gramnegative microorganisms caused 46% uti in outpatients and 61.8% in hospitalized patients in india. this result also showed that enteric gram-negative microorganisms were the most common cause of uti in female outand hospitalized patients. therefore, e m p i r i c a l t h e r a p y a g a i n s t g r a m n e g a t i v e microorganisms can be recommended for utis in women. 0.010 7 7 outpatients hospitalized patients total 0 24.1 12.1 29 22 51 100 75.9 87.9 table 6 comparison of gram positive and non-gram positive caused uti in female outpatients and hospitalized patients pgram positive 0.7537 6 13 outpatients hospitalized patients total bacteria pattern 24.1 20.7 22.4 22 23 45 75.9 79.3 77.6 non gram positive n % n % table 7 comparison fungal and non-fungal caused uti in female outpatients and hospitalized patients pgram positive bacteria pattern non gram positive n % n % 36 rosana et al. microbiol indones in this study, klebsiella pneumoniae, a gramnegative bacteria, was found as the most common cause of uti in outpatients, while in hospitalized patients it was only the second most common. study conducted by tajbakhsh et al. (2015) showed that 8.2% of uti were caused by klebsiella pneumoniae both in outpatients and hospitalized patients. considering klebsiella pneumonia has a great potential to be resistant to many antibiotics, strategies for treatment will differ between uti in outpatients and inpatients. the choice of a specific antibiotic depends on local susceptibility patterns. uncomplicated cases caused by susceptible strains may be treated orally, while intravenous agents are used only if fever is found. e.coli, a gram-negative bacteria, was found as the most common cause of uti in hospitalized patients in this study, while in outpatients, it was the second most common cause of uti. although antibiotics are still the standard treatments for utis, some strains of e. coli, called extended-spectrum beta-lactamase (esbl) e. coli, are resistant to most drugs. esbl enzymes are able to hydrolyze most of the beta-lactam antibiotics, including third-generation cephalosporins. in addition, esbl (ec) might also express co-resistance to e. coli smx/tmp, fluoroquinolones, and aminoglycosides. carbapenems are generally considered the drug of choice for intravenous treatment of esbl-ec uti. some esbl-ec isolates will have in vitro susceptibility to piperacillin/tazobactam. however, the use of this antibiotic remains controversial. there are limited oral options for the treatment of esbl-ec cystitis. an alternative is fosfomycin, an oral antibiotic agent with broad activity against multi-drug resistant pathogens including esbl-ec. there is no significant difference in the frequency of gram-positive microorganisms in the female outpatients and hospitalized patients. however, there is a striking difference between fungal-caused uti in female outpatients and hospitalized patients. candida was the only uti-causing fungus found in female hospitalized patients in this study, while there was no fungal-caused uti in female outpatients. according to our study, the most common uti-causing microbes in female outpatients in jakarta was klebsiella pneumoniae (31%) and escherichia coli (24.1%), whereas in female hospitalized patients, the most commons were escherichia coli (31%), candida sp (24.1%) and klebsiella pneumoniae (6.8%). there was no significant difference between the occurence of gram-negative and gram-positive bacteria in outand hospitalized patients (p>0.05). however, fungal infection caused by candida sp. was only found in hospitalized patients (p=0.01). this result proved that the uti-causing microbes found in hospitalized female patients were more varied in comparison to those found in female outpatients. the results of this study suggested that periodic surveys are necessary to determine the distribution and pattern of uti-causing microorganisms and that it would be beneficial as guidance for empirical antimicrobial therapy in uti patients while waiting for the results of urine culture. acknowledgment this study was funded by zambon indonesia. we thank the teams from the department of microbiology and the department of obstetrics/ gynecology, faculty of medicine, universitas indonesia for their technical assistance, support, and cooperation. references alkhyat sh, almaqtari mh. 2014. prevalence of microorganism isolate from urinary tract infection at some hospitals in sana'a city, yaman. int j curr microb app sci. 3(6):876-85. doi: 10.1371/journal.pone. 0144266. angami s, jamir n, sarma pc, deka ac. 2015. urinary tract infection, its causative microorganism and antibiotic, nagaland. arch med health sci, the official journal of yenepoya university. 3(1); 40-3. doi: 10.4103/23214848.154943. beyene g, tsegeye w. 2011. bacterial uropathogens in urinary tract infection and antibiotic susceptibility pattern in jimma university specialized hospital, southwest ethiopia. ethiop j health sci. 21 (2): 141-6. doi: 10.4314/ejhs.v21i2.69055. duerink do, roeshadi d, wahjono h, lestari es, wille jc, de jong rm, nj nagelkerke, pj van den broek. 2006. surveillance of healthcare-associated infections in indonesian hospitals. j hosp infect. 62(2):219-29. doi: 10.1016/j.jhin.2005.08.004. foxman b. 2003. epidemiology of urinary tract infection: incidence, morbidity, and economic. dis mon. 49(2):5370. doi: http://dx.doi.org/10.1067/mda.2003.7. gatot d.2002. infeksi jamur sistemik pada pasien immunocompromised [systemic fungal infections in immunocompromised patients]. sari pediatric. 3(4), 242-3. grabe m, bartoletti r, johansen te, cai t, naber kg,koves b, et al. 2015. guidelines on urological infections. european association of urology. 9-32. hooton tm. 2000. pathogenesis of urinary tract infection: an update. j antimicrob cemother. 46 (1-7): 63-5. doi: 10.1093/jac/46.suppl_1.1. komala m. kumar k.p.s. 2013. urinary tract infection: causes, symptoms, diagnosis and its management. indian j res pharm biotechnol. 1(2):228-32. doi: 10.1136/bmj.c199. mitchell, abbas, kumar, fausto. 2002. buku saku dasar patologis penyakit (pocket book basic of pathologic disease), ed.7. new york: elsevier inc. 50-1. rajaratnam a, baby nm, kuruvilla ts, machado s. 2014. diagnosis of asymptomatic bacteriuria and associated risk factors among pregnant women in mangalore, karnataka, india. j clin diagn res. 8(9):oc23-5. doi: 10.7860/jcdr/2014/8537.4842. raz r. 2011. urinary tract infection in postmenopausal women. korean j urol. 52(12): 801–8. doi: 10.4111/kju.2011.52.12.801. robinson d, toozh-hobson p, cardozo l. 2013. the effect of hormones the lower urinary tract. menopause i n t e r n a t i o n a l . 1 9 ( 4 ) : 1 5 5 6 2 . d o i : 1 0 . 11 7 7 / 1754045313511398. samirah, darwati, windarwati, hardjoeno. 2006. pola dan sensitivitas kuman di penderita infeksi saluran kemih [microorganism and sensitivity patterns in urinary tract infection patients]. indones j clin pathol med lab. 1(3): 110-3. tajbakshs e, tajbakhsh s, khamesiour f. 2015. isolation and molecular detection of gram negative bacteria causing urinary tract infection in patient referred to shahrekord hospital, iran. iran red crescent med j. 17(5): 5-7. doi: 10.5812/ircmj.17(5)2015.24779. wilson m, gaido l. 2004. laboratory diagnosis of urinary tract infections in adult patients. clin infec dis. 38(8):1150-8. doi: 10.1086/383029. volume 10, 2016 microbiol indones 37 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 3 yogiara_411 (6).pmd volume 3, number 3, december 2009 p 109 114 issn 1978-3477 *corresponding author: phone: +62-21-5703306 ext.449, fax: +62-21-5719060, e-mail: yogiara@atmajaya.ac.id bacterial community profiles in the fluid of four pitcher plant species (nepenthes spp.) grown in a nursery andree siegara and yogiara* school of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia nepenthes is one of the indonesian tropical carnivorous plants. the plant has a pitcher-like structure containing fluid for digesting insects. there are many microorganisms growing in the pitcher fluid. different species of pitcher plants and planting sites could also contribute either to the diversity or the abundance of microorganism inhabiting the pitcher fluid. to assess the bacterial community variation in the fluid of pitcher plants grown in a nursery, amplified ribosomal dna restriction analysis (ardra) was used. four specimens of n. gracilis, n. truncata, n. veitchii and n. bicalcarata were obtained from suska nursery, ciderum village, caringin, bogor, indonesia. a total of 191 positive clones were analyzed by using ardra. a sum of 124 phylotypes was obtained, comprising 17 in n. gracilis, 7 in n. truncata, 45 in n. veitchii and 55 in n. bicalcarata. it is interesting to note that each specimen harbored unique phylotypes, meaning that no phylotypes generated from one specimen were found in any of other specimens. key words: nepenthes, nursery, bacterial community, ardra the pitcher plant (nepenthes spp.) is one of the tropical carnivorous plants of indonesia. the leaves of nepenthes species are highly specialized. the ‘leaf blade’ bears at its tip a tendril, from which arises a sharply upturned and hollow pitcher, with a more or less oblique mouth overhung by a lid, from the base of which arises a short spur. the plant has two types of pitcher, upper pitcher and lower pitcher. these two types of pitcher contain fluid of the plant’s own production. the pitcher acts as a pitfall trap for a wide range of invertebrates and, less commonly, vertebrates (cheek and jebb 2001). many small animals might be trapped in the pitcher, the fluid contained therein becomes a suitable habitat for microorganism growth. the plant secretes degradative enzymes, including ribonucleases, phosphatases (matthews 1960), proteinases (nepenthesins i and ii) (hatano and hamada 2008) and possibly chitinases (amagase 1972) as well as ions including chloride (luttge 1971) and calcium (massa 1998). these enzymes help the plant to digest preys. up to 70% of the total nitrogen needed by a plant is provided by the digestion of the prey. beside those enzymes, all nepenthes species produce also various chemical substances, such as naphthoquinones, known as a substance having an antimalarial property (rizzacasa and sargent 1987). to date there are very few publications on microbial community diversity in nepenthes. yogiara et al. (2006) collected fluid samples from several pitcher plants from various locations and found 16-39 groups of bacteria lived inside the pitchers. the bacterial community profiles were different from one specimen to the others. as yet there were no publications clearly clarifying whether the community development was affected by its local environment (habitat) or the fluid. the succession of microbial population inside the fluid has not been known properly. based on the result of his research, yogiara et al. (2006) proposed that the growth of microorganisms might have not been affected by the locations or habitats, where the pitcher plants are grown, but could have been more affected by the chemical substances released in the fluid and simultaneously developed during pitcher’s lid opening. the objectives of the present study are (i) to investigate a correlation between habitat and microbial community composition in pitchers of plant grown in a nursery, (ii) to obtain information on bacterial diversity and (iii) to gain bacterial community comparison in four pitcher fluid samples from different species of pitcher plants. materials and method fluid sampling and dna extraction. pitcher plant specimens were obtained from suska nursery in ciderum village, caringin, bogor-indonesia. about 10-25 ml of fluid collected from several pitchers was poured into centrifuge tubes. to reach that sufficient volume we had to collect the fluid from several pitchers, because each pitcher can only provide about 1-5 ml of fluid. the origin and the scientific names of species, ph and types of pitchers were recorded. collected samples were then kept in an ice box to minimize the changes of community composition during the transportation to the laboratory. bacterial dna in pitcher plant fluid was extracted using fastdna® spin kit for soil (qbiogen, usa). construction of 16s-rrna gene libraries. 16s-rrna gene was amplified using bacterial universal primers, 63f (5'cag gcc taa cac atg caa gtc-3') and 1387r (5'-ggg cgg wgt gta caa ggc-3') (marchesi et al. 1998). the pcr master mix consisted of 5 pmol of each primer, 200 µm dntp, 1x taq polymerase buffer, 2.5 u of taq polymerase, 1 µl of template and ddh 2 o up to a final volume of 25 µl. the amplification process was done using pcr geneamp® pcr system 2400 (perkin-elmer co., norwalk, conn). the following cycles used were 94 °c for 2 min then followed by 30 cycles at 94 °c for 1 min, 55 °c for 30 sec and 72 °c for 1 min. the extension was completed at 72 °c for 20 min after 30 cycles, and was held at 4 °c until further use. the pcr products 2.mi-rahayu wangsa.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.2.2issn 1978-3477, eissn 2087-8575 vol 10, no 2, juni 2016, p 48-56 *corresponding author; phone: 021-8754587/8754588, email:rahayufwputrie@gmail.com some plant species have specific pathway which allow them to survive under extreem conditions such us drought stress. the best known is the crassulacean acid metabolism (cam) plants, particularly the species of the genera opuntia, agave, and a liliaceous species, one of them is aloe. genus of aloe are known have around 400 species including aloe pollyphyla, a. vera linn syn. a. barbadensis miller, a. ferox miller, a. arborecens, a. brevifolia, a. microstigma, a. buhrii, a. hereroensis, a. humilis, a. maculata, a. chinensis baker, a. indica royle, a. perryi baker and others (rodriguez-garcia et al. 2007; ucdbc 2009; rajeswari et. al. 2012; silva et al. 2014). aloe is a cam species that naturally survive to drought conditions and high temperatures. salinity and drought stress affected to the plant height, number of leaves, leaf length, leaf thickness, aerial fresh yield, leaf fresh weight, and gel weight (shams et al. 2015). nitrogen fixation procces influenced by the ability of plants to adapt in drought condition (dinh et al. 2013; serraj 2003). drought cause a significant decreases in nodule dry weight and amount of nitrogen fixing by the plant. peanut genotypes that planted aloe is a crassulacean acid metabolism (cam) species that are known to live in extreme enviroment such as drought condition. nitrogen fixation procces influenced by the ability of plants to adapt in drought condition. endophytic bacteria from aloe and their ability for nitrogen fixation were little reported, this research aimed study the endophytic bacteria from two varieties of aloe, namely aloe barbadensis miller and aloe sp. in their ability on conducting the nitrogen fixing process . characterization of endophytic bacteria were carried out by morphological observation of colony, gram staining and molecular identification. screening of nitrogen fixation was done using nitrogen-free semisolid nfb malate medium. endophytic bacteria from aloe sp. more than a. barbadensis in their potency of nitrogen fixation which related with habitat where their planted. a total of 40% of the endophytic bacteria isolates from the leaves of the aloe var. a. barbadensis and 62.5% of isolates from var. aloe sp. are known to have a better ability to fixing nitrogen than the others. isolates a. barbadensis ab 12 and aloe sp. as 8 were the best isolates from each varieties on ability for nitrogen fixation. based on 16s rrna gene analysis those two selected isolates were similar to bacillus methalotropicus strain da 16-5 and bacillus aryabhattai strain b8w22. keyword : aloe, endophytic bacteria, nitrogen fixation lidah buaya merupakan salah satu spesies tanaman crassulacean acid metabolism (cam) yang dapat hidup pada lingkungan ekstrim seperti kekeringan. kemampuan adaptasi terhadap kekeringan dipengaruhi oleh kemampuan fiksasi nitrogen. bakteri endofit dari lidah buaya dan kemampuannya dalam memfiksasi nitrogen telah sedikit dilaporkan, namun potensi dan hubungannya antara kemampuan fiksasi nitrogen dengan ketahanan terhadap kekeringan belum dilaporkan. penelitian ini bertujuan untuk mengetahui dan membandingkan kemampuan bakteri endofit dari dua varietas lidah buaya, yaitu aloe barbadensis miller dan aloe sp. dalam memfiksasi nitrogen serta hubungannya dengan ketahanan terhadap kekeringan. karakterisasi bakteri endofit dilakukan dengan pengamatan morfologi koloni, pewarnaan gram dan identifikasi molekuler. penapisan fiksasi nitrogen dilakukan dengan menggunakan medium nitrogen-free semisolid nfb malate. bakteri endofit yang berasal dari aloe sp. lebih banyak yang dapat memfiksasi nitrogen dibandingkan dengan a. barbadensis dimana kemampuan ini memiliki hubungan dengan habitat tumbuhnya. sebanyak 40% isolat bakteri endofit dari daun lidah buaya var. a. barbadensis dan sebanyak 62.5% isolat var. aloe sp. diketahui memiliki kemampuan yang lebih dalam proses penambatan nitrogen dibandingkan dengan isolat lainnya. isolat ab 12 dan as 8 adalah isolat penambat nitrogen terbaik dari setiap varietas. berdasarkan analisis gen 16s rrna isolat tersebut mempunyai kemiripan yang tinggi dengan bacillus methalotropicus strain da 16-5 dan bacillus aryabhattai strain b8w22. kata kunci : bakteri endofit, fiksasi nitrogen, lidah buaya but potential and its relationship between the ability for nitrogen fixing with resistance to drought conditions have not been reported. and its relationship with resistance to drought nitrogen fixing potential of endophytic bacteria isolated from aloe barbadensis miller and aloe sp. rahayu fitriani wangsa putrie*, tiwit widowati, sylvia j.r. lekatompessy, and harmastini sukiman plant symbiotic microbes laboratory, research center for biotechnology, indonesian institute of sciences (lipi) jalan raya bogor km. 46, cibinong 16911, indonesia under well-watered condition and under drought stress were significantly different for nitrogen fixation. drought tolerant genotypes had higher spad chlorophyll meter reading (scmr), fixed more nitrogen and achieved higher pod yield than sensitive genotypes (dinh et al. 2013). biological nitrogen fixation (bnf) in agriculture are most promising on supporting the growth and productivity of plant. plant growth promoting rhizobacteria (pgpr) had the ability to fix atmospheric nitrogen by symbiotic and non-symbiotic mechanism and provide it to plants (saharan and nevra 2011; ahemad and kibret 2014; gupta et al. 2015). bnf were 6 contribute 180 ×10 metric tons/year globally, 80% from symbiotic association and the rest from free-living or associative systems. a number of bacterial species belonging to genera of pgpr viz. rhizobium, bradyrhizobium, sinorhizobium, mesorhizobium, azoarcus, azotobacter, acetobacter, azospirillum, burkholderia, diazotrophicus, enterobacter, gluconacetobacter, pseudomonas, and cyanobacteria (saharan and nevra 2011; gupta et al. 2015). crops inoculation by pgpr provide an integrated approach for disease management, growth promotion activity, maintain the nitogen level in agricultural soil (ahemad and kibret 2014; gupta et al. 2015) plant-growth-promoting bacterial endophytes (pgpbes) have been known for positively influencing plant growth in limited field conditions. bacterial root endophytes reside in a vast number of plant species are a part of the root microbiome. those endophyte community structure (species diversity: richness and relative abundances) were influenced by abiotic and biotic factors of environment (gaiero et al. 2013). nitrogen source in the atmosphere are known about 79% of the total atmospheric gases. although nitrogen is very abundant in nature, it was often limiting plant productivity because atmospheric nitrogen is only available to organisms symbiotically associates with higher plants and non-symbiotically (khan et al. 2008; gulati et al. 2011; ahemad and kibret 2014). the abilitiy of endophytic bacteria from various plant as plant growth promoters, to fixed nitrogen and could be support in drought stress have been reported (ngoma et al. 2013; nogkhlaw and joshi 2014; ngoma et al. 2014; miliute et al. 2015). exploration of endophytic bacteria from drought tolerant plant, specially leaves, stem and roots of aloe as a potential agents for antifungal activity againts fusarium oxysporum and a vast source of extracellular enzymes such as amylase, cellulase, chitinase, pectinase, lipase, and urease also have been reported (yadav et al. 2015). the crude and ethyl acetate fractions of the metabolites of six isolates endophytic from aloe had broad spectral antimicrobial activities against pathogenic pseudomonas aeruginosa, staphylococcus aureus, bacillus cereus, salmonella typhimurium, proteus vulgaris, klebsiella pneumoniae, escherichia coli, streptococcus pyogenes, and candida albicans (akinsanya et al. 2015a). on the other hands, the ability of endophytic bacteria from aloe as a nitrogen fixing were little reported. investigate for endophytic bacteria assosiated with aloe from the pristine subtroprical forest in meghalaya india, herminiimonas saxobsidens aa jq770186 showed that their ability for iaa production, phosphate solubilisation and nitrogen fixation which are beneficial to host plant (nongkhlaw and joshi 2014). although there have been reports related to endophytic bacteria from aloe in their ability for nitrogen fixation, but potential and its relationship between the ability for nitrogen fixing with resistance to drought conditions have not been reported. previously, we have succeeded on isolating the culturable endophytes microbes from aloe which could grown in nutrien agar (na), potato dextrose agar (pda), and cornmeal malt extract (cmm) agar. a total of 43 isolates of endophytic microbes were isolated from the leaves of the aloe var. a. barbadensis and 28 isolates from var. aloe sp., respectively. endophytic microbes from a. barbadensis more than aloe sp. as many as 58% microbes derived from a. barbadensis are bacteria, 42% fungi and aloe sp. as many as 86% are bacteria, 14% fungi. this showed that the majority culturable endophyte symbiosis on the leaves of the aloe is a bacteria (putrie and sukiman 2015). based on the case, this research aimed to study endophytic bacteria from two varieties of aloe, namely a. barbadensis and aloe sp. and identifying isolates that are known had the best ability of of nitrogen fixation from each variety also its relationship with resistance to drought condition. materials and methods morphology characteristic of endopythic bacteria colony. culturable endopytic bacteria were isolated from the leaves of the aloe var. a barbadensis and from var. aloe sp. (putrie and sukiman 2015). sample of aloe used in this research, both of them derived from an experimental garden research center for biotechnology lipi but there were differences on volume 10, 2016 microbiol indones 49 place of planted. a. barbadensis planted in pots whereas aloe sp were planted in soil directly. isolates were purified by streak quadrant on nutrient agar (na) -1 (23 g l ) subsequently incubated at room temperature 24 h to optimize the growth of culture. each of colony growth were observed. those morphological were observed include colour, size, edge of the colony, the colony shape, and condition dry or slimy of colonies. gram staining. gram staining is an important and useful technique to catagorize the bacteria included in gram-positive or gram negative based on their morphology and differential staining properties. the method of gram staining was described by bartholomew (1962). the beginning stage was done by making heat-fixed smear slides of bacteria. crystal violet as a main dye and mordant solution (lugol’s iodine) dropped for ± 1 min, respectively. ethanol 95% dropped until ethanol fall colored clear and not excessive (overdecolorize). safranin as last dye dropped for ± 45 sec. each after given dye, smear slides washed with distilled water, then drain flow. gram positive bacteria stain blue-purple and gram negative bacteria stain red. nitrogen fixing assay. endophytic bacteria as many as 25 isolates from a. barbadensis and 24 isolates from aloe sp. were inoculated in a nitrogen-1 free semisolid nfb malate medium (k hpo 0.5g l , 2 4 -1 -1 -1 mgso .7h o 0.2 g l , nacl 0.1 g l , cacl 0.02 g l , 4 2 2 -1 -1 trace element 2.0 ml l [na moo .2h o 0.2 g l , 2 4 2 -1 -1 mnso 0.235 g l , h bo 0.2 g l , cuso .7h o 0.24 4 3 3 4 2 -1 -1 g l ], bromthymol blue 0.5% 2.0 ml l solution [dissolved in 0.2 n koh], fe edta [1.64% solution] -1 -1 4.0 ml l , and vitamin solution 1.0 ml l [biotin 0.01 g -1 -1 l , pyridoxin 0.02 g l ] ph adjusted to 6.8 with koh, -1 semi solid agar 1.75 g l ) (okon et al. 1977). this assay aimed to known ability of the isolates to fix atmospheric nitrogen in medium by stab inoculation. incubation conducted for 3-5 d. score positive are showed by growth of the isolates in variable depth and change in colour under the surface medium. uninoculated nfb medium was kept as control on this assay (nogkhlaw and joshi 2014). identification based on 16s rrna gene analysis. the best isolates from each varieties on ability for nitrogen fixation procces subsequently molecularly identified. one colony of isolate were taken with a sterile toothpick then inserted into eppendorf tube containing 100 ml dh o subsequently 2 it were vortex. a total of 1 ml suspension were used for the amplification with polymerase chain reaction (pcr) technique. amplification of 16s rrna gene by pcr with primer 27 f (5’-agagtttgatcctggct cag-3’) and 1492 r (5’-ggttaccttgttacgac tt-3’) (weisburg et al. 1991) in total volume 50 µl. the pcr volume contains of 1 µl dna template, 2 µl of primer for each forward and reverse, 25 µl of 2x kapa taq ready mix (kapa biosystem) and ddh o 2 20 µl. amplification was performed for 30 cycles that included initial denaturation stage at a temperature 96 °c for 5 min, denaturation at a temperature 96 for 30 sec, annealing a temperature 55 °c for 30 sec, extension at a temperature 72 °c for 1 min, final extension at a temperature 72 °c for 7 min. dna of pcr products were purified and sequenced in two directions. sequences were analyzed by comparing the sequences with genbank database using the blastn (http://www.ncbi. nlm.nch.gov) programe of the national center for biotechnology information to determine similarity. the length of base used for blastn between 500-1500 bp (clarridge 2004). results endophytic bacteria, both of a. barbadensis and aloe sp. were optimatelly growth on 24 h after incubated. morphology of all bacteria colony from a. barbadensis and aloe sp., had several similarity (table 1, table 2 and fig 1). both of them, majority of endophytic bacteria colony were pigmented, moderate, smooth of colony edge, round shape, slimy and opaque. a total of 56% isolates ab and 79.2% isolates as out of each total bacterial isolates varieties were shown a slime colony, respectively. all isolates, both it from a. barbadensis and aloe sp. then classified with gram staining. based on the result, all isolates included to gram positive bacteria. gram staining result showed that isolates stain blue-purple. after that, for those isolates were conducted nitrogen fixing assay used nitrogen free semisolid nfb malate medium. endophytic bacteria from aloe sp. more frequently than a. barbadensis in their potency for nitrogen fixation (table 3). positive results were marked by changes in media (fig 2). a total of 92% isolates ab and 96% isolates as had abillity to nitrogen fixing in medium, but their ability for each isolates were different. only 40% out of 92% isolates ab and 62.5% out of 96% isolates as. were showed a better ability to fixed nitrogen compared to the others isolates. one isolates from each varieties that shown the best ability to fixed nitrogen, ab 12, and as 8 subsequently indentified by °c microbiol indones50 utrie et al.p molecular identification. based on 16s rrna sequence gene analysis isolat ab 12 and as 8 belonged to b. methalotropicus strain da 16-5 and bacillus aryabhattai strain b8w22, respectively (table 4). discussion morphology of endophytic bacteria colony showed that majority of colony from both of them are slimy. endophytic bacteria produce more mucus or exopolisaccharide (eps) to keep plant from water loss. abiotic factor such as drought stress tolerance in bacteria were characterized by production of exopolysaccaride (eps). production of eps were increased by bacteria during a drought as a form physiological adaptation. eps quantity and composition were influence by genus and species of bacteria, in some cases dependent on environmental conditions for growth (putrie et al. 2013). based on gram staining, all isolates from a. barbadensis and aloe sp. were gram positive bacteria. this was possible because adaptation of gram positive bacteria in extreem environmet, like as drought higher than gram negative bacteria. gram positive bacteria could be survive in drought environment by spore. drought periode could be promote of presence of spore forming bacteria (meisner et al. 2015). the ability to adapt in drought condition has been known assosiate with nitrogen fixation (zahran 1999). nitrogen is generally considered one of the major limiting nutrients in plant growth (khan et al. 2008; france et al. 2009). under drought stress, the ability to maintain high nitrogen fixation could aid peanut genotypes in maintaining high yield (pimratch et al. 2008). several mechanism of nitrogen fixation were involved in the physicologycal response to drought stress such as carbon shortage and nodule carbon metabolism, limitation of nitrogen and feedback no. isolates code part of leaf morphological colony pigment size edge of the colony shape dry/slimy transparantly 1. ab 1 pole old cream moderate smooth round dry transpatant 2. ab 2 pole milky white yellowish moderate smooth round slimy opaque 3. ab 3 pole milky white small smooth round thin slimy opaque 4. ab 4 pole old cream small smooth round dry opaque 5. ab 5 pole old cream small rough round dry opaque 6. ab 6 pole old beige moderate smooth round dry opaque 7. ab 7 pole milky white yellowish central part whiter moderate smooth round slimy opaque 8. ab 8 pole bright white milk moderate smooth round thick slimy opaque 9. ab 9 pole milky white yellowish moderate smooth round slimy opaque 10. ab 10 pole creamy white in the middle point smooth round slimy transparantly in the point 11. ab 11 pole old beige-gray (more beige than ab 6) moderate smooth round dry opaque 12. ab 12 pole dull beige moderate smooth round dry transparantly in the point 13. ab 13 pole cream small smooth round dry opaque 14. ab 14 pole cream small smooth round slimy opaque 15. ab 15 center yellow small smooth round thin slimy opaque 16. ab 16 center milky white moderate smooth round thick slimy opaque 17. ab 17 center beige-gray moderate smooth round dry opaque 18. ab 18 center white point smooth round thin slimy opaque 19. ab 19 center creamy white moderate rough round thick slimy opaque 20. ab 20 center old beige-gray moderate rough round dry opaque 21. ab 21 tip white small smooth oval dry opaque 22. ab 22 tip white point smooth round slighty dry transparantly 23. ab 23 tip milky white moderate smooth round slimy opaque 24. ab 24 tip milky white moderate smooth round thin slimy opaque 25. ab 25 center yellow point smooth round slimy opaque table 1 morphology of endophytic bacteria from aloe barbadensis volume 10, 2016 microbiol indones 51 synthesis and function of the enzyme. in diazotrophs, nif genes were typically found in a cluster of around 2024 kb with seven operons encoding 20 different proteins. the complex of molybdenum nitrogenase enzyme had two component proteins encoded by the nifdk and the nifh genes. the nifdk component were heterotetrameric (α2β2) protein formed by two αβ dimers related by a twofold symmetry. nifdk carried one iron molybdenum cofactor (femo-co) within the active site in each a-subunit (nifd) (ahemad and kibret 2014). regulation by the accumulation of nitrogen fixation products (serraj 2013). endophytic bacteria in plant had a metabolism that play a role in the resilience of host plants at extreme environmental conditions such as drought and influence the process of nitrogen fixation in plants. endophytic bacteria that inhabiting on those plants had ability for fixing nitrogen by nitrogenase enzyme. those enzyme produced by nif genes that contribute to activation of the fe protein, iron molybdenum cofactor biosynthesis, electron donation, and regulatory genes required for the no. isolates code part of leaf morphological colony pigment size edge of the colony shape dry/slimy transparantly 1. as 1 pole yellow moderate smooth round slimy opaque 2. as 2 pole cream moderate rough round slimy opaque 3. as 3 pole cream moderate rough round slimy in the middle opaque 4. as 4 pole old cream of the central part, light cream in tip moderate smooth round slimy in the middle transparantly in tip 5. as 5 pole old cream slightly yellow moderate smooth round little slime opaque 6. as 6 pole old cream moderate smooth round little slime opaque 7. as 7 pole cream slightly yellow moderate rough round slimy opaque 8. as 8 pole cream slightly yellow moderate smooth round slimy opaque 9. as 9 pole cream -gray moderate smooth round dry opaque 10. as 10 pole cream slightly yellow moderate smooth round dry opaque 11. as 11 center milky white moderate smooth round slimy opaque 12. as 12 center cream moderate smooth round little slime opaque 13. as 13 center cream slightly yellow moderate smooth round little slime opaque 14. as 14 center yellow sizeable rough round little slime opaque 15. as 15 point cream slightly yellow moderate rough round dry opaque 16. as 16 tip cream slightly yellow moderate rough round slimy opaque 17. as 17 tip milky white moderate rough round slimy opaque 18. as 18 tip yellow moderate smooth round little slime opaque 19. as 19 tip cream slightly yellow moderate rough round dry opaque 20. as 20 tip milky white moderate smooth round slimy opaque 21. as 21 tip yellow a little small rough round slimy opaque 22. as 22 pole pale white moderate rough round dry wrinkled opaque 23. as 23 pole bright white moderate smooth round slimy opaque 24. as 24 pole white moderate smooth round slimy opaque table 2 morphology of endophytic bacteria from aloe sp. microbiol indones52 utrie et al.p as 8ab 12 fig 1 morphology colony of isolates as 8 and ab 12. table 3 result of nitrogen fixing test for isolates from aloe barbadensis and aloe sp. no. isolates from a. barbadensis result no. isolates from aloe sp. result 1. ab 1 ++ 1. as 1 ++ 2. ab 2 ++ 2. as 2 + 3. ab 3 ++ 3. as 3 + 4. ab 4 + 4. as 4 5. ab 5 ++ 5. as 5 + 6. ab 6 + 6. as 6 ++ 7. ab 7 + 7. as 7 ++ 8. ab 8 ++ 8. as 8 ++ 9. ab 9 + 9. as 9 + 10. ab 10 + 10. as 10 ++ 11. ab 11 11. as 11 ++ 12. ab 12 ++ 12. as 12 ++ 13. ab 13 + 13. as 13 ++ 14. ab 14 ++ 14. as 14 ++ 15. ab 15 + 15. as 15 ++ 16. ab 16 ++ 16. as 16 ++ 17. ab 17 + 17. as 17 + 18. ab 18 18. as 18 + 19. ab 19 ++ 19. as 19 ++ 20. ab 20 + 20. as 20 + 21. ab 21 + 21. as 21 ++ 22. ab 22 + 22. as 22 ++ 23. ab 23 + 23. as 23 + 24. ab 24 ++ 24. as 24 ++ 25. ab 25 + 25. control 26. control volume 10, 2016 microbiol indones 53 from primeval forest soil in qinling mountains, china were able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media (shan et al. 2013). this result may also confirmed and supported our finding that endophytes bacteria which was identified as bacillus spp. have many potentials and one of them is nitrogen fixing ability. b. methylotrophicus strain l7 also known as efficient heterotrophic nitrificationaerobic denitrification (zhang et al. 2012). other species, b. aryabhattai strain b8w22 also known as nirogen fixing bacteria. b. aryabhattai strain b8w22 were isolated from the roots of tea (camellia sinensis (l.) o. kuntze) and nicotiana attenuata are known for their potency as diazotropic bacteria in ability to fixed nitrogen and plant growth promoters (gulati et al. 2011; meldau et al. 2012). the other hand, bacillus are dominant genera of endophytic bacteria in aloe. twenty-nine culturable bacterial endophytes were isolated from surfacesterilized root, stem and leaf tissues of a. vera based on molecularly characterized those belonged to 13 genera i.e. pseudomonas, bacillus, enterobacter, pantoea, chryseobacterium, sphingobacterium, aeromonas, providencia, cedecea, klebsiella, cronobacter, the capability endophytic bacteria isolated from aloe sp. to fixed nitrogen was higher than a. barbadensis. the high nitrogen fixing were improve the ability of adaptation to drought stress. nitrogen fixation ability related with habitat where place of the planted. physiological of the host plant in rhizobiumlegume symbiosis have been known strongly impact to n -fixing system. symbiotic n fixation of legumes is 2 2 also highly sensitive to soil water deficiency. those condition promote a maximal nitrogen fixation input to the soil system by the rhizobium-legume symbiosis (zahran 1999). isolates ab 12 and as 8 were the best isolates for nitrogen fixation procces from each varieties subsequently molecularly identified. based on 16s rrna gene analysis those isolates belonged to b. methalotropicus strain da 16-5 and b. aryabhattai strain b8w22. genus of bacillus has been known for their potency as plant growth promoters, both directly and undirectly mechanism (putrie et al. 2013; meldau et al. 2012; francis et al. 2010). it was reported that bacillus spp is one of the potential bacteria beside that could fixing nitrogen, their also could produce bioactive compound for biocontrol of numerous plant pathogenic fungi. b. methalotropicus strain bc79 were isolated isolates species most related similarity length identities gaps accession number ab 12 bacillus methalotropicus strain da 16-5 97% 1437 193/199 1/199 (0%) ku862327.1 as 8 bacillus aryabhattai strain b8w22 99% 1533 1161/1171 5/1171 (0%) nr. 115953.1 fig 2 nitrogen fixing test (a) endophytic bacteria of aloe (b) plant growth promoting rhizobacteria (pgpr) isolate collection of plant symbiotic microbes laboratory research center of biotechnology lipi. table 4 identification of ab 12 and as 8 isolates based on 16s rrna gene sequence homology by using blastn program compared with genbank sequences control (+) white ring a b as 8 ab 12 control (-) microbiol indones54 utrie et al.p side of plant-microbe interactions. env microbiol. 12(1):1-12. 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doi:10.1016/j.indcrop.2006.08.005. saharan bs, nehra v. 2011. plant growth promoting rhizobacteria: a critical review. lsmr-21. 30 p[on line]. http://astonjournals.com/lsmr. serraj r. 2013. effects of drought stress on legume symbiotic nitrogen fixation : physiological mechanism. indian j exp bio. 41:1136-1141. shams j, badi hn, zeynali h, khalighi-sigaroodii f,payam najafi p. 2015. effects of salinity and drought on morphological and chemical traits of aloe vera plant. microbiol indones56 utrie et al.p 1: 48 2: 49 3: 50 4: 51 5: 52 6: 53 7: 54 8: 55 9: 56 4 no. 274 (pujawati suryatmana).pmd improving the effectiveness of crude-oil hydrocarbon biodegradation employing azotobacter chroococcum as co-inoculant pujawati suryatmana parnadi1∗, edwan kardena2, enny ratnaningsih3 & wisjnuprapto2 1soil science and land resources department, faculty of agriculture, universitas padjajaran, jalan raya jatinangor km. 20, bandung, 40600, indonesia 2enviromental engineering study programe, 3chemical study programe, institut teknologi bandung, jalan ganesha no. 10, bandung 40132, indonesia azotobacter chroococcum has a great potential as biosurfactant producing bacteria and was used as co-inoculant to promote the rate of hydrocarbon biodegradation. the rate of hydrocarbon biodegradation were 0.01212, 0.01582, and 0.01766 per day for acinetobacter sp., bacillus cereus and the consorsium culture respectively. on the other hand, the rates of biodegradation using azotobacter as co-inoculant were 0.1472, 0.01612, and 0.02709 g per day. azotobacter chroococcum co-inoculant has the capability of increasing biodegradation efficiency of crude oil hydrocarbon. the biodegradation efficiency of petroleum hidrocarbon was increated by 13.4, 14.6, and 14.4% within the petrobacter cultures. key words: azotobacter chroococcum, biosurfactant, co-inoculant, petroleum hydrocarbon, petrobacter, rate of biodegradation _____________________________________________ the technology for exploiting the ability of og microorganism to degrade crude-oil waste is still considered as one of the most efficient, economical, and environmentally sound methods (margesin and schinner 2001). crude-oil biodegradation often encounters constraints because of its low hydrocarbon biodegradation rate. the rate of biodegradation is restricted by the mass transfer rate from solid phase to liquid phase, while the mass transfer rate is correlated with solubility level of target compound. the low solubility of hydrocarbon, which comprises crude oil, is a problem in degrading the crude oil. crude oil is always in the size of macrodroplets wich can hamper the process of substrate sorption by oil-degrading bacteria (van eyk 1997). biosurfactant or bioemulsifier compounds can increase the solubility of crude-oil hydrocarbon. rhodococcus rhodochrous produces the extracellular compound s-2 (s-2 eps), i.e. glucose, d-galactose, d-mannose, d-glucoronic acid, and fat. this compound can improve the degradation of aromatic fractions (af) in crude oil. biside functioning as a bioemulsifier for af, the eps compound can functioned as the carbon source for indigenous crude-oil degrading bacteria that to indigenous bacteria increase the growth rate of one group of the microorganisms with an extracellular membrane that can act as bioemulsifier is azotobacter. azotobacter are nitrogen-fixing, non symbiotic bacteria. for example, a. vinelandii mtcc-2459 can produce an exopolysaccharide compound (eps) that consists of glucose, rhaminose, galactose, and fucose (vermani et al. 1997), while a. chroococcum produces extracellular product that can function as bioemulsifier (wisjnuprapto et al. 2005). with the potential of this genus, a. chroococcum can be utilized as co-inoculant in biodegradation process of crude-oil hydrocarbon. azotobacter are non-petrophylic bacteria (not hydrocarbon-degrading bacteria), but they can produce biosurfactant which in turn can increase the solubility of crude oil. the objective of this research was to improve the rate and efficiency of crude-oil hydrocarbon biodegradation employing a. chroococcum as a co-inoculant. materials and methods azotobacter chroococcum and petrobacter. the strain a. chroococcum used in this research was obtained from soil biology and biotechnology laboratory, faculty of agriculture, pajajaran university, bandung. petrobacter used in this research consisted of acinetobacter sp. and bacillus cereus, while petrobacter consortium consisted of acinetobacter sp., bacillus cereus, enterobacter sp., and pseudomonas sp. this petrobacter culture was isolated from an area polluted with textile industrial waste in rancaekek, bandung, and was known to have the ability of degrading hydrocarbon compounds (data not shown). production media for a. chroococcum and petrobacter was mineral media (k 2 hpo 4 1.5 g , kh 2 po 4 0.5 g , mgso 4 0.5 g in 1.0 l aquadest) with glucose as carbon source, at ph 7. media was sterilized at 121 oc for 15 minutes. the fermentation for biosurfactant production was conducted at 28 oc for three days with 100 rpm shaking speed. biosurfactant used in this research was in the form of a threeday-old a.chroococcum culture, while for the petrobacter the culture was 36 hours old. to examine the biodegradation of crude-oil hydrocarbons, we used mineral media assay that contained crude oil as carbon source. crude oil was obtained from duri-riau field, pertamina balongan, indramayu. the media was sterilized at 121 oc for 15 minutes. biodegradation of crude-oil hydrocarbons. bacterium a. chroococcum was grown in mineral media at 28 oc for _________________ ∗ corresponding author, phone: +62-22-2504274, fax: +62-222534115, e-mail: pujawati@biotech.itb.ac.id microbiology indonesia, april 2007, p 5-10 volume 1, number 1 issn 1978-3477 three days with 100 rpm shaking speed. this culture was used as biosurfactant for the degradation of crude-oil hydrocarbons conducted in a 1.0 litre closed culture. the medium was mineral media with 1% (v/v) crude oil added as substrate. there were three kinds of petrobacter cultures used in this research, i.e. acinetobacter sp., b. cereus, and petrobacter consortium consisted of acinetobacter sp., bacillus cereus, enterobacter sp., and pseudomonas sp. this experiment was designed to be conducted in batch reactor. there were three treatments of petrobacter culture and another three treatments of petrobacter culture plus a. chroococcum as co-culture. overall, there were six combinations of treatments each with three replications. the density of the inoculant petrobacter or a. chroococum used in every closed culture was 2% (v/v). biodegradation process took place for 60 days. for the first 7 days samples were taken at 24-hour intervals. afterwards, the interval was 7 days. the ph of the culture condition was adjusted to 7 and then incubated at a 100 rpm shaking speed. variables observed during biodegradation process were: cell viability, the reduction of total petroleum hydrocarbons (tph) and residual compounds of biodegradation products. closed cultures without petrobacter or a. chroococcum were prepared as controls. total petroleum hydrocarbons and determination of its residual compounds. the n-hexane extraction method of iwabuchi (iwabuchi et al. 2002, modified) was a modified and employed to isolate the residue of total petroleum hydrocarbons from test cultures. samplings for analysis were taken at 24-hour interval for the first 7 days and there after at 7day interval. the gravimetric method was employed in tph biodegradation. first stage tph was extracted using nhexane solvent. the ratio of sample to n-hexane solvent was 1:1, then the solution was then mixed using a vortex mixer at 10 rpm. the second stage of the process was separating tph by centrifugation at 3500 x g for 10 minutes. this stage produced three layers, at the bottom was the bacterial cells, in the middle was media residue, and at the top was crude oil dissolved in n-hexane. oil and n-hexane were then poured into a bottle and the extraction process was repeated again until the solution became clear. the third stage was to recover the dissolved tph through evaporation at 70 oc. after that the weight of the tph residue was determined. after crude-oil degradation, hydrocarbon residues were analyzed using gc/ms. analyses to check the types of compounds contained in the samples before and after processing were conducted at three observation points, i.e. at the beginning of the process, 14 days after starting the process, and at the end of the process. (gc separate the compounds in a gas phase by passing the gas flow through a stationary phase). retention time in gc indicates the identity of the compounds. ms is a combination of mass analysis used to separate and identify the ions that comprise the components in a complex mixture. results crude-oil hydrocarbons and the growth of tested bacteria. the biodegradation process by three petrobacter cultures declined during the exponential growth phase of bacterial cells (figure 1). the rate of crude-oil degradation by b. cereus decreased sharply during the first 14 days of incubation (figure 1a), while with the acinetobacter sp., the decrease happened during the first 7 days (figure 1b). the reduction of total petroleum hydrocarbons by petrobacter consortium happened more quickly, also in the first 7 days when the growth was at exponential phase (figure 1c). after the exponential growth phase, the bacterial population declined, and biodegradation process continued slowly until the end of the incubation period. specific biodegradation rate and its efficiency. the specific tph biodegradation rate by b. cereus, acinetobacter sp., and petrobacter consortium increased with the addition of a. chroococcum (table 1). the same also applied to the figure 1 total petroleum hydrocarbons ( ) and bacterial cell biomass ( ) during the growth of bacillus cereus (a), acinetobacter sp. (b), and petrobacter consortium (c) in mineral media plus 1% crude oil over 60-day incubation period. 0 . 8 0 . 7 0 . 6 0 . 5 0 . 4 0 . 3 0 . 2 7 x 108 6 x 108 5 x 108 4 x 108 3 x 108 2 x 108 1 x 108 0 t o ta l p e tr o le u m h y d ro c a rb o n s ( % ) b io m a ss c o n so rt iu m ( c f u /m l) c 0 10 20 30 40 50 60 time (day) 0 . 8 0 . 7 0 . 6 0 . 5 0 . 4 0 . 3 0 . 2 6 x 107 5 x 107 4 x 107 3 x 107 2 x 107 1 x 107 0 t o ta l p e tr o le u m h y d ro c a rb o n s ( % ) b io m a ss ( c f u /m l) 0 10 20 30 40 50 60 time (day) b 0 . 8 0 . 7 0 . 6 0 . 5 0 . 4 0 . 3 0 . 2 0 . 1 4 x 108 3 x 108 2 x 108 1 x 108 0 t o ta l p e tr o le u m h y d ro c a rb o n s ( % ) b io m a ss ( c f u /m l) 0 10 20 30 40 50 60 time (day) a 6 parnadi et al. microbiol indones efficiency of crude-oil hydrocarbon biodegradation by b. cereus, acinetobacter sp., and petrobacter consortium which was higher compared to control, either with or without the addition of a. chroococcum (tabel 2). the degradation rates were determined from the linear portion of the degradation curves. the number of the hydrocarbon compounds residues after biodegradation process. the result of he analysis of hydrocarbon residual compounds qualitatively gave proven that a. chroococcum was capable encreasing the removal of tph. it can be showed that the number of the tph substances detected after biodegradated were qualitatively decreasing (the number of the hydrocarbon substances showed by the number of the peak area in figure 2, 3 & 4). the result showed that the crude-oil components comprise 52 hydrocarbon compounds (t-0) dominated by aliphatic hydrocarbons, i.e. around 90% alcane compounds and 10% aromatic compounds such as benzene and naphthalene. degradation by b. cereus during the first 14 days still left 37 hydrocarbon compounds as residue (figure 2a). the culture with a. chroococcum as co-inoculant had more effective degradation process; this was shown by 16 hydrocarbon compounds as residue (figure 2b). the residue from hydrocarbon degradation by acinetobacter sp. still contained 42 compounds, while the addition of co-inoculant made the process higher with only 15 compound detected as residue (figure 3a,b). likewise to hydrocarbon biodegradation by consortium culture, which was higher with the addition of coinoculant a. chroococcu. the residue from hydrocarbon degradation by consortium. still contained 19 compounds, while the addition of co-inoculant made the process higher with only 15 compound detected as residue (figure 4a,b). that qualitatively data suggest that the removal of the crude oil hydrocarbon was enhanced in the petrobacter culture with the addition of a. chroococcum. discussion in general, the bacteria used in this experiment actively utilized the substrate during logarithmic phase of their growth. this happened because crude oil component as the available hydrocarbons were in the form of aliphatic hydrocarbon groups that could easily be degraded and table 1 specific degradation rate of crude-oil hydrocarbons by three petrobacter isolates isolate biodegradation rate (g/l day-1) b. cereus b. cereus + a. chroococcum acinetobacter sp. acinetobacter + a. chroococcum consortium consortium + a. chroococcum 0 . 0 1 5 8 2 0 . 0 1 6 1 2 0 . 0 1 2 1 2 0 . 0 1 4 7 2 0 . 0 1 7 6 6 0 . 0 2 7 0 9 table 2 efficiency of crude-oil hydrocarbon removal using petrobacter treatment, with and without the addition of a. chroococcum (coinoculant) thp removal efficiency with a. chroococcum (%) enhancement tph removal efficiency (%) petrobacter isolate tph removal efficiency without a. chroococcum (%) control (no culture) b. cereus acinetobacter sp. consortium 26.51 + 1.33 62.17 + 2.11 57.26 + 1.87 71.18 + 1.56 39.67 + 1.98 76.79 + 1.84 70.70 + 1.53 85.57 + 2.28 13.16 + 0.65 14.62 + 0.27 13.44 + 0.34 14.39 + 0.07 figure 2. chromatogram of crude-oil hydrocarbons before and af t-14 t-60 t-0 figure 2 chromatogram of crude-oil hydrocarbons degradation on different days (t = 0, 14, 60) in the presence of b. cereus alone (a) or with suuplementation of co-inoculant (b). oil hydrocarbons before and after degradation by b. (a) with the presence of co inoculant a b immediately be used as substrate by the bacteria. the bacterial growth after the exponential phase fluctuated (figure 1). this caused of the hydrocarbons were transformed into intermediate compounds by petrobacter. these compounds could be toxic and disturb the growth and viability of petrobacter’s cells. the decline of viability could be interpreted as adaptation phase towards new substrate condition. other than the presence of intermediate compounds, fluctuations happened because of substrate (carbon source) unavailability, unlike at the beginning of exponential phase when the substrate could be used volume 1, 2007 microbiol indones 7 t-0 t-14 t-60 t-0 t-14 t-60 oil hydrocarbons before and after with the presence of coinoculant immediately. residual compounds as the result of transformation were often resistant to biodegradation process. besides, aggregation between the available hydrocarbon residual compounds frequently took place. those compounds were difficult for the bacteria to utilize. this condition, that restricts continued degradation, also correlated with enzymatic activity rate that played a role in further catabolism process of intermediate compounds (van eyk 1997). bacteria needed time to adapt to, and deal, with the new substrate condition and resistant hydrocarbon residual compounds. limiting factors in hydrocarbon degradation, among others, were low hydrocarbon solubility and slow hydrocarbon transformation from solid phase to liquid phase. biosurfactant produced by a. chroococcum could increase substrate and hydrocarbon preparation to be immediately used by microorganisms. the enhancement of the tph degradation rate was closely connected with the bacteria’s uptake capacity to utilize the substrate. the higher uptake capacity the bacteria had, the more effective biodegradation process would be. uptake process is closely related to mass transfer factor of the substrate in liquid phase. in the case of figure 3. on day (t) 0, 14, and 60. t-0 t-14 t-60 a figure 4. day (t) 0, 14, and 60. a t-14 t-60 t-0 8 parnadi et al. microbiol indones t-0 t-14 t-60 t 0 t 1 4 t 6 0 oil hydrocarbons before and after with the presence of coinoculant oil hydrocarbons before and after with the presence of coinoculant a figure 3 chromatogram of crude-oil hydrocarbons degradation on different days (t = 0, 14, 60) in the presence of acinetobacter sp. alone (a) or with suuplementation of co-inoculant (b). b a b figure 4 chromatogram of crude-oil hydrocarbons degradation on different days (t = 0, 14, 60) in the presence consortium alone. (a) or with supplementation of co-inoculant (b). oil hydrocarbons before and after (a) with the presence of co oil hydrocarbons before and after (a) with the presence of co-inoculant (b) on b oil hydrocarbons before and after (a) with the presence of co-inoculant (b) on b crude-oil hydrocarbons, mass transfer factor in liquid phase refers to solubility and dispersion level of oil droplets. dispersion level is the level when large oil droplets can be emulsified and form oil micelles with maximum size < 1µm which will make substrate absorption by petrobacter easier. co-inoculant a. chroococcum could consistently improve biodegradation rate and the efficiency of crude-oil hydrocarbon biodegradation (tabel 1 & 2). this bacterium had been confirmed acting as bioemulsifier that could increase the effectiveness of petrobacter in the biodegradation process. this could happen because the extracellular substance produced by co-inoculant a.chroococcum could act as bioemulsifier. this bioemulsifier could improve dispersion and solubility of crude-oil hydrocarbons by forming oil micelles measuring 0.5 µm< 1 µm. these sizes of oil micelles were the suitable and desirable substrate size for petrobacter cell to absorb them immediately so that hydrocarbon degradation process could be more effective. this was proven by the resistance and low solubility in hydrocarbon compound degradation process which happened after the first week. the hydrocarbon degradation process was improved with the presence of a. chroococcum as co-inoculant, it could increase the solubility of residual hydrocarbon compounds, which were difficult to dissolve and degrade. co-inoculant a. chroococcum, a biosurfactant producing bacteria, could function as a bioemulsifier reservoir in an environment containing hydrocarbons. the biosurfactant mechanism in crude oil biodegradation is shown in figure 6. the a. chroococcum functioned as biosurfactant reservoir by forming oil micelles from large oil droplets. the outer layers of oil micelles were biosurfactant that had hydrophylic sequence, while the inner layer had hydrophobic sequence, these oil micelles acted as transport vehicles to form aggregates and performed crude-oil absorption process through bacterial cell wall (van eyk 1997). the bioemulsifier produced by a. chroococcum consisted of various fatty acid compounds acting as bioemulsifier that had amphiphatic characteristics (suryatmana et al. 2006). these organic compound groups acted as ligan taxi system (transport vehicles) in the uptake process of hydrocarbons by petrobacter. therefore, petrobacter could utilize the substrate more effectively. the effect of biosurfactant on hydrocarbon degradation was based on increased hydrocarbon solubility and dispersion, and a change of the affinity between bacterial cell and hydrocarbon through induction that resulted in increased hydrophobicity of cell surface (zhang & miller 1995). with that mechanism, the rate and effectiveness of hydrocarbon adhesion was increased. the adhesion process was a very vital starting process in substrate uptake. consortium isolate showed the most effective characteristics in crude-oil hydrocarbon degradation. this was indicated by the highest biodegradation rate and efficiency. likewise, petrobacter consortium (more than one bacterial species) had higher probability of metabolic diversity compared to one bacterial species. in the consortium culture, combined performance in utilizing carbon source could happen between consortium members. a consortium of bacillus cereus, acinetobacter sp., enterobacter sp., and pseudomonas sp. was a mix live that could positively interact and cooperate in using diverse substrate sources contained in crude oil. every petrobacter isolate had a different level of ability in degrading aliphatic hydrocarbons contained in crude oil from duri, like b. cereus and acinetobacter sp. wich were able to degrade aliphatic hydrocarbon c7 to c18, while the consortium was able to degrade aliphatic hydrocarbons c7 to c22 (figure 5). with those various metabolic abilities, the consortium culture prove more effective in tph degradation activity compared to a single culture. the result was indicated that intermediate compounds formed in the degradation process were in the form of various organic compounds dominated by fatty acids. the fatty acids might be an intermediate substances formed in alcane hydrocarbon degradation process by all the petrobacter used. the hypothetic of the alcane (docosane) biodegradation pathway by the consortium was showed in figure 5 the degradation pathway patern of n-alcane: (1). the forming of docosanol (2), aldehyde formed from primary alcohol; (3) aldehyde was changed to the fatty acid (docosanoic acid). note: the docosanoic acid and the fatty acids are the predominantly intermediate substances detected after 14 days biodegradation process. figure 6 model mechanism for crude-oil transportation and solubility by biosurfactant (van eyk 1997). hydrophobic chains hydrophylic end large oil droplet microbial cell 0.05 µm < micelle < 1 µm 3 µm (ch2)19 ch2 ch3ch3 docosane (ch2)19 ch2 ch2ohch3 choch3 docosanoic acid (ch 2 ) 19 ch2 coohch3 (ch2)19 ch2 ch3ch3 oh (ch 2 ) 19 ch 2 ch3ch3 o acetic acid fatty acid (c 18 ) primary alcohol docosanol (prymary alcohol) secundery alcohol nadnadh o 2 h2o nadnadh metylcetone nadnadh (ch2)19 ch2 1 2 3 h 2 o acetyl ester h2o nadnadh aldehyde docosane ch 3 (ch 2 ) 19 ch 2 ch 3 docosanol (prymary alcohol) ch 3 (ch 2 ) 19 ch 2 ch 2 oh secundery alcohol ch 3 (ch 2 ) 19 ch 2 ch 3 oh nad nadh metylacetone ch 3 (ch 2 ) 19 ch 2 ch 3 nad nadh aldehyde (ch 3 (ch 2 ) 19 ch 2 cho nad nadh nad nadh h 2 o acetyl ester docosanoic acid (ch 3 (ch 2 ) 19 ch 2 cooh primary alcohol acetic acid fatty acid (c 18 ) h 2 o h 2 o o 2 o volume 1, 2007 microbiol indones 9 ( 3 ) ( 2 ) ( 1 ) figure 5. that hypothetic pathway was constructed based on the predominantly substances detected and was correlated by the aliphatic pathway of the hofrichter’s patern. the process from docosane (aliphatic hydrocarbon) may involved oxygenase enzyme systems. alcane monoterminal oxidation pathway involves alcohol dehydrogenase formed docosanol. the alcohol dehydrogenase is intracellular enzyme which works together with electron carrier systems. following aldehyde compounds (docosanal) formed from docosanol, that involve aldehyde dehydrogenase. the reaction process is followed by fatty acids (docosanoic acid) formation that requires nad+. the next process is where fatty acid assembled in cellular lipid and plays a role in provide energy through the β−oxidation pathway. the activation of fatty acid and coa results in the release of acetyl-coa, forming shorter fatty acids than alcane from original compounds, followed by the release of carbon dioxide. the acetyl coa then enters anabolic pathways to form new cells. in subterminal pathway reactions, alcane is changed into secondary alcohol before changed again into primary alcohol, and then transformed into fatty acid. acknowledgments we thank ministry of research and technology of republic of indonesia, for supporting this research through riset unggulan terpadu xi (rut xi) 2004-2006; reginawanti hindersyah, dept. of soil science, faculty of agriculture, padjadjaran university (unpad), for permission to use her isolate (azotobacter chroococcum) in this research; made rhena yasa (head of development laboratory), pt pertamina indonesia, balongan-indramayu production unit, for the assistance given during this research. references fritsche w, hofrichter m. 2000. aerobic degradation by microorganisms. in: rehm hj, reed g (ed). biotechnology. 2nd ed. weinheim: wiley-vch. iwabuchi n et al. 2002. extracellular polysaccharides of rhodococus rhodochrous s-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria. appl environ microbiol 68:2337. margesin s. 2001. bioremediation (natural attenuation and biostimulation) of diese-oil-contaminated soil in an alpine glacier skiing area. appl environ microbiol 67:3127-3133. suryatmana p, kardena e, ratnaningsih e, wisjnuprapto. 2006. [the characterization of biosurfactant from azotobacter chroococcum]. [in indonesian]. j mikrobiol indones 11:30-34. van eyk j. 1997. petrolium bioventing. roterdam-brookfield: a.a.balkema. vermani mv, kelkar sm, kramat my. 1997. studies in polysaccharide production and growth of azotobacter vielandii mtcc 2459, a plant rhizosfir isolate. soc appl bacteriol lett appl microbiol 24:379-383. wisjnuprapto, kardena e, parnadi ps, gladys s, kristanti n. 2005. bioremediation of petroleum oil contaminated soils. p 31-34. proceedings of the coe joint symposium on environmental engineering between hokkaido university, chungbuk national university and bandung institut of technology. sapporo-japan, 2-4 feb 2005. zang, miller. 1995. effect rhamnolipid (biosurfactant) structure on solubilization and biodegradation of n-alkanes. appl environ microbiol 61:2247-2251. 10 parnadi et al. microbiol indones guide for author.cdr guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. 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oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 and color photo(s) will be charged idr 600 000 per page. non-permi members. page charge is idr 000 (usd 850 100) per printed article up to 4 pages. additional page will be charge idr 250 000 (usd 30) per printed page without 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doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 and color photo(s) will be charged idr 600 000 per page. non-permi members. page charge is idr 000 (usd 850 100) per printed article up to 4 pages. additional page will be charge idr 250 000 (usd 30) per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 (usd 50) and color photo(s) will be charged idr 600 000 (usd 60) per page. open choice beside the normal publication process, in which acceses to th article is limited to customers who have purchased a subscription, there is also open choice article that is made available publicly through microbiology indonesia on line. for open choice articles, additional price will be charged to the authors. proof print and reprints proofs will be sent to the corresponding author to be edited and approved for publication. please return the proofs immediately. the author is required to sign the proof print as approval. final editing, without changing the content, is written directly on the proof print. author will receive ten offprints free of charge. reprints are available with a minimum order of 50 copies. guide for author microbiol indones page 1 page 2 4.mi694-hanies ambarsari available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.7.3.4issn 1978-3477, eissn 2087-8575 vol 7, no 3, september 2013, p 113-123 *corresponding author; phone/fax: +62-21-75791381/7563 116 , email: ummhamna@yahoo.com the cost of raw materials has been known to be the major limitation on the economic feasibility of acetone-butanol-ethanol (abe) production. about 6070% of the total abe production cost is the cost for substrates (madihah et al. 2001). therefore, many researches have been conducted to find the most economical substrates for abe production, for examples those who used simple sugars such as glucose, lactose, galactose, and xylose (shinto et al.2008; keis et al. 2001; bahl et al. 1986), or more in the effort of developing bioenergy source materials, a laboratorium study was performed to investigate the effects of substrate types and concentrations on the production of acetone-butanol-ethanol (abe) from various substrates by clostridium saccharoperbutylacetonicum n1-4 (atcc 13564) in defined tya (tryptone-yeast extract-acetate) media. the results demonstrated that the abe ratios produced by the strain n1-4 were greatly influenced by the types of substrate and the concentrations being used. it was also shown that fermentation of disaccharides (cellobiose and dextrin) by the strain n1-4 could give relatively as high butanol and abe yields as glucose fermentation after 24 h fermentation time. the strain n1-4 was also found to be able to ferment xylans from birchwood sources producing a relatively high abe concentration, especially at a prolonged incubation time (after 48 h incubation period). a subsequent experiment using several different concentrations of glucose or cellobiose revealed that the higher the concentration of the substrate being used, then the higher the total abe concentration or productivity could be obtained but not necessarily in the same increasing ratio. to the best of our knowledge, there was no other previous study about the effect of substrate type and concentration on the abe ratio by c. saccharoperbutylacetonicum n1-4 (atcc 13564) using various substrates in defined tya media, specifically by using xylans and dextrin substrates. key words: abe ratio, acetone-butanol-ethanol fermentation, biomass substrate effect, clostridium saccharoperbutylacetonicum n1-4 dalam rangka mendapatkan bahan baku bioenergi, sebuah studi laboratorium dilakukan untuk mengetahui pengaruh dari jenis dan konsentrasi substrat biomasa terhadap proses fermentasi langsung yang menghasilkan aseton, butanol, dan etanol oleh bakteri clostridium saccharoperbutylacetonicum n1-4 (atcc 13564) dalam media tya (tryptone-yeast extract-acetate) yang terdefinisi. hasil penelitian menunjukkan bahwa rasio dari aceton:butanol:etanol (rasio abe) ternyata sangat dipengaruhi oleh jenis substrat biomass dan konsentrasi yang dipergunakan dalam proses fermentasinya. data menunjukkan bahwa dengan menggunakan substrat disakarida (yang diwakili oleh dextrin dan cellobiose), strain n1-4 mampu menghasilkan abe dengan yield yang relatif sama tinggi dengan menggunakan glukosa setelah masa fermentasi 24 jam. strain n1-4 ternyata juga mampu memproduksi abe dengan konsentrasi yang cukup tinggi dengan menggunakan substrat xylan yang diekstraksi dari kayu, walaupun memerlukan masa fermentasi lebih panjang, yaitu 48 jam. percobaan dengan menggunakan konsentrasi yang berbeda dari substrat biomasa tersebut menunjukkan lebih lanjut bahwa semakin tinggi konsentrasi substrat biomasa yang digunakan, maka produksi abe akan semakin tinggi pula walaupun tidak selalu dalam rasio yang sama. sepanjang pengetahuan kami, belum ada studi yang sama dengan penelitian kami ini tentang efek dari jenis dan konsentrasi substrat biomasa terhadap rasio abe pada proses fermentasi aseton, butanol, dan etanol secara langsung oleh bakteri clostridium saccharoperbutylacetonicum n1-4 (atcc 13564) dalam media tya yang terdefinisi. kata kunci: biomasa, clostridium saccharoperbutylacetonicum n1-4, efek substrat, fermentasi asetonbutanol-etanol, rasio abe production of acetone, butanol, and ethanol as bioenergy source materials by clostridium saccharoperbutylacetonicum n1-4 (atcc 13564) using different substrates 1 2,3 hanies ambarsari * and kenji sonomoto 1 institute of environmental technology (btl), indonesian agency for assessment and application of technology (bppt), building 412 puspiptek serpong, tangerang selatan 15314, indonesia; 2 laboratory of microbial technology, division of microbial science and technology, department of bioscience and biotechnology, faculty of agriculture, graduate school, kyushu university, 6-10-1 hakozaki, higashi-ku, fukuoka 812-8581, japan; 3 laboratory of functional food design, department of functional metabolic design, bio-architecture center, kyushu university, 6-10-1 hakozaki, higashi-ku, fukuoka 812-8581, japan complex substrates such as sago starch (madihah et al. 2001), maltodextrin (formanek et al.1997), packing peanuts (jesse et al.2002), corn starch (mcneil and kristiansen 1986), maize, and potato starch (nicole et al.1993), as well as xylans from different sources of hemicellulosic residues (qureshi et al. 2006; lemmel et al. 1985), wheat straws (qureshi et al. 2008) and other residues which were used either directly in the fermentation or with different types of pre-treatments before being used in the abe fermentation process. most of such experiments employed bacterial strains of c. acetobutylicum or c. beijerinckii, consequently there are a lot of information regarding the abe production performed by such bacteria. on the other hand, the investigations employing strains of c. saccharoperbutylacetonicum, in particular the strain n1-4, were still limited. this strain was chosen in our abe production study because it is hyperbutanol producing strain which can produce a high concentration of butanol (ogata et al. 1982). this strain was first isolated by hongo et al. in 1960 and then patented under us patent no. 2945786 (hongo 1960). this strain showed unusually large clostridial forms (spore-containing cells) and differences in substrate utilization (keis et al. 2001). it was also distinguished by a higher butanol/acetone ratio than with other industrial strains, i.e. 4:1 vs. 2:1 (biebl 1999). several reports on abe production employing this strain used only glucose or xylose as the carbon source (shinto et al. 2007; shinto et al. 2008; tashiro et al. 2007) or a limited number of complex substrates such as maize and molasses fermentation media (shaheen et al. 2000), palm oil waste (lee et al. 1995) and excess sludge from a local sewage disposal facility (kobayashi et al. 2005). a previous study by shinto et al. (2008) using a kinetic modeling method with glucose or xylose as the substrate in the tya media suggested that the strain n1-4 was substrate-dependent. nevertheless, there was no sufficient information about how the substrate type and concentration could affect the abe ratio of acetone:butanol:ethanol directly produced by the strain n1-4. therefore, the objective of this experiment was to investigate the performance of the strain c. saccharoperbutylacetonicum n1-4 (atcc 13564) in production of abe from fermentation process using several carbon sources that have not been investigated previously using this strain, such as cellobiose, dextrin, as well as more complex carbohydrates such as xylan. in particular, the effect of such various substrate types and concentrations on the abe ratio will also be studied. 114 ambarsari and sonomoto et al. microbiol indones material and methods microorganism preparation. for a long-term stock culture, c. saccharoperbutylacetonicum n1-4 (atcc 13564) was previously kept as spores in sterilized sand, while for short-term storage, it was maintained in 15% pg (potato glucose) medium containing substances mentioned in previous reports (hipolito et al. 2008; tashiro et al. 2007). for refreshing the stock culture, per 1 ml of this stock was transferred into 9 ml of fresh pg medium, heatshocked in boiling water for 1 min, cooled in iced water for several min and anaerobically incubated at 30 °c for 28 h without agitation or ph control. this refresh culture was then transferred into tya (tryptone-yeast extract-acetate) fresh medium (10% v/v) to preculture the bacteria at 30 °c for 15 h without agitation. the tya medium components per liter of distilled water were 6 g of bactotryptone (difco), 2 g of yeast extract (difco), 3 g of ch coonh , 0.5 g of kh po , 3 4 2 4 0.3 g of mgso .7h o, and 10 mg of feso .7h o. the 4 2 4 2 initial ph of this tya pre-culture medium was adjusted to 6.5 with 1 n naoh or 1 n hcl, and glucose was then -1 added into the medium to constitute a 20 g l glucose concentration before it was sterilized at 115 °c for 15 min. main culture fermentation. main culture media for the fermentation experiment were prepared similarly to the pre-culture medium except the use of various substrates as the carbon resources, in addition to glucose as the control substrate. during the experiment dextrin, cellobiose, and xylan were used in addition to glucose as the control substrate. each of such carbon sources was a laboratory graded chemical and was added as a concentrate and diluted with distilled water to a final total reducing sugar -1 concentration of 10 or 20 g l . a separate experiment was also performed to investigate if the multiplication of increasing substrate concentration will also affect the multiplication of the increasing abe ratio by applying different concentrations of glucose and -1 cellobiose (i.e. 10, 20, and 40 g l of glucose; 10, 20, -1 and 40 g l of cellobiose). the main culture medium for each carbon substrate was prepared in a 500 ml flask with the working volume of 250 ml and the initial ph of 6.5 adjusted by adding 1 n naoh or 1 n hcl, after which was sterilized at 115 °c for 15 min. the inoculum was 10% (v/v) of the culture volume. following inoculation, the broth was sparged with filtered oxygen-free nitrogen gas for 20 min to maintain strict anaerobic conditions. all cultivations were static batch fermentations conducted anaerobically at 30 °c without ph control and agitation for a total fermentation period of 24 h or 48 h. sampling and analysis. sampling was performed periodically at every 6 h. one set of the samples was centrifuged with 15 000 rpm (or 20 400 g) at 4 °c for 10 min using a high speed refrigerated micro centrifuge (tomy mx-300; tomy tech, u.s.a. inc., tokyo, japan) and supernatants were obtained. the supernatants were analyzed for the acids and abe concentrations using a gas chromatograph (6890a; agilent technologies, palo alto, ca, usa) equipped with a flame ionization detector and a 15-m capillary column (innowax; i.d. 0.53 mm; 19095n-121; agilent technologies) using isobutanol as the internal standard with 1 m hcl (tashiro et al. 2004). the glucose concentration in the supernatant was determined with a glucose analyzer (biosensor bf-5; oji scientific instrument, osaka, japan). another set of the samples was directly analyzed for the ph, the bacterial density and the total sugar concentration. the ph values were measured using the ph-meter. the bacterial growth was monitored over time as the culture turbidity (od 562 nm) with a spectrophotometer (v-530; jasco, tokyo, japan). the residual total sugar was measured by a spectrophotometer (v-530) applying the phenolsulfuric-acid method described in detail elsewhere (dubois et al. 1956). calculation. as mentioned previously (jesse et al. -1 2002), the abe concentration (g l ) was defined as the difference between the abe concentration at the indicated fermentation time and that at the beginning of fermentation period. the abe yield was calculated as -1 the abe (g l ) produced at the indicated fermentation -1 time divided by the total sugar (g l ) being utilized at the same period (formanek et al. 1997). these definitions were also used to calculate the concentration of fermentation products (acetone, butanol, or ethanol) and the butanol yield. the abe productivity was -1 defined as the abe concentration (g l ) produced per hour. the abe ratio was defined as the ratio of acetone:butanol:ethanol by using butanol as the standard of calculation. results effect of substrate type and concentration on direct abe fermentation by the strain n1-4. the data clearly show that the abe ratio obtained in the fermentation by the strain n1-4 using different substrate types but the same concentrations, as well as by using the same substrate type but different concentrations, generally could be varied (table 1). particularly between glucose and cellobiose, the abe ratio of -1 0.22:1:0 using 10 g l glucose could be changed into -1 0.29:1:0.05 ratio using 20 g l glucose, further which -1 could be changed into 0.30:1:0.06 ratio by using 10 g l -1 cellobiose or into 0.41:1:0.07 ratio by using 20 g l cellobiose. the difference of abe ratio between glucose and dextrin fermentation was not relatively significant, -1 that was 0.22:1:0 with 10 g l of either glucose or -1 dextrin and 0.29:1:0.05 with 20 g l of glucose -1 compared to 0.24:1:0.04 with 20 g l of dextrin. on the other hand, the abe ratio between glucose fermentation and xylans fermentation was significantly varied, that -1 -1 was 0.26:1:0 or 0.44:1:0 using 10 g l or 20 g l of xylans, respectively, compared to those of glucose fermentation, that was 0.22:1:0 or 0.29:1:0.05 using 10 g -1 -1 l or 20 g l of glucose, respectively. there was also a difference in the product concentrations of the strain n1-4 between glucose and cellobiose fermentation (fig 1 and table 1). the abe concentration produced by the strain n1-4 using both -1 -1 concentrations of cellobiose (10 g l or 20 g l ) as the -1 -1 substrate was higher (4.1 g l or 9.5 g l ) than those -1 using the same concentrations of glucose (3.5 gl or -1 8.9 g l ). all concentrations of acetone, butanol, and ethanol produced in cellobiose fermentation were higher than those in glucose fermentation, except the -1 butanol concentration in the fermentation of 20 g l -1 glucose (6.6 g l ) which was slightly higher than that -1 -1 in the fermentation of 20 g l cellobiose (6.4 g l ). in addition, the yields of butanol and total abe as well as the abe productivity at 24 h incubation period were all relatively higher in cellobiose fermentation than those in glucose fermentation. the results of this experiment also confirmed that the strain n1-4 could utilize the xylans from birchwood. the xylans fermentation by this strain n14 was producing lower concentrations of acetone, butanol, and ethanol than the glucose fermentation after 24 h incubation period (table 1 and fig 1). interestingly, there was a significant increase in the fermentation products between 24 and 48 h incubation period using the xylans by the strain n1-4. after 24 h incubation period, the strain n1-4 could only produce -1 -1 0.8 g l and 1.4 g l total abe concentrations by using -1 -1 10 g l and 20 g l xylans, respectively (table 1). however, after 48 h incubation period, the total abe -1 concentrations increased up to 1.1 and 3.2 g l (or -1 increase up to 36% and 124%) by using 10 and 20 g l xylans, respectively (data not shown). volume 7, 2013 microbiol indones 115 acids using different substrates (fig 3). the acetate reutilization in xylans fermentation was much lower than those of the other substrates (fig 3a). on the other hand, the butyrate production was much higher in xylans fermentation than in the fermentation of other substrates being used in this experiment (fig 3b), especially up to 24 h, after which the butyrate started to being used. nevertheless, the acetate and butyrate were still present in the media until the end of fermentation time. fig 3c shows the reutilization of total organic acids using different substrates. effect of substrate increasing concentrations on abe production by the strain n1-4. the results show that the increasing substrate concentrations could significantly increase the total abe concentration, but not necessarily in the same increasing ratio. in glucose fermentation, the total abe concentration could -1 -1 increase from 3.5 g l to 8.1 and 12.4 g l (131% and 254% increase) by increasing the initial substrate from -1 -1 10 g l to 20 and 40 g l , respectively (table 2). similarly, in cellobiose fermentation using the there was different pattern of total sugar consumption and the bacterial density (od ) in xylans 562 fermentation by the strain n1-4 compared to those of the other substrates (fig 2a,b). there was a long lag period up to 12 h before the strain started to grow exponentially until 24 h incubation period and then entered the stationary phase. in contrast, by using the other substrates the strain n1-4 could directly grow exponentially until 18 h incubation period before the bacterial density declined. the ph change in the xylans medium was also different from those in the other media (fig 2c). the ph in xylans medium was relatively constant up to 12 h incubation period and start decreasing until 24 h incubation period before increasing again, while the ph in the other substrates media showed the similar decrease up to 6 h of incubation period which was then followed by the similar increase until the end of incubation period. there was different performance of strain n1-4 in the utilization or production of acids and total organic microbiol indones116 ambarsari and sonomoto et al. c-sources acetone -1 (g l ) butanol -1 (g l ) ethanol -1 (g l ) abe -1 (g l ) consumed substrate -1 (g l ) a:b:e ratio butanol yield -1 (ggt s ) abe yield -1 (ggt s ) abe productivity -1 -1 (g l h ) glucose10 0.6 2.9 0 3.5 10 0.22:1:0 0.28 0.34 0.15 glucose20 1.9 6.6 0.4 8.9 22 0.29:1:0.05 0.31 0.41 0.37 cellobiose10 0.9 3.0 0.2 4.1 10 0.30:1:0.06 0.29 0.39 0.17 cellobiose20 2.6 6.4 0.4 9.5 20 0.41:1:0.07 0.32 0.47 0.40 dextrin10 0.5 2.2 0 2.6 7.6 0.22:1:0 0.28 0.35 0.11 dextrin20 1.3 5.6 0.2 7.1 22 0.24:1:0.04 0.26 0.33 0.30 xylan10 0.2 0.6 0 0.8 4.8 0.26:1:0 0.13 0.16 0.03 xylan20 0.4 1.0 0 1.4 7.8 0.44:1:0 0.13 0.18 0.06 table 1 effect of substrate type on abe production and abe ratio by clostridium saccharoperbutylacetonicum n1-4 atcc -1 13564 after 24 h (substrate concentration 10 or 20 g l ; working volume 250 ml) note: number following the substrate indicate the concentration of the substrate c-sources acetone -1(g l ) butanol -1(g l ) ethanol -1(g l ) abe -1(g l ) consumed substrate -1 (g l ) a:b:e ratio butanol yield -1 (ggt s ) abe yield -1(ggt s ) abe productivity -1 -1 (g l h ) glucose10 0.6 2.9 0 3.5 10 0.28 0.34 0.15 glucose20 1.7 6.1 0.3 8.1 20 0.30 0.40 0.34 glucose40 2.8 9.0 0.6 12.4 31 0.29 0.40 0.52 cellobiose10 0.9 3.0 0.2 4.1 10 0.29 0.40 0.17 cellobiose20 1.9 6.0 0.4 8.3 20 0.300 0.41 0.34 cellobiose40 3.0 8.8 0.5 12.3 37 0.22:1:0 0.28:1:0 0.31:1:0.07 0.30:1:0.06 0.31:1:0.06 0.35:1:0.06 0.240 0.34 0.51 table 2 effect of glucose or cellobiose concentrations on the abe fermentation by clostridium saccharoperbutylacetonicum n1-4 atcc 13564 after 24 h (working volume 250 ml) -1 note: number following the substrate indicate the concentration of the substrate in g l production, in both batchand continuous-culture systems (jones and woods 1986; amador-noguez et al. 2010; heluane et al. 2011; ambarsari and sonomoto 2012). in batch culture, it was reported in several previous studies employing c. acetobutylicum strains that only acids were produced when the concentration of the carbon source was limited (long et al. 1984; monot et al. 1982). in particular, when glucose was present below the threshold level of about -1 10 g l , then no shift to solvent production was obtained since all the glucose was consumed during the acidogenic phase and cell yield was reduced (long et al. 1984). similar results were also obtained in continuous cultures, such that it is now generally accepted that, under conditions of carbon source limitation, there is insufficient amount of acid end products which can be generated to reach the threshold concentration needed to induce the solvent production (jones and woods 1986). nevertheless, as far as we know, there were no other studies previously done to investigate the effect of substrate type and volume 7, 2013 microbiol indones 117 -1 increasing initial substrate concentrations from 10 g l -1 to 20 and 40 g l , the total abe concentrations could -1 -1 be enhanced from 4.1 g l to 8.3 and 12.3 g l , or there was an increase of 102% and 200%, respectively. the abe ratio in glucose fermentation was also significantly affected by the increasing initial substrate concentration, but not in the case of cellobiose fermentation. the yields of butanol and total abe were also not affected by the increasing initial substrate concentrations, but more likely to be influenced by the total sugar (substrate) being consumed by the strain -1 n1-4. by using 40 g l of substrate, the total sugar utilization by the strain seemed to be incomplete, as there was some residual substrate left in the glucose or cellobiose media. discussion there have been a number of previous studies about the effect of nutrient, including the carbon substrate, on the onset and maintenance of solvent a b c d 0 1 2 3 0 10 20 30 40 50 fermentation time (h) -1 a ce to n e co n ce n tr at io n ( g l ) 0 0.2 0.4 0.6 0 2 4 6 8 0 10 20 30 40 50 fermentation time (h) 0 3 6 9 12 0 10 20 30 40 50 fermentation time (h) fermentation time (h) -1 e th an o l co n ce n tr at io n ( g l ) -1 b u ta n o l co n ce n tr at io n ( g l ) -1 a b e c o n ce n tr at io n ( g l ) 0 10 20 30 40 50 fig 1 concentrations of acetone (a), butanol (b), ethanol (c), and total abe (d) directly produced by c. saccharoperbutylacetonicum n1-4 atcc 13564 using different substrates. fermentation conditions: initial ph = 6.5; working volume = 250 ml; -1 -1 -1 static batch culture without ph control. symbols: (◇) glucose 10 g l ; (◆) glucose 20 g l ; (△) cellobiose 10 g l ; (▲) -1 -1 -1 -1 -1 cellobiose 20 g l ; (□) dextrin 10 g l ; (■) dextrin 20 g l ; (○) xylan 10 g l ; (●) xylan 20 g l . moreover, although it was reported previously that in the fermentation broth employing c. acetobutylicum or c. beijerinckii, the typical abe ratio was 3:6:1 (qureshi et al. 2008; qureshi et al. 2006) or 0.5:1:0.17 with butanol as the major product, however it can be noted in our experiment that the strain n1-4 could vary the abe ratio depending on the type and concentration of the substrates being utilized. therefore, it can be hypothesized that the abe ratio obtained in an abe fermentation by a certain bacterial strain could be changed by changing the substrate type and concentration. our hypothesis was supported by the results from our experiments employing c. saccharoperbutylacetonicum n1-4 atcc 13564, also from previous works which suggested that according to the culture conditions, butanol and acetone production from c. acetobutylicum (matta-el-ammouri et al. 1987) or c. thermocellum (brener and johnson 1984) could be different. it was also confirmed in our experiment that the concentration using dextrin and xylans on the abe ratio from the fermentation process by the strain c. saccharoperbutylacetonicum n1-4 (atcc 13564). it was found in our experiment that the abe ratio produced by the strain n1-4 was very dependent on the type and concentration of substrates being used. these results were in accordance with other previous studies which suggested that depending on the nature of the carbohydrate and the culture conditions, the ratio of conversion to solvents can vary (monot et al. 1982; taha et al. 1973; bahl et al. 1986). our results were also in agreement with those of awang et al. (1992) employing c. acetobutylicum p262. they found that butanol concentration was more related to the type and amount of carbohydrate utilized. a previous report from our laboratory by applying a sensitivity analysis using glucose and xylose as the substrates also revealed that the strain n1-4 could produce higher butanol in a substrate-dependent pathway (shinto et al. 2008; shinto et al. 2007). microbiol indones118 ambarsari and sonomoto et al. a 0 7 14 21 28 0 10 20 30 40 50 fermentation time (h) -1 t o ta l s u g ar c o n ce n tr at io n ( g l ) b 0 3 6 9 0 10 20 30 40 50 fermentation time (h) b ac te ri al d en si ty ( o d ) 5 6 2 c 0 2 4 6 8 fermentation time (h) p h o n m ed ia 0 10 20 30 40 50 fig 2 total sugar consumption (a), bacterial density (a), and ph (c) during direct abe fermentation by c. saccharoperbutylacetonicum n1-4 atcc 13564 using different substrates. fermentation conditions: initial ph = 6.5; -1 working volume = 250 ml; static batch culture without ph control. symbols: (◇) glucose 10 g l ; (◆) glucose 20 g -1 -1 -1 -1 -1 -1 l ; (△) cellobiose 10 g l ; (▲) cellobiose 20 g l ; (□) dextrin 10 g l ; (■) dextrin 20 g l ; (○) xylan 10 g l ; (●) xylan -1 20 g l volume 7, 2013 microbiol indones 119 employing c. thermocellum (ng and zeikus 1982) and a cellulolytic mesophilic clostridium sp. strain h10 (giallo et al. 1983). in those studies, it was demonstrated that by c. thermocellum the substrate uptake rates in cellobioseand glucose-grown cell -1 suspensions were 18 and 17 nmol min mg (dry weight), respectively and the molar yields were 38 on cellobiose and 20 on glucose, while the cellulolytic mesophilic clostridium sp. strain h10 presented a better adaptation to cellobiose than to glucose, producing higher fermentation products in cellobiose than in glucose fermentation. it was suggested from those studies that the better yield obtained on cellobiose may be the result of a higher level of phosphorylation if a cellobiose phosphorylase occurs. we suggested that a further experiment was then needed to investigate the type of enzyme involved in the cellobiose direct utilization by the strain n1-4, whether it is cellobiase as detected previously in c. acetobutylicum (lee et al. 1985) or cellobiose phosphorylase as detected in c. thermocellum or in the -1 concentrations of acetone, butanol, and ethanol as well as the butanol yield, total abe yield and productivity in cellobiose fermentation by the strain n1-4 were indeed relatively higher than those in glucose fermentation. on the other hand, by using dextrin or xylan, the strain n1-4 could produce such products in lower values than by using glucose. the higher production in cellobiose fermentation than in glucose fermentation by the strain n1-4 was similar to the results obtained in a previous study (awang et al. 1992) employing c. acetobutylicum p262. in that study, they demonstrated that fermentation of cellobiose by c. acetobutylicum p262 led to conditions resulting in complete acid reutilization and the highest butanol concentration, while glucose had a greater enhancing effect on the sporulation process than starch and cellobiose which in turn could lower the butanol production. the better performance of our strain in cellobiose fermentation than in glucose fermentation was also in accordance with the results from other previous studies 0 0.6 1.2 1.8 0 10 20 30 40 50 fermentation time (h) -1 b u ty ra te c o n ce n tr at io n ( g l ) 0 1 2 3 4 0 10 20 30 40 50 fermentation time (h) -1 a ce ta te c o n ce n tr at io n ( g l ) 0 1.5 3 4.5 0 10 20 30 40 50 fermentation time (h) -1 t o ta l o rg an ic a ci d s (g l ) a b c fig 3 acetate concentration (a), butyrate concentration (b), and consumption of total sugar (c) during abe fermentation by c. saccharoperbutylacetonicum n1-4 (atcc 13564) using different substrates. fermentation conditions: initial ph = 6.5; -1 -1 working volume = 250 ml; static batch culture without ph control. symbols: (◇) glucose 10 g l ; (◆) glucose 20 g l ; (△) -1 -1 -1 -1 -1 -1 cellobiose 10 g l ; (▲) cellobiose 20 g l ; (□) dextrin 10 g l ; (■) dextrin 20 g l ; (○) xylans 10 g l ; (●) xylans 20 g l . employing various clostridial strains, in which the ethanol was always the lowest amount produced during the abe fermentation (jones and woods 1986). our previous report also indicated that the ethanol production in the presence of butyric acid was lower than that in its absence (tashiro et al. 2004). the data in table 2 clearly indicated the effect of increasing substrate concentrations on the abe production during fermentation process by the strain n1-4. our results show that the higher the substrate concentration, the higher the total abe concentration could be produced, but not necessarily in the same increasing ratio. however, the yields of butanol and total abe were not affected by the increasing substrate concentrations, especially in cellobiose fermentation. as indicated in our another separated experiment, even -1 by using the excessive cellobiose (80 g l ), the yields of butanol and total abe were the lowest, whilst on the -1 other hand, by using 5 g l (or 0.5%) the yields of butanol and total abe were the highest, which was similar with the result from another previous study by brener and johnson (1984) employing c. thermocellum to ferment a broad range of cellobiose concentrations. in that study, it was found that the bacteria did not grow at 5.0% cellobiose and the ethanol accumulation was 9 maximal (38.3 mol/10 cells) in cultures using an initial cellobiose concentration of 0.8%, but only 17.3 mol using 2.0%. our results obtained by using the increasing glucose concentrations were similar to the results obtained by shaheen et al. (2000), in which the increasing glucose concentrations caused the increasing solvent concentration and yield produced by c. saccharoperbutylacetonicum n1-4, and the glucose -1 concentration of 4% (or 40 g l ) was suggested to be the optimum concentration for glucose fermentation by this strain. cellobiose was chosen as the carbon source in this separate experiment because it is a hydrolysis product of cellulose which mostly contained in the abundant lignocellulosic materials, and the use of this carbohydrate could illustrate the potential to ferment sugars derived from hydrolysates of agricultural residues to butanol (qureshi and ezeji 2008). in contrast, the concentrations of acetone, butanol, and ethanol (fig 1) as well as the total sugar consumption (fig 2a) in xylan fermentation by the strain n1-4 were much lower than those in glucose, cellobiose, and dextrin fermentations. the butyrate production was much higher even though needed a longer period in xylan fermentation than that in the other substrates fermentation (data not shown). these cellulolytic mesophilic clostridium sp. strain h10. reutilization of acetate by the strain n1-4 using glucose, cellobiose, and dextrin was relatively in a similar rate (fig 3a), even though the production of acetone using such substrates varied significantly (fig 1a). these suggested that the acetate reutilization in this strain was partly contributing the production of acetone by using such substrates. in other words, it was suggested that the strain n1-4 could produce acetone by not only reutilizing the acetate in the media, but also by consuming the total sugars still available during the same period of fermentation (fig 2a). this suggestion was supported by early workers who had observed that once the solventogenesis occurred, the acids produced during the acidogenesis phase were reassimilated, however, the uptake of acetate and butyrate only happened when sugars were metabolized concomitantly (jones and woods, 1986). butyrate uptake in cellobiose fermentation until 24 h incubation period was higher than that in glucose and dextrin fermentation (fig 3b), which could be the cause of the higher production of acetone and butanol in cellobiose fermentation than in glucose and dextrin fermentation (fig 1a, fig 1b, and fig 2a). these results supported the conclusion made in previous study (hartmanis et al. 1984) stating that the uptake of acids and the formation of acetone are coupled and that there could not be any uptake of acetate or butyrate without the formation of an equivalent amount of acetone. also it was suggested before that in contrast to acetic acid (acetate), which specifically increases acetone formation, butyric acid (butyrate) increases both acetone and butanol formations (matta-el-ammouri et al. 1987). the previous report from our laboratory using the same strain with glucose as the substrate in a ph-stat continuous culture system also demonstrated that the presence of butyrate in the broth could induce and promote the butanol production (tashiro et al. 2004). another recent study also indicated that addition of 4 g -1 l butyric acid to tya medium could strongly improve the butanol and total abe production, suggesting that butyric acid could be potentially used as co-substrate for butanol production without contributing any remarkable inhibitory effects on cell growth (al-shorgani et al. 2012). ethanol concentrations in abe fermentation by the strain n1-4 using all utilized substrates in these experiments were lower than acetone concentrations (table 1 and 2) and required longer time than acetone and butanol production (fig 1c). these results were similar with the results from other previous experiments microbiol indones120 ambarsari and sonomoto et al. suggested that the different performances on abe production during fermentation process by the strain n1-4 using various types and concentrations of the substrates, in particular between the glucose and cellobiose fermentation, might be related to the different substrate uptake systems in the strain n1-4 as previously demonstrated in c. thermocellum. previous kinetic studies on c. thermocellum supported our hypothesis, which indicated that cellobiose and larger cellodextrin were taken up by a common uptake system, while glucose entered via a separate mechanism (strobel et al. 1995). they proposed a revised model for carbohydrate transport and utilization which incorporated the elements of atpdriven transport and internal phosphorylytic cleavage of intact-linked carbohydrates. their model could predict higher yields on oligosaccharides compared with that on the monosaccharide, if one assumes that an atp molecule is required for uptake of either glucose or other larger carbohydrates. it was also proposed in another study that phosphorylytic cleavage of cellobiose partially conserves the energy of the glycosidic bond as glucose-1-phosphate, and only one atp per cellobiose is required to form two activated glucose molecules. in contrast, hydrolytic cleavage of cellobiose leads to the consumption of two atp for the activation of the resulting glucose. thus, 0.5 atp per activated hexose is theoretically conserved through phosphorylytic cleavage, and this comparison predicts that bacterial yields on cellobiose would be greater than on glucose (strobel 1995). past work with c. thermocellum did indeed demonstrated that higher yields (ng and zeikus 1982) and lower maintenance energy coefficients were seen in cells grown on cellobiose versus glucose (strobel 1995). additionally, it was previously suggested that it was more efficient to take up an intact oligomer rather than cleave it extracellularly and transport the monomer sugar. it was then proved in c. thermocellum that the cellobiose and cellodextrin phosphorylase activities were detected in the cytosol and were not associated with cell membranes, meaning that the phosphorylation of carbohydrates occurred intracellularly (strobel et al. 1995). a recent study performed by servinsky et al. (2010) reported that c. acetobutylicum atcc 824 can uptake cellobiose directly by the phosphotransferase systems (ptss) and hydrolyzes cellobiose to glucose by the intracellular βglucosidases for further metabolism (noguchi et al. 2013). finally, a further experiment needs to be performed in c. saccharoperbutylacetonicum n1-4 volume 7, 2013 microbiol indones 121 results were compliant with those of a previous study which pointed out that the accumulation of organic acids (acetate and butyrate) greatly influenced the solvent production, in which the higher amounts of such acids appear to inhibit cell growth, sugar consumption and hence, reduce the total solvent production (madihah et al. 2001). our results in xylan fermentation were also similar those obtained previously by other researchers employing c. acetobutylicum (atcc 39236), in which the organic acids were mainly produced with only traces of acetone, butanol, or ethanol if xylans were directly used (lemmel et al. 1986). they also found that xylans fermentations were characterized by long lag times, slow fermentation rates (i.e. 190-240 h total fermentation time compared to 48-72 h when using starch as the carbohydrate source), and low xylanase activities. in addition, they never observed the complete consumption of xylans even though different types of xylans were used in their experiment, indicating that the limited amount of xylans fermented is probably due to the nature of the xylanase activity produced, not the growth conditions or media being used. interestingly, the bacterial curve obtained in the -1 fermentation of 20 g l xylans showed a diauxie growth with much lower bacterial density than the others (fig 2b). this phenomenon was maybe due to the effect of the slower total sugar consumption (fig 2a) in xylans fermentation. the first phase of bacterial growth (during the 6 h fermentation period) in xylans fermentation might use the little amount of solubilized xylans available in the beginning of fermentation period due to the autoclaved auto hydrolysis, such in the same way as the xylans solubilization due to the hot-liquidwater (lhw) pretreatment previously mentioned in a review on lignocellulosic pretreatments, which stated that due to the high water input in lhw pretreatment, the yield of solubilized (monomeric) xylans is generally also high (hendriks and zeeman 2009). this assumption was also supported by the data obtained in our separated experiment, which show that by using the very small working volume (10 ml) the consumed xylans after 24 h fermentation was much lower than that in the experiment using a higher working volume (250 ml) (ambarsari et al. 2010). the second phase of bacterial growth was then suggested to be caused by the concurrent consumption of total sugar and acids in xylans fermentation as happened previously in another study (monot et al. 1984). based on the results of our experiment, it could be tonicum` dsm 2152 and clostridium acetobutylicum dsm 792. 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saccharoperbutylacemicrobiol indones122 ambarsari and sonomoto et al. volume 7, 2013 microbiol indones 123 without carbon catabolite repression from mixed sugars with clostridium saccharoperbutylacetonicum n1-4. j biosci bioeng. 116(6):716-721. doi:10.1016/j.jbiosc.2 013.05030. ogata s, yoshino s, okuma y, hayashida s. 1982. chemical composition of autoplast membrane of clostridium saccharoperbutylacetonicum. j gen applied microbiol. 28(4):293-301. doi:10.2323/jgam.28.293. qureshi n, ezeji tc. 2008. review: butanol, `a superior biofuel` production from agricultural residues (renewable biomass): recent progress in technology. biofuels bioprod bioref. 2:319-330. doi:10.1002/bbb.85. qureshi n, li xl, hughes s, saha bc, cotta ma. 2006. butanol production from corn fiber xylan using clostridium acetobutylicum. biotechnol prog. 22(3):673-680. qureshi n, saha bc, hector re, hughes sr, cotta ma. 2008. butanol production from wheat straw by simultaneous saccharification and fermentation using 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caldwell fc, dawson ka. 1995. carbohydrate transport by the anaerobic thermophile clostridium thermocellum lqri. appl environ microbiol. 61(11):4012-4015. tashiro y, shinto h, hayashi m, baba s, kobayashi g, sonomoto k. 2007. novel high-efficient butanol production from butyrate by non-growing clostridium saccharoperbutylacetonicum n1-4 (atcc 13564) with methyl viologen. j biosci bioeng. 104(3):238240. doi:10.1263/jbb.104.238. tashiro y, takeda k, kobayashi g, sonomoto k, ishizaki a, yoshino s. 2004. high butanol production by clostridium saccharoperbutylacetonicum n1-4 in fedbatch culture with ph-stat continuous butyric acid and glucose feeding method. j biosci bioeng. 98(4):263268. doi:10.1263/jbb.98.263. keis s, shaheen r, jones dt. 2001. emended descriptions of clostridium acetobutylicum and clostridium beijerinckii, and description of clostridium saccharoperbutylacetonicum sp.nov. and clostridium saccharobutylicum sp.nov. intl j syst evol microbiol. 51(pt 6):2095-2103. kobayashi g, eto k, tashiro y, okubo k, sonomoto k, ishizaki a. 2005. utilization of excess sludge by acetone-butanol-ethanol fermentation employing clostridium saccharoperbutylacetonicum n1-4 (atcc 13564). j biosci bioeng. 99(5):517-519. doi:10.1263/jb b.99.517. lee sf, forsberg cw, gibbins ln. 1985. cellulolytic activity of clostridium acetobutylicum. appl environ microbiol. 50(2):220-228. lee tm, ishizaki a, yoshino s, furukawa k. 1995. production of acetone, butanol and ethanol from palm oil waste by clostridium saccharoperbutylacetonicum n1-4. biotech lett. 17(6):649-654. doi:10.1007/bf001 29394. lemmel sa, datta r, frankiewicz jr. 1985. fermentation of xylan by clostridium acetobutylicum. enzyme microb technol. 8(4):217-221. doi:10.1016/0141-022 9(86)90091-8. long s, jones dt, woods dr. 1984. initiation of solvent production, clostridial stage and endospore formation in clostridium acetobutylicum p262. appl microbiol biotechnol. 20(4):256-261. doi:10.1007/bf00250635. madihah ms, ariff ab, sahaid km, suraini aa, karim mia. 2001. direct fermentation of gelatinized sago starch to acetone-butanol-ethanol by clostridium acetobutylicum. world j microbiol biotechnol.17(6): 567-576. doi:10.1023/a:1012351112351. matta-el-ammouri g, janati-idrissi r, junelles am, petitdemange h, gay r. 1987. effects of butyric and acetic acids on acetone-butanol formation by clostridium acetobutylicum. biochimie 69(2):109115. mcneil b, kristiansen b. 1986. the acetone-butanol fermentation. advances in applied microbiology. allen i. laskin (ed.) 31:61-90, academic press, inc. monot f, engasser jm, petitdemange h. 1984. influence of ph and undissociated butyric acid on the production of acetone and butanol in batch cultures of clostridium acetobutylicum. appl microbiol biotechnol. 19(6):422426. doi:10.1007/bf00454381. monot f, martin jr, petitdemange h, gay r. 1982. acetone and butanol production by clostridium acetobutylicum in a synthetic medium. appl environ microbiol. 44(6):1318-1324. ng tk, zeikus jg. 1982. differential metabolism of cellobiose and glucose by clostridium thermocellum and clostridium thermohydrosulfuricum. j bacteriol. 150(3):1391-1399. noguchi t, tashiro y, yoshida, t zheng, j sakai, k, sonomoto k. 2013. efficient butanol production issn 1978-3477, eissn 2087-8575 volume 11, number 2, june 2017 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: 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escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up 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mechanism of puccinia abrupta var. partheniicola urediniospores during summer season mohamad taufik fauzi faculty of agriculture, universitas mataram, jalan majapahit 62, mataram 83125, nusa tenggara barat, indonesia phone/fax: +62-370-640744, email: taufikfz@telkom.net the success of applying plant pathogens as biological control agents of weeds relies on the ability of the biological control agent to persist at all parts of the year and to successfully go through all of its life cycle stages in the course of a single year. one important aspect of any plant disease life cycle is the ability to survive during unfavorable parts of the season. the research aimed at understanding the mechanism involved in the survival of puccinia abrupta var. partheniicola, a potential biological control agent of parthenium weed (parthenium hysterophorus), during simulated summer conditions had been undertaken in the field plot at the alan fletcher research station, brisbane. the urediniospores that were placed either on plant debris or on intact plants were exposed to summer conditions and were regularly tested their viability after being exposed. the results showed that the urediniospores could only survive for less than six weeks, which is less than half of the queensland summer where the weed is abundant. this indicates that the rust spores do not survive on infected parthenium weed debris during summer season. key words: survival, puccinia abrupta var. partheniicola, biological control of weeds the strategy used in implementing the rust puccinia abrupta var. partheniicola, a potential biological control agent of parthenium weed (parthenium hysterophorus l.), a very troublesome weed of agricultural areas in tropical and sub tropical regions such as australia, india, taiwan, vietnam and china, onto the weed is commonly referred to as ‘classical’ (tebeest et al. 1992). in this strategy, the establishment of the disease depends solely on the innate ability of the plant pathogen to establish (survive) and then to disperse. the success of this approach therefore relies on the ability of the biological control agent to persist at all parts of the year and to successfully go through all of its life cycle stages in the course of a single year. one important aspect of any plant disease life cycle is the ability to survive during unfavorable parts of the season, for example, soil-borne urediniospores of puccinia recondita rob. f.sp. tritici erics., the causal agent of leaf rust on wheat (triticum aestivum l.) are able to over summer on volunteer wheat plants and therefore are able to serve as a source of inoculums for the primary infection on autumn sown wheat (hassan et al. 1986). the survival of rust pathogenic biocontrol agents during adverse season (summer or winter) may well be on infected plant debris. the survival strategy of puccinia abrupt var. partheniicola a macrocyclic and autoecious rust, in the central queensland summer is unknown. however, since the rust’s teliospores do not germinate under normal field conditions (evans 1987), it is believed that this rust may survive by means of its urediniospores produced in an asexual life cycle phase. such spore may then remain on plant debris or in the soil. the objective of the present study was to determine whether the rust usediniospores could oversummer on the parthenium weed host or its debris under simulated queensland summer conditions. materials and methods summer survival of spores on plant debris. parthenium weed seeds collected from plants growing in the clermont region of central queensland were sown to a depth of 1 cm in seedling trays containing a peat and sand (1:3; v/v) potting compost enriched with complete fertilizer. the compost was then wetted to field capacity with tap water and placed in a glasshouse at 30/26 + 5 °c (day/night). when the seedlings were 10 days old they were transplanted to 20 cm diameter pots (one plant per pot) containing the same potting compost as described above. six-weeks-old parthenium weed plants were inoculated with the solution of urediniospores in 1 l sterile distilled water containing two drops of tween 20 at the rate of 20 000 spores ml-1 as determined using a haemocytometer. each plant was inoculated with 10 ml urediniospore solution using a wattyl jet-pack spray unit (wattyl australia pty., canada bay, nsw). the inoculated plants were then individually covered with previously misted (with distilled water), black plastic bag and placed in a dark growth chamber at 15 + 1 °c for 12 h. after this, the plastic bags were removed and the plants were placed in naturallylit glasshouse with a day/night temperature regime of 32/25 + 5 °c. the first symptoms of the rust disease appeared 10 days after inoculation. three weeks after the first symptom appeared, leaves had become heavily infected with the rust. infected leaves from some of the plants were removed and enclosed in 10 x 10 cm2 packet consisting of plastic mesh (sold as insect screen by kmart pty ltd., brisbane). these packets containing the infected leaves were then placed on the soil surface or hung on sticks 1 m above the soil surface in field plot at the alan fletcher research station (afrs), brisbane. a final treatment consisted of infected leaves that had been left attached to senesced parthenium weed plants persisting on land in the same field site. the field plot in which this experiment was undertaken had been previously cleared of other form of vegetation using a knock down herbicide. the 5 x 5 m2 field plot was fenced to the height of 150 cm with a thick plastic mesh. during the course of exposure of the leaf samples in the field, samples of infected leaves or packets were removed every 2 weeks. these samples were washed lightly to remove any adhering soil, the leaves then cut into 2 x 2 cm2 pieces microbiol indones 97volume 3, 2009 and placed into glass beakers (one sample per beaker) containing sterile distilled water. the beakers were then shaken gently to release spores from the leaves. the water washes were then spread over the surface of a water-agar medium and urediniospore germination counted 2 days later. this germination value was taken as indication of the spore viability. transfer of spores in the summer months. parthenium weed plants were produced by first sowing seed onto the soil surface of a 5 x 5 m2 fenced field plot. this plot had been prepared in the same way as in the first experiment. in addition, to these plants others were produced in 25 -cm diameter black plastic pots containing potting compost at afrs, brisbane. after seedling emergence and at the rosette stage of development, the plants in pot were inoculated with urediniospores. once infection has developed, these plants were then placed in the middle of the mature healthy plants, thick growing parthenium weed stand that had been produced in the fenced field plot. infection of some of the healthy plants was observed as soon as 3 weeks after the introduction of the infected plants. several branches bearing newly infected leaves were then randomly tagged and monthly observation taken on the formation of new pustules over a winter to a summer period (july to february). results summer survival of spores on plant debris. the viability of the urediniospores when first collected and prior to all the treatments ranged from 90 to 95%. this was done by spreading newly harvested urediniospores on the surface of a water agar medium and the urediniospore germination was counted. however, upon exposure to the different treatments viability decreases at different rates (fig 1). the lowest decrease in viability, after 2 and 4 weeks exposure, was found with urediniospores that remained on intact, but dead plants. this loss in viability (68 and 49 % urediniospore germination after being exposure for 2 and 4 weeks, respectively) was lower than that seen on leaves hanging or on the soil surface. the maximum period any urediniospores survival in the southern queensland summer was for between 4 and 6 weeks. transfer of spores in the summer months. three weeks (august) after the placement of the infected parthenium weed plants into the field plots containing the healthy parthenium weed plants, transfer of spore took place. a few pustules (>5 pustules per tagged branch) were observed on few previously healthy plants within several days. the number of new pustules forming was decreased as the summer temperature increased, three to five pustules developing in september, while only one pustule developed in october and november. the size of these newly formed pustules in the warmer months was also smaller than those formed earlier. no new pustules were observed on plants in december through february when the experiment was terminated. discussion the use of intact parthenium weed plants or their leaf debris in the present urediniospore survival study was based on the idea that p. abrupt var. partheniicola is an autoecious rust (parmelee 1967; evans 1987), able to complete its life cycle on just one host. this view suggests that the rust must survive, at all times of the year, on the weed host or in its debris and not on a secondary host. even though resting body spores (teliospores) have been found on plants growing in upland areas of mexico, they have not been noted to germinate under normal field conditions (20-30 °c) nor have they been noted to infect parthenium plants under any conditions (evans 1987). these observations therefore indicate that teliospores are unlikely to be a method of the rust survival in the field. the rust urediniospores used in the present study could only survive for a very short time (between 4 and 6 weeks; fig 1) on parthenium weed or its debris in the field during a southern queensland summer. while a typical queensland summer lasts for around 4 months, it seems that from the present studies that the rust urediniospores can only survive for less than half of this time. this result may due to the high temperatures during the mid summer (december to january) which could reach 30-35 °c. the urediniospores of this rust could not germinate at the incubation temperature of 30 °c (fauzi 1998). several rust pathogens had been reported to only survive for short period during the summer, for example, groundnut rust (p. arachidis speg.) could only survive for 20 days under the field conditions (25-28 °c) (sunkad and kulkarni 2007). similar result had been reported on the viability of p. jaceae var. solstitialis, a biocontrol agent of yellow starthistle, where urediniospores only survive for two to three weeks at warm summer temperatures (fisher et al. 2008). the field observation made by staff at the queensland department of natural resources the alan fletcher research station while working in central queensland have also indicated that no oversummering pustules have been observed (tomley, personal communication). the latest time that new pustules could be formed on new parthenium weed plants in the field was early summer, november. this observation made in this study is similar to that seen in the field in central queensland where new infections are not observed from november to march (moolayember creek, 90 km south of rolleston, central queensland). this indicated that the parthenium rust did fig 1 field survival of p. abrupt var. partheniicola urediniospores exposed to southern queensland summer conditions. urediniospores were either on parthenium weed plant, on leaves hanging in mesh bag, or on leaves placed in mesh bags on the soil surface. vertical bars represent the standard deviation. , soil surface; , plant; , hung. exposure time (weeks) g e rm in a ti o n ( % ) 1 0 0 9 0 8 0 7 0 4 0 3 0 2 0 1 0 0 2 4 6 6 0 5 0 0 microbiol indones98 fauzi not survive on infected plant debris nor did on visible infected parthenium plants (fig 1). it is therefore possible that other oversummering mechanisms, not studied at present, may be those that are used by the rust’s urediniospores. such mechanisms could involve survival on plants that are growing in cooler, wetter microclimatic regions that then provide long range dispersal of spores to other regions in autumn. such a mechanism has been seen for a yellow rust infecting wheat (rapilly 1979). this hypothesis is unlikely for parthenium weed since the availability of plants growing in autumn in other region into which the spores are dispersed is not commonly seen in the field in central queensland. another possible mechanism for oversummering is that the urediniospores survive on seed (as a seed-borne pathogen), in the soil, similar to that observed for colletotrichum gloeosporiedes f.sp. aeschynomene which has been found to re-infest northern joint vetch (aeschynomene virginica; tebeest and brumley 1978) from seed infested with c. gloeosporiodes. this survival mechanism for oversummering is unlikely to occur in the field in central queensland since there have been no reports of newly emerged parthenium weed seedlings that are already infected. this would be expected if the seed was carrying the pathogen. a third suggested mechanism for oversummering that is not supported by the present findings, is that the urediniospores oversummer on infected parthenium weed debris. a further possible mechanism for survival of the spores over summer involves the concept of latent infection. a latent infection mechanism is seen with the rust p. striiformis on wheat (dennis 1987). hungerford (1923) reported a similar mechanism for the rust p. glumorum on elymus glaucus plants over the summer period from june to september. adam and line (1984) had suggested a possible involvement of a latent infection as another survival mechanism of p. chondrillina, a successful biocontrol agent of the rush skeleton weed (chondrilla juncea l.), during the winter of 1979-1980 in washington. the possible involvement of latent infections might explain the survival mechanism of the rust under study in the field. the infection of the rust urediniospores on parthenium leaves did not form any visible symptoms because of the unfavorable environmental conditions of the hot and dry summer. however, they may have survived as mycelia on leaves, with pustules only beginning to appear when the cool autumn conditions appear. this hypothesis could be used to explain the new autumn infection of the rust p. abrupt var. partheniicola which only occurred on parthenium weed plants at moolayember creek, a site where the weed was present all the year round (tomley, personal observation). acknowledgement i thank ausaid australia for providing the scholarship to the author. i also thank alan fletcher research station, queensland department of natural resources, brisbane for the use of the field plot, glasshouse and laboratory and other facilities for running this experiment. references adam eb, line rf. 1984. biology of puccinia chondrillina in washington. phytopathology 74:742-5. dennis jl. 1987. effect of high temperatures on survival and development of puccinia striiformis on wheat. trans br mycol soc 88:91-6. evans hc. 1987. life-cycle of puccinia abrupta var. partheniicola, a potential biological control agent of parthenium hysteroporus. trans br mycol soc 88:105-11. fauzi mt. 1998. biological control of parthenium weed by puccinia abrupta var. partheniicola. [ph.d. thesis]. brisbane: the university of queensland. fisher jf, woods dm, smith l, bruckart iii wl. 2008. latent period and viability of puccinia jaceae var. solstitialis urediniospores: implication for biological control of yellow starthistle. biol control 45:146-53. hassan zm, kramer cl, eversmeyer mg. 1986. summer and winter survival of puccinia recondita and infection by soilborne urediniospores. trans br mycol soc 86:365-72. hungerford cw. 1923. studies on the life history of stripe rust, puccinia glumarum (schm.) erikss & henn. j agric res 24:6072 0 . parmelee ja. 1967. the autoecious species of puccinia on heliantheae in north america. can j bot 45:2267-328. rapilly f. 1979. yellow rust epidemiology. annu rev phytopathol 17:59-73. sunkad g, kulkarni s. 2007. studies on perpetuation and carry over of groundnut rust (puccinia arachidis speg.) in northern karnataka. karnataka j agric sci 20:297-300. tebeest do, brumley jm. 1978. colletotrichum gloesporioides borne within the seed of aeschynomene virginia. plant dis rep 62:6758 . tebeest do, yang xb, cisar cr. 1992. the status of biological control of weeds with fungal pathogens. annu rev phytopathol 30:637-57. mi661-17-07-12 (wahyuntari) skin (hide) contains several substances including collagen protein which is associated with keratin, elastin, albumin, globulins, mucoids, lipids, carbohydrates and mineral salts (joseph 1989). those substances exist in skins or hides in three layers. on the surface of intact skin are the keratinous epidermal layers which consist of epidermis, hairs, and hair root sheaths. below the epidermal layers is the basement membrane which is attached to the dermis or corium. available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.2.4issn 1978-3477, eissn 2087-8575 vol 6, no 2, june 2012, p 77-82 *corresponding author, phone: 21-7566922 ; e-mail:budiasih.wahyuntari@bppt.go.id +62-21-7560536, fax: +62properties of a bacillus megaterium dsm319 extracellular protease which are related to its application for depilating hides were investigated. the properties observed were optimum temperature and ph, the type of protease, and type of the protein which could be hydrolyzed by the enzyme. the enzyme was produced in a 3.5 o liters lkb jar fermentor in a medium containing (2.0% v/v molasses and 1.3% w/v urea at 37 c, ph 7.5, aeration 1 vvm, agitation 250 rpm for 24 h). the enzyme solution was concentrated by means of membrane ultrafiltration followed by ammonium sulfate precipitation at 70% w/v saturation. optimal temperature and ph were observed o at 30 c and ph 8, respectively. two mm pmsf and edta inhibited the enzyme leaving level of activity of 84.5 and 4.3% correspondingly, indicated that the crude enzyme might be a metal requiring serine protease. the presence of 0.5, 2.0 and 3.5 mm cacl caused an increase of the enzyme activity of 73, 88, and 79% 2 respectively. the enzyme was able to hydrolyze na-benzoyl-dl-arginine p-nitro anilide, a specific amino acid sequence cleaved by trypsin at a reaction rate of 0.024 absorbance value at 405 nm per min. the enzyme was capable of hydrolyzing bovine serum albumin, hemoglobin, and gelatin, and to hydrolyze alkaline soluble collagen and keratin. the k value of the enzyme for hydrolysis of bovine serum albumin and gelatin was 3.44 m -1 -1 and 1.65 mg ml , whereas v values were 8.09 and 55.24 mg ml , respectively. the experimental data showed max that the crude enzymes have potential for dehairing of cow hides. key words: depilating, metalloprotease, protease, serine penelitian ini mengamati sifat-sifat protease ekstrasel bacillus megaterium dsm 319 yang berkaitan dengan pemanfaatannya sebagai penghilang bulu pada kulit ternak. sifat-sifat yang diamati adalah suhu dan ph optimum, tipe protease dan jenis protein yang dapat dihidrolisis oleh enzim tersebut. enzim protease diproduksi dalam fermentor lkb 3,5 liter menggunakan medium dengan komposisi sebagai berikut: molase 2,0%, urea 1,3% b/v pada 37 ºc, ph 7,5 dengan aerasi 1 vvm, agitasi 250 rpm selama 24 jam. larutan enzim kasar dipekatkan dengan membran ultra filtrasi dilanjutkan pengendapan dengan ammonium sulfat pada tingkat kejenuhan 70% b/v. suhu dan ph optimal enzim teramati masing-masing pada 30 ºc dan ph 7,5. pmsf dan edta 2 mm menghambat aktivitas enzim hingga aktivitasnya masing-masing tinggal 84,5 dan 4,3%, yang mengindikasikan bahwa larutan enzim kasar antara lain mengandung protease serin yang memerlukan logam untuk aktivitasnya. adanya cacl 0,5 mm, 2,0 mm, dan 3,5 mm meningkatkan aktivitas protease masing-2 masing 73,88 dan 79%. protease yang diamati mampu menghidrolisis na-benzoyl-dl-arginine p-nitro anilide suatu deret asam amino spesifik yang dipotong oleh tripsin dengan laju reaksi sebesar nilai absorbansi 0,024 pada λ 400 nm per menit. protease ini juga mampu menghidrolisis bovine serum albumin, hemoglobin dan gelatin dan juga collagen dan keratin yang terlarut dalam basa. nilai k enzyme dalam menghidrolisis bovine serum albumin m -1 -1 dan gelatin masing-masing 3,44 dan 1,65 mg ml , sedangkan v nya masing-masing 8,09 dan 55,24 mg ml .max kata kunci : metalloprotease, penghilang bulu, protease, serin the dermis consists of a special type of collagen as well as various glycoproteins and protein glycans which interlinked in a close network (frendrup 2000). in leather processing, the corium is transformed into leather by removing all non collagenous substances. the common beam house operations are soaking, dehairing, liming, deliming, bating and pickling. the use of enzymes in the soaking and dehairing operations was reported to accelerate the processes. some microbial proteolytic enzymes have been reported as useful in leather processing (joseph 1989, dayanandan properties of an extracellular protease of bacillus megaterium dsm 319 as depilating aid of hides 1 2 budiasih wahyuntari * and hendrawati 1 division of biocatalyst technology, center for technology of bioindustry, agency for the assessment and application of technology, jalan mh thamrin 8, bppt bld ii/15, jakarta 10340, indonesia; 2 faculty of science and technology, syarif hidayatullah islamic state university, jalan ir h juanda 95, jakarta 15412, indonesia observed by assaying the activity in a temperature range of 20-60 ºc at ph 7.5 (the ph of enzyme production); whereas the ph-activity profile was studied by assaying protease activity in a ph range of 611 in universal buffer at 30 ºc (the optimal temperature resulted from previous experiment-the temperatureactivity profile). effect of protease inhibitor on enzyme activity. to determine the type of protease, prior to assaying activity, the enzyme was pretreated with protease o inhibitors at room temperature (~28 c) for 1 hour. the inhibitors used were phenyl-methyl-sulfonyl-flouride (pmsf) and ethylene diamine tetra acetic acid (edta). pmsf is a serine protease inhibitor and edta is a metalloprotease inhibitor. the concentrations of inhibitors tested were 2 and 5 mm. effect of divalent ions on enzyme activity. the effect of various divalent metal ions on enzyme activity was studied by incubating the enzyme in the presence of different concentrations of metal ions at room temperature and the residual activity was determined after 1.0 h. the metal salts studied were cacl , zncl , 2 2 mncl , mgcl and fecl at final concentration of 0.5 , 2 2 2 2 , 3.5, and 5 mm. hydrolytic activity of enzyme toward trypsinspecific substrate, some proteins, and kinetics parameters of the enzyme. the hydrolytic ability of the enzyme cleaving a trypsin chromogenic substrate and various proteins was observed. the chromogenic substrate used for the experiment was benzoyl-dlarginine p-nitroanilide (bapna). a 100 mg aliquot of bapna was dissolved in 100 ml 50 mm phosphate buffer ph 7. a 50 µl aliquot of crude enzyme was then added to 450 µl of substrate solution. the reaction mixture was incubated at 30 ºc and every 2 min the absorbance (a) of the reaction mixture in 10mm cuvette was measured at λ 405 nm. the activity of the enzyme increased the absorbance (a ).405 the proteins studied were bovine serum albumin, casein, gelatin, collagen, hemoglobin and keratin. casein, albumin and gelatin were dissolved in tris-cl buffer at ph 8. collagen, keratin and hemoglobin were dissolved in 1 % w/v urea in 0.1n naoh and the ph was then adjusted to ph 8 using 0.1 n hcl. an 800 µl aliquot of 1% protein suspension was combined with 200 µl enzyme solutions and then the mixture was incubated at 30 ºc for 1 h. the reaction was stopped by immersing the reaction mixtures in boiling water. the controls were run by combining the protein suspension with buffer solution (without extract enzymes) in the same proportion as the reaction mixtures. the et al. 2003; tang et al. 2004; macedo et al. 2005; pillai and archana 2008). the important factors in choosing enzymes for leather processing includes the fact that the enzyme should not attack collagenous materials and should stable under processing conditions. this experiment was to study the properties of a protease produced by b. megaterium dsm 319 that is related to its utilization in leather processing. the properties investigated were optimal ph and temperature of the enzyme activity, type of protease, and hydrolytic activity of the enzyme towards different kinds of proteins. materials and methods microorganism. b. megaterium dsm319 was used throughout the experiment. stock cultures were maintained in 50% (v /v) glycerol. materials. molasses (“gunung madu”, lampung, indonesia) and technical grade urea were used for enzyme production. all chemicals used for enzyme activity, protein assay and enzyme stability study were analytical grade. enzyme preparation. crude enzyme of bacillus megaterium dsm319 was produced in 2 l media containing 2% w/v molasses and 1% w/v urea in a biostat fermentor. fermentation conditions were: o agitation at 250 rpm, aeration 1 vvm, at 37 c and ph 7.5 for 24 h. the bacterial cell was separated by means of microfiltration and the enzyme solution was concentrated by membrane ultrafiltration followed by (nh ) so precipitation at 70% saturation. 4 2 4 assay of proteolytic activity. proteolytic activity was assayed by modification of the walter (1984) method. 400 µl of 1% w/v casein (hammarsten, merck) dissolved in tris/cl buffer ph 8 was incubated with 50 µl crude enzyme at 30 ºc for 10 min. units of enzyme activity were defined as the amount of enzyme that produced an equivalent to one mole of free tyrosine under standard reaction conditions (temperature and ph). relative enzyme activity is defined as % ratio of enzyme activity at certain conditions (various ph, temperature, inhibitors, ions) to the optimal condition (ph & temperature) without addition of inhibitor or ions). protein determination. protein assays were performed according to the bradford method (1976) using bovine serum albumin v fraction as protein standard. effect of ph and temperature on enzyme activity. the temperature-activity profile was 78 wahyuntari et al. microbiol indones hydrolytic activity was observed by sds-page by comparing the bands of proteins of the enzymatic reaction mixtures with their respective controls. enzyme kinetics using gelatin and albumin. gelatin and albumin concentrations used in the -1 experiments were 0.7, 0.8, 1.0, 2, and 4.0 mg ml . the enzymatic reactions were carried out under optimal conditions based on previous experiments. lineweaver-burk plots of the experimental results were made. application of the protease on dehairing cow hide. at our laboratory, the cow hide was immersed in enzyme containing phosphate buffer ph 8 for 2 h at 30 ºc, in a shaking incubator. then the mixture was kept at room temperature overnight. the amount of enzyme added was 5 % of the hide wet weight and the final -1 enzyme concentration used was 10 u ml . the hair was then removed mechanically. the experiment was carried out without any na s addition.2 the enzyme was also tested in commercial leather processing at “ud.sumber kulit” in magetan, east java. the dehairing process was in a horizontal drum with a capacity of 250 kg salted cow hide. the hide was added with water and 1% v/v enzyme (the enzyme final -1 concentration used was 10 unit ml ) and the drum was rotated for 1 hour. after this 1.5% w/v cao then added with rotation for 30 minutes. then 0.5% w/v n s (50% 2 of normal concentration used) was added with rotation for 1 h, then we added 1.5% cao w/vand rotated for 30 min, finally added with 100% water and kept overnight and washed in the following day. results effect of temperature and ph on enzyme activity. the assay was done at ph 7.5, the same ph as the initial ph of the fermentation (enzyme production conditions). the optimum activity was at 30-36 ºc. increasing the temperature up to 50 ºc resulted in decreasing relative enzyme activity down to 40% (fig 1). based on experimental data (fig 1), when the enzyme was incubated at temperature above 55 ºc, the activity was not measurable. at 30 ºc, the optimum ph of the enzyme activity was observed at ph 8 (fig 2). the enzyme was more stable at ph 7-8. at ph higher than 8, the enzyme assayable activity decreased drastically. effect of protease inhibitors and divalent ions on the enzyme activity. phenyl methyl-sulfonylflouride (pmsf), a serine inhibitor, at the concentration used, only partly inhibited the enzyme (fig 3). however ethylene diamine tetra acetate (edta) at 5 mm almost totally inhibited the enzyme (fig 3). at 2.0 mm pmsf decreased the enzyme activity to 84.5%, whereas 5 mm pmsf decreased the activity to 47.7%. in the presence of 2 mm edta, residual enzyme activity was only 4.3%. effect of some divalent cations on the protease 2+ activity. all divalent ions tested except ca ion decreased enzyme activity (fig 4). the calcium ion at 0.5 mm and 2.0 mm increased the enzyme activity to 2+ 63.3 and 68.7%. however, at 3.5 and 5.0 mm the ca ion decreased the enzyme activity to 71.0 and 72.0% respectively. volume 6, 2012 microbiol indones 79 fig 1 effect of temperature on protease activity in 50mm tris-cl buffer ph 7.5. fig 2 effect of ph on proatease activity at 30 ºc in universal buffer. hydrolytic enzymic activity on chromogenic substrate of trypsin and some other proteins. the crude protease was able to digest specific chromogenic substrate for trypsin. the equation of p2 nitroanilide release was y= 0.024 t + 0.664, r = 0.971 (fig 5). bovine serum albumin (bsa) and gelatin were completely dissolved in tris-cl buffer at ph 8 (fig 6 sds/page well 1a and 3a). hemoglobin was temperature ( )ºc -1 a ct iv it y ( u l ) 8 7 6 5 4 3 2 1 0 2515 20 30 35 40 50 60 6545 55 ph -1 a ct iv it y ( u l ) 0 1 2 3 4 5 6 5 6 7 8 9 10 11 1/s (1/%) 1 /v ( m g /m l) application for cow hide dehairing at our laboratory, the hide was soaked with enzyme only, without addition of n s. there were some hairs remaining on the hide. 2 experimental results of dehairing in commercial leather processing at “sumber kulit” in magetan, east java showed that the hide was completely dehaired (fig 8a2, b2). completely dissolved in 1% urea in 0.1n naoh ph 8 (fig 6 sds/page well 6a), but collagen and keratin were only partly dissolved (fig 6 sds/page well; 2a and 4a). the enzyme was able to hydrolyzed bovine microbiol indones80 wahyuntari et al. fig 3 effect of phenyl methyl sulfonyl flouride (pmsf) and ethylene diamine tetra acetate (edta) on enzyme activity at ph 8, 30 ºc. fig 4 the effect of divalent ions on protease activity. serum albumin, gelatin, and hemoglobin completely (fig 6 sds/page well 1a-1b; 3a-3b and 6a-6b). dissolved collagen and keratin was also hydrolyzed by the enzyme (fig 6 sds/page well 2a-2b and 4a-4b), but an insoluble fraction remained intact in the reaction tube after enzymatic hydrolysis. crude proteases extract (fig 6 sds/page well 5) was used as a control protein bands. enzyme kinetics on gelatin and albumin. the hydrolytic rate of the enzyme towards albumin and gelatin was observed. the experimental results, shown in indicates that hydrolysis rate of the protease on albumin 2 [1/v=160.73(1/s)+55.235, r =0.92] was much higher 2 than that of on gelatin. [1/v=49.072(1/s)+8.0948, r = 0.99] (fig 7). application of the protease on dehairing of cow hide. fig 8 shows the experimental enzyme incubation time (min) a 4 0 0 n m fig 5 protease hydrolyzed specific chromogenic substrate for trypsin, y= 0.024 t + 0.664, r2 = 0.971. fig 6 sds/page of hydrolysis products of proteases from b. megaterium dsm 319 on some proteins. 1a, bsa; 1b, hydrolyzed bsa; 2a, collagen; 2b, hydrolyzed collagen; 3a, gelatin; 3b, hydrolyzed gelatin; 4a, keratin; 4b, hydrolyzed keratin; 5, protease protein (control); 6a, hemoglobin; 6b, hydrolyzed hemoglobin. fig 7 kinetics of b. megaterium dsm319 proteases on gelatin ( ) and albumin (the lineweaver-burk .(‫ equation, r: 1/v=49.072(1/s)+8.0948, r2= 0.99; £: 1/v=160.73(1/s)+55.235, r2=0.92. δ 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0 control pmsf edta inhibitor (mm) a ct iv it y ( u /l ) 700 600 500 400 300 200 100 0 ca fe mg mn zn -1 a ct iv it y ( u l ) divalent salt 0 mm 0.5 mm 2 mm 3.5 mm 5.0 mm discussion experimental results (fig 1 and 2) showed that the enzyme was a neutral protease. it was active in the temperature range of 30-45 °c and ph 7-8, with optimum activity at 30 °c, ph 8. therefore the enzyme can be used at room temperature with slightly alkaline conditions. in tropical areas the soaking of hides is usually carried out at ambient temperature, which is around 28-32 ºc. since the enzyme activity decreased drastically at ph's higher than 8, the application of the enzyme needs a metal cofactor for its activity. the effect of some divalent ions on enzyme activity was observed. only the calcium ion enhanced protease activity, whereas other divalent ions (fe, mg, mn, and zn) decreased activity (fig 4). protease produced by bacillus pumilus that used for dehairing was also reported as a serine protease which needed a metal cofactor (wang et al. 2006). the presence of a trypsin like protease in the crude extract was also investigated. the experimental result showed that the crude protease of b. megaterium dsm volume 6, 2012 microbiol indones 81 fig 8 application of proteases from b. megaterium dsm 319 in dehairing of cow hide. a: raw hides, b: dehaired hides; a1 & b1: at our laboratory; a2 & b2 at sumber kulit in magetan, east java. enzyme should be done before alkalization (swelling) of the hides or in soaking process. a similar process has been practiced for more than a decade as reported by bezak et al. (1989) and brady et al. (1990). recent studies reported that a slightly alkaline protease produced by pseudomonas aeruginosa mcm b-326 had the potential to be used for depilating buffalo hide (zambare et al. 2011; pandeeti et al. 2011). this enzyme was active in the ph range of 7-9 and temperature range of 20-50 °c, with optimum activity at ph 8 and temperature 35 °c. inhibition studies of our enzyme showed that the protease produced by b. megaterium dsm 319 was slightly inhibited by pmsf. however, the enzyme was totally inhibited by edta, which indicates that the crude enzyme contains serine proteases and that the 319 contained trypsin-like protease as shown in fig 5. early reports on application of enzymes on the dehairing of hides mentioned that the enzyme used was trypsin (zugno 1992). the process of using trypsin in dehairing resulted in increased hide permeability that would promote the soaking process and so produce leather which would have a very clean hair pocket and grain. the most important consideration in selecting protease for dehairing of hide, that the enzyme should digest the epidermis and basement membrane but should not attack the collagen of the hide (dayanandan et al. 2003; wang et al. 2007). the basement membrane consists of globular glycoproteins, proteoglycan and non fibrous collagen which are target of proteolytic enzymes in the depilatory process (brady et al. 1990). dayanandan a, kanagaraj j, sounderraj l, govindaraju r, rajkumar gs. 2003. application of an alkaline protease in leather processing: an ecofriendly approach. j cleaner production. 11(5): 533–536. doi: 10.1016/s0959-6526(02)00056-2. tang xm, lakay fm, shen w, shao wl, fang hy, prior ba, wang zx, zhuge j. 2004. purification and characterisation of an alkaline protease used in tannery industry from bacillus licheniformis. biotechnol lett. 26(18): 1421–1424. doi: 10.1023/b:bile.0000045642.19299.3f. macedo aj, beys da silva wo, gava r, driemeier d, henriques jap, termignoni c. 2005. novel keratinase from bacillus subtilis s14 exhibiting remarkable dehairing capabilities. app environ microbiol. 71 (1): 594–596, doi:10.1128/aem.71.1.594–596. pandeeti evp, pitchika gk, jotshi j, nilegaonkar ss, kanekar pp, siddavattam d. 2011. enzymatic depilation of dnimal hide: identification of elastase (lasb) from pseudomonas aeruginosa mcm b-327 as a depilating protease. plos one 6(2): e16742. doi:10.1371/journal.pone.0016742.t001. pillai p, archana g. 2008. hide depilation and feather disintegration studies with keratinolytic serine protease from a novel bacillus subtilis isolate. appl microbiol biotechnol. 78(4): 643–650. doi 10.1007/s00253-0081355-z. walter h-e. 1984. in : h. u. bergmeyer and m. grassl (ed) proteinases in methods of enzymatic analysis. floridabasel: verlag chemie. p: 271-276. wang hy, liu dm, cheng cf, ma qy, huang q, zhang yz. 2007. screening and mutagenesis of a novel bacillus pumilus strain producing alkaline protease for dehairing. lett. app microbiol. 44(1): 1-6. doi: 10.1111/j.1472-765x.2006.02039.x. zambare vp, nilegaonkar ss, kanekar pp, 2007, production of an alkaline protease by bacillus cereus mcm b-326 and its application as a dehairing agent. world j microbiol biotechnol. 23(11): 1569-1574. doi 10.1007/s1274-007-9402-y. zugno la. 1992. the effect of trypsin on soaking of salt cured hides. j am leather chem assn. 87(6): 207-218. the experiment results showed that proteases produced by b. megaterium dsm 319 were able to digest albumin, gelatin, hemoglobin and urea soluble collagen (fig 6). application of the enzyme on the dehairing of cow hide showed that mechanical removal of the hair after soaking the hide with crude enzyme, without addition of n s, could not remove the hair completely (fig 8 2 b1). the trial application of the crude enzymes in commercial leather processing, addition of 50% n s of 2 normal used could remove the hair satisfactorily. recent study on the use of protease for depilation of hide show these proteases mostly belong to the keratinolytic group (macedo et al. 2005; pillai and archana 2008; cai et al. 2008) and elastases (pandeeti et al. 2011). the keratinolytic enzymes were reported that they could be used as hair-save depilating agent without addition of na s (macedo et al. 2005 ; cai et 2 al. 2008), whereas our enzymes for removing the hair completely still need addition of na s.2 based on the data and dehairing trial at commercial leather processing in magetan, east java, the crude protease produced by b. megaterium dsm319 can be applied for dehairing of cow hide. acknowledgements the authors express thanks to aldi sadana, alumni of faculty of animal sciences, institut pertanian bogor for his assistance in some of the laboratory work. references bezak a, matyasovsky j, janci v. 1989.the use of enzyme in soaking. j soc leather technol chem. 72: 145-147. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. j anal biochem. 72: 255-260. brady d, duncan jr, russell ae. 1990. a model for proteolytic depilation of skins. j am leather chem assn. 85(9): 334-343. cai cg, chen js, qi jj, yin y, zhengv xd. 2008, purification and characterization of keratinase from a new bacillus subtilis strain. j zhejiang univ sci b. 9(9):713-720. doi: 10.1631/jzus. b0820128. joseph kt. 1989. biotechnologies for beam house operation. j indian leather technol assoc. may: 157-162. fendrup w. 2000. hair-save unhairing methods in leather processing (report on regional programme for pollution control in the tanning industry in south east asia). united nation industrial development organization. 32 pp. microbiol indones82 wahyuntari et al. 1: 1 2: 2 page 3 page 4 page 5 page 6 04 lg.cdr a-amylase (1,4-a-d-glucan glucanohydrolase, ec 3.2.1.1) belongs to the hydrolase family of enzymes, which hydrolyze 1,4-a-glycosidic bonds of starch or glycogen in a random manner, producing various lengths of oligosaccharides with reducing glycosidic groups in an a-configuration (janecek and balaz 1992). the enzyme plays an important role in industries that use starch conversion, such as in glucose-syrup production and in the food, paper and textile industries, and in renewable energy development e.g. bioethanol production, or in biomass degradation (nielsen and borchert 2000; van der maarel et al. 2002; shigechi et al. 2004). biomass degradation requires an enzyme that is able to act on raw starch substrates, a characteristic that recently has been demonstrated for a-amylase from the yeast saccharomycopsis fibuligera r64 (sfr64) (hasan et al. 2008). a structure–function study revealed that a-amylase from sfr64 (sfamy) can degrade without being adsorbed onto raw starches (hasan et al. 2008). this finding was rather unique because raw starch degrading a-amylases are usually characterized by the presence of a starch binding domain, i.e. the c domain that interacts with the substrate (mahovic and janecek 2006). structural investigation of raw-starch degrading vol.10, no.1, march 2016, p 23-29 doi: 10.5454/mi.10.1.4 heterologous expression of a-amylase from saccharomycopsis fibuligera r64 and its tyr401trp mutant in pichia pastoris 1‡ 2§ ‡ 3‡ riezki amalia , wangsa tirta ismaya * , fernita puspasari , 4,5 1 3,5 1 khomaini hasan , toto subroto , dessy natalia , and soetijoso soemitro 1 laboratory of biochemistry, department of chemistry, faculty of mathematics and natural sciences, universitas padjadjaran, jalan singaperbangsa 2, bandung 40133, indonesia; 2 dexa laboratories of biomolecular sciences, industri selatan v, blok pp no. 7, kawasan industri jababeka ii, cikarang 17550, indonesia; 3 biochemistry research division, faculty of mathematics and natural sciences, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia; 4 faculty of medicine, universitas jendral achmad yani, jalan terusan jendral sudirman, cimahi 40285, jawa barat; 5 bioscience and biotechnology research center, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia a-amylase from saccharomycopsis fibuligera r64 is a non-adsorbing raw-starch degrading enzyme, a unique characteristic. this character is difficult to explain in the absence of its three-dimensional structure. here we discuss the expression of a-amylase from saccharomycopsis fibuligera in pichia pastoris and the effect of site directed mutagenesis on its activity. a model based on the structure of its homologs suggested mutation of codon of tyr401 into that of a trp residue. an activity study using whole cells p. pastoris showed similar substrate degradation rates by cells carrying either the native or mutant amylase encoding gene. however, the purified enzyme of the mutant strain showed faster starch hydrolysis. key words: a-amylase, pichia pastoris, raw-starch degrading enzyme, saccharomycopsis fibuligera r64, site directed mutagenesis a-amylase dari saccharomycopsis fibuligera r64 adalah enzim pendegradasi pati yang tidak teradsopsi ke pati mentah, suatu karakter yang unik. karakter yang unik ini sulit untuk dijelaskan karena ketidakadaan struktur tiga dimensinya. dalam artikel ini ditampilkan ekspresi enzim ini di ragi pichia pastoris dan efek mutagenesis terarah terhadap aktivitas enzim. model berdasarkan struktur homolognya menyarankan mutasi codon tir401 menjadi trp. uji aktivitas secara langsung menggunakan sel p. pastoris menunjukkan sel inang yang membawa gen pengkode enzim natif dan mutan mampu mendegradasi pati, tetapi kemampuan mengikat pati mentahnya tidak membaik. namun demikian, ketika menggunakan enzim murni, ditemukan bahwa mutan mampu menghidrolisis pati lebih cepat. kata kunci: a-amilase, bioinformatik, enzim pendegradasi pati mentah, mutagenesis terarah, saccharomycopsis fibuligera r64, studi struktural microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 ‡ these authors contributed equally *corresponding author; phone: +62-21-84891901, fax: +6221-89841905 ; email: wangsa.ismaya@dexa-medica.com amylolytic enzymes suggested that the capability to bind and to degrade raw-starch substrates is facilitated by a pair of tryptophan and/or of tyrosine residues, respectively (sorimachi et al. 1997). the presence of these structural motifs in sfamyr64 is not yet clear, therefore structural studies of the enzyme is of paramount importance. purification of sfamyr64 from s. fibuligera is challenging because of the presence of glucoamylase that shares very similar characteristics (ismaya et al. 2012), thereby requiring purification by hydrophobic interaction chromatography with low protein recovery. heterologous expression of sfamyr64 appears to solve the issues with contamination with the glucoamylase and low protein production. moreover, overexpression of sfamyr64 in pichia pastoris allows modification of the enzyme by means of gene mutagenesis to obtain a sfamyr64 recombinant with the desired properties. following this approach, the gene coding for sfamyr64 has successfully been cloned into the heterologous yeast p. pastoris km71h (natalia et al. 2015). following that success, we overexpressed the enzyme also in p. pastoris . gs115 furthermore, the encoding gene was modified, resulting in expression of modified sfamyr64 that demonstrated faster degradation of raw starch substrates. this paper discusses overexpression of sfamyr64 in p. pastoris and site directed mutagenesis of codon of tyr401 was changed into trp, resulting in a recombinant enzyme that degraded the raw starch substrate better. materials and methods microbial strain and plasmids. pichia pastoris gs115 and escherichia coli top10f' were obtained from invitrogen (carlsbad, ca – usa). the ppicza and pgemt vectors were obtained from invitrogen (carlsbad, ca – usa) and promega (madison, wi – usa), respectively. restriction enzymes, taq dna polymerase, and t4 dna ligase were obtained from fermentas (vilnius, lithuania). primers were synthesized by research biolabs (singapore). cloning and expression of sfamyr64 in pichia pastoris. the gene coding for sfamyr64 has previously been cloned into yep-secretex for e x p r e s s i o n o f r e c o m b i n a n t s f a m y r 6 4 i n saccharomyces cerevisiae (ismaya et al. 2012). the cloning was performed using the forward and reverse p r i m e r s d e s i g n e d f o l l o w i n g t h e p u b l i s h e d sfamyhut7212 sequence (itoh et al. 1987) to result in expression of the mature sfamyr64 (residues 26-494), and the genomic dna of s. fibuligera r64 as the template. the gene was inserted between the ndei and xbai restriction sites. the yep-secretex-sfamyr64 plasmid was employed as the template for cloning into a vector for expression in p. pastoris in the current work. the gene coding for sfamyr64 with additional ecori and xbai restriction sites was amplified by pcr, using gaattcgtgactctattcaaaagagaaa and tctagacatgaacaatgtcagaagc as the forward and reverse primers, respectively. the pcr product was then ligated to the pgemt vector to result in pgemt-sfamyr64. insertion of sfamyr64 into pgemt was confirmed by sequencing. the pgemtsfamyr64 plasmid was isolated and then digested with ecori and xbai. the resulting sfamyr64 fragment was sub-cloned into ppicza vector at the same restriction sites to result in ppicza-sfamyr64. this plasmid was then transformed into p. pastoris by means of the tm easycomp method according to the manufacturer's instruction ( , carlsbad, ca – usa). the p. invitrogen pastoris carrying ppicza-sfamyr64 was screened on yeast peptone dextrose (ypd) plates supplemented -1 with 100 mg ml zeocin and the overexpressed recombinant enzyme was designated as rsfamyr64. production, isolation, and purification of rsfamyr64. production of the recombinant enzymes was conducted according to the protocol from invitrogen ( , carlsbad, ca – usa). briefly, invitrogen a single colony of p. pastoris was inoculated in 10 ml o buffered glycerol complex medium (bmgy) at 28 c with a constant agitation of 200 rpm. after 18-20 h, the yeast cells were collected by centrifugation at 5 000 g o and 4 c for 10 min. the cells were resuspended in 250 ml buffered minimal methanol-histidine (bmmh) o medium and grown at 28 c at constant agitation of 200 rpm for 3 d with daily methanol supplementation. the recombinant enzyme in the overproduction media was separated from the yeast cells by centrifugation at 10 o 000 g for 5 min at 4 c; the supernatant was designated as the crude extract. enzyme activity was directly measured using this crude extract. the enzyme was recovered from the crude extract by precipitation with o ammonium sulphate at 70% saturation (w/v) at 4 c for 2-3 h with constant stirring. the precipitate was o collected by centrifugation at 10 000 g at 4 c for 10 min, the supernatant was decanted and the precipitate was resuspended in ±10 ml 20 mm potassium phosphate buffer, ph 6.0. the ammonium salts were o removed by overnight (15-18 h) dialysis at 4 c against 24 amalia et al. microbiol indones volume 10, 2016 microbiol indones 25 ±1 l of the same buffer. the ammonium sulphate free fraction was used for the characterization and subsequent analysis. characterization and protein analysis of rsfamyr64. the characterization of the recombinant sfamyr64 was performed as previously reported (hasan et al. 2008; ismaya et al. 2013). briefly, the enzyme activity was assayed using both soluble and raw starch substrates, based on measuring the formation of a starch – iodine complex (fuwa 1954) or the production of reducing sugars (miller 1959). the characterization involved establishing the optimum ph and temperature for reaction, the specific activity, adsorption onto substrates, and enzyme kinetics. all tests performed following procedures used with the wild type enzyme (hasan et al. 2008; ismaya et al. 2013). a qualitative assay was performed on ypd agar plates containing 1% or 2% starch and 0.5% methanol, where the residual starch was visualized with an iodine solution (fuwa 1954). the expression of the recombinant proteins was also evaluated using an sds-page analysis. bioinformatics analysis. amino acid sequences of sfamy analyzed were those of r64 (genbank: hq172905) (ismaya et al. 2013), hut7212 (genbank: p21567) (itoh et al. 1987), and kz (genbank: add80242.1) (hostinová et al. 2010). however, only the amino acid sequence of sfamyr64 was employed in the further structural analysis. the amino acid sequences of the a/b and c domains of sfamyr64 were independently subjected to a blast analysis (johnson et al. 2008) against the amino acid sequence of any proteins in the database and of proteins with known tertiary structure. in silico model of sfamy was prepared based on the structure of aspergillus niger a-amylase. the model was built using the program swiss model, and subsequently checked and validated using the programs lsqkab from the ccp4 package (collaborative computational project number 4 1994) and procheck (laskowski et al. 1993), respectively, and were finally manually evaluated using the program coot (emsley et al. 2010). the programs procheck and molprobity (chen et al. 2010) were employed for validation. the graphic representation of the model was prepared by the program pymol (delano 2008). residues important for the binding and degradation of raw starch substrates were evaluated by scanning for tryptophan and tyrosine residues and compared to the structures of a-amylase from barley (pdb: 1p6w), human saliva (pdb: 1mfv) (hostinová et al. 2010), thermoactinomyces vulgaris (pdb: 1uh4) (abe et al. 2004) and bacillus halodurans (pdb: 2c3h) (boraston et al. 2006). site directed mutagenesis of the gene coding for tyrosine to that of tryptophan. ppiczaasfamyr64 was used as the template for site-directed mutagenesis, which was carried out using quick-change site-mutagenesis according to the manufacturer's protocol (stratagene, la jolla, ca – usa). the mutagenic forward and reverse primers employed were c c a g a a a c g c c g c c g t tt g g c a a g a c t caagc and gcttgagtcttg-cca-aacggcg gcgtttctgg, respectively. the pcr conditions o included one cycle of one minute at 95 c, and sixteen o o cycles of 30 s at 95 c, one minute at 55 c, ten minutes o o at 68 c, and finally one cycle of five minutes at 68 c. the pcr product was transformed into e. coli top10f', which was further screened against low-salt -1 -1 lb containing 25 mg ml zeocin and 50 mg ml tetracycline. ppiczaa-sfamyr64 tyr401trp was isolated from positive clones and the mutation was confirmed by nucleotide sequencing analysis. the mutated plasmid was then transformed into p. pastoris for the overproduction of rsfamyr64 tyr401trp, which was designated as msfamyr64. production, isolation, purification, and characterization of the tyr401trp mutant were performed in the similar manner to rsfamyr64. results cloning and overexpression of rsfamyr64 in p. pastoris. the gene coding for sfamyr64 has successfully been cloned and overexpressed in p. pastoris gs115. p. pastoris colonies carrying ppiczaa-sfamyr64 appeared to grow well on plates supplemented with soluble starch (fig 1a). clear zones around colonies indicate starch degradation by rsfamyr64, whereas the wild type yeast itself is unable to perform starch degradation. furthermore, in expression tests at small scale, p. pastoris secreted 25 times more sfamy than the natural host s. fibuligera r64 and six times more than s. cerevisiae (fig 1b). however, the amylolytic activity of rsfamyr64 decreased during the production of rsfamyr64, particularly after the third day. an sds-page analysis of rsfamyr64 indicated the appearance of additional protein bands of lower molecular masses, which had not been observed in the first three days (data not shown). this observation suggests that enzyme degradation occurred therefore the enzyme was 26 amalia et al. microbiol indones harvested at the third day of fermentation. the molecular weight of purified rsfamyr64 (at ±56 kda) appeared higher than that of the wild type sfamyr64 (at ±54 kda). its optimum ph and o temperature for activity were 6.0 and 50 c, respectively. while the optimum temperature is the same as that of the wild type enzyme (from s. fibuligera r64), its optimum ph is slightly higher (up from 5.5). site directed mutagenesis and subsequent cloning and overexpression of the mutant. mutation of codon for tyr to that of trp was confirmed by sequence analysis, which showed the presence of the codon cca at the position 430 (fig 2a) instead of ata in the anti-sense sequence (tryptophan is coded by tgg while tyrosine is coded by tat). further, msfamyr64 was also expressed in p. pastoris at similar amount to rsfamyr64, as suggested by a qualitative enzyme activity assays (fig 2a). like rsfamyr64, the amylolytic activity decreased, also after the third day of fermentation. this result had been expected because the mutation was engineered not to alter the structure of the a-amylase, the enzyme responsible for the amylolytic activity of the p. pastoris clone. thus, mutation of tyr401trp has no effect on the amylolytic activity. characteristics of the tyr401trp a-amylase. in fig 1 (a) screening of pichia pastoris colonies that carry sfamyr64 encoding gene: numbered 1 is the untransformed p. pastoris. (b) comparison of sfamyr64 production with different hosts as compared to the natural producer. a b fig 2 (a) confirmation of tyr401trp mutation by sequencing analysis. (b) qualitative measurement of activities of rsfamyr64 (a1, b1) and msfamyr64 (a2, b2) on ytd plates containing 1% (a1, a2) and 10% (b1, b2) soluble starch. a b volume 10, 2016 microbiol indones 27 order to understand the effect of the mutation at protein level, the activities and kinetics of the purified rsfamyr64 and msfamyr64 were compared. interestingly, the activity of msfamyr64 on both soluble and raw-starch substrates was slightly higher that that of rsfamyr64. the highest activity of msfamyr64 with soluble and raw-starches were 7.8 -1 and 4.9 u ml , respectively, about ~10% higher than -1 those of rsfamyr64 (7.1 and 4.5 u ml , respectively). this 10% increase was not observed during the qualitative amylolytic assay on plates because high expression of sfamyr64 recombinants had masked the assay. the optimum ph and temperature for both recombinant and mutant enzymes were the same. the v values of rsfamyr64 and msfamyr64 for max soluble and raw-starches were identical, i.e. 7.7 and 2.1 -1 u min , respectively. the k values were also identical cat 1.1 per min and 0.5 per min for the soluble and rawstarches, respectively. the k of msfamyr64 for m -1 soluble and raw-starches were 14.2 and 2.7 mg ml , respectively, lower than the k of rsfamyr64 at 15.2 m -1 and 2.8 mg ml , respectively. consequently, the k /k cat m ratios of msfamyr64 for soluble and raw-starches were slightly better than that of rsfamyr64, which -1 were 0.07 and 0.17 ml (mg.min) , respectively (as -1 opposed to 0.08 and 0.18 ml (mg.min) , respectively). this observation suggests that the higher activities of msfamyr64 can be attributed to a better interaction with the starch substrates. furthermore, similar to the wild-type sfamyr64, both rsfamyr64 and msfamyr64 did not adsorb onto raw-starch. thus, the mutation of a tyrosine residue to tryptophan was insufficient to alter the capability of the enzyme to bind raw starch. subsequently, this mutation failed to improve the capacity of p. pastoris carrying msfamyr64 to degrade raw starch. discussion p. pastoris has widely been used in the production of recombinant enzymes (macauley-patrick et al. 2005) because of various advantages over other expression systems, especially its capability to perform post translational modification and high protein expression. our results also demonstrate the superiority of this expression system over the well know yeast s. cerevisiae, and is even much more powerful than the natural producer s. fibuligera. this provides an excellent opportunity to perform genetic modification of the enzyme to obtain amylolytic enzymes and/or yeast strains with the desired properties for applications. furthermore, proteolytic degradation upon expression of recombinant proteins in p. pastoris has often been reported, and, in fact, is a major drawback in the use of the system (potvin et al. 2012). to prevent this degradation, the enzyme was harvested after 3 d of fermentation. strains of s. fibuligera have been reported to secrete amylolytic enzymes with different thermal stability and raw-starch binding capacity. amazingly, these variations originate from slight differences in the amino acid sequences in strains r64 (ismaya et al. 2013), hut7212 (itoh et al. 1987), and kz (hostinová et al. 2010). mutations in the amino acid sequence of glucomyr64 remarkably cause the enzyme to adopt both the raw-starch binding capability of hut7212 and the thermal stability of kz (natalia et al. 2011). furthermore, the mutations in the amino acid sequences did not include bulky residues necessary for the binding of starch or raw-starch substrates; therefore, both sfamys from strains hut7212 and kz are likely also a raw-starch degrading but nonadsorbing enzymes. we have previously shown that sfamyr64 is a rawstarch degrading but non-adsorbing enzyme (hasan et al. 2008). this finding was later confirmed with a similar study performed with sfamykz (hostinová et al. 2010); this characteristic appears to be conserved among sfamys. structurally, a-amylase consists of three domains sequentially named a, b and c. the a/b domain is responsible for the catalytic activity (janecek et al. 1997), whilst the c domain is organized separately and is hypothesized to be the substrate binding domain (janecek and sevcik 1999; hasan et al. 2008). the model of sfamyr64 (fig 3) shows the arrangement of domains in the enzyme. two binding sites for raw-starch substrate were observed in the structure of glucoamylase from a. niger (sorimachi et al. 1997), which involve a rigid pair of tryptophan residues (binding site 1) and a flexible pair of tyrosine residues (binding site 2). two tryptophan residues in the first binding site are responsible for the binding of raw-starch, whilst one of the tyrosine residues in the second binding site is responsible for guiding linear starch chain to the active site, and is thereby responsible for raw-starch degradation (penninga et al. 1996). in sfamyr64, tyr401 and tyr488 have similar positions to serve in carbohydrate binding, but tryptophan does not. as the presence of the two binding sites for the binding of carbohydrate is fundamental (giardina et al. 2001), the absence of the tryptophan residue (the binding site 1) fig 4 ribbon representation of the structure of sfamy r64. 28 amalia et al. microbiol indones explains the inability of sfamyr64 for adsorption onto raw-starch granules. therefore, a tyr401trp mutant was prepared in order to introduce the capability of raw-starch degradation. introducing the raw-starch binding capability to carbohydrate acting enzymes is an attractive proposition, because it increases the local concentration of the substrate in the active site, thereby increasing the rate of the starch hydrolysis reaction (juge et al. 2002). this alteration increases the capacity of the enzyme and subsequently the p. pastoris clone to degrade raw-starch. in conclusion, we have successfully produced sfamyr64 in p. pastoris and its derivative that is engineered to degrade raw-starch. although the improvement was not obvious upon testing with whole cells, the engineered enzyme catalyzed the raw-starch degradation at a considerably higher rate. this result initiates further work to obtain p. pastoris carrying gene coding for sfamyr64 with the desired properties. acknowledgment we thank the indonesian research council (drn) and the directorate general for higher education (dikti) of the ministry of national education for their financial support. references abe a, tonozuka t, sakano y, kamitori s. 2004. complex structures of thermoactinomyces vulgaris r-47 α amylase 1 with maltooligosaccharides demonstrate the role of domain n acting as a starch-binding domain. j mol biol. 335: 811–822. doi: 10.1016/j.jmb.2003.10.078. boraston ab, healeym, klassen j, ficko-blean e,van bueren al, law v. 2006. a structural and functional analysis of α-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a 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glucoamylase bound to beta-cyclodextrin. structure 5: 657-661. van der maarel mjec, van der veen b, uitdehaag jcm, leemhuis h, dijkhuizen l. 2002. properties and applications of starch-converting enzymes of the alphaamylase family. j biotechnol. 94: 137-155. doi: 10.1016/s0168-1656(01)00407-2. volume 10, 2016 microbiol indones 29 page 1 page 2 page 3 page 4 page 5 page 6 page 7 02 fitri.cdr extended-spectrum beta-lactamase (esbl) is a group of enzymes responsible for the resistance of gram negative bacteria towards beta-lactam antibiotics. these enzymes are mainly produced by m e m b e r s o f t h e g r a m n e g a t i v e f a m i l y enterobacteriaceae such as escherichia coli and klebsiella pneumoniae, but other species such as acinetobacter baumanii and pseudomonas aeruginosa vol.9, no.4, december 2015, p 150-156 doi: 10.5454/mi.9.4.2 antibiotic use is not a risk factor of infection by extended-spectrum beta-lactamase producing bacteria in dr. soetomo hospital surabaya 1 2 1 nadhya nur fitri *, musofa rusli , and manik retno wahyunitisari 1 departement of medical microbiology, faculty of medicine, universitas airlangga, jalan mayjen prof. dr. moestopo 47 surabaya 60131, indonesia; 2 departement of internal medicine, faculty of medicine, universitas airlangga, jalan mayjen prof. dr. moestopo 47 surabaya 60131, indonesia infection by extended-spectrum beta-lactamase (esbl) producing bacteria confers a major challenge for clinicians due to limited treatment options and poor prognosis. inappropriate antibiotic use is thought to cause the emergence of this resistant strain through selective pressure mechanisms. this study aims to describe the proportion of esbl-producing bacteria and characteristics of patients with esbl-producing bacterial infection, and to analyze the risk factors of infection by esbl-producing bacteria in dr. soetomo hospital, surabaya. a cross-sectional study was conducted on medical records of inpatients of internal medicine ward of dr. soetomo hospital. samples were classified into esbl-positive or esbl-negative groups. demographic data, clinical data and previous antibiotic use of 66 samples (33 in each group) were retrospectively obtained. as many as 30 patients (45.5%) were male. mean age of patients in the esbl-positive and negative group were 53.57 (±16.77) and 54.27 (±14.88) years, respectively (p>0.05). the median pre-infection length of stay was 4 and 3 days for esbl-positive and negative group, respectively (p>0.05). type 2 diabetes mellitus was the most common comorbid disease (33.3%). the most frequent bacteria obtained from clinical isolates was escherichia coli (49.3%). proportion of esbl producers amongst e.coli and k. pneumoniae isolates were 75% and 38.5%, respectively. the most frequently prescribed empirical antibiotic was ceftriaxone. none of the antibiotic used were risk factors for infection by esbl-producing bacteria. although none of the assessed variables were risk factors for esbl-infection was discovered, this study finds a significantly larger proportion of esbl-e. coli compared to non-esbl producing e. coli. further studies should include larger sample size and quantitatively measured antibiotic use. key words: antibiotic resistance, antibiotic use, esbl, escherichia coli infeksi oleh bakteri penghasil extended-spectrum beta-lactamase (esbl) menjadi sebuah tantangan bagi klinisi karena terbatasnya pilihan terapi antibiotic dan prognosis pasien yang buruk. penggunaan antibiotik secara tidak tepat diduga menyebabkan berkembangnya melalui tekanan seleksi. penelitian ini bertujuan untuk mendeskripsikan prevalensi bakteri penghasil esbl dan karakteristik pasien yang terinfeksi oleh bakteri penghasil esbl serta menganalisis faktor risiko infeksi oleh bakteri penghasil esbl di rsud dr. soetomo, surabaya. sebuah studi cross-sectional dilakukan menggunakan rakam medic pasien rawat inap di instalasi rawat inap medik penyakit dalam rsud. dr. soetomo, surabaya. sampel dikelompokkan menjadi kelompok esbl-positif dan esbl-negatif. data demografis, klinis dan riwayat penggunaan antibiotik dari 66 sampel (33 sampel pada masing-masing kelompok) dilihat secara retrospektif. sebanyak 30 (45.5%) pasien berjenis kelamin laki-laki. rata-rata usia pada kelompok esbl-positif ialah 53.57(±16.77) tahun, sedangkan pada kelompok esbl-negatif ialah54.27 (±14.88) tahun (p>0.05). nilai median lama rawat inap sebelum infeksi pada kelompok esbl-positif dan esbl-negatif masing-masing 4 dan 3 hari (p>0.05). penyakit komorbid tersering yang dijumpai pada pasien adalah diabetes mellitus (33.3%). bakteri yang terbanyak didapatkan pada isolat klinis ialah escherichia coli (49.3%). prevalensi e. coli penghasil esbl adalah 75% dan 38.5% pada klebsiella pneumoniae. antibiotik yang paling sering diberikan pada pasien ialah cephalosporin generasi-3 (ceftriaxon). tidak didapatkan hubungan antara penggunaan antibiotik dengan infeksi oleh bakteri penghasil esbl. isolat e. coli yang menghasilkan esbl secara signifikan lebih banyak daripada isolat yang tidak menghasilkan esbl. penelitian berikutnya diharapkan dapat melibatkan sampel yang lebih besar serta mengukur penggunaan antibiotik secara kuantitatif. kata kunci: esbl, escherichia coli, penggunaan antibiotik, resistensi antibiotik. *corresponding author; phone: +62-31-5020251, fax: +6231-5022472; email: nadhya.nf@gmail.com microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 are also known to produce esbls (dhillon and clark 2012). esbls are able to hydrolize and inactivate betalactam antibiotics, including all penicillins, cephalosporin, and aztreonam, but has no activity against cephamycin and carbapenem (tham 2012). this strain also commonly shows co-resistance towards fluoroquinolones and aminoglycosides, therefore limiting treatment options (kaya et al. 2013). limited susceptible antibiotic options have become the main challenge in treating infection by esbl producing bacteria, causing an increase in medical costs, morbidity and mortality (tumbarello et al. 2006). the prevalence of esbl producing bacteria varies among countries; however, an increasing trend is reported (shaikh et al. 2015). the prevalence of esblproducing bacteria could be as low as 7.5% in north america to as high as 22% in the asia-pacific region (dhillon and clark 2012). hadi et al. (2013) reported that the prevalence of esbl-producing bacteria in institutions participating in the amrin (antimicrobial resistance in indonesia: prevalence and prevention) study increased from 22% in 2010 to 53% in 2012. one of the notorious risk factors for infection by esbl-producing bacteria is inappropriate antibiotic use. antibiotic use is thought to drive selection pressure, eliminating susceptible bacteria and enabling the growth of resistant bacteria (wright 2010). however, results obtained from previous studies vary and analytical studies on this topic are still limited in indonesia. this study is conducted to describe the proportion of esbl-producing bacteria; the clinical characteristics of patients with esbl-producing bacterial infection, and to analyze the risk factors of infection by esbl-producing bacteria in dr. soetomo hospital, surabaya. this study is by far the most recent analytical study on risk factors of infection by esbl producing bacteria in a tertiary hospital in indonesia. materials and methods study design and population. this cross sectional study involved medical records of patients with bacterial infection hospitalized at the internal medicine ward of dr. soetomo hospital, a tertiary referral hospital in surabaya, indonesia. the inclusion period was october 2014 to may 2015. the eligible population consists of patients with a diagnosis of bacterial infection from which clinical specimens have been collected for bacterial identification and antibiotic susceptibility testing at the clinical microbiology laboratory of dr. soetomo hospital. the minimum age of patients included in the study was 18 years old. incomplete medical records were excluded. samples were selected consecutively and were classified into two groups, esbl-negative and esbl-positive with a 1:1 ratio. bacterial identification was conducted with the microbacttm system (medvet diagnostics, thebarton, adelaide, australia), esbl-positive group consisted of patients with confirmed bacterial identification from clinical specimens, in which the identified bacteria was confirmed as esbl-producing, while esbl-negative group consisted of patients infected by a non-esbl-producing bacteria. esbl production by gram negative bacteria screening was done with disk diffusion testing using cefotaxime or ceftazidime based on reccomendations of the clinical and laboratory standards institute (clsi) guidelines or by the bd phoenix automated microbiology system (bd diagnostic systems, sparks, md) machine used in clinical microbiology laboratory of dr. soetomo hospital. data collection. medical records were reviewed to obtain necessary data. studied variables were patient age and sex; comorbid disease and length of hospital stay (los) prior to infection; antibiotic use and the type of clinical specimen collected. antibiotic use was defined as any class of antibiotic given to the patient before date of clinical specimen collection, with a minimum duration of antibiotic use of 48 h. statistical analysis. bivariate analysis on categorical variables was performed with chi square test or fisher's exact test when appropriate. independent samples t-test was used to analyze association of age with infection by esbl-producing bacteria. independent samples mann whitney u test was used to compare pre-infection length of hospital stay in the esbl-positive and esbl-negative group. all statistical analyses are significant at p<0.05; 95% confidence interval. all statistical analysis was conducted with spss version 20. ethical clearance. the study protocol was approved by the medical research ethics committee of dr. soetomo hospital, surabaya, indonesia [komite etik penelitian kesehatan rsud dr. soetomo] (ethical clearance approval number 135/panke.kke/ii/2015). results samples that met the inclusion criteria consisted of 66 medical records, 33 in each esbl-positive and esbl-negative group. as many as 30 patients (45.5%) volume 9, 2015 microbiol indones 151 were male. demographic and clinical characteristics of samples are shown (table 1). there are no significant differences between the demographic characteristics of the esbl-positive and esbl-negative group (p>0.05). the most common comorbid diseases in samples were type 2 diabetes mellitus and malignancy. none of the comorbidities were found to be risk factors of infection by esbl-producing bacteria. from the 66 patients, 73 clinical specimens were positive for bacterial cultures. the majority of isolates were from urinary specimens (63.01%) other clinical s p e c i m e n s o b t a i n e d a n d e s b l p r o d u c t i o n characteristics are shown (table 2). from 73 bacterial isolates, the most common species obtained was escherichia coli (47.95%). from the 33 esbl-positive isolates, as many as 27 (81.8%) isolates were escherichia coli, five (15,2%) were klebsiella pneumonia and one (3%) was enterobacter aerogenes (table 3). the number of esbl-positive e. coli is significantly larger than the number of esblnegative isolates (p=0.000). the proportion of esblproducing isolates was 75% in escherichia coli and 38.46% in klebsiella pneumoniae. antibiotics used by patients were classified based its classes, including cephalosporin, beta-lactam/beta lactamase inhibitor, fluoroquinolones, and other types of antibiotics. patients with combination of antibiotics is catagorized as those who receive more than one types of antibiotic simultaneously. the most frequently prescribed antibiotic is the third generation cephalosporin, ceftriaxone. bivariate analysis of antibiotic uses and infections by esbl-producing bacteria shows that none of the antibiotics were risk factors for infection by esblproducing bacteria (table 4). discussion infection by extended-spectrum beta-lactamase producing bacteria has become a major challenge for clinicians due to increasing prevalence, limited antibiotic options, and poor prognosis. it is therefore table 2 sources of clinical specimens esbl sputum esbl + esbl total urine pus blood 26 (56.5%) 20 (43.5%) 46 (100%) 5 (41.7%) 7 (68.3%) 12 (100%) 1 (20%) 4 (80%) 5 (100%) clinical specimens 1 (10%) 9 (90%) 10 (100%) characteristics esbl-positive n=33 demographic ** p value generated with independent samples mann whitney u test gender (male) *** p value generated with independent samples t test mean age (±sd) diabetic nephropathy comorbidities renal disease median pre-infection los (days) malignancy type 2 diametes mellitus liver disease rheumatic diseases *p value generated with fisher's exact test 12 (36.37%) 53.57 (16.77) 6 (18%) 6 (18%) 4 8 (24%) 4 (12%) 1 (3%) 2 (6%) esbl-negative n=33 18 (54.54%) 54.27 (14.88) 4 (12%) 4 (12%) 3 9 (27%) 7 (21%) 2 (6%) 1 (3%) p 0.138 0.859*** 0.492 0.492 0.547** 0.778 0.322 1.000* 1.000* table 1 characteristics of samples 152 fitri et al. microbiol indones table 4 analysis of antibiotic use in esbl-positive and esbl-negative group table 3 distribution of bacteria in the esbl-positive and esbl-negative group ciprofloxacin other classes levofloxacin fluoroquinolones meropenem *rr=relative risk 6 6 12 0 0.757 0.757 0.614 0.495* ceftazidime amoxicillin-clavulanic acid beta lactam-beta lactamase inhibitor cefixime cefoperazone-sulbactam 3 1 2 1 1 1.000* 1.000* 0.492* 1.000* 1.000* ceftriaxone cefotaxime cephalosporin 25 0 29 1.000 1.000* 1.000 antibiotic esbl-positive esbl-negative p cedecea lapagei enterococcus faecalis pasteurella mucocida acinetobacter junii staphylococcus epidermidis total 0 0 0 0 0 33 4 1 1 1 1 40 4 (5.5%) 1 (1.4%) 1 (1.4%) 1 (1.4%) 1 (1.4%) 73 (100%) proteus mirabilis acinetobacter baumanii pseudomonas aeruginosa enterobacter cloacae acinetobacter iwolfii 0 0 0 0 0 1 4 6 1 1 1 (1.4%) 4 (5.5%) 6 (8.2%) 1 (1.4%) 1 (1.4%) klebsiella pneumoniae enterobacter aerogenes escherichia coli 5 1 27 8 2 9 13 (17.8%) 3 (4.1%) 36 (49.3%) bacteria esbl-positive esbl-negative total (%) 1.098 1.098 0.882 1.000 1.000 1.000 rr azithromycin combination of antibiotics cotrimoxazole *p value generated with fisher's exact test 1 10 1 1.000* 0.786 1.000* 1.000 0.535-1.609 0.245-4.085 0.619-1.947 0.619-1.947 0.545-1.429 0.432-2.315 0.570-1.756 0.477-2.094 95%ci (lower-upper) 7 7 14 1 3 0 0 0 0 25 1 27 0 9 1 volume 9, 2015 microbiol indones 153 important to study the epidemiology and risk factors for acquisition of this resistant strain to provide adequate infection control in the hospital. a continuous surveillance should also be conducted because the prevalence usually changes overtime and differs in each health centers. demographic characteristics of patients obtained in this study showed that neither gender nor age were significant risk factors for infection by esblproducing bacteria. however, other studies with larger samples have found differing results. older age is found to be a risk factor in a multinational survey on risk factors for infection by esbl-producing bacteria (or=2.4; 95%ci=1.6–3.6) (ben-ami et al. 2009). however, a contrasting result was found by soraas et al., (2013) that younger age was found to be a risk factor for infection by esbl-producing bacteria. male sex was found to be a risk factor for infection by esblproducing bacteria in a multinational survey (ben-ami et al. 2009) (or=2.5; 95%ci=1.7-3.7). another study by tuon et al. (2010) in brazil also reported similar results (or=2.62; 95%ci=1.16-5.93). however, lee et al. (2010) reported that female sex was a risk factor for urinary tract infection by esbl-producing bacteria (or=1.44; 95%ci=1.062-1.951; p= 0.019). length of hospital stay previous to infection is also not a risk factor in this study. this is in concert with studies by tuon et al., (2011) and huang et al., (2007) which did not found that preinfection length of hospital stay as risk factors for infection by esblproducing bacteria. however a case control study in an australian tertiary hospital found that pre-infection length of hospital stay was a risk factor for colonization by esbl producing bacteria (or=1.2; 95%ci=1.01.3) (osthoff et al. 2015). none of the comorbidities were risk factors of infection by esbl-producing bacteria. however, tuon et al. (2010) reported that diabetes mellitus is a risk factor for bacteremia by esbl-producing k. pneumoniae (p=0.043). silva et al. (2006) reported in his study that diabetes mellitus and malignancy are risk factors for infection by esbl producing bacteria (p=0.02). diabetes mellitus is a well-known risk factor towards infection. patients with diabetes have underoptimal immune system function, rendering them susceptible towards infectious agents (casqueiro et al. 2012). an interesting finding in this study is that the proportion of esbl-producers among e. coli isolates reaches 75%. this result differs from a study of microbial resistance pattern in dr. soetomo hospital by kuntaman et al. (2006). the study reported prevalence of esbl-producers among e.coli was 34.86% and among k. pneumonia isolates reaches 35.35%. the study involved larger sample size and isolates from wards other than internal medicine; therefore it represented a larger population. meanwhile pajariu et al. (2010) reported the prevalence of esble. coli was 54.32% and esbl-k. pneumonia was 50% in dr. kariadi general hospital, semarang. microbial epidemiology usually varies among health institutes and changes overtime. however, the small sample size involved in this study might cause sampling bias and the results might not accurately describe population characteristics. changes of bacterial population might also be the case, however no report was available and data on microbial pattern distribution is lacking. further surveillance is needed to confirm whether there is a changing trend in bacterial distribution in dr. soetomo hospital. esbl gene may be harbored in the bacterial chromosome or in plasmid. severin et al. (2009) reported that the most predominant esbl isolate from dr. soetomo hospital was the producer of ctx-m-15, of which the gene is most commonly harbored in plasmid. unfortunately, molecular esbl analysis was not conducted in this study; hence it is not known whether the strains of esbl-producing bacteria in this study were from clonal spread or horizontal plasmid transfer. finally, none of the classes of antibiotics used by patients is found to be a risk factor for infection by esbl-producing bacteria. osthoff et al. 2015 and huang et al. 2007 reported that the use of third generation cephalosporin is a risk factor for infection by esbl-producing bacteria. the use of beta lactambeta lactamase inhibitor (chopra et al. 2015; harris et al. 2007) and fluoroquinolones (soraas et al. 2013) were also found as risk factors for infection by esblproducing bacteria. the failure to obtain an association between antibiotic use and infection by esblproducing bacteria in this study might be caused by involvement of small sample size. another reason would be because exposure towards antibiotics in this study is measured as a categorical variable (exposed/unexposed) rather than as a continuous variable such as measuring defined daily dose (ddd), length of therapy (lot) or duration of therapy (dot). schechner et al. (2013) reported that an association of antibiotic use with infection by resistant organisms is more likely to be obtained if the antibiotic used was measured as a continuous variable 154 fitri et al. microbiol indones rather than a categorical variable. in conclusion, age, gender, pre-infection length of hospital stay, comorbidities and antibiotic use were not risk factors for esbl-infection was discovered, however, this study finds a significantly larger proportion of esbl-e. coli compared to non-esbl producing e. coli. further studies should include larger sample size and quantitatively measured antibiotic use. acknowledgment the authors would like to thank the dean of faculty of medicine universitas airlangga and the director of dr. soetomo hospital whose support made this study possible. references ben-ami r, rodrıguez-bano j, arslan h, pitout jdd, quentin c, calbo es, azap ok, arpin c, pascual a, livermore dm, garau j, carmeli y. 2009. a multinational survey of risk factors for infection with extended-spectrum blactamase–producing enterobacteriaceae in nonhospitalized patients. clinl infect dis. 49(5):682-690. doi: 10.1086/604713. casqueiro j, casqueiro j, alves c. 2012. infections in patients with diabetes mellitus: a review of pathogenesis. indian j endocrinol metab. 16(1) s27s36. doi: 10.4103/2230-8210.94253. chopra t, marchaim d, johnson pc, chalana ik, tamam z, mohammed m, alkatib s, tansek r, chaudhry k, zhao jj, pogue jm, kaye ks. 2015. risk factors for bloodstream infection caused by extended-spectrum blactamase producing escherichia coli and klebsiella pneumoniae: a focus on antimicrobials including cefepime. american j infect control. 43(7):719-723. http://dx.doi.org/10.1016/j.ajic.2015. 02.030. dhillon rhp, clark j. 2012. esbls: a clear and present danger? 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bmc biology. 8:123. doi: 10.1186/1741-7007-8-123. shaikh s, fatima j, shakil s, rizyi smd, kamal ma. 2015. antibiotic resistance and extended spectrum betalactamases: types, epidemiology and treatment. saudi j biol sci. 22(1):90-101. doi: 10.1016/j.sjbs.2014.08. 002. 156 fitri et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 12 256 (iman rusmana).pmd effects of temperature on denitrifying growth and nitrate reduction end products of comamonas testosteroni isolated from estuarine sediment iman rusmana department of biology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia phone/fax: +62-251-622833, e-mail: rusmana13@yahoo.com predictions of seasonal changes in n 2 o emission that occur in natural estuaries are important to anticipate the future implications of global warming. this study showed the effect of temperature on denitrifying growth and nitrate reduction end product of comamonas testoteroni isolated from estuarine sediment using both batch and continuous cultures. the µµµµµ max values of comamonas testosteroni grown in anaerobic batch culture were increased with increasing temperature, and the highest µµµµµ max was found at 26 oc. concentrations of nitrate reduced (mg-1 dried weight cells) were higher at low temperature. concentrations of n 2 produced were higher at low temperature and the production of n 2 was higher than both no 2 and n 2 o productions. key words: denitrification, nitrate reduction, nitrous oxide, comamonas testosteroni _____________________________________________ denitrification and dissimilative nitrate reduction to ammonium are the major processes of nitrate removal in aquatic environments. both processes have an implication for global warming, due to their emission of n 2 o, a potential greenhouse gas. n 2 o concentration is currently increasing at 0.2-0.3% per year (conrad 1995), and 90% of global n 2 o emission comes from biotic processes. many studies have been conducted to determine whether denitrification or dissimilative nitrate reduction to ammonium is the process more responsible for nitrate removal and n 2 o production. in situ measurement of microbial activities can give valid information about undisturbed microbial activities in their natural condition, but this approach cannot describe the physiological process of microbial interaction due to the complexity of microbial communities. microbial activities may also be studied under laboratory conditions, thereby allowing us to control experimental conditions. this laboratory experiment may help to get better understanding of the effect of temperature on denitrifying growth and end product of its nitrate reduction of comamonas testoteroni isolated from estuary sediment. characterization of denitrifying bacteria isolated from estuary sediment indicated that c. testosteroni has complete denitrification enzymes. it has narg, napa, nirs, and nosz genes. the end-product of nitrate/nitrite reduction in 20 oc was 100% n 2 (rusmana 2006). predictions of seasonal changes in dissimilative nitrate reduction process outcome that occur in natural environments are important to anticipate the future implications of global warming, this is especially so as denitrification can produce n 2 o. some in situ measurement studies suggested that denitrification was the dominant process at low temperature, whereas dissimilative nitrate reduction to ammonium at high temperature (king & nedwell 1984; herbert & nedwell 1990; ogilvie et al. 1997). in contrast, zimmerman and benner (1994) and nowicki et al. (1997) showed the opposite results. other experiments suggested that denitrification was the predominant process at low c/n ratio, whereas drna predominated at high c/n ratio environments (koike & hatori 1978; nedwell 1982). in additional kandeler et al. (2006) sugested that nutrient availability may have contributed more to nitrate-reducing activities than to the density of the nitrate reducer community. therefore this study was conducted to determine the effect of temperature on denitrifying growth and end product of its nitrate reduction of c. testoteroni isolated from estuary sediment. materials and methods effect of temperature on end products of nitrate/nitrite reduction by c. testosteroni in batch culture. the medium composition comprise as follow (g l-1): nacl 20.0, kcl 0.5, na 2 hpo 4 5.5, nh 4 cl 0.5, k 2 so 4 1.75, nah 2 po 4 0.775, na 2 edta 0.078, mgso 4 ·7h 2 o 0.01, na-acetate 0.68, kno 3 0.17, wolin’s trace elements (7 ml). sterile medium (10 ml) was dispensed aseptically into sterile optically standardized tubes, triplicate for each temperature, capped with sterile suba-seals, and then gassed with ofn to make anaerobic by inserting a syringe needle through the suba-seal into the medium. an in-line sterile membrane filter (0.2 µm pore size, sartorius) was connected to the needle for sterilizing the ofn. to one of the three tubes was added 10% (v/v) c 2 h 2 to inhibit nitrous oxide reductase enzyme for determining total n 2 end product. the tubes were placed in the holes across the temperature gradient block with triplicate tubes in each temperature. the temperature r a n g e w a s s e t b e t w e e n 6 3 2 oc w i t h a p p r o x i m a t e l y 2 oc increments. after temperature equilibration, the tubes were inoculated with 200 µl of exponential phase starter culture. bacterial growth was monitored periodically using a nephelometer (eel unigalvo ds29, diffusion systems, london) to measure the od in each tube. when the culture had reached stationary phase, 200 µl of the headspace gas was injected into the gc to measure n 2 o gas. the culture was then centrifuged at 12,000 x g for 15 min, and the supernatant was frozen and placed in microbiology indonesia, april 2007, p 43-47 volume 1, number 1 issn 1978-3477 the -20 oc freezer for further analyses of nitrate, nitrite, and ammonium. the maximum specific growth rate (µ max ) of the bacterium for each temperature was calculated by least square regression analysis of the slope of the linear part of semilogaritmic plot of turbidity versus time (pirt 1975; nedwell & rutter 1994). end-products of nitrate reduction by c. testosteroni in steady state chemostats. the continuous culture was conducted at four different temperatures, 5, 10, 15, and 20 oc, using a chemostat that consisted of a glass vessel with a volume of 500 ml, sealed with a rubber bung. to minimize adherence of bacteria, “repelcote” (bdh) was used to coat the vessel glass wall. sterile medium was pumped continuously using a peristaltic pump (multiperpex; lkb, broma, sweden) with dilution rate 0.02 h-1 for incubation at 10, 15, and 20 oc, and 0.01 h-1 for incubation at 5 oc. a double line breaker connecting the reservoir vessel with the chemostat was used to prevent back contamination of sterile medium in the flow line. an aerobic condition was maintained by gassing the vessel and medium with sterile ofn. the ofn flow rate, 6 ml min-1, was monitored by a calibrated flow meter (rsi type) and the ofn was passed through a chromous acid trap to remove out any o 2 contained in trace amounts. a magnetic stirrer mixed the chemostat culture and the temperature in the chamber was maintained by circulating coolant through a glass jacket around the growth vessel that regulated by a thermo-circulator (grant cfc25, grant instruments, cambridge ltd.) attached to a fh15 flow heater (grant instruments, cambridge ltd.). to calculate the µ max , the dilution rate was increased so that the cells were washed out from the vessel faster than bacterial growth. the od was monitored using a spectrophotometer at 550 nm. the slope of a plot of ln od 550 versus time was calculated, then the µ max was also calculated using the following formula (pirt 1975): µ max = d washout slope where d washout = the dilution rate during washout. the half saturation constant for nitrate (k no3 -), was calculated using the following formula (pirt 1975): k no3 = s*(µ max d)/d where s* = residual nitrate concentration at steady state, d = dilution rate during steady state. the specific affinity (a a ) for nitrate was calculated for each temperature of chemostat run using the following formula: a a = (µ max / k no3 -) nitrous oxide analysis in acetylene block samples. the gas chromatograph was used to analyse n2 o as describe in trimmer et al. (1998), the presence of c 2 h 2 in injected samples can damage the ecd of the gas chromatograph, and therefore it was necessary to separate c 2 h 2 from n 2 o using an external pre-column. a stainless steel (2 m x 2 mm i.d.) packed with paropak q (80-100 mesh) was used at 25 oc, (millipore corporate, milliford, u.k.), with ofn as a carrier gas (80 ml min-1). the retention time of n 2 o was shorter than c 2 h 2 , and therefore n 2 o could be collected using a liquid nitrogen cryo-trap (-196 oc, 200 µl stainless steel sample loop) before the c 2 h 2 came off the pre-column. after n 2 o was eluted, the c 2 h 2 was switched to waste and the pre-column brought on-line into the main gc column. the cryo-trap n 2 o was mobilized into the main gc column by immersing the pre-column loop in warm water (trimmer et al. 1998). results growth kinetics of c. testosteroni batch culture in temperature gradient block. the µ max values of c. testosteroni batch culture in the temperature gradient block with acetate as a substrate is shown in figure 1. the µ max values were increased with increasing the temperatures of incubation, and the highest µ max was found at 26 oc. the natural log of µ max plotted with the reciprocal of the absolute temperature shows that the plot was linear between 12 oc and 22 oc (p < 0.001) (figure 2). these results indicated that c. testosteroni is a mesophilic bacterium. concentrations of nitrate reduced mg-1 dried weight cells were higher at low temperature, < 10 oc. similar with nitrate reduced, concentrations of n 2 produced at temperature < 10 oc (figure 3) were higher at low temperature. n 2 production from c. testosteroni was higher than both no 2 and n 2 o productions. however, there was a peak of high nitrite concentration at 10 oc compared with the other temperatures. n 2 o was only produced by c. testosteroni in small amount in all temperatures. the percentages of nitrate reduced to n 2 gas were higher than that reduced to no 2 and n 2 o gas, between 35.46 and 65.26%, at 30 and 10 oc respectively (figure 4 & table 1). figure 1 the maximum specific growth rate (µ max ) values of comamonas testosteroni batch cultures with acetate as a substrate in a temperature gradient block incubator. µ m a x ( h -1 ) temperature (oc) figure 2 an arrhenius semilogarithmic plot of the ln µ max of comamonas testosteroni versus the reciprocal of the absolute temperatures. a least square regression line was fitted to a linear section of the plot. in ( µ m a x ) 44 rusmana microbiol indones figure 6 concentrations of nitrate ( ) reduced, nitrite ( ) and n 2 o ( ) produced in steady state condition of comamonas testosteroni continuous culture at 5, 10, 15, and 20 oc. bars indicate standard errors. nitrate reduced to nitrite was between 0.15 and 8.69%, at 28 and 10 oc respectively, and only between 0.78% (at 26 oc) and 2.34% (at 8 oc) were reduced to n 2 o. the total nitrate recovery was between 36.58 and 75.70%, at 30 and 10 oc respectively (table 1). growth kinetics of c. testosteroni in nitrate-limited continuous culture with acetate as a substrate at 5, 10, 15, and 20 oc. cell density at steady state in anaerobic continuous cultures with acetate as a substrate is shown in figure 5. the ell density at steady state increased with increasing temperature, which were 1.58 + 0.12, 2.70 + 0.57, 7.77 + 1.04, and 29.75 + 3.21 x 106 cfus ml-1, at 5, 10, 15, and 20 oc respectively. total concentrations of nitrate reduced also increased with increasing temperature, which were 40.00 + 8.02, 253.94 + 69.54, 816.96 + 39.88, and 995.31 + 2.74 µm at 5, 10, 15, and 20 oc (figure 6). however, the highest amount of nitrate reduced per bacterial cell basis was at 15 oc, which table 1 cell dried weight, nitrate utilisation, and production of nitrite, dinitrogen, and nitrous oxide from c. testosteroni batch culture in temperature gradient block (+ standard error) amounts of no 3 reduced no 2 produced n 2 produced n 2 o produced (µmol) (%) (µmol) (%) (µmol-n) (%) (µmol-n) (%) temperature (oc) cell dried weight (mg) cellular n from ammonium (µmol) 17.33 + 0.02 13.00 + 0.04 17.67 + 0.03 17.33 + 0.03 14.00 + 0.06 10.33 + 0.002 11.67 + 0.02 17.33 + 0.01 16.67 + 0.04 15.00 + 0.01 15.67 + 0.01 7.67 + 0.001 2.67 + 0.001 2.33 + 0.01 32 30 28 26 24 22 20 18 16 14 12 10 8 6 29.84 + 1.20 29.89 + 3.47 29.93 + 1.28 29.97 + 0.90 29.97 + 2.12 29.95 + 2.68 29.96 + 1.58 29.91 + 3.94 29.99 + 0.69 30.00 + 0.00 30.00 + 0.00 19.80 + 1.47 18.40 + 2.25 15.60 + 1.65 99.47 99.63 99.77 99.90 99.90 99.83 99.88 99.69 99.96 100.00 100.00 66.00 61.33 52.00 0.05 + 0.002 0.05 + 0.001 0.04 + 0.004 0.06 + 0.009 0.06 + 0.013 0.05 + 0.005 0.06 + 0.005 0.05 + 0.001 0.06 + 0.002 0.56 + 0.101 1.85 + 0.405 1.72 + 0.131 0.39 + 0.134 0.20 + 0.014 0.16 0.16 0.15 0.19 0.19 0.16 0.18 0.18 0.21 1.85 6.18 8.69 2.11 1.29 10.74 10.60 18.28 18.20 16.74 15.42 16.75 18.18 17.36 16.78 14.28 12.92 8.12 7.03 35.98 35.46 61.04 60.72 55.86 51.50 56.02 60.80 57.88 55.94 48.28 65.26 44.08 45.20 0.28 + 0.036 0.28 + 0.029 0.26 + 0.018 0.24 + 0.001 0.32 + 0.010 0.46 + 0.010 0.50 + 0.007 0.28 + 0.007 0.68 + 0.013 0.32 + 0.080 0.28 + 0.011 0.34 + 0.033 0.44 + 0.006 0.32 + 0.016 0.94 0.96 0.86 0.39 1.08 1.52 1.64 0.94 2.28 1.08 0.92 1.76 2.34 2.06 7.54 + 0.008 5.65 + 0.016 7.68 + 0.012 7.54 + 0.012 6.09 + 0.024 4.49 + 0.002 5.07 + 0.008 7.54 + 0.004 7.25 + 0.016 6.52 + 0.004 6.82 + 0.004 3.34 + 0.002 1.16 + 0.002 1.01 + 0.004 37.09 36.58 62.09 61.69 57.14 53.17 57.88 61.91 60.38 57.87 55.38 75.70 48.53 48.55 no 3 recovery (%) c e ll d e n si ty ( x 1 0 6 c f u m l1 ) temperature (oc) figure 5 cell density of comamonas testosteroni continuous cultures in steady state at 5, 10, 15, and 20 oc. bars indicate standard errors. c o n c e n tr a ti o n s o f n o 3 re d u c e d ( µ m ) n o 2 a n d n 2 o p ro d u c e d ( µ m ) temperature (oc) n it ra te u p ta k e ( µ m o l/ m g -1 d ry c e ll s) n it ri te , n 2 , a n d n 2 o p ro d u c ti o n (µ m o ln /m g d ry c e ll ) figure 3 temperature effect on nitrate ( ) uptake and nitrite, ( ) dinitrogen ( ), and nitrous oxide ( ) production per dried weight cells from comamonas testosteroni batch cultures with acetate as a substrate. temperature (oc) n 2 /n o 3 ra ti o ( % ) n o 2 / n o 3 a n d n 2 o /n o 3 ra ti o ( % ) temperature (oc) figure 4 percentage ratios of the nitrite ( ), dinitrogen, ( ) and nitrous oxide ( ) production per nitrate removal basis from comamonas testosteroni batch culture in a temperature gradient block incubator. volume 1, 2007 microbiol indones 45 x table 2 kinetic growth rate parameters of c. testosteroni in nitrate-limited continuous culture experiments at 10, 15, and 20 oc temperature µ max residual nitrate k (no3-) a a(no3-) (oc) (h-1) (µmol 1-1) (µmol 1-1) (1 µmol-1 h-1) 0 . 2 2 1 7 6 0 . 0 9 2 7 6 0 . 0 3 2 7 6 2 0 1 5 1 0 1 0 4 . 6 9 2 8 3 . 0 3 8 4 6 . 0 5 1 0 5 6 . 0 9 1 0 2 9 . 6 7 539.78 0 . 0 0 0 2 1 0 . 0 0 0 0 9 0 . 0 0 0 0 6 table 3 the specific affinity (a a ) for nitrate by nitrate respiring bacteria in anerobic nitrate-limited chemostats a a (1 µmol-1 h-1) at a temperature (oc) 5 10 15 20 substrate organism glucose acetate k. pneumoniae* aeromonas sp.* klebsiella oxytoca* citrobacter sp.* klebsiella oxytoca* citrobacter sp.* c. testosteroni** 0.00003 0.00012 0.00118 0.0033 0.00011 0.00014 0.00006 0.00488 0.0015 0.00009 0.013 0.00006 0.00059 0.00004 0.00021 0.00021 0.00021 *from nedwell (1999), **this study was 105.18 x 10-9 µmol cfu-1 (figure 7). the highest concentrations of nitrite and n 2 o produced in both total and per bacterial cell basis were found at 10 oc, which were 77.99 + 1.11 µm or 2.96 x 10-9 µmol cfu-1 and 2.92 + 0.34 µm or 1.08 x 10-9 µmol cfu-1 respectively (figure 5 & 7). the kinetic growth rate parameters calculated using the formula reported by pirt (1975) are shown in table 2. the maximum specific growth rate (µ max ) values at 10, 15, and 20 oc were 0.03276, 0.09276, and 0.22176 h-1 respectively, and the specific affinity (a a ) for nitrate at 10, 15, and 20 oc were 0.00006, 0.00009, and 0.00021 l µmol-1 h-1 respectively (table 3). discussion the µ max values of c. testosteroni grown in anaerobic batch culture with acetate as a substrate indicated that c. testosteroni is a mesophilic bacterium with optimum temperature for anaerobic growth at 26 oc (figure 1). the arrhenius plot of ln µ max with 1/toa shows that the linear/ normal range temperature was between 12 and 22 oc. in this linear range the microbial growth can be predicted by extrapolation of the arrhenius relationship (ingraham et al. 1983) as follow: ln µ = (-∆e*/gas constant) (1/t) + constant where, µ is microbial growth rate, ∆e* is the activation energy, and t is temperature in kelvin. the temperature characteristic value, which is the slope of the arrhenius plot of c. testosteroni in acetate/nitrate, indicated that c. testosteroni has small response of growth to increasing temperature. there is a correlation of temperature characteristic with the energy activation as a following formula (ingraham et al. 1983): temperature characteristic = (-∆e*/gas constant) where ∆e* is the activation energy. n o 3 re d u c e d ( x 1 0 -9 µ m o l c f u -1 ) n o 2 a n d n 2 o p ro d u c e d (x 1 0 -9 µ m o l c f u -1 ) figure 7 amounts of nitrate ( ) reduced, nitrite ( ) and n 2 o ( ) produced per bacterial cell basis (cfu-1) in steady state condition of comamonas testosteroni continuous culture at 5, 10, 15, and 20 oc. temperature (oc) total nitrate reduced at low temperature, in both batch and continuous cultures, was lower than at high temperature (table 1) indicating temperature limitation on bacterial nitrate uptake. low temperature inhibits active transport of substrate and nutrient by reducing the fluidity of the membrane, which then influences transporters molecules in the membrane, presumably because stiffening of the membrane by lowered temperature repress transport protein efficiency (nedwell 1999). moir and wood (2001) suggested that there are two different mechanisms of nitrate transport; (i) nitrate/nitrite antiporter and (ii) nitrate/proton symporter. protein transporter of nitrate transport is nark (zumft 1997; moir & wood 2001; sharma et al. 2006). currently it is reported that there are two different forms of nark, nark1, and nark2 (moir & wood 2001; sharma et al. 2006). in paracoccus denitrificans nark1 is a nitrate/proton symporter and nark2 is nitrate/nitrite antiporter (wood et al. 2002). sharma et al. (2006) suggested that only the nark2 protein is required as a nitrate/nitrite transporter by pseudomonas aeruginosa under denitrifying conditions. in the present experiments, this nark efficiency was probably repressed by low temperature with stiffing of the membrane, so that nitrate uptake was repressed. the bacterial growth is correlated with substrate and nutrient uptake. in nitrate-limiting medium, nitrate is a limiting factor that controls bacterial growth. therefore, the bacterial growth yield at low temperature was lower than at high temperature (table 1). the ability of an organism to scavenge (via its transporters) a substrate is dependent upon its affinity for that substrate. if affinity is described by specific affinity (a a ) the higher the value, the greater is this affinity for this substrate (nedwell 1999). the specific affinity (a a ) value for nitrate of c. testosteroni decreased with decreasing temperature below the growth optimum (table 2). this trend of decreased affinity for nitrate probably indicates decreasing functional efficiency of nark as temperature declines. nedwell (1999) termed that any substrate taken up by some from of active transport will be less available as temperature decreases because microorganism ability to seize the substrate declines at low temperature as a result of “temperature-modulated substrate affinity”. the specific affinity (a a ) value for nitrate of c. testosteroni with acetate as a substrate at 10 oc was lower than klebsiella oxytoca with acetate and k. pneumoniae, aeromonas sp., k. oxytoca with glucose, as well as at 15 oc with k. pneumoniae and aeromonas sp. with glucose, and at 20 oc with k. pneumoniae and k. oxytoca with glucose as a substrate. however there was similar a a value with k. 46 rusmana microbiol indones oxytoca and citrobacter at 20 oc with acetate as a substrate (table 3). in steady state of nitrate limiting chemostats, the ability of a bacterial cell to seize nitrate from the environment might be controlled by the affinity of the transport protein (nedwell 1999). this a a values reflect the ability of one organism to outcompete the others. acknowledgements this work was supported by a studentship grant from the quality for undergraduate education (que) project, department of biology, bogor agricultural university, indonesia to i.r. thanks also due to d.b. nedwell department biological sciences, university of essex, cholchester, united kingdom for his great support and advice. references conrad r. 1995. soil microbial processes involved in production and consumption of atmospheric trace gases. in: jones jg (ed). advance in microbial ecology. vol 14. new york: plenum pr. p 2 0 7 2 5 0 . herbert ra, nedwell db. 1990. role of environmental factors in regulating nitrate respiration in intertidal sediments. in: revsbech np, sorensen j (ed). denitrification in soil and sediment, fems symposium. new york: plenum publ. p 77-99. ingraham jl, maaloe o, neidhardt fc. 1983. growth 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fems microbiol ecol 30:101-111. nedwell db, rutter m. 1994. influence of temperature on growth rate and competition between two psychrotolerant antartic bacteria: low temperature diminished affinity for substrate uptake. appl environ microbiol 60:1984-1992. nowicki bl, requintina e, van keuren d, kelly jr. 1997. nitrogen losses through sediment denitrification in boxton harbor and massachusetts bay. estuaries 20:626-639. ogilvie bg, rutter m, nedwell db. 1997. selection by temperature of nitrate-reducing bacteria from estuarine sediments: species composition and competition for nitrate. fems microbiol ecol 23:11-22. pirt sj. 1975. in principle of microbe and cell cultivation. blackwell: oxford. rusmana i 2006. gaseus end products of nitrate and nitrite reduction by denitrifying pseudomonads isolated from estuarine sediment. j mikrobiol indones 11:63-66. sharma v, noriega ce, rowe jj. 2006 involvement of nark1 and nark2 proteins in transport of nitrate and nitrite in the denitrifying bacterium pseudomonas aeruginosa pao. appl environ microbiol 72:695-701. trimmer m, nedwell db, sivyer db, malcolm sj. 1998. nitrogen fluxes through the lower estuary of the river great ouse, england: the role of the bottom sediments. mar ecol prog ser 163:109124. wood nj, alizadeh t, richardson dj, ferguson sj, moir jwb. 2002. two domains of a dual-function nark protein are required for nitrate uptake, the first step of denitrification in paracoccus pantotrophus. mol microbiol 44:157-170. zimmerman ar, benner r. 1994. denitrification, nutrient regeneration and carbon mineralisation in sediments of gaverston bay, texas, usa. mar ecol prog ser 114:275-288. zumft w. 1997. cell biology and molecular basis of denitrification. microbiol mol biol rev 61:533-616. volume 1, 2007 microbiol indones 47 01. charis (molecular).cdr molecular phylogeny of salmonellae: relationships among salmonella species determined from gyra, gyrb, parc, and pare genes charis amarantini* and dhira satwika faculty of biotechnology, duta wacana christian university jalan dr. wahidin sudirohusodo 5-19, yogyakarta 55224, indonesia study on molecular characteristics of salmonella from clinical isolates was done in order to find out its relationship, especially those isolated from indonesia. partial sequence of genes belonging to qrdr region, i.e. gyra, gyrb, parc, and pare were employed. specific primer pairs covering those genes are used to amplify the bacterial dna obtained. the amplicons were then analyzed by means of sequencing, and the sequences are analysed bioinformatically to find out similarities and build phylogenetic trees. by comparing all of the phylogenetic tree from qrdr region, this study revealed gyra as the most suitable gene for rapidly identify member of salmonellae as it gives better separation of samples being analysed. however, the use of parc is recommended as it gives a consistent and reliable value to separate member of salmonella and other enterobacter. further studies are under way to include member of this group, like e. coli, and the use of full sequence of qrdr genes region to verify this report. key words: gyra, parc, qrdr, salmonella penelitian ini dilakukan untuk mencari kekerabatan dan hubungan filogeni salmonella yang berasal dari isolat klinis yang diisolasi dari indonesia. selama ini sudah dilaporkan pemanfaatan gen-gen yang berada pada daerah qrdr dapat digunakan sebagai penanda untuk menentukan hubungan kekerabatan tersebut. partial sequence gen-gen pada daerah tersebut, yaitu gyra, gyrb, parc dan pare digunakan untuk tujuan tersebut. pasangan primer yang mengamplifikasi urutan parsial setiap gen digunakan dalam reaksi pcr, selanjutnya hasil amplifikasi dna isolat sampel dianalisa dengan melakukan sekuensing. setelah urutan parsial dna setiap gen diketahui, dilakukan analisa secara bioinformatis dengan melakukan alignment dna sampel dengan database yang tersedia. analisa ini memberi data berupa indeks kesamaan nukleotida hingga pembuatan pohon filogeni, yang dapat digunakan untuk melacak dan mencari kekerabatan setiap isolat. hasil penelitian menunjukkan bahwa gyra merupakan gen yang dapat digunakan untuk identifikasi cepat anggota salmonella karena memberi resolusi paling baik. meskipun demikian, parc merupakan gen yang direkomendasikan untuk memisahkan anggota salmonella maupun kelompok bakteri yang erat kekerabatannya, misal kelompok enterobacter, karena konsistensi pemisahan yang baik. kata kunci : gyra, parc, qrdr, salmonella *corresponding author; phone: +62-274-563929, fax: +62274-513235, e-mail: charis@staff.ukdw.ac.id salmonellae are a diverse group of gram-negative bacteria and consist of a number closely related organisms belonging to the family enterobacteriaceae. for a long time, the taxonomic classification and nomenclature of this group changed several times. the last classification systems have been validly proposed for two-species systems, salmonella enterica and salmonella bongori (chang et al. 1997). currently, shelobolina et al. (2004) discovered salmonella subterranea sp. nov. as a new species belongs to the genus salmonella. in addition to the taxonomic classification of twospecies systems, the salmonellae were categorized by serotype. there are over 2,500 serotypes associated with gastroenteritis and typhoid fever in human. the majority of these serotypes are often extremely difficult to be separated based on its biochemical characteristics. therefore, it would be useful to classify these groups based on molecular typing methods. molecular phylogenetic approach for classification of salmonellae is important due to the increased spread of salmonella strains, especially salmonella enterica serovar typhi (s. typhi) with fluor oquinol one reduced susceptibility. reports already available mentioning the use of genetic traits to differentiate this bacteria (for example: tajbakhsh et al. 2011, muthu et al. 2014). nalidixicacid resistant of s. typhi (narst) strains with decreased susceptibility -1 to ciprofloxacin (0.125-1 µgl ) becomes a major problem in the indian subcontinent (capoor et al. 2007), and molecular analysis showed mutations of vol.9, no.1, march 2015, p 1-8 doi: 10.5454/mi.9.1.1 some genes in quinolone resistance-determining region (qrdr). it was reported, a point mutation in qrdr of the gyra gene at various sites, especially at codons coding for serine at position 83 and aspartate at position 87 (afzal et al. 2013). other mutation was observed in the genes coding for dna gyrase (gyra or gyrb) or topoisomerase (parc and pare) in the resistant strain (muthu et al. 2014). it was reported earlier some s. typhi strains are resistant to nalidixic acid. the taxonomic classification of these strains based on 16s rrna gene showed the sensitive and resistant isolates can not be separated into different clades (amarantini and budiarso 2013). d e t a i l e d a n a l y s i s f o r b a c t e r i a l p h y l o g e n e t i c relationships through the qrdr of the gyra and gyrb subunits of dna gyrase and the parc and pare subunits of topoisomerase iv might be possible for a better classification than 16s rrna regions. the present study was done to assess the use of gyra and gyrb genes in qrdr and parc and pare genes in topoisomerase iv for determining the phylogenetic relationship among sensitive and resistant s. typhi isolates. materials and methods bacterial strains. four isolates used in this study were obtained from the previous research (amarantini and budiarso 2013); i.e. two nalidixic acid-resistant and two nalidixic acid-sensitive isolates. two isolates from reference collection of pt biofarma (s. typhi nctc 786) and blk yogyakarta (s. typhi o) were also included. dna isolation. all isolates were cultured in brain heart infusion broth at 37 °c for 18 h prior to dna extraction. to isolate chromosomal dna, 1 ml of overnight culture were put into a 1.5 ml centrifugetube and were centrifuged at 5000 rpm, 15 min to obtain the cells. isolation of dna was carried out in accordance with the phenol-chloroform-isoamyl alcohol method (sambrook et al. 1989). pcr experiments of qrdrs of the gyra, gyrb, parc, and pare genes of s. typhi. dna isolated from bacterial strains were amplified by pcr using spesific primer (table 1) for gyra, gyrb, parc, and pare (ling et al. 2003). the pcr was performed using tm dreamtaq green pcr master mix in total reaction tm volume of 50µl containing 25µl of 2x dreamtaq green pcr master mix, 1 µl of 1 µm primer stocks, and 1 µl of template dna. the pcr reaction mixtures were amplified for 35 cycles with initial-denaturation at 95 ºc for 5 min, denaturation at 94 ºc for 1 min, annealing at 52 ºc for 1 min, and extention at 72 ºc for 1 min, with a final extension at 72 ºc for 5 min. an aliquot of 5 μl of each amplified product was electrophorese in 1.2% (wt/vol) agarose gel using 1x tbe buffer gel stained with sybr® safe dna stain (life technologies). a 100-bp dna ladder (fermentas, germany) were included as molecular weight marker. dna sequencing and phylogenetic analysis. pcr products were purified and sequenced by outsourcing the samples to macrogen inc, korea. the nucleotide sequences were edited and assembled using seqman and editseq (dna star, laser gene 6, m a d i s o n , w i , u s a ) . p h y l o g e n e t i c t r e e w a s constructed with mega v5 (tamura et al. 2011) with the neighbor-joining algorithm (saito and nei 1987). the evolutionary distance matrix for the neighborjoining method was generated in accordance with the description introduced by jukes and cantor (1969). t h e m a t r i x o f t h e n u c l e o t i d e s i m i l a r i t y a n d difference was generated with phydit software (chun 1999). results we have amplified and sequenced two dna fragments of s. typhi containing gyra and gyrb qrdrs. we also used two pairs of primers to amplify table 1 primers used to sequence gyrb/gyra and pare/c (ling et al. 2003) primer name gyra gyrb parc pare 2 amarantini microbiol indones primer sequence size (bp) f, 5’ -tgtccgagatggcctgaagc -3' 347 r, 5'-taccgtcatagttatccacg -3' f, 5' -caaactggcggactgtcagg-3' 345 r, 5'-ttccggcatctgacgataga-3' f, 5' atgagcgatatggcagagcg 3' 412 r, 5' tgaccgagttcgcttaacag 3' f, 5' gaccgagctgttccttgtgg 3' 272 r, 5' agcagagtagcgatatgcaa 3' the parc and pare q r d rs. as expected, two amplification products of 435 and 297 bp were obtanined and their nucleotide sequences were determined. from these sequence data, we construct the phylogenetic structure and determine the genetic relationship among the nalidixic acid-sensitive and resistant isolates. as shown in figure 1, the phylogenetic analysis determined by gyra sequence clearly separate the sensitive and resistant isolates into different clusters. two resistant isolates (bpe 127.1 mc and bpe 122.4 cca) were delineated using the gyra gene. the percentage similarity within each strains ranged from 73.25-93.35% (table 2). in contrast to the gyra dendogram, the phylogenetic structure of salmonella on the basis of gyrb gene sequences (345 bp) were not able to distinguish the sensitive and resistant isolates (fig 2). the resistant isolates were grouped as the same cluster with the sensitive isolates. unlike the gyra dendogram, the similarity percentage between each samonella typhi o (blk yogyakarta) samonella typhi bpe 122.1 cca samonella typhi bpe 122.4 cca r* samonella typhi bpe 127.1 mc r* samonella typhi nctc 786 samonella typhi rsk 5.1 ssa salmonella newport strain sn71 (kc121321.1) salmonella typhi strain st73 (hq176366.1) escherichia coli(ay323807.1) salmonellatyphi strain st80 (hq176368.1) 13 100 55 22 41 52 0.1 fig 1 phylogenetic tree of salmonella strains based on gyra sequences analysis. the tree was constructed by neighborjoining method. bar, 1 substitution per 10 nucleotides. table 2 nucleotide similarity values (%) and the number of nucleotide differences between salmonella strains based on gyra sequence s . t y p h i o ( b l k y o g y ak ar ta ) s .t y p h i b p e 1 2 2 .1 c c a s .t y p h i b p e 1 2 2 .4 c c a r * s . t y p h i b p e 1 2 7 .1 m c r * s . t y p h i n c t c 7 8 6 s . t y p h i r s k 5 .1 s s a h q 1 7 6 3 6 8 .1 h q 1 7 6 3 6 6 .1 k c 1 2 1 3 2 1 .1 a y 3 2 3 8 0 7 .1 s. typhi o (blk yogyakarta) --25/340 23/346 30/343 38/331 49/350 192/335 192/335 158/284 136/232 s.typhi bpe 122.1 cca 92.65 --41/363 38/355 33/325 126/471 249/423 249/423 155/284 132/232 s.typhi bpe 122.4 cca r* 93.35 88.71 -- 25/358 32/329 67/370 202/356 202/356 156/283 135/232 s. typhi bpe 127.1 mc r* 91.25 89.30 93.02 -- 25/328 51/359 194/348 194/348 153/283 134/232 s. typhi nctc 786 88.52 89.85 90.27 92.38 -- 39/330 178/324 178/324 154/282 131/228 s. typhi rsk 5.1 ssa 86.00 73.25 81.89 85.79 88.18 -- 262/427 262/427 154/284 132/232 hq176368.1 42.69 41.13 43.26 44.25 45.06 38.64 -- 0/434 3/290 16/238 hq176366.1 42.69 41.13 43.26 44.25 45.06 38.64 100.00 -- 3/290 16/238 kc121321.1 44.37 45.42 44.88 45.94 45.39 45.77 98.97 98.97 -- 18/235 ay323807.1 41.38 43.10 41.81 42.24 42.54 43.10 93.28 93.28 92.34 --volume 9, 2015 microbiol indones 3 samonella typhi bpe 127.1 mc r* samonella typhi bpe 122.4 cca r* samonella typhi o (blk yogyakarta) samonella typhi rsk 5.1 ssa samonella typhi nctc 786 escherichia coli strain 408/65-1(dq447149.1) burkholderia cepacialmg1222 (ay996867.1) salmonella typhi strain 208 (ef064853.1) salmonella typhi strain atcc 19430 (ay370864.1) 100 99 0.1 fig 2 phylogenetic tree of salmonella strains based on gyrb sequences analysis. the tree was constructed by neighborjoining method. bar, 1 substitution per 10 nucleotides. table 3 nucleotide similarity values (%) and the number of nucleotide differences between salmonella strains based on gyrb sequence d q 4 4 7 1 4 9 . 1 s . t y p h i o (b l k y o g y ak ar ta ) s . t y p h i r s k 5 .1 s s a s . t y p h i b p e 1 2 2 .4 c c a r * s . t y p h i b p e 1 2 7 .1 m c r * s . t y p h i n c t c 7 8 6 a y 9 9 6 8 6 7 . 1 a y 3 7 0 8 6 4 . 1 e f 0 6 4 8 5 3 .1 dq447149.1 --16/296 13/267 20/301 17/287 16/284 233/762 278/492 420/685 s. typhi o (blk yogyakarta) 94.59 --0/267 1/296 1/283 3/284 68/296 150/288 150/288 s. typhi rsk 5.1 ssa 95.13 100.00 -- 0/267 0/267 2/267 59/267 131/258 131/258 s. typhi bpe 122.4 cca r* 93.36 99.66 100.00 -- 1/287 3/284 71/301 152/292 152/292 s. typhi bpe 127.1 mc r* 94.08 99.65 100.00 99.65 -- 2/284 66/287 145/278 145/278 s. typhi nctc 786 94.37 98.94 99.25 98.94 99.30 -- 65/284 145/277 145/277 ay996867.1 69.42 77.03 77.90 76.41 77.00 77.11 -- 297/492 765/1156 ay370864.1 43.50 47.92 49.22 47.95 47.84 47.65 39.63 -- 0/506 ef064853.1 38.69 47.92 49.22 47.95 47.84 47.65 33.82 100.00 --samonella typhi rsk 5.1 ssa samonella typhi nctc 786 samonella typhi bpe 127.1 mc r* samonella typhi bpe 122.1 cca samonella typhi bpe 122.4 cca r* samonella typhi o (blk yogyakarta) salmonellatyphi strain 1474 (fj222661.1) klebsiella pneumoniae subsp. rhinoscleromatis (af303654.1) escherichia coli (eu561348.1) escherichia coli gj0706-186 (eu513000.1) 83 36 100 99 0.1 fig 3 phylogenetic tree of salmonella strains based on parc sequences analysis. the tree was constructed by neighborjoining method. bar, 1 substitution per 10 nucleotides. 4 amarantini microbiol indones strains were nearly equal ranging from 98.94 100% (table 3). figure 3 shows the phylogenetic structure of salmonella based on parc gene sequences. the phylogenetic analysis determined by parc sequence give rise to a tree structure which clustered all the test strains into a cluster, with control strain s. typhi o blk separated into different cluster as a sole species. this finding is supported by the nucleotide similarity index value above 99.0% (table 4). an almost similar phylogenetic tree structure (fig 4) was revealed when we analyzed the isolates based on pare gene sequences. all the tested isolates are separated into a cluster which also included some member of enterobacter. however, a lower nucleotide similarity index was observed compared to the one produced which is based on parc gene sequences (table 5). a lower bootstrap value was also observed, emphasizing the consistency of isolates separation based on parc gene. table 4 nucleotide similarity values (%) and the number of nucleotide differences between salmonella strains based on parc sequence s a m o n el la t y p h i r s k 5 .1 s s a s a m o n el la t y p h i b p e 1 2 2 .1 c c a s a m o n el la t y p h i n c t c 7 8 6 s a m o n el la t y p h i b p e 1 2 2 .4 c c a r * s a m o n el la t y p h i b p e 1 2 7 .1 m c r * s a m o n el la t y p h i o ( b l k y o g y ak ar ta ) s a lm o n el la t y p h i st ra in 1 4 7 4 (f j2 2 2 6 6 1 .1 ) samonellatyphi rsk 5.1 ssa --0/334 0/331 0/331 3/334 49/330 133/272 samonellatyphi bpe 122.1 cca 100.00 --0/332 0/332 3/335 48/330 132/271 samonellatyphi nctc 786 100.00 100.00 --0/337 1/337 47/328 132/270 samonellatyphi bpe 122.4 cca r* 100.00 100.00 100.00 --0/336 47/328 132/270 samonellatyphi bpe 127.1 mc r* 99.10 99.10 99.70 100.00 --50/333 135/274 samonellatyphi o (blk yogyakarta) 85.15 85.45 85.67 85.67 84.98 --142/279 salmonellatyphi strain 1474 (fj222661.1) 51.10 51.29 51.11 51.11 50.73 49.10 --samonella typhi bpe 127.1 mc r* samonella typhi bpe 122.1 cca samonella typhi rsk 5.1 ssa samonella typhi o (blk yogyakarta) salmonella typhi (ab072701.2) salmonella typhi (am283478.1) samonella typhi bpe 122.4 cca r* samonella typhi nctc 786 escherichia coli strain 90(jn565721.1) streptococcus pneumoniae strain 5305(ay157686.1) salmonella typhi strain 35 (hq698252.1) 77 96 48 58 0.1 fig 4 phylogenetic tree of salmonella strains based on pare sequences analysis. the tree was constructed by neighborjoining method. bar, 1 substitution per 10 nucleotides. volume 9, 2015 microbiol indones 5 discussion dna sequence analysis has become increasingly popular in determining the evolutionary relationships of bacteria(tajbakhsh et al. 2011). in the present study we therefore determined the phylogenetic structure for six strains belonging to s. typhi based on partial gyra and gyrb sequences. the test strains comprised of two groups, resistant and susceptible to nalidixic acid. results of phylogenetic structure showed that all of the test strains were sharply separated, but the topology of the tree based on gyra gene was very different to that of the gyrb. the phylgenetic tree based on gyra gene showed that the resistant strains were organized into different clusters. our data indicated that the gyra nucleotide sequences showed much higher variations than gyrb. the maximum and minimum nucleotide similarityamong s. typhi strains based on gyra sequence was 93.35% and 73.25%, respectively (table 2). the phylogenetic tree based on gyrb gene grouped all six s. typhi strains togetherin a single cluster (fig 2). data showed that the nucleotide sequences from these groups were very similar (table 3) ranged from 98.94 100%.these factsindicated that they exhibited the closest relationship. souza et al. (2011) observed that non-fluorine quinolones such as nalidixic acid may be sufficient to generate mutations that alter the susceptibility of salmonellaspp to fluoroquinolones. mutations have rarely been reported in the gyrb gene (ling et al. 2003). so, it is clear that the higher genetic variation in term of nucleotide similarity of grya in the test strains may be mainly due to mutations. in general, the phylogenetic tree based on gyra and gyrb genes showed the separation isolates into several clades with a better separation compared to the one based on 16s rrna gene from the previous research (amarantini and budiarso 2013). the major difference is the position of the resistant isolates (bpe 122.4 cca * * r and bpe 127.1 mc r ) which clustered with the sensitive isolates in their analysis (fig 5). the similarity values of 16s rrna were higher than the gyra and gyrb sequences (table 6). they showed the closest 16s rrna relatedness values ( 99.42 % ≥ similarity) among all of the test strains. comparison of gyra and gyrb sequences and 16s r r n a s e q u e n c e s f o r p h y l o g e n e t i c a n a l y s i s demostrated that taxonomy based on 16s rrna typing methods may not be enough for the delineation phylogenetic differences at the spesies level. the 16s table 5 nucleotide similarity values (%) and the number of nucleotide differences between salmonella strains based on pare sequence s a m o n el la t y p h i r s k 5 .1 s s a s a m o n el la t y p h i b p e 1 2 7 .1 m c r * s a m o n el la t y p h i n c t c 7 8 6 s a m o n el la t y p h i b p e 1 2 2 .4 c c a r * s a m o n el la t y p h i b p e 1 2 2 .1 c c a s a m o n el la t y p h i o (b l k y o g y ak ar ta ) s a lm o n el la t y p h i (a m 2 8 3 4 7 8 .1 ) s a lm o n el la t y p h i (a b 0 7 2 7 0 1 .2 ) s a lm o n el la t y p h i st ra in 3 5 (h q 6 9 8 2 5 2 .1 ) samonellatyphi rsk 5.1 ssa --0/198 4/199 4/196 0/197 2/197 1/199 1/199 107/193 samonellatyphi bpe 127.1 mc r* 100.00 -- 4/198 4/196 0/197 2/197 1/198 1/198 107/192 samonellatyphi nctc 786 97.99 97.98 -- 0/200 4/197 6/200 4/207 4/207 113/203 samonellatyphi bpe 122.4 cca r* 97.96 97.96 100.00 -- 4/196 6/196 3/198 3/198 105/193 samonellatyphi bpe 122.1 cca 100.00 100.00 97.97 97.96 -- 2/196 1/197 1/197 106/191 samonellatyphi o (blk yogyakarta) 98.98 98.98 97.00 96.94 98.98 -- 3/200 3/200 108/194 salmonella typhi (am283478.1) 99.50 99.49 98.07 98.48 99.49 98.50 -- 1/481 134/233 salmonella typhi (ab072701.2) 99.50 99.49 98.07 98.48 99.49 98.50 99.79 -- 134/233 salmonella typhi strain 35 (hq698252.1) 44.56 44.27 44.33 45.60 44.50 44.33 42.49 42.49 -- 6 amarantini microbiol indones samonella typhi bpe 127.1 mc r* samonella typhi bpe 122.4 cca r* samonella typhi bpe 122.1 cca samonella typhi nctc 786 samonella typhi atcc 19430t ( z47544) samonella typhi rsk 5.1 ssa samonellabongori (af029227) escherichia coli atcc 25922 (x80724.1) 65 100 0.005 fig 5 phylogenetic tree of salmonella strains based on 16s rrna sequences analysis. the tree was constructed by neighborjoining method. bar, 5 substitution per 5000 nucleotides. table 6 nucleotide similarity values (%) and the number of nucleotide differences between salmonella strains based on 16s rrna s . t y p h i b p e 1 2 2 .4 c c a ( r * ) s . t y p h i b p e 1 2 7 .1 m c ( r * ) s . t y p h i b p e 1 2 2 .1 s . t y p h i r s k 5 .1 s s a s . t y p h i n c t c 7 8 6 z 4 7 5 4 4 a f 0 2 9 2 2 7 x 8 0 7 2 4 .1 s. typhi bpe 122.4 cca (r*) --3/1383 0/1381 2/1382 3/1381 5/1381 33/1377 45/1374 s. typhi bpe 127.1 mc (r*) 99.78 -- 3/1381 5/1382 6/1381 8/1381 36/1377 48/1374 s. typhi bpe 122.1 100.00 99.78 -- 2/1381 3/1381 5/1381 33/1377 45/1374 s. typhi rsk 5.1 ssa 99.86 99.64 99.86 -- 3/1381 5/1381 33/1377 45/1374 s. typhi nctc 786 99.78 99.57 99.78 99.78 -- 6/1381 34/1377 46/1374 z47544 99.64 99.42 99.64 99.64 99.57 -- 36/1497 48/1446 af029227 97.60 97.39 97.60 97.60 97.53 97.60 -- 39/1442 x80724.1 96.72 96.51 96.72 96.72 96.65 96.68 97.30 --rrna genes couldn't be used as a suitable marker for classification of closely related bacterial spesies (tajbakhsh et al. 2011). it was useful only for describing phylogenetic relationships between distantly related enterobacteriaceae and can not be applied for intrageneric relationship (dauga 2002). in the present study, we also noted that the a p p l i c a t i o n o f g y r a t y p i n g s h o w e d a b e t t e r classification than gyrb. the gyra gene exhibited high variation in nucleotide sequences (73.25 93.35% similarity). because of this, the gyra gene provided higher resolution than the gyrb gene. it is, therefore encouraging that gyra was found to be the best marker for classification. based on these results, it is clear there are nucleotide polymorphisms occur among gyra, gyrb, parc, and pare which result in defined clustering of the tested isolates. nonetheless, it is recommended to employ parc as a preferred gene to distinguish salmonella and its close relative, like member of enterobacter, as it is sensitive and specific. a further study need to be done to ensure this assumption as full sequence of each qrdr genes were not included in this study. acknowledgments this research was funded by the directorate general of higher education, department of national education (hibah fundamental) 2014, contract no:1348/k5/kl/2013 date 14-05-2014. references afzal a, sarwar y, ali a, maqbool, salman m, habeeb ma, haque a. 2013. molecular evaluation of drug resistance in clinical isolates of salmonella enterica serovar typhi from pakistan. j infect dev ctries. 7(12): 929-940. doi: 10.3855/jidc.3154. amarantini c, budiarso ty. 2013. 16s rdna typing of volume 9, 2015 microbiol indones 7 salmonellatyphi strains from different geographical locations in sumba island east nusa tenggara 8 amarantini microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 vol.1 , no. , 202 , p -6 2 december 2 31 36 doi: 10.5454/mi.1 . .6 2 31-36 design of adenovirus 5 vector with adenovirus 26 hexon hypervariable region sequence using approachin silico afina firdaus syuaib , ernawati arifin giri-rachman , aluicia anita 1 2 and artarini 1* 1 pharmaceutical biotechnology laboratory, department of pharmaceutics, school of pharmacy, institut teknologi bandung, west java, indonesia; 2 department of genetics and molecular biotechnology, school of life science and technology, institut teknologi bandung, west java, indonesia. adenovirus type 5 (ad5) is one of the vaccine vectors, including the covid-19 vaccine. pre-existing immunity to ad5 may suppress the immunogenicity and efficacy of adenovirus vectored vaccine. the neutralizing antibodies are directed specifically toward seven hypervariable regions (hvr) of hexon proteins located on the outer surface of the capsid. this study aims to design an ad5 vector that may circumvent anti-ad5 immunity by designing a chimera ad5 vector with the sequence of ad26 hvr (ad5hvr26) using approach.in silico substitution of the ad5 hvr dna sequence may affect the alternative splicing process of adenovirus mrna, which then influence the protein product. the splice site prediction of ad5hvr26 chimera vector was found at hvr5, 6, and 7. the codon change in the splice site was performed to decrease the possibility of incorrect splicing, while retaining the original amino acid sequence. the hvr substitution in chimera vector ad5hvr26 may also affect the interaction of hexon in the capsid. the hvr2 and hvr4 hexon proteins individually interact with other hexon proteins and ix protein. thus, two designs of the ad5hvr26 chimera vector were created in this research. the first design was the ad5 chimera vector with complete substitution of hvr hexon by ad26 sequence, with codon modification on the splice site. the second design was ad5hvr26 chimera vector without the hvr2 and hvr4 substitution to maintain the hexon protein interaction with the capsid proteins. production of the designed vectors are needed to prove the reduction of vector neutralization by pre-existing immunity. key words: adenovirus type 5, chimera vector, hexon protein, hypervariable region (hvr), neutralizing antibodies, protein interaction, splice site adenovirus tipe 5 (ad5) merupakan salah satu vektor vaksin yang digunakan, termasuk untuk vaksin covid-19. keberadaan terhadap ad5 dapat menurunkan imunogenisitas dan efikasipre-existing immunity terhadap vektor adenovirus. antibodi netralisasi yang terbentuk diketahui mentarget secara spesifik terhadap tujuh daerah hipervariabel ( , hvr) protein hekson pada permukaan kapsid adenovirus.hypervariable regions penelitian ini bertujuan untuk merancang vektor ad5 yang mampu menghindar dari imunitas anti-ad5 melalui pendekatan vektor ad5 khimera dengan hvr ad26 (ad5hvr26) dengan pendekatan . substitusi urutanin silico dna hvr ad5 diprediksi dapat mempengaruhi proses splising alternatif dari mrna adenovirus, yang akan mempengaruhi protein yang dihasilkan. hasil prediksi situs splising vektor ad5hvr26 menunjukkanchimera keberadaan situs baru pada hvr5, 6, dan 7. penggantian kodon pada situs splising baru kemudian dilakukan untuk menurunkan kemungkinan terjadinya proses splising yang salah, dengan tetap mempertahankan asam amino yang dihasilkan. substitusi hvr pada vektor chimera ad5hvr26 juga diprediksi dapat mempengaruhi interaksi hekson pada kapsid adenovirus. hvr2 dan hvr4 pada protein hekson masing-masing berinteraksi dengan protein hekson yang lain dan protein ix. oleh sebab itu, dua rancangan vektor khimera ad5hvr26 dibuat pada penelitian ini. desain pertama adalah vektor chimera ad5 dengan substitusi lengkap dengan hvr ad26, dengan modifikasi kodon pada situs splising. desain kedua adalah vektor chimera ad5hvr26 tanpa substitusi hvr2 dan hvr4 untuk mempertahankan interaksi hekson pada kapsid. produksi vektor khimera ini perlu dilakukan untuk membuktikan penurunan yang mengakibatkan netralisasi vektorpre-existing immunity adenovirus. kata kunci: ,adenovirus tipe 5 antibodi netralisasi, daerah hipervariabel ( , hvr),hypervariable regions interaksi protein, protein hekson situs splising, vektor chimera, microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: e-mail: anita@itb.ac.id therapy and vaccine, but it has high pre-existing immunity that may suppress the immunogenicity and efficacy of the adenovirus vectored vaccine (sumida et al. 2005). several strategies can be performed to avoid this high pre-existing immunity (padilla 2016,et al. kreppel and hagedorn 2021). neutralization by vaccination is one of the strategies to control the spread of the covid-19 pandemic. adenovirus type 5 (ad5) is the most common viral vector used in gene neutralizing antibodies can be avoided by removing the specific epitope present on ad5. hypervariable regions (hvr) is located on the outer surface of the hexon protein and is a specific epitope against different adenovirus serotypes (mizuta . 2009, gallardoet al et al. 2021). adenoviruses are a family of viruses that lack an envelope with a linear double-stranded dna genome that has a vertebrate host (greber 2020). the main component of the adenovirus capsid consists of hexon, penton base, and fiber proteins, while the minor components consist of iiia, vi, viii, and ix proteins. adenovirus capsid with icosahedron symmetry has 20 sides composed of 12 hexon protein trimers and 12 corners, each of which consists of a penton pentamer bound to one or more fiber proteins (flatt and butcher, 2019). antibodies to ad5 consist of antibodies to fiber, penton, and hexon. however, the most dominant antibody response that can suppress ad5 vectoredvaccine efficacy is the antibody to hexon (bradley .et al 2012). hexon is the target of neutralizing antibodies because it is much more abundant than other proteins present in adenovirus capsid. this study aims to design an ad5 vector that may circumvent anti-ad 5 immunity by designing a chimera ad5 vector with the sequence of hypervariable region of ad26 hexon. adenovirus 26 was chosen because it has low seroprevalence and does not induce tumors in rodents (pacesa 2016, yi 2022). in addition,et al. vaccines with a combination of ad5 and ad26 vectors are expected to have high efficacy as in the sputnik v vaccine (logunov . 2021). the ad5hvr26 vectoret al could not be produced in previous research reported by bradley 2012. in this study, analysis waset al., in silico carried out to estimate the cause of ad5hvr26 vector cannot be made. ad5-specific epitope recognized by neutralizing antibody was determined based on a literature study. the ad5hvr26 chimera vector was constructed by replacing the hvr hexon ad5 amino acid sequence with the ad26 amino acid sequence. changes that occur in the dna sequence and the amino acid sequence of the hexon protein can affect the viability of the chimera vector. in this study, splice site analysis and analysis of hexon proteins interaction was performed to ensure that the modification would not affect the production of the chimera vector. modifications were made to the dna sequence of the ad5hvr26 chimera vector to obtain a chimera vector design that can be produced based on in silico analyses. materials and methods substitution of ad5 hexon hvr with ad26. genome and amino acid sequences of the ad5 (ncbi reference sequence: ac_000008.1) and ad26 (genbank: ef153474.1) were obtained from national center for biotechnology information (ncbi) online databases (https://ncbi.nlm.nih.gov/). the amino acid sequences of the ad5 and ad26 hexon were aligned using clustal omega multiple sequence alignment (https://www.ebi.ac.uk/tools/msa/clustalo/) to determine the hvr substitution site. the conserved area and the hvr of the ad5 hexon protein refer to the study of roberts (2006). to obtain the gene encoding the hexon chimera protein (ad5hvr26), substitution of hvr was carried out at the nucleotide. hexon gene in ad5, ad26, and ad5hvr26 was translated in silico (https://web.expasy.org/translate/) and aligned to prove that the hvr replacement was appropriate. splice site prediction. detection of the ad5 and ad5hvr26 gene splice sites was carried out using the alternative splice site predictor (assp) website (http://wangcomputing.com/assp/). the ad5hvr26 splice site was compared to ad5 to determine different splice sites found in ad5hvr26. additional acceptor splice sites in ad5hvr26 were removed by nucleotide base substitution until they reached lowest score. replacement of nucleotide bases is carried out while paying attention to the codon sequence so that there is no change in amino acids. determination of hexon protein interaction. literature study was conducted to determine the location of the interaction between hexon ad5 protein and other capsid proteins, namely penton base, iiia, vi, viii, and ix protein. protein interactions involving hvr were visualized based on the three-dimensional structure of the capsid (pdb id: 6cgv) using pymol. hexon protein modelling and superimposition. modeling of the ad5hvr26 hexon structure was carried out with the swiss-model web server (https://swissmodel.expasy.org/) using the hexon amino acid sequence of ad5 and ad5hvr26 as target proteins. the protein used as template is ad5 hexon monomer with highest sequence identity. the study of hexon chimera structure was carried out by visualizing the three-dimensional structure and superimposition of hexon chimera proteins using pymol. 32 syuaib et al. microbiol indones volume 1 , 2026 2 microbiol indones 33 results substitution of ad5 hexon hvr with ad26. the gene encoding for hexon ad5hvr26 was obtained by substitution of ad5 hexon hvr with ad26. the replacement of hvr sequence was carried out at the nucleotide level to obtain the dna encoding for ad5hvr26 hexon protein. alignment results showed that the hvr hexon of ad5hvr26 was different from ad5 but the same as ad26 hvr sequence (fig 1). splice site prediction. replacement of the dna sequence of hvr ad5 to ad26 could cause alternative splicing errors during mrna processing. to predict the presence of additional splice sites on ad5hvr26, prediction of splice sites was performed using assp software. predictions were made on the l3 gene, which was the gene encoding for the hexon protein. the software predicted the presence of a new splice site in l3 ad5hvr26 gene that was not present in the l3 ad5 gene. additional splice sites in the l3 ad5hvr26 gene were found, i.e. five new donor splice sites and three new acceptor splice sites (table 1). the substitution of codons at the splice site was performed by changing the codons around the splice site to reduce prediction score (table 2). the higher the score, the predicted splice sites sequence was closer to the consensus sequence. after codon substitution, the splice site score was lower and might reduce the possibility of splicing. determination of hexon protein interaction. replacement of amino acid sequence of hvr ad5 to ad26 could cause changes in the three-dimensional structure of the hexon protein, thus interfering the interaction of hexon protein with other proteins. hexon protein was known to interact with each other to form a capsid structure. one of the interactions occured at amino acids 186-193, which was the hvr2 domain, and at amino acids 250-258, which was the hvr4 domain. the carboxy terminal of protein iiia was known to interact with hexon at amino acids 250 – 258 (hvr4) (fabry . 2005, san martin 2012). proteinet al ix was also known to interact with hexon proteins at amino acids 252 – 256 (hvr4) (liu . 2010). hvr2et al and hvr4 was known to interact with other proteins to make a stable capsid during adenovirus capsid assembly. thus, the substitution of hvr2 and hvr4 of hexon ad5 with hvr from ad26 might impact the formation of the capsid. hexon protein modelling and superimposition. the effect of hvr replacement on protein interaction was predicted by comparing the hexon protein ad5 with ad5hvr26 structure. the structural models of the ad5 and ad5hvr26 hexon monomers were made using the swiss model with the ad5 hexon template (pdb id: 1p30). the hvr4 of modelled hexon from ad5hvr26 showed slight change as compared to the ad5 hexon monomer (fig 2). the superimposition of the modelled structure to the template showed that hvr2 of ad5hvr26 was longer than ad5, while length of hvr4 was the same (fig 3). the structural similarity of hvr2 and hvr4 between ad5 and table 2 codon substitution of splice acceptor site table 1 splice site(s) in ad5hvr26 position (bp) putative splice site sequence hvr5 13629 alt. isoform/cryptic donor cctccagcaggtggtagtgg 13630 alt. isoform/cryptic acceptor cctccagcaggtggtagtgg hvr6 13732 alt. isoform/cryptic acceptor ggaacttcagataacagttc hvr7 14091 alt. isoform/cryptic donor acttatcaaggtgtaaagat 14092 alt. isoform/cryptic acceptor acttatcaaggtgtaaagat 14109 alt. isoform/cryptic donor attacaaatggtaatgatgg 14118 alt. isoform/cryptic donor ggtaatgatggtgctgaaga 14130 alt. isoform/cryptic donor gctgaagaaagtgagtggga position before codon substitution after codon substitution sequence score* sequence score* 13630 cct cca gca ggt ggt 3.8 cct cca gcg ggt ggt 0 13732 gga act tca gat aac 3.3 gga act tcc gat aac 0 14092 tat caa ggt gta aag 4.2 tat caa ggc gta aag 3.3 *the score reflects splice site strength 34 syuaib et al. microbiol indones fig 1 hexon of the ad5, ad26, and ad5hvr26. hvr 1-7 are highlighted inalignment of amino acid sequence order: r , g , p l e.ed reen ink, ight blue, yellow, blue, and purpl fig 2 modelling of hexon had5hvr26 monomer. the 3d structure modelling was performed using hexon ad5 structure (pdb id: 6cgv). hexon protein shown in grey, hvr4 in red, and ix protein in blue. fig 3 superimposition of ad5 and ad5hvr26 hexon monomer showing the structural difference between hvr ad5 and ad5hvr26. each hexon ad5 and ad5hvr26 monomer were shown in blue and orange, respectively. hvr hexon ad5 and ad5hvr26 were shown in purple and brown, respectively. volume 1 , 2026 2 microbiol indones 35 ad5hvr26 could provide similar protein interactions in the capsid formation process. chimera vector design. based on the splice site prediction and amino acid interaction analyses, two designs of the ad5hvr26 chimera vector were created in this study. the first design was the ad5 chimera vector sequence with complete substitution of 7 hvr hexon by ad26 sequence, with codon modification on the splice site. the second design was ad5hvr26 chimera vector without the hvr2 and hvr4 substitution in the sequence to maintain the hexon protein interaction with the capsid proteins. discussion the ad5 chimera vector is formed by replacing all or part of the amino acids in the hexon with amino acids from another serotype of adenovirus. changes in the dna sequence in the hexon hvr can cause the appearance of a sequence of nucleotide known as splice sites. an alternative splicing error is thought to be one of the reasons why the ad5hvr26 chimera vector could not be obtained in previous studies (bradley . 2012). alternative splicing errors in l3et al gene can lead to gene expression errors that lie downstream l3. gene downstream l3 are l4 and l5. the l4 gene encodes 100k, 33k, 22k and viii protein which plays a role in capsid structure stability and transcriptional regulation. the l5 gene encodes fiber protein for vector internalization into the cell. introns in pre-mrna have two distinct nucleotides at either end, gt and ag each in 5' and 3' of intron sequence. the splice site analysis was carried out with the principle of identifying the similarity of the nucleotide base sequence with the consensus sequence of the splice site. the higher the score, the sequence of splice sites is closer to the consensus order (thanaraj and stamm 2003). in adenovirus mature mrna formation, the donor splice site always occurs at position 3717. additional acceptor splice sites at positions 13630, 13732, or 14092 can lead to the formation of mature mrna that expresses incorrect capsid-building proteins. this can lead to failure of the virion structure assembly and cause the ad5hvr26 vector to not be generated. therefore, codon substitutions were carried out at position 13630 (gca→gcg), 13732 (tca→tcc), and 14092 (ggt→ggc) to avoid alternative splicing error (table 2). by removing additional acceptor splice sites, the chimera vector design was predicted to be produced without problem in capsid protein production. the structure of the adenovirus capsid consists of three major proteins (hexon, penton base, and fiber) and four minor proteins (iiia, vi, viii, ix) which interact to form an icosahedral capsid (reddy and barry 2021). the capsid-forming proteins that interact with hexon are penton base proteins, iiia, vi, viii, and ix. proteins iiia and ix are located on the exterior of the capsid, while proteins vi and viii are located on the interior of the capsid (reddy and nemerow 2014). stable protein interactions can form protein complexes that are physically and functionally stable. interactions between proteins occur at certain amino acid residues. thus, changes in amino acids in hexon proteins, especially in hvr2 and hvr4 (fig 1), can affect the formation of protein complexes that form the capsid. if the amino acid changes in the hvr region of the hexon affect the interaction of the hexon protein with other proteins, the adenovirus capsid structure cannot be formed. in conclusion, two hexon sequences were designed to produce the ad5hvr26 chimera vector. production of the designed vectors is needed to prove the reduction of vector neutralization by pre-existing immunity. if both chimera vector designs can be produced, then a vector neutralization test could be carried out to determine which vector is better. vaccines with the first design ad5hvr26 vector is predicted to be more immunogenic and have higher efficacy 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liu w, zheng x, ma q, sun x, zhang y, yu x, he m, chen l, feng y. 2022. seroprevalence of neutralizing antibodies against adenovirus type 26 and 35 in healthy populations from guangdong and shandong provinces, c h i n a . v i r o l s i n . 3 7 ( 5 ) : 7 1 6 7 2 3 . d o i : 10.1016/j.virs.2022.06.006. 5.mi665-rina imaniar available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.3.5issn 1978-3477, eissn 2087-8575 vol 6, no 3, september 2012, p 124-129 *corresponding author; phone: +62-22-2504825, e-mail: retnoningrum@indo.net.id cyclodextrin (cd) is a cyclic oligosaccharides enzymatically derived from starch. three kinds of cd have been identified, namely α-cd, β-cd and γ-cd which consists of six, seven, eight glucose molecules, respectively (endo and ueda 2004). in the cd molecules, glucopyranose units are linked by 1,4 glycosidic bonds which are unique characteristic of linier starch molecule. cd forms a structure with hydrophilic property at outer surface and hydrophobic property at inner cavity, which makes cd is used for many applications, such as in food, chemistry, pharmacy, analytical, diagnostics, cosmetics and agriculture (szejtli 1997). it works by trapping the non polar molecule with suitable size through non covalent interaction. amongst the three cd molecules, β-cd has been used mostly at industrial scale due to its inner cavity size which is more suitable for many applications. due to the need of cd at industrial level, cyclodextrin glycosyltranferase (cgtase, ec 2.4.1.19) has been studied for many years. cgtase is an extracellular enzyme that converts starch and βglucan with 1,4 glycosidic bonds to cd. cgtase belongs to family 13 glycoside hydrolase. cgtase that catalyzes the conversion of starch to cd with β-cd as main products is called β-cgtase. the β-cgtase gene from bacillus sp. a2-5a with accession number of ab015670 (genbank, ncbi) has been cloned and expressed in bacillus subtilis ana-1 (ohdan et al. 2000). the cgtase gene has 2115 bp which encodes a 704 amino acids preprotein (accession number cyclodextrin (cd) is a cyclic oligosaccharide molecule and depending on the number of glucose molecules, three types of cds are commonly used, α-cd, β-cd, and γ-cd. cds can be produced enzymatically using starch as substrate catalyzed by cd glycosyltransferase (cgtase). in current research, recombinant cgtase production from the synthetic gene was optimized for its production using three growth media and two induction temperatures. the highest yield was obtained in luria bertani medium at 25 c. the rcgtase protein was affinity purified as a 76.39 kda protein which showed β-cyclization and starch hydrolysis activities using zymography method. the optimum temperature, ph, and incubation time was 55 c, 6, and 24 h, respectively. the enzyme was stable at a wide phs in the range of 5-10, retained its half activity at 56 c for 30 min and had cyclization ratio for α-cd: β-cd: γ-cd was 4 : 81 : 15. an amount of 542 mg β-cyclodextrin was produced from 100 ml reaction of 1% (b/v) sagoo starch using 38.4 µg rcgtase in optimum condition. this work reports for the first time the character of rcgtase from bacillus sp. a2-5a using sagoo starch as a substrate. key words: bacillus sp. a2-5a, β-cyclodextrin, characterization, rcgtase, sagoo starch siklodekstrin (cd) merupakan molekul oligosakarida siklik. berdasarkan jumlah molekul glukosa yang dikandungnya, tiga tipe cd yang biasa digunakan yaitu α-cd, β-cd, dan γ-cd. cd dapat diproduksi melalui konversi enzimatik pati menggunakan siklodekstrin glikosiltransferase (cgtase). pada penelitian ini dilakukan optimasi jenis medium dan suhu induksi yang digunakan untuk produksi cgtase rekombinan dari gen sintetik. rendemen rcgtase tertinggi didapatkan dengan penggunaan medium luria bertani (lb) pada suhu 25 c. protein rcgtase dimurnikan menggunakan kolom afinitas dan menghasilkan protein berukuran 76,39 kda yang menunjukkan aktifitas siklisasi-β dan hidrolisis dengan metode zimografi. suhu, ph, dan waktu inkubasi optimum aktivitas rcgtase berturut-turut adalah 55 c, 6, dan 24 jam. enzim memiliki stabilitas pada rentang ph 5,0-10,0 dan mempertahankan 50% aktivitasnya pada 56 c selama 30 menit. rasio siklisasi α-cd, β-cd, dan γ-cd pada penggunaan pati sagu berturut-turut adalah 4 : 81 : 15. cd sebanyak 542 mg dihasilkan dari 100 ml reaksi antara pati sagu 1% (b/v) terpregelatinasi dengan 38,4 µg rcgtase pada kondisi optimum. penelitian ini melaporkan untuk pertama kali karakter rcgtase dari bacillus sp. a2-5a dengan pati sagu sebagai substrat. kata kunci: bacillus sp. a2-5a, karakterisasi, pati sagu, rcgtase, siklodekstrin-β ° ° ° ° ° ° enzymatic characterization of recombinant cyclodextrin glycosyltransferase from bacillus sp. a2-5a using sagoo starch as substrate 1 1 2 rina imaniar , catur riani , dessy natalia , 1 and debbie soffie retnoningrum * 1 school of pharmacy, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia; 2 faculty of mathematics and science, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia baa31539, ° -1 ° genbank, ncbi) with the first 29 amino acids predicted as a signal sequence. the native cgtase has an optimal ph of 5.5 and an optimal temperature of 50-55 with product specificity of cd-α : β : γ with ratio of 5 : 77 : 18 and the conversion rate was 50% from soluble starch (kelly et al. 2009; ohdan et al. 2000). in our previous research, the cgtase gene of bacillus sp. a2-5a was codon optimized for high level expression in escherichia coli and synthetically constructed. sagoo starch was the best substrate in an assay using horikoshi medium. this research was aimed to optimize the rcgtase overproduction and characterize the enzyme using sagoo starch as substrate. materials and methods bacterial strain and growth condition. e. coli bl21(de3)/pjexpress_401_cgtase (www.dna20.com) was grown in luria bertani (lb) medium containing 25 µg ml kanamycin at 37 °c. all experiments were done using sagoo starch from riau island, indonesia as substrate. overproduction and purification. three growth media, namely luria bertani (lb), terrific broth (tb) and super optimum broth (sob) were used to overproduce rcgtase at two temperatures, 16 and 25 , in the presence of 0.1 mm of iptg for 6 h. the rcgtase was affinity purified using resin in column contain nickel and tris (carboxymethyl) ethilene diamine (ted) (protino, germany). first, the column was equilibrated with 4 bed volumes of lysisequilibration-wash buffer, then allow the column to drain by gravity. the supernantant was added to the pre equilibrated column and allow the column to drain by gravity. the column was washed twice with 4 bed volumes of lysis-equilibration-wash (lew) buffer and allow column to drain by gravity. the rcgtase was eluted in for fractions. elution was done by the addition of 4 x 3 bed volumes of elution buffer containing 250 mm imidazole, ph 8 and fractions were collected separately. the overproduction and purification processes were monitored by 10% sdspage analysis. rcgtase was separated on 10% polyacrilamide gels for 60 minutes at 125 v. the gel was flooded by coomassie blue staining and destained with destaining solution to visualize rcgtase band on gel. the concentration of rcgtase was determined by densitometry method compared to bovine serum albumin (bsa) concentration using imagej c c (gallagher 2010). the purified rcgtase was used in the enzyme characterization. activity assays. the hydrolysis and cyclization activity were monitored by zymography (pakzad et al. 2004). native page was performed with 10% polyacrylamide gels. three wells were used to separate 10 µl rcgtase each. one half of the gel was incubated in 3% soluble starch at 37 °c for 30 min. the gel was washed with distilled water, and stained with a solution containing 0.1% i in 1% ki. the clear band 2 in the blue context of the gel was indicated of amylolytic activity. for phenolphthalein indicator gel method, the indicator gel was prepared by mixing 0.24 g soluble starch, 0.14 g agar in 16 ml of 0.2 m phosphate buffer (ph 8). after the mixture, 0.5 ml of 0.4% phenolphthalein was added and the whole mixture was cooled about 50 ºc. the indicator gel was poured on the second half of the polyacrylamide gel. after a 5 min incubation at 37 °c, the indicator gel was flooded with a 0.1% sodium carbonate solution until the context of the gel turned into red. after visualization of β-cgtase activity, the third half polyacrylamide gel was subjected to conventional coomassie blue staining method. the β-cyclization activity of rcgtase was measured by spectrophotometer using phenolphtalein method (goel and nene 1995). the reaction mixtures containing 1% (w/v) gelatinized sagoo starch in various phs. rcgtase of 0.384 µg was incubated at various temperatures for 30 min at temperature and ph as described below. the β-cd concentration was measured based on the decrease in color intensity of phenolphtalein at 550 nm. optimum temperature and thermostability. the optimum temperature of purified rcgtase was determined by incubating 0.02 unit activity (ua) rcgtase with 1% (w/v) gelatinized sagoo starch in 50 mm tris-cl ph 6.0 at different temperatures, ranging from 26-80 for 30 min and the reaction was stopped using 1.2 m hcl. the gelatinized sagoo starch was prepared by heating sagoo starch suspension at 80 ̊ c for 15 min. before the β-cd was measured, the reaction was neutralized using 1.2 m naoh. the reactions were carried out using the rcgtase assay procedure mentioned above (goel and nene 1995). the temperature stability of the enzyme was measured by incubating 0.02 ua rcgtase with an equal volume of 50 mm tris-cl buffer (ph 7.0) for 30 min, followed by incubation with 1% (w/v) gelatinized sagoo starch for 30 min. residual activities were measured with the standard assay as mentioned above (goel and nene 1995). °c volume 6, 2012 microbiol indones 125 optimum ph and ph stability. three buffers were used to characterize optimum ph and ph stability. as the enzyme activity can be different depending on the buffer system, normalization process is necessarily required. the optimum ph of the purified rcgtase was determined by reacting 0.029 ua enzyme with 1% (w/v) gelatinized sagoo starch in various phs, ph 4 and 5 using 50 mm sodium acetate buffer, phosphate buffer (ph 6-8) and glycine-naoh buffer (9-10) at optimum temperature for 30 min. the reactions were stopped by boiling for 5 min then neutralized by 75 mm naoh for acid condition and 75 mm hcl for base condition. then, the subsequent steps were done according to the rcgtase assay described above (goel and nene 1995). the ph stability of the rcgtase was measured by incubating 0.029 ua enzyme at various phs (4-10) in buffers mentioned above without substrate for 30 min. then the enzyme was reacted with 1% (w/v) gelatinized sagoo starch in optimum temperature and ph. the remaining activity of the enzyme was assayed by the standard assay method (goel and nene 1995). time course β-cd production. after the optimum ph and temperature were determined, 0.001 ua enzyme was reacted with 1% (w/v) gelatinized and raw sagoo starch in optimum ph and temperature for 0.5-72 h. the β-cyclization activities were measured with the standard assay as mentioned above. product specificity. 0.029 ua enzyme was reacted with 1% (w/v) gelatinized sagoo starch in optimum ph, temperature and incubation time. the cyclization ratio of different cds produced was analyzed using hplc with refractive index detector and nh column (waters, sperisorb). the flow was set 2 at 1 ml/min with 70 : 30 acetonitrile-water as mobile phase (kinalekar et al. 2000). small scale β-cd production. the β-cd was produced in 100 ml reaction containing 0.15 ua rcgtase and 1% (w/v) gelatinized sagoo starch in optimum ph, temperature and incubation time. the βcd was measured by phenolphtalein method (goel and nene 1995). results in order to find the best growth condition for rcgtase production, optimization was done using three growth media (lb, tb, and sob) and two temperatures (16 and 25 ) were used for induction. in all media and both temperatures, rcgtase demonstrated as a 76.39 kda protein was produced but at different level. the highest level was obtained when lb medium was used °c and induction was carried out at 25 for 6 h (fig 1a). for further experiment, the overproduction of rcgtase was carried out using lb medium and induction was done at 25 for 6 h. for enzyme characterization, rcgtase was required in the purified form. the rcgtase was copurified with a 45 kda protein (fig 1b), identified previously as laci protein. 8 ml of partially purified -1 rcgtase in concentration 0.038 µg ml was produced from 4 x 250 ml culture. the 76.39 kda protein displayed both starch hydrolysis and β-cyclization activities as determined by zymography method (fig 2). this demonstrated that the rcgtase produced in this ° ° c c 25 116 66,2 45 35 kda m tb lb sob i n i n i n a b fig 1 10% sds-page analysis of rcgtase in overproduction and purification. (a) rcgtase overproduction using lb medium at 25 c for 6 h. (b) purified rcgtase. m; protein marker; n: no iptg induction; i: iptg induction; tb: terrific broth; lb: luria bertani; sob: super optimum broth; c: crude extract; ft: flowthrough; w: wash; e: elution. the arrow showed rcgtase protein band. ° 126 imaniar microbiol indones 0.15 ua rcgtase at optimum ph, temperature and incubation time. discussion in present work, we reported better rcgtase overproduction condition and some characters of the rcgtase that are central to its use in the β-cd production using sagoo starch. induction at 25 for 6 h in lb medium was the best condition for rcgtase overproduction using the starch. a number of characteristic of rcgtase of bacillus sp. a5-2a including its optimal temperature and ph, its thermostability and ph stability demonstrated that this enzyme has good thermostability and works at quite wide range of phs. using gelatinized sagoo starch as substrate, the enzyme maintains its product specificity with β-cd as a predominant product and in 100 ml of culture, the amount of β-cd produced in optimal condition was 524 mg. the enzyme is unable to act on raw starch and then gelatinized sagoo starch should be used for the β-cd production. rcgtase was formed in the cytoplasm of e. coli in the form inclusion body. in this present work, induction was performed at 16 in attempt to obtain higher amount of soluble rcgtase. our current result showed that the yield of soluble rcgtase was the same as that of previous work at 25 and the yield of the total rcgtase, in the form of soluble and inclusion body was the highest using lb as growth medium. optimum temperature and thermostability. the optimum temperature of rcgtase using sagoo starch was the same as that of native cgtase a2-5a (komentani et al. 1994) and rcgtase a2-5a in b. subtilis ana-1 (ohdan et al. 2000), ranging of 50-55 . the t of rcgtase using sagoo starch (56 ) was 50 lower than native cgtase a2-5a (64.4 ) (kelly et al. 2009). the difference was probably due to the presence of cacl in the assay activity of native cgtase a2-5a 2 (kelly et al. 2009). moreover, in this research the reaction was done at ph 7.0, while the ph stability assay of native cgtase a2-5a was carried out at ph 5.5 which was close to optimum ph of rcgtase at ph 6. optimum ph and ph stability. the ph optimum of rcgtase using sagoo starch (ph 6.0) as substrate was slightly different from native cgtase a2-5a (ph 5.5) (ohdan et al. 2000). the difference of optimum ph was probably caused by the difference of incubation temperature. in this research, the incubation temperature was at 55 while native cgtase a2-5a was at 40 . the cgtase which has optimum activity ° ° ° ° ° ° ° ° c c c c c c c c work was active. although the purified rcgtase was still contaminated with laci, there was only rcgtase band that showed hydrolysis and β-activity. our previous work showed that sagoo starch was the best substrate for cgtase of bacillus sp a2-5a. the characteristic of this cgtase using sagoo starch as substrate has not been reported previously. therefore, this current work focused on the partial characterization the enzyme using sagoo starch. the first character to be studied was its optimum temperature and its thermostability. the purified rcgtase exhibited the highest β-cyclization activity at 55 (fig 3a). the activity increased from 26 to 55 , decreased above 50 °c and was almost no activity (10%) at 80 . however, it displayed 90% activity at 50-60 °c (fig 3a). in terms of its thermostability, the temperature half life (t ) was shown to be 56 for 30 min incubation 50 (fig 3b). rcgtase exhibited biphasic phenomenon of its activity with two optimum phs, ph 6 and ph 9 (fig 3c). it displayed no activity at ph 4 but then its activity increased and reached the first optimum ph, at ph 6. from ph 6 to ph 8, its activity declined and reached the lowest activity (about 60%) in the range at ph 8. with regards to its ph stability, rcgtase retained its activity (>80%) at wide range of phs (5-10) (fig 3d). the production of β-cd in the gelatinized starch was maximum at 24 h, rcgtase did not show βcyclization activity for raw sagoo starch (fig 4a). but using gelatinized sagoo starch, the β-cd produced per min was highest at 30 min incubation and reduced considerably until 72 h incubation (fig 4b). the ratio of α, β, and γ-cd using 1% w/v gelatinized sagoo starch was 4 : 81 : 15, respectively, while using 10% w/v raw sagoo starch was 5.3 : 76.4 : 18.3. in terms of β-cd production, 542 mg was produced in 100 ml reaction using 1% w/v gelatinized sagoo starch and ° ° ° ° ° c c c c c fig 2 result of zymography assay. (a) coomassie blue staining. (b) ki/i staining. (c) phenolphtalein 2 staining. volume 6, 2012 microbiol indones 127 a rcgtase b c fig 4 time course of β-cd production using purified rcgtase bacillus sp. a2-5a using 1% (w/v) gelatinized sagoo starch as substrate. the reaction was carried out at 55 c in 100 ml of 50 mm phosphate buffer ph 6.0. (a) amount β-cd produced during 72 h. (b) mg β-cd produced per min during 72 h. ° -1 m g m l β -c d 3.00 2.50 2.00 1.50 gelatinized sagoo starch raw sagoo starch 1.00 0.50 0.00 0 12 24 36 48 60 72 time (h) m g β -c d p er m in u te 0.050 0.045 0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 0.000 0 6 12 18 24 30 36 42 48 54 6660 72 time (h) a b fig 3 properties of the purified rcgtase a2-5a using sagoo starch as substrate. (a) temperature profile of purified rcgtase. for this temperature profile, enzymatic activity was measured in tris-cl ph 7.0. (b) thermal stability of purified rcgtase. thermostability was determined by preincubating the enzyme in tris-cl ph 7.0 at designed temperatures for 30 min. (c) ph profile purified rcgtase. the reaction phs were adjusted to 4-10 with the following buffers: na-acetat (ph 4.0-5.0), na-phosphate (ph 6.0-8.0), and glycine-naoh (ph 9.0-10.0). (d) ph stability purified rcgtase. ph stability was determined by preincubating enzyme at following buffer na-acetat (ph 4.0-5.0), na-phosphate (ph 6.08.0), and glycine-naoh (ph 9.0-10.0) for 30 min. the given values in all activity assays are the means of triplicates, and the error bars indicate the standard deviation of these triplicates of independent experiment ph ph 0 20 40 60 80 100 % r el at iv e ac ti v it y 20 30 40 50 60 70 80 temperature (˚c) 0 20 40 60 80 100 120 % r el at iv e ac ti v it y 4 5 6 7 8 9 10 % r es id u al a ct iv it y 120 100 80 60 40 20 0 4 5 6 7 8 9 10 a % r es id u al a ct iv it y 100 90 80 70 60 50 40 30 20 10 0 40 50 60 70 temperature (˚c) b c d 128 imaniar microbiol indones at ph 6 is from bacillus sp. ts1-1, g1 and 17-1 (rahman et al. 2006; ong et al. 2008; kaneko et al. 1989). rcgtase retained >50% of its activity at phs 5.0-10.0, while native cgtase a2-5a retains 50% of its activity at phs 9.0-10.0 (komentani et al. 1994). time course experiment of β-cd production. rcgtase using sagoo starch produced maximum β-cd at 24 h reaction time, in contrast, the β-cd produced per minute was highest at 30 min incubation time. it showed that the activity of the rcgtase decreased in longer period of reaction. in cds ratio assay using 1% (w/v) gelatinized sagoo starch, the α, β, and γ-cd ratio was 4 : 81 : 15, respectively. the α, β, and γ-cd ratio from native rcgtase is 5 : 77 : 18 (kelly et al. 2009), respectively and β-cd was still the predominant product. in other research using cgtase from bacillus circulans and sagoo starch as substrate, β-cd is 65% from total cds produced (charoenlap et al. 2004). the -1 5.42 ± 0.75 g l β-cd produced from 100 ml reaction at optimum condition had not purified yet. in conclusion, the difference of substrate did not influence of some characteristics of rcgtase a2-5a such as optimum temperature and ph but the β-cd yield was higher when using sagoo starch as substrate than using soluble starch. further study of the rcgtase and β-cd production followed by β-cd purification should be done in the near future to obtain purified rcgtase and β-cd in larger scale using sagoo starch; therefore their use in industrial scale can be applied. acknowledgment we thank to research center for food, health, and drugs of itb for the financial support of the research. references charoenlap n, saovanee d, sarote s, sittiwat l. 2004. optimization of cyclodextrin production from sago s t a r c h . b i o r e s o u r te c h n o l . 9 2 ( 1 ) : 4 9 5 4 . doi:10.1016/j.biortech.2003.07.007. endo t, ueda h. 2004. large ring cyclodextrins-recent progress. fabad j pharm sci. 29: 27-38. volume 6, 2012 microbiol indones 129 gallagher, sr. 2010. digital image processing analysis with imagej. current protocols essential laboratory technique.a.3c.1-a.3c.24. doi goel a, nene sn. 1995. modifications in the phenolphthalein method for spectro-photometric estimation of beta cyclodextrin. starch 47(10): 399-400. doi: 10.1002/star.19950471006. kaneko t, song kb, hamamoto t, kudo t, horikoshi k. 1989. construction of a chimeric series of bacillus cyclomaltodextrin glucanotransferase and analysis of the thermal stabilities and ph optima of the enzyme. j gen microbiol. 135(12): 3447-3457. doi: 10.1099/00221287135-12-3447. kelly r, dijkhuizen ml, leemhuis h. 2009. the evolution of cyclodextrin glucanotransferase product specificity. appl microbiol biotechnol. 84(1): 119-133. doi: 10.1007/s00253-009-1988-6. kinalekar ms, kulkarni sr, vavia pr. 2000. simultaneous determination of α, β, and γ cyclodextrins by lc. j pharm biomed anal. 22(4): 661-666. doi: 10.1016/s0731-7085(99)00299-x. ohdan k, kuriki t, takata h, okada s. 2000. cloning of the cyclodextrin glukanotransferase and the impact for biotechnological applications. appl microbiol biotechnol. 85: 823-835. ong ri, goh km, mahadi nm, hassan o, rahman rnrza, illias rm. 2008. cloning, extracellular expression and characterization of a predominant β-cgtase from bacillus sp. g1 in e.coli. indian j microbiol biotechnol. 35(12): 1705-1714. doi : 10.1007/s10295008-0462-2. pakzad sr, ajdary sn, moazami, haghighi s. 2004. a novel method to detect β-cyclodextrin glucosyl transferase (β-cgtase) activity on polyacrylamide gels. j iran biomed. 9(2): 87-90. rahman k, illias rm, hassan o, mahmood nan, rashid naa. 2006. molecular cloning of a cyclodextrin glucanotransferase gene for alkalophilic bacillus sp. ts1-1 and characterization of the recombinant enzyme. e n z y m e m i c r o b te c h n o l . 3 9 : 7 4 8 4 . d o i : 10.1016/j.enzmictec.2005.09.014. szejtli j. 1997. utilization of cyclodextrins in industrial products and processes. j mater chem. 7(4): 575-587. doi: 10.1039/a605235e. :10.1002/9780470089 41.eta03cs03. 1: 124 2: 125 3: 126 4: 127 5: 128 6: 129 mi658-17-07-12 (yasmon) available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.2.3issn 1978-3477, eissn 2087-8575 vol 6, no 2, june 2012, p 69-76 *corresponding author; phone: +62-21-3100806, fax: +6221-3100810; e-mail: andi.yasmon@ui. ac.id nine ha genes of influenza a (h1n1) viruses originating from swine which were detected in 2009 in jakarta, indonesia, were characterized in this study. nasopharyngeal and/or pharyngeal samples were extracted to obtain viral rna genomes. amplification of the ha segment was performed by using the reverse transcription-polymerase chain reaction (rt-pcr), and followed by nested pcr in cases of rt-pcr negative. dna sequencing was performed by using eight overlapping primers. all the jakarta strains were closely related to vaccine strain a/california/07/2009. nine amino acid changes were found in the jakarta strains, and 5 (p100s, s220t, g239d, r240q, and i338v) of those were unique to all jakarta strains with respect to strain a/california/07/2009 used to produce vaccine. an i338v substitution was detected in a cleavage site of ha and no amino acid changes were detected in potential sites for n-linked glycosylation. for seven sites (positions 131, 158, 160, 183, 187, 222, and 227) playing an important role in viral attachment to host receptor, none of the expected amino acid changes was detected; however, a s220t substitution close to amino acid 222 was found in all the jakarta strains. all amino acid changes potentially affect the pathogenicity of the viruses and the efficacy of strain a/california/07/2009 in neutralizing the jakarta strains. h1n1, hemagglutinin, influenza a, pathogenicity dalam penelitian ini telah dilakukan karakterisasi sembilan gen ha (hemagglutinin) virus influenza a (h1n1) origin babi yang dideteksi pada tahun 2009 di jakarta, indonesia. genom rna virus diperoleh dengan cara mengekstraksi sampel swab nasofaring dan/atau faring. reverse transcription-polymerase chain reaction (rt-pcr) dilakukan untuk mengamplifikasi segmen ha. nested pcr dilakukan untuk sampel yang negatif rtpcr. segmen dna ha hasil amplifikasi kemudian disekuensing menggunakan delapan primer yang saling tumpang tindih. analisis hasil sekuensing menunjukan semua strain virus jakarta berkorelasi dekat dengan strain a/california/07/2009 yang digunakan untuk produksi vaksin. ditemukan sembilan substitusi asam amino pada strain virus jakarta yang dibandingkan dengan strain a/california/07/2009, dimana 5 (p100s, s220t, g239d, r240q, and i338v) dari 9 substitusi tersebut unik untuk semua strain virus jakarta. dari semua substitusi asam amino tersebut, tidak dijumpai perubahan pada situs yang berpotensi mengalami glikosilasi (n-linked glycosylation). pada situs pembelahan ha (cleavage site of ha), dideteksi substitusi i338v. selain itu tidak dijumpai perubahan asam amino pada tujuh situs yang berperan penting dalam pelekatan virus pada reseptor sel hospes, yaitu posisi asam amino 131, 158, 160, 183, 187, 222, dan 227. namun, satu substitusi (s220t) yang berdekatan dengan asam amino 222 dideteksi pada semua strain virus jakarta. secara keseluruhan, semua substitusi asam amino tersebut berpotensi mempengaruhi patogenisitas virus dan efikasi strain a/california/07/2009 dalam menetralisasi strain virus jakarta. h1n1, hemagglutinin, influenza a, patogenisitas, substitusi asam amino key words: amino acid substitution, kata kunci: influenza a viruses (iav) belong to the family of orthomyxoviridae. they are grouped into sub-types based on differences of hemagglutinin (ha) and neuraminidase (na) proteins. the iav have a broad host range, including humans, pigs, horses, marine mammals, domestic and wild birds (webster et al. 1992), explaining why iav are more frequently reassorted in certain hosts particularly in pigs and are responsible for the emergence of novel sub-types causing occasional pandemic events (cox and subbarao 2000). an influenza pandemic occurred in 2009 that was caused by the influenza of swine-origin a/h1n1 (soi/09). the virus has re-assorted in pigs (kou et al.2009), consisting of: 1) swine originating ha, na, mp, np and ns segments; 2) avian originating pb2 and pa segments; and 3) human originating pb1 segment. since pigs are susceptible to avian and human iav, they are known as a vessel for mixing iav enabling reassortment of genes (khiabanian et al. 2009). the soi/09 spread from swine to humans might be initiated five unique amino acid residues of hemagglutinin (ha) proteins of swine influenza a (h1n1) detected in 2009 in jakarta, indonesia 1 1 2 1 andi yasmon *, yulianty muhayar , vivi setiawaty , beti ernawati dewi , 1 1 budiman bela , and fera ibrahim 1 departemen mikrobiologi, fakultas kedokteran, universitas indonesia, jalan pegangsaan timur 16, jakarta 10320, indonesia; 2 national institute of health research and development, ministry of health, jalan percetakan negara no. 23, jakarta 10560, indonesia materials and methods clinical samples. nasopharyngeal and/or pharyngeal samples were taken from suspected patients by using cotton dacron swabs. the swabs were placed in cryotubes containing 1 ml of transport medium consisting of minimum essential medium (mem; sigma, st. louis, mo.,usa) with 1% (v/v) penicillinstreptomycin. the samples were immediately shipped to the laboratory by using a special box containing 4 or o 6 ice packs to keep the temperature of samples at 2-8 c. nine samples included in this study were soi/09 positive by a real time rt-pcr, a routine test of surveillance study by influenza-like-illness network, ministry of health, indonesia. primers. primers for amplification and overlapping dna sequencing covering full length of ha gene (table 1) were determined by homological analysis of 22 ha genes retrieved from genbank (http://www.ncbi.nlm.nih.gov/genomes/flu/), with the following accession numbers: cy064700.1, c y 0 6 4 8 9 5 . 1 , h m 5 6 9 7 4 0 . 1 , c y 0 6 5 0 2 7 . 1 , c y 0 6 4 9 8 7 . 1 , c y 0 6 5 8 4 8 . 1 , c y 0 6 5 7 9 2 . 1 , c y 0 6 5 9 5 2 . 1 , c y 0 6 5 9 2 8 . 1 , h m 6 2 4 0 8 6 . 1 , c y 0 6 5 7 6 2 . 1 , c y 0 6 5 7 7 0 . 1 , a b 5 3 9 7 3 9 . 1 , h m 5 8 1 9 2 3 . 1 , h m 5 6 9 6 5 9 . 1 , c y 0 6 4 9 3 1 . 1 , c y 0 6 4 4 0 0 . 1 , c y 0 6 4 3 9 3 . 1 , c y 0 6 4 3 9 7 . 1 , cy064392.1, cy064391.1, and cy064394.1. the primers were designed by primer designer version 2, scientific & educational software-1991. amplification of ha genes. viral rna(s) were extracted from nasopharyngeal and/or pharyngeal samples by qiaamp viral rna mini-kit (qiagen) following to manufacturer's instruction with 60 µl of o elution volume and stored at -80 c not more than 1 week. one-step reverse transcription and polymerase by exposure of virus infected swine to people (lange et al. 2009) and then the infection was globally transmitted from human to human (michaelis et al. 2009), covering more than 212 countries with more than 15 290 deaths as of february 7, 2010 based on w h o d a t a a t h t t p : / / w w w. w h o . i n t / c s r / d o n /2010_02_12/en/index.html. the virus was also detected in 24 provinces in indonesia with total number of confirmed cases of about 1033 people of which 6 died (moh-indonesia 2011). the ha proteins of iav play an important role in viral pathogenesis. the protein is cleaved by host protease enzymes into two subdomains, ha1 and ha2. the ha2 is a trans-membrane protein, while the ha1 is a spike protein that directly interacts with the viral receptor on a target cell. amino acid changes of ha have been involved in virulence of viruses (kido et al. 2008). severe cases of soi/09 have also been associated with a d222g substitution of ha (kilander et al. 2010; mak et al. 2010; miller et al. 2010; potdar et al. 2010). in another study it was reported that there were three amino acid changes (d131e, s186p and a198e) of soi/09 ha, 2 of those were also found in 1918 viruses and were sufficient to confer virulence of ca/04 in mice (ye et al. 2010). moreover, suphaphiphat et al. (2010) demonstrated amino acid changes in residues 186 and 194 of soi/09 ha that can improve viral replication in cell culture and chicken eggs. based on the evidence, no conclusive amino acid changes affecting the pathogenicity of virus have been reported. therefore, we characterized the ha genes of soi/09 strains detected in 2009 in jakarta, indonesia that are useful for study of epidemiology, development of vaccines, and an understanding of complex viral pathogenicity. 70 yasmon et al. microbiol indones primer names primer sequences purposes 13f 5'-aatga aggcaata ctag tagtt-3' one-step rt-pcr 1734r 5'-atgcttctgaaat cctaat g-3' one-step rt-pcr 18f 5'-aggcaata ctag tagtt ctgct-3' nested pcr and d na sequencing 1708r 5'-acat attctacactgtag agacc-3' nested pcr and d na sequencing 380f 5'-agct cag tgtcatcattt gaa-3' dn a sequencing 770f 5'-ccggg agacaaaat aacattc-3' dn a sequencing 1171f 5'-g aatgccattg acg agatt ac-3 ' dn a sequencing 403r 5'-cctttcaaatg atga cactg-3' dn a sequencing 773r 5'-ccggctctactagtg tccag-3' dn a sequencing 1186r 5'-ctcgtcaatg gcattctgtg-3' dn a sequencing f: forw ard. r: reverse. primer positions in h a gene were indicated by primer names table 1 primers used for one-step rt-pcr, nested pcr, and dna sequencing reactions a b 3054 2036 1636 1018 506 bp 1722 bp m 1 2 3 4 65 m 1 2 3bp 3054 2036 1636 1018 506 1691 bp chain reaction (rt-pcr) was performed in 50 µl of the following reaction mixture: 1x reaction mix, 0.6 µm of each primer, 2 µl of superscript iii rt/platinum dna taq (invitrogen), and 12.5 µl of viral rna(s). the reaction was performed under the following o o conditions: 50 c for 30 min; 94 c for 2 min; 40 cycles o o o of 94 c for 30 sec, 58 c for 30 sec, and 68 c for 2 min; o and 68 c for 5 min. the rt-pcr negative samples were followed by nested pcr (platinum taq dna polymerase high fidelity [invitrogen]): 1x hifi pcr buffer, 0.2 µm of dntp mix, 0.2 µm of each primer, 80 µm of mgso , 1u of platinum taq high fidelity, 0.5-1 4 µl of rt-pcr products. the thermal cycler of nested pcr was performed with the following conditions: 94 o o c for 2 min; 30 cycles of 94 c for 30 sec, for 30 sec, o o and 68 c for 2 min; and 68 c for 5 min. the products of rt-pcr or nested pcr reactions were purified from agarose gels and then sequenced by using overlapping primers (table 1). phylogenetic tree. the neighbor‐joining method (saitou and nei 1987) was used to prepare a phylogenetic tree of ha amino acid sequences using mega5 (tamura et al. 2011). the tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were computed using the maximum composite likelihood method (tamura et al. 2004) and are in the units of the number of base substitutions per site. fifty-two viruses detected in 2009 around the world were involved in this analysis. genetic characteristics. four aspects influencing viral pathogenicity, namely a cleavage site of ha, residues involving a receptor-binding site, n-linked glycosylation, and general variability of ha amino acids were characterized in this study. the alignment of amino acid sequences and homology of ha amino acids were performed by the bioedit version 7.0.0. the residues involving the receptor-binding site were determined according to weis et al. (1988). the nlinked glycosylation sites were determined by the netnglyc1.0 program ( ). rt-pcr and nested pcr. the amplification products of rt-pcr and nested pcr were defined by dna bands corresponding to 1722 bp and 1691 bp, respectively (fig 1). using rt-pcr and nested pcr techniques, all nine ha segments were successfully amplified. the rt-pcr showed double/multiple dna fragments, while nested pcr showed specific amplification (fig 1). the annealing temperature of rtpcr had been optimized, but the double/multiple dna fragments were always detected. this effect may be caused by primers that are cross reacting with other dna sequences. even though the rt-pcr gave double/multiple dna fragments, the results can still be used for dna sequencing reaction because one of those fragments is an expected dominant fragment; thus, the primers can be used for amplification of ha segments of soi/09 viruses. phylogenetic tree. based on the phylogenetic analysis, 52 viral strains involved in this analysis were clustered into lineages i, ii, iii, and iv (fig 2). the jakarta strains and strain used to produce vaccine (a/california/07/2009) were clustered in lineage iv and were related to the viruses that have been circulating in europe, russia, asia, and america. the http://www.cbs.dtu.dk/ services/netnglyc/ results volume 6, 2012 microbiol indones 71 fig 1 results of rt-pcr (a) and nested pcr (b) with expected dna bands of 1722 bp and 1691 bp, respectively. lanes 1-6 and a-c: amplification products of rt-pcr and nested pcr, respectively. lane m: dna ladder. bp: base pair. fig 2 a phylogenetic tree based on ha amino acid sequences of influenza a viruses detected in 2009 around the world (52 strains were involved in this analysis). viral strains are named in accordance with accession numbers and strain names in genbank database. : jakarta strain. i-iv: lineages i-iv. viral progenitor (lineage i) for all viral strains analyzed in this study. all jakarta strains are dispersed into five sublineages of lineage iv (fig 2). strain a/jakarta/008/ viruses in lineage iv have evolved from virus (lineage iii) detected in lyon, northeast france. the virus detected in lyon has evolved from virus (lineage ii) detected in mexico city. the virus detected in turkey is microbiol indones72 yasmon et al. v iral strains amino acid positions in ha 39 100 176 202 211 220 239 240 338 a/california/07/2009 k p p s n s g r i a/jakarta/001/2009 . s . . . t d q v a/jakarta/002/2009 . s . n . t d q v a/jakarta/003/2009 . s q . . t d q v a/jakarta/004/2009 . s . . . t d q v a/jakarta/005/2009 . s . . . t d q v a/jakarta/006/2009 . s . . . t d q v a/jakarta/007/2009 . s . . . t d q v a/jakarta/008/2009 r s . . . t d q v a/jakarta/009/2009 . s . t t d q v 2009 was closely related to viruses detected in nepal, malaysia. strain a/jakarta/009/2009 was closely related to virus detected in argentina. two strains (a/jakarta/001/2009 and a/jakarta/003/2009) were closely related viruses detected in china, korea, and thailand. other strains (a/jakarta/005/2009, a/jakarta/006/2009, a/jakarta/004/2009, and a/jakarta/007/2009, and a/jakarta/002/2009) were closely related to viruses detected in athens and taiwan, berlin and thailand, budapest and perth, ireland and barcelona, and madrid and texas, respectively. most of the jakarta strains were very closely related to viruses detected in asia. among jakarta strains, a/jakarta/002/2009 is very closely related strain a/california/07/2009. amino acid changes in ha. different amino acid residues in the jakarta strains compared with strain a/california/07/2009 used to produce vaccine are shown in table 2. a/jakarta/008/2009, a/jakarta/003/ 2009, a/jakarta/002/2009, and a/jakarta/009/2009 showed amino acid changes, namely lysine to arginine (k39r), proline to glutamine (p176q), serine to asparagine (s202n), asparagine to threonine (n211t), respectively. five amino acid changes (p100s, s220t, g239d, r240q, and i338v) were shown by all jakarta strains and were unique to the jakarta strains. substituted amino acids shared the same polarities, except for p176q and p100s that have changed from non-polar to polar amino acids. molecular characteristics. for molecular characteristics, strain a/california/07/2009 used for vaccine was used this analysis as a reference sequence (data not shown). the question is to know whether the vaccine strain is reasonably applied for protecting people from soi/09l infection, particularly the viral strains found in jakarta. the cleavage site of ha is from amino acid 338 to 346 (vpsiqsr gl). the site consisted of a single basic amino acid (r [arginine]). all jakarta strains showed an i338v substitution in cleavage site of ha. seven potential n-linked glycosylations, amino acids 28 (nst), 40 (nvt), 104 (ngt), 293 (ntt), 304 (nts), 497 (ngt), and 557 (ngs), showed no amino acid changes for all jakarta strains. for seven amino acids (positions 131, 158, 160, 183, 187, 222, and 227) playing an important role in viral attachment to receptor, no amino acid changes in those sites were detected for any of the jakarta strains. however, a s220t substitution close to amino acid 222 was found in all jakarta strains. to our knowledge, no sequences of ha genes of soi/09 viruses from indonesia have been reported. the availability of the sequences is very useful in the study of epidemiology, development of vaccines, and our understanding of viral pathogenicity. here, we reported the evolutionary relationship and molecular characteristics of the soi/09 viruses detected in 2009 in jakarta, indonesia. based on phylogenetic analysis, the jakarta strains were closely related genetically to the vaccine strain (a/california/07/2009) and viruses circulating in europe, russia, asia, and america. the viruses have evolved from viruses from mumbai-india ( a / m u m / n i v 3 9 8 / 2 0 0 9 ) a n d t h a i l a n d (a/swine/thailand/cu-m8_2/2009). based on geographical location, there were no obvious clusters of strains. this phenomenon was also observed by others (furuse et al. 2010; galiano et al. 2011). compared with the vaccine strain a/california/07/2009, the jakarta discussion volume 6, 2012 microbiol indones 73 table 2 amino acid changes of ha of 9 soi/09 viruses detected in jakarta indonesia with respect to the vaccine strain (a/california/07/2009) antigens provide evolutionary advantage to re-infect the same hosts (carrat and flahault 2007). one important target for antibodies for neutralizing or preventing viral infection is the receptor-binding site of ha. there are seven amino acids playing an important role in viral attachment to the host receptor, those are located at 131, 158, 160, 183, 187, 222, and 227 (weis et al.1988). in this study, no substitution was detected for those amino acids; however, we found a single amino acid change (s220t) close to amino acid 222 (table 2). the amino acid change potentially affects the efficacy of neutralizing antibodies that has been formed from previous infections by different strains of iav or vaccine types; however, this issue should be addressed in the future study. regarding amino acid changes at the receptorbinding site influencing the viral pathogenicity, many researchers have reported an amino acid substitution at position 222 that were associated with severe clinical outcome (kilander et al. 2010; mak et al. 2010; miller et al. 2010; potdar et al. 2010). the d222g and d222e substitutions appeared in 5 of 25 and in 6 of 25 fatal cases respectively, suggesting a possible role for polymorphisms at position 222 in the pathogenicity of viruses (galiano et al. 2011). even though we found no amino acid changes in the receptor-binding site, a single amino acid change (s220t) close to amino acid 222 (table 2) could influence viral pathogenicity. in this regard, rogers et al. (1983) reported a l226q substitution (close to amino acid 227) that was responsible for altering specificity of ha for saα2, 3 gal to saα2, 6gal linkage. the specificity alteration might be caused by the change from leucine (l) to glutamine (q) in which both amino acids have different polarities; thus, the substitution leads to altered protein configuration so that specificity of receptor binding is also altered. in this study, serine and threonine (s220t) have the same polar features; however, the substitution is still capable of influencing pathogenicity of virus. this thought is based on a report by yamaguchi et al. (2001) that a single amino acid change at position 394 in vp1 protein was crucial for the pathogenicity of chicken anemia virus (cav) and substitution from glutamine to histidine (both amino acids have the same polar features) led cav to have low pathogenicity. all the jakarta viruses showed an i338v substitution and a single basic amino acid (r [arginine]) in cleavage site of ha (data not shown). numbers of basic amino acids in cleavage site of ha have been associated with pathogenicity of iav (kawaoka and webster 1988). the highly pathogenic influenza a strains clustered in lineage iv; indicating that the vaccine has the potential to cross-react with the jakarta strains. however, use of this vaccine in indonesia should be decided on comparing the indonesia strains circulating in 2010 and 2011 and which were isolated from representative areas in indonesia. of the nine amino acid changes detected in the jakarta strains, two (p176q and p100s) showed amino acid changes with different polarities (non-polar to polar amino acids) (table 2). the amino acid changes might affect the globular protein structure because the non-polar and polar amino acids are hydrophobic and hydrophilic, respectively. moreover, five mutations (p100s, s220t, g239d, r240q, and i338v) were unique to all jakarta strains, compared with the strain used to produce vaccine (a/california/07/2009). note that a single amino acid change can cause the alteration of protein function and can have drastic phenotypic consequences (ng and henikoff 2006). therefore, a neutralization study using anti-ha of a/california/07/2009 antibodies for jakarta strains should be tested in the future. comparison of ha1 and ha2 showed that all amino acid changes occurred in ha1 and that no changes were detected in ha2 (table 2). the iav to which the human population is exposed are known to undergo certain changes in their antigenicities, refered to as a antigenic drift. it results from the accumulation of a series of amino acid changes in the antigenic regions of the ha molecule that are necessary for changing the receptor specificity. moreover, the amino acid changes could be as a way by which viruses escape from the host immune system, particularly antibodies secreted by b cells. it is known that ha1 is expressed as trimers at surface virions and mediates viral entry into target cells through binding to terminal sialic acid groups on cellular membrane proteins (wilson et al.1981). interaction of ha1 with terminal sialic groups leads to internalization of virus into an endosome whose acidic environment allows ha2 proteins to mediate fusion between viral and endosomal membranes, thereby releasing the viral genomic rna into the target cell (matlin et al.1981; rust et al. 2004). since ha1 is most important in viral pathogenesis in initiating infection, the proteins have been a primary target for antibodies to neutralize the virus and/or to accelerate the viral phagocytosis. in order to escape from the antibodies, the ha1 proteins of iav have shown cumulative genetic drifts allowing distinction between 'new' and 'old' iav (escorcia et al. 2008). influenza outbreaks could be associated with antigenic drifts on ha1 in which the changes in viral microbiol indones74 yasmon et al. h5n1 virus is one that is characterized by availability of polybasic amino acids in the cleavage site of ha (zhou et al. 2007). the ha proteins with polybasic amino acids are very efficiently cleaved by the host proteases (kawaoka and webster 1988), leading to more viral production and dissemination to other cells of the host. like cleavage site of ha, n-linked glycosylation on ha is also important leading viral ability to impart various advantages to virus survival and virulence (vigerust and shepherd 2007). levels of potential glycosylation also affect disease severity and outcomes of infection in naive animals, in which the morbidity and mortality, as well as viral lung titers, were low while the level of potential glycosylation of viruses increased (vigerust et al. 2007). an amino acid change (t160a) of ha protein, resulting in the loss of glycosylation at 158–160, was responsible for ha binding to sialylated glycans and was critical for h5n1 virus transmission in guinea pigs (gao et al. 2009). in this study, we found no amino acid changes for any of the jakarta strains in seven potential sites for n-linked gycosylation (data not shown); indicating that the viral virulence has not changed from point of view of nlinked glycosylation. in conclusion, all jakarta strains are closely related to strain a/california/07/2009 used to produce vaccine. five amino acid changes (p100s, s220t, g239d, r240q, and i338v) are unique to all the jakarta strains. for molecular characteristics, an i338v substitution is detected in cleavage site of ha and no polymorphisms are detected in amino acids that have a potential to be nlinked glycosylation. even though there are no amino acid changes in seven sites playing an important role in viral attachment to the host receptor, a s220t substitution close to amino acid 222 was found in all jakarta strains. overall, the effects of the amino acid changes on viral pathogenicity and cross-reaction of strain a/california/07/2009 to the jakarta strains should be addressed in future studies. references carrat f, flahault a. 2007. influenza vaccine: the challenge of antigenic drift. vaccine 25(39-40): 6852-6862. cox nj, subbarao k. 2000. global epidemiology of influenza: past and present. annu rev med. 51: 407421. escorcia m, vazquez l, mendez st, rodriguez-ropon a, lucio e, nava gm. 2008. avian influenza: genetic evolution under vaccination pressure. virol j. 5:15. doi:10.1186/1743-422x-5-15. furuse y, suzuki a, oshitani h. 2010. evolutionary analyses on the ha gene of pandemic h1n1/09: early findings. bioinformation 5(1): 7-10. galiano m, agapow pm, thompson c, platt s, underwood a, ellis j, myers r, green j, zambon m. 2011. evolutionary pathways of 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m, rothfeder m, mandl cw, dormitzer pr, mason pw. 2010. mutations at positions 186 and 194 in the ha gene of the 2009 h1n1 pandemic influenza virus improve replication in cell culture and eggs. virol j. 7: 157. doi: 10.1186/1743-422x-7-157. tamura k, nei m, kumar s (2004). prospects for inferring very large phylogenies by using the neighbor-joining method. proc natl acad sci usa. 101(30). 1103011035. tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol biol evol. 28(10): 2731-2739. microbiol indones76 yasmon et al. 1: 67 2: 68 3: 69 4: 70 5: 71 6: 72 7: 73 8: 74 6 no. 297 (diana e. waturangi).pmd identification of class 1 integron of escherichia coli from street foods in jakarta frisca, bibiana widyati lay, and diana elizabeth waturangi∗ faculty of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia a total of 43 escherichia coli isolates were identified from school street foods located in northern and southern jakarta. the isolates were examined for antibiotic resistance using five antibiotics discs (ampicillin, kanamycin, streptomycin, trimethoprim, tetracycline) and screened for the class 1 integron with specific conserved region primer using pcr amplification. the antibiotic diffusion test revealed three isolates (7%) with resistance to multiple antibiotics. pcr detection of integron regions showed one isolate possessed a class 1 integron bearing one gene cassette with ~700bp amplicon size. dna sequencing showed that the gene cassette was resistant to trimethoprim determinant type v (dhfrv). the integron bearing e. coli strain could become a threat for the widespread distribution of an antibiotic resistance gene especially for pathogenic bacteria. key words: escherichia coli, street foods, antibiotic, integron _____________________________________________ poor sanitation of school’s street foods (that are obtained from street vendors outside schools), which is commonly found in indonesia, may lead to the disease that could risk millions of children health. therefore, escherichia coli is an important food hygiene indicator to access the quality of street foods. the presence of e. coli also shows the probability of contamination by enteric pathogenic bacteria, like salmonella sp., vibrio sp., or enteropathogenic e. coli (epec) (stiles and king 1981). resistance to antibiotics is highly prevalent in bacterial isolates worldwide, particularly in developing countries (okeke et al. 2000). normal intestinal microbiota, including e. coli, usually act as a reservoir for resistance determinants. they are often associated with mobile genetic vectors like plasmids, transposons, and integrons. integrons are gene acquisition and expression systems formed via site specific recombination. the units of dna captured by integrons are termed gene cassettes. gene cassettes are dna mobile elements composed of a gene, most commonly encoding antibiotic resistance, and an integrase specific recombination site known as 59-base element (59be) (stokes et al. 2001). four types of integron have been identified to date according to homology sequences of their integrase genes, of which the class 1 integron is the most prevalent among clinical isolates, especially enterobacteriaceae (chang et al. 2000). more than 60 distinct cassettes have been shown to be carried within the class 1 integron. the aim of our study was to search for the presence of the class 1 integron in e. coli isolated from street foods and to characterize their gene cassette assortments. the finding of an integron could become a threat for creating widespread antibiotic resistance genes especially in pathogenic bacteria. _________________ ∗ corresponding author, phone: +62-21-5703306 ext 335, fax: +62-21-5719060, e-mail: diana.waturangi@atmajaya.ac.id materials and methods collection of samples. a total of 20 foodstuffs and beverages samples were collected from vendors outside five elementary schools in northern and southern jakarta respectively. two kinds of foods and beverages which were those commonly consumed by children, were taken from each location. street food samples obtained were immediately placed in a cooler box during transportation to the laboratory. samples were collected from july through to august 2005. isolation of bacteria. food samples were blended and weighted aseptically. an amount of 25 g of blended food sample was added to 250 ml of tryptone soya broth medium and then incubated for 3 hours at 37 oc. beverages samples (250-300 ml) were aseptically filtered using a micro-filter vacuum pump containing a 0.2 µm filter membrane (millipore). the filter membrane was then added to 50 ml of tryptone soya broth and incubated at 37 oc. serial dilution (10-4-10-6) was made from the inoculated broths and immediately subcultured to eosin methylene blue agar (emb). identification of e. coli. identification used biochemical tests, triple sugar iron agar (tsia), imvic (indole, methyl red, voges proskauer, citrate), and mug (4methylumbelliferyl-ß-d-glucuronide) according to sni 012332-1991. antibiotic resistance test (agar diffusion). a susceptibility test to 5 antibiotics which are frequently used in therapy, and commonly found in gene cassette form, was tested using the disc diffusion method on mueller hinton agar. those antibiotics were ampicilin (10 µg); kanamycin (30 µg); streptomycin (10 µg); trimethoprim (5 µg); and tetracycline (30 µg) (oxoid, hampshire, england). the susceptibility tests were interpreted according to document m100-s9 (who 1999). detection of class 1 integron. whole-cell dna of all 43 e. coli isolates, were screened for the presence of class 1 integron using pcr amplification (perkin elmer, usa) according to the procedure of lavesque et al. (1995). specific conserved class 1 integron region primers, 5’cs (5’-ggca microbiology indonesia, april 2007, p 15-18 volume 1, number 1 issn 1978-3477 tccaagcagcaag-3’) and 3’cs (5’aagcagactt gacctga-3’) (proligo) were used to amplify any integrated gene cassettes. e. coli obtained from varanus spp. feces (waturangi et al. 2003) was used as positive control. the pcr product was separated by agarose gel electrophoresis and purified using qiaquick gel extraction kit (qiagen). the pcr product was sequenced by using big dye®terminator v3.1 cycle seuencing kit (applied biosystems usa). the product was analyzed with an abi prism 377 automated dna sequencer. for comparison with known sequences, the basic local allignment search tool (blast) computer search program was used. nucleotide sequence accession number. the nucleotide sequence data reported in this paper will appear in the genbank nucleotide database under the accession number dq520731. results a total number of 69 bacteria isolates were collected from street food samples in north and south jakarta, from which 21 isolates were obtained from food samples while 48 other isolates came from beverages. thirteen isolates from foods and 30 isolates from beverages, showed biochemical characteristics of e. coli. the characteristics gave ++-results of imvic tests and positive mug according to sni 01-23321991. escherichia coli contamination was not found in food samples from northern jakarta while 20% of food samples from southern jakarta were contaminated. some 50% of northern jakarta beverage samples and 40% from southern jakarta were contaminated with e. coli. the highest level of antibiotic resistance, from the 43 e. coli isolates attained, were found toward tetracycline (11.6%). on the contrary, none of the e. coli isolates was found to be resistant to kanamycin. some 4.7% of total isolates showed resistance to each of ampicillin, streptomycin, and trimethoprim (figure 1). pcr detection of class 1 integron showed one isolate (coded fc12a) from northern jakarta beverage sample, possessed class 1 integron bearing one gene cassette with ~700 bp of amplicon size (figure 2). dna sequencing showed that the gene cassette was resistant to trimethoprim determinant type v (dhfrv) (figure 3). discussion a total of 43 e. coli isolates from street foods were identified based on their biochemical characteristics. those isolates showed similar phenotypes and were categorized as variety-1 e. coli based on their imvic tests results ++-according to sni 01-2332-1991. in this study, two areas of sampling, northern jakarta which has poor sanitation was compared to southern jakarta. the results showed there was no significant difference between the percentages of the e. coli present in street food samples from northern jakarta and southern jakarta. this information indicated that the level of hygiene of street foods samples from southern jakarta was not better than those from northern jakarta. the presence of e. coli shown in this study is an important public health issue, especially for elementary school students in northern and in southern jakarta. the presence of e. coli in beverage samples is particulary noteworthy because the acceptable standard for e. coli in drinking water is 0 per 100 ml according to international and national regulations (who 1982). the antibiotic diffusion test revealed five isolates from a total of total 43 e. coli isolates (11.6%) from beverage samples resistant to at least one of 5 antibiotics tested. moreover, 3 isolates were resistant to multiple antibiotics i.e. the isolates were resistant to all of antibiotics tested except kanamycin. the resistant strains could be present in beverage samples via contamination from animal or human feces in beverage ingredients (especially water) which have been exposed to antibiotics and then contaminated by the street vendors. the incidence of resistance to ampicillin, streptomycin, tetracycline, and trimethoprim in e. coli supports the findings 5 0 6 0 table 1 percentage of contaminated food samples in northern and southern jakarta location contaminated food (%) not contaminated food (%) north jakarta south jakarta 5 0 4 0 1 4 1 2 1 0 8 6 4 2 0 r e si st a n c e ( % ) ampicillin (10 µg) streptomycin (10 µg) trimethoprim (5 µg) tetracycline (30 µg) antibiotic figure 1 measurement of resistance by diffusion agar method. figure 2 dna electrophoresis of pcr amplification using specific conserved region class 1 integron (5’cs and 3’cs) primer in 1% (w/v) agarose gel. well 1 contained dna marker (1 kb ladder); well 5 contained positive control (pcr amplicon from e. coli origin of varanus spp.), and well 6 contained pcr amplicon from fc12a isolate (750 bp). 16 frisca et al. microbiol indones 750 bp of other studies on antimicrobial agent resistance of e. coli from different sources throughout the world (okeke et al. 2000; schroeder et al. 2002; roe et al. 2003; sayah et al. 2005). the highest levels of resistance were observed for tetracycline (11.6%) while resistance to ampicillin, streptomycin, and trimethoprim showed the same percentage (4.7%). therefore, these isolates may play a key role as an acceptor and donor of transmissible antimicrobial resistance mechanisms. this is a matter of concern since the indiscriminate use of antibiotics, along with poor hygiene and inadequate infection control (risk factors for antibiotic resistance in bacteria) are highly prevalent in developing countries, including indonesia (okeke et al. 2000; tjaniadi et al. 2003). tetracycline, a first line broad spectrum antibiotic, has been used extensively worldwide in therapy in animals and humans since 1940 (walsh 2003). the high level of prevalence of resistance to tetracycline may be due to widespread and lengthy use of tetracycline considering its easy use orally and the low human toxicity of this antibiotic. in addition, tetracycline is a naturally derived compound; bacteria can be exposed to these agents in nature and outside of normal human use e.g. for treatment of livestock to promote growth rates (sayah et al. 2005). the existence of e. coli with low levels of resistance toward the antibiotics indicated that most of the e. coli strains were susceptible. street foodstuffs, which are not intentionally exposed to large of quantities of antibiotics, exhibited a significantly lower prevalence of antibiotic resistance than from other sources, e.g. human’s gut or feces, as studied by mulyastuti (1996). this is consistent with the hypothesis that exposure of commensal gut microbiota to antibiotics selects for resistant bacterial strains (sayah et al. 2005). susceptibility to five antibiotics by most of the e. coli isolates examined, indicates that the antibiotics are still effective for use in treatment of diarrhea, which is commonly caused by this pathogenic bacterium. pcr detection of a class 1 integron from 43 e. coli isolates revealed one isolate (coded fc12a), from northern jakarta, yielded an amplicon ~700 bp in size. sequence analysis showed that this amplicon contained a dhfr5 gene cassette for trimethoprim resistance. the predicted product of the dhfr5 reading frame comprised 157 amino acids and proved to be identical (100%) to the type 5 dihydrofolate reductase protein (dhfr5) (e. c. 1.5.1.3) according to the study of e. coli from india (accession no. aj620333.1) by mukherjee and chakraborty (2006). waturangi et al. (2003) also found the dhfr5 gene cassette in a class 1 integron from e. coli isolated from indonesian varanus spp. finding the class 1 integron bearing a trimethoprim resistance determinant gene cassette in this study supports the report of adrian et al. (1999). this showed that the gene cassettes which are commonly found integrating in the class 1 integron coded for resistance to trimethoprim and aminoglycoside antibiotics. the extensively use of trimethoprim and sulfonamide in combination since 1968 might select for the dhfr gene cassette obtained here and explain the emergence of trimethoprim resistance which is linked to the class 1 integron. antibiotic resistance could be mediated by a plasmid, a transposon, a cassette integrated in an integron, or by chromosomal mutation (huovinen et al. 1995). in this study, resistance to trimethoprim in fc12a which is encoded by the dhfr5 gene, was integrated as a gene in the class 1 integron. the other resistant isolates might be linked to other classes of integron. this study suggests that an integronbearing e. coli strain could be present in contaminated street food. this finding could potentially play a role in the transfer of antibiotic resistance genes to other human microbiota because e. coli is a commensal bacteria in the human intestine. this sounds a warning because the class 1 integron bearing e. coli, which was found in this study, could become a threat for widespread distribution of antibiotic resistance in pathogenic bacteria. volume 1, 2007 microbiol indones 17 5’cs dhfr5 1 r 1 l 2 l 59-base element 0.0 0.1 0.2 0.3 0.6 0.4 0.7 0.5 dhfr5 cassette 3’cs ttaacccggaaccaaaattgtgaaa -----dhfr5----ggaaacaaagctatgcaattgacggtaaaaagctt cgttcgc n cgccat annngggtcgttcgct 67 619 start stop figure 3 characterization of gene cassette structure (dhfr5) which is integrated in the class 1 integron fc12a. open reading frame dhfr5 gene was shown in box. start and stop translation codon were underlined. putative 59 be region, 1r, 1l, and 2l for integrase binding domains were shown with arrows. acknowledgment this research was part of the jakarta in focus project funded by the research institute of universitas katolik indonesia atma jaya, jakarta, indonesia. references adrian pv, thomson cj, klugman kp, amyes sgb. 1999. new gene cassettes for trimethoprim resistance, dfr13, and streptomycinspectinomycin resistance, aada4, inserted on a class1 integron. antimicrob agents chemo 44:355-361. chang cy et al . 2000. two new gene cassettes, drf17 (for trimethoprim resistance) and aada4 (for spectinomycin/ streptomycin resistance), inserted in an escherichia coli class 1 integron. j antimicrob chemo 46:87-89. huovinen p, sundstrom l, swedberg g, skold o. 1995. trimethoprim and sulfonamide resistance. antimicrob agents chemo 39:279289. mukherjee s, chakraborty r. 2006. incidence of class 1 integrons in multiple antibiotic-resistant gram-negative copiotrophic bacteria from the river torsa in india. j res microbiol 157:220-226. mulyastuti t. 1996. perbedaan profil kekebalan bakteri flora usus pada pengambilan sampel satu, tiga, dan lima strain, setiap tinja [lap pen]. surabaya: fakultas kedokteran, universitas airlangga. [nccls] national committee for clinical laboratory standards. 1999. performance standards for antimicrobial susceptibility testing; ninth informational supplement. wayne, pennsyslvania: nccls. document m100-s9, vol. 19. no. 1, table 2i. okeke in, fayinka st, lamikanra a. 2000. antibiotic resistance in escherichia coli from nigerian students, 1986-1998. emerg infect dis 6:393-396. roe mt, vega e, pillai sd. 2003. antimicrobial resistance markers of class1 and class 2 integron-bearing escherichia coli from irrigation water and sediments. emerg infect dis 9:822-826. sayah rs, kaneene jb, johnson y, miller ra. 2005. patterns of antimicrobial resistance observed in escherichia coli isolates obtained from domesticand wild-animal fecal samples, human septage, and surface water. appl environ microbiol 71:13941404. schroeder cm et al. 2002. antimicrobial resistance of escherichia coli o26, o103, o111, o128, and o145 from animals and humans. emerg infect dis 8:1406-1414. stiles me, king ln. 1981. biochemical characteristics and identification of enterobacteriaceae isolated from meats. appl environ microbiol 41:639-645. stokes hw et al. 2001. gene cassette pcr: sequence-independent recovery of entire genes from environmental dna. appl environ microbiol 67:5240-5246. tjaniadi p et al. 2003. antimicrobial resistance of bacterial pathogens associated with diarrheal patients in indonesia. am j trop med hyg 68:666-670. walsh c. 2003. antibiotics actions, origins, resistance. washington dc: asm pr. waturangi de, suwanto a, schwarz s, erdelen w. 2003. identification of class 1 integron-associated gene cassettes in escherichia coli isolated from varanus spp. in indonesia. j antimicrob chemo 51:175-177. [who] world health organization. 1982. bacteriological examination in: examination of water pollution control. new work: academy pr. [who] world health organization. 1999. laboratory methods for the diagnosis of epidemic dysentery and cholera. document no. m100-s9 who/cds/csr/edc/99.8. 18 frisca et al. microbiol indones 5.mi-laksmi hartayanie new available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.2.5issn 1978-3477, eissn 2087-8575 vol 10, no 2, juni 2016, p 71-77 *corresponding author; phone: +62-24-8441555, email:laksmi@unika.ac.id utilization of bacteria beneficial to human health in the food world is now more advanced. basically the bacteria can be divided into two groups, the beneficial bacteria and harmful bacteria. lactic acid bacteria (lab) belong to the beneficial bacteria for producing lactic acid. lab has made a great contribution in the development of fermented foods, particularly in foods that contain probiotics (lindayani and hartayanie 2013). lab includes microorganisms of the genera lactobacillus, leuconostoc, streptococcus, and pediococcus. the lactic acid produced from the metabolic activity of lab causes the condition of raw materials (food) becomes acidic. this is particularly advantageous, given the low ph condition that inhibits the damage caused by other microbes in almost all food (food spoilage microbes). according to josephsen and jespersen in hui et al. (2004), lab produces metabolites that function as antimicrobial compounds including organic acids (lactic acid and acetic acid), bacteriocins, hydrogen peroxide, diacetyl, and co .2 bamboo shoots are the young shoots of bamboo plants that appear at the bottom surface of the clump. the young shoots of bamboo can be consumed, thus classified as vegetable. bamboo shoots that can be consumed are usually derived from species dendrocalamus asper (yellow betung), gigantochloa verticillata, dendrocalamus litiforus (yellow bamboo), and bambusa aldhami (green bamboo) (andoko 2003). raw bamboo shoots have water content of about 90% net weight (rai 2007). bamboo shoots fermentation process takes place spontaneously. the fermentation is done by adding brine. brine prevents the liquid contained in bamboo shoots drawn out through the process of osmosis and also serves to inhibit the growth of pathogenic bacteria, so only the lactic acid bacteria are expected to grow. the final result of fermentation process is often called pickled bamboo shoots. the aim of this study is to isolate and identify the lactic acid bacteria of pickled yellow betung bamboo (d. asper) shoots fermented at colder temperatures (15 °c) and 2.5% salt content, and to investigate the antimicrobial activity of the lactic acid bacteria. lactic acid bacteria (lab) produces natural antimicrobial compounds that can inhibit and prevent the growth of spoilage bacteria. lab can be isolated from fermented food such as pickles, which ferment at cool temperature. the objectives of this research were to isolate and to obtain lab from yellow betung bamboo (dendrocalamus asper) shoots pickles that has antimicrobial activity against escherichia coli and staphylococcus aureus. it was made by submerging yellow bamboo shoots in 2.5% brine solution and keeping them in sealed container, then fermenting them at cool temperature (15 °c) for 10 days. lab was isolated using mrs agar and identified based on their morphological, physiological and biochemical characteristics. the result showed that lab isolates identified as lactobacilli and had antimicrobial activity against escherichia coli and staphylococcus aureus. all lactobacilli (21 isolates) isolated from fermentation at 15 °c were homofementative. bakteri asam laktat (bal) menghasilkan senyawa antimikroba sehingga dapat menghambat dan mencegah pertumbuhan bakteri pembusuk. bal dapat diisolasi dari makanan fermentasi seperti acar yang difermentasi suhu dingin. tujuan penelitian ini adalah mengisolasi dan mendapatkan isolat bal dari acar rebung yang mempunyai aktivitas penghambatan terhadap escherichia coli dan staphylococcus aureus. rebung difermentasi dalam larutan garam 2.5% dalam wadah tertutup pada suhu 15 °c selama 10 hari. bal yang berhasil diiolasi diidentifikasi sebagai lactobacillus yang homofermentatif dan mempunyai aktivitas antimikroba terhadap escherichia coli dan staphylococcus aureus. kata kunci: acar rebung, aktivitas antimikroba, bakteri asam laktat, suhu fermentasi key words: antimicrobial activity, bamboo shoots pickles, fermentation temperature, lactic acid bacteria antimicrobial activity of lactic acid bacteria from bamboo shoot pickles fermented at 15 °c laksmi hartayanie*, lindayani, and monika palupi murniati unika soegijapranata, jalan pawiyatan luhur iv/1, semarang 50234, indonesia materials and methods the pickling of bamboo shoots. two hundred fifty grams yellow betung bamboo shoots was washed, cut and put in a jar, then 2.5% (w / v) saline solution was added and then the jar was sealed and kept in the incubator. the fermentation process lasted for 10 d at a cold temperature (15 °c) (rahayu 2003 modified). isolation and purification of lactic acid bacteria. twenty five ml liquid from pickled bamboo shoots was diluted with 225 ml sterile 0.1% peptone -1 water (10 × concentration). the dilution process was conducted serially until the liquid was diluted down to -7 10 × the original concentration. 0.1 ml sample was taken from each dilution and inoculated into mrs agar medium containing 1% caco by spreading and 3 incubated at 37 °c for 48 h. colonies that formed clear zone was further purified using the same methods and media (yuliana and dizon 2011). morphological identification. lactic acid bacterial isolates were identified by microscopic observation based on the shape of the bacteria using gram staining and spore staining (sneath et al. 1984). motility test. isolates were taken aseptically using a needle and inoculated by puncturing into motility medium (mrs with 0.7% agar). incubation was performed at 37 °c for 48 h. non-motile isolates would have grown only around the puncture, while motile isolates would have spread farther (rahayu and margino 1997). catalase test. one loop of isolate was smeared onto an object glass that had been cleaned with alcohol. then, drops of 3% h o solution were added. the 2 2 formation of gas bubbles indicated that isolates were catalase positive and vice versa (gawad et al. 2010). gas production test. fifty μl lactic acid bacteria inoculum was used to inoculate 5 ml mrs broth with 6.5% and 18% nacl concentrations. each medium that had been inoculated with lactic acid bacteria was subsequently incubated at 37 °c for 48 h. observations of growth were done by measuring the absorbance value at 700 nm wavelength at 24 and 48 h (rahayu and margino 1997). growth at various ph (4.4 and 9.6). 50 μl lactic acid bacteria culture was inoculated into 5 ml mrs broth media ph 4.4 and ph 9.6 and then incubated at 37 °c for 48 h. observation of growth were done by measuring the absorbance value at 700 nm wavelength at 24 and 48 h (rahayu and margino 1997). growth at various temperatures (10 °c, 45 °c, and 50 °c). 50 μl lactic acid bacterial culture was used to inoculate 5 ml mrs broth media. then, each media that had been inoculated was incubated at either 10 °c, 45 °c or 50 °c for 48 h. observations of growth were done by measuring the absorbance value at 700 nm wavelength, 24 and 48 h after inoculation (rahayu and margino 1997). growth on different nacl levels (6.5% and 18%). 50 μl lactic acid bacterial culture was used to inoculate 5 ml mrs broth media with different nacl concentrations (6.5% and 18%). each medium that had been inoculated with lactic acid bacteria was subsequently incubated at 37 °c for 48 h. observations of growth were done by measuring the absorbance value at 700 nm wavelength, 24 and 48 h after inoculation (rahayu and margino, 1997). antimicrobial activity. measurement of antibacterial activity was conducted using wells (well assay). lactic acid bacterial culture was diluted in 0.85% nacl solution with turbidity level adjusted according to mcfarland standard number 5 (iniguezpalomares et al.2007). 0.1 ml of the diluted culture was taken and added to 10 ml liquid mrs medium (khunajakr et al. 2008) for further 24 h incubation at 37 °c. pathogenic bacteria was grown for 24 h then diluted in 0.85% nacl solution with turbidity level adjusted to mcfarland standard number 3 (iniguezpalomares et al. 2007). 10 ml of the diluted pathogenic bacterial culture was added to 10 ml of na medium and poured into a petri dish and let to solidity. then, a hole with 5 mm diameter was made and filled with 50 ml of lactic acid bacterial culture that had been grown in mrs broth media. subsequently, the petri dish was stored at 4 °c for 3 h to make sure the bacteria had diffused into the media. after 3 h, the petri dish was moved to 37 °c and further incubated for 24 h. clear zone formed was measured using a caliper. results isolation and identification of lactic acid bacteria. pickled yellow betung bamboo (d. asper) shoots, was used as source of microbial isolation using mrs containing 1% caco and 10 ppm na-azide as 3 selective medium. na-azide serves to inhibit aerobic microbial growth by binding to the free o . pure culture 2 was obtained by separating the colonies from one another. twenty one isolates (fig 1), which gave clear zone, were found. all 21 isolates were subsequently identified based on their morphological and physiological characters. the identification was done by testing the 72 hatayanie et al. microbiol indones morphological character by gram and spore staining, catalase test, motility test and gas production (table 1). all isolates were identified more specifically to determine the genus of the lactic acid bacteria. the identification was done by testing the ability to grow at different temperatures (10 °c, 45 °c, and 50 °c), different concentrations of nacl (6.5% and 18%), and at different ph (4.4 and 9.6). it was known that all isolates could grow at 6.5% nacl, ph 4.4 and 45 °c. most isolates can grow at 10 °c and a small percentage of isolates can grow at 50 °c. however, none of these isolates could survive at ph 9.6 and 18% nacl. these results show that all the tested isolates belong to the genus lactobacillus (table 2). antimicrobial activity of lactic acid bacteria. lactic acid bacteria isolates were tested for antimicrobial activity. antimicrobial activity test was conducted using agar diffusion method and using two bacterial pathogens, escherichia coli strain atcc 252 922 (gram-negative) and staphylococcus aureus strain atcc 252 923 (gram-positive) (fig 2). isolate a12 had the highest antimicrobial capability against s. aureus with inhibition zone diameter of 11.07 mm, while isolate a20 had the highest antimicrobial capability against escherichia coli with inhibition zone diameter of 10.93 mm (fig 3). these results indicate that both isolates have very strong antimicrobial ability. some isolates (a1, a13, a19, and a23) showed no inhibition against s.aureus dan e.coli. the isolates which had antimicrobial activity showed larger clear zone against s. aureus than e. coli except a20, a24, and a31 (table 3). discussion in this research, shoots of yellow betung bamboo (d. asper) was used for fermentation. the fermentation process was carried out for 9 d at 15 °c and was completed when the ph decreased to below 4.5, because lactic acid bacteria will stop growing at lower ph (rehm and reed 1996). fermentation performed at low temperatures takes longer than the fermentation process in general. the lower temperature can lead to inhibition of the growth of lactic acid bacteria, so that limited amount of lactic acid was produced due to slower fermentation process. clear zone around the colony arised due to the reaction between the acid produced by bacteria with caco in media resulting 3 soluble ca-lactate in the media and seen as a clear zone. table 1 showed that all isolates have characteristics similar to the common characteristics of lactic acid bacteria. they were gram-positive, rod-shaped or cocci, catalase negative, non-spore-forming, nonmotile and produced acids (mainly lactic acid and acetate) (ghiasi 2011; aly et al. 2006). all isolates were identified as lacotobacillus (table 2). lactobacillus growth characteristics include capability to grow at suitable temperatures 10 °c-50 °c, 6.5% nacl and ph 4.4, but unable to grow at 18% nacl and ph 9.6 (rahayu and margino 1997). this result is quite different from the research conducted by the tamang and sarkar (1996) in choudhury et al, (2012), whostated that lactobacillus were not the only lab found in bamboo shoots fermented at room temperature, but also pediococcus and leuconostoc. fig 1 colonies of lactic acid bacteria form a clear zone (isolate a17). volume 10, 2016 microbiol indones 73 antimicrobial activity or inhibitory ability against pathogens as indicated by the appearance of clear zone around the holes was filled with isolate (fig 2). this is due to the differences in the composition and structure of the cell wall of gram-positive bacteria (s. aureus) and gram-negative bacteria (e.coli). cell wall structure of gram-positive bacteria is more simple, single-layered with a low lipid content (1-4%), to facilitate the bioactive material transport into the cell. on the other hand, gram negative bacteria have more complex structure, consisting of a three-layered this indicates that the fermentation temperature has an influence on the growth of lab. e. coli strain atcc 252 922 (gram-negative) and s. aureus strain atcc 252 923 (gram-positive) were used for antimicrobial test because both bacteria were the most common pathogens attacking human. s. aureus is a gram-positive that lives as a saprophyte in the membrane channels of the human body, the surface of the skin, sweat glands, and the intestinal tract, while e. coli is a gram-negative commonly found in the human colon as normal flora. all isolates have isolates clear zone gram staining spore staining bacteria form catalase motility gas production result a 1 + + b + b a 2 + + b + b a3 + + b + b a11 + + b + b a12 + + b + b a13 + + b + b a15 + + b + b a16 + + b + b a17 + + b + b a18 + + b + b a19 + + b + b 4 20 + + b + b a23 + + b + b a24 + + b + b a27 + + b + b a31 + + b + b a32 + + b + b a33 + + b + b a34 + + b + b a43 + + b + b a44 + + b + b table 1 identification of lactic acid bacteria in pickled betung bamboo shoots (dendrocalamus asper) the bacterial form of basil: “-” = negative / no form: “+” = positive / form: “b” = the lactic acid bacteria. 72 hatayanie et al. microbiol indones cannot stand low ph would be inhibited. although gram-negative bacteria have lipopolysaccharide layer, the lactic acid can still inhibit it . this is because lactic acid is a water-soluble molecule that can permeate into the outer membrane. lipopolysaccharide layer was damaged by lactic acid so that other antimicrobials such as diacetyl, bacteriocins and hydrogen peroxide can enter the cells (alokomi et al. 2000 in afriani 2012). from this study, it can be concluded that twenty one isolates of lactic acid bacteria found in pickled outer layer lipoprotein, lipopolysaccharide middle layer which acts as a barrier to the entry of antibacterial compounds, and peptidoglycan inner layer with a high lipid content (11-12%) (mckane and kandel 1996). the ability of lactic acid bacteria in inhibiting the growth of pathogenic bacteria was due to the antimicrobial components produced by lactic acid bacteria, one of which is lactic acid. the lactic acid caused a decrease in ph so that the growth of grampositive ( + ) and gram negative ( ) bacteria which isolates temperature (°c) nacl (%) genus ph 4 .4 9 .6 10 45 50 6. 5 18 a 1 + + + + lactobacillus a 2 + + + + lactobacillus a3 + + + + lactobacillus a11 + + + lactobacillus a12 + + + + lactobacillus a13 + + + + lactobacillus a15 + + + + lactobacillus a16 + + + + lactobacillus a17 + + + + lactobacillus a18 + + + + lactobacillus a19 + + + + lactobacillus 4 20 + + + + lactobacillus a23 + + + + lactobacillus a24 + + + + lactobacillus a27 + + + + lactobacillus a31 + + + + lactobacillus a32 + + + + lactobacillus a33 + + + + lactobacillus a34 + + + + lactobacillus a43 + + + + la ctobacillus a44 + + + + lactobacillus table 2 results identification of lactic acid bacteria growth bacteria based capabilities at various ph, temperature, and + = isolates grow = isolates did not grow volume 10, 2016 microbiol indones 75 inhibition zone diameter 10.93 mm acknowledgments this research can take place with financial support from the directorate general of higher education and laboratory facilities of the faculty of agricultural bamboo shoots fermented at 15 °c were identified as lactobacillus. seventeen isolates had antimicrobial activity that can inhibit the growth of pathogenic bacteria e. coli and s. aureus. isolate a34 has the highest antimicrobial activity against s. aureus with inhibition zone diameter 14.53 mm and isolate a20 had the highest antimicrobial ability against e. coli with fig 2 the test results on the antimicrobial activity of isolate a20 pathogenic bacteria escherichia coli (a); a12 on staphylococcus aureus (b). k: control (medium without the addition of isolates; 1: isolates test (i repeat); 2: isolates test (replications ii); 3: isolates test (replications iii). table 3 antimicrobial activity a b isolates code s.aureus e.coli diameter clear zone (mm) a1 0.00 0.00 a2 9.77 9.03 a3 6.67 10.5 a11 10.67 10.53 a12 11.07 7.97 a13 0.00 0.00 a15 9.00 8.20 a16 10.40 9.17 a17 0.00 0.00 a18 8.47 7.90 a19 0.00 0.00 a20 9.70 10.93 a23 0.00 0.00 a24 7.23 8.70 a27 7.60 7.27 a31 6.20 9.57 a32 10.27 7.47 a33 6.90 0.00 a34 14.53 9.30 a43 10.70 9.67 a44 427 400 72 hatayanie et al. microbiol indones iniguez-palomares c, perez-morales r, acedo-felix e. 2007. evaluation of probiotic properties in lactobacillus isolated from small intestine of piglets. revista latino americana de microbiologi 49(3-4):46-54. khunajakr n, wongwicharn a, moonmangmee d. 2008. screening and identification of lactic acid bacteria producing antimicrobial compounds from pig gastrointestinal tracts. journal of kmitl science technology. 8(1):8-17. lindayani, hartayanie l. 2013. the mapping of lactid acid bacteria from fermentation of local foods (semarang): tempoyak, mandai and yellow bamboo th shoot pickles. oral presentation in the 4 international l conference of indonesian society lactic acid th th bacteria (islab). yogyakarta, 25 -26 january 2013. mc kane l, kandel j. 1996. microbiology: essentials and applications. mc graw hill. rahayu, es. 2003. lactic acid bacteria in fermented foods of indonesian origin. agritech. 23(2) : 75-84. nd rehm hj, reed g. 1996. biotechnology 2 edition volume 6 : p r o d u c t s o f p r i m a r y m e t a b o l i s m . v c h verlagsgesellschaft mbh, weinheim. germany. sneath pha, mair ns, sharpe me and holt jg. 1984. bergeys manual of systematic bacteriology. vol 2. williams &wilkins. baltimore. yuliana n, dizon ei. 2011. phenotypic identification of lactic acid bacteria isolated from tempoyak (fermented durian) made in the philippines. int j biol. 3(2): 145151. doi:10.5539/ijb.v3n2p145. technology-soegijapranata catholic university. thanks for the help (ardelia, amelia, cynthia, agatha, and lorentia) and the support of all parties involved. references afriani. 2012. kualitas dan aktivitas antimikroba produk dadih susu sapi pada penyimpanan suhu rendah. agrinak 2(1):11-16. aly s, cheik at, imael hn, alfred ts. 2006. bacteriocins and lactic acid bacteria-a minireview. afr j biotechnol. 5(9):678-683. andoko a. 2003. budidaya bambu rebung. kanisius, yogyakarta. battcock m, azam-ali s. 1998. fermented fruits and vegetables, a global perspective. fao agricultural services bulletin no. 134. choudhury d, sahu jk, sharma gd. 2012. bamboo shoot: microbiology, biochemistry and technology of fermentation-a review. indian journal of traditional knowledge 11(2):242-249. de vuyst l, vandamme ej. 1994. bacteriocins of latic acid bacteria. blackie academic & professional. gawad ae, fatah ae, rubayyi a. 2010. identification and characterization of dominant lactic acid bacteria isolated from traditional rayeb milk in egypt. journal of american science. 6 (10):728-735. ghiasi f. 2011. predominant lactic acid bacteria isolated from the intestines of silver carp in low water temperature. afr j biotechnol. 10(59):12747-12751. volume 10, 2016 microbiol indones 77 1: 71 2: 72 3: 73 4: 74 5: 75 6: 76 7: 77 mi-anastasia asri available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.9.3.3issn 1978-3477, eissn 2087-8575 vol 9, no 3, september 2015, p 113-119 *corresponding author; phone: +62-21-5703306, e-mail: bibiana.lay@atmajaya.ac.id ropey bread is a bacterial spoilage condition of bread. the spoilage is characterized by a sweet fruity odor, similar to that of over-ripe melons or pineapples. subsequently enzymatic degradation result in patchy discoloration and the bread eventually becomes very soft and sticky to the touch due to breakdown of starch and proteins by microbial amylases and proteases, and by the production of extracellular slimy polysaccharides (yibar et al. 2012). in the bread making process, the raw materials such as flour, yeast starter, butter, water, salt, sugar, and egg, are mixed and knead until the dough becomes smooth. the dough is then rounded and left to rise by fermentation of the yeast starter. the loaf is subsequently baked at 160 °c and thereafter the loaf is cooled before slicing or wrapping. if the raw materials contaminated by ropeproducing bacteria, the high temperature used in the baking process will cause spore formation. furthermore, the moist environment after the baking process will enhance spore germination and growth of vegetative cells of the bacteria (yibar et al. 2012). the bacteria involved in ropiness are from genus bacillus, primarily bacillus subtilis (formerly referred to as b. mesentericus). however, b. licheniformis, b. megaterium (sorokulova et al. 2003), b. pumilus, b. cereus (valerio et al. 2012), and b. subtilis can also be the causative agents of ropey bread (rumeus and turtoi 2013). bacillus species are aerobic, gram-positive, and able to form endospore that are resistant to high temperature. these bacteria are common soil inhabitants and may contaminate raw materials or ropey bread is a bacterial spoilage condition of bread. the spoilage involved in ropey bread is primarily due to bacillus subtilis. several studies have shown that rope spoilage can be controlled using antimicrobial agents such as acetic acid, lactic acid, and quaternary ammonium cations (qacs/quats). this research consisted of five main steps: isolation, identification, confirmation, and molecular characterization of bacteria from raw materials contaminated by rope-producing bacteria, and antimicrobial test against rope-producing bacteria. the confirmation test was done in order to determine the rope-producing ability of isolates suspected as bacillus sp. there were two different treatments in this test. in the first treatment, the inoculums were mixed with bread dough. in the second treatment, each slice of loaf was placed into a petri dish, uniformly soaked with inoculums. the first treatment did not show rope spoilage for all of the loaves, however, 6 of 11 loaves in the second treatment developed rope. the largest inhibition halos for antimicrobial test were produced by quats. this means quats is the strongest antimicrobial agent against rope-producing bacteria. the molecular characterization showed that all of the suspected isolates had 98-99% similarity to b. subtilis. key words: antimicrobial agents, bacillus subtilis, identification, isolation, ropey bread ropey bread merupakan kerusakan pada roti yang disebabkan oleh bakteri. bakteri yang terlibat dalam kerusakan ropiness berasal dari genus bacillus, terutama bacillus subtilis. beberapa studi menunjukkan bahwa kerusakan ropiness dapat dikontrol menggunakan agen antimikrob seperti asam asetat, asam laktat, dan quaternary ammonium cations (qacs/quats). penelitian ini dibagi menjadi lima tahapan, yaitu isolasi, identifikasi, konfirmasi, dan karakterisasi molekuler bakteri dari bahan-bahan mentah yang terkontaminasi bakteri penyebab ropey bread, serta uji antimikrob terhadap bakteri penyebab ropey bread. konfirmasi bakteri yang dilakukan ini dibagi menjadi dua perlakuan. pada perlakuan pertama, inokulum dicampurkan dengan adonan roti. pada perlakuan kedua, tiap-tiap potongan roti ditempatkan dalam cawan petri steril yang telah berisi inokulum. perlakuan pertama tidak menunjukkan kerusakan ropiness pada semua potongan roti, tetapi 6 dari 11 potongan roti pada perlakuan kedua memberikan hasil yang positif terjadinya kerusakan. zona bening terbesar pada uji antimikrob diproduksi oleh quats. dengan demikian, quats merupakan agen antimikrob terkuat terhadap bakteri penyebab ropey bread. identifikasi bakteri penyebab ropey bread secara molekuler menunjukkan bahwa seluruh isolat yang dipilih memiliki kemiripan 98-99% dengan b. subtilis. kata kunci: agen antimikrob, bacillus subtilis, identifikasi, isolasi, ropey bread isolation and identification of bacteria from raw materials contaminated by rope-producing bacteria anastasia asri widyasari, stella magdalena, and bibiana widiyati lay* faculty of biotechnology, atma jaya catholic university of indonesia jalan jenderal sudirman 51, jakarta 12930, indonesia bakery equipments used. the raw materials commonly used are flour, yeast, butter, and water. all types of flour were contaminated with bacillus species, although flour is generally considered as microbiologically safe product as it is a low water-activity commodity (yibar et al. 2012). bacterial spores cause major problems in the food industry due to their stress resistance. spores that have retained their viability during baking can germinate and cause ropiness if they are exposed to a warm and moist environment. the water-activity (a ), ph, and w temperature during storage may also play important roles in spore germination and growth of vegetative cells of bacillus species (yibar et al. 2012). ropiness occurs particularly when warm (25-30 °c) and humid (water-activity, ≥0.95) environmental conditions allow spore germination. moreover, rope-producing bacteria are also characterized by faster development and enhanced proteases and amylases production during growth in the bread crumb (pepe et al. 2003). rope-producing bacteria have the potential to cause disease in humans. b. subtilis can cause conjunctivitis (alloyna 2011). b. pumilus are capable of causing food borne illness (matarante et al. 2004). b. licheniformis causes bacteremia, peritonitis, gastrointestinal disease, and eye infection (haydushka et al. 2012). b. megaterium produces enterotoxin that is also important in the symptoms of food borne illness (lopez & alippi 2010). b. cereus produces two types of toxins that have been associated with diarrheic (heat-labile) and emetic (heat-stable) toxins (yibar et al. 2012). the usual recommended control procedures such as sanitation of bakery equipments, stringent temperature control during baking, and testing of raw materials may reduce the initial spore counts in dough, but do not prevent germination and growth of bacillus spores in non-acidified bread without added preservatives (antimicrobial). several studies were made for inhibition of ropiness including usage of antimicrobial agents such as acetic acid, lactic acid, and quaternary ammonium cations (qacs) (erem et al. 2009). however, acetic acid, used singly, has a negative effect on the organoleptic qualities of the baked product. reducing the concentration of acetic acid, and using it in combination with other antimicrobial agents such as lactic acid, may thus be an alternative approach. lactic acid is commonly used as flavor enhancers and humectants in meat products, but has not been evaluated as bread preservatives (pattison et al. 2004). quaternary ammonium cations, frequently called 114 widyasari et al. microbiol indones the quats are used most frequently as antimicrobial agents. quats with low concentration are able to inhibit bacteria growth, but do not kill the bacteria. previous study (malek and malek 2012) showed that quats were capable used as antimicrobial agents against b. subtilis that is one of rope-producing bacteria. the aims of the present study were to isolate and identify the bacteria from raw materials contaminated by rope-producing bacteria, and to test antimicrobial agents against rope-producing bacteria. materials and methods samples. the raw materials of bread such as flour, yeast starter, water, and butter were collected from a local bakery that produced ropey bread in jakarta. isolation of bacteria from raw materials contaminated by rope-producing bacteria. to isolate the bacteria, 1 g of each sample (1 ml water) were aseptically weighed and homogenized with 9 ml sterile aquades. ten-fold serial diution of the 0 -1 -2 -3 -1 suspensions (10 , 10 , 10 , and 10 g ml ) were prepared with the same diluent, and 100 µl aliquot of each dilution was inoculated on nutrient agar (na) using spreader and incubated at 37 °c for 24 h. the -1 colonies were counted (cfu ml ). as many as 6-8 chosen colonies were streaked on na, and incubated at 37 °c for 24 h in order to obtain pure isolates (erem et al. 2009). the contaminated raw materials in this study were also compared to raw materials that were sold in the market. therefore, flour sample from local market was collected to isolate the suspected bacteria and the chosen isolates were subsequently treated in conjunction with the other suspected isolates from contaminated raw materials. identification of rope-producing bacteria. identification was performed using biochemical tests: gram staining, spore staining, microscopy observation (phase contrast), protease, amylase, formation of indole, catalase, oxidase, voges-proskauer test, growth at 50 °c, and anaerobic growth (erem et al. 2009). confirmation test for the determination of rope-producing bacteria. there were two different treatments in this test. in the first treatment, bacillus species were grown at 37 °c in nutrient broth (nb) for 72 h diluted to an od of 0.350, and the inoculums 600 were mixed with bread dough and cooked at 160 °c for 15 min. in the second treatment, each slice of loaf (cooked at 160 °c for 15 min) was placed into a petri dish, uniformly soaked with 1 ml of inoculums. after cooling, all treated loaves were stored at 30 °c for 96 h and examined for rope production (thompson et al. 1998). molecular characterization of rope-producing bacteria. genomic dna extraction was conducted using colony pick pcr. a single colony of each isolate was picked and dissolved in 10 µl ddh o in order to be 2 amplified. the pcr conditions used with 2 µl dna template, 2 µl primer 63f and 1387r, 25 µl mytaq hs red mix (bioline) 2χ, and 19 µl ddh o. the pcr was 2 performed with the following optimized thermal profile: initial denaturation at 95 °c for 120 s followed by 40 cycles with 60 s of denaturation at 95 °c, 60 s of primer annealing at 55 °c and 90 s of primer extension at 72 °c and as last step final elongation at 72 °c for 600 s. amplification size of about 1300 bp was checked by electrophoresis on agarose gel (1%) at 80 v for 70 m. a 1 kb dna ladder was used as the molecular weight marker (ryzinska-paier et al. 2011). the restriction enzyme pst i was used for digestion in ardra (amplified ribosomal dna restriction analysis) assay in order to distinguish the band patterns of isolates. these isolates with different profiles were sent to genetika science, malaysia for sequencing. data analysis nucleotide sequence was analyzed using basic local alignment search tool (blast) from national center for biotechnology information (ncbi) for identification. antimicrobial test against rope-producing bacteria. the antimicrobial test was carried out by agar well diffusion method. mueller hinton agar (mha) was swabbed (sterile cotton swab) with 24 h old-broth culture of bacillus species (diluted to an od of 0.132). wells (10 mm) were made in mha 600 using sterile cork borer. ten-fold serial dilution of 0 -1 -2 -3 -1 antimicrobial agents (10 , 10 , 10 , and 10 mg ml ) such as acetic acid, lactic acid, and quats were prepared with the same diluent. about 50 µl aliquot of each dilution was added into the wells and allowed to diffuse at room temperature for 2 h. the plates were incubated at 37 °c for 24 h. the diameter of the inhibition zone was measured. results isolation and identification of rope-producing bacteria. the selected isolates were chosen based on gram staining, spore staining, and microscopy observation (phase contrast) that showed characteristics of bacillus species, gram-positive, and endospore forming (fig 1). the biochemical test results in this study are summarized in table 1. as many as 11 isolates were suspected as bacillus species. the most widely isolates were obtained from water. the various results between biochemical tests were shown in protease test, amylase test, and growth at 50 °c. high protease activities were shown by isolate gk5 and mp7, whereas high amylase activities were shown by gk1, a2, and a3. however, there were no significant differences between all isolates based on protease and amylase activity, as well as the capability of the isolates to grow at 50 °c. confirmation test for the determination of rope-producing bacteria. the first treatment of confirmation test did not show rope spoilage for all of the loaves, however, 6 of 11 loaves in the second treatment developed rope (fig 2). the rope spoilage was shown by discoloration and sweet fruity odor from the loaves. the other five loaves did not show negative results. the fifth loaves also showed ropiness, however the discolorations did not show significant results. molecular characterization of rope-producing bacteria. all suspected isolates showed the same band patterns according to ardra assay with psti restriction volume 9, 2015 microbiol indones 115 fig 1 gram staining (a), spore staining (b), and microscopy observation results for suspected species (c). a b c -2 capability to inhibit until the highest dilution (10 ) and have the largest inhibition halos, depicted by isolate m2 and gk3. discussion microbial spoilage is the major problem causing economic losses as well as possible food borne illness risks. rope spoilage is one of the frequent problems, occurring most frequently in baking industry and usually caused by bacillus species. the objectives of this study were to isolate and to identify the bacteria from raw materials contaminated with rope-producing bacteria, and to test antimicrobial agents against ropemicrobiol indones116 widyasari et al. table 1 biochemical test results of the suspected isolates source code gram spore proteas amylase indole catalase oxidase vp growth at 50 °c anaerobic growth flour gk1 + + a13 b9 + + +++ flour gk3 + + 13 8 + + +++ flour gk5 + + 14 8 + + +++ flour gk6 + + 12 5 + + +++ yeast starter mp7 + + 14 8 + + +++ water a2 + + 12 9 + + +++ water a3 + + 13 9 + + +++ water a4 + + 13 5 + + +++ water a5 + + 13 5 + + +++ water a6 + + 12 6 + + ++ butter m2 + + 13 5 + + ++ a b fig 2 confirmation test result with first treatment (a) which showed negative results and second treatment (b) which showed positive results. enzyme (fig 3). this means all of the suspected isolates belonged to genus bacillus with the same species. since the results of ardra assay showed the same profiles, the molecular characterization only used two isolates (gk1 and gk3) for representation. the suspected isolates were determined as b. subtilis (table 2). the suspected isolate (bs1) from flour sample (raw materials) was also identified based on molecular characterization and the result was it had also similarity profile to b. subtilis. antimicrobial test against rope-producing bacteria. the antimicrobial test results in this study are summarized in table 3. the largest inhibition halos (diameters, cm) were produced by quats due to the volume 9, 2015 microbiol indones 117 isolate code species similarity accession number gk1 bacillus subtilis 99% jx944823.1 gk3 bacillus subtilis 98% ab905422.1 bs1 bacillus subtilis 98% jf262038.1 fig 3 ardra assay results. table 2 dna sequencing analysis of 16s rrna gene of suspected isolates table 3 antimicrobial test results of the suspected isolates. na-no activity; a-values, including diameter of well (10 mm), are means of two replicates; b± standard deviation code a diameters of inhibition halos (cm ) acetic acid (aa) lactic acid (la) quats (q) distilled water 0 10 -1 10 -2 10 0 10 -1 10 -2 10 0 10 -1 10 -2 10 gk1 2.4 ± 0.0 b 0.9 ± 0.1 na 2.9 ± 0.7 1.6 ± 0.3 0.2 ± 0.2 2.5 ± 0.9 1.9 ± 1.3 1.2 ± 1.1 na gk3 3.2 ± 0.2 1.0 ± 0.1 na 3.1 ± 0.1 0.8 ± 1.1 0.1 ± 0.1 3.4 ± 0.4 3.2 ± 0.6 2.2 ± 0.3 na gk5 2.7 ± 0.2 0.5 ± 0.6 na 3.3 ± 0.9 1.6 ± 0.1 0.3 ± 0.4 2.6 ± 1.0 1.8 ± 1.6 1.5 ± 1.3 na gk6 1.7 ± 0.7 0.3 ± 0.4 na 2.3 ± 1.3 1.4 ± 0.6 0.2 ± 0.2 2.7 ± 1.3 2.0 ± 1.5 1.3 ± 1.8 na mp7 3.8 ± 0.0 0.9 ± 0.0 na 3.4 ± 0.1 2.0 ± 0.6 na 3.3 ± 0.0 3.1 ± 0.1 2.0 ± 0.2 na a2 3.0 ± 0.0 0.2 ± 0.0 na 3.7 ± 0.2 2.0 ± 0.3 0.5 ± 0.1 1.8 ± 0.0 1.0 ± 0.0 0.4 ± 0.0 na a3 2.8 ± 0.4 1.1 ± 0.4 na 3.2 ± 0.1 1.9 ± 0.4 0.2 ± 0.3 3.1 ± 0.1 2.8 ± 0.3 2.1 ± 0.3 na a4 4.0 ± 0.0 1.3 ± 0.0 na 3.6 ± 0.0 3.2 ± 0.0 na 3.3 ± 0.1 3.0 ± 0.1 1.2 ± 1.6 na a5 2.9 ± 1.0 1.0 ± 0.4 na 3.2 ± 0.1 2.3 ± 1.1 0.2 ± 0.2 3.1 ± 0.1 2.8 ± 0.1 1.9 ± 0.0 na a6 2.8 ± 0.0 1.1 ± 0.0 na 3.2 ± 0.0 2.2 ± 0.0 0.4 ± 0.0 3.2 ± 0.0 3.0 ± 0.0 2.0 ± 0.0 na m2 3.8 ± 0.0 1.1 ± 0.0 na 3.2 ± 0.0 2.3 ± 0.7 0.5 ± 0.1 3.9 ± 0.8 3.2 ± 0.3 2.5 ± 0.1 na of all of the isolates were b. subtilis, which belonged to be one of species associated with ropiness in bread. all of the bacteria isolated were also characterized to determine their amylase and protease activities, and heat resistance. the hydrolysis of starch and proteins by microbial amylases and proteases encourage rope formation (pepe et al. 2003). the antimicrobial test revealed that acetic acid, lactic acid, and quats were effective inhibitors of b. subtilis strains examined. lactic acid was more effective inhibitor compared to acetic acid due to the concentration of the solution. the lactic acid had higher concentration (80-90%, almost pure) than acetic acid (25%, about the concentration of domestic vinegar) that was used in this study. even though, pundir and jain (2011) showed acetic acid was more effective than lactic acid due to the amount of undissociated acid present. this means that the effectiveness of organic acids as antimicrobials differ widely based on concentration and the amounts of undissociated acids. other than that, ph and molarity are also capable to affect the effectiveness of antimicrobial agents. acetic acid and lactic acid are bactericidal agents that can lower the ph of outer surface by releasing the proton causing membrane disruption, enzyme denaturation, and changing the permeability of the cells. these acids can be toxic for molds and bacteria due to inability of the affected organisms to metabolize these acids. however, in mammals, acetic acid and lactic acid are metabolized in a manner similar to that of other fatty acids, and it has not been shown to cause any toxic effects at the levels utilized. both of these antimicrobial agents are organic acids that contain carboxyl functional group. the undissociated forms of these carboxylic acid antimicrobial agents are active, and the range of effectiveness extends up to ph 5.0 in most applications (pundir and jain 2011). between three antimicrobial used in this study, quats was found to be best antimicrobial agents. quats are positive compounds that are good penetrants and thus have value for porous surfaces. quats are membrane active agents with target site predominantly at the cytoplasmic (inner) membrane in bacteria (carmona-ribeiro and carrasco 2013). quats are often used to disinfect and sterilize medical tools or used in the foodservice industry as sanitizing agents (ohta et al. 2008). quats are generally more effective in the alkaline ph range, whereas carboxylic acids are most ideally performed in the ph range of below 3. carboxylic acids producing bacteria. in this study, water was the source of the most 6 -1 widely isolates that were obtained (2.31 10 cfu ml ) 5 -1 followed by flour (2.33 × 10 cfu ml ), whereas yeast starter and butter had almost no isolates. moreover, only 4-7 colonies were obtained from the isolation of bacteria from raw flour sample. water and flour have been contaminated largely by rope-producing bacteria because bacillus species are soil inhabitants. although flour is generally considered as microbiologically safe product as it has low water-activity commodity. all of these bacteria were not removed completely in spite of thorough cleaning during milling process of the flour (npcs 2011). the purpose of the confirmation test was to confirm the isolates from raw materials were surely the causative agents of ropey bread by using two treatments, the mixing inoculation of bread dough and the direct inoculation of loaf slices (fig 2). a problem with this approach was the presence of very low numbers of bacillus spores within the purchased flour. this was indicated by the absence of ropiness in the mixing inoculated treatment (fig 2a). however, ropiness was detected in the direct inoculation of loaf slice (fig 2b). in this study, raw flour sample also contained b. subtilis (table 2). b. subtilis is one of the causative agents of ropey bread. it was expected that the mixing inoculation treatment (first treatment) would have similar spore contamination levels with the direct inoculation treatment (second treatment) because the two treatments used similar raw materials. however, the loaves in the first treatment did not develop rope, whereas the second treatment showed positive results. there were several factors that enhanced the rope development. besides temperature and ph, wateractivity or humidity can also enhanced the spore germination and vegetative cells growth of ropeproducing bacteria. in this study, the temperature and the storage condition had been attempted to resemble the conditions of ropey bread production. however, humidity cannot be managed in this study. therefore, spore formation which is caused by temperature of baking process was not enhanced by humidity and thus spore germination and growth of vegetative cell were disrupted. the positive results of the second treatments showed that rope spoilage can be caused from environment or the condition after the baking process since inoculation were carried out in direct manner (npcs 2011). the results of molecular characterization method × microbiol indones118 widyasari et al. artisanal cured sausages. appl environ microbiol. 70(9):5168-5176. doi:10.1128/aem.70.9.51685176.2004. ohta y, kondo y, kawada k, teranaka t, yoshino n. 2008. synthesis and antibacterial activity of quaternary ammonium salt-type antibacterial agents with a 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perfringens strains isolated from water. j microbiol methods. 87(2):189-194. doi:10.1016/j.mimet.2011.0 8.006. saranraj p, geetha m. 2012. microbial spoilage of bakery products and its control by preservatives. int j pharm biol arch. 23: 38-48. sen a, batra a. 2012. evaluation of antimicrobial activity of different solvent extracts of medicinal plant: melia azedarach l. int j curr pharm res. 4(2):67-73. sorokulova ib, reva on, smirnov vv, pinchuk iv, lapa sv, urdaci mc. 2003. genetic diversity and involvement in bread spoilage of bacillus strains isolated from flour and ropy bread. appl microbiol. 37(2):169-173. doi:10.1046/j.1472-765x.2003.01372.x. thompson jm, waites wm, dodd cer. 1998. detection of rope spoilage in bread by bacillus species. j appl microbiol. 85(3):481-486. doi:10.1046/j.13652672.1998.853512.x. valerio f, de bellis p, di biase m, lonigro sl, giussani b, visconti a. 2012. diversity of spore-forming bacteria and identification of bacillus amyloliquefaciens as a species frequently associated with the ropy spoilage of bread. int j food microbiol. 156(3):278-285. doi:10.1016/j.ijfoodmicro.2012.04.005. yibar a, cetinkaya f, soyutemiz ge. 2012. detection of rope-producing bacillus in bread and identification of isolates to species level by vitek 2 system. j biol environ sci. 6(18):243-248. are food grade that can be mixed with the bread dough in baking industry. however, these acids are also able to be used as sanitizing agents and acid rinse. carboxylic acid sanitizers on baking equipments are noncorrosive to stainless steel, provide a good shelf life, and are cost effective. unlike carboxylic acids, quats are not food grade and thus they can only used as antiseptics, disinfectants, or sanitizers on foodhandling and food-processing equipments (marriott & gravani 2006). acknowledgments this research was funded by faculty of biotechnology, atma jaya catholic university of indonesia and faculty grant from institute research and community service. reference [npcs] niir project consultancy services. 2011. handbook on fermented foods and chemicals. new delhi: asia pasific business. alloyna d. 2011. prevalensi konjungtivitis di rumah sakit umum pusat haji adam malik tahun 2009 dan 2010 [dissertation]. medan (id): universitas sumatera utara. carmona-ribeiro am, carrasco ldm. 2013. cationic antimicrobial polymers and their assemblies. int j mol sci. 14(5):9906-9946. doi:10.3390/ijms14059906. erem f, certel m, karakas b. 2009. identification of bacillus species isolated from ropey breads both with classical methods and api identification kits. akdeniz universitesi ziraat fakultesi dergisi. 22: 201-210. haydushka ia, markova n, atanassova m. 2012. recurrent sepsis due to bacillus licheniformis. j glob infect dis. 4(1):82-83. doi:10.4103/0974-777x.93768. lopez ac, alippi am. 2010. enterotoxigenic gene profiles of bacillus cereus and bacillus megaterium isolates recovered from honey. rev argent microbiol. 42:216225. malek nann, malek ns. 2012. modification of synthetic zeolites by quaternary ammonium compounds and its antibacterial activity against bacillus subtilis. elsevier. 3:134-139. doi:10.1016/j.apcbee.2012.06. 059. marriott ng, gravani rb. 2006. principles of food sanitation. new york: springer. doi:10.1007/978-14757-6263-1 . matarante a, baruzzi f, cocconcelli ps, morea m. 2004. genotyping and toxigenic potential of bacillus subtilis and bacillus pumilus strains occurring in industrial and volume 9, 2015 microbiol indones 119 1: 113 2: 114 3: 115 4: 116 5: 117 6: 118 7: 119 guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed 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spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd 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helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com formulir berlangganan (subscription form) microbiology indonesia nama (name) institusi (institution) alamat pengiriman (address) phone/fax/email pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) ) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com page 1 05 lg.cdr endophytic microorganisms exist within the living tissues of most plant species. endophytic fungi, which colonize plants internally without apparent adverse effects, occur ubiquitously in plants and do not have pathogenic effects on its hosts. they are important sources of bioactive natural products with enormous potential for the discovery of new molecules for drug discovery, industrial use and agricultural applications (strobel 2006; qadri et al, 2013; ramesha and srinivas 2014). strobel et al. (2005) mentions several criteria must be considered in plant selection strategy. one of the strategy: plant that have an ethnobotanical history (use by indigenous peoples) that are related to the specific uses or applications of interest are selected for study. one of the interesting facts about the endophytic fungus is its ability to produce the bioactive compounds, same with the host or not (elfita et al. 2011a; elfita et al. 2012a). sambiloto (andrographis paniculata) is a plant that has been widely used for traditional medicine in indonesia. rao et al. (2004) and jada et al. (2007) write in their publication that andrographis is a genus of the acanthaceae family comprising of about 40 species several members of which enjoy a reputation in vol.9, no.2, june 2015, p 82-88 doi: 10.5454/mi.9.2.5 chemical constituens of an endophytic fungus aspergillus sp (sbd5) isolated from sambiloto (andrographis paniculata nees) 1 2 1 1 elfita , munawar , muharni , and ihsan ivantri* 1 department of chemistry, faculty of mathematics and natural sciences , universitas sriwijaya, jalan raya palembang prabumulih km 32, indralaya, ogan ilir, south sumatera 30662, indonesia; 2 department of biology, faculty of mathematics and natural sciences , universitas sriwijaya, jalan raya palembang prabumulih km 32, indralaya, ogan ilir, south sumatera 30662, indonesia medicinal plants and their endophytes are important resources for discovery of natural products. endophytic fungi isolated from medicinal plants more likely exibit pharmaceutical potentials. in the present study, an endophytic fungus aspergillus sp (sbd5) was isolated from leaves of sambiloto (andrographis paniculata). the fungus isolate was cultivated in potato dextrose broth (pdb) medium for 7 weeks in static, then extracted with ethyl acetate followed by thin layer chromatography (tlc) test. the results displayed three major spots. ethyl acetate extract was further separated by column chromatography and recrystallization to obtain three pure compounds. their structures were determined on the basic of spectroscopic analysis. the compounds is one new benzochromen derivative, 1-(3,8-dihydroxy-4,6,6-trimethyl-6hbenzochromen-2-yloxy)propan-2-one (1), together with two known compounds 5-hydroxy-4-(hydroxymethyl)-2h-pyran-2-one (2) and (5-hydroxy-2oxo-2h-pyran-4-yl)methyl acetate (3). the antibacterial activities of the compounds were tested using the disc diffusion method against staphylococcus aureus (atcc 25923), escherichia coli (atcc 25922), shigella dysenteriae and salmonella typhi. compound 1 has the highest antibacterial activity followed by compound 2 and 3. key words: andrographis paniculata, antibacterial activity, chemical constituens, endophytic fungus tumbuhan obat dan jamur endofitiknya merupakan sumber penting untuk mendapatkan senyawa organik dari bahan alam. jamur endofitik yang diisolasi dari tumbuhan obat lebih berpotensi untuk dikembangkan dalam bidang farmasi. dalam penelitian ini, sebuah jamur endofitik aspergillus sp (sbd5) telah diisolasi dari daun tumbuhan sambiloto (andrographis paniculata). isolat jamur tersebut telah dikultivasi dalam medium potato dextrose broth (pdb) selama 7 minggu dalam keadaan statis, kemudian diekstraksi dengan etil asetat yang dilanjutkan dengan uji pada kromatografi lapis tipis (tlc). hasilnya menunjukkan bahawa terdapat tiga noda mayor pada plat tlc. ekstrak etil asetat selanjutnya dipisahkan dengan kromatografi kolom dan rekristalisasi hingga diperoleh tiga senyawa murni. struktur molekul senyawa-senyawa tersebut ditentukan berdasarkan analisis spektroskopi. diperoleh satu senyawa baru turunan benzokromen yaitu 1-(3,8-dihidroksi-4,6,6-trimetil6h-benzokromen-2-iloksi)propan-2-on (1), dan dua senyawa yang telah dikenal yaitu 5-hidroksi-4(hidroksimetil)-2h-piran-2-on (2) dan (5-hidroksi-2-okso-2h-piran-4-il)metil asetat (3). aktivitas antibakteri senyawa-senyawa tersebut diuji dengan metode difusi cakram terhadap bakteri uji staphylococcus aureus (atcc 25923), escherichia coli (atcc 25922), shigella dysenteriae, dan salmonella typhi. senyawa 1 memiliki aktivitas antibakteri paling tinggi yang diikuti oleh senyawa 2 dan 3. kata kunci: aktivitas antibakteri, andrographis paniculata, jamur endofitik, kandungan kimia *corresponding author; phone: +62-711580056, fax: +62711580268 ; email: elfita69@gmail.com available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia traditional medicine. andrographis paniculata nees is a herb commonly used in india, china, and southeast asia for the treatment of a large variety of illnesses, which include meningitis, acute hepatitis, acute inflammatory, influenza, dysentry, malaria, antipyretic, detoxicant, and analgesic agent for the treatment of acute infections of the gastrointestinal tract, respiratory organ and urinary system. as part of a phytochemical study of our research, many bioactive and/or new compounds were isolated. elfita et al. (2011b) was reported one alkaloid 7hidroksipiranopiridin-4-on which is active as antimalarial from an endophytic fungus which was isolated from the stem of sambiloto. the paper was reported the isolation of nine endophytic fungi from the stems and leaves of sambiloto, but the aspergillus sp (sbd5) was not found in the study. the purpose of the present study was to isolate and elucidate three compounds produced from aspergillus sp (sbd5) an endophyitic fungus from leaves of sambiloto and how antibacterial activity of this compounds. materials and methods material and chemicals. the leaves of sambiloto were collected on january 2014 from the indralaya, ogan ilir, south sumatra. material for isolation and cultivation of endophytic fungi using oxoid: potato dextrose agar (pda), chloramphenicol, and potato dextrose broth (pdb), for sterilization: ethanol 70% and naocl. material for separattion: analytical thin layer chromatography (tlc) using merck (art.5554) silica gel 60 f , and column 254 chromatography using si gel 60 (70-230 mesh). organic solvents (hexane, ethyl acetate, methanol) were technical grade and distilled before use and ampicillin (sigma chemical). instrumentations. the apparatus in the research were colony counter, autoclave, incubator, water bath, microscope, magnetic hotplate, uv lamp, column chromatography and general apparatus in organic and microbiology laboratory, melting point, uvshimadzu, ftir-perkin elmer-spectrum one, and 1 13 nmr –agilent dd2 500 mhz ( h) and 125 mhz ( c), uv light at 254 nm and 365 nm. isolation and identification of endophytic fungi. the fungi was isolated from the leaves of sambiloto collected from the indralaya, ogan ilir, south sumatra using the reported methods of barik et al. (2010) and elfita et al. (2013). the endophytic fungal strain was identified based upon colony morphology and microscopic observation of mycelia and sexual spores according to the method described in the literature (liu et al. 2010). cultivation of pure fungal strain. thirty bottles (1 l) containing 300 ml pdb medium per bottle were o autoclaved for 45 min at 121 c. the purified fungi (a small park) were transferred under sterile conditions to the bottles under sterile condition. the cultures were then incubated at room temperature (no shaking) for 7 weeks (elfita et al. 2011c; elfita et al. 2012b). e x t r a c t i o n , i s o l a t i o n , a n d s t r u c t u r e elucidation of secondary metabolites. mycelium biomass was separated and culture broth repeatedly extracted with etoac (1:1). the extract was filtered and concentrated using rotary evaporator at a o temperature ± 45 c. the etoac extract (2 g) was applied to column chromatography on silica gel (40 x 1 cm) and eluted with n-hexaneetoac-methanol gradient and collected in 86 vials each containing 10 ml. the tlc analysis showed the presence of seven column fractions (f1-f7). fraction f2, f3, and f6 showed a major compound and further separation by column chromatography. fraction f3 (0.24 g) was rechromatographed over silica gel (30 × 0,7 cm) and eluted with n-hexane-etoac (5:5) to yield compound 1 (26 mg). fraction f6 (0.58 g) was rechromatographed over silica gel (30 × 0,7 cm) and eluted with n-hexaneetoac (4:6) to yield compound 2 (120 mg). fraction f2 (0.35 g) was rechromatographed over silica gel (30 × 0,7 cm) and eluted with n-hexane-etoac (7:3) to yield compound 3 (33 mg). the isolation of the compounds from ethyl acetate extract of aspergillus sp (sbd5) from the leaves of sambiloto is described in figure 1. structures of the afforded compounds were confirmed on the bases of different spectroscopic 1 13 means (uv, ir, h-nmr, c-nmr, hmqc, hmbc, and cosy) and comparison (elfita et al. 2014). antibacterial activity test. each of the three compounds dissolved in ethanol (concentration: 500 g -1 ml ). the mha (15 ml) was poured into petri dishes and inoculated with 100 μl of the suspension 5 −1 containing 1 × 10 cfu ml of bacteria. ampicillin was used as the positive control and discs treated with ethanol were used as the negative control. sterile paper discs (6 mm) loaded with 20 μl of the samples were placed onto the surface of the agar. the plates were then o placed in an incubator at 37 c for 24 h, after which the diameter of the zone of inhibition around each of the discs was measured and recorded. each experiment was performed in triplicate (choi et al. 2011). volume 9, 2015 microbiol indones 83 results the fungal strain was identified as aspergillus sp (sbd5) by the sekolah ilmu dan teknologi hayati, institut teknologi bandung, indonesia. the fungus aspergillus sp (sbd5) was cultivated on 9 l of pdb medium for 7 weeks at room temperature, the culture broth extracted by solvent partition with etoac (1:1), followed by evaporation. from the results obtained by evaporation of 10.8 g of etoac extract. the extract showed three major spot on tlc. the isolation of the compounds from ethyl acetate extract of aspergillus sp (sbd5) from the leaves of sambiloto is described in fig 1. compound 1 (1-(3,8-dihydroxy-4,6,6-trimethyl6h-benzochromen-2-yloxy)propan-2-one) was o obtained as a white crystal, mp. 150-152 c. uv (meoh) λ (log ε) nm: 220 (4.31), 273 (4.35). ir max -1 (kbr) ν cm : 3446.8; 3429.4 (oh), 3016.7 (ch-max aromatic), 2927.9 (ch-aliphatic), 1735.9; 1701.2 (c=o), 1633.7 (c=c aromatic), 1045.4 (c-o alcohol): 1 13 h nmr (cdcl , 500 mhz) δ ppm; c nmr (cdcl , 3 h 3 125 mhz) δ ppm (table 1).h compound 2 (5-hydroxy-4-hydroxymethyl-2hpyran-2-one) was obtained as a white crystal, mp. 155o -1 156 c; ir (kbr) ν cm : 3267.4 (oh), 3097.7 and max 3070.7 (ch-aromatic), 2924.1 and 2854.7 (chaliphatic), 1658.8 (c=o esther), 1626.0-1579.7 (c=c aromatic), 1224.8 (c=o esther), 1072.4 (c-o alcohol): 1 13 h nmr (cd od, 500 mhz) δ ppm; c nmr 3 h (cd od, 125 mhz) δ ppm (table 2).3 c compound 3 (5-hydroxy-2-oxo-2h pyran-4yl)methyl acetate) was obtained as a white crystal, mp. o -1 88-89 c; ir (kbr) ν cm : 3348.4 and 3263.6 (oh), max 3109.2 (ch-aromatic), 2960.0 (ch-aliphatic), 1730.2, 1662.6 (c=o esther), 1629.8 (c=c aromatic), 1255.7 1 and 1236.4 (c=o esther), 1037.7 (c-o alcohol); h 13 nmr (cdcl , 500 mhz) δ ppm; c nmr (cdcl , 3 h 3 125 mhz) δ ppm (table 3).c the antibacterial activity of three compounds was produced from aspergillus sp (sbd5) an endophyitic fungus from leaves of sambiloto against s. aureus, e. coli, s. dysenteriae, and s. typhi was evaluated by the disc diffusion method via determination of the diameter zones of inhibition. the antibacterial activity of the compounds was determined by the disc diffusion method (table 4). discussion 1 ( 3 , 8 d i h y d r o x y 4 , 6 , 6 t r i m e t h y l 6 h benzochromen-2-yloxy)propan-2-one (1). compound 1 was isolated from the etoac extract of liquid cultures of aspergillus sp (sbd5) as brown crystal (26 mg). the structures was determined on the basic of 1 13 spectroscopic analysis including uv, ir, h-nmr, cnmr, hmqc, and hmbc. it displayed uv absorbances at λ (meoh) 273 nm (log ε = 4.35) max indicating the presence of aromatic chromophore. the ir spectrum of 1 exhibited absorptions for hydroxyl -1 -1 (3446.8; 3429.4 cm ), c-h aliphatic (2927.9 cm ), c-1 h aromatic (3016.7 cm ), carbonyl (1735.9; 1701.2 o o ooh o ch3 1 2 3 4 5 6 7 8 9 o o ohoh 1 2 3 4 5 6 7 plant segments into pda a spergillus sp(sbd5) culture of a spergil lus sp (sbd5)compound 1compound 2compound 3 o h ho h h h3c ch3 ch3 o ohh ch3o 4 6 3 8 2 1 5 7 9 10 11 12 13 14 1' 2' 3' etoac extractthree pure compounds a : conidia b : phialide c : metula d : vesicle e : stipe sambiloto fig 1 isolation of the compounds from ethyl acetate extract of aspergillus sp (sbd5) from the leaves of sambiloto. 84 elfita et al. microbiol indones 1 13 table 1 the nmr data of compound 1, recorded at h-500mhz; c-125 mhz in cdcl3 1 13 table 2 the nmr data of compound 2 recorded at h-500mhz; c-125 mhz in cd od 3 1 13 table 3 the nmr data of compound 3 recorded at h-500mhz; c-125 mhz in cdcl 3 volume 9, 2015 microbiol indones 85 no. c dc (ppm) dh (ppm), sh, multiplicity, j (hz) hmbc 1 2 3 4 6 7 8 9 10 11 12 13 14 1’ 2’ 3’ 4-ch3 6-ch3 (a) 6-ch3 (b) 131.5 167.8 165.0 132.3 66.1 123.2 164.8 120.3 105.0 126.4 152.7 125.6 141.6 54.1 201.7 30.4 26.5 26.9 26.9 7.57 (1h; s) 8.03 (1h; s) 7.60 (1h; d; 6 hz) 6.90 (h; d; 6 hz) 4.25 (2h; s) 2.38 (3h; s) 3.46 (3h; s) 1.92 (3h; s) 1.92 (3h; s) 141.6 66.1 ; 120.3 ; 125.6 ; 126.4 105.0 120.3 167.8 ; 201.7 201.7 ; 54.1 141.6 ; 165.0 152.7 ; 26.9 ; 66.1 152.7 ; 26.9 ; 66.1 no. c dc (ppm) 2* dh (ppm), sh, multiplicity, j (hz) 2 hmbc 2 dc (ppm) 2 2 3 4 6 7 176.8 110.7 147.3 141.0 61.2 5 170.4 176.8 110.7 147.3 141.0 61.2 170.4 6.50 (1h; s) 7.95 (1h; s) 4.41 (2h; s) 147.3; 170.4 147.3; 170.4; 176.8 110.7; 170.4 * ref. elfita et al., (2014) no. c dc (ppm) 3* dh (ppm), sh, multiplicity, j (hz) 3 hmbc 3 dc (ppm) 3 2 3 4 6 7 173.9 111.2 145.9 138.0 61.4 5 162.8 173.9 111.2 145.8 137.9 61.4 162.9 6.50 (1h; s) 7.85 (1h; s) 4.93 (2h; s) 61.4; 145.9; 162.8 145.9; 162.8; 173.9 111.2; 162.8; 169.8 * ref. elfita et al. (2014) 9 20.6 8 169.8 20.6 169.8 2.16 (3h; s) 169.8 -1 -1 cm ), c=c aromatic (1633.7 cm ), and c-o alcohol -1 13 (1045.4 cm ). the c nmr spectrum, and was supported by hmqc spectrum showed 19 signals consisting af four methyls, one methylene, four methines, and ten quarternary carbons. the hmqc spectrum supplied complete assignment of all protonated carbons. the fourth methyl group include the presence of gem dimethyl groups that supported the existence of a signal at δ 1.92 ppm (6h, s) attached to h δ 26.9 ppm. two of these signal were assigned to c carbon atoms bearing hydroxyl aromatic groups (δ c 164.8 and 165.0 ppm) and one quarternary carbon binding cyclic ether group at δ 66.1 ppm. one of the c signal is the aromatik carbon bearing o-ether group as side chains (δ 167.8 ppm) and one signal is the c carbonyl carbon at the side chains (δ 201.7 ppm). c thirteen are in the chemical shifts above 100 ppm indicate the presence of seven double bonds, one of the double bond carbonyl carbon and six double bond of two aromatic rings. these data indicate that compound 1 has a structure of two aromatic rings and a cyclic ether in which the side chain attached to the aromatic ring which has ether groups and carbonyl groups. two hydroxyl and one methyl groups is also bonded to the aromatic ring. while the other two methyl groups attached to the quaternary carbon in the cyclic ether ring. the hmbc spectrum showed that the gem dimethyl correlated each other and bonded to quaternary carbon at δ 66.1 ppm and another one c methyl group attached to the aromatic ring were correlated with c-5 and c-14. correlation from h-9 (7.60 ppm; d; 6 hz) to c-10 and h-10 (6.90; d; 6 hz) to c-9 showed that both the aromatic protons in the ortho position. while the other two aromatic protons, namely h-1 (7,57 ppm; s) correlated with c-14 and h-7 (8,03ppm; s) correlated with c-6, c-9, c -11, and c-13. further hmbc correlation from methylene proton h1' (4.25 ppm; s) to c-2 and c-2' and methyl proton h-3' (2.38 ppm; s) to c-2' and c-1' indicated that the yloxypropan-2-one group bound to the c-2. the hmbc correlation and δ-assignment of compound 1 showed figure 2. the compound 2 (5-hydroxy-4-hydroxymethyl2h-pyran-2-one) and compound 3 (5-hydroxy-2-oxo2h pyran-4-yl)methyl acetate) were identified by ir, 1d and 2d nmr and in comparison with the literature data (elfita et al. 2014). the nmr data of compound 2 1 13 and 3, recorded at h-500mhz; c-125 mhz in dmso and cdcl and the literature 3 data showed (table 2, 3).3 the discovery of three secondary metabolites from an endophytic fungal species of medicinal plants sambiloto suggests that an endophytic fungus has a high ability to synthesize more than one secondary metabolites. seen that endophytic fungi can modify the structure of the resulting secondary metabolites such as compound 3 is the result of esterification of compound 2 according to the mode of enzymatic esterification. table 4 antibacterial activity (as inhibition zone diameters) of three compounds was produced from aspergillus sp (sbd5) an endophyitic fungus from leaves of sambiloto and ampicillin against s. aureus, e. coli, s. dysenteriae, and s. typhi fig 2 the hmbc correlation and δ-assignment of compound 1. 86 elfita et al. microbiol indones o h ho h h h3c ch3 ch3 o ohh ch3o 4 6 3 8 2 1 5 7 9 10 11 12 13 14 1' 2' 3' o h ho h h h3c ch3 ch3 o ohh 8.03 7.57 6.90 7.60 1.92 1.92 3.46 4.25 2.38 123.2 131.5 120.3 105.0 54.1 30.4 26.9 26.9 26.5 66.1 201.7 167.8 165.0 164.8 152.7 141.6 132.3 126.4 125.6 ch3o samples -1 (500 mg ml ) diameter of clear zone (mm) s. aureus e. coli s. dysenteriae s. typhi compound 1 11.6 ± 0.3 12.3 ± 0.4 11.8 ± 0.4 12.1 ± 0.3 compound 2 11.2 ± 0.1 10.1 ± 0.3 9.3 ± 0.3 9.7 ± 0.2 compound 3 9.3 ± 0.2 8.9 ± 0.2 8.1 ± 0.3 8.2 ± 0.3 ampicillin (positive control) 16.8 ± 0.3 18.5 ± 0.4 17.9 ± 0.3 18.1 ± 0.3 ethanol (negative control) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 compound 2 and 3 also produced from endophytic fungus trichoderma sp of brotowali (tinaspora crispa) (elfita et al. 2014). this fact indicates that the two compounds are not specifically produced by endophytic fungi of sambiloto, whereas compound 1 is a benzochromen derivative that may be produced by a typical from endophytic fungi of sambiloto. compound 1 was not have the same biogenesis pathway with compounds 2 and 3 so that it is possible to find other secondary metabolites from endophytic fungi of different species of the sambiloto. antibacterial activity test of all three compounds showed that they all have antibacterial activity below ampicillin activity as positive control. harlina et al. (2013) reported on the interpretation of antibacterial activity as followed: the diameter of clear zone > 15.0 mm was considered as strong; 10.0 to 14.5 mm as moderate and <10 mm as weak. among the three compounds, compound 1 has moderate antibacterial activity and the highest of the compounds 2 and 3. this fact may result from molecular structure of compound 1 which is identified as derivative of phenolic compound. phenolic derivative is well known for high antibacterial activity. compounds 2 and 3 have a moderate to weak antibacterial activity. the lowest activity is showed by compound 3 which appears that it is due to compound 3 was derived from compound 2 precursors that undergoes esterification. the test result above indicate that reduce of free oh groups of a compound can decrease its antibacterial activity. compared to similar compound mevalolactone (isolated from endophytic fungus aspergillus sp ejc08 of bauhinia guianensis), that has high antibacterial activity against escherichia coli, pseudomonas aeruginosa, bacillus subtilis, and staphylococcus aureus (pinheiro et al. 2013). the compound has a saturated ring are suggested to responsible for increase in antibacterial activity. other similar compounds, vermopyrone (isolated from unidentified fungus no.2533 of avicennia marina forssk) is inactive antibacterial (gunatilaka 2006). this compound has a unsaturated ring and no hydroxyl group. acknowledgment the authors are grateful to the directorate general of higher education for research funding through the hibah fundamental grant in 2014. references barik bp, tayung k, jagadev pn, dutta sk. 2010. phylogenetic placement of an endophytic fungus fusarium oxysporum isolated from acorus calamus rhizomes with antimicrobial activity. eur j biol sci. 2:8-16. choi jg, kang oh, lee ys, chae hs, chang y, brice oo, kim ms, sohn dh, kim hs, park h, shin dw, rho jr, kwon dy. 2011. in vitro and in vivo antibacterial activity of punica granatum peel ethanol extract 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phytochemical analysis of crude extracts of endophytic fungi isolated from plumeria acuminata l. and plumeria obtusifolia l. eur j exp biol. 4(2):35-43. rao yk, vimalamma g, rao cv, tzeng ym. 2004. flavonoids and andrographolides from andrographis paniculata. phytochemistry 65:2317–2321. doi: 10.1016/j.phytochem.2004.05.008. strobel g, daisy b, castillo u. 2005. the biological promise of microbial endophytes and their natural products. plant pathol j. 4(2):161-176. doi: 10.3923/ppj.2005. 161.176. strobel g. 2006. harnessing endophytes for industrial microbiology. curr op microbiol. 9:240-244. doi: 10.1016/j.mib.2006.04.001. 88 elfita et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 02 lg.cdr tempeh is traditional indonesian food derived from soybeans fermented by rhizopus sp. tempeh contains vitamins b , phytochemicals, antioxidative 12 (keuth and bisping 1994; astuti et al. 2000), essential fatty acid (utari 2010), and isoflavon compounds (haron et al. 2009). some report also stated that tempeh have many health benefits such as can prevent the free radicals (utari 2010), decreased risk of heart disease and strokes, osteoporosis, cancer, and menopause (babu et al. 2009). production of tempeh is done in different ways by different producers, and generally in uncontrolled conditions. the quality of tempeh is determined by three factors i.e raw material, microorganisms, and environment. the function of rhizopus provides substrates for bacteria in synthesizing components of taste and other nutritional components (seumahu 2012). some of bacteria were known to contribute to increase quality of tempeh, such as citrobacter freundii and klebsiella pneumoniae (keuth and bisping 1994), lactobacillus reuteri (taranto et al. 2003), bacillus sp. (barus et al. 2008) and lactic acid bacteria (efriwati et al. 2013). besides that, in tempeh vol.9, no.2, june 2015, p 58-64 doi: 10.5454/mi.9.2.2 genetic profiles of escherichia coli isolated from indonesian tempeh based on enterobacterial repetitive intergenic consensuspolymerase chain reaction (eric-pcr) 1 1,2 2 qurrota a'yun , antonius suwanto , and tati barus* 1 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 2 department of biology, faculty of biotechnology, universitas katolik atma jaya, jalan jenderal sudirman no 51, jakarta 12930, indonesia tempeh is a famous indonesian fermented food derived from soybeans inoculated with rhizopus sp. tempeh production varies depend on the producers and often conducted in an uncontrolled condition. this condition could lead to the growth of escherichia coli which is known as bacterial indicators of environmental hygiene. some strains of e. coli could induce diarrhea, acute gastroenteritis or gastrointestinal tract infections. the aim of this study was to compare genetic diversity of e. coli isolates from tempeh with medical isolates employing eric-pcr method. in this study, 63f and 1387r primers were used to amplify 16s rrna genes, and eric 1r and eric 2 primers were used for eric-pcr analysis. tempeh samples were obtained from four producers in bogor. thirty-three isolates of e. coli were successfully isolated from tempeh samples produced by only two producers, we could not obtain e. coli isolates from the other two producers. in addition, the same tempeh samples could carry different genotypes of e. coli. on the other hand, the same genotypes could be found in different tempeh samples. based on phylogenetic tree analysis, e. coli from tempeh could be separated from medical isolates. we showed that e. coli isolates derived from tempeh were genetically different from those of medical or pathogenic isolates. key words: eric-pcr, escherichia coli, tempeh tempe merupakan makanan khas indonesia yang berasal dari fermentasi kacang-kacangan yang diinokulasi menggunakan rhizopus sp. pembuatan tempe bervariasi tergantung pengrajin dan sering pada kondisi tidak terkontrol. kondisi yang tidak higienis dapat mengakibatkan tumbuhnya escherichia coli yang dikenal sebagai bakteri indikator kebersihan lingkungan. beberapa strain e. coli dapat menimbulkan diare, gastroenteritis akut atau infeksi saluran pencernaan. tujuan penelitian ini adalah untuk membandingkan keragaman genetik isolat e. coli dari tempe dengan isolat medis menggunakan metode eric-pcr. pada penelitian ini, primer 63f dan 1387r digunakan untuk mengamplifikasi gen 16s rrna, dan primer eric 1r dan eric 2 digunakan untuk analisis eric-pcr. sampel tempe diambil dari empat pengrajin tempe di bogor. tiga puluh tiga isolat e. coli telah berhasil diisolasi dari sampel tempe yang diproduksi oleh dua pengrajin, isolat e. coli tidak ditemukan dari dua pengrajin lainnya. lebih lanjut, sampel yang sama dapat membawa genotip e. coli yang berbeda. disisi lain, genotip yang sama dapat ditemukan dalam sampel tempe yang berbeda. berdasarkan analisis pohon filogenetik, e. coli dari tempe terpisah dengan isolat medis. penelitian ini menunjukkan bahwa e. coli yang berasal dari tempe secara genetik berbeda dengan isolat medis atau e. coli patogen. kata kunci: eric-pcr, escherichia coli, tempe *corresponding author; phone: +62-21-5719060, fax: +6221-5727615 ; email: antoniussuwanto@gmail.com available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia also found enterobacterial group namely escherichia coli (barus et al. 2008). unhygienic conditions led to the growth of e. coli in water provides an indication of fecal contamination. normally e. coli is found in the digestion of both humans and animals, but e.coli also have an important role in the spread of zoonotic diseases through food. certain serotypes can cause disease such as o157:h7 which produces shiga toxin (todar 2004). some strains of e. coli could induce diarrhea, acute gastroenteritis or gastrointestinal tract infections. strains of e. coli p a t h o g e n c o n s i s t s o f f i v e g r o u p s n a m e l y : enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enterohemorrhagic e. coli (ehec), enteroaggregative e. coli (eaggec) and enteroinvasive e. coli (eiec) (nataro and kaper 1998). genetic differences may affect the characteristics of e. coli, particularly in relation to the medical fields. therefore, the study of genetic diversity is important for the identification, characterization and to study evolution and epidemiology of the pathogenicity of the bacteria (rademarker and de bruijn 1997). one of the many molecular techniques to study genetic diversity is enterobacterial repetitive intergenic consensuspolymerase chain reaction (eric-pcr). eric sequences are short sequences, which is usually 126 bp long with replications area that conserve and in the non-coding area, sequences that are not encoded into proteins (lupski and weinstock 1992). this technique was used because it is fast, simple, discriminative (meacham et al. 2003) and has been successfully used to analyze the diversity of some groups of bacteria such as lactobacillus (stephenson et al. 2009), v. cholerae (waturangi et al. 2012), and klebsiella spp. (barus et al. 2013). materials and methods medical isolates of e. coli. three medical isolates were used for comparison with e. coli from tempeh i.e. e. coli atcc 25922 (collection of microbiology laboratory, faculty of medicine, university of indonesia, jakarta), e. coli o157 (collection of microbiology laboratory, faculty of medicine, atma jaya indonesia catholic university, jakarta) epec k.1.1 (collection of microbiology laboratory, faculty of mathematics and natural science, bogor agricultural university, bogor) and e. coli dh5α was used as control positive (collection of microbiology laboratory, faculty of technobiology, atma jaya indonesia catholic university, jakarta). e. coli isolates from tempeh. fresh tempeh obtained from producer emp, drg, wjb and clr in bogor, west java, indonesia. tempeh sampling were conducted in four month i.e. december 2013 until march 2014. a total of 10 g of each fresh tempeh was homogenized in 90 ml of sterile physiological salt -1 0.85% (w/v) (nacl). dilution was made from 10 until -5 -2 -3 -4 10 . a total of 100 µl from dilution of 10 , 10 , 10 , -5 and 10 were spread on eosin methylene blue (emb) o agar (oxoid) and incubated at 37 c for 24 h. colonies with characteristic metallic green on emb agar were purified by quadrant method. these colonies were further analyzed by cultivating them on simmon's o citrate agar (sca) (difco), and incubated at 37 c for 24 h. amplification of 16s rrna genes e. coli. genom of each e. coli was isolated using cetyl trimethyl ammonium bromide (ctab) method (sambrook and russell 2001). the 16s rrna gene was amplified employed a pcr machine (applied biosystems, 2720 thermal cycler) using 63f (5'cag gcc taa cac atg caa gtc-3') and 1387r primers (5'-ccc ggg aac gta ttc acc gc-3') (marchesi et al. 1998). pcr master mix (50 µl) consists of 25 µl ® gotaq green master mix (promega), 2 µl primer -1 forward and reverse (25 pmol µl ), 19 µl nucleas free water and 1 µl dna template. the pcr protocol was o as follows: initial denaturation at 94 c for 5 min, o o denaturation at 94 c for 30 s, annealing at 55 c for 30 o s, elongation at 72 c for 1 min, and post extention at 72 o c for 20 min. the cycle was repeated for 35 times. a total of 5 µl of pcr amplification products were further verified by electrophoresis in 1% agarose (bioline) for 60 min, 80 v and visualized by using uv transluminator (uvb 36 ultralum, carson, california). sequencing of pcr product were performed in first base sequensing int, malaysia, and were analyzed using mega 5.2 program. sequencing results were compared to the database with the basic local alignment search tool nucleotide (blastn) program which is provided by national center for biotechnology information (ncbi). analysis of genetic profiling of e. coli isolates by eric-pcr. eric sequence of e. coli was amplified using eric 1r (5'atgtaagctcctgg g g at t c a c 3 ' ) a n d e r i c 2 p r i m e r s ( 5 ' aagtaagtgactggggtgagcg-3') (versalovic et al. 1991). pcr master mix (25 µl) consists of 12.5 ® µl gotaq green master mix (promega), 1 µl of each -1 other primer (25 pmol µl ), 9.5 µl nuclease free water, volume 9, 2015 microbiol indones 59 and 1 µl dna template. the pcr protocol was as o follows: initial denaturation at 95 c for 7 min, o o denaturation at 95 c for 30 s, annealing at 49 c for 1 o min, elongation at 65 c for 8 min, and post extention at o 65 c for 16 min. the pcr cycle was repeated for 30 times. a total of 3 µl of pcr product was verified by electrophoresis in 1.5% agarose (w/v) for 120 min and 113 v and visualized by using uv transluminator (uvb 36 ultralum, carson, california). band profiles were then compared as binary number and analyzed using mega 5.2 software. phylogenetics tree construction was performed employing unweighted pair groups method analysis (upgma). results e. coli in tempeh. eighty-one isolates of e. coli were isolated from emp, drg, and clr tempeh (table 1). the colonies showed metallic green on emb medium. however, further analysis on simmon's citrate medium showed only 33 isolates showed positive characters of e. coli, i.e the e. coli colonies showed not be able to utilize citrate. 16s rrna gene of each isolate have been successfully amplified using 63f and 1387r primers with a size of dna fragments about 1.3 kb (fig 1). based on blastn, sequence of 16s rrna gene showed similarity with e. coli with maximum identities for each isolate ranging from 96% to 100%, with e-value 0.0. e. coli was not always found in tempeh samples. in this study, e. coli was found only in the emp and clr tempeh with different population (table 1) of four different times for sample collections. e. coli was found in emp tempeh from second until fourth sampling and only once was found in the clr tempeh. genetic profiling of e. coli isolates. eric sequences of 33 e. coli isolates from tempeh and 3 medical isolates were successfully amplified using eric 1r and eric 2 primers (fig 2). visualization of the eric-pcr profiles shows that dna banding patterns of e. coli from tempeh were different to that of medical isolates. band profiles of e. coli isolates from tempeh showed consistencies of size 0.25 kb and 1.0 kb. however, we found inconsistencies in the dna bands of e. coli from medical isolates. eric-pcr profiles isolates of e. coli from clr tempeh showed similar pattern while e. coli isolates from emp tempeh showed more heterogenome patterns (fig 2). although genetic profiles of e. coli isolates in emp tempeh more varied than clr tempeh, we found no identical profiles when isolates from tempeh compared to e. coli dh5α. phylogenetic tree based on eric-pcr profiles showed the relation between e. coli from tempeh and medical isolates (fig 3). it showed that e. coli from tempeh were genetically different from those of medical isolates. e. coli from tempeh formed a separate group (group i-iv), while medical isolates formed group vi and e. coli dh5α formed group v. table 1 the frequency of e. coli presence in fresh tempeh tempeh samples numbering of sampling emb agar (number of isolates) simmon’s citrate agar (number of isolates) emp 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 drg wjb clr 20 12 4 23 20 3 17 11 3 3 total of isolates 81 33 note ( ) = colonies which are not green metallic on emb agar or did not utilize citrate in simmon's citrate agar 60 a’yun et al. microbiol indones fig 2 genetic profiles eric-pcr isolates of e. coli from tempeh and medical isolates. molecular markers (m), isolates from clr tempeh (c3.12a, c3.23a), isolates from emp tempeh (e2.13a-e4.33a), negative control (k-), e. coli dh5α (k+), e. coli atcc 25922 (medis 1), epec k.1.1 (medis 2), and e. coli o157 (medis 3). fig 1 amplification results of 16s rrna genes e. coli from tempeh. molecular markers (m), isolates from tempeh (c3.12a-e4.33a), positive control (k+), negative control (k-). discussion the presence of e. coli in tempeh have been reported by barus et al. (2008). e. coli from tempeh were varied in each sampling of the same producers (table 1). e. coli was not always found in tempeh even the ones produced by the same producers despite of in the same tempeh. e. coli in tempeh may be affected by various sources such as raw materials, tools, people, and surrounding environment. the spread way of e. coli in tempeh can be occurred due to cross contamination directly (by hand) and indirectly (through the water) during processing (antara and gunam 2002). the presence of e. coli in food indicated contamination due to non-hygienic food processing or handling. therefore, e. coli is often used as an indicator of sanitary (githiri et al. 2009). molecular approach can be used to detect the presence of e. coli by amplifying the dna sequences which encoded 16s rrna gene. 63f and 1387r primers volume 9, 2015 microbiol indones 61 fig 3 phylogenetic tree constructed from of eric-pcr profiles using upgma method through mega 5.2 program with 1000x bootstrap. e. coli dh5α, e. coli atcc 25922 (medis 1), epec k.1.1 (medis 2), e. coli o157 (medis 3), and group (i-vi). were used to amplify 16s rrna gene sequences of e. coli. during the amplification process, 63f primer starts to amplify the dna template on the position of 43-63, meanwhile 1387r primer were amplified the dna on the position of 1387–1370, so that the resulting product approximately 1.3 kb in size (marchesi et al. 1998). the 16s rrna sequences were used as moleculer markers because of their ubiquity and their identical function in all cellular-based organisms in addition to their conservative and varied sequence (pangastuti 2006). analysis of bacterial virulence can be recognized from the phenotype (stenutz et al. 2006). some serotypes of e. coli known to have virulence factors are o (somatic), h (flagellar), and k (capsular) surface antigen profiles (nataro and kaper 1998). serotype of o157:h7 can induce the secretion of body fluids excessively and continuously that can lead to diarrhea or meningitis. e. coli o157 harbors vt1 and vt2 genes, generally found on pig (suardana et al. 2007). in this study, the multiple dna fragments of all e. coli strains generated from eric primers were composed of 4-13 bands ranging in sizes between 0.25 kb and 5 kb (fig 3a). the results indicated the extent of 62 a’yun et al. microbiol indones genetic diversity in e. coli. meacham (2003) stated that the difference in the number and size of the eric-pcr profiles described genetic diversity among bacterial isolates. eric-pcr technique employs consensus primers during pcr to amplify dna sequences located between successive repetitive sequence. it is often used for subtyping gram-negative enteric bacteria (hulton et al.1991). isolates of e. coli from tempeh showed similar characteristics in emb media. however, the genomic profiles of e. coli from tempeh were different from those of medical isolates. upgma phylogenetic tree (fig 3) showed that isolates of e. coli from tempeh formed separates group from medical isolates. isolates of e. coli from clr tempeh did not showed genetic diversity and closely related to some isolates of emp tempeh which clustered in group iv. in contrast to e. coli isolates from clr tempeh, e. coli from emp tempeh showed more genetic diversity. e. coli isolate from emp tempeh are both closely and distantly related. eric-pcr technique was successfully employed to determine genetic profiles of e.coli from tempeh and medical isolates and it showed more discriminatory power than 16s rrna genes analysis. previous study reported that eric-pcr was employed to obtain eric sequences of klebsiella spp. from tempeh and it showed more discriminative results than 16s rrna genes analysis (barus et al. 2013). ayu et al. (2014) reported that eric-pcr was able to differentiate genetic profiles of tempeh-derived k. pneumoniae isolates from those of medical isolates. our results showed that e. coli isolates from tempeh could be a distinctive non-pathogenic groups. not all tempeh contain e. coli and the presence of e. coli in tempeh was not always found in every sample and every batch. based on the analysis of genetic dna profile using eric-pcr showed that e. coli from tempeh were different from e. coli pathogens or associated with medical isolates. however, there is no single report that consumption tempeh which contain e. coli will cause disease. non aesthetic traditional tempeh production may contain some e. coli isolates. however, it does not mean necessarily contain dangerous bacteria. acknowledgment this research was supported by thesis scholarship lembaga pengelola dana pendidikan (lpdp) and dana dipa ipb 2014 to antonius suwanto. references antara s, gunam ibw. 2002. dunia mikroba (bahaya mikrobiologis pada makanan). pusat kajian keamanan pangan. denpasar (id): universitas udayana. astuti m, meliala a, dalais fs, wahlqvist ml. 2000. tempe, a nutritious healthy food from indonesia. asia pac j clin nurt. 9(4):322-325. ayu e, suwanto a, barus t. 2014. klebsiella pneumoniae from indonesian tempeh were genetically different from that of phathogenic isolates. j microbiol indones. 8(1):9-15. doi: 10.5454/mi.8.1.2. babu pd, bhakyaraj r, vidhyalaksmi r. 2009. a low cost nutritious food “tempeh”. world j dairy food sci. 4(1): 22-27. barus t, suwanto a, wahyudi at, wijaya h. 2008. role of b a c t e r i a i n t e m p e b i t t e r t a s t e f o r m a t i o n ; 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2 geomicrobiology-biomining & biocorrosion laboratory, microbial culture collection laboratory, biosciences and biotechnology research center (bbrc), institut teknologi bandung, jalan ganesha 10, bandung 40132, west java, indonesia; 3 department of mining engineering, faculty of mineral technology, universitas pembangunan nasional (upn) veteran, yogyakarta, indonesia the bioleaching of an indonesian complex copper sulfide ore was studied in shake flasks over a period of 14 days using an iron-oxidizing indigenous bacterium at room temperature (28 °c) and various pulp densities (5% and 20%). the bioleaching suspensions were periodically analyzed for cu and fe concentrations as well as eh, ph and do values. cu bioleaching efficiencies at 5% pulp density were higher than those at 20% pulp density, which correlated with fe concentration in solution. over a period of 14 days, the ph of bioleaching suspension + was in the range of 5 ~ 9, indicating that cu bioleaching was greatly influenced not only by proton h dan ferric ion but also by extracellular polymeric substances (eps) generated by the bacterium. the current study may improve our better understanding on the bacterial action for bioleaching of complex copper sulfide ores that remains debated so far as refractory ores. key words: biohydrometallurgy, bioleaching, complex copper sulfides, chalcopyrite, iron-oxidizing bacteria bioleaching bijih sulfida tembaga komplek dari indonesia diteliti pada percobaan menggunakan labu yang digoyang selama 14 hari dengan memanfaatkan bakteri indigen pengoksidasi besi yang dilakukan pada suhu ruang (28 °c) dan variasi pulp density ((5% and 20%). suspensi bioleaching diamati dan diukur secara periodik yaitu konsentrasi cu dan fe serta eh, ph dan do. hasil penelitian menunjukkan bahwa efisiensi bioleaching cu pada 5% pulp density lebih besar dibandingkan dengan efisiensi bioleaching cu pada 20% pulp density dimana nilainya berkorelasi positif dengan konsentrasi fe total yang terlarut. selama 14 hari percobaan bioleaching, nilai ph suspensi bioleaching berkisar 5~9 yang menunjukkan bahwa bioleaching cu terjadi tidak hanya disebabkan + oleh proton h dan konsentrasi fe (iii) yang terlarut tapi juga sangat dipengaruhi oleh extracellular polymeric substances (eps) yang diproduksi oleh bakteri. hasil penelitian ini mungkin meningkatkan pemahaman kita tentang peranan bakteri dalam melakukan bioleaching bijih sulfida tembaga komplek yang masih merupakan perdebatan sampai saat ini sebagai bijih yang sangat sulit untuk di-leaching atau di-bioleaching. kata kunci: bakteri pengoksidasi besi, bijih sulfida tembaga komplek, biohidrometalurgi, bioleaching, kalkopirit microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-22-2502239, fax:+6222-2504209; email: skchaerun@metallurgy.itb.ac.id have found that the primary sulfide minerals of copper have been difficult to leach for the purpose of direct copper extraction (in particular chalcopyrite, cufes ) 2 due to passivation phenomena under a variety of oxidative leaching conditions (dreisinger 2006; watling 2006; arce and gonzález 2002; herrera et al. 1989). correspondingly, many trials have been conducted to bioleach copper and overcome the passivation of chalcopyrite using different genera of prokaryotes (wang et al. 2016; zhao et al. 2015a; zhao et al. 2015b; gu et al. 2013; zhao et al. 2013; yu et al. 2011; qiu et al. 2005; bevilaqua et al. 2002). those studies employed chemolithotrophs such as acidithiobacillus ferrooxidans (qiu et al. 2005; zhao et al. 2013), it is well known that bioleaching is a low-cost, effective, environmentally friendly technology that has been applied worldwide to recover metals from both high-grade and low-grade ores as well as tailings, mine wastes and urban solid wastes. the most challenging application of the bioleaching is for processing complex copper ores which contain both copper sulfides and oxides. owing to the complexity in the mineralogical characteristics of the ores, most efficient metal extraction from the ores can be achieved through bioleaching method. some researchers have studied the bioleaching of complex copper sulfide ores and acidithiobacillus thiooxidans (qiu et al. 2005), leptospirillum ferriphilum (wang et al. 2016; zhao et al. 2015a; zhao et al. 2015b; gu et al. 2013), acidithiobacillus caldus (wang et al. 2016; zhao et al. 2015b), and sulfobacillus thermosulfidooxidans (wang et al. 2016). as a matter of fact, naturally occurring complex ores are always found in association with not only inorganic matter but also organic matter. hence, the mixotrophic bacteria which are capable of using both organic and inorganic matters are needed to be employed in bioleaching processes, since chemolithotrophs are very sensitive to organic matters. in addition to the mechanism of bioleaching copper sulfides, microbially produced extracellular polymeric substances (eps) which facilitates the attachment of microbes to the mineral surface can sequester metals (herein fe and cu) into soluble metalligand complexes by chelation, thus enhancing copper dissolution (sand and gehrke 2006). therefore, the current work studied the bioleaching of complex copper sulfide ores from indonesia using an ironoxidizing indigenous bacterium capable of oxidizing iron and producing eps in order to provide a better understanding of bioleaching copper sulfides that are recalcitrant to bioleaching. materials and methods ore samples. complex copper sulfide ore was obtained from the sungai mak copper deposits (gorontalo, indonesia) and was ground to obtain a particle size of d = 75 μm. an x-ray powder 80 diffractometry (xrd) analysis showed that the ore contained quartz (as the dominant mineral), muscovite, kaolinite, and alunite with various forms of copper minerals including covellite, chalcocite, chalcopyrite, cuprite/chalcotrichite, digenite, azurite, malachite, and chalcanthite. bacterium and growth medium. a mixotrophic bacterium used in this study was isolated from an indonesian mine site (identified as alicyclobacillus sp.), which has the abilities of oxidizing iron and sulfur and producing a high amount of extracellular polymeric substances (eps) (mubarok et al. 2017). the growth medium used was the modified luria–bertani (lb) -1 -1 medium, containing 10 g l peptone, 5 g l yeast -1 -1 extract, 10 g l nacl, supplemented with 0.5 g l -1 na s o .5h o, and 0.25 g l feso .7h o. the 2 2 3 2 4 2 modified lb medium was used in this study to provide organic carbon source in addition to iron and sulfur for bacterium in order to produce a high amount of eps since the bacterium was more favorable to generate eps under high organic carbon concentration (unpublished data). the photomicrographs of bacterial cells and its colonies are shown in fig 1a and 1b. e x p e r i m e n t a l p r o c e d u r e . b i o l e a c h i n g experiments were carried out in duplicate in sterile 300 ml erlenmeyer flasks containing 150 ml of the modified -1 lb medium supplemented with 10% (vol vol ) inoculum of the bacterium and complex copper sulfide ores (a particle size of d = 75 μm) at temperature of 28 80 °c. they were subsequently incubated for 14 d at a rotation speed of 180 rpm under various bioleaching parameters of solid to liquid ratio (pulp density) (5% and -1 20%), nacl addition (10 g l ) to prevent passivation. the ph, eh (redox potential) and do (dissolved oxygen) in bioleaching suspension were monitored periodically, and the solution (2 ml) was removed for measuring dissolved copper and iron concentrations by using atomic absorption spectrophotometer (aas). the data are presented as the averages obtained from the duplicate experiments. eps production by the bacterium during bioleaching was assayed by determining the emulsifying activity index (ei, %) as described by francy et al. (1991) with modification, which estimated the ability of eps to emulsify liquid hydrocarbons such as coconut oil, corn oil, and kerosene. results physicochemical characteristics of bioleaching suspension. figures 2a-c depict the physicochemical characteristics (ph, redox potential, and dissolved oxygen) of bioleaching suspension over a 14-day bioleaching experiment. the ph values of suspension tended to increase over time from ~5 to ~9 (fig 2a), + indicating that there was h consumption during bioleaching processes. as the ph increased, the redox potential (eh) values in bioleaching suspension also tended to increase from 248 mv (vs. she) to 403 mv (for 5% pulp density) and from 365 mv to 488 mv (for 20% pulp density) (fig 2b). this suggests that the bacterium creates a certain potential environment that greatly affects the leaching of copper species, since the leaching of each copper species is largely dependent on the electrochemical potential of the leaching solution. in addition, the dissolved oxygen levels over 14 days of bioleaching experiment were in the range of 7.1~8.3 -1 mg l (fig 2c), indicating a sufficient o supply for 2 viability and activity of leaching microbes. copper and iron bioleaching. figures 3a-b represent the dissolution of copper and iron in solution 2 chaerun et al. microbiol indones at pulp density of 5% and 20% over a 14-day bioleaching experiment. the copper bioleaching efficiencies from the ores increased rapidly during the first 1 d and subsequently increased slightly for another 13 d, which achieved the extraction levels of ~31% (for 5% pulp density) and ~27% (for 20% pulp density) (fig 3a). these increases were concomitant with an increase in iron bioleaching efficiencies (fig 3b). for 5 % pulp density, iron dissolution (with a maximum iron extraction of 4%) increased rapidly during the first 2 d and subsequently decreased rapidly for the remaining bioleaching time. iron dissolution for 20% pulp density also increased for the first 3 d, subsequently remained relatively constant for another 4 d and finally decreased up to the end of bioleaching experiment, which reached levels of only 0.9%. compared to copper extraction, the iron dissolution was very low. eps production during bioleaching. figure 4 represents the eps generated by an iron-oxidizing indigenous bacterium over a period of 7 days. three hydrocarbons (coconut oil, corn oil and kerosene) were evaluated for eps production, which was represented as emulsifying activity index (ei). it appeared that eps production tended to increase over time, thus providing the evidence of eps generation during bioleaching processes. discussion a variety of biological and chemical leaching processes have been studied by many researchers for enhancing copper leaching from complex copper sulfides as well as overcoming the passivation of chalcopyrite. one of the biological leaching processes employed is by the application of thermophilic prokaryotes to increase the solubilization of copper more efficiently (plumb et al. 2002). however, hightemperature bioleaching process suffers from high electrical energy and capital cost. in addition, most studies on copper bioleaching utilize chemolithotrophs that are very sensitive to organic matters. since complex copper sulfides are always associated with organic compounds, the indigenous bacterium employed in the present study grows well at room temperature (20 ~ 40 °c) and belongs to mixotrophic group, which is capable of utilizing both organic and inorganic compounds as well as oxidizing iron and producing eps. by having such capacities, the bacterium benefits copper bioleaching from complex copper ores both for accelerating copper dissolution and for preventing passivation phenomenon. during the bioleaching process, passivation layers formed by the jarosite precipitation on the mineral surface are thought to cause slow copper dissolution, and the passivation layer hinders greater copper extraction by restricting the flow of bacteria, nutrients, oxidants, and reaction products to and from the mineral surface (zhang et al. 2009; watling 2006; stott et al. 2000). depending on the ph, redox potential (eh), temperature, ionic composition, and fe(iii) concentration, the solubility of ferric iron is controlled by precipitation of schwertmannite and jarosite (bevilaqua et al. 2002; bigham et al. 1996). from the results of the present study, the eh continued to rise during the bioleaching period (fig 2b), indicating continued bacterial ferrous ion oxidation to ferric ion. this high ferrous-oxidizing activity of the bacterium in this study caused a steady increase in the eh over time, thus generating the amount of ferric ion in solution (fig 3b). the copper was leached rapidly from the ore during the first 1 d (fig 3a) which was concomitant with a high iron dissolution at that period for any pulp densities (fig 3b), suggesting that ferric ions enhanced copper dissolution. however, since the ph of bioleaching suspension was higher than 5 over time (fig 2a), ferric iron was in insoluble form as precipitates, bringing about passivation which in turn resulted in low copper bioleaching (only a maximum copper extraction of ~31%) (fig 3a). moreover, the bacterium used in this study is able to produce a huge amount of eps grown in lb medium, thus the eps enables insoluble ferric iron to be in soluble form, which in turn help contribute to the enhancement of copper dissolution (fig 3a). copper dissolution rate is strongly dependent on the reduction potential (eh) in solution and this parameter is far more influential than the number or activity of bacterial cells (third et al. 2000), where maximum chalcopyrite dissolution has been reported to occur at about 650 mv (vs. she) (kametani and aoki 1985). since the eh values of bioleaching suspension in this study ranged from 206 to 488 mv (vs. she) (< 650 mv), the passivation phenomena in this study could be reduced. in conclusion, the present study has shown that an iron-oxidizing bacterium indigenous to a mine site of indonesia is capable of bioleaching copper from a complex copper sulfide ore through three roles creating the reduction potential (eh) in solution which is favourable for copper leaching, generating ferric ions at a suitable rate that are needed for continued leaching of the ores, and producing eps to concentrate ferric ions by complexation at the mineral surface microbiol indones 3volume 12, 2018 -1 fig 2 the values of ph (a), redox potential (eh) in mv (vs. she) (b) and dissolved oxygen (do) in mg l (c) of bioleaching suspension containing an iron-oxidizing indigenous bacterium at various pulp densities (5% and 20%) over a period of 14 days. 4 chaerun et al. microbiol indones fig 3 copper (a) and total iron (b) dissolution (%) by an iron-oxidizing indigenous bacterium at various pulp densities (5% and 20%) over a period of 14 days. fig 1 photomicrographs of bacterial cells of an iron-oxidizing bacterium (identified as alicyclobacillus sp.) (a) and its colonies (b). which thus allow an oxidative attack on the sulfide as well as to sequester metals (herein cu) into soluble metal-ligand complexes by chelation. these in turn result in copper dissolution. the results obtained from this study may thus contribute to the development of the bioleaching technology for processing complex copper sulfide ores as refractory ores. acknowledgment this work was financially supported by a grant from the ministry of research, technology and higher education of the republic of indonesia to skc. we thank pt. gorontalo minerals indonesia for providing the copper sulfide ores. we also thank the editor and two anonymous reviewers for their constructive comments. references arce em, gonzález i. 2002. a comparative study of electrochemical behavior of chalcopyrite, chalcocite and bornite in sulfuric acid solution. int j min proc. 67(1), 17-28. doi: 10.1016/s0301-7516(02)00003-0. bevilaqua d, leite allc, garcia o, tuovinen oh. 2002. oxidation of 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iv/1, semarang 50234, indonesia six selected lactic acid bacteria (lab) isolates from pickled yellow betung bamboo shoots were grown in the mann rogosa sharpe-broth (mrsb) media with different supplementation combination. the cell-free supernatant were evaluated for their ability to produce bacteriocin by adjusting its ph to 6.0 in order to remove organic acid effects. the bacteriocin activity was assayed by agar-well diffusion method. the inhibitory activity 2 -1 calculated in activity unit (au in mm ml ) of bacteriocins. the aims of this paper is to explore the effect of different medium compositions on bacteriocin production and its inhibitory activity against pathogenic bacteria (listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091). supplementations of carbon and nitrogen sources induced production of bacteriocins. lab isolates grown in media without supplementation could not produce bacteriocins. growth of isolate d44 in the presence of 2% of -1 glucose and 2% of yeast extract yielded the largest bacteriocin inhibitory activity levels of 3179 au ml against -1 listeria monocytogenes fncc 0156, 4663 au ml against staphylococcus aureus fncc 0047, and 3109 au -1 ml against escherichia coli fncc 0091. key words: bacteriocin, lactic acid bacteria, pickled bamboo shoots, supplementation enam isolat bakteri asam laktat terpilih (lab) yang diisolasi dari acar rebung bambu kuning ditumbuhkan di media mann rogosa sharpe-broth (mrsb) dengan kombinasi suplementasi yang berbeda. supernatan bebas sel diatur ph menjadi 6,0 untuk menghilangkan efek asam organik dan dievaluasi aktivitas bakteriosinnya. aktivitas bakteriosin diuji dengan metode difusi agar-well. aktivitas penghambatan dihitung dalam unit aktivitas (au 2 -1 dalam mm ml ) bakteriosin. tujuan dari penelitian ini adalah untuk mengeksplorasi pengaruh komposisi medium yang berbeda pada produksi bakteriosin dan aktivitas penghambatannya terhadap bakteri patogen (listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, dan escherichia coli fncc 0091). suplementasi sumber karbon dan nitrogen menginduksi produksi bakteriosin. isolat lab yang tumbuh di media tanpa suplementasi tidak dapat menghasilkan bakteriosin. pertumbuhan isolat d44 dengan adanya 2% glukosa -1 dan 2% yeast extract menghasilkan aktivitas penghambatan bakteriosin terbesar, yaitu 3179 au ml terhadap -1 listeria monocytogenes fncc 0156, 4663 au ml terhadap staphylococcus aureus fncc 0047, dan 3109 au -1 ml terhadap escherichia coli fncc 0091. kata kunci: acar rebung, bakteriosin, bakteri asam laktat, suplementasi microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-24-8441555, email: laksmi@unika.ac.id often regulated by some factors, such as growth ph, temperature, and nutrient sources (mahrous et al. 2013; todorov and dicks, 2005; zhou et al. 2014). bacteriocin production requires suitable culture medium containing all essential nutrients in suitable amounts. growth medium compositions, especially the sources and concentrations of carbon and nitrogen strongly affect production of bacteriocins (khay et al. 2013; todorov and dicks (2005). the effect of medium compositions on the bacteriocin inhibitory activity of lab isolated from fermented bamboo shoot pickle is still unknown. the aims of this paper are to to compare the effect of different medium compositions on bacteriocin production and its inhibitory activity against pathogenic bacteria (listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091). bamboo shoots can be processed into fermented product such as vegetable pickle using lactic acid b a c t e r i a ( l a b ) . l a b f r o m d i ff e r e n t s a l t concentrations and temperatures have been isolated. isolates were prepared from each fermentation conditions: fermentation in 2.5% salt solution at 15 °c (21 isolates), 5.0% salt solution at 15 °c (22 isolates), 2.5% salt solution at 30 °c (22 isolates), and 5.0% salt solution at 30 °c (27 isolates). all combinations of salt concentration and fermentation temperature yielded isolates with probiotic capability and antimicrobial activity (lindayani et al. 2015). some lactic acid bacteria can produce bacteriocin (salminen et al. 2004). many studies conducted on bacteriocins found that the production of bacteriocin is materials and methods bacterial strains and culture conditions. eleven lactic acid bacteria (lab) strains isolated from pickled yellow bamboo shoots (lindayani et al. 2015; hartayanie et al. 2016) were used in this study. the lab were propagated in mrsb (merck, germany) containing 0.2% glucose (merck, germany) and incubated at 37 °c for 24 h. the microorganisms were propagated and maintained in nb (nutrient broth) medium. three indicator microorganisms used in antibacterial activity tests were listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091. the indicator microorganisms were propagated in nutrient broth (merck, germany) at 37 °c for 24 h. antibacterial activity. antibacterial activity was tested by using the well diffusion method (ivanova et al. 2000). ten µl of pathogen inocula equivalent to mcfarland 3 was inoculated with 10 ml of nutrient agar (na, merck, germany) as a pour plate and was allowed to solidify. twenty µl of cell-free supernatant was inoculated in 5.5 mm diameter wells and incubated for 3 h at 4 °c to let it be absorbed. sterile mrsb was used as a negative control. antibacterial activity was tested against pathogenic bacteria (staphylococcus aureus fncc 0047 and escherichia coli fncc 0091). clear zone around the wells was measured after 24 h incubation at 37 °c. antibacterial activity was expressed as arbitrary units (au) per ml. one au was defined as the reciprocal of the highest dilution showing a clear zone of growth inhibition. the calculation of the antibacterial activity was the same as the bacteriocin activity calculation. identification of bacteriocin–producing species. the lab was identified based on its physiological and biochemical characteristic using api 50 chl test strips (biomereux, marcy-l'etiole, france). the identified isolates were kept at -18 °c in mrs broth containing 15% of glycerol (merck, germany). bacteriocin bioassay. bacteriocin activity testing was performed by using the well diffusion method (ivanova et al. 2000). ten µl of pathogen inocula equivalent to mcfarland 3 was inoculated into 10 ml of nutrient agar (na, merck, germany) as a pour plate and was allowed to solidify. cell-free supernatant was neutralized by adjusting ph to 6.0 with 1 m naoh in order to prevent the inhibitory effect of lactic acid (karthikeyan and santosh 2009). twenty µl of neutralized cell-free supernatant was inoculated in 5.5 mm diameter wells and incubated for 3 h at 4 °c to let it absorbed. sterile mrsb was used as a negative control. bacteriocin activity was tested using pathogenic bacteria (listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047 and escherichia coli fncc 0091). the clear zone around the wells was measured after 24 hours incubation at 37 °c. bacteriocin activity was expressed as arbitrary units (au) per ml. one au was defined as the reciprocal of the highest dilution showing a clear zone of growth inhibition. key: 2 az : clear zone area (mm ) 2 as : well area (mm ) v : sample volume (ml) effect of medium composition on bacteriocin production. the lab isolates that have bacteriocin activity against pathogenic bacteria (listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047 and escherichia coli fncc 0091) were grown in mrsb at 37 °c for 48 h. the isolates adjusted to mcfarland 5. five hundreds µl of isolate was mixed with 500 µl of the following media: (i) mrsb without supplementation; (ii) mrsb supplemented with 2% glucose; (iii) mrsb supplemented with 2% glucose and 2% tryptone; (iv) mrsb supplemented with 2% glucose and 2% yeast exctract; and (v) mrsb supplemented with 2% glucose, 1% tryptone, and 1% yeast extract. then isolates were incubated in an incubator at 37 °c for 24 h. after incubation, the cells were separated by centrifugation at 11000 g for 10 min at 4 °c. after centrifugation the supernatant was collected in a fresh sterile tube and the pellet was discarded. the cell free supernatant (cfs) was neutralized by adjusting ph to 6.0 using 1 n naoh for bacteriocin assays against three pathogenic bacteria (listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047 and escherichia coli fncc 0091). results all isolates that primarily confirmed as lab from pickled yellow betung bamboo shoots were tested for antibacterial activity. the isolates showed antibacterial activity against listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091 (table 1). different treatment in salt concentration and 8 hartajanie et al. microbiol indones bacteriocin activity 2 mm ml = az as v( ) temperature of fermentation gave various lab (table 2). however, leuconostoc mesenteroides ssp. cremoris was found from fermentation condition a (a43) and c (c18 and c29). lactobacillus pentosus was found from fermentation condition b (b1) and d (d44). isolates with significant values more than 80% showed very good identification results (a20, b1, b31, b32, c19, d11, d20, and d44). the results of api kit identification of a43, c18, and c29 could not be accepted because the significant value was less than 80% (www.biomerieux-usa.com). some rod-shaped isolates were acquired i.e., b1 as lactobacillus pentosus (99.7%), c19 as lactobacillus plantarum (99.6%) and d44 as lactobacillus pentosus (96%). cocci-shaped were found from isolate a20, b3, and b32. a20 was identified as leuconostoc mesenteroides ssp. mesenteroides/dextranicum 2 (96.8%), b31 was identified as enterococus durans (86.15%) and b32 was identified as lactococcus lactis ssp. lactis 1 (96.5%). table 3 showed that there was no bacteriocin activity due to absence of supplementation. all isolates showed vary bacteriocin inhibitory activity against pathogen when they were grown in supplemented mrsb. c18 showed higher bacteriocin activity when it was cultured in mrsb supplemented with glucose and tryptone rather than in mrsb supplemented with glucose. bacteriocin production of c19, c29, and d44 w a s i m p r o v e d b y c a r b o n a n d n i t r o g e n supplementation. tryptone was better supplement than yeast extract to improve bacteriocin production of c19. but on c29, yeast extract was better than tryptone. bacteriocin production of d11 could only be induced by carbon supplementation. bacteriocin d44 could inhibit all pathogenic bacteria when produced in mrsb supplemented with combinations of carbon and nitrogen sources (tryptone table 1 result of antimicrobial activity table 2 result of api software identification (24 hours incubation) key: a = 2.5% of salt concentration at 15 °c b = 5.0% of salt concentration at 15 °c c = 2.5% of salt concentration at 30 °c d = 5.0% of salt concentration at 30 °c key: * = significant < 80 % a = 2.5% of salt concentration at 15 °c b = 5.0% of salt concentration at 15 °c c = 2.5% of salt concentration at 30 °c d = 5.0% of salt concentration at 15 °c fermentation condition isolate code species identification significant level (%) a a20 leuconoctoc mesentroides ssp mesentroides/dextranicum 2 10.93 ± 2.47 a43 * leuconostoc mesentroides ssp cremoris 9.67 ± 1.81 b b1 lactobacillus pentosus 9.50 ± 1.73 b31 enterococcus durans 9.87 ± 1.60 b32 lactococcus lactis ssp lactis 1 8.27 ± 1.10 c c18 * leuconostoc mesentroides ssp cremoris 8.90 ± 0.00 c19 lactobacillus plantarum 1 11.90 ± 0.00 c29 * leuconostoc mesentroides ssp cremoris 9.80 ± 0.00 d d11 lactobacillus fermentum 1 10.80 ± 0.00 d20 lactobacillu s plantarum 7.10 ± 0.00 d44 lactobacillus pentosus 10.90 ± 0.00 fermentation condition isolate code diameter of inhibition zone (mm) s. aureus fncc 0047 e. coli fncc 0091 a a20 9.70 ± 1.21 10.93 ± 2.47 a43 10.70 ± 0.92 9.67 ± 1.81 b b1 9.37 ± 1.00 9.50 ± 1.73 b31 12.40 ± 0.00 9.87 ± 1.60 b32 12.70 ± 0.00 8.27 ± 1.10 c c18 14.70 ± 0.00 8.90 ± 0.00 c19 10.70 ± 0.00 11.90 ± 0.00 c29 12.90 ± 0.00 9.80 ± 0.00 d d11 10.80 ± 0.00 10.80 ± 0.00 d20 10.73 ± 2.06 7.10 ± 0.00 d44 8.80 ± 0.00 10.90 ± 0.00 volume 12, 2018 microbiol indones 9 or yeast extract or a combination both of them). it had a broader zone of inhibition against pathogenic bacteria than other bacteriocins used in this study (fig 1). the presence of 2% of glucose and 2% of yeast extract resulted in the largest bacteriocin inhibitory activity -1 levels of 3179 au ml against listeria monocytogenes -1 fncc 0156, 4663 au ml against staphylococcus -1 aureus fncc 0047, and 3109 au ml against escherichia coli fncc 0091 (table 3 dan fig 2). lab isolates had different response for each treatment of supplementations as shown on table 3. carbon and nitrogen supplementation gave different response to induce bacteriocin inhibitory activity against listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091. bacteriocin from isolate d44 showed the largest inhibitory activity against pathogens when it was grown in mrsb supplemented with 2% glucose and 2% yeast extract. each isolate had different result on bacteriocin inhibitory activity for each 2 -1 supplementation. au of 3179 mm ml against listeria 2 -1 monocytogenes fncc 0156, 4663 mm ml against 2 -1 staphylococcus aureus fncc 0047, and 3109 mm ml against escherichia coli fncc 0091. compared to the other tested pathogenic bacteria, the impact of this bacteriocin was less against escherichia coli fncc 0091 and listeria monocytogenes fncc 0156. discussion the inhibitory effect of lab isolates may be due to the production of several antimicrobial compounds (sifour et al. 2012). different temperatures of fermentation may affect the potential of lab in producing bacteriocins. higher temperature altered the fermentation to homofermentative (harris, 1998 in salminen et al. 2004). since the fermentation type of lactobacillus pentosus is homofermentative (fleming 1991 in salminen et al. 2004), higher temperature (fermentation condition d) could be a better condition for the growth of lactobacillus pentosus than fermentation condition b. indirectly, maximum growth of lactobacillus pentosus may affect its potential to produce bacteriocins. leuconostoc mesenteroides belonged to heterofermentative bacteria showed a better bacteriocin inhibitory activity when isolated from higher temperature of fermentation (fleming 1991 in salminen et al. 2004). tryptone and yeast extract contain various amino acid which can help to increase the biomass (salminen et al. 2004). the inhibition activity of bacteriocin against pathogens depends on nutrient addition (table 3). specific nutrients are required to improve bacteriocin production (todorov and dicks 2005b). bacteriocins were only produced when nutrients were 2 -1 table 3 effect of adding some nutrient components in mrs-b medium on bacteriocin inhibitory activity (au mm ml ) by lab isolates which isolated from yellow betung bamboo shoot pickle (c and d) lab isolate pathogenic bacteria activity unit of bacterio cins (mm2 ml-1) medium compositions of bacteriocin production mrs-b mrs-b supplemented with glucose mrs-b supplemented with glucose and tryptone mrs-b supplemented with glucose and yeast extract mrs-b supplemented with glucose, tryptone, and yeast extract c18 1 2 3 323 867 394 831 1061 750 537 353 226 337 c19 1 2 3 173 337 415 c29 1 2 3 173 293 517 d11 1 2 3 1815 2704 2575 142 d20 1 2 3 173 94 208 77 0 d44 1 2 3 100 113 265 245 3179 4663 3109 795 3250 1558 key: 1 = l.monocytogenes (fncc 0156); 2 = s.aureus (fncc 0047); 3 = e.coli (fncc 0091) = isolates showed no bacteriocin inhibitory activity against pathogenic bacteria 10 hartajanie et al. microbiol indones 2 -1 fig 1 effect of medium compositions on bacteriocin inhibitory activity (au mm ml ) of c and d isolates against l. monocytogenes (fncc 0156), s. aureus (fncc 0047), and e.coli (fncc 0091). fig 2 effect of medium compositions on bacteriocin d44 inhibitory activity test using agar-well diffusion method against l. monocytogenes (fncc 0156) (a), s. aureus (fncc 0047) and e. coli (fncc 0091) (c) ; negative control (-) mrs-b (1) mrs-b supplemented with 2% of glucose (2) mrs-b supplemented with 2% of glucose and 2% of tryptone (3) mrs-b supplemented with 2% of glucose and 2% of yeast extract (4) mrs-b supplemented with 2% of glucose, 1 % of tryptone and 1% of yeast extract (5). available during the incubation period. it has been proved that the composition of the growth medium is very important for the production of individual bacteriocins (todorov and dicks 2005b). yeast extract is a rich source of vitamin and provides excellent growth conditions for more microorganisms. it is probable that the yeast extract may take a part in deactivation of an inhibitor of bacteriocin synthesis (fukushima et al. 1983). it was observed that all bacteriocins had different inhibitory effect on listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091. surprisingly, the same lab species from different fermentation conditions had different bacteriocin inhibitory activity (table 1). some bacteriocins inhibited not only pathogenic volume 12, 2018 microbiol indones 11 bacteriocin against pathogenic bacteria* mrs-b (control) mrs-b supplemented with 2% of glucose and 2% of tryptone mrs-b supplemented with 2% of glucose, 1% of tryptone, and 1% of yeast extract mrs-b supplemented with 2% of glucose mrs-b supplemented with 2% of glucose and 2% of yeast extract key: *1 = l. monocytogenes (fncc 0156); 2 = s. aureus (fncc 0047); 3 = e. coli (fncc 0091) but also gram-positive and gram-negative in general. bacteriocins are ribosomally synthesized compounds produced by bacteria in order to inhibit the growth of other bacteria. bacteriocins have a narrow killing spectrum and thus they are generally able to kill only bacteria closely related to the producing strain (cleveland et al. 2001 in salminen et al. 2004). moreover, gram-negative bacteria was mostly resistant to the bacteriocins of lab. bacteriocin produced by lactococcus lactis kca2386 and plantaricin 35d produced by lactobacillus plantarum had inhibitory activity against gram-negative bacteria (ivanova et al. 2000). this is quite different from most of the bacteriocins which inhibit only closely related strains (hata et al. 2010). inhibitory effects of bacteriocin on tested bacteria vary (table 3). the differences between the inhibition zone against each pathogenic bacteria associated with the components of the cell structure. inhibition mechanism by antibacterial compounds is by destroying the bacterial cell wall. the response of lab in damaging the peptidoglycan component is stronger on neighbor cells (gram-positive bacteria). lipopolysaccharide (lps) layer on gram-negative bacteria cells can interfere the ability of bacteriocin to inhibit gram-negative pathogenic bacteria. some exceptions were found, bacteriocin of c19 and c29 had a broader inhibitory activity against escherichia coli fncc 0091 than staphylococcus aureus fncc 0047 and listeria monocytogenes fncc 0156. this exceptions could have occured because of original growth condition of isolates and bacteriocin diffusion on agar-media. in conclusion, carbon and nitrogen supplemention were able to induce the production of bacteriocin in some lab. bacteriocin of lab from pickled yellow betung bamboo shoots shows inhibition towards listeria monocytogenes fncc 0156, staphylococcus aureus fncc 0047, and escherichia coli fncc 0091. acknowledgment this work was supported by a grant from directorate general of indonesian higher education. this research a part of the second year of penelitian unggulan perguruan tinggi (pupt) 2016/2017 entitled “efek probiotik dan mikrostatik dari bakteri asam laktat yang berperan dalam fermentasi acar rebung”, grant number 011/k6/km/sp2h/riset terapan/2016. references aasen im, møretrø t, katla t, axelsson l, storro i. 2000. influence of complex nutrients, temperature and ph on bacteriocins production by lactobacillcus sakei ccug 42687. j appl microbiol biotechnol. 53(2):159-166. doi: 10.1007/s002530050003. nd atlas rm. 2006. 2 handbook of microbiological media for the examination of food. taylor and francis group. chen h, hoover dg. 2003. bacteriocins and their food applications. comp rev food sci food safety. 2:82–100. doi: 10.1111/j.1541-4337.2003.tb00016.x. chouwdhury d, sahu jk, sharma gd. 2012. bamboo shoot: microbiology, biochemistry and technology of fermentation. a review. indian j tradit know. 11(2):242-249. cleveland j, montville tj, nes if, chikindas l. 2001. bacteriocins: safe, 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todorov sd, dicks lmt. 2005a. growth parameters influencing the production of lactobacillus rhamnosus bacteriocins st461bz and st462bz. j microbiol. 55(4):283-289. todorov sd, dicks lmt. 2005b. effect of growth medium on bacteriocin production by lactobacillus plantarum st194bz, a strain isolated from boza. food tech biotechnol j. 43(2):165-173. www.biomerieux-usa.com. (accessed on february 24 2016; 08:00 p.m.). www.fda.gov/food/scienceresearch/laboratorymetho ds/bacteriologicalanalyticalmanualbam/default.h tm. (accessed on january 22 2016; 12:58 p.m.). zhou f, zhao h, bai f, dziugan p, liu y, zhang b. 2014. purification and characterization of the bacteriocin produced by lactobacillus plantarum, isolated from chinese pickle. j food sci. 32(5):430-436. 14 hartajanie et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 05 saskiawan.cdr cellulose is the most abundant organic polymer and represent about 50% of the cell wall material of 12 plants. it represents about 1.5 x 10 tons of the total annual biomass production through photosynthesis process especially in the tropical area. it is considered to be an almost inexhaustible source of raw material from different products such as forest products, agriculture, and food processing (sukumaran et al. 2005; hussain et al. 2012). a promising technique for efficient utilization of cellulose resources is the microbial hydrolysis of cellulose material to produce a smaller sugar component. cellulases are a group of hydrolytic enzymes which has ability to hydrolyze cellulose. many microorganisms are capable of producing extra-cellular cellulase. important among these are the filamentous fungi which digest cellulosic wastes rapidly through their high metabolic rates. the biological degradation of cellulose has been studied for many years, and a number of cellulolytic enzymes, especially cellulases produced by fungi and bacteria have been isolated and characterized (yin et al. 2005; bakare et al. 2005; khokhar et al. 2012). bioconversion of cellulosic material to produce edible mushroom can play an important role in managing organic waste. in indonesia, paddy straw mushroom (volvariella volvacea) is one of the important edible mushrooms which is cultivated in paddy straw (chang and miles 1997). the aim of the study was to conduct screening, purification and characterization of cellulase from fungus which was isolated from used paddy straw mushroom substrate. vol.9, no.4, december 2015, p 171-177 doi: 10.5454/mi.9.4.5 screening, purification, and characterization of cellulase from fungi isolated from used mushroom substrate iwan saskiawan* and nur hasanah research center for biology, indonesian institute of sciences (lipi), jalan raya jakarta bogor km. 46 cibinong 16911, indonesia a large number of microorganism especially filamentous fungi has ability to degrade cellulose. the purpose of this study was to conduct screening, purification, and characterization of cellulase from fungi which was isolated from used paddy straw mushroom substrate. screening of cellulytic activity using cmc medium shown that 11 out of 20 isolates of fungi produced a clearing zone surrounding fungal colony. among them isolate number jmf 12 showed the highest cellulase activity and was further used for purification and characterization. the cellulase was purified to electrophoretical homogeneity by ammonium sulfate precipitation, dialyzed by novagen d-maxi tubetm dialyzer, and sephadex g-100 gel filtration chromatography. the recovery and purification fold was 3.82 % and 1.98 respectively, after sephadex g-100 gel filtration chromatograph. the o purified cellulase had an optimal ph and temperature at 6 and 45 c. the k and v of cellulase was 11.43 mm m max -1 + ++ ++ and 0.006 µmol min respectively. the purified cellulase was activated by na and zn but inhibited by ca , ++ ++ ++ co , fe , and hg . key words: cellulase, used mushroom substrate beberapa jenis mikroba terutama jamur tingkat rendah mempunyai aktivitas selulase yang cukup tinggi. penelitian ini bertujuan untuk melakukan seleksi beberapa isolat jamur yang mempunyai aktivitas selulase. isolat jamur tersebut diisolasi dari limbah media budidaya jamur merang. selanjutnya juga dilakukan pemurnian dan pengungkapan karakter enzim selulase dari isolat terpilih. hasilnya menunjukkan bahwa isolat dengan kode jmf 12 mempunyai aktivitas selulase paling tinggi diantara 20 isolat lainnya. enzim tersebut kemudian dimurnikan dengan metode pengendapan amonium sulfat, didialisis dengan novagen d-maxi tube tm dialyzes dan sephadex g-100 gel chromatography. aktivitas selulase mengalami recovery dan peningkatan aktivitas sebanyak 3.82% dan 1,98 setelah dimurnikan dengan gel kromatografi. selulase yang diperoleh mempunyai aktivitas optimal pada o -1 ph 6 dan suhu 45 c. nilai k dan v berturut turut adalah 11.43 mm dan 0.006 µmol min . aktivitas selulase m max + ++ ++ ++ ++ ++ meningkat terhadap pengaruh na dan zn dan menurun terhadap pengaruh ca , co , fe dan hg . kata kunci: limbah media tanam jamur merang, pemurnian enzim, selulase *corresponding author; phone: +62-21-8765066, fax: +6221-8765062 ; email: iwansaskiawan@gmail.com available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia materials and methods microorganisms. 20 isolates of fungi isolated from used mushroom substrate of rice straw mushroom (volvariella volvaceae) in indramayu, indonesia was deposited on indonesian culture collection (ina cc), research center for biology, indonesian institute of sciences. isolates was cultured on potato dextrose agar (pda) medium. screening of cellulolytic activity. cellulolytic activity of the fungal isolates was determined by using plate screening medium of carboxy methyl cellulose -1 -1 (cmc). the medium contained 0.4 g l cmc, 0.5 g l -1 -1 mgso .7h o, 0.03 g l kno , 1.0 g l k hpo , 4 2 3 2 4 -1 -1 0.0008 g l , feso .7h o, 0.08 g l yeast extract, 2 g l4 2 -1 -1 nano and 18 g l agar. the 5 mm in diameter of agar 3, blocks from one-week old fungal colony grown on pda plates were inoculated in the center of the cmc media plates. the plates were incubated at room temperature for 2 d. cellulolytic activity was observed by clearing zone diameter surrounding the fungal colonies. observation was done by staining the fungal colony with 0.1% congo red dye, followed by destaining with 1 % nacl solution. cellulase activity on medium cmc was determined by the index of relative enzyme activity (icmc). it was recorded as the ratio of clearing zone diameter to the diameter of colony (khokhar et al. 2012). cellulase activity assay. the cellulase activity assay was done by using cmc as a substrate. the crude enzyme solution (125 µl) was incubated with 875 ml of 0.5 % cmc (w/v) in 0.5 mm sodium phosphate buffer (ph 6.0) at 37 °c for appropriate time. after incubation, the enzymatic reaction was added with 1 ml of dns (3,5di nitro salicylic acid) and the reaction was terminated by boiling in water bath for 5 min. subsequently, the sample was withdrawn and the absorbance was measured at 540 nm (miller 1959) using a shimadzu 1700 spectrophotometer. glucose -1 was used at a concentration of 0.10 – 0.25 mg ml in accordance with the standard curved for cellulase activity measurement. one unit of the enzyme activity was defined as the amount of enzyme which released -1 1mmol of glucose per minute (u ml ). determination of protein concentration. the protein concentration of the crude enzyme as well as that of the purified one was determined by bradford method (bradford 1976) using bovine serum albumin (bsa) as protein standard. purification of cellulose. based on the screening of cellulolytic activity, fungi with the highest cellulase activity was grown at a temperature of 37 °c for 4 d on rotary shaker (120 rpm) in a 500 ml erlenmeyer flask containing 300 ml of medium potato dextrose broth (pdb). the broth, then was centrifuged at 9500 rpm for 30 min and passed through whatman filter paper grade 42 to remove cells. the crude cellulase was precipitated by 10-80% saturation of ammonium sulfate. the precipitate was centrifuged at 9500 rpm for 30 min and dialyzed (novagen d-maxi tubetm dialyzer, mwo 6-8 kda) against 10 mm phosphate buffer (ph 6.5) overnight. the resulted crude enzymes were eluted by sephadex g-100 gel filtration chromatography (2.0 x 30.0 cm) with 10 mm -1 phosphate buffer (ph 6.5) at flow rate 1.0 ml min . fractions with cellulase activity were collected and used for the characterization and determination of kinetic parameters. determination of kinetic parameter. the apparent kinetic parameters (v and k ) of the max m cellu las e w er e d eter min ed b y v ar y in g th e concentration of carboxy methyl cellulose (cmc) from 0.05 to 0.30% in 0.5 mm sodium phosphate buffer (ph 6.5). the apparent kinetic parameters were determined from double reciprocal plots (lineweaver and burk 1934). effect of ph, temperature, and cations on cellulase activity. in order to reveal the optimum ph value for purified cellulase, the activity of the enzyme was assayed on various ph values from 4.5 to 7.5. furthermore, the activity was also determined by incubated the enzyme with substrates at different temperatures ranging from 25 to 55 °c. the effect of cations on cellulase activity was determined by adding ++ + ++ ++, ++ ++ cations: ca , na , co , fe zn , and hg at concentration 10 mm on the reaction mixture. all of cellulase activity assay was carried out by mean of dns assay. results screening of cellulolytic activity. clearing zone surrounding fungal colonies on the plate screening medium indicated that the fungus has cellulase activity (fig 1). among 20 fungal isolates, there were 11 isolates shown clearing zones. determination of indexs of relative enzyme activity on cmc medium (icmc) was shown in figure 2. it showed that fungal isolate jmf 11 had the highest icmc of 0.66, following by jmf 12 and jmf 6 with the same value of 0.50. furthermore, the fungi of jmf 11, jmf12, and 172 saskiawan et al. microbiol indones jmf 6 were grown on pdb medium to produce crude cellulase. their activity was determined using cmc as a substrate. the results showed that isolate of jmf 12 -1 had the highest activity of 2.78 u mg (fig 3). for that reasons, fungal isolate jmf 12 was used in this study for production of cellulase. purification of fungal cellulose. the results of purification of cellulase produced by the fungi jmf 12 were summarized in table 1. ammonium sulphate precipitation gave purification fold of 1.32. furthermore, the crude enzyme was dialyzed using novagen d-maxi tube tm dialyzer and the purification 1.76 fold was achieved. the dialyzed enzyme was subjected to sephadex g-100 gel filtration chromatography and achieved the purification fold to 1.98. figure 5 shows the elution profiles (chromatogram) of cellulase from jmf 12. a large protein molecules of cellulase was eluted in a fraction collector no 2 and obtained a single peak at fraction collector no 3. however, a small peak of protein also appeared at fraction collector no. 8. the pattern of total protein in that chromatogram was also similar to that of cellulase activity. determination of kinetic parameter. the determination of michaelis-menten constants, k and m v of the purified cellulase for carboxy methyl max cellulose (cmc) were calculated from the double reciprocal plot of the data obtained for cellulase activity at various substrate concentrations. the k and m -1 v was 11.43 mm and 0.006µmol min respectivelymax characterization of cellulase activity. the optimum ph for cellulase activity of purified enzyme produced by fungi jmf 12 was at ph 6.0. it was 3.39 -1 -1 unit ml . the lowest activity of 0.77 unit ml was at ph 7.5 (fig 4). on the other hand, the enzyme activity o from fungi jmf 12 was optimum at 45 c of 3.74 unit -1 o ml and at 25 c the enzyme was shown the lowest -1 activity of 2.75 unit ml (fig 5). furthermore, as shown in figure 6, addition of some cations to the reaction mixture affected the cellulase activity. the + ++ results showed that addition of cations na and zn increased cellulase activity of 4.97% and 27.18% ++ ++ ++ respectively. the addition of cations ca , co , fe , ++ and hg decreased of the enzyme activity from 2.95 to 23.41%. discussion m o s t a g r i c u l t u r a l r e s i d u e s a r e r i c h i n lignocellulosic compounds whose handling and disposal are often problematic, because of their chemical structure and decomposition properties (philippoussis et al. 2001). mushrooms are well known for their delicacy, nutritional and economical values but the substrate released after mushroom crop harvest, better known as “used mushroom substrate” is also the subject of great importance. the substrate in mushrooms cultivation is a mixture of cellulosic waste such as agricultural, poultry, or industrial waste and ca b fig 1 clearing zone surrounding fungal colonies of jmf 6 (a), jmf 11(b), and jmf 12(c). table 1 summary of the purification of cellulase fom jmf 12 cellulase activity -1 (u ml ) total protein (mg) specific activity -1 (u mg ) 45.56 7.64 5.74 1.47 2.81 2.09 100.00 22.93 22.18 purification fold purification steps culture filtrate ammonium sulphate precipitation dialysis novagen d-maxi tubetm dialyzer yield (%) 1.00 total volume (ml) 180.00 21.60 28.08 total activity (unit) 264.60 60.69 58.68 1.32 1.76 0.88 0.24 3.82sephadex g-100 42.12 10.11 1.98 5.81 7.94 10.22 11.50 volume 9, 2015 microbiol indones 173 fig 4 elution profile of partially purified cellulase from jmf 12 on sephadex g-100 gel filtration -1 -1 chromatography (●= cellulase activity,u ml ; ○�= protein total, mg ml ). fig 3 the cellulase activity of fungal isolates jmf 6, jmf 11 and jmf 12 grown on pdb medium. fig 2 the index of relative enzyme activity on cmc medium icmc of twenty numbers of fungal isolates. 174 saskiawan et al. microbiol indones -1 s p ec if ic a ct iv it y ( u m g ) fungal isolates -1 s p ec if ic a ct iv it y ( u m g ) -1 s p ec if ic a ct iv it y ( u m g ) -1 a ct iv it y ( u m l ) fraction no -1 s p ec if ic a ct iv it y ( u m g ) -1 t o ta l p ro te in ( m g m l ) prepared by controlled fermentation process. the used substrate obtained after crop harvest have all essential character of organic manure. however, to obtain more, appropriate condition, the used substrate still needs to be recomposted. the recomposting processes of used m u s h r o o m s u b s t r a t e r e q u i r e s c e l l u l o l y t i c microorganisms to enhance hydrolysis of the cellulose. in this study, among 20 fungal isolates from used straw mushrooms substrate, 11 fungal isolates were identified having cellulolytic activity. they have been fig 5 effect of ph on the activity of cellulase from jmf 12. fig 6 effect of temperature on the activity of cellulase from jmf 12. fig 7 effect of the addition of cations on the relative activity of cellulase from jmf 12. volume 9, 2015 microbiol indones 175 -1 s p ec if ic a ct iv it y ( u m g )-1 c el lu la se a ct iv it y ( u m l ) -1 s p ec if ic a ct iv it y ( u m g )-1 c el lu la se a ct iv it y ( u m l ) -1 s p ec if ic a ct iv it y ( u m g ) r el at iv e a ct iv it y ( % ) metal ion temperature (°c) ph shown by the clearing zones on the cmc medium where they were grown. the detection of cellulolytic activity using cmc medium is reliable for rapid screening of fungal cellulase (teather and wood 1982; bradner et al. 1999; khokhar et al. 2012). determination of cellulase activity of crude enzyme produced by 11 fungal isolates using cmc as a substrate showed that isolates number jmf 12 had the highest activity and it was used for the purfication and characterization of cellulase. fungi are well known agents of decomposition of organic matter in common and of cellulosic substrate in particular (lynd et al. 2002). the supernatant of jmf 12 with cellulase activity -1 -1 of 1.47 u ml and specific activity of 5.81 u mg was used as crude enzyme solution and subjected to partial purification by ammonium sulfate precipitation in 10-1 80% saturation. the cellulase activity of 2.81 u ml , -1 specific activity of 7.94 u mg , yield 22.93 unit and purification fold of 1.32 was achieved on ammonium sulfate precipitation of 30% saturation (data not shown). ammonium sulfate is the most common inorganic salts that can be utilized for the precipitation of proteins. the concentration of ammonium sulfate required for precipitation varies from protein to protein and should be determined empirically (deutscher and murray 1990). dialysis using novagen d-maxi tube tm dialyzer was applied after ammonium sulfate precipitation. the precipitated protein of cellulase can be separated from small molecules by dialysis through a semipermeable membrane, such as a cellulose membrane with pores. molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis tube, whereas smaller molecules and ions traverse the pores of such a membrane and emerge in the dialysate outside the tube. this technique is useful for removing salts or other small size molecules, but it will not distinguish proteins effectively. the dialyzed fraction referred to as partially purified cellulase was then loaded on to sephadex g100 gel filtration column. by gel filtration the enzyme was purified to 1.98 fold with a yield and specific -1 activity of 3.82 % and 11.5 u mg respectively. the similar method for purification of cellulase from trichoderma viride was done by iqbal et al 2011. gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a column. on the other hand, small molecules such excess salts during ammonium sulfate precipitation can be easily separated. purification of cellulase from jmf 12 using sephadex g-100 gel filtration showed two single peak of enzyme activity as seen in figure 4. it happened because cellulase is a complex enzyme that are classified into three types such as endoglucanase, exoglucanase and bglucosidase. furthermore, the method of cellulase assay used in this study was not able to determine single types of complex cellulase specifically. the cellulase activity of jmf 12 appeared to depend on ph value. the result that was illustrated by figure 5 shows that cellulase activity gradually increase and the highest activity was reached -1 maximum at 3.40 u ml as the ph value of 6. it was also noted that the activity was decreased at ph value of 6.5 – 7.5. effect of ph on cellulase activity of jmf 12 supports the finding of okoye et al (2013) on cellulase activity of aspergillus fumigatus. furthermore, temperature is also an important factor that influences the cellulase activity. the temperature profile of the cellulase of jmf 12 showed an increase in activity as the temperature increased o from 25 – 40 c. the optimum activity was obtained at o -1 45 c of 3.72 u ml . temperature not only affects the activity of enzyme but also the stability of the enzyme. high temperature will change the protein structures leading to denaturation. many studies have been reported that the optimal temperature for cellulase production depended on the strain variation of the microorganism (murao et al 1988; lu et al 2003; sherief et al 2010; solomon et al 1997; wiseman 1995). addition of some cations to the reaction mixture affected the cellulase activity. the presence of these + ++ cations na and zn increased the enzyme activity of the jmf 12. on the other hand, the addition of cations ++ ++ ++ ++ ca , co , fe , and hg decreased the activity. the results partially corresponded to the study of metal ion effect on cellulase activity (wang et al 2012). another reasons of change in cellulase activity is that ions generally have a noticeable effect on the intensity of dns color and/or the reducing power of glucose which can affect the cellulase activity determined. so, in the colorimetric determination of cellulase activity in a salt treated solution the percentage inhibition may be related to negative effect of ions on the method of assessment (sinegani and emtiazi 2012). the study of cellulase activity of fungi isolated from used mushroom substrate was carried out and it has potential use in preparation of mushroom substrate during mushroom cultivation. however, further study 176 saskiawan et al. microbiol indones in identification of fungi jmf 12 is needed to explore its character. acknowledgment this research was supported by a research grant from indonesian government funded projects (dipa) 2014 of research center for biology, indonesians institute of sciences. references bakare mk, adewale io, ajayi a, shonukan. 2005. purification and characterization of cellulase form the wild-type and two improved mutants of pseudomonas fluorescens. afr j biotechnol 4 (9): 898-904. doi: 10.5897/ajb2005.000-3178. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein using in utilizing the principle of protein-dye binding. anal biochem 72: 248-254. doi:10.1016/0003-2697(76) 90527-3. bradner jr, gillings m, nevalainen kmh. 1999. qualitative assessment of hydrolytic activities in atartic microfungi grown at different temperatures on solid media. world j microbiol biotechnol 15, 131–132. doi: 10.1023/a:1008855406319. chang st and miles g. 1997. mushroom biology. world scientific 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scale solid state fermentation by aspergillus sulphurous. enzyme microbiol technol. 32: 305-311. doi: 10.1016/s0141-0229(02)00292-2. lynd lr, weimer pj, van zyl wh, pretorius is. 2002. microbial cellulose utilization, fundamentals and biotechnology. microbiol mol biolo reviews. 66: 506577. doi: 10.1128/mmbr.66.3.506-577.2002. miller gl. 1959. use of dinitrosalicycylic acid reagent for determination of reducing sugar. anal chem 31( 3): 426-428. doi: 10.1021/ac60147a030. murao s, sakamoto r, arai m. 1988. cellulase of aspergillus aculeatus. in: methods in enzymology, academic press inc., london. 275-284. okoye ig, ezugwu al, udenwobele di, eze soo, anyawu cu, chilaka fc. 2013. production and partial characterization of cellulases from aspergillus fumigates using two distinct parts of corn cob as carbon sources. nig j biotech. 26 : 50-59 . philippoussis a, zervakis g, diamantopoulou p. 2001. bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms agrocybe aegerita, volvariella volvacea, and pleurotus spp. world j microb biotech. 17: 191-200. doi: 10.1023/ a:1016685530312. sherief a, el-tanash ab, atia n. 2010. cellulase production by aspergillus fumigatus grown on mixed substrate of rice straw and wheat bran. research j. microbiol. 5(3): 199-211. doi: 10.3923/jm.2010.199.211. sinegani aas, emtiazi g. 2006. the relative effects of some elements on the dns method in cellulase assay. j. appl sci environ mgt. 10(3): 93-96. doi: 10.4314/ jasem. v10i3.17326. solomon o, amigun b, betiku e, ojumu tv, layokun sk. 1997. optimization of cellulase production by aspergillus flavus linn isolates nspr 101 grown on bagass. j nigerian society chem engineering. 16: 6168. sukumaran rk, rani r, pandey a. 2005. microbial cellulases production, applications, and challenges. j sci ind res 64: 832-844. teather rm, wood pj. 1982. use of congo redpolysacharide interactions in enumeration and characterization of cellulolytic bacteria in the bovine rumen. appl environm microbiol. 43: 777-780. wang g, zhang x, wang l, wang k, peng f, wang l. 2012. the activity and kinetic properties of cellulases in substrates containing metal ions and acid radicals. adv biol chem. 2: 390-395. doi: 10.4236/abc.2012.24048. wiseman a. 1995. the application of enzymes in industry. in: handbook of enzyme biotechnology 3rd edition. yin lj, lin hh, xiao zr. 2010. purification and characterization of a cellulase from bacillus subtilis yj1. j mar sci tech taiw 18(3): 466-471. volume 9, 2015 microbiol indones 177 page 1 page 2 page 3 page 4 page 5 page 6 page 7 04 testamenti.cdr vol.12, no.1, march 2018, p 23-29 doi: 10.5454/mi.12.1.4 detection of antibody to burkholderia pseudomallei in captive and wild macaques in west java and bali, indonesia 1 1,2 3 vincentius arca testamenti , diah iskandriati , aris tri wahyudi , 1,2,4* and joko pamungkas 1 primatology study program, graduate school, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; 2 primate research center, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; 3 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, jalan meranti, kampus ipb darmaga, bogor 16680, indonesia; 4 department of animal diseases and veterinary public health, faculty of veterinary medicine, institut pertanian bogor, jalan agatis, kampus ipb darmaga, bogor 16680, indonesia. melioidosis is an emerging zoonotic disease caused by the saprophytic burkholderia pseudomallei, which infects human and a wide range of animal species. melioidosis may lead to septicemia and pneumonia in human patients, which can be fatal if the patient is not treated accordingly. the disease is spread in tropical areas and is highly endemic to southeast asia and northern australia. however, melioidosis is poorly reported in indonesia, especially in the veterinary field. this research provides serological evidence of antibodies to b. pseudomallei in both captive and wild nonhuman primates. plasma samples were taken from a total of 390 monkeys in captivities and wild habitats in west java and bali, indonesia. enzyme-linked immunosorbent assay (elisa) showed that the seroprevalence was 42.21% for macaca fascicularis and 43.59% for macaca nemestrina. furthermore, the seroprevalence was 53.41% for captive macaques and 17.83% for wild macaques. the findings showed that exposure to b. pseudomallei happened in both captive and wild macaques. based on this serosurveillance results, further studies such as comprehensive culture and clinical study are required to discover the clinical burden of the disease in nonhuman primates. key words: burkholderia pseudomallei, melioidosis, nonhuman primate melioidosis adalah penyakit zoonotik yang disebabkan oleh bakteri saprofit burkholderia pseudomallei, yang dapat menginfeksi manusia dan berbagai jenis hewan. melioidosis dapat berujung pada septikemia dan pneumonia yang berpotensi fatal jika pasien tidak ditangani dengan tepat. penyakit ini tersebar di daerah tropis, terutama di daerah asia tenggara dan bagian utara australia, namun sangat sedikit didokumentasikan di indonesia. penelitian ini memaparkan data temuan serologis berupa antibodi terhadap b. pseudomallei pada satwa primata, baik yang berasal dari penangkaran maupun habitat liar. spesimen plasma darah diambil dari total 390 satwa primata di penangkaran dan habitat liar di jawa barat, bali, dan labuan. hasil enzyme-linked immunosorbent assay (elisa) menunjukkan seroprevalensi sebesar 42,21% untuk macaca fascicularis dan 43,59% untuk macaca nemestrina. seroprevalensi pada monyet di penangkaran dan di habitat liar berturut-turut adalah sebesar 53,41% dan 17,83%. temuan ini mengindikasikan bahwa paparan terhadap b. pseudomallei terjadi baik pada monyet di penangkaran maupun di habitat liar. penelitian lebih lanjut yang melibatkan kultur dan studi klinis secara komprehensif perlu dilakukan untuk mengungkap kejadian penyakit melioidosis pada satwa primata. kata kunci: burkholderia pseudomallei, melioidosis, satwa primata microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8128433208, email: jpipssp@indo.net.id underreported in a few countries in southern america and africa. furthermore, there is a list of tropical and subtropical countries where melioidosis is predicted to be endemic, but cases have not been documented (limmathurotsakul et al. 2016). indonesia has been recognized as endemic area th since early of the 20 century. but for the past few decades, melioidosis cases remain underreported in the country (tauran et al. 2015). recent case reports in human medicine include cases of four tsunami survivors in banda aceh, sumatra (athan et al. 2005), melioidosis is a potentially fatal disease caused by gram-negative bacilli, burkholderia pseudomallei, which can be found in water and soil in endemic areas (cheng and currie 2005). the disease spreads in tropical areas, and is highly endemic in southeast asia and northern australia (cheng and currie 2005). the latest global assessment by limmathurotsakul et al. (2016) indicates that melioidosis is also endemic but and three cases from a hospital in makassar, sulawesi (tauran et al. 2015). however, in a retrospective study focusing on unpublished culture-confirmed melioidosis cases, 45 unpublished cases have been identified from hospitals in jakarta, banda aceh, medan, banjarmasin, bandung, samarinda, makassar, kupang, and pekanbaru (tauran et al. 2018). reports of melioidosis in veterinary medicine in indonesia are limited, including cases in 3 cynomolgus monkeys imported to the britain (dance et al. 1992), 1 pig-tailed monkey imported to the us (ritter et al. 2013; johnson et al. 2013), 1 cynomolgus monkey in a primate research center (prabandari 2013), and 1 orangutan in a rehabilitation center (lestari 2017). melioidosis is an emerging threat to human and animal health because the disease causes considerable morbidity and mortality, and is often misdiagnosed. furthermore, the causative agent is classified as tier 1 select agent by the cdc. its persistence in the environment, high prevalence of severe sepsis, and three routes of infection are some of the key features contributing to the bioterrorism potential of b. pseudomallei (gilad et al. 2007). in a highly endemic area in thailand, the mortality rate of melioidosis was about 40% in all human patients, and even much higher (90%) in patients with severe sepsis (wiersinga et al. 2012). it is concerning that a disease with considerable case fatality rate and bioweapon potency is not yet classified as a notifiable disease in indonesia. in human medicine, melioidosis is often difficult to diagnose due to its non-characteristic, multisystemic lesions, including respiratory infection (meumann et al. 2012), genitourinary infection (morse et al. 2009), skin and soft tissue infection (gibney et al. 2008), bacteremia, neurologic lesions (saipan 1998), and bone and joint disease (morse et al. 2013). the challenge is even greater in veterinary medicine since clinical manifestations of melioidosis may differ from one animal species to another. furthermore, even if a physician or veterinarian is suspecting melioidosis based on a nonspecific presentation, collection of the right samples is problematic. in healthcare services where the awareness is low, melioidosis is very likely to be misdiagnosed and misidentified. that is particularly true in healthcare services where lesion or blood culture is not mandatory before treating bacterial infection. in order to increase the awareness about melioidosis, information regarding the disease prevalence, morbidity and mortality rate, best diagnostic techniques, as well as pathogen characteristics is needed. increased awareness would lead to improvement in diagnostic capabilities and will support healthcare professionals to perform early identification and proper treatment. those data will also contribute in showing the magnitude of the disease in indonesia. this paper describes the first milestone in revealing melioidosis cases in veterinary medicine in indonesia through a serosurveillance. this assessment will reveal whether captive and wild macaques in west java and bali have been exposed to b. pseudomallei. all reported veterinary cases of melioidosis in indonesia were from nonhuman primates (nhps), a fact that amplifies the importance of melioidosis surveillance in nhps. furthermore, nhps are beneficial to study as melioidosis in nhps share many similar clinical manifestations and epidemiological features with human melioidosis. materials and methods sample collection. sample collection in captive macaques was performed from january to april 2016. captive macaques in this study were 159 macaca fascicularis and 101 macaca nemestrina from both sexes and various ages in breeding facilities in west java (table 1). samples from wild macaques include 52 plasma samples from tinjil island, west java, and 78 archived plasma samples taken from monkeys in various locations in bali island and labuan island (table 1). all procedures in animals were carried out using protocol reviewed and approved by the institutional animal care and use committee, primate research center, bogor agricultural university, number acuc ipb prc-15-b0011. all animals were -1 anesthetized using ketamine (10 mg kg body weight) prior to physical examination. heart rate, respiratory rate, body temperature, skin turgor and any abnormality (if presented) were examined and recorded. approximately 3 ml of blood were taken from the femoral vein of each animal. elisa. elisa was conducted in microbiology and immunology laboratory, ipb primate research center. plasma separation was performed in bsl-2 in biosafety cabinet class ii type a/b3 (nuaire nu 407600). elisa assay was performed using plates coated with b. pseudomallei lipopolysaccharide, which were kindly provided by the university of florida. 300 μl of blocking buffer (5% blotto) was added to each well, and the plates were incubated at room temperature for 2 h. plates were washed 4 times with wash buffer (pbs with 24 testamenti et al. microbiol indones 0.05% tween-20). the heat-inactivated plasma were diluted at 1:200 in blocking buffer and then added to elisa plates. the plates were incubated at 37 °c for 1 h, and then washed 4 times with wash buffer. 100 μl of secondary antibody (goat anti-monkey horseradish peroxidase conjugate, diluted at 1:1000 in blocking buffer) was added into each well, and then the plates were incubated at 37 °c for 1 h. after washing the plates with wash buffer, 3,3',5,5'-tetramethylbenzidine (tmb) solution was added into each well. plates were incubated at room temperature for 15-20 min in dark area. the reaction was stopped by adding 50 μl of 1n sulphuric acid. the color development was measured using elisa plate reader at 450 nm measurement wavelength and 595 nm reference wavelength. based on the results of the first plate, two samples were used as positive controls, and two other samples were used as negative controls for the next plates. the positive controls were plasma samples that show similar antibody levels to those of aerosol-induced macaques, whereas the negative controls were plasma samples that show a similar antibody levels to those of noninfected macaques (michael norris, personal communication). cutoff value was determined for each plate according to the method by frey et al. (1998) as follows: where x = mean of optical densities (ods) of negative controls, sd = standard deviation of ods of negative controls, n = number of negative controls, t = student's t distribution value with p = 0.0001 and degree of freedom = n – 1 results seroprevalence to b. pseudomallei in macaques from various locations is shown in table 2. a remarkably high seropositivity was detected from cynomolgus monkey in darmaga facility that recently arrived from tinjil island in west java. monkeys from several locations in west java showed 48-70% positivity, whereas monkeys from various places in bali island and labuan island showed relatively low positivity (0-63%). the seroprevalence was 42.21% for all macaca fascicularis and 43.59% for all macaca nemestrina. furthermore, the seroprevalence was 53.41% for captive macaques and 17.83% for wild macaques. some of the seropositive monkeys showed antibody levels similar to those of aerosol-challenged macaques at 14 and 21 days post-infection (michael norris, personal communication). this may indicate that the macaques are potentially infected or have been exposed to b. pseudomallei. discussion this paper is the first report describing seroprevalence of b. pseudomallei infection in nonhuman primates in indonesia. the seropositivity was considerably high, and some animals had antibody levels similar to those of aerosol-induced macaques (michael norris, personal communication). however, seropositive animals in this study did not show any significant clinical manifestations associated with melioidosis. it suggests that exposure to b. pseudomallei does not necessarily cause melioidosis. the seropositivity observed in this study may reflect asymptomatic infection, or reflect exposure that happened in the past. the high seroprevalence observed in many sampling locations also indicates that exposure to the bacteria in the environment happen easily. the macaques from several locations in west java (tinjil island, jonggol, darmaga, and lodaya) showed considerably higher seroprevalence compared to the macaques from bali island, indonesia and labuan island, malaysia. the only exception is the karangasem area in bali where 7 of 11 animals were seropositive. this is in line with current reports of melioidosis in the region. to date, melioidosis cases have never reported in bali and labuan. a review by tauran et al. (2015) showed that melioidosis cases have only been officially reported in west java, aceh, south sulawesi, and central sulawesi. however, a more recent retrospective study has discovered that there were 45 unpublished cases from hospitals in jakarta, banda aceh, medan, banjarmasin, bandung, samarinda, makassar, kupang, and pekanbaru (tauran et al. 2018). this amplifies a hypothesis that melioidosis is underreported in indonesia. current reports are predicted to be only the tip of an iceberg and does not reflect the actual national burden. exposure to b. pseudomallei may occur through inoculation, ingestion, and inhalation. the high prevalence in several sampling locations is presumably related to inoculation from soil and ingestion of contaminated, non-chlorinated ground water. soil and volume 12, 2018 microbiol indones 25 water have been proven to be important in melioidosis acquisition (cheng and currie 2005). however, b. pseudomallei cannot survive exposure of uv light (tong et al. 1996), and therefore is unlikely to present in the surface layer of the soil. higher incidence of melioidosis has been reported in association with the rainy season (choy et al. 2000). contact between macaques and the bacteria most likely happen after rainwater exposed the deeper layer of the soil. water supplies in several captivities in this study are untreated and may also contribute to the exposure. drinking untreated (non-chlorinated) water is related to increased risk of acquiring melioidosis through ingestion route in human (limmathurotsakul et al. 2013). even though the mode of exposure in these animals has not been elucidated yet, preventive measures should be taken into consideration. preventive measures in semi-outdoor nhp captivity may be implemented by refining housing design and chlorinating the water supply. efforts to minimalize contact with contaminated soil may include replacing soil floor with corals, building more perches, and designing paved floor in the shelter area. this preventive measure would reduce the exposure to the bacteria, especially in rainy season when the incidence is usually higher (kasantikul et al. 2015, choy et al. 2000). water chlorination is also important to reduce the exposure from a contaminated water source. immunoassay is not the primary method in diagnosing melioidosis, but is very useful in p r e l i m i n a r y a s s e s s m e n t o f e x p o s u r e t o b . pseudomallei. furthermore, the whole process of culture and microbiological identification of b. pseudomallei from clinical samples may take several days. less time-consuming and less labor-intensive table 1 number of animals from each sampling location sampling location habitat number of animals m. fascicularis m. nemestrina jonggol, west java* captivity 149 0 darmaga, west java* captivity 77 lodaya, west java* captivity 10 24 tinjil island, west java* wild 52 alas kedaton, bali wild 12 karangasem, bali wild 11 sangeh, bali wild 4 tabanan, bali wild 17 teluk terima, bali wild 5 ubud, bali wild 9 uluwatu, bali wild 10 labuan island wild 10 1 *breeding facilities species sampling location type of habitat total number of animals seropositive seropositivity (%) m. fascicularis jonggol captivity 149 72 48.32 lodaya captivity 10 7 70.00 tinjil* w ild 52 32 61.54 teluk terima w ild 5 0 0.00 alas kedaton w ild 12 0 0.00 uluwatu wild 6 0 0.00 sangeh w ild 8 1 12.50 karangasem w ild 11 7 63.64 ubud w ild 9 1 11.11 tabanan w ild 17 2 11.76 pulau labuan w ild 10 0 0.00 subtotal 289 122 42.21 m. nemestrina darmaga captivity 77 32 45.33 lodaya captivity 24 12 50.00 subtotal 101 48 47.52 total 390 170 43.59 1 table 2 seropositivity of antibody to b. pseudomallei lps in captive and wild macaques *sampling was performed in darmaga on animals that recently arrived from tinjil island 26 testamenti et al. microbiol indones immunoassays have been developed, including iha (harris et al. 2011), elisa (nualnoi et al. 2017; suttinsunhakul et al. 2016), and rapid latex agglutination test (suttinsunhakul et al. 2015). those assays rely on the detection of antibody to immunogenic components of b. pseudomallei, i n c l u d i n g l y p o p o l y s a c c h a r i d e s ( l p s ) , o polysaccharide part of lps (ops), and capsular polysaccharides (cps). the sensitivity and specificity of those assays varies. ops-elisa performed better with clinical samples from endemic areas, whereas ops-latex agglutination test performed better with c l i n i c a l s a m p l e s f r o m n o n e n d e m i c a r e a s (suttinsunhakul et al. 2016). the elisa plates used in this study were kindly provided by dr. apichai tuanyok in the university of florida, and utilized b. pseudomallei lps as the coating immunogen. this serosurveillance enabled us to discover whether the macaques have ever been exposed to b. pseudomallei, and to decide whether follow-up actions are necessary. for surveillance targeting actual presence of the bacteria, antigencapture immunoassays will be beneficial. a monoclonal antibody based immunoluorescent assay has been developed for rapid detection and it showed a very high sensitivity and specificity in detecting b. pseudomallei in blood culture (chantratita et al. 2013). culture remains to be the gold standard; b. pseudomallei grows on routine media such as blood agar and macconkey agar. however, the yield is significantly higher when samples are cultured on selective media such as ashdown agar and selective broth (wuthiekanun 1990). a quantitation study by wuthiekanun et al. (2007) showed that median b. pseudomallei counts in blood, urine, and respiratory -1 4 -1 secretion were 1.1 cfu ml , 1.5 x 10 cfu ml , and 5 -1 1.1 x 10 cfu ml , respectively. molecular assays in detecting melioidosis have also been developed, including conventional pcr targeting 23s rrna (shahin and dorsch 2003), sequencing of 16s rrna (gee et al. 2003), qpcr targeting type iii secretion system (novak et al. 2006), and loop-mediated isothermal amplification (chantratita et al. 2008). to date, qpcr of tts-1 is considered to be the best performing molecular identification of b. pseudomallei. even though zoonotic transmission of melioidosis is extremely rare, melioidosis study in nhp may contribute to human medicine in terms of epidemiology model, animal model development and vaccine development. inhalation of b. pseudomallei by african green monkey and rhesus monkey resulted in similar clinical manifestations (fever, leukocytosis, neutrophilia, bacteremia, and severe dyspnea) and pathological lesions (pneumonia, lymphadenitis, myelitis, and splenitis) to those of humans, suggesting that agm is a valuable model for melioidosis (yeager et al. 2012). a culture-confirmed melioidosis case in a cynomolgus monkey in ipb primate research center also showed similar clinical and pathological lesions to human, including difficulty in breathing due to severe bronchopneumonia, splenic pericapsular abscessation, and suppurative hepatitis (unpublished data). this research is the first milestone regarding b. pseudomallei infection in indonesian nonhuman primates. based on the results of this study, we ascertained that more studies focusing on m i c r o b i o l o g i c a l i d e n t i f i c a t i o n , m o l e c u l a r identification, and environmental aspect of melioidosis in indonesia are required. hopefully this study may encourage fellow veterinarians and scientists to discover the burden of melioidosis in indonesia. acknowledgement this research was funded by crdf global through biorisk management engagement grant to dr. diah iskandriati. the authors thank dr. apichai tuanyok and dr. michael norris in university of florida for kindly providing the elisa plates and for the advices given. references ashdown lr. 1979. an improved screening technique of pseudomonas pseudomallei from clinical specimens. pathology. 11(2):293-297. athan e, allworth am, engler c, bastian i, cheng ac. 2005. melioidosis in tsunami survivors. emerg infect dis. 11(10):1638-1639. doi: 10.3201/eid1110.050740. brown c. 2004. emerging zoonoses and pathogens of public health significance – an overview. rev sci tech off int epiz. 23(2): 435-442. 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[oie] office international des epizooties. 2015. glanders. in: oie terrestrial manual 2015. chapter 2.5.11. ritter jm, sanchez s, jones tl, zaki sr, drew p. 2013. 28 testamenti et al. microbiol indones neurologic melioidosis in an imported pigtail macaque (macaca nemestrina). vet pathol. 50(6):1139-1144. doi: 10.1177/0300985813485249. saipan, p. 1998. neurological manifestations of melioidosis in children. southeast asian j trop med public health. 29:856–859. shahin s and dorsch m. 2003. polymerase chain reaction for the rapid detection of burkholderia pseudomallei. cbrn defence centre platforms sciences laboratory dsto-tr-1509. suttinsunhakul v, chantratita n, wikraiphat c, w u t h i e k a n u n v, d o u g l a s z , d a y n p j , limmathurotsakul d, brett pj, burtnick mn. 2015. evaluation of polysaccharide-based latex agglutination assays for the rapid detection of antibodies to burkholderia pseudomallei. am j trop med hyg. 93(3):542-546. doi: 10.4269/ajtmh.15-0114. suttinsunhakul v, wuthiekanun v, brett pj, khusmith s, day npj, burtnick mn, limmathurotsakul d, chantratita n. 2016. development of rapid elisa for detection of antibodies to burkholderia pseudomallei. j c l i n m i c r o b i o l . 5 4 ( 5 ) : 1 2 5 9 1 2 6 8 . d o i : 10.1128/jcm.02856-15. tauran pm, sennang n, rusli b, wiersinga wj, dance d, arif m, limmathurotsakul d. 2015. emergence of melioidosis in indonesia. am j trop med hyg. 93(6):1160-1163. doi: 10.4269/ajtmh.15-0292. tauran pm, wahyunie s, saad f, dahesihdewi a, graciella m, muhammad m, lestari dc, aryati a, parwati i, loho t, et al. 2018. emergence of melioidosis in indonesia and today's challenges. trop med inf dis. 3(1):32. doi: 0.3390/tropicalmed3010032. tong s, yang s, lu z, he w. 1996. laboratory investigation of ecological factors influencing the environmental presence of burkholderia pseudomallei. microbiol immunol. 40:451–453. [who] world health organization. 2010. report of the first meeting of the leptospirosis burden epidemiology r e f e r e n c e g r o u p [ i n t e r n e t ] . av a i l a b l e a t : http://whqlibdoc.who.int/publications/2010/97892415 99894_eng.pdf. ______. 2015. dengue and severe dengue [internet]. available at: http://www.who.int/mediacentre/ factsheets/fs117/en/. wu t h i e k a n u n v, d a n c e d a b , wa t t a n a g o o n y, supputtamongkol y, chaowagul w, white nj. 1990. the use of selective media for the isolation of pseudomonas pseudomallei in clinical practice. j med microbiol. 33:121-126. wuthiekanun v, limmathurotsakul d, wongsuvan g, chierakul w, teerawattanasook n, teparrukkul p, day np, peacock sj. 2007. short report: quantitation of b. pseudomallei in clinical samples. am j trop med hyg. 77(5):812-813. yabuuchi e, kosako y, oyaizu h, yano i, hotta h, hashimoto y, ezaki t, arakawa m. 1992. proposal of the burkholderia gen. nov. and transfer of seven species of the genus pseudomonas homology group ii to the new genus, with the type species burkholderia cepacia (palleroni and holmes 1981) comb. nov. microbiol immunol. 36:1251-1275. wiersinga wj, currie bj, peacock sj. 2012. melioidosis. n eng j med. 367:1035-1044. doi: 10.1056/nejmra1204699. volume 12, 2018 microbiol indones 29 page 1 page 2 page 3 page 4 page 5 page 6 page 7 02. yanti.cdr vol.11, no.4, december 2017, p 117-122 doi: 10.5454/mi.11.4.2 identification and characterizations of potential indigenous endophytic bacteria which had ability to promote growth rate of tomato and biocontrol agents of ralstonia solanacearum and fusarium oxysporum fsp. solani * yulmira yanti , warnita, reflin, and munzir busniah faculty of agriculture, universitas andalas, padang 25163, west sumatera, indonesia among plant growh promoting rhizobacteria (pgpr) groups, endophytic bacteria considered as one of the options to control vascular wilt disease because of its ability to live and colonized internal roots of plants without causing any damages. our previous research had screened 9 isolates which had best ability to promote growth rate and increase yields of tomato and biocontrol agents of ralstonia solanacearum and fusarium oxysporum f.sp solani in planta condition. in order to know its abilities, those isolates need to be characterized. this research purposed to characterize those isolates abilities to produce indole-3-asetic acid (iaa), phosphate solubilizing, siderophore production, cyanide production, nh production, and ability to colonize endophytically and 3 identified the isolates using 16s rrna. result shown that all isolates can produce iaa, where tle1.1 produce highest iaa concentration (42.5 ppm). isolates e1ab1.3, tle 1.1 and tle2.2 can dissolved phosphate. none of the isolates produced hcn and nh . only tle 2.3 isolate can produce siderophore. all of 9 isolates were 3 identified using 16s rrna gene using 27f and 1492r primers. all isolates were identified as different species, i.e. bacillus toyonensis strain bct-7112 (epl1.1.3), serratia nematodiphila strain dz0503sbs1 (tle2.3), bacillus anthracis strain atcc 14578 (epl1.1.4), bacillus cereus atcc 14579 (tle1.1), bacillus cereus strain jcm 2152 (sne2.2), enterobacter cloacae subsp. dissolvens strain atcc 23373 (e1.ab1.2), serratia marcescens strain nbrc 102204 (e1ab2.1), klebsiella michiganensis strain w14 (tle2.2), and chryseobacterium rhizoplanae strain jm-534 (kle3.3). key words: 16s rrna, characterization, endophytes, pgpr diantara kelompok plant growth promoting rhizobacteria (pgpr), bakteri endofit dianggap sebagai salah satu pilihan untuk mengendalikan penyakit layu karena kemampuannya untuk hidup dan mengkolonisasi perakaran tanaman secara internal tanpa menimbulkan kerusakan. berdasarkan hasil penelitian sebelumnya, telah didapatkan 9 isolat yang memiliki kemampuan terbaik dalam memacu pertumbuhan dan hasil tanaman tomat serta sebagai agen biokontrol ralstonia solanacearum dan fusarium oxysporum f.sp solani pada kondisi in planta. untuk mengetahui kemampuan isolat-isolat tersebut perlu dlakukan karakterisasi. penelitian ini bertujuan untuk mengetahui kemampuan isolat untuk menghasilkan asam indol asetat (iaa), pelarut fosfat, produksi siderofor, produksi sianida, produksi nh , kemampuan untuk mengkolonisasi internal perakaran 3 (endofit) dan mengidentifikasinya dengan menggunakan 16s rrna. semua isolat dapat menghasilkan iaa, dan isolat tle1.1 menghasilkan iaa dengan konsentrasi tertinggi (42.5 ppm). isolat e1ab1.3, tle 1.1 dan tle2.2 mampu melarutkan fosfat. tidak terdapat isolat yang menghasilkan hcn dan nh . hanya isolat tle 2.3 yang 3 mampu menghasilkan siderofor. semua isolat diidentifikasi dengangen 16s rrna menggunakan primer 27f dan 1492r. semua isolat teridentifikasi sebgai spesies yang berbeda, yaitu bacillus toyonensis strain bct-7112 (epl1.1.3), serratia nematodiphila strain dz0503sbs1 (tle2.3), bacillus anthracis strain atcc 14578 (epl1.1.4), bacillus cereus atcc 14579 (tle1.1), bacillus cereus strain jcm 2152 (sne2.2), enterobacter cloacae subsp. dilolvens strain atcc 23373 (e1.ab1.2), serratia marcescens strain nbrc 102204 (e1ab2.1), klebsiella michiganensis strain w14 (tle2.2), dan chryseobacterium rhizoplanae strain jm-534 (kle3.3). kata kunci: 16s rrna, endofit, karakterisasi, pgpr microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-812-6710-676; email: mira23@agr.unand.ac.id exert its beneficial effects (santoyo et al. 2016). they can contribute an important role in agriculture by conferring advantages for plant (mengoni et al. 2003). many studies had demonstrated that endophytic bacteria can produce beneficial effects on host plants, such as growth promoting substances, or prevent the negative impact caused by pathogens (bulgari et al. 2009). as plant growth-promoting bacteria (pgpb), endophytic bacteria can also enhance plant growth by activating a number of similar mechanisms, including endophytic bacteria are groups of bacteria that capable to live within the inner tissues of healthy plants without causing any disease symptoms (golinska, et al. 2015). endophytic bacteria had advantage over any rhizospheric inhabiting bacteria, because this bacteria can live within tissue plants, had opportunity to always contact with plant cells and have a better chance to indole-3-acetic acid (iaa) production, phosphate solubilization, siderophores production and nitrogen fixation (lugtenberg et al. 2013). from previous research, we had screened 9 potential endophytic bacteria which had ability to promote growth of tomato and control both ralstonia solanacearum and fusarium oxysporum fsp. lycopersici in in planta conditions (yanti et al. 2017). although their ability in in planta conditions had been shown good result, their pgpr traits must be characterized and identified to know its mechanisms to control pathogens and promote growth of tomato. this research purposed to identify isolates and characterized the ability of the 9 potential endophytic bacteria isolates to control r. solanacearum and f. oxysporum fsp . lycopersici and promote growth rate of tomatoes in in vitro conditions. materials and methods this research has been done in microbiology laboratory, department of agriculture, universitas andalas, padang, indonesia from march to june 2017. indole acetic acid production. indole acetic acid (iaa) productions was determined using quantitative methods of patten and glick (2002). isolates cultured in -1 king's b broth supplemented with the 1g ml of ltryptophan. after 42 h of incubation, the density of each culture was measured spectrophotometrically at 600 nm, and then the bacterial cells were removed from the culture medium by centrifugation (5500 × g, 10 min). a 1-ml aliquot of the supernatant was mixed vigorously with 4 ml of salkowski's reagent (150 ml of concentrated h so , 250 ml of distilled h o, 7.5 ml of 2 4 2 0.5 m fecl ·6h o) and allowed to stand at room 3 2 temperature for 20 min before the absorbance at 535 nm was measured. the concentration of iaa in each culture medium was determined by comparison with a standard curve. phosphate solubilizing activity. phosphate solubility was assayed using pikovskaya agar based on methods of wahyudi et al. (2011). each isolate was streaked to the surface of pikovskaya agar contain tricalcium phosphate and the phosphate solubilizing activity was estimated after 5 d of incubation at room temperature. phosphate solubilization activity was determined by the development of the clear zone around bacterial colony. s i d e r o p h o r e p r o d u c t i o n . s i d e r o p h o r e productions was determined using chrome azurol sulphonate (cas) agar medium described by alexander and zuberer (1991). each isolate was streaked on the surface of cas agar medium and incubated at room temperature for 3 d. siderophore production was indicated by orange halos around the colonies after the incubation. hydrogen cyanide production. hydrogen cyanide (hcn) production was assayed using methods of lorck (1948). isolates cultured on nutient agar -1 supplemented with glycine (4 g l ). the production of cyanide was detected 48 h after inoculation, using picrate/na co paper fixed to the under side of the 2 3 petri-dish lids which where sealed with parafilm before o incubation at 28 c. a change from yellow to orange, red, brown, or reddish brown was recorded as an indication of cyanide production. ammonia production. ammonia (nh3) production was assayed according to methods of cappuccino and sherman (2004) using peptone water o (10ml), incubated for 48-72 h at 28 + 2 c and nesseler's reagent (0.5 ml) added after incubations. the color change to brownish yellow indicated positive result. root colonization. root colonization ability of endophytic bacteria were assayed by reisolations of resistance isolates mutated with rifampicin from root of tomato. bacterial isolates were mutated with sequentially streaked on tsa with concentration of 0, 10, 20, 50 and 100 ppm of rifampicin each for 24-36 h. the mutant isolates growth from 100 ppm rifampicin culture then regrowh on the same media for 24-36 h, then 6 -1 were suspensed with sterile aquadest (10 cell ml ). seedlings of tomato introduced with the mutant strains by dipped the seedlings for 5 min into the bacterial solutions before planted in sterilized soil. roots of tomatoes were harvested after 4 weeks and surface sterilized with naocl 2%. sterilized roots were -4 macerated and diluted to 10 , 0.1 ml of each homogenized with tsa contained 100 ppm rifampicin and plated to petri dishes for 48 h. bacterial colonies growth on the medium with the same morphologies as its mutant culture then were counted. bacteria identification using 16s rrna. endophytic bacterial isolates was identified by 16s rrna gene. bacterial dna was extracted using the protocol of purelink genomic dna mini kit (invitrogen, thermo scientific inc. usa). the extracted dna then used as pcr template and amplified using universal primer 27f (5' aga gtt tga tcm tgg ctc ag'3) and 1492r (5'cgg tta cct tgt tac gac tt '3). the pcr conditions were o following xiong et al. (2014), denaturation at 94 c for 118 yanti et al. microbiol indones volume 11, 2017 microbiol indones 119 o o 1 min, annealing at 54 c for 30 s and extension at 72 c for 1 min for 30 cycles and final extention for 30 min. the 16s rrna fragment then assayed qualitative and quantitative by electrophoresis on agarose gel 1%. dna fragment sequenced in macrogen inc. (korea). the 16s rrna sequence data then compared with the sequences from the genbank using blast analysis (http://ncbi.nlm.nih.gov). results growth promotion characters of endophyte isolates. we aimed to characterize selected endophytic bacteria associated with the roots of tomato from previous reearch which have potential as biocontrol of r. solanacearum and f. oxysporum f.sp solani and promote growth of tomato in field conditions. all isolates shown varies ability to produce iaa, produce nh3 and solubilize phosphate (table 1). all isolates can produce iaa. isolate tle 1.1 had shown highest iaa productions, 42.5 ppm, then e1ab2.1, 38.9 ppm and tle2.2, 36.5 ppm. however, not all isolates such as epl1.1.3, tle2.3, epl1.1.4, sne2.2, e1ab2.1 and kle3.3 can solubilize phosphate. only isolates tle1.1, e1ab1.2, and tle2.2 had shown ability to solubilize phosphate. all isolates were also found could not produce nh3 which one the main source of nitrogen for plants. biocontrol characters of endophyte isolates. besides the growth promotion ability by productions of hormone indole acetic acid (iaa) and phosphate solubility, the endophytic bacteria isolates also had ability as biocontrol r. solanacearum and f. oxysporum f.sp solani which characterized by siderophore production, cyanide production and the ability of bacteria to colonize roots of tomato. from all isolates characterized, only tle2.3 can produce siderophore, and no isolates can produce cyanide. all isolates can persistent in the roots tissue until 4 weeks after introduction. r. solanacearum is one of the most devastating pathogens that attack plants from roots and invade the vascular tissues of plants. the good ability of the endophytic bacteria isolates to inhabit and persistent in roots tissue may prevent the attack of r. solanacearum both from direct mechanisms such as competitions and antimicrobial substance productions, or from indirect mechanisms such as induced systemic resistance (isr). molecular identifications. dna amplificons shown that all sequences were compatible with 27f and 1492 primers. all fragments shown on parallel with aproximately 1500 bp markers, as expected results of 27f and 1492r primers amplificons (fig. 1). electrophoresis results shown all amplicons acquired were a single dna band indicated that the primers can specifically amplified the expected fragment. sequence analysis from the 16s rrna gene of endophytic bacteria shown a variable species of bacteria (table 3). all isolates were identified as different species as seen in table 3. out of 9 isolates only 3 isolates shown high similarities between 97% to 100% with the databases in genebank. another isolates shown 96% (3 isolates), 95% (1 isolates) and 94% (2 isolates) isolates of similarity with those in genebank. discussion r. solanacearum and f. oxysporum f.sp solani are the most devastating pathogens that attack plants from roots and invade the vascular tissues of plants. the good ability of the endophytic bacteria isolates to inhabit and persistent in roots tissue may prevent the attack of r. solanacearum and f. oxysporum f.sp solani both from direct mechanisms such as competitions and antimicrobial substance productions, or from indirect mechanisms such as induced systemic resistance (isr). in this study we have characterized and identified endophytic bacteria which are shown potential activity to control r. solanacearum from tomato roots. some of strains were characterized (tle1.1, e1.ab1.2 and tle2.2) also had ability to solubilize phosphate which is one of essential substance for plant growth. moreover, all isolates also produce iaa. endophytic bacteria can enhance of plant growth through synthesis of plant auxin iaa (chen et al. 2010). iaa can stimulate growth by cell elongations or cell division (patten and glick 2002). however, ability to produce nh which also essential substance of 3 growth tested negative for all isolates. this can be concluded that the ability of the endophyte bacteria to promote growth rate of tomato are mainly by iaa productions and phosphate solvent ability. endophytic bacteria has been found inhabit most plant species and have been isolated from variety of plants. we found that all isolates were well estabilished in plant root tissues. the capability of bacteria to colonize plant tissues is desirable because its greater chance of influencing development of host plants (kuklinsky et al. 2004). the colonizations of the fig 1 results of dna amplification of 165 rrna gene from endophytic bacteria using 27f and 1492r primers. 120 yanti et al. microbiol indones table 1 iaa productions, phosphate solubility and nh productions of endophytic bacteria isolates3 (c) table 2 characterizations of endophytic bacteria isolates to produce siderophore, cyanide (hcn), ammonia (nh ) and ability to colonize tomato plant root3 table 3 identification results of endophytic bacteria using 16s rrna sequence isolates sequence analysis r esults % of similarity accession number epl1.1.3 bacillus toyonensis strain bct -7112 96 nr_121761.1 tle2.3 serratia nematodiphila strain dz0503sbs1 94 nr_0443 85.1 epl1.1.4 bacillus anthracis strain atcc 14578 97 nr_041248.1 tle1.1 bacillus cereus atcc 14579 97 nr_074540.1 sne2.2 bacillus cereus strain jcm 2152 97 nr_113266.1 e1.ab1.2 enterobacter cloacae subsp. dissolvens strain atcc 23373 96 nr_118011.1 e1ab2.1 serratia marcescens strain nbrc 102204 94 nr_114043.1 tle2.2 klebsiella michiganensis strain w14 95 nr_118335.1 kle3.3 chryseobacterium rhizoplanae strain jm -534 96 nr_134711.1 1 isolates ia a production (ppm) phosphate solubility a m m oniac (n h 3) production epl1.1.3 34.6 t le2.3 29.8 epl1.1.4 36.7 t le1.1 42.5 + sn e2.2 28.7 e1.ab 1.2 26.8 + e1ab 2.1 38.9 t le2.2 36.5 + k le3.3 30.9 1 isolates siderophore production cyanide (hcn) production endophyte colonization (10^5) 4 weeks after introduction epl1.1.3 15 tle2.3 + 20 epl1.1.4 17 tle1.1 35 sne2.2 28 e1.ab1.2 16 e1ab2.1 1.7 tle2.2 20 kle3.3 9 1 volume 11, 2017 microbiol indones 121 endophytes will barrier the pathogen attack to plants and increase the competence in the rhizosphere which lead to lower the possibility of pathogens infection. out of all isolates, only tle2.3 (serratia nematodophilla) can produce siderophore. siderophore are organic molecules that had high affinity for fe ions, prevent another pathogenic microbe to get fe as one of nutrient source. we had successfully identified all 9 isolates using 16s rrna sequences. from the bacillus genus, we have identified 3 species, i.e b. toyonensis, 2 strain of b. cereus and b. antrachis. b. toyonensis could be a valuable strain for further studies because of its ability to promote growth and control pathogens. there are not much publications of b. toyonensis ability as biocontrol agents and growth promoting agents. rocha et al. (2017) found 9 isolates of b. toyonensis which can control fusarium oxysporum f. sp. lycopersici. b. toyonensis also reported had antagonistic activities against meloidogyne incognita (xiang et al. 2017). b. cereus is one of the well studied species for plant growth promotions and biocontrol activity among of bacillus genera. b. cereus known to promote plant growth by produce growth hormone such as gibberellin (joo et al. 2004), produce antibiotic compounds (silosuh et al. 1994) and induce systemic resistance (niu et al. 2011). we also found two isolates from serratia genera which are s. marcesens and s. nematodiphila. another rarely studied strains we found in this research are s. nematodiphila. although serratia spp. have been classified as opportunistic human pathogens (grimont and grimont 2009), s. nematodiphila had reported as growth promotor, gibberellin producer and biocontrol agents of chili (kang et al. 2015), enhanced heavy metal phytoremediation (wan et al. 2012), and control xanthomonas oryzae pv. oryzae. the isolates e1ab1.2 was identified as enterobacter cloacae subsp. dissolvens. this species were not a common plant growth promoting species, but had been reported can promote plant growth by regulatory of antifungal compounds, phenolic compounds and iaa (slininger et al. 2004). all isolates showed various characters which led to growth promotions of plants and biocontrol activity. we had identified all 9 isolates from genera bacillus, s e r r a t i a , k l e b s i e l l a , e n t e r o b a c t e r, a n d chrysobacterium. this research found new pgpr strains and further research need to be done related to b. toyonensis, k. michiganensis and c. rhizoplanae. however, all isolates shown the similarity under 99% compared to genebank database indicated that these bacteria are likely to be a novel strains or species. bosshard et al. (2003) defined ≥99% similarity from 16s sequence can be considered as new species. turenne et al. (2001) also designated 16s sequence similarity range under 0.8 to 2% can might be suggested as new species. we suggest further genomic research to confirm the novelty of these isolates. acknowledgment this research was funded by 'penelitian terapan unggulan perguruan tinggi' batch 2019 contract no. rd 059/sp2h/lt/drpm/iv/2017 april 3 2017 from ministry of research, technology and higher education of the republic of indonesia. references alexander db, zuberer da. 1991. use of chrome azurol s reagents to evaluate siderophore production by rhizosphere bacteria. biol fertil soils. 12(1): 39-45. bosshard pp, abels s, zbinden r, bottger ec, altwegg m. 2003. ribosomal dna sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation). j clin microbiol. 41:4134-4140. doi: 10.1128/jcm.41.9.4134-4140.2003. bulgari d, casati p, brusetti l, quaglino f, brasca m, daffonchio d, bianco pa. 2009. endophytic bacterial diversity in grapevine (vitis vinifera l.) leaves described by 16s rrna gene sequence analysis and length heterogeneity-pcr. j microbiol. 47(4): 393401. doi: 10.1007/s12275-009-0082-1. cappuccino j, sherman n. microbiology: a laboratory manual. 2004. person education, usa. chen l, luo s, xiao x, guo h, chen j, wan y, liu c. 2010. application of plant growth-promoting endophytes (pgpe) isolated from solanum nigrum l. for phytoextraction of cd-polluted soils. appl soil ecol. 46(3): 383-389. doi: 10.1016/j.apsoil.2010.10.003. golinska p, wypij m, agarkar g, rathod d, dahm h, rai m. 2015. endophytic actinobacteria of medicinal plants: diversity and bioactivity. antonie van leeuwenhoek. 108(2): 267-289. doi: 10.1007/s10482-015-0502-7. grimont f, grimont pad, 2009. genus xxxiv. serratia bizio 1823, 288al. in: garrity gm, brenner dj, krieg nr, stately jt. 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biological control of meloidogyne incognita by spore-forming plant growthpromoting rhizobacteria on cotton. plant dis. 101(5): 774-784. doi: 10.1094/pdis-09-16-1369-re. yanti y, warnita, reflin, busniah m, 2017. indigenous endophyte bacteria ability to control ralstonia and fusarium wilt disease on chili. international seminar on biodiversity, medan, north sumatra, 4-5 nov 2017. 122 yanti et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 11 no. 301 (akhmaloka).pmd 16s ribosomal rna-based analysis of thermophilic bacteria in gedongsongo hot spring agustina lulustyaningati nurul aminin1, fida madayanti1, pingkan aditiawati2, and akhmaloka1∗ 1biochemistry research division, faculty of mathematics and natural sciences, 2school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia denaturing gradient gel electrophoresis was used to identify the bacterial community at the gedongsongo (wgs-2) hot spring. the bacterial samples were obtained from both culture dependent and independent strategies. partial 16s rrna genes were amplified by a set of primers to produce at around 400 bp fragments, including the highly variable v9 region of the 16s rrna genes. the dgge profiles showed that there were a few distinct bands, namely g1-g3, and g8-g12, which represent the predominant bacteria in natural habitat and the medium. further analysis of these bands showed that most of them, except for g7, have a high homology to the 16s rrna gene sequences of thermus sp. as for g7, the highest homology was shown to unculturable bacteria. in addition to the distinct bands in dgge, there were other three thin bands, namely g4, g5, and g6, which possibly represent non dominant microorganisms in the natural habitat, but could grow on gs-a medium. further analysis of these bands showed that g6 has 80% similarity to the 16s rrna of burkholderia sp., while g4 and g5 have a high homology to each other but only contained 10-15% homology to the sequences of 16s rrna from unculturable microorganisms. the phylogenetic analyses of the last organisms showed that there was branching from burkholderia. from all the data obtained it was suggested that the wgs-2 hot spring was predominantly occupied by the genus thermus. in addition, there were a few novel microorganisms found in the hot spring. key words: thermophiles, 16s rrna, dgge _____________________________________________ _________________ ∗corresponding author, phone:+62-22-2502103, fax: +62-22-2504154, e-mail: loka@chem.itb.ac.id one of the challenges in modern microbial ecology is how to effectively and accurately assess microbial diversity. a common thread described in numerous published studies concerning microbial diversity is that, in most environments, only 0.1 to 1.0% of bacteria detected by direct microscopic enumeration can be recovered on even the most general laboratory media (staley and konopka 1985). as a result, microbial ecologists generally are of the opinion that the vast majority of microbial diversity remains uncharacterized due to this gap between cultivable and direct estimation of microbial biomass and diversity. this concern has spurred on the development of molecular approaches for studying microbial communities, usually based on analysis of nucleic acids directly extracted from environmental samples (pace et al. 1986). the application of molecular phylogenetic to study natural microbial ecosystems without the traditional requirement for cultivation has resulted in the discovery of many unexpected evolutionary lineages; members of some of these lineages are only distantly related to known organisms but are sufficiently abundant that likely to have impact on the chemistry of biosphere (pace 1997). the classical approach to determine the microbial diversity in a natural or artificial ecosystems starts with culturing of the microorganisms in a sample. culture-based approaches to isolate microorganisms from any natural environment do not provide comprehensive information on the composition of microbial communities (bull and hardman 1991). this technique also fails to determine the majority of microorganisms in nature, which typically not cultivated using standard techniques (amann et al. 1995). it has been realized that only a minor fraction of the microorganisms (1-5%) are amenable to standard culturing techniques (schmidt et al. 1991; liesack and stackebrandt 1992; fuhrman et al. 1993). due to this difference between cultivable and in situ diversity, it is often difficult to assess the significance of cultured members in resident microbial communities. in order to overcome the limitations associated with cultural approaches, a molecular alternative has been developed. the development of techniques for the analysis of 16s rrna sequences in natural samples has enhanced our ability to detect and identify bacteria in nature (pace et al. 1986). this involves dna extraction of community directly from water, soil or sediments followed by pcr amplification and then sequencing of 16s rrna genes, which are known to be one of the established phylogenetic markers (woese 1987). such approaches have been successfully applied for hot spring (ferris et al. 2003), compost (ueda et al. 2001), marine bacteriaplankton (fuhrman et al. 1993), soil (nakatsu et al. 2000) and as well as hydrothermal environment (moyer et al. 1995) samples. muyzer et al. (1993) presented a molecular approach for analyzing the genetic diversity of complex microbial populations called denaturing gradient gel electrophoresis (dgge) at the first time. separation fragments in dgge based on the decreased electrophoretic mobility of partially melted double-stranded dna molecules in polyacrylamide gels containing a linear gradient of dna denaturants. this technique is now routinely used in many microbiological laboratories as a tool to compare the diversity of microbial communities and to monitor population dynamics (muyzer 1999). this approach has now been variously combined with group-specific amplification (heuer et al. 1997), membrane transfer and hybridization (stephen et al. 1998), band excision microbiology indonesia, april 2007, p 37-42 volume 1, number 1 issn 1978-3477 and sequence analysis (kowalchuk et al. 1997; mccaig et al. 1999), and with culturing methods (watanabe et al. 1998). in this report we will describe the bacterial community inhabiting a hot spring at gedongsongo, ungaran, central java. materials and methods sampling. sampling was carried out on one of the hot spring, namely wgs-2, at gedongsongo that located along the southern flank of ungaran volcano, central java (110o20’23.4"e; 07o12’08.5"s; and the altitude 1400 m). this hot spring has temperature at 70.2 oc and a ph 5.85. for the filtration procedure, the water sample was kept in a sterile plastic container and brought to the laboratory immediately within 2 hours. afterwards, cells were harvested by filtration of 1 l volumes of spring water gently through 0.2 µm millipore filters. filtrates containing bacterial communities were stored at -20 oc until dna was extracted. the cultivation procedure was carried out using two minimal media. the gs-a medium contains 0.15% (w/v) tryptone and 0.15% (w/v) yeast extract, while the gs-b medium contains 0.5% (w/v) peptone, 0.25% (w/v) yeast extract, and 0.5% (w/v) nacl. both media, which used natural hot spring water, were incubated at 60 oc without shaking for 24 hours. dna extraction. cells from spring water that had been collected on a membrane filter were harvested by putting the filter in a sterile erlenmeyer that contained 10 ml sterile distilled water. the erlenmeyer was then shaked gently and the membrane surface was scrubbed aseptically using ose needle for a couple times until almost all of the cells were suspended in water. each microbial sample was pelleted by centrifugation at 8000 x g for 15 min. dna was extracted using slight modification of the klijn et al. (1991) method. the pellet was suspended in 200 ìl of 10 mm tris hcl buffer (ph 8.0) containing 8 mg ml-1 of lysozyme and incubated at 37 oc for 1 h. the cells were lysed by adding 200 ìl lysis buffer containing 2% (w/v) sds, 0.8 mg ml-1 proteinase k and 200 mm edta ph 8.0. the lysis process was carried out at 50 oc for 30 min. the purification step was performed by adding 200 µl of chloroform:isoamyl-alcohol (24:1), vortexed and then centrifugated at 12,000 x g, for 30 sec. the upper solution was moved to a clean tube. this step was done 3x. subsequently, the dna was precipitated by adding 60 ìl of 3 m sodium acetate and 1 ml of 96% (v/v) ethanol (stored at -20 oc). after centrifugation at 12,000 x g for 15 min, the dna pellet was washed with 70% (v/v) ethanol and finally dissolved in 50 ìl of te buffer (10 mm tris hcl [ph 8.0], 1 mm edta). amplification of 16s rrna gene fragments. partial 16s rrna genes was amplified as described previously by ferris et al. (1996). a set primer (p1 and p2) was used to amplify the gene. one primer (p1: 5’ atggctgtcgtcagct 3’) complements on the conserved region among the bacteria domain of escherichia coli 16s rrna at positions 1055 to 1070. the other primer (p2: 5’ cgcccgccgc gccccgcgcc cggcccgccg cccccgcccc acgggcggtgtgtac 3’) is based on a universal conserved region of the e. coli 16s rrna at positions 1392 to 1406, with the addition of 40-bases of gc clamp. the specificity of this primers is imparted by the underlined regions. pcrs were performed by using cloned pfu dna polymerase according to the instructions provided by the manufacturer (promega). the temperature cycle for the pcr was 1 min of denaturation at 94 oc, 1 min of annealing, and 1 min of primer extension at 72 oc. during an initial touchdown cycle, the annealing temperature was continually decreased from 53 to 43 oc in intervals of 1 oc per cycle; 20 additional annealing cycles were done at 43 oc. the final extension at 72 oc was performed for 10 min. denaturing gradient gel electrophoresis. all reagents and techniques were performed as described by ferris et al. (1996) with small modification. acrylamide gels (8%) were prepared and run with 0.5x tae buffer (0.04 m tris base, 0.02 m sodium acetate, and 1.0 mm edta; ph adjusted to 7.4). an ingeny phor u gel electrophoresis unit was used with glass plates (16 by 18 cm), 1.0-mm spacers, and 1-cmwide loading wells. a 15 liter aquarium served as the lower buffer chamber. dgge gels contained a 35 to 60% (w/v) gradient of urea and formamide (uf) solution increasing in the direction of electrophoresis. a 100% uf solution is defined as 40% (v/v) formamide plus 7.0 m urea. dgge was conducted at 60 oc, firstly at 20v for 10 min and then at a constant voltage of 200 v for 3 hours. the gel was stained using a silver staining (bassam et al. 1991). re-pcr of dgge bands. the bands (from the fresh gel) were cut and added 50 ìl sterile deionized water and allowed dna to passively diffuse into the water at 4 oc overnight and then 5 ìl of eluted fraction was used as template dna in a pcr (ferris et al. 1996; nakatsu et al. 2000). for the gels that keeps in dry form and storage for a couple months, these dry gels were placed in vial, added water and heated at boiling water for 5 minute. after that, the gels were punched using the end of tips and allowed to passively diffuse into the water at 37 oc overnight. sequence analysis. all confirmed dgge bands were subjected to double-stranded dna sequencing. sequencing were carried out in an abi prismr 3100 genetic analyzer (applied biosystem) by the macrogen sequencing service (korea). the sequencing reaction was performed with the bigdye terminator cycle sequencing ready reaction kit (applied biosystem) using forward primer (p1). the sequencing results were subjected to analyzing and comparing to the genbank (ncbi) using the blast n program (atschul et al. 1997). the phylogenetic tree was constructed by clustalw methods from megalign program of dna star. results chromosomal dna and 16s rrna gene fragments. the cells from both filtration and cultivation were lysed to extract their total chromosomal dna. according to examination on ethidium bromide-stained agarose gels, both cultivated and filtered cells were successfully extracted from the chromosomal dna (data not shown). total chromosomal dna from both filtration and cultivation methods were used as templates to amplify partial 16s rrna gene using primers p1-p2. these primers should amplify dna fragment with the size 392 bp (including gc clamp). the pcr products of gsa, gs-b, and gs-f cells were examined on a ethidium bromide38 aminin et al. microbiol indones stained agarose gel, and the results showed a single band of the expected size as expected (figure 1). profiles of 16s rrna gene fragments. the profiles of the bacterial community of the wgs-2 hot spring from filtration and cultivation were shown in figure 2. in order to get the finest profile of the bands, it has been carried out several electrophoresis procedures using different range of gradients. the best dgge profile that is showed in this report was obtained using gels containing a 35 to 60% (w/v) gradient of urea and formamide. the dgge patterns showed that there were three distinct bands that appear in each well (gs-f, gs-a, and gs-b). the other three weak bands (g4, g5, and g6) appeared in the gs-a well (figure 2). the fragment dna from g1-g12 bands was recovered from the dgge gel and reamplified using primers p1 and p3. the sequence of p3 is similar to that of p2 but without the gc clamp (5’ cacgggcggtgtgtac 3’). the re-pcr amplicons appeared as single bands with the size as expected (data not shown). dna fragments on dgge could be stored as dry gels and were stable until a few months for reamplification (figure 3). dna sequences and homology of 16s rrna gene fragments. all of the re-pcr results (g1 to g12) have been sequenced and analyzed. those sequences have been compared with another 16s rrna gene sequence of domain bacteria using the program blastn program at ncbi website. most of the bands (g1, g2, g3, g8, g9, g10, g11, and g12) except for g7, showed high homology to each other and were closely related to 16s rrna gene of thermus species (figure 4). however, homological study of the last four bands (g4, g5, g6, and g7) revealed some unique sequences that closely related to uncultured bacteria (table 1, 2, 3, and 4). the nucleotide sequences of g4 and g5 are very similar to each other. of 300 bp that has been compared around 15% of these sequence have match with 138 and 109 sequences of 16s rrna gene to g4 and g5 respectively, at the downstream of the gene fragment. the sequence of g6 has 80% similarity to that of burkholderia sp. ccbau23014 (table 3). the 300 bp of g7 sequence has only a 10% match figure 1 electrophoregram of pcr product. lane 1 shows plasmid dna puc19 digested with hinfi; lane 2, 3, and 4 show pcr products from gs-a, gs-b and gs-f dna respectively. 1 2 3 4 392 bp 1 4 1 9 5 1 7 3 9 6 2 1 4 g7 g11 g12 g2 g1 g3 g4 g5 g6 gs-b gs-a gs-f figure 2 dgge patterns of pcr-amplified 16s rdna fragments. well 1, 2, and 3 are 16s rdna fragment from gs-b, gs-a, and gs-f respectively, while g1-g12 are bands position on dgge. g8 g9 g10 figure 4 comparison analysis of the sequence of g1, g2, g3, g8, g9, g10, g11, and g12 with some thermus sp. using megalign program. the difference are shown by dashed line box. a. the upstream part of 300 nucleotides. there are some deletion of g1, g2, and g3 that differ to other sequences. b. the downstream part of 300 nucleotides. there are some substitution of g8, g9, and g10 that differ to other sequences. a b 1 4 1 9 5 1 7 3 9 6 2 1 4 352 bp figure 3 electrophoregram of re-amplified dgge bands (without gc clamp) using modified method of different age of dry gel. lane 1 shows plasmid dna puc19 digested with hinfi; lane 2 from gel age 3 months that stored in water and keep in -20 oc until 10 months, lane 3 from gel age 10 months and lane 4 showed fragment dna from gel age 3 months. 1 2 3 4 volume 1, 2007 microbiol indones 39 with some 16s rrna gene sequences from uncultured bacteria (101 related sequences), all of them showed a consistent match to upstream portion of the g7 sequence. discussion the microbial community was performed using culturedependent and culture-independent methods. the culturedependent method used enrichment media gs-a and gs-b. both of them are not specific media. the use of these media was design to pick various kinds of microbes. the dgge profile shown in figure 2 revealed that three distinct bands from each well are aligning to each other and possibly mean that these bands represent the same and predominant bacteria that survive in this spring. however, the other three bands (g4, g5, and g6) in the gs-a well do not appear in gs-f, possibly due to fact that these bands represent microorganism that is not dominant in the spring but has high grown rate on gs-a medium. from the results of blastn program analysis on 300 bp of each band, three distinct dgge bands from each well, except for g7, are closely related to thermus sp., the highest similarity of these sequences belongs to thermus rh 0401 (accession number: ay731822). the analysis showed that figure 5 phylogenetic tree of dgge bands. the bands of g1, g2, g3, g8, g9, g10, g11, and g12 are closely related to thermus sp. while g4, g5, g6, and g7 making some new branch. g8-p1_dgge band.seq g11-p1_dgge band.seq g12-p1_dgge band.seq g9-p1_dgge band.seq g10-p1_dgge band.seq g1-p1_dgge band.seq g2-p1_dgge band.seq g3-p1_dgge band.seq thermus sp. rh-0401.seq thermus sp. y55-12.seq thermus igniterrae.seq thermus aquaticus yt-1.seq thermus oshirnai.seq kartchner caverns bac hi-04.seq escherichia coli.seq uncult alteromonadaceae cl 150-0348.seq uncl gamma proteobacterium b2m60.seq burkholderia sp. ccbau23014.seq burkholderia cepacia.seq g4-p1_dgge band.seq g5-p1_dgge band.seq g6-p1_dgge band.seq g7-p1_dgge band.seq 23.4 20 15 10 5 0 40 aminin et al. microbiol indones table 1 the comparison study of g4 sequence. about 15% of 300 bp at downstream position of g4 sequence have a consistency match with about 138 sequences of 16s rrna gene of mostly uncultured bacteria. this table only shows ten of the highest homology sequence accession number af223299 dq419338 ab167222 dq341037 af005092 dq458039 dq458033 dq458000 af391977 ay 5 11 7 4 2 uncultured gamma proteobacterium b2m60 16s ribosomal rna gene, partial sequence uncultured gamma proteobacterium clone st9-55d 16s ribosomal rna gene, partial sequence acinetobacter sp. c61 gene for 16s rrna, partial sequence uncultured desulfohalobiaceae bacterium clone 2.88m_24b 16s ribosomal rna gene, partial sequence clostridium sp. sp3 16s ribosomal rna gene, partial sequence uncultured bacterium clone dx49 16s ribosomal rna gene, partial sequence uncultured bacterium clone dx63 16s ribosomal rna gene, partial sequence uncultured bacterium clone dx53 16s ribosomal rna gene, partial sequence uncultured thermal soil bacterium clone ynpffp50 16s ribosomal rna gene, partial sequence uncultured bacterium clone gc113.16 16s ribosomal rna gene, partial sequence 95.6 95.6 95.6 95.6 95.6 89.7 89.7 89.7 89.7 89.7 sequence name score (bits) table 2 the comparison study of g5 sequence. about 15% of 300 bp at downstream position of g5 sequence have a consistency match with about 109 sequences of 16s rrna gene of mostly uncultured bacteria. this table only shows ten of the highest homology sequence accession number af223299 af005092 dq458039 dq458033 dq458000 dq463208 dq463183 dq419338 ab167222 aj863184 uncultured gamma proteobacterium b2m60 16s ribosomal rna gene, partial sequence clostridium sp. sp3 16s ribosomal rna gene, partial sequence uncultured bacterium clone dx49 16s ribosomal rna gene, partial sequence uncultured bacterium clone dx63 16s ribosomal rna gene, partial sequence uncultured bacterium clone dx53 16s ribosomal rna gene, partial sequence uncultured bacterium clone tls3-13 16s ribosomal rna gene, partial sequence uncultured bacterium clone tls1-5 16s ribosomal rna gene, partial sequence uncultured gamma proteobacterium clone st9-55d 16s ribosomal rna gene, partial sequence acinetobacter sp. c61 gene for 16s rrna, partial sequence uncultured bacterium partial 16s rrna gene, clone 20bsu24 93.7 93.7 89.7 89.7 89.7 89.7 89.7 89.7 89.7 89.7 sequence name score (bits) uncultivable bacteria. this sequence has the highest similarity (80%) to burkholderia sp. ccbau23014 16s ribosomal rna gene (accession number: ay839565.1) (table 3). actually, burkholderia is a diverse genus with diverse species that live in diverse ecological niches. the molecular and physiological baackground of this diversity and adaptability are largely unknown. multiple biotechnologically interesting strains belong to as yet uncharacterized taxa (coenye and vandamme 2003). the other bands (g4, g5, g7) have a low similarity (about 10-15%) to some 16s rrna gene sequences of unculturable bacteria. however, these sequences showed at consistent position with some 16s rrna genes (table 1, 2, and 4). even though the similarity of these bands is only at around 1015%, but all of them are similar to 16s rrna genes and none of them represent another gene sequences (138 sequences for g4, 109 sequences for g5, and 101 sequences for g7). this data suggests that the g4, g5, and g7 sequences are part of the16s rrna gene. from the result of the phylogenetic tree (figure 5), the g4, g5, g6, and g7 made separate branch in the tree. the reason for the forming separated branch might due to some unique bases that unreaveld in in other 16s rrna gene that compiled in blast. these unique bases include some substitutions along the sequence and the glaring difference of the insertion and deletion of the bases at the middle position of the sequences (the relative position of the e. coli at position 1250-1280). surprisingly, although g6 has the highest similarity with the burkholderia sp. ccbau23014, this sequence did not share the same branch as some burkholderia sp. in the tree. it suggested that g6 volume 1, 2007 microbiol indones 41 table 4 the comparison study of g7 sequence. about 10% of 300 bp at upstream position of g7 sequence have a consistency match with about 101 sequences of 16s rrna gene of mostly uncultured bacteria. this table only shows ten of the highest homology sequence ay868092 dq219828 ay048188 ay048191 ay869139 ay869123 ay869663 ay869137 ay869136 ay869134 uncultured gamma proteobacterium clone i50-0430 16s ribosomal rna gene, partial sequence uncultured bacterium isolate dgge band 5 16s ribosomal rna gene, partial sequence uncultured xanthomonas sp. 16s ribosomal rna gene, partial sequence uncultured xanthomonas sp. clone pr-vfa19 16s ribosomal rna gene, partial sequence uncultured alteromonadaceae bacterium clone i50-0558 16s ribosomal rna gene, partial sequence uncultured alteromonadaceae bacterium clone i50-0411 16s ribosomal rna gene, partial sequence uncultured alteromonadales bacterium clone i3k-0547 16s ribosomal rna gene, partial sequence uncultured alteromonadaceae bacterium clone i50-0531 16s ribosomal rna gene, partial sequence uncultured alteromonadaceae bacterium clone i50-0520 16s ribosomal rna gene, partial sequence uncultured alteromonadaceae bacterium clone i50-0489 16s ribosomal rna gene, partial sequence 71.9 69.9 69.9 69.9 69.9 69.9 67.9 67.9 67.9 67.9 accession number sequence name score (bits) table 3 the comparison study of g6 sequence. this sequence has matches to 163 sequences of 16s rrna gene. the 300 bp of g6 sequence has the highest similarity (at around 80%) to the burkholderia sp. ccbau23014 accession number ay839565 ay839129 af223299 af005092 dq228373 ay842149 ay839125 ay839234 ay271791 dq444991 burkholderia sp. ccbau23014 16s ribosomal rna gene, partial sequence uncultured bacterium clone pb 357-359/ 17 16s ribosomal rna gene, partial sequence uncultured gamma proteobacterium b2m60 16s ribosomal rna gene, partial sequence clostridium sp. sp3 16s ribosomal rna gene, partial sequence uncultured bacterium clone bg.d4 16s ribosomal rna gene, partial sequence endophyte bacterium ss14 16s ribosomal rna gene, partial sequence uncultured bacterium clone pb 357-359/ 25 16s ribosomal rna gene, partial sequence pseudomonas aurantiaca strain yc4963 16s ribosomal rna gene, partial sequence p. aurantiaca vkm b-816t 16s ribosomal rna gene, partial sequence pseudomonas sp. eur1 9.41 16s ribosomal rna gene, partial sequence 1 3 1 1 1 9 1 1 1 1 1 1 1 0 9 1 0 9 1 0 9 1 0 9 1 0 9 1 0 9 sequence name score (bits) these bands have high similarity (97-99%) to at least 100 sequences of 16s rrna genes of both culturable and unculturable of thermus sp. the comparison of g1-g3 and g8-g12 sequences toward some thermus sp. sequences using megalign program revealed some differences from each sequences (figure 4). in spite of different position at the dgge and there are some different nucleotides (substitution), the three bands from each well i.e.: g1-g2-g3, g8-g9-10, and g11-g12 seemed to represent the same species. a possible reason for the formation of more than one band from one species is that the universal primers amplified more than one operon (schmalenberger et al. 2001). it is well known that several bacterial species contain more than one 16s rrna gene in their genomes (klappenbach et al. 2001). the heterogeneity of 16s rrna gene between multiple copies within one species hampers pattern analysis (mccaig et al. 2001), and can confuse the interpretation of diversity from sequences retrieved from banding patterns. although there is single or more differences on base sequence of these dgge bands to each other, all of them showed the same highest similarity with thermus rh 0401. this evidence could not bring into the conclusion whether these dgge bands sequence lead to the same species or not, but it could not be opposed that these bands represent thermus species. the phylogenetic study that constructed using clustalw method revealed that the sequence of eight distinct bands are close to thermus group. this evidence could be seen in phylogenetic tree (figure 5). these data suggested that this hot spring was possibly occupied by thermus predominantly. in addition, data from both culture media are also dominated by these species. the analysis g6 sequence revealed similarity to at least 163 sequences of 16s rrna gene both cultivable and did not belong to 16s rrna of burkholderia species. this is supported by the analysis result which only showed similarity to burkholderia sp. ccbau23014 and not to another burkholderia. in fact, the blast analysis of g6, as shown in table 3 was moderately similar to pseudomonas sp. based on the phylogenetic tree and comparison study profile, the last four bands (g4, g5, g6, and g7) are highly recommended as 16s rrna genes sequence from novel species. the far branch at phylogenetic tree for these four bands suggest that these bands representing not only a new species but possibly a new genus or family. these sequence analyses of rrna genes from natural microbial communities have identified a broad diversity of previously unknown microorganisms. currently, about half of the >70,000 rrna sequences in the public databases represent uncultured microorganisms (cole et al. 2003). more than onethird of the 40 to 50 main relatedness group, natural divisions, of the domain bacteria are known only from detection of rrna gene sequences and have no described cultivated representatives. these division-level clades with no cultured representatives, typically known from only limited numbers of rrna sequences, have been termed candidate divisions to reflect the limited documentation that describes them (hugenholtz et al. 1998). acknowledgement this study was funded by “proyek peningkatan penelitian pendidikan tinggi (p4t)”, department of national education, indonesia (contract no. 321/p4t/dppm/hptp/iv/2004) and bpps scholarship to alna. references altschul sf. 1997. 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populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing. appl environ microbiol 64:2958-2965. ueda k et al. 2001. distribution and diversity of symbiotic thermophiles, symbiobacterium thermophilum and related bacteria, in natural environments. appl environ microbiol 67:3779-3784. watanabe k, teramoto m, futamata h, harayama s. 1998. molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge. appl environ microbiol 64:4396-4402. woese cr. 1987. the universal ancestor. proc natl acad sci usa 95:6854-6859. 42 aminin et al. microbiol indones mi635-17-07-12 (astuti) available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.2.1issn 1978-3477, eissn 2087-8575 vol 6, no 2, june 2012, p 49-56 *corresponding author; phone: +62-21-7560536, fax: +6221-7566922; e-mail: anpratomo@yahoo.com in cases of pancreatic disease, trypsin deficiency often occurs due to reduced expression of trypsin in the pancreas. patients with pancreatic problem can be treated with a supplement containing digestive enzymes, including trypsin. however, most of the enzymes currently used for the treatment are derived from porcine and bovine sources. on the other hand, lactic acid bacteria are also known to show trypsin-like activity. in the previous work, our group screened 11 lactic acid bacteria isolates, which had previously been proven to show serine protease activity, for trypsin-like activity. the strains were initially grown in mrs (de mann, rogosa and sharpe) medium before being transferred directly to the production medium to produce trypsin. during the previous study, the initial ph of the production medium was set at 6 (the same as the mrs medium ph), which is the optimum ph for the cell growth of lactic acid bacteria. however, most trypsin has an optimum ph of around 8. in this study, we altered the production medium ph to 8 and we harvested the lactic acid bacteria from mrs medium by centrifugation prior to their inoculation to the production medium. observation of the culture growth and enzyme activity indicated that the new strategy improved the enzyme activity expressed by some strains. key words: pada kasus-kasus gangguan pankreas, defisiensi tripsin seringkali terjadi karena berkurangnya ekspresi tripsin di pankreas. pasien dengan masalah pankreas dapat ditangani dengan pemberian suplemen enzim pencernaan. meskipun demikian, sebagian besar enzim yang digunakan untuk pengobatan saat ini didapatkan dari babi atau sapi. di sisi lain, bakteri asam laktat juga diketahui menunjukkan aktifitas seperti tripsin. dalam penelitian di grup kami sebelumnya, dilakukan skrining terhadap 11 isolat bakteri asam laktat untuk mendapatkan isolat yang menunjukkan aktifitas seperti tripsin. isolat-isolat ini sebelumnya telah terbukti memiliki aktivitas protease serin. isolat-isolat tersebut ditumbuhkan dalam medium mrs (de mann, rogosa and sharpe) sebelum dipindahkan ke medium produksi untuk memproduksi tripsin. pada penelitian sebelumnya, medium produksi diatur pada ph awal 6 (sama dengan ph medium mrs). namun demikian, kebanyakan tripsin bekerja pada ph optimum 8. dalam penelitian kali ini, kami mengubah ph medium produksi menjadi 8 dan melakukan pemanenan sel dengan cara sentrifugasi sel starter dari medium mrs sebelum digunakan untuk menginokulasi medium produksi. observasi terhadap pertumbuhan kultur dan aktivitas enzim menunjukkan bahwa strategi baru ini meningkatkan aktifitas enzim yang dihasilkan oleh beberapa isolat. kata kunci: aktivitas seperti tripsin, bakteri asam laktat, tripsin lactic acid bacteria, trypsin, trypsin-like activity trypsin is a protease enzyme produced by the pancreas and secreted to the duodenum, where it hydrolyses proteins into peptides during the digestion of food. trypsin is a member of the serine protease family, which digests protein from the carboxyl terminal (c-terminal) of the amino acids lysine (lys) and arginine (arg), unless these amino acids are bound to proline (pro) on their c-terminal (whitcomb 2007). in cases of pancreatic insufficiency, the capability to produce and/or to transport digestive enzymes, including trypsin, is not sufficient, thus affecting the digestion processes. for this reason, patients with pancreatic insufficiency often show symptoms of and lowe malabsorption, malnutrition, vitamin deficiency and weight loss, or in children shown by lack of weight gain (pezzilli 2009). pancreatic enzymes have often been used to treat patients with digestion problems caused by a pancreatic defect (waljee et al. 2009). it has been reported that digestive enzyme treatment was proven to increase the fat absorption coefficient of the patients (waljee et al. 2009). lactic acid bacteria (lab) have long been used in food processing. those which are most commonly used are lactococci, lactobacillus bulgaricus (courtin et al. 2002; oberg et al. 2002), l. rhamnosus (haq et al. 2006), l. casei (piuri et al. 2003), l. paracasei (bintsis et al. 2003; haq and mukhtar 2006), l. helveticus (oberg et al. 2002), l. delbrueckii (oberg et al. 2002), l. brevis, l. cellobiosus, l. fermentum, and l. plantarum (haq and mukhtar 2006). in addition, the effect of growth medium ph towards trypsin-like activity produced by lactic acid bacteria dyah wulansari, budiasih wahyuntari, trismilah, and astutiati nurhasanah* laboratory for the development of agroindustrial and biomedical technology (laptiab), bppt, gedung 611, kawasan puspiptek serpong, tangerang selatan, indonesia fig 1 the methods used in the cultivation of lactic acid bacteria to produce trypsin-like protease. squares shaded in grey show the two different methods used. a) method 1: production medium ph 6, starter culture transferred directly to the production medium as described in previous publication (wulansari and wahyuntari 2010); b) method 2: production medium ph 8, starter cultures were centrifuged prior to transferred to the production medium. materials and methods strains and media. the strains used in this study (listed in table 1) were from the collection of centre for bioindustrial technology, bppt, indonesia. the medium used for growth of the lactic acid bacteria was the mrs medium (oxoid cm0359), which was autoclaved at 121 °c for 15 min. the media used for trypsin production contains glucose 0.3%; yeast extract 1%; skim milk 2%; ph 6 or 8, and was autoclaved at 121 °c for 15 min. culture. strains from 40 μl glycerol stocks were regenerated in 4 ml mrs medium broth incubated at 37 °c, shaking at 50 rpm for 18 hours. the culture was then used to inoculate fresh mrs medium broth (1% inoculum) which was incubated at 37 °c with shaking at 50 rpm for 4-6 hours. this starter was then used to inoculate the trypsin production medium. two kind of production media and methods were used and compared (fig 1). method 1. starter was used to inoculate production several bacteria from the genus lactobacillus are known to have proteolytic activity (caplice et al. 1999; haq and mukhtar 2006). proteolysis of milk proteins by lab plays an important role in providing peptides and amino acids to cover the demand of the bacteria and previously several lab known to have trypsin-like activity have been isolated (wulansari et al. 2010). the screening was performed on isolates previously known to show serine protease activity (wulansari and wahyuntari 2010). the strains used to produce the enzyme were cultured in two stages, the growth stage and the production stage. during the growth stage, the strains were cultured in mrs (de mann, rogosa and sharpe) medium, whilst during the production stage, the strains were cultured in the production medium (glucose 0.5%, yeast extract 0.1%, skim milk 1%, ph 6) (wulansari and wahyuntari 2010). in this project, a medium with ph 8 was used and prior to inoculation to the production medium, cells were harvested from the growth medium by means of centrifugation. 50 wulansari et al. microbiol indones a method 1 harvest: centrifugation at 4 c, 5800 g (hitachi cr216, rotor r10a2), 15 min lactic acid bacteria strains in glycerol stock (proven to produce serine protease) regenerate in 10 ml mrsb (37 c, 50 rpm, 18 h) make starter in mrsb (10% inoculum size) 37 c, 150 rpm, 4-6 h trypsin production medium (glucose 0.3%, yeast extract 1%, skim milk 2%, ph 6) inoculum size 10% incubate at 37 c, 50 rpm, 18-24 h supernantant was used to measure trypsin activity (khantaphant et al. 2010) and protein concentration (bradford 1976) b method 2 make starter in mrsb (10% inoculum size) 37 c, 150 rpm, 4-6 h starter culture was centrifuged at 4 c, 1400 g (hitachi cr216, rotor r10a2), 5 min pellet was then used to inoculate production medium (glucose 0,3%; yeast extract 1%; skim milk 2%; ph 8) incubate 37 c, 50 rpm, 18-24 h starter culture vol. : production medium vol. = 1:2 harvest: centrifugation at 4 c, 5800 g (hitachi cr216, rotor r10a2), 15 min supernantant was used to measure trypsin activity (khantaphant and benjakul 2010) and protein concentration (bradford 1976) regenerate in 10 ml mrsb (37 c, 50 rpm, 18 h) lactic acid bacteria strains in glycerol stock (proven to produce serine protease) lp 027 fs2 kl3 db1 lb021 strains -1 u g 3 2.5 2 1.5 1 0.5 0 medium (glucose 0.3%, yeast extract 1%, skim milk 2%, ph 6) with inoculum size 10%. the culture was then incubated at 37 °c with stirring at 50 rpm for 18-24 h. method 2. starter culture was centrifuged at 4 °c, 1400 g (hitachi cr216, rotor r10a2) for 5 min and the pellet was then used to inoculate the production medium (glucose 0,3%, yeast extract 1%, skim milk 2%, ph 8). the production culture was then incubated at 37 °c, with 50 rpm stirring for 18-24 h. the ratio of starter culture volume : production medium volume is 1:2. each method was performed at least twice on each strain studied, thus the error bars, as shown in the figures, represent the errors obtain from these repeated experiments. the supernatants were separataed from cells by centrifugation (4 °c, 5800 g (hitachi cr216, rotor r10a2), 15 min) and the enzyme activities in the supernatants were measured. trypsin activity assay. trypsin activity was measured using α-n-benzoyl-dl-argininenitroanilide (bapna) as substrate, using a modified khantaphant and benjakul method (khantaphant and benjakul 2010). for the assay, 50 μl enzyme was mixed with 50 μl milliq water and 250 μl reaction buffer, and left for 5 min at 37 °c . the reaction buffer used was either trishcl 0.05m ph 8 or citrate buffer 0.05m ph 6. the ph of the reaction buffer used for the assay depends on the medium ph used for the enzyme production. the reaction was initiated by addition of 50 -1 μl bapna (sigma b4875) 2 mg ml and was allowed to continue for 20 min at 37 °c. to stop the reaction, 50 r μl 30% acetic acid was added to the reaction mixture. production of r-nitroaniline was measured by measuring the absorbance of the reaction mixture at 410 nm. a blank reaction was conducted in the same manner, except that the sample was added after addition of the acetic acid. trypsin activity was calculated as follows: -1 -1 where 8800 (cm m ) was the extinction coefficient of nitroaniline; a and a were the sample and blank 0 absorbances at λ 410, respectively. one unit of enzyme activity was defined as the activity that causes the release of 1 nmol r-nitroaniline per minute. trypsin inhibition assay. the trypsin inhibition assay was performed using soybean trypsin inhibitor -1 (sbti) with activity 1000 u mg (sigma, cat. number 93620). the assay was performed by dissolving 0.5, 1, 1.5, 2, 2.5 mg sbti in 1 ml enzyme and then left to incubate at room temperature for 15 min. then, the trypsin activity of the mixture was assayed. the percent inhibition was calculated by the following formula : ph and temperature profile. the assay to determine the ph and temperature pofile was performed as described previously by khantaphant and benjakul (2010). the optimum temperature as determined by the temperature profile was then used for the measurement to determine the ph profile. trypsin stability assay. stability assays were performed to check trypsin stability in the range of ph and temperature values tested. the assays were performed based on khataphant and benjakul (2010) method . trypsin stability was determined by measuring the remaining activity after the enzyme was treated at various ph and temperature values. for thermal stability, the enzyme was incubated at temperatures 24, 35, 45, 50, 55, 60, 65, and 70 °c for 30 min before transferred to ice. then, the residual activity was measured. for ph stability, trypsin was mixed with an equal volume of the appropriate buffer, and left for 30 min at room temperature. the buffer used was either 0.05m acetic acid-sodium acetate buffer for ph 4-6 or 0.05m tris hcl buffer for ph 7-9 (khantaphant and benjakul 2010). the activity was then assayed at the optimal ph and temperature using (benzoyl arginine rnitroanilide) bapna as the substrate. volume 6, 2012 microbiol indones 51 activity activitynot-inhibited inhibited activitynot-inhibited x 100% trypsin activity (unit/ml) = (a-a ) x mixture volume (ml) x 10000 8800 x reaction time (min) x 0.2 fig 2 specific trypsin like activities produced by the isolates. isolates lp027, fs2, kl 3, and db1 showed differences in activities when produced using different conditions, and thus studied further. isolate lb021 expressed similar activity whether produced using method 1 or 2. trypsin-like activity when produced using method 1; trypsin-like activity when produced using method 2. and trypsin-like activity were observed every six hours and the values were plotted against time (fig 3 and 4). the figures clearly show that the change of method used for culturing seemed to have affected not only the culture ph, but also the maximum trypsin-like activity shown by the cultures, although the maximum cells numbers seemed unchanged. since method 2 seemed to produce higher trypsin-like activity, this method was then selected as the better of the two methods. therefore, the characteristics of enzymes produced using method 2 were further explored. trypsin-like activities shown by the four strains were inhibited by soybean trypsin inhibitor (sbti). soybean trypsin inhibition assays were performed on enzymes produced using method 2 to prove that the activity was really caused by trypsin. when treated with sbti, the trypsin like-activity of the four strains seemed to be considerably inhibited (fig 5). the percent inhibition also increased, although not to the same extent, with the increase of sbti concentration. ph and temperature profiles and stabilities. the ph and temperature profiles showed that for all strains, the optimum ph was observed around ph 8 and the optimum temperature was observed around 36-38 °c (fig 6). the trypsin-like activity seemed to be retained above 60% when enzymes were heated at temperature range 37-38 °c (fig 7). the ph stability assay shows somehow more divergen result. although the trypsin-like activity expresed by fig 2-k13 seemed to be most unaffected when incubated at ph 8 for 30 min prior to activity measurement, this ph value considerably reduced the activity expressed by strains lp027 and db01. on the other hand, the trypsin-like activity expressed by lp027 seemed to be unaffected after incubation at ph 7.5, whereas, db1 retained the activity the best at ph 7.0. discussion previous publication described the selection of strains producing trypsin-like activity from lab collections known to express serine protease activity (wulansari and wahyuntari 2010). all the trypsin-like activites observed in the previous publication and in the c u r r e n t s t u d y w e r e b a s e d o n e x t r a c e l l u l a r measurements. as described by liu et al. (2010), there are three major components of lab proteolytic systems, which are the cell wall bound proteinases initiating the degradation of the extracellular caseins, the peptide transporters taking up the peptides into the results changing the ph of the production medium and inoculation method changed the ph of the culture. the different methods used for trypsin production seemed to affect the medium ph at the time of inoculation and at the time of harvest. before used for inoculation, the ph of starter cultures range between 4.87-5.74. the initial ph of the production cultures inoculated using method 1 ranged between 4.95-5.65 and went down to between 3.15-3.85 at the time of harvest. however, when the method was altered, the ph at time of inoculation increased to between 7.07-7.93 and retained around the level of 6.24-7.15 at the time of harvest. harvesting was performed when trypsin-like activity reached maximum levels during culture growth (table 1), as previously optimized. trypsin-like activity of the cultures were assayed and the results showed that when the bacteria were cultured using method 2, four strains, namely lp027, db1, fs2, and kl3, showed a considerable increase of trypsin likeactivity. one strain, however, lb021, showed exactly the same activity whether the enzyme was produced using method 1 or 2. the rest of the strains tested showed no considerable trypsin-like activity, whether cultured using method 1 or 2. changing the ph of the production medium and inoculation method affected the growth rate and the specific trypsin-like activity. the four strains that showed considerable increase of activity were then cultured using two different methods (method 1 and 2) as described earlier. the culture ph, number of cells microbiol indones52 wulansari et al. *growol is an indonesian traditional food made of cassava **dadih is an indonesian traditional drink made of fermented buffalo milk. it is similar to yoghurt in taste and appearance strains o rigin harvest time (hours afte r inoculation) lc 262 fer mented glutino us rice 18 lp 027 growol* 18 fs 2 infa nt fae ces 18 kj 1 che ese 18 sk 3 horse m ilk 18 kl 3 co conut water 21 db 1 dad ih** 21 t 3 earth 21 lb 021 fer mented glutino us rice 24 kj 3 che ese 24 kl 2 co conut water 24 table 1 strains used in the study, their origins (susanti et al. 2007) and harvest time cells, where they would be further digested by various intracellular peptidases (liu et al. 2010). searching the ncbi protein database using keywords trypsin and lactic acid bacteria several proteins from lab reported to show trypsinlike activity were identified. this, obviously, is relevant to the observed trypsin like activity observed in some of the strains screeed earlier (wulansari and wahyuntari 2010). in the previous work, production medium with ph 6 was used (wulansari and wahyuntari 2010). this ph value was used because the cultures were initially grown in mrs medium (ph 6) prior to inoculation to the production medium. in addition, ph around 6 was considered the optimum ph required for the growth of lactic acid bacteria (hutkins et al. 1993). the production medium was inoculated by directly transferring 10% inoculum previously grown in mrs edium (method 1), without separating the cells from the growth medium. using this method, the production medium ph immediately dropped to between 4.98-6.19 (fig 3). the ph further decreased until it reached between 3.25-3.71 at the end of the fermentation (fig (http://www.ncbi.nlm.nih.gov/ protein) 3), which is probably due to the secretion of lactic acid by the bacteria. this condition is, of course, not good for the survival of the bacteria, since they are very prone to cell damage when the final medium ph goes below ph 5.0 (hutkins and nannen 1993). the low ph phenomenon was observed on cultures grown using method 1. the low ph of the cultures could have considerably reduced cell viability, since lactic acid bacteria in general grow at ph range 4.5-7.0 (hutkins and nannen 1993). regardless of the cells viability, low ph might also have affected the trypsin-like activity. as is widely known, trypsin works optimally at ph around 7.5-8.5 (koutsopoulos et al. 2007). at ph lower or higher than this range the activity would most likely be reduced, thus the lower ph might have considerably reduced the trypsin-like activity expressed by the strains tested. harvesting the cells by centrifugation was aimed at isolating cells from the acidic environment of the growth medium (see table 2 for ph of the starter culture). by harvesting the cells prior to their use for inoculation, the amount of growth medium, i.e. acidic medium, being carried over to the production medium volume 6, 2012 microbiol indones 53 fig 3 changes of ph, cell number and trypsin specific activity of strains (a) lp027, (b) db1, (c) fs2, and (d) kl3, cultured using method 1, the culture ph is represented in bar and the value for each time point is written above the bar, cell 8 -1 -1 numbers (x10 cells ml ), specific activity (u g protein). 8 -1 c el l d en si ty ( x 1 0 c el l m l ) a s p ec if ic a ct iv it y ( u g p ro te in ) -1 8 -1 c el l d en si ty ( x 1 0 c el l m l ) c s p ec if ic a ct iv it y ( u g p ro te in ) -1 time (hour) time (hour) b d (i.e. 18 h), it seems that the difference in cell numbers was unlikely to be the reason, because at 18 h the cell densities of all cultures, whether grown at ph 6 or 8, 8 were approximately the same at around 7 x 10 (fig 3 and 4). to further prove that the enzyme activities observed were really produced by trypsin, trypsin inhibition assays using soy bean trypsin inhibitor was minimised. thus, the sudden decrease of production medium ph immediately after inoculation as demonstrated when method 1 was used (ph after inoculation 4.98-6.15) was prevented. since during their growth lactic acid bacteria produce lactic acid, the end ph of the production medium was 3.25-3.71 when method 1 was used. on the other hand, when method 2 was used, the production medium ph was retained at around 7 at the beginning and around 6 at the end of the fermentation. although the extent of ph decrease in method 1 and 2 was more or less equal (i.e. around 1-2 ph unit), the end ph in method 2 remained above 6. the relatively more stable ph seemed to have been able to retain the trypsin like-activities expressed by the culture when produced using method 2. there could be two underlying reasons why the trypsin like-activities were higher in method 2. it was possible that at the higher ph, more bacteria could survive and so more enzymes were produced, or that at higher ph the enzyme was more stable compared to at lower ph, that the typsin like-activities were higher. looking at the numbers of cells at the time of harvest microbiol indones54 wulansari et al. fig 4 changes of ph, cell number and trypsin specific activity of strains (a) lp027, (b) db1, (c) fs2, and (d) kl3, cultured using method 2, the culture ph is represented in bar and the value for each time point is written above the bar, cell -1 numbers (x108 cells/ml), specific activity (u g protein). 8 -1 c el l d en si ty ( x 1 0 c el l m l ) a time (hour) 8 -1 c el l d en si ty ( x 1 0 c el l m l ) c -1 s p ec if ic a ct iv it y ( u g ) -1 s p ec if ic a ct iv it y ( u g ) time (hour) fig 5 percent inhibition by soybean trypsin inhibitor (sbti) of enzyme activity produced using method 2, lp027, db1, fs2, kl3. in h ib it io n ( % ) -1 sbti concentration (mg ml ) 100 90 80 70 60 50 40 30 20 10 0 control 0.5 1.5 2.0 2.51.0 b d t ry p si n r es id u al a ct iv it y ( % ) a temperature ( )°c 100 80 60 40 20 0 35 36 37 38 39 40 (sbti), were preformed (fig 5). the assays indicated that the activities expressed by all strains were inhibited by sbti, although to a different extent with different cultures. the amount of sbti required for the inhibition, however, was higher in comparison to the amount required to inhibit trypsin from other sources volume 6, 2012 microbiol indones 55 fig 6 temperature(a) and ph (b) profile of the trypsin like activity of the enzymes produced using method 2. the assays were performed as described previously by khantaphant and benjakul (2010). the optimum temperature as determined by the temperature profile was then used for the measurement to determine the ph profile. lp027, db1, fs2, kl3. -1 t ry p si n a ct iv it y ( u l ) a -1 t ry p si n a ct iv it y ( u l ) b temperature ( )°c ph fig 7 residual activity of trypsin produced by method 2, after treatment with various temperature (a) and ph (b). for thermal stability, the enzyme was incubated at temperatures 25, 35, 45, 50, 55, 60, 65, and 70 °c for 30 min before transferred to ice. then, the residual activity was measured. for ph stability, trypsin was mixed with an equal volume of the appropriate buffer (either 0.05m acetic acid sodium acetate buffer for ph 4 6 or 0.05m tris hcl buffer for ph 7 9 (khantaphant and benjakul 2010)), and left for 30 min at room temperature. the activity was then assayed at the optimal ph and temperature (as determined by temperature and ph profile analyses) using bapna as the substrate. lp027, db1, fs2, kl3. 34 35 36 37 38 39 40 41 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 34 35 36 37 38 39 40 41 b ph t ry p si n r es id u al a ct iv it y (% ) 100 80 60 40 20 0 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 haq iu, mukhtar h. 2006. biosynthesis of protease from lactobacillus paracasei: kinetic analysis of fermentation parameters. indian j biochem biophys. 43(6): 377-381. hutkins rw, nannen nl. 1993. ph homeostasis in lactic acid bacteria. j. dairy sci. 76: 2354 2365. khantaphant s, benjakul s. 2010. purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (lutjanus vitta). food chemistry. 120(3): 658-664. koutsopoulos s, patzsch k, bosker wte, norde w. 2007. adsorption of trypsin on hydrophilic and hydrophobic s u r f a c e s . l a n g m u i r 2 3 : 2 0 0 0 2 0 0 6 . d o i : 10.1021/la062238s. liu m, bayjanov j, renckens b, nauta a, siezen r. 2010. the proteolytic system of lactic acid bacteria revisited: a genomic comparison. bmc genomics. 11(1): 36. oberg cj, broadbent jr, strickland m, mcmahon dj. 2002. diversity in specificity of the extracellular proteinases in lactobacillus helveticus and lactobacillus delbrueckii subsp. bulgaricus. lett appl microbiol. 34(6): 455-460. pezzilli r. 2009. chronic pancreatitis: maldigestion, intestinal ecology and intestinal inflammation. world j gastroenterol. 15(14): 1673-1676. piuri m, sanchez-rivas c, ruzal sm. 2003. adaptation to high salt in lactobacillus: role of peptides and proteolytic enzymes. j appl microbiol. 95(2): 372-379. struthers bj, macdonald jr. 1983. comparative inhibition of trypsins from several species by soybean trypsin inhibitors. j nutr. 113(4): 800-804. susanti i, kusumaningtyas rw, ilaningtyas f. 2007. uji sifat probiotik bakteri asam laktat sebagai kandidat bahan pangan fungsional. [probiotic character assays of lactic acid bacteria as candidates of functioned food sources]. j teknologi dan industri pangan. 18(2): 89-95. takada k, hirasawa m. 2000. expression of trypsin-like activity by the genera corynebacterium, actinomyces in canine periodontitis. j med microbiol. 49(7): 621625. waljee ak, dimagno mj, wu bu, schoenfeld ps, conwell dl. 2009. systematic review: pancreatic enzyme treatment of malabsorption associated with chronic pancreatitis. aliment pharmacol ther. 29: 235-246. doi: 10.1111/j.1365-2036.2008. 03885.x. whitcomb dc, lowe me. 2007. human pancreatic digestive enzymes. dig dis sci. 52: 1-17. doi: 10.1007/ s10620-006-9589-z. wulansari d, wahyuntari sb. 2010. selection of trypsin likeprotease producing lactic acid bacteria and characterisation of the enzyme. in: wahyuntari b, pawiroharsono s, setyahadi s, helianti i, trismilah, editors. conference on industrial enzyme and biotechnology; 2010 august 3, serpong, indonesia. p 30-36. (struthers and macdonald 1983), including bacteria (takada et al. 2000; hansen et al. 2005). the trypsin like-activities of the strains were stable at range between 36-38 °c and the effect of temperature treatment was least when the enzymes was treated at 37 °c for 30 min prior to measurement. the ph stability studies showed that the trypsin like-activities were still detectable after treatment at ph range 5.5-8 for 30 min prior to activity measurement (fig 7). the ph and temperature profiles also showed more or less the same patterns, with optimum temperature around 36-38 °c, optimum ph aroun 7-8. this is relevant to the general knowledge that trypsin has an optimum temperature of 37 °c and an optimum ph between 7.5-8.5 (koutsopoulos et al. 2007). considering the results of inhibition experiments, as well as the patterns of temperature and ph profile and stability, shows that the activities observed were really expressed by trypsin from the lactic acid bacteria. n-terminal sequencing would be necessary to be entirely sure the protein is pure enzyme. in conclusion, it is clear that the methods used for inoculation of production medium, in addition to the production medium ph, affect the trypsin-like activity. as clearly and thoroughly presented above, using production medium with ph 8 and separation of cells from growth medium prior to inoculation to the production medium, increased the trypsin-like activity produced. this is a better method for trypsin production from lactic acid bacteria. references bintsis t, vafopoulou-mastrojiannaki a, litopouloutzanetaki e, robinson rk. 2003. protease, peptidase and esterase activities by lactobacilli and yeast isolates from feta cheese brine. j appl microbiol. 95(1): 68-77. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding. anal biochem. 72: 248-254. caplice e, fitzgerald gf. 1999. food fermentations: role of microorganisms in food production and preservation. int j food microbiol. 50(1-2): 131-149. courtin p, monnet v, rul f. 2002. cell-wall proteinases prts and prtb have a different role in streptococcus thermophilus/lactobacillus bulgaricus mixed cultures in milk. microbiology. 148(pt 11): 3413-3421. hansen kk, sherman pm, cellars l, andrade-gordon p, pan z, et al. 2005. a major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis. proc natl acad sci u s a. 102: 83638368. doi: 10.1073/pnas.0409535102. microbiol indones56 wulansari et al. 1: 1 2: 2 page 3 page 4 page 5 page 6 page 7 page 8 3.mi-maryati surya available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.2.3issn 1978-3477, eissn 2087-8575 vol 10, no 2, juni 2016, p 57-64 *corresponding author; phone: +62-251-8320417, email: atie@indo.net.id hepatitis b virus (hbv) infection is globally distributed and infected approximately two billion people in the world population (who 2009). an estimation of 240 million people chronically infected with hbv(who 2015) in the world. it is the most common type of hepatitis found in developing countries, including indonesia which served as the third highest prevalence country in the world. the viral infection can cause acute and chronic hepatitis, cirrhosis, hepatocellular carcinoma (hcc) and can lead to death. in the united states and europe, approximately 10% of patients infected with hbv lead to a chronic disease with the consequences of development into liver cancer. while an effective vaccine is available for preventing hbv infection, there is no effective therapy for patients worldwide that are already chronically infected by hbv. therefore, we need a treatment strategy to reduce deaths (cdc 2015). the primary obstacle to conduct research for drug hepatitis b virus (hbv) is a dna virus with liver as primary target organ. this virus caused chronic infection that can progress to cirrhosis, liver cancer and even death. in vitro model system of hepatocyte cultures is important and widely used to study a variety aspects of hepatitis b. development of small animal tupaia sp. for the in vitro model system is an alternative to the existing hepatocyte cultures. the specific purpose of the study is to develop tupaia javanica hepatocytes culture for hbv replication, and in a broader spectrum to answer the need for in vitro model of hepatocytes. primary t. javanica hepatocytes (pth) culture was successfully maintained for 14 days to reach 80% confluence, and infection of javan gibbon hbv (gihbv) and orangutan hbv (ouhbv) onto the culture on day 15 showed viral replication for up to eight days as measured by polymerase chain reaction (pcr). pcr quantification indicated that the highest copy number of dna virus was detected on day two and decreased until day 8 after infection. cell receptor for hbv attachment, known as sodium taurocholate cotransporting polypeptide (ntcp) was expressed on the surface of pth and shown as green luminenscent when observed by immunofluorescence assay. sequence of partial s gene from the apes hbvs after the viruses have been infected to the pth showed amino acid identity to their wildtype as high as 99.29% for gihbv and 95.71% for ouhbv. this study suggested that the primary t. javanica hepatocytes culture can support the replication of gihbvand ouhbv. key words: hepatitis b virus, hepatocytes culture, in vitro model, tupaia javanica virus hepatitis b (vhb) adalah virus dna dengan organ hati sebagai target utama. virus ini menyebabkan infeksi kronis yang dapat berkembang menjadi sirosis, kanker hati bahkan kematian. kultur hepatosit adalah model in vitro yang penting dan banyak digunakan dalam mempelajari berbagai aspek terkait dengan hepatitis b. pengembangan model in vitro dari hewan kecil tupaia sp. merupakan alternatif kultur hepatosit yang sudah ada. tujuan dari penelitian ini adalah mengembangkan kultur hepatosit tupaia javanica (pth) untuk replikasi vhb sehingga secara luas dapat menjawab kebutuhan akan model in vitro hepatosit. kultur hepatosit primer t. javanica berhasil mencapai kepadatan 80% pada hari ke-14, dan infeksi vhb javan gibbon (gihbv) dan vhb orangutan (ouhbv) ke dalam kultur pada hari ke-15 menunjukkan replikasi virus hingga hari ke-8 berdasarkan pemeriksaan polymerase chain reaction (pcr). kuantifikasi pcr mengindikasikan bahwa jumlah kopi dna virus tertinggi terdeteksi pada hari ke-2 dan menurun hingga hari ke-8 setelah infeksi. pada pewarnaan imunofluoresensi, reseptor sel tempat pelekatan vhb, yaitu sodium taurocholate cotransporting polypeptide (ntcp) terekspresi pada permukaan hepatosit yang terlihat sebagai pendaran hijau. penjajaran sebagian gen s dari vhb kera setelah virus diinfeksikan ke dalam kultur primer hepatosit menunjukkan kesamaan asam amino terhadap virus wildtype sebesar 99.29% untuk gihbv dan 95.71% untuk ouhbv. penelitian ini mengindikasikan bahwa kultur hepatosit primer t. javanica mampu mendukung replikasi gihbv dan ouhbv. kata kunci: kultur hepatosit, model in vitro, tupaia javanica, virus hepatitis b primary tupaia javanica hepatocytes culture as in vitro replication system for ape hepatitis b viruses 1,2 1,2 2 1,2 maryati surya *, diah iskandriati , silmi mariya , uus saepuloh , 2 1,2,3 1,2,3 permanawati , dondin sajuthi , and joko pamungkas 1 primatology program, graduate school, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; 2 primate research center, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; 3 faculty of veterinary medicine, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia development and gene-based therapy for hbv infection is the lack of good in vitro as well as in vivo systems using small animal model, which can be infected by the virus and assure the replication occur in the system (guha et al. 2004). in the development of in vitro model, hepatocytes cultures have been demonstrated using hepatocytes of human origin, both primary cell cultures or hepatoma cells cultures. however, the use of hepatocytes culture of human origin is limited due to ethical factors. this limitation led researchers to develop hepatocyte cultures of small animal origin. the primary hepatocytes cultures of woodchuck and peking duck have been successfully developed, but these cultures were only capable in supporting woodchuck hepatitis virus (whv) and duck hepatitis b virus (dhbv) replication (tuttleman et al. 1986; theze et al. 1987; witt-kehati et al. 2016). tupaia belangeri, a small mammal, can be infected by hbv experimentally although so far there is no data found regarding natural infection with hbv in tupaia sp. (guhaet al. 2004). t. belangeri have been used as an animal model to study the effectiveness of the vaccine, the safety of blood product, the effectiveness of chemotherapy and disinfectants against viral hepatitis. human hbv can infect hepatocytes of t. belangeri both in vitro and in vivo. in addition, primary hepatocytes of t. belangeri can also be infected with woolly monkey’s hbv (wmhbv). this virus was able to synthesize mrna and cccdna, and release hepatitis b surface antigen (hbsag) and hbeag into the culture medium (walter et al. 1996; kocket al. 2001; glebe et al. 2003). the suceptibility of t. belangeri against human hbv infection in the in vivo and in vitro systems making these animals good model of liver cancer caused by hbv and aflatoxin b1 (cao et al. 2003). t. belangeri is found in south-east asia, north of the isthmus of kra, including thailand, bangladesh, burma, far north-eastern india and nepal, southern china, cambodia, lao pdr, viet nam, and associated coastal islands, including hainan (han et al. 2008a). the limitations to use these animals out of their habitat is an obstacle for the development of antiviral drugs and vaccine research in indonesia. it is necessary to find an attempt to replace it with other species of animals that have a genetic relationship with t. belangeri. tupaia javanica, one of several indonesian tupaia, is distributed in bali, java, nias, and western sumatera (han et al. 2008b; roberts et al. 2011). until now there has been no report of the use of this animal in biomedical research. t. javanica has genetic similarity to t. belangeri up to 91 % (analyzed using basic local alignment search tool). it becomes an opportunity to explore the ability of t. javanica hepatocytes to support the replication of hbv as occurs in hepatocytes t. belangeri. the purpose of this study was to developed t. javanica hepatocytes cultures to assess the ability of the hepatitis b virus replication in cell culture. it is expected to answer the need for hepatocytes in vitro model for biomedical research related to hepatitis b. materials and methods isolation and primary culture of tupaia hepatocytes. three wild adult t. javanica horsfield 1822 (identified by the indonesian institute of science number 269/iph.i.03/ks.02/ix/2013, fig 1) were maintained in the quarantine facility of primate research center, bogor agricultural university (ipb prc). all animals procedures were performed by veterinarians at the ipb prc, and animals protocols have been evaluated and approved by ipb prc institutional animal care and use committee (iacuc) number prc-ipb-13-d004. procedure of hepatocytes isolation was refered to glebe et al. (2003) with the following modification: pre-perfusion washing solution (hanks balanced salt solution from invitrogen, usa; cat# 14170-112, 5 mm egta, 0.25 -1 µg ml amphotericin b), and perfusion solution (dmem, 100× cacl , 1% colagenase ii) was used to 2 remove red blood cells from the liver.this step was done using 50 ml syringe until the liver was semisolid and pale. livers then were minced and incubated at 37 °c for 5-10 min, centrifugated at 300-500 g for 5 min, and washed using saline buffer to obtain cell 4 pellets. cells were plated in 24-well culture plate at 10 cells per well using growth media (hbm and hcm hepatocytes media from lonza usa supplemented with 20% fetal bovine serum) at 37 °c and 5% co .2 immunofluorescence of ntcp. the assay was performed after the cells reach 80% of its confluence. the cells were washed with saline buffer followed by fixation with methanol and acetone. cells were then stained with ntcp polyclonal antibody (h-42, from santa cruz biotechnology, inc usa) and incubated at 37 °c for 1 h. the cells were washed with saline buffer and incubated with anti-rabbitigg fluorescein isothiocyanate (fitc) (sigma usa) for 1 h at 37 °c. the cells images were captured with nikon fluorescence microscope. ntcp unstained cells were served as negative control. virus infection on the cells. ouhbv and gihbv 58 surya et al. microbiol indones isolates were obtained from the viruscollection of ipb -1 prc. hbsag titers (iu ml ) of the isolates were 60 -1 -1 000 iu ml (gihbv), and 125 000 iu ml (ouhbv).virus suspension from both isolates were added to pth on day 15 after the cells reach 80% confluence followed by 18 h of incubation at 37 °c with 5% co . the cells were washed with saline buffer 2 to removed excess virus and later growth media were added. the released viruses were collected from infected cells media on days 1 to 8 after infection. detection and quantification of hbv replication. detection of released hbv in the media was performed using polymerase chain reaction (pcr) and the number of dna virus were calculated using real time pcr (qpcr). dna was extracted using qiamp blood dna mini kit (qiagen, usa) according the manufacture’s procedures. the primer set used in this method was described by warren et al. (1999). this primer set amplified the partial s gene of hbv and yielded aproximately 456 bp fragment of this gene. pcr amplification conducted using conventional pcr (applied biosystem 9700, usa) at the conditions of 94 °c for 10 min, followed by 40 cycles at 94 °c for 30 s, 55 °c for 30 s and 72 °c for 2 min, with a final extension at 72 °c for 10 min. pcr reaction at 25 µl total reaction -1 containing 10 pmol ml of each primer, 12.5 ul kappa hotstartreadymix (kapa biosystems, usa), 5 µl dna and adjusted with nuclease free water to 25 µl. amplicons (5 µl) were visualized on 1% agarose gel and sized against a 100-bp dna ladder (invitrogen, usa). for hbv replication quantification, we used iq5 real time pcr (bio-rad, usa). real time pcr amplification were performed by following protocol: denaturation for 2 min at 98 °c, amplification for 40 cycles consisting of 5 sec at 98 °c and 10 sec at 55 °c. -1 the reactions were 10 pmol ml of each primer, 12.5 µl ssofastevagreensupermix (bio-rad, usa), 5 µl dna and adjusted with nuclease free water to 20 µl. sequencing and alignment of wildtype and post-infection hbvs. hbv dna from wildtype and post-infection were extracted using dna purifying kit (qiagen, usa). sequencing of nucleotides were st carried out in applied biosystems at 1 base malaysia using the primer set mentioned above. alignment of the nucleotide sequences was done with clustalw (embl-ebi). results the liver organ was collected from adult male t. javanica horsfield, 1822 (fig 1) and prepared for primary hepatocytes culture. on day 1, cells showed individual elongated-shaped for 3 d and then the cells transformed into polygonal shape and began to form colony. from the observation the stage of hepatocytes proliferation on day 1, 4, 7, and 14. cells reached 80% confluence on day 14 (fig 2). immunofluorescence assay was carried out to confirm that t. javanica hepatocytes has ntcp receptor for hbv entry into the cells. fig 3 showed the expression of ntcp indicated by the greenish fluoresceinated color under fluorescence microscope while the unstained cells showed no fluorescein coloring. the greenish fluoresceinated cells resulted from the reaction of ntcp protein bound to its antibody which later reacted with anti-rabbit igg conjugated to fitc. the result showed 70% field view ntcp. the presence of virus did not indicate any change in cells morphology or cytophatic effect of the cells (fig 4). there were no differences in hepatocytes fig 1 tupaia javanica horsfield 1822 (identified by the indonesian institute of science number269/iph.i.03/ks.02/ix/2013), photographed by walberto sinaga. volume 10, 2016 microbiol indones 59 the amplification of hbv partial s gene indicated that viral replication occurred from day 1 to day 8. in this study, the peak of viral dna copy number (3 835 and 4 825 viral copy number) was on day 2 for both gihbv and ouhbv. the viral copy number decreased after day 2 for both viruses, although ouhbv tends to increased until day 5. fig 6 showed viral dna copy number as measured by real time pcr. morphology between the infected and uninfected pth. the cells morphology was colonized polygonal-shaped. fig 5 showed detection of amplified hbv dna by conventional pcr assay. it showed the presence of both gihbv and ouhbv dna from day 1 to day 8 after infection. the 456 bp fragment was shown by dna band with different thickness indicating the initial concentration of released hbvs in the culture medium. fig 2 cells morphology of pth cultures: (a) day 1, individual elongated shape cells, (b) day 4, colony of polygonal shape cells at approximately 30% confluence, (c) day 7, colony of polygonal shape cells at approximately 50% confluence;(d) day 14, colony of polygonal shape cells at approximately 80% confluence. bar = 200 µm. fig 3 detection of ntcp receptors in pth culture using immunofluorescence assay; (a) negative control (without ntcp antibody),(b) hepatocyte ntcp(green objects, 70% field view). bar = 100 µm. a b a dc b 60 surya et al. microbiol indones in ouhbvthere were 14 points of nucleotide mutation (t2875c, a2918c, a2967c, a2977c, t3022a, t3071a, t3077c, a3112g, g3116t, a7g, g10t, a20g, a28g, and a44t) resulted 96,67 % identity between wildtype and post-infection. out of the 14 nucleotide mutations occurred, only a change of 6 amino acids had occurred (identity 95.7%): thr40asn, asp43glu, thr75ser, ser90ala, ala119thr, and phe130tyr. fig 7 showed the aminoacid alignment to see if the virus infection to tupaia cells causes any mutation of hbv, the sequencing for both viruses from wildtype and post-infection were carried out using applied biosystems. in the gihbv partial s gene occurred one point of mutation at nucleotide position 78 (g78a) with identity up to 99.76% between wildtype and post-infection viruses, that caused amino acid changes, glycine to glutamate (gly138glu) with identity 99.29%. on the other hand, c b a fig 4 cells morphology of pth culture preand post-infection with apes hbv: (a) pth on day 14, pre-infection, (b) pth on day 22, post-infection with gihbv, (c) pth on day 22, post-infection with ouhbv. bar = 200 µm. fig 5 replication of hbv dna detected in culture medium. apes hbv were used to infect pth. detectionof partial s gene was done by conventional pcr and visualized on 1% agarose gel, resulted 456 bp (m= marker; 1-8=supernatant days 1-8 after infection): (a) gihbv replication, (b) ouhbv replication. 300 pb m m m m 8 7 5 6 1 2 4 3 1 2 3 4 5 6 8 7 a b 100 pb 500 pb 400 pb 200 pb volume 10, 2016 microbiol indones 61 study were slower; the proliferation of hepatocytes reached to its 80% confluence after 14 d. animals condition might be one of the factors causing this difference. animals used in the study of glebe et al. (2003) were captive bred with known age, meanwhile the animals used in this study were wild caught, in between wildtype and post-infection virus. discussion compared to what was described by glebe et al. (2003), the growth of t. javanica hepatoyctes in this v ir al c o p y n u m b er day 1 2 3 4 5 6 7 8 5000,00 4500,00 4000,00 3500,00 3000,00 2500,00 2000,00 1500,00 1000,00 500,00 0,00 fig 6 quantification of gihbv and ouhbv replication from pth culture medium using real-time pcr based on viral copy number. gihbv ( ), ouhbv ( ), and pth ( ). fig 7 alignment of partial s amino acid sequences between wildtype and post-infection apes hbv onto pth culture: (a) gihbv, (b) ouhbv. note : conserved sequences (*), conservative mutation (:), semi-conservative mutation (.), nonconservative mutation ( ). a b 62 surya et al. microbiol indones (2003). decreased ability to support the replication of cells after the eighth day probably caused by cell death or reduced cell proliferation ability. declining of viral copy number after day 2 probably was caused by the reduction of cells number. primary cell cultures are cultures of cells obtained directly from normal tissue or organ, and have not been subcultured through passages. cells in primary culture have a limited lifespan and at one stage cells will not multiply and die. primary cell cultures are still carrying cell genotype origin, and primary hepatocyte culture can lose their phenotype and further cellular mechanisms of the cell cannot support further viral replication (lin et al. 2007). generally, the replication graphic trend was decreasing two days post-infection, the fact that there was an increase of viral copy number of ouhbv on day 5 was still unknown. sequences of gihbv partial s gene from wildtype and post-infection were aligned and showed one nucleotide change from g to a at position 78 (g78a, identity 99.76%) and caused amino acid changes, from glycine to glutamate (gly138glu) with identity 99.29% (fig 7a). meanwhile, in ouhbv there were 14 nucleotides changes in partial s gene sequences from wildtype and post-infection to the cells (identity 96.67%): t2875c, a2918c, a2967c, a2977c, t3022a, t3071a, t3077c, a3112g, g3116t, a7g, g10t, a20g, a28g, and a44t. nucleotides changes resulted in the changes of six amino acids (identity 95.7%): thr40asn, asp43glu, thr75ser, ser90ala, ala119thr, and phe130tyr (fig 7b). these changes might have occurred due to the fact that tupaia hepatocytes are not the original host for apes hbv, so the changes occurred was thought as adaptation process for the hbv to replicate in a new host. the primary t. javanica hepatocytes culture developed in this study has similar characteristic to t. belangeri hepatocytes that has been described previously with polygonal-shape morphology. the pth in this study was able to proliferate in vitro and reach 80% confluence in growth medium after 14 d of incubation. the t. javanica primary hepatocytes also showed evidence for the presence of ntcp receptors, as captured by immunofluorescence assay. there were amino acid sequences mutations of partial s geneafter the infection of apes hbv onto pth culture. considering the above mentioned findings, we conclude that t. javanica hepatocytes can be utilize as an alternate in vitro system for the replication of apes hbv (gihbv and ouhbv). to this date, this report provides the first which the age of the animals were determined by estimation. the number of hepatocytes was influenced by the liver size and weight, so it depends on the age of the animal. to obtain hepatocytes, glebe et al. (2003) described the use of pump to control the speed and pressure of the solution to minimize cells damage caused by high pressure during perfusion stages. in our study, perfusion was performed manually using syringe thus more damaged cells might have occurred which lead to the decreasing of cells number. despite the difference of the perfusion methods, in this study t. javanica hepatocytes were successfully isolated and cultured showing different morphologic appearance during cell proliferation on day 1, day 4, day 7, and day 14. on day 1, hepatocytes appeared as individual elongated-shape cells, while on day 4 it formed colony of polygonal-shape cells, and on day 14 the surface of the substrate covered by hepatocytes with 80% confluence. the polygonal-shape of the pths showed in this study was similar to the morphology of the hepatocytes from the study published by glebe et al. (2003). similar to the study conducted by yan et al. (2012) on t. belangeri, in this study we demonstrated by immunofluorescence assay that t. javanica hepatocytes also carry ntcp as cell surface receptor to facilitate the entry of hbv into the cell. in humans, the hepatocytes have a surface protein ntcp specifically interacts with the large surface protein of hbv, thereby functioning as a viral entry receptor. the expression of ntcp correlates with the susceptibility of the target cells. experimental reduction of ntcp expression markedly inhibited hbv and hdv infection on known susceptible cells (iwamoto et al. 2014; yan et al. 2014). both ape hepatitis viruses (gihbv and ouhbv) infected the pth up to eight days post infection, as demonstrated by the amplification of hbv s gene from supernatant of infected cells. this results were also supported by the viral copy number that can be detected up to the eighth day using quantification pcr method. hbv binds to its receptor (ntcp) and enters the cell through endocytosis and fuses to the endosome, thus releasing its nucleocapsid. a comprehensive illustration of replication of the hepadnaviral genome was described by beck and nassal (2007). the existence of ntcp, and the ability to amplify its gene indicated the capability of t. javanica hepatocytes in supporting the replication of non human primate hbv without causing cythopathic effect. this is in accordance to the study conducted by glebe et al. volume 10, 2016 microbiol indones 63 lin m, chen q, yang ly, li wy, cao xb, wu jr, peng yp, chen mr. 2007. hepatitis b virus infection and replication in primarily cultured human fetal hepatocytes. world j gastroenterol. 13(7):1027-1031. doi:10.3748/wjg.v13.i7.1027. pichard l, raulet e, fabre g, ferrini jb, ourlin j-c, maurel p. 2006. human hepatocyte culture. methods in molecular biology. cytochrome p450 protocols 320:283-293. doi: 10.1385/1-59259-998-2:283. roberts te, lanier hc, sargis ej, olson le. 2011. molecular phylogeny of treeshrew (mammalia :scandentia) and the timescale diversification in southeast asia. mol phylogenet evol. 60(3):358-372. doi:10.1016/j.ympev.2011.04.021. theze n, gripon p, fourel i, hantx o, trepo c, guguenguillouzo c. 1987. maintenance of woodchuck hepatitis virus activity in woodchuck hepatocyte primary culture. j gen virol. 68:1029-1039. doi:10.1099/0022-1317-68-4-1029. tuttleman js, pugh jc, summers jw. 1986. in vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis b virus. j virol. 58(1):1725. walter e, keist r, niedero st b, pult i, blum he. 1996. hepatitis b virus infection of tupaia hepatocytes in vitro and in vivo. hepatology 24(1):1-5. warren ks, heeney jl, swan ra, heriyanto, verschoor ej. 1999. a new group of hepadna viruses naturally infecting orangutans (pongo pygmaeus). j virol. 73(9):7860-7865. witt-kehati d, alaluf mb, shlomai a. 2016. advances and challenges in studying hepatitis b virus in vitro. viruses 8:21. doi:10.3390/v8010021. world health organization. 2009. hepatitis b vaccines. we e k l y e p i d e m i o l o g i c a l r e c o r d . 4 0 : 4 0 5 420.<http://www.who.int/wer/2009/wer8440.pdf>. [20 july 2015]. world health organization. 2015. manual for the development and assesment of national viral hepatitis plans. <http://apps.who.int/iris/bitstream/10665/18372 6/1/9789241509350_eng.pdf?ua=1>. [20 october 2015]. yan h, zhong g,xu g, he w, jing z, gao z, huang y, qi y, peng b, wang h, fu l, song m, chen p, gao w, ren b, sun y, cai t, feng x, sui j, li w. 2012. sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus. elife 1-28. doi:10.7554/elife.00049. yan h, peng b, liu y, xu g, he w, ren b, jing z, sui j, li w. 2014. viral entry of hepatitis b and d viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide. j virol. 88(6):3273-3284. doi:10.1128/jvi .03478-13.2016. evidence for the existence of a novel in vitro model of apes hbv infection on indonesian primary tupaia hepatocytes culture, which can be explored further for the potential study of immunotherapeutic approaches against viral hepatitis b. acknowledgment the authors would like to express their gratitude to the ministry of research, technology, and higher education of the republic of indonesia for the funding support of the research grant provided through the research scheme of university excellent research (pupt). the authors would also like to specially thank i nengah budiarsa and mr. walberto sinaga for the animal procurement in this study. references beck j, nassal m. 2007. hepatitis b virus replication. world j gastroenterol. 13(1):48-64. doi:10.3748/wjg.v13.i1.48. cao j, yang eb, su jj, li y, chow p. 2003. the tree shrews: adjuncts and alternatives to primates as models for biomedical research. j med primato.l 32(3):123-130. doi:10.1034/j.1600-0684.2003.00022.x. cdc <http://www.cdc.gov/vaccines/pubs/surv-manual/chp t04-hepb.pdf>. [20 october 2015]. glebe d, aliakbari m, krass p, knoop ev, valerius kp, gerlich wh. 2003. pre-s1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus. j virol. 77(17):9511-9521. doi:10.1128/jvi.77.1 7.9511-9521. guha c, sung wl, chowdhury nr, chowdhury jr. 2004. cell culture models and animal models of viral hepatitis. part ii:hepatitis c. lab animal 34(2):3947.doi: 10.1038/laban0205-39. han kh, duckworth jw, molur s. 2008. tupaia belangeri. the iucn red list of threatened species2008: e.t41492a10468101. doi:10.2305/iucn.uk.2008.rl ts.t41492a10468101.en. han kh, maharadatunkamsi d. 2008. tupaia javanica. the iucn red list of threatened species 2008: e.t41496a10469813. doi:10.2305/iucn.uk.2008.rl ts.t41496a10469813.en. iwamoto m, watashi k, tsukuda s, aly hh, fukasawa m,fujimoto a, suzuki r, aizaki h, ito t, koiwai o, kusuhara h,wakita t. 2014. evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells over expressing a membrane transporter ntcp. biochem bioph res comn. 443(3):808-813. doi: 10.1016/j.bbrc.2013.12.052. kock j, nassal m, macnelly s, baumert tf, blum he, von weizsacker f. 2001. efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis b virus. j virol. 75(11): 5084-5089. doi:10.1128/jvi.75.11.5084-5089. 64 surya et al. microbiol indones 1: 57 2: 58 3: 59 4: 60 5: 61 6: 62 7: 63 8: 64 02 camesi.cdr biofilms produced by pathogenic bacteria are important issues in a number of chronic infections and diseases such as atherosclerosis, chronic sinusitis, chronic wounds, cystic fibrosis, endocarditis, inner ear infections, kidney stones, leptospirosis, osteomyelitis, osteonecrosis, osteomyelitis of the jaw, periodontal disease, prosthetic joints and heart valves, urinary tract infections, and also veterinary diseases (skogman 2012). these naturally existing biofilms are major threats to humans as they are far more resistant to antibiotics and can particularly escape from host immune system (skogman 2012). bacterial attachment and biofilm formation are important steps in the establishment of chronic infections, thus, making them causative agents of some diseases (skogman 2012). biofilms can also appear in drinking water systems and serve as a significant environmental reservoir for pathogenic microorganisms (skogman 2012). biofilm is defined as a complex structure of microbial cells aggregate covered by self-synthesized matrix consist of extracellular polysaccharides, dna, and proteins. these cells aggregate then permanently attached into biotic or abiotic surfaces. biofilm formation is associated with the virulence factor of pathogenic microbes and cell protection of the vol.10, no.3, september 2016, p 87-94 doi: 10.5454/mi.10.3.2 screening of antibiofilm activity from marine bacteria against pathogenic bacteria 1 1 alianda budhiriani rossati camesi , agustina lukito , diana elizabeth 1* 2 waturangi , hwang jae kwan and 1 faculty of biotechnology, universitas katolik atmajaya, jalan jenderal sudirman 51, jakarta 12930 , indonesia; 2 biomaterial research laboratory, department of biotechnology, yonsei university, 50 yonsei-ro, seodaemun-gu, seoul 120-749, korea bacterial biofilms produced by pathogenic bacteria have become a serious issue in several chronic diseases such as atherosclerosis, cystic fibrosis, endocarditis, inner ear infections, and kidney stones. thus, inhibition and destruction of bacterial biofilm from pathogenic bacteria is needed. the purpose of this study is to analyze biofilm inhibition and destruction activities of marine bacteria associated with hard and soft corals isolated from several oceanic regions in indonesia. fifteen marine isolates collected from several regions in indonesia such as bali province, south east sulawesi province, east java province, lampung province, and banten province were tested using static biofilm assay against several pathogenic bacteria. biofilm of the pathogenic bacteria tested were stained using 0.4% crystal violet. several isolates were sequenced using 16s rrna pcr method. most of marine isolates presented higher inhibition and or destruction activity at 10% crude concentration. few isolates were further identified using 16s rrna and proven to have antibiofilm activity against several pathogenic bacteria. in conclusion, marine bacteria have broad applications in medical and pharmaceutical industries and the oceanic regions of indonesia are promising sources for the discovery of novel bacteria with antibiofilm activity. key words: antibiofilm compounds, biofilm, chronic diseases, marine isolates, pathogenic bacteria biofilm bakteri yang diproduksi oleh bakteri patogen saat ini sudah menjadi masalah serius penyebab penyakit kronis seperti aterosklerosis, sistik fibrosis, endocarditis, infeksi telinga bagian dalam, dan batu ginjal. oleh karena itu, inhibisi dan destruksi biofilm bakteri patogen sangat diperlukan. tujuan dari penelitian ini adalah menganalisis aktivitas inhibisi dan destruksi bakteri laut yang berasal dari hard coral dan soft coral di beberapa perairan indonesia. sebanyak lima belas isolat bakteri laut yang dikoleksi dari beberapa perairan indonesia seperti provinsi bali, sulawesi tenggara, jawa timur, lampung, dan banten diuji terhadap biofilm bakteri patogen menggunakan metode biofilm statis. biofilm bakteri patogen yang digunakan diwarnai menggunakan kristal violet dengan konsentrasi 0.4%. beberapa isolat kemudian di-sekuens dengan metode 16s rrna pcr. sebagian besar isolate bakteri laut menunjukan aktivitas inhibisi dan destruksi yang lebih tinggi pada konsentrasi crude 10%. beberapa isolate kemudian diidentifikasi lebih lanjut menggunakan metode 16s rrna dan terbukti memiliki aktivitas antibiofilm terhadap beberapa bakteri patogen. bakteri laut memiliki aplikasi yang luas dalam bidang medis dan industri obat-obatan, dan wilayah lautan indonesia merupakan sumber yang menjanjikan dalam penemuan bakteri dengan aktivitas antibiofilm. kata kunci: bakteri pathogen, biofilm, isolate bakteri laut, penyakit kronis, senyawa antibiofilm microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-573-1740; fax: +6221-571-9060; email: diana.waturangi@atmajaya.ac.id pathogens which result in antibiotic resistance (dheilly et al. 2010). therefore, inhibiting or destroying pathogenic bacterial biofilms could reduce the resistance, and research efforts are focused on developing novel antibioflm compounds. the marine environment hosts a large biodiversity of many organisms which are important for source of food-related resources, energy, or medicine (parraga et al. 2011). despite this importance, thousands of marine microorganisms and their roles in the ecosystem still remain undiscovered (parraga et al. 2011). during the last decades, marine organisms have provided huge advantages to human life, by producing antioxidant and antimicrobial substances that help prevent the spread of serious diseases in human body (ngo et al. 2012). marine organisms are believed to be a potential source not only for biologically active substances for the development of pharmaceuticals, but also essential substances for the inhibition of biofilm formation by pathogenic bacteria (ngo et al. 2012). the bioactive compounds produced by marine bacteria are usually secondary metabolite compounds generated in response of external pressure such as competition for nutrient or space for example demonstrated antibiofilm activity from coral-associated bacteria againsts streptococcus pyogenes biofilm formation (nithya et al. 2010). the aim of this study was therefore to assess marine-associated bacteria from indonesian waters as potential sources for novel antibiofilm compounds. materials and methods marine isolates preparation. fifteen out of fifty six marine isolates from previous study (nataprawira 2014; putra 2014) which did not have antimicrobial activity were chosen to test their antibiofilm activity. marine isolates from several indonesian regions as shown in table 1 were inoculated using loop or sterile tooth pick onto marine agar (ma) (difco) and o incubated at 28 c for 2 days to produce single, wellisolated colonies. pure colony were then refreshed into o new ma medium and incubated at 28 c for 2 d to obtain sufficient amount of isolates. refreshed isolates o in ma medium were stored at 4 c. pathogenic bacteria preparation. seven pathogenic bacteria (staphylococcus haemolyticus, s t r e p t o c o c c u s p n e u m o n i a at c c 4 9 6 1 9 , staphylococcus aureus atcc 29213, pseudomonas aeruginosa atcc 27853, vibrio cholera c43, e n t e ro t o x i g e n i c e s c h e r i c h i a c o l i ( e t e c ) , enteropathogenic escherichia coli (epec)) were streaked onto brain heart infusion agar (bhia; oxoid) to obtain single pure colonies. pure colonies from bhia were picked and cultured in new sterile culture tubes containing 5-8 ml of brain heart o infusion broth (bhib; oxoid) and incubated at 37 c overnight. pathogenic bacteria being used were also adjusted to 0.5 mcfarland (600 nm = 0.132) with dilution methods using bhib. each diluted pathogen was placed in a sterile conical tube for further use. crude preparation. pure colonies from ma were picked and inoculated in sterile culture tubes containing 5-8 ml brain heart infusion broth (bhib) (oxoid) supplemented with 1% d-glucose and incubated aerobically using waterbath shaker (lab o companion, bs-21 ) for 3 d at 28 c. the optical d e n s i t y o f i s o l a t e s w a s m e a s u r e d u s i n g spectrophotometer (genesys 20, thermo spectronic). 1 ml of broth with marine bacteria was adjusted to 0.5 mcfarland (600 nm = 0.132) with dilution methods using bhib. each diluted marine isolate was placed in sterile conical tube which then be centrifuged at 8000 rpm for 10 min using sorvall legend rt centrifuge. supernatants were transferred to new sterile tubes and then centrifuged again at 8000 rpm for 10 min. supernatants were separated from pellets to smaller conical tubes and were then ready to use (kwasny and opperman 2010). supernatants obtained from the process above were used as crudes for antibiofilm test. antibiofilm activity assays of marine isolates. the methods were divided into two, to see whether the isolates inhibit the formation of biofilm from pathogenic bacteria (inhibition activity), or whether the isolates destruct the biofilm once it is formed (destruction activity). the assays were all under taken using 96-well polystyrene assay plates (round bottom; iwaki) with lids under sterile conditions inside a laminar hood. each isolate was used in two concentrations, 5% and 10%. for the inhibition activity, marine crudes (10 µl and 20 µl) were put first into the sterile 96-well assay plate. then, 200 µl of pathogens were transferred to 96-well microplate. pathogens alone were used as positive controls. bhib medium without pathogens was used as negative o control. microplates were incubated at 37 c overnight. for the destruction activity, 200 µl of pathogens were transferred to the plate first, and then incubated at o 37 c overnight. after that, 10 µl and 20 µl for 5% and 10% of marine crudes respectively were placed into the o plates and incubated at 37 c for 30 min. all the assays were done under sterile condition inside a laminar airflow. once finished, each plate was covered with the 88 camesi et al. microbiol indones lid, which increased biofilm formation by creating an environment with reduced oxygen tension. biofilms of the bacteria usually appears at the bottom of each assay well (moscoso et al. 2006). biofilm staining. after incubation, planktonic cells and spent media were discarded from microplates and adherent cells were gently rinsed with deionized water (dh2o) twice and allowed to air dry before being stained. the biofilms were stained by 200 µl crystal violet solution (0.4% w/v; merck) for 30 minutes. after that, crystal violet was removed from the plates and the wells were rinsed with deionized water 3-5 times. rinsed plates were dried before the addition of 200 µl 96% analytical grade ethanol (merck). after addition of ethanol, microplates were incubated for 30 min at room temperature. ethanol solubilized the remaining crystal violet in the microplates. after the incubation period, ethanol from each well was transferred to new microplates and optical density measured at 595 nm using biorad 680 microplate reader (moscoso et al. 2006). absorbance attained was then used to calculate the antibiofilm activity using the following formula (sun et al. 2005): dna sequencing. six out of fifteen isolates were further sequenced to identify the bacteria. the amplification of dna was performed using 16s rrna pcr (hoa et al. 2000). dna extraction was done using ctab and nacl (aris et al. 2013) which further tm amplified by pcr (100 termocycler,biorad) using forward primer 63f (5'cag gcc taa cac atg caa gtc-3') and reverse primer 1387r (5'ggg cgg wgt gta caa ggc-3') (aris et al. 2013). the pcr conditions were as followed: 94 °c, 2 min of predenaturation, 92 °c 30 s of denaturation, followed by 55 °c 30 s of annealing, continued with elongation at 72 °c 1 min (30 cycles). next was post pcr at 75 °c, 20 min and stopped at 4 °c (aris et al. 2013). the pcr products were run in electrophoresis using 0.8% agarose gel. then samples were sent for further sequencing to gentika science and the results were aligned using bioedit and blast program from ncbi. sequences obtained were submitted to genbank to obtain accession numbers. the accession numbers obtained from genbank were then used to construct phylogenetic tree using mega 7 program. results marine isolates were analyzed for their potential to inhibit and destruct biofilm formation of several pathogenic bacteria. for the inhibition activity, it can be seen from table 2 that most samples showed an increased inhibition activity when using crudes with concentrations of 10%. these results indicated that the more crudes are added, the higher the inhibition activity of marine crudes against pathogenic biofilms. however, in some samples like bc 11-1 against staphylococcus aureus and bf 08-4 against pseudomonas aeruginosa, though not significant according to the t-test value, the inhibition activity diminished as the crude concentration increased. some crudes such as bc 11-2, bc 11-3, and bf 14-1 showed no inhibition activity to several pathogens like staphylococcus haemolyticus and vibrio cholera at 5% nor 10%. this phenomenon might show up when higher crude concentration was needed to reach optimum inhibition activity or else they do not have activity at all. overall, results showed that all marine isolates had different inhibition activity against several pathogens with the most positive results against pseudomonas aeruginosa, and the least against vibrio cholera. beside inhibition activity, marine crudes are thought to possess destruction activity against mature pathogenic biofilms (amador et al. 2003). from table 3, most samples showed an increased destruction activity when using crudes with concentration of 10%. most samples showed good destruction activity against several pathogenic bacteria with the most positive destruction activity against staphylococcus aureus and least activity against vibrio cholera. the results showed some marine samples performed inhibition activity only such as bf 13-5 against staphylococcus haemolyticus and bb 07-7 against streptococcus pneumonia while others such as bf 09-7 and bc 11-3 performed destruction activity only against staphylococcus haemolyticus and streptococcus pneumonia, respectively. based on the results of inhibition and destruction activity, six out of fifteen marine isolates with the most stable inhibition or destruction activity were chosen for sequencing. the six isolates were bc 11-1, bf 04-1, bf 13-2, bf 13-5, bc 12-3, and bf 14-2. after the sequences were obtained, nucleotide blast program from ncbi was used to find out what bacteria are those compared to the highest former sequenced bacteria. the results are presented in table 3. nucleotide blast results showed that marine isolate bc 11-1 has 99% similarity with erythrobacter sp. marine isolate bf 04-1 has 98% similarity with enterobacter cloacae, volume 10, 2016 microbiol indones 89 % activity= positive absorbance sample absorbance positive absorbance negative absorbance 100%˟ 90 camesi et al. microbiol indones table 1 colony and cell morphology of endospore-forming rhizobacteria from rhizosphere of cabbage isolates sample sample type origin of sample bc 11-1 acropora desalwi hard coral bali north, bali province bc 11-2 acropora desalwi hard coral bali north, bali province bc 11-3 acropora desalwi hard coral bali north, bali province bb 08-3 lobophytum sp. soft coral bali province bf 08-4 scleronephthya sp. soft coral kapuran, east java province bf 09-7 heteroxenia soft coral bali province bf 13-2 studeriotes sp. soft coral kapuran, east java province bf 08-1 scleronephthya sp. soft coral kapuran, east java province bf 13-5 studeriotes sp. soft coral kapuran, east java province bf 14-1 nephthyigorgia sp. soft coral kapuran, east java province bf 14-2 nephthyigorgia sp. soft coral kapuran, east java province bc 12-3 acropora echinata hard coral kendari, south east sulawesi province bf 04-1 scleronephthya sp. soft coral lampung province bb 07-4 tubastrea micrantha hard coral cilegon, banten province bb 07-7 tubastrea micrantha hard coral cilegon, banten province bf 13-2 has 99% similarity with bacillus jeotgali, bf 13-5 has 99% similarity with paracoccus marcusii, bc 12-3 has 96% similarity with micrococcus luteus, and bf 14-2 has 97% similarity with cobetia marina. those marine isolates had already given the accession number from genbank (ncbi), kp763639, kp 763640, kp763641, kp763642, kp763643, and kp7763644 respectively. marine sequences from the nucleotide blast were then used to analyze genetic relationship of each individual sample using mega7 program. the phylogenetic tree was constructed using neighborjoining algorithm. from the phylogenetic tree, it can be seen that 4 samples (bc 11-1, bf 04-1, bf13-5, and bf fig 1 phylogenetic tree of marine isolates based on 16s rrna gene sequences compared to existing marine bacteria. volume 10, 2016 microbiol indones 91 14-2) showed close relationship with existing marine bacteria from genbank, while the other two samples (bc 12-3 and bf 13-2) showed distant relationship with marine bacteria (fig 1). discussion from the inhibition and destruction activity, most of the results showed higher activity when concentrations of 10% were used (table 1 and table 2). this indicates that 10% crudes concentration might be the optimum concentration for several crudes against many pathogenic bacterial biofilms. some other isolates showed no activity at both concentrations, 5% and 10%. this could happen if higher concentration of crudes were needed. another possibility was that some crude were not suitable for pathogens biofilm inhibition or were specific to certain pathogens only (kalpana et al. 2011). mechanisms between inhibition and destruction were different. biofilm control can be achieved in several ways, such as, reduction of the planktonic table 2 inhibition activity of marine isolates (%) crude crude concentration pathogens sh sp sa pa vc etec epec bc 11-1 5% 27.6 45.1 58.9 10% 50.6 27.4 39.7 67.0 130.4 bc 11-2 5% 57.6 38.6 29.0 60.2 10% 40.2 59.3 39.5 66.1 bc 12-3 5% 12.8 55.1 10% 21.5 22.0 32.3 45.5 61.0 162.7 bf 13-2 5% 70.8 32.7 5.4 53.5 59.9 1.1 10% 37.1 42.1 11.5 57.8 72.9 bf 14-2 5% 6.0 28.0 13.4 55.7 10% 20.9 31.9 61.1 bf 04-1 5% 5.1 14.5 5.4 10% 30.1 18.8 8.4 37.9 61.0 bf 08-1 5% 15.4 12.4 10.0 30.3 10% 33.4 14.9 8.7 16.9 120 7.0 bf 09-7 5% 63.3 14.5 1.9 8.7 10% 43.7 45.0 37.5 108.7 bf 14-1 5% 18.5 39.0 39.5 66.7 77.3 10% 16.2 47.6 49.6 48.0 bf 13-5 5% 46.1 36.1 32.8 53.9 1.9 0.4 10% 49.9 22.1 22.5 47.1 bb 07-7 5% 27.7 25.2 2.7 28.9 10% 33.8 17.8 0.6 13.0 13.4 bb 07-4 5% 28.2 117.4 8.7 10% 15.4 8.7 bb 08-3 5% 32.1 4.5 7.9 83.6 10% 33.1 30.4 28.3 19.7 67.5 84.5 bf 08-4 5% 4.5 55.7 32.2 10% 4.5 38.9 36.7 6.7 bc 11-3 5% 35.2 9.3 27.1 39.3 10.2 10% 14.0 42.1 41.1 49.0 sh: staphylococcus haemolyticus; sp: streptococcus pneumonia; sa: staphylococcus aureus; pa: pseudomonas aeruginosa; vc: vibrio cholerae; etec: enterotoxigenic escherichia coli; epec: enteropathogenic escherichia coli 92 camesi et al. microbiol indones population, prevention of the initial adhesion of cells to the surface, and removal of the established biofilm (jorge et al. 2012). in inhibition activity; marine crudes would inhibit the surface attachment so that biofilms could not be formed. whereas, in destruction activity, marine crudes may exhibit bioactive compounds, mainly from extra polymeric polysaccharides or proteins which can destroy and remove mature biofilms from the surface (jorge et al. 2012). one of the components assembling biofilm formation is the production of an extracellular matrix composed of 90% water and 10% extracellular polymeric substances (eps) (flemming and wingender 2010). the eps mediates cell to cell and cell to surface interaction that are needed for biofilm formation and stabilization. the eps matrix consists of cell-surface proteins, proteinaceous pili, dna, rna, lipids, and polysaccharides (flemming and wingender 2010). many different biofilm matrix polysaccharides have been characterized, for instance pel and psl produced by pseudomonas aeruginosa, poly-nacetylglucosamine (pnag) produced by escherichia coli, and glucans produced by streptococcus mutans (rendueles and kaplan 2012). this building agent of crude crude concentration pathogens sh sp sa pa vc etec epec bc 11-1 5% 37.9 13.3 15 71.4 53.9 10% 15.7 58.4 23.6 16.4 56.3 95.7 35.2 bc 11-2 5% 17.8 43.1 30.4 10.9 20.6 10% 30.3 24.2 20.7 0.3 5.1 bc 12-3 5% 16.4 11.4 4.8 56 10% 8.1 4.7 28.2 23.8 15.8 23.1 10.4 bf 13-2 5% 18.8 48.6 10% 12.7 37.2 15.6 bf 14-2 5% 16.9 14.1 64.2 10% 33.2 14.2 49.6 84.2 bf 04-1 5% 2.9 80.1 28.1 28.7 10% 19.5 82.1 3.2 69.4 56.8 bf 08-1 5% 43.8 12.8 15.2 25.2 36.6 12 10% 35.6 37.4 34.4 3.7 42.5 134 bf 09-7 5% 26.6 16.3 3.4 35.9 10% 12.3 6.2 3.6 9.7 bf 14-1 5% 32.8 9.4 18.2 18.4 10% 35.5 1 3.4 14 bf 13-5 5% 3.6 0.8 67.7 11.2 0.4 10% 0.7 35 43.5 27 bb 07-7 5% 81.9 75.3 78.3 55.5 10% 12.8 81.3 26.9 69.6 bb 07-4 5% 26.4 27.7 19.3 16.2 11.8 7.6 28.1 10% 36 9.6 18 3.4 53.4 17.3 bb 08-3 5% 11.6 8.5 45 4.6 10% 22 10.4 1.8 21.3 0.3 bf 08-4 5% 18.5 75.2 55.2 20.4 10% 11.6 4.2 81.7 1.1 17.9 bc 11-3 5% 35.5 28 27.3 5.2 10% 48.7 30.9 12.9 13.3 sh: staphylococcus haemolyticus; sp: streptococcus pneumonia; sa: staphylococcus aureus; pa: pseudomonas aeruginosa; vc: vibrio cholerae; etec: enterotoxigenic escherichia coli; epec: enteropathogenic escherichia coli table 3 destruction activity of marine isolates (%) volume 10, 2016 microbiol indones 93 biofilm formation is important to maintain the biofilm integrity. however, some bacterial exopolysaccharides can perform functions that inhibit or destabilize the biofilm (kostakioti et al. 2013). this confirms the polysaccharide nature of the antibiofilm activity. culture supernatants derived from several marine bacteria have been shown to exhibit antibiofilm activity (gopal et al. 2013).the biofilm inhibition activity can be due to a high molecular weight polysaccharide, for example the sp1 eps from bacillus licheniformis which can inhibit biofilm formation by several other bacteria such as e.coli, acinetobacter sp., s.aureus, b.cereus, listeria monocytogenes, salmonella typhimurium, shigella sonnei, bacillus amyloliquefaciens, bacillus pumilus and bacillus subtilis without inhibiting growth (sayem et al. 2011). other study said that there are several mode of actions for antibiofilm activity which likely to be mediated by mechanisms other than growth inhibition (kostakioti et al. 2013). first mode is that antibiofilm polysaccharides act as molecules that modify the physical characteristics of bacterial cells and abiotic surfaces (kostakioti et al. 2013). second, some studies have indicated that polysaccharides might act as signaling molecules that modulate gene expression of recipient bacteria (kim et al. 2009). another possibility is the competitive inhibition of multivalent carbohydrate-protein-interactions (wittschier et al. 2009). antibiofilm activity of bacteria can also be isolated from biofilms which exhibit an antagonist effect over competing microorganisms (rendueles et al. 2012). beside the exopolysaccharide, lipopolysaccharides from gram negative bacteria can alter biofilm structure and reduced adhesion, hence inhibit biofilm formation of competing strains (lau et al. 2009). for example lps from vibrio cholerae is able to partially inhibit in vitro adhesion on colonic cell lines ht29-18n2 (benitez et al. 1997). based on the sequencing results and phylogenetic tree, samples bc11-1 (erythrobacter sp.), bf04-1 (enterobacter cloacae), bf13-5 (paracoccus marcusii), and bf14-2 (cobetia marina) showed close relationship to existing marine bacteria (figure 1). a previous study on antibiofilm activity of cobetia marina conducted by trentin et al. 2011 showed that cobetia marina reduced the number of adherent bacteria in sterile 96-well microtiter plates containing s.epidermidis compared to the untreated biofilm (control). the study also suggested that the size of aggregates were also reduced to small cluster or even single cells and very little eps appeared to have been formed (trentin et al. 2011). the results corroborate with the inhibition observed in the biofilm assay. though the mechanism was still unclear, due to a connection between cell density and the eps production which is quorum sensing (qs)-regulated, cobetia marina was thought to modulate the qs of pathogenic bacteria and thereby prevent eps production, blocking the irreversible adhesion to a surface, and consequently, the formation of biofilm (trentin et al. 2011). meanwhile, there have not been any findings in scientific research on how the other 3 marine isolates perform antibiofilm activity. the other two samples; bc 12-3 (micrococcus luteus) and bf 13-2 (bacillus jeotgali), however, showed distant relationship to existing marine bacteria. further studies should be conducted in order to analyze the genetic relationship between those two samples and to observe their antibiofilm activity mechanisms against pathogenic bacteria. most marine isolates tested have antibiofilm activity against several pathogens either with inhibition or destruction mechanism. from these results, it can be concluded that marine bacteria are great potential sources of novel antibiofilm compounds. acknowledgements we appreciated the help of previous researcher, adrian eka putra and shirley nataprawira, who had isolated the marine isolates sample, and also the help of biotechnology faculty technicians of atma jaya university, indonesia. this study was supported by yonsei university and pukyong university, korea. references amador m, jimeno j, paz-ares h, cortes-funes h, hidalgo m. 2003. progress in the development and acquisition of anticancer agents from marine microbes. ann oncol. 14(11): 1607-1615. doi: 10.1093/annonc/ mdg443. aris m, sukenda, harris e, sukadi mf, yuhana m. 2013.molecular identification of pathogenic bacteria and pcr specific primer design. budidaya perairan.1(3): 43-50. benitez ja, spelbrink rg, silva a, phillips te, stanley cm, boesmann-finkelstein m. 1997. adherence of vibrio cholerae to cultured differentiated human intestinal 94 camesi et al. microbiol indones cells: an in vitro colonization model. infect immun. 65:3474–3477. dheilly a, soum-soutera e, klein gl, bazire a, compere c, haras d, dufour a. 2010. antibiofilm activity ot the 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against staphylococcus epidermidis rp62a accumulation associated protein. clin vaccine immunol 12:93-100. doi: 10.1128/cdli.12.1.93-100.2005. trentin ds, gorziza df, abraham wr, antunes als, lerner c, mothes b, termignoni c, macedo aj. 2011. antibiofilm activity of cobetia marina filtrate upon staphylococcus epidermidis catheter-related isolates. braz j microbiol. 42: 1329-1333. doi: 10.1590/s151783822011000400013. wittschier n, faller g, hensel a. 2009. aqueous extracts and polysaccharides from liquorice roots (glycyrrhiza glabra l.) inhibit adhesion of helicobacter pylori to h u m a n g a s t r i c m u c o s a . j e t h n o p h a r m a c o l . 125:218–223. doi: 10.1016/j.jep.2009.07.009. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 9 no. 294 (cahya prihatna).pmd phenotypic, metabolic, and genetic diversity of the indonesian isolates of rhizopus oligosporus cahya prihatna1 and antonius suwanto2∗ 1research and development center, charoen pokphand indonesia, p.o. box 14430, jakarta, indonesia 2department of biology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia fifteen rhizopus oligosporus isolates were isolated from a number of tempeh samples obtained from mataram, jember, and bogor, indonesia; and subjected for characterization based on phenotypic, metabolic, and genetic fingerprinting through internal transcribed spacer (its) regions and amplified fragment length polymorphism (aflp). based on the growth on solid medium, they can be divided into three groups. firstly, isolates that produced thick mycelia, dumpy sporangiophore, and scarce spores in agar culture, the second group is isolates that produced thin mycelia, stretched sporangiophore, with abundant spores in agar culture. the third group that only comprises one isolate, fb-06, is morphologically intermediate of the first and the second groups. these characters correlated with their range of temperature tolerance. the first group is less tolerant to high temperature (45 oc) compared with the second group, and the third group is the most tolerant to temperature up to 45 oc. metabolic fingerprinting showed a very high polymorphism. in general, the result may explain a correlation in which isolates obtained from the same locations shared similar patterns. there is no correlation found between metabolic fingerprints and their phenotypic fingerprints. rhizopus oligosporus readily dominated the niche and utilized nearly all carbon sources given demonstrate the versatile nature of this fungus. its regions identification revealed single nucleotide polymorphisms in four representative isolates examined, whereas aflp fingerprinting determined each of representative isolates as individually unique. furthermore, this aflp profile seemed to agree with their phenotypic characters. key words: tempeh, rhizopus oligosporus, genetic diversity _____________________________________________ table 1 list of isolates with their origins based on morphology and culture type isolate name fb-1 fb-2 fb-3 fb-4 fb-5 fb-6 fb-7 fb-8 fb-18 fb-19 fb-20 fb-21 fb-22 fb-23 fb-24 mataram, nusa tenggara barat monjok, mataram, nusa tenggara barat abian tubun, mataram, nusa tenggara barat lembaga ilmu pengetahuan indonesia, bandung, west java taman baru, mataram, nusa tenggara barat jember, east java jember, east java kekalik, mataram, nusa tenggara barat warung jambu, bogor, west java warung jambu, bogor, west java darmaga, bogor, west java empang, bogor, west java guga sari, bogor, west java malabar, bogor, west java ciluer, bogor, west java origin tempeh is an indonesian traditional daily food made of solid-state fermentation of soybean employing foodborne fungus rhizopus oligosporus. this fungus has undergone a very long employment in that indigenous or traditional “biotechnology” practices in indonesia. therefore, it would be of importance to elucidate its biodiversity. however, little has this fungus been studied for its microbiological nature in indonesia. tempeh is usually made in farms, in such plain conditions that even the farmers do not have any knowledge regarding the microbiological aspects. strain characterization is a preliminary and essential effort in industrial microbiology that can lead to strain improvement and development since the hallmark of such purposes is economic (crueger and crueger 1984). besides of tempeh, r. oligoporus has also been used for fermentation of some agricultural by-product to produce nutritionally-improved feed. one may customize strains in such rational manner that fits industrial efficiency since wild strains do not perform well in facilitating the economical aspects of industrial microbiology. the objectives of this research were to elucidate some biological properties and the biodiversity in terms of morphology, physiology, and genetic polymorphisms of several rhizopus oligosporus isolates from indonesia. materials and methods culture and microorganisms. fourteen rhizopus oligosporus isolates were isolated from several tempeh samples that were collected from several locations in bogor, jember, and mataram, and one isolate was isolated from commercial inoculum from lembaga ilmu pengetahuan indonesia (lipi), bandung (table 1). all isolates were grown in potato dextrose agar at 32 oc for a week and maintained in malt extract agar. one-week-old cultures on malt extract agar were used for morphology, culture, and metabolic fingerprinting examinations. dna isolation. for total dna isolation, spores suspensions were inoculated into potato sucrose broth and incubated for 3 days at 32 oc in an orbital shaker (175 rpm). mycelia were collected by passing them through sieves and air-dried. approximately 0.2 g of dried mycelia was ground in mortar using liquid nitrogen. one ml extraction buffer (50 mm tris-hcl ph 8.0, 125 mm edta ph 8.0, 50 mm nacl, 0.1% (v/v) mercaptoethanol, and 2% (w/v) sodium n-lauryl sarcosinate) was poured into mycelia mats (ground mycelia) _________________ ∗ corresponding author, phone/fax: +62-251-315107, e-mail: asuwanto@indo.net.id microbiology indonesia, april 2007, p 27-32 volume 1, number 1 issn 1978-3477 5.8ss its4 its1 its2 26s 18s its5 figure 1 representation of the ribosomal dna (rdna) and internal transcribed spacers (its) regions. sites of its4 and its5 primers are indicated with arrows. and gently ground for the second time until a slurry mat formed. the suspensions were transferred into 1.5-ml tubes and leaved at bench for 30 minutes. dna was separated by consecutive extraction using 0.5-volume tris-saturated phenol and followed by 0.5-volume chloroform. the aqueous phase was transferred into a new tube and extracted for the second time using 0.5-volume chloroform. dna was precipitated with cold isopropanol and sodium acetate followed by cold ethanol wash and stored in tris-edta buffer ph 8.0 at 4 oc. morphology and culture fingerprinting. all isolates were grown in malt extract agar for a week at 32 oc in the dark. morphology observation was performed using light microscopy. the key characteristics examined were sporangiophore, sporangium, sporangiospore, and colony appearance on plates. as a guideline for morphology examination, an atlas for rhizopus identification was used (liou et al. 1990). culture growth characteristics in different temperatures were also observed by growing the isolates in an agar-solidified media of certain feed formulation at 32, 45, and 50 oc. metabolic fingerprinting. metabolic fingerprinting is based on utilization of an array of certain specific carbon sources resulting in formation of unique pattern of reactions. reaction and analysis were done using biolog, micrologtm system, release 4.2 (hayward, ca, usa). spores from a week malt-extract-grown cultures were collected and spore density was adjusted to 75%t. spore suspensions were inoculated into 96-wells biolog microplatetm and incubated for 7 days at 32 oc. plates were read in biolog reader machine and analysed with micrologtm software. internal transcribed spacer regions identification. the resulting dna was used as a template to amplify the ribosomal dna its regions. the its primers used were its4 (5’-tcctccgcttattgatatgc-3’) and its5 (5’ggaagtaaaagtcgtaacaagg-3’) (abe et al. 2003) (figure 1). dna was amplified using 1 × pcr buffer, 0.1 ml taq polymerase (new england biolabs, usa), and 100 pmol of each its4 and its5 primers. the pcr reaction was carried out for 35 cycles of denaturation at 95 oc for 1 minute, annealing at 50 oc for 1 minute, and extension at 72 oc for one minute using an applied biosystem 2400 machine. the pcramplified fragments were purified with mobio pcr dna purification kit and sequenced in an abi prism 3100 avant genetic analyzer. amplified fragment length polymorphism. approximately 300 ng of dna was subjected for endonuclease ii digestion using ecori and msei. the resulting fragments were ligated after the addition of adapters in the ecori and msei restriction sites. pre-amplification was performed with one-base-extended primers for each adapted sites of ecori and msei (ecori+1 primer: 5'-gac tgc gta cca att ca-3' and msei+1 primer: 5'-gat gag tcc tga gta ac-3'). a touch-down pcr was performed to selectively amplify the resulting pre-pcr products. the primers used were a tga-base extended of msei+1 primer and one-baseextended of ecori primer (see pre-amplification ecori primer sequence). pcr was carried out for 20 cycles of 15 seconds denaturation at 94 oc, annealing at 66 oc for 15 seconds, with the temperature for each subsequent cycle was lowered by 0.5 oc, and extension at 72 oc for one minute. cycling was continued for 30 cycles at an annealing temperature of 56 oc. after completion of the cycles, an additional incubation for 10 minutes at 72 oc was performed before the reaction mixture was cooled down. a 100-fold dilution of the resulting pcr products were sequenced in an abi prism 3100 avant genetic analyzer, and analysed with genotyper 3.7 and genescan 3.7 softwares. results morphologically, the isolates can be clearly divided into three groups in accordance to their growth ability in different temperatures. isolates possessing thin mycelia, dark sporangium, elongated sporangiophore, and abounding spores, were able to grow at 45 oc. in contrast, isolates with thick mycelia, brighter or light sporangium, dumpy sporangiophore, and scarce spores, grew poorly at 45 oc. the third group, which possesses intermediate morphology and culture features between the first and the second groups, belongs to only one isolate, fb-06. interestingly, the isolate was the most tolerant to temperature of 45 oc. none of the isolates was able to grow at 50 oc. figure 2 represents sporangium colour of fb-01 and fb-06 isolates. individual metabolic fingerprinting showed a very high polymorphism (table 2). even though, a specific pattern seemed to correlate with the origin of the isolates. isolates from bogor, jember, and mataram showed a distinct pattern, but isolates from the same locations shared a similar pattern. however, among all isolates, only fb-01, fb-02, fb-03, fb04, and fb-05 were identified as rhizopus oligosporus saito, and the remaining isolates did not indicate the characteristics of any rhizopus species. this result revealed a high variance in the physiological properties of r. oligosporus. community fingerprinting analyses indicated that almost all carbon sources were completely utilized in a fermented materials whether the raw materials were sterilized or not (table 3). however, some carbon sources were not utilized in sterilized materials, while they were utilized in unsterilized materials. although the pattern was considerably different in both raw materials, major changes appeared during fermentation resulting in quite similar pattern. the its regions identification found single nucleotide polymorphisms in the its regions of ribosomal dna. figure 3 shows the result of multiple alignment of pcr products of its regions of four representative isolates. in the isolate 28 prihatna and suwanto microbiol indones figure 2 sporangium colour of rhizopus oligosporus isolates fb-01 and fb-06. sporangium of fb-01 is brownish, whereas sporangium of fb-06 is blackish. table 2 metabolic fingerprint of several indonesian rhizopus oligosporus isolates volume 1, 2007 microbiol indones 29 table 2 continued 30 prihatna and suwanto microbiol indones table 3 fingerprint of microbial community-level of feed sample before and after fermentation using rhizopus oligosporus isolate fb-01 carbon source 10 20 30 40 50 60 70 80 90 1 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| fb-02 ccacttgacttcagatcatagttaaagatcattaagatatctgctggctagcagaacccctagattatatgttttttggttggaccaaaaaa fb-08 ...............................................n........................................................................ fb-07 -----------------------............a.................................................................................... fb-03 -....................................................................................................................... 130 140 150 160 170 180 190 200 210 220 230 240 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| fb-02 atgaaaattacaaagaggctgtattttagacaatcggtataataattaaatttaaccgagcttgtccatcaccacataaaataaatttt fb-08 ............................................................y........................................................... fb-07 ........................................................................................................................ fb-03 ........................................................................................................................ 250 260 270 280 290 300 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.. fb-02 cgtactctatagaagatccatagagtgcaa-gctgcgttcaaagactcgatgattcacgaatatgcaattcacacta-gttatcgcacttt fb-08 ..............................-..............................................-................. fb-07 ..............................-..............................................-................. fb-03 ..............................a...................................c..........a................. 370 380 390 400 410 420 430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| fb-02 agagatccattgttaaaagttgttattatattatactttcaattctgaattcatggtatatggtaaagggtaccaggcaccttccttccc fb-08 ........................................................................................................................ fb-07 ........................................................................................................................ fb-03 ..--------------------------------------------------------------------------------------------------------------------- 490 500 510 520 530 540 550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| fb-02 gatcaaaccccagaacaggcctacccattatagcctatatgtcctgagtctctcccgaaggtcagttacgaccttcatcgccagaggtt fb-08 ........................................................................................................................ fb-07 ........................................................................................................................ fb-03 ----------------------------------------------------------------------------------------------------------------------- 610 620 630 640 650 ....|....|....|....|....|....|....|....|....|....|....|. fb-02 aaatcccagtaaagtgccaatacattagttaatgatccttccgcaggttcacctac fb-08 ........................................................ fb-07 ........................................................ fb-03 ------------------------------------------------------- figure 3 multiple alignment of rdna its regions of rhizopus oligosporus isolates fb-02, fb-03, fb-07, and fb-08. table 4 the summarized fingerprint of amplified fragment length polymorphism result of rhizopus oligosporus isolates fb-01, fb05, fb-06, and fb-08 fb-07, the 36th base was replaced by a, and in the isolate fb03, the 271st base was replaced by a and the base was lost in the others, 307th base by c, 318th base by a and the base was lost in the others, and the 336th base by a and also the base was lost in the others. the amplified fragment length polymorphism analysis showed high polymorphism among four representative isolates (table 4). the result showed that each of representative isolates was individually unique. isolates fb05 and fb-08 share closer relationship compared with the others. isolate fb-06 exhibits a distinct fingerprint as to a number of fragments was not amplified. discussion rhizopus oligosporus and its allies have undergone extensive uses in fermentation to produce many kinds of traditional foods such as tempeh, kecap, and tauco. despite its extensive employment for hundreds of years in indonesia, recognition of its microbiological diversity still needs provisioning. conventional morphology identification, however time-consuming and laborious, still provides a reliable and rapid characterization for fungi since it is designated for phenotypic typing (guarro et al. 1999). in addition, some specific structures such as zygospore, asci, conidiogen, and conidia are easily recognizable and still considered as the most important sets of characteristics to be observed in some fungi species. in general, from the microscopic and macroscopic observations, in accordance with their growth ability in high temperature, all isolates were divided into three groups. it is interesting to note about the correlation of a certain morphological pattern with the high temperature tolerance. morphology divergence might be a result of culture degeneration (spontaneous mutation) or sexual interaction between strains of opposite sex that can lead to hyphal morphology modification in the progeny (mehta and cerdáolmedo 2001). these mutation and sexual interaction are assumed, in turn, also modified the genetic trait of high temperature tolerance. it is also known that some species in the phylum zygomycota have considerable morphological variation (benny 1994; weitzman et al. 1995) and several species are thermophilic (de hoog and guarro 1995). biochemical and physiological tests have also been used for identification of zygomycetes especially for clinically important strains. nevertheless, there are some major volume 1, 2007 microbiol indones 31 drawbacks restraining the approach, which is rather complicated. the procedure is both time-consuming and labour-intensive and usually requires special media, some of which are not commercially available (kaufman et al. 1990). here, we used a relatively simple, reliable, and high throughput identification kit, biolog, micrologtm system, release 4.2 (hayward, ca, usa). in our experiment, physiological characterization did not elucidate its correlation with the morphology fingerprint. however, it explicated an itinerary to the conjecture that the same origin of location of the isolates has the similar metabolic fingerprint. even though some of the isolates were not recognized as rhizopus oligosporus according to the biolog, micrologtm system test, and only of those isolates from mataram were identified as r. oligosporus, it demonstrated that there is a remarkable metabolic diversity of r. oligosporus. recalling that biolog uses database-based identification, we assumed that isolates from bogor and jember have undergone major changes in their physiological properties throughout their evolutionary flow. evolution on type of nutrition appears to have progressed in the order mucorales, resulting in great diversity of nutritional requirements (hesseltine 1955). recalling the rapid growth nature of r. oligosporus, it seems conceivable that this fungus has encountered relatively rapid evolution. analysis on community level concluded that similar fingerprint after fermentation of unsterilized and sterilized raw materials revealed that r. oligosporus outstandingly outcompete the niche and readily predominate the utilization of available nutrition. however, it is important to note that even in sterilized raw materials it still produced positive reactions, indicating the presence of highly heat-resistant microorganisms. we assumed that these microorganisms might be endospore-forming bacteria. this approach can also provide a vast amount of information that might be useful for other applications. once the community fingerprint in solid-state fermentation employing r. oligosporus has been established in such optimal conditions, one may use the information for microbiological quality control of the fermentation process by monitoring changes occurred in the fingerprints. phenotypic as well as metabolic characters are not always collinear with their genetic designation, the so-called epigenetic phenomenon. the distinct pattern between isolate fb-03 and isolates fb-02 and fb-07, which are obviously similar in their morphology and metabolic fingerprint, may be explained by the fact that rdna its regions do not encode functional proteins and are spliced out after transcription. abe et al. (2003) also found that certain strain of amylomyces rouxii shares completely the same its sequence with rhizopus oryzae. the its regions are much more variable, but sequences can be aligned with confidence only between closely related taxa. these regions are generally used for species differentiation but may also demonstrate patterns of microevolution (gräser et al. 1999). regardless of some weaknesses, the aflps may provide the most determinative in term of genetic resolution produced (mueller and lareesa wolfenbarger 1999; brugmans et al. 2003). in our experiment, there is no single isolate that has the same pattern with others. it is interesting to note that the aflp fingerprints of isolates fb-01, fb-05, fb-06, and fb08 differentiated these isolates accordingly to their phenotypic (morphology and temperature tolerance) differentiation. isolates fb-05 and fb-08 are morphologically similar, but isolates fb-01 and fb-06, each of them is morphologically different to the others. the aflp result revealed that isolates fb-05 and fb-08 have similar genetic fingerprints. on the other hand, isolates fb-01 and fb-06, each of them has dissimilar aflp fingerprints to the others. unlike the rdna its regions, aflp result may also evidence the phenotype, because aflp fingerprints the whole genome. reliable genetic resolution greatly depends on appropriate primers and pcr methods selections. the use of tga bases extension in combination with specific touch-down pcr method has proven to generate sufficient genetic resolution. references abe a, sone t, sujaya i nengah, saito k, oda y, asano k, tomita f. 2003. rdna its sequence of rhizopus oryzae: its application to classification and identification of lactic acid producers. biosci biotechnol biochem 67:1725-1731. benny gl. 1994. classical morphology in zygomycete taxonomy. can j bot 73:725-730. brugmans b, van der hulst rgm, visser rgf, lindhout p, van eck hj. 2003. a new and versatile method for the successful conversion of aflp markers into simple single locus markers. nucleic acid research 31:1-9. crueger w, crueger a. 1984. biotechnology: a textbook of industrial microbiology. sunderland: sinauer associates. de hoog gs, guarro j. 1995. atlas of clinical fungi. baarn: centraalbureau voor schimmelcultures. gräser y, el fari m, presber w, sterry w, tietz hj. 1998. identification of common dermatophytes (trichophyton, microsporum, epidermophyton) using polymerase chain reactions. br j. dermatol 138:576-582. guarro j, gené j, stchigel am. 1999. developments in fungal taxonomy. clin microbiol rev 12:454-500. hesseltine cw. 1955. genera of mucorales with notes on their synonymy. mycologia 47:344-363. liou gy, chen cc, chien cy, hsu wh. 1990. atlas of the genus rhizopus and its allies. mycological monograph no. 3. taiwan: food industry research and development institute. mehta bj, cerdá-olmedo e. 2001. intersexual partial diploids of phycomyces. genetics 158:635-641. mueller ug, lareesa wolfenbarger l. 1999. aflp genotyping and fingerprinting. tree 14:389-394. weitzman is, whittier j c, mckitrick, della-latta p. 1995. zygospores: the last word in identification of rare and atypical zygomycetes isolated from clinical specimens. j clin microbiol 33:781-783. 32 prihatna and suwanto microbiol indones issn 1978-3477, eissn 2087-8575 volume 10, number 1, march 2016 high prevalence of occult hepatitis b infection (obi) and its molecular characteristics among pregnant women in surabaya, indonesia detection of hepatitis b virus x gene mutation from local clinical samples utilization of bacillus pumilus and citrobacter youngae as flotation bioreagents in the microflotation of chalcopyrite, pyrite, and silica h e t e r o l o g o u s e x p r e s s i o n o f aa m y l a s e f r o m saccharomycopsis fibuligera r64 and its tyr401trp mutant in pichia pastoris comparison of microbial pattern causing urinary tract infection in female out and hospitalized patients in jakarta meilani, takako utsumi, juniastuti, mochamad amin, soetjipto, yoshitake hayashi, and maria inge lusida anita artarini, hanary gaby jessica, raden rini kartikasari, catur riani, and debbie sofie retnoningrum edy sanwani, dita hidayati, and siti khodijah chaerun riezki amalia, wangsa tirta ismaya, fernita puspasari, khomaini hasan, toto subroto, dessy natalia, and soetijoso soemitro yeva rosana, dwiana ocviyanti, anis karuniawati, and syadza rhizky putri akhmad 1 9 15 23 30 issn 1978-3477, eissn 2087-8575 volume 10, number 1, march 2016 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000, 200 000,-5 institutional rate (institution or library) (idr) 240 000, 400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print maggy thenawijaya suhartono, 2016 maria inge lusida, 2016 neung tiaamroeng, 2020 norio kurosawa, 2020 kartini kramadibrata, 2020 diana e waturangi, 2020 endang purwantini, 2020 raija laiho, 2016 wellyzar sjamsuridzal, 2020 yuan kun lee, 2016 yaya rukayadi, 2020 friedhelm meinhardt, 2016 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi 424-1142-1-pb (1) cover depan (6) 4 dinar 407.pmd volume 3, number 3, december 2009 p 115 120 issn 1978-3477 *corresponding author, phone: +62-0315992446, 5965304; fax: +62-031-5992445, 5965305; e-mail: ddriaty@yahoo.co.id leprosy is a chronic infectious disease caused by mycobacterium leprae. the disease mainly affects the skin, the peripheral nerves, mucosa of the upper respiratory tract and also the eyes, apart from some other structures. leprosy has afflicted humanity since immemorial time. it once affected every continent and it has left behind a terrifying image in history and human memory-of mutilation, rejection and exclusion from society (who 2001). since the introduction of multidrug therapy (mdt) by the world health organizations (who) for the treatment of leprosy in 1982, the worldwide prevalence rate of leprosy has significantly decreased (brosch et al. 2000). the mdt regimen was based on rifampicin, clofazimin and dapsone (dds). however, rifampicin, ofloxacin and minocycline (rom) and also clarithromycin were prescribed to patients who refused to take clofazimine fearing skin discoloration (you et al. 2004). it is found in only several countries in africa, asia and south america, and is mainly present in limited numbers. on the contrary, the number of new cases of leprosy has remained unchanged over the last 10 years. this situation could inhibit our ability to target drug therapy campaigns and to improved control strategies (williams et al. 1994). current recommended control measures for treatment of leprosy with mdt are designed to prevent the spread of drug resistant m. leprae. however, drug resistant m. leprae strains have been reported, and this might become the problem for eliminating leprosy (maeda et al. 2001). maeda et al. (2001) studied the mdt-resistance from the dna sequences from particular regions of m. leprae folp1 (accession no. al023093), rpob (z14314) and gyra (z70722). molecular detection of dapsone and rifampicin resistance on mycobacterium leprae from leprosy patients in east java dinar adriaty*, ratna wahyuni, cita rosita s. prakoeswa, ni putu susari, indropo agusni and shinzo izumi leprosy study group-institute of tropical disease, universitas airlangga, kampus c, mulyorejo, surabaya 60115, east java, indonesia the drug resistant problem of mycobacterium leprae has been developing since the last decade and this has become a leprosy elimination problem in several countries, including indonesia. using biological on molecular methods, it is now possible to test for drug resistant cases in relatively simple and less time consuming ways. the purpose of the study is to analyze the prevalence of drug resistance m. leprae to dapsone and rifampicin in east java based on the detection of mutations in the folp and rpob genes. all samples were obtained from multibacillary leprosy patients in east java, who have admitted to the dr sutomo hospital surabaya in 2003-2005. isolates were analyzed by pcr, and the presence of nucleotide sequence of the folp and rpob genes from m. leprae were confirmed by direct sequencing. of 94 specimens which were collected, all were analyzed for their folp and rpob genome. from 94 isolates, 70 showed a positive result by the folp1-folpr test and 77 out of 94 isolates showed positive by the rpobf-rpobr test. from 70 isolates for folp gene examination, there were 3 isolates which had mutation in the amino acid at codon 53; 2 cases threonin (acc) became alanin (gcc) and 1 case threonin (acc) became arginin (aga). these mutations are responsible to dapsone resistance. for the rpob gene, no mutation was found. the result suggested that 3 isolates (4.3%), 1 from a new case and 2 from relapse cases in this experiment, were resistant to dapsone and all isolates (100%) were susceptible to rifampicin. key words: resistance, mycobacterium leprae, dapsone, rifampicin, east java all the genes were responsible for resistance to dapsone (dds), rifampin and ofloxacin respectively. several m. leprae isolates showed point mutations in these genes. these results suggest the need for a multidrug resistance (mdr) study for leprosy. kai et al. (1999) has shown that substitution of amino acids at 53rd and 55th position in dihydropteroate synthase (dhps), which are coded by the folp1 gene, cause resistance against dds in mycobacterium leprae. whereas, in m. tuberculosis and m. leprae, resistance to rifampicin involved mutation in the rpob gene encoding the β-subunit of the rna polymerase of these species (williams et al. 2000). it was revealed that mutation at the 513, 516, 526, 531, and 533 positions of amino acids confer rifampin resistance (maeda et al. 2001). jin and gross (1988) have also shown the same causes for resistance against rifampicin after cloning in escherichia coli. the east java province still has the highest number of leprosy patients (3.27 per 10 000 inhabitants), and new cases still occur (2.43 per 10 000 inhabitants), although treatment was undertaken and the patients were all cured (dinkes jatim 2008). about 35% of leprosy patients in indonesia were in east java (depkes ri 2006). the present study is to observe the prevalence of drug resistance m. leprae for dapsone (dds) and rifampicin in east javaindonesia based on the detection of mutations in the folp and rpob genes. materials and methods dna templates preparation. ninetyfour m .leprae isolates were taken from the lesion of multibacillary (mb) patients, categorized based on the criteria of the world health organization (who). there are 48 isolates (51%) 3 irene meitiniart (51-54).pmd products of orange ii biodegradation by enterococcus faecalis id6017 and chryseobacterium indologenes id6016 vincentia irene meitiniarti1∗, endang sutariningsih soetarto1, kris herawan timotius2, and eko sugiharto3 1biology faculty, universitas gadjah mada, bulaksumur, yogyakarta 55281, indonesia 2biology faculty, universitas kristen satya wacana, jalan diponegoro 52-60, salatiga 50711, indonesia 3department of chemistry, universitas gadjah mada, bulaksumur, yogyakarta 55281, indonesia chryseobacterium indologenes and enterococcus faecalis were isolated from activated sludge of textile wastewater treatment plant. these bacteria had the ability to decolorize several azo-dyes. degradation of azo dyes was initiated by decolorization (reduction of azo bond) which occurred in anaerobic condition. in this study, w e focussed on biodegradation of orange ii by pure culture of c. indologenes id6016 and e. faecalis id6017, and to determine the metabolite products of orange ii degradation. the degradation of orange ii by both bacteria was carried out in batch experiments using liquid medium containing 80 mg/l orange ii, under sequential static agitated incubation. during the bacterial growth under static incubation (6 h), 66.1 mg/l orange ii were decolorized by 35.54 mg/l biomass of e. faecalis id6017, but no decolorization found with c. indologenes id6016. based on hplc results, the decolorized orange ii products were identified as sulfanilic acid and amino-naphthol. these metabolites were probably used or degraded by c. indologenes id6016 under agitated incubation. key words: enterococcus faecalis id6017, chryseobacterium indologenes id6016, orange ii, biodegradation _____________________________________________ _________________ ∗ corresponding author, biology faculty, universitas kristen satya wacana, jalan diponegoro no 52-60, salatiga 50711, indonesia; phone: +62-298-321212 ext. 305, fax: +62-298-321433, e-mail: irene_meiti@yahoo.com orange ii is one of synthetic azo dyes, the largest chemical class of dyes with the greatest variety of colors, which has been widely used for textile, food, and cosmetics. the compound has an azo bond (r 1 -n=n-r 2 ), where r 1 and r 2 are aromatic groups, which in some cases, can be substituted by sulphonated groups (zollinger 1987). orange ii is synthesized by diazotized reaction of sulfanilic acid with amino naphthol. several others of azo dyes are either toxic, mutagenic or carcinogenic, and have a potential health hazard (chung et al. 1981; gottlieb et al. 2003). in the dyeing process, approximately 10-15% of the dyes are released into the environment through effluent of industry. the azo dyes are generally unaffected by conventional activated sludge process under aerobic condition and usually they are found in the effluent of wastewater treatment (wwt) (supaka et al. 2004). in several cases, conventional waste water treatment of azodyes containing wastewater combined with physical or chemical treatment, such as biosorption, chemical coagulation, and electrochemical method, could produce decolorized effluent (lin and peng 1996; lin and chen 1997; kargi and ozmihei 2004). however, those approaches often have problems, caused by chemical sludge which is produced from these treatments. therefore, there is still a demand to develop alternative means of dye decolorization, such as innovative biological methods that are able to provide a more natural and complete clean-up of the pollutants in a more economical way (coughlin et al. 2003). although the azodyes are usually resistant to microbial degradation under conditions normally found in wastewater treatment plants, several microorganism are able, under certain environmental conditions, to transform azodyes to non-colored products or even to completely mineralize them (stolz 2001). several species of bacteria, either mixed or single culture, for examples pseudomonas luteola (hu 2001; chang et al. 2001), sphingomonas sp. (kudlich et al. 1996; coughlin et al. 1997; keck et al. 1997; russ et al. 2000), bacillus subtilis (zissi and lyberatos 1996), and coculture of hydrogenophaga palleronii and agrobacterium radiobacter (dangmann et al. 1996; blümel et al. 1998) have been reported to have the ability to decolorize azodyes. the bacterial ability to decolorize azo dyes varies from 20 to 200 mg/l azodye concentration. sphingomonas sp. strain 1cx has an ability to decolorize 20 mg/l orange ii, acid orange 8, acid orange 10, acid red 4, or acid red (coughlin et al. 1997). whereas, pseudomonas luteola has an ability to decolorize reactive red up to concentration of 200 mg/l (chang et al. 2001). chryseobacterium indologenes and enterococcus faecalis were used as bacterial models for dye decolorization. those bacteria were isolated from activated sludge of textile wastewater treatment plant (liem 1997; setiabudi 1997). the bacteria have the ability to decolorize several dyes such as amaranth, reactive red, yellow, and blue (meitiniarti and timotius 2003). in the mixed culture of c. indologenes and e. faecalis, various kinds of azodyes were decolorized faster than in a single bacterial culture. c. indologenes was not able to decolorize these dyes (timotius et al. 2002). there is no information of the roles of both bacteria on the azodyes degradation. orange ii was used as a model substrate of azodye degradation. coughlin et al. (1999) reported that orange ii can be degraded into sulfanilic acid, which can easily be detected chromatographically, and 1-amino-2naphthol, which undergoes rapid auto oxidation. chemical structure of orange ii and products of orange ii decolorization by orange ii reductase were shown in fig 1. microbiology indonesia, august 2007, p 51-54 volume 1, number 2 issn 1978-3477 in this study, we focussed on biodegradation of orange ii by pure culture of c. indologenes id6016 and e. faecalis id6017, grown on a semi-synthetic medium. the aims of this research were to investigate degradation ability of both bacteria and determine the metabolite products of orange ii degradation. materials and methods microorganism and culture condition. e. faecalis id6017 and c. indologenes id6016 were obtained from laboratory of microbiology, faculty of biology, satya wacana christian university, salatiga, central java, indonesia. both bacterial culture were maintained and cultivated on slant media of trypticase soy agar (tsa) at room temperature and sub-cultured every two weeks. the bacteria were grown on liquid basal media consists of 0.90 g of glucose (glucose was omitted for c. indologenes culture), 0.25 g of mgso 4 ·7h 2 o, 1.98 g of (nh 4 ) 2 so 4 , 5.55 g of k 2 hpo 4 , 2.13 g of kh 2 po 4 , 0.25 g of yeast extract, and 1 l of aquadest. orange ii (merck, ci acid orange 7, ci 15510,mw= 440.41)was used in concentration of 80 mg l-1. bacterial cultivation for dye degradation assay. each of 48 h tsa slant culture e. faecalis id6017 and c. indologenes id6016 was inoculated in basal medium without orange ii in 500 ml erlenmeyer flasks. cultures were incubated 24 h on shaker with 150 rpm agitation to reach od 600nm = 0.3. these cultures were called as pre-cultures. the growing vessels contained of basal medium with orange ii were inoculated with 10% (v/v) of each pre-culture separately and closed with rubber stopper. cultures were incubated at room temperature, under static condition for 6 h, followed by 150 rpm agitation for 12 h. before agitation, 80 ml of the e. faecalis culture was filtered using membrane filter in 2 µm diameter and then inoculated aseptically using 20 ml of c. indologenes id6016 48 h pre-culture. samples were harvested every two hours followed by centrifugation at 3326 g for 30 min to separate supernatant and cell mass (biomass). the supernatant was used for determining the dye concentration. the dye concentration was determined by spectrophotometric method at λ max (482 nm). the cell mass after twice washing, was resuspended into initial volume and measured by turbidimetric method at λ 600nm . the dye concentration was determined by a standard curve of absorbance against dye, meanwhile, biomass concentration was determined by a standard curve of optical density against biomass concentration (cell dry weight). both measurements of absorbance and optical density were done in a shimadzu uv–vis 1201 spectrophotometer. the ability of both bacterial strains to decolorize orange ii was determined by subtracting the initial dye concentration with the lowest concentration from samplings. the growth of e. faecalis id6017 and c. indologenes id6016 was determined by calculating its specific growth rate and biomass production. in the initial time of culture, after decolorization (t=6 h), and at the end of culture (t=12 h), the culture supernatant was analyzed to detect product degradation of orange ii by hplc. hplc analysis of degraded metabolites (modification of supaka et al. 2004). the degraded metabolites were analyzed using hplc shimadzu model lc-3a chromatograph, equipped with uv-detector and ods column (150 mm x 8 mm). the supernatant culture of e. faecalis id61017 and c. indologenes id61016 was injected to hplc. the mobile phase composed of methanol and acetic acid 0.6% (vol/vol) = 60:40 with the flow rate of 0.7 ml min-1 was applied. the eluent was monitored by uv absorption at 276 nm. results the growth of e. faecalis id61017 and c. indologenes id61016 and their ability to degrade orange ii. e. faecalis id6017 and c. indologenes id6016 could grew in orange ii containing media, under static incubation. as shown in table 1, e. faecalis grown faster (µ = 0.19) and produced more biomass (35.54 mg) than c indologenes (µ = 0.16 and produced 24.11 mg biomass). after static incubation and agitation, the growth curve of e. faecalis decreased. on the contrary, the growth curve of c. indologenes was constant with specific growth rate of 0.05 (fig 2 and 3). surprisingly, c. indologenes could grow in supernatant culture of e. faecalis (fig 2), without lag phase. table 1 the growth characteristic and orange ii decolorization ability of e. faecalis and c. indologenes on orange ii containing media under static and agitated incubation species of bacteria e. faecalis c. indologenes static agitated static agitated the growth characteristics and orange ii decolorization ability spesific growth rate (ì h-1) biomass production (mg l-1) decolorization rate (mg h-1) decreased of orange ii concentration (mg/l) 0.19 35.54 11.02 66.10 nd nd nd nd 0.16 24.11 0.19 1.16 0.05 17.18 nd nd nd = not determined. fig 1 chemical reaction of orange ii decolorization by orange ii reductase (zimmermann et al. 1982). (orange ii) so 3 n n oh (orange ii) azoreductase 2(nad(p)h+h+) 2nad(p)+ so 3 (sulfanilic acid) nh 2 nh 2 oh (1-amino-2naphthol) 52 meitiniarti et al. microbiol indones mi-supiana available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.9.3.6issn 1978-3477, eissn 2087-8575 vol 9, no 3, september 2015, p 136-139 *corresponding author; phone: +62-81335278182, email: diantbn@yahoo.co.id typhoid fever, also known as abdominal typhoid, is caused primarily by salmonella enterica. cases of hepatic involvement with typhoid fever have been reported by khosla (1990), which known as typhoid hepatitis. coinfection of typhoid fever with hepatitis a and e also have been reported in previous studies (bhat et al. 2008; zhang et al, 2015; tulara 2013). this coinfection makes the diagnosis and management a challenging task. our previous paper reported that hepatitis b virus (hbv) was found in abdominal typhoid patients in tuban. from 30 serum samples of abdominal typhoid patients, we found hbv dna in 5 samples using nested polymerase chain reaction technique (nurtjahyani 2013). hepatitis b virus (hbv) infection is commonly known worldwide to causes more than one million deaths every year (who 2015). this disease is also still a health problem in indonesia. the prevalence of hbv infection in indonesia is around 9.4% (riskedas 2007). hepatitis b virus is a small dna virus belonging to the hepa dna viridae family. the genome size of this virus is around 3.2 kilo bases (kb) with a diameter of 42-47 nm (dane et al. 1970; summers et al. 1975). the hbv virion (dane particle) consists of an outer protein envelope and an inner protein core (kott 2010). hepatitis b virus genome consisted of 4 orfs (open reading frames) encoding for the surface (s), pre core and core (pre-c and c), x and polymerase (p) proteins (quarleri 2014). hbv has been classified into 10 genotypes: a, b, c, d, e, f, g, h, i, and j. the prevalence of hbv genotypes varies markedly between different regions. genotype a is more abundantly found in the northern and middle europe, compared to the north of america and africa. genotype b and c are found in asia, genotype d is distributed and detected mostly in mediterranea, whereas genotype e is found in the west africa nations. typhoid fever can cause liver disorder and may result in complication. studies revealed hepatic involvement in thypoid known as typhoid hepatitis. our previous paper reported the existance of hepatitis b virus (hbv) coinfection in the serum of patients with abdominal typhoid using nested-polymerase chain reaction (pcr). based on its genomic sequence divergence, hbv has been classified into 10 genotypes (a-j),which used in the prediction of therapeutic response and correlates with the clinical outcome of chronic hbv infection. this study was conducted to determine the genotypes of hbv intyphoid patients coinfected with hbv in tuban. sera were obtained from 5 typhoid patients with positive hbv infection (proven by nested pcr). study was performed by direct sequencing using bigdye v1.1 terminator cycle sequencing kit and abi prism 310 genetic analyzer. bioinformatic analysis had been done using the genetixmac version 10 software to create the phylogenetic tree. phylogenetic analysis showed 3 samples as genotype b and 2 two samples as genotype c. key words: coinfection, genotype, hepatitis b virus demam tifoid sering dikenal sebagai abdominalis tifus dapat menyebabkan gangguan hati dan penyakit komplikasi. penyakit ini sering dikenal sebagai hepatitis tifoid. dari penelitian sebelumnya, ditemukan adanya koinfeksi virus hepatitis b (vhb) pada pasien abdominalis tifus di kota tuban dengan menggunakan teknik polymerase chain reaction (pcr). virus hepatitis b diklasifikasikan menjadi sepuluh genotipe (a-j) dan diketahui memiliki peran dalam keberhasilan terapi. tujuan dari penelitian ini adalah untuk mengetahui genotip pada pasien tifoid yang koinfeksi dengan vhb di tuban. metode dalam penelitian ini adalah melakukan sekuencing langsung dari lima sampel pasien yang positif terinfeksi vhb. analisis filogenetik dari hasil sekuensing langsung sampel berhasil mengidentifikasi tiga sampel dikategorikan sebagai vhb genotipe b dan dua sampel termasuk sebagai vhb genotipe c. kata kunci: genotip, koinfeksi, virus hepatitis b genotype of hepatitis b virus coinfection in typhoid patients 1 2 supiana dian nurtjahyani * and retno handajani 1 faculty of teaching and education science, department of biology science, universitas ronggolawe tuban, jalan manunggal no. 61, tuban, jawa timur, 62381, indonesia; 2 faculty of medical and tropical disease center, universitas airlangga, mulyorejo, surabaya, 60115, indonesia short communication genotype f is specifically found to infect the aboriginal australia. genotype g is found in united states and france, genotype h is found to be restricted to nothern latin america, whereas genotype i was identified in vietnam and laos, and genotype j was found in ryukyu island in japan (lin 2010; arankalle et al. 201; zainal et al. 2009; tatematsu et al. 2009). it is necessary to study the genotypes of hbv in typhoid patients coinfection in tuban. it is reported that hbv genotypes correlated with the clinical characteristics of the infection, thus influencing the outcome of hbv infection, and the response to antiviral therapies (okamoto et al. 1998; norderet al. 1994; stuyver et al. 2000; arauz-ruizet al. 1997; lusida et al. 2008; tatematsuet al. 2009; orito et al. 2003; akuta et al. 2005). based on the previous studies, this study was conducted to determine the genotypes of hbv in typhoid patients with hbv coinfection in tuban district. written informed consent was obtained from all participants and this study was approved by the ethical committee of koesma hospital, medika and muhammadiyah hospital indonesia. samples were 5 typhoid patients with positive hbv as proven by nested pcr from our previous study at 2013. blood samples without anticoagulant were placed in sterile centrifuge tubes, allowed to stand for 30 min then centrifuged to obtain serum. the serum was transferred to 1.5 ml eppendorf sterile tube and stored at -80 °c. method, hepatitis b virus was extracted from sera of typhoid patients with known positive hbv using ® dnazol reagent kit (invitrogen, usa) as described in the manual book. the extracted dna was stored in elusion buffer at -80 °c until being used as a dna source for nested-pcr. hbv dna used as template in nested-pcr with 35 cycles. the pcr mix consisteds of 10× buffer solution, tth dna polymerase, and dntps. we used two sets of primers. the primers for the first-step pcr reaction were p1 (sense): 5’-gtggtggacttctct caattttc-3’ and p2 (antisense): 5’cggta(a/t)a aaactgggca(a/c)gat-3’ (originally named p7 and p8 (lindh, 1997)) which target the s genomic regions of hbv (542 nucleotides product including primers). the primers for the second-step nested-pcr reaction were hbs -1 (sense): 5’-caaggtatgttgc c c g t t3 ’ a n d h b s 2 ( a n t i s e n s e ) : 5 ’ gccctaaaaactcgaacc-3 (telent 1997), with 261 nucleotides product including primer used a portion of the final solution from the first-step pcr reaction as the template in the second nested-pcr reaction. the detailed nested-pcr conditions were described in our previous paper (nurtjahyani et al. 2013). the pcr products were visualized using 2% agarose gel electrophoresis in 0.5× tbe buffer solution containing ethidium bromide and documented on an ultraviolet light using digital camera (mamiya brand). nucleotide sequence of the hbv positive samples (based on nested-pcr) were further examined by sequencing usingone of the previous primers (p-1 or hbs1). direct sequencing was performed using ® bigdye terminator v1.1 cycle sequencing kit (thermo fischer cat.no.4337450) and a pro 0. 0.0226 0.0317 0.0054 0.0033 0.0091 0.0091 hbv-d m32138 222nt hbv-e ay090452 222nt hbv-e x75657 222nt hbv-f x75658 222nt 0.0000 hbv24-p7-222nt 0.0000 hbv45-p7-222nt 0.0000 hbv10-p7-222nt 0.0000 hbv-b d23678 222nt hbv-g af160501 223nt hbv-a s50225 222nt hbv-c x75656 222nt hbv2-p1-222nt hbv49-p7-222nt c b 0.0371 0.0091 0.0091 0.0153 0.0244 0.0080 0.0033 0.0219 0.0067 0.0324 0.0137 0.0001 0.0137 0.0138 fig 1 phylogenetic tree of hbv from typhoid-hbv coinfection patients. volume 9, 2015 microbiol indones 137 sequencing pcr technique (applied biosystems, usa). nucleotide sequences of hbv s gene from serum samples were compared with the nucleotide sequences of s gene from hbvs with various genotypes that had been published in the international dna data bank (ddbj/embl/genebank). the unweighted pair group method using arithmetic averages (upgma) method using genetix mac program version 10 was used to reconstruct a phylogenetic tree. the results, a phylogenetic tree was constructed based on the pcr amplication products using primers p1-p2 and hbs1-hbs2 (222 nucleotide), and the dna from five samples as templates (fig 1). the distribution of hbv genotypes in our five samples 60% (three samples) b and 40 % (two samples) c. molecular phylogenetic analysis classifies hbv into ten major genotypes, a-j (cao 2009). studies showed that hbv genotypes correlateds with distinct geographical distributions and clinical characteristics. the genotypes also playroles in the outcome of the infection, and on the patient’s response to antiviral therapy (erhardt et al. 2005; fung and lok 2004). therefore is necessary to determine the genotypes in order to better understand the hbv coinfection and also to provide effective managementtherapy. several cases of typhoid-hepatitis coinfection were reported in some countries (ahmad et al. 2010; bhat et al. 2009), however, our previous publication was the first to report the presence of hbv in thypoid patients (nurtjahyani 2015). in our current report, genotyping of hbvs isolated from 5 patients with typhoid. hbv coinfection indicated that 3 samples belonged to hbv genotype b and 2 samples belonged to hbv genotype c. this is consistent with the previous indications that genotypes band c highly prevalent in southeast asia (mans et al. 2004) and that they are present in some regions and groups in indonesia (yano et al. 2015; maria et al. 2003; utsumi et al. 2014, handajani et al. 2004). acknowledgment we would like to thank the koesma hospital, medika and muhammadiyah hospital indonesia in the process of sample collection. the authors are also grateful to all the technicians of institute of tropical disease (itd) universitas airlangga, surabaya for the laboratory facilities. references ahmed f, chowdhury k, alam j, arefeen s, alam mm. 2010. co-infection of typhoid fever with hepatitis a, hepatitis e and dengue fever: a challenge to the physicians. ds (child) hj. 26(2):122-124. akuta n, kumada h. 2005. influence of hepatitis b virus genotypes on the response to antiviral therapies. j antimicrob chemother. 55(2): 139-142. doi:10.1093/ja c/dkh533. arankalle va, gandhe ss, borkakoty bj, walimbe am, biswas d, mahanta j. 2010. a novel recombinant (genotype i) similar to vietnam/laos in a primitive tribe in eastern india. j virol hepatol. 10:1365-2893. doi:10.1111/j.1365-2893.2009.01206.x. arauz-ruiz p, norder h, visona ka, magnius lo .1997. molecular epidemiology of hepatitis b virus in central america reflected in the genetic variability of the small s gene. j infect dis. 176(4):851-858. doi: 10.1086/516507. bhat d., dhooria g.s, bains h.s. 2009. coinfection of hepatitis a and e with salmonella infection; 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biotech center-bppt, building 630, puspiptek area serpong, tangerang 15314, indonesia; deputy of technology of agroindustry dan biotechnology-bppt, jalan mh thamrin no 10, jakarta 10340, indonesia curvularia lunata cibotium barometz. c. lunata curvularia lunata, cibotium barometz curvularia lunata cibotium barometz c. lunata curvularia lunata, cibotium barometz hydroxyoctadecadienoic acid (hode) is one of hydroxy fatty acids that has anticancer activity. hode was previously produced by chemical synthesis or bioconversion from linoleic acid. this is the first paper reported production of hode by an endophytic fungi of various carbon and hydrogen sources have been tested for their effects on the production of hode by . glucose, lactose, maltose, xylose, and sucrose were used as carbon sources, while yeast extract, monosodium glutamate, urea, and nh cl were used as nitrogen sources. fermentation was done using 100 ml medium in 250 ml erlenmeyer flask at 150 rpm, 28 °c for 10 days. hode products were analyzed by high pressure liquid chromatography using c18 coloumn and eluted by gradient system of acetonitril-water from 15% to 100%. glucose and monosodium glutamate were found to be the best carbon and nitrogen source. the optimum concentration of glucose and monosodium glutamate for the production of hode were 10 mg l and 12 mg l respectively. key words: hydroxy octadecadienoic acid, , carbon source, nitrogen source, endophytic fungi asam hidroksioktadekadienoat (hydroxyoctadecadienoic acid, hode) adalah salah satu kelompok hidroksi asam lemak yang mempunyai khasiat antikanker. pada awalnya hode dihasilkan dengan cara sintesis kimia atau biokonversi dari asam linoleat. dalam tulisan ini dilaporkan untuk pertama kali produksi hode oleh kapang endofit yang diisolasi dari tanaman . telah dilakukan percobaan untuk melihat pengaruh beberapa jenis sumber karbon dan nitrogen terhadap produksi hode oleh kapang . jenis sumber karbon yang digunakan adalah glukosa, laktosa, maltose, xilosa, dan sukrosa, sedangkan sumber nitrogen yang digunakan adalah ekstrak khamir, monosodium glutamat, urea, dan nh cl. fermentasi dilakukan didalam tabung erlenmeyer 250 ml yang berisi 100 ml media pada suhu 28 °c dan kecepatan pengocokan 150 rpm selama 10 hari. hode hasil fermentasi dianalisis dengan kromatografi cair kinerja tinggi mengunakan kolom c18 dan dielusi secara gradien menggunakan campuran asetonitril-air dari 15% sampai 100%. glukosa dan monosodium glutamat masing-masing adalah sumber karbon dan sumber nitrogen terbaik untuk produksi hode. konsentrasi terbaik dari glukosa dan monosodium glutamat berturut-turut adalah 10 mgl dan 12 mgl . kata kunci: asam hidroksioktadekadienoat, , sumber karbon, sumber nitrogen, kapang endofit 4 4 -1 -1 -1 -1 hydroxyoctadecadenoic acid (hode) is a member of hydroxyl fatty acids, well known as oxylipins, which shows bioactivity. 8-hode produced by a basidiomycetes, can control pathogenic fungi dan (bowers 1986). 13-hode, a product of enzymatic conversion from linoleic acid was reported for its ability to inhibit tumor adhesion to endhothelium (liu 1991). other report by mundt (2003) showed that 13hode produced by hub 051 was able to inhibit gram positive bacteria sbug 14, sbug 16, and laetisiria arvalis, rhizoctonia solani phoma betae et al. et al. et al. oscillatoria redekei bacillus subtillis micrococcus flavus staphylococcus aureus et al. et al. (su and oliw 1996; wadman et al. et al. c. lunata c. lunata curvularia lunata sbug 11. hode can be produced by macro fungi (bowers 1986; wadman 2005) or micro fungi 2009) or cyanobacteria (mundt 2003). hode can also be produced by enzymatic or chemical conversion of linoleic acid (omar 2003; rombi and bordighera 2002). during the study of endophytic fungi, we found that could synthesize hode in liquid medium. this study reports the effects of carbon and nitrogen sources on the production of hode by in liquid fermentation system. an endophytic fungi, biomcc fe-00283, used in this study was obtained from biomcc culture collection, biotech center, bppt. *corresponding author, phone/fax: +62-21-7563120, e-mail: nuni_28@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 4, december 2011, p 187-191 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.4.6 it was isolated from a medicinal plant from east lombok, west nusa tenggara using surface sterilization method developed by tomita (2003). cultivation was started by inoculating the fungus, previously grown on one block of potato dextrose agar (pda) into 250-ml flask containing 100 ml potato dextrose yeast extract (pdy). the flask was then incubated at 150 rpm, 28 °c for 2 days. five milliliters of growing mycelia were transferred into 250 ml flask containing 100 ml fermentation medium, and then incubated at 28 °c, for 10 days with shaking at 150 rpm. fermentation medium consisted of 20% potato extract, 4 g l c from various carbon sources (glucose, lactose, maltose, xylose, or sucrose), 0.33 g l n from various nitrogen sources (yeast extract, monosodium glutamate, urea, or kno ). the optimization of carbon and nitrogen sources concentrations were performed in the range between 0-50 g l and 4-20 g l , respectively. supernatant was obtained by filtration and hode was extracted using ethyl acetate with volume 1:1 for 1 hour. the organic phase was separated from the water phase, the volume was measured and then concentrated using vaccuum centrifuge. these dry extracts were redisolved using methanol for hplc at 100 fold concentration. subsequently, these extracts were analyzed using hplc system (waters) over c18 coloumn (puresil, 150 x 4.6 mm) and eluted using gradient system of acetonitril-water from 15% to 100% for 25 min. each treatment was done in triplicate, and the concentration of 8-hode for each treatment was statistically analyzed using 2 ways anova (montgomerry 1991) this study showed that by using glucose as carbon source produced 0.18 mg l 8-hode, which cibotium barometz c. lunata -1 -1 -1 -1 -1 3 is much higher compared to using lactose, sucrose, xylose or maltose as carbon source (fig 1). when different glucose concentrations were used as carbon source in the medium, the highest 8-hode production was found at 10 gl glucose concentration. at higher concentration the synthesis of 8-hode by was depressed significantly, suggesting that at higher concentration of glucose the growth and hode synthesis were inhibited. it was reported that in fungi the fatty acid is synthesized in the similar manner with polyketide synthesis (jenie 2006). biosynthesis of fatty acid was directly connected to carbon metabolism prior to citric acid cycle. the lower energy required to metabolize carbon source will stimulate fatty acid biosynthesis in fungi. the reported biosynthesis of hydroxy fatty acid has correlation with sporulation in genus (wadman 2008). the utilisation of simple sugar as a sole carbon source better stimulates the sporulation of in comparison to complex carbon source (calvo 2002). in this study, glucose was also shown to be the best carbon source for 8-hode production by . it was proposed that glucose also stimulated biosynthesis of 8-hode in in a biosynthesis pathway similar to the one used for the production of hydroxy fatty acid in . increasing glucose concentration to more than 10 g l in cultivation media, however, decreased 8-hode production by (fig 2). rawlings (1998) reported that pyruvate conversion to acetyl co-a was catalysed by the action of pyruvate dehydrogenase under glucose limited condition during fatty acid production in . this might also be the reason why 8-hode production decreased when glucose concentration increased. the result of this study showed that glucose at the lowest concentration -1 -1 c. lunata et al. aspergillus et al. aspergillus et al. c. lunata c. lunata aspergillus c. lunata saccharomyces cerevisae a b cd cd e 0.0000 0.0200 0.0400 0.0600 0.0800 0.1000 0.1200 0.1400 0.1600 0.1800 0.2000 glucose lactose sucrose xylose maltose carbon source fig 1 effect of carbon source on 8-hode production by . the concentration of various carbon sources was 4 g l . different of alphabet symbol shows significant different of the treatment. curvularia lunata -1 188 prabandari et al. microbiol indones (10 g l ) gave the highest 8-hode production. however, a further study is needed to examine whether even lower glucose concentration will further enhance the 8-hode production. this study also revealed that 8-hode production was significantly affected by nitrogen source. monosodium glutamate was a better nitrogen source for production of 8-hode by in comparison to urea, yeast extract or nh cl. even did not produce 8-hode when nh cl used as nitrogen source (fig 3). even though monosodium glutamate was the best nitrogen source in comparison to others, altering the concentration of monosodium glutamate did not significantly affect the 8-hode production (fig 4). increasing monosodium glutamate concen-1 c. lunata, c. lunata4 4 tration in medium affected 8-hode production by only in lower concentration (4-12 mg l ). in higher concentration (16-20 mg l ), increased monosodium glutamate concentration did not give significant effect on 8-hode production. even though at concentration 20 g l monosodium glutamate gave higher production of 8-hode, this increase was not statistically significant. production of secondary metabolite by polyketide pathway in fungi is also influenced by nitrogen source. hajjaj (2001) reported that glutamic acid and histidine fungal cell. this accumulation stimulates acetyl co-a to enter polyketide pathway rather than citric acid cycles. organic nitrogen source was also reported to c. lunata et al. -1 -1 -1 in cultivation media stimulated α-ketoglutarate accumulation inside a bcd bcd bcd e 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000 0.4500 0.5000 10 20 40 30 50 glucose concentration (g l-1 fig 2 effect of glucose concentration on 8-hode production by . different of alphabet symbol shows significant different of the treatment.curvularia lunata a b c d 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 monosodium glutamate yeast extract urea nh4cl nitrogen source fig 3 effect of nitrogen source on 8-hode production by . the concentration of various nitrogen sources was 0.33 g l . different of alphabet symbol shows significant different of the treatment. curvularia lunata -1 ) volume 5, 2011 microbiol indones 189 better increase fatty acid production by fungi in comparison to inorganic nitrogen source (hwang 2005). in this study, when nh cl employed as nitrogen source 8-hode was not detected in the culture. limited studies has been published on the qualitative production of 8-hode by fungi (wadman 2005; bowers 1986). in quantitative production, most study discussed on the trans formation of polyunsaturated fatty acid into hydoxy fatty acid. heo (2009) reported the production 0.4 g l 10-hydoxy stearic acid (10-has) by sp strain ds5. they used glucose and yeast extract as carbon and nitrogen sources and olive oil as substrate. however they did not report if other carbon and nitrogen sources were used for 10has production. while arjol (2010) reported the production oxylipins by 42a2 using fatty acids as carbon source. they detected that after 2 h incubation the concentration of monohydro xylated compound reached 4 g l . this study was the first report for quantitative 8-hode production by . et al. et al. et al. et al. flavobacterium et al. pseudomonas c. lunata 4 -1 -1 references arjol, m, galia mb, bermudo e. 2010. identification of oxylipins antifungal activity by lc-ms/ms from the supernatant of 42a2. chem phys lip. 163:341-346. doi: 10.1016/j. chemphyslip.2010.02.003. bowers, ws, hoch hc, evans ph, katayama m. 1986. thallophytic allelopathy: isolation and identification of laetisaric acid. science 232:105-106. doi: 10.1126/science.232.4746.105. calvo an, wilson ra, bok jw, keller np. 2002. relationship between secondary metabolism and fungal development. microbiol molec biol rev. 66 (2): 447-459. doi: 10.1128/mmbr.66.3.447-459.2002. hajjaj h, niederbeger p, duboc p. 2001. lovastatin biosynthesis by in chemically defined medium. appl envin microb. 67:2596-2602. doi: 10.1128/aem.67.6.2596-2602.2001. pseudo monas aspergillus terreus a bcd cde bcd cde 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000 0.4500 0.5000 4 8 12 16 20 monosodium glutamate concentration (g l-1) heo sh, hou ct, kim bs. 2009. production of oxygenated fatty acids from vegetable oils by sp strain ds5. new biotechnol. 26 (1/2):105-108. doi: 10.1016/j.nbt.2009.03.003. hwang bh, kim jw, park cy, park cs, kim ys, ryu yw. 2005. high level production of arachidonic acid by fed-batch culture of using nh oh as a nitrogen source and ph control. biotechnol lett. 27:731-735. doi : 10.107/s10529-005-53621. jenni s, leibundgut m, maier t, ban n. 2006. architecture of fungal fatty acid synthase at 5 å resolution. science 311: 1263-1267. doi : 10.1126/science.1123251. lin wy, chang jy, hish ch, pan tm. 2008. profiling the proteome during nitrogen limitation. j agric food chem. 56:433-441. doi: 10.1021/jf072420e. liu b, timar j, howlett j, diglio ca, honn kv. 1991. lipoxygenase metabolites of arrachidonic and linoleic acids modulate the adhesion of tumor cells to endhotelium via regulation of protein kinase c. cell regul. 2: 1045-1055. montgomerry dc. 1991. design and analyisis of experiments. 3 ed. new york: john wiley & sons. mundt s, kreitlow s, jansen r. 2003. fatty acid with antibacterial activity from cyanobacterium oscillatoria redekei hub 051. j appl pshycol. 14:263-267. doi: 10.1023/a:1023889813697. omar mn, moynihan h, hamilton r. 2003. scaling-up production of 13 -hydroxy-9 ,11 -octadecadienoic acid (13 -hode) through chemoenzymatic technique. bull korean chem. soc. 24:397-399. doi: 10.1002/chin.200336066. rawlings bj. 1998. biosynthesis of fatty acids and related metabolites. nat prod rep. 15 : 275-307. doi : 10.1039/a815275y. rombi m, bordighera, 2002 feb. 19. method for obtaining oil that is rich in hydoxyoctadecadienoic fatty acids (hode) or the esters thereof from a mixture containing linoleic acid or esters thereof. us patent. 6,348,609. sayyad sa, panda bp. 2007. optimization of nutrient parameters for lovastatin production by mtcc 369 under submerged fermentation using response surface methodology. appl microbiol biotechnol. 73:1054-1058. doi : 10.1007/s00253-0060577-1. tauk-tornisielo sm. viera jm, cecilia m, carneiro vs, govone js. 2007. fatty acid production by four strains of grown in plant oil and soluble carbohydrates. afric j biotechnol. 6:18401847. tomita f. 2003. endophytes in southeast asia and japan: their taxonomic and potential applications. fungal diversity 14: 187 204. flavobacterium mortierella alpine monascus pilosus s z e s monascus purpureus mucor hiemalis 4 rd fig 4 effect of monosodium glutamate concentration on 8-hode production by . different of alphabet symbol shows significant different of the treatment. curvularia lunata 190 prabandari et al. microbiol indones wadman mw, rp de vries, sic kalkhove, ga veldink, jfg vligentharat. 2009. charaterization of oxylipins and dioxygenase genes in the asexual fungus . bmc microbiol. 9(59) [on line]. doi: 10.1186/1471-2180-9-59. aspergillus niger wadman mw, van zadelhoff g, hamberg m, visser y, veldink ga, vliegenthart jfg. 2005. conversion of linoleic acid into novel oxylipins by the mushroom agaricus bisporus. lipids 40 (11): 11631170. doi: 10.1007/s11745-005-1481-2. volume 5, 2011 microbiol indones 191 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 12, number 3, september 2018 the growth of leptolyngbya hs-16 and hs-36 on 35°c with ph variation isolation and urease activity test of bacteria for calcium carbonate (calcite) precipitation (biocementation) in soil bacterial population and chemical characteristics of fermented mandai cempedak with starter induction the administration of pseudoalteromonas piscisida 1ub through artemia sp. to enhance growth performance, immune response and resistance of white shrimp (litopenaeus vannamei) larvae against vibrio harveyi the potency of aluminum hydroxide nanoparticles for dengue subunit vaccine adjuvant nurul rakhmayanti and nining prihantini hanies ambarsari and aflakhur ridlo anton rahmadi, kartika sari, nikmatul khairiyah, frio handayani, sitohang satrio, yuliani, and aswita emmawati widanarni, savini retalia sababalat, munti yuhana, and diah ayu satyari sabar pambudi, etik mardliyati, silmi rahmani, damai ria setyawati, tika widayanti, angelina gill, asri sulfianti, and whinie lestari 69 74 83 92 99 author index 106 subject index 107 adriansyah, universitas bakrie, indonesia alucia anita artarini, institut teknologi bandung, indonesia andria agusta, lembaga ilmu pengetahuan indonesia, indonesia arif nurkanto, kementrian perindustrian, indonesia astutiati nurhasanah, badan pengkajian dan penerapan teknologi, indonesia benediktus yohan, eijkman, indonesia catur riani, institut teknologi bandung, indonesia churiyah, badan pengkajian dan penerapan teknologi, indonesia debbie s retnoningrum, institut teknologi bandung, indonesia dessy natalia, institut teknologi bandung, indonesia endang purwantini, virginia polytechnic institute and state university, usa ernawati arifin giri-rachman, institut teknologi bandung, indonesia fernita puspasari, institut teknologi bandung, indonesia g i r i s h k u m a r g u p t a , m a h a r i s h i markandeshwar, india i made sudiana, lembaga ilmu pengetahuan indonesia, indonesia ihsanawati, institut teknologi bandung, indonesia is helianti, badan pengkajian dan penerapan teknologi, indonesia jignesh patel, university of texas medical branch at galveston, usa julian ransangan, university malaysia sabah, malaysia kartini kramadibrata, lembaga ilmu pengetahuan indonesia, indonesia khomaini hasan, institut teknologi bandung, indonesia mohamad yani, institut pertanian bogor, indonesia moritz müller, swinburne university of technology sarawak, malaysia n venkatesh, st. joseph's college of engineering, omr chennai india nagendra p. shah, the university of hong kong, hong kong neung tiaamroeng, suranaree university of technology, thailand niknik nurhayati, badan pengkajian dan penerapan teknologi, indonesia puspita lisdiyanti, lembaga ilmu pengetahuan indonesia, indonesia raden haryo bimo setiarto, lembaga ilmu pengetahuan indonesia, indonesia reginawanti hindersah, universitas padjajaran, indonesia rofiq sunarya, badan pengkajian dan penerapan teknologi, indonesia ruth chrisnasari, universitas surabaya, indonesia sabar pambudi, badan pengkajian dan penerapan teknologi, indonesia sarjiya antonius, lembaga ilmu pengetahuan indonesia, indonesia sartaj alam syed, the university of agriculture peshawar, pakistan shubhajit mitra, university of texas medical branch galveston texas, usa silva abraham, badan pengkajian dan penerapan teknologi, indonesia sinosh skariyachan, dayananda sagar institutions, india siti khodijah chaerun, institut teknologi bandung, indonesia tjahjani mirawati soediro, universitas indonesia, indonesia trismilah, badan pengkajian dan penerapan teknologi, indonesia valan arasu, king saud university, saudi arabia verawat, biotec national center for genetic engineering and biotechnology, thailand wangsa tirta ismaya, dexa laboratories of biomolecular sciences, indonesia woro hastuti satyantini, universitas airlangga, indonesia yaya rukayadi, universiti putra malaysia, malaysia yeva rosana, universitas indonesia, indonesia yenny meliana, lembaga ilmu pengatahuan indonesia, indonesia yoice srikandace, lembaga ilmu pengatahuan indonesia, indonesia acknowledgement volume 12 vol.12, no.3, september 2018 microbiology indonesia issn 1978-3477, eissn 2087-8575 available online at http://jurnal.permi.or.id/index.php/mioline issn 1978-3477, eissn 2087-8575 volume 12, number 3, september 2018 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000,200 000,-5 institutional rate (institution or library) (idr) 240 000,400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print neung tiaamroeng, 2020 norio kurosawa, 2020 kartini kramadibrata, 2020 diana e waturangi, 2020 endang purwantini, 2020 wellyzar sjamsuridzal, 2020 yuan kun lee, 2020 yaya rukayadi, 2020 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi iman rusmana, 2020 page 1 acknowledgement.pdf page 1 7 no. 292 (tiana milanda).pmd mutation and characterization of an albino mutant of monascus sp. isolated from the cikapundung river, bandung tiana milanda∗‡, marlia singgih wibowo, tutus gusdinar, and haryanto dhanutirto school of pharmacy, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia monascus sp. isolated from cikapundung river, bandung was mutated using ethyl methanesulfonate (2.5%, 90 min). previously, this wild type was identified as monascus purpureus itbcc-hd-f001 employing random amplification polymorphic dna (rapd). stability of the mutant was observed using color consistency and mutant stability (sub-culturing for five generations) tests. genetic variation of the mutant (m. purpureus itbcc-hdf002) was confirmed by rapd. one of the dna bands of 1150 bp was found in the albino mutant but not in the wild type, so it was considered as a genetic variation resulting from the mutation process. the albino mutant was characterized by comparing the growth curve, biomass production curve, and the monascidin a production curve of both strains i.e. wild type and the albino mutant. monascidin a production of the mutant was higher than that of the wild type. key words: monascus sp., ethyl methanesulfonate, random amplification polymorphic dna, albino mutant, monascidin _____________________________________________ ________________ ∗corresponding author and ‡present address, faculty of pharmacy, universitas padjadjaran, jalan raya bandung-sumedang km 21, jatinangor 45363, indonesia; phone/fax: +62-22-7796200, e-mail: tiana_milanda@yahoo.com monascus purpureus is a rice fermentation fungus used to produce ’angkak’. this fermentation product has been used for a long time as a food colorant, a meat preservative, and a traditional medicine, especially by the people of south china, japan, and south east asia. various secondary metabolites have been isolated from ’angkak’, such as pigments, an antihypercholesterolemic agent (monacolin k), and an antibacterial substance (monascidin a) (blanc et al. 1998a; lakrod et al. 2000). blanc et al. (1995) reported that monascidin a was citrinin, a mycotoxin that has carcinogenic, teratogenic, and nephrotoxic properties. the presence of monascidin a in products fermented by monascus raises concerns about the safety of the products. to obtain fermented products free from monascidin a, blanc et al. (1998b) tested three procedures. firstly, they conducted a selection among monascus strains to find one that did not produce monascidin a, secondly, they modified fermentation conditions, and thirdly, they degraded monascidin a found in fermentation products. these methods could suppress or eliminate monascidin a, but unfortunately the process also decreased the production of pigments and monacolin k significantly. this could happen because these three secondary metabolites were all synthesized via the polyketide biosynthesis pathway catalyzed by polyketide synthase (pks). characterization of enzymatic reactions at the branch point of the three-metabolite biosynthesis pathways is needed to properly develop a strategy to produce pigments and monacolin k free from monascidin a. (hajjaj et al. 1999). to characterize those enzymatic reactions, a study of pks enzymes and genes at the molecular level is needed. this kind of study requires a transformation system for m. purpureus that uses an albino mutant deficient in monacolin k or citrinin (monascidin a) synthesis as recipients. the use of albino mutant has a higher priority because clone selection for the recipient of pigment biosynthesis gene can be conducted from the colonies that are able to reverse the non-pigment producing capability. (blanc et al. 1998b). this albino mutant is still not available, especially one that is obtained from local indonesian isolates. in this research, we carried out the mutation of monascus sp. from local isolates and characterized a resultants albino mutant. these are the initial steps to develop an efficient transformation system for m. purpureus. materials and methods monascus sp. strains. as the parental strain we used monascus sp. isolated from the cikapundung river, bandung. two other fungal strains were used as standards, i.e. monascus purpureus cect2955t (universidad de valencia, spanyol) and monascus ruber dsm1561 (biotechnology research center, lipi, cibinong). these three fungal strains were grown on yeast extract-malt extract-peptone (ymp) agar (0.3% [w/v] yeast extract, 0.3% [w/v] malt extract, 0.6% [w/v] peptone, 2% [w/v] glucose, 2% [w/v] agar) for 7-10 days at 28 oc. the spore suspension was obtained from monascus sp. solid culture on ymp agar, and then the spore concentration was adjusted to: % transmittance (t) = 25% at wavelength (λ) = 660 nm which was equal to 2.25 x 104 spores ml-1. identification of monascus sp. employing rapd. identification of monascus sp. was carried out employing rapd according to the procedure by campoy et al. (2003), and replicated three times. five milliliters aliquots of each strain monascus sp., m. purpureus cect 2955t and m. ruber dsm1561 was inoculated into 45 ml ymp broth medium, and then shaken at 150 rpm at 28 oc for 18 h. fungal mycelium was harvested by centrifugation at 107,141x g at room temperature for three min. after that, fungal dna was isolated using a wizard genomic dna purification kit (promega). from each strain, 200 ng of dna was amplified in microbiology indonesia, april 2007, p 19-22 volume 1, number 1 issn 1978-3477 a solution containing 0.5 unit taq dna polymerase (promega), 90 nmoles magnesium chloride (promega), 8 pmoles primer crl9 (5’-cagccgcccc-3’) or crl12 (5’-cgccgcccg-3’) (proligo-sigma), 5 nmoles dntp (promega) in a 2700 thermocycler (applied-bioscience). the pcr program consisted of initial denaturation (4 min, 94 oc), followed by 44 cycles each of which consisted of denaturation (40 sec, 94 oc), hybridization (60 sec, 34 oc), and elongation (120 sec, 72 oc), and then terminated by final elongation for 10 min at 72 oc. electrophoresis was conducted on 1% (w/v) agarose gel using ix tae buffer at 70-80 volt for 90 min. mutation of parental strain m. purpureus. parental strain m. purpureus itbcc-hd-f001 was mutated using ethyl methanesulfonate (ems) according to the procedure of susilowati (1997). a 5 ml aliquot of parental strain suspension was inoculated into 45 ml ymp broth medium, and then shaken at 150 rpm at 28 oc for 64 h. ten milliliters of this liquid culture was then centrifuged at 5357 x g at 4 oc for 10 min, and then the mycelium sediment was suspended in 10 ml of 200 mm phosphate buffer ph 7.0. portions of 1.0 ml mycelium suspension and 100 µl of 2% (w/v) glucose were placed in 25 ml -erlenmeyer flasks, and then sufficient quantity of ems and 200 mm phosphate buffer ph 7.0 were added each flask to obtain a variation of ems concentrations of 0, 1, 2, 3, 4% (v/v). all flasks were shaken at 150 rpm at 28 oc for 45 and 60 min. after that, 2 ml of 5% sodium thiosulfate was added. after shaking at 150 rpm at 28 oc for 20 min, the fungal suspension was centrifuged at 5357 x g at 4 oc for 10 min. the sediment was resuspended in 1 ml of 200 m phosphate buffer ph 7.0 and this suspension was then gradually diluted. a 100 µl aliquot of each dilution was inoculated on ymp agar and incubated for 4-7 days at 28 oc. the number of colonies (both red and white) was observed, the percentage of mutant viability and mutation efficiency were determined, and then the death curve was constructed. the mutation process was repeated with ems concentrations of 0, 1.0, 1.5, 2.0, 2.5, 3.0% (v/v) with 90 min incubation periods. white colonies from the mutation process were grown again on ymp-g agar (ymp agar with 8% [w/v] glucose) for seven days at 28 oc for the color consistency test. the colonies that remained white in color were sub-cultured for five generations on ymp agar for mutant stability testing. genetic variations of m. purpureus albino mutant. genetic variation of m. purpureus albino mutant was detected employing rapd with three replications using the same procedure previously used in the identification of the parental strain. m. purpureus parental strain was used as the standard. the growth of m. purpureus parental strain and its albino mutant. five milliliters suspensions of parental strain and albino mutant was each inoculated into 45 ml ymp broth, and then shaken at 150 rpm at 28 oc. culture samples from both strains were taken every six hours until the stationary growth phase was reached. all culture samples were centrifuged at 3571 x g for 10 min to obtain a packed mycelial volume (pmv) (% w/v) and ph of the supernatant of the culture samples. both data were plotted versus fermentation time (h) on a growth curve. a 5 ml aliquot of both cultures at optimum age was used as inocula for the production process in 45 ml ymp broth medium. the culture was then shaken at 150 rpm at 28 oc. samples were taken every 24 h, after which all samples were centrifuged at 3571 x g for 10 min. the data of pmv (% w/ v) and ph of the supernatant were plotted versus fermentation time (h) on a biomass production curve. the extraction, identification, and determination of monascidin a content in fermentation samples produced by m. purpureus parental strain and albino mutant were conducted employing the procedure of blanc et al. (1995). ten milliliters aliquots from each strain were extracted three times using methanol. the resultant extraction was filtered, washed two times with isooctane, and then an equal volume of water was added. the extract was acidified using sulfuric acid to give ph of 4.5, then partitioned using methylene chloride. the bottom phase was dried, and then dissolved in methanol. a citrinin standard calibration curve was made at concentration levels of 0, 5, 10, 15, 20, 25, 30, 35, 40 µg/ml using commercial product (sigma-aldrich). identification and determination of monascidin a content were carried out using hplc (hewlett-packard) with a 214 nm uv detector. the mobile phase was methanol:water (1:1), with a 1.5 ml min-1 flow rate in a c-18 hypersil column at 40 oc column temperature. results identification of monascus sp. identification of monascus sp. employing rapd using both primers crl9 and crl12 resulted in an rapd band pattern that was identical to that of m. purpureus cect2955t. this rapd band pattern was different from the band pattern of m. ruber dsm1561. therefore, it was concluded that monascus sp. from cikapundung river was m. purpureus. this was later named m. purpureus itbcc-hd-f001 (figure 1). mutant m. purpureus itbcc-hd-f001. mutation of m. purpureus itbcc-hd-f001 using 3 and 4% (v/v) ems with a 60-min incubation time resulted in less than 10% mutant viability and unfortunately under those conditions white colonies could not be obtained from the albino mutant. later, mutation was conducted using a lower ems concentration and longer incubation time, i.e. ems concentrations of 1.0, 1.5, 2.0, 2.5, 3.0% (v/v) for 90 min. less than 10% mutant viability was obtained at an ems concentrations of 2.5 and 3.0% (v/v), but only the 2.5% (v/v) ems concentration could produce white colonies of 1.93 x 103 colonies/ml, with 6.01% mutant viability and 1.08% mutation efficiency. color consistency and mutant stability tests showed that albino mutant colonies remained white on high-glucose medium (ymp-g) and ymp solid medium over five generations. this was consistent and the stable albino mutant was later named m. purpureus itbcc-hd-f002. genetic variation of m. purpureus itbcc-hd-f002. the determination of genetic variation of m. purpureus itbcc-hd-f002 using rapd produced almost identical banding pattern compared to that of its parental strain, except for one additional band at 1150 bp produced by primer crl12. this band is the result from genetic change caused by ems mutation in the albino mutant’s dna (figure 2). the growth of m. purpureus itbcc-hd-f001 and m. purpureus itbcc-hd-f002. the two growth curves 20 milanda et al. microbiol indones figure 3 growth curve of m. purpureus parental strain ( pmv, ph ) and albino mutant. ( pmv, ph ) 1 2 3 4 5 6 7 8 4 9 7 3 4 2 8 6 1419 5 1 7 3 9 6 2 1 4 bp bp crl9 crl12 figure 1 rapd amplification results of monascus sp. dna using crl9 and crl12 as primers. 1: λ/hindiii/ecori dna marker, 2 and 5: m. purpureus itbcc-hd-f001, 3 and 6: m. purpureus cect2955t, 4 and 7: m. ruber dsm1561, 8: puc19/hinfi dna marker. 1 2 3 4 5 6 1 3 7 5 1 1 5 0 9 4 7 bp crl9 crl12 figure 2 rapd amplification results of the dna of m. purpureus parental strain and albino mutant. 1 and 6: λ/hindiii/ecori dna marker, 2 and 4: parental strain (m. purpureus itbcc-hdf001), 3 and 5: albino mutant (m.purpureus itbcc-hdf002). (figure 3) showed that the growth rate of albino mutant (m. purpureus itbcc-hd-f002) was slower than that of its parental strain (m. purpureus itbcc-hd-f001). the growth curve peak shifted from 66th h in parental strain to 144th h in the albino mutant. the decrease of ph was faster in the parental strain fermentation compared to that of its mutant. the deceleration of growth rate also caused a change in the optimum age of inocula prepared for fermentation, from 64th h in the parental strain to 102nd h in the albino mutant. the two biomass production curves (figure 4) showed that biomass production rate for albino mutant was slower than that of its parental strain, therefore production curve peak shifted from 72nd h with 12% (w/v) pmv (parental strain) to 120th h with 11.58% (w/v) pmv (albino mutant). it was also shown that medium ph fluctuated throughout both strains’ fermentation processes. monascidin a. identification employing hplc revealed that both citrinin (monascidin a) standard and fermentation extracts from the parental strain and the albino mutant produced a chromatogram peak with average retention time of 1.7 min. therefore, it was concluded that monascidin a was found in the fermentation extracts of both strains. two production curves of monascidin a (figure 5), indicated that the monascidin a production rate by albino mutant was figure 5 monascidin a production curve of m. purpureus parental strain ( ) and albino mutant ( ). m o n a sc id in a ( µ g m l1 ) volume 1, 2007 microbiol indones 21 p a c k e d m y c e li a l v o lu m e ( % w /v ) p a c k e d m y c e li a l v o lu m e ( % w /v ) figure 4 biomass production curve of m. purpureus parental strain ( pmv, ph ) and albino mutant. ( pmv, ph ) slower than that of its parental strain, resulted in the shifting of production curve peak from 96th h with 19.19 µg ml-1 monascidin a content (parental strain) to 144th h with 22.26 µg ml-1 monascidin a content (albino mutant). discussion based on microscopic morphological characteristics on various growth media, monascus sp. can be classified into m. pilosus, m. purpureus, and m. ruber (hawksworth and pitt 1983). it turns out that the identification of monascus is very difficult to do if it is based only on microscopic morphology, so identification needs to be conducted using a molecular biology technique such as rapd (lakrod et al. 2000). campoy et al. (2003) succeeded in characterizing monascus sp. employing rapd with 2 decamer (10-bases) primer, namely crl9 and crl12. the same procedure succeeded in identifying monascus sp. isolated from cikapundung river as m. purpureus, which was later named m. purpureus itbcc-hd-f001. an albino mutant was obtained through the mutation process of parental strain m.purpureus itbcc-hd-f001 using 2.5% (v/v) ems with a 90 min incubation time. this mutant was produced when both mutant viability and mutation efficiency were below 10%, as reported by susilowati (1997) during the mutagenesis of saccharomyces cerevisiae. mutation under those conditions resulted in the substitution of base pairing in pks gene for pigment biosynthesis. employing consistency and stability tests, albino mutant colonies were shown to be consistent and stable over five generations. unstable albino mutants will turn red again on ymp-g medium because a high concentration of glucose can reverse the mutation. the consistent and stable albino mutant from this experiment was later named m. purpureus itbcc-hd-f002. analysis of genetic variation caused by mutation was conducted employing rapd. this analysis produced an additional 1150 bp band. this band was the result of an amplification process by primer crl12 on one dna segment of the albino mutant chromosome, which was not present in parental strain dna. this amplification happened because the an increased homology of this dna segment to crl12, which led to base pair substitution caused by ems. characterization of the albino mutant through the comparisons of growth, biomass production, and monascidin a production curves revealed that the growth, biomass production, and monascidin a production rates are slower compared with those of its parental strain. this indicated that ems mutation decreased the ability of albino mutant to adapt to its growth surroundings. the increase of monascidin a production by the mutant at the peak of the production curve was caused by the accumulation of pigment precursor, which was also the precursor of monascidin a. all of the precursors were shifted to the monascidin a biosynthesis pathway, resulting in an increase of monascidin a production by the mutant. acknowledgment this research was funded by science and technology assessment and research project of the indonesia directorate general of higher education, department of national education, through postgraduate team research grant (hptp) ii, 2004-2006. reference blanc pj, hajjaj h, loret mo, goma g. 1998a. control of production of citrinin by monascus. in: the symposium on monascus culture and applications. toulouse-france: laboratoire biotechnologies-bioprocedes insa. 8-10 jul 1998. blanc pj, loret mo, goma g. 1995. production of citrinin by various species of monascus. biotech lett 17:291-294. blanc pj, loret mo, goma g. 1998b. pigments and citrinin production during cultures of monascus in liquid and solid media. in: advance in solid state fermentation. toulouse-france: laboratoire biotechnologies-bioprocedes insa. p 393-406. campoy s, perez f, martin jf, gutierrez s, liras p. 2003. stable transformants of monascus purpureus obtained by protoplast transformation and agrobacterium-mediated dna transfer. cur genet 43:447-452. hajjaj h et al. 1999. biosynthesis pathway of citrinin in the filamentous fungi monascus ruber as revealed by 13c nuclear magnetic resonance. appl environ microbiol 65:311-314. hawksworth dl, pit ji. 1983. a new taxonomy of monascus species based on the cultural and microscopical characters. aus j bot 31:51-61. lakrod k, chaisrisook c, yongsmith b, skinner dz. 2000. rapd analysis of genetic variation within a collection of monascus spp. isolated from red rice (angkak) and sofu. mycol res 104:403408. susilowati pe. 1997. isolasi dan karakterisasi mutan resesif sal4 di ragi saccharomyces cerevisiae [thesis]. bandung: institut teknologi bandung. 22 milanda et al. microbiol indones 01 prihantini 8 hal.cdr vol.12, no.2, june 2018, p 35-42 doi: 10.5454/mi.12.2.1 influence of temperature variations on growth of nostoc (cyanobacteria) hs-5 and hs-20 isolated from indonesian hot springs 1 1 1 nining betawati prihantini *, cahya guslyani , ratna yuniati , 1,2 and wellyzar sjamsuridzal 1 department of biology, faculty of mathematics and natural sciences, universitas indonesia, kampus ui depok 16424, west java, indonesia; 2 center of excellence for indigenous biological resources-genome studies, faculty of mathematics and natural sciences, universitas indonesia, kampus ui depok 16424, west java, indonesia the research aims to know the effect of variation temperature to the growth of nostoc hs (hot spring)-5 and hs-20. strain of nostoc hs-5 was isolated from ciseeng hot spring which has habitat temperature range of 30-43 °c, and nostoc hs-20 was isolated from pancar mountain hot spring which has temperature range of 46-69 °c. the research was done by measuring biomass weight and chlorophyll content on day-1, 2, 3, 4, 7, 10, 14, 17, and 21. the temperatures used were 20 °c, 35 °c, and 50 °c. the growth medium used was bold basal medium (bbm) with ph 6.6. each treatment was made in four replications. non-parametric statistical analysis used were the friedman test (a=0.05) and spearman test (a=0.01). the result showed there were significant differences on the biomass weight of nostoc hs-5 and hs-20 grown at temperature of 20 °c, 35 °c, and 50 °c. the average amount of biomass highest weight for nostoc hs-5 and hs-20 occurred in both strains were grown at 35 °c. besides that, there was no correlation between the weight of biomass and chlorophyll content of nostoc hs-5 and hs-20. key words: biomass weight, cyanobacteria, hot spring, nostoc, temperature penelitian ini bertujuan untuk mengetahui pengaruh variasi suhu terhadap pertumbuhan nostoc hs (hot spring)-5 dan hs-20. galur nostoc hs-5 diisolasi dari sumber air panas ciseeng yang memiliki rentang suhu habitat 30-43 °c, dan nostoc hs-20 yang diisolasi dari sumber air panas pancar mountain yang memiliki rentang suhu 46-69 °c. penelitian dilakukan dengan menimbang berat biomassa dan mengukur kandungan klorofil pada hari ke 1, 2, 3, 4, 7, 10, 14, 17, dan 21. suhu yang digunakan adalah 20 °c, 35 °c, dan 50 °c . media pertumbuhan yang digunakan adalah bold basal medium (bbm) dengan ph 6,6. setiap perlakuan dibuat dalam empat ulangan. analisis statistik non parametrik yang digunakan adalah uji friedman (a= 0,05) dan uji spearman (a= 0,01). berdasarkan hasil kualitatif, ada perbedaan yang signifikan pada berat biomassa nostoc hs-5 dan hs-20 yang ditanam pada suhu 20 °c, 35 °c, dan 50 °c. jumlah rata-rata berat biomassa yang paling tinggi untuk nostoc hs-5 dan hs-20 terjadi pada kedua strain tersebut yang ditumbuhkan pada suhu 35 °c. selain itu, tidak ada korelasi antara berat biomassa dengan kadar klorofil nostoc hs-5 dan hs-20. kata kunci: berat biomassa, cyanobacteria, nostoc, suhu, sumber air panas microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-7270163, fax:+6221-7270012; email: nining@ui.ac.id then forms new colony (komarek et al. 2014). the genus of nostoc can be found in many habitats. the nostoc habitats generally in terrestrial and subaerial, spread in alkaline soil and moist stone. nostoc can also be found in various temperature areas, i.e. hot spring (hogg 2005). nostoc has an adaptation activity to survive in high temperature areas, i.e. making a mat-structure, heat shock proteins (hsps), and etc. environmental is the key factor of microorganism growth. temperature is one of the environmental factors which affects the growth of cyanobacteria (olsson-francis 2012). the example of cyanobacteria genus which can grow in hot temperature is nostoc. prihantini (2015) reported that two strains of nostoc n o s t o c i s a h e t e r o c y s t o u s f i l a m e n t o u s cyanobacteria genus from order of nostocales and family of nostocaceae (bold and wynne 1978), has a chain-like structure and each cell is covered by sheath. nostoc has a differentiated vegetative cell that called heterocyst and akinete. heterocyst is a place where nitrogen fixation is occurred and akinete is a place where reproduction is occurred (lee 2008). the asexual reproduction of nostoc commonly occurs with production of hormogonia. hormogonium is a cell which produces by heterocyst or decayed small filaments of nostoc which is separated from parent cell found in two indonesia hot springs, i.e. hs-5 strain from ciseeng hot spring and hs-20 strain from pancar mountain hot spring. hs-5 and hs-20 are the isolate codes of both strains, while hs stands for hot spring. the growth of nostoc is performed by increasing of cells amount on certain time unit which describe in growth curve. the growth phase of nostoc is generally similar with another microorganism, i.e. adaptation phase (lag phase), exponential phase (log phase), stationer phase, and death phase (madigan et al. 2012). the growth measurement of nostoc is done by measuring the biomass weight of nostoc during the treatment days. research was conducted to determine the growth of nostoc isolated from hot spring ciseeng (hs-5 strain) and isolated from the red crater of pancar mountain (hs-20 strain) on temperature variations. materials and methods microorganisms and growth medium. two filamentous cyanobacteria strains of nostoc, i.e nostoc hs-5 and hs-20 strains were used in this study. both strains were isolated by pipette method by prihantini (prihantini 2015). the two nostoc strains, hs, were isolated from hot spring in indonesia, i.e. ciseeng (nostoc hs-5 strain) and pancar mountain (nostoc hs20 strain). all strains were cultured in bold basal medium (bbm). culturing of cyanobacteria starter and harvesting of cultures. the cyanobacteria strains were culturing to obtain an optimal biomass on bbm broth. the biomass was prepared as starter cultures and harvested at logarithmic phase or exponential phase on the 14 d of incubation. then, 10 mg of starter culture were inoculated to 200 ml of fresh medium in erlenmeyer flask, and incubated in three separated incubators which has temperature 20 °c, 35 °c, and 50 °c. the treatment was given at 3 temperatures, namely 20 °c, 35 °c, and 50 °c, with 4 replication each. observation was done for 21 d, and the observed parameters were biomass weight, ph, temperature of the incubators, and cyanobacterial morphology. measurement of biomass weight of nostoc. biomass weight measurement of nostoc strain was done by counting the different weights of empty microtube and microtube which contains pellet. measurement was done by growth data collection every day, which were day of 0, 1, 2, 3, 4, 7, 10, 14, 17, and 21. growth curve was made by comparing the biomass weight in y axis and harvesting day in x axis. the growth curve could determine the adaptation phase (lag phase), exponential phase (log phase), stationary phase, and death phase. measurement of chlorophyll content of nostoc. chlorophyll content measurement of nostoc strains was done by spectrophotometric method. chlorophyll was extracted from the biomass with 80% acetone and t h e c h l o r o p h y l l c o n t e n t w a s m e a s u r e d b y spectrophotometer (nanodrop uv-vis 1000, thermo fisher scientific, usa) at 645 and 663 nm of wavelength. the chlorophyll content was calculated using arnon formula, i.e. 12.7 (d663 nm) – 2.69 (d645 nm) (meeks 1974). measurement of chlorophyll content was carried out by collecting data on days 0, 1, 2, 3, 4, 7, 10, 14, 17, and 21. results macroscopic and microscopic observations of nostoc hs-5 and hs-20 were performed by observing the color of culture (macroscopic observation), and the shape and diameter of nostoc thallus (microscopic observation). observation of the culture color of nostoc biomass was done by comparing the culture color on the faber castell color indicator table. the inoculum used comes from a starter culture age of 21 days. based on the color chart, both the color of the starter culture and the color of the culture during observation on nostoc hs-5 were green juniper (juniper green), while the color of the culture on the nostoc hs-20 was light brown (bistre) (fig 1). in addition to macroscopic observations, microscopic observations were also carried out. the thallus shape of nostoc on starter culture and treatment culture were no different. based on cell diameter measurements, one nostoc hs-5 had a length of 3.36 mm, while one nostoc hs20 had a length of 3.69 mm (fig 2). nostoc hs-5 and hs-20 culture growth was seen in changes in the amount of biomass weight. measurement of biomass weight of the culture was done on day 0 (t0), day 1 (t1), day 2 (t2), day 3 (t3), day 4 (t4), day 7 (t7), day 10 (t10), day 14 (t14), day 17 (t17), and day 21 (t21). the inoculum used is derived from a 21-day starter culture that was in a stationary phase. the inoculum used was 20 mg for each test culture. the biomass weight data can be seen in table 1. in addition to the weight of biomass, the measurement of cyanobacteria growth rate can be done by calculating the chlorophyll content in cyanobacteria cells. chlorophyll is a green pigment produced by cyanobacteria and has an ability to photosynthesize. the measurement of chlorophyll content is generally 36 prihantini et al. microbiol indones performed on the filament cyanobacteria because it is difficult to calculate the number of cyanobacteria cells. calculation of chlorophyll content for nostoc hs-5 and hs-20 culture was performed on the same day with the measurement of culture biomass weight. the average of chlorophyll content data can be seen in table 2. discussion in both strains (fig 2), there were no heterocyst and akinete. this might be caused by enough nitrogen content in environmental conditions which affecting heterocyst and akinete production. the heterocyst and akinete production are affected by environmental conditions with limited nitrogen content (ogawa and carr 1969). the color of both nostoc hs-5 and hs-20 cells had moss green color. based on the macroscopic observation of cultures (fig 3), noticeable color change was apparent between nostoc hs-5 and hs-20 incubated at 20 °c, 35 °c, and 50 °c. nostoc hs-5 incubated at 20 °c appeared to be moss green color, while nostoc hs-20 was golden brown color. at an incubation temperature of 35 °c, visible color differences were quite markedly compared to at 20 °c. the nostoc hs-5 cultures appeared dark green (sap green) color, while the nostoc hs-20 look dark brown (bistre) color. in contrast to cultures incubated at 20 °c and 35 °c, the nostoc hs-5 and hs-20 cultures incubated at 50 °c experienced very noticeable color changes. nostoc hs-5 and hs-20 which incubated at 50 °c experienced color change to yellowish white (ivory), with a colorless or translucent culture medium. these color changes could be caused by photobleaching that continued until finally changing the color of the culture to white. color changes that occurred in cultures, especially microbiol indones 37volume 12, 2018 table 1 the average biomass weight ratio of nostoc hs-5 and nostoc hs-20 at incubation temperature was 20 °c, 35 °c, and 50 °c at observation time for 21 days time (day) biomass w eight (mg ml-1) 20 °c 35 °c 50 °c hs-5 hs-20 hs-5 hs-20 hs-5 hs-20 t0 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 t1 0.20±0.08 0.12±0.05 0.22±0.09 0.12±0.05 0.10±0.08 0.12±0.12 t2 0.20±0.20 0.10±0.00 0.22±0.15 0.07±0.05 0.07±0.05 0.15±0.10 t3 0.15±0.06 0.10±0.00 0.18±0.09 0.10±0.17 0.10±0.08 0.10±0.00 t4 0.20±0.08 0.10±0.08 0.20±0.08 0.12±0.12 0.17±0.22 0.05±0.06 t7 0.43±0.17 0.30±0.18 0.53±0.12 0.32±0.05 0.20±0.08 0.22±0.19 t10 0.35±0.13 0.20±0.00 0.38±0.09 0.22±0.05 0.12±0.05 0.15±0.06 t14 0.35±0.13 0.12±0.05 0.32±0.38 0.40±0.022 0.10±0.14 0.12±0.08 t17 0.20±0.08 0.10±0.08 0.30±0.27 0.30±0.24 0.07±0.09 0.07±0.09 t21 0.15±0.13 0.07±0.09 0.27±0.09 0.15±0.06 0.05±0.05 0.05±0.05 time (day) average of chlorophyll content (mg l-1) 20 °c 35 °c 50 °c hs-5 hs-20 hs-5 hs-20 hs-5 hs-20 t0 0.13±0.06 0.11±0.07 0.13±0.03 0.10±0.03 0.15±0.02 0.10±0.04 t1 0.23±0.06 0.13±0.04 0.40±0.22 0.17±0.07 0.21±0.12 0.18±0.03 t2 0.22±0.06 0.16±0.07 0.28±0.11 0.12±0.04 0.13±0.10 0.19±0.08 t3 0.14±0.07 0.17±0.03 0.17±0.05 0.17±0.05 0.15±0.003 0.14±0.03 t4 0.14±0.06 0.11±0.04 0.25±0.10 0.17±0.07 0.14±0.06 0.11±0.04 t7 0.16±0.17 0.12±0.06 0.13±0.02 0.12±0.07 0.10±0.08 0.12±0.07 t10 0.45±0.30 0.23±0.08 0.33±0.12 0.21±0.05 0.16±0.03 0.13±0.09 t14 0.30±0.21 0.10±0.04 0.25±0.08 0.24±0.04 0.12±0.06 0.14±0.03 t17 0.1±0.04 0.23±0.02 0.13±0.23 0.16±0.06 0.13±0.02 0.13±0.03 t21 0.37 ±0.07 0.15±0.09 0.32±0.06 0.11±0.04 0.16±0.05 0.12±0.06 table 2 average of chlorophyll content of nostoc hs-5 and nostoc hs-20 incubated in 20 °c, 35 °c, and 50°c -1 biomass weight (mg ml ) -1 average of chlorophyll content (mg l ) 38 prihantini et al. microbiol indones fig 3 macroscopic visualization of nostoc hs-5 and hs-20 on day 21. a. hs-5 at a temperature of 20 °c; b. hs5 at 35 °c; c. hs-5 at 50 °c; d. hs-20 at 20 °c; e. hs-20 at 35 ° c; f. hs-20 at 50 °c. in cultures of nostoc hs-5 and hs-20 incubated at 50 °c were caused by photobleaching. photobleaching causes the cell to turn green caused by high temperatures so that the chlorophyll pigment in the cell is damaged (hsieh et al. 2013). all nostoc strains which were used in this study performed adaptation phase (lag phase), exponential phase (log phase), and death phase, but there's stationer phase in hs-5 incubated in 20 °c and hs-20 incubated in 35 °c in 21 d (fig 4). all nostoc strains showed a fluctuation on their growth curve. based on figure 4, there was any peak difference between the strains. fig 2 microscopic characters of nostoc hs-5 (a) and nostoc hs-20 (b) in starter cultures (bar = 50 mm). fig 1 macroscopic visualization of starter cultures of nostoc hs-5 (a) and nostoc hs-20 (b). microbiol indones 39volume 12, 2018 almost all strains performed peak on day-7 (t7), except hs-20 incubated in 35 °c on day-14 (t14). the highest peak occurred in hs-5 incubated in 35 °c that had an -1 average biomass weight of 0.53 mg ml . the difference between each strain could be caused by the different metabolism activities between each strain in three kinds of temperatures. based on the growth curve of nostoc hs-5 and hs20 (fig 4) at the three treatment temperatures, there were a different growth phase between each test culture. the difference in the duration of the adaptation phase could be caused by the ability of each nostoc strain to its new environment. the nostoc hs-5 culture adaptation phase was occurred most rapidly at an incubation temperature of 50 °c, but the amount of weight of the biomass was not apparent. this could be caused because the growth rate of the culture was affected by the high temperature stress founded in the incubator at a temperature of 50 °c so that the weight gain of the biomass was not apparent. unlike the culture grown at 50 °c, the nostoc hs-5 culture was grown at 35 °c experienced a slower adaptation phase fig 4 eps (extracellular polymeric substances) production by an iron-oxidizing indigenous bacterium as represented by emulsifying activity index (ei) over a period of 7 days. error bars represent standard deviation from the mean (n = 2-3). 40 prihantini et al. microbiol indones but experienced a significant increase in biomass weight. this could be caused by environmental temperature conditions that support the growth of the culture. unlike nostoc hs-5, the nostoc hs-20 adaptation phase was occurred most rapidly at an incubation temperature of 35 °c. this was supported by the apparent weight gain of biomass compared to 20 ° c and 50 ° c. this could be caused by the nostoc hs20 culture which was suitable for 35 °c environmental conditions so that the adaptation process at that temperature was quite short. the difference in the exponential phase of each treatment culture could be caused by the metabolic activity of each culture to the environmental conditions. the difference in metabolic activity was likely to be caused by differences in genetically fig 5 graphic of correlations between the mean weight of biomass and the chlorophyll content of nostoc hs-5 and nostoc hs-20 incubated in 20 °c, 35 °c, and 50 °c. different species. the exponential phase in nostoc hs5 was occurred at an incubation temperature of 50 °c. the culture experienced a weight increase in biomass which was less noticeable compared to 20 °c and 35 °c. nostoc hs-20 culture undergone an exponential phase at an incubation temperature of 35 °c which is seen in the weight gain of biomass which looks quite different. based on the graph of the growth curves of both strains (fig 4), it was seen that nostoc hs-5 cyanobacteria tend to be mesophilic, while nostoc hs20 is probably a cyanobacteria that tends to be thermophilic. this was shown in the presence of the stationary phase in both strains. the stationary phase in nostoc hs-5 was seen at 20 °c, whereas in nostoc hs20 there was a stationary phase at 35 °c. in addition, nostoc hs-5 comes from ciseeng hot springs which have a lower temperature (30-40 °c) than the hs-20 which comes from the hot spring of pancar mountain (46-69 °c). both nostoc hs-5 and hs-20 strains were likely to be thermotolerant. it was based on the growth curve of both strains at 50 °c but the growth was very slow, unlike at 20 °c and 35 °c. in addition, the two strains were found in two indonesian hot springs which had a high enough temperature. according to matshushita et al. (2015), thermotolerant is a microorganism that can l i v e a t h i g h t e m p e r a t u r e s . t h e r m o t o l e r a n t microorganisms are generally found in hot water sources, so that microorganisms have adapted to high temperature environment, but their growth is less optimum (matshushita et al. 2015). beside of biomass weight, growth measurement of cyanobacteria can be done by measuring chlorophyll content. chlorophyll content in nostoc hs-5 and hs20 had different peaks in each treatment. the highest peak in nostoc hs-5 occurred in 20 °c, although nostoc hs-20 occurred in 35 °c. based on the average measurement of chlorophyll content, there was no significant difference in the nostoc hs-5 and hs-20 chlorophyll content. the graphic showed that the increasing of the biomass weight was not followed by the increasing of the chlorophyll content (fig 5). it could be concluded that there was no correlation between biomass weight and the chlorophyll content in each strain. besides that, based on the spearman test conducted for both strains and each incubation temperature, the results obtained were that there was no correlation between the weight of biomass and chlorophyll content. temperature variations affected the growth of nostoc hs-5 and hs-20 based on the weight of biomass. nostoc hs-5 and hs-20 grown at 35 °c produced the highest average biomass weight. the highest average chlorophyll content data on both nostoc were different. the highest average chlorophyll content in nostoc hs-5 was found in nostoc grown at 20 °c, while in nostoc hs-20 was found in nostoc grown at 35 °c. there was no correlation between biomass weight and chlorophyll content in nostoc hs5 and hs-20. acknowledgment this work was funded by hibah penelitian unggulan perguruan tinggi (pupt) 2017 to nining betawati prihantini, grant no. 2708/un2.r3.1/ hkp05.00/2017. authors thank to the center of excellence (coe) for indigenous biological resources-genome studies, universitas indonesia, depok for the facilities provided. references bold hc, wynne mj. 1985. introduction to the algae structure and reproduction. prentice-hall, inc., englewood cliffs, new jersey: xiv + 706 pp. faber castell. 2012. faber castell polychromos artist color p e n c i l s . 5 p p . h t t p : / / w w w. f a b e r c a s t e l l . c o m / service/color-charts, accessed on 08 february 2017, at 8:15 pm. st hogg s. 2005. essential microbiology, 1 ed. john wiley and sons ltd, usa: 468 pages. hsieh yy, hsieh yy, hung ph, leu jy. 2013. hsp90 regulates non-genetic variation in response to environmental stress. mol cell. 50(1): 82-92. komarek j, kastovsky j, mares j, johansen jr. 2014. taxonomic classification of cyanoprokaryotes (cyanobacterial genera) 2014, using a polyphasic approach. preslia. 86: 295-335. lee re. 2008. phycology. cambridge university press, new york: ix + 534 pp. li y, lin y, loughlin pc, chen m. 2014. optimization and effects of different culture conditions on growth of halomicronema hongdechloris – a filamentous cyanobacterium containing chlorophyll f. original research article. 5(67): 1-12. madigan mt, martinko jm, stahl da, clark dp. 2012. th brock biology of microorganisms: 13 ed. benjamin cummings, san fransisco: xxviii + 1155 pp.meeks jc. 1974. chlorophylls. in: stewart, wdp. (ed). 1974. algal physiology and biochemistry. university of california press, 161-175. microbiol indones 41volume 12, 2018 42 prihantini et al. microbiol indones olsson-francis k, simpson ae, wolff-boenisch, cockel cs. 2012. the effect of rock composition on cyanobacterial weathering of crystalline basalt and rhyolite. geobiology. 10: 434-444. ogawa re carr jf. 1969. the influence of nitrogen on heterocyst production in blue-green algae. limnol oceanogr. 14: 342-351. prihantini nb. 2015. polyphasic taxonomy of culturable cyanobacteria isolated from hot spring in west java, indonesia. doctoral dissertation. dept. of biology fmipa ui, depok: vii + 213 pp. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 135-139 (wiza).pmd agricultural and agro-industrial wastes, which are mostly in the form of lignocelluloses, basically have no economic value may be even be considered of negative value because they still need further treatment. lignocelluloses consist of hemicelluloses, celluloses, and lignin. celluloses are the biggest component and can be transformed into energy sources, paper, single-cell protein, glucose, and sorbitol (putarau 1969; coral et al. 2002). one of the goals in biotechnological development is to open the way to utilize microbes in waste bioconversion. microbe used to treat cellulose-containing wastes could produce extra-cellular enzymes that were able to degrade cellulose material into their smaller components (bedford and partridge 2001). the potency of utilizing cellulase is varied. however, there are some constraints in producing it such as the unavailability of superior microbial strains and a lack of knowledge in enzyme production technology. on the other hand, experts from developed countries acknowledge that countries rich in biodiversity, including indonesia, are a potential source of microbes for bioprocessing (fox 1994). cellulase is usually produced by bacteria and fungi. at present, fungi are usually needed in producing cellulase and for the bioconversion process to improve animal feed quality, but there is a constraint arising from the increase of crude fiber content due to the presence of hyphae which is counted as crude fiber (coral et al. 2002) . one of the alternatives is using forest litter decomposer bacteria i.e. bacillus spp. bacillus spp. have the biggest number of bacteria, can be found in almost every location, and when chemically tested, they were the most active out of seven genera of bacteria (jusfah et al. 1995; yusuf 2000). litter is organic material residue from dead plants that can be found on the earth’s surface or buried in its soil minerals. the speed of the litter decaying process by decomposer microbiology indonesia, december 2007, p 135-139 volume 1, number 3 issn 1978-3477 selection and identification of cellulase-producing bacteria isolated from the litter of mountain and swampy forest wizna1*, hafil abbas2, yose rizal1, abdi dharma3, and i putu kompiang4 1department of animal feed and nutrition, 2department of livestock production, faculty of animal husbandry, 3department of chemistry, faculty of mathematics and natural sciences, universitas andalas, kampus limau manis, padang 25163, indonesia 4research institute for animal production, departemen pertanian, ciawi, bogor 16002, indonesia the isolation and selection of cellulase-producing bacteria was conducted to identify the species of cellulolytic bacillus. the bacteria were isolated from the litter of swampy forest in pesisir selatan and mountain forest in lembah anai tanah datar. these bacteria were cultivated in selective media to obtain bacteria from the genus bacillus. six bacillus isolates were obtained from swampy forest and three bacillus isolates from mountain forest. these isolates were cultivated in agar medium with carboxymethylcellulose as the carbon source. colonies which produced clear zones were assumed to be cellulolytic bacillus. based on biochemical and morphological examinations the result indicated that these two isolates were bacillus coagulans and b. amyloliquefaciens. the cellulase activity of b. coagulans and b. amyloliquefaciens were 0.812 and 1 200 unit ml-1 to c 1 (β-exoglucanase) respectively, 0.368 and 0.488 unit ml-1 to c x (β-endoglucanase) respectively. key words: identification, cellulolytic, bacillus, litter _____________________________________________ ________________________ * corresponding author, phone: +62-751-71464 , e-mail: wiznazhari57@yahoo.com microorganisms (decomposition) depends on the enzymes produced (spurr and barnes 1980). bacillus spp. has been known to be the producer of various enzymes such as cellulase, hemicellulase, protease, α-amylase, urease, xylanase, and chitinase (cowan 1974; alexander 1977). these enzymes are expected to be able to transform and change complex molecules, especially lignocellulose which is a limiting factor in animal feed, into simpler molecular components. the objective of this research is to select and obtain a collection of cellulase-producing bacillus sp. based on their characteristics. materials and methods isolation and selection of cellulolytic bacillus. sampling locations used were several places in the forest litter area of lembah anai tanah datar and the swampy forest litter in pesisir selatan, west sumatera. one gram of soil sample from each litter was diluted to 10-5 cfu ml-1 using fisiologis nacl solution. one ml of this dilution was spread on solid medium of bacillus that contained 0.25% bacto agar, 0.6% peptone, 0.3% pancreatic digest of casein, 0.3% yeast extract, 0.3% beef extract, and 0.001% mgso 4 . 7 h 2 o for 24 h at 37 °c (cowan 1974). well-grown colonies were chosen to obtain a pure culture of the bacteria using two loops of an inoculating needle to transfer and inoculate bacillus sp. onto a petri dish containing na medium (cappucino 1987). the selection of cellulolytic bacillus sp. was conducted based on the ratio of clear zone to colony diameter after 48 h on carboxy methyl cellulase (cmc) medium (sigma) (cowan 1974). to be able to identify the clear zone more distinctly, a qualitative test was conducted by pouring five ml of 0.1% congo red on cmc medium after 24 h incubation. a clear zone indicated that the isolate was a pure culture of cellulolytic bacillus sp. after that, each of the grown colonies was recultured again on medium specific for bacillus and incubated for 24 h at 37 °c. the colonies were observed 136 wizna et al. microbiol indones table 1 isolation and selection of baccillus from the litter of swampy forest pesisir selatan (gt) isolate gt 1 gt 2 gt 3 gt 4 gt 5 gt 6 evaluation staining cell shape spore location gram test morphological observation liquid medium colony shape elevation edge motility biochemical test catalase oxidase glucose fermentation starch hydrolysis indole urease citrate gas h 2 s mr (methyl red) vp (voge prokauer) nitrate reduction casein hydrolysis gelatin hydrolysis tsia rod center positive aerobic irregular flat serrated motile + + + + + + + + r/r rod center positive aerobic irregular flat serrated motile + + + + r/y rod center positive aerobic irregular flat serrated motile + + + + r/y rod center positive aerobic irregular flat serrated motile + + r/y rod center positive aerobic irregular flat serrated motile + + y/y rod center positive aerobic irregular flat serrated motile + + y/y r = red, y = yellow. under microscope. the colonies that looked uniform and grew well indicated that the isolates obtained were pure cultures of cellulolytic bacillus sp. identification of cellulolytic bacillus sp. further identification of these species was conducted by means of macroscopic and microscopic examinations, and biochemical characterization. macroscopically, the observed characteristics were: color, shape, colony surface, and edge. microscopically, the tests included: gram reactions, shape, and cell size. biochemical tests included: carbohydrate utilization, indole, h 2 s, urease, citrate utilization, catalase activity, motility, and environmental tolerance test by growing the isolate on medium with ph 4.0 and 7.0 in the presence of nacl at a final concern of 5.0 and 7.0%. the reactions observed in chemical tests were compared to the identification keys (buchanan and gibbons 1974). determination of cellulase activity of bacillus sp. cellulase activity can be measured from the fermentation results by two selected species of bacillus and are compared with that of trichoderma harzianum (as the control) in a mixed substrate from sago pulp and rumen content (7:3). inoculum dosage, fermentation time, fermentation temperature, thickness of substrate, water content, and particle size for the determination of cellulase activity were 2%, 48 h, 40 °c, 2 cm, 60%, and 1 mm respectively. isolation of cellulase crude extract. sampling was conducted an a daily bases to observe the development of cellulase activity. culture medium was homogenized by adding 75 ml citrate buffer 0.05m (ph 6) (5x medium weight) for each sample, followed by shaking and filtering. the filtrate was obtained by placsing the homogenate inside a 250 ml erlenmeyer flask which was placed inside a 500 ml glass beaker filled with ice cubes. during filtration, the homogenate was stirred at low speed and the temperature was kept at 4 °c. the filtrate was centrifuged at 4 500 g for around 15 min (twice) under refrigeration and then filtered using whatman no. 1 to obtain crude extract of the enzyme. determination of cellulase activity. the cellulase activity test was conducted employing the somogy-nelson method (bergmeyer et al. 1981). a volume of 0.5 ml 1% (v/w) cmc substrate was pippeted into a reaction tube and was then preincubated for 5 min in 50 °c water bath. after adding 0.5 ml enzyme filtrate, the incubation was continued for 45 min (chaabouni et al. 1994). enzymatic reactions were stopped by heating the mixture in a boiling water bath for 15 min. then 1 ml of somogy-nelson reagent was added, followed by 15 min heating. finally, 1 ml of arsenomolibdic reagent was added into the solution. this mixture was shaken until no more gas was produced, and then diluted with 7 ml distilled water. after the filtrate was separated from its sediment, its absorbance was read at 620 nm wavelength. control treatments were the same, except the enzyme was first inactivated. to determine the reducing sugar content from the above enzyme reactions we used a standard curve calibrated with glucose monohydrate. cellulase activity was defined as the quantity of enzyme that can release 1 mmol glucose min-1 (chaabouni et al. 1994). results isolation and selection of cellulolytic bacillus. from the isolation and selection results conducted in the laboratory of research institute for animal diseases (bpph) baso bukittinggi, there were six isolates of bacillus sp. (gt1, gt2, gt3, gt4, gt5, and gt6) from swampy forest litter and six isolates of bacillus sp. (la1, la2, la3, la4, la5, and volume 1, 2007 microbiol indones 137 table 2 isolation and selection of bacillus from the litter of lembah anai forest (la) isolate la 1 la 2 la 3 la 4 la 5 la 6 evaluation staining cell shape spore location gram test morphological observation liquid medium colony shape elevation edge motility biochemical test catalase oxidase glucose fermentation starch hydrolysis indole urease citrate gas h 2 s mr (methyl red) vp (voge prokauer) nitrate reduction casein hydrolysis gelatin hydrolysis tsia rod center positive aerobic circular convex serrated motile + + + + + + r/r rod center positive aerobic circular convex serrated motile + + + + r/y rod center positive aerobic circular convex serrated motile + + + + + r/y rod center positive aerobic circular convex serrated motile + + r/y rod center positive aerobic circular convex serrated motile + + + + y/y rod center positive aerobic circular convex serrated motile + + + + + y/y r = red, y = yellow. fig 1 clear zones of bacillus sp. on cmc medium. sp.3/gt2/la2 sp.2/la2 sp.1/gt1 table 3 clear zone diameters of isolates obtained from isolation and selection on cmc medium (48-hour inoculation) isolate species clear zone diameter (mm) (gt1) (la1) (gt2/la2) (gt3/la4) (gt4/gt5gt6) (la3/la6) (la5) sp.1 sp.2 sp.3 sp.4 sp.5 sp.6 sp.7 37.40 12.40 09.32 la6) from lembah anai mountain forest litter (table 1 and 2). from the classification based on “bergey’s manual of determinative bacteriology”, there were seven different isolates out of 12 selected isolates, i.e. (gt1), (gt2/la2), (gt3/la4), (gt4/gt5 gt6), (la1), (la3/la6), and (la5). after that, selection on the ability to degrade was conducted qualitatively by inoculating the above isolates on cmc medium. after 48 h, three out of seven isolates were found to produce clear zones. to examine further about the clear zones and their diameters as depicted in fig 1 and table 3. sufficiently wide clear zones were obtained from bacillus sp.1 (gt1) and bacillus sp.2 (la1) with diameters 37.4 mm and 12.40 mm respectively. these two isolates with wide clear zones were then examined to determine their species in the microbiology laboratory, department of biology, faculty of mathematics and natural sciences, institut teknologi bandung. identification of bacillus sp. species observation by means of macroscopic and microscopic evaluations as well as biochemical reactions on gt1 and la1 showed some similarities and some differences. the similarities are: staining tests indicated gram positive, rod shaped, produced elliptical endospores. from the morphological observation, growth on liquid medium formed a pellicle (aerobic), serrated colony edge, and motile. from biochemical tests, these microbes hydrolyzed starch and casein: sugar fermentation was positive, produced no h 2 s, produced catalase, indicated as alkaline by litmus milk. the differences were: spore location of gt1 in the center of vegetative cell, irregular colony shape with flat elevation, positive hydrolysis of gelatin, and nitrate reduction. whereas, spore location of la1 were in the center to the end of vegetative cell, circular colony shapes with convex elevation, negative hydrolysis of gelatin, and negative nitrate reduction. the caracteristic of gt1 and la1 as well as the defferences of b. amyloliquefaciens and b. coagulans at table 4. cellulase activity. average values of cellulase activities of bacillus and t. harzianum in mixed substrate containing sago pulp and rumen content were as follows. cellulase activity of c x and c 1 of b. amyloliquefaciens were 0.368 and 0.812 unit ml-1 respectively, b. coagulans were 0.488 and 1 200 unit ml-1 respectively, and t. harzianum 0.6550 and 0.3070 unit ml-1 respectively (table 5). 138 wizna et al. microbiol indones rod center positive aerobic irregular flat serrated motile + + + + + + alkaline table 4 characteristics of bacillus sp.1 (gt1), bacillus sp.2 (la1), b. amyloliquefaciens and b. coagulans isolate bacillus sp.1 (gt1) bacillus sp.2 (la1) bacillus amyloliquefaciens bacillus coagulans staining cell shape spore location gram test morphological observation liquid medium colony shape elevation edge motility biochemical test catalase glucose fermentation lactose fermentation sucrose fermantation starch hydrolysis indole urease citrate triple sugar-iron h 2 s methyl red voges-proskauer nitrate reduction casein hydrolysis gelatin hydrolysis litmus milk rod center positive aerobic irregular flat serrated motile + + + + + + + + + alkaline rod center-end positive aerobic circular convex serrated motile + + + + + + + alkaline rod center-end positive aerobic circular convex serrated motile + + + + evaluation table 5 average value of cellulase activities of bacillus amyloliquefaciens, b. coagulans and trichoderma harzianum average value of cellulase activity (unit/ml)* c x (β-endoglucanase) c 1 (β-exoglucanase) isolates b. amyloliquefaciens b. coagulans t. harzianum 0.4880 + 0.046 0.3680 + 0.043 0.6550 + 0.045 1.2000 + 0.150 0.8120 + 0.145 0.3070 + 0.013 *cellulase activity is based on cellulase crude extract. discussion the results of this research are in line with the characteristics determined by buchanan and gibbons (1974) and holt et al. (1994) based on qualifications from “bergey’s manual of determinative bacteriology”, that a bacillus is classified into the kingdom of prokaryotes, division bacteria, class schyzomycetes, order eubacteriales, family bacillaceae, and genera bacillus. the characteristic of bacillus sp. cultivated on na medium was that the clear zone of bacillus sp.1 (gt1) was wider than that of penicillium on cmc medium as reported by dharma (1998) (37.4 mm vs 15.60 mm). identification results of these two species were in line with buchanan and gibbons (1974) and holt et al. (1994), that b. amyloliquefaciens was gram positive, rod shaped, produced elliptical endospores, and located in the center of vegetative cell, growth on liquid medium formed pellicle (aerobic), irregular colony shape with flat elevation, motile, positive hydrolysis of gelatin and nitrate reduction, and b. coagulans was gram positive, rod shaped, produced elliptical endospores, and located in the center to the end of vegetative cell, growth on liquid medium formed pellicle(aerobic), circular colony shapes with convex elevation, motile, negative hydrolysis of gelatin, and negative nitrate reduction. from the results of biochemical reactions, gt1 is b. amyloliquefaciens and la1 is b. coagulans. the value of the cellulase activity of both bacillus species were lower compared to those of enzymes produced by bacillus subtilis strain-cbtk 106 cultivated for 72 h on banana peel medium completed with c and n sources and several minerals, such as carboxy methyl cellulase (cm case) 9.6 iu gds-1 (gram dry matter substrate). filter paperase (fpase) 2.8 iu gds-1 and cellobiase 4.5 iu gds-1 (chundakkadu 1999). likewise, the cellulase activity in the fermentation of oil palm empty fruit bunch using penicillium sp. was higher for c x , it was 1 457 unit ml-1 in an 8-day fermentation and for c 1 0.01 unit ml-1 in a 14-day fermentation (dharma 1998). references alexander m. 1997. introduction to soil microbiology. 2nd ed. new york: john wiley. bedford mr, partridge gg. 2001. enzyme in farm animal nutrition. marlborough wiltshire: finnfeeds cab publ. bergmeyer hu, bergmeyer j, grab m. 1981. methods of enzymatic analysis 2. amsterdam: verlag chemie. buchanan re, gibbons ne. 1974. bergey’s manual of determinative bacteriologi. 9 t h ed. california: the williem and wilkins company. cappucino jg, sherman n. 1987. microbiology a laboratory manual. 2th ed. california: the benjamins columning publ company. chaabouni sm,taieb nh, mosrati r, radhouane e.1994. preliminary assestment of pennicillium occitanis cellulase. j enzyme microbiol 16:538-542. chundakkadu k. 1999. production of bacterial cellulases by solid state bioprocessing banana wastes. j biores technol 69:231-239. volume 1, 2007 microbiol indones 139 coral g, arikan b, unaldi mn, guvenmes h. 2002. some properties of crude carboxymethyl cellulase of aspergillus niger z10 wildtype strain. turki j biology 26:209-213. cowan st. 1974. cowan and steel’s manual for the identification of medical bacteria. 2nd ed. ames: cambridge university pr. dharma b. 1998. production of cellulases by penicilium sp. on different substrate solid state plant at different time [thesis]. padang: universitasn andalas. fox jk. 1994. biodiversity promises great prospecting. j biotechnol 13:544-545. holt jg, krieg nr, sneath pha, staley jt, williams st. 1994. bergeys manual of determinative bacteriology. 9 th ed. baltimore: the williams and wilkins company. mo. usa. jusfah j, rangkuti d, muchtar e. 1995. inventory microorganism as litter decomposer in lembah anai. in: annual report of project japan international cooperation agency (jica). universitas andalas. 7:105-109. putarau jm. 1969. by-product of cane sugar industry. in: an introduction to their industrial utilization. 1 st ed. new york: elsevier publ, company. spurr sh, barnes bv. 1980. forest ecology. 3rd ed. new york: john willey and sons. yusuf s. 2000. litter decomposer bacteria found in swampy forest. an observation considering the factors of open and closed area [thesis]. padang: andalas university. vol.1 , no. , 202 , p -6 2 december 2 x x doi: 10.5454/mi.1 . .6 2 x igg subclasses identification of immunized mice sera with dengue tetravalent dna vaccine based on prm-e genes beti ernawati dewi , 1 rizka andhitia mentari putri , 2* tjahjani mirawati sudiro 1 , and fithriyah sjatha 1 1 department of microbiology, medical faculty, universitas indonesia, jalan pegangsaan timur 16, jakarta 10320, indonesia; 2 master programme of biomedical science, faculty of medicine, universitas indonesia, jalan salemba raya no.6, jakarta pusat 10430, indonesia. dengue fever is still a serious health problem in the world. denv consists of 11 kb of single positive-stranded rna encoding three structural proteins and seven non-structural proteins. prm and e proteins are the main targets of the antibody response that rich of epitopes and able to induce protective immunity. there are four denv serotypes that have similar antigenic structures in the amino acid sequence of protein e. in our previous study, we successfully constructed a recombinant tetravalent dna vaccine candidate consisting pumvc4a-based expression plasmid for prm-e protein of all denv serotypes (pumd1, pumd2, pumd3 and pumd4). it has been proved that the vaccine candidate was able to induced anti-dengue igg as well as neutralization antibody to all denv serotypes. this study aims to determine igg subclasses of immunized mice with recombinant tetravalent dna vaccine candidates based on prm-e genes of all serotypes. key words: dengue vaccine, dna vaccine, igg subclass, recombinant, tetravalent demam berdarah masih menjadi masalah kesehatan dunia. penyakit yang disebabkan oleh virus dengue (denv) ini, . denv terdiri dariditransmisikan oleh aedes sp 11 kb rna untai positif tunggal yang mengkode 3 protein struktural dan 7 protein non struktural. protein struktural pr-m dan e ( ) merupakan target utamaenvelope dari respon antibodi yang kaya akan epitope-epitope imunologis dan berkontribusi pada induksi imunitas protektif. terdapat empat jenis denv yang memilikiserotipe kesamaan struktur antigenik pada sekuens asam amino protein e. dalam penelitian sebelumnya, kami telah berhasil mengonstruksi vaksin dna tetravalent yang terdiri dari plasmid berbasis pumvc4a dengan insert gen prm-e dari semua serotipe denv (pumd1, pumd2, pumd3 dan pumd4). kandidat vaksin terbukti mampu menginduksi igg anti-denv dan antibody netralisasi terhadap semua serotipe denv. penelitian ini bertujuan untuk mengetahui subkelas igg dari mencit yang diimunisasi dengan kandidat vaksin berbasis dari semua serotipe denvdna rekombinan gen sisipan prm-e secara tetravalent. kata kunci: rekombinan, subkelas igg, tetravalent, vaksin dengue, vaksin dna microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 081215300246;: +62 e-mail:rizkaandhitiap gmail@ .com dengue fever cases. the case fatality rate (cfr) due to dhf is considered high, although the death rate in 2017 decreased from the previous year. there were 3 provinces with the highest cfr which are gorontalo (2.18%), north sulawesi (1.55%) and southeast sulawesi (1.47%) .(kemenkes ri 2017) denv belongs to the flaviviridae family, which is a single-stranded positive-sense enveloped rna viruses. it is an spherical virus and 40 to 50 nm in diameters. the rna genome consists of approximately 11 kb nucleotides that encode large polyproteins containing three structural proteins (capsid ©, prm, and envelope (e)) and seven non-structural proteins (ns1, ns2, ns3, ns4a, ns4b and ns5). denv is divided into four serotypes, denv-1, denv-2, denv-3 and denv-4. these share about 70% amino acid sequence identity in e protein, that cross-reactive anti genetically. the e dengue fever (df) is serious health problem in the world, especially in tropical and subtropical regions. df is caused by dengue virus (denv) that transmitted by arthropods, several factors such asaedes sp mosquito. rainfall, temperature, humidity, level of urbanization and vector control can influence the spread of mosquitoes . denv distribution has(bhatt et al. 2013) occurred globally, world health organization (who) estimates there are 390 million denv infections per year, of which 96 million showed clinical manifestation with disease severity . indonesia is the(who 2017) second endemic country with the largest dengue hemorrhagic fever (dhf) case among 30 countries. in 2017, 80% of cities/districts were reported to have protein is the outer part of flaviviruses which contributes for adhesion, membrane fusion, and virus assembly. it is the main immunogen that determines tropism and virulence, and also become the main target of antibodies that stimulates the formation of neutralizing and protective antibodies. it has three domain which are edi, edii and ediii. the prm protein is a chaperon protein that helps prevent e protein from error folding through the cell secretion pathway .(gallichotte . 2015; fahimi . 2018)et al et al currently, there are several dengue vaccines that is still under development. one type of vaccine that potential for dengue vaccine is recombinant dna vaccine. it is a vaccine that genetically engineered, containing the gene of interest inserted into plasmid. compared to conventional vaccines based on protein/peptide, dna vaccine is considered to be nonvirulent, easy manufacturing, cost-efficient and high in stability .(stachyra, góra-sochacka and sirko 2014) konishi (2006), have succesfully constructed aet al. dengue tetravalent dna vaccine consisting of pcdna3-based plasmids expressing prm-e genes of the four serotypes and was evaluated in mouse model. (konishi, kosugi and imoto 2006). the result did not show any detectable interference and could induced neutralizing antibody responses to all serotypes. sjatha et al. (2014) constructed chimeric dna vaccines consist of denv diii on a jev prm-e backbone. the result showed that their vaccine candidate induced strong neutralizing antibody responses with reduced ade activity .(sjatha . 2014)et al in our previous study, we have successfully constructed recombinant dna tetravalent vaccine candidate consisting pumvc4a-based expression plasmid for prm-e protein of all denv serotypes (pumd1, pumd2, pumd3 and pumd4). the denv strains used are strains that circulate in indonesia (yunita 2012; rachmayanti 2013; putri 2015). from in vivo study, showed lower viremia titers for almost 70 times compared with unimmunized mice .(putri 2015) furthermore, it has been proved that vaccine candidate capable to induce anti-dengue igg as well as neutralization antibody to all denv serotypes. rodrigo et al. (2009) suggest that denv neutralization is modulated by combined fc region of igg subclasses virion avidity of binding and fcγr preference. sjatha et al. (2014) in their study, suggest that isotypic profile of antibody related to their affinity to complements affect antibody responses in mouse sera. to examine this possibility, this study assesses the profile of subtype igg of the vaccine candidate in mice model. materials and methods ethics statement and mice experiments. ten balb/c mice were divided into two groups: (1) five mice were immunized with pumvc4a as control and (2) five mice were immunized with the dengue tetravalent vaccine candidate (100 µg/mouse) by intramuscular injection using needle-free jet injector (shimajet: shimadzu, kyoto, japan). mice were vaccinated three times, within three-week interval. blood were drawn before vaccination, two weeks post vaccination as well as termination blood. all protocols within this experiment have been ethically approved by health research ethics committee of the faculty of medicine universitas indonesia-rscm number: ket.550/un2.f1/etik/ppm.00.02/2019. the experimental animal was carried out in the animal care facility of the department of microbiology, faculty of medicine, universitas indonesia with a closed cage system and ad libitum feed-drink. viruses. the virus used as antigen for in-house indirect elisa was dengue virus serotype-2 (denv2) ngc strain harvested at 5 d.p.i with viral titer was 3.5 x 10 ffu m . 7 l -1 propagation dh5α harboringeschericia coli plasmid. eschericia coli dh5α competent carrying recombinant plasmids (pumd1, pumd2, pumd3 and pumd4) was propagated in 5 m of liquid lurial bertani (lb) medium containing kanamycin (50 μg m °c in a shaker incubator (200l -1 ) and incubated at 37 rpm) for 18-20 hours. after bacterial growth occurs, 4 m of liquid culture of each plasmid was replanted inl 200 m of liquid lb medium containing kanamycinl ( ) and incubated at50 μg 37lm °c in a shaker -1 incubator (200x rpm) for 18-20 hours. each of the culture then was harvested using centrifuge at 4000 rpm for 5 minutes in °c.4 identification of pumd1 and pumd3. colony pcr was performed to confirm the gen insert of recombinant plasmids of pumd1 and pumd3. a small amount of cells from a single colony of e. coli dh5α competent carrying plasmids were picked up by using sterilized toothpick and resuspended into 50 µl distillated water. a 2 suspension was used asµl template in pcr reactions. pcr reactions were performed in 25 volume following pcr master mixµl kit (qiagen, catalog no. 201443) standard procedure. .identification of pumd2 and pumd4 confirmation of pumd2 and pumd4 was performed using restriction enzymes digestion method. enzymes that used in this study were xbai (thermo fisher, x dewi et al. microbiol indones volume 1 , 2026 2 microbiol indones x catalog no. 0682) for pumd2, while pumd4 were used double digest with two restriction enzyme bamhi (thermo fisher, catalog no. er0051) and xbai (thermo fisher, catalog no. 0682). reaction were performed: tango1 5 µ buffer (10x), 0 µ. of .5 ofl l xbai µ , µ nuclease free wat , µ(10 u ) 8 of er and 2l l l -1 (100 ng ) pure plasmid of pumd2, then incubatedlµ -1 for 2 hours. for pumd4, the reaction were perfomed: 1 5 µ buffer (10x) 0 25 µ xbai µ. of , . of (10 ul l ltango 1 1 ) . of (10 u ) of, 0 25 µ bamhi µ , 9 µ nuclease freel l l wat , µ 4er and 4 pure plasmid of pumd then incubatedl for 1 hour. in vitro xpression nalysis of protein ee a . the expression of prm-e protein of pumd1, pumd2, pumd3 and pumd4 was confirmed in vero cells using lipofectamine ltx with plus reagents (invitrogen). in brief, vero cells were grown in 24 wellplate to 80% confluence. first, each plasmid was diluted to in opti-mem medium (gibco-10 ng µl -1 brl) . plasmids were incubated with10 ng µl 1 µl -1 reagent plus for 5 minutes (room temperature). next, 3 µl lipofectamine-ltx was added into the mixture and incubated for 30 minutes (room temperature). the mixture then added to the cells and incubated for another 48 hours at . after incubation, cells were37 °c fixed using absolute ethanol and500 µl/well incubated for 1 hour at . then, results were4 °c visualized with immunostaining method. plasmid isolation. plasmid isolation was performed based on hispeed® plasmid midi kit (qiagen) with modification. pure dna plasmid were confirmed by gel electrophoresis then stored at -30 °c. the concentration of pure dna plasmid were measured with spectrophotometer (nanodrop 2000c, thermofisher, catalog no. nd-2000). immunization of mice. five 4-6 week-old of balb/c mice per immunization group were immunized three times into quadriceps muscle with 100 µg of candidate plasmid (tetravalent) or pumvc4a using needle-free injector (shimajet, japan). immunization was carried out within threeweeks interval, retro-orbital blood was drawn twoweeks post immunization as well as termination blood. pre-immunization blood of all groups were also taken. blood samples were centrifuge at 3000 rpm °c4 for 10 min, then collected sera were stored at -30 until°c further examination. igg subclasses measurement with in-house indirect elisa. pre-immunization and termination sera from both groups were measured for igg1, igg2a, igg2b and igg3. in this study, we were only using denv-2 (ngc) as coating antigen on elisa plates. denv-2 (3.5 x 10 ffu m ) were diluted into 1:10 in 7 l -1 coating buffer. in brief, denv-2 were coated in 96 well elisa plate (nunc, thermofisher catalog no. 442404-21) at 4 overnight. followed by blocking with°c 100 μ of 37 °cl 5% (w/v) skim milk then incubated at for 1 hour. then, plates were washed using pbs containing 0.05% tween-20. a 100 μ of seruml samples (diluted 1:50) were added into the plates and incubated 37 °c for 1 hour. after incubation platesat were washed then 100 μ each of diluted anti mouse-l igg subclasses (1:1000; biologend) were added into the plates then reincubated 37 °c for 1 hour. platesat were washed and 100 μ of diluted (1:1000) hrp-l avidin were added into the plates and incubated forat 1 hour. after incubation, plates were washed and 50 lμ of tmb substrate (1-step ultra tmb-elisa substrate solution, catalog no: 34028) was added followed by incubation in dark with gentle agitation for 20 minutes at . finally, 20 of stop solution was added and37 °c lμ absorbancies were measured at 450 nm. results identification of lasmid ecombinantp r . pcr colony was performed to confirm the insert of prm-e genes corresponds to its construction in pumd1 and primers sequences description d1 2063 s 5’-tacatcgtggtaggagcagg-3’ denv-1 d1 2510 c 5’-gctctgtccaggtgtgaact-3’ dv3kf-eco 5’-cgcgatatcaccatgaacaggagacgcagaact-3’ denv-3 dv3kr 5’-tgctctagattaggcctgcaccataactcc-3’ table 1 primers used in this study pumd3. the product that was expected for pumd1 was 448 bp, while pumd3 was 2009 bp . as(putri 2015) shown in fig 1, the presence of prm-e were detected both in pumd1 and pumd3. the inserted gene for pumd2 and pumd4 were confirmed using double digest with enzyme restriction method. the insert gene products that was expected for pumd2 and pumd4 was around ±2000 bp according to the gene map illustration shown in fig 2(yunita 2012; rachmayanti 2013). as shown in fig 3, the presence of prm-e genes insert were detected both in pumd2 and pumd4. expression of prm-e protein in vero cells. transfection of plasmids pumd1, pumd2, pumd3 and pumd4 was performed to analyze the protein expression in vero cells for 48 hours. then immunostaining were performed to confirm the ability of the recombinant plasmids. we used antibody from denv infected patients that may contain antibody to e protein to analyze prm-e protein expression of candidate vaccine. the vero cells that were transfected with pumd1, pumd2, pumd3, pumd4 were stained brown indicating positive results (fig 4). in this experiment we used pumvc4a vector plasmids as negative control and show no cells were stained indicating there was no e protein expression. also, it was confirmed that the transfection and staining process was done correctly. igg subclasses titer. termination individual sera from both tetravalent and pumvc4a groups were profiled to measure the igg subclasses titers. then, the mean groups titer of each group for anti-denv-2 igg1, igg2a and igg2b were satistically analyzed with independent t-test, while igg3 was analyzed with mann-whitney u test. the mean groups titer is shown in table 2. as seen in fig 5, the mean titers of igg1, igg2a and igg2b showed significant difference (p<0.05), while igg3 did not. also, the results showed that igg2a was the main subclass induced in immunized mice followed by igg1, igg2b and igg3. the igg2a titer in tetravalent group was 2.5 fold higher than the pumvc4a control group, while other subclasses were 2 fold higher in recognizing denv-2. the results indicates that vaccine candidates was able to induce humoral immune response in mice. discussion in our previous study, we have successfully constructed and proven our recombinant plasmids were able to induced humoral response in mice. we used prm-e as insert genes to pumvc4a as recombinant plasmid. the prm-e proteins are the main targets of the antibody response to denv infection. the glycoprotein e is rich in immunological epitopes and contributes to the induction of protective immunity. in line with sjatha (2014), suggest thatet al the antibody response to serotype-specific diii of protein e, resulted in reduced cross neutralization and did not increase virus infection or in other words there are less possibility of ade .(sjatha 2014)et al. in this study, we administered the vaccine by intramuscular injection which targets mycocytes and keratynocytes directly, including antigen presenting cells (apcs). theoritically, after dna is internalized, the dna will translocate to the nucleus to be transcribed and followed by translation in the cytoplasm, expressed into proteins that have been converted into peptide chains. in addition, intramuscular administration of dna vaccines can be targeted directly at apcs located around the injection site. in this pathway, the encoded antigen will be expressed and after transcription, the antigen peptide will be presented by mhc class i and ii against apc. then the apc will migrate to the lymph nodes and induce cd8+ and cd4+ t helper cells. in other words, these interactions will initiate humoral and cellular immune responses (abbas 2010; yam-puc .et al. et al 2016). the igg subclasses profile result was shown that the highest antibody induced by the tetravalent vaccine was igg2a followed by igg1, igg2b and igg3. it is known that the production of igg2a, igg2b and igg3 is stimulated by th1 immune response whereas igg1 is x dewi et al. microbiol indones table mean of igg subclasses titre tetravalent and pumvc4a groups2 antibody tetravalent group (mean ± sd) pumvc4a group (mean ± sd) igg1 0.920±0.203 0.490±0.051 igg2a 1.004±0.154 0.409±0.108 igg2b 0.281±0.022 0.144±0.029 igg3 0.392±0.069 0.346±0.079 volume 1 , 2026 2 microbiol indones x fig 1 colony pcr screening for pumd1 and pumd3. (a) m: λ hind/iii, 1-3: recombinant pumd1, k-:negative control; (b) m: λ hind/iii. 24: recombinant pumd3, k+: positive control for pumd3 insert cloned to pumvc4a. white box showed target band. fig illustration of plasmid construction for pumd1, pumd2, pumd3 and pumd4. (a) pumvc4a vector2 plasmid, (b) plasmid recombinant. fig screening for pumd2 and pumd4 using restriction enzyme digestion method (a) m: hind/iii, 1:3 λ pumd2, 2: pumvc4a; (b) m: λ hind/iii, 1: pumd4, 7: pumvc4a. fig expression of prm-e protein in vero cells using hrp detection system, positive results were indicated by4 brown stain (1) vero cells transfected with pumd1; (2) vero cells transfected with pumd2; (3) vero cells transfected with pumd3; (4) vero cells transfected with pumd4; (5) vero cells transfected with pumvc; (6) negative control (not transfected). stimulated by a th2 immune response (wang .et al 2019). igg2a response in homologous mice to human igg1, where the levels are the most abundant in human serum. both subclasses have the equivalent responses to the same fcγr class . in(mehlhop 2007)et al. humans, the presence of igg subclasses strongly triggers complement-dependent cytotoxicity (cdc), antibody-dependent cell-mediated cytotoxicity ( a d c c ) a n d a n t i b o d y d e p e n d e n t c e l l u l a r phagocytosis (adcp)) .(brezski and georgiou 2016) igg2a antibodies in the serum that are usually associated with the th1 subset of cd4+ t cells secrete gamma interferon (ifn-γ) and tumor necrosis factor (tnf) which induce the induction of cell-mediated immune responses. the igg2a response was reported to be correlated with neutralizing antibody titres. the igg1 antibody in mice (migg1) has a binding pattern comparable to human igg2 (higg2). this was proven by overdijk (2012), that higg2 was able toet al compete with migg1 in binding mfcγriib and mfcγriv . yamanaka(overdijk . 2012)et al et al (2013) in their study mention that the binding affinity of dengue antibodies to fcγr affects the activity of neutralizing and enhancing antibodies. igg2 antibodies in mice are known to have stronger binding affinity to complements than igg1 (yamanaka, kotaki and konishi 2013). whereas in humans, it is known that igg1 and igg3 can bind complements the most effectively, compared to igg2 and igg4 (rodrigo .et al 2009). in this study, the profiling of igg subclasses levels were only carried out against denv-2 as an antigen by elisa. therefore, in further research it is necessary to evaluate other serotypes. in addition, there has not been an evaluation of the potential antibody response to vaccine candidates in inducing ade activity. this is recommended to determine the safety of the vaccine candidate. acknowledgments this research was funded by indonesian dengue vaccine consortium. references abbas ak, lichtman ah, pillai s, baker dl. 2010 b cell. activation and antibody production cellular and. molecular immunology. elsevier: 243–268. doi: 10.1016/b978-1-4160-3123-9.50017-6. bhatt s, gething pw, brady oj, messina jp, farlow aw, moyes cl, drake jm, brownstein js, hoen ag, sankoh o, myers mf, george db, jaenisch t, wint gr, simmons cp, scott tw, farrar jj, hay si. 2013. the global distribution and burden of dengue. nature, 496(7446):504-7. doi: 10.1038/nature12060. brezski, r. j. and georgiou, g. 2016. immunoglobulin isotype knowledge and application to fc engineering. current opinion in immunology. elsevier ltd, 40(may 2015): 62–69. doi: 10.1016/j.coi.2016.03.002. fahimi h, mohammadipour m, haddad kashani h, parvini f, sadeghizadeh m. 2018. dengue viruses and promising envelope protein domain iii-based vaccines. appl microbiol biotechnol. 102(7): 2977–2996. doi: 10.1007/s00253-018-8822-y. gallichotte en, widman dg, yount bl, wahala wm, durbin a, whitehead s, sariol ca, crowe je jr, de x dewi et al. microbiol indones fig comparison of mean groups titer of each group for all igg subclasses from termination sera.5 silva am, baric rs. 2015. a new quaternary structure epitope on dengue virus serotype 2 is the target of durable type-specific neutralizing antibodies. mbio. e d i t e d b y w. i . l i p k i n , 6 ( 5 ) : 1 – 8 . d o i : 10.1128/mbio.01461-15. kemenkes ri (2017) profil kesehatan indonesia. 351.077 in. edited by t. kurniawan, rudy; yudianto; hardhana, boga; soenardi. jakarta: kementerian kesehatan r e p u b l i k i n d o n e s i a . a v a i l a b l e a t : https://pusdatin.kemkes.go.id/download.php?file=do wnload/pusdatin/profil-kesehatan-indonesia/profilkesehatan-indonesia-2016.pdf. konishi e, kosugi s, imoto j. 2006. dengue tetravalent dna vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice. v a c c i n e , 2 4 ( 1 2 ) : 2 2 0 0 – 2 2 0 7 . d o i : 10.1016/j.vaccine.2005.11.002. mehlhop e, ansarah-sobrinho c, johnson s, engle m, fremont dh, pierson tc, diamond ms. 2007. complement protein c1q inhibits antibodydependent enhancement of flavivirus infection in an igg subclass-specific manner. cell host microbe, 2(6): 417–426. doi: 10.1016/j.chom.2007.09.015. overdijk mb, verploegen s, ortiz buijsse a, vink t, leusen jh, bleeker wk, parren pw. 2012. crosstalk between human igg isotypes and murine effector cells. j i m m u n o l , 1 8 9 ( 7 ) : 3 4 3 0 – 3 4 3 8 . d o i : 10.4049/jimmunol.1200356. putri dh. 2015. pengembangan vaksin dna tetravalen dengue berbasis gen prm-e denv strain indonesia$: uji imunogenesitas vaksin dalam menginduksi antibodi netralisasi dan respon anamnestik untuk semua serotipe denv di mencit. universitas indonesia. rachmayanti n. 2013 konstruksi dan ekspresi gen premembrane dan envelope virus dengue serotype 4 strain indonesia sebagai kandidat vaksin dna. universitas indonesia. rodrigo ww, block ok, lane c, sukupolvi-petty s, goncalvez ap, johnson s, diamond ms, lai cj, rose rc, jin x, schlesinger jj. 2009. dengue virus neutralization is modulated by igg antibody subclass and fcγ receptor subtype. virology. elsevier inc., 394(2): 175–182. doi: 10.1016/j.virol.2009.09.024. sjatha f, kuwahara m, sudiro tm, kameoka m, konishi e. 2014. evaluation of chimeric dna vaccines consisting of premembrane and envelope genes of japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infectionenhancing antibody response. microbiol immunol. 58(2): 126–134. doi: 10.1111/1348-0421.12125. stachyra, a., góra-sochacka, a. and sirko, a. 2014. dna vaccines against influenza. acta biochimica polonica, 61(3): 515–522. doi: 10.18388/abp.2014_1873. wang r, zheng x, sun j, feng k, gao n, fan d, chen h, jin x, an j. 2019. vaccination with a single consensus envelope protein ectodomain sequence administered in a heterologous regimen induces tetravalent immune responses and protection against dengue viruses in mice. front microbiol. 10(may): 1–14. doi: 10.3389/fmicb.2019.01113. who. 2017. epidemiology. yam-puc jc, cedillo-barrón l, aguilar-medina em, ramos-payán r, escobar-gutiérrez a, flores-romo l. 2016. the cellular bases of antibody responses during dengue virus infection. front immunol, 7(june): 1–12. doi: 10.3389/fimmu.2016.00218. yamanaka, a., kotaki, t. and konishi, e. 2013. a mouse monoclonal antibody against dengue virus type 1 mochizuki strain targeting envelope protein domain ii and displaying strongly neutralizing but not enhancing activity. j virol, 87(23): 12828–12837. doi: 10.1128/jvi.01874-13. yunita r. 2012. imunogenisitas kandidat vaksin dna rekombinan dengan gen insersi premembran dan envelope virus dengue tipe 2 pada mencit. universitas indonesia. volume 1 , 2026 2 microbiol indones x juniastuti.pmd volume 3, number 2, august 2009 p 85-90 issn 1978-3477 *corresponding author: phone:+62-31-5030252 ext. 159 fax: +62-31-5022472, e-mail: koeraisindewi@yahoo.co.id the relatedness between hepatitis b virus from non-papuan blood donors in jayapura and the papuan clusters juniastuti1*, victor eka nugrahaputra2, mochamad amin3, ni made mertaniasih1, and maria inge lusida1,3 1department of microbiology, faculty of medicine, universitas airlangga, jalan mayjen prof. dr. moestopo 47, surabaya 60131, indonesia; 2department of microbiology, faculty of medicine, universitas cendrawasih, abepura campus, jalan sentani-abepura, jayapura 99351, indonesia; 3institute of tropical disease, universitas airlangga, jalan mulyorejo, surabaya 60115, indonesia the genotypes (a-h) and subtypes (adw2, adw4, adrq-, adrq+, ayw1-4, ayr) of hbv show distinct geographical distributions, which have been associated with anthropological history. the novel finding of the hbv subgenotypes c6 and d6 from papuans formed a specific cluster distinct from the previous hbv subgenotypes c1-c5 and d1-d5. in this study we determined the most recent genotype-subtype patterns of the hbv from non-papuan blood donors who live in jayapura and their phylogenetic relatedness, especially with the papuan clusters. fifteen hbsag-positive serums were obtained from non-papuan blood donors including from people in java (46.7%), maluku (26.7%), sulawesi (20%) and east nusa tenggara (6.7%). s gene of all hbv serum isolates were partially sequenced and analyzed. most hbv isolates (53.3%) were classified as genotype b, followed by genotype c (26.7%) and d (20.0%). the subtype adw2 (33.3%) was predominant, followed by adrq+ (26.7%) and ayw1/ayw2 (20.0%). all hbv isolates with subtype adw2 and ayw1 belonged to genotype b, while adrq+ belonged to genotype c and ayw2 belonged to genotype d. the most predominant hbv genotype-subtype (b/adw2) was consistent with the ethnic background (mostly from java people). nevertheless, based on the phylogenetic relatedness, many non papuan isolates (40%) were classified into hbv/c6 and hbv/d6 of the papuan clusters. other isolates were classified into hbv/c1, hbv/b3 and hbv/b7. in conclusion, many hbv isolates from non-papuans in jayapura belonged to the papuan clusters, but others had different genotype-subtype patterns with frequencies dependent on ethnicity. key words: hepatitis b virus, genotypes, subtypes, phylogenetic relatedness hepatitis b virus (hbv) infection is a serious global health problem. in 2000, core working party for asiapasific consensus on hepatitis b and c reported that indonesia belonged to the moderate-to-high hepatitis b endemic region (khan et al. 2004). carrier rates among blood donors ranged from 2.1 to 9.5% in 11 large cities and even higher at 17.5% in jayapura, the papua province (sastrosoewignjo et al. 1991). eight genotypes (from a to h) of hbv have been identified worldwide, based on a divergence of 8% or more of the complete genome, and more than 4% at the level of the s gene (okamoto et al. 1988; norder et al. 1994; magnius and norder 1995; stuyver et al. 2000; arauzruiz et al. 2002; kramvis et al. 2005). nine hbv subtypes (adw2, adw4, adrq-, adrq+, ayw1-4, ayr) were defined by two mutually exclusive determinant pairs, d/y and w(w1-4)/r, and a common determinant ‘a’ of hbsag (magnius and norder 1995; kramvis et al. 2005). the hbv isolates of different genotypes and subtypes show d i f f e r e n t g e o g r a p h i c a l d i s t r i b u t i o n s , v i r o l o g i c a l characteristics, and possibly clinical outcomes (kao et al. 2000; orito et al. 2001; kao 2002; kidd-ljunggren et al. 2002). they could also provide historical information o n t h e m i g r a t i o n p a t t e r n o f t h e a n c e s t o r o f l o c a l population (magnius and norder 1995; orito et al. 2001) the subgenotypes have been identified for certain hbv genotypes. hbv/c has been classified into six subgenotypes (c1 to c6) (sugauchi et al. 2001; huy et al. 2004; chan et al. 2005; sakamoto et al. 2006; lusida et al. 2008), hbv/d into six subgenotypes (d1 to d6) (norder et al. 2004; bozdayi et al. 2005; banerjee et al. 2006; schaefer 2007; lusida et al. 2008) and hbv/b into seven subgenotypes (b1 to b7) (sugauchi et al. 2001; nagasaki et al. 2006; sakamoto et al. 2006; sakamoto et al. 2007; nuraeny et al. 2008). as hbv genotypes, the hbv s u b g e n o t y p e s s e e m t o b e a s s o c i a t e d w i t h t h e i r geographical distributions. previous studies of genotype-subtype distributions of hbv in jayapura, especially those from papuans, reported that c/adr was predominant (sastrosoewignjo et al. 1991; mulyanto et al. 1997; lusida et al. 2008). in 1997 mulyanto assumed that the papuan ancestor came from new caledonia (hbv/c3), where adr is largely found. besides, based on anthropological origin, papuans resemble australian aborigines, because the mainlands of the two tribes were one continent before it was separated 7000 years ago (sugauchi et al. 2001). however, sugauchi et al. (2001) found the c/ayw3 isolates from australian aborigine (hbv/c4), which had a different hbv subtype from that of papuans. recently, lusida et al. (2008) reported that most hbv isolates from papuan blood donors in jayapura belonged to the novel c subgenotype ( h b v / c 6 ) , s e p a r a t e d f r o m t h e p r e v i o u s h b v c subgenotypes, including the new caledonia (hbv/c3) and australia clusters (hbv/c4). they also reported that other papuan isolates belonged to the novel hbv d subgenotype, namely d6. the aim of this study was to determine the most recent genotype-subtype patterns of hbv based on part of the s gene in hbsag-positive blood donors from non-papuans who live in jayapura, the papua province. their phylogenetic relatedness was also analyzed, especially with hbv of papuan clusters. materials and methods collection of field samples. serum samples were taken from non-papuan blood donors who visited the blood transfusion unit-indonesian red cross in jayapura from july 2006 to september 2006, and were screened for hbsag by using the immunochromatography method (entebe hbsag strip, hepatika laboratory, mataram). all serum samples were stored at -20 °c until transported to the institute of tropical disease in surabaya, where they were stored at -80 °c. ethical clearance of this study was obtained from the ethics committee of the faculty of medicine, airlangga university in surabaya, indonesia. all blood donors signed an agreement to participate in this study. viral dna extraction, pcr amplification and sequencing. genomic dna of hbv was extracted from serum samples by using the dna zol reagent (invitrogen) following the manufacturer’s guidelines. the extracted dna was used as a template for the amplification of the respective gene regions. pcrs were performed with the pcr mastermix (fermentas). the reactions contained 25 µl pcr mastermix, 10 ml dna and 0.5 µl of each primer with a concentration of 20 pmol µl-1, in a total reaction volume of 50 µl. the thermocycling conditions included a 5-min denaturation step of 94 °c; followed by 40 cycles of 1 min at 94 °c, 1 min at 55 °c and 2 min at 72 °c. partial s gene was amplified in the first round by using primers p7 (5’-gtg gtg gac ttc tct caa ttt tc-3’, nt 256 to 278) and p8 (5’-cgg taw[a/t] aaa ggg act cam[a/c] gat-3’, nt 796 to 776). if the first round pcr was negative, therefore the second round pcr was performed by using primers hbs1 (5’-caa ggt atg ttg ccc gtt tg-3’, nt 455 to 474) and hbs2 (5’-aaa gcc ctg cga acc act ga-3’, nt 713 to 694) (lusida et al. 2003). pcr products were sequenced using the big dye terminator v1.1 cycle sequencing kit (applied biosystems, usa) and abi prism 310 genetic analyzer (perkin elmer). analysis of sequence. sequence analysis was performed with genetyx-mac version 9 (software development co., ltd., tokyo, japan). the hbv genotype of the samples was determined based on the homology percentage of >96% in the s gene (magnius and norder 1995; arauz-ruiz et al. 1997). after aligining the sequences of the hbv nucleotide and converting it into amino acid sequences, the hbv subtype of the samples was assigned by analyzing the amino acid substitutions at positions 122, 127, 134, 159, 160, 177 in the s gene (okamoto et al. 1988; norder et al. 1994; kramvis et al. 2005). the phylogenetic tree was constructed by means of unweighted pair group method using the clustering of arithmetic averages (upgma). results a total of 15 hbsag-positive serum was obtained from non-papuan blood donors (14 male and 1 female; age 18-52). the blood donors were people from java (46.7%), maluku (26.7%), sulawesi (20%) and east nusa tenggara (6.7%) living the distribution of the hbv genotypes among the 15 serum in non-papuans indicated that most isolates (53.3%) were classified into genotype b, followed by genotype c (26.7%) and d (20%) (table 2). the hbv subtypes were determined by aligning 15 hbv sequences from non-papuans and 70 reported hbv sequences (a-h genotypes) including hbv of the papuan clusters, at the amino acids positions 117-180 of the s gene (fig 1). by analyzing amino acid substitutions at positions 122, 127, 134 and 160, it was found that the subtype adw2 (33.3%) was predominant, followed by adr (26.7%), and ayw1/ ayw2 (20%) (table 2). all isolates with adr subtype had alanine (a) at position 159 and valine (v) at position 177 in hbv, and this combination is considered important for the q determinant (fig 1). all hbv isolates with adw2 and ayw1 belonged to genotype b, subtype adr(q+) belonged to genotype c, and ayw2 to genotype d (table 2). no c/adrqisolates were found here. the genotype subtype patterns of hbv from various ethnics of non-papuan blood donors obtained in jayapura, were summarized in table 3. hbv b/adw2 was predominant in javanese, followed by b/ayw1 or c/adrq+. hbv b/adw2 was also more than b/ayw1 in sulawesi people. hbv d/ ayw2 was commonly found in moluccans, followed by c/adrq+, but only hbv c/adrq+ was found in east nusa tenggara people. the phylogenetic analysis of part of the s region (nt 503 to 694) of the hbv genome revealed that many hbv isolates (40%) from non-papuans (javanese and molluccans) were classified into papuan clusters, such as hbv/c6 (6np, 27np and 49np) and hbv/d6 (7np, 25np, 26np). some isolates from javanese and sulawesi people (30np, 37np and 57np) were classified into hbv/b3. two isolates from javanese (9np and 18np) were classified into hbv/b7 (alor, east nusa tenggara). one isolate from east nusa tenggara people table 1 ethnics of non-papuan blood donors in jayapura ethnics of non papuan blood donors number java sulawesi maluku east nusa tenggara to t a l 7 3 4 1 1 5 table 2 hbv genotype and subtype distribution patterns from non-papuan blood donors in jayapura hbv genotypes/subtypes b/adw 2 b/ayw1 c/adrq+ d/ayw2 to t a l number of cases in non-papuan blood donors 5 3 4 3 1 5 microbiol indones86 juniastuti et al. in jayapura. a sequence analysis of part of the s gene of hbv serum isolates was performed (table 1). fig 1 alignment of amino acid sequences of hbv isolates from 15 non-papuan blood donors in jayapura, indonesia (code: np) and 70 reported sequences of genotypes a to h including the papuan clusters, c6 and d6 (lusida et al. 2008). the sequences correspond to amino acids 117 to 180 of the s gene. microbiol indones 87volume 3, 2009 (12np) was classified into hbv/c1 (south east asia). some hbv/b isolates from javanese and sulawesi people (14np, 39np and 55np) were not definitely classifiable on the basis microbiol indones88 juniastuti et al. table 3 hbv genotype-subtype patterns from various ethnics of non-papuan blood donors in jayapura ethnics of non-papuan blood donors java maluku sulawesi east nusa tenggara no. of cases b/adw2 3 2 b/ayw1 2 1 c/adrq+ 2 1 1 d/ayw2 3 to t a l 7 4 3 1 1 5 of the partial s gene sequencing alone. all hbv/c isolates were from non-papuans separated from far east asia (hbv/ c2), new caledonia-polynesia (hbv/c3), australia (hbv/c4) and the philippines (hbv/c5) clusters (fig 2). discussion papua island is in the eastern part of indonesia with papuans as its native people. beside the natives, many other ethnic groups in indonesia, like javanese, sumatrans, moluccans, and people from sulawesi and east nusa tenggara descent live in papua. here, hbv isolates from non-papuans who live in jayapura, papua province, were presented. fig 2 phylogenetic analysis of hbv isolates obtained from 15 non-papuan blood donors (code: np) compared with the reported of all genotypes and subgenotypes of hbv, including the subgenotypes c6 and d6 of the papuan clusters (lusida et al. 2008); on the basis of the partial s genes (503-694). the genotypes are indicated on the branches and the subgenotypes are on the right. this study demonstrated that most (53.3%) hbv isolates from non-papuan blood donors were classified into genotype b, followed by genotype c (26.7%) and d (20%) (table 2). no hbv isolates were found with genotypes a, e, f, g or h from the non-papuans. each hbv genotype has characteristic geographic distribution. in some cases, differences in the distribution of the genotypes can be found within a single country, as has been observed in china, india, usa and indonesia as well. the genotype distributions can be influenced by the ethnic background and the country origin of the individual carriers of the virus (kramvis et al. 2005). indonesia is a country that has more than 300 ethnic groups and over 250 languages (sofro 1982). it is characterized by its national motto “bhinneka tunggal ika” or “unity in diversity”. the diversity in this country can now be assessed also in the light of the hbv genotypes. in the western part of indonesia, the hbv genotype b was predominant, while the hbv genotype c was predominant in the eastern part of indonesia, especially in papua (sastrosoewignjo et al. 1991; mulyanto et al. 1997; usuda et al. 1999; lusida et al. 2008). the results revealed that most predominant hbv genotype of non-papuans isolates was genotype b. the samples were mostly obtained from people of the western part of indonesia (javanese) (table 1 and 3). hbv genotype b was also found more in sulawesi people, which was consistent with the report of sastrosoewignjo et al. (1991). like other regions in most developing countries where vertical transmission is predominant, the distributions of hbv genotypes-subtypes in papua seemed to be dependent on ethnicity. the subtype adw2 (33.3%) was the most predominant in all hbv isolates from non-papuan blood donors, followed by adrq+ (26.7%) and ayw1/ayw2 (20%) (table 2). each hbv subtype markedly varied in different geographical regions in indonesia. the most predominant hbv subtype in the western part of indonesia, such as java, is adw (mulyanto et al. 1997). beside that, they concluded that sulawesi was the mixed hbv subtype zone, including adw. it was consistent with our results that the subtype adw2 was mostly obtained from javanese, followed by sulawesi people (table 3). nevertheless, it was different with the predominance of the adr subtype in papuans. sastrosoewignjo et al. (1991) showed that 50% of 14 hbv isolates from blood donors in jayapura belonged to the subtype adr. mulyanto et al. (1997) also reported that 76.9% of the 13 isolates of adult population of papuan in jayapura belonged to the subtype adr. lusida et al. (2008) found that 85.2% of the 23 isolates from blood donors (mostly papuans) in papua belonged to the subtype adr, too. our results confirmed the difference of the most predominant hbv subtype from papuans with non-papuans even though they lived in the same places. all hbv isolates with subtype adw2 and ayw1 belonged to genotype b, adrq+ to genotype c and ayw2 to genotype d. no hbv c/adrqisolates were found here. this was consistent with the hbv genotypes and subtypes relationship. in comparison to our results, the subtype adr is rarely found in genotype b, subtype adw2 can be found in genotypes a, c, f, g and subtype ayw2 can be found in genotypes a and c (kramvis et al. 2005). by analyzing the phylogenetic relatedness on part of the s gene, it was found that many hbv isolates (40%) from non-papuans (javanese and moluccans) belonged to the papuan clusters, hbv/c6 (6np, 27np and 49np) and hbv/ d6 (7np, 25np, 26np). surprisingly, it seemed that there could be a horizontal transmission of hbv from papuans to nonpapuans living in papua, in an adequate number. two isolates from javanese and one isolate from sulawesi people (30np, 37np and 57np) were classified into hbv/b3 (java and papua). the reported hbv/b3 isolates from papua by lusida et al. (2008), were possibly obtained from javanese living in papua. two isolates from javanese (9np and 18np) were classified into hbv/b7 (alor, east nusa tenggara). the possible reason was the fact that there were many east nusa tenggara people living in papua, especially in transmigration areas. besides, one isolate (12np) from east nusa tenggara people was classified into hbv/c1 (south east asia). again, it might be caused by horizontal transmission, because there were many thailand seamen who came to papua. interestingly, some hbv/b isolates from javanese and sulawesi people (14np, 39np and 55np) were not definitely classifiable into the previous subgenotype b (b1-b7). they had possibly distinct clusters which reflected the specificity of the ethnicity in the hbv distributions, but futher investigation about other regions, such as the pre-s or even the whole region of hbv dna is needed. finally, all hbv/c isolates from non-papuans are separated from far east asia (hbv/c2), new caledoniapolynesia (hbv/c3), australia (hbv/c4) and the philippines (hbv/c5) clusters (fig 2). this confirmed that the existence of hbv genotypes-subtypes in papua was unique. overall, it could be concluded that many hbv isolates from non-papuans in jayapura belonged to the papuan clusters, but others had different genotype-subtype patterns which frequencies were dependent on ethnicity. there could be a domination of hbv from papuans, but the existence of hbv from non-papuans must be considered as another important role to determine the circulation of the hbv genotypes-subtypes in papua. acknowledgements the authors are grateful to regina h. hutabarat, head of the blood transfusion unit-indonesia red cross, jayapura, yoes prijatna dachlan, head of institute of tropical diseases for his cooperation; izumi, ni nyoman tri puspaningsih and eduardus bimo for their invaluable supports; and koen poedjiati for her technical assistance. we also deeply appreciate the blood donors at the blood 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[dissertation]. canberra: australian national university. stuyver l, gendt s de, geyt c van, zoulim f, fried m, schinazi rf, rossau r. 2000. a new genotype of hepatitis b virus: complete genome and phylogenetic relatedness. j gen virol 81:67-74. sugauchi f, mizokami m, orito e, ohno t, kato h, suzuki s, kimura y, ueda r, butterworth la, cooksley wge. 2001. a novel variant genotype c of hepatitis b virus identified in isolates from australian aborigines: complete genome and phylogenetic relatedness. j gen virol 82:883-92. usuda s, okamoto h, iwanari h, baba k, tsuda f, miyakawa y, mayumi m. 1999. serological detection of hepatitis b virus genotypes by elisa with monoclonal antibodies to type-specific epitopes in the pres2-region product. j virol methods 80:97-112. microbiol indones90 juniastuti et al. 8 widodo_347 (39-43) vegetative compatibility groups within fusarium oxysporum f. sp. cepae in hokkaido-japan widodo1*, norio kondo2, kiroku kobayashi2, and akira ogoshi2 1department of plant protection, faculty of agriculture, institut pertanian bogor, kampus darmaga, bogor 16680, indonesia 2graduate school of agriculture, hokkaido university, sapporo 060-8589, japan in hokkaido, fusarium basal rot, caused by fusarium oxysporum f. sp. cepae is one of the important constrains since 1973 which contributes to a significant loss in onion production, either in the fields or during storage. development of resistant cultivars is suggested as one of the effective control measures against the disease, however, this should be accompanied with the better understanding of the pathogen’s population dynamics. this study was performed to investigate the population structure of f. oxysporum f. sp. cepae based on vegetative compatibility groupings (vcgs). vegetative compatibility groups of f. oxysporum f. sp. cepae were characterized using nitrate non-utilizing (nit) mutants. four vcgs and 2 single self-compatible (ssc) isolates were identified among 48 isolates, designated as vcg 0420 (33 isolates), 0421 (9 isolates), 0422 (2 isolates), 0423 (2 isolates), and 042-(2 isolates). vcg 0420, to which 4 atcc isolates out of 6 belonged, was the predominant group within the growing region encompassing hokkaido japan. vcgs 0421 and artificial vcg 042were found less frequently. four isolates from welsh onion were not compatible with any recovered vcgs and were assigned to 2 distinct vcgs (vcg 0422 and 0 4 2 3 ) . key words: allium cepa, fusarium basal rot of onions, fusarium oxysporum f. sp. cepae, population _____________________________________________ ________________________ *corresponding author, phone/fax: +62-251-8423048, e-mail: widodo@ipb.ac.id hokkaido, the northernmost island of japan, has the largest area under onion cultivation (allium cepa) and accounts for 48% of national production. fusarium basal rot, caused by fusarium oxysporum f.sp. cepae, is an important soilborne disease of onion which occurs in hokkaido. this fungus also causes seedlings damping-off and wilt. this disease was first reported in japan in 1911, in the ishikari district of western hokkaido, but had no significant economic impact for many years (kodama 1983). in 1970, the disease was reported in some areas of the central and eastern parts of hokkaido i.e. furano and kitami (kodama et al. 1974). by 1973, the disease was observed in most of the onion fields in hokkaido (kodama et al. 1974). although treating seedlings with fungicide (1-2% benomyl) prior to transplantation has significantly reduced losses (kodama 1983), the planting of resistant onion cultivars has been suggested as a valuable control measure (abawi and lorbeer 1971; simizu and nakano 1995; thornton and mohan 1996). often, however, the use of cultivars resistant to f. oxysporum is compromised by the appearance of new pathogenic strains. f. oxysporum’s ability to overcome host resistance may be related to its various strains existence and unstable genetic character (abawi and lorbeer 1965; ploetz and correll 1988; jacobson and gordon 1991). since pathogens must constantly adapt to changes in their environment to survive (abawi and lorbeer 1972), an understanding of their population dynamics would be helpful in developing resistant cultivars. puhalla (1981) first made efforts to differentiate and classify formae speciales of f. oxysporum isolated from various plant species into vegetative compatibility groups (vcgs), using nitrate non-utilizing (nit) mutant (generated on media containing chlorate) that lacked the ability to use nitrate as a nitrogen source. this was refined by correll et al. (1987). since that time, numerous investigators have classified f. oxysporum isolated from various host plants into distinct vcgs using nit mutants (elmer and stephens 1989; larkin et al. 1990; elias and schneider 1991; katan et al. 1991; loffler and rumine 1991; venter et al. 1992; fiely et al. 1995; woudt et al. 1995; woo et al. 1996; harveson and rush 1997; kondo et al. 1997; lori et al. 2004; vakalounakis et al. 2005). five vcgs of f. oxysporum f. sp. cepae from onion have been identified in colorado, usa, and one of the vcgs was present in all three regions of colorado (swift et al. 2002). the use of vcgs has also been performed to determine the diversity of nonpathogenic populations of f. oxysporum isolated from carnation fields in argentina (lori et al. 2004). beside for determining the vcgs of fusarium, the use of nit mutant combining with selective media was also useful in studying population dynamics of pathogenic f. oxysporum f. sp. spinaceae in soil (takehara et al. 2003). in the special case of f. oxysporum, the vegetative compatibility group (vcg) approach can be an effective diagnostic technique when the number of vcgs in formae speciales of interest is known to be relatively small since the tester strains needed are usually easy to obtain (summerell et al. 2003). the objectives of this study were to investigate the population type and distribution of f. oxysporum f. sp. cepae from hokkkaido island, japan, based on vegetative compatibility groups. materials and methods collection of isolates and pathogenicity testing. fusarium oxysporum were collected from infested-field-soil or diseased onion stem-plates from 6 locations (mikasa, furano, kitami, kunnenpu, tsubetsu, and tanno) in hokkaido. additional isolates were obtained from individuals (4 isolates of welsh onion from yakumo and 3 isolates of onion from furano supplied by dr. h. abe and dr. f. kodama, respectively) and culture collections (american type culture issn 1978-3477 volume 2, number 1, april 2008 p 39-43 collection). infected stemplates with brown discoloration were surface sterilized using cotton wipes wetted with 70% ethanol and allowed to dry. each stem-plate was cut with a sterile knife into small blocks (2 x 2 x 2 mm3). five pieces of each bulb were placed in komada’s medium (km) (komada 1975) and incubated at 26 °c for 5 to 7 days. the entire stemplates were used for isolates recovered from damped-off seedlings. soil dilution was used for isolation from soils. after 5-7 days incubation colonies, which were tentatively identified as f. oxysporum on the basis of colony morphology, were transferred to fresh plates of km. spores from 5-7-day-old cultures growing on km were streaked onto water agar (20 g l-1). after 18 h a single germinated spore was removed from the water agar and transferred to potato sucrose agar (psa: potato 200 g, sucrose 15 g, agar 20 g, and distilled water 1000 ml). identification was based on mycological characteristics as described by nelson et al. (1983). pathogenicity tests. each isolate was tested for pathogenicity using a susceptible onion cultivar (kitawase no. 3). a conidial suspension was prepared from 7-10-dayold cultures growing on psa plates and used as inoculum. the suspension was filtered through 4 layers of cheesecloth to separate conidia from mycelium and adjusted to 1.0 x 106 spores ml-1. sixto 8-week-old seedlings were pulled up and shaken to remove excess soil, and then cut 5 mm below their stem plate. the roots of 15 seedlings were dipped for 24 h into 20 ml of a conidial suspension and then transplanted into a soil (pot-ace, katakura chikkarin k. k., tokyo, japan)/ vermiculite mixture (3:1 v/v) in 12-cm-diameter plastic pots in triplicate. each pot contained 5 seedlings. other seedlings were dipped in sterile water and planted as above to act as controls. the number of damping-off plants was recorded over 5 weeks after transplantation. after 5 weeks, longitudinal sections of unwilted plants were observed for discoloration of stem-plates. these tests were conducted in a greenhouse with temperatures ranging from 20 to 28 °c. of the isolates that induced plant damping-off more than 30% were considered as pathogenic. re-isolation from the experimentally symptomatic plants was performed to verify that the recovered isolates had the same characteristics as the isolates in the initial inoculation. recovery of nitrate non-utilizing (nit) mutants. mutants were selected using the method of puhalla (1985). each isolate of f. oxysporum was grown on psa for 5-7 days at room temperature. four small mycelial blocks (2 mm3) of the fungus were placed onto a plate containing corn-mealagar modified by adding 15 g l-1 kclo 3 (cmac). many isolates failed to form mutants on this medium, so chlorate (kclo 3 ) concentrations were often doubled to 30 g l-1. the plates were incubated at 26 °c for 15 days and inspected periodically for the appearance of fast growing hyphae developing from the initial colony. these fast growing hyphae were then transferred to a minimal medium (mm) that contained nano 3 (2 g l-1) as the sole nitrogen source (puhalla 1985). mutants that grew on mm as thin expansive colonies with no aerial mycelium were considered as nit mutants. nit mutant phenotypes. the phenotypic classes of all nit mutants were determined by their growth on media containing nitrate (2 g l-1), nitrite (0.5 g l-1), hypoxanthine (0.2 g l-1), or ammonium tartrate (1 g l-1) as the sole nitrogen source (correll 1991). a small block of each mutant colony was transferred onto each medium. the plates were incubated as described above and colony growth was scored relative to the parental wild-type after 4 days growth. the nit mutants were placed into phenotypic classes (klittich and leslie 1988; correll 1991) based on their ability to utilize the various nitrogen sources (fig 1). complementation test. prior to complementation tests among isolates, the vegetative self-compatibility of each isolate was examined by the method of jacobson and gordon (1988). ten isolates were initially selected for vegetative compatibility tests. they were paired in all possible combinations and grown on minimal medium to determine the number of vegetative compatibility groups (vcgs). vegetatively compatible nit mutants that carry the mutated gene on different loci may complement one another by the formation of a dense aerial wild-type mycelium at the position where mycelia of nit1 and nitm colonies touch. a nitm from one isolate in each of the vcgs thus identified was selected to serve as a tester. after vcgs were established for the initial 10 isolates, the remaining isolates were treated in the same manner. for isolates that did not match with any of the established vcgs, a tester for a second group was obtained and the procedure was repeated. results when the isolates of f. oxysporum inoculated into cultivars kitawase no. 3 using the root dip method were examined, some of them induced a progressive yellowing and die back from the tips of their leaves. in these cases the aerial part died completely within two or three weeks after inoculation. all the isolates inducing such symptom were taken to be as f. oxysporum f. sp. cepae based on the description of kodama (1983). the degree of disease incidence induced within isolates having this symptom varied from 20-100%. a total of 38 isolates of f. oxysporum pathogenic to onion and 173 nonpathogenic isolates were recovered from soil samples and diseased onion bulbs (table 1). table 1 geographical location and pathogenicity tests of isolates of fusarium oxysporum collected from onion fields f. oxysporum population (cfu g-1 dry soil) no. of isolates tested no. of pathogenic isolates no. of nonpathogenic isolateslocation source (year) mikasa furano ta n n o kitami tsubetsu kunnenpu soil (1996) bulb (1997) soil (1997) soil (1997) soil (1997) bulb (1997) 5.3 x 103 1.2 x 103 1.9 x 103 0.4 x 103 7 7 5 3 2 6 2 4 6 2 5 1 1 6 2 3 1 1 5 6 6 4 7 2 4 2 1 5 1 0 40 widodo et al. microbiol indones nit mutants emerged from restricted growth on corn meal agar medium amended with chlorate after 4-15 days. most of the isolates produced mutants when generated on medium containing chlorate 15 g l-1. some other isolates had to be generated on media containing 30 g l-1 to obtain the mutants. all the isolates used in this study are able to produce all the 3 mutant phenotypes as indicated on their ability on using different nitrogen source (fig 1). the 38 isolates of f. oxysporum f. sp. cepae recovered from soils in hokkaido island-japan along with the ten known isolates of f. oxysporum f. sp. cepae were grouped into 4 distinct vcgs and 2 ssc isolates based on complementation of nit1 and nitm mutants. they consisted of one large vcg with 33 members, including 4 isolates from atcc. this one large group was first reported by yoo et al. (1993), and was numbered using puhalla’s numbering system (puhalla 1985) as vcg 0420 by katan and di primo (1999). therefore, the three other vcgs and two isolates of single self-compatibility were an additional group for this formae speciales. these three other vcgs were then designated as vcg 0421 (9 isolates), vcg 0422 (2 isolates), and vcg 0423 (2 isolates), respectively. the two isolates of ssc with members of atcc 11850 and atcc 46076 were assigned to artificial vcg 042(table 2). the population of f. oxysporum f. sp. cepae assigned as vcg 0420 was mostly distributed throughout the sampling area in hokkaido (fig 2). among the atcc isolates tested, atcc 46076 and atcc 46077 were originally isolated from hokkaido, japan, in the 1970’s. complementation assays of atcc 46077 placed it into the largest vegetative compatibility group, while atcc 46076 was unable to form heterokaryons with any other isolates collected from hokkaido, japan (table 2). the atcc 46076 isolate was also not vegetatively compatible with the most predominant isolates collected from hokkaido in this study as showed in the complementation tests with the isolate sm 025 (fig 3). fig 1 growth of wild-type parental strain (sm 025) of fusarium oxysporum f. sp. cepae and its three nitrate non-utilizing (nit) mutant phenotypes on four different nitrogen resources. a. wild–type, b. nit 1, c. nitm, d. nit 3. dense aerial mycelium growth indicating the ability on using nitrate resources. nitrate nitrite hypoxanthine ammonium table 2 vegetative compatibility groups among isolates of fusarium oxysporum f. sp. cepae vcga source/origin no. of isolates 0 4 2 0 0 4 2 1 0 4 2 2 0 4 2 3 042-b mikasa furano kitami kunnenpu tsubetsu atcc mikasa kitami ta n n o yakumo yakumo atcc 5 (sm 024, sm 025, 1-1026, 1-2015, 12017) 6*(nk 041, tr 001, tr 005, 93-37-3, 9337-4, nkf 900) 2 (ktm 517, ktm 519) 15 (kars 9801, kn-o-01, kn-o-02, kno-04, kn-o-06, kn-o-10, kn-o-11, kn-o-12, kn-o-14, kn-o-17, kn-o18, kn-o-19, kn-h-03,989-01, 9890 2 ) 1(9812-04) 4 (atcc 11711, atcc 46077, atcc 62592, atcc 60768) 6(sm 037, sm 044, sm 045, sm 046, mh 019, 3-1024) 1 (ktm 503) 2(tn 98-01, k-3-19) 2** (wo-f-41 and wo-f-46) 2** (wo-f-10 and wo-f-34) 2 (atcc 11850 and atcc 46076) * including three isolates from dr. f. kodama (nkf-900, 93-37-3, and 93-37-4); ** isolates of welsh onion from dr. h. abe; avcg’s are numbered according to puhalla’s numbering system as proposed by katan and di primo (1999); b042is an artificial group containing isolates that are single member of a vcg. ishikari district 100 km fig 2 distribution of fusarium oxysporum f. sp. cepae population in hokkaido based on vegetative compatibility groups. a. mikasa, b. furano, c. kunnenpu, d. kitami, e. tanno, f. tsubetsu, g. yakumo. vcg 0420, vcg 0421, vcg 0420 and 0421, vcg 0422, vcg 0423. fig 3 heterokaryon formation between complementary nitrate non-utilizing mutants of fusarium oxysporum f. sp. cepae on minimal medium containing nitrate as the sole source of nitrogen. a. complementation reaction between isolate sm 025 and 1-1026, b. complementation between isolate sm 025 and nkf 900, c. no complementation between isolate sm025 and atcc 46076. volume 2, 2008 microbiol indones 41 discussion thirty eight isolates of f. oxysporum f. sp. cepae collected from seven locations in hokkaido and 10 isolates from the atcc which included 4 isolates from hokkaido, were grouped into 4 vcgs and 2 ssc isolates. within 42 isolates of f. oxysporum f. sp. cepae from hokkaido, one predominant vcg containing 33 isolates was identified. the isolates obtained from onion were widely distributed across most of the sampling sites (fig 1). the presence of this predominant group in different onion fields indicates that this population has been selectively maintained. a similar phenomenon has been observed for other formae speciales of f. oxysporum (elias and schneider 1991; katan et al. 1996; ahn et al. 1998). the other vcg of onions from hokkaido (vcg 0421) and one of the members of artificial vcg 042(atcc 46076) may lack the competitive traits necessary to insure their dispersal and survival. these results also showed that population structure of f. oxysporum f. sp. cepae based on vcgs was dynamic over time as indicated by replacing the predominant population of hokkaido’s isolate in 1970’s (atcc 46076) grouped into vcg042with the new group vcg 0420 identified in this study. the change population predominance of f. oxysporum f. sp. cepae in hokkaido might be affected by the shift of onion cultivars planted and other abiotic factors. isolates within formae speciales are genetically similar, probably originating from a single pathogenic genotype or clonal origin. kistler (1997) proposed that a vcg within a population of f. oxysporum indicates a distinct clonal lineage as reported by harveson and rush (1997) on f. oxysporum f. sp. betae. they showed that vcgs of f. oxysporum f. sp. betae represented distinct isolated populations indigenous to their respective areas. other researchers (bosland and williams 1987; woo et al. 1996), however, reported that the formae speciales they were investigating did not exhibit a correlation between vcg and geographical origin. although welsh onion isolates tested in this study was not comprehensive, these isolates tended to be distinct vcgs from onion isolates and were found to be limited in their geographical origin. swift et al. 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m, hagiwara h. 2003. use of nitrate non-utilizing mutant and selective media to examine population dynamics of fusarium oxysporum f. sp. spinaceae in soil. phytopathology 93:1173-1181. thornton mk, mohan sk. 1996. response of sweet spanish onion cultivars and numbered hybrids to basal rot and pink root. plant dis 80:660-664. vakalounakis dj, doulis ag, klironomou e. 2005. characterization of fusarium oxysporum f. sp. radicis-cucumerinum attacking melon under natural conditions in greece. plant pathol 54:339346. venter sl, theron dj, steyn pj, ferreira di, eicker a. 1992. relationship between vegetative compatibility and pathogenicity of isolates of fusarium oxysporum f. sp. tuberosi from potato. phytopathology 82:858-862. woo sl, zoina a, del sorbo g, lorito m, nanni b, scala f, noviello c. 1996. characterization of fusarium oxysporum f. sp. phaseoli by pathogenic races, vcgs, rflps, and rapd. phytopathology 86:966-973. woudt lp, neuvel a, sikkema a, van grinsven mqjm, de milliano waj, campbell cl, leslie jf. 1995. genetic variation in fusarium oxysporum from cyclamen. phytopathology 85:1348-1355. yoo sj, watanabe h, kobayashi k, ogoshi a, kodama f. 1993. vegetative compatibility grouping of formae speciales of fusarium oxysporum pathogenic to liliaceae. ann phytopathol soc jpn 59:3-9. volume 2, 2008 microbiol indones 43 issn 1978-3477, eissn 2087-8575 volume 11, number 4, december 2017 the utilization of modified cassava flour (mocaf) industry waste and peat as carrier of nitrogen-fixing bacteria and phosphate solubilizing bacteria inoculant identification and characterizations of potential indigenous endophytic bacteria which had ability to promote growth rate of tomato and biocontrol agents of ralstonia solanacearum and fusarium oxysporum fsp. solani molecular identification of thermally-tolerant symbiotic dinoflagellates from hard coral (scleractinia) in biawak island, indonesia isolation and identification of ethanol and glucose tolerance yeasts strain from tacca leontopetaloides potential degradation of sara (saturated, aromatics, resinics, asphaltenes) fractions of crude oil by reservoir indigenous bacteria from south sumatera retno rosariastuti, sumani, supriyadi, muhammad ardian nursetyawan, and pramusita yoga daniswara yulmira yanti, warnita, reflin, and munzir busniah evina tami roriris, mochamad untung kurnia agung, sri astuty, and yeni mulyani gemilang lara utama, wahyu kristian sugandi, elazmanawati lembong, and edy suryadi d e a i n d r i a n i a s t u t i , i s t y a d h i t y a purwasena, pingkan aditiawati, indiani sani, tutuka ariadji, and muhammad hidayat abqory 111 117 123 129 137 author index 147 subject index 148 issn 1978-3477, eissn 2087-8575 volume 11, number 4, december 2017 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing 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lee, 2020 yaya rukayadi, 2020 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi iman rusmana, 2020 alucia anita artarini, institut teknologi bandung, indonesia ardiansyah michwan, universitas bakrie, indonesia astutiati nurhasanah, badan pengkajian dan penerapan teknologi, indonesia catur riani, institut teknologi bandung, indonesia churiyah, badan pengkajian dan penerapan teknologi, indonesia debbie s retnoningrum, institut teknologi bandung, indonesia endang purwantini, virginia polytechnic institute and state university, usa eny riyanti, balai besar penelitian dan pengembangan bioteknologi dan sumberdaya genetik pertanian, ministry of agriculture, indonesia ernawati arifin giri-rachman, institut teknologi bandung, indonesia fernita puspasari, institut teknologi bandung, indonesia hanies ambarsari, badan pengkajian dan penerapan teknologi, indonesia hideki takahashi, tohoku university, japan i made sudiana, lembaga ilmu pengetahuan indonesia, indonesia ihsanawati, institut teknologi bandung, indonesia is helianti, badan pengkajian dan penerapan teknologi, indonesia jignesh patel, university of texas medical branch at galveston, usa julian ransangan, university malaysia sabah, malaysia kartini kramadibrata, lembaga ilmu pengetahuan indonesia, indonesia khomaini hasan, institut teknologi bandung, indonesia maelita ramdani moeis, institut teknologi bandung, indonesia moritz müller, swinburne university of technology sarawak, malaysia n venkatesh, st. joseph's college of engineering, omr chennai india nagendra p. shah, the university of hong kong, hong kong neung tiaamroeng, suranaree university of technology, thailand niknik nurhayati, badan pengkajian dan penerapan teknologi, indonesia puspita lisdiyanti, lembaga ilmu pengetahuan indonesia, indonesia rofiq sunarya, badan pengkajian dan penerapan teknologi, indonesia sabar pambudi, badan pengkajian dan penerapan teknologi, indonesia sarijiya antonius, lembaga ilmu pengetahuan indonesia, indonesia sartaj alam syed, the university of agriculture peshawar, pakistan shubhajit mitra, university of texas medical branch galveston texas, usa silva abraham, badan pengkajian dan penerapan teknologi, indonesia sinosh skariyachan, dayananda sagar institutions, india siti khodijah chaerun, institut teknologi bandung, indonesia trismilah, badan pengkajian dan penerapan teknologi, indonesia valan arasu, king saud university, saudi arabia verawat, biotec national center for genetic engineering and biotechnology, thailand wangsa tirta ismaya, dexa laboratories of biomolecular sciences, indonesia woro hastuti satyantini, universitas airlangga, indonesia yaya rukayadi, universiti putra malaysia, malaysia yeva rosana, universitas indonesia, indonesia acknowledgement volume 11 vol.11, no.4, december 2017 microbiology indonesia issn 1978-3477, eissn 2087-8575 available online at http://jurnal.permi.or.id/index.php/mioline new-637-1456-1-pb cover depan 3 263 (aris tri wahyudi).pmd rapid and simple amplification of genomic dna sequences flanking transposon aris tri wahyudi department of biology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia phone/fax: +62-251-622833, e-mail: aristri2003@yahoo.com a rapid and simple method to amplify genomic dna sequences flanking mini-tn5 transposon insertion was developed. this technique can be used to determine the location and orientation of the transposon insertion within genomic dna of the bacteria. based on the mini-tn5km1 transposon sequence, pcr primers can be designed to specifically amplify the dna sequences flanking mini-tn5 transposon by inverse polymerase chain reaction (inverse pcr) directly, upstream and downstream of the transposon insertion. the method involves: (i) digestion with a restriction enzyme that does not cut mini-tn5km1 sequence; (ii) self-ligation under conditions favoring the production of monomeric circles; and (iii) inverse pcr reaction using primers designed from minitn5km1 sequence to amplify the dna sequences flanking mini-tn5km1 transposon insertion. feasibility and reliability of this method were demonstrated with mini-tn5km1 mutants of the microaerobic magnetic bacterium magnetospirillum magneticum amb-1 which are defective in magnetosomes synthesis. the inverse pcr products amplified from these mutant genomes showed the correct fragments as determined through southern hybridization and dna sequence analysis. key words: inverse pcr, transposon mini-tn5km1, southern hybridization, dna sequencing _____________________________________________ polymerase chain reaction (pcr) is a technique for the in vitro amplification of specific region of dna without conventional cloning procedures. however, limitation of this method is it amplifies only the region of dna between two convergent primers. any method for the in vitro amplification of the dna sequences flanking a known sequence of dna would have many useful applications in genetics such as amplification and identification of the sequence flanking a transposable element (transposon) (ochman et al. 1988). a mini-tn5 transposon has been constructed that simplifies substantially the generation of insertion mutants of variety of gram negative bacteria (delorenzo et al. 1990). this transposon has become a popular tool to study molecular genetics of gram negative bacteria because it stably inserts into the genome (hererro et al. 1990). thus, this is an important genetic tool to localize the gene of interest to be isolated. isolation of the dna sequences or gene interrupted by the transposon is usually achieved by cloning the dna containing the transposon, by the ligation-mediated polymerase chain reaction (lmpcr) (prod’hom et al. 1998) or by the inverse polymerase chain reaction (inverse pcr) (rich and willis 1990). the inverse pcr has been used to amplify dna sequences flanking tn5 transposon insertion such as genomes of hypomicrobium facilis (feseveldt et al. 1997), e. coli (ochman et al. 1988), pseudomonas abietaniphila (martin and mohn 1999), and pseudomonas fluorescens (mirleu et al. 2000). in this study, in order to amplify the genomic dna sequences flanking mini-tn5 transposon, an inverse pcr technique was developed based on the mini-tn5km1 sequence to design the primers directed outward from the transposon. to demonstrate this method, non-magnetic mutants of magnetospirillum magneticum amb-1 were generated by mini-tn5km1 transposon mutagenesis (wahyudi et al. 2001) that were unable to synthesize magnetosomes, membrane-bound magnetic particles of magnetite (fe 3 o 4 ) synthesized in the cell. dna sequences flanking the transposon were directly isolated by inverse pcr. the inverse pcr products obtained were confirmed by southern hybridization and dna sequence analysis. materials and methods bacterial strains, plasmids, and media. escherichia coli cells were grown in luria broth (lb) medium (tryptone 10.0 g l-1, nacl 5 g l-1, yeast extract 5.0 g l-1) at 37 oc. antibiotics were added when required with the following concentration (µg ml-1): kanamycin, 25; ampicillin 50. magnetospirillum magneticum amb-1 (matsunaga et al. 1991) culture was grown at 25 oc in magnetic spirillum growth medium (msgm) (blakemore et al. 1979), and non-magnetic mutants of amb1 were grown in msgm supplemented with kanamycin 5 µg ml-1 and incubated microaerobically. plasmid pgemt-easy (~3 kb) was used as a cloning vector of inverse pcr products. mini-tn5 transposon mutagenesis. fourteen nonmagnetic mutants of m. magneticum amb-1 defective in magnetosome synthesis, were obtained from transconjugation between amb-1 with e. coli s17-1 (λ pir) harboring putminitn5km1 (delorenzo et al. 1990; hererro et al. 1990). transconjugants were selected on msgm agar plate supplemented with kanamycin 5 µg ml-1. kanamycin resistant colonies were subsequently cultured in a liquid medium of msgm containing kanamycin 5 µg ml-1 for further analysis. southern hybridization analysis. genomic dna from non-magnetic mutants (approximately 3 µg) were digested with ecorv, bstxi, or apai and electrophoresed on 1% agarose gel. the digested dna in the gel was denatured two times with denaturation solution (0.02 n naoh, 0.5 m nacl) for 20 minutes and neutralized two times with neutralization solution (0.2 m tris-hcl, 0.5 m nacl) for 15 minutes prior to blotting. transfer of dna onto a membrane microbiology indonesia, april 2007, p 1-4 volume. 1, number 1 issn 1978-3477 figure 1 strategy for amplification of dna sequence flanking mini-tn5 transposon by inverse pcr. genomic dna of non-magnetic mutant containing inserted transposon was digested with apai, bstxi, or ecorv. is: insertion sequence of the mini-transposon, 19 base pairs (inner and outer). filter was carried out overnight using 10 x salt sodium citrate (ssc) solution (3 m nacl, 0.3 m sodium citrate, ph 7.0) as the transfer solution, and the dna was fixed onto membrane by uv cross-linking. blotted membrane was hybridized overnight at 42 oc with 1.8 kb of mini-tn5km1 transposon containing kanamycin resistance gene as a probe labeled with digoxigenin (boehringer manheim, germany). membrane was washed two times in 2 x wash solution (2 x ssc, 0.1% sodium dodecyl sulfate (sds) for 5 minutes at room temperature, and then two times in 0.1 x wash solution (0.1 x ssc, 0.1% sds) for 15 minutes at 68 oc. detection of hybridization was conducted using x-ray film. preparation of dna templates for inverse pcr. the strategy for inverse pcr is shown in figure 1. dna templates for inverse pcr were prepared from approximately 1 µg of genomic dna from non-magnetic mutants digested with ecorv, bstxi, or apai (these restriction enzymes do not digest mini-tn5km1). digested dna was purified by phenol/ chloroform extraction followed by chloroform/isoamyl alcohol treatment and the dna was precipitated with 100% ethanol and washed two times with 70% ethanol. dna pellets were diluted in 5 µl of sterile milli q and ligated with the same volume of dna ligation kit version 2 solution i (takara, tokyo, japan). the ligation reaction was incubated overnight at 15 oc and the circularized dna was precipitated with 100% ethanol, washed with 70% ethanol, and the dna pellets were diluted in 10 µl milli q. conditions for inverse pcr. the circularized dna sequences flanking mini-tn5 transposon (approximately 500 ng), was amplified using gene amp pcr 2400 (perkin elmer, usa) in a total volume of 50 µl containing 2.5 mm dntp mixture, gc buffer ii, la taq dna polymerase (takara, japan), and 10 picomol of each primer designed from minitn5km1 near insertion sequence (is). to amplify both upstream and downstream of the dna sequences flanking mini-tn5km1 were used primer 1: 5’-acactgatga atgttc-cgttg-3’ and primer 2: 5-‘acctgcaggcatgca agcttc-3’ (figure 1). a gene amp pcr system 2400 (perkin elmer, usa) was used to amplify dna by: denaturation at 95 oc for 2 minutes; primer annealing at 60 oc for 1 minute; primer extension at 72 oc for 1 minute for 30 cycles and 10 minutes at 72 oc for the last cycle. dna sequencing. the inverse pcr products were purified using gene clean iii kit (bio 101) and subsequently cloned in pgem-t easy (promega, usa) and transformed into e. coli dh5α. the recombinant plasmids were extracted from e. coli and used as templates for cycle sequencing by pcr machine 9400. dna sequencing was carried out using the dye terminator cycle sequencing kit (applied biosystems, usa) and m13 universal and m13 reverse primer were used as cycle sequencing primers. sequencing of the dna was performed using an automatic dna sequencer abi prism 377 (perkin elmer, usa). results mini-tn5 transposon mutagenesis generated eight nonmagnetic mutants of m. magneticum amb-1 defective in apai, ecorv, or bstxi dna amplified hindii hindii xhoiis (i) econi sspi mini-tn5km1 primer2 is (o) primer1 ecorv digest dna amplified apai, ecorv, or bstxi mini-tn5km1 is (o)is (i) ligation (circularization) ipcr amplification primer1 ecorv ecorv primer2 primer1: 5'-acactgatgaatgttccgttg-3' primer2: 5'-acctgcaggcatgcaagcttc-3' 2 wahyudi microbiol indones magnetosome synthesis. frequency of transconjugation was about 2.7 x 10-7 cells per recipients. all the dna fragments of m. magneticum amb-1 genome flanking mini-tn5km1 transposon could be amplified by inverse pcr (figure 2). analysis of eight genomic dna of non-magnetic mutants by southern hybridization, using kanamysin resistance gene (km1) as a probe, revealed the presence of a single mini-tn5 transposon insertion in the mutant’s genomic dna, as shown in figure 3. sub-cloning of inverse pcr products using pgem-t easy as a cloning vector, and their sequence analysis revealed the presence of all insertion sequences (is) coming from the mini tn5km1 transposon (19 bp inner and 19 bp outer), as well as 9 bases of the target sites of the transposon insertion were also identified (table 1). southern hybridization analysis of ecori digestion of many non-magnetic mutant genomes indicated that minitn5km1 inserts in the genomic fragment of approximately 12 kb in size (data not shown). this makes it difficult to clone the fragments containing the transposon into a plasmid vector, such as puc18, puc19, or prk415 (data not shown). therefore, the inverse pcr technique was developed to rapidly amplify the genomic dna sequences of m. magneticum amb-1 flanking the mini-tn5km1 transposon. primers oriented outward from the transposon permit directly amplification of those fragments, upstream, and downstream of the transposon. flanking dna sequence of m. magneticum amb-1 up to 4.2 kb can be amplified by inverse pcr with ecorv digestion. discussion in this study, the dna fragments from restriction enzyme digestion were diluted and ligated under conditions that favor the formation of monomeric circles (collin and weissman 1984). the resulting ligation products are used as substrates for enzymatic amplification by pcr using primers near the end of the core sequence (mini-tn5km1) oriented outward from the known sequence region. the product resulting from amplification is a linear doublestranded dna molecule including fragments situated both 5’ and 3’ to the known sequence region. the junction between the original upstream and downstream regions, can be identified as the restriction site of the restriction enzyme, in this case apai, bstxi, or ecorv, that was used to produce the linear fragments prior to self ligation. southern hybridization analysis of ecori or psti digested from several non-magnetic mutant genomes indicated that mini-tn5 inserted into the genomic dna as a fragment of > 12 kb in size (data not shown). such size renders difficulty in cloning a genomic fragment containing mini-tn5 transposon into plasmids puc19, puc18, or prk415 (data not shown). therefore, we developed this simple technique to amplify the genomic dna sequences flanking mini-tn5 transposons. primers oriented outward from the transposon permit directly amplification of those fragments, upstream and downstream of the transposon insertion. the expected size of the inverse pcr products generated by inverse pcr can be calculated based on the hybridization size minus the transposon mini-tn5km1 size (1.8 kb). digestion of non-magnetic mutant 10 (nma10) genome using apai yielded a 1.3 kb of inverse pcr product (3.1-1.8 kb). digestion of nma 6 and 7 genomes with ecorv, and nma 9 genome with bstxi yielded 2.3; 2.3; and 2.1 kb, respectively (4.1-1.8; 4.1-1.8; 3.9-1.8 kb; respectively). on the other hand, nma 15, 17, 19, and 41 genomes which were digested with ecorv yielded 4.2; 2.8; 3.2; and 1.5 kb inverse pcr products, respectively (6.0-1.8; 4.8-1.8; 5.0-1.8; 3.2-1.8; respectively) (figure 2 and 3; table 1). to confirm consistence of the expected of the correct inverse pcr products generated in this study, dna sequencing of the (kb) figure 2 agarose gel electrophoresis of 8 inverse pcr products amplified from non-magnetic mutant genomes by inverse pcr. m: marker 1 kb dna ladder. numbers above of the figure indicate number of non-magnetic mutant genomes. m 6 7 9 10 15 17 19 41 (kb) 4 . 2 3 . 2 2 . 8 2 . 3 2 . 1 1 . 5 1 . 3 1 2 3 2 1 . 6 1 0 . 5 figure 3 southern hybridization analysis of non-magnetic mutant genomes digested with ecorv, except no 9 and 10 were digested with bstxi and apai, respectively. m: marker 1 kb dna ladder. p: plasmid putmini-tn5km1 digested with eco ri and psti. kanamycin resistance gene (km1, 1.8 kb) isolated from putmini-tn5km1 was used as a probe. m p 6 7 9 10 15 17 19 41 (kb) 1.8 kb 6 . 0 5 . 0 4 . 8 4 . 1 3 . 9 3 . 2 1 . 8 volume 1, 2007 microbiol indones 3 table 1 size of dna fragments flanking mini-tn5km1 transposon amplified from non-magnetic mutant genomes by inverse pcr, and confirmation by southern hybridization. nine bases of target sites of the transposon insertion were identified no. of nma* inverse pcr southern target site genome product (kb) hybridization band (kb) (9 base) atc cag cgc atc cag cgc ggg cag tcc ggc cag ggc gtc ctg ggg atc caa ggc gga cct gcg ata tgg ctc 6 7 9 1 0 1 5 1 7 1 9 4 1 2 . 3 2 . 3 2 . 1 1 . 3 4 . 2 2 . 8 3 . 2 1 . 5 4 . 1 4 . 1 3 . 8 3 . 0 6 . 0 3 . 6 5 . 0 3 . 2 *nma: non-magnetic mutant of amb-1 inverse pcr products was performed. the inverse pcr products were cloned into pgem-t plasmid vector and sequenced. sequence analysis indicated that all of the inverse pcr products contained a 19 bp of insertion sequences coming from mini-tn5km1 transposon i.e. ctgtctcttgat cagatct (inner) and acttgtgtata agagtcag (outer) (delorenzo et al. 1990). the presence of these insertion sequences of inverse pcr products indicated that the products of inverse pcr were correctly amplified because primers designed outward from the transposon passed the 19 bases insertion sequences of the transposon. thus, these results suggest that inverse pcr based on this method can be used to rapidly amplify both upstream and downstream of the genomic dna fragment flanking mini-tn5 transposon insertion of the bacterial genomes interrupted by this transposon. the inverse pcr method permits the rapid amplification of regions of unknown sequence flanking specified segments of dna. since only regions of limited size can be enzymatically amplified by pcr, and since primers should be synthesized from known sequences, the inverse pcr approach is not amenable to proceeding very long distances in the genome. however, the technique described here is a simple and rapid method as an alternative to amplify and isolate genomic dna sequences flanking mini-tn5 transposon insertion. furthermore, the inverse pcr products amplified by this method can be used to quickly obtain dna sequence information on the region of the mini-tn5 transposon insertion without sub-cloning of mutant genomic dna containing transposon. in addition, we have successfully used these inverse pcr products as probes to isolate cosmid recombinant clones from a genomic library of wild-type m. magneticum strain amb-1 (wahyudi 2005). this method has also been successfully applied to amplify genomic dna fragments flanking mini-tn5km1 transposon from acid-al sensitive mutants of bradyrhizobium japonicum resulted by transposon mutagenesis (data not shown). acknowledgement part of this work was conducted at tokyo university of agriculture and technology, japan, at the department of biotechnology (matsunaga-takeyama laboratory). therefore, i grateful to tadashi matsunaga and haruko takeyama for the laboratory facilities and their valuable support. references blakemore rp, maratea d, wolf rs. 1979. isolation and pure culture of a fresh water magnetic spirilum in defined growth medium. j bacteriol 140:720-729. collin fs, weissman sm. 1984. directional cloning of dna fragments at a large distance from an initial probe: a circularization method. proc natl acad sci usa 81:6812-6816. delorenzo v, herrero m, martinko jm, parker j. 1990. tn5 transposon derivatives for insertion mutagenesis, promotor probing and chromosomal insertion of cloned dna in gramnegative eubacteria. j bacteriol 172:6568-6572. feseveldt a, poetsch m, gliesche cg. 1997. development of a species specific gene probe for hypomicrobium facilis with the inverse pcr. appl environ microbiol 63:335-337. hererro m, delorenzo v, timmis kn. 1990. transposon vectors containing non antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram negative bacteria. j bacteriol 172:6557-6567. martin vjj, mohn ww. 1999. an alternative inverse pcr (ipcr) method to amplify dna sequences flanking tn5 transposon insertions. j microbiol met 35:163-166. matsunaga t, sakaguchi t, todokoro f. 1991. magnetite formation by a magnetic bacterium capable of growing aerobically. appl microbiol biotechnol 35:651-655. mirleu p et al. 2000. fitness in soil and rhizosphere of pseudomonas fluorescens c7r12 compared with c7r12 mutant affected in pyoverdine synthesis and uptake. fems microbiol ecol 34:344 4 . ochman h, gerber as, hartl dl. 1988. genetic application of an inverse polymerase chain reaction. genetics 120:621-623. prod’hom g et al. 1998. a reliable amplification technique for the characterization of genomic dna sequences flanking insertion sequences. fems microbiol lett 158:75-81. rich jj, willis dk. 1990. a single oligonucleotide can be used to rapidly isolate dna sequences flanking a transposon tn5 insertion by the polymerase chain reaction. nucleic acids res 18:6673-6676. wahyudi at. 2005. [construction of genomic library of magnetospirillum magneticum amb-1 and screening of genes involved in magnetosome synthesis]. [in indonesian]. j mikrobiol indones 10:91-95. wahyudi at, takeyama h, matsunaga t. 2001. isolation of magnetospirillum magneticum amb-1 mutants defective in bacterial magnetic particles synthesis by transposon mutagenesis. appl biochem biotechnol 91:147-154. 4 wahyudi microbiol indones 404 not found 03 astuti.cdr vol.12, no.1, march 2018, p 15-22 doi: 10.5454/mi.12.1.3 physiological profiling and microorganism community analysis of cirebon tm shrimp paste fermentation “terasi” using biolog ecoplate dea indriani astuti*, intan taufik, dini achnafani, and ezra suci priscila department of microbiology, school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung, 40132, west java, indonesia terasi or shrimp paste is an indonesian traditional seasoning made from fermented small shrimp or krill. different indigenous microorganism community exhibit different physiological function due to lack standard in its materials and processing. this study aimed to determine physiological profiles and microorganism tm community in cirebon shrimp paste fermentation. biolog ecoplate was used to obtain microbial physiological function of the krill and 2-months old shrimp paste. microorganisms were later isolated from ecoplate substrate to determine its community structure. average well color development (awcd) from krill was thirty times higher than shrimp paste. interestingly, this study revealed a shift of carbon source utilization at day-28 of fermentation from amino acid and polymer to phenolic compound. in addition, awcd index increased in accordance with increased of microorganism community complexity at day-28. within 56 days of fermentation there was a slightly increase in water, fat, and carbohydrate content. in contrast, there was decrease in protein, ash content, and acidity level from neutral to acid, with salinity level resulted in between 16.26% to 21.42%. we conclude that there is a change of microorganism community within shrimp paste fermentation corresponding to metabolism activity which affects the product quality. tm key words: biolog ecoplate, microbial community, physiology, shrimp paste terasi udang merupakan salah satu contoh penyedap rasa alami dari indonesia dengan bahan baku udang rebon yang difermentasi. tahap pembuatan serta bahan baku yang tidak sama membuat komunitas mikroorganisme serta aktivitas metabolisme yang terlibat akan berbeda. dalam penelitian ini akan ditentukan profil fisiologi dan komunitas mikroorganisme selama proses fermentasi terasi udang dari cirebon, jawa barat. tm analisis fungsi fisiologi dilakukan dengan menggunakan biolog ecoplate pada sampel udang rebon dan tm terasi setiap 2 minggu selama 2 bulan fermentasi. mikroorganisme diisolasi dari substrat biolog ecoplate tm untuk menentukan struktur komunitasnya. nilai average well color development (awcd) biolog ecoplate dari sampel udang rebon lebih tinggi 30 kali daripada sampel terasi udang. selain itu, terlihat pergantian penggunaan kelompok substrat dari polimer dan asam amino menjadi senyawa fenolik pada terasi berumur 28 hari. nilai awcd pada sampel terasi mengalami penurunan pada hari ke-28 dan kembali mengalami kenaikan hingga hari ke-56 seiring dengan adanya peningkatan kompleksitas dari komunitas mikroroganisme. selama 56 hari fermentasi, terjadi sedikit pengingkatan kadar air, lemak, dan karbohidrat. sebaliknya, kadar protein dan abu mengalami penurunan dan rentang ph dari netral menuju asam. kadar garam berfluktuatif pada rentang 16,26% hingga 21,42%. melalui penelitian ini dapat disimpulkan bahwa adanya perubahan komunitas mikroorganisme pada fermentasi terasi udang melalui perbedaan kemampuan penggunaan substrat karbon. tm kata kunci: biolog ecoplate, fungsi fisologis, komunitas mikrooganisme, terasi udang microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-22-2511575, email: dea@sith.itb.ac.id (standar nasional indonesia sni, 1992), shrimp paste is a food seasoning with distinctive odor from fermentation of shrimp or fish or mixture of both with salt or other ingredients. exact procedures and ingredients of shrimp paste varies according to region. however, similar step of grinding, drying, and salt addition are always present. addition of salt prevent spoilage and creating a suitable environment for certain microorganism (lee et al. 2016). breakdown of protein compounds into amino acids and various chemicals corresponds to umami taste and some antioxidant properties (murwani et al. 2015). since fermentation occurred naturally, both functional and non-functional microorganisms might terasi or shrimp paste is one of indonesian traditional fermented product made from shrimp that undergo natural fermentation by the work of indigenous microorganism (kobayashi et al. 2003; murwani et al. 2015). similar product as food seasoning are also found in many asian countries, such as china, malaysia, bangladesh, and korea (kim et al. 2014). in indonesia, shrimp paste produced (by generation to generation) in many coastal areas where source of raw materials such as shrimp, krill, and fish are abundant. according to indonesian national standard contribute in the process (tamang et al. 2016). different manufacturing process will certainly give unequal results for each product, because physiology of the m i c r o o rg a n i s m a r e s t r o n g l y i n f l u e n c e d b y environment. to date, various microorganisms such as bacillus, micrococcus, tetragenococcus, sarcina, pseudomonas, sporolactobacilus, pediococcus, streptococcus, halomicrobium, and streptococcus group are recorded to be found in shrimp paste with different physiological properties such as proteolytic, halophilic, and lactic acid production (chukeatirote 2016; kobayashi et al. 2003; sobhi et al. 2012; surono and hosono 1994). microorganism community implies its functional physiology property by its catabolic processes as indicated by its capability to adapt to different metabolic condition (preston-mafham, boddy, and randerson 2002). the microorganism community level tm physiological profile can be assessed using biolog tm ecoplate introduced by garland and mills. biolog ecoplate consist of 96-well with 31 different carbon substrates based on redox reaction. reduction of tetrazolium dye in each well produces color change and are used as an indicator of respiration. the redox reaction are exploited for rapid assessment or characterization of microbial communities (garland and mills 1991). study on physiological function and dynamics of microorganism community within shrimp paste fermentation has not been reported. studies on community level of microorganism and its physiology that contribute to determine shrimp paste quality is limited. this study is intended to assess physiological function of microorganism community in shrimp paste t m fermentation using biolog ecoplate by determining the microbial community dynamics and its physiological activity especially in cirebon shrimp paste fermentation. materials and methods sample. traditionally produced cirebon shrimp pastes were collected from samadikun, cirebon, west java. according to producer, shrimp or krill were dried for 1 d under the sun, then pounded and dried again for 1 d. salt and sugar were added using krill : salt : sugar ratio of 5:1:1 (w/w). a 500 mg of mixture were wrapped with waxed paper and then stored at room temperature to undergo fermentation. shrimp paste was sampled every 14 d for 2 m. community level physiological profile. sample tm was prepared and inoculated into the biolog ecoplate based on previous method (wang et al. 2007) with some modifications. briefly, 4 g of crushed shrimp paste or krill was suspended in 36 ml of saline phosphate buffer solution or pbs; ph 7.2. homogenate was filtered using sterile gauze to remove large particles and was diluted by introducing 4 ml of homogenate into 36 ml of pbs. a 150 μl of -3 homogenate from 10 dilution was inoculated into each tm biolog ecoplate well. incubation was performed at 37 °c for 168 h. optical density was measured every 24 hours at 590 nm and 750 nm. measurements at 590 nm were to detect color changes due to formazan formation and turbidity, while measurements at 750 nm were for turbidity correction. negative value was corrected to zero. mean of each substrate density value from three repetitions was used for further calculations. the average well color development (awcd) value for each observation time was calculated according to following formula (garland and mills 1991): information: c (t) = optical density value of i substrate at a given time; r (t) = optical density value of control at a given time; i = number of substrates. total plate count. krill and shrimp paste homogenate were inoculated on nutrient agar (na) media and incubated at 37 °c for 24 h. enumeration was performed to obtain total plate count number -1 expressed in colony forming unit per ml (cfu ml ). i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f microorganism. microorganism isolation was performed from each well of ecoplate with positive value after 168 h. a 100 μl of sample composite was inoculated to nutrient agar (na) media and incubated at 37 °c for 24 h. single isolate is characterized based on colony morphology and microscopic observations by means of gram staining according to previous method (prescott, klein, and harley 2002). statistical analysis. multivariate analysis was performed by principle component analysis (pca) using xlstat. analysis was performed to differentiate krill and shrimp paste, physiological function and microorganism community during fermentation. the pca was determined based on pearson type, where number of microorganism, optical tm density value of each biolog ecoplate substrates, and isolates obtained were used as variables. 16 astuti et al. microbiol indones volume 12, 2018 microbiol indones 17 measurement of salinity and acidity. salinity was measured by argentometric according to mohr method (hong, kim, and czae 2010) with some modifications. briefly, 4 g sample was added to 36 ml sterile water and filtered by whatman paper no. 1 before titration. meanwhile, acidity level was measured using a calibrated ph-meter. proximate analysis. proximate analysis was conducted to obtain carbohydrate, protein, fat, ash, and lipid content from samples. it was performed in accordance to sni 01-2891-1992. results samples obtained from krill and traditionally fermented shrimp paste at five time-points were tm assessed by biolog ecoplate, total plate count, and chemical characteristic. samples were first assessed by tm biolog ecoplate to obtain physiological function at community level. total plate count performed to verify microorganism density that might contribute to turbidity changes in ecoplate. microorganism tm isolation from each biolog ecoplate utilized substrates was performed for community analysis. average well color development (awcd) value represented metabolic activity of microorganism community from metabolized substrates. the awcd of krill sample after 168 h incubation showed the highest value among all samples at the value of 0.404, thirty times higher than other samples (fig 1a). meanwhile, awcd value of shrimp paste are lower at the value of 0.013; 0.007; 0.004 for day-0, -14, and -28 of shrimp paste with slight increase at day-42 and 56 at the value of 0.008 and 0.009 respectively (fig 1b). in general, the result implied that microbial community of shrimp paste fermentation had lower metabolic activity than that of krill, whereas the slight increase at day-42 indicate changes in the fermentation process. the numbers of microorganism involved during fermentation were determined by culturing the microorganism on nutrient agar (na) media. samples were enumerated and expressed in colony forming unit -1 per ml (cfu ml ). total plate count of microorganism enumeration in shrimp paste samples were significantly lower than that of microorganism in krill samples (table 2). this result strongly contributed to principle component analysis (pca) plot where krill and shrimp paste samples were obviously located at different area (fig 2). this strongly suggest that krill and shrimp paste samples were significantly different in term of number of microorganism. tm mean substrate utilization on biolog ecoplate showed dynamics of carbon source group utilization during shrimp paste fermentation. utilization of carbohydrate, polymer, and carboxylic acid from day-0 to -28 showed decreasing pattern, except for amino acid (fig 3a). interestingly, amino acid and polymer utilization was not found at day-28 and -42 respectively and were replaced by phenolic utilization. corresponding to substrate group utilization, microorganism dynamics were also found during fermentation (fig 3b). the number of isolates found at day-0, -14, -28, -42, and -56 were 8, 8, 7, 8, and 7 isolates respectively. each isolate were obtained from different carbon source that can be grouped into amino acid (aa), carbohydrate (ch), carboxylic acid (ca), polymer (po), and phenolic (ph) with total number of isolates for each carbon group were 3, 16, 3, 4, and 3 respectively. aa1, aa2 and ch1 were found in day-0 and -14, while ca1 were always present at day-0 to -28. gm1 and ga5 were found at day-28 and -42 of fermentation, and po1, po2, po3, and po4 were found in day-14 and -28. as the figure 3 show microbial shifting occurred when ch10, ch11, ch12, ph1, ph2, and ph3 were obtained from carbohydrate and phenolic substrates at day-42 and -56. pca plot for physiological function and microorganism community clearly separated the physiological function at day-0, 14, and -28 of fermentation, meanwhile day-42 and -56 of fermentation are adjacent (fig 4a). in contrast, opposite pattern is found when comparing microorganism community, where day-0, -14, -28 of fermentation are plotted close to each other and distinct from day-42 and -56 (fig 4b). as shown by the analysis, physiological activity and microorganism dynamics affect nutritional and chemical characteristic of shrimp paste during fermentation (table 2). however, the dynamics are not reflected by its acidity or ph level which is generally at neural-acid range. ph level slightly increases at the beginning of fermentation from 7.02 to 7.08 and decreased to 6.74 at the last day of fermentation. salt content fluctuated in the range of 16.26% to 20.48%. water content drastically decreased from shrimp sample to day-0, from 86.70% to 41.74%. nevertheless, there are no significant changes in water content during fermentation process. fat and carbohydrate content increased from 1.32% and 17.26% to 1.47% and 19.94%, respectively. in contrast, protein and ash content decreased from 12.43% and 27.25% to 10.46% and 25.76%, respectively. fig 1 potential metabolic activity of microorganism community in krill sample (a) and shrimp paste (b) by tm awcd values from biolog ecoplate. krill and five time-points of shrimp paste fermentation samples were inoculated into each ecoplate well and measured every 24 hours for 7 d. based on 168 hours of incubation, krill sample exhibit highest awcd value among all samples. in general, awcd value for day0 (●), -14 (○), -28 (♦), -42 (■) and -56 (▲) of shrimp paste were lower than of krill sample. error bar on shrimp paste samples were not shown to simplify data visualization. a b 18 astuti et al. microbiol indones fig 2 principle componenet analysis (pca) plot of krill and shrimp paste samples. analysis was based on total plate count of microorganism on krill and shrimp paste samples isolated grown on nutriet agar media. day-0 (●), -14 (○), -28 (♦), -42 (■) and -56 (▲) of shrimp paste were adjacent to each other and distinct to (□) krill sample. table 1 total plate count enumeration of microorganism on krill and shrimp paste samples during fermentation grown on nutrient agar media sample total plate count (cfu ml-1) krill 3.7 x 108 day-0 shrimp paste 2.2 x 104 day-14 shrimp paste 3.1 x 104 day-28 shrimp paste 3.0 x 104 day-42 shrimp paste 4.2 x 104 day-56 shrimp paste 1.5 x 104 volume 12, 2018 microbiol indones 19 a b fig 3 dynamics of relative carbon source group utilization (a) and microorganism abundance (b) within 56 d of fermentation. carbon group utilization data was obtained from relative sum of mean utilization for each tm carbon group to whole substrate utilized from biolog ecoplate after 168 h incubation. microorganism abundance shown during fermentation were characterized by colony morphology and gram staining. changes of carbon group utilization were shown at day-28 when polymer and amino acid were replaced by phenolic group utilization. corresponding to carbon group utilization, 3, 16, 3, 4, and 3 isolates used amino acid (aa), carbohydrate (ch), carboxylic acid (ca), polymer (po), and phenolic (ph) substrates, respectively. fig 4 pca observation plot for (a) physiological analysis and (b) community analysis on shrimp paste sample within 56 days of fermentation. day-0 (●), -14 (○), -28 (♦) of fermentation are adjacent in microorganism community, but distinct in physiological function. meanwhile, day-42 (■) and -56 (▲)of fermentation are adjacent in physiological function, but distinct in microorganism community. a b table 2 chemical and nutritional characteristics of shrimp paste during fermentation sample ph salt (%) water (%) ash(%) protein (%) fat(%) carbohydrate (%) shrimp nd1 nd1 86.70 1.83 7.68 0.73 2.93 drying i nd1 nd1 39.72 nd1 nd1 nd1 nd1 drying ii nd1 nd1 35.36 nd1 nd1 nd1 nd1 day-0 7.02 19.77 41.74 27.25 12.43 1.32 17.26 day-14 7.08 16.26 41.76 nd1 nd1 nd1 nd1 day-28 6.83 21.41 41.92 24.73 10.34 2.02 20.99 day-42 6.89 20.48 42.91 nd1 nd1 nd1 nd1 day-56 6.74 20.48 42.73 25.76 10.46 1.47 19.94 1nd = not determined 20 astuti et al. microbiol indones discussion tm biolog ecoplate provides rapid and complex community level profiling on physiological function to characterize microorganism community. the assessment is based on carbon source utilization coupled to tetrazolium dye reduction as an indicator of respiration (garland and mills 1991). average well tm color development (awcd) from biolog ecoplate reflect the rate of substrate utilization by microorganism community (preston-mafham et al. 2002). in addition, inoculum density and different community structure may result in higher absorbance values (preston-mafham et al. 2002). awcd value of krill sample was thirty times higher than that of shrimp paste (fig 1a and 1b) suggests that rate of catabolic activity from krill was higher than shrimp paste. this might be due to the number of microorganism in krill that were significantly higher than that of shrimp paste (table 1). drying process in shrimp paste production reduce the water content and implicate towards low number of microorganism. salt addition also provide suitable condition only for certain microorganism to grow and prevent putrefaction (anggo et al. 2015). tm biolog ecoplate can reveals catabolic diversity from samples, in which each microorganism pursue its own substrate utilization pattern, which in turn indicative of the community species diversity. as much as 31 carbon or sole substrate sources are available in ecoplate and can be further categorized into several substrate group: 10 carbohydrates, 4 polymers, 8 amino acids, 2 phenolics, and 7 carboxylic acids (deng et al. 2011). based on these groups, microorganism in shrimp paste utilized carbohydrate, polymer, amino and carboxylic acid in higher level in the early 28 days of fermentation. however, amino acid and polymer utilization were replaced by phenolic utilization after 28 days (fig 2a). the dynamics of substrate utilization corresponds to microorganism dynamics found in each substrate (fig 2b). amino acids utilization was found in the early 28 days of fermentation, which corresponds to the decreasing of protein at day-28. nevertheless, further fermentation does not alter protein content (table 2). this suggests that proteolytic microorganism, those possessing protease enzymes to breakdown protein into other peptides, amino acids and other volatile compounds (khairina et al. 2016; murwani et al. 2015), are present in shrimp paste. the amino acids utilized in ecoplate were l-arginine, l-threonine, lasparagine, and l-serine. several isolates, such as aa1 and aa2 are found to utilize l-arginine and lasparagine at day-0 and -14 of fermentation, respectively. aa1 has circular white opaque colonies with coccus-shaped gram negative while aa2 has circular white transparent colonies with rod-shaped gram negative. carbohydrates and carboxylic acids utilization, which could be derived from sugar degradation, were always detected throughout fermentation. the use of tm biolog ecoplate were able to identify changes of carbohydrate used, n-acetyl glucosamine replaced by β-methyl-d-glucoside and d-mannitol at day-42 and 56. it is known that n-acetyl glucosamine is chitin monomer presented in 1,4-β-glycoside bond (beier and bertilsson 2013) found in krill. isolate ca1 was isolated from the well with d-galacturonic-acid at day0 and -14, pyruvic acid methyl ester at day-14, and tween 80 at day-28. the isolate is characterized by coccus-shaped, gram positive, white opaque colonies and circular shape. ch10; ch11; and ch12 were found in n-acetyl-glucosamine with following characteristics: circular white opaque colonies with coccus-shape gram negative; circular white opaque colonies with coccus-shaped gram positive; irregular cream opaque colonies with coccoid-shaped gram positive, respectively. polymer utilized in ecoplate were tween-40 and tween-80 at day-0 and -28. tween-80 and tween-40 are fatty acid derivatives from oleic acid and palmitic acid, respectively (ilko et al. 2015; takeno et al. 2013). oleic acid and palmitic acid compounds could be a source of carbon in the growth of microorganism through the reaction of β-oxidation. in the other hand, these two fatty acids were dominantly found abundant in krill or shrimp (li, sinclair, and li 2011). the isolates po1; po2; po3; and po4 were found and characterized by irregular white opaque colonies with rod-shaped gram negative; irregular white opaque colonies with rodshaped gram negative; circular white opaque colonies with coccus-shaped gram negative; and circular yellow opaque colonies with coccus-shaped gram negative, respectively. phenolic compounds were used in last stage of fermentation and influenced the flavor development (mcmurrough, roche, and cleary 1984) and antioxidant properties of shrimp paste (sobhi et al. 2012). the phenolic compound of 4-hydroxy-benzoicacid were used as the main carbon source by the halophile group halobacteria of archaea (fairley et al. 2002). utilization of aromatic compounds in the last day of fermentation is in accordance with a study by wittanalai, rakariyatham, and deming (2011) that examined kapi products from thailand and found that the compounds involved in the formation of a distinctive aroma were obtained in large amounts after 30 d of fermentation. the isolates ph1; ph2; and ph3 are found at day-56, and characterized by large-sized opaque white colonies with coccus-shaped gramnegative; circular large white colonies with rod-shaped gram negative; and large white colonies with coccusshaped gram positive, respectively. pca plot for functional and community structure clearly separate different sampling time indicative of different physiological function and community structure during shrimp paste production (fig 4). microorganism functional characteristic of day-0, -14, and -28 were dispersed and shown to be distinct between each other, but its community characteristics were plotted adjacent to each other indicative of its similarity or closeness. in opposite, microorganism from sample day42 and -56 showed close functional characteristics with distinct microorganism community. this suggests that the functional of microorganism community at the beginning of fermentation change more dynamically than at the end of fermentation period. in contrast, microorganism involved in early stage was not drastically changed within 28 d of fermentation. other causes of differences in pca plots are due to isolation and identification of isolates derived from ecoplate which might cause a reduction number of microorganism in isolation medium. microorganism growth were dependent on media culture (vieira and nahas 2005) while ecoplate has a specific substrate as main carbon source for microorganism to grow which not presented in media. chemistry and nutrition parameters of shrimp paste within 2 months of fermentation showed an interesting result. the ph level during early stage of fermentation increase because of the activity of microorganism that metabolize amino and amine compounds to produce nh compounds (daroonpunt 3 et al. 2016). later, the lower ph level at day-28 to -56 were due to the production of acid compounds, where lactic acid bacteria might contribute in this process, as previously shown kobayashi et al. (2003) and surono and hosono (1994). lactic acid bacteria might be found from carbohydrate utilization microorganism, where they utilize sugar compound and release lactate as metabolite product (justé et al. 2008). fluctuating values ​​of salt content might be due to uneven stirring process during shrimp paste making and causes different salt content in packaging. however, above 10% of salinity content is considered to help preservation process (christianti 2006). besides, the drastic decrease of water content by means of drying process also contribute in product preservation by inhibiting the growth of undesired microorganism (anggo et al. 2015). badan standardisasi nasional (bsn) applied a standard for shrimp paste products through standar nasional indonesia (sni) number 2716: 2016. water content, protein content, and salt content are designated maximum at 45%, minimum at 15%, and between 12-20%, respectively. the result of proximate analysis shown that the nutritional level were out of standard. changes in microorganism community and no standard in raw materials and procedures such as drying process and different salt addition might contribute to these results. the most intriguing result from this study was physiological activity performed during fermentation, where metabolic shifting occurred between polymer and amino acid to phenolic utilization. in addition, nutritional content also affected by which microorganism performed different physiological activity. this study has successfully described the physiological function of the microorganism community during fermentation and its effect on nutritional content, thus can be used as the basis for shrimp paste quality improvement. references anggo, apri d, widodo fm, fronthea s, laras r. 2015. changes of amino and fatty acids in anchovy (stolephorus sp) fermented fish paste with different fermentation periods. procedia environ sci. 23:58–63. doi: 10.1016/j.proenv.2015.01.009. badan standardisasi nasional. 1992. sni 01-2891-1992: cara uji makanan dan minuman. badan standardisasi nasional. 2016. sni 2716:2016: terasi udang. beier s, bertilsson s. 2013. bacterial chitin degradationmechanisms and ecophysiological strategies. front microbiol. 4(jun):1–12. doi: 10.3389/fmicb.2013.00149. christianti, agustin. 2006. isolasi dan karakterisasi bakteri halotoleran pada terasi [isolation and characterization halotolerant bacteria in shrimp paste]. 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1 ) : 6 7 7 6 – 8 3 . d o i : 10.1128/aem.02003-13. tamang jp, shin dh, jung dj, chae sw. 2016. functional properties of microorganisms in fermented foods. fron microbiol. 7(apr). doi: 10.3389/fmicb.2016.00578. wang gh, jin j, chen xl, liu jd, liu xb, herbert sj. 2007. biomass and catabolic diversity of microbial communities with long-term restoration, bare fallow and cropping history in chinese mollisols. plant soil environ. 53(4):177–85. wittanalai s, rakariyatham n, deming rl. 2011. volatile compounds of vegetarian soybean kapi , a fermented thai food condiment. african j biotechnol. 10(5):821–30. doi: 10.1007/s13197-015-2142-3. 22 astuti et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 mi-ringga novelni available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.9.3.5issn 1978-3477, eissn 2087-8575 vol 9, no 3, september 2015, p 129-135 *corresponding author; phone: +62-823-8576-1188; email: ringga.novelni@gmail.com infectious disease is one of the major problem in the world. most countries, especially developing ones are facing this problems. infectious disease is caused by microbial pathogens that so dinamic (darmadi 2008). one of the infectious diseases that cause increasing of the number of morbidity, mortality, and care cost at the hospital is diseases caused by the microbial pathogens that produce metallo-βlactamase (mbls) enzyme such as pseudomonas aeruginosa (amudhan et al. 2011). strain of p. aeruginosa who produced the metalloβ-lactamase (mbls) enzyme was firstly reported in japan in 1991 and since then, they has been found in asia, eropa, australia, south america, and north america (kaleem et al. 2010). p. aeruginosa that able to produce metallo-βlactamase (mbls) enzyme is a threat to the infection in the hospital, especially in the intensive care unit (icu). this negative gram bacteria has the high level of resistance to the β-lactam antibiotics because mbls they produced can hydrolyze the ring of β-lactam from the antibiotics of β-lactam group including carbapenem. icu inpatients commonly suffer the severe disease and are in the immunocompromised condition (adisasmito et al. 2006). the reasearch conducted by jamshidi et al. from 2005 to 2006 on icu inpatients of iran hospital, detected some bacteria like p. aeruginosa (43.2%), klebsiella spp. (33.7%), and staphylococcus aureus (39.2%). these pathogens were isolated from sputum sample, blood, urine, foley catheters, nasogastric the aim of this research is to detect bla and bla the resistant genes against meropenem in pseudomonas vim imp aeruginosa from sample of hospitalization patients at the internal medicine hcu of rsup dr. m. djamil padang. firstly, bacterial isolate of p. aeruginosa were isolated from the sputum samples of patients who suffered bronkopneumonia. the isolation were started with samples cultivation to the cetrimide agar media which was a selective media for p. aeruginosa. to determine the species of the bacteria, the identification using gram staining, triple sugar iron agar (tsia) test, citric test, urease test, methyl red/voges-proskauer (mr/vp) test, and molecular marker of 16s rrna genes have been conducted. the isolation and identification result showed that from 20 sputum samples of the patients there were just 10 (50%) samples were positively containing p. aeruginosa. from the p. aeruginosa isolates, the resistant genes against meropenem bla and bla were vim imp amplified using pcr. the result showed that all these p. aeruginosa isolates have positively genes encoding for metallo-β-lactamase (mbls). key words: bla gene, bla gene, meropenem, metallo-β-lactamase (mbls) enzyme, pseudomonas vim imp aeruginosa, rsup dr. m. djamil padang penelitian bertujuan untuk mendeteksi gen bla and bla yang resisten terhadap meropenem pada vim imp pseudomonas aeruginosa dari sampel pasien rawat inap di hcu penyakit dalam rsup dr m. djamil padang. pertama, isolat bakteri p. aeruginosa diisolasi dari sampel sputum pasien yang menderita bronkopneumonia. isolasi diawali dengan penanaman sampel pada media cetrimide agar yang merupakan media selektif untuk bakteri p. aeruginosa. untuk menentukan spesies bakteri, dilakukan identifikasi dengan pewarnaan gram, uji tsia, uji sitrat, uji urease, uji mr/vp, dan deteksi gen penanda 16s rrna. hasil isolasi dan identifikasi menunjukkan dari 20 sampel sputum pasien hanya 10 (50%) sampel positif mengandung bakteri p. aeruginosa. dari isolat yang positif p. aeruginosa dilanjutkan dengan pendeteksian gen bla dan bla penyebab resisten vim imp terhadap meropenem dengan menggunakan pcr. hasil penelitian menunjukkan bahwa semua isolat p. aeruginosa positif memiliki gen yang mengkode enzim metallo-β-laktamase (mbls). kata kunci: enzim metallo-β-lactamase (mbls), gen bla , gen bla , meropenem, pseudomonas vim imp aeruginosa, rsup dr. m. djamil padang determination of bla and bla resistant genes againts meropenem of vim imp pseudomonas aeruginosa isolated from hcu bronkopneumonia inpatients at internal medicine rsup dr. m. djamil padang 1 2 2 ringga novelni *, marlina , and raveinal 1 biomedical laboratory faculty of medicines, universitas andalas, jalan perintis kemerdekaan, padang 25127, indonesia; 2 faculty of pharmacy, universitas andalas, limau manis campus, padang 25163, west sumatera, indonesia tubes, and endotracheal tubes (adisasmito et al. 2006, jamshidi et al. 2009). in surgical wards of dr. m. djamil padang hospital the prevalence of p. aeruginosa as the cause of quiet high infectious around 7.32% in 2010 (lestari et al. 2010) and 6.7% in 2011 (raihanna et al. 2011). besides, based on the resistance test result, those bacterial isolates have resistance to the various of antibiotics like ceftriaxon, ceftazidime, ciprofloxacin, and meropenem (lestari 2010; raihana 2011). imipenem and meropenem are the antibiotics that often used to medicate the infection that caused by the multiresistance bacteria in icu and wards inpatients who need the intensive care. furthermore, karbapenem is selective antibiotics to cure the infection that caused by p. aeruginosa. however, resistance prevalence of p. aeruginosa to carbapenems increase in all around the world (kali et al. 2013). the number of meropenem and imipenem resistance prevalence to p. aeruginosa were 22.16% and 17.32% respectively (gupta et al. 2006). based on mentioned back ground, therefore in this research we tried to determine the resistant genes against resistance to meropenem from p. aeruginosa isolated from the inpatients at internal medicine hcu of rsup dr. m. djamil padang with pcr method. this research is useful for the health workers in order to be able to handle and prevent the resistance in the future with using wisely antibiotics to the patient so the patients will get the best and effective medication. materials and methods sampling. sampling was conducted prospectively to the in patients at internal medicine hcu of rsup dr. m. djamil padang. p. aeruginosa cultures were isolated from sputum sample of 20 bronkopneumonia inpatients. other inpatients with incomplete medical records and those unable to take his/her sputum because of their condition were excluded. bacterial isolation. the isolation was started with the sample cultivation to the cetrimide agar media which the selective media to p. aeruginosa bacteria. to determine the bacteria species, the following methods gram colouring, triple sugar iron agar (tsia) test, citric test, urease test, methyl red/voges-proskauer (mr/vp) test, and detection of 16s rrna gene have been done. dna extraction. dna extraction was performed ® based on protocol of vivantis kit. several colonies of p. aeruginosa were obtained from cultures which were 130 novelni et al. microbiol indones prepared in cetrimide media, then added to eppendorf tubes which contained 500 µl pbs solution. the samples were centrifuged for 2 min at 13 000 rpm. the precipitate were added with 100 µl buffer r1 and 10 µl lysozyme, then homogenized and incubated for 20 min at 37 °c. after the incubation, the samples were centrifuged again for 1 min at 13 000 rpm. then, 180 µl buffer r2 and 20 µl proteinase k were added. the samples then homogenized and incubated at 65 °c for 20 min. after the incubation, 20 µl rnase was added. then, the samples were homogenized and incubated at 37 °c for 5 min. the samples then added with 400 µl buffer rg, then homogenized and incubated at 65 °c for 10 min (repeated 2 times). after the incubation, 200 µl cold ethanol 96%, then homogenized (repeated 2 times). after the homogenization, dna binding was performed. the samples then centrifuged at 12 000 rpm for 1 min (repeated 2 times). the supernatant was removed, then dna pellets were added with 650 µl wash buffer, then centrifuged at 12 000 rpm for 1 min. the precipitate was obtained, then centrifuged at 12 000 ×g for 1 min. the precipitate was added with 100 µl elution buffer, then leaved for 15 min. the samples were centrifuged at 13 000 rpm at 3 min. the supernatant was obtained, then stored at -20 °c. pcr amplification for 16s rrna. the primers were used to amplify 16s rrna fragment specific for p. aeruginosa are 16s rrna -f (5’-gacgggtgag taatgcct-3’a) and 16s rrna -r (5’-cactggtg ttccttcctata-3’), which amplifies 617 bp. the running condition per cycle are 95 °c for 2 min for hot start followed by 30 cycles (92 °c for 60 sec, 57 °c for 1 min or 30 sec depends on the primer, and 72 °c for 1 min), the last extension is 72 °c for 10 min. the pcr products were analyzed by electrophoresis on agarose gel 1.5% and red gel. determination of antibiotics resistance. as much as 2 ose of test bacteria was suspended into physiological nacl in the sterile test tube and homogenized with vortex. then it was compared of its muddiness from the suspension with mcfarland standard. bacterial suspension was taken with sterile cotton swab and cultivated to mueller hinton agar media with spreading evenly on the media, then meropenem antibiotics disc (10 µg) was placed carefully on the media and pressed slowly with sterile tweezer so it would contact to the bacteria on the media. space between disc and the petri dish edge was 15 mm and space among discs was 24 mm. the culture was incubated at temperatures 37 °c for 24 h. characterization was done with measuring and comparing the obstacle area diameter against the standard table. sensitive (s) and resisten (r) against the antibiotics was concluded based on the obstacle clear area diameter around the antibiotics disc. phenotype detection of mbls. the bacteria suspension was spreaded evenly on the mueller hinton agar media. then, the meropenem disc (10 µg) and meropenem disc (10 µg ) + 10 µl edta 0,5 m placed carefully on the bacteria culture. the culture was incubated at temperature 37 °c for 24 h. there is mbls activity if the deviation of meropenem disc obstacle diameter (10 µg) + 10 µl edta 0.5m ≥ 7 mm than meropenem disc obstacle diameter (10 µg). mbls genotype detection (bla , bla ) using vim imp polymerase chain reaction (pcr) method. in one eppendorf tube containing a mixture of mixture 2 µl template dna, 12.5 µl go taq mastermix, 1 µl forward and reverse primers. the steril water was added to make 25 µl volume. the sequence of the primers used are bla -f (5’-gtccgtgatggtgvim atgagt-3’) and bla -r (5’-attcagccagatcvim agcatc-3’) for bla gene, and bla -f v i m i m p catggtttggtggttcttgt and bla -r i m p ataatttggcggactttggc for bla gene, imp which amplifies 437 bp and 448 bp. the running condition per cycle are 94 °c for 4 min for hot start followed by 30 cycles (94 °c for 30 sec, 52 °c for 1 min or 30 sec depends on the primer, and 72 °c for 1 min), the last extension is 72 °c for 5 min. the pcr products were analyzed by electrophoresis on agarose gel 1.5% and red gel. results from the sputum sample isolated from 20 inpatients who suffered bronkopneumonia at internal medicin hcu of rsup dr. m. djamil padang, 10 samples (50%) were positively contained isolates p. aeruginosa. the existence of these bacteria are also shown by the results of 16s rrna gene identification using the pcr method. the positive result of 16s rrna gene were obtained, which is spesific for the identification of gram negative bacteria, one of which is p. aeruginosa (fig 1). based on the resistance test result of 10 bacterial cultures against the meropenem antibiotics showed that all p. aeruginosa isolates were resistant to meropenem (table 1). the p. aeruginosa of isolates were able to produce metallo-β-lactamase (mbls) enzyme, as shown from mbl phenotype detection result, where the deviation of meropenem disc obstacle diameter + 10 µl edta 0.5 m larges than 7 mm, bigger compared to meropenem disc obstacle diameter. meropenem disc obstacle diameter was about 5-6 mm and meropenem disc obstacle diameter + 10 µl edta 0.5 m was about 20-26 mm (table 2). by the detection result of isolates resistance cause gene of p. aeruginosa against the meropenem antibiotics with pcr, there were obtained 10 positive cultures of bla and bla gene (fig 2).vim imp discussion the samples that were used in this research were volume 9, 2015 microbiol indones 131 samples p. aeruginosa culture obstacle area diameter (mm) obstacle area diameter standard (mm) resistance ≤ intermediate sensitive ≥ sample 1 r e s i s t a n c e 13 14-15 16 sample 2 13 16 sample 3 13 16 sample 4 13 16 sample 5 13 16 sample 6 13 16 sample 7 13 16 sample 8 13 16 sample 9 13 16 sample 10 13 16 14-15 14-15 14-15 14-15 14-15 14-15 14-15 14-15 14-15 table 1. resistance test data of pseudomonas aeruginosa against meropenem antibiotics microbiol indones132 novelni et al. sample meropenem disc obstacle diameter (mm) (a) meropenem disc obstacle diameter + edta (mm) (b) mbl positive if b ≥ 7, a ≥ sample 1 6 20 + sample 2 5 24 + sa mple 3 5 21 + sample 4 5 22 + sample 5 5 23 + sample 6 5 21 + sample 7 5 20 + sample 8 5 22 + sample 9 5 22 + sample 10 5 26 + table 2. test result of pseudomonas aeruginosa phenotype bacteria 617 bp 100 bp fig 1 617 bp pcr products of 16s rrna which was specific for pseudomonas aeruginosa were identified in all samples in 1.5% agarose gel electrophoresis. fig 2 photo of agarose gel that showed bla gene (left) and bla gene (right) that were mbls marker gene of pseudomonas vim imp aeruginosa bacteria which was isolated from sputum sample. volume 9, 2015 microbiol indones 133 citric test, because of this bacteria was able to use citric as its only one carbon source. on the urease test of p. aeruginosa bacteria showed the positive result and that meant that this bacteria could produce the urease enzyme which could decompose the urea micromolecule ((nh ) co) to be 2 2 carbondioxyde (co ) and ammonium (nh ). 2 3 ammonium was the nitrogen source that used to biosynthesize amino acids and another molecule that contained n atom. ammonium that was produced would increase media ph to over 6.8 so there would be media changing from pink to yellow. methyl red (mr) test was used to determine the existence of mix acid product from glucose fermentation through the mix acid fermentation track that were generally lactic acid, acetic acid, formic acid, and succinic acid. on the other hand, voges-proskauer (vp) test was to determine the existence of acetoin that was neutral metabolites from glucose fermentation through the butanediol track (brown 2001). in methyl red (mr) test, the positive result was shown with red ring formation to the media by adding the red menthyl indicator as the effect of media ph decreasing because the acid products were produced in a big number from glucose fermentation. the negative result would be obtained if there was no red ring formation and also it indicated that there was a little or none of organic acid remained in the media. this thing was happened because those acids has been changed to be the neutral products that could be detected with voges-proskauer (vp) test (brown 2001). in the glucose fermentation through the butanediol track, there was resulted that next was changed to be acetolactate (acetylmethylcarbinol) and carbondioxide. acetolactate was enzymatically changed to be acetoin or first oxidized to be diacetyl, then reduced to be acetoin. neutral acetoin was reduced to be butanediol next (brown 2001). in voges-proskauer (vp) test, there was added the reagents of barritt’s a (α-naphtol) and barritt’s b (potassium hidroxide). the positive result was shown by the red complex which formed from the reaction between alkaline diacetyl and creatinine (brown 2001). acetoin would react with α-naphtol reagent and potassium hydroxide to form the red ring in the media. p. aeruginosa provided the negative result in voges-proskauer (vp) test and positive result in methyl red test. after the gram colouring and biochemistry test, then inspected the genetic characterization with pcr method. this aims to make sure that growth isolates in cemitride agar media was p. aeruginosa bacteria. this inpatient’s sputum who suffered bronkopneumonia hospitalized at internal medicine hcu wards of rsup dr. m. djamil padang. the isolation was started with sample cultivating to the cetrimide agar media as the selective media for p. aeruginosa bacteria. cetrimide was the quarter ammonium salt that could hamper another bacteria growth with denaturation its bacteria cell protein (king 1954). p. aeruginosa produced piosianin pigment, so there would be green colour on the cetrimide agar media. the isolates which were grown from the selective media, were confirmed with identification test, such as gram colouring and biochemistry test. the gram colouring was used to determine the type of gram positive and negative bacteria. p. aeruginosa bacteria was gram negative bacteria. gram negative bacteria would become red because of lipid on its cell wall dissolved in time of abstersion with alcohol so the pores and cell wall permeability would be bigger and cause release of violet crystal complex which was absorbed previously, and gram negative bacteria was red after it has been given safranin. from observation result under microscope of gram colouring, there were negative red gram bacteria that rod shaped or bacilli, those ends were spherical or oval, pairs or sometimes chain-shaped that suspected as p. aeruginosa. the biochemistry test was used to determine the species of bacteria (lay 1994). the first test was triple sugar iron agar (tsia) test. reaction in the tsi agar media was used to differentiate enteric organisms based on the ablity of fermentation of glucose, sucrose, and lactose. the media which was used contained glucose, sucrose, and lactose as sugar that would be fermented, phenol red as ph indicator, sodium thiosulfate as sulfur source, and ferrous sulfate as h s 2 indicator (duncan 2005). the bacteria which was able to ferment glucose would form yellow on the base part of media, while the bacteria which was able to ferment lactose and/or sucrose would formed yellow on the slant part (brown 2001). p. aeruginosa was only able to ferment glucose to acid in the medium so there was a colour changing of phenol red indicator to be yellow on the base part and red on the slant part because of alkaline reaction. citric test was used to see the ability of microorganisms used citric as its one and only carbon source. citric utilization based on the activity of citrase enzyme that was produced by bacteria. the positive result was shown with the existence of bacteria growth and colour changing of media from green to blue. p. aeruginosa bacteria provided the positive result to 2010; adisasmito 2006). this was one of the high prevalence cause of bacteria which multi drug resistance (mdr) in the hospital, especially in the intensive care unit like hcu. one of the third generation of antibiotics from the carbapenem class that potential empirically and definitively fight against serious infection that caused by multi drug resistance (mdr) was meropenem. moreover, this antibiotics was the selective antibiotics to medicate the infection by p. aeruginosa. however, the bacteria resistance prevalence of p. aeruginosa against the carbapenem has increased in the whole world (kali et al. 2013). enzyme that was produced by p. aeruginosa was one of the main cause of resistance to the meropenem antibiotics. this enzyme was able to hydrolize βlactam ring from the antibiotics class of β-lactam including meropenem, so this cause disfunction of antibiotics (amudhan et al. 2011). to know whether p. aeruginosa produce the mbls enzyme or not the phenotype detection has been done. this process was started with making bacteria suspension then it was spreaded evenly on the mueller hinton agar media. thereafter, they were placed carefully on the bacteria culture, meropenem disc and meropenem disc + 10 µl edta 0.5m. then it was incubated at temperature 37 °c for 24 h. if the deviation of meropenem disc obstacle diameter + 10 µl edta 0.5m ≥ 7 mm than meropenem disc obstacle diameter (erfani 2013), then there is mbls activity based on the mbls phenotype detection that has been done, there was result that all cultures of p. aeruginosa bacteria that has been isolated from positive sputum samples produced mbls enzyme. this event was shown with the deviation of meropenem disc obstacle diameter + 10 µl edta 0.5m ≥ 7 mm than meropenem disk obstacle diameter. mbls enzyme was one of the cause p. aeruginosa resistance against meropenem antibiotics. encoding gene of mbls enzyme that cause resistance against meropenem mostly detected from p. aeruginosa were bla dan bla gene (doosti et al. vim imp 2013). for the correct and effective medication to the infectious diseases patients who caused by p. aeruginosa which could result the mbls enzyme, we have to know the resistance gene type from that p. aeruginosa isolates. resistance gene type from p. aeruginosa isolates was detected with pcr method. this ampilification process was done at the temperature and reduplication cycle that has been determined and could be set on the pcr machine. the pcr result showed that 10 pure et al. metallo-β-lactamase (mbls) pcr method was used to detect 16s rrna gene sensitively and specifically. this gene was one of the genetic regulatory factors and specific gene of negative gram bacteria (hasan 2012). from the detection of gram colouring result and biochemistry test, it can be concluded that 10 of the growth samples in cetrimide agar media were p. aeruginosa. p. aeruginosa was opportunistic bacteria that utilizing the damage of host defense mechanism to begin the infection, so this was a threat to the immunocompromised patients, especially in intensive care unit (hcu) that commonly with the severe disease and immunocompromised. infection by p. aeruginosa associated significantly with bad prognosis from patient who was in treatment in the hospital, where this could increase the probability of another infection and aggravating the exist infection (adisasmito et al. 2006). patients, especially in intensive care unit (hcu) that commonly with the severe disease and immunocompromised. infection by p. aeruginosa associated significantly with bad prognosis from patient who was in treatment in the hospital, where this could increase the probability of another infection and aggravating the exist infection (adisasmito et al. 2006). hcu inpatients generally get the atibiotics therapy in a long time. the longer patients get the antibiotics therapy, there will be easier to onset the colonization with microbes that have antibiotics resistance. besides, usually patients get the antibiotics therapy including ceftriaxone, ciprofloxacin, azithromycin, and meropenem. antibiotics exposure especially the third generation of cephalosporins like ceftriaxone, would spur the production of β-lactamase enzyme (waterer et al. 2001). one of the β-lactamase enzyme was metallo-βlactamase (mbls) enzyme that was produced by p. aeruginosa. 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phone: +62-21-7560729, email:silvaabraham@yahoo.com natural products derived from microorganisms have been used for insect control. most of the pesticides from microorganisms have been isolated from entomopathogens and the terrestrial environment (brakhage and schroeckh 2011). metabolites from fungal genera, such as metarhizium, trichophyton, chrysosporium and lagenidium, as well as some actinomycetes, and several basidiomycetes, have shown potential insecticidal activity (bucker et al. 2013). actinomycetes, the gram positive filamentous bacteria, constituting a significant component of the microbial population in most soils. among actinomycetes, around 7600 compounds are produced by streptomyces species (balachandran et al. 2015; velayudam and murugan 2015). marine environment is still under-explored and is considered to be a prolific resource for the isolation of less exploited microorganisms. recent study on mangrove and marine microorganisms have focused mainly on the discovery of human drugs, whereas limited information about mangrove microorganisms possessing insecticidal activities have been reported mangrove fungi are known as sources of biological active compounds. the study and the report of secondary metabolites of mangrove fungi as insecticides is very limited in indonesia. this study assess the insecticidal activities of ethyl acetate extract of indonesian mangrove fungus emericella nidulans bpptcc 6038 against spodoptera litura neonate larvae and pupae. the fungus e. nidulans bpptcc 6038 was isolated from leaves of mangrove rhizophora mucronata and identified based on its rdna sequence data, with the genbank accession number kp165435, and confirmed with morphological observation. this fungus strain was grown on malt extract broth for 14 days on rotary shaker at 65 rpm, and incubated at room temperature. mortalities of s. litura were observed on larvae fed on artificial diet containing ethyl acetate extract of e. nidulans at concentrations of 625-5 000 ppm. the lethal concentration of the extract which causes 50% mortality of larvae (lc value) was 1 102.27 ppm. the other effects of fungus extract on s. litura were decrease in growth rate, 50 longer larval period, inhibition on pupal development and absence in adult emergence. the hplc analysis of extract showed that the crude extract contained three major compounds. this study provides evidence that the extract of e. nidulans possesses insecticidal activities against s. litura. keywords: emericella nidulans, insecticidal activity, mangrove fungi, spodoptera litura kapang mangrove telah dikenal sebagai sumber senyawa aktif. penelitian dan laporan mengenai aplikasi senyawa metabolit sekunder kapang mangrove sebagai insektisida di indonesia masih sangat terbatas. penelitian ini mengkaji aktivitas ekstrak etil asetat kapang mangrove emericella nidulans bpptcc 6038 terhadap larva neonate dan pupa spodoptera litura (lepidoptera, noctuidae). kapang e. nidulans bpptcc 6038 diisolasi dari daun tumbuhan mangrove rhizophora mucronata dan diidentifikasi berdasarkan data sekuens its rdna dengan kode akses genbank kp165435. kapang ditumbuhkan dalam medium malt extract broth selama 14 hari dengan agitasi 65 rpm pada suhu kamar. mortalitas larva s. litura diamati pada perlakuan dengan penambahan ekstrak etil asetat yang dihasilkan oleh e. nidulans ke dalam pakan buatan dengan konsentrasi 625-5000 ppm. konsentrasi ekstrak yang menyebabkan kematian larva sebesar 50% (nilai 50% of lethal concentration lc50) adalah 1 102,27 ppm. pengaruh lain dari ekstrak kapang e. nidulans terhadap s. litura adalah penurunan laju pertumbuhan, penambahan waktu periode larva, penghambatan pembentukan pupa, dan tidak ada serangga dewasa yang dihasilkan. hasil hplc terhadap ekstrak e. nidulans menunjukan bahwa ekstrak tersebut terdiri dari beberapa senyawa dengan tiga senyawa utama. penelitian ini membuktikan bahwa ekstrak yang dihasilkan oleh kapang e. nidulans memiliki aktivitas insektisida terhadap s. litura. kata kunci : aktivitas insektisida, emericella nidulans, kapang mangrove, spodoptera litura (lepidoptera, noctuidae) insecticidal activities of ethyl acetate extract of indonesian mangrove fungus emericella nidulans bpptcc 6038 on spodoptera litura 1 2 1 silva abraham *, adi basukriadi , suyanto pawiroharsono , 2 and wellyzar sjamsuridzal 1 center for bioindustrial technology, badan pengkajian dan penerapan teknologi, gedung laptiab, puspiptek serpong, tangerang selatan 15314, indonesia; 2 department of biology, faculty of mathematics and natural sciences, universitas indonesia, kampus ui depok, 16424, indonesia (arasu et al. 2013). mangrove fungi are the second group of marine derived fungi which produces new chemical compounds (chen et al. 2011). mangrove associated fungi provide a broad variety of bioactive secondary metabolites with unique structure, including alkaloids, benzopyranones, chinones, flavonoids, phenolic acids, quinones, steroids, terpenoids, tetralones, xanthones, and others (joel and bhimba 2013). new active compounds which isolated from mangrove fungi culture media exhibited toxicity for insects helicoverpa armigera and sinergasilus sp. (chen et al. 2006). several species of aspergillus fungi from the mangrove plants such as aspergillus sp., a. flavus, and a. niger have known produce several active compounds (chaeprasert et al. 2010; chen et al. 2011). abraham et al. (2015) reported that five species of aspergillus and emericella isolated from rhizophora mucronata mangrove plant exhibited larvacidal activity on artemia salina, neurotoxicity on s. litura larvae and acetylcholinesterase inhibition activity. spodoptera litura is the polyphagous insect attacking more than 150 different host species and affect the agricultural crops yield (arasu et al. 2013). s. litura plays a major role in damaging the agricultural crops and therefore considered as the most economically important insect pests in many countries including india, japan, china, and southeast asia. the chemical insecticides usually used for controlling these polyphagous insect. the usage of different varieties of chemical insecticides to control insects has resulted in emergence of insecticides resistance in the pests. due to this reason, many researchers have involved on invention of alternative control methods for insect's pests. microbial insecticides are having advantage over chemical pesticides by its highly effective, safe, ecologically acceptable and pose fewer hazards (arasu et al. 2013; dhanasekaran and thangaraj 2014). these microbial insecticides tend to be highly selective and established as an alternative to eco-destabilizing chemical insecticides especially against lepidopteran insect (dhanasekaran and thangaraj 2014; arasu et al. 2013). this work explores the presence of mangrove fungal secondary metabolite substances with biological properties against s. litura larva with the long term objective for developing them as bioinsecticides. the aim of this work was to evaluate the insecticidal properties of crude ethyl acetate extract of secondary metabolites from the emericella nidulans mangrove fungus against one of the most important polyphagus insect pests, s. litura. 98 abraham et al. microbiol indones material and methods isolation and identification of mangrove fungus. the leaves of mangrove plant r. mucronata were collected in august 14, 2012 at mangrove rehabilitation and ecotourism of prof. dr. sedyatmo angke kapuk, jakarta, indonesia (abraham et al. 2015). the isolation and identification of mangrove fungus were followed the methods described by abraham et al. (2015). the leaf samples were washed in sterile artificial sea water (höller 1999) to remove dust and soil particles and were cut into 1 cm × 1 cm segments using a sterile razor blade. the leaf segments were surface sterilized following the procedures described by ananda and sridhar (2002). the surfacedisinfected leaf segments (1 cm × 1 cm) were pressed on to the surface of pda medium to ascertain the efficacy of surface sterilization procedure (schulz et al. 1993). ten gram of sample was added with 100 ml of sterile aquadest and crushed with blander. the sample suspension was placed on the milipore membrane (sartorius, gottingen, germany; pore size 0.45 µm) in buchner filter apparatus under vacuum condition (vacuubrand gmbh + co type me2, wertheim, germany). the milipore membrane with sample on the membrane surface was placed on the modification of sda media (32.5 g sda-oxoid; 500 -1 ml artificial sea water; 0.5 ml from 0.6 g ml -1 streptomycin; 0.5 ml from 0.05 g ml , tetracycline; -1 -1 0.5 ml from 0.1 g ml dodine; 2.5 m l from 0.05 g -1 ml cyclohexamide) and incubated at the room temperature until mycelia appeared. the identification of the fungi was conducted by amplification of the its1 and its4 region of ribosomal dna. fungus dna was extracted from mycelium tm using kit from prepman ultra (applied biosystems, foster city, ca, usa). the obtaining dna was amplified using the polymerase chain reaction (pcr) apparatus (bio-rad mycycler thermal cycler, bio-rad laboratories, inc., hercules, ca, usa) with its1 and its4 primers (kapa2g robust hotstart readymix, kapa biosystems, inc., wilmington, ma, usa). the amplification cycle consisted of an initial denaturation step of 95 °c for 1 min followed by 40 cycles of (i) denaturation (94 °c for 1 min), (ii) annealing (60 °c for 1 min), and (iii) elongation (72 °c for 1 min), and a final elongation of 72 °c for 5 min (michaelsen et al. 2006). the pcr products were sequenced using an automated multicapillary dna sequencer (abi prism 310 genetic analyzer, applied biosystems, foster city, ca, usa). the sequences data were aligned with sequences from the dna genbank hosted by ncbi (http://blast.ncbi.nlm.nih.gov) using blastp tool. the sequence data of its rdna of the fungus strain was deposited into genbank under the accession number kp165435. the macroscopic and microscopic observation on fungus morphology was conducted to confirm the molecular identification. culture of fungus 500 ml conical flask containing 180 ml fresh me medium 5 g peptone; 1 000 ml artificial sea water) and incubated for 14 d on rotary shaker (heidolph unimax 2010, heidolph instruments gmbh & co., schwabach, germany) at 65 rpm and kept 100 ml of ethyl acetate using separation funnel. the water fraction (upper layer) was collected and re-extracted (three times) with ethyl acetate. the ethyl acetate fraction (bottom layer) was collected and the (heidolph laborta 4000efficient, heidolph instruments gmbh & co., schwabach, germany). insect collection and rearing. larvae of s. litura were collected from the villager's capsicum and corn plant in bogor (bojong, semplak, leuwi liang and ciapus), west java, indonesia and reared in a plastic box container and fed with artificial diet (supriyono, 1997) comprised of dried yeast powder the laboratory reared larvae were used for bioassay and the s. litura culture was maintained throughout the study period. larvacidal activity of the fungus secondary metabolite. larvacidal activity of fungus secondary metabolite was evaluated using feeding nochoice method described by supriyono (1997) -1 -1 ml negative control and deltametrhin 25 g ml (decis, bayer cropscience, jakarta, indonesia) was fermentation and extraction of fungus secondary metabolite. the fermentation and extraction of fungus metabolites were followed the procedures described by abraham et al. (2015). the strain was cultivated in (30 g malt extract; at room temperature. the culture was filtered through whatman no.1 filter paper to obtain the aqueous filtrate. the aqueous filtrate was extracted with solvents were removed by vacuum rotary evaporation 150 g of soya bean (soaked in 460 ml of aquadest for 24 h; 3 g of l-(+)-ascorbic acid; 3 g of nipagine (p-hidroxyibenzoacidethylester), 11 g of ; 180 mg of gentamicyn sulphate; 1 ml of paraformaldehyde; 10 g of agar and 315 ml of aquadest. dietary . the freeze dried artificial diet powder (0.735 g) in 2.2 ml of agar solution (78 mg of agar in 2.2 ml of aquadest) supplemented with fungus ethyl acetate extract in different concentrations (5, 4.5, 4, 3.5, 3, 2.5, and 1.25 mg used as positive control. the experiment was performed at room temperature for 6 d. larval mortality, larval weight and larval consumption of artificial diet were observed and recorded after 6 d of treatment. the larval growth rate was calculated according formula of supriyono (1997): growth rate = the larval mortality was calculated according formula of arivoli and tennyson (2013) and corrections were made when necessary using abbott’s formula. per cent larval mortality = corrected mortality (%) = where %mt = % larval mortality in treatment; %mc = % larval mortality in control. probit analysis (finney 1971) was conducted to calculate median lethal concentration (lc ). one-way 50 analysis of variance (anova) was used to compare the treatment means of feeding dietary bioassay to s. litura larvae. a post-hoc tukey's honestly significant difference (hsd) test, with a significance level of α = 0.05, was performed when a significant difference between treatment means was detected. all statistical analyses were performed using ibm spss statistics ver. 21 software (ibm corp., armonk, ny, usa). larval and pupal durations. the larvae which survived from larvacidal activity treatment (at 5 000 ppm concentration) were continuously fed with normal artificial diet, without ethyl acetate extracts addition, until they became pupae and adults. the larval growth and mortality were observed every day. the larval duration after the treatment was recorded. pupal period was calculated from the day of pupation to the day of adult emergence. preliminary chemical characterization of the active extracts. waters co., milford, ma, usa all amounts of the secondary metabolite extract were loaded onto a c18 column, in 20 the active ethyl acetate extract were analyzed by high performance liquid chromatography (hplc, waters hplc-uv vis detector, ) according the procedure described by abraham et al. (2015). volume 9, 2015 microbiol indones 99 average of weight from viable larvae average of weight from control larvae × 100 (%mt %mc) (100 %mc){ {× 100 number of dead larvae total number of treated larvae × 100 treatment. the addition of ethyl acetate extract to the artificial diet demonstrated the influence of the extracts to the growth rate and larval mortality. the correlation between the concentration of the extracts to larval growth rate and mortality is showing in fig 3. larval and pupal durations. the ethyl acetate extract demonstrated the influence to larval period, inhibition in pupation and adult emergence process. tabel 2 shows the influence of the extract to the time to reach pupation, the number of larvae which reach pupation and the percentage of adult emergence. the hplc profile from ethyl acetate extract of secondary metabolite produced by e. nidulans (fig 4) were exhibited several peaks with three major peaks. the profilles indicated that the extract containing several compounds with three major compounds. discussion isolation and identification of mangrove fungus. based on result from database in ncbi blast tool for its1 and its4 sequences, the fungus isolate was identified as e. nidulans, teleomorph of aspergillus genus. several studies reported that aspergillus fungi like a. flavus; a. niger; a. versicolor and a. nidulans have been isolated from leaves, twig, and root of several mangroves rhizophoraceae e.g. rhizophora mucronata; r. stylosa and r. apiculata and produce the wide variety of secondary metabolites which have activities against human microbial pathogens; cancer hep2 and mcf7 cell lines, and acetylcholinesterase inhibition avtivity (bhimba et al. 2012; chun et al. 2013; abraham et al. 2015). the fungus a. oryzae obtained from the marine red alga preliminary chemical characterization of the active extracts. µl injection volume. elution was performed using a linear gradient consisting of double distilled water (ddh o) and acetonitrile; an isocratic step was initially 2 employed for 3 min at 85% water, followed by a moderate increase in acetonitrile to reach 100% in 20 -1 min, at a flow rate of 1 ml min . the second isocratic step was employed for 5 min with 100% of acetonitrile. results isolation and identification of mangrove fungus. the macroscopic and microscopic observation on fungus colonies exhibited the sexual structures i.e hulle cells, cleistothecia, ascocarp and ascospores, which confirmed the emericella species, a teleomorphic state of aspergillus. the identification of fungus isolate using its1 and its4 region produced sequences with at least 700 nucleotide base pairs. the blast result from ncbi shown the isolate sequences posses 100% homology with e. nidulans species found in the genbank database. the phylogenetic tree which shows the relationship of species with the other species from genbank is showed in fig 1. larvacidal activity of the fungus secondary metabolite. in the present study, ethyl acetate extract from secondary metabolite derived from e. nidulans revealed insecticidal activity against s. litura neonate larvae. table 1 shows mean of insecticidal activity of ethyl acetate extract from each concentration. the lethal concentration of the ethyl acetate extract which causes 50% mortality of larvae (50% of lethal concentration or lc value) was 1 102.27 ppm. fig 250 shows the regression line for the larval mortality induced by each concentration of ethyl acetate extract to monitor the elution profile of secondary metabolites extract, absorption at 254 nm was used. microbiol indones100 abraham et al. ay373888.1 emericella nidulans nrrl-2395 fj878647.1 emericella nidulans uoa/hcpf-10384 fj878641.1 emericella nidulans uoa/hcpf-9186 kp165435 emericella nidulans bpptcc 6038 ab248999.1 emericella dentata ifm-42024 t ay373889.1 emericella quadrilineata atc 16816 hm849679.1 rhizomucor variabilis cbs-384 jn206422.1 mucor fragilis cbs-236.35 100 100 0.1 fig 1 the neighbor-joining phylogenetic tree (shown as a rectangular cladogram) of the mangrove fungus emericella nidulans bpptcc 6038. no. fungus extract mean of mortality (%) s. litura neonate from different extract concentrations (ppm) 5000 4500 4000 3500 3000 2500 1250 625 1. e. nidulans drm3m3 76.67 ± 68.33 ± 55.00 ± 51.67 ± 53.33 ± 58.33 ± 51.67 ± 55.00 ± 12.01 b 4.41 ab 2.89 ab 3.3 ab 1.67 ab 3.33 ab 1.67 ab 2.89 ab 2. no extract (control -) 0 0 0 0 0 0 0 table 1 percent larvacidal activity of ethyl acetate extracts from five mangrove fungi against spodoptera litura (mean ± se) p ro b it 0.6 0.4 0.2 0.0000 -0.2 -0.4 log of concentration 2.50 2.75 3.00 3.25 3.50 3.75 fig 2 the linear regression of probit mortality against log concentration of ethyl acetate extract from emericella nidulans bpptcc 6038 ethyl acetate extract on spodoptera litura larvae. fig 3 effects of emericella nidulans bpptcc 6038 ethyl acetate extract on mortality and growth rate of litura neonate larvae. ( ) mortality (%), ( ) growth rate (%). spodoptera volume 9, 2015 microbiol indones 101 stylosa produced six new dihydroquinolone derivatives along with the related aflaquinolone a and a part of those compounds shown toxic activity against artemia salina (chun et al. 2013). two strains of e. nidulans which isolated from root and leaves of the mangrove plant r. mucronata also shown toxic activity against a. salina (abraham et al. 2015). larvacidal activity of the fungus secondary metabolite. in the present study, ethyl acetate extract from secondary metabolite derived from e.nidulans revealed strong larvacidal activity (76.67%) against s. litura neonate larva at 5 000 ppm concentration and was statistically significant over controttable 1). the ethyl acetate extract from e. nidulans secondary metabolite exhibited constant insecticidal activity for almost concentrations ranges, caused at least 55% larval mortality from 625 ppm to 4 000 ppm (table 1 and fig 2). the study conducted by abraham et al. (2015) reported that ethyl acetate extract from two strains of e. nidulans culture filtrate shown acute toxicity on s.litura larvae. it’s probably due to toxic substances containing in the e. nidulans secondary heterosiphonia japonica produced two new indoloditerpene derivatives, known as tremorgenic mycotoxins, which exhibit potent insecticidal, antiinsectan, and antibiotic activities (qiao et al. 2010). the mangrove fungus a. oryzae isolated from r. mucronata reported produced secondary metabolite that has larvacidal, insecticidal and acetylcholinesterase inhibition activities (abraham et al. 2015). in marine and estuarine environment, aspergillus also the one of fungal genus that often isolated from the different host, from the marine organisms to mangrove plant (nofiani et al. 2012). the endophytic fungus emericella sp. isolated from the mangrove plant aegiceras corniculatum led to isolation of six isoindolones derivatives termed as emerimidine a and b and emeriphenolicins a and d, and six previously reported compounds named aspernidine a and b, austin, austinol, dehydroaustin, and acetoxydehydroaustin with antiviral activity detected in almost compounds (zhang et al. 2011). the asexual state of e. nidulans, a. nidulans which isolated from fresh leaves of the mangrove plant rhizophora no. fungus extract numbers of larval observed larval duration (in days) pupal duration (in days) adult emergence (%) from larvae from pupae 1. emericella nidulans bpptcc 6038 3 > 23 0 2. no extract (control -) 20 12-17 2-5 0 90 0 100 [mau] 150 100 50 0 0 v o lt a g e 5 10 15 20 25 [min]time table 2 effects of the emericella nidulans bpptcc 6038 ethyl acetate extract to litura larval and pupal growth and development spodoptera fig 4 hplc profile from ethyl acetate extract of secondary metabolites produced by emericella nidulans bpptcc 6038. microbiol indones102 abraham et al. after 6 d treatment with artificial diet containing 5 000 ppm extract. between growth stimulating and growth inhibiting hormones ( . . the results obtained from the present investigation suggests that further studies on isolation and identification of the active insecticidal compound and on mode of action needs to develop the promising e. nidulans secondary metabolite as an alternative method or tool for the control of s. litura. arivoli and tennyson 2013) insect growth regulation properties of fungal secondary metabolite extracts are very unique in nature, since insect growth regulator works on juvenile hormone. the enzyme ecdysone plays a major role in shedding of old skin and the phenomenon is called ecdysis or moulting (packiam and ignacimuthu 2012). when the active fungal compounds enter into the body of the larvae, the activity of ecdysone is suppressed and the larva fails to moult, remaining in the larval stage and ultimately dying. the inhibition to the larval growth and development might be due to the interference of toxic substances that were present in the ethyl acetate extracts on the growth and developmental processes of the test insect. hplc profile of ethyl acetate extracts from e. nidulans which shown insecticidal activities exhibited several peaks and three major peaks (fig 4). the profile indicated the several substances with three major substances in the extract. each peak of ethyl acetate extract could be represented the single compound which has different toxicity and its need the further study to identified the each compound and examine toxicities degree of each fraction to obtain the best toxic fraction. through the optimization of fermentation and extraction process, the quantity of the toxic fraction could be increased so the insecticidal activity of the crude extract also could be increased. in conclusion, ethyl acetate extracts of e. nidulans secondary metabolite showed insecticidal activity and inhibition on larval growth and development on s. litura acknowlegments the authors gratefully acknowledge the ministry of research and technology republic of indonesia for providing financial support through the major research project. we appreciate center of exellence indigenous biological resources-genome studies (coe ibr-gs) universitas indonesia for providing facilities to identification process of the fungus isolates in this research. we also wish to express the gratitude to dr. agus supriyono from the badan pengkajian dan preliminary chemical characterization of the active extracts. metabolite. several studies indicate that mostly e. nidulans strains produced styrigmatocystin, the most highly toxic, mutagenic, and carcinogenic compounds which natural products known (frisvad et al. 2004). the study conducted by matasyoh et al. (2011) reported that strerigmatocystin had larvacidal activity against the lc value of the extract was 1 102.27 ppm 50 (fig 2), relatively equivalent with lc values of 50 commercial insecticides chlorpyrifos and deltamethrin (4 180 ppm and 3 990 ppm respectively) against s. litura iii instar larvae (tong et al. 2013). high larval mortality normally indicates potential insecticidal activity of fungal secondary metabolite extract. secondary metabolite compounds act as insecticides by poisoning or by production of toxic molecules after ingestion (jeyasankar et al. 2014). larval mortality may be attributed to direct insecticidal action (as a contact poison) or due to feeding inhibition or gustatory repellency or impairment in the food assimilation (jeyasankar et al. 2014). the growth rate of s. litura neonate larva treated with the fungus extract were lower than the control and generally exhibited the tendency to increased proportionally with the decreased of concentrations. the larval growth rate demonstrated the exception in 2 500 ppm and 1 250 ppm concentrations, the growth rate tend to increased compared with the lower concentrations (fig 3). the increasing of growth rate indicated the attractant substances in the extracts that induced larva to eat the artificial diet containing those substances. the further study, e.g. choice test and detection of volatile compound in the extract which attracts the larva to feed, is required to confirm the possibilities of attractant substances in the extract. larval and pupal durations. after treatment with ethyl acetate extract, the larval developmental period were increased significantly table 2), mostly all of the larvae were not able to go into further instars. the interference of toxic substances in the moulting process triggers the larval duration. larval developmental period was increased in treatment (more than 23 d) when compared to the control (12 to 17 d). the pupation process was inhibited and eventually there was no imago emergence from the metamorphosis process. in general, prolonged in larval duration was directly proportional to the increase in pupacidal activities (arasu et al. 2013). anopheles gambiae third instars larvae. the extension of larval period, failure in pupation and adult emergence could either be due to the presence of toxic ingredients in the extract or the imbalances volume 9, 2015 microbiol indones 103 technol. 102:8179-8184. doi:10.1016/j.biortech.2011. 06.048 chun ya, xiao ml, han l, chun sl, ming hw, gang mx, bin gw. 2013. 4-phenyl-3,4-dihydroquinolone derivatives from aspergillus nidulans ma-143, an endophytic fungus isolated from the mmangrove plant rhizophora stylosa. j nat prod. 76(10):1896-1901. doi:10.1021/np4004646. dhanasekaran d, thangaraj r. 2014. microbial secondary metabolites are an alternative approaches against insect vector to prevent zoonotic diseases. asian pac j trop dis. 4(4):253-261. doi:10.1016/s2222-1808(14)60569-7. th finney d j. 1971. probit analysis. 3 edition. london: cambridge university press. frisvad jc, samson ra, smedsgaard j. 2004. emericella astellata, a new producer of aflatoxin b1, b2 and sterigmatocystin. lett appl microbiol. 38:440-445. doi:10.1111/j.1472-765x.2004.01520.x. höller u. 1999. isolation, biological activity and secondary metabolite investigations of marine-derived fungi and selected host sponge [dissertation]. braunschweig (id): universität carolo-wilhelmina. jeyasankar a, premalatha s, elumalai k. 2014. antifeedant and insecticidal activities of selected plant extracts against epilachna beetle, henosepilachna vigintioctopunctata (coleoptera: coccinellidae). adv entomol. 2(1):14-19. doi:10.4236/ae.2014.21003. joel el, bv bhimba. 2013. biological activity of secondary metabolites isolated from mangrove fungi neurospora crassa. j environ biol. 34:729-732. michaelsen a, pinzari f, ripka k, lubitz w, pinar g. 2006. application of molecular techniques for identification of fungal communities colonizing paper material. int biodet biodeg. 58:133-141. doi:10.1016/j.ibiod.2006. 06.019. nofiani r, brilliantoro r, ardiningsih p. 2012. effect of culture media and incubation time on biological activities from ethyl acetate extract of marine fungus, aspergillus brevipes rk06. drug inv today. 4(6):381384. packiam sm, ignacimuthu s. 2012. effect of ponneem# on spodoptera litura (fab.) and its compatibility with trichogramma chilonis. braz arch biol technol. 5(2):291-298. doi:10.1590/s1516-89132012000200016. qiao mf, ji ny, liu xh, li k, zhu qm, xue qz. 2010. indoloditerpenes from an algicolous isolate of aspergillus oryzae. bioorg med chem lett. 20:56775680. doi:10.1016/j.bmcl.2010.08.024. schulz b, wanke u, drager s, aust hj. 1993. endophytes from herbaceous plants and shrubs: effectiveness of surface sterilization methods. mycol res. 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(lepidoptera: pyralidae) and phytotoxicity on several vegetables. j med plants res. 5(21):5301-5306. tong h, su q, zhou x, bai l. 2013. field resistance of spodoptera litura (lepidoptera: noctuidae) to organophosphates, pyrethroids, carbamates and four volume 9, 2015 microbiol indones 105 1: 97 2: 98 3: 99 4: 100 5: 101 6: 102 7: 103 8: 104 9: 105 05. astuti.cdr vol.11, no.4, desember 2017, p 137-146 doi: 10.5454/mi.11.4.5 potential degradation of sara (saturated, aromatics, resinics, asphaltenes) fractions of crude oil by reservoir indigenous bacteria from south sumatera 1* 1 1 dea indriani astuti , isty adhitya purwasena pingkan aditiawati , , 1 2 3 indiani sani , tutuka ariadji , and muhammad hidayat abqory 1 school of life science and technology, institut teknologi bandung, jalan ganesa 10, bandung 40132, indonesia; 2 faculty of mining and petroleum engineering, institut teknologi bandung, jalan ganesa 10, bandung 40132, indonesia; 3 pt. pertamina ep, jakarta, indonesia. meor (microbial enhanced oil recovery) technology utilizes metabolic activity of microorganisms such as degradation of hydrocarbon fractions which alters oil characteristics to facilitate and increase oil recovery from reservoir. this research focused on isolation of indigenous hydrocarbonoclastic bacteria that were capable of degrading sara (saturated, aromatic, resinic, asphaltenes) fractions of crude oil to be utilized in meor. sequential isolation of hydrocarbonoclastic bacteria were conducted using nutrient broth and stone mineral o salt solution medium supplemented with 2% (v/v) crude oil and 0.1% (w/v) yeast extract and incubated at 50 c, 120 rpm agitation. isolates retrieved were screened based on its activity to degrade crude oil, indicated by resazurin assay result. physical and chemical characteristics of crude oil altered by selected isolates were observed using column chromatography, biometric test, gc-ms analysis, ift, and viscosity measurements. isolates with the best degradation activity were identified with 16s rrna gene sequencing. among thirty-one bacterial isolates obtained from sequential isolation, six isolates exhibited high oil degrading ability. sara assay showed degradation activity of those isolates to each of sara fraction were around 6-70%. this degradation was followed by significant co production ranging from 2000-4000 mg (value of p<0.05). microbial 2 degradation activity exhibited changes in chemical and physical characteristics of hydrocarbons showed by changes in composition of sara fraction, decreased viscosity and ift of crude oil 17-31%. this research identified three isolates with the best hydrocarbon fraction degrading ability were identified as different strain of bacillus licheniformis and confirmed their high potential to be utilized in meor technology. key words: bacillus licheniformis, crude oil, gc-ms, meor, sara teknologi meor (microbial enhanced oil recovery) memanfaatkan aktivitas metabolisme mikroorganisme seperti degradasi fraksi hidrokarbon yang mengubah karakteristik minyak dan meningkatkan perolehan minyak dari reservoar. penelitian ini difokuskan pada isolasi bakteri hidrokarbonoklastik yang dapat mendegradasi fraksi sara (saturated, aromatic, resinic, asphaltenes) minyak bumi yang akan digunakan di meor. bakteri hidrokarbonoklastik diisolasi secara bertahap dengan medium nutrient broth dan stone mineral o salt solution ditambahkan dengan minyak bumi 2% (v/v) dan ekstrak ragi 0.1% (b/v), diinkubasi pada suhu 50 c dengan agitasi 120 rpm. isolat yang diperoleh diskrining aktivitasnya dalam mendegradasi minyak bumi, ditunjukkan dari uji resazurin. karakteristik fisik dan kimia minyak bumi yang berubah oleh isolat terpilih diamati dengan menggunakan kromatografi kolom, uji biometrik, pengukuran ift, viskositas, dan analisis gcms. isolat dengan aktivitas degradasi terbaik diidentifikasi melalui sekuensing gen 16s rrna. tiga puluh satu isolat bakteri diperoleh melalui isolasi bertahap dan enam isolat yang memiliki kemampuan degradasi minyak bumi tinggi. uji sara menunjukkan aktivitas degradasi isolat fraksi sara minyak bumi sekitar 6-70% per fraksi. degradasi diikuti oleh produksi co berkisar antara 2000-4000 mg secara signifikan (nilai p <0,05). 2 aktivitas degradasi mikroba menunjukkan terjadinya perubahan karakteristik kimia dan fisika hidrokarbon ditunjukkan melalui perubahan komposisi fraksi sara minyak bumi dan penurunan viskositas dan ift minyak turun sekitar 17-31%. penelitian ini mengidentifikasi tiga isolat dengan kemampuan degradasi fraksi hidrokarbon terbaik, teridentifikasi sebagai strain bacillus licheniformis yang berbeda dan terkonfirmasi memiliki potensi tinggi untuk digunakan dalam teknologi meor. kata kunci: bacillus licheniformis, gc-ms, meor, minyak bumi, sara microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-22-2511575, fax: +6222-2534107, email: dea@sith.itb.ac.id secondary recoveries and thus should be extracted using enhanced oil recovery (eor) techniques (safdel et al. 2017). chemicals such as surfactants, emulsifiers, polymers, acids, and solvents have been used in tertiary recovery techniques to improve crude it is estimated that over two-thirds of crude oil in a mature reservoir remains untouched after primary and oil yield (patel et al. 2015). however, chemical enhanced oil recovery methods are environmentally hazardous and expensive, and leave non-degradable residues because the chemical pathways through which these products are generated often use toxic chemicals (lazar et al. 2007; gudiña et al. 2012; patel et al. 2015). microbial enhanced oil recovery (meor) is a lowpriced technique with high potential in which different microorganisms and their metabolic products are exploited to recover the remaining trapped oil in reservoir (lazar et al. 2007). meor is widely applicable in different types of reservoir such as sandstone and carbonate reservoirs with light/heavy crude oil and low/mid and high permeabilities (long et al. 2013). the use of microbial cells, and microbial p r o d u c t s s u c h a s b i o s u r f a c t a n t s , b i o f i l m s , biopolymers, and biologically-produced acids and solvents in meor have been shown to improve crude oil recovery. this method has advantages over traditional eor techniques because these bioproducts can be produced with cheaper substrates, and are highly biodegradable and nontoxic (lazar et al. 2007). the main factors responsible for the poor oil recovery are low permeability of some reservoirs and inherent characteristics of heavy crude oil such as high viscosity, which limits its mobility, and high interfacial tension between hydrocarbon and aqueous phases, which results in high capillary forces that entraps oil in small pores within reservoir rock (sen 2008; brown 2010). nowadays, with the rising price of crude oil, declining reserves of medium and light crude oil and the abundance of unconventional crude oil (i.e. heavy oil), causing exploitation of heavy oil is favoured. heavy oil are composed usually of viscous oils, high carbon-to-hydrogen atomic ratios, and display a greater content of asphaltenes and resins than conventional oils (speight 1991), which directly impacts recovery, transport and refining processes. the rheological properties of heavy oils and the characteristics of their reservoirs make its production a challenge in the oil industry. in the past, heavy crude oil production was considered to be uneconomic, because of the intensive cost of recovery, transportation, refining and low market value (santos et al. 2014). thermal methods are common in technologies used for the production of heavy oils (shah et al. 2010). thermal methods are based on supplying heat to the reservoir. in this way, the improvement in oil recovery is mainly due to the reduction of the oil viscosity and, consequently, to the improvement of the mobility ratio. the main processes that use thermal methods for heavy oil recovery are steam methods, like cyclic steam stimulation (css), steam flooding (sf) and steam assisted gravity drainage (sagd) (santos et al. 2014). the major limitation of thermal method is it leaves considerable amounts of oil in the reservoir that can only be recovered by drive processes and it is observed that less than 30% (usually less than 20%) of the initial oil in place can be recovered. these steam methods are also damage the underground well structure and the equipment, endanger lives of the workers, expensive (i.e. add extra operational costs since larger volumes of liquids must be handled), and need an extra dehydration facility to allow for adequate separation of oil and water before oil shipment (muggeridge et al. 2014). therefore, the use of microbial to enhance oil recovery is an alternative way to produce high viscous crude oil with low-priced and environmental friendly. microbial activity can improve sweep efficiency of heavy oil, thus improves oil recovery by lowering crude oil viscosity through degradation and increasing pressure due to gas production. as demonstrated by previous studies, oildegrading bacteria has provided many opportunities to improve oil recovery. the challenge is to find an appropriate isolates that can alter physical and chemical characteristics of crude oil, so that it suitable for use in meor technology. this research aimed to isolate indigenous hydrocarbonoclastic bacteria from in south sumatera's well and further explored its ability to degrade petroleum using sara (saturated, aromatic, resinic, asphaltenes) assay to determine its meor potential. materials and methods o sample. crude oil (oil characteristics: 22-28 api) samples were taken from petroleum reservoir, south sumatera. media. stone mineral salt solution (smss) was used to isolate potential oil-degrading bacteria. the composition of medium was as follows (g/l): nh no 4 3 (2.5); mgso .7h o (0.5); mncl .4h2o (0.2); caco 4 2 2 3 (0.5); na hpo 7h o (1); kh po (0.5) (sharpley 2 4. 2 2 4 1966). the medium was supplemented with 2% (v/v) of crude oil (sterile) as the sole carbon source with the addition of 0.1% (w/v) yeast extract as nitrogen source. bacterial growth was observed in nutrient agar (na) tm difco medium (cappucino 2008). 138 astuti et al. microbiol indones volume 11, 2017 microbiol indones 139 sequential isolation of oil-degrading bacteria. in the first stage of isolation, 2% (v/v) crude oil sample was inoculated into smss media enriched with 0,1% (w/v) yeast extract and 2% (v/v) sterilized crude oil. o the culture was incubated at 50 c with 120 rpm agitation for 7 d. then, the culture was serially diluted 7 -1 to 10 cfu ml , and plated into na and smss agar using spread method. plates of culture were incubated o at 50 c for 24-48 h. emerging colonies were isolated and purified using four way streak method on na and smss plates. in second stage of isolation, the carbon source was substituted with remaining-oil recovery degradated (rod) from the first stage. isolation of stage ii was performed in the same way as the first stage. (halim et al. 2008; munawar et al. 2012). screening of oil-degrading ability using resazurin assay. screening of oil-degrading ability was carried out in two stages. in the first stage, bacterial isolates were initially cultivated in 10 ml of nutrient broth (nb) and incubated for 48 h at 50 °c with 120 rpm agitation. after incubation, the bacterial cell was centrifuged at 15,000 rpm for 10 min to obtain pellet. the pellet was washed with 5 ml of stone mineral salt solution (smss), centrifuged for another 10 min, and resuspended in 4 ml of smss medium. resazurin assay was conducted by mixing 10 ml of smss medium, 1 ml sterilized crude oil, 1 ml of resazurin, and 50µl of bacteria isolate. color change of resazurin from blue, pink to colorless indicate degradation rate. the isolates were incubated for 7 d in the incubator o shaker at 50 c and agitated 120 rpm. (modified from benedek et al. 2010). growth curve. the growth of isolates with high oil degrading ability were studied using total plate count. sampling time was conducted with 12 h interval (cappucino 2008). saturates, aromatics, resins and asphaltenes (sara) separation of crude oil. components of crude oil from each culture (before and after treatment) was separated using silica gel column chromatography (muhammad et al. 2013). total petroleum hydrocarbons (tph) extract was dissolved in 10 ml of hexane and the insoluble fraction (asphaltene) was removed using whatman filter paper and weighed. the soluble fraction was loaded on top of silica gel g (70120 mesh) column (2 cm x 30 cm) and eluted with solvents of different polarities. the alkane fraction was eluted with 20 ml of hexane; aromatic fraction was eluted with 100 ml of toluene, and resinics fraction was eluted with 20 ml of methanol:toluene (90:10). all sample fractions were evaporated and weighed to calculate the weight of each fraction. biodegradation of crude oil in a biometric system for mineralization test. biometric flask was filled with 1 ml of a 48 h old activated inoculum, 28 ml of smss medium and 2% (v/v) hydrocarbon substrate. the sidearm of biometric flask was filled with 10 ml of 0.1 m koh. flasks were incubated at 50 o c with 120 rpm agitation. the culture was sampled every 24 h for 96 h from the sidearm of the flask and measured for its carbon dioxide content with colorimetric titration and gravimetric analysis. the control sample contained 10 ml of koh, 1 ml of barium chloride and 0.1 ml of phenolphthalein. the amount of co produced was calculated using the 2 equation below: co generated(mg) = (vb va) .mco 2 2 co generated(mg) = 2x mhclx cf2 vb = volume of hcl (0.1m) used to titrate the blank (ml) va = volume of hcl (0.1m) used to titrate the treatment (ml) -1 mco2 = molar mass of carbon dioxide (g mol ) -1 m hcl = molarity of hcl standard solution (mol l ) cf = correction factor for acid/base molarity (m hcl/m koh). all the experiments were conducted in triplicate and analyzed statistically (modified from benedek et al. 2010; maier 2008; biodia et al. 2010). gc-ms analysis of residual oil components after biodegradation. liquid medium after biodegradation treatment was used as the sample, and non-inoculated medium without crude oil solution was used as a control. after 7 d of biodegradation, the medium was mixed with petroleum ether and centrifuged, the residual oil components for both the samples and the controls were measured by agilent gcmsd (6980n-5973) with temperature kept at 80 °c for -1 4 min, then increased at a rate of 5 °c·min until 250 °c and maintained at 250 °c for 20 min. identification of isolates using 16s rrna analysis. selected hydrocarbonoclastic bacteria were sequenced using colony-based method. the sequencing used universal primer 785f (5'ccagcagccgcggtaatacg-3') and 907r (5'ta c c a g g g tat c ta at c c 3 ' ) d e s i g n e d b y macrogen, korea. the result was then analyzed using mega 6.0, to generate a phylogenetic tree with neighbor joining method (tamura et al. 2011). the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates). the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were computed using the maximum composite likelihood method (tamura et al. 2004) and are in the units of the number of base substitutions per site. the analysis involved 27 nucleotide sequences. codon positions included were 1st+2nd+3rd+noncoding. all positions containing gaps and missing data were eliminated. there were a total of 1355 positions in the final data set. results sequential isolation of microorganisms and screening of oil-degrading ability using resazurin assay. sequential isolation of hydrocarbon degrading bacteria obtained 12 isolates and 19 isolates from the first stage and second stage of isolation respectively, yielding a total of 31 isolates. both gram negative and gram positive bacteria were observed among the isolates and the majority had rod-like shape. all isolates possessed the ability to degrade and utilize hydrocarbon, but six isolates namely ds1, ds3, ds19, ds31, ds32, and ds44 were found to exhibit higher efficiency of oil degradation as indicated by faster rate of resazurin color change. growth rate and crude-oil degrading of selected isolates. in general, logarithmic phase on 12 h to 48 h periods of incubation, stationary phase on 72 to 84 h and death phase at 96 h. were observed as the growth pattern of all isolates. growth curve measurement was used to determine specific growth rate and the optimum time to use each bacteria as inoculum on further assays. the optimum growth rate was achieve at 12 h for isolates ds1, ds3, ds31 and ds32, and 24 h for isolate ds19 and ds44 (fig 1). separation of sara components of crude oil fractionated by column chromatography. sara assay result is shown in the fig 2. in general, all isolate degraded resin fraction into aromatics and saturated fractions. the highest degradation ability was observed on treatment by ds31 and followed by isolate ds44, ds3 and ds1. biodegradation of crude oil in a biometric system for mineralization test. based on fig. 3, most of the six tested isolates produced high amount of co gas after 96 h of incubation. the highest co 2 2 production at 96 h incubation was shown by ds31 17000 mg. co production by ds1 7333 mg, ds3 2 11000 mg, ds19 8800 mg, ds32 7333 mg and ds44 5000 mg. crude oil gc-ms analysis. gc-ms analysis results (fig 4) detected new compounds such as (3,5) diosgenin, naphthalene, bicyclo (4.1.0) heptane, 1methyladamantane, 1,6-dimethyl decalin, trans-1,6dimethylspiro (4.5) decane, 2-isopropenyl-5-methyl4-hexenyl acetate, cyclohexene, salvialane, decahydro-4,4,8,9, 10-pentamethylnaphthalene, cyclododecanone, fukinan, benzene, and azulene. interfacial tension and viscosity reduction after incubation experiment. fig 5 shows that biodegradation of heavy oil fractions caused lighter fraction to accumulate, hence reducing oil viscosity and interfacial tension (ift). viscosity reduction in a range of 11 – 22% (p ≈ 0.05) occurred in all treatment and high viscosity reduction was observed in ds1 and ds3 treatment. meanwhile, ift reduction of 21 – 32% (p ≈ 0.05) occurred and high ift reduction was observed in ds31 and ds1 treatment. phylogenetic analysis and identification using 16s rrna sequences. identification of isolates using 16s rrna approach showed that isolate ds1, ds3, and ds31 were most closely related to bacillus sp strain fig 1 growth of bacterials coded ds1, ds3, ds19, ds31, ds32, and ds44 at different periods (h) incubation (p ≈ 0.05). 140 astuti et al. microbiol indones volume 11, 2017 microbiol indones 141 fig 2 abundance percentage of sara fraction after 7 days treatment with selected bacterial isolates coded ds1, ds3, ds19, ds31, ds32, and ds44. fig 3 carbon dioxide released (mg) during biodegradation of crude oil in a biometric flask by isolates coded ds1, ds3, ds19, ds31, ds32, and ds44 at different periods (h) incubation (p 0.05). ≈ fig 4 gc-ms analysis of biodegradation of untreated crude oil and treated crude oil by ds1, ds3 and ds31 (after 7 -1 days of incubation). based on gc-ms analysis, gained tph content of control approximately 16,072 (µg g ) by calculation of residual saturated and aromatic fractions (showed by peak chromatogram). tph content of -1 -1 -1 treatment by ds1, ds3 and ds31 sequentially are 7,510 (µg g ), 8,040 (µg g ), and 8,020 (µg g ). the highest peak chromatogram is defined as internal standard on retention time 12 min (showed by black circle). fig 5 interfacial tension and viscosity reduction (%) after seven days incubation experiment (p 0.05).≈ fig 6 phylogenetic tree of isolates ds1, ds3 and ds31 based on 16s rrna gene sequencing analysis constructed by neighbor-joining. isolates coded ds1, ds3 and ds31 were identified using blast. based on blast result from database ncbi (http://www.ncbi.nlm.nih.gov) showed that isolate ds1 has identity 99% and query cover 100% (nr118996.1), isolate ds3 has identity 99% and query cover 99% (kr018738.1), and isolate ds31 has identity 99% and query cover 99% (jx068644.1). 142 astuti et al. microbiol indones volume 11, 2017 microbiol indones 143 klp1 (fig 6). based on phylogenetic tree constructed, it could be seen that isolate ds1, ds3, and ds31 were present in the same clade as bacillus licheniformis (fig 6). discussion petroleum is a complex mixture of hydrocarbon and is generally classified into four groups: saturates, aromatics, resins, and asphaltenes. the convenience of microbes in degrading four classes of hydrocarbon fractions also varies. the vulnerability of microbial to degrade hydrocarbons can be generally ranked as follows: linear alkanes > branched alkanes > small aromatics > cyclic alkanes. some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons (pahs), may not be degraded at all (das and chandran 2011). therefore, biodegradation of crude oil could only be completed by different microbes with special functions. sequential isolation was performed to obtain hydrocarbonoclastic bacteria with various ability to degrade different fractions of oil. the results of sequential isolation from of south sumatera's well were grouped according to the stages of isolation. stage i was expected to isolate light oil fraction-degrading bacteria while stage ii was expected to isolate heavy oil fraction-degrading bacteria. stage i and ii obtained 12 and 19 isolates respectively. resazurin has a non-flouresen blue color that can be reduced to pink. the use of this dye is based on its ability as an intermediate electron acceptor in the electron transport chain without interrupting normal electron transport. resazurin assay indicated oil degrading ability because this assay could screen oxygenase enzyme activity of hydrocarbonoclastic bacteria. when o in growth media decreases as it is 2 used by enzyme oxygenase in degradation hydrocarbon fraction, resazurin will be reduced and change color from purple to nones. this indicates high oxygenase enzyme activity in biodegradation process of hydrocarbon fraction. screening test by resazurin assay indicated that ds1, ds3, ds19, ds31, ds32 and ds44 have high oil degrading ability because they could use oxygen as inisiator for hydrocarbon oxidation by oxygenases complexes enzyme. the initial intracellular attack of hydrocarbon is an oxidative process and the activation as well as incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases enzyme. peripheral degradation pathways convert hydrocarbon step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle (benedek et al. 2010). crude-oil degrading ability was significantly affected by growth of oil-degrading bacteria. fig 1 and fig 2 showed that there was a correlation between optimum growth-rate and degradation of sara fraction, especially for degradation of heavier crude oil fraction. mostly, all selected isolates, exhibited the same pattern of crude oil degradation. firstly, the isolates degraded the most readily degradable hydrocarbons. as their concentration decreased and only heavy oil fraction remained, the degradation rate slowed down. therefore, the composition ratio of hydrocarbons changes when degradation rate of some oil hydrocarbons exceeds the degradation of others (gailiūtė et al. 2014). this research exhibited degradation of crude oil altered sara composition of crude oil. sara analysis of crude oil was used to examine the changes of oil fractions before and after degradation. this method can determine the relative content of each fraction in the oil sample (guizhou et al. 2013). degradation of hydrocarbon fractions can be occurred because of other mechanisms involved are (1) attachment of microbial cells to the substrates and (2) production of biosurfactants, besides mediated by spesific enzyme (das and chandran 2011). biosurfactants that produce by microbials can act as emulsifying agents by decreasing the surface tension and forming micelles. the microdroplets encapsulated in the hydrophobic microbial cell surface are taken inside and degraded (bordoloi and konwar 2009). as seen on fig 5 there is an interfacial tension (ift) reduction of crude oil after incubation experiment treated by six tested isolates. reduction of ift can occurred through production of biosurfactant by each isolates to mediated degradation of insoluble hydrocarbon fraction. isolate ds31 that was obtained from isolation ii showed highest ability to degrade resins and asphaltenes fraction, leaving only few resins fraction and less asphaltenes fraction unutilized (fig 2). in addition, isolate ds1 and ds3 showed high ability to degrade aromatic fraction and were able to increase the accumulation of saturated fraction after degradation of heavy hydrocarbon fractions. accumulation of light crude oil fraction reduce oil viscosity (fig 5), which increase its mobility to production well. from this research, known that degradation of crude oil can affect chemical and physical characterization of crude oil (i.e. increment of light crude oil fraction and declining of ift and viscosity crude oil). chemical change of crude oil was observed through mineralization test using biometric system and gc-ms analysis. fig 3, showed that isolate ds31 produced the highest concentration of co (17600 mg). 2 c o p r o d u c t i o n b y h y d r o c a r b o n o c l a s t i c 2 microorganisms is produced through mineralization of hydrocarbon by microbial extracellular enzyme i.e. monoand dioxygenases (maier 2008). fig 4, showed that new saturated and aromatics compounds. these new compounds are calculated and gained lower total petroleum hydrocarbon than untreated crude oil. based on chemical and physical characterization test, isolate ds1, ds3 and ds31 were concluded to have the highest oil degrading abilities. phylogenetic analysis, showed the majority of the selected bacteria belonged to different strain bacillus licheniformis (fig 6). bacillus licheniformis is a gram positive bacteria, and has the ability to produce lipopeptide biosurfactant ( et al. garcía-alcántara 2016). previous studies revealed that various species of pseudomonas and bacillus are common inhabitants of petroleum ecosystems (munawar et al. 2012; ismail et al. 2013; guizhou et al. 2013; gailiūtė et al. 2014). however, studies b. licheniformis's ability to degrade crude oil, especially from south sumatera were limited and no reports exist for these strains. bioinformatics analysis using kegg (http://www. genome.jp/) b. licheniformis could degrade benzene, x y l e n e , s t y r e n e , d i o x i n , c h l o r o b e n z e n e , chlorocyclohexane, naphthalene and benzoate compounds. this ability was affected by its ability to produce of biosurfactant. biosurfactants have the ability to reduce surface tension through solubilisation of fatty acids present in crude oil which leads to proficient exploitation of hydrocarbon by microbes. therefore, biosurfactant production allowed the utilization of hydrocarbons by microorganisms, reduction of ift, and implementation of other meor potential in oil industry (bordoloi and konwar 2009; lazar et al. 2007). this great potential has also been investigated by garcía-alcántara et al. (2016) who reported that b. licheniformis consortium was 6 times higher compared with that obtained with five oil-degrading microorganisms: achromobacter (alcaligenes) xylosoxidans, b. cereus, b. subtilis, brevibacterium luteum, and pseudomonas pseudoalcaligenes. this research, demonstrated that sequential isolation method could successfully obtain hydrocarbonoclastic bacteria that can could degrade saturated, aromatic, resin, and asphaltene fractions. in addition, isolates with the highest ability to degrade crude oil ds1, ds3 and ds31, were identified as different strains of the species bacillus licheniformis. high crude-oil degrading ability was confirmed by sara analysis, co production, ift and viscosity 2 measurement and gc-ms analysis. degradation of heavy crude oil (resin fraction) altered physical characteristic of crude oil as indicated by reduction ift and viscosity 17-31% and chemical characteristic of crude oil as seen by an increment of lighter crude oil fractions, and 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mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol biol evol. 28(10):2731-2739. doi:10.1093/molbev/msr121. yan p, lu m, yang q, zhang hl, zhang zz, chen r. 2012. oil recovery from refinery oily sludge using a rhamnolipid biosurfactant-producing pseudomonas. bioresour technol. 116:24–8. doi: 10.1016/j.biortech. 2012.04.024. youssef n, simpson dr, mcinerney mj, duncan ke. 2013. in-situ lipopeptide biosurfactant production by bacillus strains correlates with improved oil recovery in two oil wells approaching their economic limit of production. int biodeterior biodegrad. 81(1):127-132. doi: 10.1016/j.ibiod.2012.05.010. 146 astuti et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 mi641-17-07-12 (malle) available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.2.5issn 1978-3477, eissn 2087-8587 vol 6, no 2, june 2012, p 83-88 *corresponding author; phone: +62-911-322653 ; e-mail: minggus@gmail.com hot springs are a common source of thermophyles which produce thermostable enzymes. the objective of this study was to isolate and identify thermostable amylase-producing bacteria from a local geothermal spring. an amylase–producing bacterium strain was isolated from this hot spring which excreted amylase after being grown on starch agar screening plates at 37 °c. it was identified as bacillus megaterium using the method of 16s ribosomal dna. the organism is a rod-shape and is a spore-forming bacterium. maximum amylase production was achieved after incubation in the production media for 72 h. preliminary analysis of the secreted amylase showed that the enzyme could bind to deae-sepharose matrix and was discharged by eluting with 0.5 m nacl. the partially purified enzyme was stable up to 75 °c, showing that this enzyme might have potential application in the starch-processing industry. bacillus, hotsprings, isolation, thermostable amylase sumber air panas merupakan habitat umum mikroba termofilik yang menghasilkan enzim-enzim termostabil. tujuan penelitian ini adalah untuk mengisolasi dan mengidentifikasi bakteri penghasil amilase termostabil dari sumber mata air panas lokal. satu strain bakteri penghasil enzim amilase berhasil diisolasi. bakteri tersebut menghasilkan enzim amilase setelah ditumbuhkan pada suhu 37 °c pada media agar yang mengandung pati. hasil analisis gen subunit 16s rrna menunjukkan bahwa bakteri tersebut adalah bacillus megaterium. bakteri tersebut berbentuk batang dan menghasilkan spora. produksi enzim amilase maksimum diperoleh setelah bakteri dikultivasi pada media produksi selama 72 jam. analisis pendahuluan enzim yang disekresi menunjukkan bahwa enzim tersebut terikat pada matriks deae-sepharose dan terlepas dari matriks setelah dielusi dengan larutan nacl 0,5 m. amilase dari fraksi nacl 0,5 m tetap aktif hingga suhu 75 °c. hal ini menunjukkan bahwa enzim amilase ini mungkin memiliki potensi untuk digunakan pada industri pengolahan tepung pati. amilase termostabil, bacillus, isolasi, air panas key words: kata kunci: sumber starch is a common reserve energy store of plants and is one of the most abundant biopolymers on earth. it is a polymer of á-glucose units connected with á-1,4 and á-1,6-glycosidic linkages. starch consists of amylose, a linear polymer of glucose, (15-25%) and amylopectin, a branched glucose polymer, (75-85%). due to its complex structures, hydrolysis of starch requires a combination of hydrolytic enzymes to release glucose units and small oligosaccharides (bertoldo and antranikian 2001). to hydrolyze starch completely into 6-carbon glucose units, at least 2 different enzymes are needed, namely, (1) an enzyme which breaks down á-1,4 linkages such as á-amylase, á-glycosidase, and â-amylase; and (2) enzyme that hydrolyzes á-1,6 linkages such as pullulanase and isoamylase (van der maarel et al. 2002). enzymatic hydrolysis (saccharification) of starch is usually carried out at relatively high temperature conditions between o 80-90 c. hence, heat-resistant enzymes are required (burhan et al. 2003). thermostable amylase has various commercial applications related to starch processing including brewing, natural sweetener production, textile and paper production, bioethanol production, and detergent manufacture. this type of enzyme constitutes about 25% of the total enzymes needed in industrial processes (sidhu et al. 1997; pandey et al. 2000). due to its promising applications, the search for new sources of thermostable amylase continues. bacteria have become important sources of thermostable amylase for industrial application because they produce amylases with greater short communication isolation and identification of a thermostable amylase-producing bacterium from hatuasa hotspring 1 2 2 dominggus malle *, junus picarima , laury chara huwae , indra 3 3 rahmawati , and wahyu purbowasito 1 faculty of agriculture, pattimura university, jalan ir m putuhena, kampus poka ambon, ambon 97233, indonesia; 2 faculty of mathematics and natural sciences, pattimura university, jalan ir m putuhena, kampus poka ambon, ambon 97233, indonesia; 3 center for biotechnology , bppt, puspiptek-serpong, tangerang selatan, indonesia agagtttgatcctggctcag-3’ and 1492r (reverse) 5'-ggttaccttgttacgactt-3’ were used to amplify the target gene (gee et al. 2003). pcr amplification was carried out using a pcr thermal cycler (takara, japan) with a thermal profile: denaturation of 96 ºc for 3 min, and 30 cycles of 96 ºc for 45 s (denaturation), 56 ºc for 30 s (annealing) and 72 ºc for 2 min (polymerization), followed by the last cycle of polymerization at 72 ºc for 7 min. the pcr product was electrophoresed on 1% w/v agarose gel and visualized after ethidium bromide staining. the pcr band was excised and extracted using a geneaid kit following the procedure provided. the purified pcr product was sequenced using abi prism 3130x1genetic analyzer (applied biosystem, usa). pcr amplification showed that a fragment about 1500 bp long appeared on 1% w/v agarose gels using its genomic dna as template. sequencing analysis of this fragment resulted in a partial sequence of about 996 bp long. the partial sequence of the 16s rrna gene obtained was compared with known bacterial 16s rrna genes using blast at ncbi , showing 99% homology with bacillus megaterium. prior to phylogenetic analysis, the nucleotide sequence was aligned with other related bacteria using clustalw. an evolutionary distance tree was generated using the neighbor-joining algorithm as implemented in the mega version 5.0 software (tamura et al. 2011). to evaluate enzyme production, 5 ml of fresh culture of the isolate which was grown overnight was transferred into 100 ml of sterilized production media containing 6 g bacto trypton, 0.5 g mgso .5h o, 0.5 g 4 2 kh po , 0.5 g nacl, and 1.0 g starch (vidyalakshmi et 2 4 al. 2009). the production media was incubated at 30 ºc with orbital shaking at 100 rpm for 6 days. a sample (www.ncbi.nih.nlm.gov/blast/) thermostability properties as compared to fungal amylases. in addition, the genera bacillus is the major source for the production of thermostable amylase and they have been widely used in commercial applications (prakash and jaiswal 2010). bacillus megaterium has attracted attention from biotechnologists due to the scientific fact that this bacterium is non-pathogenic and found in the soil and hot water springs. this species has become of special interest because of its promising biotechnological applications such as enzyme production (oyeleke et al. 2010; gurudeeban et al. 2011; sajitha et al. 2011), recombinant protein production (stammen et al. 2010) and recombinant antibody fragment productions (jordan et al. 2007). another advantage of this bacterium is that it does not produce alkaline proteases (rygus and hillen 1991). the province of maluku possesses a lot of hot springs. hence, it is possible that bacteria which can produce thermostable amylase exist there. to our knowledge, there is no investigation yet regarding the study of thermophyles isolated from those locations. in this study, we aimed to isolate and identify a bacterium that produced a thermostable amylase from a maluku hot spring. microbes were isolated from hatuasa hotsprings, tulehu village, district of central maluku. a 50 µl aliquot of sample was spread on luria-bertani (lb) agar (difco) plate sumpelented with 1% w/v of starch (from potato; sigma, usa) as the sole carbon source and then incubated at 37 °c overnight. a colony that produced the largest clear zone after iodine staining was isolated and grown onto another starch-containing lb agar plate for futher isolation and regeneration. the morphological and biochemical characterizations of the amylase-producing isolate were determined by the electron microscope and enzymatic tests, respectively. the isolated bacterial strain was designated as “hat1”. this bacterium has capability of hydrolyzing starch in lb-agar plate containing 1% w/v soluble starch as indicated by the presence of a clear zone on the plate after iodine staining. the colony forming the largest clear zone was taken for further isolation steps to obtain a single colony. the target colony was assayed for its morphological properties which showed that it is a rodshape and spore-forming bacterial strain (fig 1). for further identification, the 16s rrna gene of the isolate was amplified using polymerase chain reaction (pcr) method from the genomic dna. genomic dna was isolated using instagene kit following the manufacturer's instructions. two universal primers for bacterial 16s rrna gene, 8f (forward) 5'84 malle et al. microbiol indones fig 1 morphology of the isolate hat1 showing a rod-shape type under the scanning electron microscope. was taken every 24 h for amylase activity evaluation. amylase activity was determined using the dinitrosalicylic (dns) acid method according to miller (1959) with slight modification. briefly, a reaction mixture containing buffer solution and starch substrate was incubated at 30 ºc for 10 min. then, a 100 µl aliquot of enzyme solution was added and incubation was continued for additional 3 min. the reaction was stopped by the addition of 1 ml of dns solution. subsequently, the mixture was incubated in boiling water for another 10 min. after that, the mixture was put into an iced-water bath. the absorbance of the mixture was measured spectrophotometrically at ë 540 nm. the protein concentration was measured at ë 280 nm and by the method of bradford (1976). the amylase was partially purified using ion exchange chromatography according to the method of scopes (1994). the crude extract of amylase was applied to a deae-sepharose fast flow column (ge life science, england) which had been previously equilibrated with two volumes of 50 mm acetate buffer (ph 5.6). the column was then washed with two volumes of the same buffer to remove all unbound proteins. the bound proteins were eluted fractionally with a linear gradient of nacl from 0-0.5 m. all fractions were analyzed for their protein content and amylase activity was assayed following the methods as described above. the fraction with highest amylase activity was further analyzed for its optimum ph and thermostability properties according to takeuchi et al. (2006). to determine the optimum ph, a series of ph solutions ranging from 4.08.0 were prepared. then, the amylase activity of the fractions was assayed using these prepared buffer solutions. to determine thermostability, the amylase activity of the fraction was assayed at the optimum ph with different temperatures ranging from 40-80 ºc (with a 5-degree scale interval). the amylase secreted by the isolate bound to deae-sepharose fast flow column and almost completely discharged with 0.5 m nacl (fig 2). the amylase had an optimum ph at 6.5. volume 6, 2012 microbiol indones 85 blank ce ub 0.1 m 0.3 m 0.4 m 0.5 m fig 2 amylase activity of the deae-sepharose fractions after dns assay. the 0.5 m nacl fraction shows the highest amylase activity as compared to other fractions. (ce=crude extract; ub=unbound). fig 3 (a) effect of ph on partially-purified amylase activity; (b) effect of temperature at the optimum ph on partially-purified amylase activity. -1 enzyme dropped quickly to 3961.55 u mg protein at 80 ºc (fig 3). the isolate was capable of degrading soluble starch in the isolation media, as indicated by the formation of a clear zone after the iodine test. the clear zone indicated that the starch had been hydrolyzed into simple sugars, thus losing the capability of starch to bind to iodine leading to the disappearance of blue color. morphological analysis revealed that the isolate hat1 at low ph (4.0), the activity of the amylase remained -1 low which was about 2826.47 u mg protein, and this -1 increased to a maximum activity of 4529.14 u mg protein at ph 6.5. however, the activity suddenly -1 decreased to 2712.88 u mg protein at ph 8.0. in terms of temperature, the activity of the amylase which was -1 about 3848.05 u mg protein at 40 ºc and increased -1 until the temperature reached 70 ºc (5550.71 u mg protein). beyond this temperature, the activity of the microbiol indones fig 4 phylogenetic tree constructed using the neighbor-joining method between the isolate hat1 and several related bacillus sp. obtained from genbank. 86 malle et al. is a rod-shaped, gram positive, spore-forming strain. recently, similar work has been undertaken by gurudeeban et al. (2011), who successfully isolated a rod-shaped, spore-forming and gram positive amylase producing bacterium b. megaterium from white mangrove (avicennia marina) leaves. also, sajitha et al. (2011) isolated an amylase producing bacterium b. megaterium from an estuarine ecosystem. these studies prove that b. megaterium is a potential source of bacterial amylase. the blast analysis of the 16s rrna gene sequence confirmed that the isolate hat1 is most closely related to bacillus sp. with 99% of homology. phylogenetic analysis using distance-based method of the known bacterial 16s rrna genes revealed that the isolate has high similarity with bacillus megaterium (fig 4). this has been confirmed that the p-distance among the b. megaterium strains is zero, indicating that they are all closely related. amylase secreted by the isolate was partially purified using ion exchange chromatography on a deae-sepharose fast-flow column. after loading the crude extract solution, the pass-through fraction (eluant) showed very weak amylase activity, indicating that the amylase was bound to the matrix. the binding of the target protein to the column shows that the enzyme protein has a total negative charge at its surface. the bound amylase was almost completely discharged from the column after elution with 0.5 m nacl. this fraction showed highest amylase activity after dns assay among other fractions (fig 2). most of the known bacterial amylases were purified using anion exchange chromatography (shih and labbe 1995; hagihara et al. 2001; ballschemiter et al. 2006). the ph optimum of the enzyme was almost similar to that of the hotspring water (6.7), while the enzyme could retain its thermal stability above the temperature of the hotspring, i.e. 60 ºc. it is clear that beyond the optimum temperature (70 ºc), the activity of the amylase sharply decreased, indicating that denaturation of the enzyme may have begun to occur. this optimum thermal property of the amylase is higher than those of an estuarine b. megaterium, 35 ºc (sajitha et al. 2011) and of b. megaterium from white mangrove leaves which is also 35 ºc (gurudeeban et al. 2011). this thermostability property might be due to fact that the isolated strain inhabits a thermophilic environment as compared to the two former strains. based on the 16s rrna gene sequence analysis, and the multiple sequence alignment with other related bacillus sp, the isolate hat1 is similar to b. megaterium. the partial nucleotide sequence of the 16s rrna gene of the isolate has been deposited in the genbank with the accession number: jn995603. based on our experimental results, the properties of the partially purified enzyme indicate that the enzyme might have potential application in starch processing. however, for scientific reasons, pure enzyme must first be obtained. thus, further studies based on enzyme purification and characterization are recommended. references ballschemiter m, futterer o, liebl w. 2006. identification and characterization of a novel intracellular alkaline áamylase from the hyperthermophilic bacterium thermotoga maritima msb8. appl environ microbiol. 72(3):2206-2211. doi:10.1128/aem.72.3.22062211.2006. bertoldo c, antranikian g. 2001. amylolytic enzymes from hyperthermophiles. methods enzymol. 330:269-289. bradford, mm. 1976. a rapid sensitive method for quantitition of microgram quantities of protein utilizing the principle of protein dye binding. anal biochem. 72(1-2):248-254. burhan a, nisa u, gokhan c, omer c, ashabil a, osman g. 2003. enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic bacillus sp. isolate ant-6. process biochem. 38(10):1397-1403. doi: gee je, sacchi ct, glass mb, de bk, weyant rs, levett pn, whitney am, hoffmaster ar, popovic t. 2003. use of 16s rrna gene sequencing for rapid identification and differentiation of burkholderia pseudomallei and b. mallei. j clin microbiol. 41(10): 4647-4654. doi:10.1128/jcm.41.10.4647-4654.2003. gurudeeban s, satyavani k, ramanathan t. 2011. production of extracellular á-amylase using bacillus megaterium isolated from white mangrove (avicennia marina). asian j biotechnol. 3(3):310-316. doi: hagihara h, igarashi k, hayashi y, endo k, kitayama ki, ozaki k, kawa s, ito s. 2001. novel á-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic bacillus isolate ksmk38. appl environ microbiol. 67(4):1744-750. doi: jordan e, hust m, roth a, biedendieck r, schirrmann t, jahn d, dübel s. 2007. production of recombinant antibody fragments in bacillus megaterium. microb cell fact. 6:2. doi:10.1186/1475-2859-6-2. miller gl. 1959. use of dinitrosalycilic acid reagent for determination of deducing sugars. anal chem. 31(3): 426-428. doi: oyeleke sb, auta sh, egwim ec. 2010. production and characterization of amylase produced by bacillus megaterium isolated from a local yam peel dumpsite in 10.1016/s00329592(03)00037-2. 10.3923/ajbkr.2011.310.316. 10.1128/aem.67.4.1744-1750.2001. 10.1021/ac60147a030 volume 6, 2012 microbiol indones 87 sidhu gs, sharma p, chakrabarti t, gupta jk. 1997. strain improvement for the production of a thermostable áamylase. enzyme microb technol. 21: 525-530. stammen s, muller bk, korneli c, biedendieck r, gamer m, franco-lara e, jahn d. 2010. high-yield intraand extracellular protein production using bacillus megaterium. appl environ microbiol. 76(12): 40374046. takeuchi a, shimizu-ibuka a, nishiyama y, mura k, okada s, tokue c, arai s. 2006. purification and characterization of an á-amylase of pichia burtonii isolated from the traditional starter “murca” in nepal. biosci biotechnol biochim. 70: 3019-3024. tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum persimonymethods. mol biol evol. doi:10.1093/molbev/msr121. van der maarel mj, van der veen b, uitdehaag jc, leemhuis h, dijkhuizen l. 2002. properties and applications of starch-converting enzymes of the á-amylase family. j biotechnol. 94(2): 137-155. vidyalakshmi r, paranthaman r, indhumathi j. 2009. amylase production on submerged fermentation by bacillus spp. world j chem. 4(1): 89-91. minna, niger state. j microbiol antimicrob. 2(7): 8892. pandey a, nigam p, soccol cr, soccol vt, singh d, mohan r. 2000. advances in microbial amylases. biotecnol applied biochem. 31(2): 135-152. prakash o, jaiswal n. 2010. á-amylase: an ideal representatives of thermostable enzymes. appl b i o c h e m b i o t e c h n o l . 1 6 0 ( 8 ) : 2 4 0 1 1 4 . d o i : 10.1007/s12010-009-8735-4. rygus t, hillen w. 1991. inducible high-level expression of heterologous genes in bacillus megaterium using the regulatory elements of the xylose-utilization operon. appl microbiol biotechnol. 35(5): 594-599. doi: 10.1007/bf00169622. sajitha n, vasanthabharathi v, lakshminarayanan r, jayalakshmi s. 2011. amylase from an estuarine bacillus megaterium. curr res j biol sci. 3(2): 110115. scopes rk. 1994. protein purification: principles and practice. 3rd edition. new york: springer-verlag. shih nj, labbe rg. 1995. purification and characterization of an extracellular á-amylase from clostridium perfringens type a . appl environ microbiol. 61(5): 1776-1779. microbiol indones88 malle et al. 1: 1 2: 2 page 3 page 4 page 5 page 6 13 no 259 (siswa setyahadi).pmd screening of lactic acid bacteria for the purpose of chitin recovery processing siswa setyahadi biocatalyst production technology division, center for bio-industrial technology, badan pengkajian dan penerapan teknologi, gedung 2 lantai 15, jalan m h thamrin 8, jakarta 10340, indonesia phone: +62-21-3169513, fax: +62-21-3169510, e-mail: siswa59@yahoo.com lactic acid fermentation has been studied as an alternative method of chitin recovery from the natural chitin compounds, such as shrimp waste. the purpose of this research was to identify a potential lactic acid bacterium which can produce large amount of lactic acid and has the ability to release chitin (demineralization) from shrimp-shell-waste. among 11 bacteria tested, strain 15 was the strongest lactic acid producer yielding 1.09% (v/v) lactic acid in the media with a ph of 4.15 after 6 days incubation at 37 oc. after that, strains 17 and 23 produced 0.79 and 0.74% of lactic acid respectively. these three strains were selected for further experiments on various kinds of media using two-day incubation periods. no strains produced lactic acid well in mrs media containing lactose. the best medium for lactic acid production by strains 15 and 17 was mrs containing glucose, molasses or mixture of molasses and shrimp shell waste. fermentation of shrimp shell waste using strain 15 caused an increase of viscosity reflecting an increase of soluble chitin in the media. key words: chitin, lactic acid bacteria, recovery processing, lactic acid _____________________________________________ recent interest in chitin and chitosan in industrial use is reflected by an increasing number of meetings in europe, asia and america, and papers published on chitin-chitosan solubilization (george et al. 1999). the potential use of chitin and chitosan is widely recognised, and many new applications have been developed. currently, chitin, chitosan and their derivatives are widely used in chemical, medical, pharmaceutical, cosmetics, food technology and water treatment industries. however, the use is restricted to specific applications because of the high cost of technical-grade chitin and chitosan. greater industrial use of chitin will be possible, if its manufacturing processes were made cheaper. chitin and the by-products e.g. carotene-protein, could be recovered from crustacean waste products. marine by-products rich in chitin and protein are renewable resources present in large amounts in many countries. shrimp production in indonesia is around 240,000 metric tons per year (brkp 2005), and non-edible wastes such as carapace and exoskeleton can make up to 50-60% of this volume. due to their high availability and their chemical composition (27% chitin, 40% protein, and 35% minerals), shrimp residues are prime candidates for use as a raw material for producing chitin-chitosan. shrimp shell waste is partially used as animal feed but most of it is discarded causing serious ecological problems. it has also been used as an ingredient in culture media; for extraction of pigments and proteins to formulate animal feed; for reducing nematode populations in soils; for producing single-cell protein, and for enzyme immobilization. the traditional methods for commercial preparation of chitin from crustacean shell waste have remained essentially unchanged since first proposed (rigby 1934). the traditional processes are mechanical grinding, demineralization with 1 n hydrochloric acid and deproteinization with 3-5% sodium hydroxide at 90-100 oc. these chemical treatment procedures potentially pollute the environment and the resulting wastes have to be treated before being discarded. the treatment has been identified as the most important factor contributing to the high price for chitin and chitosan products (muzzarelli 1990). thus, in order to increase chitin use and diminish the emission of pollutants, a less expensive and more environmentally friendly method for large scale extraction of chitin needs to be developed. lactic acid fermentation combined with chemical treatment has been studied as an alternative method of chitin recovery that reduces the amount of alkali and acid needed. the removal of protein and calcium from shells is by a combination of enzymatic activity and mineral solubilization by organic acid produced in bacteria growth (shirai et al. 1997; zakaria et al. 1998; hsu and wu 2002; luis et al. 2003). the purpose of this research was to identify a potential lactic acid bacterium which could produce large amounts of lactic acid for demineralization in the shrimp-shell-waste process. materials and methods materials. bacterial culture media including deman rogosa and sharpe (mrs), mrs broth, mrs agar and ingredients including yeast extract, peptone, and glucose were purchased from merck kgaa, darmstadt, germany. lactobacilli broth aoac was purchased from difco laboratories, usa. molasses was obtained from pakistan. microorganisms and growth conditions. the lactic acid bacteria tested were six strains (3, 4, s5, 13, 15, and 17) isolated from indonesian traditional food (purchased from bppt culture collection) and five strains (5, 9, 23, 95, and 135) isolated from tempe (fermented soybean cake) purchased from the food microbiology laboratory, hamburg university. the lactic acid bacteria were maintained on mrs broth containing 30% v/v glycerol as cryoprotectant and kept at -80 oc. mrs agar slopes were prepared and stored at 4 oc. microbiology indonesia, april 2007, p 48-50 volume 1, number 1 issn 1978-3477 the inoculum was prepared using mrs broth with a loopful of cells from a slope of mrs agar and incubated at 37 oc for 24 hours. shrimp shell fermentation. screening the best lactic acid bacteria for demineralization of the shrimp shell was carried out on mrs medium at ph 7.0 which contained 6% of shrimp shell and molasses instead of glucose. an inoculum of 70 ìl of each cell suspension was subcultured into 7.0 ml medium. the culture was incubated on a shaking water bath (gyrotory shaking water bath model g 76, new brunswick scientific, usa) at 37 oc for six days. samples for determination of ph and lactic acid production were collected every 24 hours. in further experiments the lactic acid production of the three selected bacteria was observed on the mrs media containing molasses, lactose, or shrimp shell waste, incorporated with either meat extract or peptone or k 2 hpo 4 or mgso 4 . analytical procedures. the moisture content of shrimp shell waste was measured by oven drying samples at 105 oc for 90 min. ash content was determined by burning samples in a crucible at 800 oc in a muffle furnace for 90 min. mineral content was measured by titration with titrisol (titriplex solution b, merck). protein content was calculated using the following equation: % protein = 1 (% ash without naoh treatment % ash with naoh treatment) viscosities are determined using a suspended level ubbelohde viscometer equipped with three bulbs situated at different heights above the bottom of the capillary. the system dmac (n,n-dimethylacetamide)/licl (lithium chloride) is prepared by weighing rapidly the salt and adding the solvent required. chitin solutions in 5% (w/w) licl/dmac were prepared as follows; a dry chitin sample (5 mg) was suspended in 20 ml of 5% licl/dmac, and then the mixture was stored at room temperature with occasional shaking until complete dissolution was achieved. the ph of samples and fermentation broth was measured using a ph electrode (ph-mv-meter, knick). lactic acid concentrations were determined by hplc (hitachi model d-2500) after heating at 80 oc for 10 min and centrifugation at 13,000 x g at room temperature for 10 min. five µl aliquots of the filtrate were injected onto a liquid chromatography using a ion gard orh 801 column and eluted with 180 µl h 2 so 4 in 1.0 l milliq water. operationing conditions were 0.6 ml min-1 flow rate and 60 oc. the chromatograph was fitted with a ri detector (lachrom ri detector l-7490, merck). a fourier transforming infra-red (ft-ir) spectrometer was employed to confirm the structure of chitin. results shrimp shell waste. ash and chitin contents in shrimp shell waste from indonesia were 21.67 and 26.13% respectively. the results indicate that they were lower than the minced waste from cold water areas (george et al. 1999). table 1 shows the characteristics of shrimp shell waste obtained from the shrimp fishing industry in lampung, indonesia. screening of lactic acid bacteria (lab). the eleven strains tested produced varying amounts of lactic acid. after 6 days fermentation at 37 oc, strain 15 was identified as the strongest lactic acid producer with a ph 4.15 and it yielded 1.09% (v/v) lactic acid in the medium (table 2), while strains 17 and 23 produced 0.79 and 0.74% (v/v) lactic acid, respectively. these three strains were selected for further experiments. the changes of ph during fermentation process. in the course of the fermentation the ph gradually decreased from 7.0 to 4.7 in the media containing shrimp shell and molasses. results were not in agreement with those observed in mrs broth media containing glucose that the ph decreased from 5.5 to 3.5 after three days incubation. however, in all cases the decrease of ph reflected the amount of lactic acid production. lactic acid production in various media. three strains were tested in various kinds of media for two days incubation at 37 oc (table 3). no strain produced well lactic acid in the lactose media (media h). the best medium for growing strains 15 and 17 is mrs broth, which is a selective medium for producing lactic acid. strain 15 was also the best lactic acid producer when grown on medium containing molasses and table 1 characteristics of the shrimp shell waste from indonesia variable % w/w moisture ash p r o t e i n mineral chitin 28.21 21.67 37.84 36.03 26.13 table 2 lactic acid production in mrs broth media* at 37 oc for 6 days incubation time as a function of bacterial strain strain final ph lactic acid (% v/v) 3 4 s 5 5 9 13 15 17 23 95 1 3 5 5.20 5.00 5.20 5.55 5.40 > 8.00 4.15 4.45 4.60 4.80 4.85 0 . 3 0 0 . 3 5 0 . 1 9 0 . 1 6 0 . 2 3 0 . 0 7 1 . 0 9 0 . 7 9 0 . 7 4 0 . 5 7 0 . 5 1 *glucose was replaced by molasses table 3 production of lactic acid on various media by 3 bacterial strains for 2 days % (v/v) lactic acid in media a b c d e f g h strain 1 5 1 7 2 3 1 . 4 1 1 . 4 2 0 . 2 5 1 . 2 2 0 . 5 9 0 . 7 6 0 . 7 0 0 . 7 5 0 . 3 7 0 . 6 9 0 . 7 2 0 . 2 0 0 . 4 7 0 . 0 8 0 . 1 7 0.33 0.33 0.10 0 . 2 2 0 . 2 0 0 . 0 6 0.05 0.06 0.03 a: media is mrs broth; b: media is mrs broth but replacing glucose by molasses; c: media is molasses (50 g l-1) and meat extract (5 g l-1); d: media is molasses (50 g l-1) and peptone (10 g l -1); e: media is molasses (50 g l-1), k 2 hpo 4 (2 g l-1), and mgso 4 (0.2 g l-1); f: media is molasses (50 g l-1); g: media is molasses (50 g l-1) and shrimp shell waste (160 g l-1); h: media is lactose (20 g l-1) volume 1, 2007 microbiol indones 49 shrimp shell waste, reflecting the ability of the strain to digest the shrimp shell waste. fermentation of shrimp shell waste. results from ftir spectrometer for fermented shrimp shell waste with strain 15 show that peaks of o-h; n-h; ch 3 –ch 2 ; ketone and 1,4 glucosamine were at 3496.1 cm-¹; 3272.4–3218.8 cm-¹; 2954.9 cm-¹ ; 1659.2–1543.8 cm-¹ and 1074.6 cm-¹, respectively (figure 1b). those results were similar with those observed in commercial chitin (figure 1a). discussion lactic acid fermentation is one of the methods currently used in the biological procedures for the demineralization of chitin. it purifies chitin by hydrolysis of the protein, which are bonded to the chitin in the shell waste (zakaria et al. 1998; george et al. 1999). during the fermentation, based on hplc analysis strain 15 had the ability to produce well lactic acid from glucose than the medium containing lactose. this result confirmed that strain 15 is a lactic acid bacterium. fermentation of shrimp shell waste using strain 15 resulted to increase the viscosity up to 60 times compare that of unfermented one. the solubility of purified chitin from protein of shrimp shell waste increased the viscosity in the fermentation media. strain 15 is a lactic acid bacterium producing lactic acid in the glucose media. the increase of viscosity in the fermentation of shrimp shell waste show that strain 15 may be a good candidate for demineralization chitin from shrimp shell waste (george 1999). further work is needed in order to establish whether a process based on lactic acid fermentation could be good quality and economically feasible. acknowledgement i am grateful to the daad for financial supporting through a daad scholarship granted to the author through a special programme for biosciences 2003. the author would like to thank bernward bisping, university of hamburg for assistance, providing the laboratory facilities and kindness. finally, i would like to gerd mueller von der haegen and to acknowledge the assistance and support of all the seelab and food microbiology laboratory members. references [brkp] badan riset kelautan dan perikanan. 2005. statistika perikanan budidaya. jakarta: brkp. george mh, carole lr, zakaria z. 1999. fermentation of prawn waste by lactic acid bacteria. in: proceeding of the 3rd international conference of the european chitin society. potsdam. germany, 31 aug-3 sept. p 633-638. hsu yl, wu wt. 2002. a novel approach for scaling-up a fermentation system. biochem eng j 11:123-130. luis sjt, moldes ab, alonso jl, vazquez m. 2003. optimization of lactic acid production by lactobacillus delbrueckii through response. surface methodology. j food sci 68:1454-1458. muzzarelli r. 1990. chitin and chitosan: unique cationic polysaccharides. proc. symp. towards a carbohydrate based chemistry. amies, france, 23-26 oct 1989. p 199-231. rigby gw. 1934. us patent 2,040,879. shirai k, guerrero i, huerta s, saucedo g, rodriguez g, hall g. 1997. aspects in protein breakdown during the lactic acid fermentation. adv chitin sci 2:56-63. zakaria z, hall m, shama g. 1998. lactic acid fermentation of scampi waste in a rotating horizontal bioreactor for chitin recovery. proc biochem 33:1-6. a 50 setyahadi microbiol indones figure 1 ft-ir of commercial chitin (a) and chitin from lactic acid bacteria fermentation strain no. 15 on shrimp shell waste (b). a b 3600 3200 2800 2400 2000 1600 1200 800 400 wave number 7 0 6 0 5 0 4 0 3 0 t ra n s m it ta n c e (% ) t ra n s m it ta n c e (% ) 100 90 80 70 60 50 3600 3200 2800 2400 2000 1600 1200 800 400 wave number 01. rosariastuti.cdr vol.11, no.4, desember 2017, p 111-116 doi: 10.5454/mi.11.4.1 the utilization of modified cassava flour (mocaf) industrial waste and peat as carrier of nitrogen-fixing bacteria (nfb) and phosphate solubilizing bacteria (psb) inoculant * retno rosariastuti , sumani, supriyadi, muhammad ardian nur setyawan, and pramusita yoga daniswara department of soil science, faculty of agriculture, universitas sebelas maret jalan ir. sutami 36a, surakarta 57126, central of java, indonesia biofertilizer is organic fertilizer with addition of specific microorganisms. carrier material play an important role in maintaining the viability of microorganisms during storage period. solid waste of modified cassava flour (mocaf) has great potency as carrier material of good biofertilizers, because of its nutrient content. peat has also been used as a biofertilizer carrier for a long time. the aim of this study was to determine the potential of mocaf solid waste in its combinations with peat as the carrier, added by addesive materials (starch and clay), in supporting the growth of nfb and psb during the storage period and carriers quality. it was factorial experimental using completely randomized design (crd) as the based design, consist of two factors: carrier formulation (c: c1, c2 and c3); and incubation time (t: t1, t2,… t5). all materials were mixed and sterilized, than inoculated by 8 -1 nfb (rhizobium sp., azotobacter sp.) and psb (bacillus sp. dan pseudomanas sp.), 10 cfu g carrier, than incubated for 60 days at room temperature. bacteria colony were analyzed every 15 days and the quality of carrier (ph, moisture, n-total and c-organic) were analyzed at the beginning and the end of incubation. the results 8 showed the increasing growth of nfb and psb until day 60 incubation time in all formulas carriers, reach of 10 9 -1 10 cfu g . it showed that carriers could support the growth and viability of nfb and psb. all formula of carriers had fulfilled the quality standard for biofertilizer as assigned by minister of agriculture republic of indonesia no. 70 year 2011. key words: biofertilizer, carrier, mocaf, nfb, peat, psb pupuk hayati adalah pupuk organik dengan penambahan mikroorganisme spesifik. bahan dasar dalam bahan pembawa inokulum mikroorganisme, memegang peranan penting dalam mendukung kehidupan mikroorganisme selama periode penyimpanan. limbah padat indusri modified cassava flour (mocaf) memiliki potensi tinggi sebagai bahan pembawa inokulum mikroorganisme pupuk hayati yang baik, karena kandungan nutrisinya. gambut juga sejak lama telah digunakan sebagai bahan dasar bahan pembawa inokulum mikroorganisme. tujuan penelitian ini adalah mengetahui potensi limbah padat industri mocaf dalam kombinasinya dengan gambut sebagai bahan pembawa inokulum bakteri dengan penambahan bahan perekat tepung gandum dan lempung, dalam mendukung pertumbuhan bakteri penambat n (bpn) dan bakteri pelarut fosfat (bpf), selama periode penyimpanan dan kualitas bahan pembawa. rancangan percobaan adalah faktorial dengan rancangan dasar rancangan acak lengkap, terdiri dari dua faktor yaitu formula bahan pembawa (c: c1,c2 dan c3); dan lama penyimpanan (t: t0, t2,…t5). semua bahan dicampur kemudian disterilisasi, selanjutnya diinokulasi bpn (rhizobium sp., azotobacter sp.) dan bpf (bacillus sp. dan pseudomanas sp.) 8 -1 sejumlah 10 cfu g bahan pembawa, kemudian diinkubasi selama 60 hari pada suhu kamar. pengamatan dilakukan terhadap koloni bakteri setiap 15 hari sekali dan kualitas bahan pembawa (ph (h o), kadar air, n-total 2 dan kandungan corganik) pada awal dan akhir percobaan. hasil penelitian menunjukkan terjadinya 8 peningkatan pertumbuhan bakteri hingga periode penyimpanan hari ke 60, dengan koloni bakteri sejumlah 10 9 -1 10 cfu g . artinya bahwa bahan pembawa inokulum bakteri ini dapat mendukung pertumbuhan dan kehidupan bpn dan bpf. selanjutnya diketahui bahwa ke tiga formula bahan pembawa telah memenuhi standar baku mutu kualitas pupuk hayati yang ditetapkan oleh menteri pertanian republik indonesia no. 70 th 2011. kata kunci: bahan pembawa, bpf, bpn, gambut, mocaf, pupuk hayati microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82185455812; email: retnobs@staff.uns.ac.id and medium enterprises (smes) produce fertilizer derived from agricultural waste and livestock, so the development of organic fertilizer and biofertilizer also increase (simanungkalit et al. 2006). according to sivasakthivelan and saranraj (2013), there were many research about formulation of biofertilizer, because formulation of biofertilizer plays a vital role in helping fertilizer demand for agriculture increases 5-10% per year. to support the increasing needs of fertilizer so many studies about utilization of agricultural biomass and industrial waste were done. large number of small to solve many problems in agricultural field in making an organism effective in the field. carrier material may have allowed better survival of organism. many materials has been investigated for use as carrier of bacteria, they are charcoal, farmyard manure (fym), compost, coconut chell powder, vermiculite clay, teak leaf powder, powdered peanut shell, corn cobs, coffee waste, black ash, paddy husk, etc. m o c a f i n d u s t r i a l a c t i v i t i e s o f t e n c a u s e environmental of pollution caused by its solid, liquid and gas waste. the accumulation of bad odor due to the fermentation process indicated that the mocaf waste is the right nutrient for growth of microorganisms (subagio 2007). mocaf solid waste has high carbohydrate content reaching 63%-68% and moisture contents level is 20% (ogbo 2010; atika and apsari 2011). the high carbohydrate (subagio 2007) and moisture contents (alexander 1976; foth 1995; makan et al. 2013) in the waste can support microbial activities. by certain treatment the waste could become useful product and not pollute the environment (chardialani 2008). the waste could be processed into more useful products in agricultural product. solid waste of agricultural industry which has high nutrient, potential used as based material of microorganisms carrier, by combined with other material. peat has been used as the carrier material for seed inoculation for a long time (simanungkalit et al. 2006; raharjo et al. 2007; forsmann and kjaergaard 2014). peat characteristics are moisture and humid which generate good environmental conditions in the growth of microorganisms. peat also has sufficient permeability for air and water exchange (somasegaran and hoben 1994; allaire et al. 1994). peat is generally as most dependable carrier because of its high content of organic matter and water holding capacity (sivasakthivelan and saranraj 2013), so peat is well utilized as a carrier material for seed inoculation. rosariastuti et al. (2013) explained the mocaf waste characteristics similar with peat and the nutrient content mainly nitrogen and phosphate as a source of nutrients for microorganisms, and it is better than peat. there is no research about the use of solid waste of mocaf industry as carrier of bacteria. therefore, the potency of mocaf solid waste combined with peat as carrier of functional bacterial is insteresting to be investigated. the purpose of this study was to determine the potency of mocaf solid waste combined with peat as carrier in supporting the growth of nitrogen fixing bacteria (nfb) and phosphate solubilizing bacteria (psb) during the incubation. materials and methods materials and equipment. materials used include: mocaf solid waste obtained from the sme community in wonogiri indonesia, peat from histosols soil of rawapening, ambarawa indonesia, and a bacterial inoculum collection of microbiology department, faculty of agriculture, gadjah mada university in yogyakarta indonesia. specific media (nfb symbiotic in yma media, nfb non-symbiotic in ashby media, psb in pikovskaya media and total bacterial in natrium agar media) and other chemicals for laboratory analysis. research design and data analysis. this research was a laboratory experimental research. the design experimental was completely randomized design (crd) consist of two factors, first was formulas of the carrier (c) = : c1: 75% peat +25% mocaf +70g -1 starch kg carrier, c2: 75% peat+25% mocaf+(35g -1 starch + 35g clay) kg carrier, and c3: 50% peat+50% -1 mocaf+70g clays kg carrier, second was incubation time (t): t1: day 0, t2: day – 15, t3: day – 30, t4: day 45, t5: day 60) with 2 replications. each carrier was added by bacterial inoculum : rhizobium sp., azotobacter sp. (nfb), bacillus sp. and pseudomanas 8 -1 sp. (psb), as much as 10 g of carrier material. data analized by statistical analysis using anova, followed by duncan's multiple range test (dmrt) to compare the mean of treatment combinations. variables observations. variables observations including the viability of bacterial growth (colony number), and quality of carrier: ph, moisture (water content), total nitrogen, total phospate, and organic carbon. results chemical analysis of treatment formulation. three formulas carrier chemical composition (final) analysis were that total nitrogen and total phosphate were accordance with the quality standards of macro nutrients (standard n, p o5, k o minimum 4%); the 2 2 organic carbon treatment was appropriate quality standard of organic carbon (standard > 15%), the moisture appropriate with quality standards of water content (standard ≤35); ph only c1 = 5.08 and c2 = 5.05 were appropriate with quality standards ph (standard 5.0-8.0) (table 1). based on mocaf in combination with peat potentially used as carrier material of nfb and psb, and can produce good biofertilizer which has high quality. 112 rosariastuti et al. microbiol indones volume 11, 2017 microbiol indones 113 the number symbiotic of nitrogen-fixing bacteria (nfb). the colony numbers of nfb symbiotic (rhizobium sp.) growth in the carrier, at 60 8 day incubation were 8.311 9.471 log 10 (2.05x10 9 -1 3x10 ) cfu g . the results of anova, after 60 days incubation showed that the carrier material formulations (p = 0.178) had no significantly effect, whereas the incubation time (p = 0.005) had very significantly affected to the colony number of nfb. during 60 days incubation, there were fluctuation growth of nfb symbiotic rhizobium sp. (fig 1). at this stage bacterial growth were in exponential phase, which the multiplication of cells was not disordering or because of the availability of nutrients. the number of non-symbiotic nitrogen-fixing bacteria (nfb). the colony numbers nfb nonsymbiotic (azotobacter sp.) lived in the carrier were 6 6 -1 6.387 6.903 log 10 (2.6x10 8x10 ) cfu g . based on the results of the f testlevel of 95%after 60 days incubation showed that the carrier material formulations (p = 0.715) had no significantly effect, whereas the incubation time (p = 0.047) significantly affected to the number of nfb. based on figure 2, it could be seen that during 60 days of incubation there were the fluctuation nfb non-symbiotic azotobacter sp. growth (fig 2). the decrease of the number of azotobacter sp.because of the adaptation process (lag phase). in environment bacteria needs to adjust the environmental conditions. the number of phosphate-solubilizing bacteria (psb). each population of psb microbial isolate inoculated into the carrier formulations can be associated with the ability of phosphate dissolve and ph changes. the observation of microbial populations of phosphate solubilizing bacteria are presented (fig 3). based on anova, incubation showed highly significantly (p = 0.001) influenced on the number of psb, while the formulas of carrier material and the interaction between incubation time and formulas were not significantly effected (p = 0.484) to the number of psb. based on the dmrt showed that there were significantly differences in the changing of the number of psb. figure 3 showed that the incubation time, increase of the number of bacterial colonies. it can be seen on the 30th day of incubation, the number of 9 colonies reached 9.61610.571 log 10 (4.1x10 10 -1 3.7x10 ) cfu g . bacillus sp. and pseudomonas sp. isolates could degrade the nutrients present in a carrier material, so the bacterias still in increasing growth until the 60th day of incubation. the number of total bacterial. the next observation was analyzing the total bacterial viability consisting of a consortium bacteria such as rhizobium sp., azotobacter sp., bacillus sp. and pseudomonas sp. growth in all carrier formulations. the colony numbers of total bacterial lived in the carrier were 9.613 10.571 9 10 -1 log 10 (4.09x10 1.6x10 ) cfu g . the results of anova, after 60 days incubation showed that the table 1 results of chemical analysis of carrier in treatment formulation note. (ö) means qualified and (x) means not qualified by biofertilizer quality standard according to minister of agriculture indonesia no. 28 year 2009 and minister of agriculture of indonesia no. 70 year 2011. no variable treatment time early (day 0) end (day 60) value criteria value criteria 1 total nitrogen (%) c1 1.01 ö 1.16 ö c2 0.64 ö 0.69 ö c3 0.68 ö 0.77 ö 2 total phospate (%) c1 8.49 ö 8.54 ö c2 8.45 ö 9.11 ö c3 8.31 ö 9.14 ö 3 organic carbon (%) c1 43.84 ö 26.92 ö c2 36.91 ö 19.92 ö c3 39.28 ö 21.53 ö 4 water moisture (%) c1 34.99 ö 26.21 ö c2 23.46 ö 31.75 ö c3 33.80 ö 21.74 ö 5 ph c1 4.79 x 5.08 ö c2 4.52 x 5.05 ö c3 4.43 x 4.93 x fig 1 histogram number colony of rhizobium sp. during 60-days incubation. 114 rosariastuti et al. microbiol indones fig 2 histogram the number colony of azotobacter sp. during 60-days incubation. fig 3 histogram number colony of psb during 60-days incubation. fig 4 histogram number of total colony bacterial during 60-days incubation. volume 11, 2017 microbiol indones 115 formulas of carrier material (p = 0.757) had no significantly effected, whereas the incubation time (p = 0.005) had very significantly effected to the total bacterial. the observation of total bacterial is presented in figure 4. incubation time had highly significantly effected (p = 0.005) to the total number of bacterial colonies, whereas the formulas of carrier material and the interaction between incubation time and formulas of carrier material are not significantly affected (p = 0.757) to the total number of bacterial colonies. discussion there were many research about formulation of biofertilizer for example charcoal + soil mixture is the best carrier for bpf; fym + soil, fym + charcoal, soil + fym + charcoal, were good for azospirillum carrier, etc. (sivasakthivelan and saranraj 2013). in indonesia, characteristics of carrier materials in biofertilizer should refer to the government quality standards of the minister of agriculture of republic of indonesia no. 28 year 2009 and no. 70 year 2011 about organic fertilizer, biofertilizer and soil improvement. the growth rate was determined by the composition of the media and environmental factors, while rhizobium sp. population decline due to nutrient reduced, resulting the competition among the nfb to acquire nutrients (mansur et al. 2003). moreover, mocaf waste would be energy source of rhizobium sp. that could increasing the population. this was same with the statement of ahmad et al. (2011), that the high content of protein, starch and total sugars in the waste water of mocaf flour industry made the waste became source of carbon and nitrogen for microbial growth. one of the factors of population decline of rhizobium sp. was ph. ph between 4.5 to 5.1 on all treatments cause declining bacterial populations. according to martani and margino (2005), most rhizobium sp. bacteria have good growth at neutral ph and optimum at a ph of 5.5 to 7.0. according to kawuri research (1997), rhizobium sp. has the ability to adapt within the low ph (4.0 to 5.7) by realizing response called acid tolerance response (atr). in this research, low ph causes the bacteria undergo to adaptation process (lag phase), followed by the increasing growth of rhizobium sp. which have ability to live in the low ph environment. according to wyss et al. (1961), azotobacter needs a break from the vegetative cells to survive in adverse environmental factors. after the environment become in optimum conditions, including certain ph value, carbon source and new vegetative cells, azotobacter sp. would grow and return to the exponential phase (log phase). the increase of bacteria population growth in each carrier formulation cause by the availability of energy source of bacteria in the form of simple sugars such as sucrose and glucose. according to stella and sivasak (2009) the addition of nutrients such as glucose and sucrose led the increasing of the viability of microbes in biofertilizer. viability of azotobacter sp. increased due to the sources energy derived from mocaf waste. chun and vidaver (2001) found bacillus sp. reached to a lag phase quickly relatively and the process to reached rapid exponential phase was also quickly relatively. simultaneously on the 30th days incubation the number of colonies on all formulations of carrier material were increasing. na media is not selective media, but could be used as an indicator of microbial growth. na media has rich of protein, nitrogen, vitamins and from beef extract and peptone (simanungkalit 2001). the result of total bacterial viability test during incubation time could be used as a marker of the carrier material quality. good carrier materials are expected to maintain the viability and the effectiveness of microbial inoculant during storage (rao 1982). in accordance with the biofertilizers, quality standards of biofertilizers regulated by the minister of agriculture of republic of indonesia no. 28 year 2009 and no. 70 year 2011 about organic fertilizer, biofertilizer and soil improvement, biological fertilizers consortium must 7 -1 have at least 10 cfu g , ph value between 5.0 to 8.0 with a moisture of <30% in a state of solid fertilizer. the carrier formulation in this research potential to support the growth and sustain the life of functional nfb and psb bacterial. so they were potential use as carrier material in making biofertilizers. functional bacterial in specific media accordance with quality standards. the ph value during incubation period was within the proper criteria and the viability of total bacteria supported the development of carrier material formulations. these carrier formulation are new, because they have never investigated before. acknowledgment this work was supported by a grant provided by the institute of research and public services (lppm) sebelas maret university, indonesia, through the grant application (hibah terapan) 2016 with the project number of 418/un/8/lppm/2016. references achmad s, wiwik sw, didiek h. 2011. pengembangan zero waste processing dari modified cassava flour (mocaf) guna meningkatkan spinoff klaster kepada masyarakat sekitar [developing zero waste processing from modified cassava flour (mocaf) spinoff cluster into improve neighborhood to society]. 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an n-terminal domain, a central hydrophobic domain and a c-terminal domain. in order to elucidate its structure and function, a set of nuclear genes encoding subunit 8 variants was designed to incorporate a flag tag at the c-terminus and a mitochondrial signal peptide at the n-terminus. each gene was cloned into a yeast expression vector and then allotopically expressed in a yeast strain lacking endogenous subunit 8. structural and functional analysis showed that the hydrophobic character of the central hydrophobic domain of subunit 8 is critical for the atp synthase function. subunit 8 is sensitive to charge manipulation at the c-terminus. the positively charged residues at the c-terminal domain are important for subunit 8 assembly and hence its function. key words: allotopic expression, atp synthase, mitochondria, yeast _____________________________________________ mitochondrial atp synthase (e.c.3.6.1.3) also known as f 0 f 1 -atpase, is key enzyme which plays a major role in the formation of atp used to drive cellular processes. the yeast mitochondrial atp synthase is a multisubunit complex composed of at least 17 subunits grouped into two sectors, viz. f 1 and f 0 sectors. the f 1 sector lies at the inner (matrix) surface of the inner mitochondrial membrane and is comprised of subunits α, β, γ, δ, and ε, with a stoichiometry of 3:3:1:1:1, all of which are encoded at the nucleus (cox et al. 1992). the f 0 sector spans the membrane and is composed of subunits b, oscp, d, e, f, g, h, i/j, k which are encoded by nuclear genes, and subunits 6, 8, and 9, which are encoded by mitochondrial genes (stephens et al. 2000). in the inner mitochondrial membrane, the atp synthase complex can form a dimer (fronzes et al. 2006). subunit 8 of yeast mitochondrial atp synthase is a small hydrophobic polypeptide of 48 amino acids encoded by the atp8 gene (macreadie et al. 1983). analysis of its primary structure has led to the identification of three distinct domains; e.g., an n-terminal domain, a central hydrophobic domain (chd), and a c-terminal domain. the chd which spans residues 14 to 32, has been predicted to act as a transmembrane stem (nagley et al. 1990). as a mitochondrially encoded protein, subunit 8 is transcribed, translated, and transported within the organelle. subunit 8 is present in eukaryotic atp synthases but not in prokaryotic enzyme complexes (cox et al. 1992). this means that bacterial atp synthase naturally functions without the existance of subunit 8. the immediate question therefore is how this subunit functions in the enzyme complex. detailed analysis of the subunit 8 structure and function is still lacking. although subunit 8 has been considered to participate in proton translocation (nagley et al. 1988), the exact role of subunit 8 in this function remains unclear. in order to elucidate its structure and function, an allotopic expression system for subunit 8 has been developed. allotopic expression is the deliberate relocation of organellar genes to the nucleus and delivery of the gene products from the cytoplasm to the corresponding organelle. for allotopic expression of subunit 8, a nuclear version of subunit 8 gene to be expressed in the nucleocytosolic system has been designed. to ensure that the cytoplasmically synthesized subunit 8 was imported into mitochondria, sequences encoding a mitochondrial signal peptide were fused to the n-terminus of the gene (gearing et al. 1985). the allotopic expression system has been applied to study various aspects of the subunit 8 molecular biology. this system has also been successfully used to express flag tagged-subunit 8 protein (artika 2006). the allotopically expressed flag tagged-subunit 8 protein was imported into mitochondria and assembled into a functional atp synthase complex. the main purpose of flag tag addition to subunit 8 protein was to allow the use of immunochemical methods to detect subunit 8 protein. however, since the flag tag is highly charged, its incorporation into c-terminus region of subunit 8 changes the nature, number, and distribution of the charged residues that may affect the structure and functioning of subunit 8. in the present study a set of subunit 8 variants containing either double negative charges or double positive charges within the chd was flag tagged and then allotopically expressed in a mutant yeast strain lacking endogenous subunit 8. structural and functional consequences of the introduction of flag tag residues to subunit 8 variants are discussed. materials and methods materials. saccharomyces cerevisiae strain m31 [atp8, mit¯ , his6, ade1], a collection strain of the department of biochemistry and molecular biology, monash university, has previously been described (nagley et al. 1988). strain ym2 is strain m31 expressing non-tagged-subunit 8 gene fused with a mitochondrial signal peptide (roucou et al. 1999). the set of gene constructs encoding subunit 8 variants microbiology indonesia, april 2007, p 33-36 vol. 1, no. 1 issn 1978-3477 fused with a mitochondrial signal peptide has been described previously (papakonstantinou et al. 1996). the yeast expression vector ppd72 used for allotopic expression has been described (law & devenish 1988). the vector has the yeast ade1, leu2, and ura3 genes as selectable markers. gene modification and molecular cloning. the flag epitope tag was incorporate into each variant by using a pcr-based mutagenesis technique (artika 2006). the primers used were designed to incorporate additional nucleotide sequences encoding hexapeptide (dykddd) representing the flag epitope tag at the c terminus of the gene with two serine residues functioning as a bridge between the subunit 8 and the flag tag. cloning of the gene constructs into the yeast expression vector was carried out using standard methods (sambrook et al. 1989). yeast transformation. introduction of recombinant plasmid vector into yeast strain m31 was done as described by klebe et al. (1983). determination of generation time. generation time of each strain was determined as described by gray et al. (1996). isolation of mitochondria. intact mitochondria were prepared using the glass bead method (lang et al. 1977). the protein concentration of isolated and washed mitochondria was determined using the bio-rad protein micro-assay procedure based on the method of bradford (1976). protein analysis. sds polyacrylamide gel electrophoresis (sds-page) was performed as described by laemmli (1970) using a dual adjustable slab gel unit. following sds-page, the proteins were transferred onto an immobile-p membrane (pvdf). the membrane was then incubated overnight at 4 oc in blotting solution containing primary antibody. after washing away the unbound primary antibody, the membrane was incubated for 1 hour at room temperature in blotting solution containing secondary antibody (alkaline phosphatase conjugate). proteins were visualized using a vistra alkaline phosphatase conjugate substrate kit (amersham life science, bucks, u.k.). results flag tagged-subunit 8 variants assembled into functional mitochondrial atp synthase complexes. the addition of nucleotide sequences encoding the flag epitope tag to the c-terminus of six different subunit 8 variants resulted in six flag tagged-subunit 8 variants as shown in table 1. in order to examine their functionality in vivo, the genes encoding the flag tagged-subunit 8 variants fused with mitochondrial signal peptide were cloned into a yeast expression vector ppd72. the resultant recombinant plasmids were then allotopically expressed in yeast cells strain m31. the strain m31 lacks of endogenous subunit 8 due to mutation in the atp8 gene. since subunit 8 is an essential subunit of the mitochondrial atp synthase complex, the strain m31 is therefore unable to form functional mitochondrial atp synthase complex. consequently, strain m31 cannot use respiratory substrates such as ethanol to support growth. strain m31, however, can be grown on complete glucose medium. as a fermentative substrate, glucose allows yeast cells to generate atp through substrate-level phosphorylation. following transformation of the m31 host cells with the resultant recombinant plasmids, transformants were plated on solid selective glucose medium at 28 oc for three days. in order to examine whether the allotopically expressed flag tagged-subunit 8 variants rescued the ethanol negative phenotype of strain m31, transformant colonies were transferred onto solid complete-ethanol-medium. growth of transformants on the ethanol medium was observed after 4 days of incubation (figure 1). growth of flag taggedsubunit 8 variants on the ethanol medium indicated that the corresponding flag-tagged subunit 8 variant proteins had successfully assembled into functional enzyme complexes. the present results showed that the three flag taggedsubunit 8 variants were assembled into functional enzyme complexes as indicated by the growth ability of the corresponding mutant strains (df66, df68, and df71) on ethanol medium. the other three variants (df67, df69, and df70) were found to be nonfunctional as indicated by the growth inability of the corresponding strains. basic growth characteristics of strains expressing flag tagged-subunit 8 variants. the growth properties of the four strains expressing functional flag taggedsubunit 8 (a wild-type and three variants) were examined by determining their generation times for growth on liquidethanol-medium. generation time or doubling time is the time needed for the population to double. the generation time (table 2) was calculated from the growth curve of each strain. the generation time reflects the performance of the corresponding subunit 8 variant in the enzyme complex. flag tagged-subunit-8 variant proteins detected using flag monoclonal antibody. following mitochondrial isolation from strain ftc2, df66, df68, and df71, an aliquot of 100 µg mitochondrial protein of each variant was separated on sds-page. mitochondrial proteins prepared from strain ym2 expressing subunit 8 protein without flag tags were included as controls. the proteins were then transferred to pvdf membrane. the membrane was cut into two portions. one portion of the membrane containing subunit 8 protein was probed with anti-flag m2 antibody as the primary antibody. the second portion of the membrane containingtable 1 flag epitope tagged-subunit 8 variants of yeast mitochondrial atp synthase variant/strain mutation ftc2 df66 df67 df68 df69 df70 df71 wildtype, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d g16 à d, f17 à d, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d g16 à r, f17 à r, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d l23 à d, l24 à d, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d l23 à r, l24 à r, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d q29 à d, f30 à d, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d q29 à r, f30 à r, 49s, 50s, 51d, 52y, 53k, 54d, 55d, 56d table 2 generation times of strains expressing flag taggedsubunit 8 variants strain generation time (h) ftc2 df66 df68 df71 6.9 + 0.4 8.2 + 0.5 11.1 + 0.5 7.2 + 0.5 34 artika microbiol indones subunit γ protein was probed with anti-subunit γ antibody. detection of subunit γ was employed as a positive control. results (figure 2) showed that the flag tagged-subunit 8 variant proteins could be detected using anti-flag m2 monoclonal antibody. subunit γ was also detectable in each sample. as expected, the non-tagged-subunit 8 protein isolated from the ym2 strain was not detectable. discussion the present study attempted to elucidate the significant interaction of the chd with the lipid bilayer of the inner mitochondrial membrane as well as to analyze the effects of addition of charges residues at the c-terminus region of subunit 8 variants. in principal, the membrane spanning domain of subunit 8 should be disrupted upon the introduction of charged amino acids because the lipid bilayer is unable to accommodate unshielded charged residues. the present results show that among the functional flag tagged-subunit 8 variants, the variant df68, which has double charged residues in the middle of the chd, displays the most functional defects in atp synthase activity as indicated by the slowest growth rate (table 2) of this strain on ethanol medium. these observations support the previous data (roucou et al. 1999) obtained from non-flag taggedsubunit 8 variants suggesting that the hydrophobic characteritics of residues 23 and 24 of subunit 8 is critical for maximal atp synthase activity. subunit 8 is sensitive to manipulation of charged residues at the c-terminus (grasso et al. 1991). from the present study it is clear that addition of the charged hexapeptide flag tag at the c-terminus of subunit 8 variants affected the function of the flag tagged-variants. three (df67, df69, df70) out of six flag tagged-double charged variants failed to restore growth of m31 cells on ethanol (figure 1). the highly charged flag tag changes the nature, number, and distribution of the charged residues along the c-terminal region. the flag tag has mostly negative charged residues. the failure of the df70 variant to function, as measured by growth on ethanol (figure 1), might be due to the concentration of unfavourable negative charges along the c-terminal region of the df70 variant protein. it should be noted here that the distribution of the negatively charged residues within the chd is critical. when the adjacent negatively charged residues are not too close to the cterminus region (asp23, asp24 in df68), the flag residues seemed to be more tolerated as is indicated by the ability of the df68 variant to assemble a functional atp synthase complex, despite some deficiency in performance. moreover, when the double negatively charged substitution is further away from the c-terminus region (asp16, asp17 in df66), the flag residues are well tolerated. this is well reflected by the growth rate of df66 which is faster than the growth rate of df68, based on growth measured by generation time (table 2). to this end, the present studies suggest that a concentration of negatively charged residues at the cterminal region of subunit 8 has an adverse effect on subunit 8. this may be due to the presence of unfavourable chargecharge interactions with the positively charged residues along this region. the interaction of the adjacent positively charged residues with the residues of the flag tag seems to exhibit a gradient of opposite direction compared to that of the negatively charged residues. when the adjacent positively charged residues are closest to c-terminus (arg29, arg30 in df71) the flag residues were well tolerated as indicated by the relatively fast growth rate of df71. when the double positively charged residues are further away from the cterminus region (arg16, arg17 in df67 and arg23, arg24 in df69) the flag tag residues are not tolerated. it seems therefore that the presence of additional positively charged residues closest to the c-terminus of subunit 8 is favourable to counterbalance the presence of negatively charged flag tag residues. all of the flag tagged-subunit 8 variant proteins (df66, df68, df71) can be detected in a western blot analysis using anti-flag m2 monoclonal antibody. as shown in figure 2, however, for the same amount of mitochondrial lysate analyzed, the signal detected for the df68 lysate is much weaker. at this stage, it is not clear as to whether this particular variant protein undergoes proteolytic degradation or whether there is an assembly defect associated with this particular glucose medium ethanol medium figure 1 functional assessment of allotopically expressed flag tagged-subunit 8 variants. transformants (ftc2, df66, df67, df68, df69, df70, df71) were grown on solid selective glucose medium (left) and then transferred onto solid ethanol medium (right). the growth of the flag tagged-variants on ethanol medium indicated that the corresponding flag tagged-subunit 8 variant proteins are successfully imported into mitochondria upon their translation in the nucleocytosolic system, and are assembled into functional mitochondrial atp synthase complexes. figure 2 detection of the flag tagged-proteins of subunit 8 variants. flag tagged-subunit 8 variants contained in mitochondrial lysates were detected in a western blot analysis using the anti-flag m2 monoclonal antibody. mitochondrial lysates prepared from a strain expressing wildtype subunit 8 without flag tag (ym2) were included as controls. ftc2 is strain expressing flag tagged-wildtype subunit 8. df66, df68, and df71 are strains expressing flag taggedsubunit 8 variants. γ = subunit γ of yeast mitochondrial atp synthase, 8 = subunit 8 of yeast mitochondrial atp synthase. ym2 ftc2 df66 df68 df71 γ 8 volume 1, 2007 microbiol indones 35 df71 df70ftc2 df66 df67 df68 df69 df71 df70ftc2 df66 df67 df68 df69 variant causing a lesser amount of subunit 8 to be present in the complex. the flag-tagged variant protein of df66 showed an altered mobility in that this variant protein moves slightly faster in sds-page compared to the wildtype. a similar alteration in mobility is also clearly shown by the mutant protein of df68. it is possible therefore that this altered mobility is due to the presence of the negatively charged aspartate residues within the chd of both df66 and df68. acknowledgements this work was supported by the ausaid. i would like to thank rodney j. devenish and phillip nagley of the department of biochemistry and molecular biology, monash university, for guidance and providing facilities. references artika im. 2006. allotopic expression of a gene encoding flag tagged-subunit 8 of yeast mitochondrial atp synthase. hayati 13:36-38. bradford m. 1976. a rapid sensitive method for the quantities of proteins utilising the principal of protein-dye binding. anal biochem 72:248-254. cox gb, devenish rj, gibson f, howitt sm, nagley p. 1992. the structure and assembly of atp synthase. in: ernster l (ed). molecular mechanisms in bioenergetics. amsterdam: elsevier. p 2 8 3 3 1 5 . fronzes r, weimann t, vaillier j, velours j, brethes d. 2006. the peripheral stalk participates in the yeast at p synthase dimerization independently of e and g subunits. biochemestry 45:6715-6723. gearing dp, mcmullen gl, nagley p. 1985. chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus. biochem int 10:907-915. grasso dg, nero d, law rhp, devenish rj, nagley p. 1991. the cterminal positively charged region of subunit 8 of yeast mitochondrial atp synthase is required for efficient assembly of this subunit into the membrane f 0 sector. eur j biochem 199:203209. gray re, law rhp, devenish rj, nagley p. 1996. allotopic expression of mitochondrial atp synthase genes in the nucleus of saccharomyces cerevisiae. methods enzymol 264:369-389. klebe rj, harris jv, sharp zd, douglas mg. 1983. a general method for chemically induced transformation of bacteria and yeast. gene 25:333-341. laemmli uk. 1970. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227:680-685. lang bf et al. 1977. a simple method for the large-scale preparation of mitochondria from microorganism. anal biochem 77:110121. law rhp, devenish rj. 1988. expression in yeast of antisense rna to ade1 mrna. biochem int 17:673-679. macreadie ig et al. 1983. biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial atpase subunit 8 in saccharomyces cerevisiae. nucl acids res 11:44354451. nagley p et al. 1988. assembly of functional proton-translocating atpase complex in yeast mitochondria with cytoplasmically synthesised subunit 8, a polypeptide normally encoded within the organelle. proc natl acad sci usa 85:2091-2095. nagley p et al. 1990. subunit 8 of yeast mitochondrial atp synthase: biochemical genetics and membrane assembly. in: kim ch, ozawa t (ed). bioenergetics, molecular biology, biochemistry and pathology. new york: plenum. p 305-325. papakonstantinou t, law rhp, nesbitt ws, nagley p, devenish rj. 1996. molecular genetic analysis of the central hydrophobic domain of subunit 8 of yeast mitochondrial atp synthase. curr genet 30:12-18. roucou x, artika im, devenish rj, nagley p. 1999. bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial atp synthase. the hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex. eur j biochem 261:444-451. sambrook j, fritsch ef, maniatis t. 1989. molecular cloning − a laboratory manual. 2nd edition. new york: cold spring harbor. stephens an, roucou x, artika im, devenish rj, nagley p. 2000. topology and proximity relationships of yeast mitochondrial atp synthase subunit 8 determined by unique introduced cysteine residues. eur j biochem 267:6443-6451. 36 artika microbiol indones mi-andi kurniawan biofilm is a predominant habitat of microbe and ubiquities in aquatic environments. biofilm is formed when bacteria and other microorganisms attach on a surface and then replicate with producing extracellular polymers (costerton et al. 1995). biofilm has high sorption capacities and low production cost that attracted attention of many specialists in the field of water treatment (chubar et al. 2008; gadd 2009). however, the study concerning the utilization of biofilm formed in natural environment in development of water purification technologies such as biosorption of pollutant ions has been rarely conducted. the recent works mainly focus on the adsorption of heavy metal ions to bacteria or biofilm formed under artificial condition (almaguer-cantú et al. 2011; fang et al. 2010; joo et al. 2010; pérez silva et al. 2009; quintelas et al. 2009; vijayaraghavan and yun 2008). heavy metal ions have been one of the biggest water contamination problems (chubar et al. 2008; volesky 2007). one of the heavy metals that recently become a serious pollutant is lithium. lithium is utilized as an important compound of various things such as high-performance grease, heat-resistant ceramics, flux for welding, batteries, pharmaceuticals and nuclear fusion furnace (kaneko and takahashi 1990; miyai et al. 1978; dang and steinberg 1978; harlty et al. 1978). therefore, the cleaning up of lithium contamination in the environment is an important subject particularly in the development of environmentally and economically water purification technology in order to support sustainable development (tsuruta 2005). in this study, the polymer characteristics of biofilms collected from the surface of the stone in a lake biwa as one of the natural biofilm were clarified. the biofilms formed on stones were used for following considerations: 1) stone is the substrate that exist nearly in all aquatic environments, 2) the condition of stone as biofilm substrate is relatively stable during seasonal changes, and 3) to collect sufficient amounts of biofilms for the experiments. the utilization of this natural biofilm for biosorption of lithium was also investigated. the purpose of the use of the natural biofilm is to improve the application possibility in the real aquatic ecosystem. moreover, the comparison of lithium adsorption capacity of biofilms formed in vol.9, no.3, september 2015, p 106-112 doi: 10.5454/mi.9.3.2 this study examined the biosorption of lithium using biofilm matrices of natural microbial consortiums collected from lake biwa, japan. the characterization of the biofilm polymer as a suggested binding site of biofilm was also revealed in this study. the followings were observed as results of this study: 1) biofilm has both negatively and positively charged sites; 2) lithium adsorption by biofilm matrix is a physicochemical process mainly promoted by the electrostatic interaction between the ion and the charged sites of biofilm polymers; 3) the adsorbing lithium ion promote the desorption of ions from biofilms through ion exchange mechanism; 4) biofilms components changed seasonally and seems to affect the ability of biofilm to adsorb ions. according the results of this study, natural biofilm may become a promising biosorbent in the biosorption of lithium ion. key words: biofilm, biofilm polymer, biosorption, lithium ion studi ini mengkaji biosorpsi ion lithium dengan menggunakan matriks biofilm dari konsorsium mikroba yang tumbuh alami di danau biwa, jepang. karakteristik polimer biofilm yang diduga menjadi area pelekatan ion juga diungkapkan dalam studi ini. berikut ini adalah hasil-hasil yang didapat studi ini: 1) biofilm memiliki area bermuatan listrik positif dan negatif; 2) adsorpsi lithium oleh matriks biofilm adalah proses fisika-kimia sebagai hasil interaksi elektrostatis antara ion lithium dan muatan listrik di polimer biofilm; 3) penyerapan ion lithium mengakibatkan pelepasan ion dari biofilm melalui mekanisme pertukaran ion; 4) komposisi biofilm berubah sesuai musim dan tampak mempengaruhi kemampuan biofilm untuk menyerap ion. berdasarkan hasil dari studi ini, biofilm alami bisa menjadi biosorben yang menjanjikan dalam proses biosorpsi ion lithium. kata kunci: biofilm, biosorpsi, ion lithium, polimer biofilm available online at http://jurnal.permi.or.id/index.php/mioline issn 1978-3477, eissn 2087-8575 *corresponding author; phone: +62-81235352299, fax:; email: andi_k@ub.ac.id 1 faculty of fisheries and marine science, universitas brawijaya, jalan veteran, malang, indonesia; 2 college of life science, universitas ritsumeikan, 1-1-1 noji-higashi, kusatsu, shiga, japan; 3 coastal and marine research centre, universitas brawijaya, jalan veteran, malang, indonesia 1,3 2 andi kurniawan * and tatsuya yamamoto biosorption of lithium using biofilm matrix of natural microbial consortium different seasons was also conducted. materials and methods sampling site and sample preparation. the samples used in this study were collected from the shore of the southern basin (akanoiwan) of lake biwa. this lake is the largest lake in japan located in the central part of japanese archipelago (lat. 35° 15’ n, long. 136° 05’ e). samples of the biofilm were collected in winter (november 2009) and spring (april 2010) as the previous study founded that the biofilm thickness reach the maximum amount in these seasons (tsuchiya et al. 2009). stones were taken from the depth of 70-100 cm and brought back to the laboratory in a plastic container filled with nearby lake water; the container was maintained at 4 °c. the biofilms on the surfaces of the stones (ca. 100 stones in each sampling) were removed using a toothbrush and suspended in sterilized distilled water. the biofilm pellets were prepared by centrifuging (8 000 ×g at 4 °c for 10 min) the biofilm suspensions. dry weight of biofilms. the pellet of biofilms (approximately 1 wet-g) were taken and then dehydrated for 3 d until the weight was stable to give a dry weight (sissons et al. 2000). electrophoretic mobility. biofilm was washed three times with 10 mm nacl aqueous solution by centrifugation (8 000 ×g at 4 °c for 10 min), and the supernatant was discarded. the obtained biofilm pellet (ca. 0.03 g) was suspended in 1 ml of 10 mm nacl aqueous solution. the suspension was mixed vigorously with a vortex for 5 min, then sonicated (2510j-mt, yamato scientific, tokyo, japan; 42 khz, 125 w) for 10 min, followed by the vortex for 10 s. the obtained suspension was mixed with 10 mm of pbs at a ratio of 1:19 and used to analyze the electric charge of the biofilm polymer. the electrophoretic mobility (epm) of the biofilm was measured on a zetasizer nano-z (malvern instruments, ltd., worcestershire, england) in phosphate-buffered saline (pbs) varying in ph values from 2.0 to 9.0. the ph of the buffer was adjusted with 20 mm of hcl or naoh. the phs of pbs before and after the addition of biofilm and after measurement of epm were recorded. acid-base titration. biofilms were washed six times with distilled water by centrifugation. one gram of the pellet of biofilm was placed in a plastic cup, and distilled water was added to give a total weight of 40 g. then, 10 mm of hcl or 10 mm of naoh aqueous solution was titrated onto the samples using an automatic titration machine (dl50, mettler, toledo, oh, usa). the ph changes were recorded and analyzed. distilled waters (40 gram) were also subjected to titration. from the results, the adsorbed + amounts of h and oh by biofilms were calculated (freifelder 1985) as follows: + + where, c(h ) is the uptake capacity of h by unit -1 weight of biofilm (µmol wet-g ), ph(dw) and ph(bf) is the ph value of distilled water and biofilm suspension when hcl solution was added, respectively, v is the volume of sample solution (l) and w is the wet-weight of biofilm (gram). when naoh solution was added, the uptake capacity of oh ion, c(oh ), by unit weight -1 of biofilm (µmol wet-g ), was given by the similar equation as follows: + by the above equations the uptake capacity of h and oh ions by biofilm was calculated. biosorption experiment. the procedures of the biosorption experiment in this study were modified from previous works (vijayaraghavan and yun 2008 and gadd 2009). biofilms were washed six times with 5 mm phosphate-buffered saline (pbs) of ph 7 by centrifugation. the biofilm pellets were stored at -40 °c until ion adsorptions analyses were conducted. 1 wet-g of the biofilm pellet was resuspended in 50 ml of 5 mm pbs of ph 7. the suspension was mixed vigorously with a vortex for 5 min, and then sonicated for 10 min, followed by the vortex for 30 s. then, 5.0 ml of 20 mm of solution of reagent grade licl, prepared by dilute the chemical compound (wako pure chemical industries, osaka, japan) in 5 mm pbs of ph 7, was added to the suspension. the temperature of the suspension was maintained at 25 °c using a water thermostat, and mixed well using magnetic stirrer. the aliquots of the suspension were taken after 1 min-300 min, and then centrifuged (15 000 ×g at 4 °c for 1 min) to separate the supernatant and the pellet. the ion concentration in the solution was measured using a capillary electrophoresis method (capi-3300, otsuka electronics, osaka, japan). the adsorbed amount of ion to biofilm was calculated from the difference between ion concentration in the supernatant and in the control (only pbs and ion). the desorbed ion (different species from added ion) from biofilm due to addition of ion was also investigated. as a control of investigation of desorbed ion, same amount of biofilm was resuspended in 50 ml of 5 mm pbs of (1) (2) + -ph(dw) ph(bf) c(h )=(10 -10 )v/(w) -ph(bf) ph(dw) c(oh )=(10 -10 )v/(w) volume 9, 2015 microbiol indones 107 ph 7, and then was treated with the same treatment described above but without ion addition. the adsorption experiment was also conducted using one dry-g of strong acidic resin (sp-650m; toyopearl, tokyo, japan) and weak acidic resin (cm-650m; toyopearl). results electrophoretic mobility. the epm of biofilm was shown as a function of ph (fig 1). at ph 7, the biofilm showed negative epm values, whereas these values decreased at smaller ph values, especially with greater extent around ph 4. at ph 2, the biofilm showed positive epm value. the ph values of the buffer solution shifted to a greater value from its original value when biofilm samples were added to the buffer solution; e.g., the original ph values of 4.0 and 5.0 shifted to 5.6 and 5.9, respectively after adding biofilm samples. acid-base titration. acid base titrations to the biofilm and distilled water were conducted (fig 2). the acid-base titration curve of biofilm was different from that of distilled water (as a background solution). from the results of acid-base titration (fig 2), the uptake + capacities of h and oh by biofilm were calculated (fig 3). the maximum difference of phs in the acidbase titration curve between biofilm and distilled water were at around ph 4 for hcl addition and at around ph 11 for naoh addition. similarly, the maximum uptake capacity of biofilm appeared at ph 3 4 for proton adsorption and ph 10 11 for hydroxyl ion adsorption (fig 3). biosorption of lithium. the time course of lithium ion biosorption using biofilm was shown in fig 4. the adsorption process was very fast as the adsorption amount reached, within 1 min, to as much value as observed in the later stage. in order to evaluate the potential of biofilm as an adsorbent in biosorption of lithium ion, the adsorbed amount of ions to biofilms was compared with strongly and weakly cation exchange resins as one of the common adsorbents in the adsorption of heavy metal ions such lithium (table 1). the adsorbed amount of ions was greater to biofilm than those to resins for a dry gram of these substrates. moreover, various kinds of ions were suggested to be desorbed from biofilms or cation exchange resins by adsorbing ions (table 2). the time course of adsorption of lithium to biofilm sampled in spring was also studied (fig 5). the result was compared with the adsorption to biofilm sampled in winter. the adsorbed amount of ions to the biofilms -1 sampled in winter (85.3 µmol dry-g ) was greater compared with those of the biofilms sampled in spring -1 (19.4 µmol dry-g ). discussion electric charges and functional groups of biofilm. a biofilm suspension was put in an electric field, and the electrophoretic velocity was measured under various ph conditions. the electrophoretic mobility (epm) was normalized by dividing with the electric field strength to obtain the epm values (fig 1). table 1 adsorbed of lithium to biofilm and cation exchange resins adsorbent + -1 absorbed li (µmol dry-g ) biofilm (winter) 85.3 18.2 33.2 strongly cation exchange resin weakly cation exchange resin table 2 adsorbed and desorbed amount of ions to and from biofilm + absorbed amount of li -1 ( µmol dry-g ) desorbed ions -1 ion amount (µmol dry-g ) 2+ ca 14.6 33.01 42.8 2+ mg + na + k 10.012 85.3 108 kurniawan et al. microbiol indones + fig 3 uptake capicity of h (black column) or oh (white column) per unit weight of the biofilm. a b fig 1 epm of biofilm measured in various ph. -8 2 1 -1 e p m ( x 1 0 m v s ) ph 2 1 0 -1 -2 -3 2 4 6 8 10 p h 0 2 4 6 8 10 12 14 volume of hcl added (ml) 0.25 0.5 0.75 1 1.25 1.5 1.75 2 p h 0 2 4 6 8 10 12 14 volume of naoh added (ml) 0.25 0.5 0.75 1 1.25 1.5 1.75 2 ph -1 u p ta k e ca p ac it y ( µ m o l w et -g ) 2. 22. 5 2. 23. 0 3. 03. 5 3. 54. 0 4. 04. 5 4. 55. 0 5. 05. 5 8. 08. 5 8. 59. 0 9. 09. 5 9. 510 .0 5. 56. 5 10 .0 010 .5 10 .5 -1 1. 0 11 .0 -1 1. 5 20 25 15 10 5 0 fig 2 (a) acid titration to biofilm and distilled water; (b) base titration to biofilm and distilled water. : distilled water and : biofilm. volume 9, 2015 microbiol indones 109 the decrease of negative epm values from ph 7 to smaller phs particularly with greater extent around ph 4 seems to be due to the depression of ionization of functional groups, such as carboxylic group whose pka is around ph 4, at the acidic condition (freifelder 1985). positive epm value at ph 2 indicates the existence of positively charged functional group, supposed to be amino group, in the biofilm matrix. the shifted of the original ph values after adding biofilm samples suggested the decreasing in the concentration of proton by adding the biofilm sample. it seems that proton was used to de-ionize acidic functional group in the biofilm. carboxylic acid is the most probable as the functional group as discussed above and further confirmed by the buffer action as a result of acid-base titration (freifelder 1985; vijayaraghavan and yun 2008). the differences of acid-base titration curves of biofilm to that of distilled water (fig 2) reveal the presence of ionizable functional groups associated with biofilm polymer. the results show that the maximum difference of phs in the acid-base titration curve between biofilm and distilled water were at around ph 4 for hcl addition and at around ph 11 for naoh addition. similarly, the maximum uptake capacity of biofilm appeared at ph 3-4 for proton adsorption and ph 10-11 for hydroxyl ion adsorption (fig 3). it seems that at around ph 4 and ph 11 there are some functional groups in biofilm polymer having ability to adsorb proton and hydroxide ions respectively. the peak at ph 4 may indicate the existence of carboxyl groups (pka = ca. 4), and that at ph 11 the existence of amino groups (pka = ca.11). the existences of the functional groups explain fig 4 lithium accumulation to winter biofilm. the experiments were repeated 3 times, independently (average values are shown). bars represent the standard deviation. fig 5 lithium accumulation to spring biofilm. the experiments were repeated 3 times, independently (average values are shown). bars represent the standard deviation. 0 50 100 150 200 250 300 350 time (min) 100 90 80 70 60 50 40 30 20 10 0 -1 l i a cc u m u la te d ( µ m o l d ry -g ) 0 100 100 80 70 60 50 40 30 20 10 0 -1 l i a cc u m u la te d ( µ m o l d ry -g ) 0 50 100 150 200 250 300 350 time (min) 110 kurniawan et al. microbiol indones well the epm change along with ph as shown in fig 1. in case of greater amount of negative charge than positive charge in the biofilm, the biofilm would have a net negative charge. the negative epm of biofilm at ph 7 corresponds to this case (greater amount of negative charge than positive charge). thus, the biofilm matrix carries both positive and negative charge in the environment of lake water (ph value ca. 7) (tsuchiya et al. 2009). biosorption of lithium. the time course of lithium ion biosorption using biofilm was shown in fig 4. the adsorption process was very fast as the adsorption amount reached, within 1 min, to as much value as observed in the later stage. this indicates that the lithium adsorption by biofilm is a physicochemical process where the electrostatic interaction between ions and the charged sites in biofilm matrixs was a driving force to adsorb ions to inside biofilm. in order to evaluate the potential of biofilm as an adsorbent in biosorption of lithium ion, the adsorbed amount of ions to biofilms was compared with strongly and weakly cation exchange resins as one of the common adsorbents in the adsorption of heavy metal ions such lithium (table 1). the adsorbed amount of ions was greater to biofilm than those to resins for a dry gram of these substrates. this indicates more charged sites in the biofilm or greater attractive force by biofilm matrixs compared to ion exchange resins. this result suggests that biofilm may become a promising adsorbent for the biosorption of pollutant ions such as lithium ions (tsuruta 2005; volesky 2007). various kinds of ions were desorbed from biofilms (table 2). it seems that adsorbing lithium replaced the previously adsorbed ions that bound to the negatively charged site of the biofilms. the adsorbing ion seemed to promote desorption of ion from biofilms through ion exchange mechanisms. comparison to spring biofilm. the time course of adsorption of lithium to biofilm sampled in spring was also studied (fig 5). the result was compared with the adsorption to biofilm sampled in winter. the adsorbed amount of ions to the biofilms sampled in -1 winter (83.5 µmol dry-g ) was greater compared with those of the biofilms sampled in spring (19.4 µmol dry-1 g ). this indicated that more adsorption sites in biofilm sampled in winter compared with biofilms sampled in spring. this result is correlated well with the result of microscopic observation, that reveal the biofilms sampled in winter contained more polymer like substances (fig 6), it seems that the adsorption sites of cation (negatively charge sites) was mostly exist in the polymer like substances. thus, the biofilms sampled in winter (that have more polymers like substances) (tsuchiya et al. 2009) could adsorb greater amount of ions than biofilms sampled in spring. the difference of composition of biofilms indicates that the biofilms components change seasonally along with the change in the environmental factors, such as temperature and light intensity (hiraki et al. 2009; tsuchiya et al. 2009). this should be considered in the utilization of biofilm in the biosorption of pollutant ions. one of the characteristics of the natural biofilm matrix that important to be elaborate more is the microbial composition inside the biofilm matrix. therefore, the microbial composition in the natural biofilm matrix is subjected in the further study. in conluson, the followings were observed as results of the present study: 1) biofilms have both negatively and positively charged sites; 2) the lithium adsorption by biofilm is a physicochemical process where the electrostatic interaction between the ion and the charged sites in biofilm polymers is a main driving force; 3) the adsorbing ion promote the desorption of ions from biofilms through ion exchange mechanism; 4) the biofilm components change seasonally and fig 6 microscopic images of biofilm matrix samples. 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lithium using various microorganisms. journal of biosci. and bioeng. 100(5):562-566. doi:10.1263/jbb.100.562. vijayaraghavan k, yun ys. 2008. bacterial biosorbents and biosorption. biotecnol adv. 26(3):266-291. doi:10.101 6/j.biotechadv.2008.02.002. volesky b. 2007. biosorption and me. water res. 41(18): 4017-4029. doi:10.1016/j.watres.2007.05.062. 112 kurniawan et al. microbiol indones 1: 106 2: 107 3: 108 4: 109 5: 110 6: 111 7: 112 6 rev dyah (61-64).pmd formation and pathogenicity variation of oospores of phytophthora capsici infecting black pepper dyah manohara indonesian research institute for medicinal and aromatic crops, pusat penelitian dan pengembangan perkebunan, jalan tentara pelajar no 3, bogor 16111, indonesia phone: +62-251-321879, fax: +62-251-327010, e-mail: dyah_manohara@yahoo.com phytophthora capsici leonian is the causal agent of foot rot disease of black pepper (piper nigrum l.). foot rot disease is the most destructive disease which can cause significant economic losses of black pepper. two mating types of p. capsici were found in black pepper plantations in lampung. this research was aimed at examining the effects of temperature, light, and polycarbonate membrane on oospore formation. also the effect of light on germination and determining both mating types of 30 randomly selected progenies. the results showed that oospores were produced through either hyphal contact or without any contact. oospores were produced abundantly on v8 agar in the dark at 16-24 oc. oospores germinated after 3-weeks incubation in the dark followed by 1-week incubation under tl fluorescent light. all progeny were heterothallic, and consisted of 16 isolates of a1 mating type and 14 isolates of a2 mating type. there was no correlation between mating type categories and their pathogenicities on black pepper leaves. twenty-six progeny isolates may infect unwounded pepper leaves, whereas another four isolates may only infect wounded pepper leaves. pathogenicities of all progenies were lower than those of their parents. key words: phytophthora capsici, oospore, black pepper, lampung _____________________________________________ phytophthora capsici is a pathogenic causal agent of foot rot disease on black pepper (piper nigrum l.). the attack of p. capsici on the root and collar region causes sudden plant death. foot rot disease on black pepper was found for the first time in lampung in 1885 (muller 1936). at present this disease has spread to all black pepper plantations in indonesia and is the main constraint in black pepper production. p. capsici is a heterothallic fungus. sexual reproduction can occur when there are two compatible mating types (a1 and a2) for oospore formation. two mating types of p. capsici from black pepper plants were reported to have been found in lampung (manohara and sato 1992). ko (1980) used polycarbonate membrane to prove that oospore formation of p. cinnamomi, p. parasitica, and p. palmivora could take place without contact hyphal of the two compatible mating types. the formation of oospores of phytophthora is affected by several environmental factors such as temperature and light. harnish (1965) and brasier (1969) stated that light inhibited the formation of oospores but stimulated sporangia formation. optimum temperature for the formation of oospores of phytophthora differed, depending on the species. oospore germination is affected by light, oospore maturity (age), temperature, enzyme treatment and chemical treatment (ribeiro 1983). in general, the quantity of germinating oospores will increase in line with the increase of an oospore’s age. according to hord and ristaino (1991), the germination rate of oospores of p. capsici originating from chili pepper plants improved by 3% at the age of 14 days, and became 20% at the age of 60 days. the light and temperature requirement during the oospore germination process varied, depended on the species of phytophthora. oospores are very important in epidemiology and etiology of phytophthora because they are able to survive in extreme environmental conditions. the oospore is also the stage where genetic recombination occurs which can create a new individual more pathogenic than its parents (hausbeck and lamour 2004). the objectives of this research are to study: (i) the effects of temperature and light on oospore formation, (ii) oospore formation with polycarbonate membrane, (iii) oospore germination, and (iv) mating types and pathogenicities of progeny isolates. materials and methods fungal material. p. capsici isolates were obtained from diseased black pepper plants in north lampung, i.e. isolate n2 (a1 mating type), and in south lampung, i.e. isolate n4 (a2 mating type). the two isolates are assumed to have the same polyploidy i.e. diploid. these isolates were grown and maintained in v8 agar (200 ml v8, 1.0 g caco 3 , 20 g agar, and 800 ml aquadest). the effect of temperature on oospore production. culture disks of each of the isolates, n2 and n4 (diameter 0.5 cm), were placed in pairs facing each other on v8 agar with 5-cm distance from each other, and then incubated at 16, 20, 24, 28, and 32 oc. every treatment was replicated three times. colony growth was observed every day until both isolates made contact. after the culture reached the age of 14 days, the number of oospores formed was counted by taking two samples of media disks (diameter 0.5 cm) from each petri dish. the sample was placed on object glass, stained using lactophenol trypan blue with simultaneous heating, and then covered with a cover glass. the number of oospores was counted employing light microscope at 200 x magnification. every disk was examined at three randomly chosen places. the effect of light on oospore production. culture disks of isolates n2 and n4 (diameter 0.5 cm) were placed facing microbiology indonesia, august 2007, p 61-64 volume 1, number 2 issn 1978-3477 fig 1 the oospores of p. capsici and their germination. a. young oospore, b immature oospore, c. mature oospore stained with lactophenol tryhpan blue, d. hypha formation of germinating oospores, e and f. sporangium was formed by germinating oospores. table 1 incubation treatment for the germination of oospores of p. capsici incubation in the dark (20 oc) incubation under the light (26-28 oc) 2 weeks 2 weeks 2 weeks 2 weeks 3 weeks 3 weeks 3 weeks 3 weeks 1 week 2 weeks 3 weeks 4 weeks 1 week 2 weeks 3 weeks 4 weeks table 2 the effect of temperature on the formation of oospores of p. capsici incubation temperature (oc) colony contact (day) number of oosporesa 1 6 2 0 2 4 2 8 3 2 4 3 3 3 4 44.16 85.67 46.67 14.33 1.00 athe average of 18 microscope view area with 200x magnification. each other on v8 agar at a 5-cm distance, and then incubated under several light treatments using a tl fluorescent lamp with 600 lux intensity and placed at a 40-cm distance from the petri dish lid. the durations of light exposure are 0, 6, 12, 18, and 24 h respectively. temperature inside the incubation room ranged between 26-28 oc. each treatment was replicated three times. the determination of oospore number was carried out as given above. oospore production with polycarbonate membrane. isolates n2 and n4 were grown on v8 agar and incubated for 3 days in the dark. culture disks of isolate n2 (diameter 1.0 cm) were obtained from the edge of a growing colony then placed in the middle of a sterile petri dish, covered with a polycarbonate membrane(cpr, 0.2 µm, 90 mm diameter; nucleopore corporation, pleasanton, california, usa). on top of this was placed another culture disk of isolate n4. for comparison, an opposite treatment was conducted using the same isolates (ko 1980). incubation was carried out in the dark at 20 oc. after 7 days, the number of oospores formed during incubation was observed. oospore germination. oospores were released from agar media by crushing in a blender at 5000 rpm for 5 min. the oospore suspension was then centrifuged at 2000 rpm for 5 min. the sediment was filtered employing a gradual sieving filter with pore diameter of 75, 38, and 20 µm respectively. the remaining oospores left in 20 µm filter were then separated and washed with 0.5% (wt/vol) kmno 4 for 20 min. the oospore suspension was then immersed in warm water (36-38 oc) for 2.5 h (wahyuno et al. 1995). the oospore suspension was then diluted to obtain about 20 oospores per inoculating loop tip, and then streaked on basalt media (20 mg glucose, 100 mg lecithin, and 10 ml basalt solution). the incubation treatment consisted of 8 lighting combinations (table 1). the percentage of oospore germination was estimated visually. mating types and pathogenicities of progeny isolates on black pepper leaves. thirty progeny isolates were obtained from randomly selected germinating oospores (single spore isolation), isolated on v8 agar. the mating types of progeny isolates were determined using pairing method by pairing the isolates with known-tester isolates (a1 or a2 mating type) on v8 agar. the presence of oospores was observed. pathogenicities of progeny isolates were tested by inoculating them on black pepper leaves. these progeny isolates were grown on v8 agar and incubated for 7 days under continuous light. an inoculum disk (diameter 0.5 cm) was placed on the lower side of unwounded black pepper leaf surface and then one drop of sterile water was added. these leaves were placed inside a plastic container whose base had been covered by moist tissue paper. measurement of leaves spots formed was conducted after 4-day incubation at room temperature. results optimum temperature for oospore formation. oospore formed within the temperature range of 16-32 oc (table 2). when incubated at 16-24 oc, sexual reproduction structures began to appear one day after both colonies made contact. however at 28 and 32 oc, the structures were formed 4 days after both colonies made contact. the antheridia were amphigynous and colorless (hyaline). oogonia had spherical or subspherical form and were colorless (hyaline). oospores were colorless (hyaline) at the young stage and became brownish when mature (fig 1a,b). oospores were generally globose in shape with thick walls (fig 1c). the optimal temperature for oospore formation was 20 oc (table 2). the role of light in oospore production. oospore production was very sensitive to light. the contact of both colonies occurred 4 days after incubation, and on the fifth day, several reproduction structures had been formed a b c d e f 62 manohara microbiol indones table 3 the effect of the duration of light exposure on the formation of oospores of p. capsici light exposure (h) number of oosporesa 0 6 1 2 1 8 2 4 51.00 3.33 0.00 0.00 0.00 athe average of 18 microscope view area with 200x magnification table 4 formation of oospores of p. capsici with polycarbonate membrane combinationa number of oosporesb top x bottom top bottom n 2 n 4 n 2 n 4 n 4 n 2 n 2 n 4 89.5 58.5 0 0 52.4 86.4 0 0 apolycarbonate membrane was inserted between the two isolates, bthe average of 18 microscope view area with 200x magnification. table 5 oospore germination of p. capsici incubation treatment in the dark (20 oc) under the light (25-27 oc) oospore germination (%) 2 weeks 2 weeks 2 weeks 2 weeks 3 weeks 3 weeks 3 weeks 3 weeks 1 weeks 2 weeks 3 weeks 4 weeks 1 weeks 2 weeks 3 weeks 4 weeks 0.00 0.00 0.00 0.00 29.02 39.27 52.10 20.56 (in the dark). the treatment of a 6-hour exposure to light inhibited oospore formation, while exposure to light for more than 6 hours caused no oospore formation (table 3). the effect of polycarbonate membrane. this is impenetrable by the hyphae of p. capsici. this can be proven by inserting the membrane between culture disks of p. capsici and fresh v8 media. it turned out that oospores continued to be formed on isolate disk n2 and n4 even though polycarbonate membrane had been inserted between them (table 4). this result revealed that oospores formation took place without direct contact between the hyphae of two compatible mating types. oospore formation seemed to be induced by the presence of compounds that could penetrate the membrane. compared with isolate n4, the isolate n2 always produced the most oospores regardless of the position of pairing (table 4). oospore germination. oospore germinated when they were first incubated for 3 weeks in the dark, followed by exposure to continuous light for 1 week or more (table 5). oospores germinated to form germ tubes that penetrated oospore walls or passed through antheridia. the number of germ tubes produced could be more than one. the germ tubes grew longer and became hypha, or created sporangium at the tip. (fig 1d, e, f). the treatment of incubation in the continuous dark caused no oospore germination (data are not shown). a longer light treatment increased the ability of oospore to germinate. the number of germinating oospores increased in line with the duration of light exposure for up to the 3 weeks (table 5). four-weeks of exposure to light resulted in a lower number of germinating oospores compared to 3-week light exposure (table 5). further assessment found that this treatment produced many empty oospores, probably resulting from lysis. mating types and pathogenicities of progeny isolates. all progeny isolates were heterothallic; they formed oospores with their compatible tester. sixteen progeny isolates belonging to the a1 mating type formed oospores with isolate n4, while the remaining 14 isolates belong to the a2 mating type, formed oospores with isolate n2 (table 6). the symptom of infection on black pepper leaves could be seen as small spots which were brown-blackish color and which appeared 24 h after inoculation with parental isolates (n2 or n4). on the 4th day, the leaves spot areas are 17.45 cm2 and 15.87 cm2 respectively. eight progenies (hy 9, hy 11, hy 14, hy 18, hy 26, hy 27, hy 29, and hy 30) caused symptoms similar to their parents on the 2nd day after inoculation. on the 4th day, the leaves spot area ranged between 7.53-10.45 cm2. four progenies (hy 10, hy 13, hy 16, and hy 19) did not show any symptoms. however, if the leaves were wounded before inoculation, these four isolates were then able to infect and produce small spots after 4-days incubation. from the 30 progenies tested, it was evident that none of the isolates had the same or more pathogenicity compared to their parental isolates. most of them (18 progenies) produced symptom less than 3.00 cm2 (table 6). discussion the formation of oospores of p. capsici in vitro is clearly affected by temperature and light. light was a constraining factor for oospore formation. in the dark, oospores could be produced within the temperature range of 16-32 oc. the optimum temperature for oospore formation was 20 oc. this temperature was lower than the optimum temperature for vegetative growth, i.e. 24-28 oc (manohara 1988). this findings are similar to brasier’s (1969) results which revealed that the optimum temperature for vegetative growth of table 6 the variety of 30 progeny isolates of p. capsici and their pathogenicities on black pepper leaves progeny progeny isolates isolates (f1) (f1) hy 1 hy 2 hy 3 hy 4 hy 5 hy 6 hy 7 hy 8 hy 9 hy 10 hy 11 hy 12 hy 13 hy 14 hy 15 hy 16 hy 17 hy 18 hy 19 hy 20 hy 21 hy 22 hy 23 hy 24 hy 25 hy 26 hy 27 hy 28 hy 29 hy 30 n2 (parent) n4 (parent) a 1 a 2 a 1 a 1 a 2 a 2 a 2 a 1 a 1 a 2 a 1 a 1 a 2 a 2 a 1 a 1 a 2 a 2 a 1 a 1 a 2 a 1 a 2 a 1 a 1 a 2 a 1 a 2 a 2 a 1 a 1 a 2 2.10abc 6.63def 1.48ab 5.66de 1.20ab 0.78a 0.52a 0.39a 9.31fg 0.00a 9.32fg 2.82abc 0.00a 9.44fg 4.53cd 0.00a 0.93ab 7.85efg 0.00a 0.27a 0.53a 1.15ab 0.77a 0.11a 0.10a 7.53efg 9.62fg 3.90bcd 8.25efg 10.45g 1 7 . 4 5 h 1 5 . 8 7 h mating t y p e leaf spot (cm2)a mating t y p e leaf spot (cm2)a number followed by the same letters are not significantly different at 5% dmrt, aunwound inoculated volume 1, 2007 microbiol indones 63 p. palmivora was 27.5-30 oc, while sexual reproduction occurred in the range of 16-20 oc. oospore formation could occur without hyphal contact between the two mating types. it was probable that the two isolates produced compounds that induced each other to form gametangium and then oospores. this compound was then declared to be a sexual hormone. the a1 mating type produced α1 sexual hormone whereas the a2 mating type produced α2 sexual hormone (ko 1980). furthermore shen et al. (1983) reported that this sexual hormone was not specific. for example, p. infestans with an a1 mating type will produce oospores if paired with an a2 mating type such as p. parasitica, p. capsici, and p. palmivora besides with p. infestans itself. jee et al. (2002) found that phytol was highly stimulatory to oospore formation of p. cactorum and p. parasitica. elliot (1983) suspected that the role of the sexual hormone was in the inhibition of steroid synthesis which controls sexual reproduction. production of sexual hormone was affected by temperature and light. yu et al. (1981) reported that the optimum temperature for p. colocasiae to produce the hormone was 25 oc, at which temperature it would be able to stimulate p. parasitica to form oospores in large quantity. my research reported in this paper showed that oospores of p. capsici from black pepper plants would be mature after 3-weeks incubation in the dark, but that germination would require light. one-week exposure to light resulted in 29.02% germination. increasing the duration of the exposure to up to 3 weeks would increase the percentage to 52.10%. germinating oospores formed germ tubes that would develop into hyphae and mycelia or sporangia which would in turn form zoospores. according to banihashemi (2004), oospore germination is determined by the level of maturity of oospores as the internal factor, and an external factor such as exposure to light, whose duration depended on the species of phytophthora. observation by banihashemi (2004) proved that p. cactorum needed dark conditions during the formation and maturation of oospores. the germination would then occur if incubated under continuous fluorescent illumination, but would not occur without the exposure to light. the germination of oospores of p. cactorum was only 3% if incubated in the dark, while-light exposure would increase it to 38%. oospores of p. infestans germinated up to 70% under cool white fluorescent light (chang and ko 1991). the opposite happened in the germination process of oospores of p. capsici obtained from chili pepper plants. germinating oospores would be abundant if light treatment was applied during both formation and germination process (hord and ristaino 1991). my work reported here, shows that the pathogenicities of progenies seemed to have no correlation with mating types. pathogenicities of all progenies were lower than those of their parents. the same result was described by mayton et al. (2000) who did the mating with p. infestans. from 53 progenies, 52 isolates had lower pathogenicities compared to their parents. abu-el samen et al. (2003) found a genotypic variability among the asexual progenies of p. infestans. although oospores as the sexual reproduction have been considered as the important resting spore, the stage where genetic recombination occurs and the primary source of inoculum in the field, but little information is reported about the influence of some factors which stimulate the formation, germination and the pathogenicity of their progenies. thus it is an important precedent for research in p. capsici especially on black pepper. references abu-el samen pm, secor, ga, gudmestad. 2003. genetic variation among asexual progeny of phytophthora infestans detected with rapd and aflp markers. plant pathol 52:314-325. banihashemi z. 2004. light-dependent oospore germination of phytophthora cactorum in the presence of susceptible host plant. j phytopathol 152:683-686. brasier cm. 1969. formation of oospores in vivo by phytophthora palmivora. trans br mycol soc 52:273-279. chang tt, ko wh. 1991. factors affecting the germination of oospores of phytophthora infestans. j phytopathol 133:29-35. elliott cg. 1983. physiology of sexual reproduction in phytophthora. in: erwin dc, bartnicki-garcia s, tsao ph (eds). phytophthora: its biology, taxonomy, ecology, and pathology. st. paul: american phytopathological society. p 71-80. harnish wn. 1965. effect of light on production of oospores and sporangia in species of phytophthora. mycologia 57:85-90. hausbeck mk, lamour kh. 2004. phytophthora capsici on vegetable crops: research progress and management challenges. plant dis 88:1282-1303. hord mj, ristaino jb. 1991. effects of physical and chemical factors on the germination of the germination of oospores of phytophthora capsici in vitro. phytopathology 81:1541-1546. jee hj, tang cs, ko wh. 2002. characterization of phytochemicals stimulatory to sexual reproduction in phytophthora cactorum and p. parasitica. bot bull acad sin 43:203-210. ko wh. 1980. hormonal regulation of sexual reproduction in phytophthora. j gen microbiol 116:459-463. manohara d. 1988. ekobiologi phytophthora palmivora (butler). penyebab penyakit busuk pangkal batang lada (piper nigrum l.) [dissertation]. bogor: institut pertanian bogor. manohara d, sato n. 1992. morphological and physiological observation on phytophthora isolates from black pepper. indust crops res j 4:14-19. mayton h, smart cd, moravec bc, mizubuti esg, muldoon ae, fry we. 2000. oospore survival and pathogenicity of single oospore recombinant progeny from a cross involving us-17 and us-8 genotype of phytophthora infestans. plant dis 84:1190-1196. muller hra. 1936. het phytophthora-voetrot van pepper (piper nigrum l.) in nederlanch-indie. mededelingen van het institut voor plantziekten, no. 88, p 79. ribeiro ok. 1983. physiology of asexual sporulation and spore germination in phytophthora. in: erwin dc, bartnicki-garcia s, tsao ph (eds). phytophthora: its biology, taxonomy, ecology and pathology. st. paul: american phytopathological society. p 55-70. shen cy, bower la, erwin dc, tsao ph. 1983. formation of sex organs in the a1 mating type of phytophthora infestans induced chemically by a2 isolates of other species of phytophthora. can j bot 61:1462-1466. wahyuno d, manohara d, mogi s. 1995. menginduksi perkecambahan oospora phytophthora capsici. in: strengthening research on disease of industrial crops in indonesia [annual report]. bogor: research institute for spice and medicinal crops. p 37-42. yu jy, chang hs, ko wh. 1981. factor affecting the induction of sexual reproduction in phytophthora parasitica by phytophthora colocasiae. j gen microbiol 123:249-252. 64 manohara microbiol indones tresnawati_370 apparent induction of xylanase by bacillus pumilus pu4-2 using pretreated substrates sherly widjaja1, tresnawati purwadaria2*, and pius pertumpun ketaren2 1faculty of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia 2indonesia research institute for animal production, pobox 221, bogor 16002, indonesia bacillus pumilus pu4-2 produces xylanase (β-1,4-d-xylan xylanohydrolase; ec 3.2.1.8) in wheat pollard with high activity. water and naoh-soaked pollard were used in this research to enhance the production of assayable enzyme. enzyme activity was produced in minimal media containing 3% w/v untreated or water or naoh-soaked pollards in 250 ml flasks incubated on shaker incubator at 30°c and 150 rpm for 36 h. the production was also compared to untreated oatmeal known as an inducer substrate. the highest xylanase activity was obtained by using untreated pollard as a sole carbon source. the enzyme activity was 157 u ml-1 with specific activity at 718 u mg-1. xylanase production using different soaking time for water pretreated pollard also confirmed that untreated pollard was the best inducer. the production was not influenced by different water soaking times used to remove reducing sugar. although pretreatment decreased the reducing sugar, the reduction did not enhance assayable enzyme levels. the production was best induced by the soluble oligosaccharides of untreated pollard. we conclude that b. pumilus pu4-2 was able to produce xylanase with reducing sugar content up to 660 ppm present in production medium. with this reducing sugar level, repression of enzyme production was not detected in the production medium. key words: xylanase, bacillus pumilus pu4-2, wheat pollard, pretreatment _____________________________________________ ________________________ *corresponding author, phone: +62-251-240752/3, fax: +62-22-2504154, e-mail: tpurwadaria@yahoo.co.uk plant cell walls are the major reservoir of fixed carbon sources in nature. they have three major components consisting of cellulose (insoluble fibers of β-1,4-glucan), hemicellulose (noncellulosic polysaccharides including glucans, mannans, and xylans), and lignin (a complex polyphenolic structure) (wong et al.1988; tuncer 2000). these structures commonly referred as lignocellulose with each concentration a variable; cellulose 30-45, hemicellulose 30, and lignin 15-30% w/ w , respectively (tuncer 2000). xylan is one of the major polysaccharides, along with cellulose of higher plants. the abundance of xylan clearly indicates that xylanolytic enzymes can play an important role in bioconversion. the composition and structure of xylan varies according to the sources. however, all the xylans are heteropolysaccharides with homopolymeric backbone chains of â-1,4-d-xylopyranose units. the xylose backbones are substituted with mainly acetyl, arabinosyl, and glucuronosyl residues, depending on the source (tuncer 2000). as a result of this molecular complexity, the simultaneous and synergistic action of a range of bioactive enzymes is required to complete the degradation process. the enzyme that plays a key role on xylan degradation is endo-β-1,4-d-xylanase (β-1,4-d-xylan xylanohydrolase; ec 3.2.1.8) that randomly cleaves β-1,4-glycosidic bonds to produce xylooligosaccarides and -1,4-d-xylosidase (β-1,4-d-xyloside xylohydrolase; ec 3.2.1.37) that cleaves the terminal part of short xylo-oligosaccharides to produce free xylose (flores et al. 1997). kinds of substrate including monosaccharides, oligosaccharides or poly saccharides influence the production of xylanase by sclerotium rolfsii (sachslehner et al. 1998). the highest assayable xylanase activity is observed from ácellulose or bacterial cellulose, while other polysaccharides such as xylan birchwood, carboxymethyl cellulose or galactomannan induces lower activities. disaccharides, such as xylobiose and cellobiose induced more enzyme production than monosaccharides (xylose, glucose, and mannose). xylanase was successfully produced in the minimal medium containing wheat pollard (purwadaria et al. 2004a). wheat pollard used as substrate in this research was naturally found as a by-product from the flour milling process. as a biomass residue, wheat pollard can be used to produce other material with higher economic value such as a source for enzyme production, animal feed or even as a biofuel. ketaren et al. (2002) reported that pollard supplemented with xylanase could be used as a feed ration for animal diets. the application of enzymes such as xylanase in poultry diets could eliminate anti-nutritive properties of cereal grains presently being used as major ingredients of animal feed (chocht 2006). it was reported that pretreatments, especially soaking in sodium hydroxide, enhanced the cellulase production (purwadaria et al. 2004b). this result correlated with lignocellulose content that had been altered by sodium hydroxide where it breaks the lignin shield and disrupts the crystalline structure of cellulose (shin et al. 2000; mosier et al. 2005). soaking pollard in water might reduce the reducing sugar availability in pollard or decrease the catabolite repression of enzymes. meanwhile, it was reported that oatmeal was able to minimize cholesterol levels in the blood due to its high fiber content. this material could also be used as substrate for enzyme production (irawan 1999). as a material with high fiber content, oatmeal could be preferably used as substrate for xylanase production. bacillus pumilus was reported able to produce xylanase, β-xylosidase, acetylxylanesterase (degrassi et al. 1998), and cellulase (kotchoni et al. 2003). besides these abilities, b. pumilus has been employed in industry for alkaline protease production, in the environmental volume 2, number 1, april 2008 p 44-48 issn 1978-3477 decontamination of dioxins (hong et al. 2001), in the baking industry (nuyens et al. 2001), and in the adsorption of halogenated aromatic pollutants (choi et al. 2003). b. pumilus pu4-2 was a strain which was isolated by purwadaria et al. (2004a) from the gut of termitidae that had xylanolytic activity. besides microorganisms, plants (lima et al. 2001) can also produce xylanase. microbial xylanases are preferred because of their high specificity in enzyme reactions, the mild reaction condition used and the absence of substrate loss due to chemical modifications (wong et al. 1988). the objective of this study was to investigate the effect of pretreatment methods on the endo-β-1,4-d-xylanase production by b. pumilus pu4-2. it was of special interest to analyze the correlation between reducing sugar and inducer molecules and their effect on the assayable enzyme levels. materials and methods material. wheat pollard, used as substrate for this research, was obtained from ism bogasari flour mills. oat spelt xylan was purchased from sigma aldrich. organism and culture. bacillus pumilus pu4-2 isolated from gut of termitidae (purwadaria et al. 2004a) is from a collection of the indonesian research institute for animal production. the bacteria were maintained on nutrient agar containing 0.1% oat spelt xylan. experimental procedures. pollard used as substrate for the first experiments were (i) pollard pretreated by soaking in water, (ii) pollard pretreated by soaking in sodium hydroxide, (iii) untreated pollard as control, and (iv) oatmeal as a comparator. water-soaked pollard was obtained by soaking pollard (5% w/ v ) using water over one hour. at the end of one hour, the pollard was filtered using cotton cloth and dried overnight in an oven at 45°c. the dried pollard is then ready to use as substrate for enzyme production. sodium hydroxide soaked-pollard was obtained by soaking pollard (5% w/ v ) in 0.5% w/ v sodium hydroxide solution. this mixture was boiled for one hour. after this time the pollard was then washed with water until it reached neutral ph. the pollard was then filtered and dried. both water and naoh-soaked pollard were ground in a blender. in the second group of experiments, the enzyme activity in untreated pollard (control) and pollard soaked in water for 5, 10, 15, and 20 minutes were carried out. all treatments were repeated three times and enzyme levels were assayed after the treatment periods. enzyme production. eighteen-hours old cultures of b. pumilus pu4-2 grown on agar slants were resuspended by adding 5 ml of nacl 0.85% w/ v . xylanase was produced by adding 2 ml of inoculum to 50 ml pm liquid minimal medium (purwadaria et al. 2004a) which added with 0.05% w/ v yeast extract, 0.075% w/ v peptone, and 3% w/ v pollard. the suspensions in shake flasks, were incubated on a shaker incubator at 30°c and 150 rpm for 36 h. before the enzyme being assayed was separated from the biomass, 0.2% sodium azide was added. biomass was then separated by centrifugation at 12 000 x g for 20 min. the clear supernatant was used to estimate enzyme activities and stored at -20°c. enzyme activity and reducing sugar assays. xylanase activity was measured according to the method of rickard and laughlin (1980). all assays were carried out in mcilvaine (citrate-phosphate) buffer ph 7.2. endo-β1,4-d-xylan xylanohydrolase activity was assayed by using 1% solution of oat spelt xylan as the substrate. reducing sugars produced by the reaction were assayed by the dns method (miller 1959). one unit of enzyme activity is defined as the amount of enzyme producing 1 μmol xylose equivalents per minute under the given conditions. sugar composition (saccharides) of untreated and pretreated pollards was determined by analytical laboratory of indonesian research institute for animal production using hplc. sugar pack column from waters was used for the hplc, while double distilled h 2 o was used as the mobile phase and run at 90°c. oligosaccharide composition of pretreated pollards was determined, while oat meal was not. protein concentration assay. protein concentration was determined by the dye-binding method of bradford (1976) with bovine serum albumin as the standard. lignocellulose content. the lignocellulose content was determined by analytical laboratory of indonesian research institute for animal production, bogor, according to the method of van soest and robertson (1968). statistical analysis. analysis variance of enzyme activity, protein concentration, and specific activity on pollard-treated and oat was carried out using one way analysis (minitab® 15). the correlation values of reducing sugar content of pollard treated over different soaking period and the specific enzyme activity obtained were also analyzed. results come from three replicates. results results from different kinds of substrates show that the highest enzyme activity was observed from untreated-pollard (table 1) that was not significantly different with that from water-soaked pollard (157 u ml-1 vs 128 u ml-1). since the protein content in water-soaked pollard was higher than that in untreated-pollard, the specific enzyme activity in untreated-pollard (718 u mg-1) was significantly the highest compared with in other substrates (p<0.05). producing xylanase using oatmeal as a sole carbon source was not an effective way because, the enzyme activity and specific table 1 enzyme activity, protein concentration, and specific activity of xylanase produced in pretreated pollards (untreated, water, and naoh) and oatmeal kinds of pretreatment enzyme activity (u ml-1) protein (μg ml-1) specific activity (u mg-1) untreated-pollard (control) water-soaked pollard naoh-soaked pollard oatmeal 157c* 128b 5a 6a 219bc 261c 194ab 147a 718c 493b 26a 41a *different alphabet in the same column shows significantly different (p<0.05). volume 2, 2008 microbiol indones 45 activity was only 6.0 u ml-1 and 41 u mg-1, respectively. compared to all substrates used in this research, sodium hydroxide-soaked pollard had the lowest activity and specific activity, 5 u ml-1 and 26 u mg-1, respectively (p<0.05). lignocellulose content and reducing sugar in all substrates were determined to investigate the correlation between the enzyme production and the substrate composition. as predicted before, pretreatments decreased the reducing sugars (table 2 and 3). the highest lignocellulose contents were observed on naoh-soaked pollard that the only pollard where xylan was detected. it seems that the treatment opens the lignocellulose compounds or solubilized the hemicellulose, resulting the xylan was not detected in pretreated-pollards in hplc. results from different soaking period experiment showed that the highest enzyme activity was still obtained from untreated pollard. the reducing sugar available in pollard was decreasing when the washing period increased (fig 1). it also gave an interesting result that the longer the washing period, the lower the specific enzyme activity. the statistical analysis showed that there was a highly significant (p< 0.0001) correlation between reducing sugar and washing period and r2 = 0.9155. discussion bacillus pumilus pu4-2 has received considerable attention as an industrial aid, particularly in producing xylanase and probiotic for monogastric animals. recently, this microorganism was reported to be capable of producing xylanase using pollard as the sole carbon source. as a by-product from flour mills, pollard is an economically useable as a substrate in enzyme induction. the results from the first experiments were unexpected. pretreatment methods did not produce the optimum activity in this research. additionally, oatmeal was not a viable medium to increase assayable xylanase. meanwhile, untreated pollard used as a control showed the highest enzyme activity and also specific enzyme activity. from our lignocellulose and hplc data, it was concluded that pretreatment methods washed out some soluble oligosaccharides involved in the presumed enzyme induction. soaking pollard in water was undertaken to reduce the reducing sugar that may repress the presumed gene expression. apparently, it was not only reduced the reducing sugar but it also reduced the water-soluble inducer molecules, the oligosaccharide content of water-soaked pollard was lower than untreated-pollard (0.05 vs 0.34% w/w). pretreatment using sodium hydroxide maximized all of the lignocellulose components. it is assumed that this high alkali pretreatment might have dissolved all of the small oligosaccharides, those washed out during water rinsing for neutralizing the substrate to ph 7. this explains why the lignocellulose content was the highest among other substrate because there are only long-chains oligosaccharides present in this substrate, making it harder for the hemicellulolytic microbes to digest. however, this condition should still be suitable for cellulase production, since the crystalline structure of cellulose was disrupted. the disruption opened the hemicellulose molecules (xylan) to be detected in hplc. oatmeal had the lowest lignocellulose content (table 1). compared to other substrates, hemicellulose available in this material was the lowest and this is apparently not enough to induce the xylanase production in assayable quantities. oatmeal probably could induce amylase which is not produced by b. pumilus pu4-2. this explains why the activity gained using this material as carbon source was very low. generally, a control is run to standardize a set of results. surprisingly, the control in this research gave the best result. the fact that having the best result from the control was not a common occurrence. therefore a trial was done to confirm table 2 reducing sugar and lignocellulose contents in pretreated-pollards and oatmeal table 3 the composition of mono, oligo and poly-saccharides in pretreated-pollards fig 1 correlation relative specific activity of xylanase detected in different period of water-soaked pollards towards their relative reducing sugar content. period of soaking: 0, 5, 10, 15, and 20 minutes. 120.0 90.0 60.0 30.0 0.0 0.0 30.0 60.0 90.0 y= 0.8423x + 9.932 r2= 0.9115 120.0 kinds of pretreatment oligosaccharide content (% w/w) monosaccharide oligosaccharide xylan untreated pollard (control) water-soaked pollard naoh-soaked pollard 2.11 1.28 0.12 0.34 0.05 0.20 nd nd 0.14 nd: not detected. kinds of pretreatment reducing sugar (% w/w) lignocellulose content (% w/w) lignin hemicelluloses cellulose untreated pollard (control) water-soaked pollard naoh-soaked pollard oatmeal 2.2 1.3 1.1 1.2 2.78 2.40 9.05 0.95 29.15 26.40 43.99 9.00 8.17 7.99 29.13 1.62 46 purwadaria et al. microbiol indones the result obtained from the first findings. substrate used in this following trial was water-soaked pollard with various soaking period compared to untreated pollard. in the following trial, untreated pollard still gave the highest activity. with this result, it is obvious that reducing sugar had an insignificant influence compared to inducer molecules. this hypothesis was supported with the correlation experiment of reducing sugar content toward specific enzyme activity (fig 1). it was reported by sachslehner et al. (1998) that xylanase was still produced by s. rolfsii in the media containing 540 ppm kind of monosaccharides (glucose, xylose or mannose). our untreated and water-soaked pollard media contained respectively mono-saccharides 660 and 430 ppm, those concentration did not repress the formation of xylanase. the interesting result could be observed in the following trial, the period of soaking time in water-soaked pollard, was the more washing period, the lower reducing sugar, and specific activity obtained. the lower reducing sugar occurred due to the more soluble and metabolizable oligosaccharide washed out when the pollard was filtered using cotton cloth. the reducing sugar content available in production medium has a highly significant correlation with the specific activity in this following experiment (p<0.0001; r2 = 0.9115) (fig 1). it is believed that the soluble molecules washed out by pretreatment methods were important due to lower enzyme activity and specific activity compared to untreated pollard. the enzymes involved in substrate degradation were generally inducible. they were formed only when the corresponding substrate was present in the media. fungal, bacterial and actinomycete xylanases were generally induced, whether induced by xylan, xylobiose, xylose or by lignocellulosic residues that contain xylan (lemos and pereira 2002). since the polysaccharides were far too large to pass through the cell membrane and triggered the response in the microbial cell leading to the enhanced synthesis of xylanase. it was generally believed that low-molecular-mass soluble catabolites which were released from the polymeric compounds of the action of low, constitutive, amounts of this hydrolase and which could easily enter the cell, provided signal of the presence of an extracellular substrate and the stimulus for the accelerated synthesis of the enzyme (sachslehner et al. 1998). the same authors also noted that xylobiose was the best inducer to produce xylanase by s. rolfsii and it is clearly indicated that the xylobiose content washed out by pretreatment methods influenced the enzyme produced by b. pumilus pu 4-2. the protein content of the filtrate from the culture shows that different substrate produced different protein content. however, the highest protein not came from the highest enzyme activities, the protein content in the filtrate may be from the soluble protein of the substrate or other enzymes such as cellulose and glycosidases. untreated pollard as the best substrate for xylanase production by b. pumilus pu4-2 gave a lot of advantages. by using untreated pollard, it would be economically more efficient than using pretreated pollard and also save more time because there were no steps needed to prepare this material as substrate for enzyme production. another advantage, untreated pollard was more environmentally friendly than sodium hydroxide-soaked pollard because there were no sodium hydroxide residues that could pollute the environmental, especially rivers where it could affect people that still using water from rivers to fulfill their primary needs. acknowledgement authors wish to thank indonesian research institute for animal production for their financial support on this research. the funding was from the indonesia agricultural department budgett. references bradford m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the protein-dye binding. anal biochem 72:248-254. chocht m. 2006. enzyme for the feed industry. world poultry sci j 62:515. choi sd, hong hb, chang ys. 2003. adsorption of halogenated aromatic pollutants by a protein released from bacillus pumilus. water res 37:4004-4010. degrassi g, okeke bc, bruschi cv, venturi v. 1998. purification and characterization of an acetyl xylan esterase from bacillus pumilus. appl environ microbiol 64:789-792. flores me, perez r, huitron c. 1997. β-xylosidase and xylanase characterization and production by 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wheat pollard. biotropia 23:1-12. rickard pad, laughlin ta. 1980. detection and assay xylanolytic enzymes in a cellulomonas isolate. biotechnol lett 2:363-368. volume 2, 2008 microbiol indones 47 sachslehner a, nidetzky b, kulbe kd, haltrich d. 1998. induction of mannanase, xylanase, and endoglucanase activities in sclerotium rolfsii. appl environ microbiol 64:594-600. shin y, son k, yoo di. 2000. structural changes in tencel by enzymatic hydrolysis. j appl polym sci 76:1644-1651. tuncer m. 2000. characterization of endoxylanase activity from thermonospora fusca bd25. turk j biol 24:737-752. van soest pj, robertson jb. 1968. system of analysis for evaluating fibrous feeds. in: wj pigden (ed). standardization of analytical methodology for feed. cent. toronto, canada idrc. 134e. wong kky, tan lul, saddler jn. 1988. multiplicity of 3-1,4-xylanase in microorganisms: functions and applications. microbiol rev 52:305317. 48 purwadaria et al. microbiol indones istianto et al24042012 issn 1978-3477, eissn 2087-8587 vol 6, no 1, march 2012, p 42-47 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.7 short communication application of phenol pretreatment for the isolation of rare actinomycetes from indonesian soil 1 1 1 yudhie istianto *, raden setyo adji koesoemowidodo , harmoko saputra , 2 1 1 yoshio watanabe , hardaning pranamuda , and bambang marwoto 1 center of biotechnology bppt, kawasan puspiptek-serpong, tangerang selatan 15340, indonesia; 2 bioresorces laboratories, micro biopharm japan, co. ltd.1808 nakaizumi, iwata, shizuoka 438-0078, japan phenol treatment was applied for isolation of rare actinomycetes using 25 soil samples collected from pulau seribu, tanjung redep, manokwari, and halmahera. the samples were air-dried and suspended in 1.5 % (w/v) o phenol solution at 30 c for 30 minutes, and subsequently cultured on plates of humic acid-vitamin agar (hva) -1 -1 medium supplemented with cycloheximide (50 µg ml ) and nystatin (50 µg ml ). a total of 61 isolates were obtained and the most dominant isolates were not streptomyces (only 24.6%), whereas other genera such as micromonospora, actinomadura, microbispora and polymorphospora were isolated with ratios of 49.2%, 13.1%, 9.8%, and 3.3%, respectively. key words: phenol method, rare-actinomycetes, selective isolation method, soil sample perlakuan phenol diterapkan untuk mengisolasi actinomycetes selain dari streptomyces dari 25 sampel tanah yang diperoleh dari pulau seribu, tanjung redep, manokwari, dan halmahera. sampel dikering anginkan o dan disuspensi dalam 1,5% larutan fenol pada suhu 30 c selama 30 menit, kemudian dikultur pada media asam -1 -1 humat-vitamin agar (hva) yang dilengkapi dengan cycloheximide (50 µg ml ) dan nistatin (50 µg ml ). sebanyak 61 isolat diperoleh dengan komposisi: streptomyces hanya 24,6%, sedangkan marga lain seperti micromonospora, actinomadura, microbispora dan polymorphospora diperoleh masing-masing dengan rasio 49,2%, 13,1%, 9,8%, dan 3,3%. kata kunci: actinomycetes, metode isolasi selektif, perlakuan fenol, sampel tanah actinomycetes (actinobacteria) is a group of prokaryotic organisms belonging to the subdivision of the gram-positive bacteria. most of them are in the subclass actinobacteridae and order actinomycetales. all members of this order are characterized in part by the high g+c content (>55 mol%) in their dna (stackebrandt et al. 1997). actinomycetes are a group of microbes, of which many of the members can produce antibiotics and antitumors (chantongcome et al. 2009). a total of 7899 bioactive compounds have been identified up to 1988; 67% from actinomycetes, 12% from bacteria, and 20% from fungi. today, known bioactive metabolites are more than 22 000, 10 100 of which are produced by actinomycetes. so far, 140 to 160 antibiotics have been used in human therapy and agriculture, and 100 to 120 of them are produced by actinomycetes (berdy 2005). new antimicrobial agents are desperately needed to combat the increasing number of antibiotic resistant pathogenic microbial strains. natural products remain the most propitious source of novel antibiotics. it is widely accepted that actinomycetes are prolific producers of natural bioactive compounds. the efficiency of research to discover new compounds with novel chemical structures can be increased with intensive efforts in isolating and screening rare genera of microorganisms (takahashi, 2004 ; kumar et al. 2010). rare actinomycetes are usually regarded as strains whose isolation frequency much lower than those strains isolated by conventional methods. past and present efforts in the isolation of rare actinomycetes have discovered some genera, such as actinomadura, actinoplanes, micromonospora, microtetraspora, that had been recovered from many soil samples (lazzarini et al. 2001; hayakawa et al. 2010). although researchers have utilized numerous approaches to isolate new microbes, the number of microorganisms that have been successfully grown represents only a small portion of the total that exists (khana et al. 2011). hawksworth (1997) reported that the number of known bacteria comprises not more than 10% of the estimated total number of species in the world. the isolation of unknown microbial strains thus *corresponding author, phone/fax: +62-21-7560155, e-mail: yudhie_primer@yahoo.com represents an area of great potential. i n d o n e s i a i s k n o w n a s a c o u n t r y w i t h megadiversity of microorgaisms, i.e. it lies geographically on a tropical region with the highest level of biodiversity that it has high potential as a source of novel microbial bioactive metabolites. efforts to isolate and evaluate microbial bioactive compounds from indonesia, especially from actinomycetes, have expanded significantly in recent years. however, most of the isolates recovered on agar plates have been identified as genus streptomyces, which are the dominant actinomycetes in soil. lisdiyanti (2006) reported, a total of 982 actinomycetes isolated from indonesia in 2003 and 2004 using three isolation methods including sodium dodecyl sulfate yeast extract (sds-ye), rehydration-centrifugation, and dry heating. her study revealed that on the basis of 16s rrna, 66.1% isolates were identified as streptomyces and 33.8% isolates were non-streptomyces groups. for the purpose of screening rare bioactive molecules, the adoption of improved methodologies for isolating the uncommon and less studied, rare actinomycetes is required to avoid re-isolating strains that produce known bioactive metabolites and to improve the quality of the natural products screened (takahashi and omura 2003; berdy, 2005; khana et al. 2011). over the past year, we have attempted various methods to isolate novel actinomycetes, using three isolation methods including wet method, acid treatment, dry heating on the basis of 16s rrna, 79.5% isolates were identified as streptomyces and 20.5% isolates are as non-streptomyces groups. in this paper we focused on the application of phenol pretreatment for isolating rare actinomycetes from indonesian soil sample. this method selectively isolated streptomyces violaceusniger cluster (hayakawa et al. 2004), micromonospora (hayakawa et al. 1991), dactylosporangium, microbispora (takahashi and omura, 2003), and actinomadura (hayakawa et al.1995) from japan soil sample. a total of 25 soil samples were collected from pulau seribu (west java), tanjung redeb (east kalimantan), manokwari (papua), and halmahera (north maluku). samples were passed through a 2 mm mesh sieve and air-dried at room temperature for 1 week. one gram air-dried sample were mixed with 10 ml sterilized water and vigorously stirred for 2 min. after allowing the tube to stand for 1 min, 1 ml of supernatant was transferred to 9 ml of 1.5% phenol solution. then o the mixture was maintained at 30 c with occasional stirring. after incubation for 30 min, aliquots of mixture were spread onto humic acid-vitamin agar -1 supplemented with cycloheximide (50 µg ml ) and -1 nystatin (50 µg ml ). the experiments were done in o replicates and then all plates are incubated at 28 c for 1 to 3 weeks. a total of 61 actinomycete isolates were successfully isolated from 25 soil samples. twenty seven isolates were isolated from halmahera, 21 isolates from manokwari, 8 isolates from pulau seribu, and 5 isolates from tanjung redep (table 1). colonies having characteristic features such as powdery appearance with convex, concave or flat surface and color ranging from white, gray to pinkish and yellowish were selected. colonies observed on the first and second days were eliminated because actinomycetes were considered as slow grower (currie 2006). rare actinomycetes have usually been regarded as genera of actinomycetes whose isolation frequency by conventional methods is much lower than that of streptomyces. consequently, basic knowledge on the habitat, physiology and productivity of molecules of rare actinomycetes develops rather slowly. due to the discovery of actinomycetes with ecologically significant properties the screening extends into uncommon environments. for the purpose of screening novel bioactive molecules, several factors must be considered: pretreatment, selective medium and recognition of candidate colonies on a primary isolation plate. production of spore mass was detected on the 21day old cultures on humic vitamin agar. spore ornamentation was observed by olympus bx51 microscope. the dna was isolated using instagene matrix kit for dna isolation. the pellet was lysed using a lysing matrix, combined with 100µl, and centrifugation for 3 min at 15 000 x g. identification using the 16s rdna was conducted by pcr using a 9f forward primer, gagtttgat (c/t)(c/a)tggctcag; and a 1541r reverse primer, aaggaggtg(a/t)tcca(a/g)cc. the reaction mixture (50 μl total volume) contained 30 µl ddh2o, 5 µl of mgcl2, 5 µl of 10x buffer 4 µl of deoxynucleoside triphosphates, 1 µl of each primer, 0.25 µl of takara taq polymerase (takara bio inc, japan), and 5 μl of cell lysis as the template. pcr conditions were as follows: denaturation at 98 °c for 20 s, annealing at 52 °c for 45 s, and elongation at 72 °c for 2 min. a total of 30 cycles were performed, followed by a final elongation for 4 min at 72 °c. pcr products were purified with a geneaid pcr fragments extraction kit according to the manufacturer's microbiol indones 43volume 6, 2012 44 istianto et al. microbiol indones instructions (geneaid, taiwan). amplicon was sequenced with an automatic sequence analyzer (applied biosystems 3130 dna analyzer; applied biosystems, ca, usa) using the bigdye_ terminator v3.1 cycle sequencing kit (applied biosystems). related sequences were identified by performing sequence database searches using blast. sequence data for related species were retrieved from genbank. phylogenetic analysis was performed using clustal w software. phylogenetic analysis by 16s rdna sequencing (1500 bp) showed a various similarity between the strains (fig 1). table 1 shows that the most frequent genus found was micromonospora sp. (30 isolates) followed by streptomyces sp. (15 isolates), actinomadura sp. (8 isolates), microbispora sp. (6 isolates), and polymorphospora sp. (2 isolates). analysis of sequence of 16s rdna from the 61 fig 1 phylogenetic trees showing the relationship between the isolates and related microorganism in the order actinomycetales based on 16s rrna sequences. bar : 1% estimated sequence divergence. 0.01 microbiol indones 45 isolated actynomycetes strain indicated that a total 5 strains were novel actinomycetes strains exhibiting low similarity (<98%) with their close relatives (table 2). to compare and confirm the identification by genetic analysis, these isolated actynomycetes strains were identified by morphological examination. the colonies were categorized by microscope observation. from a total of 61 isolates of actinomycetes, 15 isolates were identified as streptomyces (spore chains with coiling, spiral and looped) (fig 2a), 8 isolates actinomadura (spore chains were straight and open hooked) (fig 2b), 6 isolates microbispora (longitudinal pair of spores on aerial mycelium) (fig 2c), and 30 isolates micromonospora (clusters of single conidia on substrate mycelium) (fig 2d). when the soil samples were cultured without pretreatment, the most of the colonies recovered were bacteria other than actinomycetes, followed by s t re p t o m y c e s , f u n g i , a n d n o n s t re p t o m y c e s actinomycetes. when the soil was air-dried, the number of other bacteria decreased, whereas the number of streptomyces colonies increased. over the past year, we applied various methods to isolate rare actinomycetes, such as: (1) wet method (streptomyces 65%, nocardia 29%, amycolatopsis 3.4%, kitasatospora 1.6%, and micromonospora 0.2%.), (2) acid treatment (streptomyces 89%, kitasatospora 5.5%, nocardia 4.5 %, amycolatopsis 0.6%, actinomadura 0.4%), and (3) dry heating (streptomyces 95%, nocardia 2 %, kitasatospora 2 % , micromonospora 0.5 % , and amycolatopsis 0.5 %). pretreatment with 1.5% phenol reduced the number of non-actinomycetes bacteria while significantly increased the number of micromonospora-like colonies (qiu et al. 2008). phenol treatment of soil suspension lowered the number of fungi and other bacteria, but the actinomycetes were less affected, thus 65% of the colonies belonged to rare actinomycetes. the phenol sampling place number of strain streptomyces micromonospora actinomadura microbispora polymorphospora pulau seribu 8 2 5 1 0 0 tanjun g redeb 5 0 0 1 4 0 man okwari 21 6 11 3 0 1 halmahera 27 7 14 3 2 1 total 61 15 30 8 6 2 table 1 correlation between sampling place and isolated actinomycetes genus number of strain <98% 98-99% 99-100% 100% streptomyces 15 3 4 6 2 micromonospora 30 0 9 19 2 actinomadura 8 1 1 5 1 microbispora 6 1 1 4 0 polymorphospora 2 0 1 1 0 total 61 5 16 35 5 table 2 correlation number between isolated actinomycetes and their homologies from database volume 6, 2012 a b c d fig 2 cell morphology of isolated actinomycetes (100x). (a) streptomyces sp.; (b) actinomadura sp.; (c) microbispora sp.; (d) micromonospora sp. 46 istianto et al. microbiol indones pretreatment of the soil killed bacteria and streptomycetes in the samples, while keeping micromonosporae and microbisporae a live (hayakawa et al. 1991). micromonospora (49.18%), streptomyces (24.59%), actinomadura (13.11%), microbispora (9.83 %), and polymorphospora (3.27 %). this effect was also suggested in previous works (hayakawa et al.1991; hayakawa, 2008; kim et al. 1994). phenols have been reported to exhibit antibacterial activities with distinguished characteristics in their reactivity with proteins related polyamides polymers (haslam et al. 1996). the inhibition of microorganisms by phenolic compounds may be due to iron deprivation or hydrogen bonding with vital proteins such as microbial enzymes (scalbert et al. 1991). the mechanism thought to be responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds possibly through reaction with sulfhydryl groups or though more nonspecific interaction with proteins. phenolic compounds exert antimicrobial activity by injuring lipid-containing plasma membranes, causing leakage of cellular contents. the cell wall of rare actinomycetes are rich in lipids, making them susceptible to the activity of phenol derivatives. the dominance of other bacteria and fungal contamination inhibited the colonization of actinomycetes on isolation medium. when antifungal -1 agents such as nystatin (50 µg ml ) and cycloheximide -1 (50 µg ml ) were supplemented into the isolation medium, the number of fungi decreased. thus, the isolation medium was supplemented with these antibiotics in succeeding experiments (seong et al. 2001). rare actinomycetes' growth was disadvantaged when cultured on agar media together with other fast growing microorganism. thus, the media for isolating them was designed to reduce growth of competitive microbes that it would not affect their growth. hayakawa and nonomura formulated humic acidvitamin (hv) agar, a medium containing humic acid as the sole carbon and nitrogen source. humic acid was extremely heterogeneous crosslinked polymers, which is resistant to biological decomposition. however, actinomycetes can utilize it as nutrient source and also use it to supported sporulation, while it restricts the growth of non-filamentous bacteria colonies (seong et al. 2001). humic acid vitamin agar (hva) was used for the selective isolation of actinomycetes. the black color of hva made it suitable to determine the morphology of white actinomycetes colonies. rare actinomycetes as well as streptomyces grew well on hva. although the growth rate of actinomycetes is low, discrimination of typical morphology of colonies was easy on hva. the activation of spore germination by humic acid was considered as one of the causes that increases the number of diverse actinomycetes colonies on hv agar (hayakawa and nanomuraea 1987). phenol pretreatment as one of the improved selective isolation methods were applied successfully for isolating rare actinomycetes from indonesian soil. this method had been used for the selective isolation of genera micromonospora (49.18%), streptomyces (24.59%), actinomadura (13.11%), microbispora (9.83 %), and polymorphospora (3.27 %). it was indicated that many novel actinomycetes may be obtained by their exhibition of low similarity (< 98%) with their close relatives. some of them may also be candidates for future potential novel bioactive compound producers. references berdy j. 2005. bioactive microbial metabolites, a personal view. j antibiotic. 58(1):1-26.doi:10.1038/ja.2005.1. currie cr, poulsen m, mendenhall j, boomsma jj, billen j . 2006. coevolved crypts and exocrine glands support mutualistic bacteria in fungus-growing ants. science 311: 81-83.doi:10.1126/ sc.1119744. chantongcome j, surattana a, chaiyo c, samboon t. 2009. f e r m e n t a t i o n a n d a n t i b a c t e r i a l a c t i v i t y o f micromonospora strains isolated from sea sand. j health res. 23 : 93-97. haslam e.1996. natural polyphenols (vegetable tannins) as drugs: possible mode of action. j nat prod. 59: 205215. hayakawa m, nonomura h. 1987. humic acid-vitamin agar (hva), a new medium for the selective isolation of soil actinomycetes. j ferment technol. 65 : 501-509. hayakawa m, sadaka t, kajiura t, nonomura h. 1991. new methods for the highly selective isolation of micromonospora and microbispora. j ferment technol 72 : 320-326. hayakawa m, momose y, kajiura t, yamazaki t, tamura t, hatano k, nonomura h. 1995. a selective isolation method for actinomadura viridis in soil. j ferment bioeng. 79 : 287–289. hayakawa m, yoshida y, iimura y. 2004. selective isolation of bioactive soil actinomycetes belonging to the streptomyces violaceusniger phenotypic cluster. j appl microbiol. 96 : 973–981, doi:10.1111/ j.13652672.2004.02230.x. hayakawa m. 2008. studies on the isolation and distribution microbiol indones 47 of rare actinomycetes in soil. actinomycetologica 22:12–19. hayakawa m, yamamura h, sakuraki y, ishida y, hamada m, otoguro m, and tamura t. 2010. diversity analysis of actinomycetes assemblages isolated from soils in cool-temperate and subtropical areas of japan. actinomycetologica 24: 1-11. hawksworth dl. 1997. fungi and international biodiversity initiatives. biodivers conserv. 6 : 661-668. khana m, solanki r and lal r. 2011. selective isolation of rare actinomycetes producing novel antimicrobial compounds. int j adv biotechnol res. 2 : 357-375. kim cj, lee kh, kwon os, shimazu a, yoo id. 1994. selective isolation of actinomycetes by physical pretreatment of soil sample. j appl microbiol biotechnol. 22 : 222-225. kumar n, singh rk, mishra s.k. ,singh a.k. pachouri u.c. 2010. isolation and screening of soil actinomycetes as source of antibiotics active against bacteria. int j microbiol res. 2 : 12-16. lazzarini a, cavaletti l, toppo g, marinelli f. 2001. rare genera of actinomycetes as potential sources of new a n t i b i o t i c s . a n t o n i e va n l e e u w e n h o e k . 7 8 : 399–405.doi:10.1023/ /s.10482-009-9337-4. lisdiyanti p. 2006. diversity of actinomycetes in indonesia. puspita lisdiyanti, annual reports of international center for biotechnology. osaka university. lipi pr. scalbert, a. 1991. antimicrobial properties of tannins. phytochemistry 30: 3875-3883. seong cn, choi jh, baik ks. 2001. an improved selective isolation of rare actinomycetes from forest soil. j microbiol. 39: 17-23. stackebrandt, e., f. a. rainey, and n. l. ward-rainey. 1997. proposal for a new hierarchic classification system, actinobacteria clasis novel. int j syst bacteriol. 47:479–491. tanaka y, omura s. 1990. metabolism and products of actinomycetes, an introduction. actinomycetologica. 4 : 13–14. takahashi y, omura s. 2003. isolation of new actinomycete strains for the screening of new bioactive compounds. j gen appl microbiol. 49 : 141–154. takahashi y. 2004. exploitation of new microbial resources for bioactive compounds and discovery of new actinomycetes. actinomycetologica 18 (2) : 54–61. qiu d, ruan j, huang y. 2008. selective isolation and rapid i d e n t i f i c a t i o n o f m e m b e r s o f t h e g e n u s micromonospora. j appl enviro microbiol. 74 (17) : 5593–5597. doi:10.1007/ s.00253-011-3862-6. volume 6, 2012 1: 42 2: 43 3: 44 4: 45 5: 46 6: 47 4.mi-sony suhandono available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.10.2.4issn 1978-3477, eissn 2087-8575 vol 10, no 2, juni 2016, p 65-70 *corresponding author; phone: +62 22 251-1575; +62 22 250-0258, fax: +62 22 253-4107, email:nyoman@sith.itb.ac.id coffee is an important commodity and one of the most popular beverages in the world. over 2.25 billion cups of coffee are consumed in the world every day (ponte 2002). in 2012, indonesia ranked third after brazil and vietnam in the global production of green coffee beans (usda 2012). five species of coffee that are commonly cultivated in the world include coffea arabica, c. canephora, c. robusta, c. liberica, and c. excelsa (doyle et al., 2001). in indonesia c. robusta and c. arabica are the species mostly planted (neilson, 2008). due to its high market demand, coffee processing has developed to produce various flavors to satisfy the consumers. one type of coffee that is increasingly popular is luwak coffee (kopi luwak) which is produced exclusively by the indonesian palm civet or luwak (paradoxurus hermaphrodites ssp.). luwak is a small mammal (carnivora, viverridae) living in the rain forests of java and sumatera, although its various subspecies are intensely distributed in south east asia (patou 2010). the distinctive taste of this coffee has been recognized since coffee plantation started in indonesia during the dutch colonial era. the luwak eats fruits, including coffee fruits or coffee cherries. only the best and mature coffee cherries would be eaten by the luwak. this process will produce coffee with distinctive flavor. the price of luwak coffee is relatively high because only a small quantity of this coffee is produced (marcone 2004). once ingested by luwak, coffee cherries will pass through its entire digestive system. this process removes the outer layers of the fruit. after digestion, the remaining coffee beans were collected from the forest floor, cleaned, roasted, and ground; with the same process as the harvested coffee beans. thus the unique flavor of kopi luwak comes from the digestion process in luwak, which includes mechanical, biochemical, and fermentation processes (marcone 2004). microorganisms, especially bacteria, are closely associated with the digestive process in monogastric animals (kraatz 2010). several studies have been conducted to determine the types of bacteria involved in the fermentation process, for example by trying to isolate the bacteria from faeces of civet and the luwak’s coffee beans (fauzi 2014). currently, there is no information about the bacterial diversity in parts of luwak coffee is a highly-priced coffee produced exclusively by the palm civet or luwak (paradoxurus hermaphrodites ssp.). the purpose of this study was to determine the diversity of culturable bacteria in the gastro intestinal tract of luwak. the bacterial isolates were phenotypically characterized by their morphology and molecularly by analysis of their 1 500 bp 16s rdna sequence. the results showed that enterobacter cloacae and lactobacillus brevis were found all over luwak’s digestive tract. e. cloacae was the most common species. the most diverse bacterial population was found in small intestine. seven bacterial genera were successfully identified from the small intestine and colon, compared to only five genera found in the stomach. key words: coffee, culturable bacteria, kopi luwak merupakan kopi yang dihasilkan secara eksklusif oleh musang atau luwak (paradoxurus hermaphrodites ssp.) tujuan penelitian ini adalah untuk mengetahui keragaman bakteri yang dapat dikultur dari saluran pencernaan luwak. fenotipe isolat bakteri diidentifikasi secara morofologi dan secara molekuler dengan menggunakan sepasang primer yang dapat mengamplifikasi sekuen 16s rdna. hasil penelitian menunjukkan bahwa enterobacter cloacae dan lactobacillus brevis merupakan bakteri-bakteri yang ditemukan pada seluruh saluran pencernaan luwak. e. cloacae teridentifikasi merupakan bakteri yang paling umum. populasi bakteri yang paling beragam ditemukan dalam usus kecil. tujuh genus bakteri lainnya berhasil diidentifikasi dari usus kecil dan usus besar, sedangkan lima genus ditemukan di dalam perut luwak. kata kunci : 16s rdna, bakteri yang dapat dikultur, kopi, luwak, saluran pencernaan 16s rdna, gastrointestinal tract, luwak diversity of culturable bacterial in various parts of luwak’s (paradoxurus hermaprodithus javanica) gastrointestinal tract sony suhandono, heri setiadi, tati kristianti , ali budhi kusuma, andini warih wedaringtyas, demi tristan djajadi, and i nyoman pugeg aryantha* school of life sciences and technology, research center for nanosciences and nanotechnology, institut teknologi bandung, jalan ganesha 10 bandung, jawa barat 40132, indonesia the civet’s digestive organs. civet coffee fermentation process is highly dependent on the animal. on the other hand, the existence of luwak is diminishing and the ability of civet in the process of fermentation is limited. this condition can lead to decreased production of civet coffee. therefore, it is necessary to find other alternatives that can replace the role of civet in the process of civet coffee fermentation. in vitro fermentation can be an option that may replace civet’s role in the production of civet coffee. the research on bacterial diversity in the digestive tract of the mongoose is important as the basis in determining the types of bacteria involved in the in vitro fermentation process of civet coffee. the objective of this study is to investigate the diversity of culturable bacteria in different parts of luwak’s gastrointestinal (gi) tract. materials and methods sampling, isolation, and enumeration of bacteria. adult civet (p. hermaphrodites javanica) specimen was obtained from natural forest in purwakarta, west java. the animal was then aseptically dissected to separate the gi tract into parts. the whole gi tract (stomach, intestine, and colon) with samples of digested food were separately removed under sterile conditions. samples from each gi part were serially -1 -9 diluted in phosphate buffered saline (from 10 to 10 ), plated onto lb (luria broth) and mrs (de man rogosa and sharp) agar media followed by incubation at 37 °c for 36 h. concentrations of culturable bacteria were estimated by counting the average cfu (colony forming units) per milliliter on each agar plate. colonies with different morphologies were selected using fourways streaking on the plates and stored in 50% (v/v) glycerol at -80 °c. phenotypic identification. phenotypic analysis of the isolates from luwak digestive system was conducted according to the bergey’s manual of systematic bacteriology using trypticase soy agar (tsa), trypticase soy broth (tsb) and nutrient agar (na). media for identification used were broth contaning glucose, lactose, sucrose and manitol, mr-vp (methyl red-voges proskauer), sim (sulfur-indole-motility), and simmon's citrate and nutrient gelatin media. the staining, morphology, and biochemical properties were determined as described by cowan (cowan 1974). the staining was performed using crystal violet, safranin, lugol’s iodine, barritt’s, and kovac’s reagents, red methyl, p-aminodimetilanilin oxalate, and 3% of h o .2 2 dna extraction. dna was extracted by boiling method (queipo-ortuño 2008). overnight bacterial suspension was harvested by centrifugation at 5000 rpm for 10 min. the pellet was then washed 3 times using 1.5 ml acetone. each preparation was centrifuged at 9000 g for 10 min at 4 ° remove potential pcr inhibitors. the pellet was suspended in 200 μl te buffer 10 mmol/l tris-hcl (ph 8.0), 1 mmol/ledta, and the mixture was briefly mixed in a vortex mixer. the suspension was placed in boiling water bath for 1 min, subjected to 3 freeze-thaw cycles alternating between liquid nitrogen for 3 min and 100 °c for 2 min, and then centrifuged at 13000 rpm for 5 min. a 100 μl aliquot of the supernatant was transferred to a sterile tube and stored at -20 °c until pcr testing. amplification of 16s rdna gene fragments. bacterial 16s rdna gene was amplified by pcr using universal primers specifically designed for bacterial d n a , 1 6 s r d n a f o r w a r d p r i m e r 8 f ( 5 ’ agagtttgatcctggctcag-3’); reverse primer 1492 (5’-ggttaccttgttacgactt-3’) from baker et al. (2003) in 50 μl reactions each containing 25 μl dream taq green pcr mix 2x (fermentas), 4 μl dimethyl sulfoxide (dmso), 2.5 μl of each primer for final concentration 0.1 μm, and 2 μl dna template. the pcr program was as followed: initial denaturation at 95 °c for 10 min, followed by 25 cycles, consisting of 95 °c for 30 s, 45 °c for 30 s and 72 °c for 2 min, and a final extension cycle 72 °c for 10 min (2720 thermal cycler applied biosystem). the amplified 16s rdna gene from the isolate was eluted and purified from the gel slice using gel extraction kit (geneaid, usa). purified pcr products were directly sequenced using both primers by automated dna sequencing (macrogen inc., seoul, korea) to obtain the complete nucleotide sequences of the 16s rdna gene. sequence analysis and taxonomic classification. the 16s rdna gene sequences were compared with the available sequences in genbank using the blastn bioinformatics program from the national center for biotechnology information (ncbi). based on the maximum identity score, the first ten sequences were selected and aligned using multiple alignment software program clustalw. distance matrix was generated using rdp-ii database and the phylogenetic tree was constructed according to the neighbour-joining method (saitao 1987), using mega 5.1 molecular evolutionary genetics analysis software version 5.1 (tamura et al. 2011) for the inquiry of the evolutionary relationship. the bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history c in order to 66 suhandono et al. microbiol indones of the taxa analyzed. the evolutionary distances were computed using the kimura 2-parameter method (kimura 1980) and are in the units of the number of base substitutions per site. results the total plate counts of aerobic bacteria found in the samples of digested food isolated from the digestive track on lb and mrs agar (expressed in -1 log(cfu ml ) were between 0 9 (fig 1). the total plate count of bacteria isolated from the stomach were 6.1 non lactid acid bacteria and 7.06 lactic acid bacteria. the total bacterial number in the colon was higher compared to stomach and small intestine (9.58 -1 log (cfu ml )). the largest population of lactic acid bacteria was found in the small intestine (9.36 log -1 (cfu ml )). morphological, physiological, and molecular analyses suggested that enterobacteriaceae was the dominant culturable bacteria in the digestive tract of luwak. other genera such as bacillus, pseudomonas, pantoea, escherichia, lactobacillus, ochrobactrum, and kocuria were also observed in the cultures. the 16s rrna gene sequence analysis was performed after pcr amplification in order to identify the isolates. the amplified products of 16s rdna were presented as single bands of 1 500 bp. the purified (purification kit, geneaid, usa) fractions were then quantified by 1 kb dna mass ruler standard fermentas (fig 2). phylogenetic tree was constructed from the 25 isolates. the phylogenetic tree suggested that l2lab1 0 1 2 3 4 5 6 7 8 9 10 stomach small intestine colon 9.2 9.589.36 9.1 7.06 6.1 fig 1 number of bacteria in the digestive tract of luwak (paradoxurus hermaprodithus var javanica). non lactic acid bacteria ( ) and lactic acid bacteria ( ). fig 2 agarose gel electrophoresis visualization of 16s rrna pcr product of l1uh, l3l6, l4uh, l10uh, m2, and m4 isolates. amplification products of 16s rdna genes 250 bp 500 bp 750 bp 1000 bp 1500 bp 2000 bp l1uh l3l6 l7uh l10uh m2 m4 volume 10, 2016 microbiol indones 67 palustris (l9uh), ochrobactrum sp. (ub3lab1), e. coli (uh3lab1), and l. brevis (m2) . discussions this study is the first recorded diversity study of the culturable bacteria from the digestive tract system of civet (p. hermaphrodites java). the total number of isolates and colony forming unit (cfu) presented in culture was closely related to bacillus subtilis. the same analysis was also used to identify the other isolates. bacterial identification and distribution in the digestive tract of luwak from blast analysis and phylogenetic analysis of the 16srdna gene are shown in fig 3. in the stomach of luwak, we found bacillus methylotrophicus strain py5 (l2lab1), enterobacter cloacae jq.993364.1 (l3l8), enterobacter sp. (l3l6), e. cloacae (l9l1), e. cloacae jq.993364.1 fig 1 indicated that the number of bacteria increased gradually from the jejunum (small intestine) to the colon (large intestine) and were most numerous in the colon. peristalsis and antimicrobial effects of stomach acid made the stomach and the proximal part of the small intestine habitable only to certain bacteria. according to baron (1996), genera reportedly found at this location include lactobacillus, enterococcus, streptococcus, and some acid-resistant gram-positive bacteria. most aerobic bacteria are in the jejunum, while the large intestine was dominated by aerobic and facultative anaerobic bacteria. the 16s rdna fragment analysis showed that e. cloacae and l. brevis were found all over the gastrointestinal system, indicating that they can survive (l6l9), cedecea davisae jq396389.1 (l8l4), enterobacter sp. 1054 (l7l9), enterobacter agglomerans strain a123 (l7l8), enterobacter turicensis (l5l9), bacillus sp. (l10l4), and lactobacillus brevis (m2). meanwhile, in the small intestine, we found bacillus substilis (uh3lab3), escherichia coli (uh3lab1), enterobacter sp. (l10uh), e. cloacae (l1uh), e. cloacae (l2uh), bacillus pumilus (l3uh), pseudomonas aeruginosa (l4uh), e. cloacae strain t137 (l5uh), e. cloacae (l8uh), kocuria palustris (l9uh), ochrobactrum sp.(ub3lab1) and lactobacillus brevis (m2). in the colon, we found e. cloacae (l1uh), e. cloacae (l2uh), b. pumilus (l3uh), p. aeruginosa (l4uh), e. cloacae strain t137 (l5uh), e. cloacae (l8uh), k. fig 3 phylogenetic tree by neighbour joining based on alignment 16s rdna fragment l2lab1isolate. l2lab1 bacillus methylotrophicus strain py5 b. amyloliquefaciens cy21 b. methylotrophicus l07 bacillus sp. b. amyloliquefaciens ba22 b. subtilis strain y1 b. amyloliquefaciens hs10 b. vallismortis b.amyloliquefaciens plantarum b. subtilis strain me2.jq900623.1 bacillus sp. hdly01.gu292815.1 b. amyloliquefaciens subsp. amyloliquefaciens jq963644 b. amyloliquefaciens subsp. amyloliquefaciens hq339952 b. subtilis dsm10.aj276351 b. licheniformis yw1258.af516176 b. cohnii ab023412 bacillus sp. no.51.ab066344 b. thuringiensis atcc33679.af290549 b. mycoides z84582 enterobacter cloacae 98 82 46 99 95 99 94 0.02 68 suhandono et al. microbiol indones methods. 55(3):541-555. doi:10.1016/j.mimet.2003.08. 009. th baron s. 1996. medical microbiology, 4 edition, university of texas medical branch at galveston, galveston, texas. bevilacqua a, cannarsi m, gallo m, sinigaglia m, corbo mrs. 2010. characterization and implications of enterobacter cloacae strains, isolated from italian table olives “bella di cerignola” j food sci. 75(1) :m53m60. doi:10.1111/j.1750-3841.2009.01445. cowan s. 1974. cowan and steel's manual for the identification of medical bacteria 2nd ed. cambridge university press, london, united kingdom. . 10.1128/aem.71.2.826834.2005 doyle mp, beuchat lr, montville tj. 2001. food microbiology:coffee and cocoa american society for microbiology press, washington dc. guarner f, malagelada jr. 2003. gut flora in health and disease. lancet. 361(9356):512-519. doi:10.1016/s014 0-6736(03)12489-0. fauzi m. 2014. isolasi dan karakterisasi bakteri asam laktat biji kopi luwak (civet coffe). digital repository universitas jember. kimura m. 1980. a simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. j mol evol. 16(2):111-120. doi:10.1007/bf01731581. kraatz m. 2010. lactobacilli and other lactic acid-related bacteria in the mucosal proximal gastrointestinal tract of pigs: a review of ecology for two derivative approaches for isolation of novel species, p. 674-686. in méndez-vilas a. 2010. current research, technology, and education topics in applied microbiology and microbial biotechnology. vol. 1. formatex, badajoz. marcone mf. 2004. composition and properties of indonesian palm civet coffee (kopi luwak) and ethiopian civet coffee. food res int. 37(9):901-912. doi:10.1016/j.foodres.2004.05.008. pham vht, kim j. 2012. cultivation of unculturable soil bacteria. trends biotechnol. 30(9):475-484. doi:10.1016/j.tibtech.2012.05.007. patou ml, wilting a, gaubert p, esselstyn ja, cruaud c, jennings ap, fickel j, veron g. 2010. evolutionary history of the paradoxurus palm civets-a new model for asian biogeography. j biogeogr. 37(11):2077-2097. doi:10.1111/j.1365-2699.2010.02364.x. pawar kd, banskar s, rane sd,charan ss, kulkarni gj, sarwant ss, ghate hv, patole ms, shouche ys. 2012. bacterial diversity in different regions of gastrointestinal tract of giant african snail (achatinafulica). microbiology open 1(4):415-426. doi:10.1002/mbo3.38. ponte s. 2002. the ‘latterevolution’? regulation, markets and consumption in the global coffee chain. world dev. 30(7):1099-1122. doi:10.1016/s0305-750x(02)00032-3. davis ker, joseph sj, janssen ph. 2005. effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria appl environ microbiol. 71(2):826-834. doi: in that condition. bevilacqua et al. (2009) reported that e. cloacae could be cultivated by fermentation at temperatures between 10-37 °c and ph between 4-5 and 8-10). meanwhile, l. brevis was a lactic acid bacteria that carries out the reactions, that is the conversion of carbohydrate to lactic acid plus carbon dioxide and other organic acids, without needing oxygen. low bacterial diversity in the stomach might be attributed to the physiology and ph of the stomach. the low bacterial diversity in the stomach may result from acidic environment and extracellular enzymatic digestion (pawar et al. 2012). the bacterial communities in the digestive tract of this animal may have important role in the digestion of the mucilage layer of coffee beans. we were assuming that the different digestive tract regions such as the esophagus, stomach, intestine, and colon are highly specialized compartments, and each could have distinct roles to play in the digestion. these functionally specialized tract regions may be unique microenvironments that harbor unusual bacterial communities. research suggested that the relationship between gut flora and monogastric animal is not merely commensal (a non-harmful coexistence), but rather a mutualistic relationship (sears 2005). the microorganisms may function as fermentation agent inducing the immune system, and competitor for harmful, pathogenic bacteria (guarner and malagelada 2003). the (pham and kim 2012). in this research, the lauria broth and mrs were used as isolation media for bacteria from luwak’s digested tract. therefore, our finding was limited only for bacteria that grow on these media. to obtain larger bacterial diversity, metagenomic method or more diverse selections of media might be used in the future. david et al. (2005) found that extended incubation times were able to isolate many members of rarely group bacteria. acknowledgements we would like to thank itb for their financial support through program riset dan inovasi kk itb 2013 during the project. references baker gc, smith jj, cowan da. 2003. review and reanalysis of domain-specific 16s primers. j microbiol type of cultured bacteria is dependent on the techniques and media used it is possible to accommodate some of the bacteria that have long incubation periods. volume 10, 2016 microbiol indones 69 sears cl. 2005. a dynamic partnership: celebrating our gut flora. anaerobe 11(5):247-251. doi: 10.1016/j.anaerobe. 2005.05.001. tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol biol evol. 28(10):2731-2739. doi:10.1093/molbev/msr121. usda.2012. coffee: world markets and trade, foreign agricultural service office of global analysis, united states department of agriculture. queipo-ortuño mi, colmenero jds, macias m, bravo mj, morata p. 2008. preparation of bacterial dna template by boiling and effect of iimmunoglobulin g as an inhibitor in real-time pcr for serum samples from patients with brucellosis. clin vaccine immunol. 15(2):293-296. doi; 10.1128/cvi.00270-07. saitou n, nei m. 1987. the neighbor-joining method a new method for reconstructing phylogenetic trees. mol biol evol.4(4):406-425. 70 suhandono et al. microbiol indones 1: 65 2: 66 3: 67 4: 68 5: 69 6: 70 01. rosa.cdr vol.13, no.2, june 2019, p 43-49 doi: 10.5454/mi.13.2.1 chemotactic motility and growth of pseudomonas fluorescens towards glucose concentration 1* 2 3 endah rosa , ummi mardhiah batubara , and suparjo 1 department of biology, faculty of science and technology, universitas jambi, jalan jambi-ma. bulian km 15 mendalo darat jambi 36361, indonesia; 2 department of marine science, faculty of marine sciences and fisheries, universitas riau; 3 department of feed technology and industry, faculty of animal sciences, universitas jambi. pseudomonas fluorescens is plant growth promoting rhizobacteria (pgpr) often inoculated on plants as natural biocontrol agent capable of protecting the plants from soil-borne pathogens. chemotactic motility allows populations of p. fluorescens to rapidly search for nutrients and is an important factor determining their competitive success to colonize plant root. therefore, we investigated various glucose concentrations from 0% to 1% (w/v) to enhance chemotactic motility and growth of this rhizobia. chemotactic motility was evaluated using swim plate assay and bacterial growth was measured using uv-vis spectrophotometer in lb and m9 medium. glucose with low concentration (0.05%) showed to have optimum response in p. fluorescens chemotactic motility with colony diameter 38.3 mm in lb medium and 12.8 mm in m9 medium. highest growth of p. fluorescens was 7 -1 seen in control condition of lb medium reaching a peak at 0.0246 od (~±1.44x10 cfu ml ) while growth in 600 7 -1 m9 medium supplemented with 1% glucose was just slightly lower with 0.0227 od (~±1.32x10 cfu ml ). 600 glucose in high concentration showed to repress chemotactic motility and first growth phase of p. fluorescens in lb medium due to catabolite repression. key words: catabolite repression, chemotactic motility, glucose, pgpr, pseudomonas fluorescens pseudomonas fluorescens adalah bakteri pgpr yang sering diinokulasikan ke tanaman sebagai agen biokontrol alami yang mampu melindungi tanaman dari serangan patogen pada tanah. pergerakan kemotaktik memungkinkan populasi p. fluorescens mencari sumber nutrisi dan merupakan salah satu faktor penting yang menentukan keberhasilan bakteri dalam mengkolonisasi akar tanaman. glukosa diketahui sebagai kemoatraktan paling kuat bagi p. fluorescens. oleh karena itu dilakukan pemeriksaan mengenai konsentrasi glukosa yang dapat meningkatkan pergerakan kemotaktik dan pertumbuhan bakteri rhizobia ini mulai dari konsentrasi 0% hingga 1% (w/v). pergerakan kemotaktik dievaluasi melalui uji swim plate dan pertumbuhan bakteri diukur dengan spektrofotometer uv-vis pada media lb dan m9. glukosa dengan konsentrasi rendah (0,05%) menunjukkan respons yang paling baik terhadap pergerakan p. fluorescens dengan diameter koloni sebesar 38,3 mm pada media lb dan 12,8 mm pada media m9. pertumbuhan p. fluorescens paling baik terjadi pada perlakuan kontrol di media 7 -1 lb dengan absorbansi 0,0246 od (~±1,44x10 cfu ml ) sementara pertumbuhan pada media m9 dengan 600 7 -1 glukosa 1% hanya sedikit lebih rendah yakni 0,0227 od (~±1,32x10 cfu ml ). glukosa dalam konsentrasi 600 tinggi mampu menghambat pergerakan kemotaktik dan fase awal pertumbuhan p. fluorescens pada media lb karena adanya represi katabolit. kata kunci: represi katabolit, pergerakan kemotaktik, glukosa, pgpr, pseudomonas fluorescens microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82375496335; fax: -; email: endah.rosa@gmail.com successful inoculation of p. fluorescens in establishing a symbiotic relationship with the plant root is determined by the motility of the bacteria to colonize the plant root's surface. chemotaxis is the ability of motile bacteria to find source of nutrients that can be used to support growth. which means that bacterial chemotaxis and growth play very crucial role in the process of root colonization. glucose is known as the strongest chemoattractant that can induce p. fluorescens chemotactic motility among other forms of sugar (arora and gupta 1993). however, previous studies mentioned that glucose in high concentration could inhibit chemotaxis and growth of e. coli (dobrogosz the rhizosphere harbors a vast number of prokaryotic and eukaryotic organisms that interact and compete with each other and with the plant root, thus affects the plant's health. pseudomonas fluorescens is one of the best colonizing group of pgpr (plant growth promoting rhizobacteria) often used as a research model for the study of root colonization and is inoculated on plant as biocontrol agent replacing the application of chemical fertilizer, in order to protect the plant from soil-borne pathogens (capdevila et al. 2004; de weert et al. 2002 and vicario et al. 2015). however, and hamilton, 1971; madigan et al. 2015) and motility of a. hydrophila (jahid et al. 2013), due to the catabolite repression phenomenon. meanwhile, the effect of glucose concentration on motility and growth of p. fluorescens needs further investigations. this research is aimed to study optimum concentration of glucose required for the induction of chemotactic motility as well as increasing the growth of p. fluorescens. materials and methods bacterial strain. the strain used for the study was pseudomonas fluorescens (fncc 0070) obtained from center of food and nutrition studies, gadjah mada university (cfns, yogyakarta, indonesia). culture media and growth conditions. the media used for the chemotaxis and growth assay were -1 -1 lb (10 g l casein enzyme hydrolysate, 5 g l yeast -1 extract, 10 g l sodium chloride) and m9 medium -1 -1 -1 (12.8 g l na hpo .7h o, 3 g l kh po , 0.5 g l 2 4 2 2 4 -1 nacl, 1 g l nh cl) containing various glucose 4 concentrations (0%, 0.05%, 0.2%, 0.5%, 1%) (w/v). chemotactic motility assay was carried out in growth -1 medium fortified with 3 g l bacto agar. p. fluorescens was then subcultured into 10 ml of lb for 18-20 hours of incubation in 30ºc. bacteria were incubated in aerobic conditions with shaking overnight at 125 x g prior to the day of activity. motility assay. chemotactic motility assay was performed using swim plate method as described by deloney-marino (2013) following the design by kearns and losick (2003) with slight modifications (figure 1). for motility assay, 1.5 l aliquots of p. fluorescens from overnight cultures that had reached 9 -1 1.7 od (equivalent to 10 cfu ml ) were spotted at 600 the center of plates containing ±25 ml lb or m9 with bacto agar and various glucose concentrations. petri plates were incubated in 30°c for 6 hours. the assay was performed in triplicate. after incubation, the diameter of the area of motility of the strain was measured, and the plates were photographed. bacterial growth measurement. 100 l aliquots 7 of p. fluorescens cultures 0.5 od (equivalent to 10 600 -1 cfu ml ) that had previously diluted (2:9) were inoculated into erlenmeyer flasks containing 13 ml of lb or m9 with glucose. effect of glucose concentration towards bacterial growth was measured using uv-vis spectrophotometer with optical density (od) at 600 nm (od ). measurement was performed 600 every 1 hour with 3 hours of total incubation period. the result was visualized in a bacterial growth curve. glucose utilization during bacterial growth. erlenmeyer flasks containing bacterial cultures from growth measurement activity were centrifuged at 10.000 x g in 30°c for 10 minutes. the supernatant was obtained using a sterile syringe and stored at 4°c until its analysis. analysis of the amount of glucose consumed by the bacteria was performed using titration method with benedict quantitative solution and estimated based on frais' conversion factor (frais 1972). statistical analysis. all experiments were performed using a randomized design. for chemotaxis experiment, the data were subjected to one-way analysis of variance (anova) with multiple comparison variables by least significant difference (lsd) test. while the data of bacterial growth measurement were subjected to two-way anova with comparison of means using tukeys test. differences between means were considered to be significant at p ≤ 0.05. data analysis and graph visualization were presented using software spss v.23. results chemotaxis is a directed movement of bacterial swimming motility towards chemoattractant in growth medium. the movement of bacteria which induced by glucose would create a colony phenotype as seen in halo zones in fig 2. p. fluorescens migrated chemotactically away from the point of inoculation towards another end of the uncolonized agar, which was still rich in nutrients. optimum concentration of glucose to induce chemotactic motility of p. fluorescens was 0.05% with colony diameter 38.3 mm. the colony expansion started significantly decreased as glucose concentration was raised from 0.2 % (34.5 mm), 0.5% (19.3 mm) and 1% (21.2 mm) respectively. the colony diameter in control plate was 37.1 mm. glucose concentration above 0.05% in lb medium had shown significant effect (p < 0.05) in reducing colony diameter of p. fluorescens (fig 4). while addition of glucose on m9 medium didn't show any significant effect in the reduction of p. fluorescens colony diameter (fig 3). colony diameter started to decrease in 0.2% concentration, but variation of glucose concentrations didn't show significant differences in each treatment (p > 0.05). mean diameters of swimming motility in m9 medium were 0% (10.7 mm), 0.05% (12.8 mm), 0.2% (10.8 mm), 0.5% (9.6 mm), and 1% (8.3 mm) respectively (fig 5). 44 rosa et al. microbiol indones volume 13, 2019 microbiol indones 45 fig 1 modified design of chemotaxis assay using swim plate method in bacillus subtilis (kearns and losick, 2003). 1) inoculation zone, 2) colony expansion zone, 3) uncolonized zone. fig 2 chemotactic motility of p. fluorescens (30ºc) in lb medium after 6 hours incubation. fig 3 chemotactic motility of p. fluorescens (30 ºc) in m9 medium after 6 hours incubation. fig 4 colony diameter of p. fluorescens in lb medium after 6 hours incubation. values on the histogram are the mean ± standard deviation of the mean of independent experiment. 46 rosa et al. microbiol indones addition of glucose as main carbon source in m9 medium could not induce optimum swimming motility of p. fluorescens, as the halo diameters were smaller than in lb medium (fig 6). p. fluorescens examined in lb had greater haloes than m9 with average diameter differences 19.64 mm. b e s i d e s c h e m o t a x i s , e ff e c t o f g l u c o s e concentration on the growth of p. fluorescens was also evaluated in lb and m9 media. variation of glucose concentration in lb medium showed significant effect (p < 0.05) on the growth of p. fluorescens. adding glucose to nutrient-rich medium such as lb caused p. fluorescens to have slower growth in its first phase (lag phase). during first and second hour of incubation, lb supplemented with high concentration of glucose (1%) showed lowest growth among other concentrations. bacteria grown in lb with 1% glucose during last hour of incubation was shown to peak at 0.0169 od 600 6 -1 (~±9.6x10 cfu ml ). increased exponential bacterial growth during the third hour was also followed by -1 exponential glucose depletion from 9.5 mg ml to 7 mg -1 ml which implied that the bacteria had started to consume glucose during that period. however, control condition of p. fluorescens showed highest peak at 7 -1 0.0246 od (~±1.44x10 cfu ml ).600 in contrast with lb, adding glucose to m9 medium could increase growth of p. fluorescens significantly (p < 0.05) higher than control. during third hour of incubation, bacterial growth in m9 broth supplemented with 1% glucose reached highest peak at 0.0227 7 -1 (~±1.32x10 cfu ml ). that value surpassed control 6 broth which only reached a peak at 0.0045 (~±2.7x10 -1 cfu ml ) (fig 8). glucose depletion in m9-1% glucose during third hour of incubation was strikingly -1 greater reaching 5 mg ml than that in lb-control -1 which only fell to 7 mg ml . meanwhile, p. fluorescens growth rate in m9-1% glucose was almost similar with lb-control in which both medium could increase exponential bacterial growth within short period of time (last hour of incubation) (fig 7-8), faster than burkholderia terrae reported by yang et al. (2017). discussion from the chemotactic motility assay, swimming motility of p. fluorescens showed optimum result at 0.05% glucose in lb medium, because low concentration of glucose was more quickly to deplete causing the bacteria to progressively migrate across the end of the agar. this condition was a signal for chemotaxis receptor (mcp) to produce more flagella that enabled the bacteria to form a wider circular band compared to other concentrations. inhibition of bacteria swimming motility at 0.2% glucose to 1% could be caused by catabolite repression, as downregulated intracellular cyclic-amp (camp) also inhibited biosynthesis of flagella as well as activation of fleq gene. fleq gene is a master regulator of flagellar synthesis (redondo-nieto et al. 2008; patrick and kearns 2012; alsohim et al. 2014). if master regulator could not be activated, the number of flagella synthesized would also decrease. previous study (jahid et al. 2013) reported that inhibition of swimming motility of a. hydrophila was initially observed at 0.25% glucose and motility was completely inhibited by addition of 1% glucose. while in p. fluorescens, 1% glucose concentration could not inhibit the movement of the bacteria thoroughly, though it appeared to be restricted. this strengthen a priori of knight and gregory (2014) who had previously mentioned that p. fluorescens could survive high osmotic pressure. inhibition of swimming motility due to increased glucose concentration had also been observed in some gram-negative bacteria such as e. coli and vibrio vulnificus (dobrogosz and hamilton, 1971; park et al. 2016). diameters of the halo of p. fluorescens in m9 medium supplemented with glucose appeared to be reduced and smaller than in lb medium. this is not surprising as m9 medium only contained least amount of nutrients that was less sufficient in supporting motility of the bacteria. nevertheless, hidalgo et al. (2011) showed completely different result in e. coli cft073 which motility appeared to be undisturbed by minimum nutrient composition in the medium. while this explained that m9 was a poor medium for p. fluorescens motility expression, it also showed that the bacteria required another carbon source or stimulus to induce a more progressive motility. some stimulus that could induce bacteria motility in minimal medium such as glycerol, casamino acids, yeast extract and tryptone (cong et al. 2011). from this observation we could suggest that the type of media used for the assay contributed in determining swimming motility of p. fluorescens. lb medium supported chemotactic motility of p. fluorescens better than m9 medium due to its energyrich composition which contained yeast extract, casein enzyme hydrolysate and tryptone as a source of amino acids and protein. in comparison, m9 medium only contained glucose as its main energy source that made the process of flagella synthesis less effective. as a volume 13, 2019 microbiol indones 47 result, the cells' ability to migrate across the agar became restricted and diameters of the colony appeared smaller. previous investigation by sridhar and steele-mortimer (2016) also reported that salmonella typhimurium motility was more invasive when grown in lb than in m9. significant effect of glucose concentration was also observed in the first growth phase of p. fluorescens in lb medium. addition of glucose in nutrient-rich lb did not appear to give beneficial effect for growth of p. fig 5 colony diameter of p. fluorescens in m9 medium after 6 hours incubation. values on the histogram are the mean ± standard deviation of the mean of independent experiment. fig 7 growth curve of p. fluorescens in lb medium towards glucose concentrations was measured using uv-vis spectrophotometer od . growth reached highest peak in control condition during last hour of incubation 600 followed by glucose depletion. statistical analysis was two-way anova with tukeys test (p < 0.05). fig 6 variation of p. fluorescens chemotactic motility in lb and m9 medium. values on the histogram are the mean ± standard deviation of the mean of two independent experiments. 48 rosa et al. microbiol indones fluorescens. conversely, glucose presence in lb caused catabolite repression to take place. although glucose was not the repressing factor in this case, bacterial growth in 1% glucose showed to be slower than control condition due to the interference of utilization of one carbon source. this led the bacteria to undergo increased lag times as well as the diauxic growth. pseudomonas species has reversed regulatory process of carbon catabolite repression and is different from e. coli, where the repressing carbon sources are organic acids or amino acids, and not glucose (rojo 2010). this investigation suggested a priori that glucose could not be consumed optimally to support higher growth as it was repressed by casein enzyme hydrolysate as the preferred carbon source for p. fluorescens. furthermore, siddique and zalik (2016) also mentioned that addition of glucose to nutrient-rich medium did not appear to be beneficial for microbial culturing practices if the intent is to increase growth rates. on the other hand, control condition of p. fluorescens in m9 medium was shown to be very low due to the fact that m9 is a minimal media containing only traces of mineral and nitrogen without any carbon sources. addition of glucose into m9 making glucose as the only carbon source that could be utilized by the bacteria to support growth. therefore, no additional carbon source would play a role as repressing factor and intervene the utilization of another carbon source. p. fluorescens could focus its catabolism process on one carbon source to promote its growth, which in turn made glucose consumption in m9 medium was greater than that in lb. nevertheless, p. fluorescens growth was still seen higher in lb-control as the medium contained casein enzyme hyrolysate as preferred carbon source for the bacteria. sridhar and steelemortimer (2016) and yang et al. (2017) also had reported that bacterial growth was seen higher in lb medium rather than in m9-glucose. p. fluorescens is one of the species that has the capability to grow well in minimal media. this catabolic versatility has enabled p. fluorescens to survive in extreme environment and to become an important biotechnological organism (palleroni 1984; mailloux et al. 2011). acknowledgment the authors thanked cindy r. deloney-marino for assistance and helpful discussions during the completion of this work. this work was funded by directorate general of higher education (dikti) republic of indonesia through student creativity program – research. references alsohim as, taylor tb, barrett ga, gallie j, zhang x, altamirano-junqueira ae, johnson lj, rainey pb, jackson rw. 2014. the biosurfactant visconsin fig 8 growth curve of p. fluorescens in m9 medium towards glucose concentrations was measured using uv-vis spectrophotometer od . growth reached highest peak in 1% glucose during last hour 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ihm, kuiper i, hendrickx n, bloemberg gv, vanderleyden j, mot rd, lugtenberg bjj. 2002. flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by pseudomonas fluorescens. mol plant microbe interact. 15(11): 1173-1180. doi: 10.1094/mpmi.2002.15.11.1173. deloney-marino cr. 2013. observing chemotaxis in vibrio fischeri using soft agar assays in an undergraduate microbiology laboratory. j microbiol biol educ. 14(2): 271-272. doi:10.1128/jmbe.v14i2.625. dobrogosz wj, hamilton pb. 1971. the role of cyclic amp in chemotaxis in escherichia coli. biochem biophys res commun. 42(2):202-207. doi:10.1016/0006291x(71)90088-x. frais f. 1972. practical biochemistry: an introductory course. london: butterworths. hidalgo g, chan m, tufenkji n. 2011. inhibition of escherichia coli cft073 flic expression and motility by cranberry materials. appl environ microbiol. 77(19): 6852-6857. doi:10.1128/ aem.05561-11. jahid ik, lee n, kim a, ha s. 2013. influence 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pennsylvania state york university. sridhar s, steele-mortimer o. 2016. inherent variability of growth media impacts the ability of salmonella typhimurium to interact with host cells. plos one. 11(6): e0157043. doi: 10.1371/journal.pone.0157043. vicario jc, dardanelli ms, giorando w. 2015. swimming and swarming motility properties of peanut-nodulating r h i z o b i a . f e m s m i c r o b i o l l e t t . 3 6 2 : 1 6 . doi:10.1093/femsle/fnu038. yang p, zhang m, van elsas jd. 2017. role of flagella and type four pili in the co-migration of burkholderia terrae bs001 with fungal hyphae through soil. sci rep. 7: 2997. doi:10.1038/s41598-017-02959-8. page 1 page 2 page 3 page 4 page 5 page 6 page 7 8 no. 309 (pratiwi sudarmono).pmd hospital acquired bacterial infection in burns unit at cipto mangunkusumo hospital, jakarta pratiwi sudarmono∗ & veronica wiwing department of microbiology, medical faculty, universitas indonesia, jalan pegangsaan timur 16, jakarta 10320, indonesia burn injury causes mechanical disruption to the skin, which allows environmental microbes to invade the deeper tissues. a prospective study of infections in burn patients has shown that the incidence of hospital acquired bacterial infection in burn wounds was high. in the burns unit, cipto mangunkusumo hospital, jakarta, 94 patients were hospitalized from january to july 2004. the objective of this study was to evaluate the hospital acquired infections in burn wounds. using a cross sectional study, 49 patients were included. the specimens for bacterial investigation were obtained from clean eschar which has healthy tissue taken at day 1, day 5 and day 10. at the same time, bacterial investigations were conducted from the air and the water, as well as from the hand and nasal swabs of hospital personnel. the results show that klebsiella pneumoniae is the most prominent bacterium found in the wounds, but it is also found in the air. pseudomonas aeruginosa was the number two causative bacteria which caused a change of the bacterial infectivity on day 5 and 10. these bacteria were always found when we conducted bacterial investigations from the water resource of the burns unit. methicillin resistant staphylococcus aureus is also found in the nasal swab of hospital personnel. using the antibiogram pattern, there were similarities between bacteria found in the wounds and in bacteria found in the air and water. in conclusion, hospital acquired burn wound infection in burns unit, cipto mangunkusumo hospital is as high as 62%. the surveillance data are very important for developing good clinical practice guidelines in burn injury treatment and management. key words: burn wound infection, burn injury management _____________________________________________ _________________ ∗corresponding author, phone: +62-21 3160492 ext. 10, fax: +62-21 3100810, e-mail: pratiwi@cbn.net.id burns are one of the most common and devastating forms of trauma. patients with serious thermal injury require immediate specialized care in order to minimize morbidity and mortality (church et al. 2006). burn injury causes mechanical disruption at the skin, which allows environmental microbes to invade the deeper tissues. the usual skin barrier to microbes is replaced by a moist, protein-rich, avascular eschar that fosters microbial growth. the burn wound surface is sterile immediately following injury; however, it is repopulated quickly with gram-positive organisms from hair follicles, skin appendages, and the environment during the first 48 hours. more virulent gram-negative organisms replace the gram-positive organisms after 5-7 days. gramnegative organisms have greater motility, possess many antibiotic resistance mechanisms, and have the ability to secrete collagenases, proteases, lipases, and elastases, enabling them to proliferate and penetrate into the subeschar space. the risk of burn wound infection is directly correlated to the extent of the burn and is related to impaired resistance resulting from disruption of the skin’s mechanical integrity and generalized immune suppression. (bowler et al. 2001; agnihotri et al. 2004). the criteria for admission to the burn care unit are : children with burns involving at least 10% or adults with burns involving at least 20% of their total body surface; burns affecting face, perineum or feet; suspected or proven airway injury; electric or chemical burns; age less than one year or more than 50; or pre-existing disease regardless of the extent of the burns (santucci et al. 2003). when the burn is very large, the patient will be seriously ill, and usually will die after seven days. for those surviving, the fiercest fight is against bacterial pathogens, which usually will increase the duration of hospitalization to 40-148 days (schlager et al. 1994; nasser et al. 2003; taneja et al. 2004). during hospitalization, the nosocomial infection occurs, and the pathogens can come from endogenous as well as exogenous sites (chambers 1997). the bacteria can infect the wound by the airborne route, direct contact from the hands of paramedics or contamination by non sterile equipment (samy et al. 2003). there is very limited data in this hospital about the sources of bacterial infection causing nosocomial infection. although the nosocomial infection rate in any hospital indicates good clinical practice in patient management, even if the rate is low, the burns unit needs a very specific condition since all patients are immunocompromised. in cipto mangunkusumo hospital, jakarta, no data are available about the existence of pathogens in the environment, making it very difficult when a doctor must choose the right antibiotic if infection is detected in burn wounds. besides, any data on burn infections will be very useful, because burn wound treatment is very complex and varies from one patient to another. the objective of this study was to describe infections in a specialized burns intensive care unit in cipto mangunkusumo hospital, jakarta, from january to july 2004. this study was conducted mainly to determine the source of infection from the air and the water which had been used by the patients during their hospitalization and the characteristics of secondary bacterial infection, together with its antibiotic resistance pattern. the data will be very useful for prevention of hospital acquired bacterial infection at the burns unit of cipto mangunkusumo hospital, and to review the standard operational procedures for burn treatment management. materials and methods materials. we used the observational cross-sectional method, so a control group is not necessary. the population comprised all patients with burn injuries hospitalized in this microbiology indonesia, april 2007, p 23-26 volume 1, number 1 issn 1978-3477 burns unit, cipto mangunkusumo hospital from januaryjuly 2004. for statistical calculation, we used the formula: n = zα2 x p x q where : d2 p = the condition which infection is likely happened = 50%; q is 1-p = 50%; d is precision absolute = 15%, and zα = 1.96; 43 patients were used in this study. the study had also been conducted to identify the bacterial pathogens from the air, instruments, linen, gloves, and from the skin and nasal swab of the paramedics. samples. patient samples were obtained from burn wound eschar which still contains healthy tissues. the samples were taken aseptically using a sharp scalpel after wound cleansing in day 1, day 5, and day 10 of hospitalization. samples from day 1 were taken to prove the existence of bacteria at the time of injury. those from day 5 were taken to look for a changing microbial population in the wound and those from day 10 were taken to understand the continuity of infection. the samples were excluded from the study if the specimens were taken using cotton swab (because there will be no healthy tissues) or when the specimens were contaminated by pus. all specimens were transported to the department of microbiology for bacterial culture and identification using brain heart infusion broth (difco, usa) and when bacteria were identified, the antibiograms were conducted following nccls standard. bacterial examination from air. airborne specimens were obtained using an air sampler “mas”100 (merck’s air sampler mas-100) which has been set to take the samples from 10% of room volume. the burns unit at cipto mangunkusumo hospital has one large room whith a volume of 150 m3 so the air sampler was set to take samples from 15 positions in the room. the air samples captured were inoculated in blood agar and endo agar (difco, usa) and incubated at 36 oc for 24 hours. the growing bacteria were gram stained, and then identified and characterized. bacterial examination from water. bacterial examination was conducted to search for bacterial contamination in the water used for washing and cleaning patients’ bodies. a 1.0 l aliquot of water was obtained from previously sterilized tap water. the method used to calculate the bacterial count was by most probable number(mpn). the water specimens were 10 times serial diluted, and inoculated in triplicate with 10 ml lactose broth agar followed by incubation at 35 oc for 24 hours (presumptive test). when fermentation was detected, the specimens were continuously inoculated in brilliant green lactose broth (bglb) as a confirmation test. this was followed by inoculation of 0.5 ml aliquots in endo agar for species identification. linen, instrument, and gloves. linen and gloves were cut using sterile scissors and placed in a glass tube contained thioglycolate solution. instruments such as scalpels and pinsets, which has been used to treat the wound, were also dipped in a flask containing the thioglycolate solution. all tubes and containers were incubated at 35 oc for 24 hours, and the bacteria grown in the solution were then identified further. hands and nasal swabs. the nasal swabs and the swabs from hands of doctors and paramedics were taken and cultured directly in the blood agar for further bacterial identification. ethical approval. this research had been approved by the internal review board of the medical faculty, university of indonesia. informed consent was clearly obtained from each patient. results analysis of surviving and terminal patients. from january to july 2004 there were 94 patients who had been hospitalized in the burns unit, cipto mangunkusumo hospital, jakarta. some 60 persons were men and 34 were women. the causes of burns were fire, hot water, hot food, electricity and hot kerosene. fortyfour (46.8%) patients were cured and 39 (41.5%) patients died while the remainder left hospital on their own before treatment was complete. data were analyzed using the kruskal-wallis test for three variables and the mann-whitney test for two variables (tables 1 & 2). using the kruskal-wallis test, the data showed a positive correlation between the age of patients and the cure rate. the average age of the patients who died was higher than the average age of the cured patients group (p = 0.003). the level of burn injury of the terminally-ill patients was significantly larger than that of the cured patients (p = 0.000).the length of hospitalization for patients who died was significantly shorter than for patient who recovered (p = 0.017). the second analysis was performed using the mann-whitney test. using this method the average age of the terminal patients was higher than for the patients who recovered (p < 0.006). the extent of burn injury of the patients who died was significantly larger than for the cured patients group (p < 0.000). the length of hospitalization for patients who died was significantly shorter compare to the patients who survived (p < 0.006). table 1 characteristics of patients hospitalized in the burns unit, cipto mangunkusumo hospital, from january to july 2004 sex no.patient % patient male female cause of burn injury flame hot water hot food electricity hot kerosene patient condition cured died premature release from hospital 6 0 3 4 4 4 2 8 9 8 3 4 4 3 9 1 1 63.8 36.2 46.8 29.8 9 . 6 8 . 5 3 . 2 46.8 41.5 11.7 table 2 data analysis using kruskal-wallis method patients cured premature release from hospital died variable p age the width of injury days of hospitalization 14.9 + 12.3b 24.1 + 13.2c 32.9 + 20.0d 19.1 + 12.3 20.7 + 10.1 31.1 + 9.5 24.3 + 15.3b 46.1 + 19.1b 21.9 + 14.7d 0.003 0.000 0.017 24 sudarmono and wiwing microbiol indones since cipto mangunkusumo hospital, jakarta is the national referral hospital; the patients who were hospitalized did not always arrive on the first day of their injury. forty two patients (85.7%) were referred by other hospitals, and these arrived mainly on the second or third days of injury (65.4%). in two cases patient arrived on day 10 or day 11, both with very large burn injuries. infecting bacteria and their sources. forty nine patients were included in this study on day 1, but four patients had died by day 5, and another four patients had died by day 10. thus, only 41 patients completed this study. from the eschar culture, klebsiella pneumoniae was the most prominent bacterium isolated from day 1, day 5, and day 10, from 26.5, 36.7, and 40.8% patients respectively. the second most prominent bacterium isolated was pseudomonas aeruginosa. this was found from only 5 (10.2%) patients on day one, but on day 5 it was isolated from 16 (32.7%) patients, and on day 10 it was isolated from 14 (28.6%) patients (table 3). the increase in p. aeruginosa isolates during hospitalization may be caused by the use of water which was also contaminated with the same bacterium (data not shown). of the other bacteria species isolated, one was from water (citrobacter freundii), and one from linen/bed sheets (bacillus sp.). from the air, k. pneumoniae was dominant, followed by the growth of staphylococcus aureus, enterobacter sp., acinetobacter sp., and bacillus sp. there were no bacteria isolated from gloves and medical instruments. of the more significant findings was the isolation of bacteria such as bacillus sp., c. freundii, and staphylococcus sp. from the hands of doctors and paramedics. even worse, bacteria were isolated from nasal swabs of some hospital personnel: 15 persons were contaminated with s. aureus and 8 with methicillin resistant s. aureus (mrsa). changes in bacterial population over time. cross tabulation was conducted for all bacteria obtained on day 1, day 5, and day 10. only in 19 (37.9%) patients, the burn wounds were inhabited by the same bacteria, while in 30 (62.1%) patients, the bacteria obtained in day 1, day 5 and day 10 had changed or were different. only 13 patients on day 1 were infected with k. pneumoniae but on day 5, k. pneumoniae was identified in 18 patients which were originally (day 1) inhabited by staphylococcus sp., k. oxytoca, p. aeruginosa, enterococcus sp., and aeromonas sp. for 2 patients whose wounds were initially (day 1) inhabited by k. pneumoniae, by day 5 superinfection occurred being caused by p. aeruginosa. to prove that k. pneumoniae isolated from the wound on the day 5 was from airborne contamination, the patterns of antibiograms were compared. this showed that the antibiogram patterns of k. pneumoniae isolated both from the air and from the wounds were similar. for p. aeruginosa a similar phenomenon was observed. on day 1, only 5 patients were infected by this bacteria, but by day 5 some 12 patients, which on day 1 were infected by s. aureus, k. oxytoca, enterococcus sp., aeromonas sp., acinetobacter sp., c. freundii, proteus mirabilis, shigella sp., and bacillus sp., were now superinfected by p. aeruginosa. the bacteria colonies isolated from the water used in the burns unit were also identified as p. aeruginosa. moreover, both groups showed similar antibiogram patterns. all antibiograms were compared using 23 different antibiotics (tables 4 & 5). discussion the burn wound surface is sterile immediately following injury; however, it is repopulated quickly with gram-positive organisms from hair follicles, skin appendages, and the environment during the first 48 hours. more virulent gramnegative organisms replace the gram-positive organisms after 5-7 days. in this study we took specimens from eschars at day 1, day 5, and day 10 to detect any changes in bacterial infection or superinfection of the burn wounds. santucci et al. (2003) surveyed 320 patients admitted to a burns-intensivetable 3 bacteria from burn wounds in patients at burns unit, cipto mangunkusumo hospital isolated at day 1, day 5, and day 10 from hospitalization microorganism day 1 ó (%) day 5 ó (%) day 10 ó(%) klebsiella pneumoniae bacillus sp. k. oxytoca pseudomonas aeruginosa staphylococcus sp. enterococcus sp. citrobacter freundii aeromonas sp. acinetobacter sp. proteus mirabilis shigella sp. vibrio sp. proteus vulgaris proteus sp. to t a l 13 (26.5) 13 (26.5) 6 (12.2) 5 (10.2) 3 (6.1) 3 (6.1) 2 (4.1) 1 (2.0) 1 (2.0) 1 (2.0) 1 (2.0) 49 (100.0) 18 (36.7) 7 (14.3) 1 (2.0) 16 (32.7) 1 (2.0) 1 (2.0) 4 (8.2) 49 (100.0) 20 (40.8) 5 (10.2) 14 (28.6) 1 (2.0) 1 (2.0) 8 (16.3) 49 (100.0) table 4 the similarity in antibiogram of klebsiella pneumoniae isolated from burn wounds compared to k. pneumoniae isolated from the air aml amc tic ce cl ctm cxm cec cro ctx cfm fep cpo cn ak c sxt cip mxf gat va iem ox 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 source air p-109 p-112 p-203 p-207 p-211 p-403 p-503 r r r r r r r r i i r i r i i i i i r i r i i i r r r r r r r i i r r i i i i i r r r r r i r s i r r i i i r i i r r i i r r i i r r i s r r r i r r i i r r i i r r i i r r i i s r i r r r i s s r s r r i i r r r r r i r i r r r r s s r i r r r r r s r r r r r r r r r r i i i i s s i s s s s s s s s i s s s s r s s i s i r s s s s i s s s s s s s i s s s s s s s s 1 = aml: amoxycilin, 2 = amc: amoxycillin + clav. acid, 3 = tic: carbenicillin, 4 = ce: cephradine, 5 = cl: cephalexin, 6 = ctm: cephotiam, 7 = cxm: cephuroxime, 8 = cec: cephaclor, 9 = cro: cephtriaxone, 10 = ctx: cephotaxime, 11 = cfm: cefixime, 12 = fep: cefepime, 13 = cpo: cefpirome, 14 = cn: gentamisin, 15 = ak: amikacin, 16 = c: chloramphenicol, 17 = sxt: cotrimoxazole, 18 = cip: ciprofloxacin, 19 = mxf: moxifloxacin, 20 = gat: gatifloxacin, 21 = va: vancomycin, 22 = iem: imipenem, 23 = ox: oxacillin. volume 1, 2007 microbiol indones 25 care unit in brazil, of whom one hundred and seventy-five (55%) developed hospital-acquired infections. the microorganisms causing infections were s. aureus (24%), p. aeruginosa (18%), acinetobacter spp. (14%), and coagulase-negative staphylococci (12%). during the first three days of hospitalization in the burns intensive care unit there were eight infections caused by s. aureus and three of these were resistant to oxacillin (mrsa). those data were almost similar with the data obtained from the present study conducted at the burns unit at cipto mangunkusumo hospitals, jakarta. the rate of hospital-acquired infections in our study is 62.1%, somewhat higher than in brazil. the patterns of bacterial infections are almost the same, except for k. pneumoniae which was consistently isolated from the air of our burn intensive care units as well as from the burn wounds. in sweden, appelgren et al. (2002) also conducted a 3-year prospective study of all infections presented in the burns unit of a university hospital. some 230 adult patients were included. of these 83 patients had a total of 176 infections, giving an infection rate of 48 per 1000 patient days including both nosocomial and community-acquired infections. the most common microorganisms were coagulase-negative staphylococci and methicillin-sensitive s. aureus. seventy-two patients had 107 burn wound infections. comparison of data from indonesia, brazil and sweden showed that hospital-acquired burn wound infection occurred, although with a different level of prevalence. the very high prevalence in indonesia showed that potential infection did not come solely from the endogenous bacteria, but also from bacteria from the air and water which played an important role in secondary infection. antibiotics in burn therapy can play a double role, as prophylaxis as well as for treatment. in indonesia, all patients in our burns unit received antibiotics, while in sweden antibiotics were given to only 50% of the burns patients, including 96% of the patients with infection and 26% of those without infection. the overuse of antibiotics in indonesia indicates the overanxiety of the doctors, since the quality of water and air in the hospital, as well as the hygiene of the medical personnel, is not controllable by their hospital management. however, as table 5 the similarity in antibiogram of pseudomonas aeruginosa isolated from burn wounds compared to p. aeruginosa isolated from the water aml amc tic ce cl ctm cxm cec cro ctx cfm fep cpo cn ak c sxt cip mxf gat va iem ox 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 source water p-115 p-410 p-218 p-317 p-606 p-608 p-204 p-212 p-604 p-301 p-607 p-611 r r r r r r r r r r r r r r r r r i i i r i r r r r r r r r s r r r i r r r r r r r r r i i r r r r r r r r r r r i i r r r r r r r r r r r r r r r r r r r r r r r r i i r r r r r r r r r r r i i r r r r r r r r i r r s s r i r r r r r r r r i i i r i r r r r r r r r i i i r s r r r r r i r r s s s r s r r r r r r r r s i i r s r r r r r r r r r s s r r r r r r r i r r r s s r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r r i r i i i s s i s i i i i s i i s s i i s s s s s s s r i s s i i s i s s s s r s r r i s s r r r r r r s s s s s s s s s s s s s r s s s s s s r s r r s s 1 = aml: amoxycilin, 2 = amc: amoxycillin + clav. acid, 3 = tic: carbenicillin, 4 = ce: cephradine, 5 = cl: cephalexin, 6 = ctm: cephotiam, 7 = cxm: cephuroxime, 8 = cec: cephaclor, 9 = cro: cephtriaxone, 10 = ctx: cephotaxime, 11 = cfm: cefixime, 12 = fep: cefepime, 13 = cpo: cefpirome, 14 = cn: gentamisin, 15 = ak: amikacin, 16 = c: chloramphenicol, 17 = sxt: cotrimoxazole, 18 = cip: ciprofloxacin, 19 = mxf: moxifloxacin, 20 = gat: gatifloxacin, 21 = va: vancomycin, 22 = iem: imipenem, 23 = ox: oxacillin. mentioned above, since cipto mangunkusumo hospital in jakarta has no database about the clinical epidemiology of nosocomial infection, any new data will be very useful. the database can be used to evaluate the effects of changes in burn treatment, staffing and design of burn units, and antimicrobial resistance development in relation to antibiotic usage. these data provide background information regarding extensive burn patients on which decisions for control and prevention of hospital-acquired infections can be made. acknowledgement we are most grateful to yefta moenadjat, the head of the burns unit cipto mangunkusumo hospital, for their guidance and attention which made this study possible. references agnihotri n, gupta v, joshi rm. 2004. aerobic bacterial isolates from burn wound infections and their antibiogram, a five-yearstudy. burns 30:241-243. applegren p, bjornhagen v, bragderyd k, jonsson ce, ransjo u. 2002. prospective study of infections in burn patients. burns 28:39-46. bowler pg, duerden bi, armstrong dg. 2001. wound microbiology and associated approaches to wound management. clin microbiol rev 14:244-269. chambers hf. 1997. methicillin resistant staphylococcus aureus: molecular and biochemical basis and clinical implications. clin microbiol rev 4:781-791. church d, el-sayed s, reid o, winston b, lindsay r. 2006. burn wound infection. clin microbiol rev 19:403-434. nasser s, mabrouk a, maher a. 2003. colonization of burn wound in ain shams university burn unit. burns 29:229-233. samy a, shehab e, el-sayed i, mohammad r. 2003. methicillin resistant staphylococcus aureus, a problem in the burn unit. egypt j plast reconstr surg 27:1-10. santucci sg, gobara s, santos cr, fontana c, levin as. 2003. infections in a burn intensive care unit: experience of seven years. j hosp infect 53:6-13. schlager t, sadler j, weber d, donowitz l, lohr j. 1994. hospitalacquired infections in pediatric burn patients. south med j 87:481-4. taneja n, emmanuel r, chari ps, sharma m. 2004. a prospective study of hospital-acquired infections in burn patients at a tertiary care referral center in north india. burns 30:665-669. 26 sudarmono and wiwing microbiol indones 404 not found 4 any fitriani (55-60).pmd evidence for a link between pathogenicity and the role of imp bacterial transport effector proteins in soybean infection by xanthomonas axonopodis pv. glycines any fitriani1‡, antonius suwanto1∗, aris tri wahyudi1, and budi tjahjono2 1department of biology, 2department of plant protection, institut pertanian bogor, darmaga campus, bogor 16680 xanthomonas axonopodis pv. glycines (xag) is the causal agent of bacterial pustule disease of soybeans. a nonpathogenic mutant of xag (m715) was constructed employing transposon mutagenesis which showed similar epiphytic survival in planta to its wild type strain (yr32). the objective of this work was to identify and to analyze genes involved in pathogenicity in xag yr32. inverse polymerase chain reaction (ipcr) was used to isolate the dna flanking transposon insertion. a 1.3 kb flanking dna fragment was sequenced and analyzed employing blast program to study homology, the position of transposon insertion and to predict the structure and function of the gene. one of the open reading frames (orfs) shared homology with inner membrane proteins (imps) of xanthomonas axonopodis pv. citri (genbank accession no. nc 003919). northern blot analysis revealed that an imps gene was monocistronic and the size of imps mrna in yr32 was slightly longer than in m715. reverse transcriptasepcr analysis demonstrated that the imps transcript in m715 was much less abundant than in the wild type yr32. transposon (mini-tn5-kmr-tpr) was determined to be inserted close to the end of c-terminal region of imps gene and might be sufficient to destabilize the imps transcript in m715 and so influence effectors transportation from xag to plant cell. key words: xanthomonas axonopodis, transposon insertion, non-pathogenic mutant, imps _____________________________________________ _________________ ‡ present address: department of biology education, universitas pendidikan indonesia, jalan dr. setiabudhi 229, bandung 40154 ∗ corresponding author, phone/fax: +62-251-315107, e-mail: asuwanto@indo.net.id bacterial pustule disease of soybeans could decrease soybean productivity. a number of researchers have tried to study the mechanism of pathogenicity in xanthomonas axonopodis pv. glycines (xag). in our laboratory, this research was initiated by mesak et al. (1994) thorough the establishment of a modified soybean cotyledon bioassay (hwang et al. 1992). rukayadi et al. (2000) obtained a nonpathogenic mutant (m715) which failed to cause disease in planta but with survival rate of fitness on the soybean phyllosphere similiar to that of wild type. this mutant was generated from tn5 mutagenesis of wild type strain yr32 (rukayadi 1998). akhdiya (2000) amplified dna flanking the transposon in a recombinant plasmid with primer km-tn903 and m13f. an amplicon with size 0.7 kb was obtained by pcr. pratiwi (2004) has been identified as a flanking dna transposon with a size of 1.8 kb. the dna fragment was sequenced and analyzed with bioinformatics. there are three frames, (i) frame 1, 68 nucleotides resembles the c-terminal region of type ii secretion system protein in xanthomonas axonopodis pv. citri str. 306, (ii) frame 2, resembles the end sequence of aeo11699 (gene of type ii secretion system protein) in x.axonopodis pv. citri str. 306, (iii) frame 3, resembles the gene of iron that encoded the tonb dependent receptor in x. axonopodis pv. citri str. 306. all of these studies have not revealed the genes involved in the lost of pathogenicity in m715. the objectives of this study were to identify the gene disrupted by the transposon in m715 and analyse the transcript of the disrupted gene in m715 and its wild type (yr32). materials and methods bacterial strains and plasmid. bacteria used were e. coli strain dh5α (flaczrm15 reca hsdr17 gyra thi) (sambrook and russell 2001) and x. axonopodis pv. glycines strain yr32 (wild type, rifr ) (rukayadi 1998) and strain m715 (rifr kmr, pat) (rukayadi 1998; widjaja et al. 1999). the plasmid used was pft3551 (ampr, imp-cp-xag, pgem-t easy). growth conditions and media. x. axonopodis pv. glycines yr32 and m715 were grown routinely in luria bertani broth (lb) at ph 7.0 or on ydca (10 g of yeast extract, 5 g of dextrose, 20 g of caco 3 , and 20 g of agar added to 1 litre) at 30 oc. escherichia coli strains were cultured at 37 oc in lb. antibiotics were supplemented when appropriate at concentrations of 25 (kanamycin = km), and 100 (ampicillin = amp, rifampicin = rif, ) µg ml-1. total genomic dna and plasmid isolation. total genomic dna isolation was carried out as described by lazo et al. (1987). plasmid isolation and digestion were carried out as described by sambrook and russell (2001). inverse polymerase chain reaction (ipcr). the strategy for inverse pcr was as far wahyudi et al. (2001). dna templates for inverse pcr were prepared from approximately 1 µg of x. axonopodis pv. glycines genomic dna digested with ecorv. the digested dna was further purified by ethanol precipitation. dna pellets were diluted in 10 µl ddh 2 o and ligated by ligase (new england biolabs, beverly, usa). the ligation reaction was incubated overnight at 16 oc and the dna was then precipitated with ethanol, and the resultant dna pellets diluted in 10 µl ddh 2 o. the circularized dna generated from the ligation mixture was amplified using gene amp pcr 2400 (perkin elmer, usa) in a total volume of 25 µl containing 2.5 mm dntp mixture, gc buffer i, la taq dna polymerase (takara, japan), and 10 pmoles of each primer designed from known sequence (primer microbiology indonesia, august 2007, p 55-60 volume 1, number 2 issn 1978-3477 1-f: 5’-atccttgccattg acctg-3’ primer 1-r: 5’ccaccgaac ttgaactggtc-3’). dna was amplified by pcr consisting of denaturation at 95 oc for 2 min, primer annealing at 62 oc for 1 min, primer extension at 72 oc for 1 min for 30 cycles and 10 min for the last cycle at 72 oc. pcr product was purified by dna purification methods employing centrifugation (wizard sv gel and pcr clean-up system, promega, madison, usa). gene cloning. purified pcr products were inserted into pgem-t easy vector (promega, madison, usa). ligated reaction was incubated at 16 oc overnight. competent e. coli dh5α was obtained using cacl 2 methodology for 30 min. dna was transformed into e. coli dh5α using heat shock treatment at 42 oc for one min. dna sequencing and analysis. dna sequencing was carried out using the dye terminator cycle sequencing kit. m13 forward and m13 reverse primer were used as cycle sequencing primers. sequencing of the dna was performed using an automatic dna sequencer abi prism 3100-avant genetic analyzer (california, usa). dna sequences were analyzed employing blast (ncbi) (altschul et al. 1997). rna isolation and reverse transcriptase-polymerase chain reaction (rt-pcr). total rna was isolated from liquid culture xag yr32 and m715 after incubation for 28 h (a 600 = 0.7). total rna was isolated use trizol® reagent (invitrogen, california, usa). the quality of the isolated rna was verified by running gel electrophoresis using 1.5% w/v denaturing agarose gels stained with 0.5 µm ethidium bromide, and the amount of isolated rna was determined by spectrophotometry at 260 nm and 280 nm. total rna samples (5 µg) were reverse-transcribed by m-mulv reverse transcriptase (protoscript first strand cdna synthesis kit, new england biolabs, beverly, usa) from anchored reverse gene specific primer using standard methods in a reaction volume of 20 µl. cdna was amplified by pcr using primer (imp-forward and imp-reverse) and prepcr at 95 oc for 3 min, denaturation at 95 oc for 1 min, annealing at 62 oc for 1 min, synthesis at 72 oc for 1 min, and postpcr at 72 oc for 7 min. the amplicon was run by agarose gel electrophoresis using tae buffer. 16s rdna was amplified by pcr using 16s rdna universal primer (63f and 1387r). northern hybridization and analysis. the total rna samples (yr32 and m715) (5 µg) were run on 1% w/v denaturing agarose gel for 2 h at 65 v. the gel was stained for 30 min with 0.5 µm ethidium bromide. the gel was blotted onto a nylon membrane (amersham life-science, usa) overnight in 20x ssc. the membrane was washed in 6x ssc at room temperature with agitation for 15 min and dried on blotting paper (amersham life-science,usa), followed by baking at 80 oc for 2 h. a probe consisting of a 375 bp pcr amplified fragment of the inner membrane proteins gene from yr32 was labeled with non-radioactive neblot™ phototope™ kit (new england biolabs, beverly, usa) according to manufacturer’s instructions. hybridization was performed overnight at 42 oc in hybridization solution (5 ml formamide, 1.0 ml 50x denhardts solution, 2.5 ml 20x sspe, 0.1 ml 10% sds, 50 µl 1 µg ml-1 salmon sperm dna (sigma, usa). the membrane was washed twice in 1x ssc, 0.1% sds for 10 min at room temperature and twice in 0.5x ssc, 0.1% sds for 10 min at 50 oc and then dried on blotting paper (amersham life-science, usa). nucleic acids were detected using the phototope™ detection kit (new england biolabs, beverly, usa) according to the manufacturer’s instructions. the membrane was exposed to x-ray (hyperfilm™ mp, amersham life-science, usa) in dark room. x-ray film was processed in high performance x-ray film developer (fuji photo film co., ltd, japan) and washed in water. x-ray film was washed in x-ray film fixer (fuji photo film co., ltd, japan) and then washed in water and air dried. result inverse pcr generated two dna bands, i.e.: 1.3 kb and 3.0 kb (fig 1). it is surprising that the same nucleic acid sequence in xag chromosome could result in more one band in pcr product. it is possible that the primer could be complemented with two sites on the chromosome until it formed two amplicons. for this study, we only analyzed 1.3 kb fragment because the dna sequence has aligned nucleotide with previous dna sequence (pratiwi 2004). inverse pcr product was purified and inserted to pgemt easy (3.015 kb), giving pft3551 (fig 2). plasmid verification was done with restriction analysis which showed that the insert was 1.3 kb long and the nucleotide did not have ecori, psti and saci sites (data not shown). the dna sequence was analyzed employing blast program (ncbi). blastn analysis revealed that 1.3 kb nucleotides was homologous with xanthomonas axonopodis pv. citri str. 306. alignment analysis of 1.3 kb nucleotides showed a 99% identity with xanthomonas axonopodis citri str. 306, and e-value of zero. nucleotides of 1.3 kb encoding inner membrane proteins (imps) with an identity of 90% and cystein proteases (cps) with an identity of 99% in xanthomonas axonopodis pv. citri str. 306. open reading frame (orf) analysis revealed the presence of two genes in different operons. imps and cps consisted of 182 and 153 amino acids, respectively (data not shown). genes of imps are involved in pathogenicity (von heijne 1992). the genes of imps were found at the start codon (atg), but the stop codon (tga) was as found in the pratiwi (2004) sequence after assembly. frequency for tga in xanthomonas axonopodis citri str. 306 is about 70.89% (http://rice.tigr.org/tigr_scripts/cmr2/codon_tables). putative rbs (ribosome binding site) and the promoter of imps genes (tang 1991; katzen et al. 1996; baldini et al. 1999) have been found (fig 3). the transposon inserted at the c-terminal region of the genes (fig 3 and 4). the quality of isolated total rna was confirmed by denaturing agarose gel electrophoresis. two intact ribosomal bands characteristic of undegraded rna were observed. the sample rnas also had a normal spectra (a 260 /a 280 = 2.0) and could be used for cdna synthesis and rt-pcr. two intact ribosomal bands showed that from upper to lower, 23s rrna, 16s rrna. usually, sizes of ribosomal rnas in bacteria are 1.5 kb and 2.9 kb for 16s rrna and 23s rrna respectively (data not shown). northern hybridization analysis revealed that inner membrane protein coding 0.6 kb was on a monocistronic gene. the size of transcript mrna was the same as the size of the gene at 600 bp (fig 5). the size of the transcript mrna in m715 was lower than in yr32, which means that the gene of imps in m715 was interrupted with a transposon. 56 fitriani et al. microbiol indones fig 2 plasmid pft3551. spel a m p r f1 ori lacz ecori notl apal 4315 bp p f t 3 5 5 1 bstz17i xcml imps/cps nsil alul hincll msel kpnl sall notl sacl pstl notl ecori m 1 3.0 kb 1.3 kb fig 1 agarose gel electrophoresis profile of ipcr product. m: 1 kb dna ladder (new england biolabs,beverly, usa). 1: ipcr. fig 3 dna sequence of imps genes, amino acids, and position of transposon insertion (black arrow). -35, -10: promoter, rbs: ribosome binding site; start: start codon; stop: stop codon. acagtggcggtcgtcgaataaccgg -35 gtcgcgcacttcaccttcgcttcaaattcgcctcaaacctgcgac -10 accggcgcagcgcaccctggccacctcccaagatgaggatggccg rbs atgaaatccctgaaactgttattgcggttcgccaccatcggtggg m k s l k l l l r f a t i g g start ctgatcctgctgttgctgattccgctgctcctgatccgtggcgcg l i l l l l i p l l l i r g a gtgcaggaccgcgcgcgctaccgcgacgaggcggtggagcgggtg v q d r a r y r d e a v e r v gcgcagagcaaggctggcgagcagcagttcatcgcgccggtgcgg a q s k a g e q q f i a p v r gtactgccgtataccgaagacgtgcaggtcaccgagccggacgag v l p y t e d v q v t e p d e cagggcaaccagcgcaaggtccggcgcaagcgcgaagggacgctg q g n q r k v r r k r e g t l ctgcaaacgccgcgtcgcctgaaactcagcggcgaaatggtgccc l q t p r r l k l s g e m v p tcggtgcgcgaggtgggcttgtaccgggtgcaggtgtattcctgg s v r e v g l y r v q v y s w aaagccaccttgcatgccgaatacgactccttcgactacgcggct k a t l h a e y d s f d y a a gcgccgacccgtgcgtatggccagccgtacctggcaatcggtatg a p t r a y g q p y l a i g m tccgacgtgcgcgggttggtgggcacgccgcgcttgcaggtcaat s d v r g l v g t p r l q v n ggcggcaaggatcgggtgcgcttccagagcgctatcgaacgcttt g g k d r v r f q s a i e r f cgaaagtgactgttgacacgactcttagagaccataagaatcaac r k * terminator terminator stop tccactgaattggtactttccagtcagggccgttgatcagcaatg pft3551 as the positive control. distilled water, y16s and m16s are the negative control and have no amplicon. yield of amplicon of yr32 was much more than of m715. the gene for inner membrane proteins has been successfully transcribed in yr32, but not in m715 (fig 6). in this research, we used of universal primers for 16s rdna. the aim was to examine the quality of first strand cdna synthesized. no pcr product after amplification by primers shows that the cdna formed originated from transcript mrna of inner membrane protein genes in xag. pft3551 (4.3 kb) which is a dna plasmid that possesses inner membrane proteins and cystein proteases genes from xag. pft3551 was amplified by inner membrane protein primers and has the same product length (about 375 bp). this is demonstrates that the oligonucleotides have the correct sequence for dna coding for bacterial of inner membrane proteins. discussion pustule disease is one of the five main diseases in soybeans. pustule disease attacks soybean’s leaves and the symptom of pustule disease is chlorosis with a yellow spot in the centre. pustule disease in soybeans causes a decrease in productivity in indonesia. in our laboratory, the mechanism of pathogenicity has been studied since 1995. rukayadi (1998) have been constructed the mutant m715. m715 has a phenotype of de lorenzo et al. (1990) described that mini tn5-kmr has stop transcription points at two dna flanks of the resistance antibiotic gene. the signal of the mrna transcript in m715 was weaker than inyr32. the level of transcription in m715 was lower than in yr32. first strand cdna synthesis was carried out by using total rna, reverse transcriptase and imp-reverse primer. the cdna was successfully amplified by using a combination of primer imp-forward and imp-reverse, with products lengths about 375 bp. amplicon pcr was shown in yr32, m715, and volume 1, 2007 microbiol indones 57 0 . 6 1 . 5 2 . 9 kb m 1 2 3 4 1 2 3 4 kb a b fig 5 rna electrophoresis and rna-blot analysis of yr32 and m715. a. rna electrophoregram of yr32 and m715, b. rna-blot of yr32 and m715 with inner membrane proteins probe. lane 1 and 2: yr32, lane 3 and 4: m715; lane m: 1 kb dna ladder (new england biolabs, beverly, usa). 58 fitriani et al. microbiol indones minitn5-km r-tp r fig 4 physical map of the genes and position of transposon insertion orf 1: cystein proteases. orf2: inner membrane proteins, orf3: tonb dependent-receptor. orf3 orf2 bstz17i xcml nsil orf1 alul hincll msel kpnl sall notl 2198 bp 375 bp m yr32 m715 ddh 2 o y16s m16s pft3551 fig 6 rt-pcr of inner membrane proteins from total rna sample. m: nugen marker; yr32: xag yr32 (rt of yr32, pcr with imps primer); m715: mutant m715 (rt of m715, pcr with imps primer); ddh 2 o: negative control (distillated water, pcr with imps primer); y16s: negative control (rt of yr32, pcr with 16s rdna primer); m16s: negative control (rt of m715, pcr with 16s rdna primer); pft3551: positive control (pft3551, pcr with imps primer). kb 1.25 0 . 7 0 . 4 non-pathogenicity in soybean and no hypersensitive reaction in tomato. mutant m715 was constructed by transposon mini tn5-kmr-tpr. our research revealed that mini tn5-kmrtpr was inserted in the inner membrane proteins genes. imps genes were involved in the protein transportation apparatus especially of virulence, toxins, and pathogenicity factor determinants transported from the cell to the environment (von heijne 1992). in e. coli, imps function in colicin transportation (marchler-bauer and bryant 2004). quinaud et al. (2005) and edqvist et al. (2003) revealed that in pseudomonas aeruginosa and yersinia, imps was a part of the type iii secretion system (t3s) that plays key roles in pathogenicity and are employed to inject toxin (effectors) directly into the cytoplasm of target cells. analysis of mrna showed that inner membrane proteins was successfully transcribed in xag yr32 but not in m715. the amplicon formed in m715 was weaker than in yr32. this phenomenon strongly indicated that some factor interfered in m715 expression and we consider that this is because m715 was interrupted by a transposon. the mrna of imps in m715 was not stable and had shorter half-life. additionally the size of translated protein differed from that of the wild type. transposon mutagenesis caused a change of nucleotide sequence in the position of transposon insertion. in our research, we used the composite transposon mini tn5-kmr-tpr ,derived from tn5, which has terminator transcription regions at two flanking antibiotic resistance genes (de lorenzo et al. 1990). bioinformatics analysis revealed that the transposon was inserted in the c-terminus of the imps gene. this conclusion was strongly supported by northern blotting analysis. northern blotting indicated the inner membrane protein gene system was monocistronic, because size of its the transcript was the same as for the genes (about 600 bp). this phenomenon supported bioinformatics analysis which showed that in the dna sequence of imps genes was a terminator stop transcription. in addition to the size of the transcript mrna in m715 was shorter than in yr32 (fig 5). if the transposon inserted close to the end of c-terminus in m715, then the result would be that rna polymerase could be stopped in stop transcription of the transposon at the cterminal end of the genes. the c-terminal region of proteins has a functional role in whole protein structure. tateno et al. (2006) described the c-terminus deletions and site-directed mutagenesis in membrane calcium channel would result in misfolding of the c-terminus and/or inaccessibility to trafficking/sorting machineries. takazaki et al. (2006) explained that mutation at the c-terminus of a human anion transporter affected the rate of conformational change of this protein. they concluded that the c-terminal region has a functional role in the conformational change capacity that is necessary for anion transport. han et al. (2006) reported the c-terminal region of a potassium channel plays a critical role in the localization and gating of the channel. the mutagenesis transposon in imps has a similar result. mutation at the c-terminus of imps affected protein functionality. a mutant imps was recognized by the signal recognition particle (srp) at the hydrophobic n-terminal and transported to the bacterial inner membrane. when studying protein coded in the seca-secyeg complex, mutant imps have a changed configuration and this influences localization in the inner membrane. changed localization and configuration of mutant imps affects functionality of coded proteins. in conclusion, the phenotype of m715 was not pathogenic and gave no hypersensitive reaction in tomato plants (rukayadi et al. 2000). here we conclude that this phenotype is a result of damaged bacterial proteins imps which have a role as effectors in protein translocation. we consider a defective (protein) transportation system is the reason for the loss of pathogenicity by xag. acknowledgement this report is part of af dissertation and primarily funded by the research center for microbial diversity, bogor agricultural university. we thank yul kurniarun and research and development pt. charoen phokphand indonesia for the laboratory facilities used in rna analysis. reference akhdiya a. 2000. gene cloning that involved pathogenicity in xanthomonas axonopodis pv. glycines [thesis]. bogor: bogor agricultural university. altschul sf, gish w, miller w, myers ew, lipman dj. 1997. basic local alignment search tool. j mol biol 215:403-410. baldini rl, tahara st, rosato yb. 1999. a rolling circle miniplasmid of xanthomonas campestris pv. glycines: the nucleotide sequence and its use as a cloning vector. plasmid 42:126-133. de lorenzo dv, herrero m, jakubzi u, timmis kn. 1990. mini-tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned dna in gramnegative eubacteria. j bacteriol 72:6568-6572. edqvist pj et al. 2003. yscp and yscu regulate substrate specificity of the yersinia type iii secretion system. j bacteriol 185:22592266. han w, nattel s, noguchi t, shrier a. 2006. c-terminal domain of kv4.2 and associated kchip2 interactions regulate functional expression and gating of kv4.2. j biol chem (in press). hwang pl. harsono hd, shaw pd. 1992. use of detached soybean cotyledons for testing pathogenicity of xanthomonas campestris pv. glycines. plant dis 76:182-183. katzen f, becker a, zorreguita a, puhler a, lelpi l. 1996. promoter analysis of the xanthomonas campestris pv. campestris gum operon directing biosynthesis of the xanthan polysaccharide. j bacteriol 178:4313-4318. lazo gr, roffey r, gabriel dw. 1987. conservation of plasmid dna sequences and pathovar identification of strains of xanthomonas campestris. phytopathology 77:448-453. marchler-bauer a, bryant sh. 2004. cd-search: protein domain annotations on the fly. nucleic acids res 32:327-331. mesak fm, suwanto a, tjahjono b, guhardja e. 1994. modifikasi bioesei kotiledon kedelai untuk uji patogenisitas xanthomonas campestris pv. glycines. j il pert indon 4:77-82. pratiwi e. 2004. analysis of dna sequence involved in pathogenicity and design of specific pcr primer of xanthomonas axonopodis pv. glycines [dissertation]. bogor: bogor agricultural university. quinaud m et al. 2005. the psce-pscf-pscg complex controls type iii secretion needle biogenesis in pseudomonas aeruginosa. j biol chem 280:36293-36300. rukayadi y. 1998. construction of partial genetic map and epiphytic survival of non-pathogenic mutant of xanthomonas axonopodis (campestris) pv. glycines yr32 [dissertation]. bogor: bogor agricultural university. rukayadi y, suwanto a, tjahjono b, harling r. 2000. survival and ephiphytic fitness of a nonpathogenic mutant of xanthomonas campestris pv. glycines. appl environ microbiol 66:1183-1189. sambrook j, russel dw. 2001. molecular cloning. a laboratory manual. vol. 1:1.32. new york: cold spring harbor laboratory pr. volume 1, 2007 microbiol indones 59 takazaki s et al. 2006. the functional role of arginine 901 at the cterminus of the human anion transporter band 3 protein. j biochem 139:903-912. tang j. 1991. genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in xanthomonas campestris pv. campestris. mol genet 226:409-441. tateno t, nakamura n, hirata y, hirose s. 2006. role of c-terminal of kir7.1 potassium channel in cell-surface expression. cell biol int 30:270-277. von heijne g. 1992. membrane protein structure prediction. hydrophobicity analysis and the positive-inside rule. j mol biol 225:487-494. wahyudi at, takeyama h, matsunaga t. 2001. isolation of magnetospirillum magneticum amb-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis. appl biochem biotechnol 91:147-154. widjaja r, suwanto a , tahjono b. 1999. genome size and macrorestriction map of xanthomonas campestris pv. glycines yr32 chromosome. fems microbiol lett 175:59-68. 60 fitriani et al. microbiol indones 04 lg.cdr tempoyak is a fermented dish that originated from sumatera island. it is mainly made from unpasteurised durian (durio zibethinus) flesh mixed with 2.5% salt (w/v) and placed in a sealed container (urnemi et al. 2010). dominant taste of tempoyak is acid due to fermentation process in durian flesh. tempoyak is usually consumed as a side dish when eating time. tempoyak is not only found in sumatera but also in other parts of kalimantan island with different names. in addition, this food is also known in malaysia as well (amiza et al. 2006). the main lactic acid bacteria (lab) strains isolated from tempoyak is lactobacillus spp (leisner et al. 2001; coeuret et al. 2003). recently, many investigators have shown that various fermented foodisolated lactobacillus plantarum species isolated could be considered as molecular resources for various end products such as bacteriocins, biomass, polysaccharides, and lipid for instant l. plantarum klds 1.0391 isolated from “jiaoke” (gong et al. 2010); l. plantarum s34 from “bekasam” (mustopa et al. 2010); l. plantarum lb-b1 isolated from “koumiss” (xie et al. 2011); l. plantarum gj7 isolated from “kimchi” (chang and chang 2011), and lactobacillus isolated from cheeses and yogurts (yang et al. 2012). the most end products from these lab which could be benefit for medicinal purpose is bacteriocins (gillor et al. 2005). this is secondary metabolites of lactic acid bacteria are ribosomally synthesized peptides that are active against similar or genetically close related bacteria (hata et al. 2010). salmonella enterica serotype typhi (s. typhi) is the bacteria that caused typhoid faver (bhan et al. 2005). typhoid fever occurs both in the tropical and subtropical countries, especially in developing countries where improperly sanitary conditions and inadequate health facilities commonly found (kidgell et al. 2002). the incidence of typhoid fever in the vol.9, no.2, june 2015, p 73-81 doi: 10.5454/mi.9.2.4 inhibitory activity of lactobacillus plantarum u10 isolated from tempoyak (fermented durian) made in indonesia against salmonella typhi 1 2 1 2 sogandi , apon zaenal mustopa , i made artika , and bugi ratno budiarto* 1 department of biochemistry, institut pertanian bogor, dramaga campus, bogor 16680, indonesia; 2 research centre for biotechnology, indonesian institute of science (lipi), jalan raya bogor km 46, cibinong, bogor 16911, indonesia lactobacillus plantarum u10 produced bacteriocin was isolated from a traditionally fermented food “tempoyak” from sumatera island in indonesia. production of the bacteriocins started at early exponential phase and reached maximum level at early stationary phase. furthermore, plantaricins u10 was purified by ammonium sulphate precipitation followed by gel filtration chromatography. l. plantarum u10 produced two bacteriocins with a molecular mass of approximately 4.5 and 9.8 kda by sds-page. the mode of action of plantaricins u10 was identified as bactericidal agents against salmonella typhi atcc25241 as proven by cfu counting and sem micrographs that showed differences in cell structures between treated cells and the non-treated control. sem examination also confirmed structural destruction of membrane cells integrity and considerable morphological alteration of s.typhi. key words: bacteriocin, l. plantarum u10, mode of action, purification lactobacillus plantarum u10 yang diisolasi dari makanan tradisional fermentasi “tempoyak” berasal dari pulau sumatera di indonesia menghasilkan bakteriosin. produksi bakteriosin dimulai pada awal fase eksponensial dan mencapai titik maksimum pada awal fase stasioner. selanjutnya, plantarisin u10 dimurnikan melalui presipitasi amonium sulfat dilanjutkan dengan kromatografi filtrasi gel. l. plantarum u10 menghasilkan dua bakteriosin dengan bobot molekul berkisar 4,5 dan 9,8 kda berdasarkan sds-page. mekanisme aksi plantarisin u10 telah berhasil diidentifikasi yaitu memiliki sifat bakterisidal terhadap salmonella typhi atcc25241 yang dibuktikan dengan jumlah cfu dan sem mikrograf yang menunjukkan perbedaan struktur sel antara sel dengan perlakuan dan kontrol. pemeriksaan menggunakan sem juga menunjukkan kerusakan integritas struktur membran sel dan perubahan morfologi sel. kata kunci: bakteriosin, l. plantarum u10, mekanisme aksi, pemurnian *corresponding author; phone: +62-21-8754587, fax: +6221-8754588 ; email: azmustopa@yahoo.com available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 microbiology indonesia world is estimated at more than 2.16 million cases with a mortality rate of 216000 and more than 90% of them occur in asia. in indonesia itself the estimated incidence is 180 / 100000 per year in children aged 515 years (ochiai et al. 2008). l.plantarum u10 from tempoyak (fermented durian fruit) has previously been isolated in our lab (urnemi et al. 2010). moreover, this strain also showed broad adaptive response to environmental stressors (margareta et al. 2015). furthermore, this strain exhibited good antibacterial activity against pathogenic bacteria such as e. coli nbrc 14237, staphylococcus aureus nbrc 13276 and bacillus subtilis btccb 612, thus this strain has potential application as natural antibacterial agent (urnemi et al. 2010). however, until recently nothing is known about the antibacterial activity of l.plantarum u10 against typhoid infection. therefore, the objective of this study was to purify the bacteriocin from the bacterial strain and to provide a preliminary investigation of its mode of action againts s. typhi. materials and methods bacterial strains and growth conditions. strain u10 (collection of research center for biotechnology, indonesian institute of science), the bacteriocin producer strain used in this study, was isolated from tempoyak, a traditionally fermented food from indonesia. it was cultured in deman rogosa sharpe (mrs) broth (oxoid, england), and incubated at 37 ºc. changes in ph and optical density (600 nm) were recorded every 2 h for 32 h without agitation. bacteriocin activities were measured at the same time intervals. pathogenic bacteria used is s. typhi atcc25241 grown in nutrient agar (na) containing pepton 1% (oxoid, england), beef extract 0.3% (himedia, india), nacl 0.5% (merck, germany), and agar 1.5% (oxoid, england) as slab cultured stock were stored at 20 ºc. subcultured twice in the media nutrien broth (nb) containing pepton 1% (oxoid, england), beef extract 0.3% (himedia, india), and nacl 0.5% (merck, germany) and was incubated at 37 ºc for 14 h with agitation before use. bacteriocin activity. bacteriocin activity was determined by the well-diffusion assay. cell-free supernatants adjusted to ph 6.5 by the addition of sterile 1n naoh using a digital ph meter (eutech -1 ph510), and treated with catalase (1 mg ml ) to exclude the inhibition due to hydrogen peroxide production. three hundred supernatants were then spotted onto paper discs (diameter 6 mm; filtres fioroni, france) and loaded onto soft agar plates. soft agar media containing the gram-positive and gramnegative bacteria strains were then poured into the plates. these plates were incubated at 37 ºc and examined for inhibition zones. in some experiments, antibacterial activity was expressed as arbitrary units -1 (au). to obtain the titer (au ml ), serial dilutions of bacteriocin were prepared and dispensed in wells. the -1 titer (au ml ) was defined as the reciprocal of the highest dilution which gave a definite zone (xie et al. 2011). production of crude bacteriocin. cell-free supernatants for antibacterial assay was prepared by growing l. plantarum u10 isolates (1%, v/v) into 300 ml mrs broth medium and incubated at 37 ºc for 20 h. the cells were harvested with centrifuged at 12000 g, 4 ºc for 15 min (xie et al. 2011). the plantaricins were purified from the cell free supernatant by ammonium sulphate precipitation and gel filtration sephadex g-50 (ge healtcare, sweden). ammonium sulphate was added until 80% saturation at 4 ºc. the precipitated protein was centrifugated at 12000 g, 4 ºc for 15 min and the resulting pellets were solubilized by buffer tris-hcl 10 mm ph 7.4. purification of plantaricin u10 and sds-page. for column chromatography, crude plantaricin u10 was applied to a sephadex g-50 column (25 cm with diameter 2 cm) equilibrated with 50 mmol tris-hcl buffer (ph 7.4) at 4 ºc. the column was eluted into 5 -1 ml fractions at a flow rate of 1 ml min . protein concentrations in collected fractions were determined by measuring the absorbance at 280 nm and bacteriocin activities in the collected fractions that were measured. the molecular weight of the purified plantaricin u10 was determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (sds-page) (laemmli 1970). gels consisted of a 3.9% (v⁄v) stacking gel and 16% (v⁄v) separating gels that were 1 mm thick, 9 cm long, and 9 cm wide. low range protein ladder (thermo, spectra multicolor, eu) was used as the molecular size standard and stain with silver staining kit (fermentas, eu). determination of protein. determination of protein concentration performed quantitatively using bicinchoninic acid (bca) protein assay kit (thermo fisher scientific, us) with bovine serum albumin (bsa) as the standard protein using a microplate. microplate was incubated at 37 °c for 30 min, then the reaction proceeds read at a wavelength of 540 nm using an elisa reader (pierce 2013). 74 sogandi et al. microbiol indones mode of action plantaricin u10. to study the effect of the antibacterial compound on s. typhi atcc25241, 10 ml cell-free supernatants of l. plantarum u10 was adjusted to ph 6.5. a treatment -1 with catalase (1 mg ml ) was added to the 50 ml culture of s. typhi atcc25241 at early exponential phase and then incubated at 37 ºc. samples were taken every 2 h to record the optical density at 600 nm and -1 determine the viable cells (cfu ml ) by counting cfu on nutrient agar plates after incubation at 37 ºc overnight. all experiment were carried out in triplicate (xie et al. 2011). scanning electron microscope (sem) analysis. s. typhi atcc25241 bacteria was suspended in plantaricin and later incubated for 24 h at 37 °c. the solution was centrifuged, the supernatant discarded, 2% glutaraldehyde added and soaked for several hours. after the solution was centrifuged, the supernatant was discarded, added a solution of 2% tannic acid was added and soaked for 2 h. the solution was centrifuged, fixative solution was discarded, caccodylate buffer was added and soaked for 20 min. then solution was centrifuged, buffer was discarded, 1% osmic acid was added and soaked for 1 h and subsequently dehydrated with a graded ethanol series. the samples were lyophilized, coated with gold in an ion coater, and then examined by scanning electron microscopy jeol jsm-5310 lv series (zengin and baysal 2014; wang et al. 2014). results a n t i b a c t e r i a l a c t i v i t y s p e c t r u m . t h e antibacterial activity spectrum of cell-free supernatant obtained from 20 h l. plantarum u10 culture was assayed using wells diffusion agar methods against a wide range of microorganisms (table 1). the antibacterial activity of cell-free supernatant not only give clear zone for gram-positive bacteria but also for gram-negative bacteria. based on this observation l. plantarum u10 was able to inhibit several bacteria and food-borne pathogens, such as bacillus subtilis; escherichia coli, and pseudomonas aeroginosa. particulary salmonella typhi and staphylococcus volume 9, 2015 microbiol indones 75 table 1 antibacterial activity spectrum of plantaricin u10 produced by lactobacillus plantarum u10 species strain 1 source 2 diameter (mm) of zone of inhibition salmonella typhi atcc 25241 atcc 11.33 ± 1.15 staphylococcus aureus atcc 6538 atcc 11.00 ± 0.00 bacillus subtilis atcc 19659 atcc 9.33 ± 0.58 pseudomonas aeroginosa atcc 15442 atcc 9.67 ± 0.58 escherichia coli mc mobitech escherichia coli dh5a mobitech 9.33 ± 0.58 escherichia coli top10 mobitech 8.67 ± 0.58 staphylococcus epidermis lab stock 12.67 ± 0.58 salmonella typhi lab stock 10.33 ± 0.58 listeria monocytogenes btcc b693 btcc 10.67 ± 0.58 lactobacillus plantarum s34 lab stock lactobacillus fermentum u11 lab stock lactobacillus plantarum s12 lab stock lactobacillus acidophilus c9-9 lab stock pediococcus pentosaceus gr3 lab stock pediococcus acidilactici s23 lab stock pediococcus acidilactici r24 lab stock lactoccocus lactis lac 2 lab stock lactoccocus lactis nz3900 mobitech 1 atcc, american type culture collection; btcc, biotechnology type culture collection. 2 wells (6 mm in diameter) were filled with 25 µl samples cell-free supernatant which ph neutralized to 6.5 and h o eliminated; mean counts of tri-trials (mean ± sd); ''–”no inhibitory zone was observed.2 2 aureus both of them were more sensitive to l. plantarum u10's cell-free supernatant treatment. biomass and bacteriocin production. biomass measurement and bacteriocin production result are shown. l. plantarum u10's cell density increased from 0.1 up to 3.6 (od ) during 20 h at 37 ºc and gradually 600 decreased until 2.7 (od ) when incubation was 600 continuous until 32 h. for ph medium, it decreased rapidly from 6.5 to 4.0 at the same period of measurement. furthermore, plantaricin u10 production was started at the early exponential phase -1 and reached maximum (800 au ml ) after 20 h of incubation (early stationary phase) when cells biomass was at optimum od (fig 1). purification and molecular weight of plantaricin u10. plantaricin from cell-free supernatant was collected by centrifugation, then proteins were concentrated by 80% ammonium sulphate precipitation, followed by sephadex g-50 gel filtration column. all procedures were done in cold room. forty fractions (f1-f40) with 2 ml each were succeeded to be eluted from a sephadex g-50 column and showed two distinct peaks at fraction number 8 and 14 respectively (fig 2) and were further sub-fractioned into six sample named si (f8 and f9), sii (f10 and f11), siii (f12-f14), siv (f15). sv (f16 and f17), and svi (f18-f20). antagonistic activity of each sample was assayed using s. typhi atcc25241 as bacterial indicator, yet only in sample ii (f10-11) showed inhibitory activity as by clear zone formation (data not shown). purification indicators after two steps of purification was 61.07 fold and 33.25% for yield, as summarized in table 2. the partially purified plantaricins was analysed by the sds-page for fig 1 bacteriocin production during the growth of lactobacillus plantarum u10 in mrs broth at 37 ºc. the optical density (absorbance at 600 nm: ●) and ph (▲) of culture were measured at the time intervals as -1 indicated. the antibacterial activity (■) was also assayed and expressed as au ml . fig 2 elution profile of purified plantaricin u10 on sephadex g-50 column monitoring by absorbance at 280 nm. the purified plantaricin is shown by a circle. 76 sogandi et al. microbiol indones determination of the molecular weight (fig 3). it clearly exhibited two protein bands with a molecular mass of approximately 4.5 and 9.8 kda. mode of plantaricin action. after the addition of plantaricin u10 to an exponentially growing of s. typhi atcc25241 culture, od values of the treated culture demonstrated no significant increment or relatively constant. on the other hand, the addition of plantaricins u10 into the same condition caused dramatically 8 decrease in viable cells number (from 2.2 x 10 cfu -1 6 -1 ml to 3.1 x 10 cfu ml ) over the following 9 h observation (fig 4). these results suggested that the mode of plantaricins u10 action was bactericidal effect. morphological changes of plantaricin u10 exposured s. typhi. s. typhi atcc25241 were -1 treated for 24 h with plantaricin (133 au ml ) then observed by sem to examine morphological changes table 2 purification of plantaricin u10 fig 3 sds-page analysis of bacteriocin produced by lactobacillus plantarum u10. fermentas silver stained gel, lane 1: partially purified crude extract plantaricin, and lane 2: rainbow molecular mass markers. fig 4 mode of plantaricin u10 against salmonella typhi atcc 25241. ▲ and δ, optical density measured at 600 -1 nm presence plantaricin and absence plantaricin, respectively; ■ and □ , log cfu ml counts of the indicator strain treated presence plantaricin and absence plantaricin, respectively. the arrow indicates the time of addition of the plantaricin (4 h). the experiment was conducted in triplicate, the error bars were standard deviation (sd). volume 9, 2015 microbiol indones 77 step cell free supernatant ammonium sulfhate precipitation gel filtration volume (ml) 300 6 3 total activity (au) 800 400 133 total protein (ml) 5286 177.60 14.39 specific activity -1 (au mg ) 0.15 2.25 9.24 purification (fold) 1 14.88 61.07 yield (%) 100 5.0 33.25 o d 6 0 0 lo g c fu /m l 1 0 time (h) of the appearance of the treated cells. sem micrographs showed difference in cell structures between treated plantaricins u10 group and the nontreated control. non-treated cells were intact (coccus shaped or regular rod) and showed of smooth surfaces. however bacterial cell treated with plantaricins u10 underwent considerable structural changes cell that can be differentiated (fig 5). sem examination confirmed the structural destruction integrity of the cells and considerable morphological alteration bacterial cell. this finding showed cells death by the plantaricins u10 treatment was probably through pores formation on the outer membrane of s.typhi. discussion in this study, we described the plantaricins u10 production from early growth of the cells, purification and its mode of action. the activity of plantaricins u10 againsted gram-negative bacteria such as s.typhi is a rare phenomenon. according to todorov (2009), ineffective bacteriocin against gram negative bacteria due to physical barrier of phatogenic bacteria's outer membrane that masked the site for bacteriocin action. most bacteriocins produced by l. plantarum inhibited gram-positive bacteria, e.g. plantaricin w (holo et al. 2001) and plantaricin lr14 effectively killed micrococcus luteus (tiwari and srivastava 2008). more importantly, plantaricin c19 (atrih et al. 2001), plantaricin ama-k (todorov et al. 2008), plantaricin na (mills et al., 2011), plantaricin lb-b1 (xie et al. 2011), and plantaricin y (chen et al. 2014) have been experimentally proven to be potential as natural biopreservative to control/prevent food contamination from listeria monocytogenes. in accordance with our result, others investigators showed bacteriocins activity against broad range of gram-negative bacteria. such as reported by, bacteriocin ama-k produced by l. plantarum ama-k can inhibit e. coli (todorov et al. 2007). plantaricin lp31 produceed by l. plantarum lp31 have broad inhibitory spectrum such as pseudomonas sp, s. aureus, l. monocytogenes, b. cereus, b. megaterium, and b. subtilis (muller et al. 2008). plantaricin mg produced by l. plantarum klds1.0391 can inhibit l. monocytogenes, s. aureus, and s. typhimurium (gong et al. 2010). muhialdin et al. (2012) reported lactic acid bacteria which isolated from tempoyak in malaysian showed antibacterial activity against tested strains of gram positive and gram negative bacteria such as (b. subtilis, e.coli, s. aureus, s. epidermidis, klebsiella pneumoniae, and s. typhimurium). plantaricins u10 production during growth of l. plantarum u10 in mrs broth at 37 ºc have maximum activity at stationary phase of growth. the highest activity of a bacteriocins produced during stationary phase of growth was also similar as found at plantaricin lb-b1 (xie et al. 2011), plantaricin mg (gong et al. 2010), bacteriocin la-14 (todorov et al. 2011), plantaricin st194bz (todorov and dicks 2005), and plantaricin st71ks (martinez et al. 2013). the decrease in the antibacterial activity at the later stationary phase (i.e. after 24 h) could be ascribed to plantaricins u10 degradation by proteolytic enzymes released during cell lyses, or binding of the peptide to proteins or producer cells (gong et al. 2010). during the 32 h of growth, the ph decreased from ph 6.3 to 4.0. the optical density cell (od 600nm) increased from 0.15 to 2.74 (fig. 1). a similar results were reported for bacteriocin lb-b1 produced by l. plantarum lb-b1 (xie et al. 2011), bacteriocin st4sa produced by enterococcus mundtii (todorov and dicks 2009), bacteriocin st13br produced by l. plantarum st13br (todorov et al. 2004). fig 5 morphological changes under sem micrographs caused by plantaricin u10. a: before treated plantaricin, b: after treated 24 h with plantaricin u10. 78 sogandi et al. microbiol indones plantaricins u10 in our study were obtained from partially purification steps (ammonium sulphate and size-exclusion chromatography) so the protein bands which were assumed as bacteriocins appeared as 2 bands with different molecule size (approximately 4.5 and 9.8 kda) and those predicted palntaricins were bacteriocin class ii group. this finding was within the range of most bacteriocins reported (3.0 kda bacteriocin l. plantarum st341ld and 14.0 kda bacteriocins l. plantarum st23ld) (todorov and dicks 2005). further purification steps is need to be done to obtain single protein band that refer to targeted plantaricin. according to hata et al. (2010) and gong et al. (2010) additional steps after size-exclusion chromatography such as cation-exchange or reversephase hplc are simplest way to obtain the more pure and single band protein obtained. addition of cell-free supernatant u10 into a 4-hold culture (exponential phase) of s.typhi atcc25241 resulted in growth inhibition for 12 h (fig. 4), suggesting that the mode of activity of plantaricins u10 is bactericidal on sensitive strain as proven by cell count decrease in the number of viable cell (from 2.2 x 8 -1 6 -1 10 cfu ml to 3.1 x 10 cfu ml ) of s. typhi atcc25241. a similar mode of actions also reported from other studies, e.g. plantaricin lb-b1 (xie et al. 2011), bacteriocin st4sa (todorov and dicks, 2009) and bacteriocin la-14 (todorov et al. 2011). in general, bacteriocins function produced by gram-positive bacteria is primarily directed to eliminate or reduce other competitors gram-positive bacteria (ennahar et al. 2000). under normal circumstances, bacteriocins produced by grampositive bacteria do not have a bactericidal effect on gram-negative species. however, in some cases, their bacterial activity against some sensitive gramnegatives can be directly observed under sem as membrane rupture (wang et al. 2014). in agreement, our result also proved that plantaricins u10 have ability to disturb integrity of membrane of s.typhi which was believed to be sensitive to our plantaricins u10. according to mcauliffe et al. (2001), class ii of bacteriocins exert their action mostly on cells targets by pore formation on membrane of target cells that induce proton motive force (pmf). to conclude, in this study, plantaricins u10 against s.typhi produced by l. plantarum u10 isolated from the traditional fermented food “tempoyak” have been proven. plantaricins u10 have molecular mass of approximately 4.5 and 9.8 kda by sds-page. plantaricins u10 production reached maximum at 20 h -1 incubation in media mrs broth 37 ºc (800 au ml ) at early stationary phase of the growth organism and its mode action was bactericidal, as challenged against s. typhi atcc25241. according to the sem micrographs, it showed bacteriocins form pores in the membranes of target cells. these results provide theoretical foundation for the application of plantaricins u10 as antibacterial peptide in the pharmaceutical industry. acknowledgment authors would like to thank to team in laboratory for applied genetic engineering and protein design 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bacteriocin produced by lactobaccilus plantarum lb-b1 isolated from koumiss, a traditionally fermented dairy product from china. food control. 22: 1027-1031. doi: 10.1016/j.foodcont.2010.12.007. yang e, fan l, jiang y, doucette c, fillmore h. 2012. antimicrobial activity of bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts. amb express. 1: 48. zengin h and baysal a. 2014. antibacterial and antioxidant activity of essential oil terpenes against pathogenic and spoilage-forming bacteria and cell structureactivity relationships evaluated by sem microscopy. m o l e c u l e s . 1 9 : 1 7 7 7 3 1 7 7 9 8 ; d o i : 1 0 . 3 3 9 0 / molecules191117773. volume 9, 2015 microbiol indones 81 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 02 ambarsari.cdr vol.12, no.3, september 2018, p 74-82 doi: 10.5454/mi.12.3.2 isolation and urease activity test of bacteria for calcium carbonate (calcite) precipitation (biocementation) in soil hanies ambarsari*and aflakhur ridlo pusat teknologi lingkungan (ptl), badan pengkajian dan penerapan teknologi (bppt), gedung 820 geostech kawasan puspiptek serpong, tangerang selatan 15314, banten the use of bacterial calcium carbonate (calcite) precipitation (biocementation) has recently become popular as a ground-improvement technique. ureolytic bacteria having highly urease activities were known to have important roles in calcium carbonate precipitation process. one of our research objectives is to isolate and to select as many as possible such ureolytic bacteria from indonesian soils to be further utilized for calcium carbonate (calcite) precipitation process in the soil for strengthening the soil structure. isolation was performed anaerobically in selective media containing 40% urea. four isolates with different morphologies were purified and coded as tk1, tk2, tk3, and tk4. each of them was tested for its urease activity either as a pure culture or as a mixture of several cultures. the urease activity was measured based on the ammonia concentration produced in the growth media up to 7 x 24 hours. it was known that isolate tk4 had the highest urease activity on incubation period 6 (d6), whilst a mixture of isolate cultures coded as tkc did not show a better urease activity than the isolate tk4. hence, it could be concluded that the isolate tk4 was the best candidate to be used for further research on the calcium carbonate (calcite) precipitation process (biocementation) to strengthen the soil structure. key words: biocementation, calcite, calcium carbonate, precipitation, urease, ureolytic penggunaan presipitasi kalsium karbonat bakteri (biocementation) baru-baru ini menjadi populer sebagai teknik perbaikan tanah. bakteri ureolitik yang memiliki aktivitas urease tinggi diketahui memiliki peran penting dalam proses pengendapan kalsium karbonat tersebut. salah satu tujuan penelitian kami adalah untuk mengisolasi dan untuk memilih sebanyak mungkin bakteri ureolitik tersebut dari tanah indonesia untuk digunakan lebih lanjut dalam proses pengendapan kalsium karbonat di dalam tanah untuk memperkuat struktur tanah. isolasi dilakukan secara anaerobik menggunakan media selektif yang mengandung 40% urea. empat isolat dengan morfologi yang berbeda dimurnikan dan dikodekan sebagai tk1, tk2, tk3, dan tk4. masing-masing isolat diuji untuk aktivitas urease baik sebagai kultur murni atau sebagai campuran beberapa kultur isolat. aktivitas urease diukur berdasarkan konsentrasi amonia yang diproduksi di media pertumbuhan hingga 7 x 24 jam. diketahui bahwa isolat tk4 memiliki aktivitas urease tertinggi pada periode inkubasi ke-6 (d6), sementara campuran kultur isolat yang dikodekan sebagai tkc tidak menunjukkan aktivitas urease yang lebih baik daripada isolate tk4. oleh karena itu, dapat disimpulkan bahwa isolat tk4 adalah kandidat terbaik yang akan digunakan untuk penelitian lebih lanjut tentang proses pengendapan kalsium karbonat (biocementation) untuk memperkuat struktur tanah. kata kunci: biosementasi, kalsit, kalsium karbonat, presipitasi, urease, ureolitik microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: 62-21-75791377 ext. 4089; fax: 62-21-75791381; email: hanies.ambarsari@bppt.go.id paassen et al. 2010a and 2010b; van wijngaarden et al. 2010). it was presented as a new and environmentally friendly method (dejong et al. 2006) which has an advantage over conventional chemical treatments having a limited injection distance and can be toxic to the environment (karol 2003). this new method was also known to be cost-effective in comparison to chemical treatments (ivanov and chu 2008). bacterial calcium carbonate precipitation or cementation has been applied to a variety of civil engineering applications, such as to repair cracks in rock and concrete, to improve the bearing capacity, to reduce permeability, to increase dilative tendencies, and to increase the strain stiffness in sand (al qabany and soga, 2013; dejong, 2013; martinez et al. 2013; rusu et al. 2011). construction on an organic ground involves the risk of bearing capacity failure and excessive settlement (canakci et al. 2015). to prevent such risks, the geotechnical properties of organic soil are improved by ordinary improvement techniques, such as deep mixing with cement or lime, vertical drains, sand columns, and dry jet mixing (celik and canakci 2011; jelisic and leppanen 2003). many previous studies have reported that the microbial-induced calcium carbonate precipitation technique is very effective in increasing the shear strength and in decreasing the permeability of sandy and gravelly soil (canakci et al. 2015; van der star et al. 2011; van volume 12, 2018 microbiol indones 75 soil bacteria having high urease activities play important roles in calcite precipitation (dejong et al. 2010). the main role of these bacteria in the calcite precipitation process has been to generate an alkaline environment through different physiological actions. the main nutrient and chemical compounds for soil cementation by bacteria contain nahco , nh cl, 3 4 cacl , urea, and a nutrient broth (mixture of peptone, 2 yeast extract, beef extract, and nacl). urease (urea amidohydrolase: ec 3.5.1.5) is an enzyme that hydrolyze urea into one mole of carbonate and two moles of ammonia per mole of urea as shown in the following reaction (burbank et al. 2012): 2 reaction #1: (nh ) co + 2h o co + 2nh2 2 2 3 3 through the hydrolysis of urea, the bacteria then release carbonate ions into soil or water that can bind with calcium to form calcium carbonate and its metastable polymorph, calcite, as seen in the next reaction: 2+ 2 reaction #2: ca + co caco3 3 al thawadi and cord-ruwisch (2012) and burbank et al. (2012) demonstrated that indigenous ureolytic bacteria could be enriched from a variety of soils and that population of bacteria could precipitate calcite or other calcium carbonate mineral species in quantities sufficient to alter the engineering characteristics of the soil. several bacteria able to form the calcium carbonate mineral are such as bacillus licheniformis (helmi et al. 2016), acinobacter sp. (sanchez-moral et al. 2003), escherichia coli (bachmeier et al. 2002), microbacterium sp. (xu et al. 2017), myxococcus xanthus (gonzalez-munoz et al. 2000), agrobacterium tumefaciens and lactobacillus sakei (yuliani 2015), also bacillus pasteurii that has been reclassified as sporosarcina pasteurii (canakci et al. 2015). to participate optimally in the hydrolysis of urea for calcite precipitation, the bacteria must constitutively and inducibly produce urease. the purpose of our study is to isolate and select the ureaseproducing bacteria from indonesian soils to be used further in our research of biocementation or calcium carbonate precipitation process for strengthening the soil structure. materials and methods materials used in the research were agar, nutrient broth, d-glucose, nacl, potassium dihydrogen phosphate, phenol red, urea, potassium citrate, hcl, phenol, ethanol, acetone, naoh, naocl, (nh ) so , 4 2 4 toluene, filter paper whatman no. 41, alcohol 70%, aquades, aluminium foil, wrapping, cotton, tissue paper, petri disks, erlenmeyer flaks, drugalsky bar, measuring pippets, micropippets, measuring beaker glasses, reaction tubes, autoclave, incubator, colony c o u n t e r, m i x e r, p h m e t e r, t h e r m o m e t e r, haemocytometer, spectrophotometer uv-vis jasco v530, microscope olympus, laminar air flow, analytical balance, ose needles, and measuring flask. the biocementation research was performed in laboratory of environmental microbiology, center for environmental technology (ptl, pusat teknologi lingkungan), agency for assesment and application of technology (bppt, badan pengkajian dan penerapan teknologi) in puspiptek serpong, tangerang selatan, banten, indonesia from march until june 2018. the soil sample for this experiment was obtained from the mangrove forest of tritih kulon, cilacap, central java. this experiment was conducted using the split plot experimental design. the treatments to be investigated were the isolate factor (i) with six treatment levels as the following: tk0 = without addition of the isolate as the control treatment tk1 = with addition of the isolate 1 tk2 = with addition of the isolate 2 tk3 = with addition of the isolate 3 tk4 = with addition of the isolate 4 tkc = with addition of the mixture culture of isolate 1, 2, 3, and 4 and the incubation period factor (d) with eight treatment levels as the following: d0 = incubation period of 0 x 24 hours d1 = incubation period of 1 x 24 hours d2 = incubation period of 2 x 24 hours d3 = incubation period of 3 x 24 hours d4 = incubation period of 4 x 24 hours d5 = incubation period of 5 x 24 hours d6 = incubation period of 6 x 24 hours d7 = incubation period of 7 x 24 hours such treatments were carried out using the completely random experimental design which were repeated three times. the parameters measured were + the nh residue concentration, the ph, the bacterial 4 population density, and the medium temperature. bacterial isolation and purification. one gram of mud sample taken from the mangrove forest of tritih kulon, cilacap, central java was dissolved in 9 ml of distilled water in a test tube. suspense dilution was -4 carried out until the 10 dilution level which was then spread over a plate of solid selective media (harley and prescott, 1993) consisting of 1 gram of peptone, 1 gram of d-glucose, 2 grams of potassium dihydrogen phosphate, 5 grams of nacl, 0.012 gr phenol red, 50 ml 40% urea solution, 15 gr agar and distilled water until the volume became 1 l. all ingredients except urea were put in distilled water until the final volume reached 950 ml and heated until dissolved then sterilized using autoclave at 121 c, pressure 2 atm for 15 minutes and chilled. after being chilled, 40 ml of 40% urea solution was added, then sterilized with uv light at a wavelength of 270 nm using uv light at lafc. according to gaudy and gaudy (1981), ultraviolet light with a wavelength between 200 300 nm for 15 minutes was bactericidal so that it could be used as a disinfectant. to test the urease activity a liquid selective medium was also prepared just like the solid selective medium, but without the addition of agar. incubation was carried out at 35 °c for 2 x 24 hours. bacteria having urease activity will show changes in color on the media from red to orange to red purple around the colonies. four colonies having different morphologies from the culture were streaked on the selective medium each until pure culture was obtained. after obtaining a pure culture, the isolate was stored in nutrient agar (na) media incubated at 35 c as the isolate stock. p re p a r a t i o n o f b a c t e r i a l i s o l a t e s f o r experiment. isolates were rejuvenated by transferring the cultures from the old na media to the new na media and then incubated at 35 °c for 24 hours. one young growing culture was then put into 10 ml 0.85% nacl solution, then 1 ml was taken and inoculated into a liquid selective medium and incubated at 35 °c for 2 x 24 hours. calculation of the number of growing cells was done by using a haemocytometer or a colony 7 counter. culture with a density of approximately 10 -1 cells ml was ready to be used as an inoculum. this was in accordance with alexander's statement (1977) which estimated that the number of soil microbes 5 7 capable of ammonification was around 10 10 organisms per gram of soil urease activity test. the reaction tube was filled with 10 ml of a liquid selective medium and added with 1 ml of bacterial isolate then incubated at about 35 °c anaerobically. for the mixture, first take each part of a single isolate and mix it until it is homogeneous and then put 1 ml into the test medium. the treatment was repeated 3 times. determination of urease activities (tabatai and bremner 1969 in iswandi 1989). the urea activity was determined by measuring the ammonia (nh ) 3 formed during hydrolysis of urea added to the media. urea activity was expressed in μmol ammonia nitrogen formed within 1 hour in 1 gram of medium sample given the urea at 37 °c. a total of 10 ml of the inoculated bacterial medium was put into a 100 ml erlenmeyer flask and then added with 15 ml toluene and homogenized. after 15 minutes it was added with 10 ml of 10% urea solution and 20 ml of ph 6.7 buffer citrate solution and shaken. erlenmeyer flask was clogged with a rubber plug and then incubated in a 37 c incubator for 3 hours. after that, distilled water was added to a volume of 100 ml by keeping the toluene under the 100 ml terra mark on the erlenmeyer flask and then clogged and shaken well. the suspension was filtered using whatman paper no. 41, then in the erlenmeyer flask 1 ml filtrate, 10 ml distilled water, 4 ml na-phenol solution, 3 ml naocl solution were added, shaken and let stand for 20 minutes before being added with distilled water until the volume became 50 ml and shaken again. light intensity was measured at a wavelength of 590 nm with a spectrophotometer jasco v530. the urea activity was calculated based on the standard curve equation whose formula is: concentration = absorbance (a) b where a = 0.0010 and b = 0.0107, which is a liner calibration curve with regression coefficient = 0.9941. results from the isolation process of mangrove forest sludge samples from tritih kulon, cilacap, central java, four bacterial isolates with urease activity had been obtained. after following the gram staining procedure as described by misiolek et al. (2018), four bacterial isolates were observed to have different morphological characteristics (table 1). the four isolates were then given the code names tk1, tk2, tk3 and tk4. each isolate was then tested on a medium containing 40% urea. testing on the 40% urea medium was also carried out on the mixture of the four isolates (tkc) to determine its enzymatic ability to transform urea to ammonia (fig 1). the graph in figure 1 can explain that the urease activity of each bacterial isolate was different with one another. the tk0 treatment as a control showed no activity. isolates tk1, tk4 and tkc had urease activities with the same curve patterns, while tk2 shows similarities of curve patterns with tk3. these isolates showed their urea activities starting at d3, 76 ambarsari et al. microbiol indones fig 1 the urease activity of bacterial isolates from tritih kulon, cilacap, central java expressed as the + concentration of ammonium (nh ) detected from the medium.4 table 1 morphological characteristics of bacterial isolates from tritih kulon, cilacap, central java isolate code colony morphology gram stain cell form motility form margin elevation color surface tk1 small circular entire convex brownish yellow smooth &shiny coccus non motile tk2 filamentous circular filiform umbonate (elevated convex) white dry + coccus non motile tk3 irregular undulate raised (low convex) whitish yellow smooth & shiny + coccus non motile tk4 filamentous circular entire umbonate (elevated convex) grey dry as powder + coccus non motile rd which was a measurement on the 3 day, because at the second day measurement there was no ammonia detected in the medium of each isolate. in general, all isolates reached the peak of urease activity in d5 d6 which at that time tk4 had the highest activity of 56.82 ppm at d5 and 57.32 ppm at d6. based on the data in figure 1 it can also be seen that the tolerance limit for the ammonia accumulation of each isolate differed from 38.57 ppm from tk1 on d6 to 57.32 ppm from tk4 on d6. figure 1 also explains that tk4 was an isolate that had the highest urease activity. this isolate began to show urease activity in d3, ie measurements on the third day with 28.86 ppm ammonia levels and reached its peak activity on d6 with measured ammonia levels of 57.32 ppm. tk4 isolates were coccus-shaped bacteria which in solid media showed gray tk4 colonies forming a regular circle of filamentous and surface-like powder. gram staining on all these isolates showed that this isolate tk4 was gram positive. in general, the bacterial isolates had decreased urease activity in d6 and d7 however tk3 began to experience a decrease in activity at d5. this decrease was suspected due to several things, among others, because bacterial cells still needed time to adapt to their new environment or bacteria still use n nutrient sources other than urea, peptone, which was also found in the media. this could be seen in tk2 which continued to experience cell growth which continued to increase 11 -1 12 -1 from 8.5 x 10 cfu ml on d3 to 1.4 x 10 cfu ml on d4 (fig 2) although there is a change in the ph value from 5.2 on d3 to 5.6 on d4 (fig 3 ph value). while in tk3 there were also indications that even though the urea activity decreases, there was still growth in the cells. this could be seen from the increase in the 11 -1 number of cells from 1.5 x 10 cfu ml on h3 to 4.3 x 12 -1 10 cfu ml on d4. but at this tk3 the ph value decreased from 5.2 on d3 to 4.8 on d4. graph of cell growth in figure 2 shows that in d5 and d6 the highest number of bacteria was seen, when compared to the graph of urease activity in figure 1 it could be seen that the number of cells and urease volume 12, 2018 microbiol indones 77 activity were directly proportional. this indicated that an increase in cell numbers would increase urease activity. at d7 there was a decrease in the number of cells which in turn was followed by a decrease in urease activity seen from a decrease in ammonia concentration on the medium. the graph in figure 2 also shows that tk4 was an isolate that had the highest cell number during this urease activity test. this isolate was the best growth isolate. with this high cell number, the urea activity was also the highest. however, when viewed from the ability per individual cell of each colony, seen from the comparison of the number of cells with urea activity, tk2 and tk3 were better than tk4. likewise with tkc, a mixture of these four isolates had much better urease activity than isolate tk4, because with far fewer cells but tkc urease activity was not much different from tk4. in general, however, isolate tk4 was an isolate that had the highest urease activity because in the same incubation time the isolate was able to convert urea to ammonia in the highest concentration. during the treatment there was a change in ph value (fig 3). there was a decrease in the ph value from d0 to d2 which might be caused by bacteria using the simplest nutrition first, namely glucose as a carbon source contained in the medium. in d3 there was an increase in ph due to an increase in ammonia levels in the medium. but in d7 when the ph value was the highest value during the treatment, it actually decreased the measured ammonia level. this could be caused by the increase in ph affecting the growth and performance of bacterial urease. the graph of temperature changes (fig 4) showed that the longer the test time caused the temperature of the medium increased. in the urease activity test, the temperature increase had been caused by an exogenic reaction. temperature increase was generated from cellular reactions, namely from the use of atp and + + nadph + h to nadp . this reaction occurred when urea was hydrolized to ammonia. in this urease test, each isolate began to experience a decrease in activity ranging from 31 °c to 32 °c and it could also be explained that the urease enzyme produced by each isolate was optimum at the temperature range of 30 °c, because at this temperature the hydrolysis process of urea to ammonia reached the peak activity. discussion the unmeasured ammonia at d0, d1, and d2 did not mean that there was no urea transformation to ammonia at all, but it was suspected that the activity was still low or the bacteria still needed time to adapt because moreover it was seen from the growth or number of cells still in the lag phase, so that the resulting ammonia was small and not detected by the device used, namely spectrophotometer jasco v530 which has a limit of ammonia detection to a concentration of 0.1 ppm. alexander (1977) explained earlier that bacteria able to hydrolyze urea to ammonia and used it as a source of energy for growth were mostly in the form of coccus, including the genus micrococcus. schlegel and schmidt (1994) had classified bacteria capable of using + nh4 as electron donors and called them lithotrophic organisms which, according to stanier et al. (1984) the results of ammonia metabolism are glutamate acid, asparagine and glutamine, all three of which are direct protein starters. payne (1980) and fardiaz (1994) + added that nh as a result of the reaction of the enzyme 4 urease was used for the formation of amino acids which were then converted to form cell mass, the formation of cell membranes and enzymes. according to alexander (1977), the speed of decomposition of urea into ammonia and its accumulation and immobilisation depended on the type of microbe, type of substrate, and its environment. hardiyanto (2002) stated that collaboration between microorganisms would accelerate the degradation of organic compounds, but in this study tkc which was a mixed treatment of isolates tk1, tk2, tk3, and tk4 turned out to have lower urease activity than isolate tk4. this might be due to the i n t e r a c t i o n o f a n t a g o n i s m b e t w e e n t h e s e microorganisms. in relation to antagonism one type of bacteria will destroy or inhibit the growth of other bacteria so that the population decreases. like the previous statement of atlas and bartha (1998), the microbes living together in one environment will interact between populations. these interactions can be positive (neutral, commensalism, synergism, or mutualism) and negative (antagonism, competition and predation) which can cause the destruction of some microbial populations. positive interactions will improve the life ability of some microbial populations. in the soil there is a dynamic process in the release of nitrogen from the organic n form through microbial decomposition called ammonification. at the same time, there is an inorganic n binding which is transferred to organic n by microbial activity. because of this microbial activity in nature, the level of ammonia and nitrate in the media becomes non78 ambarsari et al. microbiol indones fig 3 ph value of the medium during the urease activity testing of bacterial isolates from tritih kulon, cilacap, central java. fig 2 growth of bacterial isolates from tritih kulon, cilacap, central java during the urease activity testing. fig 4 temperature of the medium during the urease activity testing of bacterial isolates from tritih kulon, cilacap, central java. volume 12, 2018 microbiol indones 79 constant. in the wild when inorganic nitrogen is added to the environment, some will be transferred into organic bonds while others will be released in an inorganic form (stanley 1995). david et al. (1999) stated that ammonia concentration in soil was 1.25 mg -3 m while according to stevenson (1959) in stanley (1995), the equivalent ammonia concentration per kg of soil was about 5 to 15 ppm. the process of nitrogen immobilization involves + combining nh into amino acids. this depends on the 4 + growth of microbial cells. nh ions are immobilized 4 or accumulated in the medium depending on the need for nitrogen for microorganisms to grow. bacteria need nitrogen to form cytoplasmic components and peptidoglycan cell walls. cellular nitrogen levels in bacterial constant cells are at the ratio of c:n between 3:1 to 5:1 (paul and clark, 1989). meanwhile, according to elsas et al. (1997), the transformation of several pulses in the soil such as urea, depending on soil microbes, and these microbes produce urease to hydrolyze urease to ammonium carbamate. the decrease in cell numbers was thought to be due to the results of metabolism in the form of ammonia which accumulated in the medium which was toxic to microorganisms. like the statement of brock and madigan (1994), the excessive accumulation of ammonia as a result of metabolism was toxic to microorganisms. normal bacterial growth was limited by the availability of nutrients and the results of toxic metabolism in the medium and environmental conditions such as ph and temperature. temperature and medium ph at d7 were the highest temperature and ph during the urease activity test. this caused a decrease in growth speed and even microbial cell death. another thing that caused a decrease in growth speed was the depletion of nutrients in the medium, especially the content of c and n. yulinah et al. (1990) stated that the main factors that determine growth were nutrition. growth or increase in cell mass indicates the presence of nutrients that can be consumed by cells. if nutrients are reduced, growth will decrease and if nutrients are depleted, the cell growth will stop. in this study, the reduced number of cells caused a decrease in urease activity because with fewer cells, the amount of enzyme produced was reduced. whilst, the decrease of ph value for tk3 might be caused by tk3 using glucose in the medium first to grow before using other compounds, so that the results of glucose metabolism in the form of acid resulted in a decrease of the medium ph and this fermentation of glucose caused an increase in temperature. glucose is as simple as the simplest so it can be directly absorbed and most easily metabolized by bacteria. hadiutomo (1990) stated that chemoorganotrophs especially bacteria would use simple carbohydrates in the form of glucose because these compounds are the simplest and most easily degraded compared to other compounds. in an anaerobic atmosphere, glucose will be fermented to produce acids that can reduce the ph value in the bacterial growth medium. wijendra (1989) stated that urea underwent a breakdown and produced an amine group which when reacting with water would become nh oh which was alkaline so that during the treatment 4 time there would be an increase in ph value. the activity of each organism is affected by the ph of the environment to achieve the maximum activity of an enzyme that is required by an environment with a certain optimum ph range. the ph value of the medium directly affects microbial cellular activity and affects growth (yulinah et al. 1990). according to timothius (1993), ph affected the action of enzymes + and changed cell permeability. ions h and oh ions are the most mobile ions, therefore a small change in the + concentration of h ions and oh ions can cause major changes in the cellular system of microorganisms. according to schlegel and schmidt (1994), urea decomposing bacteria were more suitable to live in environments with slightly alkaline ph (ph values 6 to 8) and if there was an increase in ph it would cause interference with the cell system which could cause cell death. the decrease in urease activity of tk3 since d5 could occur due to several factors, among others due to a decrease in the number of bacterial cells which was triggered by a fairly high increase in ph and ammonia accumulation in a medium that was already outside the tolerance limit for bacteria. temperature will affect all processes that occur in microorganisms and will limit their reproduction. high t e m p e r a t u r e s w i l l d e n a t u r a t e p r o t e i n s a n d microorganism enzyme systems, while low temperatures will deactivate all cellular systems (hawker and linton, 1979). temperature is one of the factors that greatly affect growth, propagation and resistance of microorganisms. girindra (1993) explained that most enzymes would experience decreased activity at 45 °c and would lose their activity altogether at 55 °c, but lehninger (1990) argued that the loss of biological functions of enzymes was due to the influence of specific temperatures for each enzyme. suriawiria stated (1986) that in the reaction of metabolism a chemical reaction would occur, then the increase in temperature to a certain value could 80 ambarsari et al. microbiol indones increase the reaction, but beyond that limit would cause the cessation of the cellular process. according to sutedjo (1997), bacterial urease activity would reach a maximum at 30 °c. bacteria having urease activity would experience decreased activity at temperatures below 30 °c and above 35 °c , however there would still be certain types of bacteria that were able to grow and move up to 45 °c. acknowledgment this research was jointly funded by the agency for assessment and application of technology (bppt) and ministry of research technology and 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teaching material. pau biotechnology, ipb bogor. 82 ambarsari et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 2. mi-windi silvani.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.11.2.2issn 1978-3477, eissn 2087-8575 vol 11, no 2, june 2017, p 46-54 *corresponding author; phone: +62-22-2511621, fax: +6222-2534115, email: windisilvani@gmail.com stable environment in indonesia all year is good for mushroom growth (aryantha 2005). many mushrooms in indonesia are often used as food, one of which is volvariella volvacea (v. volvacea) (chen and buswell 2004). v. volvacea has distinct and delicious flavor, soft, elastic, makes it highly demand (chen and buswell 2004). demand of v. volvacea is high approximately 50-60% of total national production, with average of elevated demand was 20-25%/year (maji 2007). production of v. volvacea peak at 70% that was from west java, middle java, east java, and lampung (tridjaja 2005; sumiati and djuariah 2007). in 2015, mushroom demands gradually increase that peaked 17500 ton/year, whereas only 13825 ton/year has been fullfiled (maji 2007). obstacles commonly encountered during production of v. volvacea is decreased yields due to high contamination (sinaga 2012). approach increasing growth using mushroom growth promoting bacteria (mgpb) are therefore needed. young et al. (2013) found bacteria that produces metabolite in growth medium of agaricus blazei in dissolving metal, calcium, and nitrogen fixation that paddy straw mushroom (volvariella volvacea) contains high protein content and delicious flavor, makes it highly demand each year. production of v. volvacea does not merit the requirements due to its limited production. therefore, approach in increasing production using mushroom growth promoting bacteria (mgpb) are needed. this study aims to obtain mgpb isolate as potential agent to increase v. volvacea strain ww-08 growth. this is experimental research in laboratory that consisted of indigenous bacteria isolation, mgpb screening with dual culture, mgpb inoculum optimization, molecular identification of selected mgpb using 16s rrna, protein profiling with sds-page, and fruting body production. indigenous bacteria obtained 6 -1 from growth medium were 58 isolate, and w34 bacteria at concentration of 10 cell ml showed most significant result on micellium growth. sequence of 16s rrna region showed w34 bacteria is bacillus cereus.visualization of sds-page showed new protein in result of interaction between b. cereus and v. volvacea strain ww-08 with molecule weight of 17 kda. average of fruting body of v. volvacea strain ww-08 in treatment of b. cereus harvested for 7 days, was 240.19 g, whereas that without treatment of b. cereus was 82.15 g. these findings indicate treatement of b. cereus strain w34 increase v. volvacea ww-08 growth by 300%. key words: bacillus cereus, mushroom growth promoting bacteria (mgpb), volvariella volvacea ww-08 jamur merang (volvariella volvacea) mengandung protein tinggi dan rasa yang lezat, yang menyebabkan permintaan meningkat setiap tahunnya. produksi v. volvacea ini sangat terbatas sehingga tidak mencukupi kebutuhan di masyarakat. oleh karena itu salah satu cara yang dilakukan dalam meningkatkan produksi dengan menggunakan pendekatan mushroom growth promoting bacteria (mgpb). penelitian ini bertujuan untuk mendapatkan isolat mgpb sebagai agen potensial untuk meningkatkan pertumbuhan v. volvacea strain ww-08. penelitian ini merupakan penelitian eksperimental di laboratorium yang terdiri dari isolasi bakteri, penyaringan mgpb dengan metode dual culture, optimasi inokulum mgpb, identifikasi molekuler mgpb terpilih dengan menggunakan 16s rrna, profil protein dengan menggunakan sds-page, dan produksi tubuh buah jamur. bakteri yang diperoleh dari hasil isolasi media pertumbuhan sebanyak 58 isolat, dan bakteri w34 pada 6 -1 konsentrasi 10 sel ml menunjukkan hasil yang paling signifikan pada pertumbuhan misellium. urutan basa rrna 16s menunjukkan bakteri w34 adalah bacillus cereus. visualisasi sds-page menunjukkan terdapatnya protein baru hasil interaksi antara b. cereus dan v. volvacea strain ww-08 dengan berat molekul 17 kda. ratarata tubuh buah v. volvacea strain ww-08 dengan perlakuan b. cereus yang dipanen selama 7 hari, seberat 240,19 g, sedangkan tanpa perlakuan b. cereus berat 82,15 g. temuan ini menunjukkan perlakuan strain b. cereus w34 mampu meningkatkan pertumbuhan v. volvacea ww-08 sebesar 300%. kata kunci : bacillus cereus, mgpb, volvariella volvacea ww-08 potential mushroom growth promoting bacteria (mgpb) in optimizing paddy straw mushroom production 1 1,2 windi silvani jemsi *and i nyoman pugeg aryantha 1 department of biology, school of life science and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia; 2 bioscience and biotechnology research center, institut teknologi bandung, jalan tamansari 126, bandung 40132, indonesia take place in growth production. it has been reported that ochrobactrum pseudogrignonense is able to induce and increase mushroom growth of pleurotus ostreatus with significant increasing yield (maryana and aryantha 2013). young et al. (2012) found six soil bacterial strain that increases agaricus blazei growth. studies regarding biogical factors such as induction by indigenous mgpb are rarely done. this study aims to determine effect of mgpb induction on mushroom growth. results are expected to provide solution for indonesia farmers in increasing v. volvacea growth that can merit market demand. materials and methods identification of v. volvacea. mushroom confirmed with moleculer analysis using internal transcribed spacer (its) marker, is v. volvacea strain ww-08 (raju et al. 2014) isolation of indigenous bacteria. indigenous bacteria was isolated from several growth medium of v. volvacea in west java. referring to kumala and siswanto (2010), isolation was performed with dilution method. samples from growth medium of 1 g was diluted in eight dilution series. samples was cultured in medium (na oxoid cm3), incubated for 24 h. bacteria colony was further subcultured to obtain single colony based on its morphology. single colony obtained was cultured in stock medium (jagessar et al. 2008) indigenous bacteria screening. screening was performed on micellium growth of v. volvacea with dual culture using potato dextrose agar (pda) medium (aryantha and maryana 2012). increase in micellium growth was measured each day. data was analyzed with one way (anova) with spss. indigenous bacteria that shows highest increasing was further subcultured in na and number of colony was measured with total plate count (tpc) (suarsa et al. 2011). identification selected indigenous bacteria. identification was conducted based on morphology characters (shape, color, and surface of colony) (chen and zhang 2009), and gram staining. moleculer identification was performed using 16s rrna. dna isolation was performed with boiling method. sample in sterile deionized was boiled for 1 m at 96 °c and incubated for 3 min at -22°cin triplicate. samples were centrifuged for 5 min at 14 000 rpm, and supernatant was taken to undergo polymerase chain reaction (pcr) (langille et al. 2013). primer used was universal primer of 16s rrna: 63f (5’-caggcctaacacatg caagtc-3’) and 1387r (5’-gggcggwgtgtaca aggc-3’). pcr protocol include following: predenaturation at 95 °c for 3 min, denaturation at 95 °c 30 s; annealing 45 °c 30 s, elongation 72 °c 2 min, post ° pcr 72 °c for 7 min and stop pcr 4 c. pcr products were electrophoresed through 1% agarose. pcr products were sequenced and analyzed with basic local alignment search tool nucleotide (blastn) and at gene bank ncbi (http://www.ncbi.nlm.nih.gov). sequences of 16s rrna from databese was aligned multiple alignment clustal w using molecular evolutionary genetics analysis (mega 5.1). phylogenetic tree was constructed with distance neighbor joining (nj analysis) (tamura et al. 2011). screening of inoculum. bacteria inoculum screening was performed with dilution. bacteria -8 -9 -10 inoculum at concentration of 10 , 10 , and 10 were cultured in pda. micellium of 3 mm was cultured grown in medium and incubated for 5 d, measurement of micellium length was conducted each day (suarsa 2011). data was analyzed with one-way anova (analysis of variance) using software statistical package for the social sciences (spss) (santoso 2012). characterization of protein profil (sdspage). both bacteria and mushroom were grown in pdb. two days-aged sample were centrifuged at 13 000 rpm for 10 m at room temperature. referring to rajala (2013), supernatant was precitipated with addition of sodium deoxycholate (doc) 25 μl, vortexed and left at 10 m, and incubated overnight. samples were centrifuged at 13 000 rpm for 10 m at 4 °c. pellet was taken and washed with 1 ml aceton, in duplicate. buffer solutionof 6 × 50 ml of 20 ml (trishcl ph 6.8 50mm 2,5 ml, sodium dodesil sulfat (sds) 2% of 10% 2.5 ml stock, bromfenol blue 0.1 % 0.5 ml, glycerol 10% 5 ml, β-mercaptoethanol 100 mm 0.36 ml) was added, heated at 96 °c for 4 m. running buffer used was 5 × 100 ml (tris base 1.51 gr, glycine 9.4 g, sds 10% 5 g, added deionized water to reach final volume of 100 ml). gel was composed of 30% acrylamid mix (acrylamid 2.92 g, metilen bis acrylamid 0.08 g, deionized water 10 ml), consisting of staking gel 4% (de ion 1.67 ml, tris ph 8.8 1.25 ml, sds 10% 50 μl, acrylamid mix 2 ml, ammonium persulfate (aps) 25 μl, tetra etil metil ethilene diamine (temed) 5 μl) and separating gel 12% (de ion 3.05 ml, tris ph 6.8 1.25 ml, sds 10% 50 μl, acrylamid mix 0.65 ml, aps 25 μl, temed 5 μl). gel was soaked with 200 ml staining solution (cbbr 0.5 g, metanol 100% 90 ml, pure acetetae acid 20 ml, deionized water 90 ml), and washed with destaining 400 ml (metanol 100% 180 ml, deionized water 180 volume 11, 2017 microbiol indones 47 ml, pure acetic acid 40 ml). result of sds-page was visualized by bands or press dry gel to obtain better result. result of sds-page was analyzed with imagej to show detected protein peak (susnea 2011). test of selected bacteria on v. volvacea generative phase. selected bacteria grown in na was subcultured in nb, and harvested after 24 h. cultivation in mushroom house was modified using plastic pot of 30 cm diameter and height of 15 cm, referring to aryantha and maryana (2012). each pot was filled with 5 logs composed of cotton as basic medium, bran of 2.5% and kapok of 1%, with log plastic of 2 kg. treatment consisted of selected bacteria and watering, were sprayed to mushroom each day on growth medium surface. treatments were conducted for 15 d. humidity was maintaned with automatic water sprayer of 70-80%, and temperature was set to 26-28 °c. initiation of fruting body emerged was measured at harvesting. number and weight of fruitng body was measured at mature phase. data was analyzed with anova using software spss (jackson et al. 2014). results isolation of indigenous bacteria. isolation of indigenous bacteria obtained 58 isolates (table 1). bacteria isolated from subang were the highest among others, which was 25 originate from 3 mushroom house with different medium. whereas 12 isolates was obtained from karawang in 3 mushroom house, 12 isolates from sukabumi in 2 houses, and 7 isolates from cikampek in 2 houses (table 1). screening of indigenous bacteria. screening of 58 isolates was performed with dual culture. results showed there were 2 bacteria that exhibited inhbition of mushroom, and bacteria that induced mushroom growth. there were 10 isolates obtained that possess ability to induce mushroom growth (fig 1). the highest micellium growth induced by bacteria, was exhibited by c bacteria, namely w34. w34 isolates was isolated from houses in sukabumi, treated with sago medium and covering soil. highest micellium growth rate influenced by w34, was about 13-15 mm each day, whereas lowest rate obtained at treatment of g bacteria b41, was about 5-7 mm. growth rate in control without bacteria treatment, was about 3-5 mm each day. statistical analysis showed results were significant among treatments, that suggested each bacteria exhibited significant effects on mushroom growth. w34 was able to highly enhance micellia growth of cold strain v. volvaceae, makes it chosen for further analysis. other 48 isolates were considered to inhibit mushroom growth indicated by halo zone (fig 2). identification of selected bacteria. identification was conducted based on morphology and gram staining of w34 (fig 3). colony appeared was milky white slightly cloudy, colony surface was glisten/oily, unshaped edge, and round shaped. this bacteria is ocassionally motile in certain environment that appears separated and form new colony. bateria staining showed bacteria was gram positive, and bacil, categorized as bacillus. bacillus sp. is commonly about 3-4 mm, dull, unshaped edge, flat, and glossy surface. molecular identification was performed using 16srna as house keeping gene. the pcr product showed the target size (fig 4). optimization of indigenous bacteria inoculum. the highest micellia growth was 1 ml addition of 6 -1 bacteria age of 24 h with density of 10 cell ml source growth medium number of isolates subang (1) paddy straw 7 subang (2) soil 9 subang (3) cotton waste 9 karawang (1) cotton waste and paddy straw 5 karawang (2) cotton waste 5 karawang (3) soil 4 sukabumi (1) sago waste 3 sukabumi (2) sago waste 9 cikampek (1) paddy straw 3 table 1 indegenous bacteria obtained from volvariella volvacea growth medium 48 jemsi et al. microbiol indones 1 4 3 2 fig 1 screening of indigenous bacteria with dual culture. (1-2) bacteria that induce mushroom growth, (3-4) bacteria that inhibit mushroom growth. fig 2 micellia growth induced by indingenous bacteria (a) 17, (b) 32, (c) 34, (d) 35, (e) 39, (f) 40, (g) 41, (h) 42, (i) 47, (j) 54, and (k) control. fig 3 morphology and gram staining of bacteria. (a) colony, (b) gram staining. 40 35 30 25 20 15 10 5 0 a b c d e f g h i j k bacterial treatment l en g th o f m ic el li u m ( m m ) a b volume 11, 2017 microbiol indones 49 treatment of inoculum l en g th o f m y ce li u m ( m m ) 40 35 30 25 20 15 10 5 0 a b c d e non-treated mushroom was sprayed with water once a day to maintain its water supply. highest result was obtained in day 3 that produced 188 g of 10 fruiting body, followed by 101.04 g of 9 fruiting body at day 5, 83.48 gram of 6 fruiting body at day 4, 54.87 g of 7 fruiting body at day 2, 55.54, g of 5 fruiting body, 10.02 g of 4 fruiting body. these results indicate treatment of bacteria enhance mushroom production of 200% compared to non-treated mushroom. discussion isolation was performed to obtain promising bacteria. indigenous bacteria obtainedwas different each area. these results might be due to various houses and growth medium used. study conducted by nannipieri et al. (2003) shows that soil is complex and dynamic system biology. bacteria diversity is present due to nutrient content contained in soil such as c, n, (treatment a) (fig 5). characterization of protein (sds-page). protein obtained from isolate was performed with sdspage. sodium dodesil sulfat (sds) is ionic detergent that dissolves hidrophobic resulting negative muatan in overall protein structure and further visualized with imagej. visualization of sds page with imagej is shown in fig 6. treatment of selected bacteria in generative phase of v. volvacea. weight and number of fruting body were measured for 7 harvesting days. fig 7 showed fruiting body and mycelium with and without treatment. highest result treated with bacteria, was at 3rd day or 7th day after mushroom seeds cultivated, that resulted 681.77 g of 29 fruitng body. weight harvested at 5th day was 406.65 g of 21 fruiting body, 308.43 g of 16 fruiting body at day 4,246.84 g of 18 fruiting body at day 2, 217.75 g of 15 fruiting body at day 6, and 66.06 g of 13 fruiting body at last day. ~1482 bp 28 28 28 28 18 92 34 34 20 92 23 30 45 10.000 8.000 6.000 5.000 4.000 3.000 2.500 2.000 1.500 1.000 750 500 250 fig 4 the pcr product of 16s rrnagene of w34 isolate. fig 5 optimized inoculum of selected bacteria a=10.6, b=10.5, c=10.4, d=10.3, e=10.10. median of treatments followed by similar leter, is insignificant difference value (p>0.05) analyzed with duncan post hoc test. 50 jemsi et al. microbiol indones 260 135 95 72 52 42 34 26 17 10 1 2 3 4 1 2 3 4 fig 6 sds-page (a) visualization of protein peak (b) protein bands. (line 1) ladder (spectra broad range pre-stained), (line 2) jvc mushroom (line 3) jvvc + w34, and (line 4)w34 bacteria. fig 7 volvariella volvacea. (a) fruiting body, (b) mycellium +34, (c) mycellium -34. fig 8 production of volvariella volvacea fruiting body. : bacteria, : without bacteria. c b a 200 180 160 140 120 100 80 60 40 20 0 b o d y w ei g h t o f th e fr u it ( g ) harvesting day 10 2 3 4 5 6 volume 11, 2017 microbiol indones 51 and tang 2007). several b. cereus strain potetially act as probiotic, mesophilic, optimal at 20 °c 40 °c, and adapt to various environments (vilain et al. 2006). rajkovic (2006) reported highest cell obtained from b. cereus is at 24 h, in which approximately 59 × 6 -1 10 cfu ml , indicated as stationary phase. further 5 -1 treatment with bacteria of 10 cell cfu resulted length 4 -1 of 34.5 mm. treatment of 10 cell cfu resulted length of 32.5 mm. lowest decreased mycellium was 30.5 mm obtained in treatment of bacteria (f) with 3 -1 inoculum of 10 cell cfu . mycellium of 28.5 mm was 10 -1 obtained for 5 d of growth with cells of 10 cfu ml . based on measurement, induction by b. cereus toward cold v. volvacea strain was significantly different among treatments. the new protein are formed by interaction between mushroom and bacteria weighs around 17-26 kda. in this interaction between mushroom and bacteria also obtained a new extracellular proteins which previously were not present at each isolates, with molecular weight about 17 kda. research hao-chiet al. (1997) showed that a successful immunomodulatory protein purified from v. volvacea, resulted in protein of about 17-18 kda. protein molecule is not activated when the fungus stands alone, but further activated upon interaction with bacteria. protein b. cereus obtained on the value 34-42 kda. referring to berber (2004), protein with a molecular weight of approximately 2945 kda protein is believed to originate from b. cereus. based on the data in fig 7 can be seen that the treatment of bacteria produce fruiting bodies are heavier in higher mass. suharnowo and isnawati (2012) states that the high growth of fruiting bodies is due to the secondary metabolites produced by bacteria that this compound could help increase the formation of fruiting bodies. bacteria generally produce metabolites that can be utilized by fungi to protect themselves from environmental stress, heavy metal stress and are also useful in inducing the growth of fungus. production of biofertilizer bacteria can form compounds that can increase the solubility of minerals and nitrogen fixation. research conducted by zarenejad et al. (2012) showed that mgpb is able to dissolve phosphate to be easily digested by fungus as a source of energy (atp) and growth. another role as biostimulan is production of phytohormones such as auxin, cytokinin and ethylene that enhance the growth of fungi (payapanon et al. 2011; ahemad et al. 2015). harni et al. (2015) reported that b. cereus isolated from the growth media patchouli is able to enhance plant growth by producing indole acetic acid (iaa). p, s, and other compounds. composition of substrate as growth medium also affect bacteria diversity (fig 1). referring to poyedinok et al. (2008) dan sales-campos et al. (2011), bacteria growth is strongly influenced by environmental factors such as temperature, ph, oxygen supply, as well as internal factors such as nutrient content and other compounds to feed microorganism. bacteria also has antagonist effect in inhibiting mushroom growth or other microorganism. as shown in fig 2, selected bacteria enhanced micellium growth indicated by growth to the edge. enhancement of mushroom bacteria is commonly associated with ability of bacteria to produce secondary metabolite (kimet al. 2008). there were 10 bacteria chosen based on its ability to enhance highest micellia growth. bacteria has different response to micellia growth, either synergistic or antagonistic (frey-klett et al. 2011). micellia growth relies upon secondary metabolite secreted by bacteria, either growth factor or antibiotic (antifungi) (berendsenet al. 2012). several interactions occurred between mushrooms and bacteria is physical association consisting of planktonic, mixed biofilm, and intrahphal colonization. molecullar communication such as antibiotis, metabolite alteration, chemotaxis signaling, metabolite convertion, phsiochemical changes, adhesion, protein production, and genetical alteration. such interactions is commonly employed by both bacteria and mushroom to induce growth, development, and reproduction, transportion, gen aquisition, survival, and mutualistic and pathogenic simbiosis (frey-klett et al. 2011). bacteria induce growth of v. volvacea belongs to gram negative and gram positive. according to allen (1995), both grampositive and gram-negative bacteria possess ability to intake metal cation present in soil. gram-positive bacteria such as bacillus sp., thiobacillus sp. and gram-negative such as pseudomonas sp., commonly found in nature, are also able to dissolve phospate and utilized by other organism growth (sharma et al. 2013). phylogenetic tree shows relationship among species with similarity value shown in bootstrap. results showed w34 has close relationship to b. cereus (dharmayanti 2011). outgroup used was paenibacillus brasiliensis. w34 is located near b. cereus, sbtbc-001 strain, jn66, dan etc. these results are in accordance with blast that indictae selected indigenous bacteria belongs to b. cereus. b. cereusis gram-positive bacteria, bacil, aerobic, and facultative anaerobic, motile, ocassionally produce spora. this bacteria is abundantly found in nature, and soil (huang 52 jemsi et al. microbiol indones counting and classification system. information syst fronti. 11(4):349-368. doi:10.1007/s10796-009-91490. dharmayanti ni. 2011. filogenetika molekuler metode taksonomi organisme berdasarkan sejarah evolusi. balai besar penelitian veteriner. bogor. frey-klett p, burlinson p, deveau a, barret m., tarkka m, sarniguet a. 2011. bacterial-fungal interactions: hyphens between agricultural, clinical, environmental, and food microbiologists. microbiol mol biol rev. 75(4):583-609. doi:10.1128/mmbr.00020-11. harni r, supramana s, sinaga ms, giyanto g, supriadi s. 2015. mekanisme bakteri endofit mengendalikan nematoda pratylenchus brachyurus pada tanaman nilam. buletin penelitian tanaman rempah dan obat. 23(1). hao-chi hsu, chyong-ing hsu, rong-hwa lin, chianliang kao, jung-yaw lin. 1997. fip-vvo, a new fungal immunomodulatory protein isolated from volvariella volvacea. biochem j. 323(2):557-565. doi:10.1042/bj3230557, huang wc, tang ic. 2007. bacterial and yeast culturesprocess characteristics, products and applications. bioprocessing for value-added products from renewable resources. 185-224. hussein aha, a’al nia. 2011. morphological and microscopical comparison features of bacillus cereus isolates. kufa j vet med sci. 2(1):27-37. jackson dj, makrinioti h, rana bm, shamji bw, trujillotorralbo mb, footitt j, del-rosario j, telcian ag, nikonova a, zhu j, aniscenko j. 2014. il-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo. american journal of respiratory and critical 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indonesia (maji). 2007. berbisnis jamur [mushroom commercialization]. http://berbisnisjamur.com/. maryana y, aryantha inp. 2013. induction of pleurotus ostreatus mycelial growth with bacteria. prosiding watering also should not be done too often in which excess water on the media causes mycelium subemerged and rotting. according to li et al. (2014), excess water in media causesaltered colors, resulting in death. research conducted by mishra (2011), reported that the fungus will flourish on the materials that have been weathered or decomposed. organic materials containing cellulose and lignin in large numbers supports the growth of mycelium and fruiting body development related to enzymes produced by the fungus. temperature also influences the growth of fungal mycelium especially for v. volvacea cold strain. the temperature of the growth of v. volvacea or button mushrooms ranged between 24-30 °c, and optimum growth at 27°c (thakur and singh 2014). acknowledgment we thank the directorate general of higher education (dikti) for research grants. we also thank the microbiology laboratory of biological sciences centre bandung institute of technology for providing materials research. references ahemad m. 2015. phosphate-solubilizing bacteria-assisted phytoremediation of metalliferous soils: a review. 3 biotechnol. 5(2):111-121. 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6, no 3, september 2012, p 89-97 *corresponding author; phone: +62-251-8622932, e-mail: hefni_effendi@yahoo.com terrestrial fungi served an enormous resource for the discovery of novel natural product in the past 60 years, many of them being potential targets for biomedical developments. the discovery of penicillin in 1929 started the era of fungal antibiotic and was followed by other important fungal metabolites like cephalosphorins, cyclosporins, and griseofulvins. until now, fungi have only been surpassed by actinomycetales as sources for biologically active metabolites. the terrestrial fungal biodiversity seems to be nearly exhausted. however, there are many fungal endophytes for example that have not been characterized. thus, nowadays, researchers throughout the world pay increasing attention toward the potential of marine microorganism as an alternative source to isolate novel metabolites (anke and erkel 2002; biabani and laatsch 1998; pietra 1997). natural products, such as secondary metabolites, isolated from plants, animals, and microbes are important sources for bioactive molecules that, in many cases, have been developed into medications . some of the natural products isolated from marine invertebrates have been shown to be, or are suspected to be, of microbial origin and this is now thought to be the case for the majority of such molecules. marine microorganisms, whose immense genetic and biochemical diversity is only beginning to be appreciated, seem likely to become a rich source of novel chemical entities for the discovery of more effective drugs (haefner 2003). the marine environment has proven to be a very rich source of (villa and gerwick 2010) this study was aimed at pursuing new biologically active secondary metabolites of microfungus species, lecanicillium evansii, isolated from sponge callyspongia sp. collected from west bali sea, indonesia. sponges were collected by scuba diving. a tiny piece of sponge was inoculated on the surface of malt agar plates and incubated at 27 °c. in order to get a pure mono-culture of the fungus, repeated sub-culturing onto fresh malt agar plates were performed. the collected fungi were maintained on malt agar plates using the wickerham medium. mass cultivation of the fungus l. evansii (10 l) was carried out in 30 erlenmeyer flasks in wickerham medium. after 10 days incubation, without shaking under constant room temperature (20 °c), fungal mycelia were separated from the culture broth. the mycelia were extracted with methanol and ethyl acetate was added to the media. both methanol-added mycelia and ethyl acetate-added media were left overnight. seven compounds were isolated from l. evansii. those compounds comprised phenolic compounds (terphenylin, deoxyterphenylin, terprenin 2, terprenin epoxide), bipeptide (cyclo-tyrosylprolyl), and simple aromatic compounds (acetyl hydroxybenzamide, 4-hydroxybenzaldehyde). detailed analysis by nmr and mass spectrometry enabled their identification to be new deoxyterphenylin, new terprenin 2, and new terprenin epoxide. key words: callyspongia, deoxyterphenylin, lecanicillium evansii, terprenin 2, terprenin epoxide penelitian ini bertujuan untuk mencari bahan bioaktif metabolit sekunder dari mikrofungi lecanicillium evansii yang diisolasi dari bunga karang callyspongia sp. yang dikoleksi dari perairan laut bali barat, indonesia. bunga karang dikoleksi dengan penyelaman (scuba). potongan kecil bunga karang diinokulasi pada media agar dan diinkubasi pada 27 °c. untuk mendapatkan kultur tunggal dari mikrofungi, dilakukan beberapa kali sub kultur. kultur massal mikrofungi l. evanssi (10 l) dilakukan pada 30 botol erlenmeyer yang diisi medium wickerham. setelah 10 hari inkubasi tanpa goyangan pada suhu kamar 20 °c, miselium dan media dipisahkan. miselium diekstraksi dengan metanol, sedangkan pada media ditambahkan etil asetat. keduanya didiamkan selama semalam. tujuh senyawa berhasil diisolasi dari l.evansii. senyawa-senyawa tersebut terdiri dari: fenolik (terfenilin, dioksiterfenilin, terfenilin 2, terprenin epoksida), bipeptida (siklo-tirosilprolil), dan senyawa aromatic sederhana (asetil hidroksibenzamida, 4-hidroksibenzaldehida). analisis lanjut menggunakan nmr dan lcms mengungkapkan bahwa dioksiterfenilin, terprenin 2, and terprenin epoksida merupakan senyawa baru yang belum pernah dilaporkan sebelumnya. kata kunci: callyspongia, dioksiterfenilin, lecanicillium evansii, terprenin 2, terprenin epoksida phenolic compounds of sponge-associated fungi (lecanicillium evansii) hefni effendi department of aquatic resources management, faculty of fisheries and marine sciences, institut pertanian bogor, kampus darmaga, bogor 16680, indonesia extremely potent compounds that have demonstrated significant activities as antitumors, antiinflammatories, analgesics, immunomodulators, antiallergies, and antivirals (newman and cragg 2004; blunt et al. 2008; simmons et al. 2005). although approximately three to four thousands known fungal secondary metabolites have been isolated, possibly not more than five to seven thousands taxonomic species have been studied in this respect. genera such as: aspergillus, penicillium, fusarium, and acremonium are among fungi highly capable of producing high diversity of secondary metabolites (dreyfuss and chapela 1994). the number of secondary metabolites isolated from marine-derived fungi has been increasing. this proves that they are rich sources of bioactive compounds with therapeutic potential. almost one thousand marine natural product were discovered since 1990, with a pronounced increase between decades (leal et al. 2012). the success story of cephalosporin isolation, which is now widely used in antibiotic therapy, from the fungus cephalosporium sp. derived from the microbial flora of sea water collected from cagliari, italy, in the 1940's marked the 90 effendi microbiol indones this study was aimed at pursuing new biologically active secondary metabolites of new fungi species (lecanicillium evansii strain 1), isolated from sponge callyspongia. materials and methods isolation of the new fungal species. the strain of new fungal species of l. evansii was isolated from the sponge callyspongia sp. microfungi was collected from west bali sea, indonesia. sponges were collected by scuba diving. a small piece of the inner part of the sponge was sliced under sterile conditions. this tiny piece was then inoculated on the surface of malt agar plates and incubated at 27 c. in order to get a pure mono-culture of the fungi, purification through several sub-cultures onto fresh malt agar plates were repeatedly carried out. the collected fungi were maintained under malt agar plates using the wickerham medium (table 1). to eliminate bacterial contaminants, chloramphenicol -1 -1 (0.2 g l ), streptomycin sulphate (0.1 g l ), and -1 penicillin g (0.1 g l ) were added to the medium. fungal species identification was conducted by the centraalbureau voor schimmelcultures, netherlands, ° substances amounts yeast extract (sigma) 3.0 g malt extract (merck) 3.0 g pepton (merck) 5.0 g glucose monohydrate (caelo) 20.0 g agar (merck) 16.0 g artificial sea water (biomarine) 24.4 g distilled water 1000 ml naoh or hcl for ph adjustment (7.2 7.4) several drops onset of interest of pursuing natural product from marine fungi (faulkner 2000). cephalosporin c becomes the only compound from a fungus isolated from a marine source that up to now has been established as a medication or a source for partial synthetic derivatives (biabani and laatsch 1998). ziconotide (prialt; elan pharmaceuticals), a peptide originally discovered in a tropical cone snail, was the first marine-derived compound to be approved in the united states in december 2004 for the treatment of pain. then, in october 2007, trabectedin (yondelis; pharmamar) became the first marine anticancer drug to be approved in the european union (molinski et al. 2009). and stored at the institute of pharmaceutical biology and biotechnology, duesseldorf university, germany. mass cultivation of the fungus l. evansii (10 l) was carried out in 30 erlenmeyer flasks in wickerham medium. after 10 d incubation, without shaking, under constant room temperature (20 c), fungal mycelium were separated from the culture broth. the mycelia were extracted with methanol, and ethyl acetate was added to the media. both methanol-added mycelia and ethyl acetate-added media were left overnight. extraction and isolation of the secondary metabolites. extraction and isolation of the secondary metabolites of l. evansii are presented in fig 1. an unknown natural product sample often contains a ° table 1 wickerham medium for marine fungi culture mixture of many components. the components must be separated from one another so that each component can be analysed individually. many organic liquids are immiscible with water. when such a liquid is added to water, two layers are formed. whether the organic layer is in the upper or lower layer depends upon the relative density of organic liquid and water. analysis of the secondary metabolites. thin layer chromatography (tlc) was always conducted to each fraction prior to further chemical work, to get the overview of the identity of the fraction and the qualitative purity of theisolated compound. band separation in tlc would also be very helpful in optimising the solvent system that would be applied later in column chromatography. the use of analytical and semi preparative hplc were intended to analyse the peaks distribution, either from raw extracts or fractions, as well as to evaluate the purity of isolated compounds. ei-ms (electron impact mass spectrometry), fab-ms (fast atom bombardment mass spectrometry), and esi-ms (electron spray ionisation mass spectrometry) were applied to identify molecular weight. proton (1h) and carbon (13c) nmr spectra were recorded at 300 °k on bruker dpx 300, arx 400 or avance dmx 600 nmr spectrometers (500 mhz). volume 6, 2012 microbiol indones 91 fig 1 isolation scheme of secondary metabolites from l. evansii marine sponge callyspongia sp. marine fungus l. evansii (strain 1) l. evansii (strain 1) 300 ml stand culture wickerham medium in aritificial sea water 8 days at 27 °c isolation of fungal strains etoac extract (167 mg) h2o extract fraction 1 (40 mg) fraction 3 (15.3 mg) fraction 3.3 deoxyterphenylin (4.3 mg) fraction 3.4 terprenin 2 (5.0 mg) semi prep. hplc fraction 2 (30.3 mg) fraction 4 (10.6 mg) sephadex (lh 20) column 100 % meoh semi prep. hplc fraction 2.1 cyclo tyrosylprolyl (7.7 mg) fraction 2.3 acetyl hydroxy benzamide (5.1 mg) fraction 2.2 4-hydroxy benzaldehyde (5.2 mg) fraction 2.5 terprenin epoxide (6.0 mg) semi prep. hplc fraction 4.1 terphenylin (4.9 mg) high resolution spectrum displayed a molecular weight -1 of 322.1195 g mole . this was also confirmed by several ion peaks in the ei-ms spectrum at m/z 322 [m]+, 307 [m-ch3]+ (fragment 1) 292 [m-2(ch )]+ 3 (fragment 2), and 276 [m-ch -ch o]+ (fragment 3) 3 3 (fig 2). by analysing the chemical shifts, signal multiplicities, and coupling constants (7.25-8.82 hz) of protons displayed in the 1h nmr spectrum, one mono-substituted benzene (ring a) δ 7.61 (h2, h6), 7.42 (h3, h5), 7.34 (h4), and one para-substituted phenyl (ring c) δ 7.19 (h2'', h6''), 6.80 (h3'', h5'') (table 2, fig 3) were identified. the cosy correlations between h2''/6'' and h3''/5'' and h6'' indicated an aa'bb' spin system and revealed the presence of a para-substituted phenyl in results seven compounds were successfully isolated from the fungus l. evansii. the compounds were deoxyterphenylin (new compound), terprenin 2 (new compound), terprenin epoxide (new compound), t e r p h e n y l i n , c y c l o ( t y r o s y l p r o l y l ) , a c e t y l hydroxybenzamide, and 4-hydroxybenzaldehyde. only three new elucidated compounds would be discussed further in this paper. deoxyterphenylin. the esi-ms spectrum of compound deoxyterphenylin presented intense ions at m/z 323.4 [m+h]+(positive) and 321.8 [m-h] (negative) determining a molecular weight of 322 g -1 mole and suggesting a molecular formula of c h o 20 18 4 which corresponded to deoxyterphenylin. the ei-ms microbiol indones92 effendi table 2 nmr data of deoxyterphenylin fig 3 cosy and roesy correlations of deoxyterphenylin (in meod) (in dmso) 2 7.61 (d, 7.3) 7.61 (d, 8.5) h3 3 7.42 (t, 7.3, 7.9) 7.46 (t, 7.4, 7.8) h2, h4 4 7.34 (t, 7.3, 7.6) 7.37 (t, 7.3, 7.5) h3, h5 5 7.42 (t, 7.3, 7 .9) 7.46 (t, 7.4, 7.8) h4, h6 6 7.61 (d, 7.3) 7.61 (d, 8.5) h5 5‘ och 3.68 (s) 3.65 (s) 6‘ 6.46 (s) 6.45 (s) 2‘‘ 7.19 (d, 8.8) 7.11 (d, 8.5) h3‘‘ 3‘‘ 6.80 (d, 8.8) 6.76 (d, 8.6) h2‘‘ 4‘‘ och 3.36 (s) 3.30 (s) 5‘‘ 6.80 (d, 8.8) 6.76 (d, 8.6) h6‘‘ 6‘‘ 7.19 (d, 8.8) 7.11 (d, 8.5) h5‘‘ h3, h4 h2, h4 h3, h5 h4, h6 h4, h5, h6’ h5’och3 h3’’ h2’’ h3”, h5” h6’’ h5’’ position h (ppm), multiplicity ( j in hz) (in meod) h (ppm), multiplicity ( j in hz) (in dmso) cosy roesy fig 2 hypothetical fragmentation of deoxyterphenylin in the ei-ms spectrum fragmentation 1 fragmentation 2 fragmentation 3 ho oh ch o o ho oh ch o o ho oh c o o a b c roesy cosy ho oh ch3 ch3 o o irradiation of the other methoxy group protons at the higher field (δ 3.36, h4”) however did not cause any observable effects on other protons. thus, a 2droesy (rotational frame nuclear overhauser effect spectroscopy) experiment was conducted. again a correlation between h6' and 5'-och was clearly 3 observed, while the correlation of 4”-och and h3”/5” 3 allowed for positioning of the second methoxy function. thus, the structure of compound deoxyterphenylin was established as depicted in fig 3. terprenin 2. the esi-ms spectrum of compound terprenin 2 showed the ion peak at m/z 407[m+h]+ -1 suggesting a molecular weight of 406 g mole and a correlations to both oxygenated aromatic carbons (δ 138.50 and δ 153.72) that in turn were correlated to the methoxy signals. long range correlation in the hmbc spectrum confirmed the position of prenyl group through a number of prominent connections between h1''' and c3, c2, c4 (fig 5). the correlation of h1''' and h2 in the roesy spectrum also assured the location of the prenyl group (fig 6). likewise, the isolated proton of h5' displayed correlations with h2”/h6”, and 6'och , thus supporting the structure of terprenin 2 3 (table 3). terprenin epoxide. the molecular formula of volume 6, 2012 microbiol indones 93 fig 4 hypothetical fragmentation of terprenin 2 in the ei-ms spectrum ring c. the second spin system of a mono-substituted benzene in ring a was also distinctly observed in the cosy spectrum (fig 3). one penta-substituted phenyl (ring b) was clearly determined by one isolated singlet at δ 6.46 (h6'). in addition, two methoxy singlets (δ 3.68 and 3.36) were observed. to determine the positions of the methoxy groups, noe (nuclear overhauser effect) experiments were performed. irradiation of methoxy protons (δ 3.68, h5') resulted in the intensification of the singlet proton (h6'), assuring that the position of isolated singlet proton (h6') was adjacent to this methoxy group (h5'). molecular formula of c h o . the molecular weight 25 26 5 was also confirmed by the ei-ms spectrum with several intense ion peaks at m/z 406 [m]+, 340 [mprenyl group]+ (fragment 1), 322 [m-prenyl grouph o]+ (fragment 2), 177 [m-prenyl group-ring a]+ 2 (fragment 3), 69 [m of prenyl group]+ (fragment 4) (fig 4). the esi-ms high resolution spectrum displayed an ion peak at m/z 429.1673 [m+na]+, supporting the assignments of molecular weight and molecular formula. the placement of the two methoxy groups in ring b (positions 3' and 6') was identified by close inspection of the hmbc spectrum. in terprenin 2, h5' displayed fragmentation 1 fragmentation 3 fragmentation 4 loss of prenyl group h c3 o ch3 h c3 oh ch3 o o loss of prenyl group fragmentation 2 oh ho o ch3 oh ch3 h c3 o h c3 loss of the main skeleton h c3 o o ho ch3 oh oh ch3 h c3 loss of prenyl group and ring a ho o ch3 oh h c3 ch3 oh o h c3 oh), 5.90 (2'-oh), 4.85 (4''-oh)], and contained three phenyl rings with different substitution patterns as described above for terprenin epoxide (table 4, fig 7). the presence of an epoxy group was assured by the molecular formula and by the hmbc correlations between h4a''' and h4b''' with c2''' and c3'''. the downfield shifts of the latter could only be explained by oxygen substitution, while the molecular formula only accounted for one additional oxygen atom in comparison to terprenin 2. terprenin epoxide and molecular weight were -1 established as c h o and 422 g mole , respectively, 25 26 6 by the esi-ms (m/z 423.3 [m+h]+, 421.6 [m-h]-). the high resolution of esi-ms indicated the molecular -1 weight of 422.1713 g mole . the 1h nmr data analysis indicated that terprenin epoxide had an epoxy prenyl side chain [δ 3.28 (h1'''a), 3.15 (h1'''b), 4.63 (h2'''), 1.19 (h4a'''), 1.14 (h4b''')], two methoxy groups [δ 3.37 (3'-och ), 3.70 3 (6'-och )], three phenolic hydroxyl groups [δ 4.85 (4-3 microbiol indones94 effendi fig 5 cosy and hmbc correlations of terprenin 2 (in cdcl )3 fig 6 roesy correlations of terprenin 2 (in acetone) fig 7 cosy and hmbc correlations of terprenin epoxide h c3 hmbc cosy h c3 4b 4a ho o oh oh ch3 o h c3 o oho ch3 h c3 4b 4a ohho abc roesy h c3 o oh o ch3 h c3 ch3 oho oh abc cosy hmbc with the aid of proton multiplicity and coupling constants observed in the 1h nmr spectrum, it was determined that the three phenyl rings possessan abx spin system on ring a δ 7.17 (h2), 6.67 (h5), 7.08 (h6), a one proton system in ring b (δ 6.46 (h5'), an aa'bb' spin system on ring c [δ 6.92 (h3'', h5''), 7.50 (h2'', h6'')]. an abx spin system was also found in the side chain of epoxy prenyl group (table 4, fig 7). proton signals of the 1h nmr spectrum recorded in cdcl and methanol also supported the assignment of 3 these three spin system types. the epoxy group brought about a centre of asymmetric at position 2'''. consequently the methylene protons at position 1''' resonated as double doublet signals as clearly seen in the 1h nmr spectrum recorded in acetone. an h/d exchange experiment using methanol as solvent to eliminate the hydroxyl protons that existed in the 1h nmr spectrum recorded in chloroform was conducted. signals of the hydroxyl groups at positions 4, 2', and 4” disappeared. cosy correlations also supported the assignment of the four spin systems in the molecule (fig 7). likewise, hmbc correlations assigned the position of the isolated proton h5' and the position of two methoxy groups in ring b. the side chain of epoxy prenyl group was bound to ring a through the position 3. this side connection was verified by the cosy correlations of h1''', h2''', and h2. the long range correlations between h1''' and c2 as well as between h2''' and c3 in the hmbc spectrum also clarified the side chain position (fig 7). discussion deoxyterphenylin has never been quoted in the literature before. takahashi et al. (1976) isolated a related deoxyterphenylin from aspergillus candidus. both methoxy groups of this compound, however, are located at positions 2' and 5', whereas the methoxy groups of the new deoxyterphenylin at positions 5' and 4”. antimicrobial assay conducted using this newly elucidated deoxyterphenylin demonstrated no antimicrobial activity. para-terphenyl metabolites showing a typical phenolic nature are found rarely as natural products. however, terphenylquinones were mostly isolated from basidiomycetes (thomson 1971). volume 6, 2012 microbiol indones 95 table 3 nmr data of terprenin 2 1 δ h (ppm), (in meod) multiplicity (j in hz) (in acetone) 1 δ h (ppm), multiplicity (j in hz) (in cdcl )3 position δ 13c(ppm) (in cdcl )3 1 δ h (ppm), multiplicity (j in hz) cosy (h  h) (in cdcl )3 hmbc (h  c) (in cdcl )3 roesy (h  h) (in acetone) 1 129.94 (s) 2 132.36 (d) 6.95 (d, 1.9) 7.13 (d, 2.5) 7.25 (d, 2.1) h6 c1, c4, c1‘‘‘ h1‘‘‘ 3 126.40 (s) 4 153.72 (s) 5.15 (oh) c3, c4, c5 5 115.50 (s) 6.67 (d, 8.2) 6.83 (d, 8.2) 6.89 (d, 8.7) h6 c3, c4, c6, c1‘‘‘ h6 6 125.23 (d) 6.91 (dd, 1.9; 8.2) 7.05 (dd, 1.9; 8.2) 7.22 (dd, 2.1; 8.5) h2, h5 c1,c2 h5 1‘ 115.64 (s) 2‘ 147.31 (s) 5.88 (oh) c1’, c2’, c3’ 3‘ 138.50 (s) 3‘och3 56.04 (q) 3.35 (s) 3.38 (s) 3.46 (s) c3’ 4‘ 130.00 (s) 5‘ 103.83 (d) 6.34 (s) 6.46 (s) 6.45 (s) h6’(och )3 c6, c1’, c2’, c3’, c6’, c1“, c2’’, 6’’ h6’(och )3 6‘ 153.72 (s) 6‘och3 60.73 (q) 3.66 (s) 3.69 (s) 3.74 (s) h5’ c6’ h5’ 1‘‘ 130.16 (s) 2‘‘ 132.12 (d) 7.36 (d, 8.2) 7.50 (d, 6.3) 7.53 (d, 6.6) h3’’ c4’, c4’’, c6’’ h3’’, h4’’,h5’’ 3‘‘ 115.41 (d) 6.75 (d, 8.2) 6.92 (d, 6.3) 6.92 (d, 6.5) h2’’ c1’’, c4’’, c5’’ 4‘‘ 155.13 (d) 4.86 (oh) c3’’, c4’’, c5’’ 5‘‘ 115.41 (d) 6.75 (d, 8.2) 6.92 (d, 6.3) 6.92 (d, 6.5) h6’’ c1’’, c3’’,c4’’ 6‘‘ 132.28 (d) 7.36 (d, 8.2) 7.50 (d, 6.3) 7.53 (d, 6.6) h5’’ c4’, c2’’, c4’’ h5’, h3’’, h4” h5’’ 1‘‘‘ 30.02 (t) 3.35 3.36 (d, 6.9) 3.41 (d, 7.3) h2’’’, h4a’’’, h4b’’’ c2, c3, c4, c2’’’, c3’’’ h2, h2’’’ 2‘‘‘ 121.90 (d) 5.35 (m) 5.39 (m) 5.40 (m) h1’’’, h4a’’’, h4b’’’ c4a’’’ h1’’’ 3‘‘‘ 135.00 (s) 4a‘‘‘ 25.85 (q) 1.72 (s) 1.78 (s) h1’’’, h2’’’, h4b’’’ c2’’’, c3”’, c4b’’’ h2’’’ 4b‘‘‘ 17.93 (q) 1.71 (s) 1.77 (s) h1’’’, h2’’’, h4a’’’ c2’’’, c3”’, c4a’’’ h2’’’ against mouse spleen lymphocytes stimulated with con a and lps. the ic values of terprenin, 3-50 methoxyterprenin, and 4''-deoxyterprenin were -1 calculated as 1.2, 2.0, and 5.6 ng ml against con a-1 induced proliferation and 4.5, 8.0, and 15.6 ng ml against lps-induced proliferation (kamigauchi et al. 1998). stead et al. (1999) quoted that terprenin possesses a potent cytotoxicity against balb/mk and other hyperproliferative cell lines. it is assumed that the existence of an oxygen-linked isoprene substituent brings about such an effect on cytotoxicity potency of this compound. this effect likely occurs through the inhibition of pyrimidine biosynthesis. however no antimicrobial activity against bacteria and fungi was reported. antimicrobial assay conducted using terprenin epoxide as the test substance demonstrated no biological activity. acknowledgments this work was supported by the daad, germany and the marine natural product project of peter proksch takahashi et al. (1976) working with aspergillus candidus reported five p-terphenyl derivates. -1 terphenylin (3-10 g ml ) caused a specific toxicity on hela cells. certain quinones containing alkylating group have anti tumor activity. cain (1961) performed several cytotoxicity tests on some quinone derivatives to find out the features of the quinone molecule responsible for antitumor activity. terprenin 2 has never been reported in the literature. a related compound with the prenyl group attached to the benzene ring at the position four through an oxygen bridge (terprenin) was reported by kamigauchi et al. (1998) and stead et al. (1999). there was no antimicrobial activity of this compound identified after carrying out a number of antimicrobiological assays. terprenin epoxide differs from the known terprenin derivatives in their side chains. the epoxy group on the side chain makes the terprenin epoxide a new natural product. this unusual characteristic of prenyl and epoxy group side chains in terprenin has never been mentioned in the literature before. terprenins possessed very strong proliferations microbiol indones96 effendi table 4 nmr data of terprenin epoxide 1.14 (s) 1.25 (s) c2‘‘‘, c3‘‘‘, h4a‘‘‘ 2 128.0 7.17 (br s) 7.25 (d) h5, h6, h1‘‘‘ 3 136.0 4 4.85 (oh) 5 6.67 (d, 8.2) 6.87 (d, 8.2) h6 6 7.08 (d, 8.2) 7.20 (dd, 8.2, 2.0) h2, h5 1‘ 131.0 2‘ 5.90 (oh) 3‘ 139.0 3‘och3 3.37 (s) 3.45 (s) c3‘ 4‘ 5‘ 6.46 (s) 6.45 (s) 6‘-och 3 c1‘, c3‘, c6‘ 6‘ 154.0 6‘och3 3.70 (s) 3.74 (s) h5‘ c6‘ 2‘‘ 7.50 (d, 8.8) 7.53 (d, 6.7) h3‘‘ 3‘‘ 6.92 (d, 8.8) 6.90 (d, 6.7) h2‘‘ 4‘‘ 4.85 (oh) 5‘‘ 6.92 (d, 8.8) 6.90 (d, 6.7) h6‘‘ 6‘‘ 7.50 (d, 8.8) 7.53 (d, 6.7) h5‘‘ 1‘‘‘ h1‘‘‘a: 3.28 (dd, 15.7, 8.8) h1‘‘‘b: 3.15 (dd, 15.7, 9.5) 3.21 (dd, 19.6, 9.8) h2‘‘‘ c2 2‘‘‘ 72.0 4.63 (t, 9.5, 8.8) 4.65 (t, 9.3) h1‘‘‘ c3 3‘‘‘ 91.0 4a‘‘‘ 24.0 1.19 (s) 1.38 (s) h4b‘‘‘ c2‘‘‘, c3‘‘‘, h4b‘‘‘ 4b‘‘‘ 26.0 h4a‘‘‘ (in acetone) (in cdcl ) position 13 δ c (ppm) (in acetone) 1 δ h (ppm), multiplicity ( j in hz) (in acetone) 1 δ h (ppm), multiplicity ( j in hz) (in cdcl 3) cosy (h  h) hmbc (h  c) 3 of heinrich-heine university, düsseldorf, germany. the measurement of 1d and 2d nmr spectra was carried out by w. peters at the nmr service, heinrichheine university, düsseldorf, and victor wray at the institute for biotechnology, braunschweig, germany. gratitudes were due to rainer ebel, ruangelie edrada ebel, and victor wray for their help in the structure elucidation. references anke t, erkel g. 2002. non-β-lactam antibiotics. in: esser k, bennett jw, editors. the mycota, a comprehensive treatise on fungi as experimental systems for basic and applied research. x. industrial application (osiewacz hd, editor). heidelberg: springer. p 93-108. biabani ma, laatsch h. 1998. advances in chemical studies on low-molecular weight metabolites of marine fungi. j prakt chem. 340(7): 589-607. doi: 10.1002/prac.19983400702. blunt jw, copp br, hu wp, munro mh, northcote pt, prinsep mr. 2008. marine natural product. nat prod rep. 25(1): 35-94. cain bf. 1961. potential anti tumor agents. part i. polyporic acid series. j chem soc. 936-940. doi: 10.1039/jr9630000356. dreyfuss mm, chapela ih. 1994. potential of fungi in the discovery of novel, low molecular weight pharmaceuticals. in: the discovery of natural products with therapeutic potential. boston: butterworthheinemann. p 49-80. faulkner dj. 2000. highlights of marine natural product chemistry (1972-1999). nat prod rep. 17(1):1-6. doi: 10.1002/chin. 200025294. haefner b. 2003. drugs from the deep: marine natural products as drug candidates. drug discov today. 8(12). doi: 10.1016/s1359-6446(03) 02713-2. kamigauchi t, skazaki r, nagashima k, kawamura y, yasuda y, matsushima k, tani h, takahashi y, ishii k, suzuki r, koizumi k, nakai h, ikenishi y, terui y. 1998. terprenins, novel immunosuppressants produced by aspergillus candidus. j antibiotics. 51(4): 445-450. doi: 10.1002/chin.199839281. leal mc, puga j, serodio j, gomes ncm, calado r. 2012. trends in the discovery of new marine natural products from invertebrates over the last two decades-where and what are we bioprospecting? plos one 7(1). 15p [on line]. doi:10.1371/journal.pone.0030580. molinski tf, dalisay ds, lievens sl, saludes jp. 2009. drug development from marine natural products. nat rev drug discov. 8(1): 69-85. doi:10.1038/nrd2487. newman dj, cragg gm. 2004. marine natural products and related compounds in clinical, advanced preclinical trials. j nat prod. 67(8):1216-1238. doi: 10.1021/np040031y. pietra f. 1997. secondary metabolites from marine microoorganism: bacteria, protozoa, algae, and fungi. achievements and prospects. nat prod rep. 14(5): 453-463. doi: 10.1039/np9971400453. simmons tl, andrianasolo r, mcphail k, flatt p, and gerwick wh. 2005. marine natural products as anticancer drugs. mol cancer ther. 4(2): 333-342. stead p, affleck k, sidebottom pj, taylor nl, drake cs, todd m, jowett a, webb g. 1999. isolation and characterization of a prenlated p-terphenyl metabolite of aspergillus candidus possessing potent and selective cytotoxic activity; studies on mechanism of action. j antibiotics. 52(2): 89-95. takahashi a, nunozawa t, endo t, nozoe s. 1992. isolation of 1-ß-d-arabinofuranosylcytosine from the mushroom xerocomus nigromaculatus hongo. chem pharm bull. 40(5):1313-1314. takahashi c, yoshihira k, natori s, umeda m. 1976. the structures of toxic metabolites of aspergillus candidus. i. the compounds a and e, cytotoxic p-terphenyls. chem pharm bull. 24(4): 613-620. thomson rh. 1971. naturally occurring quinones. london: academic press. p 153 . villa fa, gerwick l. 2010. marine natural product drug discovery: leads for treatment of inflammation, cancer, infections, and neurological disorders. immunopharmacol immunotoxicol. 32(2):228-37. volume 6, 2012 microbiol indones 97 1: 89 2: 90 3: 91 4: 92 5: 93 6: 94 7: 95 8: 96 9: 97 guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can be used as long as there is consent from its authors. all pages, including the pages with figures and tables at the end of the paper, must be numbered consecutively. research paper may occupy up to 4500 words, some figures and tables; or 15 pages maximum. reviews, written as continuous articles without the subheadings such as materials and methods, results, and discussion should not exceed 3500 words or 12 pages maximum including figures and tables. short article which contains research material for unique interest but not sufficient to form regular research article will be published as a short communication. the manuscript should be prepared in a single section without sub-headings such as materials and methods, results, and discussion; not exceed 3500 words. for regular research papers, the presentation of the manuscripts should be as follows. title. the title page should include the title, the author's name(s), each author's institution and address, and a footnote containing the address to which all correspondence should be sent, complete with telephone and fax numbers as well as e-mail address. please provide the title of the work in indonesian also (for indonesian author). for international author, editors of mi will translate the text. abstract. it must not exceed 250 words. abstract contains a brief summary of the text, covering the whole manuscript without being too elaborate on every section. avoid any abbreviation, unless it is a common knowledge or has been previously stated. key words consisting of three to five words are listed in order of importance and placed below the abstract. please provide the abstract and the key words of the work in indonesian also (for indonesian author). for international author, editors of mi will translate the text. introduction. this section must give sufficient background in order that readers can understand and evaluate the results without having to read previous publications related to the topic. use only references that really support the discussion. introduction includes the background and the objective of the research. uncommon name of organism (local name) must be followed by its scientific name at the first use. materials and methods. this section should contain sufficient technical information to enable the experiments to be reproduced successfully. if a special condition is needed, for example in centrifugation, it should specify the name of the machine, rotor model, temperature, time, and centrifugal speed aims and scope microbiology indonesia provides a unique venue for publishing original researches in microbiology, and ensures that authors could reach the widest possible audience. mi publishes a wide range of research disciplines on bacteria, archaea, fungi, protozoa, and virus. topics include (but are not limited to) methods in microbiology, environmental microbiology, food microbiology, plant-microbe interaction, animal-microbe interactions, microbial community, microbial genetics, virology, comparative and functional microbial genomics, and gene expression in microbes. submission of manuscripts manuscripts to be published in microbiology indonesia include research papers, short communications, reviews, and features. the authors guarantee that the articles have not been published elsewhere. the papers should be written in english using appropriate format and corrected by an english language expert. the editor will not accept manuscripts that are deficient in these respects. the editor is not obligated to return the rejected articles. on line submission. authors are encouraged to submit on line by accessing the following website: http://jurnal.permi. or.id/index.php/mionline/ and simply click the guideline button and then follow the instruction to submit on line. on line submission will reduce the overall editorial and reviewing process, and finally accelerate the publication. email submission. the soft copy of manuscript also can be sent directly to microbiology.indonesia@permi.or.id. hard copies submission. in the case that authors cannot do manuscript submission on line, the conventional submission can be chosen. three hard copies of original articles and one soft copy of the file should be submitted. the file is prepared in microsoft word 2007. please indicate the author's name and file name. it is the author's responsibility to check the readability of the soft copy. manuscripts will not be accepted if these requirements are not fulfilled. please send the manuscript or its soft copy to: editors of microbiology indonesia room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia 15314 phone/fax: +62-21-7560536 ext. 7119/ +62-21-7560694 e-mail: microbiology.indonesia@gmail.com manuscript submission must be accompanied by a covering letter from the corresponding author. the letter should clearly state the name of corresponding author, complete mailing address, phone fax number, and e-mail address. corresand ponding author is responsible for the originality of the research and the content of the manuscript. other authors (if any) must approve of the subttmedi manuscript. to expedite the review process, authors must recommend at least two reviewers who are not members of their institution(s) and have never been volume 12, number 1, march 2018 issn 1978-3477, eissn 2087-8575 microbiology indonesia (in x g). for widely recognized materials and methods such as media and determination of protein concentration, it is sufficient to mention the reference only. if there are commonlyused modified techniques, the authors must explain the technique briefly. for example, it is better to write cells are “ separated employing ultrasonic treatment as reported by wiranto (1988) instead of only cells are separated employing ” “ treatment reported by wiranto (1988) . explain thoroughly if ” the method is new, likewise for uncommon equipment, materials, and microbial strain. if there are many strains or mutants used, present them in a table that contains the identities (name, code, collection number, source, and characteristics (physiological and genetic) of the strains, mutants, bacteriophages, plasmids. the authors agree that any plasmids, viruses, and living materials such as microbial strains and cell lines described in the article are available for academic purposes. any members of the scientific community can ask for those materials at reasonable cost for noncommercial purposes. please also write the measurement unit in international standard units and type g l -1 rather than g/l. results. this section only contains research results, please state the data clearly and precisely in a past tense. do not state references, or discuss the result either. do not repeat the details of experiment in this section, which were already mentioned in the materials and methods section. do not use too many tables and graphs if it can be explained briefly in the main text. means in tables and graphs should be given standard deviation. limit the use of photograph; present only the ones that clearly represent the results. figures and tables must be numbered consecutively. every figure and table showed must be cited on the text. authors that used microsoft excell program need to give the raw data. add measurement scale on photos if needed, arrows should be given to point certain objects. discussion. this section contains the interpretation of the results and discussion that is related to previously reported results. restating the methods, results, and other information presented in the introduction must be avoided. acknowledgment(s). this section can be used to acknowledge financial support received in funding the research and to appreciate the institutions or persons for their assistance in the research or in the writing process of the manuscript. references. mi strongly recommends to use primary references only. references are arranged in alphabetical order using harvard referencing style. please use the standard abbreviation of a journal's name according to the issn list of title wordabbreviations and refer to : [cse] council of science editors. 2006. scientific style and format: the cse manual for authors, editors, and publishers. 7th ed. reston (va): the council. journal suwanto a, kaplan s. 1989. physical and genetic mapping of the 2.4.1 genome: presence of rhodobacter sphaeroides tw o unique ci rcular chromosomes. j b act er iol. 171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 and color photo(s) will be charged idr 600 000 per page. non-permi members. page charge is idr 000 (usd 850 100) per printed article up to 4 pages. additional page will be charge idr 250 000 (usd 30) per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 (usd 50) and color photo(s) will be charged idr 600 000 (usd 60) per page. open choice beside the normal publication process, in which acceses to th article is limited to customers who have purchased a subscription, there is also open choice article that is made available publicly through microbiology indonesia on line. for open choice articles, additional price will be charged to the authors. proof print and reprints proofs will be sent to the corresponding author to be edited and approved for publication. please return the proofs immediately. the author is required to sign the proof print as approval. final editing, without changing the content, is written directly on the proof print. author will receive ten offprints free of charge. reprints are available with a minimum order of 50 copies. guide for author microbiol indones formulir berlangganan (subscription form) microbiology indonesia nama (name) institusi (institution) alamat pengiriman (address): phone/fax/email : pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com formulir berlangganan (subscription form) microbiology indonesia nama (name) institusi (institution) alamat pengiriman (address): phone/fax/email : pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com guide for author issn 1978-3477, eissn 2087-8575 volume 11, number 3, september 2017 in vitro phytochemical and inhibitory potential tests of buton forest onion extract (eleutherine palmifolia) on vibrio harveyi oil recovery test using bio surfactant of halo tolerant bacteria brevundimonas diminuta and bhurkholderia glumae at variation of nacl salt concentrations the antibacterial potential of pineapple (ananas comosus (l.) merr) core extract against methicillin-resistant staphylococcus aureus (mrsa) performance optimization of microbes from shrimp pond sediment by adding em4 in nitrification process for the treatment of wastewater containing high ammonia concentration the dynamic growth and chemical change of mixed cultures inoculation on tapioka fermentation waode munaeni, arman pariakan, munti yuhana, mia setiawati, and la ode baytul abidin bambang yudono, muhammad said, sri pertiwi estuningsih, and aulia karima boby pratama putra, danti nur indiastuti, and deby kusumaningrum h a n i e s a m b a r s a r i a n d m u h a m m a d rahmadi harahap maria erna kustyawati, sri setyani, azhari rangga, and irfa rista mutia 75 81 89 94 103 issn 1978-3477, eissn 2087-8575 volume 11, number 3, september 2017 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000, 200 000,-5 institutional rate (institution or library) (idr) 240 000, 400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print neung tiaamroeng, 2020 norio kurosawa, 2020 kartini kramadibrata, 2020 diana e waturangi, 2020 endang purwantini, 2020 wellyzar sjamsuridzal, 2020 yuan kun lee, 2020 yaya rukayadi, 2020 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi iman rusmana, 2020 cover depan (11) editorial board sept (1).pdf page 1 guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can be used as long as there is consent from its authors. all pages, including the pages with figures and tables at the end of the paper, must be numbered consecutively. research paper may occupy up to 4500 words, some figures and tables; or 15 pages maximum. reviews, written as continuous articles without the subheadings such as materials and methods, results, and discussion should not exceed 3500 words or 12 pages maximum including figures and tables. short article which contains research material for unique interest but not sufficient to form regular research article will be published as a short communication. the manuscript should be prepared in a single section without sub-headings such as materials and methods, results, and discussion; not exceed 3500 words. for regular research papers, the presentation of the manuscripts should be as follows. title. the title page should include the title, the author's name(s), each author's institution and address, and a footnote containing the address to which all correspondence should be sent, complete with telephone and fax numbers as well as e-mail address. please provide the title of the work in indonesian also (for indonesian author). for international author, editors of mi will translate the text. abstract. it must not exceed 250 words. abstract contains a brief summary of the text, covering the whole manuscript without being too elaborate on every section. avoid any abbreviation, unless it is a common knowledge or has been previously stated. key words consisting of three to five words are listed in order of importance and placed below the abstract. please provide the abstract and the key words of the work in indonesian also (for indonesian author). for international author, editors of mi will translate the text. introduction. this section must give sufficient background in order that readers can understand and evaluate the results without having to read previous publications related to the topic. use only references that really support the discussion. introduction includes the background and the objective of the research. uncommon name of organism (local name) must be followed by its scientific name at the first use. materials and methods. this section should contain sufficient technical information to enable the experiments to be reproduced successfully. if a special condition is needed, for example in centrifugation, it should specify the name of the machine, rotor model, temperature, time, and centrifugal speed aims and scope microbiology indonesia provides a unique venue for publishing original researches in microbiology, and ensures that authors could reach the widest possible audience. mi publishes a wide range of research disciplines on bacteria, archaea, fungi, protozoa, and virus. topics include (but are not limited to) methods in microbiology, environmental microbiology, food microbiology, plant-microbe interaction, animal-microbe interactions, microbial community, microbial genetics, virology, comparative and functional microbial genomics, and gene expression in microbes. submission of manuscripts manuscripts to be published in microbiology indonesia include research papers, short communications, reviews, and features. the authors guarantee that the articles have not been published elsewhere. the papers should be written in english using appropriate format and corrected by an english language expert. the editor will not accept manuscripts that are deficient in these respects. the editor is not obligated to return the rejected articles. on line submission. authors are encouraged to submit on line by accessing the following website: http://jurnal.permi. or.id/index.php/mionline/ and simply click the guideline button and then follow the instruction to submit on line. on line submission will reduce the overall editorial and reviewing process, and finally accelerate the publication. email submission. the soft copy of manuscript also can be sent directly to microbiology.indonesia@permi.or.id. hard copies submission. in the case that authors cannot do manuscript submission on line, the conventional submission can be chosen. three hard copies of original articles and one soft copy of the file should be submitted. the file is prepared in microsoft word 2007. please indicate the author's name and file name. it is the author's responsibility to check the readability of the soft copy. manuscripts will not be accepted if these requirements are not fulfilled. please send the manuscript or its soft copy to: editors of microbiology indonesia room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia 15314 phone/fax: +62-21-7560536 ext. 7119/ +62-21-7560694 e-mail: microbiology.indonesia@gmail.com manuscript submission must be accompanied by a covering letter from the corresponding author. the letter should clearly state the name of corresponding author, complete mailing address, phone fax number, and e-mail address. corresand ponding author is responsible for the originality of the research and the content of the manuscript. other authors (if any) must approve of the subttmedi manuscript. to expedite the review process, authors must recommend at least two reviewers who are not members of their institution(s) and have never been volume 11, number 3, september 2017 issn 1978-3477, eissn 2087-8575 microbiology indonesia (in x g). for widely recognized materials and methods such as media and determination of protein concentration, it is sufficient to mention the reference only. if there are commonlyused modified techniques, the authors must explain the technique briefly. for example, it is better to write cells are “ separated employing ultrasonic treatment as reported by wiranto (1988) instead of only cells are separated employing ” “ treatment reported by wiranto (1988) . explain thoroughly if ” the method is new, likewise for uncommon equipment, materials, and microbial strain. if there are many strains or mutants used, present them in a table that contains the identities (name, code, collection number, source, and characteristics (physiological and genetic) of the strains, mutants, bacteriophages, plasmids. the authors agree that any plasmids, viruses, and living materials such as microbial strains and cell lines described in the article are available for academic purposes. any members of the scientific community can ask for those materials at reasonable cost for noncommercial purposes. please also write the measurement unit in international standard units and type g l -1 rather than g/l. results. this section only contains research results, please state the data clearly and precisely in a past tense. do not state references, or discuss the result either. do not repeat the details of experiment in this section, which were already mentioned in the materials and methods section. do not use too many tables and graphs if it can be explained briefly in the main text. means in tables and graphs should be given standard deviation. limit the use of photograph; present only the ones that clearly represent the results. figures and tables must be numbered consecutively. every figure and table showed must be cited on the text. authors that used microsoft excell program need to give the raw data. add measurement scale on photos if needed, arrows should be given to point certain objects. discussion. this section contains the interpretation of the results and discussion that is related to previously reported results. restating the methods, results, and other information presented in the introduction must be avoided. acknowledgment(s). this section can be used to acknowledge financial support received in funding the research and to appreciate the institutions or persons for their assistance in the research or in the writing process of the manuscript. references. mi strongly recommends to use primary references only. references are arranged in alphabetical order using harvard referencing style. please use the standard abbreviation of a journal's name according to the issn list of title wordabbreviations and refer to : [cse] council of science editors. 2006. scientific style and format: the cse manual for authors, editors, and publishers. 7th ed. reston (va): the council. journal suwanto a, kaplan s. 1989. physical and genetic mapping of the 2.4.1 genome: presence of rhodobacter sphaeroides tw o unique ci rcular chromosomes. j b act er iol. 171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 and color photo(s) will be charged idr 600 000 per page. 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darmaga campus, bogor 16680, indonesia protease of two streptomyces sp. strain were chosen for characterization because of the large clear zone surrounding the colony in nutrient agar media containing 1% (w/v) skim milk. extracellular protease from the two isolates slw 8-1 and 45i-3 were characterized following incubation of the isolate in nutrient broth media containing skim milk or chicken feather (1%). the optimum activity of the protease slw 8-1 was at ph 9 and 80 ºc, whereas that of the keratinase was at ph 6.5 and 70 oc. protease of strain 45i-3 showed its optimum activity at ph 7.5 and 50 ºc whereas the keratinase was at ph 8.5 and 80 oc. key words: streptomyces sp., protease, keratinase _____________________________________________ _________________ ‡present address, pt. panjang jiwo, jalan daan mogot km. 19, tangerang, tanah tinggi 15122, indonesia ∗ corresponding author, phone/fax: +62-251-622833, e-mail: ameryandini@yahoo.com proteases are one of the important enzymes for industry. the sale of proteases constitutes around 60% of the total sales of industrial enzymes (adinarayana et al. 2003) and around 500 tons of protease enzyme are produced every year to fulfill demand coming from industries (crueger and crueger 1984). many species of bacteria are known in relation to their ability to produce protease, such as bacillus subtilis, escherichia coli, clostridium bifermentans, and streptomyces sp. (bockle et al. 1995; rao et al. 1998; petinate et al. 1999b; adinarayana et al. 2003; enggel et al. 2004). besides protease, streptomyces sp. is also able to produce keratinase, the enzyme that degrades keratin (letourneaeu et al. 1998; bressolier et al. 1999; moreira et al. 2001). keratin is abundant in the nature. it is usually found in feather, hair, nail, and horn (ignatova et al. 1999). the presence of many cystein bridges or disulfide bonds, hydrogen bonds, and hydrophobic interactions in keratin structure make this substrate very stable, rigid, and it can hardly be degraded by common proteolytic enzymes (lin et al. 1992; bockle et al. 1995; bressolier et al. 1999). the test on keratinolytic activity is usually conducted by using flour made from chicken feathers. lintang (2003) stated that the amino acid content in chicken feather flour is very similar to the amino acids contained in keratin, e.g. the amount of the amino acid serine, arginine, and proline. keratin in chicken feathers contains some nutrients, which are 81% crude protein, 7% crude fat, 1% crude fiber, 0.33% calcium, and 0.55% phosphorus. the high nutrient content in chicken feathers suggest this is a good feeding substance. the constraints faced in using chicken feathers for feed is the difficulty in digesting the feathers because keratin is not water soluble. a treatment which is usually applied to make the feathers more easily digestable is to use high pressure and high temperature. this treatment, however, needs a very large amount of energy and causes a loss of a large amount of its amino acid contents (ignatova et al. 1999). a biological approach to treat the waste of chicken feathers can be seen as an alternative solution. materials and methods screening of proteolytic streptomyces strains. culturing a strain from the microbiology laboratory, department biology, institut pertanian bogor was conducted by growing the strains on nutrient agar with 1% skim milk (nas media). the culture was then incubated for 7-8 days at room temperature. strains which produced protease were characterized by formation of clear zone around their colony in nas media. enzyme production. pure cultures of the strains were transferred into the nas media and incubated for 7-8 days at room temperature. the colonies which grew in the nas media were then harvested using cookborers (with 0.5 cm of diameter) and inoculated to 100 ml of nutrient broth media (3 g beef extract and 5 g peptone in 1 l of destilled water) that contained 1% (w/v) skim milk (nbs) in erlenmeyer sized 500 ml. the cultures were incubated at room temperature using a shaking incubator with an agitation speed of 240 rpm. from the 3rd day of incubation, the protease activities of the cultures were assessed every 24 h. the results, which were conducted until day 14th, were aimed at determining the optimum time to yield the crude protease activity produced. the crude extracts of protease were separated from the cell mass by centrifugation (8,000 g for around 5 min). protease activities. protease activities were determined using a modified walter (1984) method. as much as 100 ml crude protease extract was added with 0.5 ml of 1% (wt/vol) casein or 1% (wt/vol) chicken feather and 0.5 ml of 200 mm buffer tris-hcl ph 7.5. the solution was then incubated at 37 oc for 10 min. the reaction was stopped by adding 1 ml of 10% (wt/vol) trichloroacetic acid (tca) with continued incubation at 10 oc for 10 min. the next step was centrifugation of the reaction solution at 8,000 g for 10 min. the supernatant (0.75 ml) was added to 2.5 ml na 2 co 3 0.5 m and 0.5 ml folin ciocalteau reagent (1:2), strongly shaken, and incubated for 10 min at room temperature and its absorbance read at 578 nm. one unit (iu) of the enzyme microbiology indonesia, august 2007, p 69-73 volume 1, number 2 issn 1978-3477 activity is defined as the amount of enzyme that produced 1 µmol soluble tyrosine per min. one unit is equal to 16.67 nkat (dybkaer 2001). protein concentration. amounts of protein (mg ml-1) were determined by the bradford method (bradford 1976). the protein standard was bovine serum albumin (bsa). specific activity of the protease (nkat mg-1 protein) is the ratio between protease activity (nkat ml-1) and total protein (mg ml-1) of an extract. enzyme characterization. the determination of optimum ph for protease and keratinase activities was conducted by assessing the extract of crude enzyme from ph 5 until ph 9 with interval of 0.5 units. if the optimum activity of the crude enzyme was ph 9, the assessment was continued to ph 10, 11, and 12. the buffer that was used included 200 mm citric acid (ph 5.0-6.5), 200 mm tris-hcl (ph 7.0-9.0), 200 mm glycine-naoh (ph 10.0), and 200 mm phosphate-naoh (ph 11 and 12). the effect of ph on enzyme activities for each kind of substrate was monitored by using an extract of crude enzyme, based on the highest activities in their activity curve. the determination of optimum temperature of protease and keratinase of the crude extract was conducted through assessment of enzyme activity with various incubation temperatures, which started from 30 oc until 90 oc using 10 oc intervals. the analyses were done at optimum ph. results proteolytic strains. a description of the six isolates of streptomyces sp. grown in nas media is presented in table 1. based on the data shown, isolates slw 8-1, and 451-3 had a bigger clear zone compared to that of the other four isolates. protease activities. on the 3th day, the strain slw 8-1 had not shown any protease activity. the activity started to be detected on the 4th day. the highest activity was on the 7th day and started to decline on the 9th day, and with a bump in activity on the 10th day (fig 1). the strain 45i-3 had a less varied patterns for protease activity. on day 3, the enzyme activities of this strain had been detected. the peak of the activity was on day 4, continued with the declining activities until day 6, and increased again until day 11 (fig 1). enzyme characterization. the extract of crude protease from strain slw8-1 shows activity at ph between 8.5 and 10.0. the highest activity, which is 0.59 nkat mg-1 protein, falls at ph 9 (fig 2). the highest activity of keratinase for this strain (3.337 nkat mg-1 protein) falls at ph 6.5 (fig 3). protease of strain 45i-3 shows activity between ph 6.5-8, with the highest activity of 0.49 nkat mg-1 protein at ph 7.5 (fig 4). the highest keratinase activity of strain 45i-3 is 27.59 nkat mg-1 protein, which was obtained at ph 8.5. this strain also shows high keratinase activity at ph 7.0. the activity is 24.31 nkat mg-1 protein (fig 5). as a function of temperature, protease activity produced by strain slw8-1 has three peaks i.e 0.63 nkat/mg protein (40 oc), 0.56 nkat mg-1 protein (50 oc), and 0.73 nkat mg-1 protein (80 oc) (fig 6). keratinase produced by strain strain slw8-1 has optimum temperature at 70 oc with an activity of 4.559 nkat mg-1 protein (fig 7). table 1 description of 6 isolates grown nutrient agar with 1% skim milk media after 12 days of incubation isolate clear zone proteolytic index pd 3-27 slw 8-1 2 3 4 p 1 6 ks-1 ps-4-11 45i-3 yes yes n o yes yes yes < 1.00 2.46 < 1.00 1.30 1.50 fig 2 protease activities from strain slw8-1 as a function of ph at 37 oc. fig 3 keratinase activity from strain slw8-1 as a function of ph at 37 oc. fig 1 activity curves from slw8-1 and 45i-3 proteases measured at ph 7.5 and 37 oc. protease 451-3, protease slw8-1 (u ml-1). protease of strain 45i-3 has optimum temperature at 50 oc with protease activity 0.48 nkat mg-1 protein (fig 8). keratinase produced by strain strain 45i-3 has optimum temperature at 80 oc with activity 71.65 nkat mg-1 protein (fig 9). both isolates produced protease and keratinase with a higher keratinase activity if cultured on nas media (fig 10). fig 4 protease activities from strain 45i-3 as a function of ph at 37 oc. fig 5 keratinase activities from strain 45i-3 as a function of ph at 37 oc. fig 6 the effect of temperature on protease activity from strain slw8-1 at ph 9. temperature (oc) temperature (oc) fig 7 the effect of temperature on keratinase activity from strain slw8-1 at ph 6.5. temperature (oc) fig 8 the effect of temperature on protease activity strain 45i3 at ph 7.5 temperature (oc) fig 9 the effect of temperature on keratinase activity strain 45i-3 at ph 8.5. kasein (slw8-1) feather (slw8-1) feather (451-3) kasein (451-3) substrat fig 10 relative protease and keratinase activity (%) from streptomyces sp. slw8-1 and 45i-3 measured at optimum ph and temperature of each enzyme. discussion although the diameter of the clear zone in solid media is not always correlated with the high activity of protease production in liquid media, the method is often used to precisely identify whether or not the isolates produce extracellular protease. many bacteria are known for their ability to produce protease (rao et al. 1998). each of the bacteria isolates of the same genus produces a different quality and of protease having different characteristics. the addition of 1% (w/v) skim milk to the na media was intended to induce the bacteria to synthesize protease. beyond its optimum time, protease activities continued to decline. the decrease was considered to result from autolysis process of the protease itself. decreasing amounts of substrate, due to hydrolysis that occurred by extracellular protease produced previously, is also considered to contribute some effect to the decline of the protease activities beyond its optimum period. feather meal can be used to induce keratinase activity. vibrio sp. strain kr6 and chryseobacterium sp. produced higher yields of keratinase using feather meal or raw feather as a casein substrate (sangali and brandelli 2000; brandelli and riffel 2005). protease of strain slw8-1 belongs to the alkaline proteases, while its keratinase belongs to the ph neutral group of enzyme. strain 45i-3, on the other hand produces, a neutral protease and an alkaline keratinase. a protease which is active in alkaline condition has also been reported by petinate et al. (1999a) for enzymes produced by streptomyces cyaneus (ph 9), letourneau et al. (1998) for protease produced by streptomyces sp. s.k 1-02 (ph 10), and adinarayana et al. (2003) for protease produced by bacillus subtilis pe-11 (ph 10). protease with optimum activity at a neutral ph was reported by lin et al. (1992) for b. licheniformis, bressolier et al. (1999) for s. albidoflavus, and moreira et al. (2001) for s. clavuligerus. the several peak of activities which occurred in the optimum temperature determination may be due to the presence of isozymes. isozymes are enzymes which catalyze the same reaction but shows different physical and chemical characteristics (for example isoelectric point, optimum ph, substrate affinity, or inhibitor effect) when they are synthetised via different genes. letourneau et al. (1998) also reported that streptomyces sp. s.k 1-02 will rapidly produced a protease mixture with high keratinolytic activity if cultured on a simple medium supplemented with feather meal. some studies have also reported the ability of one strain to produce different types of protease such as streptococcus suis which is able to produce 4 isozymes (jobin and grenier 2003). streptomyces albidoflavus has been reported to produce at least 6 isozymes of protease after the exponential growth phase is completed. the production of several proteases in streptomyces is perhaps due in-situ degradation of mycelium proteins by protease during different stages of morphological differentiation (bressolier et al. 1999). ginther (1979) also reported that in streptomyces lactamdurans the production of an antibiotic and protease is closely associated with the sporulation process. the protease of strain 45i-3 has optimum temperature at 50 oc with protease activity 0.48 nkat mg-1 protein (fig 8). the similar result was reported by moreira et al. (2001) for a protease partially purified from s. clavuligerus 3585, and lin et al. (1992) for protease activity of b. licheniformis. in our study, the protease of strain slw 8-1 shows optimum activity at ph 9 and 80 oc, while its optimum keratinase activity is reached at h 6.5 and 70 oc. also ph 7.5 and 50 oc is needed by strain 45i-3 to show its optimum protease activity, and ph 8.5 and 80 oc are needed for its optimum keratinase activity. acknowledgment the authors wish to thank yulin lestari for the isolates. references adinarayana k, ellaiah p, prasad ds. 2003. purification and partial characterization of thermostable serine alkaline protease from a new isolated bacillus subtilis pe-11. aaps pharm sci tech 56:19 . bockle b, galunsky b, muller r. 1995. characterization of a keratinolytic serine proteinase from streptomyces pactum dsm 40530. appl environ microbiol 6:3705-3710. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantitaties of protein in utilizing the principle of protein-dye binding. ann biochem 72:248-254. brandelli a, riffel a. 2005. production of an extracellular keratinase from chryseobacterium sp. growing on raw feathers. biotechnol 8:35-42. bressollier p, letourneau f, urdaci m, verneuil b. 1999. purification and characterization of a keratinolytic serine protease from streptomyces albidoflavus. appl environ microbiol 65:25702576. crueger a, crueger w. 1984. biotechnology: a textbook of industrial microbiology. in: brock td (ed). sunderland: minauer associater inc. dybkaer r. 2001. unit “katal” for catalytic activity. j pure appl chem 73:927-931. enggel j, meryandini a, natalia l. 2004. karakterisasi protease ekstraseluler clostridium bifermentans r14-1-b. j mikrobiol indones 9:9-12. ginther cl. 1979. sporulation and the production of serine protease and cephamycin c by streptomyces lactamdurans. antimicrob agents chemotherapy 15:522-526. ignatova z, gousterova a, spassov g, nedkov p. 1999. isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain thermoactinomyces candidus. j can microbiol 45:217-222. jobin mc, grenier d. 2003. identification and characterization of four proteases produced by streptococcus suis. fems microbiol lett 220:113-119. letourneau f, soussotte v, bressollier p, branland p, verneuil b. 1998. keratinolytic activity of streptomyces sp. s.k 1-02 : a new isolated strain. appl environ microbiol 26:77-80. lin x, lee c, casale es, shih jch. 1992. purification and characterization of keratinase from a feather-degrading bacillus licheniformis strain. appl environ microbiol 58:3271-3275. lintang raj. 2003. keratinase pendegradasi bulu ayam dari bakteri termofilik l-23 asal sulawesi utara [thesis]. bogor: program pascasarjana ipb. moreira ka, calvalcanti mth, duarte hs, tambourgi eb, de melo ehm, silva vk, porto a l f, filho jldel. 2001. partial characterization of protease from streptomyces clavuligerus using an inexpensive medium. j brazilian microbiol 32:215-220. petinate sdg, branquinha mh, coelho rrr, vermelho ab, de simone sg. 1999a. purification and partial characterization of an extracellular serine-proteinase of streptomyces cyaneus isolated from brazilian cerrado soil. appl microbiol 87:557-563. petinate sdg, martins rm, coelho rrr, meirelles mnl, branquinha mh, vermelho ab. 1999b. influence of growth medium in proteinase and pigment production by streptomyces cyaneus. j mem inst oswaldo cruz rio de janeiro 94:173-177. rao mb, thanksale am, ghatge ms, desphande vv. 1998. molecular and biotechnological aspects of microbial protease. j microb mol biol rev 62:597-635. sangali s, brandelli a. 2000. feather keratin hydrolysis by a vibrio sp. strain kr2. j appl microbiol 89:735-743. walter he. 1984. proteinase (proteins substrates). method with haemoglobin, casein and azocoll as substrate. in: bergmeyer j, grass m (eds). methods of enzymatic analysis. 3th ed. weinheim germany: verlag chemie. p 270-278. 01 tuberculosis (tb) is still one of the high prevalence diseases in the world. latent infection of mycobaterium tuberculosis (mtb) is believed to infect almost one third of the world population (koul et al. 2011). no existing anti-tubercular drugs can be used to against this latent infection. recently, poor control of anti-tubercular drugs intake leads to the emergence of resistance (araújo-filho et al. 2008; lawn and zumla 2011). phor-phop, a two-component system signal transduction in mtb, is known to regulate 114 genes related to the virulence of mtb (walters et al. 2006; gonzalo-asensio et al. 2008). the currently available anti-tubercular drugs recognise only single targets. using this system as target is very useful, since it will block and shut down multiple targets simultaneously. previous studies showed that in vivo disruption of phor-phop drastically attenuated mtb (walters et al. vol.8, no.4, december 2014, p 141-146 doi: 10.5454/mi.8.4.1 cloning, overexpression, and purification of phor cytoplasmic domain protein from mycobacterium tuberculosis strain h37rv 1 1 1 oktira roka aji , dyshelly nurkartika pascapurnama , fenryco pratama , 2 1 1* ihsanawati , maelita ramdani moeis , and ernawati arifin giri-rachman 1 school of life sciences and technology, institut teknologi bandung, jalan ganesha 10 bandung, west java, 40132, indonesia; 2 department of chemistry, faculty of mathematics and life sciences, institut teknologi bandung, jalan ganesha 10, bandung, west java, 40132, indonesia tuberculosis is still a major health problem in the world. this infectious disease is caused by mycobacterium tuberculosis (mtb). novel anti-tubercular drug is urgently needed to counter multidrug resistant cases and mtb's spread. the cytoplasmic domain of phor histidine kinase, a part of the two-component system phor-phop in mtb, is one of the potential candidates for anti-tubercular drug target. three dimensional structure (3d-structure) of the protein (drug target) is needed to screen potential drug candidate using rational drug design approaches. previous studies have successfully characterized and isolated putative cytoplasmic domain of phor (cytophor) from mtb strain h37rv. this study aimed to clone, overexpress, and purify cytophor protein. cytophor was fused with thioredoxin protein in pet32b expression vector and overexpressed in escherichia coli (e.coli) bl21(de3) as soluble fraction by induction with 1 mm iptg. purification of his-tagged cytophor was carried out using imac ni-nta agarose his-tag affinity column. sds-page analysis showed that another protein was co-purified (~35 kda) along with the cytophor protein. subsequent protein purification using deae-ion exchange column generated a strong single band of 37 kda on sds–page which was identified as cytophor protein. the purified cytophor protein was successfully obtained and could be used for further analysis to determine the 3d-structure of cytophor protein. key words: mycobacterium tuberculosis, rational drug design, tuberculosis, two-component system tuberkulosis (tb) masih menjadi masalah kesehatan utama di dunia. penyakit menular ini disebabkan oleh bakteri mycobacterium tuberculosis (mtb). obat anti-tb baru sangat dibutuhkan untuk menanggulangi kasus multi-resisten obat tb yang ada saat ini dan penyebaran mtb. domain sitoplasmik protein phor histidin kinase yang merupakan bagian dari two component system phor-phop di mtb, adalah salah satu kandidat yang sangat potensial untuk dijadikan sebagai target baru obat anti-tb. pada studi rational drug design, struktur tiga dimensi protein target obat dibutuhkan untuk menyeleksi kandidat obat yang potensial ini. studi sebelumnya telah berhasil mengkarakterisasi dan mengisolasi domain sitoplasmik phor (cytophor) dari mtb strain h37rv. penelitian ini bertujuan untuk mengkloning, mengekspresikan, dan memurnikan protein cytophor. cytophor disubkloning dalam vektor ekspresi pet32b. protein berhasil diekspresikan sebagai fraksi terlarut dalam e. coli. pemurnian cytophor dilakukan dengan menggunakan imac ni nta agarosa kolom afinitas his-tag. analisis sds-page pada hasil pemurnian menunjukkan bahwa protein kontaminan masih terbawa(~ 35 kda). selanjutnya, pemurnian protein dilakukan dengan menggunakan kolom penukar ion deae menghasilkan satu band berukuran 37 kda pada analsis sds–page. dengan demikian, protein cytophor telah berhasil dimurnikan dan dapat digunakan untuk analisis lebih lanjut dalam menentukan struktur tiga dimensi protein cytophor. kata kunci: mycobacterium tuberculosis, rational drug design, tuberkulosis, two-component system *corresponding author; phone: +62-22-2511575, 2500258, fax: +62-22-2534107 ; email: erna@sith.itb.ac.id 2006; gonzalo-asensio et al. 2008). homology study revealed that phor is different from other prokaryotic proteins (suwanto and giri-rachman 2012) also, this signal transduction system is not found in human (suwanto 2012). therefore, the drug candidate hopefully has high selectivity and limited toxicity. one potential drug target is the dimerization domain of phor. dimerization plays a key role in the activation of the transduction system phor-phop. the dimerization domain is located in the cytoplasmic domain of phor (cytophor) (ryndak et al. 2008; yamada and shiro 2008). 3d-structure of cytophor is needed to screen novel drugs using rational drug design method. rational drug design is a method for developing new drugs based on structural simulation. although the 3d-structure of the protein target can be used to screen hundreds of molecules that can potentially inhibit the protein targets, currently, the 3dstructure of phor in mtb has not been determined. to determine the protein structure, large quantities of purified protein are required. previous studies have successfully isolated and cloned cytophor coding sequence from mtb strain h37rv into prset expression vector (kurnia 2012). in this study, we subcloned, overexpressed, and purified cytophor recombinant protein in e. coli bl21 (de3). the focus of our research is to produce and purify cytophor protein in soluble form. thus, it will be useful for determination of cytophor 3d-structure. materials and methods subcloning of phor cytoplasmic domain into vector expression pet32b. a dna fragment containing the putative cytoplasmic domain of phor was previously isolated from genomic dna of mtb strain h37rv (kurnia 2012). the dna fragment was subcloned into pet32b expression vector by pcr (pet32b-cytophor). the forward primer used contained a kpni site upstream of the start codon: 5‟g g t a c c at g c g a c a g t t c at c a c c 3 ‟ (cytophor-forward primer). the reverse primer contained an ecori site and a stop codon downstream of cytophor: 5'-gaattctcacaaccccagtc cggt-3' (cytophor-reverse primer). the pcr amplified fragment was ligated into pet32b vector. the plasmids were introduced into e. coli bl21(de3) by transformation using heat shock method (sambrook and russel, 2001). the ligation product (pet32bcytophor) was examined by 1% agarose gel electrophoresis and sequenced. the construct was validated by restriction digestion using kpni-ecori, pcr amplification, and dna sequencing. dna sequence and translation analyses were carried out using bioedit© and clc genomic® programs. overexpression of recombinant phor cytoplasmic domain. e. coli bl21(de3) containing pet32b-cytophor plasmid were grown in lb agar containing 100 ppm ampicillin. a single colony was inoculated into 5 ml lb broth containing ampicillin. the culture was incubated at 37 °c, with 200 rpm agitation overnight. then, the culture was used to inoculate 1 liter lb broth and ampicillin. when the optical density (od ) reached 0.4-0.7, the culture 600nm was induced with 1 mm iptg (isopropyl-1-thio-galactopyranoside) for 4 hours. cells were harvested by centrifugation at 7000xg, 4°c, for 30 minutes. the cell pellet was washed with 20 mm tris cl (ph 8.0), then resuspended in 2 ml lysis buffer (20 mm tris cl ph 8.0, 1 mm pmsf), followed with 10 minutes sonication . the insoluble material and cell debris (pellet) were separated by centrifugation at 12000xg, 4 °c, for 10 minutes. the cell lysate containing soluble fraction of his-tagged cyto-phor was filtered using 0.22 μm millipore filter and used for protein purification. protein purification using ni-nta affinity chromatography (imac). protein was purified using ni-nta-agarose system at 4 °c. ni-nta agarose was first equilibrated using buffer containing 20 mm triscl ph 8.0, 20 mm imidazole, and 300 mm nacl. the cell lysate was applied on ni-nta agarose for 1 hour at 60 rpm, 4 °c. purification was carried out in the native state at 4 °c with a linear flow rate of 0.5-1 ml -1 min , using 50 ml of econo column (biorad). the column was washed with binding buffer (20 mm triscl ph 8.0, 300 mm nacl, 50 mm imidazole) several times to remove unbound proteins. cytophor protein was eluted with elution buffer containing 250-500 mm imidazole. the base-buffer used in this purification contained 20 mm triscl ph 8.0 and 300 mm nacl. eluted fractions were pooled and concentrated to about 5 ml using microsep™ advanced centrifugal filter with 10 kda mw cut off (pall). the sample was dialyzed against buffer containing 20 mm triscl ph 8.0, and 200 mm arginin, at 4 °c overnight. protein purification using ion exchange chromatography. final purification was achieved by ion exchange chromatography using deae (diethylaminoethyl) resin. the partially purified histagged cyto-phor protein was applied to deae on 50 ml econo column (biorad). the column was eluted 142 aji et al. microbiol indones with 0-800 mm nacl gradient using 20 mm tris-cl buffer ph 8.5 flow rate of 0.5-1 ml at 4 °c. the eluted fraction were collected and pooled in one falcon before being dialyzed against buffer containing 20 mm triscl ph 8.0, 200 mm nacl, and 200 mm arginin, and concentrated by ultrafiltration using microsep™ advanced centrifugal filter with 10 kda mw cut off (pall). cytophor concentration was determined using bradford assay (bradford 1976), with bovine serum albumin (bsa) as protein standard. cytophor protein was then run on 12% sds-page gel according to laemmli method (laemmli 1970). protein bands were visualized by coomassie brilliant blue staining and silver staining. results subcloning of the cytoplasmic domain of phor into pet32b expression vector. the specific primers (cytophor-forward and reverse) were designed to amplify cytophor gene fragment. the pcr amplified fragment was inserted via its unique kpni and ecori restriction sites into the corresponding sites in pet32b vector. cytophor was fused with another protein in this expression vector. a genetic map of pet32bcytophor (fig 1) shows thioredoxin, his-tag, and stag located upstream from cytophor. pcr analysis using cytophor-forward and reverse primer was performed to verify cytophor in pet32b. the specific amplicon (~561 bp) in the electropherogram confirmed the presence of cytophor in the pet32b (fig 2). sequencing analysis was performed by bioedit© and clc genomic® programs, indicating the insertion of cytophor in the correct direction without shift in the reading frame. this result indicated that cytophor had been successfully inserted in frame within pet32b. overexpression of the cytoplasmic domain of phor recombinant protein. the expression system in pet32b was driven by t7 promoter and lac operator upon induction with iptg. no expression of the protein of interest was observed in the non-induced sample. calculation of total molecular weight using mw prediction program (http://www.expasy.org/ compute_pi/mw) revealed that this fusion protein has molecular weight of 36.8 kda. sds-page analysis of the cytophor fusion protein showed the presence of a band approximately ~37 kda (fig 3). this result was consistent with the prediction of its molecular weight. protein purification using ni-nta affinity chromatography (imac). in this study, we used imac ni-nta to purify the overexpressed cytophor fusion protein, making use of the his-tag present in this recombinant protein. purification process was performed under native condition, since cytophor fusion protein was overexpressed as soluble fraction. sds-page analysis showed that several impurities volume 8, 2014 microbiol indones 143 fig 1 genetic map of pet32b-cytophor vector. location and transcriptional orientation of bla coding sequence, β-lactamase for ampicillin resistance; ori (origin of replication); laci, laci repressor; t7 promoter (upstream) and t7 terminator (downstream); trxa (thioredoxin); 6xhis-tag; s-tag; and f1 origin of replication are shown. the amplified dna fragment of cytophor gene (561 bp) was ligated into kpni/ecori site of pet32b expression vector under control of t7 promoter. cytophor was fused with thioredoxin protein, 6xhis-tag and s-tag. were co-eluted with cytophor (fig 4a). other nontagged but histidine rich proteins might also interact with the ni-nta resin. this non-specific binding was reduced by adding low concentration of imidazole in wash buffer and during pre-treatment of protein. protein purification using ion exchange chromatography. partially purified cytophor was applied to deae ion exchange chromatography column and eluted using nacl gradient (0-800 mm). peak fraction containing the highest levels of protein was eluted at 400 mm nacl. sds-page analysis showed that cytophor had been purified to near homogeneity (fig 4b) discussion cytophor contains several conserved motif from histidine kinase: h-box (helr), n-box (nlvana), g1-box (ddgpg), f-box (ferf) dan g2-box (gtglgl) (kurnia 2012; sundari 2010). histidine kinase phor from mtb strain h37rv was categorized as class i of histidine kinase (yamada and shiro 2008). h-box in this histidine kinase is located in the dimerization domain (jung et al. 2012; yamada and shiro 2008). this domain plays a key role in the activation of signal transduction. blockade of the dimerization domain will block the signal transduction pathway and subsequently stop the expression of virulent genes. in this study, thioredoxin was used as fusion partner to increase protein solubility. recombinant proteins that were produced as fusion with thioredoxin had been proven to be soluble (la vallie 1994). in silico analysis using protein-solubilization prediction software ( ) indicated that http://www.biotech.ou.edu/#r cytophor fusion protein is produced in soluble form in e.coli . soluble protein is usually easier to purify than protein that is expressed in inclusion bodies. several e.coli proteins rich of histidine residue had been reported to co-purify in ni-nta agarose affinity chromatography system, for examples peptidyl prolyl cis-trans isomerase (slyd) 27 kda; class carbonic a n h y d r a s e ( c a n ) 2 5 k d a ; u d p l a r a 4 n formyltransferase (arna) 74.3 kda; subunit e1 multienzyme complex pyruvate dehydrogenase (acee) 99.7 kda; and l-glutamine dfructose-6phosphate aminotransferase (glms) 67 kda ) (öberg et al. 2004; robichon et al. 2011). these proteins bound non-specifically to ni-nta resin and hardly removed. therefore, another purification system must be performed in order to remove all contaminant proteins. in this experiment, the purification step had successfully removed almost all impurities. however, we found that cytophor fusion protein easily formed aggregate at high protein concentration. this suggests that protein storage and analysis must be performed carefully. addition of 200 mm arginine was aimed to prevent rapid aggregation. amino acids sequence analysis and 3d prediction revealed that this protein had three cysteine residues. the presence of these cysteines may interfere with protein solubility, since non-reducing sds-page analysis showed that this protein formed a trimer (data not shown.). in conclusion, the cytoplasmic domain of phor has successfully been cloned and expressed in e. coli bl21(de3) as soluble form. the protein has also been successfully purified using chromatography affinity (imac) ni-nta, followed by ion exchange 144 aji et al. microbiol indones fig 2 cytophor gene amplification using specific primer cytophor-forward and primer cytophor-reverse. the pcr produced ~561 bp bands on agarose gel (1% w/v) representing the cytophor gene. left lane: dna ladder 1 kb (thermo scientific); middle lane: 1, 2, 3 cytophor gene ~561 bp; right lane: negative control. fig 4 the purification of cytophor recombinant proteins extracted from e. coli bl21(de3) using ni-nta affinity chromatography (imac) followed by ion exchange chromatography. protein samples were separated in 12% sds-page gels. (a) sds–page analysis of partially purified cytophor protein stained with coomassie brilliant blue. 6xhis-tagged cytophor proteins from e. coli bl21(de3) were purified under native conditions using affinity chromatography (imac) ni-nta. lane m : broad range protein markers, ei : the eluent collected from affinity chromatography columns. (b) sds–page analysis of purified cytophor protein stained with silver-stain. protein was eluted using nacl gradient (0-800 mm) on deae-ion exchange column. eluted protein of deae-ion exchange chromatography produced specific band at ~37 kda. sds-page analysis showed that purification process successfully remove almost all impurities proteins. lane m : broad range protein markers (thermo scientific), e2 : the eluent collected from ion-exchange columns. fig 3 overexpression of cytophor protein in e. coli bl21(de3) was analyzed by 12% sds-page gel. the recombinant e. coli was grown overnight then induced with 1 mm iptg for 4 h. left lane: low range protein markers (thermo scientific). non-ind: cell lysate without iptg induction. ind: cell lysate with iptg induction containing his-tagged cytophor. volume 8, 2014 microbiol indones 145 chromatography. the purified protein would be used for further crystallization studies as a target for structure-based discovery of novel anti-tubercular drugs. acknowledgment this research was supported by twas the academy of sciences for the developing world 20122013 to ernawati arifin giri-rachman. references araújo-filho jad, vasconcelos-jr ac, sousa emd, silveira cd, sousa ptp, severo ka, vieira lf, kipnis a, junqueira-kipnis ap. 2008. multidrug-resistant tuberculosis : case reports study in a central state of brazil. braz j infect dis. 12(1): 94–98. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal biochem. 254: 248–254. gonzalo-asensio j, mostowy s, harders-westerveen j, huygen k, hernández-pando r, thole j, behr m, gicquel b, martín c. 2008. phop: a missing piece in the intricate puzzle of mycobacterium tuberculosis virulence. plos one 3 (10) (january): e3496. doi:10.1371/journal.pone.0003496. jung k, fried l, behr s, heermann r. 2012. histidine kinases and response regulators in networks. curr opin microbiol. 15 (2) : 118–24. doi:10.1016/j.mib. 2011.11.009. koul a, arnoult e, lounis n, guillemont j, andries k. 2011. the challenge of new drug discovery for tuberculosis. nature 469 (7331): 483–90. doi:10.1038/nature09657. kurnia is. 2012. pengembangan sistem penapisan a n t i t u b e r k u l a r b a r u p a d a e s c h e r i c h i a c o l i bl21(de3)plyss melalui fusi phor domain sitoplasmik mycobacterium tuberculosis h37rv dengan protein represor iclr. [thesis] bandung (id) : institut teknologi bandung. laemmli uk. 1970. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227: 680–685 la vallie e, mccoy j. 1994. expression and purification of thioredoxin fusion proteins. curr protoc mol biol. 16.8.1-16.8.14. doi: 10.1002/0471142727.mb1608s28. lawn sd, zumla ai. 2011. tuberculosis. the lancet 378 (9785) : 57–72. doi:10.1016/s0140-6736(10)62173-3. öberg k, andersson lc, lundqvist j, anderson l, stålbrand h. 2004. effect of flow rate , molecular weight of ( his ) 6 -tagged proteins, and expression system on the performance of histrap hp columns: 4–6. robichon c, luo j, causey tb, jack sb, samuelson jc. 2011. engineering escherichia coli bl21(de3) derivative strains to minimize e.coli protein contamination after purification by immobilized metal affinity chromatography. appl environ microbiol. 77(13):4634. doi:10.1128/aem.00119-11. ryndak m, wang s, smith i. 2008. phop, a key player in mycobacterium tuberculosis virulence. trends microbiol. 16 (11): 528–34. doi:10.1016/j.tim.2008.08. 006. sambrook j, russell dw. 2001 : molecular cloning: a rd laboratory manual. 3 edition. new york : cold spring harbor laboratory press. sundari s. 2010. pengembangan sistem penapisan antibakteri baru dengan target phor mycobacterium tuberculosis. [thesis] bandung (id) : institut teknologi bandung. suwanto as, giri-rachman ea. 2012. molecular cloning of phor sensor domain from mycobacterium tuberculosis for structure-based discovery of novel anti-tubercular. j life sci 6: 268–275. suwanto as. 2012. karakterisasi dankkloning dna pengkode sensor domain protein phor mycobacterium tuberculosis sebagai molekul target obat anti tuberkular baru.[thesis] bandung (id): institut teknologi bandung. walters sb, dubnau e, kolesnikova i, laval f, daffe m, smith i. 2006. the mycobacterium tuberculosis phopr two-component system regulates genes essential for virulence and complex lipid biosynthesis. mol microbiol. 60 (2): 312–30. doi:10.1111/j.13652958.2006.05102.x. yamada s, shiro y. 2008. structural basis of the signal transduction in the two-component system. adv exp med biol. 631:22-39. doi: 10.1007/978-0-387-788852_3. 146 aji et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 01 rakhmayanti.cdr vol.12, no.3, september 2018, p 69-73 doi: 10.5454/mi.12.3.1 o the growth of leptolyngbya hs-16 and hs-36 on 35 c at different acidity * nurul rakhmayanti, nining betawati prihantini department of biology, faculty of mathematics and natural sciences, universitas indonesia, kampus ui depok 16424, indonesia cyanobacteria are prokaryotic organisms belonging to the kingdom eubacteria. cyanobacteria can be found in hot spring. leptolyngbya is one genus of cyanobacteria that can be found in hot spring. the observation of o leptolyngbya growth on temperature of 35 c with initial ph variation had been done. the study was experimental trial. the study aimed to determine the best initial growth ph for leptolyngbya hs (hot spring)-16 and hs-36. leptolyngbya hs-16 was isolated from pancar mountain hot spring, while leptolyngbya hs-36 was isolated from maribaya hot spring. the acidity (ph) of pancar mountain and maribaya hot spring was 7. each strain was grown o in blue green medium number 11 with variation of initial ph (6, 7, 8 and 9) and incubated at 35 c. parameters was wet biomass weight of leptolyngbya in each strain. the results of 15 days observation showed that the best initial ph for growing leptolyngbya hs-16 is 7, while leptolyngbya hs-36 is 9. from this study it could be seen that leptolyngbya hs-16 and hs-36 could be cultured with alkaline condition. key words: hot spring, leptolyngbya, ph cyanobacteria merupakan organisme prokariotik yang berasal dari kingdom eubacteria. cyanobacteria dapat ditemukan pada sumber air panas. salah satu genus dari cyanobacteria yang ditemukan dalam sumber air panas, o yaitu leptolyngbya. pengamatan pertumbuhan leptolyngbya pada suhu 35 c dengan variasi ph awal telah dilakukan. penelitian ini merupakan penelitian eksperimental. penelitian ini bertujuan untuk mengetahui ph pertumbuhan awal terbaik untuk leptolyngbya hs-16 dan hs-36. leptolyngbya hs-16 diisolasi dari sumber air panas di gunung pancar, sedangkan leptolyngbya hs-36 diisolasi dari sumber air panas di maribaya. derajat keasaman (ph) air dari sumber air panas di gunung pancar dan maribaya adalah 7. masing-masing strain ditumbuhkan pada medium blue green nomor 11 dengan variasi ph awal (6, 7, 8 dan 9) dan diinkubasi pada suhu o 35 c. parameter yang diteliti adalah berat basah biomassa leptolyngbya pada masing-masing strain. pengamatan dilakukan selama 15 hari dengan 11 sampling. hasil pengamatan 15 hari menunjukkan bahwa ph awal terbaik untuk pertumbuhan leptolyngbya hs-16 adalah 7, sedangkan leptolyngbya hs-36 adalah 9. berdasarkan penelitian dapat diketahui bahwa leptolyngbya hs-16 dan hs-36 dapat dibiakkan dengan kondisi alkalin. kata kunci: leptolyngbya, ph, sumber air panas microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-81297776638; fax: +62-21-7270012; email: nining@ui.ac.id environmental condition and their population increased twice of their first population. the population of microorganisms increased because they could used source of their new environmental. stationary phase occurred after exponential phase. stasioner phase is the phase that microorganisms could not do doubling cell. this phase occurred because the nutrients present in the growth medium are insufficient for doubling cell and accumulation of the metabolic waste of the microorganisms. death phase will occured after stationer phase (madigan et al. 2015). cyanobacteria can be found in soil, rocks and waters (bold et al. 1978). another source where cyanobacteria can be found is hot spring. maribaya hot spring and pancar mountain hot spring are the example of source whereas cyanobacteria can be found. maribaya hot spring has ph 6 to 7, while pancar mountain hot spring has ph 7. leptolyngbya is one genus of cyanobacteria that can be found in maribaya and pancar mountain hot spring (prihantini 2015). cyanobacteria are prokaryotic organisms belonging to the kingdom eubacteria (van den hoek 2002). these organisms has photosynthetic apparatus that plays role in producing energy (scholnick et al. 2006). cyanobacteria has photosynthetic pigment named phycobilin. these organisms stores glycogen as its food storage (markou et al. 2014). the growth phases of cyanobacteria are same with other microorganisms. the growth curve of population in microorganisms known as exponential growth. exponential growth in microorganisms consist of lag phase, exponential phase, stationary phase and death phase. lag phase occur when microorganisms has been innocculated into a new medium and adaptation with new environmental with different condition of their habitat. exponential phase happened when microorganism could adapted with their new leptolyngbya have filamentous as the form of the colony. characteristic of these organisms have a thin filament with 0.5 to 3.5 mm wide with simple trichome, and some species have sheath (komarek 2007). leptolyngbya can be found in environmental condition with ph 7 to 8.5 (olsson-francis et al. 2012) leptolyngbya have many benefit for our life. it could be seen leptolyngbya could produce lipid as biofuel feedstock, produce secondary metabolites as antibiotic, and could react as bioremediator in dairy waste (abazari et al 2012; beetul, 2014; khemka et al 2015). in order to beneficial of leptolyngbya, it is important to cultivated leptolyngbya. the acidity (ph) in the environment can affect the growth rate of cyanobacteria especially leptolyngbya. cyanobacteria can also live in environmental conditions with a wide range of ph, but some species are sensitive to acidic conditions (gerloff-elias et al, 2005). cyanobacteria could barely find in freshwater with range of ph 4 to 5 (bold et al. 1978). variation of initial ph in growth medium will affect the growth of cyanobacteria. the leptolyngbya hs-16 and hs-36's best initial growth ph have not known yet. the aim of this study is to determine the best initial growth ph for leptolyngbya hs-16 and hs-36 in blue green number 11 medium (bg-11). materials and methods microorganisms and growth medium. the microorganisms used in this study were cyanobacteria genus leptolyngbya strain hs-16 and hs-36. leptolyngbya hs-16 was isolated from pancar mountain hot spring, while leptolyngbya hs-36 was isolated from maribaya hot spring. those strains were grown in blue green number 11 medium/ bg-11 (nies 2007) with variations of ph value 6, 7, 8 and 9. the bg-11 medium were made as reported by prihantini (2015). cyanobacteria cultivation in bg-11 medium. the first step of cyanobacteria cultivation was inoculated 30 mg biomass of each strain into 100 ml growth medium in 250 ml erlenmeyer flask. before inoculation of cyanobacteria into medium, the medium had been adjusted the variation of ph value into 6, 7, 8 and 9. the treatment of variation ph value was repeated twice in each strain. those strain were o incubated at temperature 35 c. measurement the weight of wet biomass leptolyngbya hs-16 and hs-36. measurement of leptolyngbya hs-16 and hs-36 biomass were done in 15 days with 11 times of sampling. sixteen of sterile eppendorf tube 2 ml was measured at analytical measurement tool. biomass of those strain were taken aseptically with sterile micropipet amount of 2 ml. eppendorf tube with biomass of those strain inside were centrifuged with biofuge primo r machine in room temperature for 10 minute (in 10.000 rpm). the supernatant of those strain were taken out and wet biomass weight were measured with analytical measurement tool. the growth curves were made by comparrasion between wet biomass weight as the ordinate axis y with observed time as absisca x. the growth curves were made by microsoft excel. results the study of growth leptolyngbya hs-16 and hs36 had been done. it took 15 days with 11 times of sampling. the result produced growth curve of leptolyngbya hs-16 and hs-36. the growth curve showed the growth of both strains in the adaptation stage to the ph condition of the medium. it could be seen at the growth curve of leptolyngbya hs-16 and hs-36, which each strain produce the growth curve unstable. the age of inoculum that used in this study were 5 months old. macroscopic observation of leptolyngbya were observed. color appereance of leptolyngbya based on faber castle standard color. color appereance of leptolyngbya hs-16 was emerald green, while leptolyngbya hs-36 was brown ochre at day 0 (t0). at day-15 (t15), the color appereance of leptolyngbya hs-16 in ph 9 was changed from emerald green into apple green, while leptolyngbya hs-36 in ph 6, 7 & 8 were changed from brown ochre into apple green. the average of wet weight biomass of leptolyngbya hs-16 and hs-36 are shown on table 1. the growth curve of leptolyngbya hs-16 shown on figure 3, while the growth curve of leptolyngbya hs36 shown on figure 4. the growth curve of leptolyngbya hs-16 and hs-36 were made based on their wet weight of biomass. after 15th day of incubation, both leptolyngbya were able to grow on initial ph 6 medium, with wet -1 -1 weight 0.0295 g l for hs-16 and 0.02905 g l for hs36. the growth curve of those strain in initial ph 6 th th were slightly rise at 2 until 10 day, and drasticaly th th th increased at 13 until 14 day, but decreased at 15 day. on the other medium with initial ph 7, leptolyngbya hs-16 were able to grow and produced maximum amount of wet weight than other initial ph, but 70 rakhmayanti et al. microbiol indones volume 12, 2018 microbiol indones 71 fig 1 the color appereance of leptolyngbya hs-16 at day-15 (t15). fig 2 the color appereance of leptolyngbya hs-36 at day-15 (t15). fig 3 the growth curve of leptolyngbya hs-16. fig 4 the growth curve of leptolyngbya hs-36. leptolyngbya hs-36 produced minimum amount of wet weight than other initial ph. the wet weight of 1 leptolyngbya hs-36 was 0.0404 g l and -1 leptolyngbya hs-36 was 0.01995 g l . the growth curve of leptolyngbya hs-16 in initial ph 7 was th th th decreased at 2 until 4 day then increased at 3 until th th th 13 day and decreased at 14 until 15 day, while th leptolyngbya hs-36 was increased constantly at 1 th th until 14 day and decreased at 15 day. wet weight of leptolyngbya hs-16 and hs-36 in growth medium -1 -1 with initial ph 8 were 0.03725 g l and 0.05345 g l . both of those strain well adapted in medium with initial growth ph 8, especially leptolyngbya hs-36. th th leptolyngbya hs-16 was increased at 1 until 13 day th th then slightly decreased at 14 until 15 day, while th th leptolyngbya hs-36 was increased at 1 until 15 day. wet weight of leptolyngbya hs-16 and hs-36 in -1 growth medium with initial ph 9 were 0,02735 g l -1 and 0,05995 g l . leptolyngbya hs-36 produced maximum amount of wet weight than other initial ph. th th leptolyngbya hs-16 was decreased at 1 until 3 day th th th then increased at 6 until 13 day and decreased at 14 th until 15 day, while leptolyngbya hs-36 was th th th decreased at 1 until 3 day then increased at 6 day, but th th slightly decreased again at 7 until 8 day and increased th again until 15 day. discussion based on the result of this experiment, the color of leptolyngbya hs-16 changed on growth medium with initial ph 9, and leptolyngbya hs-36 changed on growth medium with initial ph 6, 7, and 8. it hapened because of their physiology adaptation mechanism in new environmental. the physiological adaptation caused the alteration phycobilin content (muster et al. 1983). as we can see on the growth curve, both strains still in lag phase on growth medium with initial ph 6, 7, 8, and 9. it proven that the curve still in unstable stage. lag phase sometimes could be the longest phase for some microorganisms because in this phase, microorganisms must adapt with new environmental conditions like new source of nutrient, ph and temperature (hogg 2005). both of these strains were able to grow in initial growth ph 6 medium, but not as good as in alkaline condition. leptolyngbya were often be found in neutral to alkaline condition (madigan et al. 2015). leptolyngbya hs-16 had the most average in growth medium with ph 7 than other ph. it could be leptolyngbya hs-16 was already adapted in growth medium with ph 7, because leptolyngbya hs-16 was isolated from pancar mountain, which has ph 7 (prihantini 2015). leptolyngbya hs-16 was well adapted in medium growth with ph 7, which coud be grouped into neutrophile organisms (madigan et al. 2015). leptolyngbya hs-36 were well adapted in growth medium with initial ph 8 and 9, especially leptolyngbya hs-36 had the most average in growth medium with initial ph 9. it happened because leptolyngbya were often found in environmental with ph 8 (olsson-francis et al. 2012). it could be also that 72 rakhmayanti et al. microbiol indones -1 table 1 the average of wet weight leptolyngbya hs-16 and hs-36 (g l ) wet weight of biomass (g l-1) t hs-16 hs-36 6 7 8 9 6 7 8 9 0 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 1 2 3 6 7 8 10 13 14 15 0.01375 0.01345 0.00445 0.01305 0.01535 0.0107 0.0132 0.02525 0.0348 0.0295 0.00685 0.01075 0.00655 0.0151 0.01555 0.0179 0.02695 0.0681 0.06415 0.0404 0.00755 0.00755 0.00835 0.01245 0.0126 0.01465 0.033 0.05635 0.0362 0.03725 0.00645 0.00705 0.0061 0.01515 0.0188 0.02485 0.0285 0.05005 0.031 0.02735 0.00615 0.0101 0.00555 0.0129 0.0077 0.00755 0.0086 0.02285 0.0325 0.02905 0.0069 0.00545 0.0058 0.01 0.0108 0.0127 0.01675 0.01765 0.02345 0.01995 0.0046 0.0052 0.0064 0.01075 0.01205 0.01525 0.02685 0.0284 0.04965 0.05345 0.00925 0.00745 0.00645 0.0166 0.01705 0.0163 0.03555 0.0548 0.0596 0.05995 leptolyngbya was alkalophile, which microorganisms that could live with ph 8 to 10 (madigan et al. 2015). th those strains were decreased at 15 day of observation. it could be happened because the nutrients in the growth medium had been reduced. the nutrients in the growth medium used those strain for metabolisms to their growth (madigan et al. 2015). based on the th observation of the 15 day and the discuccion that had been done, the best growth of leptolyngbya hs-16 was on growth medium with initial ph 7, while the best growth of leptolyngbya hs-36 was on growth medium with initial ph 9. acknowledgment this work was fully funded by hibah publikasi internasional terindeks untuk tugas akhir mahasiswa (pitta) 2017 to nining betawati prihantini, grant no. 667/un2.r3.1/hkp.05.00/2017. references abazari m, gholamreza z, iraj r. 2013. antimicrobial potentials of leptolyngbya sp. and its synergistic effects with antibiotics. jundishapur j microbiol. 6:1-6. bold, h.c., wynne m.j. 1985. introduction to the algae structure and reproduction. prentice-hall, inc., englewood cliffs, new jersey: xiv + 706 pp. beetul k, sadally sb, taleb-hossenkhan n, bhagooli r, puchooa d. 2014. an investiagtion of biodiesel production from microalgae found in mauritian waters. biofuel res j. 2:58-64. gerloff-elias a, elly s, thomas p. 2005. effect of external ph on the growth, photosynthesis and photosynthetic electron transport of chlamydomonas acidophila negoro, isolated from and extremely acidic lake. plant cell environ. 28:1218-1229. st hogg s. 2005. essential microbiology, 1 ed. john wiley and sons ltd, usa: 468 pages. khemka a, meenu s. 2015. phycoremediation of dairy wastewater coupled biomass production using leptolyngbya sp. j environ sci water res. 2: 104-111. komarek j. 2007. phenotype diversity of the cyanobacterial genus leptolyngbya in the maritime antarctic. polish polar res. 3:211-231. lee, r.e. 2008. phycology. cambridge university press, new york: ix + 534 pp. madigan m, martino j, bender k, buckley d, stahl d. 2015. brock biology of microorganisms, usa: pearson education. markou g, vandamme d, muylaert k. 2014. microalgal and cyanobacterial cultivation: the supply of nutrients. water res. 65:186—202. muster p, binder a, schneider k, bachofen r. 1983. influence of temperature and ph on the growth of the thermopilic cyanobacteria mastigocladus laminosus in continous culture. plant cell physiol. 24:273-280. nies-collection. 2007. list of strains: microalgae and protozoa 7th ed. nissei eblo co., ltd. tsukuba: v + 159 pp. olsson-francis k, simpson ae, wolff-boenisch, cockel cs. 2012. the effect of rock composition on cyanobacterial weathering of crystalline basalt and rhyolite. geobiology. 10:434-444. prihantini nb. 2015. polyphasic taxonomy of culturable cyanobacteria isolated from hot springs in west java, indonesia [dissertation]. depok (id): universitas indonesia. scholnick s, nir k. 2006. metal homeostasis in cyanobacteria and chloroplasts. balancing benefits and risks to the photosynthetic apparatus. plant physiol. 141:805-810. van den hoek c, mann dg, jahns hm. 2002. algae: an introduction to phycology, usa: cambridge university press. volume 12, 2018 microbiol indones 73 page 1 page 2 page 3 page 4 page 5 isolation and characterization of new antibiotics from indonesian coastal marine bacteria veronica , bibiana w. lay , stella magdalena1 1 1*and 1faculty of biotechnology, universitas katolik atma jaya indonesia jalan jenderal sudirman 51, jakarta 12930, indonesia antibiotics are organic compounds produced by various microorganisms and have the ability to inhibit the growth or kill other microorganisms. however, the irrational application of antibiotics lead to resistance of microorganisms so that they become ineffective. the objectives of this study were to isolate and characterize new antibiotics from indonesian coastal marine bacteria. in this study, a total of 141 isolates consisting of seven streptomyces sp. isolates and 134 isolates of other marine bacteria, were obtained from indonesian coastal regions. based on antimicrobial activity assay, four sp. and five marine bacteria isolates showed streptomyces antimicrobial activity towards and aureus with the diameter of inhibition of 3-12 bacillus cereus staphylococcus mm. further, antimicrobial compounds were produced successfully extracted with six organic solvents, such as 1-butanol, dichloromethane, n-hexane, chloroform, and toluene. the best solvent to extract antimicrobial compounds from sp. isolates was 1-butanol, while the best solvent to extract antimicrobial streptomyces compounds from other marine bacteria isolates could not be specified. antimicrobial compounds were successfully separated by thin layer chromatography with mobile phase used were 1-butanol, acetic acid, and water at a ratio of 4:1:2 and retention values obtained at 0.50 and 0.63. key words: antibiotics, marine bacteria, sp., extraction, thin layer chromatographystreptomyces antibiotik merupakan senyawa organik yang dihasilkan oleh berbagai mikroorganisme dan mempunyai kemampuan untuk menghambat pertumbuhan maupun membunuh mikroorganisme lain. namun, cara penggunaan antibiotik yang kurang tepat telah memicu terjadinya resistensi mikroorganisme, sehingga penggunaan antibiotik menjadi tidak efektif lagi. tujuan dari studi ini ialah mengisolasi dan mengkarakterisasi antibiotik baru dari bakteri laut daerah pantai indonesia. dalam studi ini, sebanyak 141 isolat yang terdiri dari tujuh isolat sp. dan 134 isolat bakteri laut lainnya, berhasil diisolasi dari daerah pantai indonesia. streptomyces berdasarkan uji aktivitas antimikroba, empat isolat sp. dan lima isolat bakteri laut lainnya streptomyces menunjukkan aktivitas antimikroba terhadap dan dengan diameter zona bacillus cereus staphylococcus aureus penghambatan sekitar 3-12 mm. selanjutnya, senyawa antimikroba yang dihasilkan berhasil diekstraksi dengan enam pelarut organik, yaitu 1-butanol, diklorometana, heksana, kloroform, dan toluena. pelarut terbaik untuk mengekstraksi senyawa antimikroba dari isolat sp. adalah 1-butanol, sedangkan pelarut terbaik streptomyces untuk mengekstraksi senyawa antimikroba dari isolat bakteri laut lainnya tidak spesifik. senyawa antimikroba tersebut berhasil dipisahkan dengan metode kromatografi lapis tipis dengan fase gerak yang digunakan adalah 1butanol, asam asetat, dan akuades dengan rasio 4:1:2 dan diperoleh nilai faktor retensi sebesar 0.50 dan 0.63. kata unci : k antibiotik, bakteri laut, sp., ekstraksi, kromatografi lapis tipisstreptomyces vol.8, no.3, september 2014, p 87-93 doi: 10.5454/mi.8.3.1 *corresponding author; phone: +62-812-1101925, fax: +6221-5719060, email: stella.magdalena@atmajaya.ac.id antibiotics are organic compounds produced by various microorganisms and have the ability to inhibit the growth or kill other microorganisms. however, the irrational application of antibiotics lead to resistance of microorganisms so that they become ineffective (sunaryanto 2009). in 2003, antibiotics used for et al. treatment of diseases were 84% of hospitalized patients in indonesia. meanwhile, in 2012 the results of surveillance showed decline of inappropriate use of antibiotics, however extended-spectrum β-lactamaseproducing escherichia coli and methicillin-resistant staphylococcus aureus increased. in addition, 81% of the hospitalized patients carried multiresistant , e. coli such as resistance to ampicillin (73%), trimethoprim (56%), chloramphenicol (43%), ciprofloxacin (22%), and gentamicin (18%) (hadi . 2013). this triggers et al research to renew the mechanism of existing antibiotics and to seek and obtain new antibiotics from new resources, such as coastal region. almost 70% of antibiotics originated from actinomycetes streptomyces, mostly from the genera . actinomycetes has the ability to synthesize secondary metabolites, such as enzymes and antibiotics. one of the countries with largest coastal region is indonesia mailto:stella.magdalena@atmajaya.ac.id 88 veronica et al . microbiol indones an archipelago which has sea covering up two-thirds of its entire region. moreover, sea has greater biodiversity than land so that the type of microorganisms obtained from the coastal region will also be more varied compared to soil microorganisms. however, the potential of marine and other marine actinomycetes bacteria producing antimicrobial compounds has not been explored (sunaryanto 2009; gokulkrishnan et al. et al. 2011). the objectives of this study were to isolate and characterize new antibiotics from indonesian coastal marine bacteria. materials and methods . samples were collected from samples collection 22 coastal regions in java, sumatera, kalimantan, and sulawesi. sea sediments and water samples (approximately 80-100 cm below sea level) were collected and stored at 4 °c. bacterial isolation. sediment samples were heated at 65 °c for 60 min to suppress gram-negative bacteria and to optimize the growth of marine streptomyces et al sp. (sunaryanto . 2009). one gram of dried sample were inoculated into 100 ml modified starch casein broth and incubated at 28 °c with 120 rpm agitation for 7 d. after 7 d incubation, samples were diluted to 10 and spread to modified -2 starch casein agar (sca) and marine agar (ma) (difco). both media were incubated at 28 °c for 7 d. colonies with different morphologies were picked and cultivated in sca for sp. and ma for streptomyces other marine bacteria (poorani . 2009).et al preliminary screening of antimicrobial activity. each isolate was inoculated into 10 ml brain heart infusion (bhi) (oxoid) and incubated at 28 °c with 120 rpm agitation for 6 days for streptomyces sp. and 3 days for other marine bacteria. after incubation, the medium were centrifuged at 13 684 ×g (thermo scientific) for 15 min. the supernatant was collected and used in the antimicrobial activity assay. test bacteria used for the antimicrobial activity assay were foodborne pathogens. the assay was done using agar well diffusion against bacillus cereus, staphylococcus aureus (atcc 25923), salmonella typhi pseudomonas aeruginosa,, and enteropathogenic all of them were obtained e. coli. from atma jaya culture collection. the clear zone around each well were interpreted as positive results of antimicrobial activity. pure extraction of antimicrobial compound. cultures that have antimicrobial activity were inoculated into 60 ml bhi and incubated at 28 °c with 120 rpm agitation for 6 d for sp. and 3 streptomyces days for other marine bacteria. after incubation, the medium were centrifuged at 5 857 ×g (thermo scientific) for 15 min. the supernatant was collected and transferred to a new tube. extraction of antimicrobial compound was done with method of augustine . (2005). the organic solvents used et al were 1-butanol, dichloromethane, chloroform, nhexane, and toluene (merck). the organic phase was collected, air-dried overnight, and dissolved in 1 ml phosphate buffered saline (pbs). the crude extract are to be used in the next step. secondary screening of antimicrobial activity. crude extract from previous step was used in the antimicrobial activity assay. the antimicrobial activity assay was done using agar well diffusion against bacillus cereus, staphylococcus aureus (atcc 25923), ,salmonella typhi pseudomonas aeruginosa, e. coli. and enteropathogenic positive results of antimicrobial activity were determined by measuring the sizes of inhibitory zone around the wells. about separation of antimicrobial compounds. 2 µl of crude extract was spotted on silica 60 f254 (merck). the plate was placed in tlc chamber which contains the mobile phase. several mobile phase combination were used, such as 1-butanol:acetic acid:water (4:1:2) (dharmaraj . 2010), et al ethanol:water:chloroform (4:4:2) (augustine et al. 2005), methanol:dichloromethane (1:10) (zheng . et al 2005a), and methanol:acetic acid:benzene (3:1:1) (selvendran and babu 2013). the plate was dried and visualized under ultraviolet (uv) light or sprayed with h so . the retention factor (rf) value for each spot 2 4 were measured using standard formula. r f value = distance travelled by solute distance travelled by solvent. results bacterial isolation. a total of 141 isolates with different morphologies from 22 coastal regions were obtained from sca and ma medium (table 1). from 141 isolates, only seven isolates were actinomycetes and all of them were isolated from sindhu sanur beach, bali. volume 8, 2014 microbiol indones 89 preliminary screening of antimicrobial activity. from 141 isolates, only nine isolates showed antimicrobial activity. there were four streptomyces sp. isolates and five isolates of other marine bacteria from sindhu sanur beach, bali. four streptomyces sp. isolates showed antimicrobial activity towards , , and , while b. cereus s. aureus e. coli five isolates of other marine bacteria showed antimicrobial activity only towards and b. cereus s. aureus (table 2). table 1 isolates obtained from 22 coastal regions in indonesia region sample code origin of location number of isolates java a1 carita beach 7 a2 pari island (near fish cages) 7 a3 pari island (near coral reef) 7 a4 indrayanti private beach 5 a5 indrayanti public beach 3 a6 alam indah beach 5 a7 pangandaran beach 4 a8 burung indah island 3 a9 kongsi island 3 a10 pasir perawan beach 6 a11 pasir perawan beach (near mangrove) 7 sumatra b1 tanjung pendam beach 9 b2 pasir padi beach 7 b3 mabay beach 5 b4 lengkuas beach 4 sulawesi c1 akarena beach 7 kalimantan d1 singkawang beach 6 bali-nusa tenggara f1 bali beach 9 f2 kuta beach 3 f3 sindhu sanur beach 23 f4 gili island 3 f5 tuban beach 8 table 2 antimicrobial activity of nine isolates from sindhu sanur beach isolate diameter of inhibition (mm) b. cereus s. aureus s. typhi p. aeruginosa e. coli epec f3.1 7.5 ± 0.7 6.5 ± 0.7 f3.5 5.5 ± 0.7 4.0 ± 0.0 f3.6 2.5 ± 0.7 f3.7 5.0 ± 1.4 3.0 ± 0.0 f3.8 5.0 ± 0.0 f3.s1 5.5 ± 0.7 15.5 ± 0.7 4.5 ± 0.7 f3.s2 3.5 ± 0.7 14.5 ± 0.7 f3.s3 3.5 ± 0.7 16.0 ± 0.0 4.0 ± 0.0 f3.s6 9.5 ± 0.7 results are mean ± standard deviation (n=2). table 3 antimicrobial activity towards after extractionb. cereus isolate diameter of inhibition (mm) cell -free supernatant extracted by 1-butanol dichlorometh ane n-hexane chloroform toluene f3.1 2.0 ± 0.0 4.5 ± 0.7 5.5 ± 0.7 5.0 ± 0.0 4.5 ± 0.7 5.0 ± 0.0 f3.5 4.5 ± 0.7 10.0 ± 0.0 9.0 ± 0.0 12.0 ± 0.0 8.5 ± 0.7 8.5 ± 0.7 f3.6 2.5 ± 0.7 5.5 ± 0.7 6.5 ± 0.7 6.0 ± 1.4 3.5 ± 2.1 6.0 ± 1.4 f3.7 3.0 ± 0.0 5.5 ± 0. 7 7.5 ± 0.7 8.5 ± 0.7 4.5 ± 0.7 5.5 ± 0.7 f3.8 7.0 ± 0.0 12.0 ± 0.0 8.0 ± 0.0 9.0 ± 0.0 10.0 ± 0.0 8.5 ± 0.7 results are mean ± standard deviation (n=2). microbiol indones secondary screening of antimicrobial activity. those antimicrobial compounds from nine isolates were extracted using various organic solvents and showed antimicrobial activity towards and b. cereus s. aureus (table 3, table 4). almost all isolates depicted greater inhibition zones after extraction using organic solvents. separation of antimicrobial compounds. antimicrobial compound from those isolates were separated by tlc with various mobile phase combination. the result showed different number and position of spots, and also different rf value (table 5). the result of tlc with 1-butanol:acetic acid:water (4:1:2) as mobile phase, were two spots with different rf values (table 5). f3.1 was chosen as a representative isolate producing two antimicrobial compounds. each spot from isolate f3.1 was dissolved by pbs and used for antimicrobial activity assay. b. the second spot with 0.63 rf value can inhibit cereus, while the first spot with 0.50 rf value cannot inhibit . diameter of zone inhibition was b. cereus 3 mm (fig 1). table 4 antimicrobial activity towards after extractions. aureus isolate diameter of inhibition (mm) cell -free supernatant extracted by 1-butanol dichloromethane n-hexane chloroform toluene f3.8 3.0 ± 0.0 4.0 ± 0.0 5.0 ± 0.0 6.0 ± 0.0 7.0 ± 0.0 6.0 ± 0.0 f3.s1 3.0 ± 0.0 9.0 ± 0.0 6.0 ± 0.0 5.5 ± 0.7 3.5 ± 0.7 f3.s2 2.0 ± 0.0 5.5 ± 0.7 4.0 ± 0.0 5.0 ± 0.0 5.0 ± 0.0 f3.s3 3.0 ± 0.0 5.5 ± 0.7 3.5 ± 0.7 2.5 ± 0.7 3.5 ± 0.7 3.0 ± 0.0 f3.s6 1.0 ± 0.0 3.0 ± 0.0 results are mean ± standard deviation (n=2); showed no inhibition zone. table 5 rf value from various mobile phase combination isolate rf value from various mobile phase combination a b c d f3.1 0.50 ; 0.63 0.81 0.06 0.71 f3.5 0.50 ; 0.63 0.88 0.06 0.71 f3.6 0.63 0.88 0.06 0.71 f3.7 0.50 0.88 0.06 0.71 f3.8 0.63 0.88 0.06 0.75 f3.s1 0.50 0.88 0.04 0.75 f3.s2 0.50 0.88 0.05 0.75 f3 .s3 0.50 0.88 0.06 0.75 f3.s6 0.50 0.88 0.08 0.73 (a) 1-butanol:acetic acid:water (4:1:2), (b) ethanol:water:chloroform (4:4:2), (c) methanol:dichloromethane (1:10), (d) methanol:acetic acid:benzene (3:1:1). f3.1 fig 1 antimicrobial activity by second spot with 0.63 rf value from isolate f3.1. bottom: first spot with 0.50 rf value; top: second spot with 0.63 rf value. 90 veronica et al . discussion the microorganisms living and growing in the ma ri ne e nvi ro nme nt a r e me ta boli ca lly a nd physiologically more diverse compared to terrestrial microorganisms. however, the potential of marine microorganism has not been explored, especially marine microorganisms in indonesian's marine e n v i r o n m e n t . t h e m o s t s t u d i e d m a r i n e microorganisms are , while other marine actinomycetes bacteria have not yet been extensively studied. from pre vious study, mar ine with ac tinom yce tes antimicrobial activity were successfully isolated from three beaches in indonesia, such as cirebon desa gebang beach, anyer beach, and kukup gunung kidul yogyakarta beach (sunaryanto . 2009). in et al this study, the objective was to isolate actinomycetes and other marine bacteria from different location in indonesia possessing antimicrobial activity. from 141 marine bacteria that were successfully isolated from 22 coastal regions in indonesia, only seven isolates were . previous study also actinomycetes obtained similar results (sunaryanto 2009). the et al. number of that were successfully actinomycetes isolated from the marine environment tends to be less than the number of that were isolated actinomycetes from soil and brackish water. it suggests soil structure, humidity, and ph in soil and brackish water are optimum condition for growth.actinomycetes furthermore, only nine from 141 isolates (6%) showed antimicrobial activity in this study. similar result were also obtained by a previous study reporting that only five from 77 isolates (6%), from sediment and sea water, have antimicrobial activity (zheng . et al 2005b). usually, bacteria are associated with marine invertebrates and seaweeds have greater activity than bacteria isolated from sediment and sea water. these bacteria could acquire the necessary nutrition from their hosts, while on the other hand, they could excrete products such as antibiotic and toxin to improve the chemical defense capability of the hosts. the result of antimicrobial activity assay showed that the isolates were active against gram-positive bacteria ( and ) compared to gram-b. cereus s. aureus negative bacteria. it is caused by the structural differences between these microorganisms. gramnegative bacteria having an outer polysaccharide membrane that makes the cell wall impermeable to lipophilic solutes, while gram-positive bacteria having only a peptidoglycan layer which is not an effective permeability barrier (khajure and rathod 2011). throughout secondary screening, all isolates showed different activities compared to preliminary screening. some isolates lost their activities, such as isolate f3.s1 and f3.s3 were only inhibited by s. aureus s. aureus, whereas previously they could inhibit b. cereus, e. coli and (table 4). according to khajure and rathod (2011), during screening of antimicrobial c omp ou nds , ma r in e ba c te r ia o fte n sh ow e d antimicrobial activity on agar but not in liquid medium. however, f3.8 showed improved activity depicting greater inhibition zone after extraction (table 3, table 4). when compared with marine actinomycetes isolates obtained by sunaryanto (2009), marine et al. actinomycetes in this study had a lower activity. their isolates could inhibit , e. coli, s. aureus, p. aeruginosa and . to get a better activity, isolates in bacillus subtilis this study should be grown in a richer medium and incubated for longer time. based on the results, the antimicrobial compounds can be extracted with 1-butanol, dichloromethane, nhexane, chloroform, and toluene. these results differ from previous study reporting that antimicrobial compounds can only be extracted with n-hexane and petroleum ether, and cannot be extracted using other solvents such as n-butanol, ethyl acetate, chloroform, benzene, and xylene (augustine . 2005). it showed et al that antimicrobial compounds should be extracted with different solvents depending on the suitability of antimicrobial compounds and solvents polarity. the best solvent to extract antimicrobial compounds from sp. isolates was 1-streptomyces butanol, however the best solvent to extract antimicrobial compounds from marine bacteria isolates cannot be specified. in this study, solvents with higher polarity yield better extraction of antimicrobial compounds. similar result was also obtained by previous study that used a range of solvents like nhexane, chloroform, ethyl acetate, benzene, n-butanol, and ethanol to extract the antimicrobial compounds from (rabah 2007). they reported actinomycetes et al. that n-hexane and chloroform were poor solvents, while n-butanol was a good solvent to extract the antimicrobial compounds. the reason is that n-butanol is more polar compared to n-hexane and chloroform. in this study, inhibition zones produced from crude extract is greater than inhibition zones from cell free supernatant. similar result were also obtained by previous study that inhibition zones after extraction with ethyl acetate is greater than before extraction (jafarzade . 2013). extraction of antimicrobial et al volume 8, 2014 microbiol indones 91 compounds will increase the purity and therefore will result in a better zone of inhibition since the impurities contained in the cell free supernatant could interfere in the antimicrobial activity. separation using tlc with various mobile phase combinations showed different number and position of spots, and also rf values. it depends on the suitability of antimicrobial compounds, stationary phase, and mobile phase polarity. the best mobile phase combination is 1-butanol, acetic acid, and water because it produced two spots that showed there are two compounds in one solution. the second spot with 0.63 rf value can inhibit while the first spot b. cereus, with 0.50 rf value cannot inhibit . it showed b. cereus that the second spot is an antimicrobial compound that has a high purity because it is still able to inhibit bacterial growth with low concentration (marked with a thin spot on tlc plate). however the first spot may not necessarily be an antimicrobial compound. the first spot may be an antimicrobial compound that loses its activity because both of these compounds are synergistic so that when they were separated it will reduce their activity. in addition, the first spot may have antimicrobial activity against other test bacteria (mangunwardoyo . 2009).et al other mobile phase combination is ethanol, water, and chloroform with ratio 4:4:2. polarity between antimicrobial compound and mobile phase is almost similar so that the compound was carried by the mobile phase. other mobile phase combination is methanol and dichloromethane with ratio 1:10. polarity between antimicrobial compound and mobile phase is not similar so that the compound is not carried by the mobile phase and not separated properly. these results differ from previous studies. it showed that the antimicrobial compounds and rf value were different (zheng . 2005a; selvendran and babu 2013).et al four sp. and five marine bacteria streptomyces isolates from sindhu sanur beach, bali showed antimicrobial activities towards and b. cereus s. aureus with the diameter of inhibition about 3-12 mm. all of the solvents can be used to extract the antimicrobial compounds depending on the suitability of antimicrobial compounds and solvents polarity. the best solvent to extract the antimicrobial compound from sp. isolates was 1-butanol, while the streptomyces best solvent to extract antimicrobial compound from marine bacteria isolates could not be specified. the best mobile phase combination on tlc for separating the antimicrobial compounds is 1-butanol, acetic acid, and water (4:1:2) with 0.50 and 0.63 rf values. acknowledgement this study was supported by the faculty of biotechnology, atma jaya catholic university of indonesia. we also would like to thank tresnawati purwadaria for her advice during this study. references augustine sk, bhavsar sp, kapadnis bp. 2005. a nonpolyene antifungal antibiotic from streptomyces albidoflavus pu 23. j biosci. 30(2):201-211. dharmaraj s, ashokkumar b, dhevendaran k. 20 . 10 isolation of marine and the evaluation of streptomyces its bioactive potential. afr j microbiol res 4 :240-. (4) 248. gokulkrishnan k, kusuma s, boopalan k. 2011. antimicrobial activity of marine bacteria isolated from the mangalore coast, west coast of india. rec res sci tech 3 :15-17.. (4) hadi u, kuntaman, qiptiyah m, paraton h. 2013. problem of antibiotic use and antimicrobial resistance in indonesia: are we really making progress? indones j trop infecti dis. 4(4):5-8. jafarzade m, yahya na, mohamad s, usup g, ahmad a. 2013. isolation and characterization of pigmented bacteria showing antimicrobial activity from malaysian marine environment. mal j microbiol. 9(2):152-160. khajure pv, rathod jl. 2011. antimicrobial and cytotoxic potential of the compound secreted by marine bacteria collected from the karwar coast, west coast of india. i j biotech. 16(1):60-65. mangunwardoyo w, cahyaningsih e, usia t. 2009. ekstraksi dan identifikasi senyawa antimikroba herba meniran ( l.). jurnal ilmu phyllanthus niruri kefarmasian indonesia. 7(2):57-63. poorani e, saseetharan mk, dhevagi p. 2009. lasparaginase production and molecular identification of marine sp. strain epd 27. ijib streptomyces . 7 :150-155.(3) rabah fl, elshafei a, saker m, cheikh b, hocine h. 2007. screening, isolation, and characterization of a novel antimicrobial producing actinomycete, strain raf10. biotechnology. 6(4):489-496. selvendran m, babu m. 2013. studies on novel bacteriocin like inhibitory substance (blis) from microalgal symbiotic spp mmb2 and its activity against vibrio . aquatic bacterial pathogens. japs 3 :169-175.. (2) doi:10.7324/japs.2013.30230. microbiol indones92 veronica et al . sunaryanto r, marwoto b, irawadi tt, mas'ud za, hartoto l. 2009. isolasi dan penapisan aktinomisetes laut penghasil antimikroba. ilmu kelautan 14 :98-. (2) 101. zheng l, chen h, han x, lin w, yan x. 2005 . a antimicrobial screening and active compound isolation from marine bacterium nj6-3-1 associated with the sponge . world j hymeniacidon perleve microbiol biotechnol 21:201-206.. doi:10.1007/ s11274-004-3318-6. zheng l, chen h, han x, lin w, yan x. 2005b. marine bacteria associated with marine macroorganisms: the potential antimicrobial resources. ann microbiol. 55(2):119-124. volume 8, 2014 microbiol indones 93 vol.1 , no. , 202 , p -6 2 december 2 24 30 doi: 10.5454/mi.1 . .6 2 24-30 the qpcr assay for detecting the presence and relative abundance of pseudomonas aeruginosa aada2and antibiotic resistance gene in hospital wastewater of national reference hospital rida tiffarent , rosdiana irawati , conny riana tjampaksari , 1* 2 3 fitriyah sjatha , windi muziasari , anis karuniawati 3 4 3 and 1 national research and innovation agency (brin), master programme in biomedical sciences, faculty of medicine, universitas indonesia, jakarta, dki jakarta, indonesia; 2 departement of waste water treatment plants of rscm. jakarta, dki jakarta, indonesia; 3 departement of microbiology, faculty of medicine, universitas indonesia, jakarta, dki jakarta, indonesia; 4 department of food and environmental sciences, division of microbiology and biotechnology, university of helsinki, viikinkaari 9, helsinki, 00014, finland; resistomap oy, helsinki, finland. antimicrobial resistance is one of the top 10 global health threats. the hospital wastewater (hww) potentially becomes the reservoir and dissemination of antibiotic resistance gene (arg) and bacterial pathogens. in indonesia, the protocol to monitor the args form hww has not been established. this study aimed to detect the presence and find the relative abundance of and gene from national reference hospitalp. aeruginosa aada2 (nrh) inlet and outlet wastewater through qpcr assay. the primers used were supported by resistomap. the study revealed that the qpcr assay was able to detect the ct value of and . the genep. aeruginosa aada2 aada2 was found in all waste water samples, meanwhile was only found in some of inlet samples.p. aeruginosa aada2 had the highest relative abundance and this gene's mobility uses plasmids and integrons that potentially enhance the acquired antimicrobial resistance (amr) mechanism. this study implicated that qpcr assay was capable to detect pathogenic bacteria and arg, and arg could be released to the environment even though the wastewater samples have been proceeded in wastewater treatment plants (wwtp). the qpcr assay can be used as the method to monitor the amr status in a hospital and the spreading potency to the environment using the hww. key words: antibiotic resistance genes, antimicrobial resistance, environment, extrinsic resistance mechanism, wastewater resistensi antimikroba adalah salah satu dari 10 ancaman terbesar untuk kesehatan global. air limbah rumah sakit (hww) berpotensi menjadi reservoir dan penyebaran gen resistensi antibiotik (arg) dan bakteri patogen. di indonesia, protokol untuk memantau arg dari hww belum ditetapkan. penelitian ini bertujuan untuk mendeteksi keberadaan dan nilai gen dan dari air limbah inlet dan outletrelative abundance p. aeruginosa aada2 rumah sakit rujukan nasional (nrh) melalui uji qpcr. primer yang digunakan dalam studi didapatkan dari resistomap. studi ini mengungkapkan bahwa uji qpcr mampu mendeteksi nilai ct dan .p. aeruginosa aada2 gen ditemukan pada semua sampel air limbah, sedangkan hanya ditemukan pada beberapaaada2 p. aeruginosa sampel inlet. memiliki nilai tertinggi dan mobilitas gen ini menggunakan plasmid danaada2 relative abundance integron yang dapat berpotensi meningkatkan mekanisme resistensi antimikroba (amr) dapatan. penelitian ini mengimplikasikan bahwa uji qpcr mampu mendeteksi bakteri patogen dan arg, serta kemungkinan dilepaskannya arg ke lingkungan meskipun sampel air limbah telah diproses di instalasi pengolahan air limbah (ipal) sebelumnya. uji qpcr dapat digunakan sebagai metode untuk memantau status amr di rumah sakit dan potensi penyebarannya ke lingkungan menggunakan hww. kata kunci: air limbah, gen resisten antibiotik, lingkungan, mekanisme resisten ekstrinsik, resistensi antimikroba microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 * c o r r e s p o n d i n g a u t h o r : p h o n e ;: + 6 2 e m a i l :rida003@brin.go.id 2019). antibiotic resistance in bacteria can occur because these bacteria have or overexpress the genes that encode the resistance characteristics. mechanisms of antibiotic resistance can occur through both intrinsic and acquired mechanisms. the mechanism of intrinsic resistance is related to the genetics of bacteria that are normally present in these bacteria. the mechanism of acquired resistance is related to the horizontal transfer of antibiotic resistance genes so that resistance in 2019, the world health organization (who) declared antimicrobial resistance as one of the top 10 global health threats (holmes 2016; who worldet al. health organization 2021). overuse of antimicrobials is a major driver of resistance of infectious pathogens (cdc centers for disease control and prevention characteristic from one bacteria can be transferred to another (holmes 2016; peterson and kaur 2018).et al. pseudomonas aeruginosa is a rod shaped gramnegative bacteria with a genome length of 5.5 – 7 mbps. it is known as an opportunistic pathogen associated with nosocomial infections and ventilatorassociated pneumonia (pachori 2019; panget al. et al. 2019). is one of the eskape groupp. aeruginosa (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumanii, pseudomonas aeruginosa enterobacter, and species) which is a group of pathogenic bacteria that responsible for infections in hospitals with their ability to escape from antibiotics (helen w boucher, 2020). the results of the latest surveillance conducted by the european center for disease prevention and control (ecdc) showed that nearly 31% of p. aeruginosa isolates were resistant to at least one group of tested antibiotics (european centre for disease prevention and control. 2018). the multidrug resistance (mdr) p. aeruginosa arises concern because of the nature of the bacteria which is easy to adapt to various environmental conditions and can spread widely (helen w bouche, 2020). detection canp. aeruginosa be conducted by conventional bacterial culture methods, immunological assays, and molecular assays (tang 2017). in this study, we conducted theet al. detection of through molecular assay, thep. aeruginosa qpcr, targeting the complete genome at certain region of as referred to stedtfeld et al.'s studyp. aeruginosa (2018). wastewater treatment plants (wwtp) are the important reservoir of antimicrobial resistance issue (pärnänen 2019). study from oliveiraet al. et al.(2018) reported the finding of 11% (from total 27 isolates) resistant isolated from wwtpp. aeruginosa rio para located in the city of divinópolis, southern brazil. data from brazil showed a high profile of antibiotic resistance in isolated fromp. aeruginosa hospital wastewater (hww) treatment (hwwtp), the discovery of mdr were 85.4% in passop. aeruginosa fundo, 82% in rio de janeiro, and 60% in manaus. the p. aeruginosa isolates (15 isolates) from clinical specimens in national reference hospital dr. cipto mangunkusumo (rscm) from april to november 2015 were resistant to ceftazidime, ciprofloxacin, amikacin, and carbapenems thorough in vitro vitek 2 compact et al.test (prasetyo 2022). amikacin is one of the aminoglycosides antibiotic group and this group is also one of drug of choice for treatment p. aeruginosa infection (clsi clinical and laboratory standards institute, 2020). however, there have been no studies reporting and analyzing the profiles of pathogenic bacteria, resistance genes, and antibiotic residues contained in hospitals wastewater especially national reference hospital (nrh). this research aimed to detect and find the relative abundance of and antibiotic resistancep. aeruginosa gene (arg) , for the antimicrobial resistanceaada2 monitoring study in hospital wastewater (hww). materials and methods the research design is a descriptive study to detect certain gene of bacteria and arg in hww and approved by rscm ethic committee no. 21-09-0905. hospital wastewater (hww) samples collection. samples were collected from oct 25th – nov 27th, 2021 from nrh's wastewater area inlet and outlet every 3 days. total samples were 24 (12 samples each inlet and outlet). the inlet and outlet wastewater area were collected each 1 liter and placed in alcohol 70%-disinfected bottle. the wastewater samples were transported to laboratory and filtrated immediately using polyethersulfone (pes) 0.22 µm membrane with 47 mm diameter. after the filtration, filter membranes were stored in -20 °c freezer until the dna extraction step performed. the volume of filtered hww samples were 50 ml for inlet and 100 ml for outlet. the reset of hww samples were stored in -30 °c. dna extraction. dna extraction from filter membrane was using kit dneasy powerwater kit™ (qiagen) and following manufacture instructions. at the final step, the pellet from extraction was homogenized using elution buffer and measured for dna concentration and purity using nanodrop with 260/280 nm wavelength. qpcr assay. qpcr quantification was using sensifast™ sybr ® no-rox kit (bioline) with 10 μl total volume each reaction. one reaction consists of: 5 μl 2x master mix, 0.4 μm forward primer, 0.4 μm reverse primer and 1 μl dna (0.2 ng μl ) as -1 template. the primers were used in this study can be seen in table 1. the primer pair targeting the p. aeruginosa was designed to detect the species-specific complete genome of p. aeruginosa from ncbi reference sequence, nc_002516.2 region 14103321410412, “agcgttcgtcctgcacaagttc gacggcctgtcccaggtcgaagtggccgagc gcatgggaatctccctgagcatggtgga”. real-time cycling conditions included 2 min enzyme activation at 95 °c followed by 40 cycles of volume 1 , 2026 2 microbiol indones 25 denaturation at 95 °c for 5 sec and annealing 60 °c for 10 sec and elongation at 72°c for 10 sec. each sample was tested duplo in qpcr assay. relative abundance. the relative abundance was obtained from normalize each gene with 16s rrna gene using livak formulation relative abundance = 2 −δct (δct = ct of gene – ct of 16s rrna gene). results waste water samples collection and dna extraction. the hww treatment used in nrh was activated sludge with extended aeration system. the wastewater samples were collected from two spots as can be seen in hww treatment diagram in figure 1. the dna concentrations from extraction process were 31.00-162.30 ng µl and 1.90-25.20 ng µl from inlet -1 -1 and outlet hww samples, respectively (table 2). qpcr assay and relative abundance. based on qpcr assay (table 3) the was detected atp. aeruginosa certain date in inlet wastewater samples and not detected at all in outlet wastewater samples. the aada2 gene was detected in all inlet and outlet samples with cycle threshold (ct) values 17.650 to 35.285. the relative abundance value can be seen in table 4. gene forward primer reverse primer p. aeruginosa pao1, complete genome region 14103321410412 agcgttcgtcctgcacaagt tccaccatgctcagggagat aada2 caatgacattcttgcgggtatc gacctaccaaggcaacgctatg 16s rrna gggttgcgctcgttgc atggytgtcgtcagctcgtg 1 sampling dna concentration (ng µl-1) purity inlet 25-oct 81.90 1.91 28-oct 96.80 1.95 31-oct 75.90 2.01 3-nov 142.90 1.98 6-nov 31.00 1.99 9-nov 47.00 1.95 12-nov 162.30 1.93 15-nov 123.90 1.96 18-nov 57.30 1.97 21-nov 46.80 1.90 24-nov 150.30 1.93 27-nov 57.20 1.95 outlet 25-oct 25.20 1.83 28-oct 4.80 1.22 31-oct 4.40 1.88 3-nov 1.90 1.87 6-nov 4.20 1.66 9-nov 3.20 1.76 12-nov 4.00 1.75 15-nov 3.00 1.55 18-nov 7.60 1.68 21-nov 2.00 1.38 24-nov 2.70 2.00 27-nov 3.41 2.41 1 table dna concentration from hww samples2 table 1 sp. prevalence in 2017-2021staphylococcus 26 tiffarent et al. microbiol indones generally has higher relative abundance thanaada2 p. aeruginosa. the relative abundance was visualized in heat map (table 5). the darker colour shows higher value of relative abundance. discussion the gene was found constantly inaada2 wastewater samples inlet and outlet, meanwhile the p. table 3 cycle threshold (ct) values and standard deviation (stdev) of and in qpcrp. aeruginosa aada2 assay sampling p. aeruginosa aada2 16s rrna ct value stdev ct ct value stdev ct ct value stdev ct inlet 25-oct 36.245 0.460 19.885 0.163 17.095 0.021 28-oct 35.625 2.029 19.265 0.035 17.015 0.078 31-oct 22.170 0.339 17.400 0.057 3-nov 21.675 0.502 17.690 0.209 6-nov 22.740 0.495 18.085 0.007 9-nov 22.050 0.071 17.585 0.262 12-nov 17.650 0.368 17.380 0.028 15-nov 34.945 0.615 21.080 0.042 17.295 0.095 18-nov 17.740 0.523 17.505 0.191 21-nov 36.160 0.948 17.650 0.028 17.240 0.338 24-nov 36.335 0.021 20.925 0.163 17.205 0.021 27-nov 33.983 0.508 21.350 0.269 17.508 0.365 outlet 25-oct 22.305 0.304 19.755 0.187 28-oct 25.140 0.014 22.425 0.064 31-oct 24.335 0.163 21.725 0.148 3-nov 25.570 0.085 25.395 0.007 6-nov 32.640 0.170 25.628 0.321 9-nov 27.588 0.484 23.615 0.247 12-nov 35.285 1.549 25.640 0.354 15-nov 32.485 0.021 25.425 0.007 18-nov 23.330 0.099 21.105 0.064 21-nov 27.495 0.361 27.880 0.071 24-nov 24.330 0.085 21.035 0.078 27-nov 25.115 0.035 21.515 0.332 1 fig 1 the diagram of nrh hww treatment, red asterisk signs were the sampling spots. volume 1 , 2026 2 microbiol indones 27 sampling p. aeruginosa aada2 inlet 25-oct 0.000002 0.144586 28-oct 0.000002 0.210224 31-oct 0.036651 3-nov 0.063153 6-nov 0.039692 9-nov 0.045279 12-nov 0.82932 15-nov 0.000005 0.072544 18-nov 0.849685 21-nov 0.000002 0.752623 24-nov 0.000002 0.075887 27-nov 0.000011 0.06971 outlet 25-oct 0.170755 28-oct 0.152301 31-oct 0.163799 3-nov 0.885768 6-nov 0.007745 9-nov 0.063703 12-nov 0.001249 15-nov 0.007494 18-nov 0.213899 21-nov 1.30586 24-nov 0.101884 27-nov 0.082469 1 table 4 relative abundance and genep. aeruginosa aada2 table 5 . heat map relative abundance and genep. aeruginosa aada2 sampling p. aeruginosa aada2 inlet 25-oct 28-oct 31-oct 3-nov 6-nov 9-nov 12-nov 15-nov 18-nov 21-nov 24-nov 27-nov outlet 25-oct 28-oct 31-oct 3-nov 6-nov 9-nov 12-nov 15-nov 18-nov 21-nov 24-nov 27-nov value:1 0.000002 1.30586 2 28 tiffarent et al. microbiol indones aeruginosa was found only in inlet samples. this indicated that the system of wastewater treatment has not completely eliminated the arg. the arg in outlet has the potency to contaminate the environment when it was released. the args in studies that have been reported found in hww were aada25, dfra16, dfra5, macb, mexf, mexw, smee, blaveb, aada2, blages, blavim, aac.6…30/aac.6…ib., baer, cpxa, crp, dfra1, emra, qach, tet36, ugd et al.(majlander 2021; cai 2021). this should be a concern because theet al. mobility characteristic of gene. according toaada2 the comprehensive antibiotic resistance database (card) (alcock 2020), is anet al. aada2 aminoglycoside nucleotidyltransferase gene encoded by plasmids and integrons in ,k. pneumoniae salmonella corynebacterium glutamicum c.spp., , freundii aeromonas, and spp. this gene expresses enzyme aminoglycosides 3'-adenyltransferase and causes the resistance to streptomycin and spectinomycin. the mobility of this gene uses plasmids and integrons that means this gene can be transferred intraspecies or interspecies (horizontally) in acquired resistance mechanism. is also one ofp. aeruginosa bacteria that was reported carrying the gene.aada2 the isolates from patients in 3 generalp. aeruginosa hospitals in tehran, iran were found carrying aada2 gene with prevalence 47.6% (salimizadeh 2018).et al. this study shows that the qpcr assay is capable to detect the presence of potential pathogen bacteria and arg in hww. the method can be used as the monitoring of antimicrobial resistance (amr) status in hospital and the prevention and controlling of amr spreading to environment. further study can be developed to optimize the controlling of amr such as the pathogenic bacteria isolation from hww and then the bacteria will be assessed phenotypically (such as antibiotic susceptibility) and genotypically (pcr followed by sequencing) so the args can be confirmed to be expressed in bacteria and can be traced back to see the potency of horizontal transfer. in conclusion, the qpcr assay can be a method for monitoring pathogen bacteria and args to describe the amr status in hospital using wastewater samples. acknowledgments we thank the resistomap for funding and supplying some of the laboratory materials and primers in this study. references alcock bp, raphenya ar, lau tty, tsang kk, bouchard m, edalatmand a, huynh w, nguyen alv, cheng aa, liu s. 2020. card 2020: antibiotic resistome surveillance with the comprehensive antibiotic r e s 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perspective. rsc adv. 7:5178951800. doi: 10.1039/c7ra09064a. who world health organization, 2021. 10 global health issues to track in 2021. 30 tiffarent et al. microbiol indones 05 kustyawati.cdr vol.11, no.3, september 2017, p 103-109 doi: 10.5454/mi.11.3.5 the dynamic growth and chemical change of mixed cultures inoculation on tapioka fermentation maria erna kustyawati*, sri setyani, azhari rangga, and irfa rista mutia department of post harvest technology, faculty of agriculture, universitas lampung, jalan s. brojonegoro 1, bandar lampung, indonesia. saccharomyces cerevisiae and lactobacillus plantarum possess several extracellular and intracellular enzymes beneficial for cassava fermentation. tapioka (cassava starch) has limited uses in food industries due to its low pasting properties, therefore, biomodification by the use of fermentation is needed. the research was aimed to monitor the growth of s. cerevisiae and l. plantarum during tapioca fermentation, and to evaluate the chemical change of the fermented tapioka. mixed cultures was inoculated at the designed concentration into tapioca o suspension and incubated at room temperature (30±2 c) in facultative aerobic condition for 0, 24, 48, 60, 72, 96, 120, and 144 h. the growth change of s. cerevisiae and l. plantarum was monitored, and the change of ph, residual sugar, and starch granule was investigated. the result showed that s. cerevisiae had longer lag phase as well as stationary than l. plantarum did; nevertheless, they both reached log phase at the same time. coinoculated mixed cultures did not affect the change on ph and reducing sugar but increased pronouncely protein content at stationary period. besides, there was sign of erosion to the structure of cassava starch granules which was an indication of changes in the pasting property of the cassava starch. key words: chemical change, mixed culture co-inoculation, saccharomyces cerevisiae, starch granule saccharomyces cerevisiae dan lactobacillus plantarum memiliki berbagai enzim ekstraseluler dan intraseluler yang sangat mungkin memberikan manfaat pada modifikasi tapioka. tapioka memiliki kegunaan terbatas pada industri makanan karena sifat pasting yang rendah, oleh karena itu, biomodifikasi dengan menggunakan fermentasi sangat dibutuhkan dalam upaya meningkatkan karakteristik tapioka. kultur campuran pada konsentrasi tertentu diinokulasikan ke dalam suspensi tapioka dan diinkubasi selama 0, 24, 48, 72, 96, dan o 120 jam pada suhu kamar (30±2 c) pada kondisi fakultatif aerobik. pertumbuhan s. cerevisiae dan l. plantarum dimonitor, dan perubahan ph, gula residu dan granula tapioka diamati. hasil pengamatan menunjukkan bahwa s. cerevisiae mempunyai fase lag dan fase stasioner yang lebih lama dibanding l. plantarum. namun s. cerevisiae dan l. plantarum mencapai fase log pada waktu yang sama. inokulasi kultur campuran s. cerevisiae dan l. plantarum tidak mengakibatkan perubahan terhadap nilai ph dan gula reduksi, tetapi meningkatkan protein secara nyata pada fase stasioner. disamping itu, terdapat erosi pada struktur granula tapioka yang mengindikasikasikan adanya perubahan sifat pasta tapioka. kata kunci: inokulasi, saccharomyces cerevisiaefermentasi tapioka, kultur campuran, perubahan kimia, microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone/fax: +62-721-783821, email: maria.erna@fp.unila.ac.id have potential use in the food products as they contribute to the desired flavor (schwan et al. 2007). the role of amylolytic yeast in producing yeast biomass from starch, and producing foods with low carbohydrates have much to do, such as production of amylase in fermentation of sticky rice, and cassava tape (fleet 2001). yeast great potential and still very necessary, especially in food diversification through a fermentation process to produce a new type of food or modification of existing products with better nutritional value, as well as aroma and texture adapted to the people's will. tapioca (cassava starch) has limited uses in the food industries due to its low in pasting properties. researchers have focused on fermenting cassava with additional nutrients for improving the quality of cassava flour (uboh and akindahu 2005; subagio saccharomyces cerevisiae has a very important role as a starter in the fermentation of various foods and beverages known as brewer's yeast, distillers yeast, and baker's yeast (kurtzman and fell 1998). in indonesia, the use of yeast to produce traditional foods and fermented foods has not been so entrenched in comparison to fungi such as mucor spp., rhizopus spp., penicillium spp., and aspergillus spp., or the use of lactic acid bacteria lactobacillus casei, lactobacillus lactis, acetobacter xylinum, acetobacter aceti, due to lack of knowledge in the utilization as a starter or as an agent in the fermentation process. yeast has amylolytic properties in starch degradation that is capable for producing amylase. amylolytic yeast may 2006). however, a challenged method to improve the properties of tapioca has been attracting most scientists. one of the techniques was modification of physical, chemical, and pasting characteristic of tapioca by fermentation with the use of starter culture. abriba (2012) investigated the microbial succession during the controlled fermentation of cassava tubers and isolated 8 potensial microorganisms, these were b a c i l l u s s u b t i l i s , l a c t o b a c i l l u s p l a n t a r u m , c o r y n e b a c t e r i u m m a n i h o t i , l e u c o n o s t o c mesenteroides, enterobacter aerogenes, aspergillus niger, geotrichum sp., and saccharomyces cerevisiae. nevertheless, l. plantarum and l.pentosus were the dominat bacteria found in the beverage cauim produced from cassava and rice fermentation (oguntoyinbo et al. 2010). fermenting cassava with addition of with mixed cultures lactobacilus plantarus, saccharomyces cerevisiae, and rhizopus oryzae produced the cassava flour having protein increased and reduced starct content (gunawan et al. 2015). the addition of saccharomyces cerevisiae during the fermentation of tapioca enriched the mineral and protein of the strach, as well as reduced the starch gelatinization temperature (kustyawati et al. 2013). it was chalenged that inoculation of s. cerevisiae and l. plantarum can be used as starter cultures in submerged fermentation of tapioca slurry, since the mold such as rhizopus sp. was most suitable to the solid fermentation. the objective of the work was to study the growth of saccharomyces cerevisiae and l. plantarum during tapioca fermentation, to investigate the chemical change, and their effect on the pasting properties of the starch. materials and methods materials. pure culture of saccharomyces cerevisiae and lactobacillus plantarum were purchased from the culture collection of gadjah mada university, broth malt extract broth, malt extract agar (difco™, becton and dickinson company, sparks, usa), saline (0.85 % nacl), oxytetracycline and chloramphenicol, and reagents for chemical analysis were obtained from sigma chemicals company (st.louis, mo. white cassava tubers (manihot utilisima var kasetsart) were obtained from the institute for agricultural research and technology (bptp) bandar lampung. culture preparation. single colonies from pure cultures of each species were inoculated separately into 5 ml of me broth and mrs borth medium (difco™, becton and dickinson company, sparks, usa) and then incubated at 25 °c and 32 °c for 24 h for s. cerevisiae and l. plantarum, respectively. after this period, tubes were centrifuged, the pellets washed with sterile saline solution (0.9% nacl, wt/vol) centrifuged and re-suspended again in sterile saline solution to -1 obtain a concentration of about 7 log 10 cfu ml . growth medium preparation. fermentation experiments were carried out in submerged fermentation method of an extracted cassava. briefly, 200 ml of extracted cassava slurry was placed into a 250 ml flask. the sugar concentration (10% glucose) was adjusted in distilled water and heated at 100 °c for 15 min to prevent sugar caramelization then added to the flask. the flask was independently inoculated with 50 µl of the yeast saline suspension to reach an initial concentration of inoculum of about 10.50±0.21 log10 -1 cfu ml for s. cerevisiae, and 10.36±0.32 log10 cfu -1 ml for l. plantarum. flasks were incubated without shaken. another flask was served as control without inoculation. the flasks were covered by cotton to create an microaerophilic condition then fermented at o room temperature (30±2 c) for 0, 24, 48, 72, 96, 120, 144, and 168 h. the filtrate was taken for analysis. chemical analysis. chemical analysis included ph, protein content, and reducing sugar. the ph of filtrate obtained from the fermentation was determined using kent ph meter (kent industry measurement limited, surrey, england) model 7020 equipped with a glass electrode. protein content expressed by soluble n was analyzed by the method of kjedahl (aoac 2009). estimation of reducing sugar was done by nelsonsomogyi method (gusakov et al. 2011). sugar that have the characteristic of being reducing sugars, as they contain functional groups capable of being oxidised and therefore causing reduction of other species under specific conditions. structurally, reducing sugars must contain a free aldehyde or an alpha-hydroxy ketone capable of being oxidised. microbiological analysis. were taken samples from the fermentations daily. one ml of sample was -4 -6 taken from the flask and serially diluted to 10 to 10 with sterile distilled water into the test tubes. following homogenizing the sample with the vortex, one ml of diluted sample was taken and spread plated onto petridishes with designated media that is malt extract agar (mea) for growth of s. cerevisiae, and deman rogosa sharpe agar (mrs) is for l. plantarum. chloramphenicol 0.5% and oxytetracyclin 0,5% were added to inhibit the bacteria. the plates were incubated o aerobically at 29±2 c for 24-48 h for s. cerevisiae and 104 kustyawati et al. microbiol indones volume 11, 2017 microbiol indones 105 o 37 c for 48 h for l. plantarum. counts were expressed -1 as cfu ml . effect of mixed culture inoculation on starch granule and pasting properties. structure change of starch granules was analized microscopically followed the method by mcmaster (1994). briefly, fresh sample of starch slurry (0,5% v/v) was taken and diluted into sterile aquades. after homogenizing an appropriate amount of diluted sample was dropped on to the haemocytometer that has connection to the computer. selested granules were then photographed. the pasting properties of native tapioca and modified tapioca (72 h fermentation) were observed and compared using the brabender micro visco-amylograph versoin 2.4.9 to evaluate gelatinization properties of starch during the process of cooking. results microbial growth. four phases were detected in this experiment, they were adaptation phase (lag phase), growth phase (exponential phase), static phase (stationary phase), and mortality phase (death phase); even thogh, the growth phase of l. plantarum was not clear noted. the lag phase of s. cerevisiae was longer than l. plantarum; however, both of the cultures obtained the log phase at the incubation time of 72 h. the death phase of both l. plantarum and s. cerevisiae started at 120 h (fig 1a). ph change. ph is one of the most important factors for maximizing growth of microorganisms. coinoculation of s. cereviseae did not affect the ph of the substrate. in the medium where either mixed cultures or s. cerevisiae alone was inoculated into, the ph decreased to 4 – 4.3 (fig 1b). however, it decreased much lower than that of the other inoculated cultures when l. plantarum was co inoculated. of all the measurements, the ph of the substrate started to decrease at 48 h fermentation, and stayed at the same value untill the fermentation was ending when it was co inoculated with s. cerevisiae. reducing sugar. the ability of the mixed cultures to break down sugars is very little, of which it was also occurred to the control one (fig 1c). either control or mixed cultures started metabolizing sugar at the first 24 h, which was showed by sharply decreased the reduced sugar. however, the profile of reduced sugar was not variably changed at the following hours except at the first 72 h. protein content. the protein content experienced to gradual augment with the fermentation time and that of more pronounced at 96 h of fermentation, thought as a stationary period. the contol had 1.32% and fermented one contained 1.82% at 96 h fermentation (fig 1d). the change of granule. granules of native starch hillum and lamellae of granules were noted in the native tapioca starch as seen in fig 2a. they has hillum steadily stayed in the center of the granule and has bire fringence. granules has birefringence characteristic under analyzes of polarization microcopic, and reflects the black and white color. the colors are as an indication of amilose structures in the starch. where there was a notice of irregulated shape and cleaned appearance in the native strach, it could be due to the collition between granules during processing which could result in the breakage of granule structure. on the other hand, co-inoculated starch granules at 72 h incubation had irregulated shape (fig 2b), lossed their birefringence characteristics, its hillum as well as lamellae was ruptured, and there was an indication of errosion on the periphere structur of granules. discussion microbial growth. submerged fermentation of co-culturing s. cerevisiae and l. plantarum was applied in this study. the growth pattern of s. cereviseae and l. plantarum on cassava fermentation was shown in fig 1. four phases were detected, adaptation phase (lag phase), growth phase (exponential phase), static phase (stationary phase), and mortality phase (death phase). the lag phase of s. cerevisiae was longer than l. plantarum; however, both of the cultures obtained the log phase at the incubation time of 72 h. the environment condition such as the acidity of the substrate where the cultures were inoculated may comfort for the initial grow of l. plantarum. the long of lag phase experienced by s. cerevisiae indicated that the medium was unfavored. it was either lack of nutrition or excess of nutrient so that the cell needed the time to produce enzim suited to hydrolize the nutrients (mahreni and suhenry 2011). this may explain that l. plantarum had short time of the lag phase. at the stationary phase, wheather, l. plantarum or s. cerevisiae produced their metabolites products which stayed up to 120 h of incubation. this study was in agreement with arroyo-lopez et al. (2009) where yeasts had a very short lag period under the conditions included in the medium containing 50% glucose+50% fructose, ranged from 0.30 to 16.7 h. in correlation with the ph of the medium, it showed that the growth of s. 106 kustyawati et al. microbiol indones fig 1 (a) saccharomyces cerevisiae and lactobacillus plantarum changes in the population of during fermentation. (b) effect of mixed culture co-inoculation on ph during fermentation. (c) effect of mixed culture co-inoculation on reducing sugar during fermentation. (d) effect of co-inoculated mixed culture on the protein content during fermentation. a b d c cerevisiae may favor with low of ph 3-4 (fig 1b). wahono et al. (2003) found that optimum ph for s. cerevisiae was 4-4.5. ph affects the rate fermentation of s. cerevisiae of which the optimal value was 4-4.5 (frazier and westhoff 1978). the change of ph. it could be that the production of acid causing the decrease of ph was more carried out by l. plantarum than s. cerevisiae. the results agree with the other findings that the optimum ph levels for addition of s. cerevisae were from 3.5 to 6.0 (manikandan and viruthagri 2010; polyorach et al. 2013). in correlation with the growth of bacteria (fig 1a), it showed that the ph of the medium lowered as the increase of growth of l. plantarum. the metabolism products of the l. plantarum may greatly affect the ph of the fermentation media. reducing sugar. this research showed that at the longer fermentation, reducing sugar increased. it was assumed that extracellular amylase activities was getting higher as the fermentation was longer resulting in more starch was hidrolysed which increased the reducing sugar content. this was in agreement with the research done by kartikasari et al. (2016). protein change. the protein content of the fermentation medium innoculated with mixed culture (1.82 %) was higher than that of the control one (1.32%). it was likely that yeast biomass contributed to the protein since there was no nitrogen source added to the fermentation medium. the microbial biomass product which was formed during the fermentation contributed to the protein increase since the cultures remained in the fermentation substrate. in addition, the production of microbial enzymes during the fermentation may also contribute to the increase of protein. this finding was egreement to the study done by yuangklang and ) where crude wachirapakorn (2011 protein of cassava pulp fermented by s. cerevisiae th increased at the 5 day of fermentation, as a result of the production of single cell protein during the fermentation process. the change of starch granules. the analysis of starch granules was carried out to find out wheather the addition of mixed culture s. cerevisiae and l. plantarum was able to improve the pasting properties of cassava starch. fig. 1c showed that the starch granules had lossed their bierefringence which were indicated by the ruptured of the granule lamellae and undetected hillus. the extracellular amylolytic enzyme produced by the cultures hidrolyzed the liberated starch granules especially in the granule surface and resulted in the formation of hole like looked at the granule surface that contributed to the possible liberation of starch from the granules. when the starch granules were degraded and the starch was released, the pasting properties of the strach such as starch gelatinization, viscosity, and other rheological properties of the starch could have been changed. the granules that losses their birefringence characteristic may have changes in their pasting properties due to the change in the amylose structure. the reasons beyond volume 11, 2017 microbiol indones 107 fig 2 effect of co-inoculated mixed cultures on the starch granules. microscopic study at 100 magnification. 1. signed of errosion, 2. ruptured granules, 3. hillus, 4. lamellae. table 1 amylograph properties of native tapioca and modified tapioca fermented for 72 h samples native tapioca modified tapioca start gelatinization temperature (°c) 71.2±0.15 71.8±0.18 maximum viscosisy (bu) 1089±0.25 1150±0.15 breakdown (bu) 761±0.2 624±0.15 setback (bu) 305±0.32 588±0.15 this process could have been the enzymatic activity of cultures that hydrolyzed carbon backbone chain of the oligosaccharide in the starch. this study was agree with done by kustyawati et al. (2016)previous study that an errotion occured in the starch granules fermented with s. cerevisiae only. the growth of lactic acid bacteria during cassava fermentation produces enzym hidrolyzing starch material resulting in the changes of its functional properties. the characteristic differences of such as granular shape, ratio amylose/amylopectin, molecular starch and the existence of other components influenced the ability of starch to form the final product characteristics which are desired (copelan et al. 2009). pasting viscosity is an important characteristic of starch during heating of water-starch suspension as this was the basic thought when the starch was applied to food industries. in this experiment (table 1), the increase in temprerature gelatinization of modified tapioca may be due to the changed of granule structure and the more complex compounds containing in the starch. the increase in protein and reducing sugar, and low ph may lead to more energy needed for gel formation. breakdown is an indication of how easier the the rupture or breakdown the granule structure is (varavinit et al. 2003). high breakdown value leads the strach to bear cohesiveness characteristic which less use in food industries. the modified tapioca had the low breakdown value meaning more aplicable to food industries. another pasting property correlated to viscosity is setback. setback value indicates the occuring of retrogradation or sineresis of the starch. the modified tapioca in this experiment had high setback value which meant it was easily undergo retrogradation. this may be influenced by high protein content in the starch. the presence of protein, lipid, ash, fiber, and as well amylose contribute to the retrogradation (eliasson 2004). this finding was in agreemnet with et al. (2004) where starch lindeboom with high protein and amylose will retrogade more because amylose entraps more water and undergoes recrystalization. in conclusion, s. cerevisiae and l. plantarum were growth quite well during tapioca fermentation, and resulted in the chemical characteristic changes of modified tapioca. the increase in protein content and reducing sugar, as well as the occuring of granula erosion in the starch contributed to the improving of pasting properties of modified tapioca. s. cerevisiae and l. plantarum was important for improvement of tapioca pasting properties which was useful in food industries. acknowledgment this work was supported by a grant provided by the institute of research and public services (lppm) university of lampung, indonesia, through the grant application (hibah terapan) 2016 with the project number of 418/un/8/lppm/2016. references abriba c, henshaw ee, lenox j, eja m., ikpoh s, bassey e, agbor be. 2012. microbial succession and odour reduction during the controlled fermentation of cassava tubers for the production of 'foofoo', a staple food consumed popularly in nigeria. j microbiol biotech res. 2 (4):500-506. arroyo-lópeza, n., sandi orlić s, querol a, barrio e. 2009. effects of temperature, ph and sugar concentration on the growth parameters of saccharomyces cerevisiae, s. kudriavzevii and their interspecific hybrid. int j food microbiol. 131:120–127. doi: 10.1016/j.ijfoodmicro. 2009.01.035 aoac (official methods of analyses of the association of official analytical chemists). 2009. seventeenth ed. vol. 2. association of official analytical chemists, gaithersburg. pp. 915-922. copeland l, blazek j, salman h, tang mc. 2009. form and functionality of starch. food hydrocoll. 23: 1527-1534. eliasson c and ann. 2004. starch in food (structure, fuction and applications). woodhead publishing limited, cambridge england. fleet gh. 2001. wine. in: doyle mp, beuchat lr, montville tj. (eds.), food microbiol fundamentals and frontiers. asm press, washington, dc, pp. 747–772. frazier wc and westhoff dc. 1978. food microbiology, tata mc graw hill book pub!. co. ltd., new delhi. gunawan s, widjaja t, zullaikah s, ernawati l, istianah n, aparamarta hw, prasetyoko d. 2015. effect of fermenting cassava with lactobacillus plantarum, saccharomyces cereviseae, and rhizopus oryzae on the chemical composition of their flour. int food res j. 22(3): 1280-1287. kartikasari sn, sari p, subagio a. 2016. characterization of chemical properties, amylograpic profiles (rva) and granular morphology (sem) of biologically modified cassava starch. j agroteknologi. 10(1):12-18. kurniawati li, gunawan s, aida n, widjaya t. 2012. pembuatan mocaf dengan fermentasi menggunakan lactobacillus plantarum, saccharomyces cerevisiae, dan rhizopus oryzae [making mocaf by fermentation using 108 kustyawati et al. microbiol indones lactobacillus plantarum, saccharomyces cerevisiae, dan rhizopus oryzae]. j tek pomits. 1(1): 1-6. kustyawati me, hayati, t. 2013. effect of fermentation by saccharomyces cerevisiae on the biochemical characteristics of tapioca starch. agritech, issn 0216-0455 33(3):281-288. kustyawati me, setyani s, rangga a. 2016. the role of saccharomyces cerevisiae as modification agents on the cassava starch. conference proceeding in uisfs, 117124, august 23-24 bandar lampung, indonesia. kurtzman c, fell j. 1998. the yeasts—a taxonomic study, 4th ed. elsevier, amsterdam. mahreni and suhenry s. 2011. growth kinetics of saccharomyces cerevisiae in the extract medium banana peels. seminar rekayasa kimia dan pangan. issn: 1411-4216, july 26. manikandan k. and viruthagiri t. 2010. optimization of c/n ratio of the medium and fermentation conditions of ethanol production from tapioca starch using co-culture of aspergillus niger and saccharomyces cereviseae. int j chem technol res. 2: 947-955. oboh g. and akindahunsi aa. 2005. nutritional and toxicological evaluation of saccharomyces cereviseae fermented cassava flour. j food compost anal. 18: 731738. oboh g and elusiyan ca. 2007. changes in the nutrient and antinutrient content of micro-fungi fermented cassava flour produced from lowand medium cyanide variety of volume 11, 2017 microbiol indones 109 cassava tuber. afr j biotechnol. 6 (18):2150-2157. oguntoyinbo fa, dodd cer. 2010. bacterial dynamics during the spontaneous fermentation of cassava dough in gari production. food control. 21:306-312. polyorach s, wanapat m, wanapat s. 2013. enrichment of protein content in cassava (manihot esculenta crantz) by supplementing with yeast for use as animal feed. emirates j agricul food chem. 25: 142-149. subagio a. 2006. ubi kayu substitusi berbagai tepungtepungan [cassava as substituton of various starch]. food review. 1 (3): 18-22. schwan rf, almeida eg, souza-dias ma, jespersen l. 2007. yeast diversity in rice-cassava fermentations produced by the indigenous tapirapé people of brazil. fems yeast res. 7:966-972. vavarinit s, shobsngob s,varanyanond w, chinachoti p. naivikul o. 2003. effect of amylase content on gelatinisation, retrogradation and pasting properties of flour from different cultivars of thai rice. starch-starke. 55 (9): 410-415. wahono sk, damayanti e, rosyida vt. 2011. growth rate of saccharomyces cerevisiae on the fermentation process for producing alcohol from sorghum bicolor l. seminar rekayasa kimia dan proses. issn: 1411-4216. yuangklang c, wachirapakorn c, paengkoum p. 2011. protein enrichment of cassava pulp fermentation by s. cerevisiae. j anim vet adv. 10(18):2434-2440. doi: 10.3923/java. 2011.2434.2440. page 1 page 2 page 3 page 4 page 5 page 6 page 7 05 hartajanie.cdr vol.12, no.2, june 2018, p 65-68 doi: 10.5454/mi.12.2.5 lactobacillus fermentum llb3 improves antioxidant activity of bitter melon (momordica charantia) juice laksmi hartajanie*, lindayani, angela novita, emilia triviana sutanto, and agata apriliana sundoro unika soegijapranata, jalan pawiyatan luhur iv/1, semarang 50234, indonesia momordica charantia (bitter melon) contains substances with antidiabetic properties such as charantin, vicine, and polypeptide-p, as well as other unspecific bioactive components such as antioxidants. it is suitable for functional drink and need further studies to elaborate its functional properties. lactobacillus fermentum llb3 isolated from bamboo shoot pickle was used to ferment bitter melon juice. the aim of this study was to evaluate changes in antioxidant activity of bitter melon juice during fermentation. study has been carried out by fermenting bitter melon juice with l. fermentum llb3. the free radical scavenging activity of the phenolics were done using 2,2-diphenyl1-picrylhydrazyl (dpph). antioxidant activity of bitter melon juice increased during 24 hours fermentation. in addition, the sugar content and ph decreased compared with the baseline value. the fermentation of bitter melon juice by l. fermentum llb3 increased its antioxidant activity. these result suggest that fermented bitter melon juice is a promising agent for diabetes management. key words: antioxidant activity, bamboo shoot pickle, bitter melon, diabetes management, lactobacillus fermentum llb3 momordica charantia (pare) mengandung zat dengan khasiat antidiabetes seperti charantin, vicine, dan polypeptide-p, serta komponen bioaktif spesifik lainnya seperti antioksidan, sehingga cocok untuk diolah menjadi minuman fungsional. lactobacillus fermentum llb3 yang diisolasi dari acar rebung digunakan untuk memfermentasi jus pare. tujuan dari penelitian ini adalah untuk mengevaluasi perubahan aktivitas antioksidan jus pare selama fermentasi. penelitian dilakukan dengan memfermentasi jus pare dengan l. fermentum llb3. aktivitas antioksidan diukur menggunakan 2,2-difenil-1-pikrilidrazil (dpph). aktivitas antioksidan jus pare meningkat selama 24 jam fermentasi. selain itu, kadar gula dan ph menurun dibandingkan dengan nilai awal. fermentasi jus pare oleh l. fermentum llb3 dapat meningkatkan aktivitas antioksidannya. hasil ini menunjukkan bahwa jus pare fermentasi merupakan produk yang menjanjikan untuk manajemen diabetes. kata kunci: acar rebung, aktivitas antioksidan, lactobacillus fermentum llb3, manajemen diabetes, pare microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-24-8441555, email: laksmi@unika.ac.id active compounds and for its functional properties (majekodunmi et al. 1990; matsuda et al. 1998; begum et al. 1997). the fruit extract had the highest value of antioxidant activity and that gallic acid was the predominant phenolic compound in the fruit extract (kubola and siriamornpun 2008). the folk medicinal properties associated with bitter gourd includes treatment for various chronic and degenerative diseases, including, diabetes mellitus (nerurkar et al. 2006; aboa et al. 2008; nerurkar et al. 2010) coronary heart disease and cancer (anilakumar et al. 2015). lactic acid fermentation is considered as one of the most suitable tool to exploit the biogenic/functional potential of plant matrices and to enrich them with bioactive compounds (pellati et al. 2004). the fermentation by selected lactic acid bacteria was largely used to enhance the antimicrobial, antioxidant and immune-modulatory features of several cereal, pseudocereal and leguminous flours (coda et al. 2012) as well as of medicinal plants like echinacea spp. (rizello et al. foods such as fruits, vegetables and grains are reported to contain a wide variety of antioxidant c o m p o n e n t s , i n c l u d i n g p h y t o c h e m i c a l s . phytochemicals, such as phenolic compounds, are considered beneficial for human health, decreasing the risk of degenerative diseases by reduction of oxidative stress and inhibition of macromolecular oxidation (karovicova and kohajdova 2003). bitter melon (momordica charantia l.) is an important vegetable grown in tropical and sub-tropical regions (aboa et al. 2008; wu and ng 2008). the fruits are eaten while still green and unripe. they have been used for generations by indigenous populations in africa, china, india, japan, and latin america for food and folk medicine (khan and anderson 2003; aboa et al. 2008; matsuda et al. 1998). bitter melon has been the subject of intensive investigations for biologically 2013). lactic acid fermentation can help to improve the safety, shelf life, and nutritional and sensory properties of vegetables. strains of several lactobacillus species have been proven to exert a range of health-promoting activities such as immunomodulation, enhancement of resistance against pathogens and reduction of blood cholesterol levels and are used as probiotics (karovicova and kohajdova 2003). in this study, bitter melon juice was fermented using lactobacillus fermentum bll3 over a period of 72 h and investigated the changes in antioxidant activity, sugar content, and ph. to the aims of this research is evaluate changes in antioxidant activity of bitter melon juice during fermentation. materials and methods microbial strains. probiotic lactic acid bacteria (lactobacillus fermentum llb3) was isolated from pickled bamboo shoots based on the previous study of hartayanie et al. (2016). bacterial culture was stored frozen at-20 ºc in mrs medium (merck, germany) containing 20% glycerol. the strains were reactivated by means of double passage on mrs when needed. bitter melon juice preparation. immature bitter melon (momordica charantia) was obtained from pasar bandungan, ambarawa. the fruits were washed with deionized water, drained at ambient temperature and cut in halves, lengthwise. the seeds were removed and flesh was extracted using a commercial food processor (philips) under room temperature. the bitter melon juice was placed into bottle and then pasteurized for 5 min at 80 ºc. the fermentation of bitter melon juice. lactobacillus fermentum llb3 was grown in the mrs broth incubated at 37 ºc for 24 h and reached an od 600 6 -1 of 1.0, which is equivalent to 10 cfu ml . twenty mililiter of the cultivated mrs broth was added aseptically into 180 ml of pasteurized bitter melon juice. the fermentation process took place in a incubator (memmert, germany) at 37 ºc for 72 h. the fermented bitter melon juice was harvested at 24, 48, and 72 h and compared with those of their unfermented control (un-inoculated) counterparts denoted as 'fermentation time 0'. dpph free radical scavenging activity. the dpph assay was conducted according to (brandwilliams et al. 1995) with some modifications. stock -1 solution of 0.6 mol l dpph (sigma-aldrich) in methanol (merck) was prepared and kept at −20 ºc until used. fresh working solution was prepared for each assay by mixing 10 ml of stock solution with 45 ml of methanol to obtain an absorbance of 1.1 ± 0.01 units at 515 nm. each appropriately diluted bitter melon extract and standard solution (100 μl) was mixed and incubated with 3900 μl of working solution for 30 min in dark. all of the samples and blank solution (3,9 ml dpph + 0,1 ml methanol) were measured at 515 nm wavelength by using spectrophotometer. the percentage of free radical scavenging activity was calculated as follows: chemical analysis. the ph of the samples were measured by using digital ph meter (ec-phtestr30kit). the total soluble solid analysis for determining the sugar concentration of the samples were determined by using brix refractometer (hanna instruments sucrose refractometer). statistical analysis. the experiment was done in triplicate for each substance. the results were expressed as percentage decrease with respect to control values and compared by one-way anova and duncan test. a difference was considered statistically significant if p<0.05.performed in accordance to sni 01-2891-1992. results the percentage of dpph scavenging activity in bitter melon juice (0 h) was 72.46% and increased to 82.44% at 24 h. afterwards, it remained constant (table 1). the fermentation process is characterized by a decrease in sugar content and ph of the foods due to the production of organic acids. the ph decreased from 5.55 to 4.8 and the sugar content also decreased from 2.7% to 2.3% (table 2). discussion dpph assay has been widely used to determine the free radical scavenging activity of various plant extracts (gil et al. 2000; pellati et al. 2004; rizello et al. 2013). antioxidant activity was measured by dpph-linked free radical scavenging activity assay at 0, 24, 48, and 72 h of fermentation time. antioxidant molecules in the sample scavenge the free radical dpph and the color from the dpph assay solution becomes light yellow resulting in a decrease of the absorbance at 517 nm. l. fermentum llb3 can 66 hartajanie et al. microbiol indones scavenging effect (%) = [1 a517 nm sample a517 nm blank ] x 100% volume 12, 2018 microbiol indones 67 increased antioxidant activity of bitter melon juice. lactic acid bacteria are used as starter culture in many fruit and vegetable fermentations due to their ability to produce β-glucosidase enzyme. this enzyme is an important catalyst in the liberation of aromatic compounds from glucoside precursors present in fruits and their fermentation products (michlmayr and kneifel 2013; denkova et al. 2013). as shown in table 1, the dpph scavenging effect of bitter melon juice increased significantly after 24 h fermentation. the fermentation process is characterized by a decrease in sugar content and ph of the foods due to the production of organic acids. l. fermentum llb3 was able to utilise bitter melon juice for lactic acid production without nutrient supplementation. the reducing sugar concentration rapidly decreased from -1 2.7 to 2.5 g l within 24 h of fermentation period. between 24 and 72 h of fermentation, the rate of sugar utilization slowed down and its concentration -1 decreased slightly to 2.3 g l . between 24 and 72 hr fermentation, slightly changes in sugar concentration was observed (table 2). the bitter melon fermented juice is safe for diabetic consumption because no sugar adding in fermentation process. l. fermentum llb3, used in this study was found capable of rapidly utilising sugar in vegetable juice medium for lactic acid production. rapid decrease in ph was also observed during the first 24 hr of fermentation period where ph decreased from 5.55 to 4.61 (table 2). sligthly ph changes were observed from 24 to 72 h fermentation period. the decrease in ph was concomitant to the increase in viable cell count during the first 24 h of fermentation. a rapid decrease of ph during the early stage of fermentation is an important indicator of end product quality. the increase in acidity during lactic acid fermentation can minimizes the activities of spoilage bacteria and contributes to the pleasant taste and desirable aroma (breidt et al. 2013). from this study, it can be concluded that the fermentation of bitter melon juice by l. fermentum b3 increased its antioxidant activity. this result suggest that fermented bitter melon juice is a promising agent for diabetes management. acknowledgements this work was supported by a grant from directorate general of indonesian higher education. this research a part of the third year of penelitian unggulan perguruan tinggi (pupt) 2017 entitled “efek probiotik dan mikrostatik dari bakteri asam laktat yang berperan dalam fermentasi acar rebung”, grant number 011/k6/km/sp2h/penelitian/2017. table 1 change in scavenging effect during fermentation of bitter melon juice time (hour) scavenging effect (%) 0 b 80.47 ± 3.74 b 82.48 ± 3.28 b 82.44 ± 1.73 72 48 24 a 72.46 ± 2.86 * values ​​with different superscripts showed significant differences between treatments (p <0.05) based on the one-way anova test. the differences among different treatments were determined by duncan's multiple range test at the 0.05 probability level. table 2 change in ph and sugar content during fermentation of bitter melon juice bitter gourd juice o brix 0 h fermentation a 2.36 + 0.08 a 2.37 + 0.05 b 2.55 + 0.10 72 h fermentation 48 h fermentation 24 h fermentation c 2.72 + 0.08 * values ​​with different superscripts showed significant differences between treatments (p <0.05) based on the one-way anova test. the differences among different treatments were determined by duncan's multiple range test at the 0.05 probability level. ph c 5.55 + 0.01 b 4.81 + 0.04 b 4.81 + 0.02 a 4.61 + 0.04 68 hartajanie et al. microbiol indones references aboa k, fred-jaiyesimi a, jaiyesimi a. 2008. ethnobotanical studies of medicinal plants used in the management of diabetes mellitus in south western nigeria. j ethnopharm. 115:67-71. anilakumar kr, kumar gp, ilaiyaraja n. 2015. nutritional, pharmacological and medicinal properties of momordica charantia. international journal of nutrition and food sciences 4(1):75-83. doi: 10.11648/j.ijnfs.20150401.21 begum s, ahmed m, siddiqui bs, khan a, saify zs, arif m. 1997. triterpenes, a sterol, and a monocyclic alcohol from momordica charantia. phytochem. 44(7):13131320. brand-williams w, cuvelier me, berset c. 1995, “use of a free radical method to evaluate antioxidant activity”. lwt-food sci technol. vol. 28, pp. 2530. breidt f, mcfeeters rf, 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ng l. 2008. antioxidant and free radical scavenging activities of wild bitter melon (momordica charantia linn. var. abbreviata ser.) in taiwan. lwt-food sci. technol. 41:323–330. page 1 page 2 page 3 page 4 uus saepuloh.pmd volume 3, number 2, august 2009 p 91-95 issn 1978-3477 *corresponding author, phone: +62-251-8347519, fax: +62-251-8310033, e-mail: uussaepuloh@yahoo.com expression of simian retrovirus type d serotype 2 envelope in insect cell using baculovirus expression vector system uus saepuloh1, diah iskandriati1, mohamad sadikin2, and joko pamungkas1,3 1primate research center, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; 2department biochemistry and molecular biology, faculty of medicine, universitas indonesia; jalan salemba raya 6, jakarta 10430, indonesia; 3faculty of veterinary medicine, institut pertanian bogor, darmaga campus, bogor 16680, indonesia simian retrovirus type-d (srv) is a causative agent of simian acquired immunodeficiency syndrome in asian macaques, and can serve as a viral model in understanding of retrovirus infection because of some similarities to human aids pathogenesis. study of infection and pathogenesis of srv in macaques could be a strategy of vaccine and antiviral development for preventive and therapeutic purposes. we expressed the srv-2 envelope gene using baculovirus expression vector system and transfected it to spodoptera frugiferda insect cell line for srv-2 recombinant protein production. analysis using pcr and sequencing technique of recombinant in the passage-3 viral stock indicated the occurrence of recombination between srv-2 envelope and baculovirus genome. purification using immobilized metal ion affinity chromatography ni2+-nta to recombinant protein could minimize the presence non-specific proteins. the sds-page analysis showed a specific protein for srv-2 gp70 envelope. western blot analysis of this purified protein indicated a specific reaction with anti-srv-2 antibody positive of macaca fascicularis serum shown as srv-2 gp70 envelope band. keywords: srv-2, baculovirus, sf9 cell, macaca fascicularis the simian retrovirus (srv) is a betaretrovirus capable of causing an aids-like disease in asian macaques. of the five simian retrovirus neutralization serotypes identified (srv-1 to srv-5), three (srv-1 to srv-3) have been molecularly cloned and genomically sequenced (daniel et al. 1984; marx et al. 1984; gardner et al. 1988). disease caused by the more commonly found srv-2 infection in macaque is characterized by diarrhea, chronic weight loss, anemia and sometimes retroperitoneal fibromatotis (moazed and thouless 1993). srvs have emerged as significant pathogens in captive macaques following recognition of their etiologic role in outbreaks of immunodeficiency disease at several regional primate research centers in the us in early 1980 (daniel et al. 1984; gardner et al. 1988). serological studies in macaca fascicularis, macaca nemestrina and pongo pygmeus in indonesia showed the presence of antibodies to srv-2, leading to the assumption that these animals have been infected with srv or similar agent (iskandriati et al. 1998a; iskandriati et al. 1998b; warren et al. 1998). the high prevalence of disease among wild populations pose some problems for breeders providing a population of srv-free macaques, since macaques are frequently used in biomedical researches (lerche and osborn 2003). regarding potentially active infection and immune abnormality affected by this virus, srv-2 is a pathogenic agent that should be eliminated in the macaca breeding colony (marx et al. 1984; lerche et al.1995; morton et al. 2008). in this study, we developed srv-2 envelope (env) recombinant protein production method using baculovirus expression vector system (bevs). this recombinant protein could be used as antigen sources for serological test against antibody anti-srv-2 or as srv-2 recombinant protein vaccine candidate. recombinant baculoviruses are widely used to express heterologous genes in insect cells. the bevs has some advantages, such as the capacity for large inserts of dna and high yield of recombinant protein. protein produced in bevs is very similar to naturally occurring human proteins in terms of post-translational modifications, biological activity and protein stability (possee 1997; joshi et al. 2000). for this reason, bevs is widely used in academia and industry for expression of a variety of recombinant protein in insect cells (kost and condreay 1999). materials and methods determination of entry clone srv-2 env. the srv-2 provirus isolated from peripheral blood mononuclear cells (pbmcs) of indonesian m. fascicularis (provided by pssp lppm ipb) was pcr amplified using specific primers srv-2 bacnterm 5823u to srv-2 ltr90l that produced the blunt end pcr product. this pcr product was cloned to pentr/ d/topo vector (invitrogen), which facilitated the entry into baculovirus genome. the plasmid pentr/d/topo was then analyzed using sequencing methods with specific primers (srv mf-mn 5737-ltr: 216u20, 727u20, 1284u20 and 1770u20). spodoptera frugiferda (sf9) insect cell preparation. sf9 cells were seeded into a six-well plate (8x105 cells/well) in 2 ml of complete grace’s insect medium (gibco) supplemented with 10% fbs (fetal bovine serum), 100u ml-1 penicillin, and 100 µg ml-1 streptomycin. the cells were incubated at 27°c for one hour to allow the cells fully attach to the bottom of the plate and then were verified by inspecting them under an inverted microscope. lr recombination. the lr recombination reaction was prepared by adding pentr/d/topo plasmid as entry clone (100-300 ng), baculodirect linear dna (300 ng), 5x lr clonase reaction buffer, te buffer and lr clonase enzyme mix. the mixture was incubated at 25 °c for one hour, then added with microbiol indones92 saepuloh et al. 2 ml of protease k solution and incubated for 10 min at 37 °c. lr recombination reaction and cellfectin (invitrogen) reagent were combined in 800 ml grace’s insect medium unsupplemented to make the transfection mix. the medium in the cells was removed and added to the entire tranfection mix dropwise onto the cells, then incubated at 27 °c for 5 h. the transfection mixture was later on removed and added with 2 ml of complete growth medium with antibiotics and 100 um ganciclovir to each well. the plate was incubated at 27 °c for 96 h and visual inspection of the cells was daily conducted to observe signs of the infections using inverted microscope. once the transfected cells demonstrated signs of infection, the medium was collected from each well and transferred to a sterile snap cap tube. this was used as the p1 viral stock, kept at 4 °c and protected from light. the recombination reaction was performed according to standard procedure by invitrogen. high-titer viral stock preparation. the sf9 cells at density 8x105 cells per well was seeded in 2 ml of complete growth medium with 100 mm ganciclovir in a six-well tissue culture plate. the cells were incubated at 27 °c for 72 h, then the medium was collected and centrifuged at 1000 x g (beckman gs-6r) for 5 minutes. the supernatant is p2 viral stock and kept at 4 °c. to generate the high titer viral stock p3, the sf9 cells was seeded at density 8x105 cells ml-1 in 20 ml growth medium in t75 flask. cells was incubated in 27 °c for 48 h then infected with 0.5 ml p2 viral stock and incubation was continued for 72 h. the supernatant was collected as p3 viral stock. pcr and sequencing analysis of p3 viral stock dna recombinant. dna was isolated both of supernatant and cells p3 viral stock using qiaamp dna minikit procedure (qiagen). dna recombination between baculodirect n-term and srv-2 env was amplified using polyhedrin forward primer and srv-2 env 5974l and 6243l. pcr product was purified using qiagen gel extraction kit (qiagen, usa) and cloned into pcr 2.1 topo 10 (invitrogen, usa). nucleotide sequence was carried out on automatic dna sequencing (abi, usa) and alignment of the obtained sequences was performed with computer software blast program (ncbi). recombinant protein purification. sf9 cells were grown in t225 flask to a density of 8 x 105 cells ml-1 and infected with p3 viral stock. media were harvested at 72 h post-infection and centrifuged at 528 x g for 10 min at 4 °c (beckman gs-6r). the collected supernatant were concentrated by tangential flow filtration with pellicon xl device (millipore) then centrifuged at 191 000 x g for 3 h at 4 °c (beckman xl90 optima, fixed angel ti90). the pellet were diluted in 1 ml pbs and loaded onto a 2 ml probond chelating column (invitrogen) charged with nicel-nitriloacetic acid (ni2+-nta) and equilibrated with native binding buffer. after washing the column with 8 ml native washing buffer supplemented with 20 mm imidazole, recombinant protein were eluted with elution buffer containing 250 mm imidazole. fractions were collected in 2 ml and the protein concentrations were analyzed by bicincroninic assay kit (pierce). the ni2+-nta purification procedure was referred to probond purification system from invitrogen. sds-page and western blot analysis. the purified srv-2 env recombinant protein was detected by electrophoresis on a ready gel 4-15% gradient tris-hcl sdspage (biorad) and then either stained in a gel with coomassie blue or transferred onto a nitrocellulose membrane. the membrane was rinsed with pbst 0.1%, blocked with blotto (5% skim milk in pbst 0.1%) for 2 h at room temperature and cut into strips. individual strips were incubated with m. fascicularis plasma/serum containing primary antibody anti-srv-2 diluted in blotto for overnight. after being washed three times with pbst 0.1%, the membrane was incubated with anti-human igg alkaline phosphatase conjugate (1:5000 dilution in blotto). the membrane was washed and the bands were developed by reaction with 5bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (bcip-nbt) as substrate. result the srv-2 env provirus was isolated from pbmcs of indonesian m. fascicularis using specific primer srv-2 bacnterm 5823u (upper primer) and srv-2 ltr90l (lower primer) with pfu polymerase enzyme, resulting in blunt pcr product about 2000 bp (fig 1). in order to facilitate the insertion of the gene of interest srv-2 env to baculovirus vector, we used pentr/d/topo cloning kit (invitrogen) as entry clone. this entry clone utilized a highly efficient method to clone a blunt end pcr product directly into a baculovirus vector with no additional ligation or restriction enzyme required. in this system, pcr products are directionally cloned by adding four bases to the forward primer (cacc). the overhang in the cloning vector (gtgg) invades the 5’end of the pcr product, anneals to the added bases and stabilized the pcr product in the correct orientation (fig 2). sequencing to the pentr/d/topo plasmid was done using m13 forward primer and some specific srv-2 env primers (216u20, 727u20, 1284u20, 1770u20). analysis of the sequencing result with nucleotide alignment using blast program indicated the presence of the gene of interest srv-2 env in pentr/d/topo plasmid with proper orientation (data not shown). this plasmid was then recombined to 2000 bp 1 2 3 4 fig 1 amplification of srv-2 env provirus isolated from indonesian macaca fascicularis using specific primer bacnterm 5823u-ltr90l. 1, 1 kbp dna ladder (invitrogen); 2-3, pbmcs cell; 4, reagent control. microbiol indones 93volume 3, 2009 baculovirus genome using lr recombination reaction with lr clonase enzyme mix. in this case, we used linear baculodirect baculovirus expression system (fig 3) containing the entry clone to transfect onto the sf9 cells using cellfectin cationic-lipid, n,ni,nii,niii-tetrametyln,ni,nii,niii-tetrapalmitylspermine (tm-tps) with dioleoyl phosphatidylethanolamine (dope). the srv-2 env gene recombinant was expressed using polyhedrin promoter and proliferated in sf9 cells. negative selection of the recombinant with correct expression was done using ganciclovir. infection of the sf9 cells typically displayed the specific characteristic of cell morphology as observed from visual inspection using inverted phase microscope. characteristics of infected cells were shown with increased cell diameter, cessation of cell growth, detachment and cell lysis. in order to verify the recombination between srv-2 env with baculovirus genome in sf9 cells, we amplified the passage-3 (p3) viral stock using pcr technique with specific primer of polyhedrin forward to srv-2 5974l and srv-2 6243l. the specific bands about 400 bp and 600 bp was indicated the presence of recombination between srv-2 env and baculovirus genome (fig 4). additionally, to ensure the recombination of srv-2 env with baculovirus genome, the pcr product then cloned to pcr 2.1 ta topo cloning kits (invitrogen), and the plasmid was then sequenced using m13 forward primer. analysis of the sequencing result indicated the presence of recombination between srv-2 env and baculovirus genome in correct direction (data not shown). the srv-2 env recombinant protein expression was demonstrated by specific bands for envelope glycoprotein (gp70) and gp20 on sds-page analysis, although there were fig 2 schematic diagram of pentr/d/topo that will facilitated the srv-2 env as entry clone to baculovirus genome. fig 3 schematic diagram of genetic linear dna baculodirect n-term. 400 bp 600 bp 1 2 3 1 2 3 fig 4 pcr analysis of p3 viral stock of supernatant and pellet cells using specific primer: panel a, primer polyhedrin forward to srv2 env 5974l and panel b, primer polyhedrin forward to srv-2 env 6243l. 1, 100 bp dna ladder (invitrogen); 2, supernatant cell; 3, pellet cell. p ph p ie-1(0) p p10 bsu36i some non specific bands (fig 5). the purification to the viral stock using affinity chromatography purification system containing metal chelating resin ni2+-nta was minimizing the presence of non-specific protein, specifically designed to purify 6xhis-tagged protein. it was shown by sds-page analysis with the specific band of srv-2 gp70 env (fig 6). western blot analysis of this purified protein indicated a specific reaction with anti-srv-2 antibody positive of m. fascicularis serum shown by srv-2 gp70 env band (fig 7). discussion the gene of interest to be expressed using baculovirus vector is srv-2 env isolated from indonesian m. fascicularis. this gene will be expressed to surface glycoprotein and transmembrane that will first be recognized by host receptor in the initial infection (brody et al. 1994; rasko et al. 1999). this srv-2 env protein is very stable suggesting high degree of adaptation of srv-2 to its host (staheli et al. 2006). we used pentr directional topo cloning kit (invitrogen) that facilitated the recombination of srv-2 env gene to a b microbiol indones94 saepuloh et al. fig 7 western blot analysis of ni2+-nta purified srv-2 env recombinant protein probed with positive antibody anti-srv-2 of m. fascicularis serum (a) and negative antibody anti-srv-2 of m. fascicularis serum (b). 1, 10, kaleidoscope protein standard; 2, 9, srv-2 antigen virus; 3, 8, binding buffer fraction; 4, 7, washing buffer fraction; 5, 6, elution buffer fraction. baculovirus genome. this is a universal cloning method using the site-specific recombination properties of bacteriophage lambda (landy 1989). baculodirect has evolved from gateway technology (invitrogen, usa) platform and enables the direct transfer of the gene into a baculovirus genome without the need for the propagation of the recombinant bacmid dna. briefly, attr1 and attr2 gateway sites have been introduced into the viral genome to enable the recombinatorial cloning of gene interest from the gateway entry clone directly into baculovirus dna. this feature make baculodirect system ideal for protein expression system, since it immediately removes one of the more-time consuming stages of the entire process (hunt 2005). the specially engineered baculodirect linear dna (fig 3) contains a strong polyhedrin promoter (p ph ) for high level protein expression. att recombination sites for efficient recombination with any attl-flanked gateway entry vector and herpes simplex virus thymidine kinase (hsv-1 tk) and lacz genes located between attr sites for the selection of recombinant bavaculovirus. during the lr reaction with gateway entry clone, the lacz and tk genes are recombined out as by-products. sf9 cells are then placed under selection with ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl) guanine], which is enzymatically phosphorylated by hsv-1 tk. once phosphorilated, the active analog incorporates into dna and inhibits dna replication. ganciclovir selection has been used in sf9 cell to purify the recombinant viruses, therefore eliminating any remaining parental non-recombinant virus (hunt 2005). we observed the srv-2 env expression both in pellet and in supernatant cells indicated by specific bands of gp70 and gp20 after sds-page analysis (fig 5). it means that the recombinant protein has already been released to the cell supernatant. in this study, we used the full-length gene of srv-2 env, and the presence of gp20 band indicated that the srv-2 env protein has been cleaved by cellular protease enzyme, although the band was thinner compared to whole srv-2 antigen (brody et al. 1994). it was assumed that to produce both gp70 and gp20 recombinant protein the use of full-length srv-2 env protein was not efficient. the expression level of full-length env protein was lower compared to truncated env protein (yao et al. 2000). k d 205.0 49.5 32.5 27.5 18.5 6 . 5 1 2 3 4 5 6 7 8 9 10 fig 5 expression of srv-2 env recombinant protein using sds-page technique. 1, broad low marker protein standar; 2, whole srv-2 antigen; 3, supernatant cell p3 viral stock; 4, pellet cell p3 viral stock. k d 205.0 116.5 80.0 49.5 32.5 27.5 18.5 6 . 5 k d 205.0 116.5 80.0 49.5 32.5 27.5 18.5 6 . 5 fig 6 ni2+-nta purification analysis of srv-2 env recombinant protein in p3 viral stock by sds-page staining with coomassie brilliant blue. 1, protein standard broad low marker; 2, whole srv-2 antigen virus; 3, elution buffer fraction; 4, washing buffer fraction; 5, binding buffer fraction; 6, recombinant protein pre-purification. 1 2 3 4 5 6 116.5 80.0 1 2 3 4 a b gp70 env gp20 env gp70 env microbiol indones 95volume 3, 2009 the recombinant protein was purified using immobilized metal ion affinity chromatography (imac) containing metal chelating resin ni2+-nta to bind the 6x his-tagged protein. this his-tagged protein will compete with imidazole and will be removed from the column when the concentration was increased to 250 mm in elution buffer. this purification system has already removed some non-specific proteins, shown by elution buffer fraction in sds-page analysis (fig 6). we could not observe the gp20 env protein, probably it was also removed during the early elution process due to its low level protein expression. in this study, we observed the antigenicity of this purified srv-2 env recombinant protein on western blot analysis using antibody anti-srv-2 positive of m. fascicularis serum. it was shown by a specific band of srv-2 gp70 env (fig 7). thereby, this gp70 env recombinant protein retained its antigenicity and could be used as alternative antigen source for serological test in eia (enzyme immunosorbant assay) or western blot analysis against antibody srv-2 and could be applied for routine diagnostic purposes in supporting the macaca specific pathogen free (spf) colony program. in the future, the development of srv-2 recombinant protein will substitute the use of srv-2 active viral protein as antigen source due to its pathogenicity and biosafety level requirement. references brody ba, rhee ss, hunter e. 1994. post-assembly cleavage of retroviral glycoprotein cytoplasmic domain removes a necessary fusion activity. j virol 68:4620-7. daniel md, king nw, letvin nl, hunt rd, seghal pk, desrosiers rc. 1984. a new type d retrovirus isolated from macaques with an immunodeficiency syndrome. science 223:602-5. gardner mb, luciw p, lerche n, marx p. 1988. nonhuman primate retrovirus isolates and aids. adv vet sci comp med 32:171-90. joshi l, davis tr, mattu ts, rudd pm, dwek ra, shuler ml, wood ha. 2000. influence of baculovirus-host cell interactions on complex n-linked glycosilation of a recombinant human protein. biotechnol prog 16:650-6. hunt i. 2005. from gene to protein: a review of new and enabling technologies for multi-parallel protein expression. protein expr purif 40:1-22. iskandriati d, pamungkas j, suparto ih, grant r, agy mb, morton wr, sajuthi d. 1998a. evidence for retroviruses infection in captive orangutans (pongo pygmeus) returned to indonesia. j med primatol 27:173-80. iskandriati d, pamungkas j, surya m, mariya s, budiarsa in, sajuthi d. 1998b. prevalensi antibodi simian retrovirus tipe-d serotipe-2 (srv-2) pada macaca fascicularis dan macaca nemestrina di empat propinsi di indonesia. jpi 2:5-8. kost ta, condreay jp. 1999. recombinant baculovirus as expression vectors for insect cells and mammalian cells. curr opinion biotechnol 10:428-33. landy a. 1989. dynamic, structural, and regulatory aspect of sitespecific recombination. annu rev biochem 58:913-49. lerche nw, heneine w, kaplan je, spira t, yee jl, khabbaz rf. 1995. an expanded search for human infection with simian type d retrovirus. aids res hum retrovir 11:527-9. lerche nw, osborn kg. 2003. simian retrovirus infection: potential confounding variables in primate toxicology studies. toxicol pathol 31:103-10. marx pa, maul dh, osborne kg. 1984. simian aids: isolation of type d retrovirus and disease transmission. science 223:1083-6. moazed tc, thouless me. 1993. viral persistence of simian type-d retrovirus (srv-2/w) in naturally infected pigtailed macaques (macaca nemestrina). j med primatol 22:382-9. morton wr, agy mb, capuano sv, grant rf. 2008. specific 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(short com) ... short communication screening of quorum quenching activity of bacteria isolated from ant lion billy christianto yogiara*and faculty of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia g b . key words: g k . kata kunci: acterial intercellular communication or quorum sensing controls the pathogenesis of many medically important organisms. therefore, it is important to isolate bacteria that can disintegrate the communication, in a process called quorum quenching. bacteria from ant lions ( sp.) were grown on luria agar, and approximately 1.85 x 109 cfu ml was obtained. seven morphologically different colonies were screened for quorum quenching activity using wild type as an indicator. one isolate (myr7) was found to possess quorum quenching activity, was later identified as by employing 16s rrna ant lion, quorum sensing, quorum quenchin omunikasi intersel bakteri atau telah diketahui mengendalikan sifat patogen dari beberapa organisme yang penting secara medis. oleh karena itu, isolasi bakteri yang dapat memutus rangkaian komunikasi intersel-yang disebut dengan -menjadi sangat penting. bakteri dari undur-undur ( sp.) yang ditumbuhkan pada media agar-agar luria, menghasilkan sejumlah 1.85 x 109 cfu ml . sebanyak tujuh koloni yang berbeda secara morfologi ditapis aktivitas -nya dengan sebagai bakteri indikator. penapisan tersebut menghasilkan satu isolat (myr7) yang memiliki aktivitas yang diidentifikasi sebagai menggunakan gen penyandi 16s rrna undur-undur, , myrmeleon chromobacterium violaceum aeromonas quorum sensing quorum quenching myrmeleon quorum quenching chromobacterium violaceum quorum quenching, aeromonas quorum sensing quorum quenchin -1 -1 antimicrobial agents, including antibiotics and related medicinal drugs, have long been used for treatments to reduce the threat posed by infectious diseases. unfortunately, many pathogenic bacteria have become resistant to several antibiotics, for i n s t a n c e , p e n i c i l l i n r e s i s t a n t (bacquero 1995; goldstein and garau 1997), vancomycin-resistant enterococci (dixson . 1985), methicillin-resistant (sutherland and rolinson 1964; kareiviene 2006; martins and cunha 2007), multi-resistant salmonellae (newell . 2010), and multi-resistant (espinal . 2001). the increasing resistance of pathogenic bacteria to antibiotics may lead to public health risk. to overcome this problem, innovative strategies were needed to discover novel antibiotic targets or antivirulent drugs as alternatives to classical antibiotics (baron 2010). it has been known that the nature of bacterial pathogenesis is controlled by quorum sensing mechanisms (kievit . 2000; williams 2000). to be able to communicate with other cells, the bacteria produce, detect, and respond to a small signal molecule called autoinducer. the autoinducer is responsible to s t re p t o c o c c u s pneumoniae et al staphylococcus aureus et al. et al mycobacterium tuberculosis et al et al et al. induce particular gene expression, including virulent genes. according to finch . (1998), quorum sensing mechanism can be disrupted and has become a potential target for antiinfection therapy. antiquorum sensing activity is often called quorum quenching. this activity can be useful to prevent colonization of pathogenic bacteria that use quorum sensing to regulate virulent genes. in recent years, quorum quenching enzyme and inhibitor from various sources have been studied, both from prokaryotic and eukaryotic organisms. there are two types of prokaryotic quorum quenching enzymes such as ahllactonase and ahl-acylase. ahl-degrading enzymes from eukaryotic organisms can be found on pig kidney (acylase i) and on airway epithelian humans (lactonase) (dong and zhang 2005). in this study we used ant lion ( sp.) whose bacterial community and potential activity are underexplored. dunn and stabb (2005) performed culture-independent 16s rrna gene sequence analysis on the bacteria associated with the tissues of an ant lion, . all 222 sequences obtained by dunn and stabb (2005) were identified as these sequences could be subdivided into two main groups, the with 75 clones similar to spp. and the with 144 clones similar to the family et al myrmeleon myrmeleon mobilis proteobacteria. proteobacteria wolbachia proteobacteria α γ*corresponding author, phone: +62-21-5731740, fax: +62-21-5719060, e-mail:yogiara@atmajaya.ac.id issn 1978-3477, eissn 2087-8575 vol 5, no 1, march 2011, p 46-49 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.1.8 e n t e ro b a c t e r i a c e a e enterobacteriaceae wolbachia et al. bacillus cereus b. sphaericus, morganella morganii, serratia marcescens, klebsiella chromobacterium violaceum et al chromobacterium violaceum et al et al et al c. violaceum . t h e y f o u n d t h a t t h e -like 16s rrna gene sequences were most commonly isolated from gut tissue, and -like sequences were predominant in the head and body tissues. nishiwaki (2007) has reported insecticidal activity of bacterial isolates of ant lion. isolated , and species killed 80% or more cutworms when injected at a dose of 5 x 10 cells per insect. this study explored the possibility of finding antiquorum sensing molecule from ant lion-associated bacteria. the aim of this study is to obtain bacterial isolates that possess quorum quencing activity. about 20 ant lions were rinsed and surfacesterilized three times using sterile 0.85% of nacl and vortexing. the specimen was put into a 50 ml conical tube and ground. about 1 ml of 0.85% of nacl was a d d e d a n d s e r i a l d i l u t i o n s w e r e a p p l i e d . approximately 100 µl sample was spread onto modified luria agar (0.25% (w/v) trypton, 0.125% (w/v) yeast extract, 0.25% (w/v) nacl, and 1.5% (w/v) bacteriological agar), and then incubated at 30 c for 2 3 d. plate screening assay was used to evaluate quorum quenching activity with as an indicator (adonizio . 2006). in brief, luria agar plates spread with followed by spotting of tested bacteria. plates were incubated for 2 d at 30 °c, and quorum sensing inhibition was detected by a ring of colorless, but viable, cells around the bacterial isolate colony (adonizio . 2006). molecular identification using 16s rrna gene sequencing was carried out at eijkman molecular biology institute, jakarta-indonesia. dna sequences were aligned to 16s-rrna gene database provided by ribosomal database project (rdp) web site (http://rdp.cme.msu.edu/index.jsp) (cole . 2007, 2009). phylogenetic tree was constructed using treebuilder software provided by rdp and viewed by mega4 software (tamura . 2007). a total of approximately 1.85 x 10 cfu ml bacteria were observed and seven isolates were successfully isolated from ant lions. one out of seven isolate were detected to possess quorum quenching activity (fig 1). both isolate produced extracellular compound that may degrade the signal molecule required for quorum sensing activity. the degradation was indicated by colorless surroundtested isolates. these colorless bacteria were confirmed to produce purple pigment when streaked back onto another plate (data not shown). partial 16s-rrna gene sequences of isolate myr7 wes submitted to genbank database 5 9 -1 o (www.ncbi.nlm.nih.gov) under accession number hq453362. molecular identification showed that isolate myr7 had similarity to the genus . the isolate myr7 had 98% similarity to strain m10 dq200865. phylogenetic tree analysis (fig 2) showed phylogenetic position of isolate myr7 among members of order. the tree revealed that the isolate myr7 was clustered in the genus cluster and shared the same branch with subsp. ( d s m 3 0 1 8 7 ) a n d s t r a i n m 1 0 (dq200865). these results are different from the study conducted by dunn and stabb (2005), who successfully identified the family from the south american ant lion as stated above. quorum quenching activity was found in several bacteria, both gram positive and gram negative. it was reported that (leadbetter and greenberg 2000) and (uroz . 2005) had the ability to use ahl molecules as nitrogen and carbon sources, respectively, as well as energy sources. six bacteria isolated from the leaf surface of , i.e. , , , and , were proved to actively degrade acylhomoserine lactone as a signal molecule in quorum sensing mechanism (morohoshi 2009). the quorum quenching activity were also shown by other bacteria including sp. strain 240b1 , , sp. ibn110., , strain pai-a, pao1, strain xj12b (dong and zhang 2005). not only prokaryotic organisms possess anti quorum aeromonas aeromonas aeromonadales aeromonas aeromonas hydrophilla hydrophilla a e ro m o n a s enterobacteriaceae variovorax paradoxus rhodococcus erythropolis et al solanum tuberosum agrobacterium larrymoorei r. erythropolis, b. silvestris microbacterium testaceum b. cereus, eschericia coli bacillus , b. thuringiensis, b. cereus, b. mycoides, b. anthracis a. tumefaciens arthrobacter k. pneumoniae pseudomonas p. aeruginosa ralstonia t fig 1 screening of quorum quenching activity of isolates myr7 and myrb. dh5 was used as a negative control.escherichia coli volume 5, 2011 microbiol indones 47 sensing activity, eukaryotic organisms have also been known to have a quorum quenching activity. extracts of pea plants ( ) and crown vetch ( ) (teplitski 2000), , , , , and (adonizio 2006) have been tested of their abilities to inhibit the expression of quorum sensing activity byahl inactivation. spp. has not been reported to possess quorum quenching ability. therefore, this result was the first of such findings. despite the ability to inhibit quorum sensing, member of the genus has been known to its quorum sensing machinery such as ahyri and asari that are homologous to luxri. produces c4-hsl and n-hexanoylhomo serine lactone (c6-hsl) (swift . 1997). in conclusion, we have successfully isolated two bacterial isolates that possess antiquorum sensing activity against quorum sensing-regulated pigment production of . according to partial 16srrna gene analysis, the bacterial isolate had similarity pisum sativum coronilla variations et al. c. erectus, chamaecyce hypericifolia callistemon viminalis bucida burceras tetrazygia bicolor quercus virgiana et al. aeromonas aeromonas a. hydrophila et al c. violaceum to spp. this finding was not expected because is one of the bacteria known to use quorum sensing mechanisms to regulate certain gene expression, but not for its antiquorum sensing activity. aeromonas aeromonas references adonizio al, downum k, bennett bc, mathee k. 2006. anti-quorum sensing activity of medicinal plants in southern florida. j ethnopharmacol. 105:427-435. baquero f. 1995. pneumococcal resistance to -lactam antibiotics: a global geographic overview. microb drug resist. 1(2):115-120. baron c. 2010. antivirulence drugs to target bacterial secretion systems. curr opin microbiol. 13(1):100-105. cole jr, wang q, cardenas e, fish j, chai b, farris rj, kulam-syedmohideen as, mcgarrell dm, marsh t, garrity gm, tiedje jm. 2009. the ribosomal database project: improved alignments and new tools for rrna analysis. nucleic acids res. 37:d141-d145. cole j r, chai b, farris rj, wang q, kulam-syed-mohideen as,mcgarrell dm, bandela am, cardenas e, garrity gm, tiedje jm. 2007. the ribosomal database project (rdp-ii): introducing space and quality controlled public data. nucleic acids res. 35:d169-d172. myrdp doi:10.1016/j.jep.2005.11.025. doi:10. 1089/mdr.1995.1.115. doi:10.1016/j.mib. 2009.12.003. doi: 10.1093/nar/gkn879. doi: 10.1093/nar/gkl889. fig 2 phylogenetic tree of isolate myr7 between the genus and the other genera within aeromonadales order. the tree was constructed using weighted neighbor-joining tree building algorithm. the numbers in front of the branch point of the tree are bootstrap values with 100 replicates. the bar below the tree represents a distance scale. the code following underscore is genbank accession number. aeromonas 48 hristiantoc et al. microbiol indones dixson s, brumfitt w, hamilton-miller jm. 1985. in vitro activity of six antibiotics against multiresistant staphylococci and other grampositive cocci. eur j clin microbiol. 4(1):19-23. dong yh, wang lh, xu jl, zhang hb, zhang xf, zhang lh. 2001. quenching quorum-sensing-dependent bacterial infection by an nacyl homoserine lactonase. nature. 411(6839):813-817. dong yh, zhang lh. 2005. quorum sensing and quorum quenching enzymes. j microbiol 43(1):101-109. dunn ak, stabb ev. 2005. culture-independent characterization of the microbiota of the ant lion (neuroptera: myrmeleontidae). appl environ microbiol. 71(12):8784-8794. espinal ma, laszlo a, simonsen l, boulahbal f, kim sj, reniero a, hoffner s, rieder hl, binkin n, dye c, williams r, raviglione mc. 2001. global trends in resistance to antituberculosis drugs. n engl j med. 344(17):1294-1303. finch rg, pritchard di, bycroft bw, williams p, stewart gsab. 1998. quorum sensing: a novel target for anti-infective therapy. j antimicrob chemother. 42(5):569-571. goldstein fw, garau j. 1997. 30 years of penicillin-resistant : myth or reality? lancet. 350(9073):233-234. d kareiviene v, pavilonis, a, sinkute, g, liegiute s, gailiene g. 2006. resistance to antibiotics and spread of phage types. medicina. 42(4):332-339. kievit tr de, iglewski bh. 2000. bacterial quorum sensing in pathogenic relationships. infect immun. 68(9):4839-4849. leadbetter jr, greenberg ep. 2000. metabolism of acyl-homoserine lactone quorum-sensing signals by . j bacteriol. 182(24):6921-6926. martins a, cunha m de l. 2007. methicillin resistance in and coagulase-negative staphylococci: epidemiology and molecular aspects. microbiol immunol. 51(19):787-795. morohoshi t, someya n, ikeda t. 2009. novel n-acylhomoserine lactonedegrading bacteria isolated from the leaf surface of and their quorum-quenching properties. biosci biotechnol biochem. 73(9):2124-2127. myrmeleon mobilis s. pneumoniae staphylococcus aureus variovorax paradoxus staphylococcus aureus solanum tuberosum doi:10.1038/35081101. doi:10.1128/aem.71.12.8784-8794.2005. oi:10.1016/s0140-6736(05)62222-2. doi:10.1271/bbb.90283. newell dg, koopmans m, verhoef l, duizer e, aidara-kane a, sprong h, opsteegh m, langelaar m, threfall j, scheutz f, van der giessen j, kruse h. 2010. food-borne diseases-the challenges of 20 years ago still persist while new ones continue to emerge. int j food microbiol. 139:s3-s15. nishiwaki h, ito k, shimomura m, nakashima k, matsuda k. 2007. insecticidal bacteria isolated from predatory larvae of the antlion species myrmeleon bore (neuroptera: myrmeleontidae). j invertebr pathol 96:80-88. doi:10.1016/j.jip.2007.02.007. sutherland r, rolinson, gn. 1964. characteristics of methicillin-resistant . j bacteriol. 87(4):887-899. swift s, karlyshev av, fish l, durant el, winson mk, chhabra sr, williams p, macintrye s, stewart gs. 1997. quorum sensing in and : identification of the luxri homologs ahyri and asari and their cognate nacylhomoserine lactone signal molecules. j bacteriol. 179(17):52715281. tamura k, dudley j, nei m, kumar s. 2007. mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol biol evol. 24(8):1596-1599. teplitski m, robinson jb, bauer wd. 2000. plants secrete substances that mimic bacterial n-acyl homoserine lactone signal activities and affect population density-dependent behaviors in associated bacteria. mol plant microbe interact. 13(6):637-648. uroz s, chhabra sr, cámara m, williams p, oger p, dessaux y. 2005. nacylhomoserine lactone quorum sensing molecules are modified and degraded by w2 by both amidolytic and novel oxidoreductase activities. microbiology. 151:3313-3322. waters cm, bassler bl. 2005. quorum sensing: cell-to-cell communication in bacteria. annu rev cell dev biol. 21:319-346. williams p, camara m, hardman a, swift s, milton d, hope vj, winzer k, middleton b, pritchard di, bycroft bw. 2000. quorum sensing and the population dependent control of virulence. philos trans r soc london ser b. 355(1397):667-680. doi:10.1098/rstb.2000. 0607. staphylococci aeromonas hydrophila aeromonas salmonicida rhodococcus erythropolis doi:10.1016/j.ijfoodmicro. 2010.01.021. doi:10.1093/molbev/msm092. doi:10.1094/mpmi.2000.13. 6.637. doi:10.1099/mic.0.27961-0. doi: 10.1146/annurev.cellbio. 21.012704. 131001. volume 5, 2011 microbiol indones 49 7 maria mahata_344 (34-38) chitinases (ec 3.2.1.14) are enzyme that catalyze the degradation of chitin into the monomer n-acetyl-dglucosamine (park et al. 1997; yi-wang et al. 2001). while chitosanases (ec 3.2.1.132) are glycosyl hydrolase that catalyze the degradation of chitosan into α-d-glucosamine monomers. chitin is a linear polymer of n-acetyl-dglucosamine units linked through α (1-4) glycosidic bonds and distributed widely in nature as the skeletal materials of crustaceans and insects (minoru et al. 2002), and also as a cell wall component of bacteria and fungi. chitosan is a partially or fully deacetylated chitin. the α 1.4 glycosidic bond at linear polymer of n-acetyl-d-glucosamine of chitin is very strong and the chitinase or a specific chitosanase can catalyze degradation of the bond into a simple monomer. chitin combined with protein and (organic salt) caco3 form the skeletal material of crustacean and insects and this structure is involved in self defence mechanism against pathogenic bacteria and evaporation (yamasaki 1993). the use of protein of skeletal crustacean as a protein source in poultry feed is inhibited by chitin compounds, because the poultry’s digestive tract does not produce chitinase to hydrolyze chitin. therefore, before adding to poultry feed, the crustacean skeleton should be hydrolyzed by chitinase into simple monomers, so that poultry can then digest. generally, bacteria use their chitinase for degrading chitin as their carbon source, but some of them use chitinases for their self defence mechanism against pathogenic microorganisms. the characterization of chitinase from some bacteria has been undertaken, for example from bacillus circulance wl-12, enterobacter sp. g1, stenotrophomonas maltophilia c3, bacillus sp. nctu2, aeromonas sp. 10 s-24, enterobacter sp. nrg4, (park et al. 1997; watanabe et al. 1999; zhang et al. 2001; min-wen et al. 2002; ueda et al. 2003; dahiya et al. 2005). in nature, some bacterial species can produce chitosanases which hydrolyze chitin (shimosaka et al. 1995) and cellululose (reyes and corona 1997) as their substrates. mahata et al. (2005) reported that isolate 99 could produced both chitosanase and chitinase (at first, it was found that this bacterium could only produced chitosanase, whose activity was lower than that of isolate 97). both chitosanase activities from isolate 99 and 97 were higher than the activity of matsuebacter chitosanotabidus 3001 (a novel chitosanase bacterium isolated from water from matsue city, japan) used as control bacterium. the growth rates of isolate 99 in colloidal chitin solution and solid colloidal-chitin-agar medium were higher than that of other chitinase producing bacteria (119, 130, 136, and enterobacter sp. g-1) in the same medium. wide clear zones were produced by isolate 99 in solid colloidal chitin agar media. so far, we do not known how many types of chitinases and chitosanases are present in nature. the aims of this research was to characterize the chitinase from isolate 99 compared with the characteristics of chitinase from chitinolytic bacterium enterobacter sp. g-1 as a control bacterium since it produces both chitinase and chitosanase. materials and methods materials. the bacteria used in this experiment were isolate 99 and enterobacter sp. g-1 from the laboratory collection of the department of biochemistry and biotechnology, faculty of life and environtmental science, shimane university, japan. bacteria culture and chitinase production. single colonies of isolate 99 and enterobacter sp. g-1 were cultured in 2 ml of luria bertani medium (1% polypepton, 0.5% yeast extract, and 1% nacl) at ph 7.2 and 30°c for 24 h. one ml of each bacteria culture (isolate 99 and enterobacter sp. g-1) were characterization of extracellular chitinase from bacterial isolate 99 and enterobacter sp. g-1 from matsue city, japan maria endo mahata1*, abdi dharma2, irsan ryanto1, and yose rizal1 1department of animal nutrition and feed science, faculty of animal science, 2 department of chemistry, faculty of mathematics and natural science, universitas andalas, kampus limau manis, padang 25163, indonesia one hundred and twenty isolates of chitosanase producing bacteria were screened from water and soil from localies around matsue city, japan. in previous experiments, four isolates (isolates 96, 97, 99, and 100 strain ) were analyzed for their chitosanase characteristics, and one of the isolates (99) was detected as being both a chitosanase and a chitinase producer. characteristics of the chitinase enzyme were analyzed in this study. chitinase from bacterial isolate 99 showed higher activities compared to that enterobacter sp. g-1 (isolated from water in matsue city, japan), the activity was 0.039 u/ml and the specific activity was 0.56 u/mg protein, while those from enterobacter sp. g-1 were 0.029 u/ml and 0.48 u/mg protein respectively. chitinase from isolate 99 was stable in a ph range between 4-7, while that from enterobacter sp. g-1 was stable in ph range 3-7. optimum ph of the chitinase produced by isolate 99 was 5 whereas the chitinase from enterobacter sp. g-1 it was ph 7. chitinase from isolate 99 was stable at temperature 20-60°c, while that from enterobacter sp. g-1 at 20-50°c. chitinase secreted by isolate 99 showed optimum temperature of 50°c while chitinase from enterobacter sp. g-1 was optimal at 40°c. several ions (fe2+, ba2+, co2+) increased the activity of the enzyme from isolate 99 whereas ca2+ and co2+ increased activity of the enterobacter sp. g-1 chitinase.. key words: chitinase activity, ph, temperature, metal ion _____________________________________________ ________________________ corresponding author, phone/fax: +62-751-71464, e-mail: mariamahata2002@yahoo.com * issn 1978-3477 volume 2, number 1, april 2008 p 34-38 taken from luria bertani medium and cultured in 200 ml colloidal chitin solution. every day, 2 ml of each bacteria culture were taken and centrifuged at 3 500 rpm for 5 min. after centrifugation, the supernatant was separated from the pellet (bacteria) and kept at 4°c for the analysis of enzyme activity, specific activity, optimum temperature, and stability, optimum ph and stability and also to analyze the effect of some metal ions on chitinase activity. chitinase activity and specific activity. chitinase activity was determinated employing modified schales method with colloidal chitin as substrate (imoto and yagashita 1971). a mixture of 0.5 ml colloidal chitin solution (ph 5.2), 1.48 ml mcilvaine buffer ph 7.0 and 20 μl chitinase sample from isolate 99 and enterobacter sp. g-1 were incubated at 30°c for 30 min in shaking incubator. the reaction was stopped by boiling the mixture (100°c) for 15 min, and then centrifuged at 3 500 rpm for 5 min. as much as 1.5 ml of the supernatant was placed in a testube into which 2 ml schales reagent was added. the reducing sugar (product of the reaction) in the supernatant was detected spectrophotometrically (spectronic 21) at a 420 nm. one unit of chitinase activity equals the amount of chitinase needed to produce 1 μmol reducing sugar which was equivalent to n-d, acetyl glucosamine per min. specific activity is measured by comparing chitinase activity (u) with protein content of the enzyme (μg protein), the protein content is measured by lowry et al. (1951) method. optimum temperature and stability. optimum temperature for chitinase activity from isolate 99 and enterobacter sp. g-1 were determinated at 20, 30, 40, 50, 60, and 70°c, and enzyme stability was determined at 20, 30, 40, 50, 60, 70°c for 60 min. optimum ph and stability. optimum ph of chitinase from isolate 99 strain and enterobacter sp. g-1 were measured at ph 2 to 8 by using mcilvaine buffer. the chitinases sample from both bacteria were added to colloidal chitin substrate and incubated at 30°c for 30 min after which chitinase activity was analyzed. the ph of chitinase stability was measured at ph 2-8 and 30°c for 60 min and the colloidal chitin substrate was added before measuring chitinase activity. the effect of metal ions on chitinase activity. the effect of metal ions on chitinase activity produced by isolate 99 and enterobacter sp. g-1 were determined employing schales method (imoto andyagashita 1971). the chitinase sample from both bacteria were preincubated with certain ions (mg 2+ , na 2+ , zn2+, cu 2+ , and fe 2+ ) in mcilvaine buffer ph 7.0 at 30° c for 30 min. the final concentration of metal ion in mixed solution was 1 mm. all ions were as chloride with the exception for cu 2+ which was a sulphate. results chitinase activity and specific activity. the highest chitinase activity from isolate 99 was found on fourth day incubation while that from enterobacter sp. g-1 on the third day (fig 1). chitinase activity from isolate 99 was 0.039 u/ml and its specific activity was 0.56 u/mg protein. chitinase activity from isolate 99 and its specific activity were higher than those of enterobacter sp. g-1 which were 0.029 u/ml (fig 2) and 0.48 u/mg protein (fig 3). optimum temperature and stability. the optimum temperature of chitinase from isolate 99 was 50°c, and the temperature stability range was 20-60°c. the optimum temperature of chitinase from enterobacter sp. g-1 was detected at 40°c and its temperature stability was at 20-50°c after 60 min incubation (fig 4 and 5). optimum ph and stability. the optimum ph of chitinase from isolate 99 was 5 and its ph stability range was 4 to 7, while the optimum ph of chitinase from enterobacter sp. g-1 was 7 and its ph stability was 3 to 7 after 60 min incubation (fig 6 and 7). the effect of metal ions on chitinase activity. several ions (fe 2+ , ba 2+ , co 2+ ) increased the activity of enzyme from 0.4 0.1 0.2 0.3 0.6 0.5 0 1 20 543 6 7 e n zy m e ac ti v it y (u /m g p ro te in ) incubation period (day) f i g 3 c h i t i n a s e s p e c i f i c a c t i v i t y f r o m i s o l a t e 9 9 a n d e n t e ro b a c t e r s p . g 1 : — — c h i t i n a s e 9 9 , — — c h i t i n a s e enterobacter sp. g-1. fig 1 the clear zone of chitinase activity from isolate 99 on a solid chitin colloidal media on day 3 incubation. right the clear zone of 10 ul of isolate 99 culture with diameter 1.10 cm. left the clear zone of 10 ul chitinase from isolate 99 with diameter 0.90 cm. e n zy m e ac ti v it y ( u /m l) incubation period (day) 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 1 20 543 6 7 fig 2 chitinase activity from isolate 99 and enterobacter sp. g-1: —— chitinase 99, —— chitinase enterobacter sp. g-1. volume 2, 2008 microbiol indones 35 discussion this study revealed that the activity of chitinase from isolate 99 (0.039 u/ml) was higher than that of enterobacter sp. g-1 (0.029 u/ml), but its activity was lower than chitinase activity from paneibacillus illinoisensis (3.4 u/mg) which was isolated from a garden soil containing crab shell on the west coast of korea (hwan et al. 2006). the chitinase from isolate 99 later will be used for hydrolyzing chitin in shrimp waste, and then the shrimp waste is used for poultry feed. yamasaki et al. (1993) reported that enterobacter sp. g-1 was both a chitosanase and a chitinase producing bacterium, but its chitinase activity was lower than its chitosanase activity. the highest chitinase specific activity from isolate 99 was 0.56 u/mg protein which was higher than the highest chitinase activity from enterobacter sp. g-1 (0.48 u/mg protein). this fact indicated that chitinase protein content from both bacteria was equivalent with its activity. compared with other bacteria, chitinase specific activity from isolate 99 in this experiment was higher than that of stenotrophomonas maltophilia c3 (0.14 u/mg protein), but lower than purified chitinase from pseudomonas aeruginosa strain 385 (1.12 u/mg protein) (thompson et al. 2001), 80% deacetylated chitosan 70% deacetylated chitosan 90% deacetylated chitosan 100% deacetylated chitosan 0.5% colloidal chitin 100 50 31 8 0 100 118 30 13 21 100 0 1 20 0 substrate relative activity (%) m. chitosanotabidus 3001 99 97 table 2 substrate specificity of chitosanase from isolate 97, 99, and matsuebacter chitosanotabidus 3001 e n zy m e ac ti v it y (u /m l e n zi m ) 0.1 0.02 0.06 0.04 0 0.08 0.12 2 3 5 6 84 7 ph fig 7 chitinase ph stability from isolate 99 and enterobacter sp. g-1: —— chitinase 99, —— chitinase enterobacter sp. g-1. ion bacteria chitinase activity (u/ml) before incubation with ion chitinase activity (u/ml) after incubation with ion percentage increase or decrease* after incubation with ion fe2+ ba2+ ca2+ cu2+ co2+ isolate 99 enterobacter sp3 g-1 isolate 99 enterobacter sp3 g-1 isolate 99 enterobacter sp3 g-1 isolate 99 enterobacter sp3 g-1 isolate 99 enterobacter s3p g-1 0.039 0.029 0.039 0.029 0.039 0.029 0.039 0.029 0.039 0.029 0.055 0.040 0.089 0.023 0.040 0.064 0.040 0.049 0.221 0.246 41.03 37.93 128.21 20.69* 2.561 20.69 2.56 68.97 466.67 748.28 table 1 the effect of metal ions on chitinase activity from isolate 99 and enterobacter sp. g-1 0.08 0.02 0.04 0.12 0.06 0 0.1 0.14 50 704020 6030 fig 4 the effect of temperature on chitinase activity from isolate 99 and and enterobacter sp. g-1: —— chitinase 99, —— chitinase enterobacter sp. g-1. temperature (°c) c h it in as e ac ti v it y (u /m l e n zi m ) e n zy m e ac ti v it y (u /m l e n zi m ) 0.1 0.02 0.06 0.04 0 0.08 0.12 2 3 5 6 84 7 fig 6 the effect of ph upon chitinase activity from isolate 99 and e n t e ro b a c t e r s p . g 1 : — — c h i t i n a s e 9 9 , — — c h i t i n a s e enterobacter sp. g-1. ph isolate 99 while ca 2+ and co 2+ increased activity of the enterobacter sp. g-1 chitinase (table 1). 0.04 0.05 0.06 0.03 0.01 0.02 0 50 704020 6030 temperature (°c) e n zy m e ac ti v it y (u /m l e n zi m ) fig 5 chitinase temperature stability (°c) curve from isolate 99 and enterobacter sp. g-1: —— chitinase 99, —— chitinase enterobacter sp. g-1. 36 mahata et al. microbiol indones and purified chitinase from aspergillus fumigatus y j407 (3.36 u/mg protein) (xia et al. 2001). this experiment showed that isolate 99 was a potential chitinase producing bacterium. its ability to produce chitinase was better than that enterobacter sp. g-1, and it was also a potential chitosanase producing bacterium because its chitosanase activity was higher than that of matsuebacter chitosanotabidus 3001 in previous experiments (table 2) (mahata et al. 2005). this experiment found that the isolate 99 is a bacterium that can produce both chitinase and chitosanase. the optimum temperature of chitinase from isolate 99 was 50°c which was higher than the optimum temperature of chitinase from enterobacter sp. g-1 (40°c). this data shows that chitinase from isolate 99 was more tolerant to high temperature than chitinase from enterobacter sp. g-1, but that its activity in optimum temperature was lower than that of enterobacter sp. g-1. yi -wang et al. (2001) stated that the optimum temperature of exochitinase from b a c i l l u s c e re u s w a s 3 5 ° c w h o i t w a s l o w e r t h a n chitinase from isolate 99, but relatively similar to chitinase from enterobacter sp. g-1. min-wen et al. (2002) also found that the optimum temperature range of chitinase from bacillus sp. nctu2 was 50 to 60°c, and that the chitinase optimum temperature from isolate 99 was within in this range. this experiment also found that the temperature stability (incubated at 60 min) of chitinase from isolate 99 was 20 to 60°c , and for enterobacter sp. g-1 it was between 20 to 50°c. this data showed that chitinase from isolate 99 was more tolerant and stable at high temperature compared with chitinase fom enterobacter sp. g-1, but that its activity was not as high as chitinase activity from enterobacter sp. g-1. yi-wang et al. (2001) reported bacillus cereus had a wide range of temperature stability, the range was from 4 to 70°c. the temperature stability of chitinase from isolate 99 and enterobacter sp. g-1 in these studies were within this range. apparently, the chitinase from isolate 99 can be characterized as a thermotolerant enzyme and its stability is adequate for industrial application. in acid conditions, chitinase from isolate 99 degraded chitin more actively than chitinase from enterobacter sp. g-1. in general, the optimum ph of chitinase from microorganisms (bacteria, yeast, fungi) is 3.5 to 8 (koga et al. 1999), and the optimum ph of chitinase from isolate 99 and enterobacter sp. g-1 in these experiments were within the range reported. chitinase activity from isolate 99 was stable at ph 4 to7, while enterobacter sp. g-1 was stable at ph 3 to 7. chitinase from both bacteria were stable in acid or alkaline conditions. the ph stability of chitinase from bacillus cereus and bacillus sp. nctu2 were 2.5 to 8 (yiwang et al. 2001 and min-wen et al. 2002). the ph stability of chitinase from isolate 99 and enterobacter sp. g-1 in this experiment was within this reported range. chitinase activity from isolate 99 was increased to 41.03, 128.21, and 466.67% respectively after being incubated with fe 2+ , ba 2+ , and co 2+ , while chitinase activity from enterobacter sp. g-1was increased up to 120 % after being incubated with ca 2+ and 748.28% with co 2+ . chitinase activity from enterobacter sp. g-1 was decreased to 20.69 % after being incubated with ba 2+ . in general chitinase activity was inhibited by hg 2+ and ag + , while cu 2+ could increase or decrease chitinase activity. in some fish species and microorganism like pseudomonas aeruginosa, their chitinase activities are increased by cu 2+ (jolle‘s and muzzarelli 1999). some chitinase enzymes are very sensitive to metal ions. for example, chitinase activity from aspergillus fumigatus was inhibited strongly by hg 2+ , pb 2+ , ag + , fe 2+ , mn 2+ , and zn 2+ (xia et al. 2001). chitinase activity from bacillus brevis was inhibited by ag + after incubated in 1 mmol l-1 ag + at ph 8 and 30°c for 30 min and only 60% of the enzyme activity remained (sheng et al. 2004). howard et al. (2004) reported that chitinase b of microbulbifer degradans 2-40 has two catalytic domains (gh18n and gh18c), and the activity of each domain was not affected by a 10 mm concentration of various chloride salts: mg 2+ , mn 2+ , ca 2+ , k + , but the activity of gh18n was reduced 36% by ni 2+ , 8% by sr 2+ , and 41% by cu 2+ , while the activity of gh18c was reduced 14% by ni 2+ , 5% by sr 2+ and 53% by cu 2+ . hg 2+ completely inhibited the activities of both domains. the metal ion sensitivity of chitinase from isolate 99 strain and enterobacter sp. g-1 in this experiment was not the same as reported previously, its activity was not inhibited by fe 2+ , ba 2+ , ca 2+ , cu 2+ , and co 2+ , except that for chitinase from enterobacter sp. g-1 which was decreased by ba 2+ (table 1). its response to other ions is still unknown. in conclusion, the chitinase characteristics (enzyme activity, temperature, ph, and the effect of metal ion on chitinase activity) from isolate 99 were better and it was more tolerant than those of enterobacter sp. g-1. several ions (fe 2+ , ba 2+ , co 2+ ) increased the activity of the enzyme from isolate 99, while ca 2+ and co 2+ increased the activity of chitinase from enterobacter sp. g-1 but ba 2+ decreased its activity. acknowledgements this experiment was supported by a bpps scholarship from directorate general of higher education, department of national education republic of indonesia. i thank to kawamukai of shimane university, japan for his given me permission for using the bacteria collection at the biochemistry and biotechnology laboratory, faculty of life and environmental science, shimane university. references dahiya n, tewari r, tiwari rp, hoondal gs. 2005. chitinase from enterobacter sp. nrg4: its purification, characterization, and reaction pattern. electr j biotechnol 8(2). 10 p. [on line]. http:// www.ejbiotechnology.info/content/vol 8/issu 2/full/6/index.html. howard mb, ekborg na, taylor le, weiner rm, hutcheso sw. 2004. chitinase b of microbulbifer degradans 2-40 contains two catalytic domains with different chitinolytic activities. bacteriol 186:12971303. imoto t, yagashita k. 1971. a simple activity measurement of lysozyme. agric biol chem 35:1154-1156. jolle‘s p, muzzarelli raa. 1999. chitin and chitinase. birkhauser verlag basel, switzerland 98:111-123. koga d, mitsutomi m, kono m, matsumiya m. 1999. biochemistry of chitinases in chitin and chitinase. jolle’s p, muzzarelli raa (eds). birkhauser verlag: basel, switzerland 98:111-123. lowry oh, rosebrough hpj, farr al, randall rj. 1951. protein measurement with the valin phenol reagent. biochem 193:256-275. volume 2, 2008 microbiol indones 37 mahata me, yun cs, abdi d, irsan r, yose r, kawamukai m. 2005. karakterisasi kitosanase ekstraseluler dari bakteri asal air dan tanah di kota matsue, jepang dan dibandingkan dengan kitosanase dari matsuebacter chitosanotabidus 3001. j mikrobiol indonesia 10:21214. min-wen c, tseng cs, cheng cy, li, yk 2002. purification characterization and cloning of a chitinase from bacillus sp. nctu 2. biotechnol appl biochem 35:213-219. minoru m, hiroyuki s, yoshihiro s. 2002. control of function of chitin and chitosan by chemical modification. mini review, in trends in glycoscience and glycotechnology 14:205-222. park jk, morita k, fukumoto i, yamasaki y, nakagawa t, kawamukai m, matsuda h. 1997. purification and characterization of the chitinase (chia) from enterobacter sp. g1. biosci biotechnol biochem 61:684689. reyes mp, corona fg. 1997. the bifunctional enzyme chitosanasecellulase produced by the gram negative microorganism myxobacter sp. al-1 is highly similar to bacillus subtilis endoglucanases. arch microbiol 168:321-327. sheng l, zhi-an z, ming l, zhen-rong g, chen bai, weida h. 2004. purification and characterization of a novel chitinase from bacillus brevis. acta biochim biophys sinica 34:690-696. shimosaka m, nogawa m, wang xy, kumehara m, okazaki m. 1995. production of two chitosanases from a chitosan-assimilating bacterium, acinetobacter sp. strain chb101. appl environ microbiol 61:438-442. thompson se, smith m, wilkinsons mc, peek k. 2001. identification and characterization of a chitinase antigen from pseudomonas aeruginosa strain 385. appl environ microbiol 67:4001-4008. ueda m, kojima m, yoshikawa t, mitsuda n, araki k, kawaguchi t, miyatake k, arai m, focalize t. 2003. a novel type of family 19 chitinase from aeromonas sp. no. 10 s-24 cloning, sequence, expression, and the enzymatic properties. eur j biochem 270: 25132520. watanabe t, oyanagi w, suzuki k, tanaka h. 1999. chitinase system of bacillus circulans wl-12 and importance of chitinase a1 in chitin degradation. bacteriol 172:4017-22. xia g, chunsheng j, ju z, shoujun y, shuzheng z, cheng j. 2001. a novel chitinase having a unique mode of action from aspergillus fumigatus yj-407. eur biochem 268:4079-4085. yamasaki y. 1993. studies on chitinolytic and chitosanolytic enzymes produced by a bacterium and their application [thesis]. japan: shimane university. yi-wang s, anne lm, george t, shaw jw, robert dl, narendra k, singh. 2001. purification and characterization of a bacillus cereus exochitinase. enzyme microb technolo 28:492-498. zhang z, yuen gy, sarath g, penheiter ar. 2001. chitinase from the plant disease biocontrol agent, stenotrophomonas maltophilia c3. the american phytopalthological society. 38 mahata et al. microbiol indones 7 rev caecil (65-68).pmd the dynamics of bacterial communities during traditional nata de coco fermentation cecilia anna seumahu1‡, antonius suwanto2∗, debora hadisusanto3, and maggy thenawijaya suhartono3 1graduate school of biotechnology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia 2department of biology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia 3pt. niramas utama, jalan raya bekasi tambun km 39.5, bekasi 17510 , indonesia 3department of food technology and nutrition, institut pertanian bogor, darmaga campus, bogor 16680, indonesia one of the important problems in traditional nata de coco (nata) fermentation is production inconsistency due to strain or genetic variability reflecting mixed microbial communities involved in this process. this research was aimed at examine the population dynamics of the bacterial community during the fermentation processes. samples were collected daily for six days from fermentation media derived from “good” and “bad” nata fermentation. we compared the levels of bacterial diversity through amplified 16s-rrna (ardra). dna was extracted directly from the fermentation media and 16s-rrna gene was amplified employing universal bacterial primers. the amplicons were cloned into pgem-t easy vector, and restriction enzymes haeiii and rsai were used to generate ardra profiles. ardra phylotypes of dna extracted from the fermentation medium obtained from different nata qualities were compared. phylotype profiles demonstrated unique bacterial community profiles for different conditions of nata quality, which could be developed as a parameter to monitor nata quality during fermentation. in this research we found that the dynamics of the bacterial population involved in nata fermentation were a crucial factor for determining traditional nata quality. key words: nata de coco, bacterial community dynamics, ardra _____________________________________________ _________________ ‡present address, universitas pattimura, jalan ir. m. putuhena, kampus poka, ambon 97233, indonesia ∗ corresponding author, phone/fax: +62-251-362830, e-mail: asuwanto@indo.net.id traditional nata fermentation process is not yet fully studied nor comprehended at the molecular level, although the process has enough reason to be explored. randazzo et al. (2002), have studied community activity and the dynamics of bacterium populations during cheese production. ampe et al. (2001), also studied the “sour cassava” fermentation process and they found that the dynamics of microorganisms was sequentially changed during the fermentation process. we consider that if we studied the dynamics of the microbial population, we could have better understanding on the role of microorganisms in the fermentation processes so that we can systematically establish a consistent nata starter culture. population dynamics need to be studied through community analysis because it represents an excellent approach to comprehending the function and structure of a community. this analysis gives the opportunity to identify dominant and unique strains in a controlled environment (marsh et al. 2000). giraffa and neviani 2001, reported that the first step to comprehend these concepts in food microbiology is to analyze microbiological profiles and community structures and dynamics; and also their functions in altering the environment and biologic conditions in food. the challenge is the difficulty in cultivating all microorganisms from food on laboratory standard media since most microorganisms from nature are not yet culturable. (giraffa and neviani 2001). ampe et al. (1999), have compared the standard microbiological techniques, and technique that do not depend on cultivated processes, to examine microbial populations. they found that the culture-independent technique is the most suitable to depict population dynamics. weisburg et al. 1991, found that amplification of the 16srrna gene, cloning and sequencing it represents one of the important methods to identify microorganisms directly from nature (culture-independent). this technique is essential in studying the dynamics of the microbial population in nata fermentation due to the extreme ph of media via the acid cultivation conditions (ph 2-3). our previous investigations indicated that not all of the bacteria involved in nata fermentation could be cultured. this research aims to study the diversity and dynamics of the bacterial population during traditional nata de coco fermentation. materials and methods nata media solution from the fermentation processes with the bad and good outcomest were sampled at days 5, 6, 7, 8, 9, and 11 for ardra. nata media solution was categorized as “good” if it yielded nata with a thick and smooth texture. in contrast, a nata media solution was categorized as “bad” if it yielded nata with the hard texture that had bubbles of gas trapped in it. one of the a. xylinum collection strain ib-1 was marked by molecular marker to become a xylinum strain ib-1nal-r. this was used to analyse growth of a. xylinum and the roles of other bacteria in fermentation media. samples of media and strains of a. xylinum used in this study were collected from a nata de coco company in jakarta. growth analysis. acetobacter xylinum strain ib-1, which is initially sensitive to nalidixic acid, was screened for spontaneous nalidixic-resistant mutans by cultivating it repeatedly at media suplemented with that antibiotic. a. xylinum microbiology indonesia, august 2007, p 65-68 volume 1, number 2 issn 1978-3477 strain ib-1nal-r, which is resistant to nalidixic acid, was subsequently grown in media without antibiotic supplementation as control ( meds), the medium with the addition nalidixic acid 20 µg ml-1 (nal), and the heat-treated medium by boiling at 10 min (blc) to eliminate most vegetative bacterial cells. bacteria populations grown in different treatments were examined on the first, fifth and tenth days of cultivation. media used for the growth were as described previously (seumahu 2005). dna isolation. the isolation of dna from samples was conducted as reported previously by ampe et al. (1999). amplification and cloning of the 16s-rrna gene. amplification of the 16s-rrna gene was conducted employing 63f: 5'-caggcctaacacatgcaagtc-3' and 1387r: 5'gcggwgtgtacaaggc-3' for the bacteria domain group (marchesi et al. 1998).16s-rrna amplicons were purified employing wizard sv gel and the pcr clean-up system. the purified dna’s were ligated into pgem-t easy vector (promega, madison, wi, usa) and transformed into escherichia coli dh5á . transformants were selected on luria agar media (la)+ampicilin (100 µg m-1) supplemented with x-gal (40 µg ml-1). 16s-r-rna gene in recombination plasmid library (collection of 16s-rrna genes in pgem-t vector) were amplified again using m13f and m13r primer (moffett et al. 2000) to obtain individual 16s-rrna genes. this step would ensure that the amplified 16s-rrna genes were from recombination plasmids and not from the bacterial host 16s-rrna gene. amplified ribosomal dna restriction analysis (ardra). the 16s-rrna gene, amplified from recombination plasmids, was digested with restriction enzymes rsai or haeiii, to yield a specific pattern representative of the existing bacteria and designated as profile 1, profile 2, etc. the percentage of specific patterns calculated on each day of fermentation and was depicted as a population dynamic curve. result growth of a. xylinum in the presence of microbial community. refering to the obtained data, we made a cell growth histogram depicting the growth profiles of a. xylinum strain ib-1nal-r in fermentation media with different treatments (fig 1). fig 1 indicates that fermentation media with a blanching treatment (blc) did not enhance the growth of strain ib-1nal-r compared to that of the control medium (nata fermentation medium without any treatment). in the fermentation medium with nalidixic supplementation, the growth of ib-1nal-r became very depressed. although the population did increase, the sum of the cell count was not as high as that in the control medium or blanching treatment. this result suggested that the pre-existing bacterial population in the media were essential for successful nata fermentation and migt have positive or synergistic effect to the growth of strain ib-1-nal-r. ardra reveals bacterial profiles during nata fermentation. results of ardra analysis showed the existence of at least twenty two different bacterial group during nata fermentation (fig 2). each ardra profile found in nata fermentation was calculated as a percentage to the total profiles every day starting from the fifth day up until eleventh day. five profiles were considered to be unique because their presence could only be found over certain specific days of fermentation (fig 3). unique ardra profiles include profile 1 to 5. profile 6 to 22 was not depicted in a growth curve because we found them only on certain days and they did treatment and days of cultivation fig 1 acetobacter xylinum ib-1nal-r growth in fermentation media with different treatments (med = media without treatment, blc=heated media, nal = media with nalidixic acid supplementation 20µg/ml, the number following the name of the media indicates the day of fermentation). the numbers following each treatment indicated the days of nata fermentation (0.5 and 10 days). 66 seumahu et al. microbiol indones not show significant percentage numbers of the total population (data is not presented at this article). discussion a. xylinum with antibiotic resistance marker was employed, in the laboratory scale, to examine the influence of some media treatment on the growth of a. xylinum during nata fermentation. three kinds of media were used, i.e. media without treatment, media with blanching for 10 minutes to eliminate as many as possible contaminants in the media, and media with nalidixic acid supplementation (20 µg ml-1) to suppress the growth of other bacteria sensitive to this antibiotic. the growth of contaminants was expected to be suppressed to give enable to a. xylinum to ‘outcompete’ and yield pellicle of good nata gel. strain ib-1 was marked for the purpose of cell estimation when they were re-grown on media with nalidixic acid supplementation. we assumed that if strain ib-1nal-r could be maintained as dominant population, this isolate will grow fast and produce excellent nata gel. the results showed that nata which was produce during the fermentation process, when the natural contaminat population was suppresed with either a blanching treatment or by nalidixic acid supplementation, was inferior in quality compared to that of the control media. therefore, the presence of foreign bacteria at the control media might have a synergistic effect and stimulate rapid growth of the a. xylinum population. in traditional nata fermentation, media preparation was often conducted under sterile conditions. however, the fermentation did not always fail or result in ‘bad nata’. this might explain why the preexisting bacteria in the media preparation and also during the fermentation process, could enhance nata production and might possibly show exhibit symbiosis or excrete essential factors required for cellulose biosynthesis. in this study we define ‘good nata’ fermentation as one which will generate a thick (1.5-2 cm), homogenous cellulose gel with high transparency; while ‘bad nata’ fermentation will generate frothy, thin (frequently less than 0.5 cm), soft with white or opaque color nata gel after 8 days of fermentation (seumahu 2005). in this study, ardra was employed to better understand the bacterial community involved in the production of ‘bad’ and ‘good nata’. this analysis is based on direct extraction of total dna from both cultured and uncultured bacteria. specific bacterial strains in the bacteria domain could be identified by their specific profiles generated from the electrophoregrams of 16s gene digested with restriction enzymes haeiii or rsai. results of the ardra indicated that in a traditional nata fermentation process, a. xylinum represented seeding during a process which could also have symbiosis, or association with other bacteria present, in either coconut water or coconut milk that might generate a mutual effect in ‘good nata’ production or an antagonistic effect in ‘bad nata’ production. bacterial growth pattern shown in fig 3, indicated a sharp fluctuation for profile 2, 3, 4, or 5 during the course of fermentation. in fermentation generating the ‘bad nata’, this a b fig 2 ardra profile of haeiii and rsai from bacteria group which emerged during nata fermentation (a=haeiii; b=rsai) (the numbers above every column show individual recombinant 16s-rrna genes analysed, various type of bacterial profiles m = molecular marker size). 1 0 0 0 5 0 0 3 0 0 bp 1 0 0 0 5 0 0 3 0 0 bp fig 3 growth pattern curve of 5 dominant ardra profiles. a. profiles from bad, and b. good nata fermentations (. ) profil 1, ( ) profile 2, ( ) profile 3, ( ) profile 4, ( ) profile 5. a b volume 1, 2007 microbiol indones 67 growth profile was very erratic, while at fermentation with a good outcome, this type of profile was less erratic and tended to stabilize over time. profile 1 did not show any difference of their population dynamics either in the ‘bad’ or ‘good nata’ production. profile 2 in ‘bad’ fermentation tends to show fluctuation while in good fermentation it tends to stabilize with a low percentage of variability. on the other hand, profile 4 in both ‘good’ and ‘bad’ fermentation did not show the existence of different population dynamics. this fermentation process showed the existence of unique profiles, i.e. profile 3 and 5 from the bacteria domain. profile 3 showed rather sharp difference between ‘bad’ and ‘good’ nata in a fermentation resulting in ‘bad nata’, this profile tend to fluctuate and rise in the final fermentation process. on the other hand, in fermentation with the ‘good nata’ the existence of this profile tended to be minimal or was not visible. profile 5 on fifth day of the fermentation process for the ‘bad’ result showed the highest percentage (70%), while on the same day this profile showed only 25% in a fermentation process until yielded the ‘good nata’. in a fermentation process with the ‘bad’ result, this pattern was not detected on subsequent day, while for the fermentation process with a ‘good’ result, this pattern was still detectable in spite of its low amount (5%) on eleventh day. we conclude that community unique profiles represent one of the key factor which in this study can be considered essential indicators for ‘bad’ or ‘good’ nata fermentation. this analysis could be more dramatic if the sampling had not been limited to begin from day 5 where cellulose pellicles start to emerge. another unique matter is the amount of ‘other’ profiles present in relative small numbers and the flat spreading during the fermentation process with the ‘good’ result as compared to fermentation process yielding the ‘bad’ result (data not presented). a possible explanation for the existence of different types of bacteria at different steps represent a symbiosis process, where a different set of bacteria are required at different steps of fermentation to provide essential nutrients to a. xylinum for its cellulose biosynthesis. the presence of these bacteria could supply otherwise deficient, but essential, nutrients which are important for the growth of a. xylinum for ‘good nata’ production. this result also indicates that traditional nata fermentation, which tends to be semi-aseptic, might be required to provide some beneficial bacterial inocula for ‘good nata’ production (fig 1.) since a. xylinum grew better in medium without antibiotic supplementation or having blanching treatment. other factors which might have an effect to bacterium population dynamics is the change of ph of the fermentation medium during the process. at the start of fermentation, the ph of the media is 3.9. this value droped over time until it reached approximately ph 2.0 at the end of the fermentation. this might have effect on the complexity of the bacterial community profiles. acknowledggements this research was supported by research center for microbial diversity, faculty of mathematics and natural sciences, bogor agricultural university, bogor, and pt niramas utama in jakarta. this research is part of cas thesis. references ampe f, ben-omar n, moizan c, wacher c, guyot j. 1999. polyphasic study of the spatial distribution of microorganisms in mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations. app environ microbiol 65:5467-5473. ampe f, sirvent a, zakhia n. 2001. dynamics of microbial community responsible for traditional sour cassava starch fermentation studied by denaturing gradient gel electrophoresis and quantitative rrna hybridisation. intl j food microbiol 65:455 4 . giraffa g, neviani e. 2001. dna-based, culture independent strategies for evaluating microbial communities in food-associated ecosystems. intl j food microbiol 67:19-34. marchesi jr, sato t, weightman aj, martin ta, fry jc, hiom sj, wade wg. 1998. design and evaluation of useful bacteriumspecific pcr primer that amplify genes coding for bacterial 16s rrna. appl environ microbiol 64:795-799. marsh tl, saxman p, cole j, tiedje j. 2000. terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. appl environ microbiol 66:3616-3620. moffet bf, walsh ka, harris ja, hill tcj. 2000. analysis of bacterial community structure using 16s rdna analysis. anaerobe 6:129-131. randazzo cl, toriani s, akkermans dadl, de vos wm, vaughan ee. 2002. diversity, dynamics and activity of bacterial communities during production of an artisanal sicilian cheese as evaluated by 16s rrna analysis. appl environ microbiol 68:1882-1892. seumahu ca. 2005. analisa dinamika populasi bakteri selama proses fermentasi nata de coco menggunakan ampilfied ribosomal dna restriction analysis (ardra) [thesis]. bogor: bogor agruculture university. weisburg wg, barns sm, pelletier da, jane dj. 1991. 16s ribosomal dna amplification for phylogenetic study. j bacteriol 173:697-703. 68 seumahu et al. microbiol indones 6 akhmaloka_340 (27-33) cell lysis method affects assessment of microbial diversity based on ribotyping analysis heni yohandini1, fida madayanti1, pingkan aditiawati2, and akhmaloka1* biochemistry research division, faculty of mathematics and natural sciences1, school of life sciences and technology2, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia the microbial community in kawah hujan, kamojang, west java, indonesia, was analyzed using 16s-rrna-gene-sequencing combining with denaturing-gradient-gel electrophoresis (dgge) technique. two different cell lysis methods, enzymatic-based, and physical treatment-based dna extraction, were used to isolate chromosomal dna for 16s rdna gene-fragment amplification. the dgge profiles showed some differences in banding pattern that were obtained from both cell lysis methods. the dna sequence analysis of the individual dgge bands revealed that most of the band sequences obtained by physical treatment were close to 16s rrna gene fragments from the bacterial domain, while most of band sequences performed by enzymatic method had high homology with 16s rrna gene fragments from archaeal domain. further analysis of the sequences from both methods performed by comparisons with the ribosomal database project showed that some of dgge sequences from kawah hujan consisted unique 16s rdna sequences. keywords: kamojang, microbial diversity, 16s rrna, denaturing-gradient-gel electrophoresis _____________________________________________ an accurate measurement of microbial diversity in the environmental samples has long challenged the microbiologist and the microbial ecologist. few microbes have sufficiently distinct cellular morphologies to be identified by microscopic techniques. however, conventional cultivations of microorganisms are laborious, time consuming and most importantly, selective and biased for the growth of specific microorganisms. some microbes could only be cultivated if their metabolic and physiological requirements were reproduced in vitro (nadkarni et al. 2002; zengler et al. 2002). methods used to characterize microbial diversity in the environment that is independent of cultivation have been developed in recent times (marsh et al. 2000; sekiguchi et al. 2002; nakagawa and fukui 2003; ashby et al. 2007). one such method is the molecular phylogenetic analysis of the small subunit ribosomal rna (ssu rrna) by sequencing, dgge, or restriction fragment length polymorphism analyses. this approach was used for the study of marine, soils, thermal, acidic, alkaline, and hyper-saline habitats and has resulted in the discovery of new phylogenetic groups of both bacteria and archaea (bintrim et al. 1997; nakagawa and fukui 2003; sait et al. 2006; spear et al. 2007). these studies showed that the diversity of microbial ecosystems is typically 100 to 1 000 times greater than that found in cultivation alone (skirnisdottir et al. 2001; spear et al. 2007). dgge is one technique that is frequently used to measure complexity of microbial communities and to study their dynamics and to infer the phylogenetic relationship of the community members (muyzer et al. 1993; heuer et al. 1999; lohr et al. 2006). dgge analysis involves the separation of pcr-amplified 16s rrna-gene-segments in an acrylamide gel denaturing gradient. the separation is based on differences in melting characteristics of the double-stranded dna segments, which are in turn dependent upon sequence differences. the result is the simultaneous detection of many individual 16s rrna molecules as a profile made up of bands, each of which can be re-amplified and then sequenced (ward et al. 1998; aminin et al. 2007). extraction of pool of dna from the community in reasonable quantity is an important step in molecular a n a l y s i s o f m i c r o b i a l c o m m u n i t i e s . n o u n i v e r s a l method for extraction of community dna from samples of varied origin is available. comparative studies have been performed to analyze the efficiency of methods for extraction and purification dna from soil and sediment (zhou et al. 1996; chauduri et al. 2006). the differences in dgge/tgge profile caused by different lysis methods have been reported previously (muyzer and smalla 1998). dgge/ tgge patterns of pcr products from bacterial genomic dna extracted from soil samples using a harsh lysis method (i.e. lysozyme plus sds and bead-beating) gave more bands and more intense bands than profiles of pcr products obtained from the same sample by using a soft lysis method (i.e. lysozyme and alkaline sds). differences in dgge patterns were also found by comparing two different lysis methods, i.e., beadmill homogenization alone versus a combination of freeze-thawing, lysozyme and sds treatment, and beadmill homogenization (liesack et al. 1997; muyzer and smalla 1998). however, the impact of the extraction method on the outcome of indigenous microbial community analysis has not yet been clearly established (kresk et al. 1999). in this report we describe the genomic dna differences in microbial community using samples from the kawah hujan crater, west java, extracted by using two differences cell lysis, enzymatic, and physical treatment-based methods. materials and methods sampling. water samples were collected from one of kawah hujan craters (e 107°48’14.38", n -7°8’21.7" and the altitude 1 690 m), kamojang, west java. the crater has ________________________ *corresponding author, phone: +62-22-2515032, fax: +6222-2502360, e-mail: loka@chem.itb.ac.id issn 1978-3477 volume 2, number 1, april 2008 p 27-33 a temperature at 90°c and ph 1.9. microorganisms in 1 l of water sample were collected on a 0.22-μm-pore-size millipore membrane filter by filtration within 4 h after sampling. the cells on the membrane were re-suspended in 25 ml of ste buffer [10 mm tris-hcl (ph 8.0), 0.1 m nacl, 1 mm edta] and pelleted by centrifugation. the pellets containing microbial communities were stored at -20°c until dna extraction. bead beating/sds-based dna extraction. the pellets containing microbial cells were mixed with 350 μl of dna extraction buffer [100 mm tris-hcl (ph 8.0), 100 mm sodium edta (ph 8.0), 100 mm sodium phosphate (ph 8.0), and 1.5 m nacl], 0.2 g glass beads and 20 μl of proteinase-k (10 mg ml -1 ) in microcentrifuge tubes by vortexing at medium speed for 15 min at room temperature. after the mixing treatment, 30 μl of 20% sds was added, and the samples were incubated at 65°c for 2 h with gentle end-over-end inversions every 15 to 20 min. supernatants were mixed with an equal volume of chloroform isoamylalcohol (24:1 v/v). the aqueous phase was recovered by centrifugation and precipitated with 0.6 volume of isopropanol at room temperature for 1 h. the pellet of crude nucleic acids were obtained by centrifugation at 16 000 g for 20 min at room temperature, washed with cold 70% ethanol, and re-suspended in sterile deionized water, to give a final volume of 50 μl. lysozyme/sds-based dna extraction. the pellet cells were suspended in 200 ìl of 10 mm tris hcl buffer (ph 8.0) containing 8 mg ml-1 of lysozyme and incubated at 37°c for 1 h, the cells were lysed by adding 200 ìl lysis buffer containing 2% sds, 0.8 mg ml -1 proteinase k and 200 mm edta ph 8.0. the lysis process was carried out by incubation at 50°c for 30 min. 150 ìl ice cold potassium acetate and acetic acid glacial mixed solution were added and the denatured proteins were precipitated by centrifugation. crude dna’s were processed as for the bead-beating method above. amplification of 16s rrna gene-fragments. the amplification of fragments of dna coding for 16s rrna was performed by touch-down pcr using primer as stated in table 1. one primer complements a region conserved among bacteria (corresponding to positions at 1 055 to 1 070 in the e. coli dna sequence of the 16s rrna gene). the other primer was based on a universally conserved region (corresponding to positions at 1 392 to 1 406 in the e. coli sequence, with additional 40-base gc clamp). these primers were designed to amplify 16s rrna gene fragments in the bacteria domain (ferris et al. 1996). pcrs were performed by using cloned taq dna polymerase according to the instructions provided by the manufacturer (promega). the temperature cycle for the pcr was 1 min of denaturation at 94°c, 1 min of annealing, and 1 min of primer extension at 72°c. during an initial touchdown cycle, the annealing temperature was continually decreased from 53 to 43°c in intervals of 1°c per cycle; 20 additional annealing cycles were performed at 43°c. the final primer extension was for 10 min at 72°c. dgge and re-amplification of 16s rrna-genefragments. dgge was performed by using d-code systems (bio-rad laboratories) with a 1.5-mm gel. approximately 100to 500-ng portions of pcr products were applied directly onto 8% (w/v) polyacrylamide gels with denaturing gradients from 30 to 40% [100% denaturant was 7 m urea and 40% (v/v) deionized formamide]. electrophoresis was performed with 0.5x tae buffer (20 mm tris, 10 mm acetic acid, 0.5 mm edta, and ph 8.3) at 200 v and 60°c for 4 h. after electrophoresis, the gels were stained using the silver staining method (bassam et al. 1991). each band in the dgge gel was excised with a razor blade and then placed in 50 μl of tris-edta buffer and incubated overnight at 37°c. the eluted dna was re-amplified using the same primers as previous but without gc clamps. pcrs were performed at the following conditions: an initial denaturation step at 94°c for 5 min, followed by 30 cycles of 1 min at 94°c, 1 min at 50°c, 1 min at 72°c, and a final extension step of 10 min at 72°c. all of re-amplification results were used for dna sequencing. sequence analysis. the sequencing results were compared with dna sequences from genbank database at ncbi (national centre of biotechnological information) through web site http://www. ncbi.nlm.nih.gov using the blast program (altschul et al.1990) for screening sequence similarity. sequence alignments were performed by the clustelx program. phylogenetic reconstruction was accomplished using the phylogeny inference package (phylip version 3.62). evolutionary distances were calculated by the kimura 2-parameter method with the dnadist program (felsenstein 1989). phylogenetic trees were constructed from distance matrices by the neighbor-joining method (saitou and nei 1987), which was implemented using the neighbor program. the node reproducibility for tree topology was estimated by bootstrap analysis, which included 1 000 replicate data sets. results chromosomal dna and 16s rrna-gene-fragments. based on the examination of et-br stained agarose gels, chromosomal dnas extracted by the bead beating-based dna extraction method gave more intense bands compared to that the lysozyme method (fig 1) when comparing equal amount of the samples. however, the dna supernatant from the first method was darker in color, being a yellowish-brown (data not shown). chromosomal dna extracted by the lysozyme-based method requires additional purification step besides the chloroform-isoamylalcohol extraction to remove protein from the supernatant. this protein contamination often inhibited dna amplification. total chromosomal dnas from both methods were used as a dna template for the amplification of partial 16s-rrna-genes without further purification. the amplification of 16s-rrna-gene fragments had been successfully carried out, resulting in single bands of sizes expected (fig 2). dgge profiles of 16s-rrna-gene-fragments. 16s rrna genes fragments were separated by dgge. the table 1 the primer sequences primer primer sequence (5’ ! 3’) p1 p2 atggctgtcgtcagct cgcccgccgcgccccgcgcccggcccgccgcccccgccc cacgggcggtgtgtac 28 yohandini et al. microbiol indones profiles of bands representing the microbial community of kawah hujan using both dna extraction methods are shown in fig 3. different chromosomal dna isolation methods gave different dgge profiles. the bands from the physical treatment method appeared at the upper and lower areas of the gel (fig 3, lane 1), while the bands from the lysozyme treatment method were distributed throughout the gel (fig 3, lane 2). variations in denaturant gradient concentration gave a different pattern of band separation (data not shown). however, the best separation of the bands was obtained at a concentration of 30-40% of denaturant. phylogenetic and homological analysis of 16s-rrnagene-sequences. phylogenetic trees of 16s-rrna-gene sequences were constructed based on the distance matrix methods as stated in the methodology. the tree showed that most of the sequences obtained by physical treatment were clustered on separated branch (fig 4). one band (k3-z-4) was a new branch compared to most of others and close to xenorhabdus chiersii. two bands (k3-z-9 and k3-z-8) were similar to each other and close to enterobacter sp., while k 3 z 1 5 w a s f a r a w a y f r o m o t h e r s b u t c l o s e t o pseudomonas (fig 4). however, all of band sequences from the physical treatment were bacterial 16s-rrna-gene sequences. in contrast, most of bands recovered from enzymatic treatment had no similarity to 16s rrna gene sequences from the bacterial domain (fig 5b), except for k3-k-11 and k3-k-12 which were close to the glacial ice bacterium and pseudomonas (fig 5a). the rest of the band sequences made clusters similar t o e a c h o t h e r a n d w e r e s i m i l a r t o s u l f o l o b u s yangmingensis (fig 5b). one band, k3-k-13 (fig 5b), formed a different branch far away from the others but still close to the branch of other archaeal types. further analysis of the homological sequences made by comparing the sequences performed by using enzymatic lysis to the closest archael 16s-rrna-sequences from the database (genbank) showed that many nucleotide substitutions were present in the kamajong sequences (fig 6). this substitution also revealed that the sequences performed by physical treatment (fig 7) were similar, although the subsitutions were less frequent compared to that for the enzymatic lysis. discussion the microbial community from one of the kawah hujan craters was assessed by ribotyping analysis. the bands pattern from dgge analysis showed that there were differences between the pattern obtained from enzymatic and physical extraction methods (fig 3). this suggested that the pcr amplicons represented different communities. further analysis by re-amplification and sequencing of each band proved that each single band represented a different strain of microorganism (fig 4 and 5). as stated previously, most of sequences from chromosomal dna extracted by physical treatment represented that of bacterial groups, while that extracted by enzymatic treatment more closely represented archaeal groups. this result was surprising, that the two methods gave meaningful differences at the domain level. chromosomal dnas extracted by enzymatic treatment were less abundant than that the other extraction method. according to frostegard et al. (1999), the low 1 419 bp 517 bp 396 bp 214 bp 75 bp 1 2 3 4 fig 2 amplification product of 16s rrna gene fragments. 1, puc19/ hinfi; 2, contamination control of the pcr process; 3, pcr products from chromosomal dna extracted by bead beating-based method; 4, pcr product from chromosomal dna extracted by lysozyme-based method. fig 1 electrophoregram of the chromosomal dna. 1, λ/hindiii markers; 2, chromosomal dna extracted by bead beating-based method; 3, chromosomal dna extracted by lysozyme-based method. 1 2 3 23 130 bp 9 416 bp 6 557 bp 4 361 bp 2 322 bp 2 027 bp volume 2, 2008 microbiol indones 29 a b1 2 21 fig 3 a, dgge profiles of kawah hujan microbial community from which individual bands were excised; b, numbering pattern of bands which were excised and reamplified; 1, performed by using bead beating-based cell lysis method; 2, performed by using lysozyme-based cell lysis method. efficiency of dna extraction was mainly due to incomplete cell lysis in by addition to the fact that dna was adsorbed onto soil or mud particles during the extraction. however, physical treatment could improve the efficiency of dna extraction compared with enzymatic treatment due to its better ability to disrupt the cells, even some of dna was absorbed onto the particles. we used a set of primers to amplify 16s-rrna-genefragments that were designed to recover all of the 16s rrna gene sequences from bacterial domain. these primers have been reported to recover sequences from members of cyanobacteria, green sulfur and green non-sulfur bacteria, proteobacteria, gram-positive, and thermus 16s rrna genes (ferris et al. 1996; aminin et al. 2007). according to homological analysis of dgge band sequences, the microbial community extracted by lysozyme-based methods mostly belonged to the archaeal domain, whereas the bead beating-based method extracted the microbial community in the bacterial domain. this result suggested that the primers also amplified 16s rrna gene from members of archaea, besides bacteria. in our results there was no obvious difference in the intensity of bands of the pcr product, but the dgge patterns of both amplicons were completely different. 16s-rrna-gene fragments from the physical treatment gave more bands than did the profiles of pcr products obtained from the same sample using the enzymatic method. these results support previous reports (muyzer and smalla 1998; chaudhuri et al. 2006). in most cases, physical-based methods were able to disrupt a wide range of cell types with high dna yields, but could shear the chromosomal dna to smaller fragments (liesack et al. 1997). however, enzymatic-based methods were usually more selective and resulted in higher molecular weight of the chromosomal dna (muyzer and smalla 1998). we expected that physical treatment could recover dna from a more wide-ranging diversity of microbes compared to the enzymatic treatment. in spite of this, the results did not provide evidence for this assumption. we suggest that differences in the lysis method select different members of the microbial community. the enzymatic-based fig 4 phylogenetic tree of sequences performed by using bead beating-based cell lysis method. squares ( ) show position of the samples. 30 yohandini et al. microbiol indones 1000 fig 5 phylogenetic tree of sequences performed by using lysozyme-based cell lysis method. the trees were separated into i and ii due to the long distance between the archaeal and bacterial domains. squares ( ) show position of the samples. method had limited ability to isolate chromosomal dna of unexposed microbes in the spring water, for example by entrapping microbes in sludge. in this study most of microbes identified by enzymatic-based method were close to the archaeal domain. this was suggested by the fact that these organisms grew exposed in the spring water. meanwhile the physical treatment methods had possibility to release unexposed microbes so that the chromosomal dna could be isolated. in our results, the physical treatment method recovered most of bacterial domain (fig 4). volume 2, 2008 microbiol indones 31 1000 i ii this was suggested by the fact that the bacteria grow in association to sludge in the springs. this is probably due to the fact that the spring was not an ideal habitat for these organisms. meanwhile, for most of the archaea such as sulfolobus, the spring water provided suitable conditions for growth (lohr et al. 2006). most of the 16s-rdna sequences from both the enzymatic and physical extraction methods were unique. many substitutions occurred in the sequences from enzymatic extraction (10-15%) compared to that the closest archael sequences from the genbank. on the other hand, using physical extraction these substitutions were less frequent. it is a well known that the differences above 5% on the 16s rrna gene sequences are believed to be due to different strains (ashby et al. 2007). this is suggested by the fact that most of the sequences from enzymatic treatment belong to novel archaea strains, probably unique to kawah hujan, kamojang. from all of data obtained in this study, we proposed that the kawah hujan, kamojang habitat contains a lot of novel thermophilic microorganisms. we also recommend using more than one lysis method for assessing microbial communities in nature. acknowledgement this research was funded by grant from program riset kk, fakultas matematika dan ilmu pengetahuan alam, institut teknologi bandung to akhmaloka and insentif riset dasar, kmrt, to fida madayanti. references altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j mol biol 215:403-410. fig 6 alignment analysis of the sequences performed by using enzymatic-lysis with some archaea sequences from genbank the bottom sequences was the consensus, ( ) no substitution. fig 7 alignment analysis of the sequences performed by using physical-lysis with some proteobacter sequences from genbank. the bottom sequences was the consensus, ( ) no substitution. 32 yohandini et al. microbiol indones aminin aln, madayanti f, aditiawati p, akhmaloka. 2007. 16s r i b o s o mal rna-based analysis of thermophilic bacteria in gedongsongo hot spring. microbiol indones 1:37-42. ashby mn, rine j, mongodin ef, nelson ke, dimster-denk d. 2007. serial analysis of rrna genes and the unexpected dominance of rare members of microbial communities. appl environ microbiol 73:45324542. bassam bj, anolles cg, greshoff pm. 1991. fast and sensitive staining of dna in polyacrylamide gels. anal biochem 196:80-83. bintrim sb, donohue tj, handesman j, robert gp, goodman rm. 1997. molecular phylogeny of archaea from soil. proc natl acad sci 94:277282. chaudhuri sr, pattanayak ak, thakur ar. 2006. microbial dna extraction from samples of varied origin. current science 91:16971700. felsenstein j. 1989. phylipphylogeny inference package. cladistic 5:164-166. ferris mj, muyzer g, ward dm. 1996. denaturing gradient gel electrophoresis profiles of 16s rrna-defined populations inhabiting a hot spring microbial mat community. appl environ microbiol 62:340-346. frostegard a, courtois s, ramisse v, clerc s, bernillon d, legall f, jeanin p, nesme x, simonet p. 1999. quantification of bias related to the extraction of dna directly from soils. appl environ microbiol 65:5409-5420. heuer h, hartung k, wieland g, kramer i, smalla k. 1999. polynucleotide probes that target a hypervariable region of 16s rrna gene to identify bacterial isolates corresponding to bands of community fingerprints. appl environ microbiol 65:1045-1049. kresk m, wellington emh. 1999. comparison of different methods for the isolation and purification of total community dna from soil. j microbiol methods 39:1-16. liesack w, janssen ph, rainey fa, ward-rainey nl, stackebrandt e. 1997. microbial diversity in soil: the need for a combined approach using molecular and cultivation techniques. in: van elsas jd, trevors jt and wellington emh (eds) modern soil microbiology. new york: marcel dekker. pp 375-439. lohr aj, laverman am, braster m, van straalen nm, roling wfm. 2006. microbial communities in the world’s largest acidic volcanic lake, kawah ijen in indonesia, and in the banyupahit river originating from it. microbial ecology 52:609-618. marsh tl, saxman p, cole j, tiedje j. 2000. terminal restriction fragment length polymorphism analysis program, a web-based r esearch tool for microbial community analysis. appl environ microbiol 66:3616-3620. muyzer g, de wall ec, uitterlinden ag. 1993. profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna. appl environ microbiol 59:695-700. muyzer g, smalla k. 1998. application of denaturing gradient gel electrophoresis (dgge) and temperature gradient gel electrophoresis (tgge) in microbial ecology. antonie van leeuwenhoek 73:127-141. nadkarni ma, martin fe, jacques na, hunter n. 2002. determination of bacterial load by real-time pcr using a broad-range (universal) probe and primers set. microbiology 148:257-266. nakagawa t, fukui m. 2003. molecular characterization of community structures and sulfur metabolism within microbial streamers in japanese hot springs. appl environ microbiol 69:7044-7057. sait m, davis ker, janssen ph. 2006. effect of ph on isolation and distribution of members of subdivision 1 of the phylum acidobacteria occurring in soil. appl environ microbiol 72:1852-1857. saitou n, nei m. 1987. the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol biol evol 4:406-425. sekiguchi h, watanabe w, nakahara t, xu b, uchiyama h. 2002. succession of bacterial community structure along the changjiang river determined by denaturating gradient gel electrophoresis and clone library analysis. appl environ microbiol 68:5142-5150. skirnisdottir s, hreggvidsson go, holst o, kristjansson jk. 2001. a new ecological adaptation to high sulfide by a hidrogenobacteri sp. growing on sulfur compound but not on hydrogen. microbiol res 156:41-47. spear jr, barton ha, robertson ce, francis ca, pace nr. 2007. microbial community biofabrics in a geothermal mine adit. appl environ microbiol 73:6172-6180. ward dm, ferris mj, nold sj, bateson mm. 1998. a natural view of microbial diversity within hot spring cyanobacterial mat communities. microbiol mol biol rev 62:1353-1370. zengler k, toledo g, rappé m, elkins j, mathur ej, sort jm, keller m. 2002. cultivating the uncultured. proc natl acad sci 99:15681-15686. zhou j, bruns ma, tiedje jm. 1996. dna recovery from soils of diverse composition. appl environ microbiol 62:316-322. volume 2, 2008 microbiol indones 33 7 okky karnaradjasa.pmd ecological role of a softcoral-associated bacterium arthrobacter sp. on marine biofilm-forming bacteria ocky karna radjasa* and agus sabdono department of marine science and center for tropical coastal and marine studies, universitas diponegoro, jalan prof. sudarto sh no. 1, semarang 50275, indonesia a marine bacterium species associated with softcoral sinularia sp. collected from vicinity of peucang island, ujung kulon, west java, was successfully screened for estimating its ecological role through inhibiting marine biofilm-forming bacteria isolated from the surrounding colonies of sinularia sp. and was identified as closely related to arthrobacter nicotianae based on its 16s rdna structure. the bacterium was found to inhibit the growth of four biofilm-forming isolates (vibrio harveyi, v. fortis, staphylococcus sciuri, and tenacibaculum marilutum) indicating the significance of secondary metabolite production which may provide important defensive functions against fouling microorganisms. the isolate was capable of amplifying gene fragments of non-ribosomal peptide synthetases. a 416 bp long dna fragment was obtained and the deduced amino acid sequence showed conserved signature regions for the peptide synthetases and revealed a high similarity to that of actinoplanes teichomyceticus (62.5% identity). key words: ecology, softcoral, bacteria, biofilm _____________________________________________ ________________________ *corresponding author, phone: +62-24-7460038, fax: +62-24-7460039, e-mail: ocky_radjasa@yahoo.com issn 1978-3477 volume 2, number 2, august 2008 p 84 88 softcorals are an important and diverse group of colonial invertebrates belonging to the phylum coelenterata (cnidaria), class anthozoa, subclass octocorallia. one of the major groups, the order alcyonacea, consists of hundreds of different species including the member sinularia which can dominate many indo-pacific reefs (coll and sammarco 1986). furthermore, one of the reasons for the evolutionary success of the alcyonacean softcorals in the indo-pacific is considered to be the high level of secondary metabolites which are commonly found in their tissues (sammarco and coll 1992). one of the most interesting aspects of the softcorals is that fouling organisms usually do not colonize their surfaces and it is believed that antifouling represents another ecological role of secondary metabolites in the alcyonacea (sammarco and coll 1992). in general, secondary metabolites are thought to enhance the fitness of the producing species (engel et al. 2002). marine biofouling, is a natural process resulting from organism growth on underwater surfaces which causes huge economic losses to marine industries. in seawater, the microbial population on surfaces produce primary biofilm, which is generally thought to be a prerequisite for the attachment and metamorphosis of fouling organisms (callow and callow 2002). it has been widely reported that many biologically active natural products from marine invertebrates have striking similarities to metabolites of their associated microorganisms including bacteria (proksch et al. 2002; thiel and imhoff 2003). thus, it is important to highlight the possible ecological role of marine bacteria associated with softcoral in providing protection of the host from fouling microrganisms. bacterialsoftcoral association that occurs on the softcoral surface then could be of great interest to search for potential use as commercial antifouling compounds. non-ribosomal peptides represent one of the large families of secondary metabolites and numerous natural products belonging to these groups. they are widely used as pharmaceuticals, industrial agents or agrochemicals. this type of peptide is sythesized by extremely large polyfunctional enzyme systems within the protein known as non-ribosomal polypeptide sythetases (nrps) (silakowski et al. 2000). advanced techniques of molecular biology such as the polymerase chain reaction (pcr), in particular the application of degenerated primers of nrps to amplify gene fragments from peptide producers, have allowed the screening for the presence of non-ribosomal peptides among secondary metabolite-producing microorganisms (marahiel et al. 1997; ayuso-sacido and genilloud 2004; radjasa et al. 2007a,b). in this work, we report the ecological relevance role of marine bacteria associated with softcoral sinularia sp. for the production of secondary metabolites which are antagonistic toward marine biofilm-forming bacteria. this is coupled with pcr based-screening for the presence of nonribosomal polypeptide synthetases. materials and methods sampling and isolation of softcoral-associated bacteria. colonies of soft coral sinularia sp. were collected from the vicinity of peucang island, ujung kulon, west java, indonesia by scuba diving. upon collection softcoral colonies were put into sterile plastic bags (whirl-pak, nasco, usa. the tissues were then rinsed with sterile seawater (filtered by using 0.2 µm membrane filter and autoclaved) and scraped off with a sterile knife. the resultant tissues were serially diluted, spread on 50% strength zobell 2216e marine agar medium and incubated at room temperature for 48 hours. on the basis of morphological features, colonies were randomly picked, and purified by making streak on plates (madigan et al. 2000). isolation of marine biofilm-forming bacteria. bacterial isolation was carried out by using a method modified from harder et al. (2003). four pre-sterilized glass slides had been deployed in 4 different directions around softcoral colony for a week. the biofilm developed in these glass slides were then collected and put into sterile petri dishes, rinsed with sterile seawater and scrapped off with a sterile knife. the resultant mixture was diluted. a 100 µl aliquot of each dilution was spread onto 50% strength zobell 2216e and incubated at room temperature for 48 hours. colonies with distinguishing feature were selected and purified. inhibitory interaction test. the ability of softcoralassociated bacteria to inhibit the growth of marine biofilmforming bacteria was performed by using an overlay test method. aliquots culture of each marine biofilm-forming bacterium in the logarithmic growth phase (ca. 109 cells ml-1) was mixed with tsb soft agar medium (1% v/v), which were then poured on to the respective agar surface, previously inoculated with softcoral-associated bacteria, and incubated for 4 d. the plates were then incubated at room temperature for 48 hours. antibacterial activity was defined by the formation of inhibition zones around the bacterial colonies. pcr-based screening of nrps-producing bacterial strains. genomic dna of secondary metabolite producingstrains for pcr analysis were obtained from cell materials taken from an agar plate, suspended in sterile water (sigma, germany) and subjected to five cycles of freeze (-80oc) and thaw (95oc). amplification of peptide synthetase gene fragments was carried out with the nrps degenerated primers a2gamf (5´-aag gcn ggc gsb gcs tay stg cc-3´) and a3gamr (5´-ttg ggb ikb ccg gts gin ccs gag gtg-3´) designed from conserved regions of adenylation domains of various bacterial peptide synthetase sequences (genbank accession numbers: aak81824, aak81827, aak81826, aac82549, caa40561, cac48362, caa11796, cac48369, cac48369, aaf42473, bab69322, cab38518, aag02364, aag02355, aag02356, caa67248, cab93684, cab93684, cab93683, aac68816, aac44129, caa65394, aag05812, aag05789, aag05789, aaf40220, aad51026, cac11137, aab96629). the sequence of the reverse primer was based on the signature sequence of the superfamily of adenylate forming enzymes tsgxtgxpk (motif a3) found in peptide synthetases, but also in acetyl-coa synthetases. the sequence of the forward primer, based on the motif kaggay(lv)p (motif a2), is highly conserved for peptide synthetases which are involved in non ribosomal peptide synthesis (radjasa et al. 2007a). nrps-pcr was performed with a thermal cycler (eppendorf inc. germany) as follows: 1 µl template dna, and 1 µl of each of the appropriate primers, which were then put into puretaq ready-to-go pcr beads (amersham biosciences europe gmbh, germany). each pcr run comprised 40 cycles, with denaturing conditions for one minute at 95oc, annealing for one minute at 70oc and extension for two minutes at 72oc, respectively. pcr amplification and sequencing of 16s rrna gene fragments. pcr amplification of partial 16s rrna genes of active strains and marine biofilm-forming bacteria, purification of pcr products and subsequent sequencing analysis were performed according to the method of radjasa et al. (2007b). the determined dna sequences of strains were then compared for homology to the blast database (sabdono and radjasa 2008). cloning and sequencing of the putative peptide synthetase domain. the amplified pcr-products were purified using the high pure pcr product purification kit (roche diagnostics, mannheim, germany) following the manufacturers protocol. the big dye terminator cycle sequencing ready reaction kit (applied biosystems) was used for subsequent sequencing on an abi 310 analyzer (perkin elmer applied biosystems, foster city, usa). phylogenetic analysis. a phylogenetic tree was constructed using maximum-likelihood analysis. alignment positions with < 50% of sequences of the entire set of data having the same residues were excluded from the calculations to prevent uncertain alignments within highly variable positions of the 16s rdna. phylogenetic analysis was performed with the paup software package. results softcoral isolate, sfnb.5 was found to effectively inhibit the growth of 4 marine biofim-forming bacteria. this was identified as shown table 1. molecular identification indicated that sfnb.5 showed highest similarity to the member of genus athrobacter (table 2). identification of biofilm-forming bacteria is presented in the table 3. this indicates that these bacteria belonged to the members of vibrio, staphylococcus, and tenacibaculum. pcr-based screening by using specific primers nrps revealed that bacterial isolate sfnb.5 was capable of amplifying the gene fragments of nrps as shown in the fig 1. to investigate the genetic potential of isolate sfnb.5 to produce secondary metabolites, a 416 bp long dna fragment was obtained. the deduced amino acid sequence did indeed show conserved signature regions for peptide synthetases (table 4). table 1 inhibitory interaction of marine biofilm-forming bacteria by softcoral bacterial isolate sfnb.5 biofilm-forming bacteria bfb1.7 bfb1.8 bfb2.1 bfb2.6 isolate sfnb.5 + + + + table 2 molecular identification of softcoral bacterium sfnb.5 isolate closest relative similarity (%) acc. number sfnb.5 arthrobacter nicotianae 9 7 aj315492 table 3 molecular identification of marine biofilm-forming bacteria isolate closest relative similarity (%) acc. number bfb2.1 bfb1.8 bfb1.7 bfb2.6 vibrio fortis staphylococcus sciuri vibrio harveyi tenacibaculum marilutum 9 9 9 7 9 7 9 7 aj514916 s83569 aj672389 ay661693 volume 2, 2008 microbiol indones 85 a comparison of the 16s rrna gene sequence of strain sfnb.5 with sequences from genbank demonstrated that this isolate is affiliated to the genus arthrobacter. the phylogenetic tree shown in fig 2 shows that isolate sfnb.5 is most closely related with a. nicotianae with a similarity of 97%. discussion within minutes of immersing a clean surface in seawater, it adsorbs a molecular conditioning film, consisting of dissolved organic material. bacteria colonize this within hours, along with unicellular algae and cyanobacteria. these early colonizers form a biofilm, an assemblage of attached cells sometimes referred to as microfouling. a macrofouling community may then develop and overgrow the initial microfouling (callow and callow 2002). accumulating evidence that invertebrate-associated microbes contribute to the production of bioactive compounds has prompted the search for antimicrobial compounds from these microoorganisms (webster et al. 2001; proksch et al. 2002). growth inhibition of marine biofilmforming bacteria by isolate arthrobacter sfnb.5 demonstrates the so far uncharacterized secondary metabolites of this isolate lead to antagonistic activity and hence they may lead to advantages in the competition for space colonization of the softcoral surface. this assumption is supported by the fact that the nrps positive strain sfnb.5, exhibited antibacterial activity against four marine biofilmforming bacteria. the efficient inhibition of marine biofilmforming bacteria by strain sfnb.5 may be beneficial not only for the respective bacterium but also for the softcoral host since it further protects the softcoral from fouling microorganisms. fig 1 pcr amplification of nrps gene fragments. lane 1, dna markers; lane 2-3, sfnb.5; lane 4, control pseudomonas dsm 50117. table 4 characterization of nrps gene fragments strain closest gene fragment similarity (%) acc. number sfnb.5 actinoplanes teichomyceticus glycopeptide gene 62.5 x 5 6 9 2 8 it is interesting to note that the active softcoral isolate sfnb.5, which is closely related to arthrobacter species, is indeed very distantly related to the member of marine biofilmbacteria (vibrio, staphylococcus, and tenacibaculum) isolated from the surrounding soft coral colony, despite the fact that both were exposed to the same ecological conditions. wahl (1989) reported that biotic surfaces frequently harbor species-specific microbial communities that can be highly variable and distinct from those found in the surrounding environment. furthermore, engel et al. (2002) reported that there is evidence that there are symbiotic microbes which can chemically defend the host microbial colonization. the members of the genus arthrobacter has been known to produce various chemicals such as acetylchloline having pharmacological significance (mohapatra and bapuji 1998); cold-active antimicrobial compounds (o’brien et al. 2004) and enzymes (fujita et al. 1990). most non-ribosomal peptides from microorganisms are classified as secondary metabolites, since they rarely play a role in primary metabolism, such as growth or reproduction but have evolved to somehow benefit the producing organisms (neilan et al. 1999). products of the microbial nonribosomal peptide synthesis include the immunosuppressant cyclosporine and common antibiotics such as gramicin s, tyrocin a and surfactins (kleinkauf and von doehren 1996). the comparison of the derived amino acid sequence of the putative non ribosomal peptide synthetase of strain sfnb.5 revealed a high similarity to sequence fragments of known glycopeptide synthetases of actinoplanes teichomyceticus. the highest similarity was found with sequences of organisms belonging to the actinomycetes, from which many genera possess non-ribosomal peptide synthetase genes (jensen et al. 2005). a. teichomyceticus produced the glycopeptide teicoplanin, which is used for the treatment of serious infections caused by gram-positive pathogens (borghi et al. 1989; sosio et al. 2004) in conclusion, the softcoral bacterium sfnb.5 exhibited secondary metabolites which inhibit the growth of marine biofilm-forming bacteria. the present study highlights the ecological role of secondary metabolite producers amongst colonizers of softcoral sinularia sp. 86 radjasa and sabdono microbiol indones 1 2 3 4 this work was partly supported by a grant from ministry of research and technology within the competitive research grant scheme (rut xi) no. 011.27.sk.rut.2004 awarded to as. the work was also part of a research grant provided by the international foundation for science (ifs), sweden awarded to okr (contract no. 3965-1). references ayuso-sacido a, genilloud o. 2004. new pcr primers for the screening of nrps and pks-i systems in actinomycetes: detection and distribution of these biosynthetic gene sequences in major taxonomic groups. microb ecol 49:10-24. 6 0 7 9 9 9 9 8 7 2 7 5 9 9 6 0 7 8 6 9 5 3 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 phormidium corrallyticum arthrobacter arilaitensis cip 108037 sfnb5 isolate arthrobacter nicotianae arthrobacter ardleyensis an25 arthrobacter russicus bacillus algicola pseudoalteromonas luteoviolacea pseudoalteromonas ruthenica pseudoalteromonas elyakovii pseudomonas putida azohydromonas australica rhodobacter maris rhodobacter vinaykumarii ja 123t rhodobacter changlensis alpha proteobacterium d21 rhodobium gokarnense strain ja 173 myroides odoratimimus myroides odoratus halomonas variabilis halomonas elongata halomonas meridiana flavobacterium odoratum flavobacterium frigoris flavobacterium columnare flavobacterium degerlachei flexibacter aurantiacus flavobacterium sp. fig 2 phylogenetic affiliation of soft coral isolate sfnb.5. borghi a, antonini p, zanol m, ferrari p, zerilli lf, lancini gc. 1989. isolation and structure determination of two new analogs of teicoplanin, a glycopeptide antibiotic. j antibiotic 13:361366. callow me, callow ja. 2002. marine biofouling: a sticky problem. biologist 49:1-4. coll jc, sammarco pw. 1986. soft corals: chemistry and ecology. oceanus 29:33-37. engel s, jensen pr, fenical w. 2002. chemical ecology of marine microbial defense. j chem ecol 28:1971-1985. fujita k, hara k, hashimoto, kitahata s. 1990. purification and some properties of ß-fructofuranosidase from arthrobacter sp. k-1. agric biol chem 54:913-919. harder t, lau sck, dobretsov s, fang tk, qian p-y. 2003. a distinctive epibiotic bacterial community on the soft coral dendronephthya sp. and antibacterial activity of coral tissue extracts suggest a chemical mechanism against bacterial epibiosis. fems microbiol ecol 43:337-347. volume 2, 2008 microbiol indones 87 acknowledgement jensen pr, mincer tj, williams pg, fenical w. 2005. marine actinomycete diversity and natural product discovery. antonie van leeuwenhoek 87:43-48. kleinkauf h, von doehren h. 1996. a nonribosomal system of peptide biosynthesis. eur j biochem 236:335-351. madigan mt, martinko jm, parker j, brock td. 2000. biology of microorganisms. new jersey: prentice-hall. marahiel ma, stachelhaus t, mootz hd. 1997. modular peptide synthetases involved in nonribosomal peptide synthesis. chem rev 97:2651-2673. mohapotra br, bapuji m. 1998. characterization of acetylcholinesterase from arthrobacter ilicis associated with the marine sponge spirastrella sp. j appl microbiol 84:393-398. neilan ba, dittmann l, rouhiainen ra, bass v, schaub k, sivonen, borner t. 1999. nonribosomal peptide synthesis and toxigenicity of cyanobacteria. j bacteriol 181:4089-4097. o’brien a, sharp r, russell nj, roller s. 2004. antartic bacteria inhibit growth of food-borne microorganismes at low temperatures. fems microbiol ecol 48:157-167. proksch p, edrada ra, ebel r. 2002. drugs from the sea-current status and microbiological implications. appl microbiol biotechnol 59:125-134. radjasa ok, martens t, grossart hp, brinkoff t, sabdono, simon a. 2007a. antagonistic activity of a marine bacterium pseudoalteromonas luteoviolacea tab4.2 associated with coral acropora sp. j biol sci 7:239-246. radjasa ok, salasia sio, sabdono a, wiese j, imhoff jf, lammler c, risk mj. 2007b. antibacterial activity of marine bacterium pseudomonas sp. associated with soft coral sinularia polydactyla against streptococcus equi subsp. zooepidemicus. int j pharmacol 3:170-174. sabdono a, radjasa ok. 2008. phylogenetic diversity of organophosphorous pesticide-degrading coral bacteria from midwest coast of indonesia. biotechnolology 7:694-701. sammarco pw, coll jc. 1992. chemical adaptation in the octocorallia: evolutionary considerations. mar ecol prog ser 88:93-104. silakowski b, nordsiek g, kunze b, blöker h, müller r. 2000. novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium stigmatella aurantiaca sg a151. chem biol 53:11 1 . sosio m, kloosterman h, bianchi a, de vreugd p, dijkhuizen l, donadio s. 2004. organization of the teicoplanin gene cluster in actinoplanes teichomyceticus. microbiology 150:95-102. thiel v, imhoff jf. 2003. phylogenetic identification of bacteria with antimicrobial activities isolated from mediterranean sponges. biomol eng 20:421-423. wahl m. 1989. marine epibiosis. 1 fouling and antifouling: some basic aspects. mar ecol prog ser 58:175-189. webster ns, wilson kj, blackkall ll, hill rt. 2001. phylogenetic diversity of bacteria associated with the marine sponge rhopaloeides odorabile. appl environ microbiol 67:434-444. 88 radjasa and sabdono microbiol indones 3. naely revisi.cdr exploration, isolation and quantification of -carotene from bacterial symbion of sp. β acropora naely kurnia wusqy , leenawaty limantara , ferry fredy karwur 1* 2 1 and key words: 16s rdna b . 1 2 satya wacana christian university, salatiga, 50711, central java, indonesia; ma chung research center for photosynthetic pigments, universitas ma chung, malang 65151, east java, indonesia acropora acropora acropora erythrobacter flavus erythrobacter flavus acropora nasuta erythrobacter flavus acropora erythrobacter flavus in the microbial world, pigments are one of the most conspicuous traits. marine bacteria associated with sp. collected from taka cemara, karimunjawa islands were screened for the production of a yellow pigment. the isolation of bacterial symbionts from sp. on zobell 2216e medium resulted in one bacterium, kj5, positively synthesized carotenoids. by reverse phase hplc analysis, one peak of the pigment types was identified as a peak which appeared at 60.24 min. then, sample of the was collected and identified according to their spectral characteristics and compared with the published data in different types of solvent. based on the hplc analysis, the total carotene contents were calculated by converting the broad absorption of carotene. molecular identification of the bacterium kj5 using 16s rdna showed that bacterium kj5 was closely related to with 96% homology value. , sp., , , hplc akteri laut berwarna kuning telah berhasil diisolasi dari karang lunak yang berasal dari taka cemara, karimunjawa, jawa tengah. dari hasil skrining pada media zobell 2216e ditemukan satu isolat kj5 yang diduga mengandung karotenoid. identifikasi karoten dengan fase terbalik kromatografi cair kinerja tinggi (kckt) berhasil mengidentifikasi -karoten yang muncul pada menit ke 60,24. -karoten murni diperoleh dengan menampung hasil kckt yang selanjutnya dianalisa dengan uv-tampak. spektra diidentifikasi sesuai dengan karakteristiknya dan dibandingkan dengan referensi lain dalam berbagai jenis pelarut. identifikasi spesies bakteri yang dilakukan menggunakan reaksi berantai polimerase, menunjukkan bahwa isolat bakteri kj5 memiliki homologi sebesar 96% dengan kata kunci : sp., , hplc, 16s rdna β-carotene β-carotene βββ-carotene keberadaan β β β-karoten, vol.8, no.2, june 2014, p 58-64 doi: 10.5454/mi.8.2.3 *corresponding author; phone: +62-85842278789, email: naely_wusqy@yahoo.com natural pigments with an annual growth rate of 510%, have now comprised 31% of the worldwide colorant market compared to 40% for synthetic colorants (downham and collins 2000; mapari . 2010). nd pro-vitamin activities which provide additional value to the products (paz . 2012). recently, the price of extracted and purified natural carotene is much higher than that of synthetic carotene ($1000 to $2000 kilogram-1 for natural versus $400 to $800 kilogram-1 for synthetic). the price difference reflects that the consumers prefer the natural products to the synthetic carotene (caswell and zilberman 2000). et al et al natural -carotene is an orange-yellow pigment of carotenoid family that is widely used as a food colorant. -carotene is very attractive as natural food colorant due to its antioxidant a β β β β β many of the heterotrophic bacteria that synthesize carotenoids have been isolated from coastal and oceanic waters (du 2006). the widespread occurrence of carotenoids in non-phototrophic bacteria suggests that theirpresence is crucial for the viability of these organisms in their natural environment. due to the absence of photosynthetic apparatus, the importance of the carotenoids in these microorganisms lies mainly in protecting the microbes from photooxidative damage and in absorption of visible light (britton 1995). in this work, we reported the identification of a marine bacterium associated with soft coral and its potential for the productionof carotene. . colonies of soft coral were collected et al. et al. acropora nasuta acropora nasuta βmaterials and methods collection of samples and bacterial isolation from taka cemara karimunjawa waters manually. upon collection colonies were put into sterile plastic bags (whirl-pak, nasco, usa) and put into a cool-box. the tissues were then rinsed with sterile seawater and cut with a sterile knife. the resultant tissues were serially diluted, spread on ½ strength zobell 2216e marine agar medium and incubated at room temperature for 48 h. on the basis of morphological features, 9 colonies were randomly picked and purified by making streak plates (madigan . 2000). . the universal primers 27f (5'-agagtttgatcmtggc tcag-3') and primer 1492r (5'-tacggytacctt gttacgactt-3') were used to amplify 16s-rdna gene (long and azam 2001). the temperature cycle of amplification was as follows: initial denaturation at a temperature of 94 °c for 2min, and then successive denaturation (94 °c for 1 min), annealing (55 °c for 1 min), andextension (72 °c for 2 min). series of denaturation, annealing and extension wererepeated until 45 cycles. electrophoresis was done on 2% agarose. sequencing was done according to radjasa (2007). homology search and dna data bank by blast (atschul 1997). . ten plates of bacterial symbiontswhichwere cultured on zobell agar medium were collected and their pellets moisture was measuredby balancing moisturizer. then, 10 ml of et al et al. et al. 16s rdna polymerase chain reaction extraction of pigments 100% acetone were added into pellets for extraction (khalil and varananis 1996), with the aid of a sonicator (britton . 1995). . was identified and analyzed by using high performance liquid chromatography in reverse phase column ab with ods, c18, diameter of 4 mm x 25 mm. the method used consists of an elution gradient of methanol, acetone and ammonium acetate solution (1 m), similar to the method used by hegazi . (1998). the flow-rate was 1ml min , and the gradient protocol lasted approximately 80 min. all these steps were carried out at room temperature. peak of which appeared at 60.24 min was collected a n d i d e n t i f i e d a c c o r d i n g t o t h e i r s p e c t r a l characteristics and compared with the published data in different types of solvent. samples used in this study were taken from taka cemara, karimunjawa islands (fig 1a). one isolate, namely kj5 (fig 1b) which was found to produce yellow pigment was expected as source of carotenoids. a total of 200 g wet weight of kj5 isolate were extracted, resulted in 0.42 g of yellow pigments with 47.10% moisture. et al et al . acropora nasuta identification and analysis of β-carotene βcarotene βcarotene -1 results sampling and isolation of bacterial symbionts a b fig 1 sample of from taka cemara, karimunjawa, jepara (a) ; sample of kj5 isolate (b).acropora nasuta volume 8, 2014 microbiol indones 59 m (+) (kj5) 2.000 1.000 500 fig 2 pcr amplification of 16s rdna fragment. m: marker ; (+): positive control; kj5: sample. strain sw-46 16s ribosomal rna, partial sequenceerythrobacter flavus b36 gene for 16s rrna, partial sequencealpha proteobacterium strain fa2 16s ribosomal rna gene, partial sequencesphingomonas phyllosphaerae sp. mbic4118 gene for 16s rrna, partial sequenceerythrobacter strain 2pr56-3 16s ribosomal rna gene, partial sequenceerythrobacter flavus strain rkhc-1 16s ribosomal rna gene, complete sequenceerythrobacter citreus partial 16s rrna gene, isolate amv17erythrobacter gaetbuli 0.002 kj5 fig 3 phylogenetic tree based on 16s rrna gene sequences of strain kj5 and representative members of related species of the genus .erythrobacter table 1 molecular identification of kj5 isolate code length closest relative homology kj5 1440 bp erythrobacter flavus 96 % dna amplification of isolate kj5 using 16s rdna pcr showed positive results with the presence of bacterial dna isolate kj5 with the appropriate base length of approximately 1500 bp (fig 2). phylogenetic tree shown in fig. 3 shows the phylogenetic affiliation of bacterial isolate with other microorganisms. molecular identification, by two directions sequencing of the pcr product, showed that isolate kj5 has the highest percentage of similarity with strain with a 96% level value (table 1). from the results of hplc analysis during 80 -carotene absorption wavelength of 427, 449 and 477 nm at 60.24 min (fig 8) (hegazi . 1998). the spectra of hplc pigment pattern of kj5 erythrobacter flavus et al min, we found the β 60 wusqy et al. microbiol indones fig. 4 high-performance liquid chromatogram of acetone extract from at 450 nm wavelength (a); 2 dimension of extract pigment of (b); at 60,24 min by uv-vis (c). erb. flavus erb. flavus spectroscopyabsorption maxima of β-carotene extract fig. 5 the spectra of hplc pigment pattern of kj5 isolate at three different solvents; acetone (..); ethanol (--) and hexane (-) at 300-500 nm wavelengths. volume 8, 2014 microbiol indones 61 isolate at three different solvents (fig 5), and table 2 gives the absorption maximum of the pigments in various solvents. based on the hplc analysis results -carotene for this sample was in at a t 59.73 60.65 (fig 4) carotene extracted from the sample was 0,421 gr. the carotene per gram of can be calculated according to the following formula: (limantara . 2013) the concentration of total -carotene per gram of wet weight of is 30.01 g/g or 56.74 μg/ g of dry weight. was proposed by yoon ., (2003). the characteristics of these bacteria are nonspore-forming rods, gram-staining reaction is negative, and motile by means of a single polar flagellum. colonies are yellow, smooth, glistening, circular, convex with entire margins and 10-15 mm in diameter after 3 d cultivation at 30 °c on ma. optimal temperature for that the maximum absorbance of , the total -carotene contents were calculated. the total concentration of total β β β β β r erb. flavus erb. flavus et al erb.flavus erythrobacter flavus et al yield (μg/ml) = 0.0108x + 12.677 discussion growth is 30-37 °c. growth occurs at 10 and 42 °c, but not at 4 °c or above 43 °c. optimal ph for growth is 6.5-7.5 (yoon . 2003). most species of this genus contain bacteriochloro simidu 1982; yurkov . 1994). the hplc absorbant chromatogram is shown highlighting the separation of the pigment from . uv-visible absorption spectra of carotenoid pigments are of immense importance, since they aid a great deal in determining the structure of carotenoids (medicharla . 1991). from the hplc profile and uv-visible absorption spectra, we can conclude that produces a carotene type of carotenoid pigment that has many potential benefits. from the data it was evident that . produced less than carotene as compared to other microorganisms e.g. (prieto . 2011), fungi (murillo . 1978), and ( 1997). however, the carotene content reported in this study was higher than sp. which produced 4.88 μg per 100 gram (baskar . 2010), has been shown to have a potential 56. l et al et al erb.flavus et al erb.flavus erb flavus dunaliella salina et al phycomyces blakesleeanus et al blakeslea trispora mutant mehta et al. streptomyces et al erb.flavus . phyll α and carotenoids (shiba and strain for β-carotene production. the maximal -carotene yield was 74 μg g of dry weight. these results suggest that strain kj5 is worthy of further study for -carotene industrialization β β β β β -1 references acetone ethanol hexane eluent britton .et al (1995) 425,450, 478 jeffrey .et al (1997) 426,453,480 (iii/ii=21%) 427,449, 475 422,450,477 (iii/ii=36%) 425, 453, 476 (iii/ii=22%) hegazi .et al (1998) 425,449, 477 428,4 52, 476 results 429,451,480 (iii/ii=20%) 429,454,479 (iii/ii=11%) 426,450,476 (iii/ii=33%) 427,449, 477 (iii/ii=13%) table 3 broad absorption, yield and dry weight of β carotene broad absorption ( x) y ield conc (μg/g dry weight) 1166. 17 12.64 56.74 table 2. data in the mobile phase and different solvents. where x is the broad absorption l list of β-carotene spectral data from several of the β-carotene, y is the concentration ( m ) -1 μg 62 wusqy et al. microbiol indones acknowledgment references we wish to acknowledge the advice and all support of ocky k. radjasa for this manuscript. we would also like to thank majid khoeri for some of the sequencing, indriatmoko and lia kusmita for pigment analysis. this work was supported by the indonesian ministry education and culture. a b ltschul sf, madden tl, schaffer aa, zhang j, zhang z, miller w, lipman dj. 1997. gapped blast and psiblast: a new generation of protein database search p r o g r a m s . n u c l e i d a c i d r e s . 2 5 : 3 3 8 9 3402.doi10.1093/nar/25.17.3389 askar v, madhanraj p, kanimozhi k, panneerselvam a. 2010. characterization of carotenoids from sp. of marine and fresh water environment. arch appl scie res .2 (6): 380-388 britton g pfander . 1995 carotenoi birkäuser verlag basel boston berlin. caswell m, zilberman d. 2000. algolculture economic research service, usda department of agricultural and resource economics, university of california at berkeley: 1-10. downham a, collins p. 2000. colouring our foods in the last and next millennium. int j food sci technol. 35(1):5-22. doi:10.1046/j.1365-2621.2000. 00373.x. du hl, jiao nz, hu yh, zeng yh. 2006. diversity and distribution of pigmented heterotrophic bacteria in marine environments. fems microbiol ecol. 57(1): 92-105. doi: 10.1111/j.1574-6941.2006.00090.x. hegazi mm, perez-ruzafa a, almela l, candela me. 1998. separation and identification of chlorophyll and carotenoids from caulerpaprolifera, janiarubens, and padinapavonica by reversed-phase high-performance liquid chromatography. j chromatography. 829 :153159.doi0021-9673/98/$. khalil ia, varananis fr. 1996. carotenoid extraction and analysis by reversed phase hplc system. sarhad j agric. 105(67): 15-21. limantara l, heriyanto, indriatmoko, mucko p, adhiwibawa mas, indrawati r, priharyanti mnu, brotosudarmo thp. 2013. variation on chlorophylls and carotenoids composition from and grown in three different locations in indonesia (jepara beach, madura island and maluku island). j appl phys. (submitted). . streptomyces . kappaphycus alvarezii padina australis . , liaaen-jensen s, h . d. . . long ra, azam f. 2001. antagonistic interactions among marine pelagic bacteria. appl environ microbiol. 67 (11): 4975-4983.doi10.1128/aem.67.11.49754983.2001. madigan mt, martinko jm, parker j. 2000. . prentice hall. new jersey. mapari sas, thrane u, meyer as. 2010. fungal polyketideazaphilone pigments as future natural food colorants? trends in biotechnol 28 (6): 300-307. doi: 10.1016/j.tibtech.2010.03.004. jagannadham mv, rao vj, shivaji s. 1991. the major carotenoid pigment of a psychrotrophic micrococcus roseus strain: purification, structure and interaction with synthetic membranes. j bacteriol. 173:79117917.doi: 0021-9193/91/247911-07$02.00/0. mehta bj, salgado lm, bejarano er, cerdá-olmedo e. 1997. new mutants of for -carotene production. appl environ microbiol. 63: 3657–3661.doi: 10.1128/aem.69.7.40434048.2003. murillo fj, calderon il, lopez-diaz i, cerdá-olmedo e. 1 9 7 8 . c a r o t e n e s u p e r p r o d u c i n g s t r a i n s o f phycomyces. appl environ microbiol. 36 (1978) 639–642.doi: 0099-2240/78/0036-0639$02.00/0. paz eda, martin a, estrella a, rodríguez-rojo s, matias aa, duarte cmm, cocero mj. 2012. formulation of carotene by precipitation from pressurized ethyl acetate-on-water emulsions for application as natural c o l o r a n t . f o o d h y d r o c o l l o i d s 2 6 ( 1 ) : 1 7 2 7 . doi:10.1016/j.foodhyd.2011.02.031. prieto a, canavate jp, garcia-gonzalez m. 2011. assessment of carotenoid production by in different culture systems and operation regimes. j biotechnol. 151(2):180-185. doi: 10.1016/j.jbiotec.2010.11.011. radjasa ok, martens t, grossart hp, brinkoff t, sabdono a, simon m. 2007. antagonistic activity of a marine bacterium tab4.2 associated with coral sp. j biol sci. 7(2): 239246. doi: 10.3923/jbs.2007.239.246. shiba t, simidu u. 1982. gen. nov., sp.nov., an aerobic bacterium which contains bacteriochlorophyll . int j syst bacteriol. 32:211–217.doi: 10.1099/00207713-32-2-211. yoon jh, kim h, kim hg, kang kh, park yh. 2003. sp. nov., a slight halophile from the east sea in korea. int j syst evol microbiol. 53, 1169–1174.doi 10.1099/ijs.0.02510-0. yurkov vv, stackebrandt e, holmes a, fuerst j, hugenholtz p, golecki j, gad'on n, gorlenko v, brock biology of microorganisms phycomyces blakesleeanus dunaliella salina pseudoalteromonas luteoviolacea acropora erythrobacter longus a erythrobacter flavus β β volume 8, 2014 microbiol indones 63 kompantseva e, drews g. 1994. phylogenetic positions of novel aerobic, bacteriochlorophyll acontaining bacteria and description of gen. nov., sp. nov., gen. nov., sp. nov., and sp. nov. int j syst bacteriol. 44:427–434. doi: 0020-7713/94/$04.00+0 roseococcus thiosulfatophilus erythromicrobium ramosum erythrobacter litoralis . 64 wusqy et al. microbiol indones nurcholis et al_24042012 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.1issn 1978-3477, eissn 2087-8575 vol 6, no 1, march 2012, p 1-8 *corresponding author, phone: +62-21-7560536 ext 124, fax: +62-21-7566922, e-mail : nikniknur@gmail.com arabinofuranosidase (abfa) is one of the most important enzymes involved in degradation of lignocelullose biomass. two genes encoding α-l-arabinofuranosidase (abfa), each from bacillus subtilis db104 (abfaa1) and an indigenous indonesian b. licheniformis cw1 (abfab3), were cloned by the pcr approach and expressed in escherichia coli. sequences analysis of abfaa1 and abfab3 revealed that each consists of 1721 and 1739 base pairs long dna, respectively. each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an abfa protein of 500 and 502 amino acids for abfaa1 and abfab3, respectively. the deduced amino acid sequence of abfab3 shares 75% identity to that of abfaa1. the recombinant enzymes were expressed constitutively in e. coli. partial characterization of those enzymes revealed that the abfaa1 and abfab3 were optimally active at 50 ºc and 60 ºc at ph 6, respectively. thermostability studies of the recombinant enzymes with p-nitrophenyl α-l-arabinofuranoside at their optimal conditions showed that up to 50% abfaa1 activity was lost after 5 h incubation at 50 ºc, whereas the abfab3 retained its activity over 75% after 12 h pre-incubation o at 60 c. this thermostability study of recombinant abfab3 showed for the first time that the arabinofuranosidase from b. licheniformis is a thermostable enzyme. the recombinant enzyme showed a higher optimal reaction temperature (60 ºc) in comparison to the previously reported thermostable arabinofuranosidase. the thermostable abfab3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides. key words : α-l-arabinofuranosidase, bacillus, cloning, thermostability arabinofuranosidase merupakan salah satu enzim penting dalam degradasi biomassa lignoselulosa. gen yang mengkodekan α-l-arabinofuranosidase (abfa) masing masing dari bacillus subtilis db104 (abfaa1) dan isolat asli indonesia b. licheniformis cw1 (abfab3) telah diklon dengan pendekatan pcr dan diekspresikan dalam escherichia coli. analisa sekuen abfaa1 dan abfab3 menunjukan bahwa masing-masing klon secara berurutan mengandung dna berukuran 1721 dan 1739 bp. masing-masing klon dna terdiri dari satu hipotetis kerangka pembacaan terbuka berukuran 1503 dan 1509 bp yang mengkodekan protein abfa berukuran 500 dan 502 asam amino secara berurutan untuk klon abfaa1 dan abfab3. urutan asam amino hasil deduksi abfab3 mempunyai kemiripan 75% terhadap abfaa1. enzim rekombinan diekspresikan secara konstitutif pada e. coli. karakterisasi parsial dari enzim tersebut menunjukkan bahwa abfaa1 dan abfab3 masing-masing bekerja secara optimal pada suhu 50 ºc dan 60 ºc pada ph 6. studi termostabilitas enzim rekombinan menggunakan para-nitrophenyl α-l-arabinofranoside pada kondisi optimalnya memperlihatkan bahwa aktivitas abfaa1 turun hingga 50% setelah diinkubasi selama 4 jam pada suhu 50 ºc, sementara abfab3 mempertahankan aktivitasnya hingga lebih dari 75% setelah 12 jam inkubasi pada suhu 60 ºc. studi termostabilitas abfab3 rekombinan ini memperlihatkan untuk pertama kalinya bahwa arabinofuranosidase dari b. licheniformis merupakan enzim termostabil. enzim rekombinan ini memperlihatkan suhu optimal reaksi yang lebih tinggi dibandingkan dengan enzim termostabil yang telah dilaporkan sebelumnya. abfab3 termostabil ini potensial untuk digunakan dalam proses degradasi biomassa lignoselulosa secara sinergis dengan enzim xilanase termostabil dalam proses produksi xilooligosakarida. kata kunci: α-l-arabinofranosidase, bacillus, kloning, termostabilitas in the last few decades, bioconversion of lignocellulosic biomass has received a great deal of attention because of its potential application in various agro-industrial processes especially in the production of renewable biofuel (keshwani and cheng 2009; sanchez and cardona 2008) and chemicals such as xylo-oligosaccharides (akpinar et al. 2009), protein (bals et al. 2007), 2,3-butanediol (saha and bothast 1999), and ferulic acid (hunhammar et al. 1997). alpha-l-arabinofuranosidases (abfa, ec 3.2.1.55) are exo-type enzymes that catalyze the hydrolysis of the cloning of a comparative study of recombinant arabinofuranosidase originating in bacillus subtilis db104 and newly isolated bacillus licheniformis cw1 1,2 1 1 1 mochamad nurcholis , niknik nurhayati *, is helianti , maria ulfah , 1 2 budiasih wahyuntari , and agustin krisna wardani 1 center for bioindustrial technology, badan pengkajian dan penerapan teknologi, jalan mh thamrin 8, jakarta 10340, indonesia; 2 department of food technology, faculty of agricultural technology, universitas brawijaya, jalan veteran, malang 65145, indonesia α-l-arabinofuranosidase genes and its expression in escherichia coli: ag-3') and 1510r (5'gttac(g/c)ttgttacgac tt-3') (dhiaf et al. 2008). the partial 16s rrna gene sequence was analyzed and compared with known bacterial sequences in the ncbi genbank using the blast program (http://blast.ncbi.nlm.nih.gov /blast.cgi) and submitted to genbank (www.ncbi. nlm.nih.gov/genbank). cloning of α-l-arabinofuranosidase-encoding genes. genomic dna was each extracted from b. subtilis db104 and b. licheniformis cw1 according to the phenol/chloroform protocol developed by sambrook and russel (2001). the genes encoding α-larabinofuranosidases were each amplified by pcr from the genomic dna using the following primer pairs: abfabs-f (5'gactagttagttcggtcgaaa gaaatgtttacgc-3') and abfabs-r (5'-gaagat cttta tgactgttttttcaggcggatcac3') for abfaa1, abfabl-f (5'atggtacctaacgctcccaa tcggactgt3') and abfabl-r (5'gcgagatcttc attgtttcttcattctgat3') for abfab3. primers used were based on nucleotides sequences of α-larabinofuranosidase genes of b. subtilis 168 (genbank nc_000964) and b. licheniformis dsm13 (genbank nc_006322). the amplified genes were each cloned into pgem-t easy vector (promega, madison) according to the manufacturer's protocol. positive clones were verified by restriction endonuclease analysis of the recombinant plasmids by using ecori and assays of their expressed recombinant protein a g a i n s t s p e c i f i c s u b s t r a t p a r a n i t r o p h e n y l arabinofuranoside. sequence analysis. identification of the 16s rdna and arabinofuranosidase genes were performed by using basic local alignment search tool (blast, national center for biotechnology information) available at http://blast.ncbi.nlm.nih.gov/blast.cgi with the standard parameters. nucleotide sequences were aligned using clustal w version 11 (thompson et al. 1994) that is accessable as public web server at http://align.genome.jp/. the alignments were visually edited when necessary using genedoc (nicholas and nicholas 1997). the nucleotide sequence of the clone b3 containing the α-l-arabinofuranosidase gene of b. licheniformis cw1 was deposited in the genbank database under accession number jn967641. enzyme production. e. coli transformants harbouring recombinant plasmids containing arabinofuranosidase gene were cultivated in 50 ml lb media containing ampicillin. cells were harvested from the overnight culture by centrifugation at 4025 x g (rotor r10a, hitachi) 4 ºc for 5 min. the pellet non reducing terminal α-l-arabinofuranosidic linkage in hemicellulose such as arabinoxylan, arabinan and other l-arabinose containing polysaccharide. these enzymes act synergistically with other hemicellulases, such as mannanase, xylanase, and acetyl xylan esterase to degrade the hemicellulose backbone completely (ross et al. 1992; gilead and shoham 1995). as a debranching enzyme α-l-arabinofuranosidase is one of hemicellulases that seems to be critical in the early steps of hemicellulose degradation (jeffries 1990). various attempts have been developed to obtain arabinofuranosidase enzymes including direct purification of those native enzymes (kaneko et al. 1994; gilead and shoham 1995; degrassi et al. 2003; raweesri et al. 2008) as well as over-expressing their recombinant enzymes (whitehead and hespell 1995; morales et al. 1995; pei and shao 2008). this view is supported by the genetic information accessible from genbank (www.ncbi.nlm.nih.gov/ genbank). gene cloning and its expression in host cells such as e. coli, bacillus, or yeasts have become a promising method of choice. the objectives of the current study were to clone the two genes encoding α-l-arabinofuranosidase from bacillus subtilis db104 and a newly isolated and identified strain b. licheniformis cw1, as well as to express the cloned genes in e. coli. characteristics of both recombinant enzymes were compared to select for a thermostable arabinofuranosidase having an optimum reaction temperature not less than 60 ºc, which has the potential to be applied synergistically along with thermostable xylanases in xylooligosacharide production from lignocellulosic biomass. this is usually conducted in a reaction temperature not less than 60 ºc. materials and methods bacterial strains and culture conditions. b. subtilis db104 and b. licheniformis cw1 were obtained from bppt culture collection. the strains o were grown at 37 c, shaken at 150 rpm for 16-18 h in lb medium (ph 7.2) containing 10 g soy peptone, 10 g nacl, and 5 g yeast extract per liter. for gene cloning and protein expression e. coli strain dh5α was employed and grown in lb medium containing -1 ampicillin (100 µg ml ). 16s rdna-based identification. bacillus cw1 isolate was identified by 16s rdna-based amplification using universal primers for eubacterial rrna genes, 9f (5'-agagtttgatc(c/a)tggctc 2 nurcholis et al. microbiol indones obtained was resuspended in 5 ml citrate buffer ph 6, and disrupted by sonication (heat system xl ultrasonicator) at the maximum frequency (20 khz) for o 20 s on and 20 s off repeatedly for 5 minutes at 4 c. the crude extracts containing recombinant enzyme were then recovered by centrifugation for 10 min at 5800 x g. enzyme assay. the arabinofuranosidase assay was based on the hydrolysis of para-nitrophenyl α-larabinofuranoside (pnp-a) (sigma-aldrich, usa) described by martinez et al. (2006) with slight modification. the routine assay contained 175 µl of appropriately diluted enzyme sample, 175 µl of 50 mm citric acid-na hpo buffer (ph 6) containing 1 2 4 o mm of pnp-a. mixtures were incubated at 40 c for 20 min, and the reaction terminated by the addition of 700 µl of 1 m na co . the colour intensity of the released 2 3 para-nitrophenol was measured at 405 nm. one unit of activity was defined as the amount of enzyme that produces 1 µmol of p-nitrophenol per min under the assay conditions used (martinez et al. 2006). the unit activity of enzyme is expressed in units of activity per mg of total protein (specific activity). the protein quantification was measured by the bradford method with bovine serum albumin (bsa) as standard (bradford, 1976). e f f e c t s o f p h a n d te m p e r a t u r e o n arabinofuranosidase activity. the effect of temperature on the activity of α-l-arabinofuranosidase was investigated in the reaction mixtures containing 50 mm citric acid-na hpo buffer (ph 6) by measuring 2 4 the activity using pnp-a at specific temperatures ranging from 30-70 ºc. the activity of α-larabinofuranosidase over a ph range of 5-9 was analyzed by reacting the enzyme at their optimal temperatures in three different buffers (50 mm): citric acid-na hpo (ph 5 to 6), phosphate buffer (ph 7 to 8), 2 4 tris-hcl buffer (ph 9) (gilead and shoham 1995) using the routine assay. the results were expressed as a percentage of the activity obtained at either the optimum ph or the optimum temperature (canakci 2007). thermostability. temperature stability of α-larabinofuranosidase enzymes was studied by incubating them at their optimal temperatures in the absence of the pnp-a substrate. at various time intervals, an aliquot of the enzyme was removed and placed on ice. the residual enzymatic activity of each enzyme aliquot was determined by routine assay. the results were expressed as a percentage of residual activity calculated on the basis of the unheated sample. e n z y m e t h e r m o s t a b i l i t y w a s e v a l u a t e d b y determination of decimal reduction time (d-value) and half life (t ). d-value is defined as the time exposure 1/2 required to reduce 90% of initial residual activity of arabinofuranosidase at a constant temperature (wahyuntari and suhartono 2002). the d-value was determined from the negative reciprocal of the slopes of the regression lines, using the linear portions of the residual activity versus time of exposure at constant temperature (as per equation 1): log (a) = log (a ) – t/d (1)0 in which, a is the actual enzyme activity, a is the 0 enzyme activity before exposure to heat, t is length of time of heating, and d is decimal reduction time. the time at a specified temperature required for loss of 50% activity (half life) was calculated based on the equation 1 (wahyuntari and suhartono 2002). results 16s rdna-based identification. an indigenous bacterium cw1 isolate collected by bppt-cullture collection was used as the genetic source in this work. this bacterial strain was isolated from ciseeng hot spring, west java, indonesia. partial sequencing of the amplified 16s rdna of bacillus cw1 isolate revealed 100% similarity to the 16s rdna of bacillus licheniformis. this molecular identification confirms the biochemical identification of the cw1 isolate, as previously identified as b. licheniformis (unpublished data). the partial sequence of the 16s rdna has been submitted to the genbank with accession number jn967640. c l o n i n g a n d e x p r e s s i o n o f α l arabinofuranosidase gene. in this work we have isolated α-l-arabinofuranosidase genes from genomic dna of two bacillus species by means of the p c r c l o n i n g a p p r o a c h . a m p i l i f i c a t i o n o f arabinofuranosidase genes of b. subtilis db104 (abfaa1) and b. licheniformis cw1 (abfab3) revealed 1721 and 1739 bp dna fragments, respectively. restriction endonuclease analysis of the recombinant plasmids resulted in three dna fragments consisting of undigested recombinant plasmids of 4736 or 4740 bp, a vector fragment of 2997 bp and the inserted fragment of 1739 or 1743 bp confirming succesfull gene cloning (fig 1a). the cloned genes were each expressed and assayed to confirm their abfa activity. the recombinant enzymes demonstrated positive activity towards the specific substrate paranitrophenyl α-l-arabinofuranoside indicated by the volume 6, 2012 microbiol indones 3 below 50 ºc, but it decreased drastically at temperatures above 50 ºc (fig 2b). in contrast, enzymatic activity of abfab3 increased along with the increased reaction temperature to reach its maximum activity at 60 ºc. when incubated at their optimal phs and temperatures, abfaa1 (50 ºc) retained less than 60% activity after 4 h (fig 2c) while abfab3 (60 ºc) maintained more than 75% activity after 12 h. employing the equation (1), the following inactivation equations of abfaa1 and abfab3, respectively were obtained: log (a) = -0.0475 (t) + 0.3385 for abfaa1 at 50 ºc (2) log (a) =-0.0095 (t) + 0.6000 for abfab3 at 60 ºc (3) based on equations (2) and (3), the d-value of abfaa1 at 50 ºc and abfab3 at 60 ºc are 8 and 63 h, respectively. the half life of both abfas estimated by the corresponding equations (2) and (3) revealed t 1/2 values of 2 h (abfaa1) and 19 h (abfab3). the predicted t value of abfaa1 seemed to be shorter than 1/2 the actual experimental data by about 4 h (fig 2c). discussion a few arabinofuranosidases have been purified and characterized from the genus bacillus. examples are b. pumilus (degrassi et al. 2003), b. polymyxa (morales et al. 1995), b. stearothermophilus (gilead and shoham 1995; bezalel et al. 1993), and b. subtilis (kaneko et al. 1994). to our knowledge, this is the first report on an arabinofuranosidase characterization isolated from b. licheniformis. in this work, an arabinofuranosidase gene, abfab3, was successfully isolated and cloned from b. licheniformis cw1. identification of the arabinofuranosidase gene worked well using gene color change of the reaction mixtures from clear to a slightly yellow (fig 1b). two positive clones (a1 and b3) were chosen for further analysis of their nucleotide sequences and biochemical properties of the expressed abfas. sequence analysis of abfaa1 and abfab3. sequence analysis of the cloned abfaa1 and abfab3 revealed the presence of an open reading frame of 1503 bp (abfaa1) and 1509 bp (abfab3) for genes encoding a hypothetical protein of 500 and 502 amino acids of abfaa1 and abfab3, respectively. identification of those proteins by database enquiry using blastp tools (http://blast.ncbi.nlm.nih.gov/blast.cgi) determined that the 500 and 502 amino acids proteins were α-l-arabinofuranosidases which showed 100% identity to putative arabinofuranosidases of b. subtilis 168 and b. licheniformis atcc 14580, respectively. the deduced amino acid sequence of abfab3 shares 75% identity with that of abfaa1 (fig 3). no putative signal sequence was detected when amino acid sequences of the abfaa1 as well as abfab3 were analysed using signal prediction tools signalp v1.1 (available as public web server at http://www.cbs.dtu. d k / s e r v i c e / s i g n a l p / ) , i n d i c a t i n g t h a t b o t h arabinofuranosidases are intracellular enzymes. partial characterization of recombinant arabinofuraosidases. effect of temperature and ph on activity and stability of the recombinant abfa were studied. the abfaa1 and abfab3 were active at ph range of 6-8 and temperature of 30-70 ºc. abfab3 was relatively more tolerant to ph in comparison to abfaa. abfab3 retained its enzymatic activity in the ph range of 6-9, whereas that of abfaa1 was active in the ph range of 6-8; however both enzymes showed maximum activity at ph 6 (fig 2a). the enzymatic activity of abfaa1 seemed to be relatively stable at temperatures a 3 kb à 1.7 kb à a1 m b3 c a a1 c b3 b fig 1 verification of recombinant pgem t easy plasmid harbouring abfa of b. licheniformis cw1 (a; line b3) and b. subtilis db104 (a; line a1) by means of ecori restriction endonuclase analysis (a) and qualitative verification of the recombinant abfa activity by means of assays against specific substrate para-nitrophenyl arabinofuranoside (b). m: 1 kb dna marker; c: negative control. 4 nurcholis et al microbiol indones 1995), or butyryvibrio fibrisolvens (50 ºc; hespell et al. 1992), but lower than that of b. stearothermophilus l1 and t-6 (70 ºc; bezalel et al. 1993; gilead and shoham 1995), geobacillus caldoxylolyticus tk4 (7580 ºc; canakci et al. 2007), thermotoga maritima msb8 (90 ºc; miyazaki 2005), and thermobacillus xylanilyticus (90 ºc; debeche et al. 2000). based on these data it can be concluded that in terms of temperature stability, abfab3 is the second best enzyme among bacillus arabinofuranosidase with a dvalue of 63 h and half-life of 19 h. the primary structures of abfaa1 and abfab3 share 71% and 75% identity respectively to that of the characterized thermostable abfa of g. caldoxylolyticus tk4 (canakci et al. 2007). when the amino acid sequence of abfaa1 and abfab3 were compared to that of g. cladoxylolyticus tk4 (abfatk4) in terms of cysteine content, abfaa1 has one less cysteines (6 cys) than abfab3 and abfatk4 (7 cys). all of them contain four conserved cys residues (cys147, cys174, role. however, in the case of b. licheniformis cw1 abfa, cell lysis during exponential growth or during entry into the stationary phase cannot be excluded as a possible reason for the abfa activity detected in the extracellular millieu. further analyses such as subcellular localization or a control study using a cytoplasmic marker protein, such as isocitrate dehydrogenase (gilead and shoham 1995), should be conducted to discriminate the abfa activity caused by cell lysis or secretion. gonzales-pastor et al. (2005) observed that b. subtilis is able to initiate lysis to surrounding cells as a mechanism to postpone sporulation. the activity of the two enzymes towards para-nitrophenyl-arabinofuranoside in various ph and temperature conditions shows that abfab3 is more stable at higher temperature (60 ºc) in comparison to abfaa1 (50 ºc). abfab3 is optimally active at 60 ºc which is higher than the optimal temperature reaction of a thermostable abfa from b. pumilus (55 ºc; degrassi et al. 2003), b. polymyxa (55 ºc; morales et al. fig 3 alignment of the deduced amino acid sequences of the α-l-arabinofuranosidase of b. subtilis db104 (abfaa1) and b. licheniformis cw1 (abfab3). identical amino acids are indicated by black shades. hyphens indicate gaps. consensus sequence indicated below is based on the allignment of abfab3, abfaa1 and putative abfas of b. amyloliquefaciens ta208 (ncbi-gi: 328554491), b. halodurans (ncbi-gi: 15614424), b. atrophaeus (ncbi-gi:311066968) as well as the known abfas of geobacillus stearothermophilus (ncbi-gi:122937809) and g. cladoxylolyticus (ncbigi:113374907). the blue arrow head (▼ ) indicates the cys positions assumed to be crucial in the protein stabilization. the red blocked c (c) indicates conserved cys whereas the yellow blocked cys (c) indicates the unique cys of the corresponding arabinofuranosidase. 6 nurcholis et al microbiol indones agricultural wastes. food and bioprod process. 87(2): 145-151. doi:10.1016/j.fbp.2008.09.002. antelmann h, tjalsma h, voight b, ohlmeier s, bron s, van dijl jm, hecker m. 2001. a proteomic view on genomebased signal peptide predictions. genome res. 11:14841502. doi: 10.1101/gr.182801. bals b, teachworth l, dale b, balan v. 2007. extraction of proteins from switchgrass using aqueous ammonia within an integrated biorefinery. appl biochem biotechnol. 143(2):187-198. bendtsen jd, kiemer l, fausboll a, brunak s. 2005. nonclassical protein secretion in bacteria. bmc microbiol. 5. [on line]. doi:10.1186/1471-2180-5-58. bezalel l, shoham y, rosenberg e. 1993. characterization and delignification activity of a thermostable α-larabinofuranosidase from bacillus stearothermophilus. appl environ microbiol. 40:57-62. bradford mm. 1976. a rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal biochem. 72:248-254. canakci s, belduz ao, saha bc, yasar a, ayaz fa, yayl n. 2007. purification and characterization of a highly t h e r m o s t a b l e α l a r a b i n o f u r a n o s i d a s e f r o m geobacillus caldoxylolyticus tk4. appl microbiol biotechnol. 75:813-820. doi:10.1007/s00253-0070884-1. debeche t, cummings n, connerton i, debeire p, o'donohue mj. 2000. genetic and biochemical characterization of a highly thermostable α-la r a b i n o f u r a n o s i d a s e f r o m t h e r m o b a c i l l u s xylanilyticus. appl environ microbiol. 66(4):17341736. pmcid: pmc92053. degrassi g, vindigni a, venturi v. 2003. a thermostable alpha-arabinofuranosidase from xylanolytic bacillus pumilus: purification and characterisation. j biotechnol. 101(1): 69-79. dhiaf a, bakhrouf a, witzel k. 2008. resuscitation of eleven-year vbnc citrobacter. j water health. 6(4): 565-568. fenel f, leisola m, jänis j, turunen o. 2004. a de novo designed n-terminal disulphide bridge stabilizes the trichoderma reesei endo-1,4-β-xylanase ii. j biotechnol. 108:137-143. gilead s, shoham y. 1995. purification and characterization of alpha-l-arabinofuranosidase from bacillus stearothermophilus t-6. appl environ microbiol. 61(1):170-174. doi: 0099-2240/95/04.0010. gonzalez-pastor je, hobbs ec, losick r. 2003. cannibalism by sporulating bacteria. science 301(5632):510-513. hespell rb, o'bryan pj.1992. purification and characterization of an α-l-arabinofuranosidase from butyrivibrio fibrisolvens gs113. appl environ m i c r o b i o l . 5 8 ( 4 ) : 1 0 8 2 1 0 8 8 . d o i 0 0 9 9 2240/92/041082-07$02.00/0 hirose i, sano k, shioda i, kumano m, nakamura k, yamane k. 2000. proteome analysis of bacillus subtilis cys216, and cys351). the remaining non conserved cys residues are located in different positions within each abfa. one cysteine residue (cys198) has a location which indicates it is essential in stabilizing the abfa of g. cladoxylolyticus. this cys198 is altered to alanine (ala198) which is conserved among seven different abfa’s including abfaa1 and abfab3 (fig 3). this alteration (a198c) may lead to a possible formation of disulfide bond between cys198 and cys216 which could stabilize the protein conformation of abfatk4 resulting in 10 or 20 ºc higher thermostability in comparison to abfab3 or abfaa1, respectively. a similar phenomenon is observed between abfab3 and abfaa1 sequences. one cysteine residue (cys335) which is conserved in abfatk4 as well as abfab3, is altered to alanine (a335) in abfaa1. this alteration (c335a) is most probably responsible for the lower thermostability of abfaa1 in comparison to abfab3, as the result of a possible loss of the disulfide bond formed between cys335 and cys351. however further studies on site directed mutagenesis of either abfaa1 or abfab3 should be carried out to support this hypothese since cysteine content is not the only factor to contribute to protein thermostabilization. the important role of cys in the protein thermostability has been reported in the case of α-amylase of pyrococcus furiosus (savchenko 2002), endoglucanase cel12a of humicola grisea (sandgren et al. 2003), and manosidase of aspergilus satoi (tatara et al. 2005). in all these studies it is evident that substitution at cys residue greatly decreased thermostability of the enzyme. in other studies, cysteine residues were introduced to form de novo disulfidebridges in order to improve thermostability of endoxylanase of t. reesei (fenel et al. 2004) and b. stearothermophilus (jeong 2007). cysteine frequently forms a disulfide bond that stabilizes proteins by lowering their conformational entropy compared with their unfolded state (zhang et al. 2011). based on its relatively higher thermostability, the newly isolated and characterized arabinofuranosidase of b. licheniformis cw1 is potential to be applied in the degradation process of lignocellulose biomass in combination with thermostable xylanases, which usually conducted at high temperature (not less than 60 ºc) to produce xylooligosaccharides. references akpinar o, erdogan k, bostanci s. 2009. enzymatic production of xylooligosaccharide from selected volume 6, 2012 microbiol indones 7 purification, characterization and its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural residues. bioresourtechnol. 99(18):8981-8986. rosch j, caparon m. 2004. a microdomain for protein secretion in gram-positive bacteria. science 304:15131515. doi: 10.1126/science.1097404. ross nw, johnson kg, braun c, mackenzie cr, schneider h. 1992. enzymatic hydrolysis of water-soluble lignincarbohydrate complexes from populus deltoiedes: effect of combinations of b-mannanases, xylanase and acetyl xylan esterase. enzyme microb technol. 14:90–95. saha bc, bothast rj.1999. production of 2,3-butanediol by a newly isolated enterobacter cloacae. appl microbiol biotechnol. 52:321-326. doi: 10.1007/s002530051526. sambrook j, russell dw. 2001. molecular cloning a rd laboratory manual vol 1. 3 ed. cold spring harbor laboratory press: cold spring harbor, new york. sanchez o,cardona ca. 2008. trends in biotechnological production of fuel ethanol from different feedstocks. bioresour. technol. 99:5270–5295. doi:10.1016/ j.biortech.2007.11.013. sandgren m, gualfetti ppj, paech c, paech s, shaw a, gross ls, saldajeno m, berglund gi, jones ta, mitchinson c. 2003. the humicola grisea cel12a enzyme structure at 1.2 å resolution and the impact of its free cysteine residues on thermal stability. prot sci. 12:2782-2793. savchenko a, vieille c, kang s, zeikus jg. 2002. pyrococcus furiosus a-amylase is stabilized by calcium and zinc. biochemistry 41: 6193-6201. tatara y, yoshida t, ichishima e. 2005. a single free cysteine residue and disulfide bond contribute to thermostability of aspergillus saitoi 1,2-α-mannosidase. biosci b i o t e c h n o l b i o c h e m . 6 9 ( 1 1 ) : 2 1 0 1 2 1 0 8 . doi:10.1271/bbb.69.2101. thompson jd, higgins dg, gibson tj. 1994. clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. nucleic acids res. 22(11):4673-4680. doi: 10.1093/nar/22.22.4673. wahyuntari b, suhartono m. 2002. thermostability of an extreme thermophilic extracellular protease bacterium bacillus tps2d from tangkuban perahu. hayati 9(4):114-118. whitehead tr,hespell rb. 1990. the genes for three xylandegrading activities from bacteroides ovatus are clustered in a 3.8-kilobase region. j bacteriol. 172(5):2408-2412. zhang l, chou cp, moo-young m. 2011. disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by escherichia coli expression system. biotechnol adv. 29: 923–929. doi:10.1016/ j.biotechadv.2011.07.013. extracellular proteins: a two-dimensional protein electrophoretic study. microbiology 146(1):65-75. hunhammar m, faulds cb, artolome b, williamson g. 1997. novel biotransformations of agro-industrial cereal waste by ferulic acid esterases. ind crops prod. 6:367-374. doi:10.1016/s0926-6690(97)00027-7. inacio jm, correia1 il, de sa´-nogueira i. 2008. two distinct arabinofuranosidases contribute to arabinooligosaccharide degradation in bacillus subtilis. microbiology 154:2719–2729. doi: 10.1099/mic.0. 2008 /018978-0. jeffries tw. 1990. biodegradation of lignin-carbohydrate c o m p l e x e s . b i o d e g r a d a t i o n 1 ( 2 3 ) : 1 6 3 1 7 6 . doi:10.1007/bf00058834. jeong my, kim s, yun cw, choi yj, chob sg. 2011. engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from bacillus s t e a ro t h e r m o p h i l u s n o . 2 3 6 . j b i o t e c h n o l . 127(2):300–309. kaneko s, sano m, kusakabe i. 1994. purification and some properties of alpha-l-arabinofuranosidase from bacillus subtilis 3-6. appl environ microbiol. 60(9):3425-3428. doi:0099-2240/94/04.00+0. keshwani dr, cheng jj. 2009. switchgrass for bioethanol and other value-added applications: a review. bioresour technol. 100: 1515-1523. doi.org/10.1016/j.biortech. 2008.09.035. martinez c, gertosio c, labbe a, perez r, ganga ma. 2006. production of rhodoturola glutinis: a yeast that secretes α-l-arabinofuranosidase. electron j biotechnol. 9(4):407-413. [on line]. doi:10.2225/vol9-issue4fulltext-8. m i y a z a k i k . 2 0 0 5 . h y p e r t h e r m o p h i l i c α l arabinofuranosidase from thermotoga maritima msb8: molecular cloning, gene expression, and characterization of the recombinant protein. extremophiles 9(5):399-406. doi:10.1007/s00792-0050455-2. morales p, sendra jm, gonzales p. 1995. purification and characterization of an arabinofuranosidase from bacillus polymyxa expressed in bacillus subtilis. appl m i c r o b i o l b i o t e c h n o l . 4 4 ( 1 2 ) : 1 1 2 1 1 7 . doi:10.1016/0141-0229(94)00062-v. nicholas kb, nicholas hb. 1997. genedoc: a tool for editing and annotating multiple sequence alignments. http://www.psc.edu/biomed/genedoc. park jm, han ns. 2007. rapid detection and isolation of known and putative alpha-l-arabinofuranosidase genes using degenerate pcr primers. j microbiol biotechnol. 17(3):481-489. pei j, shao w. 2008. purification and characterization of an extracellular alpha-l-arabinosidase from a novel isolate bacillus pumilus ara and its over-expression in escherichia coli. appl microbiol biotechnol. 78(1):115-121. doi:10.1007/s00253-007-1295-z. r a w e e s r i p p, r i a n g r u n g r o j a n a . 2 0 0 8 . a l p h a l arabinofuranosidase from streptomyces sp. pc22: 8 nurcholis et al microbiol indones 1: 1 2: 2 page 3 page 4 page 6 page 7 page 8 abidin, laode baytul, 75 abinawanto, 1, 62 abqory, muhammad hidayat, 129 aditiawati, pingkan, 129 agung, mochamad untung kurnia, 123 ambarsari, hanies, 103 anggiani, milani, 62 ariadji, tutuka, 129 aryantha, i nyoman pugeg, 46 astuti, dea indriani, 129 astuty, sri, 123 armiyanti, yunita, 23 budisatria, rachel, 55 busniah, munzir, 117 cahyani, martha eka, 1 daniswara, pramusita yoga, 111 dwiyantari, 28 estuningsih, sri pertiwi, 81 harahap, muhammad rahmadi, 103 helianti, is, 1, 35, 62 herlina, leni, 28 indiastuti, danti nur, 89 jap, lucy, 55 jan, tan tjie, 55 jemsi, windi silvani, 46 karima, aulia, 81 kristiawan, onie, 28 kristianti, tati, 28 kustyawati, maria erna, 94 kusumaningrum, deby, 89 lembong, elazmanawati, 137 lusiastuti, angela mariana, 69 munaeni, waode, 75 mulyani, yeni, 123 nurhayati, niknik, 1, 62 nuryady, mohammad mirza, 23 nusetyawan, muhammad ardian, 111 pariakan, arman, 75 pratama, lisa, 35 purwasena, isty adhitya, 129 putra, boby pratama, 89 putrie, rahayu fitriyani wangsa, 18 rangga, azhari, 94 ratih, nine kirana, 11 reflin, 117 rohmat, riesa kw, 28 roriris, evina tami, 123 rosaria, 55 rosariastuti, retno, 111 said, muhammad, 81 sani, indiani, 129 sari, cut nanda, 28 senjarini, kartika, 23 setyani, sri, 94 soka, susan, 11 stephanie, 11 sugandi, wahyu kristian, 137 suhandono, sony, 28 sukiman, harmastini, 18 suliandri, ken sawitri, 28 sumani, 111 supriyadi, 111 suryadi, edy, 137 suryani, ani, 35 suwanto, antonius, 11 ulkhaq, mohammad faizal, 69 usman, 28 utama, gemilang lara, 137 utomo, sugeng setyo, 23 wahyuntari, budiasih, 35 warnita, 117 widjajati, sri mumpuni wahyu, 23 widowati, tiwit, 18 yanti, yulmira, 117 yudono, bambang, 81 yuhana, munti, 75 author index volume 11 vol.11, no.4, december 2017, p 147 microbiology indonesia issn 1978-3477, eissn 2087-8575 16s rrna, 117 28s nrdna gene, 123 activated sludge, 94 aeromonas hydrophila, 69 akkermansia muciniphila, 11 antibacterial, 89 antibiotics, 69 anopheles sundaicus, 23 bacillus cereus, 46 bacillus licheniformis, 129 banana, 55 bawang hutan bulbs extract, 75 bhurkholderia glumae, 81 biofertilizer, 111 brevundimonas diminuta, 81 characterization, 117 chemical change, 103 clarias gariepinus, 69 crude biosurfactant, 81 crude oil, 129 dilution susceptibility test, 89 dinoflagellates, 123 elisa, 11 em4, 94 endophytes, 117 enhanced oil recovery, 28 enzyme, 35 ethanol tolerance, 137 indigenous yeasts, 137 fungi, 35 iaa production, 18 iga, 11 igg, 23 indigenous halo tolerant bacteria, 81 inhibitory potential, 75 gc-ms, 129 glucose tolerance, 137 interesterification, 35 lactic acid, 55 lipase, 35 malaria, 23 meor, 129 methicillin-resistant staphylococcus aureus (mrsa), 89 mixed culture co-inoculation, 103 mocaf waste, 111 mushroom growth promoting bacteria (mgpb), 46 mutation, 18 nitric acid, 18 nitrification, 94, nitrogen fixing-bacteria, 111 oligosaccharides, 55 original signal peptide, 62 overlapped pcr method, 28 pas, 55 peat carrier, 111 pgpr, 117 phosphate solubilizing bacteria, 111 pichia pastoris, 1, 62 pineapple core, 89 prebiotics, 55 phytochemical, 75 ppiczα a, 1, 62 recovery oil, 81 resistence, 69 rhizomucor miehei, 1 rt-pcr, 11 saccharomyces cerevisiae, 103 salivary, 23 sara, 129 shrimp pond sediment, 94 starch granule, 103 surfactant peptide, 28 symbiodinium, 123 synthetic lipase gene, 1, 62 tacca leontopetaloides, 137 tempeh, 11 thermally-tolerant, 123 thermomyces lanuginosus, 62 vancomycin, 89 vibrio harveyi, 75 volvariella volvacea ww-08, 46 subject index volume 11 vol.11, no.4, december 2017, p 148 microbiology indonesia issn 1978-3477, eissn 2087-8575 cloning of synthetic lipase gene from rhizomucor miehei with original signal peptide in pichia pastoris. martha eka cahyani, is helianti, niknik nurhayati, abinawanto…………....... effect of tempeh supplementation on the profiles of human intestinal immune system and gut microbiota. stephanie, nine kirana ratih, susan soka, antonius suwanto….............. studies for iaa (indole-3-acetic acid) production by isolates h6 with nitric acid mutation. rahayu fitriyani wangsa putrie, tiwit widowati, harmastini sukiman…………..... analysis of human immune response against salivary glands protein extract of anopheles sundaicus in malaria endemic area. mohammad mirza nuryady, sugeng setyo utomo, yunita armiyanti, sri mumpuni wahyu widjajati, kartika senjarini……………........... construction and expression of single recombinant peptide surfactant for eor application. cut nanda sari, usman, riesa kw rohmat, leni herlina, ken sawitri suliandri, onie kristiawan, diwiyantari, tati kristianti, sony suhandono…………................................... isolation, characterization, and production of lipase from indigenous fungi for enzymatic interesterification process. lisa pratama, is helianti, ani suryani, budiasih wahyuntari………………………………………………………………….............................. potential mushroom growth promoting bacteria (mgpb) in optimizing paddy straw mushroom (volvariella volvacea ww-08) growth. windi silvani jemsi, i nyoman pugeg aryantha………………….......................................................................................................... in vitro and in vivo prebiotic activities of purified oligosaccharides derived from various local bananas (musa sp.): tanduk, uli, raja sereh, and cavendish. rachel budisatria, rosaria, lucy jap, tan tjie jan……………………..................................................................... cloning of lipase gene from thermomyces langinosus into pichia pastoris with its original signal peptide. milani anggiani, is helianti, niknik nurhayati, abinawanto.............. resistance test on aeromonas hydrophila isolated from african catfish (clarias gariepinus) against some antibiotics groups. mohammad faizal ulkhaq, angela mariana lusiastuti………………………………………………………………………......................... in vitro phytochemical and inhibitory potential test of bawang hutan bulb extract (eleutherine palmifolia) on vibrio harveyi. waode munaeni, arman pariakan, laode baytul abidin, munti yuhana…………………………………….......................................................... oil recovery test using biosurfactant of halo tolerant bacteria brevundimonas diminuta and bhurkholderia glumae at variation of nacl salt concentrations. bambang yudono, muhammad said, sri pertiwi estuningsih, aulia karima………................................................. antibacterial potentiality testing of pineapple core extract (ananas comosus (l.) merr) against methicillin-resistant staphylococcus aureus (mrsa) with vancomycin control. boby pratama putra, danti nur indiastuti, deby kusumaningrum……………........................... performance optimization of microbes from shrimp pond sediment by adding em4 in nitrification process for the treatment of wastewater containing high ammonia concentration. hanies ambarsari, muhammad rahmadi harahap............................................ the dynamic growth and chemical change of mixed cultures inoculation on tapioka fermentation. maria erna kustyawati, azhari rangga, sri setyani………................................ the utilization of modified cassava flour (mocaf) industry waste and peat as carrier of nitrogen-fixing bacteria and phosphate-solubilizing bacteria inoculant. retno rosariastuti, sumani, supriyadi, muhammad ardian nursetyawan, pramusita yoga daniswara. identification and characterizations of potential indigenous endophytic bacteria which had ability to promote growth rate of tomato and biocontrol agents of ralstonia solanacearum and fusarium oxysporum fsp. solani. yulmira yanti, warnita, reflin,munzir busniah………………………………………………................................................................ molecular identification of thermally-tolerant symbiotic dinoflagellates from hard list of contents volume 11 1 11 18 23 28 35 46 55 62 69 75 81 89 94 103 111 117 microbiology indonesia issn 1978-3477, eissn 2087-8575 coral (scleractinia) in biawak island, indonesia. evina tami roriris, mochamad untung kurnia agung, sri astuty, yeni mulyani………………………................................................... isolation and identification of ethanol and glucose tolerance yeasts strain from tacca leontopetaloides. gemilang lara utama, wahyu kristian sugandi, elazmanawati lembong, edy suryadi………………………………………….................................................................. potential degradation of sara (saturated, aromatics, resinics, asphaltenes) fractions of crude oil by reservoir indigenous bacteria from south sumatera. dea indriani astuti, isty adhitya purwasena, pingkan aditiawati, indiani sani, tutuka ariadji, muhammad hidayat abqory......................................................................................................................................... author index .............................................................................................................................. subject index ............................................................................................................................. 123 129 137 147 148 volume 11, 2017 microbiol indones guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of 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vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on 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thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com author index 05 lindayani.cdr vol.12, no.1, march 2018, p 30-34 doi: 10.5454/mi.12.1.5 short communication probiotic potential of lactic acid bacteria from yellow bamboo shoot fermentation using 2.5% and 5% brine at room temperature lindayani*, laksmi hartajanie, and monika palupi murniati unika soegijapranata, jalan pawiyatan luhur iv/1, semarang 50234, indonesia yellow bamboo shoot is a popular food material especially in semarang because it is used as filling for lumpia (authentic food of semarang). beside used as filling, yellow bamboo shoot commonly known by indonesian people. considering bamboo shoot often processed into fermented traditional food, yellow bamboo shoot also potential to be examined as source of fermented food producing lactic acid bacteria. lactic acid bacteria still become the most beneficial microorganisms associated with gastrointestinal system and moreover for obesity diet. the aim of this research is to study the probiotic potential of lactic acid bacteria produced from yellow bamboo shoot fermentation in 2.5% and 5% brine under room temperature (30 °c). from isolation, 22 single colonies obtained from 2.5% brine and 27 isolates obtained from 5% brine. the morphology and physiology analysis resulted in lactobacillus and streptococcus genus. all isolates were tested subsequently for probiotic potential. based on the result, more than 50% identified isolates have probiotic potential. key words: lactic acid bacteria, probiotic, yellow bamboo shoot rebung bambu kuning merupakan bahan makanan yang populer khususnya di semarang karena digunakan sebagai pengisi untuk lumpia (makanan asli semarang). selain digunakan sebagai pengisi, rebung bambu kuning umum dikenal masyarakat indonesia. diketahui bahwa rebung umumnya diproses sebagai makanan fermentasi secara tradisional, rebung bambu kuning berpotensi diteliti sebagai sumber makanan fermentasi yang menghasilkan bakteri asam laktat. bakteri asam laktat menjadi mikroorganisme yang menguntungkan berasosiasi dengan sistem pencernaan dan juga berguna untuk diet bagi orang yang kegemukan. tujuan dari penelitian adalah untuk mempelajari potensi probiotik bakteri asam laktat yang dihasilkan dari fermentasi rebung bambu kuning dalam 2,5% dan 5% larutan garam pada 30 °c. berdasarkan hasil isolasi, diperoleh 22 koloni tunggal dari fermentasi 2,5% larutan garam dan 27 koloni tunggal dari 5% larutan garam. hasil analisa morfologi dan fisiologi diperoleh genus lactobacillus dan streptococcus. semua isolasi dilakukan uji untuk mengetahui potensi probiotik. berdasarkan hasil ujinya diketahui 50% isolasi mempunyai potensi probiotik. kata kunci: bakteri asam laktat, probiotik, rebung bambu kuning microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-24-8441555, email: lindayani@unika.ac.id 2015; rahayu and margino 1997). lab has contributed in fermented food development especially fermented food with probiotics (lindayani and hartayanie 2013). the following genera considered the principal lab i.e. lactobacillus, leuconostoc, streptococcus pediococcus. the growth of lab is greatly influenced by intrinsic and extrinsic factors. different fermentation temperature, salt concentration, and area condition may allow for differences of lab despite the same source or material used (usmiyati and marwati 2007). vegetable and fruit fermentation usually use salting method without addition of starter culture. beside to extend shelf life, salting also affect the flavor formed by microorganism which occurs during fermentation, especially lab (rahayu 2003). lab produced lactic acid as metabolite causing acidity to raw material (food). this condition gives advantage as low ph lactic acid bacteria (lab) are group of beneficial microorganisms which important in food industry i.e. food fermentation, thus can extend the shelf life of food product (othman et al. 2017; lindayani and hartayanie 2013). lab fermentation produces lactic acid and smaller amount of acid i.e. acetic acid, ethanol, and co which inhibit spoilage and 2 pathogenic bacteria. the example is antimicrobial activity of lab isolated from beef (al-allaf et al. 2009), fermented milk (savadogo et al. 2004), and traditional fermented beverage from ethiopia (tadasse et al. 2005). the benefit of lab in food industry has promoted further exploration into bacteria strain from various sources like fruit and vegetable fermentation (nuraida inhibits nearly all food spoilage microorganisms. according to magala et al. 2015; josephsen and jespersen cit. hui et al. (2004), lab produces lactic acid and other antimicrobial substance which can inhibit the growth of spoilage bacteria. probiotic is living microorganisms that, when administered in adequate amounts confer health benefit on the host such as maintaining the balance of gut microbiota i.e. lactobacillus acidophilus and protein. probiotics produce lactic acid, acetic acid, hydrogen peroxide, lactoperoxidase, lipopolysaccharides, and other antimicrobial such as bacteriocins. bacteriocin has ability to inhibit pathogenic bacteria with close relation to the producing strain. bamboo shot is young bamboo bud that comes out of the ground. this young bud generally edible, thus included in group of vegetable, i.e. betung bamboo shoot (dendrocalamus asper), temen bamboo shoot (gigontochloa verticillata), yellow bamboo shoot (dendrocalamus litiforus), dan green bamboo shoot (bambusa aldhami) (andoko 2003). raw bamboo shoot has 90% water content per gram material (rai 2007). f e r m e n t a t i o n o f b a m b o o s h o o t o c c u r s spontaneously. the process carried on by adding salt solution (brine). salt solution has functions to draw the liquid inside the bamboo shoot out through osmosis and to inhibit pathogenic bacteria, allowing only lab to grow. the final product of fermentation often called yellow bamboo shoot pickle. salt effects the growth of lab. in addition, salt has important role in fermentation process, where during fermentation, only lab that grow. in a short fermentation, the salt addition is preferably 2.5-10% to allow intermediate to fast fermentation rate. low salt concentration (<2.5%) may cause proteolitic bacteria to grow. too high salt concentration (>10%) may result on wrinkled end fermentation product and may allow halophilic bacteria to grow or even no fermentation occurs. in indonesia, research about lab is required scientifically because there is still lack of data's about lab which isolated from crop fermentation, whereas it is known that the source of crop is abundant. in this research, selection of bamboo shoot has come from consideration that bamboo shoot is one of food material which get less attention and mostly used only for filling traditional food like lumpia in semarang. either edible or non-edible bamboo shoot are available in great amount and lab which obtained from bamboo shoot fermentation has a bigger opportunity to be developed to industrial scale to support fermentation product which can contributes better quality of human living. this research was done at microbiology laboratory, faculty of agricultural technology, soegijapranata catholic university, semarang. materials and chemicals used i.e. yellow bamboo shoot which obtained from semarang , mrs broth and agar (merck), alcohol 95%, naoh solution, hcl solution, gram staining solution (violet crystal, lugol's iodine solution, alcohol / ethanol 95%, safranin), hydrogen peroxide (h o solution) 3%, sterile aquadest, bile salt 2 2 (zigma-aldrich) and bacteria culture (staphylococcus aureus, eschericia coli). equipment used i.e. petridish, reaction tubes, microscope, object glass, ose, hockey stick glass, gram staining tools, spectrophotometer (shimadzu), incubator, pipette, micropipette, autoclave, sterile filter discs, and durham tubes. bamboo shoot fermentation was done for 7 d. based on isolation and morphological test, 22 isolates were obtained from 2.5% fermentation under room temperature (code c) and 27 isolates from 5% fermentation under room temperature (code d). all bacteria sample were purified subsequently. each colony which has clear zone was tested subsequently to morphological test. based on morphological test, all identified isolates give negative result on catalase test and non-motile (fig 1 and 2). based on gas formation test, some isolates produced gas; some isolates produced no gas (no growth) (fig 3). according to tadesse et al. (2005), lactic acid bacteria have characteristic i.e. rod or cocci, gram positive, some producing co (heterofermentative), 2 some not producing co (homofermentative).2 physiological analyses were tested including growth at different nacl concentration (6.5% and 18% of nacl), growth at different ph (4.4 and 9.6), and growth at different temperature (10 °c, 45 °c, and 50 °c). the result shown that all isolates code c were not growth at different nacl concentration, whereas for isolates code d, only one isolate able to grow. at different ph, in ph 4.4 medium found ten isolates from code c were grown, whereas only six isolates were grown from isolate code d. in ph 9.6 medium, two isolates were grown for code c whereas for d code only one isolate was grown. based on growth at 10 ºc, 45 ºc, and 50 ºc were found that all isolates were able to grow at 10 ºc incubation temperature, except one volume 12, 2018 microbiol indones 31 isolate from code c which not grow. similarly at 45 ºc, all isolates were able to grow. but, at 50 ºc, some isolates were not growing. based on morphological and physiological analyses, lactic acid bacteria were categorized as lactobacillus and streptococcus. lactobacillus bacteria generally found in plants, vegetables, dairy-based product, meat product, inside gastrointestinal tract of human and animal (ray and bhunia 2008; karovičová and kohajdová 2005). all isolates (22 code c and 27 code d) were tested subsequently to probiotic potential i.e. tolerance to acid (ph 3 and 7 at 0, 1.5 and 3 h incubation) and bile salt (0.3% and 0.5% concentrate at 0, 2, and 4 h incubation), and antimicrobial test against staphylococcus aureus and eschericia coli. from result of bile salt tolerance test, it is found that for code c, 12 isolates were not able to grow in 0.3% bile salt medium at 0, 2, 4 h incubation, while in 0.5% bile salt medium at 0, 2, 4 h incubation 13 isolates were not able to grow. for code d, 5 isolates were not able to grow in 0.3% bile salt medium at 0, 2, 4 h incubation, while in 0.5% bile salt medium at 0, 2, 4 h incubation 11 isolates were not able to grow. from result of acid tolerance test, isolates code c and d shown different fig 1 catalase test of lactic acid bacteria fermented in 5% brine under room temperature. fig 2 motility test of lactic acid bacteria. the growth in stabbing area shows that the isolates are non-motile. fig 3 isolate shows no gas production (homofermentative). 32 lindayani et al. microbiol indones response. isolates code c in ph 3 medium at 0, 1.5, 3 h incubation shown four isolates were not able to grow, while in ph 7 medium at 0, 1.5, 3 h incubation found 3 isolates were not able to grow. isolates code d in ph 7 medium at all incubation times were able to grow and in ph 3 medium at all incubation times shown 2 isolates were not able to grow. based on antimicrobial test against staphylococcus aureus and eschericia coli, the result shown four isolates code c able to inhibit growth of staphylococcus aureus and eschericia coli while 21 isolates from code d were able to inhibit the growth of staphylococcus aureus and eschericia coli. according to saarela et al. (2000), probiotics should have characteristics i.e. tolerance to bile salt, able to survive in human gastrointestinal system, has antagonistic properties against pathogenic bacteria, have antimutagenic and anticarsinogenic properties. screening bacteria with probiotic characteristic has been performed by ability to survive at low ph, tolerance to bile salt and antibacterial activity against pathogen (e. coli and s. aureus). lactic acid bacteria should have ability to survive at low ph because lactic acid bacteria will enter human gastrointestinal tract which has low ph. almost all isolates were able to survive at ph 3 and 7. according to research reported by jay (2000) cit. mavhungu (2005); battcock and azam-ali (1998) which stated that almost all bacteria include lactobacillus genus, able to grow at neutral ph. ability to survive of lactic acid bacteria is effected by bile salt concentration. higher the concentrate of bile salt, the ability to survive of lactic acid bacteria is lower. bile salt causes destruction to bacterial cell membrane, where the main components of cell membrane are lipid and fatty acid. based on research done by klayraung et al. (2008), lactobacillus genus which isolated from fermented pork, fermented fish, fermented tea, and garlic pickle are able to survive at 0.3%-1% bile salt environment. in overall, isolates code d have more inhibition ability against two indicator pathogens (e. coli and s. aureus). antimicrobial activities by lactic acid bacteria is caused by the ability of lactic acid bacteria producing acid which inhibit pathogenic bacteria, or because another substance which called bacteriocins which produced by that bacteria (josephsen and jespersen cit. hui et al. 2004). to conclude, pure isolates from yellow bamboo shoot pickle in brine 2.5% and 5% in room temperature are 22 and 27 isolates, respectively. according to morphological and physiological, all isolates have been identified as lactobacillus and streptococcus. lactic acid bacteria which obtained from yellow bamboo shoot pickle have probiotic potential. therefore, yellow bamboo shoot, has an opportunity to be probiotic bacteria source which can contributes to better quality of human living. acknowledgment the researcher would like to thank “direktorat jendral pendidikan tinggi untuk penelitian unggulan perguruan tinggi (pupt) 2014-2015” for financial support. thank you also to amelia juwana, cynthia christine, lorentia santoso, agata apriliana sundoro and all parties which have been helping this research. references andoko a. 2003. budidaya bambu rebung. 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[selection and optimation of process of bacteriocin production from lactobacillus sp.]. jurnal pascapanen. 4(1) 2007: 27-37. http://pascapanen.litbang.pertanian.go.id/assets/media/pu blikasi/jurnal/j.pascapanen.2007_1_4.pdf 34 lindayani et al. microbiol indones page 1 page 2 page 3 page 4 page 5 7 523 16s dissemination (joko... dissemination in pigtailed macaques after primary infection of dengue-3 virus joko pamungkas , diah iskandriati , uus saepuloh , moses affandi , esther arifin ,yasmina paramastri , fitriya nur anisa dewi , dondin sajuthi 1,2* 1,3 1 4 1,3 1,3 3 1,2 and 1 2 3 4 primate research center, institut pertanian bogor, jalan lodaya ii/5, bogor 16151, indonesia; faculty of veterinary medicine, institut pertanian bogor, jalan agatis darmaga, bogor, indonesia; pt. bimana indomedical, jalan kh rd abdullah bin nuh 3, sindangbarang, bogor 16115, indonesia; faculty of biotechnology, unika atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia macaca nemestrina macaca nemestrina macaca nemestrina one step reverse transcription real time pcr macaca nemestrina nonhuman primates (nhps) play as indispensable animal model in biomedical research for studying a variety of human health issues, diseases and disorders, therapies, and preventive strategies. since the immunological and physiological responses of nhps, at some extent, to experimental viral infections are similar to humans, it is possible that studies of dengue infection in nhps may aid understanding of dengue infection in humans. in this study, we used pigtailed macaques ( ) as the experimental animal to study dengue-3 (den-3) virus infection. we evaluated den-3 viral distribution and replication sites after a primary infection in all collected tissues. sequential localization in tissue of den-3 virus was studied in pigtailed macaques euthanized three days post viral inoculation (10 pfu ml ). pigtailed macaque that was inoculated subcutaneously or intravenously; showed the highest viremia (62.94 pfu ml and 58.62 pfu ml ) detected by one step reverse transcription real time pcr. the virus inoculated in pigtailed macaques by subcutaneous injection was rapidly disseminated from the inoculation site to the lymph nodes, adrenal glands, kidneys, heart, thyroid, liver, prostate gland, and seminal vesicles. meanwhile, dissemination of dengue virus in pigtailed macaques inoculated intravenously was detected in lymph nodes, thymus, salivary glands, liver, and prostate gland. this study suggested that the above mentioned-tissue specimens are involved or affected by den-3 virus replication and the route of infection seemed to have influenced the virus dissemination. key words: , animal model, dengue-3 virus, dissemination, rt-pcr satwa primata merupakan hewan model yang sangat penting dalam penelitian biomedis untuk memperlajari berbagai permasalahan kesehatan manusia, terutama terkait pengobatan dan strategi pencegahannnya. mengingat keadaan respons fisiologi dan respons kebal satwa primata terhadap infeksi virus mirip dengan yang dijumpai pada manusia, sangat dimungkinkan bahwa penelitian infeksi virus dengue pada satwa primata bisa membantu pemahaman tentang infeksi virus dengue pada manusia. pada penelitian ini, digunakan beruk ( ) sebagai hewan percobaan untuk mempelajari infeksi virus dengue serotipe-3 (den-3). setelah diinfeksi dengan virus den-3, distribusi dan tempat replikasi virus den-3 dievaluasi pada semua jaringan tubuh yang diambil; tahap lanjutan lokalisasi dari replikasi virus den-3 pada jaringan dipelajari pada beruk yang di-etanasi tiga hari setelah diinfeksi virus den-3 (10 pfu ml ). beruk yang diinokulasi melalui rute subkutan atau rute intravena menunjukkan titer viremia tertinggi (62.94 pfu ml dan 58.62 pfu ml ) yang dideteksi dengan metode . virus yang diinokulasikan ke beruk melalui rute subkutan secara cepat mengalami diseminasi ke kelenjar limfa, kelenjar adrenal, ginjal, jantung, tiroid, hati, kelenjar prostat, dan kandung kemih. sementara diseminasi virus pada beruk yang diinokulasi melalui rute intravena dapat dideteksi pada kelenjar limfa, timus, kelenjar air liur, hati, dan kelenjar prostat. hasil penelitian ini menunjukkan bahwa replikasi virus den-3 terjadi pada berbagai jaringan tersebut dan perbedaan rute infeksi diindikasikan berpengaruh terhadap diseminasi virus. kata kunci: , hewan model, virus dengue-3, diseminasi, rt-pcr 7 -1 -1 -1 7 -1 -1 -1 dengue (den) viruses are mosquito-borne rna viruses, which belong to the family , and are grouped into four antigenically distinct types, den-1, den-2, den-3, and den-4. dengue virus infection in recent decades has become a major international public health concern and it has been reported to cause several clinical consequences in humans; dengue fever (df) is an acute, self limited febrile illness, but infection might also result in a more severe form, dengue hemorrhagic fever (dhf) with thrombocytopenia and capillary leakage and might flaviviridae process to a life threatening hypovolemic shock, dengue shock syndrome (dss) (peng 2004; bente and rico-hesse 2006). according to who (2009), the incidence of dengue infection has grown dramatically around the world; about 2.5 billion people which are two fifths of the world's population are now at risk from dengue infection. who estimates there may be 50 million dengue infections worldwide every year. understanding the dengue virus pathogenesis has been extremely difficult because only human seem to develop this disease clinically. target cells and organs for den replication in humans remain unclear. the involvement of liver cells in the pathogenesis of den et al. *corresponding author, phone: +62-251-8313637, fax. +62-251-8360712, e-mail: jpi-pssp@indo.net.id issn 1978-3477, eissn 2087-8575 vol 5, no 2, june 2011, p 94-98 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.2.7 virus infection has been indicated by abnormal liver function, pathological findings, and detection of viral antigen in hepatocytes at biopsy from human tissue and mice model (peng . 2004; basilio-de-oliveira . 2005, paes 2009). strong tissue alteration associated with dhf/ dss are observed in the liver, bone marrow, lymphoid organ, heart, lung, and kidney of tissues collected at autopsy (basilio-de-oliveira 2005) to better understand the pathogenesis of den virus infection, animal model is necessary to reveal the detailed symptoms and distribution of the virus in tissues, as well as the histopathological findings associated with den virus infection. the genetic similarity of nhps to human had been reported to have consequences in their role as hosts to the same pathogens; this could mean that there is (are) critical molecule(s) shared by nhps and human being used by pathogens in their life cycle. thus the nhps often are the only species that can be infected by human pathogen (bennet 1998). the detection of dengue antibodies in sera from nhps in sylvatic or rural setting of asia and africa suggest that they are involved in den virus transmission (de silva 1999). several studies using nhp models have been done in the past six decades. although there were no clinical signs detected in den infected-nhps, researchers could show that these animals were susceptible to den infection with viremia level reported to be lower than 100 pfu ml (bente and rico-hesse 2006). it is very unfortunate that until recently there is limited information regarding the target cells and organs for virus replication in this model. recent study by onlamoon . (2010) described the hemorrhage induction in rhesus macaques ( ) by den-2 virus infection. it is the intention of this study to show the susceptibility of pigtailed macaques ( ) to den infection and to show that the infection caused the dissemination of den virus in organs of pigtailed macaques infected with den-3, as well as analyzing the pathological finding in various organ. . den-3 virus 98900645 was isolated from human case of dss in indonesia by passaging five times in c6/36 cells (kindly provided by dr maya williams of the viral disease program, us namru2, jakarta, indonesia) study was carried out using protocol reviewed and approved by the institutional et al et al et al. et al. et al. et al. et al macaca mulatta macaca nemestrina -1 materials and methods viruses animals and housing. animal care and use committee (pt. bimana indomedical number p.09-08-ir). fourteen pigtailed macaques at 4 to 6 year of age were obtained from the colony maintained by the primate research center at bogor agricultural university, in bogor, indonesia. during the study, pigtailed macaques were housed in individual stainless steel cages in climate-controlled room (24 2 c and 30-70% humidity). they were fed with commercial monkey chow and supplemented with fruits. water was available . environmental enrichment was given to the pigtailed macaques to assure that their welfare issue was not neglected. pigtailed macaques used in this study were negative to den-1, -2, -3 and -4 viral antibodies as previously screened by elisa (human gesellschaft fur biochemia und diagnostica mbh, germany). prior to virus inoculation and each blood collection, pigtailed macaques were anesthetized with intramuscular injection of ketamil® (ketamine hydrochloride 10%; troy laboratories pty. ltd., nsw, australia). daily observation was conducted intensively to assess pigtailed macaque's clinical condition. . pigtailed macaques were randomly divided into three treatment groups (4 animals per-group) and a control group (2 animals). anesthetized pigtailed macaques were inoculated subcutaneously (sc) on the upper back, intradermally (id) on the left and right abdomen and intravenously (iv) on saphenous veins of the left and right legs with 1 ml of den-3 virus at 10 -10 pfu ml . control pigtailed macaques received 1 ml of media (dmem) subcutaneously. . blood samples were collected from femoral vein of all pigtailed macaques on day 0, 1, 2, and 3 post inoculation (p.i). plasma samples were obtained and titrated for den-3 virus by real-time rt-pcr (iq5 multicolor real time pcr, biorad) using primer and probe (taqman). the values in pfu per milliliter for each rna standard was based on the original titer of stock virus as determined by plaque assay. both the real time pcr protocol and pfu per milliliter conversion were done as previously described by callahan (2001). . six treated pigtailed macaques (2 animals per group) were humanely euthanized with intravenous injection of euthal® (sodium pentobarbital and phenytoin; a croesus pharma inc, philippines) on day two p.i for macroscopic evaluation. liver, spleen, adrenal gland, kidney, stomach intestines, reproductive organs (ovary, testicle, uterus, prostate gland, seminal vesicle), urinary bladder, lymph nodes (axillary, + o 7 8 -1 ad libitum infection of pigtailed macaques viremia level detection pathology and histopathology volume 5, 2011 microbiol indones 95 96 pamumgkas et al. microbiol indones mesenteric, inguinal, submandibular), thyroid gland, heart, lung, aorta, trachea, esophagus, brain, pituitary gland, eye, oral mucosa, abdominal skin, skin of inoculation site, and mammary gland (if applicable) were fixed in 10% nbf and stained with routine hematoxylin-eosin (he) for light microscopic evaluation. . tissue samples for rna isolation were all collected organs from necropsy. 200 µl extraction buffer (phosphate buffer saline, edta, sds, and protease) was added into each minced tissue to destruct the cells and protein. tissues were placed in vials and incubated overnight at 56 °c. qiaamp rna viral mini kit (qiagen) was used to extract the viral rna from samples. . primers set used in the pcr were adapted from callahan (2001). the primers produced 124 base pair (bp) of amplicon. 50 ul of one step reverse transcriptase (qiagen) was added to 10 pmol each of the primers and dna template. positive, negative controls, and one or more reagent controls were included in each run. pcr amplification was carried out in geneamp 9700 thermal cycler under the following conditions: 50 °c for 15 min and 94 °c for 30 min to produced cdna from samples, followed by amplification of 45 cycles at 94 °c for 30 sec for denaturation, at 60 °c for 30 sec for primers annealing, at 72 °c for 30 sec for elongation then ended with extension for 10 min at 72 °c and finally stored at 4 °c. pcr product was analyzed by 2% of agarose gel electrophoresis and visualized using gel doc (biorad geldoc 2000). the den-3 98900645 induced viremia in all 14 infected pigtailed macaques with duration from 1 to 4 days with peak titer on day 2 p.i (table 1). all pigtailed macaques remained healthy and did not show any signs of disease although viremia was detectable. viremia levels on day 2 p.i. were at 62.94 pfu ml (1.5799, sc), 10.68 pfu ml (f9004, sc), 47.98 pfu ml (1.6087, id), 15.64 pfu ml (1.6136, id), 58.62 pfu ml (m9008, iv), and 4.09 pfu ml (1.5808, iv); these were the highest and the lowest viremia levels in each treatment group. these pigtailed macaques were submitted for necropsy immediately after euthanasia. all pigtailed macaques were in healthy condition with adequate body fat stores; significant gross lesions were not seen in any of the examined pigtailed macaques. mild to moderate erythema and skin crusting at the inoculation tissue processing reverse transcriptase polymerase chain reaction results et al. -1 -1 -1 -1 -1 -1 sites were present inconsistently. histologically, perivascular lymphocytic infiltrates were present in variable degrees of severity in multiple tissues; lungs, urinary bladder, and abdominal skin were the most consistently involved tissues. mild lymphocytic hepatitis with or without oval cell hyperplasia was present in all six pigtailed macaques. cellular apoptosis on intestines was prominent in 4 pigtailed macaques (m9008, i.5808, i.6087, i.6136). skin lesions at the inoculation sites were varied from minimal perivascular dermatitis to moderate necrotizing epidermitis on pigtailed macaques receiving intra-dermal and subcutaneous viral inoculation. the dissemination of den-3 into distant organs was traced by reverse transcriptase pcr two days after inoculation using primers targeted to amplify the capsid region of den-3 in the position of 118-221 of its nucleotide. we detected the virus on variety of tissues in five of six pigtailed macaques (table 2). in one subcutaneously infected pigtailed macaque (f9004), all tissues were negative by pcr. of the pcr positive pigtailed macaques, viral rna was detected in spleen, lung, adrenal gland, kidney, heart, thyroid gland, live and seminal vesicle from pigtailed macaque 1.5799, in the abdominal skin, spleen, thymus, axillaris lymph node, submandibular lymph node, pancreas, adrenal gland, salivary gland and thyroid gland from pigtailed macaque 1.6087, in abdominal skin, inguinal lymph node and salivary gland from pigtailed macaque 1.6136, in bone marrow, spleen, salivary gland, liver and mammary gland from pigtailed macaque m9008 and in mesenteric lymph node and liver from pigtailed macaque 1.5808. table 1 den-3 virus viremia level in detected by reverse transcripatse real time pcr (pfu ml ) macaca nemestrina -1 monkey id day 0 day 1 day 2 day 3 day 4 1.1.3251 1.5799 f9004 9350 1.6087 1.8258 9173 1.6136 m9008 1.5808 9188 8015 9336 2.4192 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.46 <1.00 <1.00 <1.00 1.61 <1.00 <1.00 1.93 9.37 8.73 7.50 11.57 1.62 3.85 3.66 1.89 <1.00 1.34 <1.00 <1.00 <1.00 12.19 62.94 10.68 14.56 47.98 24.34 39.65 15.64 58.62 4.09 48.13 9.98 <1.00 <1.00 1.47 <1.00 6.53 3.90 <1.00 35.07 28.35 4.94 <1.00 <1.00 3.82 2.94 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 discussions the experiment was designed to examine the dissemination of den-3 in a nonhuman primate (nhp) model during acute phase of primary infection. reports have shown that viremia was detected in nhp after inoculation of den viruses through subcutaneous route (scott 1980; kochel 2005; koraka 2007; bernardo 2008). in this study, three ways of inoculation routes (subcutaneously, intradermally, and intravenously) were performed, in which the subcutaneous and intradermal routes of infection mimic the natural way of transmission of this virus in human. it is somewhat interesting that pigtailed macaques infected intravenously appeared to have the least viral dissemination in tissues, even in pigtailed macaque with high level of viremia. this may be related to the fact that in humans, subcutaneous and intradermal routes of den virus infection involved the role of skin dendritic cells (dc) that is believed to be important in the virus replication with dc-sign (dcspecific icam3-grabbing on integrin ) serving as coreceptor for viral entry (green and rothman 2006). previous reports have shown that den viruses viremia was found starting day 2 post inoculation in (koraka 2007; bernardo 2008), (scott 1980) and (wijaya 2010). increasing viremia levels detected from day 0 to day 2 p.i. in all pigtailed macaques in this study proved that den-3 infection and viral replication had occurred in the inoculated pigtailed macaques. to prove that the den-3 viral rna detected in the plasma samples was not a result from unwashed-remaining concentration of virus inoculum, we showed that pigtailed macaques inoculated intradermally and subcutaneously demonstrated high viremia levels, similar to those receiving intravenous viral inoculation. et al. et al. et al. et al. macaca fascicularis et al. et al. m. mulatta et al. m. nemestrina et al. in this study, we confirmed the presence of the den-3 viral rna in various organs by real time rtpcr. this evidence showed that the den-3 had undergone replication in their target cells and organs. the variety of viremia levels in pigtailed macaques within the same group showed that dendritic receptor may play roles in viral replication. pigtailed macaques with high level of viremia had broader viral dissemination in various organs; the high titer of viruses resulted from replication might be distributed to a wider range of organs. pigtailed macaques inoculated subcutaneously and intradermally showed wider range of involved organs including spleen, adrenal gland, kidney, thyroid gland, liver, and seminal vesicles, while pigtailed macaques inoculated intravenously had limited viral dissemination in spleen, thymus, salivary gland and prostate gland. regardless the viremia level detected in examined tissues, none of the monkeys in this study demonstrated significant pathological changes like that had been described in humans (basilio-de-oliviera 2005); hepatitis only present in minimal to mild degree. this supported the fact that none of the pigtailed macaques had any sign of disease during the experiment. several human cases of dengue virus infections showed to involve the findings of liver abnormality and viral antigens were detected in patients with hepatic failure secondary to dengue hemorrhagic fever. this study had shown similar condition of hepatitis which was also emphasized with the presence of viral rna in liver tissue. to the authors' knowledge, this is the first report of systemic dengue viral dissemination within 2 days after single dose of inoculation in infected . one study has reported the use of as animal model for dengue virus infection (taweechaisupapong 1996), in where the study et al. m. nemestrina m. nemestrina et al. volume 5, 2011 microbiol indones 97 table 2 den-3 virus were detected in various organ by reverse transcriptase pcr 1.5799 (m) f9004 (f) 1.6087 (m) 1.6136 (f) m9008 (m) 1.5808 (m) abdominal skin + + bone marrow + spleen + + + thymus + lungs + axilaris lymph gland + inguinalis lymph gland + messenterica lymph gland + routes of inoculation subcutaneous intradermal intravenoustissues m, male; f, female. emphasized on epidermal change due to intradermal inoculation of den-2. although there was no evidence for hemorrhagic condition in the infected pigtailed macaques as compared to the rhesus macaques (onlamoon 2010), we still detected the viremia level and viral rna distribution in tissues after den-3 virus infection. we concluded that is a good alternate model to evaluate replication of dengue virus in tissues. furthermore, by understanding the target cells and organs for den-3 replication, such study to reveal the pathogenesis of dengue virus infection that might lead to severe form of disease could be performed. the authors would like to thank i nengah budhiarsa and permanawati for their technical assistance in the husbandry and veterinary care of the animals during the initial stage of the study. this study was partially supported by the state ministry of research & technology, republic of indonesia, through the incentive program research grant to jp (contract no. 068/rt/d.psiptn/insentif/ppk/i/2009). m. nemestrina acknowledgements references basilio-de-oliveira ca, aguiar gr, ms baldanza, barth om, eyer-silva wa, paes mv. 2005. phatologic study of a fatal case of dengue-3 virus infection in rio de janeiro brazil. braz j infect dis. 9(4): 341-347. doi:10.1590/s1413. bernardo l, izquierdo a, prado i, rosario d, alvarez m, santana e, castro j, martinez j, rodriguez r, morier l, guillen g, guzman mg. 2008. primary and secondary infections of monkey with asian and american genotypes of dengue virus 2. clin vaccine immunol. 15(3): 439-446. doi: 10.1128/cvi.00208-07. bennett bt, abel cr, henrickson r. 1998. nonhuman primates in biomedical research: diseases. san diego: academic press. bente ad, rico-hesse r. 2006. model of dengue virus infection. drug discov today dis models. 3(1):97-103. doi: 10.1016/j.ddmod. 2006.03.014. macaca fascicularis callahan jd, wu shuenn-jue l, schultz ad, mangold be, peruski lf, watts dm, porter kr, murphy gr, suharyono m, king chwanchuen, hayes cg, temenak jj. 2001. development and evaluation of serotype and group-spesific fluorogenic reverse transcriptase pcr (taqman) assay for dengue virus. j clin microbiol. 39(11): 4119-4124. doi: 10.1128/jcm.39.11.4119-4124.2001. de silva am, dittus wpj, amerasinghe ph, amerasinghe fp. 1999. serologic evidence for an epizootic dengue virus infecting toque macaques ( ) at polonnaruwa, sri langka. am j trop med hyg. 60(2): 300-306. green s, rothman a. 2006. immunophatological mechanisms in dengue and dengue hemorrhagic fever. curr opin infect dis. 19(5): 429-436. kochel tj, watts dm, gonzalo as, ewing df, porter kr, russell kl. 2005. cross-serotype neutralization of dengue virus in monkeys. j infect dis. 191(6): 1000-1004. doi:10.1086/427511. koraka p, benton s, van amerongen g, stittelaar k, osterhaus adme. 2007. characterization of humoral and cellular immune responses in cynomolgus macaques upon primary and subsequent heterologous infections with dengue viruses. microbes infect. 9(8):940-946. doi:10.1016/j.micinf.2007.03.012. onlamoon n, noisakran s, hsiao hm, duncan a, villinger f, ansari aa, perng gc. 2010. dengue virus-induced hemorrhage in a nonhuman primate model. blood. 115(9):1823-1834. doi:10.1182/blood-200909-241990. paes mv, lenzi hl, nogueira acm, nuovo gj, pinhao at, mota em, basilio-de-oliveira ca, schatzmayr h, barth om, de barcelos arves am. 2009. hepatic damage associated with dengue-2 virus replication in liver cells of balb/c mice. lab invest. 89:1140-1151. doi:10.1038/labinvest.2009.83. peng t, junlei zhang, jing an. 2004. the animal models for dengue virus infection. dengue bul. 428:168-173. scott mr, nisalak a, eckels kh, tingpalapong m, harrison vr, gould gj, chapple fe, russel pk. 1980. dengue-2 vaccine: viremia and immune response in rhesus monkeys. infect immun. 27(1):181-186. taweechaisupapong s, sriurairatana s, angsubhakorn s, yoksan s, bhamarapravati n. 1996. and studies on the morphological change in the monkey epidermal langerhans cells following exposure to dengue 2 (16681) virus. southeast asian j trop med public health. 27(4): 664-672. widjaja s, winoto i, sturgis j, maroef cn, listiyaningsih e, tan r, pamungkas j, iskandriati d, blair pj, sajuthi d, porter kr. 2010. and dengue virus infectivity: a potential model for evaluating dengue vaccine candidate. microbiol indones. 4(2): 49-54. [who] world health organization. 2009. dengue and dengue haemorrhagic fever [internet]. [cited 2009 may 6]. available from: http://www.who.int /mediacentre/ factsheets/ fs117/en/ index.html. macaca sinica aotus nancyme in vivo in vitro macaca nemestrina 98 pamumgkas et al. microbiol indones 5. waode.cdr effect of micro-encapsulated synbiotic at different frequencies for luminous vibriosis control in white shrimp ( )litopenaeus vannamei waode munaeni, munti yuhana*, widanarniand department of aquaculture, faculty of fisheries and marine science, institut pertanian bogor campus darmaga, bogor 16680, indonesia litopenaeus vannamei vibrio harveyi bacillus ipomea batatas in vivo v. harveyi bacillus . v. harveyi litopenaeus vannamei, vibrio harveyi litopenaeus vannamei vibrio harveyi bacillus ipomea batatas spray drying vibrio harveyi survival rate specific growth rate feed conversion ratio bacillus total bacterial count v. harveyi count total vibrio count litopenaeus vannamei vibrio harveyi the aim of this study was to evaluate the effect of micro-encapsulated synbiotic application at different frequencies for luminous disease control in white shrimp ( ). the luminous disease is caused by . in this experiment, a synbiotic which was a combination of the probiotic sp. np5 rf and the oligosaccharides from sweet potato ( l.) jago variety was applied. the synbiotic was encapsulated by spray drying method. the experiment was conducted by supplementing the shrimp's diet with the micro-encapsulated synbiotic for 40 d. treatments included the administration micro-encapsulated synbiotic at different frequencies i.e. once a week (a), twice a week (b), daily (c), and without microencapsulated synbiotic (control treatment). the control treatment consisted of positive (k+) and negative (k-) controls. after 30 d period, all of the shrimp were challenged by intramuscular injection of pathogenic rf at a cell concentration of 10 cfu ml except the negative control. the treatment c resulted in significantly higher survival rate (sr), specific growth rate (sgr), and immune responses than those of the controls, whereas the feed conversion ratio (fcr) was lower than the controls. in addition, the population of sp np5 rf and total bacterial count (tbc) in the intestines increased, whereas the population of rf and the total vibrio count (tvc) were lower compared to the controls. key words: frequencies, micro-encapsulated, synbiotic, penelitian ini dilakukan untuk mengkaji efek pemberian mikrokapsul sinbiotik dengan frekuensi berbeda pada udang vaname ( ) yang diinfeksi . sinbiotik yang digunakan merupakan kombinasi dari probiotik sp. np5 rf dan oligosakarida dari ubi jalar ( l.) varietas jago yang dienkapsulasi dengan metode . percobaan dilakukan selama 40 hari dengan menambahkan mikrokapsul sinbiotik pada pakan. perlakuan meliputi pemberian mikrokapsul sinbiotik dengan frekuensi satu kali seminggu (a), dua kali seminggu (b), setiap hari (c), dan tanpa mikrokapsul sinbiotik (kontrol). perlakuan kontrol terdiri dari kontrol positif (k+) dan kontrol negatif (k-). setelah 30 hari periode percobaan, semua udang diuji tantang dengan rf konsentrasi sel 10 cfu ml kecuali kontrol negatif. hasil penelitian menunjukkan bahwa, perlakuan c memberikan nilai (sr), (sgr), dan respon imun yang lebih tinggi serta (fcr) yang lebih rendah dibanding kontrol. perlakuan juga mampu meningkatkan populasi sp. np5 rf dan (tbc) di usus serta mampu mengurangi rf dan (tvc) dibanding perlakuan kontrol. kata kunci : frekuensi, , mikrokapsul, sinbiotik, r r 6 -1 r r r r 6 -1 r r vol.8, no.2, june 2014, p 73-80 doi: 010.5454/mi.8.2.5 *corresponding author; phone: +6281320006175; email: yhnmymy@yahoo.co.id white shrimp ( ) has currently become one of the major aquacultural commodities in south east asian countries including indonesia. the application of intensive cultivation to increase shrimp production has resulted in deterioration of water quality which influences disease susceptibility. one of the important diseases in shrimp is vibriosis (chiu . 2007). this disease which is mainly caused by luminous (phuoc . 2009) can cause mass mortality from shrimp larvae (chrisolite . 2008) including the nauplius, zoea, mysis and post larvae stadia to adult shrimp in the litopenaeus vannamei et al vibrio harveyi et al et al fattening ponds (soto-rodriguez . 2012). one of the biological control strategies to improve growth and disease resistance in aquaculture organisms is synbiotic application. a synbiotic is a nutritional supplement which is combination of probiotics and prebiotics (cerezuela . 2011). probiotics are living microbial cells which are beneficial to the host and improve the quality of the environment as well as the host's immune responses against diseases (newaj-fyzul . 2014). prebiotics are food ingredients that cannot be digested by the host but can be selectively metabolized by bacteria (ringo . 2010). the application of probiotics and prebiotics simultaneously has proven to improve the survival rate and to enhance the immune system in et al et al et al et al 74 munaeni et al. microbiol indones shrimp (li . 2009), koi carp ( koi) (lin . 2012), and nile tilapia ( ) (putra 2010). application of fresh culture synbiotics has some drawbacks such as a limited storage period because the probiotic cells are easily degraded by unfriendly enviromental conditions. one method to protect the probiotic bacteria cells is micro-encapsulation. there are several techniques used in microencapsulation of probiotics including spray-drying, e x t r u s i o n , e m u l s i o n , a n d p h a s e s e p a r a t i o n (kailasapathy 2002; mu˜noz-celayaa . 2012). spray drying is relatively simple compared to other methods (jyothi . 2010). the genus which is resistant to high temperatures and high pressures (lin . 2012) was used in this study and oligosaccharides from sweet potato ( l.) starch was used as the source of the prebiotic (marlis, 2008). a previous study showed that the application of 2% micro-encapsulated synbiotic made from sp. np5 combined with oligosaccharides from sweet potato ( l.) has improved the survival rate, immune system, and disease resistance in white shrimp ( ) infected by (zubaidah . 2014). the effectiveness of synbiotics was influenced by several factors such as the host species, dosage, duration or frequency of administration, and the type of probiotic and prebiotic administered (merrifield . 2010; nayak 2010; cerezuela . 2011). different synbiotic administration frequencies may affect the induction of the host's immune response (nayak 2010). previous research has showed that the application of fresh culture synbiotic at different frequencies resulted in different effects on the immune response of white shrimp ( ) (oktaviana . 2014). based on the facts above, this study was aimed to evaluate the effect of micro-encapsulated synbiotic application in different frequencies for luminous disease control on white shrimp ( ). the prebiotic used in this study is a product of sweet potato ( l.) var. jago flour extraction. the preparation of the prebiotic made from sweet potato ( l.) flour refers to the method by marlis (2008). fresh sweet potatoes ( l.) from the jago variety were peeled and cut into ± 1 mm thick slices. then the sweet potato slices were dried in et al cyprinus carpio et al oreochromis niloticus et al et al bacillus et al ipomea batatas bacillus i. batatas l. vannamei v. harveyi et al et al et al l. vannamei et al l. vannamei i. batatas i. batatas i. batatas materials and methods the preparation of the prebiotic and probiotic bacteria. an oven at 55 c for 5 h. the dried slices were then grinded using a willey mill then flour sifted through a size 60 mesh. five hundred grams of the sweet potato flour was then mixed with water at a ratio of 1:1 (w/v) then steamed for 30 min at 100 c. after steaming, the dough was made into flour again and was extracted for its oligosaccharides based on the method by muchtadi (1989). the extraction used 70% ethanol and the mixture was thickened in a vacuum evaporator at 40 c. the concentration of the oligosaccharide used was 5% (marlis 2008). the probiotic bacteria used was np5 which was isolated from nile tilapia ( ) (putra 2010) and had been made resistant using 50 μg ml of the antibiotic rifampicin (rifampicin 0.25 g, 9.5 ml absolute ethanol, 0.5 ml ) ( sp np5 rf ). the sp np5 rf cells were cultured in an swc-agar medium (sea water complete: 5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 750 ml sea water, 250 ml aquadest, 20 g bactoagar) and incubated for 24 hours at 29 c. the cells were inoculated to the liquid swc medium and incubated in waterbath shaker (julabo sw-20c) for 18 hours at 29 c at a speed of 140 rpm. the gel suspension was transfered to a 25 ml corning tube and was centrifuged at a speed of 5.000 rpm for 10 min. the cells were then rinsed with 25 ml of pbs (phosphate buffered saline: 8 g nacl, 0.2 g kh2po4, 1.5 g na2hpo4, 0.2 g kcl, 1000 ml ), homogenized, and centrifuged at a speed of 5,000 rpm for 10 min. the pellets produced were homogenized in 25 ml pbs. one percent of sp. np5 rf cells at a concentration of 10 cfu ml 2% of prebiotic (mahious . 2006) were mixed with a maltodextrin coating agent and milk whey until homogenous using a mixer. the ratio of synbiotic, whey and maltodextrin was 1:1:1 (v/v/w). then this mixture was micro-encapsulated using the spray dryer method (mini bunchi 190) at an inlet temperature of 150 c and an outlet temperature of 70 c. the 2.6 ± 0.3 g of white shrimp larvae were obtained from balai budidaya air payau (bbap-the brackish water cultivation research station), situbondo, east java. the larvae were allowed to adapt for 2 weeks in 20 holding tanks sized 60 x 40 x 40 cm (30 liter volume) at a density of 10 shrimp/tank. during the keeping period, siphoning was done daily and 50% of the water was replaced o o o -1 r r o o r 8 -1 o o 3 the micro-encapsulation of the synbiotic. research design. bacillus sp. o. niloticus aqua bidestilata bacillus . bacillus . aquadest bacillus et al volume 8, 2014 microbiol indones 75 afterwards. the water quality parameters were maintained at temperatures of 25-26 c, ph 7.7-8.0, salinity 32-35 g l , do 7.3-79 ppm and tan 0.5-0.9 mg l . the test feed was gold coin 932p commercial pellet with a protein content of 31.90%, fat 7.55%, and betn 36.85%. the dosage of the micro-encapsulated synbiotic was 2% (zubaidah . 2014) and the mixture was sprayed with 2% egg white as a binder. the control was also sprayed with the binder. feed was given 4 times a day starting at 07.00, 11.00, 15.00, and 19.00 western indonesia meantime with a feeding rate (fr) of 10% of the shrimp's biomass. the treatment consisted of feeding the microencapsulated synbiotic once a week (a), twice a week (b), every day (c), and without any microencapsulated synbiotic for the positive control (k+) and negative control (k-). the feeding was done for 30 d, and then all the shrimp were injected with rf on day 31 except for the negative controls which were injected with . the shrimp were then kept for a further 9 d. the specific growth rate (sgr) and feed conversion rate (fcr) were calculated from day 0 to day 30, the survival rate (sr) was calculated from the start till the end of the study using this equation: sr (%) = [(nt/no) x 100%]; sgr (%) = [100 x (ln we ln ws)/t]; fcr = [(f) / (bt bo)] (zokaeifar 2012). where: is the number of live shrimps at the end of the study (individuals), is the number of live shrimps at the beginning of the study (individuals), is the average weight of the shrimps at the end of the study (grams), is the average weight of the shrimps at the beginning of the study (grams), is the duration of the study (days), is the amount of feed, is the shrimp biomass at the end of the study (grams), is the biomass of the shrimp at the beginning of the study (grams). the blood sampling was done according to the method by chiu . (2007). an amount of 0.1 ml shrimp haemolymph was drawn from the base of the first swimmeret using a 1 ml syringe which had been filled with 0.9 ml anticoagulant (30 mm trisodium citrate, 0.34 m sodium chloride, 10 mm edta). the mixture of haemolymph and anti-coagulant was observed and the number of cells counted using a haemocytometer and a microscope at a 400x magnification. o -1 -1 r et al at satiation v. harveyi pbs et al. nt no we ws t f bt bo et al observation parameters. survival, growth, and feed conversion rates. immune response. the phenoloxidase activity measurement was done based on the formation of dopachrome produced by the l-dopa. the standard solution containing 100 μl haemocyte suspension, 50 μl cacodylate buffer (0.01 m sodium cacodylate, 0.45 m sodium chloride, 0.01 m calcium chloride, 0.26 magnesium chloride) and 50 μl l-dopa (3 mg ldopa, 1 ml cacodylate buffer) was used to measure the po background activity in the test solution. the measurement of the optical density (do) was done using a spectrophotometer with a wavelength of 492 nm (chiu . 2007). the observation of the immune response was done on day 0 (before treatment), day 30 (before the challenge test), day 32 (1 day post challenge test), day 36 (4 ds post challenge test), and day 40 (9 ds after challenge test). the intestines from 2 shrimps from each treatment were weighed and 0.01 g was taken from each sample and homogenized with 0.99 ml pbs. this mixture was then spread onto specific tcbs (thiosulphate citrate bile-salt sucrose) agar media with the antibiotic rifampicin for the rf count and tcbs without the antibiotic for the total vibrio count (tvc) the sp. np5 rf count was done by spreading the mixture onto swc agar media with antibiotics and for the total bacterial count (tbc) the mixture was spread onto swc agar media without antibiotics. the media were then incubated for 24 h at 35 c. the observations of the bacteria population in the intestines were done on days 0, 30, 32, 36, and 40. the data were analyzed using microsoft excel 2010 and tested using anova (analysis of variants); if it had a significant effect, it was continued with a duncan test using the spss (statistical program software system) version 16. the survival rate (sr) before the challenge test was 100%. after the challenge test, the highest sr was shown by treatment c at 63.33 ± 5.77 % and this was significantly different (p < 0.05) from treatment a, k+ and k-, but was not significantly different (p > 0.05) from treatment b at 56.66 ± 5.77% (fig 1). the highest sgr was shown by treatment c at 3.35 ± 0.25% which was significantly different from treatment a, k+ and k-, but was not significantly different from treatment b (fig 2a). the lowest fcr was shown by treatment c at 1.63 ± 0.03 which was significantly different (p < 0.05) from all other et al v. harveyi . bacillus l. vannamei bacterial population in the intestines. data analysis. r r o results 76 munaeni et al. microbiol indones treatments (fig 2b) thc (fig 3a) and po (fig 3b) in the increased after 30 days of treatment and decreased after day 32 and increased again at day 36 and day 40. the highest thc and po during the study were shown by treatment c. the thc for treatment . l. vannamei c during the duration of the study was, respectively, 1.99 ± 0.32 x 10 cells ml , 5.25 ± 0.91 x 10 cells ml , 4.65 ± 0.51 x 10 cells ml , 5.07 ± 0.70 x 10 cells ml , and 5.99 ± 0.46 x 10 cells ml . the po for treatment c during the duration of the study was, respectively, 0.28 ± 0.05, 0.56 ± 0.10, 0.49 ± 0.14, 0.44 ± 0.10, 0.66 ± 7 -1 7 -1 7 -1 7 -1 7 -1 fig 2 spesific growth rate (sgr) (a); and feed conversion ratio (fcr) (b) of . different letters over each treatment bar (mean ± sd) indicated significant difference (duncan; p < 0.05). negative control (k-); positive control (k+); supplementation of micro-encapsulated synbiotic diet at once a week (a); twice a week (b); daily (c). l. vannamei fig 1 survival rate (sr) of . different letters over each treatment bar in same colours (mean ± sd) indicated significant difference (duncan; p < 0.05). negative control (k-); positive control (k+); supplementation of microencapsulated synbiotic diet at once a week (a); twice a week (b); daily (c). l. vannamei (a) (b) fig 3 total haemocyte count (thc) (a) and phenoloxidase (po) (b) of . different letters over each treatment bar in same day (mean ± sd) indicated significant difference (duncan; p < 0.05). negative control (k-); positive control (k+); supplementation of micro-encapsulated synbiotic diet at once a week (a); twice a week (b); daily (c). l. vannamei (a) (b) 0.03. . after 30 days of treatment, the sp. np5 rf count (fig 4a) and tbc (fig 4b) increased; the highest was showed by treatment c (7.29 ± 0.02 and 8.82 ± 0.04) log 10 cfu g . these numbers decreased after day 32, then increased again at day 36 and day 40. one day after the challenge test (day 32), the highest rf count (fig 4c) and tvc (fig 4d) were shown by treatment a (6.49 ± 0.10 and 6.88 ± 0.24) log 10 cfu g which were significantly different from the other treatments. after day 36 and 40, the rf count and tvc for treatments c and b showed a significant decrease compared to the other treatments the high sgr and low fcr for treatments c and b in this study are suggested to be an effect of the increased sp np5 rf (fig 4a) in the shrimp's intestines. the sp np5 rf are believed to bacillus v. harveyi v. harveyi bacillus . bacillus . r -1 r -1 r r r discussion have the ability to secrete exogenous enzymes which can catalyze macro molecules in feed into simpler molecules, making it easier for the host to digest the feed. other studies have also reported that the shrimp's increased growth is believed to be caused by the increased digestive enzymes which were induced by probiotic bacteria (wang 2007) such as protease and amylase which can stimulate and increase the host's digestion rate (zhang . 2010). the probiotic bacteria sp. np5 which had been isolated from nile tilapia ( ) are amylolytic bacteria which have an important role in digestion (putra 2010). besides resulting in a higher growth performance, treatments c and b can also improve the white shrimp's immune response and resistance in comparison to the controls. this can be seen in the increased thc (fig 3a) and po (fig 3b). haemocytes have an important role in the immunity system; they remove foreign particles in the haemocoel through phagocytosis, encapsulation, and nodular aggregation (rodriquez and le muollac 2000, smith 2003, kakoolaki et al bacillus o. niloticus et al. et volume 8, 2014 microbiol indones 77 (a) (c) fig 4 np5 sp. rf (a); total bacterial count (tbc) (b); rf (c); and total vibrio count (tvc) (d) of . different letters over each treatment line in same day (mean ± sd) indicated significant difference (duncan; p < 0.05). negative control (k-); positive control (k+); supplementation of micro-encapsulated synbiotic diet at once a week (a); twice a week (b); daily (c). bacillus count v. harveyi count l. vannamei r r al. 2010, et al. 2012, hao et al. 2014dantas-lima ). the supplementation of probiotics can increase the thc which in turn will improve the immune response during times of duress due to pathogenic infections in shrimp (chiu . 2007). one day after the challenge test, the thc in all the treatments decreased. the decrease in the number of haemocytes is an effect of the shrimp's immune system mechanisms such as haemocyte infiltration to infected tissues or haemocyte death due to apoptosis (costa 2009). the phenoloxidase (po) enzyme activity is also very important against the microbial infections (vargas-albores and yepiz-plascencia 2000, chiu . 2007; cerenius . 2010; ). po in the haemolymph functions as an inactive pro-enzyme or also known as propo. transformation from propo to po involves several reactions in the propo activing system (rodriquez and le moullac 2000). the propo system can be activated by several microbial polysaccharides and specific pattern recognition proteins (prps) such as . 2007). treatments c and b in this study were able to increase the po value before the challenge test compared to the controls. the decrease of po could cause lowered immunity in shrimps (cerenius . 2010; 2014). the high immune response in treatments c and b sumable as the effect of the increase population of sp. np5 rf (fig 4a) and tbc (fig 4b) in the shrimp's intestines. the probiotic can directly improve immunity by passing through the epithelial cells in the intestines and having direct interaction lymphoid tissues then activating the immune response (immunostimulant). the indirect way is through contact between the epithelial cells in the intestines and galt (gut associated limphoid tissue) which activates the cytokines, enabling communication among cells to activate an immune response (immunoregulator) (nayak 2010). the decrease in rf count and tvc in treatments c and b in this study are believed to be influenced by the administration of the microencapsulated synbiotic. probiotic bacteria can produce antibacterial molecules such as bacteriocin which directly inhibit other bacteria, actively fight infection, inhibit the movement of other bacteria on the intestinal wall (translocation), improve the mucosal barrier by increasing production of non-specific immune responses (cerezuela . 2011; cerezuela . 2013). application of prebiotics could also inhibit the et al et al. et al et al hao et al. 2014 et al et al hao et al. bacillus v. harveyi et al et al lps-and-β 1, 3-glucanbinding protein (lgbp) and peptidoglycan-binding proteins (pgbp) (wang r r growth of pathogens and improve the immune system (mahious . 2006). in conclusion, the administration of microencapsulated synbiotic treatment c (supplementation of micro-encapsulated synbiotic diet at daily) is able to produce a low feed conversion ratio (fcr) value and can increase the survival rate (sr), specific growth rate (sgr), and immune response and resistance to in white shrimp ( ) compared to the controls. in addition, treatment c could also increase the population of sp np5 rf and the total bacterial count (tbc) and decrease rf and total vibrio count (tvc) in the shrimp's intestines compared to the controls. the research was financially supported by boptn from dikti scheme (2013) strategic research bogor agricultural university to munti yuhana with contract number : 134/it3.41.2/l2/spk/2013. et al v. harveyi l. vannamei bacillus . v. harveyi r r acknowledgment references cerenius l, jiravanichpaisal p, hai-peng l, söderhälz i. 2010. crustacean immunity in invertebrate immunity. bioscience. 13:239-259 cerezuela r, meseguer j and esteban ma. 2011. current knowledge in synbiotic use for fish aquaculture. review. j aqua res dev. http://dx.doi.org/10.4172/ 2155-95546.s1-008. cerezuela r, fumanal m, tapia-paniagua st, moriñigo ma, esteban ma. 2013. changes in intestinal morphology and microbiota caused by dietary 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dieguez al, romalde jl. 2012. virulence of responsible for the “brightred” syndrome in the pacific white shrimp . j invert pathol. 109:307-317. sritunyalucksana k, wongsuebsantati k, johansson m, söderhäll k. 2001. peroxinectin, a cell adhesive protein associated with the propo system from the black tiger shrimp, . dev comp immunol. 25(5):353-363. vargas-albores f, yepiz-plascencia g. 2000. beta glucan binding protein and its role in shrimp immune response. aquaculture 191:13-21. wang yb. 2007. effect of probiotics on growth performance and digestive enzyme activity of the shrimp . aquaculture 269:259-264. doi:10.1016/ j.aquaculture.2007.05.035. vibrio harveyi litopenaeus vannamei penaeus monodon penaeus vannamei zhang q, ma h, mai k, zhang w, liufu z, xu w. 2010. interaction of dietary and fructooligosaccharide on the growth performance, nonspecific immunity of sea cucumber, . fish shellfish immunol. 29(2):204-211. doi:10.1016/j.fsi.2010.03.009. zokaeifar h, balcázar jl, saad cs, kamarudin ms, sijam k, arshad a, nejat n. 2012. effects of on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, . fish shellfish immunol. 33:683-689. doi:10.1016/j.fsi.2012.05.027. zubaidah a, yuhana m, widanarni. 2014. addition of microcapsulated synbiotic with different dosages on feed vaname shrimp ( ) for tackling infection with . hayati j bios. submitted. bacillus subtilis apostichopus japonicus bacillus subtilis litopenaeus vannamei litopenaeus vannamei vibrio harveyi 8 munaeni et al.0 microbiol indones 2 459 dyah.cdr glucose biosensor using selected indonesian bacteria dyah iswantini , novik nurhidayat , trivadila1 2 1* and 1 2 department of chemistry, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; research center for biology, lembaga ilmu pengetahuan indonesia, jalan raya jakarta-bogor km. 46 cibinong, bogor 16911, indonesia escherichia coli e coli bacillus subtilis, thermus filiformis e coli e. coli, b. subtilis, t. filiformis e coli e. coli e coli escherichia coli bacillus subtilis thermus filiformis escherichia coli e coli bacillus subtilis, thermus filiformis e oli b. subtilis t. filiformis. e oli b. subtilis t. filiformis e coli e oli e coli escherichia coli bacillus subtilis thermus filiformis microbial glucose sensors have been developed using bacterial strains from japan. however, there is interest in developing local bacteria as glucose sensors in indonesia. in this research, the stability and the potential of a selected number of indonesian bacteria as glucose biosensors was explored. results of this study indicate that three of them, . , and exhibit properties of high viability and stability at high temperature (3060 ºc). spectrophotometrical and electrochemical measurements showed significant absorbance values and highly stable current features for . as indicated by its high capacity to produce glucose dehydrogenase. and produced currents of 3.25 µa, 0.2 µa, and 0.02 µa respectively, and . also produced a much higher activity of glucose dehydrogenase. electrochemical measurement using -modified carbon paste electrode allowed the determination of glucose concentration of up to 20 mm. therefore, indonesian . has a high stability and can be used as a glucose biosensor. key words: glucose biosensor, electrochemistry, bacteria, , , asal jepang telah digunakan sebagai sensor glukosa. pengembangan menggunakan bakteri asal indonesia yang berpotensi sebagai biosensor glukosa perlu dieksplorasi. hasil seleksi menunjukkan ada tiga bakteri, yaitu . , dan yang stabil pada suhu tinggi (30-60 ºc). uji enzim glukosa dehidrogenase menggunakan metode spektrofotometri dan elektrokimia menetapkan . c sebagai bakteri yang berpotensi paling baik dalam menghasilkan serapan cahaya dan stabilitas arus dibandingkan dengan dan . c dapat menghasilkan arus sebesar 3.25 µa, sedangkan dan berturut-turut hanya 0.2 µa dan 0.02 µa, maka . dapat menghasilkan aktivitas glukosa dehidrogenase yang lebih besar daripada bakteri lain. secara elektrokimia, . c yang diimobilisasikan di atas permukaan elektroda pasta karbon dapat mendeteksi konsentrasi glukosa sampai 20 mm. oleh karena itu, . asal indonesia ini mempunyai peluang untuk dikembangkan sebagai biosensor glukosa. kata kunci: biosensor glukosa, elektrokimia, bakteri, , , the total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030, and the top four countries are india, china, united states and indonesia for both 2000 and 2030 (wild 2004). hence, the need for sensors that can quickly measure blood glucose level is high. currently, a disposable glucose sensor is available in the market. however, the enzyme sensor is expensive and has a low stability. recent developments show that microorganisms can also be used to produce glucose sensors (ohfuji . 2004). the sensor is called a microbial sensor. compared to enzyme sensors, microbial sensors have the advantages of increased length of sensor lifetime, and lower cost because the active enzyme does not need to be isolated and purified (lobanov 2001). microbial biosensors have been developed for the measurements of glucose (richardson . 1991), succinate (richardson 1991), lactate (smutok . 2007), and nicotinic acid (takayama 1995). et al. et al et al. et al et al. et al et al. other related studies to glucose biosensor using microbe as biocatalyst had been reported by ikeda . (1998) and iswantini . (1998), while electrochemical method had been successfully applied to study the kinetics and thermodynamics of the holoenzyme formation and to the measurements of the enzymatic activity by k12 (ifo3301) cells. ikeda . (2001) reported that freeze-dried cells had been prepared and utilized for the construction of the whole cell-based glucose sensor, meanwhile yu and liu (2003) used god enzyme with flow injection analysis. recent research indicates that glucose dehydrogenase (gdh) derived from can function as an enzymatic biofuel cell, which can operate as a glucose biosensor (chau . 2009). previous research has explored non-pathogenic indonesian microbes in their ability to produce gdh enzyme nevertheless, this research in indonesia has focused on gdh enzyme farming. the aim of this research was to obtain indonesian bacteria to be used as glucose biosensors using the electrochemical method. et al et al in vivo e. coli et al e. coli e. coli et al . *corresponding author: phone and fax: +62-251-8624567 e-mail: dyahprado@yahoo.co.id issn 1978-3477, eissn 2087-8575 vol 5, no 1, march 2011, p 9-14 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.1.2 materials and methods microbial cells and reagents. spectrophotometrical measurements. preparations of the bacterial cell modified electrode. electrochemical measurements. , , and were isolated from semurup hotspring area, kerinci-jambi, sumatera. the bacteria were selected based on glucose utilization and gram staining (gram negative for , gram positive spore forming for and temperature resistance of the thermophile . . the bacteria were grown on heterotropic liquid medium (1.5% tryptone, 0.5% yeast extract, 0.5% nacl) for 2 h at 37 °c for and , and 50 °c for . to a late logarithmic phase. they were then incubated to reach the od of 0.5. the cells were harvested by centrifugation washed twice with a saline solution (0.85% nacl) and were kept refrigerated at 5 °c. p q q ( 2 , 7 , 9 t r i c a r b o x y l 1 h p y r r o l o [ 2 , 3 f]quinoline-4,5-dione) and q (2,3-dimethoxy-5methyl-1,4-benzoquinone) were purchased from sigma chemical co. all other chemicals used were of high purity. 75 l of each cell suspension was added to phosphate buffer containing pqq (0.3 m and 0.6 m) and mgso as prosthetic group and activator, respectively. 175 l of 1 mm glucose was added as a substrate. absorbance was measured at 340 nm every 15 sec for 3 min. enzyme activity is equal to the concentration of nadph produced per ml per min. carbon paste electrodes were constructed by packing a 2:1 ratio of graphite powder and paraffin liquid into one end of glass tubing, and the surface was smoothed using a piece of waxed paper. a 10 l of aliquot of the cell suspensions was applied onto the surface of each carbon paste electrode. the solution was allowed to evaporate. next, the surface was covered with a dialysis membrane, and fixed with nylon fibre. the finished electrodes, referred to as whole cell electrodes in the following text, were used for enzyme-electrochemical measurements. electrochemical measurements were carried out with basi ec epsillon voltammetry analyzer under anaerobic conditions, in which an ag agcl sat k l electrode, a platinum disk, and a carbon paste electrode were used as a reference electrode, a counter electrode, and a working electrode, respectively. the measurements were conducted at room temperature (25 °c) with an electrolysis cell containing 1 ml of basal solution of phosphate buffer at a previously determined optimum of ph 6.5. the test solution was stirred with a magnetic stirrer and was deaerated by passing over argon gas, unless stated otherwise. e. coli b. subtilis t. filiformis e. coli b. subtilis, t filiformis e. coli b. subtilis t filiformis 610 0 4 μ μ μ | | c results selection of a high lifetime stability indonesian microbe. spectrophotometrical gdh activity measurement. electrochemical measurement of gdh activity. from a number of microbial species, , and were screened for temperature resistance from 25 °c to 50 °c over a 1-3 month period. microbes showed temperature resistance and long stability (tables 1 and 2). bacterial gdh enzyme activity was determined spectrophotometrically at 340 nm. the results showed that gdh activity increased up to 200 times when in the presence of pqq and mgso . the activity of gdh in the presence of na edta was much higher than that without na edta ( table 3). using the reactivation of the -modified carbon paste electrode the capability of microbes as glucose biosensor was electrochemically measured by examining the effects of pqq (table 4) and mg ions and determination of the gdh activity. the currenttime curve measured at 213 mv with the modified electrode is shown in fig 1, while fig 2 shows the relation between the current and the ph of the solution, fig 3 and 4 show the current-time curve with and . modified electrodes. e. coli p. fluoresens, enterobacter, bacillus, bactotermo, s. stearothermophyllus in vivo e. coli e. colib. subtilist filiformisin vivo in vivo 4 2 2 2+ table 1 the life span of several indonesian microbes microbes life span escherici coli bacillus subtilis thermus filiformis pseudomonas. fluorescens enterobacter streptococcus 2 weeks 5 years 2 weeks 2 days 2 days 1 week table 2 temperature resistance of several indonesian microbes microbes maximum temperature resistance (ºc) eschericia coli bacillus subtilis thermus filiformis pseudomonas fluorescens enterobacter streptococcus 37 42 60 37 37 30 table 3 activity of gdh enzyme in (spectrophotometrically) eschericia coli gdh activity (µmol nadph ml -1 min -1 ) without pqq and mgso4 +pqq +pqq+mgs o4 containing na2edta 6.7524 x 10 -4 4.0514 x 10 -2 3.4727 x 10 -2 without na2edta 9.6463x 10 -4 9.6463 x 10 -4 1.9293 x 10 -2 buffer 10 swantinii et al. microbiol indones measurement of gdh activity using -modified carbon paste electrode at 213 mv showed significant current in the presence of pqq and mgso , the maximum current attained for 60 sec, the cyclic voltammetry of -modified carbon paste electrode as shown in fig 6. fig 7 shows the comparison of gdh activity in , and . and fig 8 shows the glucose concentration dependence of the gdh activity in . e. coli e. coli e. coli b. subtilis, t. filiformis e. coli 4 fig 1 current-time curve measured at 213 mv with the modified electrode. a, starting point of measurement, addition; of 5 mm q ; , ; , in the phosphate buffer ph 6.0. eschericia coli b, 0 4 and 10 mm glucose c 4.6 μm pqq and d 10 mm mgso fig 2 curve of ph dependence of the current magnitude. , glucose dehydrogenase activity. a b c d e fig 3 current-time curve measured at 213 mv with the modified electrode. a, 5 mm q , , , were added to the phosphate buffer ph 6.5. bacillus subtilis 0 4 ; b 10 mm glucose; c 4.6 μm pqq; and d 10 mm mgso fig 5 current-time curve measured at 213 mv with the –modified electrode. a, starting point of experiment; b, 5 mm q , , , were added to the phosphate buffer ph 6.5. e. coli 0 4 ; c 10 mm glucose; d 4.6 μm pqq; and e 10 mm mgso fig 6 cyclic voltammetry of -modified carbon paste electrode. escherchia coli c u rr e n t ( a ) μ 2.37 1.79 1.20 0.61 0.02 0.1 5.0 10.0 15.0 20.0 time (min) a b c d ph phosphate buffer solution c u rr e n t ( a ) μ 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 4 5 6 7 1.24 1.09 2.20 2.99 3.46 c u rr e n t ( a ) μ 1.50 0.88 1.19 0.57 0.26 0.1 7.6 15.1 22.5 30.0 time (min) 3.5 c u rr e n t ( a ) μ 2.6 1.8 0.9 0.0 0.3 time (min) 6.5 12.7 18.8 25.0 a b c d e c u rr e n t ( a ) μ 46.8 -9.2 8.8 -37.2 -180-406 47 273 potential (mv) [1] [2] volume 5, 2011 microbiol indones 11 table 4 dependence of pqq concentration on the current [pqq] (μm) current (μa) time (min) 0.1 0.5 1.2 2.3 4.6 6.0 1.5 1.0 1.6 3.0 5.5 4.5 30.0 30.0 14.5 6.0 6.0 5.5 stability of gdh activity. discussion the stability of gdh activity was determined on immobilized and . cells on the surface of the carbon paste electrode. the treated carbon paste electrodes were stored in nacl 0.85% at room temperature. after 6 h the activity of gdh was measured. the results indicated that had the greatest stability with 74.04% of the gdh activity remaining after 6 h, followed by at 58% and at 27.61%. of the microbes tested, the isolated , , and had glucose utilization e. coli, b. subtilis, t filiformis b. subtilis e. coli bactotermo e. coli b. subtilis t. filiformis c u rr e n t ( a ) μ fig 4 current-time curve measured at 213 mv with the –modified electrode. a, starting point of experiment; b, 5 mm q , , on the phosphate buffer ph 6. thermus filiformis 0 4; c 10 mm glucose; d 4.6 μm pqq; and (e) 10 mm mgso 0.098 0.075 0.052 0.020 0.000 c e d a b 0.2 time (min) 7.6 15.0 22.4 29 characters essential for this glucose biosensor experiment. their nature of cell wall and temperature respect would then give more on physiological characters on the biosensor output and higher stability with respect to temperature resistance and life span at room temperature than other microbes. and had the highest life span and stability on temperature resistance, respectively. although had a lower life span and temperature resistance than that of and , but only to be potentially used in biosensor in this research supported by previous research (ikeda . 2001) proved that could be used as a glucose biosensor. therefore all of them were used in the experiment. the biosensor capacity is based on oxidation of glucose by gdh enzyme produces gluconate and nadph with nadp as an electron acceptor. glucose + nadp gluconate + nadph b. subtilis t. filiformis e. coli b. subtilis t. filiformis e. coli et al e. coli + + gdh fig 7 the activity of in , , and which measured electrochemically escherichia coli bacillus subtilis thermus filiformis . glucose dehydrogenase fig 8 the glucose concentration dependence of the activity of gdh in eschericia coli. fig 9 stability of bacteria activity for 6 h time period glucose dehydrogenase . e. coli b. subtilis t. filiformis c u rr e n t ( a ) μ 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 e. coli b. subtilis t. filiformis 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 glucose (mm) 20 40 600 g d h a c ti v it y ( a ) μ 9 8 7 6 5 4 3 2 1 0 1.2 1.91 2.74 4.52 7.63 8.03 12 swantinii et al. microbiol indones a c ti v it y s ta b il it y ( % ) absorbance values resulted from spectrophoto metrically measurement indicated that the gdh activity was produced by , , and . it was known that the oxidation of glucose by gdh depended on the concentration of pqq as a prosthetic group in the presence of mg ions as an activator of gdh. meanwhile edta could decrease the activity of gdh in by chelating mg ions (ikeda 1998; iswantini . 1998). this prompted study on the effect of the addition of pqq and mgso on the activity of gdh in the presence or absence, of na edta. cells had the highest gdh activity compared to and . (table 3). had low gdh activity because this is a gram positive bacterial having cell wall higher in containing peptidoglican much higher than that of gram negative ( ) (demchik . 1996). the presence and absence of na edta in the buffer affected the activity of gdh. from electrochemical measurements, a current of the fixed potential at 213 mv was applied to follow the appearance of gdh activity (fig 1). a 5 mm q , 10 mm glucose, 4.6 were added to the buffer solution in that order. a small current started to appear when q and glucose were added. it started to increase only after the addition of pqq, because apo-gdh (inactive gdh) in the cells was converted to holo-gdh (active gdh) by incorporating pqq added to the solution, and the hologdh catalyzed the oxidation of glucose with q as an electron acceptor to produce a catalytic anodic current. when mgso was added to the solution, the current started to increase again and approached a significant magnitude, then continued to increase gradually and reached the steady state until at least 7 min. this result indicated that could produce gdh enzyme that could oxidize glucose to be gluconate, and the g d h a c t i v i t y c o u l d b e d e t e r m i n e d e a s i l y spectrophometrically and electrochemically, at optimum ph of 6.5 (fig 2) and 4.6 m of pqq (table 4). electrochemically, gdh activity in could be detected, but unstable current had been produced after the addition of q and glucose (fig 3). it may be due to the peptidoglican content of the cell wall of positive bacteria that could not excrete gdh enzyme from its periplasm easily (demchik . 1996). measurement of gdh activity in modified carbon paste electrode had been carried out at optimum condition (ph 6.5 of phosphate buffer and ) as shown in fig 5. addition of q and glucose to the buffer solution had little observable effect on the current. it increased significantly about 1.8 a after the addition of pqq and attained a steady-state. when e. coli b. subtilis t. filiformis in vivo e. coli et al et al e. coli b. subtilis t filiformis b. subtilis e. coli et al e. coli e. coli in vivo b. subtilis b. subtilis et al e. coli 2+ 2+ 4 2 2 0 4 0 0 4 0 0 μm pqq, and 10mm mgso 4.6 μm pqq mgso was added to the solution, the current started to increase again and approached 0.8 a and attained a steady state for 60 sec while the current was stable for 8 min. this result indicated that measurement of gdh activity in -modified carbon paste electrode at optimum condition gave the highest activity of gdh compared to that of the other microbes. the result indicated that among several indonesian microbes, , , and had high stability with respect to temperature resistance and life span. measurement of gdh activity using modified carbon paste electrode at 213 mv gave significant current in the presence of pqq and mgso , the maximum current attained for 60 sec, the cyclic voltammetry of -modified carbon paste electrode as shown in fig 6. constant current could be maintained for 8 min. the capability of for glucose biosensor were good when it was done at ph 6.5 of phosphate buffer and 4.6 m of pqq concentration. electrochemically, gdh in gave a highest activity among three bacteria tested ( , and ) as shown in fig 7. the activity of gdh and the number of gdh in bacteria were supposed depend on the structure of their cell wall. dependence of glucose concentration on the current produced by -modified carbon paste electrode indicated that this method allowed the detection of glucose concentration up to 20 mm (fig 8). this result similar to amperometric biosensor for continuous glucose monitoring based on the mwcnts/graphite/ gox packed needle-type electrode (jia . 2008). this indicated that can be developed as glucose biosensor, because the detection limit was comparable to carbon nanotube glucose biosensor. also, this result showed that using microbe cell as glucose biosensor has similar capability compared to purified enzyme. this method provide less costly because the active enzyme does not need to be isolated and purified. this result was supported by recent research that gdh derived from could be used as the enzymatic biofuel cell, which could operate as a glucose biosensor (chau . 2009). the glucose biosensor sensitivity activity of was observed to reach the steady state for as long as 120 sec at 58% of maximum activity after the addition of glucose. this stability is lower than that of gdh activity resulted by k-12 (ifo3301) which shown to reach 70% of maximum activity (iswantini . 2000). fig 9 showed the comparison of the stability of gdh activity resulted that among the 3 bacteria, the activity of gdh in was the highest, while the lowest was that of . this might be due to the ability of this bacteria to form 4 4 e. coli e. coli b. subtilis t. filiformis e. coli e. coli e. coli e. coli e. coli b. subtilis, t. filiformis e. coli et al e. coli e. coli et al e. coli e. coli et al b. subtilis t. filiformis volume 5, 2011 microbiol indones 13 endospore. bacteria that have a high ability to form endospore have longer life spans than others. therefore, a longer life span would result in higher gdh stability. the low gdh stability of might be due to the storing temperature: it is a thermophilic bacteria with an optimum temperature of 50-70 °c. storing at less than these temperatures may cause a degradation of gdh activity. because, gdh in gave a highest activity among three bacteria tested, electrochemically (fig 7), however, indonesian . can be used as a glucose biosensor with high stability, even the stability still less than the purified enzyme biosensor that has excellent stability with almost 90% of its bioactivity maintained after storage at 4 °c in phosphate buffer solution for ten days (wang . 2008). this work was funded by the indonesia toray foundation research grant 2005. we would like to thank tokuji ikeda from laboratory of bioanalytical physical chemistry, department of agricultural 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biosensor for glucose detection utilizing a periplasmic receptor protein. electrochem solid-state lett. 8(8):h61-64. wang h, zhou c, liang j, yu h, peng f, yang j. 2008. high sensitivity glucose biosensor based on pt electrodeposition onto low-density aligned carbon nanotubes. int j electrochem sci. 3(11):1258-1267. wild s, roglic g, green a, sicree r, hilary k. 2004. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetic care. 27(5):1047-1053. pseudomonas fluorescens e. coli . doi:10.1016/j.bios.2007.06.021. doi:10.1016/0022-0728(99)03651-1. doi:10.1016/so9565663(03)0019-4. doi:10.1149/ 1.1943549. doi:10.2337/diacare.27.5.1647 4 482 lita tritarna ok.cdr genome-shuffling-improved acid tolerance and lactic acid production in for commercializationlactobacillus plantarum lita triratna , budi saksono , linda sukmarini , asep suparman 1 1 1 2 * and 1 2 research center for biotechnology, lembaga ilmu pengetahuan indonesia, jalan raya bogor km 46, cibinong 16911, indonesia department of food science and technology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia lactobacillus plantarum l. plantarum lactobacillus plantarum genome shuffling lactobacillus plantarum shuffling genome shuffling genome shuffling l. plantarum genome shuffling, lactobacillus plantarum ; we applied genome shuffling to improve the acid tolerance of , while simultaneously enhancing lactic acid production. the starting populations were mutant libraries generated by gradual low ph adaptation and ultraviolet irradiation and then which were subjected to recursive protoplast fusion. a library of shuffled mutants (fusants) with genetic exchange is achieved by repetition of this process. after three rounds of genome shuffling, we obtained the best performing fusant that grow better at low ph 4.0 and consume glucose faster than does the wild type. in addition, lactic acid production of this fusant was 64% higher than that of the wild type. these results demonstrated that the genome shuffling has been successful in engineering with multiple beneficial improved phenotypes. in the future, this technology is a promising candidate to accelerate poorly characterized strains for commercialization. key words: genome shuffling, , lactic acid, acid tolerance teknik telah diaplikasikan untuk memperbaiki toleransi asam dan secara simultan meningkatkan produksi asam laktatnya. populasi awal yang digunakan ialah pustaka mutan yang didapat dengan menurunkan ph medium pertumbuhan secara bertahap dan iradiasi sinar ultra violet. pustaka mutan kemudian digunakan dalam proses fusi protoplas secara berulang. hasil dari pustaka mutan disebut fusan. paska pengulangan yang ke-3 dari didapatkan fusan unggul yang mampu tumbuh dengan baik pada ph 4.0 dan mengonsumsi gula lebih cepat dibandingkan dengan galur liar. produksi asam laktat oleh fusan ini meningkat 64% dibandingkan dengan galur liar. hasil ini menunjukkan bahwa teknik telah berhasil merekayasa yang memiliki keunggulan fenotipe. di masa mendatang teknik ini mampu menjadi kandidat yang menjanjikan dalam komersialisasi galur mikrob yang belum dikarakaterisasi dengan baik. kata kunci: , asam laktat, toleransi asam lactic acid has been widely used in the food, pharmaceutical and cosmetics industries. it has recently emerged as an important source for the production of other chemicals, particularly polylactic acid for biodegradable plastic (singh 2006). most lactic acid is manufactured by fermentation process as opposed to chemical synthesis since it is environmentally friendly (john . 2009). fermentation at low ph contributes to the economics of the lactic acid purification process. thus, the commercial goal of improving the growth and lactic acid production of microorganisms at acid condition could decrease waste and reduce the cost of production. moreover, it also reduces potential contamination (porro . 1999; carlson and peters 2002; singh . 2006). is of commercial interest for lactic acid production, but the effect of acid stress on the bacterium is a complex and poorly understood process. as a result, it is difficult to improve lactic acid production with rational engineering or direct genetic manipulation. on the other hand, classical strainimprovement-methods have succeeded in obtaining et al. et al et al et al l. plantarum many industrial strains, but this is a time-consuming and laborious process because of many repeated rounds of random mutation and selection methods, especially for engineering complex phenotypes (zhang 2002). thus, an efficient technology named genome shuffling has been presented as a novel whole-genome engineering approach for the rapid improvement of cellular phenotypes. this approach uses recursive protoplast recombination throughout the entire genome without the necessity for genome sequence data or network information (patnaik 2002; stephanopoulos 2002). at present, genome shuffling has been successfully applied for the improvement acid tolerance in (patnaik 2002), acid and glucose tolerance in (wang 2 0 0 7 ; yu 2 0 0 8 ) , d e g r a d a t i o n o f pentachlorophenol in (dai and copley 2004) and the production of hydroxycitric acid in (hida 2007). however, shuffling of an industrial strain has not been reported. thus, in the present study, we applied genome shuffling to improve the acid tolerance of while simultaneously enhancing lactic acid production. et al. et al. lactobacillus et al. l. rhamnosus et al. e t a l . sphingobium chlorophenolicum streptomyces et al. l. plantarum l. plantarum *corresponding author: phone: +62-218754587, fax: +62-21-8754588, e-mail: saksonobudi@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 1, march 2011, p 21-26 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.1.4 materials and methods bacterial strains and media. adaptation of to low ph. strain mutagenesis and mutant screening. protoplast preparation and genome shuffling. was obtained from biotechnology type culture collection (btcc) research center for biotechnology-lipi, indonesia. the strain was stored in a deman rogosa and sharpe (mrs) broth with 10% (w v ) glycerol at -70 °c. the strain was grown in mrs broth from a 1% inoculum with 18 h incubation at 37 °c. at least two generations of precultures were required just before the experiments. yeast extract fermentation medium (ye) contained 100 g l glucose and 15 g l yeast extract. regeneration medium (rm) was mrs agar without tween 80 and supplemented with 0.5% bsa, 25 mm mgcl , 25 mm nacl , and 0.5 m sucrose (patnaik 2002). culture adaptation was achieved by modification of the procedure from patnaik (2002), through a continuous and controlled decrease in broth medium ph 6.2 to ph 5.0 and ph 4.0. for scenarios in which the imposed ph was not tolerated, and the culture steady-state could not be maintained, the culture was replaced with fresh mrs broth of ph lower than 4.0. adapted wild-type strain in ph 4.0 medium was then used as the parental, or starter, for genome shuffling. the cells of wild-type strain were grown in a 10 ml mrs broth at 37 °c for 18 h. cultures were centrifuged at 4500 x for 10 min. uv irradiation was performed by exposing samples of the wild-type strain in uncovered petri dish directly to uv light at a distance 20 cm with 120 mj cm of intensity for 30 s using uv crosslinker (hoefer uvc500, usa). the mutants were then spread on mrs agar plates containing 2% (w v ) caco and incubated at 37 °c for 48 h. the fast growing colonies, which had bigger transparent haloes compared to colony diameter, were picked off and selected as the starter for the genome shuffling. protoplast formation was carried out as described by cocconcelli . (1986) with modification. strains were cultured at 37 °c for 18 h in mrs broth. the cells were harvested by centrifugation at 4500 and 4 °c for 10 min, and washed twice with distilled water. the cells from a 5 ml culture were suspended in protoplast b l tris hcl 1 m ph 6.3, 2.5 ml sucrose 1 m, 2 ml cacl 50 mm, and 450 l distilled water) containing lysozyme (2.0 x 10 u ml ) and incubated at 37 °c for 2 h for protoplast formation. lactobacillus plantarum et al. et al. l. plantarum g et al x g lactobacillus -1 -1 -1 -2 -1 5 -1 2 2 3 2 l. plantarum uffer (lpb) (50 μ μ et al. et al the starting populations for genome shuffling were mutants-library-generated by gradually low ph adaptation and ultraviolet (uv) irradiation. fusions between protoplast preparations were generated by mixing an approximately equal number of protoplasts from two populations as described by patnaik with slightly modification (patnaik 2002). the shuffled mutants, named fusants were then cultivated in regeneration medium. each culture was incubated at 37 c for 6 h and processing continued by spreading in selection medium of mrs agar plates containing 2% (wv ) caco , ph 4.0, and then incubated at 37 c for 3-4 d. subsequent colonies were selected randomly to carry out further selection with small-scale fermentation analysis in 2 ml ye broth culture at ph 4.0. the cultures were incubated at 37 c for 48 h. profiles of both growth and lactic acid production for each culture were then followed. the value of both growth and lactic acid production then were analyzed using spss program to identify the fusants that have a significant change in their growth and lactic acid production compared to that of control. the formation of protoplasts, their fusion, and subsequent regeneration was repeated three times with the pooled regenerated cells from one fusion being the inoculum for the subsequent protoplast culture. samples from each of the pooled fusion libraries f1-f3 (first/f1, second/f2, third/f3) were saved for later analysis. non-shuffled (unshuffled) controls were prepared by the recursive formation and regeneration of protoplast without exposure to peg. the best performing selected shuffled strain, f3-33, after three rounds of genome shuffling (f3) was cultured in 10 ml mrs broth before being transferred into 100 ml ye medium at ph 4.0 in shaking flasks (250 ml) at 100 rpm, 37 °c. the growth, ph, residual glucose, and lactic acid production were periodically monitored up to 96 h. cell growth was monitored by measuring cell number via the optical density at 600 nm (a ). supernatants of the samples for assay were collected by centrifugation. total lactic acid for fusant selection was estimated according to the titration method, and the total sugar concentration was determined using the phenol-sulphuric acid method, as described by dubois . (1956). furthermore, total lactic acid of the selected f3 fusant was then determined by reversed-phase highperformance liquid chromatography (hplc) (shimadzu lc-6ad, japan) using a c18 silica column (hplc packed column ug120 shiseido, japan) with uv detector set at 220 nm. the column was eluted with 20 mm nah po (ph 2.55) as a mobile phase at a flow o -1 o o 3 600nm 2 4 shaking flask analysis. analytical methods. 22 triratna et al. microbiol indones rate of 1.0 ml min . all of the measurements were performed in duplicate from two independent experiments. the first population of the adapted acid-mutant-library was achieved by slowly decreasing the ph from 6.2 to 4.0 over a period of 840 h. a stable population of was able to grow at ph 4.0, a ph that severely inhibits growth of the wild-type strain. the second population was enriched for acid tolerance of the wild-type strain using uv mutant library selected on mrs-agar-plates at ph 4.0. ten colonies which had bigger transparent haloes compared to colony diameter were obtained (unpublished data). the resulting new population (shuffled strains/fusants) represented a larger combinatorial library of the original genetic diversity. after the first fusion, 56 colonies were picked off randomly for further selection in small-scale fermentation. selection was done by spss program based on their growth and lactic acid production values. fusants with higher and lower lactic acid production respectively were shown (fig 1, 2, 3). first-round shuffling also resulted 29 fusants with growth-faster compared to that of wild-type (unpublished data). among those fusants, 4 fusants, i.e. f1-2, f1-16, f1-48, and f1-50, showed the value of relative lactic acid production per a higher than 1.0 (control) and 2 fusants, i.e., f1-6 and f1-30 showed relative growth-fastest (120%). relative lactic acid production is the value of total lactic acid produced by sample divided by total lactic acid produced by control, whereas relative growth is the value of cell density of sample divided by that of control at a . two fusants from the selected f1 fusants library, with higher relative lactic acid production/ a (p < 0.05) to that of wild type, f1-30 and f1-16 (shown as red bar) were used as resources for the next fusion (fig 1a). figure 1b showed 4 fusants (f2-3, f2-22, f2-26, and f2-65) and 10 fusants (f2-64, f2-77, f2-78, f2-79, f2-88, f2-89, f2-90, f2-91, f2-109 and f2-116) with lower and higher lactic acid production, respectively. second round of shuffling also resulted 87 of fusants with growth faster compared to that of f1-30 as control (unpublished data). among second shuffled library f2, 4 fusants, namely f2-78, f2-88, f2-89, and f2-91 were selected and used for the next fusion (fig 1c). these fusants were selected due to their higher relative lactic acid production to that of f1-30 as control (p < 0.05). the result showed 4 fusants (f3-33, f3-118, f3-119, and f3-176) and 34 fusants with higher and lower -1 results isolation of initial strains. genome shuffling. l. plantarum 600nm 600nm 600nm volume 5, 2011 microbiol indones 23 a b c fig 1 relative lactic acid production to the control and relative lactic acid production per a . a, f1 fusants library; b, f2 fusants library; c, f3 fusants library . wild-type strain (wt), f130 fusant, and f2-89 fusant is used as the control for f1, f2, and f3fusants library respectively (for more detail information see result session of genome shuffling). x-ordinate represents colonies numbers from each fusant library. bar represents relative lactic acid production of fusants, line represents relative lactic acid production per a . red bar indicated the fusants were choose for next rounds. 600nm 600nm lactobacillus plantarum fig 2 relative growth of f3 fusants library to f2-89 fusant as a control. x-ordinate represents colonies numbers from f3 fusants library. lactobacillus plantarum 140 120 100 80 60 40 20 0 l a c ti c a c id p ro d u c ti o n r e la ti v e ( % ) l a c ti c a c id /o d 6 0 0 m m r a ti o 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 fusant number 1 5 9 1 3 1 7 2 1 2 5 2 9 3 3 3 7 4 1 4 5 4 9 5 3 5 7 6 1 6 5 6 9 7 3 7 7 8 1 8 5 8 9 9 3 9 7 1 0 1 1 0 5 1 0 9 1 1 3 1 1 7 1 2 1 1 2 5 1 2 9 1 3 3 1 3 7 1 4 1 1 4 5 1 4 9 1 5 3 1 5 7 1 6 1 1 6 5 1 6 9 1 7 3 1 7 7 f 2 -8 9 r e la ti v e l a c ti c a c id p ro d u c ti o n p e r a 6 0 0 n m1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1 5 9 1 3 1 7 2 1 2 5 2 9 3 3 3 7 4 1 4 5 4 9 5 3 5 7 6 1 6 5 6 9 7 3 7 7 8 1 8 5 8 9 9 3 9 7 1 0 1 1 0 5 1 0 9 1 1 3 1 1 7 1 2 1 1 2 5 1 2 9 1 3 3 1 3 7 1 4 1 1 4 5 1 4 9 1 5 3 f2 fusants 140 120 100 80 60 40 20 0 r e la ti v e l a c ti c a c id p ro d u c ti o n ( % ) r e la ti v e l a c ti c a c id p ro d u c ti o n p e r a 6 0 0 n m1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 140 120 100 80 60 40 20 0 r e la ti v e l a c ti c a c id p ro d u c ti o n ( % ) 1 5 9 1 3 1 7 2 1 2 5 2 9 3 3 3 7 4 1 4 5 4 9 5 3 5 7 6 1 6 5 6 9 7 3 7 7 8 1 8 5 8 9 9 3 9 7 1 0 1 1 0 5 1 0 9 1 1 3 1 1 7 1 2 1 1 2 5 1 2 9 1 3 3 1 3 7 1 4 1 1 4 5 1 4 9 1 5 3 1 5 7 1 6 1 1 6 5 1 6 9 1 7 3 1 7 7 f 2 -8 9 f3 fusants 1 5 9 1 3 1 7 2 1 2 5 2 9 3 3 3 7 4 1 4 5 4 9 5 3 5 7 6 1 6 5 6 9 7 3 7 7 8 1 8 5 8 9 9 3 9 7 1 0 1 1 0 5 1 0 9 1 1 3 1 1 7 1 2 1 1 2 5 1 2 9 1 3 3 1 3 7 1 4 1 1 4 5 1 4 9 1 5 3 1 5 7 1 6 1 1 6 5 1 6 9 1 7 3 1 7 7 f 2 -8 9 f3 fusants r e la ti v e g ro w th ( % ) 120 100 80 60 40 20 0 , yield., laktat; relative lactic acid production, respectively (fig 1c). among third shuffled library, f3-33 showed highest relative lactic acid production (136%) compared to that of f2-89 as control (p < 0.05). the relative growth performance to the control of 180 colonies from third shuffled library (f3) which were picked off randomly. figure 2 showed 109 and 6 of fusants with growth faster and slower, respectively. the relative growth of f3 fusants library was mostly higher compared with that of the f2-89 fusant as control (fig 2), while relative lactic acid production of f3 fusants library was mostly lower than that of control (fig 1c). the best performing lactic acid production of fusants in each round (f1-30, f2-89, f333) were compared for their relative lactic acid production to that of the wild-type (fig 3). it is shown that f3-33 fusant in the third round genome shuffling performed the highest in lactic acid production. the performance of f3-33 was further compared with the wild-type strain in shaking flasks (fig 4a-c). consistent with smallscale fermentation results, the genome shuffled strain f3-33 produced about a 64.5 % improvement of lactic acid production (12.0 g l ) over the wild-type for 48 h (fig 4a). this lactic acid production increased up to 48 h, after which no significant improvement was found. moreover, the growth characterization and glucose consumption were consistent with the lactic acid production. the fusant f-33 grew 53% faster compared with the wild-type (fig 4b), and also consumed glucose faster (fig 4c). as lactic acid fermentation at low ph (at or below the of lactic acid, ph 3.8) can reduce the cost shaking flask analysis. discussion -1 pka 24 triratna et al. microbiol indones 180 160 140 120 100 80 60 40 20 0 r e la ti v e l a c ti c a c id p ro d u c ti o n ( % ) wt f1-30 f2-89 f3-33 fusant a ab bc cd fig 3 relative lactic acid production of the best performing fusants from each of three rounds of genome shuffling (f1-f3) to the wild type; wt is wild type strain of . f1-30 is fusant from colony number 30 of first fusion round, f2-89 is fusant from colony number 89 of second fusion round, while f3-33 is fusant from colony number 33 of third fusion round. bar with different letters are significantly different by dunnet test at p<0.05. lactobacillus plantarum a b c fig 4 characterization of lactic acid production: a, cell growth; b, and residual sugar (glucose) of fusant f3-33 compared to that of wild type (wt) . ,lactobacillus plantarum production and decrease waste, acid tolerance phenotypes meet the performance criteria for industrial strain commercialization. thus, the strains tolerant to acid conditions are desirable industrial phenotypes (patnaik 2002). environmental tolerance of ph is a complex and yet poorly understood phenomenon, therefore, therefore we applied genome shuffling approach to improve lactic acid production of local . this approach has successfully been used to improve acid tolerance in through fusion between a low-ph-adapted mutant library population and nitrosoguanidine (ntg) mutant-library-population (patnaik . 2002), and in through uv irradiation and ntg mutagenesis (wang . 2007). here, we used an et al. l. plantarum lactobacillus et al l. rhamnosus et al time (h) time (h) a 6 0 0 n m time (h) 14 12 10 8 6 4 2 0 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 f3-33. 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 1.10 1.00 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 00 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 wt; , volume 5, 2011 microbiol indones 25 adapted low-ph-mutant library population and an uvirradiationmutant library population, instead of a nitrosoguanidine (ntg) mutant library population, as the starter of genome shuffling. since genome shuffling practically mimics the features of natural evolution through recursive genetic recombination, it requires a diverse population of mutants with an improvement of the desired phenotype compared with the wild-type as a starting point (yu 2008). the present study showed more than 50% of fusants with change in their relative lactic acid production and growth. the percentage of fusants with different performing compared to that of their control was increased to 60% (9% and 51% with their lactic acid production and growth, respectively), 66% (9% and 57% with their lactic acid production and growth, respectively) and 85% (21% and 64% with their lactic acid production and growth, respectively) in first, second, and third rounds, respectively. the results of the present study also proved that genome shuffling is an effective strategy for generating strains which become more tolerant to the acid and result in the improvement of lactic acid production. it was shown that the relative lactic acid production of the best performing fusants (f1-30, f2-89, and f3-33) from each of three rounds of genome shuffling were higher than that of the wild-type (120%, 144%, and 167.8% respectively) (fig 3), corresponding with the higher growth of those fusants compared with the wild-type (unpublished data). after three rounds of genome shuffling, we obtained f3-33 fusant which had the highest lactic acid production and capable of growing at ph 4.0. in this regard, f3-33 fusant was further characterized by comparison with the wild-type strain. in agreement with small-scale fermentation, the lactic acid production of f3-33 fusant was 64.5% higher compared with the wild-type strain in shaking flasks (fig 4a). after 48 h of growth at ph 4.0, the wildtype strain had produced 7.74 g l of lactic acid, while the f3-33 fusant produced 12 g l . characterization of f3-33 fusant and the wild-type in the shaking flask showed the lactic acid production was closely correlated with the growth and glucose consumption ( fig 4b and 4c). the best performing fusant grew faster, consumed glucose faster, and produced more lactic acid, and also brought the medium to a lower ph (unpublished data), as reported by patnaik . (2002) and wang (2007). however, these previously published results showed that the fusants produced three-fold more lactic acid than the wild-type at ph 4.0. the improvement of lactic acid of our fusant was lower than that of these previous reports. this result may due to the fact that the starting et al. et al et al. -1 -1 population for each round of fusion had low diversity. the fusant library f1 and f2 were generated from two colonies/ fusants of f0 and f1 fusants library, respectively, while the fusant library f3 was generated from 4 fusants of f3 fusants library. from f1, f2, and f3 fusants library, the percentage of fusants which had changed in their lactic acid production were 9%, 9% and 21%, respectively. these achievements were agreed with the number of fusants used as resources to generate fusants library. thus, we concluded that the low genetic diversity within a selected population resulted in low recombination and combinatorial library. these results, however, demonstrated that we had been succeeded in applying the genome shuffling to engineer with multiple beneficial improved phenotypes including acid tolerance, lactic acid enhancement, and glucose/substrate consumption at one time. in the future, it is promising that this simple technology is a promising candidate to accelerate poorly characterized strains for commer cialization. we would like to thank yantyati widyastuti for providing btcc strain. funding for this project was provided in part by the indonesian ministry of education and culture program 2009. l. plantarum lactobacillus plantarum acknowledgements references carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002. low ph lactic acid fermentation. united state patent us 6,475,759. cocconcelli ps, morelli l, vescobo m, bottazzi v. 1986. intergeneric protoplast fusion in lactic acid bacteria. fems microbiol lett. 35 (2-3):211-214. dai mh, copley sd. 2004. genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by atcc 39723. appl environ microbiol. 70(4):2391 2397. dubois m, gilles ka, hamilton jk, rebers pa, smith f. 1956. colorimeteric method for determination of sugars and related substances. anal chem. 28(3):350-356. hida h, yamada t, yamada y. 2007. genome shuffling of sp. u121 for improved production of hydroxycitric acid. appl microbiol biotechnol. 73(6):1387-1393. john rp, anisha gs, nampoothiri km, pandey a. 2009. direct lactic acid fermentation: focus on simultaneous saccharification and lactic acid production. biotech adv. 27(2):145-152. patnaik r, louie s, gavrilovic v, perry k, stemmer wpc, ryan cm, del cardayré s. 2002. genome shuffling of for improved acid tolerance. nat biotechnol 20(7):707-712. porro d, bianchi mm, brambilla l, menghini r, bolzani d, carrera v, lievense j, liu cl, ranzi bm, frontali l, alberghina l. 1999. replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts. appl environ microbiol. 65(9):4211-4215. sphingobium chlorophenolicum streptomyces lactobacillus . doi: 10.1111/j.1574-6968.1986.tb01529.x. doi: 10.1128/aem.70.4.2391-2397.2004. doi: 10.1021/ac60111a017. doi: 10.1007/s00253-006-0613-1. doi: 10.1016/j.biotechadv. 2008.10.004. doi: 10.1038/nbt0702-707. 26 triratna et al. microbiol indones singh sk, ahmed su, pandey a. 2006. metabolic engineering approaches for lactic acid production. process biochem. 41(5):991-100. stephanopoulos g. 2002. metabolic engineering by genome shuffling. nat. biotechnol. 20(7): 666-668. wang y, li y, pei x, yu l, feng y. 2007. genome-shuffling improved acid tolerance and l-lactic acid volumetric productivity in .jbiotechnol.129(3):510-515. lactobacillus rhamnosus doi: 10.1016/j.procbio.2005.12.004. doi: 10.1038/nbt0702-666. doi:10.1016/j.jbiotec.2007.01.011. yu l, pei x, lei t, wang y, feng y. 2008. genome shuffling enhanced llactic acid production by improving glucose tolerance of . j biotechnol. 134(1-2):154-159. zhang dx, perry k, vinci va, powell k, stemmer wpc, cardayre sd. 2002. genome shuffling leads to rapid phenotype improvement in bacteria. nature. 415(6872): 644-646. lactobacillus rhamnosus doi: 10.1016/j.jbiotec. 2008.01.008. doi: 10.1038/ 415644a. 1 454 the relation (purwati).cdr the relation between levels of tnf a and pla with the severity degree of dengue hemorrhagic lpha, il-1β, pge2 2 purwati , nasronudin , endang retnowati kusumowidagdo , fedik abdul rantam 1* 2 3 and 2 1 2 3 infectious and tropical disease division, department of internal medicine dr. soetomo hospital, school of medicine, universitas airlangga, jalan mayjen prof. dr. moestopo 6-8, surabaya 60115, indonesia; institute of tropical disease, universitas airlangga, jalan mulyorejo, surabaya 60115, indonesia; department of clinical pathology, dr. soetomo hospital, school of medicine,universitas airlangga, jalan mayjen prof. dr. moestopo 6-8, surabaya 60115, indonesia thromboxane leucotrien, cross sectional correlate enzyme assay the pathogenesis of dengue virus infection is still being debated. based on the existing data, there is a strong evidence that the immunopathological mechanism plays a role in dengue virus infection with various complications. some unknown immune responses play a role in the pathogenesis of dengue virus infection. researchers are trying to establish the role of several inflammatory mediators such as pla , il-1 , tnf, pge , pgi2, thromboxane a , leucotrien and mptp, in relation to the severity degree of the dengue virus infection. the aim of this study is to recognize the relation between the severity degrees of dengue hemorrhagic fever (dhf) patients and the immunological profile in the sub-cellular level, such as pla , il-1 , tnf, pge and pla 2. the collected data was processed and presented analytically. the relation between each parameter ( pge and the degree of dhf was analyzes, using spearman's correlation analysis, ordinal regression. it was shown that there was dhf grade 1 and 3, and al here were increased levels of the four parameters in dengue grade 1 to 2, but decreased levels in grade 3. this can be caused by inflammatory processes, but the severity degree of dhf can also be influenced by complement, thromboxane, and leukotrien. key words: dengue hemorrhagic fever, cytokine, pla , il-1β, tnf , pge patogenesis terjadinya infeksi virus dengue hingga saat ini masih diperdebatkan. terdapat bukti yang kuat bahwa mekanisme imunopatologi berperan dalam terjadinya infeksi virus dengue dengan berbagai komplikasi. beberapa penelitian mencoba membuktikan peran beberapa mediator inflamasi seperti spla , il-1, tnf-, pge , a , dan mptp dalam hubungannya dengan derajat keparahan dari infeksi virus dengue tersebut. penelitian observasional ini dilakukan dengan rancang bangun . terdapat 45 penderita yang dirawat di ruang penyakit infeksi rsud dr. soetomo yang memenuhi persyaratan. berdasarkan pemeriksaan il-1 ( dan spla dengan metode eia diketahui tidak didapatkan korelasi yang bermakna antara pengaruh il-1 dan spla terhadap derajat keparahan deman berdarah dengue (dbd). terdapat peningkatan kadar pada dbd derajat 2 dibandingkan dengan derajat 1, tetapi pada derajat 3 terdapat penurunan kadar dari semua parameter. perbedaan yang bermakna ditemukan atkan antara dbd derajat 1 dan 3 serta antara dbd derajat 2 dan 3. tidak didapatkan hubungan linear antara il-1 , dan spla dengan n 3, serta derajat 2 dengan 3. hal ini mungkin disebabkan penurunan proses inflamasi pada derajat 3, tetapi beratnya derajat dbd mungkin dapat dipengaruhi oleh komplemen, tromboksan, leukotrien, atau faktor lain yang mempengaruhi permiabilitas endotel dan kerusakan endotel kapiler. kata kunci: demam berdarah dengue, sitokinin, pla , il-1 , tnf , pge 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 b a b a tnfα, spla ,il-1β) no relation between the levels of tnfα, pge2, il-1β, and spla2 in patients with various degrees of dhf, but there were significant differences between so 2 and 3, on il-1β. t -α β ), tnf-α, pge β, tnf-α, pge hanya pada pengaruh il-1β terhadap derajat keparahan dbd. perbedaan bermakna didap β, tnf-α, pge derajat keparahan dbd, tetapi pada il-1β terdapat perbedaan bermakna antara derajat 1 denga β -α 2 2, dengue hemorrhagic fever (dhf) is one the health problems in the tropical region. in southeast asia, with a total population of 1.5 billion, nearly 1.3 billion people are at risk of dengue virus infection. dengue is a major cause of hospitalization and death in children. dhf was first reported in indonesia in 1968, namely jakarta and surabaya. the dhf incidence rate continues to increase (nasronudin 2007). dengue virus infection is actually a self-limiting disease, but is often accompanied by dangerous complications, such as plasma fluid leakage characterized with haemo-concentrations (increased hematocrytes), shock and bleeding (who 2008; garcia . 2010) the pathogenesis of dengue virus infection is still being debated. based on the existing data, there is strong evidence that the immunopathological mechanism plays a role in dengue virus infection with various complications as mentioned above. some immune responses are known to play a role in the pathogenesis of dengue virus infection. researchers are trying to establish the role of several inflammatory mediators, such as pla , il-1 , tnf, and pge2, in et al . 2 b a *corresponding author, phone: +62-31-5035925, fax: +62-31-5035975, e-mail: purwatipanpan@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 2, june 2011, p 51-55 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.2.1 relation to the degree of severity of the dengue virus infection. so far, not many researchers have been interested in studying the above mentioned inflammatory mediators (khrisnan . 2007; miller . 2008). . the analytic study in this research was an observational study with crosssectional design. the research was conducted from february to november 2009 at the infectious diseases ward of dr. soetomo hospital: selection and blood sampling, institute of tropical disease, airlangga university, the department/installation of clinical pathology airlangga university school of medicine, dr. soetomo hospital: plasma separation, platelet tests, hematocryte, dengue igg and igm, ns1, plasma sp samples were taken from 45 hospitalized patients at the tropical infectious diseases ward at dr. soetomo hospital, surabaya, with dhf as a diagnosis based on who criteria in 1997, and positive results of serological tests and/or igg-anti-dengue igm and/or ns-1 antigen, meeting the criteria for inclusion and exclusion. . (i) based on the 1997 who criteria, diagnosis of dhf is established when all of the following components are met: fever or history of acute fever, between 2-7 days, usually biphasic, at least one of bleeding manifestations, such as (positive rumple-leede test), ptechiae, ecchymosis, or purpura, mucosal bleeding (epistaxis or gum bleeding), or bleeding from other sites (haematemesis or melena), thrombocytopenia (platelets <100 000 ed with the previous value, other plasma leakage signs such as pleural effusion, ascites or hypoproteinemia). (ii) with positive result of igg anti-dengue, and/or igm anti-dengue and/or ns-1 (non-structural-1) antigen dhf patient (mcbride 2009; thomas . 2010). (iii) age ≥ 14 years. (iv) willing to participate in this study and signed an informed consent. patients younger than 21 years can be represented by their parents in signing the informed consent. . patients with 2-7 days of fever caused by other than dengue infection, the presence of severe primary diseases (hematological disorders, diabetes mellitus, chronic kidney failure, heart failure, liver failure, liver cirrhosis), and age <14 years. et al et al et al materials and methods sample preparation inclusion criteria exclusion criteria la2 activity, prostaglandins 2 plasma, il-1β, tnf-α. μl ), at least one of plasma leakage signs (such as increased hematocryte > 20% compared to standard according to age and sex, decrease in hematocryte > 20% after fluid replacement therapy compar -1 studied variables data analysis enzyme immunometric assay for results characteristic of subjects . independent variables were levels of necrosing t f ; were a s imprecision of the examined sample duplicate (within run). control accuracy was not done due to lack of accuracy control materials. . enzyme immunometric assay. . after pipetting 100 ncubate at 37 °c for 2 h. after medium discharge, empty the contents of the wells and wash by adding 400 °c for 1 h. remove again the content of the well °c for 30 min. finally, empty the content of the wells and wash by adding 400 on to every well and pipette 100 n s y at 450 nm, preferably with correction between 570 nm and 590 nm. if the plate reader cannot be blanked against the blank wells, manually substrate the mean optical density of the blank wells from all the readings relation of four parameters with the degree of dhf (not linear), using anova. . umor actor-α, interleukin-1 β, s e c r e t o r y p h o s p h o l i p a s e a 2 p l a s m a , a n d prostaglandin-2 and dependent variables severity degrees of dengue hemorrhagic fever. quality ssurance examination of tnfα, spla2, pge2, il1β with precision control, which seek the collected data will be processed and presented analytically: the relation of each parameter (tnfα, spla2, pge2, il-1β) with the degree of dhf, using spearman's correlation analysis, the relation of four parameters with the degree of dhf, using multiple ordinal regress μl of standard diluents into s0 (0 pg ml ) standard wells, and pipetting 100 μl of standard 1 through 7 into appropriate wells, take 100 μl of samples into appropriate wells and tap the plate gently to mix the contents. seal the plate and i μl of wash solution to every well, then repeat the wash 3 more times for a total of 4 washes. after the final wash, remove or aspirate the wells and firmly tap the plate on a lint free paper towel to remove any remaining wash buffer. then pipette 100 μl of yellow antibody into each well, except the blank then seal the plate and incubate at 37 s and wash by adding 400 μl of wash solution to every well and add 100 μl of blue conjugate to each well, except the blank and seal the plate and incubate at 37 μl of wash soluti μl of substrate solution into each well after 30 mi for incubation at room temperature. take and pipette 100 μl of top solution 2 to each well. blank the plate reader against the blank wells, read the optical densit this research is an observational analytic study with a case-control design tnfα, spla2, pge2, il-1β -1 52 purwati et al. microbiol indones in order to determine the relation between tnf-α levels, il-1β, pge2 and pla2 and the severity degree of dhf. research subjects were dhf patients who met the criteria for the samp levels of tnf-α, il-1β, pge2 and pla a-2 the plasma was stored in the with an age range of 14 48 years old, and the mean was 21 years old. the levels of tnf-α were 73.2 to 809.7 pg ml , with a mean of 199.7 pg ml ; the levels of il-1 β were 12.7 l l l l quality assurance of levels of tnfα, spla2, pge2, il-1β was performed by seeking imprecision wit , le acceptance. samples were taken at the tropical disease ward, dr. soetomo hospital, surabaya, from march to september 2009. during the collection of samples until the examination of , ; tissue bank at dr. soetomo hospital, surabaya, and the institute of tropical diseases, airlangga university. a -70 °c storage was needed in order to maintain bioactivity. based on the results, there were 45 dhf patients with the following details: 7 patients with dhf grade 1 (15.6%), 35 patients with dhf grade 2 (77.8%) and 3 patients with dhf grade 3 (6.7% ). dhf grade 4 was not found in this study. most of the cases were dhf grade 2. of the 45 patients, 31 were male (68.9%) and 14 were female (31.1%), to to 45.9 pg m , with a mean of 31.6 pg ml ; the levels of pge2 were 39.1 to 1350 pg m , with a mean of 238.09 pg m , and the levels of pla a-2 were 37.8 to 195.9 u ml , with a mean of 97.48 u m . the characteristics of samples can be seen in table 1. . hin run absorbance. deter mination imprecision was performed on different patient samples by double or duplicate examinations, and the result can be seen in in table 2. -1 -1 -1 -1 -1 -1 -1 -1 quality assurance table 1 characteristics of research subjects characteristics mean sd age (y.o.) tnf-α (pg ml )-1 il-1β (pg ml )-1 pge2 (pg ml ) -1 pla a-2 (u ml )-1 21.3600 199.7378 31.6333 238.0911 97.4933 6.2050 143.63442 6.45720 207.44473 30.06433 n sd cv (%) tnfα spla2 pge2 il-1β 10 10 5 10 0.00732 0.01570 1.34000 0.000803 2.00 2.92 4.97 2.1 the research findings will be meaningless if the data is not valid. in this study, in order to indicate the data was performed with valid quality assurance, the volume 5, 2011 microbiol indones 53 table 2 imprecision within run absorbance , pgetnfα, spla ,il-1β2 2 parameter examination of the levels of tnfα, spla2,pge2, il1β was performed with precision control to measure imprecision. in this study, the linear correlation between tnfα levels and the severity degree of dhf was not found, with no significant difference (p = 0.922). it was discovered in dhf grade 1 that the mean level of tnfα was 139.9571 pg m it was discovered in dhf grade 1 that the il-1β mean level was 28.9571 pg m , dhf grade 2 was 33.2143 pg m , and gra il-1β this study found a significant difference in il-1β relation between levels of il-1β, tnf-α, pge2 and pla2 with the severity degree of dhf the influence of il-1β, tnf-α, and pla2 on the severity degree of dhf was not found significant, with each p = 0.402 p = 0.589 p = 0.959, as well as the influence , dhf grade 2 was 216.4629 pg m and grade 3 was 144.1000 pg m there were elevated mean levels in dhf grade 1 and 2, but decreased levels in dhf grade 3. in this study, the spla2 levels were not linearly correlated with the severity degree of dhf, with no significant difference (p = 0.709). it was discovered in dhf grade 1 that the spla2 mean level was 87.5429 u m , dhf grade 2 was 100.3486 u m , and dhf grade 3 was 87.4000 u m . there were elevated levels of spla2 in dhf grade 1 and 2, but decreased levels in grade 3. in this study, the pge2 levels were not linearly correlated with the severity degree of dhf, with no significant difference (p = 0.929). it was discovered in dhf grade 1 that the pge2 mean level was 175.8571 pg ml , dhf grade 2 was 258.5171 pg m , and 3 dhf grade 3 was 145.0000 pg m . there were elevated mean levels of pge2 in dhf grade 1 and 2, but decreased levels in grade 3. ificant difference (p = 0.929). de 3 was 19.4333 pg m . there were elevated levels of in dhf grade 1 and 2, but decreased levels in grade 3. (p = 0.000) against the severity degree of dengue by using anova. significant differences were found between dhf grade 1 and 3 with a mean difference of 9.5238, with a standard error of 3.7653 (p = 0.040); between dhf grade 2 and 3 with a mean difference of 13,7809, with a standard error of 3,2825 (p = 0.000). linear correlation was not found. . relation etween the evels of tnfα with the everity egree of dhf relation etween evels of il-1β and the everity egree of b l s d . relation between the levels of spla with the severity degree of dhf. relation between levels of pge2 and the severity degree of dhf. b l s d l l l l l l l l il-1β l l l -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 1 -1 2 dhf. in this study, levels were not linearly correlated with the severity degree of dhf, with no sig discussion of il-1β, tnf-α, and pge2 on the imprecision of tnfα, spla2, pge2, il-1β was below 5%. imprecision is a deviation of the results compared to the aver in this study, the linear correlation between tnfα levels and the severity degree of dhf was not found. the une response to viral infection which produced tnfα as a result of stimulation in b lymphocytes. b lymphocytes respond to denv-3 antigen, denv antigen-1, denv-2, and denv-4 even though in lower levels. this allows a cross response between each different se severity degree of dhf, was not found significant with each p = 0.332, p = 0.594, p = 0.696. age values, the lower the deviation (which is determined by a standard deviation or coefficient of variation), the more similar result from a series of examinations. an acceptable deviation for particular examination parameters was stated as the coefficient of variation. generally, the coefficient of variation should not exceed 5%, except for certain parameters which are allowed up to 10% . condition was due to a decrease of inflammatory processes in dhf grade 3, and the severity degree of dhf may be influenced by complement, thromboxane, leucotrien or other factors that affected endothelial permeability and capillary endothelial damage, resulting in more severe plasma leakage (gulati and maheshwari. 2007; whitehorn and farrar 2010). the spla2 levels were not linearly correlated with the severity degree of dhf. the condition was due to a decrease of inflammatory processes in dhf grade 3, and the severity degree of dhf may be influenced by complement, thromboxane, leukotrien or other factors that affect endothelial permeability and capillary endothelial damage, resulting in more severe plasma leakage. the situation can also be caused by a specific imm rotype, which makes a variation of responses to the activation of t lymphocytes (chaturvedi 2000). the pge2 levels were not linearly correlated with the severity degree of dhf. this condition can be attributed to the decrease of inflammatory processes in dhf grade 3, and the severity degree of dhf may be influenced by complement, thromboxane, leukotrien or other factors that affect endothelial permeability and capillary endothelial damage, resulting in more severe plasma leakage. pge2 was affected by spla2 and this study showed the same pattern of increase and decrease of these parameters on the severity degree of dhf. it is concluded that there was a spla2 influence on pge2. although the level of pge2 not linearly correlated with the severity degree of dhf, but there were found et al. 54 purwati et al. microbiol indones decreasing increasing level from dhf grade i to grade 2, but the level decrea (p = 0.000) against the severity degree of dengue by using anova. the reason of that is the same above. based on the data above, there was no linear correlation between these parameters and the severity degree of dengue, even though there were increased levels in grade 1 and 2, but then decreased in grade 3. (p = 0.000) against the severity degree of dhf by using anova. significant differences between dhf grade 1 and 3 were found with a mean difference of 9.5238, with a standard error of 3.7653 (p = 0.040), between dengue 2 and 3 degrees with a mean difference of 13.7809, with a standard error of 3.2825 (p = 0.000). there was not any linear relation. the condition was due to a decrease of inflammatory processes, but the severity degree of dhf can be influenced by complement, thromboxane, leukotrien or other factors that affect endothelial permeability and capillary endothelial damage, resulting in more severe plasma leakage. there were similar patterns against the severity degree of dhf on four parameters above, which were higher in grade 2 compared to grade 1, and lower in grade 3. there were elevated levels of the four parameters in dhf grade 1 to 2, but decreased levels in grade 3. the condition was due to a decrease of inflammatory processes, but the severity degree of dhf can be influenced by complement, thromboxane, and leukotrien. in this study, the cytokine level ( ) elevated in dhf garde 1 to grade 2, but that parameters decreased in grade 3. this condition may due to a decrease of inflammatory processes in dhf grade 3. so in this study increasing the cytokines level ( ) not linier with severity degree of dhf, its may due to decreasing inflammatory in grade 3. we would like to thank suharto for his continuous support and ideas. we would also like to thank our residents at the department of internal medicine (musofa rusli, widi hersana, agus suprapto) and clinical pathology (liana, binawati), dr. soetomo teaching hospital, faculty of medicine, airlangga university, surabaya. this study was funded by the national strategy, dp2m, directorate general of higher education. se in grade 3 that maybe due to inflammatory decreasing in grade 3. this study found a significant difference in il-1β this study found a significant difference only in il-1β, tnfα, spla2, pge2, il-1β tnfα, spla2, pge2, il-1β acknowledgement references chaturvedi uc, agarwal r, elbishbishi ea, mustafa as. 2000. cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis. fems immunol med microbiol. 28(3):183-188. doi: 10.1111/j.1574695x.2000.tb01474.x. garcia g, sierra b, perez ab, aguirre e, rosado i, gonzalez n, izquierdo a, pupo m, danay diaz, dr, sanchez l, marcheco b, hirayama k, guzman mg. 2010. asymptomatic dengue infection in a cuban population confirms the protective role of the rr variant of the fc{gamma}riia polymorphism. am j trop med hyg. 82(6):1153-1156. doi:10.4269/ajtmh.2010.09-0353. gulati s, maheshwari a. 2007. atypical manifestations of dengue. trop med int health. 12(9):1087-1095. doi:10.1111/j.1365-3156.2007. 01891.x. krishnan mn, sukumaran b, pal u, agaisse h, murray jl, hodge tw, fikrig e. 2007. rab 5 is required for the cellular entry of dengue and west nile viruses. j virol. 81(9):4881-4885. doi:10.1128/jvi. 02210-06. miller jl, dewet bjm, martines pl, radcliffe cm, dwek ra, pauline m, rudd, siamon g. 2008. the mannose receptor mediates dengue virus volume 5, 2011 microbiol indones 55 infection of macrophages. plos pathog. 4(2):e17. doi:10.1371/ journal.ppat.0040017. mcbride wjh. 2009. evaluation of dengue ns1 tests kits for the diagnosis of dengue fever. diagn microbiol infect dis. 64(1):31-6. doi: 10.1016/j.diagmicrobio.2009.01.002. n . thomas l, najioullah f, verlaeten o, martial j, brichler s, kaidomar s, moravie v, cabie a, cesaire r. 2010. relationship between nonstructural protein 1 detection and plasma virus load in dengue patients. am j trop med hyg. 83(3):696-699. doi: 10.4269/ajtmh. 2010.10-0138. whitehorn j, farrar j. 2010. dengue. br med bull. 95:161-173. [who] world health organization. 2008. dengue and dengue hemorrhagic fever. publication no. 117. geneva (ch): who. asronudin. 2007. imunopatofisiologi molekuler infeksi virus dengue [immunopathophysiology of molecular dengue virus infection]. in: nasronudin, hadi u, arvijanto v, triyono, bramantono ea, suharto, soewandojo e, editors. penyakit infeksi di indonesia [tropical and infectious diseases in indonesia]. surabaya (id): airlangga univ pr. p 45-46 03 rahmadi.cdr vol.12, no.3, september 2018, p 83-91 doi: 10.5454/mi.12.3.3 bacterial population and chemical characteristics of fermented mandai cempedak with starter induction * anton rahmadi , kartika sari, nikmatul khairiyah, frio handayani, sitohang satrio, yuliani, and aswita emmawati department of agricultural products technology, faculty of agriculture, universitas mulawarman, jalan kuaro, samarinda ulu, samarinda, east kalimantan 75119, indonesia traditionally fermented foods can be improved by introducing starter and hygienic production. the study observes the changes in population of lactic acid bacteria (lab), ph, polyphenolic levels, and antioxidant activity of spontaneous and lactobacillus casei induced mandai cempedak fermentation at 37 °c for seven days. the hygienic process included two steps boiling of inner-skin of cempedak at 80-90 °c for 15 minutes. lab and nonlab growth were quantified with plate count. phenolic substances were spectrophotometrically quantified. gallic acid (gae), tannic acid (tae), and catechin (ce) were used as standards. dpph method was employed to measure antioxidant activity. as the result, the lab dominated bacterial population during the course of -1 fermentation. the lab grew from 3.3±0.5 to 8.8±0.6 log cfu ml for spontaneous fermentation and from 3.3±0.4 -1 to 9.0±0.5 log cfu ml for starter induced fermentation. the population of lab in spontaneous and l. casei induced fermentation grew in almost similar pattern and can be approached by linear regression. the degree of acidity increased during the fermentation process and achieving ph 3.5 at the sixth day of fermentation. the fermentation process increased the phenolic contents both in spontaneous and l. casei induced fermentation, and resulting in enhancing the antioxidant activity. the phenolic contents, except total tannins, were higher in starter induced fermentation, thus lowering ic of inhibitions of dpph reduction. hence, l. casei produced fermented 50 products with better antioxidant activity in comparison to spontaneously fermented products. from these parameters, l. casei was successfully used as starter for mandai cempedak and optimum fermentation at 37 °c was 6 days. key words: antioxidant, bacterial population, mandai cempedak, phytochemicals makanan fermentasi tradisional dapat ditingkatkan kualitasnya dengan menggunakan biakan pemula dan produksi yang higienis. penelitian ini bertujuan untuk mengamati perubahan populasi bakteri asam laktat (lab), ph, kadar polifenol, dan aktivitas antioksidan dari fermentasi spontan dan biakan pemula l. casei dari mandai cempedak pada suhu 37 °c selama tujuh hari. proses persiapan higienis dilakukan dengan dua tahapan perebusan kulit cempedak bagian dalam pada suhu 80-90 °c selama 15 menit. pertumbuhan populasi lab dan non-lab dikuantifikasi dengan angka lempeng total. kadar fenolik dikuantifikasi secara spektrofotometri. asam galat (gae), tanat (tae), dan katekin (ce) digunakan sebagai standar. dpph digunakan untuk mengukur aktivitas antioksidan. bakteri asam laktat mendominasi populasi bakteri selama waktu fermentasi. dari studi ini didapat -1 hasil sebagai berikut, populasi lab tumbuh dari 3,3±0,5 ke 8,8±0,6 log cfu ml untuk fermentasi spontan dan dari -1 3,3±0,4 ke 9,0±0,5 log cfu ml untuk fermentasi dengan biakan pemula. populasi lab dalam fermentasi spontan dan yang diinduksi l. casei berkembang dengan profil yang serupa dan dapat diprediksi dengan regresi linier. tingkat keasaman meningkat seiring dengan proses fermentasi dengan ph terendah adalah 3,5 pada hari ke-6. fermentasi meningkatkan kadar fenolik baik untuk fermentasi spontan maupun yang diinduksi dengan l. casei, dan hal ini meningkatkan aktivitas antioksidan. semua fenolik, kecuali total tanin, terdapat dalam konsentrasi yang lebih tinggi pada fermentasi dengan biakan pemula, sehingga fermentasi cempedak meningkatkan kapasitas pereduksi dpph menjadi lebih baik. fermentasi dengan induksi l. casei memiliki aktivitas antioksidan yang lebih baik dibandingkan pada fermentasi spontan. sebagai kesimpulan, l. casei dapat digunakan sebagai biakan pemula pada fermentasi mandai dengan waktu optimum fermentasi pada suhu 37 °c adalah enam hari. kata kunci: antioksidan, mandai cempedak, populasi bakteri, phytochemicals microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: 62-541-741118; fax: 62541-747479/732870 ; email: arahmadi@unmul.ac.id traditional fermented products, spontaneous fermentation is commonly performed at room temperature (nuraida 2015). mandai cempedak is made from the inner-skin of cempedak (or jackfruit) by utilizing salt to promote lab growth (nur 2009; rahmadi et al. 2013; emmawati 2015). lactobacillus plantarum and leuconostoc sp. are predominant bacteria that can be isolated from these products. in kalimantan, in addition to be consumed as fresh fruit and seed, the inner-skin of cempedak or tiwadak (artocarpus champeden) fruit can be processed into a fermented food called mandai or dami (emmawati et al. 2015). to produce mandai cempedak and other stages of processing including peeling the outer skin of the fruit, removing the epidermis, and immersing in salt water in order to preserve and soften the texture. the length of fermentation is observed from several hours to one month. the disadvantage of the traditional fermentation process includes the use of salt in high concentrations (15-25% w/v) that leads to an increase in the body's salt intake (nacl). high salt diet has health implications (song et al. 2017). the use of high salt content also provides off and bitter taste in the traditionally fermented products as a result of mineral accumulation in the product (panagou et al. 2011). in community, hygienic practice during fermentation is often neglected, resulting in dissimilar quality between batches and potential pathogenic microorganism contamination in the mandai cempedak product (blana et al. 2014). there should be an effort to improve the process of traditional food fermentation. lactobacillus plantarum is traditionally found in plant-based fermented products. this is due to the versatility of l. plantarum enzymes in comparison to other labs (siezen et al. 2012). lactobacillus plantarum produces amylase, b-glucosidase, decarboxylase, lactate dehydrogenases, peptidases, phenolic acid decarboxylases, phenol reductase, proteinase, and tannase enzymes. on the other hand, regardless more commonly found in dairy-based products, l. casei may be employed for plant-based fermentation, as it is recorded to produce amylase, lactate dehydrogenases, peptidases, and proteinase enzymes (hur et al. 2014). the lab starter utilized in this research was prepared from lactobacillus casei shirota strain. this study aims to compare changes in bacterial populations, ph, phenolic (total phenolics, total tannins, and flavonoids) contents, and total antioxidants without addition of salt by spontaneous and starter induced fermentation of mandai cempedak at temperature of 37 °c. materials and methods mandai cempedak fermentation. the part of cempedak fruit used was the inner-skin that has been sorted and cleaned. the inner-skin of cempedak was 3 diced to the size of 3-5 cm . the pieces of the inner-skin of cempedak were boiled at temperature of 80-90 °c for 15 minutes to remove the sap and then the water was drained. the inner-skin of cempedak was separated into sealed containers, each was weighted at 100 g. potable water was poured into the container until the entire dices were submerged. the samples were boiled once more at temperature of 80-90 °c for 15 minutes. half of the samples were inoculated with l. casei at a 6 8 concentration of 4x10 to 4x10 cfu, or approximately 4 ml of the concentrated l. casei inoculum for each container. the samples were further fermented for seven days at 37 °c. observations on total bacteria, total lab, ph, phenolic contents, and antioxidant activity potency were performed daily until the seventh day. total plate count. total plate count (tpc) was performed as described by fardiaz (1993). nutrient agar (na) medium (accumedia, usa) was used for total bacteria count, while de mann rogosa sharpe agar (himedia, india) medium used for lab count. the media was sterilized at 121 °c for 15 minutes, then the media was cooled to 60 °c. the warm media was poured aseptically in a sterile petri dish which was then homogenized to spread evenly across the surface. petri dishes containing media were allowed to solidify. as many as 0.1 ml of samples were poured into a petri dish and flattened. the samples were incubated in the incubator in reversed position at 37 °c for 24 hours. the calculation based on the number of colonies ranges -1 from 25 to 250 cfu ml , considering dilution factor. the calculation of non-lab population was performed by subtracting the log of total bacterial population obtained from the na medium with log of lab population of the mrsa medium. ph. the degree of acidity was measured using sudarmadji et al. (2007) method. about ± 50 ml of sample product was placed into a small jar and the ph was measured in duplicate for each product. prior to analysis, the ph meter had been checked and calibrated in buffer ph 4 and 9. total phenolics. phenol analysis was performed by spectrophotometric method employing folinciocalteu (mu'nisa et al. 2012; nurhayati et al. 2012). gallic acid (sigma-aldrich, usa) was used as the standard. the total content of phenolics in the extract was expressed in gallic acid equivalent (gae). each sample was weighed as much as 5 mg, then dissolved in 2 ml of 95% ethanol. further, about 5 ml of aquades and 0.5 ml of folin-ciocalteau (sigma-aldrich, usa) 50% (v/v) were added. samples were allowed to stand for 5 minutes and a 5% (w/v) of na co (sigma-2 3 aldrich, usa) solution was added to make 10 ml of total volume. the solution was homogenized in dark room for one hour. the solution was measured at a wavelength of 752 nm (genesys 20, thermo-fischer, 84 rahmadi et al. microbiol indones volume 12, 2018 microbiol indones 85 usa). total tannins. total tannins were tested according to malangngi et al. (2012). a total of 0.5 g of the product was extracted with 10 ml of diethyl ether (merck, usa) for 20 hours, then filtered. the residue obtained was boiled in 100 ml of aquadest (soil science laboratory, mulawarman university, indonesia) for two hours, then cooled and filtered. the extract obtained was added with distilled water until the extract volume reached 100 ml. a total of 0.1 ml of the extract was added with 0.1 ml of folin ciocalteu reagent (sigma aldrich, usa) and was vortexed. about 2 ml of na co (sigma aldrich, usa) was 2 3 added and the sample was vortexed once more. the absorbance was read at 760 nm of wavelength (genysis 20, thermo fischer, usa) after incubation for 30 min at room temperature (28±2 °c). the results obtained were plotted against the standard curve of tannic acid (sigma aldrich, usa) that was prepared in the same procedure. the total content of tannins was expressed as tannic acid equivalent (tae). total flavonoids. total flavonoids were measured by the method of zou et al. (2004). about 1 mg of extract was dissolved in 10 ml of 95% of ethanol (kimia farma, indonesia) and 0.7 ml of distilled water was added. about 0.1 ml of 5% nano (sigma 2 aldrich, usa) was added into the mixture. after 5 minutes, 0.1 ml of alcl 10% (sigma aldrich, usa) 3 was added. after 6 minutes, 0.5 ml of 1 m naoh (merck, usa) was added. all ingredients were mixed evenly and the samples were incubated for 10 minutes. the absorbance was measured at a wavelength of 510 nm, after 1 ml of sample was replaced with 1 ml of 95% ethanol solvent. the results obtained were plotted against standard curve of catechin (sigma aldrich, usa) that was prepared in the same way. total flavonoids were expressed as catechin equivalent (ce). antioxidant activity. total antioxidants were performed by spectrophotometric method using 2,2diphenyl-1-picrylhydrazyl (dpph) (sigma-aldrich, usa) reduction principle (farhan et al. 2012). a total of 1 ml of diluted extract in ethanol (kimia farma, indonesia) was added to 1 ml dpph (prepared at 0.15 mm in ethanol) and at the same time, a control consisting of 1 ml of dpph with 1 ml of ethanol was prepared. the reaction mixture was vortexed and then incubated in the dark at room temperature for 30 minutes. the absorbance was measured at 519±2 nm (genesys 20, thermo-fischer, usa). vitamin c (sigma aldrich, usa) was used as a positive control and ethanol was used as the substract. the ability to inhibit dpph reduction of the extract was calculated by comparing the absorbance of the reduced control of sample divided by the absorbance of the control. the total antioxidant value was then plotted in a linear regression equation: [antioxidant potential] = a [ingredient in ppm] + b to obtain its ic value. 50 results total bacteria and lab. the growths of microbes in mandai cempedak without addition of salt were quantified in seven days of fermentation course with -1 initial microbial concentration of 3,3±0,3 log cfu ml -1 for spontaneous fermentation and 3,5±0,5 log cfu ml for starter induced fermentation. the dominance of the lab in mandai cempedak fermentation was estimated in linear equations presented in figure 1 and table 1. the lab grew to achieve the density of 8,8±0,6 log cfu -1 ml for spontaneous fermentation and 9,0±0,5 log cfu -1 ml for starter induced fermentation at the end of the observation period (fig 2). until the seventh day, lab continued to grow and dominate in both spontaneous and starter induced fermentation (fig 2). confirmation of lab growth was performed with mrsa medium and partial biochemical tests including gram positive confirmation, microscope observation, the absence of spore, non-motility, and positive catalase. lab and non-lab growths were observed from day one to day seven at 37 °c (fig 1). in the spontaneous mandai cempedak fermentation, l. plantarum was dominated microbial growth, while in the starter induced fermentation, there were at least two distinct lab isolates observed under microscope. however, we are yet to determine the species. this data proved that lab was able to survive in the heat process applied in the initial processing of mandai cempedak. lab is known to produce lactic acid in an amount that is sufficient to increase the acidity of the fermented product. the increase of acidity of mandai cempedak started from the second table 1 the linear equation of bacterial growth in mandai cempedak fermentation with and without starter linear equation spontaenous fermentation l. casei induced fermentation total bacteria y = 0.9029*x + 2.687 y = 0.8717*x + 2.681 total lab y = 0.9008*x + 2.149 y = 0.9128*x + 2.081 fig 1 linear regression of bacterial growth in mandai cempedak fermentation with and without starter. a: the linear regression of growth of bacteria and lab in spontaneous fermentation b: the linear regression of growth of bacteria and lab in l. casei induced fermentation. * significantly different in the multiple ttest. sem is symbolized with (┬) located above and below the observation point. 86 rahmadi et al. microbiol indones a b fig 2 the lab and non-lab population in mandai cempedak fermentation with and without starter. a: the lab and non-lab population in spontaneous fermentation b: the lab and non-lab population in l. casei induced fermentation. * significantly different in the multiple t-test. sem is symbolized with (┬) located above and below the observation point. a b volume 12, 2018 microbiol indones 87 day of the fermentation course to the last observed day. the lowest ph (3.5) was observed on the sixth day of fermentation. overall, there was no significant difference of the lowest ph of spontaneous and starter induced mandai cempedak, although slight variations of ph on day to day observation were measured (fig 3). fermentation is proven to increase the phenolic contents in certain food products, and as resulting in enhancing the antioxidant activity. these phenomena were highlighted in mandai cempedak fermentation (fig 4). the difference of total phenolic and flavonoid contents between spontaneous and starter induced fermentation products was observed, in which the use of l. casei induced fermentation resulting in higher concentrations of total phenolic and flavonoid contents. in addition, there was no difference of total tannins for both products in any day of observation (fig 4). hur et al. (2014) stated that antioxidative activity may be increased in fermented plant-based food products as a result of microbial enzymatic hydrolysis of phenolic compounds. discussion the result is in line with the research of de angelis et al. (2004) and fiocco et al. (2007) which stated that several strains of lab were able to survive after mild heat processing because they produced proteins that were protective to heat. the inner-skin of cempedak contained fibrous materials that provide an extent of supports for cell immobilization, giving protection to the viability of lactobacilli (lye et al. 2012). this also explained the survivability of the lab during mild heat treatment. it is noted that cempedak medium promoted lab growth and domination. heat treatment of mandai cempedak may resulted in the release of nutritious compounds such as free amino, nitrogen, and sugars into the fermentation media, leading to their increased availability to be utilized by lab. teh et al. (2010) postulated that the sugars from the skin of durian (durio zibethinus), cempedak, and mangosteen were assimilated by lactobacilli and utilized for growth. acidity. from the lowest achieved ph, both optimum fermentations at 37 °c were 6 days. the ph is used as general indication of optimum lab fermentation as stated by mousavi et al. (2010). rhee et al. (2011) reported a decrease in ph as a result of lactic acid production, which was also an indicator of the success of fermentation of traditional food products by lab. the l. plantarum and l. casei are regarded to be facultatively heterofermentative bacteria (ashraf et al. 2011; zago et al. 2011), therefore produces a variety of organic acids to reduce the ph. cueva et al. (2013) stated that during the in vitro fermentation of up to 48 h of grape seeds by lactobacilli, organic acids such as 4hydroxyphenylacetic acid, phenylpropionic acid, 3hydroxyphenylacetic acid, phenylacetic acid, 3-(4hydroxyphenyl)-propionic acid, and 4-hydroxy-5(phenyl)-valeric acid were significantly increased by the fermentation process. these compounds, along with lactic acid, caused the increasing acidity of the fermented mandai cempedak. phytochemistry and antioxidant activity. lactobacillus casei and l. plantarum are known to fig 3 ph of mandai cempedak fermentation with and without starter. * significantly different in the multiple ttest. sem is symbolized with (┬) located above and below the observation point. 88 rahmadi et al. microbiol indones fig 4 the total phenolic, tannins, flavonoid contents and antioxidant activity in fermentation of mandai cempedak with and without starter. a: the total phenolic contents in spontaneous and l. casei induced fermentation. b: the total tannin contents in spontaneous and l. casei induced fermentation. c: the total flavonoid contents in spontaneous and l. casei induced fermentation. d: the ic of inhibition of dpph 50 reduction in spontaneous and l. casei induced fermentation. * significantly different in the multiple ttest. sem is symbolized with (┬) located above and below the observation point. a b c d have -glucosidase and -galactosidase activities that increase flavonoids in fermented food products, i.e. in soybean fermentation (marazza et al. 2009). in this regard, hur et al. (2014) proposed that lab fermentation causes formation of organic acids that influences ph, maillard reaction, and pentose phosphate pathway which subsequently attenuate redox balance and radical scavenging activity. the fermentation also causes activations of -glucosidase, galactosidase, tannase, and phosphoketolase that assist hydrolysis and de-polymerization of phenolics. the downstream processes have an implication of liberation of phenolic substances which in turn provides hydrogen or electron donor and positively modulates metal ion chelation activity. as a result, radical scavenging activity may increase. quercetin and gallic acid were significantly higher during lab fermentation of graptopetalum paraguayense in any stage of fruit maturity (wu et al. 2011). hole et al. (2012) concluded that fermentation of cereal products with specific probiotics exhibited significant increase of free phenolic acids, i.e. caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, 5,5-diferulic acid, 8-o-4-diferulic acid, and 8,5-diferulic acid. selected labs were able to increase free phenolic acids due to high feruloyl esterase activity (fae). in solid state fermentation, the increase of antioxidant activity with regard to modulation of polyphenolic content is also proven. lee et al. (2008) reported that an increase of antioxidant activity is observed when employing different starter to ferment koji bean, which was attributed to the increase of phenol and anthocyanin contents. further, the increase in phenolic substances were measured during fermentation of cooked grass pea seed, wheat grains, and soybean products (starzynska-janiszewska et al. 2008; bhanja et al. 2009; singh et al. 2010; dajanta et al. 2013). however, the changes of phytochemical contents are medium and strain specific (martins et al. 2011). in other condition, i.e. tea fermentation, it is stated that the monomeric flavonoids were transformed to polymeric derivatives as the tea leaves were further fermented (kim et al. 2011). as a result, polyphenolic contents were lessened as the higher degree of fermentation occurred. further, external factors affecting the changes of phytochemical contents are duration of fermentation course, temperature, ph, inhibitors, stimulators, and the composition of atmosphere in the fermentation chamber (hur et al. 2014). the change in ic values of dpph reduction 50 inhibition is limitedly influenced by ph if the difference of the ph is less than 1. however, if the difference of ph at range of greater than 2, the ic 50 value of dpph reduction inhibition between products will differ significantly (pekal and pyrzynska 2015). in this study, the comparison of ic inhibition of dpph 50 reduction was performed only between the same day fermented of mandai cempedak products, where the difference of ph was less than 1 between spontaneous and starter induced fermented products. it was concluded that ic inhibitions of dpph reduction of 50 starter induced fermentation were lower than the values p r o d u c e d f r o m s p o n t a n e o u s f e r m e n t a t i o n . lactobacillus casei produced fermented products with better antioxidant activity in comparison to l. plantarum fermented products. as conclusion, the growths of bacteria in mandai -1 cempedak initially started from 3,3±0,3 log cfu ml -1 and 3,5±0,5 log cfu ml for spontaneous fermentation and starter induced fermentation, respectively. the growth curves of the lab in spontaneous and l. casei induced mandai cempedak fermentation in the seven day of fermentation at 37 °c were in log-linear pattern. 9 -1 the lab grew to achieve the density of 10 cfu ml for both types of fermentation at the end of the observation period. there was no significant difference of the lowest ph of spontaneous and starter induced mandai cempedak, although slight variations of ph on day to day observation were measured. the starter induced fermentation products contained higher total phenolic and flavonoid in comparison to spontaneous fermentation products, while there was no difference of total tannins for both products in any day of observation. lactobacillus casei produced fermented products with better antioxidant activity in comparison to l. plantarum fermented products, indicated by ic 50 inhibitions of dpph reduction. from these parameters, l. casei was successfully used as starter for mandai cempedak and optimum fermentation at 37 °c was 6 days. acknowledgment the authors would like to thank the indonesian directorate general of research and development strengthening (drpm) ministry of research, technology and higher education, republic of indonesia for financing this research through penelitian strtegis nasional institusi (psni) scheme. microbial population, flavonoid levels, and total antioxidants in spontaneous fermentation have been volume 12, 2018 microbiol indones 89 90 rahmadi et al. microbiol indones presented in indonesian association of food technologists (patpi) national seminar on 10-11 october 2017 in university of lampung. references ashraf r, shah np. 2011. selective and differential enumerations of lactobacillus delbrueckii subsp. bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, lactobacillus casei and bifidobacterium spp. in yoghurt 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2department of agronomy and horticulture, faculty of agriculture, institut pertanian bogor, jalan meranti, darmaga campus, bogor 16680, indonesia bacterial canker, caused by clavibacter michiganensis subsp. michiganensis is a newly introduced disease of tomatoes in indonesia. its existence was first officially reported in 2004. the objective of this research was to monitor the existence of c. michiganensis subsp. michiganensis in various tomato production centers in sumatra and java. tomato samples showing symptoms of c. michiganensis subsp. michiganensis infection were collected from various tomato production centers in sumatera and java and the causal agents were isolated from these samples. based on the occurrences of typical symptoms of c. michiganensis subsp. michiganensis infection in tomato, the incidence of suspected c. michiganensis subsp. michiganensis infection ranged from 1-20%. from a total of 74 tomato plants sampled, 24 bacterial isolates exhibiting similar colony m o r p h o l o g y t o c . m i c h i g a n e n s i s s u b s p . m i c h i g a n e n s i s w e r e o b t a i n e d . a f t e r v a r i o u s p h y s i o l o g i c a l , h ypersensitive response, and pathogenicity tests, 18 isolates derived from 14 tomato production centers in eight provinces in sumatera and java were identified as c. michiganensis subsp. michiganensis. although the incidence was still low, results of these experiments further indicated that c. michiganensis subsp. michiganensis existed in indonesia and had spread in a number of tomato production centers in sumatra and java. positive results of c. michiganensis subsp. michiganensis identification from suspected tomato samples should be a warning sign for all tomato production stakeholders in indonesia, especially those responsible in regulating the seed trade, importation, and plant quarantine. key words: bacterial canker, new disease in tomato, clavibacter michiganensis subsp. michiganensis, isolate identification, field survey _____________________________________________ ________________________ *corresponding author, phone: +62-751-72776, fax: +62-751-72702, e-mail: aswaldi@yahoo.com clavibacter michiganensis subsp. michiganensis, which causes bacterial canker disease of tomato, is a seed-transmitted bacterial pathogen. bacterial canker has caused serious damage in tomato plants (hayward and waterston 1964). yield reduction due to c. michiganensis subsp. michiganensis infection in tomatoes could be as high as 80% (chang and pataki 1992; vasinauskienë 2002). growth of c. michiganensis subsp. michiganensis and development of the disease were optimal in hot weather with a temperature of 26-28°c (hayward and waterston 1964). bacterial canker is a new disease in indonesia since this country was reported free from this disease up to 2002. the detection of c. michiganensis subsp. michiganensis among commercially traded tomato seeds in indonesia was first reported by anwar et al. (2004a,b). there is a possibility that this pathogen has spread to a number of tomato production centers in indonesia. c. michiganensis subsp. michiganensis could have been introduced into production centers through infected seeds. it could quickly spread among tomato plants through various means and large scale infection of c. michiganensis subsp. michiganensis in tomato crops could take place over only a few seasons. it would be difficult to eradicate c. michiganensis subsp. michiganensis once the pathogen has been introduced and established in certain areas (ark 1994; fatmi and schaad 2002). therefore, monitoring of the existence of this pathogen and how wide it has spread in the field is a necessary step in preventing further spreading of bacterial canker. direct collection of suspected c. michiganensis subsp. michiganensis from infected field grown tomato followed by analysis using standard laboratory procedures for c. michiganensis subsp. michiganensis identification needs to be conducted. the aim of this research was to monitor the existence of c. michiganensis subsp. michiganensis in various tomato production centers in sumatra and java. the specific objectives of this research were (i) to collect samples of tomato fruits and plants exhibiting symptoms of c. michiganensis subsp. michiganensis infection, (ii) to estimate disease incidences in the field based on symptoms of c.michiganensis subsp. michiganensis infection, (iii) to i d e n tify the presence of c. michiganensis subsp. michiganensis among collected tomato samples, and (iv) to determine the distribution of c. michiganensis subsp. michiganensis in various tomato production centres in sumatra and java. materials and methods collection of c. michiganensis subsp. michiganensis infected tomato samples. samples of c. michiganensis subsp. michiganensis from infected tomato plant tissues and fruits were collected up to july 2006. the collected samples consisted of leaves, stems, and fruits showing various symptoms associated with c. michiganensis subsp. michiganensis infection. samples were collected using stratified-purposive-random-sampling from various major tomato production centers in sumatra and java. see results for the locations of tomato sample collection. collected plant materials were taken from each location and brought back directly or sent by express mail service (titipan kilat-tiki) from the location to padang, west sumatra. the isolation of bacterial pathogens from tomato issn 1978-3477 volume 2, number 2, august 2008 p 63 68 64 zainal et al. microbiol indones s a m p l e s w a s c o n d u c t e d a t t h e b a c t e r i o l o g y l a b , phytopathology department, faculty of agriculture, andalas university, west sumatra. the incidence of suspected c. michiganensis subsp. michiganensis infection was evaluated by direct field observation in each location. the number of tomato plants showing symptoms of c. michiganensis subsp. michiganensis infection was recorded and percentages of symptom occurrences among tomato plantations were calculated. isolation of suspected c. michiganensis subsp. michiganensis from tomato samples. collected tomato samples (5 g) were dipped for 15 min in 15 ml of phosphate buffer tris (pbt) at 4°c and homogenized using a mortar and pestle. after centrifugation at 1 844 x g for 5 min, the supernatant plant extract was transferred into sterile micro-centrifuge tube and labeled as undiluted stock. serial dilution of 10-1 and 10-2 were made from each of the undiluted stock using sterile pbt. subsequently, each of the undiluted and diluted extracts (100 µl) was plated twice onto nutrient agar (na) medium and the plates were incubated at 23-27°c. occurrences of bacterial colonies on the na medium were evaluated 2 weeks after plating. all bacterial colonies showing characters similar to that of a reference c. michiganensis subsp. michiganensis colony were transferred onto ydc medium. selected bacterial colonies growing on ydc medium showing similar characteristics of reference c. michiganensis subsp. michiganensis colony (yellow, wet, and mucoid) were isolated and evaluated for various physiological characters. suspensions of c. michiganensis subsp. michiganensis isolate 542 (anwar et al. 2004a; anwar et al. 2005) at 102, 103, and 104 cfu ml-1 were plated on both na and ydc medium and used as the control. identification of suspected bacterial isolates. suspected c. michiganensis subsp. michiganensis colonies identified from ydc medium were subjected to the gram reaction (suslow et al. 1982; fatmi and schaad 1988; kritzman 1991), the production of fluorescent pigment, pectinase activity, oxidation, and nitrate reduction tests, respectively. the suspected isolates were also grown on ttc medium and tested for their tolerance against 6% nacl. c. michiganensis subsp. michiganensis isolate 542 was also subjected to the same tests and used as the control. after a series of physiological tests, bacterial isolates physiologically identified as similar to c. michiganensis subsp. michiganensis were subjected to the hypersensitive reaction (hr) test using leaves of nicotiana tabacum and mirabilis jalapa (gitaitis 1990; alarcon et al. 1998) and tested for their pathogenicity using tomato seedlings. the hr tests were conducted by injecting tested bacterial isolates into the leaves of n. tabacum and m. jalapa using 1 ml needle syringe (alarcon et al. 1998; anwar et al. 2005). the occurrence of necrotic lesions on injected leaves after 24 h indicated a positive result for the hr test. pathogenicity tests were conducted using four-week-old seedlings of c. michiganensis subsp. michiganensis susceptible-tomato cv. marta. inoculation of the seedlings with the tested bacterial isolates was conducted by cutting their epicotyls at 1 cm above their cotyledons using scissors. the scissors had previously been dipped in a suspension of the tested bacteria (107 cfu ml-1). inoculated seedlings were covered with a transparent plastic bag to maintain humidity and incubated for 48 h in a glasshouse. occurrences of typical symptoms associated with c. michiganensis subsp. michiganensis infection such as wilting of the cotyledons, tissue discoloration, and the wilting or death of seedlings were recorded and used to determine pathogenicity of the tested isolates against tomato plants. table 1 location of collected tomato samples, observed symptoms of collected samples, disease incidence and number of bacterial isolates positively identified as clavibacter michiganensis subsp. michiganensis from various tomato production centers in sumatra and java province: location, district north sumatera peceran, berastagi/karo west sumatera danau kembar, solok lembah gumanti, solok tanjung baru, tanah datar baso, agam banuhampu, agam iv angkek, agam bengkulu selupu rejang, rejang lebong talang rimbo, kepahiang west java cipanas, cianjur pacet, cianjur central java pejawanan, banjarnegara wanayasa, banjarnegara east java matus, kediri kepung, kediri pujon, malang ngantung, malang observed symptoms on collected tomato samples dwarf, blackened tomato stem bird’s eye spot symptom split stem secretion wilting leaves and necrosis at leaf perimeters wilting leaves and necrosis at leaf perimeters stem necrosis (tissue discoloration) wilting leaves and necrosis at leaf perimeters wilting leaves and necrosis at leaf perimeters, stem necrosis dwarf, blackened tomato stem wilting leaves and necrosis at leaf perimeters split stem secretion stem necrosis, wilting leaves, and necrosis at leaf perimeters split stem secretion and showing pith discoloration wilting leaves and necrosis split stem secretion and showing pith discoloration stem necrosis, wilting leaves, and necrosis at leaf perimeters stem necrosis disease inci-dence (%)* no. of bacterial isolates** 2-10 1 3 0.8 1 4 2-11 2-10 1 2 0.8 0.8 1 5 1 7 2-20 5-10 2 4 2 3-20 5-12 4 2 1 1 1 1 1 1 1 2 1 3 2 2 1 *disease incidences were calculated based on number of suspected symptoms observed in the field, **identification of suspected bacterial isolates as cmm was conducted based on morphological and physiological characteristics of the isolates. volume 2, 2008 microbiol indones 65 plants from a number of tomato production centers are presented in table 1. representative symptom exhibited by tomato plants and a fruit corrected from solok, kediri, banjarnegara, and cianjur are presented in fig 1. incidence of suspected c. michiganensis subsp. michiganensis infection in the field. interviews with local farmers indicated that most of them did not recognize the symptoms of c. michiganensis subsp. michiganensis infection in tomatoes. surveys conducted based on the occurrence of typical symptoms of c. michiganensis fig 2 representative results of physiological tests on suspected isolates of clavibacter michiganensis subsp. michiganensis obtained from various tomato production centers in sumatra and java; a, morphology of the bacterial colony suspected as c. michiganensis subsp. michiganensis on tsa medium 24 hours after plating; b, positive results of gram test using koh solution on isolate slk 11 from danau kembar, solok, west sumatra; c, ectinase test on isolate agm-3 from baso, agam, west sumatra; and d, oxidation test on isolate rjl-74 from talang rimbo, kepahiang, bengkulu. fig 1 examples of tomato showing typical symptoms of suspected clavibacter michiganensis subsp. michiganensis in fection observed in the field. a, tomato fruit showing bird’s eye spot symptoms (arrow) from danau kembar, solok, west sumatera and tomato plants showing; b, split stem secretion and showing pith discoloration (arrow) from lembah gumanti, solok, west sumatera; c, wilting leaves and necrosis at the leaf perimeters from matus, kediri, east java; d, blackened stem, wilting leaves and necrosis at the leaf perimeters from wanayasa, banjarnegara, central java; and e, stem necrosis (tissue discoloration) from cipanas, cianjur, west java. results distribution of c. michiganensis subsp. michiganensis in various tomato production centers. surveys conducted in various locations in java and sumatra (table 1) led to the collection of 74 tomato samples exhibiting symptoms associated with c. michiganensis subsp. michiganensis infection. the samples consisted of leaves or stems (72 samples) and fruits exhibiting bird’s-eye-spot symptoms (2 samples). various symptoms exhibited by tomato b c d e a a b c d 66 zainal et al. microbiol indones subsp. michiganensis infection in tomato plants indicated that the incidence of suspected c. michiganensis subsp. michiganensis infection ranged from 1-20% (table 1). the highest disease incidence (up to 20%) was observed at pejawanan, banjarnegara, central java, and at pujon, malang, east java (table 1). disease incidence ranging from 10-12% was observed at ngantung, malang, east java; wanayasa, banjarnegara, central java; baso and banuhampu, agam, west sumatra; and peceran, berastagi/ karo, north sumatra. an incidence of up to 5-7% was observed at cipanas and pacet, cianjur, west java. other locations surveyed indicated that the incidence of suspected c. michiganensis subsp. michiganensis infection was less than 5% (table 1). in the various locations surveyed, the tomato cv. marta was the most common of the tomato cultivars that exhibited symptoms of c. michiganensis subsp. michiganensis infection. this cultivar is recommended for cultivation at high altitude. other tomato cultivars exhibiting symptoms of c. michiganensis subsp. michiganensis infection in the field were the tomato cvs. permata, montera, and cosmonot. these four tomato cultivars were the most commonly grown cultivars in north sumatra, west sumatra, bengkulu, east java, and west java. isolation of c. michiganensis subsp. michiganensis from tomato samples. bacterial isolates grown on na medium exhibited many different colony morphologies. of the colonies evaluated, only 24 indicated similar morphologies as the reference c. michiganensis subsp. michiganensis isolate 542 and these 24 isolates were selected for further experiments. to obtain a pure single colony, the 24 isolates were spread onto ydc medium and incubated for 24-48 h at room temperature. the expected c. michiganensis subsp. michiganensis colonies on ydc medium should be slow growing, mucous, and yellow to pale-orange in color (anwar et al. 2005). results of the single colony purification showed that six isolates were not c. michiganensis subsp. michiganensis and only 18 isolates exhibited a yellow color and were mucous and wet colonies (fig 2a). the other six bacterial isolates were fast growing and neither showed mucous nor yellow to pale colonies on ydc medium. identification of bacterial isolates. various physiological tests were conducted to confirm the identity of the isolates as c. michiganensis subsp. michiganensis. results of evaluation indicated 18 selected isolates were gram positive (table 2). results also indicated those 18 isolated all showed similar results of physiological tests as the c. michiganensis subsp. michiganensis isolate 542 (table 2), indicating they were c. michiganensis subsp. michiganensis the control c. michiganensis subsp. michiganensis isolate 542 clearly induce positive hr response in leaves of both n. tabacum and m. jalapa (table 2). results of the hr tests showed that 17 out of 18 isolates also induced the hr response in leaves of both species (table 2). one isolate (kar19), although physiologically exhibited similar characters to c. michiganensis subsp. michiganensis isolate 542, did not induce hr response on either n. tabacum or m. jalapa. results of the pathogenicity tests further confirmed the identity of the isolates as c. michiganensis subsp. michiganensis inoculation of 11 out of 18 isolates onto tomato seedlings resulted in symptoms similar to that of c. michiganensis subsp. michiganensis isolate 542 (table 2) and these 11 isolates were positively identified as c. michiganensis subsp. michiganensis. although showing inconsistent results, pathogenicity tests of the other six isolates also positively identified them as c. michiganensis subsp. michiganensis. after the inoculation table 2 characteristics of bacterial isolates suspected as clavibacter michiganensis subsp. michiganensis from various tomato production centers in sumatera and java isolate code number origin of suspected bacterial isolates characteristics of isolate a b c d e f g h i kar-15 kar-17 kar-19 kar-22 rjl-74 agm-1 agm-3 agm-7 slk-9 slk-11 slk-13 tnd-5 cjr-45 cjr-53 bjn-28 mlg-65 mlg-66 kdr-68 clavibacter michiganensis subsp. michiganensis 542 peceran, berastagi/karo peceran, berastagi/karo peceran, berastagi/karo logumba, berastagi/karo talang rimbo, kepahiang iv angkek, agam baso, agam banuhampu, agam danau kembar, solok danau kembar, solok lembah gumanti, solok tanjung baru, t. datar cipanas, cianjur pacet, cianjur wanayasa, banjarnegara pujon, malang kepung, kediri matus, kediri pri, wageningen-holland + + + + + + + + + + + * + + + + + * + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + * + + + + + + * + + + + + + * + + + + + + * + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + a, gram reaction test; b, pectinase test; c, oxidation test; d, hypersensitive response (hr) test; e, pathogenicity test on seedlings of tomato cv. marta; f, fluorescent pigment test on king’s b medium; g, nitrate reduction test; h, growth on ttc medium; i, tolerance against 6% of nacl; *showed inconsistent hr test results among tomato seedlings tested. volume 2, 2008 microbiol indones 67 of tomato seedlings with these six suspected isolates, some seedlings showed symptoms typical of c. michiganensis subsp. michiganensis infection while the others did not. moreover, the inoculated seedlings also exhibited various severities of symptoms. although isolate kar19 did not induce the hr response, tomato seedlings inoculated with this isolate exhibited typical symptoms of c. michiganensis subsp. michiganensis infection in at least one tomato seedling. such results indicate that isolate kar19 was also c. michiganensis subsp. michiganensis michiganensis subsp. michiganensis. inoculation of the bjn28 isolate onto tomato seedlings resulted in negative symptoms for c. michiganensis subsp. michiganensis infection (table 2). however, physiological characters of isolate bjn28 were similar to that of c. michiganensis subsp. michiganensis isolate 542 and it did induce the hr response in n. tabacum and m. jalapa (table 2). further pathogenicity tests need to be conducted to confirm the identity of isolate bjn28. this remains to be done. six bacterial isolates identified as non-c. michiganensis subsp. michiganensis based on their colony morphologies also exhibited different physiological characteristics compare to that of c. michiganensis subsp. michiganensis i s o l a t e 5 4 2 . t h e s i x n o n -c. michiganensis subsp. michiganensis bacterial isolates neither induced the hr response in leaves of n. tabacum and m. jalapa nor the typical symptoms of c. michiganensis subsp. michiganensis infection in tomato seedling (data not presented). such results confirmed the identity of these isolates as nonc. michiganensis subsp. michiganensis. discussions the introduction of a new disease into certain regions or countries such as indonesia is one of the many consequences of trading and exchanging seeds between countries (chang et al. 1989; chang et al. 1991). free seed importation to indonesia and germplasm exchange for breeding programs in the year 2000 might possibly have introduced certain pathogens previously absent in indonesia, such as c. michiganensis subsp. michiganensis the results of this experiment further indicated that c. michiganensis subsp. michiganensis, the causal agent of bacterial canker in tomato, have existed in indonesia, and spread in a number of tomato production centers in sumatra and java. p o s i t i v e r e s u l t s o f c . m i c h i g a n e n s i s s u b s p . michiganensis identification from suspected tomato samples should be seen as a warning sign for all tomato production stakeholders in indonesia, especially those responsible in regulating the seed trade, importation and plant quarantine. in a number of locations, c. michiganensis subsp. michiganensis infection occurred only in one or two plants, indicating that the spreading of this pathogen was still limited. however, if not managed correctly, such conditions might develop into wide spread infections among tomato plantations and result in the outbreak of bacterial canker that would cause substantial losses to tomato growers. therefore, tomato growers in the surveyed regions should be aware of this newly introduced tomato disease in order to prevent possible outbreaks of bacterial canker. outbreaks of bacterial canker due to c. michiganensis subsp. michiganensis infection has been reported in various countries (hausbeck et al. 2000; sahin et al. 2002). o b s e r v e d s y m p t o m s o f c . michiganensis subsp. michiganensis infection in the field include wilting leaves and necrosis at the leaf perimeters (basim et al. 2004), fruit showing bird’s eye spot symptoms (medina-mora 2001), as well as split stem and stem canker (burokienë et al. 2005). wilting-leaf-symptoms in the tomato plant occur because c. michiganensis subsp. michiganensis infection develops unilaterally from the lower position to an upper one on the stem and finally damages all of the leaves. in these experiments, bird’s eye spot symptoms were observed at danau kembar, solok, west sumatera; split stem secretions and pith discoloration were observed at lembah gumanti, solok, west sumatera; wilting leaves and necrosis at the leaf perimeters at matus, kediri, east java; blackened stem, wilting leaves and necrosis at the leaf perimeters were observed at wanayasa, banjarnegara, central java; and stem necrosis (tissue discoloration) was observed.at cipanas, cianjur, west java. the inconsistencies between field survey and laboratory observations could not rule out the fact that c. michiganensis subsp. michiganensis has positively been identified in a number of surveyed locations. symptoms of c. michiganensis subsp. michiganensis infection in the field were often similar to that of xanthomonas spp. (gram negative bacterium) infection in tomato. in such cases, field surveys based on symptoms might result in higher disease incidences than that positively identified as c. michiganensis subsp. michiganensis infection based on laboratory studies. any mistakenly identified symptoms in the field caused by xanthomonas spp. could easily be eliminated in laboratory by the simple gram reaction test, since xanthomonas spp. is gram negative while c. michiganensis subsp. michiganensis is gram positive. furthermore, the hr test using leaves of n. tabacum and m. jalapa (gitaitis 1990) could be used as quick indicators to verify the presence of c. michiganensis subsp. michiganensis using these hr tests, positive c. michiganensis subsp. michiganensis identification could be done only after 12 h of inoculation of the suspected bacterium into the leaves of n. tabacum and m. jalapa. on the other hand, pathogenicity testing exhibited varied results in this research. the expected positive identification of c. michiganensis subsp. michiganensis was inoculated tomato seedlings showing wilting symptoms for cotyledons, hypocotyls, death of the inoculated seedlings (gitaitis et al. 1991; anwar et al. 2005). however, in some cases in these experiments, such clear cut results were not observed and the inoculated tomato seedlings did not exhibit clear symptoms of c. michiganensis subsp. michiganensis infection. the inconsistencies in the pathogenicity test might be because (i) environment conditions might not be optimum for disease development after c. michiganensis subsp. michiganensis inoculation, (ii) tomato cv. marta might not be the best indicator for the pathogenicity test, and (iii) the tested isolates might exhibit different degrees of 68 zainal et al. microbiol indones virulence against tomato cv. marta. in this research, the pathogenicity test was conducted in a glasshouse. the temperature in the glasshouse sometimes reaches 30°c. such temperature might not be suitable for disease d e v e l o p m e n t a f t e r c . m i c h i g a n e n s i s s u b s p . michiganensis infection. the most common tomato cultivar used for the p a t h o g e nicity test against c. michiganensis subsp. michiganensis is the highly susceptible tomato cv. money maker. this tomato cultivar, however, is not available in indonesia. therefore, the commonly grown tomato cultivar marta was used as the indicator in this research. the response of tomato cv. marta against c. michiganensis subsp. michiganensis infection has not been reported elsewhere. a number of suspected c. michiganensis subsp. michiganensis isolates were tested and the results of pathogenicity test were inconsistent. however, results of this research indicated that the tomato cultivar ratna was susceptible to infection of most of the c. michiganensis subsp. michiganensis isolates identified. differences in virulence among c. michiganensis subsp. michiganensis isolates might result in inconsistencies in pathogenicity testing (berry et al. 1989). it seems possible that the identified c. michiganensis subsp. michiganensis isolates from the various tomato production centers in sumatra and java consisted of many different isolates with different degrees of virulence against the tomato cv. marta. to test this hypothesis, however, further analysis of the identified c. michiganensis subsp. michiganensis isolates using molecular markers is required. further identification of isolated c. michiganensis subsp. michiganensis isolates will be conducted and the results will be presented in later reports. acknowledgement part of this research was supported by competitive grant (hibah bersaing) xiv, entitled: management of new tomato disease (bacterial wilt and bacterial canker) in indonesia, contract no. 005/sp3/pp/dp2m/ii/2006, date: february 01, 2006, from the department of national education, republic of indonesia, coordinated by aa. the authors would like to acknowledge s ilyas and giyanto as part of the primary author’s phd graduate program advisory committee. az was supported by bpps scholarship from department of national education, republic of indonesia to pursue phd. degree at bogor agricultural university (ipb), bogor, indonesia. references alarcon c, castro j, munoz f, arce-johnson p, delgado j. 1998. protein(s) from the gram-positive bacterium clavibacter michiganensis subsp. michiganensis induced a hypersensitive response in plants. phytopathology 88:306-310. anwar a, ilyas s, sudarsono. 2004a. deteksi bakteri clavibacter michiganensis subsp. 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infected tomato stem under natural field conditions in california, ohio and marocco. plant pathol 51:149154. gitaitis rd. 1990. induction of hypersensitive reaction in four-o’clock by clavibacter michiganensis subsp. michiganensis. plant dis 74:58-60. gitaitis rd, beaver rw, voloudakis ae. 1991. detection of clavibacter michiganensis subsp. michiganensis in symptomless tomato transplants. plant dis 75:834-838. hausbeck mk, bell j, medina-mora c, podolsky r, fulbright dw. 2000. effect of bactericides on population sizes and spread of clavibacter michiganensis subsp. michiganensis on tomatoes in the greenhouse and on disease development and crop yield in the field. phytopathology 90:38-44. hayward ac, waterston jm. 1964. corynebacterium michiganense. description of pathogenic fungi and bacteria no. 19. cab international, wallingford, uk. kritzman g. 1991. a method for detection of seedborne bacterial diseases in tomato seeds. phytoparasitica 19:133-141. medina-mora cm, hausbeck mk, fullbright dw. 2001. bird’s eye lesions of tomato fruit produced by aerosol and direct application of clavibacter michiganensis subsp. michiganensis. plant dis 85:88-91. sahin f, uslu h, kotan r, donmez mf. 2002. bacterial canker, caused by clavibacter michiganensis subsp. michiganensis, on tomatoes in eastern anatolia region of turkey. plant pathol 51:399. suslow tv, schroth mn, isaka m. 1982: application of rapid method for gram differentiation of plant pathogenic and saprophytic bacteria without staining. phytopathology 72:917-918. vasinauskienë m. 2002. bacterial diseases of greenhouse-grown tomatoes. biologiya (viln.) 1:29-31. 7. 631 character (maria).cdr short communication characterization of a new thermoalkalophilic xylanase-producing bacterial strain isolated from cimanggu hot spring, west java, indonesia maria ulfah, is helianti*, budiasih wahyuntari, niknik nurhayatiand center for bioindustrial technology, laboratorium of bioindustrial technology, laptiab bppt puspiptek -serpong, tangerang selatan 15314, indonesia bacillus halodurans bacillus halodurans bacillus halodurans bacillus halodurans bacillus haloduran bacillus halodurans alkalophilic bacteria and their enzymes are important for industrial applications. therefore, finding out new strains of alkalophilic bacteria from indonesian microbial diversity is still required. in this study, a thermoalkalophilic bacterium was isolated from sediment of local hot spring, cimanggu, west java. the temperature of the spot was 60 °c and the ph was 8. the bacterium could live at ph 11 and temperature 55 °c, and produced xylanase that have optimal activity at alkaline ph 9 and high temperature 70 °c. based on the analyses of 16s rrna sequence similarity and biochemical characteristics, this strain was clustered into the same group of species with 99% identity to c-125. the isolate also showed other enzyme activities such as amylase, protease, and gelatinase, promising its potential use as an industrial enzymes producer. key words: 16s rrna, , biochemical properties, thermoalkalophilic xylanase. bakteri alkalofil dan enzim yang dihasilkannya bermanfaat dalam proses industri. karena itu, penemuan bakteri baru dari sumber biodiversitas mikrob lokal indonesia masih diperlukan. pada penelitian ini, satu galur bakteri alkalotermofil berhasil diisolasi dari endapan sumber air panas, cimanggu, jawa barat, indonesia. sumber air panas ini mempunyai ph 8 dan suhu 60 °c. bakteri yang yang telah diisolasi dapat bertahan hidup pada ph 11 dan suhu 55 °c, dan menghasilkan xilanase yang mempunyai ph dan suhu optimum masing-masing 9 dan 70 °c. hasil karakterisasi menggunakan analisis sekuens dna dari gen 16s rrna dan secara biokimia menunjukkan bakteri ini satu kelompok dengan dan mempunyai homologi 99% dengan s c-125. galur bakteri ini juga potensial menghasilkan enzim industri lainnya seperti protease, amylase, dan gelatinase. kata kunci: 16s rrna, , karakter biokimia, xilanase termoalkalofil. alkalophiles have made a great impact in industrial application, for their capability to produce alkalophilic o r a l k a l i n e s t a b l e e n z y m e s . a l k a l o p h i l i c microorganisms can be isolated from normal environments such as garden soil, although viable counts of alkalophiles are higher in samples from alkaline environments (horikoshi 2004). a thermoalkalophilic jb99 was reported as a source of alkaline thermostable keratinolytic protease for dehairing process in leather industry (shrinivas and naik 2010). s7 was also reported as a producer of thermostable alkaline endo-β-1-4-xylanase, which is potential for application in pulp and paper industry (mamo 2006). in pulp and paper industry, enzymatic processes are attractive futuristic alternatives to the current harsh chemical procedures. the search for alternative procedures to reduce persistent use of toxic chemicals has been going on for many years. pulp bleaching bacillus halodurans bacillus halodurans et al. process is conducted at high temperature and alkaline environment; therefore, the use of thermostable alkaline xylanase is very attractive from the economical and technical point of view (mamo 2006). a number of bacteria were isolated from various environments and screened for potential producers of xylanolytic enzymes. i5 is an isolated endoxylanase producer, and from this strain an endoxylanase gene has been cloned (helianti . 2008). aq1, which was isolated from sediment of an aquarium, was also reported as e n d o x y l a n a s e p r o d u c e r. t h e o r i g i n a l a q 1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression in , however this endoxylanase showed less activity in alkaline condition (helianti . 2010). recently, we isolated a thermoalkalophilic bacterium from sediment of mineral rich, local hot spring, cimanggu, west java. the temperature of the spot was 60 °c and the ph was 8. about 0.5 ml of material from colloidal sediment was inoculated to alkaline medium for selection and incubated at 50 °c et al. bacillus licehniformis et al bacillus subtilis e.coli et al dh5α *corresponding author, phone: +62-217560536 ext 124, fax: +62-21-7566922, e-mail: is.helianti@bppt.go.id issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 139-143 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.7 with shaking. after 18-20 hours incubation, the culture was spread on the same solid medium containing xylan. the selection medium was based on the previous work (ohta . 1974) with the following component: polypeptone 10 g, meat extract 10 g, yeast extract 3 g, glucose 3 g, na co (anhydrous) 10 g in 1 l deionized water with range ph 10 to 11. solid medium was prepared by the addition of 2% (w v ) agar. a pure bacterial strain that grew at ph 10 and 50 °c and produced clear zone on solid medium containing 0.5% xylan was isolated (fig 1a). it was designated as cm1. the supernatant (obtained by centrifugation 5600 g, 4 °c) of the culture grown in liquid medium containing 0.5% xylan was used to assay the xylanase activity. the effect of temperature on xylanase activity was measured in temperature range of 60-100 °c at ph 9 tris-hcl buffer. the effect of ph on the activity was measured at 70 °c at ph range 7-11 using the following buffers: 50 mm phosphate buffer (ph 7), 50 mm tris-hcl (ph 8-9), and 50 mm tris-glycine buffer (ph 10-11). the thermostability was measured by incubating the enzyme at 60 °c and ph 9 for 15, 30, 45, and 60 min. xylanase activity was measured duplicates using the miller (1959) method using dinitrosalicylic acid to quantify reducing sugar. d-xylose was used as a l of 1% oat spelt xylan in 50 mm of buffer at the indicated ph. the mixture was then incubated at the indicated temperature for 5 min subsequently dns reagent (1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulfite, and 1% sodium hydroxide, 20% (w v ) potassium sodium tartrate) was added to stop the reaction. then the mixture was boiled at 100 °c for 5 min, and kept at room temperature. afterwards 250 l of water was added and the mixture was centrifuged to obtain a clear supernatant. the absorbance was measured at 540 nm. as blank we used the same mixture as in the above sample; however, enzymes were added following addition of dns into the reaction mixture. one unit xylanase activity was defined as the amount of enzyme that releases et al 2 3 -1 -1 standard. 50 μl crude extract at appropriate dilutions in phosphate buffer was mix with 450 μ 750 μl 1 μmol of xylose per min under the assay condition. . , μ the optimum ph and temperature for the enzyme was ph 9 and 70 °c, respectively, showing thermoalkalophilicity of the xylanase produced by this cm1 isolate. the activity at the ph 9 and temperature 70 °c was 126.67 ± 0.85 u mg . the ph profile showed that the crude enzyme was active in the range of ph 7 11. the crude enzyme still retained 65% of its activity after incubation at 60 °c and ph 9 for 30 min (fig 2a, b, and c). other enzyme activities of this strain were investigated with cmc, skim milk, starch, and gelatin. the strain was streaked on the luria bertani agar medium containing 0.5% of each substrate. the isolate showed protease, gelatinase, and amylase activity, but no cellulose activity (fig 1 b, c, d). -1 a b c d fig 1 isolate cm1 in luria bertani media containing specific substrate: a, xylan; b, skim milk; c, starch; d, gelatin. to make the clear zone looked clearer, ki reagent was added to starch hydrolizing medium.2 fig 2 temperature and ph profile, and thermostability of crude xylanase from isolate cm1: a, the effect of temperature on cm1 crude xylanase activity. for this temperature profile, enzymatic activity was measured in 50 mm sodium phosphate ph 9. b, the effect of ph on cm1 crude xylanase activity. for this ph profile, enzymatic activity was measured in temperature 70 . the reaction phs were adjusted to 7-11 with the following buffers: 50 mm phosphate buffer (ph 7), 50 mm tris-hcl (ph 8-9), and 50 mm tris-glycine buffer (ph 10-11). c, of cm1 crude xylanase. the enzyme used for the determination of ph stability was diluted in 50 mm tris-hcl ph 9 for the designed time periods before the activity was assayed at 60 for 5 min. the assay results shown are the means of duplicated independent experiments, and the error bars indicate the standard deviation of these results thermostability in incubated at 60 . °c °c °c 160 140 120 100 80 60 40 20 0 60 70 80 90 100 110 temperature (°c) 140 120 100 80 60 40 20 0 7 8 9 10 11 12 ph 100 10 1 r e si d u a l a c ti v it y (% ) time (min) 0 10 20 30 40 50 60 140 ulfah et al. microbiol indones for further characterization of the isolates, the analysis of 16s rrna gene was conducted. genomic dna was extracted by the standard phenolchloroform-isoamyl alcohol method as described previously (helianti 2008). the 16s rrna gene fragment was amplified by pcr with universal primers 16s-27f (5'-gagtttgatcctggctcag3') and 16s-1525r (5' -agaaaggaggtgatccagcc3'). the pcr was conducted using dna taq polymerase (kappa, usa) under the following conditions: denaturation at 94 °c for 1 min, annealing 61 °c 35 sec, extension 72 °c 2 min for 30 cycles followed by elongation at 72 °c for 5 min using a thermal cycler (eppendorf, germany). it was ligated to pgem-t easy vector at 4 °c over night. the white/blue screening with x-gal/iptg on the lb agar medium containing ampicill ml was done. the positive clones were then further verified using restriction enzyme and the confirmed clone was sequenced and analyzed. the sequencing was performed by abi 3100 dna sequencer. the dna sequence was compared to other bacterial 16s rrna sequences in ncbi genbank using blast program. then, the dna sequence and the analysis result were submitted to the genbank. the accession number of the sequence was jn903769 we characterized the strain based on this partial 16s rrna sequences and its position in phylogenetic tree. the 1544 bp length of sequence of 16s rdna showed 99% identity to c-125, and phylogenetic tree analyses showed that this isolate belongs to the group of this species (fig 3). to complete the characterization of the isolate, morphology, biochemical characteristics, and carbohydrate metabolic profile was also investigated standard microbiological techniques were applied to determine microbiological characteristics such as et al. bacillus haloduranss in 100 μg -1 . . colony, spore, cell shape gram staining, and voges-proskauer reaction. carbohydrate source utilization experiments were carried out applying the api 50 chb system (bio merieux, nurtingen, germany). a single colony from 16-18 h culture incubated in 55 °c was inoculated to alkaline medium. one mililiter culture was then centrifuged at 5800 g, 4 °c for 15 min. the pellet was resuspended in 2 ml nacl 0.85% then used to inoculate, dropwise, 5 ml nacl 0.85% until the turbidity reached 2 mcfarland. then, twice the inoculum volume was used to inoculate 10 ml of api 50 chb medium. the cell suspension was used to fill the wells of the api 50 chb strips. the strips were then incubated at 37 °c for 24 48 h. a positive test corresponds to acidification revealed by the phenol red indicator contained in the medium changing to yellow. so far, there was no report on the complete biochemical properties of except c-125. there is a report about the purification and characterization of endoxylanase from strain which had 100% identity of 16 rrna to c-125 (mamo 2006). however, they did not describe the detailed biochemical properties. isolate c125 was at first identified as (aono 1995) however it was reidentified as c-125 (takami and horikoshi 1999) mainly due to the 16s rrna sequence and dna-dna hybridization data. among strains, only this one has been investigated biochemically therefore, these biochemical properties of the newly isolated bacterial strain were compared to those of c-125. the newly isolated cm1 was less tolerant against nacl (could not resist nacl concentration above 7%) and metabolized sorbitol, whereas the , , , . . bacillus halodurans bacillus halodurans bacillus halodurans bacillus halodurans et al. bacillus lentus bacillus halodurans bacillus halodurans bacillus halodurans bacillus halodurans fig 3 the position of cm1 strain in the phylogenetic tree of genus . the tree was constructed using neighbor joining method. the bar below the tree represents a distance scale. the code in the bracket is a genbank accession number. bacillus volume 5, 2011 microbiol indones 141 b. halodurans-c125(ab013373.1) cmi(jn903769) b. halodurans-ah101(ab027713.1) b. halodurans-xjrmli(ef466141.1) b. halodurans-us193(am295056.2) b. halodurans-34(af542086.1) b. subtilis-aq1(fj644629.1) b. licheniformis-sp5(jn409454.1) b.megatorium-en2(jn642548.1) b.circulans-bp9-5b(jn644554.1) 0.1 c-125 was more tolerant to nacl and could not degrade sorbitol (table 1). in conclusion, we have isolated an alkalophilic bacterium from mineral rich local hot spring, cimanggu, west java. 16s rrna sequence, morphology, and biochemical properties indicated that this new strain was cm1. the new bacterial strain produced alkalothermophilic xylanase which demonstrated optimum activity at ph 9 and 70 °c. this enzyme still retained 65% residual activity after 30 min incubation at 60 °c, thus promising its potential application in pulp and paper industry. furthermore, this strain is also a potential producer of other enzymes such as protease, amylase, and gelatinase, so that the strain would be a good candidate of enzyme workhorse in industries. , bacillus halodurans table 1 characteristic of cm1 compare to c-125bacillus halodurans bacillus halodurans acknowledgements references authors thank dian fajar vitianingrum for technical assistance in enzymatic assay. aono r. 1995. assignment of facultatively alkalophilic bacterium sp. strain c-125 to group 3. j syst bacteriol. 45(3):582585. doi:10.1099/00207713-45-3-582. helianti i, niknik n, budiasih w. 2008. cloning, sequencing ndonesian strain i5 in . world j microbiol biotechnol 24(8):1273-1279. doi:10.1007/s11274-007-9601-6. helianti i, niknik n, maria u, budiasih w, siswa s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated aq1 in . j biomed biotechnol. 12 p [on line]. doi:10.1155/2010/980567. bacillus bacillus lentus bacillus licheniformis escherichia coli bacillus subtillis escherichia coli , and expression of a β-1-4-endoxylanase gene from i . characteristics strain cm1 strain c-125 cell shape rod rod* cell size 2.7 -5.5 0.6-0.7 x 2.5-4 μm* colony color white to brownish cream-white** endospores gram staining + + + ** + * growth in anaerobic agar + + * voges-proskauer * growth at 40 ºc + + * 50 ºc + + * 55 ºc + + * growth at ph 5 * ph 7 + + * ph 11 + + ** growth in nacl 5% + + * nacl 7% + + * nacl 8% + * nacl 10% + * hydrolysis of xylan + +.** skim milk + n.d casein + +. ** starch + + ** gelatin + + ** cmc l-arabinose ribose d-xylose fructose galactose mannose melibiose lactose sucrose trehalose rafinos sorbitol + + + + + + + + + + + + n.d + * + ** + * + * + ** + ** + * * + ** + ** + ** + ** ** 142 ulfah et al. microbiol indones nd: not described; *takami and horikoshi 1999;**aono 1995. horikoshi k. 2004. alkaliphiles. proc jpn acad ser b phys biol sci. 80:166178. mamo g, rajni hk, mattiason b. 2006. a ther ylanase from s7: purification and characterization enzyme microb technol. 39(7):1492-1498. doi:10.1016/j.enzimtec.2006.03.040. miller gl. 1959. use of dinitrosalycylic acid as reagent for the determination of reducing sugars. anal chem 31(3):208-218. doi: 10.1021/ac60147a030. mostable alkaline active endo-β1-4 x bacillus halodurans . . ohta k, kiyomiya a, koyama n, nosoh y. 1974. the basis of the alkalophilic property of a species of . j gen microbiol. 86(2):259-266 shrinivas d, naik gr. 2010. characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic jb 99 exhibiting dehairing activity. int biodeterior biodegradation. 65(1):29-35. doi:10.1016/j.ibiod.2010.04.013. takami h, koki h. 1999. reidentification of facultatively alkalophilic sp. c-125 to j biosci biotechnol biochem. 63(5):943-945 doi:10.1271/bbb.63.943.. bacillus bacillus halodurans bacillus bacillus halodurans. . . volume 5, 2011 microbiol indones 143 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 13, number 3, september 2019 the use of agrobacterium sp. i and compost as chelator 3 combined by npk fertilizer and mendong plant (fimbristylis sp.) in bioremediation of paddy soil contaminated by lead (pb) the utilization of arbuscular mycorrhizal fungi for planting agarwood (aquilaria spp.) seedling in open land antibacterial potential of radish extract (raphanus sativus l.) against fish spoilage bacteria levels of cxcl10 chemokine in dengue infected hepatocyte huh 7 it-1 cell line co-cultured with peripheral 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budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi debbie s retnoningrum, 2020 iman rusmana, 2020 page 1 editorial board.pdf page 1 3. 491 polydnavirus (endang) ... polydnavirus symbiont detected from calyx tissues wasps of three lepidopteran cabbage pests endang sri ratna*, damayanti buchori, teguh santosoand department of plant protection, faculty of agriculture, bogor agricultural university jalan kamper kampus ipb darmaga, bogor 16680, indonesia snellenius manilae cotesia diadegma semiclausum s. manilae d. semiclausum cotesia snellenius manilae cotesia diadegma semiclausum s. manilae d. semiclausum cotesia parasitoid wasps are a potent biological control agent in the field. the successful of parasitism are determined by several factors, among them by the presence of polydnavirus (pdv) symbiont that could break down the immunity mechanism of its host. we explored the existence of pdv on wasps , sp., and , a group of parasitoid on cabbage pests in indonesia. morphological study of pdv was done by preparing ultrasectioned calyx tissues and negative stained of extracted calyx fluid of adult parasitoids. virogenic stroma resulted from differentiated calyx epithelial cells appeared on all three wasps. bracovirus and ichnovirus were detected from the calyx tissues of and . the electron dense materials of pdv were distributed within nucleus and vacuolated cytoplasm of calyx cells, calyx lumen and on the surface of eggs wasps. pdvs particles were also shown in the extracted calyx fluid of sp. key words: calyx tissues, parasitoids, polydnavirus, ultrastructure parasitoid merupakan salah satu musuh alami penting di lapang. kesuksesan parasitisme ditentukan oleh beberapa faktor, antara lain polidnavirus (pdv), yaitu simbion yang dapat mematahkan sistem pertahanan inang, sehingga parasitoid dapat berkembang biak dengan sukses pada inangnya. dalam penelitian ini, telah dieksplorasi keberadaan pdv pada tiga jenis parasitoid, , sp., dan , yang merupakan musuh alami hama penting pada kubis. studi morfologi pdv dilakukan dengan membuat irisan ultra preparat dari jaringan kaliks dan melakukan pewarnaan negatif pada ekstrak cairan kaliks dari parasitoid dewasa. stroma virogen dari sel epitel yang telah terdiferensiasi tampak dari ketiga parasitoid yang diuji. bracovirus dan ichnovirus juga terdeteksi dalam jaringan kaliks dari dan . materi padat elektron dari pdv tampak terdistribusi dalam inti dan sitoplasma jaringan kaliks, rongga kaliks, dan permukaan telur parasitoid. partikel pdv juga tampak dalam cairan kaliks dari parasitoid sp. kata kunci: jaringan kaliks, parasitoid, polidnavirus, ultrastruktur lepidopteran larval pests are known to cause significant damages to cabbage crops. these pests harbors a complex of natural enemies, e.g. the ichneumonid wasp that are well known to control the diamondback moth larvae (sastrosiswojo and sastrodihardjo 1986; momanyi 2006). several other braconid wasps such as and (hymenoptera: braconidae) have been recorded as a natural enemies of the larval pests , and , respectively (grasela 2008; ratna 2009). one of the successes key to parasitism is the ability of the parasitic wasp to avoid the defense mechanism of its hosts (mcneil 2010). several studies have shown that prevention of encapsulation can be done by the symbiosis of insect with polydnavirus (pdv) (suzuki and tanaka 2006; mahmoud 2011; provost 2011). the case of mutualism between viruses and eukaryotic cells of ovaries tissues was reported on braconid and ichneumonid wasps (fleming 1992; drezen 2003). pdv originated from braconids and ichneumonids wasps are termed as diadegma semiclausum plutella xylostella et al. snellenius manilae cotesia marginiventris spodoptera litura trichoplusia ni et al. et al. et al. et al. et al. bracovirus and ichnovirus respectively (beckage and gelman 2004; kroemer and webb 2004). studies have shown that presence of pdv symbiont inside the reproductive organ of parasitic wasps prevents the encapsulation process, through the manipulation of the larval host's physiology (lavine and beckage 1995; drezen 2003; pruijssers 2009). pdv is composed of a large segmented multiple circular double stranded dna genome included in a virus particle (drezen 2003; kroemer and webb 2004). proviral dna or pdv segment is integrated in the wasp's genome and can pass through vertical or horizontal transmission (lawrence 2005) and were replicated in the nucleus of calyx epithelial cells of the wasp ovaries (drezen 2003). the mature virions are injected into the larval host along with eggs during oviposition (stoltz 1986; schmidt and schuchmann-feddersen 1989). bonvin (2004) reported that viral transcript of braconid was present in the hemocytes, fat body and nervous tissue of larval host . chen (2007) reported that pdv genomes of ( ) was expressed in the hemolymph, brain and midgut of parasitized . the viral transcript of ichneumonid was also identified in the extracted tissues of parasitized larvae et al. et al. et al. et al. et al. c. inanitus spodoptera littoralis et al. c. vestalis = p. plutellae p. xylostella hyposoter didymator*corresponding author, phone: +62-251-8629364, fax: +62-251-8629362, e-mail: esratna@gmail.com issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 113-119 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.3 s. frugiperda et al apanteles melanoscelus a. paleacritae cardiochiles nigriceps c. inanitus c. texanus microplitis croceipes et al. et al. et al glyptapanteles indiensis c. plutellae microplitis rufiventris et al. campoletis sonorensis h. exiguae s. manilae cotesia d. semiclausum s. manilae s. litura cotesia d. semiclausum t. ni p. xylostella et al. et al. et al. et al. (clavijo . 2011). these viruses disrupt the host immunosystem, thus avoiding the juvenile parasitoid from being encapsulated (blumberg 1997). the presence of this pdv were shown by ultrastructures studies in several braconid wasps e.g.: , , , , and (stoltz 1976; stoltz and vinson 1977, 1979; hamm 1992; marti . 2003), (chen and gundersen-rindal 2003), (bae and kim 2004), (hegazi 2005), and ichneumonid wasp e.g.: (stoltz and vinson 1979) and (krell and stoltz 1980). in this study, we determined the existence of bracovirus and ichnovirus on the wasp's , sp., and . wasp obtained from parasitized larvae was collected from taro plantation at bogor area, while sp., and obtained from parasitized larvae and pupae and , respectively were collected from cabbage crops at cianjur area. all larval hosts were fed on cabbage leaves and maintained in the laboratory under room temperature 25-27 °c and 87100% humidity. the developed pupal parasitoids of each group were isolated into a cage made of wooden (20 cm ) covered with a general white-net cloth (mesh: 50 holes/inch). new emerging adult female parasitoids was paired with two day old male to allow mating. the pair was put into a plastic cylinder cage covered by a white-net cloth (diam. 10 cm, height 25 cm). into each cage, 30 second instar larval host were introduced. larval hosts were exposed to a pair of wasp for a period of two days. wasps were fed with 40% honey solution that was absorbed on cotton pad. one day prior to dissection, the wasps were designed to severe from host exposure to allow accumulation of large number of pdvs in their calyx's fluids (beckage 1994). morphological study of pdv was carried out by preparing ultrasectioned calyx tissues and negative stained of extracted calyx fluid isolated from the wasps (hamm 1992; hegazi 2005). female reproductive tracts taken from 3 days old wasps were dissected and were put into fixative solution prior to ultrastructure preparation according to hegazi (2005). these ovaries were fixed in a primary fixative solution materials and methods source of insects. ultra structural preparation of pdv from wasp's calyx tissues. 3 consisting of 8% glutaraldehyde in 0.2 m cacodylate buffer (ph 7.3) (1:1, v/v) for 3 h at 0-5 °c. after fixation, they were washed out by immersing in buffer solution for 10 min. these samples were post-fixed in 2% osmium tetroxide in 0.2 m cacodylate buffer solution (1:1, v/v) for another 1 h at 0-5 °c. they were rapidly dehydrated using a grades series of ethanol solutions (concentration ranging from 30, 50, 70, and 95%, 15 min each) and were subsequently washed in ethanol absolute (three changes) for 10 min at 0-5 °c. the samples were then transferred to propylene oxide solutions for 15 min (three changes), and infiltrated with mixed solution of propylene oxide and spurr's low-viscosity embedding resin (1:1; v/v) for overnight followed by 100% spurr's resin for 2 h. infiltration process was carried out by placing it on the agitator and given high speed agitation at room temperature. each specimen was transferred to embedding block filled with fresh spurr's resin and polymerized in the oven at 60 °c for overnight. polymerized blocks were trimmed and sectioned on reichert-jung ultra cut type 701704 ultramicrotome using 45 degree angle glass knives prior to provide the ultra thick (1 μm) and ultra thin (125 nm) slices of the calyx tissues. section thickness was determined by interference colours produced by reflected light (grey for thick sections and gold for thin sections). the ultra thick sections were mounted on glass slides and stained with 1% toluidine blue in 1% of borax within 3-5 min and viewed under light microscope. the ultra thin sections were mounted on 200-mesh copper grid and stained with 2% uranyl acetate in 70% ethanol solution for 20 min. they were then washed with distilled water and stained in 4% lead citrate solution ready used for another 20 min. these grids were further floated over a drop of 0.05 m naoh solution to reduced lead carbonate contamination and washed thoroughly with distilled water and leave it to dry. the pdv were observed from this sectioned under hitachi transmission electron microscope. pdv's were isolated from parasitoid by extraction of its calyx fluid (hamm 1992). seventy five ovaries were each isolated from 3-4 days old wasps , sp., and , in cold ringer solution. these ovaries were washed in 2% nahclo solution and put in 1.5 ml an eppendorf tube containing 15 uid was obtained by maceration of reproductive tract tissues using a pestle pipette glass followed by centrifugation at 2500 rpm within 8 min. the supernatant contained pdv was transferred into another tube and centrifuged at 12000 rpm within 1 h. the second supernatant was removed and the pellet was resuspended by distilled water prior to negative staining preparation. negative staining of calyx fluid. et al. s. manilae cotesia d. semiclausum μl ringer solutions on crushed ices. calyx fl 114 sri ratna et al. microbiol indones one drop of suspension was mixed with one drop of 1.5% fosfotungstic acid solution which was placed on a 400 mesh grid layered with 1% formvar film. grid was viewed under the same electron microscope as above. pdv's were assumed to be replicated in a part of ovaries of both ichneumonid and braconid wasps. our study revealed the results structure of wasps ovaries. enlargement at the base of calyx tissues of three days old female wasps (fig 1 and fig 2). oogenesis was carried out within 2 sets of paired ovarioles or egg tubes. the penultimate oocytes were located on ovarial reservoirs, then move down gradually to a pear-shaped calyx and tubular lateral oviduct. both tubes fused to form the short common oviduct as a place of mature eggs and ended with the ovipositor. our result showed the presence of ultra thick sectioned calyx tissue that consisted of the outer part of volume 5, 2011 microbiol indones 115 fig 1 the reproductive tracts of adult female (a) and (b) . et: egg tube; or: ovarial reservoir; c: calyx; lo: lateral oviduct; co: common oviduct: o: ovipositor: pg: poison gland. bars = 0.5 mm. s. manilae d. semiclausum fig 2 enlargement picture part of ovaries . et: egg tube; or: ovarial reservoir; cb: calyx based; bar = 0.5 mm. s. manilae tissues, layered by basal lamina, followed with epithelial cells (fig 3). in the center region, a lumen calyx contained eggs are surrounded by calyx fluids. virogenic stroma from the epithelial cells of appears to have a bud stalk-like shaped which supported a group of cells developed into calyx lumen and almost half of lumen filled with eggs (fig 3a). these cells mostly consisted of relatively big nucleus that is thought as a place of replicated pdv. a different appearance was seen in the wasp sp., i.e. the layers of vacuolated calyx epithelial cells that are more clearly surrounding the calyx lumen which are filled with less egg (fig 3b). bud cells was not shown in this sectioned. as in here we saw virogenic stroma with obviously protruded nucleus, which fulfills almost three-part of the calyx lumen. in contrast, in ichneumonid wasps , the virogenic stroma was not spread out circularly around the lumen calyx, but instead laid on spot in a certain area (fig 3c). the vacuolated cytoplasm of calyx epithelial cell with small nucleus deeply inserted between eggs. result of low magnification of the electron microscope convinced that active calyx epithelial cells are vacuolated. the nuclei have irregular shapes. the electron dense materials of pdv appeared within calyx s. manilae cotesia s. manilae d. semiclausum pdv in calyx tissues and calyx fluid. nuclei and cytoplasm. it also seems to spread in the surface of the eggs of both parasitoids and (fig 4a and 4b). fig 4c also showed that the pdv materials had been lysed from the cells spreading within the vacuoles of calyx lumen. under high magnification it is shown that cytoplasm of the calyx epithelial cells contained different morphological structure of pdv particles that differs between the two group species of wasps (fig 5). pdvs from braconids wasp are typically bracovirus which is recognized as a group of s. manilae d. semiclausum s. manilae 116 sri ratna et al. microbiol indones fig 3 transversal sections of calyx tissues of wasps (a) , (b) sp., and (c) . ec: epithelium calyx cells; be: budding epithelium; vs: virogenic stroma; e: eggs, lc: lumen calyx; bars: 1 mm. s. manilae cotesia d. semiclausum fig 5 pdv particles of the calyx tissues (a) (b) . bars = 0.5 m s. manilae d. semiclausum μ fig 6 polydnavirus particles in calyx fluid of (a) pdvs with and without tails (b) pdvs within the sack. t: tail or protrusion membrane; sm: enveloped membrane are being lysis; bars = 0.2 m. cotesia sp. μ cylindrical nucleocapsid particles each surrounded by a single unit membrane, compared with pdvs from ichneumonid wasp known as ichnovirus which is a single particle of lenticular nucleocapsid surrounded by double membranes. the result of investigation under negative staining of calyx fluid extracts revealed that the pdv shape appeared as a circular dna particles surrounded by a membrane (fig 6a). two shapes of virion existed with and without membrane protrusion or tail-like appendages. fig 6b showed that capsid of bracovirus of sp. was bursting from its envelope. our result showed that the ovarial reservoirs were full of eggs, which is indicated by the enlargement of based calyx. the swollen tissues consisted of developed epithelial cells producing pdv, and the lumen calyx filled with eggs and the released pdvs d. semiclausum cotesia discussions fig 4 calyx tissues (a) , (b) , and (c) calyx lumen ). cl: calyx lumen; cp: cytoplasm; e: eggs; n: nucleus; pdv: particles dense virion; v: vacuolated cytoplasm; bars = 1 m. s. manilae d. semiclausum d. semiclausum μ volume 5, 2011 microbiol indones 117 (stoltz 1976; marti . 2003). this is consistent with the result of hegazi (2005) which revealed that pdv started to appear in the calyx cells of the midage pupae of the braconid wasp and it is very abundant in the calyx fluid of pharate adult. here, we observed the expansion of calyx epithelial layers forming clusters of cells possessing large irregular shaped nuclei and vacuolated cytoplasm in the side region beneath the lamina basal that is considered as a virogenic stroma. this virogenic stroma was more developed in the braconids and sp. as indicated by the presence of pdv in almost half or two-third areas of its lumen. it is interesting to note that those tissues originated from the growth point of epithelial cells, protruding in one side, and then formed a bud-shape like pedicel. this was not seen in the ichneumonids calyx tissues. bracovirus seems to be replicated in these cells producing enlargement of nuclei, occupying most of the cell volume at the end of virus replication (drezen 2003; marti 2003). the virus particles are then released into and dispersed within a calyx lumen (beckage and gelman 2004). the budding structures were also reported in calyx epithelium cells of and that seems to characterize ichnovirus (stoltz 1976; stoltz and vinson 1979). using high magnification, the electron dense materials appeared in the nucleus and vacuolated cytoplasm of the epithelium calyx cells and also in the egg's surface and calyx fluid of all three wasps. there is indication that pdvs are being replicated within the cells followed by lysis and then discharged into the calyx lumen. hegazi (2005) also mentioned that vacuolated cytoplasm appeared in the wasp calyx cells and pdvs were also found on the surface of egg chorion on the lumen of lateral oviduct. two different ultrastructures of ichnovirus and bracovirus particles found in the lumen of oviduct or calyx of the epithelium cells was elaborated by several authors. ichnovirus particles found in the calyx fluid of consist of a nucleocapsid seen as electron dense materials surrounded by inner and outer membranes (krell and stoltz 1980). on the other hand, bracovirus particles is a package containing a single large virus particles (drezen 2003). the spherical shape of mature virion (braconidae) were covered individually by nucleocapsids that consist of a single unit membrane (hegazi 2005). this bracovirus was also discovered in the calyx fluids (stoltz 1976). our result showed that several nucleocapsid particles of sp. were located within the sack and the individual capsid covered with a single membrane. some of them had a protrusion of tail-like shaped. this is similar to what et al. et al et al. m. rufiventris s. manilae cotesia d. semiclausum et al. et al. c. nigriceps m. croceipes et al. et al. h. exigua et al. m. rufiventris et al. c. texanus et al. cotesia was found by stoltz and vinson (1977), where the electron-dense nucleocapsids particles per envelope appeared in the nucleus of calyx epithelial cells of braconids wasps, (stoltz and vinson 1977) and (drezen 2003). in general, pdvs could be integrated in the calyx cells, but the nature ancestral virus in braconids and ichneumonids was probably specific between families (drezen 2003; federici and bigot 2003; kroemer and webb 2004). the morphology of nucleocapsid of both virions reflected the differences of original ancestral virions. kroemer and webb (2004) explained that the multiprotein double membrane of bracovirus was recognizably different compared to lipid membrane found in ichnovirus. a characterized protrusion membrane were shown in bracovirus , and and ichnovirus wasps (stoltz 1976; stoltz and vinson 1977; beckage 1994). however, this protrusion membrane was not shown in bracovirus (hamm 1992). the evidence of bursting pdv from the sack was shown in our study. according to stoltz and vinson (1977), nucleocapsid particles of calyx fluid were visible within envelopes and the n e g a t i v e s t a i n e d n o n p r o t r u s i o n m e m b r a n e baculovirus-like particles were released from disrupted envelopes. the envelope structures contained one or several pdvs have also been reported in and it seems a characteristic of bracovirus where the virion is released through a lysis process (hamm 1992). the different process of released pdv has been explained by several authors that cell lyses from envelopes was found in the case of pdv that was released in bracovirus and that budding process happens without damaging the cells in the case of pdv released in ichnovirus (stoltz and vinson 1979; fleming 1992; drezen 2003; wyler and lanzrein 2003; bonvin 2004). observation of the negative staining from the calyx fluid extracts showed that pdv is only found in the wasp sp. it might be possible that the critical time for a calyx tissues extraction has influenced the production of a mature released pdvs. hegazi (2005) explained that the ovarial reservoir of can be clearly distinguished at 3 days old pupae and pdv is visible in the calyx lumen of pharate adult or one day prior to adult emergence. pdv began to replicate during the late stages of pupal development that coincides with melanization of the pharate adult cuticle (kroemer and webb 2004) or in a newly emergence wasp (lavine and beckage 1995). in the ovary of , pdv was reported to be present on 5-day old wasp (bae and kim 2004). beckage a. paleacritae c. congregata et al. et al. c. congregata c. melanoscela m. croceipes c. sonorensis et al. et al. c. marginiventris et al. a. melanoscelus c. marginiventris et al. et al. et al. cotesia et al. m. rufiventris c. plutellae et al. (1994) elaborated that newly emerging wasp aged less than 24 h had significantly less virus compared to 3-4 days old mature females. based on our result, the epithelial calyx tissues of 3 days old appeared to be intact and has no vacuolated cytoplasm. this result suggested that the virus has not fully grown in the wasp, or that the mature virion had not been released into calyx lumen due to the fact that its epithelial cell differentiation is still in process. however, the evidence showed in the wasp , showed that the virogenic stroma calyx fluid started to be visible prior to adult eclosion. at this time, the lumen contained one or at least a single egg and this stroma is continuously producing during the whole life span of the adult females (drezen 2003). this study reveals that bracovirus and ichnovirus wasp symbionts of the lepidopteran cabbage pests are very important in preventing encapsulation. these findings can be used to increase the success of biological control agent in the 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doi:10.1242/jeb.030635. ratna es. 2009. efisiensi parasitisasi inang (f.) oleh endoparasitoid ashmead di laboratorium [eficiency of parasitization on larval host, (f.) by an endoparasitoid (= ) ashmead in the laboratory]. j hpt tropika. 8(1):8-16. sastrosiswojo s, sastrodihardjo s. 1986. status of biological control of diamondback moth by introduction of parasitoid in indonesia. in: talekar ns, griggs td, editors. diamondback moth management. 1 international workshop; 1985 march 11-15. tainan(tw). tainan:avrdc publication no. 86-248. p 185-194. schmidt o, schuchmann-feddersen i. 1989. role of virus-like particles in parasitoid-host interaction of insects. subcell biochem. 15: 91-119. stoltz db, vinson sb. 1979. viruses and parasitism in insects. adv virus res. 24:125-171. doi:10.1016/s0065-3527(08)60393-0. pseudoplusia includens. spodoptera litura snellenius manila spodoptera litura , snellenius microplitis manilae diadegma eucerophaga st stoltz db, vinson sb, mackinnon ea. 1976. baculovirus-like particles in the reproductive tracts of female parasitoid wasps. can j microbiol. 22(7):1013-1023. doi:10.1139/m76-148. stoltz db, vinson sb. 1977. baculovirus-like particles in the reproductive tracts of female parasitoid wasps ii: the genus . can j microbiol. 23(1):28-37. doi:10.1139/m77-004. stoltz db. 1986. interactions between parasitoid-derived products and host insects: an overview. j insect physiol. 32:347-350. suzuki m, tanaka t. 2006. virus-like particles and venom of induce host hemocyte apoptosis. j insect physiol. 52(6):602-613. doi:10.1016/j.jinsphys.2010.02.009. wyler t. lanzrein b. 2003. ovary development and polydnavirus morphogenesis in the parasitic wasp . ii. ultrastructural analysis of calyx cell development, virion formation and release. j gen virol. 84(5):1151-1163. doi:10.1099/vir.0. 18830-0. apanteles meteorus pulchricornis chelonus inanitus volume 5, 2011 microbiol indones 119 1 langkah sembiring.pmd review ecological approach to unravel streptomycete diversity as an unsurpassed sources of natural bioactive products langkah sembiring1* and michael goodfellow2 1laboratory of microbiology, faculty of biology, universitas gadjah mada, jalan teknika selatan, sekip utara, kampus bulaksumur, yogyakarta 55281, indonesia; 2school of biology, newcastle university, ridley building, newcastle upon tyne, ne1 7ru united kingdom search and discovery for natural bioactive products have been so important to control the emergence of antibiotic resistant microbial pathogens. therefore, novel microorganisms that produce such metabolites is extremely needed. the capacity of members of the genus streptomyces to produce commercially significant bioactive metabolites, notably antibiotics remains unsurpassed. however, it is acknowledged that discovering commercially useful secondary metabolites from streptomycetes is becoming more difficult due to lack of knowledge on the ecology and complexity of streptomycete systematics. in fact, those are fundamental aspects for developing strategy and method for isolation. in order to devise an appropriate program for successful selective isolation of sreptomycetes, it is fundamentally important to understand their occurance and activity in nature. a multistep extraction procedure designed for representative sampling, called dispersion, and differential centrifugation technique in combination with the incorporation of antibiotics into isolation media has become one of the most important selective method for the isolation of streptomycetes from natural habitats. the availability of new procedures to selectively isolate representative of streptomycetes from natural habitats opens up the possibility to determine the extent of streptomycete diversity from various habitats. hence, the capacity of well characterized streptomycete isolates to produce commercial novel active metabolites could be further assessed appropriately. key words: ecological approach, streptomycete, diversity, natural bioactive _____________________________________________ ________________________ *corresponding author, phone/fax: +62-274-580839, e-mail: lsembiring@yahoo.com issn 1978-3477 volume 2, number 2, august 2008 p 49-56 the search and discovery of new microorganisms that produce novel secondary metabolites is extremely important not least because of the need to find new pharmacologically active compounds to control the emergence of antibiotic resistant microbial pathogens (bérdy 1995; demain 1998; demain and elander 1999). it is widely acknowledged that some microorganisms are better sources of bioactive compounds than others. amongst bacteria, the ability of members of the genus streptomyces to produce commercially significant, pharmacologically active metabolites, notably antibiotics, remains unsurpassed (bérdy 1995; sanglier et al. 1996; garrity and holt 2001). however, it is becoming increasingly difficult to discover commercially useful secondary metabolites from these organisms as known streptomycetes are being isolated and screened with increasing frequency with the result that the same kinds of bioactive compounds are being rediscovered at great expense. this situation raises the question whether streptomycetes are exhausted as a source of new bioactive compounds and hence should lose their pre-eminance in screening programmes designed to detect novel natural products. to some extent, the answer to this question depends on the extent of the untapped taxonomic and genetic diversity that is encompassed in the genus at specific and infraspecific levels. the primary aim of this review is to unveil the potential of the members of the genus streptomyces as an unsurpassed source of bioactive products as well as the constraint that usually prevent the succesful discovery of novel strain that produce commercial bioactive product. the strategy and method developed on the basis of ecological and taxonomical selective isolation approach in order to overcome the problems of discovering novel bioactive producing streptomycetes are therefore also discussed accordingly. study of streptomycete diversity in natural habitats the analysis of dna extracted from environmental habitats shows that the genetic diversity of microorganisms is much greater in natural habitats than was previously recognized (embley and stackebrandt 1997; head et al. 1998; bull et al. 2000). the genus streptomyces accommodates an unusually high degree of natural diversity with almost 600 validly described species (goodfellow et al. 2007). nevertheless, a steady flow of new streptomycete species are being described to accommodate either organisms isolated from diverse habitats (kim et al. 1998; al-tai et al. 1999; kim et al. l999, 2000; sembiring et al. 2000; goodfellow et al. 2007; ambarwati et al. 2009) or existing strains redescribed in the light of the application of modern taxonomic techniques (labeda and lyons l991a,b; labeda et al. 1997). it is clear from such studies that new streptomycete species should be circumscribed using a combination of genotypic and phenotypic data and that strains isolated from unexplored habitats are likely to form new centres of taxonomic variation. there is a strong circumstantial evidence that the discovery of previously unknown natural products occurs when novel organisms are examined in either established or new pharmacological 50 sembiring and goodfellow microbiol indones screening programmes (nolan and cross 1988; omura 1992; woodruff 1999). it is highly probable that the genus streptomyces is underspeciated partly because of the historical difficulties in isolating and characterising a representative sample of the streptomycete community found in natural habitats. however, the availability of new procedures to selectively isolate and characterise representative of streptomycetes from natural habitats opens up the possibility of determining the extent of streptomycete diversity associated with neglected habitats, such as the rhizosphere of tropical trees (sembiring et al. 2000; ambarwati et al. 2009). it is becoming increasingly apparent that streptomycetes are widely distributed in the root systems of a broad range of plants (upton 1994; katsifas et al. 1999; atalan et al. 2000; sembiring et al. 2000) though little is known about the extent of their taxonomic diversity, activities or interactions with other organisms found in and around plant roots. nevertheless, there is evidence that streptomycetes or their products can be used to suppress root-infecting fungi in vivo (lui et al. 1995; you et al. 1996; trejo-estrada et al. 1998a). an example of biosystematic studies on members of three putatively novel streptomyces species isolated from rhizosphere soil show that a coherent strategy is available to determine the species richness of cultivable streptomycetes isolated from environmental samples (atalan et al. 2000). representative strains from selective isolation plates can be grown on oatmeal and peptone-yeast extractiron agars and assigned to groups based on aerial spore mass colour, substrate mycelial pigmentation, the colour of any diffusible pigments and the ability to produce melanin pigments. the resultant colour-groups can be evaluated by examining representative strains by using curie-point pyrolysis mass spectrometry (goodfellow et al. 1997a; sembiring et al. 2000) and/or by 16s rdna sequencing and dna:dna relatedness studies (goodfellow et al. 1997b). a similar strategy has been used to highlight potentially novel rhodococci that were selectively isolated from deep sea sediments in the north-west pacific ocean (colquhoun et al. 1998a,b, 2000). ecology and strategy to selectively isolate streptomycetes little is known about the geographical distribution of streptomyces species (goodfellow and simpson 1987; goodfellow and o’donnell 1989; bull et al. 1992) or about fluxes in streptomycete populations due to seasonal and climatic changes or to human intervention as in agriculture and farming practices (atalan 1993; upton 1994). this lack of knowledge can be partially attributed to the complexity of streptomycete systematics, notably to the lack of reliable identification schemes. the identification of streptomycetes below the genus level remains difficult and has been rarely attempted in ecological studies (goodfellow and dickinson 1985; upton 1994; manfio 1995; atalan et al. 2000) even with the availability of computer-assisted identification procedures (williams et al. 1983; langham et al. 1989; kämpfer and kroppenstedt 1991). the literature on the occurrence and activity of streptomycetes in nature is as extensive as it is diffuse (williams 1982; goodfellow and williams 1983; williams et al. 1984a,b; goodfellow and simpson 1987; mccarthy and williams 1990; korn-wendisch and kutzner 1992). however, streptomycetes are common in both aquatic and terrestrial environments; most are strict saprophytes though members of a few species form parasitic associations with animals and plants. little is known about the role of streptomycetes in natural habitats though composts, fodder and soil seem to be primary reservoirs. innumerable “non-selective” media have been recommended for the isolation of streptomycetes (williams and davies 1965; williams et al. 1984a). many of these contain glucose, glycerol, mannitol, or starch as the carbon source and arginine or asparagine as the nitrogen source. chitin has also frequently been used as a source of carbon and nitrogen. such “non-selective” media are now known to favour the isolation of a narrow ranges of streptomycetes and do not support the growth of actinomycetes with more exacting growth requirements (cross et al. 1976; williams et al. 1984a). selective isolation procedures are necessary to determine the numbers and types of streptomycetes occurring in natural habitats. selective media favour the growth of target microorganisms but not that of unwanted organisms. a number of approaches based on some aspect of the biology of individuals or groups of organisms can be used to selectively isolate actinomycetes from environmental samples. the organisms may be selected by plating serial dilutions of environmental samples onto nutrient media containing compounds which inhibit the growth of unwanted bacteria but not that of the target streptomycetes, by enriching the environmental substrate prior to selective isolation or by treating it using either chemical and/or physical methods which favour the isolation of streptomycetes but not that of unwanted bacteria and fungi. the incorporation of antibiotics into isolation media has become one of the most important selective techniques for the isolation of streptomycetes (porter et al. 1960; gregory and lacey 1963; williams and davies 1965; williams and mayfield 1971; orchard and goodfellow 1974; labeda and shearer 1990). the antifungal antibiotics cycloheximide and nystatin are routinely incorporated into media selective for streptomycetes, at approximately 50 µg ml-1 each, to eliminate or control the growth of fungi on isolation plates. media supplemented with antibacterial antibiotics are often used to good effect though streptomycete counts as well as those of unwanted bacteria may be reduced (williams and davies 1965; davies and williams 1970). it is always difficult to know which antibiotic or combination of antibiotics are likely to be the most effective for the isolation of target organisms. one approach which has been applied with some success is to determine the antibiotic sensitivity patterns of representatives of a specific taxon and to supplement media with antibiotic(s) that inhibit unwanted bacteria but not that of the target streptomycetes. williams and davies (1965) screened members of 45 streptomyces spp. against four antibiotics at five different concentrations and found that the least inhibitory antibiotics were polymixin b sulphate (5.0 µg ml-1) and sodium penicillin (1.0 µg ml-1). they supplemented starch-casein agar with these antibiotics and found a decrease in the total number of streptomycetes from soil though the plates were cleaner and streptomycete colonies easier to recognise and isolate than on control plates lacking antibacterial antibiotics. the high streptomycetes counts associated with habitats such as soil need to be interpreted with care as most colonies growing on isolation plates originate from spores. the growth of streptomycetes in soil is similar to that of many other microorganisms in this habitat where supplies of nutrients are discontinuous. it seems that streptomycetes live in soil for long periods as arthrospores that germinate in the presence of exogenous nutrients, the lack of which prevents germination of most or all spores added to sterile soil (mayfield et al. 1972). these investigators estimated the mean doubling time of streptomycetes in soil to be 1.7 days. this protracted doubling time probably reflects the stop-go nature of the streptomycete life-cycle. the specific growth rates and genetration times of streptomycetes grown in batch culture are roughly intermediate between those of bacteria and fungi ( flowers and williams 1977). the survival capacity of streptomycete spores appear to be greater than that of hyphae (williams et al. 1972). the walls of spores are usually thicker than those of hyphae (sharples and williams 1976) and are also more hydrophobic (ruddick and williams 1972) due to the presence of an outer sheath that envelopes the spore wall (williams et al. 1973). streptomycete spores have a net negative surface charge at low ph levels (douglas et al. 1971), a relatively low endogenous metabolism (ensign 1978) and generally show more resistance to heat than corresponding hyphae (goodfellow and simpson 1987). they are dispersed above soil by wind or rain (lloyd 1969) and within soil by arthropods and water movements (ruddick and williams 1972). the major factors governing the distribution and activity of streptomycetes in soil are nutrient availability, moisture content, temperature and ph though soil type and seasonal change also have an influence (williams et al. 1972; williams 1978; atalan 1993; upton 1994). streptomycetes can grow in soil at low oxygen levels, but not when carbon dioxide concentrations exceed 10%. in arid soils, streptomycete counts decrease sharply at moisture tensions above pf 4.0, but their relative proportion to other bacteria may be greater as their spores are more resistant to desiccation than vegetative cells of bacteria. optimal counts from neutral soils and optimal radial growth of streptomycetes inoculated into sterile soil occur at moisture tensions between pf 1.5 and 2.5. at these tensions, soil pores are partially filled with available water but still contain sufficient air for the growth of the aerobic microbiota. halophilic (hunter et al. 1981) and osmophilic streptomycetes (wong and griffen 1974) have been reported. soil reaction is an important factor determining the distribution and activity of streptomycetes. acidophilic and neutrotolerant streptomycetes, which grow between ph 3.5 and 7.5 but optimally around ph 5.5, are common in acidic soils (williams et al. 1971; khan and williams 1975; goodfellow and dawson 1978; goodfellow and simpson 1987). these organisms produce chitinases (williams and robinson 1981) and diastases (williams and flower 1978) with ph optima lower than those of en-zymes from neutrophilic streptomycetes which grow between ph 5.5 and 8.0, but optimally around 7.2. the presence of low numbers of neutrophilic streptomycetes in acidic soils has been attributed to their ability to grow in less acidic microsites (williams and mayfield 1971). it has been shown that when nitrogen containing substrates, such as chitin or dead fungal mycelium, are added to poorly buffered acidic soil, a succession of acidophilic to neutrophilic streptomycetes occurs that parallels ammonification and the resultant rise in ph (williams and robinson 1981). little is known about the growth of most streptomycetes in situ. it seems unlikely that they grow optimally in temperate soils as most strains are mesophilic under laboratory conditions. however, temperature can indirectly be implicated in examples of the influence of seasonal and climatic factors in the size and composition of streptomycete populations. it has been reported that streptomycete counts in grassland were highest in summer and that the distribution of “streptomyces malachiticus” is restricted to subtropical and tropical soils (küster 1976). clay and humic colloids can influence the activity of streptomycetes at the micro-environmental level. streptomycete spores are readily adsorbed to kaolin but not to montmorillonite except at low ph (ruddick and williams 1972). addition of calcium montmorillonite or calcium humate to cultures of streptomycetes can accelerate their growth and respiration (mara and oragui 1981). it has also been shown that sites of adsorption with humic material can lead to microsites of increased ph in acidic soils (williams and mayfield 1971). streptomycete as a potential source of natural bioactive products wide range of marketed microbial agents with therapeutic which are produced by the streptocetes including antibacteria (cephamycins, carbapenems, clindamycin, quinupristin, and streptomycin), antifungal (nystatin, cycloheximide), antineoplastics (daunorubicin, doxorubicin), immunomodulator (tacrolismus, rapamycin), and antiparasitic (ivermectin, abamectin, doramectin, moxidectin (kuo and garrity 2002). however, in general, streptomycetes are not considered to have a significant role in plant root systems (williams et al. 1984b). however, it is now apparent that streptomycetes are widely distributed in the root systems of diverse plants (rangaswami and vasantharajan 1962; bernhard 1967; watson and williams 1974; vruggink 1976; buti 1978; miller et al. 1990; sardi et al. 1992; upton 1994; katsifas et al. 1999; atalan et al. 2000) though little is known about the extent of their diversity, activities, or interactions with other organisms in the root environment. positive rhizosphere effects have been reported for streptomycetes in several root systems, such as those of maize, perennial ryegrass, soya, tomato, and wheat (abraham and herr 1964; upton 1994). volume 2, 2008 microbiol indones 51 there is a growing interest in using members of the streptomycete rhizosphere community to enhance plant growth and production, and to inhibit root infecting fungi (hettiarachi and penninckx 1990; trejo-estrada et al. 1998a,b). two mechanisms have been proposed to explain the inhibition of fungal pathogens in the rhizosphere by biocontrol agents. antibiosis occurs when one or more diffusible compounds inhibit growth or development changes in the pathogen thereby impairing its ability to colonise the rhizosphere and establish disease. mycoparasitism is a different process which is initiated by physical destruction of the fungal cell wall mediated by the action of hydrolytic enzymes produced by the biocontrol agent (adams 1990). most actinomycetes considered to suppress the growth of root infecting fungi are streptomycetes (table 1). antibiotics produced by actinomycetes have been used directly or assumed to be responsible for the biocontrol potential of the producing strains. examples of such metabolites include aminoglycosides (qin et al. 1994), macrolide benzoquinones (rothrock and gottlieb 1984), nucleosides (hwang et al. 1994) and polyenes (smith et al. 1990; raatikainen et al. 1994). streptomyces violaceusniger strain yced-9 is an antifungal biocontrol agent which produces three different antibiotics, namely geldanamycin, nigericin, and a complex of macrocyclic lactone antibiotics (trejo-estrada et al. 1998a,b). this organism, which was isolated from soil by crawford et al. (1993), was selected for its potential to suppress dumping-off disease of lettuce caused by pythium ultimum, and for its ability to antagonize the growth of many fungal pathogens in vitro and in vivo (crawford et al. 1993; crawford 1996). streptomyces strain table 1 actinomycetes reported to be antagonistic towards fungal root pathogens actinomycete genus/species fungal pathogen reference actinoplanes missouriensis actinoplanes micromonospora rhodococcus s. diastatochromogenes s. griseoalbus s. griseus s. hygroscopicus subsp. geldanus s. violaceusniger strain a50 s. violaceusniger yced-9 streptomyces streptomyces strain 385 not stated aphanomyces sp. phytophthora sp. pythium sp. phytophthora sp. phytophthora sp. phytophthora capsici pythium spp. phytophthora sp. gaeumannomyces graminis pythium debaryanum phellinus weirii fomes annosus phytophthora cinnamoni rhizoctonia solani phomopis sclerotioides rhizoctonia sp. botryosphaeria dothidea phytophthora capsici rhizoctonia solani rhizoctonia solani fusarium oxysporum phymatotrichum omnivorum rhizoctonia solani verticillium alboatrum gaeumannomyces graminis rhizoctonia bataticola aspergillus spp. ‘wood infecting fungi’ phytophthora cinnamoni fusarium oxysporum fusarium oxysporum fusarium moniliforme sclerotium rolfsii fusarium oxysporum aspergillus parasiticus fusarium tricictum fusarium oxysporum phytophthora spp. rhizoctonia solani fusarium culmorum fusarium udum corticum salmonicolor phytophthora capsici phytophthora cinnamoni fusarium oxysporum pythium ultimum sutherland et al. (1984) sneh et al. (1977) sutherland and lockwood (1984) sutherland and papavizas (1991) khan et al. (1993) sutherland and lockwood (1984) renwick et al. (1991) kaspari (1973) rose et al. (1980) merriman et al. (1974a, 1974b) ebben and spencer (1978) rothrock and gottlieb (1984) hwang et al. (1994) trejo-estrada et al. (1998a) whaley and boyle (1967) smiley (1978a, 1978b) sing and mehrota (1980) stabi et al. (1980) blanchette et al. (1981) murray (1987) sabaou and bounagu (1987) huber et al. (1989) kalappanavar and hiremath (1990) plakshappa et al. (1990) chung and hong (1991) borghi et al. (1992) singh et al. (1999) keast and tonkin (1983) kundu and nandi (1985) kempf and wolf (1989) guar and sharma (1991) joseph et al. (1991) ahn and hwang (1992) stirling et al. (1992) abdel-moneim et al. (1993) crawford et al. (1993) 52 sembiring and goodfellow microbiol indones 385 suppresses fusarium wilt of cucumber (cucumis sativus) caused by fusarium oxysporum when used in combination with paenibacillus strain 300 (singh et al. 1999). natural bioactive substances, notably which are produced by microorganisms have been the subject for many studies due to their importance in the fields of both medicine and agriculture. such natural microbial bioactive products are including antibiotics, anticancer, antiviral, immunomodulator as well as antiparasitic. in the field of medicine, antibiotics have been used as the main agents to control the emergence of antibiotic resistant microbial pathogens, while in the field of agriculture antibiotics and antihelminth have also been utilized to control plant pathogen microbes as well as plant pest nematodes, respectively. since amongst bacteria, the ability of members of the genus streptomyces to produce commercially significant, pharmacologically active metabolites, 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microorganisms, an odyssey revisited. actinomycetologica 13:58-67. 56 sembiring and goodfellow microbiol indones you mp, sivasithamparam k, kurtboke di. 1996. actinomycetes in organic mulch used in avocado plantations and their ability to suppress phytophthora cinnamomi. biol fertil soils 22:237-242. 2. 580 selection (dwii sugipr... selection of yeasts antagonists as biocontrol agent of mango fruit rot caused by botryodiplodia theobromae dwi sugiprihatini , suryo wiyono , widodo 1 2* 2 and 1 2 balai besar uji standar karantina pertanian, jalan pemuda no 64 kav16-17, rawamangun, jakarta 13220, indonesia department of plant protection, faculty of agriculture, institut pertanian bogor, darmaga campus, bogor 16680, indonesia botryodiplodia theobromae b. theobromae cryptococcus albidus aerius pichia guilliermondii debaryomyces hansenii b. theobromae c. albidus aerius c. albidus c. terreus candida edax candida edax cryptococcus luteolus botryodiplodia theobromae cryptococcus albidus botryodiplodia theobromae b. theobromae cryptococcus albidus aerius pichia guilliermondii debaryomyces hansenii b. theobromae c. albidus aerius c. albidus c. albidus c. terreus candida edax candida edax c. luteolus cryptococcus albidus botryodiplodia theobromae ; fruit rot caused by is one of the most important post harvest disease of mango in indonesia. study on biological control on the disease is required to develop environmentally-sound control technology. the research objectives were to study the potency of yeasts in controlling post harvest mango disease i.e. fruit rot caused by and mechanism involve in the biocontrol. total yeast isolates used for screening were twenty one, four from collection of plant clinic of institut pertanian bogor, and twenty one isolated from healthy mango skin. all of yeast isolates were characterized and identified using biolog. bioassay on antagonistic activity of yeasts against fruit rot, in-vitro dual culture test and chitinolytic activity were carried out. var. wsw1, k1, and k12 were the three most effective antagonistic yeasts against with effectiveness of 70.83, 45.83, 37.50% respectively. in vitro bio-assay showed that var. wsw1, wsw2, c. albidus k6, ysw1, osw1, k13, and k2 had high antibiosis activity. biocontrol activity of tested yeasts against fruit rot of mango did not correlate to its antibiosis and chitinolytic activity. key words: biocontrol, , , mango, yeast busuk buah yang disebabkan merupakan salah satu penyakit pasca panen mangga terpenting di indonesia. penelitian tentang pengendalian hayati penyakit tersebut diperlukan untuk mengembangkan teknologi pengendalian yang ramah lingkungan. penelitian ini bertujuan mengkaji potensi khamir antagonis dalam pengendalian busuk buah mangga yang disebabkan dan beberapa mekanisme yang terlibat. isolat khamir diperoleh dari koleksi klinik tanaman institut pertanian bogor (ipb), yang diisolasi oleh salah seorang penulis dan diisolasi dari buah mangga sehat. isolasi khamir dilakukan dengan pencucian kulit buah yang dilanjutkan dengan penanaman pada media pda ph 5.5. jumlah isolat khamir yang diseleksi semuanya ada 21, 4 dari koleksi klinik tanaman ipb dan 17 dari hasil isolasi penulis. semua isolat khamir identifikasi dengan biolog. semua isolat khamir dikaji kemampuan antagonisnya terhadap busuk buah, uji koloni ganda in-vitro dan uji aktivitas kitinase. var. wsw1, k1, dan k12 adalah tiga isolat khamir antagonis paling efektif terhadap dengan tingkat penekanan secara berturut-turut 70.83, 45.83, dan 37.50%. uji koloni ganda in-vitro menunjukkan bahwa var. wsw1, wsw2, k6, ysw1, osw1, k13, dan k2 mempunyai aktivitas antibiosis yang tinggi. keefektifan pengendalian hayati khamir yang diuji terhadap busuk buah mangga tidak berkorelasi dengan aktivitas antibiosis dan kitinase khamir tersebut. kata kunci: , , khamir, mangga, pengendalian hayati mango is one of important tropical fruit commodity for many tropical countries for domestic trade and -also export. one major post harvest disease in indonesia is fruit rot caused by , causing serious damage in storage and shipping. infection rate of disease on mango fruit arumanis was average 54% (yulianingsih . 1990). there are no effective control measures against this disease. existing control measures is the application of fungicide after harvest. the fungicide use has low consumer's acceptance due to environment and health issues. biological control using yeasts is a promising alternative to control post harvest diseases of fruits and botryodiplodia theobromae cv. et al vegetables. the advantages using yeast for biocontrol agent it grows fast, dry tolerance; do not produce mycotoxin and allergenic spores (droby and chalutz 1994). previous research showed that yeasts can be applied to delay fruit ripening process and disease control as well (janisiewicz and korsten 2002). delay of fruit ripening by yeast is through inhibition of ethylene production (droby 1997). some research showed that some yeasts are effective biocontrol agent of post harvest diseases (fan and tian 2001; janisiewicz and korsten 2002) the use of was effective to control anthracnose of chili caused in storage (chanchaichaovivat 2007). in addition, was also effective biocontrol agents against anthracnose, a post harvest disease of mango (kefialew and ayalew 2008). i.e. et al. pichia guilliermondii by colletotrichum gloeosporioides et al. candida membranifaciens *corresponding author, phone/fax: +62-251-8423048, e-mail: suryow@hotmail.com issn 1978-3477, eissn 2087-8575 vol 5, no 4, december 2011, p 154-159 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.4.2 b. theobromae b. theobromae cv. b. theobromae b. theobromae et al. information on the use of yeast to control fruit rot of mango is not available. the objectives of this research were to investigate the potency of yeasts as biocontrol agent of fruit rot of mango caused by and to examine mechanism involved in the biocontrol. twenty one yeasts isolates were used in this study. four yeasts isolates (wsw1, wsw2, osw1, and ysw1) were obtained from plant clinic of institut pertanian bogor. seventeen other yeast isolates were obtained from healthy mango fruit originated from cirebon, west java. yeast isolation from fresh fruit of mango was done by skin washing than serial dilution technique up to 10 000 and continued by plating onto potato dextrose agar (pda) ph 5.5. the isolated yeasts then purified and identified physiologically up to species level by using biolog tm (micro log tm system, release 4.2). the pathogen was isolated from infected mango fruit by tissue plating technique and cultivated on pda ph 5.5 and identified morphologically by based on watanabe (2002). . selection of yeast isolates for biocontrol effectiveness was conducted according to modified technique of kefialew and ayalew (2008). all of yeast were cultured on potato dextrose broth (pdb) ph 5.5 and shaken at 100 rpm for 3 days. mango “gedong gincu” were obtained from farmer's field in cirebon, west java and kept under cool box and stored in refrigerator before handled. mango fruits were disinfected by sodium hypochloride 1% for 1 min and rinsing by sterilized water and air dried. thereafter mango fruit was dipped by yeast suspension at density of 10 cells ml for 1 min, then air dried. skin fruit was taken up by knife up to size of 8 cm x 3 cm, put and placed into moistened 30 cm x 20 cm x 4 cm plastic pans. preliminary research shows that disease severity produced by artificial inoculation of on mango fruit skin highly correlated to whole fruit inoculation. then conidia of 7-day old of grown on pda at the rate of 10 ml as 50 µl was pippeted on the yeast-treated mango skin, then mangos skin was incubated under dark for 24 h then incubated for 5 days. disease severity was assessed by necrotic percentage of skin (kefialew and ayalew 2008). biocontrol efficacy (be) of yeasts was calculated using formulae of chanchaichaovivat (2007) as follow: be = (dc-dt)/dc x 100%; where be, biocontrol efficacy (%); dc, disease severity of control; dt, disease severity of treatment. materials and methods yeast used in this study. biocontrol assay by o testin-viv 6 -1 6 -1 fruits dipping with sterilized water and fungicide with active ingredients thiram (tiflo 80 wp) at the rate of 0.1% (w/v) for 1min were applied for negative and positive control respectively. the experiment was carried out using randomized complete designed with 4 replications. . dual culture assay was performed to determine if biocontrol mechanism is antibiosis (wisniewski 2007). all of yeasts isolates were tested against fruit rot fungi yeast was streaked forming line in the middle of 9-mm petridish containing pda. agar plug ø 3 mm-7day old was seeded on the right and left of yeast streak, therefore line connecting two fungal colony centers was perpendicular to the yeast streak. inhibition zone was assessed after 3 days of incubation and expressed in cm. the size of inhibition zone indicated antibiosis of yeasts against tested fungus (spadaro 2002; indriatmi 2008). all of treatments was arranged in randomized complete design and three times replicated. . all yeast isolates were grown on chitin agar, containing 0.5% colloidal chitin for 3 days (shanmugaiah 2008). chitinolytic activity was indicated by formation of clear zone surrounding yeast colony. identification using biolog resulted that there was variability of yeast originated from fructoplane of mango and plant clinic of ipb collection (from shallot's leaves). four yeast isolates from plant clinic collection were identified as three species var. wsw1, var. wsw2, osw 1. only one isolate of plant clinic was a different species as from mango fructoplane ysw1. moreover, seventeen yeast isolates from mango consisted of 11 species , var. and . bioassay on biocontrol effectiveness showed that some yeasts isolates had ability to suppress fruit rot caused by (table 2). three isolates had disease biocontrol effectiveness rate over 35% which were var wsw1, k1 k 12 with biocontrol effectiveness as 70.83, 45.83 and 37.5%, respectively. such effectiveness rate was comparable, even higher than standard fungicide tiflo 80 wp with active ingredient thiram that had 35% (table 2, fig 1). other important et al. b. theobromae. b. theobromae et al. i.e. cryptococcus albidus aerius c. albidus aerius candida edax i.e. c. terreus i.e. candida edax, candida mexicana, candida terestre, bulleromyces albus c. albidus aerius, cryptococcus amylololentus, cryptococcus l u t e o l u s , d e b a r y o m y c e s h a n s e n i i , p i c h i a guilliermondii, rhodotorula aurentica, r. glutinis b. theobromae c. albidus . aerius p. guilliermondii , d. hansenii dual culture assay chitinolytic activity results volume 5, 2011 microbiol indones 155 finding was even though some isolates belong to the same species they had very different antagonistic activities. for instance, species var. only isolates wsw1 had high antagonistic activity (70.83%), other isolates wsw2, k6, and k 10 which had no antagonistic activity. the yeasts had variability in antibiosis indicating by the size of inhibition zone (table 3). some yeasts had strong antibiosis against such as var. . -dual culture test showed that var. wsw1, c. albidus aerius i.e. in-vitro b. theobromae c. albidus aerius in-vitro c. albidus aerius c. albidus wsw2, osw1, ysw1, k2, k13, k6 had high antibiosis activity. furthermore, other yeast isolates had lower antibiosis activity. interestingly, there was no correlation between antibiosis activity and biocontrol efficacy. among yeasts having high efficacy rate against fruit rot disease, only var wsw1 was effective yeasts and had high antibiosis activity. other effective yeasts k1 and k7 had low antibiosis activity (table 3). ca. edax c. terreus c. luteolus ca. edax c. albidus in vitro c. albidus . aerius p. guilliermondii d. hansenii table 1 identification of yeasts isolates using biolog tm table 2 biocontrol effectiveness of yeasts against fruit rots of mango isolates origins identified species probability (%) wsw1 plant clinic (shallot, brebes) cryptococcus albidus var. aerius 100 wsw2 plant clinic (shallot, brebes) cryptococcus albidus var. aerius 100 osw1 plant clinic (shallot, brebes) candida edax 100 ysw1 plant clinic (shallot, brebes) cryptococcus terreus 84 k1 mango, cirebon pichia guilliermondii 94 k2 mango, cirebon cryptococcus luteolus 91 k3 mango, cirebon debaryomyces hansenii 99 k4 mango, cirebon candida terestre 54.7 k5 mango, cirebon candida edax 94 k6 mango, cirebon cryptococcus albidus var. aerius 99 k7 mango, cirebon bulleromyces albus 99 k8 mango, cirebon rhodotorula glutinis 100 k9 mango, cirebon pichia guilliermondii 99 k10 mango, cirebon cryptococcus albidus var. aerius 99 k11 mango, cirebon rhodotorula aurantica 99 k12 mango, cirebon debaryomyces hansenii 98 k13 mango, cirebon candida edax 99 k14 mango, cirebon pichia guilliermondii 99 k15 mango, cirebon candida mexicana 99 k16 mango, cirebon cryptococcus amylololentus 84 k17 mango, cirebon debaromyces hansenii 99 isolate codes yeast species disease severity (%) biocontrol efficacy (%) wsw1 cryptococcus albidus var. aerius 29.17 ± 5.13 g 70.83 wsw2 cryptococcus albidus var. aerius 87.50 ± 4.54 b 12.50 osw1 candida edax 91.67 ± 4.37b 8.33 ysw1 cryptococcus terreus 75.00 ± 5.81 cd 25.00 k1 pichia guilliermondii 54.17± 4.69 f 45.83 k2 cryptococcus luteolus 91.67 ± 6.53 b 8.33 k3 debaryomyces hansenii 91.67± 6.53 b 8.33 k4 candida terestre 95.83 ± 4.46 ab 4.17 k5 candida edax 100.00 ± 0.00 a 0.00 k6 cryptococcus albidus var. aerius 95.83 ± 4.46 ab 4.17 k7 bulleromyces albus 95.83± 4.46 ab 4.17 k8 rhodotorula glutinis 70.83 ± 6.72 d 29.17 k9 pichia guilliermondii 75.00 ± 4.85 cd 25.00 k10 cryptococcus albidus var. aerius 79.17± 4.48 c 20.83 k11 rhodotorula aurentica 75.00 ± 4.35 cd 25.00 k12 debaryomyces hansenii 62.51 ± 6.53 e 37.50 k13 candida edax 70.83 ± 5.72 d 29.17 k14 pichia guillermondii 91.67 ± 6.53 b 6.33 k15 candida mexicana 87.50 ± 7.44 b 12.5 k16 cryptococcus amylololentus 70.83± 6.53 d 29.17 k17 debaryomyces hansenii 100.00 ± 0.00 a 0.00 untreated (water) 100.00± 0.00 a fungicide a.i. thiram 65.00 ± 7.11 e 35.00 note: numbers followed by same symbols are not significantly different according dmrt test at α=0.05 156 sugiprihatini et al. microbiol indones bulleromyces albus p. guilliermondii among yeasts tested, only k7 and k 14 had chitinolityic activity (table 3). those two yeasts had low biocontrol efectiveness. fructoplane and phylloplane are rich source of microbes, in which yeast is dominant group followed discussion fig 1 effectiveness of antagonistic yeasts against fruit rot disease of mango caused by in bioassay using mango skin. note: a. k1, b. var. wsw1, c. fungicide (thiram), d. untreated botryodiplodia theobromae pichia guilliermondii cryptococcus albidus aerius code species inhibition zone (cm) chitinolytic activity wsw1 cryptococcus albidus var. aerius 2.10 ± 0.23a wsw2 cryptococcus albidus var. aerius 1.97± 0.16ab osw1 candida edax 1.80± 0.12bc ysw1 cryptococcus terreus 1.83 ± 0.12bc k1 pichia guilliermondii 0.37 ± 0.11ij k2 cryptococcus luteolus 1.67 ± 0.12 c k3 debaryomyces hansenii 0.50 ± 0.21i k4 candida terestre 1.20 ± 0.26 de k5 candida edax 0.70 ± 0.33 j k6 cryptococcus albidus var. aerius 1.47 ± 0.18 d k7 bulleromyces albus 0.50± 0.18 i + k8 rhodotorula glutinis 0.47 ± 0.19i k9 pichia guilliermondi 1.00± 0.10 fg k10 cryptococcus albidus var. aerius 1.17± 0.15 ef k11 rhodotorula aurentica 0.50±0.20 i k12 debaryomyces hansenii 0.40± 0.22 i k13 candida edax 1.43 ± 0.21d k14 pichia guilliermondii 1.03± 0.89 fg + k15 candida mexicana 1.07 ± 0.25f k16 cryptococcus amylololentus 0.97±0.14 fg k17 debaryomyces hansenii 0.57 ± 0.14 i table 3 antibiosis of yeasts against and their chitinolytic activitybotryodiplodia theobromae note: + forming clear zone, numbers followed by same symbols are not significantly different according dmrt test at α=0.05 by bacteria and filamentous fungi (elmer and reglinski 2002). the number of isolated yeasts from fructoplane of mango in this study was 11 species , var. and . some of isolated yeasts were potential as biocontrol agent i.e. var. i.e. ca. edax, ca. mexicana, ca. terestre, b. albus c. albidus aerius, c. amylololentus, c. luteolus, d. hansenii, p. guilliermondii, r. aurentica, r. glutinis c. albidus aerius wsw1, p. volume 5, 2011 microbiol indones 157 guilliermondii d. hansenii pichia, p. anomala p. membranifaciens, p. guilliermondii et al debaryomyces et al. c. albidus . aerius cryptococcus cryptococcus c. infirmo-miniatus c. laurentii c. laurentii et al. c. albidus c. albidus aerius candida membranifaciens ca. membranifaciens et al. c. albidus . aerius c. albidus . aerius, k1, and k12 indicated by having biocontrol efficacy rate over 35%. yeasts belong to the genus such as , and have been reported as effective biocontrol agents against various post harvest diseases (elmer and reglinski 2001; fan and shiping 2002; chancaichivat . 2008). moreover sp. is also reported by lesser extent as biocontrol agent of post harvest diseases (mclaughlin 1990). the most effective yeast antagonist obtained in the study is var wsw1. even though there are not many report on biocontrol using , some species of were reported as biocontrol agent, e.g., and against pear rot disease (benbow and sugar 1999), against blue mold of peach (zhang 2007), and against blue and grey mold of apple (fan and tian 2010). it is interesting that one yeast isolate, var. wsw1 was very effective against fruit rot of mango (70.83% of biocontrol efectiveness) (fig 1, table 2), it was even higher than fungicide tiflo 80 wp with active ingredients thiram that provide 35%. as comparison, research of kefialew and ayalew (2008) showed that the use as biocontrol yeasts on mango anthrachnose provided efficacy rate of 82%, on grey mold 61 % 81 % (gholamnejad 2010). this effectiveness of var wsw1 used in the study was very high and promising, because it was achieved under extreme disease conducive environment, in which diseases pressure is very high (conidia density 10 ml ), and incubated in moistened (100 % relative humidity) and darkened environment. in standard storage condition, the effectiveness is expected to be higher. further test on combination of the yeast and real storage condition is necessary other technique to increase efficacy are combining with other species, and or application and formulation optimization (janisiewicz 1996). based on data obtained in this experiment, even isolates wsw1, wsw2, k6, and k10 referred to species var they were very different in their antagonistic activity, only wsw1 had effective biocontrol activity. this shows that biocontrol effectiveness of tested yeasts is isolate-base and not species-base. even though yeast isolates belong to same species, may have different antagonistic activity, therefore biocontrol activity test is necessary for each isolates. mechanism of biocontrol is necessary to be recognized, therefore a biocontrol agent can further 6 -1 developed and optimized. there are various mechanism involve in biocontrol using yeasts competition, antibiosis, lysis and resistance induction (janisiewicz and korsten 2001; de ingeniis 2004; wisniewski 2007). weak correlation of antibiosis activity and biocontrol efectiveness such as depicted in table 2 and table 3, show that antibiosis is not main mechanism underlying biocontrol of yeasts. in this research, antibiosis is valid only for biocontrol mechanism of var. . even though other previous researcher reported that chitinolytic and other lytic enzyme activity involve in mechanism of biocontrol using yeast (spadaro 2002; masih and paul 2002), this study showed no relation between chitinolytic activity and biocontrol effectiveness of yeasts. nutrients competition and induced resistance which involve in biological control control using yeast (janisiewicz and korsten 2001; el-tarabily 2004; yao and tian 2005) were not investigated in this study. further study to examine the role of induced resistance and competition of the three antagonistic yeasts is required. the study yielded three potential antagonistic yeasts effective against fruit rot of mango. some of mechanism of antagonism has also been investigated. thus one of important initial step in developing biocontrol agent has been carried out. further study is needed to develop them as biocontrol agents: other mechanism involved, efficacy with real storage condition, environment and nutritional affecting factors, mass production and formulation technology. the first author would like to indonesian plant quarantine agency for providing opportunity and post graduate scholarship in institut pertanian bogor, for financial and laboratory support for conducting the research et al. in-vitro c. albidus aerius . 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(boca raton us): crc pr. wisniewski m, wilson c, droby s, chalutz e, el ghaouth a, stevens c. 2007. postharvest biocontrol: new concepts and applications. in: vincent c, goettel ms, lazarovits g, editors. biological control: a global perspective. wallingford (uk):cab international. p 262-273. yao hj, tian sp. 2005. effects of a biocontrol agent and methyl jasmonate onpostharvest diseases of peach fruit and the possible mechanisms involved. j app microbiol. 98:941950. doi:10.1111/j.13652672.2004.02531.x yulianingsih, broto w, sabari sd. 1990. inventarisasi penyakit pasca panen mangga cv. arumanis, gedong dan cengkir [inventory of post harvest disease of mango cv. arumanis, gedong dan cengkir]. hortikultura 29(1): 46-49. zhang h, zheng x, yu t. 2007. biological control of postharvest diseases of peach with food control 18: 287-291. doi:10.1016/j.foodcont.2005.10.007. pichia membranefaciens. botrytis cinerea candida guilliermondii candida oleophila . bacillus laterosporous cryptococcus laurentii. volume 5, 2011 microbiol indones 159 5. 545 induction (churiyah).cdr induction of var. hairy roots using atcc 15834 for production of bioactive protein trichosanthes cucumerina anguina agrobacterium rhizogenes churiyah , gustaf adolf wattimena , bambang pontjo priyosoeryanto1* 2 3and 1 th 2 3 center for pharmaceutical and medical technology, agency for the assessment and application of technology (bppt), bppt building ii, 15 floor, jalan mh. thamrin no.8, jakarta 10340, indonesia; department of agriculture, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; department of veterinery, institut pertanian bogor, darmaga campus, bogor 16680, indonesia trichosanthes cucumerina . anguina agrobacterium rhizogenes trichosanthes cucumerina . anguina rolb agrobacterium rhizogenes, trichosanthes cucumerina trichosanthes cucumerina . anguina agrobacterium rhizogenes brine shrimp lethality tes in vitro rol b in vitro agrobacterium rhizogenes trichosanthes cucumerina in the recent years, hairy roots have become a useful model system for the study of bioactive secondary molecule production and protein expression. hairy roots of var were obtained by direct inoculation of the plantlet with atcc 18534 strain agropine. six root clones of var hairy roots th1, th2, th3, th4, th6, and th8 were obtained from the induced hairy roots. the hairy root proteins were extracted with phosphate buffer saline, then fractionated by ammonium sulphate precipitation (80%), dialysis, and gel filtration. the toxicity of the proteins was analyzed using brine shrimp lethality test followed by cytotoxicity test (3(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2h-tetrazolium bromide assay) using hela and k-562 cancer cell line. the th2 root clone showed the highest protein yield (0.83%) and most toxic on bslt with lethal concentration 50 was 5.76 µg ml . pcr analysis indicated the integration of gene to the genome of th2 root clone which was showed by a dna band of 780 bp on the electrophoretic agarose gel. protein fractionation of the th2 root clone resulted in four fractions, one of which theth2-3 protein fraction revealed the highest yield (0.29%) and toxicity on bslt with l up to 0.92 µg ml . the cytotoxicity test of the th2 protein and th2-3 protein fraction indicated that both of proteins inhibited the proliferation of hela and k-562 cell with lc up to 49 µg ml . key words: bioactive protein hairy roots, . kultur akar rambut telah banyak digunakan sebagai model untuk mempelajari produksi berbagai senyawa metabolit sekunder serta ekspresi protein. penelitian yang dilakukan bertujuan untuk mendapatkan kultur akar rambut dari var yang dapat memproduksi protein bioaktif melalui infeksi strain atcc 15834. protein dari kultur akar rambut diekstraksi menggunakan dapar fosfat ph 7, dan difraksinasi dengan menggunakan pengendapan ammonium sulfat (80%), proses dialisis, dan kromatografi filtrasi gel. protein dan fraksinya diuji toksisitasnya secara (bslt), kemudian protein dan fraksi protein yang aktif diuji sitotoksisitasnya secara menggunakan sel kanker hela (human cervic carcinoma) dan sel k-562 (human erythro leukemia) dengan 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2h-tetrazolium bromide. dari hasil penelitian diperoleh 6 klon kultur akar rambut yaitu th1, th2, th3, th4, th6 dan th8 yang tumbuh stabil. diantara 6 klon tersebut, klon th2 menghasilkan rendemen protein paling tinggi sekitar 0.83% dari total akar rambut yang diekstrak dan paling aktif dalam uji bslt dengan ld 5.7 µg ml . hasil analisis secara pcr dari klon th2 menunjukkan terdapatnya pita dna 780 bp pada ekeltroforesis gel agarose, yang membuktikan terintegrasinya gen pada genom akar rambut th2. dari fraksinasi protein klon th-2 secara kromatografi gel diperoleh empat fraksi protein di mana th23 yang paling tinggi rendemennya yaitu 0.29% dari total protein yang difraksinasi, dan paling aktif pada uji bslt dengan ld 0.92 µg ml . uji sitotoksisitas secara dari protein klon th2 dan fraksi protein th2-3 menunjukkan kedua protein tersebut menghambat proliferasi sel hela dan sel k-562 dengan ic paling tinggi, yaitu 49 µg ml . kata kunci: , kultur akar rambut, protein bioaktif, . -1 -1 -1 -1 -1 -1 c50 50 50 50 50 , is a common soil organism capable of entering the plant through a wound which results in the transfer of part of the ri (root inducing) plasmid from bacterial cell to plant genome, causing the proliferation of adventitious root (hairy roots) at the site of infection. these hairy roots can be excised and cultivated indefinitely under sterile conditions (ercan . 1999). the active compound from a plant can be directly extracted from intact plants or produced by culture. the advantages of the culture are that the culture is agrobacterium rhizogenes et al in vitro in vitro sterile and the environmental conditions are controllable. the hairy root culture, resulting from plant transformation using have attracted considerable attention, because they are usually able to produce the same compounds that can be found in the normal roots of the parent plant (park and fucchini 2000). the patterns of alkaloid-like compounds obtained by thin layer chromatography in some hairy roots of several species of mexican cacti were qualitatively similar between the transformed and non-transformed roots (gonzales-diaz . 2006). hairy roots have many potential advantages for bioactive protein production, including a low risk of contamination with potential animal pathogens, having agrobacterium rhizogenes et al *corresponding author, phone/fax: +62-21-3169505, e-mail: churiyah_zafrollah@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 125-131 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.5 the proper protein modification machinery and involving an inexpensive scale-up for commercial production (ko 2006). to elaborate hairy roots, as a distinctive production strategy for the commercial production of valued natural products, will also contribute to the environmental safety and resourcesparing technology for valued natural products for which native plant stocks may be limited (bolivar 2007). var. is a tropical or subtropical plant. its popular name is snake gourd. in indonesia it is called paria ular. the plant is used as a vegetable and traditional medicine, the fruit is considered to be an anthelmintic, an emetic, and a purgative agent. the seed is said to have a cooling effect, and peptides in the plant are commonly used as an abortifacient (gildemacher 1994). previous researchers found several bioactive proteins from this plant. tap 29 (trichosanthes anti-hiv protein, 29 kda), which was isolated and purified from , is capable of inhibiting hiv-1 infection and replication (huang . 1991). karasurin, an abortifacient protein has been isolated from fresh root tubers of maximowicz var. japonicum kitamura (toyokawa 1991). a type-i ribosome-inactivating protein (rip) designated trichoanguin was purified from seeds, which strongly inhibited protein synthesis in a rabbit reticulocyte lysate (chow 1999). a genomic clone encoding trichobakin, a type i ribosome-inactivating protein has been isolated from the plant and was found to inhibit luciferase mrna translation in the rabbit reticulocyte cell-free system (chi . 2001). a putative ribosome inactivating protein (rip) named t 33 was isolated from dried roots of maximovicz (fei . 2004). in order to elucidate other bioactive proteins, and to provide a system for large scale production of the target protein, hairy root induction using was investigated. the research was conducted to verify the possibility of obtaining hairy root lines of using , that are capable of producing stable bioactive protein for medication. . . strain atcc 15834 was grown on yma medium which consist of 10% (w/v) mannitol, 0.5% kh po , 0.2% mgso 7h o, 0.1% nacl, 0.4% yeast extract, solidified with 12% bacto agar. this culture was incubated at room temperature for 3 days. seeds of were obtained from the experimental field of institut pertanian bogor. the seeds were et al. trichosanthes cucumerina anguina et al. trichosanthes kirilowii et al t. kirilowii et al. trichosanthes anguina et al. trichosanthes sp. et al t. kirilowii et al a. rhizogenes t. cucumerina a. rhizogenes a rhizogenes . t. cucumerina materials and methods agrobacterium culture establishment of hairy root cultures 2 4 4 2 surface sterilized in a 30% domestic bleaching agent (1% of sodium hypochlorite) for 2 minutes. seeds were then washed three times with sterile distilled water followed by soaking for 1 min in 70% ethanol. subsequently seeds were transferred to a 20% solution of domestic bleach (1% of sodium hypochlorite) containing 50 µl of tween 20 and soaked for 15-20 min followed by three times washing with sterile distilled water. the seeds were then cultured on basal murashige and skoog (ms) medium containing 0.9% (w/v) agar. the seed culture was incubated for 30 days at 25 °c in a light/dark regime of 16/8 hours under a light intensity of 15 lux until the development of plantlets. hairy root formation was induced by direct inoculation of plantlet with . the adventitious roots arising on the wound site were excised and transferred to ms solid medium containing 250 mg l cefotaxime for eliminating the . . protein was extracted according to the procedure developed by di-toppi . (1996), with some modification. hairy roots were washed with tap water and ground in cold phosphate buffered saline, consisting of 0.14 mm nacl in 5 mm ph 7 na-phosphate buffer, homogenized in the same buffer in the ratio of 1:4 (w/v). each homogenate was stirred overnight at 4 °c, and then filtered. filtrate was then centrifuged at 10 000 g for 25 minutes. the protein was precipitated using 80% saturated ammonium sulfate, and the extracted protein was dialyzed against 5 mm ph 7 na phosphate buffer using cellophane. after the insoluble material had been removed by centrifugation, the dialysates were then lyophilized. . plant genomic dna for polymerase chain reaction (pcr) analyses was isolated according to hamill and lidgett (1991). isolated dna was analyzed by pcr for gene, and dna plasmid from strain atcc 15834 was used as a positive control. the primers designed to a m p l i f y w e r e 1 ( 5 at g g at c c c aaattgctattcc-cccacga-3) and 2 (5-ta ggcttctttcattc-ggtttactgcagc-3). pcr for was carried out by amplification under the following conditions. initial denaturation at 94 °c for 2 min and then denaturation at 94 °c for 1 min, annealing at 45 °c for 1 min and extension at 72 °c for 1 min for 30 cycles, with a final extension at 72 °c for 5 min. the amplicons were analyzed by electrophoresis on 2% agarose gel with 1000 bp molecular markers. . protein fractionation using gel filtration chromatography was done following the procedure of di-toppi . (1996). crude protein was diluted on 5 mm ph 7 na-phosphate t. cucumerina a. rhizogenes agrobacterium et al rolb a. rhizogenes ro l b ro l b rolb rolb et al -1 extraction of proteins pcr analysis of transformation gel filtration chromatography 126 churiyah et al. microbiol indones buffer, and subjected to gel filtration on sephadex g75, equilibrated and eluted in the same buffer. the excluded protein was detected on the basis of their absorbance at 280 nm wavelength, then the same fraction was pooled and lyophilized. a batch of hatching brine shrimps was set up with the temperature between 28 and 30 °c, under conditions of 30-35 ppm salinity, ph range of 8-9, and strong aeration under continuous light condition. after these conditions were fulfilled, about one teaspoon of brine shrimp eggs was added into the system. after approximately 48 hours of hatching, the phototropic nauplii were collected with a pipette from the lighted side and concentrated in a small beaker and used for toxicity testing. different concentrations (0, 10, 100, and 1000 µg ml ) of the protein samples were prepared. these protein samples were placed in separated test tubes, and twenty brine shrimps were transferred to each test tube using micro pipettes. after 24 hours the test tubes containing different concentrations of protein samples were observed, and the number of surviving naupliiin each test tube was counted. the % (percentage) lethality of nauplii was calculated with each concentration represents each sample. the value of lethal concentration (lc ) was calculated using a finny computer program. . hela (human servic carcinoma) cells were maintained as monolayer cultures in rpmi 1640 medium supplemented with 1% antibiotics (50 iu ml penicillin and 50 µg ml streptomycin) and 10% fetal bovine serum in a humidified incubator containing 5% co at 37 °c. subcultures were obtained by trypsin treatment of confluent cultures. the k-562 (human erythro leukemia) cell line was grown in suspension in the same medium. the cells were plated in 100 µl of medium in 96 microwell plates at a density of 10 cells/well for hela and 5 x 10 cells/well for k-562, and the plates were placed in a 37 °c, 5% co incubator. next day the cell culture medium was mixed with 100 µl/well medium containing the indicated concentrations (0,10, 20, 30,40 and 50 µg ml ) of protein in triplicate. after 72 hours of treatment, the cells were harvested. we compared the different concentration of each protein which inhibited the growth of both cell lines, using the mtt [3-(4,5dimethyl-2-thiazolyl)-2, 5-diphenyl-2h-tetrazolium bromide] dye-uptake method. to each cell was added with 10 ul of 0.5mg ml mtt and this maintained at 37 °c, in 5% co incubator for 4 hours to allow mtt to be converted into formazan crystals by reacting it with metabolically active cells. the reaction was stopped by adding acidic isopronanol (34 µl hcl in 10 ml isopropanol), and cell viability was measured at brine shrimp lethality test (bslt). cytotoxicity assay -1 -1 -1 5 4 -1 -1 50 2 2 2 570 nm using a plate reader (wang . 2000). the cells without treatment were used as controls. an infection of explants with atcc 15834 strain agropin resulted in the formation of hairy roots in 70% of the explants. in this research, root tips were individually inoculated in the ms medium containing 250 mg ml of the antibiotic cefotaxime. after several subcultures, the bacteria-free clones obtained were transferred to the medium without antibiotics. the aseptic root clones which showed the most favorable growth characteristics were chosen for further investigation, those were six hairy root clones namely th1, t 2, th3, th4, th6, and t 8. . the result of protein extraction was described by protein yield, that is the ratio between the protein which resulted from the extraction process and the extracted material. the protein yields ranged from 0.5-0.8 % (table 1), with the highest yield was 0.83% resulted from th2 root clone, and the lowest one was 0.51% from th6 root clone. . pcr performed using primer specific for the sequences in the gene resulted in the amplification of a single amplicon with the expected size of 780 bp (fig 1). the amplicons were detected on the lane 2 (dna of ) and lane3 (dna of th2 hairy root clone) but not detected on lane 4 (dna of non-transformed root). this 780 bp amplicon obtained from pcr amplification confirmed the presence of the gene which indicating integration of ri t-dna into the genomic dna of th2 root clone. whereas non-transformed root (normal root) as a negative control did not show any positive signal for the gene . t h e chromatography elution profile showed the fractionation of th2 protein with separated into four peaks (fig 2). all protein fractions was examined for their toxicities through bslt, and the best fraction was selected according to the highest yield and toxicity et al t. cucumerina a. rhizogenes rolb a. rhizogenes rol b rolb results establishment of hairy root cultures. extraction of proteins pcr amplification . g e l f i l t r a t i o n c h r o m a t o g r a p h y -1 h h table 1 protein yields of the six hairy root clones of var. and their activities on brine shrimp lethality test (bslt). t.cucumerina anguina root clone yield (%) lc50 (µg ml -1 ) th1 th2 th3 th4 th6 th8 0.70 0.83 0.65 0.56 0.51 0.74 7.54 5.76 16.48 14.55 10.11 5.63 volume 5, 2011 microbiol indones 127 on bslt. the selected protein fraction was th2-3 protein fraction which resulted in a protein yield of 0.29%, and its lc value was 0.92 µg ml (table 2). the lc of the protein is defined as the concentration which kills, or inactivates by 50% of the tested brine shrimp. lc is inversely proportional to the toxicity of a compound, means that the lower is the lc , the higher is the toxicity. as listed in table 1, each of the samples tested were toxic within 24 hours in different concentrations. the rate of lethality or mortality increased by increasing the concentration of the protein samples. the results indicated that before fractionation, protein 50 50 50 50 -1 brine shrimp lethality test (bst). table 2 protein yields of the protein fraction of th2 root clone and their activities on brine shrimp lethality test (bslt). protein fraction yield (%) lc50 (µg ml -1 ) th2-1 th2-2 th2-3 th2-4 0.056 0.061 0.290 0.061 5.60 5.36 0.92 4.99 780 bp fig 1 pcr amplification of genomic dna from the th2 root clones. (1) molecular marker size, (2) genomic dna of , (3) dna of th2 hairy root clone, (4) dna of the normal root. a. rhizogenes fig 3 comparison of the cytotoxicity of the th2 protein and th2-3 protein fraction on cancer cell line hela and k-562 using 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide (mtt) method. in vitro 128 churiyah et al. microbiol indones protein concentration ( g/ml-1)µ 1 2 3 4 5 c e ll v ia b il it y (% ) untreated cell k562-th2-3 hela-th2 k-562-th2 hela-th2-3 100 90 80 70 60 50 40 30 20 10 0 extracted from th2 root clone was the most toxic protein against the brine shrimp nauplii compared with those extracted proteins from the other root clones. after fractionation the th2-3 protein fraction was more active compared to the three other protein fractions. in the control test, all of the nauplii were alive after 24 hours. so it can be concluded that the toxicity that was found in the experiment might be due to the toxic property of the protein. . the correlation between concentration of proteins and their cytotoxic effect on hela and k-562 cells was investigated by mtt assay. the protein at concentrations ranging from 1 to 5 µg ml was effective in inhibiting the growth of the both cell lines tested for 72 hours. the th2 protein inhibited proliferation of the hela cell in the range of 3.08 26.39 % and the k-562 cell in the range 5.68-31.66 % compared to the untreated cell. while the th2-3 protein fraction showed more active in inhibiting of hela cell proliferation in the range of cytotoxicity on cancer cell line -1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 protein fractions a b s o r b a n s o n 2 8 0 n m fig 2 the chromatography elution profile of t 2 proteins on the chromatography gel filtration. h t 2h -1 t 2h -2 t 2h -3 t 2h -4 8.58 37.69 % and k-562 cell 12.28-61.15% compared to untreated cells. the inhibitory effect of the protein was dose-dependent (fig 3). the protein additions gave different mortality rates at different concentrations, which increased with increasing concentration of protein. we have observed that, among plants that produce bioactive compound, plant produced bioactive protein with highest yield and most active against cancer (churiyah and darusman 2009; churiyah and harran, 2010; churiah and sumaryono 2010). the research also showed that highest yield of proteins was from the seeds, then was from the root, and the lowest was from the fruit of var. (churiah and sumaryono 2010). therefore this plant was chosen as a model for inducing hairy root. the hairy root cultures have excellences including stable in genetic, free of contaminants microbes, the environmental condition unrestrained, easily for extracting the proteins because have no chlorophyll as in the leaves of a plant, and not containing fat as on the seeds of plants. , the causative agent of hairy root syndrome, is a gram-negative soil bacterium, capable of entering plant through a wound and causing the proliferation of secondary roots. when the bacterium infects the plant, the t-dna between the tr (t-dna right) and tl (t-dna left) regions of the ri-plasmid in the bacterium is transferred and integrated into the nuclear genome of the host plant. the transformation process produces a valuable by-product, hairy roots, which will form at or near the site of infection (ercan . 1999). infection resulting in the induction of roots that exhibited typical phenotypic characteristics of hairy roots such as intense hairiness, plagiotrophic growth, higher lateral branching and faster growth than the normal roots. above all, the roots were able to grow well on hormone-free medium indicating the hormone-independent nature of roots that generally results is due to their transformed nature (hamill and lidgett 1997). the symptoms observed with are suggestive of auxin effects resulting from an increase in cellular auxin sensitivity rather than changed auxin production (ercan . 1999). atcc 15834 strain agropin that was used in this research was found to be an effective means of inducing hairy-root formation. previous studies have also revealed that the induction frequency of hairy root formation using this strain was higher than other strains. hairy root formation in some discussion trichosanthes cucumerina trichosanthes cucumerina anguina a. rhizogenes et al a. rhizogenes a. rhizogenes et al a. rhizogenes agrobacterium volume 5, 2011 microbiol indones 129 rubia tinctorum et al a. rhizogenes papaver somniforum agrobacterium rhizogenes in vitro agrobacterium rhizogenes et al a. rhizogenes rol rol aux rol populations have been induced by using many strains of agrobacterium, in which the strain agropin 18534 showed the formation of hairy roots at the high frequency of up to 33-75% compared to the other strain r1000 which induced hairy roots at a lower frequency, about 15-50% (ercan . 1999). the infection frequency of the strain 15834 on the opium popy ( ) resulted in hairy root formation up to 85% (park and fucchini 2000). in our present study, of the six hairyroot clones established, there was a clear-cut difference from one clone to another in terms of protein production (protein yield) and the toxicity on bslt (table 1). previous researchers evaluated the susceptibility of several species of mexican cacti to . stem discs taken from cultured plants were inoculated with a4 agropine-type strain. the frequency of hairy roots formation, the number of roots per explant and its growth rates were variable among the tested species (gonzales-diaz . 2006). these differences may be attributed to secondary variation in the t-dna insertion, the number of t-dna copies that get integrated, and the size and location of integration of tdna of ri -plasmid into the plant genome. since the hairy root clone obtained by each transformed plant cell might result in a different clone, it is expected that each clone behaves differently with respect to growth and protein production. therefore, the selection of a hairy root clones having properties of high biomass accumulation and protein production becomes one of the important and unexploited strategies to obtain a stable and high level of protein production the best clone th2 established in the present study produced protein about 0.85% from fresh weight extracted material, and its lc was 5.76 µg ml on bslt. furthermore, after fractionation of proteins from th-2 clones using gel-filtration chromatography the resultant th2-3 protein fraction gave a high yield and was the most active in bslt test than the other protein fractions. the yield was 0.29% from the weight of protein fractionated and its the lc was 0.92 µg ml . is known to lodge dna plasmids having t-dna region that gets transferred into the higher plant genome. the t-dna has several genes, of which the important ones are the genes ( a, b, c, d), genes involved in auxin metabolism and opine synthesizing genes. since auxin is also synthesized by higher plants, the presence of the other genes such as genes and opine synthesis, the character of hairy roots are generally tested to confirm the integration of t-dna into the host genome. in most . 50 50 -1 -1 of the earlier studies, the transformation of plant tissues by was generally confirmed by using a biochemical method to detect opines in plant tissues. in the present study, the hairy root clones were tested for transformation and integration of t-dna into the genome by means of pcr amplification using gene-specific-primer. thus the presence of a specific band upon pcr amplification clearly established the proof for successful transformation (fig 1). the gene was used to confirm the transformation of chicory hairy root genome (park . 2002), and the -gene also used to prove the successful transformation of hairy root cultures of red beet ( ) (rudrappa . 2005). another research group demonstrated the presence of the genes in the dna of the transformed roots (gonzalesdiaz 2006). to succeed in establishing a hairy root culture system for a certain plant species, several essential conditions should be taken into consideration. these conditions include the bacterial strain of , an appropriate explants, a proper antibiotic to eliminate bacteria, and a suitable culture medium (bi hu and min du 2006). stable transformation and expression of transgene was achieved in witloof chicory ( ) using an mediated system. transformation frequency varies with the use of different types of strains of , concentration of and age of explants. park (2002) found a stable transformation and expression of transgene in pokeweed ( ) hairy root and produce novel ribosomeinactivating protein in it culture. the results of mtt assay indicated that both of the proteins were effectively inhibiting the growth of hela and k-562 cell, although the mechanism of action was not clear yet. previous research reported that the active compound cucurbitacin isolated from acts as an inhibitory component on tyrosinase activity and melanin synthesis of b16/f10 melanoma cells (hyuncheol 2002) and induced apoptosis through caspase-3 and phosphorylation of jnk in hepg-2 hepatocellular carcinoma cells (takashaki . 2009). in conclusion, the present study demonstrates that 15834 strain agropin used with established hairy root cultures that can be useful for stable bioactive protein production. a. rhizogenes rolbrolb rola et al rola beta vulgaris et al rolb et al. a. rhizogenes cichorium intybus a. rhizogenes a. rhizogenes a. rhizogenes et al. phytolaca americana trichosanthes kirilowii et al. et al a.rhizogenes t. cucumerina references bolivar fm, condori j, rimando am, hubstenberger j, shelton k, o'keefe sf, bennett s, dolan mc. 2007. production and secretion of resveratrol in hairy root cultures of peanut. phytochemistry 68:19922003. 130 churiyah et al. microbiol indones chi pv, truong ho, ha nt, won-li c, binh lt. 2001. characterization of trichobakin, a type i ribosome-inactivating protein from sp. biotechnol and app biochem. 34(2): 85-92. chow lp, chou mh, ho cy, chuag cc, pan fm, wu sh, lin jy. 1999. purification, characterization and molecular cloning of trichoanguin, a novel type i ribosome-inactivating protein from the seeds of . biochem j. 338:211-219. churiyah, darusman lk. 2009. bioactive protein from (thunb.) cogn. hayati j biosc. 16(4):161-164. churiyah harran s. 2010. fraksi protein bioaktif dari bagian tanaman (l.) voigh dan 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vol.12, no.2, june 2018, p 43-48 doi: 10.5454/mi.12.2.2 partial purification and characterization of glucose oxidase from aspergillus niger ipbcc.08.610 1 1* 2 aninda indriani , laksmi ambarsari , and dewi seswita zilda 1 department of biochemistry, institut pertanian bogor, jalan raya darmaga, kampus ipb, bogor 16680, west java, indonesia; 2 research center for marine and fisheries product processing and biotechnology, jalan ks tubun, petamburan vi, slipi, 10260, central jakarta, indonesia glucose oxidase is an enzyme which catalyzes β-d-glucose to gluconic acid and hydrogen peroxide. glucose oxidase from aspergillus niger ipbcc.08.610 was isolated, purified, and characterized. the enzyme was purified by ammonium sulphate precipitation and dialysis. the specific activity and yield of dialysis fraction -1 o were 19.766 u mg and 4.744%. the optimum ph and temperature were 6 and 30 c. the stability of the enzyme at optimum ph and temperature decreased 50% at 25 minutes. the k and v values for enzyme were 27 mm m max -1 and 0.986 u mg . key words: aspergillus niger ipbcc.08.610, characterization, glucose oxidase, purification glukosa oksidase adalah enzim yang mengkatalis β-d-glukosa menjadi asam glukonat dan hidrogen peroksida. glukosa oksidase dari aspergillus niger ipbcc.08.610 diisolasi, dimurnikan, dan dikarakterisasi. enzim dimurnikan dengan pengendapan amonium sulfat dan dialisis. fraksi dialisis memiliki aktivitas spesifik -1 o 19,766 u mg dan menghasilkan 4,744%. ph dan suhu optimum adalah 6 dan 30 c. stabilitas pada ph dan suhu -1 optimum menurun 50% pada menit ke-25. nilai k dan v enzim adalah 27 mm dan 0,986 u mg .m maks kata kunci: aspergillus niger ipbcc.08.610, glukosa oksidase, karakterisasi, pemurnian microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-81288080715, email: laksmi@apps.ipb.ac.id is the best source to produce glucose oxidase, because god from a. niger is more stable than god from p. amagasakiense (holland et al. 2012). god of a. niger is an intracellular enzyme and it found in the mycelium of a. niger (singh and verma 2013). the mycelium of a. niger produce glucose oxidase that had a thermal stability in various mediums (bhatti and saleem 2009). the local isolate of a. niger ipbcc with code 08.610 which was isolated from dryobalanops fence litter in tarakan, north kalimantan had several studies related to glucose oxidase production. putri (2011) showed a. niger ipbcc 08610 could produce glucose oxidase as an intracellular enzyme. the optimum incubation time for god production was 48 hours (triana 2013). the purification process by using ammonium sulfate precipitation method produced enzyme with activity -1 12.71 u ml (rohmayanti et al. 2016). currently, the study related to the characterization of glucose oxidase very limited. characterization of god is needed because the enzyme activity was influenced by several factors such as optimum ph, optimum temperature, enzyme kinetics, enzyme stability at ph and optimum temperature. information regarding the enzyme characterization was required by the industry to improve the product quality and process efficiency. therefore, this study aimed to purify and glucose oxidase (ec 1.1.3.4) is an enzyme that catalyzed the oxidation of β-d-glucose using oxygen as an electron acceptor to produce hydrogen peroxide and gluconic acid (singh and verma 2010). glucose oxidase (god) used as a food preservative, component of glucose sensor, and fuel cell applications (bio-fuel) (bankar and bule 2009). biosensors using god could help the diabetic patient to monitor their blood sugar level. glucose oxidase is a standard enzyme for biosensors. it had a relatively high-selectivity of glucose, easy to obtain, cheap, resistant to ph, ion, and temperature changes. these properties allowed unfavorable conditions in manufacturing and storage processes of the enzyme, especially for new users biosensors (yoo and lee 2010). naturally god produce by several fungal species with different characteristics and activities. penicillium amagasakiense (p. amagasakiense), aspergillus niger (a. niger), and penicillium notatum (p. notatum) are fungi to produce god in industrial scale (zia et al. 2007). a. niger is widely used as a major source of glucose oxidase commercially. fiedurek et al (1986) explained that aspergillus niger characterize glucose oxidase. the information regarding purification and characterization of glucose oxidase from a. niger ipbcc 08.610 could be utilized as an important reference source to produce an effective and efficient industrial glucose oxidase. materials and methods production and isolation of god. the media for -1 glucose oxidase production contained 0.4 g l -1 -1 (nh ) hpo , 0.2 g l kh po , 0.2 g l mgso .7h o, 4 2 4 2 4 4 2 -1 40 g l caco 3.3% sucrose, and 0.35% glucose. the 3, medium was adjusted to ph 5.5 with the addition of 1 m h po . the medium incubated at 200 rpm at 30 °c for 3 4 48 hours. after incubation, mycelium was separated from the production media by gauze. the filtered biomass was homogenized by using quartz sand and 0.1 m sodium phosphate buffer ph 6.0 and then it was centrifuged at 12000 rpm for 15 min. the supernatant was a crude extract of glucose oxidase. the protein concentration and enzyme activity of crude extracts were analyzed. afterward, the crude extract was purified. determination of protein concentration and enzyme activity. for determination of protein concentration, 20 μl samples were added by 1000 μl of bradford reagents, incubated for 10 min, and measured the absorbance at 595 nm. the standard curve was made by using bovine serum albumin (bsa) solution with -1 concentration around 0.1-0.5 mg ml . the linear regression equation of standard curve was used to determine the protein concentration the sample. glucose oxidase activity was the amount of glucose oxidase used to catalyze 1 μmol β-d-glucose to h o 2 2 per minute. to determine the enzyme activity, 170 μl 10% glucose (w/v), 800 μl odianicidine (6.6 mg odianicidine (100 ml buffer potassium phosphate 0.1 m -1 ph 7), 30 μl horseradish peroxidase (hrp) 60 u ml , and 30 μl god were mixed. the oxidation reaction of o-dianicidine was analyzed by measuring the absorbance at 436 nm. purification of glucose oxidase. crude extract was mixed and homogenized with 80% saturated ammonium sulfate for 1.5 hours at 4 °c, and centrifuged at 12000 rpm for 15 minutes. the precipitate fraction was separated and dissolved in phosphate buffer 0.1 m ph 6, and purified by using dialysis process. dialysis process used molecular weight cut off 12 kda. the fraction was put into the dialysis tube and dialyzed for 9 hours in 0.001 m phosphate buffer ph 6 solution. the buffer solution was replaced every 3 hours. the dialysis process was stopped when the enzyme fraction did not produce a white precipitate if bacl was added into the buffer 2 solution. d e t e r m i n a t i o n o f o p t i m u m p h a n d temperature. phosphate citrate buffer with concentration 0.1m ph 4 to 7 with interval 0.5 were used to determine the optimum ph. the buffer from assay determination was replaced by the test buffer, and the absorbance was measured at 436 nm. for optimum temperature determination, god assay reagents were incubated for 10 min at some temperature conditions (25, 30, 35, and 40 °c) and the absorbance was measured at room temperature at 436 nm. enzyme stability at optimum ph and temperature. the thermal stability of god was determined by incubating the reagent enzyme assay at optimum temperature and ph for 10, 20, 40, 50, and 60 minutes. the enzyme activity of the treatments was compared with pre-incubation activity. the stability of the enzyme was expressed as a percentage of residual activity and it was calculated by using the following equation: residual activity(%) = x 100% kinetics of glucose oxidase. determination of enzyme kinetics by measuring the enzyme activity at several substrate concentrations. the substrate solution was glucose with concentration range 10-100 mm. the result obtained was applied into the michaelis-menten curve. through the michaelismenten curve, a range of substrate concentrations which showed a significant increase in enzyme activity was selected to create lineweaver-burk plot and determine k and v .m max results production and isolation of god. glucose oxidase was produced by growing the mycelium of a. niger. the mycelium had white color and round shape. quartz sand was added to the mycelium to take glucose oxidase from the cell. after mycelium was homogenized by using quartz sand, the sample was centrifuged. pellets and supernatant were separated after the centrifugation process. the supernatant was a crude extract of glucose oxidase. purification of glucose oxidase. the purification results showed an increase in the purity of glucose 44 indriani et al. microbiol indones enzyme activity after incubation enzyme activity before incubation oxidase. the specific activity of crude extract was -1 0.248 u mg . the results showed that the activity of glucose oxidase from ammonium sulfate precipitation -1 method was 1.168 u mg , and it was increasing 4,709 times if it compared to the activity of crude extract. the protein concentration of ammonium sulfate fraction was 6.84 mg, and it was decreasing if it compared to the protein content of crude extract. the ammonium sulfate fraction was purified by dialysis process. the dialysis process showed a significant increase in the purity of the sample was 79.701 times bigger than the initial (table 1). optimum ph and temperature. the ph and the optimum temperature were determined at the highest activity. the activity of glucose oxidase was increasing -1 at ph 4 to ph 6. the highest activity was 1.438 u ml at ph 6 of (fig 1). at ph 6.5 and 7, the activity was decreasing by 72% of the highest activity. at temperature 30 °c, the activity was increasing, and it was starting to decrease as the temperature was higher than 30 °c. the activity at temperature 30 °c was increasing 282% than the activity at 25 °c, start from -1 -1 1.062 u ml and increased into 2.992 u ml . at temperatures of 35 °c and 40 °c, the activities were -1 -1 decreasing, respectively 1.477 u ml and 1.168 u ml (fig 1). stability at optimum ph and temperature. figure 2 shows the residual activity at optimum ph and temperature. the residual activity was decreasing at treatment time was increasing. in 25 minutes of treatment time, the residual activity was decreasing 50% of the initial. in 60 minutes of treatment time, residual activity was decreasing 37.5% of the initial. kinetic glucose oxidase. the michelis-mentens curve showed the rate increase until 0.03 m glucose concentration. the michelis-mentens curve plot in the range 0.01 to 0.03 m of substrate concentration was transformed into the linewave-burk curve. through the lineweaver-burk curve, k and v values were m max -1 obtained, respectively 27 mm and 0.986 u mg (fig 3). discussion production and isolation of glucose oxidase. aspergillus niger was a fungus with the genus aspergillus, and the spores were black or dark brown (gautam et al. 2011). the production of glucose oxidase from aspergillus niger ipbcc 08.610 used a liquid medium contain (nh ) hpo , kh po , 4 2 4 2 4 mgso .7h o, caco , sucrose, and glucose. 4 2 3 (nh ) hpo served as a source of nitrogen and medium 4 2 4 of ph control (khursid et al. 2011). the concentration of mgso .7h o used to be very low, because excess 4 2 2+ mg ions would decrease god production (hamid et al. 2003). sucrose and glucose were used as carbon sources that could affect the formation of biomass mycelium and cell metabolites. the concentration of sucrose was higher than glucose, because sucrose was a substrate that could produce god more optimal. the excessive glucose concentration could affect the ph culture, it could decrease the mass of mycelium, and the concentration of god produced (bankar and bule 2009). caco was added to induce god formation due 3 to a shift of metabolism from glycolysis to the phosphate pentose pathway. caco could inhibit the 3 synthesis of 6-phosphofructokinase and the concentration of glucose-6-phosphate dehydrogenase was increasing, so the production of god was also increasing (moorthi 2009). environmental ph might affect to the nutrient solubility, enzyme synthesis rate, and reduction oxidation reactions (bankar and bule. 2009). the optimum ph for the production of god was 5.5 (singh and verma 2010). the types of isolates, medium of nutrients, and physiological conditions, the table 1 purification table from intracellular glucose oxidase from a. niger ipbcc 08.610 volume 12, 2018 microbiol indones 45 fraction activity (u ml -1) [protein] (mg ml -1) volume (ml) total activity (u) total protein (mg) specific activity (u mg -1) yield (%) increasing ratio, compared to crude extract sample crude extract 0.617 2.445 50 30.838 122.25 0.248 100 1 ammonium sulfate 1.998 1.71 4 7.992 6.84 1.168 25.916 4.709 dialysis 2.151 0.109 0.68 1.463 0.074 19.766 4.744 79.701 incubation time was also affecting the production of glucose oxidase (sabir et al. 2007). according to triana (2013), the god activity of a. niger ipbcc 08.610 was reaching maximum after 48 hours of incubation. it was indicated by the god activity 48 hours was higher than 72 hours of incubation. the enzyme activity decreased due to the accumulation of the hydrolysis product (purkan et al. 2014). glucose oxidase was produced from aspergillus niger metabolites (haq et al. 2013). aspergillus niger ipbcc 08.610 produced glucose oxidase as an intracellular enzyme (putri 2012). it was indicated by the enzyme activity of intracellular enzymes was higher than the extracellular enzymes. glucose oxidase as an intracellular enzyme was presented in the mycelium (bhat et al. 2013). glucose oxidase was obtained by the breakdown of the a. niger mycelium and the cell membranes, so that enzymes could be extracted and obtained as the crude extracts. crude extract of glucose oxidase was purified by ammonium sulfate precipitation. purification of glucose oxidase. crude extract god was purified by using two steps of purification processes, they were ammonium sulfate precipitation and dialysis process. the crude extract still contained carbohydrates, lipid and protein. so, it needed a 46 indriani et al. microbiol indones fig 1 determination of (a) optimum ph and (b) optimum temperature. a b fig 2 stability at optimum ph and temperature. fig 3 the lineweaver-burk curve. 3.5 4 4.5 5 5.5 6 6.5 7 7.5 purification process to get the enzyme with appropriate activity (wahidah et al. 2017). the crude extract was purified by ammonium sulfate precipitation method. ammonium sulfate precipitation was the purification method based on the interactions between water, proteins, and salts. at certain saturation levels, the water molecules bonded to the salt, so the proteins would be settled down (abelson et al. 2014). the saturation level of ammonium sulfate for glucose oxidase purification was optimized first. ammonium sulfate with 80% of saturation level produced the highest glucose oxidase activity (triana 2013). the ammonium sulfate with 80% of saturation level could be interpreted that the protein was more hydrophilic (setiawan et al. 2013). the fraction of the ammonium sulfate was purified by dialysis process. dialysis process was performed to remove the ammonium sulfate in extract, so the fraction only containing proteins (nelson and cox 2013). the membrane size used was 12 kda, so the molecules with size less than 12 kda could be removed and the desired protein would be remained inside the membrane (sattayasai 2012). optimum ph and temperature. temperature and ph could affect the enzyme activity. ph and temperature could damage the structure of the enzyme. if the protein structure was changed, then the active side of the enzyme would be disturbed. the changed active side could decrease the enzyme ability to bind with the substrate (voet and voet 2010). ph and temperature that produced the maximum enzyme activity were called the optimum ph and temperature. enzyme activity at the optimum ph and temperature reaches the maximum value because amino acids on the active side are at the appropriate conformation to bind to the substrate. so it could bind optimally with the substrate. the activity at ph lower and higher than the optimum condition were decreasing due to ph could affect the ionic bonds and hydrogen bonds on the catalytic side (reece et al. 2010). stability at optimum ph and temperature. the god from a. niger ipbcc 08.610 had optimum o activity at ph 6 and 30 c. the information of stability at optimum ph and temperature were used to determine the decrease of activity before the enzyme was becoming inactive. the glucose oxidase was not o active effectively at 30 c without stabilization. the immobilization process could stabilize god from a. niger (simpson et al. 2007). the most of protein molecule were denatured at high temperature and long exposure of heating time. the denaturation of enzymes accompanied by disruption hydrophobic interactions. that was effect by the dissociation subunit, so the enzyme activity was decreasing (singh and verma 2013). kinetic constants. the rate of hydrolysis or the decomposition reaction was called velocity (v). the speed of the reaction was increasing with the increasing of substrate concentration. the speed in linear correlation was called the maximum speed (v ). the km value indicated that a half of the enzyme max active side was binding to the substrate (nelson and cox 2013). the v and k values could be affected by enzyme max m amount, purity level, and environmental conditions, such as ph and temperature. the low v value was max obtained due to enzyme concentration was too low, and the formation of the enzyme substrate complex was limited, so the reaction rate was decreasing (iswantini et al. 2009). the low k value indicated that the glucose m oxidase had a high affinity to the substrate, so the bond of the enzyme and substrate was getting stronger and causing the equilibrium of the reaction toward the formation of enzyme substrate complex (campbell et al. 2004). to conclude, glucose oxidase was isolated from mycelium a. niger ipbcc 08.610 as intracellular enzyme. god was purified by ammonium sulfate precipitation method and dialysis process with enzyme -1 activity and yield respectively 19.77 u mg and 4.74%. o the optimum ph and temperature were 6 and 30 c. at the optimum ph and temperature showed the stability of enzyme was decreasing 50% at 25 minutes. the k m and v values for the enzyme were 27 mm and 0.986 max -1 u mg . acknowledgements this research was funded by “program penelitian unggulan perguruan tinggi (pupt)” with contract number 1396/1t3.11/pn/2017 and 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saeed mk, andaleeb f, rajoka mi, sheikh ma et al. 2007. thermal characterization of purified glucose oxidase from a newly isolated aspergillus niger uaf-1. j clin biochem nutrit. 41(2):132-138. 48 indriani et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 03. diah (administration).cdr administration of microencapsulated probiotic at different doses to control streptococcosis in tilapia (oreochromis niloticus) 1 2 2 diah ayu satyari utami , widanarni *, and m. agus suprayudi 1 study program of aquaculture, graduate school; 2 department of aquaculture , faculty of fisheries and marine science faculty institut pertanian bogor, dramaga campus, bogor 16680, indonesia this study aimed to produce microencapsulated probiotic and determine its optimal dose to control r streptococcosis in tilapia (oreochromis niloticus). the probiotic used in this study was bacillus sp. np5 rf that was encapsulated by sterile 10% maltodextrin solution and dried by spray dryer. the experimental fish were reared 28 d and fed by the administration of microencapsulated probiotic in feed with different doses (0.5% (a), 10 -1 1% (b), and 2% (c)) which were 10 cfu g as the concentration and control without administration of microencapsulated probiotic, including negative (k-) and positive (k+) control. on day 30, all of the fish except -1 5 -1 kwere challenged by injecting 0.1 ml fish of streptococcus agalactiae (10 cfu ml ) by intra-peritoneal (ip) route. this study showed that administration of 0.5% microencapsulated probiotic was effective to control streptococcosis in tilapia with higher post-challenge survival rate, better hematological parameter values, and could inhibit s. agalactiae growth in the host target organs. key words: probiotic, microencapsulated, streptococcosis, tilapia penelitian ini bertujuan untuk menghasilkan mikrokapsul probiotik dan menentukan dosis terbaik untuk pengendalian streptococcosis pada ikan nila (oreochromis niloticus). probiotik yang digunakan pada penelitian r ini adalah bacillus sp. np5 rf yang disalut dengan larutan maltodekstrin 10% steril dan dikeringkan dengan spray dryer. ikan uji dipelihara selama 28 hari dan diberi pakan dengan pemberian mikrokapsul probiotik pada 10 -1 pakan dengan dosis yang berbeda 0,5% (a), 1% (b), dan 2% (c) dengan kepadatan 10 cfu g dan kontrol tanpa pemberian mikrokapsul probiotik meliputi kontrol negatif (k-) dan positif (k+). pada hari ke-30, semua ikan -1 5 -1 kecuali kdiuji tantang dengan streptococcus agalactiae melalui injeksi 0.1 ml ekor (10 cfu ml ) secara intra-peritoneal (ip). penelitian ini menunjukkan bahwa pemberian 0.5% mikrokapsul probiotik efektif untuk mengendalikan streptococcosis pada ikan nila dengan tingkat kelangsungan hidup yang lebih tinggi setelah uji tantang, nilai parameter gambaran darah yang lebih baik, dan dapat menghambat pertumbuhan s. agalactiae pada organ target inang. kata kunci : ikan nila, mikrokapsul, probiotik, streptococcosis *corresponding author; phone: +62-8129357404; e-mail: widanarni@yahoo.com tilapia (oreochromis niloticus) is a commodity that is widely cultivated in the world. developing countries, especially asia such as china, indonesia, philippines, and thailand is a major producer of tilapia (josupeit 2005). in 2012, the production of tilapia by the aquaculture sector in asia increased to 3.3 million tonnes (fao 2014). intensive culture is conducted to fulfill high demand of tilapia. otherwise, intensive culture has some risks include disease emergencies. one of the diseases that attack of tilapia is streptococcosis caused by streptococcus agalactiae. in experimental infection, s. agalactiae could cause mortality up to 90% at 6 days after injection (evans et al. 2006b). some antibiotics such as erythromycin, doxycycline, kitasamycin, and lincomycin are effective to control streptococcosis. currently, fish disease control through the use of antibiotics has been prohibited, because it requires a long time for antibiotic residues withdrawal from fish body (iregui et al. 2014). the presence of antibiotics in water and sediment can affect normal flora, plankton, and animals, which can induce changes in microbiota diversity and ecological balance (cabello 2006). the use of antibiotics can initiate bacteria resistance. some strains of streptococcus have been known able to develop resistance properties to an antibiotic (darwish and hobbs 2005). furthermore, its resistance properties will lead to the ineffectiveness of antibiotics to control the disease, although it is given in high concentration (yanong 2013). probiotics are live or dead microbes or microbial components that give some benefits to the host (lazado and caipang 2014). based on study by tanbiyaskur vol.9, no.1, march 2015, p 17-24 doi: 10.5454/mi.9.1.3 (2011), oral application of 1% fresh culture bacillus sp. np5 in tilapia that challenged by s. agalactiae had higher survival rate (80.56%) than positive control (13.89%). bacillus sp. np5 is a probiotic that was isolated from tilapia's digestive tract (putra 2010). the main aspects that must be considered in probiotic preparation are the viability of probiotic during preparation and storage, whereas those are the weakness of the use of fresh culture probiotic (wang et al. 2008). therefore, it needs a method that can protect a n d m a i n t a i n c e l l v i a b i l i t y i n a l o n g t i m e . microencapsulation has been applied in many food products to extend the storage time of probiotic and its cell viability in the digestive tract of the host through the protection of probiotic cells in microcapsule beads (krasaekoopt et al. 2003). based on the previous study, administration of microencapsulated bacillus showed better growth and survival rate than control in white shrimp larvae (nimrat et al. 2011). one of the factors that affect probiotic performance is dose (nayak 2010). in addition, application of microencapsulated probiotic in aquaculture has not been studied widely, especially for streptococcosis control in tilapia. the fresh cultre dose applicated for tilapia still using 1%, so the aims of this study were to produce microencapsulated probiotic and determine the best dose to control streptococcosis in tilapia. materials and methods preparation of microencapsulated probiotic. probiotic used in this study was bacillus sp. np5. the probiotic was given antibiotic rifampicin resistant r marker (bacillus sp. np5 rf ). probiotic cells were cultured in lbb (luria bertani broth) and incubated in water bath shaker (140 rpm; 29 °c) for 18 h. fresh culture was harvested by centrifugation (6000 7000 rpm) for 20 min to get probiotic biomass and encapsulated by sterile 10% maltodextrin solution. the mixture was homogenized, and then dried in buchi mini spray dryer at inlet temperature of 120 °c and an outlet temperature of 70 °c (utami et al. 2015). the viability of product after drying was monitored to ensure probiotic count within the product. viability test was carried out by the spread plate technique using lba (luria bertani agar) that was supplemented with -1 50 mg ml rifampicin (lba + rf). preparation of pathogen for the challenge test. pathogen used for the challenge test was s. agalactiae nk1 that was obtained from instalation of research and fish diseases control development (irfdcd) depok, west java, indonesia. this pathogen is nonhemolytic bacterium that has characteristics include thin growth on solid medium, white to yellowish, slimy liquid, and hard to be taken (hardi 2011). bacteria stock was cultured in bhia (brain heart infusion agar). bacteria cells were cultured in 5 ml bhib (brain heart infusion broth), then incubated in waterbath shaker at 29 °c with a speed of 140 rpm for 24 h, so that would be obtained s. agalactiae inoculant 7 -1 with concentration of 10 cfu ml . serial dilution 5 was conducted to get the inoculant concentration of 10 -1 cfu ml which is the ld50 of s. agalactiae nk1 infection in tilapia (aryanto 2011) and would be used in the challenge test. experimental design. the fish strain used in this study was tilapia nirwana strain that was obtained from c e n t e r o f f i s h b r e e d i n g r e s e a r c h ( c f b r ) sukamandi, west java, indonesia. the 6.38±0.05 g fish 3 were reared in aquarium sized 60x30x30 cm at a density of 10 fish per aquarium. this study was conducted in crd (completely randomized design) consist of 5 treatments with 5 replications, with administration of microencapsulated probiotic in diet with different doses (0.5% (a), 1% 10 -1 (b), and 2% (c)) which were 10 cfu g as the concentration and control without administration of microencapsulated probiotic (utami et al. 2015), including negative (k-) and positive (k+) control. the test feed was commercial pellet (hi-provite 781-1) with 30.18% protein, 5.25% fat, 52.53% carbohydrate, 9.12% ash, and 2.92% crude fiber contents. the microencapsulated probiotic added to the diet and mixed with 2% egg white as a binder. the fish were fed three times a day (08:00, 12:00, 16:00) by at satiation for 28 d. on day 30, 8 fish from each treatment except kchallenged by injecting 0.1 ml per fish of s. 5 -1 agalactiae (10 cfu ml ) using a sterile syringe by intra-peritoneal (ip) route. furthermore, fish were reared for 10 d. replacement of water in the rearing tanks as much as 80% of total volume were conducted every 4 d to maintain water quality, whereas the water quality was maintained in the normal range for freshwater culture, according to boyd (1990) that -1 dissolved oxygen > 5 mg l , temperature at 24-30 °c, ph of 6.5 9.5, and total ammonia nitrogen (tan) < 0.52 ppm. survival rate observation. survival rate (sr) was calculated after the challenge test with the following equation: sr (%) = [(nt/n0) x 100%] where: nt is the number of fish that live at the end of 17 widanarni microbiol indones the study (individual), n0 is the number of fish at the beginning of the study (individual). measurement of hematological parameters. blood samplings were done on day 0, 4, 8, and 10 of the challenge test. the fish were anaesthetized using ms222 and then 0.2 ml blood of the fish was drawn using a sterile syringe which previously had been rinsed by anticoagulant (na citrate). blood samples were used for measurement of hematocrit (ht), hemoglobin (hb), erythrocyte count (ec), leukocyte count (lc), and phagocytic activity (pa). hematocrit was measured by taking blood sample using microhematocrit tube and centrifuged at the speed of 5000 rpm for 5 min. hematocrit was determined by comparing the length of blood cells and the length of total blood volume in microhematocrit tube. hemoglobin was measured by sahli method using sahlinometer (wedemeyer and yasutake 1977), ec and lc following the procedure by blaxhall and daisley (1973). phagocytic activity was assessed by making blood smear slide from the blood sample that was mixed with the staphylococcus aureus suspension 7 -1 (10 cfu ml ) and incubated for 20 min. the slide was dried, fixed with methanol for 5 min and dried again, then stained by immersion in giemsa for 20 min. the slide was observed using a microscope with 400x magnification to determine pa based on percentage of 100 phagocyte cells which showed phagocytosis. enumeration of total s. agalactiae in target organs. the enumeration was carried out by the spread plate technique using bhia with target organ samples of streptococcosis (liver, kidneys, eyes, and brain). colonies of s. agalactiae were distinguished with other bacteria based on the characteristics which are spherical, small with a thin growth on solid medium, white to yellowish and eradiate bluish color when exposed to light. enumerations of total s. agalactiae in target organs were done on day 0, 7, and 10 of the challenge test to know the infection peak and recovery period in week 1 and week 2. statistical analysis. all data were tabulated using microsoft excel 2007 and analyzed using one wayanova, then continued by duncan multiple range test with a 0.05 significance using spss 20. results the highest survival rate (sr) after the challenge test with s. agalactiae was shown by treatment a (75±12.5%) that was not significantly different (p>0.05) with k(87.5±12.5%), but was significantly different (p<0.05) with k+, b, and c (fig 1). a showed the highest hb, ht, and ec before the challenge test (fig 2a, b, c.), where hb was (7.20±0.20 g%) followed by b (6.47±0.31 g%), k(6.00±0.80 g%), c (5.80±0.20 g%), and k+ (5.47±0.31 g%). a also showed the highest ht (22.90±0.10%) that was volume 9, 2015 microbiol indones 19 fig 1 survival rate of tilapia after the challenge test. different letters on each bar (mean±sd) indicated significant differences (duncan; p<0.05). negative (k-); positive (k+) control; administration of microencapsulated probiotic in feed at a dose of 0.5% (a); 1% (b); 2% (c). significantly different (p<0.05) with k-, k+, and c, but was not significantly different (p>0.05) with b. this was also reflected in ec, where a showed the highest 6 of ec before the challenge test (2.75±0.08x10 cells -3 mm ) that was significantly different (p<0.05) with k-, k+, and c, but was not significantly different (p>0.05) with b. a also showed the highest of hb after challenge test, whereas hb of a after challenge test on day 4, 8, and 10 were, 5.33±0.42; 5.40±0.20; 6.60±0.20 g%, respectively. ht levels also declined and the peak occurred on day 8, where ht was in the range of 10.83±1.47-18.13±1.90%. a also showed the highest of ht on post-challenge test that was not significantly different (p>0.05) with k-, but significantly different (p<0.05) with k+. this is also reflected in ec, where ec declined on day 4 of the challenge test, then increased again on day 10, where the highest ec of the fish that were challenged by s. agalactiae was shown 6 by a was, 1.26±0.10; 1.41±0.08; 1.55±0.04x10 cells -3 mm , respectively. otherwise, the administration of microencapsulated probiotic was not significantly different (p>0.05) in tilapia's lc and pa before the challenge test (fig 2d, e.). a showed the increasing of lc and the 5 -3 peak occurred on day 8 (0.95±0.06x10 cells mm ) that was significantly different (p<0.05) with k+, b, and c, and then decreased on day 10. pa increased on day 8 and decreased again on day 10. a showed the highest of pa after challenge test was, 74.74±3.18; 67.14±6.23; 65.56±5.09%, respectively. the lowest total s. agalactiae in almost target organs after the challenge test was shown by a (fig 3a, b, c, d.). on day 7 and 10, the lowest total s. agalactiae in the liver was shown by a (5.80±0.02; -1 4.90±0.03 log cfu g ) was significantly different (p<0.05) with k+, b, and c. the lowest total s. agalactiae in the kidneys on day 7 was shown by a -1 (5.16±0.01 log cfu g ). the lowest total of s. agalactiae in the eyes showed in a (5.83±0.02; 20 widanarni microbiol indones -1 4.79±0.02 log cfu g ). the highest total of s. agalactiae in the brain on day 7 was shown by k+ -1 (6.46±0.01 log cfu g ) was significantly different (p<0.05) with a, b, and c, while the lowest total of s. agalactiae in the brain on day 7 was shown by a -1 (5.85±0.02 log cfu g ). total s. agalactiae in target organs almost decreased on day 10. discussion administration of probiotic is able to increase the host resistance against pathogen as reported by brunt and austin (2005) showed that a. sobria gc2 could i m p r o v e t h e r e s i s t a n c e o f r a i n b o w t r o u t t o (d) (e) fig 2 hemoglobin (a); hematocrit (b); erythrocyte count (c); leukocyte count (d); phagocytic activity (e) of tilapia during the challenge test. different letters on each bar on the same day (mean±sd) indicated significant differences (duncan; p<0.05). negative (k-); positive (k+) control; administration of microencapsulated probiotic in feed at a dose of 0.5% (a); 1% (b); 2% (c). lactococcosis and streptococcosis compared to controls. it was similar to the results of this study, in which administration 0.5% of microencapsulated r bacillus sp. np5 rf improved tilapia resistance against s. agalactiae. it happened because one of probiotic modes action is to inhibit pathogen infection by enhancing the host immune response (denev et al. 2009) through non-specific and cellular immune stimulation (fyzul et al. 2014). furthermore, it also happened because the probiotic and its components or products interact with gut associated lymphoid tissue (g a lt) to induce the host immune response (dimitroglou et al. 2011). h o w e v e r, b e t t e r r e s u l t s d i d n o t o c c u r i n administration of the higher doses of microencapsulated probiotic. dose is one of the limiting factors to achieve the optimum effect of probiotics on the host immune system, whereas the dose of 6 10 probiotics used in aquaculture vary from 10 -10 cfu -1 g (nayak 2010). nikoskelainen et al. (2001) stated that low doses of probiotic may fail to stimulate the fish immune system, but high doses of probiotic can cause interference in the host, in which it has been reported 12 that administration of l. rhamnosus in high dose (10 -1 cfu g feed) in o. mykiss showed higher mortality 9 -1 than lower dose (10 cfu g feed). in this study, administration of microencapsulated probiotic affected hb, ht, and ec before the challenge test. there is a link between hb, ht, and ec, where the higher ec, will show the higher hb and ht. blaxhall and daisley (1973) stated that ec, hb, and ht are the parameters that indicate anemia in fish, where ec in this study was in the normal range, which 5 -3 is 10-30x10 cells mm (takashima and hibiya 1995). the higher ht in the administration of microencaps u l a t e d p r o b i o t i c t r e a t m e n t s i n d i c a t e d t h a t microencapsulated probiotic was safe and could improve fish health status as reported by aly et al. (2008), where the administration of bacillus subtilis and lactobacillus acidophilus could improve ht in tilapia. a positive result from the administration of probiotic on hb, ht, and ec also occurred in catla catla (hamilton) which were supplemented with lactobacillus acidophilus in diet, and it was related to probiotic ability to improve hematological parameter values as a result of haemopoetic stimulation (renuka et al. 2014). fluctuation of hb, ht, and ec occurred after the challenge test, where hb decreased on day 4 and 8 of the challenge test, then increased again on day 10. the decreasing of hb, ht, and ec indicated that fish were volume 9, 2015 microbiol indones 21 anemic. symptoms of anemia in fish can be assessed by packed cell volume (pcv) value or ht, where ht is less than 20% is a sign of anemia in teleostei (clauss et al. 2008). s. agalactiae is a gram-positive bacteria that cause septicemia, where the suffering organs due to s. agalactiae infection are brain, kidneys, and eyes (abdullah et al. 2013). anterior portion of the kidney is the main organ of blood production in teleostei (arnold 2009). problems in the kidneys will cause blood production disorders which lead to decrease of erythrocyte production, then it will cause fluctuations of hb and ht. lc and pa values were not significantly different between treatments before the challenge test indicated that fish were not stress and exposed to infection. the differences between lc and pa occurred after the challenge test. leukocytes play a major role in nonspecific immunity during inflammation and the amount can be an indicator of fish health status (secombes 1996). lc improvement after infection associated with inflammatory response mediated by leukocytes against bacterial infections (roberts 1978). based on the results, it could be concluded that the administration of microencapsulated probiotic help to recover the hematological parameter changes due to infection, improved nonspecific immune response and resistance to disease, especially streptococcosis. the higher lc in the administration of microencapsulated probiotic treatments showed that leukocytes were produced in great numbers against s. agalactiae infection. leukocytes are blood cells that play major role in phagocytosis process. its role is played by monocytes and neutrophils that exhibited by pa. monocytes and neutrophils are the components that produce superoxide anion (o ), hydrogen peroxide 2 (h o ), nitric oxide (no), peroxynitrite (onoo ), 2 2 hypochlorous acid (hocl) and hydroxyl radical (oh ) that have a very high microbiocidal ability (ellis 2001; secombes 1996). the ability of probiotic to inhibit s. agalactiae growth in tilapia could be determined by total of s. agalactiae in target organs (liver, kidneys, eyes, and brain). on day 0 (before the challenge test), colonies of s. agalactiae were not found in target organs, as well as the negative control after the challenge test. it indicated that the fish were free from s. agalactiae infection. the highest total of s. agalactiae in all target organs on day 7 after the challenge test associated with fish mortality, where in this study mortality begun to occur on day 3 and reached the peak on day 8. it was related to yanong and floyd (2002) statement that streptococcus infection in fish can cause high mortality (> 50%) on day 3 to day 7 post-infection. the presence of s. agalactiae in the eyes caused some clinical signs such as purulens, opacity, and exopthalmia, while the presence of s. agalactiae in the brain caused abnormal swimming like gasping, tilted swimming even whirling (hardi et al. 2011). these changes can lead to death, especially changes that show damage in the structure and function of organs which play a role in physiological processes in fish body, such as the liver, kidneys, and brain, while the symptoms that exhibited in the eyes are symptoms that occur in chronic phase (evans et al. 2006a). furthermore, the mortality was found in the fish that had shown abnormal swimming and drastic reduction in hematological parameters, while the experimental fish that showed symptoms in the eyes still looked active. total of s. agalactiae in target organs were lower in the administration of microencapsulated probiotic treatments than k+ and total of s. agalactiae in target organs decreased on day 10 associated with phagocytic activity and incubation period of s. 22 widanarni microbiol indones fig 3 total s. agalactiae in the liver (a); kidneys (b); eyes (c); brain (d) of tilapia during the challenge test. different letters on each bar on the same day (mean±sd) indicated significant differences (duncan; p<0.05). negative (k-); positive (k+) control; administration of microencapsulated probiotic in feed at a dose of 0.5% (a); 1% (b); 2% (c). agalactiae in fish body, which is that time the body did homeostasis activity as indicated by hb, ht, and ec improvement, lc and pa reduction. at that moment, the infection rate also reduced. in conclusion, administration of 0.5% microencapsulated probiotic was effective to control streptococcosis in tilapia after the challenge test with higher survival rate, better hematological parameter values, and could inhibit s. agalactiae growth in the host target organs. acknowledgement thanks to directorate general of higher education (dghe) that has provided master program scholarship for the first author by program beasiswa unggulan in 2012. references abdullah s, omar n, yusoff sm, obukwho eb, nwunuji tp, hanan l, samad j. 2013. clinicopathological features and immunohistochemical detection of antigens in acute experimental streptococcus agalactiae infection in red tilapia (oreochromis spp.). springerplus. 7p [on line]. doi:10.1186/2193-1801-2286. aly sm, ahmed yag, ghareeb aaa, mohamed mf. 2008. studies on bacillus subtilis and lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of tilapia nilotica (oreochromis niloticus) to challenge infections. fish shellfish immunol. 25: 128-136. doi:10.1016/j.fsi.2008.03.013. arnold je. 2009. hematology of fish: wbc and rbc cell morphology. proceeding of the acvp/asvcp concurrent annual meetings; 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7.3 (light) and 7.0 mg l (shadowy). oxygen supports were significantly different at the light and shadowy conditions, whether from diffusion process 71.71 and 79.37%, respectively, or from periphyton photosynthesis, 21.73 and 15.30%, respectively. the compositions of periphytons living in the two conditions were similar; mostly composed by group of diatom, with the same dominant species of sp. the difference in periphyton growth was shown by its density. in the light condition, the diatom tends to grow at higher density in comparison to the shadowy condition. the role of periphyton to support oxygen in upstream waters is light dependent. the higher the light intensity (photosynthetic active radiation ranged), the higher the support of oxygen will be. key words: diffusion, oxygen support, periphyton, upstream waters sebagian besar kandungan oksigen terlarut di sungai berasal dari proses fotosintesis dan difusi. proses fotosintesis di perairan mengalir dilakukan oleh organisme autotrof, khususnya komunitas mikroalga perifitik yang hidup menempel pada batuan atau substrat lainnya, dan proses difusi intensif yang berlangsung bersamaan dengan aliran air. sungai bagian hulu dicirikan dengan dasar berbatu dan berarus deras. penelitian ini dilakukan untuk mengkaji peran oksigen hasil fotosintesis oleh perifiton dan hasil proses difusi di sungai. pengamatan eksperimental lapangan dilakukan di hulu sungai cisadane (600 m di atas permukaan laut) dengan dua kondisi cahaya alami berbeda, yaitu terpapar cahaya atau terang (24 630-104 240 lux) dan terlindung atau teduh (11 120-65 300 lux). kedua kondisi cahaya tersebut (terang dan teduh), secara berturut-turut, memiliki kandungan oksigen terlarut aktual yang relatif sama, yaitu 7.3 dan 7.0 mg l . sokongan oksigen dari proses difusi pada kondisi terang (71.71%) dan teduh (79.37%), secara signifikan berbeda dari hasil fotosintesis perifiton pada kedua kondisi, terang (21.73%) dan teduh (15.30%). komunitas perifiton pada kedua kondisi cahaya didominasi oleh kelompok yang sama, yaitu diatom dengan spesies dominan sp. perbedaan pertumbuhan perifiton ditunjukkan oleh kepadatannya. kondisi yang terang mendukung pertumbuhan diatom untuk mencapai kepadatan yang lebih tinggi dari kondisi teduh. peran perifiton dalam menyokong oksigen di hulu sungai bergantung kepada cahaya. semakin tinggi intensitas cahaya (dalam kisaran radiasi untuk aktivitas fotosintesis), maka akan semakin tinggi sokongan oksigen yang dihasilkan. kata kunci: difusi, hulu sungai, perifiton, sokongan oksigen -1 -1 oxygen is an essential element for living, both in terrestrial and aquatic ecosystems. dissolved oxygen in aquatic ecosystem is very important to support ecological processes within, such as decomposition and respiration. the variation of oxygen content in water depends on temperature, salinity, water turbulence, and atmospheric pressure. the higher the water temperature and altitude, the lower the atmospheric pressure and dissolved oxygen in the water will be. the dissolved oxygen content in water also depends on mixing, turbulence; photosynthesis, respiration, and decomposition process; and pollution inflow. the source of dissolved oxygen in running water (lotic) water is influenced by atmospheric diffusion and autotrophic photosynthesis. the atmospheric oxygen diffusion is directed by agitation or turbulence of water mass caused by current or water fall. the liquid film transfer coefficient for oxygen expressed per unit volume of water is termed as the reaeration coefficient, k (d-1). the magnitude of k is importantly influenced by internal turbulence, which acts to reduce the thickness of the diffusional layer. surface turbulence of most streams is primarily by flow-induced turbulence (gelda . 1996). in fact, the process ofet al *corresponding author, phone: +62-8129053354, fax: +62-251-862 2932, e-mail: niken_tmpratiwi@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 4, december 2011, p 182-186 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.4.5 plastic rope was used as artificial substrate in the main research. the main research was conducted to determine the oxygen support from photosynthesis and diffusion process into running water ecosystem. modified winkler method was used to ascertain oxygen content as the result of net primary production (npp) through photosynthesis. furthermore, the values of actual oxygen, saturated oxygen, and oxygen reaeration coefficient were used to determine the value of oxygen from diffusion process (o'connor and dobbins 1958). photosynthetic oxygen content or npp was produced by periphyton grown on selected artificial substrate. npp was measured as photosynthetic oxygen in four hours period of incubation. selected substrates were placed in light and dark incubation bottles that were tied to holders (fig 1). the constructions were located at light site (station 1) and shadowy site (station 2) for observation. photosynthesis is not only performed by periphyton, but also phytoplankton. unfiltered water samples were placed in the dark and light incubation bottles to determine the photosynthetic oxygen of the phytoplankton. the content of dissolved oxygen from atmospheric diffusion (d) was calculated as the result of multiplication of re-aeration coefficient (k) and the difference between values of saturated and actual oxygen (o'connor and dobbins 1958). the value of k is influenced by velocity and depth of water. d = k (saturation do -actual do) (owens . 1964) where: u = velocity in average (m s ); h = depth in average (m). re-aeration rate of the actual temperature is formulated by churchill . (1962) as: k (t°c) = k (20°c) x 1.0238 et al et al -1 (t-20) 2 2 volume 5, 2011 microbiol indones 183 oxygen diffusion from the atmosphere is time consuming, even in a high turbulence condition. as a consequence, the autotrophic photosynthesis plays a very important role as oxygen source in running water system, which is controlled by nutrients, temperature, light, and flow (uehlinger . 2000). therefore, periphyton and phytoplankton community could play roles as autotroph in supporting oxygen of lotic water through photosynthesis process. this project is a preliminary research to compare oxygen from photosynthesis by autotrophic organism (periphyton and phytoplankton) and atmospheric diffusion, in running water system. the periphyton community was grown on artificial substrates exposed to direct solar radiation (light condition) and indirect solar radiation (shadowy condition). the field observation was carried out in cisadane river, 6°32'59"s and 106°41'23"e, ciampea udik, tenjolaya, west bogor district, west java. the research was prepared as a field experimental observation in simple random design with solar radiation exposure as treatment factor. this research consists of two parts, pre-observation and main research. there were several supporting data, comprising of aquatic physical, chemical, and biological measurement. the aim of pre-observation was to determine the appropriate artificial substrate in 'nature-like' growth composition of periphyton. the materials used as artificial substrates were pieces of pvc, plastic rope, glass, and glass fiber, each sized 5x1 cm . substrates were exposed to experimental conditions for fourteen days to grow the community of periphyton. the artificial substrate with sufficient density of periphyton will be used in main research. illumination condition was considered as major factor in selection of observation site. bright illumination sites were located under 24 630-114 500 lux of light intensity, and dark illumination sites were located under 11 120-98 360 lux. the other factors are depth and current condition which should be assumed as homogeneous. based on current condition, the observation sites were categorized as middle velocity current (janauer . 2010). pre-observation showed that plastic rope was colonised by periphyton in similar composition to the natural condition. the community of periphyton on the plastic rope was dominated by diatoms comprising of and with total density of 2160 ind cm at the light site, and 1560 ind cm at the shadowy site. meanwhile, the natural periphyton consists of and . by this means, et al et al thallasiothrix, melosira, stauroneis, navicula thallasiothrix, melosira, stauroneis, navicula 2 -2 -2 fig 1 design of incubation bottles in holder construction. 184 pratiwi et al. microbiol indones t h e a p p r o a c h o f r e a e r a t i o n c o e ff i c i e n t determination refers to approximate delta method. the “approximate delta method” is a simple procedure for simultaneous calculation of the stream re-aeration coefficient, primary production rate, and respiration rate from a single-station stream diurnal profile of dissolved oxygen (do) (mcbride and chapra 2005). for stream where temperature variability is small, the delta method of chapra and di toro (1991) is useful for scoping do reaction-rate parameters. this method enables a simple graphic fit to reaeration, production, and respiration rates, and is thus widely used for preliminary interpretation of diel do studies. this simplicity, however, comes at a price, as the method assumes that reaeration, respiration, and saturation do concentration are all constant, while production can be described as a simple sinusoidal function for daylight hours (butcher and covington 1995). the observation of periphyton community was performed on scraped sample from 5x1 cm of substrate. the sample was preserved by lugol's iodine solution (takano . 2004), and observed to determine the composition using identification guidelines for freshwater microalgae. the densities were determined using standard equation for periphyton analysis. the measurement of light intensity, water temperature, current velocity, turbidity, and nutrients (nitrogen from nh , no , and no , phospate from po ) were held along with the observation of oxygen condition. besides the nutrients, the other parameters were measured in situ. analysis of variance was used to analyze the difference of oxygen support at two sampling sites with different light conditions, and to demonstrate that there is no difference in oxygen support at two sampling sites with different current velocity. the difference between periphyton densities and proportion of oxygen support in both sites were 2 et al 3 2 3 4 analyzed with t-test. the rich-riffle upstream water of cisadane river is characterized by relatively steep sloping riverbank, small to big stones of bottom, and high speed of water current. the ranges of temperature are 23.2027.2 °c at the light sites (station 1) and 22.40-24.5 °c at the shadowy sites (station 2). the light intensity for both light and shadowy conditions ranged around 24 630-104 240 and 11 120-65 300 lux. ciampea river has a high variation of current speed. it is slow at the break of day, which ranged between 0.5-1 ms , and higher at noon, of which the condition depends on elevation, depth, and bottom width. high current flow creates a disturbance for periphyton community. increasing levels of disturbance would systematically remove these rare taxa. the result showed that periphyton community of the two light conditions was mostly composed of several types of diatoms, which are sp., sp., sp., and sp. (fig 2). phytoplankton community consists of sp., sp., and sp. (fig 3). the densities of algae at station 1 were relatively higher than those at station 2, especially for periphyton community (p(t<=t)<0.05) (fig 2 and fig 3). it indicates that light intensity has important role in supporting the growth of periphyton. algal photosynthesis is conducted at specific range of light intensity (photosynthetic active radiation par). the range is species-specific. each kind of algae will produce organic matter and oxygen as the result of photosynthesis. the product of the photo-synthesis in an algal community will increase with the increase of light intensity. the net primary oxygen production of periphyton was on average 2760.06±1223.75 mg o m d in 430 l at station 1 and 2322.70±1133.35 mg o m d in 430 l at station 2. the other autotrophic community that contributes in oxygen production aside from periphyton is phytoplankton. however, the -1 -2 -1 -2 -1 thallasiothrix melosira stauroneis navicula navicula nitzschia diatoma 2 2 fig 2 composition of periphyton on artificial substrate at two sampling sites. thalasiothrix melosira stauroneis navicula station 1 station 2 1200 1000 800 600 400 200 0 volume 5, 2011 microbiol indones 185 contributions by phytoplankton were relatively low, 833.40±321.19 mg o d in 430 l at station 1 and 809.22±340.33 mg o d in 430 l at station 2. rich riffle river water create water mass turbulence that generate oxygen content through diffusion process. the oxygen support by diffusion process was calculated using the d value, which was assessed purely by physical process. in this observation, oxygen support from diffusion process was 9106.75±1665.18 mg o d in 430 l for station 1 and 12 050.46±1905.01 mg o d in 430 l for station 2. based on those results, it is shown that oxygen support at both light and shadowy conditions from periphyton photosynthesis were 21.73 and 15.30%, and from diffusion process was 71.71 and 79.37%, respectively. these values indicated that diffusion gave higher proportion of oxygen support than photosynthesis (p(t<=t)<0.05) in lotic system at the bright illumination site, and (fig 4). autotrophic photosynthesis process of aquatic ecosystem is highly influenced by light intensity in water column. on the other hand, light condition in shallow-river is relatively similar at the surface and at 2 2 2 2 -1 -1 -1 -1 vice versa the bottom. these conditions are suitable for algae as the major component of micro-algae community. in a shallow stream, periphyton plays more important roles than plankton in utilization of nutrients and in biomass and oxygen production (flipo . 2004; lutscher . 2007). gjerløv and richardson's (2010) study on the light reduction experiment resulted in a significant reduction of oxygen production when streams were shaded in contrast to their full exposure to sunlight. the biofilm (periphyton) biomass production was also lower under shaded conditions. as a consequence, photosynthesis will proceed as effectively at the bottom of the river, performed by periphytic algae, especially diatoms. the 14 days exposure of artificial substrates shows a relatively great amount of oxygen for a short diatom natural growth period. it is supposed that in natural substrates, the photosynthesis process by the attached algae could supply higher amount of oxygen to the water column of shallow-river. in certain conditions, physical processes give low oxygen input to the river, as a consequence, oxygen source is mostly emanating from biological process (nakova . 2009). et al et al et al fig 4 proportion of oxygen support by photosynthetic and diffusion process at: a) station 1 and b) station 2, at lotic system. 6.56% fig 3 composition of phytoplankton on artificial substrate at two sampling sites. diffusion process 71.71% phytoplankton periphyton 21.73% diffusion process 79.37% phytoplankton 5.33% periphyton 15.30% a) b) navicula nitzschia diatoma station 2station 1 20 18 16 14 12 10 8 6 4 2 0 186 pratiwi et al. microbiol indones on the other hand, the gradient of fast-flowing rivers running from the mountainous areas to lowland rivers has a large influence towards the diatom assemblages. the influence is rather complex because variables such as slope, elevation, concentration of nutrients, land-use, and temperature are correlated to one another and these environmental factors determine the diatoms' distribution and composition patterns (potapova and charles 2002; mendes . 2009). furthermore, the decrease in the number and varieties of species along the disturbance may have been due to the loss of rare taxa or loss of habitat. physical and chemical modifications in the habitat of benthic diatoms produced changes in the assemblage. benthic diatoms' density decreased immediately after the dredging and other physical disturbances (luttenton and baisden 2006; licursi and gómez 2009). the interference of structuring diatom assemblages will reduce the photosynthesis intensity, and reduce the oxygen production. aeration process into water body is one a characteristic of gas concentration fluctuation in water mass. the process occurred when the kinetic energy of turbulence is strong enough to ward off surface tension and gravitation. the water capacity to absorb gas depends on temperature and atmospheric tension. at certain time, water will reach at a saturated condition. saturated condition of dissolved oxygen is reached when the concentration is balance with the atmospheric oxygen concentration, that the diffusion process will no longer take place. the result showed that both photosynthesis and diffusion processes took place at both sampling sites. the proportions of oxygen between both processes were significant statistically (p<0.05). it showed that intensive diffusion process supplied higher dissolved oxygen than the photosynthesis process. the support of oxygen from photosynthesis process tends to increase by the increase of light intensity. it is shown by the relatively high correlation coefficient between oxygen production and light intensity (0.9439) and between periphyton density and light intensity (0.9739). in certain conditions, oxygen from diffusion is relatively low although the water turbulence is intensive. on the other hand, it supposed that photosynthesis by attached algae in natural substrates could support higher amount of oxygen to the water column of shallow-river. authors thank to laboratory of aquatic productivity and environment, department of aquatic , et al acknowledgements resources management, faculty of fisheries and marine sciences, institut pertanian bogor in facilitating this research. references butcher jb, covington s. 1995. dissolved-oxygen analysis with temperature dependence. j environ eng. 121(10):756-759. churchill ma, elmore hl, buckingham ra. 1962. the prediction of stream reaeration rates. asce j sanit eng div. 88(sa4):1-46. chapra sc, di toro dm. 1991. delta method for estimating primary production, respiration, and aeration in 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doi:10.1016/j.ecoinf.2009. 09.015. owens m, edwards rw, gibbs jw. 1964. some reaeration studies in streams. int j air water pollut. 8:469-486. o'connor dj, dobbins we. 1958. mechanisms of reaeration in natural streams. transactions of asce. 123:641-684. potapova mg, charles df. 2002. benthic diatoms in usa rivers: distributions along spatial and environmental gradients. j biogeogr. 29:167-187. takano k, igarashi s, hino s. 2004. seasonal changes in silicon content of diatoms estimated from the ratio of particulate silicon to diatom volume under silicon sufficiency in diatom-rich lake barato. jap j limnol. 5(2):115-120. doi:10.1007/s10201-004-01176. uehlinger u, konig c, reichert p. 2000. variability of photosynthesisirradiance curves and ecosystem respiration in a small river. freshw biol. 44:493-507. mi651-17-07-12 (chaerun) available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.2.2issn 1978-3477, eissn 2087-8575 vol 6, no 2, june 2012, p 57-68 *corresponding author; phone: +62-87878590709; e-mail: skchaerun@gmail.com. in the present study, ten mercury-resistant heterotrophic bacterial strains were isolated from mercurycontaminated gold mine sites in bandung, west java province, indonesia. the bacteria (designated strains skcsh1skcsh10) were capable of growing well at ~200 ppm of hgcl except for strain skcsh8, which was 2 able to grow at 550 ppm hgcl . the bacteria were mesophilic and grew optimally at 1% nacl at neutral ph with the 2 o optimal growth temperature of 25-37 c. phenotypic characterization and phylogenetic analysis based on the 16s rrna gene sequence indicated that the isolates were closely related to the family xanthomonadaceae, aeromonadaceae, and pseudomonadaceae and they were identified as pseudomonas spp., stenotrophomonas sp., and aeromonas sp. eight bacterial strains were shown to belong to the pseudomonas branch, one strain to the stenotrophomonas branch and one strain to the aeromonas branch of the γ-proteobacteria. phylogeny based on their 16s rrna gene sequences indicated that four of the isolates (skcsh1, skcsh4, skcsh7, skcsh9) could be classified as representatives of four novel species in the genus pseudomonas that were allocated to p. moraviensis (96.96% similarity) and p. plecogossicida (94.53, 96.61, and 96.73% similarity). four other isolates could be allocated to p. plecogossicida (97.57 and 98.66% similarity) and p. hibiscicola (99.97% similarity), one isolate to stenotrophomonas africana (99.69% similarity), and one other isolate to aeromonas hydrophila subsp. ranae (99.43% similarity). the findings of this study provide the first information of the phylogenetically-diverse hg-resistant bacteria in the hg-polluted sites of indonesia that may be highly useful for developing in situ bioremediation or detoxification of hg-contaminated sites in indonesia. key words: 16s rrna gene sequences, in situ bioremediation, mercury (hg), phenotype, phylogenetic analysis tujuan penelitian ini adalah untuk mempelajari secara filogenetika bakteri-bakteri heterotrof yang resisten terhadap merkuri (hg) yang diisolasi dari daerah pertambangan emas yang tercemar hg di bandung, jawa barat, indonesia. ada 10 isolat bakteri resisten hg yang telah diisolasi dari daerah pertambangan tersebut. kesepuluh -1 strain adalah mesofilik dan mampu tumbuh pada lb medium dengan konsentrasi hgcl sampai sekitar 200 mg l 2 -1 (kecuali strain skcsh8 mampu tumbuh sampai konsentrasi hgcl sebesar 550 mg l ) dengan kondisi 2 o pertumbuhan optimum: konsentrasi nacl 1%, ph netral, dan pada kisaran suhu 25-37 c. karakterisasi secara fenotip dan analisis sekuen gen 16s rrna menunjukkan bahwa delapan strain bakteri termasuk dalam genus pseudomonas, satu strain dalam genus stenotrophomonas, dan satu strain lagi dalam genus aeromonas dari kelas γproteobacteria. analisis filogenetika dari sekuen gen 16s rrna mengindikasikan bahwa ke-empat isolat (skcsh1, skcsh4, skcsh7, skcsh9) mempunyai peluang sebagai spesies baru dari genus pseudomonas yaitu pseudomonas moraviensis (dengan similaritas 96,96%) dan pseudomonas plecogossicida (dengan kemiripan 94%, 53%, 96,6%, dan 96,73%). empat isolat yang lain mempunyai kesamaan gen 16s rrna sebesar 97,57% dan 98,66% terhadap pseudomonas plecogossicida dan sebesar 99,97% terhadap pseudomonas hibiscicola, serta dua isolat lagi mempunyai kesamaan gen 16s rrna sebesar 99,69% terhadap stenotrophomonas africana dan sebesar 99,43% terhadap aeromonas hydrophila subsp. ranae. hasil penelitian ini memberikan informasi pertama tentang keanekaragaman secara filogenetika bakteri resisten hg pada lahan yang tercemar hg di indonesia, yang mungkin dapat bermanfaat dalam mengembangkan bioremediasi secara in situ lahan-lahan yang tercemar hg di indonesia. kata kunci: analisis filogenetika, bioremediasi in situ, fenotip, merkuri (hg), sekuen gen 16s rrna mercury (hg) pollution is a significant problem in traditional mining, in particular that located in indonesia, with respect to abandoned hg mines and residual hg from gold mining operations that largely mercury (hg)-resistant bacteria in hg-polluted gold mine sites of bandung, west java province, indonesia 1,2 1 3 siti khodijah chaerun *, sakinah hasni , edy sanwani , 4 and maelita ramdani moeis 1 laboratory of biogeosciences, mining and environmental bioengineering, research division of genetics and molecular biotechnology, school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia; 2 center for life sciences, institut teknologi bandung, indonesia 3 department of metallurgical engineering, faculty of mining and petroleum engineering, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia; 4 research division of genetics and molecular biotechnology, school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia (barkay et al. 2003; chiu et al. 2007). they have an essential role in mercury bio-geochemistry and recycling. the mechanism of their mercury resistance is mediated through a cytoplasmic mercuric reductase 2+ 0 by which soluble hg is converted into insoluble hg , followed by volatilization of the relatively non-toxic 0 hg . the mercuric reductase is encoded by the mera gene (nascimento and chartone-souza 2003; fantozzi et al. 2009). essa et al. (2002) reported that several different mechanisms can make microbes able to survive in the presence of high concentrations of hg. by having evolved resistance mechanisms to detoxify several chemical forms of mercury, resistant microbes may play an important role in mercury biogeochemistry in mercury-contaminated environments (dzairi et al. 2004; gustin and stamenkovic 2005; ni chadhain et al. 2006). thus, it is expected that exploration of mercury-resistant microbes may potentially be beneficial for detoxifying mercurycontaminated sites. hence, the aim of this work was to study the phylogenetic diversity of indigenous hg-resistant bacterial strains at a traditional gold mine of indonesia combined with phenotypic analysis (the so-called polyphasic characterization). it is expected that the molecular ecological approaches to studying the 16s rrna gene would exhibit the presence of phylogenetically novel bacterial sequences at these sites as well as novel bacterial isolates resistant to hg. such information may be highly useful for developing in situ bioremediation or detoxification of hgcontaminated sites in indonesia. materials and methods s t u d y s i t e s , s a m p l e c o l l e c t i o n , a n d physicochemical analysis. the study site was located at a traditional gold mining in highly hg-contaminated environment and overburdened site in bunikasih, pangalengan, west java province, indonesia. river sediment samples were collected from 20 random positions of the bunikasih river, pangalengan (coded as sediment i-iv) and one soil sample from tailings (coded as tailing soil) in november 2009. for river sediment samples, the site was divided into four plots. at each plot one composite mixed sample was obtained by pooling 4-6 sediment samples from random locations throughout the plot. all samples were transferred to the laboratory and stored at -20 until required for analysis. sample ph was measured by potentiometer in suspension (1:1 w/v) according to o c contribute a significant loading of hg to the watersheds each year, e.g. traditional gold mining in bunikasih, pangalengan, west java province, indonesia. correspondingly, mercury is known as one of the most toxic heavy metals and its toxicity is due to its ability to bind readily to thiol group of protein, thus inactivating vital cell functions (wagner-dobler 2003). most of the hg in the atmosphere is in the form of elemental hg 0 (hg ), which is volatile and is oxidized to the mercuric 2+ ion (hg ) as a result of its interaction with ozone in the presence of water (chiu et al. 2007; zhu et al. 2008). 2+ the main form of hg in the aquatic environment is hg (green-ruiz 2006; ariya et al. 2009; mason 2009). 2+ 0 when both inorganic mercury forms (hg and hg ) are present in aquatic systems, they are converted into highly toxic organic mercury (methylmercury (mehg)) that is subsequently bioaccumulated through all levels of the food chain (mason 2009). thus, the bioaccumulation and biomagnification of mercury in food chains pose a risk to consumers at the upper trophic levels (garcia-sanchez et al. 2006; ni chadhain et al. 2006; yang et al. 2009). importantly, mercury contamination caused by the amalgamation of gold in small-scale gold mine regions is also a current environmental problem worldwide, particularly in the mining areas of developing countries. the mining operations generally involve the extensive use of mercury to recover gold through the amalgamation process (ambers and hygelund 2001; lecce et al. 2007; tian et al. 2009). the open-pan amalgamation, when pasty amalgam is heated to vaporize the mercury and separate the gold which is left behind, causes leaving residual mercury to be left in the gold mining sites. up to the present, mercury contamination of stream sediments caused by gold mining operations have been reported on a worldwide basis (ambers and hygelund 2001; miller et al. 2003; lecce et al. 2007; tian et al. 2009). although mercury has been known as an environmental pollutant for many decades, the release of mercury to the environment from anthropogenic sources is of growing concern (us epa, 2003). in north sulawesi province of indonesia, in 1998 approximately 22000 small-scale gold miners were active and produced an estimated 10 tonnes of gold bullion. the mercury concentration which was employed in this operation reached 1 kg out of 30 kg of ore (ayhuan et al. 2003). from a microbiological standpoint, mercuryresistant bacteria, which include aerobes or facultative anaerobes, are frequently isolated from a variety of environmental niches such as water, soil and sediment 58 chaerun et al. microbiol indones din iso 10390 (din iso 1997), and total mercury content was analyzed according to din iso 38414-s (din iso 1983). isolation and cultivation of mercury (hg)resistant bacteria. bacterial strains were isolated from both samples (sediment and tailing soil) on luria bertani (lb) broth supplemented with three 2+ -1 concentrations of hg as hgcl (25, 50, 100 mg l ) at 2 -1 ph 7 and supplemented with antifungal (30 mg l nystatin) to prevent fungal growth. the flasks were o incubated at room temperature (25 c) for 4 d with agitation of 120 rpm, and samples were subsequently transferred onto lb agar plates supplemented with 25 -1 mg l of hgcl using a 4-way streak method. ten 2 morphologically different colonies were randomly chosen from each type of hg-amended agar plates and subcultured on hg-supplemented lb agar six times for 3-4 d to purify the isolates. eventually, the pure cultures were subcultured onto hg-amended lb agar plate and hg-amended lb broth for stock cultures. minimum inhibitory concentration (mic) of 2+ hg . the assay aimed for bacterial growth at the 2+ highest hg concentration in the form of hgcl . 2 briefly, bacterial cultures in lb broth were transferred to a fresh medium and supplemented with hgcl at 2 -1 concentrations ranging from 25 to 600 mg l . the cultures were then incubated at room temperature and their optical density (od) was measured at the time of inoculation and afterward up to 4 d with spectrophotometer at λ at 600 nm. minimal inhibitory 2 + concentration (mic) was the minimum hg concentration that inhibited growth, i.e., no increase in λ at 600 nm in 4 d. phenotypic characterization. phenotypic characterization was conducted on the analysis of bacterial cell morphology, biochemical tests, tests of the ability to grow on the variations of temperature, salinity, and ph (cappuccino and sherman 2005). all strains were tested at 25 °c for the following key characteristics. analysis of bacterial cell morphology used gram stain, endospore staining and capsule staining, where bacteria were viewed routinely by phase contrast microscopy at ×1000 magnification. biochemical tests included utilization of catalase, oxidase and citrate, indole production, h s production, 2 mrvp test (methyl red-voges proskaur), urease test, nitrate reduction and hydrolysis of gelatin, casein, lipid, starch and tween 80. in addition, tests of the bacterial ability to use different sugars as a carbon source were performed. these included glucose, galactose, fructose, mannosa, arabinose, sucrose, maltose, and lactose. all strains were tested for their ability to grow on lb agar at various temperatures (4, 25, 37, and 50 ) and on lb broth at different ph values (3, 5, 8, and 10) adjusted by adding 1n hcl or 1n naoh. tolerance to salinity was determined in nutrient agar (na) supplemented with 0.5, 3, and 5% nacl. phylogenetic characterization of hg-resistant b a c t e r i a l s t r a i n s . c h r o m o s o m a l d n a f o r phylogenetic characterization was extracted and tm purified by using the ultraclean microbial dna isolation kit (mo bio laboratories, inc.) according to the manufacturer's instructions. the 16s rrna gene was amplified by pcr with primers 27f (5'agagtttgatcctggctcag-3') and 1492r (5'ggttaccttgttacgactt-3') as described previously (chaerun et al. 2004). the 16s rrna gene was amplified from genomic dna by pcr (2720 thermal cycler applied biosystems) in a final volume of 25 µl, consisting of 2.5 µl of 10 x kapa taq buffer, 5 µl of 1 mm deoxynucleoside triphosphate (dntps), 1 µl of 10 µm each of primers 27f and 1492r, 0.1 µl of kapa taq dna polymerase, 1 µl of the template dna, and 14.4 µl of deionized h o. amplifications 2 were carried out at 95 °c for 3 min, 30 cycles at 95 °c for 1 min, 56 °c for 45 s, 72 °c for 1 min 30 s, and the final extension for 4 min at 72 °c. pcr products were checked by 1% agarose gel electrophoresis and stained with ethidium bromide (etbr). the amplified products were purified and sequenced by the direct sequencing st method at the 1 base bioengineering technology service co., ltd. (singapore). the sequences of the 16s rrna genes from each isolate were used as query to determine the genus and species of its closest prokaryotic relative using blastn (altschul et al. 1990). sequences were aligned using clustal x program (version 1.83) (thompson et al. 1994). phylogenetic trees were inferred by the neighbourjoining method (saitou and nei 1987) with the phylogenetic analysis package paup* 4.0 (swofford 2002) and treeview was utilized to plot the tree topologies. a bootstrap analysis was performed using 1000 trial replications to provide confidence estimates for branch support (felsenstein 1985). reference 16s rrna sequences were obtained from the genbank that were included in the phylogenetic analysis. results s a m p l e c h a r a c t e r i s t i c s ( p h a n d h g concentration). sample characteristics were performed to provide a detailed description of sampling o c volume 6, 2012 microbiol indones 59 be isolated from sediment i. the failure to isolate bacterial strains in this sample was because the colonies growing on agar plates were dominated by fungal colonies (data not shown). a high level of resistance to hgcl was noted with all bacterial strains, capable of 2 -1 growing in the presence of at least 25 mg l of hgcl 2 with the most resistant strain growing in the presence of -1 550 mg l of hgcl (table 2). of the ten isolates, six 2 -1 isolates were able to grow at ≥ 200 mg l of hgcl (one 2 -1 strain was highly resistant to hgcl of 550 mg l , while 2 the four remaining isolates grew below 200 ppm of hgcl ). strain skcsh8 (from sediment ii) was the 2 most resistant strain to hgcl . the most sensitive 2 strains were found for strains skcsh6 and skcsh7 (from sediment iv), which were inhibited by 60 and 80 -1 mg l of hgcl , respectively (table 2).2 phenotypic characterization of hg-resistant bacteria. phenotypic characteristics of all ten hgresistant bacterial strains are given in table 3. all ten strains (skcsh1~skcsh10) were gram-negative and had no capsule. cells of all strains were rod-shaped in all growth phases in both liquid and agar media, except for strains skcsh7 and skcsh8 which were coccoid and curved-rods, respectively. when they were grown on lb agar, colonies of all strains were white, except that strain skcsh7 which was yellow-colored. catalase activity was present for all strains, while oxidases were only present for strains skcsh1, skcsh4, skcsh7, skcs8, and skcsh9. negative results were obtained for indole production, h s 2 production from thiosulfate, and urease activity, and positive results for vp reaction for all strains. assays for mr reduction were negative for all strains with the exception of strains skcsh1 and skcsh4. citrate utilization was also negative for all strains except for strains skcsh1, skcsh2, skcsh4, and skcsh9. hydrolysis of lipid and tween 80 was detected for all bacterial strains, while none of them could hydrolyze gelatin with the exception of strain skcsh2. of the ten strains, only strains skcsh2 and skcsh7 were able to hydrolyze casein. likewise, only four strains (skcsh2, skcsh4, skcsh8, and skcsh9) were capable of reducing nitrate to nitrite. nacl tolerance was also studied with nacl added at final site conditions with respect to the ph and hg concentration that are listed in table 1. both samples (sediments and tailing soil) had slightly alkaline phs (7.4-8.2). both samples were also characterized by high -1 hg concentrations (27.6-60.7 mg l ), suggesting that the bacteria residing the site might be resistant to hg. the highest hg concentration was found in sediment iv and the lowest was obtained in the tailing sample (table 1). isolation and cultivation of hg-resistant b a ct eria a n d t h eir min im u m i n h ib it o ry 2+ concentrations (mic) of hg . of the sediment and tailing soil samples, ten morphologically different bacterial colonies were successfully isolated and they were designated as strains skcsh1-sksh10 (table 2). only one isolate was obtained from tailing soil sample and from sediment iii. the more highly polluted samples (sediment ii and iv) had the highest number of mercury-resistant aerobic heterotrophs (table 2). two isolates (strains skcsh2 and skcsh8) were recovered from sediment ii and six isolates (strains skcsh4-7 and skcsh9-10) from sediment iv whose sediment mercury concentrations were 46.1 -1 and 60.7 mg l , respectively. in the case of the tailing soil, only one isolate was identified due to the appearance of the same colonies on agar plates. moreover, no hg-resistant bacterial strains were able to microbiol indones characteristics sampling site sediment i sediment ii sediment iii sediment iv tailing soil ph 7.8 7.9 7.8 7.4 8.2 hg conc. (mg l1) 28 .4 46.1 44 60.7 27.6 table 1 ph and mercury (hg) concentration of samples table 2 mercury-resistant bacterial strains and their 2+ minimum inhibitory concentrations (mic) of hg from the bunikasih traditional gold mine, pangalengan, west java province, indonesia isolates mic of hgcl2 (mg l -1 ) sampling site skcsh1 250 tailing soil skcsh2 225 sediment ii skcsh3 175 sed iment iii skcsh4 225 sediment iv skcsh5 100 sediment iv skcsh6 60 sediment iv skcsh7 80 sediment iv skcsh8 550 sediment ii skcsh9 250 sediment iv skcsh10 200 sediment iv 60 chaerun et al. concentrations in the range of 0.5-5% (w/v). all strains were able to grow at nacl levels of 0.5-3% nacl (optimum 1% nacl), except for strain skcsh9 which did not grow at 3% nacl. moreover, no growth occured at concentrations greater than 3%, with the exception of strains skcsh1, skcsh2, skcsh4, and skcsh7. volume 6, 2012 microbiol indones 61 table 3 phenotypic characteristics of mercury-resistant bacterial strains isolated from the bunikasih traditional gold mine, pangalengan, west java province, indonesia characteristics isolates 1 2 3 4 5 6 7 8 9 10 gram capsules cell shap e rods rods rods rods rods rods cocci curved rods rods rods colon y colour white white white white white white yellow white white white catalase a ctivity + + + + + + + + + + oxidase activity + + + + + imvic t est: indole production mr reduction + + vp reactio n + + + + + + + + + + citrate u tilization + + + + h2s production urease activity hydrolysis of : gelatin + starch casein + + lipid + + + + + + + + + + tween 80 + + + + + + + + + + nitrate reduction to nitrite + + + + growth at/in : 0.5% nacl + + + + + + + + + + 3% n acl + + + + + + + + + 5% n acl + + + + 4oc 25 o c + + + + + + + + + + 37 o c + + + + + + + + + + 55oc w w w w w growth on ph: 3 + 5 + ++ +++ ++ + +++ 8 +++ +++ +++ +++ +++ +++ +++ +++ +++ + 10 + + + + + + +++ +++ carbo n source: glucose +(a) galactose +(a) +(a) +(a) fructose mannose arabinose sucrose malto se lactose note: (-): negative result; (+): positive result; (++): high growth; (+++): extremely high growth; (w): weak growth; (a) acid production; (mr): methyl red; (vp): voges-proskauer; (isolates 1-10): skcsh1-skcsh10 16s rrna gene sequences of strains skcsh6 and skcsh10 all had the signature nucleotides and nucleotide pairs to indicate that they belonged to the family pseudomonadaceae. the 16s rrna gene sequences from strains skcsh6 and skcsh10 were almost 100% identical to each other, and were 99.97% identical to that of pseudomonas hibiscicola. phylogenetic analyses placed these two bacteria in a group that included our other hg-resistant bacterial strain (skcsh2), pseudomonas hibiscicola, pseudomonas geniculata, stenotrophomonas maltophilia and stenotrophomonas africana (fig 2). s t r a i n s k c s h 2 b e l o n g e d t o t h e f a m i l y xanthomonadaceae and the 16s rrna gene sequence of this strain was 99.69% identical to that of stenotrophomonas africana. these three bacteria (strains skcsh2, skcsh6 and skcsh10) grouped together with more than 99% similarity. in addition, strain skcsh8 formed a distinct phylogenetic group of the family aeromodaceae within the genus aeromonas with aeromonas hydrophila subsp. ranae as its closest neighbour (99.43% sequence similarity) (fig 3). discussion all hg levels in samples taken were above the maximum permissible concentration of hg in -1 sediments and soils of 0.04 mg l (swedish environmental protection agency 1967), while the maximum permitted hg concentration in water is 0.005 -1 mg l (government decree of the republic of indonesia no. 82, 2001). hence, methods for cleaning up mercury-polluted environments in a cost-effective way are urgently required, since mercury is a highly toxic metal. thus, by finding the mercury-resistant bacteria in this study, they might be beneficial to be all strains grew at 25 and 37 , with no or weak growth at 55 . most strains grew over a ph range of 5 -10 (optimum ph 7-8), but none of strains did grew at ph 3 (below ph 5) with the exception of strain skcsh3. in addition, all strains were incapable of producing acid from the oxidation or the fermentation of the following carbohydrates: gluclose, galactose, fructose, mannose, arabinose, sucrose, maltose and lactose. however, some strains had the ability to produce acid from oxidation or fermentation of glucose (skcsh1) and galactose (skcsh4, skcsh6, skcsh7). phylogenetic analysis of hg-resistant bacteria. in order to identify the bacterial isolates, their 16s rrna genes were amplified and sequenced. strain skcsh1 was affiliated to pseudomonas moraviensis ( 9 6 . 9 6 % s i m i l a r i t y ) , s t r a i n s k c s h 2 t o stenotrophomonas africana (99.69% similarity), strains skcsh3, skcsh4, skcsh5, skcsh7, skcsh9 to pseudomonas plecogossicida (97.57%, 94.53%, 98.66%, 96.61%, 96.73% similarity, respectively), strains skcsh6 and skcsh10 to pseudomonas hibiscicola (99.97% similarity), and strain skcsh8 to aeromonas hydrophila subsp. ranae (99.43% similarity) (table 4). in an inferred phylogenetic tree, these three novel species (skcsh4, skcsh7, skcsh9) together with our two other bacterial strains (i.e., skcsh3 and skcsh5) formed one distinct phylogenetic which was distantly related to recognizable species of the genus pseudomonas, with pseudomonas plecogossicida as the closest relative (fig 1). the 16s rrna gene sequences of each of the five strains were more similar to each other (> 99%) than to any other sequence in genbank. in addition, strain skcsh1 branched off most distantly and placed this strain within the genus pseudomonas with pseudomonas moraviensis as its closest neighbour. the o c o c microbiol indones table 4 mercury-resistant bacterial strains isolated from the bunikasih traditional gold mine, pangalengan, west java province, indonesia sampling sites stra in species seque nce simila rity (%) tailing soil skcsh1 pse udomonas morav iensis 96.96 sediment ii skcsh2 stenotrophomonas africana 99.69 sediment iii skcsh3 pseudomonas ple cogossicida 97.57 sediment iv skcsh4 pseudomonas ple cogossicida 94.53 sediment iv skcsh5 pseudomonas ple cogossicida 98.66 sediment iv skcsh6 pse udomonas hibiscicola 99.97 sediment iv skcsh7 pseudomonas ple cogossicida 96.61 sediment ii skcsh8 aeromonas hydrophila subsp. ranae 99.43 sediment iv skcsh9 pseudomonas ple cogossicida 96.73 sediment iv skcsh10 pse udomonas hibiscicola 99.97 the name of bacterial strains was a result of 16s rrna sequencing analysis. 62 chaerun et al. biological agents for environmental applications. application of these bacteria to mercury-polluted environments may lead to a potential cleanup technology which may be capable of bioremediating soil, water, or sediments contaminated with mercury in an environmentally friendly way. the role of the bacteria in mercury bioremediation is likely to have high levels of efficacy due to their mer operon-based resistance mechanism, which functions by active enzymatic reduction of mercury ions to water-insoluble metallic mercury (vetriani et al. 2005; omichinski 2007; poulain et al. 2007). therefore, further investigation of these bacterial mercury resistance is greatly needed in an effort to develop environmentally friendly, cost-effective bioremediation technology in indonesia. correspondingly, current environmental issues regarding mercury contamination in indonesia are due to the illegal mining of gold using the amalgam process in which mercury is employed. both wastewater and waste produced in the amalgam process were discharged into rivers, where mercury can be persistent over long periods of time, which in turn is a risk to humans because of its accumulation in the food chain (laperdina 2002; gustin et al. 2003; wang et al. 2004; kelly et al. 2006). the persistence of mercury in these mining disposals would enable us to acquire a high diversity of mercury-resistant bacteria. from the sediment and tailing soil samples of the bunikasih river from which our mercury-resistant bacteria were isolated, ten heterotrophic bacteria were isolated with their level of resistance to mercury (hgcl ) of 60-550 2 -1 mg l (table 2). their mercury resistance, which was over the hg concentrations of the samples (27.6-60.7 -1 mg l ) (table 1), indicates that the bacteria might have adapted to mercury accumulated in sediments of the bunikasih river for a long period of time due to their volume 6, 2012 microbiol indones 63 fig 1 phylogenetic tree of strains skcsh1, skcsh3, skcsh4, skcsh5, skcsh7, and skcsh9, based on 16s rrna gene sequences. the branching pattern was produced by the neighbour-joining method. numbers at nodes are percentage bootstrap values based on 1000 iterations; only values above 50% are shown. genbank accession numbers are given in parentheses. bar, 1 substitutions per 100 nucleotides. (lpsn); http://www.bacterio.cict.fr/). of the ten bacterial strains, eight strains were identified as pseudomonas that was predominant within the hgresistant bacterial diversity in the sites (table 4). the dominance of the genus pseudomonas is not surprising, since the genus pseudomonas has been recognized for its ability to utilize a broad spectrum of environmental pollutants such as organics and heavy metals (tvrzova et al. 2006). the genus pseudomonas is also known as a large and widely diverse bacterial group and members of the genus pseudomonas are ubiquitous in a wide variety of habitats such as soil, water and sediments (young and park 2007). of the members of the genus pseudomonas, pseudomonas plecoglossicida predominated in all samples (sediments iii and iv) from which five strains of p. plecoglossicida were successfully isolated (table 4). the species p. plecoglossicida has been reported to be a causative agent of fish disease (nishimori et al. 2000). two other pseudomonas species were also obtained in this study; two strains of the species pseudomonas hibiscicola from sediment iv sample and one strain of the species superior traits (i.e. a high and constitutive expression of the mercury resistance genes) (omichinski 2007). in mic experiments (table 2), the bacterial strains were grown on lb medium containing ionic mercury (hgcl ), where their growth greatly depended on the 2 hg(ii) bioavailability and toxicity. nutrients in lb medium that contained sulfhydryl group (i.e. yeast extract) and a negatively charged ion (i.e. chloride) which binds to ionic mercury, thus altering its bioavailability and toxicity (essa et al. 2002; barkay et al. 2003). therefore, the ability to grow in the presence of mercury has been demonstrated here to be a important feature of mercury-resistant cells for the application in bioremediation processes. the 16s rrna gene sequence analysis revealed that ten mercury-resistant bacterial strains were identified as pseudomonas spp., stenotrophomonas sp., and aeromonas sp. (table 4). to date, the genera pseudomonas, stenotrophomonas and aeromonas comprise 198, 13 and 30 species, respectively, with validly published names, at the time of writing (list of prokaryotic names with standing in nomenclature microbiol indones fig 2 phylogenetic tree of strains skcsh2, skcsh6, and skcsh10, based on 16s rrna gene sequences. the branching pattern was produced by the neighbour-joining method. numbers at nodes are percentage bootstrap values based on 1000 iterations; only values above 50% are shown. genbank accession numbers are given in parentheses. bar, 1 substitutions per 100 nucleotides. 64 chaerun et al. pseudomonas moraviensis from tailing soil sample (table 4). the first strain of the species p. moraviensis was isolated from soil contaminated with nitroaromatis in moravia, in the czech republic (tvrzova et al. 2006). in addition to the genus pseudomonas, one species of the genus aeromonas (i.e., aeromonas hydrophila subsp. ranae) was also recovered from sediment ii sample (table 4). members of the genus aeromonas have been recognized as opportunistic human pathogens, and strains of aeromonas hydrophila are also pathogenic to amphibians (huys et al. 2003). they are also widely distributed in freshwater environments (holmes et al. 1996) and some strains of aeromonas hydrophila are reported to be resistant to heavy metals (miranda and castillo 1998). apart from the genera pseudomonas and aeromonas, one member of the genus stenotrophomonas (i.e., stenotrophomonas africana) was present in the sediment ii sample (table 4). the genus stenotrophomonas was created in 1993 to accommodate xanthomonas maltophila (formerly pseudomonas maltophila) (palleroni and bradbury 1993), and stenotrophomonas africana is a later synonym of stenotrophomonas maltophila (coenye et al. 2004). the species s. africana is an essensial cause of nosocomial infection that is present in a wide range of environmental niches (drancourt et al. 1997). the genus stenotrophomonas is frequently found in soils and especially in the plant rhizospheres which are particularly contaminated with potentially toxic metals such as copper, platinum, mercury, gold, cadmium, lead, chromium, silver, and selenium salts (barkay et al. 2003). although large numbers of the genus pseudomonas are capable of being resistant to mercury, they are not representatives of the type strains of mercury-resistant volume 6, 2012 microbiol indones 65 fig 3 phylogenetic tree of strain skcsh8, based on 16s rrna gene sequences. the branching pattern was produced by the neighbour-joining method. numbers at nodes are percentage bootstrap values based on 1000 iterations; only values above 50% are shown. genbank accession numbers are given in parentheses. bar, 1 substitutions per 100 nucleotides. ayhuan d, atteng o, dondokambey a, randuk m. 2003. mercury pollution on district of dimembe river system, north sulawesi, indonesia, due to traditional gold mining activities. j physique iv france. 107: 79-82. barkay t, miller sm, summers ao. 2003. bacterial mercury resistance from atoms to ecosystems. fems microbiol rev. 27(2-3): 355–384. cappuccino jg, sherman n. 2005. microbiology: a laboratory manual. massachusetts: addison-wesley publishing company. p 81-86. chaerun sk, tazaki k, asada r, kogure k. 2004. bioremediation of coastal areas 5 years after the nakhodka oil spill in the sea of japan: isolation and characterization of hydrocarbon-degrading bacteria. environ int. 30(7): 911-922. chiu hh, shieh wy, lin sy, tseng cm, chiang pw, wagnerdobler i. 2007. alteromonas tagae sp. nov. and alteromonas simiduii sp. nov., mercury-resistant bacteria isolated from a taiwanese estuary. int j syst evol microbiol. 57(6):1209-1216.doi: doi:10.1099/ijs.0.64762-0. coenye t, vanlaere e, falsen e, vandamme p. 2004. stenotrophomonas africana drancourt et al. 1997 is a later synonym of stenotrophomonas maltophilia (hugh 1981) palleroni and bradbury 1993. int j syst evol microbiol. 54(4):1235-1237.doi:10.1099/ijs.0.630930. din iso 38414-s. 1983. deutsche einheitsverfahren zur wasser-, abwasser-, und schlammuntersuchung; schlamm und sediment. beuth, berlin, wien, zurich. din iso 10390. 1997. bestimmung des ph-wertes. beuth, berlin, vienna, zurich. drancourt m, bollet c, raoult d. 1997. stenotrophomonas africana sp. nov., an opportunistic human pathogen in africa. int j syst bacteriol. 47(1):160-163. dzairi fz, zeroual y, moutaouakkil a, taoufik j, talbi m, loutfi m, lee k, blaghen m. 2004. bacterial volatilization of mercury by immobilized bacteria in fixed and fluidized bed reactors. annals microbiol. 54(4): 353-364. essa amm, macaskie le, brown nl. 2002. mechanisms of m e r c u r y b i o r e m e d i a t i o n . b i o c h e m s o c i e t y transactions. 30(4): 672-674. fantozzi l, ferrara r, frontini fp, dini f. 2009. dissolved gaseous mercury production in the dark: evidence for the fundamental role of bacteria in different types of mediterranean water bodies. sci total environ. 407: 917-924. felsenstein j. 1985. confidence limits on phylogenies: an approach using the bootstrap. evolution 39(4): 783791. garcia-sanchez a, contreras f, adams m, santos f. 2006. atmospheric mercury emissions from polluted mining areas (venezuela). environ geochem health. 28(6): 529-540.doi: 10.1007/s10653-006-9049-x. government decree of the republic of indonesia no. 82. 2001. water quality management and water pollution control. jakarta, indonesia. green-ruiz c. 2006. mercury (ii) removal from aqueous bacteria. until presently, there are only two type strains of mercury-resistant bacteria in the world which were t alteromonas tagae sp. nov. (type strain at1 = bcrc t t 17571 -jcm 13895 ) and alteromonas simiduii sp. t t t nov. (type strain as1 = bcrc 17572 – jcm 13896 ) isolated from water samples of the er-jen river estuary, tainan, taiwan (chiu et al. 2007). thus, our findings of hg-resistant bacteria (pseudomonas moraviensis strain skcsh1 and pseudomonas plecogossicida strains skcsh4, skcsh7, and skcsh9) could be regarded as the type strains of four novel species in the genus pseudomonas that will add to the diversity of hgresistant bacteria worldwide. the distinct nature of their phylogenetic position suggests that these four isolates have the high possibility to be novel species of the genus pseudomonas due to their 16s rrna gene sequence similarities below 97% (the threshold recognized as delineating a genospecies (stackebrandt and goebel 1994; prakash et al. 2007; tindall et al. 2010)). in conclusion, the present study has provided the first evidence of the phylogenetically-diverse hgresistant bacteria in the hg-polluted sites of indonesia in which the genus pseudomonas predominates. likewise, the 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environ poll. 131(2):323-336. yang y, chen h, wang d. 2009. spatial and temporal distribution of gaseous elemental mercury in chongqing, china. environ monit assess. 156(14):479-489.doi:10.1007/s10661-008-0499-8. microbiol indones68 chaerun et al. young jm, park dc. 2007. probable synonymy of the nitrogen-fixing genus azotobacter and the genus pseudomonas. int j syst evol microbiol. 57(12):28942901.doi:10.1099/ijs.0.64969-0. zhu w, fu x, feng x, lu jy. 2008. annual time-series analyses of total gaseous mercury measurement and its impact factors on the gongga mountains in the southeastern fringe of the qinghai-tibetan plateau. j mountain sci. 5(1): 17-31. doi:10.1007/s11629-0080017-z. 1: 1 2: 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 02 cellulose is nature's most abundant polysaccharide consisting of β-1, 4-linked glucose units. as the major constituent of plant cell walls, the polymer represents the most important resource for production of bioethanol and other fine chemicals (lynd et al. 2005; chandel et al. 2012). the use of cellulolytic enzymes rather than acid hydrolysis ensures environmentally friendly glucose formation, a prerequisite of a number of biotechnological applications including the above mentioned ones. with respect to the catalyzed reaction, there are three types of cellulolytic enzymes: (i) endocellulases (ec 3.2.1.4), also known as glucanases, which randomly cleaves internal bonds at amorphous sites, thereby generating new chain ends; (ii) exocellulases (ec 3.2.1.91), which cleaves cellulose two to four units from the ends of the exposed chains, releasing oligosaccharides such as cellotetraose or cellobiose, and (iii) cellobiases (ec 3.2.1.21) or betaglucosidases, which splits the above oligosaccharides into monosaccharides (zhang et al. 2006). the demand for cellulases steadily rises due to their usefulness for food processing, in the textile and pulp and paper industry, as feed additives, and, as mentioned previously, for lignocellulose based bioethanol production (maki et al. 2009). endo-1,4-β-glucanases (1, 4-β-d-glucano-glucohydrolases; e.c. 3.2.1.4) are rather widespread. by randomly hydrolyzing internal β-1, 4-d-glycosidic bonds, they decrease the polymer length, concomitantly increasing the concentration of reducing sugars (onsori et al. 2005). although currently commercialized cellulases are predominantly produced by fungi (maki et al. 2009; chandel et al. vol.8, no.4, december 2014, p 147-160 doi: 10.5454/mi.8.4.2 cloning, sequencing, and expression of the gene encoding a family 9 cellulase from bacillus licheniformis f11 in escherichia coli and bacillus megaterium, and characterization of the recombinant enzymes * is helianti , maria ulfah, niknik nurhayati, and lina mulyawati center of bioindustrial technology, agency for the assessment and application of technology (bppt), gedung 611, laptiab-bppt, puspiptek-serpong, tangerang selatan, banten, indonesia a gene encoding cellulase belonging to the glycosyl hydrolase family 9 along with its native promoter was isolated from bacillus licheniformis f11, cloned in escherichia coli dh5 α and subcloned by transconjugation to bacillus megaterium ms941. functionality of the encoded protein was proven both in heterologous hosts, e. coli and b. megaterium. in the latter, the gene product was found in the extracellular fraction expressing a high specific activity; whereas in e. coli the protein was not secreted into the medium, and rather, showed a lower specific activity. the optimum temperature of the recombinant enzyme expressed in the hosts range from 65-75 ºc; whereas the optimum ph is 6. the recombinant enzyme was stable between 50-60 ºc and in a broad ph 2+ 3+ 2+ range (ph 5 9). addition of ca and fe enhanced the enzyme activity, whereas edta and cu had the opposite effect. lichenin, rather than carboxyl methyl cellulose, is the preferred substrate. key words: bacillus licheniformis, bacillus megaterium, cloning, e. coli, expression, family 9 cellulase, transconjugation sebuah gen penyandi selulase (glikosil hidrolase keluarga protein 9) bersama dengan promotor aslinya diisolasi dari bacillus licheniformis f11 dan dieskpresikan pada escherichia coli dh5 α dan disubklon secara konjugasi ke bacillus megaterium ms941. protein yang dikodekan oleh gen tersebut terbukti dapat berfungsi dengan baik di kedua host heterolog, e. coli dan b. megaterium. pada b. megaterium, produk gen disekresikan dalam fraksi ekstraseluler (supernatatan) dengan aktivitas spesifik yang tinggi; sedangkan pada e. coli produk gen (enzim) ditemukan dalam fraksi intraseluler dan memiliki aktivitas spesifik yang lebih rendah. suhu optimal enzim rekombinan di kedua inang berkisar 65 75 ºc; sedangkan ph optimum adalah 6. enzim rekombinan ini 2 + 3 + stabil pada kisaran suhu 50 60 ºc dan dalam kisaran ph yang luas (ph 5 sampai 9). penambahan ca dan fe 2+ meningkatkan aktivitas enzim, sedangkan edta dan cu memiliki efek sebaliknya. lichenin adalah substrat yang paling disukai oleh selulase rekombinan ini. kata kunci: bacillus licheniformis, bacillus megaterium, e. coli, ekspresi, kloning, selulase keluarga 9, transkonjugasi *corresponding author; phone: +62-21-7560536 ext 7119, fax: +62-21-7566922 ; email: is.helianti@bppt.go.id 2012), studies about bacterial cellulases are quite f r e q u e n t l y p e r f o r m e d o n b a c i l l u s s u b t i l i s , p a e n i b a c i l l u s , c l o s t r i d i u m c e l l u l o l y t i c u m , thermobifida fusca, and clostridium thermocellum (singhania et al. 2010). the investigation of cellulase genes from bacillus encoding family 5 enzymes was most often performed by cloning and expression in e. coli without the respective native promoter (bischoff et al. 2005; qiao et al. 2009; jung et al. 2010). compared to cellulase family 5, the cellulase family 9 is less studied. cellulase family 9 (gh9) is known to have both endocellulolytic and exocellulolytic activities and it also shows synergism with both endocellulases and exocellulases (qi et al. 2008). there was a report describing that a single gh9 cellulase is essential for microbial cellulose degradation, and that gh9 alone can perform cellulose degradation (tolonen et al. 2009; wilson 2009). cloning of family 9 members from bacteria other than bacillus, such as thermobifida halotolerans, had been reported (zhang et al. 2011); also, liu et al. (2004) reported the cloning of cellulase genes (family 12 and family 9) from bacillus licheniformis gnxii in e. coli using pet expression system (again without the respective promoter). however, no information available regarding the characteristics of the recombinant enzymes. in this study, a cellulase gene of the glycosyl hydrolase family 9 along with its original promoter was pcr-amplified from bacillus licheniformis f11, which was previously isolated from indonesian shrimp waste (waldeck et al. 2006). the obtained fragment was subcloned into a conjugative e.coli-bacillus shuttle vector allowing expression not only in e. coli but also in bacillus megaterium ms941 (wittchen and meinhardt 1995). the key properties of the recombinant enzymes present in the different hosts were determined. materials and methods strains, plasmids, and media. the bacterial strain originally used as host for obtaining and maintaining the recombinant plasmid was e. coli dh5 α. e. coli s17-1 served as the donor in conjugation experiments, and bacillus megaterium ms941 as the recipient. the plasmid used was pbbre194, an e.colibacillus conjugative shuttle vector constructed from pe194 and pbbr mcs3 (meinhardt laboratory, muenster university, germany). the mobilizable vector carries two origins of replication as well as two antibiotic resistant genes (erythromycin and tetracycline). lb medium or lb supplemented with carboxyl methyl cellulose (cmc) and tetracycline (12. -1 -1 5 µg ml ) or erythromycin (5 µg ml ) was used to select the transformants. genomic dna of b. licheniformis f11 (waldeck et al. 2006) served as the source of the cellulase gene. dna extraction and primers design. b. licheniformis f11 was cultivated as previously described (waldeck et al. 2006). the chromosomal dna was extracted essentially as described in helianti et al. (2010). all genetic experiments were performed according to the protocols in sambrook and russel (2001). the cellulase gene was amplified by using a pair of oligonucleotides, 5'ggggtaccgggc tgtcagatctgttgacaataaataaac -3' as the f o r w a r d a n d 5 ' c c g c t c g a g t t a g t a accgggctcatgtccgaaaacgag-3' as the reverse primer. concomitantly designed kpni and psti restriction sites, which were used for cloning, are underlined. primers were designed manually by retrieving and analyzing the genome of b. licheniformis dsm13 (nc_006322.1). the promoter regions were predicted using the promoter prediction s e r v e r ( h t t p : / / w w w. f r u i t f l y. o r g / s e q _ t o o l s / promoter.html). polymerase chain reaction (pcr). after the initial 3-min hot start at 95 °c, the mixture was subjected to 25 cycles, each consisting of 20 s at 98 °c, 15 s at 71 °c, and 90 s at 72 °c, followed by 5 min at 72 °c to complete the elongation. the thermal cycler from eppendorf (germany) and the high fidelity phusion dna polymerase (neb, uk) were used. the amplified fragment was purified using the geneaid pcr clean r up kit (geneaid, china), cut with kpn i and pst i and subsequently ligated into pbbre194, which was linearized by the same restriction enzymes. the ligation mixture was used to transform e. coli dh5 α. dna sequencing. sequencing was performed with fluorescence –labeled dideoxynucleotides (big dye terminator v3.1 kit, applied biosystems, foster city, usa) and the abi prism 3730 capillary dna sequencer (applied biosystems, foster city, usa). sequencing was performed using the pcr primers and two internal primers (5'-tcggcaa acggagtatatgc-3' and 5'agagtaaga agaatctgtcg-3'). the primers used in the amplification of the cellulase gene and its promoter were designed based on the bacillus licheniformis dsm13 genome sequence (http://www. ncbi.nlm.nih.gov) (veith et al. 2004). the sequencing results were analyzed using genetyx software (sci-ed software, north carolina, usa). 148 helianti et al. microbiol indones bacterial conjugation. the recombinant plasmid pbbre194-cel9 was isolated from recombinant e. coli dh5 α and used to transform e. coli s17-1, which would serve as the donor for the conjugative transfer. the bacterial conjugation from e. coli s17-1 to b. megaterium ms941 was conducted based on the protocol developed by aquino de muro and priest (2000), which was optimized by richhardt et al. (2010) (fig 1b). for conjugation process, b. megaterium ms941 cells were initially grown in lb broth without antibiotics, whereas e. coli s17-1 containing the recombinant plasmid pbbre194-cel9 was cultivated at 30 ºc overnight in lb medium with tetracycline. two 250-ml erlenmeyer flasks, each containing 50 ml of lb medium, was subsequently inoculated with either1 ml of the overnight b. megaterium or e. coli culture. the cultures were then grown at 30 ºc until od reached 0.6-0.8. the cells were then harvested 600nm by centrifugation (3000 x g, 4 ºc) and washed twice in 15 ml holding buffer (12. 5 mm kh po , 12.5 mm 2 4 k hpo , 1 mm mgso , ph 7.2), pelleted by 2 4 4 centrifugation and, after resuspension in holding buffer, mixed with the donor cells. using syringe and filter, the mixture was then compressed on a sterile nitrocellulose 0.45 µm filter to ensure close contact of donor and recipient cells. the filter was placed on sporulation agar for 48 h at 30 ºc (schaeffer et al. 1965) with the side containing cells facing upwards. counter selection of bacillus transconjugants against e. coli was performed by pasteurization, where cells were collected from the filter by suspending in 900 µl holding buffer and then incubated for 20 min at 80 ºc, before subsequently spread on lb agar plates containing erythromycin. transconjugants were further analyzed with respect to their cellulase activity and plasmid verification by restriction enzyme analysis. the procedure of cloning and transformation is shown schematically in fig 1b. zymogram and sds/page analyses. the molecular mass was determined by zymogram analysis using 10% polyacrylamide gels (page), containing 0.1% sodium dodecyl sulfate (sds), and 0.05% cmc for clear zone detection. pageruler protein ladder with molecular weight ranging from 10 to 200 kda (fermentas, germany) was used as standard. the part of the gel with the protein marker was stained with coomassie brilliant blue (cbb), the remaining part of the gel was used for zymogram analysis. the zymogram assay was conducted based on a previous report (sunna et al. 1997). congo red, nacl, and hcl were added sequentially to detect the clearing zone. enzyme preparations from recombinant e. coli dh5 α. a single recombinant colony containing pbbre194-cel9 was used to inoculate 5 ml media (lb and lb containing cmc and tetracycline) and grown overnight. the culture was then transferred into an erlenmeyer flask with 50 ml medium and shaken for 24 h, 150 rpm at 37 °c in a kühner shaker (kühner, switzerland). in 6 h intervals, the cell density was determined and the cellulase activity in both supernatant (obtained by pelleting the cells) and intracellular fraction were measured. to obtain the latter, cell pellet was resuspended in 5 ml of 50 mm p h o s p h a t e b u ff e r c o n t a i n i n g 1 m m o f 2 m e r c a p t o e t h a n o l . c e l l s w e r e d i s r u p t e d b y ultrasonication according to the previously reported method (helianti et al. 2008) and the debris was removed by centrifugation to obtain the crude enzyme extract serving as the intracellular fraction. as a control, a single recombinant e. coli colony containing empty pbbre194 was used as inoculum and subjected to the same procedure. enzyme preparation from recombinant b. megaterium ms941. procedures were essentially performed as previously explained. however, both lb and lbcmc media containing erythromycin, instead of tetracycline, were used. as a control, a single recombinant b. megaterium colony containing empty pbbre194 was used as inoculum and subjected to the same procedure. partial purification of the recombinant 2+ enzymes. to measure the effect of cu , sds, and tween 80 on the cellulase activity, partially purified enzyme was used. the purification procedure was as follows: crude enzymes from e. coli and b. megaterium fermented in lb-cmc were concentrated by membrane filtration (millipore membrane with 10 kda cut off). the concentrated enzymes were then subjected to q-sepharose column (1 ml packed column). the target cellulase was eluted by 0-1 m nacl in 20 mm -1 phosphate buffer with 0.5 ml min flow rate. the fractions with cellulase activity were pooled. the buffer was replaced with 20 mm phosphate buffer by membrane filtration, then the enzyme was subjected to assay as described above. enzymatic activity assay . the cellulase activity was measured (each sample in triplicate) by the method from sanchez-torres et al. (1996) using dinitrosalicylic acid to quantify the reducing sugars. d-glucose was used as standard. 100 μl of 1% cmc was mixed with equal volume of the enzyme preparation in 0.4 m phosphate buffer at ph 7. the volume 8, 2014 microbiol indones 149 mixture was incubated at 50 °c for 10 min, after which, 3 ml of dns reagent (1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulfite, and 1% sodium hydroxide, 20% (w/v) potassium sodium tartrate) (miller 1959) was added. to stop the reaction, the mixture was boiled (100 °c for 5 min). after addition of 2 ml water, samples were centrifuged to obtain clear supernatants. for each sample, the absorbance at 520 nm was measured at the indicated ph and temperature , however, the enzymes were added following the addition of dns. the protein concentration was determined by bradford method using bovine serum albumin (bsa) as standard (bradford 1976). one unit (u) of activity is defined as the amount of enzyme that produces 1μmol glucose per minute. effect of ph and temperature on cellulase activity. the effect of temperature on cellulase activity was measured (each sample in triplicate) at temperature range between 30-80 °c, ph 7 in phosphate buffer. the effect of the ph on the cellulase activity was measured (each sample in triplicates) in a range between 5-10 at 60 °c using 50 mm of the following buffers; citrate buffer (for ph 5, 6), sodium phosphate buffer (for ph 6-8), tris-hcl buffer (ph 89), and glycin-naoh buffer (ph 9-11). ph and temperature stability. for checking the temperature (in)stability, the enzyme was preincubated without substrates at 50, 60, 70, and 80 ºc, for 20, 40, and 60 min, respectively. subsequently, the activity was determined at 60 ºc ph 7. to check the influence of ph on the stability, the enzyme preparations without substrates were preincubated at ph 5, 6, 7, 8, and 9 at 60 ºc for 20, 40, and 60 min, respectively, and then the activity was determined at 60 ºc at the respective ph. effects of various additives and substrates on cellulase activity. effects of additives on cellulase activity were examined by adding various metal ions 2+ 2+ 3+ 2+ 2+ (ca , cu , fe , zn , and mg ), chelator and detergents (edta, sds, triton x-100, tween 80) and substrates (birchwood xylan, oatspelt xylan, beechwood xylan, lichenin, corncobs, empty bunch oil palm, bagasse, filter paper). a mixture with cmc1% as substrate without additives was used as a control. results cloning and expression in e. coli and b. megaterium. we successfully amplified the specific 1.9 kb dna fragment using primers designed based on the cellulase family 9 from b.licheniformis dsm13, with b. licheniformis f11 chromosomal dna as template, and then cloned this specific fragment in pbbre194 vector. the cloned fragment, including the putative glucanase family 9 gene (cel9), contains an upstream non-coding region of 304 bp with a potential promoter (bold face in fig 1a) and 1965 bp encoding a the predicted glucanase (fig 1a). the sequence is available at genbank under the accession number kc663680. the cel9 gene was expressed in e. coli dh5 α as well as in bacillus megaterium ms941from the shuttle plasmid as can already be seen from the clearing halos around the positive colonies in lb medium containing cmc (fig 2). the calculated molar mass of the native protein without its signal peptide is 69.91 kda, which agrees with the result of the zymogram analysis (fig 3). concerning the growth curves of the recombinant e. coli and b. megaterium strains harboring pbbre194-cel9 (in lb and lb medium containing cmc), there was no significant difference with respect to the cell densities (fig 4). the cellulase activity of the recombinant b. megaterium was found exclusively in the culture medium. intracellular cellulase activity was not detected. up to 12 hours of growth, no enzyme activity was detected in the supernatant of the recombinant e. coli. however, there was some activity in the supernatant after 18 and 24 h (data not shown). the finding of extracellular activity after prolonged cultivation agrees with the plate assay, where old cells lysed and the enzyme was set free, that the activity could be measured in the supernatant. after 24 h of cultivation, the specific activities of the e. coli intracellular recombinant enzyme in lb and lb-cmc -1 reached 4.3±0.1 and 2.6±0.1 u mg , respectively, whereas the specific activities of the b. megaterium -1 extracellular form were 54.8±1.5 and 78.8±4.7 u mg , respectively (fig 4). when e. coli and b. megaterium containing empty pbbre194 plasmid were cultivated in lb medium, we could not detect any cellulase activity produce by any recombinant cells (fig 4a). however, in lb-cmc medium e. coli and b. megaterium showed a faint intrinsic cellulase activity -1 of 0.14 and 0.11 u mg , respectively (fig 4b). recombinant e. coli and b. megaterium harboring empty pbbre194 could not grow well in lb – cmc medium. the cell density only reached od600 0.5 in the saturated condition (fig 4b). hence, the results suggest that the promoter is recognized in e. coli. indeed, the proposed promoter (bold face in fig 1a) and the spacing of both motifs clearly agree with the e. coli -35 and -10 consensus 150 helianti et al. microbiol indones a -35 -10 gggctgtcagatctgttgacaataaataaacaatcatgttagaatttccaaaatataacacttcgtttggaatgtgctgtctattagattt ctactctcataacttagtttattgaacaaataaactaagttacttatcaaattcctcgcttgcagtcgtgtgctgatttaatgtgcaatcaat atcttcggtttttcaactttggccttgttttgttcgccggcaaatctaaaaggaggtgagcatgttgtagacagatggagttgcttgatctt aacgaacatgatggggaggaagcagtac 10 20 30 40 50 60 gtgaaacagaaagtatttttaaaaatgaaagcgctttgtttggcacttttagtgatcttc m k q k v f l k m k a l c l a l l v i f 70 80 90 100 110 120 tctatgagcatagcgtcgttttcagaaaagacccgtgcagcttctgctgaagaatatcct s m s i a s f s e k t r a a s a e e y p 130 140 150 160 170 180 cataattatgctgaactgctgcaaaagtctttgttattttatgaagcacagcgctcggga h n y a e l l q k s l l f y e a q r s g 190 200 210 220 230 240 agacttccggaaaacagccggctgaattggagaggagactccgggcttgaggacggaaaa r l p e n s r l n w r g d s g l e d g k 250 260 270 280 290 300 gacgttggcctcgatttaacgggagggtggtatgatgccggcgaccacgtgaagttcggt d v g l d l t g g w y d a g d h v k f g 310 320 330 340 350 360 ctgccgatggcttattctgccgcaatcctgtcatggtcggtctatgagtaccgagatgcc l p m a y s a a i l s w s v y e y r d a 370 380 390 400 410 420 tacaaagaatcgggtcagcttgatgcggcgctggacaatattaaatgggcgacagactac y k e s g q l d a a l d n i k w a t d y 430 440 450 460 470 480 tttcttaaagcccatacggctccttatgaattgtggggccaagtcggaaatggcgctcta f l k a h t a p y e l w g q v g n g a l 490 500 510 520 530 540 gaccacgcatggtgggggccggccgaagtaatgccgatgaagcgccctgcctataagatc d h a w w g p a e v m p m k r p a y k i 550 560 570 580 590 600 gatgccggctgtccggggtcagaccttgctggtggtacagccgcagcgctagcatcagca d a g c p g s d l a g g t a a a l a s a 610 620 630 640 650 660 tcaattattttcaagccgacagattcttcttactctgaaaaattactggctcatgccaag s i i f k p t d s s y s e k l l a h a k 670 680 690 700 710 720 caattgtatgattttgccgaccgctaccgcggcaaatattcagactgcattacagacgca q l y d f a d r y r g k y s d c i t d a 730 740 750 760 770 780 cagcaatattataattcgtggagcgggtataaagatgaactgacatggggagctgtctgg q q y y n s w s g y k d e l t w g a v w 790 800 810 820 830 840 ctctacttggcaacagaagaacaacaatatttggataaagcccttgcttcggtctcagat l y l a t e e q q y l d k a l a s v s d volume 8, 2014 microbiol indones 151 850 860 870 880 890 900 tggggcgatcccgcaaactggccttaccgctggacgctttcctgggatgacgtcacttac w g d p a n w p y r w t l s w d d v t y 910 920 930 940 950 960 ggagcacagctgctgctcgctcgtctgacaaacgattcccgttttgtcaaatctgtcgaa g a q l l l a r l t n d s r f v k s v e 970 980 990 1000 1010 1020 cgcaatcttgattattggtcgacaggctacagtcataatggaagcatagaacggatcacg r n l d y w s t g y s h n g s i e r i t 1030 1040 1050 1060 1070 1080 tatacgccgggcggtttggcctggcttgagcagtggggatcattgcgatacgcttcgaat y t p g g l a w l e q w g s l r y a s n 1090 1100 1110 1120 1130 1140 gccgcttttctcgctttcgtttattccgattgggtggatacagaaaaagcgaaaagatat a a f l a f v y s d w v d t e k a k r y 1150 1160 1170 1180 1190 1200 cgggattttgctgttcggcaaacggagtatatgctaggagataatccgcagcagcgaagc r d f a v r q t e y m l g d n p q q r s 1210 1220 1230 1240 1250 1260 tttgtcgttggatacggtaaaaatccgccgaaacatccgcatcaccgtacagcacacggt f v v g y g k n p p k h p h h r t a h g 1270 1280 1290 1300 1310 1320 tcatgggccaatcagatgaatgtgcctgaaaaccatcgccataccctatacggcgcatta s w a n q m n v p e n h r h t l y g a l 1330 1340 1350 1360 1370 1380 gtcggcggtccgggaagggacgattcgtaccgagatgacataacagattatgcgtcaaac v g g p g r d d s y r d d i t d y a s n 1390 1400 1410 1420 1430 1440 gaagttgcgatcgattataatgccgcttttaccggcaacgtagcgaaaatgtttcagctg e v a i d y n a a f t g n v a k m f q l 1450 1460 1470 1480 1490 1500 ttcgggaaaggccatgttccgctgcctgattttccggagaaggaaacacctgaggacgaa f g k g h v p l p d f p e k e t p e d e 1510 1520 1530 1540 1550 1560 tattttgcagaggcatcaatcaacagctccggaaacagctatactgaaatccgggcgcag y f a e a s i n s s g n s y t e i r a q 1570 1580 1590 1600 1610 1620 ctcaataaccgttcgggatggccggcaaagaaaaccgatcaattgtctttccgctactac l n n r s g w p a k k t d q l s f r y y 1630 1640 1650 1660 1670 1680 gttgacttgacggaagctgtagaagcgggatattccgccgaagatataaaagtcacagcc v d l t e a v e a g y s a e d i k v t a 1690 1700 1710 1720 1730 1740 ggctataacgaaggggcctcggtatcagagctgaagccgcatgacgcttcaaagcacatt g y n e g a s v s e l k p h d a s k h i 152 helianti et al. microbiol indones 1750 1760 1770 1780 1790 1800 tactatacagaagtcagcttcagcggggttttgatttatccaggcggtcaatccgcccat y y t e v s f s g v l i y p g g q s a h 1810 1820 1830 1840 1850 1860 aaaaaagaagtgcagttccgcctttcggcaccagacggaacgtctttttggaacccggaa k k e v q f r l s a p d g t s f w n p e 1870 1880 1890 1900 1910 1920 aatgaccactggtatcagggtctgtcacatgcgcttctgaagacgcggtatattccaacg n d h w y q g l s h a l l k t r y i p t 1930 1940 1950 1960 gccgccggccagcggctcgttttcggacatgagcccggttactaa a a g q r l v f g h e p g y * b fig 1 (a) dna and the predicted protein sequences of the cellulase family 9 gene of bacillus licheniformis f11. the putative promoter (with its -35 and -10 region, respectively) is highlighted in bold face. the putative shine/dalgarno sequence or ribosome binding site, as well as the corresponding start codon (gtg), are in italics. amino acids are given in the one letter codes. underlined amino acids refer to the predicted signal peptide. the translational stop is marked with an asterisk. (b) schematic representation of the cloning procedure. the noncoding region of the cloned fragment is given in blue. the coding region, including the promoter, is in red (the figure not drawn to scale). volume 8, 2014 microbiol indones 153 microbiol indones fig 2 the colonies of recombinant e. coli and bacillus megaterium on lb agar medium containing cmc 1% stained with congo red. fig 3 the zymogram of the protein crude extract. the protein marker is pageruler protein ladder, the ladder is shown in figure. 1) negative control (supernatant recombinant bacillus megaterium ms941 with empty vector); 2) supernatant of recombinant bacillus megaterium ms941 (pbbre194 -cel9) cultivated in lbcmc; 3) supernatant of recombinant b. megaterium ms941 (pbbre194 -cel9) cultivated in lb; 4) sonication extract of recombinant e. coli containing (pbbre194 -cel9) cultivated in lb-cmc; 5) sonication extract of recombinant e. coli (pbbre194 -cel9) cultivated in lb. 154 helianti et al. microbiol indones sequences. the promoter is probably constitutive, since in medium without the substrate, the gene was expressed well. the signal peptide is probably also recognized in the gram negative host but secretion is solely possible through the plasma membrane, thereby delivering (and capturing) the recombinant protein to the periplasmic space. characterization of the recombinant gene product from two hosts. the properties (ph and temperature profiles) of the gene products expressed in the different hosts in different media were, as expected, rather similar. the activity of the recombinant cellulases was optimal at ph 6 (data not shown) and 6575 ºc (fig 5), the enzymes were stable at 50 and 60 ºc at ph 5-9 (fig 6). the stability at 50 and 60 ºc in a wide ph range (5-9) of the recombinant extracellular enzyme expressed by b. megaterium ms941 is a distinct character of cellulase family 9. 2+ most of the tested metal ions, except for cu , enhanced or had no negative effected on the activity of the crude extract of recombinant cellulases when we . investigated the crude extract of the enzymes further, 2+ we found that cu , sds, and tween 80 had different 2+ effects on cellulase activity of the crude extract. cu , sds, and tween 80 increased the cellulase activity of the intracellular fraction/crude extract of the recombinant e. coli, while decreasing the cellulase activity of the supernatant of recombinant b. megaterium (data not shown). to clarify, we conducted partial purification. after purification of the crude kda 200 150 120 100 85 70 60 10 40 30 50 15 m54321 volume 8, 2014 microbiol indones 155 fig 5 effect of temperature on activity of recombinant cellulase produced in e. coli and b. megaterium in lb and lb-cmc, respectively. for this temperature profile, cellulase activity was measured at indicated temperature with temperature range 30-80 °c for 10 min at ph 7 using phosphate buffer. each sample was in triplicates. the error bars showed the standard deviation of three values of independent experiment. the symbols : are the supernatant of recombinant bacillus megaterium ms941 (pbbre194 -cel9) cultivated in lb; : supernatant of recombinant bacillus megaterium ms941 (pbbre194 -cel9) cultivated in lbcmc; : sonication extract of recombinant e. coli containing (pbbre194 -cel9) cultivated in lb; : sonication extract of recombinant e. coli containing (pbbre194 -cel9) cultivated in lb-cmc. fig 4 the growth of recombinant bacillus megaterium ms941 and recombinant e. coli containing pbbre194 cel9 and their cellulase productivity cultivated in lb (a) and lb-cmc medium (b). the solid line with symbols : cell density of recombinant b. megaterium (pbbre194 -cel9) ; : cell density of recombinant e.coli (pbbre194 -cel9); : cell density of recombinant b. megaterium (empty pbbre194); : cell density of recombinant e. coli (empty pbbre194). the dotted line with symbols : specific activity of supernatant of recombinant b. megaterium (pbbre194 -cel9); : specific activity of intracellular fraction of recombinant e. coli (pbbre194 -cel9); : specific activity of supernatant of recombinant b. megaterium (empty pbbre194); : specific activity of intracellular fraction of recombinant e. coli (empty pbbre194). a b 20 30 40 50 60 70 80 90 0 20 40 60 80 100 120 r el at iv e ac ti v it y ( % ) temperature (°c) extract , the specific activity of the enzymes produced by e. coli and b. megaterium cultivated in lb-cmc -1 increased to 23.94 and 298 u mg , respectively. using the partially purified enzymes, it was confirmed that 2+ cu and tween 80 reduced the activity significantly, whereas sds only moderately decreased the activity (table 1). the crude extract of recombinant cellulase displayed substrate preferences, where the specificity against lichenin was the highest in each case (table 1). discussion bacillus licheniformis f11 is known to harbor a cellulase family 5 gene and, indeed, displayed cellulase activity (waldeck et al. 2006). however, there was no information related to the cellulase family 9 gene. furthermore, compared to cellulase family 5, the cellulase family 9 is less studied. cellulases family 9 is potential for many applications since they are known to have both endocellulolytic and exocellulolytic activities on processing cellulose, and also show synergism with both endocellulases and exocellulases (qi et al. 2008). the cellulase family 9 gene we cloned had 99% similarity to the gene of b. licheniformis atcc14580 or dsm13 (veith et al. 2004; rey et al. 2004). when we conducted blast analysis against genbank data base, the results showed that there were only a few dna sequence information retrieved for this cellulase family 9 gene. the analysis of amino acid sequences deduced from the genes indicated that the table 1 effect of additives and different substrates on recombinant cellulase activity *a: supernatant of recombinant bacillus megaterium ms941 (pbbre194 -cel9) cultivated in lb-cmc; b: sonication extract of recombinant e. coli (pbbre194 -cel9) cultivated in lb-cmc; c: partially purified recombinant cellulase samples were used. *a mixture solution without additional substance and cmc1% as substrate were used as control cacl2 control metal ions (5mm) relative activity a (%) relative activity b (%) c cuso4 fecl3 zncl2 mgcl2 c sds detergent (0.25%) triton x-100 c tween 80 chelator (10 mm) edta 10 mm substrate (1%) birchwood oatspelt beechwood lichenin corncobs empty bunch oil palm bagasse 100 100 120.2±3.6 118.4±3.6 41.3±5.3 34.6±2.9 114.7±4.1 128.9±5.3 103.8±9.5 128.9±9.5 108.9±8.0 104.7±2.6 195.8±5.1 198.9±3.4 114.0±10.0100.7±2.4 82.2±4.4 78.2±3.8 57.9±8.6 53.2±3.1 0±3.6 12.1±6.0 29.4±5.9 0±5.4 39.6±9.4 21.1±9.4 123.0±7.7 126.8±5.7 1.3±6.8 40.2±9.5 32.3±8.8 24.2±10.0 26.0±10 42.9±10 156 helianti et al. microbiol indones enzyme consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from nterminal to c-terminal as liu et al. reported (2004). the gram-negative bacterium e. coli is the most frequently used organism for heterologous protein production since this bacterium is well known and its genetic manipulation methods are well established (tempe 2006). there are several reports describing the cloning and expression of cellulase genes from gram positive bacteria in e. coli. they reported that the protein was found solely intracellular (bischoff et al. 2007; qiao et al. 2009; jung et al. 2010). in our study, a clearing halo around the e. coli colony was observed, although the enzyme is not expected to occur outside the outer membrane, since the cellulase activity in the supernatant was not significant (fig 2). this might be due to cell lysis occuring when we overexpressed a protein in the periplasm of e.coli. this phenomenon had previously been observed, for example with glucanase overexpression in paenibacillus macerans (borriss et al. 1990). compared to other cellulase family 9 from other resources, this cellulase has several different properties. the cellulase of the b. licheniformis f11 family 9 is stable over a wide ph range (5-9) at 50 ºc and 60 ºc. to our understanding, this character is distinct from other cellulases family 9. it seems to be more thermoand acidophilic than that of thermobifida halotolerans, which has an optimum activity at 55 ºc and ph 8 (zhang et al. 2011), and that of the cellulase family 9 enzyme from a german grassland soil metagenomic library with an optimal activity at 55 ºc and ph 7 (nacke et al. 2012). all metal 2+ ions, except for cu , enhanced the activity of recombinant cellulases from both hosts. consistent with the findings that metal ions enhanced the activity, edta was found to inhibit the protein, suggesting the cellulase being a metalloenzyme as for the cellulase family 9 from thermobifida halotolerants (zhang et al. 2011). interesting results were also obtained with 0.25% sds. sds usually decreased the activity. however, in this recombinant cellulase, it had only slight moderate negative effect on the activity (table 1). sds was shown to positively affected the activity of recombinant bacillus lehanus protease expressed in e. coli (joshi and satyanarayana 2013). sds is also known to have dual interaction through binding as both a denaturant and a recovery reagent (xiang et al. 2006). from our experiments, we found that the intrinsic cellulase activity of e. coli and b. megaterium were extremely faint. although, the genomic information demonstrated that e.coli str. k-12 substrain mg1655 (genbank: u00096.3), which is the parent of e. coli dh5 α, had at least two families of glycosyl hydrolase gene (family 25 and 65), no data confirmed whether any of these glycosyl hydrolases was cellulase. on the other hand, the genomic information of bacillus megaterium dsm319 (genbank: cp001982.1; wittchen and meinhardt 1995, eppinger et al. 2011), which is the parent of b. megaterium ms941, demonstrated that the strain had 3 families of glycosyl hydrolase; family 31, 5, and 18, which, especially family 5, was predicted to be cellulase. the reason why this intrinsic cellulase gene family 5 was not expressed well in the b. megaterium ms941 is still unclear. in other report, it was described that the newly isolated b. megaterium had cellulase activity (shobharani et al. 2013). the specific activities of the intracellular form of the e. coli recombinant cellulase cultivated in lb and lb-cmc were low; whereas, those of the b. megaterium extracellular form were high (fig. 3). the recombinant b. megaterium expressed cellulase activity exclusively in the culture medium and no intracellular cellulase activity was detected. such findings agree with the presence of a signal peptide for the general secretory pathway (tjalsma et al. 2000). these results showed b. megaterium as the preferred host for production of secreted heterologous enzymes. previously, we overexpressed the bacillus subtilis aq1 xylanase in e. coli and found significant extracellular enzyme activities (helianti et al. 2010). the rather low extracellular cellulase activities found in this study may be due to the different promoter used to regulate the level of gene expression and different signal peptide that determine the protein allocation (takemori et al. 2012). in this study we used b. megaterium ms941 as bacillus host. due to the deletion of the nprm gene, b. megaterium ms941 had almost completely lost its extracellular proteolytic activities and additionally displays stable plasmid maintenance (wittchen and meinhardt 1995). indeed, highly efficient expression of homologous and heterologous genes was reported in b. megaterium (meinhardt et al. 1989, biedendieck et al. 2011) and currently it is a rather popular host for a number of applications (vary 2004). since transformation of the species is difficult to perform (vary et al. 2007), we have chosen the conjugational approach developed by richhardt et al. (2010). the presented work confirmed that the cellulase volume 8, 2014 microbiol indones 157 gene from b. licheniformis was expressed succesfully in both e. coli and b. megaterium. however, the extracellular expression in b. megaterium can be more efficient in terms of enzyme recovery and downstream process (for example removing the cell disrupting step), since the gene product is secreted into the medium. the conjugative transformation protocol applied readily generated the respective transformants and thus again proved useful for genetic manipulation of bacillus (richhardt et al. 2010). combining our approach with a bacillus high copy number plasmid will probably facilitate efficient overexpression of foreign proteins via conjugative plasmid transfer. acknowledgment the part of the work was funded by national innovation system research grant of incentive (insinas) program from indonesian ministry of research and technology no rt 2013-1363 granted to ih. the authors thank prof. 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2006. outlook for cellulase improvement: screening and selection strategies. biotechnol adv. 24(5): 452-481. 160 helianti et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 04. gurning.cdr vol.13, no.4, december 2019, p 129-133 doi: 10.5454/mi.13.4.4 the effect of aeration rate on the growth of blue green microalgae by aspergillus oryzae in buffalo dung as alternative media edwin yonathan gurning, amos imanuel, nina juliana roberta turnip, and 1* adelina manurung bioprocess engineering study program, faculty of biotechnology, institut teknologi del, jalan sisingamangaraja, laguboti, toba samosir 22381, north sumatera, indonesia. the high demand of arthrospira platensis as a veritable protein source encourages its mass production worldwide. currently, mass production of arthrospira platensis is hindered by the relatively high price of the growth media. recently, it is discovered that arthrospira platensis can be cultivated using buffalo dung as an alternative medium. buffalo dung is an excellent source of nitrogen and phosphorus which are principal macronutrients for the growth of arthospira platensis. in addition to nitrogen and phosphorus, carbon is also a macronutrient that is important to the growth of microalgae. the carbon source used by the microalgae is carbon dioxide, which is consumed through photosynthesis. carbon dioxide can be derived directly from the atmosphere as atmospheric co existing as much as 0.04%-v/v in air, which can be provided directly using an aeration pump 2 into the growth medium microalgae. during the aeration process, co mass transfer occurs from the gaseous 2 phase into the liquid phase. this research aims to investigate the effect of the aeration rate on the growth of the blue-green microalgae arthrospira platensis using buffalo dung media as an alternative medium. arthrospira platensis will be cultivated on buffalo dung media using various aeration rates to determine the effect of aeration on the specific growth rate (µ). the air will also be pumped into the growth medium without arthrospira platensis at the specific aeration rates to determine the mass transfer coefficient (k a) that occurs from the air leading to l growth medium. analysis of mass transfer coefficient (k a) of carbon dioxide will be conducted using the sulfite l method. variation of aeration that used in this research are 0.2 vvm; 0.4 vvm; 0.6 vvm; 1.2 vvm; 2.4 vvm that has -1 -1 -1 -1 mass transfer coefficient dan specific growth rate are 0.005 min and 0.1987 day ; 0.009 min and 0.2279 day ; -1 -1 -1 -1 -1 0.012 min and 0.2044 day ; 0.034 min and 0.1918 day ; 0.035 min , and µ in 2.4 vvm can't be determined, respectively. key words: aeration rate, arthrospira platensis, buffalo dung, mass transfer, sulfite method permintaan biomassa arthrospira platensis semakin meningkat. hal ini dilatarbelakangi oleh kandungan protein arthrospira platensis yang cukup tinggi, sehingga mendorong produksi arthrospira platensis semaksimal mungkin. namun harga medium pertumbuhan yang digunakan relatif mahal. dewasa ini, arthrospira platensis diketahui dapat ditumbuhkan pada medium alternatif kotoran kerbau. kotoran kerbau dijadikan sebagai sumber nitrogen dan fosfor yang merupakan makronutrien oleh arthrospira platensis. selain nitrogen dan fosfor, sumber karbon juga merupakan makronutrien penting bagi pertumbuhan mikroalga. sumber karbon yang digunakan oleh mikroalga adalah karbondioksida. karbon dioksida dibutuhkan dalam proses pertumbuhan arthrospira platensis. senyawa ini dapat berasal dari atmosfer berupa karbon dioksida atmosferik dengan menyalurkan udara melalui sistem aerasi ke dalam medium pertumbuhan mikroalga. pada aerasi, terjadi perpindahan massa karbon dioksida dari fasa gas menuju fasa cair. penelitian ini bertujuan untuk mengetahui pengaruh laju aerasi terhadap pertumbuhan arthrospira platensis pada media alternatif kotoran kerbau. arthrospira platensis ditumbuhkan pada medium kotoran kerbau dan dilakukan aerasi dengan laju alir tertentu untuk menentukan laju pertumbuhan spesifik. analisis konsentrasi karbon dioksida menggunakan metode sulfit. variasi laju alir yang digunakan adalah 0.2 vvm; 0.4 vvm; 0.6 vvm; 1.2 vvm; 2.4 vvm yang memiliki koefisien transfer volumetrik dan laju -1 -1 -1 -1 pertumbuhan spesifik secara berturut turut 0.005 menit and 0.1987 hari ; 0.009 menit and 0.2279 hari ; 0.012 -1 -1 -1 -1 -1 menit and 0.2044 hari ; 0.034 menit and 0.1918 hari ; 0.035 menit and µ in 2.4 vvm tidak dapat dihitung. kata kunci: arthrospira platensis, kotoran kerbau, laju aerasi, metode sulfit, transfer massa microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82167244711; email: adelia.manurung@del.ac.id vitamins, and iron (hadiyanto and azim 2012). biomass production of arthrospira platensis depends on various factors such as the composition of the medium, temperature, light intensity, ph of the medium, amount of inoculum, salt, and aeration rate (pandey et al. 2010). in general, arthrospira platensis is grown on zarrouk medium, which is a synthetic medium and is arthrospira platensis or often called "spirulina" is produced around 15,000 tons/year, especially in open pond systems (eumofa 2018). arthrospira platensis has a high nutrient content such as proteins, essential amino acids, fatty acids, polysaccharides, carotenoids, relatively expensive. only recently has it been found that buffalo dung can be used as an alternative medium for growing arthrospira platensis microalgae. buffalo manure is used as a source of nitrogen and phosphorus which is a macronutrient for arthrospira platensis (simangunsong and sinaga 2018). however, the growth of microalgae is also influenced by carbon sources to produce biomass using light energy. generally, carbon sources are available in inorganic forms, namely carbon dioxide gas (tebbani et al. 2014). carbon dioxide is the main element that is important in the process of photosynthesis. carbon dioxide is needed for the formation of carbohydrate compounds that will be converted into biomass. microalgae biomass consists of about 50% carbon and almost entirely from carbon dioxide gas (lam et al. 2012). in the process of cultivating arthrospira platensis, aeration is carried out by flowing air into the microalgae growth medium. the air content is dominated by nitrogen and oxygen. in addition, air also contains 0.04% v/v of carbon dioxide gas needed by microalgae as a substrate in photosynthesis (richmond 2003). thus, aeration is an important parameter that needs to be considered in increasing arthrospira platensis production. aeration has advantages such as avoiding the deposition of microalgae, homogenizing the culture medium so that the cell can have adequate nutrients and light, avoiding temperature differences and facilitating the transfer of carbon dioxide gas from the air to the medium. therefore, the phenomenon of carbon dioxide gas mass transfer needs to be reviewed. the phenomenon of mass transfer of carbon dioxide is considered important to know in order to obtain the appropriate aeration rate so that microalgae biomass is obtained as much as possible. this is useful for increasing arthrospira platensis production on a laboratory scale. so far there have been no studies on the effect of aeration rates on the growth of arthrospira platensis blue-green microalgae on alternative media for buffalo dung. materials and methods strain and culture medium. arthrospira platensis was produced form the culture collection of algae, institut teknologi del, indonesia. the inoculum was grown in zarrouk medium. the -1 composition of inoculum on a g l basis was as follows: ca cl .2h o, 0.04; feso , 0.01; k so , 1; 2 2 2 4 2 4 k hpo , 0.5; mgso , 0.2; na -edta, 0.08; nacl, 1; 2 4 4 2 nahco , 16.8; nano , 2.5; na s o , 0.05. 3 3 2 2 5 additionally, the medium contained trace metal -1 solution, the composition on a g.l basis was as follows: h bo , 2.86; mgcl , 1.81; na moo , 0.01; 3 3 2 2 4 cuso , 0.07; znso , 0.22. the buffalo medium 4 4 -1 contained 4 g.l buffalo dung. additionally, the buffalo medium contained nahco 8.5 mg and 3 na s o 5 g to oxidize toxic compounds and keep ph in 2 2 5 base condition. photo bioreactor design and operation. the photobioreactor was a glass bubble column. the operating volume of the reactor was 0.8 l. the air flow rate was adjusted using a gas flow meter. the air was sparged through a cross-shaped pipe sparger via 1 mm holes. the light intensity of 3000-4500 lux mantained during the operation. all photobioreactor were run at room temperature (24 °c). a 10% inoculum (9-day-old culture grown under similar condition) was used to inoculate in buffalo medium. to highlight the suitable aeration rate, the experiment was performed in the bubble column photo bioreactor with variation continuous flow rate. the cultivation study were conducted at 5 different flow rates (0.2, 0.4, 0.6, 1.2, and 2.4 vvm) in a cycle 24 h. analytical methods. uv/vis spectrophotometer was employed to determine the absorbance of culture at 560 nm. the sample was taken 2 ml each day to known the absorbance of biomass. the biomass concentration was determined by recorded the absorbance of variation biomass dilution that diluted with medium. the biomass then filtered cells with whatman gf/c filters (1.2 µm). the filtered cells were dried at 70 °c at oven until a steady weight was recorded for dry weight measurement. the specific growth rate was calculated using a graphical method. the specific growth rate determined by the natural logarithm value of the biomass concentration (corresponding to the logarithm phase) of the algae was plotted against time, and the slope was used to derive the specific growth rate. the mass transfer coefficient volumetric (k a) of l carbon dioxide analysis by sulphite method. buffalo medium added with excess sulphite then aerated at certain aeration rate. the sample taken every 15 minutes. the sample mixed kio , ki, and h so then 3 2 4 titrated with na s o . the quantity of sulphite is 2 2 3 equivalent to the quantity of reduced iodine and since the quantity of sodium thiosulfate used in the titration is equivalent to the quantity of remaining iodine. the difference between the total iodine and the volume of x gurning et al. microbiol indones volume 13, 2019 microbiol indones x sodium thiosulfate is a measure of the sodium sulphite concentration. the oxygen dissolved determined form the sodium sulphite concentration that reduces. the natural logarithm of difference bulk liquid (henry law) and oxygen dissolved was plotted against time. the slope was used to derive the mass transfer coefficient volumetric. results -1 alternative media with a concentration 4 g l of buffalo dung given different variations in aeration rate (0.2, 0.4, 0.6, 1.2, and 2.4 vvm) which has co 2 volumetric mass transfer coefficient of 0.0054; 0.0095; -1 0.0121; 0.034; and 0.0349 min . the graph of the relationship between the aeration rate and the volumetric mass transfer coefficient of co (k a) is 2 l shown. the value of the co volumetric mass transfer 2 coefficient (k a) in the photobioreactor will increase l with increasing air aeration rate (fig 1). low air aeration rates will form smaller and slower bubbles so that the flow regime in the photobioreactor will form a laminar flow. this phenomena results in a low value volumetric mass transfer coefficient of co (k a).2 l arthrospira platensis was grown in five (5) variations in the aeration flow rate on alternative buffalo dung mediums namely 0.2, 0.4, 0.6, 1.2, and 2.4 vvm. the arthrospira platensis growth curve at various aeration flow rates of 0.2, 0.4, 0.6, and 1.2 vvm is stated. the lag phase experienced by arthrospira platensis is very short (fig 2). this is caused by the inoculum in alternative media buffalo manure already in the exponential phase. if the initial culture conditions have reached the stationary phase, then the lag phase in the new medium will last long because the cell will adjust to the new conditions of the media. the specific growth rate of arthrospira platensis increased at a flow rate of 0.2 and 0.4 vvm. an increase in the specific growth rate of 0.2 vvm towards 0.4 vvm occurs significantly (fig 3). the greater the aeration rate given to arthrospira platensis culture on alternative buffalo dung media, the higher the specific growth rate but only up to 0.4 vvm, above 0.4 vvm the specific growth rate will decrease. increasing the aeration rate which results in an increase in the specific growth rate in arthrospira platensis due to the equilibrium between the negative effects caused by shear stress and the positive effect caused by an increase in aeration rate and co mass transfer and the 2 positive influence is more dominant. shear stress is a force that causes the deformation of a material by producing slippage along a parallel plane. discussion co absorption into the medium is greater as the 2 value of the co volumetric mass transfer coefficient 2 (k a) increases. the greater the surface area of the gas l and liquid that are in contact with each other, the absorption of co will be greater because the surface of 2 the gas and liquid that are in contact with each other will increase the chance of co to diffuse into the 2 medium. in the study, the value of (k a) in buffalo dung l -1 -1 medium was 0.0054 min to 0.0349 min (0.2 vvm to 2.4 vvm) which showed that the value of (k a) gets l lower with the flow rate increasing. the growth of arthrospira platensis in several variations of the aeration rate experiences an exponential phase until on the 9th day or the 10th day. previous studies conducted grew arthrospira platensis on alternative buffalo dung media and the results th showed that the stationary phase had occurred on the 9 day. growth at the aeration rate of 2.4 vvm does not th produce a stationary phase until the 20 day until the remaining 100 ml medium is obtained and cannot be re-aerated. the aeration rate of 2.4 vvm is considered very unsuitable for use in bubble reactors on a lab scale of 800 ml with one aeration hole without condenser. the condenser is needed in condensing the evaporated medium during the aeration process. the evaporated medium will then be condensed into a liquid form again by the condenser. an aeration rate that is too high causes a decrease in the amount of medium due to the high rate of evaporation (simangunsong and sinaga 2018). there are two phenomena that contribute when there is an increase in evaporation of the liquid. first, evaporation is a function of the rate at which water vapour is released from water. when air bubbles burst on the surface of the liquid, the burst bubbles will add momentum to the air which will then increase the rate at which moist air will be released from the surface of the liquid. second, when air is injected into water, bubbles will form and steam will diffuse from water into the air (helfer et al. 2012). thus, increasing the aeration flow rate will increase momentum which will push moist air out. the effect of large bubbles on the rate of evaporation conclude that the greater the rate of aeration in the media, the greater the evaporation of the liquid that will be caused a decrease in the amount of water (garcia 2017). thus, the aeration rate that is too high makes the measurement of cell concentration be inaccurate, this is because evaporation will reduce the amount of media volume which results in measurements of cell concentration will always increase. the decrease of specific growth rate is caused by excess shear stress which occurs due to an overly high flow rate which causes the cell to experience a decrease in color and a decrease in the value of cell absorbance (converti et al. 2006). the cell will experience stress at a flow rate that is too high and then lose the color of arthrospira platensis cells which indicates cell death. the shear stress can cause a decrease in the rate of cell growth, decreased cell productivity, damage to cells and lysis in cells (rodriguez et al. 2011). the highest specific growth rate (µ) in this study -1 was 0.2279 days at a flow rate of 0.4 vvm. the value of the specific growth rate (µ) is higher than the value of the specific growth rate (µ) (1.2 vvm) growth medium is in the form of bicarbonate-enriched sot medium with a type of photo bioreactor glass bubble column with a volume of 20 l (ronda et al. 2012). the specific x gurning et al. microbiol indones fig 1 relationship of flow rate with co volumetric mass transfer coefficient (k a).2 l fig 2 arthrospira platensis growth curve at various aeration rates of 0.2, 0.4, 0.6, and 1.2 vvm. fig 3 spesific growth rate of arthrospira platensis at various flow rates in buffalo dung media. volume 13, 2019 microbiol indones x growth rate (µ) of the buffalo dung medium was lower than the zarrouk medium in the 8 l bubble bubble photo reactor which was an aeration rate of 0.5 vvm of 1 0.3 days (oncel and akpolat 2006). with mathematical models, the flow rate of 0.36 vvm or less than 0.36 vvm is the aeration rate suitable for the growth of arthrospira platensis in airlift-circulated tubular pbr (converti et al. 2006). the results of the research stated above provide information that the aeration rate, type of photo bioreactor, and type of medium influence the value of the specific growth rate (µ) of arthrospira platensis. in conclusion, the relation between co that 2 flowed into the growth medium, the mass transfer coefficient of volumetric and specific growth rate has been described. the results showed that the flow rate significantly affects the operational conditions of photo bioreactor that are observed through microalgae growth rate, and the mass transfer coefficient of volumetric. aeration flow rate contains co that 2 supplied to the system is inversely proportional to the specific growth rate from 0.4 vvm. flow rate is too high causes sheer stress on the cells. the aeration rate condition is 0.4 vvm also gives that the value of the mass transfer coefficient of volumetric is maximum for the growth of arthrospira platensis. acknowledgements this work was supported by a grant from lppm it del. references converti a, lodi a, borghi ad, solisio c. 2006. cultivation of spirulina platensis in a combined airlift-tubular reactor system. biochem eng j. 32(1): 13-18. doi: 10.1016/j.bej.2006.08.013. eumofa. 2018. blue bioeconomy: situation report and perspectives. european market observatory for fisheries and aquaculture products. directorate-general for maritime affairs and fisheries of the european commision. brussels. doi: 10.2771/053734. garcia hfl. 2017. mathematical modeling as a research tool in the cyanobacteria cultivation. [thesis]. erlangennurnberg: friedrich-alexander-universitat. hadiyanto, azim m. 2012. mikroalga: sumber pangan dan energi masa depan. [book]. semarang: upt undip press. isbn: 978-602-097-298-3. helfer f, lemckert c, zhang h. 2012. influence of bubble plumes on evaporation from non-stratified waters. j hydrol. 438-439: 84-96. doi: 10.1016/j.jhydrol. 2012.03.020. lam mk, lee kt, mohamed ar. 2012. current status and challenges on microalgae-based carbon capture. int j greenh gas control. 10: 456-469. doi: 10.1016/ j.ijggc.2012.07.010. maier rm, pepper il, gerba cp. 2009. environmental nd microbiology, 2 ed. academic press. isbn: 978-0-12370519-8. oncel ss, akpolat o. 2006. an integrated photobioreactor system for the production of spirulina platensis. biotechnology 5(3): 365-372. issn: 1682-296x (print); 1682-2978 (online). pandey jp, pathak n, tiwari a. 2010. standardization of ph and light intensity for biomass production of spirulina platensis. j. algal biomass utln. 1(2): 93-102. richmond a. 2003. handbook of microalgal culture: biotechnology and applied phycology. [book] john wiley & sons. isbn: 978-0-632-05953-9. rodriguez jjg, miron as, camacho fg, garcia mcc, belarbi eh, chisti y, grima em. 2011. carboxymethyl cellulose and pluronic f68 protect the dinoflagellate protoceratium reticulatum against shear-associated damaged. bioprocess biosyst. eng. 34: 3-12. doi: 10.1007/s00449-010-0441-7. ronda sr, bokka cs, ketineni c, rijal b, allu pr. 2012. aeration effect on spirulina platensis growth and γlinolenic acid production. braz j. microbiol. 43(1): 1220. doi: 10.1590/s1517-83822012000100002. simangunsong l, sinaga b. 2018. studi kinetika pertumbuhan pada mikroalga hijau-biru arthrospira platensis dengan media alternatif kotoran kerbau. growth kinetic study of blue-green microalgae arthrospira platensis using buffalo dung as alternative media. [thesis]. laguboti: institut teknologi del. tebbani s, filali r, lopes f, dumur d, pareau d. 2014. co 2 biofixation by microalgae: modeling, estimation and control. [book]. london: wiley. isbn: 978-1-84821598-6. page 1 page 2 page 3 page 4 page 5 8 nur richana (74-80).pmd the process of xylanase production from bacillus pumilus rxaiii-5 nur richana1‡*, tun tedja irawadi1, m. anwar nur1, illah sailah2, and khaswar syamsu2 1department of chemistry, faculty of mathematics and natural sciences 2department of agroindustrial technology, faculty of agroindustrial technology institut pertanian bogor, darmaga campus, bogor 16680, indonesia the optimum conditions for the growth of bacillus pumilus rxaiii-5 (a potential xylanase producer) were sought, these included temperature, ph, aeration, and agitation of the culture batch. afterwards a mathematical model based on the parameter of cultivation kinetics was formulated. at the same time, the rheology of the fluid used for bacterial cultivation in a bioreactor was studied. the data obtained was used for estimating the ‘scaling up’ of enzyme production. the results of the study indicate that the optimum condition for processing in 50 ml erlenmeyer flask are used temperature of 35 oc (308 ok), ph 7, and an agitation rate of 140 rpm. the highest xylanase activity and its specific activity are 297.132 u.ml -1 and 655.32 u.g -1protein, respectively. subsequent experiments in a bioreactor using all of the experiment parameters mentioned above, except for the agitation rate, shows that the results are as follows. the highest specific growth was at 0.082 hour -1 at an aeration and agitation rate of 0.5 vvm and 150 rpm, respectively. based on the data of the cultivation kinetics, the optimum conditions for the fermentation in biostat 2l-bioreactor is 1 vvm and 200 rpm of aeration and agitation, respectively. the efficiency of substrate (yp/s) and of cell biomass (y p/x ) to produce xylanase is 50.744 u.g -1 and 43.906 u.g -1, respectively. the efficiency of substrate to cell production (y x/s ) is 1.178g.g -1. the liquid cultivation-medium has non-newtonian properties. based on a mathematical model it is found that the consistency index (k constant) and index of liquid behavior (n value) are 0.179 g.cm -1.second -1 and 0.3212, respectively. becouse the value of 0<n<1 and the constant k>0, the culture liquid is categorized as pseudo plastic one. the rheynold number (nre) is 6.9 x 103 which indicates it has turbulent characteristics. from a calculation it is found that the power required t o r u n a s u i t a b l y s i z e d i m p e l l e r i s 0 . 2 2 8 h p ( h o r s e p o w e r ) a n d t h e p o w e r c o n s u m p t i o n p e r u n i t v o l u m e i s 0.2265 hp.m -3. all these values were used for scaling up xylanase production in the bioreactor. key words: bacillus pumilus, xylanase, production _____________________________________________ _________________ ‡present address and *corresponding author, indonesian center for agricultural postharvest research and development, pusat penelitian dan pengembangan pertanian, jalan tentara pelajar no 12, bogor 16114, indonesia; phone: +62-251-7177064, fax: +62-251-321762, e-mail: r1ch4n4@yahoo.co.id xylanase is a group of extracellular enzymes which are able to hydrolyze hemicelullosic materials into xylose and xylo-oligosaccharides. xylanases show great potential for industrial applications mainly for the bioconversion of lignocelluloses to sugar, ethanol, and other useful substances, clarification of juices and wines, improving the nutritional quality of silage and green feed and the de-inking processes of waste papers (viikari et al. 2001). the enzymes have been grouped based on the type of substrate hydrolized. xylanases are categorized into three groups: β-xylosidase, exoxylanase and endoxylanase. endoxylanase is the main enzyme responsible for the cleavage of the linkages within the xylan backbone (belfaquih et al. 2002). only a few microorganisms are capable of producing xylanase extracelullarly. some findings indicated that bacteria (gilbert and hazlewood 1993; sunna and antranikian 1997), fungi (tonukari et al. 2002; kheng and omar 2005), actinomycetes (ball and mccarthy 1989; begg et al. 2001), and yeast (hrmova et al. 1984; liu et al. 1999) were capable of producing xylanase. xylanase-producing alkaliphillic bacteria can be used as bleaching agent for paper processing (ruiz-arribas et al. 1995). cultivation of xylanase-producing microorganisms in a liquid medium has been applied widely in producing the enzyme. there are many advantages in using a liquid medium, i.e. the type of component and composition of the medium are easy to adjust to obtain the optimum conditions for microbial growth; more efficient consumption of substrate easier in adjusting the microorganism growth rate; and the risk of contamination is less compared to using a solid medium. however, the cultivation in liquid medium required skills and expertise in operating a bioreactor for obtaining a high level of enzyme production. by shaking the liquid medium, it is expected that temperature, ph, oxygen, nutrient supply as well as other environmental factors are homogenous throughout the medium in the bioreactor. the engineering on the processing of xylanase production is based on the information obtained through studying on the optimization process, the strategy on the studying optimum level of substrate, and then the modeling competency of the fermentation industry. all these cover the studies on the cultivation kinetics based on three rates i.e. the rates of biomass production, of substrate consumption and of enzyme production. production of xylanase from isolate bacteria capable of utilizing xylan for growth substrate as carbon source. in general, the agro-residues such as wheat bran, sugar cane bagasse, corn cob, paddy straw, rice husks, and cassava wastes were found to be more suitable like xylan (saurabh et al. 2001; richana et al. 2004). saha (2002) reported structure microbiology indonesia, august 2007, p 74-80 volume 1, number 2 issn 1978-3477 of xylan in corb fiber is commercially available enzyme preparation. this work was conducted to study the ability of bacillus pumilus rxaiii-5 in producing xylanase using corn cobs as a substrate. optimum conditions such as agitation, aeration, and the time course for bacterial cultivation are designed to favor bacterial growth. from the study it is expected that the parameters of cultivation kinetics will be obtained. there can be used as a basic strategy to optimize the amounts of substrate added. the study of rheology of the liquid medium in cultivation is also conducted for the purposes of scaling up of a bioreactor system. materials and methods the experiment was carried out over the period december 2002 to september 2003. to determine the effect of ph and temperature on the growth of b. pumilus rxaiii-5, the temperature used were 25-50 oc (298-323 ok), ph 7-11 and agitation 100-160 rpm. ten percent of inoculants were placed in 100 ml erlenmeyer flask. composition of growth medium was 0.125% (wt/vol) bactopeptone, 0.05% (wt/vol) yeast extract, 3.04% (wt/vol) corn cob xylan, 0.08% (wt/vol) kh 2 po 4 , and 0.02% (wt/vol) mgso 4 ·7h 2 o (richana et al. 2004). surface response method box-behnker experimental design using 3 variables was used for finding the optimum conditions for cultivation. observations were carried out on the biomass production, xylanase activity, soluble protein content and substrate residue. cultivation kinetics in biostat b-21 bioreactor. xylanase production using 1 liter of medium in a bioreactor was conducted at the optimum condition (t= 35 oc/308 ok, ph 7) obtained from the previous experiments. in this experiment the agitation rate was varied between 150, 200, and 250 rpm and the aeration was adjusted between 0.5 and 1.0 vvm (air volume per medium volume per minute). the ph of the medium was controlled by automatic addition of naoh and hcl. silicone was also added as an antifoaming agent. sample of the culture were withdrawn at 0, 2, 4, 6, 8, 12, 16, 20, 24, 32, 36, 40, 44, and 48 h after the inoculation. observations were conducted on the cell biomass, enzyme activity and the soluble protein content. cell biomass was separated by pelleting using centrifugation at 10,000 x g (centrifugal force). soluble protein was determined by the method of bradford (1976) using 100-800 ppm bovine serum albumin (bsa) is standard. absorbance of the reaction mixture was determined by spectrophotometrically at λ=600 nm. xylanase activity was determined by the ability of enzyme to hydrolyze xylan into invert sugar according to the method of winterhalter and liebl (1995). analyses of invert sugar used the dns (3,5-dinitro salysilic acid) method and spectrophotometry at ë=550 nm. one unit of xylanase activity is the amount of enzyme which is able to convert xylan to produce 1 ìmol invert sugar (or xylose) per min. (kubata et al. 1992). a series of xylose standards was used. combination of agitation and aeration rates was used to calculate the parameter kinetics, in which a mathematical model was developed to explain the system dynamics on the transformation of cell, substrate, and products during cultivation. in this experiment the monod (1949) mathematical equation was used as follow: dx/dt = ì x (cell biomass) ì = ì max .s/(k s + s) or 1/ì = (k s /ì max )(1/s) +1/ì max ds/dt = y p/s ds/dt (substrate) dp/dt = y p/x dx/dt (xylanase activity) y p/s = -dp/ds = the efficiency of substrate consumption to produce enzyme y x/s =-dx/ds = the efficiency of substrate consumption to biomass y p/x = dp/dx = the efficiency of biomass to produce enzyme, where x: cell concentration (g. l-1), s: substrate concentration (g. l-1), p: product concentration, t: fermentation time (hour), u: specific growth rate (l.hour-1), k s : constant (g. l-1), ì max : specific maximum of growth rate (l. jam-1). parameter kinetics consisting of ì max , y p/s , y x/s , y p/x were determined based on experimental data using a nonlinear regression technique (scragg 1991). the rheology of the liquid culture. the density of the liquid culture was determined using picnometer (weight per volume), and its viscosity using abrookfield viscometer at 6, 12, 30, 60 rpm having a spindle number of 2. data of viscosity, shear rate and shear strength were recorded. from these, the consistency index (k) and liquid index (n) was calculated. the rheynolds number (nre) was determined as follows: nre = (n 1 d 1 2 ñ) / ì a n=shaking speed, d=diameter of impeller, ρ=density, ì a= viscosity, n liquid index. these data, combined with the data obtained from the run of biostat-21 bioreactor, enabled the power needed to run the impeller to be calculated. power consumption for the impeller was then calculated using the equation presented as follows: p = (ñ n3 d 1 5 np) /g c p= power consumption, np = power number, g c = gravitation = 9.81 g cm/g sec2. bioreactor scaling up was calculated based on the power consumption per unit of volume. result optimum conditions for the growth of bacillus pumilus rxaiii-5. this work was designed to study the effect of temperature, ph and the rate of agitation in an effort to obtain optimum conditions for the growth of the bacterium. the quadratic-response-curve model was used in the analyses of variance (table 1). it was found that there was no significant effect of temperature, ph and the rate of agitation on soluble protein content. analyses of variance of xylanase activity indicates that the mathematical model used fits very significantly. the coefficient of determination (r2) is 0.962 which means there is less than 4% of variance which does not fit the model. volume 1, 2007 microbiol indones 75 variation of temperature, ph and shaking speed is greatly affected xylanase activity. interaction effects upon xylanase activity is shown only by the two parameter of ph and shaking speed. application of the quadratic response model shows an empirical relationship between xylanase activity and the test variable and unit code with a regression equation as follows: y = 1010.958 – 22.639 x1 – 365.38 x2 + 22.128 x3 + 0.178 x1 * x1 + 0.872 x2 * x1 + 21.735 x2 * x2 – 0.0108 x3 *x1 – 0.872 x3 * x2 – 0.0536 x3 * x3, where x1: temperature, x2: ph, and x3: rpm. maximum xylanase activity is 297.13 u.ml-1 at ph 7, temperature 35 oc, and an agitation rate of 140 rpm (fig 1). minimum xylanase activity is 42.71 u.ml-1 at ph 9.9, 42 oc, and 120.89 rpm for ph, temperature and agitation, respectively. these data indicate the minimum conditions in which b. pumilus rxaiii-5 can survive and produce xylanase. the results of the analyses of variance on specific activity are similar to those of xylanase activity, i.e. the presumed model fits very well. there is a highly significantly effect of ph on xylanase activity and specific activity for both the linear as well as the quadratic models. however, the effect of the interaction between ph and agitation rate on both xylanase activity and specific activity is small. application of quadratic response model shows an empirical relationship between xylanase activity and test variable and unit code with its regression equation as follows: y = 2217.857 – 37.77 x1-846.99 x2+ 48.03 x3 + 0.399 x1*x1+0.74 x2*x1+ 52.61 x2*x2-0.05 x3*x11.84 x3*x2-0.12 x3*x3, where x1: temperature, x2: ph, x3: rpm. maximum specific activity is 650.11 u.g-1 at ph 7 and 35.2 oc calculated from the quadratic mathematical model (fig 1). the optimization of agitation and aeration in a 2 l bioreactor. the work on the optimization of agitation and aeration of the liquid medium was conducted in a 2 l bioreactor. the aeration rates were 0.5 and 1.0 vvm and the agitation rates were 200, 150, and 100 rpm. sample were taken to estimate on the dry weight of the cell mass, protein content and xylanase activity. dry weight of cell. fig 2. indicates that the adaptation phase for the bacterial growth lasted for 6 h for the culture in all treatments. the same goes for the exponential phase, the phase of the culture of all treatments lasted for 30 min. the highest bacterial growth (2.802 g l-1) of liquid culture was obtained at 200 rpm and 1 vvm of agitation and aeration rates, respectively. this was followed (2.523 g l-1) by 150 rpm and 0.5 vvm of agitation and aeration rates respectively. results mentioned above fit the theory that the oxygen consumption rate by aerobic microorganisms is high enough, but insufficient oxygen supply will decrease yield. soluble protein. total soluble protein content is used as one of the parameters to measure enzyme production. the table1 analysis of variance for soluble protein, xylanase activity and specific activity of liquid culture of b. pumilus rxaiii-5 source of variance protein xylanase activitya specific activityb regression model linear quadratic r2 mean x1 = temperature x2 = ph x3 = rpm (x1)(x2) (x1)(x3) (x2)(x3) (x1)(x2)(x3) ns ns ns 0.778 0.433 ns ns ns ns ns ns ns ** ** ** 0.96 1 0 5 . 9 2 ns ** ns ns ns * ns ** ** ** 0.97 245.90 ns ** * ns ns * ns *significantly different, **very significantly different, ns = not significantly different, axylanase activity: unit/ml, bspesific ativity: unit xilanase/mg protein. p h temp (oc) agitation (rpm) temp (oc) agitation (rpm) p h fig 1 interaction effect among treatments (temperature, ph, agitation) on the xylanase activity of b. pumilus rxaiii-5. 76 richana et al. microbiol indones enzyme activity 266 191 117 48 11.00 9.67 8.38 7.00 35.00 40.67 48.33 55.00 enzyme activity 69.54 52.77 35.99 19.22 140 126.67 113.33 35.00 100 41.67 48.33 55.00 enzyme activity 265 182 99 16 140 126.67 113.33 100,00 7.00 8.33 9.67 11.00 data obtained showed a similar increased pattern of total soluble protein content, cell dry weight and xylanase activity. the highest total soluble protein content was recorded using agitation rate of 150 and aeration rates at 0.5 and 1.0 vvm (fig 3). xylanase activity. xylanase activity of b. pumilus rxaiii5 in the liquid culture was not detectable before 10 h after inoculation. in almost all treatments, the optimum xylanase activity was at 24 to 30 h after inoculation, and from then on the xylanase activity decreased. the highest xylanase activity was produced at an aeration rate of 0.5 and 1.0 vvm and at agitation rate of 200 rpm compared to those of the other combinations treatments (fig 4). the highest xylanase activity, 92.52 u ml-1, was detected at 24 h after inoculation with an agitation rate of 200 rpm and an aeration rate of 1 vvm. the smallest xylanase activity was recorded at 100 rpm and 0.5 vvm of agitation and aeration rates, respectively. this was due to the lower aeration rate resulting in a lower oxygen supply which slowed metabolism there by producing less enzymes, including xylanase. cultivation kinetics of b. pumilus rxaiii-5. the data on the effect of aeration and agitation was processed similarly to the above using the monod equation (monod volume 1, 2007 microbiol indones 77 fig 2 several growth curves of b. pumilus rxaiii-5 with various agitation rates and two aeration rates (a: 0.5 vvm and b: 1 vvm). cell dry weight is plotted against time. ( ) 200 rpm, ( ) 150 rpm, ( ) 100 rpm. a b fig 3 curves of total soluble protein at aeration rates of a: 1.0 vvm and b: 0.5 vvm using three agitation rates. ( ) 200 rpm, ( ) 150 rpm, ( ) 100 rpm. a b fig 4 curves of xylanase activity at aeration rates of a: 1.0 vvm and b: 0.5 vvm using three agitation rates. ( ) akt (200 rpm), ( ) akt (150 rpm), ( ) akt (100 rpm). a b 1949) as described above (fig 5). calculated results are shown in table 2. rheology of the cultivation liquid. the data obtained shows that the viscosity of the medium decreases over the course for culturing the bacterium (fig 6). this would table 2 value for yp/s, yp/x, and yx/s of liquid culture of b. pumilus rxaiii-5 at various aeration and agitation rates aeration (vvm) agitation (rpm) specific growth (ì h-1 ) yx/s (g g-1 substrate) yp/s (u g-1 substrate) yp/x (u g-1 biomass) 0 . 5 0 . 5 0 . 5 1 1 1 2 0 0 1 5 0 1 0 0 2 0 0 1 5 0 1 0 0 0.078 + 0.003 0.082 + 0.013 0.067 + 0.012 0.081 + 0.002 0.081 + 0.001 0.072 + 0.007 1.057 + 0.123 0.757 + 0.055 0.757 + 0.082 1.178 + 0.132 0.956 + 0.096 0.962 + 0.093 26.295 + 2.643 12.535 + 2.473 9.665 + 1.186 50.744 + 7.706 39.124 + 6.799 22.090 + 3.674 25.272 + 2.370 9.176 + 1.812 11.755 + 1.771 43.906 + 7.127 42.075 + 7.490 20.088 + 3.307 +: standard deviasi, yx/s: the efficiency of substrate consumption for the cell biomass production, yp/s: the efficiency of substrate consumption for the enzyme production, yp/x: the efficiency of the enzyme production by cell biomass. influence the flow performance of the medium in a bioreactor. flow pattern changes of the liquid would alter mass and heat transfer and can cause inappropriate oxygen distribution in the bioreactor medium. fig 6b shows that the liquid culture be haves as a nonnewtonian fluid, its viscosity changes with the change of shear rate. as the liquid is non-newtonian, then it is necessary to give special attention to the consistency index (k) and the index (n) of liquid behavior to determine the properties of the liquid culture. these linear regression relationship between the log of sheared rate and the log of viscosity show a slope of n-1, with an intercept of antilog k (fig 7). discussion in addition to growth medium, bacterial cultivation required particular environmental conditions for supporting their good growth. the environmental factors include 78 richana et al. microbiol indones fig 5 linear regression of y p/s (a), y x/s (b), and y p/x (c) for liquid culture of b. pumilus rxaiii-5. a b c fig 6 rheology of liquid culture of b. pumilus rxaiii-5 (a: relationship of viscosity and time of fermentation, b: relationship of viscosity and shear rate). a b fig 7 plot of logarithmic of viscosity and logarithmic of shear rate of a liquid culture of b. pumilus rxaiii-5. log of shear rate (sec-1) physical factors such as temperature and agitation, as well as chemical environment which can influence the physical properties of cells. cell membranes are greatly affected by temperature, while their permeability depends on the content and type of lipid in the membrane. a temperature increase of 5-10 oc over the optimum temperature may cause lysis and kill the cells of microorganisms. the total content of soluble protein begins to increase at the start of the exponential growth phase. high total enzymes (xylanase activity) were also produced during this phase. this phenomenon shows that xylanase is actually a primary metabolic product that is associated with cell growth. the enzyme is used for cell growth as well as for hydrolyzing xylanase as its substrate. the results also indicate that even tif the growth conditions of a bacterial isolate is made basic (high ph) from the start, the experimental results indicate that it does not automatically require a high ph for optimum growth. bacillus pumilus rxaiii-5 still survivies at ph 9.9 while the optimum growth is at neutral ph (ph 7). a similar phenomenon was reported by yang et al. (1995). in his experiments with bacillus sp., which were able to survive at ph 11.5, resulted in xylanase activity of 49 u.ml-1 with an optimum of ph 7. this result agrees with funding of rawashdeh et al. (2005) who reported a xylanase from streptomyces sp (strain lb 24d) with a ph optimum of ph 6-7. to scale-up xylanase production it is necessary to conduct a study on the mathematical model for cultivation kinetics. this model was obtained by entering data of the kinetics parameters into the monod equation (monod 1949), and then excecuting it to give a validation. four parameters i.e. specific-growth, biomass production, substrate consumption, and enzyme production (xylanase activity) rates were studied in the cultivation kinetics. the resulting data are useful for determinating the optimum conditions, i.e. the strategy for obtaining optimum substrate additions and studying mathematical models for the fermentation industry. the specific growth rate (ì) was not constant asit depends on the physical and chemical environment. the specific growth rate is a slope obtained from a linear relationship between time of cultivation and natural logarithm (ln) of dried cell weight of a bacterial culture. in this experiment 6 treatments resulted in specific growth rates (ì) of 0.0670.082 an hour (table 2). the highest specific growth rate was obtained at 150 rpm and 0.5 vvm for agitation and aeration rates, respectively, and the minimum specific growth rate was obtained at 100 rpm and 0.5 vvm for the agitation and aeration rates. lower aeration reduced the specific growth rate. at an aeration rate of 0.5 and 1.0 vvm, with agitation rate of 100 rpm, the specific growth rates were 0.067 and 0.072 an hour, respectively. microorganism growth and xylanase activity is closely related to substrate consumption. this phenomenon was shown by the equilibrium of substrate consumption, microorganism growth rate and xylanase activity (enzyme production). the efficiency of each of the three parameters was shown as the yield i.e. yp/x defined as the efficiency of substrate consumption for the enzyme production, yx/s as the efficiency of substrate consumption for the cell biomass production, and yp/x as the efficiency of the enzyme production by cell biomass. the value of the three parameters was calculated by the monod equation (monod 1949), and the result are presented in fig 5. the highest efficiency for substrate consumption for cell biomass production (yx/s) was 1.18 g g-1 at an agitation rate of 200 rpm and an aeration rate of 1.0 vvm (table 2). the increase in the efficiency of the substrate consumption (yx/s) and also will be increases the efficiency of enzyme production by cell biomass (yp/x). the enzyme production was greatly affected by cell biomass production. biomass production was directly related to substrate consumption. in these experiments, the highest biomass was achieved at levels of aeration with agitation rates of 1.0 vvm and 200 rpm, respectively. this indicates that increasing the aeration and agitation rates will increase the availability and facilitate the distribution of oxygen supply in the bioreactor. the lag phase could be shortened with a faster rate of aeration. the oxygen consumption by microorganisms was related to the aeration and agitation of the cultivation process. low levels of oxygen supply would reduce enzyme, antibiotic, and organic acid production by the microorganism. based on the data on the cultivation kinetics of b. pumilus rxaiii5 in liquid culture, an optimum condition was selected to obtain the highest efficiency of xylanase activity using biomass as a parameter. an optimum condition was obtained at the aeration and agitation rates of 1.0 vvm and 200 rpm, respectively with a recorded yx/s 1.18 g g-1 substrate, yp/s 50.74 u g-1 and yp/x 43.91 u g-1 biomass. our study on the flow behavioural and properties of liquid culture medium (rheology) are very useful for bioreactor design. the properties of the fluid flow of a liquid culture medium affected the homogeneity of the liquid culture components. fluid viscosity and flow properties affected significantly the transfer of components entering and leaving the cell. fermentation broth was found to behave as shear thickening fluid (n= 0.3212), and consistent index (k = 0.179 g cm-1 second-1). liquid culture is a non-newtonian and mathematical calculation giving n= 0.32 and k = 0.18 g cm-1. second-1. this means that the k > 0 and 0 < n <1 which indicates the liquid has pseudoplastic properties. this result differ from funding of mora-alvarez (1999) who reported the rheological characterization of fermentation broth from xylanase production by trichoderma longibrachiatum broths with n ranged from 1.14 to 2.15. this experiment also means that the viscosity decrease is due to an increase in the shear rate. increasing the speed of shaking would reduce the liquid viscosity. thus, the flow of liquid around the impeller is much more rapid than that at the surface as well as around the wall of the bioreactor. therefore, the rheology of the liquid medium influences the energy required for the operation of a bioreactor. to agitate a non-newtonian liquid requires higher energy as compared with that of a newtonian liquid. the calculated rheynold number (nre) of the liquid was 6.9 x 103 i.e. larger than 103. the value of nre indicates that the liquid has volume 1, 2007 microbiol indones 79 turbulent properties (doran 1995). the power value (np) was determined by fitting the nre value on the curve of power function versus nre. for a six-blade-turbine impeller the np value obtained was 5.0. the energy requirement to run the impeller with 0.228 hp (horse power) for aerating the system per unit volume was 0.2665 hp m-3. the value is then incorporated into the calculation for scaling up the bioreactor. due to the complexity of the properties of the liquid medium, the bioreactor design for bacterial culture is very critical. ones the course of the reaction, the rheology of the liquid medium changed from pseudoplastic to newtonian due to the decomposition of xylan (polymer) to xylose (monomer). references ball as, mccarthy aj. 1989. production and properties of xylanases from actinomycetes. j appl bacteriol 66:439-444. beg qk, kapoor m, mahajan l, hoondal gs. 2001. microbial xylanases and their industrial applications; a review. j appl micribiol biotechnol 56:326-338. belfaquih n, jaspers c, kurzatkowski w, penninckx mj. 2002. properties of streptomyces sp. endoxylanases in relation to their applicability in kraft pulp bleaching. world j microbiol biotechnol 18:699-705. bradford mm. 1976. a rapid and sensitive methods for quantitative proteins utilizing the principles of protein dye binding. anal biochem 72:248-354. doran pm. 1995. bioproses engineering principles. academic pr limited. london. p 51-103. gilbert hj, hazlewood gp. 1993. bacterial cellulase and xylanases. j gen mikrobiol 139:187-194. hrmova m, biely p, vrsanska m, petrakova e. 1984. induction of cellulose and xylanase-degrading enzyme complex in the yeast of trichosporon cutaneum. j arch microbiol 138:371-376, kheng pp, omar ic. 2005. xylanase production by alocal isolate, aspergillus niger usm ai 1 via solid state fermentation using palm kernel cake (pkc) as substrate. songklanakarin. j sci technol 27:325-336. kubata kb, horitsu hk, kawai, takamizawa k, suzuki t. 1992. xylanase i of aeromonas caviae me-1 isolated from the intentine of a herbivorous insect (samia cyrithia pryeri). j bioschi biotech biochem 56:1463-1464. liu w, lu y, ma g. 1999. induction and glucose repression of endoβ-xylanase in the yeast trichosporon cutaneum sl409. process biochem 34:67-72. monod j. 1949. the growth of bacterial cultures. ann rev microbiol 3:371-374. rawashdeh r, saadoun i, mahasneh a. 2005. effect of cultural condition on xylanase production by streptomyces sp. (strain lb 24d) and its potential to utilize tomato pomace. african j biotechnol 4:251-255. richana n, lestina p, irawadi tt. 2004. karakterisasi lignoselulosa: xilan dari limbah tanaman pangan dan pemanfaatannya untuk pertumbuhan bakteri rxa iii-5 penghasil xilanase. j penelitian pertanian 23:171-176. ruiz-arribas a, fernandez-abalos jm, sanches p, gardu al, santamaria ri. 1995. over production, purification and biochemical characterization of xylanase i (xys 1) from streptomyces halstedii. jm8. appl environ microbiol 61:24142419. saha bc. 2002. production, purification, and properties of xylanase from a newly isolated fusarium proliferatum. process biochem 37:1279-1284. saurabh g, kuhad rc, bharat b, hoondal gs. 2001. improved xylanase production from a haloalkalophilic staphylococcus sp. sg-13 using inexpensive agricultural residues. world j microbiol biotechnol 17:5-8. sunna a, antranikian g. 1997. xylanolytic enzyme from fungi and bacteria. crit rev biotechnol 17:39-67. tonukari nj, scott-craig js, walt jd. 2002. influence of carbon source on the epression of cochliobulus carbonum xylandegrading enzyme genes. african j biotechnol 1:64-66. viikari l, kantelinen a, sundqvist j, linko m. 2001. xylanases, in bleaching: from an idea to the industry. fems microbiol rev 13:335-350. winterhalter c, liebl w. 1995. two extremly thermostable xylanase of the hyperthemophilic bacterium thermotoga maritima msbb. app environ microbiol 61:1810-1815. yang vw, zhuang z, elegir g, jeffries tw. 1995. alkaline-active xylanase produced by an alkaliphilic bacillus sp. (vi-4) isolated from kraft pulp. j ind microbiol 15:434-441. 80 richana et al. microbiol indones 07 lactic acid bacteria (lab) are “generally regarded as safe” (gras) organisms that are widely utilized as starter culture, food preservative, and flavour enhancer in the food and beverage industry. recently, lab have been used for unconventional purposes including production of heterologous proteins, metabolic engineering, and vaccine delivery (de vos and hugenholtz 2004). the vast applicability of these lactic acid producer bacteria because of their ability to resist and well adapt in variable environments (belfiore et al. 2013; song et al. 2014;wu et al. 2014). the principal protein that responsible for wide adaptability of lab in different environment which is a group protein called heat shock proteins (hsp) (broadbent et al. 1997). these chaperon proteins, althoguh not all hsp contain chaperonin activity, will correct the expressed proteins to prevent it from misfolding event, allowing resumption of normal and physiological activity and leading to a higher level of stress tolerence when lab face environmental stress conditions (haslbeck et al. 2002; narberhaus 2002). depend on the type of stress, expression of these chaperon proteins by lab will also differ. generally the small heat shock proteins (shsp) have a major role in lab adaptation and survival among the others (guzzo 1996; koponen et al. 2012). the molecular mass of shsp range from the smaller 18 kda up to 30 kda. for instance, shsp expressed by leuconostoc o oenos (heat shock 42 c, acidic shock ph 3, and ethanol vol.8, no.4, december 2014, p 191-198 doi: 10.5454/mi.8.4.7 characterization of chaperone-like activity of small heat shock protein (shsp) isolated from indonesian traditional food (tempoyak ) lactobacillus plantarum u10 1,2 1* 1 haslia margareta , apon zaenal mustopa , bugi ratno budiarto , 2 and utut widyastuti 1 research center for biotechnology-indonesian institute for science (lipi), jalan raya bogor km.46 cibinong 16911 bogor, indonesia; 2 school of biotechnology, institut pertanian bogor, jalan raya darmaga kampus ipb darmaga bogor 16680, indonesia the characterization of small heat shock protein (shsp) from tempoyak-originated lactobacillus plantarum was investigated. the heat adaptive response proteins were ranging from 18 kda to 51 kda. interestingly, the intercellular protein (ip) fraction of heat shocked-l.plantarum u10 exhibited chaperone like activity by the ability to prevent loss of proteinase k activity from denaturation. furthermore, the shsp gene that related to the predicted shsp ±18 kda protein were successfully identified by pcr method and this gene has 423 bp size. the shsp gene has 140 amino acids (with unique motive at c-terminus t-l-p-k amino acid sequence) and has closely 100% identity with those of l.plantarum isolated from food or non-food environment. moreover, the gene encoding shsp ±18 kda protein was indeed up-regulated after l.plantarum u10 treated by heat shocking as proven by reverse transcriptase-pcr. this result suggested that shsp ±18 kda in our study may confers a survival advantage on lactobacillus plantarum and capable of protecting the cell against under temperature stress. key words: chaperone assay, heat shock protein, lactobacillus plantarum u10, rt-pcr karakterisasi gen small heat shock protein (shsp) dari lactobacillus plantarum u10 yang diisolasi dari makanan tradisional indonesia tempoyak telah diteliti. protein yang berhubungan dengan respon adaptif panas mempunyai ukuran yang bervariasi antara 18 kda sampai 51 kda berdasarkan pemisahan sds-page. fraksi interselular protein (ip) dari l. plantarum yang telah diberikan kejutan panas menunjukkan bahwa protein pendamping (chaperon) dibuktikan oleh kemampuan untuk mencegah hilangnya aktivitas proteinase k dari denaturasi dengan uji chaperon. selain itu, gen shsp yang diprediksi shsp ±18 telah berhasil diidentifikasi dengan metode pcr dan memiliki ukuran 423 bp dengan 140 asam amino dengan motif khas pada c-terminal (urutan asam amino t-l-p-k) dan memiliki identitas mendekati 100 % dengan l. plantarum lain yang diisolasi dari makanan atau lingkungan. gen penyandi shsp ±18 dengan pemberian kejutan panas dibuktikan dengan reverse transcriptase pcr (rt-pcr). hasil ini menunjukkan bahwa shsp ±18 kda dapat memberikan manfaat untuk kelangsungan hidup l. plantarum dan mampu melindungi sel dari tekanan panas. kata kunci: chaperone assay, heat shock protein, lactobacillus plantarum u10, rt-pcr *corresponding author; phone: +62-21-87545787, fax: +62218754568 ; email: azmustopa@yahoo.com shock 12%) has molecular mass of 18 kda (guzzo et al. 1997), shsps expressed by lactobacillus o o plantarum (heat shock 37 or 40 c, cold shock 12 c, solvent shock ethanol 12% or buthanol 1%) have molecular mass of 18.5 kda, 18.55 kda and 19.3 kda (capozzi et al 2012; fiocco et al. 2007). based on their primary structure, the shsp is classified into two classes, a and b. the b class has longer n-terminal region and shorter c-terminal region than the a class (münchbach et al. 1999). l. plantarum becomes one of the most applicable bacterium in food industries. for instance, the ability of this bacteria to produce natural antibiotics has gradually replaced the use of synthetic antibiotic as a food preservative (da silva sabo et al. 2014). l. plantarum u10 from traditional indonesia fermented food named tempoyak (fermented durian fruit) has been isolated and characterized (urnemi et al. 2010). this strain exhibited excellent antibacterial activity against broad pathogenic bacteria, thus this strain has potential application as natural antibacterial agent (urnemi et al. 2010). to the best of our knowlegde, there are few studies on the function of shsp and respective gene on the growth and surival of l. plantarum especially those which habitating fruitbased fermented foods. therefore, we aim to characterize the function of the chaperone-like activity conntaining small heat shock protein expression under in vitro heat stress condition and to elucidate its respective gene. materials and methods heat shock treatment. l.plantarum u10 were cultured in 100 ml mrs (de man, rogosa, sharpe) medium and incubated at 37 ºc overnight. cells were harvested by centrifugation at 10 000 × g when the od was ~0.6. the pellets were resuspended with 20 600 o ml mrs medium. the heat shock treatment at 42 c : (a) control (without heat shock), (b) heat shock 30 s, (c) heat shock 45 s. after the treatment, the cells were kept at room temperature for 20 min. the survival rate of cells was monitored by counting cfu on agar after incubation at 37 ºc overnight. all experiments were carried out in triplicate (delmas et al. 2001; guzzo et al. 1997). cell-free supernatant extraction. the method followed as described by birdsell and cota-robles -1 1967. cells pellet was mixed with 20 µg µl of lysozyme, 0.5 m of sucrose, and 1 mm edta and let stand for 1 h at 37 ºc to disrupt the cell membrane enzymatically. afterward, the disrupted cells were subjected for freeze thaw treatment to increase cell lysis for three times with each repetition for 1 h (30 min of freeze and 30 min thaw) before pellets was sonicated. then, 100 ml of buffer b (tris hcl 10 mm ph 8.5, nacl 100 mm, and tween-20 0.25%) was added into the pellet , and sonicated for 15 s, interval one minutes with five times repeation. the extract was isolated by centrifugation at 17 000 ×g, 4 ºc for 20 min. the supernatant or extracellular fluid was move to sterile falcon then pellets subjected to sds-page. chaperon activity assay the method followed methods from collada et al. 1997 and kim et al. 1998 by modification. chaperon activity assay of intracellular proteins fraction (ip) of l. plantarum u10 was conducted on 0.2% gelatin-containing agarose using proteinase k. a total of 0.25 grams of agarose was dissolved in 50 mm tris-hcl ph 7.4 and then heated. into the agarose solution then added 2.5 ml of 2% gelatin, stirred until homogeneously mixed. a 25 ml of agar solution was poured to the plate, let it harden and create wells using tips. there are several combinations for each sample: sample 1; proteinase k mixed with ip (1:1, w/w) then denaturated for 15 min at o 100 c, sample 2; proteinase k mixed with ip (1:1, w/w) without denaturation, sample 3; ip without -1 denaturation, sample 4; protease k only (4 µg µl ) o denaturated for 15 min at 100 c, sample 5; proteinase -1 k only (8 µg µl ). the agarose plate was incubated for 24 h at 37 ºc. the chaperon activity was detected by proteinase k activity and was shown as clear zone formation. the statistics analysis were caried out using programs minitab15 software. isolation of genomic dna and pcr. the total genomic dna of l. plantarum u10 was isolated from o 5 ml culture in mrs broth (oxoid) grown at 37 c overnight. bacterial cells were collected by centrifugation at 11 000 × g for 10 min. the genomic dna was obtained according to the method with modification (zhu et al. 1993). the pellet was resuspended with 500 μl te buffer (10 mm tris–hcl -1 ph 8 0, 1 mm edta) containing 60 µg µl lysozyme o and then incubated at 37 c for 1 h. after incubation, 200 μl of 10 % sodium dodecyl sulfate, 100 μl of 5 m nacl and 80 μl of 10 % ctab were added. the o mixture was then incubated at 68 c for 30 min and an equal amount of chloroform (1:1, v/v) was added. centrifugation was conducted at 13 000 rpm for 10 min. the supernatant was collected and 1:1 (v/v) ethanol was added and then centrifuged at 13 000 rpm for 10 min. dna was dissolved in te buffer containing 192 margareta et al. microbiol indones -1 10 µg µl rnase. the shsp gene was amplified using nested-pcr with first round pcr using primer 5'tgaaatttgaaagggga'3 and 5'gggccgct cacttgttact'3 and for the second round pcr using primer 5'atggctaatactttaatgaat cgg'3 and 5'ttattgaatttcgatttga ccg'3. those primers were retrieved from the nucleotide sequences of putative small heat shock gene on l. plantarum wcfsi (genbank accession number al935259) (spano et al. 2004). the shsp gene was amplified in 20 μl volumes each containing 20 ng -1 template dna, 1 unit µl of genomic dna were isolated from l. plantarum and added to a 20 μl pcr mixture containing 0.5 μl kapa tak polymerase, 0.5 μl of 10 mm dntp mix, 1x pcr buffer 4 μl, 2 mm mgcl 1 μl, 0.5 μl of each primer, and ddh o 12.5 μl. 2 2 the pcr was run following this program: predenaturation 5 min at 94 ºc followed by 35 cycles of 1 min at 94 ºc, primer annealing temperature for 1 min at o 55 ºc, and 72 c extension for 30 s, followed by a final o extension step at 72 c for 5 min. pcr products were analyzed on gel electrophoresis carried out by applying 20 μl of sampel to 1.5 % agarose gel. gel was run for 30 min at 100 v in tbe 1x buffer. the standard marker was used 1kb dna ladder promega. after electrophoresis the gel was stained with ethidium bromide for 10 min, and then wash with aquadest and thereafter visualized using uv light source. analyses of dna and amino acid sequences were caried out using blast programs on ncbi (www.ncbi.nlm. nih.gov). total rna isolation and rt-pcr analysis. the total rna of heat shocked-l. plantarum u10 was obtained using ribo-pure bacteria kit ambion as manual instruction. l. plantarum u10 were cultured in mrs medium and grown for 13 h at 30 ºc. afterward, the cell was transfered into fresh mrs medium then incubated at the same condition until od reached 0.6. 600 the cells were harvested by centrifugation at 13 000 × g. the heat shock treatment was done with added 1 ml fresh mrs to the pellets and then heated in 42 ºc for 15 min and control without heat shock treatment, then allowed at room temperature for 20 min. two hundreds fifty μl of ice-cold zirconia beads was poured to each sample. the cells were centrifuged, and then the supernatant was removed. the next step, the cells were resuspend in 350 μl rnawiz by vortexing vigorously for 10-15 s. the cells were transferred in rnawiz to a tube containing 250 μl zirconia beads, then it was vortexed for 10 min to lyse the cells. the zirconia beads were separated by centrifugation at 13 000 ×g, 5 min at 4 ºc, then the supernatant was kept to a fresh 1.5 μl tube. into the supernatant 0.2 volumes choloform was added , mixed well and incubated 10 min at room temperature. the mixtures was spun 5 min at 4 ºc, 16 000 × g and then the supernatant was transferred to fresh tube. finally, rna purification was done by adding 0.5 volumes of 100 % ethanol to the supernatant, and the sample was moved to the filter cartridge and was centrifugated for 1 min, the flow through was discarded and filter was washed with 700 μl wash solution 1 (warm at 37 ºc before used), then centrifuged for 1 min. after that the filter sample was washed with 2x500 μl wash solution 2/3, then centrifuged for 1 min. the sample was centrifuged for 1 min to remove excess wash solution from the filter. rna was eluted by applying 25-50 μl elution solution (preheated to 95-100 ºc) to the center of the filter. the dnase treatment was done with addition of 10x dnase buffer to the rna, and then incubation for 30 min at 37 ºc.the quality of rna samples was checked on 1.2% agarose gel, and the concentration was determined spectrophotometrically at 260 nm. about 150 ng of total rna were used in a final volume of 25 μl for the rt-pcr experiments. the rt-pcr program was as follow: 45 ºc, 30 min (reverse transcriptase reaction) ; 94 ºc , 5 min ; 94 ºc, 30 s (denaturation) ; 52 ºc, 1 min (annealing) ; 72 ºc, 1 min ; 72 ºc, 5 min (extention). the pcr fragments were visualized on 1.2% agarose gel. results the effect of heat shock treatment on profile of the protein expression in l.plantarum u10. to elucidate the diversity and function of heat shock protein on the growth and survival of l.plantarum u10, the bacteria was treated by incubation at different temperature counted for its cell viability after treatment. o the prominent effect of heat stress (42 c) on protein expressions was observed for 30 min induction that clearly shown the abundant protein expressed with diverse molecular weight (18.16 kda, 34 kda, 40.48 kda and 51.93 kda) (fig 1a). while prolonged induction time reduced significantly some of protein expressed, it may due to the difference in turn-over of protein degradation within cell. to confirm whether heat stress could reduce cells viability that impact on the quantity of protein, we have done cells counting for each treatment. there is no significantly differences 10 -1 between heat-treatments groups (3.3x10 cfu µl for 10 -1 30 min induction, and 3.2x10 cfu µl for 45 min volume 8, 2014 microbiol indones 193 10 -1 induction) and control group (3x10 cfu µl ) (fig 1b). this result confirm that the elevated protein expression in l.plantarum u10 is due to solely the effect of heat stress treatment. chaperone activity of cell-free extract from heat shocked-l. plantarum u10. to confirm the involvement of shsp in l.plantarum u10 resistency to heat stress, we tested the chaperone activity within cellfree extract (ip) of heat shocked-l.plantarum u10 by observing the proteinase k activity. ip fraction exhibited chaperonin-like activity as proven by residual proteinase k activity that could be still observed on sample no 1 after denaturation treatment while sample no 4, proteinase k alone with denaturation, loss its protease activity (1.3±0.11 cm vs 0.73±0.11 cm) (fig 2). proteinase k alone without denaturation showed broad clear zone formation (3.36±0.11 cm). on the other hand, protease activity was only slight observed in sample 3 (1.06±0.11 cm). this result indicated that the heat resistence of l.plantarum u10 may due to chaperonin activity within its cells. shsp gene identification of l.plantrarum u10 and its identity to other l.plantarum strains. here, we focused to identify ±18 kda protein at gene level with assumption that the targeted protein may belong to 194 margareta et al. microbiol indones treatment -1 cfu ml 10 -1 3.3x10 cfu ml 10 -1 3.3x10 cfu ml 10 -1 3.3x10 cfu ml control heat shock 42 ºc, 30 min heat shock 42 ºc, 45 min fig 1 profile of l. plantarum intracellular protein expression after heat shock treatment.(a) bacteria was o incubated in 42 c for 30 or 45 min, then the intracellular protein was collected and the proteins profile (50 µg of total protein for each samples) was separated on 12% sds-page (b) the population of bacteria was -1 counted before and after heat shock treatment and shown as cfu ml . sampel clear zone formation (cm) 1 2 3 4 5 1.3±0.011 3.1±0.11 1.06±0.11 0.73±0.11 3.36±0.11 o fig 2 chaperon activity assay of intracellular proteins fraction (ip) of (42 c, 30 min) heat shockedl. plantarum on 0.2% gelatin-containing agarose using proteinase k. sample 1: proteinase k mixed with ip (w/w) then o denaturated for 15 min at 100 c; sample 2: proteinase k mixed with ip (w/w) without denaturation; -1 sample 3: ip without denaturation; sample 4: proteinase k only (4 µg µl ) denaturated for 15 min at 100 o -1 o c; sample 5: proteinase k only (8 µg µl ) only. the agarose plate was incubated for 24 h at 37 c. proteinase k activity was shown as clear zone. (a) (b) volume 8, 2014 microbiol indones 195 shsp protein based on their molecular mass.the shsp gene was obtained by pcr with size of 423 bp (140 amino acid) and theoretically molecular mass of respective protein was 15.44 kda, respectively (fig 3a). furthermore, our shsp has 100 % protein identity with l.plantarum jdm1, l.plantarum zj316 and l.plantarum 16 while has 99 % with l.plantarum wcsf1and the amino acid sequence (t-l-p-k) that shows identity of shsp also observed at 124 to 127 position (fig 3b). the gene expression of heat shockedl.plantarum u10 protein. the expression of the gene encoding shsp ±18 of l. plantarum u10 was expressed after the environmental temperature for cell o o growth was shifted from 30 c to 42 c as shown by rtpcr result with size was ±423 bp and no band related to shsp ±18 in non-heat shock cell (fig 4). this result indicated the heat shock protein gene was expressed under heat stress environment. discussion several studies have shown the role of shsp in lab adaptation and survival under certain rush enviromental condition. factors such as abiotic stress (heat or cold shock, densitty of cells, ethanol and buthanol solvents) greatly impact on level expression of shsp mrna and further quantity of shsp produced (fiocco et al. 2007). in agreement with this finding, we also detected some of proteins to be upregulated after o l.plantarum was heat-shocked at 42 c for 30 min (od ~0.6). interesting finding was that the heat-600 treatment in our study could up-regulated several proteins with protein mass ranging from 18 kda up to 50.93 kda, which might be predicted as group of heat shock proteins. guzzo (1996) revealed that the proteins pattern of cells-free extract of l.oenos has molecular fig 4 rt-pcr analysis of l. plantarum hsp ±18 gene expression, line 1; sample obtained from heat shockedl.plantarum u10, line 2; sample without heat shock treatment, m; 100 bp molecular marker. the band corresponding to shsp ±18 mrna was pointed by yellow arrow. fig 3 pcr result of shsp gene (shsp 18.55) from genomic dna of l.plantarum and its amino acid sequence similarity with other lactobacillus plantarums hsps. the red box is shsps conserve motif (t-l-p-k amino acid sequence). (a) (b) 196 margareta et al. microbiol indones mass of 75, 66, 64, 24, 18, and 14.5 kda which was o previously treated by heat shock (42 c, od ~0.4) 600 using 2d-sds page. among proteins expressed only protein with molecular mass of 18 kda, named lo18, showed remarkable expression under heat, acid, and solvent treatment. moreover, cross reactivity study (using antisera against dnak, groel and e18.5 stress proteins) to the other proteins also pointed out that only protein with 64 kda gave positive result against groel. in our study, the protein expression could also be classified into one of group shp. but, we need to verify this classification. it is well known that heat shock proteins contain chaperone activity in which these proteins under stress condition will help in preventing intracellular protein from action of cellular degradation mechanism (ehrnsperger et al. 1997; veinger et al. 1998; lee and vierling 2000). here, we developed a simple method to detect chaperonin activity within ip fraction l.plantraum using proteolytic enzyme activity test on agarose. this method was based on protective action of predicted chaperonin-like proteins on proteolytic enzyme degradation due to heat treatment instead of measuring the residual enzyme activity after heat treatment in present of chaperone protein containing sample (collada et al. 1997; kim et al. 1998). chaperone assay clearly shown that ip fraction contained chaperon-like protein activity based on their ability to protect proteinase k from thermal inactivation.we assume that direct interaction between chaperon-like protein (within ip fraction) with proteinase k caused this proteolytic enzyme resist to heat-denaturation. whether the ip fraction could protect other enzymes from heat denaturation in vitro need further investigation. as proven by leroux coworkers (1997) and lee and co-workers (1997), direct interaction of several shsp with their target proteins to stabilize proteins, which agregate during heat or chemical treatment, is by forming a molecular chaperons. it seems that some of chaperone-like proteins within ip fraction co-operate each other to perform not only in preventing aggregation of proteinase k but also in reactivating the proteolytic activity of proteinase k during heat treatment. a study by delmas and co-workers (2001) showed lo18 protein (after sequentially purified by affinity chromatography and ion-exchange chromatography) could only reactivate catalytic function of citrate o synthase after challenged with heat treatment (45 c for 90 min) while the effect of this protein on lactate dehydrogenase only prevented it from heat-induced aggregation without restoring enzyme activity. it seems that some factors acted on refolding enzyme hence restroring its activity. in accordance with our result, the ip fraction of heat shocked-l.plantarum may contains such those factors in helping the proteinase k refolding. heat shock proteins especially those with molecular mass of ±18 kda have been intensively studied due to their pivotal role in miroorganism growth, adaptation and survival in response to environmental stress (sugimoto et al. 2008; tsakalidou and papadimitriou 2011; de angelis and gobbetti 2011). in food industry, these group of proteins exhibit significant impact on lab strain improvement especially those which are used as starter strains (carvalho et al. 2004; ricciardi et al. 2012). wineoriginating l.plantarum shsp genes, named hsp18.5 (420 nucleotide long) and hsp19.3 (444 nucleotide long) have been cloned. their respective proteins (upregulated under heat, cold and ethanol stress) have molecular mass of 18.483 and 19.282 kda and level of those proteins under different stress induction was strongly linked to inverted repeat sequence (ttagcactc-n -gagtgctaa) homologue to the 9 circe elements found to the upstream regulatory region of heat shock operons (spano et al. 2004). other shsp gene, called shsp 18.55, also succesfully cloned with only 27% identity with hsp18.5 and hsp19.3 though this shsp 18.55 protein is involved in the general stress response in wine l. plantarum (spano et al. 2005). tempoyak-isolated l.plantarum contains a gene enconding shsp 18.55 in its chromosal dna as shown by pcr result (fig 3) with size approximately 423 base pair and its amino acid sequence identity almost near the same to those of l.plantarum reference strains that habitating either food or non-food enviroments. furthermore,t-l-p-k amino acid sequence found in tempoyak-isolated l.plantarum's shsp ~18 protein is also found in lab species such as oenococcus. oeni and in positive-gram bacteria such as clostridium acetobutylicum and streptococcus thermophilus (spano et al. 2005). the exact role of shsp ±18protein on l.plantarum wcsf1 has been elucidated (capozzi et al. 2011) deletion effect of this gene significantly impact on cells morphology (the mutant clumped together, had rough surfaces and shrunken empty appearance), membrane fluidity and physicochemical surface properties. interestingly, l.plantarum u10 in our study has an ability to express shsp ±18 protein after heat shocked as proven by rtpcr. volume 8, 2014 microbiol indones 198 recently, the application of shsp protein originated from bacteria in food biotechnology gains much interest for stress biomarker in developing bacterial starter/probiotics and manufacture of foods (guzzo et al. 2012). currently, we attempt to use shsp ±18 gene as an alternative selectable marker to replace antibiotics genes in food grade expression vectors to answer regarding the safety issue in the recombinant protein production. in summary, the group of shsp which is contain chaperone-like activity in tempoyak-isolated l. plantarum has been expressed by heat shock treatment. the shsp mainly with molecular mass of ±18 kda was predicted playing an important role in heat-resistant of proteinase k in vitro the gene correspondent to shsp protein from this bacteria was successfully cloned and showed similarity properties with those of shsp originated from other l. plantarum. furthermore, the gene enconding ±18 protein has been proven to be expressed by l. plantarum u10 as respons to o temperature shift (30 to 42 c). references belfiore c, fadda s, raya r, vignolo g. 2013. molecular basis of the adaption of the anchovy isolate lactobacillus sakei crl1756 to salted environments through a proteomic approach. food res int. 54:13341341. doi : org/10.1016/j/foodres.2012.09.009. birdsell dc, cota-robles eh. 1967. production and ultra structure of lysozyme and ethylenediaminetetraacetatelysozyme spheroplast of escherichia coli. j bacteriol. 93(1):427-437. broadbent jr, oberg cj, wang h, wei l. 1997. attributes of the heat shock response in three dpecies of dairy lactobacillus. appl microbiol. 20:12-19. capozzi v, russo p, ladero v, fernández m, fiocco d, alvarez ma,grieco f, spano g.2012. biogenic amines degradation by lactobacillus plantarum: toward a potential application in wine. front microbiol.3:122. doi : 10.3389/fmicb.2012.00122 carvalho as, silva j, ho p, teixeira p, malcata fx, gibbs p. 2004. relevant factorsfor the preparation of freezedried lactic acid bacteria. int dairy j. 14:835–847. doi : 10.1016/j.idairyj.2004.02.001 collada c, gomez l, casado r, aragoncillo c. 1997. purification and invitro chaperone activity of a class i small heat-shock protein abundant inrecalcitrant chestnut seeds. plant physiol.115:71–77 delmas f, fabrice p, coucheney cd, jean g. 2001. biochemical and physiological studies of the small heat shock protein lo18 from the lacic acid bacterium oenococcus oeni. j mol microbiol biotechnol.3(4):601610. da silva sabo s, michele v, jose m, ricardo p. 2014. overview of lactobacillus plantarum as a promising bacteriosin producer among lactic acid bacteria. food res int. 64:527-536. doi:10.1016/j.foodres.2014.07.041 de angelis m, gobbetti m. 2011. stress responses of lactobacilli. in: tsakalidou, e., papadimitriou, k. 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lh. 1993. isolation of genomic dnas from plants, fungi and bacteria using benzyl chloride. nucl acids res. 21:5279-5280. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 cloning, expression and bioinformatic analysis of human papillomavirus type 52 l1 capsid gene from indonesian patient sony suhandono , dewi ayu kencana ungu , tati kristianti ,1* 1 1 edhyana sahiratmadja , herman susanto2 3and 1school of life science and technology, institut teknologi bandung, jalan ganeca 10 bandung 40132, indonesia; 2department of biochemistry, faculty of medicine, universitas padjadjaran, jalan eijkman 38, bandung 40161; 3department of obstetrics and gynaecology, faculty of medicine universitas padjadjaran/ dr. hasan sadikin general hospital, jalan pasir kaliki 190, bandung, indonesia; human papillomavirus (hpv) type 52 is the most prevalent type for causing cervical cancer in indonesian population. cervical cancer becomes the most common cancer suffered by indonesian women. prevention of hpv infection can be achieved using hpv virus-like particle (vlp) vaccine derived from l1 major capsid protein. this study aimed to clone and analyze hpv-52 l1 gene. dna obtained from biopsy of a cervical cancer patient was amplified using specific primers designed from asian originated hpv-52 l1 gene available in the genbank. the isolated hpv-52 l1 gene sequence was submitted to genbank with accession number kf225497. expression of hpv-52 l1 gene was performed using prset/emgfp expression vector. we escherichia coli analyzed and compared the hpv-52 l1 gene expressions from recombinant bl21 (de3) that had been e.coli induced for 3 hours with 1 mm iptg and without induction. the protein was expressed in insoluble form. we performed the following bioinformatic analyses: construction of phlyogenetic tree, t-cell epitopes prediction and 3d proteins structure modelling. we utilized the following softwares: mega5 for phylogenetic tree, iedbann for mhc prediction, clc dna workbench 6.5 for hydrophobicity analysis, and pdb-viewer deep for 3d protein structure analysis. the phylogenetic tree which was developed based on kf225497 sequence showed that it shared a branch with asian countries (philippines and thailand). the deduced amino acid sequences of the predicted epitopes that were consistent in all of the programs were 259gtlgdpvpgdlyiqgs274 and 345kkestykne353. this information may be useful to design diagnostic strategies and vaccine suitable for indonesian population. key words: epitope, hpv-l1 52 gene, human papillomavirus l1 protein human papillomavirus ( hpv ) tipe 52 merupakan jenis penyebab kanker serviks yang paling umum pada penduduk indonesia, terutama kaum perempuan. pencegahan infeksi hpv dapat dicapai dengan menggunakan vaksin ( vlp ) hpv yang berasal dari l1 protein kapsid utama. penelitian ini bertujuan untuk virus like particle mengkloning dan menganalisa l1 gen hpv-52. dna yang diperoleh dari biopsi pasien kanker serviks diamplifikasi menggunakan primer spesifik yang dirancang dari l1 gen hpv-52 berasal dari asia yang tersedia di genbank. urutan l1 gen hpv-52 yang terisolasi didaftarkan ke genbank dengan nomor akses [kf225497]. ekspresi l1 gen hpv-52 dilakukan dengan menggunakan vektor ekspresi escherichia coli prset/emgfp. kami menganalisis dan membandingkan l1 gen hpv-52 dari bl21 (de3) rekombinan e.coli yang telah diinduksi selama 3 jam dengan 1 mm iptg dan tanpa induksi. protein diekspresikan dalam bentuk terlarut. kami melakukan analisis bioinformatika: pembuatan pohon filogenetik, prediksi epitop sel t, dan modeling struktur 3d protein. kami menggunakan software mega5 untuk pohon filogenetik, iedbann untuk prediksi mhc, clc dna workbench 6.5 untuk analisis hidrofobik dan pdb -viewer deep untuk 3d analisis struktur protein . pohon filogenetik yang dihasilkan berdasarkan pada urutan kf225497 menunjukkan bahwa sekuen l1 gen hpv 52 berbagi cabang dengan negara-negara asia (filipina dan thailand). urutan asam amino epitop yang diprediksi konsisten dalam semua program adalah 259gtlgdpvpgdlyiqgs274 dan 345kkestykne353. informasi ini dapat digunakan untuk merancang strategi diagnostik dan vaksin yang cocok untuk penduduk indonesia. kata unci : k epitope, gen hpv-l1 52 , protein l1 human papillomavirus l1 vol.8, no.3, september 2014, p 94-102 doi: 10.5454/mi.8.3.2 * co r r e s p o n d i n g a u t h o r; ph o n e: + 6 2 2 2 2 5 11 5 7 5 fax +6222-2534107, e-mail: sony@sith.itb.ac.id persistent human papillomavirus (hpv) infection is associated with higher risk of cervical cancer development. about 79.14 million indonesian women aged above 15 have risk of cervical cancer. based on world health organization (who) report (2010), 13262 women are diagnosed with cervical cancer and 7493 die from the disease every year. precaution act using vaccination can suppress the cervical cancer occurrence. traditionally most prophylactic vaccines are made from live attenuated or inactivated virus mailto:sony@sith.itb.ac.id volume 8, 2014 microbiol indones 95 with hpv-52 (panigoro 2013). patient diagnosis et al. was performed by linear array hpv genotyping test (roche). genomic dna for pcr template was isolated from a cervical cancer patient with qiaamp dna mini kit (qiagen). primer was designed based on asian originated hpv-52 l1 sequence available in genbank. the hpv52 l1 gene was amplified using forward primer 5'ccactgtgtacctgcctcct3' and reverse primer 5'atgcagggcgttttagtttg-3'. pcr carried out with dreamtaq green pcr master mix ™ (fermentas). pcr amplification was conducted using abi® thermal cycler. touchdown pcr has been used to amplify hpv l1 gene with the following condition: initial denaturation at 95 °c for 3 min followed by 10 cycle of 95 c for 30 s, 57 °c for 30 s, 72 °c for 2 min, o 10 cycle of 95 °c for 30 s, 57-47 °c (1 c decreased o every cycle) for 30 s, 72 °c for 2 min, 10 cycle of 95 °c for 30 s, 47 °c for 30 s, 72 °c for 2 min. the cycles were followed by final extension at 72 °c for 7 min and holding at 4 °c. the amplified dna was extracted from gel electrophoresis using gel/pcr dna fragments extraction kit (geneaid). the pcr product was then cloned to pgem -t easy vector (promega) and ® transformed into straie. coli n dh5α using heat shock method. confirmed recombinant plasmid was sequenced using t7 and sp6 primers by macrogen inc., south korea. sequence analysis. forward and reverse sequences obtained from sequencing by macrogen inc. was trimmed and overlapped in order make a dna contig using clc dna workbench 6.5 software. to check hpv-52 l1 gene homology, the sequence was analyzed using basic local alignment search tool (blast) available online at http://ncbi.nlm.nih.gov. protein sequence was obtained using (swiss sib institute of bioinformatics) expasy dna translate tool available online from http://web.expasy.org/ translate/(gasteiger 2003). expression vector construction. the hpv-52 l1 gene was cloned into prset/emgfp (invitrogen). restriction sites were added to hpv l1 gene by pcr using hpv-52 l1 specific primers with restriction site sequence added at the 5' end. the primers were forward primer 5'tccactgtgtacctgcct 3' ggatcc and reverse primer 5'ttagattatgcagaattc gggcgttttag -3'. stop codon was added at the 5' end of the reverse primer. restriction sites used for gene insertion to prset-emgfp expression vector were i and i. pcr was carried out withbamh ecor (varsani 2003). however, no recent report has et al. shown successful attempt on growing papillomavirus in media culture (smidkova 2010). major protein et al. l1 and minor protein l2 that composed hpv icosahedral capsid, can be used as vaccine. l1 capsid protein alone has been proven to self assembles into virus-like particle (vlp) (smidkova 2010). hpv et al. vlps proved to induce high titer of neutralizing antibodies in animal model (suzich 1995 and et al. bretburd 1995). various expression systems such et al. as animal cells (zhou 1991), insect cells (rose et al. et al et al.. 1993), yeast (sasagawa 1995), plant cells (biemelt 2003 and warzecha 2003) and et al. et al. bacterial cells (seo 2009) have been used to et al. produce vlps. antigenic epitope recognition by t-cell epitope is the key molecular event that triggers the immune response in infected organism (mohatbakar 2007). epitope identification was also conducted to assure that the vlps generated from escherichia coli expression system were suitable for indonesian patient. papillomaviruses have been detected in a various animals as well as in humans. over 150 types of hpv had been fully characterized (chen 2011). not all et al. hpv types can infect genital part such as cervix, vagina, vulva, penis and anus. only thirty hpv types are known as genital types. these genital types can be divided into two types: low and high risk types, according to the virus's association with genital cancer. low-risk hpv types include types 6, 11, 42, 43,and 44, which usually cause benign genital warts. high-risk hpv types include types 16, 18, 31, 33, 34,35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70, which cause genital cancer (gomez and santos 2007; de villiers 2004). even though hpv types 16 and 18 had been shown to have high prevalence in cervical cancer infection in indonesia, current study had also shown that hpv-52 also had high prevalence in certain regions of indonesia (vet 2008).this study suggested et al. incorporation of hpv 52 in indonesian hpv vaccine design. in this study, we aimed to clone, express and analyze hpv-52 l1 collected from indonesia. materials and methods the hpv-52 l1 gene isolation and cloning. study protocol was approved by medical faculty of universitas padjadjaran ethical committe no. 190/fkup-rshs/kepk /kep./ec/2010. biopsy was performed from one cervical cancer patient infected http://ncbi.nlm.nih.gov. http://web.expasy.org/ 96 s uhandono et al . microbiol indones fig 1 aligment result of prset/emgfp-hpv52-l1 amino acid sequence [1] and hpv-52 l1 amino acid sequence [2]. red: histag; yellow: xpress epitope; green: enterokinase binding site. kapa2g fast hot start ready mix (kapabiosystem). touchdown pcr has been used to amplify hpv l1 gene with the same cycling parameters used in cloning. to generate the recombinant plasmid prset/emgfp hpv52l1, both pcr product and plasmid were digested using i and i. both digested bamh ecor fragment were ligated using t4 dna ligase overnight at 4 °c. the prset/emgfp-hpv52l1 sequencing was performed in macrogen, korea. restriction analysis and sequencing proved that both recombinant plasmids were constructed correctly. recombinant hpv-52 l1 expression and inclusion bodies isolation. the bl21(de3) e.coli containing recombinant plasmid prset/emgfp hpv52l1, were grown in 5 ml lb with ampicillin (10 µg ml ) overnight. the overnight culture was 5 -1 inoculated to 500 ml lb medium with ampicillin (10 µg ml ) and grown at 37 °c, with 200 rpm shaking 5 -1 until exponential phase (od = 0.8). the hpv-52 l1 600 expression was induced by addition of 1 mm iptg and then incubated for 3 h at 37 °c, with 200 rpm shaking. the induced cell was harvested by centrifugation at 5000 g for 20 min. the cell pellet was resuspended in 6 ml 20 mm tris-hcl ph 8, sonicated on ice (6x10 s) and centrifuged at 5000 g or 10 minute at 4 °c. then, the cell pellet was resuspended in 3 ml cold solution containing 2m urea, 20 mmtris-hcl, 0.5 m nacl, 2% triton x-100 ph 8 before being sonicated again on ice (6x10 s) and centrifuged at 5,000 g for 10 minute at 4°c. afterwards, the cell pellet was washed twice with urea containing-buffer and once with urea lackingbuffer (20 mmtris-hcl). the cell pellet was then stored frozen for later use. phylogenetic tree construction. phylogenetic tree was constructed based on the maximum likelihood with mega 5.05 software. hpv-52 l1 gene sequences fig 2 sds-page prset/emgfp-hpv52-l1. the size of protein of interest is 48 kda polyacrylamide gel 12%. a:pellet-induced ;b: supernatant-induced; c: pelletnon induced ; d: supernatantnon induced volume 8, 2014 microbiol indones 97 fig 3 hpv-52 l1 phylogenetic tree. this phylogenetic showed that hpv-52 l1 gene from asian countries including indonesia forms its own cluster. table 1 t-cell epitopes on sequence of hpv-52 l1 from indonesia hla-drb1*12:02 hla-drb1*15:02 hla-drb1*07:01 dslffflrreqmfvr slffflrreqmfvrh gdslffflrreqmfv asledtyrfvtstai sledtyrfvtstait ledtyrfvtstaitc edtyrfvtstaitcq dtyrfvtstaitcqk tyrfvtstaitcqkn yrfvtstaitcqknt lrreqmfvrhffnra rreqmfvrhffnrag reqmfvrhffnragt eqmfvrhffnragtl qmfvrhffnragtlg mfvrhffnragtlgd fvrhffnragtlgdp asledtyrfvtstai sledtyrfvtstait ledtyrfvtstaitc edtyrfvtstaitcq dtyrfvtstaitcqk tyrfvtstaitcqkn yrfvtstaitcqknt tsiyyyagssrlltv srtsiyyyagssrll rtsiyyyagssrllt siyyyagssrlltvg vsrtsiyyyagssrl iyyyagssrlltvgh yyyagssrlltvghp hla-a*11:01 and hlaa*33:03 andhla-b*15:02 alleles. human leukocyte antigen (hla) class ii sequences used for this study were hla-drb1*07:01, hla-drb1*12:02 and hla-drb1*15:02. these alleles had been chosen because of their high frequency of occurence in western javanese (indonesia) population (yuliwulandari 2008). hydrophobicity and antigenicity graph was created using clc dna workbench 6.5. l1 protein structure modelling was done using homology modelling: automated mode which can be accessed through http://swissmodel.expasy.org. l1 protein model then analyzed using sib:deep viewer (expasy). results hpv-52 l1 gene isolation, cloning and expression vector construction. hpv-52 l1 gene was isolated from a cervical biopsy tissue of an used to construct phylogenetic tree were obtained from ncbi genbank database [http://www.ncbi.nlm.nih. gov]. the sequence origins and accession numbers of hpv-52 l1 genes used in the construction of the phylogenetic tree were costa rica [hq537740], china [gq472848], thailand a [hq537743], thailand b [ h q5 37 74 2 ], z a mbi a [ h q5 37 7 36 ] , rw a n da [hq537735], panama [u45923], philippine a [u45922], philippine b [u45921], germany [u45920], bolivia [u45919], and spain [u45918]. epitope prediction. further analyses had also been conducted using iedbann software in order to determine the epitope regions. this software is based on artificial neural network (ann) algorithm. iedbann is available online at http://tools.immuneepitope.org/ analyze/html/mhc_binding.html. this tool was chosen because it mightpredict peptide binding to hla class i and ii molecules (gustiananda 2011). human leukocyte antigen (hla) class i sequences that had been used for this study were hla-a*24:07, http://swissmodel.expasy.org. http://www.ncbi.nlm.nih. http://tools.immuneepitope.org/ 98 s uhandono et al . microbiol indones fig 5 hydrophobicity and antigenicity plot of hpv-52 l1 protein. this plot showed regions of hydrophobicity and antigenicity on hpv-52 l1 protein sequence. higher number means higher hydrophobicity and antigenicity property on that particular amino acid position. fig 6 indonesian hpv-52 l1 3d model structure. red: loop; green: ; blue: nd. a: 46tssgngkkvl55, b: α-helix β-stra 124etsnkyagkpgidnr138, c: 67tpcnnnsgnpgdc179, d: 259gtlgdpvpgdlyiqgs274 and e: 345kkestykne353. fig 4 t-cell epitope candidate site on sequence of indonesian hpv-52 l1. a: hla-drb1*07:01; b: hla-drb1*12:02; c: hla-drb1*15:02. purple band represent the peptide sequence of epitope candidate on hpv-52 l1 sequence. volume 8, 2014 microbiol indones 99 epitope prediction. we combined three analytical methods to determine hpv-52 l1 epitope. the first method that had been used was mhc analysis using iedbann. peptides between 8 12 mers length were obtained. we restricted the analysis only to peptides with ic50<50nm or netctlpan score <1% to ensure they bound strongly to the hla molecules. peptide with strong affinity to hla molecule would most likely become t-cell epitope. list of peptide with strong affinity towards hla molecule is provided in table 1. hla class ii alleles that had been used in this study were hla-drb1*12:02, hla-drb1*15:02 and hla-drb1*07:01. these alleles had been chosen because they have high frequency in western javanese (indonesia) population (yuliwulandari 2008).the alleles hla-drb1*15:02 covered 23%, hladrb1*07:01 covered 37.8% and hla-drb1*12:02 covered13.1% of western javanese population (gustiananda 2011). we found that the t-cell epitope candidates have different affinity level towards each hla class we studied. in figure 3, we described the hpv-52 l1 sequence parts that have high affinity towards hla molecule. peptide with strong affinity to hla molecule would most likely become t-cell epitope. we found out that our t-cell epitope candidates of hpv-52 l1 have strong affinity towards both hla-drb1*15:02 and hla-drb1*07:01. the t-cell epitope candidates mainly reside around 400aa position. the second method that has been used is hydrophobicity and antigenicity analysis. in this analysis, we searched for sequences with low hydrophobicity value and high antigenicity value. hydrophobicity and antigenicity graphs of hpv-52 l1 proteins were obtained from clc dna workbench 6.5. fig. 5 shows that hydrophobicity peaks are inversed from antigenicity peaks. regions of antigenicity are 100-120aa, 295-310aa, 340-350aa, and 410-430aa. we suggest that in these areas resides t-cell epitope. the third and final method is 3d protein structure modelling. we used protein model from protein data bank [pdb id: idzl] as modelling template. this template was chosen because it has deviation value of rms 0.7å. rms deviation values under 1å indicates good aligment between model and template. using sib:deep viewer, 5 loop regions in the model of the hpv-52 l1 were determined. the loop regions were determined by the analysis of polar amino indonesian patient using specific primers designed for this study. hpv-52 l1 capsid gene was successfully cloned to pgem -t easy cloning vector and ® prset/emgfp expression vector. recombinant plasmid sequence analysis showed that the hpv-52 l1 fragment was in-frame with prset/emgfp start codon. to identify the dna fragment obtained, bl as t a na ly si s wa s c o nd uc te d o nl in e a t http://blast.ncbi.nlm.nih.gov. the result shows 99 % similarity with the sequence in genbank with accession number jn874433 the cloned hpv-52 l1 . sequence did not contain 99 bp of the 5' and 159 bp of the 3' regions of the genbank sequence jn874433. the hpv-52 l1 gene obtained (1444 bp) was registered to genbank with accession number kf225497. recombinant hpv-52 l1 expression and inclusion bodies isolation. the prset/emgfphpv52l1 was transformed to bl21(de3). e.coli after the culture reached exponential phase, the expression of hpv-52 l1 protein was induced for 3 hours by 1 mm iptg for 3 hours at 37 °c, with 200 rpm shaking. induction started when the transformed e.coli bl21(de3) culture reached exponential phase (od = 600 0.8). these expression conditions have been optimized for high hpv-52 l1 protein yield. the hpv-52 l1 was expressed as fusion protein with histidine tag. sdspage analysis of the cell lysate showed an approximately 48 kda protein presents in the cell pellet. however, no significant protein band was found in the supernatant. this result showed that the protein expressed in insoluble form. inclusion bodies isolation was performed to isolate the hpv-52 l1. phylogenetic tree construction. phylogenetic tree was constructed using mega 5.05. maximum likelihood method was used for construction of the phylogenetic tree (mathura and kagueane 2009). hpv-52 l1 gene sequences used for constructing phylogenetic tree were obtained from ncbi genbank database [http://www.ncbi.nlm.nih.gov]. we used 17 different sequences from different geographic locations to visualize the evolutionary relationship of the indonesian hpv-52 l1 gene with other known hpv-52 l1 genes. genbank has limited hpv-52 l1 dna sequence that has complete origin country data. unfortunately we were unable to find other hpv-52 l1 originate from indonesia. we compared the hpv-52 l1 sequence with other hpv-52 l1 sequences from asia (philippine, thailand, and china), africa (zambia and rwanda), america (panama, bolivia, and costa rica), and europe (germany and spain). the result is presented in fig. 3. http://blast.ncbi.nlm.nih.gov. http://www.ncbi.nlm.nih.gov 100 s uhandono et al . microbiol indones acids and the accessibility of the protein groups. the loop regions obtained are 46tssgngkkvl55, 124etsnkyagkpgidnr138, 67tpcnnnsgnpg dc1 79, 25 9gt lgd pv pgdly iq gs274 a nd 345kkestykne353.we found three structure differences after we compared the resulting hpv-52 l1 model with its template. all difference we discovered located on the loop regions at 23-32aa, 269279aa, and 349-358aa. discussion human papillomavirus (hpv) can enter target cells through minor damage of the genital mucosa. if this high risk-hpv infection becomes persistent, it can lead to cervical cancer (burd 2003). vaccination can be a feasible solution to prevent cervical cancer development caused by high risk hpv. hpv vaccine can be made using hpv capsid protein called l1 protein. in this study we successfully cloned, expressed and analyzed hpv-52 l1 gene isolated from an indonesian cervical cancer patient. the hpv-52 l1 obtained was registered to genbank under accession number kf225497. expression vector, prset/emgfp, was e.coli used as a vector for hpv-52 l1 expression. expression of his-hpv52 l1 fusion protein was induced with 1mm iptg at 37 c when the culture reached o exponential phase (od =0.8). induction at mid 600 exponential phase (od =0.8) gave the best result 600 compare to induction at late exponential phase (od =1.6 or 2.3). his-tag was fused to hpv-52 l1 600 protein to facilitate protein purification. his-tag binds to nickel or cobalt ions that immobilized on a support matrix such as nta (nitrilotriacetic acid) (bornhorst 2000). ni-nta affinity chromatography has been proven to produce higher protein yield than gst-tag (scheich 2003). the position of his-tag at a et al. recombinant protein determines the expression level of recombinant protein. his-tag at n-terminus could increase stability of translation initiation region on mrna structure (block 2009).the expressed et al. his-hpv52 l1 fusion protein was found in insoluble form. this occurred when the protein was expressed at 37 c. higher solubility might have been achieved if o protein had been expressed in cold condition (seo et al. 2011). the phylogenetic tree showed that the variation of hpv-52 l1 viral dna occurs geographically. these variations may influence viral infection and development of cervical cancer. mutation can end up with amino acid substitution that can affect viral assembly, carcinogenic potential and host immune responses. human immune mechanism against hpv infection was not fully study yet. we cannot be certain that vaccines developed from certain hpv types can protect against infection of other types hpv. l1 gene variations can influence the persistence of hpv infection to cause cervical cancer (frati et al. 2011). the phylogenetic tree indicates that the hpv-52 l1 indonesia is located at the same branch with other asian countries (thailand and philippines), while not too close to its counterparts derived from european isolates. according to chen (2011) et al. hpv variants may differ in terms of pathogenicity despite its phylogenetic proximity. hpv type 18, 16 and 52 variants of non-europeans are commonly found in cancer tissues and cervical injuries (vet et al. 2008 and chen 20). hpv vaccine is commercially et al. available from cervarix and gardasil. both vaccines are made of l1 protein of hpv types 18, 16, 11 and 6,which came from europe (who 2010). our phylogenetic studies suggest that these vaccines may be less suitable for indonesian. both vaccines have been licensed for use in indonesia since 2009; however there has been no report regarding their degree of efficacy and effectiveness (who 2010). designing va ccine candidate by epitope prediction can save production cost. in this in-silico study we used three methods to determine epitopes of hpv-52. they were mhc analysis, hydrophobicity, antigenicity and protein 3d structure. mhc class i and ii is associated with cd8 + t-cells and cd4 + in cell-mediated immune response (cell mediated immune response / cmi) respectively. both molecules mhc class i and ii have different molecular structures, but both have a peptide binding region. in humans, major histocompability complex (mhc) are also known as human leukocyte antigen (hla) (murphy 2008). hla class i and class ii genes et al. have the highest rate in the human genome polymorphism. in this study, only hla genes class ii with high allelic frequency in indonesian population had been used for epitope prediction. the class ii hladr,-dq and -dp are primarily expressed by antigen presenting cells, like dendritic cell, and can present processed antigen to cd4 t-cells (stern 2004). cd4+ + cells are important to help b cells to differentiate to antibody secreting plasma cells and memory b-cells (stanley 2008). predicted t-cell epitopes were plotted based on volume 8, 2014 microbiol indones 101 antigenicity for antibodies of other hpv types. therefore, application of vaccines designed for one type may not be able to prevent hpv infection byother hpv types. it is imperative to add other types of hpv to the vaccine design to achieve broader cases coverage. addition of hpv type 31, 33, 35, 45, 52 and 58 to the hpv 16/18 vaccine design are estimated to prevent 90% of worldwide cervical cancer burden, which equal to 440,000 cases (clifford 2006). the incorporation of the data from the mhc analysis, hydrophobicity, antigenicity and protein 3d structure, the epitope regions of the hpv-52 l1 protein isolated from i n d o n e s i a u s e d i n t h i s s t u d y a r e 259gtlgdpvpgdlyiqgs274 and 345kkestyk ne353. acknowledgement this research was supported by grant from stranas dikti. references biemelt s, sonnewald u, galmbacher p, willmitzer l, muller m. 2003. production of human papillomavirus type 16 virus-like particle in transgenic plants.j virol . 77(17): 9211-9220. doi: 10.1128/jvi.77.17.92119220.2003. block h, maertens b, spriestersbach a, brinker n, kubicek j, fabis r, labahn j, schäfer f. 2009. immobilized-. metal affinity chromatography (imac): a review. method enzymol 463: 439474.. bordoli l, kiefer f, arnold k, benkert p, battey j, schwede t 2009. protein structure homology modelling using . swiss-model workspace. nat protoc. 4: 1-13. doi:10.1038/nprot.2008.197. bornhorst ja, falke jj. 2000. purification of protein using polyhistidine affinity tags. methods enzymol 336: . 245-254. breitburd f, kirnbauer r, nl. hubbert, b. nonnenmacher, c. trindinh-desmarquet, g. orth, jt. schiller, dr. lowy. 1995. immunization with viruslike particles from cottontail rabbit papillomavirus (crpv) can protect against experimental crpv infection. j. 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the dna double chain (burd 2003). l1 protein can form a virus like particle (vlp) that is structurally and immunogenically similar to the infectious hpv. vlp composed of l1 protein has the ability to bind monoclonal antibodies. hydrophobicity and antigenicity plot (fig 5) indicated that the hydrophobicity values are inversed of antigenicity values. this result is supported by the fact that most often antigen determinants (epitopes) are located in polar hydrophilic areas, so it can easily be recognized by antibody (mohatbakar 2007). antigen determinants are less likely located in the hydrophobic area because this area tends not to be exposed at the surface. hpv-52 l1 protein 3d structures (fig 6) were made using automated mode homology modelling t h a t c a n b e a c c e s s e d o n l i n e a t t h e s i t e ht tp ://s w iss mod e l. e xp a sy. org . t hi s p r og ra m automatically creates a protein 3d model based on the homology of amino acid sequences (input) with amino acid sequence 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[10 summary report 2010 june 2013]. available at www.who.int/ hpvcentre. yuliwulandari r, kashiwase k, nakajima h, uddin j, susmiarsih tp, sofro sm, tokunaga k. 2008. polymorphism of hla genes in western javanese ( i n d o n e s i a ) : c l o s e a ff i n i t i e s t o s o u t h e a s t asianpopulation.tissue antigens. issn 0001-2815. zhou j, sun xy, stenzel dj, frazer i. 1991.expression of vaccinia recombinant hpv 16 l1 and l2 orf proteins in epithelial cells is sufficient for assembly of hpv virion-like particles. virology 185: 251257. http://www.who.int/ dewi16042012 available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.1.3issn 1978-3477, eissn 2087-8587 vol 6, no 1, march 2012, p 15-22 cloning and expression of nonstructural protein ns1 of dengue virus serotype 2 beti ernawati dewi*, fithriyah, andriansjah rukmana, paisal, deka larasati, and tjahjani mirawati sudiro department of microbiology, faculty of medicine, universitas indonesia, jalan pegangsaan timur 16, jakarta 10320, indonesia early diagnosis of dengue virus (denv) infection is affirmative for patient management and control of the disease. detection of nonstructural-1 (ns1) antigen has been proven to provide early detection of denv infection. commercial ns1 antigen assays are available in indonesia with variable sensitivity. in an attempt to develop an ns1-based diagnostic test, we successfully cloned ns1 gene of denv2 to a glutathione stransferase-based vector pgex6p-1 in escherichia coli system. the recombinant protein (pg2ns12) was o expressed in e. coli bl21. after induction with isopropyl-β-d-thiogalactoside 0.1 mm for 4 h at 25 c a recombinant protein gst-ns1 with molecular size of approximately 75 kda was obtained. the fusion protein -1 was insoluble and found in the pellet fraction of the cell lysate. addition of lysozyme (10 mg ml ) and dnase-i -1 (7.2 mg ml ) in the lysis buffer was necessary to collect proteins from the pellet fraction. the proteins in the cell pellet were fractionated through sephadex-g100 column, and gst-ns1 was further purified with glutathionesepharose 4b beads. to obtain pure recombinant ns1 protein to be used in the immunization of mice, the fusion protein was cut with prescission protease® by addition of 0.075% triton-x 100 was necessary to cut the fusion protein. we found that antibodies that recognized the recombinant ns1 protein and denv2 virus were produced in mice immunized with purified ns1 protein. therefore, our recombinant ns1 could be used to produce antibody that is potentially useful for developing diagnostic assay to determine the presence of dengue virus ns1 antigen in patient sera. key words: cloning, dengue virus, expression, non structural protein ns1 diagnosis dini infeksi virus dengue (denv) amat penting dalam penatalaksanaan penderita dan pengendalian penyakit ini. deteksi antigen nonstructural 1 (ns1) telah terbukti mampu mendeteksi dini infeksi denv. uji antigen ns1 komersial telah tersedia di indonesia dengan berbagai tingkat sensitivitas. dalam usaha mengembangkan uji diagnosis berdasarkan ns1, kami telah berhasil mengklona gen ns1 denv2 pada vector pgex6p-1 yang berbasis glutathione s-transferase pada system escherichia coli. protein rekombinan pg2ns12 diekspresikan di e. coli bl21. setelah induksi dengan isopropil-β-d-tiogalaktosida 0.1 mm selama o 4 jam, pada suhu 25 c didapatkan ekspresi fusi protein rekombinant gst-ns1 berukuran sekitar 75 kda. -1 -1 penambahan lisozim (10 mg ml ) dan dnase-i (7.2 mg ml ) dalam dapar pelisis diperlukan untuk mendapatkan protein rekombinan dari fraksi pelet sel. protein rekombinan difraksinasi menggunakan kolom sephadex-g100 dan dilanjutkan dengan purifikasi menggunakan gluthatione-sepharose 4b. untuk mendapat protein ns1 murni yang digunakan dalam imunisasi mencit, protein fusi gst-ns1 dipotong dengan prescission protease® dengan penambahan 0.075% triton-x 100 pada reaksi. imunisasi mencit dengan protein ns1 menginduksi antibodi yang dapat mengenali protein ns1 rekombinan dan virus denv2. hasil ini menunjukkan bahwa ns1 rekombinan dapat digunakan untuk memproduksi antibodi yang berpotensi dalam pengembangan suatu uji diagnostik untuk mendeteksi keberadaan antigen ns1 dalam serum penderita. kata kunci:, ekspresi, kloning, protein non struktural ns1, virus dengue dengue virus (denv) infection is a major problem in indonesia (setiati et al. 2006). it can manifest as mild dengue fever (df), severe dengue hemorrhagic fever/dengue shock syndrome (dhf/dss), or even fatal. early diagnosis of denv infection is affirmative for patient management and control of the disease. diagnosis of denv infection is now mainly based on clinical observation and routine hematologic tests. rapid serological test is available, but it gives good results only after the formation of igg/igm, i.e. five days after the onset of illness. rapid early diagnostic test, such as rt-pcr test is also available, but they are expensive and need special equipment to conduct (shu and huang 2004). thus, despite the availability of those tests, development of new rapid early diagnostic test to detect denv infection remains a challenge. detection of ns1 antigen has been proven to provide early detection of denv infection (alcon et al. 2002; dussart et al. 2008). ns1 protein is a nonstructural protein which is important in the replication of denv (perera and kuhn 2008). it can be found *corresponding author, tel: +62-21-3100806, fax: +62-21-3100810, e-mail: betied@yahoo.com 16 dewi et al. microbiol indones intracellularly, or in association with micro particles but not with virions and extracellulary. to develop an ns1-based diagnostic test, we attempted to clone ns1 protein of denv2 in e.coli system. the glutathione s-transferase system is chosen because it provides integrated system for expression, purification and detection, and already widely used to produce various viral proteins (matusan et al. 2001; ma et al. 2008). in the process, we still faced some problems in the expression and purification of the recombinant protein. here, we described the construction of the recombinant protein gene in a vector and several methods to purify the protein to find the best way to obtain pure ns1 protein, and also test of the antigenicity of the protein in mice. materials and methods rna extraction. rna denv-2 was obtained from patient plasma with code number of ds31/06 in jakarta in 2006. rna was extracted from 140 µl of plasma using qiaamp® viral rna kit (qiagen, ici americas inc.) according to the manufacturer's instruction. complementary dna (cdna) strands were reverse-transcribed using super script ii first strand synthesis system with random hexanucleotide primer according to the manufacturer`s instructions (invitrogen, california, usa). the pcr amplification of the entire genome was performed using the cdna tm products in a ptc-100 programmable thermal cycles (mj research, inc). recombinant plasmid. pgd ns12 plasmid was 2 constructed by inserting ns1 dv2 gene and its 72 bp upstream fragment into pgex6p-1 plasmid (amersham pharmacia biotech, 1997). first, cdna was obtained as stated above. sequencing of e and ns1 region of the virus was done preceeding the cloning process. full length of ns1 gene was amplified by pcr with sense primer (d2 2329sbam: 5'cgcga ggatcctggataggaatgaattcacgc-3') and anti sense primer (d2 ns-1-350 csal : 5'tccgct gtcgactcaggctgtgaccaaggagtt-3'). the primers contained upstream bamhi and downstream sali restriction sites (underlined nucleotides). to improve protein expression, the amplification of ns1 region included 72 bp up stream of ns1 (falgout et al. 1989). for cloning, pcr product was purified with a qiaquick® pcr purification kit (qiagen, ici americas inc) and digested with bamhi and sali enzymes. after purification with gel extraction kit (qiagen), the ns1 fragment was inserted into pgex-6-1 using t4 dna ligase (promega) and the ligation mixtures were transformed into competent e. coli top 10 (invitrogen, california, usa) (ausubel et.al. 1994). selection of recombinant bacteria was done on lb agar containing -1 ampicillin 60 µg ml . the size and orientation of the recombinant plasmid pgex-6-1-ns1 (fig 1) was confirmed by bali restriction enzymes digestion and pcr with primer pair of ns1 sense and antisense primers recognizing the vector part. the recombinant pgex-6-1-ns1 was sequenced to confirm the cloned ns1 gene nucleotide sequence. the nucleotide sequence of the cloned ns1 gene and its deduced amino acid sequence were aligned to the sequence of the original virus as well as to the published sequences for den-2 strain existed in genbank database. g s td v 2 n s 1 p ro t e i n e x p re s s i o n . f o r expression purposes, the recombinant plasmid was subcloned into e. coli bl21. to optimize expression, several conditions were carried out, i.e.: 1) iptg inducer concentrations (0.05, 0.1, 0.25, 0.5, 0.75, and 1 mm); 2) induction time (1, 2, 3, and 4 h); 3) induction temperature (room temperature (25 ºc) and 37 ºc). iptg concentration and time of induction were considered in relation with the amount of protein product and the possibility of inclusion bodies formation which can interfere the purification of recombinant protein. the recombinant e. coli bl21 carrying were fig 1 construction of pgst-ns1 dv2. ns1 dna fragment with 72 bp upstream sequence was inserted into bamhi and sali sites of vector pgex-6p-1. lac pro lac z orf3 gst bali precision ns1 ampr-pro ampicillin pbr322 origin pgex6p-1-ns1 6123 bp lacl 1 bali volume 6, 2012 microbiol indones 17 cultured in luria bertani (lb) agar supplemented with -1 o 60 µg ml ampicillin at 37 c for 18 h, followed by -1 subculture in lb broth containing 60 µg ml o ampicillin at 37 c shaked at 200 rpm. after 18 h, it was subcultured again with starting od 0.01, and 600 o incubated in rotary shaker at 37 c until it reached od 600 0.5. expression of the fusion protein was induced with isopropyl-1-thio-β-d-galactopyranosine (iptg) 0.05 o to 1 mm, 1 to 4 h, 37 c. four hours after iptg induction, the bacteria were resuspended in phosphate buffer saline (pbs) ph 7.3 -1 containing lysozyme (10 mg ml ) and dnase-i (7.2 -1 o mg ml ) and incubated 5 min, 37 c. the bacterial cells were lysed by freeze-thaw method (30 cycles of dry ice o and 60 c bath) or by sonication (30 cycles of 30 output 30 s pulse on and 10 s pulse off). the lysate was centrifuged at 13 000 x g, 10 min. then the supernatant was collected and the pellet was resuspended in pbs, and stored in -80 ºc until tested. the pellet and supernatant fractions were examined by sds polyacrylamide gel electrophoresis 8% (sds-page). purification of gst-ns1 denv2. to reduce the cellular proteins found in the cell pellet, the suspension of the cell pellet was fractionated through sephadexg100 colum, and the fractions were analysed by sdspage 8%. the fractions with high concentration of the expected protein were taken and gst-ns1 denv-2 protein was further purified using glutathionesepharose 4b beads (bulk gst purification modules, ge healthcare) by modified manufacturer's method. the fraction containing recombinant protein was added with 50% gluthatione sepharose 4b and was incubated at 4 ºc for 18 h. the mixture was passed through bulk gst. here, the protein bound to the glutathionsepharose, and then eluted with elution buffer. excission of gst-ns1 with prescission protease. we used prescission protease ® (ge healthcare) to cut the ns1 protein from gst-ns1 by modified manufacturer's protocol. several conditions were carried out to optimized excition proceses, i.e.: 1) in column or in solution; 2) duration of reaction; 3) with or without addition of triton-x100. in column, treatment was done by passing the protein into the 120 glutathione-sepharose 4b (ge healthcare) column, followed by adding the prescission protease and then mix them. the reaction was incubated at 5 °c for 6 to 20 h. the colum was then centrifuged at 500 g for 5 min at room temperature. in solution, treatment was done by incubating the fusion protein with the prescission o protease at 5 c for 6 to 20 h. after incubation, the reaction mixture was passed through the glutathionsepharose column. the gst fragment of the fusion protein and the protease would bind to the glutathione, but the ns1 fragment would pass during elution. immunogenicity of recombinant ns1 denv2 protein. immunogenicity study was done in mice. immunization of mice was carried out by method described previously (ausubel 1994) with minor modifications. six mice balb/c aged 6 to 8 weeks were immunized. for the first immunization either gstns1 fusion protein or purified ns1 protein were injected intraperitoneally into four mice and two mice, respectively. a hundred µg protein in the suspension of complete freund's adjuvant were injected to each mouse. three and two weeks later, respectively, the animals were given the first booster using 25 µg of purified ns1 protein in suspension of incomplete freund's adjuvant. this procedure was repeated as the second booster two weeks later. a week after the second booster samples of mice sera were taken from tail vein and tested for anti-ns1 antibody by elisa. elisa was done using purified ns1 protein and denv2 virus as antigen. sds-page, western blotting, and elisa. sdspage and western blotting were used to determine whether rns1 protein was expressed. the e. coli -1 pellets were added with 100 ng ml lysozyme in tris edta and incubated for 5 min at 37 ºc. the mixture was dissolved in loading buffer (1% sds, 1% of 2–mercaptoethanol, and 200 mm dtt) and then boiled for 5 min, chilled on ice for 5 min and separated by 8% sds-page in vertical electrophoresis unit (biorad, california, usa). two gels were run at a time. one of the gels was stained with coomasie blue staining solution and the molecular size was determined using the molecular weight standard (sigma-aldrich co). the other gel which was unstained was transferred onto nitrocellulose membrane (amersham pharmacia) by a transblot tm cell (biorad, california, usa) filled with methanol-tris glycine buffer. to confirm protein transformation, membrane was stained with ponceau s solution for 5 minutes at rt and destained with washing buffer (500 ml 1xpbs and 100 µl tween 0.1%). the membrane was blocked with 5% skim milk in pbs for 18 h at 4 ºc. ns1 protein expression was determined by incubating the membrane with dengue positive sera and monoclonal antibody anti-gst-hrp conjugate. ns1 protein was visualized by addition of dab substrate with h o for 5 min at rt.2 2 indirect elisa was done according to the method described by igarashi a (2000) in technical manual of arbovirus study with special emphasis on japanese microbiol indones enchepalitis and dengue viruses. elisa plates were -1 coated with 100 µl ns1 (2.3µg µl ) in 1:25 coating o buffer and incubated overnight at 4 c. after adding 300 µl blocking buffer containing 5% low fat milk (tropicana slim, pt nutrifood indonesia, jakarta) in pbs ph 7.3 for 1 h at room temperature, they were rinsed with 300 µl washing buffer (pbs/tween 20) three times. after that, the test sera diluted 1:50 were o added and incubated for one hour at 37 c, followed by rinsing with washing buffer. the secondary antibody (goat antimouse igg hrp or goat antihuman igg hrp) (sigma aldrich, missouri) diluted 1:5000 in skim milk o 1% was added and incubated for one hour at 37 c, before the third rinsing. for detection, 100 µl substrate h o + tmb (3,3′,5,5′-tetramethylbenzidine) 2 2 (kirkegaard & perry laboratories, maryland) 1:1 was incubated for 10 min at room temperature in the dark. the reaction was stopped by addition of 100 µl h so 2 4 3n, and the od was read by elisa reader (bio-rad 450 model 550, california). results construction of pgst-ns1 dv2. the amplified and purified ns-1 gene fragment was inserted into expression vector pgex-6p-1 to generate a recombinant plasmid pgex-ns1. the purified and digested ns-1 gene was cloned in the correct frame with the gst at c-terminus of pgex-6p-1 for high level of protein expression in e. coli. transformation of ligation mixture into e. coli top10 resulted in about 120 colonies. random 16 colonies were chosen for further analysis. recombinant clones (pgd ns9 and 2 pgd ns12) with correct size were selected for protein 2 expression (fig 2). sequencing of the inserts showed that no mutations found in four known b-cell epitopes of ns1 of the selected recombinant plasmid (fig 3). expression of gst-ns1 dv2. the expected 75kd protein of gst-ns1 dv2 can be seen after induction of e. coli bl21 containing pgd ns12 (fig 4 and 5). the 2 best result was obtained when induction was done with 0.1mm iptg for 4 h at room temperature (data not shown), but the protein was mainly insoluble, and located in the pellet fraction of the cell lysate. the recombinant protein might be expressed as inclusion bodies. to increase the solubility of the protein in supernatant phase we tried other lysis buffers such as ntt buffer (1.5% n-lauroysarcosine, 1% triton x100, 150 mm nacl, 10 mm tris, ph 8.0), or lysis buffer containing 10 mm tris ph.8,0; 0.1% triton-x; 0.5 mm pmsf ; 0,1% lysozyme, and 5 mm imidazole). but these buffers seemed to interfere with the affinity of the fusion protein to the glutathione-sepharose beads during purification step. so we did not use additional reagents for further processess, and instead, we focused on optimizing the purification of the protein from the pellet fraction. addition of lysozyme and dnase into pbs in lysis buffer significantly improved cell lysis as could be seen by translucent appearance of the final results and after centrifugation the pellet could be easily resuspended in pbs. purification of gst-ns1 dv2 fusion protein. the gst-ns1 protein was in gluthatione-sepharose 4b slurry and the protein was cut with prescission protease® with the protocol provided by the manufacturer. cleavage was performed in both solution and in column, however the protease failed to cut the protein. addition of a new enzyme did not improve the cleavage. so we tried to add triton x-100 into the reaction, and the results showed that an addition of 0.075% triton x-100 cut the fusion protein successfully. both cleavage in solution and in glutathione-sepharose column gave similar results. however, cleavage in glutathione-sepharose column had the advantage of reducing the amount of enzyme used. after cleavage, gst fragment and the protease remained bound to the glutathione and the ns1 protein passed through the column. immunogenicity of ns1 protein. mice were m 1 2 3 4 5 6 6557 bp 2027 bp 564 bp 846 5287 cut bali uncut fig 2 analysis of recombinant clones. m: λ hindiii ; lane1 and 4. pgex-6p-1 wt; lane 2 and 5. pg2ns9; lane 3 and 6: pgns12. lane 1 to 3: the plasmids were cut with bali; lane 4 to 6: the uncut plasmids. 18 dewi et al. volume 6, 2012 microbiol indones 19 immunized intraperitoneally with ns1 protein. one week after the second booster, samples of mice sera were tested by elisa using purified ns1 and denv2 as antigen. the immunized mice sera recognized both the recombinant antigen and the dengue virus (fig 6). these results suggested that the ns1 protein expressed in e.coli could be served as a good antigen to induce antibody anti-ns1 in mice. this antigen can be used further in the production of antibody, and also can be used as antigen in the detection of antibody. discussion rapid and early diagnostic of dengue virus fig 3 deduced amino acid of ns1 insert in pgst-ns1 dv2 in comparison to ns1 sequence of the original ds31/06 sequence. squares showed the positions of b-cell epitopes in ns1 protein. 104 80 104 80 m 1 2 3 4 5 6 7 m 1 2 3 4 5 6 33 33 gst-ns1 a b fig 4 analysis of the expressed fusion protein gst-ns1 denv2 after induction with iptg (a) on sds-page 8%; (b) westernblot using anti gst antibody. arrows show the expected product gst-ns1 (75 kda) and gst (26 kda). lane m: protein marker; lane 1: e. coli bl21 without induction; lane 2: e. coli bl21 with induction; lane 3: e. coli bl21 containing pgex-6p-1 without induction; lane 4: e. coli bl21 containing pgex-6p-1 with induction; lane 5: e. coli bl21 containing pgd ns12 without induction; lane 6: e. coli bl21 containing pgd ns12 with induction; lane 7: 2 2 denv2. kda kda microbiol indones infection can lead to early therapeutic intervention and significantly related to the recovery of the patients. in several viral infections, virus load is greatest during the early symptomatic phase and immediately following the onset of symptome. in dengue virus infection, the peak of dengue viral load was before onset the fever (vaughn et al. 2000). dengue ns1 antigen detection is suggested as a helpful tool for the early diagnosis of dengue infection after the onset of fever in primary and secondary infection. it has been reported that ns1 antigen was found circulating from the first day after the onset of fever up to day 9, once the clinical phase of the disease is over (shu et al. 2002). the ns1 protein could be detected even when viral rna was negative in reverse transcriptase-pcr or in the presence of immunoglobulin m antibodies (alcon et al. 2002). the circulating ns1 in acute phase serum sample is within -1 -1 the range of 10 ng ml to 50 µg ml , which does not differ significantly in primary or secondary infection (alcon et al. 2002). recently, commercial diagnostic ns1 kits are available in indonesia with various specificity and sensitivity values. the sensitivity of some ns1 antigen assays ranged from 29 to 88%, and the specificity ranged from 89 to 100% (guzman et al. 2010; wang and sekaran 2010). the reasoning behind the different sensitivities for different kits, different serotypes and different geographical sites requires further study. the difference may reflect different levels of avidity of the test mabs for the relevant epitope(s) in ns1 from different serotypes, and potentially, different lineages from the same serotype, as well as the different virus burden caused by different serotypes (guzman et al. 2010). the other limitation of of ns1 diagnostic kit is unability to distinguish within dengue serotypes. qiu et al developed ns1 antigen assay using monoclonal antibody which successfully differentiated denv-2 from other serotypes by 83.3% sensitivity and 100% specificity (qiu et al. 2009). overall, results of these studies suggest that the currently available ns1 antigen detection kits still need to be improved, mainly in sensitivity. in this study we cloned ns1 from denv-2 virus isolated in jakarta. we expected that using locally circulating strain as source may improve test sensitivity fig 5 western blot analysis of expressed gst-ns1 after purification of pellet and supernatant phase with glutathione-sepharose 4b slurry. m: protein marker; lane 1: pellet; lane 2: supernatant. (a) western blot done using anti-gst monoclonal antibody. (b) western blot done using serum of patient with denv2 infection. 104 kda 80 kda 75 kda a b m 1 2 1 2 3 2.5 2 1.5 1 0.5 0 1:1000 1:500 1:100 1:50 k+ kns1 dv2 fig 6 elisa results to test sera from mice immunized with recombinant ns1 protein. od value was the average of two tests. 450 recombinant ns1 and denv-2 whole virus were used as antigen. k+ is positive control; kis negative control. 20 dewi et al. volume 6, 2012 microbiol indones 21 if the test is to be used in indonesia. however, this must be further investigated. in this study, we used 72 bp upstram of ns1 region to express ns1 protein. expression of ns1 dengue virus gene products involves specific proteolytic cleavages of a precursor polyprotein. falgout et al. showed that the 24-residue hydrophobic sequence preceding ns1 was necessary and sufficient for the production of glycosylated ns1 and that this sequence was cleaved from ns1 in the absence of most dengue virus proteins. this hydrophobic sequence serves as an n-terminal signal sequence that is cleaved by signal peptidase . recombinant ns1 denv2 in this study was expressed in a gst system in e. coli. many eukaryotic genes can not be expressed efficiently in e. coli host due to the difference in codon preference as well as toxicity of foreign protein or mrna. it is also known that heterologously expressed eukaryote protein are not post-translationally modified when it is expressed in e. coli. it is also difficult to express soluble protein or facilitate the secretion of expressed protein into culture media. furthermore, proteins expression in large amounts tend to precipitate, forming inclusion bodies (das et al. 2009) and present a difficulty in the purification. on the other hand, fusion proteins produced in this system have several advantages: they are produced at high level, are relatively stable, and can be easily be purified (nasoff e al. 1991). in infected cells, monomeric ns1 is hydrophilic, but upon dimerization ns1 becomes more hydrophobic and membrane-associated (winkler et al. 1989). in this study, gst-ns1 protein was also insoluble and remained in the pellet phase of the cell lysate. we failed to improve the solubility of the fusion protein into the supernatant fraction of the cell lysate. by adding lysozyme and dnase in the lysis buffer and gluthatione-sepharose purification system, the gstns1 could be isolated from the pellet fraction. despite this success of isolation, improvement of our method to increase the pure protein yield is still necessary. for diagnostic purpose, actually gst-ns1 can be used without cleavage (nasoff et al. 1991). however, to improve the specificity of antibody anti-ns1 we produced, we think it is necessary to purify ns1 protein. for cleavage of gst-ns1, an addition of triton x-100 was necessary. this result suggested that the secondary structure of the fusion protein may cover the cleavage site of the protease. immunization of mice with our recombinant ns1 also showed that the protein retained its ability to induce antibody that recognize both recombinant ns1 and denv2 virus. acknowledgements this research was in part funded by risbin iptekdok 2008, universitas indonesia reseach grant (ruui) 2009 (407m/drpm-ui/a/n1.4/2009), and competitive grant dghe republic of indonesia (hibah riset berdasarkan prioritas nasional) batch ii 2009 (744a/drpm-ui/a/n1.4/2009). references alcon s, talarmin a, debruyne m, falconar a, deubel v, flamand m. 2002. enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein ns1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. j clin microbiol. 40(2): 376-381. doi: 10.1128/jcm.40.2. 376-381.2002. ausubel fm, brent mr, kingston re, moore dd, sedman jg, smith ja, struhl k. 1994. current protocol in molecular biology. vol 3. john willey & son inc. das d, mongkolaungkoon s, suresh mr. 2009. super induction of dengue virus ns1 protein in e. 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production of lactic acid bacteria from pickled yellow bamboo shoots (dendrocalamus asper) physiological profiling and microorganism community analysis tm of cirebon shrimp paste fermentation “terasi” using biolog ecoplate detection of antibody to burkholderia pseudomallei in captive and wild macaques in west java and bali, indonesia probiotic potential of lactic acid bacteria from yellow bamboo shoot fermentation using 2.5% and 5% brine at room temperature siti khodijah chaerun, frideni yushandiana putri, wahyudin prawita minwal, zela tanlega ichlas, and mohammad zaki mubarok laksmi hartajanie, lindayani, and lorentia santoso dea indriani astuti, intan taufik, dini achnafani, and ezra suci priscila vincentius arca testamenti, diah iskandriati, aris tri wahyudi, and joko pamungkas lindayani, laksmi hartjanie, and monika palupi murniati 1 7 15 23 30 issn 1978-3477, eissn 2087-8575 volume 12, number 1, march 2018 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000, 200 000,-5 institutional rate (institution or library) (idr) 240 000, 400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print neung tiaamroeng, 2020 norio kurosawa, 2020 kartini kramadibrata, 2020 diana e waturangi, 2020 endang purwantini, 2020 wellyzar sjamsuridzal, 2020 yuan kun lee, 2020 yaya rukayadi, 2020 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi iman rusmana, 2020 cover depan (12) 655-1552-1-pb cover depan 149-150 (padlan).pmd i collect cartoons. there is one by nick downes that i clipped a few years ago that shows a professor being told (as a thug is getting ready to shoot him): “surely you were aware when you accepted the position, professor, that it was “publish or perish”. you see, “publish or perish” is used in academic circles to mean that if you do not publish (papers) you do not get tenure or you do not get promoted; in other words, you do not get recognition. these days, the number and quality of one’s publications is taken as a measure of one’s productivity, of one’s worth. it even matters in which journals one publishes. there is an insistence, for example, that publications be “peer-reviewed”, i.e., judged worthy of publication by others in the field: non-”peerreviewed” publications do not count as much. in the philippines, it is a big plus to publish in international journals. in fact, some institutions even give monetary rewards for publishing in international journals. (in marked contrast, the national academy of science and technology (nast) philippines, bless its collective soul, yearly gives awards to outstanding scientific papers published in local journals). not all scientific results are of international interest. for example, the association of gastrointestinal disorders with the amount of rainfall (probably a fictitious association) in the town of puling (a fictitious town) in the province of bugat (a fictitious province) in the philippines, would be of little interest to the rest of the world and will most probably not be accepted for publication in an international journal. however, that finding would be of great interest to the inhabitants of puling and other towns in the philippines and will almost surely be a feature article in the puling bugat journal of science (a fictitious journal). how do we decide what is a worthy publication and what is not? one publication is often cited as one that has made a tremendous impact on the lives of billions and that has been reprinted and quoted innumerable times, but which was never “peer-reviewed” nor published in an international journal the bible. scientists publish their experiments and experiences for a purpose, although that purpose has changed over the years. originally, scientists published their data, the analysis of those data, and their theories based on their results, so as to share with their colleagues (the other scientists in the world) the knowledge that they had gained from their experiments. like other endeavors, science builds on previous work the more work had been done and the more findings are made available to the public, the easier it would be for others in the field to know which new path to take, what else needs to be done, what mistakes to avoid, etc. further, if we knew what microbiology indonesia, december 2007, p 149-150 volume 1, number 3 issn 1978-3477 short communication on scientific publications eduardo agustin padlan the marine science institute, college of science, university of the philippines, diliman, quezon city 1101, philippines others are doing, we would not waste our time and resources by duplicating their work. collaborations may even get started by the sharing of ideas and experiences. the sharing of knowledge is not only through publications. results and hypotheses are often shared during personal visits to other labs, at meetings, through letters, even in phone conversations. it is all in the spirit of sharing of cooperation. that was the norm when i started doing science in the mid-1960s, although i soon learned that not everyone played fair there was some competition and results were kept secret. but by and large, sharing was the prevailing attitude. it is very different now. with the start of biotech companies in the mid-1980s, secrecy became more and more prevalent. you cannot blame the companies. they exist for corporate profits. how could they possibly compete if they disclose their trade secrets? there was a meeting held in the late 1980s in which a representative from a biotech company discussed at length their results on a molecule they were working on, but would not name the molecule! the moderator, a scientist of the old school, led the audience in a round of hissing. that was how the science community felt about secrecy at the time. that has changed. nowadays, many (most?) scientists keep their findings secret until their commercial value has been assessed and secured (through patents). scientists have gone corporate! as howard schachman wrote in a recent article, scientists nowadays have a new creed: “patent and prosper”. more and more, the spirit of sharing, of cooperation, is being replaced by competition. even “peer review” of publications sometimes suffers. remember that your “peers”, those who would be passing judgment on your papers, are the other scientists in your field, i.e., your competitors! so, one hears stories of papers whose publication had been “hindered”. and, of course, your competitors get to find out from your manuscripts what you are up to and what your latest findings are. one can only hope that those instances are rare. personally, i prefer a system in which all papers are published. science is self-correcting. if you keep publishing results that are wrong or experiments that were sloppily done, people will stop paying attention to your work. and there are few things worse that could happen to a scientist, or to anyone for that matter, than to be ignored by one’s peers. all self-respecting scientists will publish only “good” papers. in my opinion, every scientific experience should be made known to everyone, including and especially experiments that went wrong or that were extremely difficult to perform 150 padlan et al. microbiol indones (but not sloppy work). we learn from our mistakes; we can learn from the mistakes of others, too. in a dissenting opinion, dr. giselle concepcion, who read an early version of this article, points out that there still has to be some form of review. “while expert scientists can discern what is good or bad science, that may not be true of students. bad science could be damaging to our youth”, she correctly states. “self-correcting science, learning from one’s or others’ mistakes, like natural selection and evolution, will be wasteful and will take too much time,” she adds. i guess i have to agree. but i still think that every scientific experiment, data, interpretation, theory, hypothesis, etc., provided it is not fraudulent or incompetently done, should be made available to others. every piece of knowledge is important. there is a university in the us whose library plans to get a copy of every written piece of work in history. there is a church denomination that is currently compiling the genealogy of every man and woman on earth. there is a continuing effort to catalog every plant and animal species in the world. on the occasion of his 250th birthday, musicians in his native austria played every note that mozart had ever written. the key word in all of these efforts is “every”. and these are almost surely not the only endeavors in which every bit of data on a given subject is being collected. i am certain that soon every scientific paper, even the ones published in the puling bugat journal of science, will be accessible through the internet. when that happens, the original purpose of publishing scientific findings will be achieved. when that happens, there will be no distinction between an international publication and a local one. but until then, here in our country, a measure of a scientist’s worth, his productivity, is the number of his (or her) international publications. to be productive in science, one has to be creative. but being creative is often not enough. one may be very creative, but he (or she) would have a hard time producing publishable results if he (or she) is saddled with loads of teaching and administrative responsibilities a situation in which most scientists in the philippines find themselves. further, it would be difficult to produce if one does not have the funds, the equipment and other necessities to do the work. clearly, most scientists in the philippines are at a great disadvantage. we are not lacking in creativity, but we usually do not have the resources to accomplish what we are capable of doing. we may have ideas that are of international importance and which can compare with those of the best scientists in the world (just look at how well many filipinos abroad are faring), but we here (with some notable exceptions) simply cannot work on those ideas and compete with the rest of the world because of lack of resources. but there are lots of important problems local problems for which we do not have to compete with the rest of the world. and we can publish our results in local journals. those of us who work on local problems are doing the country a lot of good. of course, we do not get much recognition from those who insist that we publish in international journals. maybe we can make our local journals internationally “visible” and thus be included in international indices (the sign of international recognition). that may not be too difficult to achieve. if we could convince our more productive local scientists and our compatriots abroad, who are able to produce internationally competitive results, to publish seminal papers or review papers in local journals and afterwards cite those (local) papers in their other (international) publications, then the international community will become aware of our local science and our local journals. issn 1978-3477, eissn 2087-8575 volume 10, number 2, june 2016 i n d o n e s i a accredited at level “a” until februari 2019 no. / /201040 p 4 patron siswa setyahadi, 2020 chief editor debbie s retnoningrum, 2020 editorial board members antonius suwanto, 2020 brett neilan, 2020 dessy natalia, 2020 managing editor is helianti, 2020 astutiati nurhasanah, 2020 electronic editor iman rusmana, 2020 is helianti, 2020 business manager diana nurani, 2020 editorial office indonesian society for microbiology (sekretariat permi) room 124/tmc 2 drn, puspiptek-serpong, tangerang selatan , indonesia15314 phone: +62-21-7560536 ext 7119 fax: +62-21-7560694 e-mail: microbiology.indonesia@gmail.com url: http://jurnal.permi.or.id/index.php/mionline publisher indonesian society for microbiology published in march, june, september, and december. subscription prices for one year, not including shipping and handling indonesian overseas individual rate (idr) 1 0 000, 200 000,-5 institutional rate (institution or library) (idr) 240 000, 400 000,bank bank mandiri cabang menara thamrin, jakarta, acc permi; acc no 103-0002080774 printed by: cv. istiqom print maggy thenawijaya suhartono, 2016 maria inge lusida, 2016 neung tiaamroeng, 2020 norio kurosawa, 2020 kartini kramadibrata, 2020 diana e waturangi, 2020 endang purwantini, 2020 raija laiho, 2016 wellyzar sjamsuridzal, 2020 yuan kun lee, 2016 yaya rukayadi, 2020 friedhelm meinhardt, 2016 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2015-2019 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi 440-1145-1-pb (3) a.front cover 10(2)-juni cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 9, number 4, december 2015 isolation, identification and screening of antimicrobial properties of the marine-derived endophytic fungi from marine brown seaweed antibiotic use is not a risk factor of infection by extended-spectrum beta-lactamase producing bacteria in dr. soetomo hospital surabaya orchid mycorrhizae fungi: identification of rhizoctonia in west kalimantan heavy metals biosorption by copper resistant bacteria of acinetobacter sp. irc2 screening, purification and characterization of cellulase from fungi isolated from used mushroom substrate changi wong, peter proksch, lee tung tan, samuel lihan, aazani mujahid, and moritz muller nadhya nur fitri, musofa rusli, and manik retno wahyunitisari rosa suryantini, reine suci wulandari, and rina sri kasiamdari wahyu irawati, adolf jan nexson parhusip, and raden nida sopiah iwan saskiawan and nur hasanah 141 150 157 163 171 author index subject index 178 179 page 1 03 putra.cdr the antibacterial potential of pineapple (ananas comosus (l.) merr) core extract against methicillin-resistant staphylococcus aureus (mrsa) 1* 2 3 boby pratama putra , danti nur indiastuti , and deby kusumaningrum 1 2 3 school of medicine; department of pharmacology and therapeutics; department of medical microbiology, faculty of medicine, universitas airlangga, jalan prof. dr. moestopo no. 47, surabaya 60132, east java, indonesia. staphylococcus aureus is one of major pathogens causes serious infection. penicillin antibiotic is one of therapies against staphylococcus infection. however, inadequate and irrational use of antibiotic causes resistance and emerges incidence of methicillin-resistant s. aureus (mrsa). herbal medicine from pineapple, especially from its core extract, is expected can reduce the incidence of antibiotics resistance because it contains bromelain, flavonoid, saponin, and tannin, which have antibacterial effect. this research was conducted to investigate the antibacterial potentiality of pineapple core extract against mrsa. this research is true experimental with posttest controlled group design. pineapple core was extracted by maceration method. pineapple core extract's -1 concentrations used were 750, 500, 250, 187.5, 125, and 62.5 mg ml . the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) were determined by broth dilution test with five replications. vancomycin was used as control group. mic was determined visually by comparing turbidity of o o solutions after incubation at 37 c for 24 h. then these solutions were cultured on nutrient agar plates at 37 c for 24 h. mbc was determined visually by inspecting the presence of bacterial colonies growth. the minimum inhibitory concentration (mic) could not be determined due to no turbidity changes. vancomycin control cannot be used for determining mic. cultures on nutrient agar plates had no colonies growth in concentrations of 750 -1 and 500 mg ml . in summary, pineapple core extract has antibacterial potentiality against methicillin-resistant s. -1 aureus (mrsa) with mbc of 500 mg ml . key words: antibacterial, dilution susceptibility test, methicillin-resistant staphylococcus aureus (mrsa), pineapple core, vancomycin staphylococcus aureus merupakan salah satu patogen yang menyebabkan infeksi serius. penisilin merupakan salah satu terapi infeksi staphylococcus. penggunaan antibiotika yang tidak tepat dan irrasional mengakibatkan resistensi dan munculnya methicillin-resistant s. aureus (mrsa). obat herbal dari nanas, terutama ekstrak dari bonggolnya, diharapkan dapat menurunkan insidensi resistensi antibiotika karena mengandung bromelain, flavonoid, saponin dan tanin, yang memiliki efek antibakteri. penelitian ini dilakukan untuk menguji potensi antibakteri ekstrak bonggol nanas terhadap mrsa. penelitian ini merupakan eksperimental murni dengan post-test controlled group design. bonggol nanas diesktraksi dengan metode maserasi. konsentrasi ekstrak bonggol nanas yang digunakan antara lain 750, 500, 250, 187,5, 125, dan 62,5 mg -1 ml . konsentrasi hambat minimum (khm) dan konsentrasi bunuh minimum (kbm) ditentukan dengan uji dilusi sebanyak lima replikasi. vancomycin digunakan sebagai kelompok kontrol. khm diamati secara visual o dengan membandingkan kekeruhan suspensi setelah inkubasi pada suhu 37 c selama 24 jam. tiap suspensi o dikultur pada media agar nutrien pada suhu 37 c selama 24 jam untuk melihat media yang tidak ada pertumbuhan koloni bakteri agar dapat menentukan kbm. konsentrasi hambat minimum (khm) tidak dapat diamati karena tidak ada perbedaan kekeruhan. kontrol vancomycin tidak dapat digunakan untuk menentukan khm. kultur pada media agar nutrien menunjukkan tidak ada pertumbuhan koloni pada konsentrasi 750 dan 500 -1 mg ml . ekstrak bonggol nanas memiliki potensi antibakteri terhadap methicillin-resistant s. aureus (mrsa) -1 dengan kbm 500 mg ml . kata kunci: antibakteri, bonggol nanas, methicillin-resistant staphylococcus aureus (mrsa), uji dilusi, vancomycin vol.11, no.3, september 2017, p 89-93 doi: 10.5454/mi.11.3.3 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8170528825, email: boby_pratama_putra@yahoo.co.id lactam antibiotics. however, inadequate and irrational use of the antibiotics causes s. aureus resistances against beta-lactam antibiotics and emerges incidence of methicillin-resistant s. aureus (mrsa). metaanalytical study by tacconelli et al. (2008) shows correlation between frequency of antibiotic use and incidence of antibiotic resistance. based on the infectious disease is one of global health problems especially in tropical areas. staphyloccocus aureus is one of the commonest and potentially dangerous human pathogens (miller and diep 2008). one of therapies against s. aureus infection is using betaresearch conducted by jarvis et al. (2012) involving 67,412 hospitalized patients in the united states, the prevalence of mrsa colonization or infection is 66.4 per 1000 patients, as many as 61.8% has been colonized by mrsa and 38.2% were infected by mrsa. mrsa infects mostly the integument system and soft tissue, and is largely nosocomial infection. mrsa infection epidemiologically has higher morbidity and mortality that infection caused by methicillin-sensitive s. aureus (mssa) (gordon and lowy, 2008). in addition, decrease in incidence of mrsa infection would reduce the treatment cost (rubin et al. 1999). therefore, it is necessary to search for alternative medicines from natural materials to reduce the incidence of mrsa, one of them is pineapple core extract. pineapple is one of the commodities in indonesia that has been consumed by many people. however, pineapple core is often discarded as waste, while gautam et al. (2010) through experiments came to the conclusion that bromelain activity in pineapple stem tissue is higher than the activity of the enzyme in the pineapple fruit. pineapple core contains proteolysis enzyme called bromelain that lyse bacterial cell wall (ali et al. 2015). besides, pineapple core also contains flavonoid, saponin, and tannin that have antibacterial effect. materials and methods extraction. pineapple in the study was obtained from pineapple plantation in ponggok district, blitar regency, east java, indonesia. the extraction method used was maceration method. a total of 400 grams of pineapple core powder was added 500 ml of ethanol 96% then homogenized at 120 rpm for one hour, then left for 24 h. after 24 h, the solution was filtered using a buchner filter and repeated three times. the filtrate was evaporated by using a rotary evaporator at 70°c to obtain crude extracts. vancomycin stock preparation. one vial of the vancomycin 500 milligrams was dissolved with aqua pro injection solution of 10 ml obtained a -1 concentration of 50 mg ml . the solution is dissolved with serial dilution to obtain a concentration of 0.005 -1 -1 mg ml or 5 μg ml . the final step was making -1 vancomycin stock with concentration of 2 μg ml by -1 dissolving 4 ml vancomycin 5 μg ml in 6 ml aqua pro injection. methicillin-resistant staphylococcus aureus ( m r s a ) p re p a r a t i o n . m e t h i c i l l i n r e s i s t a n t staphylococcus aureus (mrsa) colony was obtained from laboratory of microbiology, faculty of medicine, universitas airlangga. mrsa culture with age of 24 h was taken amount of 0.1 ml 0.5 mcfarland 8 -1 (1.5 x 10 cfu ml ) then placed in tube containing sterilized broth dilution. anti-bacterial dilution susceptibility test. the method used in the research was broth dilution test with five replications. the first serial is pineapple core -1 extract with concentration of 1500 mg ml then diluted -1 until obtained concentration of 375 mg ml , but the -1 tube with concentration of 750 mg ml was discarded. the second serial is pineapple core with concentration -1 of 1000 mg ml then diluted until achieved -1 concentration of 125 mg ml . all the test tube was added 1 ml of inoculum thus obtained final concentration of 750, 500, 250, 187.5, 125, 62.5 mg -1 ml . the sterility control consists of 1 ml extract and 1 ml sterile medium, the growth control consists of 1 ml inoculum and 1 ml sterile medium, and antibiotic control consists of 1 ml vancomycin stock and 1 ml inoculum. then, both test and control tubes were incubated at 37 °c for 24 h for determining the mic. after observation, each suspension was streaked on nutrient agar medium then incubated at 37 °c for 24 h for determining the mbc. results extraction. 400 grams of pineapple core powder was extracted with maceration method and obtained 150 grams of pineapple core crude extract that has characteristics of viscous and dark brown-colored. the contamination test was negative. anti-bacterial dilution susceptibility test. dilution susceptibility test was used for determining mic was summarized in the table 1. from the result, the mic could not be determined because the extract's dark brown color and high turbidity interfere the interpretation. after each suspension was cultured on nutrient agar plate, the result was summarized in the table 2. the result showed no colonies growth in -1 concentrations of 750 and 500 mg ml . thus, the mbc of pineapple core extract against the mrsa is 500 mg -1 -1 ml . test tubes with concentration of 250 mg ml could not be referred as the mbc because only killed the bacterial colonies only on the first replication, while the other four replications did not. 90 putra et al. microbiol indones extract concentration (mg ml-1) bacterial growth on n utrient agar plates replication 1 replication 2 replication 3 replication 4 replication 5 750 500 250 + + + + 187,5 + + + + + 125 + + + + + 62,5 + + + + + s g+ + + + + + a + + + + + extract concentration (mg ml-1) observation bacterial growth in sterilized broth dilution replication 1 replication 2 replication 3 replication 4 replication 5 750 x x x x x 500 x x x x x 250 x x x x x 187.5 x x x x x 125 x x x x x 62.5 x x x x x sx x x x x g+ x x x x x a x x x x x volume 11, 2017 microbiol indones 91 discussion in this research, the mic could not be determined because there was no significant difference of turbidity between before and after treatment. the use of vancomycin control was hopefully could be used to determine the mic by comparing the bacterial colonies growth on nutrient agar plate between the control and test tubes. the dose of vancomycin used was based on -1 literature study that vancomycin 2 μg ml is the mic of vancomycin against mrsa (sakoulas et al. 2004). unfortunately, there was no significant similarity of culture results between antibiotic control and test tubes thus the mic still could not be determined. while the -1 mbc in this research was 500 mg ml . for further investigation especially for determining mic, we can consider using agar dilution method or microdilution method. the pineapple core extract used in this research is crude extract. based on literature studies, pineapple core contains some antibacterial substances; they are bromelain, tannin, saponin, and flavonoid. bromelain is proteolysis enzyme that disrupt the peptide bond on bacterial cell wall thus lyse the bacterial wall (ali et al. 2015). pineapple bromelain extract can inhibit -1 streptococcus mutans with concentration of 2 mg ml , porphyromonas gingivalis with concentration of 4.15 -1 mg ml , and aggregatibacter actinomycetemcomitans -1 with concentration of 16.6 mg ml (praveen et al. 2014). besides, pineapple contains flavonoid that inhibits peptidoglycan synthesis on bacterial cell wall and induces protein denaturation on bacterial membrane cell (eumkeb et al. 2012). flavonoid also disrupts bacterial cell wall induces bacterial + metabolites leakage and h ions from flavonoid will react with polar groups so alters the phospholipid on membrane cell (retnowati, 2011). audies (2015) states that pineapple contains tannin, which is a phenol compound and works at the polypeptide wall of bacteria causes shrinkage walls of bacteria. based on other literature, pineapple core also contains saponins which can increase the permeability of the bacterial cell table 1 dilution susceptibility test for determining minimum inhibitory concentration (mic) table 2 dilution susceptibility test culture for determining minimum bactericidal concentration (mbc) -1 (note: s: sterility control, g+ : growth control, a : vancomycin 2 μg ml , x : cannot be assessed). (note: s: sterility control, g+ : growth control, a : vancomycin 2 μg ml-1, + : viable growth, : no viable growth). membrane so that it can alter the structure and function of membrane, disrupt the surface tension of the cell wall which allows the antibacterial substances enter easily into cells then interfere bacterial metabolism, and cause denaturation of proteins on bacterial membrane (karlina et al. 2013). retnowati (2011) also stated that the damage to the bacterial membrane cause the release of enzymes and metabolites resulting in decreased metabolism that decreases atp production resulting in bacterial cell death. purification of each substance from the crude extract may be considered for further investigation thus the mechanism and concentration of each substance for antibacterial against mrsa can be achieved. in summary, pineapple core extract has antibacterial potentiality against methicillin-resistant staphylococcus aureus (mrsa) with mbc of 500 mg -1 ml , while mic can be determined in further investigation using agar dilution method or microdilution method. acknowledgment the authors are so thankful to the laboratory of phytochemistry and pharmacognosy, faculty of pharmacy, universitas airlangga, who helped in extraction process. besides, the authors would like to thank the staffs of the laboratory of microbiology, faculty of medicine, universitas airlangga, who helped in preparing for the dilution susceptibility test. references ali aa, milala ma, gulani ia. 2015. antimicrobial effects of crude bromelain extracted from pineapple fruit (ananas comosus (linn.) merr.). adv biochem. 3(1):1–4. doi: 10.11648/j.ab.20150301.11. audies a. 2015. uji efektivitas antibakteri ekstrak kulit nanas (ananas comosus. l) terhadap pertumbuhan streptococcus mutans penyebab karies gigi [antibacterial effectivity testing of pineapple peel extract (ananas comosus. l) against streptococcus mutans causing dental caries] [undergraduate thesis]. padang (id): universitas andalas. caesarita dp. (2011). pengaruh ekstrak buah nanas (ananas comosus) 100% terhadap bakteri staphylococcus aureus dari pioderma [the effect of pineapple extract (ananas comosus) 100% on staphylococcus aureus bacteria from pioderma] [undergraduate thesis]. semarang (id): universitas diponegoro. eshamah h. 2013. antibacterial effects of proteases on different strains of escherichia coli and listeria monocytogenes [dissertations]. clemson (us): clemson university. eumkeb g, siriwong s, phitaktim s, rojtinnakorn n, sakdarat s. 2012. synergistic activity and mode of action of flavonoid isolated from smaller galangal and amoxicillin combinations against amoxicillinresistant escherichia coli. j app microbiol. 112(1):5564. doi: .10.1111/j.1365-2672.2011.05190.x gautam ss, mishra sk, dash v, goyal ak, rath g. 2010. comparative study of extraction, purification and sstimation of bromelain from stem and fruit of pineapple plant. thai j pharm sci. 34(2):67–76. gordon rj, lowy fd. 2008. pathogenesis of methicillinresistant staphylococcus aureus infection. cli infect dis. 460(5):350–359. doi: 10.1086/533591. jarvis wr, jarvis aa, chinn ry. 2012. national prevalence of methicillin-resistant staphylococcus aureus in inpatients at united states health care 2010. am j infect control. 40(3):194-200. doi: 10.1016/j.ajic.2012. 02.001. karlina cyy, ibrahim m, guntur t. 2013. aktivitas antibakteri ekstrak herbal krokot (portulaca oleraceae l.) terhadap staphylococcus aureus dan escherichia coli [antibacterial activity of herbal krokot extract (portulaca oleraceae l.) against staphylococcus aureus dan escherichia coli]. j lentera bio. 2(1):87-93. miller lg, diep ba. 2008. colonization, fomites, and virulence : rethinking the pathogenesis of communityassociated methicillin-resistant staphylococcus aureus infection. cli infect dis. 46(1):752–760. doi: 10.1086/526773. praveen nj, rajesh a, madan m, chaurasia vr, hiremath nv. 2014. in-vitro evaluation of antibacterial efficacy of pineapple extract (bromelain) on periodontal pathogens. j int oral health. 6(5):96-98. retnowati y, balangi n, posangi nw. 2011. pertumbuhan bakteri staphylococcus aureus pada media yang diekspos dengan infus daun sambiloto (andrographis paniculata) [staphylococcus aureus bacterial growth on media exposed with infused bitter leaves (andrographis paniculata)]. saintek. 6(2):1-9. rubin rj, harrington ca, poon a, dietrich k, greene ja, moiduddin a. 1999. the economic impact of staphylococcus aureus infection in new york city hospitals. emerg infect dis. 5(1):9–17. sakoulas g, moise-broder pa, schentag j, forrest a, moellering rc, elipoulos gm. 2004. relationship of mic and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant staphylococcus aureus bacteremia. j clin microbiol. 42(6):2398-2402. doi: 10.1128/jcm.42.6.23982402.2004. 92 putra et al. microbiol indones tacconelli e, angelis gd, cataldo ma, pozzi e, cauda r. 2008. does antibiotic exposure increase the risk of methicillin-resistant staphylococcus aureus (mrsa) isolation?: a systematic review and meta-analysis. j a n t i m i c r o b c h e m o t h e r. 6 1 ( 1 ) : 2 6 3 8 . d o i : 10.1093/jac/dkm416. volume 11, 2017 microbiol indones 93 page 1 page 2 page 3 page 4 page 5 short communication modified slide culture method for faster and easier identification of dermatophytes yeva rosana *, tetsuhiro matsuzawa , tohru gonoi ,1 2 2 and anis karuniawati1 1department of microbiology, medical faculty, university of indonesia jl. pegangsaan timur no.16, jakarta 10320, indonesia 2medical mycology research centre, chiba university, japan basic slide culture as a morphological identification was known as the most common method for the identification of pathogenic mold fungi. this method preserved the morphological features relatively undisturbed compared with adhesive tape preparations. however, it was necessary to modify method of basic slide culture to improve its usability and shorten the time it needed to identify mold fungi. there were four kinds of method carried out in this study; two kinds of modified slide culture, one kind of direct culture on slant agar plate, and a basic slide culture for identifying mold fungi, which result would be compared with each other. these four methods were tested to 4 species of dermatophytes which were known as mold fungi that could infect skin, hair, and nails in human; those were , , , trichophyton mentagrophytes microsporum canis microsporum gypseum and . result of this study showed that both modified slide culture and direct culture on epidermophyton floccosum slant agar plate could visualize the structure of dermatophytes faster than basic slide culture method. these methods were also easier to prepare compared to basic culture method. conclusion of this study showed that basic slide culture method needed to be modified for better identification of mold fungi. key words: identification, dermatophytes modified slide culture , slide culture konvensional sebagai suatu identifikasi morfologi dikenal sebagai metode yang paling umum untuk identifikasi jamur kapang patogen. metode ini relatif lebih tahan lama untuk menyimpan gambaran morfologi dibandingkan dengan menggunakan metode selotip. walaupun demikian, perlu dilakukan modifikasi metode konvensional untuk meningkatkan kegunaannya dan mempersingkat waktu yang slide culture diperlukan untuk mengidentifikasi jamur kapang. pada penelitian ini dilakukan empat metode; dua metode slide culture slide culture slide culture yang dimodifikasi, satu metode pada plat agar miring, dan satu metode konvensional untuk jamur kapang, yang hasilnya akan dibandingkan satu sama lain. keempat metode ini diujikan pada 4 spesies dermatofita yang dikenal sebagai jamur kapang yang bisa menginfeksi kulit, rambut, dan kuku pada manusia; yaitu , , , dan trichophyton mentagrophytes microsporum canis microsporum gypseum epidermophyton floccosum slide culture . hasil penelitian menunjukkan bahwa kedua metode yang dimodifikasi dan langsung pada plat agar miring dapat memvisualisasikan struktur dermatofita lebih cepat slide culture dibandingkan metode konvensional. metode ini juga lebih mudah pada tahap persiapannya slide culture dibandingkan konvensional. kesimpulan dari penelitian ini menunjukkan bahwa metode slide culture slide culture konvensional perlu dimodifikasi untuk identifikasi jamur kapang yang lebih baik. kata kunci : jamur dermatofita, identifikasi, modifikasi slide culture vol.8, no.3, september 2014, p 135-139 doi: 10.5454/mi.8.3.7 *corresponding author; +6221 3160492. fax: +6221 3100810, e-mail : yeva.rosana@ui.ac.id superficial fungi was known to live on the dead horny layer of the skin and elaborate an enzyme that enabled them to digest keratin, causing the superficial skin to scale and disintegrate, nails to crumble, and hairs to break off. superficial fungi were also capable of eliciting an allergic or id reaction. superficial fungal infections were common skin diseases, affecting millions of people worldwide. these infections occurred in both healthy and immunocompromised patients, and their etiologic agents consisted of dermatophytes, yeasts and nondermatophyte molds (mims . 2008; richardson . 2000).et al et al dermatophytes were responsible for most superficial fungal infections. dermatophytes could be classified according to their natural habitats into three categories: (1) geophilic, which normally live in the soil, (2) zoophilic, which primarily parasitize the body surfaces of animal, (3) anthropophilic, which generally infect human (hiruma . 2003). there were three et al mailto:yeva.rosana@ui.ac.id 136 rosana microbiol indones genera of dermatophytes could infect the human, i n c l u d in g , a n d tr i c h o p h y t o n m i c ro s p o r u m e p i d e r m o p h y t o n m i c ro s p o r u m . o n l y a n d tr i c h o p h y t o n i n v a d e d t h e h a i r , w h e r e a s epidermophyton trichophyton and caused nail infections. these species of dermatophytes were correlated with the clinical diseases, which are classified according to the location of the infection; the clinical lesion, the species of fungi, and therapy varied for different sites (pakshir . 2009). identification of et al this species was very important because of their chronic nature had a risk for significant morbidity. microscopic examination using koh preparation was a simple laboratory method for detection of fungal organisms present in skin and nails or hair. it was accomplished by scraping the skin, nail lesion or hair and examining the material with the microscope. using this method we could show the two structural elements of fungi such as the spore and hyphae, but it could not identify the species of dermatophytes (larone 2011). to identify or confirm the diagnosis of superficial fungal infections, fungal culture must be done using appropriate medium (difco 2003). it was best to examine dermatophytes microscopically when the culture had form conidia or spores. the best method for preserving and observing the actual structure of dermatophytes were the slide culture (fujita 2013). this method preserved the morphological features relatively undisturbed compared with tease mounts and cello-tape mounts (hughes . 2004). slide culture et al was not a rapid method, but it was unsurpassed as a routine means of studying the fine points of the microscopic morphology of dermatophytes (patrick et al et al. 2010, wijedasa . 2012). because basic slide culture method was quite difficult to carry out and requires a long time to get a result, it was needed to modify slide culture method to gain a better result. in this study, there were carried out four methods plate to identify dermatophytes fungi that could infect skin, hair, and nails in human: a basic slide culture method, two kinds of modified slide culture and one kind of direct culture on slant agar. although basic slide culture was a standard method for mold identification, but it needs space to incubate and not easy to prepare. the method of modified slide culture, agar block was put directly to a plate agar to produce of reproductive structure more rapid. it did not require bent glass rod and glass slide, and without adding sterile water on the petri plate. moreover it could be put more than two slides. the difference between two kinds of modified slide culture were time to put agar block, the one was placed on the sterile medium while the other was put after the growth of the dermatophytes. method of direct culture on slant agar only need half volume of standard media for culture, and can visualize structure the dermatophytes directly under the microscope. these four kinds method were tested to 4 species of d e r m a t o p h y t e s , w h i c h w e r e tr i c h o p h y t o n mentagrophytes microsporum canis microsporum , , gypseum epidermophyton floccosum, and . basic slide culture. a bent glass rod was placed in sterile petri plate side, and a sterile glass slide was put on the glass rod. a 1-by-1-cm block potato dextrose agar (pda) cut with a sterile scalpel was then transferred to the glass slide (fig. 1a). using sterile wire needle, the fungus would then be inoculated from culture plate to the four sides of the agar block. sterile coverslip was put over the block with slight pressure to ensure adherence. approximately 2 ml of sterile water was put on the bottom of petri plate, and then the plate cover was replaced. the whole procedure was repeated for each of culture used in this study. when everything set, the plates were put on clean plastic basket and incubated at 30 celcius.0 modified slide culture. a 1-by-1-cm block pda cut with a sterile scalpel was transferred to a plate of pda (fig. 1b). fungus was inoculated using sterile wire needle onto the four sides of the agar block. sterile coverslip was put over the block with slight pressure to ensure adherence, and the plate cover was replaced afterwards. the whole procedure was repeated for each of culture used in this study. after it finished, the plates were put on clean plastic basket and incubated at 30 0 celcius. another modified slide culture method used fungus that was grown on potato dextrose agar (fig. 1c). a 1-by-1-cm block pda was put on fungus culture. sterile coverslip was put over the block with slight pressure to ensure adherence, then the plate cover was replaced. the whole procedure was repeated for each of culture used in this study. then, the plates were put on clean plastic basket and incubated at 30 0 celcius. direct culture on slant agar plate. approximately 10 ml pda was poured on sterile petri plate and was spread on the plate. then the petri plate was put on its cover to gained about half of the plate covered with thin layer of pda. after potato dextrose agar was solid, the petri plate was covered and kept in room temperature. volume 8, 2014 microbiol indones 137 fig 1 basic slide culture, agar block pda on the glass slide put on the bent glass rod (a); modified slide culture 1, agar block pda was put on the sterile medium (b); modified slide culture 2, agar block pda was put after the growth of the dermatophytes (c); visualize structure the dermatophytes directly under the direct culture on slant agar plate, microscope (d). fig 2 macroconidia of (a); spiral hyphae and microconidia of (b);microsporum canis trichophyton mentagrophytes macroconidia of floccosum (c), macroconidia of (d).epidermophyton microsporum gypseum fungus was directly inoculated on slant agar plate. the whole procedure was repeated for each of culture used in this study. when done, the whole plates were put on clean plastic basket and incubated at 30 celcius.0 reading and interpretation of the results. the growth of fungus was examined periodically. the fungus could grow on the surface of the slide and also under the surface of the coverslip. the closed petri plate could be placed on the microscope stage, and the slide culture could be examined with the low-power (10x) objective. when reproduced structures had developed well, forceps was used carefully to remove the coverslip and to put it on a second slide filled with a drop of lpcb. agar block was removed using flamed wire needle and put into container of antifungal disinfectant. a drop of lpcb was put on the remaining slide and the slide was then covered with a new coverslip. each of the microscopic slide culture was sealed around the edges with the nail polish and kept on room temperature. for direct culture plates, the examination was done on the slant agar plate under a microscope (fig. 1d). the dermatophytes structures which are key features for species identification were observed through thin layer of pda directly. earlier visualization of structures related to identification was possible for all 4 species as shown in figures 2. all of the methods allowed observation of very delicate fungal structures of diagnostic significance. this method could overcome three major drawbacks that were associated with adhesive tape preparations. first, fungal structures were be prevented from crushed and damaged during the preparation procedures, affecting the accurate identification of the fungus. second, as a result of the smear was not dry, slide culture can be used for microscopic examination for long period from the initial preparation. third, fungal structures embedded in the agar can be observed. slide cultures was sealed around the edges with the nail polish were also suitable for long-term storage . on room temperature table 1 comparison of basic slide cultures, modified slide sulture and direct culture on slant agar plate species * day 5 day 7 day 10 microsporum canis bsc macroconidia (-) macroconidia (-) macroconidia (+) msc1 macroconidia (-) macroconidia (+) msc2 macroconidia (-) macroconidia (+) dsc macroconidia (-) macroconidia (+) trichophyton mentagrophytes bsc spiral hyphae (-) macroconidia (-) microconidia (-) spiral hyphae (-), macroconidia (-), microconidia (+) spiral hyphae (+), macroconidia (+), microconidia (+) msc1 spiral hyphae (-) macroconidia (-) microconidia (+) spiral hyphae (+) macroconidia (+) microconidia (+) msc2 spiral hyphae (-) macroconidia (-) microconidia (+) spiral hyphae (+) macroconidia (+) microconidia (+) dsc spiral hyphae (+) macroconidia (+) microconidia (+) epidermophyton floccosum bsc macroconidia (-) macroconidia (-) macroconidia (+) msc1 macroconidia (-) macroconidia (+) msc2 macroconidia (-) macroconidia (+) dsc macroconidia (+) microsporum gypseum bsc macroconidia (-) macroconidia (-) macroconidia (+) msc1 macroconidia (-) macroconidia (+) msc2 macroconidia (-) macroconidia (+) dsc macroconidia (-) macroconidia (+) * bsc (basic slide cultures); (msc1) modified slide sulture 1; (msc2) modified slide culture 2; dsc (direct culture on slant agar plate) 138 r osana microbiol indones volume 8, 2014 microbiol indones 139 the comparison of each method could be seen in table 1. basic slide culture required more days to visualize morphology of dermatophytes, compared to modified slide culture 1, modified slide culture 2, and direct culture on slant agar plate. microsporum canis required the longest time to visualize compared to others species in this study. in addition, as the sides of the agar block were exposed to the environment during the observation, contamination of the slide culture was always a possibility. similarly, due to the thickness of the agar block, focusing on the slide culture agar block under x 40 was not possible in many instances. modified slide culture 1 and 2 was the best method for storage slide purpose. it could visualize faster than basic slide culture, but only cover slide could be stained and visualized. direct culture on slant agar plate took the shortest time to visualize all of the dermatophytes species in this study compared to the other methods. although the direct culture method did not allow staining to visualize structures using lactophenol cotton blue, it seems the direct culture on slant agar plate was the best method to rapid identification for diagnoses purpose, but it was not suitable for storage. direct examination of the agar plate under a microscope was particularly useful for observation of dermatophytes structure which was the key features for species identification. in conclusion, this study demonstrates that basic slide culture method visualized structure of dermatophytes longer than modified slide culture and direct culture on slant agar plate. basic culture method needed to be modified for identification of dermatophytes. acknowledgement this study is funded by takeda science foundation. we thanks to team of microbiology department faculty of medicine university of indonesia and medical mycology research centre chiba university japan for technical assistance and supporty. references difco laboratories. 2003. difco and bbl manual of microbiological culture media. maryland: becton dickinson and company. fujita s. 2013. simple modified method for fungal slide preparation. med mycol j. 54(2):141-146. hiruma m, yamaguchi h. 2003. dermatophytes. in anaissie ej, mcginnis mr, pfaller ma, editors. clinical mycology. usa: churchill livingstone. 370-379. hughes ad, lorusso gd, greer dl. 2004. the 'double-layer tape prep': an improvement to a standard technique. j med microbiol. 53: 455. larone dh. 2011. medically important fungi. 5 edition. th usa: asm press. mims c, dockrell rv, goerin. 2008. medical microbiology. 4 edition. toronto: mosby.th pakshir k, bahaedinie l, rezaei z, sodaifi m, zomorodian k. 2009. activity of six antifungal drugs against in vitro clinically important dermatophytes. jundishapur j microbiol. 2(4): 158-163. patrick cyw, antonio h y, chui hk, susanna k p, lau yy, kwok yy. 2010. agar block smear preparation : a novel method of slide preparation for preservation of native fungal structures for microscopic examination and long term storage. j clin microbiol. 48(9):3053 3061. richardson m, elewski b. 2000. superficial fungal infections. oxford: fine print. wijedasa mh, liyanapathirana lvc. evaluation of 2012. an alternative slide culture method for the morphological identification of fungal species. sri lanka j infec dis . 2(2): 47-52. 4 hery winarsi_351.pmd a supplement based on zn-enriched virgin coconut oil as an antifungal agent for vaginal candidiasis patients hery winarsi1*, hernayanti1, and agus purwanto2 1biology faculty, universitas jenderal soedirman, kampus karangwangkal, purwokerto 53122, indonesia; 2 margono soekarjo hospital, purwokerto 53146, indonesia this research was conducted to investigate the amount of candida albicans in vaginal secretion of vaginal candidiasis patients administered with zn-enriched virgin coconut oil. thirty respondents were selected based on several criteria as follows: the number of c. albicans colonies in the vaginal secretion was more than 105 cfu.ml-1, voluntary, healthy, willing to sign the informed consent and resided in purwokerto. in group a, 10 women were administered 2 tablespoons per day of zn-enriched virgin coconut oil. in group b, 10 women were administered 1 tablespoon per day of zn-enriched virgin coconut oil; and in group c, 10 women served as control group. vaginal secretions were taken 3 times, before intervention (at baseline time), at 1 month and 2 months after intervention. samples were taken by collecting vaginal secretions from the vaginal proximal region using a sterile cotton bud, which was then put into a tube containing sterile carrier media. the vaginal secretions were tested for the number of total c. albicans using pour plate method. two months after treatment, the number of colonies decreased from 4.4x106 to 2.5x106 cfu ml-1 (p=0.03) in group a. there was no significant difference in the number of colonies between group a and group b, the number of c. albicans colonies was still above the normal range. therefore, the recommended dosage of intervention with zn-enriched virgin coconut oil is one tablespoon a day. key words: candida albicans, vaginal candidiasis, virgin coconut oil, zn _____________________________________________ ________________________ *corresponding author, phone: +62-281-638794, fax: +62-281-631700, e-mail: winarsi@yahoo.com issn 1978-3477 volume 2, number 2, august 2008 p 69 72 candida albicans is a normal microbiota that is commonly found in the vagina. however, excessive amount of this yeast can cause discomfort, pruritis, and pain. this condition is known as leukorrhea or vaginal candidiasis (vc). many conditions can trigger the emergence of vc, such as trichomoniasis, diabetes mellitus, vaginitis senilis, inflammatory chronical inflammation of the pelvis, virus infection, disturbances in immune function, and candidiasis. the last two conditions were commonly experienced by women (sobel 2005; winarsi et al. 2006). transmorphism of c. albicans from yeast to mycelia is potentially pathogenic. the mycelium of c. albicans is capable of binding to the epithelium of the hosts cells mycelia and penetrating the surface with mycelium protein, which is then tightly crosslinked with the cells epithelium. proteins contain amino acids that are able to act as a transaminase substrate of mammalian cell keratin. binding of c. albicans enzymes to the epithelium of host cells leads to pathogenic process. proteinase and phospholipase secreted by the mycelium are capable of digesting epithelium cells, which then facilitates the invasion by the mycelium. c. albicans itself may perform phagocytosis on the endothelium of the host cell. this ability enhances the virulence of c. albicans. winarsi et al. (2006) reported that vc incidence in purwokerto was 38%, which occured among women of 15-53 years of age that experienced zn deficiency (winarsi et al. 2005). zn is a co-factor for enzyme that has antifungal, antiadenoma, antiprostatitis, and immunostimulator potency. it is important for maturation, activation, proliferation, and differentiation of t-cells. therefore, the occurrence of vc is likely to be worse in women with zn deficiency. the application of clinical medicine might cause immunosuppresor effect. therefore, people prefer to use natural remedies such as virgin coconut oil (vco). vco not only contains lauric acid, but also capric, caprilic, and myristic acids (ingle et al. 1999). these fatty acids have antifungal, antibacterial, as well as antiviral potency, and they help maintain the immune system (bergsson et al. 2001). lauric acid can kill c. albicans and repair the metabolism energy (portillo et al. 1998). the aim of this research was to investigate the growth of c. albicans in vaginal secretions of vc patients treated with zn-enriched vco. materials and methods zn-enriched vco has been formulated by winarsi et al. (2006). thirty vc patients were selected based on the following criteria: number of c. albicans in the vaginal secret (more than 105 cfu ml-1), resident in purwokerto, willing to volunteer for the research and sign an informed consent form. subjects were divided into 3 groups with 10 patients in each group. those in group a were treated with 2 tablespoons per day, those in group b were treated with 1 tablespoon per day, while those in the group c were given a placebo and served as control group. treatments were carried out for two months. vaginal secretion were sampled at three periods: i.e at baseline and then continued by one and two months after treatment. sample was taken by sweeping the vaginal proximal area using a sterile cotton bud, which was then put in a tube containing sterile carrier media. the sample was then tested for the total number of colonies of c. albicans using pour plate method. results the amount of c. albicans at baseline time was above the normal range, being 8.38x106-1.24x 107 cfu ml-1 (fig 1). one month after beginning treatment, the number of c. albicans 70 winarsi et al. microbiol indones colonies decreased from 8.4x106 to 4.8x106 cfu ml-1 (p = 0.022) group receiving 2 tablespoons of vco enriched with zn per day (group a). however, the number of colonies remained above normal. there was no significant difference on the amount of c. albicans between group a and group b (p = 0.32). because the amount of c. albicans was still high, the time of treatment was lengthened. after 2 months of intervention, the number of c. albicans colonies in group a decreased from 4.4x106 to 2.5x106 cfu ml-1 (p = 0.03). the growth of c. albicans is influenced by ph. measurements of ph of vaginal secretions of vc patients are presented in table 1. the ph at baseline time was relatively neutral approximately 6.0. one month after intervention, the ph decreased from 5.8 to 5.5, and continued to decrease 2 months after treatment, from 5.3 to 5.0 (table 1). discussion at baseline time, the amount of c. albicans was similar among the 3 groups (p = 0.45), indicating that the groups were homogeneous before treatment. therefore, a change in the number of colonies after treatment reflects the effect of zn-enriched vco. vc is a pathological infection condition. one factor causing vc is the excessive amount of c. albicans in vaginal secretions. in this case, the host immune system is disrupted by c. albicans. this causes rapid proliferation of yeast cells, which results in increasing amount of c. albicans. fungal cells secrete enzymes that facilitate their invasion. secreted aspartyl proteinase (sap) produced by c. albicans increases the microorganism’s ability to colonize and penetrate into the host tissue. in the host’s body, the yeast cells are sometimes unrecognized by the host’s immune system (zeppelin et al. 1998). sap induces the release of mannan (a component of the fungal cell wall), which then inhibits and modulates the host’s immune system. this enzyme suppresses the host’s immunoglobulin and complement levels (naglik et al. 2003). sap hydrolyzes the mucous secreted by the host’s digestive tract, so that candida cells may directly penetrate the mucous cells (chaffin et al. 1998). candida cells are still able to secrete sap even after they have been subjected to phagocytosis by macrophages. therefore, the activity of candida cells is stronger than what the host’s body expected. this situation leads to suppression of the host’s immune system and thus causes infection. mannoproteins and enolase (metabolites of c. albicans) are antigens that are able to stimulate the host’s humoral immune response. thus, mannoprotein modulates the host’s immune response. it makes c. albicans unrecognized to the host’s immune system, so that the cells are not opsonicated or phagocytosized. the other mechanism by which c. abicans makes itself unrecognizable to the host immune system is through adhesion with components of the host’s cells, including thrombocyte, and complement of ic3b. this triggers phospholipase release by c. albicans, which assists the penetration to the host’s tissue and crushes the cell membrane. the activity of phospholipase is the primary factor affecting virulence in c. albicans. candidiasis incidence is a consequence of c. albicans virulence, which is influenced by the metabolites of c. albicans and a predisposition factors of the host’s body. regarding the immunocompetence between the organism and the host’s normal immune system, c. albicans acts as a normal microbiota on the skin, mucous surfaces, digestive tract, urethra, and genitalia. accompanied by other normal microbiota, c. albicans balances the formation of colonies, so that the growth of the pathogenic microbes may be prevented and a balanced ph is maintained. in normal amounts, c. albicans is not pathogenic, because it can be controlled by the immune system and other normal microbiota. the existence of c. albicans and other normal microbiota have a competitive effect, especially on the adhesion and nutrition absorption ability of the host’s cells. under certain conditions, the amount of c. albicans cells in the body increases when the activity of immune cells decreases. this condition disturbes the balance among other normal microbiota or other factors which triggers the growth of c. albicans. candidiasis represents an opportunist i n f e c tion, so that infection usually occurs in immunocompromized individuals (fridkin and jarvis 1996). the decrease in the number of c. albicans colonies after 1 month of intervention with zn-enriched vco was related to the components of organisms cell walls. chaffin et al. (1998) and marcilla et al. (1998) stated that the cell walls of c. albicans were composed of glucan, chitin, mannoprotein (mannan binding to protein), protein, fat, and inorganic salts as minor components. these components build up the yeast cell walls and mycelium in the relatively same amount. glucan builds up cell structure, while chitin maintains integrity of the cell wall structure (marcilla et al. 1998). mannoprotein and other proteins are predominant in the external layer of the cell wall with only small amounts being found in the 1.40e+07 1.20e+07 1.00e+07 8.00e+06 6.00e+06 4.00e+06 2.00e+06 0.00e+00 0 1 2 a m o u n t o f c . a lb ic a n s ( c fu m l1 ) fig 1 the amount of candida albicans colonies. a, group treated with zn-enriched vco, 2 tablespoons/day; b, group treated with znenriched vco, 1 tablespoon per day; c, control group. a b c a a b b c c table 1 the average ph of vaginal secretion ph in the period of intervention (month) group 0 1 2 a b c 6 . 1 6 . 1 6 . 0 5 . 8 5 . 6 5 . 5 5 . 3 5 . 1 5 . 0 a, group treated with zn-enriched vco, 2 tablespoons per day; b, group treated with zn-enriched vco, 1 tablespoon per day; c, control group. period of intervention (month) volume 2, 2008 microbiol indones 71 internal layer. mannoprotein is covalently bound to β-glucans and protein chains (chaffin et al. 1998). mannoprotein is reported to trigger host immune responses to candidiasis, because this compound is thought to be involved in the changing of cell morphology. mannoprotein has immunomodulator potency to the host’s body. it generally controls the host’s immune system, including natural killer, phagocytes (macrophage), as well as cellular and humoral immune cells (chaffin et al. 1998; marcilla et al. 1998). znenriched vco may attract mannoproteins, the primary component of c. albicans exterior cell walls, causing destruction of the cell walls. this will cause glucan and chitin, other components of c. albicans cell walls, unable to maintain the integrity of the entire cell walls, thus making the cell walls very brittle. a brittle cell wall can be easily lysed, and unable to maintain c. albicans structure. brittleness of c. albicans cell walls may be related to glycoprotein because the ability of the external layer of cell walls to act as an adhesion mediator on host epithelial cell surface is interrupted (chaffin et al. 1998). these conditions could therefore suppress the growth of c. albicans. goyal and khuller (1992) reported that the cell membrane of c. albicans consisted of a phospholipid layer, which was one of its energy sources. when c. albicans is in contact with lauric acid in the host body, the lipid compounds of cell wall will be lysed. as the lipid content of the membrane is destroyed, the cell content leaks out. therefore, the growth of c. albicans is inhibited and the c. albicans can even be killed. caprilic acid derivatives of vco could also kill vc causing c.albicans. the potency of vco as an antifungal agent is shown by the activity of lauric and caprilic acids. lauric acid in the body is converted into monolaurine, while capric acid is converted into monocaprine compounds. these compounds are monoglycerides which have antimicrobial activities capable of regulating the growth of c. albicans (bergsson et al. 2001). zn also has an antimicrobial potency, especially towards pathogenic microbes. in the form of free ions, they can directly attack microbes. zn is also a component of human membrane cell structure and it is an essential antioxidant for cells with short half-life, such as immune cells (filipe et al. 1995). therefore, zn fortified by vco will improve the capacity of the host immune and defense systems. specific protein compilers of the cell wall are responsible for c. albicans dimorphism (marcilla et al. 1998), but this dimorphism also depends on temperature, ph, and co 2 concentration (ernst 2000). c. albicans optimally grows at 37°c and neutral ph. at neutral ph (at baseline time), the organism in a mycelial state, but at lower ph (after 1 and 2 months of treatment) it is in yeast state (molero et al. 1998). in the yeast form, adhesion of fungal cells to the host epithelial cells is stronger. protein and glycoprotein components of the c. albicans cell wall surface are also important in adhesion (senet 1998), which is the first step of colonization and infection (chaffin et al. 1998). adhesion occurs at a minimum ph of 3-4, and is optimal at ph 6 (sundstrom 2000). at relatively neutral ph (at baseline time), cell colonization could occur, so that the number of c. albicans colonies were higher than normal. at decreased ph, although not significant, the number of c. albicans colonies declined. kanbe and cutler (1994) argued that c. albicans was capable of producing a lactic acid compound in vaginal secretion. lactic acid causes the cell wall of c. albicans, which consists of mannan, mannoprotein and chitin were denaturated, and reduce the level energy of this microorganism. this finding supports the statement of klotz and smith (1995), that some microorganisms are more sensitive to acidic environment. as proteins, enzymes will be denatured by acid. this causes physiological disturbance and a decrease of the microbe’s life-time. a decrease of ph will also improve the inhibition of c. albicans growth by vco. by the end of the treatment (2 months), the amount of c. albicans in the vaginal secret were still above the normal range. a greater effect may be achieved by lengthening the intervention period, so that the normal amount of vaginal microbiota would be recovered. after 2 months of intervention, both dosage given did not show any significant difference (p = 0.26). therefore, based on technical and economical considerations, the recommended dosage is 1 tablespoon of zn-enriched vco per day. acknowledgement the author sincerely thanks ditbinlitabmas dirjen dikti for financial funding through the hibah bersaing project xiv/1-xiv/2 budget for 2006 and 2007. references bergsson g, arnfinnsson j, steingrimsson o, thormar h. 2001. in vitro killing of candida albicans by fatty acids and monoglycerides. antimicrob agents chemother 45:3209-3212. chaffin wl, lopez-ribot jl, casanova m, gozalbo d, martinez jp. 1998. cell wall and secreted proteins of candida albicans: identification, function, and expression. microbiol mol biol rev 62:130-180. ernst jf. 2000. regulation of dimorphism in candida albicans. contrib microbiol 5:98-111. filipe pm, fernandes ac, manso cf. 1995. effects of zinc on copper-induced and spontaneous lipid peroxidation. biol trace elem res 47:51-56. fridkin sk, jarvis wr. 1996. epidemiology of nosocomial fungal infections. clin microbiol rev 9:499-511. goyal s, khuller gk. 1992. phospholipid composition and subcellular distribution in yeast and mycelial forms of candida albicans. j med vet mycol 30:355-362. ingle dl, driedger a, traul ka, nakhasi dk. 2006. dietary energy value of medium-chain triglycerides. j food sci 64:960-963. kanbe t, cutler je. 1994. evidence for adhesin activity in the acid-stable moiety of the phospho-mannoprotein cell wall complex of candida albicans. infect immunol 62:1662-1668. klotz sa, smith rl. 1995. gelatin fragments block adherence of candida albicans to extracellular matrix proteins. microbiology 141:2681-2684. marcilla a, valentin e, sentandreu r.1998. the cell wall structure: developments in diagnosis and treatment of candidiasis. int microbiol 1:107-116. molero g, diez-orejas r, navarro-garcia f, monteoliva l, pla j, gil c, sanchez-perez m, nombela c. 1998. candida albicans:genetics dimorphism and pathogenecity. int microbiol 1:95-106. 72 winarsi et al. microbiol indones naglik jr, challacombe sj, hube b. 2003. candida albicans secreted aspartyl proteinases in virulence and pathogenesis. microbiol mol biol rev 67:400-428. portillo mp, s e r r a f, s i m o n e . 1 9 9 8 . e n e rg y r e s t r i c t i o n w i t h high-fat diet enriched with coconut oil gives higher ucp1 and lower white fat in rats. int j obes rel met dis 22:974-979. rink l, kirchner h. 2000. zinc-altered immune function and cytokine production. j nutr 130:1407s-1411s. senet jm. 1998. candida adherence phenomena from commensalism to pathogenicity. int microbiol 1:117-122. sobel jd. 2005. genital candidiasis. medicine 33:62-65. sundstrom p. 2002. adhesion in candida spp. cell microbiol 4:461469. winarsi h, hernayanti, purwanto a, sukanto. 2006. profile and antioxidant status of vaginal candidiasis patient in purwokerto. media medika indones 41:108-112. winarsi h, muchtadi d, zakaria fr, purwanto a. 2005. effect of zn supplemented to immune status premenopausal women intervented with isoflavoned drinking. j biosains 12:82-85. zepelin mb, beggah s, boggian k, sanglard d, monod m. 1998. the expression of the secreted aspartyl proteinases sap4 to sap6 from candida albicans in murine macrophage. mol microbiol 28:543-554. guide for author.cdr guide for authors associated with them or their laboratory (ies); please 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(choose of subscription, not including package and postage) ) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com page 1 page 2 blanko pendaftaran anggota.pdf page 1 form berlangganan 2019.pdf page 1 cover belakang.pdf page 1 8. 489 isolation (dwi).cdr short communication isolation and characterization of chitinolytic bacteria and their potential to inhibit plant pathogenic fungi dwi suryanto*, netti irawati, erman munirand department of biology, faculty of mathematics and natural sciences, universitas sumatera utara, jalan bioteknologi 1, medan 20155, indonesia ganoderma boninense fusarium oxysporum, penicillium citrinum g. boninense f. oxysporum p. citrinum p. citrinum ganoderma boninense fusarium oxysporum penicillium citrinum g. boninense f. oxysporum p. citrinum p. citrinum a study on the isolation and characterization of chitinolytic bacteria and their potential to inhibit plant pathogenic fungi has been done. the bacteria were isolated from the soil of karo, langkat, and bangka, sumatra. , and of the stock cultures in laboratory of microbiology faculty of mathematics and natural sciences, universitas sumatera utara were used for growth inhibition assay by the isolated bacteria. kr05 and lk08 shared similar morphological and physiological characters; like wise, kr07 shared property similarities with bk08. all bacterial isolates inhibited the growth of , , and at a different extent. lk08 showed the highest inhibition rate followed by bk07 and bk09. however, was inhibited more by bk07 and bk09. the crude enzyme preparation of the latter isolate exhibited the highest chitinase activity. the result suggested that their swarming activity seemed to contributed to inhibition of fungal growth. key words: chitinolytic bacteria, growth inhibition, pathogenic fungi, chitinase activity. telah dilakukan isolasi dan karakterisasi bakteri kitinolitik dan kajian tentang potensi bakteri ini dalam menghambat pertumbuhan jamur pathogen tanaman. bakteri diisolasi dari contoh tanah karo, langkat, dan bangka, sumatra. , dan merupakan koleksi laboratorium mikrobiologi, fakultas matematika dan ilmu pengetahuan alam, universitas sumatera utara digunakan dalam uji daya hambat oleh bakteri kitinolitik. kr05 dan lk08, serta kr07 dan bk08 memiliki sifat morfologi dan fisiologi yang sama. semua isolat bakteri menghambat pertumbuhan , , dan . lk08 menunjukkan daya hambat terbesar diikuti oleh bk07 dan bk09. namun demikian kelihatannya lebih terhambat oleh bk07 dan bk09. enzim kasar dari isolat bk09 menunjukkan aktivitas kitinase tertinggi. hasil menunjukkan bahwa aktivitas keriap memberikan sumbangan terhadap kemampuan menghambat pertumbuhan jamur patogen. kata kunci: bakteri kitinolitik, penghambatan pertumbuhan, jamur patogen, aktivitas kitinase. the use of biological sources for plant disease control remains an important potential alternative to the use of pesticides. this method has been proposed for the replacement of chemical control of plant diseases. biological control using microorganisms has been studied intensively since there is not many alternatives to control left (kotan . 2009; oskay 2009). health problems, environmental concerns, development of resistance in target populations also contribute to developing biological control using natural enemies (ningthoujam . 2009; mejía . 2008). antagonistic microorganisms, by their interactions with various soil-borne plant pathogens, play a major role in microbial equilibrium and serve as powerful agents for the control of biological diseases (alabouvette . 2006; ozbay and newman 2004). the antagonism may operate through antibiosis, competition, predation, or parasitism (alabouvette . 2006; ozbay and newman 2004). antibiosis is et al et al et al et al et al the antagonism resulting from the production of secondary metabolites by one microorganism that is toxic to other microorganisms. antibiosis is a very common phenomenon responsible for the activity of many biological control agents, such as fluorescent spp., spp., spp. and spp. (alabouvette . 2006), and aj-7 that suppressed the growth of , the causal agent of phytophthora blight in red-peppers (joo 2005). parasitism involves the production of several hydrolytic enzymes that degrade cell walls of pathogenic fungi (alabouvette . 2006; ozbay and newman 2004). the lytic activity of bacteria is one of many mechanisms that has been implicated in biocontrol (anitha and rabeeth 2010; patel 2007; gohel . 2006). a number of fungi are particularly susceptible to degradation by microorganisms (kim 2008). mycolytic enzyme-producing microorganisms have a great potential in solving such problems (patel 2007; gohel . 2006). investigations on lytic activity of biocontrol agents have focused mainly on the characterization of enzyme systems capable of pseudomonas bacillus streptomyces trichoderma et al s. halstedii phytophthora capsici et al et al. et al et al. et al. et al *corresponding author, phone: +62-61-8223564, fax: +62-61-8214290, e-mail: d.suryanto@lycos.com issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 144-148 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.8 degrading fungal cell wall components, of which chitinases are among the most intensively studied (anitha and rabeeth 2010; patel 2007; gohel . 2006). fungi and bacteria are important degraders of chitin in the soil ecosystem and contribute to the recycling of vital carbon and nitrogen resources (metcalfe . 2002; tsujibo . 2002). combined chitinolytic bacteria of strain c-1, sp. strain c-61, and strain c-3 inhibited the growth of , , and spp. (kim . 2008). and suppressed the growth of (susanto 2005). crude chitinase enzyme extracted from showed zone of inhibition on , , and 2 isolates of (anitha and rabeeth 2010). for the purpose of employing such microorganisms for biological control of fungal diseases of plant, isolation of bacterial candidates, and assay on their ability to inhibit fungal growth and their chitinase activity are one of the important steps to be done prior other steps. screening of chitinolytic bacteria was conducted by inoculating soil samples of langkat, karo, and bangka in modified salt medium (0.7 g k hpo , 0.3 g kh po , 0.5 g mgso .7h o, 0.01 g feso .7h o, 0.001 g znso , and 0.001 g mncl in 1000 ml) containing 2% (w/v) chitin colloidal (msmc) agar aerobically. after 4-5 days of incubation, a clear zone around the bacterial colony was formed, indicating that bacteria have chitinolytic activity. the colony was then transferred into different dishes several times until a pure culture was obtained. all cultures were cultivated at 30 °c. all media were adjusted to ph 6.8. unless mentioned otherwise, the seed cultures for all bacterial inoculation were taken from a 2-day-old culture of either msmc broth or agar. the cultures were grown with the initial cell concentration of ≈ 10 cfu ml . all fungal cultures, , and were of the collection of the laboratory of microbiology, department of biology, universitas sumatera utara, medan. the fungal cultures were maintained in potato dextrose agar (pda). cell shape and gram staining were evaluated microscopically, while the colony was observed directly. motility was observed using swarming activity. physiological properties including catalase, oxidase, gelatinase, citrate-, starch-, and sugarutilization tests were observed using 3% h o and gelatine nutrien medium, simmons citrate agar (sca), starch agar (sa), and triple sugar indole agar (tsia), respectively. et al. et al et al et al serratia plymuthica chromobacterium lysobacter enzymogenes p. capsici rhizoctonia solani fusarium et al t. koningii t. harzianum ganoderma boninense et al. s. griseus f. oxysporum alternaria alternate rhizoctonia solani, f. solani aspergillus flavus g. boninense f. oxysporum penicillium citrinum , bactident oxidase (merck), 2 4 2 4 4 2 4 2 4 2 2 2 8 -1 ® swarming activity was observed by inoculating 5 μl of bacterial culture at the center of nutrient agar (na) or msmc agar dish. swarming activity was measured every day as colonial expansion. bioassays against different fungal species were conducted to determine the antifungal activity of chitinolytic bacterial isolates. an agar plug (ø 5-mm) of , and from the margin of an actively growing culture was inoculated at the center of petri dishes containing 20 ml of msmc agar. a well (ø 5-mm) was made at the edge of petri dishes opposite to the fungal inoculation at a distance of 3.5 cm from the center. after 1 day incubation, 30 μl of bacterial seed solution was inoculated into the wells. the plates were incubated at 25-30 °c for 3 to 8 days until there was an inhibition of fungal growth. antagonistic activity was measured as radius of uninhibited mycelia substracted by radius of inhibited mycelia by bacterial chitinolytic activity. one ml of chitinolytic isolate grown in msmc broth was reinoculated into 20 ml of msmc broth and incubated for 96 hours. crude enzyme was obtained by spinning bacterial culture at 6 000 g for 20 minutes. a supernatant containing crude enzyme mixed with substrate of 1% (w/v) chitin colloidal and incubated for 60 minutes. reaction was stopped by placing the reaction mixture in boiling water for 15 min. the mixture was spinned at 6.000 rpm for 20 minutes. the chitinase activity was measured by comparing free n-acetyl glucosamine (glcnac) that was treated with untreated chitin, with chitinase. glcnac was measured by the colorimetric method of reissig (1955). one unit of chitinase activity was defined as the amount of enzyme which produces 1 μmol glcnac per hour. six isolates were characterized based on their morphological and physiological traits. the isolates showed different characters, except kr05 and lk08, and kr07 and bk08 (table 1), indicating different species of bacteria, and all isolates were aerobe. some isolates exhibited catalase activity, an important enzyme produced by aerobic bacteria. five isolates were gram-negative and one was gram-positive. chitinolytic bacteria spread among gram-negative and gram-positive (anitha and rabeeth 2010; kotan . 2009; kim . 2008). bacterial genera and along with bacteria from the g r o u p a n d t h e family (donderski and (anitha and rabeeth 2010; prapagnee . 2009) are reported to produce chitinase. all isolates were able to grow in chitin colloidal media. chitin colloidal is one of the substrates g. boninense f. oxysporum p. citrinum was et al et al achromobacter, bacillus, chromobacterium, pseudomonas vibrio, f l a v o b a c t e r i u m c y t o p h a g a enterobacterioceae streptomyces et al brzezińska 2001), and volume 5, 2011 microbiol indones 145 commonly used to induce hydrolytic enzymes such as chitinase (nandakumar . 2007). chitinolytic bacteria were often characterized by their ability to produce a clear zone around their colony in chitin containing media. the clear zone was formed because chitin was hydrolized into its soluble monomer or derivates, mainly glcnac by extracellular chitinase produced by the bacteria (nandakumar . 2007). finally, the degradation products are then taken up by the cells as carbon and nitrogen sources (metcalfe . 2002; tsujibo . 2002). instead of the sim test, motility of isolates was examined through their swarming activity. the swarming activity was tested with chitin availability in the media. the isolates showed different responses of growth in na and msmc. the bacterial isolates swarmed on the agar surface, colonizing agar plate at different expansion rates (table 2). the swarming activity test indicated that the isolates moved at different rates in different media. all isolates expanded rapidly in chitin containing media, except that of bk07. the isolate showed more swarming activity in complete media such as na. kr07 showed optimum swarming activity both in na and in msmc agar with colony expansion of 34.20 and 29.07 mm respectively in 7-days incubation time, followed by bk09 (table 1). this indicated that the movement of isolates was stimulated by chitin availability. an et al et al et al et al autoinduction phenomenon might be involved in this swarming activity (eberl 1999). other factors affecting swarming activity were not examined in this study. however, senes . (2002) showed that increasing mannitol concentration from 2 to 20 mm in swim tryptone agar (tra) containing 0.25% agar inhibited the movement of . the swarming response of this strain, on the contrary, did not exhibit any changes when the mannitol concentration in swarm tra (1% agar) plates was varied from 0.2 to 20 mm. these results suggested that chemotaxis itself, at least toward mannitol, is unlikely to play a role in . swarming motility. swarming activity in solid media was observed as an indication of the colonization ability of the bacteria in the environment. interestingly, the ability to inhibit fungal growth seemed in line with the swarming activity, except for that of kr07 and bk08, hence the swarming activity test might be one useful step in selecting biological control agent. swarming activity may indicate the colonization rate of microorganism in the environment. swarming, therefore, is thought to be a successful strategy developed by flagellated microorganisms to ensure their rapid expansion in the natural environment, where microbial activities are often associated with solid surfaces (senes . 2002). efficacy of all isolates in inhibiting fungal growth was examined by growing the isolate next to the fungi et al. et al bacillus cereus b cereus et al characters isolates kr05 kr07 lk08 bk07 bk08 bk09 colony shape circular circular circular circular circular circular colony color transparent cream transparent transparent transparent transparent cell shape rod rod rod rod coccus rod physiological traits gram + motility + + + + + + catalase + + + oxydase + + + + + + starch + + + citrate + + + + + gelatin + + + + table 1 morphological and physiological traits of the chitinolytic bacterial isolates isolates media colony expansion (mm) of days4 5 6 7 kr05 msmc 6.63 7.85 8.90 9.60 na 9.18 12.15 13.07 14.13 kr07 msmc 31.67 32.85 33.13 34.20 na 27.38 27.82 28.53 29.07 lk08 msmc 8.60 9.60 10.65 11.58 na 4.00 5.00 5.00 6.00 bk07 msmc 6.58 6.73 7.40 8.18 na 9.28 10.11 10.98 11.58 bk08 msmc 16.75 18.75 19.38 20.07 na 8.07 8.78 9.25 10.10 bk09 msmc 19.75 24.58 24.70 25.65 na 4.68 6.15 6.85 7.65 table 2 swarming activity of the chitinolytic bacteria on chitin colloidal msmc agar and na 146 suryanto et al. microbiol indones in chitin-containing media to induce extracellular chitinase. the isolate ability in controlling fungal growth varied during cultivation (table 3). although lk08 showed optimum inhibition rate to all fungi, bk07 exhibited more inhibition to the growth of . different ability of chitinolytic bacterial isolates to inhibit fungal growth was previously observed (matroudi . 2009). in general, all isolates were more capable of inhibiting growth rather than that of and . this variation might be caused by species specific, different bacterial chitinase activity, chitin composition of the fungal mycelium, the growth rate of the bacterial and the fungi, and other antifungal metabolites. the fungal cell walls are usually which is bound to chitin in an amorf structure. since nsible for fungal cell wall lytic and degradation (anand and reddy 2009; gohel . 2006). the presence of other metabolites in addition to chitinase is also responsible for inhibiting fungal growth (prapagdee . 2008; getha and vikineswary 2002). all six isolates were grown in msmc broth to find out their ability to utilize chitin. the result showed that their chitinase activity varied (table 4). although crude chitinase activity of bk09 was relatively high, this isolate was not as capable as lk08 to inhibit fungal growth. the high chitinase activity seemed not to correlate with the ability of an isolate to inhibit fungal p. citrinum et al g. boninense f. oxysporum p. citrinum et al et al composed not only of chitin but also of other sugars such as β-1,3 glucan, fungal cell wall is made up of mainly of glucan and chitin, the β1,3-glucanase and chitinase are key enzymes respo growth. this is probably caused by the structural difference of the substrates. the structure of chitin in fungal cell walls, however, was more complex compared to that of chitin colloidal used as carbon source in chitinase activity assay. the mycelia of and is rather hard to be lysed since the cells contain protein and lipid, which act as a barrier to hydrolytic enzymes (sivan and chet 1989). this research was supported by a grant from dp2m, directorate general of higher education, indonesian ministry of education through hibah bersaing. fusarium penicillium acknowledgements references alabouvette c, olivain c, steinberg c. 2006. biological control of plant diseases: the european situation. eur j plant pathol. 114(3): 329-341. doi: 10.1007/s10658-005-0233-0. anand s, reddy j. 2009. biocontrol potential of sp. against plant pathogens. int j agric sci. 1(2): 30-39. anitha a, rabeeth m. 2010. degradation of fungal cell walls of phytopathogenic fungi by lytic enzyme of afr j plant sci. 4(3): 61-66. trichoderma streptomyces griseus. table 3 diameters of the inhibition zone as a result of antagonism assays of the chitinolytic bacteria against fungi isolates fungi inhibition zone (mm) of days4 5 6 7 kr05 ganoderma boninense 5.13 15.15 17.22 12.61 fusarium oxysparum 0 0 1.98 1.93 penicillium citrinum 0 0 0 1.34 kr07 ganoderma 0 3.00 3.00 5.00 fusarium oxysparum 0 3.67 3.67 4.84 penicillium citrinum 0.19 0.19 0.85 0.85 lk08 ganoderma boninense 0 13.60 18.87 25.34 fusarium oxysparum 0 6.96 14.98 21.96 penicillium citrinum 0.30 2.43 2.45 0.70 bk07 ganoderma boninense 15.14 15.14 17.22 12.61 fusarium oxysparum 0 0 4.83 4.83 penicillium citrinum 2.25 2.25 3.30 8.12 bk08 ganoderma boninense 0.94 0.94 0.63 0.31 fusarium oxysparum 0 0 1.37 2.37 penicillium citrinum 0.25 0.25 0.95 0.32 bk09 ganoderma boninense 5.52 5.52 7.84 18.13 fusarium oxysparum 0 0 2.31 2.49 penicillium citrinum 2.52 2.52 3.49 7.92 table 4 crude chitinase activities of the chitinolytic bacteria isolates activity (unit ml ) -1 kr05 0.088 kr07 0.036 lk08 0.060 bk07 0.092 bk08 0.363 bk09 0.726 volume 5, 2011 microbiol indones 147 donderski w, brzezińska ms. 2001. occurrence of chitinolytic bacteria in water and bottom sediment of eutrophic lakes in iławskie lake district. polish j environ studies. 10(5): 331-336. eberl l, molin s, givkov m. 1999. surface motility of mg1. j bacteriol. 181(6): 1703-1712. getha k, vikineswary s. 2002. antagonistic effects of strain g10 on f.sp. race 4: indirect evidence for the role of antibiosis in the antagonistic process. j ind microbiol biotechnol. 28(6): 303-310. gohel v, singh a, vimal m, ashwini p, chhatpar hs. 2006. bioprospecting and antifungal potential of chitinolytic microorganisms. afr j biotechnol. 5(2): 54-72. joo g-j. 2005. production of an anti-fungal substance for biological control of causing phytophthora blight in red-peppers by biotechnol lett. 27(3): 201-205. kim yc, jung h, kim ky, park sk. 2008. an effective biocontrol bioformulation against phytophthora blight of pepper using growth mixtures of combined chitinolytic bacteria under different field conditions. eur j plant pathol. 120(4): 373-382. doi: 10.1007/s10658007-9227-4. kotan r, dikbas n, bostan h. 2009. biological control of post harvest disease caused by on stored lemon fruits. afr j biotechnol. 8(2): 209-214. matroudi s, zamani mr, motallebi m. 2009. antagonistic effects of three species of sp. on the causal agent of canola stem rot. egyptian j biol. 11: 37-44. mejía lc, rojas ei, maynard z, van bael s, arnold ae, hebbar p, samuels gj, robbins n, herre ea. 2008. endophytic fungi as biocontrol agents of pathogens. bio control. 46(1): 4-14. metcalfe ac, krsek m, gooday gw, prosser ji, wellington emh. 2002. molecular analysis of a bacterial chitinolytic community in an upland pasture. appl environ microbiol. 68(10): 5042-5050. doi: 10.1128/aem.68.10.5042-5050.2002. serratia liquefaciens streptomyces violaceusniger fusarium oxysporum cubense phytophthora capsici streptomyces halstedii. aspergillus flavus trichoderma sclerotinia sclerotiorum, theobroma cacao nandakumar r, babu s, raguchander t, samiyappan r. 2007. chitinolytic activity of native strains. j agric sci technol. 9: 61-68. ningthoujam ds, sanasam s, tamreihao k, nimaichand s. 2009. antagonistic activities of local actinomycete isolates against rice fungal pathogens. afr j microbiol res 3(11): 737-742. oskay m. 2009. antifungal and antibacterial compounds from strains. afr j biotechnol. 8(13): 3007-3017. ozbay n, newman se. 2004. biological control with spp. with emphasis on . pakistan j biol sci. 7(4): 478-484. patel b, gohel v, raol b. 2007. statistical optimization of medium components for chitinase production by strain jd2. ann microbiol. 57(4): 589-597. prapagdee b, kuekulvong c, mongkolsuk s. 2008. antifungal potential of extracellular metabolites produced by against phytopathogenic fungi. int j biol sci. 4(5): 330-337. reissig jl, strominger jl, leloir lf. 1955. a modified colorimetric method for the estimation of n-acetylamino sugars. j biol chem. 27: 959-66. senes s, celandroni f, salvetti s, beecher dj, wong acl, ghelardi e. 2002. swarming motility in and characterization of a mutant impaired in swarm cell differentiation. microbiol. 148(6): 1785-1794. sivan a, chet i. 1989. degradation of fungal cell walls by lytic enzymes of . j gen microbiol. 135(3): 675-682. susanto a, sudharto ps, purba ry. 2005. enhancing biological control of basal stem rot disease ( ) in oil palm plantation. mycopath. 159(1): 153-157. tsujibo h, orikoshi h, baba n, miyahara m, miyamoto k, yasuda m, inamori y. 2002. identification and characterization of the gene cluster involved in chitin degradation in a marine bacterium, sp. strain o-7. appl environ microbiol. 68(1): 263-270. doi: 10.1128/aem.68.1.263270.2002. pseudomonas fluorescens . streptomyces trichoderma t. harzianum paenibacillus sabina streptomyces hygroscopicus bacillus cereus fliy trichoderma harzianum ganoderma boninense alteromonas 148 suryanto et al. microbiol indones 9 siti herlinda.pmd selection of isolates of entomopathogenic fungi and the bioefficacy of their liquid production against leptocorisa oratorius nymphs siti herlinda1*, sri indah mulyati2 and suwandi1 1department of pest and discase, faculty of agriculture, universitas sriwijaya, kampus inderalaya, jalan raya palembang-prabumulih km 32, ogan ilir, inderalaya 30662, indonesia; 2dinas tanaman pangan dan hortikultura, provinsi sumatera selatan, jalan kapten t. tendean 1058, palembang 30139, indonesia entomopathogenic fungi are fungi pathogenic to insects and are widely used as biocontrol agents for insect pests. the aim of this research was to study the virulence of beauveria bassiana and metarhizium sp. isolates and to evaluate the efficacy of liquid production of those fungi against leptocorisa oratorius (rice bug). twelve isolates of b. bassiana and five isolates of metarhizium sp. were used in this research. selection result of b. bassiana isolates on third-instar rice bug nymphs showed that the isolate kbc caused the highest mortality rate (93%), while the lowest (46%) was caused by the isolate bby 725. the shortest time needed to produce 50% mortality (lethal time, lt 50 ) was 3.52 days (isolate kbc). the longest time (10.36 days) was produced by isolate slss. the mortality of rice bug nymphs caused by metarhizium isolates was only 50-62%. the shortest lt 50 of metarhizium (5.75 days) was produced by isolate mtm, while the longest (7.46 days) was produced by isolate mpx. bioefficacy tests on six kinds of liquid formations of entomopathogenic fungi indicated that all were effective, mostly with lt 50 d” two days. the mortality rates of rice bug nymphs caused by bioefficacy of fungus liquid production was generally above 85% up to 100%. the liquid media for entomopathogenic fungi performed better compared with solid media (sda), as indicated by the greater mortality rate and shorter lt 50 . key words: entomopathogenic fungi, isolate, liquid production, leptocorisa oratorius _____________________________________________ volume 2, number 3, december 2008 issn 1978-3477 p 141-146 ________________________ * corresponding author, phone: +62-711-580663, fax: +62-711-580276, e-mail: linda_hasbi@mail.pps.unsri.ac.id entomopathogenic fungi are lethal to insects, and at present these fungi are used as biocontrol agents for insects. known entomopathogenic fungi, such as beauveria, can kill insect pests of the order lepidoptera (soetopo 2004), coleoptera (lord 2001; wraight and ramos 2002) and homoptera (wraight et al. 1998), whereas metarhizium is effective in killing insects of the orders orthoptera (santiago et al. 2001), diptera (moraga et al. 2006), and hemiptera (liu et al. 2002). the effectiveness of toxins of either fungus against insects from the order hemiptera, especially the rice bug (leptocorisa oratorius), has not been reported. l. oratorius feeds on developing rice to reduce grain size. fungi from both genera have also been developed as granular bioinsecticide formulations (knudsen et al. 1990; geden and steinkraus 2003), but the possibilities for developing a liquid bioinsecticide containing beauveria and metarhizium as active agents to kill rice bugs has not yet been investigated. in constructing liquid bioinsecticide, factors that influence the virulence of fungi isolates during the process need to be considered so as to obtain effective products. the virulence of entomopathogenic fungi isolates is affected by a number of factors, such as the medium used in spore germination and production of the bioinsecticides (alves et al. 2002; geden and steinkraus 2003; thompson et al. 2007). during the process of bioinsecticide production with pathogenic fungi as the active agents, culture media such as maize, rice, and sabouraud dextrose-broth (sdb) are believed to modify the effectiveness of bioinsecticide production because of the different chemical compounds they contain. in addition to the cultivation medium, medium used in production is also thought to have an influence on the effectiveness of the bioinsecticide. in this research, the media added to the liquid composition of entomopathogenic fungi were combinations of shrimp shell compost extract (ssce) and sucrose. the goal of this research was to study the virulence of b. bassiana and metarhizium sp. isolates and to evaluate the efficacy of those liquid production on l. oratorius. materials and methods isolate preparation. insects infected by b. bassiana and metarhizium were collected from various locations in south sumatera and other provinces in indonesia. each fungus was then cultured and sub-cultured to obtain specific isolates (table 1). the isolation method used for these soil-borne fungi followed the method of liu et al. (2002). b. bassiana and metarhizium were collected from infected insects, and then grown on sabouraud dextrose-agar (sda) containing 100 ppm streptomycin. isolate selection. this was conducted by growing the spores from each isolate on sda and suspending these at a concentration of 106 spores ml-1. the concentration of the spores was measured by the method of moraga et al. (2006). spores were inoculated topically; each isolate was inoculated on 10 third-instar rice bug nymphs with a dose of 10 ml inoculum per nymph, according to the procedure of thalib et al. (2005), with three replications. the best isolates were then selected based on lowest lt 50 and highest mortality produced by b. bassiana and metarhizium. this gave one best isolate for b. bassiana and another for metarhizium. they were then cultivated on maize, rice and sdb media for producing liquid bioinsecticide. spore cultivation on maize media and production process. the best isolates of b. bassiana and metarhizium from the previous evaluation were cultivated separately on media of broken maize mixed with 20% (v/v) ssce and 30% (v/v) sterilized water per 250 g medium. scce was prepared according to suwandi (2004). the mixed medium was sterilized and then inoculated with 10 pieces (each measuring 0.5 x 0.5 cm) of pure fungus culture of the fungi for every 250 g cultivation medium, followed by incubation at room temperature for 10 days. each culture of b. bassiana (code a) and metarhizium (code d) cultivated on maize media were separately mixed with scce solution that had been maintaned at 60oc for two hours. scce was poured over the culture to obtain a final spore concentration of 109 spores ml-1. this mixture was then crushed in a blender and then filtered using a strainer of 1 mm hole diameter. each suspension was then added with 30% sugar as preservative to prevent spore germination. this liquid bioinsecticide was then placed in a heat-proof clear glass jar (5 cm diameter and 500 ml volume) that had been sterilized and covered with aluminum foil to be ready for application or storing. from here on, bioinsecticide with b. bassiana as the active agent will be referred to as bioinsecticide a, while those with metarhizium as active agent will be referred to as bioinsecticide d. the bioinsecticides were stored a month before testing with storage under ambient condition (23-25oc and 90% humidity). spore cultivation on rice media and production process. the spores of b. bassiana and metarhizium were cultivated separately on media of broken rice mixed with 20% scce (v/v) and 30% (v/v) sterilized water per 250 g medium as previously described for the maize media. each culture of b. bassiana (code b) and metarhizium (code e) cultivated on rice media were separately mixed with scce solution that had been kept at 60oc for two hours. scce was poured into the culture to obtain a spore concentration of 109 spores ml-1. the mixture of rice medium, scce and fungus was then macerated in a blender, and then filtered using a strainer with 1 mm hole diameter. each suspension was then combined with 30% (w/v) sugar as spore preservative. this liquid bioinsecticide was placed in clear heat-proof glass jars (5 cm diameter and 500 ml volume) that had been sterilized, covered with aluminum foil to be ready for application or storing. solution with b. bassiana as active agent will be referred to as bioinsecticide b, while solution with metarhizium as the active agent will be referred to as bioinsecticide e. the solution were stored a month before testing with storage under ambient condition (23-25oc and 90% humidity). spore cultivation on sdb media and production process. the spores of b. bassiana and metarhizium were cultivated separately on sdb media (30 g l-1 medium). the medium was inoculated with 10 pieces (each measuring 0.5 x 0.5 cm ) of pure b. bassiana culture for every 1.0 liter of cultivating medium. this procedure was repeated for metarhizium sp. incubation was at room temperature for 10 days using shaking incubator to obtain the optimal number of spores. each culture of b. bassiana (code c) and metarhizium (code f) grown on sdb media was mixed and blended separately and filtered through a strainer with 1 mm hole diameter. each suspension was then made to 30% (w/v) sugar as spore preservative. from here on, the mixture of sdb and sugar with b. bassiana as active agent will be referred to as bioinsecticide c, while the mixture with metarhizium will be referred to as bioinsecticide f. the bioinsecticide were stored a month before testing with storage under ambient condition (23-25oc and 90% humidity). preparation of test insects. rice bugs, adults and nymphs were collected from rice fields in various rice production centers, such as ogan komering ulu timur (okut), musi banyuasin (muba), ogan ilir (oi), ogan komering ilir (oki), and banyuasin. nymphs were then transferred to and maintained in the laboratory. they were placed inside a mesh cage (30 x 30 x 100 cm). inside the cage, some rice plants in the generative state (milky rice grains) were also placed as feed and to provide a surface for egglaying. every day, the first instar nymphs produced were moved into a plastic cage (30 x 50 x 50 cm) that contained fresh feed. for the bioefficacy tests, second (f2) or later generations of rice bug nymphs were used. bioefficacy tests of bioinsecticide against rice bug. liquid solutions with the active ingredient from b. bassiana (code a, b, c) , and the solution with the active ingredient from metarhizium (code d, e, f) were tested for their effectiveness by conducting bioefficacy tests at three different concentrations (103, 105, 107 spores ml-1), and a control (sterilized water). bioefficacy tests were conducted by applying 10 ml bioinsecticide topically to third-instar rice bugs. each level of concentration was applied to 10 test nymphs with three replications of each. after application of a bioinsecticide, the third-instar nymphs were placed into plastic cylinders (8.5 cm diameter and 15 cm high) covered with muslin and a stem of a rice plant with milky grains was placed inside the cylinder. during the nymph stage, the number of dead nymphs was recorded every three hours, while the number of the nymphs that transformed into imagos was recorded every day until each nymph had transformed. table 1 the entomopathogenic fungi isolates collected and used isolate code source insect insect location beauveria bassiana t b bby 715 kbc cpjw8 p d 2 p d 1 bts3 btss7 la bby 725 ua slss metarhizium sp. mla mbl m t m m t m t m p x thrips tabaci hypothenemus hampei chrysodeixis chalcites chrysodeixis chalcites chrysodeixis chalcites plutella xylostella tenebrio molitor tenebrio molitor leptocorisa acuta conopomorpha cramerella setora nitens spodoptera litura leptocorisa acuta bronstispa longissima tenebrio molitor tenebrio molitor plutella xylostella bogor jember curup (bengkulu) cipanas pagaralam pagaralam palembang inderalaya jember jember jember inderalaya inderalaya manado palembang inderalaya palembang 142 herlinda et al. microbiol indones data analysis. mortality data and death time of rice bug nymphs were analyzed using lt 50 , calculated by means of probit analysis employing the program sas-stat in sas 6.12. results results of the selection for isolates of b. bassiana on third-instar rice bug nymphs showed wide variation in mortality, with a range of 46-93.33% (table 2). the highest rice bug mortality (93%) was produced by isolate kbc, while the lowest was produced by isolate bby 725. the mean of (lt 50 ) values indicated that the shortest (3.52 days) was produced by isolate kbc, while the longest (10.36 days) was produced by isolate slss. isolate kbc was obtained from c. chalcites, isolate bby 725 from conopomorpha cramerella, and isolate slss from spodoptera litura. the mortality of rice-bug nymphs produced by metarhizium isolates showed different tendencies compared with that of b. bassiana isolates. there was little variation in mortality, this ranging only between 50 to 62% (table 3). the shortest mean lt 50 values (5.75 days) resulted with isolate mtm, while the longest value (7.46 days) was associated with isolate mpx. isolate mtm was collected from t. molitor while mpx was collected from p. xylostella. isolate kbc of b. bassiana and isolate mtm of metarhizium were selected for processing into solutions of entomopathogenic fungus. results from the bioefficacy testing of six kinds of solutions on rice bug nymphs indicated that all were effective, mostly with lt 50 values d” 2 days, except for bioinsecticide a at concentration of 103 conidia ml-1 and bioinsecticide b at 107 conidia.ml-1 (table 4). lc 50 values in this research could not be calculated because there was no significant effect of spore concentration on the mortality or lt 50 for nymphs. the lt 50 for nymphs with either b. bassiana or metarhizium sp. in the liquid bioinsecticides were shorter than values for the same fungi isolates on sda (table 2, 3). in the bioefficacy tests, no bioinsecticide of the fungi cultured on maize media (code a and d), rice media (code b and e), or sdb (code c and f) produced a mortality rate or lt 50 values better than any other (table 4, 5). therefore, those three media are all suitable for the cultivation of table 2 selection results of entomopathogenic fungal isolates (beauveria bassiana) with rice-bug as test insect lt 50 (days) mean lowest highest isolate code mortality (%) 8.30 7.30 3.11 6.51 7.90 7.40 4.70 6.97 5.30 9.18 7.23 9.64 13.12 11.20 3.91 9.04 13.60 11.20 11.85 11.63 7.20 11.04 9.11 11.33 t b bby 715 kbc cpjw8 p d 2 p d 1 bts3 btss7 la bby 725 ua slss 46.67 53.33 93.33 56.67 46.67 50.00 65.00 50.00 63.33 46.00 64.00 52.00 10.07 8.80 3.52 7.60 9.80 8.90 6.80 9.70 6.20 9.96 8.05 10.36 table 3 selection results of entomopathogenic fungus isolates (metarhizium) with rice bug as test insect lt 50 (days) mean lowest highest mla mbl m t m m t m t m p x 62.00 60.00 62.00 54.00 50.00 7.05 6.96 5.75 6.93 7.46 6.29 6.28 5.06 5.82 6.38 8.00 7.78 6.56 5.60 9.20 isolate code mortality (%) 3.09 1.94 1.35 2.02 1.62 2.35 1.99 1.79 1.71 a b c control 1 x 103 1 x 105 1 x 107 1 x 103 1 x 105 1 x 107 1 x 103 1 x 105 1 x 107 0 1 0 0 . 0 0 1 0 0 . 0 0 96.67 90.00 1 0 0 . 0 0 86.67 90.00 90.00 80.00 0 2.68 1.69 1.14 1.75 1.41 2.04 1.62 1.52 1.44 2.31 1.44 0.92 1.47 1.18 1.73 1.25 1.24 1.14 table 4 bioefficacy test results of liquid bioinsecticide (with active ingredient beauveria bassiana) on rice bugs at three different concentrations 95% confidence level (days) lowest highest bioinsecticide source spore concentration (spores ml-1) mean mortality (%) mean lt 50 (days) 1 x 103 1 x 105 1 x 107 1 x 103 1 x 105 1 x 107 1 x 103 1 x 105 1 x 107 0 90.00 90.00 1 0 0 . 0 0 90.00 90.00 93.33 86.67 90.00 90.00 1.41 1.39 1.60 1.31 1.26 1.38 1.46 1.58 1.30 1.13 1.12 1.30 1.03 0.98 1.10 1.17 1.30 1.02 1.67 1.64 1.89 1.57 1.53 1.65 1.73 1.85 1.56 d e f control table 5 bioefficacy test results of liquid bioinsecticide (with active ingredient metarizium) on rice bugs at three different concentrations 95% confidence level (days) lowest highest bioinsecticide source spore concentration (spores ml-1) mean mortality (%) mean lt 50 (days) volume 2, 2008 microbiol indones 143 entomopathogenic fungi. mortality rates of rice-bug nymphs caused by liquid bioinsecticides were also higher compared to those of isolates grown on sda. mortality rates of ricebug nymphs resulting from the fungal-derived suspension was mostly above 85%, with the highest at 100% (table 4, 5). as shown by the lt 50 values, mortality rates of rice bug nymphs resulting from the fungal-derived suspension was not affected by spore concentration treatment. discussion results of b. bassiana isolates on rice-bug nymphs showed that isolate kbc (from c. chalcites) produced the best bioinsecticide, while of the isolates from metarhizium sp. mtm was the best. therefore, isolates kbc and mtm were selected as active agents for the liquid bioinsecticide preparations of entomopathogenic fungi. those two isolates were selected because they had the highest capabilities and were the quickest to kill rice bug nymphs, i.e. the shortest lt 50 . the ability of those entomopathogenic fungal isolates to produce the highest mortality rates and lowest lt 50 values might be caused either by their genetic characteristics, or by their the viability of the spores. according to soetopo (2004), a high viability of fungal spores tended to cause a high mortality rate on host insects, but it was not the main cause. the main cause here was genetic characteristics of the strains. b. bassiana and metarhizium sp. isolates needed 3.83 days and 3.52 days, respectively, as the shortest time to kill their insect hosts. the time needed was relatively long because the spores attached to the integuments had to germinate first. fuxa and richter (2004) stated that hyphae from metarhizium sp. spores entered the host’s body with the help of enzymes or mechanical pressure. in the end, the host was covered all over with propagules and the soft parts of the body were penetrated so hyphal growth could be observed outside the host insect’s body. external hyphal growth would produce conidia which would be spread spores into the environment upon reaching maturity. these then infect other healthy insects. in this research, host insects infected by b. bassiana showed symptoms such as loss of appetite, slow movement and finally died. after death, white colored fungal hyphae appeared from their stiff and dry bodies. dead insect hosts infected by metarhizium sp. showed the same symptoms as those infected by b. bassiana, except for the color of the hyphae, which was greenish white. during the inoculation process of fungal spores, the humidity under the cage cover was kept above 90% and room temperature was adjusted to 23-25oc. this was to prevent poor spore germination. bidochka et al. (2000) stated that the optimum temperature needed for entomopathogenic fungal spores to germinate was 22-27oc with optimum humidity above 90%, and that the greater the humidity the more virulent the fungi would be. at under 86% humidity, the virulence would decrease continously. in bioefficacy tests of the liquid solution from entomopathogenic fungi, the mortality of the insect host was greater and occurred faster when the fungus was in liquid state compared to isolates grown on solid media (sda). in other words, the liquid state of the fungus was able to increase their effectiveness. akbar et al. (2005) reported that entomopathogenic fungi in liquid fungus tended to have higher viability compared to those on solid media. consequently, they are also more virulent. three kinds of media (maize + ssce, rice + ssce and sdb) were used in the processing of liquid bioinsecticide from entomopathogenic fungi were better for fungal culturing than were the solid media used for culturing the same isolates. the liquid fungus caused higher mortality and faster killing rate compared to sda. the better the media for fungi culturing were, the more mycotoxin would be produced by entomopathogenic fungi (klinger et al. 2006). according to akbar et al. (2005) entomopathogenic fungi grown on liquid media produced mycotoxins and spores with greater viability and virulence compared with those established on solid media. in short, liquid processing of entomopathogenic fungi kill through two processes, firstly by enhanced growth of fungal spores, and secondly by the mycotoxin contained in the resulting suspension resulted in greater mortality (shorter lt 50 ). acknowledgements financial support of this research was provided by the project of incentive research fund, ministry of research and technology, republic of indonesia, budget year 2007 with contract number: 94/rt/insentif/ppk/i/2007, 15th january 2007. we thank hartono for his assistance in the laboratory work. references akbar w, lord jc, nechols jr, loughin tm. 2005. efficacy of beauveria bassiana for red flour beetle when applied with plant essential oils or in mineral oil and organosilicone carriers. j econ entomol 98:683-688. lord jc. 2001. desiccant dusts synergize the effect of beauveria bassiana (hyphomycetes: moniliales) on stored-grain beetles. j econ entomol 94:367-372. alves sb, rossi ls, lopes rb, tamai ma, pereira rm. 2002. beauveria bassiana yeast phase on agar medium and its pathogenicity againts diatraea saccharalis (lepidoptera: cerambidae) and tetranychus urticae (acari: tetranychidae). j invert pathol 81:70-77. bidochka mj, kamp am, decroos jna. 2000. insect pathogenic fungi: from genes to populations. fungal pathol 42:171-193. fuxa jr, richter ar. 2004. effects of soil moisture and composition and fungal isolate on prevalence of beauveria bassiana i n laboratory colonies of the red imported fire ant (hymenoptera: formicidae). environ entomol 33:975-981. geden cj, steinkraus dc. 2003. evaluation of granular formulations of beauveria bassiana for control of lesser mealworm and hide beetle in georgia poultry houses. j econ entomol 96:1602-1607. klinger e, groden e, drummond f. 2006. beauveria bassiana horizontal infection between cadavers and adults of the colorado potato beetle, leptinotarsa decemlineata (say). environ entomol 35:992-1000. knudsen gr, johnson jb, eschen dj. 1990. alginate pellet formulation of a beauveria bassiana (fungi: hyphomycetes) isolate pathogenic to cereal aphids. j econ entomol 83:22252228. 144 herlinda et al. microbiol indones liu h, skinner m, parker bl, brownbridge m. 2002. pathogenicity of beauveria bassiana, metarhizium anisopliae (deuteromycotina: hyphomycetes), and other entomopathogenic fungi against lygus lineolaris (hemiptera: miridae). j econ entomol 95:675-681. moraga eq, garcia ar, alvarez cs. 2006. laboratory evaluation of entomopathogenic fungi beauveria bassiana and metarhizium anisopliae against puparia and adults of ceratitis capitata (diptera: tephritidae). j econ entomol 99:1955-1966. santiago dr, castillo ag, arapan rs, navasero mv, eusebio je. 2001. efficacy of metarhizium anasopliae (metsch.) sor. againts the oriental migratoria locust, locusta migratoria manilensis meyen. philipp agric sci 84:26-34. soetopo d. 2004. efficacy of selected beauveria bassiana (bals.) vuill. isolates in combination with a resistant cotton variety (psb-ct 9) againts the cotton bollworm, helicoverpa armigera (hübner) (lepidoptera: noctuidae). [dissertation]. philippines: university of the philippines los banos. suwandi s. 2004. effectiveness of shrimp shell compost extract for suppression of leaf diseases on cowpea, chilli pepper and cabbage. pest trop j 1:18-25. thalib r, adam t, suwandi s, pujiastuti y, herlinda s. 2005. fitness of beauveria bassiana isolates from sumatera and java on plutella xylostella larvae. in: crop security for food safety and human health. proceedings of the first international seminar and conference of security. malang, indonesia, sept 20-22, 2005. thompson sr, brandenburg rl, roberson gt. 2007. entomopathogenic fungi detection and avoidance by mole crickets (orthoptera: gryllotalpidae). environ entomol 36:165-172. wraight sp, carruthers ri, bradley ca, jaronski st, lacey la, wood p, wraight sg. 1998. pathogenicity of the entomopathogenic fungi paecilomyces sp. and beauveria bassiana againts the silverleaf whitefly, bemisia argentifolii. j invertebr pathol 71:217-226. wraight sp, ramos me. 2002. application parameter affecting field efficacy of beauveria bassiana foliar treatments againts colorado potato beetle, leptnotarsa decemlineata. biol contr 23:164178. volume 2, 2008 microbiol indones 145 6 512 16s rdna (anaindrayati)... 16s rdna-based identification of novel superoxide dismutase producing bacteria isolated from indonesia ana indrayati , valentina yurina , laras ajeng pitayu , debbie soefie retnoningrum 1 2 2 2* and 1 2 university of galuh, jalan re martadinata 150, ciamis 46251, indonesia; school of pharmacy, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia flavobacterium okeanokoites escherichia fergunosii e. coli pantoea escherichia bacillus, pectobacterium pseudomonas polymerase chain reaction flavobacterium okeanokoites, escherichia fergunosii e. coli pantoea escherichia bacillus, pectobacterium pseudomonas superoxide dismutase (sod) has therapeutic importance because of its antioxidant activity and protects cells from reactive oxygen species attack. this research was intended to screen bacteria isolated from indonesia for producing novel sods and to identify the producers using 16s rdna approach. intracellular proteins were each extracted and assayed for their inhibition reduction activity by colorimetric method and by zymography for the presence of sod protein band(s). for species identification, each of 16s rdna genes was amplified by polymerase chain reaction from genomic dna followed by sequencing, blast, multiple alignment and phylogenetic analyses. all 16 intracellular proteins gave inhibition reduction percentage in the range of 15 to 70% and in zymography, their sod profiles were quite diversed with at least one intense sod band present in most isolates. the sod producers were assigned to three species, , , and and to four genera, , , and . the remaining five were grouped in gamma-proteobacterium cluster and two formed a cluster with . three marine and four soil isolates could be attractive candidates for novel sods based on unique properties of sod producers. in conclusion, 16s rdna-based identification of bacteria isolated from indonesia reveals that seven isolates might be attractive candidates for novel sod producers to be applied in pharmaceutical fields in the future. key words: 16s rdna, superoxide dismutase, zymography, marine bacteria, soil bacteria superoksida dismutase (sod) penting untuk terapi karena aktivitas antioksidannya dan sel dilindungi dari radikal oksigen species. penelitian ini bertujuan melakukan penapisan terhadap isolat bakteri dari indonesia yang menghasilkan sod baru dan melakukan identifikasi spesies bakteri dengan pendekatan 16s rdna. protein intrasel diisolasi dan diuji aktivitas penurunan inhibisinya secara kolorimetri dan dianalisis dengan zimografi untuk mendeteksi keberadaan protein sod. untuk identifikasi spesies, setiap gen 16s rdna diamplifikasi dengan dari dna genom, dilanjutkan dengan analisis urutan nukleotida, blast, pensejajaran urutan nukleotida, dan pembuatan pohon filogenetika. sebanyak 16 protein intrasel memberikan inhibisi reduksi antara 15 dan 70% dan dengan zimografi, profil sod-nya cukup beragam dengan setidaknya satu pita tebal sod pada sebagian besar isolat. penghasil sod diidentifikasi sebagai tiga spesies, yaitu dan dan empat genus, yaitu , , dan . lima isolat dimasukkan dalam kelompok gamma-proteobakteri, sedangkan dua isolat ke dalam kelompok . tiga isolat dari laut dan empat isolat dari tanah berpotensi sebagai kandidat penghasil sod baru berdasarkan pada keunikan sod yang dihasilkan. identifikasi berbasis 16s rdna bakteri dari indonesia menunjukkan ada tujuh isolat kandidat yang menarik sebagai penghasil sod baru yang dapat diterapkan di bidang farmasi di masa depan. kata kunci: 16s rdna, superoksida dismutase, zimografi, bakteri laut, bakteri tanah superoxide dismutases (sods) [e.c. 1.15.1.1] are key enzymes that play a role in protecting cells and tissues from toxic effects of free radicals (pan 1999). they catalyze the conversion of toxic superoxide radical (o -) to less toxic hydrogen peroxide (h o ) and oxygen (o ) (mccord and fridovich 1969). a number of aerobic bacteria and archaea has been reported to produce sods. as many as 57 sod homolog genes were found in 138 bacterial and archaeal genomes (banci . 2005). sods from prokaryotes require metal ions as cofactors and based on their cofactors, sod from prokaryotes are classified as (cu, zn)sod, fe-sod, mn-sod, and ni-sod (wuerges . 2004). several bacterial sods have been thoroughly investigated and sods from et al. et al et al 2 2 2 2 psychrophilic, mesophilic, and thermophilic bacteria have also been reported (valderas . 2001; castellano . 2006; he . 2007). sods have been used in pharmaceutical field in particular for therapeutic and cosmetic purposes. it was reported that sod is useful as therapeutic agent for treatment of inflammatory disorders (yasui and baba 2006). another work indicated that the inclusion of sod in combination with catalase in cosmetic preparations prevents skin aging (lods . 2000). some reports demonstrated that decrease of sod gene expression is associated with apoptosis and a number of neurodegenerative diseases including parkinson's disease and alzheimer (choi . 2005; nikam . 2009). our laboratory is interested in finding novel sods for therapeutic and cosmetic applications. due to high microbial diversity, indonesia has potential to contain et al et al et al et al et al et al *corresponding author, phone/fax: + 62222504852, email: retnoningrum@indo.net.id issn 1978-3477, eissn 2087-8575 vol 5, no 2, june 2011, p 88-93 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.2.6 sod-producers from previously unidentified microorganisms. the number of prokaryotes on earth was predicted to be 4-6 x 10 and only minority of them has been explored for sod activity (curtis . 2004). our current work focused on the exploration of novel sod-producing bacteria isolated from soil and marine sources in indonesia. our preliminary screening using a qualitative assay indicated that 16 of 40 bacterial isolates from our collection demonstrated sod activity measured in a qualitative assay. in present research, we intended to measure the sod activity from the 16 isolates in a more accurate quantitative assay, to confirm the presence of sod protein using zymography and to identify sod producers using 16s rdna-based method. all isolates were obtained from our culture collection and previously isolated from marine and soil sources in indonesia. stla, bl6, sck, and sck2 were isolated from sea water located at cilaut eureun, garut, west java; mks11 and mks13 were isolated from sea water close to makassar, sulawesi; others were isolated from soil at north bandung, west java. when first isolated, microbes from sea water were enriched in sea water broth then isolated using sea water agar. microbes from soil were enriched in liquid nutrient medium and isolated using nutrient agar. luria bertani (lb) broth was used to grow all isolates in sod measurement assay except nutrient broth was used for isolates stla, cm2, and mks11. 7.5% (v/v) overnight culture grown in 37 ºc was added to fresh liquid lb medium or nutrient broth and incubation was continued until the optical density of 580 nm of 0.4-0.6 was reached. bacterial suspension was centrifuged at 1600 g, 10 ºc for 10 min; cell pellet was washed with phosphate buffer saline (pbs), resuspended in 1.5 ml of pbs and phenyl methyl sulphonyl fluoride was added until final concentration of 1 mm. cells was lyzed using sonication (fisher sonic dismembrator, model, 300) at 4mhz on ice 30 sec for each cycle, with 30 sec break for each cycle, until the bacterial suspension opacity significantly reduced. after sonication, the lysate was centrifuged at 1400 g, 6 ºc for 10 min. total protein extract was concentrated using freeze-drying technique until half of original volume; the concentrated protein was analyzed for its sod activity. assay was done using colorimetric and zymography methods. in colorimetric assay, sod activity was determined for its ability to inhibit nitro 30 et al materials and methods bacteria and growth media. total protein preparation. sod assay. blue tetrazolium (nbt) reduction by riboflavin and tetramethylethylenediamine (temed), according to published method using (sun . 1988). the presence of sod activity in protein extracts was confirmed by zymography method as previously described using 3 µg of intracellular proteins (kim 2000). the 16s rdna of the 16 microbes were amplified by pcr using a pair of specific primers, unibi (5'-ggttac ( g c ) t t g t ta c g a c t t3 ' ) a n d b a c t f i (5'agagtttgatc(ac)tggctcag-3') (weisburg 1991). the pcr products were analyzed using 1% agarose gel electrophoresis, purified from gel using gfx pcr dna and gel band purification kit (amersham) and directly sequenced using both primers by an automated dna sequencer (macrogen, korea) to obtain complete nucleotide sequences of 16s rdna gene. the sequences were then analyzed for i d e n t i t y u s i n g n c b i b l a s t ( h t t p : / / w w w. ncbi.nlm.nih.gov) and wu-blast program (http://www.ebi.ac.uk) to 16s rdna sequences of species deposited in ncbi and embl databases. identification to species level was defined as sequence identity of minimum of 99% to sequences deposited in those databases; assignment at genus level was defined as sequence identity of 97-99% to sequences of previously characterized bacterial species. microbial isolate could not be assigned to any taxa when similarity score lower than 97% with those present in the databases at the time of analysis (harris and hartley 2003; janda and abbott 2007). interpretation of sequence data was valid only if a percentage of position ambiguity was less than 1%. multiple sequence alignment was done using clustalw2 to each isolate d i s p l a y i n g s i m i l a r p e r c e n t a g e i d e n t i t y ( h t t p : / / w w w. e b i . a c . u k / to o l s / m s a / c l u s t a l w 2 / ) . phylogenetic analysis was first performed to each isolate showing the similar percentage identity using clustalw2-phylogeny program based on neighborjoining clustering method to determine taxonomic relationships (http://www.ebi.ac.uk). the analysis was repeated to all isolates displaying percentage identity lower than 99% simultaneously after multiple alignments with 16s rdna sequences showing the closest taxonomic relationships obtained from individual phylogenetic analysis. our preliminary data showed that all 16 isolates displayed sod activity in a qualitative assay. in current work, our semiquantitive assay showed that the reduction 50 μl extract protein et al et al. et al. 16s rdna-based identification. results bacterial isolates obtained from indonesia. volume 5, 2011 microbiol indones 89 sku2 inhibition value was in the range of 14.95 to 70.42 % confirming that all isolates produced sod with variable degree (table 1). however, the colorimetric assay has limitation because other substances besides sod may interfere the value of % reduction inhibition. to rule out this possibility, zymography was performed to demonstrate the presence of protein band with sod activity. electrophoretic mobilities, the number and the intensity of sods from each isolate seemed to be quite variable. multiple sods (more than 1 sod bands) were observed in several isolates while others only produced one sod (fig 1, table 1). with regards to their sod protein band intensity, almost all isolates produced one intense sod band but in some of them, one or two additional bands were also present. three isolates only produced one intense sod band, stla, bl6 and bcd3. in general, the presence of intense sod band agreed with high sod activity measured by % reduction inhibition and vice versa (table 1). however, some isolates contained weak sod band in zymography gave high activity in colorimetric assay and several isolates contained at least one predominant sod band showed low activity in the semiquantitative assay. the size of 16s rdna pcr products was approximately 1410 base pairs (bp) which agreed to its theoretical size (fig 2). results of the identification of soil and marine isolates producing sod activity. blast analysis of two-directions sequencing of 16s rdna gene combined with individual phylogenetic analysis for percentage identity and microbial assignment are depicted in table 1. based on the criteria for taxa assignment ( materials and methods), three bacteria were identified to species level, stla to (renamed as ) and , bbk to , and tkc to . another four isolates were only assigned to genus level, tw to , sku2 to mks 13 to , and vr1 to . however, the remaining seven isolates exhibited lower sequence similarity in the range of 85-94% and two isolates gave more than 1% nucleotide ambiguity; therefore, they could not be further analyzed. phylogenetic analysis to 11 unidentified isolates demonstrated that they were grouped into four clusters (fig 3). two marine isolates, mks 13 and sc2, formed a cluster with and . vr and sku2 isolated from soil were positioned in one group with soil bacteria, and . bcd, a soil bacterium and bl6, a marine isolate were clustered in one group of and sp. three soil isolates (bdg1, cm4, and tw) and one marine bacterium (mks11) were grouped with gammaproteobacteria. see planomicrobium okeanokoites flavobacterium okeanokoites staphylococcus equorum escherichia fergusonii e. coli escherichia pantoea, bacillus pectobacterium bacillus subtilis b. mucilaginosus pectobacterium carotovorum pantoea vagans pseudomonas fluorescens pseudomonas name of isolates taxon assignment and percentage identity microbial sources reduction i (%)nhibition sod band band intensity stla planomicrobium okeanokoites staphylococcus equorum (99%) 66.81 1 intense bbk escherichia fergusonii (99%) 28.74 1 2 intense weak bl6 pseudomonas sp. igcar-26/07 (82%) 70.42 1 intense cm2 na* 40.74 1 weak sck na* 47.06 1 2 intense weak bdg 2 erwinia pyrifolia (85%) 51.52 1 2 intense weak bcd pseudomonas fluoresc ens (90%) 62.87 1 intense bdg1 gamma-proteobacterium (90%) 36.54 2 weak tw escherichia coli (98%) 61.90 1 weak vr pectobacterium carotovorum (97%) 14.95 1 2 intense weak sc2 bacillus mucilaginosus (95%) 25.81 4 weak pantoea vagans c9-1 (98%) 36.36 3 2 intense weak tkc escherichia coli (99%) 33.33 2 intense mks 11 shigella sp.(92%) 37.50 1 weak mks 13 bacillus subtilis (98%) 39.29 2 1 intense weak cm4 shigella flexneri (94%) marine soil marine soil marine soil soil soil soil soil marine soil soil marine marine soil 62.96 1 1 intense weak table 1 sod activity of 16 microbial samples determined by semiquantitative method based on 16s rdna-based approach *percentage of ambiguity > 1% 90 indrayati et al. microbiol indones 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 discussion our result showed the sod profiles were quite variable in all isolates and this phenomenon agrees with that of other investigations (yun and lee 2002). in general, the sod activity measured by percentage inhibition reduction agreed with the results from zymography. however, several protein preparations showed disagreement between the two results. the total proteins may contain other substances that lowered the percentage reduction inhibition or on the other hand, sod inhibitors may be present in the protein preparations resulting high percentage reduction inhibition. our 16s rdna gene sequencing provided genus identification only 43.7% and even less for species assignment, 18.7%. this result was in contrast with other studies where 16s rdna-based analysis yielded better species and genus identification rates of 62 to 91% and 88.7%, respectively (janda and abbott 2007). in the present work, 56.3 % of isolates remained unidentified and the number was significantly higher than previously reported, 1 to 14% (janda and abbott 2007). a previous work indicated that a different rate of higher than 0.5% could be used a consideration for assignment of a new species within a known genus (drancourt . 2000). therefore, based on this concern, 11 isolates discovered in this study may be novel species under certain genera. other housekeeping genes such as a or b should be used for species identification in future experiment. based on blast analysis (table 1) and phylogenetic analysis of individual isolate (data not et al sod rpo fig 1 sod activity of bacterial intracellular total proteins from mid-log phase determined by zymography. 1, stla; 2, bbk; 3, bl6; 4, cm2; 5, sck; 6, bdg2; 7, bcd3; 8, bdg1; 9, tw; 10, vr; 11, sc2; 12, sku2; 13, tkc; 14, mks11; 15, mks13; and 16, cm4. isolates stla, bbk, and tkc were identified as , and , respectively. planomicrobium okeanokoites escherichia fergusonii, e. coli 1000 bp 2000 bp fig 2 representative of 16s rdna gene sod-producing isolates. lanes 1-4, isolates bcd, vr, tw, and stla, respectively; 5, negative control; 6, 100 bp dna marker. fig 3 neighbor-joining tree based on 16s rdna sequences analysis. eleven isolates are clustered into four groups, two with bacilli, two with , two with and , and the remaining are in gamma proteobacterium groups. pseudomonas pantoea pectobacterium mks13 bacillus mucilaginosus s-2 bacillus subtilis sc2 vr pectobacterium carotovorum_pc1 erwinia pyrifoliae pantoea vaganas-c9-1 sku2 bcd pseudomonas flourescens_jv bl6 bdg1 pseudomonas sp. igcar-26/07 gamma-proteobacterium cb24 cm4 mks11 shigella flexneri escherichia coli cft073 shigella sp. abo12-1 tw volume 5, 2011 microbiol indones 91 shown), stla exhibited 99% identity to two species, and . is present in human and certain animal skin and also reported to be isolated from processed food. on the other hand, was reported to be present in marine sediment and grew well in the presence of 07% of nacl (dai . 2005). it is a gram variable depending on the presence of nacl in the growth medium (nakagawa . 1996). in our gram staining, stla was a gram negative bacterium and since it was isolated from marine sediment; therefore, it was assigned as . since the temperature used for microbial growth was 37 c, it was not surprising that all isolates identified belonged to mesophilic microbes. our isolate, tkc and bbk identified as and , respectively were isolated from soil and later grown at 37 c. it has been known that they are distributed in water and soil. all other 11 isolates were also taxonomically close to mesophilic bacteria. since we are interested in sods to be applied for human use, the sods from mesophilic bacteria is suitable considering that they are stable at human temperature, 37 c. except for , sods from species identified in current work have not been studied extensively (gregory . 1973; steinman . 1994). particularly interesting are sods from marine bacteria since they might have unique properties. was reported to grow well in the presence of 0-7% of nacl (nakagawa . 1996); hence stla might be a source of novel salt resistant sod. the information of sod from was currently unavailable; however, our zymography demonstrated that stla produced one major sod (fig 1, lane 1). grows between 25-35 c and its main habitat is soil; however, most bacilli isolated from marine origin were . a marine isolate of was demonstrated to resist high temperature (50-55 ºc) and high nacl (10%) and it can grow at broad range of phs, 5.7 11.5 (ivanova . 1999). our isolate, mks 13, has a potency to produce unique sod since it was obtained from marine source and its taxonomy is close to . three genes were found in genome of subsp. strain 168. in our zymography experiment, mks13 was shown to produce three sod proteins with one major sod band, probably due to the response to aerobic condition in our experiment. sods from marine bacilli have not been investigated and therefore, mks13 is an attractive bacterium for producing novel sods. s. equorum f. okeanokoites s. equorum f. okeanokoites et al et al f. okeanokoites e. coli e. fergunosii e. coli et al et al f. okeanokoites et al f. okeanokoites b. subtilis b. subtilis b. subtilis et al b. subtilis sod b. subtilis subtilis o o o o e. coli pseudomonas p. fluorosence pseudomonas p. aeruginosa pantoea pectobacterium four isolates, bdg1, cm4, mks11 and tw were grouped in gamma-proteobacterium (fig 3). the amino acid sequences of sods of the members of this group share high homology to those of ; therefore, they are not attractive candidate for further study. two isolates, bl6 and bcd were clustered with sp. and (fig 3). in , only sod from has been studied. therefore, both bacteria especially bl6, a marine isolate might produce sods with novel properties. isolates sku2 and vr were assigned in the genera of and , respectively and sods from both genera have not been explored. this research was funded by science and technology research grant 2007 from indonesia toray science foundation to 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giovania , and bibiana w lay 1 department of master of biotechnology, faculty of biotechnology, universitas katolik atma jaya, jalan jenderal sudirman 51, jakarta 12930; 2 department of biology, faculty of biotechnology, universitas katolik atma jaya, jalan raya cisauk, bsd city, tangerang, banten 15345. tempeh is the most famous traditional fermented food in indonesia. tempeh fermentation consists of two stages. in the first stage, the acidification of soybeans used bacteria around 24 hours. lactic acid bacteria are found in tempeh. therefore, this study is aimed to investigate the diversity of lab from tempeh based on 16s rrna gene sequences and to study their function in tempeh fermentation. in this study, twenty-two lab isolates were obtained from tempeh. the isolates were closely related to lactobacillus agilis, lactobacillus fermentum, weissella confusa, and lactobacillus delbrueckii. l. fermentum (13 isolates) were the most abundant in tempeh, followed by l. agilis (7 isolates). it was found lab important for the acidification of soybeans which the ph of soybean soaking water decreased from ph 7 to ph 4.4-4.9. key words: diversity, lab, lactobacillus, tempeh, weissella tempe merupakan makanan fermentasi dari kedelai asli dari indonesia. tahap pertama dalam fermentasi tempe di indonesia merupakan pengasaman kedelai oleh bakteri yang berlangsung sekitar 24 jam. salah satu jenis bakteri yang berperan dalam proses pengasaman tersebut adalah bakteri asam laktat (bal). oleh karena itu, penelitian ini bertujuan mengkaji keragaman bal dari tempe berdasarkan sekuens gen 16s rrna dan mendapatkan potensinya dalam menurunkan ph saat perendaman kedelai pada fermentasi tempe. dalam penelitian ini, dua puluh dua isolat bal telah diisolasi dari tempe. isolat tersebut berkerabat dekat dengan lactobacillus fermentum (13 isolat) paling banyak terdapat pada tempe, disusul lactobacillus agilis (7 isolat), dan yang lainnya weissella confusa, dan lactobacillus delbrueckii. ditemukan bal dapat menurunkan ph air rendaman kedelai dari ph 7 menjadi ph 4,4-4,9. kata kunci: bal, keanekaragaman, lactobacillus, tempe, weissella microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-5703306; fax: +6221-5719060 ; email: tati.barus@atmajaya.ac.id (aderibigbe dan osegboun 2006). tempeh fermentation consists of two stages. the first stage lasts about 24 hours, in which soybean acidification occurs through bacterial activities. throughout this stage, the ph will decrease from 7 to 4 (barus et al. 2008). this condition is important for the growth of rhizopus spp. in the second stage, which happens ± 48 hours. rhizopus can be obtained from commercial inoculums or traditional inoculums (barus et al. 2019a). one of the most important microorganisms that mediate acidification is the lactic acid bacteria (lab) group, as they produce lactic acid as their main fermentation product from the culture medium (konings et al. 2000). lab belongs to a group of gram-positive bacteria and they are catalase negative. however only little research on lab involved in tempeh fermentation has been reported. currently, various molecular biology approaches are available and applied to study bacterial diversity. among these molecular methods, the 16s rrna gene sequences have been used extensively to study the tempeh is the most popular fermented soybean product in indonesia as an important source of protein at low prices for indonesians. it has been reported that tempeh reduced the adhesion of etec to intestinal epithelial cells of pig and human origin (roubos et al. 2009) and showed antibacterial activity against bacillus cereus (roubos et al. 2010). tempeh can prevent diarrhea (roubos-van den hil et al. 2009) and anemia (astuti 1999). consuming tempeh is good for the gut microbiota (stephanie et al. 20170). it has also been reported that tempeh contains vitamin b12 (keuth and bisping 1994) and antioxidant compounds (esaki et al. 1996). anti-nutritional compounds were found to be lower than soybeans (hong et al. 2004). such beneficial effects of tempeh consumption have been reported and thus it is widely known as part of the vegetarian or vegan diet in many countries, including japan, the netherlands, australia, and the usa diversity of bacteria in the environment (pisa et al. 2011; koike et al. 2003; barus et al. 2013; barus et al. 2017). in this study, we explored the diversity of lab from tempeh based on the 16s rrna gene sequences and then used for the analysis of the role of lab in determining the quality of tempeh. materials and methods isolation of lactic acid bacteria. the sample of tempeh was collected from the local market in jakarta, lembang, and tangerang, indonesia. aseptically, 5 g of each sample was added into 45 ml of sterile 0.85% (w/v) nacl (oxoid) solution and mixed thoroughly. then, it was made a dilution series of 10-1 to 10-7. furthermore, dilutions in the series 10-5-10-7 were taken as 1 ml in each and directly poured in duplicate on the de man rogosa sharpe (mrs) agar (oxoid ltd, basingstoke, uk) with 1% calcium carbonate (caco3) (w/v) and were incubated at 37°c for 48 h (assohoundjeni et al. 2016; sirichokchatchawan et al. 2018) under aerobic conditions. each olony with a halo zone was isolated and observed in terms of characters: nonmotile, endospores negative, catalase negative, and gram-positive bacteria. furthermore, fresh overnight cultures of each isolate were grown in luria-bertani (lb) broth and stored in the freezer at −80 °c in 20% glycerol for further tests. genomic extraction. lab isolates were grown for 0 18 hours at 30 c on luria-bertani broth. cells were harvested by centrifugation at 12,000 × g for 4 min. the cell pellet was resuspended in 1 ml of 10 mm tris–hcl, ph 8.0, 10 mm edta, 100 mm nacl, 2% (w/ v) sds, followed by genomic extraction using the genomic dna purification kit (fermentas®, lithuania) based on the manufacturer's protocol. genomic extracts were stored in te 1x buffer (10 mm tris-cl ph 8.0, 1 mm edta ph 8.0) at -20°c. amplification of 16s rdna. for molecular identification of lab isolates based on the 16s rdna gene, we amplification of partial 16s rrna gene was conducted via the geneamp® pcr system 2700 (applied biosystems, carlsbad, ca, usa) using the universal primer pair 63f (5ʹ-cag gcc taa cac atg caa gtc-3ʹ) and 1387r (5ʹ-cag gcc taa cac atg caa gtc-3ʹ) (marchesi et al. 1998). the pcr reaction mixture (25 µl) was prepared as follows: 12.5 µl gotaq green (promega, madison, usa), 9.5 µl nuclease free water (promega, madison, usa), 1 µl of 10 pmol of each primer, and 1 µl dna extraction (±100 ng). pcr conditions were as follows: initial 0 denaturation at 95 c for 5 minutes; 30 cycles consisting 0 0 of denaturation at 95 c for 1 minute, annealing at 57 c 0 for 1 minute, extension at 72 c for 1 minute; and post 0 extension at 72 c for 10 minutes. pcr products were visualized on 1% (w/v) agarose gel (promega, madison, usa) and then stained with ethidium bromide (sigma aldrich, usa). sequence analysis. pcr products of all lab isolates were then partially sequenced using the 63f and 1387r primer at macrogen inc., south korea. dna sequences were matched to genbank through base using nucleotide basic local alignment search tool (blastn) at the national biotechnology information center (ncbi) at www.ncbi.nlm.nih.gov to the database of 16s ribosomal rna sequences (bacteria and archaea). phylogenetic tree analysis using molecular evolutionary genetics analysis x (mega version x). determined ph of soybeans soaking water. the soybean soaking was done using a method described by barus et al. (2019b) with modification. modification in terms of replacing chemical acidification using acetic acid with acidification using the species of lactic acid bacteria found in this study. each of the soybeans (80 grams of wet weight) soaked in 240 ml of sterile water then inoculated with 1.8 ml suspension of each lab 7 (10 cells ml ) (keuth and bisping 1994). results after the purification process, as much as twentytwo lab isolates with form halo zone have been successfully isolated from tempeh samples in the mrs agar medium supplemented with 1% caco3 incubated 0 at 37 c (table 1). the genome of 22 lab isolates and amplification of 16s rdna have been successfully obtained from the isolates. the amplicon of 16s rdna sequences using primer pair 63f and 1387r yielded a single dna band (±1,300 bp) for each isolate (figure 1 as representative). blastn results of the partial sequence of 16s rrna genes (approximately 950 nucleotides) showed among our isolates with various lab (table 1). seven isolates similar to lactobacillus agilis 96%-99% (tbte1-tbte4, tbte11-tbte12, tbte18). thirteen isolates similar to lactobacillus fermentum 97% -100% (tbte5, tbte7-bte10, tbte13tbte17, tbte19-tbte21). two isolates similar to weissella confusa 96% (tbte6), and lactobacillus delbrueckii 98% (tbte22). filogram genetic diversity among lab isolated 150 barus et al. microbiol indones volume 14, 2020 microbiol indones 151 from tempeh based on the 16s rdna sequence has been successfully constructed (figure 2). the lab isolates were divided into four major clusters. the first and second clusters were related to w. confusa and l. delbrueckii, respectively. the third cluster was formed with l. fermentum, while the fourth cluster was related to l. agilis (figure 2). discussion the growth temperature of lab in the spontaneous 0 fermentation was reported 35-37 c (manini et al. 2016). all lab isolates in this study could grow to a temperature of 37°c. the appearance of halo zones was due to the dissolution of caco in the mrs agar 3 fig 1 pcr amplification sequences of 16s rdna sequences of lab isolates from tempeh using primer 63f and 1387. marker: 1 kb dna lambda ladder. fig 2 filogram genetic diversity among lab isolated from tempeh based on 16s rdna.. isolate code organism accession number identity (%) jakarta tbte1 lactobacillus. agilis strain c1-3-2 kp979478.1 98 jakarta tbte2 l. agilis strain aac59 ky810569.1 99 jakarta tbte3 source ab911458.1 97 tangerang tbte4 l. agilis strain m17 kc561119.1 98 jakarta tbte11 l. agilis strain dspv009p ku295178.1 99 jakarta tbte12 l. agilis strain tb-a07 ab425914.1 96 jakarta tbte18 l. agilis strain dspv005c gq231439.1 98 jakarta tbte5 lactobacillus fermentum strain eipw-5a kf932274.1 97 lembang tbte7 l. fermentum strain sm44 kj690759.1 98 jakarta tbte8 l. fermentum strain lab-21-ittg ky574532.1 98 jakarta tbte9 l. fermentum strain imau60221 fj915666.1 98 jakarta tbte10 l. fermentum strain 10 mf066936.1 99 jakarta tbte13 l. fermentum strain nl31 kj958422.1 99 jakarta tbte14 l. fermentum strain chchm7.1an (jq jq446535.1 97 jakarta tbte15 l. fermentum strain 6704 kx218444.1 100 jakarta tbte16 l. fermentum strain klab15 km485578.1 95 jakarta tbte17 l. fermentum strain kf5 kt159934.1 99 jakarta tbte19 l. fermentum strain klds 1.06 eu419592.1 98 jakarta tbte20 l. fermentum strain bcs27 eu547298.1 98 jakarta tbte21 l. fermentum strain saba5 kx599357.1 97 lembang tbte6 weissella confusa strain sl3 ku060304.1 96 jakarta tbte22 lactobacillus delbrueckii strain smn1-6 ky007528.1 98 1 152 barus et al. microbiol indones table 1 identity of lab isolates from tempeh based on 16s rdna sequences medium by acid production (pisol et al. 2015). all isolates were negative endospores, non-motile, negative catalase, and gram-positive bacteria. the character of all isolates in this study is in line with the character of lactic acid bacteria (lab) were reported by nurhikmayani et al. (2019) and pisol et al. (2015). the amplicon of 16s rdna sequences using primer pair 63f and 1387r yielded a single dna band (±1,300 bp) for each isolate (figure 1 as representative). the size of this amplicon is in line with wahyudi et al. (2011) and barus et al. (2017). blastn results of the partial sequence of 16s rrna genes (approximately 950 nucleotides) showed among our isolates with various lab (table 1). schlaberg et al. (2012) explained that the percentage of similarity of isolates was categorized in the taxonomic level of the same species, genus, family if the similarity values were ≥99%, 97% to <99%, 95% to <97% respectively. the 16s rdna sequence variations can show evolution among bacterial species because each w. confuse, l. delbrueckii, l. fermentum and l. agilis occupy in different clusters. the 16s rrna gene sequence is currently the most used in assessing bacterial diversity in a particular habitat because the 16s rrna gene is distributed in all bacteria. (větrovský et al. 2013). the 16s rdna sequences also have variable and conserved regions, and this region reflects the phylogenetic relationship between species. however, variations in the 16s rdna sequence are not enough to compare bacteria to strain levels. this can be seen in figure 2 that each cluster is occupied by bacteria with different strains. three strains of w. confusa (cluster 1), three strains of l. delbrueckii (cluster 2), two strains of l. fermentum (cluster 3), and three strains of l. agilis are exactly occupied the same cluster. the results of this study are in line with those previously reported (clarridge et al. 2004; sapalina et al. 2020). lab plays an important role in many fermented foods in the world. bal in fermented foods is important for health (caggianiello et al. 2016; choi et al. 2015; adeniyi et al. 2015; nuraida 2015). lab has been reported to play a role in determining the quality of fermented food (han et al. 2016; o'sullivan et al. 2002; settanni et al. 2010) and is important as a probiotic for health (angmo et al. 2016; mokoena et al. 2016; argyri et al. 2016). huang et al. (2018) reported that tempeh volume 14, 2020 microbiol indones 153 contains important lab to prevent diabetes mellitus. each of 80 g wet weight of soybeans soaked in 240 ml of sterile water then inoculated with 1.8 ml 7 -1 suspension of each isolate lab (10 cell ml ). all the isolates were added for acidification in soaking soybeans. it was found that the acidity of soybean soaking water decreased from ph 7 to ph 4.4 4.9. in this condition, tempeh will be successfully produced in good quality because this acidic condition is suitable for the growth of rhizopus. barus et al. (2008) reported that ph 7 of soybean soaking water will drop to around ph 4 after 24 hours. this acidic condition is needed for the germination of rhizopus as the main microbe of tempeh. medwid et al. (1984) have reported that rhizopus olygosporus germinates well under acidic conditions with ph 4-5. in tempeh fermentation, acidic conditions can be made by adding organic acids such as lactic acid (huang et al. 2018) or acetic acid (kartawiria et al. 2018). however, tempeh fermentation in indonesia does not use organic acids but acidic conditions occur naturally by various types of bacteria. this can cause the quality of tempeh to be inconsistent and sometime it can cause a bitter taste of tempe (barus et al. 2008). therefore, to get a consistent quality of tempeh, it is necessary to add a good selection of microbes to obtain good tempeh quality. therefore, lab can add to create acidifications in tempeh fermentation. abundant bacteria have been reported in tempeh (barus et al. 2008; barus et al. 2017; barus et al. 2013; ayu et al. 2014; efriwati et al. 2013; nurdini et al. 2015). therefore, research on the role of bacteria in tempeh still needs to be continued. conflict of interest 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2 ) , 3 4 4 0 . d o i : 10.1155/2015/901656. page 1 page 2 page 3 page 4 page 5 page 6 page 7 characterization and pathogenicity of as the causal agentfusarium oxysporum of fusarium wilt in chili ( l.) capsicum annuum rejeki siti ferniah , budi setiadi daryono , rina sri kasiamdari ,1* 2 2 and achmadi priyatmojo3 1department of biology, diponegoro university, jl. prof. sudharto sh tembalang semarang, indonesia; 2 , faculty of biology, gadjah mada university, jl teknika selatan sekip utara yogyakarta indonesia; 3faculty of agriculture, gadjah mada university, sekip unit 1 yogyakarta, indonesia; fusarium wilt is a serious disease attacking chili plants in central java which cause lost of chili productivity. fusarium wilt is caused by pathogenic fungi which is host specific. the objectives of this fusarium oxysporum, research were to characterize the pathogenic as the causal agent of fusarium wilt in chili plants and f. oxysporum to observe the virulence of the pathogen. fungal pathogen was isolated from tawangmangu as an endemic area of fusarium wilt in central java. the fungi was characterized morphologically and identified molecularly by its internal transcribed spacer regions (its regions). pathogenicity test was done to observe the virulence of the pathogen. one pathogenic strain was isolated from tawangmangu, karanganyar and was identified morphologically and molecularly as f. oxysporum. key words: characterization, pathogenicity, , fusarium oxysporum capsicum annuum layu fusarium merupakan salah satu penyakit tanaman yang sering dikeluhkan oleh para petani cabai di jawa tengah karena dapat menyebabkan gagal panen. penyakit tersebut disebabkan oleh jamur fusarium oxysporum yang bersifat spesifik inang. penelitian ini bertujuan untuk menentukan karakter morfologis dan identifikasi secara molekular patogen tanaman cabai dan mengetahui virulensi patogen tersebut. patogen f. oxysporum diisolasi dari tawangmangu sebagai daerah endemis layu fusarium di jawa tengah. jamur pathogen dikarakterisasi secara morfologis dan diidentifikasi secara molekular menggunakan gen its. isolat f. oxysporum yang didapat diuji patogenitasnya pada tanaman cabai. hasil penelitian mendapatkan satu isolat jamur yang bersifat pathogen serta secara morfologis dan molekular adalah jamur f. oxysporum. kata unci : k karakterisasi, patogenitas, , fusarium oxysporum capsicum annuum vol.8, no.3, september 2014, p 121-126 doi: 10.5454/mi.8.3.5 *corresponding author; phone: +62-24-76480923, ferniah_mikro@yahoo.com chili is a potential horticulture in indonesia and produced mainly in java. indonesia produced 954.360 ton red chili in year 2012 and 48% of the plantation was located in java (anonim 2013). central java has the second biggest of chili plantation but many plants have been attacked by fusarium wilt especially in the rainy seasons. the symptoms of fusarium wilt are wilting, vein clearing in younger leaflets, epinasty, stunting and yellowing of older leaves (agrios 2005). according to agrios (2005), fusarium wilt in plants was usually caused by . this species can fusarium oxysporum infects more than 100 species of plants and causes the wilt symptoms. was the main fusarium oxysporum disease in thailand chili crops and one of the causal agents of wilting chili in india, china, and indonesia (ali 2006). pathogenicity of in many plants have f. oxysporum been studied for many decades. it is because the pathogen has a systemic reaction in plants that causes the death of the infected plants. fusarium oxysporum as the causal agent of wilting banana was reported by g ro e ne wa ld ( 20 05 ). ge ne ti c div e rs ity a nd pathogenicity of was studied in japanese f. oxysporum onion (dissanayake 2009) and in indonesian et al. onion (choiruddin 2010). pathogenic f. f. oxysporum sp was determined in cotton roots by pcr vasinfectum based (abd-elsalam 2006). the fusarium wilt had et al. also been observed in melon (herman and perl-treves 2007; oumouloud 2008). in solanaceae,et al. f. oxysporum was reported as the causal agent in tomato (hibar 2007; jacobs 2013), potato (bayona et al. et al. et al. et al. et al.2011; du 2012), and eggplant (altinok 2014). studies of fusarium wilt in chili were done morphologically (zahara and harahap 2007), biologically (nugraheni 2010), and enzymatically (chaiyawat 2008; wongpia and lomthaisong et al. 2010). many chili crops in indonesia have problems by fusarium wilt but the causal agent have not been determined yet. in this research a pathogen causing the mailto:ferniah_mikro@yahoo.com fusarium wilt in chili was isolated and identified morphologically and molecularly. molecular identification confirmed the morphological character. in this study, isolation of the pathogen was done from the endemic area of fusarium wilt in tawangmangu, karanganyar, central java. materials and methods wilting plant materials were plant materials. collected from chili plantation in tawangmangu karanganyar. treaty plants were tm999 cultivar (seeds produced by seminis, monsanto, korea) and gantari cultivar (seeds produced by balai penelitian dan pe ngembanga n hor tikultura ngipiksari, yogyakarta, indonesia) grown in sterile soil. fungi were fungal isolation and identification. isolated from the browning vascular of plants. the stem was cut on the border of brown vascular and healthy vascular. a five millimeter slice of the stems were surface sterilized in 70% ethanol for one minute, rinsed in sterile water and dried with tissue, and grown on the potato dextrose agar (pda). the cultures were incubated at 25 °c with 12 h dark and 12 h light cycle for 2 days. each colony was transferred into new pda and incubated for 5 days to get pure culture of isolate. characterization was done based on the morphology of colonies and cells. when fusarium species is present, the isolate was grown on synthetic nutrient-poor agar (sna) to analyse the s h a p e o f ma c r o c o n id i a a n d mi c r o c o n i d ia . identification was done by comparing the morphology with the atlas of (leslie and summerellfusarium 2006; and samson 2008). a single spore of et al. fusarium oxysporum was used for further test (as master isolate). pathogenicity test. the was grown f. oxysporum in potato dextrose broth (pdb) for 4 days. conidia densities were calculated with haemacytometer and adjusted to 10 conidia/ml. the inoculum was 6 inoculated in chili plant by root dip method (herman and perl-treves 2007; karimi 2010). one month et al. old of healthy chili plants were taken from the soil. the root were rinsed in water, soaked in 1% chlorox for 1 minute, rinsed with sterile water, and then soaked in fungal suspension for 30 minutes. this treatment were done for 10 plants. roots of healthy plant were soaked in sterile water was done as control. each plant was planted in sterile soil in polybags. disease symptoms were observed every odd days after inoculation (dai). symptoms were remarked by the stunting, chlorosis, and/or wilting of the leaves which determined by scoring. score 0 = no symptom, 1 = lower height compared to control, 2 = lower height and chlorosis, 3 = 10% chlorosis and/or 10% wilting, 4 = 11 25% wilting, 5 = 26 50% wilting, 6 = 51 100% wilting and dead.the disease severity index (dsi) w a s d e t e r m i ne d a c c or d i n g t o wo n g p ia & lomthaisong, 2010: dsi = (disease severity scale x number of plants in each scale) the highest numerical scale index x total number of plants pathogen was re-isolated from the symptomatic plant. the pathogen was compared morphologically and molecularly with the master isolate. . master isolate and molecular identification isolate from symptomatic plant were identified molecularly. molecular identification of fungi was done using its regions (abd-elsalam . 2006; toju et al et al. 2012). the its rdna of the isolate was amplified by polymerase chain reaction (pcr). pair of primers used were its1 and its4. sequences of the primers were forward its1 5'-tccgtaggtgaacctgcg : g 3' and reverse its4: 5’tcctccgcttattgata tgc 3', with target size 540 570 bp. pcr was done with kapa2g fast pcr kit. pcr program for its rdna was 95 °c 3 minutes for pre-denaturation, 95 °c 10 second for denaturation, 52 °c 10 second for annealing, and 72 °c 10 second for polimeration. the cycle was repeated 39 times, and the final extention was 72°c 5 minutes. pcr products were verified in 2% agarose gel electrophoresis and the dna was sequenced. the sequencing product was analysed by blastn in the genebank database using mega 5.1 software. results tawangmangu was endemic of fusarium wilt in the rainy season 2013. there were wilting leaves and browning or discoloration in the stem of the diseased plants caused by fusarium wilt. one strain of fusarium oxysporum, which was then called p1a, was successfully isolated from the diseased plants in tawangmangu. a specific fungi was successfully isolated from the stems. figure 1 showed the morphological characters of the fungi. the observed morphological characters were as follows: colony with white cottony aerial ∑ 12 f2 erniah et al . microbiol indones mycellium and purple on the reverse with 4 5 cm diameter at 5 days incubation on pda. conidia was grown from short phialid with a false head. macroconida was straight fussiform, pedicellate basal cell, 27 46 x 3 4,5 µm, with 3 5 septates. microconidia were abundant, ellipsoid or fussiform without or with 1 2 septates, 5 15 x 2,2 3,5 µm. chlamydospore was formed terminally or intercallary, single or in pairs. the morphology has previously been described by leslie and summerell (2006), and samson (2008), as the et al. characteristics , thus confirming the f. oxysporum identification of the isolate. the pathogenicity test showed that p1a caused wilting in chili. figure 2 showed that the symptom increased significantly 15 dai. at 19 dai, the fungi caused wilting of tm999 cultivar and gantari cultivar with dsi scores 0.4 and 0.63, respectively. so the p1a isolate was pathogenic to both cultivars. re-isolation from the symptomatic chili plants obtained one isolate that is morphologically similar to p1a isolate. the isolate was named p1a'. both isolates were identified molecularly. fig 1 morphology of . a. colony, b. reverse colony, c. phialid , d. macroconidia, e. microconidia, f. fusarium oxysporum chlamydospore. fig 2 disease severity index of the chili plants by tm : tm999 cultivar, gtr : gantari cultivar. f. oxysporum. 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 h+1 h+3 h+5 h+7 h+9 h+11 h+13 h+15 h+17 h+19 days after inoculation (dai) tm gtr a b c d e f volume 8, 2014 microbiol indones 123 molecular analysis showed similarity in the dna sequences of p1a and p1a'. figure 3 showed the pcr products of of p1a and p1a' rdna. the alignment of these sequences showed 100% similarity to fusarium oxysporum accession number kf998987.1. figure 4 showed the phylogenetic tree of the sequences with f. oxysporum rdna sequences obtained from the genebank database. discussion fusarium wilt in tawangmangu chili plantation covered 30 40% area and caused loss of crops. the pathogen attacked plants in young and mature plants. symptoms in young plants were stunting, chlorosis, wilting, and finally death. if the pathogen attacked a mature plant, flowers and fruits of the plant not develop normally. the disease was named fusarium wilt because wilting is the common observable symptoms. wilting is actually a secondary symptom. the primary symptom was browning or discoloration in the basal stem, that is only observable by cutting the stem. macroscopical and microscopical characterization confirmed that the isolate was f. oxysporum. molecular identification was needed to proof the name of the species. the rdna its regions showed that p1a was the same as p1a', and confirmed the identity as f. fig 3 pcr products of 's its rdna. p1a (lane 1) and p1a' (lane 2) as 530 bp. m is dna marker ladder (geneaid). f. oxysporum fig 4 phylogenetic tree of pathogenic in chili (yellow box was p1a/p1a'). f. oxysporum f. oxy hq384393 f. oxy kf494093 f. oxy kf998987 f. oxy hg529205 uncultured soil fungus hm132003 p1a/p1a’ 0,0001 12 f4 erniah et al . microbiol indones oxysporum kf998987.1. the rdna its is a region c o m m o n l y u s e d i n t h e f u n g a l m o l e c u l a r characterization and taxonomic classification. however, it cannot be used in taxonomic classification below the species level, since will require more selective/specific sequences as well as specific primers for the sequence amplification, for example a pair of unique primer developed by abd-elsalam . (2006), et al which is specific to the sequence of fusarium oxysporum vasinfectum.f.sp f. oxysporumpathogenicity test showed that the from diseased chili was a pathogenic fungi and caused the same symptoms in chili plants. the fungi cause stunting and wilting with or without yellowing. the wilting started from the older leaves and spread to the younger. the stem of the plants had vascular discoloration or browning in the transverse cutting. the wilting increased 15 dai, when the pathogen had successfully infected the chili plants. the pathogen needed time to enter the plant root through wounding and therefore penetrated and colonized the vascular. pathogen colonization in vascular bundle inhibited water and nutrient transport from soil to the shoot (agrios 2005) so it caused wilting of the leaves. the time required for the infection was f. oxysporum similar to pathogenesis test in chili seedling (suryanto et al et al. 2010) and wilted rocket plant (srinivasan . 2011). the fungi needed 13 17 dai to infect a plant and show symptoms. due to its capability to grow on the nutrient medium, the is classified as a f. oxysporum non-obligate pathogen. the pathogen can grow and multiply on dead organic matter as saprophytic microorganism. in the case of , they live in fusarium soil as chlamydospores. the chlamydospores were dormant in the soil until they met a specific host to grow. planting the soil with soybean may not endangere the plants, but planting with chili will wake up the pathogen. so plants rotation is needed in the field to inactivate the pathogen. acknowledgement this research is a part of dissertation report funded by the bpps program, ministry of education republic of indonesia, year 2011 2014. references abd-elsalam ka, asran-amal a, schnieder f, migheli q, verreet j-a. 2006. molecular detection of fusarium oxysporum vasinfectum f. sp. in cotton roots by pcr and real-time pcr assay. journal of plant diseases and protection 113 : 14 19. agrios gn. 2005. plant pathology. 5 edition. elsevier th academic press. london. ali m. 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cendawan pada tanaman cabai ( ) pada capsicum annuum topografi yang berbeda. seminar temu teknis pejabat fungsional non peneliti. bogor, 21 22 agustus 2007. 12 f6 erniah et al . microbiol indones 4. tri ratna revisi.cdr population and diversity of endophytic bacteria associated with medicinal plant curcuma zedoaria tri ratna sulistiyani , puspita lisdiyanti , yulin lestari 1,2 3 1,4* and 1 2 3 4 department of biology, faculty of mathematic and natural sciences, institut pertanian bogor, dramaga campus, bogor 16680, indonesia; research center for biology, indonesian institute of sciences, cibinong science center,cibinong 16911, indonesia; research center for biotechnology, indonesian institute of sciences, cibinong science center, cibinong 16911, indonesia; biopharmaca research center, institut pertanian bogor, bogor 16151, indonesia curcuma zedoaria alphaproteobacteria, betaproteobacteria, gammaproteobacteria, firmicutes, actinobacteria, stenothropomonas, pseudomonas, enterobacter, providencia, klebsiella, dickeya, pantoea, bacillus, acinetobacter, citrobacter mycobacterium, cellulomonas, microbacterium, methylobacterium, penylobacterium, roseomonas, agrobacterium, bosea, xanthobacter, rhizobium, burkholderia, ralstonia, alcaligenes. curcuma zedoaria curcuma zedoaria nutrient agar water yeast extract agar alphaproteobacteria, betaproteobacteria, gammaproteobacteria, firmicutes, actinobacteria, stenothropomonas, pseudomonas, enterobacter, providencia, klebsiella, dickeya, pantoea, bacillus, acinetobacter, citrobacter mycobacterium, cellulomonas, microbacterium, methylobacterium, penylobacterium, roseomonas, agrobacterium, bosea, xanthobacter, rhizobium, burkholderia, ralstonia, alcaligenes. curcuma zedoaria traditionally (white turmeric) known as herbal medicine which possessing many biological activities. many endophytic bacteria live in association with their host and may play an important biological roles. the main interest of this study was to investigate the endophytic bacterial diversity associated with white turmeric. white turmerics were collected from three locations in bogor, west java, indonesia. the isolation of endophytic bacteria was carried out using 4 kinds media (nutrient agar (na), na contained white turmeric extract (nat), water yeast extract agar (wyea), wyea contained white turmeric extract (wyeat)), and 2 methods of spread plate and plant piece methods. the identification of selected isolates was conducted by molecular analysis based on 16s rdna. the suitable media and method of isolation endophytic bacteria were na and spread plate method. a total of 207 bacterial colonies were isolated from rhizomes, stems, and leaves and 73 endophytic bacteria were selected based on morphological characteristics. from them, 32% isolates from bojong gede, 22% isolates from cibinong and 46% isolates from dramaga were obtained. endophytic bacteria were predominated 38% in the rhizomes, 32% of stems, and 30% of leaves. based on 16s rdna sequence analysis, the isolates were belonging to the cluster and with twenty three different genera includes , and the plant location, age, part of plant, media and method of isolation seem to influence the endophytic bacterial communities. key words: 16s rdna, , diversity, endophytic bacteria, population secara tradisional kunyit putih merupakan tanaman herbal yang banyak digunakan untuk pengobatan penyakit terutama yang berhubungan dengan kanker. bakteri endofit banyak ditemukan hidup dalam jaringan tanaman inang dan memainkan peran biologi yang penting. penelitian ini bertujuan untuk mengkaji keragaman bakteri endofit yang berasosiasi dengan tanaman kunyit putih ( ). tanaman kunyit putih diambil dari tiga lokasi yang berbeda di bogor, jawa barat, indonesia. isolasi bakteri endofit dilakukan menggunakan 4 macam media ( (na), na dengan ekstrak kunyit putih (nat), (wyea), wyea dengan ekstrak kunyit putih (wyeat)) dan 2 metode yaitu metode sebar dan potongan tanaman. identifikasi isolat terseleksi menggunakan analisis molekuler berdasarkan 16s rdna. media dan metode isolasi bakteri endofit yang cocok adalah media na dan metode sebar. sebanyak 207 isolat telah berhasil diisolasi dari bagian akar, batang dan daun. sebanyak 73 isolat yang berbeda dari 207 isolat dipilih berdasarkan perbedaan karakteristik morfologi. tiga puluh dua persen isolat diperoleh dari bojong gede, 22% isolat dari cibinong dan 46% isolat berasal dari dramaga. bakteri endofit didominasi oleh bakteri dari rimpang yaitu sebanyak 38%, 32% dari batang, dan 30% dari daun. berdasarkan hasil analisis sekuen 16s rdna, bakteri yang diperoleh termasuk dalam kluster dan dengan 23 genus yang berbeda yaitu , and lokasi, usia, bagian tanaman, media dan metode isolasi berpengaruh terhadap komunitas bakteri endofit yang diketahui dari suatu tanaman. kata kunci : 16s rdna, bakteri endofit, , keragaman, populasi vol.8, no.2, june 2014, p 65-72 doi: 10.5454/mi.8.2.4 *corresponding author; phone: +62-251-8622833, e-mail: yulinlestari@gmail.com. the endophytic microbes have been known as secondary metabolites producers which have several potential applications in pharmaceutical industry. some of the endophytic microbes can produce the same bioactive compounds as that of the plant thus making them as a promising source of novel 66 sulistiyani et al. microbiol indones original plants sources sampling locations plant age (month) part of plants family species cim anggu, bogor bojong gede (bg) 11 rhizome, stem, leaf zingiberaceae curcuma zedoaria (christm.) roscoe rimbo panti, padang research center for biology, (cbn) 8 rhizome, stem, leaf zingiberaceae curcuma zedoaria (christm.) roscoe cimas, sukabumi biopharm aca research center, (drmg) 12 rhizome, stem, leaf zingiberaceae curcuma zedoaria (christm.) roscoe table 1 characteristics identity of white turmeric plants used in this research compounds, such as anticancer, antibiotic, antimycotic and antiviral (christina . 2013). population and profile of endophytic microbes are influenced by location of the host plants (procopio . 2009), environmental conditions, plants species, and plants age (dalal and kulkarni 2013). plants that used by human as a traditional medicine with high etnobotanical history are possessing great biodiversity of endophytic microbes. is a family of the important medicinal plants. many compounds have been detected in species, such as, turmerin, sesquiterpenes, steroid and essential oils (joy 1998). several important genera belong to the ginger family are and each having different compounds which can be used in pharmaceutical industry. one of the ginger family that interesting to be investigated for their endophytic bacteria is spp known as white turmeric is traditionally used as herbal medicine to treat diseases related to cancer. in addition, several s t u d i e s s h o w e d t h a t w h i t e t u r m e r i c h a s pharmacological effects of antibacterial (banisalam . 2011), anticancer and antioxidants (muthu-kumar . 2012). according to lakshmi (2011), essential oil from the rhizome of white turmeric has activity to inhibit the proliferation of cancer cells. several endophytic microbes have been isolated which are able to produce various bioactive c o m p o u n d s . a n d were the most commonly isolated bacterial genera and potential as a bioactive compounds producer. taxol as the world's first billion dollar anticancer drug was produced by fungus from the yew (ryan . 2007). from et al et al zingiberaceae zingiberaceae et al. curcuma, kaempferia, hedychium, amomum, zingiber, alpinia, elettaria, costus, curcuma . curcuma zedoaria et al et al et al. p s e u d o m o n a s , b a c i l l u s , burkholderia taxomyces andreanae taxus brevifolia et al bacillus amyloliquefaciens ophiopogon japonicus et al. c. zeodaria et al. actinomycetes curcuma aeruginosa et al. c. zedoaria showed antitumor activity against gastric carcinoma cell lines (chen 2013), endophytic microbes from the rhizome of produced antimicrobes compound (srikandace 2007). which was isolated from rhizome of had alpha glucosidase inhibition activity (pujiyanto 2012). natural products produced by endophytic bacteria can be applied as a foundation for the development of therapeutic agents. many researches have explored the potential of endophytic bacteria as source of bioactive compounds producer, however, information on the diversity of endophytic bacteria of a particular plant has not been studied in depth. in indonesia, the study on the diversity and activity of endophytic bacteria to produce several bioactive compounds in white turmeric has not been done. the main objective of the study was to explore the diversity of endophytic bacteria in white turmeric from three locations based on molecular identification of 16s rdna sequences. white turmerics ( ) were collected from three locations in bogor, west java, indonesia. the plants materials collected from private garden in bojong gede (bg), experiment garden of research center for biology, indonesian institute of sciences, cibinong (cbn) and garden of medicinal plants collection of biopharmaca research center, bogor agricultural university, dramaga (drmg) (table 1). white turmeric plants were identified based on the morphological characteristics at the herbarium bogoriense, indonesian institute of sciences, cibinong, indonesia. from each plant materials, rhizome, stem, and leaf were collected for endophytic bacteria isolation. materials and methods plant materials. volume 8, 2014 microbiol indones 67 surface sterilization of rhizomes, stems, and leaves isolation of endophytic bacteria dna extraction and amplification of 16s rdna . rhizomes, stems, and leaves were thoroughly washed to remove external soil and microbes using running tap water for 5-10 min. surface sterilization was done by stepwise soaking using 70% etanol solution for 3 min, 3% (v/v) sodium hypochloride for 5 min, 70% etanol solution for 30 s, and followed by three times rinsing with sterile distilled water. the samples were dried using sterilized towel tissue. . endophytic bacteria isolation was done by plant piece and spread plate methods. rhizomes, stems and leaves were cut using sterile knife approximately into 4-6 mm pieces. in the plant piece method, pieces of samples were placed on four different media, nutrient agar (na), na contained 2% white turmeric plant extract (nat), water yeast extract agar (wyea), and wyea contained 2% white turmeric plant extract (wyeat). the media were supplemented with cycloheximide 50 µg ml to avoid the growth of fungi. plates were incubated at 28 c for 2-15 days. in spread plate method, isolation of endophytic bacteria was done by grinding 1 g of samples in 9 ml of sterilized distilled water and 100 µl of 10 and 10 serial dilutions were spreaded on four different media same as above. the plates were incubated at 28 c for 2-15 days. bacterial colonies which appeared on the media in a spread plate method were counted and expressed in colony forming units (cfu) per gram, and population data were transformed to log (cfu per gram sample (cfu g ). some endophytic bacterial isolates which grew both on the media using spread plate and plant piece methods were picked up based on several phenotypic characteristics and then purified to obtain a single colony. based on their different morphological characteristics, the endophytic bacteria were selected for further studies. phenotypic characteristics which were used to observe the colony were: color, surface, the margin of the colony and gram reaction using koh test. . dna extraction was conducted by colony pcr method (packeiser 2013) using gradient pcr machines (eppendorf mastercycler gradient pcr system 5331). amplification of 16s rdna was performed by pcr using primer pair of 27f (5'agagtttgatcctggctcag-3') and 1492r (5'ggttaccttgttacgactt-3') (palaniappan 2010). the 16s rdna amplification was carried out in a total volume of 25 µl containing ultrapure water, gotaq green master mix, 10 µm of each primer, -1 o -1 -2 o -1 et al. et al. dimethyl sulfoxide (dmso), and dna template. the pcr conditions was set as follows: initial denaturation at 95 c for 90 s, followed by 30 cycles of denatura-tion at 95 c, for 30 s; annealing at 50 c, for 30 s; elongation at 72 c, for 90 s and final extension at 72 c for 5 min, finally at 4 c for 20 min. pcr products were analyzed using 1% agarose gel. gel was soaked in ethidium bromide solution (5 µgml ) for 30 min, rinsed with 1x tae buffer, and the results were detected using a uv transilluminator. . the amplified dna were partially sequenced using forward primer 27f by automated dna sequencer (abi prism 3130 genetic analyzer) (applied biosystems). the sequenced data were processed using bioedit programme. the homology of 16s rdna sequence were searched using blastn at the ncbi website and the references sequence was obtained from the genbank (www.ncbi.nlm.nih.gov). constructions of phylogenetic tree was done using neighbor-joining tree method (njt) implemented in mega 5.05 software (tamura 2011). model of k2+g+i (kimura2-parameter and gamma distributed) was selected as the best-fit substitution model for the current analysis. strength of internal branches of the phylogenetic tree was tested with boostrap analysis using 1000 replications. . observation of morphological characters of white turmeric plant was done referred to the plant identification book of flora of java and compared to herbarium specimen in the herbarium bogoriense. based on the morphological characteristics, all samples were identified as (christm.) roscoe, the member of the genus in the family of (table 1). . the population of endophytic bacteria contained in white turmeric plants differed between location, age, rhizome, stem and leaf, and also influenced by media and method of isolation. based on the data presented in fig 1, three plant materials showed to have different endophytic bacterial population ranging from 2 to 4 log (cfu g ). the number of isolates obtained by spread plate method were higher compared to plant piece method (table 2). the most effective media and method of isolation endophytic bacteria from white turmeric plant were na and spread plate method. the highest population of o o o o o o 1 -1 dna sequencing and phylogenetic analysis plant materials identity population of endophytic bacteria associated with et al. c. zedoaria curcuma zingiberaceae results c. zedoaria 68 et al.sulistiyani microbiol indones fig 1 the population of white turmeric endophytic bacteria from three sampling locations based on spread plate method, na, nat, wyea, wyeat. table 2 the number of selected endophytic bacterial isolates obtained from white turmeric using spread plate and plant piece methods na nat wyea wyeat s p s p s p s p bojong gede rhizome 6 1 3 2 1 1 stem 7 2 2 3 2 2 3 1 leaf 4 4 7 3 4 2 8 2 cibinong rhizome 2 3 3 1 1 1 1 stem 3 5 1 3 4 2 2 leaf 5 6 1 6 4 3 dramaga rhizome 4 4 4 5 5 2 4 2 stem 5 4 3 4 3 2 4 3 leaf 3 2 2 4 3 2 5 1 39 27 24 34 28 12 31 12 total 207 s: spread plate method; p: plant piece method endophytic bacteria was found in samples that collected from drmg and among the part of plant, rhizome showed the highest population of endophytic bacteria. two hundred and seven endophytic bacteria were isolated from different parts of white turmeric plants. based on the morphological characteristics of 207 isolates, 73 were selected for further studies. among the 73 selected isolates, 23 isolates from bg (32%), 16 from cbn (22%) and 34 from drmg (46%). furthermore, 28 isolates were obtained from rhizomes (38%), 23 isolates from stems (32%) and 22 isolates from leaves (30%). the gram reaction results showed that 49 isolates were gram-negative and 24 isolates were gram-positive bacteria (table 3). both of grampositive and negative bacteria were found in all samples and bacteria from the rhizomes, stems and leaves were dominated by gram negative bacteria. volume 8, 2014 microbiol indones 69 table 3 diversity of endophytic bacteria from white turmeric plants based on 16s rdna analysis ko h test bojong gede research center for biology, cibinong biopharmaca research center, dramaga total rhizome + 1. bacillus subtilis 2. cellulomonas hominis 1. bacillus safensis 1. microbacterium trichothecenolyticum 2. bacillus cereus 5 1. klebsiella pneumoniae 2. pseudomonas denitrificans 3. pseudomonas stutzeri 4. pantoea dispersa 5. klebsiella variicola 6. bosea thiooxidans 1. providencia vermicola 2. phenylobacterium koreense 3. enterobacter aerogenes 4. roseomonas mucosa 1. burkholderia cenocepacia 2. burkholderia phenoliruptrix 3. enterobacter cloacae 4. enterobacter ludwigii 5. pantoea dispersa 6. pantoea agglomerans 7. pseudomonas geniculata 8. pseudomonas gessardii 9. pseudomonas nitroreducens 10.stenotrophomonas maltophilia 11. klebsiella pneumoniae 12. klebsiella variicola 13. acinetobacter calcoaceticus 23 stem + 1. microbacterium laevaniformans 2. microbacterium trichothecenolyticum 3. microbacterium hominis 4. mycobacterium simiae 5. bacillus pumilus 1. bacillus safensis 2. mycobacterium cosmeticum 1. microbacterium resistens 2. bacillus cereus 3. microbacterium laevaniformans 10 1. klebsiella pneumoniae 2. erwinia chrysanthemi 3. xanthobacter flavus 4. enterobacter oryzae 1. stenotrophomonas maltophilia 2. pseudomonas otitidis 1. stenotrophomonas maltophilia 2. enterobacter ludwigii 3. acinetobacter calcoaceticus 4. ralstonia mannitolilytica 5. klebsiella variicola 6. citrobacter freundii 7. pseudomonas moraviensis 13 leaf + 1. microbacterium laevaniformans 2 .micrococcus yunnanensis 1. microbacterium testaceum 2. bacillus safensis 3. bacillus subtillis 1. microbacterium resistens 2. microbacterium testaceum 3. microbacterium laevaniformans 4. brevibacterium epidermidis 9 1. pseudomonas stutzeri 2. klebsiella pneumoniae 3. agrobacterium larrymoorei 4. bosea thiooxidans 1. stenotrophomonas maltophilia 2. pseudomonas denitrificans 3. enterobacter cancerogenus 4. methylobacterium organophilum 1. enterobacter cancerogenus 2. alcaligenes faecalis subsp. faecalis 3. klebsiella variicola 4. pseudomonas moraviensis 5. rhizobium tarimense 13 total 23 16 34 73 +: gram positive bacteria; -: gram negative bacteria molecular identity of endophytic bacteria based on partial sequencing of 16s rdna. based on the results of partial sequencing of about 700 1200 bp and analysis of 16s rdna, the 73 isolates showed high similarities between 97% to 100% with the data bases in genbank. the molecular identification of all isolates into species level were presented in table 3. according to phylogenetic tree analysis, the isolates widely distributed to the cluster of alphaproteobacteria, betaproteobacteria, gammaproteobacteria, firmicutes, actinobacteria, stenothropomonas, pseudomonas, enterobacter, providencia, klebsiella, dickeya, pantoea, bacillus, acinetobacter, citrobacter mycobacterium, cellulomonas, microbacterium, methylobacterium, penylobacterium, roseomonas, agrobacterium, bosea, xanthobacter, rhizobium, burkholderia, ralstonia, alcaligenes and with twenty three different genera, including , and (fig 2). 70 et al.sulistiyani microbiol indones discussion the population of endophytic bacteria differed between location, age, rhizome, stem and leaf, and their diversity was also influenced by growth media. in this study, isolation of endophytic bacteria of white turmeric plant from three locations in west java, indonesia was done using two methods and four different media. endophytic bacteria were succesfully isolated using four kinds of media, but addition of extract of white turmeric plants seemed to decrease population and diversity of endophytic bacteria. among the four kinds of media used, na was the suitable media for the endophytic bacteria isolation compared to wyea. based on the isolation method, the number of endophytic bacteria obtained using spread plate method was higher compared to plant piece method. this may caused by the differences in size and preparation of samples between the two methods. in the spread plate method, sample (4-6 mm ) were firstly ground to pieces and spread over the plate for bacterial growth. when the sample extracted using water, more microbes inside a plant moved to the water. this method seems to give more chance for endophytic bacteria to grow. while for the plant piece method the chance for endophytic bacteria to grow is limited because the sample (4-6 mm ) were directly put on media. several genera of bacteria can be found in three different locations, i.e. and . sp. and sp. are the most abundant endophytic bacteria found in the plants. they are considered easy to be cultured (seghers 2004). the presence of microbes in a host plant can be affected by the compounds contained in the host plants (strobel and daisy 2003). the plants of the same species may produce relatively similar bioactive compounds (bernhoft 2010). rhizome, stem and leaf from cbn had the lowest abundance of endophytic bacteria. the plant materials originally come from tissue culture which was subsequently domesticated in that place. it could be possible reason for limited numbers of endophytes. endophytic bacteria of white turmeric plant from drmg was more diverse compared to other samples. the differences may also be influenced by the different in ecological niche condition of the plant. the fact that more than one hundred of medicinal plants can be 2 2 microbacterium, pseudomonas, enterobacter, bacillus, stenothropomonas, klebsiella, mycobacterium, pantoea pseudomonas bacillus et al. found in drmg, may also influence the soil microbial diversity. another reason which can influence the endophytes diversity is age of host plant. sample taken from drmg was the oldest (12 months), followed by sample from bg (11 months), and sample from cbn (8 months). as a mature plant developed, all the nutrients for the endophytic bacteria may be more available and abundance thus stable endophytic population can be obtained. age of plants has been reported to influence the variation of endophytic community in the gingseng plants (vendan 2010). three plant materials of white turmeric plants showed different population of endophytic bacteria. the population on the rhizome of the plant was higher than those of the stems and leaves. the greater population found in the rhizome may be caused by the content of rhizome compounds. the plant uses the rhizome to store starch, protein, fat and other nutrients which are useful for the plant and its endosymbionts. dalal and kulkarni (2013) reported that population of endophytic microbes in roots or rhizome were the highest compared other part of plants, due to root is the earliest place for microbes entering the plant. among the 73 selected isolates, isolates belongs to the cluster of was the most dominant, followed by , , and they were 37, 16, 8, 8, 4 respectively. the genera of was dominant, followed by and . in the present study, 73 endophytic bacteria which represented 46 species were belonging to 23 different bacterial genera have been identified from variously locations, aged, and part of plant of white turmerics. cho (2007) isolated 13 different bacterial genera of 63 endophytic bacteria from gingseng roots cultivated in three different areas. vendan (2010) isolated four clusters, 9 genera in 51 isolates from variously aged gingseng plants. germida (1998) reported that isolated 18 endophytic bacterial genera in 220 isolates from root tissues of three field-grown canolas. thus, it seems that the diversity of endophytic bacteria in white turmeric plant collected from bogor, west java, indonesia was more diverse compared to others result studies. in conclusion, that there are differences in the population and number of endophytic bacteria isolates recovered from white turmeric in bogor, west java, indonesia and the isolates obtained depend on the location, age, part of plant, media and method of isolation. et al. gammaproteobacteria actinobacteria alphaproteobacteria, firmicutes betaproteobacteria, microbacterium pseudomonas, bacillus, klebsiella enterobacter et al. et al. et al. γ-proteobacteria α-proteobacteria β-proteobacteria firmicutes actinobacteria outgroup fig 2 phylogenetic tree based on 16s rdna sequences of the endophytic bacteria using neighbor-joining tree method, model kimura2-parameter and gamma distributed with 1000 replications. volume 8, 2014 microbiol indones 71 72 sulistiyani et al. references banisalam b, sani w, philip k, imdadul h, khorasani a. 2011. comparison between and antibacterial activity of from malaysia. afr j biotech. 10(55):11676-11681. doi:10.5897/ajb10.962 bernhoft a. 2010. a brief review on bioactive compounds in plants. in: symposium held at the norwegian academy of science and letters, 2008 nov 13 14. oslo. p 11-17. chen yt, yuan q, shan lt, lin ma, cheng dq, li1 cy. 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setyahadi1 2 1* and 1 2 center for bioindustrial technology, laboratoria pengembangnan teknologi agro-biomedika, badan pengkajian dan penerapan teknologi, pusat penelitian ilmu pengetahuan dan teknologi, serpong, tangerang 15314 , indonesia; faculty of fishery and marine sciences, universitas padjajaran, jalan raya jatinangor km 21, sumedang 45363, indonesia bacillus licheniformis lactobacillus acidophilus bacillus licheniformis lactobacillus acidophilus chitin extraction from shrimp shells involves two processing steps, these are the deproteination and demineralization process. the aim of this experiment was to compare the order of the chitin extraction process. the first experiment was deproteination of fresh shrimp shells followed by demineralization process and the second one was demineralization of fresh shrimp shells followed by deproteination. f11.1, a proteolytic producing bacterium, was used for the deproteination process. fncc116, a lactic acid bacterium, was used for the demineralization process. the deproteination was done in a 1 liter fermenter jar at 55 ºc, 250 rpm and 2.5 vvm aeration for 60 h. the demineralization was done in the same size fermenter at 30 ºc and 50 rpm agitation for 48 h. the experimental results showed that demineralization followed by the deproteination process resulted in a better chitin yield than when the process was conducted in the opposite order. the first process reduced 47.37% protein and 50.23% ash, whereas the second process reduced 79.61% protein and 88.65% ash. key words: microbiological chitin extraction, deproteination, demineralization ekstraksi kitin terdiri atas dua tahap proses, yaitu proses deproteinasi dan demineralisasi. tujuan penelitian ini membandingkan urutan tahapan proses ekstraksi kitin dari kulit udang. percobaan pertama adalah deproteinasi kulit udang segar dilanjutkan dengan demineralisasi kulit udang yang telah dideproteinasi. percobaan kedua adalah demineralisasi kulit udang segar dilanjutkan dengan deproteinasi kulit udang yang telah didemineralisasi. f11.1, penghasil enzim proteolitik digunakan dalam proses deproteinasi. fncc116, penghasil asam laktat digunakan untuk proses demineralisasi. proses demineralisasi dilakukan dalam tabung fermentor 1 liter, pada 55 °c, 250 rpm, aerasi 2.5 vvm selama 60 jam. proses demineralisasi dilakukan dalam fermentor yang berukuran sama pada 30 °c, 50 rpm selama 48 jam. hasil percobaan menunjukkan bahwa proses demineralisasi dilanjutkan dengan deproteinasi menghasilkan produk kitin yang lebih baik daripada proses dengan urutan sebaliknya. percobaan pertama berhasil menurunkan kadar protein sebesar 47.37% dan abu sebesar 50.23%; sedangkan proses kedua menurunkan kadar protein sebesar 79.61% dan abu 88.65%. kata kunci: ekstrasi kitin secara mikrobiologi, deproteinasi, demineralisasi chitin (poly -(1-4)-n-acetyl-d-glucosamine) is a linear polysaccharide and the second most abundant natural polymer after cellulose. in nature chitin appears as ordered crystalline microfibrils forming structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast (rinaudo 2006). chitin and its derivatives have been used in many applications including pharmaceuticals, textile, food, and cosmetics. the primary source of chitin production is from marine crustacean shell waste. shrimp shells are predominantly composed of chitin in a complex binding to 10-20% calcium and 30-45% protein (rao and stevens 2005). indonesia is one of the main shrimp producing and exporting countries. in 2007, 160 797 tons shrimps was exported and 90% of it was in the form of frozen headless shelled ones. as a consequence, there has been a lot of shell waste from frozen shrimp industries. shrimp shell waste consists of 45% of the whole shrimps (dhewanto and kresnowati 2002), therefore in 2007, the amount of shell waste was about 100.188 tons, most of which has only been used as animal feed. there has been some chitin production in indonesia, however, all of it is produced using a chemical process. the chemical method of chitin extraction from the shells involves alkali deproteination using 2.75 m naoh and acid demineralization using 1 m hcl. the chemical process may cause hydrolysis of the polymer and inconsistence of some physical properties, however, the main concern using the chemical process is the use of harsh chemicals at high temperature which causes corrosion of the equipment and environmental problems of the waste disposal (beaney 2005). to solve these problems, some biological processes including enzymatic, microbiological as well as chemical-biological combinations have been studied. some experiments of chitin extraction were done by combining chemical demineralization and enzymatic or microbiological deproteination (gagne and simpson 1993; bustos and healy 1994; oh 2000; et al. et al. *c budiasih_solichin@yahoo.com orresponding author, phone: +62-21-811153294, fax: +62-21-7560536, email: issn 1978-3477, eissn 2087-8575 vol 5, no 1, march 2011, p 39-45 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.1.7 yang 2000). other experiments used single strain o r m i x e d c u l t u r e s o f m i c r o o rg a n i s m s f o r demineralization and deproteination in a separate process or in a one step operation. rao (2000) used 541 for the demineralization and deproteination of shrimp biowaste with addition of organic acids (lactic, acetic and citric acid) and inorganic acid (hcl). a mixed cultures consisting of was used by healy (2003) to extract chitin from prawn shell waste. rao and stevens (2006) conducted a one step chitin extraction process using 541 with addition of glacial acetic acid to adjust the ph of the shrimp waste down to ph 6 at the beginning of the process. the wild type of the same bacterium ( f11) used in this experiment was used for deproteination of shrimp shells by daum (2007) in combination with different species of for demineralization. the aim of these experiments was to find the succession of microbiological deproteination and demineralization in chitin extraction of shrimp shell waste that would render the higher chitin yield. the first experiment was deproteination of fresh shrimp shells followed by the demineralization process. the second experiment was demineralization of fresh shrimp shells followed by deproteination. f11.1, a proteolytic, chitinase-defficient bacterium, was used for the deproteination process, and fncc-116, a lactic acid bacterium, was used for the demineralization process. headless shrimp shells of were obtained from a frozen shrimp processing company “pt wirontono baru” north jakarta, indonesia. the shells were washed and disintegrated into size of 5-10 mm and kept at -20 ºc before being used for the experiments. f.11-1 used for removing protein from shrimp shells was isolated from shrimp shell waste of pt laura indo, a frozen shrimp processing company, palembang, sumatera, indonesia. the bacterium was isolated and identified by waldeck (2006). recently the bacterium was genetically modified ( f.11) by hoffman (2010). the stock culture was kept in 10% glycerol and 10% skimmed milk were stored in a deep freezer (nu6520e, nuair, plymouth mn55447, usa) at -80 ºc. fncc 116, a lactic acid producing bacterium, was used for the demineralization process. et al. et al. lactobacillus plantarum l. plantarum, l. salivarius, streptococcus faecium, and pediococcus acidilacti et al. l. plantarum bacillus licheniformis et al. lactobacillus b. licheniformis lactobacillus acidophilus penaeus vannamei b. licheniformis et al. b. licheniformis pga et al. l. acidophilus materials and methods shrimp shells and microorganisms. the bacterium was obtained from the food nutrition culture collection of the faculty of agricultural technology, university of gadjah mada, yogyakarta, indonesia. the stock cultures was kept in 10% glycerol and 10% skimmed milk and were stored in a deep freezer (nu-6520e, nuair, plymouth mn55447, usa) at -80 ºc. refreshing of the frozen stock culture was conducted by transferring 1 ml stock culture into 9 ml of sterile de man rogosa and sharpe (mrs) broth, which was incubated at 37 ºc for 24 h. to prepare a starter inoculums, 10 ml refreshed culture were transferred into 90 ml mrs broth in a 250 ml erlenmeyer flask and incubated at 37 ºc until the optical density reached 0.85 at wavelength of 600 nm (u-2001, hitachi instrument inc, usa), which equals a cell concentration of about 1x 10 ml . based on previous studies, the optimum demineralization condition is at 30 ± 2 ºc and 50 rpm agitation (junianto 2009). three hundred grams frozen shrimp shell waste (69.5% moisture) were added to 900 ml liquid media and 100 ml starter inoculums. 100 ml medium contained 60 mg glucose and 0.5 mg yeast extract. the ph was adjusted to ph 7.0. the fermentation was done at 37 ºc and 50 rpm agitation for 48 h. when the demineralization process was completed the shells were separated from the broth and washed with running water until the washed water became neutral (ph 7.00) and drained. the demineralized shells were then kept in freezer -20 ºc (derby f 20u, denmark) for the following process. demineralization efficiency is defined as efficiency of ash removal that calculated as follows initial % ash subtracted by % ash after the process devided by initial % ash multiply by 100%. the frozen stock culture of f11.1 was refreshed in luria bertani (lb) broth. the culture was incubated in a shaking incubator (lab-therm, kühner, switzerland) at 55 ºc and 180 rpm for 6 h or until the optical density of the culture reached 0.9 which, based on previous experiments, equals a cell density of about 1x10 cfu ml (junianto 2009). each 100 ml fermentation medium contained 0.5g kh po , 0.5g nacl, 0.5g yeast extract 0.05g mgso and 0.1g cacl . two hundred ml of inoculums were added into 300 g of shrimp shells in 800 ml medium. the fermentation was carried out at 55 ºc, 2.5 vvm aeration and 250 rpm agitation for 60 h and the ph was maintained in a range of 7.8-8.2. after the deproteination process was completed, the shells were separated from the broth, washed, drained and kept at -20 ºc for the following process. deproteination et al. b. licheniformis et al. fermentation demineralization process. deproteination process. 9 -1 9 -1 2 4 4 2 40 wahyuntari et al. microbiol indones efficiency is defined as efficiency of protein removal that calculated as follows initial % protein subtracted by % protein after the process devided by initial % protein multiply by 100%. all fermentations were conducted in a custom made fermentor which consisted of a 2 l glass cylinder jar, equipped with a janke and kunkel rod agitator for agitation, a compressor connected to a lkb-bromma (sweden) flow meter for aeration and heated by a coil in the fermentor that was connected to a water bath for temperature control. the shrimp shells were not sterilized prior to fermentation, since the process is intended for small scale industries that mostly do not have sterilization facilities. based on preliminary experiment, the amount of the inoculums of 10 cfu ml was able to avoid overgrown of contaminated bacteria. moisture content was determined by heating samples at 110 ºc in a “kett” infrared moister meter model f-1a (tokyo, japan). ash content was determined after combustion of 5 g dried sample in a crucible at 600 ºc for 4 h (aoac 1984) in a muffle furnace fischer scientific model 182a. insoluble protein content of the shrimp shells and fermented solid samples was solubilized using 1m naoh. a half gram sample was added to 7.5 ml of 1m naoh and incubated for 24 h. the protein content of the supernatant was measured according to the lowry method (1951) using bovine serum albumin fraction iv (sigma, st louis, usa) as a standard. glucose was analyzed using method of miller (1959) and lactic acid content was analyzed using hplc (merck-hitachi, tokyo, japan), aminex hpx-87h (300mm x 7,8mm) column, at 65 ºc (l-5025-column thermostat, merck, tokyo, japan), isocratic mobile phase of 0.005 n h so with a flow rate of 0.6 ml min (l-6200apump, merck-hitachi, tokyo, japan), detector ri-71 (merck, tokyo, japan). the glucose standard used was 1% glucose (sigmaaldrich, st louis, usa) and the lactic acid standard was 10% l-lactic acid (oxoid, hampshire, england). the protease activity in fermented broth was assayed using azocasein as a substrate according to the method described by waldeck (2006). one unit was defined as the amount of enzyme releasing 1 mol azocasein per min under reaction conditions. the density of bacterial growth in fermentation broth was assayed after serial dilution by counting colony forming unit (cfu ml ) on mrs agar plate after incubation at 37 ºc, 24 h for fncc116 and on lb agar plate after incubation at 55 ºc, 24 h for f11.1. 9 -1 -1 -1 2 4 analytical procedures. differential refractometer et al. l. acidophilus b. licheniformis fig 1 deproteination process of fresh shrimp shell waste using f11.1. , cell growth; , protease production. bacillus licheniformis fig 2 demineralization of deproteinated shrimp shells using fncc116. , cell growth; , glucose consumption; , lactic acid production. lactobacillus acidophillus volume 5, 2011 microbiol indones 41 0 6 12 18 1.00e+07 1.00e+08 1.00e+09 1.00e+10 0 12 24 36 48 60 72 p ro te a se (u m l -1 ) c e ll (c f u m l -1 ) fermentation time (h) 61.0e+10 0 2 4 1.0e+7 1.0e+8 1.0e+9 0 12 24 36 48 60 c e ll (c f u m l -1 ) fermentation time (h) results deproteination process of fresh shrimp shell waste using f11.1. the process was carried out within 60 h, maximum cell growth and proteolytic activity reached after 36 h of fermentation with the maximum amount of the cell was 2.02 x 10 . initial proteolytic activity was 0.52 u ml and reached maximum activity of 15.27 u ml after 36 h then the activity decreased down to 5.03 at the end of the process. during the deproteination process, protein content of the shell decreased from 19.56% down to 5.44% at the end of fermentation process (fig 1). demineralization of deproteinated shrimp shells using fncc116. the maximum cell amount was reached after 24 h-fermentation, and almost all glucose was used and some of it was converted into lactic acid for 36 h of fermentation (fig 2). the protein and ash content of the shrimp shells at the end of the deproteination of fresh shrimp shells followed by demineralization of the deproteinated shells are shown (fig 3). the protein content of the shells was reduced from 19.56% to 5.44% at the end of deproteination process but then increased to 10.3% after demineralization process. during deproteination process the ash content was increased from 19.57% to b. licheniformis l. acidophillus 9 -1 -1 ] g lu c o se ; la c ti c a c id [g (1 0 0 m l ) -1 26.21%, but the ash content was reduced significantly down to 9.74% after demineralization proces. demineralization of fresh shrimp shell waste using fncc 11. maximum cell amount, lactic acid production was reached after 24 hs l. acidophillus 42 wahyuntari et al. microbiol indones fig 3 ash and protein content in the shrimp shells during deproteination followed by demineralization process. ---, deproteination; , demineralization; , protein; , ash. fig4 demineralization of fresh shrimp shell waste using fncc 116 , cell growth, glucose consumption g 100ml) ; lacticacidproduction (g100 ml) ph value lactobacillus acidophillus . , ( , ; , . -1 -1 15 20 25 30 15 20 25 30 (1 0 0 g ) -1 ] 1 0 0 g ) -1 ] 0 5 10 15 0 5 10 15 0 12 24 36 48 60 72 84 96 108 120 p ro te in [ a sh [g ( fermentation time (h) 0.0 2.5 5.0 7.5 1.0e+7 1.0e+8 1.0e+9 1.0e+10 0 12 24 36 48 60 c e ll a m o u n t (c f u m l -1 ) fermentation (h) fig 5 deproteination of demineralized shrimp shells using f 11.1. , cell growth; , protease production. bacillus licheniformis 0 10 20 1.0e+07 1.0e+08 1.0e+09 0 12 24 36 48 60 p ro te a se (u m l -1 ) c e ll a m o u n t (c f u m l -1 ) fermentation time (h) of fermentation. after demineralization process, the ash content of fresh shrimp shells was reduced from 19.57% down to 0.91%, however the protein content was increased from 19.17% to 26.16% (fig 4). deproteination of demineralized shrimp shells using f 11.1. the maximum cell amount was reached after 24 hs of fermentation (2.77x10 cfu ml ), however the maximum proteolytic enzyme production reached after 48 hs of fermentation. during deproteination process, the protein content of the shells decreased from 26.26% down to 4.0%, whereas, the ash content increased from 0.92% to 2.22% (fig 5). the protein and ash content of shrimp shells during demineralization followed by deproteination process during microbiological chitin extraction are shown (fig 6). fermented shrimp shells composition resulted from different fermentation methods: deproteination followed by demineralization (dp-dm), and demineralization followed by deproteination (dm-dp) are compared (fig 7). b. liheniformis 8 -1 fig 6 ash and protein content in the shrimp shells during demineralization deproteination followed by process. , demineralization; ---, deproteination; , protein; , ash. fermentation time (h) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 12 24 36 48 60 72 84 96 108 120 p ro te in [g ( 1 0 0 g ) -1 ] a sh [g ( 1 0 0 g )1 )] fig 7 comparison of fermented shrimp shells composition resulted from different fermentation methods. dp-dm: deproteination followed by demineralization, dm-dp: demineralization followed by deproteination. , ; , protein; , .chitin ash 19.51 9.74 2.22 58.5 8 78.4 5 92.8 4 0 20 40 60 80 100 fresh dp-dm dm-dp c o m p o n e n t p e rc e n ta g e (% ) fresh dp-dm dm-dp 19.28 10.3 3.99 discussion based on previous studies optimal agitation for demineralization using fncc 116 is 50 rpm and optimal agitation and aeration for deproteination using f11.1 are 250 rpm and 2.5 vvm respectively (junianto 2009). the aim of this study was to determine the succession of chitin extraction steps from shrimp shells. the first experiment was deproteination of fresh shrimp shells followed by demineralization of the deproteinated shells. the second experiment was demineralization of fresh shrimp shells followed by deproteination of the demineralized shells. in the first experiment, the fresh shrimp shells were fermented using f.11.1 for 60 h. the cell concentration as well as the protease production reached their maximum after 36 h (fig 1). the initial protein content of the shells was 19.17% (dryweight). due to the protease activity during the deproteination process, the protein content of the shells decreased down to 5.54% (dryweight) (fig 3). the main solid components in the shrimp shells are insoluble protein, chitin and minerals. in the deproteination process only protein was enzymatically hydrolyzed whereas chitin and minerals (ash) contents showed only minor changes. in the closed system (fermentation jar) the solid dry matters of shrimp shells were constant, if the insoluble protein was solubilized, and the ash and chitin content were intact, then the total amount of solid dry matter was decreased. as a result the percentage of ash in the shells increased from 19.57% (dryweight) up to 26.21% (dryweight) (fig 3). in the first experiment, after deproteination shrimp shells were demineralized. the experimental result (fig 2) shows that the cell growth entered stationary state after 12 h of fermentation. the glucose was consumed rapidly during the first 24 h and lactic acid content in the broth increased rapidlyduring this time. during the demineralization process ash content decreased from 18.5% to 10.3% at the end of 48 h fermentation (fig 3). however, the protein percentage of the shrimp shells increased from 5.54% to 10.3%. based on the data, it seems that fncc116 hardly produced proteolytic enzyme, and mainly produced lactic acid. belongs to the homofermentative lactic acid bacteria that is able to convert the majority of glucose into lactic acid (sanders and klaenhammer 2001). as a result the main product in the demineralization process was lactic acid which would react with the calcium carbonate component in the chitin fraction of the shrimp shells to l. acidophilus b. licheniformis et al. b. licheniformis l. acidophilus l. acidophilus volume 5, 2011 microbiol indones 43 form calcium lactate (rao and stevens 2005). the bacterium used only produced very little protease (0.011 u ml ), therefore the activity of the enzyme on shrimp shell protein was undetectable. this might be the reason why the protein content of the shrimp shells increased during the course of the demineralization (fig 3). after the demineralization of deproteinated shrimp shells was completed, the ash, protein and chitin content of the fermented product were 9.74, 10.3, and 78.5% (dry weight) respectively (fig 7). the deproteination process reduced the protein content of the shrimp shells from 19.57 down to 10.3% (efficiency of the protein removal was 47.37%). the demineralization process reduced the ash content of the shrimp shells from 19.17% down to 9.74% (efficiency of the ash removal was 50.23%). in the second chitin extraction process demineralization was followed by deproteination. fig 4 shows cell growth, glucose consumption, lactic acid production and ph of fermented broth during the demineralization process using fncc 116. the amount of the cells increased rapidly during 12 h of fermentation and the stationary stage lasted for the following 24 h. after that the cell amount decreased slowly. the glucose was consumed very fast during the first 24 h and seemed to be converted into lactic acid as shown by the rapid decrease of ph from 6.9 to 4.4 during the first 24 hs. later the ph decreased slowly until the end of the process was reach after 24 h to ph 4.1. the second experiment led to a higher mineral and protein removal than the first experiment (fig 3 and 6). shrimp shells matrix is formed mainly of chitin and protein hardened by mineral salts especially calcium carbonate (beaney 2005). mainly produced lactic acid by breaking down glucose creating lactic acid thereby lowering the ph of the fermentation broth and suppressing spoilage by microbial growth. the lactic acid reacted with calcium carbonate in the chitin fraction to form calcium lactate which is soluble and could be removed by washing. the following process was hydrolyzing protein in the chitin by fermentation of the proteolytic bacterium f11.1. the proteolytic enzyme production increased along with the increase of cell concentration and reached the maximum activity after 48 h fermentation; however, the protein content was reduced drastically for the first 12 h of fermentation and decreased slowly for the rest of the fermentation time (fig 5). the removal of protein content in the shrimp shells in the second process was higher since the calcium carbonate had been -1 l. acidophilus et al. l. acidophilus b. licheniformis removed, the proteolytic enzyme could contact more easily with the protein in the chitin fraction of the s h e l l s . t h e c h i t i n e x t r a c t i o n p r o c e s s b y demineralization followed by deproteination was done for 108 h or 4.5 days. the protein content of the shrimp shells was reduced from 19.57% down to 3.99% or 79.61% protein removed. the ash content was reduced from 19.51% down to 2.22% or 88.65% ash removed. (fig 7). daum (2007) used the wild strain of f11 and different species of sp. after 5.5 days, the protein and ash removed were 95% and 89.6% respectively with 2.12% protein and 2.08% ash. the slightly better results of daum 2007 even with the wild type strain might be the fact that the fermentor used was equipped with a commercial ruskton impeller stirring system instead of the much simpler design of impeller stirring system used in this work. the different design of impeller might result in different agitation and aeration effect to the fermentation system and the fermentation process was longer (5.5 days) than this experiment(4.5 days). 541, which produces lactic acid and protease was used by rao and stevens (2005) for demineralization and deproteination in a one step fermentation of shrimp biowaste in a drum reactor and a beaker. the efficiency of deproteination and demineralization in the drum reactor were 66 and 63% respectively and in the beaker 54 and 52% respectively. the ph was maintained at ph 6 by adding acetic acid during the experiment within 24 h. these experiments showed two steps chitin extraction process which was demineralization process using lactic acid bacterium followed by deproteination process using proteolytic producing bacterium (this work and daum . 2007 report) gave a better protein and ash removal than that of one step protein and ash removal done by rao and steven (2005). to get a more efficient process for protein and ash removal that could be applied in larger scale, further works have to be done to improve the process. the efficiency of bacterial fermentation depends on factors such as quantity of inoculums, initial ph, ph during the fermentation and fermentation time. further experiments will be done to optimize the conditions of the demineralization and deproteination process. to conclude, demineralization (dm) followed by deproteination (dp) of shrimp shells gave a higher chitin extraction efficiency than carrying out the et al. b. licheniformis lactobacillus et al l. plantarum et al 44 wahyuntari et al. microbiol indones process steps in opposite order (dp-dm). the dm-dp process removed 79.61% protein and 88.65% ash as compared to 47.37% protein and 50.23% ash removed by the dp-dm process. references aoac. 1984. official methods of analysis.15 ed. arlington,virginia: association of official analytical chemistry. inc. beaney p, lizardi-mendoza j, healy m. 2005. comparison of chitins produced by chemical and bioprocessing methods. j chem technol biotechnol. 80:145-150. doi: 10.1002/jctb.1164. bustos ro, healy mg. 1994. microbial/enzymatic deproteination of prawn shell waste. in: proceeding icheme research event; 1994 january 4-6, london.1994, p: 126-128. daum g, stöber h, veltrup k, meinhardt f, bisping b. 2007. biotechnological process for chitin recovery out of shrimp wast. j biotechnol. 131(1):s188. dhewanto m, kresnowati mtap. 2002. chitosan industry: an alternative for maritime industry in empowerment indonesia. in: proceedings of indonesian student scientific meeting; 2002 october 4-6; berlin, germany. istec-europe. p: 327-333. gagne n, simpson bk. 1993. use of proteolytic enzyme to facilitate the recovery of chitin from shrimp waste. food biotechnol. 7(3):253263. healy m, green a, and healy a. 2003. bioprocessing of marine crustacean shell waste. acta biotechnol.23 (2-3):151 260. doi: 10.1002/abio.200390023. hoffman k, daum g, köster m, kulicke wm, meyer-rammes h, bisping b, meinhardt f. 2010. genetic improvement of strains for efficient deproteinization of shrimp shells and production of high-molecular-mass chitin and chitosan. appl environ microbiol. 76 (24):8211-8221. junianto, mangunwidjaja d, suprihatin, mulyorini, wahyuntari b. 2009. [effect of aeration and agitation rate on protein hydrolysis of shrimp shells in microbiological chitin extraction] [in indonesia]j. bionatura. 11(2):107-117. lowry oh, roseborough nj, farr al, rundall rj. 1951. protein measurement with folin phenol reagent. j biol chem. 193(1):265275. miller gm. 1959. use of dinitrosalicylic acid reagent for determination of r e d u c i n g s u g a r s . a n a l c h e m . 3 1 ( 3 ) : 4 2 6 4 2 8 . d o i : 10.1021/ac60147a030. oh ys, shih il, tzeng ym, wang sl. 2000. protease produced by k-187 and its application in the deproteinization of shrimp and crab shell waste. enzyme microbiol technol. 27(1-2):3-10. doi:10.1016/s0141-0229(99) 00172-6. rao ms, munoz j, stevens wf. 2000. critical factors in chitin production by fermentation of shrimp biowaste. appl microbiol biotechnol. 54(6):808-813. rao ms, stevens wf. 2005. chitin production by fermentation of shrimp biowaste in a drum bioreactor and its chemical conversion to chitosan. j chem technol biotechnol. 80(9):1080-1087. doi: 10.1002/jctb.1286. 2006. fermentation of shrimp biowaste under different salt concentrations with amylolytic and non-amylolytic strains for chitin production. food technol biotechnol. 44(1):83-87. rinaudo m. 2006. chitin and chitosan:properties and application. prog polym sci. 31(7):603-632. sanders me, klaenhammer tr.2001. invited review: the scientific basis of lactobacillus acidophilus ncfm functionality as a probiotic". th bacillus licheniformis pseudomonas aeruginosa lactobacillus rao ms, stevens wf. lactobacillus j dairy sci. bacillus licheniformis 84(2):319-331. doi: 10.3168/jds.s0022-0302(01) 74481-5. waldeck j, daum g, bisping b, meinhardt f. 2006. isolation and molecular characterization of chitinase-deficient strains capable of deproteiniation of shrimp shell waste to obtain highly vicous c h i t i n . a p p l e n v i r o n m i c r o b i o l . 7 2 ( 1 2 ) : 7 8 7 9 8 5 . d o i : 10.1128/aem.00938-06. yang jk, shih il, tzeng ym, wang si. 2000. production and purification f protease from that deproteinize crustacean waste. enzyme microbiol technol. 26(5-6):406-413. bacillus subtilis volume 5, 2011 microbiol indones 45 404 not found guide for author.cdr guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can be used as long as there is consent from its authors. all pages, including the pages with figures and tables at the end of the paper, must be numbered consecutively. research paper may occupy up to 4500 words, some figures 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publishers. 7th ed. reston (va): the council. journal suwanto a, kaplan s. 1989. physical and genetic mapping of the 2.4.1 genome: presence of rhodobacter sphaeroides tw o unique ci rcular chromosomes. j b act er iol. 171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up 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vol.14, no.4, december 2020, p 129-139 doi: 10.5454/mi.14.4.2 trench construction in peat soil and the dgge analyses of nif gene and activity of dehydrogenase 1,2* 1,2 3 happy widiastuti , darmono taniwiryono , iman rusmana , 1 and galuh wening permatasari 1 indonesian research institute for biotechnology and bioindustry (iribb), bogor, indonesia; 2 masyarakat perkelapa-sawitan indonesia (maksi); 3 institut pertanian bogor (ipb), bogor, indonesia. basal stem rot (bsr) is a major disease in oil palm crops which also possible happen on peat soils. ganoderma boninense is the pathogen that causes this disease and infects the plants through the root. maintaining root health expects to reduce the intensity of the occurrence of bsr disease and its spread. in this research, trench construction and maintenance was carried out which aims to break the spread and activity of ganoderma. trench maintenance was carried out by giving empty bunches oil palm inoculated with decomposer, azotobacter inoculants and humic acid. four treatments tested were trenched (t1) and untrenched (t0) in combination with two level of ganoderma attack, which are moderate (mo) and severe (se). the soil sample were retrieved from different point, which are in the trench area (tr), harvest path zone (h), and frond stack zone (m). the soil characteristics including microbe abundance, dehydrogenase activity, and pcr-dgge of microbe community in each treatment were analyzed to reveal the effect of trench construction. the results showed that trench implementation reveals major results related to the abundance of microbes and fungi population, supported by the enhancement of dehydrogenase activity at the block with moderate ganoderma attack. in addition, the dgge study effectively separates the microbial population based on nif gene of four separate treatment into two clusters, splitting the groups depending on the ganoderma level attack. this study shows enhancement of soil characteristics biologically and nutrients status of oil palm leaves especially p, as the results of trench construction in peatland. key words: dgge, microbes, oil palm, peatland, trench busuk pangkal batang (bpb) adalah penyakit utama pada perkebunan kelapa sawit yang juga dapat terjadi pada tanah gambut. ganoderma sp. adalah patogen penyebab penyakit bpb yang menginfeksi tanaman melalui akar. dengan menjaga kesehatan akar diharapkan mampu mengurangi intensitas terjadinya penyakit bpb dan penyebarannya. dalam penelitian ini, dilakukan pembuatan galian (trench) dan pemeliharaannya untuk menghentikan penyebaran dan aktivitas ganoderma. pemeliharaan galian dilakukan dengan memberikan tandan kosong kelapa sawit yang diinokulasi dengan dekomposer, azotobacter dan asam humat. empat perlakuan yang diuji adalah dengan galian (t1) dan tanpa galian (t0) di kombinasi dengan dua tingkat serangan ganoderma, yaitu sedang (mo) dan parah (se). sampel tanah diambil dari titik yang berbeda, yaitu di daerah galian (tr), jalur panen (h), dan jalur gawangan mati (m). karakteristik tanah terdiri dari kelimpahan mikroba, aktivitas dehidrogenase, dan komunitas mikroba menggunakan pcr-dgge di setiap perlakuan dan dianalisis untuk mengungkapkan efek konstruksi galian. hasil penelitian menunjukkan efek positif dari implementasi galian terhadap kelimpahan mikroba dan populasi jamur, didukung oleh peningkatan aktivitas dehidrogenase di blok dengan intensitas serangan ganoderma tingkat sedang. selain itu, studi dgge secara efektif memisahkan populasi mikroba berdasarkan gen nif dari empat perlakuan yang terpisah menjadi dua kelompok berdasarkan tingkat serangan ganoderma. studi ini menunjukkan peningkatan karakteristik tanah secara biologis dan status nutrisi daun kelapa sawit khususnya fosfor (p), sebagai hasil dari konstruksi galian di lahan gambut. kata kunci: dgge, galian, kelapa sawit, lahan gambut, mikroba microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8159762577; email: happywidiastuti@gmail.com peatlands are created by the deposition and burying of plant detritus-derived organic matter. it grows under conditions of near-continuous soil saturation, leading to anaerobic conditions that drastically slow down the decomposition. peatland soils contain a wide variety of organic matter content and thickness while they frequently contain 80 to 100 percent organic matter that is multiple meters or thicker than others (craft 2016). the main problem in peatland could be overcome by oil palm has been planted in peat soil since 1980 due to the decline in available suitable mineral soil. the productivity of oil palm in peatlands ranged from 12 to 27 ton/ha and yield up to 23% or 2% lower than mineral landfill. in 2010, the oil palm area reached 701.868 ha in kalimantan and 500.000 ha in riau (wahyunto 2011). drainage reparation, ameliorant application, and utilizing the biological ecosystem which plays role in the quality improvement of nutrients to support plant development (rawat et al. 2018). another problem that usually finds in the peatlands is the soil pathogen, such as ganoderma. lately, a report from huan and wahidin (2009) mentioned that ganoderma sp. cases in peatlands increasing even in the first generation of oil palm. the trench construction method is one of the efforts to repair the peatland area. the field observation in the pt thip area shows that trenches with 4 x 4 x 0.75 meters of size surrounding the oil palm infected by ganoderma sp. successfully decreasing the infection rate (huan and wahidin 2009). in rhizobia, the two major groups of nitrogenfixing genes, the nif genes and fix genes, are present. the nif genes encode nitrogenase and mimic the nitrogen-fixing genes found in klebsiella pneumoniae and other microbial groups structurally and functionally (ruvkin and ausubel 1980). while most of the nif genes are present on rhizobia plasmids, it has also been mentioned in bradyrhizobium chromosomes (sa'nchez et al. 2013). the nitrogen fixation mechanism is catalyzed by the enzyme nitrogenase, either in symbiotic or nonsymbiotic microorganisms, and this enzyme complex is encoded by the genes nifdk and nifh. there are two subunits of the enzyme nitrogenase, a molybdenum iron protein (mo-fe), subunit i and subunit ii of an iron-containing protein (fe). in different classes of microorganisms there is considerable difference in the organization and complexity of nif genes (downie 1998). the nonsymbiotic k pneumoniae, according to dean and jacobson (1992), comprises at least 20 nif genes arranged in approximately eight operons. in most cases, the regulations of all nif genes are regulated by nifa (positive transcription activator) and nifl (negative regulator). nif gene regulation is determined by the concentration of both oxygen and nitrogen in the system (merrick and edwards, 1995). when soil ammonia levels (nh3 or nh4) are high, nifl slows down the fixation of nitrogen to serve as a negative controller or gene expression by stopping nifa from functioning as an activator. if the concentration of o is 2 high, then nitrogenase synthesis decreases which leads to a decrease in bnf. the diversity of nitrogen-fixing bacteria in soil is commonly studied by dgge (rusmana et al. 2014). the organic material application has proven to increase the carbon soil as well as the pathogen antagonism (scotti et al. 2015). besides, empty fruit bunches (efb) applications also increasing the ph soil which responsible to increase the soil nutrition and diversity of microbes. the organic material also improving the physical, chemical, and biological soil characteristics (haynes 2008). a report from komariah et al (1993) and nurani et al (2007) reported that the microbes' consortium and efb application successfully increase the ph and the saturation of nutrients in peatlands, also decreasing the cation exchange capacity as well as the c/n ratio. besides, yusnaini (2009) revealing that organic compost application could increase the ph from 3.56 up to 5.47. based on the previous results, we tried to make trench and maintain those trench with the addition of efb compost, azotobacter, humic acid, and analyze the soil characteristics, biological activity as well as the microbes community. materials and methods this experiment was done in the oil palm field in pasangkayu, sulawesi. the soil type used was wet soil. the treatment tested was a combination of two different factor treatments, which are trenched (t1) and untrenched (t0); in combination with the intensity of attack i.e. severe (se: >0.1) and ganoderma moderate (mo: <0.01) as shown in table 1. the percentage of attack was measured by the ganoderma counting the percentage of oil palm suffer from basal stem rot (bsr) disease caused by .ganoderma the trench size around 24 x 1 x 0.6 m was manually constructed, with 3 meters gap for every trench (figure 1), followed by the maintenance through adding empty fruit bunches (efb) oil palm, humic acid, and azotobacter inoculum (table 2). the trench was filled with one layer of efb which contained 288 fruits which previously inoculated using decomposer and ganoderma antagonist microbes directly using automatic applicator tools. the efb in trench was then buried with the soil and frond piles. at the end of incubation time, around 4 weeks, the trench was opened and 1 kg of and 150 ml of humic azotobacter acid were applied for each trench (table 2). the trench was then layered with the frond piles and soil to optimized the decomposition process. in trenching plot, the inorganic fertilizer was applied in a lower dose i.e. 75% dose of recommended. soil and oil palm leaves sample. the sample was taken two times i.e. before and after trench construction. we took the soil sample inside the trench especially for block l5 and l9 and in a similar area of 130 widiastuti et al. microbiol indones volume 14, 2020 microbiol indones 131 table 1 the data of each treatment area block year of planting wide area (ha ) production (ton ha -1) trenching ganoderma intensity attack (%) treatment code f15 1999 44 23.84 t0 0.0022 (mo) t0-mo l5 2000 34.88 26.26 t1 0.0094 (mo) t1-mo l3 1998 31.46 23.62 t0 0.025 (se) t0-se l9 1995 31.22 21.23 t1 0.033 (se) t1-se t0: untrenched; t1: trenched; mo: moderate; se:severe table 2 the time of trenching construction and product application treatment code trenching end date application of efb compost +humic acid+ azotobacter application of efb compo st +humic acid+ azotobacter 1st 2nd t0-mo t0-se t1-mo t1-se october 2011 august 2011 november 2011 september 2011 january april 2013 june -april 2013 t0: untrenched, t1: trenched, mo: moderate ganoderma attack, se: severe ganoderma attack, efb: empty fruit bunches oil palm fig 1 the trenching size and pattern applied in the oil palm crops. green triangle indicated the oil palm tree. trench for untrenched treatment (f15 and l3). a soil sample from trench was taken from the trench in the center of every 8 ha. the sample was composite of the 50 grams of soil from left, right, and the middle of the trench area. then, the soil was labelled for further analysis. as a comparison, we also took the soil sample from the harvest path zone (h) and frond stack zone (m) following the same procedure. leaves sample were taken from the same location as the soil in the form of 5 pieces of the leaves, the right and left side after the sweet spines. the parameters of leaf nutrients analyzed were n, p, and k content. dehydrogenase activity assay. to analyze the dehydrogenase activity, three soil samples were retrieved from a different point of each treatment: tr (trench area), m (frond stack zone), and h (harvest path zone). the dehydrogenase activity was measured in the darkroom. as much 5 g of soil sample was used. ttc (triphenyl tetrazolium chloride) 3% and tris-hcl were added. the vial was then vigorously mixed for 24 0 h, in 37 c. after incubation the methanol was added to each vial and shook for 2 hours, 125 rpm using a shaker. the suspension was filtered using whatman paper number 5 and measured in 485 nm using a spectrophotometer. dna extraction from soil sample. extraction was conducted by using the power soil dna isolation kit (mobio laboratories, carlsbad, ca, usa). extraction was done according to the procedures of the kit from the company. the quality of dna then checked using nanodrop 2000 (thermo scientific, wilmington, de, usa). polymerase chain reaction analyses of nif gene. dna was amplified by using polf / polr primer sets. pcr was performed by using kapa hot start readymix (kapa biosystems, wilmington, ma, usa). the primer used were f (polf-gc): cgcccgccgcgccccgcgcccggcccgccgc ccccgcccctgcgayccsaargcbgactc and r (polr): atsgccatcatytcrccgga. each pcr reaction contained 12.5 µl kapa hotstart readymix, 1.25 µl forward primer (0.5 um), 1.25 µl reverse primer (0.5 um), 3 µl template (~100 ng) and 7 µl nuclease-free water. pcr was performed using tgradient thermocycler (biometra gmbh, goettingen, germany). the pcr thermal cycling conditions were performed as follows: initial denaturation (95 °c, 1 minutes), denaturation (95 °c, 15 s), annealing (55 °c, 15 s), extension (72 °c, 15 s), post-extension (72 °c, 5 minutes), for 30 cycles. products were run at 1.5 % agarose gel for the checked correct size and stored at 20 °c until analyzed on dgge. denaturing gradient gel electrophoresis (dgge) analysis. dgge analysis was performed using the d-code universal mutation detection system (bio-rad, hercules, ca, usa). pcr products (25ul) were separated in 1 mm thick in a 8% (w/v) polyacrylamide gel [acrylamide–bisacrylamide (37.5:1)] in 1× tae buffer (40mm tris, 20mm acetic acid, and 1mm edta) with a denaturing gradient from 35 to 60% (100% denaturant corresponds to 7 m urea and 40% (v/v) deionized formamide) in 130 v, 60 °c for 6 hours. the gel was stained in the sybr safe solution (invitrogen-molecular probes, carlsbad, ca, usa) for 1 hour, and photographed by g:box (syngene, frederick, md, usa) under uv transillumination. the dgge result was then interpreted using ntsys software and gel analyzer software. upgma method was applied to calculate the phylogenetic tree based on comparing dgge band patterns. results compost characteristics. the results of the c analysis showed that the c content of compost was decrease in the range of 2.4% -12% while an increase in n ranged from 92-176% (table 3). these results indicated the decomposition of microbes that emit co2 and form proteins along with the multiplication of microbial cells. these results were in line with the decrease in cn ratio in the range of 88-149%. the interesting point was the rate of decomposition of efb in the plot with moderate ganoderma attack was higher compared to that pathogen severe attack. microbiology characteristics of soil before and after trench construction. the trench construction has been proven to increase the total of cellulolytic fungi, total bacteria, and nitrogen fixing bacteria especially in t1-mo and t1-se location. while the amount of phosphate solubilizing bacteria only increasing in the t1-mo which has a moderate level of ganoderma attack. in contrast, the total fungi in a location with trench construction showed reduction in volume (table 4). dehydrogenase activity analysis. the graph table 3 empty fruit bunches (efb) oil palm compost characteristics treatment code before after % tage of decreasing c/n ratio c (%) n (%) c/n ratio c (%) n (%) c/n ratio t1-mo 43.32 0.76 57 48.05 2.1 22.88 149 t1-se 46.01 1.17 39.32 47.1 2.25 20.93 88 132 widiastuti et al. microbiol indones treatment group before trenching application after trenching application total of fungi (cfu) lignol ytic fungi (cfu) cellulolytic fungi (cfu) total of bacteri a (cfu) phosphate solubilizing bacteria (cfu) nitrogen fixing bacteria (cfu) total of fungi (cfu) lignol ytic fungi (cfu) cellulolytic fungi (cfu) total of bacteri a (cfu) phosphate solubilizing bacteria (cfu) nitrogen fixing bacteria cfu) t0-mo 15 x 10 5 0 6 x 10 3 75 x 10 6 0 0 4.0 x 10 5 0 3.3x10 5 1.3x10 11 0 0 t0-se 24 x 10 5 0 2 x 10 3 6 x 10 6 0 0 1.2x10 4 0 3.3x10 4 1.5x10 11 1.6x10 9 0 t1-mo 1 x 10 5 0 1 x 10 3 6 x 10 5 0 0 1.5x10 5 0 1.1x10 4 2.3x10 11 3.0x10 6 2.7x10 8 t1-se 46 x 10 5 0 3 x 10 3 1 x 10 9 0 0 1.0 x 10 5 0 4.0 x 10 5 1.3x 10 11 0 2.1x 10 7 table 4 the microbes population before and after trenching construction in each treatment v o lu m e 1 4 , 2 0 2 0 m icro b io l in d o n es 1 3 3 compared dehydrogenase activity in each soil sample from each treatment from three locations, which are the frond stack zone, harvest path zone area, and trench area of oil palm crops. according to the data, the highest dehydrogenase activity showed in the trench (tr) sample in each location, range from 9.23 to 10.51 in the location without trench (t0) and 2.56 to 20.11 in the location with trench (t1). interestingly, the trench (t1) showed higher dehydrogenase activity than nontrench location (t0), specifically in blocks f15 and l5. these two blocks have a history of 's ganoderma moderate attack. in addition, the sample in the frond stack zone (m) has lower dehydrogenase activity than the harvest path zone (h). while in the l3 and l9 block, which have a severe history of , ganoderma showed no constant pattern of dehydrogenase activity. unlike in the f15 and l5 locations, the harvest path zone (h) location in the l3 and l9 block has lower dehydrogenase activity than the frond stack zone (m) sample (figure 2). dgge analysis of nif gene. the dna extraction given the results in varied yield range from 31 to 101 ng/µl, and standard purity od 260/280 range from 1.8 to 1.9 (table 5). the phylogenetic tree of dgge analysis revealing the closeness of microbes population based on the nif gene detected in each location (figure 4). there are two clusters which built in the tree. the first cluster consists of tr-t0-mo, ht1-mo, m-t0-mo, tr-t0-se, h-t0-mo, m-t1-mo, and tr-t1-mo. the phylogenetic evolution showed that the microbes population in the first cluster closer to the reference sample, which is compost, in particular the tr-t0-mo sample. the majority of cluster i member is the location with a moderate level of ganoderma attack. whilst, the second cluster consists of tr-t1-se, m-t0-se, m-t1-se, h-t1-se, and ht1-mo. the tree revealed that the location in cluster ii mostly has a severe ganoderma attack. in general, from both clusters, the microbes population from the frond stack zone (m) soil sample was closer to the trench sample (tr). in addition, the microbe population from a harvest path zone (h) location usually formed the new tree branch. the phylogenetic data also supported with the distance analysis data to strengthen the analysis (figure 5). nutrient status of leaves before and after trench construction. the results of the nutrient analysis at moderate ganoderma attacks showed increasing of n levels in the treatment without trench approximately 293% and 91% each for t0 and t1 (table 6) whereas in plots with severe ganoderma attack, the increase in leaf n levels was 338% and 103 % for t0 and t1, respectively. these results indicated that by making trench the rate of increase in n of the leaves reduced compared to that without trench. interestingly, an increase in leaf n content was higher at severe ganoderma attack rates compared to those at moderate attack rates. for leaf p levels, at moderate ganoderma attack, the increase of p was 147% (t0) and 7 times (t1) while at severe was 19% and 11 times each for t0 and t1 respectively. these data showed that trench yield the higher p leaf content compared to those untrenched. for k leaves an increase of 10 times and 6 times respectively for t0 and t1 at moderate ganoderma attack while at severe ganoderma attack was 19 and 13 times for t0 and t1 respectively. the increase in nutrient status that occured in trenched block seems to be lower compared to untrenched block. the k nutrient status of severe ganoderma attack was higher compared to that moderate ganoderma attack. it seems that the tendency of k leaf nutrient was similar with n levels. discussion this study finds that the construction of trench able to increase the existence of bacteria nitrogen fixation. this caused by the trench development contains azotobacter, humic acid, and efb compost for plant development. a study reported that obligatory aerobes such as azotobacter vinelandii able to protect nitrogenase from oxygen and remove nitrogen by using cytochrome oxidases (poole and hill, 1997). nitrogen in soil parent material is not present, given the fact that nitrogen content in the atmosphere is highest of all atmospheric gasses (hedin et al. 2009). soil nitrogen inputs for plant nutrition and crop productivity are thus largely dependent on organic matter degradation, inorganic fertilizer applications, and biological nitrogen fixation (bnf) through nitrogenase enzyme activity (vitousek et al. 2013). in addition, the trench system construction in the location where ganoderma attacks at a moderate level positively affect the dehydrogenase activity. additional of efb in the trench location expected to enhance the ph of soil. b. trisakti et al (2017) reported that the efb with piece size <1 cm to 12-15 cm have high ph range 9.15 ± 0.25 to 9.25 ± 0.18. this basic condition leads to inhibition of pathogen growth, which only survive in acidic environment (saidi et al. 2008). however, in the location with severe ganoderma attack level, trench implementation shows 134 widiastuti et al. microbiol indones fig 2 the dehydrogenase activity of each treatment tested. fig 3 the pcr-dgge result. lane 1: compost; 2: tr-t0-mo; 3: h-t0-se; 4: empty; 5: m-t1-mo; 6: tr-t1mo; 7: m-t0-mo; 8: tr-t0-se; 9: h-t0-mo; 10: tr-t1-se; 11: h-t1-se; 12: m-t0-se; 13: m-t1-se; 14: h-t1-mo. fig 4 phylogenetic tree construct of microbes community from each sample based on nif gene. volume 14, 2020 microbiol indones 135 table 5 yield and purity of dna extracted from soil sample block sample concentration (ng/µl) od (260/280) compost 42.4 1.92 f15 tr-t0-mo 60.3 1.86 m-t0-mo 101.1 1.88 h-t0-mo 90.7 1.86 l3 tr-t0-se 43 1.81 m-t0-se 32 1.9 h-t0-se 48.1 1.84 l5 tr-t1-mo 42.9 1.88 m-t1-mo 46.8 1.84 h-t1-mo 32.5 1.89 l9 tr-t1-se 48.4 1.87 m-t1-se 47.1 1.88 h-t1-se 31.4 1.92 treatment code before after % increase n (%) p (%) k (%) n (%) p (%) k (%) n p k t0-mo 0.74 0.083 0.067 2.91 0.205 0.74 293 147 1004 t0-se 0.65 0.162 0.038 2.86 0.19 0.75 338 19 1874 t1-mo 1.68 0.023 0.202 3.21 0.182 1.503 91 691 644 t1-se 1.61 0.017 0.100 3.27 0.2 1.443 103 1080 1326 table 6 the nutrient status of leaves before and after trenching construction in each treatment fig 5 genetic distance of microbes species from each sample based on nif gene. 136 widiastuti et al. microbiol indones no consistent effect of dehydrogenase activity. it may take longer time on this location with severe level of ganoderma attack to restore microbial activity. in fact, the data of biology and chemical characteristic before trench construction shows that the soil sample which has a severe level of ganoderma attack already have higher nitrogen content, total fungi, and bacteria compare to the location with moderate ganoderma attack. this might lead to the hypothesis that over-adding nutrition, in this case, trench development, to the soil that already has high nutrition could reduce the soil performance or might be reaching the saturated level of nutrition in the soil. the dehydrogenase is one of soil enzyme that becomes the indicator of ecosystem status. soil enzyme acts as mediators and catalysts of important soil function, involving organic inputs decomposition, the transformation of soil organic native, releasing the inorganic nutrient, n fixation, nitrification, 2 genitrification, and xenobiotics detoxification (dick 1997). dehydrogenase is an enzyme that transfers protons and electrons from substrates to acceptors to oxidize organic soil matter. this enzyme is known as an essential part of intact cells but does not remain in the soil (das and varma 2011). it is known that the dehydrogenase enzyme will occur if only the soil bacteria exist in the sample. pseudomonas genus observed abundantly in the soil where dehydrogenase activity is high (walls-thumma 2000). the correlation between dehydrogenase activity and ganoderma sp. level in soil could be explained by the fact that the abundance of pseudomonas sp. in the soil has inhibition activity to the ganoderma sp. (bivi et al. 2010). by coupling pcr amplification of taxonomic targets with sequence dissimilarities examined by denaturing gradient gel electrophoresis (dgge), molecular fingerprints of variety can be obtained by splitting dna fragments of equal length but of separate sequences (muyzer et al. 1993; muyzer et al. 1996), resulting in a dgge fingerprint that is unique to the sample location. a pcr-dgge is able to detect up to 95-99% of the bacterial population, and many candidate sequences can be used as genetic biomarkers, including variable regions within the 16s ribosomal dna gene (tsen et al. 1998), the 23s ribosomal dna gene, and the rpob gene (encoding the rna polymerase β subunit) (dahllof et al. 2000). the dgge analysis divides the microbial community based on the nif gene existence into two clusters. we hypothesize that if the nif gene playing role in the nitrogen fixation in the soil, then the bacteria community will be different from one another, especially our sampling area comprising various treatment group. the cluster i consist of microbial communities which found in the moderate level of ganoderma location. in contrast, cluster ii involving microbial found in the severe effect of ganoderma sp. the data shows that the microbial community in the soil with moderate and severe ganoderma attack have its microbial type. hypothetically, specific microbial communities have the ability to fight against the ganoderma with different intensity levels. interestingly, the microbial in the trenching location has a similar characteristic to the frond stack zone. this might correlate with sun exposure in the frond stack zone that could enrich the microbial population. previous studies found that the nif genes encoding active nitrogenase can be passed to non-nitrogen-fixing prokaryotes to allow the removal of ambient nitrogen gas in ammonia as a supply of nitrogen (temme et al. 2012; setten et al. 2013; wang et al. 2013; han et al. 2015). the nif gene detected by the dgge might contain culture and non-cultured bacteria. this leads to the hypothesis that the specific bacteria in each sampling location might not be cultured as reported in hadianta et al. (2014). however, we still unsure about the uncultured bacteria nif that we have identified. further study needed to be done to confirm this statement. the development of trenching in four different locations shows a significant effect related to the abundance of microbes and fungi population, supported by the enhancing of dehydrogenase activity in the location with a moderate level of ganoderma attack. also, the dgge analysis successfully clustering the microbial community by two clusters from four different locations, dividing the classification based on the ganoderma level attack. the leaf nutrient status showed that trench could induce enhancement of leaf nutrient especially for p, in contrast to n and k leaf content. the increasing of ganoderma sp attacks followed by an increase in leaf n and k nutrient status. references bivi mr, farhana ms, khairulmazmi a, idris a. 2010. control of ganoderma boninense: a causal agent of basal stem rot disease in oil palm with endophyte bacteria in vitro. int j agric biol. 12(6):833-839. craft c. 2016. creating and restoring wetlands: from theory to practice. elsevier. usa. das sk, varma a. 2011. role of enzymes in maintaining volume 14, 2020 microbiol indones 137 soil health. in: shukla, g., varma, a. (eds.) soil enzymology, soil biology 22, springer-verlag. berlin heidelberg usa. dick rp. 1997. soil enzyme activities as integrative indicators of soil health. in: pankhurst ce, doube bm, gupta vvsr (eds) biological indicators of soil health. cabi, wellingford, 121-156. hadianta ri, rusmana nr, mubarik. 2014. diversity of nitrogen fixing bacteria based on nifh gene in rice fields. adv. environ. biol. 8(14): 63-69 han y, lu n, chen q, zhan y, liu w, lu w, zhu b, lin m, yang z, yan y. 2015. interspecies transfer and regulation of pseudomonas stutzeri a1501 nitrogen fixation island in escherichia coli. j. microbiol. biotechnol. 25: 1339–1348. doi:10.4014/jmb.1502. 02027 haynes rj. 2008. soil organic matter quality and the size and activity of the microbial biomass: their significance to the quality of agricultural soils. in: huang q., huang p.m., violante a. (eds) soil mineral microbe-organic interactions springer. berlin. heidelberg. pp 201–231 hedin lo, brookshire ej, menge dn, barron ar. 2009. the nitrogen paradox in tropical forest ecosystems. annu. rev. ecol. evol. syst. 40:613–635. doi: 10.1146/annurev.ecolsys.37.091305.110246. huan lk, wahidin u. 2009. ganoderma basal and middle stem rot and its management on first generation oil palms planted on peat. proceedings of agriculture, biotechnology & sustainability conference : pipoc 2009, international palm oil congress : palm oil : balancing ecologics with economics: kuala lumpur (malaysia), 9-12 nov, 2009, p. 562-581 komariah, prihartini s, suryadi me. 1993. microorganism activity on peatland reclamation in: microorganism activity on peatland reclamation. proceedings of technical meeting on soil fertility and production. research centre for soil and agroclimate. bogor. pp. 105-113. (in indonesian) nurani d, parmiyanti s, purwanta h, angkoso g, koesnandar. 2007. increase ph of peatsoil by microbial treatment. international symposium and workshop on tropical peatland. yogyakarta, 27-31 august 2007. poole rk, hill s 1997. respiratory protection of nitrogenase activity in azotobacter vinelandii roles of the terminal oxidases. biosci rep. 17(3):303-17. rawat j, jyoti s and pankaj s. 2018. biochar: a sustainable approach for improving plant growth and soil properties. intech open. doi: 10.5772/intechopen.82151 hadianta r,rusmana i, nisa r. mubarik. 2014. diversity of nitrogen fixing bacteria based on nifh gene in rice fields. advanced in environmental biology 8(14), 6369. scotti r, bonanomi g, scelza r, zoina a, rao ma. 2015. organic amendments as sustainable tool to recovery fertility in intensive agricultural systems. j. soil sci. plant nutr. 15(2): 333-352. doi:10.4067/s071895162015005000031 setten l, soto g, mozzicafreddo m, fox ar, lisi c, cuccioloni m, angeletti m, pagano e., díaz-paleo a, ayub nd. 2013. engineering pseudomonas protegens pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. plos one. 8(5):e63666. doi:10.1371/ journal.pone.0063666 temme k, zhao d. & voigt c. a. 2012. refactoring the nitrogen fixation gene cluster from klebsiella oxytoca. proc natl acad sci usa. 109: 7085–7090. doi:10.1073/pnas.1120788109 vitousek pm, menge dn, reed sc, cleveland cc. 2013. biological nitrogen fixation: rates, patterns and ecological controls in terrestrial ecosystems. philos trans r soc lond b biol sci. 368(1621): 20130119. wahyunto rs, nugroho k, sukarman hc & tafakresnanto, c. 2011. peta lahan gambut indonesia skala 1:250.000 (indonesian peatland map at the scale 1:250,000). indonesian center for agricultural land resources research and development, bogor, indonesia walls-thumma d. 2000. dehydrogenase activity in soil bacteria. http://www.gardenguides.com/130633dehydrogenase-activity-soil-bacteria.html accessed on july 25, 2020. wang l, zhang l, liu z, zhao d, liu x, zhang b, xie j, hong y, li p, che, s, dixon r, & li j. 2013. a minimal nitrogen fixation gene cluster from paenibacillus sp. wly78 enables expression of active nitrogenase in escherichia coli. plos genet. 9:e1003865. doi: 10.1371/journal.pgen. 1003865 yusnaini, s. 2009. keberadaan mikoriza vesikular arbuskular pada pertanaman jagung yang diberi pupuk organik dan inorganik jangka panjang. j. tanah trop. 14(3): 253-260 g. muyzer, e. c. de waal, and a. g. uitterlinden, “profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna,” applied and environmental microbiology, vol. 59, no. 3, pp. 695–700, 1993. g. muyzer, s. hottentrager, a. teske, and c. wawer, “denaturing gradient gel electrophoresis of pcramplified 16s rdna—a new molecular approach to analyse the genetic diversity of mixed microbial communities,” in molecular microbial ecology manual, g. a. kowalchuk, f. j. bruijn, i. m. head, a. d. akkermans, and j. d. van elsas, eds., pp. 1–23, 138 widiastuti et al. microbiol indones kluwer academic, nowell, mass, usa, 1996. h. y. tsen, c. k. lin, and w. r. chi, “development and use of 16s rrna gene targeted pcr primers for the identification of escherichia coli cells in water,” journal of applied microbiology, vol. 85, no. 3, pp. 554–560, 1998. i. dahllöf, h. baillie, and s. kjelleberg, “rpob-based microbial community analysis avoids limitations inherent in 16s rrna gene intraspecies heterogeneity,” applied and environmental microbiology, vol. 66, no. 8, pp. 3376–3380, 2000. volume 14, 2020 microbiol indones 139 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 2.mi655-oslan jumadi atmosphere. analysis of gas trapped in polar ice shows that in the pre-industrial era it was only ca. 0.7 µmol -1 mol (ippc 2007b). ch is an important component of the global 4 carbon cycle and contributes to enhanced greenhouse effect as well as n o. paddy cultivation is considered 2 as one of the main human-related sources of ch , and 4 has been predicted that ch emission from paddy 4 cultivation will increase as rice production increase by -1 -1 65 % from end 1990s of 460 mt yr to 760 mt yr in 2020 (neue 1997). ch emission from paddy 4 -1 cultivation contributes about 20 to 150 tg ch yr to 4 the global budget (crutzen 1995). larger variation in the magnitude of ch emission from that source is 4 attributed to soil parameters (ph, redox potential, organic matter content, temperature, clay) or agronomic condition (rice cultivars and farming practices) (fumoto et al. 2008). the magnitude of ch emission from paddy field 4 reflects the balance between methanogenesis and methanotrophy. ch production is the final process in 4 the anoxic microbial degradation pathway in paddy available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.3.2issn 1978-3477, eissn 2087-8575 vol 6, no 3, september 2012, p 98-106 *corresponding author; phone:+62-411-840610, fax :+62411-841504, e-mail: oslanj@gmail.com global atmospheric concentration of methane (ch ) and nitrous oxide (n o) have increased due to 4 2 natural process, biological processes and also by anthropogenic activities. ch concentration in the 4 atmosphere has increased from early 90s of about 1732 ppb to 1774 ppb in the 2005, and that concentration has increased up to 40% since pre-industrial time, while the global atmospheric concentration of n o increased 2 from 270 ppb in pre-industrial time to about 319 ppb in 2005 (ippc 2007a). more than one third of all ch emissions come 4 from soils, as a result of the microbial breakdown of organic compounds in strictly anaerobic conditions. this process occurs in natural wetlands, in flooded rice fields, and in landfills rich in organic matter, as well as in the gut of some species of soil dwelling termites. the rice field and landfill sources make up an important part of anthropogenic methane emissions, which have led to the more than doubled concentration in the the potential of methane and nitrous oxide productions and community structure of methanogenic archaea has been characterized by incubation studies and pcr-dgge approaches, respectively. the results showed that addition of straw into the soil was the most important factor that influenced ch production in this incubation 4 experiment, while n o production was observed higher in soil without straw amendment under saturated 2 condition. most of the clone sequences revealed from dgge band was associated with a archaea lineage, methanocellales ord. nov (rice cluster i). other sequences belong to methanomicrobiales, methanosaetales, and rice cluster 2 (rc-2). the study also showed that the hydrogenotrophic methanogens were the dominant members in methanogenic archaea community obtained from tropical paddy field soil, while acetoclastic methanogens present in relatively minor quantity in the soil. key words: ch , methanogenic archaea, n o, paddy soil4 2 telah dilakukan karakterisasi struktur komunitas archaea metanogenik dari tanah persawahan dengan metode pcr-dgge serta potensinya dalam menghasilkan gas metana dan nitrous oksida secara teknik inkubasi. hasil memperlihatkan bahwa pemberian jerami merupakan faktor yang sangat mempengaruhi dalam produksi gas metana, sementara produksi gas nitrous oksida tertinggi pada tanah berkondisi air jenuh tanpa jerami. secara umum klon sekuen dari dgge band teraffiliasi dengan archaea methanocellales ord. nov (rice cluster i). sekuen lainya adalah methanomicrobiales, methanosaetales, dan rice cluster 2 (rc-2). studi ini juga menunjukkan bahwa kelompok metanogen hidrogenotrofik adalah struktur komunitas archaea metanogen yang dominan, sedangkan komunitas metanogen asetoklastik merupakan struktur komunitas yang minor pada tanah persawahan di maros, sulawesi selatan. kata kunci: archaea metanogen, ch , n o, tanah sawah4 2 methane and nitrous oxide productions and community structure of methanogenic archaea in paddy soil of south sulawesi, indonesia 1 2 oslan jumadi * and kazuyuki inubushi 1 department of biology, faculty of mathematics and natural science, universitas negeri makassar, jalan daeng tata raya, parangtambung, makassar, south sulawesi 90224, indonesia; 2 graduate school of horticulture, chiba university, matsudo, chiba 271-8510, japan field soil, while, methane oxidation occurs at anaerobic-aerobic interface with available oxygen (o ) 2 and ch concentration. the quantity of ch actually 4 4 emitted depends on both production and oxidation of ch . therefore, basic knowledge on the underlying 4 processes is required when designing strategies to mitigate and control ch emission from paddy field 4 (henckel et al. 2000; bodegom et al. 2001). ch is produced by microbial community consisting 4 of various fermenting microorganisms that degrade organic matter and ultimately to acetate, h and co . the 2 2 most actual production of ch is brought about by 4 methanogenic archaea, which either convert acetate to ch and co (acetoclastic methanogenesis) or convert h 4 2 2 plus co to ch (hydrogenotrophic methanogenesis) 2 4 (conrad et al. 2006; sakai et al. 2008). in paddy field soil, the taxonomy of methanogenic archaea members consisted of methanosaetaceae and methanosarcinaceae as acetoclastic methanogens type. while, the type of hydrogenotrophic methanogens are including members of methanomicrobiales, methanobacteriales, and rice cluster1 (rc-i) lineage with new name methanocellales ord.nov (sakai et al. 2007 and 2008) . not many studies have been conducted to measure the potential of ch and n o productions and 4 2 characterize the responsible microbe of methanogenic archaea from tropical paddy soil. therefore, the current paper showed the potential of ch and n o productions 4 2 and methanogenic archaea structure from paddy soil in south sulawesi, indonesia that approached by incubation experiments and pcr-dgge, respectively. materials and methods soil sampling and analysis descriptions. soil was sampled in maros district, south sulawesi province, indonesia (5m asl., 05°00.419` s 119°31.219` e), which is a rice-growing area (6513 ha). soil was classified as typic haplusterts according to u.s. soil taxonomy, 1998. the soil was alluvial type according to center for soil and agro-climate research, indonesia, 2000. soil samples were taken from each micro-plot either before transplanting or after plowing at a depth of 0-15 cm (plowed layer) and sieved as moist condition through 2 mm mesh and stored at 4 °c until soils were used in laboratory incubation experiment. the soil had a ph (h o) of 7.22, 2 -1 -1 ec of 0.17 ds m , total carbon content of 22.5 g-c kg , -1 total n content of 2.30 g-n kg dry soil, and c/n ratio of 9.78, measured by electrode methods and a c/n analyzer (mt 700, yanaco, kyoto, japan), respectively. determination of potential production of ch 4 and n o. the greenhouse gas production potential 2 (ch and n o) of paddy field soil sample were 4 2 determined by incubation method in the laboratory. a composite portion of 100 grams soil were pre incubated at 30 °c for seven days in sealed 1000 ml glass bottle for pre-incubation purpose. the soil samples were then added with inorganic fertilizer -1 (urea) at a rate of 120 mg-n kg . then, these samples were set as two factorial design with 3 replications, and the first factor with organic amendments (with 1.0 g of rice straw or without), and the second factor was water regime (ws=water saturated, wss=water saturated plus straw). all soil samples were incubated at 30 °c and gas in the headspace of each bottle were taken every five days interval to quantify the concentrations of ch and n o. the concentrations of 4 2 ch and n o in the samples were quantified using gas 4 2 chromatographs (shimadzu, gc 14b, kyoto, japan) equipped with a flame ionization detector (fid) and an electron capture detector (ecd), respectively. after gases were sampled the bottle stopper were opened for 30 min to change the air in side the bottle and soils were incubated continually until 35 d. means and standard deviations of the data were calculated. means were compared using the least significant differences (lsd=0.05) value by spss software (ver.11.0 for windows, spss inc., chicago, usa). dna extraction and polymerase chain reaction (pcr) amplification. at the end of incubation (35 d), soil from surface (0.5 cm) and subsurface (1.0 cm below surface) of each treatment was collected carefully and subjected for extraction of dna from soil. soil dna was extracted using a fastdna spin kit for soil (bio 101, inc. vista, ca, usa) following the manufacturer's instructions. in this procedure, cell lyses was performed by vigorous shaking in a mini-beadbeater (biospec product, wakenyaku, co., tokyo, japan) with intense speed of 4.8 for 30 sec. dna extracts were stored at -20 °c before used as a template for subsequent pcr reaction. one µl of dna extract was used as template for pcr amplification with primers 0357f (5`-ccctac ggggcgcagcag-3`; escherichia coli position 340-357) and 0691r (5`-ggattacargatttcac3`; e.coli position 707-691) (watanabe et al. 2004, 2006 and 2007). the primer pair was used for pcr amplification of 16s rrna gene fragment of methanogenic archaea. pcr amplification was performed in a total volume of 50 µl reaction mixture volume 6, 2012 microbiol indones 99 2+ containing 5 µl of 10x ex taq buffer (20 mm mg ), 4 µl of dntp mixture with final concentration of 2.5 mm of each, 0.5 µl of each primers (80 pmol µl) and -1 0.3 µl of ex taq dna polymerase (5 u µl , a hot start version) and made to volume using a sterile distilled water (otsuka pharm., japan). ex taq dna polymerase, dntp mixture, and 10x ex taq buffer were purchased from takara bio inc. shiga, japan. the pcr reaction was run using a dna thermocycler (takara bio inc. model tp600, japan). the thermal profile regime was a hot start at 94 °c for 3 min (denaturation) and 35 cycles at 94 °c for 1 min, followed by 53 °c for 1 min, and then 72 °c for 2 min. a final extension step was at 72 °c for 5 min. the quality of the pcr products were tested by running 5μl of it on a 2% agarose for 30 min at 100 v in 1 x tae and visualized by uv transillumination (atto printgraph, model ae-6932) after staining with ethydium bromide for 30 min and washing with distilled water. denaturing gradient gel electrophoresis (dgge), sequencing, and phylogenetic analysis. dgge analysis was performed on the d-code system (bio-rad, hercules, calif.). pcr products were applied directly into 7% (wt/vol) polyacrylamide gels in 1x tae (40 mm tris base (ph 7.2), 20 mm acetic acid, 0.5 m edta 1 mm) with 35-60% denaturing gradient (urea and formamide). one hundred percent denaturing acrylamide was defined as 7 m urea and 40% formamide. a top gel without denaturant was cast above the denaturing gel before the polymerization started. 25 µl of pcr products were loaded into each lane and run at 60 v for 14 hours at constant temperature of 60 °c and visualized after staining with ethydium bromide for 30 min and washed twice with distilled water. prominent dgge bands were excised from the gel with a sterile razor blade and placed in 1.5 ml plastic tube with 50 µl of sterile distilled water and vortex gently for 5 seconds and then incubated overnight at 4 ˚c. after centrifugation at 2000 rpm for 10 seconds, 1 µl of liquid was used as template for pcr reactions performed under the conditions described above and then separated on dgge again after only a single band appeared. the single band was excised as mentioned above, and then subjected as template for pcr reactions performed under the conditions described before without gc clamp on forward prime. the quality of dna recovered from gel acrylamide after pcr reactions were determined on agarose gel electrophoresis as explained above. the remaining pcr products were purified using tm suprex (takara bio. inc. shiga, japan) according to manufacturer's instructions. one µl of purified pcr product was used as a template for cycle sequencing reaction performed with a dna sequencing kit tm bigdye terminator v3.0 (applied biosystem, foster city, ca, usa) with forward and reserve primers run separately. the sequencing reaction was performed for 30 cycles at 95 °c for 30 sec (denaturation), at 60 °c for 30 sec (annealing) and at 75 °c for 95 sec (extension). prior to dna sequence analyses with an abi 3100 genetic analyzer (applied biosytems, usa) the reaction products were purified with centri-sep columns (princeton separation, nj) following manufacturer's instructions. phylogenetic relationships of the aligned sequences were inferred using the neighbor-joining (saitou and nei 1987). a bootstrap analysis with 1000 replicates was made for all database sets to evaluate the stability of phylogeny (felsenstein 1985). the evolutionary distances were computed using the kimura 2-parameter method (kimura 1980). phylogenetic analyses were conducted in mega 4 (tamura et al. 2007). the nucleotide sequences in this study have been deposited in the dna databank of japan (ddbj, url: ) under accession no. ab 440226 through ab440235. results change in production of greenhouse gas (ch and 4 n o) during soil incubation is shown in fig 1a and b, 2 respectively. the production of ch with straw 4 amendment under flooded condition was significantly higher than the other treatments (fig 1a). the straw amended soil under flooded condition had produced -1 -1 20.6 µg ch -c g dry soil after 5 days of incubation 4 and its almost ten times higher compared with water -1 -1 saturated (2.6 µg ch -c g dry soil ) and peaked on 10 4 -1 -1 day incubation (103 µg ch -c g dry soil ), then 4 gradually declined thereafter. meanwhile, ch4 productions in the other treatments were almost negligible. on the other hand, the n o production was 2 observed higher in soil without straw amendment and under unflooded water condition at 5 d incubation compared to the other treatments, and then rapidly decreased on 10 d incubation. after 15 d of incubation small amount of n o was produced and then decline 2 after the end of incubation (fig 1b). soil incubated with straw amendment and under saturated water http://www.ddbj.nig.ac.jp/ 100 jumadi microbiol indones acetoclastic methanogenesis (methanosaetales and methanosarcinales) was as low as 10 %. discussion the production of ch during incubation was 4 much higher in flooded condition with straw amendment due to the pathway of ch formation. this 4 ch production pattern is a typical ch kinetics. this is 4 4 similar to the results found in comparable experimental systems under continuous production for several approaches with a primer set (0357f-gc/0691r). the pair primer is suitable for investigating methanogenic archaeal community in paddy field soil. extraction of the soil dna revealed that from soils incubated after 35 d at different layer indicated fine reproducibility of the dgge banding patterns (fig 2). the dgge band showed that no apparent change of archaea community structure in all soil treated, even in the soil amended with straw which had more than 30 times higher ch4 production potential (fig 1a). most clones were, revealed from the dgge band, condition produced small amount of n o at 5 d of 2 incubation and then declined thereafter. the other treatments were almost not producing n o during the 2 incubation period. the community structure of methanogenic archaea in paddy field soil in tropical region such as indonesia were investigated with pcr-dgge method affiliated with a clone lineage of archaea, rice cluster i (rc-i). other sequences belong to methanomicrobiales, methanosaetales and rice cluster 2 (rc-2) (fig 3). all paddy field soil incubation exhibited a high frequency (70%) of sequences characteristic of hydrogenotrophic m e t h a n o g e n s ( m e t h a n o c e l l a l e s ( r c i ) a n d methanomicrobiales), whereas the frequency of 0.0 0.5 1.0 1.5 2.0 2.5 3.0 5 10 15 20 25 30 35 b incubation time (day) n o 2 -1 p ro d u ct io n  g -n g d ry s o il a c h 4 -1 p ro d u ct io n  g -c g d ry s o il 0 30 60 90 120 0 3 6 5 10 15 20 25 30 35 control unflooded control flooded straw+unflooded straw+flooded fig 1 change in production of (a) ch and (b) n o during incubation of soil samples. the error bars indicate the standard 4 2 deviation of the results. volume 6, 2012 microbiol indones 101 total ch production, respectively (conrad et al. 2002). 4 chin et al. (2003) suggested that ch was initially 4 produced from reduction of co resulting in the 2 accumulation of acetate and at this stage the relative abundance of acetoclastic methanogens was low. later on, the production of ch was originated from acetate 4 by 40% with relative increase of population of methanosarcina spp. lueders and freidrich (2000) observed that the population structure of methanogenic archaea remained remarkably constant over time after flooding in the paddy field soil even though they observed a shift in the activity of the methanosarcina-like populations. similar observation by watanabe et al. (2006) showed that the community structure of methanogenic archaea in japanese paddy field soil did not change throughout a year even at the mid-season drainage and upland cropping time. however, sugano et al. (2005) suggested that methanogenic archaea communities involved in the decomposition of rice straw under flooded condition were affected by mid-season drainage irrespective of plant part on the rice straw (leaf sheath or leaf blade). in addition, using different sources of soil samples, nicol et al. (2003) showed a change of the methanogenic archaea community in the managed grassland rhizosphere soil compared to the natural one after incubated for 28 d in anaerobic condition. weeks as described previously (frenzel et al. 1996; yagi et al. 1997; inubushi et al. 1992 and 2002). flooded condition allows the soil environment to become anoxic, which is the required environmental condition for the methanogens to produce ch by 4 converting substrate acetate or h plus co .2 2 acetate or h plus co are sub-products of various 2 2 fermenting microorganisms that degrade straw. therefore, the ch production was not detected in soil 4 incubated without straw even under flooded condition, while small amount of ch production occurred during 4 the early stages incubation in saturated soil condition with straw amendment, which then decreased to almost nil value at 35 d (fig 1a). hence, the incorporation of straw into the soil was the most important factor that influenced ch production in this incubation 4 experiment. conrad and klose (2006) reported that the addition of straw enhanced the intermediate production of h 2 and acetate that served as the methanogenic substrates. also some fatty acid was determined increased transiently upon straw addition. they also suggested that the copy number of bacteria and archaea 16s rrna genes increased at the end of their experiment. in addition, under steady state condition, cellulose would methanogenically be degraded to 50% co + 2 50% ch , while methanogenesis from acetate and h 4 2 plus co theoretically contribute >67% and <33% to 2 fig 2 denaturing gradient gel elecrophoresis (dgge) band patterns of pcr products with 0357f-gc and 0691r primer. lane 1-1: without straw saturated-surface soil; lane 1-2: without straw saturated-subsurface soil; lane 2-1: without straw flooded-surface soil; lane 2-2: without straw saturated-subsurface soil; lane 3-1: straw saturated-surface soil; lane 3-2: straw saturated-subsurface soil; lane 4-1: straw flooded -surface soil; lane 4-2: straw flooded -subsurface soil. 1-1 1-2 2-1 2-2 3-1 3-2 4-1 4-2 102 jumadi microbiol indones we determined a few changes of bacteria community structure by pcr-dgge using pairs of universal bacteria primers of 341f/534r (data not shown). the explanation of these results might be due to the soil chemical properties during incubation that have the present work indicated that soil incubated at flooded or saturated condition with and without straw did not alter the community structure of methanogenic archaea at different layers of soil sampled. but, in another work that used the same soil dna extraction, m.archaeon sanae (ab196288) rc1 (af225686) rc1 (af225577) band 1(ab440226) clone soil 113a 20 (ay487185) rice soil clone as17-02 (af225665) rc1 (af225672) m.archaeon et1-2 (aj244278) rc1 (af225675) rc1 (af225658) rc1 (aj227921) band 4(ab440229) band 5(ab440230) band 3(ab440228) band 8(ab440233) band 9(ab440234) soyang 1af-1100ar (af056361) methanospirillum hungatei (m60880) band 6(ab440231) ch4micro (af225674) ch4micro (af225642) methanoculleus palmolei (y16382) methanoculleus bourgensis (ab065298) methanoculleus chikugoensis (ab038795) methanoculleus thermophilus (ab065297) methanomicrobium mobile (m59142) methanocorpusculum parvum (m59147) methanogenium cariaci (m59130) methanoplanus limicola (m59143) rc3 (aj227929) rc3 (af225643) rc3 (aj227959) band 2(ab440227) band 10(ab440235) rc2 t 0357f/0691r(ab199852) rc2 (af225582) rc2 etoh (y18069)6 rc2 (aj227931) methanohalophilus mahii (m59133) as08-34 (af225663) as01-17 (af225619) methanosarcina mazei (ab065295) as00-27 (af225596) band 7 (ab440232) as08-32(af225661) methanothrix thermophila (m59141) s15-24(af236505) rot18 (y18091) s15-8 (aj236489) methanosaeta concilii (m59146) as17-20 (af225680) abs11 (y15391) as00-17(af225587) 70 100 44 37 39 99 52 75 88 80 76 36 9 13 47 71 61 68 60 57 55 55 37 21 24 20 25 51 44 44 44 43 39 36 13 17 21 15 41 40 32 23 0.02 rice cluster i = methanocellales methanomicrobiales methanosaeta rice cluster 2 methanosarcinales rice cluster 3 fig 3 phylogenetic tree based on partial nucleotide acid retrieved from the dgge bands. bands obtained from this study are marked with triangle shape ( ). values in the figures are bootstrap values, and branching corresponding to partitions reproduced in less than 50% bootstrap replicated are collapsed. the scale of bar indicates 2 changes per 100 nucleotide acid. volume 6, 2012 microbiol indones 103 favorable environment for the methanogenic archaea communities to survive under all the treatments used. meanwhile, the fermenting microorganisms (dominantly bacteria) might have a shift of population during the straw degradation. another possibility was the incubation time was not long enough to induce the appearance of other methanogenic population particularly acetoclastic methanogenesis type as suggested by kruger et al. (2005). the methanogenic archaea is also known to tolerate higher oxygen and have some enzymes to detoxify oxygen-related toxic compounds, hence, methanogens are proposed to be more resistant to o 2 (lueders and friedrich 2000; erkel et al. 2006). the study also suggested that the production of ch by 4 methanogenic archaea seems not correlated with the band patterns but mainly due to the soil environments that enhanced methanogenesis such as in soil-flooded with straw amendments. similar to other reports, this study showed that hydrogenotrophic methanogens were dominant members in methanogenic archaea community retrieved from temperate paddy field soil (lueders and friedrich 2000; sugano et al. 2005; watanabe et al. 2006), while acetoclastic methanogens were relatively minor in abundance in the paddy field soil. we speculated that the members of community structure of hydrogenotrophic methanogens, methanocellales (rc-i), was dominant in the soil used that took from the tropic region, of indonesia. the methanocellales (rc-i) lineage has been found in almost every paddy field soil samples, particularly on rice roots and also detected dominant appeared in the prolonged incubation at 50 °c (chin et al. 2003; conrad et al. 2006; wu et al. 2006). in addition, due to the capacities and a unique set of antioxidant enzyme and dna repair, methanocellales should have a competitive superiority over the other hydrogenotrophic methanogens in rice rhizosphere and other methanogenic environments with oxic episodes, such as boreal peat lands and tropic soils (erkel et al. 2006). more over ramakrishnan et al. (2001) also reported that methanocellales or rc-i occurred in such soils from various geographic regions. in addition, lu and conrad (2006) reported that methanocellales (rci) were found to be highly active and play key role in ch production from plant-derived carbon and, 4 therefore, the group was responsible for microbial ch 4 emission from paddy field (erkel et al. 2006). methanocellales (rc-i) has been identified as a different genotypic group from the orders of methanosarcinales and methanomicrobiales due to low sequence similarity value with those of the two aforementioned orders (großkopf et al. 1998). the physiological properties of this members is also not similar with members of the methanosarcinales and methanomicrobiales, which utilize acetate and simple methylated compound, while this member of methanocellales (rc-i) use h plus co to produce 2 2 ch (conrad 1996; erkel et al. 2006; sakai et al. 2008).4 two bands (band 2 and 10) were revealed as rice cluster 2 (rc-2). the rc-2 were placed between the orders methanosarcinales and methanomicrobiales (fig 3), where it has also been reported by großkopf et al. (1998) based on the level of rdna similarity to these o r d e r s . o n l y 1 b a n d o f e a c h m e m b e r o f methanosaetales and methanomicrobiales was retrieved from dgge as band 7 and band 6, respectively, while no member of methanosarcinales or rice cluster 3 were identified from dgge band (fig 2), großkopf et al. (1998) and fey and conrad (2000) reported that methanosaetales was hardly detected in roots but commonly appeared in anoxic bulk soil, especially at low acetate concentration. since the paddy field soils used was taken as a bulk soil before transplanting time, it is also possible that member of methanosaetales was detected in soil used. n o gas is emitted in paddy soils via nitrification and 2 denitrification or nitrate accumulated when soils have an aerobic surface layer and anaerobic condition, respectively (akiyama et al. 2005; inubushi et al. 1996; jumadi et al. 2008). the main pathway of n o production 2 from paddy-soil system depends on the soil water status. therefore, current study also shows that n o production 2 in almost neglected monitored even in closed system experiment. in addition, at saturated condition the n o 2 released by denitrification increase with decreasing o 2 partial pressure. therefore, after five days incubation n o 2 production at control flooded was monitored higher and then decrease thereafter substantially to lower concentration. that might be due to the availability of no and reduced o concentration.3 2 the study also showed that n o emissions were 2 suppressed in all the treatments and it could be predicted that n o emission was almost not existent. 2 the field scale studies in the tropical region of indonesia showed that n o was almost not emitted. 2 however, in incubation experiment the n o production 2 by nitrification occurred even in moist soil (50-70% of soil moisture) and it was also influenced by the number of responsible microbe such as ammonium oxidation 104 jumadi microbiol indones fey a, conrad r. 2000. effect of temperature on carbon and electron flow and on the archaea community in methanogenic rice field soil. appl environ microbiol. 66(11): 4790-4797. doi:10.1128/aem.66.11.47904797.2000. felsenstein j. 1985. confidence limits of phylogenies: an approach using bootstrap. evolution 39(4): 783-791. 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working group i to the fourth assessment report of intergovernmental panel on climate change. cambridge: cambridge university press. ipcc. 2007b. technical summary. in: climate change 2007: mitigation. contribution of working group iii to the fourth assessment report of the intergovernmental panel on climate change. cambridge: cambridge university press. jumadi o, hala y, inubushi k. 2005. production and bacteria and nitrite oxidation bacteria (hadi et al. 2008; inubushi et al. 2003; jumadi et al. 2005; jumadi et al. 2008b; jumadi et al. 2012). the study showed that the hydrogenotrophic m e t h a n o g e n s w e r e d o m i n a n t m e m b e r s i n methanogenic archaea community obtained from tropical paddy field soil of south sulawesi indonesia, while, acetoclastic methanogens were relatively minor presence in the soil sample. study also suggested that n o production almost neglected in paddy soil with 2 saturated condition. acknowledgments this study was supported by the ghg-sscp 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conrad r. 2006. diversity and ubiquity of thermophilic methanogenic archaea in temperate anoxic soils. environ microbiol. 37:175186. yagi k, tsuruta h, minami k. 1997. possible option for mitigating methane emission from rice cultivation. nutr cyc agroecos. 49(1):213-220. doi:10.1023/a:1009743909716. 106 jumadi microbiol indones 1: 98 2: 99 3: 100 4: 101 5: 102 6: 103 7: 104 8: 105 9: 106 05. sinaga.cdr vol.14, no.4, december 2020, p 156-162 doi: 10.5454/mi.14.4.5 growth kinetic study of blue-green microalgae arthrospira platensis using buffalo manure as alternative media 1 1 2 brian a. sinaga , lianty simangunsong , andy trirakhmadi , monita 3 1* pasaribu , and merry meryam martgrita 1 bioprocess engineering study program, faculty of biotechnology, institut teknologi del jalan sisingamangaraja, laguboti, toba 22381 north sumatera, indonesia; 2 pt aimtopindo nuansa kimia, kawasan industri de primatera blok b4 no. 6, jalan raya sapan tegalluar, bojongsoang, kabupaten bandung 40287 west java, indonesia; 3 department of mineral chemical engineering, polytechnic ati makassar, jalan sunu no. 220, suangga, talo, makassar 90152 south sulawesi, indonesia. north sumatera is one of the provinces in indonesia with the highest buffalo population, which is responsible for the high accumulation of buffalo manure that can cause environmental and aesthetic problems if left untreated. one of the possible alternatives for solving this issue is by implementing buffalo manure as growth media for microorganisms, e.g. microalgae. in this research, buffalo manure was used as alternative media for arthospira platensis cultivation. buffalo manure was taken from sitoluama village, laguboti, toba regency of north sumatra province. research steps included media and culture preparation, cultivation, sampling, sample analysis, and verification of constructed models and validation. buffalo manure concentration in media is varied from 1 g.l 1 -1 -1 -1 to 8 g.l which is analogous to nitrogen content of 0.002 mg.l to 0.018 mg.l . growth data was used for growth -1 -1 kinetic modelling, which was most satisfactory for monod model (µ = 0.5915 day , k = 0.421 g.l ). max s key words: alternative media, arthrospira platensis, buffalo manure, growth kinetics sumatera utara merupakan salah satu propinsi di indonesia dengan populasi kerbau terbesar, yang menyebabkan tingginya akumulasi kotoran kerbau yang dapat menyebabkan masalah lingkungan dan estetika bila tidak ditangani. salah satu alternatif solusi untuk masalah ini adalah dengan memanfaatkan kotoran kerbau sebagai media pertumbuhan untuk mikroorganisme, misalnya mikroalga. dalam penelitian ini, kotoran kerbau digunakan sebagai media alternatif untuk kultur arthospira platensis. kotoran kerbau diambil dari desa sitoluama, laguboti, kabupaten toba, propinsi sumatera utara. tahapan penelitian meliputi persiapan media, persiapan kultur, kultivasi, mengambilan sampel, analisis sampel, serta verifikasi dan validasi model kinetika. -1 -1 konsentrasi kotoran kerbau dalam media bervariasi dari 1 g.l hingga 8 g.l yang analog dengan kandungan -1 -1 nitrogen 0,002 mg.l hingga 0,018 mg.l . data pertumbuhan digunakan untuk menentukan model kinetika -1 -1 pertumbuhan, yang menunjukkan kesesuaian tertinggi dengan model monod (µ = 0,5915 hari , k = 0,421 g.l ).max s kata kunci: arthrospira platensis, kinetika pertumbuhan, kotoran kerbau, media alternatif microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-87825663415; email: merry.martgrita@del.ac.id (irshad et al. 2013; ghimire et al. 2017). this has been causing the accumulation of manure in many places, especially in rural areas. it can cause terrible odour, carries out diseases, and even worse, river pollution and eutrophication in lakes when the manure is leached to water bodies. despite the problems it poses, buffalo manure possesses high organic matter content, which makes it a sound option as an alternative growth medium for several species of microalgae, especially arthrospira platensis. arthrospira has a high market value due to its potential as food supplement or feed due to its high protein content of 50-70% dry weight (michael, kyewalyanga, and lugomela 2019). commonly, a. platensis grows very well in synthetic media such as zarrouk's media, but due to its considerable material cost, many researchers were driven to discover per 2016, north sumatera was the province in indonesia with third-highest buffalo population, which equals 116,403 (badan pusat statistik 2016). toba district in north sumatera was no exception, with the total buffalo population of 3,681 in the same year (badan pusat statistik kabupaten toba samosir 2013). the high population of buffalo corresponds to the high buffalo manure production, which is still underutilized despite its possibilities for use as organic fertilizer and feedstock for biogas production (karttek et al. 2014). mostly toba district people use buffalo manure as organic fertilizer due to its three highest content needed for plants growth, those are nitrogen (2 g/kg), phosphor (2 g/kg), and potassium (10.1 g/kg) alternative media, such as tofu liquid waste (syaichurrozi and jayanudin 2016), tapioca liquid waste (insan et al. 2018), chicken manure (yilmaz, sezgin, and duru 2015) and cow manure (astiani, dewiyanti, and mellisa 2016). there are other microalgae biomass productions that also use waste as alternative growth media, such as aquaculture wastewater for chlorella minutissima biomass production (hawrot-paw et al. 2019), aquaculture biological waste for ankistrodesmus gracilis (sipaubatavares, florencio, and scardoeli-truzzi 2018), and diluted composting fluids for selenastrum sp. (marika et al. 2017). up to this research, there has been no prior research that discusses the kinetic study of the growth of a. platensis on alternative buffalo manure media. the discovery of the aforementioned kinetic parameters would be fundamental in developing a closed-nutrient cycle between buffalo farming and a. platensis aquaculture. materials and methods culture preparation. arthrospira platensis was cultivated from stock culture into zarrouk's media (michael et al. 2018) for 9 days. during cultivation, culture was provided with 3000-4500 lux light using led lamps and aeration. medium preparation. buffalo manure was collected directly from the cage, not from the manure shelter. all of the buffalo manure can be utilized to reduce the overflow of manure waste according to the purpose of this research and utilize it as substrate in microalgae growth media. buffalo manure was sundried for 1-2 days, cooled and sieved twice using 60 fine mesh. 100 g of this manure were dissolved in 1 l sterilized distilled water and left for 3 days, with -1 addition of 8.5 mg.l sodium metabisulfite on the first -1 day and about 5 g.l sodium bicarbonate on the second day. this mixture was furthermore filtered using filter paper and taken 10-80 ml, each to be diluted with distilled water to 720 ml. variation. the concentration of buffalo manure -1 was varied between 1-8 gl . this variation aimed to collect sufficient data for fitting to various growth kinetic models. cultivation. cultivation of a. platensis was carried out in 1 l erlenmeyer flasks with total liquid volume of 800 ml, consisting of 720 ml prepared buffalo manure medium and 80 ml prepared microalgae culture (10% -1 v/v inoculum with microalgae concentration of 2 g.l ). each flasks were aerated continuously in order to prevent carbon dioxide acting as limiting substrate. in order to ensure the photoautotrophic growth of a. platensis, light was provided by a similar fashion as during culture preparation. these experiments were conducted in two repetitions. sampling and sample analysis. liquid samples of the cultures were taken on daily basis for 10–11 days. each sampling was carried out twice. taken samples were examined for biomass concentration and nitrogen content measurement. biomass concentration was determined by measuring the sample absorbance in 560 nm wavelength and calculating the biomass concentration using the standard curve, which was previously prepared by measuring the absorbance values of liquids with known biomass concentrations. biomass cells were harvested from the sample by centrifugation in 3000 rpm for 15 minutes, followed by o decantation, drying in 60 c oven and dehydration using desiccator. data interpretation. biomass cell concentration data obtained through sample analysis was used to determine the specific growth rate (µ) achieved in the logarithmic phase, which follows the following firstorder rate form: equation (1) -1 x�= cell concentration over time (g.l ) -1 x �=�initial cell concentration (g.l )o t�=�cultivation time (day) -1 µ�=�specific cell growth rate (day ) specific growth rates of a. platensis in varied buffalo manure concentrations were fitted to five conventional growth kinetic models: monod: contois: verhulst: tessier: -1 k � = saturation constant (g.l )s -1 µ � =�maximum specific growth rate (day )max -1 x � =�maximum cell concentration (g.l )max results growth curves of a. platensis cultivated in various buffalo manure concentrations are shown in figure 1 volume 14, 2020 microbiol indones 157 158 sinaga et al. microbiol indones and figure 2, which exhibit no correlation between buffalo manure concentration and final biomass concentration. highest biomass concentration was -1 obtained in 8 g.l . specific growth rates of a. platensis cultivated in various buffalo manure concentrations are shown in figure 3. by linear regression, kinetic parameters of various growth kinetic models of a. platensis were determined and can be seen at table 1. whereas the comparison of µ for a. platensis from max different researches is stated in table 2. discussion figure 1 until figure 8 show that lag phase of a. platensis cell growth is very short and almost unseen. this condition is caused by the phase of inoculum when it was inoculated to buffalo manure alternative media is in the exponential phase. it is supported by maier, pepper and gerba (2009), that if the inoculum condition is in the stationary phase then the lag phase will be clearly seen, because cells need time for adaptation in a new medium condition that physiologically the cell phase will move from stationary phase to exponential phase. other parameters that affect lag phase of a. platensis are ph, light intensity, and temperature. condition of ph, light intensity, and temperature of inoculum medium and buffalo manure alternative media are the same, those are ph 10, light intensity 3-4.5 klux, and temperature 25-27°c. in each concentration of buffalo manure media, in general exponential phase is started at day-0, day-1, or day-2. this condition is proven that lag phase of a. platensis growth occurred in a short period. the research conducted by syaichurozzi and jayanudin (2016) about a. platensis growth in liquid tofu waste media and the research conducted by astiani, dewiyanti and mellisa (2016) about utilization of bovine, birds, and buffalo manure as a. platensis growth media, also give the results that lag phase took place in a very short period that was in day-0. exponential phase is occurred until day-6 or day-7, and after that a. platensis cells enter deceleration phase until death phase at day-10. substrate concentration in buffalo manure media affects specific growth rate of a. platensis cells, as seen on figure 9. to calculate a. platensis specific growth rate, cell concentration data from each points of sampling time of both repetition experiments was averaged, and specific growth rate value of a. platensis cell in the batch system is determined by the equation (1). figure 9 shows the increasing specific growth rate of a. platensis cells in buffalo manure concentration of 1 -1 -1 g.l until 4 g.l , and then the specific growth rate is -1 decrease insignificantly at concentration 5 g.l . the higher the buffalo manure concentration in the media the higher the specific growth rate of a. platensis to the -1 limit concentration of 5 g.l . the decreasing specific growth rate is due to the color of media that was getting -1 darker in the concentration of 5 g.l . the value of specific growth rate in each variation concentration of substrate is used to determine growth kinetic models such as monod, contois, tessier, and verhulst (table 1). parameters determined based on those kinetic models are µ to know the value of max maximum growth rate of a. platensis and k to know the s rate of a. platensis cells to reach the saturation point. in -1 buffalo manure concentration more than 5 g.l the media condition is more murk, therefore in the determination of growth kinetic model of a. platensis, the specific growth rates of a. platensis in concentration -1 media more than 5 g.l was not included. there was a table 1 parameter kinetics for several growth kinetic model table 2 comparison of µ for a. platensismax µmax (day-1) medium reference 0.4 zarrouk’s medium wang, fu and liu (2006) 0.22 modified bristol’s medium sydney et al. (2010) 0.4 swine manure mezzomo et al. (2008) 0.5915 buffalo manure current research kinetics model kinetic parameters 2 r monod -1 µ = 0.5915 daymax -1 k = 0.421 g.ls 0.9775monod -1 µ = 0.5915 daymax -1 k = 0.421 g.ls 0.9775 contois -1 µ = 0.6695 daymax -1 ks = 5.143 g.l 0.4602 tessier -1 µ = 0.417 daymax -1 ks = 16.92 g.l 0.7227 verhulst -1 µ = 0.3924 daymax -1 x = 0.2317 g.lmax 0.6866 -1 fig 1 arhtospira platensis growth curves in buffalo manure concentration 1 g.l . -1 fig 2 arhtospira platensis growth curves in buffalo manure concentration 2 g.l . -1 fig 3 arhtospira platensis growth curves in buffalo manure concentration 3 g.l . -1 fig 4 arhtospira platensis growth curves in buffalo manure concentration 4 g.l . volume 14, 2020 microbiol indones 159 -1 fig 5 arhtospira platensis growth curves in buffalo manure concentration 5 g.l . -1 fig 6 arhtospira platensis growth curves in buffalo manure concentration 6 g.l . -1 fig 7 arhtospira platensis growth curves in buffalo manure concentration 7 g.l . -1 fig 8 arhtospira platensis growth curves in buffalo manure concentration 8 g.l . 160 sinaga et al. microbiol indones growth inhibition in murky media because the light was obstructed therefore the photosynthesis process of a. platensis cell was inhibit (cheunbarn and peerapornpisal 2010). lineweaver-burk linearization is determined by comparing the value and substrate concentration at exponential phase. the equation obtained is used to determine the monod kinetic parameters, those are µmax and ks based on the equation, gradient value of 0.7124 . indicates ks/µ value and intercept value of 1.6905 is max equal to 1/µ , therefore it is obtained the µ value of max max -1 -1 0.5915 day and ks value of 0.421 g.l . contois kinetic model is different from monod's due to both substrate and cell concentration are independent variables. in general, contois model is used to determine the correlation of microbe cell growth rate and the concentration of substrate and cell mass (abdullah et al. 2016). according to the linearization result for contois -1 model, it is obtained µ value of 0.6695 day and ks max -1 value of 5.143 g.l . based on linearization for tessier model, gradient value of 0.0591 indicated 1/ks value and intercept value of 0.8732 is equal to ln µ , therefore max -1 it is obtained the µ value of 0.417 day and ks value of max -1 16.92 g.l . different from monod, contois, and tessier kinetic models, verhulst kinetic model involves kinetic parameters of µ and x whereas x constitutes max max, max maximum dry cell weight. according to linearization for verhulst model, it is obtained gradient value of 0.5905 indicates µ /x value and intercept value of max max 0.3924 that is equal to µ value, therefore the x value max max -1 -1 is 0.2317 g.l and µ value is 0.3924 day . the most max suitable growth kinetic model of a. platensis is chosen 2 based on the correlation coefficient (r ) that is close to one. correlation coefficient is an important value to describe the data accuracy and to consider the accuracy of regression model (abdullah et al. 2016). based on data in table 1, monod kinetic model is the most suitable growth kinetic model for a. platensis because it has the highest correlation coefficient (0.9775) compared to the other kinetic models. the µ value of monod model obtained from max -1 current research (0.5915 day ) is higher compared to µ value obtained by wang, fu and liu (2007) that max observed the effects of light intensity variation to a. platensis growth rate, µ value obtained by sydney et max al. (2010) about a. platensis strain leb-52 growth in modified bristol media, and µ value obtained by max mezzomo et al. (2010) about the cultivation of a. platensis in swine manure, as shown in table 2. due to the existence of inhibition in higher buffalo manure concentrations, it is obvious that the growth of a. platensis in this medium would not satisfy any conventional growth kinetic models. however, growth rates in concentration up to 4 g.l-1 was deemed normal enough for fitting to conventional models. by statistic comparison, it was concluded that monod model fits the experimental growth rates significantly better than other models. monod model is the most fundamental model for biomass growth rate and only suitable for low substrate concentration. however, it is frequently found that this model is relatively representative in many situations, which has been proved in this study. this research confirmed the possibility of using buffalo manure as an alternative growth medium for a. platensis. in buffalo manure -1 concentrations lower than 5 g.l , the growth followed the monod model while in higher concentrations, growth inhibition was detected. it is highly advised that further studies investigate more on the effect of other limiting substrate, such as carbon dioxide. acknowledgments this work was supported by a grant from lppm it del. fig 9 arhtospira platensis growth rates at various buffalo manure concentrations media volume 14, 2020 microbiol indones 161 references abdullah nah, nayan na, amirra nn, kamaludin nhi, idris zm, tompang mf. 2016. cell growth kinetics of aspergillus oryzae in industrial natural rubber effluent serum. arpn journal of engineering and applied sciences, 11(4): 2687-2692. issn 1819-6608. astiani f, dewiyanti i, mellisa s. 2016. pengaruh media kultur yang berbeda terhadap laju pertumbuhan dan biomassa spirulina sp. effect of different culture media on growth rate and biomass of spirulina sp. jurnal ilmiah mahasiswa kelautan perikanan unsyiah, 1(3): 441–447. issn 2527-6395. badan pusat statistik. 2016. populasi kerbau menurut provinsi, 2009-2016. buffalo 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advances 14(5): 125–129. doi: 10.3923/javaa.2015.125.129. 162 sinaga et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 10 andi utama (86-90).pmd generation and characterization of temperature resistant mutant of recombinant pj156/cav-17 virus andi utama1∗ and hiroyuki shimizu2 1research center for biotechnology, lembaga ilmu pengetahuan indonesia, jalan raya bogor km. 46, cibinong 16911, indonesia; 2department of virology ii, national institute of infectious diseases, 4-7-1 gakuen, musashimurayama, tokyo 208-0011, japan previous study revealed that a recombinant virus between poliovirus (isolate pj156) and coxsackie a virus serotype 17 (cav-17), namely pj156/cav-17, was temperature sensitive. it is well known that two amino acids in 3d region (his-73 and ile-362) are determinants for temperature sensitivity of poliovirus, in particular for sabin 1 strain. however, it is not known whether those amino acids affect the temperature sensitivity for other enteroviruses. sequence analysis of 3d region of pj156/cav-17 showed that amino acid in 3d-73 and -362 were tyr and ile, respectively, similar with the sequence of parental cav-17 virus. since amino acid in 3d-73 of pj156/ cav-17 was not his, it is suggested that the temperature sensitivity of the pj156/cav-17 was associated with the ile-362. to confirm this suggestion, the temperature-sensitive escape mutants of pj156/cav-17 were generated by blind passaging at 39.5 oc. the escape mutants were then recovered and plaque purified, and the sequence of 3d region was determined. it was found that the amino acid in 3d-362 was thr, instead of ile. consequently, ile362 was proved to be involved in temperature sensitivity of pj156/cav-17. full sequences of both viruses were also determined and compared. furthermore, the characteristics of the temperature resistant of pj156/cav-17 variant were analyzed. it is confirmed that the recovered pj156/cav-17 virus could grow well at 39.5 oc, and there was strong correlation between temperature sensitivity and attenuation. key words: poliovirus, cav-17, recombinant virus, temperature-sensitive _____________________________________________ _________________ ∗ corresponding author, phone: +62-21-8754587, fax: +62-21-8754588, e-mail: andiutama@yahoo.com it is well known that poliovirus (pv) can recombine with other viruses. in fact, genetic recombinations of pv have been found in excreted viruses, including viruses from vaccine-associated paralytic poliomyelitis (vapp) cases and healthy vaccinees. the recombination does not occurr only among different serotypes of the vaccine strains, but also between vaccine strains and wild type pv (guillot et al. 2000; liu et al. 2000; dahourou et al. 2002). it is generally assumed that natural circulation of vaccine-strain derivatives is strictly limited in time. therefore, such derivatives are believed to be unable to survive in nature long enough to evolve into highly transmissible neurophatogenic variants. however, incidents of paralytic poliomyelitis outbreaks due to circulating vaccine-derived poliovirus (cvdpv) were reported; respectively in the dominican republic and haiti (kew et al. 2002), the philippines (shimizu et al. 2004), in egypt (yang et al. 2003), and in madagascar (rousset et al. 2003). a sequence analysis showed that the above cvdpvs were recombinant viruses between pv and unidentified enterovirus that underwent the recombination in the noncapsid region. moreover, we recently demonstrated that the recombination could occur between pv (isolate pj156) and coxsackie a virus serotype 11 (pj156/cav-11) (utama and shimizu 2005) and cav-17 (pj156/cav-17) (utama and shimizu 2006). the characteristics of resultant recombinant v i r u s e s v a r y, a n d s u g g e s t e d t o b e d e t e r m i n e d b y characteristics of both parental viruses. the recombinant pj156/cav-17 virus was found to be temperature sensitive; the virus could not grow at 39.5 oc (utama and shimizu 2006), similar to previous report (semler et al. 1986). the virus did not exhibit any virulence on the pv receptor-transgenic (tgpvr) mice (utama and shimizu 2006). these suggest the correlation between temperature sensitivity and attenuation of the virus. from the study of pv sabin 1 strain, it is suggested that the temperature sensitive effect associated with 3’-terminal part of the sabin 1 genome are results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the his-73 and ile-362 codons of 3d, which coding rna polymerase, and nucleotide g-7441 of 3’-untranslated region (bouchard et al. 1995; georgescu et al. 1995). since the 3’-terminal part of the pj156/cav-17 was originated from cav-17, the temperature sensitivity of this recombinant virus is supposed to be due to the sequence in 3’-terminal part of cav-17. in this study, temperature resistant mutants were generated in order to determine the sequence contributed to the temperature sensitive (ts) phenotype of the recombinant pj156/cav-17. furthermore, the temperature resistant (tr) pj156/cav-17 virus was characterized, including its neurovirulence to confirm the correlation between temperature sensitivity and attenuation. materials and methods generation of temperature resistant pj156/cav-17. to generate temperature sensitive (ts) escape mutant pj156/ cav-17, the virus was infected to hep-2 cells and cultured at 39.5 oc in dulbecco’s modified eagle’s medium (dmem) supplemented with 2% bovine calf serum (maintenance medium). the cytophatic effect (cpe) was observed for 5 days. if the cpe was negative, the virus was then blind passaged until the virus develops complete cpe. afterward, the virus was recovered and plaque purified. the temperature resistant (tr) phenotype was finally confirmed by culturing the virus at 39.5 oc in the maintenance medium. microbiology indonesia, august 2007, p 86-90 volume 1, number 2 issn 1978-3477 plaque purification of temperature resistant pj156/ cav-17. plaque purification was performed on an hep-2 cell monolayer as described previously (arita et al. 2005; utama and shimizu 2005; utama and shimizu 2006). a tenfold serial dilution of viruses prepared in the maintenance medium were inoculated in hep-2 cells using 6-well plates, and incubated at 35.0 oc for 30 min. the cells were covered with 2 ml of 0.5% agarose-me in dmem with 5% bovine calf serum. after incubation at 35.0 oc for 3 days, plates were stained with 2 ml of 0.5% neutral-red in maintenance medium containing 0.5% agarose-me. plaque size was measured, and plaque numbers were calculated after incubation at 35.0ºc for a further day. identification of determinant sequence associated with the temperature sensitivity. to identify the amino acids responsible for temperature sensitivity, the viral rnas were extracted from both ts and tr pj156/cav-17 viruses, respectively, using high pure viral rna kit (roche, germany). the viral rna was used as template for amplification of partial 3d region (approximately 1.5 kb) by rt-pcr using access rt-pcr system (promega, madison, usa). primer ug7 (5’-tttgaaggggtgaaggaaccagc-3’) and uc12 (5’-tcaattagtctggattttccctg-3’) were used for amplification. rt-pcr was carried out in 50 µl reaction mixture containing 2 µl rna template, 10 µl amv/tfl buffer, 1 µl dntp (10 mm), 4 µl mgcl 2 (25 mm), 1 µl amv reverse transferase, 1 µl tfl dna polymerase, 2 µl each primer, and 27 µl ddh 2 o. reverse transcription was performed at 48 oc for 45 min, followed by thirty cycle of pcr reaction; 94 oc, 10 sec; 50 oc, 10 sec; 65 oc, 1 min. amplified cdna fragments were purified with wizard sv gel and pcr clean-up system (promega, madison, usa), and sequenced on genetic analyzer abi 3100 (applied biosystem, usa) using primer ca-3d1s (5’-gagcgggccagtgtggtggag-3’). in addition, full length sequence (7.44 kb) of both viruses was also determined. one-step growth-curve and temperature sensitivity analyses. one-step growth-curve experiments were conducted by infecting a monolayer of hep-2 cells with viruses at a multiplicity of infection (moi) of 10 ccid 50 per cell (shimizu et al. 2004; utama and shimizu 2005; utama and shimizu 2006). at different times post-infection, the cells and supernatant were collected, frozen and thawed three times, and then centrifuged (10,000 x g, 5 min) to remove cell debris. virus titers in the supernatants were determined by the end-point dilution method in hep-2 monolayer-cultures in 96-well plates at 35.0 oc. to test temperature sensitivity, one-step growth experiments were carried out at 35.0 oc and at 39.5 oc, respectively. neurovirulence test. groups of eight (four male and four female) 5-week-old pv receptor-transgenic (tgpvr) mice were inoculated intracerebrally with 30 µl virus solution (shimizu et al. 2004; utama and shimizu 2005; utama and shimizu 2006). tenfold dilutions of virus solution were made in mem with 2% bovine calf serum so that each mouse received approximately 101.3 to 106.3 of ccid 50 . mice were examined over 14 days for paralysis and/or death. the amount of virus that caused 50% paralysis and/or death dose (pd 50 ) was calculated. results generation of temperature-resistant pj156/cav-17. the recombinant pj156/cav-17 virus showed complete cpe at 39.5 oc in day-4 after twelve time blind passage. the virus was then plaque purified. plaque assay showed that the plaque size of pj156/cav-17 (tr) mutant was slightly bigger (1-2 mm) as compared with original pj156/cav-17 (ts) (<1 mm) (fig 1). ten purified isolates were cultured at 39.5 oc in maintenance medium, and was confirmed to be able growth at 39.5 oc. identification of amino acids determined temperature sensitivity. previous studies revealed that two amino acids in 3d region (his-73 and ile-362) were determinants of temperature sensitivity (bouchard et al. 1995; georgescu et al. 1995). a sequence analysis of 3d region of pj156/cav-17 showed that amino acid in 3d-73 and -362 were tyr and ile, respectively, similar with the parental cav-17 sequence. since amino acid in 3d-73 was not his, it is suggested that the temperature sensitivity of the virus was due to the ile-362. to confirm this suggestion, the pj156/cav-17 (tr) viruses were recovered, and the sequences of partial 3d region from eight plaque-purified isolates were determined. it is found that the amino acid in 3d-362 in all isolates was thr, instead of ile (fig 2). full length sequences (7.44 kb) of pj156/cav17 (ts) and pj156/cav-17 (tr) were determined and compared. it is found that 35 nucleotides were different between both viruses, and 17 of 35 nucleotide mutations were associated with 15 amino acids substitution (table 1). in particular, there were 3 amino acids substitution in 3d region; gly-163-arg, ala-197-val, and ile-362-thr, respectively. based on current knowledge, since no other mutations associated with temperature sensitivity, it is suggested that ile-362 was determinant for temperature sensitivity of pj156/ cav-17. one-step growth of recombinant viruses. a one-step growth experiment of the pj156/cav-17 (ts) and pj156/cav17 (tr) viruses was conducted at 39.5ºc (fig 3). pv strains such as sabin 1, mahoney, and pj156 were used for comparison. as expected, sabin 1 was temperature sensitive, while mahoney and pj156 were temperature resistant. on the other hand, recombinant pj156/cav-17 virus was temperature sensitive; it could not grow at 39.5 oc. however, two isolates of pj156/cav-17 resulted from blind passaging (pj156/cav-17 (tr-1) and pj156/cav-17 (tr-2)) could grow at 39.5 oc. although the growth those viruses were slightly a b fig 1 plaque size of the pj156/cav-17 (ts) (a) and pj156/cav17 (tr) (b). volume 1, 2007 microbiol indones 87 slow as compared to the mahoney and pj156 strains, they finally could grow well after 6 h post infection. neurovirulence test. to analyze the neurovirulence of the pj156/cav-17 (tr), groups of 8 pv receptor-expressed transgenic mice (tgpvr mice) were intracerebrally inoculated with the virus, along with sabin 1 and pj156 viruses. as shown in table 2a, the pj156/cav-17 (tr) exhibited 50% paralysis and/or death dose (pd 50 ) of 2.66, which is lower than that of parental pj156 (3.66). on the other hand, as expected, sabin 1 (vaccine strain) showed a high pd 50 value (>8.06). these results imply that the pj156/cav (tr) was more virulence than the parental pj156. in another experiment, it was shown that pj156/cav-17 (ts) possessed a high pd 50 (>6.78), which similar with sabin 1 (>7.53), whilst mahoney (wild-type strain) possessed low pd 50 value (3.41) (table 2b). these results confirmed that there was a dramatic change of neurovirulence phenotype from pj156/cav-17 (ts) to pj156/cav-17 (tr). these results also exhibited a strong correlation between temperature sensitivity or resistance and attenuation or neurovirulence. fig 2 amino acid alignment of partial 3d region. table 1 sequence different between pj156/cav-17 (ts) and pj156/cav-17 (tr) region nucleotide amino acid pj156 cav-17 position ts tr position ts tr vp4 vp2 2 a 2b 2c 2c 3b 3c 3 d 878 911 1 1 4 8 1 4 9 1 1 5 6 2 3 4 9 1 3 4 9 3 3 7 5 3 4 1 1 6 4 1 3 0 4 9 4 0 5 3 9 6 5 3 9 7 5 7 3 8 6 4 7 3 6 5 7 6 7 0 7 1 c a g c a a t t c a t a a g g c t t g a t t g c c t g c c g a a t c 46 57 67 1 8 1 2 0 5 36 1 2 3 95 3 2 7 3 9 1 0 1 1 6 3 1 9 7 3 6 2 leu ile asp ser ser asn ile t h r ser ty r lys val gly ala ile p h e val asn p h e cys asp t h r ile gly his arg ile arg val t h r discussion a recombinant virus between poliovirus (isolate pj156) and coxsackie a virus serotype 17 (cav-17) was recently constructed and characterized (utama and shimizu 2006). the resultant recombinant pj156/cav-17 virus showed a temperature sensitive phenotype. to determine the sequence contributed to the temperature sensitive (ts) phenotype of the pj156/cav-17 virus, elevated temperature escape mutants were generated by performing blind passage at 39.5 oc. the pj156/cav-17 virus could grow well at 39.5 oc in day-4 after twelve time blind passage. the pj156/cav (tr) showed a bigger plaque size (1-2 mm) as compared with original pj156/cav-17 (ts) (<1 mm) (fig 1). ten purified isolates were cultured at 39.5 oc in maintenance medium, and was confirmed to be able growth at 39.5 oc, indicating from complete cpe. one-step growth experiment showed that pj156/cav-17 (tr) could grow at 39.5 oc, although the growth was not as good as parental pj156 or mahoney strain. previous studies revealed that mutations in the 5’ncr, base 2438 of vp3, bases 2741, and 2795 of vp1, base 6203 of 3dpol, and base 7441 of the 3’ncr were associated with fig 3 one-step growth of parental and recombinant viruses. ( ) sabin 1, ( ) mahoney, ( ) pj156, ( ) pj156/cav17, ( ) pj156/cav-17 (tr-2), ( ) pj156/cav-17 (tr-1). v ir u s ti te r (t c id 5 0 .u l1 ) 88 utama and shimizu microbiol indones phenotypic reversion of the temperature sensitivity of pv, particularly sabin 1 strain (bouchard et al. 1995). in addition, two amino acids in 3d region (his-73 and ile-362) were determinants for temperature sensitivity of pv sabin 1 (georgescu et al. 1995). although it is reported that a chimeric plasmid from cdna clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive (semler et al. 1986), it is not known whether those amino acids affect the temperature sensitivity of cav, including cav-17. a sequence analysis of partial 3d region of pj156/cav-17 (ts) showed that amino acid in 3d73 and -362 were tyr and ile, respectively (fig 2). on the other hand, it was found that the amino acid in 3d-73 and 3d-362 of pj156/cav-17 (tr) were tyr and thr, respectively. consequently, ile-362 was proved to be involved in temperature sensitivity of pj156/cav-17. to identify other determinants, full sequences of both pj156/cav-17 (ts) and pj156/cav-17 (tr) viruses were also determined and compared. it is found that 35 nucleotides were different between both viruses (data not shown), and 17 of 35 nucleotide mutations were associated with 15 amino acids substitution (table 1). in particular, there were 3 amino acids substitution in 3d region; gly-163-arg, ala-197-val, and ile-362-thr, respectively. although there were several mutations in capsid and other nonstructural protein-coding regions, based on current knowledge, none of these mutations are related to temperature sensitivity of pv and other enteroviruses. hence, it is suggested that ile-362 was a determinant for temperature sensitivity of pj156/cav-17. the pj156/cav-17 (tr) exhibited a pd 50 of 2.66, which is lower than that of parental pj156 (3.66) (table 2a). sabin 1 was used as control for vaccine strain, and as expected, it showed a high pd 50 value (>8.06), which means very low neurovirulence. contrary to this, pj156/cav (tr) was high virulence, even more virulence than the parental pj156. in another experiment, it was shown that pj156/cav-17 (ts) possessed a high pd 50 (>6.78), which similar with sabin 1 (>7.53), whilst mahoney (wild-type strain) possessed low pd 50 value (3.41) (table 2b) (utama and shimizu 2006). these results confirmed that there was a dramatic change of neurovirulence phenotype from pj156/cav-17 (ts) to pj156/ cav-17 (tr). moreover, the neurovirulence of pj156/cav-17 (tr) was higher than pj156 and wild-type mahoney strain. in most pvs, there is a strong correlation between virulence or attenuation and temperature resistance or sensitivity (shimizu et al. 2004; yang et al. 2005). our results also support the fact that there is a correlation between temperature resistant and high virulence. acknowledgements this research was supported by the japanese society for promotion of sciences through a postdoctoral grant awarded to au. references arita m, shimizu h, nagata n, ami y, suzaki y, sata t, iwasaki t, miyamura t. 2005. temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys. j gen virol 86:1391-1401. bouchard mj, lam d-h, racaniello vr. 1995. determinants of attenuation and temperature sensitivity in the type 1 poliovirus sabin vaccine. j virol 69:4972-4978. brown b, oberste ms, maher k, pallansch ma. 2003. complete genomic sequencing shows that polioviruses and members of human enterovirus species c are closely related in the noncapsid coding region. j virol 77:8973-8984. dahourou g, guillot s, gall ol, crainic r. 2002. genetic recombination in wild-type poliovirus. j gen virol 83:3103-3110. georgescu m-m, tardy-panit m, guillot s, crainic r, delpeyroux f. 1995. mapping of mutations contributing to the temperature sensitivity of the sabin 1 vaccine strain of poliovirus. j virol 69:5278-5286. guillot s, caro v, cuervo n, korotkova e, combiescu m, persu a, aubert-combiescu a, delpeyroux f, crainic r. 2000. natural genetic exchanges between vaccine and wild poliovirus strains in humans. j virol 74:8434-8443. karber g. 1931. beitrag zur kollektiven behandlung pharmakologischer reihenversuche. arch exp pathol pharmakol 162:480-483. kew om, morris-glasgow v, landaverde m, burns c, shaw j, garib z, andre j, blackman e, freeman cj, jorba j, sutter r, tambini g, venczel l, pedreira c, laender f, shimizu h, yoneyama t, miyamura t, van der avoort h, oberste ms, kilpatrick d, cochi s, pallansch m, de quadros c. 2002. outbreak of poliomyelitis in hispaniola associated with circulating type 1 vaccine-derived poliovirus. science 296:356-359. liu hm, zheng dp, zhang lb, oberste ms, pallansch ma, kew om. 2000. molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in china. j virol 74:11153-11161. rousset d, rakoto-andrianarivelo m, razafindratsimandresy r, randriamanalina b, guillot s, balanant j, mauclère p, delpeyroux f. 2003. recombinant vaccine–derived poliovirus in madagascar. j infect dis 9:885-887. semler bl, johnson vh, tracy s. 1986. a chimeric plasmid from cdna clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive. proc natl acad sci usa 83:1777-1781. table 2 neurovirulence of pj156/cav-17 (ts) and pj156/cav-17 (tr) viruses on tg-mice dose (log ccid 50 mouse -1 ) 8.18 5.78 4.78 3.78 2.78 1.78 0.78 a virus pd 50 b sabin 1 pj156 pj156/cav-17 (tr) 5/8a nd nd nd nd 8/8 nd nd 8/8 nd 5/8 7/7c nd 0/8 4/8 nd 0/8 1/8 nd 0/8 nd >8.06 3.66 2.66 dose (log ccid 50 mouse -1 ) 7.28 6.28 5.28 4.28 3.28 2.28 1.28 b virus sabin 1 mahoney pj156/cav-17 (ts) 2/8a nd nd nd nd 0/8 nd nd 0/8 nd 7/8 0/8 nd 3/8 0/8 nd 1/8 0/8 nd 0/8 nd >7.53 3.41 >6.78 ano. of paralyzed or dead mice/no. of total mice, bpd 50 was calculated by the karber formula (karber 1931), cone mouse was died after injection of the virus, nd = not determined. pd 50 b volume 1, 2007 microbiol indones 89 shimizu h, thorley b, paladin fj, brussen ka, stambos v, yuen l, utama a, tano y, arita m, yoshida h, yoneyama t, benegas a, roesel s, pallansch m, kew om, miyamura t. 2004. circulation of type 1 vaccine-derived poliovirus in the philippines in 2001. j virol 78:13512-13521. utama a, shimizu h. 2005. construction of a recombinant virus between poliovirus and coxsackie a virus 11. annal bogorienses 10:19-26. utama a, shimizu h. 2006. construction and characterization of chimeric virus between poliovirus and coxsackie a virus serotype 17. j mikrobiol indones 11:77-81. yang cf, chen hy, jorba j, sun hc, yang sj, lee hc, huang yc, lin ty, chen pj, shimizu h, nishimura y, utama a, pallansch m, kew om, yang jy. 2005. intratypic recombination among lineages of type 1 vaccine-derived poliovirus emerging during chronic infection of an immunodeficient patient. j virol 79:126231 2 6 3 4 . yang cf, naguib t, yang sj, nasr e, jorba j, ahmed n, campagnoli r, van der avoort h, shimizu h, yoneyama t, miyamura t, pallansch m, kew om. 2003. circulation of endemic type 2 vaccine-derived poliovirus in egypt from 1983 to 1993. j virol 77:8366-8377. 90 utama and shimizu microbiol indones 5 486 cyclo (rofiq).cdr cyclo (tyrosyl-prolyl) produced by sp.: bioactivity and molecular structure elucidation streptomyces rofiq sunaryanto , bambang marwoto , liesbetini hartoto , zainal alim mas'ud , tun tedja irawadi 1,2* 2 1 3 3 and 1 2 3 department of agro-industrial technology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; center of biotechnology, badan pengkajian dan penerapan teknologi, kawasan puspiptek serpong, tangerang, banten 15314, indonesia; department of chemistry, institut pertanian bogor, darmaga campus, bogor 16680, indonesia streptomyces streptomyces escherichia coli pseudomonas aeruginosa staphylococcus aureus bacillus subtilis streptomyces streptomyces escherichia coli pseudomonas aeruginosa staphylococcus aureus bacillus subtilis streptomyces determination of bioactivity by minimum inhibitory concentration (mic) methods and molecular structure identification of antibiotic produced by sp. have been carried out. the antibiotic was produced by liquid culture using sp. isolate. purification of antibiotic was carried out by silica gel column chromatography and preparative hplc. molecular structure identification was carried out using esi-ms, h nmr, c nmr, and c dept nmr. pure antibiotic showed inhibition activity to gram-negative and gram-positive bacteria. mic to atcc 25922, atcc 27853 , atcc 25923, and atcc 66923 were 27.0, 68.7, 80.2, and 73.7 µg ml , respectively. identification using esi-ms showed that the molecular weight of this antibiotic was 260 g mol , and molecular formula was c h n o . elucidation of molecular structure using h nmr, c nmr, and c dept nmr showed that antibiotic was cyclo(tyrosyl-prolyl). key words: antibiotic, cyclo(tyrosyl-prolyl), inhibitory concentration, bioactivity telah dilakukan penentuan bioaktivitas antibiotik menggunakan metode konsentrasi hambatan minimum dan identifikasi struktur molekul antibiotik yang dihasilkan oleh sp. antibiotik diproduksi dengan menggunakan isolat sp. dengan kaldu fermentasi. pemurnian antibiotik dilakukan menggunakan kromatografi kolom silika gel dan hplc preparatif. identifikasi struktur molekul dilakukan menggunakan esi-ms, h nmr, c nmr, dan c dept nmr. antibiotik hasil purifikasi menunjukkan daya hambat terhadap bakteri gram-negatif dan gram-positif. konsentrasi hambatan minimum terhadap atcc 25922, atcc 27853, atcc 25923, dan atcc 66923 masing-masing ialah 27.0, 68.7, 80.2, dan 73.7 µg ml . identifikasi menggunakan esi-ms menunjukkan bobot molekul antibiotik sebesar 260 g mol , dengan rumus molekul c h n o . hasil elusidasi struktur molekul menggunakan h nmr, c nmr, dan c dept nmr menunjukkan antibiotik yang dihasilkan oleh sp. adalah cyclo (tyrosyl-prolyl). kata kunci:antibiotik, siklo (tirosil-prolil), konsentrasi hambatan, bioaktivitas 1 13 13 -1 -1 1 13 13 1 13 13 -1 -1 1 13 13 14 16 2 3 14 16 2 3 increasing microbial resistance due to the use of antibiotics and the emergence of new pathogenic microbes has inspired the search of new antibiotics from microbes. this situation encourages the growing importance of business to get cheap antibiotic materials continuously available in large quantities and has all the elements needed for the manufacture of antibiotics. approximately 800 classes of peptide antibiotics have been discovered and studied by several researchers. most of these peptide antibiotics have been included in clinical trials and has been used in several clinical drugs such as actinomycin, gramicidine, bacitracin, polymyxyn, tyrocidine, and many other peptide antibiotics (dubin . 2005). developments in science and inventions peptide antibiotics initiated new drug developments, especially for the treatment of infections (kesting . 2010). the discovery of peptide antibiotics produces broadet al et al spectrum antimicrobial activity with the potential to make peptides as anti-cancer drugs (lu and chen 2010) and anti-virus (lee 2011) or parasite infections (manfredini 2010). one member of the class of the simplest peptide antibiotics with low molecular weight was cyclo dipeptide group. although cyclo dipeptide antibiotic have a low molecular weight and simple structure, this antibiotic is known have antimicrobial activity with a broad spectrum (rhee 2004). for example antibiotics cyclo (leu-pro) has a minimum inhibition concentration (mic) 32 µg ml against 99-38, and the mic µg ml against k-01-511. cyclo(phe-pro) has a minimum inhibition concentration µg ml against k-99-38. combination of cyclo(leupro) with cyclo(phe-pro) was able to increase anti microbial activity, where the mic against 99-38 k-1 was 1 µg ml and mic against k-01-511 was 0.5 µg ml (rhee 2004). the objective of this research was to obtain information on bioactivity of an antibiotic produced by sp. and elucidate molecular structure of antibiotic. et al. et al. enterococcus faecium e. faecalis e. faecium e. faecium e. faecalis streptomyces -1 -1 1 -1 -1 *corresponding author, phone/fax: +62-21-7560208, email: rofiqsn@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 2, june 2011, p 81-87 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.2.5 materials and methods microorganism liquid culture and extraction of active substance . sp. was isolated from sediment of marine site in banten west java (sunaryanto . 2010). the isolate was deposited in strain collection of the biotechnology of microbial culture collection (bio-mcc) bppt puspiptek serpong, tangerang. . an established slant of isolate was inoculated into a 250 ml flask containing 100 ml of vegetative medium (yeme medium) consisting of: bacto peptone 5 g l , yeast extract 3 g l , malt extract 3 g l , glucose 3 g l , demineral water 25 ml, and sea water 75 ml. the ph value of the medium was adjusted to 7.6 before sterilization. the flask was incubated at 30 °c for 2 days in an incubator-shaker. fifty milliliters of the culture was transferred to 1000 ml of the fermentation medium. fermentation medium consisted of bacto peptone 15 g l , yeast extract 3 g l , fe (iii) citrate hydrate 0.3 g l , demineralized water 250 ml, and sea water 750 ml (nedialkova and mariana 2005). the ph value of the medium was adjusted to 7.6 before sterilization. the fermentation was carried out at 30 °c for 5 days in incubator-shaker. for extraction of active substance, the culture broth was centrifuged at 14 000 x g for 15 min. biomass was dried and weighed than extracted twice using methanol 500 ml. methanol extract was concentrated by evaporation under vacuum to the least possible volume, after dehydration with anhydrous na so and weighed. the broth supernatants were extracted using ethyl acetate. supernatant and the organic solvent were mixed thoroughly by shaking them in 2 l capacity separating funnel and allowed to stand for 30 min. two layers were separated; the aqueous layer and the organic layer, which contained the solvent and the antimicrobial agent. the organic layer was concentrated by evaporation under vacuum to the least possible volume, after dehydration with anhydrous na so the aqueous layer was re-extracted and the organic layer added to the above organic layer. the organic layer was concentrated by repeated cycle of evaporation under vacuum. the dry extract of the supernatant was purified using silica gel column chromatography. dry extract was injected onto the column and then eluted stepwise with chloroform-methanol solvent system as follows: first the column was eluted with 100% chloroform (fraction 1). then repeated with reducing the chloroform by 10% in each fraction while the methanol was increased by 10% in each fraction, until the percentage of methanol was 100%. thirty fractions streptomyces et al -1 -1 -1 -1 -1 -1 -1 2 4 2 4. were collected (each of 20 ml) and then concentrated and dried for testing their antimicrobial activities. the active fractions obtained from chromatography column were further purified by preparative hplc. . purification by preparative hplc was conducted using a waters 2695 hplc, photodiode array detector (pad), and column puresil 5 c18 4.6x150 mm. the sample volume was 100 l per injection under conditions of average pressure of 1267 psi, and the flow rate was 1 ml min where the mobile phase was 0-45% methanolwater and time period was 25 min. . molecular weight and formula were determined using esi-ms (lct premier-xe waters), molecular structure elucidation of active compound were determined using ftir (shimadzu 8300), h nmr, c nmr, and dept c nmr (buker av-500 (500 mhz)). . antimicrobial activity was monitored by the agar diffusion paperdisc (6 mm) method. discs were dripped with methanol solution of extract, dried, and then placed over the agar surface plates freshly inoculated with either atcc 25922, atcc25923, atcc 66923, and atcc27853 as test orga nisms. suspensions of test organisms were adjusted to 10 cfu ml . each experiment was run in duplicate, and the most potent isolates were noted for each test microorganism, based on the mean diameter of inhibition zones (bonev . 2008). rifampicin (500 ppm) was used as a control. mic determinations were performed using the agardilution methods according to methods of andrews (2001) and bonev (2008). active purified compound was dissolved in methanol (6500 g ml ) and taken as standard stock. a series of two fold dilutions each solution were dripped on paper disc 6 mm, dried, then placed over on agar surface plates freshly inoculated with either atcc 25922, atcc25923, atcc 66923, or atcc27853. minimum inhibitory concentration is defined as the lowest concentration of antimicrobial that will inhibit the visible growth of a micro-organism and determined toward a standard curve of clearing zone diameter and the concentration of active compound. the mic of tetracycline and streptomycin as positive controls were also determined and each experiment was run in duplicate. analysis was performed using hplc with an analytical sunfire c18 column (4.6 x 250 mm, shiseido co. ltd., tokyo, japan). mobile -1 1 13 13 6 -1 -1 preparative hplc elucidation of chemical structure antimicrobial activity assay minimum inhibitory concentration (mic) hplc analysis. μ μ e. coli staphylococcus aureus bacillus subtilis pseudomones aeruginosa et al . et al. e. coli s. aureus b. subtilis p. aeruginosa 82 sunaryanto et al. microbiol indones phase used methanol-water (0-100% linear gradient for 25 min and then isocratic elution with 100% methanol over 10 min), at a flow rate of 1 ml min , volume of injection 10 lper injection, and detection was at a of 210 nm. liquid culture of isolate was carried out for 5 days by using yeast-peptone medium. from a 5 l volume of culture, 4.72 g of dry biomass was obtained and after extraction by methanol 2.72 g of extract was obtained. on the other hand, 0.33 g of ethyl acetate extract was obtained from supernatant. antibacterial activity assay of the both extract against atcc 25922, atcc25923, atcc 27853 atcc 66923 showed that the extract of supernatant was active, but no activity with the extract of biomass (table 1). supernatant extract was further purified using column chromatography and preparative hplc. pure active fraction was collected and mic was determined using four bacterial testes. the active fraction had a strong inhibition against gram-positive and gramnegative (table 2). mic of the compound to atcc 25922 was 27.0 µg ml , while to atcc 27853 was 68.7 µg ml . when compared with tetracycline and streptomycin, the active fraction had the highest inhibition against atcc 25922, but lower inhibitions against atcc 2785. active fraction also had a strong inhibition against gram-positive bacteria, mic to atcc 25923 was 80.2 µg ml while to atcc 66923 was 73.7 µg ml . compared with the tetracycline and streptomycin, this active fraction had higher inhibition against atcc 25923 and atcc 6692. molecular weight, formula and structure elucidation of active compound were determined using results -1 -1 -1 -1 -1 μ l e. coli s. aureus p. aeruginosa , b. subtilis e. coli p. aeruginosa e. coli p. aeruginosa s. aureus b. subtilis s. aureus b. subtilis esi-ms, h nmr, c nmr (fig 1), and ftir respectively (fig 4). esi-ms spectra were obtained on lct premier-xe waters. esi-ms spectra showed that this active compound has molecular weight of 260.0 g mol . data base from lct premier-xe waters program showed that this molecule had 14 carbon, 16 hydrogen, 2 nitrogen, and 3 oxygen. the most possible of molecular formula was c h n o the position each carbon, nitrogen, oxygen, and hydrogen atoms were confirmed by h nmr, c nmr, and ftir. this chemical characteristics were indicated by esi-ms at 261 (m+h) (fig 1a). high-resolution h nmr spectrum were obtained on a bruker av-500 (500 mhz) with tetramethylsilane (tms) as internal standard in cdcl and give 1 13 -1 1 13 + 1 14 16 2 3. 3 m/z samples diameter of clear zone (mm) staphylococcus aureus atcc 25923 bacillus subtilis atcc 66923 pseudomonas aeruginosa atcc 27853 extract of biomass extract of supernatant 10.39 24.43 9.64 control (rifampicin 500 ppm) 21.27 44.57 10.08 escherichia coli atcc 25922 9.55 10.12 table 1 antibacterial activity of biomass and supernatant extract from sp.streptomyces diameter of disc paper: 6 mm table 2 minimum inhibitory concentration of active purified compound minimum inhibitory c oncentration µg ml -1 sample escherichia coli atcc 25922 staphylococcus aureus atcc 25923 bacillus subtilis atcc 66923 pseudomonas aeruginosa atcc 27853 active purified compound 27.0 80.2 73.7 68.7 tetracycline (positive control) 64.0 256.0 128.0 12.5 streptomycin (positive control) 50.0 130.0 120.0 61.0 table 3 spectrum data of h nmr and c nmr of active compound produced by sp. 1 13 streptomyces no δ 13 c δ 1 h (ppm) (in meod) functional group 1 170.7 95 (s) 2 3 57.926 (d) 4.358 (t) 4 166.935 (s) 5 6 60.082 (d) 4.048(dd) 7 29.421 (t) 2.088 (m) 8 22.477 (t) 1.801 (m) 9 45.942 (t) 3.518 (dd) 10 37.694 (t) 3.066 (dd) 1’ 127.651 (s) 2’ 132.135 (d) 7.031 (d) 3’ 116.315 (d) 6.690 (d) 4’ 157.699 (s) 5’ 116.315 (d) 6.690 (d) 6’ 132.135 (d) 7.031 (d) c o n r ch c o n r ch -ch2 ch2 n c r =chc oh volume 5, 2011 microbiol indones 83 (ppm) t: triplet; d, duplet; dd, double doublet; m: multiplet -ch2 -ch2 =ch=ch=ch84 sunaryanto et al. microbiol indones a b c fig 1 spectrum of bioactive compound produced by sp.: a, esi-ms m/z 261 (m+h) ; b, h nmr; c, c nmr.streptomyces + 1 13 350 000 300 000 250 000 200 000 150 000 100 000 50 000 100 0 200 300 400 500 600 700 800 900 8 8 2 .2 8 2 5 .0 8 0 2 .8 6 7 0 .4 6 7 9 .0 5 8 3 .0 5 4 1 .2 5 4 4 .2 5 4 3 .2 5 2 1 .2 max: 346 333 5 2 3 .2 5 2 2 .2 4 1 0 .0 3 7 4 .0 3 0 3 .0 2 8 4 .0 2 6 2 .0 2 2 8 .4 1 9 1 .2 methanolwaterh3’ h5’ h2’ h6’ h8 h7 h10h9 h6 h3 impurity impurity (ppm)0.51.01.52.5 2.03.5 3.04.5 4.05.5 5.06.5 6.07.5 7.08.0 0 .2 1 0 .2 1 2 .0 9 0 .4 6 2 .5 5 0 .6 2 1 .0 0 0 .2 4 1 .0 4 1 .3 8 1 .4 7 1 .2 7 1 .0 0 0 .2 4 0 .2 7 1 .4 6 2 .1 0 0 .6 4 1 .1 6 0 .4 1 0 .7 2 c2’ c6’ c3’ c5’ methanol c8c7c9 c10 c6 c3 c1’ c4’ c4 c1 170 160 150 140 130 120 110 100 (m/z) (ppm) fig 3 spectrum of dept c nmr active compound produced by sp. 13 streptomyces volume 5, 2011 microbiol indones 85 following data: δ : 4.358 (t, 1h), 4.048(1h, dd), 2.088 (2h, m), 1.801 (2h, m), 3.518 (2h, dd), 3.066 (2h, dd), 7.031 (2h, d), 6.690 (2h, d), and c spectrum : 170.795 (s), 57.926 (d), 166.935 (s), 60.082 (d), 29.421 (t), 22.477 (t), 45.942 (t), 37.694 (t), 127.651 (s), 132.135 (d), 116.315 (d), 157.699 (s). the impurities of active compound was also showed at h nmr δ 1.7 δ 0.9 (fig 1b). based on information of esi-ms, h nmr, and c nmr (fig 3) spectrums the molecular structure of active compound can be predicted as fig 2. spectrum data of h nmr and c nmr were presented on table 3. structure elucidation of active compound was also conducted using dept c nmr (fig 3). h h h 13 1 1 13 1 13 13 fig 2 molecular structure prediction of active compound produced by sp.streptomyces 13 13 two singlet carbons representing a ketone group were evident in the c spectrum at 1) and er analysis of the c spectrum revealed two other non substituted carbons [ 127.651 (c1'), 157.699 (c4')], six methine carbons [ 57.926 (c3), 60.082 (c6), 132.135 (c2'), 116.315 (c3'), 116.215 (c5'), 132.135 (c6')], and four methylene carbons [ 29.42 (c7), 22.477 (c8), 45.942 (c9), 37.694 (c10)]. dept 45° spectrum on fig 3 showed that there were 3 nonsubstituted carbon [ 127.651 (c1'), 157.699 (c4'), and 166.935 (c4)]. dept 135° and 90° showed that there were six methine carbons [ 57.926 (c3), 60.082 (c6), 132.135 (c2'), 116.315 (c3'), 116.215 (c5'), 132.135 (c6')] and four methylene carbons [ 29.42 (c7), 22.477 (c8), 45.942 (c9)]. carbons at position 3' and 5' appeared more upfield than c2' and c6'. this was due to the shielding effect of the hydroxyl group at c4' toward its ortho coupled carbon (c3' and c5'). a similar phenomenon occurred on c1' (para coupled with c4') which shifted more upfield than c2' and c6'. δ 170.795 (s) (c δ 166.935 (s) (c4) (table 3, fig 1c). furth δ δ δ δ δ δ -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 the infra red spectrum in a kbr pellet showed characteristic bands at 3383 cm (n-h), 3227 cm (oh), 2959 cm (saturated c-h), 1660 cm (c=o), 1515 cm (benzene ring), 1456 cm (methine), 1344 cm (methylene), 1232 cm (phenol), 1116 cm (c-o), 827 cm ( -disubstituted benzene ring) (fig 4).p discussion the active compounds inhibited both grampositive and gram-negative bacteria. according to rhee (2004), most of antibiotic peptides especially cyclic dipeptide has antimicrobial properties with broad spectrum. several cyclo peptides have broaddept 90 “sample 4” 3 1 /opt/topspin mercian [r e l] 1 .5 1 .0 0 .5 0 .0 160 140 120 100 80 60 40 (ppm) c1 c4 c4’ c2 c6’ dept 135 dept 45 ch ch c3’ c5’ ch ch ch ch ch ch c6 c3 c9 c10 c7 c8 “sample 4” 3 1 /opt/topspin mercian “sample 4” 4 1 /opt/topspin mercian “sample 4” 5 1 /opt/topspin mercian c1 c 2h c 2h c 2h c 2h methanol 86 sunaryanto et al. microbiol indones fig 4 infra red spectrum of active compound produced by sp.streptomyces spectrum activity and also able to act as an anti-viral and anti cancer. the same case were also presented by (lu and chen 2010; lee 2011). in addition to having antimicrobial activity with a broad spectrum, our compound also had lower mic compared with tetracycline suggesting higher activity than tetracyclin and streptomycin. the same result is shown by rhee (2004). activity of cyclo(tyrosyl-prolyl) on the is lower compared with tetracycline. it is suspected that the has a resistance to cyclo(tyrosyl-prolyl). according to macfarlane (2000) and reig . (2009), has a resistance to type of peptide and aminoglycoside antibiotics. cyclo(tyrosyl-prolyl) was peptide antibiotic and streptomycin was aminoglycoside antibiotic. has highly adaptable nature, including the ability to develop resistance. it can grow on a wide variety of substrates and alter its properties in response to changes in the environment (lambert 2002). thus, lower inhibition of cyclo(tyrosyl-prolyl) and streptomycin than that of tetracycline on might be caused by resistance. this active compound was included in group cyclo dipeptide, namely was cyclo(tyrosyl-prolyl). this active compound has the same profile such as h nmr, c nmr, infra red spectrum, and molecular weight with cyclo(tyrosyl-prolyl) that found previously but from different origin (stierle 1988; guo . 2007). previously, this compound was produced by a host-specific phytotoxin for spotted knapweed (stierle . 1988), and produced by gcm5-1a isolated from the pine wood nematode (pwn), and from (guo . 2007). et al. p. aeruginosa p. aeruginosa et al. et al p. aeruginosa p. aeruginosa p. aeruginosa et al. et al alternaria alternate and can be used as et al p. fluorescens bursaphelenchus xylophilus et al 1 13 acknowledgments references 1 13 13 we thank hardaning pranamuda, anis mahsunah and evita chrisnayani for their valuable advice and support our research, and mercian co. japan for sample analyses using esi-ms, h nmr, c nmr, and dept c nmr. first author was supported by islamic development bank scholarship. andrews jm. 2001. determination of minimum inhibitory concentration. j antimicrob chemother. 48(suppl. 1):5-16. doi: 10.1093/jac/48. suppl_1.5. bonev b, james h, judicael p. 2008. principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. j antimicrob chemother. 61(6):12951301. doi: 10.1093/jac/dkn090. dubin ap, mak, dubin g, rzychon m, stec-niemczyk j, wladyka j, maziarka k, chmiel d. 2005. new generation of peptide antibiotics. acta biochim pol. 52(3):633-638. doi:10.1093/jac/dkn090. guo q, daosen g, zhao b, xu j, li r. 2007. two cyclic dipeptides from pseudomonas fluorescens gcm5-1a carried by the pine wood nematode and their toxicities to japanese black pine suspension cells and seedlings in vitro. j nematol. 39(3):243-247. kesting mr, stoeckelhuber m, holzle f, mucke t, neumann k, woermann k, jacobsen f, steinstraesser l, wolff kd, loeffelbein dj, rohleder nh. 2010. expression of antimicrobial peptides in cutaneous infections after skin surgery. brit j dermatol. 163(1):121-127. doi: 10.1111/j.1365-2133.2010.09781.x. lambert pa. 2002. mechanisms of antibiotic resistance in j r soc med. 95(suppl. 41):22-26. lee sb, li b, jin s, daniel h. 2011. expression and characterization of antimicrobial peptides retrocyclin-101 and protegrin to control viral and bacterial infections. plant biotechnol j. 9(1):100-115. doi: 10.1111/j.1467-7652.2010.00538.x. lu j and chen z. 2010. isolation, characterization and anticancer activity of sk84, a novel glycine-rich antimicrobial peptide from . peptides. 31(1):44-50. doi:10.1016/j.peptides. 2009.09.028. macfarlane ema, kwasnicka a, hancock rew. 2000. role of phop-phoq in resistance to antimicrobial cationic peptides and aminoglycosides. microbiology. 146:25432554. pseudomonas aeruginosa. drosophila virilis pseudomonas aeruginosa volume 5, 2011 microbiol indones 87 manfredini f, beani l, taormina m, vannini l. 2010. parasitic infection protects wasp larvae against a bacterial challenge. microb infect. 12(10):727-735. doi:10.1016/j.micinf.2010.05.001. milne pj, oliver dw, roos hm. 1992. cyclodipeptides: structure and conformation of cyclo(tyrosyl--prolyl) j crystallog spect res. 22(6):643-649. doi: 10.1007/bf01160980. munekata m and tamura g. 1981. selective inhibition of sv40transformed cell growth by diketopiperazines. agric biol chem. 45: 2618-2628. nedialkova d and mariana n. 2005. screening the antimicrobial activity of actinomycetes strains isolated from antartica. j cult collect. 4: 29-35. reig s, hutha a, kalbacherb h, winfried, kerna v. 2009. resistance against antimicrobial peptides is independent of . escherichia coli acrab, mexab and nora efflux pumps. int j antimicrob agents 33(2):174-176. doi:10.1016/j.ijantimicag.2008.07.032. rhee kh. 2004. cyclic dipeptides exhibit synergistic, broad spectrum antimicrobial effects and have anti-mutagenic properties. int j antimicrob agent. 24(5):423-427. doi:10.1016/j.ijantimicag.2004. 05.005. sunaryanto r, marwoto b, irawadi tt, mas'ud za, hartoto l. 2010. isolation and characterization of antimicrobial substance from marine s p . m i c r o b i o l i n d o n e s . 4 ( 2 ) : 8 4 8 9 . d o i : 10.5454/mi.4.3.1. stierle a, cardellina jh, strobel ga. 1988. maculosin, a host-specific phytotoxin for spotted knapweed from . proc nat acad sci. 85(21): 8008-8011. pseudomonas aeruginosa staphylococcus aureus . s t re p t o m y c e s alternaria alternata 02. jatmiko.cdr vol.15, no.2, june 2021, p 45-53 doi: 10.5454/mi.15.2.2 bacterial population dynamics of natural fermentation of sumbawa mare's milk using metagenomic approach yoga dwi jatmiko , aditya ragil suharto , irfan mustafa , siska aditya 1* 1 1 2 and 1department of biology, universitas brawijaya, malang 65145, indonesia; 2 faculty of veterinary medicine, , indonesia.65145universitas brawijaya, malang naturally fermented sumbawa mare's milk is a mare's milk product that has been fermented naturally involving indigenous microbes. studies of bacterial communities during the fermentation of mare's milk based on metagenomic method remains limited. this study aimed to assess the changing of bacterial density and the physicochemical aspects during natural fermentation of sumbawa mare's milk and to evaluate the dynamics of the bacterial population during the natural fermentation using a metagenomic approach. mare's milk samples obtained from regency of dompu were fermented for 60 day. on the day 0, 7, 15, 30 and 60 mare's milk sample were collected for further analysis, such as bacterial density enumeration, nutrition content, physical properties of the milk, and total dna isolation. the total dna samples obtained were analyzed using next-generation sequencing. the density of lactic acid bacteria decreased along with fermentation periods in mrs and m17 media. meanwhile, the density of aerobic bacteria in pca medium relatively fluctuated. the physicochemical content of mare's milk also changed during fermentation periods. carbohydrate content and total sugar decreased along with the decreasing ph value. moreover, the lipid content was increased, and the protein content fluctuated. the changes in physical properties such as whey color, acidity, and gas formation were observed until the end of fermentation process. using metagenomics analysis, the bacterial diversity from each sample period was categorized as low because was dominant until the end of the fermentation. lactobacillus helveticus lactobacillus helveticus as a member of lab did not grow on isolation media on the late fermentation period (day 60). the presence of uncultivable bacteria can be detected with a metagenomic approach, fulfilling the limited information on the bacterial composition of fermented sumbawa mare's milk products. bacterial diversity, natural fermentation, next generation sequencing, sumbawa mare's milkkey words: susu kuda sumbawa terfermentasi merupakan produk susu kuda yang difermentasi secara alami dengan melibatkan mikrobia kajian tentang komunitas bakteri selama fermentasi susu kuda berdasarkan indigenous. metode metagenomik masih terbatas. penelitian ini bertujuan untuk mengetahui perubahan densitas bakteri dan aspek fisikokimia selama fermentasi alami susu kuda sumbawa, dan mengetahui dinamika populasi bakteri selama fermentasi alami dengan menggunakan pendekatan metagenomik. sampel susu kuda yang berasal dari kabupaten dompu, provinsi nusa tenggara barat difermentasi secara alami pada suhu ruang selama 60 hari. pengambilan sampel susu dilakukan pada hari ke0, 7, 15, 30 dan 60 untuk dilakukan uji lebih lanjut. uji yang dilakukan meliputi enumerasi bakteri, pengukuran kandungan nutrisi, pengamatan sifat fisik susu, dan isolasi dna total. sampel dna total yang didapatkan dilakukan analisis . densitas bakteri next generation sequencing asam laktat pada media mrs agar dan m17 agar mengalami penurunan seiring periode fermentasi. sedangkan densitas bakteri aerobik pada media pca cenderung mengalami fluktuasi. faktor fisikokimia susu kuda sumbawa mengalami perubahan selama periode fermentasi. kandungan karbohidrat dan total gula mengalami penurunan seiring dengan turunnya nilai ph. sedangkan kadar lemak mengalami peningkatan, dan kandungan protein mengalami fluktuasi seiring periode fermentasi. perubahan sifat fisik susu kuda yang diamati sampai akhir fermentasi antara lain perubahan warna menjadi kuning, aroma yang semakin asam, dan terbentuknya gas. whey hasil analisis metagenomik diperoleh informasi bahwa indeks diversitas bakteri pada sampel di setiap periode fermentasi tergolong rendah yang ditandai dengan adanya dominansi sampai akhir lactobacillus helveticus periode fermentasi. yang merupakan anggota bal tidak tumbuh pada media isolasi di lactobacillus helveticus akhir periode fermentasi. keberadaan bakteri yang tidak dapat dikulturkan ini dapat diketahui melalui pendekatan metagenomik, yang melengkapi informasi komposisi bakteri pada produk susu kuda sumbawa terfermentasi. kata kunci: diversitas bakteri, fermentasi alami, , susu kuda sumbawanext generation sequencing microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 341-575841; : +62fax: +62341-554403; e-mail: jatmiko_yd@ub.ac.id thousand tons per year (data center and information system for agriculture 2019). however, the demand for fresh milk and its derivative products continues to increase. hence, alternatives to milk other than cows are important to be introduced to consumers. a promising milk commodity is mare's milk. mare milk is rich in milk is a promising food commodity in indonesia. milk production in indonesia is still dominated by cow's milk with an increasing rate of 9.80% per year or 905.49 essential nutrients and is recommended to be the alternative to human milk because their whole protein and salt content are comparable (malacarne 2002). et al. sumbawa mare's milk is an indonesian local commodity, mainly produced as a small scale by local household in sumbawa island, especially bima, dompu, and sumbawa regency. sumbawa mare's milk is marketed nationwide as naturally fermented milk. natural fermentation of mare's milk especially sumbawa mare's milk contain an array of natural microbes and some of them exert beneficial health effects (sujaya 2008; shi 2012). the natural et al. et al. fermentation process is mainly dominated by lactic acid bacteria species such as subsp. lactococcus lactis cremoris lactobacillus casei/lb. paracasei, lb. , plantarum lb. acidophilusand . besides lactic acid bacteria activity, acetic acid bacteria and yeast also contributed to texture formation and aroma during the natural fermentation of mare's milk (spinnler et al. 2001). natural fermentation is a process that lacks hygiene, therefore, bacterial contaminants from surrounding environment were detected in this product. as discussed by mulyawati (2019), they detect the et al. p o t e n t i a l p a t h o g e n b a c t e r i a f r o m g e n e r a staphylococcus ochrobactrumand in the fermented sumbawa mare's milk. it was more likely that these bacteria gained access to the product during milking process and associated with the fermentation utensils. therefore, the information of population dynamic during natural fermentation of sumbawa mare's milk is important to be elucidated as the foundation of quality control and further development of sumbawa mare's milk product. microbial diversity analysis of milk fermentation can be done using a culture-dependent as well as metagenomics approach or using the combination of both approaches. culture-dependent is a conventional method that depends on growing microbes on conventional growth medium, this method is limited to the viable-bacteria in the medium. meanwhile, there are many non-cultivable bacteria that undetected during the cultivation. metagenomics is a method that directly isolate the genome from environment to define the diversity and functional structure of the microbe (hoyles & swann 2019). using the combination of metagenomics and culture-dependent approach can generate complex data that give a preview of the natural fermentation process of sumbawa mare's milk. this experiment was focused on the changing of bacterial density, population dynamic, and physicochemical aspect during the natural fermentation of sumbawa mare's milk. materials and methods sample collection and fermentation of sumbawa mare's milk. sumbawa mare's milk was freshly collected from sumbawa horse breeder in dompu regency, west nusa tenggara province, indonesia. after milking, the mare's milk was placed in a sterile container and transported on ice to the laboratory for further analysis. the sumbawa mare's milk (1.5 l) was transferred into five sterile glass bottles, and each bottle was filled with 200 ml of fresh mare's milk. the natural fermentation process was performed by incubating the mare's milk for 60 day at room temperature. samples of fermented mare's milk were withdrawn at five distinct times: 0 (on the day the milk was received), 7, 15, 30, and 60 days. the subsamples of 100 ml each were collected for physicochemical analyses, 25 ml sample for bacterial isolation, 25 ml sample for ph measurement, 25 ml of sample for physical change observation, and 5 ml of sample for total dna extraction. isolation and enumeration of bacteria. bacterial isolation was conducted by collecting 25 ml mare's milk sample in every fermentation period. afterward, the sample was eluted to 225 ml of saline water (nacl 0.85%), and then serially diluted 10-fold (10 to 10 ). then, 1 ml aliquot of each diluted sample -1 -7 was used to determine the bacterial counts (cfu) by pour plate method with de man, rogosa and sharp (mrs)-agar and m17 agar supplemented with caco 3 1% to isolate and group lactobacillus lactococcus respectively (de man 1960; . et al. süle 2014)et al. plate count agar (pca) was used to isolate aerobic heterotrophic bacteria. the plates were incubated at 37 c for 24-48 hours, and the total colony was o determined. lactic acid bacteria colonies were shown by the presence of clear zone around the colonies. physicochemical analyses. sample of naturally fermented mare's milk was collected from each fermentation period (0, 7, 15, 30, and 60 days) for proximate analysis including ash, carbohydrate, fat, rough fiber, and water content, and total sugar based on the aoac method. physical properties of sumbawa mare's milk including color, odor, texture, and gas formation was observed qualitatively. the ph value was measured using a digital ph meter. dna extraction. the dna for metagenomic analysis was extracted from the samples collected in every fermentation period (0, 7, 15, 30 & 60 days). a 46 jatmiko et al . microbiol indones volume 15, 2021 microbiol indones 47 total of 5 ml sample was firstly added with 45 ml trisodium citrate 2% buffer. the mixture was then incubated for 5 minutes and homogenized for 1 minute using a vortex. subsequently, the mixture was centrifuged at 12000 rpm for 1 minute at room temperature. the resulting pellet was collected and used for total dna extraction. the total dna was extracted with dneasy powerfood microbial kit (qiagen, inc), using the manufacturer's instruction. the concentration and purity of the genomic dna was checked with a nanodrop 2000 spectrophotometer (nanodrop technologies, usa) and 1.5% agarose gel electrophoresis. pure dna samples were stored at 20 c until to be used for next-generation sequencing.o n e x t g e n e r a t i o n s e q u e n c i n g a n d bioinformatic analysis. total dna with a concentration at least 0,1 ng/µl were sent to ngs facility macrogen, inc., south korea. sequencing process were using 16s rrna region v3-v4 (341f: 5'c c t a c g g g n g g c w g c a g 3 ' ; 8 0 5 r : 5'gactachvgggtatctaatcc-3') (klindworth et al. 2013). the sequencing was performed on an illumina miseq platform. the paired-end sequences were analyzed using qiime2 2019.4. and changed into a manifest. the manifest was used to show the exact location of the file so it can be imported and analyzed using qiime2 ( . the sequence bolyen 2019)et al. reads obtained were demultiplexed and quality processed using dada2 (callahan 2016). a et al. high-quality sequence reads were further considered to generate species diversity estimation using shannon diversity index. the 16s rdna reference alignment, and taxonomical assignation were based upon the greengenes database as the reference ( mcdonald et al. 2012). results physicochemical properties. after 60 day of natural fermentation process of sumbawa mare's milk, several physical changes were observed such as aroma, texture, color change, and gas formation as summarized in table 1. the fermentation process of the mare's milk created a distinct characteristic that different from other milk products. the aroma of mare's milk getting more acidic during the fermentation periods, followed by changing of the milk color on day 30 until the end of the fermentation period. the texture of the milk was watery, and the gas formation was detected at every fermentation period, marked by the increasing pressure of the gas to the bottle cap. the nutrient content value of sumbawa mare's milk varied in every period of the natural fermentation process, indicating the change in the nutrient content of the milk (table 2). the natural fermentation process of sumbawa mare's milk allowed the carbohydrate and total sugar content gradually decreased during fermentation period. it was followed by the decrease of ph from 3.66 in the initial period to 3.37 on day 60. fat content on day 0 had a value of 1.41% and decreased until day 15 up to 1.26%. in the following periods, the fat content value increased until day 60 by 1.65%. meanwhile, the protein content of sumbawa mare's milk during the natural fermentation process increased in the beginning of the fermentation, from 1.15% on day 0 to 1.88% on day 7. after day 7, the protein content gradually decreased until end of fermentation (1.07%). during natural fermentation period, ash content on the milk slightly increased from 0,39% (day 0) to 0.43 (day 60), and water content gradually increased from 91.53% on day 0 to 96.32% on day 60. ash content of sumbawa mare's milk during the natural fermentation process was low but slightly increased from 0.39% to 0.43% in the end of the fermentation. bacterial density. bacterial population dynamic during the natural fermentation process of sumbawa mare's milk can be observed through the changing of bacterial density. three groups of bacteria were observed including aerobic bacteria, rod-shaped lactic acid bacteria ( ), and coccus-shaped lactic lactobacillus acid bacteria ( ). during five periods of lactococcus natural fermentation (0, 7, 15, 30, and 60 days), each growth medium had different variations of bacterial number (table 3). bacterial density from three media had an average of 1.87 log cfu/ml, ranging from 0 log cfu/ml to 3.50 log cfu/ml. growth medium for lactic acid bacteria (mrs agar and m17 agar, both with the addition of caco 1%) had 3 high density at the early period of fermentation. the mrs agar showed the highest cell density on day 7 (3.50 log cfu/ml) and the m17 agar had the highest value on day 0 (2.62 log cfu/ml) (table 3). however, in the following periods, the bacterial cell density decreased on both media, until 0 log cfu/ml in the last period. meanwhile, bacterial cell density on pca medium relatively fluctuated. on day 0, the bacterial density was 3.02 log cfu/ml and increasing up to 3.23 log cfu/ml on day 7. but the bacterial cell density decreased until day 30 reaching 2.04 log cfu/ml and increased again on day 60 up to 2.90 log cfu/ml. 48 jatmiko et al . microbiol indones bacterial diversity in different periods of natural fermentation. genomic sequencing of five periods of the natural fermentation process of sumbawa mare's milk generated high quality reads ranged from 17852 to 48687 (average 34375.80 reads), corresponding to a total of 269 otus (average 53.80 otus). alpha diversity of five periods was determined using three indices (table 4). shannon indices, simpson indices, and margalef indices ranged from 3.46 to 3.81, 0.88 to 0.91, and 2.89 to 10.09, respectively. shannon and simpson indices indicate species diversity in a sample. according to these indices, the highest diversity was achieved on day 30, followed by day 0. based on the margalef index, it was known that on the day 60 has high species richness. the value of margelef index correlated with the number of table 1 physical properties of sumbawa mare's milk during the natural fermentation process parameters fermentation periods (day) 0 7 15 30 60 aroma sour sour strongly sour strongly sour strongly sour texture watery watery watery watery watery color white white cloudy white yellowish yellowish gas formation yes yes yes yes yes table 2 nutrient content of sumbawa mare's milk during the natural fermentation process parameter fermentation periods (day) 0 7 15 30 60 carbohydrates (%) 5.52 4.41 4.53 1.37 0.53 protein (%) 1.15 1.88 1.57 1.46 1.07 fat (%) 1.41 1.31 1.26 1.62 1.65 water (%) 91.53 92.29 92.25 95.17 96.32 ash (%) 0.39 0.38 0.39 0.38 0.43 crude fiber (%) 0.04 0.02 0.03 0.02 0.01 total sugar (%) 3.11 2.96 1.89 0.59 0.19 ph 3.66 3.59 3.37 3.4 3.37 table 3 bacterial density during the natural fermentation process of sumbawa mare's milk culture medium bacterial cell number (log10 cfu/ml) day 0 day 7 day 15 day 30 day 60 mrs agar 3.14 3.50 1.95 0 0 m17 agar 2.62 2.51 0 1 0 pca 3.02 3.23 2.23 2.04 2.90 table 4 genomic analysis and bacterial diversity during natural fermentation of sumbawa mare's milk fermentation periods no. of reads no. of otu diversity indices no. of observed species shannon simpson margalef day 0 25193 45 3.76 0.90 4.34 12 day 7 44511 32 3.66 0.89 2.89 9 day 15 35636 40 3.46 0.88 3.72 15 day 30 17852 42 3.81 0.91 4,18 16 day 60 48687 110 3.59 0.88 10.09 76 mean ± sd 34375.80 ± 12908.14 53.80 ± 31.78 3.66 ± 0.14 0.89 ± 0.01 5.04 ± 2.88 25.60 ± 28.31 volume 15, 2021 microbiol indones 49 observed species. the total observed species during the natural fermentation process of sumbawa mare's milk was 128. alpha rarefaction curve also generated based on observed otus in every fermentation period (figure 1). the plots reached the plateau indicated that the sampling depth and sequencing coverage were good for all samples. bacterial composition. a total of 110 otus containing 20 phyla, 32 classes, 44 orders, 63 families, 73 genera, and 89 species were observed. five periods of fermentation showed similar bacterial structures at the phylum level. the most dominant phylum was firmicutes with relative frequency of day 0, 7, 15, 30 and 60 were 97.71%, 98.38%, 98.56%, 98.24%, and 96.12%, respectively. there were several other phyla including proteobacteria, bacteriodes, actinobacteria, and acidobacteria (figure 2a.). bacterial structure at the genus level was dominated by during the first period lactobacillus until the last period of natural fermentation. the relative frequency fluctuated from day 0 lactobacillus to 7 with increased from 92.55% to 95.87%. however, in the subsequent periods, the relative frequency was decreased. from day 15 to 60 the relative frequency was 94.45%, 80.31%, and 91.79%, respectively (figure 2b.). on day 30, the relative frequency dropped to 80.31%, but there was an increase in family lactobacillaceae to 14.53% (figure 2b.). the increasing amount of relative frequency on the family lactobacillaceae has occured because the database cannot assign the sequence until the genus level. another lactic acid bacteria group observed was lactococcus. their relative frequency was not as high as the group, but their population lactobacillus dynamic during natural fermentation was also notable. lactococcus frequency decreased on day 0 to 7, from 4.25% to 2.512%, but their relative frequency rose again to 4.09% on day 15. in the following periods, the relative frequency of decreased until lactococcus 0.67% at day 60 (figure 2b.). at the species level, was the lactobacillus helveticus most dominant species during the natural fermentation (figure 2c). their relative frequency was fluctuated. in the early period of fermentation (day 0 to day 7), the relative frequency increased from 89.20% to 95.51%. but after day 7, their relative frequency decreased from 93.51% (day 15), 78.61% (day 30) to 91.07% (day 60) (figure 2c). besides several lactobacillus helveticus, groups of lactic acid bacteria such as , lactococcus lactobacillus zeae, lactobacillus pontis, and lactobacillus delbrueckii were observed in an insignificant relative frequency. also, on day 60, methylobacterium komagatae (0.39%) was also observed (figure 2c). besides lactic acid bacteria groups, there was other potential pathogenic bacteria from the member of family enterobacteriaceae, such as and citrobacter methylobacterium komagatae (figure 2c). a low number of uncultured and uncharacterized bacteria (others) were identified in the fermented mare's milk using ngs, with the relative frequency in the fermentation process ranging from 0.1-2.49%. the highest relative frequency of other group was achieved on day 60 (2.49%). discussion during natural fermentation, sumbawa mare's milk undergone several changes in its physicochemical properties. first, the aroma of the milk became more acidic. it was correlated with the ph value that keeps decreasing until the last period of natural fermentation. natural fermentation of the mare's milk was performed by lactic acid bacteria and acetic acid bacteria that already exist in the milk (yao 2017; gemechu et al. 2015). the longer the fermentation process, the milk color changed to yellowish. changing the color of the mare's milk could happen due to a reaction between vitamin b (riboflavin) and lactose on milk ( 2 goulding et al. 2020). therefore, this hypothesis requires to be proven because the vitamin content was not measured in this study. t he m il k f e r me n t a t i on pr o c ess u t ili z e s carbohydrates and converting them to organic acids (lactic acid and acetic acid), and co ( 2 widyastuti et al. 2014). as a result, the carbohydrates and total sugar content in the sumbawa mare's milk gradually decreased during the fermentation periods. the low carbohydrates and sugar content on the late period of natural fermentation (day 30 and day 30) limit the growth of the lactic acid bacteria group. low sources of energy and nutrient can restrain bacterial growth ( 2009). barboza et al. the total protein content of the milk decreased during the fermentation periods. lactic acid bacteria activity in the milk needs amino acid to support their existence ( 2006). in fact, the amount of savijoki et al. amino acid in the milk was insufficient. to handle this situation, lactic acid bacteria possess complex proteinase and peptidases to enable them to produce essential amino acids for their growth ( tzvetkova et al. 2007). hydrolysis protein also provides a health 50 jatmiko et al . microbiol indones benefit to humans due to the reduction of the milk allergenicity. species from the lab group such as, bifidobacterium lactis can hydrolyze -lactoglobulin, β which is the strongest antigen leading to milk allergy ( 2005).prioult et al. the total fat content on mare's milk is the lowest compare to other livestock, the average fat content in mare's milk is 1.67% ( 2010). the uniacke-lowe et al. total fat content of sumbawa mare's milk decreased from 1.41% to 1.26% on day 0 until day 15. the decreasing of fat content during this period could occur due to the lipolytic activity of bacteria mainly lactic sequencing depth day 0 day 7 day 15 day 60 fig 1 alpha rarefaction curve from five periods of the natural fermentation process of sumbawa mare's milk. fig 2 bacterial composition during natural fermentation of sumbawa mare's milk. (a) phylum level. (b) genus level. (c) species level. volume 15, 2021 microbiol indones 51 acid bacteria ( 2015). the lipolytic activity of bao et al. lactic acid bacteria can produce a distinct aroma caused by fatty acid oxidation into ketone ( 2012). hassan et al. therefore, after day 15, the fat content on sumbawa mare's milk steady increased until day 60 (table 2). the accumulation of free fatty acid due to the lipolytic activity of lactic acid bacteria was able to increase the total fat content of the mare's milk (coşkun & öndül 2004). ash content of mare's milk ranged from 0.32% to 0.61% based on the mare lactation period (schryver et al., 1986). the increasing ash content during the fermentation process can be caused by other milk component reductions such as carbohydrate and fat ( 2013). meanwhile, the water content of obadina et al. sumbawa mare's milk increased which was in contrast to the decrease of carbohydrate and total sugar content. the increasing water content occurred due to sugar was metabolized using tricarboxylic acid cycle by lactic acid bacteria and produced co and h o (2 2 mamlouk and . gullo 2013) the bacterial density in the initial of fermentation (day 0) was ranged from 2.62 to 3.14 log 10 cfu/ml. the value was different in every medium. raw mare's milk had lower bacterial density than raw cow milk, the average just 4.6 log cfu/ml (doreau & martin-rosset 2011; salimei & fantuz 2012). this value can be different due to horse type, and lysozyme and lactoferrin activity of the milk ( 2012). šarić et al. the rarefaction curve of all fermentation periods already reached its plateau, which means that the sequencing depth already covered the bacterial diversity ( 2020). zhao et al. lactobacillus helveticus was the most dominant species throughout the fermentation process of sumbawa mare's milk. lactobacillus helveticus was also found as dominant species in airag, which this species had high proteolysis ability (miyamoto 2015). et al. lactobacillus helveticus was first isolated by orla-jensen from emmental cheese ( 2006). other lactic acid naser et al. bacteria groups were also detected in a lower relative abundance representing , lactococcus lactobacillus pontis, lactobacillus zeae, lactobacillus and delbrueckii. the role of lactic acid bacteria in producing a natural antimicrobial agent which can enhance the shelf-life of the milk and serve as a beneficial microorganism ( 2000). benkerroum et al. citrobacter is a common intestinal microbiota of humans and animals, usually are harmless commensals, but several species such as citrobacter freundli capable of acting as an opportunistic pathogen ( 2018). meanwhile, fusco et al. methylobacterium komagatae are facultative methylotrophic bacteria that can be found almost in every habitat such as soil, freshwater, lake sediment and have a close association with plants (bracke 2000; nercessian 2005; et al. et al. crump & koch 2008; kato 2008; podolich et al. et al. 2009). however, the relative abundance of potential pathogenic bacteria was detected in a low number. this can happen due to the competitive advantage of lactic acid bacteria by producing some antimicrobial substances especially organic acids, and bacteriocins ( 2018). the relative proportion of identified singh bacteria was higher than the unclassified and uncharacterized bacteria suggested that the identified bacteria play an pivotal role in the natural fermentation process. the utilization of both culture-dependent and culture-independent analyses to observe the dynamic of the bacterial population of sumbawa mare's milk during natural fermentation successfully showed the dynamic of bacteria throughout the fermentation process. however, this technique still needs to be improved, due to no correlation between the result of culture-dependent and culture-independent methods. by using metagenomic approach, lactobacillus helveticus was detected in all fermentation s, however, in the culture-dependent method, the bacterial density on mrs agar and m17 agar gradually decreased until no bacterial colonies found on day 60. this can be occurred due to specific growth requirements for certain bacteria ( 2020). another example zhao et al. was several sequences cannot be annotated into species level due to discrepancy of sequence with greengenes database. if the database cannot match the sequence until the species level, it will correspond to the upper taxonomic level ( 2018). further shangpliang et al. study required to be conducted to confirm this dominant bacterium using dependent-culture method. acknowledgements this study was funded by hibah peneliti pemula (dipa-023.17.2.677512/2020), universitas brawijaya. references data centre and information system for agriculture. 2016. buku utlook omoditas eternakan: usu api [book o k p s s of utlook for ommodity nimal usbandry: cow's o c a h milk]. the general secretariat of the ministry of a g r i c u l t u r e . 6 1 p . a v a i l a b l e f r o m : 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collected in inner mongolia. j. dairy sci.103(1):106116. doi: 10.3168/jds.2019-17023. 02. mukamto (isolation).cdr isolation of oxo-degradable polyethylene degrading-bacteria of benowo landfill soil surabaya 1 1 1 2 mukamto* , yuni sri rahayu , lisa lisdiana , and hardaning pranamuda 1 department of biology, faculty of mathematics and natural sciences, universitas negeri surabaya, jalan ketintang baru, wonokromo, surabaya 60231, indonesia; 2 center for bioindustrial technology, gedung 611, laptiab, puspiptek-serpong, banten, indonesia the widespread consumption of oxo-degradable polyethylene plastics in indonesia is potentially cause pollution. this problem can be overcome by utilizing plastic degrading-bacteria as degradation agent. the present study aims to isolate and characterize of oxo-degradable polyethylene degrading-bacteria from benowo landfill soil surabaya. isolation and screening was done by the culture technique and clear zone method. total abundance of bacteria was calculated based on the total plate count method. bacterial colonies screening was done based on morphological characteristics and the diameter of clear zone. four isolates with the largest diameter of clear zone were futher characterized based on cell morphology and physiology biochemistry characters. the results showed the average of total abundance of oxo-degradable polyethylene degrading4 -1 bacteria of benowo landfill was 1.28 x 10 cfu g . the four of twelve isolates with the largest diameter of clear zone showing the highest degradation capability, that were isolates a221 (0.7 cm), a231 (1 cm), a232 (0.6 cm), and c231 (1.3 cm). colony morphology characteristics of four isolates were generally shape of circular and irregular; margin of entire and lobate; elevation of flat; optics of opaque; and pigmentation of yellow, greenish and cream. these four isolates were gram negative with the shape bacilli and cell size range of 3-4 µm. physiological and biochemical characteristics among the four isolates were resistant to acidic conditions; nonmotile; produce catalase enzyme except a231; capable to reduce glucose and mannitol; uncapable to reduce lactose; capable to produce acids, except a232; produce of 2.3-butanediol; unable reduce sodium malonate; and capable to reduce nitrate, except a232 and c231; as well as producing hydrogen sulfide except a221 and a232. the four isolates has similar properties with the genus mycobacterium. key words: benowo landfill, characterization, isolation, oxo-degradable polyethylene-degrading bacteria penggunaan plastik jenis polietilen oxo-degradable di indonesia cukup tinggi sehingga berpotensi menimbulkan pencemaran. salah satu metode untuk mengatasi pencemaran yang disebabkan oleh sampah plastik polietilen oxo-degradable adalah dengan memanfaatkan isolat bakteri yang mampu mendegradasi plastik tersebut. tujuan penelitian ini adalah mengisolasi dan mengkarakterisasi bakteri pendegradasi polietilen oxodegradable dari tanah tpa benowo surabaya. isolasi dan skrining bakteri dilakukan menggunakan teknik kultur dan pengamatan zona bening. kelimpahan total bakteri dihitung berdasarkan metode angka lempeng total. koloni bakteri diskrining berdasarkan karakteristik morfologi koloni dan zona bening yang terbentuk. empat isolat bakteri dengan diameter zona bening terbesar dikarakterisasi morfologi sel dan fisiologi biokimia. hasil 4 -1 rerata kelimpahan total bakteri sebesar 1,28 x 10 cfu g . empat dari duabelas isolat memiliki diameter clear zone terbesar yang menunjukkan kemampuan degradasi paling tinggi, yaitu isolat a221 (0,7 cm), a231 (1 cm), a232 (0,6 cm), dan c231 (1,3 cm). karakteristik morfologi koloni keempat isolat tersebut adalah bentuk circular dan irregular; tepian entire dan lobate; elevasi flat; optik opaque; serta pigmentasi kuning, kehijauan dan cream. keempat isolat bakteri merupakan bakteri gram negatif berbentuk basil dengan ukuran sel berkisar 3-4 µm. karakteristik fisiologi biokimia keempat isolat diantaranya tahan kondisi asam, non motil, menghasilkan enzim katalase kecuali a231, mampu mereduksi glukosa dan manitol, tidak dapat mereduksi laktosa, mampu memproduksi asam campuran kecuali a232, produksi 2,3-butanidiol, tidak dapat mereduksi sodium malonat, mampu mereduksi nitrat kecuali a232 dan c231, serta memproduksi hidrogen sulfida kecuali a221 dan a232. keempat isolat bakteri memiliki kemiripan dengan genus mycobacterium. kata kunci : bakteri pendegradasi polietilen oxo-degradable, isolasi, karakterisasi, tpa benowo *corresponding author; phone: +62-85648219005, e-mail: mukamtomuhammad@gmail.com during the past 25 years, plastic materials have gained widespread use in human life and substituted the role of metal and wood (zusfahair et al. 2007; raaman et al. 2012). plastics characteristics are strong, lightweight, durable, easy to set up, and anti-rust (kathiresan 2003; kavitha et al. 2014). however, plastics disadvantageous characteristics are not heatresistant, easy to damage at low temperatures, and hard to degrade (kathiresan 2003; akbar et al. 2013). vol.9, no.1, march 2015, p 9-16 doi: 10.5454/mi.9.1.2 plastics are composed of petroleum based materials called resins (e.g polythene and polypropylene). this material is resistant to biodegradation, hence potentially cause pollution in the environment (kumar et al. 2007; kavitha et al. 2014). one kind of the plastic waste that hard to degrade is p o l y e t h y l e n e . p o l y e t h y l e n e ( p e ) i s a l i n e a r hydrocarbon polymers consisting of long chains of the ethylene monomers (tokiwa et al. 2009; usha et al. 2011). polyethylene is hard to degrade, which results of highly stable c-c and c-h covalent bonds, the addition of antioxidants and stabilizers, also high molecular weight and density (arutchelvi et al. 2008; leja and lewandowicz 2009). recently, biodegradable polyethylene called the oxo-degradable polyethylene was developed. the oxo-degradable polyethylene is a polyethylene with the addition of additives in the form of transition metals (mn-stearate, cu-stearate, nistearate and fi-stearate) and has exposed to uv irradiation, so it has a lower molecular weight than conventional polyethylene (europeanbioplastics 2009). transition metals are usually employed as prooxidant additives and they catalyzed the chain scission of pe through a free radical chain reaction (suresh et al. 2011). according to tokiwa et al. (2009), the presence of additives (such as starch) on the polymer pe made it easier to degrade by microorganisms in the environment than conventional polyethylene. naturally, oxo-biodegradable polyethylene will be degraded in 2 years (usha et al. 2011; wahono 2010). continuous accumulation of plastic waste in environment can cause pollution and threat to humanity and environment (deepika and jaya 2015). in general, the plastic waste in the environment is processed by means of burn or bury. burning or burying of the plastics releases harmful toxic gases, such as co and co which is a major pollutant in 2 environment (zusfahair et al. 2007; kavitha et al. 2014). another method to reduce plastic waste is by landfilling technique, but this technique is less effective because it requires a wide area (sihaloho 2011). alternative solutions that can be offered is biodegradation. biodegradation is the chemical processes of material degradation caused by the activity of microorganisms such as bacteria, fungi, algae and others (leja and lewandowicz 2009). the microbial species associated with the degrading capability were reported as bacteria pseudomonas sp (kathiresan 2003; gupta et al. 2010; usha et al. 2011), bacillus sp (gupta et al. 2010; usha et al. 2011), staphylococcus sp (usha et al. 2011), streptomyces sp, micrococus sp, moraxella sp (kathiresan 2003), klebsiella sp, nocardia sp, mycobacterium sp (leja and lewandowicz 2009), xanthomonas sp, flavobacterium sp, agrobacterium sp (gupta et al. 2010). polyethylene degrading-bacteria capable to produce extracellular and intracellular depolimerase enzymes to catabolize the plastic polymers into monomers. hence, the plastics is easier to accumulate in the cells of microorganisms and used as the source of carbon and energy (prabhat et al. 2013). plastics monomers metabolism by aerobic bacteria produce carbon dioxide and water, while anaerobic metabolism produces methane gas, hydrogen sulfide, carbon dioxide, and water (usha et al. 2011). this research was conducted to obtain indigenous isolates of oxo-degradable polyethylene degradingbacteria of benowo landfill surabaya soil and characterize the bacterial isolates. materials and methods material. oxo-degradable polyethylene plastic powder was obtained from pusat pengkajian dan penerapan bioteknologi industri dan pertanian, puspiptek, tangerang. sample collection. soil sample were collected from three sites (a, b, c) of benowo landfill surabaya. these sites were chosen based on the major waste type (which is plastics waste) and the degradation level of the palstics waste on the sites. soil samples were taken from a depth of 5-10 cm and collected in sterile bottles, then the temperature and ph were measured and recorded. enrichment media. this research use enrichment broth and enrichment agar media. oxo-degradable polyethylene powder were added in both media as main carbon source for the bacteria. enrichment broth media contain 0.1% (nh ) so , 0.1% nano , 0.1% 4 2 4 3 kcl, 0.02% mgso , 0.01% yeast extract, 2 g oxo-4 degradable pe powder and dissolved in 1 l steril h o 2 (burd 2008). enrichment agar media contain 0.1% (nh ) so , 0.1% nano , 0.1% kcl, 0.02% mgso , 4 2 4 3 4 0.01% yeast extract, 2 g oxo-degradable pe powder -1 -1 and added 12 g l agar and 1 ml l tween 80. tween 80 were added to optimize the biodegradation process. both media were adjusted to ph 5 with 5n hcl.the growth media was sterilized at 121 °c for 20 min. isolation and screening of oxo-degradable polyethylene degrading-bacteria. isolation and screening were done by culture technique (burd 2008) 10 mukamto microbiol indones and clear zone method (usha et al. 2011). 1 g of each soil sample was dissolved in erlenmeyer flasks contain 20 ml enrichment broth, then shaken at 32 °c for 4 weeks in a waterbath shaker (200 rpm). after 4 weeks incubation, each soil suspension was taken as 1 ml in test tubes containing 9 ml of enrichment broth media -1 -1 for the 10 dilution. dilution series was done from 10 -4 to 10 dilution. 0.1 ml of the each dilution was taken and planted in the enrichment agar media with pour plate method. for each sample, two replicas maintained and kept for incubated at 32 °c for 2-7 d. the isolates capable of forming clear zone were characterized based on colony morphology. four of bacterial isolates that able to form the largest clear zone were further characterized for their cell morphology and physiology biochemistry characteristics. characterization of oxo-degradable polyethylene degrading-bacteria. degrading-bacteria were characterized by observing colony morphology, cell morphology and physiology biochemical properties. the cell morphology was observed based on cell shape, cell arrangement, gram and size of the cell. bacterial cell size was measured by using a compound microscope equipped with a camera (olympus) and analyzed by software scopeimage 9.0 (hic). physiological and biochemical test perform in this study were acid-fast staining, malonate test, and hydrogen sulfide production (benson 2001); motility test (pelczar and chan 1986); catalase test, sugar reduction, nitrate reduction, and acids fermentation test (hadioetomo 1993). results total abundance of oxo-degradable polyethylene degrading-bacteria. the total abundance of oxo-degradable polyethylene-degrading bacteria of 4 -1 different sites varied from 1.24 x 10 cfu g to 1.26 x 4 -1 10 cfu g . the average of total abundance of oxodegradable polyethylene degrading-bacteria from 4 -1 benowo landfill was 1.28 x 10 cfu g (table 1). isolation of oxo-degradable polyethylene degrading-bacteria. the four isolates with the largest d i a m e t e r o f c l e a r z o n e i n d i c a t e d t h e h i g h e s t degradation of oxo-degradable polyethylene capability, they were isolate a221, a231, a232, and c23 (fig 1). those four isolates were characterized for colony morphology, cell morphology, cell size, table 1 the total abundance of oxo-degradable polyethylene degrading-bacteria from soil sample of benowo landfill surabaya soil sample cfu/g a 1,24 x 104 b 1,32 x 104 c 1,26 x 104 average 1,28 x 104 fig 1 formation of clear zone on bacterial isolates colony were a221 (a), a231 (b), a232 (c), and c231 (d). a, b, and c were taken from the ic site of benowo landfill surabaya volume 9, 2015 microbiol indones 11 a b c d biochemical and physiological characteristics. the characteristics of four isolates has similar properties with the genus mycobacterium (table 2). discussion the average of total abundance of oxo-degradable polyethylene degrading-bacteria from benowo landfill 4 -1 was 1.28 x 10 cfu g (table 1). this result was nearly similar to the prior study of kumar et al. (2007) who 4 -1 4 -1 reported 1.07 x 10 cfu g to 1.35 x 10 cfu g bacteria was found in the mangrove soil sample at suva, fiji islands. however, total abundance of oxodegradable polyethylene degrading-bacteria from benowo landfill was lower than those reported by kathiresan (2003) and usha et al. (2011). kathiresan (2003) reported that total abundance of polyethylene degrading-bacteria from mangrove avicennia 4 -1 rhizosfer was 24.50 x 10 cfu g and rhizophora 4 -1 rhizosfer was 41.33 x 10 cfu g . usha et al. (2011) reported that total abundance of polyethylene degrading-bacteria from garbage soil in india was 4 -1 62.71 x 10 cfu g . however, such variation of total abundance of bacteria can occur between different geographical locations owing to differences in the environmental parameters (kumar et al. 2007). isolation and screening of oxo-degradable polyethylene degrading-bacteria were found 12 isolates. those four isolates able to form clear zone, they were isolate a221, a231, a232 and c231 (fig 1). colony morphology characteristics of those four isolates were generally shape of circular and irregular; margin entire and lobate; elevation of flat; optics of opaque; and pigmentation of yellow, greenish and cream. those four isolates had the characteristics of cell were bacilli, with cell arragement monobacill for a221 and a231, diplobacill for a232 and streptobacill for c231; gram negative; and cell size range of 3-4 µm (table 2). thakur (2012) and kathiresan (2003), reported that polyethylene-degrading bacteria were included as gram positive and gram negative bacteria. isolation and screening of oxo-degradable table 2 characterization of four oxo-degradable polyethylene degrading-bacteria isolates with largest diameter of clear zone from soil sample of benowo landfill surabaya 12 mukamto microbiol indones characteristics bacterial isolates a221 a231 a232 c231 colony morphology shape irregular circular circular circular margin lobate entire lobate threed-like elevation flat flat flat flat optic opaque opaque opaque opaque pigmentation cream greenish yellow ish dim yellow clear zone diameter (cm) 0,7 1 0,6 1,3 cell morphology shape bacilli bacilli bacilli bacilli arrangement monobacilli monobacilli diplobacilli streptobacilli gram size 3.077 3.094 3.610 4.375 biochemical physiology test acids-tolerant + + + + hydrolysis h2o2 + + + motility test non motile non motile non motile non motile glucose reduction + + + + lactose reduction mannitol reduction + + + + methyl red test + + + voges proskauer test + + + + malonate utilization nitrate reduction + + h2s production + + location of bacterial isolates soil sample a,b,c a,b,c a,b,c c probable genus* mycobacterium mycobacterium mycobacterium mycobacterium th * = holt jg, krieg nr, sneath pha, staley jt, williams st. 1994. bergey's manual of determinative bacteriology. 8 edition. polyethylene degrading-bacteria from benowo landfill surabaya use two method, they are culture technique (burd, 2008) and clear zone (usha et al. 2011). tokiwa et al. (2009), reported that clear zone method is a widely and effective to be used for screening polymer degrading-bacteria and assessment of the degradation potency of different microorganisms towards a polymer. the clear zone around the colonies showed polymer degrade as carbon source by bacteria. in the enrichment media was added oxo-degradable polyethylene powder as primary carbon source for bacteria, so bacteria grew in this media was bacteria with oxo-degradable polyethylene degrading capability. in addition, bacteria isolates with low degrade capability was not formed clear zone around colonies. the size of clear zone around the colonies depands on bacteria capability to degrade of oxodegradable polyethylene, availability of functional groups that increases hydrophilicity, size molecular weight and density of the polymers, structural complexity (linearity or presence of branching) in the polymers, nature and physical form of the polymers (films or powder), and presence of ester or amida bonds (arutchelvi et al. 2008). the enrichment agar media was added emulsifier of tween 80. tween 80 is non-ionic surfactants. the function of tween 80 is to enhance biodegradation of polyethylene by increasing the adhesion of the bacteria towards the polyethylene surface (albertsson et al. 1993). oxo-degradable polyethylene is biodegradable plastics. it is easier to degrade than conventional polyethylene (wahono 2010). the presence of additives on oxo-degradable polyethylene polymers such as transition metals will result in easier to oxidize process at thermal degradation and photodegradation (kalus 2007). jeon and kim (2014) reported that the transition metals of fe-stereate additives is more suitable as the photodegradation catalyst of pe than mn-stereate and co-stereate. polymers oxidation will cause lower molecular weight of pe, hence its easier to degrade by microorganisms (europeanbioplastics 2009; tokiwa et al. 2009). luckachan (2006) states that the biodegradation is divided into two stages, they are abiotic degradation and biotic degradation. abiotic degradation can occur through thermal degradation, photooxidative, hydrolytic, chemical, mechanical-chemical, and additioninduction, whereas biotic degradation is performed by bacterial degradation. at the abiotic degradation, oxidation of the carbon chains (e.g photodegradation) will cease the change of the backbone structure of oxo-degradable polyethylene polymers (leja and lewandowicz 2009). oxidation of the carbon chains polymers results in the formation of functional groups such as carboxylic acids, aldehydes, ketones, lactones, and low molecular weight hydrocarbons (chiellini et al. 2006). the formation of functional groups in the polymers properties cause the change of hydrocarbon polymers fig 2 oxo-degradable polyethylene degrading-bacteria isolates from benowo landfill soil surabaya were a221 (a), a231(b), a232 (c), and c231 (d). volume 9, 2015 microbiol indones 13 a b c d characteristics from hydrophobic to hydrophilic. the change of the hydocarbon polymers characteristics made it able to absorb water and facilitate to degrade of polymers by microorganisms (ammala et al. 2011). the next stage is biodegradation by microorganisms (bacteria). oxo-degradable polyethylene degrading-bacteria consume polyethylene as the primary carbon source. it able to reduce of partial plastic. firstly, the bacteria will colonize the oxodegradable polyethylene surfaces by forming biofilms (usha et al. 2011). the bacteria also produce extracellular enzyme to degrade the polymer backbone into smaller fragments and lower molecular weight (oligomers, dimers, monomers). the polymer backbone are degraded through some steps, involve several extracellular enzymes. the functional groups (carbonyl groups) are converted to alcohol by the monooxygenase enzyme. after that, alcohol is oxidized to aldehyde by the alcohol dehydrogenase enzyme. next, aldehyde is converted to the fatty acid/carboxylic acid, ketone or ester by dehydrogenase enzyme. the fragments are then transferred into bacteria cells (mineralization) (premraj and doble 2004; ammala et al. 2011; roy et al. 2011; usha et al. 2011). inside the cells, it is metabolized via β-oxidation cycle with the aid of intracellular enzymes (leja and lewandowicz 2009). in the β-oxidation cycle, the carbon polymer molecules are broken down to produce 2-carbon acetyl coa. then acetyl-coa are moved to citric acid cycle and produce co and h o in aerobic 2 2 conditions or co , h o, ch , and h s in anaerobic 2 2 4 2 conditions (premraj and doble 2004; leja and lewandowicz 2009). the res ults of phys iological biochemical characterization of four isolates showed isolates were acid-resistant condition, and capable to produce the catalase enzyme except isolate a231. motility test of bacteria used semisolid medium na, showed isolates was non-motile. non-motile of bacteria isolates was characterized by bacteria growth does not spread (pelczar and chan 1986). reduction test of glucose and mannitol showed positive results for 4 isolates, but it can not reduce lactose. this suggests that the bacterial isolates uncapable to produce the lactase enzyme to breakdown lactose and utilize glucose as a carbon source. these results are consistant with prior study that polyethylene degrading-bacteria isolated from soil samples do not produce the lactase enzyme. marista et al. (2013) stated that most of the soil bacteria uncapable to reduce lactose. m r-v p test is used to determine of acids fermentation presence. the bacteria with acids fermentation ability can produce co and h , because 2 2 formic hydrogenylase enzyme presence (hadioetomo 1993; benson 2001). three of four isolates showed positive results and one isolate showed a negative result, that is isolate a232. this indicated that the isolate a232 unable to reduce sugar in anaerobic conditions, hence acidic compounds is not form. the fermentation test of 2,3-butanediol in the four isolates showed positive results, showed the presence of acetoin as the precursor for 2,3-butanediol formation (hadioetomo 1993). nitrate reduction test showed two isolates were able to utilize nitrate as an electron acceptor, which are isolates a221 and a231. two other isolates unable to utilize nitrate, which are isolates a232 and c231. in the malonate utility test, showed all bacteria isolates were not utilize the sodium malonat as carbon source. it is indicated by the absence of color change in the malonate broth media. hydrogen sulfide production test done using tsia media. in sulfite, plastic polymer will be bioassimilitated to produce co , h o, h s, and biomassa by 2 2 2 bacteria (hydrogen sulfide) (roy et al. 2011). certain bacteria can produce hydrogen sulfide from the amino acid cysteine . the presence of cysteine desuffurase enzyme will transform cysteine into an alfa-amino cacrylic acid, then alfa-amino cacrylic acid is transformed to amino acid. next, amino acid is formed pyruvaic acid (benson 2011). the result of hydrogen sulfide production test were two isolates able to produce hydrogen sulfide, they are a232 and c231. this indicated that both isolates capable to degrade the polyethylene polymers. in aerobic condition, degradation process of plastics polymer will generate residues such as, co , h o, ch and h s (leja and 2 2 4 2 lewandowicz 2007). this present study only performed isolation, c h a r a c t e r i z a t i o n , a n d i n i t i a l s c r e e n i n g o f biodegradation ability of the isolates. thus, further characterization were needed to identify the isolates. analysis of the degradation potency of the isolates as microbial consortia were also needed. references albertsson ac, sares c, karlsson s. 1993. increased biodegradation of ldpe with nonionic surfactant. acta polymerica. 44 (5): 243-246. doi: 10. 1002/actp. 1993. 010440506. akbar f, anita z, harahap h. 2013. pengaruh waktu simpan 14 mukamto microbiol indones film plastik biodegradasi dari pati kulit singkong terhadap sifat mekanikalnya [the influence of storage time of biodegradable plastic film from cassava peel starch on the mechanical characteristics]. jurnal teknik kimia usu. 2 (2): 1-5. ammala a, bateman s, dean k, petinakis s, sangwan p, wong s, yuan q, yu l. 2011. an overview of degradable and biodegradable polyolefins. progress p o l y m e r s c i . 3 6 ( 8 ) : 1 1 0 6 . d o i : 1 0 . 1 0 1 6 / j . progpolymsci.2010.12.002. arutchelvi j, sudhakar m, arkatkar a, doble m, bhaduri s, uppara pv. 2008. biodegradation of polyethylene and polypropylene. indian j biotechnol. 7: 9-22. benson. 2001. microbiological application laboratory edt manual in general microbiology. 8 . the mcgraw-hill companies. burd d. 2008. plastic not fantastic. 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(10): 37223742. doi: 10.3390/ijms10093722. usha r, sangeetha t, palaniswamy m. 2011. screening of polyethylene degrading microorganisme from garbage soil. libyan agriculture research center journal internasional. 2 (4): 200-204. wahono, tri (ed.). 2010. plastik oxium terurai dalam dua tahun [oxium plastic degradation in two years]. [online] (www.hariankompas.com diakses 9 maret 2014). zusfahair, lestari p, ningsih dr, widyaningsih s. 2007. biodegradasi polietilen menggunakan bakteri dari tpa (tempat pembuangan akhir) gunung tugel kabupaten banyumas [biodegradation of polyethylene use bacterial mount tugel landfiling banyumas]. molekul. 2 (2): 98-106. 16 mukamto microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 2. 540 antimicrobial (adolf).cdr antimicrobial activity of melinjo seed and peel extract ( ) against selected pathogenic bacteriagnetum gnemon adolf jan nexson parhusip azis boing sitanggang1* 2,3and 1 2 3 department of food technology, universitas pelita harapan, mh. thamrin boulevard, 1100 lippo village, tangerang, indonesia; department of food science and technology, institut pertanian bogor, darmaga , bogor 16680, indonesia; southeast asian food and agricultural science and technology (seafast) center, institut pertanian bogor, darmaga, bogor 16680, indonesia gnetum gnemon emping aspergillus flavus bacillus cereus , staphylococcus aureus enterobacter aerogenes b. cereus s. aureus gnetum gnemon gnetum gnemon aspergillus flavus bacillus cereus , staphylococcus aureus enterobacter aerogenes minimal inhibitory concentration minimal bactericidal concentration b. cereus s. aureus gnetum gnemon melinjo ( ) is an indonesian native plant which has not been widely accepted due to its limited utilization. mainly, melinjo is consumed as an ingredient to make a vegetable dish or as raw material of ' '. the purpose of this research was to study the antimicrobial activity of the melinjo seed extract and melinjo peel extract. in this study, extraction from melinjo seed and peel was conducted by maceration using three kinds of solvent: ethanol, ethyl acetate and hexane for 24 h at room temperature. the results showed that none of the melinjo extracts (concentration from 5% 25% w/v) could inhibit the growth of ipbcc 88.030; whereas for atcc 10876 atcc 25953, and atcc 13048 there was efficient inhibition by 5% (w/v) of melinjo seed-ethanol extract. the minimal inhibitory concentration (mic) value of melinjo extract was ranged from 0.26 g ml to 1.46 g ml , whilst the minimal bactericidal concentration (mbc) value was ranged from 1.02 g ml to 6.04 g ml . the inhibitory capacity of extract had a similar level as compared to 10 ppm penicillin g on atcc 10876 as well as on atcc 25953. furthermore, as compared to 10 ppm streptomycin, the inhibitory capacity of the extract was equal for the all tested bacteria. cell wall deformation was observed using sem, and confirmed by the presence of ions (ca and k ) outside of the cells, detected by means of aas. key words: melinjo, , extraction, antimicrobial, inhibition melinjo ( ) adalah tanaman lokal indonesia yang belum dimanfaatkan secara luas. umumnya melinjo dikonsumsi sebagai komponen dalam pembuatan sayur ataupun dalam pembuatan kue kering yang dikenal dengan emping. tujuan penelitian ini adalah mengkaji aktivitas antibakteri ekstrak melinjo yang berasal dari biji dan kulit melinjo. proses ekstraksi dilakukan dengan metode maserasi menggunakan tiga jenis pelarut, yaitu etanol, etil asetat, dan heksana selama 24 jam pada suhu ruang. hasil ekstraksi biji melinjo (5% w/v) dengan menggunakan pelarut etanol sampai dengan konsentrasi 25% (w/v), ternyata tidak menunjukkan aktivitas antifungal khususnya terhadap pertumbuhan ipbcc 88.030; namun demikian ekstrak melinjo tersebut memiliki aktivitas antibakteri, khususnya terhadap atcc 10876 atcc 25953, dan atcc 13048. nilai (mic) dan (mbc) dari ekstrak melinjo untuk berbagai bakteri yang diujikan berturut-turut berada pada selang 0.26 g ml hingga 1.46 g ml , dan 1.02 g ml hingga 6.04 g ml . penghambatan ekstrak melinjo menunjukkan kemampuan yang hampir sama dengan 10 ppm penisilin g terhadap bakteri contoh atcc 10876 dan atcc 25953, sedangkan jika dibandingkan dengan streptomisin, kemampuan penghambatan ekstrak melinjo hampir mirip dalam beberapa variasi konsentrasi. uji konfirmasi berupa pengamatan struktur sel bakteri menggunakan sem menunjukkan terjadinya perubahan pada struktur dinding sel bakteri dan hal ini diperkuat dengan adanya pengeluaran sejumlah ion yang menyusun dinding sel (ca and k ) yang dikuantifikasi menggunakan aas. kata kunci: melinjo, , ekstraksi, antimikrob, penghambatan μ μ μ μ μ μ μ μ -1 -1 -1 -1 2+ + -1 -1 -1 -1 2+ + melinjo ( ) is native to indomalaya, belonging to the gnetacea family (kato . 2009; kato . 2011). the size of the melinjo tree is about 50 ft in height and it is often found in dry and humid forests of the region. particularly in indonesia, the distribution areas of this plant cover in andaman, sumatra and java island (manner and elevitch 2006). for sumatra itself, the productivity of melinjo is more than 20,000 granules per year (manner and elevitch, 2006). it is a spontaneous re-growth species in fallow forests and commonly planted as a cultivated species in both backyard gardens and orchards or as hedges. in gnetum gnemon et al et al addition to those, melinjo has been utilized as a conservation farming component at the upland watershed in semi-arid climate (hafif . 1995). for local food products, melinjo seeds are commonly used as raw material for making ' (common name of the traditional cracker in indonesia) and as supplementary soup material. the high purine content of melinjo seeds may cause an increase in uric acid production which can lead to chronic arthritis due to bone erosion (terkeltaub 2010). therefore many people are reluctant to consume it. melinjo peel, as a waste from the process of making emping are seldom used as vegetables, most likely being thrown out (siswoyo . 2011). et al emping' et al*corresponding author, phone: (+62)-21-5460901, ext 1249, fax: (+62)-21-5460910, e-mail: adolf.parhusip@uph.edu issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 103-112 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.2 several bioactive compounds are found in melinjo, such as saponins, tannins, and flavonoids (kato . 2011; santoso . 2010; siswoyo . 2011). these compounds have been studied to have potential utilization as drugs or antibody, antimicrobial, pigments or even anti-inflammatory agents (khan 2003; rauha 2000; uboh 2010). furthermore, a recent study has reported the isolation of stilbenoids isolated from the seeds of melinjo (kato 2009) which stated these stilbenoids show moderate antimicrobial activity via a diphenyl-picrilhydrazil-hydrate (dpph) radical scavenging effect, including lipase and . however, this study focused itself on isolation process of stilbenoids with complex steps and studied biological activities of the extracts generally. on the other hand, a detail investigation specifically on the antimicrobial activity of melinjo extracts has not been reported. in accordance to those findings, where the melinjo contains bioactive compounds, such as saponins, tannins, and flavonoids, and stilbenoids; there is therefore a need to investigate the antibacterial activity of melinjo seed and peel extract which can be applied further in food products or even in drugs. initially, we hypothesized the mechanism of antibacterial activity of this extract would be by interfering with the synthesis of the bacteria cell wall. therefore within this study we carried out an antibacterial comparison study between the melinjo extract and common antibiotics used as antibacterial agents (penicillin g and streptomycin). in addition, to see the cell wall deformation, we carried out sem analysis to depict clear images of cell wall changes due to the addition of extract into the growth medium. selected microorganisms used within this study were atcc 25953, atcc 10876, atcc 13048, atcc 07853 and ipbcc 88.030, which were considered to represent gram-negative, gram-positive bacteria and also mold. we used bacteria and mold within this study since their cell wall assemblies are different. consequently, this results in different responses for the cell wall deformation as antimicroorganism added to inhibit the growth of those microorganisms. several pathogenic bacteria used for this study, such as atcc 25953, atcc 10876, atcc 13048, and atcc 07853 were purchased from et al et al et al et al. et al. et al. et al. staphylococcus aureus bacillus cereus enterobacter aerogenes pseudomonas aeruginosa aspergillus flavus staphylococcus aureus bacillus cereus enterobacter aerogenes pseudomonas aeruginosa α-amylase inhibition activity materials and methods microorganisms. american type culture collection (atcc) biological research center (brc), manassas, va 20108, usa, whilst ipbcc 88.030 was purchased from bogor agricultural university, bogor, indonesia. at first glance, all bacteria were activated on each physical growth medium. for bacteria, media used were nutrient agar (na) and nutrient broth (nb), whereas for fungi ( ipbcc 88.030) media used were potato dextrose agar (pda) and potato dextrose broth (pdb). after the activation, those microorganisms were then preserved in micro-tubes (containing 0.5 ml activated culture) with addition of glycerol (50% v/v) and finally stored at -40 °c until used again. several pro-analytical organic solvents, such as ethanol, ethyl acetate, and hexane were purchased from mayky lovic tangguh perkasa, indonesia. such media, nutrient agar (na), potato dextrose agar (pda), nutrient broth (nb), and potato dextrose broth (pdb) were purchased from sigma-aldrich. especially for nb, the mdl number was mfcd01867738 and we confirmed that it does not have any sodium chloride (nacl) in its composition. this was very important, since within this study such cell leakage due to the addition of the extract would be carried out in terms of ca and k ion determinations. moreover, two antibiotics, penicillin g and streptomycin were obtained from meiji seiyaku (tokyo, japan). the ripe melinjo seed and melinjo peel (red color) used in this study were collected in september 2009 from the village of balekambang condet, east jakarta, indonesia. the plant was identified by the research center of biology, indonesian institute of sciences (lipi), where a voucher specimen was deposited in pelita harapan university (1283/iph.1.02/if.8/xii/2009). . removal of water from melinjo seeds and melinjo peels was done using a cabinet dryer. thus, the dried materials were crushed to reduce the size into powder form and sieved using an abrasive screener (ø: 35 mesh). extraction was carried out using several pro-analytical organic solvents (ethanol, ethyl acetate, and hexane) with a ratio between melinjo materials (seed and peel) to solvent being 1:4. the extraction was carried out at 30 °c, shaken at 150 rpm using an orbital shaker incubator (type hotech, taiwan) for 24 h. the mixture was then filtered using whatman filter paper no. 4 by assistance of a vacuum pump. the extraction process did not follow the study reported by sunaryanto . (2010), since the sources of bio-actives that act as antimicroorganisms were totally different. further crude aspergillus flavus aspergillus flavus et al reagents and chemicals. seed and peel samples. extraction of samples 2+ + 104 p sarhusip and itanggang microbiol indones extract isolation was done by amplification using a rotary evaporator (buchi) at 40 °c and shaken at 75 rpm in round-bottomed flask until crystal or paste appeared. eventually, the crude extracts were put in a dark bottle and stored at temperature of 4 °c. the antimicrobial assay was carried out using the well-diffusion method according to bloomfield (1991) with minor modification according to yasni . (2009) for five kinds of microorganism as pre-mentioned above. each extract resulting using several solvents (ethanol, ethyl acetate, hexane) from different parts of melinjo (seed, peel) was diluted to give several concentrations of the extract (b/v), such as 0; 5; 10; 15; 20; 25%. the mic (minimal inhibitory concentration) and mbc (minimal bactericidal concentration, bactericidal term is used for bacteria) or mfc (minimal fungicidal concentration, fungicidal term is used for fungus) were determined according to bloomfield (1991). by plotting natural logarithm (ln) of concentration (mo) in x-axis and inhibition zone square value (z ) in y-axis, the value that crosses x = 0 will be mt. furthermore, mic was then calculated as 0.25 x mt and mbc or mfc as 4 x mic. the determination of the preferred extract was based on the inhibition zone value as reported by elgayyar . (2001) and suliantari . (2008). following this method, statistical software spss 16.0 was used to run anova in giving a conclusion as to which extract would be preferred at low concentration gives higher inhibition zone. generally an anti-spore is defined as a compound that has the ability to inhibit spore activity (jenson and moir 2003). determination of anti-sporal activity from a preferred extract onto spore-forming bacteria atcc 10876 was conducted using the well-diffusion method. the inhibition zone for spores (culture was incubated for 48 h) was compared with the inhibition zone for vegetative cells of atcc 10876 (culture was incubated for 8 h). the statistical analysis was carried out employing the (p < 0.05). each experiment was run in duplicate, and mean values were calculated. a statistical package (spss version 16.0) was used for the data analysis. this test was carried out to compare the performance between preferred extract of melinjo and several antibiotics that have antibacterial capacity. the procedure was by varying the concentration of antiotics and the inhibition diameter was then evaluated using the welldiffusion method. . to determine the presence of leakage of antimicrobial assay. anti-sporal activity. antibiotics testing comparison. atomic absorption spectrophotometer (aas) analysis et al et al et al one way b. cereus b. cereus b. cereus t-test 2 bacterial cells, aas analysis was conducted within this study which focused on calcium ions (ca ) and potassium ions (k ) quantification. the bacterial strains were activated using nb for 8 h. freshly activated bacteria were collected by centrifuging the broth at 4 032 g force (rotating at 6 000 rpm; radius of rotor 100 mm) for 15 min. cells were then washed for several times using de-mineral water and finally resuspended again in 10 ml de-mineral water in a reaction tube for each bacterial strain. into the tube, a preferred extract concentration was added and then incubated for 24 h. the quantification method of those cations followed the procedure of prashar . (2003), where the suspension was then analyzed to determined ca at 422.7 nm, whilst k at 766.5 nm using aas instrument (aas type shimadzu aa-680). to determine the efficacy of extract and the morphological changes, sem studies were performed on tested bacteria treated with preferred melinjo extract. controls were prepared without melinjo extract. the bacterial samples were washed gently and gradually with 2% glutaraldehyde solution, 2% tannic acid solution, buffer solution (0.1 m sodium cacodylate containing 10 mm mgso , ph 6.7), and 1% osmium tetraoxide solution. each specimen was dehydrated using sequential exposure per ethanol concentrations ranging from 50 100%. the ethanol was replaced by tertiary butyl alcohol. after dehydration, the specimen was put onto a stub. finally, the specimen was sputter-coated with gold (aurum) in an ion coater for 5 min, followed by microscopic examinations (sem type jsm-5310 lv) with a magnification of 10,000-15,000 times. the data was analyzed using one-way analysis of variance (anova) for repeated measurements using statistical software spss 16.0 for windows. the duncan's multiple range tests was used to determine differences at each point. differences at each point were considered significant at . crude extracts obtained from melinjo seed tended to be colored as brownish yellow in color, while the extract obtained from the melinjo peel was dark red. within this study, both melinjo seed and peel could be extracted with ethanol, 10.25%, and 13.33%, respectively (table 1). conclusively, through the yield percentage of this investigation, the dominant components contained in melinjo seed or melinjo peel were polar, as reflected by higher yield using polar solvent. 2+ + 2+ + × et al p ≤ 0.05 scanning electron microscope (sem) analysis. statistical analysis. results extraction. 4 volume 5, 2011 microbiol indones 105 antimicrobial activity of seed-based and peelbased melinjo extract. the results showed that ipbcc 88.030 could not be inhibited by either melinjo seed and peel extract since even the highest concentration applied (25% w/v) into the wells, the growth of this fungus could not be inhibited. in addition, the extract inhibition towards gram-positive bacteria ( atcc 10876 and atcc 25953) was positively higher than for gram-negative bacteria ( atcc 13048 and atcc 0785 (fig 1, 2, 3, and 4). aspergillus flavus b. cereus s. aureus e. aerogenes p. aeruginosa ) 106 p sarhusip and itanggang microbiol indones table 1 yield of melinjo seed and peel extracts plant sample solvent yield (%) melinjo seeds ethanol 10.25 ethyl acetate 2.31 hexane 0.00 melinjo peels ethanol 13.33 ethyl acetate 2.43 hexane 1.51 fig 1 inhibition diameter of melinjo extracts ( ) againstsource of extract solvent used bacillus cereus. fig 2 inhibition diameter of melinjo extracts ( ) againstsource of extract solvent used staphylococcus aureus. minimum inhibitory concentration and minimum bactericidal concentration. anti-sporal activity from the mic and mbc values (shown in table 2), melinjo seed ethanol extract has the lowest mic and mbc values. therefore, conclusively, melinjo seeds ethanol extract was the most effective extract that could inhibit bacterial growth for both gram-negative and grampositive bacteria, as compared to other melinjo extracts through the exception of inhibition to mold growth ( ipbcc 88.030). there were not any mic and mfc values for . representative images to depict inhibition zone, could be seen in fig 5 which ethanol was used as the solvent and source of extract was melinjo peel. in addition, the preferred extract was chosen according to suliantari . (2008), summarized in table 3. . anti-sporal activity of preferred melinjo extract can be observed by contacting the extract with spores. the number of a. flavus a. flavus et al fig 3 inhibition diameter of melinjo extracts ( ) againstsource of extract solvent used enterobacter aerogenes. fig 4 inhibition diameter of melinjo extracts ( ) againstsource of extract solvent used pseudomonas aeruginosa. are used as a bridge for phospholipid components on cell wall. the ions ca and k can be detected after the bacteria have been in contact with the extract. this indicates that the destruction or collapse was happened on the bacteria cell membrane, which might cause the extraction of ions of ca and k from the body of a detected cell by aas. as seen on the table 4, the amount of ca that has been detected on gramnegative bacteria (73.17 mg l and 62.41 mg l ) was larger as compared to gram-positive bacteria (58.27 mg l and 67.17 mg l ). the morphological changes on the bacteria cell surface can be detected using sem. there were some morphological changes in , , and which treated with 5% melinjo seeds ethanol extract and which was treated with 15% melinjo peels ethyl acetate extract (fig 9). morphological changes that occurred include 2+ + 2+ + 2+ -1 -1 -1 -1 scanning electron microscope (sem) imaging. b. cereus s. aureus e. aerogenes p. aeruginosa spores is large when the bacterial culture is incubated for 48 hours (fig 6). according to the results of statistic analysis (p < 0.05), there was a significant difference between anti-sporal activity of extract onto spores of (10.18 mm) and anti-bacterial activity of extract towards vegetative cells of (12.83 mm). the results from comparison testing of antibiotics and preferred extracts were shown (fig 7 and 8). according to the overall results, it was known the inhibition capability of preferred extracts was nearly comparable with 10 ppm of tested antibiotics (fig 7 for comparison with penicillin g and fig 8 for comparison with streptomycin). potassium ions (k ) exist on a bacterial cytoplasm membranes which have functionality on membrane transportation process. calcium ions (ca ) and magnesium ions (mg ) on gram-negative bacteria b. cereus b. cereus antibiotics comparison testing. atomic absorption spectroscopy (aas) analysis. + 2+ 2+ volume 5, 2011 microbiol indones 107 bacterium melinjo extracts mic (μg ml -1 ) mbc (μg ml -1 ) bacillus cereus melinjo seed – ethanol extract 0.76 3.03 melinjo seed – ethyl acetate extract 0.86 3.45 melinjo peel – ethanol extract 1.40 5.58 melinjo peel – ethyl acetate extract 0.26 1.02 melinjo peel – hexane extract 0.26 1.04 staphylococcus aureus melinjo seed – ethanol extract 0.15 0.58 melinjo seed – ethyl acetate extract 1.19 4.76 melinjo peel – ethanol extract 0.90 3.58 melinjo peel – ethyl acetate extract 1.46 5.82 melinjo peel – hexane extract 0.00 0.00 enterobacter aerogenes melinjo seed – ethanol extract 0.40 1.58 melinjo seed – ethyl acetate extract 0.97 3.86 melinjo peel – ethanol extract 0.00 0.00 melinjo peel – ethyl acetate extract 1.35 5.39 melinjo peel – hexane extract 0.00 0.00 pseudomonas aeruginosa melinjo seed – ethanol extract 1.04 4.15 melinjo seed – ethyl acetate extract 1.51 6.04 melinjo peel – ethanol extract 0.00 0.00 melinjo peel – ethyl acetate extract 1.36 5.43 melinjo peel – hexane extract 0.00 0.00 tabel 2 minimal inhibitory concentration (mic) and minimal bactericidal concentration (mbc) values of melinjo extract using different sources (seed, peel) and different solvents (ethanol, ethyl acetate, hexane) fig 5 representative images of inhibition zone of ethanol extract from melinjo seed. bacillus cereus 5% of melinjo seed – ethanol extract staphylococcus aureus 5% of melinjo seed – ethanol extract enterobacter aerogenes 5% of melinjo seed – ethanol extract pseudomonas aeruginosa 15% of melinjo peel – ethyl acetate extract table 3 preferred extract for each tested bacteria leakage (fig 9f), pore formation (fig. 9h) and lysis of the membranes integrity (fig 9b, 9h), and bumps (fig 9b, 9d, 9f, 9h). cell shapes became abnormal caused by the penetration of extract into cells which caused swelling in some cells (fig 9d, 9h) and rough surfaces as compare to the smooth ones of untreated cells. 108 p sarhusip and itanggang microbiol indones bacillus cereus vegetative cells spores d ia m e t e r o f in h ib it io n ( m m ) 0 2 4 6 8 10 12 14 12.83 10.18 fig 6 diameter of inhibition resulting from 5% melinjo seed-ethanol extract against vegetative cells and spores of bacillus cereus. fig 7 diameter of inhibition of preferred melinjo extracts and penicillin g against selected bacteria. fig 8 diameter of inhibition of preferred melinjo extracts and streptomycin against selected bacteria. discussions melinjo seed and melinjo peel extract showed antibacterial activity against , , , and , but did not show antifungal activity because it could not inhibit . none of the extracts could inhibit the growth of could be explained as fungus is eukaryotic organism that has cell wall which contains very stiff chitin (yokoi . 1998). this chitin fundamentally contributes to fungal cell wall strength and stability and prevents outer material from penetrating. furthermore, it may be expected that an extract could not interfere with the permeability of cytoplasmic membrane because extracts did not have any reaction with sterol inside a mold's cytoplasmic membrane (farkas 1979). b. cereus s. aureus e. aerogenes p. aeruginosa aspergillus flavus growth a. flavus et al p. aeruginosa volume 5, 2011 microbiol indones 109 however, melinjo seed and melinjo peel extract have antibacterial activity, proven by the facts that the extract could inhibit the growth of tested bacteria as seen in the inhibition area, and moreover was supported by (i) the mic and mbc values (0.26~1.46%; 1.02~6.04% respectively), (ii) anti spore results (5% w/v of extract), (iii) having the equivalent inhibition capacity as compared to antibiotics (penicillin g and streptomycin 10 ppm). the extract has higher inhibitory effect towards gram-positive bacteria as compared to gram-negative bacteria because gram-negative bacteria have several complex layers on their cell walls. the structure of the cell wall layer on gram-negative bacteria consists of p e p t i d o g l y c a n a n d a n o u t e r m e m b r a n e (lipopolysacharide and lipoproteins) (lesage and bussey 2006). the existence of an outer membrane cell from gram-negative bacteria causing the inhibitional diffusion of antimicrobial extract inside both peptidoglycan membranes and bacteria cells and leads to inappropriate conditions for penetrating interacting with inner cell wall components (fan . 1975). in addition, approximately 90% of a gram-positive bacteriums cell wall is composed of peptidoglycan, whereas the peptidoglycan layer in the gram-negative bacterium cell wall is only 50-10%. the outer membrane of gram-negative bacteria is impermeable to penicillin g, so that bacteria growth can not be inhibited (tompsett . 1947). for antisporal activity, there was a significant difference between anti-sporal activity of extract onto spores of (10.18 mm) and anti-bacterial activity of extract towards vegetative cells of (12.83 mm). the layer difference at vegetative cell and endospore level will cause diameter dissimilarity which was produced by the extract. generally layers of endospore are exosporium layers, spore coats, cortex (atrih . 1998). an enormous layer on it along with its stiff nature, will cause the extract to be harder to diffuse into the cell and inhibit cell growth. therefore, the inhibition diameter of extract toward spores of was smaller as compared to its vegetative cells. for the preferred extracts were able to inhibit both gram-positive and gram-negative bacteria (fig 7). however, penicillin g was also found to effectively inhibit gram-positive bacteria (fig 7). according to baldwin . (1997), antibacterial activity of penicillin g was due to its interference on the synthesis process of the bacteria cell wall. though this conclusion needs further clarification, due to this finding, we hypothesize the preferred melinjo extracts should follow the mechanism of penicillin g inhibition in that they were et al et al b. cereus b. cereus et al b. cereus antibiotics comparison testing, et al bacterium total ca 2+ (mg l -1 ) total k + (mg l -1 ) bacillus cereus 58.27 196.39 staphylococcus aureus 67.17 190.03 enterobacter aerogenes 73.17 130.06 pseudomonas aeruginosa 62.41 111.26 table 4 total calcium and potassium ions detected by aas fig 9 sem photomicrographs showing the morphological changes of tested bacteria after exposure to preferred melinjo extract ( ) for each bacteria. a, control; b, treated with 5% melinjo seed ethanol extract; c, s. aureus control; d, treated with 5% melinjo seed ethanol extract ; e, control; f, treated with 5% melinjo seed ethanol extract; g, control; h, treated with 15% melinjo peel ethyl acetate extract. gnetum gnemon bacillus cereus bacillus cereus staphylococcus aureus enterobacter aerogenes enterobacter aerogenes pseudomonas aeruginosa pseudomonas aeruginosa able to influence the synthesis process of bacteria cell wall. streptomycin could inhibit gram-positive and gram-negative bacteria. according to lin (2000), the inhibition mechanism of streptomycin was by attacking the ribosome of microbes which leads to error reading of the mrna sequence taking place, resulting in the formation of polypeptide which becomes irregular and nonfunctional, and therefore inhibited bacterial growth. the anti-bactericidal activity of the each preferred extract for each bacterium was comparable with the action of streptomycin in inhibiting bacterial growth (fig 8). furthermore, we et al. 110 p sarhusip and itanggang microbiol indones suggest the overall inhibition mechanisms of melinjo extracts against bacteria were by interfering with protein synthesis in bacteria, degrading the existing present cell wall or interfering which bacterial cell wall synthesis, and damaging cell membrane of bacteria (as reported by yasni . (2009)) and proven by the analysis of mineral content and sem imaging system (fig 9). according to parhusip (2006) and rogers (1970), destruction of bacterial cell membrane may also cause the extraction of minerals the from cell inner side such as, calcium (ca ) and potassium (k ). therefore, within this study we have found the k leakage was higher for both gram-negative and positive bacteria as compared to ca leakage. moreover, in the grampositive bacteria the k leakage was higher than gramnegative ones. however, this condition was contrary to ca leakage, since the gram-negative leakage was found to be higher than the gram-positive ones. this could be explained since the outer membrane of gramnegative bacteria consists of a large number of ca ions, whereas gram-positive bacteria consist of ca which is located on its cytoplasmic membrane (farkas 1979). this was also confirmed by the results mentioned by beveridge (1999). lastly, those findings mentioned above were totally confirmed morphologically through sem imaging (fig 9). the same imaging phenomenon was also reported by al-reza (2010) who mentioned that the essential oil extract of causes the formation of pores or holes in the cell membrane and cell lysis in , which indicates the occurrence of disturbances on the structure of cell membranes. within this study the same perspective was also found. moreover, changes in morphology in this study were also similar to results of shalamanov (2005), including changes in the morphology on gram-negative bacteria ( , , and ) that had been contacted with chlorhexidine gluconate. changes that occurred were elongation, variations in shape and size of cells, the formation of bumps, grooves and wrinkles on the cell walls. these sem images were performed to provide basic supportive data on the occurrence of morphological changes in bacteria cells so that the mechanism of melinjo extract action could be deduced. finally, from these comprehensive data we could conclude that melinjo extract has real potential to be applied in food preservation against selected pathogenic bacteria. we would like to thank universitas pelita harapan (uph) for the funding, and elisa fr and jennifer octavia for the assistance. et al et al. zizyphus jujuba s. aureus enterobacter cloacae pseudomonas aeruginosa serratia marcescens 2+ + + 2+ + 2+ 2+ 2+ acknowledgements references al-reza sm, rahman a, lee j, kang sc. 2010. potential roles of essential oil and organic extracts of in inhibiting food-borne pathogens. food chem. 119: 981-986. doi:10.1016/j.foodchem.2009. 07.059. atrih a, zöllner p, allmaier g, williamson mp, foster sj 1998. peptidoglycan structural dynamics during germination of 168 endospores. j bacteriol. 180: 4603-4612. baldwin je, byford mf, clifton i, hajdu j, hensgens c, roach p, schofield cj. 1997. proteins of the penicillin biosynthesis pathway. curr opin struct biol.7(6): 857-864. beveridge tj. 1999. structures of gram-negative cell walls and their derived membrane vesicles. j bacteriol. 181(16): 4725-4733. bloomfield sf. 1991. methods for assessing antimicrobial activity. in: denyer sp, hugo wb, editors. mechanisms of action of chemical biocides their study and exploitation. blackwell scientific publication: london. elgayyar m, draughon fa, golden da, mount jr. 2001. antimicrobial activity of essential oils from plants againts selected patogenic and saprophytic microorganisms. j food protect. 64(7): 1019-1024. fan dp, beckman be, gardner-eckstrom hl. 1975. mode of cell wall synthesis in gram-positive bacilli. j bacteriol.123: 1157-1162. farkas v. 1979. biosynthesis of cell walls of fungi. microbiol rev. 43(2): 117-144. hafif b, masbulan e, suwardjo h 1995. peluang melinjo ( ) sebagai bagian tanaman konservasi pada daerah aliran air pada daerah semi arid. 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2 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 3 biopharmaca research center, institut pertanian bogor, taman kencana campus, bogor 16151, indonesiaa endophytic actinomycetes associated with medicinal plants is very important as source of various bioactive compounds. the fact that more than 99% of microbes that exist in nature may have the potency but still unexplored. published data regarding diversity of endophytic actinomycetes from tinospora crispa is mainly based on a culturable approach. this paper describes the first reported data regarding metagenomic analysis on the diversity of endophytic actinomycetes from t.crispa based on 16s rrna gene using pcr-dgge. there were some similarities amongst endophytic actinomycetes found in stems, roots, and leaves with soil actinomycetes community in the rhizosphere of t. crispa. there were a total of 21 bands found from the dgge analysis which were interpreted using phoretix 1d software. diversity of actinomycetes in the stems, leaves, roots were represented by 17, 16, and 14 bands, respectively. whereas only 10 bands represented diversity of actinomycetes in the soil rhizosphere. the 12 dominant and or different bands with 180 bp in size were molecularly sequenced. the a4 and a9 bands have 95% and 86% similarities with williamsia and streptomyces, respectively. these similarities were less than 97% thus may indicate novel actinomycetes. the other 10 sequenced bands have closed similarity ranging from 97-100% and they were closely related to the genus streptomyces, microbacterium, amycolatopsis, actinomadura, actinoplanes, actinokineospora, kibdelosporangium, williamsia, and kocuria. these findings indicate that diversity of actinomycetes can be found associated with t. crispa. key words: 16s rrna, dgge, endophytic actinomycetes, metagenomic, tinospora crispa aktinomiset endofitik yang berasosiasi dengan tanaman obat berperan penting sebagai sumber berbagai senyawa bioaktif. fakta mengindikasikan bahwa lebih dari 99% mikrob yang ada di alam diduga memiliki potensi, tetapi belum banyak dieksplorasi. keragaman aktinomiset endofit yang telah dilaporkan masih terbatas pada pendekatan culturable. data hasil penelitian ini merupakan informasi baru tentang keragaman aktinomiset endofit t. crispa yang dianalisis status metagenomiknya berdasarkan gen 16s rrna menggunakan pcrdgge. terdapat beberapa kesamaan antara aktinomiset endofit yang ditemukan di batang, akar, dan daun dengan komunitas aktinomiset tanah di rhizosfer t. crispa. total pita yang ditemukan dari analisis dgge yang menggunakan perangkat lunak phoretix 1d yaitu sebanyak 21 pita. keanekaragaman aktinomiset di batang, daun dan akar yaitu 17, 16 dan 14 pita, sementara hanya 10 pita ditemukan di tanah rhizosfer. berdasarkan profil dgge, diperoleh 12 pita yang dominan dengan panjang pita 180 bp yang kemudian disekuensing. pita a4 dan a9 masing-masing memiliki kesamaan 95% dan 86% dengan williamsia dan streptomyces. kesamaan sekuen tersebut kurang dari 97% sehingga diduga aktinomiset tersebut tergolong baru. terdapat 10 pita lainnya yang memiliki kesamaan berkisar 97-100% dan berkekerabatan dekat dengan genus streptomyces, microbacterium, amycolatopsis, actinomadura, actinoplanes, actinokineospora, kibdelosporangium, williamsia, dan kocuria. data tersebut menunjukkan bahwa beragam aktinomiset dapat ditemukan berasosiasi dengan t. crispa. kata kunci : 16s rrna, aktinomiset endofit, dgge, metagenomik, tinospora crispa *corresponding author; phone: +62-251-8622833, fax: +62251-8622833, e-mail: yulinlestari@gmail.com tinospora crispa l. miers is a medicinal plant traditionally utilized by asian people as an antidiabetic agent (puranik et al. 2010; patela and mishrab 2012). endophytic actinomycetes from t. crispa are capable of producing secondary metabolites which can also function as an antidiabetic agent (pujiyanto et al. 2012). this phenomenon is presumably the result of genetic exchange and evolution between endophytes and their host (tan and zou 2001). the biological function of t. crispa as an antidiabetic agent may be associated with the presence and diversity of actinomycetes in the host plant tissue. actinomycetes are very important microbes as they have an ability to vol.9, no.1, march 2015, p 25-34 doi: 10.5454/mi.9.1.4 26 primanita et al. microbiol indones produce various metabolite compounds. previous study done by pujiyanto et al. (2012) showed that among s everal medicinal plants w hich w ere traditionally used as antidiabetics possessed endophytic actinomycetes which may function as source of antidiabetics. amongst the 65 culturable endophytic actinomycetes isolated from those antidiabetic plants, 32 isolates were found from t. crispa. diversity of actinomycetes in t. crispa can be explored using culture dependent and or independent approaches. isolation of actinomycetes using a culturedependent method has faced several constraints such as cultivation conditions, techniques used, isolation media and cultivation time (qin et al. 2012). these partly because many members of the actinomycetes genus are relatively slow-growing and some genera are often difficult to be cultured (nimnoi et al. 2010). moreover, it is only 0.1-10% of all microbes in nature can be cultured in the laboratory (zeyaullah et al. 2009). culturable microbes can grow rapidly in nutrient-rich media, aerobic conditions and at moderate temperatures (thontowi 2009), however, more than 99% of microbes that exist in nature, which may have a very useful potential, are still not known nor utilized (zeyaullah et al. 2009). the suitable method, i.e. a culture-independent, needs to be used to explore whole diversity of endophytic actinomycetes. for that purpose, a metagenomic approach, which does not require culture, is used for genome analysis of bacterial communities (thontowi 2009). the dgge (denaturing gradient gel electrophoresis) technique is used to study microbial communities in situ. pcr-dgge result can be used to observe metagenomic data, especially from the dominant endophytic actinomycete species that colonizes plant organs and the rhizosphere (nimnoi et al. 2010). information on endophytic actinomycete diversity from t. crispa based on 16s rrna gene using a culture-independent technique has remained obscure. this research aimed to explore diversity of endophytic actinomycetes in t. crispa based on 16s rrna-based metagenomic analysis using pcr-dgge. for data comparison, diversity of actinomycetes in the rhizosphere of t. crispa was also assesed. metagenomic information on endophytic actinomycetes from t. crispa can be further used for searching of their functional consequences. materials and methods sample extraction and surface sterilization. t. crispa plants were obtained from medicinal plants collection garden, biopharmaca research center, institut pertanian bogor. the leaves, stems and roots of plants were cleaned and cut into pieces, then their surfaces were sterilized sequentially by soaking in 70% alcohol for 1 min, then in 1% sodium hypochlorite (naocl) for 5 min, followed by soaking in 70% alcohol for 1 min. in the final step, samples were rinsed 3 times with sterile distilled water. to prove that only endophytic actinomycetes genom were extracted, the last soaking water during surface sterilization process was spreaded on humic acid vitamin b (hv) medium and checked for their grown colonies (combs and franco 2003) used as negative control test. soil samples were taken from rhizosphere of t. crispa at a depth of approximately 1-5 cm from the soil surface. genomic dna extraction from endophytic actinomycetes and soil actinomycetes. genomic dna of endophytic actinomycetes in plant samples was extracted using genomic dna mini kit (plant) geneaid. soil dna was extracted using the power soil dna isolation kit (mobio laboratories, carlsbad, ca, usa). the used procedures followed the standard protocol recommended by the manufacturer. the extraction results were then quantitatively checked using nano drop 2000 spectrophotometer (thermo scientific, wilmington, de, usa). pcr for dgge. amplification was done using nested pcr technique which was conducted to obtain propagation of 16s rrna gene using actinomycetes specific primers (table 1). step 1 amplification was conducted using promega gotaq green master mix with the mixed composition consisted of 12.5 µl of master mix 27f dan 16sact1114r 60 pmol primers at 0.25 µl each, 5 µl of dna template and added with nuclease free water to a volume of 25 µl. pcr conditions used were pre-denaturation at 94 °c for 5 min, denaturation at 94 °c for 60 s, annealing at 65 °c for 45 s (a decrease at 0.5 °c per cycle was set for the first 20 cycles, the last decrease was at 55 ºc), elongation at 72 ºc for 120 s for 30 cycles and final elongation at 72 °c for 7 min (zhang et al. 2013). step 2 amplification also used promega gotaq green master mix with the mixed composition of 25 µl of promega gotaq green, p338f-gc dan p518r 100 volume 9, 2015 microbiol indones 27 pmol primers at 0.25 µl each, 1 µl of step 1 pcr products was used as a dna template and added with n u c l e a s e f r e e w a t e r t o a v o l u m e o f 5 0 µ l . amplification was conducted using t1-thermocycler (biometra, goettingen, germany) with these following optimizations: pre-denaturation at 94 °c for 5 min, denaturation at 94 °c for 60 s, annealing at 55 °c for 45 s, elongation at 72 °c for 60 s for 30 cycles and final elongation at 72 ºc for 5 min (zhang et al. 2013). the amplification result was then observed by migrating 5 µl of the amplicon on 1% agarose gel, 80 volts for 45 min. the migration result was then stained with 0.1% ethidium bromide for 15 min and then viewed under uv transiluminator and documented using gel doc. the remaining pcr products were stored at -20 ºc prior to be analysed using dgge. dgge (denaturing gradient gel electrophoresis). the dgge was conducted using the d code universal mutation detection system (bio-rad, hercules, ca, usa). the amount of 30 µl template (25 µl of pcr products + 5 µl of loading dye) was migrated at 1 mm 8% polyacrylamide gel (acrylamidebisacrylamide [37.5:1]) in a 7 l 1x tae (40 mm tris, 20 mm acetic acid, and 1 mm edta) using urea as a denaturant. the denaturant gradient of 30% -70% (100% denaturant was made with 8.4 g of 7 m urea, 8 ml of formamide, 0.4 ml of 50x tae, 4 ml of acrylamide-bisacrylamide and added with sterile distilled water to a volume of 20 ml). migration was conducted at 60 ºc and 150 volts for 5 h. once the migration was completed, the gel was soaked and s t a i n e d w i t h s y b r s a f e ( i n v i t r o g e n molecular\probes, carlsbad, ca, usa) for 1 h. gel visualization was conducted using the g:b o x (syngene, frederick, md, usa). the selected dominant and or different bands were cut and each band was put into a microtube containing 100 µl of ddh o and stored for 24 hours at 4 ºc. acrylamide gel 2 image results with g:box were analyzed using 1d phoretix software to estimate the total band appeared. dgge products was settled for 24 h, then the products were vortexed and amplified in pcr instrument using primers without g c clamp with similar p c r conditions. sequencing of 16s rrna gene and construction of phylogenetic tree. the pcr products were then sent to the sequencing service company (1stb a s e malaysia). sequencing was performed using double stranded pcr-dgge product. sequencing results were compared to the data base from the ncbi website (http://www.ncbi.nlm.nih.gov) with the basic local alignment search tool (blast). nucleotide sequence alignment and phylogenetic tree construction were conducted using mega 5.2 software. construction of a phylogenetic tree was conducted using the neighbour joining method. results pcr-dgge profile and phylogenetic tree of actinomycetes based on 16srrna genes. the last soaking water which was placed in hv test medium showed no growth, proving that only genomic dna of endophytic actinomycetes were extracted. the -1 genomic dna concentrations of 35.1 ng µl , 37.2 ng -1 -1 µl , and 30.9 ng µl were obtained from the roots, stems and leaves, respectively. the soil g enomic dna -1 concentration obtained was 3.7 ng µl . the purity of d n a obtained from this extraction with the a260/a280 parameter ranged from 1.6 to 1.7 which slightly lower than the recommended value (1.8-2.0) (sambrook and russell 2001), indicating some table 1 primer sequences primer sequences reference 27f 5'-agaptttgatcctggctcag -3' bruce et al. 1992; martina et al. 2008 16sact1114r 5'-gagttgaccccggcrgt-3' bruce et al. 1992; martina et al. 2008 p338f 5'-actcctacgggaggcagcag -3' ovreas et al. 1997 p518r 5'-attaccgcggctgctgg -3' ovreas et al. 1997 gc clamp 5'cgcccgccgcgcgcggcgggcg ovreas et al. 1997 gggcgggggcacgggggg * description: f / f, forward primer; r / r, reverse primer, gc clamp added at the end 5'primer p338f impurities. however, this impurity seem did not interfere during the dna replication process. amplification with nested pcr was conducted in 2 steps using actinomycetes specific primers i.e. 27f/16sact1114r and p338fgc/p518r. step 1 pcr was successfully amplifying the band with a length of ~1087 bp (fig 1a), while step 2 pcr was at ~180 bp length (fig 1b). in this study, 12 dominant and or different bands were obtained from dgge profile (fig 2a). there were some similarities between endophytic actinomycetes community found in stems, roots, leaves and soil. interpretation using phoretix 1d software showed that diversity of actinomycetes community were represented by 17, 16, 14 and 10 bands in the stems and leaves, roots and soil, respectively. total number of bands obtained with this software was 21 bands (fig 2b). the 12 dominant sequenced bands revealed their genetic relationship based on their phylogenetic tree (fig 2c). analysis of phylogenetic tree based on genetic distance matrix (pdistance) showed nucleotide sequence similarity in the stems and leaves, but there were several differences from the roots and soil (fig 2d). almost all endophytic actinomycetes who reside in leaves have also been found in the stems, while for roots and soil, their actinomycetes were more varied. the alignment results of 12 partial sequences (~180 bp) of endophytic actinomycetes from t. crispa plant and soil actinomycetes in the rhizosphere was analyzed using the blast program (table 2). the a2 and a10 sequences have 100% similarity with actinokineospora auranticolor strain ifo 16518 and amycolatopsis orientalis strain h c c b 10007, respectively. a1, a3, a5, a6, a7, a8, a11 and a12 sequences have ≥ 97% similarity with streptomyces acidiscabies strain at c c 49003, williamsia marianensis strain dsm 44944, kocuria halotolerans strain yim 90716, microbacterium ginsengisoli strain gsoil 259, kibdelosporangium philippinense strain a 80407, actinomadura rifamycini strain jcm 3309 and actinoplanes sp., respectively. a relatively new sequence was also found based on the percentage of a maximum identity from the blast results showing the data base sequence similarity at 95% and 86 % of the ~180bp (a4 and a9 sequences). these sequences were closedly related with williamsia marianensis strain dsm 44944 and streptomyces javensis strain b22p3, respectively. the phylogenetic relatedness of endophytic actinomycetes 16s rrna gene sequences from t. crispa and rhizosphere soil was compared with closedly related strains sequenced from database (fig 3). abundance of endophytic actinomycetes in plants and soil actinomycetes. based on the nucleotide partial sequenced at ~180 bp length, there were 12 species of endophytic and soil actinomycetes dominant in t. crispa plant. among the 12 species obtained, one species found in the soil has a closedly related with streptomyces acidiscabies strain atcc 49003 (1). meanwhile, six endophytes and soil a c t i n o m y c e t e s h a v e a c l o s e r e l a t i o n s h i p t o actinokineospora auranticolor strain ifo 16518 (2), kocuria halotolerans strain y i m 90716 (5), microbacterium ginsengisoli strain gsoil 259 (6), kibdelosporangium philippinense strain a 80407 (8), streptomyces javensis strain b22p3 (9), and actifig 1 (a) step 1 amplification results of 16s rrna gene of endophytic actinomycetes in roots, stems, leaves of t. crispa plant (a), (b), (d) and soil (t) (~1087bp) and (b) step 2 with length of ~180bp. 28 primanita et al. microbiol indones fig 2 (a) dgge profile 16s rrna gene of endophytic actinomycetes from t. crispa plant and soil actinomycetes; (b) interpretation from phoretix 1d software. numbers next to bands indicate the cut/splitted bands for re-amplification. wells from left to right: (t) soil, (d) leaves, (b) stems, and (a) roots; (c) re-amplification dgge bands; (d) p-distance analysis. table 2 sequence similarity of 16s rrna gene of endophytic actinomycetes from medicinal plant t. crispa and soil actinomycetes with reference strain (genbank) comparison sequence from database band maximum e-value accession identity streptomyces acidiscabies strain atcc 49003 a1 97% 1e-81 nr116534.1 streptomyces javensis strain b22p3 a9 86% 6e-46 nr028958.1 williamsia marianensis strain dsm 44944 a3 97% 2e-80 nr043263.1 williamsia marianensis strain dsm 44944 a4 95% 1e-76 nr043263.1 kocuria halotolerans strain yim 90716 a5 99% 8e-89 nr044025.1 kocuria halotolerans strain yim 90716 a7 99% 1e-83 nr044025.1 actinokineospora auranticolor strain ifo 16518 a2 100% 2e-89 nr040873.1 microbacterium ginsengisoli strain gsoil 259 a6 97% 1e-81 nr041516.1 kibdelosporangium philippinense strain a 80407 a8 98% 2e-85 nr025572.1 amycolatopsis orientalis strain hccb10007 a10 100% 3e-89 nr103940.1 actinomadura rifamycini strain jcm 3309 a11 99% 3e-88 nr113155.1 actinoplanesp. se 50/110 strain se 50/110 a12 98% 2e-84 nr074431.1 volume 9, 2015 microbiol indones 29 a b c d leaves stems roots soil nomadura rifamycini strain jcm 3309 (11). there were 5 species which found only as endophytes which were closedly related with williamsia marianensis strain dsm 44944 (3,4), amycolatopsis orientalis strain hccb10007 (10), kocuria halotolerans strain yim 90716 (7), actinoplane sp. strain se 50/110 (12) (fig 4a). there were several similar endophytic actinomycetes found in different plant organs (figure 4b). actinomycetes that have a close relationship with williamsia marianensis strain dsm 44944 (3,4) and actinoplanes sp. strain se 50/110 (12) were found in roots and stems, while kocuria halotolerans strain yim 90716 (5) were found in roots and leaves. the actinomycetes which were found in roots, stems and leaves were closedly related with actinokineospora auranticolor strain ifo 16518 (2), microbacterium ginsengisoli strain gsoil 259 (6), amycolatopsis orientalis strain hccb10007 (10) and actinomadura rifamycini strain jcm 3309 (11), streptomyces javensis strain b22p3 (9), kibdelosporangium philippinense strain a 80407 (8) and kocuria halotolerans strain yim 90716 (7). fig 3 phylogenetic relatedness of 16s rrna gene sequences of endophytic actinomycetes from t. crispa and soil actinomycetes of dgge product (analysis with 1000 bootstrap replicated). fig 4 (a) abundance of soil actinomycetes (t) and endophytic actinomycetes (e) (b) endophytic actinomycetes in roots (a), stems (b) and leaves (d). 30 primanita et al. microbiol indones discussion as the first published report, this study describes metagenomic diversity of endophytic actinomycetes in r o o t s , s t e m s , l e a v e s o f t. c r i s p a p l a n t a n d actinomycetes from rhizosphere soil based on 16s rrna-gene using pcr-dgge technique. dgge profiles shows each separate band which representing one of its own species community. the dominant species and the intensity of each band indicates their relative abundance (nimnoi et al. 2010). amplification with nested pcr and dgge was also successfully detects actinomycetes community in the soil, plant rhizosphere of artemisia tridentata and from roots of aquilaria crassna (heuer et al. 1997; franco et al. 2009; nimnoi et al. 2010). genetic distance (pdistance) analysis of 16s rrna gene sequences of the s e l e c t e d b a n d s i n d i c a t e s s i m i l a r i t y o f t h e actinomycetes community between stems and leaves, but has several differences from roots and soil. this phenomenon is predicted due to the migration of actinomycetes from stems to the leaves, soil and roots. differences in the nucleotide sequences based on the pdistance matrix indicates species diversity between each sample. the differences are thought to be influenced by differences in isolated parts of plant organs and the effect of actinomycetes found in phyllosphere. endophytic actinomycetes community was found t o b e m o r e d i v e r s e t h a n r h i z o s p h e r e s o i l actinomycetes. relatively low actinomycetes community in the soil was allegedly due to the high competitive niches in the soil (hibbing et al. 2010). endophytes are sheltered from environmental stresses and microbial competition by the host plant and they seem to be ubiquitous in plant tissue (nimnoi et al. 2010). endophytic actinomycetes population in the stems and leaves exhibit a more diverse than in the roots. while relatively low actinomycetes community in the roots was allegedly due to the migration of roots endophytic actinomycetes to other plant organs as a result of the old plants used in this research work. association between actinomycetes in the soil with endophytic actinomycetes occur as it is seen in figure 4a. several endophytic actinomycetes in t. crispa plant organs are also found in the soil rhizosphere. actinomycetes which abundantly live in the rhizosphere soil can colonize and penetrate into plant roots (sardi et al. 1992; nimnoi et al. 2010). with a passive penetration, microbial endophytic can also move in the open space or cracks in the root tip (nimnoi et al. 2010). actinomycetes in roots migrate to other plant organs through the intercellular and vascular system (feng et al. 2004). similar results were also reported by qin et al. (2012) that the abundance of endophytic actinomycetes on the stems and leaves of medicinal plant maytenus austroyunnanensis showed greater diversity than the roots. these phenomenon may affects the production cycle of the active compounds in the leaves and stems of t. crispa, which often used for medicinal treatment. microbial endophytics of medicinal plants have a role in the biochemical cycles and production of bioactive compounds in their host (zhao et al. 2011). the study shows that several similar endophytic actinomycetes are found in different plant organs (fig 4b), which is presumably due to the migration of the endophytic actinomycetes between plant organs (tian et al. 2007). the endophytes can come from the surrounding environments, such as rhizosphere and phyllosphere of plants which can enter the plant tissue through stomata, lenticel and physical injuries (broken trachoma) or area in which the lateral roots emerged (susilowati 2010). interaction between endophytic microbes and plants is regarded as symbiosis mutualism. under this type of interaction, endophytic microbes obtains nutrients and protection from the host plant, but they produce compounds that can trigger plant growth and suppress pathogens, phosphate s o l v e n t , a s s i m i l a t i o n o f n i t r o g e n f o r p l a n t s (rosenblueth and romeo 2006) and able to produce enzyme inhibitor (khamna et al. 2009; pujiyanto et al. 2012). based on the partial sequences similarity and phylogenetic relatedness of 16s rrna gene, there are two spesies which were closedly related to the genus s t re p t o m y c e s . s t re p t o m y c e s i s t h e d o m i n a n t actinomycetes isolated from the soil rhizosphere (khamna et al. 2009). the structure of microbial community in the soil is determined by environmental factors such as soil characteristics, type of plants and cultivation that can cause the changes in the structure of soil microbes (zhao et al. 2012). plant roots exudates stimulate the growth of actinomycetes in the rhizosphere and the synthesis of antimicrobial compounds. the diversity of endophytic actinomycetes species in roots and stems of potato samples which were analyzed with dgge showed to have a high similarity with streptomyces (sessitsch et al. 2002). on the agar medium (a culture-dependent), streptomyces are the more frequently found genus. volume 9, 2015 microbiol indones 31 streptomyces and some other genera such as micromonospora and streptosporangium are reported as the most dominating genera isolated from wheat, potatoes and several medicinal plants (sessitsch et al. 2002; coombs and franco 2003; zhao et al. 2011). previous research work on the isolation of endophytic actinomycetes from t. crispa using a culture dependent technique based on 16s rdna-gene, found that amongst the 32 isolates of actinomycetes, the majority were closely related with streptomyces (pujiyanto et al. 2012). in this paper, the diversity of endophytics actinomycetes are decribed based on metagenomic analysis which considered as a culture-independent technique. there are two sequences which are closely related with streptomyces, while others are more closedly related to rare actinomycetes. based on a culture independent approach, this work succeeds to obtain 21 dominant sequences. the relatively low number of sequences obtained may be due to the distribution of each band using dgge results represent one species in a community. in this case, different strains in one species is only represented by one band. pcr-dgge is able to separate the analyzed samples into several separate bands. each separate band represents one of its own species. the dgge profile indicates dominant species and the intensity of each band shows their relative abundance (nimnoi et al. 2010). meanwhile, in a culture-dependent technique, isolates can be derived from a different strain belongs to one species. a culture-independent technique based on 16s rrna gene using pcr-dgge are able to explore m e t a g e n o m i c s d i v e r s i t y o f e n d o p h y t i c s actinomycetes. this approach able to explore diversity of actinomycetes which are relatively difficult to be cultured or rare and even undiscovered or novel endophytic actinomycetes from t. crispa. this study found a species which was closedly related to actinokineospora auranticolor strain ifo 16518, a rare actinomycetes. actinokineospora is a rare endophytic actinomycetes which is found with cultureindependent on the leaves of medicinal plant maytenus austroyunnanensis (qin et al. 2012). actinokineospora has an ability to produce antimycoplasma (hasegawa 1991) and antitrypanosomally actinosporin a and b (harjes et al. 2014; abdelmonsen et al. 2014). analysis of soil actinomycetes community in xinjiang, china using 16s rrna based pcr-dgge discovered species with a closedly related with microbacterium (zhang et al. 2013). microbacterium was reported to have antibacterial and anti-fungal activity (qin et al. 2009) and producing β-glucosidase (park et al. 2008). using a similar approach, endophytic actinomadura was isolated from roots of aquilaria crassna (nimnoi et al. 2010). actinomadura has been reported to produce indole acetics acid (iaa) as a growth hormone and produce siderophore (khamna et al. 2009) as well as antibacterial compunds (qin et al. 2009). this work found a band which has s e q u e n c e s i m i l a r i t y w i t h k o c u r i a . k o c u r i a halotolerans strain yim 90716 is rarely found as endophytic actinomycetes. this genus is reported to have been found in seawater, marine sediment, water and desert soil (kaur et al. 2011). it has the ability to produce iaa and siderophore (khamna et al. 2009) as well as biosurfactant (sarafin et al. 2014). for williamsia which are found in this study as endophyte, this genus has also been found in roots of eucalyptus microcarpa (kaewkla & franco 2012). it has an ability to produce rdx (hexahydro-1,3,5-trinitro-1,3,5triazine) bioremediation (thompson et al. 2005). kibdelosporangium is also a rare genus reported as an endophytic (janso & carter 2010). it has a capability in producing antiviral cycloviracins (tomita et al. 1993) and kibdelomycin (philips et al. 2011). as endophytic actinomycetes, amycolatopsis and actinoplanes were isolated from uncultivatedly rice paddy roots (tian et al. 2007). amycolatopsis was found to have antibacterial and anti-fungal activity (qin et al. 2009), whereas actinoplanes produced teicoplanin, a g l y c o p e p t i d e a n t i b i o t i c f o r t h e t r e a t m e n t o f multiresistant gram-positive infection (taurino et al. 2011) and α-glucosidase inhibitors (zhang et al. 2003). meanwhile the α-glucosidase inhibitors can also be produced by streptomyces isolated from t. crispa (pujiyanto et al. 2012). it is clearly described that actinomycetes able to produce various bioactive compounds thus have open the potency to discover new bioactive compounds. acknowledgement this research work was partly supported by scholarship from directorate of higher education awarded to mona primanita and also partly funded by university priority research grant (decentralisation scheme) contract number 02/01/2014/sp dipa023.04.2.189772 / 2014 given to yulin lestari. references abdelmohsen ur, cheng c, viegelmann c, zhang t, 32 primanita et al. microbiol indones grkovic t, ahmed s, quinn rj, hentschel u, ebel re. 2014. dereplication strategies for targeted isolation of new antitrypanosomal actinosporins a and b from a marine sponge associated-actinokineospora sp. eg49. mar drugs. 12 (3): 1220-1244. doi: 10.3390/ md12031220. bruce kd, hiorns wd, hobman jl, osborn am, strike p, ritchie da. 1992. amplification of dna from native populations of soil bacteria by using the polymerase chain reaction. appl environ microb. 58: 3413-3416. doi: 10.1128/aem.71.12.8265-8272.2005. c o o m b s j t, f r a n c o c m m . 2 0 0 3 . i s o l a t i o n a n d identification of actinobacteria from surface sterilized wheat roots. appl environ microbiol. 69 (9): 56035608. doi: 10.1128/aem.69.9.5603–5608.2003. feng c, hua ss, feng cs, xian jy. 2004. migration of azospirillum brasilense yu62 from roots to stem and leaves inside rice and tobacco plants. act botanica sinica. 46: 1065-1070. franco g, hernandes r, barrios n, strapz jl, crawford dl. 2009. molecular and cultural analysis of seasonal actinomycetes in soil from artemisia tridentata habitat. phyton int j exp botany. 78: 83-90. harjes j, ryu t, abdelmohsen ur, silva lm, horn h, ravasi t, hentschel u. 2014. draft genome sequence of the antitrypanosomally active sponge-associated bacterium actinokineospora sp. strain eg49. 2014. g e n o m e a n n o u n c . 2 ( 2 ) : 1 6 0 1 6 5 . d o i : 10.1128/genomea.00160-14. hasegawa t. 1991. studies on motile arthrospore-bearing rare actinomycetes. actinomycetol. 5(2): 64-71. doi: 10.3209/saj.9_98. heuer h, krsek m, baker p, smalla k, wellington emh. 1997. analysis of actinomycete communities by specific amplification of gen encoding 16s rrna and gel electrophoretic separation in denaturing gradients. appl environ microbiol. 63 (8): 3233-3241. doi: 10.1128/aem.71.12.8265-8272.2005. hibbing me, fuqua c, parsek mr, peterson sb. 2010. bacterial competition; 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2research center for biotechnology, indonesian institute of sciences (lipi) jalan raya bogor km 46. cibinong, bogor 16911, indonesia xanthomonas oryzae oryzae xoo oryza sativa pv. ( ) is the bacterial causative agent of leaf blight in rice ( l.), the most serious bacterial disease of rice in many rice growing areas worldwide. this study aimed to identify and characterize several virulence factors of seven isolates from yogyakarta, west java, and west sumatera. the xoo identification of using 16s rrna confirmed high homology to pv. pxo99 and xoo xanthomonas oryzae oryzae a revealed three groups. the first group was xoo93229, the second group containing xoo1110, xoo1122, xoo1130, xoo7624 and xoo8024 as the same cluster with pxo99 and the third group was kacc10331 and a maff311018. the amounts of exopolysaccharide (eps) and cellulase produced were varying depending on the xoo isolates. the eps were produced more by isolate xoo1130, xoo1122 and xoo8024. all tested isolates revealed similar cellulase activity except for isolate xoo8024. the pathogenicity assay among the isolate xoo showed that all tested isolates were virulent except xoo7624. the assay revealed that the tested isolates in planta have multiplied and continued increasing the population size except for xoo1110 and xoo7624. high yield of eps, cellulase activity, more virulence, and increasing population size revealed from isolate xoo1130 and xoo1122. key words: pv. 16 rrna, virulence factorxanthomonas oryzae oryzae, pv. ( ) merupakan bakteri penyebab penyakit hawar daun padi ( xanthomonas oryzae oryzae xoo oryza sativa l.) dan merupakan penyakit yang serius di daerah pertanaman padi. penelitian ini bertujuan untuk mengidentifikasi, mengkarakterisasi dan mengukur aktivitas fenotipe beberapa faktor virulen pada tujuh isolat bakteri yang diisolasi dari yogyakarta, jawa barat, dan sumatera barat. identifikasi bakteri xoo xoo menggunakan 16s rrna diperoleh homologi yang tinggi dengan pv. pxo99 dan xanthomonas oryzae oryzae a hasil pengelompokan didapatkan tiga grup. pada grup pertama adalah bakteri xoo93229, grup kedua adalah bakteri xoo1110, xoo1122, xoo1130, xoo7624 dan xoo8024 bersama pxo99 dan pada grup ketiga adalah a kacc10331 dan maff311018. besarnya eksopolisakarida (eps) dan faktor virulen lainnya bervariasi bergantung pada isolat yang digunakan. eps pada isolat xoo1130, xoo1122 dan xoo8024 diproduksi lebih banyak. aktivitas cellulase memberikan hasil yang sama pada semua isolat kecuali pada isolat xoo8024. uji patogenisitas dan menunjukkan bahwa enam isolat yang diujikan adalah virulen kecuali xoo7624 in planta kurang virulen pada varietas ir24, peningkatan jumlah populasi bakteri pada semua isolat kecuali pada isolat xoo1110 dan xoo7624. isolat xoo1130 dan xoo1122 memberikan nilai yang tinggi pada produksi eps, aktivitas cellulase, virulensi, dan jumlah populasi bakteri. kata kunci : pv. 16 rrna, faktor virulensixanthomonas oryzae oryzae, vol.8, no.3, september 2014, p 103-111 doi: 10.5454/mi.8.3.3 *corresponding author; phone: + , fax: 62-251-8337975 + ; email: fatimahsuw@gmail.com62-251-8338820 b a c te r ia l le a f b li gh t (b lb ) c a us e d by xanthomonas oryzae oryzae xoopv. ( ) is the most devastating bacterial disease of rice production worldwide. in the fourth position of top 10 xoo bacterial plant pathogen after , pseudomonas syringae ralstonia solanacearum agrobac te rium a nd tumefacient et al xoo (mansfield . 2012). infects the rice leaf typically through hydathodes at the leaf tip, broken trichomes, leaf margins and wounds in the leaves or roots, multiplies in the intercellular spaces and enters into xylem vessels (ou 1985; nozue . et al 2011). after sufficient multiplication in the intercellular spaces of the underlying epitheme, the bacteria enter the xylem and spread in the vascular system (mew . 1993; liu . 2006).et al et al produces a range of virulence factors, xoo including eps, extracellular enzymes, and type iii effectors (liu . 2014). eps can play a critical role et al in facilitating adhesion of bacteria to the host surface during initial stages of plantpathogen interactions and disease development (subramoni 2006). cell-et al. mailto:fatimahsuw@gmail.com wall degrading enzymes, such as cellulases, pectinases, xylanases and proteases, are secreted by plant pathogens cells to break down the components of host cell walls and may play a crucial role in virulence and bacterial nutrition (temujin . 2011). many et al xanthomonads require a type iii secretion system (t3ss) for pathogenicity on plant hosts, and the requirement reflects the utilization of t3 effectors to mediate the processes of pathogen adaptation to specific host tissues, species and genotypes (buttner and he 2009; white . 2009; verdier . 2012).et al et al this study aimed to identify and characterize several virulence factors of seven from indonesia xoo and provided new insight towards indonesian xoo isolates as general feature of for the development of effective disease control methods. materials and methods identification of pv using 16s x. oryzae oryzae rrna. genomic dna of xoo was extracted from 5 ml nb cultures grown overnight. the bacterial cells were pelleted and lysed in 650 µl extraction buffer (100 mm tris ph 8, 100 mm edta, 250 mm nacl, 15% sds (w/v), 1% pvp-40 (w/v) at 65 c for 30 minutes. dna o was isolated using a modified method with 100 µl of 5 m potassium acetate and precipitated with isopropanol (george . 1997). et al for 16s rdna sequencing, primers 8f (5'agagtttgatcatggctcag-3' and 15r (5'aaggaggtgatccaaccgca-3') were used to amplify the full length of bacterial 16s rdna (chao . 2008). cr mixture contained 10 mm et al each 25 μl p tris-hcl (ph 8.3), 50mm kcl, 1.5mm mgcl2, 200μm of each dntp, 400nm of each primer, 1 u of taq polymerase, and 10 ng of the dna template. the pcr conditions were 96 °c for 5 min; 35 cycles consisting of 96 °c for 1 min, 55 °c for 3 min, and 72 °c for 1 min; and 72 °c for 7 min. the pcr products were subjected to gel electrophoresis in 1.5% agarose gel, followed by ethidium bromide staining. the pcr product was sequenced (macrogen). sequence assembly was performed with dna baser software. similarity searches with sequences were performed by online blast analysis. for phylogenetic analysis, sequences were aligned by using the clustalw software. the measurement of exopolysaccharide (eps). measurement of exopolysaccharide (eps) was conducted as described by jeong . (2008). a single et al colony of each xoo strain was inoculated in 40 ml of nb medium and incubated for 72 h at 28 c with o agitation. the optical density of the bacterial cultures was adjusted to 1.0 at 600 nm with nb. the culture supernatants were transferred into new 50-ml tubes and supplemented with 1.0% potassium chloride (w/v; final concentration). two volumes of absolute ethanol were added to each solution, and the tubes were placed at -20 c overnight. the precipitated crude eps was o collected by centrifugation for 30 min at 83,000×g. the eps pellets were dried at 55 c for 12 h and the dry o weight of each was measured. cellulase activities were assayed cellulase assay. as described (jeong . 2008). xoo strains were et al cultured in nb medium for 72 h, after which the optical density of the cultures was adjusted to 1.0 at 600 nm with nb. thirty microliters of culture supernatant were placed in a hole in the assay agar medium, which contained 0.1% carboxymethyl cellulose, 50 mm sodium phosphate (ph 7.0), 0.8% agarose, and 0.02% sodium azide, and the plate was incubated for 20 h at 28 c. the incubated plates were stained with 0.1% o congo red for 10 min and then washed several times with 1 m nacl. after washing, the cellulase activity was determined by measuring the diameter of the clear zone around the hole. pathogenicity and assays. in planta the xoo strains were grown on nb medium for 2 days at 28 °c. the bacterial cells were resuspended in sterilized water at an optical density of 600 nm (od 600) (about 10 cfu -8 ml ). bacterial blight inoculation was carried out on 6--1 week-old on resistance rice varieties irbb7 and code and susceptible rice varieties ir24 and kencana bali using the leaf-clipping method (kaufmann 1973). experiments were conducted under greenhouse conditions. for pathogenicity assay, the lesion length was measured in 7, 14 and 21 days after inoculation with 10 leaves for each strain. in planta assay was carried out on susceptible rice variety ir24. it determined xoo strains multiplication in planta at six time points after infection by leaf clipping on 3, 6, 9,12, 15 and 18 days after inoculation. the in planta was assayed as described by hu . (2007). the leaf et al pieces were then ground in 10mm mgcl2. the leaf homogenate was diluted in 10mm mgcl solution.2 serial dilutions were made and spread onto psa agar plates. the plates were incubated at 28 °c until the number of colony-forming units (cfu) per leaf was counted. 104 f atimah et al . microbiol indones results growth curve of . xoo population growth is studied by analyzing the growth curve of a microbial culture. the bacterial growth can be plotted as the logarithm of the number of viable cells versus the incubation time. the resulting curve has four distinct phases. the cell density of isolates increased xoo from 0.1 to 2.7 (od ) during 48 h of growth at 600nm 28 c (fig 1). the growth cell started at the early o exponential until 18 h and reached its maximum at the early stationary phase on 24 h then remained stable until 48 h during stationary phase. xoo1122 isolate showed increasing in its growth rate on exponential phase (18-24 h) however xoo7624 growing slow compare to other isolates. identification of using 16s rrna and xoo phylogenetic analysis. the partial 16s rrna gene sequences (1490 bp) of all strains were determined. then, the sequences were compared with related bacteria in genbank and sequence similarities were determined using the blast program. the result confirmed that all isolates showed high homology (99-100%) to pv. pxo99 xanthomonas oryzae oryzae a (table 2). the clustalw analysis of the 16s rrna gene sequences revealed three groups. the first group containing one isolate, xoo93229, the second group containing. xoo1110, xoo1122, xoo1130, xoo7624 and xoo8024 as the same cluster with pxo99 , and the a third group kacc10331 and maff311018 were xoo clustered together (fig 2). fig 1 optical density at 600 nm of seven indonesian xoo strains. table 1 bacterial strains used in this study strain collection site cultivar source source xoo93229 harjobinangun, yogyakarta cisadane icabiograd xoo7624 bogor,west java local variety icabiograd xoo8024 cianjur, west java local variety icabiograd xoo11-010 cianjur west java ciherang icabiograd xoo11-022 maninjau, west sumatera kuriak putiah icabiograd xoo11-028 maninjau, west sumatera kuriak putiah icabiograd xoo11-030 maninjau, west sumatera kuriak putiah icabiograd 3,5 3 2,5 2 1,5 1 0,5 0 12 18 24 30 36 42 48 time course (h) x00 1130 x00 1122 x00 7624 x00 8024 x0093229 x00 1110 x00 1128 volume 8, 2014 microbiol indones 105 measurement of exopolysaccharide (eps). the seven tested colonies produced eps with different xoo yield of production. isolate xoo1130, xoo1122 and xoo8024 giving more production of dry weight up to 80 mg however xoo1110, xoo7624, xoo1128 and xoo93229 showed less production around 40-50 mg (fig 3). cellulase activities. cellulase produced by the tested on medium agar plates showed the clear xoo zones around the wells refer to cellulase activities was measured (fig 4). all isolates revealed similar xoo cellulase activity (17-18 mm) except for strain xoo8024 (12 mm). fig 2 clustalw analysis of 16s rrna sequence of indonesian isolates xoo93229, xoo7624, xoo8024, xoo1110, xoo xoo1122, and xoo1130 compared with pxo99a (cp000967), maff311018 (ap008229), and kacc10331 (ae013598). table 2 identity of indonesian pv isolatesxanthomonas oryzae oryzae isolate species identiity (%) accession number xoo93229 xanthomonas oryzae pv. oryzae pxo99a 100 cp000967.1 xoo7624 xanthomonas oryzae pv. oryzae pxo99a 99 cp000967.1 xoo8024 xanthomonas oryzae pv. oryzae pxo99a 100 cp000967.1 xoo11-010 xanthomonas oryzae pv. oryzae pxo99a 99 cp000967.1 xoo11-022 xanthomonas oryzae pv. oryzae pxo99a 100 cp000967.1 xoo11-030 xanthomonas oryzae pv. oryzae pxo99a 99 cp000967.1 xoo93229 xoo7624 xoo8024 xoo11010 xoo1122 xoo1130 xo99a maff311018 kacci10331 106 f atimah et al . microbiol indones pathogenicity assay. all tested strains caused leaf blight on the surface of rice cultivar ir24 on 5 days after inoculation. the development of bacterial leaf blight symptoms in rice plants inoculated with xoo strains observed on lesions leaf of resistant cv. irbb7 and code and susceptible cv. ir24 and kencana bali on 21 d post inoculation. symptoms of blb appeared on leaves as pale-green to grey-green water-soaked streaks near the leaf tip and margin. these lesions coalesced and became yellowish-white with wavy edges. on leaf sheath of susceptible cultivars, the affected leaves will turn yellow, roll up and wilt rapidly and systemic infection that produces tannishgrey to white lesions along the vein under greenhouse inoculation (fig 5). xoo differences in virulence among the strains were quantified according to the lesion length of the necrotic area. xoo93229, xoo1122, xoo1128, xoo1130, xoo1110 and xoo8024 strains were shown to cause symptoms at day 7 after inoculation. it also showed the development of disease and increased virulence to susceptible rice cultivar ir24 at 21 d post inoculation. the average lesion length was 30, 31, 32, 32, 27, and 26 cm, respectively. strain xoo7624 showed the lowest lesion length at day 7 post inoculation and displayed the same pattern of lesion length until 21 d post inoculation (fig 6). fig 3 eps dry weights of seven indonesian xoo strain. fig 4 cellulase produced by the seven pv. on medium agar plates. the clear zones around the xanthomonas oryzae oryzae wells refer to cellulase activities of strain (a) xoo1130, (b) xoo93229, (c) xoo110, (d) xoo1122, (e) xoo8024, (f) xoo xoo7624, and (g)xoo1128. a b c d e f g volume 8, 2014 microbiol indones 107 100 90 80 70 60 50 40 30 20 10 0 xoo strain xoo 93 229 xoo 1110 xoo 1128xoo 1130 xoo 1122 xoo 8024 xoo 7624 a 1 2 3 b 1 2 3 1 2 3 4 1 2 3 4 fig 5 leaf phenotype of irbb7 and ir24 as an example showing increased susceptibility to pathogen strain (a) xoo93229 and b) xoo1122. leaf 1) irbb7, leaf 2) and 3) ir24. leaves were photographed 5 days after inoculation. arrow ( indicates position of clip-inoculation of the leaf tips. (c) lesions leaves of 21 days after inoculated with xoo93229 (left) and xoo1122 (right). resistant cultivar: leaf 1) irbb7 2). code and susceptible cultivar: 3) ir24, and 4) kencana bali. fig 6 pathogenicity assay of strains on the susceptible rice cultivar ir24. length of lesions was measured in 7, 14, and 21 xoo dai with 10 leaves for each strain. fig 7 growth curve of isolates on rice leaves after inoculation. mean cfu per leaf was calculated from two xoo independent experiments with three leaves for each strain. 108 f atimah et al . microbiol indones 45 40 35 30 25 20 15 10 5 0 days after inoculation xoo1122 xoo1130 xoo1110 xoo93229 xoo7624 xoo8024 xoo1128 7 14 21 1,00e+12 1,00e+11 1,00e+10 1,00e+09 1,00e+08 1,00e+07 1,00e+06 1,00e+05 1,00e+04 days after inoculation 0 63 9 12 15 21 xoo93229 xoo1122 xoo1110 xoo1130 xoo7624 xoo8024 xoo1128 in planta assay. similar amounts of bacteria (2.8x10 cfu) were inoculated on rice by leaf clipping. 4 the multiplied at six time points xoo strains in planta after infection. initially, the distinct difference was observed in 3 days after inoculation (dai) however xoo1110 and xoo7624 strain were less in population size (3x10 and 2x10 , respectively). xoo93229, 6 6 xoo1122, xoo1128 and xo1130 strain multiplied continued increasing until it reached a maximal population in 10 cfu and xoo1110 and xoo7624 strain 11 reached a maximal population in 10 cfu within 15 days 9 after inoculation (fig 7). discussion the identification of 16 rrna sequence, characterization of its growth curve and the important virulence factors such as cellulase and extracellular polysaccharide (eps), pathogenicity assay and in planta assay have been examines in this study using seven indonesian isolates. the amounts of eps xoo and cellulase produced were varying depending on the xoo strain. the identification of 16 rrna gene sequence was used to confirm the identity of strains. the 16s rrna gene is most typically used for classification of bacteria. in this study, the 16s rrna gene sequenced of all xoo isolates used confirmed high homology to xanthomonas oryzae pv. oryzae pxo99 (salzberg a 2008). a dendrogram depicting the estimated et al. phylogenetic relationship was based on comparisons of the available 16s rrna sequence data for the xanthomonas oryzae pv oryzae. indonesian xoo isolates was clustered as the same cluster with pxo99 a (salzberg . 2008) however the isolate xoo93229 et al was in different cluster. hauben . (1997) reported et al that a very small degree of divergence of 16s rrna gene sequences among the xanthomonas. lang . et al (2010) reported the high degree of diversity of xanthomonas isolates within and between countries. in this study, the third cluster consisted of korean strain kacc10331 (lee . 2005) and the japanese strain et al maff311018 (ochiai . 2005). it was similar with et al salzberg . (2008) which used the phylogenetic et al relationships among pxo99 and the complete a genome of the genomes of maff311018, and ka cc103 31 ge ner ate d a c lad ogra m whe re maff311018 and kacc10331 was grouped together. the xoo1130, xoo1122 and xoo8024 isolates produced high eps compare to other isolates. based on their colony morphology, these three isolates were mucoid and shiny when compared with the other isolates (data not shown). this phenotype results from the production of copious amounts of the extracellular polysaccharide (eps), known as xanthan gum. thein and prathuangwong (2010) reported that colonies of mutants were smaller when compared with the wild type that resulted from less production of eps. in this study, we used jeong . (2008) method which the et al nb medium does not contain sucrose, results in a more consistent optical density for xoo, and was used for eps assays. he . (2006) mentioned that high et al concentrations of sucrose in the medium have been reported to result in high background in assays of some extracellular enzyme activities. however it should be noted that lan . (2007) observed in long-term et al storage and culture will lead to spontaneous loss of virulence and associated with reduction of eps production. partially purified eps preparations have been found to induce rice leaf wilting. this may be due to cell membrane leakage caused by eps (yang and white 2004). the diameter of the zone of clearance indicates the ability of the bacteria to hydrolyze cellulase. the capacity of the bacterial isolates to degrade cellulase was indirectly determined using the degradation of c a r bo xy me th y lc e llu lo se (c mc) , ind ic a t ing endoglucolytic activity (soares . 2012). in this et al study, all of the xoo tested isolates giving similar cellulase activity except for isolate xoo8024. however the variation of cellulase activity using plate assay method was not related with the virulence ability. hu . (2007) reported that cellulase plate assay et al method failed to discriminate the difference of enzyme activity between mutants and wild type also mentioned by jeong . (2008) that the cellulase activities of the et al rpf mutants and the complementation strains were similar to those of the wild-type. efficient methods for recovering bacterial cells directly from plant tissues permit analyses of in vivo expression in plant-pathogen interactions (mehta 2003) and may help in the early detection of genes involved in pathogenicity (thwaites 2004). hu et al et al. (2007) showed that the expression of the gene can only be detected when xoo grows in planta but cannot be detected when it grows on psa agar. in this study, the difference of population size and the increasing of multiplication related with the ability to colonize rice volume 8, 2014 microbiol indones 109 seedlings have associated with the virulence ability. the pathogenicity assay among the xoo isolate showed severely increased virulence to susceptible rice cultivar ir24 except for xoo7624. however it assumed that our result comes from wild type compare to mutant type generated by study of feng . (2009) reported et al that the xrva mutant gxn1280 and the xrva overexpression strain gxo3098 showed a significant reduction in lesion length compared to the wild-type strain but the bacterial populations of these mutants in rice leaves were not significantly different from that of the wild-type. in this study, isolate xoo1130 and xoo1122 revealed high yield of eps, cellulase activity, virulence, and increasing population size. in contrary with xoo7624 revealed low yield of eps, less virulence, and low number of population size but high cellulase activity. xoo93229 revealed high cellulase activity, more virulence, and increasing population size but low yield of eps production. xoo8024 with high yield production of eps, more virulence and increasing population size was low in cellulase activity. initially it was presumed that all the tested isolates will have association between the virulence factor and pathogenicity. regarding phenotypic characterization, recent molecular characterization of xoo, the availability of genome sequence for rice and xoo may facilitate our research for identification of many new pathogen-associated molecular patterns (pamps) and avirulence and virulence effectors (liu . 2014).et al acknowledgment this work was financially supported by indonesian toray science foundation science and technology research grant (itsf-strg). we thank to mahrup and milannisa dwitaharyani for technical support. references buttner d, he sy. 2009. type iii protein secretion in plant pathogenic bacteria. plant physiol. 150:1656-1664. doi:10.1104/pp.109.139089. chao sy, tomii k, watanabe, tsai y. 2008. diversity of lactic acid bacteria in fermented brines used to make stinky tofu. int j food microbial. 123:134-141. doi: 10.1016/j.ijfoodmicro.2007.12.010. feng jx. song zz, duan cj, zhao s, wu yq, wang cw, dow jm, tang jl. 2009. the xrva gene of xanthomonas oryzae oryzae, pv. encoding an h-nslike protein, regulates virulence in rice. microbiology. 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syringae . mol pl a nt mi c robe int e rac t 17: 12501258. doi:10.1094/mpmi.2004. 17.11.1250 . verdier v, lindsay rt, aaron wh, rene c, r. andres c, clarice ls, adam jb, jan el. 2012. transcription activator-like (tal) effectors targeting ossweet genes enhance virulence on diverse rice ( ) oryza sativa varieties when expressed individually in a tal effector-deficient strain of . new xanthomonas oryzae phytologist. doi: 10.1111/j.1469-8137.2012. 04367. white ff potnis n. jones jb. and kloebnik r. 2009. the type iii effectors of xanthomonas. mol plant pathol. 10: 749-766. doi: 10.1111/j.1364-3703.2009.00590. yang b, white f. 2004. diverse members of the avrbs3/ptha family of type iii effectors are major virulence determinants in bacterial blight disease of rice mol plant microbe interact, 17:1192-1200. . doi.org/10.1094/mpmi.2004. 17.11.1192. volume 8, 2014 microbiol indones 111 2 roni.pmd volume 3, number 3, december 2009 p 126 132 issn 1978-3477 analysis of rumen microbial population of cattle given silage and probiotics using terminal restriction fragment length polymorphism roni ridwan1*, yantyati widyastuti1, sri budiarti2 and achmad dinoto3 1research center for biotechnology, lembaga ilmu pengetahuan indonesia, jalan raya jakarta-bogor km 46 cibinong, bogor 16911, indonesia; 2department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 3research center for biology, lembaga ilmu pengetahuan indonesia, jalan raya jakarta-bogor km 46 cibinong, bogor 16911, indonesia, rumen ecology is an important observation in evaluating the effectivity of silage and probiotic additives relating to their roles in cattle productivity. the objective of this study was to examine the effects of silage and probiotics on ruminal ecosystems in vivo using a molecular approach. terminal-restriction fragment-length-polymorphism (t-rflp) analysis was used to detect changes of ecological communities based on 16s-ribosomal deoxyribonucleic acid (16s-rdna). two rumen canulated po cattle were fed several diets i.e.; (r0) basal diet dry matter basis (pennisetum purpureum 70% and commercial concentrate 30%), (r1) silage (basal diet fermented using lactobacillus plantarum btcc570), (r2) silage + probiotics (l. plantarium str btcc531), (r3) basal diet + probiotics (l. plantarium str btcc531). digesta samples were collected 3 h after feeding for ph and t-rflp analysis. t-rflp analysis was performed using the 16s-rdna amplified from each sample. the lengths of the terminal restriction fragments were analysed after digestion with hhai, haeiii and mspi. results showed the effectivenes of silage and probiotics, given together, on the index of smith and wilson evenness applied to t-rflp ecology data (evar) with 0.89±0.04 being the highest. the diversity of rumen microorganisms is influenced by individual differences of each animal. t-rflp analysis has a potency to be used for comparisons of complex bacterial communities, especially to detect changes in community structure in response to different variables and to show rumen bacteria diversity in the rumen. keywords: silage, probiotics, rumen ecology, evar, t-rflp, 16s-rdn *corresponding author, phone/fax: +62-21-8754587/8754588, e-mail: roni001@lipi.go.id the rumen is a complex ecosystem in which animal feed is consumed by ruminants and digested by an active and diverse microbiotal population. most of the microorganisms aid digestion, but others may potentially cause pathogenesis. the implication of microbiota in the rumen for nutrition and feed conversion of the animals merits greater attention. microbiota in the rumen consists of mainly bacteria, followed by protozoa and fungi. information on number of cultured bacteria is around 1010, protozoa 106 and fungi 104 cfu ml-1 rumen content (hungate 1966). the number and composition of rumen microbiota were influenced by the composition of the feed consumed by the animals. naturally, the feed contains roughage and concentrate, but it may also contains feed additives such as directly fed microorganisms or probiotics. several microorganisms have been widely selected and used as probiotics. lactic acid bacteria (lab) are considered ‘generally recognized as safe’ (gras) and some species have been selected as probiotics. lab present in young animals are about 106 cfu ml-1 and the number decreased when the animals changed the type of feed being consumed. the presence of lab in the rumen is related to, firstly, acidosis caused by excess concentrate consumption and secondly, probiotic properties (stewart 1992). probiotics have been reported to give a positive effect to the rumen environment, prevent the gastrointestinal tract from infection, and therefore improve animal productivity (fuller 1992). silage is a fermented ruminant feed of high moisture content. the process of fermentation is called ensilage and depends on the activity of lab to convert water-solublecarbohydrates into organic acids, mainly lactic acid. lab are still alive in silage, and the consumption of silage by animals gives opportunity for lab to enter the rumen (weinberg et al. 2004). the detection of rumen bacterial population by simple culture methods may give unreliable results, since many of the bacteria present in the rumen are not able to be cultured by available methods. the molecular tool commonly used for examining microbial communities is the small-subunitribosomal-deoxyribo-nucleic-acid (ssu-rdna). terminalrestriction fragment-length-polymorphism (t-rflp) analysis is one of the procedures that can be used to track spatial and temporal changes in ssu-rdna’s from microbial communities (liu 1997). among the available procedures, trlfp is suitable to give a rapid comparison of complex bacterial communities. t-rflp has been used for detecting the bacteria in the rumen of unfaunated and faunated cattle. t-rflp patterns showed that the absence of protozoa in the rumen changes the composition of fecal bacteria (ozutsumi et al. 2008). probiotic strains ingested by humans were able to survive in the intestine and tracked in the feces as indicated by the corresponding t-rfs (bakir et al. 2008). the aim of the present study is to analyze the effect of the administration of lab from silage and probiotics on the rumen population of cattle using t-rlfp. materials and methods bacterial strains and media. lactobacillus plantarum btcc 570 and l. plantarum btcc 531 were from the biotechnology culture collection (btcc)-lipi. l. plantarum btcc 570 was used as noculants for silage fermentation and l. plantarum btcc 531 was used as a probiotic. medium deman rogosa sharpe (mrs) (de man et al. 1960) was used throughout the study. silage. elephant grass (pennisetum purpureum) was preserved as silage. a commercial concentrate 10% (w/w) was mixed during the preparation of silage. the nutrient composition of the concentrate was crude protein 14%, fat 4%, crude fiber 8%, digestible protein 11%, total digestible nutrient (tdn) 68% and ash 10%. l. plantarum btcc 570 (1010 cfu ml-1) 0.1% was used as a inoculant by spraying and mixed thoroughly with p. purpureum and concentrate. plastic bags were used as a silo for the fermentation of silage. incubation was for 30 days. the ph and population of lab was determined after fermentation. probiotics. l. plantarum btcc 531 was cultivated in mrs medium for 18 h at 30ºc and harvested by centrifugation at 16 770 x g. the resulting pellet was mixed in 10% w/v skimmed milk solution, then freeze dried overnight. the resultant probiotics powder was put in soft capsules of 400 mg. animals and sample collection. two rumen fistulated ‘peranakan ongole’ (po) cattle (cattle a and b) were reared at the animal house of the research center for biotechnology in cibinong. they were given several diets, i.e. a basal diet consisting of 70% dry matter fresh elephant grass (p. purpureum) and 30% commercial concentrate (r0), silage of p. purpureum (r1), r1+ 1 capsule of probiotics (r2) and r0 + 1 capsule of probiotics (r3). the amount of dietary material given was calculated based on the body weight of each animal. the diets were given 2 x a day at 08.00 and 13.00 hrs, while water are available ad libitum. adaptation and treatment periods for each diet were 10 and 14 days, respectively. during the treatment period, samples of rumen content was collected on days 5, 10, and 14. samples of rumen content were collected 3 h after the morning feeding through the fistula, and squeezed using a double layer of cheese cloth. samples were kept in 20 ml sterile corning tubes and stored at -20ºc until analysis. extraction of dna. 10 g of each rumen sample was squeezed using a double layer of cheese cloth and thoroughly mixed. squeezed rumen samples (200 ìl) were washed 2 x using phosphate buffer saline (800 ìl) and centrifugation at 14 534 x g for 2 min. the pellet was used for dna extraction. bacterial dna was extracted using the dneasy blood and tissue dna extraction kit (qiagen, japan) according to the manufacturer’s instructions. the purity of dna was measured using gene quant pro (amersham biosciences-england). pcr amplification. two universal primers 46f (5’gcytaacacatgcaagtcga-3’) and 1080r (5’cccaacatctcacgac) were used in pcr to amplify the 16s-rdna coding region. the 46f primer was labeled with 6carboxyfluorescein (6-fam, sigma tokyo, japan). pcr amplification was performed in a total volume of 50 ì l containing 1 ìg of dna, concentration of primer 6fam46f; 0.4 ì m, primer 1080r; 0.4 ì m and premix taq® 25 ìl (takara bio inc. otsu, japan). the pcr was conducted using a thermocycler biometra®tgradient (thermoblockgermany). the pcr conditions were based on the method of dinoto et al. (2006b) with the following modifications: heating 94ºc for 2 min, followed by 30 cycles consisting of 94ºc for 2 min, 48.5ºc for 1 min and 72ºc for 1 min and a final extension period of 72ºc for 10 min. the pcr product was purified by suprec pcr (takara bio inc. otsu japan). purified pcr products from individual dna extractions from 3 collection times (day 5, 10, and 14) from each diet were combined and digested with restriction enzymes i.e. haeiii (50 u ì l-1), hhai (10 u ì l-1) and mspi (50 u ì l-1) (takara bio inc.) at 37ºc for 24 h. the reaction was terminated by placing the microtubes with individual samples in a water bath at 65ºc for 20 min, and then put on ice immediately. the restriction digest products were subjected to ethanol precipitation and vacuum drying. t-rflp analysis. the lengths of the terminal restriction fragments (t-rfs) were determined with standard sized marker 500 lz using abi prism 3100 genetic analyzer at atmajaya university and genescan analysis 3.7 software (applied biosystem). the major t-rfs were identified by computer simulation, which was performed using 16s-rrna gene sequences registered with the ribosomal database project (rdp) ii (http://rdp8.cme.msu.edu/html/tapjava2.html). the dendrogram analysis was based on the similarity coefficient for the objective interpretation of the difference t-rfs patterns. dendrogram-type was established using the unweighted pair-group method with arithmetic mean (upgma) (blackwood et al. 2003). analysis of the data. the data obtained from the t-rflp analyses were normalized as described by sait et al. (2003). t-rfs peak areas with total areas less than 5% threshold value were excluded. a sorensen’s pairwise similarity coefficient, cs, was calculated for each pair of t-rflp profiles within a complete edited data set as follows: cs=2j/(a+b) where j is the number of t-rfs with relative areas of greater than zero common to the two profiles being compared within an edited data set, and a and b are the number of t-rfs with a relative area of greater than zero in each of the two profiles. diversity of rumen bacteria populations were base on method diversity index smith and wilson evenness (evar) described by blackwood et al. (2007) given below: microbiol indones 127volume 3, 2009 {arctan i = 1 i = 1 { [ in ( ) in ( )/s] /spi pi 2 s s s s evar = 12 results silage and probiotics. silage has been created with the following characteristics; soft texture, not moldy, sour aroma typical of lactic acid, ph 3.99, dry matter 32.78% and a total population of lab around 1.1 x 106 cfu g-1 silage. probiotic broth has been created with a population density of 7.5 x 1011 cfu ml-1 with a value of a 600 of 1.7. probiotic is further made into probiotic powder using freeze-drying to produce a population density of 1.67 x 1013 cfu g-1. extracted and amplificated dna of rumen bacteria. dna concentration of extracted cattle rumen contents with a total volume of 100 ìl in the range of 31.71-128.40 ng ì l-1 and a dna vs rna purity was measured by ∆a 260/230nm of 1.05-1.26 and dna vs protein ∆a 260/280nm of 0.98-1.20. pcr amplification of 50 ìl reaction mixture has been successfully performed from all treatments with the resulting size of 10001100 bp, in accordance with if the primer in use is 6fam-46f and 1080r (fig 1). amplicon concentration ranging between 53.00-116.40 ng ì l-1 with dna vs rna purity on ∆a 260/230nm and protein vs dna on ∆a 260/280nm each of 1.82-2.57 and 1.17-1.92. similarity coefficient (c s ). normalization is performed on each sample using a method based on the percentage of fig 1 amplified dna of rumen bacteria from all treatments. fig 2 the dendrogram illustrates the relationship of the t-rflp pattern from rumen bacteria by haeiii, hhai, and mspi restriction enzymes base on upgma clustering. area that is the comparison between the relative peak area detected by the total peak area of all communities of enzyme used multiplied by 100%. a percentage greater than 5% of the population was considered to be stable and to represent the most factual bacterial population in the community (sait et al. 2003). table 1 shows that the treatments r2 with r3 and r0 with r3, have the highest cs values. treatment r0 and r2 show the lows l-value for cs, so the bacterial population in the r2 has a difference of treatment which is far with the r0 treatment. a close relationships of organisms in the rumen can be seen from the dendrogram which uses clustalx2 through application of the t-rf pattern based on upgma. in principle clustering by upgma is almost the same as the value of the cs (fig 2). microbiol indones128 ridwan et al. tabel 1 similarity coefficient (c s ) 1000 bp diversity indexs smith and wilson evenness (evar). ecological diversity of bacterial communities from a data scaning can be seen with the index diversity use of smith and wilson evenness (evar). a third enzyme was used in trflp analysis for strengthening the data obtained by observation. given r0, r1, r2, r3 and the second cattle po showed the diversity of rumen bacteria that have a sufficient interest to the area with a population of fluorescence intensity that was around 28, 29, 26, and 25 t-rfs of all enzyme is used in this research. r0 is quite diverse with the evar value higher than those of r1 and r3 (table 2). interestingly, r2 as a treatment of silage and probiotic combination showed a synergistic effect that increases the evar value to be highest amongst all treatments. closeness of t-rf (bp) with the rdpii organisms. to know the names of the population of rumen bacteria from closeness and diversity of each treatment, the size t-rfs was next compared with genebank database on the ribosomal data project (rdp) ii on the homepage http:// rdp8.cme.msu.edu/html/tap-java2.html. data in table 3 show the closeness of t-rfs observations with prediction organisms from databases on the three restriction enzyme used. the genescan abi prism 3100 results are shown in the form of on electropherogram for all treatment of the two cattle which were digested with haeiii enzyme (fig 3). discussion the process of pcr amplification using label primer at the 5’ end of the with 6-carboxylfluorescence-amplifiedspecific-regions of bacterial genes 16s-rdna content of total community dna was succesful. the automatic genetic analyzer only read fragments of nucleic the flourescence at the 5’ end of the absorption by the sensor when loaded at the capillary (liu et al. 1997; kaplan et al. 2001). because the amount of dna loaded on the capillary cannot be accurately controlled, the sum of all t-rf peak areas in a pattern (total peak area) varied between t-rf patterns. to compensate for this variation, it was necessary to normalize peak detection thresholds and totals of peak areas (kaplan et al. 2001). osborne et al. (2006) states that to minimize variation, bias, and to ensure the size of t-rf which was found was genuine one should be normalize performance on certain thresholds. the high value of cs for the treatment had a structure similar to the organisms in the rumen. while the small value of cs showed differences between organisms that were found in the rumen. closeness of organisms in the rumen can be seen from the dendrogram also use clustalx2 through application of the t-rf pattern based on upgma (fig 2). the treatment r2 is able to alter the community of previously dominant to be a population in decline and creation of new ones. the new population shows the role of ecology in the rumen, so that the old population will not necessarily fit with the new environment. diversity of rumen microorganisms depends on feed, the condition of the cattle, and sinergism with other microbes. equality in this equation, as compared to the other indexes in the analysis, showed that a more ecological approach was closest to reality and to have a high correlation (blackwood et al. 2007). in our study, an increased diversity tabel 2 diversity indexs smith and wilson evenness (evar) table 3 closeness of t-rfs lengths (bp) with organism on ribosomal database project ii microbiol indones 129volume 3, 2009 organism t-rfs length (bp) predicted observed (haeiii,hhai,mspi) (haeiii,hhai,mspi) bifidobacterium angulatum atcc27535(t) 46, 335, 43 46,338, nd clone eh-7 152, 59, na 152, 59,55 str.rj5 152, 159, 93 152, 161, 95 clone wchbi-82 161, 331, 250 161, 331, 256 clone12-102 181, 64, 59 181, 65, 57 eubacterium cellulosolvens atcc43171(t) 201, 152, 184 201, 150, 184 syntrophomonas saporans 201, 341, 259 201, 339, 258 clone vc2.1 bac43 201, 23, 259 201, nd, 258 eubacterium ptautii dsm 4000(t) 201, 350, 260 201, 349, 258 clostridium fusiformiscm973 201, 1052,184 201, nd, 184 arcobacter cryaer ophilus ccug17801 arcobacter butzleri ccug10373 /(clonet31/clonet55/clone t95) 224, 64, 436 223, 67, 436 ruminococcus productusatcc27340/ clostridium sp. str ain dr6a 233, 150, 182 233, 150, 184 ruminococcus hansenii atcc 27752(t) 235, 151, 259 235, 150, 258 cytophaga fucicola nn015860/sw17 242, 56, 51 243, 59, 51 epulopiscium sp. str ain morphotype a2/ epulopiscium fishelsoni red. sea a2 247, 334, 244 247, 331, 241 bifidobacterium longum atcc15707(t) 247, 334, 96 247, 331, 94 metabacterium polyspora 251, 517, 248 251, nd, 248 clostridium purinolyticum atcc33906(t) 260, 160, 128 260, 161, 123 index in silage and probiotic treatment is related to the beneficial effects on cattle productivity, in which much more propionic acid is produced in rumen (ridwan unpublished data). although, the presence of l. plantarum (silage inoculant) and leuconostoc sp. (probiotic) could not be detected in the rumen five days after consumption, the data of increased bacterial diversity and rumen metabolic changes (data not shown) indicate indirect roles of those microorganisms in rumen ecology. new microorganisms might exist in low numbers in a population at early on after additives were consumed and disappear several days after due to the complex environment in the rumen. however, those microorganisms which are able to change the microbial diversity in rumen and finally support the production beneficial metabolites for host most likely exist. closeness of restriction sites of microorganisms that are contained in the database tolerate an accuracy between trf data observations with t-rf predictions of more than 3 fig 3 t-rflp patterns of 16s rdna’s from rumen samples of two peranakan ongole breed cattle from a basal diet (r0), probiotic (r3), silage (r1), and silage + probiotic (r2) treatment generated after digestion with haeiii restriction enzyme. 16s rdnas were amplified with universal primers fam-46f and 1080r. microbiol indones130 ridwan et al. bp (sakamoto et al. 2004). there was a tendency of a decline in the population of clostridium purinolyticum, which was originally found in the r0 treatment, when cattle were fed with silage (r1) or silage plus probiotic (r2) (fig 3.). the population of epulopiscium sp. morphotype a2/e. fishelsoni str red.seaa2/bifidobacterium longum atcc15707 (t) in both experimental animals decreased when treated with r1, as well as with r3. metabacterium polyspora appeared in rumen of cattle a when r1 was applied as feed. the population of ruminococcus productus atcc 27340/clostridium sp. strain dr6a on the cattle a decreased dramatically when this animal received r1, but their was an increase in the population of this organism in the r3 treatment. this is similar with that of animal b, where r2 treatment showed indications of an increased population r. productus atcc 27340/clostridium sp. strain dr6a str. thus, there are indications that a given probiotic encourages rumen stimulation based on the presence of bacteria that previously did not exist, or were scarce. this also occured in both cattle a and cattle b, with a decrease in the population of cytophaga fucicola str nn015860/str sw17. taken together, treatments of all feed consumed by the po cattle clearly shows the diversity of rumen ecology (table 3). a decline in the population of dominant bacteria in the rumen after feed-treatment stimulated the growth of other bacteria in proportions detectable by t-rflp analysis, although this new population is still relatively low. c l o n e t 3 1 / c l o n e t 5 5 / c l o n e t 9 5 / a r c o b a c t e r cryaerophilus ccug17801/a. butzleri ccug10373 in the rumen of cattle a treated with r1 eliminated the dominant bacteria in the population such as m. polyspora. metabacterium polyspora is actually a gram-positive anaerobic bacterium and was originally found in the digestive tract of pigs (esther et al. 1998). clone wchbi-82-102 and clone12 appeared significantly after decreasing r. hansenii atcc 27752 (t) and c. purinolyticum atcc33906 (t). clostridium purinolyticum is well known as gram negative bacterium that is capable of degrading protein. this bacterium grows optimally at ph 6.5-9.0 under anaerobic conditions (durre et al. 1981). this study reflects the dynamics of bacterial populations towards feed modulations. the succession of microbe close to clone wchbi-82/clone12-102, epulopiscium sp. strain morphotype a2/e. fishelsoni red sea a2/b. longum atcc15707 (t), and c. purinolyticum atcc33906 (t) was noted after reduction of levels of this organism which was followed by an increase in the population of clone eh-7/ str.rj5 and c. fucicola nn015860, str sw17. in a previous study, it was reported that the pattern of decrease and increase in the population of certain bacteria correlates with the improvement of the immune response under stress conditions, the stimulation of microbial growth in the rumen and the stabilization of rumen acidity (krehbiel et al. 2003). almost all organisms, including anaerobic bacteria, can grow at a temperature of 35-43oc, derived from the digestive tract of cattle, other ruminants and nonruminants, except for cytophaga. this organism is an aerobic bacteria, isolated from sea water, and can hydrolyze cellulose substrate (johansen et al. 1999). as shown in table 3, b. angulatum has the closest t-rf observation value when compared the to t-rf prediction values stored in the database. since b. angulatum was reported as an original organism of the human digestive tract (dinoto et al. 2006a), the existence of this organism needs to be clarified in the future using a pcr method with a specific primer, or by using a clone library approach. the diversity of rumen microorganisms is influenced by the individual differences of each animal. this can be seen with the appearance of some of the population that occurred in cattle a, but not in cattle b. in conclusion, this study clearly shows that the feed additives used influence the diversity of the microbial population in the rumen as revealed by t-rflp analysis. the highest diversity (evar smith and wilson evenness = 0.89±0.04) of rumen microbial community, which was found when cattle received silage and probiotics simultaneously. this phenomenon reflects the synergistic effects of a change of the microbial population and of metabolites in the rumen. references bakir ma, koga y, benno y. 2008. effect of four probiotic strains on human fecal microflora. int j probiotics prebiotics 3:55-60. blackwood cb, hudleston d, zak dr, buyer js. 2007. interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities. appl environ microbiol 73:5276–83. blackwood cb, marsh t, kim sh, paul ea. 2003. terminal restriction fragment length polymorphism data analysis for quantitative 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lactic acid bacteria. ed. bjb wood. new york: elsevier, pp.49-68. weinberg zg, muck re, weimer pj, chen y, gamburg m. 2004. lactic acid bacteria used in inoculants for silage as probiotics for ruminants. appl biochem biotechnol 118:1-10. microbiol indones132 ridwan et al. 02. rahmawati.cdr vol.14, no.3, september 2020, p 95-100 doi: 10.5454/mi.14.3.2 detection of liberibacter asiaticus causing citrus vein phloem degeneration from siam citrus leaves (citrus nobilis var. microcarpa) in singkawang city plantation, pontianak, west kalimantan 1 1 2 3 1* rahmawati , iliana , agus rachmat , latiffah zakaria , and mukarlina 1 department of biology, faculty of mathematics and natural sciences, universitas tanjungpura, jalan prof. dr. h. hadari nawawi, pontianak 78124, west kalimantan, indonesia; 2 research center for biotecnology, indonesian institute of sciences (ppo-lipi), cibinong, bogor 14430, west java, indonesia; 3 school of biological sciences, universiti sains malaysia, 1800 usm, pulau pinang malaysia. the objective of the-present study was to detect the presence of pathogenic fastidious bacterium, candidatus liberibacter asiaticus using pcr from leaves of siam citrus showing citrus vein phloem degeneration (cvpd) symptoms in singkawang city plantation, pontianak, west kalimantan, indonesia. citrus leaf samples were collected based on visual observation of symptoms showing cvpd infection. typical symptoms of cvpd include leaf yellowing (chlorosis), vein banding, leaves become stiff, thicker and smaller in size. the pathogenic bacterium, candidatus liberibacter asiaticus was detected using two specific primers, oi1/ oi2c amplified 16s rrna gene and a2/j5, amplified ribosomal protein gene of the rplkajl-rpobcoperon (β-operon). pcr amplification detected the presence of 1100 bp band using oi1/ oi2c primers, and 703 bp band using a2/j5 primers from symptomatic siam citrus leaves. pcr products were not detected from healthy plants serve as a control. by using two sets of specific primers to amplify 16s rrna gene and ribosomal protein gene, candidatus liberibacter asiaticus was detected in symptomatic siam citrus leaves in singkawang city, pontianak, indonesia. detection of the bacterial pathogen causing cvpd is important to prevent the spreading of the disease which could affect the production of citrus fruits. key words: candidatus liberibacter asiaticus, cvpd, pcr detection, siam citrus, singkawang city pontianak tujuan dari penelitian ini adalah untuk mendeteksi keberadaan bakteri patogen, candidatus liberibacter asiaticus menggunakan pcr dari daun jeruk siam yang menunjukkan gejala penyakit citrus vein phloem degeneration (cvpd) di perkebunan kota singkawang, pontianak, kalimantan barat, indonesia. sampel daun jeruk diambil berdasarkan pengamatan secara visual terhadap daun yang menunjukkan gejala infeksi cvpd. gejala khas cvpd memperlihatkan daun menguning (klorosis), tulang daun hijau tua (vein banding), daun menjadi kaku, lebih tebal dan lebih kecil ukurannya. bakteri patogen, candidatus liberibacter asiaticus dapat terdeteksi menggunakan dua pasang primer spesifik, yaitu, oi1/ oi2c gen 16s rrnadan a2/j5, gen protein ribosom dari operon rplkajl-rpobc (β-operon). amplifikasi pcr mampu mendeteksi keberadaan pita dna sebesar 1100 bp menggunakan primer oi1/oi2c dan pita dna sebesar 703 bp menggunakan primer a2/j5 dari daun jeruk siam yang bergejala cvpd. pita dna tidak terdeteksi dari daun tanaman sehat yang berfungsi sebagai kontrol. penggunaan dua pasang primer spesifik gen 16s rrna dan gen protein ribosom, candidatus liberibacter asiaticus terdeteksi pada daun jeruk siam yang bergejala di perkebuanan kota singkawang, pontianak, indonesia. deteksi bakteri patogen penyebab penyakit cvpd merupakan informasi penting untuk mencegah penyebaran penyakit yang dapat mempengaruhi produksi buah jeruk. kata kunci:candidatus liberibacter asiaticus, cvpd, deteksi pcr, jeruk siam, kota singkawang pontianak microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-561-739630; email: mukarlina@untan.ac.id types of diseases including citrus vein phloem degeneration (cvpd) or citrus greening which is also known as huanglongbing disease. the disease is caused by a gram-negative bacterium, candidatus liberibacter of which three species are known as causal pathogens of cvpd, namely ca. liberacter asiaticus, ca. liberacter africanus and ca. liberacter americanus. after they are famous in continent (bove, 2006). the bacteria are spread by psyllids, diaphorina citri in asia, brazil and florida, and triozaerytreain africa. in addition to insect vector, cvpd transmission citrus is one of the fruit commodities in indonesia that is very popular with the community, both as fresh fruit and in processed form. siam citrus citrus nobilis var. microcarpa) has a high economic value for citrus farmers in indonesia. in west kalimantan, the main siam citrus plantation is located in singkawang city 2. with a cultivation area of 5776 m . cultivation of siam citrus is susceptible to various usually occurs through plant seeds that come from propagation by grafting infected buds (hung et al. 2000), candidatus liberibacter asiaticus infected citrus plant by penetrating phloem tissues and vascular systems causing clogging and reducing water transportation (teixeira et al. 2005). typical symptoms of cvpd disease in citrus leaves those infected with the pathogen liberibacter will show, chlorosis symptoms or yellowish patches (blotching, mottle) irregularities on the leaves and leaf bones (zubaidah, 2010). however, these visual observation are often misleading as in some cases the symptoms may be related to other biotic and abiotic factors (lin et al. 2010) which showed similar symptoms with cvpd. among the biotic and abiotic factors that resemble cvpd symptoms including zinc deficiencies (timmer et al., 2003), stem pitting caused by citrus tristeza virus, phytophthora root rotand citrus blight (beattie and barkley, 2009). according to pereira et al. (2011), diagnostic errors based on visual observation can be higher than 30%. therefore, visual inspection of the symptoms can lead to misidentification of the correct pathogens. due to these the limitations, detection of cvpd pathogen is commonly based on pcr due to its reliability and simplicity. two specific primers are often used to detect the presence of cvpd pathogens in plant tissues, namely primers oi1/oi2c which was based on 16s rdna with a dna target of 1160 bp (wirawan et al., 2018) and primers a2 / j5 primers, based on ribosomal protein genes (rpla/rplj genes) of the β-operon sequences (hocquellet et al. 1999). primers a2 / j5 were used to distinguish between candidatus liberibacter asiaticus and candidatus liberibacter africanus by the size of the pcr products obtained (hocquellet et al. 1999). in indonesia, cvpd infection on citrus has increased in citrus producing regions. the disease incidence has increased to 62.34% in east java, 60% in north bali, and 70% in southeast sulawesi (nurhadi, 2015). typical symptoms of cvpd were also observed in siam citrus plantation in setapok village, singkawang city, pontianak, west kalimantan. thus, the aim of the present study was to determine the pathogen causing symptoms of cvpd in the plantation. materials and methods sampling of citrus leaves. symptomatic siam citrus leaves (citrus nobilis var. microcarpa) showing cvpd symptoms were collected randomly from a citrus plantation in setapok village, singkawang district, singkawang city, west kalimantan, indonesia. mild and severe symptoms of cvpd were observed in the plantation, which includes leaf yellowing (chlorosis), vein banding, leaves become stiff, thicker and smaller in size (fig 1). the symptomatic leaves were put in plastic bags and stored in a cooler box. the samples were then brought to genomics and plant quality improvement laboratory,lipi biotechnology research center, o cibinong, bogorand stored in a freezer (-80 c) until 96 rahmawati et al. microbiol indones fig 1 symptoms of cvpd on siam citrus leaves in the field. type 0: healthy leaves. type 1: partial chlorosis at the leaf margins. type 2a: chlorosis between the leaf bones showing vein banding, and the leaves become stiff (mild cvpd symptoms). type 2b: chlorosis between the leaf bones, showing vein banding, leaves are thicker and stiffer (severe cvpd symptoms). volume 14, 2020 microbiol indones 97 used. dna extraction. for dna extraction, the citrus leaves were washed with 70% alcohol for 1 min and then washed using three changes of sterile distilled water. the sterile filter paper was used to dry the leaves and cut into small pieces of about 0.5 cm. the small pieces of the leaves were put 1.5 ml micro centrifuge tube, containing liquid lb medium (luria o bertani) and incubated in an oven at 37 c for 2-3 days. lb medium is used for the growth of pure bacterial cultures to be isolated. the next step was isolation of the bacterial plasmid dna from the leaves in the lb medium. the broth was centrifuged at 12000 rpm for 2 min, the supernatant was removed and 100 μl of solution 1 (1 m tris-cl ph 8, 0.5 m edta, 0.5 m glucose) was added and incubated in ice for 15 min. after incubation, 200 ml of solution ii (10 m naoh, 20% sds) was added and incubated in ice for 15 min of which 150 μl ch3coona solution was added, and incubated again in ice for 15 min. the mixture was centrifuged at 10000 rpm for 5 min and 1 ml of 96% ethanol was added, and centrifugation was repeated in the same condition. after centrifuged, the pellet was washed with 70% ethanol and dried. the pellet was then dissolved in 20 µl tris edta buffer or distilled water and stored at 0 20 c until used. the concentration and purity of the plasmid dna were measured using nanophottometer, at a wavelength of 260 nm, and calculated based on the ratio a280/a260 and a260/a230 ng/µl. dna purity limits, theorecally the purity of dna was analysis by the ration 1.8 – 2.0 (sambrook et al. 1989). amplification of 16s rdna gene and ribosomal protein gene. for pcr amplification, extract from three leaves samples were used. pcr reaction for both genes was performed using dreamtag pcr master mix solution (thermo fisher scientific). the pcr reaction was prepared in 12.5 µl reaction containing 1 µl dna sample, 1 µl forward primer and 1 µl reverse primer, 6.25 µl pcr master mix solution and 3.25 µl water dnase, rnase-free. the specific primers used for amplification of 16s rrna gene were oi1/oi2c primers with atarget pcr product of approximately 1100 bp for candidatus liberibacter asiaticus (bove 2006) another specific . primers, a2/j5 primers were based on ribosomal protein gene of the rplkajl-rpobcoperon (β-operon) and the expected pcr product was 703 bp for candidatus liberibacter asiaticus. candidatus liberibacter africanus produces a band of 669 bp using a2/j5 primers (hocquellet et al. 1999). pcr was performed in biometra thermal cycler and pcr conditions used were as follows: initial 0 denaturation at 95 c for 5 min; 40 cycles of 0 0 denaturation at 94 c for 1 min, annealing at 55 c 0 (primers) and -60 c (primers) for 30 s, extension at 0 72 c for 1 min; and the final extension was carried out 0 at 72 c for 5 min. � pcr products were analyzed using 0.8% agarose gel electrophoresis, carried out for 60 min, 100v and 400 ma. the marker used was 100 bp dna ladder (lamda hindiii). after electrophoresis, the agarose gel was immersed in ethidium bromide for 15 min, rinses with running water and visualized using uv trans illuminator (uvitec). results the dna extraction method used in this study was a modification of the plasmid dna isolation method based on the principle of alkaline lysis solution. the principle of this method is almost the same as the dna extraction using cetyl trimethyl ammonium bromide (ctab) of which the dna of the bacteria was extracted from the leave samples. dna extraction with ctab is a common method for isolating pathogenic bacteria as members of liberibacter. research that has been done, successfully detected the presence of the pathogenic bacteria candidatus liberibacter asiaticus, using two specific primers. the positive reaction of primers oi1/oi2c produced a dna band of 1100 bp, which was shown in the leaf type 1 sample (fig 2). whereas the positive reaction of primers a2/j5 produced dna bands of703 bp, which was shown in leaf type 2b samples (fig 3). discussion plasmid dna isolation method based on the principle of alkaline lysis was applied in this study because experiments with the ctab method have been carried out, however it was not successful in extracting bacterial dna from symptomatic citrus leaf extract. in contrast, ctab method was used by ruangwon and akarapisan (2006) and taufik et al. (2010) to extract dna of candidatus liberibacter asiaticus from symptomtic citrus leaves of which pcr amplification was successfully amplified the 16s rrna gene. pcr amplification using oi1/oi2c primers produced 1100 bp band from leaves extract showing type 1 symptoms (partial yellowing on the leaf vein). 98 rahmawati et al. microbiol indones pcr amplification using a2/j5 primers produced 703 bp band (figure 3) which was only detected from leaves extract from type 2b leaves (severe cvpd symptoms). the results of the pcr amplification from the type 1 and type 2b leaves extract indicated the presence of ca. liberibacter asiaticus in the symptomatic siam citrus leaves showing symptoms of cvpd in setapok village, singkawang city, pontianak, west kalimantan. the size of the pcr products obtained was in accordance to the size of the pcr products stated by bove (2006) for oi1/oi2c primers and hocquellet et al. (1999) for a2/j5 primers for detection of ca. liberibacter asiaticus. amplification using specific primers oi1/oi2c only showed positive dna band reactions in leaf type 1, and result in negative dna bands from samples of type 2b (mild cvpd symptoms) and leaf type 2b leaves (severe cvpd symptoms). according to wirawan et al. (2004), although leaf samples showing chlorosis indicating cvpd symptoms, pcr amplification did not produce any band which may be due to low concentration or uneven distribution of the bacteria in the leaves tissues, which may affect the dna concentration from the leaves extract. huang (1979), also indicated that due to low concentration of the bacteria in citrus host, the detection of the pathogen is difficult. based on the results of measurements of dna purity in this study 1,800 ng/µl (leaf type 1), 1,724 ng/µl (leaf type 2a) and 1,865 ng/µl (leaf type 2b). other possible explanations could ca. liberibacte rasiaticus but caused by abiotic and biotic factors such as nutrient deficiencies and infection be the symptoms shown on the leaves were not related to by other microbes of which the symptoms are similar with cvpd causal pathogen. wirawan et al. (2004) never fig 2 amplification products using oi1/ oi2c primers. lane 1; type 1 symptoms leaves : partial chlorosis (positive sample ca. liberacter asiaticus); lane 2: water control (negative control ca, liberibacter asiaticus); lane 3: type 1 symptoms : partial chlorosis (positive sample ca. liberacter asiaticus); lane 4: type 2a symptoms : mild cvpd symptom (negative sample ca, liberibacter asiaticus); lane 5: type 2b symptoms : severe cvpd symptom (negative sample ca, liberibacter asiaticus). m: 100 bp marker. fig 3 pcr amplification using primers a2/ j5. lane 1 : water control (negative control ca, liberibacter asiaticus); lane 2 : healthy leaf : type 0 (negative control ca, liberibacter asiaticus); lanes 3 and 4: leaves extract from type 1 symptoms : partial chlorosis (negative sample ca, liberibacter asiaticus); lane 5 : leaves extract from type 2a symptoms : mild cvpd symptoms (negative sample ca, liberibacter asiaticus); lane 6 : leaves extract from type 2b symptoms : severe cvpd symptoms (positive sample ca. liberacter asiaticus). m: 100 bp marker. volume 14, 2020 microbiol indones 99 found two typical protein molecules in plants attacked by cvpd, namely virulent protein (toxin) from the pathogenic bacteria ca. liberibacter asiaticus and citrus plant receptor proteins, these two protein molecules interact with each other and influence ion transport into the plant cell so that plants lack mineral elements of zn, mn, and ca. the reaction of the two proteins is what is suspected, be the cause of plant leaves experiencing chlorosis. the symptoms of cvpd observed in singkawang city plantation were similar with cvpd symptoms reported by wijaya (2003) in east java citrus plantation, karangasem regency. similar cvpd symptoms were also reported by ardiartayasa et al. (2006) in eight villages, katung, belancan, bayung gede, cloud, chess, pengotan, evening and pelaga villages in denpasar, bali, indonesia, with citrus leaves showing mild to severe chlorosis. by taufik et al. (2010) also reported the same chlorosis symptoms and vein banding in the citrus plantation in konawe selatan regency, southeast sulawesi, indonesia. symptoms of chlorosis and vein banding observed in the field were in accordance with the symptoms described by wijaya (2003) of which the chlorosis in cvpd citrus leaf become irregular due to reduced formation of chlorophyll in the leave and according to nurhayati et al. (2016), to diagnose cvpd in citrus plants chlorophyll content which can be the basis of reference, namely around 47.06 spad. thus, the leaves become stiff but the leaf vein remains dark green. according to susanti et al. (2014), infection by ca. liberibacter asiaticus causing chlorosis might also indicate physiological disorders as masses of bacterial cells can inhibit the transportation of nutrients to and from the phloem leading to degeneration of phloem cells. these conditions affected the transportation of nutrients to other parts of the citrus plant. hence the reduced chlorophyll formation in leaves which experience chlorosis results in a decrease in photosynthetic activity in plants. wirawan et al. (2018), also stated that lorosis occurs through transmission of vector insect stillets in plant tissue when pathogens suck in plant fluids and are in the phloem tissue then scattered to the parts of the plant along with the translocation of organic matter, too many pathogens result in chlorosis and phloem leaf necrosis. both specific primers, oi1/ oi2c and a2/j5 have been applied in several studies related to cvpd in indonesia. for instance, studies by taufik et al. (2010) and meitayani et al. (2014) using oi1/ oi2c detected candidatus liberobacter asiaticum from infected citrus in sulawesi tenggara and karangasem regency. the pcr products obtained were 1100 bp band indicated the presence of candidatus liberobacter asiaticum. by using a2/j5 primers, 703 bp band of candidatus liberobacter asiaticum was also reported by hocquellet et al. (1999) from diseased citrus from various citrus orchards in bali, indonesia; south africa and mauritius. in a study by ruangwong and akarapisan (2006), using a2/j5 primers produced 703 bp band of candidatus liberobacter asiaticum amplified from diseased citrus plants in chiang mai, chiang rai and phrae provinces. in the present study, by using a2/j5 primers, pcr products wereonly detected from plant extract with severe symptoms of cvpd (type 2b leaves sample). whereas in leaf type 1 and leaf type 2a samples, did not show any dna bands. this is thought to be due to the smaller primary target, in accordance with the statement of hocquellet et al, 1999 that the target dna using primers a2 and j5 are smaller than the primary targets designed by other 16s rrna primers, so that dna degradation during amplification and electrophoresis can be reduced. early detection of cvpd especially in a young citrus plants is important to prevent spreading of the disease. thus, a suitable dna extraction method is important to obtain good quality dna for pcr amplications. as conclusion, pcr amplification using two specific primers oi1/ oi2c and a2/j5 were able to amplify bacterial dnafrom symptomatic leaves extract of siam citrus collected from setapok village, singkawang city, west kalimantan, pontianak, indonesia. therefore, the bacteria isolated from siam citrus leaves showing cvpd symptoms were candidatus liberibacter asiaticus. acknowledgements the authors are grateful to my tacg bioscience enterprise (sa0168756-p) for primary assistance, genomics and plant quality improvement laboratory, lipi biotechnology research center and department of biology, faculty of mathematics and natural sciences, tanjungpura university for the facilities and assistance in completing the research work. references adiartayasa w. 2006. identification of several citrus varieties and detection of cvpd pathogens by pcr in 100 rahmawati et al. microbiol indones kintamani district [thesis]. denpasar (id): udayana university. beattie gac, barkley p. 2009. huanglongbing and its vectors: a pest-specific contingency plan for the citrus and nursery and garden industries. (version 2). horticulture australia ltd., sydney. p 272. bove jm. 2006. huanglongbin: a destructive, newlyemerging, century-old disease of citrus. j plant phatol. 1(88):7-37. doi: 10.4454/jpp.v88i1.828. hocquellet a, toorawa p, bové jm, garniner m. 1999. detection and identification of the two candidatus liberobacter species associated citrus huanglongbing by pcr amplification of ribosomal protein genes of the operon. molecular and cellular probes. 13:373-379. doi: 10.1006/mcpr.1999.0263. huang ch. 1979. distribution of likubin pathogen in likubin affected citrus plant. j of agricultural research of china. 28:29-33. hung th, wu ml, su hj. 2000. identification of alternative hosts of the fastidious bacterium causing citrus greening disease. j phytopathology. 148:321-326. doi: org/10.1046/j.1439-0434.2000.00506.x. lin h, chen c, doddapaneni h, duan y, civerolo el, bai x, zhao x. 2010. a new diagnostic system for ultrasensistive and specific detection and quantification of candidus liberibacter asiaticus. the bacterium associated with citrus huanglongbing. j microbial. method. 81:17-25. meitayani, swari np, adiartayasa w, wijaya in. 2014. detection of citrus vein phloem degeneration (cvpd) disease using the polymerase chain reaction (pcr) technique on citrus plants in bali. fp unud, denpasar. j agroekoteknolgi tropica. 3(2):70-79. issn 2301-6515.2014.05. nurhadi. 2015. citrus huanglongbing disease (candidatus liberibacter asiaticus) threats and control strategies. indonesian citrus and subtropical fruit research institute. nurhayati i, adiartayasa w, bagus ign. 2016. detection of the presence of causes of citrus vein phloem degeneration (cvpd) molecularly in the siam citrus plant (citrus nobilis lour var. microcarpa hassk) based on variations in the symptoms of chlorosis. j agroekoteknologi tropika. 5(4):344-353. pereira fmv, milori dmbp, pereira-filho er, venancio al, russo mdt, cardinali mcdb. 2001. laserinduced fluorescence imaging method to monitor citrus greening disease. comput, electron, agric.79:90-93. ruangwong o, akarapisan a. 2006. detection of candidatus liberibacter asiaticus causing citrus huanglongbing disease. j of agricultural technology. 2(1):111-120. sambrook jef, fritsch t, maniatis. 1989. molecular cloning laboratory manual 3rd ed. cold spring harbour lab. press, new york. susanti h, mukarlina, linda r. 2014. leaf and twig anatomy citrus nobilis l. var. micerocarpa attacked by cvpd (citrus vein phloem degeneration.) j protobiont. 3(3):51-55. teixeira dc jl, danet s, eveillard ec, martins wc, de jesus jr, pt, yamamoto sa, lopes r, beozzo bassanezi a, juliano ayres c, saillard, bove jm. 2005. citrus huanglongbing in sao paulo state, brazil: pcr detection of the 'candidatus' liberibacter species associated with the disease. molecular and cellular probes. 19:173-179. doi: 10.1016/j.mcp.2004.11.002. taufik m, khaerani a, pakki t, gianto. 2010. detection of the presence of citrus vein phloem degeneration (cvpd) using pcr (polymerase chain reaction) techniques in southeast sulawesi. j tropical plant pests and diseases. 10(1):73-79. issn.14117525.2010.03. timmer lw, garnsey sm, broadbent p. 2003. diseases of citrus. in: disease of tropical fruit crops (ed. r.c. p l o e t z ) . a p s p r e s s , u s a : 1 6 3 1 9 5 . d o i : 10.1079/9780851993904.0000. wijaya in. 2003. diaphorina citri kuwayama (homoptera:psyllidae), biotechnology and its role as a vector of cvpd disease in the siam citrus plant [thesis]. bogor (id): bogor agricultural institute. wirawan igp, liliek s, wijaya in. 2004. cvpd disease in citrus plants [thesis]. denpasar (id): udayana. wirawan igp, pasari nkd, sritamin m. 2018. cayenne pepper plant disease detection (capsicum frutescens l.) which grows around citrus plants symptomatic citrus vein phloem degeneration (cvpd) using technique polymerase chain reaction (pcr). j agroekoteknologi tropika. 7(1):73-80. issn: 23016515. zubaidah s. 2010. capability increasing of some antibiotics for eliminate liberibacter asiaticus bacteria to find the free-cvpd citrus seedling. j basic science. 11(1):4554. page 1 page 2 page 3 page 4 page 5 page 6 untitled-1 bacteria may be living in plant tissue as pathogen, saprophytic, or endophyte. bacteria that colonize healthy plant tissue without causing or produce obvious injuries to the host is defined as an endophytic bacteria (bacon and hinton 2007). endophytic bacteria may exhibit a plant growth promotion (pgp) ability through an extensive mode of action such as acc deaminase production, phytohormones synthesis, phosphate solubilization, siderophore production and nitrogen fixation (rosenblueth and martínez-romero 2006; malfanova 2013). by colonizing plant tissues, bacterial endophytes can help their host more efficiently than those that bind exclusively to the plant's rhizosphere. of many studies on endophytic bacteria, very few shows their occurrence and functional role in plant reproductive organs such as seeds, flowers, or fruits (kukkurainen et al. 2005; de melo pereira et al. 2012; compant et al. 2011; audipudi et al. 2014). fruits are unique habitat for microbial growth in the phyllosphere region because of their sugar content. as the bacteria proliferate inside the plant tissue, they are likely to develop interaction with the host and many of them have shown plant growth-promoting (pgp) effect. these abilities were mediated through direct effect of p g p a b i l i t y o r t h r o u g h c o m p e t i t i o n w i t h phytopathogens. direct pgp effect mediated by endophytes is mostly based on providing essential n u t r i e n t s a n d p r o d u c t i o n o r r e g u l a t i o n o f phytohormones (malfanova 2013). de melo pereira et al. (2012) demonstrated that pseudomonas, enterobacter, and bacillus isolates from strawberry fruits were capable to produce indole acetic acid (iaa), fix n and secrete siderophore. these abilities also 2, have been confirmed in planta to enhance strawberry vol.8, no.4, december 2014, p 161-169 doi: 10.5454/mi.8.4.3 isolation and molecular identification of endophytic bacteria from the arils of durian (durio zibethinus murr.) var. matahari * sony suhandono and indah budi utari school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, west java, indonesia endophytes are plant-associated microorganisms able to form colonies in internal tissue and considered as an important component of biodiversity. however, information about the of naturally fruits-associated existence endophytic bacteria at different life-history stages of hosts is very limited. durian (durio zibethinus murr.) is an exotic tropical fruits with a high economical value, but the occurrence and functional role of associated endophytes remains unexplored. a total of sixteen endophytic bacterial isolates were identified by 16s rrna sequence analysis from ripe and unripe stages of durian fruits var. matahari. these isolates belonged to the genus staphylococcus, bacillus, enterobacter, moraxella, gordonia, salmonella, rhizobium, brachybacterium, kocuria, and klebsiella. this is the first report of an endophytic bacterial spesies residing in durian arils. this research also indicated potency of culturable endophytes from durian fruits in plant growth promotion. key words: 16s rrna gene, durian, endophytes endofit adalah mikroorganisme asosiatif pada tanaman yang mampu berkoloni dalam jaringan internal tertentu dan menjadi komponen penting dalam biodiversitas. akan tetapi, informasi mengenai keberadaan bakteri endofit pada bagian organ buah di tahap perkembangan yang berbeda masih sangat terbatas. durian (durio zibethinus murr.) merupakan buah tropis dengan nilai ekonomis yang sangat tinggi. namun, studi mengenai keberadaan dan peranan bakteri endofit pada buah durian masih belum tereksplorasi dengan baik. enam belas isolat bakteri endofit yang berasal dari arillus durian var. matahari mentah dan matang telah berhasil diidentifikasi dengan menggunakan analisis sikuen gen 16 rrna. isolat tersebut terdiri dari genus staphylococcus, bacillus, enterobacter, moraxella, gordonia, salmonella, rhizobium, brachybacterium, kocuria, dan klebsiella. penelitian ini adalah studi pertama mengenai identifikasi spesies bakteri endofit yang berada pada arillus buah durian. selain itu, hasil penelitian ini pun menunjukkan bahwa bakteri endofit yang berasal dari buah durian berpotensi dalam pertumbuhan tanaman. kata kunci: durian, endofit, gen16s rrna *corresponding author; phone: +62-22-2511575, fax: +6222-2534107 ; email: sony@sith.itb.ac.id growth. however, our understanding of the role and potential of endophytes under different stages of fruits development is still very limited. durian (durio zibethinus murr) is an economically important fruit in southeast asia. the major importers are taiwan and hong kong for fresh durian, while usa, australia, and hong kong for frozen durian (perez 2013). as a result, the demand for durian is always increasing every year, especially for favorite cultivars. this climacteric fruit has a strong smell and a unique taste. durian normally has five locular units and each locular contains 1–5 pulp units. the pulp unit consists of seed, which is completely covered by a creamy, white, yellow or golden yellow aril, the edible portion of the fruit. in spite of their unique characteristics, durian also has great nutritions and antioxidant activities (poovarodom et al. 2010). nevertheless, the information about microorganisms in durian is rather limited, expecially in the term of endophytic bacteria. the aim of this study was to identify endophytic bacteria found in the arils of durian fruits under natural condition using the culturedependent method and 16s rrna sequence analysis for its characterization. materials and methods samples. durian fruit (durio zibethinus murr.), var. matahari were collected from an agricultural field belonging to the kebun percobaan, balai penelitian buah, subang, west java, indonesia. the cultivar matahari was chosen because it produced high quality fruit, based on some characteristics like the thickest flesh, the highest percentage of edible portion, and yellow colour of the fruit flesh (arillus). unripe and ripe fruits were selected from the same tree and brought to the laboratory in sterile polyethylene bags. to avoid contamination and to isolate endophytic bacteria only from arillus part, the suture and the stalk of the fruits were covered with wax. isolation of endophytic bacteria. the fruits surface (epicarp) was sterilized by 70% ethanol for 4 min, 2.5% sodium hypochlorite for 4 min, and 70% ethanol for 4 min, followed by three rinses in sterile deionized water. after surface disinfection, 1 g of durian arillus from three different locular unit site were mixed with 1 ml 0.85% nacl and followed by serial dilutions. after that, 0.1 ml aliquotes from appropriate dilutions were plated into luria bertani agar medium. triplicate plates were incubated at 37 ºc for 48 h for the total plate counts. colonies with unique morphological appearence was selected from the plates. a single pure colony was obtained from approximately 11 times subcultures process. gram staining was also performed to confirm the purity of the colony. biochemical characterization. isolates were tentatively grouped according to their morphological and cultural characteristics of the colonies on plates (cappuccino and sherman 2005). for biochemical analysis, all isolates was grown aerobically on luria–bertani (lb) plates overnight at 37 ºc. a total of eighteen biochemical tests were performed to all isolates, including those for sugars fermentations, oxidase, catalase, cell motility, and imvic test using standard methods as described by cappuccino and sherman (2005). the isolated endophytic bacteria were identified by biochemical characterization based on bergey's manual of systematic bacteriology (garrity 1984). identification of endophytic bacteria using 16s rrna gene. single colony of bacteria was grown in liquid lb medium for 16-18 hours for dna extraction. bacterial dna was obtained according to the boiling procedure reported by yang et al. (2008). universal primers 8f (5'-agagtttgatcctggctcag-3') and 1492r (5'-ggttaccttgttacgactt-3') were used in order to amplify approximately 1500 bp of 16s rrna gene (turner 1999). a total pcr volume of 50 μl -1 contains 2 μl dna template (25 ng μl ), 25 μl gotaq green® master mix (promega), 1.5 μl primer forward -1 and 1.5 μl primer reverse (25 pmol μl ), and 20 μl nuclease free water. the pcr was performed with a thermal profile of 95 ºc for 3 min, followed by 30 cycles of denaturation at 95 °c for 30 s, annealing at 45 °c for 45 s, and extension at 72 °c for 2 min and a final elongation at 72 °c for 7 min (applied biosystems, 2720 thermal cycler). a 5 μl of pcr products then visualized by electrophoresis in 1% agarose (bioline) in 1x tae buffer for 25 min, 100v. pcr products were then purified using kit from geneaid. sequencing of pcr products were performed in macrogen inc., korea. the nearly fulllength 16s rrna gene sequence was compiled using clc genomics workbench 3 and compared with those from the national centre of biotechnology information (ncbi) using the basic local alignment search tool (blast) program. alignment and clustering were performed by kimura-2 parameter (tamura 1992) and neighbor-joining methods. phylogenetic trees and molecular analyses were conducted using mega version 5.2 (tamura et al. 2011), where 1000 replication of bootstrap analyses were performed (soltis and soltis 2003). 162 suhandono et al. microbiol indones bioassay of plant growth promoting (pgp) properties. furthermore, sixteen isolates then evaluated in term of pgp properties to measure their potency in growth promotion. indole-3-acetic-acid (iaa) production assay. the bacterial isolates were inoculated into 20 ml of nutrient broth supplemented with 0.2 % (v/v) of ltryptophan and incubated for 24 h at 37 ºc. after incubation, the culture was centrifuged at 3000 rpm for 20min and the supernatant was used for analyzing indole 3 acetic acid production (rahman et al. 2010). initially one ml supernatant was mixed with 2 ml of salkowski reagent and tubes were incubated in the dark for 30 min. iaa production in the cultured medium was evident by characteristic indication of reddish to pinkish colour in the solution. uninoculated growth medium was used as negative control. phosphate solubilization assay. phosphate solubilization was measured by the procedure described by jasim et al. (2013). plates containing -1 pikovskaya (pvk) medium (c h o 10 g l , cahpo 6 12 6 4 -1 -1 -1 5 g l , (nh ) so 0.5 g l , nacl 0.2 g l , mgso .7h o 4 2 4 4 2 -1 -1 -1 0.1 g l , kcl 0.2 g l , yeast extract 0.5 g l , mnso .h o 4 2 -1 -1 0.002 g l , feso .7h o 0.002 g l ) containing 2.4 mg 4 2 -1 ml bromophenol blue were inoculated with a streak of each bacterial culture. plates inoculated with the isolates were incubated for 48 h at 37 ºc and observed for the transparent “halos” around each colony due to the utilization of tricalcium phosphate present in the medium. uninoculated growth medium was used as negative control. results in order to determine the existence of endophytic bacteria from durian fruit, we isolated bacteria from three different locular units (sites) of each fruits lifehistory stages. a total of thirty five bacterial isolates were isolated from unripe and ripe stages. in contrast, no colonies appeared in both of control plates, indicating that aseptic handling and cultivation of the endophytic bacteria were successful. furthermore, we chose bacterial colonies from lb plate according to their morphological differences. sixteen morphologically different colonies were selected; four isolates from unripe stage (a1-a4) and twelve isolates from ripe stage (b1-b12). the ripe stage of durian fruits exhibited the highest number of cultivable bacteria. a total of sixteen bacterial isolates were characterized by 16s rrna gene analysis. the phylogenetic tree (fig 1) was obtained using the neighbor-joining method after calculation of a kimura two-parameter substitution model with the software mega 5.2 and evaluated by 1000 bootstrap. the most common endophytic bacteria associated with durian arillus belong to the genera klebsiella, enterobacter, bacillus, moraxella, gordonia, salmonella, rhizobium, staphylococcus, brachybacterium, and kocuria (fig 2). enterobacter and salmonella from ɣproteobacteria class could be isolated from both fruit developmental stages. meanwhile, bacillus and staphylococcus from firmicutes class were only found in ripe durian. of all isolates, 37.5% were gramnegative and 62.5% were gram-positive. all isolates showed various biochemical characteristic (table 1). these characteristics were important, as they supported the molecular identification based on some unique features of each genus. bioassays were performed to assess the plant growth promoting properties of each endophytic bacteria based on their iaa production and phosphate solubilization abilities. sixteen endophyte isolates were subjected to iaa assay using salkowski's reagent for colorimetric iaa detection in liquid culture. as many as eight isolates (50%) had been shown to produce iaa. all isolates from unripe stages were able to produce iaa, whereas only four isolates showed the ability in ripe stage. a qualitative test of inorganic phosphate solubilization was also accomplished to assess the ability of all isolates to solubilize inorganic insoluble phosphate. among these isolates, nine isolates (56.2%) had the ability to solubilize phosphate (table 2). the highest number of phosphate solubilizing bacteria was found in the ripe stage and predominated by enterobacter. discussion recently, endophytes are viewed as a new potential source of novel genes, proteins, and natural biochemical compounds. these unique bacteria showed that they can also colonize plant reproductive organs such as seeds, flowers, or fruits (kukkurainen et al. 2005; pereira et al. 2011; compant et al. 2011; audipudi et al. 2014). the present study demonstrates that healthy and field-grown durian fruits were inhabited by various endophytic bacteria. generally, thirty five colonies were isolated from both fruit stages; nine isolates from unripe stage and twenty six isolates from ripe stage. the largest population and diversity of endophytic bacteria in fruit arilli was observed during volume 8, 2014 microbiol indones 163 fig 1 neighbor-joining tree of the 16s rrna sequences of durian (cv. matahari) fruits endophyte . the tree topology was evaluated with kimura (two parameters). bar: 2 nucleotide substitutions per 100 nucleotides. ripe stage. during ripening under natural conditions, the fructose and glucose contents were significantly increased in durian arillus, which may be one of the factors that contributes to the differences in the endophyte diversities between the developmental stages (voon et al. 2006). the monosaccharide content was attributed by the hydrolysis of sucrose in the unripe stage and may lead to the enhancement of endophyte growth in the ripe stage. the isolation was performed by culture-dependent method in aerobic condition. thus, there is also a possibility to isolate endophytic bacteria anaerobically. however, low endophytic bacterial counts in our study were also largely supported by the fact that the fruit endophytic bacterial populations would decreas because of their distance from the root (compant et al. 2011; malfanova 2013). sixteen isolates were selected for further molecular identification and plant growth properties investigations. each isolate was represented by morphological characteristics that are different from the other culturable endophytic bacteria existed in the sample. 164 suhandono et al. microbiol indones table 2 growth promoting capability of the isolated endophytic bacterial isolates table 1 biochemical characteristics of the endophytic bacterial isolates no s o u rc es is o la te s g ra m s ta in in g m o ti li ty l it m u s m il k r ea ct io n fermentation l a ct o se d ex tr o se s u cr o se h s p ro d u ct io n 2 n o r ed u ct io n 3 imvic test in d o le m et h y l r ed v o g es p ro sk a u er c it ra te t es t u re a se t es t c a ta la se t es t o x id a se t es t g el a ti n l iq u ef a ct io n s ta rc h h y d ro ly si s l ip id h y d ro ly si s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 u n ri p e d u ri an r ip e d u ri an a1 a2 a3 a4 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 gram gram + gram + gram + gram + gram gram + gram + gram + gram gram + gram gram gram + gram gram + + + + + + + + + alkaline alkaline alkaline reduction reduction reduction reduction reduction reduction reduction reduction reduction reduction alkaline alkaline alkaline + + + + + + + ---+ + + + + + + + + +-++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + no sources isolates identification plant growth promoting properties iaa production phosphate solubilization 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 unripe durian ripe durian a1 a2 a3 a4 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 moraxella osloensis enterobacter hormaechei gordonia terrae salmonella bongori enterobacter sp. bacillus sp. rhizobium sp. bacillus licheniformis staphylococcus pasteuri staphylococcus sciuri klebsiella variicola staphylococcus sciuri staphylococcus sciuri brachybacterium rhamnosum kocuria kristinae salmonella enterica + + + + + + + + + + + + + + +++-----volume 8, 2014 microbiol indones 165 the molecular identification of the cultured endophytic bacteria using 16s rrna gene analysis revealed high bacterial diversity in the arillus of durian fruits. we discovered that bacterial endophytes in ripe durian arillus were more diverse than in unripe stage (fig 2). bacterial endophytes from ripe arillus included twelve isolates from eight different genera. meanwhile, only four isolates belonging to moraxella osloensis, enterobacter hormaechei, gordonia terrae, and salmonella bongori were isolated from unripe arillus. only two genera were found in both developmental stages. this difference might have been caused by various biotic and abiotic parameters (berg and smalla 2009). many identified bacterial genera, such as enterobacter have also been identified in other studies (souza et al. 2013; ferrara et al. 2011; wang 2006; madmony et al. 2004). on the other hand, our study is also the first to report the finding of some interesting genera in durian fruits, such as gordonia. the finding of this interesting genus suggests that durian fruits are host to unique endophytic bacteria that may possess significant biosynthetic potential. members of enterobacteriaceae family had been isolated from a wide variety of plants, like in maize seeds (johnston-monje and raizada 2011). our study has revealed the appearance of enterobacter genus in unripe and ripe stages of durian arillus (fig 2). based on their biochemical characteristics (table 1), enterobacter hormaechei (a2) and enterobacter sp. (b1) should be able to utilize a wide range of sugars. this might contribute to the occurence of these species in fruits. furthermore, metagenomic studies also referred to enterobacter spp. as one of the “core microbiota” in maize (johnston-monje and raizada 2011). enterobacter does not only reside in the host tissues, but also plays an important role as a plant growth promoting bacteria (asis and adachi 2003; ogbo and okonkwo 2012). salmonella bongori (a4) and salmonella enterica (b12) were also isolated from both stages of durian fruits. although salmonella is described as human pathogenic bacteria, this genus has recently been reported as a plant endophyte (schikora et al. 2012). different distribution of genes essential for surface attachment and secretion system, like spi2 type iii transport system, may indicate critical divergences between the strains that infect plants and human (fookes 2011). according to their biochemical characteristics (table 1), neither of them can metabolize sucrose nor produce hydrogen sulfide. this finding agrees with former studies (ellermeier and slauch 2006), which described specific metabolic characteristics of salmonella genus. indeed, the genes that play roles in the production of hydrogen sulfide can be used as salmonella-specific molecular markers (porwollik et al. 2002). staphylococcus was the most frequently encountered genus in this study. staphylococcus and bacillus are members of phylum firmicutes. interestingly, staphylococcus sciuri (b6, b8, b9) is the predominant firmicutes in durian arillus. this is different from many studies that showed bacillus spp. as the most common plant endophytic isolates (souza et al. 2013; kumar et al. 2011; figueiredo et al. 2009). staphylococcus sciuri is commonly present on skin and mucosal surfaces of a wide range of wild animals, like bats (vandžurova et al. 2012). since bats are well known pollinators of durian (baker 1970; morton 2004), this finding will be a very interesting topic for further study. indeed, it has been suggested that some taxa of fruit endophytes may not only be transported from the root environment but also from other sources such as the anthosphere (compant et al. 2011). of ten genera found as durian's matahari endophytes, six of them were moraxella (a1), gordonia (a3), rhizobium (b3), klebsiella (b7), brachybacterium (b10), and kocuria (b11). most of these isolates were positive for nitrate reduction test (table 1). reduction of nitrate is generally an anaerobic respiration, in which an organism derives its oxygen from nitrate. this result may explain how the bacteria adapted to low oxygen levels inside the fruits. rhizobium sp. is a well-known symbiotic nitrogenfixing bacteria, which can be found as an endophyte in numerous plants (van rhijn and van derleyden 1995). most rhizobium sp. play a role as plant growthpromoting rhizobacteria (pgpr). wei (2013) also observed that klebsiella variicola from sugarcane roots was capable to fix nitrogen. in contrast, there was not enough information about the role of the remaining spesies. however, the presence of gordonia is noteworthty, as members of this genus have the capabilities to degrade, transform, and synthesize some organic compounds (drzyzga 2012). the remaining isolates were related to the bacillus licheniformis (b4) and bacillus sp. (b2). the bacillus species have been described as endophytic species in banana (souza et al. 2013), maize (figueiredo et al. 2009), and daedok plants (kang et al. 2013). bacillus plays a role as biocontrol agent in plant and stimulates plant growth. in strawberry plant (de melo pereira et al. fig 2 distribution of endophytic bacterial isolates in durian (durio zibethinus murr) cv. matahari arillus. 166 suhandono et al. microbiol indones 2012) some bacillus species were able to produced iaa, siderophore, and proven to improve plant growth. in addition, bacillus licheniformis from daedok plants not only have the capacity to produce plant growth hormones but also showed antifungal activity against plant pathogenic fungi (kang et al. 2013). even though the presence of endophytic iaaproducing bacteria has already been reported in other plants, reports from durian fruits is very limited. interestingly, all unripe endophytic isolates gave positive colour reactions to salkowski's reagent but only one-third of the ripe isolates exhibited positive response (table 2). both of enterobacter spp. from unripe and ripe durian were able to produce iaa. our findings are relevant with the results of endohytes study from pollen pines (madmony et al. 2005), which revealed enterobacter cloacae as an iaa-producing bacteria. durian is a great source of tryptophan (leontowicz et al. 2011) that can be used as the precursor of iaa biosynthesis by microorganism (gravel et al. 2007). in tomato, iaa has been reported to have some crosstalk with ethylene during fruits maturation (mcatee et al. 2013). however, until now there has been no direct evidence linking between iaa produced by bacterial endophyte with the modulation of host fruits ripening process. availability of organic phosphate (p) for plant nutrition could be a limitation in some soils. phosphate is an essential nutrient for plant growth and has only limited bioavailability. it is considered to be important elements that limit plant growth. thus, solubilization and mineralization of p by phosphate-solubilizing bacteria (psb) is one of the most important bacterial physiological traits in soil (jeffries et al. 2003). several genera are known to be able to solubilize phosphate such as pseudomonas (park et al. 2009), bacillus (turan et al. 2007), rhizobium (chabot et al. 1996), and enterobacter (sharma et al. 2005). our result are relevant to these studies (table 2). among all endophytic bacteria, 56.25% isolates showed positive results in phosphate solubilizaton. furthermore, some interesting genera also exhibited this properties, including moraxella, klebsiella, staphylococcus, salmonella, and kocuria. although knowledge of the genetics of phosphate solubilization is still scanty, some genes involved in mineral and organic phosphate solubilization have been isolated and characterized (rodriguez et al. 2006). the ability of endophytic bacteria from durian arils to solubilize phosphate may be employed to boost organic phosphate uptake from the soil. in conclusion, the results from the study demonstrated the diverse community of endophytic bacteria associated with durian arillus. we have successfully isolated 35 endophytic bacteria from two developmental stages. to our knowledge, this study constitutes the first report on the isolation and identification of endophytes bacteria in the arillus of durian fruits, especially var.matahari. moreover, metagenomic studies are needed to unravel in depth the structure and function of the endophyte community in durian fruits. among these endophytic bacterial isolates obtained, most of them have the ability to produce iaa and perform phosphate solubilization activity. hence, these isolates can be considered to have potential use in plant growth promoting and development. further studies are needed to explore some unique isolates to determine their functional roles. acknowledgment we are grateful to santoso pj from balai penelitian buah tropika solok, west sumatra 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page 5 page 6 page 7 page 8 page 9 3. 626 isolation (muhammad).cdr isolation and identification of emestrin from and investigation of its anticancer properties emericella nidulans muhammad nursid , ekowati chasanah , murwantoko , subagus wahyuono 1* 1 2 3 and 1 2 3 research center for marine and fisheries product processing and biotechnology, kementerian kelautan dan perikanan, jalan ks tubun petamburan vi, jakarta 10260, indonesia fisheries department, faculty of agriculture, universitas gadjah mada, bulak sumur, yogyakarta 55281, indonesia; faculty of pharmacy,universitas gadjah mada, sekip utara, yogyakarta 55281, indonesia emericella nidulans , emericella nidulans emericella nidulans e. nidulans flow cytometry cell-cycle arrest epithiodioxopiperazine emericella nidulans , ; the research to isolate, identify and investigate of anticancer properties of active compound produced by marine fungus has been done. active compound was isolated from mycelium extract of the fungus. the molecular formula of active compound was established as c h n o s by lc-esi-tof-ms m/z 597.1105 [m h] . elucidation of molecular structure using ft-ir, lc-esi-tof-ms, h-nmr, c-nmr, and dept 135˚ showed that the active compound was emestrin. emestrin was found to have cytotoxic effect against t47d, hepg , c28, and hela but it was not too toxic against vero cells with ic value of 1.8 µg ml , 4.2 µg ml , 2.6 µg ml , 13.8 µg ml , and 260.9 ml , respectively. base on the cell cycle analysis by using flow cytometry, emestrin treatment at concentration of 1.0 μg ml induced cell-cycle arrest in g /g phase whereas at concentration of 3.0 ml , a sub-population of cells (sub g ) appeared. the apoptosis assay by using annexin-v-fluos revealed that most of t47d cell treated with the compound at 1.0 and 3.0 underwent apoptosis (83.6 and 92.6%, respectively). this anticancer activity of emestrin may be related to the unique of the epithiodioxopiperazine moiety with internal disulphide bond of this compound. key words: apoptosis, cytotoxic, emestrin penelitian untuk mengisolasi, mengidentifikasi, dan mengetahui aktivitas antikanker senyawa aktif yang dihasilkan oleh kapang laut sudah dilakukan. senyawa aktif dari kapang berhasil diisolasi dari ekstrak miselium. berdasarkan data spektra massa, senyawa aktif tersebut memiliki rumus molekul c h n o s m/z 597.1105 [m h] . elusidasi struktur dengan menggunakan ft-ir, lc-esi-tof-ms, h-nmr, c-nmr, dan dept-135˚ membuktikan bahwa senyawa aktif tersebut ialah emestrin. emestrin memiliki aktivitas sitotoksik terhadap sel t47d, hepg , c28, dan hela; tetapi tidak begitu toksik terhadap sel vero dengan nilai ic berturut-turut sebesar 1.8 µg ml , 4.2 µg ml , 2.6 µg ml , 13.8 µg ml , dan 260.9 μg ml . berdasarkan analisis siklus sel terhadap sel t47d dengan menggunakan , perlakuan emestrin pada konsentrasi 1.0 menyebabkan pada fase g /g sedangkan pada konsentrasi 3.0 menyebabkan munculnya subpopulasi sel g pada kromatogram. uji apoptosis dengan menggunakan annexin-v-fluos memperlihatkan bahwa hampir sebagian besar sel t47d yang diberi perlakuan emestrin pada dosis 1.0 dan 3.0 mengalami apoptosis (masing-masing sebesar 83.6 dan 92.6%). aktivitas antikanker emestrin kemungkinan besar disebabkan oleh adanya gugus yang memiliki jembatan disulfida. kata kunci: apoptosis, , emestrin sitotoksik 27 21 2 10 2 2 50 0 1 1 27 21 2 10 2 2 50 0 1 1 1 13 -1 -1 -1 -1 -1 -1 -1 -1 1 13 -1 -1 -1 -1 -1 -1 -1 -1 μg μg μg ml μg ml μg ml μg ml , marine microorganisms, particularly marine fungi, have recently drawn much attention as an important source of biologically active secondary metabolites. more recently, marine fungi have become an important research subject of natural products with significant value due to the diversity in chemical structures and biological activities (gresa . 2009). about 70-80% of the secondary metabolites that have been isolated from marine fungi are biologically active (jadulco 2002; gressa . 2009). overall, research on marinederived fungi have led to the discovery of 272 new natural products including many that have novel et al et al carbon skeletons; it is evident that marine-derived fungi have the potential to be a rich source of pharmaceutical leads compounds (bugni and ireland 2004). apoptosis or programmed cell death has, since its first description in 1972, become an important area of research due to the fact that it plays a pivotal role in embryonic development and in pathological processes. apoptosis is also important in controlling cell number and proliferation as part of normal development. apoptosis also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or noxious agents (ghobrial 2005; elmore 2007; doonan and cooter 2007). et al. *corresponding author, phone :+62-21-53650158, fax: +62-21-53650157, e-mail: muhammadnursid@gmail.com issn 1978-3477, eissn 2087-8575 vol 5, no 4, december 2011, p 160-169 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.4.3 the killing of tumor cells by diverse cytotoxic approaches, such as anticancer drugs, gammairradiations or immunotherapy, is predominantly mediated through the induction of apoptosis. apoptotic therapy has attracted many groups of investigator, and many companies had entered the race to develop the first generation of apoptotic anticancer medications. a number of anticancer agents, such as ciplastin, etoposide, mitomycin, and actinomysin d have been reported to induce apoptosis in cancer cells. thus, apoptosis in cancer cells play a critical role in the killing of tumor cells during cancer chemotherapy (genderen 2003; matsushita . 2005). in the screening of bioactive secondary metabolites, we found that marine fungus strain mfw39 obtained from ascidian collected from wakatobi marine national park, south east sulawesi, inhibited the growth of t47d (breast cancer) cell line. molecular and morphological taxonomy of this fungus revealed that the fungus was a . mycelium extract of this fungus had stronger cytotoxic activity (ic = 21.9 µg ml ) than broth extract (ic = 169.3 µg ml ) (nursid . 2011). the objective of this study was to isolate, identify, and investigate antitumor activity of active compound from mycelium produced by marine fungus. was isolated from the marine ascidia , which had been collected from the wakatobi marine national park as previously described (nursid . 2011). for production of secondary metabolites, the fungus was cultured (1l x 20) for 5 weeks (static) at 27-29 °c in sws medium containing 0.1% soytone, 1.0% soluble starch, and seawater 100%. . the mycelium extract was fractionated by vacuum column on silica gel using n-hexane-etoac (8:1), n-hexane-etoac (1:1), etoac 100%, and meoh 100%. fraction 3 was separated in silica gel vacuum column using -hexaneetoac (8:1), (5:1), (1:1), etoac 100% and etoacmeoh (5:1). fraction 3.3 and 3.4 was purified using silica gel preparative tlc. analytical tlc was performed by using silica gel aluminum plate (3 x 5 cm) whereas preparative tlc was performed by using silica gel glass plate (10 x 20 cm) and developed with -hexane etoac (1:1). analytical hplc was conducted using shimadzu hplc with photodiode array detector 2996, column ods 4.6 x 150 mm et al. et al aplidium longithorax emericella nidulans et al e. nidulans e. nidulans mfw39 a. longithorax et al n n 50 50 -1 -1 materials and methods fungal material and culture. isolation of active compound tlc and hplc condition. (shimadzu), volume injection 20 µl, flow rate 1.0 ml min , gradient (water 20% acetonitrile 100%), and time period was 45 min. . the active compound was identified by using spectroscopic analysis including infra-red (ir), mass spectra (ms), nuclear magnetic resonance (nmr, including h-nmr, cnmr, and dept 135°). ir-spectra were recorded on perkin elmer one ft-ir spectrophotometer. mass spectra were recorded using lc-esi-tof-ms (waters, column sunfire 4.6 x 150 mm, isocratic water + 0.1% formic acid : acetonitrile = 45/55 v/v, flow rate at 0.7 ml min , volume injection of 10 µl, capillary voltage 1800 v, and cone voltage 60 volt). nmr spectra were recorded under conditions as indicated on a jeol jnm eca-500 spectrometer. chemical shifts ( ) are given in parts per million downfield from tms as internal standard. t47d (breast cancer), hepg2 (liver cancer), c28 (colon cancer), hela (cervix cancer), and vero (normal cell) were cultured in rpmi 1640 medium (sigma), supplemented with 10% fetal bovine serum, 1% fungizone, and 2% penicillinstreptomycin. the cells were maintained at 37 °c in a moisture-saturated atmosphere containing 5% co . all of the cells were seeded at density of 2 x 10 cells well in 200 µl of medium and allowed to attach overnight. after the cells were grown to about 80% confluence, treatment were initiated by supplementing to get 0.4, 0.8, 1.0, 2.0, 4.8, 16.0, 32.0, and 64.0 µg ml of final media concentration of compound. cytotoxicity test was performed using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (mtt) assay according to ebada . (2008). the mtt assay is a colorimetric assay base on the cleveage of yellow water-soluble tetrazolium salt, mtt, to form water-insoluble, dark blue formazan cristals. mtt cleveage occurs only in living cells by mitocondrial enzyme succinate dehydrogenase. the c l e a v a g e p r o d u c t f o r m a z a n w a s m e a s u r e d spectrophotometrically at 570 nm using microplate reader (dynex revelation). the percent inhibition of cells growth were calculated with formula: (a-d) (b-c)/(a-d) x 100 %, where a: control cell absorbance, b: compounds absorbance, c: controls compound absorbance, and d: c o n t r o l m e d i a a b s o r b a n c e . t h e i n h i b i t i o n concentration 50 (ic ) value is defined as the concentrations of compound which inhibited 50% of the cell growth. ic value was determined by using minitab probit analysis version 16.0. . the effects of the compounds on the cell cycle were studied by flow cytometry -1 1 13 -1 4 -1 -1 compound identification cytotoxicity test. cell cycle analysis δ 2 50 50 et al volume 5, 2011 microbiol indones 161 according to rannali . (2003). briefly, the t47d cell was seeded at a final density of 7 x 100 cells well in 6-well microculture and incubated for 12 h in co incubator (37°c, 5% of co flow). the compound was added to cells at 1.0 and 3.0 g ml for 24 h. at the end of the incubation period, supernatant was collected and centrifuged at 200 rpm for 5 min to collect detach cells. the remaining cells were detached using 0.025% trypsin edta solution for approximately 5 min. pellet cells were washed with pbs twice then resuspended with 200 g ml of rnase (dnase free), 0.1% triton x-100 and 300 l propidium iodide (roche). mixtures were incubated at room temperature for 30 min and cell cycle distribution was analyzed using facscalibur (becton-dickinson) flow cytometer. doxorubicin was used as a positive control. . discrimination of apoptosis and necrosis cell was conducted using annexin-v-fluos staining kit (roche) according to elmore (2007). after t47d cells treated with 1.0 and 3.0 g ml for 24 h, the cells were trypsinized, washed with pbs, and resuspended the cell pellet in 100 l of annexin-vfluos staining kit. the cells then incubated in dark room for 10 min at 20-25 °c. typical histogram of apoptotic and necrotic cells was performed using facscalibur (becton-dickinson) flow cytometer. doxorubicin was used as a positive control. the resulting broth and mycelium were separately extracted with etoac and ch cl meoh (1:1) to obtain crude extracts of 0.46 and 37.0 g, respectively. based on mtt test, the mycelium extract showed stronger cytotoxic activity than broth extract against t47d cell. the active components et al g 5 -1 -1 -1 -1 2 2 2 2 μ μ μ μ μ apoptosis assay results i s o l a t i o n a n d i d e n t i f i c a t i o n o f a c t i v e compound. on mycelium extract were purified using assay-guided isolation to yield the active compound. the active compound appeared as yellow solid and display a single spot on tlc [( -hexane:etoac (1 : 1 v/v) ( value of 0.45)] (fig 1). the active compound was eluted at 15 min in the hplc chromatogram (fig 2). the molecular formula of active compound was established as c h n o s by lc-esi-tof-ms (m/z 597.1105 [m h] (fig 3). ir absorption at 3447 cm , 1688 cm and 1614 cm indicated the presence of hydroxyl, ester and amide groups. the h-nmr and c-nmr (fig 4) of active compound in cdcl 4.0 and 56.5), 3. 28. 5. 6.3). the c-nmr spectrum of active compound displayed 27 carbon signals consisting of 2 methyl (ch ), 13 methine (ch), and 12 quaternary carbons as judged by dept 135° spectrum (fig 5). these spectra were closely similar to the spectrum of emestrin (fig 6) that reported previously by seya . (1985); ooike . (1997); onodera . (2004); and n rf n et al et al et al 27 21 2 10 2 3 h c h c h c 3 -1 -1 -1 1 13 13 showed methoxy group (δ δ -methyl group (δ 4 and δ 0), and two hydroxyl groups (δ 5 and δ 162 nursid et al. microbiol indones rf 0,45 a b fig 1 tlc of active compound under 254 nm uv (a) and sprayed with phosphomolybdic acid (b). e. nidulans 0 5 10 15 20 25 30 35 40 45 min 0 250 500 750 1000 1250 1500 1750 2000 2250 mabs id#1 maxplot (1.00) 1 2 3 4 5 6 7 8 9 fig 2 hplc chromatogram of active compounde. nidulans . volume 5, 2011 microbiol indones 163 fig 3 lc-esi-tof-ms spectrum of active compound.e. nidulans herath . (2005). the summary of h-nmr, cnmr and reported emestrin was showed in table 1. . the mtt test used for the evaluation of cytotoxic properties of the active compound in this research. the growth-inhibitory effects were studied in five cell lines, including t47d (breast cancer), hepg2 (liver cancer), c28 (colon cancer), hela (cervix cancer) and vero (normal cell). morphological changes in the cells caused by emestrin were observed by microscope as shown in fig 7. after 24 h incubation with the compound at concentration 0.8, 2.0, and 16.0 µg ml , morphology of t47d, hepg2 and c28 cells changed, but the morphology of hela cells changed only after emestrin treatment at 16.0 µg ml . in contrast, there were no morphological changes in vero and untreated cells (control). growth inhibition of t47d, hepg2, c28 hela, and vero cells after treated with emestrin were shown in fig 8. emestrin tested showed a dose-dependent inhibition of all cells except to vero cell. probit analysis showed that emestrin had strong cytotoxicity activity to t47d, hepg2, c28, and hela cells but it was not too toxic to vero cell (table 2). in further study we used t47d cells because it was highest inhibited by emestrin. et al 1 13 -1 -1 cytotoxic activity effects on cell cycle arrest in t47d cells induced apoptosis in t47d cells . cell cycle distribution of t47d cells exposed to emestrin was investigated by flow cytometry. t47d cells were exposed to the emestrin at 1.0 and 3.0 ml for 24 h, and the fraction of cells at different phases of the cell cycle were monitored after propidium iodide (pi) staining. following the emestrin treatment at concentration 3.0 ml , an apoptotic sub-population of cells (sub g ) appears (fig 9). on the contrary, there were no sub g in the cell controls. in fig 9, also it can be seen that emestrin treatment at concentration of 1.0 ml induced cell-cycle arrest in g /g phase. . next the cells were exposed to 1.0 ml and 3.0 ml of emestrin for 24 h and the cells were stained with annexin-pi to discriminate apoptosis, necrosis and viable cells. almost t47d cells were treated with emestrin underwent apoptosis. percentage of apoptotic cells were 83.6% at 1.0 ml of emestrin concentration and increased at concentration 3.0 ml (92.6%). only 6.4% and 2.9% of dead cells were caused by necrosis at concentration 1.0 ml and 3.0 of emestrin, respectively (fig 10). μg μg μg μg μg μg μg μg μg/ml -1 -1 -1 -1 -1 -1 -1 -1 1 1 0 1 100 597.1104 % 0 300 400 500 600 700 800 900 1000200 309.0536 252.0148 310.0600 371.1167 521.1713 665.1083 795.0793 863.0673 925.0563 m/z 733.1066 619.0951 666.1152 164 nursid et al. microbiol indones fig 4 h-nmr (a) and c-nmr (b) spectrum of active compound. 1 13 e. nidulans a b volume 5, 2011 microbiol indones 165 fig 5 dept 135 spectrum of active compound o e. nidulans j coupling) c h c 1 167.1 168.0 2-nme 3.40 (3h, s) 28 3.43 28.8 3 78.5 77.9 4 163.8 165.2 5a 5.71 (1h, d, 8.4 hz) 60.9 5.93 58.9 6 4.88 (1h, td, 2.3 & 2.8 hz) 75.2 4.82 74.8 7 4.93 (1h, dd, 2.3 & 8.4 hz) 107.7 4.98 109.9 8 6.32 (1h, dd, 2.3 & 8.4 hz) 138.6 6.34 138.5 10 6.91 (1 h, d, 2.3 hz) 143.3 6.91 142.9 10a 112.5 107.8 11 4.98 (1h, s) 76.8 4.98 79.7 11-oh 5.5 (s, 1h) 5.23 11a 73.1 83.0 1' 122.7 119.4 2' 7.75 (1 h, d, 2.3 hz) 122.2 7.66 122.2 3' 145.3 146.2 4’ 154.3 154.6 4'-ome 4.0 (3h, s) 56.5 4.02 56.3 5' 7.04 (1h, d, 8.4 hz) 112.3 7.06 112.1 6' 7.79 (1 h, dd, 1.55 hz) 126.5 7.78 125.8 7' 165.3 166.3 1" 145.2 145.5 2" 148.9 152.1 2"-oh 6.32 (1h) 4.77 3" 6.92 (1 h, d, 8.4 hz) 115.3 6.95 110.6 4" 7.07 (1 h, d, 2.3 hz) 125.6 7.13 125.5 5" 126.7 128.6 6" 7.88 (1h, d, 2.3 hz) 121.1 7.68 126.9 7" 5.41 (1h, d, 12.2 hz) 74.9 5.45 76.4 note : s = singlet, d = doblet, dd = doblet-doblet, td = triple-doblet emestrin carbon number active compound table 1 h-nmr and c-nmr of active compound compare with emestrin 1 13 e. nidulans o n n oo o o o o o h s s o h o h 1 2 34 5 5a 6 8 10 10a 11 11a 7' 1' 5' 4' 3' 2' 1" 6" 5" 3" 2" 7" fig 6 molecular structure of emestrin. fig 8 growth inhibition of t47d, hepg2, c28, hela, and vero cells after treated with various concentration of emestrin for 24 h (± standard deviations). fig 7 morphological changes in t47d, hepg2, c28 hela, and vero cells after treated with emestrin (magnification 100 x under inverted microscope). 166 nursidi et al. microbiol indones concentration of emestrin 0.8 mlμg -1 2.0 mlμg -1 16.0 mlμg -1 untreated cell (control) t47d hepg2 c28 hela vero table 2 ic values of emestrin against several types of cells50 cancer cell lines ic50 (µg ml -1 ) t47d (breast cancer) 1.8 ± 0.1 hepg2 (liver cancer) 4.2 ± 0.6 c28 (colon cancer) 2.6 ± 0.3 hela (cervix cancer) 13.8 ± 1.5 vero (normal cell) 260.9 ±49.9 fig 10 apoptosis and necrosis were induced in t47d cells detected by annexin-pi staining and compared to the cells control. viable cells: lower left quadrant; apoptotic cells: lower right quadrant; and necrotic cells: upper right quadrant. fig 9 flow cytometric analysis of the dna histogram of pi-stained t47d cells. volume 5, 2011 microbiol indones 167 0 40 80 120 160 200 0 40 80 120 160 200 0 40 80 120 160 200 0 40 80 120 160 200 10 4 10 10 10 10 3 2 1 0 10 4 10 10 10 10 3 2 1 0 10 4 10 10 10 10 3 2 1 0 10 4 10 10 10 10 3 2 1 0 discussion emestrin was isolated by seya in 1985 from mycelial acetone extracts of the fungus emestrin is a member of a group epithiodioxopiperazine (etp) that are toxic secondary metabolites made only by fungi. at least 14 different etps (excluding those with minor modifications) are known. the diversity of structures stems from the amino acids of the core etp moiety, as well as the modifications of these amino acids. all natural etp isolated to date contain at least one aromatic amino acid. a diverse range of filamentous ascomycetes produce etp. five classes of ascomycetes (dothideomycetes, eurotiomycetes, lecanoromycetes, saccharomycetes and sordariomycetes) are known produce etp. at least two basidiomycetes, and a sp., produce etp epicorazine and hyalodendrin, respectively (gardiner . 2005). several natural products containing etp moieties have so far been reported to be promising as a antitumor agent such us mpc 1001 (emestrin c), gliotoxin and chaetoxin. emestrin c showed antiproliferative activities against du145 human prostate cancer cell line with ic value of 9.3 nm. the toxicity of etps is due to the presence of a disulphide bridge, that can inactivate proteins via reaction with thiol groups, and to the generation of reactive oxygen species by redox cycling (onodera . 2004; gardiner . 2005). we have demonstrated that emestrin strongly inhibits the growth of t47d, hepg2 and c28 cells. this compound had ic values of < 5 µg ml to these cells. microscopic study (fig 9) exhibited that cells morphology changed from taper to round or irregular shape. under microscopy analysis, many cell populations detached from microplate after exposed to the compound for 24 h. the cytotoxicity of emestrin to these cells related to the structure of etp moiety. when t47d cells were treated with the emestrin, etp moiety may interact with the cell membrane to alter permeability characteristics and then affect the entry or exit of amino acids and nucleotides known to regulate cellular metabolism, and thus result in cellular structural changes simultaneously with their functional changes in both physiological and pathological conditions. this effect implies that etp moietyinduced disruption could functionally and structurally damage cell membrane as well as other cellular structures and ultimately cause cell death. the formation of distinct dna fragments is a b i o c h e m i c a l h a l l m a r k o f a p o p t o s i s , w i t h et al. emericella striata. stereum hirsutum hyalodendron et al et al et al 50 50 -1 internucleosomal dna cleavage activity as a major characteristic (rannali . 2003; yu . 2005). the normal metabolic cellular activities of the g period in cell division are in preparation for mitosis, including transcription translation, and increase of cytoplasmic materials. the flow cytometry study presented in this report suggests a possible association between emestrin and cell cycle arrest activity. as shown in fig 9, the emestrin apparently affected the proliferation of t47d cells by inhibited the progression of the t47d cells through the g phase of the cell cycle. when t47d cells were treated with emestrin, apoptotic cells with high dna content apparently accumulate during the g1 period, in comparison with the untreated cells (fig 9). as a result, the synthesis of proteins involved in transcriptional regulation and cell cycle control and the completion of the s and m phases are delayed, giving rise to a plethora of cellular effects, not least of which is potential activation of pathways leading to cell cycle arrest and apoptosis (yu . 2005). when a cell undergoes apoptosis, changes occur at the cell surface. one of plasma membrane alteration is the translocation of phosphatidylserine (ps) from the inner part of the plasma membrane to the out layer, by which ps becomes exposed at the external surface of the cells. ps exposure therefore represent a useful assay for the apoptosis. ps present on the outer leaflet can be detected using annexin v (elmore 2007). annexin v is ca -dependent phospholipid-binding protein with high affinity for ps. this protein can hence be used as a sensitive probe for ps exposure upon the outer leaflet of the cell membrane and is, therefore, suited to detect apoptosis cells. necrotic cells also expose ps, and will therefore also bind annexin v. to differentiate between apoptotic and necrotic cells, pi is often used in conjunction with annexin v. pi will mark necrotic cells, but not apoptotic cells. in this assay, annexin v bind the phospholipid ps, marking apoptotic and necrotic cells, while pi bind dna, marking only necrotic cells (ranalli 2003). in this research, we assayed the ability of emestrin to induce apoptosis in cells. the double staining of annexin v-propidium iodide by annexin-v-fluos staining kit analysis showed that emestrin was a potent inducer of apoptosis in t47d cells. fig 10 showed that emestrin at 1.0 and 3.0 5.0 µg ml could induce the high amount of apoptosis in t47d cells. it were higher than doxorubicin at 5.0 µg ml . doxorubicin (14hidroxydaunorubicin) is an antracyclic antibiotic drug widely used in the treatment of a variety of cancers. doxorubicin has multiple mechanisms of action, including its interaction with the enzyme topoisomerase ii, metal ion chelation and free radical generation. et al et al et al et al. 1 1 2+ -1 -1 168 nursid et al. microbiol indones more recently doxorubicin was found to reduce the viability of cancer cells via rna damage (brilhante . 2011). base on the cytotoxicity test, cell cycle analysis and apoptosis assay, we can infer that the emestrin was potential as anticancer agent. this anticancer activity may be related to the unique of the internal disulphide bond of emestrin, although this hypothesis could be further proved. this research was supported by ministry of marine and fisheries affairs. we thanks to farid abdullah and juana nursanthi (pathology clinic lab and parasitology lab., faculty of medicine, universitas gadjah mada, yogyakarta) for the flow cytometry analysis, anis mahsunah (p biotechnology bppt) for the lc-esi-tof-ms, sofa fajriyah and akhmad darmawan (puslit kimia lipi) for the nmr data, and asri pratitis (biotechnology lab., research center for marine and fisheries product processing and biotechnology, jakarta) for the fungus isolation et al . acknowledgment references 3 bugni ts, ireland cm. 2004. marine-derived fungi: a chemically and biologically diverse group of microorganisms. nat prod rep. 21:143163. doi: 10.1039/b301926h. brilhante o, stumpp t, miraglia sm. 2011. long-term testicular toxicity caused by doxorubicin treatment during pre-pubertal phase. int j med med sci. 3(2):52-60. ebada se, edrada ru, 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338-340. saraste a, pulkki k. 2000. morphologic and biochemical hallmarks of apoptosis. cardiovasc res. 45:528-537. seya k, nakajima s, kawai k. 1985. structure and absolute configuration of emestrin, a new macrocyclic epidithiodioxopiperazine from . j chem soc chem com. 117:657-658. yu fr, lian xz, guo hy, mcguire pm, li rd, wang r, yu fh. 2005. isolation and characterization of methyl esters and derivatives from ( ) and their inhibitory effects on the human sgc-7901 cells. j pharm pharmaceut sci. 8(3):528-535. aspergillus insuetus. chlorophytum comosum emericella foveolata. cladorrhinum emericella striata euphorbia kansui euphorbiaceae volume 5, 2011 microbiol indones 169 04. ramadhani.cdr vol.14, no.2, june 2020, p 73-82 doi: 10.5454/mi.14.2.4 synergistic interaction of arbuscular mycorrhizal fungi and phosphatesolubilizing bacteria on sorghum bicolor (l.) moench growth under saline condition 1* 1* indriati ramadhani and sri widawati 1 microbiology division, research center for biology, indonesian institute of sciences-lipi, jalan raya jakarta-bogor km 46, cibinong 16911, indonesia *i ramadhani and s widawati contributed equally to this work. sweet sorghum (sorghum bicolor (l.) moench) is a food source and a raw material in biofuel-ethanol production. arbuscular mycorrhizal fungi (amf) and phosphate-solubilizing bacteria (psb) are called rhizosphere microorganisms, which are useful microorganisms that enhance plant growth. rhizosphere microorganisms also increase plant's resistance to environmental stress, while npk fertilizer used on agricultural land are found to increase crop yields only. however, its continuous application has a negative impact on the environment. therefore, this research aimed to study the synergy between amf, and psb with npk fertilizer in influencing the sweet sorghum growth in saline condition. two treatment factors were used, which are microbes combination (arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria), and npk doses without npk, 25% npk, 50% npk, and 100% npk. a complete randomized design was used as an experimental design with 3 replications for each treatment. furthermore, zeolite was used to grow sweet sorghum seeds individually and were maintained for one month in a greenhouse. pots were watered with 50% seawater (freshwater: seawater = 1:1) every day to keep the moisture. plant growth parameters were also measured, which includes amf colonization in the roots, number of amf spores, and psb population in the planting medium. the combination of amf, psb, and npk in sweet sorghum increased the plant height, number of leaves, plant fresh weight, plant dry weight, and total of plant p, although not always significant. amf+psb+25% npk produced the highest number in all parameters. therefore, the synergy between amf, and psb with npk fertilizer is able to increase the plant growth in saline condition. key words: arbuscular mycorrhizal fungi, npk, phosphate-solubilizing bacteria, saline, sweet sorghum sorgum manis (sorghum bicolor (l.) moench) merupakan sumber makanan dan bahan baku dalam produksi bioetanol. jamur mikoriza arbuskular (jma) dan bakteri pelarut fosfat (bpf) disebut sebagai mikroorganisme rizosfer, yang merupakan mikroorganisme yang berguna untuk meningkatkan pertumbuhan tanaman. mikroorganisme rizosfer juga meningkatkan ketahanan tanaman terhadap cekaman lingkungan, sedangkan pupuk npk yang digunakan pada lahan pertanian ternyata hanya meningkatkan hasil panen saja. namun, penerapannya yang berkelanjutan memiliki dampak negatif terhadap lingkungan. oleh karena itu, penelitian ini bertujuan untuk mempelajari sinergi antara jma, dan bpf dengan pupuk npk dalam mempengaruhi pertumbuhan sorgum manis dalam kondisi salin. dua faktor perlakuan yang digunakan, yaitu kombinasi mikroba (jamur mikoriza arbuskular dan bakteri pelarut fosfat), dan dosis npk tanpa npk, 25% npk, 50% npk, dan 100% npk. rancangan acak lengkap digunakan sebagai desain penelitian dengan 3 ulangan untuk tiap perlakuan. selanjutnya, zeolit digunakan untuk menumbuhkan biji sorgum manis secara individu dan dipelihara selama satu bulan di rumah kaca. pot disiram dengan 50% air laut (air tawar: air laut = 1: 1) setiap hari untuk menjaga kelembaban. parameter pertumbuhan tanaman juga diukur, yang meliputi kolonisasi jma di akar, jumlah spora jma, dan populasi bpf dalam media tanam. kombinasi jma, bpf, dan npk dalam sorgum manis meningkatkan tinggi tanaman, jumlah daun, berat basah tanaman, berat kering tanaman, dan p total tanaman, meskipun tidak selalu signifikan. jma + bpf + 25% npk menghasilkan nilai tertinggi di semua parameter pertumbuhan. oleh karena itu, sinergi antara jma dan bpf dengan pupuk npk mampu meningkatkan pertumbuhan tanaman dalam kondisi salin. kata kunci: bakteri pelarut fosfat, jamur mikoriza arbuskula, npk, salin, sorgum manis microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8567364656; email: indriatiramadhani@gmail.com carbohydrate content consists of approximately 62% carbohydrates, being a potential sweet sorghum component (barcelos et al. 2016). it is a suitable bioethanol feedstock in eastern indonesia (hasibuan and nazir 2017). previous studies showed that sweet sorghum has a higher energy output than sugarcane, sugar beet, corn, and wheat (dar et al. 2018). sweet sorghum (sorghum bicolor (l.) moench) is a plant which is widely cultivated due to its usefulness as a food source and a raw material in biofuel-ethanol production (dar et al. 2018). its sufficient soil salinity is an environmental condition which is + toxic to plants (liang et al. 2018). the high na and cl ion contents in saline soils reduce the nutrient absorption, nutritional balance, and disrupt plant metabolic processes (arzani and ashraf 2016). in addition, high soil salinity reduces soil porosity and aeration (khataar et al. 2018). also, it reduces water absorption and causes physiological dryness (saxena et al. 2017). production of alternative food substitutes for rice is needed for coastal communities in indonesia. sweet sorghum can be used as one of the potential food to be developed in coastal areas (marles et al. 2018). sweet sorghum has quite high carbohydrates, but people prefer to use it for biofuel production and feedstock (dar et al. 2018). several studies have also been conducted to find formulas that can help sweet sorghum grow in saline stress condition. one of them is the use of rhizosphere microbes such as arbuscular mycorrhizal fungi (amf), which have been shown to help sweet sorghum to grow in saline conditions (wang et al. 2019). r h i z o s p h e r e m i c r o o rg a n i s m s a r e u s e f u l microorganisms, known to increase plant growth and enhance resistance to environmental stress (de zelicourt et al. 2013). a typical amf is a symbiotic mutualism relationship between soil fungi and plant roots, which is able to assist plants in nutrient absorption and increase its tolerance to environmental stress (smith and read 2008). while phosphatesolubilizing bacteria (psb) are rhizosphere microorganisms, which assist plants in nutrient absorption, such as phosphate (awasthi et al. 2011). in addition, psb are discovered to increase the bioavailability of phosphate in mycorrhizal plants (toro et al. 1997). npk fertilizer used on agricultural land are found to increase crop yields, change the crop quality, and the soil productivity (zhong and cai 2007). however, mineral fertilizer application on soil causes shift in microbial community (chu et al. 2007). while microbial application enhances plants' tolerance to environmental stress (bhardwaj et al. 2014). thus, npk fertilizer combined with potential microbes reduces its impact on the agricultural land. previous studies showed that the combination of psb and amf was well studied in several types of plants such as helianthus tuberosus, triticum aestivum, and sorghum bicolor (rupaedah et al. 2014; minaxi et al. 2013; nacoon et al. 2020). however, little known whether the combination of psb and amf is able to increase plant growth in saline condition. therefore, this research aimed to study the synergy between amf and psb with the npk fertilizer in influencing sweet sorghum growth in saline condition. materials and methods preparation of planting media. two hundred and sixty grams of zeolite grade 1 (0.7–1.4 mm in diameter) was placed in a pot measuring 13 cm high and 10 cm in diameter, covered and sterilized in an autoclave at 121°c for 15 minutes. npk fertilizer used was blue in color and granular, easily soluble in water, and has the main content of n (16%), p (16%) and k (16%). before planting sweet sorghum, npk were first added to the media with several concentrations, namely 0% npk (0 g npk), 25% npk (0.625 g npk), 50% npk (1.250 g npk), and 100% npk (2.500 g npk). this preparation was carried out in a sterilized room. � b i o s t i m u l a n t p re p a r a t i o n , p l a n t r o o t inoculation, and planting. microbes used were arbuscular mycorrhizal fungi (amf) and phosphatesolubilizing bacteria (psb). the arbuscular mycorrhizal fungi used were obtained from the soil (ph = 5.6) of the tasikmadu sugar cane plantation, karanganyar, central java, indonesia. the phosphatesolubilizing bacteria used were pseudomonas fluorescens isolate ilpl1 which isolated from the rhizosphere amaranthus hybrydus and ipomoea aquatica on the coast of laki beach, seribu islands (widawati 2011). two treatment factors were used, which are microbes combination (amf and psb), and npk doses without npk, 25% npk, 50% npk, and 100% npk. a complete randomized design was used as an experimental design with 3 replications for each treatment. a biostimulant in the form of liquid inoculant, isolated from pseudomonas fluorescens, was made by culturing 1 ose needle into 50 ml pikovskaya media (5 g ca (po ) . 0.5 g (nh ) so ; 0.2 3 4 2 4 2 4 g nacl; 0.1 g mgso . 7h o; 0.2 g kcl; 10 g glucose; 4 2 0.5 g yeast extract; 20 g agar; mnso and a little feso , 4 4 -1 1000 l aquadest) (gaur 1981), placed in a shaker for 5 days then transferred into 500 ml liquid pikovskaya and placed in a shaker again. the inoculant was 8 homogeneous and contained bacterial 10 cfu/ml. then, the sorghum seeds were washed with 70% alcohol rinsed 3 times with sterilized distilled water and left to germinate in a petri-dish containing a sterilized filter paper. after growing 2 leaves, sweet sorghum root was inoculated with arbuscular mycorrhizal spores and a slightly wet tissue containing 74 ramadhani et al. microbiol indones volume 14, 2020 microbiol indones 55 20 amf spores,was attached to sweet sorghum roots. then, sweet sorghum root was placed into the planting hole with 5 ml of liquid psb inoculant and covered with zeolite. plants were maintained for one month in a greenhouse, while they were watered with 50% seawater (freshwater: seawater = 1:1) every day to maintain the moisture of the zeolite. fifty ml of 50% seawater is used to water the sweet sorghum seeds to grow sprouts every day. after the seedlings have grown sprouts, they were watered with 100 ml of seawater every day. thus, after 2 months, sweet sorghum plants were harvested to determine the amf colonization in the root, amf and psb population in the planting medium. the parameters measured include plant height, number of leaves, fresh weight, dry weight of plants, and the absorption of p in leaves (total p content of plants). the bacterial population in the planting mediumwere calculated by the number plate method (marschner and dell 1994) with a dilution method of -1 -7 10 to 10 . a total of 5 g of zeolite was taken from the composite where plant roots were harvested and used for 3 repeated tests. a total of 1 g of zeolite was taken and placed into a test tube containing 9 ml of sterilized -1 aquadest (10 dilution) and homogenized using vortex for 1 minute at 1000 rpm. one ml sample was taken -1 -2 from the 10 dilution and transferred to a 10 dilution -7 tube: this procedure was repeated until the 10 dilution. -3 -5 -7 also 0.1 ml sample was taken from 10 , 10 , and 10 dilutions into a sterilized petri-dish, while pikovskaya media was added to it. bacterial population calculations were carried out after incubation for 3–7 days at 28 °c. the calculation of psb population on pikovskaya media was carried out only on p. fluorescens colonies that formed the clear zone (halo zone). � analysis of root colonization. root colonization analysis by amf was carried out following the root staining method (brundrett et al. 1996). these test's procedures are as follows: the roots were washed clean 3 times with distilled water, the roots were soaked in 10% koh (w/v) heat at 60 °c for 30 minutes, the solution was removed and the roots were washed 3 times with distilled water, the roots were soaked again in 2% hcl (v/v) for 2−3 minutes, then the solution was removed and the roots were soaked the 3rd time in 0.05% (w/v) hot trypan blue dye solution at 90 °c or put in an autoclave at a pressure of 15 psi for 10 minutes, the solution was removed and the roots were soaked the 4th time in a destaining solution that was 50% (v/v) glycerol, then the roots were cut into ± 1 cm. ten pieces of roots were taken randomly and arranged in a row on the glass object. root colonization by amf was observed and calculated under a compound microscope by the slide method (giovannetti and mosse 1980). the colonization percentage of amf was calculated from the following equation: percentage of amf colonization = (root length infected/root length observed) × 100%. calculation of amf spore density in planting media. calculation of amf spore density after harvest was conducted using 10 g of growth media by the wetsieving sucrose centrifugation method (brundrett et al. 1996). the filtrate (spores) from the filtration (water mixed with spores) were added into a petri-dish with gridline attached underneath (size 0.5–1 cm). analysis of n and p content in plants. measurement of n and p contents in plants follows the method from the soil research book compiled by sulaeman et al. (2005). leaves were dried at 50 °c until moisture content was lost. a total of 0.2 g of leaves were dissolved in 2 ml of acidic solution, then were extracted using a destruction block at a temperature of 170 °c or until the solution was clear. the solution was filtered and diluted with distilled water to a volume of 10 ml. a total of 1 ml of solution was dissolved in 1 ml of p dye and added 3 ml of distilled water. furthermore, the absorbance was measured with a spectrophotometer with a wavelength of 450 nm. � data analysis. the data obtained were analyzed with anova statistical variants followed by dmrt (duncan multiple range test) at 5% using spss version 23. results growth of sweet sorghum. in general, sweet sorghum seedlings inoculated with amf, psb, different npk concentrations, and harvested after 1month have significantly increased of plant growth, though the gradual increase of npk did not always correlate with an increase in plant growth at the p<0.05 level (table 1, figure 1a). all treatments significantly increased plant height compared to the control treatments. the 25% npk treatment improved plant height more than 50% npk and 100% npk. the psb+25% npk treatment significantly increased plant height compared to psb only, psb+50% npk, and psb+100% npk. the amf+25% npk treatment significantly increased plant height compared to psb only, amf+50% npk, and amf+100% npk (table 2). the amf+psb+25% npk treatment significantly increased plant height compared to am+psb only, amf+psb+50% npk, and amf+psb+100% npk. the amf+psb+25% npk treatment significantly increased plant height compared to the control and other treatments (table 1, figure 1b). all treatments did not significantly increase the number of leaves compared to the control treatments. the 50% npk treatment significantly increased the number of leaves compared to control, 25% npk, and 100% npk. the psb+100% npk treatment did not significantly increase the number of leaves compared to control, psb only, psb+25% npk, and psb+50% npk. a similar result was also observed for amf+npk treatments. the amf+100% npk treatment also did not significantly increase the number of leaves compared to control treatments, amf only, amf+25% npk, and amf+50% npk. the amf+psb+25% npk and amf+psb+50% npk treatments significantly increased the number of leaves compared to the control and other treatments (table 1, figure 1). � all treatments did not significantly increase plant fresh weight compared to the control treatments. the 50% npk treatment significantly increased plant fresh weight compared to control, 25% npk, and 100% npk. the psb, psb+25% npk, and psb+50% npk treatments significantly increased plant fresh weight compared to the control treatments, and psb+100% npk. a similar result was also observed for amf+npk treatments. the amf+100% npk treatment also did not significantly increase plant fresh weight compared to the control treatments, amf, amf+25% npk, and amf+50% npk. the amf+psb+25% npk treatment significantly increased plant fresh weight compared to amf+psb only, amf+psb+50% npk, and amf+psb+100% npk. the amf+psb+25% npk treatment significantly increased plant fresh weight compared to the control and other treatments (table 1). all treatments did not significantly increase plant dry weight compared to the control treatments. the 50% npk treatment significantly increased plant dry weight compared to control, 25% npk, and 100% npk. the psb, psb+25% npk, and psb+50% npk treatments significantly increased plant dry weight compared to control and psb+100% npk. a similar result was also observed for amf+npk treatments. the amf+100% npk treatment also did not significantly increase plant dry weight compared with control, am only, amf+25% npk, and amf+50% npk. the amf+psb+25% npk treatment significantly increased plant dry weight compared to amf+psb only, amf+psb+50% npk, and amf+psb+100% npk. the amf+psb+25% npk treatment significantly increased plant dry weight compared to the control and other treatments (table 1). � all treatments did not significantly increase a total of plant p compared to the control treatments. the 25% npk treatment significantly increased the total of plant p compared to control, 50% npk, and 100% npk. the 100% npk treatment did not significantly increase the number of leaves compared to control treatments. the psb+25% npk treatment significantly increased the total of plant p compared to control, psb+50% npk, and psb+100% npk. the psb+100% npk treatment did not significantly increase a total of plant p compared to control. the amf+25% npk and amf+50% npk treatments significantly increased the total of plant p compared to control treatments, amf, and amf+100% npk. the amf+psb+25% npk treatment significantly increased the total of plant p compared to the control and other treatments (table 1). � microbial populations on sweet sorghum of planting medium. in general, the highest population of psb found on planting media after 1-month, was in psb inoculation without npk addition. the addition of 100% npk concentration significantly decreased the psb population. subsequently, the psb population also increased on amf and npk addition. the increased psb population started from amf addition without npk. the psb population increased gradually until the addition of amf with 50% npk. however, the psb population decreased significantly with amf and 100% npk (table 2). � the decreased npk concentration increased the colonization percentage of amf in s. bicolor roots. therefore, the highest colonization percentage of amf was produced in amf inoculation without npk but with 25% npk addition. the percentage of amf colonization decreased significantly until the addition of 100% npk. although, psb addition also increased the colonization percentage of amf in sweet sorghum roots. the highest colonization percentage of amf was produced in the psb inoculation without npk but with 25% npk addition. the colonization percentage of amf decreased along with the increase in npk concentration. thus, the lowest colonization percentage was found in the psb inoculation with 100% npk (table 2). the highest number of amf spores in the sweet sorghum planting media was in the amf inoculation with 25% npk addition but without npk. the highest number of amf spores in s. bicolor roots planting 56 ramadhani et al. microbiol indones volume 14, 2020 microbiol indones 55 treatments plant height (cm) number of leaves plant fresh weight (g) plant dry weight (g) total of plant p (%) control 16.00a 4.00a 0.50a 0.15a 0.03a 25% npk 39.60de 4.00a 1.50bc 0.45bc 17.02d 50% npk 37.33cd 5.00b 1.50bc 0.46bc 5.60b 100% npk 29.00b 4.00a 0.93ab 0.28ab 1.11a psb 42.00ef 5.00b 1.83bc 0.56bc 11.68c psb+25% npk 46.67gh 5.00b 1.83bc 0.55bc 20.23de psb+50% npk 46.33gh 5.00b 1.50bc 0.44bc 16.98d psb+100% npk 35.00c 4.00a 1.17ab 0.35ab 1.12a amf 42.00ef 5.00b 1.83bc 0.54bc 11.38c amf+25% npk 47.00gh 5.00b 1.87bc 0.56bc 17.41d amf+50% npk 44.67fg 5.00b 1.50bc 0.46bc 17.00d amf+100% npk 35.00c 4.00a 1.07ab 0.32ab 0.79a amf+psb 49.00hi 5.00b 2.33cd 0.71cd 23.02e amf+psb+25% npk 58.33j 6.00c 3.67e 1.11e 29.96f amf+psb+50% npk 50.00i 6.00c 3.00de 0.90de 17.33d amf+psb+100% npk 37.67cd 4.00a 1.17ab 0.35ab 1.17a table 1 effect of amf, psb and npk concentrations on various growth parameters of sweet sorghum growth after 4 weeks each value represents the mean of three replicates. values (along each column) sharing the same letter are not significantly different at the 5% (p≥0.05) level as determined by dmrt. media was in the amf inoculation with psb and 50% npk addition. however, the number of amf spores did not always correlate with the increase in npk concentration. the highest number of amf spores in the psb and npk was in the amf+psb+50% npk. therefore, the addition of psb and npk did not significantly affect the number of amf spores (table 2). arbuscular mycorrhizal fungi colonization on sweet sorghum. arbuscular mycorrhiza colonized s. bicolor root in treatment with amf+psb+25% npk, amf+25% npk, amf, psb+amf, amf+psb+50% npk, amf+50% npk, and amf+psb+100% npk. however, the control treatment was not found in the am colonization structure. am is able to form a new colonization structure at the root of the sweet sorghum. the structure of arbuscular mycorrhizal colonization observed is vesicular (figure 2a−h). discussion in general, the synergy between amf, psb, and npk fertilizers increase the sweet sorghum growth in saline condition.previous study has shown that the combination of amf, psb, and npk could increase the growth of sweet sorghum (ramadhani et al. 2019). salt in growth medium affect plant growth and development because salt reduces nutrient acquisition in plants (ruizlozano et al. 2012). also, the high level of salt in the soil causes very important ecological and agronomic problems (yaish and kumar 2015). hence, amf increases the host plant growth under salt pressure (zuccarini and okurowska 2008). the soil microorganisms in rhizosphere also increase the nutrient availability and enhance the iaa production (armada et al. 2015; marulanda et al. 2009). in other treatments, the combination of selected microbes, 56 ramadhani et al. microbiol indones fig 1 growth of sorghum bicolor 1 months after planting. a (1) 25% npk; (2) amf; (3) psb+amf; (4) psb; (5) amf+psb+25% npk. b (1) control; (2) psb+100% npk; (3) amf+100% npk; (4) amf+psb+25% npk. fig 2 amf colonization on sweet sorghum roots. a control. b amf+psb+25% npk.c amf+25% npk. d amf. e psb+amf. f amf+psb+50% npk. g amf+50% npk. h amf+psb+100% npk. ve vesicle. scale bars 20 µm (a−h). pseudomonas, and amf, increase their host plant growth in saline conditions (hidri et al. 2016). our result shows that amf, psb, and npk inoculation in sweet sorghum increase plant height, the number of leaves, plant fresh weight, plant dry weight, and total p compared to control treatments, though it is not always significant. in other treatments, the salinity reduces plant height, size, and yield on the crop of brassica (zamani et al. 2010). moreover, the salinity may reduce the crop yield by interfering with the water and nutritional balance of the host plant (islam et al. 2001). sweet sorghum plant in saline condition with the amf+psb+25% npk treatment showed significant increase in height, number of leaves, plant fresh weight, plant dry weight, and total p. in other treatments, 25% npk increased all growth parameters of sweet sorghum plant. however, this study was not conducted under saline condition (ramadhani et al. 2019). our result showed that the amf+psb+25% npk treatment significantly increased plant fresh and dry weight compared to the control and other treatments. g. based on the host plant growth data, the higher the npk concentration, the more inhibiting thehost plant growth. the host plantgrowth at the highest concentration of treatment, namely 100% npk alone, was more inhibited than the combination of psb, amf, and 100% npk. this is because high salt concentrations can inhibit the host plant growth by reducing chlorophyll content and disrupting the photosynthesis process. however, the presence of amf can help the absorption of mineral elements and increase the efficiency of photosynthesis, then improving the growth of the host plant (porcel et al. 2012). similarly, hajiboland et al. (2010) had reported that high salinity reduced tomatoes' production. the estimation of potential yield losses due to salinity is about 20% of total yield (ashraf and harris 2005). our result also showed that the amf+psb+25% npk treatment significantly increased the total plant p compared to the control and other treatments. similarly, the colonization of amf is well known to increase host nutrient acquisition, particularly p (smith and read 2008). moreover, the combined inoculation of amf and phosphate-solubilizing fungi gave better uptake of p (goenadi and sugiarto 2000; cabello et al. 2005). the highest population of psb was obtained in the inoculation of psb without npk addition. according to previous studies, pseudomonas is known for high salinity tolerance (vyas et al. 2009). in other treatments treatments number of viable psb cells (cell/g zeolite) colonization percentage of am (%) number of am spores (spores/10 g zeolite) control 0.0a 0.0a 0.0a 25% npk 0.0a 0.0a 0.0a 50% npk 0.0a 0.0a 0.0a 100% np k 0.0a 0.0a 0.0a psb 58.7 × 10 6 c 0.0a 0.0a psb+25% npk 36.7× 10 6 b 0.0a 0.0a psb+50% npk 33.0 × 10 6 b 0.0a 0.0a psb+100% npk 0.4 × 10 6 a 0.0a 0.0a amf 0.0a 82.0gh 21.0f amf+25% npk 0.0a 83.3gh 25.0g amf+50% npk 0.0a 50.0d 16.0d amf+100% npk 0.0a 10.0b 2.0b amf+psb 18.3 × 10 6 ab 80.0f 3.0b amf+psb+25% npk 22.7 × 10 6 ab 90.0i 3.0b amf+psb+50% npk 31.0 × 10 6 b 60.0e 18.0e amf+psb+100% npk 0.5 × 10 6 a 20.0c 6.0c table 2 effect of amf, psb, and npk concentrations on microbial populations on sweet sorghum of planting medium each value represents the mean of three replicates. values (along each column) sharing the same letter are not significantly different at the 5% (p≥0.05) level as determined by dmrt. volume 14, 2020 microbiol indones 79 pseudomonas fluorescens has been shown to have excellent plant colonization and potential plant growth promotion abilities (oteino et al. 2013). the highest colonization percentage of am was produced in the am inoculation without npk but with 25% npk. the lowest percentage of colonization was found in the inoculation of psb with 100% npk. the highest number of am spores in sweet sorghum roots planting media was in the inoculation of am with 25% npk addition but without npk. similarly, in other treatments, amf colonization was lowest in plants with the highest fertilizer dosage (omorusi and ayanru 2011). our result showed that arbuscular mycorrhizal fungi colonized sweet sorghum root in the treatment with amf+psb+25% npk, amf+25% npk, amf, psb+amf, amf+psb+50% npk, amf+50% npk, and amf+psb+100% npk. the structure of arbuscular mycorrhizal colonization observed was vesicular. similarly, new structures of amf colonization were formed at the root of host's plant such as hyphae, vesicles, arbuscules, and spores (sieverding 1991). in contrast, colonization of plants' roots by other amf species were reduced in the presence of nacl indicating that salt suppresses the formation of amf colonization structures (giri et al. 2007; sheng et al. 2008). therefore, this indicated that amf's ability to live in saline condition depends on the concentration of nacl in the soil and the amf species. � synergistic interaction of amf, psb, and npk improve the sweet sorghum growth in saline condition. the am+psb+25% npk treatment in saline condition produced the highest plant height, leaves number, plant fresh weight, plant dry weight, and total p of sweet sorghum. acknowledgements the author thanks to i made sudiana as the head of microorganism ecology research group, ety suryati, gunawan, and riski mareta ayu prisillia for technical help and data collection. author contributions all authors have reviewed the final version of the manuscript and approved it for publication. sw designed the study; sw conducted the research and collected the data; ir and sw analysed the data; ir and sw wrote and reviewed the paper. therefore, ir and sw are the main contributors to this manuscript. references armada e, azcón r, lópez-castillo om, calvo-polanco m, ruiz-lozano jm. 2015. autochthonous arbuscular mycorrhizal fungi and bacillus thuringiensis from a degraded mediterranean area can be used to improve physiological traits and performance of a plant of agronomic interest under drought conditions. plant physiol biochem. 90:64-74. doi: 10.1016/j.plaphy. 2015.03.004. arzani a, ashraf m. 2016. smart engineering of genetic resources for enhanced salinity tolerance in crop plants. crit rev plant sci. 35:146–189. doi: 10.1080/ 07352689.2016.1245056. awasthi r, tewari r, nayyar h. 2011. synergy between plants and p-solubilizing microbes in soils: effects on growth and physiology of crops. irjm. 2(12):484-503. barcelos ca, maeda rn, santa anna lmm, pereira jr n. 2016. sweet sorghum as a whole-crop feedstock for ethanol production. biomass bioenerg. 94:46-56. doi: 10.1016/j.biombioe.2016.08.012. bhardwaj d, ansari mw, sahoo rk, tuteja n. 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this section can be used to acknowledge financial support received in funding the research and to appreciate the institutions or persons for their assistance in the research or in the writing process of the manuscript. references. mi strongly recommends to use primary references only. references are arranged in alphabetical order using harvard referencing style. please use the standard abbreviation of a journal's name according to the issn list of title wordabbreviations and refer to : [cse] council of science editors. 2006. scientific style and format: the cse manual for authors, editors, and publishers. 7th ed. reston (va): the council. journal suwanto a, kaplan s. 1989. physical and genetic mapping of the rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. j bacteriol. 171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spora acaulospora foveata [the development of acaulospora foveata's spore]. j mikrobiol indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in escherichia coli. j biomed biotechnol. 12 p [on line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 and color photo(s) will be charged idr 600 000 per page. non-permi members. page charge is idr 850 000 (usd 100) per printed article up to 4 pages. additional page will be charge idr 250 000 (usd 30) per printed page without photo(s). page with black and white 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240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com gfa-backcover c.guide for author 10(2)-juni 02. fernanda.cdr vol.13, no.1, march 2019, p 9-15 doi: 10.5454/mi.13.1.2 isolation of a functional gene encoding homologous lysophospholipase from indonesian indigenous bacillus halodurans cm1 1 1 2* shanni fernanda , abinawanto , and is helianti 1 department of biology, fmipa universitas indonesia, kampus ui depok, depok 16424, indonesia; 2 center for bioindustrial technology, agency for assessment and application of technology (badan pengkajian dan penerapan teknologi), kawasan puspiptek jl. raya puspiptek serpong, tangerang selatan, 15314, indonesia lipase is a biocatalyst widely used in industry, for example detergent, pharmaceutical, food, or oil purification. one of the most widely lipase used for oil purification is lysophospholipase. as much as 50% of industrial enzyme needs are supplied from microorganisms. however, enzyme productivity from wild type microbial strain is usually limited and not applicable in industry, so that genetic engineering is necessary. cloning gene encoding for lysophospholipase from aspergillus niger and cryptococcus neoformans have been conducted, but has never been conducted from alkalothermophilic bacteria, such as bacillus halodurans. bacillus halodurans cm1 is an alkalothermophilic bacterial strain isolated previously that has many industrially potential enzymes. this study aimed to isolate one of the gene encoding lipase from bacillus halodurans cm1 and cloned into escherichia coli dh5α using the pgem-t easy vector. the gene fragment encoding lysophospholipase obtained with size 783 base pairs and had 100% similarity with gene encoding lysophospholipase from bacillus halodurans c-125 (no access genbank: ba000004.3). e. coli harbouring the recombinant plasmid with the gene also showed activity on trybutiryn medium compared to negative control. key words: bacillus halodurans cm1, cloning, lysophospholipase lipase adalah biokatalis yang banyak digunakan di industri, misalnya deterjen, farmasi, makanan, atau pemurnian minyak. salah satu lipase yang paling banyak digunakan untuk pemurnian minyak adalah lysophospholipase. sebanyak 50% kebutuhan enzim industri diperoleh dari mikroorganisme. namun, produktivitas enzim dari mikroba galur liar biasanya terbatas dan tidak fisibel di industri, sehingga diperlukan rekayasa genetika. kloning gen pengkode lysophospholipase dari aspergillus niger dan cryptococcus neoformans telah dilakukan, akan tetapi yang berasal dari bakteri alkalothermophilic, seperti bacillus halodurans, belum pernah dilakukan. bacillus halodurans cm1 adalah galur bakteri yang diisolasi sebelumnya yang memiliki banyak enzim yang potensial bagi industri. penelitian ini bertujuan untuk mengisolasi gen lysophospholipase dari bacillus halodurans cm1 dan dikloning ke escherichia coli dh5α menggunakan vektor pgem-t. plasmid rekombinan disekuensing. hasilnya didapat open reading frame (orf) lysophospholipase berukuran 783 pasangan basa dan kemiripan 100% dengan gen pengkode lysophospholipase dari bacillus halodurans c-125 (nomor akses genbank: ba000004.3). dari pengamatan zona bening di sekitar klon positif re e. coli kombinan, produk gen ini juga menunjukkan aktivitas pada medium tributirin dibandingkan dengan kontrol negatif. kata kunci: bacillus halodurans cm1, cloning, lisofosfolipase microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-7560694; email: is.helianti@bppt.go.id more easily hydrolyze the crude oil than lipase (cesarini et al. 2015). it is capable of hydrolyzing both a c y l g r o u p s o f p h o s p h o l i p i d s t o p r o d u c e phosphoglycerates and fatty acids (ramrakhiani and chand 2011). it is being used to produce useful phospholipid derivatives, to reduce the cholesterol content of food, and to refine vegetable oils, especially in terms of crude oil degumming. compared with traditional physical degumming methods, enzymatic degumming can greatly reduce the consumption of chemicals while producing very little wastewater. this leads to an economical, efficient, and stable green oil degumming process (jiang et al. 2011a; ramrakhiani and chand 2011). the lysophospholipase has been also enzymes are widely used as biocatalyst in many aspect of daily life, such as detergents, medicines, food, and oil refining. as much as 50% or more of these needs come from microorganisms, because microbial enzyme is commonly easier to cultivate. enzymes that are widely used in the oil refining industry are lipase and phospholipase (borrelli and trono 2015). during the last decade, the identification and production of phospholypase has attracted research interest because of its commercial application in various industries. phospholipase especially lysophospholipase can applied in oil refining (cesarini et al. 2015). substantial effort has been made to develop phospholipases, including three commercial phospholipases, for use in oil degumming. phospholipases have been shown to reduce the phosphorus content of oil to 5 ppm. with some phospholipases, an additional bleaching step is needed after enzymatic degumming. in addition, the supply of phospholipases is generally limited and cannot meet the increasing market demand for phospholipase (jiang et al. 2011b). in addition, the use of enzymes in oil purification industry causes oil free from harmful chemicals. the use of enzyme in this application is better. however, the use of enzymes is limited because of the availability and price. therefore, genetic engineering is required to produce high amounts of enzymes (cesarini et al. 2015). the genes encoding some of these phospholipase have been cloned and expressed, mainly in escherichia coli systems (chandrayan et al. 2008; jiang et al. 2012). for example, jiang et al. expressed the phospholypase b (plb) from pseudomonas fluorescens in e. coli bl21 and achieved a plb activity of 176.2 u·mg-1 (jiang et al., 2012). when chandrayan et al. introduced the gene encoding the plb from pyrococcus furiosus into e. coli bl21(de3) (plyss), this phospholypase was expressed as inclusion bodies and refolded using heat and denaturant treatment (chandrayan et al. 2008). lysophospholipase from aspergillus niger has also been cloned and expressed in pichia pastoris (zhu 2 0 0 7 ; c o e e t a l . ( 2 0 0 3 ) a s a l s o c l o n e d lysophospholipase gene from cryptococcus neoformans. h o w e v e r , c l o n i n g o f g e n e e n c o d i n g lysophospholipase from alkalotermophilic bacteria, such as bacillus halodurans has never been conducted. bacillus halodurans cm1 is bacterial strain of badan pengkajian dan penerapan teknologi culture collection (bpptcc) isolated from hot spring sediment in cimanggu, west java. the bacteria have a similarity of 99% with 16s rrna of b. halodurans c125. it have many industrially potential enzymes (ulfah et al. 2011). previous research has shown that this bacteria have lipase enzymes (aisyah et al. 2017), however, the study about their properties and respective gene have not been carried out. this study aimed to isolate one of the gene encoding lipase from b. halodurans cm1 and cloned into e. coli dh5α using the pgem-t easy vector. materials and methods medium. horikoshi medium was used for cultivation of bacillus halodurans cm1. luria bertani (lb) medium contain ampicilin, x-gal, and iptg were used for cultivation of recombinant e. coli dh5α. extraction genomic dna from b. halodurans cm1. extraction of the genome dna of b. halodurans cm1 performed using phenol-chloroform extraction method with modifications (saito and miura 1963). the result of extraction genome visualisation was observed in agarose 1% by electrophoresis. amplification of fragment gene encoding l y s o p h o s p h o l i p a s e . f o r w a r d p r i m e r 5 ' atgtggaaatgggaagttgctgagc-'3 dan reverse primer 5'-ctatgataattgctgttcga taaaaaaacagg-'3 were designed based on sequences of genes encoding lysophospholipase from b. halodurans c-125 on the site http://www.genome.jp and used in amplification of gene terget. the amplification was performed using kapa extra hot start taq dna polymerase based on the protocol of kapa (kapa biosystems 2017) under pcr condition 95 ºc 3 min, 95 ºc 30 sec, annealing 57 ºc 30 sec, extension 72 ºc 1 min, and continued to extra extension 72 ºc 10 min. transformation of recombinant plasmid pgem-t easy to escherichia coli dh5α ligation of the pcr fragment into pgem-t easy vector using t4 dna ligase was carried out based on protocol of promega (2015). plasmid pgem-t easy that contained lysophospholipase gene was transformed into competent cell escherichia coli dh5α by heat shock methods (hanahan 1983). screening of transformant was perfomed by screening blue-white that used lb -1 agar containing 100 µg ml ampicillin, 0.1 m isopropyl β-d-1-thiogalactopyranoside (iptg), and 4 % 5 b r o m o 4 c h l o r o 3 i n d o l y l b e t a d galactopyranoside (x-gal). the culture then was incubated over night at 37 ºc. the color of positive colonies that contains plasmid with gene encoding lysophospholipase are white, but the color of negative colonies are blue. positive colonies were cultured in a liquid lb medium containing ampicillin for extraction plasmid dna. extraction and verification of plasmid of recobinant escherichia coli dh5α. extraction plasmid from positive colonies of recombinant escherichia coli dh5α was performed by using the alkaline method (sambrook and russel 2001). the extracted plasmid was confirmed by digestion using 10 fernanda et al. microbiol indones http://www.genome.jp volume 13, 2019 microbiol indones 11 the enzyme ecori. sequencing and analysis of dna sequences. the plasmid that has been confirmed by digestion was delivered to first base for sequensing primer forward puc m13 (-40) and primer reverse m13 (-20). the sequencing result is then analyzed with bioedit and sequence scanner 2.0 (applied biosystems) software, then sequenced using clustal w program at http://www.genome.jp. sequencing analysis of their relationships with genomic databases available on genbank using the bioinformatics approach is the basic technique of the local search alignment tool (blastn) at http://www.ncbi.nlm.nih.gov/blast.cgi. qualitative test of gen products on lipid media.the escherichia coli dh5α sample containing the lysophospholipase gene from bacillus halodurans cm1 was inoculated on the lb medium to produce ampicillin and incubated at 37 °c for 24 hours. the same procedure was also done to the negative control (blue colony). the culture of the sample was incubated using an incubator shaker at a rate of 150 rpm at 37 °c for overnight, then the culture was redistributed by diverting 1 ml into 7 ml lb with ampicillin. refreshed cultures are allowed for ± 3 hours to reach various ods between 0.7 and 0.8. od measurements were performed using spectrophotometry at a wavelength of 600 nm. the od value between the culture sample and the negative control is attempted to have the same measurement value. cultures that have achieved these od values, are re-inoculated by taking 2 ml into 50 ml lb with ampicillin, 2% tributyrin and 0.1 m iptg; then incubated at the incubator shaker at 37 ºc, 150 rpm and overnight. a total of 1.5 μl sample cultures were spotted using micropipets into lb media containing tributyrin (tba) and 0.1 m iptg for qualitative assay (litthauer et al. 2010). incubation is o carried out at 37 c for 24-72 hours. lipolytic activity indicated by lypolitic index of recombinant e. coli dh5α was compared to the negative control. results g e n o m i c d n a e x t r a c t i o n a n d p c r amplification result. genomic dna could be extracted from b. halodurans cm1, and the results were visualized o na 1% agarose gel, and dna fragment more than 10.000 bp was detected (fig 1a). the genome can be extracted well, and did not contain any contaminants. by using the designed primers, the specific bands lies between 750 bp and 1.000 bp were detected (fig1b). sequencing and analysis of dna sequences. after ligation of the pcr product into pgem t easy vector, the white colonies grew on transformation plate were picked.there are 102 white colonies, however, only two clones used for futher analysesof extraction plasmid.the plasmid before (fig 2a) and after verification using ecori restriction enzyme showed 2 bands at ±3009 bp and ± 789 bp (fig 2b).the clones that has been confirmed by ecori digestion further used for sequencing. dna sequencing result showed that both clones showed open reading frame of protein. analyze of dna sequence with blast showed gene h a s 1 0 0 % s i m i l a r i t y w i t h g e n e e n c o d i n g lysophospholipase from bacillus halodurans c-125. it can be concluded, lysophospholipase gene had been isolated and cloned into plasmid pgem-t easy (fig 3). qualitative test of expression of gen products on lipid media. inoculation of positive colonies on lb tributyrin, ampicillin, and iptg agar media was carried out to determine the presence of lipase activity showed by clear zone. the addition of iptg was done to induce t7 promoters in the vector so that the gene could be translated. the positive colonies growing on the medium showed a clear zone after 3 days incubation compared to negative control (fig. 4). therefore the gene encoding lysophospholipase homolog showed the true lipase activity against tributyrin. tributyrin is one lipase substrate that can be used to measure lipolytic activity, even can be used also to measure the activity of phospholipase and lysophospholipase. alignment of the deduced amino acid with other lysophospholipase showed that, b. halodurans cm1 phospholypase has homology with other bacillus phospholipase. for example with that of bacillus pseudocaliphilus there was 57% homology, and with that of b. thuringiensis there was 43% homology (fig 5). discussion bacillus halodurans cm1 is very unique bacterial strain isolated previously from indonesia hotspring (ulfah et al. 2011). this bacterial strain is very potential in producing xylanase and the gene has been cloned (helianti et al. 2018). other than xylanase, protease, amylase, etc were also produced (ulfah et al. 2011). previous research has shown that this bacteria have lipase enzymes (aisyah et al. 2017), however, the study about their properties and respective gene have not been carried out. further investigation showed that, lysophosfig 1 genomic dna extracted frombacillus halodurans cm1 (a);and the pcr amplification of target gene (b). 12 fernanda et al. microbiol indones fig 2 histogram the number colony of azotobacter sp. during 60-days incubation. a a b a b pholipase from aspergillus niger has also been cloned and expressed in pichia pastoris (zhu 2007). the lysophospholipase gene from cryptococcus neoformans has also been cloned (coe et al. 2003). however, based on our further study none of this study related to the cloning of lysophospholipase gene from b. halodurans. based on genomic information, b. halodurans c-125 has at least 3 kinds of putative lipase genes, namely: phospholipase/carboxylesterase, acetyl esterase, and lysophospholipase (takami et al. 2000). however, we are not sure which gene from these putative that have matched our primer, and is this gene h o m o l o g o u s w i t h o u r b a c t e r i a l s t r a i n ' s lysophospholipase. therefore, we choose one of these lipase genes to be isolated using pcr approach. in this study, pgem t-easy vector was used, since this cloning is ta cloning vector that utitilize the pcr product by taq polymerase that have a-cohesive end, have blue-white screening system, has t7 or sp6 promoter system, and gave very good result in gene isolation in many reports (helianti et al. 2010; helianti et al. 2018). qualitative assay of lipase activity was confirmed by the clear zone around the colony. this result showed that this lypophospholipase gene product had true lipase, the same result was reported by ramchuran et al 2006 and sharma et al. 2018. however, lipolytic activity of lysophospholipase will be more optimal when on a specific substrate, such as agar medium containing lysolecithin or egg yolks (merino et al. 1999). using this dna vector, the target gene could be expressed (fig. 5). compared to a negative control e. coli clone with plasmid harbouring volume 13, 2019 microbiol indones 13 lysopl c-125 atgtggaaatgggaagttgctgagccgcgtggggtggtcgtcgtcattcatggggcgggagaacaccat reverse atgtggaaatgggaagttgctgagccgcgtggggtggtcgtcgtcattcatggggcgggagaacaccat forward atgtggaaatgggaagttgctgagccgcgtggggtggtcgtcgtcattcatggggcgggagaacaccat ********************************************************************* lysopl c-125 gaacaccatgggcgttatcaatggctcgcaaaaaagtttaatagcatcggattatctgtagtgatgggt reverse gaacaccatgggcgttatcaatggctcgcaaaaaagtttaatagcatcggattatctgtagtgatgggt forward gaacaccatgggcgttatcaatggctcgcaaaaaagtttaatagcatcggattatctgtagtgatgggt ********************************************************************* lysopl c-125 ttccaacagtacattgatgttgtcttggaatgggtggaagcagctaagttggagcacgtgccaatcttc reverse ttccaacagtacattgatgttgtcttggaatgggtggaagcagctaagttggagcacgtgccaatcttc forward ttccaacagtacattgatgttgtcttggaatgggtggaagcagctaagttggagcacgtgccaatcttc ********************************************************************* lysopl c-125 tgtttggccacagcatgggcggacttgtagccgttcgcacgatgattgaaggaggcacattgccagtgc reverse tgtttggccacagcatgggcggacttgtagccgttcgcacgatgattgaaggaggcacattgccagtgc forward tgtttggccacagcatgggcggacttgtagccgttcgcacgatgattgaaggaggcacattgccagtgc ********************************************************************* lysopl c-125 gtgctgtcattctttcatcaccatgctttgatttatatcagtcacctgggaaaggaaaagaattggctt reverse gtgctgtcattctttcatcaccatgctttgatttatatcagtcacctgggaaaggaaaagaattggctt forward gtgctgtcattctttcatcaccatgctttgatttatatcagtcacctgggaaaggaaaagaattggctt ********************************************************************* lysopl c-125 cgaaaatgttgcaccgagtaacgcctactttctcgcatcattcaggcattcgttccgatttagttactc reverse cgaaaatgttgcaccgagtaacgcctactttctcgcatcattcaggcattcgttccgatttagttactc forward cgaaaatgttgcaccgagtaacgcctactttctcgcatcattcaggcattcgttccgatttagttactc ********************************************************************* lysopl c-125 gaaatgaagagattcgtgaagcctacttgaaggatgagcttagagtaacaaaagtgtccacgaaatggt reverse gaaatgaagagattcgtgaagcctacttgaaggatgagcttagagtaacaaaagtgtccacgaaatggt forward gaaatgaagagattcgtgaagcctacttgaaggatgagcttagagtaacaaaagtgtccacgaaatggt ********************************************************************* lysopl c-125 attatgagttatcgaaggcgatgcgagatacccgtcgttatcctgaaaagttcccgaacgtaccattgc reverse attatgagttatcgaaggcgatgcgagatacccgtcgttatcctgaaaagttcccgaacgtaccattgc forward attatgagttatcgaaggcgatgcgagatacccgtcgttatcctgaaaagttcccgaacgtaccattgc ********************************************************************* lysopl c-125 tgttatgcaggcgggagaagattatatcacggatagaaaagcggcgtgggaatggtttaattcggttca reverse tgttatgcaggcgggagaagattatatcacggatagaaaagcggcgtgggaatggtttaattcggttca forward tgttatgcaggcgggagaagattatatcacggatagaaaagcggcgtgggaatggtttaattcggttca ********************************************************************* lysopl c-125 agtaacggaaaaggcctataaagagtggaatggactctatcatgaaatttttaatgagcctgagcggga reverse agtaacggaaaaggcctataaagagtggaatggactctatcatgaaatttttaatgagcctgagcggga forward agtaacggaaaaggcctataaagagtggaatggactctatcatgaaatttttaatgagcctgagcggga ********************************************************************* lysopl c-125 ggctgtgtttcaatacacctgtttttttatcgaacagcaattatcataa reverse ggctgtgtttcaatacacctgtttttttatcgaacagcaattatcatag forward ggctgtgtttcaatacacctgtttttttatcgaacagcaattatcatag ************************************************fig 3 the nucleotidesequence of gene encoding lysophospholipase bacillus halodurans cm1 compared to b. haloduransc-125. fig 4 clear zone around positive clones. 14 fernanda et al. microbiol indones fig 5 alignment of deduced amino acid of bacillus halodurans cm1 phospolipase compared to other amino acid phospholipase from other resources. bpseudalcaliphilus_phospholipa: lysophospholipase from bacillus pseudalcaliphilus; alteribacillus_persepolensis: lysophospholipase from alteribacillus_persepolensis; bthuringiensis_lysophospholipa: lysophospholipase from from bacillus thuringiensis. cm1_lysophospholipase ----------mwkwevaeprgvvvvihgagehhgryqwlakkfnsiglsv bpseudalcaliphilus_phospholipa ----------mwtyaskdarativlihgagehhgryewlaqkwnehgihv alteribacillus_persepolensis ---------mmknwmcdrargtvliihgagehhgryewviqylnqlrfhv bthuringiensis_lysophospholipa mkksemeesrmwnyeaeeakavivivhgameyhgryeavaemwnhigyhv * .: .:..::::*** *:****: : : * * cm1_lysophospholipase vmgdlpgqgrtrgkrghiqsfqqyidvvlewveaaklehvpiflfghsmg bpseudalcaliphilus_phospholipa imgdlpgqgktrgkrghinqfsqyidavqewvdeakkfeqpifilghsmg alteribacillus_persepolensis vsgdlpghgrtrgkrghidtfdqyintvyewykeaasyelpvflfghsmg bthuringiensis_lysophospholipa vmgdlpshgttsrnrghidsfdeyieevklwvkearkyrlpiflfghsmg : ****.:* * :****: *.:**: * * . * . *:*::***** cm1_lysophospholipase glvavrtmieggtlpvravilsspcfdlyqspgkgkelaskmlhrvtptf bpseudalcaliphilus_phospholipa glvairyvmeskakdiqglllsspclglfrpiktskdlaskvlnrltptl alteribacillus_persepolensis glvairtlmek-ympikgiilsspclglyeypskaadvaakmfhriaptf bthuringiensis_lysophospholipa glivirmmqetkredvdgiilsspclgvlagpsaplqaaskilniiapkl **:.:* : * : .::*****:.: : *:*::: ::*.: cm1_lysophospholipase shhsgirsdlvtrneeireaylkdelrvtkvstkwyyelskamrdtrryp bpseudalcaliphilus_phospholipa tvasginsnhvtrdeqirdqyvrdelrvtkvsvrwyqelhknmhlatryp alteribacillus_persepolensis kaksgirasrvtrspearaayekdefnvsvvtarwyqetlkaikrsffea bthuringiensis_lysophospholipa qfatnltvemstrnhevrdamendslflrkvsvrwyseltksieiahkki :.: . **. : * .*.: : *:.:** * * :. : cm1_lysophospholipase ekfpnvpllvmqagedyitdrkaawewfnsvqvtekaykewnglyheifn bpseudalcaliphilus_phospholipa ekmpdiplavlqagddkivskyavrdwfdsldvtekyykewkglyhevfn alteribacillus_persepolensis drfpnvpllvmqagedyivdkyaahrwfnrietadrsmkewkglyhelln bthuringiensis_lysophospholipa ddfpdvplllmqacedklvdktrvrtwfdnvkisdkafkewpncyhelln : :*::** ::** :* :..: . **: :. ::: *** . ***::* cm1_lysophospholipase epereavfqytcffieqqls-------100% bpseudalcaliphilus_phospholipa epekevvfrhavgimnlwt-------57% alteribacillus_persepolensis epereevfqfmmnfinqrl-------54% bthuringiensis_lysophospholipa eyerdeilnyiqsfteirinniietnk 43% * *:: ::.. : : false insert dna, the positive clone showed clear zone around the colony. the clear zone was could be considered appeared from the gene inserted in pgem. the extracellular expression could be come from the enzyme expressed in the cells which leaked into extra of the cells, since e. coli is usually cannot secreted the enzyme (helianti et al. 2010). the isolation of this gene is the first step to express the gene product in suitable host and produce and apply the gene product. acknowledgment part of this study was funded by insinas research incentive program of ministry of research, technology, and higher education 2018-2019 granted to ih. references aisyah a, mangunwardoyo w, trismilah, suhendar d. 2017. optimization and concentration of lipase from bacillus halodurans cm1. al kauniyah: j biology. 10(2): 114-123. borrelli gm, trono d. 2015. recombinant lipases and phospholipases 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(2001). molecular cloning: a laboratory manual. 3rd edn. cold spring harbor laboratory press, cold spring harbor, ny, usa. . sharma a,meena kr, kanwar ss 2018. molecular . characterization and bioinformatics studies of a lipase from bacillus thermoamylovorans bhk67. int j biol m a c r o m o l . 1 0 7 ( p t b ) : 2 1 3 1 2 1 4 0 . d o i : 10.1016/j.ijbiomac.2017.10.092. takamih, nakasone k, takaki y, maeno g, sasaki r, masui n, fuji f, hirama c, nakamura y, ogasawara n, kuhara s, horikoshi k. 2000. complete genome sequence of the alkaliphilic bacterium bacillus halodurans and genomic sequence comparison with bacillus subtilis. nucleic acids research 28 (21): 4317—4331. ulfah, m, helianti i, wahyuntari b, nurhayati n. 2011. characterization of a new thermoalkalophilic xylanaseproducing bacterial strain isolated from cimanggu hot spring, west java, indonesia. microbiol indones 5 (3): 139—143. zhu s. 2007. cloning and characterization of two lipases and a lysophospholipase from aspergillus niger. thesis. c o n c o r d i a u n i v e r s i t y ( c a n a d a ) , p r o q u e s t dissertations publishing: x+119 hlm. page 1 page 2 page 3 page 4 page 5 page 6 page 7 03. eveline.cdr vol.14, no.1, march 2020, p 17-24 doi: 10.5454/mi.14.1.3 antibacterial potential of star anise (illicium verum hook. f.) against food pathogen bacteria * eveline and agustin novita food technology department, faculty of science and technology, university of pelita harapan, mh. thamrin boulevard 1100 lippo village, kelapa dua, karawaci, tangerang, indonesia. star anise (illicium verum hook. f.) is commonly used as spice and flavor enhancer in food. previous research revealed the presence of active compound which could inhibit bacterial growth. thus, in order to apply star anise as natural antibacterial agent in food product, a further research concerning antibacterial activity and stability of star anise was conducted. crude extract of star anise was obtained using ethanol and acetone with maceration method for 3 days, then diluted to 10, 20, 30, 40, and 50% (w/v). well diffusion was conducted against three food spoilage bacteria (staphylococcus aureus, escherichia coli, and bacillus cereus). extract from ethanol with 30% concentration was selected as the best extract in which inhibit more than 6 mm inhibition zone with mic and mbc value: 1.59% and 6.36% (s. aureus), 1.04% and 4.18% (e. coli), and 0.59% and 2.39% (b. cereus). this selected extract was used to test the extract stability against 4 levels of heating temperature (60, 70, 80, and 90°c) for 2 levels of heating time (15 and 30 minutes), and 4 levels of ph (4, 5, 6, and 7). based on our results, different heating treatment and ph caused extract instability. star anise extract was more stable at 60°c for 15 minutes heating treatment and ph 4, which resulting the lowest inhibition zone reduction compared to control extract. star anise extract was categorized as low toxic compound (lc = 212.09 ppm). terpenoids (anethole, 2,6-dimethyl-6-(4-50 methyl-3-pentenyl)-2-norpinene, β-caryophyllene, β-bisabolene) was founded as major antibacterial compound in star anise extract; fatty acid (6-octadecenoic acid, hexadecanoic acid, stearic acid) and benzaldehyde (4anisaldehyde, p-allylanisole) were also founded as minor compound. key words: antibacterial, illicium verum hook. f., ph, stability, temperature, time bunga lawang (illicium verum hook. f.) umumnya dimanfaatkan sebagai rempah dan perisa pada makanan. penelitian terdahulu mengungkap adanya senyawa aktif yang berpotensi menghambat pertumbuhan bakteri. hal ini mendorong dilakukan penelitian lebih lanjut agar diketahui aktivitas dan stabilitas bunga lawang sebagai senyawa antibakteri alami yang dapat diterapkan dalam produk pangan. ekstrak kasar bunga lawang diperoleh dari ekstraksi dengan etanol dan aseton selama 3 hari pada konsentasi 10, 20, 30, 40, dan 50% (b/v). pengujian difusi sumur dilakukan terhadap tiga bakteri perusak pangan (staphylococcus aureus, escherichia coli, dan bacillus cereus). ekstrak 30% dengan pelarut etanol merupakan ekstrak terpilih penghasil zona hambat lebih dari 6 mm dengan mic dan mbc berurutan sebesar 1,59% dan 6,36% (s. aureus), 1,04% dan 4,18% (e. coli), 0,59% dan 2,39% (b. cereus). ekstrak terpilih digunakan dalam tahap pengujian stabilitas ekstrak terhadap suhu pemananasan (60, 70, 80, dan 90°c), waku pemanasan (15 dan 30 menit), dan ph (4, 5, 6, dan 7). perlakuan panas dan perubahan ph menyebabkan ketidakstabilan ekstrak. ekstrak bunga lawang lebih stabil pada suhu 60°c selama 15 menit dan ph 4, kondisi ini menghasilkan ekstrak dengan penurunan zona hambat terkecil terhadap nilai penghambatan ekstrak kontrol. ekstrak bunga lawang termasuk dalam kategori senyawa toksik rendah (lc 50 = 212,09 ppm) dalam fungsinya sebagai senyawa antibakteri yang mengandung senyawa antibakteri mayor berupa golongan terpenoid (anethole, 2,6-dimethyl-6-(4-methyl-3-pentenyl)-2-norpinene, β-caryophyllene, βbisabolene); dan senyawa antibakteri minor berupa golongan asam lemak (6-octadecenoic acid, hexadecanoic acid, stearic acid) dan golongan benzaldehide (4-anisaldehyde, p-allylanisole). kata kunci: antibakteri, illicium verum hook. f., ph, stabilitas, suhu, waktu microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8128187282; email: eveline.fti@uph.edu 2016; wei et al. 2014). the potency of star anise as antimicrobial agent then encouraged further research to increase the possibility to use star anise as natural antibacterial agent in food industry. � crude extract of star anise was obtained using maceration method with ethanol (polarity index 5.2) and acetone (polarity index 5.1) as solvent for 3 days. the crude extract then diluted into 5 levels of concentration (10, 20, 30, 40, and 50% (w/v)) and tested with well diffusion method against 3 species of star anise (illicium verum hook. f.) is spice which commonly used as traditional medicine. this plant is commonly found at tropical and subtropical area in asia (ahmad and youssef, 2015; sivakumar et al. 2016). star anise's essential oil consists of 89.5% transanethole, which acts as antimicrobial, antimicotoxins, antioxidants, and insecticide compound (aly et al. bacteria (s. aureus, e. coli, and b. cereus). previous research regarding solvent used in antibacterial compound extraction to apply in food product by ahmad and youssef (2015), dasgupta and klein (2014), and das and kumar (2013) showed that ethanol and acetone work effectively to extract phytochemical compound (flavonoids, alkaloids, triterpenoids, tannins, steroids, and glycosides) which can be used as antibacterial agent against e. coli, s. aureus, and b. subtilis. aguda and chen (2016) added that both ethanol and acetone were categorized as generally recognized as safe (gras) by food and drug administration (fda). � the selected concentration was then used to test extract stability against 4 levels of heating temperature (60, 70, 80, and 90 °c) for 2 levels of heating time (15 and 30 minutes), and 4 levels of ph (4, 5, 6, and 7). the heating temperature and time were determined based on previous research by peter (2001) regarding the stability of trans-anethole in fennel extract (foeniculum vulgare) at 70°c for 15 minutes heating treatment. research by handayani and sriherfyna (2016) and surono et al. (2016) also showed that bioactive compounds were commonly degraded at temperature more than 50°c, also the common temperature used in food processing starts around 63°c for 30 minutes. level of ph used in this research was determined based on the ph of food product (3.5-7.0) and the ability of pathogenic microbes to grow at ph 4.0-9.5 (surono et al. 2016). analysis about minimum i n h i b i t o r y c o n c e n t r a t i o n ( m i c ) , m i n i m u m bactericidal concentration (mbc), toxicity analysis using brine shrimp lethality test (bslt), and gas chromatography-mass spectrometry (gc-ms) were also conducted in this research. the results of this research hopefully can be used as source of information, especially regarding optimum conditions of star anise extract for application in food industry. materials and methods material. dry star anise (“sajp” brand), ethanol, acetone, alcohol, dimethyl sulfoxide (dmso), hexane, nacl 0.85%, nutrient agar (na), nutrient broth (nb), hcl 0.1 n, naoh 0.1 n, ph 4 and 7 buffer solution, and isolates of staphylococcus aureus (gram positive bacteria), escherichia coli (gram negative bacteria, and bacillus cereus (spore bacteria). sample preparation phase methods. sample preparation steps that were performed to obtain star anise powder, included size reduction (35 mesh), conducting proximate analysis, and determining optimum time of tested bacteria growth using bacterial growth curve. total colony used for well diffusion test 8 -1 in this research were 10 cfu ml . phase i methods. phase i research (fig 1) started with maceration process of star anise powder (1:10; 2025 °c) using ethanol and acetone for 3 days. extraction was executed by constant shaking at 150 rpm. filtration of the filtrate (using whatman no.1 and buchner vacuum) and evaporation (40 °c; 35 rpm; 1 hour) were carried out in order to produce antibacterial compound extract. extract was then diluted to 5 levels of concentration (10, 20, 30, 40, and 50% (w/v)). antimicrobial activity test was conducted using well diffusion method in order to determine selected extract which was the best extract concentration in this research. phase ii methods. in phase ii (fig 1), the stability test was performed by setting the extract into different temperature (60, 70, 80, and 90 °c) with different heating time (15 and 30 minutes), and ph (4, 5, 6, and 7); mic and mbc test; component analysis using gcms; and toxicity test (sangi 2012) were also performed toward selected extract from phase i research. analysis. proximate analysis in phase i research was used to determine the water content of dry star anise powder (aoac 2005). mic and mbc in phase ii research were used to analyze the minimum value needed to inhibit bacterial growth and kill 90% of tested bacteria (bloomfield 1991). in addition, to test the cytotoxicity of extract, we used bslt method (sangi 2012), whilst gc-ms test was used to analyze major antibacterial compound in extract. experimental design. experimental design used in phase i research was completely randomized design with one factor and five levels (10% [a ], 20% 1 [a ], 30% [a ], 40%, [a ]), and 50% [a ]) with three 2 3 4 5 repetition. experimental design used in phase ii research for stability against heating temperature and time was completely randomized design with two factors and three repetition. temperature factor used four levels which were: 60 (a ), 70 (a ), 80 (a ), and 1 2 3 90°c (a ); while heating time used two levels which 4 were: 15 minutes (b ), and 30 minutes (b ). stability 1 2 test against ph value was performed using completely randomized design with one factor and three repetition. levels of ph were: 4 (a ), 5 (a ), 6 (a ), and 1 2 3 7 (a ). 4 18 eveline et al. microbiol indones volume 14, 2020 microbiol indones 19 results phase i. the yield of 72 hours maceration extraction using ethanol was 15.19 ± 0.35% and 12.45±0.37% using acetone. ethanol is a polar solvent, so it is easy to attract most of the polar in star annise. the bacteria used. bacteria used in test were in log phase after 6 hours of incubation period. total 8 colony used for well diffusion in this research were 10 -1 cfu ml ; total colony of s. aureus, e. coli, and b. 8 -1 8 cereus used in test were 1.9x10 cfu ml , 1.8x10 -1 8 -1 cfu mll , and2.5x10 cfu ml . inhibition diameter based on extract concentration. table 1 showed that extract concentration significantly affecting inhibition diameter (p<0.05). the bigger extract concentration would produce larger inhibition diameter. table 1 also showed that ethanol was proven to be effective in inhibiting the growth of tested bacteria. at a concentration of 30%, ethanol was able to inhibit the bacteria with more than 6 mm inhibition diameter, it was minimal inhibition according to khalaphallah (2015); thus, the extract concentration used for the next phase was 30%. phase ii. phase ii research was done in order to determine the stability against heating temperature (60, 70, 80, and 90 °c) and heating time (15 and 30 minutes), and ph (4, 5, 6, and 7). in this phase, mic and mbc test, toxicity test, and component analysis using gc-ms were also done towards selected extract from phase i. e x t r a c t s t a b i l i t y b a s e d o n h e a t i n g temperature and time. statistical test results (table 2) showed that stability of antibacterial activity of extract on s. aureus and e. coli was affected by heating temperature and time (p<0.05), but there was no interaction between these two factors; when tested on b. cereus (table 3), stability of antibacterial activity of extract was affected by both factors (heating temperature and time) interactively (p<0.05). higher fig 1 flowchart of star anise antibacterial activity research. source: modification from ahmad and youssef (2015); badal and degoda (2017); nam et al. (2017); shete and chitanand (2014); wei et al. (2014) 20 eveline et al. microbiol indones heating temperature and time tend to produce smaller inhibition diameter. when heated for more than 60°c for 15 minutes heating period, the extract was not showing active antibacterial activity (inhibition diameter <6 mm). extract stability based on ph. changes in ph affected the inhibition diameter (p<0.05). the addition of hcl 0.1 m as the acid regulator and naoh 0.1 m as base regulator on extract gave significant difference compared to control extract with ph ~5.66 (the extract without any treatment). table 4 showed that ph escalation in extract towards neutral produced smaller inhibition diameter, and antibacterial compound in star anise extract was more stable at ph 4-5. mic and mbc. mic and mbc value were determined based on selected extract's (30% table 1 phase i analysis results (inhibition diameter based on extract concentration) concentration (%) solvent inhibition diameter (mm) s. aureus e. coli b. cereus 10 ethanol acetone 4.25 ± 0.15a 4.30 ± 0.18a 4.83 ± 0.19l 3.43 ± 0.16k 4.89 ± 0.16v 3.25 ± 0.05t 20 ethanol acetone 4.88 ± 0.21b 5.19 ± 0.21bc 5.88 ± 0.26mn 5.09 ± 0.24l 4.95 ± 0.20v 4.39 ± 0.16u 30 ethanol acetone 6.65 ± 0.04d 5.43 ± 0.19c 6.49 ± 0.28o 5.72 ± 0.28m 6.04 ± 0.08x 5.06 ± 0.14vw 40 ethanol acetone 7.35 ± 0.34e 6.42 ± 0.29d 7.07 ± 0.17p 6.18 ± 0.30no 6.15 ± 0.24x 5.40 ± 0.20w 50 ethanol acetone 7.91 ± 0.31f 7.14 ± 0.29e 8.04 ± 0.27q 6.49 ± 0.20o 6.89 ± 0.33z 6.51 ± 0.28y different notation showed that there was significant difference (p<0.05); not compared between bacteria inhibition diameter (mm) based on heating temperature inhibition diameter (mm) based on heating time (minutes) control 60°c 70°c 80°c 90°c control 15 30 s. aureus 6.36 ± 0.26b 7.03 ± 0.28a 5.66 ± 0.28c 4.61 ± 0.17d 3.97 ± 0.22e 6.36 ± 0.26h 5.46 ± 0.35hi 5.18 ± 0.36i e. coli 6.53 ± 0.18k 6.39 ± 0.33k 5.39 ± 0.26l 4.44 ± 0.32m 4.42± 0.23m 6.53 ± 0.18p 5.38 ± 0.25q 4.94 ± 0.25q different notation showed that there was significant difference (p<0.05); not compared between bacteria and heat treatment table 2 phase ii analysis results (extract stability based on heating temperature and time on staphylococcus aureus and escherichia coli) heating temperature (°c) heating time (minutes) inhibition diameter (mm) b. cereus control (no heat treatment) (~25°c, 0 minute) 6.68 ± 0.12a 60 15 6.43 ± 0.24a 30 6.00 ± 0.17b 70 15 5.43 ± 0.21c 30 5.29 ± 0.19c 80 15 4.11 ± 0.08de 30 4.30 ± 0.16d 90 15 4.22 ± 0.15de 30 3.95 ± 0.14e different notation showed that there was significant difference (p<0.05) table 3 phase ii analysis results (extract stability based on heating temperature and time on bacillus cereus) ph inhibition diameter (mm) s. aureus e. coli b. cereus control (ph ~5.66) 6.63 ± 0.20a 6.59 ± 0.23n 6.38 ±0.29a 4.0 6.24 ± 0.14b 6.83 ± 0.25m 6.54 ± 0.27a 5.0 6.22 ± 0.10b 6.67 ± 0.32l 6.40 ± 0.29a 6.0 6.10 ± 0.28b 5.86 ± 0.18k 5.59 ± 0.25b 7.0 3.42 ± 0.16a 2.44 ± 0.12k 2.13 ± 0.10c different notation showed that there was significant difference (p<0.05); not compared between bacteria table 4 phase ii analysis results (extract stability based on ph volume 14, 2020 microbiol indones 21 concentration) inhibition zone. mic value was the minimum concentration needed to inhibit 90% growth of tested bacteria, while mbc value was the minimum concentration needed to kill 90% of tested bacteria (zadrazilova et al. 2015). mic and mbc test results were 1.59% and 6.36% for s. aureus, 1.04% and 4.18% for e. coli, and 0.59% and 2.39% for b. cereus (table 1). toxicity. selected star anise extract was categorized as low toxic compound (lc = 212.09 50 ppm) in its function as antibacterial compound that contains as follows (in the order of the best antibacterial potential). gc-ms. star anise extract contained major antibacterial compounds in the form of terpenoids (anethole, β-caryophyllene, β-bisabolene, 2,6dimethyl-6-(4-methyl-3-pentenyl)-2-norpinene, βlinalool, p-allylanisole, trans-γ-bisabolene); and minor antibacterial compounds in the form of fatty acids (6-octadecenoic acid, hexadecanoic acid, stearic acid) and benzaldehyde (methoxy acetophenone, 4anisaldehyde, 3-propenylphenol). discussion star anise extract made using ethanol for 3 days extraction time at 30% concentration effectively exerted best inhibition toward three food spoiling bacteria (staphylococcus aureus, escherichia coli, and bacillus cereus). according to baldosano et al. (2015), most of the compounds in star anise tend to be polar, thus the amount extracted using ethanol solvent was greater than acetone solvent. more active compounds found in an extract would produce larger inhibition diameter (khasanah 2014). according to khalaphallah (2015), antimicrobial compound was categorized as active if the inhibition diameter is more than 6 mm. antibacterial activity test result of star anise extract using ethanol was proven to be effective in inhibiting the growth of tested bacteria. tannin and phenolic compounds were antimicrobial agent that tend to be polar, thus both were extracted more in ethanol extract and resulting greater inhibition zone than acetone extract (iloki-assanga et al. 2015; wijayanti, 2016; medini et al. 2014; chouksey et al. 2013). mic and mbc value against the three tested bacteria respectively were: 1.59% and 6.36% (s. aureus), 1.04% and 4.18% (e. coli), and 0.59% and 2.39% (b. cereus). the more susceptible the bacteria to anethole (mohammed 2009). different structure and outer cell membrane component caused different mic and mbc value in s. aureus (gram positive) and e. coli (gram negative), this was also affected by membrane sensitivity against specific antimicrobial compounds. gram negative bacteria have more complex cell wall which contain thin peptidoglycan (10-50%). antimicrobial compounds destroy outer cell membrane through porins (hydrophilic pathway) then separate phospholipids and lipopolysaccharides. gram positive bacteria don't have outer cell membrane, but it's composed with thicker peptidoglycan (90%), thus generally harder to be penetrated by antimicrobial compounds. star anise extract was unstable toward heating treatment and changes in ph value; but heating treatment at 60 °c for 15 minutes and ph 4-5 was able to exert closest result in inhibiting bacterial growth when compared to control extract. generally as the heating temperature and time escalated, the antibacterial compounds lost its stability (thermolabile) because chemical structure of the compounds was degraded during heating and the antimicrobial activity decreased (turek and stintzing 2013; sant'anna et al. 2012; and zhang et al. 2014). ardiansyah (2002) reported that in certain specific condition, antibacterial activity could also increase as the temperature increases. degradation of extract compounds formed new compounds which also have the potential to inhibit microorganism growth. oxidized anethole compound would form 4-methoxy benzaldehyde (4-anisaldehyde) and anisketone which act as antimicrobial compound (okamoto et al. 2002; fahlbusch et al. 2003; kosalec et al. 2005). escalation in ph also affected extract stability. the presence of fatty acids, such as hexadecanoic acid, 6octadecenoic acid, and stearic acid in extract made the extract more stable at ph 4-5. ph can affect the absorption of fatty acids in bacteria. as the ph increase, effectiveness of fatty acid absorption would decrease, thus the antibacterial activity also decreased (mcgaw et al. 2002). purbowati et al. (2016) and pan et al. (2014) added that phenolic compounds in the extract are damaged by increased ph and causing the antibacterial activity became less effective. in reverse, a decrease in + ph (an increase in h ions) causes bacterial cytoplasm became less stable and it needs more energy to restore the cell's internal ph to normal state. cell metabolism will be disturbed as more energy required to keep the normal state of cell's internal ph, then the bacteria cells will die over time (naufalin et al. 2006). the decrease in ph also increases the stability and effectiveness of phenolic compounds which are more hydrophobic in acidic conditions thereby facilitating the dissolution of 22 eveline et al. microbiol indones bacterial membrane fat (purbowati et al. 2016; pan et al. 2014). toxicity value (lc ) indicates the safety level of 50 extract to be applied in food products. this test was done as the first step in other complex toxicity test. based on the principle of brine shrimp lethality test (bslt), the more amount of compound needed to kill 50% of shrimp larvae, then the compound will be categorized as more non-toxic. juniarti et al. (2009) divides the toxicity level of lc into three categories which are: lc >1000 ppm 50 50 as non-toxic, 30<lc <1000 ppm as low toxic, and 50 lc <1000 ppm as toxic. test result showed that star 50 anise extract had lc of 212.09 ppm (low toxic). 50 nakamura (1996) reported that veranisatin a, b, and c were toxic compound which contributed in star anise toxicity test, but the concentration of veranisatin a, b, and c in dry star anise were low, respectively, 0.00016%, 0.00010%, and 0.000015% and it's categorized as safe to use in medicine and food products. fda already classified illicium verum hook. f. as generally recognized as safe (iofi 2017). dewi et al. (2012), soetan and oyewole (2009), and yunilla (2016) added that the natural antinutrient bioactive components formed by plant metabolism can also be toxic, such as glycosides, tannins, lignin, triterpenoids, oxalates, and amino acids. low toxicity is considered safe and harmless if consumed as herbal intake at the right dose (yunilla 2016). further in vivo research is needed as dose reference for the application in food products that are safe for humans (bpom 2014). the major antibacterial compound in star anise extract, including terpenoids (anethole, βcaryophyllene, β-bisabolene, 2,6-dimethyl-6-(4methyl-3-pentenyl)-2-norpinene, β-linalool, pallylanisole, trans-γ-bisabolene); and minor antibacterial compound, including fatty acids (6octadecenoic acid, hexadecanoic acid, stearic acid) and benzaldehyde (methoxy acetophenone, 4-anisaldehyde, 3-propenylphenol). all component were categorized as essential oil that could act as antibacterial agent (attokaran, 2017; aly et al. 2016; taniguchi et al. 2014; herman et al. 2016; andrade et al. 2015; okatomo et al. 2002; fahlbusch et al. 2003; chong et al. 2015; carneiro et al. 2017; dahham et al. 2015; mcwilliams, 2006; courtois et al. 2012; rai et al. 2011; marteau et al. 2016; karimi et al. 2015; uitterhaegen et al. 2016). okamoto et al. (2002), tisserand and young (2014), and kosalec et al. (2005) added that oxidized anethole will form anisketone which also acts as antimicrobial agent. in the recent study of alhajj et al. 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oddolam gaertn.). tangerang: universitas pelita harapan. zhang g, yuan c, sun y. 2018. effect of selective encapsulation of hydroxypropyl-β-cyclodextrin on components and antibacterial properties of star anise essential oil. molecules. 23(5): 1126. doi: 10.3390/molecules23051126. zhang j, han j, oyeleye a, liu m, liu x, zhang l. 2014. extraction methods of natural products from traditional chinese medicines. methods mol biol. 1263:177-185. doi: 10.1007/978-1-4939-2269-7_14. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 5. mi-ulkhaq.cdr available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.11.2.5issn 1978-3477, eissn 2087-8575 vol 11, no 2, june 2017, p 69-73 *corresponding author; phone: +62-21-7520482, email:faizal.ipb@gmail.com aeromonas hydrophila is a bacterium that causes motile aeromonads septicemia (mas), infecting fresh water fish, including african catfish. this disease causes a high mortality rate both on seed stage and adult stage, and leads to a great economic loss (omeje and chukwu 2012). the number of deaths caused by this disease reached 173 tons of carps in west java with a total loss of rp 126 million suffered by the farmers (in 2005). janda and abbott (2010) reported that the incidence of mas diseases caused the death of 820 tons of carp with total losses reaching 37.5 million dollars. bacteria from the genus aeromonas, are opportunistic bacteria that can be isolated from waters, including groundwater, surface water, drinking water, and wastewater. moreover, these bacteria can also be found in food, cheese, and milk (sharma et al. 2009). janda and abbott (2010) have identified twenty-one species in the genus aeromonas, but only several are known pathogen in fish, a. salmonicida, a. hydrophila, a. formicans, and a. liquefaciens (abdel-raouf and ibraheem 2008). a prevention method is commonly applied to prevent bacterial infections in fish by using antibiotics. however, a number of alternatives have been sought to replace the use of antibiotics as antimicrobial agents, such as vaccination, immunostimulants, and probiotics (guz and kozinka 2004). because the improper use of antibiotics can cause serious problems, for example increased resistance of bacterial pathogens (balcazar et al. 2006). in previous reports, costa and cyrino (2006) aeromonas hydrophila is a type of bacterium causing motile aeromonads septicemia (mas) disease, which infects fresh water fish, including african catfish, and leads to death as well as huge losses to farmers. this research aims to determine the resistance status of various antibiotic classes in a. hydrophila bacteria isolated from african catfish. bacterial isolates of a. hydrophila were taken from the liver and kidneys of infected african catfishes obtained from parung, bogor. characterization of the bacteria was carried out based on colony morphology and biochemical properties. meanwhile, bacterial resistance test was conducted using antibiotic disks with kirby-bauer method. based on colony morphology and biochemical properties, the characterization results indicated that the bacterial isolates tested were a. hydrophila. further examination of the antibiotic resistance test showed that the bacteria were resistant to penicillin antibiotics and macrolides. future researches are expected to use molecular identification for a.hydrophila bacteria mutant to known the dna base. key word : aeromonas hydrophila, antibiotics, clarias gariepinus, resistance bakteri aeromonas hydrophila merupakan bakteri penyebab penyakit motile aeromonads septicemia (mas) yang menginfeksi ikan air tawar, termasuk ikan lele dumbo dan mengakibatkan kematian dan kerugian yang besar. penelitian ini bertujuan untuk menentukan status resistensi dari berbagai golongan antibiotik pada bakteri a. hydrophila yang diisolasi dari ikan lele dumbo. isolat bakteri a. hydrophila diambil dari organ hati dan ginjal ikan lele dumbo sakit yang diperoleh dari wilayah parung, bogor. karakterisasi bakteri dilakukan berdasarkan morfologi koloni dan sifat biokimia serta uji resistensi bakteri menggunakan disk antibiotik dengan metode kirby-bauer. hasil karakterisasi bakteri menunjukkan bahwa isolat bakteri yang diuji adalah a. hydrophila berdasarkan morfologi koloni dan sifat biokimia. pemeriksaan lebih lanjut mengenai uji resistensi antibiotik menunjukkan bahwa bakteri a. hydrophila resisten terhadap antibiotik golongan penisilin beta-laktam dan makrolida. penelitian selanjutnya diharapkan dapat menguji secara molekuler untuk mengetahui susunan basa dna dari bakteri a. hydrophila resisten antibiotik. kata kunci : aeromonas hydrophila, antibiotik, ikan lele dumbo, resisten short communication resistance test on aeromonas hydrophila isolated from african catfish (clarias gariepinus) against some antibiotics groups 1 2 mohammad faizal ulkhaq* and mariana lusiastuti 1 faculty of fisheries and marine, universitas airlangga, jalan wijayakusuma 113, banyuwangi 68425, indonesia; 2 centre of research and development of freshwater aquaculture, research instalation and development centre for fish disease control, jalan perikanan 13, depok 16436, indonesia demonstrated that a. hydrophila isolated from piaractus mesopotamicus and oreochromis niloticus is resistant to antibiotics derived from amoxicillin, ampicillin, lincomycin, novobiocin, oxacillin, penicillin, and trimethoprim + sulfametoxasol. similarly, dias et al. (2012) suggests that a. hydrophila bacteria isolated from the ornamental fish are resistant to several types of antibiotics, namely tetracycline, ampicillin, erythromycin, ticarcillin, and carbenicillin. results of another study also mentioned that the resistance of a. hydrophila occurs in isolates derived from carp against ampicillin and penicillin antibiotics (guz and kozinka 2004). unfortunately, the resistance of a. hydrophila isolated from african catfish against different classes of antibiotics has not been studied. therefore, this research aims to determine the status of various classes of antibiotic resistance in a. hydrophila bacteria isolated from african catfish. isolation and characterization of bacterial isolate. a. hydrophila was isolated from the liver and kidneys of african catfish obtained from the diseased area in parung, bogor. the bacterial isolates were cultured on trypticase soy broth (tsb) media at 25 ml in a water bath shaker at 160 rpm for 24 h at 29 °c. further characterization of a. hydrophila isolates was conducted based on morphological and biochemical factors in accordance with iso (7309: 2009), including cell shape, gram stain, motility test (using sim medium), oxidation, catalation, and oxidative/fermentative test (of) using of medium. moreover, bacterial culture isolates were also conducted on rimmlershoots (rs) media, a selective medium for a. hydrophila. resistance test. resistance test on a. hydrophila was performed by kirby-bauer method using antibiotic discs commercially available. the type of antibiotic used consisted of erythromycin 15 µg (macrolides group), clindamycin 2 µg (lincosamides group), tetracycline 30 µg (tetracycline class), chloramphenicol 30 µg and 50 µg (fenikol group), enrofloxacin 5 µg (fluoroquinolones group), kanamycin 30 µg (aminoglycosides group), penicillin 10 µg and ampicillin 10 µg (beta-lactam penicillin group), as well as rifampicin 5 µg (ansamicyn group). a. hydrophila isolates were uniformly distributed in trypticase soy agar (tsa) media in a petri dish, and then placed in the midst of an antibiotic disc plate. next, they were incubated for 24 h at 29 °c in an incubator. finally, the level of bacterial resistance to antibiotics was determined by measuring the diameter of clear zone formed and categorized by nccls (2002). characterization of bacteria. characterization of the bacterial isolate based on its colony morphology and biochemical properties showed that the isolates were a. hydrophila. (table 1). this is supported by the observation that the bacterial colony in rs media was yellow with no black spots in the middle. microscopic observation showed that the bacteria was motile and the results of biochemical tests, furthermore, showed that the bacteria produced catalase and oxidase enzymes that break down the sugar anaerobically and aerobically . antibiotic resistance test. the results of antibiotic resistance test against a. hydrophila bacteria (table 2) showed that the bacterial isolates were table 1. results of characterization of a. hydrophila isolates based on colony morphology and biochemical properties parameter results a.hydrophila tested literature (sni 7309:2009) shape of colonies small spherical not described elevation convex not described edges smooth not described color beige not described shape of cells short stem cell not described rs media + not described motile + + catalase + + oxidase + + gram negative (-) negative (-) of positive o/f positive o/f 70 ulkhaq et al. microbiol indones resistant to clindamycin (a macrolide), as well as penicillin and ampicillin (beta-lactam). colony of a. hydrophila was yellow with no black spot in the middle. shotts and rimler (1973) stated that the colonies of a. hydrophila in rs media were round, in convex elevation, flat edges, and yellow. similarly, the results of a research conducted by chirila et al. (2008) stated that the colonies of a. hydrophila were white on blood agar, and bluish green on meitert istrati media. biochemical tests of this bacteria showed that the isolate used in this study is a. hydrophila. this is consistent with the results of a research conducted by jayavignesh et al. (2011) which stated that the assay results of biochemical characteristics of a. hydrophila are indole (+), mr (-), vp (+), citrate (+), catalase (+), urea (-), oxidase (+), gelatin hydrolysis (+), carbohydrate utilization of lactose (+), glucose (+), and trehalose (+). tantu et al. (2013) adds that the characteristics of aeromonas sp. are gram negative, oxidase (+), catalase (+), h s (+), motile (+), of (+), 2 and citrate (-). antibiotic resistance is one of bacterial properties indicating the nature of immune or resistance to certain antibiotics (byarugaba 2010). antibiotic resistance can occur via two processes, namely resistance that occurs due to a spontaneous mutation in the chromosome and resistance that occurs due to plasmid displacement. resistance on chromosomes is more stable and cannot be moved horizontally into other bacteria, while resistance on plasmids is not stable/easily lost and easily transferred to other bacteria that do not have the gene (cruz et al. 2012). the mechanism of bacterial resistance to antibiotics depends on the types of bacteria. for gram negative bacteria, there are several mechanisms of antibiotic resistance used as a means of resistance of bacteria to antibiotics. these mechanisms include several ways. first, resistance occurs by modification in the bacterial cell wall, thus preventing the amount of antibiotics across the cell membrane. second, resistance occurs by increasing betalactamase production, thereby damaging the structure of beta-lactam. third, resistance occurs by increasing activity of the efflux pump,so that the bacteria will pump out antibiotics before they can cause any effects. fourth, resistance occurs by modifying enzymes, so antibiotics cannot interact with receptors. fifth, resistance occurs by metabolic bypass mechanism, so antibiotics cannot across the cell membran the last one is through ribosomal modification process, thus preventing the merging of antibiotics that inhibit bacterial protein synthesis (peleg and hooper 2010). the resistant mechanism of a. hydrophila bacteria to betalactamase group (ampicillin and penicillin) occurs because a. hydrophila bacteria produce betalactamase enzyme, thus damaging the structure of beta-lactam. the structure of beta-lactam in penicillin group is a structure that inhibits bacterial cell wall synthesis (lakshmi et al. 2014). the results are consistent with the research results by odeyemi and ahmad (2015) stating that 53 aeromonas isolated from aquatic environments in malaysia have 100% resistance to ampicillin and 92.5% resistance to no. antibiotics zone diameter (mm) (nccls 2002) results of the research r i s a. hydrophila (mm) category 1. erythromycin 15 µg ≥ 23 14-22 ≤ 13 5.4 s 2. clindamycin 2 µg ≥ 21 15-20 ≤ 14 23 r 3. tetracycline 30 µg ≥ 21 15-18 ≤ 14 9.5 s 4. chloramphenicol 30 µg ≥ 18 13-17 ≤ 12 11.4 s 5. chloramphenicol 50 µg ≥ 18 13-17 ≤ 12 10.4 s 6. enrofloxacin 5 µg ≥ 23 17-22 ≤ 16 14.1 s 7. kanamycin 30 µg ≥ 18 14-17 ≤ 13 5.4 s 8. penicillin 10 µg ≥ 15 ≤ 14 16.2 r 9. ampicillin 10 µg ≥ 17 14-16 ≤ 13 18.3 r 10. rifampicin 5 µg ≥ 20 17-19 ≤ 16 13.7 s table 2. results of antibiotic resistance test against a. hydrophila bacteria volume 11, 2017 microbiol indones 71 10.1016/j.vetmic.2006.01.009. byarugaba d. 2010. 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kozinka a. 2004. antibiotic susceptibility of aeromonas hydrophila and a. sobria isolated from farmed carp (cyprinus carpio l.). bull vet institute pulawy 48: 391-395. igbinosa ih, chigor vn, igbinosa eo, obi lc, okoh ai. 2013. antibiogram, adhesive characteristics, and incidence of class i integron in aeromonas spesies isolated from two south african river. biomed res int. 8p. janda jm, abbott sl. 2010. the genus aeromonas: taxonomy, pathogenicity, and infection. clin microbiol rev. 23(1):35-73. doi:10.1128/cmr.00039-09. jayavignesh v, kannan ks, bhat ad. 2011. biochemical characterization and cytotoxicity of the a. hydrophila isolated from catfish. archives app sci res. 3(3):8593. kaskhedikar m, chhabra d. 2009. multiple drug resistance of aeromonas hydrophila isolates from chicken samples c o l l e c t e d f r o m m h o w a n d i n d o r e c i t y o f madhyapradesh. vet world. 2(1):31-32. lakshmi r, nusrin ks, ann gs, sreelakshmi ks. 2014. role of beta lactamases in antibiotic resistance: a review. int res j pharm. 5(2):37-40. doi:10.7897/2230 -8407.050207. leclercq r. 2002. mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. antimicrob. resistance 34(4): 482-492. doi:10.1086/324626. odeyemi oa, ahmad a. 2015. antibiotic resistance profiling and phenotyping of aeromonas species isolated from aquatic sources. saudi j bio sci. 24(1): 65-70. doi: 10.1016/j.sjbs.2015.09.016. omeje vo, chukwu cc. 2016. a relative prevalence of oreochromis niloticus, clarias gariepinus, and penicillin. in addition to the fish, 100% isolates of a. hydrophila isolated from chickens in india also develop resistance to ampicillin (kaskhedikar and chhabra 2009). the resistance of a. hydrophila against lincosamide class of antibiotics (clindamycin) occurs since a.hydrophila can modify receptors of antibiotics, so antibiotics cannot bind to the receptors (ribosomes). leclercq (2002) suggests that bacterial resistance to the lincosamides occurs through three ways, namely modifying the receptors on the ribosome, preventing an increase in the performance of antibiotics after binding to the receptors, and inactivating the antibiotics that move into the bacterial cell. this is supported by the research conducted by stratev et al. (2013) stating that 100% of bacterial isolates derived from a. hydrophila, b-hemolytic isolated from fish, rainbow trout (oncorhynchus mykiss), has resistance to amikacin, amoxicillin/clavulanic acid, ampicillin/sulbactam, cefazolin, cefotiam, clindamycin, erythromycin, imipenem, linezolid, meropenem, teicoplanin, and vancomycin. similar results were reported by igbinosa et al. (2013), i.e. 82.1% of aeromonas isolates isolated from the river kat and tyume in africa experienced clindamycin resistance. it can be concluded that a. hydrophila bacteria isolated from sick african catfish in bogor can develop resistance against two classes of antibiotics (beta-lactam penicillin and lincosamide). references [nccls] national committee for clinical laboratory standards. 2002. performance standards for antimicrobial disk dan dilution susceptibility tests for bacteria isolated from animals; approved standardsecond edition. nccls document m31-a2 (isbn 156238-461-9). nccls, 940 west valley road, suite 1400, wayne, pennsylvania 19087-1898, usa. [sni] standar nasional indonesia 7303:2009. metode identifikasi bakteri aeromonas hydrophila secara biokimia. jakarta. abdel-raouf n, ibrahem ibm. 2008. antibiotic activity of two anabaena spesies against four fish pathogenic aeromonas spesies. afr j biotechnol. 7(15): 26442648. issn: 1884-5315 angka sl. 2005. kajian penyakit motile aeromonas septicemia (mas) pada ikan lele dumbo (clarias sp) : patologi, pencegahan dan pengobatannya dengan fitofarmaka [study of motile aeromonas speticemia (mas) on african catfish]. [disertasi]. bogor (id): institut pertanian bogor. balcazar jl, de blas i, ruiz zarzuela i,cunningham d, vendrell d, muzquiz jl. 2006. the role of probiotics in aquaculture. vet microbiol. 114(3-4):173-186. doi: 72 ulkhaq et al. microbiol indones stratev d, vashin i, daskalov h. 2013. antimicrobial resistance of b-haemolytic aeromonas hydrophilas strains isolated from rainbow trouts (oncorhynchus mykiss). bulgarian j vet med. 16(4):289-296. tantu w, tumbol ra, longdong snj. 2013. deteksi keberadaan bakteri aeromonas sp. pada ikan nila yang dibudidayakan di karamba jaring apung danau tondano [detection of aeromonas sp. on tilapia fish cultivated in cage culture in tondano lake]. budidaya perairan 1(3):74-80. haterotis niloticus to aeromonas hydrophila in an integrated fish farm. nigerian vet j. 33(2):492-498. peleg ay, hooper dc. 2010. hospital-acquired infections due to gram-negative bacteria. the new england j med. 362:1804-1813. doi:10.1056/nejmra0904124. sharma i, kumar a, pramanik ak. 2009. review of technique on isolation and identification of aeromonas from food of animal and fish origin. assam university j sci technol: biologycal sciences. 4(1):73-85. shotts ebj, rimler r. 1973. medium for the isolation of a. hydrophila. j appl microb. 26(4):550-553. volume 11, 2017 microbiol indones 73 1: 69 2: 70 3: 71 4: 72 5: 73 3 410 shanti ratnakomala.cdr screening of actinomycetes producing an atpase inhibitor of rna helicase from soil and leaf litter samples japanese encephalitis virus shanti ratnakomala , roni ridwan , puspita lisdiyanti , abinawanto , andi utama 1 1 1 2 1 * and 1 2 research center for biotechnology lipi, jalan raya bogor km 46, cibinong 16911, indonesia; department of biology, faculty of mathematics and natural sciences, universitas indonesia, ui campus, depok 16242, indonesia japanese encephalitis virus actinoplanes philippinensis streptomyces chartreusis japanese encephalitis virus, japanese ensefalitis virus actinoplanes philippinensis streptomyces chartreusis japanese encephalitis virus actinomycetes are commercially important microorganisms for the production of antibiotics, enzymes, inhibitors of enzymes, and other bioactive secondary metabolites. some 853 isolates of actinomycetes were isolated from soil and leaf litter samples in kupang ntt and enrekang, south sulawesi. those isolates were then tested for inhibition of atpase activity of rna helicase from (jev), in order to identify a drug candidate for the treatment of jev infection. results revealed that 14 isolates have relatively high inhibition-activity on jev atpase activity of the jev-rnahelicase, which range from approximately 40.0-50.0% inhibition. the highest inhibition-activity was identified in 5-849 with 49. 9% of inhibition-activity and 5-095 with 49.2% of inhibition-activity. key words: actinomycetes, jev, rna helicase, inhibitor aktinomiset merupakan mikroorganisme yang penting secara komersial untuk produksi antibiotik, enzim, enzim inhibitor, dan metabolit sekunder bioaktif. sejumlah 853 isolat aktinomiset telah diisolasi dari sampel tanah dan serasah di kupang (nusa tenggara timur) dan enrekang (sulawesi selatan). isolat diuji kemampuannya untuk menghambat aktivitas atp-ase dari rna helikase (jev), dalam rangka mencari kandidat obat bagi pengobatan infeksi jev. hasil penelitian menunjukkan ada 14 isolat yang memiliki aktivitas inhibisi 40-50% terhadap aktivitas atpase dari rna helikase-jev, di antaranya ialah 5-849 dan 5-095. kata kunci: aktinomiset, virus, , jev, rna helikase, inhibitor japanese encephalitis virus c u l e x t r i t a e n i o r y n c h u s flavivirus flaviviridae (jev) is one of the most prevalent causative agents of viral encephalitis with high morbidity and mortality. approximately 50 000 human cases occur annually in asia. in nature, jev is transmitted between vertebrates by the mosquito , a n d c a u s e s i n f e c t approximately 10% of the susceptible population in southeast asian countries each year. in indonesia, particularly, jev is endemic in kalimantan, bali, nusa tenggara, sulawesi, maluku, papua and lombok (spicer 1997). this virus is recognized by the world health organization as a major threat to public health because of the high incidence of clinical infections, and approximately thirty percent of which are fatal and half result in neuropsychiatric sequelae. jev belongs to genus in the family whose members include several pathogens of humans and animals. although vaccines have been developed since 1960, unfortunately no effective drug is clinically available so far. several efforts have been performed to find a drug as a candidate for treatment of jev infection, including finding inhibitors of enzymes which are essential for jev replication such as , , protease, rna helicase and rna polymerase (borowski . 2002). some studies recently discovered drug candidates for , particularly (hcv) and jev, through rna helicase inhibitor screening. hatsu (2002) noted that secondary metabolites from broth cultures of sp. could be act as inhibitor to rna helicase of jev in general, the drug candidates were chemical substances such as ribavirin-5-triphosphate (borowski . 2001) and sch16 a derivatives o thiosemicarbazone (mibt) (sebastian 2008) which were able to completely inhibit jev and (wnv) replication, however the studies on the inhibition by natural products is still limited. actinomycetes are commonly found in natural substrates such as soils and litters, and play a significant role in the degradation of naturally organic substances (williams . 1984). therefore, actinomycetes have the ability to produce large amounts of secondary metabolites such as antibiotics, enzymes, enzyme inhibitors, and other bioactive compounds. about 70% of presently commercial antibiotics were found in secondary metabolites produced from actinomycetes (miyadoh 1993). et al flavivirus hepatitis c virus et al streptomyces et al , et al in vitro west nile virus et al . . . f n-methylisatin-β*corresponding author, phone: +62-21-8754587, fax. +62-21-8754588, e-mail: shanti_ratna01@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 1, march 2011, p 15-20 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.1.3 furthermore, indonesian actinomycetes have not been explored yet for screening of useful bioactive compounds such as inhibitors of jev replication. therefore, a study to find a novel strain with a novel secondary metabolite useful for pharmaceutical and industrial applications is important. in this study, we isolated actinomycetes from soil and leaf litter samples and screened the actinomycetes for inhibitor of atpase activity of jev rna helicase. the 853 isolated actinomycetes used for testing for inhibition-of atpase activity of jev rna helicase were obtained from soil and leaf litter samples in kupang, nusa tenggara timur, and enrekang south sulawesi, indonesia under collaborative research between indonesian institute of sciences (lipi), indonesia and national institute of technology & evaluation (nite), japan in 2005. the project have been done from 2004-2009. (widyastuti and ando 2009). in the project in 2005 sds-yeast extract (sds-ye) method (hayakawa & nonomura 1987), rehydration-centrifugation (rc) method (hayakawa 2000), and oil separation system (oss) method were used in this study as referenced in widyastuti and ando (2009). all the isolates have been identified at the genus level by sequencing of their 16s rdna at the project in 2005 as stated in widyastuti and ando (2009). in this study, the compiling 16s rdna data of the isolates based on their isolation sources and isolation methods were done. yeast extract-starch agar (ys agar) (nonomura and ohara 1969) was used as growth medium and contained 2 g yeast extract, 10 g soluble starch, 15 g agar added to 1 l distilled water and adjust ph to 7.3. international project 2 (isp 2) medium was used as production medium for screening the jev atpase inhibitor from actinomycetes, and contained 4 g yeast extract, 10 g malt extract, 4 g dextrose, 20 g agar added to 1 l distilled water and adjust ph to 7.3. the expression plasmids containing jev ns3 genes were independently transformed into bl21 (de3) plyss cells (stratagene, la jolla, ca, usa). after the iptg induction at 37 c for 3 h, the cells were harvested by centrifugation at 8000 rpm. the harvested cells were resuspended in buffer b (10 mm tris hcl buffer (ph 8.5), 100 mm nacl, 0.25% v/v tween 20), and disrupted by sonication for 5 min on ice. the soluble fraction of the cell lysate was mixed with talon metal affinity resin (clontech, palo alto, materials and methods microorganisms. , growth medium and production medium. expression and purification of jev ns3 proteins et al. streptomyces e. coli . o ca, usa). after gentle mixing for 1 h at 4 c, the resin was collected by a brief centrifugation and washed with buffer b. resin-bound protein was eluted with 2 volumes of buffer b containing 400 mm imidazole. eluted protein fractions were dialyzed against dialysis buffer (10 mm tris hcl (ph 8.5), 100 mm nacl, 10% v/v glycerol) at 4 c. purified jev ns3 proteins from the transformant were then used as substrate helicase enzyme foratpase assay (utama 2000a) the amount of phosphate moiety released from atp was measured. a 50 µl per well of reaction mixture containing 10 mm mops buffer (ph 6.5), 2 mm atp, 1 mm mgcl , purified ns3 protein (0.8 pmol) and 5 µl per well of culture supernatant as inhibitor substances was incubated in a 96 well microtiter plate at room temperature for 30 min. the reaction was stopped by adding 100 µl per well of dye solution (water:0.081% malachite green:5.7% ammonium molybdate in 6 n hcl:2.3% polyvinyl alcohol = 2:2:1:1, v/v). after the addition of 25 µl per well of 30% sodium citrate, the absorbance at 620 nm with a reference wavelength at 492 nm was measured. percentage of inhibition (%) was measured using the equation (a-i) /a x 100, which a was absorbance without added inhibitor substances, and “i” was absorbance with added inhibitor substances (hatsu . 2002). in this assay the amount of free phosphate moiety released from atp was measured via absorbance of rna helicase enzyme. we have measured the change of absorbance with and without added inhibitor substances. if there was no inhibitory effect from the supernatant of actinomycetes, the jev rna helicase enzyme would continue to hydrolyze atp to adp and inorganic phosphate released from this reaction will be bounded with malachite green and ammonium molibdate made a coloured complex compound of phosphomolybdate-malachite green. the absorbance of this coloured complex compound were detected using spectrophotometer. of the 853 isolates of actinomycetes used in this study, 529 isolates were defined as a group of (family ), and 324 isolates as a group of nonor so called rare actinomycetes (other family in the order ) further, from the group of non, 381 have zoospore (motile actinomycetes) and 153 do not have zoospore (nonmotile actinomycetes). based on results in origin, 716 were isolated from soil and 137 were isolated from leaf litter samples. some 488 isolates from the soil sources o o e. coli et al . et al streptomyces streptomycetaceae streptomyces actinomycetales streptomyces colorimetric atpase assay. results actinomycetes isolates. . . 2 16 ratnakomala et al. microbiol indones were identified as belonging to the family and 228 isolates were identified as group of non. a total of 137 isolates obtained from leaf litter samples were divided into 41 isolates of the family and 96 isolates of the group of nongroup (table 1). this result revealed that the genus was the dominant actinomycetes in the soils used. based on results of the isolation methods, 468 were isolated by sds-ye method resulted in as the major genus, 286 were isolated by rc method resulted as the major genus, and 99 were isolated by oil separation method which resulted as the major genus. twenty eight genera were found in kupang, nusa tenggara timur and 40 genera were found in enrekang. furthermore, 33 genera were found in soils samples and 12 genera were found in litter samples. the genera of were only found in and (table 2) the genus , , and were abundant in soil samples and the genus , , and were abundant in litter samples. all actinomycetes isolates obtained from soil and leaf litter samples in kupang and enrekang were then screened for their ability to inhibit atpase activity of jev rna helicase. of the 853 actinomycete isolates, 14 isolates produced metabolites having inhibitory activity as high as 40-50%. thirty-five isolates demonstrated inhibition to 30-40% and 37 isolates showed 20-30% inhibitory activity ( fig 1). of the 14 isolates actinomycetes which were found to have the highest potency as antiviral compound were presented in table 3, the bioactive compounds produced from secondary metabolites were released in broth cultures. the highest inhibition-activity was identified in 5-849 with streptomycetaceae streptomyces streptomycetaceae streptomyces streptomyces streptomyces actinoplanes streptomyces dietzia, kocuria, planomonospora, and sphaerosporangium kupang, and the genera of actinokineospora, cellulomonas, dermatophilus, geodermatophilus, agromyces, virgosporangium, planotetraspora were only found in enrekang . streptomyces actinoplanes nonomuraea actinoplanes streptomyces kinesoporia actinoplanes philippinensis screening of actinomycetes which have inhibition-activity on atpase of jev rna helicase. volume 5, 2011 microbiol indones 17 sampling source sampling site σ isolate streptomyces non-streptomyces motile non-motile litter kupang 59 31 23 5 enrekang 78 10 54 14 soil kupang 318 210 52 56 enrekang 398 278 42 78 table 1 actinomycetes isolated from leaf litter and soil samples compiling data from final report of collaborative research project in 2005 between lipi-nite 1 (widyastuti and ando 2009). table 2 genera of actinomycetes found in and samplessoil leaf litter soil actinokineospora 2 2 actinoplanes 47 25 72 catenuloplanes 3 7 10 couchioplanes 1 2 3 dactylosporangium 1 2 3 planotetraspora 4 4 actinomadura 1 4 5 agromyces 1 1 cellulomonas 1 1 cryptosporangium 7 11 18 dermatophilus 1 1 dietzia 1 1 geodermatophilus 1 1 isoptericola 1 2 3 kocuria 1 1 kribbella 3 13 16 microbispora 1 1 micromonospora 7 5 12 mycobacterium 7 6 13 nocardia 2 10 12 nocardioides 1 4 5 nonomuraea 16 11 27 planomonospora 4 4 planobispora 1 1 promicromonospora 2 2 4 pseudonocardia 3 1 4 saccharothrix 1 1 2 sphaerosporangium 1 1 streptacidiphilus 2 2 streptomyces 210 272 482 streptosporangium 1 1 2 virgosporangium 1 1 verrucosispora 1 1 leaf litter actinoplanes 10 45 55 catenuloplanes 1 1 couchioplanes 2 2 dactylosporangium 1 1 kineosporia 12 6 18 amycolatopsis 1 1 cryptosporangium 11 11 krasilnikovia 1 1 2 micromonospora 2 1 3 nocardia 1 1 promicromonospora 1 1 streptomyces 31 10 41 total 853 data source: compiling data from final report of collaborative research project in 2005 between lipi-nite 1 (widyastuti and ando 2009). 49. 9% of inhibition-activity and 5-095 with 49.2% of inhibition-activity. actinomycetes are prokaryotic microorganisms belonging to a group of gram positive bacteria that have high g+c contents, are saprophytic, produce large amount of sporangia and stable mycelium. actinomycetes are widely distributed in nature and in man-made environments, and play an important role in the degradation of organic matter. they are also well known as a rich source of antibiotics and bioactive streptomyces chartreusis. discussion 18 ratnakomala et al. microbiol indones molecules, and are of considerable importance in industry (seong 2001). many are well known for their economic importance as producers of biologically active substances, such as antibiotics, vitamins and enzymes (basilio 2003). this report revealed as stated also in widyastuti and ando (2009), that species diversity in the soil samples (33 genera) are higher when compared with the litter samples (12 genera), this is in line with the review of hayakawa (2008) that states soil is the natural habitat of actinomycetes. the genus was widely distributed in kupang, nusa tenggara timur, and enrekang both in sampling sources. as noted by hayakawa (2000), rare actinomycetes were divided in two groups according to the type of zoospora produced, non-motile actinomycetes and motile actinomycetes. non-motile actinomycetes formed non-flagellated zoospores, such as , etc. and motile actinomycetes formed flagellated zoospores. examples of motile zoospora are and actinomycetes isolated from et al et al streptomyces et al. nocardia micromonospora, actinokineospora, actinoplanes, catenuloplanes, couchioplanes, dactilosporangium, planotetraspora. . , . table 3 actinomycetes have highest inhibitory activity to atpase activity of jev rna helicase enzyme isolated from soils and leaf litter samples in kupang and enrekang isolate inhibition (%) name of isolate 5-087 45.1 streptomyces floridae 5-095 49.2 s. chartreusis 5-096 43.5 s. marokkonensis 5-119 47.0 streptomyces smarlab 330204 5-124 46.0 s. violens 5-217 40.9 s. albus 5-218 44.3 s. ginsengisoli 5-224 41.0 s. albus 5-264 43.6 s. cyanoalbus 5-304 44.2 s. durhamensis 5-320 41.4 s. pulveraceus 5-800 42.3 s. badius 5-849 49.9 actinoplanes philippinensis 5-1036 40.4 kribbella flavida inhibition (%) number of isolates 0 594 0.01-10 96 10.01-20 77 20.01-30 37 30.01-40 35 40.01-50 14 table 4 actinomycetes having inhibitory activity to atpase activity of jev rna helicase enzyme isolated from soils and leaf litter samples at kupang and enrekang enrekang soils and leaf litter samples were abundant with rare actinomycetes from the genus . based on the isolation method as stated in widyastuti and ando (2009) sds ye methods were useful for isolation of the genus on the other hand rc methods gave an abundance of the genus further, in this research it can be seen that the actinomycetes originating from litter sample was dominated by motile actinomycetes (the genus ), while from the soil was dominated by non-motile actinomycetes (the genus ) (table 1). the rc method is a simple enrichment method incorporating differential centrifugation for the isolation of motile actinomycetes (hayakawa 2000). the phosphate buffer-soil extract solution liberates motile zoospores and centrifugation eliminates and other nonmotile actinomycetes from the supernatant, thereby facilitating selective growth of motile actinomycetes on the isolation plates subsequent to inoculation (hayakawa 2008). the oss method is a selective isolation methods expected to favour actinomycetes from the genus , but unfortunately in this study this genus is not obtained. using oss methods the genus this may be because the isolates were putatively closely related with the genus on the basis of their mycolic acid profiles. our analysis of screening for inhibitory activities to jev rna helicase enzymes showed a high proportion of inhibitory activity was produced by species. seeking an inhibitor to jev rna helicase was performed through rnastimulated-atpase-activity (atp hydrolysis). inhibition of atp hydrolysis, was derived from an indirect inhibition of rna helicase-mediated hydrolytic processing so that the energy performed from hydrolysis atp was not available to unwind dsrna jev. colorimetric atpase assay was chosen for detection of hydrolyzed atp, because that was the easy and safe methods, without using radioactive compound which relatively unstable and caused radiation contamination (utama 2000b; boguszewskachachulska 2004). robarts 2004 and welbourn 2005 used colorimetric atpase assay to detect atp hydrolysis from rna helicase jev. screening inhibitors of jev rna helicase from actinomycetes showed a varied percentage of inhi bition among the isolates, between 0% and 49.9% (fig 1). the highest percentages of inhibition were 49.9% performed by isolate 5-849 identified as and 49.2% inhibition-activity obtained actinoplanes streptomyces actinoplanes actinoplanes streptomyces et al streptomyces rhodococcus mycobacterium was found mycobacterium rhodococcus streptomyces et al et al et al et al actinoplanes philippinensis , . , . . , . . . . . volume 5, 2011 microbiol indones 19 from isolate 5-095 identified as (table 3). isolated from leaf litter samples at enrekang was a rare actinomycetes. the role of rare actinomycetes as bioactive molecule sources became apparent as these organisms provided about 25% of the antibiotics of actinomycete origin reported during 1975 to 1980. rare actinomycetes have usually been regarded as strains of actinomycetes whose isolation frequency by conventional methods is much lower than that of strains. hatsu . (2002) noted that secondary metabolites from broth cultures of could act as inhibitor to rna helicase enzyme of jev. helicase enzyme was a great potential target for the discovery new antiviral compounds because this enzyme is essential enzyme for replication of the virus. helicase enzyme has a function as not only for helicase activity, but also rna binding activity and rna-stimulatedatpase activity, both of these activities influence helicase activity. this function is interrupted or at least depressed by the existence of an atpase inhibitor. therefore this make the enzyme an attractive target for development new antiviral drugs, because an inhibitor of helicase enzyme could be due to the discovery of an inhibitor of rna binding activity or an atpase inhibitor. in general, the active isolates showed an inhibition of atpase activity of jev rna helicase enzymes were mainly produced from genus , although some of the non isolates also had inhibitory activity. whether the activities being detected in these cases were due to single inhibitor acting on multiple microbial species or were mixtures of compounds with different specificities is unclear without chemical fractionation of the active extracts. a diverse percentage of inhibition existed among the isolates, suggesting a varied ability of microorganisms to produce inhibitor compounds. are fast growing actinomycetes, will reach their stationary phase earlier at which stage the secondary metabolites are produced. differences in incubation time to reach the stationary phase between and rare actinomycetes could act as a factor causing diverse inhibitory activity. of some of the antiviral candidates discovered, the drug candidates were proven and , such as bay 57-1293 a compound which suppresses (hsv) infection in the monkey (betz . 2000). some studies recently noted the discovery of drug candidates through helicase-inhibitorscreening, particularly for hcv and jev. in general, the drug candidates were chemically substances such streptomyces chartreusis actinoplanes philippinensis streptomycetes et al streptomyces streptomyces streptomycetes streptomyces streptomyces in vitro in vivo herpes simplex virus et al flavivirus , , , , as ribavirin -5”-triphosphate (borowski 2001), while the studies on naturally occuring inhibitors are rare or yet to be done. in the present study, we report on biologically active compound from actinomycetes which potentially have antiviral activity making them promising new drug candidates. the existence of candidate strains with high inhibition of atpase activity of jev rna helicase enzymes, suggests that indonesian actinomycetes have not been thoroughly investigated and therefore have potential as a source of novel bioactive compounds. these results not only suggested the usefulness of indonesian actinomycetes as screening sources of natural product based drug discovery programs, but also may be useful as basic data such as distribution studies. et al. references 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9(2):100-106. nonomura h, ohara y. 1969. distribution of actinomycetes in soil. vii. a culture method effective for both of preferential isolatin and enumeration of and strains in soil (part ii). j ferment technol. 47:701-709. . . . . in vitro japanese encephalitis virus streptomyces microbispora streptosporangium hcv ntpase/helicase by 1-β-d-ribofuranosyl-1,2,4triazole-3-carboxamide-5'-triphosphate (ribavirin-tp). . doi:10.1046/j.1365-2672.2003.02049.x doi:10.1128/aac.46.6.1766-1772.2002. doi:10.1128/aac.46.5.1231-1239.2002. doi:10.1128/aac.46.5.1231-1239.2002. doi:10.3209/saj.16_6 doi:10.1016/03856380(87)90001-x. doi:10.1023/a:1026579426265. doi:10.3209/saj.saj220103 doi:10.3209/saj.7_100. . . . robarts my, blouin ag, bleker s, kleinschmidt ja, aggarwal ak, escalante cr, linden rm. 2004. residues within the b' motif is critical for dna binding by the superfamily 3 helicase rep40 of adenoassociated virus type 2. j biol chem. 279(48):50472-50481. sebastian l, desai a, shampur mn, perumal y, sriram d, vasanthapuram r. 2008. n-methylisatin-beta-thiosemicarbazone derivative (sch 16) is an inhibitor of japanese encephalitis virus infection and . virol j 5(1)64:1-12 seong cn, choi jh, baik ks. 2001. an improved selective isolation of rare actinomycetes from forest soil. j microbiol. 39(1):17-23. spicer pe. 1997. japanese encephalitis in western irian jaya. j travel med. 4:146-147. utama a, shimizu h, hasebe f, morita k, igarashi a, shoji i, matsuura y, hatsu m, takamizawa k, hagiwara a, miyamura t. 2000a. role of the dexh motif of the and ns3 proteins in the atpase and rna helicase activities. virology. 273(2):316-324. in vitro in vivo . japanese encephalitis virus hepatitis c virus . doi:10.1074/jbc.m403900200. doi:10.1186/1743-422x-5-64 doi:10.1111/j.1708-8305.1997.tb00803.x. doi:10.1006/viro.2000.0417. . utama a, shimizu h, morikawa s, hasebe f, morita k, igarashi a, hatsu m, takamizawa k, miyamura t. 2000b. identification and characterization of the rna helicase activity of ns3 protein. febs lett. 465(1):748. welbourn s, green r, gamache i, dandache s, lohmann v, bartenschlager r, meerovitch k, pause r. 2005. ns2/3 processing is required for ns3 stability and viral rna replication. j biol chem. 280(33):29604-29611. widyastuti y ando k 2009. taxonomic and ecological studies of actinomycetes in indonesia. [final report joint research project between indonesian institute of sciences (lipi), representing the government research centers (grc) of the republic of indonesia and national institute of technology and evaluation (nite) of japan]. jakarta (id): lipi pr. p 709-711. williams st, lanning s, wellington emh. 1984. ecology of actinomycetes. in: goodfellow m, mordarski m, williams st, editors. the biology of the actinomycetes. london (gb):academic pr. p 481-528. japanese encephalitis virus 7 hepatitis c virus , . doi:10.1016/s00145793(99)01705-6. doi:10.1074/jbc.m505019200. 20 ratnakomala et al. microbiol indones a'yun, qurrota, 56 abraham, silva, 97 agusta, andrea, 33 aji, oktira roka, 51 amarantini, charis, 1 artika, i made, 73 barus, tati, 56 basukriadi, adi, 97 bela, budiman, 89 budiarto, bugi ratno, 73 dayamanti, tri asmira, 44 elfita, 82 fathoni, ahmad, 33 fitri, nadhya nur, 150 giri-rachman, ernawati arifin, 51 handajani, retno, 136 hasanah, nur, 171 helianti, is, 65 ibrahim, fera, 89 ihsanawati, 51 irawati, wahyu, 163 ivantri, ihsan, 82 jamal, yuliasri, 33 kasiamdari, andrina, 157 kurniawan, andi, 106 lay, bibiana widiyati, 113 lestari, yulin, 25 lihan, samuel, 141 lisdiana, lisa, 9 magdalena, stella, 113 marlina, 129 moeis, maelita ramdani, 51 muharni, 82 mujahid, aazani, 141 mukamto, 9 muller, moritz, 141 munawar, 82 mustopa, apon zaenal, 73 novelni, ringga, 129 nurhasanah, astutiati, 65 nurhayati, niknik, 65 nurkanto, arief, 33 nurtjahjani, supiana dian, 136 parhusip, adolf jan nexon, 163 patriati, arum, 51 pranamuda, hardaning, 9 praptiwi, 33 pratama, fenrico, 51 prawiroharsono, suyanto, 97 primanita, mona, 25 proksch, peter, 141 putra, edy giri-rachman, 51 putri, fentri paramitha, 65 rahayu, yuni sri rahayu, 9 raveinal, 129 rusli, musofa, 150 saskiawan, iwan, 171 satwika, dhira 1 sjamsuridzal, wellyzar, 97 sogandi, 73 sopiah, raden nida, 163 sudarmono, pratiwi pudjilestari, 120 sudiro, tjahjani mirawati, 120 sulfianti, asri, 89 sumaryono, wahono, 120 suprayudi, m agus, 17 suryantini, rosa, 157 suwanto, antonius, 56 syamsu, khaswar, 65 tan, lee tung, 141 utami, diah ayu satyari, 17 wahyudi, aris tri, 25 wahyunitisari, manik retno, 150 wibisana, ahmad, 120 widanarni, 17 widyasari, anastasia asri, 113 witono, suryo, 44 wong, changi, 141 wulandari, reine suci, 157 wulansari, dewi, 33 yamamoto, tatsuya, 106 yasmon, andi, 89 author index volume 9 vol.9, no.4, december 2015, p 178 microbiology indonesia issn 1978-3477, eissn 2087-8575 (+)-2,2'-epicytoskyrin a, 33 16s rrna, 25 acinetobacter sp. irc2, 163 andrographis paniculata, 82 antibacterial activity, 33, 82 antibiotic resistance, 150 antibiotic use, 150 antimicrobial agents, 113 antimicrobial properties, 141 bacillus megaterium btpacbd1, 65 bacillus subtilis, 113 bacteriocin, 73 benowo landfill, 9 biofilm, 106 biofilm polymer, 106 biosorption, 106, 163 blavim gene, 129 blaimp gene, 129 cacl2, 65 cellulase, 171 ccd, 65 characterization, 9 chemical constituens, 82 cmv strain, 44 coinfection, 136 copper, 163 genetic variability, 44 dgge, 25 diaporthe sp. gnbp-10, 33 emericella nidulans, 97 endophytic actinomycetes, 25 endophytic fungi, 141 endophytic fungus, 82 eric-pcr, 56 esbl, 150 escherichia coli, 56, 150 expression, 89 genotype, 136 gyra, 1 heavy metals, 163 hepatitis b virus, 136 histidine kinase component, 51 identification, 113, 157 insecticidal activity, 97 isolation, 9, 113 lactobacillus plantarum u10, 73 lipopeptide, 120 lithium ion, 106 mangrove fungi, 97 marine brown seaweed, 141 metagenomic, 25 metallo-β-lactamase (mbls), 129 meropenem, 129 microbiology, 89 microencapsulated, 17 mode of action, 73 multi-drug resistance, 51 optimization, 120 orchid mycorrhizae, 157 oxo-degradable polyethylene-degrading bacteria, 9 padina sp., 141 parc, 1 penicillin acylase, 65 probiotic, 17 pseudomonas aeruginosa, 129 purification, 73 qrdr, 1 rational drug design, 51 recombinant protein, 89 resistant, 163 response surface methodology, 120 rhizoctonia, 157 ropey bread, 113 streptococcosis, 17 salmonella, 1 soybean, 44 spodoptera litura, 97 surfactin, 120 tempeh, 56 tilapia, 17 tinospora crispa, 25 tuberculosis, 51 uncaria gambier, 33 used mushroom substrate, 171 xylose, 65 yeast, 89 subject index volume 9 vol.9, no.4, december 2015, p 179 microbiology indonesia issn 1978-3477, eissn 2087-8575 molecular phylogeny of salmonellae: relationships among salmonella species determined from gyra, gyrb, parc, and pare genes. charis amarantini, dhira satwika....... isolation of oxo-degradable polyethylene degrading-bacteria of benowo landfill soil surabaya. mukamto, yuni sri rahayu, lisa lisdiana, hardaning pranamuda............................ administration of microencapsulated probiotic at different doses to control streptococcosis in tilapia (oreochromis niloticus). diah ayu satyari utami, widanarni, m agus suprayudi............................................................................................................................ 16s rrna-based metagenomic analysis of endophytic actinomycetes diversity from tinospora crispa l. miers. mona primanita, aris tri wahyudi, yulin lestari.............................. antibacterial activity and mode of action of (+)-2,2'-epicytoskyrin a. andria agusta, dewi wulansari, yuliasri jamal, arief nurkanto, praptiwi, ahmad fathoni................................. genetic diversity of cucumber mosaic virus strain soybean from several areas. tri asmira damayanti, suryo wiyono............................................................................................... expression and purification of phor sensor-domain histidine kinase of mycobacterium tuberculosis in escherichia coli. ernawati arifin giri-rachman, fenryco pratama, oktira roka aji, arum patriati, ihsanawati, maelita ramdani moeis, edy giri-rachman putra............. genetic profiles of escherichia coli isolated from indonesian tempeh based on enterobacterial repetitive intergenic consensuspolymerase chain reaction (ericpcr). qurrota a'yun, antonius suwanto, tati barus................................................................... medium optimization for penicillin acylase (pac) production by recombinant bacillus megaterium ms941 containing pac gene from bacillus thuringiensis bgsc bd1 using response surface methodology. fentri paramitha putri, astutiati nurhasanah, niknik nurhayati, is helianti, khaswar syamsu...................................................................................... inhibitory activity of lactobacillus plantarum u10 isolated from tempoyak (fermented durian) made in indonesia against salmonella typhi. sogandi, apon zaenal mustopa, i made artika, bugi ratno budiarto............................................................................................... chemical constituen from an endophytic fungus aspergillus sp (sbd5) isolated from sambiloto (andrographis paniculata nees). elfita, munawar, muharni, ihsan ivantri............... cloning and expression of ha1 gene of h1n1 influenza virus 2009 pandemic (h1n1pdm09) indonesia strain in the pichia pastoris expression system for the development of influenza vaccine. asri sulfianti, andi yasmon, budiman bela, fera ibrahim........................................................................................................................................ insecticidal activities of ethyl acetate extract of indonesian mangrove fungus emericella nidulans bpptcc 6038 on spodoptera litura. silva abraham, adi basukriadi, suyanto pawiroharsono, wellyzar sjamsuridzal.......................................................................... biosorption of lithium using biofilm matrix of natural microbial consortium. andi kurniawan, tatsuya yamamoto.................................................................................................... isolation and identification of bacteria from raw materials contaminated by ropeproducing bacteria. anastasi asri widyasari, stella magdalena, bibiana widiyati lay............. optimization of surfactin production by bacillus amyloliquefaciens md4-12 using response surface methodology. ahmad wibisana, wahono sumaryono, tjahjani mirawati sudiro, pratiwi pudjilestari sudarmono................................................................................... determination of blavim and blaimp resistant genes againts meropenem of pseudomonas aeruginosa isolated from hcu bronkopneumona inpatients at internal medicine rsup dr m djamil padang. ringga novelni, marlina, raveinal............................... genotype of hepatitis b virus coinfection in typhoid patients. supiana dian nurtjahyani, retno handajani.......................................................................................................................... isolation, identification, and screening of antimicrobial properties of the marinelist of contents volume 9 1 9 17 25 33 44 51 58 65 73 82 89 97 106 113 120 129 136 microbiology indonesia issn 1978-3477, eissn 2087-8575 derived endophytic fungi from marine brown seaweed. changi wong, peter proksch, lee tung tan, samuel lihan, aazani mujahid, moritz muller............................................................ antibiotic use is not a risk factor of infection by extended-spectrum beta-lactamase producing bacteria in dr. soetomo hospital surabaya. nadhya nur fitri, musofa rusli, manik retno wahyunitisari.......................................................................................................... orchid mycorrhizae fungi: identification of rhizoctonia in west kalimantan. rosa suryantini, reine suci wulandari, andrina sri kasiamdari.......................................................... heavy metals biosorption by copper resistant bacteria of acinetobacter sp. irc2. wahyu irawati, adolf jan nexon parhusip, raden nida sopiah............................................................... screening, purification, and characterization of cellulase from fungi isolated from used mushroom substrate. iwan saskiawan, nur hasanah................................................................ author index.............................................................................................................................. subject index.............................................................................................................................. 141 150 157 163 171 178 179 volume 9, 2015 microbiol indones guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can 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foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 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netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com ai-si-list author index (7) 05. kurnianda.cdr vol.13, no.2, june 2019, p 70-74 doi: 10.5454/mi.13.2.5 polyoxygenated diterpene produced by the indonesian marine sponge callyspongia sp. as an inhibitor of the human pancreatic cancer cells 1,3* 1 1,3 1 viqqi kurnianda , suci faradilla , sofyatuddin karina , sri agustina , 1 1 1 maria ulfah , chitra octavina , farah syahliza , muhammad rizki 1 1 2* ramadhan , syahrul purnawan , and musri musman 1 department of marine science, faculty of marine and fisheries, universitas syiah kuala, banda aceh, 23111, indonesia; 2 department of chemistry education, faculty of teacher training and education, universitas syiah kuala, banda aceh, 23111, indonesia; 3 laboratory of marine chemistry and fisheries biotechnology, faculty of marine and fisheries, universitas syiah kuala, banda aceh, 23111, indonesia. the isolation of bioactive compound from the indonesian marine sponge callyspongia sp. based on bioassay-guided separation with several steps of chromatography has been done. there were 4 major compounds known as isocopalane diterpene derivatives together with a plausible new compound, isocopalanol, a + polyoxygenated isocopalane diterpene. isocopalanol has a molecular weight [m+h] 412.576 m/z indicate that the compound has molecular formula c h o determined by lcms-esi. this compound has bioactivity ic = 0.1 24 44 5 50 -1 μg ml against human pancreatic cancer cell. key words: bioactivity, callyspongia sp., cancer, diterpene, isocopalane senyawa bioaktif dari bunga karang laut indonesia callyspongia sp. telah diisolasi secara kromatografi dengan panduan bioasai. ada empat senyawa utama yang dikenal sebagai turunan diterpen isocopalan bersama dengan senyawa yang kemungkinan baru yaitu, isocopalanol, suatu senyawa diterpen isocopalan polioksigen. + isocopalanol berdasarkan data lcms-esi memiliki berat molekul [m+h] 412,576 m/z yang menunjukkan -1 senyawa tersebut memiliki rumus molekul c h o . senyawa ini memiliki bioaktivitas ic = 0,1 μg ml terhadap 24 44 5 50 sel kanker pankreas manusia. kata kunci: bioaktivitas, callyspongia sp., diterpen, isocopalan, kanker microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-852-6052-5207; fax: +62-651-53498; email: musrimusman@unsyiah.ac.id chemotherapy drugs have two forms, namely those consumed directly and those given by infusion (tsukamoto et al. 2000; shah et al. 2007; zeng et al. 2014). the treatment with chemotherapy makes the patients receive chemicals substances continuously for a long period of time which has side effects such as decrease the body weight (moutinho-ribeiro et al. 2017). therefore, alternative methods are needed in the treatment of cancer. the results of previous studies have reported that the secondary metabolites from the indonesian marine organisms have the activity against anticancer (romuald et al. 2010; kurnianda et al. 2018). in this study, we tried to find bioactive compounds as pancreatic cancer cell inhibitors from the indonesian's marine sponge callyspongia sp. materials and methods extraction and isolation. bioactive compound was extracted from callyspongia sp. (600 g wet weight, fig 1) by acetone and partitioned with ethyl acetate and pancreatic cancer is a disease in which there is an increase in the number of abnormal cells in the pancreas. pancreatic cancer is one cancer that is very malignant, difficult to diagnose and carry (coombs et al. 2014; sanli et al. 2017; hanada et al. 2017). generally, cancer was found at an advanced stage without obvious symptoms except by medical checkups. on the other hand, scenario in advanced cancer administration requires high-cost treatment such as surgery, chemotherapy, and radiotherapy although there is no guarantee of recovery (donadelli et al. 2007; zigeuner 2017). the appropriate treatment options can be chosen by doctors based on the patient's health status and the progress of cancer, one of which is chemotherapy (morimoto et al. 2003; romuald et al. 2010; kenner et al. 2017). to get rid of cancer cells in the body or prevent growth, patients can do chemotherapy with anticancer drugs. chemotherapy can be done before or after surgery, or even if surgery cannot be performed. water to obtain 23.86 mg of ethyl acetate portion. furthermore, it was fractionated by an open column chromatography silica gel using etoac gradient produced three major fractions. the results of viability showed that the third fraction showed the strongest -1 activity against panc-1 [15.62 mg (ic = 30 μg ml )]. 50 then, the third fraction (15.62 mg) was purified using 5 c -ms ii hplc (etoac:dcm) yielding seven 18 fractions. the fifth fraction indicated as potential -1 bioactive compound [7.6 mg (ic = 5 μg ml )] was 50 5 purified using column c -ms iii hplc using 18 meoh:h o (1:1) produced four fractions, the fraction 2 a (0.8 mg), fraction b (0.5 mg), fraction c (0.3 mg), and fraction d (0.8 mg). the fraction d [0.8 mg (ic = 0.1 μg 50 -1 ml )] showed cytotoxic to panc-1 cells. materials. the framework of skeleton from bioactive compound analyzed by nmr jeol eca500 mhz. the isolation sample based on bioassayguided separation using the esi-tof-ms brand q-tof ultima (waters co., ma, a.s.a). the ir spectrum was analyzed using the jasco ft/ir-5300 tool. the uv s p e c t r u m w a s a n a l y z e d u s i n g a u v2 4 5 0 spectrophotometer (shimadzu, kyoto, japan). the bioactive compounds purified using hplc cosmosil 5 c -ms-ii (id 10 mm × 250 mm, nacalai tesque) and 18 75 cosmosil ods ( c opn, nacalai tesque, kyoto, 18 japan). the bioassay test was conducted using bioradic spectroscopy, bioradic plates, and 96-well plastic plates. for the toxicity test a co incubator was used 2 with the following reagents, kanamycin solution 50 μg -1 ml , trypsin mc-coys medium, trypan blue, human cell panc-1, phosphate buffer buffer (pbs), fetal bovine serum (fbs), and modified dulbecco eagle medium (dmem). cell culture and toxicity test. cell culture and toxicity test were operated with reference to the procedure developed by arai et al. (2016). cultivation of panc-1 (human pancreatic carcinoma) cells was preserved in dmem (dulbecco's modified eagle medium) on heat-deactivated 10% fetal bovine serum -1 (fbs) and kanamycin (50 mg ml ) in a moistened ambiance of 5% co at 37°c. the growth of panc-1 2 cells was carried out in two different media, namely a glucose-deficient medium and a glucose medium. in means of nutrient hunger, cultivation panc-1 cells could be carried out in the glucose deficient medium containing of basal medium buffer (ph 7.4), i.e. (25 m m n ( 2 h y d r o x y e t h y l ) p i p e r a z i n e n ' 2 ethanesulfonic acid (hepes), enhanced 6.4 g/l nacl, -1 -1 -1 700 mg l nahco , 400 mg l kcl, 265 mg l 3 -1 -1 cacl .2h o, 200 mg l mgso .7h o, 125 mg l 2 2 4 2 -1 -1 nah po , 0.1 mg l fe(no ).9h o, 15 mg l phenol 2 4 3 2 red, 10 ml vitamin solution (x100) (gibco, -1 carlsbad, ca, usa), 200 mmol l l-glutamine -1 (gibco), and 50 mg l kanamycin holding 10% dialyzed fbs. the glucose medium was prepared by mixing basal medium enriched with 10% fbs and 2.0 -1 g l glucose (final concentration of 25 mm). the bioassay was carried out by comparing panc-1 cells growth in the glucose-deficient medium with the glucose medium (kurnianda et al. 2018; romuald et al. 2010). the cells were pre-incubated with the dmem containing as much as 10% fbs for 24 hours. to regulate nutritional hunger, the glucose deficient medium or the glucose medium was replaced. the medium was incubated for 12 hours, then the diluted fraction d were added, after that the incubation was continued for another 12 hours under 5% co at 37°c. 2 detection of cell proliferation was exploited the wts8 colorimetric reagent. in determining ic values can 50 be done using the growth inhibition curve. the difference of ic values between the glucose deficient 50 medium and the glucose medium was determined selectivity in the anti-proliferation (s.i) activity (kurnianda et al. 2017; coombs et al. 2014). fig 1 callyspongia sp. volume 13, 2019 microbiol indones 71 results structure identification. substances obtained from the fractions a, b, and c have been identified as isocopalane diterpene derivative compounds. the compound derived from the fraction d (compound 1) is thought to be a new compound of a polyoxygenated isocopalane diterpene. the compound 1 (colorless amorphous) has the molecular formula c h o 24 44 5 determined by lcms-esi with molecular weight + [m+h] 412.576 m/z. based on the ftir spectrum shows that the active metabolite has functional groups -1 -1 at 3419 cm as o-h streaching, 2995 cm and 2853 cm 1 as alkane skeleton of hydrocarbon, and the fingerprint -1 region of ether functional group at 1261 cm as c-o (fig 2). the results show that the active compound is an polyoxygenated hydrocarbon. discussion 1 13 the h and c nmr of compound 1 (table 1) showed the characteristics signal of methyl group observed at 0.82 (s, 3h), 0.84 (s, 3h), 0.85 (s, 3h), 1.12 (s, 3h) 1.15 (d, 3h), 1.17 (d, 3h), 1.49 (s, 3h) as the previous reported (wyk et al. 2007). the presences of the oxymethylene appears at 2.71 (br d, 1h, c-15). the signal from oxymethane appears at 3.05 (br t, 1h, c12), 4.74 (br t, 1h, c-1'), 4.81 (br t, 1h, c-1”). furthermore, cosy and hmbc correlation indicate that the active compound contain polyoxygenated moiety with diterpene skeleton (takekawa et al. 2006; kubota et al. 2007; kurnianda et al. 2017). the analysis of cosy spectrum from compound active revealed the proton-proton sequences of carbon 1-2–3, 5-6-7, 9-11-12, 1'-2' and 1”-2”. the correlation 1 13 table 1. h (500 mhz) dan c (125 mhz) data nmr (cdcl ) of compound 13 72 kurnianda et al. microbiol indones of proton-carbon from hmbc spectrum showed that the characteristics correlation at c-20 to c-1, c-17 to c8 indicated methyl group attach to bridge from 2 sixmembered rings moiety. furthermore, correlation between c-18 and c-19 as methyl singlet attached on 3 quaternary sp carbon at c-4. the presence of 2 ether moieties confirmed by hmbc and cosy correlation at c-12 to 1” and c-15 to 1' as no cross link between them (fig 3). we assumed this position contain ether functional group moiety confirmed by ir data and proton chemical shift between c-12 to c-1” and c-15 to c-1' (shah et al. 2007; zeng et al. 2014). furthermore, the absolute stereochemistry of secondary alcohol moiety determined by mosher's method. the relative configuration indicate as (5r*, 8s*, 9s*, 10r*, 12s*, 13r*, 14s*,1'r*, 1''r*)isocopalane diol. the viability against human pancreatic cancer cell in glucose deficiency medium from marine sponge callyspongia sp. showed a potential inhibitor of cell proliferation analyzed by wst-8 colorimetric reagents. the linear interpolation of the viability curve was used for determine of the ic value in glucose 50 deficiency medium. the bioactive compound indicated as anti-proliferative activity in glucose deficiency medium (romuald et al. 2010). the bioactive compound from the indonesian marine sponge callyspongia sp. has potential anti-1 proliferative against panc-1 cell with ic = 0.1 μg ml . 50 the bioactive compound indicated as secondary metabolite which has activity for alternative drug models -1 against commercial drug antimycin = 0.1 μg ml . acknowledgment authors thank to department of marine science, syiah kuala university for supporting of collecting sponges in sabang island and laboratory of marine natural products, faculty of science, university of the ryukyus, japan for purification methods and bioassay tests. references american cancer society. 2016. pancreatic cancer cell. th http://www.cancer.org. accessed on march 24 , 2019. arai m, kamiya k, shin d, matsumoto h, hisa t, setiawan a, kotoku n, kobayashi m. 2016. n-methylniphatyne a, a new 3-alkylpyridine alkaloid as an inhibitor of the cancer cells adapted to nutrient starvation, from an indonesian marine sponge of xestospongia sp. chem p h a r m b u l l ( to k y o ) . 6 4 ( 7 ) : 7 6 6 7 7 1 . d o i : 10.1248/cpb.c16-00118. coombs jr, zhang l, morken jp. 2014. enantiomerically fig 2 structure of bioactive compound 1. fig 3 cosy and hmbc correlation of bioactive compound 1. volume 13, 2019 microbiol indones 73 enriched tris(boronates): readily accessible conjunctive reagents for asymmetric synthesis. j am chem soc. 136(46):16140-161403. doi: 10.1021/ja510081r. donadelli m, costanzo c, beghelli s, scupoli mt, dandrea m, bonora a, piacentini p, budillon a, caraglia m, scarpa a, palmieri m. 2007. synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin a and gemcitabine. biochim biophys acta. 1773(7):10951106. doi: 10.1016/j.bbamcr.2007.05.002. kubota t, nishi t, fukushi e, kawabata j, fromont j, kobayashi j. 2007. nakinadine a, a novel bis-pyridine alkaloid with a β-amino acid moiety from sponge amphimedon sp. tetrahedron lett. 48(29):4983–4985. doi: 10.1016/j.tetlet.2007.05.121. kurnianda v, khairunnisa, winanda a, mauliza a, ulfah m, nurfadillah, ishida r, kobayashi m. 2017. polyhydroxy isocopalane from indonesian's marine sponge callyspongia sp. as anti–white spot syndrome virus from litopenaeus vannamei. j pharm nat prod. 3(2):140-143. doi: 10.4172/2472-0992.1000140. kurnianda v, mardiah a, karina s, agustina s, ulfah m, octavina c, syahliza f, ramadhan rm, s purnawan. 2018. inhibitory of panc-1 produced by indonesian's marine sponge endectyon delaubenfelsi adapted to nutrient starvation. iop conf. ser.: earth environ sci. 216:012032. doi: 10.1088/1755-1315/216/1/012032. morimoto y, kitao s, okita t, shoji t. 2003. total synthesis and assignment of the double-bond position and absolute configuration of (−)-pyrinodemin a. org lett. 5(15):2611-2614. doi: 10.1021/ol034700v. national cancer institute (nci). 2016. pancreatic cancer for th patients. http://www.cancer.gov. accessed on march 14 , 2019. romuald c, busseron e, coutrot f. 2010. very contracted to extended co-conformations with or without oscillations in twoand three-station [c2]daisy chains. j org chem. 75(19):6516-6531. doi: 10.1021/jo101234u. shah an, summy jm, zhang j, park si, parikh nu, gallick ge. 2007. development and characterization of gemcitabine-resistant pancreatic tumor cells. ann surg oncol. 14(12):3629–3637. doi: 10.1245/s10434-0079583-5. takekawa y, matsunaga s, soest rw, fusetani n. 2006. amphimedosides, 3-alkylpyridine glycosides from a marine sponge amphimedon sp. j nat prod. 69(10):1503-1505. doi: 10.1021/np060122q. tsukamoto s, takahashi m, matsunaga s, fusetani n, soest rw. 2000. hachijodines a−g:  seven new cytotoxic 3alkylpyridine alkaloids from two marine sponges of the genera xestospongia and amphimedon. j nat. prod. 63(5):682-684. doi: 10.1021/np9905766. woonyoung c, bogdan c, andrea o, xiaoping s, arlene sr, colin d, david jm. 2014. intrinsic basal and luminal subtypes of muscle-invasive bladder cancer. nat rev urol. 11(7):400-410. doi: 10.1038/nrurol.2014.129. wyk aww, froneman pw, bernard ks, coleman mtd. 2007. new isocopalane diterpene diester from a subantarctic marine nudibranch. j arkivoc. 9: 121-128. doi: 10.3998/ark.5550190.0008.914. 74 kurnianda et al. microbiol indones page 1 page 2 page 3 page 4 page 5 5 irene_365.pmd 26-2-2009.pmd ________________________ *corresponding author, phone: +62-298-321212, fax: +62-298-321433, e-mail: irene_meiti@yahoo.com issn 1978-3477 volume 2, number 2, august 2008 p 73 78 optimum concentration of glucose and orange ii for growth and decolorization of orange ii by enterococcus faecalis id6017 under static culture v. irene meitiniarti1*, endang s. soetarto2, eko sugiharto3, and kris herawan timotius1 1 faculty of biology, universitas kristen satya wacana, jalan diponegoro 52-60, salatiga 50711, indonesia; 2 faculty of biology, 3 department of chemistry, faculty of natural science and mathematics, universitas gadjah mada, jalan teknika selatan, sekip utara, kampus bulaksumur, yogyakarta 55281, indonesia growth and decolorization performance of bacterial grown on azodyes-containing-medium is influenced by various concentrations of carbon sources and azodyes. the optimum level of glucose and orange ii concentration for growth and orange ii decolorization by enterococcus faecalis id6017 are reported in this paper. the experiments were carried out in liquid static culture as batch experiments. glucose and orange ii concentrations used in these experiments were 0.45, 0.90, 1.80 g l -1, and 40, 80, 120 mg l-1, respectively. the specific growth rate and decolorization rate of orange ii by e. faecalis were highest on the medium which contained at least 0.90 g l-1 glucose. it is necessary to note that glucose above 0.90 g l-1 gave no significant difference. on the medium containing 0.90 g l-1 glucose and 80 mg l-1 orange ii, e. faecalis grew with the highest specific growth rate (0.28 h-1) and orange ii decolorization rate (0.47 h-1). the maximum specific growth rate of biomass (µ max ) and the halfsaturation coefficient (k s ) under optimal conditions were 0.25 h-1 and 1.5 g.l-1, respectively. the kinetics of decolorization indicated that the process followed first order kinetics with respect to the initial concentration of orange ii. the inhibition constant (k i ) was found to be 750 mg l-1 orange ii, indicating that orange ii concentration at e” 750 mg l-1 would inhibit bacterial growth to decolorize orange ii. key words: enterococcus faecalis, azodyes, decolorization, glucose, orange ii _____________________________________________ orange ii is one of synthetic azodyes which has been widely used for coloring of textiles, food, and cosmetics. orange ii (acid-orange-7 or p-(2-hydroxy-1-naphthylazo) benzenesulfonic acid sodium salt) has the molec(ular structure c 16 h 11 n 2 nao 4 s. 5h 2 0 (fig 1: merck index 1968). this azodye has been used as a model substrate for azodye degradation. orange ii can be degraded by sphingomonas sp. 1cx and e. faecalis id6017 into two main intermediate products i.e. sulphanilic acid and 1-amino-2-naphthol (coughlin et al. 1999; meitiniarti et al. 2007). both of them may be mineralized further. azodyes are relatively resistant to microbial degradation under conditions normally found in waste-water treatment plants. however, several microorganisms are reported to be able to transform azodyes into non-colored products or even to mineralized products (stolz 2001). recently, a number of studies have been focusing on microbial degradation of azodyes (kim et al. 1995). this is because physicochemical methods used for color removal of the effluents show disadvantages in terms of operational problems, high cost and sludge production (kapdan et al. 2000; kodam et al. 2005). according to rafii et al. (1990), human intestinal microbiota generally found in the effluent of waste water treatment and using of these bacteria for azodyes degradation has been reported. in industrial waste-water containing various dyes, azodye degraders should exhibit decolorizing ability for a wide range of dyes and environmental conditions (fang et al. 2005). in regard to this e. faecalis is a bacterial species which has been reported to decolorize azodyes (handayani et al. 2007). therefore the decolorization of azodyes is an important topic for study. bacterial azodyes degradation is usually initiated by cleavage of the azo bond under anaerobic conditions resulting in the formation of aromatic amine colorless products. hence the process is called decolorization (zimmermann et al. 1982; tan 2001; van der zee 2002). the performance of microbial decolorization rates depends on the activity of the microorganism through a specific enzymatic process and is affected by environmental conditions, such as substrate concentration, composition of the medium, ph, carbon and nitrogen sources, and incubation temperature. sphingomonas sp. strain 1cx has an ability to decolorize 20 mg l-1 orange ii, acid orange 8, acid orange 10, acid red 4, or acid red (coughlin et al. 1997), where as e. faecalis id6017 has an ability to decolorize acid red 27 and reactive red 2 up to concentration of 100 mg l-1 in batch system (handayani et al. 2007) and orange ii up to concentration 120 mg l-1 in fedbacth system (meitiniarti et al. 2006). meitiniarti et al. (2005) reported that e. faecalis produces an orange ii reductase. several environmental conditions, such as substrate and cosubstrate concentration, could influence the enzymes activity (tan 2001). in this paper, we discuss the optimum level of glucose as co-substrate and orange ii concentrations on the growth kinetics of e. faecalis and orange ii decolorization performance. fig 1 chemical structrure of orange ii. oh n=n so 3 na.5h 2 o 74 meitiniarti et al. microbiol indones materials and methods microorganism and culture condition. e. faecalis id6017 was used as a bacterial model for the decolorization of orange ii and was maintained on a slant media of trypticase soy agar (tsa) at room temperature (26-28°c) as a stock culture. e. faecalis was grown on liquid-basalmedium consisting of (g l-1): 0.25 mgso 4 .7h 2 o, 1.98 (nh 4 ) 2 so 4 , 5.55 k 2 hpo 4 , 2.13 kh 2 po 4 , and 0.25 yeast extract. glucose was added in concentrations of 0.45, 0.90, and 1.80 g l-1 orange ii (merck, ci acid orange 7, ci 15510, mw= 440.41) was added in concentrations of 0, 40, 80, and 120 mg l-1. inoculum preparation. 48-h-old tsa slant cultures of e. faecalis were grown on 500 ml erlenmeyer flask, containing basal medium without orange ii, for 24 h on a shaker (150 rpm). this culture was the pre-culture (inoculum). experimental design and culture conditions. the experiments were carried out in two batchs using liquid medium for 9 h at room temperature. in the first experiment, three flasks each containing 80 mg l-1 orange ii medium were prepared and each flask was added 0.45, 0.90, and 1.80 g l-1 glucose. this experiment was done to determine the glucose concentration that increased bacterial growth and orange ii decolorization rate. the glucose concentration which resulted in increasing growth and decolorization rate, was selected and used as the limited-substrate-concentration. in the second group of experiments, the selected glucose concentration was then supplemented with 40, 80, and 120 mg l-1 orange ii concentrations. experiments was carried out in triplicate. as much as 50 ml aliquots of pre-culture were transfered to 450 ml medium in a 1 l volume growing vessels (duran) with a rubber stopper, and incubated under static conditions at room temperature. at interval of one hour, 10 ml aliquots of each culture was removed up the late exponential growth-phase. parameters examined were concentrations of glucose, orange ii, cell mass (biomass) and sulphanilic acid (as a decolorization product of orange ii). analytical methods. samples were centrifused at 3 326g for 30 min to separate biomass from the supernatant. the supernatant was analyzed for glucose, orange ii, and sulphanilic acid concentrations. after being washed twice, the biomass was re-suspended into an initial volume (10 ml) using distilled water and examined spectrophotometrically (ë 600nm ). the biomass concentration in units of cell dry weight was calculated using a standard curve of optical density (od 600nm ; y-axis) against biomass concentration (cell dry weight) (mg l-1; x-axis). glucose concentrations were determined spectrophotometrically (ë 540nm ) using the dns reagent method (james 1995) and orange ii concentration were determined spectrophotometrically (ë 482nm ) (zimmermann et al. 1982). the sulphanilic acid concentration was analyzed by injecting five to ten µl sampels of supernatant into a hplc shimadzu model lc-3a chromatograph, equipped with uvdetector and ods column (150 mm x 8 mm). the mobile phase was composed of methanol and acetic acid 0.6% (v/v) = 60:40. the flow rate was 0.7 ml min-1. the eluent were monitored using a uv detector at 276 nm (chang et al. 2001; supaka et al. 2004). determination of kinetic parameters (k, µµµµµ, µµµµµ max , k s , and k i ). the ability of bacterial strains to decolorize orange ii was determined by calculating the decolorization rate used equation (1) (wuhrmann et al. 1980): note: k is decolorization rate (decrease of color intensity); c 0 and c are color concentrations (mg l-1) at t 0 and t; t 0 and t are time (h). the substrate/co-substrate consumption and decreasing of decolorization product (sulphanilic acid) was calculated using equation (2): note: s is substrate/co-substrate consumption (mg); s 0 is glucose/orange ii concentration (mg l-1) at t 0 ; s t is the lowest glucose/orange ii concentration or the highest sulphanilic acid concentration (mg l-1); s e is the lowest sulphanilic acid concentration (mg l-1); t 0 , t, and t e are time (h). the specific growth rate (µ ) is determined from the cell mass as a first order with the following equation: integration of equation 3 yields: where x and x 0 are biomass concentrations at time t and t=0. generally, the growth rate is restricted by the concentration of the growth-limiting substrate that can be described by the monod equation: where µ max is the maximum specific growth rate when s>>k s . the constant k s is the saturation constant or half-velocity constant and is equal to the concentration of the ratelimiting substrate when the specific rate of growth is equal to 50% of the maximum. in this paper, the monod equation modified by the hanes model (called the hanes-monod model) was used to analyze the values of µ max and k s (brandt 2001): a plot of against is linear with a slope of 1/u and an intercept on the y-axis of max 1 µ . when a substrate is inhibited by its own biodegradation, like orange ii, the monod model was derivatived by incorporating the inhibition constant k i . among the substrate inhibition models, the andrew’s equation (7) is most widely used (okpokwasili and nweke 2005): s = s 0 -s t or s = s t -s e sk s s + = . max µ µ maxmax µµµ s kss += ln (c 0 -c) (t-t 0 ) k = ——————— ,x dt dx µ= 0 xx = at t=0 t exx µ 0 =,ln 0 t x x µ= or volume 2, 2008 microbiol indones 75 where k i is the inhibition constant (calculated by microsoft excel software in this paper). results optimal level of glucose. the addition of various concentration of glucose (0.45 to 1.80 g l-1) did not give any significant difference in the specific growth rate, biomass production of e. faecalis or the concentration of sulphanilic acid (table 1 and fig 2). however, as shown in table 1, the decolorization of orange ii was significantly influenced by the addition of glucose. by calculating glucose consumption in three initial glucose concentrations, we showed that by increasing the glucose concentration, from 0.90 to 1.80 g l-1, this did not efficiently increased the specific growth rate, biomass production, and orange ii reduction by e. faecalis. thus, in the second experiment, we used optimal glucose concentration i.e. 900 mg l-1. optimal level of orange ii. the specific growth rate of e. faecalis in orange ii concentration from 40 to 120 mg l-1 the optimal glucose concentration was not significantly different to that of the control (fig 3). based on the specific growth rate of e. faecalis, our results shown that by increasing the orange ii concentration did not increase the specific growth rate. by calculating the kinetic parameter of growth our results show that the best specific growth rate of e. faecalis was on the 80 mg l-1 orange ii-containingmedium (table 2). in the orange ii 120 mg l-1 containing medium, the decolorization rate and capacity were higher than the lower orange ii concentration (40-80 mg l-1) (table 2). the maximum specific growth rate (µµµµµ max ), the halfmaximum saturation coefficient (k s ), and the inhibition constant (k i ). by calculating µ max and k s values using the hanes-monod model for e. faecalis growing on medium containing various glucose consentrations, we obtained values of µ max and k s of 0.22 h-1 and 0.05 g l-1, respectively (fig 4). by similar methods, the values of µ max and k s for e. faecalis growth on medium containing various orange ii concentrations could be determined. the values of µ max and k s were 0.25 h-1 and 1.5 mg l-1, respectively (fig 5). based on the kinetic parameters obtained: m (0.26, 0.28, and 0.25 h-1 for 40, 80, and 120 mg l-1 orange ii, respectively), µ max (0.25), and k s (1.5) with various initial s (orange ii) concentrations, the k i value could be calculated using microsoft excel programme by inserting the assumed k i value as a variable. after 15 iterations, the best fit (i.e between the m values calculated by hanes-monod method and the m haldane, correlation coefficient r2 ≅ 1) gave an orange ii k i value of 750 mg l-1. discussion as shown by the data in table 1 and fig 2, increasing the glucose concentration from 0.45 to 0.90 g l-1, increased specific growth rate and biomass production. however, increasing the glucose concentration from 0.90 to 1.80 g l-1 did not significantly increase the specific growth rate and i s k s sk s 2max ++ ⋅= µµ table 1 the influence of glucose on enterococcus faecalis id6017 growth and its performance for orange ii decolorization in the medium containing 80 mg l-1 orange ii and several concentrations of glucose parameter of growth and decolorization glucose concentration (g l-1) 0.45 0.90 1.80 specific growth rate (h-1) biomass produced (mg) glucose consumption (g) orange ii reduction (mg) decolorization rate (h-1) capacity of decolorization (mg substrate/mg cell d.w.) concentration of sulphanilic acid (mg l-1) 0.18 + 0.01 29.3 + 2.518.04* 0.39 + 0.005 65.9 + 4.4 0.51 + 0,1 3.65 + 0.3 32.5 + 0.3 0.20 + 0.02 30.1 + 7.422.86* 0.67 + 0.007 71.1 + 3.2 0.78 + 0.2 3.11 + 0.1 35.5 + 0.12 0.21 + 0.02 31.3 + 3.716.52* 0.79 + 0.009 72.5 + 8.6 0.77 + 0.2 4.39 + 0.5 36.7 + 0.71 *biomass produced during decolorization of orange ii. b io m a ss c o n c n ( m g l -1 ) 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 1 2 3 4 5 6 7 80 incubation time (h) y1= 13.091e0.1797x r2= 0.991 y2= 12.628e0.2042x r2= 0.9513 y3= 8.8155e0.2086x r2= 0.9845 a ( ) µ1= 0.1797 h-1; ( ) µ2= 0.2042 h-1; ( ) µ3= 0.2086 h-1 1 2 3 4 5 6 l n o ra n g e i i c o n c n ( m g l -1 ) 5 4 . 5 4 3 . 5 3 2 . 5 2 1 . 5 1 0 . 5 0 0 y3= -0.7782x + 6.1293 r2= 0.9666 y2= -0.7832x + 6.1398 r2= 0.9616 y1= -0.5039x + 5.3186 r2= 0.9479 incubation time (h) b ( ) k1= 0.509 h-1; ( ) k2= 0.7832 h-1; ( ) k3= 0.7782 h-1 fig 2 specific growth rate (a) and orange ii decolorization rate, (b) during enterococcus faecalis growing on the medium containing orange ii and 0.45 g l-1 glucose ( ), 0.90 g l-1 glucose ( ), and 1.80 g l-1 glucos ( ). 76 meitiniarti et al. microbiol indones biomass produced by the bacteria. this might be because the glucose concentration of 0.90 g l-1 is adequate to support the growth of e. faecalis. it was also shown from glucose consumption and biomass production that these were low during bacterial growth. for their growth, bacteria require c and n sources in certain proportions (thingstad and lignell 1997). in the 0.90 g l-1 glucose containing medium, the proportions of c and n sources were suitable for bacterial growth. thus, the bacteria grew fast and produced more biomass in this medium. amount of glucose was related to the rate of decolorization and the amount of reduced orange ii. the increase in orange ii concentration from 40 to 80 mg l-1 raised the specific growth rate of e. faecalis. however, when orange ii was increased up to 120 mg l-1, it decreased the specific growth rate of e. faecalis. these results show that e. faecalis did not use orange ii as a growth substrate and might be toxic for bacterial growth. the highest specific growth rate of e. faecalis was on the medium containing 80 mg l-1 orange ii. this might be due to the fact that in 80 mg l-1 orange ii concentration, the bacteria b io m a s c o n c n ( m g l -1 ) 8 0 6 0 4 0 2 0 0 2 4 8 1 060 incubation time (h) fig 3 the growth of enterococcus faecalis on the medium without orange ii ( ), with 40 mg l-1; ( ), 80 mg l-1; ( ), and 120 mg l-1 ( ), orange ii. bars indicated standard error where p=0.05. 0.25 + 0.03 33.72 + 7.50 23.90* 756.2 + 22.9 74.45 + 6.38 62.00 0.32 + 0.05 2.77 + 0.57 37.02 + 0,71 in contrast to the influence of glucose on bacterial growth, the decolorization of orange ii was significantly influenced by the addition of glucose. the consumption of glucose was related with the rate of decolorization and the amount of reduced orange ii. this means that additional glucose as an energy source is needed by the e. faecalis to decompose orange ii i.e. decolorization of orange ii is an energy consuming activity. the addition of glucose did not give any significant difference on sulphanilic acid formation. based on the concentration of sulphanilic acid at the end of cultivation, it showed that the concentration of sulphanilic acid was quite high. this is because this bacteria can not metabolise sulphanilic acid (meitiniarti et al. 2007), although the added table 2 the influence of orange ii on enterococcus faecalis id6017 growth and its performance for orange ii decolourization in the medium contained 0.90 g l-1 glucose and several concentrations of orange ii parameter of growth and decolorization orange ii concentration (mg l-1) 0 4 0 8 0 120 specific growth rate (h-1) biomass produced (mg) glucose consumption (g) orange ii reduction (mg) orange ii reduction (%) decolorization rate (h-1) dye removal capacity (mg substrate/mg cell d.w) concentration of sulphanilic acid (mg l-1) 0.28 + 0.03 51.77 + 8.10 695.6 + 12.9 n.d n.d n.d n.d 0.26 + 0.02 45.04 + 11.90 25.10* 718.2 + 38.6 33.63 + 4.70 84.00 0.95 + 0.14 1.55 + 0.19 24.2 + 2.9 0.28 + 0.02 34.1 + 7.1 22.9* 732.7 + 25.0 64.22 + 6.38 80.27 0.47 + 0.07 2.64 + 0.32 34.02 + 3.09 *biomass produced during removal of orange ii, n.d: not determined. 1 0 8 6 4 2 0 0 0 . 5 1 . 0 1 . 5 2 . 0 g lu c o se c o n c n /s p e c if ic g ro w th r a te ( s /µ ) glucose concentration (s) (g l-1) y= 4.6111x + 0.2503 r2= 0.9961 fig 4 a plot of glucose concentration/specific growth rate (s/µ) vs glucose concentration (s) resulting in a straight line with slope equal to 1/µ max = 4.61 and an intercept of k s /µ max = 0.25 for various cosubstrate concentrations on growing medium of enterococcus faecalis. o r a n g e i i c o n c n /s p e c if ic g ro w th r a te (s /µ ) 6 0 0 5 0 0 400 3 0 0 2 0 0 100 0 0 5 0 1 0 0 150 fig 5 a plot orange ii concentration/specific growth rate (s/µ) vs orange ii concentration (s) resulting in a straight line with a slope equal to 1/µ max = 3.93 and an intercept of k s /µ max = 5.89 for various substrate concentrations on growing medium of enterococcus faecalis. orange ii concentration (s) (mg l-1) volume 2, 2008 microbiol indones 77 nutrient requirement was fullfilled and they could tolerate orange ii concentration. so, they could grow favorably. based on the decolorization rate (table 2), our results show that the increase in orange ii concentration also increased the decolorization rate. this is because the reductase enzyme activity of e. faecalis to remove or reduce orange ii, was related to substrate concentration (orange ii). the kinetic analysis of decolorization indicated that the decolorization process followed first order kinetics with respect to the initial concentration of orange ii. e. faecalis had the highest orange ii decolorization rate on the medium containing 80 mg l-1 orange ii because e. faecalis could not use orange ii as a growing substrate. at a higher concentration, orange ii did inhibit e. faecalis growth. as shown on table 2, the increase in the decolorization rate at higher orange ii concentrations would not decrease sulphanilic acid concentration. it seems that although the sulphanilic acid concentration increased due to increasing of orange ii decolorization, e. faecalis could not metabolise sulphanilic acid. this would result in the accumulation of sulphanilic acid in the medium. mendez-paz et al. (2005) reported that sulphanilic acid produced by orange ii decolorization was not degrade futher and it would then be accumulated in the medium. the µ max value obtained from e. faecalis, grown on medium containing various glucose concentrations, was similar to the specific growth rate of e. faecalis which was grown on the medium contained of 0.9-1.8 g l-1 glucose concentration. this result showed that by increasing the glucose concentration up to 1.86 g l-1, it would not efficiently increase the growth of e. faecalis. a low value of k s obtained here indicated that bacteria had a very high affinity for glucose as a carbon substrate. hence, the bacterial growth would not be affected until the glucose concentration declined to a very low level. this result also showed that using glucose at an optimal concentration is quite economic when applied in waste-water-treatment plants. the µ max values of e. faecalis growth with various orange ii concentrations was similar with the specific growth rate of e. faecalis grown on the medium containing lower than 40 mg l-1 orange ii concentration. this result indicated that orange ii was not a growth substrate for e. faecalis. futhermore, when the k s value of orange ii was compared with that of glucose, it showed that bacterial affinity to orange ii was lower than to glucose. this result indicated that e. faecalis would preferentially use glucose on the medium for rapid growth (thingstad and lignell 1997). although a decrease in orange ii concentration occurred in these experiments, it is not be due to orange ii consumption as a substrate. orange ii might be used together with direct growing supporting substrate via cometabolism. based on the value (750 mg l-1) of inhibition coefficient k i obtained in this experiment, it is shown that orange ii at a concentration of ≥ 750 mg l-1 would inhibit growth of e. faecalis. the k i value of orange ii also indicated that although µ of e. faecalis on the medium contained of 120 mg l-1 orange ii decreased, orange ii concentration used in this experiment (40, 80, and 120 mg l-1) was too low to inhibit the growth of e. faecalis. based on our results, it can be concluded that by increasing the glucose (as a co-substrate) concentration up to 0.90 g l-1 and orange ii up to 80 mg l-1, we could increase the specific growth rate and orange ii decolorizatin rate of e. faecalis. an increase in orange ii concentration above 80 mg l-1 would decrease the specific growth rate of e. faecalis, and when the concentration was increased to more than 750 mg l-1, the growth of bacteria will be inhibited. although the actual dye concentration in effluent is not more than 750 mg l-1, we can predict that the specific growth rate of bacteria will decrease when the orange ii concentration was more than 80 mg l-1. moreover, our results show that e. faecalis could not consume sulfanilic acid as one of the intermediate products of azodye degradation. accordingly, it is suggested that this bacteria should not be used alone in dye-contaminated waste-water treatment, but should be mixed with other bacterial species which could consume intermediate products of azodye degradation and had the ability to degrade other kinds of azodyes. references brandt bw. 2001. realistic characterization of biodegradation. [doctoral thesis]. amsterdam: vrije universiteit. chang js, chou c, lin yc, lin pj, ho jy, hu tl. 2001. kinetic c h a r a c t e r i s t i c s o f b a c t e r i a l a z o d y e d e c o l o r i z a t i o n b y pseudomonas luteola. water res 35:2841-2850. coughlin mf, kinkle bk, tepper a, bishop pl. 1997. characterization of aerobic azodye-degradating bacteria and their activity in biofilms. water sci technol 36:215-220. coughlin mf, kinkle bk, bishop pl. 1999. degradation of azodyes containing aminonaphthol by sphingomonas sp. strain 1cx. j ind microbiol biotechnol 23:341-346. fang h, wenrong h, yuezhong l. 2004. biodegradation mechanism a n d k i n e t i c s o f a z o d y e 4 b s b y a m i c r o b i a l c o n s o r t i u m . c h e m o s p h e r e 5 7 : 2 9 3 3 0 1 . handayani w, meitiniarti vi, timotius kh. 2007. decolorization of acid red 27 and reactive red 2 by e. faecalis id6017 under batch system. world j microbiol biotechnol 23:1239-1244. james cs. 1995. analytical chemistry of foods. london: blackie academic and professional. kapdan ik, kargi f, macmullan g, marchant r. 2000 effects of environmental conditions on biological decolorization of textile dyestuff by c. versicolor. enz microbial technol 26:381-387. kim sj, ishikawa k, hirai m, shoda m. 1995 characterictics of a newly isolated fungus geotrichum candidum dec 1 which d e c o l o r i z e s v a r i o u s d y e s . j f e r m e n t b i o e n g 7 9 : 6 0 1 6 0 7 . kodam km, soojhawon i, lokhande pd, gawai kr. 2005 microbial decolorization of reactive azodyes under aerobic conditions. world j microbiol biotechnol 21:367-370. meitiniarti vi, sunardi ev, timotius kh. 2006. the influence of fed orange ii containing medium to the growth of e. faecalis id6017 and its decolorization ability. biota 11:52-58. meitiniarti vi, sutarto es, timotius kh, hedrawan jt. 2005. dekolorisasi pewarna azo orange ii oleh enterococcus faecalis id6017 dan chryseobacterium indologenes id6016. biologi 4:303-313. meitiniarti vi, sutarto es, timotius kh, sugiharto e. 2007. product of orange ii biodegradation by enterococcus faecalis id6017 and chryseobacterium indologenes id6016. microb indones 1:51-54. mendez-paz d, omil f, lema jm. 2005. anaerobic treatment of azo dye acid orange 7 under batch conditions. enz microbial technol 36:264-272. merck index. 1968. an encyclopedia of chemical and drugs. 8th ed. new york: merck and co., inc. okpokwasili gc, nweke co. 2005. microbial growth and substrate utilization kinetics. afr j biotechnol 5:305-317. 78 meitiniarti et al. microbiol indones rafii f, franklin w, cerniglia c. 1990 azoreductase activity of anaerobic bacteria isolated from human intestinal microflora. appl environ microbiol 56:2146-2151. stolz a. 2001. basic and applied aspects in the microbial degradation of azodyes. appl microbiol biotechnol 56:69-80. supaka n, juntongjin k, damronglerd s, delia ml, strehaiano p. 2004. microbial decolorization of reactive azodyes in a sequential anaerobic-aerobic system. chem eng j 99:169-176. tan ncg. 2001. integrated and sequential anaerob/aerob biodegradation of azodyes [doctoral thesis]. wageningen: wageningen university. thingstad tf, lignell r. 1997. theoritical models for the control of bacterial growth rate, abundance, diversity, and carbon demand. aqua microb ecol 13:19-27. wuhrmann k, mechsner k, kappeler t. 1980. investigation on rate-determining factors in the microbial reduction of azodyes. eur j appl microbiol biotechnol 9:325-338 zee fp van der . 2002. anaerobic azodye reduction [doctoral thesis]. wageningen: wageningen university. zimmermann t, kulla hg, leisinger t. 1982. properties of purified orange ii azoreductase, the enzyme initiating azodye degradation by pseudomonas kf46. eur j biochem 129:197-203. 8 riri prihatini.pmd phylogenetic evidence that two submerged-habitat fungal species, speiropsis pedatospora and xylomyces chlamydosporus, belong to the order jahnulales insertae sedis dothideomycetes riry prihatini1,2, nattawut boonyuen1 and somsak sivichai1 1biotec national center for genetic engineering and biotechnology, thailand science park, 113 paholyothin road, klong 1, klong luang, pathumthani, 12120 thailand; 2balai penelitian tanaman buah tropika, jalan raya solok-aripan km.8, solok 27301, sumatera barat, indonesia the genera speiropsis and xylomyces are anomorph fungi. the taxonomic address for the fungi has been unclear. in this study, observation of morphological traits indicates that they have a unique pattern of mycelia with dark-brown to black colour and thick-walled hyphae. the same culture patterns of certain fungi isolated from freshwater habitats in thailand were selected from biotec culture collection (bcc, thailand), while more species were added from centraalbureau voor schimmelcultures (cbs, netherlands). these fungi were composed of jahnula spp. (2-celled ascospores), brachiosphaera tropicalis (hyaline and 4-5 armed conidia), s. pedatospora (hyaline and branches conidia) and xylomyces sp. (dematiaceous and fusiform chlamydospores). this study was undertaken to confirm the taxonomic address for s. pedatospora and xylomyces based on phylogenetics relationships as inferred from their its rdna sequence data by using mp (unweighted and successive weighted mp), nj, ml and bayesian analysis. phylogenic analysis revealed that isolates of s. pedatospora (2 strains) was a member of the order jahnulales and clustered with jahnula spp. (5 strains) and b. tropicalis (4 strains) with >82% bootstrap support and 100% posterior probabilities. four isolates of x. chlamydosporus, x. elegans and x. aquaticus were shown to be polyphyletic within the jahnulales and pleoporales. the mp and nj showed the same topology as in the jahnulales clade obtained by ml analysis. key words: its rdna, speiropsis pedatospora, xylomyces _____________________________________________ volume 2, number 3, december 2008 issn 1978-3477 p 136-140 ________________________ * corresponding author, phone: +62-755-20137, fax: +62-755-20592, e-mail: riri_silva@yahoo.com the genus speiropsis are possessed with erect, simple, straight, septate, mononematous, aggregates in fascicles or synnematous to sporodochial conidiophores with discrete, denticulate, polyblastic conidiogenous cells and catenate conidia in branched or unbranched chains (barbosa and gusmao 2005). s. pedatospora is a mitosporic fungus found in submerged leaves in freshwater bodies (barbosa and gusmao 2005) and also a leaf pathogen in eucalyptus saligna. additionally, the genus xylomyces a mitosporic fungus, has different nodes of ontogeny which produces large, dematiaceous, thick-walled, multiseptate and fusiform chlamydospores. this anamorph fungus is mainly found in freshwater-submerged-wood (goh et al. 1997). the taxonomic position of xylomyces and s. pedatospora are still unclear. furthermore, brachiosphaera, another freshwater fungus, characterized by producing round-shaped conidia with conidial arms, mostly 4-5 each with 1-4 septa. its colonies are characterized by effuse, septate mycelia, mostly submerged in culture media. initially it is hyaline and turns into olivaceous brown when older. a few segments of hyphae become slightly constricted at the septa, and cells are enlarged, with ellipsoidal to round conidia in their clustering (chang 1994). all jahnula sp., a group of fungi that inhabit wood submerged in freshwater, are characterized by hyaline to blackish-translucent-membranous ascomata with subtending, wide-septate-brown-spreading hyphae, large angular cells of peridia, septate pseudoparaphyses, hamathecium, eightspored, clavate to cylindrical asci, one-septate, broad fusiform, brown and multiguttulate ascospores. moreover, jahnula colonies on potato dextrose agar (pda) grew slowly having dark-brown to black, effuse hyphae which are thickwalled, septate, constricted at the septa and cells of the hyphae are cylindrical to subglobose (raja and shearer 2006). these cultural traits of jahnula spp. were similar to xylomyces sp., s. pedatospora and brachiosphaera tropicalis (boonyuen and sivichai, personal observation). based on those similarities, these fungi were suspected to have same taxonomic address. since jahnula sp. and b. tropicalis. were in order jahnulales (campbell et al. 2007), the same order was suspected to be the taxonomic address for s. pedatospora and xylomyces sp. however, the limited of morphological traits of these fungi made it impossible to determine if they belonged to the same order. the molecular phylogenetics approach therefore offers the opportunity to elucidate the taxonomic position of these fungi. the two objectives of this study are: (i) to address the taxonomic position of s. pedatospora and xylomyces sp. based on its sequence data and (ii) to compare the phylogenic topologies of the order jahnulales calculated on the maximum parsimony, distance matrix method and maximum likelihood analysis. materials and methods fungal isolates. we studied a total 15 strains obtained from the biotec culture collection (bcc, thailand) and the centraalbureau voor schimmelcultures (cbs, netherlands). all strains were maintained on pda. approximately 100 mg of mycelium was used for each dna extraction. dna extraction. fungal mycelium was harvested to the 10 ml tubes and added by sterile sand with 300 ìl 0.5n naoh and griund into a fine powder. the harvested tubes were centrifuged at 11 000 g for 3 min. then 5 ìl supernatant was dissolved in 195 µl tris hcl ph 8.0. these samples were used directly as templates in pcr (modified from wang et al. 1993). dna amplification, purification and sequencing. the its region (its1, 5.8s and its2) was amplified using the pair of primers its 1 and its4 (white et al. 1990). a single 50 µl pcr reaction contained: 34 µl sterile water, 5 µl 10 times pcr buffer (final concentration 1x), 2.5 µl 50 mm mgcl 2 (final concentration 2.5 mm), 1 µl 10 mm dntp mix (final concentration 0.2 mm), 1 µl 10 µm each of the primers (final concentration 0.2 µl), 0.5 µl taq polymerase and 4 µl dna template. the pcr thermal cycling profile for the primer its 1 and its 4 included a first denaturation step at 94°c for 2 min, 35 cycles of 94°c for 1 min, 55°c for 1 min, 72°c for 1 min, and final extension step at 72°c for 2 min. analysis of the pcr products were performed on a 0.8% agarose gels stained with ethidium bromide under a 1000 v illuminator. the pcr products were purified using nucleosprin plant dna purification kit (macherey-nagel). sequencing of the amplified purified pcr product was performed by the biotec research unit (bsu). the sequences were then submited to genbank (www.ncbi.com) to obtain genbank accession numbers. phylogenetics analysis. multiple alignments were performed on the sequences generated in this study with those obtained from genbank (shown with genbank accession numbers) using the clustal w1.6 incorporated in bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). the sequences were adjusted manually to minimize the gap. the data set was exported as a nexus file for maximum parsimony, maximum likelihood and distance matrix method analyses in paup 4.0b10 (swofford 2002). the phylogenetic trees were generated using optimality criteria: maximum parsimony (mp), distance matrix method (neighbor joining/ nj), and maximum likelihood (ml) to generate trees with robust support. maximum parsimony analyses were performed by using heuristic methods with the character first defined as unordered and given equal weighting. successive weighted parsimony was executed to select the more consistent characters. relative support for resulting trees was obtained from bootstrap analysis using 1000 heuristic searches by taking a 50% or greater frequency in the consensus trees. all heuristic searches were performed by tree-bisectionreconstruction branch swapping with 100 random sequences. successive weighting was performed to ensure the stability of the weight and to evaluate the consistency of the characters. successive weighting was performed based on the maximum value of the rescaled consistency index for each character for all most parsimonious trees (farris 1989). the robustness test of the each branch was calculated by the consistency index (ci), retention index (ri), rescaled consistency index (rc) and homoplasy index (hi). the kishino-hasegawa test (kht) was performed in order to determine the best tree fit (kishino and hasegawa 1989). anisomeridium polyori and pyrenula pseudobufonia were used as the out-group. an evolutionary model was constructed by using the modeltest 3.06 (posada and crandall 1998). bayesian posterior probabilities were determined by the markov chain monte carlo sampling in mrbayes 3.0b4 (huelsanbeck and ronqvist 2001), using an estimated model of evolution. markov chains were run for 2 m generation and trees were sampled every 100th generation (resulting 20 000 total trees). the first 2000 trees were used for the burn in phase of the analyses and were discarded. the remaining 18 000 (postburning) trees were used to generate a majority-rule consensus tree. sequences used for phylogenetic analysis (accession numbers in parentheses): aliquandostipitate khaoyaensis (af201728), jahnula sunyatsenii (af201727), capnodium coffea (dq491515), capnodium salicinum (aj244240), dothidea hippophaeos (af027763), d. sambuci (dq491505), d. insculpta (af027763), westerdykella dispersa (dq468031), w. cylindrical (ay943056), elsinoe ampelina (ay826764), e. proteae (af097578), mycosphaerella fijiensis (ef666077), m. graminicola (dq019341), m. punctiformis (ay152594), alternaria alternate (ef192234), pyrenophora phaeocom (dq491507), setosphaeria rostrata (af071342), leptosphaeria maculans (dq133891), l. biglobosa (dq133893), lophiostoma arundinis (aj496633), a. polypori (dq782838), p. pseudobufonia (dq782845). results sequences partial 18s, its1 and its2 regions were aligned along with the intervening 5.8s rdna and partial 28s regions. the average size of this region was 495 bp, of which the shortest sequence was x. acuaticus cbs636.91 (465 bp) and the longest was jahnula sp. ss3792 (536 bp). the partial 18s region ranged from 5 to 49 bp, its1 region from 137 to 172 bp, its2 region from 160 to 189 bp, partial 28s region from 5 to 8 bp, while 5.8 region was conserved (156 bp). overall, s. pedatospora displayed a sequence of 486 to 487 bp, xylomyces sp. 465 to 502 bp, b. tropicalis 485 to 488 bp, jahnula sp. 487 to 536 bp, and t. aristata 487 bp. the gene position of 15 new sequences generated in this study are presented in table 1. the matrix was process to produce phylogenies based on mp (fig 1), nj (fig 2) and ml (fig 3). discussion the phylogenies were placed within the dothideomycetes, which is based on a. polypori and p. pseudobufonia (both species belong to the pyrenulales, eurotiomycetes). two strains of s. pedatospora and x. chlamydosporus were placed in the jahnulales, together with all strains of b. tropicalis and jahnula sp. meanwhile, other strains, including x. elegans, x. aquaticus and t. aristata were clade in pleosporales. the order jahnulales, dothideomycetes was introduced by pang et al. (2002), characterized by stalked/sessile, dimorphic ascomata, hyphal-stalk-cell of approximately volume 2, 2008 microbiol indones 137 table 1 gene position in nuclear partial 18s, its 1, 2 regions, 5.8 rdna, and partial 28s rdna sequence position of each region on the sequence its1 5.8s its2 partial 28s fungal species jahnula appendiculata ss3028 jahnula australiensis ss3613 jahnula sp. ss3792 jahnula granulosa ss3815 xylomyces chlamydosporus ss0807 xylomyces chlamydosporus ss2917 xylomyces elegans ss1077 xylomyces aquaticus cbs636.91 brachiosphaera tropicalis ss2522 brachiosphaera tropicalis ss2523 brachiosphaera tropicalis ss2724 brachiosphaera tropicalis ss2944 speiropsis pedatospora ss2229 speiropsis pedatospora ss2236 tetraploa aristata cbs996.70 accession no. fj887914 fj887915 fj887916 fj887917 fj887918 fj887919 fj887920 fj887921 fj887922 fj887923 fj887924 fj887925 fj887926 fj887927 fj887928 sequence length (bp) 5 1 1 4 8 7 5 3 6 5 2 3 5 0 2 4 9 7 4 9 4 4 6 5 4 8 5 4 8 8 4 8 7 4 8 6 4 8 7 4 8 6 4 8 7 partial 18s 1 5 1 5 1-49 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 6-161 6-158 5 0 1 8 6 6-177 6-162 6-159 6-145 6-144 6-156 6-159 6-158 6-157 6-158 6-157 6-166 1 6 2 3 1 7 1 5 9 3 1 4 1 8 7 3 4 2 1 7 8 3 3 3 1 6 3 3 1 8 1 6 0 3 1 5 1 4 6 3 0 1 1 4 5 3 0 0 1 5 7 3 1 2 1 6 0 3 1 5 1 5 9 3 1 4 1 5 8 3 1 3 1 5 9 3 1 4 1 5 8 3 1 3 1 6 7 3 2 2 3 1 8 5 0 6 3 1 5 4 8 2 3 4 3 5 3 1 3 3 4 5 1 8 3 1 9 4 9 7 3 1 6 4 9 2 3 0 2 4 7 6 3 0 1 4 6 0 3 1 3 4 8 0 3 1 6 4 8 3 3 1 5 4 8 2 3 1 4 4 8 1 3 1 5 4 8 2 3 1 4 4 8 1 3 2 3 4 8 2 5 0 7 5 1 1 4 8 3 4 8 7 5 3 2 5 3 6 5 1 9 5 2 3 4 9 8 5 0 2 4 9 3 4 9 7 4 7 7 4 9 4 4 6 1 4 6 5 4 8 1 4 8 5 4 8 4 4 8 8 4 8 3 4 8 7 4 8 2 4 8 6 4 8 3 4 8 7 4 8 2 4 8 6 4 8 3 4 8 7 fig 1 phylogenetics relationship of speiropsis pedatospora, xylomyces spp., brachiosphaera tropicalis and jahnula spp. based on its and 5.8s rdna sequences. the phylogram represented were the best trees obtain from kishino-hasegawa test in paup* 4b10 based on unweighted parsimony (left) and successive weighted parsimony (right). bootstrap support >50% for both trees were shown above the branches. posterior probabilities were demonstrated above the branches of unweighted parsimony tree after the bootstrap values. the unweighted parsimony yielded 2 mpts with 2204 steps, ci = 0.468, ri = 0.667, rc = 0.312 and hi = 0.532. the successive weighted parsimony analysis yielded a phylogeny with 1031 steps, ci = 0.564, ri = 0.712, rc = 0.402 and hi 0.436. 138 prihatini et al. microbiol indones fig 3 phylogenetics relationship of speiropsis pedatospora, xylomyces, brachiosphaera tropicalis and jahnula spp. based on its and 5.8s rdna sequences. the phylogram calculated on the maximum likelihood analyses in paup* 4b10. the likelihood value = -9229.03443, base frequencies were estimated as follow: a = 0.22546, c = 0.27760, g = 0.24967 and t = 0.24727, the estimated value of the proportion as invariable sites = 0.225, and the gamma shape parameter = 1.536. the maximum likelihood analysis yielded a phylogeny with 2223 steps, ci = 0.466, ri = 0.661, rc = 0.307 and hi = 0.536. fig 2 phylogenetics relationship of speiropsis pedatospora, xylomyces spp., brachiosphaera tropicalis and jahnula spp. based on its and 5.8s rdna sequences. the phylogram calculated on the neighbor joining analysis in paup* 4b10. bootstrap support >50% for neighbor joining trees were shown above the branches. volume 2, 2008 microbiol indones 139 40 µm wide, and ascospores composed of 2-cells or without appendages (pang et al. 2002). the order comprised a single family, the aliquandostipitaceae. jahnulaes are widely distributed from temperate to tropical regions. mostly the species of this order are to be found in freshwater habitats on lignicolous materials. this ecological name refers to their habitats being submerged wood (pang et al. 2002; raja and shearer 2006). moreover, phylogeny analysis confirms that s. pedatospora is closely related to b. tropicalis, with strong boostrap support (98%) and high posterior probabilities (76%). the hyline conidia could be the apomorphic character, since both s. pedatospora and b. tropicalis possessed this morphology. the phylogenies revealed that xylomyces strains comprised a polyphyletic genus. xylomyces chlamydosporus is placed in the jahnulales while x. elegans and x. aquaticus are both accommodated in the pleoporales. even though the genus xylomyces was created based on morphology and ontogeny (goh et al. 1997), their phylogenies did not support that placement. this might result from convergent evolution among the xylomyces species. convergent evolution appears as a result of ecological equivalents. since all xylomeces share an aquatic habitat, this scheme indicates that chlamydospore-morphology in this genus results from adaptation to their habitat. molecular phylogenetic studies also have confirmed the polyphyly of many anamorphic genera and species (shenoy et al. 2007), such as chalara (paulin-mahady et al. 2002), galerina (gulden et al. 2005) and zopfiella (cai et al. 2006). furthermore, jahnulales has been divided into three subclades, each one with strong bootstrap support (>95%). the first subclade is composed of j. appendiculata, a. khaoyaensis, jahnula sp. ss3792 while j. granulosa, b. tropicalis and s. pedatospora are grouped in the second subclade. the third subclade is comprised of two species being j. sunyatsenii and x. chlamydosporus. all of trees based on mp (unweighted and successive weighted parsimony), nj and ml analyses showed similar topology. those strains were placed in the three big clades, with clustering in the jahnulales, pleosporomycetidae and dothideomycetidae. the nj tree has a different branch pattern with mp and ml trees of the three big clades. the nj tree showed dothideomycetidae as a sister group of insertae sedis dothideomycetidae (jahnulales), while in other trees, dothideomycetidae appears as sister group of pleoporomycetidae. the phylogenic tree analyses obtained agrees with result of schoch et al. (2006) that the dothideomycetes was segregated into two subclasses comprised of the dothideomycetidae (dothideales, myriangiales, capnodiales) and the pleosporomycetidae (pleosporales). however several classes belonged to insertae sedis dothideomycetes. based on these phylogenies, the jahnulales is a sister group of either the pleoporomycetidae or the dothideomycetidae. we conclude that the jahnulales was belongs to the insertae sedis dothideomycetes with a 100% bootstrap support and 100% posterior probability. acknowledgement this study was supported by the unesco postgraduated inter-university course in biotechnology 2006-2007 sponsored by thailand government and japanese national commission for unesco. references barbosa ff, gusmao lfp. 2005. two speiropsis species (anamorphic fungi-hyphomycetes) from bahia state, brasil. acta bot bras 19:515-518. cai l, jeewon r, hyde kd. 2006. molecular systematic of zopfiella and allied genera: evidence from multigene sequences analyses. mycol res 110:359-368. campbell j, ferrer a, raja ha, sivichai s, shearer ca. 2007. phylogenetics relationship among taxa in the jahnulales inferred from 18s and 28s nuclear ribosomal dna sequences. can j bot 85:873-882. chang hs. 1994. notes on genus brachiosphaera from taiwan. bot bull acad sin 35:125-127. farris js. 1989. the retention index and the rescaled consistency index. cladistics 5:417-419. goh tk, ho wh, hyde kd, tsui km. 1997. four new species of xylomyces from submerged wood. mycol res 101:1323-1328. gulden o, stensrud o, shalchian-tabrizi k, kauserud h. 2005. galerina earle: a polyphyletic genus in consortium of darkspored agarics. mycologia 97:823-837. huelsenbeck jp, ronqvist f. 2001. mrbayes: bayesian inference of phylogeny. bioinformatics 17:754-755. kishino h, hasegawa m. 1989. evaluation of the maximum likelihood estimate of the evolutionary tree topologies from dna sequence data and the branching order of homonidea. j mol evol 29:170179. pang kl, abdel-wahab ma, sivichai s, el-sharouney hm, jones ebg. (2002). jahnulales (dothideomycetes, ascomycota): a new order of lignicolous freshwater ascomycetes. mycol res 106:10311042. paulin-mahady ae, harrington tc, mcnew d. 2002. phylogenetics and taxonomic evaluation of chalara, chalaropsis, and thielaviopsis anamorphs associated with ceratocystis. mycologia 94:62-72. posada d, crandall ka. 1998. modeltest: testing the model of dna substitution. bioinformatics 14:817-818. raja ha, shearer ca. 2006. jahnula species from north and central america, including three new species. mycologia 98:319-332. shenoy bd, jeewon r, hyde kd. 2007. impact of dna sequencedata on taxonomy of anamorphic fungal. fungal divers 26:1-54. schoch cl, shoemaker ra, seifert ka, hambleton s, sratafora jw, crous pw. 2006. a multigene phylogeny of the dothideomycetes using four nuclei loci. mycologia 98:1041-105. swofford dl. 2002. phylogenetics analysis using parsimony (* and other method). version 4. sinauer associates, sunderland, massachusetts. wang h, meiqing q, cutler aj. 1993. a simple method of preparing plant samples for pcr. nucleic acids res 20:4153-4154. white tj, bruns t, lee s, taylor j. 1990. amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis ma, gelfand dh, sninsky jj, white tj (eds). pcr protocols. san diego. academic pr. p 315-322. 140 prihatini et al. microbiol indones 1. 570 stability (winiati).cdr stability of viable counts of lactic acid bacteria during storage of goat milk soft cheese winiati pudji rahayu , feri kusnandar, widya eka prayitno * and department of food science and technology, faculty of agricultural engineering and technology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia lactobacillus acidophilus l. casei l. acidophilus l. casei l. acidophilus l. casei lactobacillus acidophilus l. casei l. acidophilus l. casei l. acidophilus l. casei the use of goat milk is limited in indonesia due to lack of good milking practices resulted in disliked goaty smell. one of the method to eliminate this off flavor is by processing the goat milk into soft cheese. the aim of this research was to study the stability of viable starter lactic acid bacteria cultures ( fncc-0051 and fncc-0090) during storage of goat milk soft cheese. three batches of goat milk soft cheeses were produced with different starter cultures fncc-0051 (5.0 x 10 cfu ml ); fncc-0090 (5.0 x 10 cfu ml ); and the mixture of fncc-0051 (2.5 x 10 cfu ml ) and fncc-0090 (2.5 x 10 cfu ml ). the goat milk cheeses had white color and soft. the viable lactic acid bacteria in the goat milk soft cheese reached 10 cfu g , which was stable for 8 weeks at 5 °c. panelists liked goat milk soft cheeses, especially in term of its aroma. the specific aroma produced could mask the disliked goaty smell. key words: goat milk, soft cheese, starter lactic acid bacteria susu kambing di indonesia masih terbatas pemanfaatannya karena bau kambing yang kurang disukai, terutama akibat proses pemerahan yang kurang baik. salah satu cara untuk mengurangi aroma yang kurang disukai tersebut ialah dengan mengolahnya menjadi keju lunak. penelitian ini bertujuan menentukan stabilitas kultur starter bakteri asam laktat ( fncc-0051 dan fncc-0090) selama penyimpanan keju lunak susu kambing. keju lunak diproduksi menggunakan tiga kultur starter yang berbeda, yaitu fncc-0051 (5.0 x 10 cfu ml ); fncc-0090 (5.0 x 10 cfu ml ); dan campuran fncc-0051 (2.5 x 10 cfu ml ) dan fncc-0090 (2.5 x 10 cfu ml ). keju yang dihasilkan berwarna putih dan bertekstur lunak. jumlah bakteri asam laktat pada keju lunak susu kambing mencapai 10 cfu g dan relatif stabil selama penyimpanan 8 minggu pada suhu 5 °c. para panelis menyukai aroma keju lunak susu kambing karena mampu menutupi aroma susu kambing yang kurang disukai. kata kunci: keju lunak, starter bakteri asam laktat, susu kambing 6 -1 6 -1 6 -1 6 -1 9 -1 6 -1 6 -1 6 -1 6 -1 9 -1 the goat milk production has steadily increased in indonesia since 2000; however, its usage as a healthy drink is limited due to its unpleasant smell. the goat milk is usually consumed by children and adults. goat milk provides essential nutrients, such as minerals, vitamins and easily digestible proteins with balanced amino acid profile which is important in supporting most body functions (silanikove . 2010). however, many people does not like the goaty smell of goat milk resulted in the low consumption of goat milk. the goaty smell comes from medium-chain fatty acid (caproic, caprylic and capric acids) in goat milk fat (silanikove 2010). in turkey, greece, and france, goat milk cheese has been processed commercially. in recent years, cheese becomes popular in indonesia and it is mainly consumed as a food complement. most of cheeses in indonesia are imported products, particularly, natural cheeses made from cow's milk. goat milk is potential to be processed into cheese products in order to promote the added value of goat milk. recently, world-wide interest on functional foods containing probiotic bacteria for health promotion and et al et al. disease prevention has increased remarkably (vankerckhoven . 2008). cheese product has been developed as a vehicle for probiotic bacteria. in an effort to extend the probiotic product range, a small number of researchers and companies have manufactured cheeses with a high viable count of probiotic cultures. cheese containing probiotic is a functional food. therefore, this study reports the stability of fncc-0051 and fncc-0090 as starter cultures in goat milk soft cheese during storage, and goat milk soft cheese's chemical and sensory state. . the commercial fncc-0051 and fncc-0090 were used as cheese starter cultures. the strains were activated by growing at 37 °c overnight in a sterile de mann's rogossa sharpe broth (mrsb). the goat milk was heated at 85 °c for 30 min. the two lactic acid bacteria (ml l ) were inoculated into goat milk then incubated at 37 °c for 6 h. . goat milk soft cheese was made from heated ettawa goat milk (85 °c et al lactobacillus acidophilus l. casei l. acidophilus l. casei materials and methods starter cultures goat milk soft cheese production -1 *corresponding author, phone/fax:+62-251-8626725, e-mail: wini_a@hotmail.com issn 1978-3477, eissn 2087-8575 vol 5, no 4, december 2011, p 149-153 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.4.1 for 30 min) and 5% (v/v) inoculums of the single or mixed strain starter culture was added. three batches of goat milk soft cheeses were produced with different starter cultures: fncc-0051 (5.0 x 10 cfu ml ) (batch 1), fncc-0090 (5.0 x 10 cfu ml ) (batch 2), and the mixture of fncc-0051 (2.5 x 10 cfu ml ) and fncc0090 (2.5 x 10 cfu ml ) (batch 3). each batch was made in two replicates. the use of lactic acid bacteria reduced the ph which affected the coagulation time. heated goat milk was inoculated by starter culture and was incubated at 37 °c for 6 h. the matured milk was added with liquid rennet (0.06 ml l of milk) and it was coagulated after 2 h. coagulated milk (curd) was cutted with cheeseharp into cube form (1x1x1 cm). the curds were cooked at 40 °c for 30 min. the whey was drained and the fresh cheese was salted (2%, w/w). the soft cheese was packed in plastic bag and was stored at 5 °c for 8 weeks. . the ph change of raw milk, maturated milk, curd, whey, and fresh cheese were measured. the ph of soft cheese was also measured during 8 weeks storage period. the ph was measured according to the standard procedure of aoac (1995). 10 ml of goat milk and whey were measured directly by ph meter (thermo). ten gram of curd and fresh cheese were diluted in 10 ml of aquades before measurement. . to count the viability of starter culture bacteria, twenty gram samples of maturated milk, curd, whey, as well as soft cheese were collected during production. twenty gram of cheese samples were collected each week at first month and every two weeks at second month to find out the stability of the starter lactic acid bacteria during storage. the viable lactic acid bacteria during processing was only counted for the first batch ( fncc-0051) while during storage was counted for all batches ( fncc-0051, fncc-0090, and the mixture of fncc-0051 and fncc-0090). these experiments were carried out in two replicates. the enumeration of the viable lactic acid bacteria was carried out as described by burns et al. 2008. twenty gram of cheese was placed in 180 ml of 2% (w/v) sodium citrate sterile solution. the cheese sample was crushed to bits by stomacher. decimal dilutions of the homogenates were made in 0.0043% (w/v) kh po solution. 1 ml of maturated milk and whey were directly diluted into 9 ml of 0.0043% (w/v) kh po solution. appropriate dilutions were pour-plated. lactic acid bacteria were enumerated l. acidophilus l. casei l. acidophilus l. casei l. acidophilus l. acidophilus l. casei l. acidophilus l. casei 6 -1 6 -1 6 -1 6 -1 -1 ph measurement viability of lactic acid bacteria during processing and storage of soft cheese at 5 °c 2 4 2 4 (37 °c, 48 h) on de mann's rogossa sharpe agar (mrsa). . the acceptance of goat soft cheese was evaluated according to the procedure of drake' (2007). the cheeses were evaluated based on their aroma, taste, and aftertaste by nine trained panelists. a commercial goat milk cheese was tested to know the position of the produced cheese. the soft cheese samples were placed in saucer coded by threedigit random numbers. coffee powder and water were served in between to neutralize the sense of smell. sensory evaluation used 0-15 cm-line scale (0 = dislike intensely and 15 = like extremely). the panelis scored on the line scale. the scores were obtained by measurement the length from 0 cm to the signed mark. then, the scores were analysed by anova. . cheese which has no syneresis during 8weeks storage was used for chemical and trace element analyses. the chemical analyses were moisture content (gravimetric method, bsn 1992a), ash (bsn 1992a), fat (soxhlet method, bsn 1992a), crude protein (kjeldahl method, aoac 1995), and total carbohydrate (by difference). the trace element analyses were done using aas included as, pb, cu, zn, hg, and sn (sni 01-2980-1992 method, bsn 1992b; 1998a; 1998b). . the ph value and viable counts of lactic acid bacteria during cheese production are shown in table 1. ph reduction was observed during cheese production. the viable count of lactic acid bacteria at cultured milk, curd and whey were 10 cfu g and it increased one log cycle in soft cheese (table 1). ph value of soft cheese significantly decreased during storage at 5 °c (p<0.05). the lowest ph occurred after 8 weeks storage at 5 °c was 4.37. viable counts of lactic acid bacteria was stable during storage, which remained constant at 10 cfu g after 8-weeks storage at 5 °c (fig 1). . the average sensory scores by trained panelists are presented in table 2. there was significant (p<0.05) difference in aroma score between the experimental goat milk soft cheese and commercial goat milk soft cheese. goat milk soft cheese had better aroma (11.33-11.97: like fairly well-like very well) than commercial goat milk soft cheese (6.66: like slighly). moreover, no significant (p>0.05) difference in taste and aftertaste score were observed between the cheeses. sensory quality test chemical composition and trace element analyses results ph value and stability of viable lactic acid bacteria during production and storage at 5 °c sensory evaluation 8 -1 9 -1 150 rahayu et al. microbiol indones volume 5, 2011 microbiol indones 151 fig 1 total viable lactic acid bacteria and ph in goat milk soft cheese with different starter culture during 8-week storage at 5 °c. table 2 sensory properties* of goat milk soft cheese with different lab starter culture sample ph l.acidophilus fcnn-0051 (log10 cfu g -1 ) heated raw milk 6.60±0.00 na cultured milk 6.30±0.00 8.45±0.12* curd 6.12±0.02 8.85±0.17 whey 6.15±0.07 8.47±0.09 soft cheese 5.70±0.00 9.94±0.09 na: no available data *cfu ml -1 table 1 ph values and total fcnn-0051 in goat milk soft cheese during production lactobacillus acidophilus samples aroma taste after taste cheese with l.acidophilus fcnn-0051 11.40± 2.38 a) 10.36 ± 2.50 a) 9.49 ± 3.36 a) cheese with l. casei fncc-0090 11.97 ± 2.13 a) 10.42 ± 2.95 a) 8.56 ± 2.82 a) cheese with l.acidophilus fcnn-0051 and l. casei fncc-0090 11.33 ± 2.89 a) 10.37 ± 2.69 a) 8.92 ± 3.50 a) commercial feta cheese 6.66 ± 4.47 b) 8.54 ± 4.09 a) 5.70 ± 3.06 a) * : 0 = dislike intensely and 15 = like extremely :different letters in the same column indicate significant differences (p< 0.05) a,b table 3 nutritional composition of goat milk soft cheese compared to other cheeses component (%) experimental goat milk soft cheese l.acidophilus fcnn-0051 commercial goat milk cheese a sni b fresh soft feta cheddar moisture 52.00 ± 0.67 59.80 ± 6.81 52.30 ± 1.21 41.70 ± 1.76 max. 45.0 ash 3.17 ± 0.05 1.74 ± 0.97 4.30 ± 0.27 3.60 ± 0.13 max. 5.5 fat 23.49 ± 0.68 22.50 ± 4.37 25.30 ± 1.06 26.60 ± 1.13 min. 25.0 protein 15.67 ± 0.03 18.90 ± 5.26 25.10 ± 1.56 30.30 ± 0.56 min. 19.5 a b park (1990); bsn (1992) for cheddar-processed cheese table 4 trace element content of goat milk soft cheese compared to other cheese trace elements (ppm) goat milk soft cheese l.acidophilus fcnn-0051 cheeses sni c goat milk cheese a cow milk cheese b as < 0.003 na na max. 0.1 pb < 0.01 na na max. 0.3 cu 13.53 6.46 na max. 20.0 zn 21.6 15.5 28.1 max. 40.0 hg < 0.0002 na 0.001 max. 0.03 sn < 0.01 na na max. 40.01 a b c park (1990); gambelli et al. (1999); bsn (1992) na: no available data . the selected goat milk soft cheese originated from batch 1, cultured with fncc-0051 was analyzed its chemical composition and trace element content. the chemical composition of the cheese is shown in table 3. the experimental cheese had lower fat and protein content as compared to commercial cheeses. trace element chemical composition and trace element content l. acidophilus content of goat milk soft cheese is shown in table 4. as, pb, hg, and sn level were below limit of detection (lod) of instrument, indicating the cheese was free from heavy metals. however cu and zn were detected in the goat milk soft cheese. cu and zn as essential trace elements for body function were present at 13.53 and 21.56 ppm, respectively. 7.00 6.00 5.00 4.00 3.00 1.00 0.00 2.00 p h 1 2 3 4 5 6 7 storage period (week) 11 10 9 8 7 6 5 4 3 2 1 0 l a b (l o g c fu g ) 1 0 -1 lab batch 1 lab batch 2 ph batch 1 ph batch 2 ph batch 3 lab batch 3 discussion in this research, fcnn-0051 and fcnn-0090 were used as starter cultures for manufacturing cheese. vinderola . 2009; ong and shah 2009 reported that a3 as well as 4962, l10 and 279, l26 were able to use for manufacturing cheese. the decrease of ph value was observed during cheese production due to metabolic activity of starter culture in fermenting lactose during growing, shown by higher population of viable lactic acid bacteria. the milk was fermented for 6 hours and ph value was 6.3. chymosin, which is the most important enzyme in rennet, was activated at ph 6.3. the enzyme helped in hydrolizing peptide bonds, particularly the phe-met bond between residues 105 and 106 of κcasein, which triggered protein coagulation (egito 2007). the goat milk soft cheeses had white color, soft, and crumbly texture. goat milk is reported to form a finer curd than cow milk following acidification, which mimics the conditions in the stomach, suggesting it would be more readily digested. during cooking, the temperature of the cheese curds was 40 °c. have optimal growth temperatures of 35-45 °c, while was able to grow through a wide range of temperatures (15-45 °c) (randazzo . 2004). in suitable temperature, metabolism of viable lactic acid bacteria was optimized. in soft cheese, ph change was attributed to the increase of metabolic activities of bacteria. the culture was in better environmental conditions to multiply in cultured milk affected lower ph value in soft cheese. the number of viable lactic acid bacteria (10 cfu g ) was relatively stable during 8-weeks storage at 5 °c, indicating that storage at 5 °c did not promote metabolic activity of lab in cheese. soft cheese goat milk is a suitable food for the delivery of fcnn-0051 and fcnn-0090 since the culture remained viable. similar result was reported by ong . (2006) at cheddar cheese inoculated with 4962, 279, 1941 as well as inoculated with lafti l10, lafti l26, lafti b94, survived during manufacturing process and maintained their viability of >3.2x10 cfu g at the end of ripening (6 months, 4 °c). ong and shah (2009) also showed the stability of viable counts of 4962 during ripening of cheddar cheese, which was remained viable (>10 cfu g ) at the end of 24 weeks and their viability was not affected by the ripening temperatures (4 and 8 °c). l. acidophilus l. casei et al l. acidophilus l acidophilus l. acidophilus l. casei l. casei et al. l. acidophilus lactobacillus casei et al l. acidophilus l. casei et al l. acidophilus l. casei bifidobacterium longum l. acidophilus l. paracasei b. lactis l. acidophilus 9 -1 ® ® ® 7 −1 8 −1 152 rahayu et al. microbiol indones sensory properties of goat milk cheese are an important factor for consumer in accepting and for producer in manufacturing and marketing the products (ribeiro and ribeiro 2010). in general, goat milk cheese has brighter white color than cow milk cheese due to the low β-carotene in goat milk (raynalljutovac . 2008). lack of the pressing process led to the texture of cheese becomes soft and crumble. the pressing is needed (except for soft cheeses) to achieve form a rind surface. the goat milk soft cheese was accepted by panelist. panelist gave better score to the experimental goat milk soft cheese from batch 1, batch 2, and batch 3 than the commercial one, especially in term of its aroma. the goat milk soft cheese had sour aroma that covered the goaty smell. molecular weight of lactic acid (90.08 g mol ) which is responsible for sour aroma is lower that caproic acid (116.1 g mol ), caprylic acid (144.2 g mol ), and capric acid (172.3 g mol ) which are responsible for goaty smell, so that easily reach the olfactory epithelium. smit . (2005) described that flavour compounds in cheese arise from the action of enzymes from rennet, milk, the starter and non-starter lactic acid bacteria, together with non-enzymatic conversions. moisture content of the cheese was within the range of semisoft or semihard cheese (45-55%). fat and protein composition (%) of experimental goat milk soft cheese were lower than commercial fresh soft nor feta cheese due to its milk composition. heavy metals content (as, pb, hg, and sn) of goat milk soft cheese were below the lod of aas, indicating the cheese was free from toxic substances, while cu and zn as essential trace elements for body function were present at 13.53 and 21.56 ppm, respectively. et al et al -1 -1 -1 -1 references aoac. 1995. official method of analysis of the aoac. 14 ed. arlington, virginia: aoac. badan standardisasi nasional.1992a. keju cedar olahan [cheddar cheeses processed].sni 01-2980-1992. jakarta (id): bsn. badan standardisasi nasional. 1992b. cara uji makanan dan minuman [food and beverage analyzed]. sni 01-2891-1992. jakarta (id): bsn. badan standardisasi nasional. 1998a. cara uji cemaran arsen dalam makanan [arsenic contamination food analyzed]. sni 01-4866-1998. jakarta (id): bsn. badan standardisasi nasional. 1998b. cara uji cemaran logam dalam makanan [metal contamination food analyzed]. sni 01-2896-1998. jakarta (id): bsn. burns p, patrignani f, serrazanetti d, vinderola gc, reinheimer ja, lanciotti r, guerzoni me. 2008. probiotic crescenza cheese containing and manufactured with high-pressure homogenized milk. j dairy sci. 91(2):500-512. doi:10.3168/jds.2007-0516. drake' ma. 2007. invited review: sensory analysis of dairy foods. j dairy sci. 90(11):4925-4937. doi:10.3168/jds.2007-0332. egito as, girardet jm, laguna le, poirson c, mollé d, miclo l, humbert g, gaillard jl. 2007. milk-clotting activity of enzyme extracts from th lactobacillus casei lactobacillus acidophilus sunflower and albizia seeds and specific hydrolysis of bovine -casein. int dairy j. 17(7):816-825. doi:10.1016/j.idairyj.2006.09.012 gambelli l, belloni p, ingrao g, pizzoferrato l, santaroni gp. 1999. minerals and trace elements in some italian dairy products. j food comp anal. 12(1):27-35. doi:10.1006/jfca.1998.0802. ong l, henriksson a, shah np. 2006. development of probiotic cheddar cheese containing , , and spp. and the influence of these bacteria on proteolytic patterns and production of organic acid. int dairy j. 16(5):446456. doi:10.1016/j.idairyj.2005.05.008. ong l, shah np. 2009. probiotic cheddar cheese: influence of ripening temperatures on survival of probiotic microorganism, cheese composition and sensory organic acid profiles. j food sci tech. 42(7):1260-1268. doi: 10.1016/j.lwt.2009.01.011. park yw. 1990. nutrient profiles of commercial goat milk cheeses manufactured in the united states. j dairy sci. 73(11):3059-3067. doi:10.3168/jds.s0022-0302(90)78993-x. randazzo cl, restuccia c, romano ad, caggia c. 2004. , dominant species in naturally fermented sicilian green olives. int j food microbiol. 90(1):9-14. doi:10.1016/so1681605(3)00159-4. κ . lactobacillus acidophilus lb. casei lb. paracasei bifidobacterium lactobacillus casei raynal-ljutovac k, lagriffoul g, paccard p, guillet i, chilliard y. 2008. composition of goat and sheep milk products: an update. small rum res. 79(1):57-72. doi.10.1016/j.smallrumres.2008.07.009. ribeiro ac, ribeiro sda. 2010. specialty products made from goat milk. small rum res. 89(2):225-233. doi:10.1016/j.smallrumres.2009.12. 048. silanikove n, leitner g, merin u, prosser cg. 2010. recent advances in exploiting goat's milk: quality, safety and production aspects. small rum res. 89(2):110124. doi:10.1016/j.smallrumres.2009.12.033. smit g, smit ba, engels wjm. 2005. flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products. fems microbiol rev. 29(3):591-610. doi: 10.1016/j.fmrre.2005.04.002. vankerckhoven v, huys g, vancanneyt m, vael c, klare i, romond mb, entenza jm, moreillon p, wind rd, knol j, wiertz e, pot b, vaughan ee, kahlmenter g, goossens h. 2008. biosafety assessment of probiotics used for human consumption: recommendation from the eu-prosafe project. trends in food sci tech. 19(2):102-114. doi:10.10.16/j.tifs.2007.07.013. vinderola g, prosello w, molinari f, ghiberto d, reinheimer j. 2009. growth of a13 in argentinian probiotic cheese and its impact on the characteristics of the product. int j food microbiol. 135(2):171-174. doi:10.1016/j.ijfoodmicro.2009.08.021. lactobacillus paracasei volume 5, 2011 microbiol indones 153 1. 530 antibacterial (i made)... antibacterial activity of propolis supplemented-chewing candy against streptococcus mutans i made artika , haryanto susilo , adinda virginia dwi setyo , ahmad endang zainal hasan 1* 2 2 1 and 1 2 department of biochemistry, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; department of pharmacy, faculty of mathematics and natural sciences, universitas pakuan, jalan pakuan, bogor 16143, indonesia streptococcus mutans s. mutans trigona in vitro s. mutans trigona s. mutans. streptococcus mutans, trigona streptococcus mutans s. mutans trigona in vitro s. mutans trigona s. mutans streptococcus mutans, trigona is considered to play a major etiological role in development of human dental plaque believed to related to dental caries, the most prevalent disease of the human oral cavity. the objectives of the present study were to formulate and produce propolis supplemented-chewing candy and to investigate its antibacterial activity against . propolis is a natural resinous bee-hive product thought to have antimicrobial, anti-inflammatory and immunostimulating activities. propolis was extracted from hives of bees of spp. using ethanol. the extract was coated with maltodextrine and homogenized to generate propolis microparticles. the particles were introduced into chewing candy preparations for the production of propolis supplemented-chewing candy. the candy was then subjected to antibacterial assays to test its activity against isolated from human dental plaque. results showed that the ethanol extracted propolis of spp. bee-hives can be homogenized to form propolis microparticles. the propolis microparticles could be used as a supplement in the formulation of chewing candy preparations. the propolis supplemented-chewing candy showed antibacterial activity against the candy, therefore, has the potential to be used as an antiplaque agent for prevention of dental caries. key words: antibacterial activity, propolis supplemented-chewing candy, spp. diduga memegang peran penting dalam etiologi perkembangan plak gigi pada manusia yang berhubungan dengan karies gigi, penyakit rongga mulut yang paling banyak terjadi. penelitian ini bertujuan memformulasi dan memroduksi permen kenyal berpropolis serta menentukan aktivitas antibakterinya terhadap . propolis bahan alami mengandung resin yang diperoleh dari sarang lebah dan diketahui memiliki aktivitas antibakteri, antiinflamasi, dan imunostimulasi. propolis diekstraksi dari sarang lebah spp. menggunakan etanol. ekstrak kemudian dilapisi dan dihomogenisasi untuk membentuk mikropartikel propolis. partikel ini selanjutnya dicampurkan ke dalam sediaan permen kenyal dalam pembuatan permen kenyal berpropolis. aktivitas antibakteri permen selanjutnya diuji secara terhadap yang diisolasi dari plak gigi. hasil menunjukkan bahwa ekstrak etanol propolis dari sarang lebah spp. dapat dihomogenisasi membentuk mikropartikel propolis. mikropartikel propolis dapat dijadikan suplemen dalam formulasi sediaan permen kenyal berpropolis. permen kenyal berpropolis menunjukkan aktivitas antibakteri terhadap . oleh karena itu, permen ini berpotensi untuk digunakan sebagai bahan antiplak gigi guna mencegah karies gigi. kata kunci: aktivitas antibakteri, permen kenyal berpropolis, spp. dental caries continues to be an important public health problem in some parts of the world and is considered to be the most prevalent disease affecting the human oral cavity (duailibe . 2007). the incidence is particularly high during childhood (gasparini . 1989; okada . 2005). the tooth enamel and dentin are demineralized by acids, such as lactic acid, which are produced as a by-product of carbohydrate metabolism by cariogenic bacteria in dental plaque (yoo . 2007). is the leading cause of dental caries worldwide, and is considered to be the most cariogenic amongst the oral streptococci. its etiological role in human dental decay has been extensively discussed by hamada and slade (1980). the relationship between dental plaque and the occurrence of dental caries has also been indicated by others (newman 1986). et al et al et al et al streptococcus mutans to date, the prevention and control of dental caries, is not restricted to a single procedure. in addition to traditional methods, such as periodic dental followups, brushing with fluoride-containing dentifrices, topical application of fluorides, low-sucrose diets, a more effective control procedure needs to be applied in some cases. researchers are currently also interested in the prospect that natural substances offer alternatives for the control of dental caries (duailibe . 2007). the use of natural agents against selected oral pathogens has been reported (li . 1997). propolis, a natural product from the common honeybee ( ) has been shown to exert antibacterial action against a number of oral microorganisms including (duailibe . 2007; ophori . 2010). however, the detailed mechanism of propolis antibacterial activity has yet to be elucidated. propolis has also been reported to inhibit cell adhesion as well as water-insoluble-glucan formation by (koo et al et al apis mellifera s. mutans et al et al s. mutans et *corresponding author, phone/fax:+62-251-8423267, e-mail: imart171@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 99-102 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.1 al et al a. mellifera et al a. mellifera trigona trigona campylobacter s. mutans et al trigona s. mutans s. mutans et al trigona et al . 2000). in addition, it possesses anti-inflammatory, local anesthetic, hepatic-protective, antitumor, and immunostimulating activities (bankova . 2000). the chemical composition of propolis is highly variable and depends on the local flora at the site of pollen collection. although its biological activity, especially against microorganisms is always present, samples from different geographic and climatic zones have their activities resulting from completely different chemical composistions (bankova 2005). the chemical composition of propolis produced by from different regions in java, indonesia, has also been investigated. it was found that the chemical composition of this propolis is strikingly diverse. all of the samples analyzed, however, contain phenolic acids which are thought to play roles in propolis bioactivity (syamsudin . 2009). in addition to , propolis is also produced by the stingless bee spp. these bees tend to make less honey and produce more propolis. previous studies have shown that the propolis of spp. contains flavonoids and shows activity against a number of bacterial strains including spp. and (fatoni . 2008). in the present study, propolis of spp. was introduced into chewing candy preparations and the antimicrobial activity of the candy against was then analyzed in order to elucidate as to whether propolis is still active against after being blended into a chewing candy preparation. . propolis was extracted using the method of fatoni . (2008) with slight modifications. raw propolis of spp. collected from pandeglang, banten province, indonesia was cut into small pieces, ground and extracted with 70% ethanol (1:5 w/v) in shaker (eyela, japan) at a speed of 130 rpm and at room temperature for 14 days. the clear filtrate was then decanted and followed by solvent evaporation using an evaporator. the solid extract obtained was then solubilized in 70% ethanol (1:1 w/v). the solution was used as the propolis stock. . propolis microparticles were generated using a modified method of bhaskar . (2009). maltodextrine was used as a coating agent to protect propolis bioactive chemicals from conditional factors such as heat and moisture change during the process of nanoparticle formation. a thin coating using maltodextrine was also intended to make propolis water soluble. magnesium stearate was included as a powdery anti-sticking materials and methods extraction of propolis generation of propolis microparticles agent for making the sticky surface of propolis microparticles non-adhesive. maltodextrine (85 g) and magnesium stearate (5 g) were dissolved in 100 ml water. the mixture was then homogenized at 22 000 rpm for 30 min. to the mixture was added 120 ml of 20% (v/v) propolis stock and the mixture was homogenized at 22 000 rpm for 30 min. the solution was then dried using a vacuum dryer at a temperature of approximately 45 °c. the powder obtained was ground using high energy milling to generate microspheres of less than 500 nm in diameter. the size of microparticles in the resulting powder was then examined under the scanning electron microscope. the moisture of the powder was determined using a moisture balance. chewing candy preparations were formulated to contain 11% (w/v) of gelatine, 10% (w/v) of sucrose, 0.05% (w/v) of methylparabene, 0.05% (w/v) citric acid, and a required amount of colouring essence. propolis was supplemented at three different concentrations ie. 6, 7, and 8% (w/v). preparations without propolis supplementation were also prepared to act as a control. the propolis concentration in each preparation was determined spectroscopically at a wavelength of 324 nm using a propolis solution of known concentration as the standard. in order to generate elastic chewing candy, gelatine was initially soaked in water for several minutes followed by boiling with stirring until a homogenous gelatine solution was obtained. separately, a solution containing sucrose, methylparabene, citric acid and essence was prepared and this was added to the gelatine solution. the solution was homogenized by stirring at 6 rpm, at 70 °c. the solution was incubated until the temperature fell to about 50 °c prior to the propolis addition, followed by homogenization. the solution was then poured into a candy template to obtain shaped candy of 15 g pieces. the shaped candy was then cooled at -4 °c in order to obtain an elastic texture. antibacterial activity of propolis supplemented-chewing candy was determined using the disc-diffusion method (andrew 2009). an aliquot of 50 l of culture isolated from human dental plaque was inoculated homogenously into a petri dish containing 20 ml warm pyg agar medium [1% (w/v) peptone, 1% (w/v) yeast extract, 2% (w/v) glucose, 2% (w/v) bacto agar] followed by incubation until the medium solidified. a paper disc of 5 mm in diameter was placed on top of the medium. as much as 10 l of water solubilized candy (3:4 w/v) was loaded onto the paper disc and the petri disc was incubated at 37 °c for 24 h. the diameter production of propolis supplemented-chewing candy. antibacterial activity assay. μ μ s. mutans 100 artika et al. microbiol indones of the inhibitory zone was measured using microcallipers. assays were performed in triplicate. macerate of raw propolis, which was dark in colour, resulted in a brown filtrate. following solvent evaporation, solid propolis extract having a soft consistency, being sticky, and dark brown in colour was produced. the propolis yield obtained was 13% (w/w). preparation of propolis microparticles resulted in dry propolis microparticle powder. the powder had a water content of 4.4% (w/w). under the scanning electron microscopy, using 3000 magnification, the average size of the coated particles was about 500 nm in diameter. the size of the propolis microparticles themselves is believed to be smaller. under these conditions, the smallest particle detected had a diameter of about 180 nm. the particles showed a wrinkled irregular shape with a coarse surface. this might be due to water loss from the particles during the vacuum-drying process (fig 1). propolis could be supplemented into chewing candy preparations. the candy preparation generated was elastic and transparent. the preparation could be shaped by using a candy template. the propolis supple mented-chewing candy showed antibacterial activity against . this is indicated by the formation of inhibition zones on the test plates. the highest activity was shown by candy supplemented with 8% (w/v) propolis (table 1). results generation of propolis extract. generation of propolis microparticles. production of propolis supplemented-chewing candy. antibacterial activity of propolis supplemented-chewing candy. s. mutans discussions dental caries is a disease of the teeth that can lead to pain, infection, tooth loss, and in severe cases, death. most school children and adults worldwide have experienced dental caries. the disease is most prevalent in asian and latin american countries, and has been attributed to a number of factors such as age, culture, dietary habits, race and geographical location (ophori . 2010). the goal of the present study was to develop a dental antiplaque-agent in the form of propolis supplemented-chewing candy that potentially can be used in the prevention of dental caries, especially amongst school children. for these purposes the antibacterial activity of the propolis supplementedchewing candy against was tested. propolis is generally derived from beehives of . in this study propolis was extracted from beehives of spp. collected from pandeglang district, banten province, indonesia. spp. belongs to stingless bee group the propolis from which has yet to be widely explored. prior to its use in the candy preparation, the propolis extract was homogenized to microparticles in order to improve its effectiveness as antimicrobial agent by size reduction and hence increased surface area. assays showed that the propolis microparticles have antibacterial activity against at a propolis concentration of 6, 7, and 8% (w/v) (un published data). from the present study it is clear that the antibacterial activity of the propolis supplementedchewing candy was due to the bioactive compounds of the propolis. the data also indicated that the bioactivity of the propolis active substances was successfully maintained during propolis nanoparticle preparation and candy production. the inhibitory activity of propolis from other bees on has also been reported (duailibe . 2007; ophori . 2010). ophori . (2010) showed that an ethanol extract of propolis obtained from beehives of has a strong antimicrobial activity against isolated from dental caries in an studies. similarly, duailibe . (2007) showed that extract prepared with propolis produced by the bee possesses antimicrobial activity against present in the oral cavity. although the detailed chemical composition of the propolis used in the present study has yet to be investigated, previous studies have indicated that propolis of spp. contains flavonoids and tannins thought to be responsible for its antibacterial activity (fatoni . 2008). compounds responsible et al s. mutans a. mellifera trigona trigona in vitro s. mutans s. mutans et al et al et al a. mellifera s. mutans in vitro et al melipona compressipes fasciculata in vivo s. mutans trigona et al fig 1 propolis microparticles of spp. propolis microparticles (coated samples) were observed under the scanning electron microscopy at 7 kv in a vacuum. trigona table 1 growth inhibition zone due to antibacterial activity of propolis supplemented-chewing candy streptococcus mutans propolis concentration of candy (%, w/v) diameter of inhibition zone (mm) 0 not detected (control) 6 6.3 ± 0.6 7 6.3 ± 0.6 8 8.0 ± 1.0 amoxycillin 10 mg/ml 17.3 ± 2.1 volume 5, 2011 microbiol indones 101 for propolis antibacterial activity vary considerably depending on the local flora at the site of collection. european propolis (poplar type) contains flavanones, flavones, phenolic acids and their esters found to be responsible for antibacterial activity. in brazilian propolis ( type), compounds responsible for antibacterial activity are prenylated-coumaric acids and labdane diterpenes, while in cuban propolis they are prenylated benzophenones (bankova 2005). baccharis p references andrew jm. 2009. bsac standardized disc susceptibility testing method (version 8). j antimicrob chemother. 64(3):454-489. doi:10.1093/jac/ dkp244. bankova v. 2005. recent trends and important developments in propolis research. evid based complem altern med. 2(1):29-32. doi:10.1093/ ecam/neh059. bankova v, castro sl, marcucci mc. 2000. propolis: recent advances in c h e m i s t r y a n d p l a n t o r i g i n . a p i d o l o g i e . 3 1 ( 1 ) : 3 1 5 . doi:10.1051/apido:2000102. bhaskar k, anbu1 j, ravichandiran v, venkateswarlu v, rao ym. 2009. lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, and studies. lipids health dis. 8(6):1-15. doi:10.1186/1476-511x-8-6. duailibe sac, goncalves ag, ahid fjm. 2007. effect of a propolis extract on counts . j appl oral sci. 15(5):420-423. in vitro, ex vivo in vivo streptococcus mutans in vivo fatoni a, artika im, hasan aez, kuswandi. 2008. antibacterial activity of propolis produced by spp. against spp. hayati j biosci. 15(4):161-164. gasparani r, pozzi t, fonzi l, rossolini gm, mazzini m, felagalli a, pozzi g. 1989. prevalence of and dental decay in school children from siena (italy). eur j epidemiol. 5(2): 189-192. hamada s, slade hd. 1980. biology, immunology, and cariogenicity of . microbiol mol biol rev. 44(2):331-384. koo h, rosalen pl, cury ja, ambrosano gmb, murata rm, yatsuda r, ikegaki m, alencar sm, park yk. 2000. effect of a new variety of propolis on mutans streptococci. curr microbiol. 41(3):192196. li xc, cai l, wu cdc. 1997. antimicrobial compounds from against oral pathogens. phytochemistry. 46(1):97-102. newman hn. 1986. the relation between plaque and dental caries. j r soc med. 79(14):1-5. okada m, soda y, hayashi f, doi t, suzuki j, miura k, kozai k. 2005. longitudinal study of dental caries incidence associated with and in pre-school children. j med microbiol. 54(7): 661-665. doi:10.1099/jmm. 046069-0. ophori ea, eriagbonye bn, ugbodaga p. 2010. antimicrobial activity of propolis against . afr j biotechnol. 9(31):49664969. syamsudin, wiryowidagdo s, simanjuntak p, heffen wl. 2009. chemical composition of propolis from different regions in java and their cytotoxic activity. am j biochem biotech. 5(4):180-183. yoo sy, park sj, jeong dk, kim k, lim s, lee s, choe s, chang y, park i, kook j. 2007. isolation and characterization of the mutans streptococci from the dental plaques in koreans. j microbiol. 45(3):246-255. trigona campylobacter streptococcus mutans streptococcus mutans apis mellifera ceanothus americanus streptococcus mutans streptococcus sobrinus streptococcus mutans 102 artika et al. microbiol indones 11 (sedarnawati yasni) catatan penelitian (91-93).pmd short communication antiphatogenic and anti food spoilage activities of ethylacetate and methanol extract of panax ginseng var. notoginseng sedarnawati yasni department of food science and technology, faculty of agricultural technology, institut pertanian bogor, darmaga campus, bogor 16680, indonesia phone: +62-251-626725, 356889, fax: +62-251-62672, e-mail: sedarnawati@yahoo.com javanese ginseng is a traditional herb known to possess broad health benefits that have been clinically proven. the aim of this research was to analyze the antimicrobial activity of javanese ginseng against pathogenic bacteria (escherichia coli, salmonella typhimurium, bacillus cereus, staphylococcus aureus), food spoilage bacteria (bacillus stearothermophilus and pseudomonas fluorescens) and food spoilage fungi (aspergillus flavus, fusarium graminearum, and penicillium citrinum). the result may increase the utilization of ginseng not only for health purposes but also as a natural food preservative. it may also open new possibilities for the development of natural functional foods. ethylacetate and methanol extracts, obtained by maceration, were fractionated employing vacuum liquid chromatography (vlc). fractionation using methanol and ethylacetate as solvents produced six fractions from each solvent. fractions 1 and 4 of methanol extract performed the highest growth inhibitory effects on bacillus cereus (gram-positive bacteria) and escherichia coli (gram-negative bacteria), whereas fractions 4 and fraction 5 of methanol extract effectively inhibited the growth of penicillium citrinum. key words: antimicrobial activity, extract, javanese ginseng, pathogenic bacteria, food spoilage _____________________________________________ ginseng (panax spp.) is a herb whose roots and rhizomes have been widely used as traditional medicine all over the world. more than 10 panax spp. (araliaceae) have been used, but the most popular is panax ginseng (asian ginseng). zhu et al. (2004) reported that almost 200 components of p. ginseng had been isolated and characterized, among others were ginsenosides, polyacetylenes, alkaloids, polysaccharides, oligosaccharides, oligopeptides, flavonoids, lipids, vitamins, and minerals; with ginsenosides (saponin triterpene) as the main fraction exhibiting biological activities. ginseng has been known to possess bioactive compounds and physicochemical activities that can decrease the risks of various diseases. according to a number of pharmacological studies undertaken over the last 20 years, p. ginseng extract could influence the cardiovascular system, the immune system, the endocrine system, and the nervous system (oliveira et al. 2005); causing lowered blood pressure (kim et al. 1994; han et al. 1998; jeon et al. 2000; sung et al. 2000); and playing a role as an antioxidant (kim et al. 1992). empirically, ginseng had been used as supplement to cure fatigue (attele et al. 1999), improve gastrointestinal symptoms, as a sedative, and as a tonic (takagi et al. 1972 a, b; nabata et al. 1973; kaku et al. 1975). in fact, ginseng water extract, especially ginsenosides, had the ability to accelerate small intestine transit times which meant it was effective in directly suppressing intestinal motility on muscles and inhibited the cholinergic nervous system (hashimoto et al. 2003). hexane extracts from ginseng (panax ginseng var. notoginseng) rhizomes did not show antimicrobial activities against pathogenic bacteria (escherichia coli, salmonella typhimurium, and bacillus cereus) and food spoilage bacteria (staphylococcus aureus, bacillus stearothermophilus, and pseudomonas fluorescens) or food spoilage fungi (aspergillus flavus, fusarium graminearum, and penicillium citrinum). on the other hand, ethylacetate extracts had the ability to inhibit the growth of all microbes tested, while methanol extracts were able to inhibit only the bacteria tested (unpublished data). therefore, the goal of this research was to reexamine the antimicrobial activities of ethylacetate and methanol extract fractions against pathogenic and food spoilage bacteria, as well as food spoilage fungi. sample material used in this research was javanese ginseng rhizome powder obtained from semarang (central java). bacteria tested were e. coli, s. typhimurium, b. cereus, s. aureus, b. stearothermophilus, and p. fluorescens obtained from the food microbiology laboratory, center for food and nutrition studies, institut pertanian bogor; fungi tested were a. flavus, f. graminearum, and p. citrinum all from the microbiology laboratory, indonesian institute of sciences (lembaga ilmu pengetahuan indonesia), bogor. test bacteria were prepared in 10 ml nutrient broth media, incubated at 37 oc for 24 h. the test-bacteria culture (105 cfu ml-1) was for use in the antimicrobial activity test of ginseng extract. fungal cultures were subcultured on potato-dextrose-agar medium and incubated at 28 oc for 4-7 days until spores were produced. spore suspensions were prepared by adding physiological saline solution containing 0.5% (vol/vol)tween 80 to give a concentration of 105 spores ml-1. this spore suspension would be used in antimicrobial activity test of ginseng extract. ethylacetate and methanol extracts of javanese ginseng, which were known to have antimicrobial activities, were obtained through multistage extractions without heating. the microbiology indonesia, august 2007, p 95-97 volume 1, number 2 issn 1978-3477 extraction process was conducted with the ratio of material to solvent 1:3 and repeated several times until a colorless extraction was produced. each extract was evaporated using rotavapor at 45 oc to remove solvent residue. concentrated extracts were placed into dark bottles, redried using freeze drying method, and then blown with n 2 . the extracts would be used for antimicrobial tests employing a welldiffusion method (garriga et al. 1993). in antibacterial activity test, the four wells with a 6.2 mm diameter were prepared in one petri dish, 60 µl of javanese ginseng extract was put into two wells (duplicate) and the other two wells were each filled with 60 µl dimethyl sulfoxide (dmso), and another with 60 ml solvent (both as a negative control). then the agar plate was incubated for 24 h at 37 oc. fungal antimicrobial activity tests were conducted by inoculating 60 µl javanese ginseng extract into two wells, and the other 2 wells containing 60 µl dmso, and 60 µl solvent (as a negative control) respectively. the agar plate was incubated for 48 h at 28 oc (room temp.). the fractionation of ethylacetate and methanol extracts of javanese ginseng, were known to possess high antimicrobial activities and broad spectrum inhibition against all tested microbes, was conducted employing vacuum-columnchromatography with silica gel 60 gf 254 with a particle size at less than 45 µm. the objective was to find the fractions that played a role in the inhibition of bacterial growth. initial fractionation was conducted to determine the optimal solvent system using thin layer chromatography (tlc). solvents used consisted of proanalysis grade diethylether, petroleum ether, and chloroform. the fractions obtained were evaporated using a rotavapor at 45 oc, dried using n 2 , and then weighed. a solution of each fraction (5%, wt/vol) was made by dissolving in dmso for later use in the antimicrobial activity test. ethylacetate extracts and methanol extracts were subfractioned into six fractions. all six fractions from the ethylacetate extract did not show growth inhibitory abilities against the fungi tested. the fractions 1, 2, 4, 5, and 6 from the ethylacetate extract were able to inhibit the growth of b. cereus. in addition fraction 1 was able to inhibit the growth of all bacteria tested except for b. stearothermophilus, while fractions 3-6 inclusive were able to inhibit the growth of e. coli and p. fluorescens (table 1). fraction 1-4 inclusive of methanol extract were able to inhibit the growth of all tested bacteria, whereas fraction 5 was only able to inhibit the growth of b. cereus (4.87 mm), b. stearothermophilus (4.36 mm), e. coli (3.65 mm), s. typhimurium (2.30 mm), and fraction 6 was able to inhibit the growth of b. stearothermophilus (3.00 mm) and s. aureus (0.70 mm). all six fractions of methanol extracts did not show growth inhibitory abilities against the fungus f. graminearum. in addition, fractions 1 and 2 were able to inhibit the growth of the fungus a. flavus (1.20 mm and 0.60 mm respectively), while fractions 4 and 5 were only able to inhibit the growth of p. citrinum (3.35 mm and 4.55 mm respectively) (table 2). inhibition of several test bacteria by the ethylacetate extract was lower than that of the methanol extracted fractions, despite an indication from a previous research (unpublished data) that ethylacetate extract possessed antimicrobial activities against all tested bacteria. therefore, it is safe to say that many fractions soluble in ethylacetate do not contain the active components needed to inhibit the growth of the test microbes. fractions of methanol extract of javanese ginseng contained more active components. as a result, they had more effective inhibitory activity against the bacteria tested. in general, the methanol extract contains phenols, hydroquinones, alkaloids, flavonoids, steroids, triterpenoids, saponins, and tannins (hiserdt 1998). extracted phenolic components can easily penetrate bacterial cell walls, which might explain the high inhibitory activity against bacteria tested. this mechanism could be caused by the presence of accumulated lipophilic components in their cell wall or cell membrane which changed the composition of the cell wall components. it is suspected that bacterial inhibition mechanism by methanol extract was the result of a reaction between the extract and cell membrane or components in cytoplasm. phenolic compounds could react with bacterial cell, disturb transportation processes, cause coagulation of cytoplasmic components, and disturb the proton motive force that plays a role in the cell’s energy production. based on the data of the antimicrobial activity test conducted on each six fractions of methanol and ethylacetate extracts, we can conclude that the fractions of methanol extract had broader antimicrobial capabilities compared to that of ethylacetate extract. fraction 1, fraction 2, fraction 3, and fraction 4 of the methanol extract had a broad inhibitory spectrum against all tested bacteria with the highest inhibitory ability differently for each kinds of bacteria tested. antifungal activity of the methanol-extract-fractions was shown only by fraction 1, fraction 2, fraction 4, and fraction 5, while all six fractions the methanol extract had no antifungal table 1 activities of fractions of ethylacetate extract of javanese ginseng against several tested microbes zone of inhibition (mm) of fraction 1 2 3 4 5 6 tested microbes b. cereus b. stearothermophilus e. coli s. typhimurium s. aureus p. fluorescens a. flavus p. citrinum f. graminearum 0.95 0 0.90 0.55 0.55 0 0 0 0 1.01 0 0 0 0 0 0 0 0 0 0 0.60 0 0 0.48 0 0 0 0.85 0 0.10 0 0.40 1.38 0 0 0 1.00 0 0.40 0.70 0 0.28 0 0 0 2.18 0 0.35 0 0 0.88 0 0 0 table 2 activities of fractions of methanol extract of javanese ginseng against several tested zone of inhibition (mm) of fraction 1 2 3 4 5 6 tested microbes b. cereus b. stearothermophilus e. coli s. typhimurium s. aureus p. fluorescens a. flavus p. citrinum f. graminearum 5.04 4.78 3.85 4.22 4.53 1.98 1.20 0 0 4.22 4.72 3.30 4.78 4.19 3.13 0.60 0 0 2.97 4.17 3.13 1.01 1.90 2.13 0 0 0 5.17 4.91 5.77 3.67 3.02 3.93 0 3.35 0 4.87 4.36 3.65 2.30 0 0 0 4.55 0 0 3.00 0 0 0.70 0 0 0 0 96 short communication microbiol indones capabilities against the fungus f. graminearum. ethylacetate fractions had low inhibitory abilities and inhibited few as tested bacteria, they were also unable to inhibit the growth any of the tested fungi. this research has resulted in useful scientific information to widen the utilization of ginseng, not only for health purposes, but also as natural food preservative. by combining its benefits for health and its ability to preserve food naturally, it is possible to develop a functional drink formula for special purposes without the need to add synthetic preservative agents. references attele as, wu ja, yuan c. 1999. ginseng pharmacology, multiple constituents and multiple actions. biochem pharmacol 58:16851693. garriga m, hugas m, aymerich t, monfort jm. 1993. bacteriocinogenic activity of lactobacilli from fermentation sausages. j appl bacteriol 75:142-148. han kh, choe sc, kim hs, sohn dw, nam ky, oh bh, lee mm, park yb, choi ys, seo jd, lee yw. 1998. effect of red ginseng on blood pressure in patients with essential hypertension and white coat hypertension. am j chin med 26:199-209. hashimoto k, satoh k, murata p, makino b, sakakibara i, kase y, ishige a, higuchi m, sasaki h. 2003. components of panax ginseng that improve accelerated small intestinal transit. j ethnopharmacol 84:115-119. hiserdt rd, franzblau sg, rosen rt. 1998. isolation of 6-,8and 10gingerol from ginger rhizome by hplc and preliminary evaluation of inhibition of mycobacterium avium and mycobacterium tuberculosis. j agric food chem 3:477-480. jeon bh, kim cs, park ks, lee jw, park jb, kim k-j, kim sh, chang sj, nam kj. 2000. effect of korea red ginseng on the blood pressure in concious hypertensive rats. gen pharmacol 35:135-141. jones d, gorman s, mccafferty df, woolfson ad. 1991. the effects of three non-antibiotic, antimicrobial agents on the surface hydrophobicity of certain microorganism evaluated by difference methods. j appl bacteriol 71:218-227. kaku t, miyata t, uruno t, sako i, kinoshita a. 1975. chemicopharmacological studies on saponins of panax ginseng c.a. meyer. arzneim-forsch 25:539-547. kim h, chen x, gillis cn. 1992. ginsenosides protect pulmonary vascular endothelium against free radical–induced injury. biochem biophys res commun 189:670-676. kim jm, marshal mr, cornel ja, boston jf, wei ci. 1995. antibacterial activity of carvacrol, citrals and geraniols against salmonella typhimurium in culture medium and fish cubes. j food sci 60:1365-1368. kim nd, kang sy, schini vb. 1994. ginsenosides evoke endothelium dependent vascular relaxation in rat aorta. gen pharmacol 25:1071-1077. nabata h, saito h, takagi k. 1973. pharmacological studies of neutral saponins of panax ginseng root. jpn j pharmacol 23:294 1 . oliveira, ac, perez ac, prieto jg, duarte idg, alvarez ai. 2005. protection of panax ginseng in injured muscles after eccentric exercise. j ethnopharmacol 97:211-214. sung j, han kh, zo jh, park hj, kim ch, oh bh. 2000. effects of red ginseng upon vascular endothelial function in patients with essential hypertension. am j chin med 28:205-216. takagi k, saito h, nabata h. 1972a. pharmacological studies of panax ginseng root: estimation of pharmacological actions of panax ginseng root. jpn j pharmacol 22:245-259. takagi k, saito h, tsuchiya m. 1972b. pharmacological studies of panax ginseng root: pharmacological properties of a crude saponin fraction. jpn j pharmacol 22:339-346. ultee a, skemp ra, steging g, smid ej. 2000. antimicrobial activity of carvacrol towards bacillus cereus on rice. j food prot 63:620624. zhu s, zou k, cai s, meselhy mr, komatsu k. 2004. simultaneous determination of triterpene saponins in ginseng drugs by highperformance liquid chromatography. chem pharm bull 52:995998. volume 1, 2007 microbiol indones 97 yunita n made (91-94) ok.pmd the production of tannin acyl hydrolase from aspergillus niger yunita arian sani anwar, hasim, and i made artika∗ department of biochemistry, institut pertanian bogor, darmaga campus, bogor 16680, indonesia the aim of this research was to produce tannin acylhydrolase (tannase) from aspergillus niger isolated from cacao pod. the first step of the study included determination of optimal ph, temperature, and incubation period to produce tannase. optimal conditions obtained for tannase production were ph 5.5, a temperature of 28 oc and an incubation period of 3 days. optimization of production medium was conducted. the media tested were solid and liquid wheat flour media with a concentration of tannic acid as inducer at 0, 3, 5, and 7% (wt/vol). the best production medium was solid medium with tannic acid concentration of 5% (wt/vol). key words: tannase, aspergillus niger, solid state fermentation, liquid state fermentation, tannic acid inducer _____________________________________________ _________________ ∗ corresponding author, phone/fax: +62-251-423267, e-mail: imart171@yahoo.com tannin acylhydrolase also known as tannase (ec 3.1.1.20) is a hydrolytic enzyme that catalyses the hydrolysis of (hydrolysable) tannins releasing glucose and gallic acid. tannins are natural compounds which have a number of phenolic hydroxyl groups and can precipitate protein. the ability of these compounds to precipitate protein generates some problems. tannins are capable of interacting with protein and crude-fibres and also with digestive enzymes so that they could interfere with the digestion process that can inhibit the growth of livestock (butler and rogler 1992). tannase is extensively used in food and medical industries. in the food industry, the enzyme is used in the manufacture of instant tea, as a clarifying agent of wine, fruit juices, and in reduction antinutritional effects of tannins in animal feed. in brazil, tannase has a potential use for reducing astringency of cashew apple juice. in the medical industry, tannase is used in the production of gallic acid, a substrate for the chemical synthesis of propyl gallate and trimethoprim (pinto et al. 2001). a number of microorganisms such as fungi, bacteria, and yeast are known tannase producers. species belonging to the aspergillus and penicillium genuses were reported as the best tannase producers (pinto et al. 2001). purnama (2004) found that aspergillus niger isolated from cacao pod reduces tannin levels up to 79.3% (wt/wt). hatamoto et al. (1996) isolated and characterized a tannase gene from aspergillus oryzae. the gene was found to encode a protein of 588 amino acids. the tannase gene product was translated as a single polypeptide and processed by cleavage into two tannase subunits linked by disulfide bonds. the native tannase was concluded to consist of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300,000 (hatamoto et al. 1996). sabu et al. (2005) reported that the optimum temperature for fungal tannase activity was between 30 and 40 oc. the enzyme showed optimum activity at ph value between 5.0 and 6.0. tannase is an extracellular enzyme that needs an inducer to increase the enzyme synthesis. several studies on optimum production of tannase by moulds have been conducted. tannase was found to be induced by tannic acid and some of its derivatives (aguilar et al. 2001). similarly, sanchez (2003) reported that tannase production increases when the culture media contains 3% (wt/vol) of tannic acid. the tannase activity obtained in these media reached 7.45 u ml-1. in the present study tannase was produced using a. niger isolated from local cacao pod. optimization of production medium and characterization of the resultant tannase were carried out. the optimum concentration for induction was also determined. materials and methods maintenance of culture. a strain of aspergillus niger was obtained from the stock collection of the engineering and bioprocess laboratory, institut pertanian bogor. the strain was isolated from cacao pod collected from a cacao plantation in bogor, west java. potato dextrose agar (pda) (difco) slants were used for maintenance of a. niger with incubation at 28 oc for 6 days. fully sporulated slants not in use were stored at 4 oc. the slants were subcultured routinely once every three weeks. preparation of spore inoculum. fungal spore inoculum was prepared by adding 10 ml of sterile distilled water containing 0.1% (vol/vol) tween 80 to a fully sporulated culture. the spores were dislodged using a sterile inoculation loop under strict aseptic condition and then vortexed in a slanted position. the volume of 1 ml of the prepared spore suspension was used as inoculum with concentration of 3 x 107 spores. preparation of production medium. tannase production was carried out using both solid and liquid media. the solid medium for solid state fermentation (ssf) was prepared as follows: a mass of 5 g of wheat flour was taken into a 125 ml erlenmeyer flask and moistened with 5 ml of czapeck medium (nano 3 3 g l-1, kcl 0.5 g l-1, mgso 4 ⋅3h 2 o 0.348 g l-1, feso 4 ⋅7h 2 o 0.01 g l-1, k 2 hpo 4 ⋅3h 2 o 1.301 g l-1, and tannic acid 30 g l-1). the liquid medium used for tannase production was the czapeck medium with 70 g l-1 glucose as carbon source. optimization of condition for tannase production. determination of optimal ph, temperature and incubation period for tannase production was carried out using solid state fermentation. variable parameters for the enzyme microbiology indonesia, august 2007, p 91-94 volume 1, number 2 issn 1978-3477 production were ph (4, 4.5, 5, 5.5, 6), temperature (26, 28, 30, 32 oc), and incubation period (1-5 days). extraction of crude enzyme. tannase was extracted from the fermented substrate by adding 50 ml of distilled water containing 0.01% (vol/vol) tween 80. contents were mixed well using a magnetic stirrer. crude enzyme was separated from fermented matter by centrifugation (beckman j2-21 rotor) at 7700 g, 4 oc, for 20 min. the supernatant was separated by filtration through whatman no. 1 and the filtrate was collected in bottles for further studies. tannase assay. tannase activity was determined by method of libuchi et al. (1966). the substrate solution, containing 1 ml of 0.35% (wt/vol) purified tannic acid in 0.05 m citrate buffer (ph 5) was preincubated at 30 oc for about 5 min. enzyme solution (0.25 ml) was added followed by incubation at 30 oc for 15 min. the blank solution was prepared by adding citrate buffer in place of the enzyme. into the solution, 5 ml of 95% (vol/vol) ethanol was added followed by mixing in order to stop the reaction. after this, 0.25 ml aliquots of the reaction and blank mixtures were transferred into respective test tubes. ethanol solution was added to all tubes and the tubes were mixed thoroughly. absorbance was measured at 310 nm. one unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 µmol of the ester bond in 1 min. optimization of medium for tannase production. in the present study, the effect of different tannic acid concentrations on tannase production in solid state fermentation and liquid surface fermentation was studied. for these purposed, the tannic acid concentration in the czapeck medium used for preparation of solid and liquid production media was varied from 0, 30, 50, and 70 g l-1. the culture was incubated at 28 oc on rotary shaker (150 rpm) for 72 h. tannase assay was performed using method of libuchi et al. (1966) and total soluble protein was determined by the method of bradford (1976). results effect of temperature. temperature for the growth studies was 26, 28, 30, and 32 oc. maximum tannase activity (0.167 u ml-1) was obtained at growth temperature of 28 oc. thereafter a declining trend was observed, as shown in fig 1. effect of ph. to determine effect of ph of the growth medium on tannase production, a range of ph of the medium was used being 4, 4.5, 5, 5.5, and 6. the optimum ph of medium was found to be 5.5 (fig 2). effect of incubation period. incubation period is the most important parameter for maximum tannase production. the optimum incubation period was obtained at 72 h. up to 72 h there was a rise in tannase activity, after which a decrease was observed (fig 3). effect of media and tannic acid concentration. this research tested solid and liquid media with an inducer concentration varied at 0, 3, 5, and 7% (wt/vol). the maximum enzyme activity was obtained with the solid medium with a tannic acid (inducer) concentration of 5% (wt/vol). the optimum activity of tannase obtained from solid and liquid media was 1.441 and 0.603 u ml-1 respectively (fig 4). total soluble protein in solid media was higher than liquid media (fig 5). the maximum protein content obtained at liquid and solid media when the tannic acid concentration of 5% (wt/vol) was 0.494 and 0.712 mg ml-1 respectively. discussion the present study clearly showed that various factors affect tannase production by a. niger isolated from indonesian cacao pod. tannase activity at a growth temperature of 28 oc was higher than that of growth temperature of 26 and 32 oc but was not different from that of 24 26 28 30 32 34 36 0.20 0.16 0.12 0.08 temperature (oc) t a n n a s e a c ti v it y ( u m l1 ) fig 1 effect of temperature on tannase activity at ph 5.5 and 3 days incubation period. t a n n a s e a c ti v it y ( u m l1 ) 0.17 0.16 0.15 0.14 4.0 4.5 5.0 5.5 6.0 6.5 p h fig 2 effect of ph on tannase activity at 28 oc and 3 days incubation period. t a n n a s e a c ti v it y ( u m l1 ) 0.18 0.16 0.14 0.12 0.10 0.08 0 24 48 72 96 120 144 incubation period (h) fig 3 effect of incubation period on tannase activity at 28 oc and ph 5.5. 92 anwar et al. microbiol indones 30 oc. similar results were reported for tannase from a. niger (lekha and lonsane 1997; sanchez 2003). slightly different results were reported by banerjee et al. (2005) who found an optimum growth temperature of 30 oc. this was mainly due to the difference in the strain used in the study. they used rhizopus oryzae and aspergillus foetidus as tannase producers. the lower activity of tannase observed at 26 oc could be due to a lower enzymatic reaction rate leading to lower tannase activity compared to the tannase activity obtained at optimal growth temperature (28 oc). on the other hand, at high temperature, proteins are denatured because of the disruption of their tertiary and quaternary structures, and enzymatic activities decline. in addition, at temperature above the maximal growth temperature, excretion of protease proceeds rapidly so that tannase activity decreases (mackenzie et al. 1994). apart from growth temperature, the ph value of growth medium also affects tannase activity. tannase activity was higher in the culture with growth medium with ph of 5.5 compared to the tannase activity from growth medium with other ph values. similar results were reported for tannase from a. niger (ramirez-coronel et al. 2003; sabu et al. 2005). apparently different results were reported by sanchez (2003) who found that optimum ph value for tannase production was 4. it is important, however, to note that in these experiments the ph values tested were limited to ph 4 and 7, while the ph value of 5.5 was not tested (sanchez 2003). optimization of incubation period was carried in order to determine the best time for harvesting tannase from production culture. incubation period also affects the assayable tannase level. the optimum incubation period was 3 days which was similar to that found by kar et al. (1999) and pinto et al. (2001). different optimum incubation periods, however, were reported by alberts (2002) and rana and bhat (2005). alberts (2002) found that optimum incubation period was 24 h while rana and bhat (2005) reported maximum tannase production with incubation period of 96 h. the varied optimal incubation period found by different workers might be due to differences in fungal strain and medium composition used for tannase production. solid medium was found to be better than liquid medium. this could be due to effect of catabolite repression is less significant in solid medium. the present study showed that solid state fermentation can increase tannase activity about 1.5 folds. in addition, tannase production was affected by concentration of inducer in the medium. the highest tannase activity was found in solid state fermentation with tannic acid concentration of 5% (wt/vol). tannase activity was lower at lower (3%, wt/vol) tannic acid concentration. similarly, at higher (7%, wt/vol) tannic acid concentration the tannase activity decreased indicating that the optimum tannic acid concentration was 5% (wt/vol). excessive tannic acid was reported to act as repressor and prevents synthesis mrna. in addition, the increased of tannic acid causes an increased of heat build up and reduced aeration which in turn decreased productivity of tannase (banerjee et al. 2005). similar results were reported previously by lekha and lonsane (1994) and aguilar (2001). increased productivity of hydrolytic enzyme in solid media is due rapid oxygen uptake rate which allows fungus to form abundant aerial mycelium. aerial mycelium gives a strong increase in enzyme production (rahardjo et al. 2002). in addition, solid media generate higher product stability and lower catabolic repression compared to liquid media (holker et al. 2004). moreover, protease activity in solid state fermentation was found to be lower than protease activity in liquid surface fermentation resulting in higher tannase productivity (aguilar et al. 2002). references aguilar cn, augur c, favale-torres e, viniegra-gonzalez g. 2001. induction and repression patterns of fungal tannase in solid state and submerged cultures. process biochem 36:565-570. aguilar cn, augur c, favale-torres e, viniegra-gonzalez g. 2002. cultures conditions dictate protease and tannase production in submerged and solid state cultures of aspergillus niger aa-20. appl biochem biotechnol 103:407-414. alberts eh. 2002. cloning, expression and characterization of tannase from aspergillus species. [thesis]. bloemfontei: university of the free state bloemfontei, south africa. banerjee d, mukherjee g, patra kc. 2005. microbial transformation of tannin rich substrate to gallic acid through co-culture method. bioresource technol 96:949-953. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. anal biochem 72:248-254. butler lg, rogler jc. 1992. biochemical mechanisms of the antinutritional effects of tannins. in: ho ct, lee cy, huang mt (eds). phenolic compounds in food and their effects on health volume 1, 2007 microbiol indones 93 t a n n a s e a c ti v it y ( u m l1 ) 1 . 6 1 . 2 0 . 8 0 . 4 0 . 0 0 1 2 3 4 5 6 7 8 tannic acid concentration (% w/v) fig 4 tannase activity in solid and liquid media using different tannic acid concentrations as inducer. liqiud media ( ), solid media ( ). t o ta l so lu b le p ro te in ( m g m l1 ) 8 7 6 5 4 3 2 1 0 0 3 5 7 tannic acid concentration (%) fig 5 total soluble protein in solid and liquid media using different tannic acid concentrations as inducer. liquid media ( ), solid media ( ). i: analysis, occurrence, and chemistry. washington: american chemical society. hatamoto o, watarai t, kikuchi k, misusawa hs. 1996. cloning and sequencing of the gene encoding tannase and structural study of the tannase subunit from aspergillus oryzae. gene 175:215-221. holker u, hofer m, lenz j. 2004. biotechnological advantages of laboratory scale solid state fermentation with fungi. appl microbiol biotechnol 64:175-186. kar b, banerjee r, bhattarcharyya bc. 1999. microbial production a gallic acid by modified solid state fermentation. j indust microbiol biotechnol 23:173-177. lekha pk, lonsane bk. 1994. comparative titres, location, and properties of tannin acyl hydrolase produced by a. niger pkl 104 in solid state, liquid surface and submerged fermentations. process biochem 29:497-503. lekha pk, lonsane bk. 1997. production and application of tannin acyl hydrolase: state of the art. adv appl microbiol 44:215-260. libuchi s, minoda y, yamada k. 1966. study on acyl hydrolase of microorganism iii. a new method determining the enzyme activity using a change in ultra violet absorption. agric biol chem 31:513. mackenzie da, gendron lcg, jeenes dj, archer db. 1994. physiological optimization of secreted protein production by aspergillus niger. enzyme microb technol 16:276-280. pinto gas, leite sgf, terzi sc, couri s. 2001. selection of tannaseproducing aspergillus niger strains. brazilian j microbiol 32:242 6 . purnama in. 2004. studies on potency of fungal isolates to cleave tannin linkage in caccao pod (theobroma cacao l). [thesis]. bogor: institut pertanian bogor, indonesia. rahardjo ysp, weber fj, le comte ep, tramper j, rinzema a. 2002. contribution of aerial hyphae of aspergillus oryzae to respiration in a model solid state fermentation system. biotechnol bioeng 78:539-544. ramirez-coronel a, viniegra-gonzalez g, augur c. 2003. a novel tannase from aspergillus niger with β-glucosidase activity. microbiology 149:2941-2946. rana nk, bhat tk. 2005. effect of fermentation system on the production and properties of tannase of aspergillus niger van tieghem mtcc 2425. j general appl microbiol 51:203-212. sabu a, kiran gs, pandey a. 2005. purification and characterization of tannin acyl hydrolase from aspergillus niger atcc 16620. food technol biotechnol 43:133-138. sanchez hh. 2003. optimization of aspergillus niger tannase production using taguchi methods, abstr institute of food technologists annual meeting. chicago, july 12-16, 2003. abstr no 76b-23, p 23. 94 anwar et al. microbiol indones 5 484 ratih asmana ok.cdr optimization of human interferon 2b soluble protein overproduction and primary recovery of its inclusion bodies α ratih asmana ningrum debbie sofie retnoningrum, yeyet cahyati, heni rachmawati , and * school of pharmacy, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia escherichia coli escherichia coli the hifn2b open reading frame has been constructed and overexpressed in bl21(de3). the yields of protein purified using nickel column from inclusion bodies (ib) and total soluble proteins were 3.46 mg and 2.57 mg in 1 l culture, respectively. this research was aimed to obtain optimal condition for high level overproduction of soluble as well as primary recovery of hifn2b from ib. we used two different conditions for obtaining soluble protein, i.e. induction temperatures and inducer concentrations, and three different conditions for inclusion bodies, i.e. centrifugation speeds, washing and solubilizing buffers. induction using 0.5 mm of isopropyl thiogalactopyranoside at 25 °c yielded 8.9 mg hifn2b in 1 l culture. the best recovery of ib was achieved when 10 000 g was applied for centrifugation, 1% triton x-100 in 50 mm tris cl ph 8.0 as washing buffer, and 8m guanidine hcl in 50 mm tris cl ph 8.0 containing 800 mm 2-mercaptoethanol as solubilizing buffer were used. at this optimal condition the yield of hifn2b from ib was 28.85 mg in 1 l culture. the total recovery of at optimal condition was 50% from ib and 14% from soluble protein. hifn2b from ib was refolded by 9 d dialysis in refolding buffer (0.2 mm edta, 0.25 mm ditiothreitol, 50 mm tris and 0.4 m urea ph 8.0). key words: human interferon 2b, overproduction, soluble protein, inclusion bodies, protein refolding kerangka baca terbuka hifn2b telah dikonstruksi dan diekspresikan dalam bl21(de3) pada penelitian sebelumnya. jumlah protein yang dimurnikan menggunakan kolom nikel dari badan inklusi (bi) dan protein terlarut total ialah masing-masing 3.46 dan 2.57 mg dalam 1 l biakan. penelitian ini ditujukan untuk memperoleh kondisi optimal pada overproduksi hifn 2b terlarut dan perolehan hifn 2b dari bi. dua kondisi yang berbeda digunakan untuk mendapatkan protein terlarut, yaitu suhu dan konsentrasi badan penginduksi, serta tiga kondisi yang berbeda untuk bi, yaitu kecepatan sentrifugasi, dapar pencuci, dan pensolubilisasi. penggunaan suhu 25 °c dan isopropil tiogalaktopiranosida 0.5 mm sebagai penginduksi telah menghasilkan hifn2b sebanyak 8.9 mg dalam 1 l kultur. perolehan terbaik dari badan inklusi diperoleh dengan menggunakan kecepatan sentrifugasi 10 000 g, dapar pencuci triton x-100 1% dalam tris cl 50 mm ph 8.0, dan dapar pensolubilisasi guanidine hcl 8m yang mengandung 2-merkaptoetanol 800 mm dan tris cl 50 mm ph 8.0. dengan menggunakan kondisi optimal dihasilkan hifn2b dari bi sebanyak 28.8 mg dalam 1 l biakan. renaturasi hifn2b dari bi dilakukan dalam dapar perenaturasi (edta 0.2 mm, ditiotreitol 0.25 mm, tris 50 mm, dan urea 0.4m ph 8.0) selama 9 hari. kata kunci: interferon 2b manusia, overproduksi, protein terlarut, badan inklusi, pelipatan kembali protein hifnα2b hifnα2b α α α interferon (ifn) is a cytokine produced and secreted by almost all eukaryotic cells as a response to viral, bacterial, antigen, or mitogen stimuli. based on their receptor types on the cell membrane surface, ifn is classified into type i and type ii. type i consists of ifn , ifn , ifn , and ifn . while type ii consists of ifn (wang . 2002; gao . 2004). ifn has wide range of biological activities ranging as antiproliferation, immunomodulation, and antivirus (samuel 2001). human ifn2b (hifn2b), as a subclass of ifn , is a glycoprotein consisting of 165 amino acids with size of 19 271 dalton. the molecule's oglycosylation at threonine position 106 is not important for its biological activity (nyman 1998). hifn2b has two disulfide bridges formed by cysteins (between positions 1 and 98, and between 29 and 138). previous study reported that disruption of disulfide bridges α β τ ω γ α α et al et al et al. formed by cystein 1 and 98 resulted in higher antiviral activity (neves 2004). so far hifn 2b is used as a therapeutic protein for hepatitis b and hepatitis c treatments, both as a single therapy or in combination with other nucleoside analogs (jonasch and haluska 2001). its use to treat several types of cancer, i.e. multiple myeloma, chronic myeloid leukemia, nonhodgkin's lymphoma, renal cell carcinoma, epidermoid cervical cancer, head and neck tumours, melanoma and medullary thyroid carcinoma, had also been reported as well (wang . 2002). dna manipulation has been applied to express many eukaryotic genes in prokaryotes, such as . however, the use of as an expression host often results in the formation of insoluble protein in inclusion bodies (ib). for proteins containing disulfide bridges, ib formation tends to be higher than those without disulfide bridges. in order to recover its activity several procedures including solubilization, refolding, and purification is absolutely et al. et al in vitro escherichia coli e. coli α *corresponding author: phone: +62-22-2504852, fax: +62-22-2504852 email: h_rachmawati@fa.itb.ac.id issn 1978-3477, eissn 2087-8575 vol 5, no 1, march 2011, p 27-32 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.1.5 necessary (rabhi-essafi 2007). two factors that influence ib formation are protein properties (average charge, cysteine and proline contents, hydrophilicity, and total number of residues) and environmental or culture condition (temperature, ph, and nutrient supply) (fischer and sumner 1993; rabhi-essafi 2007). several publications have reported cloning and overexpression of hifn-2b gene in with various yields (neves 2004; srivasta 2005; valente 2006). in addition, optimization of ib primary recovery of hifn-2b expressed in has been reported. the recovery of ib from optimal condition ranged from 61% to 68% (valente 2006). in our previous work (retnoningrum . 2010), we assembled synthetic open reading frame (orf) encoding for hifn-2b using thermodynamically balanced inside out method. the orf was cloned and overexpressed in bl21(de3). the recombinant hifn-2b was produced as a fusion protein of 37 kda, containing thioredoxin and polihistidine tag at its n terminus. the protein was confirmed to be hifn-2b by nano lc ms/ms resulting 80% amino acids coverage. therefore, t in soluble form and primary recovery of hifn2b from ib. optimal condition will be used to obtain highest expression level in the future. bl21(de3) harbouring pet32brecombinant plasmid from previous work was used for gene expression (retnoningrum 2010). bl21(de3) pet32bovernight culture was added (3% v v ) to 100 ml of lb broth containing 100 μg ml of ampicillin. the culture was incubated for about 1.5 h in a shaking incubator at 37 °c 200 rpm. three final concentrations of iptg (0.25, 0.5, and 1.0 mm) were each added to midlog phase of cell culture. incubation was continued for an additional 3 h. cell pellet was harvested by centrifugation at 5000 g for 10 min. the cells were resuspended in lysis buffer (50 mm nacl and 1 mm edta), the composition that was reported by valente (2006). the cells were lyzed by sonication at 2.5 hertz in the presence of 1 mm of phenylmethyl sulphonyl fluoride. to prevent temperature elevation, the cells were sequentially sonicated and cooled on ice for 10 times, each time for 30 s. soluble recombinant protein was separated from ib by centrifugation at 10 000 g. the soluble protein -1 -1 et al. et al. e. coli et al. et al. et al. e. coli et al. et al e. coli e. coli et al. e. coli ifn2b et al. his research aimed to obtain optimal condition for overproduction of hifnα2b hifnα2b materials and methods bacterial strains, plasmids, and culture media. overproduction of hifn-2b. was purified using nickel column according to the manufacturer's protocol (protino, germany). the protein isolation and overproduction steps were monitored using 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page). to determine the yield, protein concentration was measured based on coomassie blue staining using bradford method with standard concentrations of bovine serum albumin were ranging from 125 to 1000 μg ml . optimization of inclusion bodies primary recovery referred to condition as reported by valente . (2006) with some modifications. after sonication, ib was separated from soluble protein by centrifugation for 10 min. five centrifugation speeds 500, 1000, 2500, 5000, and 10 000 g were tested in this experiment. the cells were washed with washing buffer (750 μl of buffer for each 0.1 g of pellet), incubated at room temperature for 5 min and centrifuged at 12 000 g for 5 min. the washing step was repeated to remove impurities. two washing buffers were studied, i.e. buffer a (50 mm tris cl ph 8 containing 1% triton x-100) and buffer b (lysis buffer containing 0.5% triton x-100 ph 7.2). the washed pellet was resuspended in solubilizing buffer (750 μl of buffer for each 0.1 g of pellet) for 30 min at room temperature. two solubilizing buffers were tested, guanidine hcl (gdnhcl) buffer consisted of 6 m gdnhcl in 50 mm tris-hcl ph 8 containing either 2mercaptoethanol (2-me) or dithiotreitol (dtt). to observe the effect of reducing agents, various concentrations of 2-me (100, 200, 400, 650, and 800 mm) and dtt (20, 50, 100, 200, and 300 mm) were tested. subsequently, solubilized pellet was centrifugated at 15 000 g for 15 min and 4 c to separate hifn 2b and unsolubilized material. hifn 2b from ib was characterized using sds-page and its concentration was measured. dialysis was perfomed to refold hifn 2b from ib. two steps were applied, first step to remove denaturant using denaturing removal buffer and the second step to renaturate hifn 2b from ib using refolding buffer (valente . 2006). to remove the denaturant, 0.6 mg of ib was dialyzed overnight in the cellulose ester membrane (spectra/por ce, 1 ml volume capacity, mwco 1000) against 250 ml denaturing removal buffer (0.2 mm edta in 50 mm tris, ph8.0) at 4 c with gentle agitation. to renaturate the hifn 2b from ib, the hifn 2b was dialyzed in 250 ml refolding buffer (0.4 m urea, 0.2 mm edta, 0.25 mm dtt in 50 mm tris, ph 8.0) for 9 d with buffer changes every -1 o ® o optimization of inclusion bodies primary recovery. protein refolding. et al et al α α α α α α 28 ningrum et al. microbiol indones 2 d. to monitor the renaturation process, refolded hifn 2b was analyzed using non reducing sdspage. the expression profile of soluble hifn2b showed that the best expression level was achieved at 25 c with 0.5 mm iptg induction (fig 1). the purity of hifn2b after purification using nickel affinity chromatography was analyzed using 15% polyacrylamide gel as shown in α results overproduction of hifn-2b. o volume 5, 2011 microbiol indones 29 fig 1 sds-page of human interferon 2b (hifn2b) soluble protein. a, lanes 1-3: total soluble protein produced at 37 c with 0.25, 0.5, and 1.0 mm of iptg. lanes 4-6: total soluble protein produced at 25 c with 0.25, 0.5, and 1.0 mm of iptg. b, purified hifn2b soluble protein obtained from optimal condition of overproduction (at 25 c with 0.5 mm iptg). o o o a b 1 2 3 4 5 6 k ad 116.0 66.2 45.0 35.0 25,0 18.4 14.4 fig 2 the effect of centrifugation speeds on human interferon 2b (hifn2b) inclusion bodies (ib) yield. a, lanes 1-5: ib sedimented at 10 000; 5000; 2500; 1000; and 500 g. pellet weight was 0.1 g and loaded volume was 5 μl. b, amount of ib with different centrifugation speed. b a 1 2 3 4 5 m kda 116.0 66.2 45.0 35.0 25.0 18.4 14.4 8.2 17.9 37.48 76.89 129.18 0 20 40 60 80 100 120 140 160 0 2500 5000 7500 10000 centrifugation speeds (g) ib a m o u n t ( g ) μ fig 1b. under optimal overproduction condition, the yield of purified hifn2b was 8.9 mg protein in 1 l culture. the total recovery of purified hifn2b was 14% from total soluble protein. the solubilization step showed minor impurities in all centrifugation speeds used (fig 2a). the highest amount of hifn 2b from ib was achieved at 10 000 g (fig 2b). we studied two washing buffers to compare its ability to remove impurities. the impurity profiles of the two buffers looked the same (fig 3a) but buffer a showed higher ib recovery than buffer b (fig 3b). we used gdnhcl buffer containing various concentration of 2-me or dtt in solubilization step. by this mean the impurity profiles did not differ (fig 4a and b). however, higher ib recovery was achieved when highest concentration of 2-me was used (fig 4d). under this optimal condition the hifn 2b from ib that can be recovered was 28.85 mg l (50%), hence made up 64% (14% from soluble protein and 50% from ib). non-reducing sds-page was used to check the renaturation result. this method can distinguish protein electrophoretic mobility based on its disulfide bond numbers. one upper band (fig 5 lane 2) had the same size with solubilized ib and reduced soluble optimization of ib primary recovery. α α total yield of hifnα2b protein -1 fig 3 the effect of washing buffer on human interferon 2b (hifn2b) inclusion bodies (ib) yield. a, lane 1: ib washed with buffer a (50 mm tris cl ph 8 containing 1% triton x-100); lane 2: ib washed with buffer b (50 mm nacl and 1 mm edta containing 0.5% triton x-100 ph 7.2). pellet weight was 0.1 g and loaded volume was 5 l. b, amount of ib measured using bradford method in different buffer. μ a 1 2 m kda 114.0 66.2 45.0 35.0 25.0 18.4 b 266.72 178.26 0 50 100 150 200 250 300 buffer a buffer b washing buffer ib a m o u n t ( g ) μ protein (fig 5 lane 1 and 3) and lower band had the same size with unreduced soluble protein (fig 5 lane 4). this means that hifn 2b from ib was not totallyα 284.43 296.89 318.72 328.64 340.84 0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 dtt concentration (mm) 30 ningrum et al. microbiol indones fig 5 non-reducing sds-page of refolded human interferon 2b (hifn2b). lane 1: solubilized inclusion bodies; 2: refolded hifn2b ; 3: reduced soluble hifn2b; 4: unreduced soluble hifn2b. fig 4 the effect of dithiotreitol and 2-merchapethanol concentrations on the solubilization of human interferon 2b (hifn2b) inclusion bod (ib) yield. a, lanes 1-5: solubilized ib with gdnhcl containing 20, 50, 100, 200, and 300 mm dtt. b, the effect of 2-merchapethanol on the solubilization of hifn2b ib. lanes 1-5: solubilized ib with gdnhcl containing 100, 200, 400, 650, and 800 mm 2merchapethanol (2-me). the pellet weight was 0.1 g and sample volume loaded was 5 l. c, ib amount measured by bradford method in solubilizing buffer containing various concentration of dtt. d, ib amount measured by bradford method in solubilizing buffer containing various concentration of 2-me. ies μ c d a b 1 2 3 4 1 2 3 4 5 m m 1 2 3 4 5kda 114.0 66.2 45.0 35.0 25.0 18.4 14.4 294.53 319.63 360.21 291.92 384.77 0 50 100 150 200 250 300 350 400 0 200 400 600 800 2-me concentration (mm) ib a m o u n t (μ g ) ib a m o u n t (μ g ) refolded. only about 50% of hifn 2b was refolded and formed disulfide bridges. several approaches have been reported to improve the solubility of recombinant hifn2b including using different expression systems (using , murine myeloma cell, and baculovirus) and different affinity tags (thioredoxin or gluthatione s transferase) (fischer and sumner 1993). the purification of soluble hifn2b is more cost effective and less time consuming over refolding and purification from ib. hence, optimizing production step of recombinant hifn2b is the most reasonable alternative to reduce ib formation. previously, we tried to improve hifn2b solubility by using low copy number plasmid (pet32b) and constructing the hifn2b fusion protein containing thioredoxin tag. we observed that environmental factors, i.e temperature and iptg concentration, affected the yield. current results confirmed that the best expression level was achieved at 25 c with 0.5 mm iptg. nevertheless, at 37 c, soluble protein was not obtained when induction was done using 0.25 and 0.5 mm iptg. this might be due to high level of ib formation. only when induction was performed using 1.0 mm iptg soluble protein produced at low level. the result indicates that lowering temperature to 25 c whilst still using 0.5 mm iptg can decrease protein synthesis, therefore, increasing the formation of soluble hifn2b. our result is comparable with previous work showing the overproduction of gluthatione s transferase hifn2b fusion protein at 25 c with 0.5 mm iptg and gfp chey fusion protein at 25 c with 8 μm iptg (rabhi-essafi 2007; sevastsyanovich 2009). however, cirkovas and serekaite (2010) reported that overproduction conducted under low temperature 20 c improved the solubility of mink growth hormone. in this study, it was demonstrated that simultaneous decrease iptg concentration into micromolar range did not reinforce the effect of temperature. the centrifugation speeds at 5000 to 20 000 g commonly used to separate ib from homogenate (fischer and sumner 1993). since the high speed can lead to co-sedimentation of proteins, hence increasing the impurities, we studied various centrifugation speeds (500 to 10 000 g) to obtain the highest hifn 2b recovery with the lowest impurities. we observed that the impuritiy profiles from each speed did not demonstrate significantly different and α α discussion streptococcus lividans, pichia pastoris, bacillus subtilis et al. et al. e. coli o o o o o o volume 5, 2011 microbiol indones 31 the highest amount of hifn 2b from ib was obtained at 10 000 g. lowering centrifugation speeds did not lead to high hifn 2b recovery and did not have any effect on eliminating impurities. valente (2006) reported that at least 5000 g was needed to recover more than 93% of hifn 2b from ib but increasing the speed tend to give higher impurities. ib may contain protein impurities originated from such as rna polymerase, outer membrane proteins, or enzymes. membrane proteins or kanamycin phosphotransferase are soluble impurities. other ib impurities were washed using buffer containing triton x-100, sucrose or urea. triton x-100 that is normally used at concentrations ranging from 0.5% to 5% (fischer and sumner1993). we observed that varying concentrations of triton (0.5% to 1%) to remove the impurities did not affect hifn 2b purity. the higher recovery achieved by buffer a might be due to higher ph (washing condition at ph 8.0 on buffer a comparing to at ph 7.2 on buffer b) that stabilized the hifn 2b in washing step. valente (2006) compared two washing buffer, i.e. 20 mm tris in 50 mm edta ph 8.0 and 50 mm tris-cl containing 1% triton ph 8. although the latter buffer resulted in higher purity of ifn 2b, it did not have significant effects on hifn 2b recovery. generally, there are two denaturants used to break existing intramolecular and intermolecular disulfide bonds i.e. urea and gdnhcl. it has been proven (valente 2006). we examined gdnhcl buffer containing various concentration of 2me or dtt as reducing agents. higher amount of reducing agents reported might lead to cosolubilization of protein (valente 2006), however current results showed no difference in impurity profiles of each dtt and 2-me concentration. higher hifn 2b recovery was achieved when the highest concentration (800 mm) of 2-me was applied. so far, the highest concentration of 2-me that ever used is 500 mm (fischer and sumner 1993). however, there is no good explanation of why using 650 mm of 2-me resulted in lowest recovery. t was 64%, which is higher than previous study reported by srivasta et . (2005) and valente (2006). renaturation step after ib solubilization is required to obtain native conformation and to catalyze disulfide bond formation. the denaturants must be removed and ib must be renaturated in oxidizing buffer (fischer and sumner 1993). in this study, we used buffer containing tris and edta ph 8.0 to remove denaturant and α α α α that urea combined with dtt is not good for ifnα2b ib solubilization he total yield of hifnα2b protein et al. e. coli et al. et al. e. coli et al. al et al. α α α α oxidizing buffer containing urea, dtt, and edta ph 8.0. hifn 2b protein was renatured in refolding buffer for 9 days based on previous publication report (valente . 2006). the refolded hifn 2b protein profile on native gel showed two different bands. the lower band that has same size with the non-reduced soluble protein should correspond to biologically active hifn 2b. so it seems that only about 50% of hifn 2b that could be successfully refolded, so further study is still needed to increase the refolding result. valente . (2006) reported three different bands after 9 d renaturation. we suggest that thioredoxin tag in the hifn 2b facilitates the disulfide bridge formation, although sachdev and chirgwin (1998) reported that the mbp and thioredoxin fusions of pepsinogen expressed in did not necessarily facilitate native refolding, but enhanced the recovery of soluble protein. to conclude, the optimal condition to obtain hifn 2b soluble protein and primary recovery of its inclusion bodies has been established yielding 8.9 mg protein of soluble protein with 14% recovery and 28.85 mg of ib with 50% recovery from 1 l culture. this research was funded by riset unggulan institut teknologi bandung 2010 granted to heni rachmawati. α α α α α et al et al e. coli α acknowledgements references cirkovas a, serekaite j. 2010. increase in the solubility of recombinant mink growth hormone at low cultivation temperature of coli. biotechnol biotechnol equip. 24(4):2169-2171. fischer b, sumner i. 1993. isolation, 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quality of recombinant protein. fems microbiol lett. 299:86-94. doi:10.1016/j.jbiotec.2009.11.008. doi:10.1006/prep. 1997.0826. doi: 10.1128/cmr.14.4.778-809.2001. doi:10.1111/j.15746968.2009.01738.x. 32 ningrum et al. srivasta p, bhattacharaya p, pandey g, mukherjee kj. 2005. overexpression and purification of recombinant human interferon alpha2b in . protein expr purif. 41(2):313-322. valente ca, monteiro ga, cabral jms, fevereiro m, prazeres dmf. 2006. optimization of the primary recovery of human interferon 2b from escherichia coli inclusion bodies. protein expr purif. 45(1): 226-234. wang ys, youngster s, grace m, bausch j, bordens r, wyss ds. 2002. structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications. adv drug deliver rev. 54(4):547-570. escherichia coli doi:10.1016/j.pep.2004.12.018. doi:10.1016/j.pep.2005.06.014. doi:10.1016/s0169-409x(02) 00027-3. 02. muin.cdr vol.13, no.3, september 2019, p 83-89 doi: 10.5454/mi.13.3.2 the utilization of arbuscular mycorrhizal fungi for planting agarwood (aquilaria spp) seedling in open land abdurrani muin faculty of forestry, universitas tanjungpura, jl. imam bonjol pontianak, 78124, indonesia. agarwood is a type of semi-tolerant plant, so that for planting the seedlings should be grown under the shade. for planting in open land, it requires treatment in which one of them is using seedlings inoculated with arbuscular mycorrhizal fungi. the aim of the research is to obtain information on agarwood growth that has been inoculated with fungi mycorrhizal arbuscular when planted in the open land and ability to grow between agarwood seedlings inoculated mycorrhizal that was planted in the shade and in the open area. split plot randomized block design was applied with treatments: the first plot consisting of plant had been inoculated with mycorrhizae and without mycorrhizal inoculation, and the sub plot was the types of shading that consists of open land, paranet 60 % intensity and natural vegetation. to reduce variabilty of site topographical differences were separated as bloks. variables measured were: plant height (cm), stem diameter (mm), number of leaves, and survival percentage of plant. the results show that the height and diameter growth of seedlings innoculated with mycorrhizae were higher than non innoculated. the seedlings innoculated with mycorrhizal fungi were planted in the paranet shading grew better and significantly different compared to the vegetation shading. seedlings innoculated mycorrhizal that were planted in open land grew better and significantly different compared to vegetation shading. this study results indicate that planting agarwood in the open land can be done using seedlings inoculated arbuscular mycorrhizal fungi. key words: agarwood plant, arbuscular mycorrhizal fungi, shading and open land gaharu merupakan jenis tanaman yang bersifat semitoleran, sehingga hanya bisa ditanam dibawah naungan. untuk penanaman pada lahan terbuka diperlukan perlakuan dimana salah satunya adalah menggunakan bibit yang terinokulasi fungi mikoriza. tujuan penelitian adalah untuk memperoleh informasi mengenai pertumbuhan tanaman gaharu yang terinokulasi fungi mikoriza pada lahan terbuka, kemampuan tumbuh tanaman yang terinokulasi fungi mikoriza dibawah naungan dan di lahan yang terbuka. metode split plot design acak kelompok dengan perlakuan : petak utama terdiri dari bibit tanpa mikoriza dan bermikoriza. sebagai sub plot adalah tipe naungan yang terdiri dari : lahan terbuka, paranet 60 % dan vegetasi alam. variabel yang diukur adalah : tinggi tanaman (cm), diameter batang (mm) jumlah daun dan persentase hidup. hasil penelitian menunjukkan bahwa pertumbuhan tinggi dan diameter bibit yang terinokulasi mikoriza lebih baik dibandingkan dengan tanpa mikoriza. bibit terinokulasi mikoriza yang ditanam dibawah naungan paranet tumbuh lebih baik dan berbeda sangat nyata dibandingkan dengan naungan vegetasi. bibit terinokulasi mikoriza yang ditanam di lahan terbuka, tumbuh lebih baik dan berbeda nyata dibandingkan dengan dibawah naungan vegetasi. hasil penelitian ini mengindikasikan bahwa penanaman tanaman gaharu di lahan terbuka dapat dilakukan dengan menggunakan bibit terinokulasi fungi mikoriza arbuskula. kata kunci: fungi mikoriza arbuskula, naungan dan lahan terbuka, tanaman gaharu microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-816223336; email: abdurrani.muin@gmail.com from natural forests. in terms of technology to accelerate the growth of agarwood trees has been found through various studies as conducted by muin and muin et al. (muin 2009; muin et al. 2010) that must be supported by planting activities. due to the semi-tolerant properties of the agarwood plant people are not be able to plant this species in open lands. difficulties in planting in the open land were obtanined from experiences of community in the district parindu and bonti sanggau west kalimantan (muin et al. 2017). this information came up because peoples want to plant agarwood (aquilaria spp) on open land, especially in the land rehabilitation activities (gn-rhl/gerhan) and community agarwood (aquilaria spp) is a forest products produced from wood trunk section that specifically form aromatic resin. because the utilization is very wide, it becomes a forest products commodity that have high economic value. more over agarwood is one of the main of forest products in international trade. intensive logging on agarwood in natural forest, resulting in the decrease of its existence that is already endangered. one contributing factor is that genus agarwood (aquiliria spp) is already included in cites appendix ii which prohibits trade in agarwood derived plantation forest (hutan tanaman rakyat). based on the nature of the semi-tolerant tree species, the planting of agarwood could be expanded into open land. however, it require a variety of treatment, both while still in the nursery and when planted in the field. semi-tolerant seedlings planting in open fields requiring high seedling quality index, and special treatment when planted in the field, among others by using mycorrhizal seedling and providing shade with the appropriate light intensity (muin 2009). this information on the treatment was no available and becomes critically needful regarding the cultivation of agarwood using mycorrhizal seedlings and provision of appropriate shade to be planted in open land. the aims of research areto obtain information an agarwood plant growth that has been inoculated with arbuscular mycorrhizal fungi when are planted in the open land, and to search the differences between the ability to grow with and without mycorrhizal seedlings are planted in the shade and on the open land. the final goal of this research is to confirm that people can plant agarwood (aquilaria spp) on open land on a broader scale, thereby they can increase the production of agarwood aromatic resin and expand economic development, especially for the people who live around the forest. materials and methods research had been conducted at kebun percobaan universitas tanjungpura, desa pak laheng kabupaten mempawah for mycorrhizae, seedlings inoculated with mycorrhiza and paranet with intensity 60%. in this study, the data collection was done on the plant until the age of 12 weeks. the materials used for this research are agarwood seedlings which are inoculated with mycorrhizal fungi and without mycorrhizae, paranet with 60% light intensity. the research method was using experimental split plot randomized block design which seedlings as main plot that not inoculated with mycorrhizae (f ) and inoculated with mycorrhiza (f ), 1 2 and sub plot in the form of shade was consisting of without shade (n ), shade paranet intensity 60 % (n ) 1 2 and the shade of natural vegetation (n ). blocks were 3 used in this study due to the differences of site topography. data that had been collected : plant height, diameter and number of leaves and survival of plants. to determine differences in the effects of each treatment, analysis of variance was parformed according to the split plot design experiment. the least small different test (lsd) was used to determine the different effecst of the treatment given to the agarwood plant. results plant height (cm). base on analysis of variance, the micorrhizal plants and shade treatment as well as the interaction has not shown any significant effect on height variable of the agarwood plant until the age of 12 weeks (3 months). although the results of analysis of variance showed no significant effect, but the difference in height gain for 12 weeks of each treatment can be seen (fig 1, 2). the treatment without shade (n ) showed height 1 growth was slower compared to the treatment of paranet 60% (n ) at the age of up to eight weeks, but 2 became faster after more than 10 weeks old (fig 1). the height growth of agarwood plant grown in the open land was better than the one in the shade paranet (n ).2 the plants that have been inoculated with arbuscular mycorrhizal fungi (f ) growth better started 2 in week ten and up to 12 weeks height growth was 10.94 cm, while plants without mycorrhiza (f ), height 1 growth until the age of 12 weeks only 8.85 cm. is seems that the arbuscular mycorrhizal fungi began to affect th the plant on 10 weeks (fig 2). stem diameter (mm). effect of arbuscular mycorrhizal fungi and shade treatment to plant diameter growth and its analysis of variance according to split plot design experiment results was shown (table 2). based on analysis of variance that giving shade and mycorrhizal fungi treatments and the interaction of both treatment show hihgly significant effects on the growth of the agarwood plant diameter (table 2). lsd test showed that mycorrhizal agarwood plant grown in the shade paranet 60% had the best diameter growth. the differences in the growth of plants grown in the shade of forest vegetation, and in the open land and under paranet presented (fig 3). it shows the difference in diameter growth was taking place since the plant eight weeks old (measurement week 4). diameter growth of plants grown in the open land turned out to be slower compared to those grown under paranet and natural vegetation (fig 3). significant difference between the diameter growth agarwood plant with myocrrhizal and without mycorrhizal. growth of mycorrhizal agarwood plant was faster and started to take place in a six-week-old plants (measurement weeks to 3) (fig 4). amount of leaves growth. analysis of variance 84 muin microbiol indones volume 13, 2019 microbiol indones 85 fig 1 graph of mean height growth (cm) of agarwood plant with treatment in open land (n ), under shading of 1 paranet 60% (n ) and under shading of natural vegetation (n ) until age 12 weeks.2 3 fig 2 graph of mean height growth (cm) of agarwood plant without and with mycorrhizal treatments, until age 12 weeks. fig 3 graph of average agarwood diameter growth (mm) in open land (n ), under paranet shading 60% (n ) and 1 2 under natural vegetation (n ) until 12 weeks old.3 fig 4 mean of diameter growth (cm) of agarwood plant without mycorrhizal and with mycorrhizal treatment until age 12 weeks. 86 muin microbiol indones results showed that the treatment given to agarwood plants very significant effect on the increase of the amount of leaves. however, interaction of the two treatments did not affect on the increase of the amount of leaves. the difference in the leaves of 12 weeks old the agarwood plant (sixth weeks measurement) presented in table 3 which shows more leaves on mycorrhizal plants grown in the open land. mycorrhizal agarwood plant increased as much as 13 leaves, while mycorrhizal agarwood plant grown in the shade paranet only grown 5 leaves and whereas leaves grown under natural vegetation shading were 9. plant survival (%). the treatment of mycorrhiza and shade effected on the percentage of plant survival until the age of 12 weeks (table 4). the percentage of plants survival grown in open fields is lower and significantly different compared to the one planted in the shade of natural forest vegetation and paranet. dead plants on open land occured until the age of two weeks, including the mycorrhizal plant. drought and high air temperatures (40 °c) for 4 weeks during the study cause agarwood plants in the open land experiencing wilting and death. discussion the results showed the effects of treatments on the growth of mycorrhiza and shade height, diameter, number of leaves and the plant survival. significant effect only occurs in diameter and increase the number of leaves parameters, while the height growth and survival had not yet significant differences. however, high growth mycorrhizal agarwood plant grew faster since the age of the plant was 10 weeks (the fifth measurment), especially plants grown in the open land (without shade) (fig 2). meanwhile the growth of mycorrhizal agarwood plant started faster on a 6 weeks old plants (measurement to three). this result shows that arbuscular mycorrhizal fungi (amf) inoculated on seedlings in nursery can be used as an instrument in accelerating the growth of the agarwood plant after planting in the field. seedlings grown in nutrient-poor of ultisol soil with a low ph requires forming associations with mycorrhizal fungi. by nature, the type of plants forming mycorrhizal symbiosis with fungi grow on marginal land that is poor in nutrients and low ph, such as red-yellow podzolic soil (muin et al. 2016). research conducted by nurul huda, muin and fachrizal (2016) showed that the 3 year sold agarwood plants planted in the satong village has been in symbiosis with arbuscular mycorrhizal fungi. results of nurul huda et al. (2016) showed that the seedlings inoculated with arbuscular mycorrhizal fungi (amf) grew faster with higher quality index. this result indicates that the growth of agarwood from nursery to be planted in the red yellow podzolic soil requires association with mycorrhizal fungi forming. this symbiosis confers benefits directly to the host plant's growth and development through the acquisition of phosphorus (p) and other mineral nutrients from the soil by the amf (aggarwal et al. 2011). role of arbuscular mycorrhizal fungi (amf) in global sustainable development. am fungi facilitate plant uptake of mineral nutrients such as phosphorus and nitrogen by increasing the absorbing surface area and by mobilizing sparsely available nutrients. in turn, plant hosts supply am fungi with a carbon source that is essential for fungal growth (wang et al. 2017). fungal hyphae normally transport posphorous over longer distances from depleted zones compared to roots. the am especially benefit plants grown in soils where p is likely to limit plant growth. a reduced effectiveness of am colonization of roots often occurs when soluble soil p levels increase this review gives an overview on the role of mycorrhizae in absorbing phosphorous (lizadeh 2011). arbuscular mycorrhizal fungi (amf) improve the plant mineral nutrition in particular the acquisition of phosphorus and some nitrogen and minor nutrients (karti et al. 2018). arbuscular mycorrhizal fungi (amf) constitute a group of root obligate biotrophs that exchange mutual benefits with about 80% of plants and provide the host with water, nutrients, and pathogen protection, in exchange for photosynthetic products (berruti et al. 2016). arbuscular mycorrhizal fungi (amf) are ubiquitous organism that forms association with the root of most terrestrial plants and influence soil fertility through the enhancement of chemical, biological and physical content (syibli et al. 2013; armadal et al. 2016). the percentage of plants survival in the open, including the mycorrhizal plants was very low, because when planted in february 2016 (up to age 3 weeks) drought with extremely high temperatures (40 °c) was leading to high soil temperatures and increasing evaporation on the leaves. high soil temperatures (36 °c) and dry soil conditions causes the amount of water absorbed less than evaporation, so that the plant become wilted and died. the high light intensity could be disadvantage to plants, because the soil became hot, while too low light can reduce the amount of volume 13, 2019 microbiol indones 87 mycoorhizal rooting due to lack of supply of carbohydrates resulted on photosynthesis. the establishment of am symbiosis involves recognition of the two partners and bidirectional transport of different mineral and carbon nutrients through the symbiotic interfaces within the host root cells (wang et al. 2017). intriguingly, recent discoveries have highlighted that lipids are transferred from the plant host to am fungus as a major carbon source. however, by week four weather returned normal with rainfall and then a normal air temperature (28 °c 32 °c), the plants began to grow normally. in the dry season, almost all crops grew well in the shade and yet mostly in the open land, they did not but in process stagnant growth (slow). this is because the seedlings was planted in this study had a high quality index. according muin (2009) that planting semitolerant seedlings in open land requiring high seedling quality index, and special treatments when planted in table 1 height of agarwood plant (cm) with mycorrhizal and shading treatment until 12 weeks olds table 2 diameter (mm) of 12 weeks old agarwood plant with mycorrhizal and shade treatments table 3 number increasing of leaves (number of) 12 weeks old agarwood plant with mycorrhizal and shade treatments table 4 survival of agarwood plants with mycorrhiza and shade treatments untill 12 weeks old shading treatment mycorrhizal treatment without mycorrhizal with mycorrhizal average height under shading open land (n )1 10.06 11.80 10.93 paranet 70 % (n )2 8.68 9.27 8.98 natural vegetation (n )3 8.26 11.75 11.75 average height with mycorrhizal 9.37 10.94 10.16 shading treatment mycorrhizal treatment without mycorrhizal with mycorrhizal mean diameter with shading open land (n )1 0.64 1.12 0.88 paranet 60 % (n )2 1.52 2.35** 2.44** natural vegetation (n )3 1.34 1.78 1.56 mean diameter with mycorrhizal 1.17 1.75** 1.63 note: **) significant difference at 1% level shading treatment mycorrhizal treatment without mycorrhizal with mycorrhizal mean leaves number with shading open land (n )1 9.33 13.33 11.33** paranet 60 % (n )2 6.00 5.00 5.50 natural vegetation (n )3 5.33 9.33 7.33 mean leaves number with mycorrhizal 6.88 9.22 8.05 note: **) significant difference at 1% level shading treatment mycorrhizal treatment without mycorrhizal with mycorrhizal mean survival with shading open land (n )1 78.25 60 69.13 paranet 60 % (n )2 90 90 90 natural vegetation (n )3 90 90 90 average leaves number with mycorrhizal 86.08 80 83.04 88 muin microbiol indones open land as it is done on ramin (gonystylus bancanus miq. kurz) seedlings. one of the weaknesses in this study, the seedlings planted were not adapted well to field conditions. the provision of shade to new seedling planted in the field in needed so that the plant does not receive direct sunlight and soil temperatures are not too high. the soil temperature is too high (over 32 °c) will affect negatively to the development of mikroiza arbuscular fungi (muin et al. 2016). plants that are semitoleran need shade when they are in seedlings growth level and requires full light after approaching adult levels (sapling). however with mycorrhizal fungi inoculation agarwood plant (aquilaria spp) are able to grow in the open, as is the case in plants ramin peat swamp forest. arbuscular mycorrhizal fungi inhabiting soil play an important role for vascular plants. interaction between arbuscular mycorrhizal fungi, plants and soil microorganisms leads to many mutual advantages. however, the effectiveness of mycorrhizal fungi depends not only on biotic, but also abiotic factors such as physico-chemical properties of the soil, availability of water and biogenic elements, agricultural practices, and climatic conditions (jamiołkowska et al. 2018). research results of muin (2009) showed that the mycorrhizal seedlings of ramin (semitoleran) planted in the logged open land grew faster whitin the three months compared without mycorrhizae. the results of research showed that mycorrhizal fungi can enhance the growth of agarwood in the open land. most vascular plants form symbiotic relationships with arbuscular mycorrhizal fungi (amf), root symbionts that provide soil nutrients to plants in exchange for carbohydrates, which may reduce the effects of environmental stresses on plants (pischl and barber 2017). am fungi facilitate plant uptake of mineral nutrients such as phosphorus and nitrogen by increasing the absorbing surface area and by mobilizing sparsely available nutrients. in turn, plant hosts supply am fungi with a carbon source that is essential for fungal growth (wang et al. 2017). arbuscular mycorrhizal fungi (amf) are ubiquitous organism that forms association with the root of most terrestrial plants and influence soil fertility through the enhancement of chemical, biological and physical content (syibli et al. 2013). acknowledgements the research was financially support by direktorat jenderal pendidikan tinggi dan kebudayaan sceme (2016) penprinas mp3ei 2011-2025 to abdurrani muin with contract nomor :036/sp2h/lt/dprm/ii th /2016, february 17 , 2016. references aggarwal a, kadian n, tanwar a, yadav a, gupta.kk. 2011. role of arbuscular mycorrhizal fungi (amf) in global sustainable development. j app nat sci. 3(2):340-351. doi: 10.31018/jans.v3i2.211. armada e, lópez-castillo o, roldán a, azcón r. 2016. potential of mycorrhizal inocula to improve growth, nutrition and enzymatic activities in retama sphaerocarpa compared with chemical fertilization under drought conditions. j soil sci plant nutr. 16(2):380-399. doi: 10.4067/s0718-95162016005000035. berruti a, lumini e, balestrini r, bianciotto v. 2016. arbuscular mycorrhizal fungi as natural biofertilizers: let's benefit from past successes. front microbiol. 6:1559. doi: 10.3389/fmicb.2015.01559. bolduc ar, hijri m. 2010. the use of mycorrhizae to enhance phosphorus uptake: a way out the phosphorus crisis. j biofertil biopestici. 2(1):1-5. doi: 10.4172/21556202.1000104. jamiołkowska a, księżniak a, gałązka a, hetman b, kopacki m and bednarz s. 2018. impact of abiotic factors on development of the community of arbuscular mycorrhizal fungi in the soil: a review. int agrophys. 32(1): 133–140. doi: 10.1515/intag-2016-0090. lizadeh oa. 2011. role of mycorrhiza in absorbing soil phosphorous. intl res j appl basic sci. 2(10):371-375. karti pdmh, prihantoro i, setiana ma. 2018. evaluation of arbuscular mycorrhizal fungi inoculum on production and nutrient content of pennisetum purpureum. trop anim sci j. 41(2):114-120. doi: 10.5398/tasj.2018. 41.2.114. muin a. 2009. teknologi penanaman ramin (gonystylus bancanmus miq. kurz) pada areal bekas tebangan. untan press. 110p. muin a, indrayanti, artuti. 2010. penyediaan bahan induksi yang cocok dan efektif untuk pembentuklan gubal gaharu. prosiding seminar nasional dan rapat tahunan dekan bidang ilmu-ilmu pertanian, badan kerjasama perguruan tinggi negeri wilayah barat. badan penerbit fakultas pertanian universitas bengkulu. 1135-1141. muin a, fachrizal, firiana n. 2016. growth of agarwood (aquilaria spp) inoculated with arbuscular mycorrhizal fungi under shading and in open land. in national seminar on land rstoration for sustainable land productivity, seameo biotrop, bogor. 26-27 september 2016. muin a, burhanuddin, muin s. 2017. teknik budidaya dan induksi tanaman penghasil resin aromtik gaharu. untan press, pontianak. volume 13, 2019 microbiol indones 89 nurul huda, muin a, fahrizal. 2016. asosiasi fungi mikoriza arbuskula (fma) pada tanaman gaharu (aquilaria spp) di desa laman satong kabupaten ketapang. jur hut lestari. 1(4):20-25. pischl ph, barber na. 2017. plant responses to arbuscular mycorrhizae under elevated temperature and drought. j plant ecol. 10(4):692–701. doi: 10.1093/jpe/rtw075. syibli ma, muhibuddin a, djauhari s. 2013. arbuscular mycorrhiza fungi as an indicator of soil fertility. agrivita, journal of agricultural science. 35(1): 4453. doi: 10.17503/agrivita.v35i1.228. wang w, shi j, xie q, jiang y, yu n, wang e. 2017. nutrient exchange and regulation in arbuscular mycorrhizal symbiosis. mol plant. 10(9):1147-1158. doi: 10.1016/j.molp.2017.07.012. page 1 page 2 page 3 page 4 page 5 page 6 page 7 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 13, number 4, december 2019 antibacterial activity test of indigenous yeast from sapodilla fruit against staphylococcus aureus and escherichia coli citric acid production from toba banana peel (musa acuminata colla) through submerged fermentation using aspergillus niger gene cloning of xilanase glycoside hydrolase family 11 from bacillus halodurans cm1 in escherichia coli dh5α the effect of aeration rate on the growth of blue green microalgae in buffalo dung as alternative media prevalence of hepatitis b virus infection in blood donors based on titer hepatitis b surface antigen examination (hbsag) gemilang lara utama, mutiara nabila, heni radiani arifin, elazmanawati lembong, and tita rialita meva gustina e. sidauruk, surya ningsih hutauruk, merry meryam martgrita, and adelina manurung muhamad taufiqul naufal, agustin krisna wardani, and is helianti edwin yonathan gurning, amos imanuel, nina juliana roberta turnip, and adelina manurung supiana dian nurtjahyani and retno handajani 109 118 123 129 134 author index 137 subject index 138 page 1 02. limbong.cdr vol.13, no.2, june 2019, p 50-55 doi: 10.5454/mi.13.2.2 antioxidant and antibacterial activities enhancement of solid-state fermented candlenut kernels by aspergillus oryzae grace dolorosa limbong, levy nathanael nababan, adelina manurung, 1 and merry meryam martgrita bioprocess engineering study program, faculty of biotechnology, institut teknologi del jalan sisingamangaraja, laguboti, toba samosir 22381, north sumatera, indonesia. according to several studies, solid state fermentation (ssf) can enhance antioxidant and antibacterial activity of natural sources, and microorganism that is widely used in this kind of research is aspergillus oryzae. therefore, this study employed ssf by a. oryzae to enhance antioxidant and antibacterial activity of candlenut kernel. candlenut kernel powder, that has been moistened with 60% water, was inoculated with 10% (w/w) of 5-dayculture of a. oryzae, and was fermented for 9 days (until exponential phase; sample-1) and 12 days (until stationary phase; sample-2). the fermented candlenut kernels was extracted by ethanol and concentrated using rotary evaporator. total phenolic content of control (unfermented extract), sample-1, and sample-2 are 0.183 mg -1 -1 -1 gae g , 2.761 mg gae g , and 4.194 mg gae g , respectively. this results supported the ic value determined 50 -1 -1 -1 by dpph (2,2-diphenyl-1-picrylhydrazyl) method, those are 617.11 μg ml , 260.23 μg ml , and 45.29 μg ml . -1 these results revealed a very strong antioxidant activity (< 50 μg ml ) in the sample fermented until stationary phase. antibacterial assay against staphylococcus aureus resulted diameter of inhibition zone 7.17 mm, 13.51 mm, and 18.51 mm, respectively; whereas against pseudomonas aeruginosa resulted diameter of inhibition zone 6.52 mm, 11.786 mm, and 15.269 mm, respectively. from this result, ssf until stationary phase enhanced higher antioxidant and antibacterial activity compared the other treatments. key words: antibacterial, antioxidant, aspergillus oryzae, solid state fermentation, stationary phase berdasarkan beberapa penelitian, solid state fermentation (ssf) dapat meningkatkan aktivitas antioksidan dan antibakteri sumber hayati, dan mikroorganisme yang sering digunakan dalam penelitian seperti ini adalah aspergillus oryzae. oleh karena itu, dalam penelitian ini dilakukan ssf oleh a. oryzae untuk meningkatkan aktivitas antioksidan dan antibakteri biji kemiri. serbuk biji kemiri, yang dilembabkan dengan 60% air, diinokulasikan dengan 10% kultur a. oryzae berumur 5 hari, dan difermentasi selama 9 hari (hingga fase eksponensial; sampel-1) dan selama 12 hari (hingga fase stasioner; sampel-2). biji kemiri yang telah difermentasi, diekstraksi menggunakan etanol dan dikonsentrasikan menggunakan rotary evaporator. kandungan fenolik total -1 sampel kontrol (tanpa fermentasi), sampel-1, dan sampel-2 berturut-turut adalah 0,183 mg gae g , 2,761 mg gae -1 -1 g , dan 4,194 mg gae g . hasil ini mendukung nilai ic yang ditentukan menggunakan metode dpph (2,2-50 -1 -1 -1 diphenyl-1-picrylhydrazyl), yaitu secara berturut-turut 617,11 μg ml , 260,23 μg ml , and 45,29 μg ml . hasil -1 ini menunjukkan aktivitas antioksidan yang sangat kuat (< 50 μg ml ) pada sampel yang difermentasi hingga fase stasioner. uji antibakteri terhadap staphylococcus aureus, secara berturut-turut, menghasilkan diameter zona inhibisi sebesar 7,17 mm, 13,51 mm, dan 18,51 mm, sedangkan terhadap pseudomonas aeruginosa menghasilkan diameter zona inhibisi sebesar 6,52 mm, 11,786 mm, dan 15,269 mm. berdasarkan hasil penelitian ini, dapat disimpulkan bahwa ssf hingga fase stasioner dapat meningkatkan aktivitas antioksidan dan antibakteri lebih tinggi dibandingkan perlakuan yang lain. kata kunci: antibakteri, antioksidan, aspergillus oryzae, pertumbuhan fase stasioner, solid state fermentation microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-87825663415; email: merry.martgritta@del.ac.id as anti-inflammation, anti-pyretic, and is traditionally used for wound. therefore, candlenut plant can be utilized as medicine, especially due to its extract that has antioxidant and antibacterial activities (ako et al. 2005; krisnawati et al. 2011). based on the research that has already been conducted (siddique et al. 2011), ic value of 50 candlenut seed antioxidant activity using dpph -1 method is 30.37 mg ml compared to ic value of bht 50 -1 standard 15.3 μg ml . both dpph (2,2-diphenyl-1picrylhydrazyl) and bht (butylated hydroxytoluene) candlenut is plant that distributed widely in the world. parts of candlenut tree, such as leaf, seed, pericarp, sap, and flower can be utilized as natural herbal medicine. its periderm is used for curing diarrhaea and tumour. candlenut leaves for curing headache, fever, boils, arthritis, and gonorrhea. flower and fresh sap for curing sprue in children. candlenut seed contents sterol, flavonoids, and triterpene that act are stable radical compounds that can reduce chemical reaction of other radical compounds, therefore both compounds are usually used in antioxidant activity assay. based on research which was conducted by pakpahan & pangaribuan (2018), antibacterial activity of ethanol candlenut kernel extract to staphylococcus aureus resulted 19.17 mm of inhibition zone (strong inhibition response) and to pseudomonas aeruginosa resulted 6-8 mm of inhibition zone (moderate inhibition response). based on those researches, antioxidant and antibacterial activity of candlenut kernel are possible to be increased. several researches proved that antioxidant and antibacterial activity of natural sources can be enhanced after fermented using solid state fermentation method (ako et al. 2005, juan and chou 2010). solid state fermentation can also increase production rate, decrease production cost, and using more simple technique compared to submerged fermentation (juan and chou 2010). microorganisms that generally used in solid state fermentation are aspergillus oryzae, aspergillus niger, monascus purpureus, and bacillus subtilis (das and mukherjee 2007; juan and chou 2010; wen et al. 2013; jamaluddin et al. 2016). the objective of this research is to increase the activity of antioxidant and antibacterial in solid state fermented candlenut kernels by aspergillus oryzae. materials and methods substrate preparation. candlenut was peeled, washed with water, sun-dried, and the kernels were pounded into powder. microorganisms preparation. aspergillus oryzae was purchased from laboratory of microbiology, sekolah ilmu dan teknologi hayati (sith), institut teknologi bandung. a. oryzae was rejuvenated on potato dextrose agar (pda). inoculum was cultured on growth media yeast peptone glycerol o (ypg) and was incubated in 37 c for 72 h. culture on ypg media will be used to make standard curve, growth curve, and to ferment candlenut powder (lee et a l . 2 0 0 8 ) . p s e u d o m o n a s a e r u g i n o s a a n d staphylococcus aureus were rejuvenated in nutrient o broth (nb) and incubated in 37 c for 24 h (pakpahan and pangaribuan 2018). solid state fermentation. as much as 50 g candlenut powder was moistened with 30 ml distilled water until approximately 60% of humidity in fermentation flask. the fermentation flask was o autoclaved for 15 min and cooled until 25 c before inoculated with a. oryzae. as much as 10% (v/v) of 5 days aspergillus oryzae was inoculated into fermentation media, whereas for control is fermentation media without a. oryzae. fermentation o flask was incubated in 37 c. fermentation was ended when reached exponential phase time (day-9) or stationary phase time (day-12). fermentation was repeated three times to achieve accurate data. extraction. as much as 100 ml of 96% ethanol was added into each fermentation flasks, stirred using o magnetic stirrer for 24 h in 25 c. extracts were filtered using gauze and concentrated in vacuum evaporator. the concentrated extracts were determined for its total phenolic content, antioxidant, and antibacterial activity. total phenolic determination. total phenolic determination was conducted based on modification method in juan and chou (2010). as much as 0.1 ml of extract aliquot was mixed with 1 ml folinciocalteau reagent and left for 3 min reaction. three hundred µl of 1n na co was added and left for 90 2 3 o min reaction in 25 c. absorbance was measured on 725 nm. gallic acid standard curve was determined using 8 concentrations of gallic acid ranged from 0 until 100 ppm. as much as 0.01 g gallic acid in 100 ml volumetric flask was added with 1 ml ethanol, and then ethanol was added again until the indicator line. from 100 ppm stock solution was taken 1 ml and added with 1 ml reagen folin-ciocalteu in 10 ml volumetric flask. the solution was homogenized and left for several minutes. then 4 ml of 1n na co was 2 3 o added into volumetric flask and left for 15 min in 25 c. solution absorbance was measured on 725 nm. absorbance values of gallic acid were plotted to gallic acid concentrations, and count for gallic acid standard curve equation. total phenolic content will be in µg gallic acid/mg extract. antioxidant activity assay. antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (dpph) method (tristantini et al. 2016). every 10 mg sample was dissolved in 100 ml ethanol. dissolved sample was diluted into 5 concentration, those are 50 ppm, 100 ppm, 150 ppm, 200 ppm, and 250 ppm. two ml of every diluted sample was mixed with 2 ml of 50 ppm dpph. two ml of ethanol was mixed with 2 ml of 50 ppm dpph as blank. both solution were o incubated in 25 c for 30 min. absorbance was measured on 517 nm. percent inhibition of dpph will be counted using the following equation: volume 13, 2019 microbiol indones 51 52 limbong et al. microbiol indones in vitro antibacterial activity assay. antibacterial activity assay was conducted using agar disk diffusion method (pakpahan and pangaribuan 2018). as much as 14.0449 g nutrient agar (na) media was dissolved into 500 ml distilled water, poured into 1000 ml erlenmeyer flask. na media was autoclaved for 15 min. sterilized media was poured into 6 petri o dishes and cooled at 25 c. antibacterial activity was assayed to staphylococcus aureus and pseudomonas aeruginosa. one hundred l bacterial suspension of staphylococcus aureus and pseudomonas aeruginosa from exponential phase were inoculated to sterilized na media and spread using glass rod spreader. sterilized disk papers were placed on na agar and each disk paper was dripped with 10 µl sample, ampicillin (positive control), or ethanol (solvent). na media were o incubated at 37 c for 24 h. antibacterial activity was observed by measuring the diameter of inhibition zone around disk paper using calipers. ethanol showed no antibacterial activity, whereas ampicillin showed a very strong antibacterial activity with inhibition zone diameter of 36 mm to s. aureus and 32.78 mm to p. aeruginosa. results total phenolic content was increase after candlenut kernels were solid state fermented by aspergillus -1 oryzae until exponential phase (2.76 mg gae g ) and the highest total phenolic content was reached when fermentation was stopped at stationary phase (4.19 mg -1 gae g ), as showed in table 1. antioxidant activity was also increase with the increasing of total phenolic content, as showed in table 2. ic of stationary-phase fermented extract was 45.29 50 ppm, it was 13.6 times higher than the unfermented extract (617.11 ppm), and 5.7 times higher than the exponential-phase fermented extract (260.23 ppm). stationary-phase fermented extract had a very strong activity (ic < 50 ppm), whereas exponential-phase 50 fermented extract and unfermented extract had a weak activity (ic > 200 ppm).50 the fermented candlenut kernel extracts showed a strong inhibition response compared to unfermented extract (table 3), both to staphylococcus aureus and pseudomonas aeruginosa. the stationary-phase fermented extracts showed wider inhibition zone (18.51 mm and 15.26 mm) compared to exponentialphase fermented extract (13.51 mm and 11.79 mm) respectively to staphylococcus aureus and pseudomonas aeruginosa. discussion solid state fermentation (ssf) made phenolic compound extraction from natural sources more easier (dey and kuhad 2014). the phenolic compounds will be converted into a more soluble form by the enzyme of microorganism. aspergillus oryzae was the microorganism which was used in this research, and was predicted can convert phenolic compound in candlenut kernels into soluble form during the fermentation process. the prediction was proved in this research, with the increasing content of total phenol in the fermented extract. based on research conducted by rashid et al. (2019), it was stated that after solid state fermentation by a. oryzae, the total phenolic content of rice bran was increase 3.8-fold higher than the unfermented. and three types of phenolic acid detected by hplc in unfermented rice bran (coumaric, ferulic acid, and protocatechuic) was also increase in the fermented rice bran by 3.2-fold, 52fold, and 3.2-fold respectively. the increasing content of ferulic acid in solid-state fermented rice bran by rhizopus oryzae was only 23.2-fold compared to the unfermented rice bran (schmidt et al. 2014). and the total phenolic content of solid-state fermented rice bran by monascus purpureus was only increase 1.04-fold higher than the unfermented (jamaluddin et al. 2016). ssf method has an ability to produce secondary metabolites more efficient than smf (submerged fermentation) method and ideal for fungal cultivation which can grow at limited water. the limited water need gives ssf some advantages, such as reduced downstream processing and reduced stirring, therefore ssf can produce high concentration product in a lower requirement of energy (nigam 2009). martins et al. (2011) stated that phenolic compounds, that often be extracted from solid-state fermented substrate, were flavonoid, flavonol, flavone, flavanol, isoflavon, and anthocyanidin. phenolic compounds were considered as natural antioxidant and represent several important bioactive groups in foodstuff. these compounds consist in all plants which were used as foodstuff, but the types and concentration were vary depend on the plant species, genetic factors, and environmental condition. solid state fermentation process is often used to increase the number of phenolic compound in several foodstuff and to increase antioxidant activity which related to the increasing of total phenolic compound. several methods have already been conducted to increase the synthesis of bioactive metabolite from microbes (schmidt et al. 2014), one of those is solid state fermentation. phenolic compound in plants functioned as protecting mechanism and other biological function such as antioxidant activity. plants produce several bioactive compounds which have strong correlation between its antioxidant activity and its total phenolic content, and phenolic compounds have the biggest contribution to antioxidant activity (li et al. 2008). the very strong antioxidant activity of stationary-phase fermented extract was supported by its highest content of total phenolic compounds. phenolic compounds are secondary metabolites which were produced in the end of exponential phase and during the stationary phase (madigan et al. 2012). during the fermentation process, extracellular enzyme of fungi was produced to release the phenolic group from substrate matrix together with the production of phenolic compound during secondary metabolism (dey et al. 2016), and also converted phenolic compound into a more soluble form in ethanol (dey and kuhad 2014). siahaan et al. (2015) stated that the inhibition zone diameter of fermented extract in ethanol related with the components which can be extracted by ethanol. ethanol is a polar compound that is able to extract other polar compounds such as phenol, saponin, alkaloid, and terpenoid. half of those compounds have antioxidant and antibacterial activities. the wider inhibition zone diameter in antibacterial assay to staphylococcus aureus compared to pseudomonas aeruginosa, was caused by the different gram characteristic between both bacteria. staphylococcus aureus is a gram-positive bacteria which its cell wall has 80 nm of peptidoglycan thickness, but has no outer membrane (al hanif 2009). madigan et al. (2012) stated that the number of peptidoglycan layers in gram-positive bacteria is 40 layers, which composed 90% of all cell wall components. pseudomonas aeruginosa is a gramnegative bacteria which its cell wall has 10% peptidoglycan layers of all cell wall components. cell wall of gram-negative bacteria is composed mostly by outer membrane which made its cell wall more complex. action mechanism of most antibacterial compound is by inhibit the biosynthesis of peptidoglycan in the transpeptidase reaction which will cause lysis of cells. in general, antibacterial compound is difficult to pass the outer membrane and attack peptidoglycan in the cell wall of gram-negative bacteria because of the different polar characteristic. the similar profile of the increasing antioxidant and antibacterial activity with the increasing of total phenolic content in the stationary-phase and exponential-phase fermented extracts compared to table 1. total phenolic content table 3. antibacterial activity of candlenut kernel extracts to staphylococcus aureus and pseudomonas aeruginosa sample total phenolic content -1 (mg gae g ) unfermented fermented until exponential phase fermented until stationary phase 0.18 2.76 4.19 table 2. ic value and antioxidant activity level50 sample unfermented fermented until exponential phase fermented until stationary phase 617.11 260.23 45.29 ic (ppm)50 very weak very weak very strong antioxidant activity sample unfermented fermented until exponential phase fermented until stationary phase 7.17 mm 13.51 mm 18.51 mm inhibition zone moderate strong strong inhibition response 6.52 mm 11.79 mm 15.26 mm inhibition zone moderate strong strong inhibition response staphylococcus aureus pseudomonas aeruginosa volume 13, 2019 microbiol indones 53 unfermented extract, is most likely affected by the activity of β-glucosidase enzyme which convert phenolic isoflavon compound in candlenut kernels into free isoflavon called aglycones. aglycones have an effective biological activity to inhibit chronic disease such as cardiovascular and cancer. beta-glucosidase enzymes able to hydrolyze β-glucoside bonds of carbohydrate conjugates in phenolic compounds. the enzymatic hydrolysis on β-glucoside bonds of phenolic compounds seemed a promising method to increase the concentration of free polyphenol and increase the nutraceutical activity in several natural sources (georgetti et al. 2009). besides that, as stated by widowati et al. (2011), the activity of β-glucosidase to hydrolyze β-glucoside of carbohydrate conjugates will produce carbohydrates in the form of monosaccharides or disaccharides as nutrition to support a. oryzae growth. in conclusion, the present study has shown that solid state fermentation of candlenut kernels until stationary phase time by aspergillus oryzae can enhance the production of phenolic compounds and therefore increase the antioxidant and antibacterial activities. acknowledgment this work was supported by a grant from lppm it del. references ako h, kong n, brown a. 2005. fatty acid profiles of kukui nut oils over time and from different sources. ind crop prod. 22(2): 169-174. doi: 10.1016/j.indcrop.2004.07.003. al hanif ms. 2009. pola resistensi bakteri dari kultur darah terhadap golongan penisilin di laboratorium mikrobiologi klinik fakultas kedokteran universitas indonesia (lmk-fkui) tahun 2001 – 2006. bacterial resistance pattern from blood isolates against penicillins in clinical microbiology laboratory fmui year 20012006. 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2016 mar 17. yogyakarta. p g1-1-g1-7. wen yl, yan lp, chen cs. 2013. effects of fermentation treatment on antioxidant and antimcrobial activities of four common chinese herbal medicinal residues by aspergillus oryzae. journal of food and drug analysis 21:219-226. doi: 10.1016/j.jfda.2013.05.013. widowati e, andriani mam, kusumaningrum ap. 2011. study of total probiotics bacteria and antioxidant activity in yoghurt tempeh using substrate variation. jurnal teknologi hasil pertanian iv(1): 18-31. volume 13, 2019 microbiol indones 55 page 1 page 2 page 3 page 4 page 5 page 6 04. sariyanti.cdr vol.15, no.1, march 2021, p 21-26 doi: 10.5454/mi.15.1.4 identification of dermatophyte fungi causing tinea edis and tinea nguium n p u i malabero coastal communities, bengkulu mardhatillah sariyanti putri maya agustria , willujeng fanny 1*, 2 herlambang , besly sinuhaji , risky hadi wibowo , novriantika lestari , 2 1,3 4 1 enny nugraheni 1 , sipriyadiand 4 1 faculty of medicine and health sciences, university of bengkulu, indonesia; 2 medical study program, faculty of medicine and health sciences, university of bengkulu, indonesia; 3 clinical laboratory of rskj soeprapto bengkulu, indonesia; 4 departmen of biology, faculty of mathematic and natural science, university of bengkulu, indonesia. dermatophytosis cases have increased significantly in various countries, more than 20-25% of the population were infected by superficial fungal infections. malabero urban village is a coastal area in bengkulu city with high temperature and humidity. the daily activities of residents in the area are mostly fishermen, so they are in a watery or wet environment for approximately 12 hours every day. hence, this research aimed to identify dermatophyte fungi that caused tinea pedis and tinea unguium from these coastal communities. the diagnosis of tinea was based on symptoms and physical examination of the lesion area. furthermore, specimen collection was carried out by scraping the lesion area, then examined with 20% koh and fungal culture on sabouraud dextrose agar media. subjects included 79 people who had symptoms of tinea pedis and 33 people with symptoms of tinea unguium. the results of dermatophyte fungi identification were obtained, namely trichophyton mentagrophytes, trichophyton rubrum trichophyton tonsurans aspergillus niger, , and . our conclusion is the most identified dermatophyte species is trichophyton mentagrophytes. key words: coastal, dermatophyte, tinea pedis, tinea unguium kasus dermatofitosis meningkat signifikan di berbagai negara, lebih dari 20-25% populasi terinfeksi oleh jamur superfisial. kelurahan malabero merupakan salah satu kawasan pesisir di kota bengkulu dengan temperatur tinggi dan lembab. aktivitas sehari-hari penduduk di kawasan tersebut sebagian besar adalah nelayan, sehingga berada di lingkungan yang berair atau basah kurang lebih 12 jam setiap hari. oleh karena itu, penelitian ini bertujuan untuk mengidentifikasi jamur dermatofita penyebab tinea pedis dan tinea unguium pada masyarakat pesisir tersebut. diagnosis tinea didasarkan pada gejala dan pemeriksaan fisik pada area lesi. selanjutnya pengambilan spesimen dilakukan dengan pengerokan daerah lesi, kemudian diperiksa dengan koh 20% dan kultur jamur pada media (sda). subjek penelitian meliputi 79 orang yang sabouraud dextrose agar memiliki gejala tinea pedis dan 33 orang dengan gejala tinea unguium. hasil identifikasi jamur dermatofita diperoleh , , , dan . trichophyton mentagrophytes trichophyton rubrum trichophyton tonsurans aspergillus niger kesimpulan penelitian ini adalah spesies dermatofita yang paling banyak teridentifikasi adalah trichophyton mentagrophytes. kata kunci: dermatofit, pesisir, tinea pedis, tinea unguium microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ; : +62fax: 85-664924312 +62 e-mail: mardhatillahs@unib.ac.id; cross-sectional study has been conducted by ' araya, tesfaye and fente (2020), showed that the prevalence of dermatophytosis was 46.5%, but in developed countries, it was less than 5% which indicates that dermatophytosis is still a common problem in developing countries. tinea pedis infects about 10% of the world's population, whereas the prevalence of tinea unguium in asia reaches 12.1% ––––––– (behzadi, behzadi and ranjbar 2014; sigurgeirsson and baran 2014). the distribution of causative species vary with geographic region, such as , trichophyton rubrum t. m e n t a g ro p h y t e s m i c ro s p o r u m c a n i s, , a n d epidermophyton floccosum (ameen 2010). fungal disease kill more than 1.5 million and affect over a billion people, so it become a serious public health problem '— . the current (bongomin 2017)et al. report from 14 countries, 12.5% of the global population were affected by fungal disease (denning 2017). dermatophytosis is the most common infectious disease in the world, especially in developing countries, caused by dermatophyte fungi. the incidence of dermatophytosis is more than 20-25% of the population worldwide . a (havlickova, czaika and friedrich 2008) dermatophytosis has emerged as a social, psychological, and economic burden on the society regarding its chronic and recurrent course (patro, panda and jena 2019). the tinea infections are common in tropics with higher humidity geographical areas, overpopulation, and poor hygienic living conditions (weitzman and summerbell 1995). tropical climate and higher humidity conditions are good environment for the growth of dermatophyte fungi. personal hygiene is important because good personal hygiene will mi ni miz e the e nt ra nc e ( po rt de entr y) o f microorganisms into the body, so it can prevent someone from contracting the disease (richardson and edward 2000). coastal areas are often associated with health problems because coastal communities generally pay little attention to environmental cleanliness, thus impacting the quality of public health in the coastal environment . (fleming 2006; stewart 2008)et al. et al. high humidity in the coastal area is a factor that strongly supports the growth and development of pathogens, such as fungi. some aspects of health that are a problem in coastal areas are environmental health, infant and toddler health, and maternal health (heller, colosimo and de figueiredo antunes 2003). indonesia is an archipelago region, located in the equator, and have a tropical climate. these condition allow to develop infectious diseases, such as fungal infection. based on geographical location, bengkulu province has a wet tropical or type a tropical climate (rain tropical) with humidity of 81 to 91%, the maximun temperature average of 29-30ºc, and minimum temperature of 23-24ºc every month. malabero village is one of the coastal areas in the city of bengkulu with total population about 2,486 residents. the most of community working as fishermen and other activities are doing traditional fish processing (hayati and handayani 2014). therefore, it was the first research to identify the occurrence of tinea pedis and tinea unguium in malabero residents, and also using macroscopic and microscopic examination trough koh and sda culture to identify the species of dermatophyte causing both of tinea. materials and methods subject selection. the research was a cross sectional design study with population of the residents of malabero village which has the symptoms of tinea pedis and tinea unguium from physical examination. in the first stage, research subjects were selected based on inclusion and exclusion criteria. inclusion criteria included, malabero coastal communities who had symptoms of tinea pedis and tinea unguium, and were over 18 years old. exclusion criteria included, malabero coastal communities suffering from chronic diseases, such as tuberculosis, diabetes mellitus and cushing disease; use steroids for a long time (more than 2-4 weeks of administration); and experiencing immunological function disorders, such as the acquired immuno deficiency syndrome (aids). furthermore, the subjects were geft an explanation and inform concent sheet. macroscopic and microscopic identification of dermatophye fungi. identification of dermatophyte fungi was carried out at the microbiology laboratory, department of biology, faculty of mathematics and natural sciences, university of bengkulu. the skin and nail lesions of subject were cleaned with 70% ethyl alcohol using sterile cotton. research sample of scraping the lesion, then transferred to the laboratory as soon as possible for direct microscopic examination and culturing. sample for tinea pedis suspect took from the fourth toe clefts of both feet, while in tinea unguium was sampled by scraping material from the white spots on the surface of the nail. microscopic examination of specimens was performed following treatment with an aqueous solution of 20% potasium hydroxide (koh), then all samples were cultured on s a b o r o u d d e x t r o s e a g a r ( s d a ) m e d i u m . identification of fungal characters included macroscopic and microscopic examination. results the total of research subjects included 106 people who had symptoms of tinea pedis or tinea unguium in malabero coastal community. the most age group and gender characteristics of subjects with tinea pedis and tinea ungium symptoms were adult age group (26-45 years) and female group (table 1). the distribution of each tinea pedis and tinea unguium cases based on age groups classification were 35 cases accounting for 46.8% and 21 cases accounting for 55.7%. whereas, 41 females with tinea pedis representing 56,2% of total cases and 20 females representing 60.6% of the total tinea unguium cases. dermatophyte species identification that cause of tinea pedis and tinea unguium was carried out through macroscopic and microscopic examination. from all specimens, four species of dermatophytes were identified, which were the most t. mentagrophytes 22 sariyanti et al . microbiol indones volume 15, 2021 microbiol indones 23 commnon, followed by , and . t. rubrum t. tonsurans trough koh and culture examination of specimens, t. mentagrophytes was the most etiological agent of 46 tinea pedis cases accounting for 63% of total cases, and was identified in 18 tinea unguium cases representing 54,4% of total cases. was t. rubrum ranked the second most frequent dermatophyte fungi causative agent being identified in 20 cases of tinea pedis and 10 cases of tinea unguium, followed by t. tonsurans was responsible for 2 cases each of tinea pedis and tinea unguium. filamentous nondermatophytes including was rare as aspergillus niger causative agents of 6,8% and 9,1% cases each of tinea pedis and tinea unguium total cases (table 2). in figure 1 showed the macroscopic morphology of the dermatophye and non-dermatophye species had been indentified. macromorphological examination of t. mentagrophytes on sda medium showed varied of colonies colour from white to cream or yellowish with a powdery to granular surface like a pile of cotton and no pigment appears on the surface of the reverse colony. species exhibited downy to cottony t. rubrum in texture with white coloured on the surface and brownish yellow pigmentation on the reverse colonies. the macroscopic morphology of had a t. tonsurans yellowish white surface with velvety or powdery texture and feathers at the edges surface. while, aspergillus morphologically identified was niger characterized by yellowish green to olive green colonies and with uniform black to greyish black colonies on the reverse colonies. microscopic examination of t. mentagrophytes showed septate hyphae which branced conidiophores extend, numerous single-celled microconidia, often in dense clusters like grapes. macroconidia were cigar to club shaped and may exhibit some distortion. while microscopic morphology of exhibited t. rubrum hyaline septate hyphae, characterized by several tearsshaped microconidia, and macroconidia formed directly on the ends of thick hyphae singly or in groups. microscopic morphology of produced t. tonsurans septate hyphae and the most prominent feature was the numerous microconidia formed along the hyphae. the microconidia were pyriform (tear-drop) to clavate (club-like) to cylindrical shaped, while cacroconidia were rare and also showed from cylindrical to cigar shaped. whereas microscopic examination of aspergillus niger featured a large number of conidia on table 1 data of patients suffering dermatophytosis in coastal communities, malabero village according to age group table 2 distribution of dermatophyte causing tinea pedis and tinea unguium clinical type of tinea gender age groups (yr) total male/female total 18-25 26-45 46-65 tinae pedis male 2 14 16 32 73 female 5 21 15 41 tinea unguium male 1 6 6 13 33 female 1 10 9 20 total 9 51 46 45/61 106 fungal species number of case in type of tinea tinea pedis tinea unguium trichophyton rubrum male female 10 10 4 6 trichophyton mentagrophytes male female 21 25 7 11 trichophyton tonsurans male female 2 1 1 aspergillus niger male female 1 4 1 2 total 73 33 24 sariyanti et al . microbiol indones vesicles (figure 2). discussion the sex of the study subjects who experienced tinea pedis and tinea unguium were mostly women with a total of 61 people (57.5%). this condition caused by the activity of the most women in the malabero village have been doing traditional fish processing. many factors that support skin infections of the community in the area are personal hygiene, a moist and wet environment, and heavy sweating (hayati and handayani 2014). a person who is active in a hot and humid work environment will more often sweat and make the body in humid conditions. these conditions can increase risk factors for fungal infections. according havlickova, czaika and friedrich (2008), the distribution of dermatophytes varies with geographical region and with a wide range of environmental and socio-economic conditions, as well as cultural factors. differences on the global public health problems also reflect the different of community and environmental hygiene, as well as different availability of therapeutic measures.the low education level of this research subjects was one of the factors that influence poor personal hygiene in malabero coasta community. according to , good pascapurnama (2018)et al. education about the importance of foot hygiene, can help prevent and minimize the progression of tinea fig 1 macroscopic morphology of fungal species, a,b: surface & reverse , c,d: surface & t. mentagrophytes reverse , e,f: surface & reverse , g,h: surface & reverse t. rubrum t. tonsurans aspergillus niger. fig 2 . microscopic examination of fungal species, a: , b: , c: , d: t. mentagrophytes t. rubrum t. tonsurans aspergillus niger. volume 15, 2021 microbiol indones 25 pedis. study by ' olutoyin, onayemi and gabriel, (2017) was identified certain factors as being significant for the development of superficial fungal infections, included behavioral characteristics such as poor hygienic habits. most of the research subjects were in the advanced adulthood group, at the 26-45 years and 46-65 years age groups with a total of 97 people (91.5%). in the study of – , at the regional general hidayati et al. (2009) hospital, dr. soetomo surabaya in 2003-2005, also found the most age groups suffering from dermatophytosis at the age of 25-44 years (36.3%). according to , that age group was adams (2015)et al. classified as productive age or working age, so that activities that produce sweat in high intensity with poor personal hygiene, thus causing an increased risk of dermatophytosis. in another study conducted by – , in pertamina general hospital, bertus (2015)et al. prof. dr. kandou manado, the age group of 45-64 years (49.24%) were the most patient who were infected with dermatophytosis. it was thought to be caused by immune system decreasing trough the increased of age. fungal diseases can attack all age levels, but in people with immunocompromised, they are more susceptible to fungal infections. with increasing age, it also increases the risk of degenerative diseases, which are also predisposing factors for fungal infections (gow and netea 2016). according to a cross sectional study conducted on patients who attended the dermatology outpatient department, showed nail specimens positive by culture, was the major trichophyton mentagrophytes isolate followed by and trichophyton tonsurans trichophyton rubrum (balamuruganvelu, reddy and babu 2019), while the study by –gupta (2018)et al. , highlighted a rise in dermatophyte infection caused t. verrucosum which is a zoophilic dermatophyte. the most sequential transmission of dermatophytosis to humans is zoophilic, anthropic and geophilic (brown et al. 2012). the condition of malabero village which is densely populated, so it is very possible for dermatophyte transmission. the results obtained were in line with research conducted – , hidayati (2009)et al. dermatophytosis caused by had t. mentagrophytes the highest percentage of 15.7%, followed by t. rubrum with a percentage of 13.7%. aspergillus was identified to cause tinea niger pedis by 6.3%. the fungus species is often found in soil, water and decaying vegetation. because these species are often found in the soil, if walking barefoot or not cleaning the feet properly can cause infection of this species. usually attacks aspergillus niger immunocompromised patients, but can also attack immunocompetent patients. and also able to metabolize the keratin layer, but with lower efficiency than dermatophytes, this makes this genus can cause tinea pedis . tinea pedis lesions (viegas 2013)et al. caused by are similar to lesions caused by a. niger dermatophytes, called pseudodermatophytes. hot and humid environmental conditions in the malabero village can increase tinea pedis infection caused by this fungus species. according to tosti, piraccini and lorenzi (2000), tinea pedis caused by occurs a. niger more in the tropics and subtropics with hot and humid climates, such conditions are endemic areas of . .a niger acknowledgements we would like to acknowledge the support from medical study program, faculty of medicine and health sciences university of bengkulu. the authors are also deeply grateful for all participants of malabero coastal community and laboratory technicians of basic science laboratory, faculty of mathematic and natural sciences university of bengkulu. references adams, c. et al. 2015. environmental and genetic factors on the development of onychomycosis. journal of fungi. 1(2): 211216. doi: 10.3390/jof1020211. ameen, m. 2010. epidemiology of superficial fungal infections. clinics in dermatology. 197201. doi: 10.1016/j.clindermatol.2009.12.005. araya, s., tesfaye, b. and fente, d. 2020. epidemiology of dermatophyte and non-dermatophyte fungi infection in ethiopia. clinical, cosmetic and investigational d e r m a t o l o g y . 1 3 : 2 9 1 2 9 7 . d o i : 10.2147/ccid.s246183. balamuruganvelu, s., reddy, s. v and babu, g. 2019). age a n d g e n d e r w i s e s e a s o n a l d i s t r i b u t i o n o f dermatophytosis in a tertiary care hospital, puducherry, india. journal of clinical and diagnostic research. 13(2). 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concentration, nitrogen and phosphorus composition on crude oil biodegradation by epyzim and mixed cultures of pseudomonas aeruginosa and arthrobacter simplex erliza noor1* and linawati hardjito2 1department of agroindustrial technology, faculty of agricultural technology; 2department of aquatic products technology, faculty of fisheries and marine science, institut pertanian bogor, darmaga campus, bogor 16880, indonesia biological methods have gained attention as an alternative treatment for oil degradation in pollution remediation. external and internal factors have a great influence on crude oil biodegradation. this experiment studied the effect of oil concentrations and ratios of ammonium and phosphate on oil degradation in mixed cultures of local strains of pseudomonas aeruginosa and arthrobacter simplex. the oil degradation ability of this mixed culture was compared to the dormant culture of epyzim. the increase of oil concentration, from 1, 3, 5 and 10% (w/v), significantly lowered the ability of both cultures to degrade the oil i.e from 83 % for 1% oil concentration to 64% for 10% oil concentration using local strains. the local strains showed better capability compare to the dormant culture. medium composition was designed by three levels of ammonium concentration (7.6, 37.9 and 75.8 mg l-1) and two levels of phosphate concentration (2.0 and 9.9 mg l-1). the ratio of ammonium to phosphate of 3.8:1.0 in the growth media has resulted the maximum level of oil degradation, i.e 83% and 88%, for dormant and local cultures respectively. the results suggest a potential usage of local microorganisms in degrading crude oil-polluted water. key words: oil pollution, oil biodegradation, epyzim, pseudomonas aeruginosa, arthrobacter simplex _____________________________________________ ________________________ *corresponding author, phone/fax: +62-251-8621974, e-mail: erlizanoor@yahoo.com volume 2, number 3, december 2008 issn 1978-3477 p 146-149 applying chemical methods to degrade oil spill results in secondary pollutants caused by chemicals or surfactants, while physical method only concentrate the pollutant without removing it permanently. thus, both methods are less efficient and less effective in the control of pollution problems. biodegradation is an appropriate technique as the hydrocarbon pollutant is converted permanently by microorganisms into carbon dioxide and water, or converted into less toxic compounds. several hydrocarbon degraders have been isolated from the indonesian environment and some of them showed high a capability to degrade hydrocarbons. previous studies described a pseudomonas aeruginosa produced biosurfactant with enhanced hexadecanes or as a mixture of long chain n-alkanes (robert et al. 1989). n-alkanes are commonly found in nature (wakeham et al. 1986; kennicut 1988). hydrocarbon biodegradation is affected by internal and external factors. the first is the genotype of the microorganism while the second includes environmental factors such as temperature, ph, oxygen and oil concentrations. in addition, the biodegradation is also influenced by the availability of nitrogen and phosphorous compounds (ratledge 1992; margesin and schinner 2001). microorganisms are able to use hydrocarbons such as thiopene, benzothiopene and dibenzothiopene as carbon sources (fedorak and westlake 1983; arvin et al. 1988; mueller et al. 1991). the nutrient composition, especially c, n and p, are important controllable factors in oil biodegradation. the aim of this research is to investigate the influence of nitrogen and phosphorus composition on the degradation of crude oil employing a mixed culture of p. aeruginosa and arthrobacter simplex. the performance of the mixed culture was compared to commercial culture produced for the same purpose named epyzim. materials and methods bacterial cultures. pseudomonas aeruginosa and a. simplex were isolated and acclimated from an oil contaminated area in cepu, central java. the bacteria were cultured in tripticase soy agar (tsa). in addition, each bacterium was grown in tauson medium containing 1 g (nh 4 ) 2 so 4 , 0.3 g mgso 4 .7h 2 o, 0.05 g feso 4 .7h 2 o, 0.15 g kh 2 po 4 , 0.2 g k 2 hpo 4 .3h 2 o, 0.6 g caso 4 .2h 2 o and added to 1 l of distilled water. crude oil was used as a carbon source and added at concentration of 1.0 g l-1. an imported commercial microorganism preparation called epyzim was used as a reference. a 0.5 g aliquot of epyzim powder was activated in 150 ml media by adding 1.0 g l-1 of crude oil as carbon source. each culture was grown on a shaker at 120 rpm at room temperature for 48 hours. all experiments were carried out in duplicate. the growth of microorganisms was monitored by observing the turbidity of media. the optical density of the media measured by using the spectrophotometer at a wavelength of 550 nm. media composition. a 10 % (v/v) of epyzim culture was used as a starter. for the p. aeruginosa and a. simplex cultures, each was added at concentration of 5% (v/v) as a mixed starter. those starters were inoculated in tauson media with variations of ammonium and phosphate ratio (7.6:2.0, 7.6:9.9, 37.9:9.9 and 75.8:9.9) or n:p ratio of 3.8, 0.8, 3.8 and 7.6. the ammonium and phosphate sources were (nh 4 )so 4 , kh 2 hpo 4 , and k 2 hpo 4 . to each media having various n:p ratios was added 10.0 g l-1 of crude oil (virginia oil company, east kalimantan). the experiments were conducted in duplicate. the cultures were grown for 21 days at room temperature on a shaker at 120 rpm. sampling was undertaken daily during the culture growth phase. the oil residue following each treatment was analysed to determine the hydrocarbon degradation rate by applying the gravimetric method. the oil content was measured by extracting samples with hexane. the % of oil degraded at the end of each experiment was determined using the following formula: the influence of oil concentration. oil concentrations used consisted of 1, 3, 5 and 10% (w/v). the bacteria were grown in 150 ml of tauson medium agitated at 120 rpm for 21 days at room temperature. the number of cell in both cultures are similar for each inoculum and measured as an od 550 equal 1. the bacteria added at 10% of the working volume. at the end of cultivation, the oil residue was measured in duplicate to determine the degradation rate. results oil biodegradation in tauson media. a mixed culture of p. aeruginosa and a. simplex degraded oil to a lower level than epyzim. at oil concentration of 1%, epyzim degraded oil by up to 83% at n/p of 3.8. the ratio of nitrogen and phosphorus was determined based on the ammonium and phosphate salt added to the medium. at the highest n/p which was 7.6, the epyzim bacteria degraded oil up to 70.7%. at the lowest n/p (0.8), the epyzim bacteria degraded oil up to 75.0%, which is about 9.6% lower than the highest degradation value achieved at n/p of 3.8 ( table 1). an equal mixed culture of p. aeruginosa and a. simplex at the lowest n/p (0.8) resulted in oil degradation up to 80.0%. at higher n/p (3.8) resulted from the ratio of ammonium: phosphate of 2.0:7.6 and 37.9:9.9, the mixed culture of p. aeruginosa and a. simplex degraded oil by up to 87.7 and 80.7%, respectively. the mixed culture p. aeruginosa and a. simplex showed a degradation level higher than epyzim (fig 1), this also confirmed by the specific growth rate of a mixed culture that higher than epyzim for all n/p ratio as shown in table 2. n/p of 3.8 was the optimal condition for both cultures. n/p ratio did not influence the degradation time. for all n/p, a mixed culture of p. aeruginosa and a. simplex and epyzim reached stationary growth phase after 9 days and 11 days cultivation, respectively (fig 2). the investigation showed that n/p of 3.8 was the optimal condition for bacterial growth and oil degradation and the ratio was used for further experiments. the influence of oil concentration on biodegradation. in the media containing an ammonium and phosphate ratio of 7.6:2.0 at various initial oil concentrations, results showed that increasing the initial oil concentration decreased oil degradation (fig 3). in the mixed culture of p. aeruginosa and a. simplex with the initial oil concentration of 1, 3, 5, and 10% resulted in oil degradation of up to 83, 77, 74 and 65%, respectively after 21 days cultivation. for epyzim under the same conditions, oil degradation reached 80, 77, 71 and 62%. the optimum biodegradation for both cultures is presented as a histogram in fig 4. this shows that the performance of mix culture p. aeruginosa and a. simplex is slightly better than for the epyzim. both cultures were able to degrade oil at the highest concentration tested (10% w/v). the time required to degrade the various oil concentrations was between 10-15 days, where the cultures reached the stationary phase. initial oil content average oil content at stationary phase initial oil content x 100 % table 1 oil biodegradation at various ammonium phosphate ratio (n/p) at an initial oil concentration of 1% for 21 days percentage of oil degraded (%) epyzim mixed culture* nh 4 mg l-1 po 4 mg l-1 n/p 7 . 6 7 . 6 37.9 75.8 2 . 0 9 . 9 9 . 9 9 . 9 3 . 8 0 . 8 3 . 8 7 . 6 83.0 75.0 81.7 70.7 87.7 80.0 80.7 80.0 *a mixed culture of pseudomonas aeruginosa and arthrobacter simplex. the equal amounts of epyzim and mixed culture i.e 10% (v/v) at od 550 = 1 were used at an initial cultivation. 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18 20 22 t ime (days) 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18 20 22 t ime (days) o il c o n c e n tr a ti o n ( % ) o il c o n c e n tr a ti o n ( % ) fig 1 oil degradation by epyzim and mixed culture of pseudomonas aeruginosa and arthrobacter simplex. o: 2 mg l-1 po 4 + 7.6 mg l-1 nh 4 (n:p = 3.8); ¸: 9.9 mg l-1 po 4 + 7.6 mg l-1 nh 4 (n:p = 0.8); r: 9.9 mg l-1 po 4 + 37.9 mg l-1 nh 4 (n:p = 3.8); x: 9.9 mg l-1 po 4 + 75.8 mg l-1 nh 4 (n:p = 7.6). volume 2, 2008 microbiol indones 147 fig 4 the comparison of oil degradation by epyzim and mixed culture of pseudomonas aeruginosa and arthrobacter simplex. o : epyzim, n: mixed culture. discussion the highest oil degradation for both cultures was achieved at a n/p ratio of 3.8. this was produced by the ratios of ammonium and phosphate of 7.6:2.0 and 37.9: 9.9 (table 1). the results indicate that the n/p ratio is an important factor affecting the growth of bacteria and oil degradation (fig 1 and 2). the lower level of oil degradation is due to the inhibition of bacterial growth caused by unbalanced nutrients (n/p). at a n/p ratio of 0.8, resulting from the ammonium and epizym -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 0 2 4 6 8 10 12 14 17 20 t ime (days) l n o p ti c a l d e n si ty mixed cult ure -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 0 2 4 6 8 10 12 14 17 20 t ime (days) fig 2 the growth of epyzim and mixed culture of pseudomonas aeruginosa and arthrobacter simplex. o: 2 mg l-1 po 4 + 7.6 mg l-1 nh 4 (n:p = 3.8); ¸: 9.9 mg l-1 po 4 + 7.6 mg l-1 nh 4 (n:p = 0.8); r: 9.9 mg l-1 po 4 + 37.9 mg l-1 nh 4 (n:p = 3.8); x: 9.9 mg l-1 po 4 + 75.8 mg l-1 nh 4 (n:p = 7.6). epyzim 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 14 16 18 20 t ime (days) o il d e g ra d a a ti o n ( % ) mixed cult ure 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 14 16 18 20 t ime (days) fig 3 percentage of oil degradation by epyzim and mixed culture of pseudomonas aeruginosa and arthrobacter simplex during microbial growth at various oil concentrations. o: oil concentration of 1%; ¸: oil concentration of 3%; r: oil concentration of 5%; x: oil concentration of 10%. table 2 specific growth rate of epyzim and mixed culture at various ammonium phosphate ratio (n/p) specific growth rate, µ (day-1) epyzim mixed culture nh 4 mg l-1 po 4 mg l-1 n/p 7 . 6 7 . 6 37.9 75.8 2 . 0 9 . 9 9 . 9 9 . 9 3 . 8 0 . 8 3 . 8 7 . 6 0.33 0.26 0.32 0.31 0.52 0.27 0.52 0.47 148 noor and hardjito microbiol indones 0 10 20 30 40 50 60 70 80 90 1 3 5 10 oil concentration (%) o il d e g ra d a ti o n (% ) phosphate addition at concentration of 7.6 and 9.9 mg l-1, respectively, the added phosphate was excessive. similarly, at a n/p ratio of 7.6, the added ammonium (75.8 mg l-1) was higher than needed. these conditions resulted in a lower oil degradation. for biodegradation of light crude oil the n/p molar ratios between 10:1 and 20:1 were optimal for oil degradation (nagwa et al. 1999). it can be concluded that unbalance n/p results in unfavourable conditions for microbial growth that finally decreases oil degradation. the equal mixed culture of p. aeruginosa and a. simplex degraded oil better than epyzim. the oil degradation of both cultures was in the range of 62-83% (fig 3). the values obtained agreed with previous investigations, which was between 45-90% degradation (swannell et al. 1996) and 75%87.5% degradation of crude oil by addition of npk fertilizer (ijah et al. 2008). epyzim consists of dormant bacteria that require an adaptation phase before degrading oil, while p. aeruginosa and a. simplex are local cultures that are better adapted to the environmental conditions. the experiments were conducted at room temperature which is close to that of their original habitat. the data in fig 4 indicate that at various concentrations of oil, the mixed culture of p. aeruginosa and a. simplex degraded oil better than epyzim. the single culture of p. aeruginosa was able to degrade oil up to 25% at an initial concentration of 1% (w/v) over 14 days cultivation (udiharto 1992). in our experiments with a degradation time between 10-15 days, the data showed 70 to 80% oil degradation (fig 3). previous investigations indicated that a mixed culture of p. aeruginosa and a. simplex grew and degraded oil to a significantly higher level than if one used a single culture (hardjito 2002). in addition the percentages of degraded oil were 58.9, 63.8 and 68.9, respectively. the reason why a mixed culture gave better performance than a monoculture was due to the production of biosurfactant during the growth period of p. aeruginosa (robert et al. 1989).this degradation time with this mixed cultures is also less than the degradation time employing mixed cultures of p. aeruginosa, p. stutzeri and b. subtilis, which was 60 days (swannell et al. 1996). these results show that p. aeruginosa and a. simplex work better together to degrade oil. the results of our investigation indicated that mixed culture of p. aeruginosa and a. simplex has potential to be developed as a biological agent to degrade oil. in term of ease of use, epyzim is more practical, while a mixed culture of p. aeruginosa and a. simplex is better in term of biodegradation ability. it is suggested that the development of a dormant mixed culture of p. aeruginosa and a. simplex would increase the practicality of this improved procedure. references arvin e, jensen b, aamand j, jorgensen c. 1988. the potential for free living ground water bacteria to degrade aromatic hydrocarbons and heterocyclic compounds. water sci technol 20:109-118. fedorak pm, westlake dws. 1983. microbial degradation of organic sulfur compounds in prudhoe bay crude oil. can j microbiol 29:291-296. hardjito l. 2002. the treatment of petroleum industrial waste and bioremediation of contaminated site: the indonesia experience. in: proceeding of the asem conference on bioremediation. ha noi, vietnam, sep 24-27, 2002. p1-14 ijah ujj, safiyanu h, abioye op. 2008. comparative study of biodegradation of crude oil in soil amended with chicken dropping and npk fertilizer. sci world j 60:63-67. kennicut mc. 1988. the effect of biodegradation on crude oil bulk and molecular composition. oil chem pollut 4:89-112. margesin r, schinner f. 2001. bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area. appl environ microbiol 67:3127-3133. mueller jg, lantz se, blattman bo, chapman pj. 1991. bench scale evaluation of alternative biological treatment processes for remediation of pentachlorophenol and creosote contaminated materials slurry phase bioremediation. environ sci technol 25:1055-1061. nagwa ma, nagwa mm, magdy em. 1999. crude oil biodegradation by naturally inhabiting mixed bacterial culture under different environmental factors. pak j biol sci 2:1211-1218. ratledge c. 1992. mini-review compilation biodegradation and biotransformation of oil and fats. j chem tech biotechnol 55:397414. robert m, mercade me, bosch mp, parra ll. 1989. effect of the carbon source on biosurfactant by pseudomonas aeruginosa 44t1. biotechnol lett 11:871-874. swannell rpj, lee k, mcdonagh m. 1996. field evaluation of marine oil spill bioremediation. microbiol rev 60:342-365. udiharto m. 1992. aktivitas mikroba dalam degradasi minyak bumi. dalam: prosiding diskusi ilmiah vii. hasil penelitian lemigas. jakarta, indonesia, feb 11-13, 1992. wakeham sg, canuel ea, doering ph. 1986. behavior of aliphatic hydrocarbons in coastal seawater mecososm experiment with c14 octadecane and c-14 decane. environ sci technol 20:574580. volume 2, 2008 microbiol indones 149 6. supiana (short).cdr short communication role of chloramphenicol acetyltransferase (cat) enzyme for early detection of chloramphenicol resistant salmonella typhi supiana dian nurtjahyani faculty of teaching and educational science, universitas pgri ronggolawe tuban, east java, indonesia salmonella typhi salmonella typhi salmonella typhi salmonella typhi salmonella typhi salmonella typhi salmonella typhi salmonella typhi antibody. salmonella typhi salmonella typhi salmonella typhi salmonella typhi microbial resistance to antibiotic is a major problem in the treatment of infectious diseases. the purpose of this study was to identification the role of cat enzyme for early detection of chloramphenicol resistant . observation was performed on isolated from dr. sutomo hospital, surabaya health laboratory, and microbiology laboratory-airlangga university, surabaya in 2009 and had been subcultured. the sub-culture was then tested for its susceptibility to chloramphenicol by using anti-chloramphenicol acetyl transferase (cat) antibody from sigma (catalog number c.9336). susceptibility test using dilution and diffusion methods proved that resistant could change to sensitive and vice versa. it seems that the cat enzyme can bind anti-cat so reversing the resistant into sensitive and conversely, sensitive into resistant one. key words: cat enzyme, chloramphenicol, resistant resistensi mikroorganisme terhadap antibiotika merupakan masalah besar dalam penanganan penyakit infeksi. tujuan penelitian ini adalah untuk mengidentifikasi peran enzim cat untuk deteksi dini yang resisten terhadap kloramfenikol. observasi terhadap kuman yang diisolasi dari rsud dr. sutomo surabaya, laboratorium kesehatan daerah surabaya dan laboratorium mikrobiologi unair surabaya pada tahun 2009 dan telah di sub kultur lagi . hasil sub kultur kemudian dilakukan uji kepekaan dengan anti-chloramphenicol acetyl transferase (cat) hasil uji kepekaan secara dilusi dan difusi membuktikan yang resisten dapat berubah kepekaannya menjadi sensitif dan sensitif dapat berubah kepekaannya menjadi resisten. kesimpulan dalam penelitian ini anti cat berperan mengikat aktivitas enzim cat sehingga yang resisten menjadi sensitif dan sebaliknya sensitif yang diberi kloramfenikol dapat berubah menjadi resisten kata kunci : enzim cat, kloramfenikol, resisten . vol.8, no.2, june 2014, p 81-85 doi: 10.5454/mi.8.2.6 *corresponding author; phone: +62, email: diantbn@yahoo.co.id 81335278182 microbial resistance to antibiotic is a major problem in the treatment of infectious diseases. since 1 9 7 2 , r e s i s t a n c e o f t o chloramphenicol was widely reported around the world. resistance mechanisms of microorganisms to antimicrobial drugs might occur through changing of drug receptor, decreasing the drug amount that reaches the receptor, damaging or deactivating the drug, and developing resistance metabolic pathways. bacteria may have one or more abilities to establish the resistance mechanism. one of the abilities is to produce cat enzyme that can deactivate chloramphenicol (baron s 2005).the purpose of this study was to identify the role of cat enzyme for early detection of resistant to chloramphenicol. s a l m o n e l l a t y p h i salmonella typhi salmonella typhi salmonella typhi salmonella typhi observation was performed on isolated from dr. sutomo hospital, surabaya health laboratory, and microbiology laboratory-airlangga university, surabaya in 2009.the sub-cultured bacteria were then tested for their susceptibility to chloramphenicol by using anti-chloramphenicol acetyl transferase (cat) antibody from sigma catalog number c.9336). the study was conducted in the biology laboratory, unirow tuban and the institute of tropical disease, airlangga university (itdairlangga university), surabaya. fifteen isolates taken from blood specimens were firstly tested using chloramphenicol. strains that consistently showed chloramphenicol sensitivity and resistance were used for susceptibility test using dilution and diffusion methods. the chloramphenicol susceptibility was performed by administering an anticat antibody (10 µg ml ) ( -1 82 nurtjahyani microbiol indones salmonella typhi salmonella typhi and 3.125 µg of chloramphenicol to the resistant strains. sensitive strains treated without anti cat was used as control. the treatment was conducted on taken from 15 stock tubes. three isolates from tubes 3, 9, 12, which consistently showed resistance, were then coded as r3, r9, and r12. three isolates from tubes number 13, 14, 15, which consistently showed sensitivity, were then coded as s13, s14, and s15. whereas, from tubes 1,2,4,5,6,7,8,10,11 no results found. r3 r12r9 s13s15 s14 a b fig 1 dilution susceptibility test to determine the sensitivity and resistance of the strains after overnight incubation at 37 c. salmonella typhi o salmonella typhi salmonella typhi salmonella typhi salmonella typhi clear culture of means no bacteria growth, whereas turbid cultures means there was bacteria growth. resistant strains (r3, r9, and r12) were treated with anti cat 10 µg and 3.125 µg chloramphenicol ( fig 1a). cultures of resistant strains r3 and r12 turned turbid, while r9 culture remained clear. sensitive (strains s13, s14, s15 ) were treated only with 3.125 µg of chloramphenicol, without anti cat (figure 1b). cultures of sensitive strains s15 and s13 turned turbid; whereas s14 culture remained clear. clear culture of meaning means there was no bacteria growth in negative control medium (r9) and turbid cultures showed growth (r3 and r12) (fig 2). salmonella typhi salmonella typhi r12r9r3 fig 2 cultures of chloramphenicol resistant strains treated with anti-cat after overnight incubation at 37 c. salmonella typhi o volume 8, 2014 microbiol indones 83 salmonella typhi the diffusion susceptibility test results using chloramphenicol-disk concentration 30 µg (c30) method for resistant (r3, r9, r12) indicated the presence of inhibition zone, meaning the resistant bacteria change to be sensitive to chloramphenicol (fig 3). r9r12 r3 fig 3 diffusion susceptibility test results resistant the same strain using chloramphenicol-disk method (c30) after overnight incubation at 37 ºc inside petridish. salmonella typhi salmonella typhi the diffusion susceptibility test results using chloramphenicol-disk method for sensitive (s13, s14, s15), arrow indicated the presence of inhibition zone (fig 4). the inhibition zone showed that the bacteria still sensitive to chloramphenicol. salmonella typhi salmonella typhi ; no inhibition zone means the bacteria is resistant to chloramphenicol. it showed that sensitive changed to be resistant to chloramphenicol. s14 s15 s13 fig 4 difusion susceptibility test results sensitive the same strain using chloramphenicol-disk method (c30) after overnight incubation at 37 c inside petridish. salmonella typhi 0 microbiol indones salmonella typhi salmonella typhi salmonella typhi salmonella typhi . et al. salmonella typhi salmonella typhi s. typhi et al. et al. et al. salmonella typhi the results indicate that change of the sensitivity of . resistant is assumed to produce cat enzyme that after given the anti-cat, could change susceptibility become sensitive to chloramphenicol, and vice-versa. sensitive that was given chloramphenicol could change susceptibility to become resistant. it demonstrates produce cat enzymes that can inhibit chloramphenicol so that bacteria become resistant to chloramphenicol. similar results research by juwita 2013. out of 37 blood samples of typhoid fever patients in paediatric department of rsud ulin banjarmasin, 20 samples were positive of isolate and the samples had undergone sensitivity test to antibiotic chloramphenicol, amoxicillin, and cotrimoxazole. the similar results were obtained on studies strain resistance to chloramphenicol (mandal 2004).this is also shown by mirza (2000) in pakistan that at the same time genetically multidrug resistance has been reported to occure endemic, such as in bangladesh. in india it was reported that 10% were found to be multidrug resistant (mdr) (defined as resistance to ampicillin, chloramphenicol and cotrimoxazole). there was a decrease in the susceptibility to ciprofloxacin of with mic showing an upward trend µg ml (nagshetty 2002). plasmids as intermediary for resistance genes to chloramphenicol, trimethoprim and ampicillin are type i cat, type vii dihydrofolate reductase and tem-1 ßlactamase. treatment with anti-cat is based on the assumption that resistant bacteria capable of producing cat enzyme and this enzyme may be expressed to the culture media so that when anti-cat added to the media can bind the expressed cat enzyme, this is mean cat cannot function as a contributing factor in the resistance, so when exposed to chloramphenicol the bacteria will be sensitive. one out of three resistant isolates was turned into sensitive, this is supported by the statement of wain (2003) and asma (2005) that the genes responsible for resistance to chloramphenicol were brought by the plasmid and also performed by chromosomes, the catp gene. dumo and adrian (1992) studied about in vitro resistance by kirby-bauer method found resistant to chloramphenicol (3.8%), ampicillin (6.7%), ceftriaxone (5.1%) and the highest resistance against cotrimoxazol (11.4%). chloramphenicol resistance level, study is still low compared to other antibiotics so that when the cat enzyme bind to anti(0.125-4 ) -1 cat as performed in this study, there is still a chance to turn out the bacteria to be sensitive to chloramphenicol, so the anti-cat allegedly had a role in bacterial sensitivity, because it can change the resistant bacteria to be sensitive. according to the committee for veterinary medical products, the term anti-microbial resistance refers to two terms, namely microbial resistance and clinical resistance. microbial resistance is a biological process associated with various resistance mechanisms involving the role of resistance genes in bacteria. microbial resistance is affected by an enzyme that makesanti-microbial agent is not active. clinical resistance is bacterial resistance that dependent on the responds of bacterial infection to the treatment given (emea 1999; fluit 2001). we can conclude that the anti cat enzyme can shutdown cat activity, rendering the sensitivity of the resistance strains. so that cat enzyme could be used as an early detection of resistant to chloramphenicol. salmonella typhi references asthma h, abdul h, yasra s, aamir a, saira b, ayesha t, mushkoor m. 2005. identification of drug resistance genes in clinical isolates of for development of diagnostic multiplex pcr . pak j med sci. 4 (21): 402-407 baron s. 2005. medical microbiology 4 edition. the univesityof texas medical at galveston. dumo cc, dan adrian pc. 1992. in-vitro resistance pattern of . phil j microbiol infect dis 21 (2) : 76 -78. emea. 1999. discussion paper on antimicrobial resistance. fluit ac, wielder clc, verhoef j, schmiitz fj. 2001. e p i d e m i o l o g y a n d s u s c e p t i b i l i t y o f 3 , 0 5 1 isolates from 25 university hospitals participating in the european sentry study. clin microbiol. 39: 3727-3732. doi: 10.1128/jcm. 39.10.3727-3732.2001. nagshetty k, shivannavar t. channappa, gaddad sm. 2010. antimicrobial susceptibility of in india. j infect dev ctries. 4(2):070-073. mandal s, mandal md, pal nk. 2004. plasmid-encoded multidrug resistance of and some enteric bacteria in and around kolkata, india: a preliminary study. online j health allied. 4: 2. mirza s, kariuki s, mamun k z, beeching n j, hart c a. 2000. analysis of plasmid and chromosomal dna of multidrug resistant from asia. j clin microbiol. 38 (4): 1449-1452. salmonella typhi . salmonella typhi staphylococcus aureus salmonella typhi salmonella typhi salmonella enterica th 84 nurtjahyani volume 8, 2014 microbiol indones 85 juwita s, hartoyo e, yulia l, budiarti. 2013. pola sensitivitas in vitro terhadap a n t i b i o t i k k l o r a m f e n i k o l , a m o k s i s i l i n , d a n kotrimoksazol di bagian anak rsud ulin banjarmasin periode mei-september 2012 [the pattern of sensitivity in vitro of to the antibiotics chloramphenicol, amoxicillin, and cotrimoxazole in the child's hospital ulin banjarmas in salmonella typhi salmonella typhi the period may-september 2012] berkala kedokteran 9(1): 21-29. wain j, nga diem lt, claire k, keitg j, sarah f, diep st, tahir a, gaora o, parry c, parkhill j, ferrar j, white jn, dougan g. 2003. molecular analysis of inch11 antimicrobial resistance plasmids from strains associated with typhoid fever antimicrob agents chemother. 47(9): 2732-2739. salmonella serovar typhi . 12 (yaya rukayadi) catatan penelitian (94-96).pmd short communication the effects of xanthorrhizol on the morphology of candida cells examined by scanning electron microscopy yaya rukayadi∗ and jae-kwan hwang department of biotechnology, yonsei university, 134-sinchon-dong, seodaemun-gu, seoul 120-749, korea the effects of xanthorrhizol, a natural anticandidal agent isolated from the rhizome of temulawak or java turmeric (curcuma xanthorrhiza roxb.) on the morphology of four human pathogenic candida species, i.e., c. albicans, c. glabrata, c. guilliermondii, and c. parapsilosis was examined by scanning electron microscopy (sem). the sem analysis showed that, unlike control cells representing normal oval to spherical with smooth surface, treatment of candida strains with xanthorrhizol at 1 x mics (minimum inhibitory concentration) significantly affected the external morphology, exhibiting deformation, and protrusions on the cell surface. the potent anticandidal activity of xanthorrhizol may support the use of medicinal plants for the treatment of candidal infections. key words: anticandidal, candida sp., scanning electron microscopy, xanthorrhizol _____________________________________________ _________________ ∗ corresponding author, phone: +82-2-2123-4097, fax: +82-2-362-7265, e-mail: yr32@yahoo.com the incidence of invasive fungal infections, particularly those caused by candida sp., has increased over the past few decades (hsueh et al. 2005). recently, strains of candida sp., such as c. albicans, c. glabrata, c. guilliermondii, and c. parapsilosis are showing increased resistance to traditional antifungal agents (hawser and dauglas 1995; nguyen et al. 1996; barchiesi et al. 1999; dauglas 2003). this demonstrates the great importance of identifying novel antifungal agents (ficker et al. 2003). recent years have seen a growing interest in the use of natural antifungal agents isolated from the medicinal plants. xanthorrhizol, a novel bioactive compound isolated from the rhizome of an indigenous indonesian medicinal plant, temulawak or java turmeric (curcuma xanthorrhiza roxb.) has been previously reported to possess an antibacterial activity against several oral pathogens (hwang et al. 2000a, 2000b) and has the ability to prevent and remove streptococcus mutans biofilm formation (rukayadi and hwang 2006a, 2006b, 2006c). xanthorrhizol also has anticandidal activity (rukayadi et al. 2006), anti-malassezia activity (rukayadi and hwang 2007a) and antimycotic activity against opportunistic filamentous fungi (rukayadi and hwang 2007b). this short communication reports the effect of xanthorrhizol on the morphology of candida cells examined using the scanning electron microscopy (sem). the candida strains (c. albicans atcc 10231, c. glabrata atcc 50044, c. guillerimondii atcc 9058, and c. parapsilosis atcc 22019), used in this study, were obtained from the american type culture collection (rockville, md, usa). the strains were cultured on sabouraud dextrose broth (sdb) or sabouraud dextrose agar (sda) (difco, becton dickinson and company, usa) for 48 h at 35 oc. the standardized inoculum (a mcfarland standard) for each isolate used was 5 x 106 cfu ml-1 (nccls 2002). xanthorrhizol (fig 1) (hwang et al. 2000a) was isolated from the ethyl acetate fraction of the methanol extract from curcuma xanthorrhiza according to the method of hwang et al. (2000a). briefly, the rhizomes of c. xanthorrhiza (100 g) were ground and extracted with 75% meoh (v/v; 400 ml), and further fractioned consecutively with ethyl acetate (4.8 g), n-butanol (1.7 g), and water (1.1 g). xanthorrhizol was isolated from the ethyl acetate fraction using silica-gelcolumn chromatography (merk; 70-230 mesh; 5 x 43 cm; nhexane/ethyl acetate, 10:1) yielding 0.2 g. xanthorrhizol was dissolved in 10% (vol/vol) dimethylsulfoxide (dmso) to obtain 1 mg ml-1 stock solutions. dmso at 10% (vol/vol) was found not to kill candida. samples for scanning electron microscopy were provided as follows: 10 ml of cultures of c. albicans, c. glabrata, c. guilliermondii, and c. parapsilosis were exposed to 1 x mics of xanthorrhizol (15, 10, 8, and 25 µg ml-1, respectively) (rukayadi et al. 2006). after 1 hour of incubation at 35 oc, 1 ml of each culture, and a negative control of a corresponding culture were aliquoted and centrifuged at 3,900 x g for 10 min. cell pellets were resuspended in 10 ml of sterile water and fixed overnight in 4% (vol/vol) glutaraldehyde. the samples were washed twice with 2-ml portions of sterile water and centrifuged at 3,900 x g for 10 1 4 4 oh 3 2 1 7 1 5 6 5 8 9 1 0 1 2 1 1 1 3 fig 1 structure of xanthorrhizol (hwang et al. 2000a). microbiology indonesia, august 2007, p 98-100 volume 1, number 2 issn 1978-3477 min. the final pellets were then resuspended in sterile water. a drop of each suspension was transferred onto glass cover slips and fixed onto aluminium sem stubs (agar science ltd, standstead, uk). the drop was spread thinly on the slip and dried in the air for 2 hour at room temperature. graded concentrations of ethanol (70, 80, 90, and 95%, vol/vol), were applied, 2-5 min each, to ensure complete dehydration of specimens. the specimens were coated with gold in a lowpressure argon atmosphere employing a model e5000 polaron sputter coating unit (polaron equipment ltd, new haven, west sussex, uk). a jeol jsm-840 scanning electron microscope (jeol technics ltd, tokyo, japan) was used to evaluate samples, operating at accelerating voltages of 2025 kv (helal et al. 2006). sem analysis showed that, unlike control cells (antifungal-agent free) showing normal oval to spherical shapes with smooth surfaces, treatment of candida species with mic of xanthorrhizol affected the external morphology of these yeasts (fig 2). control cells displayed well-formed cells with smooth unadulterated surface (fig 2a, c, e, g). in contrast, cells incubated in the presence of xanthorrhizol demonstrated a greater tendency to clump compared with the control cultures (e.g., c. albicans fig 2b). xanthorrhizoltreated c. glabrata cells showed minor abnormalities without a smooth or a slightly awkward surface (fig 2d). xanthorrhizol-treated candida cells exhibited deformation and protrusions on the cell surface, which was more clearly demonstrated with c. guilliermondii and c. parapsilosis (fig 2f, h). electron micrographs revealed the existence of a recognizable affected external morphology of candida cells caused by xanthorrhizol. visible deformation, protrusion, or clumping was noted for each species at concentration mics for 1 h treatment. in general, candida exposed to xanthorrhizol at concentrations 1 x mics exhibited substantial ultrastructural abnormalities such as shape deformation, protrusion, rugged cells surface, and clumping. although, we were not able to identify the underlying molecular changes caused by the compounds by scanning electron microscope after 1 h treatment, we were able to show that the observable cell wall changes were generally obtained following exposure of the isolates to concentrations of xanthorrhizol equal to 1 x mics. analysis of electron micrograph at the appropriate exposure time and higher concentrations (2 x mics or 4 x mics) may result in more detailed analyses of the activities and effects of antifungal agents (klepser et al. 1998). further studies have been conducted examining the effect of xanthorrhizol on the morphology candida cells at 2 x mics and 4 x mics for 2 and 4 h of incubation. in summary, the potent anticandidal action of xanthorrhizol against strains of four human pathogenic candida species was demonstrated by scanning electron microscopy analysis. the results showed the usefulness of xanthorrhizol, a promising new antifungal agent for the topical treatment of candidiasis. fig 2 sem of candida albicans (a and b), c. glabrata (c and d), c. guilliermondii (e and f), and c. parapsilosis (g and h) after treating by xanthorrhizol at 1 x mic for 1 h of incubation. volume 1, 2007 microbiol indones 99 a b c d e f g h references barchiesi f, tortorano am, di francesco lf, cogliati m, scalise g, viviani ma. 1999. in vitro activity of five antifungal agents against uncommon clinical isolates of candida spp. j antimicrob chemother 43:295-299. douglas lj. 2003. candida biofilms and their role in infection. trends microbiol 11:20-36. ficker ce, smith ml, susiarti s, leaman dj, irawati c, arnason jt. 2003. inhibition of human pathogenic fungi by members of zingiberaceae used by the kenyah (indonesian borneo). j ethnopharmacol 85:289-293. hawser sp, dauglas lj. 1995. resistance candida albicans biofilms to antifungal agents in vitro. antimicrob agents chemother 39:2128-2131. helal ga, sarhan mm, abu shahla nk, abou el-khair ek. 2006. effect of cymbopogon citrates l. essential oil on growth and morphogenesis of saccharomyces cerevisiae ml2-strain. j basic microbiol 46:375-386. hsueh pr, lau yj, chuang yc, wan jh, huang wk, shyr jm, yan jj, yu kw, wu jj, ko wc, yang yc, liu yc, teng lj, liu cy, luh kt. 2005. antifungal susceptibilities of clinical isolates of candida species, crytococcus neoformans, and aspergillus species from taiwan: surveillence of multicenter antimicrobial resistance in taiwan program data from 2003. antimicrob agents chemother 49:512-517. hwang jk, shim js, baek ni, pyun yr. 2000a. xanthorrhizol: a potential antibacterial agent from curcuma xanthorrhiza against streptococcus mutans. planta medica 66:196-197. hwang jk, shim js, pyun yr. 2000b. antibacterial activity of xanthorrhizol from curcurma xanthorrhiza agaist oral pathogens. fitoterapia 71:321-323. klepser me, ernst ej, ernst me, messer sa, pfaller ma. 1998. evaluation of endpoints for antifungal susceptibility determinations with ly303366. antimicrob agents chemother 42:1387-1391. [nccls] national committee for clinical laboratory standards. 2002. reference method for broth dilution antifungal susceptibility testing of yeasts. approved standard m27-a2. wayne: nccls. nguyen mh, peacock je, morris aj, tanner dc, nguyen ml, snydman dr, wagener mm, rinaldi mg, yu vl. 1996. the changing face of candidemia: emergence of non-candida albicans species and antifungal resistance. am j med 100:617-623. rukayadi y, hwang jk. 2006a. in vitro activity of xanthorrhizol against streptococcus mutans biofilms. lett appl microbiol 42:400-404. rukayadi y, hwang jk. 2006b. effect coating the wells of polystyrene microtiter plate with xanthorrhizol on the biofilm formation of streptococcus mutans. j basic microbiol 46:411-416. rukayadi y, hwang jk. 2006c. effect of xanthorrhizol on streptocossus mutans biofilms in vitro. j microbiol indones 11:40-43. rukayadi y, yong d, hwang jk. 2006. in vitro anticandidal activity of xanthorrhizol isolated from curcuma xanthorrhiza roxb. j antimicrob chemother 57:1231-1234. rukayadi y, hwang jk. 2007a. in vitro anti-malassezia activity of xanthorrhizol isolated from curcuma xanthorrhiza roxb. lett appl microbiol 44:126-130. rukayadi y, hwang jk. 2007b. in vitro antimycotic activity of xanthorrhizol isolated from curcuma xanthorrhiza roxb. against opportunistic filamentous fungi. phytother res 21:434-438. 100 short communication microbiol indones guide for author.cdr guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can be used 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widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com page 1 page 2 blanko pendaftaran anggota.pdf page 1 form berlangganan.pdf page 1 cover belakang.pdf page 1 05. nurtjahyani.cdr vol.13, no.4, december 2019, p 134-136 doi: 10.5454/mi.13.4.5 short communication prevalence of hepatitis b virus infection in blood donors based on titer hepatitis b surface antigen examination (hbsag) 1 2 supiana dian nurtjahyani * and retno handajani 1 faculty of teaching and educational science, universitas pgri ronggolawe tuban, east java, indonesia; 2 faculty of medical and tropical disease center, universitas airlangga, .east java, indonesia hepatitis b remains a global public health problem. infection from hepatitis b virus (hbv) can be transmitted through a blood test or a blood transfusion. this study was conducted to identify the prevalence of hbv infection in blood donors based on examination of hbsag titers . blood donors from tuban red cross used as sample. the method used in this research is hbsag titers examination performed by elisa according to the procedure outlined in the kit. hbsag titers positive mostly found in men. in men from 13 samples (8.67%) are hbsag titers positive of 150 samples, while in woman all negative for hbsag titers from 137 samples. the average titer positive was 3.095 with a standard deviation of 0.187. while hbsag titers negative have average of 0.03 with a standard deviation of 0.14. this study showed that the prevalence of hbv infection in blood donors is most numerous in men with hbsag titers positive number of 8.67%. key words: blood donor, hbsag titer, hbv hepatitis b masih merupakan masalah kesehatan masyarakat secara global. infeksi virus hepatitis b (vhb) bisa melalui pemeriksaan darah maupun transfusi darah. tujuan penelitian ini untuk mengidentifikasi prevalensi infeksi vhb pada pendonor darah berdasarkan pemeriksaan titer hbsag. metode yang digunakan dalam penelitian ini adalah metode eksperimen laboratorium. pemeriksaan titer hbsag dilakukan dengan metode elisa sesuai dengan prosedur yang terdapat pada kit. hasil penelitian menunjukan titer hbsag positif paling banyak pada pria sebanyak 13 sampel (8,67%) dari 150 sampel sedangkan pada wanita semua titer hbsag negatif sebanyak 137 sampel. rata-rata titer positif 3,095 dengan standar deviasi 0,187. sedangkan untuk titer hbsag negatif rata-rata 0,03 dengan standar deviasi 0,14. kesimpulan dalam penelitian ini prevalensi infeksi vhb pada pendonor darah paling banyak terdapat pada pria dengan titer hbsag positif 8,67%. kata kunci: pendonor darah, titer hbsag, virus hepatitis b microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-81335278182; fax: -; email: diananin39@gmail.com hbv can also a cause of fulminant hepatitis. most patients recover completely within 6 months but when neonates 10% of adult patients and 90% of patients who are infected with hbv will become chronic. there are 80% hepatocellular carcinoma patients caused by hbv infection (sastri 2008). detection of hbsag is one of the easiest and cheapest ways to detect hbv infection, by that reason researchers interested in conducting research in the donor blood in tuban regency since hbv infection can be detected in a blood test as well as the blood transfusion process. objective of this study is to identify the prevalence of hbv infection in blood donors based on hbsag titers examination. the method used in this research was a laboratory experiment. research conducted in the laboratory at biology university ronggolawe (unirow) tuban and the institute of tropical disease airlangga university (unair itd) in surabaya from march to april 2012. hepatitis b is one of several major human diseases, and is a serious global public health problem. about two billion people (who 2000) or one: third of the world's population has been infected with hepatitis b. more than 350 million people among them are suffering with hepatitis b virus (manesis et al. 2001) located mostly in asia or africa (lavanchy et al. 2004). until today in indonesia hepatitis b virus infection (hbv) is a major health problem. in the world itself, an increase number of patients annually infected with the hepatitis b virus both acute, chronic or cirrhosis liver (handajani et al. 1997). in human hbv infection is often not causing much different, in some cases it's detected accidentally during blood test. eventhough in some infection hbv on human can also occur with symptoms of acute hbv infection. volume 13, 2019 microbiol indones 135 sample for this study were taken from tuban red cross's blood donors. hbsag examination conducted using enzyme-linked immunosorbent assay (elisa) kit from abbott, inspection done in accordance with the instructions on the kit. as many as 150 blood samples collected from the blood donors in tuban red cross. the results of the study are presented in table 1. from table 1, majority of donors are 148 men (98.7%) and 145 donors (96.7%) were age over 20 years old. donors were male as much as 148 and 2 female. from the younger than 20 years old age group as many as 3 people and older than 20 years old age group as many as 147 people. titers negative with most numbers is range on 0.021 to 0.040 (44.2%) of the total 140 samples. blood donors for this study were tested using hbsag examination. hbsag positive are 13 samples on men, while hbsag negative are 135 men and women samples are 2 samples. hbsag titer positive with most numbers is range on 1.00 to 3.00 (60%) of the total 10 sample. of the total 150 samples of blood donors pmi in tuban, there were 13 (8.67%) hbsag positive donors. this study is in line with research of yugi et al. (2006) were examine 463 blood donors with hbsag negative, and in between there are 143 with hbv dna. he also mention the correlation between titer of anti-hepatitis b core (antihbc) and hbv dna, which is the higher number of titers of anti-hbc on a line with the number of hbv dna. sastri (2008) study showed the frequency of anti-hbc positive blood donors with hbsag negative is 27%, mainly in males aged 20-29 years (44.4% from population) with blood type o. the averages and standard deviation of hbsag titers in blood donors in tuban red cross can be seen in table 4. in table 4 the hbsag titers positive from male donor has average standard deviation of 3.095 to 0.187 while the hbsag titers positive from female donor are zero. hbsag titers positive from female donor has average of 0.03 with a standard deviation of 0.14. table 1 age of donors whose blood taken as sample table 2 results of hbsag titer examination with negative result titer hbsag frequency % 0.01 0.020 17 12.4 0.021 0.040 62 45.3 0.041 0.060 18 13.1 0.061 0.080 19 13.9 0.081 0.0100 21 15.3 total 137 sex age(years) total sample (bags) (%)no. ≤20 ≥20 male 148 bags (98.7%)1. 0 2 3 145 female 2 bags (1.3%)2. total 150 bags (100%) titer hbsag frequency % 1,003,00 9 60 3,00-5,50 4 40 total 13 100 no. sex titer hbsag positive negative mean sd mean sd 1 male 3,095 0,187 0,093 0,182 2 female 0 0 0,030 0,14 table 3 results of hbsag titer examination with positive result table 4 averages and standard deviation hbsag titer in phase window period, the recovery phase, occult hepatitis b (ohb) (akahane et al. 2002; allain et al. 2006), and post infection the amount of antigen (hbsag) are less. this can cause negative result of hbsag examination, but in this phase hbv is found ) (akahane et al. 2002; chan et al. 2003). intravenously drugs users and blood transfusion recipients, including hemodialysis patients are a highrisk group for hbc((levinson et al. 2003, may et al. 2018)). the risk of hbv transmitted by blood transfusion has been reducing drastically with the screening of hbsag routinely from all blood donors (brooks et al. 2011). to prevent transmission of hbv through blood transfusion, indonesia generally imposes standard for hbsag examination to screen for hbv. based on the results of research and discussion can be summarized as follows: the prevalence of hbv infection in blood donors is based on examination of hbsag titers most men has 13 samples (8.67%) of positive hbsag titers, the average of positive hbsag titers were 3.095 with a standard deviation of 0.187 whereas in women were all hbsag negative titer. acknowledgements we thank to the blood donor unit pmi tuban who have helped in providing research samples and laboratory staff biology unirow tuban and itd airlangga university surabaya, which has provided facilities and support research. as well as kemenristek-higher education through kopertis region vii surabaya for their grants for this research funds. references akahane y, okada s, sakamoto m, wakamiya m, kitamura t, tawara a, dkk. 2002. persistence of hepatitis b viremia after recovery from acute hepatitis b: correlation between anti-hbc titer and hbv dna in serum. hepatol res. 24(1):8–17. doi: 10.1016/s1386-6346(02)00015-3. allain jp. 2006. epidemiology of hepatitis b virus and genotype. j clin virol. 36 (suppl1): s12–7. doi: 10.1016/s1386-6532(06)80003-x. brooks ea, lacey lf, payne sl, miller dw. 2011. economic evaluation of lamivudine compared with interferonalpha in the treatment of chronic hepatitis b in the united states. am j manag care. 7(7): 677-682. chan hl,wong ml, hui ay, hung lc, chan fk, sung jj. 2003. hepatitis b virus genotype c takes a more aggressive disease course than hepatitis b virus genotype b in hepatitis b e antigenpositive patients. j clin microbiol. 41(3):1277–9. doi: 10.1128/jcm.41.3.12771279.2003. handajani r soemarto, purnomo s, soetjipto, maria il, mertaniasih nm, choirul an. 1997. pemanfaatan primer spesifik untuk deteksi genotip virus hepatitis c d dalam serum. majalah kedokteran surabaya. apr-juni; 20:49 – 58. (issn: 0303-7932). handajani r, soetjipto mi, lusida mi. 2004. molecular analysis of various region of hepatitis b virus in chronic hepatitis b patients with and without lamivudine nd therapy. 2 year competitive res. grant xi. hepatitis group-tdc unair, biomedical research unit mataram hospital, jsps, kobe univ. lavanchy d. 2004. hepatitis b virus epidemiology, disease burden, treatmen, and current and emerging prevention and controlmeasures. j viral hepat: 1197-107.107. doi: 10.1046/j.1365-2893.2003.00487.x. levinson w, jawetz e. hepatitis viruses. dalam: scott d.holmberg, anil suryaprasad, john w.ward, penyunting. 2003. division of viral hepatitis, national center for hiv/aids, viral hepatitis, std and tb th prevention. medical microbiology 2 immunilogy 7 edition london: mcgraw hill. hlm.2–9. manesis ek, hadziyannis s. 2001. interferon a treatment of hepatitis b e antigen negative cronic hepatitis b. gastroenterol: 121101-109.109. may s, mandal s, keel p, haywood b, ngui sl, ramsay m, tedder rs, ijaz s. 2018. hepatitis b virus immunization and neonatal acquisition of persistent infection in england and wales. j infect dis. 2218(5):726-733. doi: 10.1093/infdis/jiy209. sastri s. uji anti-hbc pada donor darah yang sudah lolos skrining pada unit tranfusi darah pmi cabang padang.working paper. fakultas kedokteran; 2008 [diunduh 2 september 2012]. tersedia dari: http://repository.unand.ac.id/id/eprint/803. yugi h, mizui m, tanaka j, yoshizawa h. 2006. hepatitis b virus (hbv) screening strategy to ensure the safety of blood for transfusion through a combination of immunological testing and nucleic acid amplification testing japanese experience. clin virol. 36(suppl 1):56–64. doi: 10.1016/s1386-6532(06)80010-7 136 nurtjahyani et al. microbiol indones page 1 page 2 page 3 4. 649 geminiviruses (rika).cdr geminiviruses associated with the weed species , and from java, indonesia ageratum conyzoides centipeda minima, porophyllum ruderale, spilanthes iabadicensis rika meliansyah , sri hendrastuti hidayat *, kikin hamzah mutaqin1,2 1 1and 1 2 department of plant protection, faculty of agriculture, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; department of agrotechnology, faculty of agriculture, universitas pajajaran, jalan raya bandung sumedang km 2140600, jatinangor, bandung ubr 40600, indonesia geminivirus geminivirus geminivirus geminivirus ageratum conyzoides centipeda minima a. boehmerioides porophyllum ruderale spilanthes iabadicensis geminivirus geminivirus begomovirus geminivirus geminivirus geminivirus geminivirus geminivirus ageratum conyzoides , centipeda minima a. boehmerioides porophyllum ruderale spilanthes iabadicensis geminivirus geminivirus genbank geminivirus geminivirus begomovirus geminivirus has a wide host range including cultivated plants and weeds. infected weeds may play an important role in disease epidemic. unfortunately, little is known about weeds species that may serve as alternative host for . this research was conducted to identify on weeds around chili pepper field to study their potential role as virus reservoir. field surveys were conducted to chilli pepper growing area in west and central java provinces, and the special province of yogyakarta during 2009 to collect symptomatic weed plants. infection was detected using pcr technique from 9 weed samples, i.e. 5 samples from bogor (agrbgr), sukabumi (agrskm), magelang (agrmgl), sleman (agrjgy), and garut (agrgrt); from magelang (ctpmgl); from sleman (acpjgy); from bogor (prlbgr); from magelang (splmgl). further genetic analysis showed that those geminiviruses can be differentiated into 2 clusters, showing the possible genetic differences among them. they neither have a close relationship with other geminiviruses published earlier in the genbank, indicating weed infecting collected in this study is possibly a distinct . key words: , , polymerase chain reaction, weed species memiliki kisaran inang yang luas termasuk berbagai tanaman budi daya maupun gulma. hingga saat ini pengetahuan tentang jenis-jenis gulma yang berpotensi menjadi sumber masih sangat terbatas. penelitian ini dilakukan dengan tujuan mengidentifikasi pada gulma-gulma yang tumbuh di sekitar pertanaman cabai dalam upaya mempelajari perannya sebagai sumber inokulum virus. survei lapangan telah dilakukan pada tahun 2009 ke beberapa daerah penanaman cabai di provinsi jawa barat, jawa tengah, dan daerah istimewa yogyakarta untuk mengumpulkan gulma-gulma yang menunjukkan gejala. infeksi berhasil dideteksi menggunakan teknik pcr dari 9 sampel gulma, yaitu 5 sampel masing-masing dari bogor (agrbgr), sukabumi (agrskm), magelang (agrmgl), sleman (agrjgy), dan garut (agrgrt) dari magelang (ctpmgl), dari sleman (acpjgy), dari bogor (prlbgr), dari magelang (splmgl). analisis genetika lebih lanjut menunjukkan bahwa yang menginfeksi gulma-gulma tersebut dapat dibedakan menjadi 2 kelompok besar yang merupakan indikasi adanya keragaman genetika di antara mereka. yang menginfeksi spesies gulma yang berbeda tersebut tidak memiliki hubungan kekerabatan yang dekat dengan geminivirus lain yang dilaporkan dalam sehingga yang diidentifikasi dari spesies gulma tersebut merupakan yang berbeda.. kata kunci: , , polymerase chain reaction, gulma geminiviruses are single-stranded dna viruses with geminate particle morphology. they are classified into four genera ( , and ) on the basis of host range, insect vector and genome organization (fauquet and stanley 2005). most of the geminiviruses are transmitted by whiteflies and belong to the genus . these species have been reported to cause significant economic yield losses to many crops in tropical and subtropical regions of the world. member of was known to have enormous diversity resulting from their widespread geographic distribution and host adaptation (varma and malathi 2003). diseases caused by in indonesia has been reported including those infecting tobacco mastrevirus, curtovirus, topocuvirus begomovirus begomovirus begomovirus begomovirus (trisusilowati . 1990), tomato (sukamto . 2005; kon . 2006; santoso . 2008), chilli pepper (sulandari . 2006; hidayat . 2006; trisno . 2009), and also a weed species (haerani and hidayat 2003; kon . 2007). among those diseases, the most concerned one is pepper yellow leaf curl disease which induces symptoms involving foliar chlorosis and curling, reduced leaf size, inhibited fruit set and abnormal fruit. emergence of pepper yellow leaf curl disease in indonesia was first reported in 1999 in west java (rusli . 1999), and in 2003 the disease had been widely spread in java with the highest incidence and severity occurred particularly in central java (sulandari . 2006). census data for the period from 2001 2003 shows that the disease has undergone a 4.6 fold increase between 2001 and 2002 and a 2.5 fold increase between 2002 and 2003 (indonesian ministry of et al et al et al et al et al et al et al ageratum conyzoides et al et al et al *corresponding author, phone: +62-251-8629363, fax: +62-251-8629362, e-mail: srihendrastutihidayat@gmail.com issn 1978-3477, eissn 2087-8575 vol 5, no 3, september 2011, p 120-124 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.3.4 agriculture 2003, unpublished data). it was believed that two main factors may contribute to the spread and distribution of the disease in indonesia, i.e. fluctuation of whitefly population and the presence of alternative plants that may act as virus reservoirs. it was reported previously that weed species may serve as alternative hosts for geminiviruses. yang . (2008) identified a distinct species from from fujian province, china; whereas wu . (2007) was successfully isolated a monopartite geminivirus from three weed species ( spp., , and ) w e r e i d e n t i f i e d a s p o t e n t i a l 's alternative host in jamaica (roye and mclaughlin 1997); meanwhile in indonesia sulandari . (2006) reported that , , , were very susceptible to geminivirus infection. thus, to build more information on the potency of weed species as virus reservoirs especially for pepper yellow leaf curl disease in java, surveys were carried out in 2009 in west and central java. to this aim, weed samples showing leaf curl and yellow vein symptoms were collected from different chilli pepper growing area. in this paper, we report the identification of geminivirus associated with yellow disease of 4 weed et al begomovirus emilia sonchifolia et al m. coromandelianum from guangdong, china. sida macroptilium lathyroides wissadula a m p l i s s i m a begomovirus et al hyptis brevipes physalis floridana crotalaria juncea ageratum conyzoides species: , and . naturally infected weed species with yellow vein symptoms were observed in west java (bogor, sukabumi, garut), central java (magelang), and yogyakarta (sleman). the specific symptoms was observed in weed species ( , ), s p e c i e s ( ) , species ( ), and species ( ) viral dna was extracted from all samples as described by kon . (2002). total dna was extracted from symptomatic leaves according to kon (2002). the dna pellet was resuspended in 100 µl te buffer. amplification of geminivirus genome was proceeded using a pair of specific primers designed for the amplification of coat protein gene, cp protein-v1 (5' taattctagatgtcgaagcgacccgccga 3') and cp protein-c1(5' ggccgaatttcttaatttt gaacagaatca 3'). these specific primers were a. conyzoides centipeda minima, porophyllum ruderale, spilanthes iabadicensis compositae a. conyzoides, c. minima, e. prostrate, p. ruderale s. iabadicensis, g. peruviana e u p h o r b i a c e a e c r o t o n h i r t u s convolvulaceae ipomoea triloba onagraceae ludwigia peruviana . et al et al. materials and methods virus sources and dna extraction. pcr-based detection using geminivirus specific primers. volume 5, 2011 microbiol indones 121 geminivirus acronim geographic location sequence length (bp) genbank accession no. ageratum conyzoides -bogor agrbgr indonesia : bogor, west java 864 ns ageratum conyzoides -sukabumi agrskm indonesia : sukabumi, west java 993 ns ageratum conyzoides -magelang agrmgl indonesia : magelang, central java 868 ns ageratum conyzoides -sleman agrjgy indonesia : sleman, jogyakarta 843 ns spilanthes iabadicencismagelang splmgl indonesia : magelang, central java 890 ns centipeda minimamagelang ctpmgl indonesia : magelang, central java 756 ns porophyllum ruderale -bogor prlbgr indonesia : bogor, west java 832 ns bean yellow dwarf virus bydv south africa 2566 dq458791 tomato leaf curl java virus tlcjv indonesia : java 2752 ab100304 tomato leaf curl java virus-[ageratum] tlcjv[ageratum] indonesia : java 2747 ab162141 tomato leaf curl malaysia virus tlcv malaysia 2754 af327436 tomato leaf curl laos virus tlcv laos 2748 af195782 ageratum yellow vein taiwan virus ayvv taiwan 2734 af307861 ageratum yellow vein china virus-[hn2] ayvv china 2768 aj495813 chilli leaf curl virus-[multan] chilcva pakistan 2754 af336806 pepper leaf curl bangladesh virus peplcv bangladesh 2753 af314531 pepper yellow leaf curl indonesia virus pepylcv indonesia : west java 1560 ab189849 sida yellow vein vietnam virus siyvvnv vietnam 2753 dq641696 mimosa yellow leaf curl virus miylcv vietnam 2757 dq641695 malvastrum yellow vein virus-[y47] myvv china 2731 aj457824 malvastrum yellow vein yunnan virus myvvnv china 2747 aj786711 ns, the sequence has not been submitted to genbank table 1 list of geminiviruses used for viral sequence analysis obtained from asian vegetable research and d e v e l o p m e n t c e n t e r ( av r d c ) , ta i w a n . amplification with pcr technique was carried out in a 25 µl reaction mixture containing 1 µl (200 n ) of sample dna solution and 1 µl (0.2 µm) of each primer using ready to go pcr kit (amersham life science). pcr was performed in thermalcycler gen amp pcr system9700 (perkin elmer) with 30 cycles of melting, annealing and dna extension at 94 °c for 1 min, 55 °c for 1 min, and 72 °c for 2 min, respectively. pcr products were then analysed by electrophoresis in 1% agarose gels in tris-edta buffer (0.5x) and visualized under uv transilluminator (maniatis . 1982). d n a f r a g m e n t s o f approximately 760 bp, as a product of pcr amplification, was sent to macrogen inc. (south korea) for dna sequencing by the dideoxy nucleotide chain termination method. sequence data were then assembled and analyzed with the aid of bioedit programme and paup version 4.0 (swofford 2002). sequences available from genbank were used for phylogenetic analysisis (table 1). the cladogram was set up with a quantitative cladistic maximum parsimony using heuristic methods. a hundred bootstrap iterations were performed. pcr using specific primers cp protein-v1/cp protein-c1 was successfully amplified a ~ 760 base pairs of coat protein fragments from 9 symptomatic samples (fig 1) i.e. 5 samples from bogor (agrbgr), sukabumi (agrskm), magelang (agrmgl), sleman (agrjgy), and garut (agrgrt); from magelang (ctpmgl); from sleman (acpjgy); from bogor g tm et al geminivirus a. conyzoides c. minima a. boehmerioides p. ruderale s e q u e n c e a n a l y s i s . results amplification and sequencing of coat protein gene. (prlbgr); from magelang (splmgl). dna fargments were not obtained from other weed samples ( from garut from brebes, from garut, and from cianjur). major constraint for pcr-based detection using weed samples occurred on viral dna extraction. field samples tend to easily damage thus required immediate processing. in addition, inhibitor and secondary metabolites components found in weed tissues may inhibit the amplification process using pcr. nucleotide sequence data was obtained from 7 virus samples (table 1). nucleotide's length of the virus that was successfully sequenced and used for sequence comparison was in the range of 756 to 993 bp, which contains parts of geminivirus coat protein gene (santoso . 2008). coat protein fragment analysis showed that weed-infecting geminiviruses collected from this study can be differentiated into 2 groups (fig 2). the first group consists of 6 weed-infecting geminiviruses from this study (agrbgr, agrskm, agrjgy, agrmgl, splmgl, and ctpmgl) with a 100 bootstrap value, and the second group consists of one weed-infecting geminivirus from this study (prlbgr) and other geminiviruses previously reported in genbank with a 92 bootstrap value. further more, the virus isolates in the first group can be differentiated into 4 subgroups each consisting of agrbgr and agrskm, agrjgy alone, agrmgl and splmgl, and ctpmgl alone. in the second group the virus isolate prlbgr was placed in the different sub group with other weed-infecting geminiviruses. none of the weed infecting geminiviruses collected from this study has close relationship with geminiviruses previously reported from java, indonesia (tlcv, tlcv-[ageratum], pepylcv). s. iabadicensis g. parviflora , e. prostrata i. triloba l. peruviana et al analysis of genetic relationship. 122 meliansyah et al. microbiol indones fig 1 amplification product of geminiviral dna fragment using specific primers cp protein-v1/cp protein-c1. the samples consist of weed-infecting geminiviruses : k. positive control (artificially inoculated geminivirus from ); 1. -bogor; 2. -sukabumi; 3. -magelang; 4. -sleman; 5. -garut; 6. -magelang; 7. -magelang; 8. -brebes; 9. -garut; 10. -garut; 11. -jogyakarta, 12; cianjur; 13. -bogor; m. 1 kb dna a. conyzoides a. conyzoides a. conyzoides a. conyzoides a. conyzoides a. conyzoides s. iabadicencis c. minima e. prostrata g. parviflora i. triloba a. conyzoides l. peruvianap. ruderale ladder. m k 21 3 4 5 6 7 8 9 1110 12 13 1000 bp 1750 bp 760 bp discussions weeds are potential sources of primary inoculum of viruses and play an important role in their persistence and spread (hallan . 1998). weed infecting geminiviruses has been reported from different geographic location especially in the region where the geminivirus infection causing significant yield loss in important crops. roye and mclaughlin (1997) reported a distinct geminivirus species from spp., , and when they studied the role of weed species in the establishment of tomato and pepper diseases due to infection in jamaica. artificial inoculation using involving several weed species was conducted by sulandari . (2006) to determine the host range of pepper infecting geminivirus and concluded that , , , were very susceptible to pepylcv infection. yellow vein symptom or leaf netting is commonly found associated with geminivirus infection on weed species. during field survey to chilli pepper growing area in 2009, we easily found showing yellow vein symptom. evidence of infection on et al sida macroptilium lathyroides wissadula amplissima geminivirus bemisia tabaci et al h. brevipes p. floridana c. juncea a. conyzoides a. conyzoides geminivirus a. fig 2 cladogram showing the interrelationship among weed-infecting geminiviruses and relative geminivirus' species based on allignment of part of coat protein gene's nucleotide sequence. boothstrap value (100 replication) are shown on the branch of cladogram. bean yellow dwarf virus (bydv) is included as outgroup. agrbgr = bogor, agrskm = sukabumi, agrmgl magelang, agrjgy= sleman, splmgl = bydv = from south africa (dq458791), tlcv java = t from java, indonesia (ab100304), tlcv java = -[ageratum] from java, indonesia (ab162141), tlcv malaysia = t segment a from malaysia (af327436), tlcv laos = from malaysia (af195782), ayvv taiwan = from taiwan (af307861), ayvv china = from china (aj495813), chilcva = -[multan] from pakistan (af336806), peplcv = segment a from bangladesh (paf314531), pepylcv = p virus from west java, indonesia (ab189849), siyvv = from vietnam (siyvv dq641696), miylcv = from vietnam (dq641695), myvv = from china (aj457824), myvynv = from china (aj786711). a. conyzoidesa. conyzoides= a. conyzoides= a. conyzoidess. iabadicencis-magelang, ctpmgl =c. minima-magelang, prlbgr = p. ruderale-bogor, bean yellow dwarf virus omato leaf curl java virus tomato leaf curl java virus omato leaf curl malaysia virus tomato leaf curl laos virus ageratum yellow vein taiwan virus ageratum yellow vein china virus chili leaf curl virus pepper leaf curl bangladesh virus epper yellow leaf curl indonesia sida yellow vein virus mimosa yellow leaf curl virus malvastrum yellow vein virus malvastrum yellow vein yunnan virus conyzoides et al et al geminivirus geminivirus c. minima, p. ruderale, s. iabadicensis. geminiviruses et al et al a. conyzoides c. minima, p. in indonesia has been reported previously by haerani and hidayat (2003), sukamto . (2005), and kon . (2007). new infection on weed species in indonesia was reported in this paper, i.e. infecting and preliminary reports indicated that the primary infecting weeds are not the same ones that infect crops (gilbertson . 1991; mc laughlin . 1994), although it has been speculated that a number of common weeds may serve as alternate hosts for crop-infecting geminiviruses. based on phylogenetic analysis, roye and mclaughlin (1997) concluded that weed-infecting geminiviruses are not host to cropinfecting geminiviruses in jamaica. similarly, phylogenetic relationships of the weed-infecting viruses collected in this study with other geminiviruses indicate that cropand weed-infecting geminiviruses from java, indonesia are distinct, and highly diverse. despite all seven weed-infecting geminiviruses were collected from endemic area of pepper yellow leaf curl disease in java, none of them has a close relationship with pepylcv indonesia. based on our phylogenetic analysis, it is evident that , volume 5, 2011 microbiol indones 123 ruderale, s. iabadicensis ludwigia peruviana a. conyzoides s. iabadicensis p. ruderale synedrella nodiflora galinsoga parviflora et al tomato leaf curl java virus ageratum yellow vein virus and are not reservoirs for geminiviruses important on chilli pepper and tomato in java. however, our host range study showed that pepylcv was able to infect , , , , , and (data not published). earlier, kon . (2007) found evidence for interspecies recombination between (tolcjv) and a strain of (ayvv [java]). therefore, the importance of weeds as alternative hosts for cropinfecting geminiviruses in indonesia will need further investigation. these results may significantly affect the development of strategies for managing the spread of these geminiviruses. this work was supported by the australian center for international agricultural research (project no. hort/2004/048), and competitive research grant, ministry of national education, republic of indonesia (dipa no. 0041/023-04.1/-/2010). we are grateful to sri sudarmiyati tjitrosoedirdjo for her assistance in weed identification. acknowledgements references fauquet cm, stanley j. 2005. revising the way we conceive and name viruses below the species level: a review of geminivirus taxonomy calls for new standardized isolate descriptors. arch virol. 150 (10):21512179. doi:10.1007/s00705-005-0583-0. gilbertson rl, hidayat sh, martinez rt, leong 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geminivirus problems: a serious threat to crop production. ann appl biol 142(2):145-164. doi:10.1111/j.1744-7348.2003.tb00240.x. java virus ageratum yellow vein virus tomato leaf curl virus (philippines) begomovirus sida macropilium lathyroides wissadula amplissima begomovirus begomovirus geminivirus begomovirus capsicum annuum malvastrum leaf curl guangdong virus begomovirus : begomovirus emilia sonchifolia. 124 meliansyah et al. microbiol indones 04 widanarni.cdr vol.12, no.3, september 2018, p 92-98 doi: 10.5454/mi.12.3.4 the administration of pseudoalteromonas piscisida 1ub through artemia sp. to enhance growth performance, immune response and resistance of white shrimp (litopenaeus vannamei) larvae against vibrio harveyi 1 1 1 widanarni , savni retalia sababalat , munti yuhana , and diah ayu satyari 2 utami 1 department of aquaculture, faculty of fisheries and marine science, institut pertanian bogor, dramaga campus, bogor 16680, west java, indonesia; 2 department of aquaculture, politeknik kelautan dan perikanan, jembrana, desa pengambengan, kecamatan negara, jembrana 82218, bali, indonesia. this study aimed to evaluate the effectiveness of the supplementation of pseudoalteromonas piscisida 1ub through artemia sp. to enhance the growth performance, immune response and the resistance of white shrimp (litopenaeus vannamei) larvae to the infection of vibrio harveyi. the natural feed given to the white shrimp larvae r 6 7 8 -1 was artemia sp. enriched with p. piscisida 1ub at concentrations of 10 , 10 , 10 cfu ml , and a control -1 (artemia sp. without any enrichment). the experimental shrimps (0.25±0.02 mg shrimp ) were reared in the -1 aquarium (25 × 20 × 30 cm) containing 4 l sea water with a stocking density of 30 shrimps l . the experimental shrimps were fed the experimental feed from mysis 3 to pl12, and after that they were challenged with v. harveyi 7 -1 (10 cfu ml )through an immersion method. the results of this study revealed that the administration of artemia sp. enriched with p. piscisida 1ub could improve the survival, daily growth rate and absolute growth of length of white shrimp larvae. the activities of protease, lipase, and amylase of white shrimp larvae treated with probiotic were higher (p<0.05) than those of the control. after the challenge test, white shrimp larvae treated with probiotic also had better survival and immune response (total hemocyte count, phagocytic activity, phenoloxidase activity and respiratory burst activity) than those of the the positive control. the best results were obtained in the probiotic 8 -1 application with a concentration of 10 cfu ml . key words: daily growth rate, hemocyte cell, mysis, post-larvae, probiotic, survival penelitian ini bertujuan untuk mengetahui efektivitas dari suplementasi pseudoalteromonas piscisida 1ub melalui artemia sp. untuk meningkatkan kinerja pertumbuhan, respons imun, dan resistensi larva udang vaname (litopenaeus vannamei) terhadap infeksi vibrio harveyi. pakan alami yang diberikan pada larva udang vaname r 6 7 8 -1 yaitu artemia sp. yang diperkaya dengan p. piscisida 1ub pada konsentrasi 10 , 10 , 10 cfu ml , dan kontrol -1 (artemia sp. tanpa pengayaan). udang uji (0,25±0,02 mg ekor ) dipelihara di dalam akuarium (25 × 20 × 30 cm) -1 yang berisi 4 l air laut dengan padat tebar 30 ekor l . udang uji diberi pakan uji dari mysis 3 hingga pl12, setelah 7 -1 itu diuji tantang dengan v. harveyi (10 cfu ml ) melalui metode perendaman. hasil penelitian ini membuktikan bahwa pemberian artemia sp. yang diperkaya dengan p. piscisida 1ub dapat meningkatkan kelangsungan hidup, laju pertumbuhan harian dan panjang mutlak larva udang vaname. aktivitas protease, lipase, dan amilase larva udang vaname yang diberi perlakuan probiotik lebih tinggi (p<0,05) dibanding kontrol. setelah uji tantang, larva udang vaname yang diberi probiotik juga memiliki kelangsungan hidup dan respons imun yang lebih baik (total hemosit, aktivitas fagositik, aktivitas phenoloxidase, dan aktivitas respiratory burst) dibanding kontrol positif. 8 -1 hasil terbaik diperoleh pada aplikasi probiotik dengan konsentrasi 10 cfu ml . kata kunci: kelangsungan hidup, laju pertumbuhan harian, mysis, post-larva, probiotik, sel hemosit microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: 62-8129357404; email: widanarni@yahoo.com the white shrimp larvae production businesses, causing a low survival and growth. one of the diseases that attacks white shrimp is vibriosis, caused by vibrio harveyi (phuoc et al. 2009). it causes the high mortality of the shrimp larvae in hatcheries (chrisolite et al. 2008) at all stadia, from the nauplius, zoea, mysis, and post-larvae to adult shrimp in grow-out ponds (saulnier et al. 2000). various efforts have been undergone to control these diseases, for example by using antibiotics, vaccines, immunostimulants, and probiotics. disease control using antibiotics has been restricted, because it white shrimp (litopenaeus vannamei) is one of the most widely cultivated fishery commodities both in indonesia and in the world. indonesia is one of the largest white shrimp exporters in the world besides ecuador, thailand, vietnam, china, india, and malaysia (fao 2013). production of white shrimp must be supported by a sustainable supply of high quality shrimp larvae. however, various problems, especially diseases, still become the main obstacles in causes the pathogens to be resistant to the antibiotics. currently, many safe and effective biological control methods have been developed, and one of them is the application of probiotics. probiotics are beneficial for cultivated organisms, because they can modify microbial communities, improve nutritional values, improve the host's response to a disease, improve the environmental quality (verschuere et al. 2000), and improve immune response (nayak 2010). the results of several previous studies have proven the probiotic success in increasing the shrimp's growth, survival, immune response and resistance (chiu et al. 2007; nimrat et al. 2012; zokaeifar et al. 2012; nurhayati et al. 2015; widanarni et al. 2015). in this study, the probiotic used was pseudoalteromonas piscisida 1ub which had been tested and had been proven to be able to inhibit the growth of v. harveyi through in vitro tests, and its application through immersion method could increase the survival of giant tiger shrimp larvae (widanarni et al. 2009). the administration of a probiotic to shrimp larvae could be done through the enrichment of artemia, the main natural feed for shrimp larvae, due to its ideal size for the larvae, its high nutritional value, and digestibility. this study aimed to evaluate the effectiveness of the administration of p. piscisida 1ub through artemia sp. to enhance the growth performance, immune response and resistance of white shrimp larvae infected by v. harveyi. materials and methods probiotic preparation. the probiotic used was pseudoalteromonas piscisida 1ub (a wild type isolate) that had been marked with the antibiotic rifampicin (p. r piscisida 1ub ) as a molecular marker (widanarni et al. 2003). this method aimed to ensure that probiotic isolate used in this study was p. piscisida 1ub which was a collection of the fish health laboratory, department of aquaculture, faculty of fisheries and marine science, institut pertanian bogor, indonesia, or was not an isolate from other sources. this method also aimed to distinguish p. piscisida 1ub isolate with other bacterial isolates which could contaminate sample or r medium used in this study. the p. piscisida 1ub was cultured in sea water complete (swc) slant agar o medium and incubated at room temperature (28-30 c) for 24 hours. then, probiotic cells were harvested and were inoculated into a swc broth medium and those were incubated in a waterbath shaker at a temperature o of 28-29 c (140 rpm; 16 h). experimental design. the present study compared the growth performance and the immune response of white shrimp larvae fed artemia sp. nauplii r enriched with p. piscisida 1ub at different 6 7 8 -1 concentrations, i.e. 10 , 10 , 10 cfu ml and without any probiotic (control). four groups of treatments in triplicates were applied in this experiment, i.e. 1ub6, 1ub7, 1ub8 and control. white shrimp rearing. this experiment used 12 units of glass aquarium with a dimension of 25 cm × 20 cm × 30 cm filled with 4 l of disinfected seawater. water temperature throughout the experiment was o maintained at a range of 30 to 32 c by using thermostat. aeration was provided to each experimental aquarium through an aeration unit connected to an air blower. specific pathogen free white shrimp larvae at mysis 1 stage were obtained from a local hatchery (pt. suri tani pemuka, labuan, banten province, indonesia) and acclimatized at laboratory condition until they reached mysis 3 stage. during this stage, shrimp larvae were fed artemia sp. nauplii at a range of 3-4 nauplii per larvae per feeding. the larvae -1 (0.25±0.02 mg shrimp ) were randomly distributed into each experimental tank at a density of 30 white -1 shrimps l . feeding was performed using artemia sp. nauplii r previously enriched with p. piscisida 1ub at different concentrations. feed quantity was gradually increased following the developmental stage of the shrimp at a level ranging from 8 to 10 artemia sp. nauplii per larvae per feeding (nimrat et al. 2011). feeding was offered 6 times a day at 02.00, 06.00, 10.00, 14.00, 18.00, and 22.00 for 15 d of culture. enrichment of artemia sp. artemia sp. enrichment was performed following the procedures described by daniels et al. (2010) with some modifications. artemia sp. nauplii was obtained by hatching 2 g of artemia sp. cyst (supreme plus, us) in 1 l of seawater. enrichment was performed by adding r p. piscisida 1ub suspension into newly hatched artemia sp. nauplii culture medium (seawater at a -1 salinity of 30 g l ) at a concentration depending on the treatment. artemia sp. nauplii were maintained in the -1 probiotic suspension at a density of 100 individual ml for 4 hours. similar procedure was applied to artemia sp. nauplii provided for the shrimp in control treatment, except there was no probiotic added to the artemia sp. culture medium. the nauplii were collected using a plankton net, rinsed with fresh seawater and directly transferred to the shrimp larvae culture tank or kept in a volume 12, 2018 microbiol indones 93 94 widanarni et al. microbiol indones o refrigerator with a temperature of 4 c for later feeding time in the same day. growth performance parameters. white shrimp survival, growth (daily growth rate and absolute growth of length), digestive enzymes activities, and bacterial count were determined at the final day of experiment. digestive enzymes activities measured in the present study were amylase, protease, and lipase activities by pooling 25 to 30 shrimps (0.5 g) per replicate tank. protease and amylase activities were determined following the procedures described in worthington (1993), while lipase activity was determined according to the method described in borlongan (1990). the measurement of amylase, protease, and lipase activities aimed to evaluate the ability of probiotic isolate used in this study to secrete several exogenous enzymes such as amylase, protease, and lipase to promote the growth performance of white shrimp larvae. rna:dna ratio in the whole body of shrimp larvae was measured to evaluate the effect of probiotic administration on the shrimp growth potential (tanaka et al. 2007; zehra and khan 2013). three shrimps were collected from each tank and pooled together for rna and dna extractions, performed using extraction kits isogen (nippon gene, japan) and puregene (qiagen), respectively. the concentrations were subsequently measured using dna/rna quant. challenge test. after the completion of the feeding experiment, challenge test was performed using pathogenic vibrio harveyi mr5339 isolated from shrimp infected with luminescent vibriosis (a collection of the fish health laboratory, department of aquaculture, bogor agricultural university, indonesia). challenge test was carried out on 15 shrimps from each tank placed in 15 units of glass container previously filled with 1 l of disinfected seawater with the procedure previously described in merchie et al. (1998) and widanarni et al. (2014). the bacterial suspension was added to each container at an 7 -1 initial density of 10 cfu ml . on the following day, v. harveyi suspension was occasionally added to replace loss due to water replacement, that were done on day 1 and 3 of the challenge test period. feeding during challenge test was done using non-enriched artemia nauplii, which was offered five times a day. negative control used the shrimp from the control treatment applied in this test with similar treatment, except there was no v. harveyi suspension added to the container. challenge test was performed for 5 days. immune parameters. immune parameters were measured after feeding experiment (prior to challenge test) and after the completion of the challenge test. body fluid collection was conducted following the procedures previously described in tampangallo et al. (2012) with some modifications. briefly, five to six post-larvae (0.3 g) were collected from each tank, placed in a pestle and added with 900 µl of precooled anticoagulant solution (30mm trisodium citrate, 0.34 m sodium chloride, 10mm edta and 0.12 m glucose, ph of 7.55) (liu and chen 2004). each larvae was slightly compressed to let the hemolymph flowing out the shrimp's body and mixed with anticoagulant solution. the solids were subsequently removed and hemolymph mixture was transferred into a new microtube for further analysis. total hemocyte count (thc) was measured following the procedures described by yeh and chen (2009), while phagocytic index was measured according to anderson and siwicki (1995). phenoloxidase activity was measured according to liu and chen (2004) and martín et al. (2012) by measuring the formation of dopachrome at an optical density (od) of 490 nm. respiratory burst was performed according to the procedure described in song and hsieh (1994) and (2012), that martín et al. were based on the formation of formazan. respiratory burst was determined as the formation of blue formazan at an od of 630 nm per 10 µl of homogenates. water quality. water temperature in each tank was monitored daily, while dissolved oxygen (do) concentration, ph and salinity were measured weekly. all water quality parameters were in normal range for shrimp larviculture, with the ranges for temperature, o -1 do, ph and salinity were 31-32 c, 3.8-4.5 mg l , 7.3-1 8.0 and 28-32 g l , respectively. statistical analysis. all data was represented in mean and standar deviation. the data, except bacterial count data, were subsequently subjected to analysis of variance (anova). significant differences were determined by duncan post hoc test. statistical analysis was performed using statistical software spss version 23. results the shrimp larvae's survival in all probiotic treatments were significantly different (p<0.05) from the control (63.06±5.50%). probiotic administrations on white shrimp larvae resulted in higher growth as indicated by the significantly higher dgr and absolute growth of length (p<0.05). the highest growth was abbreviations are as follows: dgr = daily growth rate; al; absolute length; rna:dna = rna:dna ratio. values are presented as means±standard deviations. different uppercase letters in the same row indicate significant differences (p<0.05) among treatment groups. table 1 growth performance of white shrimp larvae (litopenaeus vannamei) administered pseudoalteromonas piscisida 1ub with different concentrations through artemia sp. observed in white shrimp larvae in 1ub8 (36.41±0.81 -1 % day ; 11.48±0.24 mm). the ratios of rna:dna in white shrimp larvae administered probiotic were significantly higher (p<0.05) than those in the control -1 (0.249±0.015 ng μl ) (table 1). the digestive enzyme activities in white shrimp larvae fed probiotic were higher (p<0.05) than those in control group (table 2). total bacterial counts at the beginning of the 6 treatment were relatively similar, 1.60-3.76 x 10 cfu -1 larvae , while at the end of the treatment (pl12), total bacterial counts increased with the highest value 8 -1 obtained in 1ub8 (2.86 x 10 cfu larvae ), the lowest 7 total bacterial count was found in control (2.13 x 10 -1 r cfu larvae ). the p. piscisida 1ub at the end of the rearing period was found in 1ub6, 1ub7, and 1ub8. r the highest total p. piscisida iub was found in 1ub7 5 -1 (2.40 x 10 cfu larvae ) (table 3). after feeding experiment, the immune parameters in white shrimp larvae supplemented with probiotic were better than those of the control groups. the similar trends were observed in immune parameters before and after challenge test, that showed white shrimp fed with probiotic-encapsulated artemia sp. nauplii showed higher (p<0.05) levels of immune responses than those of the positive control. this was confirmed by the higher white shrimp survival in probiotic treatments than that of the positive control following challenge test against v. harveyi (table 4). discussion the results of the study revealed that the r administration of p. piscisida 1ub produced better results in growth performance parameters, including survival, dgr, and absolute growth of length than those of the control. the high survival in all probiotic treatments is suspected to be caused by the ability of p. r piscisida 1ub to improve the white shrimp larvae's fitness through the improvement of the microbial community in the white shrimp larvae's body. some studies demonstrated that probiotics could increase the survival of aquatic organisms. the enrichment of artemia naupli with lactobacillus sporogenes could increase macrobrachium rosenbergii post-larvae survival (seenivasan et al. 2012). the administered probiotic bacteria had a function as a source of macro and micro nutrients (verschuere et al. 2000), improving the nutritional value of the artemia sp. fed to the white shrimp larvae, that will then lead to a better white shrimp growth. the better growth was also supported by the higher rna:dna ratios, total bacterial counts, protease activities, and amylase activities in probiotic groups at the end of the feeding experiment than those of the control. the highest results were found in 1ub8. hamsah et al. (2018) reported that the administration of p. piscisida 1ub at a 6 -1 dose of 10 cfu ml through bio-encapsulation of parameter probiotic concentration control 1ub6 1ub7 1ub8 survival (%) 63.06±5.50 a 79.44±2.39 b 89.72±4.37 c 92.78±6.32 c dgr (% day -1) 29.54±0.93 a 31.95±0.09 b 33.78±1.20 b 36.41±0.81 c al (mm) 7.54±0.15 a 9.32±0.39 b 9.83±0.14 b 11.48±0.24 c rna:dna (ng μl -1) 0.249 ±0.015 a 0.345±0.029 b 0.381±0.024 bc 0.393±0.007 c values are presented as means±standard deviations. different uppercase letters in the same column indicate significant differences (p< 0.05) among treatment groups. table 2 the digestive enzymes activity in white shrimp (litopenaeus vannamei) larvae administered pseudoalteromonas r piscisida 1ub with different concentrations through artemia sp. treatments digestive enzyme activity (u ml -1 minute-1) protease lipase amylase control 0.0280±0.0012a 0.083±0.004a 0.629±0.004a 1ub6 0.0440±0.0002b 0.108±0.001b 1.103±0.006b 1ub7 0.0490±0.0008c 0.109±0.001b 1.318±0.012c 1ub8 0.0540±0.0003d 0.112±0.001b 1.418±0.004d volume 12, 2018 microbiol indones 95 artemia sp. resulted higher growth performance and survival of white shrimp than those of the control. the r p. piscisida 1ub was found in all probiotic treatments, but it was not found in the control. it showed that probiotic used in this study could colonize in white shrimp's larvae body. probiotic commonly could improve nutritional status, modulate other microbes, colonize, and produce several exogenous enzymes (widanarni et al. 2009; labh 2015). the p. piscisida 1ub can secrete amylase protease, lipase, and mannase (hamsah et al. 2017). the p. piscisida 1ub can be combined with a prebiotic and work as synbiotic to reach a better effect to the host. hamsah et al. (2018) reported that the administration of synbiotic (a combination of p. piscisida 1ub and mannan oligosaccharide) through bio-encapsulation of artemia sp. resulted better growth performance and survival of white shrimp larvae than those of control, probiotic, and prebiotic treatments. the white shrimp's survival after the challenge test in probiotic groups, was higher than that of the positive control. this demonstrated that the administration of p. r piscisida 1ub had a positive effect in the resistance of the shrimp against the infection of v. harveyi. this was supported by higher total hemocyte counts, phagocytic activities, po activities, and rb activities in probiotic groups after the challenge test than those of positive control. probiotic is an immunogenic material (aly et al. 2008), which has β-glucan, lipopolysaccharides, and peptidoglycans in its cell wall which have an abbreviations are as follows: thc = total hemocyte count; pa = phagocytic activity; po = phenoloxidase activity; rb = respiratory burst activity. values are presented as means±standard deviations. different uppercase letters in the same parameter and observation period indicate significant differences (p< 0.05) among treatment groups. table 4 the immune response and the resistance of white shrimp (litopenaeus vannamei) larvae administered pseudoalteromonas piscisida 1ub at different concentrations through artemia sp. before and after the challenge test with vibrio harveyi parameter s probiotic concentration control (+) control (-) 1ub6 1ub7 1ub8 thc (x10 6) pre -challenge test 14.26±0.91 a 14.82±0.91 a 16.24±1.71 bc 16.79±1.44 bc 19.60±2.55 c post -challenge test 9.08±0.22 a 17.10±0.12 b 20.55±0.56 c 21.50±0.11 cd 24.10±0.73 d pa (%) pre -challenge test 17.00±1.63 a 17.70±1.25 a 22.00±1.63 b 23.50±0.41 b 28.30±0.47 c post -challenge test 15.00±3.56 a 28.00±0.82 b 36.67±1.25 c 45.67±2.49 d 54.00±0.82 e po (o.d. 490 nm) pre -challenge test 0.54±0.02 a 0.53±0.01 a 0.50±0.01 a 0.66±0.03 b 0.53±0.01 a post -challenge test 0.350±0.007 a 0.670±0.002 b 0.680±0.014 b 0.750±0.017 b 0.810±0.035 b rb (o.d. 630 nm) pre -challenge test 0.76±0.04 a 0.77±0.07 a 0.95±0.02 ab 1.03±0.09 b 1.08±0.20 b post -challenge test 0.28±0.08 a 1.04±0.03 c 1.10±0.01 c 0.75±0.02 b 1.40±0.02 c survival (%) post -challenge test 66.67±9.43 a 100.00±0.00 c 84.44±8.31 b 86.67±5.44 b 93.33±5.44 b r n.d. = not detected any colonies of pseudoalteromonas piscisida 1ub grown on the test medium. table 3 the bacterial population in white shrimp (litopenaeus vannamei) larvae administered pseudoalteromonas r piscisida 1ub with different concentrations through artemia sp. total bacteria total pseudoa lteromonas piscisida 1ub r bacteria treatment s mysis 3 (cfu larvae -1) pl12 (cfu larvae -1) mysis 3 (cfu larvae -1) pl12 (cfu larvae -1) control 3.76 x 106 2.13 x 10 7 n.d. n.d. 1ub6 1.72 x 10 6 2.24 x 10 7 n.d. 1.94 x 10 5 1ub7 1.60 x 10 6 2.20 x 10 7 n.d. 2.40 x 10 5 1ub8 1.76 x 10 6 2.86 x 10 8 n.d. 9.56 x 10 4 96 widanarni et al. microbiol indones immunostimulatory effect (smith et al. 2003; gullian et al. 2004). this would also increase the phagocytic activity of hemocyte cells and po activity. the administration of the probiotic could increase total hemocyte count, that would lead to an immune response improvement during infections by pathogens in shrimp (chiu et al. 2007). the increasing of the total hemocyte count caused the increasing of the rb activity, because all rb activities occur inside hemocyte cells, which conduct foreign particle elimination activities in the phagocytosis process (rodriguez and le muollac 2000). in summary, the administration of p. piscisida 1ub through artemia sp. could effectively improve the white shrimp larvae's growth performance. it also improved immune response and the resistance of white shrimp larvae to the infection of v. harveyi with the best results obtained in 1ub8. the results of this study can be a new strategy to enhance the production of white shrimp larvae through the application of probiotic and a protocol to create standard operational procedure of the application of probiotic through bio-encapsulation of artemia sp. in a shrimp hatchery. references aly sm, ahmed ya, ghareeb aa, mohamed mf. 2008. studies on bacillus subtilis and lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of tilapia nilotica (oreochromis niloticus) to challenge infections. fish s h e l l f i s h i m m u n o l . 2 5 ( 1 2 ) : 1 2 8 1 3 6 . d o i : 10.1016/j.fsi.2008.03.013. anderson dp, siwicki ak. 1995. basic hematology and serology for fish health programs. proceeding of the second symposium on diseases in asian aquaculture “aquatic animal health and the environment”; 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s, khan ma. 2013. dietary lysine requirement of fingerling catla catla (hamilton) based on growth, protein deposition, lysine retention efficiency, rna/dna ratio and carcass composition. fish physiol. biochem. 39(3): 503-512. doi: 10.1007/s10695-0129715-0. zokaeifar h, balcázar jl, saad cr, kamarudin ms, sijam k, arshad a, nejat n. 2012. effects of bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, litopenaeus vannamei. fish shellfish immunol. 33(4): 683-689. doi: 10.1016/j.fsi.2012.05.027. 98 widanarni et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 04. dewi.cdr vol.13, no.3, september 2019, p 97-102 doi: 10.5454/mi.13.3.4 levels of cxcl10 chemokine in dengue infected hepatocyte huh 7 it-1 cell line co-cultured with peripheral blood mononuclear cells 1* 2 1 beti ernawati dewi , eva damayanti , tjahjani mirawati sudiro , and agus 1 syahrurachman 1 department of microbiology faculty of medicine universitas indonesia – cipto mangunkusumo hospital, universitas indonesia. jl. pegangsaan timur no. 16, jakarta, indonesia,10320; 2 master programme of biomedical science, faculty of medicine, universitas indonesia, indonesia. dengue is a mosquito borne virus that spreads rapidly in the world. at present, it is estimated that more than 3.9 billion people are at risk of being infected with dengue virus (denv) and there are 96 million clinical cases that have been reported annually in 128 countries worldwide. in denv infected patients often associated with liver dysfunction which hepatocyte and kuppfer cells as the main target of viral infections. denv infection induced the expression of several chemokines, which might play an important role during the inflammatory response and pathogenesis of a disease. cxcl10 is known as a chemokine that activated lymphocytes for innate and adaptive immunity, induces tissue damage, and modulates tumor formation. therefore, we conducted an in vitro study using huh 7it-1 cells co-cultured with peripheral blood mononuclear cells (pbmcs) to investigate cxcl10 chemokine induction during denv infection. huh 7it-1 cells were grown on 96 micro well plate until a monolayer was formed. the cells were infected with denv-2 moi 0.5 ffu/cell and 1 ffu/cell in the presence of pbmcs. huh7 cell medium were used as negative control. after 2 hours of infection, cells were co-cultured with pbmcs and incubated at 37 ºc with 5% co for 48 hours. cell supernatant was collected and cxcl10 chemokine 2 levels were measured using cxcl10 quantikine elisa kit. statistical analysis was performed by spss 23. in the presence of pbmcs, cxcl10 levels from huh 7it-1 infected by denv-2 moi 0,5 ffu/cell and 1 ffu/cell -1 -1 were 552,653 ± 22,779 pg ml and 576,787 ± 16,901 pg ml , those levels were higher than negative control. as conclusion, denv-2 infected huh 7it-1 cells were able to induce the secretion of cxcl10 from pbmc. key words: cxcl10 chemokine, dengue, huh 7it-1 dengue merupakan mosquitos borne virus yang paling cepat menyebar di dunia. diperkirakan saat ini lebih dari 3,9 miliar orang berisiko terinfeksi denv, dengan 96 juta kasus klinis yang dilaporkan pertahun di 128 negara di seluruh dunia. disfungsi hati sering dikaitkan pada pasien terinfeksi denv dengan sel hepatosit dan kuppfer sebagai target utama infeksi virus. infeksi denv diketahui dapat menginduksi ekspresi beberapa kemokin, yang mungkin memainkan peran penting selama respon inflamasi dan patogenesis suatu penyakit. cxcl10 diketahui sebagai kemokin yang mengaktifkan limfosit untuk kekebalan bawaan dan adaptif, menginduksi kerusakan jaringan, dan memodulasi pembentukan tumor. oleh karena itu, kami melakukan penelitian in vitro menggunakan sel huh 7it-1 yang diko-kultur dengan pbmc untuk mengetahui profil kemokin cxcl10 selama infeksi denv. sel huh 7it-1 ditumbuhkan pada 96 micro well plate hingga monolayer. sel kemudian diinfeksikan denv-2 dengan moi 0,5 dan 1 ffu/sel dan diko-kutur bersama pbmc. medium sel kultur huh7 digunakan sebagai kontrol negatif. setelah 2 jam infeksi, sel diko-kultur dengan pbmc dan diinkubasi pada suhu 37 ºc dengan co2 5% selama 48 jam. supernatan sel kemudian dipanen dan kadar kemokin cxcl10 diukur menggunakan kit elisa quantikine cxcl10. analisis statistik dilakukan dengan spss 23. pada sel yang di ko-kultur dengan pbmc, kadar cxcl10 dari huh 7it-1 yang diinfeksi dengan denv-2 moi 0,5 ffu/sel dan 1 ffu/sel adalah -1 -1 552.653 ± 22.779 pg ml dan 576.787 ± 16.901 pg ml , kadar tersebut lebih tinggi daripada kontrol negatif. sebagai kesimpulan, sel huh 7it-1 yang diinfeksi dengan denv-2 mampu menginduksi sekresi cxcl10 dari pbmc. kata kunci: dengue, huh 7it-1, kemokin cxcl10 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-812-8172-6129; email: betied@yahoo.com are at risk of being infected with denv and there are 96 million clinical cases that have been reported annually in 128 countries worldwide (gyawali & taylor-robinson 2017). furthermore, denv infection is known to cause dengue hemorrhagic fever (dhf) with a mortality rate of 0.5% -3.5% in asia. in indonesia, denv infections cause outbreak cases every year, with the largest cases occurring in 1998 and dengue is a mosquito borne virus that spreads rapidly in the world. this virus transmitted by aedes aegypti and aedes albopictus mosquitoes and causes of febrile illnes in the tropics and subtropics region. at present, it is estimated that more than 3.9 billion people 2004 with 79,480 sufferers and over 800 deaths. in the following years the number of cases by denv infection was known to continued to increase and categorized as an epidemic disease with an increasing area (candra 2010). denv is an rna virus belongs to the flaviviridae family and consist of four serotypes, ie denv-1, denv-2, denv-3, and denv-4 (whitehorn 2012). all of serotypes cause a wide spectrum of clinical presentation, ranging from asymptomatic infections, dengue fever (dd), dengue hemorrhagic fever (dhf) or dengue syock syndrome (dss). dhf and dss are severe clinical forms due to denv infection which can cause bleeding, vascular leakage, shock and organ damage, including the liver (dhanoa et al. 2016, shah and rathi 2017). denv infection in human are often associated with liver dysfunction with hepatocyte and kuppfer cells as the main target of viral infections (samanta 2015). in addition, a number of in vitro studies have shown that denv is able to infect human hepatoma cell lines such as ha22t, hep3b, plc, chang liver cells, hlcs, huh7, and hepg2 (matsuda et al. 2005, lin et al. 2000). in vivo studies, introducing denv-2 intravenously into balb/c mice producing virus particles in the liver and showing clinical signs that are almost similar to those seen in infected humans (sakinah et al. 2017). overall, these findings support the idea that hepatocytes are likely target cells for denv. the pathogenesis of liver cell damage due to denv infection cannot yet be explained with certainty and many different mechanisms are involved, especially the direct cytopathic effect of the virus and immune response to virus infection (trung et al. 2010). one reason is the excessive production of cytokines and chemokines in infections (rathakrishnan et al. 2012). chemokine is a small protein (8-10 kda) that is commonly secreted during a viral infection. the main function of chemokine is for recruitment of leukocytes, which are important for leukocyte homeostasis and mediate immune and inflammatory responses. in addition, the different chemokine expressions in various inflammatory and infectious diseases indicate that they play an important role during the inflammatory response and pathogenesis of a disease. according to this idea, many viral infections are known to induce chemokine expression, which is most likely involved in regulating the recruitment of effector leukocytes to the site of infection. denv infection is known to induce the expression of several chemokines, i n c l u d i n g c x c l 1 / i l 8 , c c l 3 / m i p 1 , ccl5/rantes, and ccl4/mip-1 in various human cell lines, but until now, not much is known what chemokines may play a role in denv infection (chen et al. 2006). cxcl10 levels was increased in dengue patients with warning sign compared by dengue fever patients (p=0,046) (masood et al. 2018). other than that cxcl10 can be secreted from leukocytes, eosinophils, neutrophils, monocytes, endhotelial, epithelial, stroma cells and keratinocytes in response to inf-γ. cxcl10 is known to be able to induces chemotaxis, apoptosis, cell growth inhibition and angiostasis. abnormal levels of cxcl10 have been observed in body fluids of individuals infected with viruses such as ebola, hepatitis b and c, dengue, hiv, and several other viruses. it was also found to be increasing in bacterial, fungal and parasitic infections, which showed that cxcl10 played an important role in the pathogenesis of these diseases (liu et al. 2011). therefore, we conducted an in vitro study using hepatocyte cell line, huh 7it-1 cells co-cultured with pbmcs to investigate cxcl10 chemokine induction during denv infection, because cxcl10 is the most prominent chemokine and may be induced quickly after denv infection (chen et al. 2006). hence, these study results can be used as a reference for further research to explore the association of cxcl10 in causing liver cell damage. materials and methods this research was conducted in laboratory of virology and molecular biology, department of microbiology, faculty of medicine, universitas indonesia from january to july 2019. in this study we used hepatocyte huh 7it-1 cell line, vero cell, and peripheral blood mononuclear cells (pbmcs) from healthy control with ethical permission from the fkui rscm health research ethics committee, with e t h i c a l n u m b e r : k e t4 7 2 / u n 2 . f 1 / e t i k / ppm.00.02/2019. treatment group. there are 6 treatment groups in this study, which are summarized in table 1. propagation of huh 7it-1 cells. in this study, we used hepatocyte cells of huh 7it-1 cells from cryopreservation huh 7it-1 cell from virology and molecular biology, department of microbiology, faculty of medicine, universitas indonesia's collection, then resuspended with dmem medium (gibco) and centrifuged at 1200 rpm for 4 minutes at 4 ºc. the 98 dewi et al. microbiol indones volume 13, 2019 microbiol indones 99 supernatan was discarded and pellets resuspended with 5 ml dmem 10% fbs medium, transferred into the t25 flask and incubated at 37 ºc with 5% co until the 2 monolayer cell was formed. to propagate huh7 cells, passages are performed according to previous study and incubated in an incubator 37 ºc with 5% co2 (tresnaningtyas et al. 2018). propagation of denv-2 strains new guinea c. denv-2 strain new guinea c was provided from virology and molecular biology, department of microbiology, faculty of medicine, universitas indonesia's collection and propagated in vero cells on t75 flask based on previous study (tresnaningtyas et al. 2018). for denv-2 ngc propagation, mem 2% fbs medium was used, and incubation was carried out at 37 ºc with 5% co cells observed every day to see 2. the cytopathic effects (cpe). denv-2 was harvested on 5-7th days of incubation or after cpe were seen. denv-2 ngc titers were measured by focus assay method, then stored at -80 ºc until used for experiment. isolation of pbmcs. pbmcs was obtained from the blood of healthy control who have been vaccinated with the japanese encephalitis virus (jev) vaccine (dewi et al. 2008). ten ml of healthy control's blood was tested with dengue ag+ab duo (sd biosensor) rapid test to confirm that the subjects were negative anti-denv igg, anti-denv igm, and ns1 denv antigen. pbmcs isolation process was carried out by the ficoll gradient centrifugation method according to previous study (tresnaningtyas et al. 2018, dewi et al. 2008). the number of adherent pbmc cells was counted by haemocytometer, then resuspended in rpmi 1640 (sigma) with 10% fbs and 1% penstrep (gibco) to prepare the cells with a concentration of 1 x 6 -1 10 cells ml . infection of denv-2 ngc to huh 7it-1 cells. huh 5 -1 7it-1 cells with a concentration of 5 x 10 cells ml were grown on 96 microwell plates by incubating at 37 °c with 5% co for 24 hours until the monolayer cells 2 formed. the medium in 96 microwell plates was removed when the cells are monolayer and the cells were infected with 50 μl denv-2 ngc at various titers according to the treatment. infected cells were incubated at 37 ºc and 5% co for 2 hours with agitation every 30 2 minutes. in this study, we used culture cell medium as negative controls. co-cultured pbmcs on huh 7it-1 cells infected with denv-2. fifty μl of adherent pbmcs with a 6 -1 concentration of 1 x 10 cells ml and 50 μl rpmi 1640 medium was added to each well according to the treatment group. plates were then incubated for 48 h at 37 ºc with 5% co after incubation, the supernatant in 2. each well was collected and stored at -80 ºc until used for the measurement of chemokine. measurement of cxcl10 chemokine levels. cxcl10 chemokine levels in supernatan denv infected huh 7it-1 co-cultured with pbmcs were determined using human cxcl10 quantikine elisa kit (r&d system, united states). cxcl10 measurement were carried out according to the assay procedure from human cxcl10 quantikine elisa kit (r&d system, united states). absorbance values of each well were analyzed by elisa reader with a wavelength of 450 nm (elisa 2018). statistical analysis. data were analyzed using the statistical product and service solutions (spss) 23. normality of the chemokine levels data were analyzed by shapiro-wilk normality analysis and for the significance were analyzed by anova. results levels of cxcl10 chemokine. determination of cxcl10 chemokine levels were measured by the cxcl10 elisa kit (r&d system, united states). the absorbance results of each treatment were then converted into chemokine levels by converting od values into standard curves that have been made previously. denv-2 infected huh 7it-1 cells without pbmcs secreted cxcl10 in lowest levels and known to have no significant differences according to statistical analysis (p>0.05). these results are shown in the blue graph in figure 1. on the contrary, pbmcs cocultured with denv-2 ngc infected huh 7it-1 cells was secreted cxcl10 chemokine in higher levels. the cxcl10 levels produced by huh 7it-1 cells infected by denv-2 ngc moi 0.5 and 1 ffu/cell were 552,653 ± 22,779 pg/ml and 576,787 ± 16,901 pg/ml respectively. these results indicated that cxcl10 levels produced by huh 7it-1 cells infected by denv-2 moi 0.5 ffu/cell and 1 ffu/cell was higher than negative control (p<0.05). discussion denv is known to infect cells in several important organs such as bone marrow, spleen, lymph nodes, central nervous system and liver (martina et al. 2009). liver is the most common organ involved in severe denv infection. the ability of denv to infect liver cells is due to the attachment of viruses to heparan sulfate receptors (hs) on the cell surface. hs has been 100 dewi et al. microbiol indones known to be a receptor for entry of all denv strains (seneviratne et al. 2006). in addition, there are also other receptors such as grp78, dc-sign, and laminin receptors (pando-robles et al. 2014). the ability of denv to infect liver cells in humans in this study was demonstrated through in vitro experiments using huh 7it-1 cells. these cells are hepatocyte cell lines originating from human liver tumors, which are expected to be able to resemble the real situation. in addition, in this study there were also cell groups that were treated with pbmcs co-culture. pbmcs is a white blood cell consisting of b lymphocyte cells, t lymphocytes, natural killer cells (nk), and monocyte cells. these cells play an important role in humoral and cellular immunity in the human immune system (warnasih et al. 2016). the presence of monocytes and macrophages in denv infection is known to excrete dissolved mediators such as chemokines and cytokines that can influence denv infection towards disease severity (peiris et al. 1981). the existence of pbmcs in this research is expected to be able to provide an overview of the cxcl10 chemokine profile of huh 7it-1 cells which infected with denv and co-cultured with pbmcs. cxcl10, also called interferon γ-induced protein or ip-10, is a 10 kda-sized protein that belongs to the chemokine family. cxcl10 specifically activates cxcr3 receptors which are predominantly expressed by t lymphocytes, b lymphocytes, nk cells, dendritic cells, and activated macrophage cells. cxcl10 is a pleiotropic molecule that has strong biological functions, including supporting the chemotactic activity of cxcr3 cells, inducing apoptosis, regulating cell growth and ploriferation, and angiogenesis in infectious diseases, as well as inflammation and cancer. the strong cxcl10 chemotactic activity of activated lymphocytes modulates innate and adaptive immunity, induces tissue damage, and modulates tumor formation (liu et al. 2011). in this study, denv infection in huh 7it-1 cells cocultured with pbmcs showed an increase in cxcl10 chemokine production compared with control cells after 48 hours of infection. this can be seen in figure 1. which shows that infections of denv-2 moi of 0.5 and moi of 1 ffu/cells have higher cxcl10 levels when compared to control cells that are only treated with huh 7it-1 cell culture medium. this is similar to the results of in vivo studies conducted by masood et al., which showed an increase of cxcl10 levels in denv infected patients when compared to healthy controls (masood et al. 2018). improvement of cxcl10 levels also occurred in studies conducted by sung et al., in infected mice with denv-2 and showed elevated levels of cxcl10 in the serum and liver of mice. in addition, the infection also causes liver damage in mice that is associated with an increase of cxcl10 levels which is able to recruit nk cells into the liver and cause early cell death after denv infection (sung et al. 2012). the increase of cxcl10 levels in this study is thought to occur as an antiviral response to viral fig 1 levels of cxcl10 chemokine.*p value <0.05. volume 13, 2019 microbiol indones 101 infection in target cells, this is because cxcl10 is a proinflammatory chemokine induced by ifn-γ, which is highly specialized in primary cells infected with denv. cxcl10 plays a role in the antiviral response by competing to bind to heparin sulfate which is a receptor for the entry of denv on target cells, to blocking entry and replication (conroy et al. 2015). in addition, cxcl10 also has chemotactic abilities that play a role in innate immune responses, cxcl10 binds to the cxcr3 receptor, which dominantly expressed by t lymphocytes, b lymphocytes, nk cells, dendritic cells, and activated macrophage cells. cxcl10 is known to recruit t cells and nk cells that are activated to the location of denv infection to carry out the process of virus elimination (masood et al. 2018). in chen et al. study it is known that an increase of cxcl10 levels in denv infection is followed by the expression of other molecules such as perforin, granzim a and b and ifn, which is known to be produced by nk cells and causes apoptosis and target cell destruction to prevent the spread of the denv virus in infected cells. on the other hand, high cxcl10 levels have been found in the serum of patients with chronic inflammatory conditions (chen et al. 2006). thus, cxcl10 is known to have a protective role against denv, but could also be associated with a potentially destructive inflammatory response if it is produced in high quantities and uncontrolled (becerra et al. 2009). whitout pbmcs, denv infected huh 7it-1 did not increase the cxcl10 level. huh7it-1 cells are known to only produce certain cytokines and chemokines based on in vitro studies conducted by rowell et al. (1997). based on these studies it is not known whether cxcl10 chemokines can be secreted by hepatocyte cells or not, but in this study it was seen that cxcl10 levels did not increase significantly in huh 7 cells infected with denv-2 without pbmc when compared to control cells without pbmc (rowell et al. 1997). in conclusion, denv-2 infection in huh 7it-1 cells co-cultured with pbmcs is able to induce cxcl10 secretion, which may be induced as an antiviral response to denv infection. acknowledgment this research was funded by the indonesian science fund (dipi). authors thank to y. shimidzu, kobe university, graduate school of medicine for permission in using huh 7it-1 cells in this study. references angelina m, hanafi m, suyatna fd, mirawati st, ratnasari s, dewi be. 2017. antiviral effect of sub fraction cassia alata leaves extract to dengue virus serotype-2 strain new guinea c in human cell line huh-7 it-1. iop conf ser: earth environ sci. 101(1). doi: 10.1088/17551315/101/1/012004. 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(016. isolasi peripheral blood mononuclear cells (pbmcs) dari darah manusia sehat dengan metode sentrifugasi gradien ficoll. ekologia. 16(1):19–23. doi: 10.33751/ekol.v16i1.979. whitehorn j. 2012. dengue fever viruses. in: els john wiley & sons, ltd., chichester, united kingdom. doi: 10.1002/9780470015902.a0000412.pub2. page 1 page 2 page 3 page 4 page 5 page 6 guide for author.cdr guide for authors associated with them or their laboratory (ies); please provide their contact information where indicated on the submission form. format general. all parts of the papers, including abstract, titles of the tables and figures, table's footnotes, figure legends, and references should be double-spaced on quarto-size (letter) paper with 2 cm margin, using times new roman font with 12 font size. figures and tables must be placed at the end of the manuscript, each of them on separate sheets. figures and papers from previous publications can be used as long as there is consent from its authors. all 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list of title wordabbreviations and refer to : [cse] council of science editors. 2006. scientific style and format: the cse manual for authors, editors, and publishers. 7th ed. reston (va): the council. journal suwanto a, kaplan s. 1989. physical and genetic mapping of the 2.4.1 genome: presence of rhodobacter sphaeroides tw o unique ci rcular chromosomes. j b act er iol. 171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for 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microbiology.indonesia@gmail.com page 1 page 2 blanko pendaftaran anggota.pdf page 1 form berlangganan 2019.pdf page 1 5 iwan.pmd biosynthesis of polyamide 4, a biobased and biodegradable polymer iwan saskiawan research center for biology, lembaga ilmu pengetahuan indonesia, jalan raya bogor-jakarta km. 46, cibinong 16911, indonesia phone: +62-21-8765066, fax: +62-21-8765062, e-mail: iwansaskiawan@hotmail.com polyamide 4, which is composed of repeating unit of γ-aminobutyric acid (gaba), is a biobased and biodegradable polymer since it can be synthesized from renewable material instead of fossil-based material. gaba is produced by decarboxylation of glutamate (glu) using glutamate decarboxylase (gad: ec 4.1.1.15), which is produced by some microorganisms. in this study, enzymatic conversion of gaba from glutamate by lactococcus lactis and escherichia coli cell and chemical polymerization of gaba to polyamide 4 were revealed. the results show that gad activity of e. coli was higher than that of l. lactis. the treatment of e. coli cell by heating and sonication increased the gad activity and conversion rate of glutamate to gaba was up to 70.5%. the optimum temperature for this conversion is 37ºc. on the other hand, chemical synthesis of polyamide 4 was catalyzed by heating gaba at 215ºc for 2 minutes. key words: polyamide 4, biodegradable polymer, γ-aminobutyric acid, glutamate decarboxylase _____________________________________________ volume 2, number 3, december 2008 issn 1978-3477 p 119-123 much fossil fuel and its related resources have been and are being consumed to produce many kinds of products for human life. however, future reliance on fossil fuels has been questioned due to emerging concerns about greenhouse gas (ghg) emissions, particularly carbon dioxide (co 2 ) and its potential contribution to global climate change (gcc) (hansen et al. 2000; woodruff et al. 2006). recent study predicted that by 2100 the global average surface air temperature will be 1.4-5.8°c higher than in 1961-1990. for that reason, it is important to take into account full implementation of plans to reduce fossil fuel use (hebert 2005). consequently, there is a need to develop a bio-based product by using renewable materials as an alternative material to substitute fossil-based products. the one of biobased products is biopolymers. utilization of biopolymers not only reduces the consumption of fossil-based material but also solves the disposal problem due to non-degradable petroleum based polymer as means of reducing the environment impact. furthermore, the exploitation of abundant biomass resources to develop biopolymer is one of the solutions of solid waste accumulation problem. representatives of biobased-biodegradable polymers are poly d-β-hydroxybutylate (phb), poly l-lactide (pla), well known as biodegradable plastic (tokiwa and calabia 2006) and poly amino acid (obst and steinbuchel 2004). the other biobased-biodegradable polymer which has different structure is polyamide 4 also well known as nylon 4. polyamide 4 is composed of repeating unit of γ-aminobutyric acid (gaba). kawasaki et al. 2005 reported that polyamide 4 can be biodegraded in an activated sludge. on the other hand, polyamide 4 has good prospect as a novel bio-based polymer because it is synthesized from 2-pyrrolidone, a lactam of gaba, which can be made by means of decarboxylation of glutamate. this synthesis was catalyzed by glutamate decarboxylase (gad). the synthetic route for biodegradable polyamide 4 was shown in fig 1. at first, biomass is saccharified to produce glucose. then it was fermented by coryneform bacteria (eg: corynebacterium glutamicum) to produce glutamate. using microbial gad, the glutamate was decarboxylated to become gaba. finaly, the monomer unit of gaba was heated to produce 2-pyrrolidone and chemically polymerized to produce polyamide 4. this paper described the study on biosynthesis of polyamide 4. it covers the synthesis of gaba using microbial gad of lactococcus lactis and escherichia coli under different conditions and the chemical synthesis of polyamide 4 from gaba. materials and methods bacterial strain. a wild type l. lactis nbrc 100933 and e. coli nbrc 3806 were obtained from national institute of technology and evaluation-biological resource center (nbrc), japan. l. lactis nbrc 100933 was grown in medium no. 310 containing 10 g l-1 peptone, 10 g l-1meat extract, 5 g l-1 yeast extract, 20 g l-1 glucose, 1 g l-1 tween 80, 2 g l-1 k 2 hpo 4 , 5 g l-1 ch 3 coona, 2 g l-1 c 6 h 14 n 2 o 7 , 0.2 g l-1 mgso 4 ·7h 2 o, 0.05 g l-1 mnso 4 ·nh 2 o, ph 6.0-6.5. e. coli nbrc 3806 was grown in medium no. 802, which contains 10 g l-1 polypepton, 2 g l-1 yeast extract, 1 g l-1 mgso 4 ·7h 2 o, diluted in distilled water. rapid glutamate decarboxylase (gad) assay. one ml of seed culture of l. lactis nbrc 100933 growing in medium no. 310 was transferred to 50 ml of tygg medium (5 g l-1 tryptone, 5 g l-1 yeast extract, 10 g l-1 glucose and 10 mm glutamic acid) in 300 ml flask and incubated for 24 h at 30°c in a shaker on 180 rpm. the cells were harvested by centrifugation at 1 800 xg for 20 min at 4°c and washed once with pbs (0.15 m nacl, 10 mm sodium phosphate, ph 7.3). gad activity assay was carried out using gad reagent (rice et al.1993). the gad reagent consisted of 1 g l-1 l-glutamic acid, 0.05 g l-1 bromocresol green (colorimetric indicator), 90 g l-1 of nacl and 3 ml of tritonx-100 per liter. after the wash step, the cells were transferred to a test tube, 1 ml of gad reagent was added, mixed immediately, and vortexed vigorously for 30s. the tube was then incubated in a water bath at 35°c and observed hourly for 4 h. a distinct change from yellow to blue was considered as a positive response of gad activity. the similar method for gad activity assay was also applied for of e. coli nbrc 3806. gad activity assay. the determination of gad activity assay of l. lactis nbrc 100933 was referred to the method of nomura et al. (1999) with minor modification. the cell suspension of l. lactis nbrc 100933 was frozen at -50°c and lyophilized. for the enzyme assay, the cell powder was suspended in 1 ml of mm sodium acetate buffer (ph 4.7). the reaction mixture for gad activity assay consisted of 50 µl of 4 mm glutamate in 0.1 m sodium acetate buffer (ph 4.7), 25 µl of 0.4 mm pyridoxal phosphate (plp), 10 µl cell powder suspension and 15 µl of distillated water. the reaction was carried out for 8 h at 30°c. one katal of gad was defined as the amount of enzyme required to produce 1 mol gaba s-1 at 30°c and ph 4.7. the cell preparation of e. coli nbrc 3806 and measurement of its gad activity were carried as refered by plokhov et al. (2000). e. coli nbrc 3806 was grown in a medium containing 10.0 g l -1 glucose, 0.2 g l-1 kh 2 po 4 , 0.5 g l-1 mgso 4 , 1.0 g l-1 (nh 4 ) cl and 20 g l-1 peptone in distillated water (ph 7). after incubation for 48 h, the cell was harvested by centrifugation at 40 000 xg for 20 min at 4°c. the obtained wet cell biomass was frozen at -20°c. five milligrams of wet cells were introduced to 12.5 ml 0.9% nacl and stirred with a magnetic stirrer for 15 min. this suspension was then divided into 2 equal portions. the first portion was incubated for at 53°c for 45 min and sonication at 1 min, while the second portion was not treated by heating and sonicated. furthermore, both portions were centrifuged at 5 000 xg for 10 min. the pellets finally were diluted in 2.5 ml 50 mm sodium acetate buffer (ph 4.7). these solutions were used for gad activity assay. the reaction mixture for gad activity assay of e. coli nbrc 3806 consisted of 50 µl 4 mm glutamate in 0.1 m sodium acetate buffer (ph 4.7), 25 µl 0.4 mm pyridoxal phosphate (plp), 10 µl enzyme solution as prepared above and 15 ml of distilled water. the reaction was carried out at various incubation times at 30°c and terminated by the addition of 30µl 30% naoh solution. the enzyme activity was expressed in units. one unit was defined as the amount of enzyme required to produce 1 µmole gaba in 1 min at 37°c. gad specific activity was expressed in u mg-1 cell (wet weight). gaba analysis by high performance liquid chromatography (hplc). standard sample contained 2.5 µm and 5 µm of glu-gaba. a 20 µl of the sample was dried using vacuum system. then, it was added with 20 µl tea solution (ethanol:h 2 o:triethylamine=2:2:1) and evaporated by vacuum system for 10 min. the dried s a m p l e then was added by 20 µl of pitc solution (ethanol:h 2 o:tea:pitc = 7:1:1:1) and vortexed for 20 min at 25°c. furthermore, the sample was dried using vacuum system and dissolved in 200 µl phase a solution (6% acetronitrile in 60 mm sodium acetate buffer ph 6.0). the solution was then filtered using 0.45 µm filter. hplc was performed using reverse-phase column wakosil ptc (200 × 4.0 mm) at 1 ml min-1 flow rate. two mobile phase were employed, 6% acetronitrile in 60 mm sodium acetate buffer at ph 6.0 and 60% acetronitrile. detection was conducted at ultra violet spectrum (λ = 254 nm). preparation of pyrrolidone from gaba. 2-pyrrolidone, a monomer of polyamide 4 is a lactam that is produced by the cyclization of gaba. it is synthesized by dehydration of gaba. a sum of 10 mmol gaba were placed in roundbottom flask equipped with magnetic stirrer. the gaba was then heated at high temperature on oil bath under inert atmosphere until gas-bubbling stops. the products were analyzed with the jeol eca-500 nmr spectrometer in d2o solution. fig 1 the biosynthetic route of polyamide 4 synthesis, a biobased and biodegradable polymer. glucose saccharification biomass fermentation enzymatic synthesis chemical synthesis glutamic acid γ-aminobutyric acid (gaba) polyamide 4 hooc h 2 nchch 2 ch 2 cooh h 2 nch 2 ch 2 ch 2 cooh h o [-nch 2 ch 2 ch 2 c-] n pyrrolidone 120 saskiawan microbiol indones polymerization of pyrrolidone. the method of polymerization of pyrrolidone was referred to kawasaki et al. (2005). a mixture of 2-pyrrolidone (17 g, 200 mmol) and sodium (138 mg, 6 mmol) were put in round-bottom flask equipped with magnetic stirrer. the mixture was heated to 50°c using oil bath under reduced pressure. after the sodium was reacted completely with 2-pyrrolidone, sebacoyl chloride (583.5 mg, 1.5 mmol) was added into the flask and was maintained at 50°c under reduced pressure for about 3 h. the polymerization mixture was dissolved in formic acid and precipitated in acetone followed by washing with water and with methanol. nmr spectrum was obtained from chloroform/ formic acid solution (95/9%) (v/v). results production of gaba by microbial gad of lactococcus lactis and escherichia coli. the results of the rapid gad activity assay of l. lactis and e. coli was shown in fig 2. the tube containing e. coli cell in gad reagent show a darker blue color, suggesting that e. coli has higher gad activity than that of l. lactis. gad activity of e. coli nbrc 3806 cells with heating and sonication treatment was 4.15 u mg-1 cell, (wet weight) and the gad activity without treatment was 3.81 u mg-1 cell (wet weight). furthermore, we attempted to synthesize gaba using l. lactis and e.coli on medium tyg. the results showed that after 16 h incubation the production of gaba using e. coli (678.1 mg l-1) was higher than that using l. lactis (178.8 mg l-1) (fig 3). this result seems to be corresponding with the previous result of rapid gad activity assay. however, the addition of 10 mm glutamate on tyg medium had no effect in enhancing gaba production. on the other hand, the rate and level of e. coli grown on tyg medium was also higher than those of l. lactis (fig 4). to optimize the enzymatic production of gaba by gad of e. coli, it is important to reveal the substrate specificity of the enzyme. fig 5 shows the conversion of various glutamate-containing substrates to gaba by e. coli. the result shows that the highest conversion (70.5%) is obtained using l-glutamic acid. the lowest conversion (3.82%) was obtained on l-glutamate hcl substrate. the effect of temperature on the reaction rate to produce gaba was also studied. the result showed that the reaction fig 2 rapid gad assay. a, gad reagent as a control; b, gad reagent with lactococcus lactis cell and c, gad reagent with escherichia coli cell. a b c fig 3 gaba production by lactococcus lactis and escherichia c o l i o n t y g m e d i u m w i t h a d d i t i o n o f 1 0 m m g l u t a m a t e . a, e. coli on tyg medium; b, e. coli on tyg medium with addition of 10mm glutamate; c, l. lactis on tyg medium; d, l. lactis on tyg medium with addition of 10 mm glutamate. g a b a p ro d u c ti o n ( m g l -1 ) fig 4 cell growth of lactococcus lactis and escherichia coli on tyg medium with addition of 10 mm glutamate. n, e. coli on tyg medium; o, e. coli on tyg medium with addition of 10 mm glutamate; l , l. lactis on tyg medium; ¡ , l. lactis on tyg medium with addition of 10 mm glutamate. fig 5 glutamate-containing substrate specificity of gad of escherichia coli. , l-glu; , l-glu hcl; , na l-glu. volume 2, 2008 microbiol indones 121 rate on incubation at 30°c, 37°c and 50°c were similar. on the other hand, the conversion rate of glutamate to gaba was decreased when the reaction was carried out at 70°c (fig 6). chemical synthesis of polyamide 4. the chemical synthesis of polyamide 4 was initiated by preparation of pyrrolidone, a monomer unit of polyamide 4. the gasbubbling in a flask containing gaba stopped when it was heated on 215°c for 2 min. fig 7 shows 1h nmr spectra of the product and gaba. the spectrum displays characteristics signal for the methylene protons (b, c, a) of gaba at chemical shifts of 1.8, 2.2 and 2.9 ppm (fig 7a), and characteristics signal for the methylene protons (b, c, a) of pyrrolidone at chemical shifts of 2.1, 2.3 and 3.3 ppm (fig 7b). furthermore, the crude polyamide 2 was washed with methanol and applied in 1h nmr analysis. the spectra illustrate that washing the crude polyamide 4 with methanol effectively removes the remaining pyrrolidone (fig 8). discussion gaba is an important ubiquitous non-protein amino acid in both prokaryotic and eukaryotic organisms and is produced by gad (ec: 4.1.1.15) from glutamic acid. gaba is representative depressive neurotransmitter in the sympathetic nervous system and has been proved to be effective for lowering the blood pressure of experimental animals and humans. furthermore, gaba-enriched food is also used as dietary supplement and nutraceutical to help treat sleeplessness, depression and autonomic disorders, chronic alcohol-related symptoms, and to stimulate immune cells (zhang et al. 2006). many studies were conducted to produce gaba especially for the nutraceutical purpose (nomura et al. 1998; kono and himeno 2000; aoki et al. 2003; park et al. 2004; park and oh 2006). it is very important to develop the method to produce gaba in massive production. the utilization of microbial gad in transformation of glutamic acid to gaba seems to be promising method in the future. a lactic acid bacterium is well known in producing gad that is used in cheese industry to increase the content of gaba in cheese production. however, in this study, e. coli had higher activity of gad than that of l. lactis when glutamic acid was used as a substrate. gad is an intracellular enzyme and for that reason the increase of the cell wall permeability controls the substrate access to the enzyme. for that reasons, it seems that the heating and sonication treatment affecting in the cell wall permeability is a one method for increasing the gad activity of e. coli as shown in the results of this study. on the other hand, cyclization of gaba to produce pyrrolidone a monomer unit of polyamide 4 and polymerization of pyrrolidone is also play important role in biosynthesis of polyamide 4. in this study, heating of gaba on 215°c for 2 min is appropriate for cyclization of gaba. furthermore, the chemical polymerization of pyrrolidone was successfully achieved using sebacoyl chloride (0.75 mol %) as initiator reaction, followed by washing the crude polyamide 4 with methanol. this study clearly demonstrated that polyamide 4, the biobased and biodegradable polymer can be biochemically synthesized. this material has a good prospect in the future since the development of bio-based product by using renewable materials as an alternative material to substitute fossil-based products is needed. fig 8 1h nmr spectra analysis of pyrrolidone (a), crude polyamide 4 (b) and polyamide 4 after washing with methanol (c). a c b a c b a c b a b c (a) (b) (c) (-n-ch 2 -ch 2 -ch 2 -c-) n p p 5 4 3 2 1 0 oh hn o ch 2 c h 2 h 2 c (a) (b) (c) nh 2 -ch 2 -ch 2 -ch 2 -cooh a b c c b a fig 7 1h nmr spectra analysis of gaba (a) and pyrrolidone (b) as the results of heating of gaba. hn o ch 2 c h 2 h 2 c (a) (b) (c) a b 0 20 40 60 80 0 50 100 150 200 250 incubat ion t ime (min) c o n v e rs io n o f g lu t o g a b a ( % ) fig 6 effect of temperature on the conversion of glutamate to gaba. , 30oc; , 37oc; , 50oc; , 70oc. 122 saskiawan microbiol indones acknowledgement i would like to thank to japan international cooperation agency (jica), national institute of advanced industrial sciences and technology (aist) japan, for giving me a good opportunity to conduct research about biomass technology. i also would to express my sincere gratitude to naoko yamano and atsuyoshi nakayama for their guidance during my work. i also thank to sei-ichi aiba, head of bio-based polymers groups, research institute for innovation in sustainable chemistry aist and all members of his laboratory for their assistance. references aoki h, uda i, tagami k, furuya y, endo y, fujimoto k. 2003. the production of new tempeh-like fermented soybean containing a high level of γ-aminobutyric acid by anaerobic incubation with rhizopus. biosci biotechnol biochem 67:1018-1023. hansen j, sato m, ruedy r, lacis a, oinas v. 2000. global warming in the twenty-first century: an alternative scenario. proc natl acad sci usa 97:9875-9880. hebert p. 2005. the kyoto protocol: in force? (editorial). can med assoc j 172:437. kawasaki n, nakayama a, yamano n, takeda s, kawata y, yamamoto n, aiba s. 2005. synthesis, thermal and mechanical properties and biodegradation of branced polyamide 4. polymer 46:99879993. kono i, himeno k. 2000. changes in γ-aminobutyric acid content during ben-koji making. biosci biotechnol biochem 64:617-619. nomura h, kimoto h, someya y, furukawa s, suzuki i. 1998. production of γ-aminobutyric acid by cheese starters during cheese ripening. j dairy sci 81:1486-1491. nomura n, kimoto h, someya y, suzuki i. 1999. novel characteristic for distinguishing lactococcus lactis subsp. lactis from subsp. cremoris. int j syst bacteriol 49:163-166. obst m, steinbuchel a. 2004. microbial degradation of poly (amino acid)s. biomacromolecules 5:1166-1176. park kb, ji ge, park ms, oh sh. 2004. expression of rice glutamate decarboxylase in bifidobacterium longum enhances γaminobutyric acid production. biotechnol lett 27:1681-1684. park kb, oh sk. 2006. production of yogurt with enhanced levels of γ-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract. biores technol 98:1675-1679. plokhov ay, gusyatiner mm, yampolskaya ta, kaluzhsky ve, sukhareva bs, schulga aa. 2000. preparation of γ-aminobutyric acid using e. coli cells with high activity of glutamate decarboxylase. appl biochem biotech 88:257-265. rice ew, johnson ch, dunnigan me, reasoner dj. 1993. rapid glutamate decarboxylase assay for detection of escherichia coli. appl environ microbiol 59:4347-4349. tokiwa y, calabia bp. 2006. biodegradability and biodegradation of poly (lactide). appl microbiol biotechnol 72:244-251. woodruff re, mcmichael aj, hales s. 2006. action on climate change: no time to delay. global warming is real, so what are we going to do about it, who will do it, and when? med j aust 18:539-540. zhang h, yao hy, chen f. 2006 accumulation of γ-aminobutyric acid in rice germ using protease. biosci biotechnol biochem 70:1160-1165. volume 2, 2008 microbiol indones 123 05. widiastuti.cdr vol.15, no.1, march 2021, p 27-35 doi: 10.5454/mi.15.1.5 exploration of lignocellulolytic microbes in oil palm rhizosphere on peat soils and their respiration activities happy widiastuti , darmono taniwiryono , heru bagus 1 1 siswanto , 1 pulunggono , syaiful anwar , basuki sumawinata , husni mubarok 2 2 2 3 , and supiandi sabiham 2 1 indonesian research institute for biotechnology and bio-industry, and indonesian oil palm society (maksi), bogor, indonesia; 2ipb university, and peatland society of indonesia (hgi), bogor, indonesia; 3 agronomy research, pt astra agro lestari tbk, jakarta, indonesia. microbial respiration in peatlands plays a role in contributing co emissions. studies of microbial exploration 2 and respiration on peat soils in oil palm plantations have not been widely reported. this study was aimed to explore lignocellulolytic microbes found in peat soils (planted with 12-year-old oil palm, and fern vegetation peat), and compared with that found in mineral soils. exploration was done by growing the samples on specific medium for each group of microbial functions. respiration activity of the obtained culture was then analyzed based on the oxidation of peroxidase catalysis using a chromogen substrate (tetramethylbenzidine) and measured using spectrophotometry at a wavelength of 450 nm. the results showed that both in mineral (ms) and peat planted with oil palm (gs) in a depths of 0-20 cm, lignolytic fungi were found with a population of 17 x 10 . 2 similar results were also found in peatland with fern vegetation (gns) but at a depth of 20-40 cm. lignolytic bacteria (related to methylene blue degradation) can be found on peat soils planted with oil palm at a depth of 0-60 cm and the population increases with increasing depth. this lignolytic bacteria was also found on peat soils with fern vegetation and mineral soils planted with oil palm. at a depth of 0-20 cm the population of lignolytic bacteria in non-oil palm peat is highest. cellulolytic bacteria were isolated at a depth of 0-60 cm. cellulolytic bacterial populations were highest in oil palm peat at all depths compared to other samples. respiration analysis of several dominant isolates showed fairly high variation between microbial function groups and within the same function group. the lignolytic microbial group degrading methylene blue showed high respiration activity and varies greatly (0.19-1.85 mer). while the respiration activity of cellulolytic bacteria ranged from 0.45 to 0.62 mer. key words: lignolytic and cellulolytic microbes, microbial extracelluler respiration, peatlands respirasi mikroba di tanah gambut berperan dalam menyumbangkan emisi co . studi tentang eksplorasi dan 2 respirasi mikroba di tanah gambut di perkebunan kelapa sawit belum banyak dilaporkan. penelitian ini bertujuan mengeksplorasi mikroba lignoselulolitik yang terdapat di tanah gambut dan tanah mineral yang ditanami kelapa sawit berumur 12 tahun. eksplorasi dilakukan dengan menumbuhkan contoh pada medium spesifik masing masing kelompok fungsi mikroba. pada tahap selanjutnya dari kultur murni yang diperoleh dilakukan analisis aktivitas respirasi berdasarkan oksidasi katalisis peroksidase menggunakan substrat kromogen (tetramethylbenzidine) dan diukur menggunakan spektrofotometri pada panjang gelombang 450 nm. hasil isolasi menunjukkan bahwa pada gambut sawit dan mineral kedalaman 0-20 cm dijumpai jamur lignolitik (gs) (ms) dengan populasi 17 x 10 . hasil yang sama juga dijumpai pada gambut dengan vegetasi paku-pakuan 2 (gns) namun pada kedalaman 20-40 cm. bakteri lignolitik (pendegradasi methylene blue) dapat dijumpai pada tanah gambut yang ditanami kelapa sawit pada kedalaman 0-60 cm dan populasinya meningkat dengan bertambahnya kedalaman. bakteri ini dijumpai juga pada tanah gambut dengan vegetasi paku-pakuan dan tanah mineral sawit. pada kedalaman cm populasi bakteri lignolitik di tertinggi. bakteri selulolitik 0-20 gambut non sawit adalah berhasil diisolasi pada kedalaman 0-60 cm. populasi bakteri selulolitik adalah tertinggi di gambut sawit pada semua kedalaman dibandingkan dengan sampel lainnya. analisis respirasi beberapa isolat dominan menunjukkan variasi yang cukup tinggi di antara kelompok fungsi mikroba dan di dalam kelompok fungsi yang sama. kelompok mikroba pendegradasi methylene blue menunjukkan aktivitas respirasi yang tinggi dengan lignolitik variasi di antara isolat yang juga tinggi (0,19-1,85 mer). sedangkan aktivitas respirasi bakteri selulolitik berkisar antara 0,45-0,62 mer. kata kunci: gambut, mikroba lignolitik dan selulolitik, respirasi mikroba ekstraseluler microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-; fax: +62; email: happywidiastuti@gmail.com (wahyunto . 2011) and based on 2008 data on peat et al conditions indonesia stores the third largest carbon stock in the world (after canada and russia) of around 54,016 million tons (joosten, 2009). however, if peatlands are cleared/ converted and/ or drained, the indonesia, which represents tropical peatlands, has an area of ​​around 14.9 million hectares of peatlands carbon presents in the peat material and plant biomass will be oxidized to co . on cleared land, for example 2 for oil palm plantations, enhanced emissions of co2 from autotroph (ra-root) and heterotroph (rhmicrobial decomposition activities of organic matter) is suspected. the dynamics of peatland greenhouse gases involve co uptake through photosynthesis and release through 2 autotroph respiration from plants (roots), heterotroph respiration (microbes) and transport of dissolved and difficult-to-decompose organic matter (jauhiainen et al.; et al. moore 2013). heterotrophic respiration is the decomposition of organic matter by microbes that produce co , n o, and ch . in this process the 2 2 4 groundwater depth determines the boundary between aerobic conditions above and anaerobes at the bottom. changes in water availability and water content in the substrate can change the structure of microbial communities that are sensitive to the availability of water and oxygen (jaatinen 2008). organic et al. polymers on the surface of peat decompose aerobically by bacteria and fungi and when the peat becomes saturated with water, a succession occurs so that anaerobic bacteria will decompose to produce ch by 4 methanogenic microbial groups. denitrifying bacteria will produce n o in anaerobic conditions in the 2 presence of nitrates. the depth of the ground water on peat is a major factor used to explain the dynamics of ghg flux in tropical peat in addition to the vegetation above it (ipcc 2014). soil respiration is the main source of atmospheric co and its value is higher than fossil fuels. the rate of 2 soil efflux at the surface of the soil is the sum of heterotroph (microbial decomposition) and autotroph (root) respiration where the root accounts for almost half the co flux (berger 2010). in natural 2 et al. ecosystems, soil organic c consists of various carbon compounds which can be grouped in labile (dissolved organic c) and recalcitrant. therefore differences in the organic soil c fraction will affect heterotrophic respiration due to the ease of decomposition and microbial preference. non-recalcitrant substrates are probably temperature sensitive (fierer 2005; et al. davidson and janssens 2006). wang (2014), based et al. on a meta-analysis, revealed a 21% increase in heterotroph respiration with an increase in temperature of 2 °c. however, a comprehensive analysis of the magnitude of co emissions from total respiration in 2 both autotrophs and heterotrophs in oil palm land on peat soils has not been widely reported. the major microbes in pea t re lated to decomposition are cellulolytic and lignolytic microbes. these microbial groups produce extracellular enzymes that can degrade cellulose or lignin. those enzyme can be cellulase for cellulose, while and laccase, mnperoxidase and fe-peroxidase for lignin. each microbe has the ability to decompose and so do the different respiration activities. lignolytic microbes can be methylene blue degrading microbes caused by similarities or dissimilarities in the analysis of lignin degradation. methylene blue degrading microbes are like (eslami . 2017). pseudomonas auroginosa et al this study was aimed to explore lignocellulolytic microbes found in peat soils (planted with 12-year-old oil palm, and fern vegetation peat), and compared with that found in mineral soils. microbial populations of cellulose and lignin decomposition and the amount of respiration of several dominant lignocellulose isolates will be described. materials and methods research location and method of sampling. soil samples were taken from a 12 year old oil palm plantation in pt ktu (kimia tirta utama), pangkalan pisang, koto gasib, siak regency, riau, in february 2019. the peat maturity is sapric. samples were taken from 3 points for each habitat namely oil palm peat (gs), non oil palm peat (gns), and nearby mineral soil (ms) at 3 depths namely 0-20 cm, 20-40 cm, and 40-60 cm with 3 replications so that the total sample were 27. the samples were taken non-sterile using a drill in the area of ​​an oil palm which was approximately 1.5 m from the oil palm tree. samples from non-palm peat (gns) were samples taken on peat soils with fern vegetation. soil samples after being taken were put in plastic bags after being labeled and stored in ice boxes to be brought to the laboratory. isolation and characterization of lignolytic and cellulolytic microbes. microbes were isolated from soil samples by serial dilution, which were then grown in a selective medium based on microbial function groups. for bacteria, the sample suspension planted in the medium was 10 to 10 dilutions, while -7 -9 for fungi the dilution was 10 to 10 . for the isolation -4 -6 of lignolytic fungi and lignolytic bacteria, guaiacol (0,4ml/l) and methylene blue (25mg/l) medium were used respectively, while for the isolation of cellulolytic fungi and bacteria, cmc (2g/l) was added in the media. analysis of lignolytic and cellulolytic microbial respiration. respiration analysis was 28 widiastuti et al . microbiol indones volume 15, 2021 microbiol indones 29 carried out on selected dominant isolates following the method proposed by zhou . (2010). et al this method utilizes the peroxidase activity of the key components (multi-heme c-type cytochromes) of the extracellular electron transfer network. the bacterial intrinsic peroxidase-catalyzed oxidation of chromogen (e.g., tetramethylbenzidine) resulted in a measurable color change correlated with the mer (microbial extracellular respiration) ability of the tested microorganisms. results isolation and characterization of lignolytic and cellulolytic microbes. the microbes that have been isolated from three habitats are cellulolytic and lignolytic bacteria and lignolytic fungi as presented in figure 1, while populations of each of those microbial functions group are presented in table 1. cellulolytic bacteria form a clear zone around the colony, while lignolytic bacteria change the color of the medium to yellow and other isolates show a bluer colony color which may indicate absorption of methylene blue inside the cell. the table shows that in gs (peat-oil palm) at a depth of 0-20 cm cellulolytic bacteria, lignolytic bacteria, and lignolytic fungi were found, while at a depth of 20-40 cm only cellulolytic and lignolytic bacteria were found. the population of lignolytic bacteria was much higher compared to cellulolytic bacteria and its population actually increased with increasing depth, although lignin content of each depth was relatively similar. in gns soil (peat-non oil palm), lignolytic fungi were not found at a depth of 0-20 cm, but found at a depth of 20-40 cm. the population of lignolytic bacteria was very high at a depth of 0-20 cm, but decreased with increasing depth and not found at 40-60 cm depth. whereas the population of cellulolytic bacteria was almost the same at all depths although it was much lower compared to lignolytic bacteria. the content of celullase was higher with the increasing of depth. lignolytic fungi were found only at a depth of 20-40 cm. in the mineral soil of oil palm at all depths, cellulolytic and lignolytic bacteria were found. however, lignolytic fungi were only found at a depth of 0-20 cm. the population of lignolytic fungi was much lower than that of cellulolytic bacteria and lignolytic bacteria. the rasio of total fungal and bacteria of gs (peta-oil palm), gns (peat-non oil palm), and ms (mineral-oil palm) was 15 10 , 24 10 , and 38 10 . in -6 -7 -6 this research the ratio is less than 1 and the bacteria is dominant compared to fungi. comparison between populations of lignolytic bacteria (degrading of methylene blue) in 3 habitats shows that the population in gns (peat-non oil palm) is highest at 0-20 cm and decreases at subsequent depths. however, the population of these bacteria in oil palm mineral soils (ms) is higher compared to gs (peat-oil palm) especially at depths of 40-60 cm. the content of lignin was higher in gs (peat-oil palm) compared to those in ms (mineral-oil palm). cellulolytic bacterial population in gs (peat-oil palm) is slightly higher than ms (mineral-oil palm) and cellulolytic bacterial population is almost the same at a depth of 20-60 cm in all three habitats. the population of cellulolytic bacteria when compared to lignolytic bacteria is much lower at almost all depths in both gs (peat-oil palm), gns (peat-non oil palm) and ms (mineral-oil palm). analysis of lignolytic and cellulolytic microbial respiration. respiration analysis of table 1 microbe p tion of each functional group in each habitatopula sample code depth (cm) cellulolytic bacteria (cmc) lignolytic bacteria (met blue) lignolytic fungi (guaiacol) gs 0-20 2.2 106 1.3 107 17 102 20-40 65 104 33 108 0 40-60 82 104 4.9 109 0 gns 0-20 67 10 4 3.7 1012 0 20-40 53 104 9.3 109 17 102 40-60 65 104 0 0 ms 0-20 1.7 106 6 108 17 102 20-40 55 104 3.4 109 0 40-60 64 104 5.5 1010 0 notes: peat-oil palm) peat-non oil palm) mineral-oil palm)gs ( , gns ( , ms ( 30 widiastuti et al . microbiol indones several lignolytic and cellulolytic isolates is presented in figure 2. the population of each isolate analyzed is presented in table 2. the analysis was carried out on 3 functional groups microorganism namely cellulolytic bacteria, lignolytic bacteria, and lignolytic fungi. respiration ability of each isolate varies both in the same functional group and between functional groups. in general, lignolytic fungi have the lowest respiration ability and are followed by cellulolytic bacteria. among the cellulolytic bacterial groups analyzed, their respiration ability showed quite wide variations. the respiration range for cellulolytic bacteria is between 0.222 microbial extracellular respiration (mer) (peat-oil palm 40-60 cm) to 0.655 mer (mineral-oil palm 0-20 cm). similar results were also found in lignolytic bacteria. lignolytic bacterial respiration has very wide variations ranging from 0.172 to 1.831 mer. isolates derived from gns (peatnon oil palm) at a depth of 40-60 cm have the highest respiration while the lowest respiration ability is isolates from ms (mineral-oil palm) isolated from a depth of 20-40cm. the existence of lignolytic fungi of the three types of samples were very rare and found in only low population. though from the isolates obtained, the ability of those two lignolytic fungi isolates had almost the same respiration activity ranging from 0.184 to 0.213 mer. this value when compared to those other isolates shows a fairly low respiration value. discussion the table 1 shows that in gs (peat-oil palm) at a depth of 0-20 cm cellulolytic bacteria, lignolytic bacteria, and lignolytic fungi were found, while at a depth of 20-40 cm only cellulolytic and lignolytic bacteria were found. differences in oxygen content in the soil are likely to cause this difference. the oxygen content was high in the surface and lower in the deeper layers. the population of lignolytic bacteria is much higher compared to cellulolytic bacteria and its population actually increases with increasing depth, although lignin content of each depth was relatively similar. depth also strongly influenced microbial diversity and composition, while both depth and vegetation (oil palm and fern) exhibited significant impact on the archaeal communities. microbial diversity was highest at the surface, where fresh leaf litter accumulates, and so higher nutrient supply is available. the population of lignolytic bacteria is very high, but decreases with increasing depth and not found at the depths of 40-60 cm. it might be that there is no correlation with the content of lignin since the lignin content was relatively similar in each depth. meanwhile, the population of cellulolytic bacteria is almost the same at all depths although it is much lower compared to lignolytic bacteria. the content of celullase was higher with the increasing of depth. lignolytic fungi are only found at a depth of 20-40 cm. there was correlation between root weight and the oxygen concentration (boggie 1977). it might be there are dynamic of water content that affect the oxygen content since the reduction of water content increases the oxygen content (estop-aragones 2012). et al. plant roots affect the oxygen content since the root exclude the oxygen, in turn affect the microbial composition. table 2 soil chemical characteristics used in this research sample code depth (cm) ph org c (%) n total (%) c/n ratio p total (%) cellulose (%) lignin (%) total cellulose -lignin fiber content gs gns ms 0-20 20-40 40-60 0-20 20-40 40-60 0-20 20-40 40-60 3.65 3.23 3.71 3.19 3.16 3.27 4.22 3.92 3.75 52.8 55.6 52.7 54.7 56.9 55.2 4.7 2.0 0.9 0.64 0.58 0.54 0.95 0.94 0.76 0.16 0.07 0.05 82.5 146.3 97.6 57.6 60.5 72.6 29.4 28.6 17.6 0.12 0.05 0.17 0.05 0.03 0.04 0.02 0.01 0.0 36.78 54,23 58.25 38.25 64.72 53.65 16.59 16.75 16.30 21.22 20.82 19.96 21.67 23.21 21.57 10.82 15.91 15.18 58.0 75.05 78.21 59.92 87.93 75.22 27.41 32.66 31.48 33.3 46.6 65.0 33.3 53.3 66.7 notes: peat-oil palm) peat-non oil palm) mineral-oil palm)gs ( , gns ( , ms ( volume 15, 2021 microbiol indones 31 fig 1 pure culture of isolates used in respiration analyses. a) pure culture of cellulolytic bacteria (1-2) and methylene blue degrading bacteria (3-4), b) lignolytic bacteria ability in degrading methylene blue (blue medium turn to yellow) and c) cellulolytic bacteria ability in degrading cellulose (clear zones formation). fig 2 the respiration activities selected microorganism isolated from gs (peat-oil palm), gns (peat-non oil palm, fern) and ms (mineral-oil palm). fig 3 population of each bacteria used in respiration analyses. 32 widiastuti et al . microbiol indones the population of lignolytic fungi is much lower than that of cellulolytic bacteria and lignolytic bacteria. it showed that lygnolytic fungi need oxygen for their enzyme activity (eischlerova 2012). though the et al. content of lignin was higher with the depth but the oxygen content was seem likely more affect the activity of lignolityc microbe. in addition microbial population on peat is also influenced by peat origin and soil reaction. tveit . (2012) suggested that bacteria are et al dominant in bog and are slightly found in fen which has acid reactions and in peat derived from sphagnum which has neutral reactions. population ratio of fungi to bacteria is ranging from 0.31 to 0.68 while in peat originating from coniferous forests the ratio is above 1 which indicates that fungi dominate. the ratio of total fungal to bacteria of gs (peat-oil palm), gns (peatnon oil palm), and ms (mineral-oil palm) was 15 10 , -6 24 10 , and 38 10 , respectively. it means that the ratio -7 -6 is less than 1 and the bacteria are dominant than fungi. lignolytic bacteria (methylene blue degradation bacteria) population in gns is highest at 0-20 cm and decreases at subsequent depths. this study illustrated significant role of depth in microbial community structure, and thus we recommend the inclusion of this factor in future microbial ecological research in peatlands ecosystem. however, the population of these bacteria in oil palm mineral soils (ms) is higher compared to oil palm peat (gs) especially at depths of 40-60 cm. the content of lignin was higher in gs compared to those in ms. it might be there is no correlation between the population of lignolytic bacteria and the content of the lignin since there is variation of lignin chemical structure or might be dissolved organic carbon (doc) is more important than lignin content. degree of decomposition and ph were found to be the major driving factors for doc release (schalm and zeitz 2015). the results of the study of eslami (2011) et al. showed that there are two mechanism i.e . biodegradation and biosorption of methylene blue. in this study, the two mechanisms appear to occur simultaneously. at first the lignolytic isolate formed a yellow color which might indicate the degradation of methylene blue and then the colony changed to a bluer color which was thought to be the absorption of methylene blue. habibi and mehrabi (2017) also reported the degradation of methylene blue from ralstonia eutropha. its ability is greatly influenced by the presence of sugar, types of compounds n, ph and cucl . the stages in activity in 2 bacillus thuringiensis methylene blue degradation are (1) biosorption of methylene blue in bt 016 biomass through electrostatic with chelating activity, (2) methylene blue subsequently degraded through enzyme activity along with metabolic processes. this is due to the presence of lip and hrperoxidase enzymes. lip enzyme is stable at 60 c.0 respiration ability of each isolate varies both in the same functional group and between functional groups. in general, lignolytic fungi have the lowest respiration ability and are followed by cellulolytic bacteria. the respiration ability of lignolytic bacteria is strongly influenced by the origin of isolates and it seems that isolates originating from peat with non-oil palm vegetation have higher respiration ability compared to those from peat or mineral soils planted with oil palm. isolates derived from non-oil palm peat at a depth of 40-60 cm have the highest respiration while the lowest respiration ability is isolates from mineral soil isolated from a depth of 20-40 cm. soil respiration varies greatly and is influenced by availability of substrate, organic matter, temperature, humidity, availability of oxygen and nutrients, and soil characteristics (truu . 2009; bobuľská . 2015; et al et al yiqi and zhou 2010). while giorgio and gasol (2010) suggested that microbial activity is influenced by ph, temperature, rate of availability of organic compounds, their characteristics, and nutrient availability. nevertheless, this respiration analysis also showed potential isolates. an interesting result in this study was that the highest respiration ability of lignolytic bacteria was shown by isolates isolated from peat soil that were not planted with oil palm but growing ferns. these results probably indicate that oil palm cultivation does not support growth and respiratory activity especially by lignolytic bacteria. meanwhile, respiration of lignolytic bacteria isolated from mineral soils with oil palm vegetation was generally lower, except for lignolytic bacteria isolated from a depth of 20-40 cm. respiration ability of this isolates is almost equivalent to isolates isolated from peat soil with non-oil palm vegetation. the results in this study indicate that respiration is not affected by the origin of the isolate depth. the ability of bacteria originating from the surface layer, in this analysis is sometimes lower than that originating from deeper layers of soil. it supposed that the conditions when the bacteria lived more influential compared to the potential analyzed in this study. results reported by naconieczna and sypniewski (2014) and liu (2016) showed that et al. respiration activity in the surface layer was generally higher than in deeper layers because of the more available fresh materials (keller and takagi 2013; fauzi 2016). in deeper soil layers, lignin content et al. is likely to be higher than at the surface. lignin is a material that is quite resistant to decomposition. the analyses of lignin and cellulose in those three samples showed in table 1. the influence of roots can also affect the activity of respiration. the presence of roots results in higher microbial activity (peterson 2003). in non-oil palm peat with fern vegetation, the lignin might has not been decomposed compared to oil palm which has deeper roots. girkin (2020)et al. reported that roots exclude oxygen that affect the decomposition activities. lignin is a material that is quite resistant to decomposition. in addition, samples in this study were taken in the oil palm area where there are very many active root hairs that exudate amino acids and other organic compounds that are very important for microbial activity in general. also this area is an area where fertilization is a routine, so that it contains higher nutrients compared to peat with fern vegetation. in addition, samples from oil palm trees are more shaded so that they might be have lower temperatures compared to fodder vegetation that may affect in lignin decomposition. nitrogen affects the decomposition of recalcitrant compounds. the addition of n will reduce its decomposition (brouns et al. 2016). the effect of other nutrients in fertilizers such as p, k, ca and mg, may also affect decomposition and respiration activities. in this research it showed that the content of n of mineral soil was lowest compared to the other two peat soils. though the respiration activity of lignolytic bacteria isolated from gns (peat-non oil palm) was higher compared to that isolated from ms (mineral-oil palm). nitrogen was the core parameter correlating to microbial communities, but the interactive effects from various environmental variables displayed significant correlation to relative abundance of major microbial groups (too . 2018). et al quality and composition of organic matter, rather than tree species, demonstrated direct impact on the bacterial diversity (millard and singh 2010). root inputs of oxygen varied significantly between species which, combined with different litter chemistry, exerts a key limitation on rates of decomposition. differences between species are significant. root oxygen mitigates methane fluxes in tropical peatlands (girkin . et al 2020). lignolytic fungi population is very low and only found at certain depths. actually this fungus has a higher ability to decompose lignin compared to bacteria, but bacteria have a diversity of lignolytic substrate that can be decomposed compared to fungi. compared to other microorganism, it showed that there is only fungi that have extracellular lignin degrading enzymes such as laccase, li-peroxidase and feperoxidase. the activity of these lignolytic enzymes will change lignin into an available form (swift . et al 1979). lignolytic bacteria have a higher population than fungi and this is the same in all three habitats. more specifically, the population of lignolytic bacteria is much higher compared to cellulolytic bacteria. lignolytic activity will help cellulolytic microbes in their activities due to the presence of lignin on the surface of the tissue (pauly and keegstra 2008). the results of research conducted by winsborough and basiliko (2010) show that bacteria are more active than fungi in many types of peat. bacteria and fungi in their metabolism are chemoautotrophic. heterotrophic microbes get carbon and energy together with the degradation of organic compounds present in the soil, including plant residues and dead microorganisms. microbes in their activity also release extracellular enzymes into the environment that initiate the degradation of organic matter, which can be large and insoluble compounds to pass through the microbial membrane. this enzyme converts macromolecules into soluble forms that can be absorbed and metabolized by microbes (bobuľská . 2015).et al bacteria and fungi are involved in the degradation of organic material in both aerobic and anaerobic conditions but fungi are more efficient than bacteria in the degradation of recalcitrant materials such as lignin because fungi produce extracellular enzymes that are more diverse than bacteria (tveit . 2012). in et al mineral soils the population of lignolytic bacteria is more in deeper layers, possibly due to the presence of higher or accumulated lignin. lignolytic bacteria population, dominant both on the surface and to a depth of 60 cm. microbial composition of peat-oil palm, peat-non oil palm, and mineral-oil palm soils varies. the population of lignolytic fungi was rarely in contrast to lignolytic bacteria, while cellulolytic bacteria was found in all depth of each habitat. respiration activities affected by functional and the origin of the isolate. cellulolytic bacteria have low respiration activities compared to selected isolates of lignolytic bacteria. the lignolytic bacteria isolated from peat-non oil palm have the highest respiration activities. volume 15, 2021 microbiol indones 33 34 widiastuti et al . microbiol indones acknowledgements this study was part of a research funded by the indonesian palm oil plantation fund management agency, the ministry of finance of the republic of indonesia with contract no prj-68/dpks/2018. references adji, f f, hamada,u. darang, s.h. limin and r. hatano. 2014. effect of plant-mediated oxygen supply and drainage on greenhouse gas emission from a tropical peatland in central kalimantan, indonesia. soil scie plant nutr. 60(2), 216-230. berger, t. w., e. inselsbacher, and s. zechmeisterboltenstern 2010, carbon dioxide emissions of soils under pure and mixed stands of beech and spruce, affected by decomposing foliage litter mixtures, soil biol. biochem., 42(6), 986–997. boggie, r. 1977. water-table depth and oxygen content of deep peat in relation to root growth of pinus contorta. plant and soil volume 48, pages447–454. bobuľská l, 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wang j and xu s 2014 soil respiration under climate warming: differential response of heterotrophic and autotrophic respiration 20 glob. change biol. 3229–37. winsborough c, and n. basiliko. 2010. fungal and bacterial activity in northern petaland. geomicrobiology j. 27 (4), yiqi l dan x zhou 2010 soil respiration and the environment elsevier biodegradation of methylene blue from aqueous solution by bacteria isolated from contaminated soil . j adv. evironm health res. 5, 10-15. zhou, j. wen, .j. c. and q. lu. 2015. rapid measurement of microbial extracellular respiration ability using a high-throughput colorimetric assay . environmental sci and tech letters. volume 15, 2021 microbiol indones 35 5 agus rohyadi_336 (22-26) growth responses of external hyphae of arbuscular mycorrhizal fungi to acidic soil conditions and their effects on cowpea growth agus rohyadi faculty of agriculture, universitas mataram, jalan majapahit 62, mataram 83125, indonesia phone: +62-370-621435, fax: +62-370-640189, e-mail: arohyadi01@telkom.net the effectiveness of arbuscular mycorrhizal (am) fungi has often been attributed to growth of their external hyphae, whilst the hyphae themselves may be subjected to the effects of severe soil conditions. the growth of external hyphae of gigaspora margarita and glomus etunicatum and their functions in cowpea growth have been studied at low soil ph using a pot system making is possible for the hyphae to grow separately from their host’s roots. pots had two compartments, one for roots (rc) and one for hyphae (hc). the rc was a cylindrical bag made of 30 μm nylon mesh that retains the roots but allows the hyphae to pass through, placed centrally and surrounded by the hc. initially, the rc was filled with 120 g of a soil/sand mixture (ph 5.3), inoculated with g. margarita, g. etunicatum or free fungal inoculants. a pre-germinated cowpea seed was grown in the compartment for two weeks before the hc was filled with 580 g of the mix in which the ph had been adjusted to 4.6, 4.9 or 5.2. growth of the plants and of the fungal hyphae in the hc was assessed 6 weeks later. the two fungi differed in their responses to soil ph levels in their growth of external hyphae although they colonized plant roots in the same way. at ph 4.6, the hyphae of g. etunicatum grew more weakly than those of g. margarita. increasing the ph enhanced the growth of g. etunicatum’s hyphae but reduced g. margarita’s. in relation to their external hyphal functions, g. margarita was able to improve its shoot dry weight and p uptake of cowpea plants higher than g. etunicatum. these findings highlight the ability of developing an extensive external hyphal network under adverse conditions of excessive h+ ions as an important characteristic for the effectiveness of am fungi in acidic soils. key words: acid soils, arbuscular mycorrhiza, cowpea, external hyphae, gigaspora margarita, glomus etunicatum _____________________________________________ arbuscular mycorrhizal (am) fungi, members of phylum glomeromycota (schüßler et al. 2001), establish mutual symbioses with the majority of terrestrial plant species under a wide range of soil conditions. the role of the fungi as plant growth promoters is widely recognized. they contribute to plant growth by employing their hyphae in soil to enhance the uptake of nutrient elements, especially phosphorus (p) that often increases plant growth. generally, plants in symbiosis grow better than those without it, with the greatest effect on plants growing in marginal soils, particularly those deficient in p, and on plant species lacking inherent morphological and physiological mechanisms for efficient p uptake (manjunath and habte 1991). it is thought that the presence of am can be of great importance to enable plants to withstand severe soil conditions such as those found in acid soils (ph < 5.0), in which toxicity of excessive h+ ions per se and various limiting factors relating to the low ph such as deficiency and/or toxicity of some mineral elements exists (marschner 1991). improved growth of some crop species by am in their soils has been reported recently, such as cowpea, maize, and soybean (yost and fox 1979; rohyadi et al. 1988; nurlaeny et al. 1996; clark and zeto 2000). the beneficial effects of am symbioses in acid soils varies considerably with fungal species and even isolates within a species (clark and zeto 2000; clark 2002), and their preferences to optimum soil ph are considered as the main factor influencing their symbiotic effectiveness (wilson 1988). despite this, species or isolates preferring the same soil ph can also have different effects on their host plants. for example, two am fungi, gigaspora margarita and glomus etunicatum, previously reported to be favored by acidic soil conditions (borie and rubio 1999; clark 2002), showed differential effectiveness in increasing cowpea growth at soil ph 4.7-5.2; with the former being more effective than the latter (rohyadi et al. 2004). this is thought to correspond with the capacity of these fungi to produce an extensive network of external hyphae in the soil, but since there are no measurement on the hyphae reported this hypothesis needs further elucidation. the functions of fungal hyphae growing out from colonized roots in a symbiotic relationship have been well documented. besides serving as the main inoculants for new root colonization, the external hyphae mainly serve as an extension of the root system that enhances plant access to water and nutrients in bulk soils outlying from depleted root zones. despite this importance, only few observations have been carried out on the hyphae present in acidic soils so far due to problems in appropriate methods (van aarle et al. 2002). hence, how they interact with adverse conditions there remains a question. exposure to some acidity-related factors may be detrimental to the hyphae. this paper presents results of an experiment aimed at studying the influence of low soil ph, in terms of excessive h+ ion activities, on the growth of the external hyphae of g. margarita and g. etunicatum and their function in the growth and in p uptake in cowpea plants. materials and methods experimental pot system. in this experiment pots were designed (fig 1) to enable external hyphae of am fungi to grow separately from their host plant roots. each pot had two compartments, one for rc and one for fungal hc. the rc was a cylindrical bag (3 cm diameter, 8 cm high) made up of 30 μm nylon mesh that retains roots but allows hyphae to pass through, placed centrally such that it is surrounded by the hc. the significance of using this pot system was to: firstly, to allow the set-up of different treatments of soil conditions for roots and external hyphae; and secondly, to issn 1978-3477 volume 2, number 1, april 2008 p 22-26 limit the access of roots to soil resources in the rc and simultaneously to permit hyphae growing out from infected roots to develop and interact with soil conditions with different ph levels in the hc, thereby contributing maximally to the growth of the host plants. therefore, differences in plant responses to mycorrhiza were assumed to be the consequence of the functioning of the hyphae developing in the hc. the experiment comprised three levels of mycorrhizal inoculation with and without g. margarita, g. etunicatum fungal inoculants, placed in the rc; and three levels of soil ph of 4.6, 4.9, and 5.2 in the hc. the treatments were arranged in a completely randomized design with four replicates. pots without plants or inoculants were included to check changes in ph of growth media during the experiment. biological materials. the fungi used were g. margarita becker and hall (beg 34) and g. etunicatum becker and gerdemann (ut316-9, invam collection), supplied by the laboratory of soil biology, the university of adelaide, waite campus, south australia. previous work showed that both g. margarita and g. etunicatum were superior in acidic conditions (clark 1997; clark 2002; borie and rubio 1999). these isolates were raised in pot cultures of subterrainean clover (trifolium subterranean l.) in a sand and soil mixture (90:10 w/w) for 4 months. another set of pot cultures without these fungi was prepared similarly to provide a mycorrhizafree control. the plant used was the cowpea cv red caloona supplied by csiro department of tropical agriculture, brisbane, australia. growth medium. the growth medium was a 10:90 (w/w) mixture of soil and washed sand. the original soil was an acidic podzolic soil (a ph h2o of 4.9); a cultivated, grey sandy loam collected from the flaxley farm, in the adelaide hills, south australia. it was taken from 25 cm deep, air-dried; ground and sieved 5 mm mesh and then completely mixed with washed sand. the mixture is referred to as ‘soil’ hereafter. it was first fertilized in with 59.4 mg nh 4 -n, 178.2 mg no 3 -n, 36.0 mg p, 54.0 mg s, 214.2 mg k, 18.9 mg mg, 114.3 mg ca, 13.5 mg na, 8.1 mg cl, 2.7 mg fe, 0.45 mg b, 0.45 mg mn, 0.45 mg zn, 0.036 mg cu and 0.009 mo mg kg-1 soil, as given in ruakura solution (smith et al. 1983). the soil had a final ph 5.3 and was denoted as m 0 . after incubation for a week, different volumes of h 2 so 4 and/or naoh solutions were added to adjust the soil ph to 4.6, 4.9 or 5.2. the result and soils were denoted as m 1 , m 2 or m 3 respectively. the adjustment of the ph and some chemical properties of these soils followed the procedures described by rohyadi (2003). the status of some macronutrients in these soils was quite similar, while the concentrations of soluble aluminium ranged from 0.4 to 1.1 μg al3+ g-1 soil [measured by the method of close and powel (1989)], which were sub-toxic to the am fungi tested (rohyadi 2003). therefore, using this experimental set up the interfering effect of toxic al is minimized, so that variation induced by these fungi could be attributed to the soil ph per se in terms of h+ ion concentrations. lastly, these soils were autoclaved at 121°c for two periods of 1 h, separated by one day intervals prior to use. experimental procedures. growth medium m 0 and pot culture inoculants (with g. margarita, g. etunicatum or without these fungi), in a 90:10 (w/w) ratio, were mixed thoroughly and 120 g of each mix was placed into the rc. after moistening with reverse-osmosis-treated (ro) water to field capacity (0.1 g water g-1 soil), a single pre-germinated cowpea seed was then grown in the compartment for 2 weeks prior to the hc being filled with 580 g of m 1 , m 2 or m 3 soil. therefore, plant roots and external fungal hyphae were grown in different acidic soil conditions. the plants were maintained in a greenhouse at an average temperature of 28°c during the day and 19°c at night. during the experiment, soil moisture was maintained at field capacity by watering the rc with ro water, and the hc with ro water adjusted a ph 4.6, 4.9 or 5.2. they were harvested 6 weeks later by separating plant shoots from the roots. the shoots were then dried at 70°c for 48 h and weighed. further, the dried shoots were ground and digested using a 6:1 (v/v) mixture of nitric and perchloric acids, and p concentration in the digest was measured (rayment and higginson 1992) with a shimadzu uv-1601 spectrophotometer. roots were carefully pulled out from the pots, washed under a stream of water, blotted dry and then weighed to determine fresh weight. root samples were taken randomly, and after staining with trypan blue (phillips and hayman 1970), the root length and the mycorrhizal colonized root length (mcrl) was assessed using the gridline-intersect method of giovannetti and mosse (1980). for assessing growth of fungal hyphae in the hc, representative samples of soil in the hc were collected and air-dried. the assessment used the aqueous extraction and membrane-filtertechnique modified as described by rohyadi (2006). the growth was expressed as hyphal length density (hld) counted in meter g-1 soil and in meter cm-1 mcrl. mycorrhizal growth responses (mgr) and mycorrhizal p-uptake responses (mpr) expressing the functioning of the external fungal hyphae in plant growth and p uptake, were measured using following formulae (rohyadi et al. 2004): fig 1 overview of the pot system with two compartments for plant rc and external fungal hc. the rc was a cylindrical bag made of 30 μm nylon mesh. 10 cm rc hc slide view top view 9 cm volume 2, 2008 microbiol indones 23 where: s dw (m)ij: shoot dry weight of plants colonized by a fungus (i) at a ph level tested (j) s m/dw (nm)j: mean of shoot dry weight of non-mycorrhizal (control) plants at a ph level tested (j) sp cont. (m)ij: shoot p content of plants colonized by a fungus at a ph level tested sp m/cont. (nm)j: mean of shoot p content of nonmycorrhizal (control) plants at a ph level tested data were analyzed using analysis of variance followed by lsd-test for significant treatments at p<0.05. results change in soil ph. there was no significant change in soil ph measured after experiment (data not shown). the only decline of about 0.014 units, was observed for the control pots at ph 4.6. root length, mycorrhizal colonization, and external hyphal growth. there was no significant effect of mycorrhizal inoculation on the growth of plant roots irrespective of soil ph in the hc. however, changes in the soil ph in terms of increasing its level to above ph 4.6 stimulated root elongation. no mycorrhizal colonization was observed of roots of control plants. meanwhile, both the length and percentage of roots colonized by g. margarita and g. etunicatum were not significantly different (table 1). the two test fungi grew external hyphae out from the colonized roots into the hc. growth of the hyphae was significantly affected by the hc soil ph with different trends (table 1). increasing the ph from 4.6 to 4.9 and then to 5.2 steadily decreased g. margarita’s hyphal growth, but it significantly increased g. etunicatum’s. in general, however, compared to that of g. etunicatum, the hyphal length density (hld) of g. margarita, either expression in g-1 soil or in cm-1 mcrl, was higher (particularly at ph 4.6) by two or three times respectively. plant growth and responses to mycorrhiza. inoculation with the two fungi increased the growth of cowpea plants, although its effect depended on fungal isolates and the hc soil ph. control plants with no-mycorrhiza grew poorly regardless of the soil ph compared to those inoculated with g. margarita. whilst growth of g. etunicatum inoculated plants was also poor at ph 4.6, it improved as the ph increased. similarly, mycorrhizal inoculation improved shoot-p concentrations, which together with increased shoot growth increased p uptake by the mycorrhizal colonized plants (table 2). table 2 also shows the same trends for mycorrhizal contributions of these fungi to plant growth and p uptake, expressed as mgr and mpr. increasing soil ph from 4.6 to 5.2 decreased the mgr and mpr to g. margarita, but increased both responses to g. etunicatum. the data in fig 2 shows extent of the mycorrhizal contributions appears to be in line with the growth of external fungal hyphae in the hc. however, patterns were different between the two fungal isolates. inhibition of growth of g. margarita’s external hyphae by increasing soil ph corresponded to the reduction in both in the mgr and the mpr of their host plants (fig 2a). in contrast, increased growth of g. etunicatum’s increased their host plant growth and p-uptake responses (fig 2b). table 1 growth and colonization of roots of cowpea plants in the rc and growth of external hyphae of g. margarita and g. etunicatum at different soil ph in the hyphae compartment initial soil ph inoculation root length (m/plant) mycorrhizal colonization (%) mcrl (m/plant) hyphal length density (m g-1soil) (m cm-1mcrl) 4.6 4.9 5.2 nm gi. margarita g. etunicatum nm gi. margarita g. etunicatum nm gi. margarita g. etunicatum 4.73b*) 4.86ab 4.71b 4.88ab 5.04a 5.04a 5.08a 5.12a 5.05a nd 49a 46a nd 53a 54a nd 53a 56a nd 2.38ab 2.16b nd 2.62a 2.72a nd 2.71a 2.70a nd 3.14a 0.98e nd 2.65b 1.40d nd 2.46b 2.23bc nd 6.92a 2.52c nd 6.55a 2.98c nd 5.54b 5.00b * means within a column with different superscripts are significantly different based on lsd-test at p<0.05. nm: no mycorrhiza, mcrl: mycorrhizal colonized root length, nd: not detected. table 2 shoot growth and p content of cowpea plants and their responses to mycorrhiza initial soil ph inoculation shoot dw (mg/plant) mgr (%) shoot-p (%) (mg/plant) mpr (%) 4.6 4.9 5.2 nm g. margarita g. etunicatum nm g. margarita g. etunicatum nm g. margarita g. etunicatum 150c*) 271a 170bc 160bc 264a 183b 166bc 255a 244a na 81 13 na 65 14 na 53 47 0.088c 0.117a 0.105b 0.084c 0.119a 0.118a 0.085c 0.096bc 0.119a 0.13c 0.32a 0.18bc 0.13c 0.31a 0.25ab 0.14c 0.25ab 0.29a na 139 35 na 130 84 na 73 98 * means within a column with different superscripts are significantly different based on lsd-test at p<0.05. nm: no mycorrhiza, dw: dry weight, mgr: mycorrhizal growth response, mpr: mycorrhizal p-uptake response, na: not applicable. % mgr = (nm)js (nm)j s (m)ijs m/dw m/dw dw x 100 % mpr = (nm)jsp (nm)j sp (m)ijsp m/cont. m/cont. cont. x 100 24 rohyadi microbiol indones discussion results of the present study demonstrate that low soil ph significantly influenced growth of external hyphae of am fungi with a consequence of their working to benefit growth of the host plants. these findings support the hypothesis of robson and abbott (1989) that high soil acidity in terms of excessive h+ ion activities is one of the main factors limiting the growth and functioning of am fungi in acidic soils. several stages in the life cycle of am fungi such as the germination of spores, the elongation of germ tubes and the colonization of host plant roots are inhibited at low soil ph according to green et al. (1976); siqueira et al. (1985); and clark (1997). however, due to inappropriate experimental methods, the toxic effects of excessive h+ ions on the growth of the external fungal hyphae have not been described so far. using pots divided in such a way as to have a special compartment for growing fungal hyphae separately from their host plant roots, this study found that the two am fungi tested, g. margarita and g. etunicatum, exhibited different levels of external hyphal development. g. margarita produced external hyphae at a much higher rate than g. etunicatum. this indicates that although these two fungi formerly have been categorized as superior under acidic conditions, they basically need different acidity levels to develop an extensive network of hyphae in soils. g. margarita appears to be better adapted to low ph (4.6-5.2), whereas g. etunicatum required a ph 5.2 or higher. the production of hyphae formed in soils amongst ma fungi in general may vary considerably with their inherent characteristics (abbott and robson 1985) and responses to soil conditions (wilson 1988). however, mechanisms of how these fungi resist the adverse soil conditions remain in question. the adequacy of energy supplied by host plants for the fungal hyphae to grow (abbott et al. 1984), or the production of exudates by the hyphae (bago et al. 1996) that may modify soil ph surrounding them, has been proposed. results of the present study verifies in part the first mechanism by which better growth of host plants generates a greater length density of symbiotic fungal hyphae in soils or vice versa. however, my results did not support the second mechanism. i did not find any significant changes in observed ph level of bulk soils in the hc. the present study also found that the two fungi significantly improved p uptake and growth of cowpea plants. this is further evidence on the importance of am symbioses, particularly for plants encountering unfavorable conditions, such as low soil ph. my results support recent work showing beneficial effects of am on plant growth on acid soils (yost and fox 1979; rohyadi et al. 1988; nurlaeny et al. 1996; clark and zeto 2000). in spite of the above facts, variation in am effectiveness in acidic soils exists among fungal species (clark 2002). g. margarita was found much more effective than g. etunicatum, as in our previous work (rohyadi et al. 2004). however, the causes of the variation remain unknown. most studies using conventional (non-compartmented) pots pointed out that the variation was a consequence of differences mainly in the intensity of root colonization (nurlaeny et al. 1996; clark et al. 1999; clark 2002). increased length (or percentage) of root colonization increased mycorrhizal effects on plant growth and p uptake. this was, however, not the case in the present study. as might be expected, using a growing system of compartmented pots, g. margarita and g. etunicatum extensively colonized cowpea plant roots and in the same way. however, they had different effects on the growth and p uptake of the plant. this indicates clearly that symbiotic effectiveness of these fungi was not related to either length or percentage of root colonization, but on other fungal-symbiont traits, mainly growth of external hyphae in soils. therefore, in short, this study verified that the effectiveness of am symbiosis on plant growth in acid soils is closely associated with the growth of external fungal hyphae rather than with root colonization. these findings also answer the question on the cause of the different responses of the cowpea plant to these am fungi tested as reported previously (rohyadi et al. 2004). some other studies employing compartmented pot systems have also been demonstrated the significant role of external hyphae in am symbioses (jakobsen et al. 1992; smith et al. 2000; van aarle et al. 2002). these studies suggested that the fungal hyphae may function as the vital component for am effectiveness. for example, smith et al. (2000) counted that up to 80% of the total p taken up by mycorrhizal colonized plants was generated from the external hyphae of the fungus. it seems that the function of external fungal hyphae becomes more important for plants grown on fig 2 a, mycorrhizal growth (mgr); and b, p-uptake responses (mpr) of cowpea plants to hyphal length density (hld) of gi. margarita and g. etunicatum at different soil ph levels in the hc; hld = ; mgr = ; mpr = . gi. margarita g. etunicatum m p r ( % ) h l d ( m g -1 s oi l) 4 3 2 1 0 0 4.6 4.9 5.2 5.24.94.6 150 120 90 60 30 soil ph b gi. margarita g. etunicatum m g r ( % ) 120 100 80 60 40 20 00 4 3 2 1 h l d ( m g -1 s oi l) 4.6 4.9 5.2 5.24.94.6 a volume 2, 2008 microbiol indones 25 acid soils since most of them have poorly developed root systems (marschner 1991). intensive growth of the hyphae from the inhibited roots may essentially compensate their limited access to resources of nutrients and water in soils. importantly, the results of this study increase our understanding of the detrimental effects of high soil acidity on various am fungal activities. excessive h+ ions may negatively influence not only spore germination and germ tube growth, but also the development and distribution of external hyphae in the soil. as the external hyphae of the fungi are themselves subject to the negative effects of soil acidity, effectiveness of am symbioses at low soil ph may depend on their fungal component’s ability to withstand the adverse conditions in growing external hyphae. consequently, changes in soil ph affecting the growth of the hyphae may also affect plant responses to the symbioses. therefore, a species or isolate of am fungus which has the characteristic of an extensive hyphal network would seem to be an excellent candidate for inoculation purposes in acid soils. this study verified that g. margarita is tolerant to low ph, thereby it could be considered as one possible candidate. references abbott lk, robson ad. 1985. the effect of soil ph on the formation of vesicular arbuscular mycorrhiza by two species of glomus. aus j soil res 23:235-261. abbott lk, robson ad, de boer g. 1984. the effect of phosphorus on the formation of hyphae in soil by vesicular-arbuscular mycorrhizal fungus glomus fasciculatum. new phytol 97:437-446. bago b, vierheilig h, piché y, azcón-aguilar c. 1996. nitrate depletion and ph changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus glomus intraradices in monoxenic culture. new phytol 133:273-280. borie f, rubio r. 1999. effects of arbuscular mycorrhizae and liming on growth and mineral acquisition of aluminium tolerant and aluminium sensitive barley cultivars. j plant nut 22:121-137. clark rb. 1997. arbuscular mycorrhizal adaptation, spore germination, root colonization, and host plant growth and mineral acquisition at low ph. plant soil 192:15-35. clark rb. 2002. differences among mycorrhizal fungi for mineral uptake per root length of switchgrass grown in acidic soil. j plant nut 28:1753-1772. c l a r k r b , z e t o s k . 2 0 0 0 . m i n e r a l a c q u i s i t i o n b y a r b u s c u l a r mycorrhizal plants. j plant nut 23:867-902. clark rb, zeto sk, zobel rw. 1999. arbuscular mycorrhizal fungal isolate effectiveness on growth and root colonization of panicum virgatum in acidic soil. soil biol biochem 31:1757-1763. close ea, powell hkj. 1989. rapidly extracted (0.02 m cacl 2 -soluble) ‘reactive’ aluminium as a measure of aluminium toxicity in soils. aus j soil res 27:663-672. giovannetti m, mosse b. 1980. an evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. new phytol 84: 489-500. green we, graham so, schenck nc. 1976. the influence of ph on the germination of vesicular arbuscular mycorrhizal spores. mycologia 68:929-934. jakobsen i, abbott lk, robson ad. 1992. external hyphae of vesiculara r b u s c u l a r m y c o r r h i z a l f u n g i a s s o c i a t e d w i t h tr i f o l i u m subterraneum l. 1. spread of hyphae and phosphorus inflow into roots. new phytol 120:371-380. manjunath a, habte m. 1991. root morphological characteristics of host species having distinct mycorrhizal dependency. can j bot 69:671-676. marschner h. 1991. mechanisms of adaptation of plants to acid soils. plant soil 134:1-20. nurlaeny n, marschner h, george e. 1996. effects of liming and m y c o r r h i z a l c o l o n i z a t i o n o n s o i l p h o s p h a t e d e p l e t i o n a n d phosphate uptake by maize (zea mays l.) and soybean (glycine max l.) grown in two tropical acid soils. plant soil 181: 275-285. phillips jm, hayman ds. 1970. improved procedures for clearing roots and staining parasite and vesicular arbuscular mycorrhizal fungi for rapid assessment on infection. trans br mycol soc 55:158161. rayment ge, higginson fr. 1992. australian laboratory handbook of soil and water chemical methods. melbourne: inkata pr. robson ad, abbott lk. 1989. the effect of soil acidity on microbial activity in soils. in: robson ad (ed). soil acidity and plant growth. sydney: ap. p 139-166. rohyadi a. 2003. effects of aluminium on arbuscular mycorrhizal symbioses in cowpea plant growth [ph.d thesis]. adelaide: university of adelaide. rohyadi a. 2006. elevated aluminium concentrations in soil reduce growth and function of external hyphae of gigaspora margarita in growth of cowpea plants. bionatura 8:47-59. rohyadi a, fa smith, murray rs, smith se. 2004. effects of ph on mycorrhizal colonization and nutrient uptake in cowpea under conditions that minimise confounding effects of elevated available aluminium. plant soil 260:283-290. rohyadi a, modjo hs, radjagukguk b. 1988. the effects of arbuscular mycorrhiza on growth of maize on the red yellow podzolic soil (in indonesian). j bpps-ugm 2:155-162. schüßler a, schwarzott d, walker c. 2001. a new fungal phylum, the glomeromycota: phylogeny and evolution. mycol res 105: 4131421. siqueira jq, sylvia dm, gibson j, hubbell dh. 1985. spores, germination, and germ tubes of vesicular arbuscular mycorrhizal fungi. can j microbiol 3:1965-1971. smith fa, jakobsen i, smith se. 2000. spatial differences in acquisition of soil phosphate between two arbuscular mycorrhizal fungi in symbiosis with medicago truncatula. new phytol 147:357-366. smith gs, johnston cm, cornforth is. 1983. comparison of nutrient solutions for growth of plants in sand culture. new phytol 94:537548. van aarle i, olsson pa, söderström b. 2002. arbuscular mycorrhizal fungi respond to the substrate ph of their extraradical mycelium by altered growth and root colonization. new phytol 155:173-182. wilson do. 1988. differential plant response to inoculation with two va mycorrhizal fungi isolated from a low-ph soil. plant soil 110:69-75. yost rs, fox rl. 1979. contribution of mycorrhizae to p nutrition of crops growing on an oxisol. agronomy 71:903-908. 26 rohyadi microbiol indones 7 munti.pmd free-living ice-nucleating active bacteria from high mountain lake habitats munti yuhana1* and kurt hanselmann2 1department of aquaculture, faculty of fisheries and marine sciences,institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 2microbial ecology group, institute of plant biology, university of zurich, switzerland we collected the culturable heterotrophic bacteria from oligotrophic high mountain lake habitats and tested their capability to induce ice formation. direct plating was carried out using low-nutrient medium at a temperature of between 3 and 4°c. as many as 84 isolates were recovered from glacial ice and natural biofilm growing on granite rocks surface. six out of 84 isolates were capable of expressing the ice-nucleation phenotype. after autoclaving the cell suspension at 121°c for 15 min, isolate j78 was still able to retain the ability for ice formation. heat-stable ice nuclei produced by ice-nucleating active bacteria have potential applications in biotechnology. characterization of ina bacteria was performed employing live-dead gram staining and molecular methods. universal primers for bacteria (s-d-bact-0008-b-s-20 and s-d-bact-1524-a-a-18) were used for pcr to amplify almost the full length of the 16s rrna genes of selected ina isolates. restriction fragment length polymorphism analysis resulted in 2 unique patterns, as represented by j43 and j83, respectively. based on dna sequencing of 16s rrna gene, isolate j43 (genebank accession no. aj864852) was closely related to pseudomonas mephitica (99.2% sequence similarity) and janthinobacterium lividum (99% similarity), whereas isolate j83 (genebank accession no. aj864859) showed 100% sequence identity to pseudomonas fluorescens. key words: high mountain lake habitats, ice nucleation, 16s rrna gene, free-living bacteria _____________________________________________ ________________________ *corresponding author, phone: +62-251-8628755, fax: +62-251-8622941, e-mail: myhn@gmx.ch volume 2, number 3, december 2008 issn 1978-3477 p 131-134 various gram-negative bacteria are capable of catalyzing ice formation at temperatures of -2 to -12°c in nature (lindow et al. 1982; hirano et al. 1985). most of ice-nucleating-active (ina) bacteria are associated with plants (lindow et al. 1978; lindermann et al. 1982; loper and lindow 1994; waturangi et al. 2008) or animals (lee et al. 1995; worland and block 1999). other microorganisms, including several genera of fungi (pouleur et al. 1992) and lichens (kieft 1988) have been reported in their ability to induce ice formation. ice-nucleating active (ina) bacteria have been recovered from various geographical areas, such as antarctica, temperate, subtropical or tropical regions. ina bacteria, which induce frost damage in various plants, can contribute to a very devastating loss in agricultural crops production (lindow et al. 1982). ina bacteria produce outer surface membrane protein that can act as a catalyst for the unusual transition of water from liquid to its solid phase (gurian-sherman and lindow 1993). droplets of pure water (ina-free water) remain in its liquid state (supercool) up to a temperature of -40°c. the higher temperature of ice catalysis conferred by bacterial ice-nuclei makes them useful in ice-nucleation-limited processes such as artificial snow production, the freezing of some food products and possibly in future weather modification schemes (gurian-sherman and lindow 1993). up to today, reports on free living ina bacteria recovered from high mountain lake habitats remain limited. microorganisms that survive and actively grow in these habitats might be good sources of cold-active-proteins, such as ice nucleation active protein, which are catalytically efficient at low temperatures (gerday et al. 1997). in this study, we report our study on free living ina bacteria recovered from these extreme habitats. materials and methods samples collection and treatments. samples were collected from the joeri lakes catchment, located in the southeastern swiss alps at an altitude of approximately of 2 750 m a.s.l (yuhana et al. 2006). lake water and biofilm samples were aseptically collected during the snow-free season. subsurface water samples were obtained in sterile glass bottles and patches of biofilm samples were collected from the surface of submerged rocks. aliquots of 50 and 100 µl of water were spread-plated and subcultured onto minimal growth agar medium (ph 7.1), whereas biofilm samples were spread aseptically onto the medium after mechanical disruption of the biofilm with a sterile loop. growth temperature for all cultures was 4±1°c. the minimal growth medium (designated as mm) contained the following ingredients (final ph 7.1): 6 µm mgso 4 , 10 µm cacl 2 , 20 µm na 2 co 3 , 14 µm nano 3 , 10 µm nh 4 cl, 1.75 µm k 2 hpo 4 , 2.7 µm edta na salt, 10 mg l-1 yeast extract, 10 mg l-1 peptone, 15 g l-1 washed-bacteriological agar and 1 x trace elements, (added to the autoclaved basal medium). the concentrated trace elements stock solution (10 000 x) contained the following: 850 µm znso 4 ·7h 2 o, 7 100 µm mncl 2 ·4h 2 o, 86 µm co(no 3 ) 2 ·6h 2 o, 1 600 µm na 2 moo 4 ·2h 2 o, 29 750 µm citrate·h 2 o and 21 480 µm ferric ammonium citrate. when growth occurred (after 14 to 21 days of incubation in the cold), single colonies of visibly dominant and different colony morphotypes were subcultured onto new minimal medium. isolates were maintained in 10 x diluted luria bertani (lb) agar medium (ph 7.2) containing the following ingredients: 0.5 g l-1 bacto tryptone, 1.0 g l-1 yeast extract, 0.5 g l-1 nacl and 15 g l-1 bacteriological agar. cell morphology and characterization by gram staining. fluorescent staining was performed to determine the gram type, characterize cell morphology and to distinguish dead cells from living ones. staining was carried out as described by the manufacturer (molecular probes, inc.) using the viability gram staining kit (v-7023 molecular probes inc.). the treated samples on slides were observed with a zeiss axioplan microscope (carl zeiss, oberkochen germany) employing 3 different excitation filters (365-395, 450-490 and 546-580 nm) and photographed with an optronic digital camera. ice nucleation assay. the ice nucleation capacity of all 84 isolates was tested qualitatively by the tube assay (modified from hirano et al. 1985). isolates were grown in 10 ml liquid mm and incubated at 4°c until the stationary phase was reached. cells were then suspended in 10 ml autoclaved pbs solution (10 mm potassium phosphatebuffered saline, ph 7.0) and kept in a cooling-bath at -2 to -10°c for 5 to 10 min. a suspension of e. coli cells containing plasmid pjl1703 (loper and lindow 1994) was used as a positive control, whereas a cell-free pbs solution served as a negative control. the ice nucleation activity of each ina isolate was observed by droplet-freezing assay and the ice nucleation frequency was calculated by the following formula: n(t) = [(ln f) ] / v, (vali 1971) where n(t) is the frequency of ice nucleation at t temperature, f indicates the proportion of droplets unfrozen and v is the volume of individual droplets. genomic dna extraction. total genomic dna from pure isolates was extracted with cetyltrimethyl ammonium bromide (ctab) (modified from murray and thompson 1980). this provided a simple, non toxic and inexpensive method and yielded enough dna template for pcr amplification. after pelleting the cells, extraction buffer (2% w/v ctab, 100 mm tris-cl ph 8.0, 1.4 m nacl, 20 mm edta) was added and mixed. the solution was incubated at 60°c for 30 min and centrifuged at 12 000 xg (4°c) for 10 min. after transferring the supernatant into a fresh tube, an equal volume of chloroform:isoamyl alcohol (24:1) was added, mixed gently and the solution was centrifugated at 12 000 xg (4°c) for 10 min, these steps were repeated one more time. the resultant supernatant was transferred into a new tube and 1/10 volume equivalent of 7.5 m ammonium acetate was added. dna was then collected by precipitation in ethanol. dna extracts were checked using the following electrophoresis conditions: agarose gel (1% w/v), [tris-acetate-edta] (tae) buffer 0.5 x (20 mm trizma base, 10 mm glacial acetic acid, 0.5 mm edta), running time 30 min at 5 v/cm. successful extraction of dna was verified by staining the gels in a 1 µg ml-1 ethidium bromide solution. 16s rrna gene amplification by pcr. nearly full-length 16s rrna genes were amplified by pcr using the bacterial primers: s-d-bact-0008-b-s-20 (5'-aga gtt tga tcc tgg ctc ag-3') and s-d-bact-1524-a-a-18 (5'-aag gag gtg atc car ccg-3'). pcr amplification was performed in a 25 µl reaction volume with a techne thermocycler (techne ltd, oxford, uk). each reaction mixture contained (final concentration) taq buffer (1x) (sigma), 1.5-2.0 mm mgcl 2 , 0.1 mg ml-1 dnase-free bovine serum albumin (amersham, pharmacia biotech inc.), 0.2 mm dntps, 200 nm of forward and reverse primer, respectively, 40 u taq polymerase (sigma), ddh 2 o and approximately of 20-100 ng of template dna. the following touch-down pcr program was used: initial denaturation at 94°c for 2 min; 20 cycles of 94°c for 20 sec, 63°c for 30 sec with lowering temperature by 0.5°c in every cycle, 72°c for 80 sec with increasing duration by 1 sec in every cycle; another 20 cycles of 94°c for 20 sec, 53°c for 30 sec, 72°c for 100 sec with increasing period by 1 sec every cycle; followed by a final extension step at 72°c for 10 min. pcr products were analyzed by electrophoresis in 1% w/v agarose gels in 0.5x tae running buffer, stained with ethidium bromide (1 µg ml-1) and photographed under uv. for rflp analysis, 8 µl pcr products were doubledigested with 1 u restriction enzyme hinfi (5'-g/antc) and haeiii (5'-gg/cc) in a total reaction volume of 10 µl. sequencing. the pcr products of the 16s rrna genes from different rlfp patterns were purified through microcon centrifugal filter devices (microcon ym 100, millipore, bedford, mass., usa). nearly full-length 16s rrna genes were bidirectionally sequenced with a dna sequencer (abi prism 377), using abi prism® big dyetm v2.0 (applied biosystems) as described by the manufacturer with the following 6 primers: s-d-bact-0008-b-s-20 (5'-aga gtt tga tcc tgg ctc ag-3'), s-*-univ-0519-a-a-18 (5'-gwa tta ccg cgg ckg ctg-3'), s-*-univ-0519-a-s-18 (5'-cag cmg ccg cgg taa twc-3’), s-d-bact-1099-b-s-16 (5’-gya acg agc gca acc c-3’), s-d-bact-1099-b-a-16 (5’-ggg ttg cgc tcg ttr c-3’) and s-d-bact-1524-a-a-18 (5'-aag gag gtg atc car ccg-3'). for a 10 µl-single pcr reaction, 5 to 20 ng dna template, 3 µl big dye (applied biosystems) and 3 µl of 1.5 µm primer were used. after the sequencing pcr, the products were purified with sephadex g-50 (amersham, pharmacia biotech ab) and loaded onto a sequencing machine (abi prism 377 dna sequencer). the blast search tool available from ncbi (http://www.ncbi.nlm.nih.gov/blast) was used to list the closest neighbors of the sequences. phylogenetic tree construction. the phylogenetic tree was analysed online using the phylip interface, available at the ribosomal database project (http://rdp8.cme.msu.edu/ html/analyses.html). sequence data was imported and aligned to their closest neighbors by an automated alignment. the distance matrix was calculated by maximum likelihood method and a phylogenetic tree was constructed based on neighbor-joining analysis. results eighty-four isolates were recovered from lake water, glacier and biofilm growing on granite rocks surface. generally they are cream-pigmented morphotypes and some of them were capable of producing water-soluble yellowgreenish fluorescent pigments. test tube assay showed that 6 out of 84 isolates were capable of catalyzing the ice nucleation in the tubes at temperatures between -2 to -5°c. fluorescent microscopy determination after live-dead gram staining, showed that all of the 6 ina isolates were gram negative and rod shaped (fig 1). after autoclaving the cell suspension at 121°c for 15 minutes, isolate j78 still showed its ability to initiate the ice formation at lower temperatures, ranging from -8 to -10°c. other ina isolates (j43, j71, j77, j83 and j84) and autoclavedpositive control, were no longer capable of catalyzing ice formation even at lower temperatures (fig 2). 132 yuhana and hanselmann microbiol indones isolates j43, j71, j77, j78 and j84 (fig 3). 16s rrna genes of isolates j83 and j43 (as a representative of other 5 ina isolates) were selected for sequencing. the dna sequence of isolate j43 (1484 bp) showed 99.2% similarity to pseudomonas mephitica or janthinobacterium lividum. the nucleotides have been submitted to the genbank with accession no. aj864852. dna sequence of isolate j83 (1491 bp) showed 100% similarity to p. fluorescens ccm 2115 and has been submitted with accession no. aj864859. discussion all ina isolates from high mountain lake habitats showed ice nucleation activity at temperatures warmer than -5°c. similar results were shown in a previous study (kieft 1988) of ina associated with lichens isolated from high mountain habitats. his study showed that several epilithic lichen samples of the genera rhizoplaca, xanthoparmelia and xanthoria expressed ice nucleation activity at temperature as warm as -2.3°c. according to the ice nucleation proteins classification described by turner et al. (1990), all of our ina isolates are characterized as class a, which showed ice nucleation activity between temperature range of -2 to -5°c. waturangi et al. (2008) reported that fig 1 fluorescent micrographs of isolate j43 and j83. live cells fluoresce blue, whereas dead cells fluoresce green. both isolates were gram negative. j 4 3 j 8 3 19 µµµµµm the highest ice nucleation activity among ina isolates was shown by j84 with frequency of 60 nuclei ml-1 and the lowest was indicated by isolate j71 (32 nuclei ml-1). isolates j43, j77 and j83 had the same ice nucleation frequency of 38 nuclei ml-1 and isolate j78 was 46 nuclei ml-1 (table 1). the temperature of the assay was warm (-2°c). ice nucleation activity of isolate j78 after autoclaving was 46 nuclei ml-1 at -8°c. pcr amplifications of the 16s rrna gene of selected ina isolates yielded products of approximately 1500 bp. by using double restriction with hinfi and haeiii, two different rflp patterns were obtained. j83 has a unique pattern, whereas j43 shows corresponding band patterns with fig 2 ice nucleating assay tubes. (a) assay carried out before autoclaving the cell suspension at the temperature ranging from from -2 to -5°c. assay showed ice nucleation capability of isolate j43, j71, j77, j78, j83, and j84. j57 represents a non ice-nucleatingactive isolate, remaining unfrozen after 30 minutes incubation at temperatures ranging from -8 to -10°c. cell-free pbs solution (-) serves as a negative control, while cell suspension of e. coli containing plasmid pjl1703 was used as a positive control; (b) assay carried out after autoclaving the cell suspension. suspension of the isolate j78 remaining capable of inducing the ice nucleation at lower temperatures ranging from -8 to -10°c. a b table 1 ice nucleation activity of free living ina bacteria from high mountain lake habitats no of frozen droplets/ total droplets isolate j43 j71 j77 j78 j83 j84 j78 (after autoclaving) 17/20 16/20 17/20 18/20 17/20 19/20 18/20 38 nuclei/ml (-2°c) 32 nuclei/ml (-2°c) 38 nuclei/ml (-2°c) 46 nuclei/ml (-2°c) 38 nuclei/ml (-2°c) 60 nuclei/ml (-2°c) 47 nuclei/ml (-8°c) n (t), ice nucleation activity; where n, frequency of ice nucleation at temperature t. n(t) (at temperature) fig 3 rflp patterns of 16s-rrna genes from the ina isolates. lane 1, molecular marker 100 bp ladder (pharmacia, biotech); lane 2-7, j83, j43, j71, j77, j78, and j84. running conditions on spreadex el-800 gel (elchrom sci.): 70 vcm-2 for 90 min, running temperature 55°c in 30 mm tae buffer. volume 2, 2008 microbiol indones 133 tropical ina bacterial isolates showed ice nucleation activity at cooler temperature of -8°c and categorized as class b. at this temperature, the ice nucleation activity values of tropical ina isolates were between 38 and 60 nuclei ml -1 . isolate j78 showed the existance of heat-stable ina protein, since the heat treatment of 121°c for 15 min did not diminish its capability to initiate ice formation. previous studies also reported heat-stable ina protein isolated from a lichen, rizoplaca chrysoleuca (kieft 1988) and from fusarium (pouleur et al. 1992). kieft (1988) reported that control cell suspension (no heat treatment) and 10 min treatment at temperatures of 40 to 70°c, showed the ice nucleation activity at temperatures between -2.3 to -3.3°c, whereas the ice nucleation activity at a lower temperature of -14.6°c was detected after cell suspension exposed by heat treatment at temperature range of 80 to 95°c. our isolate j78 showed its ice nucleation ability at warmer temperatures than that of ina expressed by r. chrysoleuca, i.e. at temperatures ranging from -8 to -10°c. this might indicate a more stable ina protein structure present in this isolate, however, further studies are required to determine the uniqueness of the j78 ina protein. based on their dna sequences, our ina isolates phylogenetically fall into the âand ã-subgroups of proteobacteria (fig 4). isolate j83, which is closest related to p. fluorescens strain iam12022, belonging to the gammasubdivison. p. fluorescens has been reported to belong to the bacteria which are able to catalyze ice formation (lee et al. 1995) while isolate j43 falls into the âsubdivison. this isolate is closely related to p. mephitica atcc 33665t or j. lividum dsm 1522t. to our knowledge, there are no other reports on the ability of the bacteria belonging to p. mephitica or j. lividum in catalyzing the ice formation. bacteria which are able to catalyze water crystallization may have an advantage over those which cannot. those which can will be able to survive in frozen environments through slow cellular dehydration (baertlein et al. 1992). this is a very important property for microorganisms to survive in this cold and extreme habitat. ice formation on the outside of the cell allows water molecules to move from the cytoplasm across the cell membrane to join crystals of pure water nucleated extracellularly. this increases the osmotic potential inside the cells, thereby preventing freezing and cell damage by internal ice crystals. the ice nucleation process allows for ordered propagation of ice throughout the cell rather than a rapid freezing, which can result in membrane rupture and cell death (baertlein et al. 1992). since this ability was only found in 6 out of the 84 isolates, we must assume that other bacteria use different strategies to survive periods of freezing, for instance by producing anti-freeze proteins (feller et al. 1996) or other osmolytes. acknowledgements this work was supported by an annual grant from the microbial ecology group, insitute of plant biology, university of zurich. authors would like to thank to steven lindow, ucla-berkeley, usa for positive control sample of e. coli (pjl1703). references baertlein da, lindow se, panopaulos nj, lee sp, mindrinos mn, chen thh. 1992. expression of bacterial ice nucleation gene in plants. plant physiol 100:1730-1736. feller g, narinx e, arpigny jl, aittaleb m, baise e, genicot s, gerday c. 1996. enzymes from psychrophilic organisms. fems microbiol rev 18:189-202. gerday c, aittaleb m, arpigny jl, baise e, chessa jp, garsoux g, petrescu i, feller g. 1997. psychrophilic enzymes: a thermodynamic challenge. biochim biophys acta prot struct mol enzymol 1342:119-131. gurian-sherman d, lindow se. 1993. bacterial ice nucleation: significance and molecular basis. faseb j 7:1338-1343. hirano ss, baker lt, christen du. 1985. ice nucleation of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury. plant physiol 77:259-265. kieft tl. 1988. ice nucleation activity in lichens. appl environ microbiol 54:1678-1681. lee mr, lee re, strong-gunderson jm, minges sr. 1995. isolation of ice-nucleating active bacteria from the freeze-tolerant frog, rana sylvatica. cryobiology 32:358-365. lindermann j, constantinidou ha, barchet wr, upper cd. 1982. plants as sources of airborne bacteria, including ice nucleation bacteria. appl environ microbiol 44:1059-1063. lindow se, arny dc, upper cd. 1978. distribution of ice nucleationactive bacteria on plants in nature. appl environ microbiol 36:831-838. lindow se, arny dc, upper cd. 1982. bacterial ice-nucleation: a factor in frost injury to plants. plant physiol 70:1084-1089. loper je, lindow se. 1994. a biological sensor for iron available to bacteria in their habitats on plant surfaces. appl environ microbiol 60:1934-1941. murray mg, thompson wf. 1980. rapid isolation of high molecular weight plant dna. nucl acids res 8:4321-4325. 6. 526 fusarium (widodo) roba... t"!rfrq*lp[n*u $$$n ! s?ff-34??" *{$$}{ g s?-#5?5 v*$ $. ffi* 3scegsaxekr ?#e{" p 133-l3s available *l*ine sf: htp :l/wwur. penni "*c ffi *t;ma!1fu adex" php/n*i**line s*{: i s-54"54/$ei-s-3"6 fasariurn speeies assoeiated with carm rot of tarc in bogor wtrdodot *osupramana depar wtt 6 plstt protcction facvlty ofagricdrt tz, trnsritar pertuie bogpr jclan kanpa, kamprs durmttga bryer 16680, in&nesia in kor-w€st java, corm rofi disease of,8o bas &rn#ed in many cropping areas 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{tg$4} als* r€f*r*ed that &arc dryr rot caused by has been causing serious losses in miyakonojo-miyazaki, japan. since the accurate identification of the causal agent of plant diseases is the key information in the determination of control measures, the results of this study will be valuable for that purpose. the objectives of the study presented here was to determine what species of were associated with diseased taro corm in some subdistricts in bogor area and the pathogen host range. this information would be valuable and may provide some understanding for strategic management of the disease. . preliminary surveys were conducted to observed characteristic, above or below grown symptoms, and the most common cultivars planted by farmers in surveyed areas. samples of diseased corms were surface disinfected with 70 % alcohol, and incubated in moist chamber to induce the fungal sporulation. nine subdistricts, representing the large taro-growing areas in bogor were sampled (table 1). forty individual diseased plant samples were collected from eighteen villages of nine subdistricts in bogor area. one or both of two cultivars, and/or , were sampled depending on the planted cultivars during the study. . individual diseased corms were surfaced disinfected by wiping with 70 % alcohol wetted cotton, sliced transversally into 5 mm thickness with sterilized knife, and then cut in small cubes (5 mm x 5 mm x 5 mm) between the rotten and healthy tissue. five to ten pieces of the tissue were dipped in 10 % household bleach (0.525 % sodium hypochlorite) for 10 sec, rinsed in sterilized distilled water, and blotted dry on tissue paper. pieces were placed onto potato dextrose agar (pda) mixed with two drops of 20% lactic acid per petri dish and incubated at room temperature (± 27 °c) under near ultra violet (366 nm) with a 12 h photoperiod. after 5 days incubation, upon small pieces of colonised agar from actively growing margins where grew from the tissue were sub-cultured onto pda. single spore cultures fusarium oxysporum fusarium bentul hijau fusarium materials and methods field survey isolation. field survey were established onto water agar, sub-cultured again onto pda, and 7 to 10 days later. identification based on conidial size, conidiogenesis, conidial form, chlamydospores formation were determined on cultures grown on carnation leaf agar (cla) medium, and then identified according to the taxonomic system outlined by leslie and summerell (2006). the experiment was performed on two taro cultivars, cv. bentul and cv. hijau commonly planted in bogor. the corms harvested from healthy individual plants of taro in commercial farmer field were surface disinfected with 70 % alcohol, and sliced transversally into 1.5 cm thickness with sharp sterilized knife. each isolate tested were cultured on pda for 7 days in room temperature under nuv with 12 h photoperiod were plugged with cork borer (ø 5 mm) from actively growing colonies. inoculation was performed by putting plugs of each isolate on the centre of 3 taro corm slices, in which the fungal colony faced the corm t i s s u e s . ta r o c o r m s l i c e s i n o c u l a t e d w i t h pda plugs only were used as control. the treated taro corm slices were placed in plastic box moist chamber by placing cotton wetted with sterile water, covered with plastic wrap, and incubated in dark room. daily observation up to about 5 days after inoculation was done to determine the presence of rotting symptom and incubation period. pathogenicity tests was also performed using the whole unsliced corms with the same inoculation method as mentioned previously, except for the observation period, which was conducted up to 2 weeks after inoculation. taro slices cv. bentul was prepared with the methods as mentioned in the previous experiment and inoculated with conidial suspension, either in singly or combination of species recovered. conidial suspensions of and were prepared by adding sterile distilled water to 7 to 10 day's old cultures rubbing the culture surface with a sterile glass spatula, shaking thoroughly, and filtering through four sterilized layers of cheesecloth. the conidial suspensions were adjusted into 1.25 x 10 ml by means of bright line haemocytometer. the conidial suspension (0.05 ml) of fungus cultures were inoculated singly and in combination onto taro corm slices using micropipette, and placed in plastic box moist chamber, covered with plastic wrap and incubated in the dark at room temperature (± 27 °c). five days after inoculation rotting diameter on 6 corm slices of each treatments was observed. fusarium f. solani f. oxysporum inoculation on sliced taro corm. inoculation on the whole unsliced taro corms. inoculation with single and combination of isolates on sliced taro corm. 6 -1 volume 5, 2011 microbiol indones 133 subdistrict number of individual plant samples ciomas sukaraja bogor timur kemang bogor selatan cijeruk ciawi bogor barat dramaga 12 4 2 3 4 4 3 4 4 total 40 table 1 number and places of diseased plant samples collected inoculation on vegetative propagation of taro. host range tests inoculation on sliced edible corm. inoculation on ornamental inoculation on legume crops results field observation. two cultivars of taro (cv. hijau and bentul) were used in the experiment. the planting medium containing soil and goat manure (4:1 v/v) was twice heat sterilized in two consecutive days, each for 30 min at 120 °c using autoclave. conidial suspensions of was prepared using the same method as mentioned previously ten healthy plants were selected for freedom from disease, washed the attached soils with tap water, dipped in diluted commercial clorox solution (0.525 % sodium hypochlorite) for 60 sec, and then rinsed with sterile water. each set of test plants were dipped in conidial suspension of each isolates for 24 h, grown in pots containing about 2 l prepared medium, and fertilized with n:p:k (15:15:15) 3 weeks after planting. control plants were inoculated with sterile water only. development of disease symptoms was studied during 3 months. . this experiment was performed on other edible and ornamental and commonly legume crops planted by taro farmers, as follows: giant taro ( ), blue taro ( ), chinese evergreen ( ), dumb cane ( ), angel wings ( ), nephthytis ( ), long yard bean ( ), and french bean ( ). sliced edible corm and inoculation procedure was performed using the same methods as mentioned in previous experiment. . some species of ornamental ( and ) were used in the experiment. inoculation was performed on vegetative propagation using dipping method with conidial suspensions as described previously. . long yard bean and french bean commercial seeds was first surface disinfected by dipping in the diluted commercially clorox for 60 sec, rinsed in sterile water for 2 times, and then germinated in plastic box contained heat sterilized sand. germinated seeds were soaked in conidial suspension (1.25 x 10 ml ) for 24 h and planted in plastic pots contained heat sterilized planting medium as mentioned in previous experiment. development of disease development was observed once a week for 2 months. foliar symptoms include leaf curling or crinkling, leaf browning from the edge, chlorosis, shrivelling and withering of petiole, was the distinct easily detected in the fields. during the field observation, we found 2 type of above ground fusarium species fusarium araceae alocasia macrorhiza xanthosoma violaceum aglaonema commutatum dieffenbachia seguine caladium bicolor syngonium podophyllum vigna ungiculata phaseolus vulgaris araceae araceae a. commutatum, d. seguine, c. bicolor, s. podophyllum araceae araceae 6 -1 symptoms, that were plant suddenly died without changing of leaf colours (fig 1a), and preceded with leaf yellowing (fig 1b). advanced disease development caused wilting, stunting of individual plants which eventually die. when the diseased plant was cut transversally, an internal symptom includes vascular discoloration and corm rotting appeared (fig 1c and 1d). in severely infected areas, many cluster-stunted plants showed the empty hollow patches (fig 1e). diseased plant was easily pulled out when corm have already completely rotted. the similar symptoms as observed in this study have also been described on taro in japan (nishimura and kudo 1993) and on konnyaku ( ) (shibata and aoki 1994). , from several observations in preliminary studies with different individual diseased plant samples incubated in moist chamber, was consistently isolated. two species of were found among 40 cultures recovered from taro corm showed wilting and/or chlorotic symptoms in above ground from 9 different sub districts in bogor, west java (table 2). two species of identified as and were recovered from 40 samples associated with taro corm rot disease in 9 different subdistricts in bogor-west java. was observed as the predominant species recovered from 70% of the total diseased plant samples. both species of species were recovered from individual plant samples showing sudden death symptoms above ground, meanwhile only was detected from diseased plants with leaf yellowing symptom. the 2 species of recovered were identified as and based on their specific characters, especially microconidiophore and the formation of microconidium in false heads with long or short monophialides, respectively (fig 2a-b and 2e-f). these 2 species, and comprised 70% and 30% of the total samples, respectively (table 2). colonies of identified in this study was fast growing on pda after 2-3 days, white or cream with spares mycelium. numerous microand macroconidia were formed on cla medium. microconidia produced on elongate monophialide conidiogenous cells (fig 2b); ellipsoid or reniform with 0 or 1-septate (fig 2c), 8 -16 x 4-8 µm, and formed in false heads (fig 2a). macroconidia wide, straight, stout with blunt apical cell, basal cell poorly developed 3-septate, 27-39 µm x 4-7 µm (fig 2d). chlamydospores formed abundantly, either terminally or intercalary, and rapidly within 2 weeks on cla, 3.9-7.8 µm in diameter (fig 2e). amorphophallus konjach fusarium fusarium fusarium f. solani f. oxysporum f. solani fusarium f. oxysporum fusarium f. solani f. oxysporum f. solani f. oxysporum f. solani pathogen isolation, identification and pathogenicity test on taro. 134 w sidodo and upramana microbiol indones d h 10 µm10 µm75 µm 50 µm a b c 20 µm 5 µm 30 µm 10 µm e g i fusarium solani fusarium oxysporum f j 10 µm 10 µm fig 2 microscopically characters of and associated with taro corm rot based on microconidium on conidiophore, microconidium and macroconidium size on cla medium. a, falsehead of with long microconidiophore; b, microconidiophore; c, microconidia; d, macroconidia; e, chlamydospores; f. falsehead of with short microconidiophore; g, microconidiophore; h, microconidia; i, macroconidia; j. chlammydospores fusarium solani f. oxysporum f. solani f. oxysporum volume 5, 2011 microbiol indones 135 b c d e a fig 1 variation of field symptoms of taro corm rot. a, wilting and leaf curling symptom; b, leaf chlorosis; c, corm and leaf petiole discoloration; d, corm rotting and hollowing; e, cluster symptom in the field (circled). pure cultures of the other suspect pathogen identified as was fast growing, mycelia floccose with range in colour from pale to dark violet on pda. microand macroconidia abundantly produced on cla medium. microconidia abundant, formed in falsehead on short lateral monophialides (fig 2f and 2g), ellipsoidal to cylindrical, straight or kidney shape (fig 2h), 8-15 µm x 4-8 µm. macroconidia straight to slightly curved, relatively slender than apical cell tapered or curved f. oxysporum f. solani, with a slight hook, 27-39 µm x 4-5 µm (fig 2i). chlamydospores abundantly produced on cla medium within 2 weeks after inoculation, formed either terminally or intercalary in hyphae, with diameter in range of 3.9-7.8 µm (fig 2j). both species was recovered from the individual plant samples showing sudden death symptom on above ground, while isolation from the diseased plant samples with leaf yellowing only was observed. both species fusarium f. oxysporum fusarium table 3 pathogenicity of single and mixed inoculation with and on cv. bentul f. solani f. oxysporum fusarium species corm rotting diameter (cm)* f. solani f. oxysporum f. oxysporum + f. solani 4.02 ± 0.79 a** 3.80 ± 1.02 a 4.56 ± 1.07 a *5 days after inoculation, **not significantly different ( )p=0.05 a b c fig 3 inoculation test of and on taro (top), giant taro (middle), and blue taro (bottom) using sliced corm. a, ; b, ; c, non-inoculated f. solani f. oxysporum f.solani f. oxysporum 136 w sidodo and upramana microbiol indones table 2 distribution and composition of species associated with corm rot of taro in bogor, west java indonesia fusarium subdistrict number of samples number of fusarium species recovered f. solani f. oxysporum ciomas sukaraja bogor timur kemang bogor selatan cijeruk ciawi bogor barat darmaga 12 4 2 3 4 4 3 4 4 7 3 2 3 3 3 2 2 3 5 1 0 0 1 1 1 2 1 total isolates 28 12 proportion (%) 70 30 were also pathogenic and caused rotting on either cv. bentul or cv. hijau. mixed inoculation with both species of although tended to show larger rotting on sliced taro corm than single treatment, but was not significantly different (table 3). in the inoculation to whole unsliced taro corm assay, corm rotting were formed after 2 weeks incubation period. corm surface showed water soaking symptom, and when peeled rotting have spread around the inoculation site in 3 mm depth. unfortunately, fusarium the pathogenicity test by inoculation on vegetative propagation of taro did not show any specific above ground symptoms in 3 months after inoculation, however, corms rotting have slightly occurred and the 2 species were reisolated. rotting on sliced giant taro ( ) and blue taro ( ) only occurred when inoculated only by (fig 3). three months after single inoculation with or on taro vegetative propagation, all treated plants did not show any above ground symptoms, but slight rotted on corm was formed. above ground symptomatic also were not formed when both species inoculated onto long yard bean, bean, and some ornamental : chinese evergreen, dumb cane, angel wings, and . although a number of species, including and are commonly known as saprophytes (summerell 2003) from diseased roots and stem bases, our study showed that both species were capable of causing rotting symptoms on sliced taro corm. unfortunately, inoculation assay on vegetative propagation of taro did not show any above ground symptoms after 3 months incubation period. we predicted that, it might need more than 3 months of incubation for the species to initiate the above ground symptoms. in our interview with the farmers during the field survey, the above symptoms could be detected at least 5 months after planting when they use the healthy propagations. generally, is well known as a causal agent of wilt diseases of over 100 cultivated plant species, including tomato, potato, sugarcane, bean, cowpea, date and oil palm, as well as cooking and dessert bananas, but rotting symptom caused by this species was also reported on various plant species, such as rose (barguil 2009), saffron (di primo and cappelli 2000), and amaranths (chen and swart 2001) as also observed in our study. these 2 species were also revealed as causal agents on other plant, calla lily, with dwarfism, wilting, damping off, and tuber dry rotting symptoms (ciampi 2009). meanwhile tuber rot incited by was reported as the most important disease that affects ornamental caladium (knauss 1975). in other root crop such as cassava, numerous and diverse species of were associated with rotted cassava roots, with the 2 largest of the aflp groups corresponds to and (bandyopadhyay 2006). the occurrences of the same disease of taro caused by as in our study have also been reported in hawaii (raabe . 1981). in solomon islands corm fusarium a. macrorhiza x. violaceum f. solani f. oxysporum f. solani fusarium araceae syngobium fusarium f. solani f. oxysporum, et al. fusarium fusarium f. oxysporum et al. araceae et al. fusarium f. solani fusarium f. oxysporum f. solani et al. f. solani et al host range tests. discussion volume 5, 2011 microbiol indones 137 rot of taro have also been reported with various causal agents, including , and (jackson and gollifer 1975), while other researchers identified more specifically, that the causal agents of corm rot on other crop ( ) was f. sp. (nieda 1985) and f. sp. (nishimura and kudo 1994; shibata and aoki 1994). the 2 species of , and , were also recovered from storage cocoyam ( spp.) showing spoilage symptoms in nigeria and have been confirmed as causal agents of the disease in pathogenicity tests (ugwuanyi and obeta 1996), while in cameroon was one of the fungal species associated with the destructive root rot disease of cocoyam (pacumbaba 1992). was also one of the fungal consistently reisolated from the cocoyam rotting tissue arising from inoculation among 2 others fungi, and (maduewesi and onyike 1981). these 2 species were also confirmed have an association with and caused rotting symptom on white yam ( ) during storage (ezeibekwe and ibe 2010). other researcher determined that was reported as one of the causal agents of storage rot of taro in guam (wall and cruz 1991). although the colony between and often appear similar on pda, but these species easily distinguished by the presence of microconidia in chains for and the presence of chlamydospores and microconidia in false heads for as indicated in our study. the consistent recovery 2 species during our preliminary observation by incubating of several diseased corms in moist chamber and pathogenicity tests in this study, we think that those 2 species are the causal agents of taro corm rot disease in bogor. based on host range tests, only was pathogenic to all tested edible , while was more specific to . neither nor isolated from diseased taro caused any symptoms on the ornamental and legume crops tested. it is possible that the associated with taro corm rot might be a specific pathogen to as previously described by nishimura and kudo (1994) as f. sp. . cross inoculation tests of 17 formae speciales of to taro have been conducted by other researcher (nishimura and kudo 1994), and they proposed for the taro isolates as schl. f. sp. nishimura et kudo. other researcher reported that was determined as causal agent of cutting rot disease of and did not show any symptom when pythium myriotylum, p. splendens f. oxysporum araceae amorphophallus f. solani radicicola f. oxysporum colocasiae fusarium f. solani f. oxysporum colocasia f. solani et al. f. solani botryodiplodia theobromae sclerotium rolfsii fusarium dioscorea rotundata f. proliferatum f. proliferatum f. oxysporum f. proliferatum f. oxysporum fusarium f. solani araceae f. oxysporum c. esculenta f. solani f. oxysporum araceae f. oxysporum c. esculenta f. oxysporum colocasiae f. oxysporum f. oxysporum colocasiae f. solani d. maculate inoculated to other members of the family ornamental plants (chase and el-gholl 1982). from this report, it is also possible that the recovered from our research might be a specific pathogen to members of the family edible crops as reported by nieda (1985) on . further inoculation assay on living taro plants are necessary to carry out to clarify this hypothesis. we thank the quality for undergraduate education (que) project, department of plant protection, institut pertanian bogor for research grant support in conducting the studies described in this manuscript. araceae f. solani araceae amorphophallus rivieri acknowledgments references bandyopadhyay r, mwangi m, aigbe so, leslie jf. 2006. species from the cassava root rot complex in west africa. phytopathology 96(6):673-676. doi:10.1094/phyto-96-0673. barguil bm, viana fmp, anjos rm, cardoso je. 2009. first report of dry rot caused on rose ( spp.) in brazil. plant dis 93(7):766. doi: 10.1094/pdis-93-7-0766a. burdani dh, supramana, widodo. 2001. studi lini dasar penyakit busuk umbi pada talas ( ( l.) schott ) di bogor [base line study on the corm rot disease of taro ( ( l.) schott ) in bogor]. bul hama peny 13 (1):6-14. chase ar, el-gholl ne. 1982. stem rot, cutting rot, and leaf spot of 'perfection' incited by . plant dis 66 (7):595-598. doi:10.1094/pd-66-595. chen wq, swart wj. 2001. genetic variation among isolates associated with root rot of in south africa. plant dis 85(11):1076-1080. doi:10.1094/pdis.2001.85.10. 1076. ciampi lp, nissen mj, venegas ge, fuentes pr, costa ml, schöbitz tr, alvarez de, alvarado ap. 2009. identification of two species of link that cause wilting of colored callas ( (l.) spreng) cultivated under greenhouse conditions in chile. chil j agric res 69(4):516-525. doi:10.4067/s071858392009000400006. di primo p, cappelli c. 2000. preliminary characterization of f. sp. causing fusarium corm rot of saffron in italy. plant dis 84(7):806. doi:10.1094/pdis-91-9-1203b. ezeibekwe io, ibe ae. 2010. fungal organisms associated with yam ( poir) rot at owerri, imo state of nigeria. j mol gen 2(1):1-5. doi: 10.3923/jmolegene.2010.1.5. jackson gvh, gollifer de. 1975. disease and pest problems of taro ( l. scott) in the british solomon islands. pans 21:45-53. knauss jf. 1975. description and control of tuber rot of caladium. plant dis rep 59(12):975-979. leslie jf, summerell ba. 2006. the laboratory manual. 1 ed. iowa:blackwell publ, maduewesi jnc, onyike rci. 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(in japanese). nishimura n, kudo k. 1994. f. sp. n. f. sp. causing dry rot of taro ( ). ann phytopathol soc jpn 60(4):448-453. ogaraku ao, osman ho. 2008. storage rot of some yams ( spp.) in keffi and evirons, nasarawa state, nigeria. prod. agric technol 4(2):22-27. pacumbaba rp, wutoh jg, eyango sa, tambong jt, nyochembeng lm. 1992. isolation and pathogenicity of rhizosphere fungi of cocoyam in relation to cocoyam root rot disease. j phytopathol 135(4):265-273. doi:10.1111/j.1439-0434.1992.tb04312.x. raabe, rd, conners, il, martinez, ap. 1981. checklist of plant diseases in hawaii. college of tropical agriculture and human resources, university of hawaii . information text series no. 22. honolulu: trop agric human resources. shibata s, aoki t. 1994. dry rot of konnyaku ( koch) caused by . ann phytopathol soc jpn 60(6):748. (in japanese). summerell ba, salleh b, leslie jf. 2003. a utilization approach to identification. plant dis 87(2):117-127. doi:10.1094/pdis. 2003.87.2.129. ugwuanyi jo, obeta jan. 1996. fungi associated with storage rot of cocoyams ( sp.) in nsukka, nigeria. mycopathologia 134(1):21-25. doi: 10.1007/bf00437048. wall gc, cruz jf. 1991 and causing storage rots of taro in guam. plant dis 75(12):1286. doi:10.1094/pd-75-1286d.. amorphophallus konjac fusarium oxysporum fusarium colocasia lasiodiplodia theobromae fusarium proliferatum 03. tjampakasari.cdr vol.14, no.4, december 2020, p140-148 doi: 10.5454/mi.14.4.3 quality of airborne bacteria in operating theaters in several hospitals in jakarta and its surrounding areas in 2018-2019 1* 2 1 conny riana tjampakasari , nabila naura , and tjahjani mirawati sudiro 1 department of microbiology faculty of medicine university of indonesia, jakarta 10430, indonesia; 2 general physician education program faculty of medicine university of indonesia, jakarta 10430, indonesia. nosocomial infection is an infection obtained by a patient or hospital staff while in hospital. this infection plays a role in causing morbidity and mortality in hospitals and can occur in various hospitals rooms, including operating theaters. nosocomial infections can occur due to various factors, one of which is contamination from airborne bacteria. in some countries, regulations are set to limit the concentration of airborne bacteria, both in the operating theaters and other rooms in hospitals, hence the need for monitoring and supervision of air quality as areflection on the cleanliness conditions in hospitals. based on this, it is necessary to know the bacteriological air quality in the operating theaters in several hospitals in jakarta and surrounding areas as one of the steps to prevent nosocomial infections. the method uses an air sampler with the principle of impaction. air sampler works by separating the particles from the air by utilizing the inertia of the particles to force the bacteria to settle to the surface of the medium. a total of 217 examinations in the operating theaters were carried out in 17 hospitals in jakarta and surrounding areas during january 2018 to june 2019. the majority of the operating theaters in hospitals in jakarta and surrounding areas have air quality that met appropriate quality standards. in 2018, 120 of 137 (87.59%) examination in the operating theaters met the quality standar. meanwhile in 2019, 70 of 80 (87.50%) operating theaters met the standard determined by the ministry of health of the republic of indonesia. key words: air sampler method, operating theater, quality of air bacteria infeksi nosokomial adalah infeksi yang diperoleh selama pasien atau petugas rumah sakit berada di rumah sakit. infeksi ini berperan dalam menyebabkan morbiditas dan mortalitas di rumah sakir dan dapat terjadi di berbagai ruangan rumah sakit, termasuk ruang operasi. infeksi nosokomial dapat terjadi karena berbagai faktor, salah satunya adalah kontaminasi bakteri udara. di beberapa negara, regulasi ditetapkan untuk membatasi konsentrasi bakteri di udara, baik di ruang operasi maupun ruangan lain di rumah sakit, sehingga perlu adanya pemantauan dan pengawasan kualitas udara sebagai refleksi dari kondisi kebersihan di rumah sakit. berdasarkan hal tersebut, maka perlu diketahui kualitas bakteriologis udara di ruang operasi pada beberapa rumah sakit di jakarta dan sekitarnya sebagai salah satu langkah awal pencegahan infeksi nosokomial. metode pada penelitian ini menggunakan air sampler dengan prinsip impaksi. air sampler bekerja dengan cara memisahkan partikel dari udara dengan memanfaatkan inersia partikel untuk memaksa bakteri mengendap di permukaan medium. sebanyak 217 ruang operasi dilakukan pemeriksaan, berasal dari 17 rumah sakit di jakarta dan sekitarnya selama januari 2018 hingga juni 2019. sebagian besar ruang operasi di rumah sakit di jakarta dan sekitarnya memiliki kualitas udara yang memenuhi standar kualitas yang sesuai. pada tahun 2018, 120 dari 137 (87.59%) ruang operasi telah memenuhi standar. sedangkan pada tahun 2019, 70 dari 80 (87.50%) ruang operasi telah memenuhi standar yang ditetapkan oleh kementerian kesehatan republik indonesia. kata kunci: kualitas bakteri udara, metode air sampler, ruang operasi microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-3100810; fax: +6221-3100806 ; email: connyrianat@yahoo.com case was dominated by nosocomial infections (danasekaran and annadurai 2014). an estimated 30% of inpatients in europe have experienced at least one time nosocomial infection. it is known that the average prevalence of nosocomial infections in europe was 7.1% and in the united stetes 4.5% in 2002. this figure has increased significantly in developing countries, where the prevalence of nosocomial infections varies from 5.7% to 19.1%. it is estimated that out 100 inpatients, 7 of whom are invoved in nosocomial infections in developing countries nosocomial infection play a role in causing morbidity and mortality in most hospitals. this infection can occur in various rooms in hospitals, including operating theater as rooms in hospitals with high activity and patients with higher morbidity and mortality than other rooms (denina et al. 2012). according to who, based on a recent study in europe, patients who had infections in reached 51% where the (vincent et al. 2009 and leong et al. 2013). nosocomial infections can occur due to various factors, one of which is contaminationfrom airborne bacteria. in some countries, regulations are set to limit the concentration of airborne bacteria, especially in the operating theaters (eachempati 2017) although nosocomial infections are multifactorial involving patient and procedural factors, airborne bacterial are considered as one of the main source of exogenous contaminant bacteria (rajasekar et al. 2011). air is not a medium in which bacteria grow, but rather substancs that can carry particles, including bacteria. during surgical procedures, airborne particles that can carry bacteria, including textiles fibers, dust, skin fragments and respiratory aerosols can contaminate surgical instruments or directly enter areas of the body that should be sterile, namely surgical wound and cause them to become infected (hansen et al. 2005). from previous studies, it is known that some operating theaters have air quality that does not rfer to established standars, regulation of the minister of health of the republic of indonesia no. 7 of 2019 for the operating theaters and decree of the minister of h e a l t h o f t h e r e p u b l i k o f i n d o n e s i a n o . 1204/menkes/sk/x/2004 (permenkes 2019 and permenkes 2004). wismana (2016) in his research stated that 2 out of 4 operating theaters in undaan eye hospitals in surabaya did not meet the maximum air bacteria concentartions standards. the bacteria that were identified in the room were bacillus sp, grampositive rod bacteria, gram positive coccus bacteria, gram negative rod bacteria and acinetobacter sp which is pathogenic and also fungi (rachel et al. 2014). various attempts were made to prevent nosocomial infections due to airborne bacterial contamination through dust and droplets, such as the use af antiseptics, disinfectant through cleaning and sterilization and isolation of patients with highly infectious diseases (zaidi et al. 1995). however, maintenance of air quality is not good carried out routinely can be contributing factor in the in the occurence of nosocomial infections. ishida et al. (2006) report that airborne bacteria in the hospital environment have become a major source and serious problem of postoperative infections.many of these air bacteria are known to be resistant to antiseptics that are commonly used in many hospitals (landrin et al. 2005). therefore, there is a need reserach conducted to determine bacteriological air quality in the operating theaters as a room in a hospital that requires sterile conditions (yagoub and agbash 2010). materials and methods medium. nutrient agar are used to breed airborne bacteria. medium was prepared by using oxoid medium consists of lab lemco powder 1.0 gr, yeast ektraxt 2.0 gr, peptone 5.0 gr, sodium chloride 5.0 gr and agar 15 gr. the whole weighed 22.4 gr in 800 ml water then the sterilization process is carried out according to procedure (the oxoid 2006). examination airborne bacteria. the tools used for this research is air sampler (merck) with the principle of impaction. the air sampler works by separating the particles from the air by utilizing the inertia of the particles to force them settle to the surface of the medium (yagoub et al. 2010 and opertaor mas 100 2019). the first thing to do is determine the volume of the room to be inspected. the number of points for air sampling per room is 10% of the volume of the room. at each sample point 1 liter of air was taken for 10 minutes (stetzenbach 2015). at each point used four nutrient agar media (difco and bbl 2018). incubate and count colonies. the nutrients were o incubated in incubator (thermo) at 35 c for 18-24 h. the number of bacterial colonies formed was calculated using a colony counter (wtw bzg 30) and was converted to conversion table (opertaor mas 100 2019). the average value of each sample point examined was expressed as the number of airborne bacteria in a room being inspected (mara et al. 2018) the result are then compared with the criteria for the maximum number of colonies according to minister of health regulation no. 1204 of 2004 by the ministry of health and regulation of the minister of health of the republic of indonesia no. 7 of 2019. results from january 2018 to june 2019, 217 airborne bacteria were examined in the operating theater at 17 hospitals in jakarta, west java and banten. the hospitals involved in this research were a hospital , b, d, e, h, i, j, k, n and p in jakarta, d hospital, g, m and o in banten and c hospital, f, l and q in west java. the examination result in each room are divided 3 3 into several groups of air bacteria per m (cfu/m ) 3 3 which are 0-10 cfu/m , 11-35 cfu/m , 36-100 3 3 3 cfu/m , 101-180 cfu/m , 181-300 cfu/m and >300 cfu/m. in 2018, 102 out of 137 examinations are 0-10 3 3 cfu/m , 18 examination are 11-35 cfu/m , 7 3 examination are 36-100/m , 4 examination are 1013 3 180 cfu/m , 1 examination are 181-300 cfu/m and 5 volume 14, 2020 microbiol indones 141 142 tjampakasari et al. microbiol indones 3 examination are >300 cfu/m (table 1). in 2019, 60 3 out of 80 examinations are 0-10 cfu/m , 10 3 examination are 11-35 cfu/m , 5 examination are 363 3 100/m , 2 examination are 101-180 cfu/m , 1 3 examination are 181-300 cfu/m and 2 examination 3 are >300 cfu/m . the overall results these years can be seen in table 1. the result of the study in 2018, showed that, the number of air bacteria in 120 operating theaters 3 3. (87.59%) was in 0-10 cfu/m and 11-35 cfu/m these result meet the maximum concentration of airborne bacteria determined by the ministry of health of the republic of indonesia in the ministry of health regulation of the republic of indonesia 2019 no.7. in 2019, this figure is not much different, around 87.50 % of operating theaters are in the same group as the previous year. an aexample of the results of an examination of air bacteria can bee seen in fig 1. the study was also conducted to compare the average concentration of airborne bacteria in several operating theaters in several hsopitals for time to time. the hospitals are a, b, c, d, e, g, j, k, n and p. comparison cannot be done in other hospitals for hospitals f, h, i, l, m, o and q because data collection was only carried out once in the hospital in the period january 2018june 2019. during januari 2018-june 2019 of the 10 hospitals examined only 3 had an average concentration of airborne bacteria thet never exceed the ministry oh 3 health's republic indonesia (35 cfu/m ) standard, that are a hospital, b and d. the average concentration 3 of air bacteria in a hospital is 5.58 cfu/m (table 2). overall, the average monthly inspection results meet the quality standard. the average airborne bacterial concentartion of b 3 hospital was 6.19 cfu/m (table 3). in general, the average monthly examination results meet the quality standard. it was reported thet in april, the process of cleaning the room, especially the air conditioner and hepa filter was carried out routinely according to applicable procedures. this condition is considered as one of the main factors thet play role in maintaining bacteriological air quality in the operating theaters at this hospital. the average airborne bacterial concentration of d 3 hospital was 15.5 cfu/m (table 4). during 20182019, four examinations were recorded and all of them met quality standard. however, it was noted that there was only one operating theaters examined at each examination so that the bacterial air quality of the entire operating theaters could not be conclude. the average conentration of air bacteria in hospitals that did not meet the indonesian ministry of 3 health's standards was 67 cfu/m at c hospital (table 5). the results showed that the average concentration of air bacteria in each examination did not meet the quality standard. besides that the hospital that did not meet the other standards was e hospital with an average number of 3 airborne bacteria 69.75 cfu/m (table 6). examination at e hospital were only carried out during 2018 four times, two of which were carried out in march. in march, two examination results showed that the concentration of airborne bacteria did not meet the fig 1 various culture result of examination of air bacteria on nutrient agar with air sample method. a. the number 3 3 of bacteria 0-10 cfu/m . b. the number of bacteria 11-35 cfu/m . c. the number of bacteria 36-100 3 3 3 cfu/m . d.the number of bacteria 101-180 cfu/m . e. the number of bacteria 181-300 cfu/m . f.the 3 number of bacteria >300 cfu/m . v o lu m e 1 4 , 2 0 2 0 m ic ro b io l in d o n es 1 4 3 operating theaters consentration of air bacteria (cfu/m3) jan 18 feb 18 m arch 18 apr 18 m ay 18 jul 18 augt 18 sep 18 oct 18 nov 18 des 18 jan 19(1) jan 19 (2) feb 19 m arch 19 apr 19 m ay 19 june 19 1 10 2 5 7 2 2 10 16 9 3 2 23 7 4 2 6 3 2 2 9 5 4 19 2 2 9 7 6 3 18 26 6 2 3 3 7 3 5 3 8 4 3 3 4 17 7 4 4 22 3 2 4 2 2 4 6 3 5 2 2 3 4 5 3 2 3 3 3 2 2 3 5 3 3 6 6 3 2 6 6 10 3 6 23 5 2 3 3 2 6 10 7 8 10 3 3 24 10 2 2 3 3 2 3 7 4 average 7.17 3.83 6.0 8.0 2.5 2.5 9.5 10.17 6.17 2.83 6,0 16.67 5.25 2.5 2 3.5 3.33 3.33 5.58 table 1 result of air bacteria examination in operating theaters in various hospitals in jakarta surrounding areas in january 2018-desember 2018 and january-june 2019 table 2 result of air bacteria examination in the operating theaters in a hospital in january 2018-june 2019 table 3 result of air bacteria examination in the operating theaters in b hospital in january 2018-june 2019 operating theaters consentration of air bacteria (cfu/m3) jan 18 feb 18 (1) feb 18 (2) march 18 apr 18 may 18 jul 18 augt 18 oct 18 nov 18 des 18 jan 19 feb 19 m arch 19 apr 19 m ay 19 june 19 1 8 30 9 2 3 3 3 4 8 4 5 7 5 3 4 6 10 2 4 32 3 13 4 3 7 2 8 5 4 5 3 5 3 3 4 3 8 21 15 10 6 4 3 6 7 6 3 5 3 4 12 3 4 4 3 16 4 10 6 3 7 4 3 2 3 3 3 2 18 5 5 average 5.75 24.75 7.75 8.75 4.75 3.25 5 4 6.5 4.25 3.75 5 3.5 3.5 9.25 4.25 6 6.19 3 3 amount of air bacteria per m of air (cfu/m ) number of examination (n=137) number of examination (n=80) 0-10 11-35 36-100 101-180 181-300 >300 102 (74.45%) 18 (13.14%) 7 (5.11%) 4 (2.92%) 1 (0.73%) 5 (3.65%) 60 (75%) 10 (12.50%) 5 (6.25%) 2 (2.50%) 1 (1.25%) 2 (2.50%) table 4 result of air bacteria examination in the operating theaters in d hospital in 2018-2019 table 5 result of air bacteria examination in the operating theaters in c hospital in 2018-2019 table 6 result of air bacteria examination in the operating theaters in e hospital in 2018 quality standard, but in subsequent examinations the average concentration of airborne bacteria dropped significantly so that it met the quality standard. hospitals that do not meet the other standards are 3 g hospital with an average number 410.5 cfu/m (table 7). the examination was carried out once in aech year in 2018 and 2019. the results of the first examination showed thet the air bacteria was far above 3 the quality standard which is 730 cfu/m , while the results of the second examination showed a very 3 significant decrease to 91 cfu/m , although these results still do not meet quality standards. j hospital has an average of bacteria was 147.33 3 cfu/m (table 8). the examination was carried out in 2018 only. the examination showed a significant decrease concentrations from august to september, 3 3 which was 353 cfu/m to 46 cfu/m . the 3 concentrations decreased again to 43 cfu/m at subsequent in october. however, the three examonations did not show result that met the quality standard. 3 in k hospital the average was 54 cfu/m (table 9). the results showed that did not meet the quality standard but the second showed an improvement with the average that met quality standard. 3 n hospital has an average 39.25 cfu/m (table 10). the concentration at the first examination was 30 3 cfu/m , but at a subsequent an increase concentration 3 to 48.5 cfu/m . the next was p hospital with average of 309.33 3 cfu/m (table 11). all examinations showes taht did not meet quality standard. based on monitoring, the process of cleaning has been done routinely in accordance with procedures, but the tools in the room are very dense and not neatly arranged. discussion distribution of concentration into 4 groups which 3 3 3 are 0-10 cfu/m , 11-35 cfu/m , 36-100 cfu/m , 3 3 101-180 cfu/m , 101-180 cfu cfu/m , 181-300 3 3 cfu/m and >300 cfu/m are based ondecree of the minister of health of the republic of indonesia no. 1204/menkes/sk/x/2004 and regulation of the minister of health of the republic 2019 no. 7, the 3 standard quality in the operating theater is 35 cfu/m . the media used in this study was nutrient agar media. it is a comman medium that contains s source of nitrogen, vitamins and carbohydrates, used to grow various species of gram positive and negative bacteria and even fungi can grow well in this medium. in this study, the dominant bacteria found were bacillus sp, staphylococcus aureus and staphylococcus epidermidis while aspergillus sp was the dominant group of fungi found. these microbes that are most commonly found in various rooms in hospitals and even communities 3 concentration of air bacteria (cfu/m ) 1 2 march 18 may 18 nov 18 jan 19 13 operating theaters 13 6 6 12 12 31 31 15.5 average 3 concentration of air bacteria (cfu/m ) 1 2 march 18 august 18 april 19 72 operating theaters 72 63 63 66 66 67 average 3 concentration of air bacteria (cfu/m ) 1 2 march 18 may 18 jul 18 91 298.5 operating theaters 15 2 194.75 3 2.5 9 12 69.75 average 144 tjampakasari et al. microbiol indones (kandy et al. 2019 and leong et al. 2013). the discovery of these microbes shows that the room being examined needs to be carried out a more intensive sterilization process. during the examination (2018), regulation no. 7 of 2019 has not yet been issued so that so that the standard still refers to the decree of no. 1204 of 2004. based on these regulations, the maximum quality standarad is 10 3 cfu/m , so that the examination that meets the quality 3 standard in 2018 is only the 0-10 cfu/m group, which is 74.45%. however, in general the examinations have shown good results. hospitals recorded with a concentration that did not exceed the standard quality were a hospital, b, and d. in the d hospital only one operating theater was eximed at ehamination so that it could be it is considered for the hospital to examine the entire operating theater to obtain more conclusive examination results. each hospital is equipped with a heating, ventilation and air conditioning (hvac) system that has a highefficiency particulate air filter (hepa filter) as one of its componenet. hvac is an air conditionng systems that plays a role in maintaining the appropriate warmth and air quality in the room. it is works based on mechanical engineering subdiscipline by involving the principles of thermodynamics, fluid mechanics and heat transfer. table 7 result of air bacteria examination in the operating theaters in g hospital in 2018-2019 table 8 result of air bacteria examination in the operating theaters in h hospital in 2018 table 9 result of air bacteria examination in the operating theaters in k hospital in 2018 2019 table 10 result of air bacteria examination in the operating theaters in n hospital in 2019 table 11 result of air bacteria examination in the operating theaters in p hospital in 2019 3 concentration of air bacteria (cfu/m ) 1 march 18 may 18 730 operating theaters 91 730 91 410.5 average 3 concentration of air bacteria (cfu/m ) 1 augt 18 oct 18 353 operating theaters 43 353 43 147.33 average sep 18 46 46 3 concentration of air bacteria (cfu/m ) 1 march 19 apr 19 (2) 547 operating theaters 157 547 157 309.33 average apr 19 (1) 224 224 3 concentration of air bacteria (cfu/m ) 1 oct 18 march 18 105 operating theaters 3 105 3 54 average 3 concentration of air bacteria (cfu/m ) 1 jan 19 feb 19 35 operating theaters 57 30 48.5 39.25 average 2 25 40 volume 14, 2020 microbiol indones 145 hepa filters play a role in filtering air very efficiently through its use in areas that need contamination control. however, it cannot filter odorous gases and molecules. hvacs equipped with hepa filters are important in maintaining air conditioning systems in hospitals as areas that require sterile conditions but it is known that often hvca and hepa filters are contaminated so that the contaminated air also flows into the room. although every examination is equipped with an advise and input, suspected that not all hospitals do what is recommended to reduce the concentrationof airborne as happened to c hospital on february 2018, july 2018 and april 2019; e hospital on march 2018, g hospital on mei 2018 and march 2019, j hospital on august 2018, september 2018 and october 2018; k hospital on october 2018, n hospital on february 2019 and p hospital on march 2019 and april 2019. based on a study conducted by liu et al (2005), bacteriological contamination with hvac significantly higher compared to natural air ventilitaion (vua). the use of hvac which aims to maintain the air system can cause bacteriological contamination, because the hvac system is an efficient location as a place for bacterial colonization when cleaning is not done routinely (kandy et al. 2019). this finding was also reported by guo et al (2003). it is known that thi air filtration technique has a mechanism that should play a significant role in protecting air from bacterial contamination so that it is necessary to carry out regular cleaning of hvac and hepa filters to maintain good air quality (kemenkes 2012). based on observations, hospitals that have decreased concentrations of airborne bacteria such as c hospital, e, g, j, s, k, s, n and p had carried out extensive cleaning involvong sterilization of equipment, cleaning of hvac and hepa filters, lamp replacement and the use of hypochlorite as disinfectabts that are known to be most effective in eradicating microorganisms. in g hospital, the average concentration of airborne bacteria in the theaters room reaches 730 3 3 cfu/m on may 2018 and in p hospital 547 cfu/m on 3 march 2019, 224 cfu/on april 2019 and 157 cfu/m on the second examination in april 2019. it should be noted that the steps to clean the theaters room must be done more comprehensively because of the high risk of nosocomial infection. a similar study conducted by hailemariam et al (2016) in etiopia showed that the average concentration 2 in the passive and active 13.12 cfu/dm and 87.27 2 cfu/dm respectively. considered acceptable based on the fisfer index in this study, where the active theaters room quality standard is devided into 3 group, such as 2 2 optimal (0-60 cfu/dm ), acceptable (6190 cfu/dm ) 2 and unacceptable (>91 cfu/dm ), while the findings in the passive theaters room are considered unacceptable 2 based on standard quality to be optimal (0-4 cfu/dm ), 2 acceptable (5-8 cfu/dm ) and unacceptable (>9 2 cfu/dm ) (pasquarella et al. 2000). similar results were found in a study conducted in nothern etiopia by gebremariam kibrom (2015), 2 where the concentration was 91.8 cfu/dm in the active 2 theaters room and 17.2 cfu/dm in the passive theaters room, but in this study accordingly, the concentration of airborne bacteria in the active theaters room slightly more higher than the fisher index. the average concentration was known to be highest in the morning, 63.3% of the samples belong to unacceptable group. in the afternoon 45% of the samples belong to unacceptable. this is allegedly because in general, the morning is the time with the highest activity, such as changing patient clothes, bathing patients, changing diapers and gicing medicine. in general, 60% of the theaters room examines do not have good bacteriological air quality (sulistiyo et al. 2017). another study conducted by sulistiyo et al (2017) reported that the concentration of airborne bacteria in the theaters room at rsud tugurejo semarang was 3 54.57 cfu/m , where this number did not meet quality standars. after sterilization, the concentration of 3 airborne bacteria decrease to 24 cfu/m , however this number still does notmeet quality standards (hailemariam et al. 2016). the majority of theaters room in hospitals jakarta, west java and banten have met air quality standards. in 2018, 87.59% of bacterial air quality examination in the theaters room meet the quality standars whereas in 2019, 87.50% of examination in the theaters room meet the quality standars. it can be concluded that hospitals in jakarta and surrounding areas have maintained goos procedures in mainstaining sanitation and monitoring air quality in the theaters room. acknowledgements the authors thank to clinical microbiology laboratory fkui-rscm, department of microbiology fkui-rscm for the collection of air samples during the field studies. 146 tjampakasari et al. microbiol indones references danasekaran gmr and annadurai k. 2014. prevention of healthcare-associated infections: protecting patients, saving lives. int j community med public health. 1(1):67-8. doi: org/10.1016/j.jhin.2013.10.007.18. denina h, jing q, william wn, naomichi y, kyle b, hamid ry and jordan p. 2012. human cccupancy as a source of indoor airborne bacteria. plos one.17(4):1-10. doi:10.1371/journal.pone.0034867. difco and bbl manual of microbiological culture media. 2009. 2nd edition. maryland : becton, dickinson and company. eachempati sr. 2017. airborne bacteria in the operating 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disinfection and sterilization practices in mexico. j hosp infect. 31(1):25-32. doi: 10.1016/01956701(95)90080-2 148 tjampakasari et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 6 rarah ratih adjie.pmd stability of two probiotics bacteria of goat milk yoghurt in rat digestive tract rarah ratih adjie maheswari1*, i komang gede wiryawan2 and gina lesmana maduningsih1 1department of animal production and technology; 2department of animal nutrition and feed technology, institut pertanian bogor, darmaga campus, bogor16680, indonesia increased age will affect the bacterial population of the human digestive tract, in which many bacteria will adapt and colonize different sites. combining probiotics and prebiotics in what has been called a synbiotic could beneficially affect the host by improving survival and implantation/adhesion of live microbial dietary supplements in the gastrointestinal flora. probiotic bacteria are expected to survive in the digestive tract to give health effects to the host by balancing the intestinal microflora. the addition of fructo-oligosaccharides is expected to increase viability and growth of probiotics in the digestive tract. the objective of the current research is to investigate the effect of probiotic yoghurt and synbiotic yoghurt of etawa breed cross saanen (pesa) goats together with fructo-oligosaccharides, on female rats and to study the stability of probiotic bacteria in the digestive tracts. the results showed that synbiotic yoghurt intake had no significant influence (p>0.05) on ration consumption, body weight gain, feed conversion, final body weight and mortality. the synbiotic intake significantly increased the population of bifidobacterium longum (p<0.01) and lactobacillus acidophilus (p<0.05), but in contrast decreased (p<0.05) the population of escherichia coli. the yoghurt synbiotic treatment over 14 days influenced probiotic bacteria’s stability as shown by the reduced population of b. longum and l. acidophilus. key words: probiotic, prebiotic, yoghurt, digestive tract, synbiotic _____________________________________________ volume 2, number 3, december 2008 issn 1978-3477 p 124-130 ________________________ * corresponding author, phone/fax: +62-21-86283789, e-mail: rarah_maheswari@yahoo.co.id increased age will affect the bacterial population of the human digestive tract, in which many bacteria will adapt and colonize different sites. a probiotic is defined classically as a viable microbial dietary supplement that beneficially affects the host through its effects in the intestinal tract. this definition, however, was initially intended for use with animal feed. for human nutrition, the following definition has been proposed: “a live microbial food ingredient that is beneficial to health” (salminen et al. 1998). probiotic microorganisms that have a favorable influence on physiological and pathological processes of the host by their effect on the intestinal flora may play a role in improving human health (erickson and neil 2000). some evidence suggests a role for probiotics in reducing the risk of rotavirus-induced diarrhea and colon cancer. combining probiotics and prebiotics in what has been called a synbiotic could beneficially affect the host by improving survival and implantation of live microbial dietary supplements in the gastrointestinal flora. this takes place by selectively stimulating the growth, or activating, the catabolism of one, or a limited number of health-promoting bacteria in the intestinal tract, and by improving the gastrointestinal tract’s microbial balance (gibson and roberfroid 1995). human in vivo trials have established that the addition of fructo-oligosaccharides (fos) or inulin to the diet leads to an increase in bifidobacteria (gibson et al. 1995; bouhnik et al. 1996; bouhnik et al. 1999; kolida et al. 2002). several studies have described fermentation in vitro of fos in pure cultures of bifidobacterium (mckellar and modler 1989; yamazaki and dilawri 1990; wang and gibson 1993; gibson and wang 1994; hopkins et al. 1998; kaplan and hutkins 2000; perrin et al. 2001; palframan et al. 2003). the combination of probiotics and prebiotics in a synbiotic might improve the survival of the bacteria crossing the upper part of the gastrointestinal tract, thereby enhancing their effects in the large bowel. in addition, their effects might be either additive or synergistic. the bifidobacteria and lactobacilli have been extensively studied and considered as valuable native inhabitants of the colon. however, the formulation of a synbiotic using probiotics from local collections or made from goat milk have not yet been investigated. the criteria established to select suitable bifidobacterium and lactobacillus strains and to identify which strains posessing the desired traits are also the subject of many studies. several specific characteristics possessed by these bacteria are thought to be either desirable or necessary. probiotic bacteria should resist low ph and survive gastric acidity, bile salts at concentrations present in the intestinal tract, be able to adhere to intestinal mucosal cells and provide clinically proven benefits. recently, it has been suggested that the ability of probiotic bacteria to ferment oligosaccharides may be an especially important characteristic (gibson and roberfroid 1995; collins and gibson 1999). this is because of the availability of carbohydrates that escape metabolism and adsorption in the small intestine and then have a major influence on the microbiota that become established in the colon. razafindrakoto et al. (1994) showed that goat’s milk has equivalent nutritional value to cow’s milk. goat’s milk can be used for undernourished children as an alternative to cow’s milk. it may used at home for the prevention of malnutrition. treatment with goat’s milk in these experiments may be used to increase body weight of rats caused by easy absorption. also it can increase therapheutic effect of synbiotic products. the objective of this research is to investigate the effect of probiotic and synbiotic yoghurt of etawa breed cross saanen (pesa) goats as well as fos, on female rats and to study the stability of probiotic bacteria in the digestive tracts. female rats were used in this study, mainly because of their capability to have a high stress character, so the effect of probiotic and prebiotic expected to be determined easily. materials and methods bacteria strains. the organisms used in this study include 4 strains, l. acidophilus rrm-01 (la-rrm01) and b. longum rrm-01 (bl-rrm01) as probiotic bacteria, l. bulgaricus rrm-01 (lb-rrm01) and streptococcus thermophillus rrm-01 (st-rrm01) as yoghurt culture starters. the organisms were obtained from the division of dairy production collection at faculty of animal science, bogor agricultural university. preparation of milk base. fresh raw pesa goats milk was purchased from the pt fajar taurus dairy farm, and skimmed to 0.4-1% (v/v) fat using a cream separator. milk was divided into 3 batches and heated 115°c for 3 min. preparation of probiotic yoghurt and synbiotic yoghurt. the sterilized milk bases were cooled to incubation temperature, and batches of each milk base were inoculated using 5% (v/v) mixed lactic starter (s. thermophilus and l. bulgaricus, 1:1) (yoghurt), 5% (v/v) l. acidophilus starter (acidophilus milk) and 5% (v/v) b. longum starter (bifidus milk) and incubated at 37°c for 20 h. probiotic yoghurt was made by mixing yoghurt, acidophillus milk and bifidus milk (2:1:1) and synbiotic yoghurt was made from probiotic yoghurt fortified with 0.9% (w/v) fructo-oligosaccharides (cosucra, belgium). probiotic and synbiotic yoghurts were aseptically packed and stored at 4±1°c. animals. fifteen weaned female sprague dawley rats (rattus novergicus), 21 days old, with an average body weight range of 30.40-30.92 g were used. the rats were kept in the laboratory of breeding and genetics, department of animal production and technology, faculty of animal science ipb. the rats were fed with pelleted starter broiler 511. experimental design. nine rats were randomly divided into 3 treatment groups with 3 replications of each. the treatments consisted of feeding with probiotic yoghurt of goat milk, r1; synbiotic yoghurt of goat milk, r2; and water (control), r3. the treatments were given orally at 0.03% (v/w) of rat body weight with initial bacterial population of 9.25 log cfu ml-1 for l. acidophilus and 9.66 log cfu ml-1 for b. longum. observations were conducted over 42 days. observations were also conducted on 6 rats (excluding r1, r2, r3) which are divided into two treatments, i.e. 3 rats adapted with broiler 511 pellets feed for 7 days to obtain an initial population of microorganisms, with the other treatment with synbiotic yoghurt of goat’s milk for 42 days, after which treatment was stopped (withdrawal) for 14 days. the withdrawal-test of synbiotic yoghurt treatment was aimed at investigating its influence on probiotic bacterial population in the colon, consumption, body weight gain and feed conversion. adaptation. all rats were given pelleted starter broiler diet for 7 days and then each groups of rats for appropriate treatment were given appropriate diets (water, prebiotic yoghurt or symbiotic yoghurt) for 7 days prior to the main experiment. sampling. faeces were obtained by placing the rats into sterile cages without rice hull bedding in the morning to allow the rats to defecate naturally. analysis of faeces sample was conducted 2 weeks before each experiment was started and then 2, 4 and 6 weeks during each treatment for the first 3 groups of rats, and 2, 4, 6 and 8 weeks for rats with other treatments. rats colon were removed surgically after weighing in the 6th weeks of treatment for the 3 groups of treated rats (main treatment) and in the 8th week after treatment for the two other groups of rat with other treatments by placing the rats in a glass box containing cotton with 90% (w/v) chloroform for 3 minutes. preparation of samples. one gram of fresh faeces was suspended in 9 ml phosphate buffer and bacterial population (l. acidophilus, b. longum and e. coli) were estimated. statistical analysis. data were analyzed in a completely randomized design with 3 treatments and 3 replications of each treatment. further analysis for significantly difference was conducted using tukey test. parameters observed. the parameters observed were: (i) consumption of feed dry matter (g/head/day), calculated by substracting dry matter of the ration offered with dry matter of the remaining ration; (ii) body weight gain (g/head/ day), obtained by substracting the final body weight from the initial body weight and dividing by the number days of the experiment; (iii) the value of feed conversion, expressed by the ratio between daily dry matter consumption and daily body weight gain; (iv) final body weight (g/head), obtained by weighing the rats at the end of the experiment; (v) mortality rate, expressed by the number of rats that died during the experiment. l. acidophilus enumerated using mrs-im agar medium with maltose addition, whereas b. longum was enumerated using mrs-im agar medium with addition of 20% glucose, 10% dichloxallin solution, 10% licl and cystein hcl. one gram of each sampel was serially diluted 10 times in sterile buffer peptone water. enumeration was carried out using the pour plate technique. suspended samples that had been diluted in phosphate buffer were cultured on eosin methylene blue agar (emba) and incubated at 35-37°c for 24 h. colonies that gave metallic green color were identified as e. coli. results dry matter consumption, body weight gain, final body weight, feed conversion and rat mortality. the effects of the fermented milk on dry-matter consumption, body weight gain, final body weight and feed conversion are presented in table 1 and 2. the mean dry matter consumption of the yoghurt treatment group was lower than in the control and the mean body weight gain and final body weight of the yoghurt treatment group was slightly higher than those of the control, but neither difference was significant (p<0.05). although statistically the effect of treatment was not significant on feed conversion (p<0.05), we consider feed conversion of the yoghurt treatment to be a better than control because the mean of the feed conversion for the yoghurt treatment was lower than for its control. no rat mortality was observed during the experiment. this is volume 2, 2008 microbiol indones 125 because of the good environmental conditions that are suitable for the animals. population of fecal flora . the population of the bacterial flora in the faeces obtained during the control and the yoghurt treatment is shown in fig 1-3. the presence of b. longum and l. acidophilus in the faeces during these experiments was variable. the population of e. coli in the control treatment increased during the first 28 days. however, a significant decrease occurred at day 42. in contrast, constant population of e. coli was obtained in faeces of prebiotic-treated rats, while in the synbiotic yoghurt-treated rats the population of e. coli tended to decrease over 42 days. the population of e. coli in synbiotic-treated rats decreased by 1.12 log cfu g-1. the growth curve of e. coli is shown in fig 3. population of colon flora. the population of b. longum in the colon varied between 8.64-12.20 log cfu g-1. differences in the increase of b. longum population in the colon was found in all of the treated-samples and in the control. however an analysis of variance shows that pesa goat milkbased synbiotic yoghurt feeding significantly influenced the population of b. longum in the colon (p<0.01). variance analysis showed, an increase in the population of l. acidophilus was significant for the synbiotic-yoghurt intake (p<0.05). the population of e. coli in the colon in all of the treatments varied between 5.40-8.34 log cfu g-1. variance analysis showed that the synbiotic yoghurt-treated significantly decreased the population of e. coli (p<0.05). discussion effect of probiotic and synbiotic yoghurt treatments. feed consumption of rats fed with probiotic yoghurt and synbiotic yoghurt was lower than in their control. previous studies have shown that, the food consumption of the goat’s milk-based diet was lower than that for a normal diet (aliaga et al. 2003). the type of diet given influences food intake, the goat’s milk diet is consumed in smaller volumes. this might be caused by the special organoleptic characteristics of goat’s milk, which have an intense aroma and a strong flavor and a slightly salty taste (jandal 1996). despite the lower food intake in rats given the goat’s milk yoghurt diet, the body weight gain and final weight gain of rats treated table 1 the effect of probiotic yoghurt and synbiotic yoghurt treatments on consumption, body weight gain, feed conversion, and final body weight of rats treatments probiotic yoghurt synbiotic yoghurt 9.26 ± 0.77 2.74 ± 0.67 3.47 ± 0.57 181.90 ± 35.60 parameters control dry matter consumption (g/head/day) body weight gain (g/head/day) feed conversion final body weight (g) 11.14 ± 0.48 1.95 ± 0.73 6.27 ± 2.24 144.60 ± 28.39 10.64 ± 0.91 2.68 ± 0.12 3.98 ± 0.49 178.00 ± 10.44 table 2 response of synbiotic yoghurt treatment and without synbiotic yoghurt treatment parameters adaptation period without treatment for 14 days (post treatment) dry matter consumption (g/head/day) body weight gain (g/head/day) feed conversion 12.67 ± 1.05 2.98 ± 0.15 4.25 ± 0.36 12.00 ± 1.09 2.91 ± 0.67 4.33 ± 1.34 1 2 1 0 8 6 4 2 0 treatment time (days) 0 1 4 2 8 4 2 5 6 fig 3 effect of probiotic yoghurt( ), synbiotic yoghurt ( ) and control/water (n), without treatment after for 14 days ( ) on escerichia coli population in faeces. l . a c id o p h il lu s p o p u la ti o n (l o g c fu g -1 ) treatment time (days) l . a c id o p h il lu s p o p u la ti o n (l o g c fu g -1 ) 1 2 1 0 8 6 4 2 0 0 1 4 2 8 4 2 5 6 fig 2 the effect of probiotic yoghurt ( ), synbiotic yoghurt ( ) and control/water (n ), and without treatment after 14 days ( ) on lactobacillus acidophillus population in faeces. b . l o n g u m p o p u la ti o n (l o g c fu g -1 ) 1 0 9 . 5 9 8 . 5 8 0 1 4 2 8 4 2 5 6 treatment time (days) fig 1 the effect of probiotic yoghurt ( ) , synbiotic yoghurt ( ), control/water (n ) and without treatment after 14 days ( ) on bifidobacterium longum population in faeces. 126 maheswari et al. microbiol indones with goat’s milk yoghurt was slightly higher than those for the control. this fact is explained as the goat’s milk yoghurt diet have a greater food conversion efficiency due to the nutrient content of goat’s milk. aliaga et al. (2003) showed use of the goat’s milk diet led to a better food conversion efficiency with no significant differences in weight gain. the probiotic bacterias content in probiotic yoghurt and synbiotic yoghurt may influence feed consumption, body weight gain, feed conversion and final body weight. it is reported that l. acidophilus is able to increase digestibility and the utilization of protein (jin et al. 1997). the same results were obtained by mcdonough et al. (2006). they reported that rats treated with yoghurt had a higher body weight gain compared to rats given a non-fermented milk diet and that feed efficiencies for yoghurt diets were significantly higher than were efficiencies for milk diets. the composition of synbiotic yoghurt enriched with l. acidophilus and b. longum, as well as fos, strongly supported the rat growth. the fos prebiotic is used as a nutrient by l. acidophilus and b. longum for their optimum growth in the digestive tract, whereas l. acidophilus and b. longum produce metabolites beneficial to the host. these metabolite products can be used to form or increase the size of new cells which will influence body weight gain. mortality refers to the number of dead rats caused by a treatment. zero mortality is plausible since the enviromental coniditions satisfy all rat requirements. population of fecal flora. besides their desired health and clinical properties, probiotics must meet several basic requirements for the development of marketable products. the most important requirements are that probiotic bacteria survive in sufficient numbers in the product, that their physical and genetic stability during storage of the product are guaranteed, and that all of their properties essential for expressing their health benefits after consumption are maintained during manufacture and storage of the product (heller 2001). to ensure a probiotic health effect, bacterial cells must survive in intestinal passage and establish themselves in the terminal ileum or in the large intestine in sufficient numbers. this research shows high survival rates for b. longum and l. acidophilus in faeces. the growth curve of b. longum in faeces samples during our experiments behaves as a fluctuative curve (fig 1). the same result was reported by droault et al. (1999), stating that the presence of various antimicrobial peptides secreted by digestive tract of rats might play an important role to the bacterial viability inside of digestive tract itself. antimicrobial peptides disturb permeability of the cell membrane leading to change transmembrane transport by create such hole inside. this reason gave for explaining the differences of microbial content in the faeces of rats. although there were no significant differences in fecal bacterial counts, the bifidobacteria and l. acidophilus counts increased after ingestion of either the probiotic or the synbiotic yoghurt. even though feeding of yoghurt was terminated after 14 days, population of b. longum in the rat’s faeces still increased. fuller (1989) stated that b. longum tend to adhere better to the digestive tract and forms which compete with pathogenic bacteria to obtain nutricional sources and colonization. other studies showed that, l. bulgaricus and s. thermophilus might acted as bifidogenic factors on the resident colonic flora (bartram et al. 1994). it has also been shown that bifidobacteria survived the passage through the uppergastrointestinal tract after oral feeding (poschart et al. 1992). the consumption of a prebiotic regulary promotes the composition of colon microbes, therefore bifidobacteria is more likely to be viable in the colon and prominent in the faeces (gibson and roberfroid 1995). this result indicates that yoghurt consumption (in both probiotic and synbiotic forms) affects the colonic environment in a way that favors colonization with b. longum and l. acidophilus. the addition of fos may have supported the growth of b. longum and l. acidophilus. fos are not degraded in the upper gastrointestinal tract by the enzymatic digestive system, but are easily metabolized by b. longum and l. acidophilus as carbon sources. the fermentation of fos by bifidobacteria leads to the production of various organic acids. formic acid was detected at low concentration in glucose and fructose fermentations. the main products of fermentations are three carbon sources as such lactic and acetic acids (shene et al. 2005). in its competition with pathogenic bacteria, b. longum was reported to have the ability to prevent membrane disruption by pathogenic bacteria. this ability may be delivered optimally in certain times and under appropriate condition (davidson et al. 2000). the population of b. longum reached 9.91 log cfu g-1, which can be considered in the range of a probiotic population capable of promoting human health. because the minimum suggested level of viable bifidobacterium cells at the time of consumption is approximately 107 cfu g-1 of product, and the suggested daily intake is approximately 109 viable cells (around 100 g of product per day) (robinson 1987). taken together, we conclude that terminating yoghurt after 14 days did not seriously effect to the stability of b. longum in faeces. fluctuative growth was also observed in l. acidophillus in a similar way to that of b. longum. since l. acidophilus is known to use fos in its fermentation process (kaplan and hutkins 2000), population increase by day 14 might be promoted by fos fermentation in the colon. when the population of l. acidophilus was examined in the fresh product, a decrease in the population was obtained from day 21 (9.30 log cfu g-1) to day 28 (7.83 log cfu g-1). the decrease of l. acidophillus implies a decrease in bacterial intake to the digestive tract leading to a lower faeces population. however, the population of l. acidophillus population significantly increased by day 42 as shown by the population in synbiotic-treated rats of 1.04 log cfu g-1, which was higher compared to the control (0.92 log cfu g-1) and probiotic-treated rats (0.11 log cfu g-1). a decrease in the population of l. acidophilus in rat’s faeces was found in synbiotic-treated rats, when the yoghurt intake was terminated for 14 days. the maximum population was 8.34 log, which reflects a negative response to the stability of l. acidophillus in the digestive tract. molin et al. (1993) reported that the normal population of l. acidophilus in faeces is 108 cfu g-1, therefore we conclude that terminating volume 2, 2008 microbiol indones 127 the yoghurt intake for 14 days promoted of l. acidophillus population decrease. l. acidophilus and b. longum population increase in synbiotic yoghurt promotes a decline in e. coli population (table 3 and 4). those bacteria, which tend to associate with the membrane of digestive tracts, lead to an increase in the population of native lactobacilli in the digestive tract. furthermore, this situation inhibits the population of pathogenic bacteria, such as e. coli. the involvement of lactic acid bacteria in the fermentation process leads to a decreased population of e. coli in the 42-days-treated synbiotic rats. the decreasing population reached 5.90 log cfu g-1. lactic acid bacteria have a unique process in their fermentation, in which organic acid is accumulated leading to decrease ph. lactic acid tends to decrease the ph value of digestive tract in the range ph 4-5. since e. coli requires a ph value in range of 6-7 for optimal growth, the acidic condition dramatically reduce the population of e. coli. in general, the results on faeces bacterial counts found in our study indicate a great stability of the faeces flora for symbiotic treatments. this has also been found in other studies after different forms of dietary intervention (bornside 1978; bartram et al. 1994). population of colon flora. an increased population of b. longum reflects the effect of fos content in the yoghurt. the addition of fos to the yoghurt helps probiotic bacteria through promoting their viability and survivald ability in the digestive tract. this disaccharide composed of fructose and galactose is not digested by humans but is readily fermented by bifidobacteria as well as by certain bacteria of the resident colonic flora, thus enhancing the selective proliferation and colonization of bifidobacteria. fooks et al. (1999) stated that the fermentation of fructans is more favourable to bifidobacteria than of other carbohydrate sources. fructan leads to changed microflora composition which is dominated by bifidobacteria. this fact is known as the bifidogenic effect. fig 2 shows the effect of treatments to a population of b. longum. the population of b. longum in synbiotic-treated rats went as high as 11.48 log cfu g-1 leading to microfloral balance in digestive tract. we conclude that the consumption of pesa goat-milk-based synbiotic yoghurt for 42 days continuously promotes the growth of b. longum in the colon. the low number of b. longum, mp to 8.55+0.23 log cfu g-1 reflected as adaptation response in -14-days-synbiotic treated rats. it also indicated the disturbances in the stability of b. longum in the colon. a decrease of b. longum intake from synbiotic yoghurt has a vital role for delivering effectiveness. a fail in the population of b. longum leads to a reduction of their effect to human health since the population was out of standard population range (mitsuoka 1978). alternatively, at low populations of b. longum, pathogenic bacteria can become a dominant group adhering to the digestive tract’s membrane. it is suggested that yoghurt consumption must be regular and continuous to supply lactic acid bacteria to the digestive tract. because the digestion process takes around 12 h (from mouth to rectum), this means probiotic bacteria must be consumed on daily basis. the population of l. acidophilus in the colon, which increased to 1.32 log cfu g-1, was beyond the range of the normal population. molin et al. (1993) reported that the digestive tracts of an adult people was populated by lactobacilli in a range of numbers, being 102-104 cfu g-1, 104-106 cfu g-1 and 108 cfu g-1 in intestine, colon and faeces, respectively. a high population of l. acidophilus might be caused by the addition of fos to synbiotic yoghurt leading it to be used as an energy sources by bacteria to enhance their growth and promote positive effects to host’s health. table 4 shows the average value of the population in all treatments. the production of bacteriocins by lactobacilli is relatively common, which may contribute to their colonization of a wide range of habitats and their competitive edge over other bacteria (garriga et al. 1993). the antimicrobial activity of lactic acid bacteria may be due to a number of factors. among these are decreased ph levels, competition for substrates and the production of substances with bactericidal or bacteriostatic action, including bacteriocins (parente and ricciardi 1999). this was proved by the evidence in our experiments that the population of e. coli as one of pathogenic bacteria in the digestive tract was decreased by treating with synbiotic yoghurt (table 5). the low number of l. acidophilus in the colon was caused by the termination l. acidophilus intake from synbiotic yoghurt after 14 days. furthermore, it initiated inability to complete in adhesion to the digestive tract. as a consequence, pathogenic bacteria would increase, leading to a shift in the microfloral balance in the colon. in some cases, negative effects to human health will occur when such pathogenic bacteria are the dominant group in the digestive tract, i.e. salmonella, e. coli and listeria. this leads to an table 3 bifidobacterium longum population in colon treatment population (log cfu g-1) control probiotic yoghurt synbiotic yoghurt adaptation post treatment (no treatment for 14 days) 9.11 ± 0.36a 8.87 ± 0.81a 11.48 ± 0.94b the fonts printed in superscript indicate significant difference between treatments (p<0.01). table 4 lactobacillus acidophillus population in colon t r e a t m e n t control probiotic yoghurt synbiotic yoghurt adaptation post treatment (no treatment for 14 days) population (log cfu g-1) 9.56 ± 0.31a 9.28 ± 0.66a 11.32 ± 0.98b the fonts printed in superscript indicate significant difference between treatments(p<0.05). table 5 escherichia coli population in colon treatment population (log cfu g-1) 7.48 ± 0.65a 7.16 ± 1.12a 5.54 ± 0.12b control probiotic yoghurt synbiotic yoghurt adaptation post treatment (no treatment for 14 days) the fonts printed in superscript indicate significant difference between treatments(p<0.05). 128 maheswari et al. microbiol indones 8.56 ± 0.63 8.72 ± 0.27 8.72 ± 0.63 8.55 ± 0.23 8.09 ± 0.45 4.58 ± 0.33 increase of carcinogenic compounds, toxins, nh 3 , h 2 s, amines and phenolics. mitsuoka (1978) stated that e. coli can be obtained in the newly born baby and its population tends to increase along with the increase of baby’s age. a low population of e. coli in synbiotic yoghurt implies an ability of l. acidophillus and b. longum to inhibit e. coli in the colon. this is caused by the ability of those probiotics to ferment fos, as a simple sugar monosaccharide, leading to the production of various acidic compounds leading to the inhibition of e. coli. in agreement with our results, oyetayo et al. (2003) reported that rats that were treated with feed containing l. acidophilus for 3 days had a lower population of enterobacteria in their faeces. when synbiotic yoghurt intake was terminated after 14 days, positive effect of probiotic bacteria in decreasing pathogenic bacteria was detected. the results show that by the decreasing this population to 4.58+0.33 log cfu g-1 will decrease the e. coli content in the faeces (fig 3). therefore, the e. coli population may still decrease, even though yoghurt intake was terminated. it is suggested that this phenomenon is due to the residual effect of antimicrobial activity of l. acidophillus and b. longum. the presence of e. coli, either in faeces or colon, implied that l. acidophillus and b. longum were able to survive in the digestive tract leading to a decrease in the e. coli population. sufficient numbers of b. longum and l. acidophillus cells survived throughout the intestinal track. synbiotic yoghurt intake significantly increased the population of b. longum and l. acidophillus, giving a positive effect in inhibiting pathogenic bacteria. it was demonstrated by the decrease e. coli population in the intestine of rats treated with synbiotic yoghurt compared to that of control. eventhough the population of probiotic bacteria did not severly effect to rats’s performance, numerically performance of synbiotic yoghurt-treated rats are better than that of control and probiotic-treated rats. negative response was observed in population of l. acidophilus and b. longum when synbiotic yoghurt intake was terminated for 14 days. their populations in colon were similar compare to that of adaptation phase. the results showed that the termination of yoghurt intake led to the decrease of l.acidophillus and b. longum population. in addition, their performace were not as good as the group of rats treated with synbiotic yoghurt 42 days. it was shown by their feed consumption and conversion, which were higher or comparable to that of 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34-43 doi: 10.5454/mi.14.1.5 optimization of xylanase production by streptomyces costaricanus 45i-3 using various substrates through submerged fermentation 1,2 3 4 sipriyadi , aris tri wahyudi , maggy thenawidjaya suhartono , and anja 3* meryandini 1 microbiology study program, department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, kampus ipb dramaga, bogor 16680, west java, indonesia; 2 department of biology, faculty of mathematics and natural sciences, universitas bengkulu, kampus unib kandang limun, 38371, bengkulu, indonesia; 3 department of biology, faculty of mathematics and natural sciences; 4 department of food science and technology, faculty of agricultural and technology, institut pertanian bogor, kampus ipb dramaga, bogor 16680, west java, indonesia. xylanase is an important hydrolytic enzymes with many application in several industries, but to obtain enzyme derived products is not easy. thus, the optimization of efficient xylanases production is a great interest for enzyme application. this study aims to determine the type of substrate, medium composition, and optimum conditions of xylanase production by s. costaricanus 45i-3. determination of substrate type was done by growing the tested bacteria on birchwood xylan, beechwood xylan, oat spelled xylan, corn cobs xylan, and tobacco xylan substrate, meanwhile the determination of medium composition and enzyme production were done by measuring xylanase activity at various substrate concentration and replacing the carbon, nitrogen, phosphate and surfactants source. the results showed that the highest enzymatic index (ei) produced from corn cob xylan substrate at 3.60 meanwhile the second highest was beechwood xylan substrate at 2.87 ei, however this substrate is purer, thus this substrate was selected and used as xylan sources for further optimization measurement. the best xylanase activity -1 (2.29 u ml ) obtained on eighth day after inoculation on rotary incubator at 120 rpm in 28 ºc. arabinose as the -1 source of carbon generate the highest activity at 3.161 u ml meanwhile the most preferred source of phosphate is -1 na hpo (2.37 u ml ). both source of nitrogen i.e. nitrogen ammonium sulphate (nh ) so and yeast extract 2 4 4 2 4 -1 were able to produce xylanase at 2.57 and 2.36 u ml . the addition of surfactant in production medium showed -1 -1 addition of sds surfactant (0.146 u ml ) and tween 80 (0.438 u ml ) showed a negative response by decreasing the activity. the conclusion showed that the xylanase activity was increased after optimization at various c, n, and p sources, and the use of nitrogen source (nh4) so ), become a more economical alternative to replacing a 2 4 nitrogen source yeast extract so it can lower the production costs of xylanase enzyme. key words : fermentation, streptomyces costaricanus 45i-3, substrate variation xilanase merupakan enzim hidrolisis penting dengan banyak aplikasi di berbagai industri, namun untuk mendapatkan produk turunan enzim tidaklah mudah. dengan demikian, optimalisasi produksi xilanase yang efisien merupakan perhatian besar untuk aplikasi enzim. penelitian ini bertujuan untuk mengetahui jenis substrat, komposisi medium, dan kondisi optimum produksi xilanase oleh s. costaricanus 45i-3. penentuan jenis substrat dilakukan dengan cara menumbuhkan bakteri uji pada media xilan birchwood, xilan beechwood, xilan oatspelt, xilan tongkol jagung, dan xilan tembakau, sedangkan penentuan komposisi media dan produksi enzim dilakukan dengan mengukur aktivitas xilanase pada berbagai konsentrasi substrat. dan mengganti sumber karbon, sumber nitrogen, sumber fosfat dan surfaktan. hasil penelitian menunjukkan bahwa indeks enzimatik (ei) tertinggi dihasilkan dari substrat xilan tongkol jagung sebesar 3,60 sedangkan tertinggi kedua adalah substrat xilan beechwood sebesar 2,87 ei. substrat ini lebih murni sehingga dipilih dan digunakan sebagai sumber xilan untuk -1 optimalisasi lebih lanjut. aktivitas xilanase terbaik (2,29 u ml ) diperoleh pada hari kedelapan setelah inokulasi pada inkubator bergoyang 120 rpm dan suhu 28 ºc. arabinosa sebagai sumber karbon menghasilkan aktivitas -1 -1 tertinggi yaitu 3,161 u ml sedangkan sumber fosfat yang paling baik adalah na hpo (2,37 u ml ). kedua 2 4 sumber nitrogen yaitu amonium sulfat (nh )2so dan ekstrak khamir mampu menghasilkan xilanase pada 2,57 4 4 -1 -1 -1 dan 2,36 u ml . penambahan surfaktan pada media produksi sds (0,146 u ml ) dan tween 80 (0,438 u ml ) menunjukkan respon negatif dengan penurunan aktivitas. kesimpulan menunjukkan bahwa peningkatan aktivitas xilanase setelah dilakukan optimasi pada berbagai sumber c, n, dan p, serta penggunaan sumber nitrogen (nh )2so ), menjadi alternatif yang lebih ekonomis untuk menggantikan ekstrak ragi sumber nitrogen sehingga 4 4 dapat menurunkan biaya produksi enzim xilanase. kata kunci : fermentasi, streptomyces costaricanus 45i-3, variasi substrat microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-251-8625481; fax: +62251-8625708; email: ameryandini@yahoo.com xylan is the main component of hemicellulose. the main chain is composed by β-xilopironosa units with β-1,4-glycosidic bond with a subsidiary chain in volume 14, 2020 microbiol indones 35 the form of glucopyranosyl, 4-o-methyl-dglucopyranosyl, α-l-arabinofuranosyl, acetyl, or furulil and p-coumaril (kulkarni et al. 1999; li et al. 2000) endo-β-1,4-xylanase (ec 3.2.1.8) is an enzyme which is important to hydrolyze xylan perfectly thus it can produce an usable product such as xylose, and xylobiose like xylooligosaccharides (bernier et al. 1983; chakrit et al. 2006). � various microbes were reported able to produce xylanase. bacillus from bacteria, trichoderma and aspergillus from fungi, and streptomyces from actinomycetes were known to be a potential microbes producing xylanase. those microbes are known to have varied and widespread ecological niches (collins et al. 2005). bacteria from indonesia such as bacillus licheniformis strain 15 produces endo-β-1,4-xylanase which grown on banana peels substrate (helianti et al. 2007). meryandini et al. (2007) reported that the isolate of streptomyces costaricanus 45i-3 isolated from peat swamps soil in kalimantan, indonesia, has an optimum activity at ph 5.0 in 50 °c. however this study only used a single substrate i.e. oat spelt xylan and did not conduct the activity measurement of various carbon, phosphate and nitrogen sources also the effects of surfactants to produce an optimum conditions in producing extracellular xylanase. � the main nutrients for microorganisms growth were carbon sources, nitrogen and mineral components especially phosphate. the media formulation for the growth and fermentation s. costaricum is an important stage at designing an experiments in the work scale. papagianni (2004) reported that the carbon sources, nitrogen sources and the fermentation time have a significant role in determining the enzyme production level in a culture. therefore, it is important to optimize the conditions to produce enzyme inexpensively using the available nutrient sources abundantly. response surface method has been used as successful statistical tools for the optimization of media composition in the fermentation process for enzyme production (dobrev et al. 2007). therefore the aim of this study was to optimize the nutritional conditions in producing xylanase using s. costaricanus 45i-3 by conducting a series of research stages and data analysis. materials and methods bacterial strain. the bacteria used in this study was s. costaricanus 45i-3 isolated from peat swamp forest in kalimantan, indonesia, a collection of dr. yulin lestari (staff of department of biology, faculty of mathematic and natural sciences, bogor agricultural university, indonesia). these isolate was cultured on international streptomyces project no. 2 (isp2), supplemented with antibiotics nalidixic acid (1 -1 -1 mg ml ) and cyclohexamide (5 mg ml ), and incubated for 8 days at room temperature (27 °c), and this isolate was used as stock culture for the further assays. morphological characteristic of s. costaricanus 45i-3 in various growth media. the cultivation of s. costaricanus 45i-3 was conducted using various solid media, which aims to see the growth and morphological characteristics in media used; nutrient agar (na), potato dextrose agar (pda), isp, yeast soluble starch agar (ysa), international streptomyces project no. 4 (isp4), and beechwood xylan agar medium. each media (25 ml) were sterilized at 121 °c for 15 min. after sterilization, a loopful of s. costaricanus 45iwas streaked on media, then incubated at 27 °c for 8 days. the bacterial cells that showed the best growth will be used for further study. the measurement of β-xylanase activity of s. costaricanus 45i-3 based on clear zone. a total volume of 250 ml fermentation medium (g/l) which consist of yeast extract 1.25 g, kh po 0.25 g, 0.05 g 2 4 mgso ∙7h o) and 0.8% (w/v) substrate (beechwood 4 2 xylan) were added to 250 ml distilled water. after the medium mixed perfectly the sterilization was performed on 121 °c for 15 min. after sterilization, the medium then inoculated by 1% (v/v) bacterial culture and incubated at 27 °c for 8 days with 120 rpm agitation. furthermore, the culture was centrifuged at 8000 rpm for 15 min to remove bacterial cells. the supernatant then was precipitated using 80% acetone solvent in accordance with meryandini et al. (2007). xylan degradation activity assay was conducted using two layers agar method with modifications based on chen et al. (2004). the base layer of agar medium consists of 50 mm acetate buffer (sodium acetate + acetic acid) ph 6.0 and 1.7% agar. meanwhile the upper layer consists of 0.8% (w/v) xylan substrate (beechwood, birchwood, oat spelled, corn cobs, and tobacco) and 1.7% (w/v) agar in 50 mm acetate buffer ph 5.0. a total of 3, 5, 7, and 10 ml precipitated xylanase were dripped on each media with different substrates and dried subsequently at 27 °c for 15 min, then incubated at 37 °c for 4 days. after the incubation process the media were stained using 1% congo red for 15 min and rinsed using 1 mm nacl. the hydrolysis of β-xylanase indicated by the clear zone (halo) formed with an orange margin around (carder, 1986). this ei= diameter of hydrolysis zone diameter of colony halo was measured for subsequent calculation of the enzymatic index (ei) using the equation. the effect of xylanase production using various concentration of xylan. in this study a beechwood xylan was used as a carbon source. to determine the minimum concentration with the best activity in producing β-xylanase, a varying concentration of xylan were used in fermentation media. beechwood xylan was added to each fermentation medium with concentrations 0.2, 0.4, 0.6, 0.8, and 1.0% (w/v) respectively, meanwhile the 0% (w/v) medium (without the addition of beechwood xylan) used as a control. the effect of various carbon, nitrogen, phosphate, and surfactant sources towards βxylanase produced by s. costaricanus 45i-3. observation of various nutrients addition i.e. carbon, nitrogen, phosphate and surfactant sources, towards βxylanase production was done by replacing the resource of various nutrients with a variety of other sources. the production of β-xylanase with the addition of major carbon source in the form of beechwood xylan 0.8% (w/v) with various other carbon sources, such as glucose, galactose, maltose, xylose, fructose, sucrose, arabinose and corn cob xylan with 0.3% (w/v) concentration, respectively. meanwhile, the nitrogen source used were malt extract, peptone, tryptone, yeast extract, ammonium sulfate (nh )2 so , and ammonium persulfate (nh )2 s o 4 4 4 2 8 respectively 0.3% (w/v). the effect of various phosphate sources addition was done by replacing the main source of phosphate kh po with other 2 4 phosphate sources, i.e. na hpo , nah po , k hpo 2 4 2 4 2 4 and alpo respectively 0.03% (w/v). measurement of 4 xylanase activity with 0.03% (w/v) surfactants addition was performed by adding tween 80, sds (sodium dodecyl sulfate), and dmso (dimethyl sulfoxide) respectively. xylanase activity assay. the released reducing sugars were measured using dns method (miller, 1959). the dns method was conducted by reacting 0.5 ml of 0.8% beechwood xylan substrate with 0.5 ml xylanase in acetate buffer (0.05 m, ph 5.0) and incubated at room temperature for 30 min. the reaction stopped by adding 1.5 ml of 3.5 dinitrosalicylic acid and heated for 15 min at 100 ºc. the solution then allowed to cool down and the absorbance value was measured using a spectrophotometer at 540 nm. xylanase activity was calculated by using xylose standard curve. one unit of enzyme activity is defined as the amount of enzyme required to produce 1 mol xylose sugar per minute in standard conditions. results morphological characteristic of s. costaricanus 45i-3. s. costaricanus 45i-3 bacterial culture showed several morphological characteristics when grown on a various growing medium as shown in figure 1. the s. costaricanus 45i-3 colony was round-shaped hyphae, undulate margin with mealy surface, the substrate mycelia are yellow, and the aerial mycelia are yellowish white with a bright yellow pigmentation on beechwood xylan medium, meanwhile on the other media (yma, ysa, na, pda and isp4) the aerial mycelia are brown with a yellowish white to brownish gray substrate mycelium (table 1). bacterial strains isolated from peat swamp forest in kalimantan have flexous hyphae but has a spiral type on isp4 medium. the spores have a smooth morphological characteristic. s. costaricanus 45i-3 was able to grow well in six different media as shown in figure 1, but the most rapid and abundant growth were in beechwood xylan and yma medium, the growth on those media nd was started on 2 day after incubation, the growth begins with substrate hyphae formation and followed table 1 morphological characteristic of s. costaricanus 45i-3 on various media isolate code growth media color pigmentation on media hyphae type spore morphology aerial mycelia substrate mycelia 45i-3 yma grey brownish grey none flexous smooth ysa grey brownish grey none flexous smooth beechwood xylan yellowish white yellow bright yellow flexous smooth na white yellowish white none flexous smooth pda grey yellowish grey none flexous smooth isp4 grey greyish white none spiral smooth 36 sipriyadi et al. microbiol indones with aerial hyphae covering the majority of growth media which followed by sporulation process. on the other hand, the four other media showed a slower growth and the aerial hyphae which formed was thinner and did not cover the overall surface of growth media. xylanase activity on various xylan sources. this measurement aimed to determine which xylan source preferred by tested bacteria that could be seen its ei value. the highest ei value (3.6) was obtained in the solid culture which was added with corn cob xylan, while the lowest in tobacco xylan-enriched medium with a mere of 2.08 ei value. in addition, three other substrates i.e. birchwood xylan, beechwood xylan and oat spelt xylan produces an adjacent ei values respectively at 2.80, 2.87 and 2.77 (fig. 2). optimal incubation time in producing xylanase. the maximum activity of xylanase was th obtained in 8 day after incubation. the optimum th activity of extracellular enzyme was generated on 8 -1 day i.e. at 2.29 u ml (fig. 3). the effect of various concentration of substrate/beechwood xylan for xylase production. every microorganism requires a certain concentration of carbon source for their growth. the results in figure 4 shows that the addition of a carbon source ranging from 0-1% (w/v) beechwood xylan give a different effect on xylanase production. in general, it can be concluded that the addition of higher concentrations produce a greater xylanase. however, the provision of th th 0.8% (w/v) and 1.0% (w/v) carbon source at 7 and 8 day after incubation showed not significant result with a nearly same amount of xylanase production i.e. 2.29 -1 u ml . the effect of various carbon sources towards xylanase production. figure 5 shows the effect of various carbon sources usage for xylanase produced by s. costaricanus 45i-3 isolates, then different treatments were done by adding various carbon sources (0.3%) i.e. glucose, galactose, maltose, xylose, fructose, sucrose, arabinose, and corn cob xylan. the addition of 0.3% arabinose and fructose as additional -1 carbon sources produced the highest (3.16 u ml ) and -1 lowest xylanase (0.45 u ml ), respectively. the effect of various nitrogen sources towards xylanase activity. ammonium sulphate (nh ) so 4 2 4 and yeast extract are the best nitrogen sources for -1 xylanase production i.e. 2.52 and 2.36 u ml individually compared to the other organic and inorganic nitrogen sources which used, such as malt extract, peptone, tryptone and ammonium persulfate (fig. 6). the effect of various phosphate sources towards xylanase activity. the results obtained indicate that the best phosphate source for xylanase production was na hpo . the addition of na hpo can 2 4 2 4 -1 induce xylanase production up to 2.37 u ml . the effect of various surfactants towards xylanase activity. the effect of surfactant on xylanase production was tested using 3 different surfactants sources including tween 80, sds, and dmso. the results showed that both of 0.03% tween 80 and sds had a significant effect to decline of xylanase production. the addition of tween 80 decreased -1 xylanase production to 0.44 u ml and sds addition -1 also decreased xylanase production to 0.15 u ml . the addition of dmso (dimethyl sulfoxide) did not affect xylanase production which was proven with the -1 xylanase production that still reached 2.46 u ml . discussion xylan is one of the carbon sources used by microorganisms, particularly bacteria, to be reformed into a simpler molecular structure which can then be used as an energy source. several carbon sources used in this study were birchwood xylan, beech wood xylan, oat spelled xylan, corncob xylan and tobacco xylan. this may occur because xylan from corn cobs, birchwood, beechwood and oats contains many growth factors needed, vitamins and proteins which can provide source of carbon and nitrogen for the test bacteria (sasmitaloka et al. 2019; revanker and lele 2006), low lignin and silica (battan et al. 2006). based on the results, xylanase production peaked at -1 the eighth dayof incubation, accounted at 2.29 u ml . several other research results showed that the optimal incubation time to produce xylanase depends on the type of bacteria used regarding with the time of bacterial growth. gram-negative and gram-positive bacteria, which are non-actinomycetes, tend to have a shorter incubation time compared to actinomycetes group. several studies reported that bacillus subtilis cho40 merely produced xylanase during 4 days of incubation time (khandeparker et al. 2011). another study revealed the optimal extracellular xylanase production time, carried out by li et al. (2010), of actinomycetes th isolate, streptomyces rameus l2001, was on the 7 day. kavya and padmavathi (2009) reported that the xylanase produced by aspergillus niger was at 6.11 u -1 th ml on the 6 day after incubation. as can be observed th from curve, bacterial cells started to decrease on the 8 day which was the peak of exponential period. in this phase, bacterial cells grow dramatically and require volume 14, 2020 microbiol indones 37 fig 1 morphological characteristic of streptomyces costaricanus 45i-3 on various substrates. a1 (beechwood xylan), a2 (yeast solube startch), a3 (yma), a4 (isp4), a5 (pda) and a6 (na). b1 (morphology of aerial hyphae), b2 (crossed hyphae). c (morphological spore and hyphae) figure taken using scanning electron microscope (sem). fig 2 the effect of different substrates on xylanase production by streptomyces costaricanus 45i-3 after 4 days o incubation at 28 c. -1 fig 3 cell biomass of s. costaricanus 45i-3 (mg ml ) ( ) and s. costaricanus 45i-3 xylanase activity ( ) o on 0.8% beechwood xylan medium at 120 rpm in room temperature (27 c). 38 sipriyadi et al. microbiol indones fig 6 the effect of various phosphate sources towards xylanase produces by s. costaricanus 45i-3 after incubated for 8 days. fig 4 screening of isb64 isolate-producing cellulase thermophilic bacteria from the ie seuum hot spring, aceh besar. (a) clear zone, (b) bacterial colony, and (c) cmc media flooded with 1% congo red. fig 5 the effect of various carbon sources towards xylanase produced by s. costaricanus 45i-3 after incubated for 8 days. volume 14, 2020 microbiol indones 39 fig 7 the effect of various phosphate sources towards xylanase produced by s. costaricanus 45i-3 after incubated for 8 days. fig 8 the effect of various surfactant towards xylanase produced by s. costaricanus 45i-3 after incubated for 8 days. more energy source for survival than in other periods. therefore, production of xylanase was excreted abundantly in order to gain more energy in terms of degrading media-contained xylan. moreover, xylan concentration strongly related to enzyme production and for this reason, this study also monitored the effect of several concentrations of xylan to xylanase activity. based on the results presented in figure 4, the concentration used in the next optimization test was 0.8% (w / v). this result is in accordance with guha et al. (2013) which states that the best concentration to produce xylanase is to provide a carbon source (xylan) from 0.25 to 1.0% (w / v). lawrence et al. (2015) reported that the xylan concentration variation of 0.5-1% (w / v) added to the fermentation medium did not have a significant effect on xylanase production at concentration more than 1%. the administration of 0.53.0% molasses as a supplement for b. subtilis and b. megaterium can reduce xylanase production in line with the increase in the concentration of molasses given. this probably due to the nutrients contained in molasses produce catabolite suppressants in xylanase production (irfan et al. 2016). application of several additional carbon sources also shows an influence on xylanase activity. figure 5 shows the addition of fructose to the beech wood xylan production media significantly decreased the xylanase production, it was because of the xylanase produced in beech wood xylan media is 2.11 u / ml without the addition of fructose. additional fructose can inhibit xylanase production, since fructose as a simple sugar will be used first as a carbon source by s. costaricanus 45i-3 isolates or bacterial cell growth. as supposed by guan et al. (2016), simple sugars, including fructose, 40 sipriyadi et al. microbiol indones have no remarkable effect with xylanase production since, perhaps, those sugars could prevent synthesis of enzymes. this is also supported by study conducted by ajijolakewu et al. (2016) who suggested that there were repressive effects produced by simple carbohydrate molecules in producing xylanase. indeed, those were only utilized for growth. on the other hand, there is breakthrough discovering that s. thermocoprophilus tc12w was able to produce 1204.8 u/g of xylanase by using alkaline pretreated empty fruit bunch (apefb) as a carbon source, which was the first report utilizing that (sinjaroonsak et al. 2019). giving different nitrogen sources turned out to have various effects on xylanase activity, indicating that yeast extract had the effect of being the best nitrogen source on xylanase activity. bhardwaj et al. (2019) stated in their review that nitrogen is one of the most important elements for metabolisms, including enzymatic activity. a study by zuhri et al. (2013) showed that ammonium sulfate is the best nitrogen source for the cultivation of bacillus sp. m123 for the production of alkaline proteases. addition of 1% casein played role in both the highest xylanase activity and xylanse production, 1.78 -1 -1 u g and 3.69 u mgg , respectively (tai et al. 2019). sudan and bajaj (2007) reported that adding 0.3% ammonium sulfate to the production medium was able to make aspergillus niveus rs2 produce 15 u/ml xylanase after 5 days of incubation which was the second best after yeast extract. the role of nitrogen, especially ammonium sulfate, from xylanase excretion was found from anoxybacillus kamchatkensis strain nastpd13 (yadav et al., 2018). ravindran et al. (2019) reported the highest xylanase (6495.6 iu/g of dry scw) produced by a. niger was successfully resulted using media supplemented with 0.2g/g of yeast extract as nitrogen source. all nitrogen sources added to the production media showed that all of them were able to promote cell biomass growth and xylanase production except for ammonium persulfate (nh )2s o which 4 2 8 inhibited cell biomass growth and xylanase production. this is presumably because (nh )2 s o is toxic so that 4 2 8 it inhibits the growth of s. costaricanus 45i-3 cells and also inhibits xylanase production. the effect of different inorganic phosphate resources towards xylanase production has been tested and presented in figure 7, shows that s. costaricanus 45i-3 isolates was able utilizing other phosphate sources i.e. nah po , kh po , and alpo to produce xylanase, 2 4 2 4 4 it is seen from the xylanase production which were -1 relatively stable at 2.17 to 2.34 u ml . a slightly different results in the addition of phosphate sources was k hpo which only capable inducing xylanase 2 4 -1 production at 1.69 u ml . a similar result were reported by mandal (2015) which stated that na hpo is the best 2 4 phosphate sources to bacillus cereus bsa1 for xylanase production, with the production of xylanase at 5.53 u -1 ml . the phosphate salts with a certain concentration can encourage organism growth and stimulates the synthesis of extracellular enzymes in production medium chellapandi and jani (2008). microbes using phosphate source as an ingredient synthesis of nucleic acids and phospholipids on the cell wall.the addition of other biosurfactant such as 0.03% sodium dodecyl sulfate (sds) decrease xylanase activity by 13 fold -1 (0.15 u ml ) compared with the production media -1 without surfactant addition i.e. 2.11 u ml (figure 8). silva et al. (2015) reported that the addition of sds on production media decreasing xylanase activity by 7.75 -1 fold (12.9 u ml ) which is lower than controls (100 u -1 ml ). sds is a strong denaturant, the usage of sds at certain concentrations can interfere enzyme activity function and lipid solubility (wamack et al. 1983) as conclusion, from the results obtained it can be concluded that s. costraricanus 45i-3 was able to grow on a variety of media, but the best growth medium was medium containing xylan (beechwood xylan) which was produced a yellow aerial hyphae, smooth-shaped spores and produces bright yellow pigmentation. the th usage of 0.8% beechwood xylan substrate on 8 day after incubation was able to produce the highest xylanase production. the best fermentation conditions were on the addition of arabinose carbon source, and nitrogen source i.e. ammonium sulfate (nh )2 so and 4 4 yeast extract. the most preferred source of phosphate was na hpo . the addition of surfactants such as sds 2 4 and tween-80 gave negative effects because it decreased the level of xylanase production while the addition of dmso did not decrease the activity. acknowledgments this research was funded by the directorate of research and community service, directorate general for strengthening research and development, ministry of research, technology and higher education. in accordance with the letter of assignment agreement implementation research program number: 044 / sp2h / lt / drpm. sipriyadi. volume 14, 2020 microbiol indones 41 references ajijolakewu ka, leh cp, abdullah wnw, lee ck. 2016. assessment of the effect of easily-metabolised carbon supplements on xylanase production by newly isolated trichoderma asperellum usm sd4 cultivated on oil palm empty fruit bunches. bioresources. 11(1):96119627. battan b, sharma j, kuhad rc. 2006. high-level xylanase production by alkaliphilic bacillus pumilus ash under solid-state fermentation. world j microbiol biotechnol. 22(12):1281–1287. doi:10.1007/s11274-006-9173-x. bhardwaj n, kumar b, verma p. 2019. a detail overview of xylanases: an emerging biomolecule from current and future prospective. bioresour bioprocess. 6(40):1-36. doi: 10.1186/s40643-019-0276-2. bernier r, desrochers m, jurasek l, paice m. 1983. isolation and characterization of a xylanase from bacillus subtilis. appl environ microbiol. 46(2):511-514. doi: 10.1128/aem.46.2.511-514.1983. carder jh. 1986. detection and quantitation of cellulase by congo red staining of substrates in a cup-plate diffusion assay. anal biochem. 153(1):75-79. doi: 10.1016/00032697(86)90063-1. chakrit t, khin lk, khanok r. 2006. purification of xylanase from alkaliphilic bacillus sp. k-8 by using corn husk column. proc biochem. 41(12):2441-2445. doi: 10.1016/j.procbio.2006.07.004. \chellapandi p, jani hm. 2008. production of endoglucanase by the native strains of streptomyces isolates in submerged fermentation. braz j microbiol. 39(1):122–127. doi: 10.1590/s1517-838220080001000026. chen pj, wei tc, chang yt, lin lp. 2004. purification and characterization of carboxymethyl cellulase from sinorhizobium fredii. bot bull acad sinica. 45: 111-118. collins t, gerday c, feller g. 2005. xylanases, xylanase families and extremophilic xylanases. fems microbiol rev. 29(1):3–23. doi : 10.1016/j.femsre.2004.06.005. dobrev gt, pishtiyski ig, stanchev vs, mircheva r. 2007. optimization of nutrient medium containing agricultural wastes for xylanase production by aspergillus niger b03 using optimal composite experimental design. bioresour technol. 98(14):26712678. doi: 10.1016/j.biortech.2006.09.022. gomes j, gomes i, kreiner w, esterbauer h, sinner m, steiner w. 1993. production of high level of cellulasefree and thermostable xylanase by a wild strain of thermomyces lanuginosus using beechwood xylan. j biotechnol. 30(3):283–297. doi:10.1016/01681656(93)90145-d. guan gq, zhao px, zhao j, wang mj, huo sh, cui fj, j i a n g j x . 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maharjan j, korpole s, prasad gs, shani g, bhattarai t, sreerama l. 2018. production, purification, and characterization of thermostable alkaline xylanase from anoxybacillus kamchatkensis nastpd 13. front b i o e n g b i o t e c h n o l . 6 ( 6 5 ) : 1 1 3 . d o i : 10.3389/fbioe.2018.00065. zuhri r, anthoni a, yetria r. 2013. effect of carbon and nitogen sources on production of alkaline protease from thermophilic bacillus sp. m1.2.3. jurnal biologika. 2(1):40-46. volume 14, 2020 microbiol indones 43 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 404 not found 1 cahya prihatna rev.pmd volume 3, number 3, december 2009 p 99 108 issn 1978-3477 review the plant – pathogen interactions cahya prihatna research school of biological sciences, research school of biology, school of medicine, biology and environment, the australian national university, gpo box 475, canberra act 2601, australia, email: cahyaprihatna@gmail.com; cahya.prihatna@anu.edu.au interaction between plants and their pathogens is complex, involving multifaceted recognition of pathogens by the plants and, on the other hand, subtle evasion from the pathogens. plants perceive pathogens through direct recognition of common molecular patterns in microbes and direct recognition of effectors or their perturbation on cellular components by the pathogens. recognition of microbeor pathogen-associated molecular patterns triggers innate immunity that renders plants resistant to most potential microbial pathogens. recognition-dependant immunity in plants largely relies on polymorphism of resistance gene products that confer specificity towards host-specialised pathogens, which, in turn, induces more specific resistance that is effective against host-specialised pathogens. the deployment of effective resistance involves signalling of pathogen recognition through complex signalling cascades, transcriptional reprogramming, and defence-related genes, which all contribute to an arrest of pathogen growth. our current insights into effector biology and to which the plants respond, provide a detailed information on the evolutionary arms race between plants and their pathogens. these will lead to an improvement of current strategies for crop improvement and protection. key words: innate immunity, resistant, effectors, microbeor pathogen-associated molecular patterns disease in plants is rare because most plants are resistant to most pathogens. however, in extensively cultivated genetically identical plants, coevolutionary arms race between pathogens and plants is not uncommon. plants are constantly challenged by a battery of potential pathogens ranging from fungi, oomycetes, bacteria, insects, nematodes, and viruses. unlike animals, plants are sessile, unable to escape pathogen attack. plants lack mobile cells able to deliver somatically generated, adaptive immune responses to sites of infection. nevertheless, every plant cell is independently able to mount defence responses against microbial attack. moreover, in order for potential pathogens to become actual pathogens in plants, they must overcome multifaceted defence mechanisms, from physical barrier, preformed antimicrobial compounds, and recognitiondependent immunity mediated by resistance (r) genes (dangl and jones 2001). pathogens able to breach beyond the passive lines of defence layers may seek persuasive and subtle relationships with the host to dodge the surveillance system. interaction between plants and their pathogens seems to be an intricate relationship involving subtle evasion (in pathogens) and recognition (in plants) mechanisms. host plant immunity to pathogen attack largely relies on the polymorphic capacity of r gene products that perceive specific elicitors produced by pathogens (dangl and jones 2001; jones and dangl 2006; bent and mackey 2007). in theory, any pathogen-derived molecule could act as an elicitor for re-programming the transcriptional or physiological states of plants into defence mode. on the other hand, pathogens continue to evolve their virulence machinery to subvert host defence responses. here, we review both virulence system in pathogens and resistance system in plants in turn, the evolutionary arms race involving both pathogen and host, and we present updates to the field based on recent findings on molecular plant-pathogen interactions. the plant surveillance system. there are two branches of the plant immune system (jones and dangl 2006). the first system uses transmembrane pattern recognition receptors (prrs) that perceive slowly evolving microbial or pathogen-associated molecular patterns (mamps or pamps) and the other uses r proteins that perceive effectors produced by host-specialised pathogens (fig 1). pamps or mamps can trigger initiation of pamp-triggered immunity (pti) (nürnberger et al. 2004), which is probably the first perception-based response against microbial infection that makes most plants immune to most potential pathogens. pti contributes to the so-called non-host resistance or basal resistance. however, host-specialised pathogens, particularly biotrophic microbes, have evolved the ability to suppress or interfere with pti by releasing effector proteins into the plant apoplast or the plant cell cytosol. the second and more elevated deployment of host immune response is initiated by recognition of specific elicitors encoded by pathogen avirulence (avr) genes and hence, designated as effector-triggered immunity (eti). an avirulence protein refers to the recognised effector protein that renders pathogens avirulent in their host plants. avr genes and their cognate host resistance (r) genes, contribute to the basis of the so-called gene-for-gene model of host–pathogen incompatibility. a plant is resistant if it carries cognate r genes to the corresponding avr genes in the interacting pathogens. alternatively, if either is inactive or absent, disease results. thus, gene-for-gene interaction between r and avr determines the outcome of plantpathogen relationship: incompatible (resistant plant, avirulent pathogen) or compatible (susceptible plant, virulent pathogen) interactions. surprisingly, r-mediated resistance and basal resistance give similar defence responses (tao et al. 2003). here, we define basal resistance as a form of immunity in susceptible microbiol indones100 review fig 1 model for the plant immune system for bacteria. left to right, the evolution of bacterial resistance through recognition of pathogenassociated molecular patterns (such as bacterial ûagellin) by extracellular receptor-like kinases (rlks) that triggers the deployment of basal immunity that requires map kinase signalling cascades and transcriptional reprogramming in the nucleus mediated by wrky transcription factors. this type of immunity confers the plant resistant to most potential pathogens. some pathogenic bacteria have evolved the machinery to dodge the recognition by rlks by releasing effector proteins that suppress the basal immunity. in the absence of cognate plant resistance proteins, plants are susceptible and therefore the bacteria proliferate in the apoplast. plants, on the other hand, have evolved to produce resistance proteins that recognise the effectors either directly or indirectly. in the figure, the resistance proteins are represented by cc-nb-lrr and tir-nb-lrr (see text). these proteins recognise the activity of the effectors and trigger the so-called effector-triggered immunity (eti) that is efficient to limit the growth of the bacteria. hosts challenged by virulent pathogens. this similarity in defence output suggests that eti is an accelerated and magnified version of basal resistance (wise et al. 2007). however, whether the r-mediated resistance and basal resistance superimpose similar signalling cascades or not is not yet understood. in addition, even though the gene-forgene relationship may imply a simple ligand-receptor interaction between avr and r proteins, there are, in other cases, demonstrations in which r proteins recognise avr proteins indirectly (mackey et al. 2002; axtell and staskawicz 2003; mackey et al. 2003; shao et al. 2003; rooney et al. 2005). the notion of indirect recognition of avr proteins by r proteins implies that r proteins monitor the structural integrity of plant proteins that are the nominal targets of avr proteins. thus, a third component is required in this model, which could be the virulence target of avr proteins. this model is known as “guard hypothesis”, denoting r proteins guard the “guardee”, the molecular target of effector proteins. all functional r genes known so far confer resistance to fungal, bacterial, viral, and even insect and nematode pathogens with very different lifestyles. despite the ability to confer resistance against a broad diversity of pathogens, r genes encode only five classes of proteins. the largest class of r proteins contains a “nucleotide binding site and leucine-reach repeat” (nb-lrr) domains and thought to be cytoplasmic (although they could be membrane-associated). based on the n-terminal structures, the nb-lrr class can be subdivided into two subclasses: one that has homology to the intracellular signalling domains of the drosophila toll and mammalian interleukin (il)-1 receptors (tir-nblrr), and the other consists of coiled-coil domain (cc-nblrr). to a lesser extent, the other classes of r proteins consist of receptor-like kinases (rlks), membrane-anchored receptorlike proteins (rlps), and serine-threonine protein kinases that are mostly transmembrane or membraneassociated. the nb-lrr proteins function so far exclusively in disease resistance. plant nb-lrr proteins share broad similarity with mammalian caterpiller/nucleotide oligomerisation domain (nod)/nod-like receptor (nlr) proteins family (ting and davis 2005), and stand atpases (leipe et al. 2004). the most common feature of most r proteins is the presence of an lrr domain in variable length. in other proteins, lrr domains function in protein-protein interaction, protein-ligand binding, and carbohydrateprotein interaction (kajava 1998). indeed, domain swapping experiments suggest that lrr domain play a role for r proteins – effectors interaction specificity (ellis et al. 1999; luck et al. 2000). in addition, comparative sequence analyses predicted that lrr is solvent-exposed and is under diversification pressure from highly evolving pathogen effectors (michelmore and meyers 1998; kobe and kajava 2001). for instance, the flax l alleles l2 and l10, differ in amino acid sequences of their lrr domains and recognise different effectors. swapping the lrr of l10 with that of l2 changed its specificity in effector recognition (ellis et al. 1999). however, analyses of flax l proteins suggested that besides of lrr, tir domain is also under diversification pressure and this domain determines the specificity of l proteins with the corresponding avr proteins of melamspora lini (see below) (luck et al. 2000). pamp-triggered immunity effectors suppress pamp-triggered immunity effectors recognition by r proteins rlk mapkkk mapk wrlu nucleus pamp-triggered immune responses wrlu nucleus wrlu nucleus mapkkk mapkkk mapk mapk rlk effectortriggered immune responses iir-nb-lrr cc-nb-lrr resistance susceptibility resistance , kinase; , leucine-rich repeats; , nucleotide binding; , toll-interleukin-1receptor; , coiled-coil; , effectors rlk microbiol indones 101volume 3, 2009 based on animal model, the tir and cc domains from plants are suggested to be involved in protein-protein interactions that recruit host signalling partner proteins. recent work suggests the role for tir domain in stabilisation of r-avr interaction (directly or indirectly) and defence signalling based on observation in nicotiana n protein that associates with the tobacco mosaic virus (tmv) – derived effector protein, p50 (burch-smith and dinesh-kumar 2007; burchsmith et al. 2007). the n’s tir domain is necessary for n – p50 association, and the tir domain itself is sufficient to associate with p50. this domain interacts with p50 indirectly and requires other yet unknown proteins for n activation and defence signalling to ensue. the central domain of r proteins (the nb domain) has a nucleotide-binding pocket and modular functions for atpase activity to hydrolyse atp in vitro (tameling et al. 2002). mutations in the conserved motifs of nb domain impair its atpase activity, but not its binding capacity, and cause autoactivation leading to pathogen-independent hypersensitive response (hr) induction in planta (tameling et al. 2006). the lr10 leaf rust resistance gene of wheat is unusual in a way that the nterminus of the cc domain is under strong diversifying selection (caroline et al. 2009). moreover, resistance conferred by lr10 requires two related, yet unidentified ccnb-lrr proteins. very little is known about the mechanism of nb-lrr protein activation. activation of these proteins involves intraand intermolecular conformational changes and seems to be under negative regulation. mutation in the lrr domain or nb domain can lead to autoactivation and trigger autodefence responses, suggesting that intramolecular twist or domain interaction negatively regulates nb-lrr activation (bendahmane et al. 2002; shirano et al. 2002). heat shock protein 90 (hsp90) and other receptor cochaperones are involved in nb-lrr folding, rendering it signal-competent prior recognition of pathogen effectors or their targets (fuente et al. 2005). activation of nb-lrr involves avrdependent release of atpase domain from inhibition by lrr followed by multimerisation that forms a complex, which recruits additional proteins to the n terminus for signalling (deyoung and innes 2006). microbial pattern recognition triggers pti. in plants, distinguishing between self and non-self through perception to widely conserved microbial molecules is perceived by pattern-recognition receptors (prrs) that are mostly receptor-like kinases (rlks). rlks are transmembrane proteins consisting of divergent extracellular domains involved in ligand interaction and intracellular kinase domains that modulate downstream signalling. microbial patterns perception subsequently prompts pti. although the complete mechanism of pti has not been elucidated, studies on bacterial flagellin, a subunit protein of flagella, have provided invaluable information on the induction of pti. induction of pti involves mitogen-activated protein (map) kinase signalling cascade, expression of defencerelated proteins, reactive oxygen species (ros) burst, and cell wall strengthening or callose formation, all of which contribute to an arrest of pathogen growth (nürnberger et al. 2004). in addition to flagellin, gramnegative bacteria also contain lipopolysaccharide, which can also trigger pti. similarly, chitin, glucan, and ergosterol, which are major constituents of fungal cell wall, are also elicitors of pti. the nand c-termini of flagellin are more conserved among bacteria compared with the central part. a 22-aminoacid peptide (flg22) from the n-terminus of flagellin is sufficient to induce activation of many cellular responses in arabidopsis thaliana (felix et al. 1999). flg22 is perceived by flagellin sensing 2 (fls2), a receptor-like kinase (rlk) that contains extracellular lrrs and intracellular serine/threonine kinase domain (gómez-gómez and boller 2000). fls2 binds to flg22 and acts in early stages of pathogen invasion (chinchilla et al. 2006). early activation of fls2 is inferred from mutants lacking fls2 that are more sensitive to flagellin from pseudomonas syringae pv. tomato dc3000 in spray application, but not in syringe infiltration into the leaf apoplast (zipfel et al. 2004; sun et al. 2006). a rapid deployment of defence responses following perception of flg22 by fls2 requires somatic embryogenesis receptor kinase (serk)3/brassinosteroid-associated kinase (bak)1, which is also an rlk (chinchilla et al. 2007; heese et al. 2007). serk3/bak1 links signal perception and transduction in pti, not only to flg22-dependent pti, but also to some unrelated pamps-dependent pti, suggesting a role for this rlk in integrating diverse pamp perceptions into downstream responses. downstream of flagellin perception by fls2 is the activation of map kinase cascade and wrky transcription factor that culminate in induction of defence responses. however, arabidopsis plants overexpressing wrky showed enhanced resistance to both bacteria and fungi, suggesting that resistance mechanisms following flagellin perception are not specific to bacteria. alternatively, plants respond to pamps and activate defence responses using diverse map kinase cascade components and transcription factors that converge to signal multiple pamps perception (asai et al. 2002). plant perception of bacterial elongation factor and cold shock proteins (ef-tu) activates similar defence responses to flg22 (kunze et al. 2004; zipfel et al. 2006). in arabidopsis, ef-tu is perceived by another rlk protein called efr. the first 18-amino-acids of ef-tu are sufficient to induce expression of genes that are also activated upon flg22 recognition (zipfel et al. 2006). moreover, flg22 can activate transcription of efr. hence, it is likely that similar defence responses comprising pti following pamp recognition converge on common signalling pathways. however, arabidopsis double mutant lacking fls2 and efr-1 is still able to induce pti after treatment with agrobacterium cell extracts (zipfel et al. 2006). it hints a possibility that there should be additional pamps and corresponding receptors. there are over 600 rlks genes in arabidopsis thaliana, and it is possible that many are pamp receptors function to recognise a broad range of pamps. pti suppression and host resistance. logically, it would be surprising if pathogens were to carry avr genes without any other functions other than to allow recognition by the corresponding r genes. one possible explanation is that avr proteins often act as virulence factors in the absence of r proteins partner (susceptible plants). in contrast to most microbiol indones102 review r genes, effector genes are remarkably diverse (luderer and joosten 2001). an individual strain of a bacterial pathogen delivers effectors into its hosts ranging from 20 to nearly 100 effectors. they are also diverse in their biological functions that all contribute to the virulence of pathogens in plants. compared with pamps, which conceptually are surface-derived structural molecules, effectors are relatively high-evolving molecules that often function as virulence factors. plant pathogenic microbes release effectors to attain a subtle evasion to host plant defence responses. effectors from bacteria are delivered into host cell cytoplasm or apoplast through their type iii secretion system (ttss) machinery (petnicki-ocwieja et al. 2002; alfano and collmer 2004). some pathogenic fungi and oomycetes use specialised feeding structures called haustoria to facilitate intimate interface with their hosts. both systems deliver effectors that function in virulence, either by inhibiting or mimicking cellular process in plants. to achieve this, most effectors have function in suppressing pti. for example, resistance to non-host pseudomonas requires nonhost1 (nho1) that is activated upon recognition of flagellin. however, nho1 is ineffective against virulent p. syringae dc3000. dc3000, as many other strains has flagellin that is potent in triggering pti, but this form of immunity fails to halt dc3000 growth because it produces effectors that contribute to pti suppression. at least nine effectors were defined to be capable of suppressing nho1 expression (li et al. 2005). pti suppression is also evident in bean deploying more rapid and elevated defence responses against p. syringae mutant strain that is unable to inject any type iii-effectors compared with bean treated with the isogenic wild-type strain (wei et al. 2007). hence, the diminishing of pti allows pathogens to achieve successful colonisation in host plants. plants have evolved r proteins as a surveillance system for effector traffic in host plants. r-mediated responses (eti) are typically characterised by host programmed cell death (pcd), a form of hypersensitive response that is usually restricted to the site of pathogen ingress, leaving the surrounding cells unaffected (hofius et al. 2007). eti is aimed to restrict the fitness of successful pathogen capable of suppressing pti. although the mechanism of arresting pathogen growth by hr is unclear, at least in pathosystems undergoing r-avr interaction, hr is the most common and effective defence response against pathogens. moreover, hr is not always observable, nor it is per se a requirement for eti. thus, it is unclear what actually retards the growth of pathogens in resistant host plants. what biochemical functions do effectors govern to achieve successful colonisation in plants? most pathogen effectors that have been characterised biochemically exhibited proteolytic activity, suggesting that virulence mainly involves host protein degradation. however, the enzymatic activities of many effectors remain obscure since their sequence and structure predictions do not give clues to their cellular functions. some effectors have unique characteristics and are likely to act in a diverse fashion for virulence. here, we highlight only some new examples. hopu1 from the bacterial pathogen p. syringae is a monoadp-ribosyltransferase (adp-rt) that suppresses plant immunity by targeting rna-binding proteins that possess rna-recognition motifs (rrms) (fu et al. 2007). adpribosylation of rna-binding proteins affects rna metabolism and plant defence transcriptome, and eventually suppresses plant immunity. another mechanism of virulence in p. syringae is that this bacterium impedes host cell vesicle transport. the p. syringae hopm effector manipulates arfegf proteins likely to be involved in host vesicle transport (nomura et al. 2006). in fungi, exocytosis is an important step during infection as a mean of delivering a cargo of molecules required for pathogenicity including effectors. in magnaporthe grisea, an integral membrane p-type atpase, mgapt2, is required for exocytosis during infection in plants (gilbert et al. 2006). m. grisea mutants lacking mgapt2 are defective in the ability to secrete several extracellular enzymes and form abnormal golgi-like cisternae. the enzyme is also required by the fungus for successful foliar and root infection, but in incompatible interaction, it triggers a rapid induction of plant defence responses. plant pathogens may also exploit the “compatibility factor” in plants for their virulence. for instance, pthxo1, a type iii effector from xanthomonas oryzae strain pxo99a targets os8n3, a dominant susceptibility gene in rice that is induced during disease development (yang et al. 2006). pthxo1 is a member of transcription activator-like (tal) effector family. rnamediated silencing of os8n3 leads to resistance against pxo99a infection, but not to other strains. in addition to os8n3, pxo99a affects the expression of two additional plant genes, ostfx1 and ostfiiaγ1 (sugio et al. 2007). ostfx1 encodes a bzip transcription factor, whereas ostfiiaγ1 encodes a subunit of the transcription factor iia that resides in chromosome 1 of rice, respectively. expression of both genes is dependent on two type iii effectors, pthxo6 and pthxo7. an interesting evidence is found in interaction between lov1 in a. thaliana and victorin from cochliobolus victoriae. lov1 confers susceptibility in a. thaliana to c. victoriae (lorang et al. 2007). surprisingly, lov1 encodes a cc-nb-lrr protein that shares extensive similarity to the rpp8 resistance gene family. c. victoriae produces victorin, a host-selective toxin (hst), necessary for the fungus to cause victoria blight disease in oats. although lov1, in the presence of victorin, confers susceptibility to c. victoriae, it also induces the production of defence-related proteins similar to resistance-like physiology. nonetheless, alterations in known defence response pathways do not alter susceptibility to c. victoriae, suggesting that these defence response pathways are dispensable for susceptibility towards c. victoriae. these demonstrate that a plant nb-lrr gene can confer both resistance and susceptibility. some pathogens are shown to be able to subvert plant defence by suppressing r-mediated host resistance. interaction between fusarium oxysporum f.sp. lycopersici (fol) and tomato plants is race-cultivar specific. races of fol are historically named according to the r gene that counters them in order of their discovery. fol race 1 is countered by the i (immunity) gene and the unlinked i-1 gene; fol race 2 is virulent towards i and i-1 tomato plants, but is impeded by i-2; and fol race 3 is virulent on i, i-1, and i-2 tomato plants, but is stopped by i-3 (fig 2). surprisingly, avr1 that is only present in fol race 1 and recognised by the microbiol indones 103volume 3, 2009 fig 2 he gene-for-gene interaction between fusarium oxysporum f. sp. lycopersici (fol) with tomato plants. the interaction between fol with tomato plants is race-cultivar specific. arrows indicate the resistance in tomato plants conferred by the resistance genes against their corresponding avirulence genes in fol races. fol race 1 that carries all the avr1, avr2, and avr3 is countered by the i (immunity) gene and the unlinked i-1 gene; fol race 2 is virulent towards i and i-1 tomato plants, but is impeded by i-2; and fol race 3 is virulent on i, i-1, and i-2 tomato plants, but is stopped by i-3. however, some natural fol race 1 isolates are known to be virulent on i-2 and/or i-3 plants, and introduction of avr1 in race 2 and race 3 renders them gaining virulence on i-2 and i-3 plants, while removal of avr1 in race 1 leads to loss of virulence towards i-3 plants. avr1 that is present only in fol race 1 and recognised by the i and i-1 genes, suppresses the i-2 and i-3-mediated resistance. i and i-1 genes, suppresses the i-2 and i-3-mediated resistance (houterman et al. 2008). some fol race 1 strains are known to be virulent on i-2 and/or i-3 plants (mes et al. 1999; rep et al. 2005), and introduction of avr1 in race 2 and race 3 renders them gaining virulence on i-2 and i-3 plants, while removal of avr1 in race 1 leads to loss of virulence towards i-3 plants. suppression of r-mediated resistance has also been observed in bacteria (janjusevic et al. 2006; rosebrock et al. 2007) and oomycetes (dou et al. 2008). molecular events following pathogen recognition by r proteins involve ion fluxes, activation of map kinase and other protein kinases accompanied by production of reactive oxygen intermediates (rois) (ligterink et al. 1997; romeis et al. 1999) and nitric oxide (no) (delledonne et al. 1998; durner et al. 1998; delledonne et al. 2001), and transcriptional reprogramming of genes mostly involve in defence responses (kan et al. 1992). biochemical changes ensued upon elicitation by non-specific and race-specific elicitors are rapid. ion fluxes occur within minutes, rois and no are subsequently produced, and protein kinases pathways are activated. downstream of these pathways is activation of defence genes function in induction of hormonal signalling molecules, cell wall strengthening, production of antimicrobial compounds, and programmed cell death as a form of hypersensitive response, all of which contribute to resistance (hammond-kosack and jones 1996; mcdowell and dangl 2000). non-specific, systemic acquired resistance (sar) establishment is also preceded by these events (durrant and dong 2004). the deployment of defence responses is dependent on salicylic acid (sa), jasmonic acid (ja), and ethylene that act as regulators of signalling pathways mediating plant responses to pathogens with different lifestyles. sa mediates defence responses against biotrophic pathogens, whereas ja activates defence responses to necrotrophic pathogens. in compatible interaction, saand ja-dependent pathways are mutually antagonistic in a way that activation of sa as a response to a biotrophic pathogen renders a plant more susceptible to a necrotrophic pathogen when both pathogens are in close proximity (spoel et al. 2007). however, the trade-off control is weak in spatial inoculation of both pathogens and absent in incompatible interaction. the latter suggests a mechanism of plants to prevent the growth of necrotrophic pathogens in cells undergoing programmed cell death. it seems that plants tightly regulate the tradeoffs between sa and ja to prevent unfavourable signal interactions and maximise their versatility to defend themselves from multiple attackers. programmed cell death is typically accompanied by systemic acquired resistance, which is broad spectrum and long lasting. ja, but not sa, acts as a mobile signal in transmitting long-distance information and mediates the systemic acquired resistance (sar) (truman et al. 2007). transcriptional signature of ja-mediated sar overlaps with local basal defence and wounding, as well as herbivory responses, indicating that ja-dependent signalling pathways are evolutionarily conserved that mediate biotic and abiotic responses. sa is known to regulate plant defence responses mediated by r genes. interplay between sa-mediated responses and mamp-triggered responses involve overlapping functions of two sa signalling components sid2 and pad4, which in arabidopsis, disruption of these components affects mamp-triggered responses to flg22 of pseudomonas syringae pv. tomato dc3000 (tsuda et al. 2008). recognition of pathogen effectors. sequence analyses of many effectors usually do not give clues on their cellular fol races fol genotype i genes race 1 (worldwide) race 2 (worldwide) race3 (north america and australia): from early 1980’s avr1 avr2 avr3 avr2 avr3avr1 avr1 avr2 avr3 i 1 2 1 3 104 review microbiol indones functions (kamper et al. 2006; does and rep 2007). nonetheless, some can be identified as having enzymatic activities or other cellular functions. those that have cellular functions would logically target host proteins for virulence, and r proteins would recognise these perturbations on host proteins. plants recognise pathogen effectors either through direct r-avr interaction or indirectly through recognition of changes in structural integrity of host proteins following their interaction with avr proteins (jones and dangl 2006). the simplest model for r-avr interaction would be r proteins, specified by the lrr domain, recognise avr proteins directly (the receptor-ligand model). domain swapping and mutational analysis revealed that the lrr domain govern recognition specificity in r proteins. however, as will be described herein, only few demonstrations of direct r-avr interaction have been described. direct r-avr interaction is possibly best exemplified by the flax l5, l6, and l7 r proteins with the corresponding avrl567 protein in flax rust fungus, melampsora lini (dodds et al. 2006). the l loci encode nblrr proteins with different specificities to avr proteins in various strains of m. lini. in yeast, l5, l6, and l7 r proteins interact physically with avrl567 protein with specificities consistent with responses observed in planta, indicating that recognition specificity between these proteins is based on direct interaction. amino acid sequence differences in avrl variants render different recognition in both flax and yeast, and variation in specificity is associated with several polymorphic sites. direct r-avr interaction is also observed in the rice pi-ta protein and the avr-pita protein from magnaporthe grisea (jia et al. 2000). based on yeast two-hybrid system and in vitro binding assays, avr-pita binds specifically to lrr domain of pi-ta. another example is the rrs1-r protein that interacts with a nucleus-targeted type iii effector of ralstonia solanacearum, popp2 (deslandes et al. 2003). rrs1-r, a member of tir-nb-lrr is an unusual r protein because it has the wrky motif characteristic of some plant transcriptional factors. physical interaction between rrs1r and popp2 was observed in yeast although the domains in rrs1-r responsible for the interaction could not be determined. instead, a full-length rrs1-r protein is required for such interaction. nuclear localisation of rrs1-r requires interaction with popp2, and indeed, both proteins are colocalised in the nucleus of protoplasts. interestingly, rrs1s, a protein with high similarity to rrs1-r but present in susceptible ecotype also binds to popp2 in yeast and is colocalised into the nucleus. however, in many cases, simple physical interaction does not necessarily imply the detection of effectors by r proteins per se. as mentioned above, pathogen effectors may be recognised by r proteins indirectly. observation in many other pathosystems failed to detect direct r-avr interaction, and this led to the formulation of “guard hypothesis”, which proposes that an r protein detects the perturbation of a host protein by the cognate avr determinant. the host protein is the molecular target for the virulence function of the avr determinant and, on the other hand, the surveillance target of the cognate r protein for such interference. one host protein may be guarded by more than one r proteins and may be the target of several pathogen effectors as well. rin4 (rpm1 interacting 4) is required for rpm1-mediated resistance in arabidopsis against p. syringae carrying two unrelated type iii effectors, avrrpm1 and avrb (mackey et al. 2002). rin4 also mediates interaction between the rps2 (resistance to p. syringae pv. tomato 2) and the p. syringae avrrpt2 type iii effector (axtell and staskawicz 2003; mackey et al. 2003). as in the case of rrs1s and popp2 interaction (see above), rps2 coimmunoprecipitates with avrb and produces nonproductive complex (leister and katagiri 2000). the corresponding r protein for avrb is rpm1. such overlapping interactions suggest that either direct or indirect interaction of r/avr does not necessarily imply r proteins per se confer genefor-gene specificity. other component(s) are required to form active and specific complexes of effector recognition. rin4 is a 211-acylated-amino acid that possesses plasma membrane-associated domain, which is by far known to be manipulated by three different bacterial type iii effectors and “guarded” by two nb-lrr proteins. avrrpm1 and avrb both interact and phosphorylate rin4, and such modification induces activation of rpm1 (mackey et al. 2002). the p. syringae avrrpt2 is a cystein protease that is autoprocessed and activated inside host cells, which then it destroys rin4 by cleaving it at two sites (kim et al. 2005). this cleavage has two consequences: activation of rps2 on one hand, and interference of rpm1 function on the other hand. besides rin4, additional host proteins may also be the targets of avrrpm1 or avrrpt2 since the elimination of rin4 did not abolish the virulence to a weakly pathogenic strain possessing these effectors in susceptible plants (rin4 rpm1 rps2) (belkhadir et al. 2004). rin4 interacts with ndr1 (nonrace specific disease resistance 1), a gpi-anchored protein that is required for the activation of both rpm1 and rps2 (day et al. 2006). in solanaceae, the mechanism of rin4 degradation overlaps between tomato and nicotiana benthamiana (luo et al. 2009). this degradation is induced by an effector avrpto from p. syringae and dependent on resistance proteins pto and prf in tomato and n. benthamiana. analysis on effector secretome in p. syringae reveals other effectors, besides of avrrpt2, which can also elicit rin4 proteolysis. this suggests that rin4 could be a common target for several effectors from p. syringae. another example of indirect interaction has been shown in the interaction between the rps5, a cc-nb-lrr protein in arabidopsis and the avrpphb, a type iii effector from p. syringae (shao et al. 2003). rps5 is ndr1-independent, and its activation requires pbs1 (avrpphb susceptible 1), which is a serine-threonine protein kinase that is cleaved by avrpphb. both kinase activity and cleavage of pbs1 are required for rps5 to function, suggesting that cleaved pbs1 retains its enzymatic activity and contributes to the activation of rps5. similar to avrrpt2, avrpphb is a cysteine protease that is self-cleaved and activated inside host cells and is nonetheless able to cleave pbs1 at a site in the kinase activation region homologous to its own cleavage site. direct and indirect recognition of effectors by r proteins will result in different consequences on how pathogens modify their effectors in order to evade the surveillance system in their hosts. effectors that are recognised directly will undergo diversification but retain their virulence capacity. whereas microbiol indones 105volume 3, 2009 effectors that are recognised indirectly, they will be discarded by the pathogens. although discarding such effectors might be such a significant penalty for the pathogens, evidence indicated that the polymorphism in such effectors is presence or absence. in plant populations, the guarded effector targets are evolutionary unstable depending on the presence or absence of the r gene. opposing selection forces occur in the guarded effector targets predicted from evidence that many pathogen effectors have multiple targets in the host and most effectors retain their virulence despite the absence of the r gene. in the absence of the r protein, the guardee proteins will be under selective pressure to evade manipulation by effectors while, on the other hand, in the presence of r protein the guardee proteins will be under selective pressure to improve perception by the effectors. these conflicting selection forces impose evolutionary constraints on the guarded effector targets that would be relaxed in the presence of a “decoy” that mimics the operative guarded effector targets by acting as a coreceptor regulating r gene activation (hoorn and kamoun 2008). the decoy would arise from duplication of guarded effector target genes followed by subsequent evolution, or evolve independently by mimicking effector targets. decoy exclusively functions in effector perception with no association in development, disease, or resistance. distinctive to the guard hypothesis, decoy does not support pathogen fitness in the absence of r gene. evidence supporting the decoy model is inferred from several cases of effector perception involving rin4 (belkhadir et al. 2004; lim and kunkel 2004; chisholm et al. 2005; takemoto and jones 2005), rcr3 (shabab et al. 2008), pto (mucyn et al. 2006; xiang et al. 2008), and bs3 (schornack et al. 2008; zhou and chai 2008). however, the decoy model still requires experimental evidence, and therefore, it is a challenging platform for our understanding on effector perception. conclusion and conundrum. immunity of plants to pathogens is largely dependent on the polymorphism capacity of prr and r proteins that recognise pathogen molecular patterns and avr proteins or their interference on plant defence system. recognition of mamps/pamps is a prerequisite of pti that confers plants resistant to potential non-host pathogens. pti seems to be effective in protecting plants against most non-host pathogens but ineffective to host-specialised pathogens. evidence from studies of bacterial type iii effectors suggest that effector proteins contribute to the virulence of pathogens by suppressing pti. such interference is countered by plants through r proteins that recognise avr proteins (direct interaction) or their perturbation on host proteins (indirect interaction). the resulting eti is highly effective in such it is able to restrict pathogen growth and spread. however, there are still more questions than answers as to our understanding on plant immunity is still very limited. the response outputs between pti and eti are similar. do the signalling and transcriptional factors between pti and eti overlap? dissecting the interplay between them is pivotal because there, is where pathogens most likely manipulate the plant defence, and on the other hand plants regulate critical molecules that activate and/or switch these defence modes. some prr proteins are also involved in plant development as well as response to abiotic stress. some effectors mimic plant hormones to suppress pti. coronatine, a small protein produced by p. syringae mimics jasmonic acid that can suppress salicylic acid signalling pathway necessary for defence against biotrophic pathogens (zhao et al. 2003; brooks et al. 2005). taken together, plants seem to regulate the interplay between pti, eti, abiotic stress, and hormonal metabolism to counteract pathogen attack. certain plant nb-lrr proteins act in the nucleus to trigger downstream signalling and defence pathways. mildew a (mla) r proteins in barley function in the nucleus to confer resistance against the powdery mildew fungus. mla10 r protein recognises the a10 avr protein, and this recognition induces association of mla10 and wrky transcription factors in the nucleus (shen et al. 2007). the wrky transcription factors suppress the pti and mla interferes with the wrky suppression to de-repress pti, therefore defence ensues. this evidence suggests a common transcriptional factors function in pti and eti as well as an integration of mechanism of defence responses conferred by prr and r proteins to distinct pathogen signals. although in many observations hr is effective in arresting pathogens, knowledge on the mechanism of how it can arrest pathogen growth is lacking. nonetheless, in some pathosystems, necrotic hr is not involved in arresting pathogen growth. for example, potato rx protein can mediate an extreme resistance against potato virus x (pvx) without causing a necrotic hr (bendahmane et al. 1999). in other case, resistance to wilt pathogens fusarium oxysporum, which colonises plants in the vascular system, does not involve pcd distinctive to most resistance responses to foliar pathogens. thus, cell death and pathogen arrest in disease resistance might represent separable mechanisms. programmed cell death is specific to eti and is generally not associated with basal defences. several pathogen effectors can suppress pcd either by altering expression of some crucial plant genes or by interfering key components involved in signalling pathways. therefore, some pathogens target pcd as a form of hr in eti for their virulence in host plants. this indicates the evolutionary arms race between host plants and pathogens is extending beyond the first level of eti. we need to understand the coevolution of pathogen effectors and r genes in plants. why are most plants resistant to most pathogens? host specificity seems 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endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi iman rusmana, 2020 page 1 editorial board.pdf page 1 4. 621 characterization (seti... characterization and phylogenetic analysis of soybean rhizobial strains from java and sumatra setiyo hadi waluyo center for the application of isotopes and radiation technology, national nuclear energy agency (batan), jalan lebak bulus raya pasar jum'at, jakarta 12070, indonesia bradyrhizobium japonicum b. japonicum b. japonicum bradyrhizobium elkanii bradyrizobium japonicum sinorhizobium fredii b. elkanii b. japonicum s. fredii crossinoculation cross-inoculation” promiscuous bradyrhizobium japonicum b. japonicum promiscuous distinct bradyrhizobium japonicum bradyrhizobium elkanii bradyrhizobium japonicum sinorhizobium fredii. b. elkanii b. elkanii b. japonicum s. fredii ardra cross-inoculation sequencing twenty-seven and twenty-four soybean rhizobial isolates from java and sumatra, respectively,were characterized. based on cross-inoculation, eight isolates from java and nine from sumatra could be grouped as soybean specific rhizobial species , while 19 isolates from java and 15 from sumatra were promiscuous. ardra of intergenic spacer region of 16s-23s rdna showed that the isolates from java were different from those from sumatra. six soybean specific isolates from java and one from sumatra were in the same cluster with the reference strain, usda 110, thus could be classified as . one soybean specific isolate from java's has a distinct position, while the other soybean specific isolate from java was placed in another group dominated by isolates from sumatra. the nineteen promiscuous isolates from java were clustered in a different group. this group, together with the isolate with distinct position and the other group that were dominated by isolates from sumatra, were distinct from usda 110. therefore it is tempting to speculate that they represent indigenous soybean rhizobial strains. based on complete sequencing of the amplified 16s rdna of 21 selected isolates, these isolates could be divided into three groups consisting of twelve , eight and one . most of the strains were isolated from acid soils at sitiung, west-sumatra, while only two isolates were obtained from java. four isolates from java, two isolates from sitiung, and two isolates from bukit tinggi were identified as . one isolate from java with a distinct position on the ardra was identified as . key words: amplified ribosomal dna restriction analysis (ardra), cross-inoculation, rhizobia, soybean, sequencing dua puluh tujuh isolat bakteri rhizobia kedelai dari jawa dan 24 isolat dari sumatra telah diidentifikasi secara pada tanaman kedelai dan kacang hijau serta secara genetika molekuler (ardra dan pemetaan). dengan metode “ , 8 isolat jawa dan 9 isolat sumatra dapat membentuk bintil akar efektif hanya pada tanaman kedelai (spesifik), sedangkan sisanya 19 isolat jawa dan 15 isolat sumatra dapat membentuk bintil akar efektif pada tanaman kedelai dan kacang hijau ( ). metode ardra dapat membedakan antara isolat dari jawa dan sumatra. enam isolat jawa dan tiga isolat sumatra berada dalam satu grup dengan usda 110, dan diklasifikasikan sebagai . satu isolat jawa spesifik kedelai menempati posisi tersendiri, dan sisanya 1 isolat berada dalam grup yang didominasi oleh isolat-isolat sumatra. sembilan belas isolat jawa membentuk grup tersendiri, bersama dengan satu isolat yang posisinya dan grup yang didominasi oleh isolat-isolat sumatra. grup ini berbeda nyata dengan usda 110. oleh karena itu, diduga kuat bahwa isolat-isolat tersebut adalah bakteri rhizobia kedelai asli indonesia. pemetaan deret ribosomal dna 16s dari 21 isolat pilihan menunjukkan bahwa isolat-isolat tersebut dapat dibagi menjadi tiga grup, yaitu grup 12 isolat, grup delapan isolat, dan satu isolat yang khusus tersebut masuk dalam grup sebagian besar galur di peroleh dari tanah asam di sitiung, sumatra barat. hanya dua strain yang berasal dari tanah jawa. empat isolat jawa, dua isolat bukit tinggi dan dua isolat sitiung diidentifikasi sebagai . satu isolat yang khusus diidentifikasi sebagai . kata kunci: , , kedelai, rhizobia, leguminous plants nodulating bacteria were first described in 1888 (beijerinck 1888) and their initial classification as rhizobia was based on their host range specificity. their symbiotic relations with the host plants remains very important, since this is the most conspicuous feature of rhizobia and has an important practical value. however, the symbiotic and physiological properties of soybean nodulating rhizobia have been found to be more diverse than originally anticipated. the discovery of fast-growing soybean nodulating rhizobia belonging to the genus ( ) (keyser . 1982; schollasinorhizobium ensifer et al and elkan 1984; young 2003, young 2010) underlines the need to consider the symbiotic properties, particularly since representatives of this genus often fail to nodulate modern soybean cultivars. in addition, there are some rhizobia outside the genus and ( ), now designated spp., that were reported to form nodules on soybean (chen . 1995; jarvis . 1997). the ambiguous results often found with hostrange nodulation tests have driven the development of dna based determination techniques (laguerre . 1994; massol-deya . 1995; vandamme . 1996; rademaker and de bruijn 1997). in recent years, other methods, including phenotypic traits, dna:dna relatedness and bradyrhizobium sinorhizobium ensifer mesorhizobium et al et al et al et al et al *corresponding author, phone : +62-21-7690709, fax : +62-21-7513270, e-mail: shwaluyo@yahoo.com issn 1978-3477, eissn 2087-8575 vol 5, no 4, december 2011, p 170-181 i n d o n e s i a available online at: http://www.permi.or.id/journal/index.php/mionline doi: 10.5454/mi.5.4.4 molecular techniques based on polymerase chain reaction (pcr), have been included in rhizobial classification (sikora and redzepovic 2003; kwon . 2005; el-fiki 2006, martens . 2007). presently, there is a great variety of phenotypic and genotypic methods available that permit a different degree of phylogenetic classification varying from genus, species, subspecies, biovar to the strain level. however, the description of new genera and species of root-nodulating rhizobia should fulfill a minimal standard, as has been proposed by graham (1991) and novikova (1996). the development and implementation of these molecular techniques have accelerated the taxonomic evaluation of rhizobia. the current classification of rhizobia, which is mainly based on the nucleotide sequences of the small subunit ribosomal rna (rrna), are ( ), , ( ) , , , , , , and (hungria . 2006; willems 2006; raychaudhuri . 2007; rivas . 2009, lu . 2011). the importance of rhizobial strains for soybean cultivation in indonesia had already been shown since a long time ago (toxopeus 1938). meanwhile, the taxonomy of and genera is of particular interest as they consist of strains known to nodulate soybean, which is an important food crop. most of soybean nodulating rhizobial strains from the primary gene centers for soybean in china or japan have been well studied and characterised. however, while indonesia is supposed to be a second gene-centre of soybean plants, (hymowitz and newell 1981) there is only a limited information on the diversity of rhizobial strains from indonesia. there is a general lack of information on the population structure of the indigenous soybean rhizobia native to indonesian soils. however, insight in the structure of indigenous soybean rhizobia populations is one of the important aspects for the success of biological nitrogen fixation (bnf). particularly in the areas where indigenous rhizobia are present abundantly (saono 1988). as a consequence, a thorough survey is needed to study the occurrence of the bacteria in different locations in indonesia. furthermore, it is essential to characterise the isolates using reliable molecular methods. it opens the possibility to select elite indigenous soybean rhizobia under favourable conditions, such as those in java, where they are abundantly present in most soils. in addition, under acidic condition such as in sumatra, et al et al et al rhizobium agrobacterium bradyrhizobium sinorhizobium e n s i f e r , m e s o r h i z o b i u m , a z o r h i z o b i u m methylobacterium burkholderia cupriavidus devosia ochrobacterium phyllobacterium shinella et al et al et al et al bradyrhizobium sinorhizobium where the condition is unfavourable for rhizobia, the survey may be important to select rhizobial strains that are well adapted to stress conditions. in this study, therefore, a comparison was made between indigenous soybean rhizobia isolated from traditional soybean areas in java and a variety of soils in sumatra. the bacterial isolates were characterised for their symbiotic properties and classified based on amplified ribosomal dna restriction analysis (ardra) of pcr-amplified 16s rdna and 16s-23s rdna intergenic spacer region. a selected number of isolates were characterised in more detail by the amplification, cloning, and sequence analysis of the major part of their 16s rdna genes. fifty-one soybean rhizobial isolates from java and sumatra, usda 110 and cb1809 and c b 7 5 6 ( o b t a i n e d f r o m t h e l a b o r a t o r y o f microbiology, wageningen university, netherlands) were maintained in yeast extract mannitol broth (yemb) solidified with 1.0% agar and grown for inoculum preparation in yemb at 30 °c for 4 7 days (somasegaran and hoben 1995). to obtain indigenous soybean nodulating rhizobial strains, local varieties of soybean seeds ( cv. tidar) obtained from the bogor research centre for food crops, bogor, indonesia and mungbean ( cv. manyar) seeds obtained from the centre for the application of isotopes and radiation technology, national nuclear energy agency, jakarta, indonesia were used to study the host specificity of the isolates. to characterize the symbiotic properties, the rhizobial isolates were used to inoculate soybean and mungbean plants grown on a modified hoagland-n free medium as described previously (winarno and lie 1979). plants were harvested 20 days after inoculation. colour and weight of shoots, as well as weight and number of nodules were determined. the capacity to fix n was deduced by comparing the colour and weight of the control and inoculated plants, all were grown in a n-free medium. genomic dna to be used as template for pcr amplification of the 16s rrna gene and the 16s-23s rrna intergenic spacer regions was extracted from yemb-grown bacterial cells as follows. four ml of bacterial culture containing 10 cfu ml was harvested by centrifugation and suspended in 450 µl te buffer (1 mm tris-hcl, 0.1 mm edta, ph 7.4) , containing 0.5% sds, followed by incubation at 37 °c with gentle shaking for 30 minutes. dna was extracted by the addition of an equal volume of phenol materials and methods rhizobial strains and media. agronomic analysis. genetic analysis. b. japonicum bradyrhizobium spp. glycine max vigna radiata 9 -1 volume 5, 2011 microbiol indones 171 buffered in 10 mm tris-hcl and 1 mm edta, ph 7.0 to the mixture. this extraction was repeated once using an equal volume of phenol and chloroform mixture. subsequently, nucleic acid was precipitated by the addition of equal volume of ethanol 90% (-20 °c) and 10% volume of sodium acetate (3 m, ph 5.2) . the precipitate was collected by centrifugation, dried and dissolved in 50 µl sterile water. the amount of the isolated dna was determined by electrophoresis on a 1% agarose gel containing ethidium bromide with 0.1 g of dna digested with iii (gibcobrl lifetechnology, breda, netherlands) as a reference. ribosomal dna fragments were amplified using a set of primers as described previously, primer 8f [ 5'-cac gga tcc aga gtt tgat (c/t) (a/c) tag tcc ag-3'] an primer 1510r [5'-gtt aa gtt actg (c/t) tac gtt gtt acg actt-3'] for 16s rdna pcr reaction (lane 1991; laguerre et al. 1994) and primer phr [5'-tgcggctggatcacctcctt-3'] and primer p23sroi [5'-ggctgcttctaagccaac3'] for 16s-23s rdna intergenic spacer pcr reaction (massol-deya . 1995). dna amplification using approximately 50 ng of genomic dna as a template in a 100 l pcr reaction volume was performed using a unoii thermocycler, biometra, göttingen, germany as described previously (massol-deya et al. 1995). the size and amount of the amplified dna was examined by electrophoresing 5.0 µl of pcr product on a 0.7% agarose gel containing ethidium bromide and dna digested with diii as a reference. approximately 400 ng of the amplified dna was digested using restriction endonucleases i, i, iii, and i (5 units per 25 µl reaction) following instruction of the manufacturer (gibcobrl lifetechnology, breda, the netherlands) and the generated fragments were separated by electrophoresis on 3% agarose gels containing ethidium bromide at 100 v for 2 hours. ardra fingerprints of 16s rdna and 23s-16s rdna (intergenic spacer region) pcrproducts were recorded as tiff files, analysed and used to prepare a dendogram based on predictions by unweighted pair group method using arithmetic averages (upgma) by molecular analysist software (biorad 1995). based on the effectiveness and the efficiency of the soybean nodulating rhizobia strains in biological nitrogen fixation of soybean, seven isolates from java and 14 isolates from sumatra were chosen for further examination by 16s rdna sequencing. the 16s rdna pcr products from the selected strains, were purified using qiaquick pcr purification kit following instructions of the hind et al hin cfo dde hae msp cloning of the 16s rdna pcr products and plasmid dna isolation. manufacturer (qiagen, hilden, germany), and were quantified by electrophoresis on 1.2% agarose gel with known amount of dna digested with diii as a standard (gibcobrl life technology, breda, netherlands). then the pcr-amplified 16s rdna fragments were cloned in jm109 using the pgem -t vector system following a procedure provided by the supplier (promega, leiden, netherlands). for plasmid dna isolation, one colony of ampicillin-resistant transformant was used to inoculate to luria-bertani (lb) broth medium containing ampicillin (100 g ml ), and incubated at 37 °c for 24 hours (manniatis . 1982). plasmid dna was isolated using a column wizard plus minipreps dna purification system (promega, leiden, netherlands). purified plasmid dna was quantified by electrophoresis on a 1.2% agarose gel with known amount of dna digested with diii as a standard (gibcobrl life technology, breda, netherlands). the purified plasmid dna (250 ng) was used as template for sequencing reaction. infrared-labelled primers were used for reaction, for the forward reaction primer sp6 ird800 (5' -gat tta ggt gac act ata g-3') and for the reverse reaction primer t7 ird800 (5'taa tac gac tca cta tag gg-3')(mwg biotech, ebersberg, germany). pcr sequencing reactions were performed with reagents provided by the supplier (amersham pharmacia biotech, freiburg, germany) and the following temperature setting: 93 °c for 3 minutes; 30 cycles of 93 °c for 30 seconds, 45 °c for 30 seconds, 70 °c for 15 seconds; and storage at 4 °c. the products were separated and analysed on a li-cor dna sequencer 4000l (licor, lincoln, nebraska, usa). before loading the samples (1.8 µl), the gel was pre-run for 30 minutes at 1000v. after loading samples, electrophoresis was carried out at 1000 v constant voltage while the gel was heated at 50 °c. the sequences of approximately 1000-1200 nucleotides both in the forward and reverse directions were obtained, corrected manually, and combined into a single contig of 1200 1500 of unambiguous sequence by using seqman ii dna star software (dnastar inc., madison, wisconsin, usa). the obtained 16s rdna sequence data were analysed for their homology with the blastn program from the genbank network (http://www.ncbi.nlm.nih.gov/cgi-bin/blast/). a rooted phylogenetic tree (neighbour joining) was calculated using the programmes and database from ribosomal rna alignment (arb), an environment sequence analysis. identification of the soybean rhizobia on the basis of 16s rdna homology. hin escherichia coli et al hin r -1 tm 172 waluyo et al. microbiol indones for 16s/18s/23s ribosomal rna sequence data, and position 150 1114 (felsenstein correction). the arb package is a software containing combination of alignment and dendrogram tools, allowing alignment to a comprehensive 16s rdna database and detailed phylogenetic analysis. in general, rhizobial strains obtained from java were as effective as the soybeannodulating reference strains usda 110 and cb 1809. the number of nodules were 43, 29, and 46 for usda 110, cb 1809 and isolates from java, respectively, while the total weights of nodules were 0.288, 0.25, and 0.263 g for usda 110, cb 1809 and isolates from java, respectively. the average shoot weight of soybean inoculated with usda 110, cb 1809 and isolates from java were 3.36, 3.7, and 3.75 g, respectively. on the other hand, the average number and weight of nodules, as well as shoots weight, of plants inoculated with the rhizobial strains from sumatra were 37, 0.225, and 2.43 g, respectively. this is significantly lower than those from java. the difference was most significant in the weight of shoots (= 0.01, mstat-c 1988, table 3). it was observed that the first two leaves of all soybean plants inoculated with rhizobial isolates from sumatra were yellow and in most cases were lost very quickly, indicating poor n fixing capacity. the ability of the isolated rhizobial strains to form nodule on mungbean plant was also tested (fig 1). this analysis revealed that most of the strains from both java and sumatra showed promiscuous nodulation properties that were not observed with the well-known inoculant strains of usda results agronomic aspects. e. coli b. japonicum b. japonicum b. japonicum b. japonicum b. japonicum 110 and cb 1809. genomic dna of the isolated rhizobial strains (27 from java and 24 from sumatra) and that of the reference strain usda 110 were isolated and used as templates for pcr amplifications of the 16s rdna and the 16s-23s rdnas spacer regions (fig 1). the amplified 16s rdna's all showed the expected size of approximately 1.6 kb and upon digestion with four different restriction enzymes ( i, i, iii and i) a variety (up to 19) of restriction length polymorphism patterns were observed. this ardra approach allowed the grouping of the rhizobial isolates into two main clusters, while java isolate j-tgs50 and sumatra isolates s-st123 and sst414 showed rather unique position (fig 2). one cluster consists of most (25) of the rhizobial isolates from java, four isolates from sumatra and also usda 110. the other cluster consists of most (18) of the rhizobial isolates from sumatra and only one strain isolated from java (j-yg49). both clusters contained isolates that showed only nodulation of soybean as well as ones with promiscuous nodulation properties. the pcr products of the 16s-23s spacer rdna were all of the same size of approximately 2 kb. differentiation of these 16s-23s rdna amplicons by ardra using the same restriction enzymes as used for the 16s rdna amplicons, revealed that these large dna fragments showed significant sequence variations (fig 3, fig 4, fig 5, fig 6). twenty-seven composite restriction pattern types were obtained by combining data from the digestion patterns (table 1). i was the most discriminating enzyme, with twenty-one (21) genotypes detected amplified ribosomal (16s and 16s-23s) dna restriction analysis (ardra). b. japonicum cfo dde hae msp b. japonicum dde 2322 2027 564 2322 2322 2322 2027 2027 2027 bpbp 2000 1600 564 fig 1 16s rdna (above) and 16s-23s rdna (below) pcr-products. volume 5, 2011 microbiol indones 173 among the rhizobial isolates. based on these complex ardra fingerprints, the rhizobial isolates were grouped into four main clusters (fig 7). all strains isolated from java, except for j-yg49, could be grouped into two clusters, one includes usda 110. again the java isolate j-tgs50 showed a unique position. similarly, all strains isolated from sumatra, except for strains s-st224, s-st325 and s-st123, could be grouped in one very large cluster, while two strains (s-bt221 and s-bt322) formed a cluster that was distantly related to that of usda 110. the obtained 16s rdna sequences of 21 selected, soybean-nodulating rhizobial strains from those b. japonicum b. japonicum indonesian isolates were compared to those of reference strains obtained from the genbank and arb databases (table 2). the high homology of the rdna sequences indicated that all the isolates, except for j-tgs50, could be assigned to genus and are related to either . or (table 2). strain tgs50, the unique isolate from java, was found to belong to genus and its 16s rdna sequence showed 97.7 % homology to that of . a rooted phylogenetic tree was constructed,showing that all rhizobial isolates from java and sumatra, except for strain j-tgs50, could be grouped into 2 species, and (fig 8). bradyrhizobium b elkanii b. japonicum sinorhizobium s. fredii b. elkanii b. japonicum 10090807060504030 java java sumatra sumatra java java java java java java java java java java java java java sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra sumatra java origin strain java java usda java java java java java java java java sumatra sumatra +/+4 effectiveness soybean mungbean -/-0+/+3 +/+4+/+3 -/-0+/+2 +/+4+/+4 +/+5+/+4 -/-0+/+4 +/+3+/+3 +/+4+/+5 +/+3+/+4 +/+4+/+4 +/+4+/+3 +/+5+/+3 +/+4 -/-0+/+4 -/-0+/+4 -/-0+/+4 +/+4+/+4 -/-0+/-0 +/-0+/+4 +/+2+/+3 +/+4+/+3 -/-0+/+2 +/-0+/+2 +/+2+/+2 +/+1+/+2 +/+3+/+4 -/-0+/+4 +/-0+/+3 +/+4+/+1 +/+2+/+1 -/-0+/+2 -/-0+/+1 +/+2+/+2 +/+4+/+4 +/+3+/+3 -/-0+/+2 +/+3 -/-0 -/-0+/+3 -/-0+/+4 -/-0+/+3 -/-0+/+4 +/+4+/+4 +/+3+/+4 +/+4+/+4 +/+2+/+3 +/+4+/+5 +/+4+/+2 +/+5+/+4 +/+4+/+4 +/+3+/+2 -/-0+/+2 +/+5+/+4 j-dlg1 s-st24 j-tgs50 s-st325 s-st224 j-wg2 j-tm3 j-mj4 j-kh5 j-ng6 j-wk7 j-plr8 j-srg9 j-dlg10 j-bgr15 j-ctm20 j-jkt25 j-mj28 s-st123 s-st16 s-st41 s-st42 s-st29 s-st33 s-st45 s-st310 s-st18 s-st311 s-st316 s-st215 s-st219 s-st518 s-st220 s-st117 s-st17 s-st412 s-st414 j-yg49 j-pn33 j-yg38 110 j-mdn39 j-mld40 j-tgs41 j-bsk42 j-psr43 j-mj44 j-plr45 j-mj47 s-bt221 s-bt322 j-klt48java fig 2 dendogram derived from 16s rdna ardra fingerprints and symbiotic properties of rhizobial strains isolated from java and sumatra soil samples as well as usda 110. +/+, nodulating and n-fixing; +/-, nodulated and no n-fixed; -/-, not nodulated and no n-fixed. the effectiveness of n fixation is indicated by values ranging from 1 (ineffective) to 5 (effective). b.japonicum 174 waluyo et al. microbiol indones fig 3 restriction patterns of pcr-amplified fragments of 16s-23s rdna intergenic spacer digested with i. lanes 1, 10 and 19 are 100 bp dna ladder (life technology). lanes 2-9 (s-st16; s-st41; s-st42; s-st29; s-st224; s-st33; s-st24; s-st45) were derived from rhizobial isolates from sumatra. lanes 11-18 (j-mdn39; j-tgs50; j-bsk42; j-yg49; j-klt48; j-kh5; j-mld40; j-psr43) were derived from rhizobial isolates from java. msp fig 4 restriction patterns of pcr-amplified fragments of 16s-23s rdna intergenic spacer digested with i. lanes 1, 11 and 20 are 100 bp dna ladder (life technology). lanes 2-10 (s-st16; s-st41; s-st42; s-st29; s-st224; s-st33; s-st24; s-st45; s-st311) were derived from rhizobial isolates from sumatra. lanes 12-19 (j-mdn39; j-tgs50; j-bsk42; j-yg49; j-klt48; j-kh5; j-mld40; j-psr43) were derived from rhizobial isolates from java. dde fig 5 restriction patterns of pcr-amplified fragments of 16s-23s rdna intergenic spacer digested with i. lanes 1, 11 and 20 are 100 bp dna ladder (life technology). lanes 2-10 (s-st16; s-st41; s-st42; s-st29; s-st224; s-st33; s-st24; s-st45, s-st311) were derived from rhizobial isolates from sumatra. lanes 12-19 (j-mdn39; j-tgs50; j-bsk42; j-yg49; j-klt48; j-kh5; j-mld40; j-psr43) were derived from rhizobial isolates from java. cfo volume 5, 2011 microbiol indones 175 bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 11 12 19171614 15 1813 bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 11 12 19171614 15 1813 20 bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 11 12 20171614 15 1813 19 fig 6 restriction patterns of pcr-amplified fragments of 16s-23s rdna intergenic spacer digested with i. lanes 1, 11 and 20 are 100 bp dna ladder (life technology). lanes 2-10 (s-st16; s-st41; s-st42; s-st29; s-st224; s-st33; s-st24; s-st45; s-st311) were derived from rhizobial isolates from sumatra. lanes 12-19 (j-mdn39; j-tgs50; j-bsk42; j-yg49; j-klt48; j-kh5; j-mld40; j-psr43) were derived from rhizobial isolates from java. table 1 hae 16s-23s rdna genotypes and restriction patterns of the isolated rhizobial strains revealed by ardra 176 waluyo et al. microbiol indones bp 1500 2072 600 100 bp 1500 2072 600 100 1 2 3 4 5 6 7 8 9 10 1112 19171614 15 1813 20 isolate 16s-23s r dna genotype restriction pattern of amplified 16s-23s rdna digested with mspi ddei haeiii cfoi s-st16 i a* a a a s-st29 a a a a s-st18 ii a h a a s-st215 iii a q a h s-st41 iv b b b a s-st42 b b b a s-st45 b b b a s-st33 b b b a s-st311 b b b a s-st117 b b b a s-st17 b b b a j-yg49 v b a b a s-st414 vi b j b a s-st224 vii c c c c j-wg2 c c c c j-ng6 c c c c j-wk7 c c c c j-plr8 c c c c j-kh5 c c c c j-dlg10 c c c c j-dlg1 c c c c j-srg9 c c c c s-st24 viii d d a d s-st310 ix d k b i s-st412 x d k b p j-mdn39 xi e e c e j-klt48 e e c e j-mj44 e e c e j-plr45 e e c e j-bsk42 xii e n c g s-st220 xiii e p h m j-tgs50 xiv f f d f j-mld40 xv g g c g j-tgs41 g g c g isolate 16s-23s r dna genotype restriction pattern of amplified 16s-23s rdna digested with mspi ddei haeiii cfoi table 1 continued 10090807060504030 j-tgs50java origin strains j-wg2java j-kh5java j-ng6java j-dlg10java j-dlg1java j-wk7java j-plr8java j-srg9java j-mj28java s-st224sumatra s-st325sumatra j-mld40java j-tgs41java j-mj4java j-psr43java j-bsk42java j-mj44java j-plr45java j-mdn39java j-klt48java j-mj47java j-yg38java j-jkt25java j-ctm20java 110usda j-pn33java j-tm3java j-bgr15java s-st123sumatra s-bt221sumatra s-bt322sumatra s-st41sumatra s-st42sumatra s-st33sumatra s-st24sumatra s-st45sumatra s-st16sumatra s-st17sumatra s-st18sumatra s-st29sumatra s-st310sumatra s-st311sumatra s-st412sumatra s-st414sumatra s-st215sumatra j-yg49java s-st316sumatra s-st117sumatra s-st518sumatra s-st219sumatra s-st220sumatra fig 7 dendogram derived from 16s-23s intergenic spacer ardra fingerprints of soybean rhizobial strains isolated from java and sumatra soil samples as well as usda 110.bradyrhizobium japonicum volume 5, 2011 microbiol indones 177 j-psr43 g g c g s-st518 xvi h i e h j-tm3 xvii i l f j j-pn33 i l f j j-yg38 i l f j j-jkt25 i l f j j-bgr15 xviii o l f j j-ctm20 o l f j s-st123 xix l o g k s-st219 xx m p h l s-st325 xxi n k b o j-mj4 xxii j m c g s-st316 xxiii k d a n s-bt221 xxiv p r i q s-bt322 xxv q s j r j-mj28 xxvi r t k c j-mj47 xxvii s u e e *letters represent the pattern of cut dna that is presented as a finger-prints. isolates with similar letter are classified in one group within their associated restriction enzymes. table 2 numbers of nucleotide differences and % 16s rdna homologies in the aligned sequences of well-known soybean-nodulating strains (accession number beu3500), usda 110 (accession number z35330) and usda 205 (accession number d14516) bradyrhizobium elkanii bradyrhizobium japonicum sinorhizobium fredii b. elkanii usda76 b. japonicum usda 110 s. fredii usda 205 strain number of nucleotides n 1 %h 2 strain number of nucleotides n % h strain number of nucleotides n %h s-st17 1390 6 99.6 j-wg2 1089 7 99.4 j-tgs50 1440 30 97.9 s-st414 1415 3 99.8 j-dlg10 1265 28 97.8 j-kh5 1199 11 99.1 j-srg9 1443 15 99.0 j-y49 1445 7 99.5 j-tm3 1426 3 99.8 s-st316 1443 9 99.4 s-st123 1329 23 98.3 s-st29 1054 9 99.1 s-st325 1445 11 99.2 s-st33 1442 10 99.3 s-bt221 1447 21 98.5 s-st215 1348 3 99.8 s-bt322 1446 16 98.9 s-st16 1446 25 98.3 s-st518 1425 18 98.7 s-st45 1360 6 99.6 s-st117 1445 9 99.4 1 2 number of nucleotide differences. % of 16s rdna homology. speciesb. 36 20 japonicumb. japonicumb. lupinib. japonicumb. japonicumb. 28 japonicumb. elkaniib. 64 67 frediir. elkaniib. speciesb. xinjiangensiss. frediis. 62 99 0.10 47 frediis. 32 frediis. 43 sahelis. elkaniib. elkaniib. 34 elkaniib. 35 elkaniib. elkaniib. elkaniib. elkaniib. elkaniib. elkaniib. elkaniib. elkaniib. 35 japonicumb. japonicumb. japonicumb. japonicumb. japonicumb. japonicumb. japonicumb. japonicumb. escherichia coli 55s iam 12608 lmg 6138 t dsm 30140 usda 136 j-dlg10* j-srg9* s-st518** iam 13625 usda 76 lmg 10689 iam 14142 j-tgs50* lmg 6217 usda 205 lmg 7837 j-yg49* s-st33** s-st16** s-st29** j-kh5* s-st316** s-st45** s-st215** s-st17** s-st117** s-st414** usda 110 j-wg2* j-tm3* s-st123** s-st325** s-bt221** s-bt322** dsm30131t mg1655 fig 8 a rooted phylogenetic tree based on 16s rdna sequences of the indonesian rhizobial isolates (bold, * from java and ** from sumatra) and related other and spp. constructed by the arb software with 100 times bootstrap. the bar indicates the phylogenetic distance (0.1 knuc). bradyrhizobium sinorhizobium 178 waluyo et al. microbiol indones table 3 efficiency of rhizobial strains isolated from java and sumatra compared to reference strains. number and weight of nodules formed on soybean as well as shoot weight were determined strain nodule plant -1 shoot weight (g plant -1 ) number weight (g) none control 0 0 0.57±0.32 bradyrhizobium spp. @ cb756 0 0 0.30±0.03 bradyrhizobium japonicum @ usda 110 43±10 0.288±0.07 3.36±0.80 bradyrhizobium japonicum @ cb1809 29±5 0.250±0.06 3.70±0.28 java @@ 46±10a* 0.263± 0.07a** 3.75± 0.62a*** sumatra @@ 37±14 b 0.225±0.07 b 2.43±0.80 b cv # = 33% cv=29% cv=25% @ @@ # for these reference strains means were calculated from 3 replicates a total of 24 randomly choosen bacterial strains used for each set of soil samples. *, **, *** values followed by a different letter in same column are statistically different, revealed by t-test with confidence levels at = 0.10, 0.05 and 0.01 respectively. cv = coefficient variation (mstat-c, 1988) . discussion fifty-one different rhizobial strains (27 from java and 24 from sumatra) were isolated from nodules. based on their nodulation ability on both soybean and mungbean, these rhizobial strains could be classified as promiscuous strains (34) or strains that show a narrow host-range (17) and only nodulate soybean (fig. 2). saono (1988) suggested that the native soybeanrhizobia population in java appears to be dominated by promiscuous strains, and this is supported here with quantitative data. all of the soybean plants that were inoculated by rhizobial isolates from java (except j-mj44) grew vigorously in an n-free medium. in contrast, several ineffective bradyrhizobial strains were found in sumatra. it is likely that the abundance of these strains would have a negative impact on the inoculation by effective spp. inoculants. ardra of pcr-amplified 16s rdna and 16s-23s rdna spacer fragments were used to differentiate soybean-nodulating rhizobial strains. based on ardra of 16s rdna, nearly all of the soybeannodulating bacteria could be grouped into two separate large clusters comprising either the java or the sumatra isolates. ardra of 16s-23s rdna spacer fragments has been reported to be useful for a further subclassification of bacteria (jensen . 1993; masoldeya . 1995; gurtler and stanisich 1996; scheinert . 1996). this is due to the fact that the 16s-23s rdna spacer region of all prokaryotes exhibit a high degree of sequence and size variations at the level of the genus and species. the sequence variations of the 16s-23s rdna spacer region of the soybeannodulating bacteria confirmed the clustering of most sumatra isolates and allowed a further classification of the java isolates into two large distinct clusters. one of bradyrhizobium et al et al et al these clusters was found to include strains that were closely related to usda 110, a reference strain which is highly specific for soybean. based on their grouping and symbiotic properties, we assume that these java isolates and one isolate from sumatra (s-st123) are strains. although clustered at one group, the isolate s-st123 is distinct in its symbiotic property to usda 110. it formed many but ineffective soybean-nodules. one unique isolate from java, j-tgs50, showed an unique position in comparison with the clustered isolates from java and sumatra. this group and the group containing most sumatra isolates, which were also shown distinct from usda 110, nodulated both soybean and the indigeneous mungbean. hence these strains may represent the indigeneous bacterial population capable of nodulating soybean. was found to be dominant in acid soils from sitiung, west-sumatra. ten out of fourteen studied isolates from this location could be assigned to . apart from isolate s-st518 they form a homogeneous group, and notably strains s-st17, sst45, s-st117, s-st215 and s-st414 show high similarity to the reference strain usda 76 (fig 5). however in spite of their phylogenetic relation, the strains showed considerable variability in their symbiotic properties. only two out of seven strains from java (strain jkh5 and j-yg49) were found to be . in contrast to the isolates from sumatra, the n fixation capacity of these strains from java was all very high on soybean. was found dominant in java. four isolates (j-dlg10; j-wg2; j-tm3; j-srg9) from seven analysed strains could be classified as b. japonicum b. japonicum b. japonicum b. japonicum b. elkanii b. elkanii b. elkanii b. elkanii b. elkanii b. elkanii b. japonicum b. volume 5, 2011 microbiol indones 179 japonicum b japonicum b. japonicum b. japonicum b. japonicum sinorhizobium s. fredii b. japonicum b. japonicum s. fredii s. saheli s. fredii s. fredii s. saheli et al et al s. fredii aeschynomene et al. et al. . this confirms earlier results based on symbiotic properties and ardra 16s rdna and 16s23s rdna. one of these isolates, strain j-tm3, was very specific and effective on soybean. this could be due to the cultivation of imported soybean seeds contaminated with soils (toxopeus 1938) or the introduction as inoculant by researchers (newton 1962). it is not be possible to differentiate between these or other possibilities, but the observation that strain j-tm3 is highly related to usda 110, a well known inoculant isolated from japan, suggests strongly that it is not a native strain (fig 2/table 1). remarkably, the other strains (j-dlg10; jwg2 and j-srg9) have been shown to nodulate mungbean plants. this is in apparent contrast with the common phenotype of which is known to nodulate only soybean. several strains (s-st123; s-st325; s-bt221; s-bt322) were also found in sumatra soils. however, while all strains from java are highly effective, these strains from sumatra are ineffective. it is interesting to note that a strain belonging to the genus that include fast growing species, was also found in indonesia. hence, the growth properties of this strain, j-tgs50, was determined and compared to the other strains and usda 110. mean generation time (g) of j-tgs50 was almost 6-fold lower than that of usda 110 and even lower than that previously reported for . this was also apparent during growth of colonies on yem agar plates. while colonies of the strain j-tgs50 with a diameter size between 0.10 1.0 mm could already be observed 3 days after inoculation, no usda 110 colonies were detectable at the same period of time. analysis of 16s rdna sequences of strain j-tgs50 showed it to be related to and . until now, only and not strains have been reported to nodulate soybean (keyser . 1982; de lajudie . 1994; young 1996). however, strains were found to show a broad host-range and formed nodules on soybean as well as many other legumes, including cowpea, pigeon pea and mungbean (scholla and elkan 1984; stowers and eaglesham 1984; chamber and iruthayathas 1988). this contrasts with the restricted nodulation properties of strain j-tgs50 which did not nodulate mungbean. from the data presented here and the earlier results, it can be concluded that there is no relationship between the nodulation phenotype and rrna traits. this confirms earlier reports for btai1, a phototrophic symbiont of the legume (young 1991) and for peanut bradyrhizobial strains (zhang 1999). therefore, besides nodulation phenotype, other traits, eg. 16s rdna sequences, should be taken into account for identification and classification of rhizobial strains. this study demonstrates that species known to nodulate soybean, , and most likely also are present in indonesia. there appeared a great diversity in effectiveness between these bradyand sinorhizobia strains. in java, many effective strains are already present in the soil, presumably by selection of soybean over a long period of cultivation. this could be the reason for the absence of a response to soybean inoculation practice in java (saono 1988). bradyrhizobia strains are present in low amount in acid soils of sumatra and are suggested to occupy niches in the acid soils, which are not toxic to these bacteria. it is interesting that there is a large variation in n fixation capacity among the isolates from sumatra. hence, combination of 16s rdna sequence analysis as described here with nodulation phenotype trait analysis opens the possibility to 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j bacteriol. 173: 2271-2277. young j pw .1996. phylogeny and taxonomy of rhizobia. plant soil 186: 45-52. young jm. 2003. the genus name casida 1982 takes priority over chen . 1988, and wang . 2002 is a later synonym of casida 1982. is the combination ' ' (casida 1982) willems . 2003 legitimate? request for an opinion. int j syst evol microb. 53: 2107-2110. young jm. 2010. sinorhizobium versus ensifer: may a taxonomy subcommittee of the icsp contradict the judicial commission? int j syst evol microb. 60: 1711-1733. zhang a, nick g, kaijalainen s, terefework z, paulin l, tighe sw, graham ph, lindstrom k .1999. phylogeny and diversity of strains isolated from root nodules of peanut ( ) in sichuan, china. system appl microbiol. 22: 378-386. rhizobium fredii rhizobium rhizobium japonicum rhizobium rhizobium ensifer sinorhizobium et al sinorhizobium morelense et al ensifer adhaerens sinorhizobium adhaerens et al bradyrhizobium arachis hypogaea volume 5, 2011 microbiol indones 181 cover 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editorial board.pdf page 1 01. mardhia.cdr vol.14, no.3, september 2020, p 89-94 doi: 10.5454/mi.14.3.1 antibiotic sensitivity pattern of bacterial isolates among diabetic outpatients with urinary tract infection in pontianak 1* 1 2 mardhia , mahyarudin , and abror irsan 1 department of microbiology, faculty of medicine, universitas tanjungpura, jalan prof. hadari nawawi, pontianak, 78124, indonesia; 2 department of public health, faculty of medicine, universitas tanjungpura, jalan prof. hadari nawawi, pontianak, 78124, indonesia. diabetic patients are associated with a higher risk of infection. the research purposed to identify antibiotic susceptibility patterns among diabetic outpatients with urinary tract infection in pontianak. an experimental study was performed for 13 bacterial isolates of diabetic outpatients with urinary tract infection in the clinic of diabetes mellitus, sultan syarif mohamad alkadrie hospital, pontianak. the disc diffusion method was used to perform the susceptibility of antibiotics to the bacterial isolates. among 13 isolates, the most common causative agent of urinary tract infection was escherichia coli (53.85%), followed by pseudomonas aeruginosa (30.77%), klebsiella spp., and enterobacter aerogenes were 7.69%. most isolates of bacteria of the study had a high sensitivity to cefepime (92.31%), then followed by levofloxacin, amikacin, and meropenem for 84.62%. the study revealed low sensitivity of bacteria to amoxicillin/clavulanate, co-trimoxazole, cephazoline, and ceftriaxone (30.77%, 23.08 %, 23.08%, 23.08%, respectively). all bacterial isolates had high resistance to ampicillin. moreover, multidrug resistance observed among bacterial isolates. key words: antibiotic susceptibility, diabetes, urinarytract infections pasien dengan diabetes memiliki risiko tingg mengalami infeksi. penelitian ini bertujuan untuk mengetahui pola sensitivitas antibiotik pada pasien rawat jalan diabetes mellitus dengan infeksi saluran kemih di pontianak. penelitian ini dilakukan pada 13 isolat bakteri dari pasien diabetes dengan infeksi saluran kemih di klinik diabetes mellitus, rumah sakit sultan syarif mohamad alkadrie, pontianak. uji sensitivitas antibiotik dilakukan menggunakan metode difusi cakram. dari total 13 isolat bakteri, penyebab terbanyak dari infeksi saluran kemiha dalah escherichia coli (53,85%), kemudian pseudomonas aeruginosa (30,77%), klebsiella spp., dan enterobacter aerogenes sebanyak 7,69%. hampir seluruh isolate bakteri menunjukkan sifat sensitif terhadap cefepime (92,31%), kemudian levofloxacin, amikacin, dan meropenem sebesar 84,62%. sensitivitas rendah terlihat pada amoxicillin/clavulanate, co-trimoxazole, cephazoline, dan ceftriaxone (30,77%, 23,08 %, 23,08%, 23,08%, secara berurutan). semua isolate bakteri menunjukkan resistensi terhadap ampicillin dan resistensi terhadap lebih dari satu jenis antibiotik. kata kunci: diabetes, infeksi saluran kemih, sensitivitas antibiotik microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-08195452038; email: mardhia@medical.untan.ac.id asymptomatic, dysuria to pyelonephritis. diabetic patients are 15 times have a higher risk for hospitalization due to pyelonephritis (saleem and daniel 2011). therefore, it is a significant problem for patients with diabetes to get appropriate treatment. � the successful therapy for urinary tract infection depends on the identification of microbial agents and the selection of antimicrobial against them (gutema et al. 2018).the mainbacteria associated with urinary tract infection in diabetes are escherichia coli, enterococcus spp., klebsiella spp., proteus spp., pseudomonas aeruginosa, staphylococcus aureus, and coagulase-negative streptococcus. other studies showed that fungi, namely, candida spp. and actinomyces spp., also have a role as urinary tract infection agents in diabetes mellitus(borj et al. 2017; gutema et al. 2018). diabetesis a severe chronic metabolic disorder characterized by high blood glucose because of insulin production disorder, the body's inability to insulin utilization, or both. the prevalence of diabetes has increased during the last few decades (world health organization 2016; gutema et al. 2018). over time diabetesmay developorgan failure and suppressed the immune system that leads to an increase in the risk of infection. urinary tract infection remains to be the most common infection diagnosed in diabetic patients. urinary tract infection in diabetes is 46.9 per 1,000 people/year, which is higher than non-diabetes (29.9 per 1,000 people/year) (fowler 2008; hirji et al. 2012). stage of urinary tract infection ranging from antibiotic susceptibility is diverse among species and areas. therefore, determining the sensitivity of antibiotics to bacterial isolates is essential. furthermore, there is an increase in the antibiotic resistance prevalence due to the widespread and indiscriminate use of broad-spectrum antibiotics (altulaibawi 2019). data showed about 30% of urinary tract infection bacterial agents are resistant to ciprofloxacin and levofloxacin (triono and purwoko 2012; rahman 2017). thus, this study was performed to provide local data about the susceptibility pattern of antibiotics among diabetic outpatients with urinary tract infection in pontianak. materials and methods research design. an experimental study was conducted at the microscopic laboratory, faculty of medicine, universitas tanjungpura, pontianak, during the period november 2019 to july 2020. the research procedures were approved by ethics committee of the faculty of medicine, universitas tanjungpura. bacterial isolates. bacterial isolates were derived from diabetic outpatients with urinary tract infection in the clinic of diabetes mellitus, sultan syarif mohamad alkadrie hospital, pontianak. all patients were from pontianak. isolation of bacteria were inoculated in macconkey agar (merck) and identification of bacteria by biochemistry test. all procedures were done by previous study. a total of 13 bacterial isolates were inoculated in macconkey (merck) agar plates using a standard inoculating loop for bacterial regrowth and incubated (memmert) at 37 °c for 24 hours. antibiotic susceptibility test. mcfarland 0.5 of bacterial suspensions were inoculated in mueller hinton agar (merck) to undergo antibiotic susceptibility testing using the disk diffusion method. sterile cotton swab was dipped in the suspension and the excess liquid pressed. the sterile cotton swab was swab on the agar plate surface and repeated three times o by rotated 60 c of the plate. antibiotic disks were placed on inoculated agar surface and incubated at 37 °c for 24 hours. the inhibition zone was examined according to the clinical and laboratory standard institute guidelines (clinical and laboratory standards institute 2020). antibiotic agents that were used are co-trimoxazole (sxt, 25µg, oxoid), ciprofloxacin (cip, 5 µg, oxoid), levofloxacin (lev, 5 µg, oxoid), nitrofurantoin (f, 300 µg, oxoid), amikacin (ak, 30 µg, oxoid), ampicillin (amp, 10 µg, oxoid), amoxicillin/clavulanate (amc, 30 µg, oxoid), cephazoline (kz., 30 µg, oxoid), ceftriaxone (cro, 30 µg, bd bbl), cefepime (fep, 30 µg, bd bbl), gentamicin (gm., 10 µg, bd bbl), meropenem (mem, 10 µg, bd bbl), and tobramycin (nn., 10 µg, bd bbl). results generally, antibiotic susceptibility patterns for urinary bacterial isolates from diabetic patients showed high sensitivity to cefepime (92.31%), then followed by levofloxacin, amikacin, and meropenem for 84.62% each. the study revealed low sensitivity of bacteria to amoxicillin/clavulanate, co-trimoxazole, cephazolineand ceftriaxone (30.77%, 23.08 %, 23.08%, 23.08%, respectively). all bacterial isolates had high resistance to ampicillin. e. coli, as the main causative agent in the study, was sensitive to amikacin and cefepime (100%, for each). p. aeruginosa was sensitive to levofloxacin, amikacin, and meropenem (100%, for each), followed by ciprofloxacin, cefepime, gentamicin, and tobramycin (75%, for each). e. pyogenes were100% sensitive to cotrimoxazole, levofloxacin, amoxicillin/clavulanate, and cefepime. at the same time, klebsiella spp. appeared sensitive to cefepime and meropenem (100%, for each). detail of antibiotic sensitivity profile and inhibition zone diameter, as seen in table 1 and table 2, respectively. discussion globally, antibiotic resistance rates are on the increase. meanwhile, antibiotic sensitivity is a primary concern in the treatment of patients with infection. patients with diabetes are prone to have an infection, commonly urinary tract infection because of the impaired immune response, dysfunctional bladder, and other mechanisms (alrwithey et al. 2017). other studies demonstrated that diabetic patients with urinary tract infections are vulnerable to have resistant pathogens as the causative agent (nitzan et al. 2015). our study revealed bacteria that cause urinary tract infection in patients with diabetes mellitus, namely e. coli(7/13), klebsiella spp. (1/13), e. aerogenes (1/13), and p. aeruginosa (4/13). several studies reported that e. coliis the most common bacteria in urinary tract infection in diabetic or non-diabetic patients (nitzan et al. 2015; borj et al. 2017; gutema et al. 2018; al-tulaibawi 2019). 90 mardhia et al. microbiol indones v o lu m e 1 4 , 2 0 2 0 m icro b io l in d o n es 9 1 antibiotic sensitivity n (% ) bacteria *n sxt cip lev f ak am p am c fep k z cro gm m em nn escherichia coli 7 2 (28.57) 5 (71.43) 6 (85.71) 1 (14.29) 7 (100) 0 (0) 3 (42.86) 7 (100) 3 (42.86) 1 (14.29) 5 (71.43) 6 (85.71) 6 (85.71) pseudomonas aeruginosa 4 0 (0) 3 (75) 4 (100) 0 (0) 4 (100) 0 (0) 0 (0) 3 (75) 0 (0) 2 (50) 3 (75) 4 (100) 3 (75) klebsiella spp. 1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (100) 0 (0) 0 (0) 0 (0) 1 (100) 0 (0) enterobacter pyogenes 1 1 (100) (0) 1 (100) (0) (0) (0) 1 (100) 1 (100) (0) (0) (0) (0) (0) total 13 3 (23.08) 8 (61.54) 11 (84.62) 1 (7.69) 11 (84.62) 0 (0) 4 (30.77) 12 (92.31) 3 (23.08) 5 (38.46) 8 (61.54) 11 (84.62 9 (69.23) table 1 antibiotic sensitivity profile of bacterial isolates from diabetic patients with urinary tract infection *n: number of isolates sxt: co-trimoxazole, cip: ciprofloxacin, lev: levofloxacin, f: nitrofurantoin, ak: amikacin, amp: ampicillin, amc: amoxicillin/clavulanate, kz.: cephazoline, cro: ceftriaxone, fep: cefepime, gm.: gentamicin, mem: meropenem, nn.: tobramycin. 9 2 m a r d h ia e t a l . m ic ro b io l in d o n es table 2 inhibiton zone diameter of disk diffusion among bacterial isolates from diabetic patients with urinary tract infection *sensitive, **intermediate, resistant isolate no.1,5,6,12 p. aeruginosa; no.2-4,8,9,11,3 e. coli; no.7 klebsielasp; no.10 e. aerognenes sxt: co-trimoxazole, cip: ciprofloxacin, lev: levofloxacin, f: nitrofurantoin, ak: amikacin, amp: ampicillin, amc: amoxicillin/clavulanate, kz.: cephazoline, cro: ceftriaxone, fep: cefepime, gm.: gentamicin, mem: meropenem, nn.: tobramycin. isolate inhibition zone diameter (mm) sxt cip lev f ak amp amc fep kz cro gm mem nn 1 0 21.60** 22.52* 0 26.13* 7.02 9.33 28.67* 0 15.33 17.67* 25.33* 20.67* 2 23.60* 26.08* 25.60* 10.40 23.08* 0 8.18 30.27* 9.50 22.12** 11.06 29.53* 15.13* 3 18.82* 27.15* 27.29* 17.23* 20.02* 0 26.84* 33.66* 26.40* 30.60** 16.68* 30.33* 16.68* 4 0 11.28 16.08 14.66 28.67* 0 0 25.67* 0 19.33 19.17* 19.03 20.41* 5 21.02 32.18* 32.40* 0 19.16* 0 11.17 32.00* 0 30.33* 17.50* 29.00* 16.67* 6 0 24.32* 24.46* 0 17.33* 0 9.16 13.83 0 6.67 0 30.66* 9.83 7 0 14.94 20.32** 14.73 10.80 0 11.56 30.00* 8.42 28.02** 0 28.00* 10.80 8 0 27.44* 26.77* 16.17** 19.55* 11.66 10.02 34.32* 23.66* 34.84* 15.16* 29.50* 15.68* 9 0 23.31* 27.30* 15.14** 17.58* 8.52 20.32* 31.50* 0 24.00** 14.00** 30.00* 14.00** 10 21.02* 23.16 25.61* 0 16.87** 9.32 20.96* 30.20* 0 20.21 14.32** 18.04 14.06** 11 0 28.29* 27.70* 16.06** 18.03* 12.51 27.53* 31.62* 28.52* 28.59** 16.11* 29.08* 15.27* 12 0 27.68* 26.85* 0 19.57* 12.06 26.11 32.80* 0 27.38* 16.51* 30.72* 17.38* 13 0 16.16 22.34* 16.06** 19.06* 0 9.22 26.24* 9.04 21.62** 17.02* 28.32* 17.32* volume 14, 2020 microbiol indones 93 table 1 demonstrated that all bacteria isolates are resistant to ampicillin (100%). ampicillin is an antibiotic effective for gram-positive and gramn e g a t i v e m i c r o o r g a n i s m s . h o w e v e r, s o m e microorganisms develop resistance to ampicillin. studies have shown an increasing trend in ampicillinresistance (aamodt et al. 2015; richey et al. 2015). contradict to other studies, nitrofurantoin showed the second rank of the highest antibiotic resistance (92.31%) (gardiner et al. 2019; zubair and shah 2019). table 2 showed majority isolates have resistant to 5 antibiotics (30.76%) and 2 isolates demonstrated resistant to 9 antibiotics (15.38%). there is an increasing trend in antimicrobial resistance among uropathogenic. the primaryantibiotic resistance mechanism for gram-negatives are the production of βlactamases and frequently aminoglycoside modifying enzymes (khoshnood et al. 2017; bitsori and galanakis 2019). bacteria classified as enterobacteriaceae with sensitivity test results resistance or intermediate towards third-generation cephalosporin antibiotic should be tested for the production of extended spectrum ßlactamases (esbl). a previous study in dr.soetomo hospital surabaya found a more significant rate of esbl producing e.coli compare to non-esbl producing e. coli (fitri et al. 2015). pathogensthat produce esbl represent resistance to third-generation cephalosporin, monobactam, as well as to newer ßlactam antibiotics (bitsori and galanakis 2019). further test is needed to reveal esbl bacteria in this study. based on our study, cefepime was reported as an antibiotic for urinary tract infection with the highest sensitivity compared to others (92.31%). cefepime is classified as beta-lactam, fourth-generation cephalosporin antibiotic. it is used to treat uncomplicated pyelonephritis as second-line therapy and as an alternative therapy in urosepsis, renal diseases, and extended-spectrum beta-lactamases bacteria (baldwin et al. 2008; seputra et al. 2015; bonkat et al. 2018; kim et al. 2018). e. coli isolates exhibited sensitivity towards amikacin and cefepime (100% for each), followed by levofloxacin, meropenem, and tobramycin (85.71%, for each), ciprofloxacin (73.41%). less sensitivity is shown towards amoxicillin/clavulanate, cephazoline (42.86%, for each), co-trimoxazole (28.57%), nitrofurantoinm and ceftriaxone (14.29%, for each), whereas another study has shown the opposite result (gutema et al. 2018; al-tulaibawi 2019; zubair and shah 2019). p. aeruginosa was shown as the highest sensitivity towards levofloxacin, amikacin, and meropenem (100%, for each), ciprofloxacin, cefepime, gentamycin, and tobramycin (75%, for each) and less sensitivity for ceftriaxone (50%). other antibiotics do not affect p. aeruginosa. p. aeruginosa is known to have resistance towards multiple antibiotics, such as aminoglycoside, quinolones, and β-lactams through some mechanisms (pachori et al. 2019; pang et al. 2019). p. aeruginosa demonstrated intrinsically, acquired, and adaptive resistance (pang et al. 2019). further research using a larger population or samples and different hospitals should be conducted. this study results may be used as data to improve the treatment of diabetic patients with urinary tract infections based on the pattern of antibiotic susceptibility. antibiotic susceptibility pattern is required for the rational use of antibiotics and the prevention of resistant urinary pathogens. furthermore, the rise of antibiotic resistance should be a significant concern for clinicians in treating diabetic patients with urinary tract infection, as demonstrated in this study. acknowledgements we thank clinic of diabetes mellitus, sultan 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(january 2011). seputra kp, tarmono t, noegroho bs, mochtar ca, wahyudi i, renaldo j, hamid ara., yudidana iw, ghinorawa t. 2015. penatalaksanaan infeksi saluran kemih dan genitalia pria. ikatan ahli urologi indonesia. triono aa, purwoko ae. 2012. efektifitas antibiotik golongan sefalosporin dan kuinolon terhadap infeksi saluran kemih. mutiara med. 12(1):6–11. world health organization. 2016. global report on diabetes. zubair ku, shah ah. 2019. frequency of urinary tract infection and antibiotic sensitivity of uropathogens in p a t i e n t s w i t h d i a b e t e s . p a k j m e d s c i . 35(6):1664–1668. page 1 page 2 page 3 page 4 page 5 page 6 03. rini.cdr vol.15, no.1, march 2021, p 15-20 doi: 10.5454/mi.15.1.3 thermostable alkaline protease activity from ducck225 and aspergillus flavus its compatibility to local detergents almalina nabila sulistyo rini sworo ukmi ri ujiyanto, i r *, s pand department of biology, faculty of science and mathematics, universitas diponegoro jl. prof soedharto, sh, tembalang, semarang 50275, indonesia. protease important enzyme in many industries, including detergent. ducc-k225 is a aspergillus flavus thermotolerant indigenous molds isolated from madura island which is potential in producing thermostable alkaline protease enzymes. the enzyme produced by submerged culture on modified czapeks dox liquid medium containing glucose as carbon source and 1% of casein. the aims of this study were to determine the activity and stability of thermostable alkaline protease produced by ducc-k225 at various temperatures, also the a. flavus compatibility to 5 local detergents. research were using , conducted completely randomized design in triplicate with temperature variation for protease activity as treatment. the results showed that the activity of highest thermostable alkaline proteases was 214.503 u/ml, with retained activities up to 78% in 60 minutes at 55°c. th is enzyme compatible with 5 local detergents tested, with the retained activity varied 59.48%-99.48% at 29 c and o 62.83%-98.05% at 55 c. the compatibility to detergent confirmed by blood o all tested were the increament of solubility. alkaline protease, ducc-k225, compatibility, detergent, thermostablekey words: a. flavus protease merupakan enzim yang penting untuk industri, termasuk industri deterjen. aspergillus flavus ducc-k225 kapang termotoleran indigenous dari pulau madura, yang berpotensi menghasilkan merupakan enzim protease alkalis termostabil. produksi enzim dilakukan dalam kultur terendam pada medium czapexs dox broth yang mengandung glukosa sebagai sumber karbon, dan kasein 1%. tujuan dari penelitian ini untuk men aktivitas dan stabilitas ensim protease alkalis yang dihasilkan oleh ducc-k225 pada getahui a. flavus berbagai suhu, serta kompatibilitasnya terhadap 5 jenis deterjen lokal. hasil yang diperoleh menunjukkan bahwa ensim tetap stabil pada suhu yang menunjukkan bahwa ensim ini termostabil, a tertinggi ensim ini 55 c ktivitas o adalah 9 48 9,48 dan 62.83214.503 u/ml, dengan aktivitas yang dipertahankan sebesar 5 . %-9 % pada suhu 29 c %-o 98,05 ensim ini kompatibel terhadap semua deterjen yang diujikan, karena dapat % pada suhu 55 c. o meningkatkan kelarutan noda darah pada kain. kata kunci: ducc-k225 deterjen, kompatibilitas, protease alkalis, termostabil a. flavus , microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ; ;: +62-811288428 fax: +62 e-mail: mgiswororukmi@lecturer.undip.ac.id nature, including in extreme environment (coral et al. 2003). some microorganisms have the potential to produce thermostable alkaline proteases, among others are fungi that can produce many kinds of extracellular enzymes . the enzyme production from fungi has several advantages, the because enzymes are more easily obtained in fermentation eassubmerged , ily separated from the medium, and can be grown on a cheap medium (souza 2015; soares 010). et al. et al. 2 according to souza (2015 the genus et al. ) the mold of aspergillus known as a largest producer of widely alkaline proteases such as , which aspergillus flavus has been known as a species that generally has high a ability in producing extracellular protease. the production of microbial enzymes by were influenced the composition, while the enzyme activitymedium affected by the incubation temperature (packianathan et al. 2008). the aims of this study were to examined the production of thermostable alkaline protease from protease (ec 3.4) is an enzyme that able to hydrolyze the peptide bonds between amino acids in proteins (nallusami, remya & al-bahri 009). the 2 availability of proteases is important for several industries, especially the proteases which are stable at high temperature and ph according to gupta . et al (2002) chimbekujwoa ome and . (2020) set al industries need thermostable alkaline proteases which is active at ph 8-12 and temperatures of 50°c-70°c the . presence of alkaline proteases is one of the 3 major groups of enzymes needed in industrial processes, which have a contribution of 60% in the sale of enzymes worldwide (sahib 2009; lanka, anjali, & pydipalli 2 , . 2020017 chimbekujwoa ). protease enzymes et al can be obtained from various sources such as plants, animals and microorganisms that are widespread in indigenous thermotolerant fungi aspergillus flavus ducc-k225 isolated from lime soil in madura , island east java and examined using glucose as carbon source the effect of temperature on the enzyme activity, as well as the compatibility to detergents. enzyme local materials and methods c u l t u r e m a i n t e n a n c e a n d i n o c u l u m preparation. the mold ducc-k225 was a. flavus maintained on pda agar slant, and sub cultured before used in the study. the spore inoculum was prepared by adding sterile distilled water containing 1% of tween 80 into 5 days old mold culture on pda (coral et al. 2003). enzyme production. the fungal alkaline protease from ducc-k225 produced a. flavus according to coral 2003 packianathan . 2008) by et al. et al ( ) and ( submerged fermentation in the modified czapeks dox medium composed of 30 g/l glucose; 0.5 g/ l kcl; 0.01 g /l feso ; 0.5 g/l mgso ; 1 g/l k hpo ; 2 g/l 4 4 2 4 nano , and 1 % casein. the media ph was arranged at 3 9. one percent fungal spore suspension of s a. flavus ducc-k225 (10 /ml) inoculated into the medium, the 8 flasks were then incubated on rotary shaker with 120 rpm for 7 days at room temperature. the fungal cultures filtered using whatman paper. no. 1, the supernatant obtained centrifuged at 4000 rpm for 20 minutes and use as crude enzymes. protease assay. the protease activity was measured by modified method of keay . (et al 1970 in coral 003; rani & prasad 013 chimbekujwoaet al. 2 2 ; 2020) using casein as substrate. one ml of culture supernatant was mixed thoroughly with 1ml of 2% of casein solution, incubated at 37°c for 10min and the reaction was stopped by adding 2ml of 0.4m trichloroacetic acid and incubated for 20min at 37°c. the solution filtered using whatman no. 1 paper. one ml of filtrate then mixed thoroughly with 5ml of 0.4m na co and 1ml of 0.5n folin phenol reagent, 2 3 incubated at 37°c for 20min. the absorbance of the final solution was measured at 660nm. one unit of protease activity was defined as the amount of enzyme required to liberate 1 µmol of tyrosine in 20min at 37°c. t h e r m o s t a b i l i t y a s s a y. t h e e n z y m e thermostability examined according to kamoun et al. (2008) by measuring the enzyme activity for 1 hour at various temperatures with interval of 15 minutes at ph 9.8. thermal stability of enzyme was determined by incubating 2 ml of culture supernatant mixed with 2% casein solution for 60 minutes at 29 c, 40 c, 45 c, o o o 50 c, 55 c, and 60 c. the heated enzyme was used o o o un as control. the compatibility to commercial detergents. alkaline protease of ducc-k225 was tested a. flavus for its compatibility to 5 commercial detergents i.e. b, d, s, r, and j . a , according to choudhary (2012) detergent solution of 0.7g/100ml were heated at 100 c o for 1 hour, to destroy indigenous protease that might be present. the detergent solution mixed with the culture supernatant in a ratio of 1:1 (v/v) incubated at 40ºc for 20 min. the residual protease activity examined using standard assay procedure as mentioned previously. a mixture of culture supernatant and tap water (1:1) used as control 100%. the relative enzyme activity was expressed as percentage activity considering the activity of control. the enzyme activities were examined in 3 treatments, i.e. 0.3ml of culture supernatant mixed with 5.7ml of detergent solution (0.7g/100ml); 0.3ml of detergent solution (0.7g/100ml) mixed with 5.7ml of distilled water; and 0.3 ml of culture supernatant mixed with 5.7ml of distilled water. the enzyme assays conducted at room temperature (29 c) and the optimum temperature of o protease activity obtainedwhich previously (55 c). o cleansing power as additive in local detergents. the cleansing power of the enzyme carried out by based on niyonzima & more (2015) soaking a piece of 4x4cm white cloth which square have been stained with blood in 4 solutions, containing (1) 50ml of distilled water 1ml of culture supernatant , and 1ml of detergent solution (0.7g /100 ml) which has been heated before at 100 c for 1 hour; (2) 51ml of o distilled water and 1 ml culture supernatant; (3) 51ml of distilled water and 1ml of detergent solution (0.7g /100ml) heated at 100 c; (4) 52ml of distilled o and water as control. the bloodstained clothes were soaked in the solution at room temperature (29 c) and 55 c for o o 10 minutes. the clothes were then dried and visually observed for the stain removal. the blood solubility in the soaking solution were also observed by measuring the absorbance at λ . 200 results effect of temperature on protease activity. the protease activity examination at various temperatures carried out to obtain the optimum temperature of . a flavus ducc-k225 protease. figure 1 showed that the , activity of alkaline protease increased along with the temperature increment. the highest protease activity reached at 55°c with the value of 214,50u/ml, 16 rini et al . microbiol indones volume 15, 2021 microbiol indones 17 although it is statistically at par with 50 c and 60 c. o o thermostability of alkaline protease. the enzyme thermostability have been examined at the optimum temperature of 55 c, which obtained from o previous examination of temperature effect on protease activity he result showed that the enzyme was still . t active for 60 minutes with retained activity value of 77.8 – 78.8% (fig 2). compatibility with detergents. the detergent compatibility test was carried out at room temperature ( and to determine the ability of . 29°c) 55°c a flavus ducc-k225 thermostable alkaline protease as additive for commercial detergents. the enzyme compatibility to detergents determined by the retained activity value of enzyme in the detergent solutions. table 2 t retained showed hat the activity of the enzyme in the detergent solution were varied. in three commercial detergents d, j and r were , which is in slightly higher 55°c 29°c while b at than at , for and s detergent at 29 c 55 cwere slightly higher than at o o (table 2) , but not different significantly. cleansing potential as detergent additive. the effect of alkaline protease produce by . ducc-a flavus k225 on detergent's cleansing power was carried out by soaking the blood stained fabrics in the detergent solution mixed with enzyme at two different temperature. the visually examination found that the enzyme increased the detergent's removing power of the blood stain from the fabrics (table 3). the measurement of soaking solution's absorbance at λ 200 were to ensure that the blood dissolved to carried out the water. the obtained of absorbance values were higher 55°c than 29°c indicated that more at at dissolved blood occurred at higher temperature (fig 3). the highest absorbance showed on the enzyme and s detergent mixed solution, this result in line with the detergents* protease activity (u/ml) retained activity (%) 29°c 55°c 29°c 55°c enzyme 66.76 ± 1.46 111.77 ± 2.70 100.00 100.00 b 61.55 ± 2.83 102.23 ± 1.33 92.19 91.47 d 57.01 ± 1.29 99.42 ± 2.19 85.39 88.96 j 48.11 ± 1.78 85.43 ± 2.83 72.07 76.44 r 39.71 ± 2.70 70.22 ± 3.10 59.48 62.83 s 66.41 ± 2.66 109.58 ± 1.66 99.48 98.05 table 2 the of ducc-k225 alkaline proteas various detergentscompatibility e to a. flavus table 3 the of ducc-k225 alkaline proteas various detergentscompatibility e to a. flavus control (water) water + detergent 29oc 55oc water + enzyme + detergent 29oc 55oc no detergent no detergent s s d d b b r r j j 18 rini et al . microbiol indones value of detergent's compatibility (table 2). discussion aspergillus flavus ducc k225 is an indigenous mold isolated from lime soil of madura island. the temperature of the sampling site was 38 c with the ph 0 of 8.3. this fungus can grow up to 45 c at the ph 8 on o pda medium and showed proteolytic activity with the index of 1.38. based on the data obtained, study in the production of alkaline thermotolerant protease from this isolate were conducted, to examine on the thermotolerant characteristic and the compatibility to comercial detergent. the result of protease activity from ducc k225 examination showed that a. flavus the enzyme is a thermostable one. the enzyme activity at 55 c-60 c higher than at lower temperatures o o (fig.1.), this indicates that the enzyme structure remains stable up to 60 c. according to yeoman o et al., (2010) the thermostable enzymes can theirmaintain fig 1 the effect of temperature on the alkaline protease activity of . ducc-k225.a flavus remarks: the same superscripts show no significant difference (p<0.05ly ). fig 2 the stability of alkaline protease enzyme produced by . ducc-k225 at 55°c.a flavus fig 3 the blood stain solubility in the soaking water. wwater; e – enzyme; b,d,j,r,s detergents. volume 15, 2021 microbiol indones 19 structural integrity at above 55 c coral . 2003 o . ( ) et al reported the thermostable alkaline protease produced by an strain stable at 40 c, aspergillus niger which is o while alkaline protease from as2 a. flavus studied by rani & prasad 2013 stable at 55 c. ( ) found based on o previous data obtained in this study, the temperature of 55 c was stated as optimum temperature of this 0 protease. the observation at showed that this 55 c o enzyme still active for 60 minutes with 78% retained activity (fig. 2.). this result supported the previous indication that the alkaline protease produced by . a flavus ducc-k225 was thermostable. the similar result also found in previous study on the alkaline proteinases produced by which aspergillus fumigatus fresenius tku003 and which aspergillus terreus is thermostable at 50°c and 60°c (nirmal 2011). . et al the enzyme retained activity value of the alkaline protease produced by . ducc-k225a flavus in five commercial indicated that detergents (fig 3), this enzyme compatible with all detergents tested. choudhary 2012 niyonzima & more 201( ), ( 5) and devi 2008 the alkaline et al ( ) have been reported that protease from several species aspergillus were compatible to detergents. the stability of enzyme was strong related to the retained activity in detergent solutions. s the difference in enzyme stability of a. flavus ducc k225 in the five detergent solution used, may corelated with detergent . surfactant is ingredient one of the main ingredient in commercial detergents, which have the ability to interact with proteases in increasing or inhibit protease activity (zhang & ing zhang 2015). the detergent s, b, and d contain ed sodium alkyl benzene sulfonate as surfactant with the concentrations of 22%, 20% and 15% respectively, while j and r detergents 16% and 19%contained surfactant respectively, but the types of surfactant were not clearly defined samanta & mitra . according to ( ) t2004 here are 4 groups of surfactants, i.e. anionic surfactants, non-ionic surfactants, cationic surfactants, and amphoteric surfactants an anionic surfactants . such as sodium alkyl benzene sulfonate also known as linear alkyl benzene sulfonate (las) have the greatest on protease activity, compared to other effect anionic surfactants such as sodium duodesyl sulfate (sds) and sodium lauryl sulfate (sls) (zhang & zhang 2015). the protease activity will be increased or persists constantly when the concentration of anionic surfactants increases (barberis 2013). the low et al concentration of las in water solution (<20%) caused low protease activity, whereas in the high concentration caused enzyme denaturation. (>45%) the highest compatibility of the alkaline protease from a flavus. ducc-k225 shown in s detergent which is contained , 20% las as surfactant with the activity retained 9 . % and 9 . % at 29 c and 55 c 9 48 8 05 o o respectively the ability of alkaline protease of . . a flavus ducc-k225 in removing blood stains on cloth indicates that this enzyme has the potential to be used in detergent formulas (tambekar & tambekar 2013). shahid & ahmed 2016 roteases capable ( ) stated that p in removing proteinic stains such as blood, often used in various detergent industries. the alkaline protease of a flavus. ducc-k225 showed highest compatibility to s detergent, hence the a flavus. ducc-k225 alkaline protease potentially as developed to be applied detergent additives. references barberis s, quiroga e, barcia c liggieri c 2013 'effect of , . . laundry detergent formulation on the performance of alkaline phytoproteases', 16: electro. j. biotechnol., 1-15, doi:10.2225/vol16-issue3-fulltext-1. chimbekujwoa ja'afarua adeyemo ki, mi, om. 2020. purification, characterization and optimization conditions of protease produced by aspergillus brasiliensis strain bcw2 scientific african 8. :1-9. https://doi.org/10.1016/j.sciaf.2020.e00398. choudhary 2012, 'compatibility with commercial v. detergents and stain removal capability of aspergillus versicolor j. acad. indus. res protease' ., 1(1): pp. 301-305. . issn: 2278-5213 coral g, arikan b, unaldi mn guvenmez h 2003, , 'thermostable alkaline protease produced by an aspergillus niger ann. microbiol. strain', 53:491-498. devi mk, banu ar, gnanaprabhal gr, pradeep b v p a l a n i s w a m y m 2 0 0 8 ' p u r i f i c a t i o n , , . . characterization of alkaline protease enzyme from native isolate and its compatibility aspergillus niger with commercial detergents', ind.. j. sci. technol., 1:1-6, doi:10.17485/ijst/2008/v1i7.8. gupta, r, beg, q, khan, s chauhan, b 2002 'an overview . . . on fermentation, downstream processing and properties of microbial alkaline proteases', appl. m i c r o b i o l . b i o t e c h . , 6 0 : 3 8 1 3 9 5 , doi:10.1007/s00253-002-1142-1. kamoun as, hadar a, ali ne, ghorbel b, kanoun s nasri , m 2008 'stability of thermostable alkaline protease . . from rp-1 commercial solid bacillus licheniformis laundry detergent formulations', microbiol. res., 163: 299-306, doi:10.1016/j.micres.2006.06.001. lanka s, anjali ch pydipalli m 2017 'enhanced . . . production of alkaline protease by aspergillus niger 20 rini et al . microbiol indones def 1 isolated from dairy form effluent and determination of its fibrinolytic abilility', afr j. . m i c r o b i o l . r e s . 1 1 : 4 4 0 4 4 9 , doi:10.5897/ajmr2016-8379. nallusami sr, remya al-bahry s 2009, 'partial a, . characterization of proteases produced by three fungal isolates from the rhizosphere of wild yam dioscorea wallicnii', j. appl. biol. sci., 3(3):71-75. nirmal np, shanka s, laxman rs 2011 'fungal proteases: . . an overview', 1: 1-40.int. j. biotech & biosci., niyonzima, fn more, ss 2015 'purification and , . . characterization of detergent-compatible protease from gr', ., 5: 61-70, aspergillus terreus biotech doi:10.1007/s13205-014-0200-6. packianathan, c, devanathan, v, anbu, p, ponnuswamy, mn, kalaichelvan, pt hur, bk 2008, 'purification, , . characterization and crystallization of an extracellular alkaline protease from ha-10', aspergillus nidulans j. b a s . m i c r o b i o l . 4 8 ( 5 ) : 3 4 7 – 3 5 2 , doi:10.1002/jobm.200800043. rani, mr prasad, nn 2013, 'studies on purification of , . alkaline protease from a mutant aspergillus flavus as2', ., 8(3): 58-66.res. j. biotechnol sahib ra 2009 'study of optimum condition for alkaline . . protease production from local isolate of aspergillus niger', iraqi j. sci., 50: 476-481. samanta ak mitra s 2004, 'efficacy of selective , . surfactants/detergents as washing agents on soiled white and dyed cotton fabrics', ind. j. fiber text. res., 29: 223-232. shahid smt & ahmed k 2016, 'enzyme proteases used in . laundry detergents engineering', 28: 2711-sci. int. 2717. . issn 1013-5316 soares fef, fabio rb, hugo lag, jackson vdea, sebastiao rf, juliana ma, alexandre ot, vinicus, lrv jose hq 2010, 'optimization of medium , . composition for protease production by paecilomyces marquandi in solid-state-fermentation using response surface methodology', . af j. microbiol. resr. 4: 2699-2703. . issn 1517-8382 \souza pm, bittencourt mla, caprara cc, freitas m, almeida rpc, silveira d, fonseca ym, filho exf, junior a magalhaes po 2015, 'a biotechnology , . perspective of fungal proteases', 46 2j. microbiol. ( ): 337-346, doi:10.1590/s1517-838246220140359. tambekar sd tambekar dh 2013, 'compatibility and , . wash performance analysis of alkaline protease from bacillus pseudofirmus (jq337958) with commercial detergents', 3: 738-int. j. pharma. chem. biol. sci., 744. issn: 2249-9504. yeoman cj, han y dodd d, schroeder cm, mackie ri , , cann, iko 2010, 'thermostable enzymes as . biocatalysts in the biofuel industry', adv. appl. microbiol.70: 1-55, elsevier inc. issn 0065-2164, doi:10.1016/s0065-2164(10)70001-0. zhang j zhang j 2015, 'study on the interaction of , . alkaline protease with main surfactants in detergent', coll. polym. sci., 124: 1-9, doi:10.1007/s00396-0153777-3. 7 agatha winny_416.pmd volume 3, number1, april 2009 p 33-36 issn 1978-3477 *corresponding author, phone/fax: +62-251-8625588, e-mail: awsanjaya@yahoo.com detection of listeria monocytogenes in pasteurized milk sold in bogor and its relationship with human health agatha winny sanjaya1*, mirnawati sudarwanto1, and kibuuka robert2 1faculty of veterinary medicine, institut pertanian bogor, darmaga campus, bogor16680, indonesia; 2faculty of veterinary medicine, umutara polytechnic university, p.o. box 57, nyagatare, rwanda many countries have established a zero tolerance policy, under which ready-to-eat foods are contaminated with listeria monocytogenes at a detectable level. the research was done in two parts. the first part was to qualitatively identify the presence of l. monocytogenes in pasteurized milk (n=32 samples) sold in different supermarkets in bogor. the method was adopted from the bacteriological analytical manual/food and drug administration. all samples tested resulted negative to l. monocytogenes. the second part of the research was to evaluate the growth of l. monocytogenes in sterilized milk stored in an incubator set at 4oc and monitored for 7 days. the original l. monocytogenes culture at a concentration of 1x109 cfu ml-1 was diluted with buffered phosphate water 0.1% to reach a cell concentration of approximately 1.0 x 102 cfu ml-1. growth was observed on the first, second, third, fourth and fifth day. on the sixth and seventh day, the numbers of colony forming units observed were almost similar (2.5-2.8 x 105 cfu ml-1). a population of 10 cells is sufficient to cause serious listeriosis infection in human. key words: listeria monocytogenes, pasteurized milk, human health listeria monocytogenes is a foodborne bacterium recognized as pathogenic for human and animals, and is able to persist in the food industry environment for several years. post-processing contamination of food with l. monocytogenes is a critical problem of public health. several outbreaks of listeriosis were linked with the consumption of ready-to-eat foods (rte) with a long shelf-live (fraizer et al. 1988). many countries have established a zero tolerance policy, under which rte foods contaminated with l. monocytogenes at a detectable level are deemed adulterated. the disease mainly affects pregnant women, newborn babies and adults who have a deficiency in their immune system. this fatal disease has a mortality rate of 25% and a hospitalization rate of 92%. in human, l. monocytogenes is carried asymptomatically in the faeces of 2-6% of the general population. it is shed in high numbers (>104 g-1) in faeces of infected people (mc lauchlin 1990). the organism is also considered to be potentially present in all raw foods and ingredients. therefore, pasteurization is an important process in reducing bacteria present in milk so that it is safe for consumption. much of the burden of illnesses was a result of inappropriate basic sanitation that occurs in food production, processing, retailing and handling at consumers’ homes. the objective of this research was to detect the presence of l. monocytogenes in different brands of pasteurized milk sold in bogor supermarkets, and the second research was to determine the number of colony-forming units in the sterile milk sample after 7 consecutive days kept in the refrigerator at 4 °c. materials and methods sample collecting and handling. samples collected were those of pasteurized high temperature short time liquid milk locally processed and packed in indonesia. samples were placed in an insulated cooler with ice packs, with a temperature range of 4 °c to 10 °c. samples of four brands (a,b,c and d) were collected from four supermarkets, namely bogor plaza (g), hypermart (h), ekalokasari plaza (e) and jambu plaza (j). the collections were carried out twice in two weeks. number of collection in every treatment was 8. the research was done in two parts. the first part was to qualitatively identify the presence of l. monocytogenes in pasteurized milk sold in bogor supermarkets. standard procedures were taken to ensure that all samples had not expired and that they were eligible for further process. the second part of the research was to evaluate the growth of l. monocytogenes in sterilized milk stored in an incubator set at 4 °c and monitored for 7 days. the original l. monocytogenes culture (field stem-laboratory of veterinary public health, faculty of veterinary medicineipb), at a concentration of 1x109 cfu ml-1 was diluted with buffered phosphate water 0.1% to reach a cell concentration of approximately 1.0x102 cfu ml-1. qualitative analysis of listeria monocytogenes. the method used to detect l. monocytogenes was adopted from the bacteriological analytical manual/food and drug administration (fda 2003). it began with an enrichment of the sample in a listeria enrichment broth, a buffered medium supplemented with oxoid sr 141. after 24 h and 48 h of incubation at 30 °c, the enrichment culture was streaked with a listeria selective media the oxford medium (oxa), which contains polymyxin b, acrilflavin and ceftazidime. after 24-48 h and seven days of incubation (37 °c), the oxa suspect colonies were seen as a black halo as a result of esculin hydrolysis, after which the five suspected colonies were streaked with tryptose soy agar and yeast extract (tsa oxoid cm131), and incubated at 37 °c for 24 h. presumptive listeria isolates are based on a standard series of biochemical tests (oxoid sr 141). the confirmation sugar test of l. monocytogenes isolates are rhamnose positive, xylose negative and camp test positive (staphylococcus aureus atcc 25723) with β-hemolysis enhanced in the vicinity of s. aureus. the suspected colonies were also subjected to potassium hydroxide, a motility test using sim medium (oxoid cm 0435) to detect the umbrella-like growth, microbiol indones hydrogen peroxide and mannitol. the colonies from tsa with yeast extract were again re-streaked with tryptose soy broth (oxoid cm 0435). a control positive was involved in picking a loop full of l. monocytogenes isolated from the field (laboratory of veterinary public health, faculty of veterinary medicine-ipb) and mixed with listeria enrichment broth in a clean and steril erlenmeyer. a control negative was involved in the use of a loop full of staphylococcus aureus with listeria enrichment broth, and broth controls were run in the same manner as the test samples. inoculation of listeria monocytogenes in sterile milk. a serial dilution of listeria monocytogenes (isolated from the field) was done with buffered phosphate water (bpw 0.1% v/v) to reach a cell concentration of approximately 1.0 x 102 cfu ml-1. one hundred milliliters of sterile milk was used as a test sample in each of the 8 clean and sterile erlenmeyers. then a 0.1 ml of 1.0 x 102 cfu ml-1 suspension was aseptically mixed by using a pipette into each of the 7 erlenmeyers, coded e1 to e7. code e8 was reserved for the control sample and was not inoculated. all the erlenmeyers were stored in a refrigerated incubator set at 4 °c. erlenmeyer e1 was removed after 24 h of incubation from the refrigerated incubator together with e8 as the control. then 1 ml of e1 was aseptically poured into sterile petri dishes labeled 100, 10-1 and 10-2 (all in duplicates), and 9 ml of bpw 0.1% was also prepared for dilution and 1 ml of e8 (control) was also prepared. each of the petri dishes was poured with a nutrient agar (merck 105450) and homogenized thoroughly. all the plates were incubated at 37 °c for 24 to 48 h. the control (e8) was returned to the refrigerator. after 24 to 48 h of incubation the colonies were counted and results recorded. on the second day 1 ml of e2 was aseptically poured into petri dishes labeled 100, 10-1 and 10-2, using dilution of 9 ml of bpw 0.1%. the erlenmeyer e8 (control) was also prepared and all dishes were poured with nutrient agar, homogenized and then incubated at 37 °c for 24 to 48 h. the research was continued, repeating the method of total plate count for e3 until the seventh day for e7 compared to the e8 as control. descriptive statistics were used to describe the results. the results of the colonies counted per day were converted into logarithm and plotted against time in days. results qualitative analysis. in this research, l. monocytogenes was not detected in all samples tested. it can be concluded that l. monocytogenes was not present in the milk. quantitative analysis. the growth of inoculated l. monocytogenes in a commercial sterile whole milk product was monitored at 4 °c for a period of one week, and the results obtained were shown in table 1. on the first, second, third and fourth day there was no significant increase in the number of colony-forming units. however, on the fifth day a dramatic increase in the number of colony-forming units was observed. on the sixth day, a slight decrease in colony-forming units was noted when compared to that on the fifth day. on the seventh day the population of colony-forming units shot up, it is noted that an increase in the number of days has a strong relationship with an increase in the amount of l. monocytogenes. discussions qualitative analysis. pasteurized milk is known to be one of the vehicles through which l. monocytogenes is transferred to human. therefore many countries have initiated a zero tolerance policy prohibiting the sale of processed rte food products contaminated with l. monocytogenes. this policy designates l. monocytogenes as a contaminant. despite the imposed policy, the bacteria are constantly found in these foods. vanderlinde and grav (1991) found 2.4% cases of listeria spp. as a secondary contamination in different supplements. dominguez et al. (1985) and garcia and vitas (2004) found a higher incidence of l. monocytogenes in pasteurized milk, 45.3% and 44.7% respectively, while gül et al. (1994) from turkey reported 1.1% found in samples of pasteurized milk samples. farber et al. (1988) reported that 18.2% of raw milk in ankara, turkey, contained l. monocytogenes. sharif and tunail (1991) tested samples of pasteurized milk and they were all reported negative of l. monocytogenes. based on all the findings mentioned above, it can be concluded that l. monocytogenes has a very low occurrence in milk. this does not mean that all the samples were free of l. monocytogenes. other confounding factors may have played a significant role. mesophilic aerobic bacteria and lactobacilli overwhelming flora that are seen may prevent proliferation of listeria. some authors reported that lactobacillus sake (lb.706) and similar spp., produce bacteriosin and play a role in inhibiting proliferation of listeria spp. (johnson et al. 1989; weis 1989). stopforth et al. (2005) reported that l. monocytogenes does not proliferate in food with a high total amount of microorganism. the inhibitory effect on the growth of l. monocytogenes is mainly related to the microbiological composition of the raw milk, in terms of thermophilic lactobacillus and yeast. inhibition of l. monocytogenes is probably due to the interrelationship between the microbiological and chemical factors. it is also important to note that this research may not get positive result because the number of samples tested was small (n=32). it was difficult to calculate the right sample size because the prevalence of l. monocytogenes in indonesia remains unknown. it is also important to point out that this research should use an additional method such as the conventional cultural method (polymerase chain reaction and enzyme linked immunosorbent assay) concurrently. those method are said to be of higher sensitivity. moreover, the control of l. monocytogenes 34 sanjaya et al. table 1 total colony counts for l. monocytogenes in commercial steril milk stored in the refrigerator at 4 °c for 7 days sample periode time (days) storage at 4 °c total microbes (cfu ml-1) e 1 e 2 e 3 e 4 e 5 e 6 e 7 1st 2nd 3rd 4th 5th 6th 7th 5.9 x 102 1.0 x 102 7.0 x 104 1.0 x 104 7.4 x 105 2.5 x 105 2.8 x 105 microbiol indones 35volume 3, 2009 in pasteurized milk via haccp is focused mainly on the selection of raw milk and the control of the processing, packaging, distribution and storage condition. even though the pathogen is effectively controlled during pasteurization, its presence in the final product is possible because of a post-pasteurization contamination from various sources at the plant environment. at present, however, it is presumed that l. monocytogenes can be killed by heating to 72 °c for at least 15 sec. quantitative analysis. listeria monocytogenes maintained its population relatively well in sterile milk stored at 4 °c (table 1). this is because it is a psychrotrophic organism which grows at lower temperatures. the reason of the late growth of l. monocytogenes as shown in the graph is likely caused by initial shock which may lead to delayed recovery and growth. in this study the author also attributes this observation to the alteration of the physiological state of the organism caused by the sudden cold-shock which may result in additional lag phase. the cells become easier to recover as they adapt to the environment at the later stages. this model provides a dairy industry with a useful tool for effective management and optimization of product safety, and it can lead to more realistic estimations of pasteurizedmilk safety related risks. it can therefore be presumed that l. monocytogenes concentrations on the fifth, sixth and seventh days of milk storage in refrigeration temperature (4 °c) can be dangerous to human health in both the immunecompromised and the healthy individuals. a population of 10 cells is sufficient to cause serious listeriosis in the nonhealthy individuals (invasive form), but in healthy individuals (non-invasive form) it requires over 105 cells. doyle et al. (2001) reported that l. monocytogenes grows in pasteurized milk with the amount increasing 10-fold in 7 days at 4 °c; therefore, milk fluid that becomes contaminated after pasteurization and stored under refrigeration temperature may attain very high population of l. monocytogenes after one week. therefore an inappropriate setting of temperature may further enhance the multiplication of bacterial cells. in relation to the above condition, hayes (1996) noted that the organism reach a significant increase on the fourth and fifth days. whereas the research obtained a drastic increase starting the fifth day, and the number increased unreasonably high on the sixth day. however, on day seven there was no difference in the number of colony forming units, when compared with those calculated on the sixth day. this could have been due to exhaustion of nutrients in the media and lack of space for multiplication. the importance of public health and sanitary handling during processing the rte food, and to keep food under refrigeration temperature is one way of controlling organisms. a population of 10 cells is sufficient to cause serious listeriosis infection in human. effect on human health. according to the research findings, it can be assumed that the growth of l. monocytogenes is not affected by low temperature. inoculation of 10 cells of l. monocytogenes into sterile commercial milk and then stored in a refrigerator at 4 °c for 7 d, showed significant growth of the bacteria. cells multiplication occurred on the first day; however, there was a decrease on the second day. on the third, fourth, fifth and sixth days, an exponential and rapid increase in the number of cells was observed. the number of cells on the 7th day was not significantly different from that on the 6th day (table 1). this phase is known as the stationary phase. the minimum number of cells required to cause listeriosis is just 100 cfu (mc lauchlin 1990) when compared with the cell populations obtained in the experiment. listeriosis almost always occurs because of the patient’s predisposed immune deficiency. currently the acquired immune deficiency syndrome (aids) could have the highest risk for listeriosis in the world. patients who have had organ transplants, malignancy undergoing chemo therapy or chronic liver disease, will have a higher risk for listeriosis compare to the normal population. however, listeriosis can occur in healthy individuals, particularly during an outbreak. consumption of a product of this nature, which is contaminated with a few l. monocytogenes cells can result in an immediate outbreak of listeriosis. adult patients with meningo encephalitis will develop headache, fever and confusion. there is an unusual predilection for the development of disease in the posterior fossa with cerebral and brain stem involvement (dee and lober 1986). multiple cranial nerve palsies and ataxia frequently develop. gastrointestinal symptoms were also common beside fever, vomiting and diarrhea prior to the development of invasive listeriosis (schwartz et al. 1989). the mortality rate for listeriosis is still high (25-30%) while in perinatal cases, the infant mortality rate is approximately 50% (mclauchlin 1990). all pasteurized milk samples collected from different supermarkets sold in bogor which were local products were negative of l. monocytogenes based on the fda method. the absence of the bacteria most likely happened because of the effective pasteurization process and the fact that there is no post-pasteurization contamination. the risk assessment reinforces past conclusions that food borne listeriosis is moderately rare even though it can lead to a severe disease. listeria monocytogenes can resist cold temperatures, as low as 4 °c. such temperature cannot inactivate the organism, even though other organisms can not survive. every effort should be made to ensure that rte foods with longer shelflife are totally free of l. monocytogenes, considering that few cells can multiply to very high numbers under refrigeration temperature. the results of this study could assist the government to evaluate the adequacy and focus of current prevention programs. moreover, the result could support the development of new initiatives to ensure that these programs protect the public health appropriately and the effectiveness of new strategies in minimizing public health impact on food borne listeriosis. references dee rr, lober b. 1986 brain abscess due to listeria monocytogenes: case report and literature review. rev infect dis 13:1108-10. dominguez rl, fernandez garayzabal jf, vazquez boland ja, rodiquez ferri e, suarez fernandez g. 1985. isolation de microorganismos du genre listeria à partir de latí cru destinë à la consommation. can j microbiol 31:938-41. doyle me, mazzotta as, wang t, wiseman dw, scout vn. 2001. heat resistance of listeria monocytogenes: jfp 64:410-29. farber jm, sanders gw, malcom sa. 1988. the presence of listeria spp. in raw milk in ontario. can j microbiol 34:95-100. [fda] food and drug administration. 2003. foodborne pathogenic microorganisms and natural toxins handbook. washington dc: fda. fraizer wc, westhoof dc. 1988. food microbiology international. singapore: mc graw hill. garcia-jva, vitas ai. 2004. occurrence of listeria monocytogenes in fresh and processed foods in navarra (spain). int j food microbiol 90:349-56. gül k, suay a, elçi s, özerdem n. 1994. incidence and isolation of listeria species in raw milk. türk mikrobiyol cem derg 24:22-5. hayes pr. 1996. food microbiology and hygiene. 2nd ed. london: elsevier. johnson jl, doyle mp, cassens rg. 1989. survival of listeria monocytogenes in ground beef. int j food microbiol 6:243-7. mclauchlin j. 1990. human listeriosis in britain, 1967-1985, a summary of 722 cases. j epidemiol infect 104:181-9. microbiol indones36 sanjaya et al. schwartz b, hexter d, broom cv. 1989. investigation of an outbreak of listeriosis. j infect dis 159:680-5. sharif a, tunail n. 1991. investigation on listeria monocytogenes contamination of raw milk obtained from different regions of anatolia and pasteurized milk sold in ankara. mikrobiyol bült 25:15-20. stopforth jd, skandamis pn, sofos jn, davidson pm. 2005. naturally occurring compounds-animal sources. in: davidson pm, sofos jn and branen al, editors. antimicrobials in food. 3rd ed. boca raton: crc. p 453-506. vanderlinde pb, grav fh. 1991. detection of listeria in meat and environmental samples by an enzyme-linked immunosorbent assay (elisa). jfp 54:230-1. weis j. 1989. vorkommen von listerien in hack-fleish. tierärztl umsch 44:370-5. 03. nasir.cdr vol.15, no.2, june 2021, p 54-60 doi: 10.5454/mi.15.2.3 molecular identification of hg-resistant bacteria and their potential in reducing mercury contamination soraya fitria nasir*, ani m. hasan**, aryati abdul, yuliana retnowatiand department of biology, faculty of mathematics and natural sciences, gorontalo state university, jalan prof bj habibie, bone bolango regency, 96554, indonesia. the aim of this research was to obtain and determine the identity of hg-resistant bacteria in soil contaminated with gold processing waste and test its ability to reduce mercury contamination. soil samples as a source of hg resistant bacterial isolates were obtained from the gold processing location in ilangata village, anggrek district, north gorontalo regency. the research was conducted at the microbiology laboratory, department of biology, faculty of mathematics and natural sciences. mercury analysis was carried out at the laboratory of fisheries product quality development and testing (lppmhp), gorontalo province, and bacterial identification was carried out at the hasanuddin university medical research center research unit. the parameters observed were the types of hg resistant bacteria and the ability of the bacteria to reduce mercury contamination. the results showed that there were four bacterial isolates on the soil contaminated with 4.5 ppm mercury, which were named ilb01, ilb02, ilb03, and ilb04. molecular identification showed that ilb01 was closely related to stenotrophomonas sp. sb67 enterobacter cloacae and ilb02 close to strain cm 1, these strains were not resistant to mercury contamination; while ilb03 which is similar to strain bs0591 and ilb04 which is similar to bacterium bacillus albus strain sq30 16s could be resistant and was able to reduce mercury contamination by 99% at 10 ppm levels. heavy metals, hg-resistant bacteria, molecular identification, mercurykey words: tujuan penelitian ini untuk mendapatkan dan menentukan identitas bakteri resisten hg pada tanah terkontaminasi limbah pengolahan emas serta menguji kemampuannya untuk menurunkan cemaran merkuri. sampel tanah sebagai sumber isolat bakteri resisten hg diperoleh dari lokasi pengolahan emas desa ilangata, kecamatan anggrek kabupaten gorontalo utara. penelitian dilaksanakan di laboratorium mikrobiologi jurusan biologi fakultas matematika dan ilmu pengetahuan alam. analisis merkuri dilakukan di laboratorium pembinaan dan pengujian mutu hasil perikanan (lppmhp) provinsi gorontalo dan identifikasi bakteri dilakukan di unit penelitian hasanuddin university medical research center. parameter yang diamati adalah jenis bakteri resisten hg dan kemampuan bakteri dalam menurunkan cemaran merkuri. hasil penelitian menunjukkan terdapat empat isolat bakteri pada tanah terkontaminasi 4.5 ppm merkuri yang diberi nama ilb01, ilb02, ilb03, dan ilb04. identifikasi molekuler menunjukkan ilb01 memiliki kedekatan dengan stenotrophomonas sp. sb67 enterobacter cloacae dan ilb02 dekat dengan strain cm 1, dua galur bakteri tidak resisten terhadap cemaran merkuri; sementara ilb03 yang mirip dengan strain bs0591 dan ilb04 bacterium yang mirip dengan strain sq30 mungkin resisten terhadap merkuri dan mampu menurunkan bacillus albus cemaran merkuri sebesar 99% pada kadar 10 ppm. kata kunci: bakteri resisten hg, identifikasi molekuler, logam berat, merkuri microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 82293272739;: +62 e-mail: oyafitria@gmail.com, animhasan@ung.ac.id mercury is a type of pollutant that needs attention because it is a heavy metal that has strong toxic properties. mercury that enters water bodies has an impact on the environment. mercury that enters the environment undergoes precipitation and is converted by microorganisms into methyl mercury. the presence of methyl mercury in the waters has a bad impact because it will accumulate in the body of the biota that live in the waters and has the potential to cause disturbances in the body's organs and metabolism of the biota. saleh . (2014), reported methyl mercury that et al enters the waters will accumulate in zooplankton or phytoplankton which then through the food chain will accumulate in fish bodies. this accumulation of the mining industry is an important industry that contributes economic and social benefits and accelerates development in an area. one of the potential gold mines that is still operating today is in north gorontalo regency which is dominated by traditional mining. traditional gold mining activities have big potential to produce waste that will damage the environment. the process of refining gold using mercury or amalgamation can cause environmental pollution because the leaching results are discharged into water bodies. mercury causes dysfunction in the body parts of the fish and even causes death. therefore, prevention efforts are needed from an early age to overcome pollution. according to haritash and kaushik (2009), methods of remediation of polluted environments in conventional ways, namely physically (incineration and washing) and chemically (chemical reactions, dilution and extraction) are considered only as a process of moving a pollutant from one place to another. the biological method of remediation (bioremediation) is a method that is environmentally friendly and occurs naturally. bioremediation is an alternative solution to environmental problems due to heavy metal pollution in mining areas. various types of bacteria isolated from contaminated areas are known to be potential good bioremediator agents because they can reduce high levels of heavy metals in laboratory experiments. some bacteria that have the ability to adapt to environments contaminated with heavy metals can help the process of restoring the environment with a remediation mechanism. this is because these bacteria have the ability to resist high contamination of heavy metals. freetes (2019) explained that bacteria that et al. are able to survive at high concentrations of heavy metals can eliminate toxic effects in contaminated areas. bacteria that have the ability to eliminate these toxic effects have the potential to act as bioremediating agents in the environment. various types of bacteria can be used to remediate heavy metals as stated by lutfi . (2018), obtained by et al bacteria isolated from mining morganella morganii areas and can be a bioremediator agent against mercury because it can reduce mercury levels by up to 92.46% at a hg concentration of 130 ppm. in rondonuwu's (2012) study, there were 10 bacterial isolates that could grow and reduce mercury in farming luria media. four of the isolates were from the sp. with the ability to bacillus reduce levels of 52% to 98%. apart from that, from the gram-negative group of bacteria, namely micrococcos luteus, brevibacillus morganella morganii, sp., eschericia pseudomonas and sp. able to reduce mercury levels by 53% to 98%. the purpose of this study was to determine the potential bacteria as a bioremediator agent in soil contaminated with gold mining waste in ilangata village, anggrek district, north gorontalo district and the ability of these bacteria to reduce mercury contamination. materials and methods soil sampling was carried out at the soil sampling. gold processing plant in ilangata village, anggrek district, north gorontalo regency at three points using purposive sampling technique which has high mercury content because it is located very close to the gold processing waste disposal site. soil samples were taken and composited then put into sterile adhesive plastic samples. isolation and purification of bacteria. bacterial isolation was carried out at the microbiology laboratory, department of biology, f.mipa, gorontalo state university by following the procedure of amelia, (2016) by taking a sample that has been et al weighed as much as 1 gram and diluted with 9 ml of distilled water. the dilutions were carried out in a series from 10 to 10 . the sterilized na media is -1 -5 poured sufficiently into the petri dish. after that, as much as 0.5 ml from the 10 dilution to the 10 put into a -1 -5 petri dish containing the medium and then homogenized. the petri dishes were wrapped in paper and incubated for 48 hours at 37 c. furthermore, the ᵒ bacterial suspension was prepared by diluting it with sterile distilled water. sterile aquades were put into each test tube as much as 10 ml and then put in 4 ose bacterial colonies and then homogenized. after that, the four tubes were equalized with a standard solution of 0.5 (1x10 cfu / ml) (quelab 2005). mc farland 8 the growing bacteria were then purified by the scratch method on media.nutrient agar bacterial resistance test. the resistance test was carried out using heavy metal mercury by inserting 1 ml of bacterial suspension which had a cell density of 10 8 into a petri dish containing a medium with heavy metal mercury in concentration of 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm. this resistance standard was taken based on the results of the analysis of the soil content of gold mining waste by the gorontalo province lppmhp which was at 4.5 ppm. petri dishes were wrapped in paper and incubated for 48 hours at 37 c. ° bacteria identification. the identification of bacteria was carried out at the hasanuddin university medical research center research unit with a dna extraction process then carried out by the dna amplification process with a pcr machine (polymerase chain reaction). dna extraction was carried out using the spin column method. the dna amplification process was carried out by taking 25 µl of kappa brand pcr master mix (composition: enzyme taq polymerase, buffer, dntp, mgcl ), 1.5 2 volume 15, 2021 microbiol indones 55 56 nasir et al . microbiol indones µl forward primer (63f: caggcctaacatgc aagtc), and 1.5 µl of reverse primer (1387r: gggcggtgtgtacaaggc), 16 µl of nuclease free water and 6 µl of isolated dna into each 0.2 ml tube (pcr tube). close the pcr tube to then spin down then insert it into the machine (pcr thermal cycler machine) with the pcr machine protocol, namely: initial denaturation of 1 cycle at 95 ° c for 5 minutes. then denaturation at 95 ° c for 10 seconds, annealing at 60 ° c for 5 seconds and extention at 72 ° c for 5 seconds. these three processes occur for 40 cycles. finally, final extension at 72 ° c for 5 minutes for 1 cycle. the electrophoresis process was carried out to see the length of the bands on each bacterial isolate using agarose and then visualized it under uv light. the results of pcr products that have bands are carried out sequencing analysis at first base-singapore then the sequencing results are carried out by blast analysis to compare with the existing data base on gen bank by accessing the web: https://www.ncbi.nlm.nih.gov . test the ability of bacteria to reduce mercury contamination. the bacterial isolates were then tested for their ability to reduce mercury contamination by inoculating 10 ml of bacterial suspension in nutrient broth medium containing a concentration of 10 ppm hgcl then incubating in an incubator shaker at a speed 2 of 100 rpm for 24 hours. furthermore, centrifugation was carried out for 5 minutes at a speed of 5000 rpm. the supernatant was analyzed by aas to determine the metal concentration that had decreased (amelia , et al 2016). testing for mercury levels is carried out by testing for levels of mercury (hg) carried out at the laboratory of fisheries product quality development and testing (lppmhp) in gorontalo province using sni 01-2354.6-2006 standards for solid samples and sni 6989.78: 2011 standards for liquid samples. the principle of the test for mercury levels is that the hg 2+ will be reduced by sn to hg. then the atoms were 2+ analyzed quantitatively by cold-vapor atomic absorption spectrophotometry or mercury analyzer using a wavelength (λ) of 253.7 nm. calculation of the decrease in mercury levels. the reduction in heavy metal content can be calculated using the dimawarnita formula (2017): where: α is the percentage of metal reduction; cs is s the final metal concentration (ppm); co is the initial metal concentration (ppm). results macroscopic characteristics of bacteria. based on the results of isolation carried out on soil contaminated with heavy metal mercury, four bacterial isolates were obtained which were morphologically different based on macroscopic observations (fig 1). in ilb01 the colony obtained was yellow with a texture that was stickier than the other bacterial isolates obtained. the colonies are round in shape with intact edges. in ilb02, bacterial colonies appear white with intact and rounded edges. the colony surface looks flat and has a hard and dry texture whereas ilb03 and ilb04 have almost the same morphology, namely white with wavy edges, flat surface and hard and dry texture, but in terms of shape ilb03 has a similar shape to roots while ilb04 has irregular colony forms (table 1). i d e n t i f i c a t i o n o f b a c t e r i a . m o l e c u l a r identification of bacteria can be done using the polymerase chain reaction (pcr) technique. the sequencing process is to determine the nucleotide sequence present in the dna fragment detected by pcr amplification. the nucleotide sequence obtained was then analyzed by blast by matching it to the gen bank. from the four bacterial isolates, it was found that the bacterial isolates were detected to have a band at 996 bp (fig2). based on the reconstruction of the phylogenetic tree, it is known that ilb01 and ilb03 are in the same branch or originate from the same ancestor but then form different branches. ilb01 is related to ilb03 but is not in the same clade. ilb01 is phylogenetically located in the same clade as sp. stenotrophomonas sb67. meanwhile ilb03 seems to form its own branch which looks closer to in bacterium strain bs0591. other branches also form a branch that separates the two nodes. one node forms a branch that connects the clade between ilb02 and strain enterobacter cloacae cm 1 16s while the other node shows the closeness between ilb04 and strain sq30. bacillus albus however, these two bacteria come from the same ancestor as seen from their main branching (fig 3). bacterial resistance test. the results showed that ilb03 and ilb04 could grow in media containing 10 ppm hgcl but at higher concentrations they could not 2 survive. meanwhile ilb01 and ilb02 were unable to grow at the all hgcl concentrations given (table 2). 2 the ability of bacteria to reduce mercury contamination. based on the results of the analysis of the reduction in mercury contamination carried out by inoculating two isolates of mercury resistant bacteria, namely ilb03 and ilb04 into medium containing nutrient broth 10 ppm mercury, it was found that the two isolates could reduce the mercury content by 99% for 24 (table 3). discussion gold processing using the amalgamation method is one of the gold laundering methods using mercury metal. these activities produce waste that contains mercury and is released into the environment. this mercury metal pollution causes water and soil quality to decline and can affect the biota that lives around it. the land for disposal of waste from gold processing is known to have a large mercury content because the gold laundering process is still carried out by amalgamation using mercury. the results of the lppmhp analysis of gorontalo province show that the soil around the gold processing is contaminated with high mercury because it contains mercury levels between 2.6 4.5 ppm. this concentration is quite high because according to alloway in mirdat (2013), et al. the concentration of heavy metal hg in soil and plants is in the normal range is 0.01 0.3 ppm, while in the critical range of 0.3 0.5 ppm. based on molecular identification, it was found that ilb01 has a kinship relationship with stenotrophomonas sp. sb67 (acc. dm065750.1). ilb02 can be seen to have a kinship relationship with enterobacter cloacae strain cm 1 16s (acc. kc920908.1) because it is in one node that forms the same clade. in ilb03, a phylogenetic tree is obtained, fig 1 morphology of colony bacterial isolates from soil samples; a) bacteria ilangata 01 (ilb01); b) ilangata bacteria 02 (ilb02); c) ilangata bacteria 03 (ilb03); d) ilangata bacteria 04 (ilb04). fig 2 electrophoresis results of bacterial isolates with 63f and 1378f dna marker primers of 100bp. a b c d volume 15, 2021 microbiol indones 57 table 1 colonies bacterial isolates character in macroscopic table 2 resistance test for bacterial isolates of soil samples table 3 decrease in mercury contamination for 24 hours without replication isolate name characteristics of colonies colour edge shape surface texture ilb01 yellow whole round-flat arise soft ilb02 white whole round flat hard and dry ilb03 white wavy similar roots flat hard and dry ilb04 white wavy irregular flat hard and dry isolate name concentration of hgcl2 (ppm) 0 10 20 30 40 50 ilb01 + ilb02 + ilb03 + + ilb04 + + isolate name initial hg(ppm) final hg(ppm) percentage of reduction (%) ilb03 10 0.0342 99.658% ilb04 10 0.0403 99.597 % fig 2 neighbor-joining phylogenetic trees. bootstrap value shows the percentage of 1000 replications. information: + = growing bacteria 58 nasir et al . microbiol indones which shows that ilb03 forms its own branches and is not on the same branches or nodes as the bacteria present in the phylogenetic tree. ilb03 forms a branch that is closer to the bacterium strain bs0591 (acc.mk823779.1). meanwhile, ilb04 seen from the existing phylogenetic tree has a very close relationship with strain sq30 because it is in the bacillus albus same clade. the potential of bacteria in reducing mercury contamination can be seen from their resistance ability in conditions containing mercury metal contamination. four isolated bacterial isolates were subjected to resistance tests at levels of 10 to 50 ppm for 48 hours on nutrient agar media to show that ilb03 which is similar to and ilb04 which bacterium strain bs0591 has similarities to is bacillus albus strain sq30 mercury resistant bacteria because it shows growth in media containing 10 ppm hgcl but at higher 2 concentrations cannot survive. meanwhile, ilb01 has similarities with and ilb02 stenotrophomonas sp. which has similarities with enterobacter cloacae strain cm 1 showed no growth at the five concentrations of hgcl given. the ability of ilb03 and 2 ilb04 resistance can be caused because these bacteria can develop resistance mechanisms against heavy metals such as absorption of heavy metals in the cell wall, metal immobilization in the bioleaching mechanism and the involvement of operon genes in the biotransformation mechanism. meanwhile, bacteria that are unable to grow at higher levels is thought to be because bacteria have a limited ability to tolerate mercury metal. when bacteria are unable to develop a resistance mechanism against heavy metals, death will occur due to not being able to tolerate the presence of mercury contamination. this difference in resistance ability can also occur because each bacterial isolate has a different response mechanism to keep growing in mercury-contaminated conditions. bacterial isolates that are not resistant are thought to be due to inhibited cell metabolic processes so that growth does not occur and even death. the ability to develop a resistance mechanism against heavy metals makes these bacteria have the ability to reduce contamination. this resistance mechanism can occur in several mechanisms such as biosopsy or bioaccumulation. in addition, it can also occur with the involvement by the gene operon. mer ilb04 which has similarities with bacillus albus also has a biosorption or bioaccumulation mechanism as well as a mechanism involving the mer operon gene. in sholikhah and nengah (2012), the ability of the genus can occur through the biosorption and bacillus bioaccumulation processes. the interaction between metal ions and the cell wall of gram-positive bacteria shows the role of carboxyl groups on peptidoglycan and phosphoryl groups in the secondary polymers of teichoic and theicuronic acids. in addition, it can occur because hg binds to the negatively charged ions that 2+ make up peptone and meat extract in media nutrient broth nutrient broth . the components of this media are protein groups. one of the functional proteins is the metalloprotease enzyme, which is a protein degrading which requires metal ions in its metabolic activity so that the metal ions in the medium are reduced. in prasetya (2017) also obtained results that in the et al. genus tested there was the involvement of the bacillus enzyme mercury reductase encoded in mer a with the help of nadh. the mechanism of mercury reduction, which is an enzymatic reduction process or change to a non-toxic compound, has something to do with the involvement of mercury-resistant genes or operon mer. this is because the operon mer consists of genes that can convert toxic compounds such as methyl mercury into enzymatically non-toxic compounds. hg will be 2+ reduced to hg assisted by nadph as a cofactor for 0 enzymatic processes. according to vetriani et al. (2005), mercury resistant bacteria have operon mer which codes for flavoenzymes then mercury reductase reduces hgions to hg which is non-toxic and 2+ 0 volatile. the ability of bacterial resistance to contaminated environmental conditions by developing resistance mechanisms such as absorption by means of biosorption or bioaccumulation and biotransformation mechanisms by involving the mer operon gene causes the bacteria to be able to reduce large mercury contamination so that it can make these bacteria potential as bioremediator agents for environmental recovery especially in mining areas. references amelia, titik fadilah, ace baehaki and herpandi. 2016. aktivitas reduksi merkuri pada bakteri yang diisolasi dari air dan sedimen di sungai musi. 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[dissertation]. bogor (id): bogor agricultural university. saleh, baskara adam., boedi setya rahardja and muhammad arief. 2014. studi kandungan logam berat merkuri (hg) dan prediksi kandungan metil merkuri (ch hg) pada organ kerang darah (3 anadara granosa) di kecamatan sidayu dan kecamatan bayuurip, pantai utara gresik, jawa timur [study of heavy metal mercury (hg) content and prediction of methyl mercury (ch hg) in blood shell organ 3 ( in sidayu and bayuurip districts, anadara granosa) gresik north coast, east java]. fisheries and marine scientific j 6 (2): 210-212. 10.20473/jipk.v6i2.11310.. sholikhah, umi and nengah dwianita kuswytasari. 2012. uji potensi genera sebagai bioakumulator bacillus merkuri [potential test of genera as bacillus bioaccumulator of mercury] journal of the november . ten institute of technology. ventriani, costantino., yein s. chew, susan m. miller, jane yagi, jonna coobs, richard a. lutz and tamar barkay. 2005. mercury adaptation among bacteria from a deep sea hydrothermal vent. appl environ microbiol. 71 (1): 224. doi: 10.1128/aem.71.1.220–226.2005. 60 nasir et al . microbiol indones 03. harti.cdr vol.13, no.3, june 2019, p 56-63 doi: 10.5454/mi.13.2.3 antimicrobial bioactive compounds of snail seromucoid as biological response modifier immunostimulator 1* 2 3 agnes sri harti , nony puspawati , and rahajeng putriningrum 1 departemen of nursing, stikes kusuma husada surakarta, jalan jaya wijaya no. 11 surakarta 57127, indonesia; 2 faculty of health science, universitas setia budi surakarta, jalan letjen. sutoyo, surakarta 57127, indonesia; 3 departemen of midwery, stikes kusuma husada surakarta, jalan jaya wijaya no. 11 surakarta 57127, indonesia. anti-microbial bioactive compounds from snail (achatina fullica ferussac) contained in snail seromucoid. it contains bioactive compounds such as glycans, peptides, glycopeptides, and chondroitin sulfate which can function as biological response modifiers (brm) immunostimulators. immunostimulators are compounds that can increase cellular immune responses in various ways, namely increasing the number and activity of t cells, nk cells, and macrophages and releasing interferons and interleukin. immunostimulators are compounds that can increase cellular immune responses in various ways, namely increasing the number and activity of t cells, nk cells, macrophages and releasing interferons and interleukins.the purpose of this study was to analyze antimicrobial bioactive seromucoid compound of snail (achatina fullica ferrusac) as biological response modifiers (brm) immunostimulators. the research methods based on experimental laboratory results with research stages including snail seromucoid isolation; antimicrobial activity; characterization physicochemical and profile of snail seromucoid proteins. the results of antimicrobial activity showed that 100% seromucoid concentrations had mic (minimal inhibition concentration) in staphylococcus aureus, candida albicans, and pseudomonas aeruginosa. the physicochemical examination results showed specific gravity of 1.010; ph 8, -1 -1 -1 glucose 16 mg dl ; 9 mg dl cholesterol; protein 2.8 mg dl and negative heavy metals (pb, cu, hg, al). the results of the analysis of protein profiles showed that there were 3 subunits of proteins, range from 55 to 72 kda and 1 specific protein sub unit of 43 kda which was thought to be antimicrobial and biological response modifiers (brm) immunostimulators. key words: antimicrobial, biological response modifier, immunostimulator, seromucoid, snail senyawa bioaktif anti mikrob bekicot (achatina fullica ferussac) terdapat dalam seromukoid bekicot. seromucoid bekicot mengandung senyawa bioaktif seperti glycans, peptida, glikopeptida dan chondroitin sulfat yang dapat berfungsi sebagai biological response modifiers (brm) immunostimulator. imunostimulator merupakan senyawa yang dapat meningkatkan respon imun seluler dengan berbagai cara yaitu meningkatkan jumlah dan aktivitas sel t, sel nk dan makrofag serta melepaskan interferon dan interleukin. tujuan penelitian adalah menganalisis senyawa bioaktif antimikrobial seromukoid bekicot (achatina fullica ferrusac) sebagai biological response modifiers (brm) immunostimulator dan karakterisasi fisika kimiawi serta profil protein seromukoid bekicot. metode penelitian berdasarkan hasil eksperimental laboratorium dengan tahapan penelitian meliputi isolasi seromukoid bekicot; karakterisasi fisika kimiawi, mikrobiologis, dan profil protein seromukoid bekicot. hasil uji mikrobiologis menunjukkan seromukoid konsentrasi 100% bersifat mic (minimal inhibition concentration) terhadap staphylococcus aureus, candida albicans, dan pseudomonas aeruginosa.. hasil -1 pemeriksaan fisika kimiawi seromukoid bekicot menunjukkan berat jenis 1.010; ph 8, glukosa 16 mg dl ; -1 -1 kolesterol 9 mg dl ; protein 2,8 mg dl dan logam berat (pb, cu, hg, al) negatif. hasil analisis profil protein menunjukkan adanya 3 sub unit protein yaitu kisaran 55 – 72 kda dan 1 sub unit protein spesifik 43 kda yang diduga bersifat antimikrob dan biological response modifiers (brm) immunostimulator. kata kunci: antimikrob, bekicot, biological response modifier, imunostimulator, seromukoid microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8179453831; email: agnessriharti@yahoo.com can increase the immune response in various ways, namely increasing the number and activity of t cells, nk cells and macrophages and releasing interferons and interleukins to increase cellular defense. materials that can stimulate an increase in immune response are called immunostimulators (levinson and jawetz 2003). groups of compounds that have potential immunostimulation activity are alkaloids, terpenoids, immunostimulation can increase the body's specific and non-specific defense mechanisms as well as the occurrence of non-specific induction of both cellular and humoral defense mechanisms including inflammation. immunostimulators are compounds that volume 13, 2019 microbiol indones 57 quinones; simple phenolic compounds; group of polysaccharides, peptides, glycoproteins and nucleotides. the use of bioactive compounds as immunostimulators aims to reduce intracellular microbial infections, overcome immunodeficiency, or to trigger the growth of body's defense cells in the immune system. materials that can stimulate the immune system are called biological response modifiers (brm) immunostimulators. snail hemolymph is the main fluid in the body of the snail as a seromucoid that functions for heart contractions and leg movements. bioactive compounds in hemolymph are glycans, peptides, glycopeptides, and chondroitin sulfate. the snail c h o n d r o i t i n s u l f a t e c a n f u n c t i o n a s a n immunomodulation and immunosuppressant. snail (achatina fulica) mucus 100% concentration and snail mucus cream 5% have an effective effect on the duration of second degree (a) burn healing (mandala and agnes 2014). the content of bioactive compounds in seromucoid can stimulate the function of cellular immunity, namely lymphocyte proliferation and the production of reactive oxygen intermediated macrophages (viera et al. 2004). the results of the study by harti et al. 2018 showed that snail mucus and chitosan were able to increase lymphocyte proliferation, but the characterization of protein profiles of bioactive compounds that play a role in the process was unknown. immunostimulatory effects found in snail seromucoid can be used to increase body immunity against facultative intracellular bacterial infection. the increasing incidence of infectious and degenerative diseases and the absence of appropriate therapies for the use of natural bioactive compounds, research on the physical, chemical, microbiological, and biomolecular characteristics of snail seromucoid as biological response modifiers immunostimulators or effective anti-inflammatory drug candidates need to be conducted. the purpose of this study was to analyze antimicrobial bioactive seromucoid compound of snail (achatina fullica ferrusac) as biological response modifiers (brm) immunostimulators. materials and methods snail sample handling. local snail samples (achantina fulica) with an average weight of 19 g and a height/width of 25/43 mm were obtained from local cultures placed in plastic containers at room o temperature of 20-22 c and fed with cabbage (fig 1). snail seromucoid isolation. seromucoid samples were obtained from local achatina fulica ferussac snails, from 10 to 50 snails. the end of the shell was opened and using an injection syringe needle was inserted and turned so that the liquid that comes out was placed in a sterile conical tube. the liquid that comes out was clear to brownish yellow as seromucoid and then centrifuged 3000 rpm for 30 minutes. antimicrobial activity.in vitro microbiological examination includes antimicrobial activity testing of diffusion and dilution methods to determine mic (minimal inhibition concentration) or mkc (minimum killing concentration) (soleha 2015). antimicrobial activity test of the dilution method was performed to determine the mic or mkc seromucoid concentration of 100% against staphylococcus aureus, candida albicans and pseudomonas aeruginosa isolates obtained from the microbiology laboratory of the university of setia budi surakarta. seromucoid samples prior to diffusion and dilution tests were fig 1 local snail samples (achatina fulica). filtered aseptically using a disposible filter membrane. characterization of chemical and biochemical physics. physical examination, namely color, odor, consistency, and viscosity. chemical tests were ph and h e a v y m e t a l s . b i o c h e m i c a l t e s t s i n c l u d e d measurements of glucose, cholesterol, and total protein. protein profile of the sds-page method. snail seromucoid protein profile purification and characterization with sodium dodecyl sulphate polyacrilamide gel electrophoresis (sds-page) to obtain dominant bands with a certain molecular weight were done.characterization was carried out using the sds-page technique (wayan 1991) with a 10% separating gel (1.2 g acrylamid; 0.032 g bis-acrylamid; 3 ml 1.5 m tris ph 8.8; 0.12 ml sds 10%; 8.88 ml aquadest, 7 µl temed and 80 µl 10% aps) and 3% stacking gel (0.9 g acrylamid; 0.024 g bis-acrylamid; 2.52 ml 1.5 m tris ph 6.8; 0.3 ml sds 10%; 17.18 ml aquadest, 3.5 µl temed and 50 µl aps 10%). gel plate was added with a solution of separating gel 10% vertically, then the top was added to butanol and allowed to polymerize. the next process is the addition of 3% stacking gel put into the glass until it is full then installed a comb and left until polymerization occurs. the gel-filled plate was mounted on a minigel twin g-42 slab and poured with electrophoresis buffer (3g 0.0248 m tris; 14.4 g glycine 0.19 m; 10 ml 0.1% sds 10%) where the electrodes were poured. a total of 50 µl samples in ependorf plus 5 x sds samples buffer (red prob-b) were 12.5 µl (2.5 ml 1.5 m tris ph 6.8, 2 g sds, 0.5 g dithiothretol (dtt) ) in 5 ml merchaptoethanol; 10 mg bromphenol blue; 10 ml of glycerin and 2.5 ml of aquadest). the sample is then boiled for 2 minutes removed and immediately put ice, poured little by little into each well of stacking gel. as a marker used protein with molecular weight in the range of 10 180 kda, brand vivantis. the power supply is turned on by the electric current used by 99.9 volts, 50 ma and 12 w. if the sample has reacted to the bottom, then the process is stopped. the plate is opened and separated and then washed with buffer and painted with methylene blue (berniyanti and suwarno 2007). results the recovery of snail seromucoid. the average seromucoid volume obtained for each snail varies from 1.0 to 4.0 ml when isolating the snail seromucoid. samples were further treated, namely mucomucoid or mucus macerated with water for 24 hours at 40° c. the obtained supernatant is said to be wsf (water soluble fraction). the mucin fraction of wsf obtained then precipitated with a ratio of 1: 3 ethanol is a general method of isolation from mucus. wsf and the mixture were centrifuged at 2900 x g for 30 min. the precipitation obtained was reconstituted with tris -cl and mucin fraction was obtained. seromucoid is centrifuged 3000 rpm for 30 minutes as a hemolymph fluid (fig 2). antimicrobial activity test. the test results of snail seromucoid antimicrobial activity diffusion method on muller hilton medium were shown to be antimicrobial (fig 3 and table 1). physical-chemical and biochemical test. the results of physicochemical and biochemical test as show in table 2. snail seromucoid profile with sds-page method. the characterization of snail seromucoid protein profiles as fig 4 was carried out with sodium dodecyl sulphate polyacrilamide gel electrophoresis (sds-page) to obtain dominant bands with certain molecular weights. discussion the results of antimicrobial seromucoid activity test of snail using diffusion method on muller hilton medium (soleha 2015) did not show the existence of inhibition; this is likely due to seromucoid can not be diffused completely in the media so that it does not show any inhibitory power. therefore continued dilution test with liquid culture and cell suspension according to 0.5 8 -1 mcfarland standard (1.5 x 10 cfu ml ) to determine mic (minimum inhibition concentration) or mkc (minimum killing concentration). there is a difference in the variation of antimicrobial activity compared to previous studies that show a difference in results. this is influenced by the different inoculum strains used which are related to the level of microorganism resistance as well as the type of antibacterial achasin protein that are the result of genetic expression of each snail strain that is different (dolaskha et al. 2014). various types of proteins or achasin proteins in snails have important biological functions as bacterial (enzyme) binding receptors (dang et al. 2015). the results of the study by berniyanti and suwarno (2007) show that snail mucus is capable of being antibacterial to streptococcus mutans and escherichia coli. anggraeni et al. (2018) stated that snail mucus was able to inhibit the growth of methicillin resistant staphylococcus aureus (mrsa). the 58 harti et al. microbiol indones concentration of 100% snail mucus effectively inhibits the growth of gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (salmonella typhosa) (huda and marhamah 2016). the inhibitory potential and antibacterial snail mucus on the isolates of staphylococcus sp, streptococcus sp and pseudomonas sp bacteria were varied (etim et al. 2015). antibacterial and antifungal test results from meat protein extracts of 7 different snail types showed variations in antibacterial and antifungal activity by diffusion and dilution methods, because it is influenced by the ecological conditions of snails (ulagesan and hak jun kim 2018). the results of physicochemical and biochemical examinations as in table 2 show that seromucoid or snail hemolymph has physical and chemical characteristics such as liquid consistency, transparent white to brownish yellow color, odorless or slightly fishy, s​​ pecific gravity 1,010; ph 8; heavy metals (pb, fig 2 the results of seromucoid snail isolation. fig 3 the result antimicrobial activity diffusion method of candida albicans, staphylococcus aureus, and pseudomonas aeruginosa. fig 4 profile of seromucoid snail protein with sds-page method. marker volume 13, 2019 microbiol indones 59 -1 hg, cu, al) negative; glucose 16 mg dl ; cholesterol 9 -1 -1 mg dl and protein 2.8 mg dl . snails secrete mucus from the glands of the legs are used as a mechanical function, especially locomotor or adhesion movement for viscoelasticity on the substrate with a thickness of 10-20 um and containing water 99.7% by weight and in dry conditions in forming a solid and thin film layer. the content in mucus gel is composed of mucopolysaccharides and glycoproteins. the mucus composition of each snail species varies and is influenced by external or ecological factors, namely temperature, humidity, light intensity and food supply. the results of chemical analysis on mucus snails eobania vermiculata, theba pisan and monacha obstructa, contain three main compounds namely oxime, methoxy-phenyl and cyclotrisiloaxane, hexamethyl (zhuang et al. 2015). snails have a humoral and cellular immune system found in hemolymph. hemolymph is composed of plasma and hemocytes. snail plasma or mucomucoid contains a number of proteins that function to eliminate pathogens directly and hemocytes function as immune effector cells that play an important role in killing pathogenic microbes through various methods, namely phagocytosis, encapsulation and cytotoxic reactions. gastropod hemocytes play an important role in cell d e f e n s i v e r e a c t i o n s n a m e l y p h a g o c y t o s i s , encapsulation, nodulation and neutralization of parasites, the process of blood coagulation and wound healing. snail hemolymph bioactive compounds as drug derivatives have been used in the medical field including skin smoothing, treatment of respiratory infections, burns (bismili et al. 2013). snail seromucoid or hemolymph contain bioactive compounds such as glycans, peptides, glycopeptides and chondroitin sulfate. the snail chondroitin sulfate can function as an immunomodulation and immunosuppressant. the content of glycoaminoglycans (gags), heparin, heparin sulfate, chondroitin sulfate, sulphate dermal and hyaluronic acid in table 1 results of examination of seromucoid antimicrobial activity test for diffusion method table 2 physicochemical and biochemicals test of seremucoid snail no material diameter of resistance (mm) staphylococcus aureus pseudomonas aeruginosa candida albicans 1 2 average 1 2 average 1 2 average 1 snail slime 18 16 17 18 18 18 19 19 19 2 seromucoid 16 18 17 15 14 14.5 18 17 17.5 3 snail slime cream 32 20 26 22 21 21.5 16 18 17 4 positive control 20 20 20 21 21 21 19 20 19.5 5 negative control 0 0 0 0 0 0 0 0 0 1 examination t ypes results physicist consistency color smell d ensity liquid transparent white to brownish yellow odorless a little fishy 1.010 chemical ph h eavy metals (pb, h g, cu, al) 8 negative b iochemistry g lucose cholesterol protein 16 mg dl-1 9 mg dl-1 2,8 mg dl-1 1 60 harti et al. microbiol indones hemolymph and snail mucus function as primary biological modifiers that act as stabilizer cofactors, and or coreceptor for growth factors, cytokines, and chemokines; enzyme activity regulator; signifying or labeling molecules in response to cellular damage, such as wound healing, infection, and tumorigenesis; targets for bacterial, viral, parasitic virulence factors; and the immune system (sallam et al. 2009). a number of protein lectins are known to be contained in snails, namely selectin, galectin, c-type lectins, and fibrinogen-related proteins (freps) secreted by snails after infection that plays a role in the process of pathogen agglutination (dolaskha et al. 2015). also the presence of aldolase and myosin are identified as proteins that play a role in the regulation of hemosite migration and have an impact on the process of killing pathogens by cytotoxic reactions and phagocytosis (suwannatri et al. 2016). immunostimulatory effects found in snail and chitosan seromucoid can be used to increase body immunity against facultative intracellular pathogenic bacterial infections (harti et al. 2018). this shows that the presence of bioactive compounds in snail hemolymph is very potential as an antibiotic candidate, namely the presence of bioactive compounds that are antioxidant, anticancer, antiviral and antiinflammatory. the content of bioactive compounds in snail seromucoid can stimulate the function of cellular immunity, namely lymphocyte proliferation and the production of reactive oxygen intermediated macrophages. the bioactive component of natural ingredients is to activate macrophage cells or work as an immunostimulant so that it is useful as adjunctive therapy for inflammation including cancer patients in relation to increasing the non-specific immune system of patients against infection, increasing the effectiveness of therapy through the tumorisidal effector system. humoral immune response can be known from the antibody titer that is secreted. cellular immune response is played by t lymphocytes and activated effector cells. this response can be known from the lymphocyte proliferation response. humoral immune responses play a role in host defense against extracellular microbes, which are mediated by antibodies. whereas defense against intracellular microbes requires an immunecellular response. immunomodulatory activity can be carried out using the haemagglutination test method in vivo by measuring the concentration of tumor necrosis factor-α (tnf-α) as a proinflammatory cytokine and interleukin -10 (il-10) as an anti-inflammatory cytokine. an increase in il-10 production can suppress the production of tnf-α production and affect the b lymphocyte cells to produce antibodies. stimulated t lymphocytes will produce cytokines in the form of interferon-γ (ifn-γ) and interlukin-2 (il-2). ifn will play a role in macrophage cell activation and can i n d u c e t h e e x p r e s s i o n o f c l a s s i i m a j o r histocompatibility complex (mhc) molecules in macrophage cells, thereby helping macrophage cell function in lymphoid follicles to recognize antigens. macrophage cells can also release cytokines, il-1, which play a role in stimulating the proliferation of th and b cells. whereas il-2 does not only play a role in the expansion of t lymphocyte clones after being recognized as antigens, but also increases the proliferation and differentiation of other immune cells such as nk cells and b cells (levinson and jawetz 2003). based on figure 4, that the results of the characterization of the snail seromucoid protein profile sds-page method showed that there were 3 sub-units of protein, namely the range 55 72 kda and 1 specific protein sub-unit 43 kda. from the results of this research that has been carried out shows that the 3 subunits of the protein are achasin sulfate proteins that act as antimicrobials. while 1 sub-unit of spefysical protein with a molecular weight of 43 kda is thought to be related to adhesin protein. the results of the analysis of total protein using the bradford method with bsa (bovine serum albumin) as a standard and measured spectrophotometrically at ƛ 595 nm, the concentration -1 of protein content was obtained 6.99 µg ul . the difference in the protein profile of the results of research conducted with previous studies is due to variations in snail strains so that the type and amount of bioactive compounds in hemolymph is the result of gene expression of bioactive compounds. the results of the research that have been carried out show that snail bioactive compounds are able to inhibit the growth of methicillin resistant staphylococcus aureus (mrsa) and the results of protein analysis show that there are 4 subunits of protein, 87.59kda; 77.66 kda, 70.97 kda and 49.46 kda (anggarini et al. 2018). protein profiles of snail mucus obtained at basal and tip shells are different. helix aspersa shows protein bands of 82.97 and 175 kda (greistorfer 2017), whereas in arion sub fuscus snails there are 6 proteins in the range of 10 200 kda with 2 specific proteins 15 kda and 61 kda in the mucous adhesive. in water snails lottia sp showed 68 volume 13, 2019 microbiol indones 61 kda protein in the upper mucus and 80 kda and 118 kda while in adhesive or lower mucus (zhuang et al. 2015). in littoria sp in the upper mucus showed protein profiles 59 and 65 kda; and mucus under the presence of proteins 36 and 41 kda (etim et al. 2015). to conclude, based on the results of the antimicrobial activity test the dilution method that has been carried out shows that the potential of the snail mic seromucoid antibacterial at 100% concentration against staphylococcus aureus, candida albicans and pseudomonas aeruginosa. the physicochemical characterization of snail seromukoid shows specific gravity of 1,010; ph 8, -1 -1 glucose 16 mg dl ; cholesterol 9 mg dl ; protein 2,8 -1 mg dl and heavy metals (pb, cu, hg, al) are negative. the protein profile of anti-microbial bioactive compounds in seromucoid or snail hemolymph (achatina fullica ferrusac) shows that there are 3 achasin sulfate protein sub units with molecular weight in the range of 55 72 kda and 1 specific protein sub unit of 43 kda which is suspected as biological response modifiers (brm). acknowledgements acknowledgments to the directorate of research and community service of the ministry of research and technology of indonesia and all parties who have assisted in this research. this article is part of the pdupt (higher education basic research) drpm program which is funded by directorate of research and community service of the ministry of research and technology of indonesia in 2019 with contract number 074 / l6 / ak / sp2h.i / penelitian / 2019. references anggraini d, sri darmawati, endang twm. 2018. protein profile and anti-microbial power of snail slime (achantina fulica) against methicillin resistant staphylococcus aureus (mrsa). proceedings of the unimus student national seminar. e-issn: 2654-766x vol. 1, 2018 hal 73 – 77. berniyanti t, suwarno. 2007. characterization of local isolate snail slime (achasin) protein as an antibacterial factor. j veter media. issn 2015-8930 , hal 139-144 vol. 23, no. 3, september 2007. bislimi k. behluli a, halili j, mazreku i, osmani f, halili f. 2013. comparative analysis of some biochemical parameters in hemolymph of garden snail (helix pomatia l.) of the kastriot and ferizaj regions, kosovo. dec 2013. vol. 4, no. 6 pp 11 18 issn 2305-8269; 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doi: 10.3390/app8081362 vieira tc, costa-filho a, salgado nc, allodi s, valente ap, nasciutti le, silva lc. 2004. acharan sulfate, the new glycosaminoglycan from achatina fulica bowdich 1822. structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix. eur j biochem. 271(4):845-854. doi: 10.1111/j.1432-1033.2004.03989.x. wayan ta. 1991. genetic engineering. inter university center for biotechnology, gajah mada university, jogjakarta. zhuang j, coates cj, zhu h, zhu p, wu z, xie l. 2015 identification of candidate antimicrobial peptides derived from abalone hemocyanin. dev comp immunol. 49(1):96-102. doi: 10.1016/j.dci.2014.11.008. volume 13, 2019 microbiol indones 63 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 03. eveline.cdr vol.13, no.3, september 2019, p 90-96 doi: 10.5454/mi.13.3.3 antibacterial potential of radish extract (raphanus sativus l.) against fish spoilage bacteria 1* 2 eveline and chikita wini tanumihardja 1 2 lecturer; alumnus food technology department, faculty of science and technology, universitas pelita harapan, mh thamrin boulevard 1100 lippo village, kelapa dua, karawaci, tangerang, indonesia. radish (raphanus sativus l.) root is commonly used as flavor enhancing additive or side dish. previous research revealed the presence of active compound in which could inhibit bacterial growth. thus, a research concerning natural antibacterial for fish products that are categorized as high-risk food being contaminated by spoilage bacteria (pseudomonas aeruginosa, bacillus cereus, and staphylococcus aureus) was done. radish root extraction was made by using ethyl acetate (semi polar) for 3 days. well diffusion was performed using 4 extract concentration (10, 20, 30, and 40% (w/v)) against three fish spoilage bacteria. based on our results, 30% concentration was the best concentration which inhibit more than 10 mm in inhibition zone with minimum inhibitary concentration (mic) and minimum bactericidal concentration (mbc). the scores were of 0.06% and 0.24% (p. aeruginosa), 0.13% and 0.50% (s. aureus), and 0.12% and 0.48% (b. cereus). moreover, based on stability test against heating temperature showed that this extract concentration was more stable in 80 °c with duration times for 5 minutes and ph 3 which resulting the lowest inhibition zone reduction compares to control extract. selected radish extract was categorized as low toxic compound (lc = 839.52 ppm) and by gcms test its 50 functioning in antibacterial compound containing major antibacterial compound (bis(2-ethylhexyl) phthalate, 1,2-benzenedicarboxylic acid, 9,12,15-octadecatrienoic acid), fatty acid (n-hexadecanoic acid, butanedioic acid), carboxylic acid (isobutyric acid, malic acid, oleic acid), and minor antibacterial compound (nhydroxymethylacetamide, 2,4-bis(1,1-dimethylethyl), 2,4-pentanedione,2-cyclohexen-1-one, hydrazine, cyclohexene oxide, gamma-sitosterol). key words: antibacterial, ph, raphanus sativus, stability, temperature, time umbi lobak (raphanus sativus) umumnya dimanfaatkan sebagai bahan tambahan peningkat rasa dan aroma atau pendamping makanan utama. penelitian terdahulu mengungkap adanya komponen aktif yang dapat menghambat pertumbuhan bakteri. hal ini mendorong dilakukannya penelitian mengenai senyawa antibakteri alami untuk produk ikan yang merupakan jenis pangan berisiko tinggi terkontaminasi bakteri pembusuk (pseudomonas aeruginosa, bacillus cereus, dan staphylococcus aureus). ekstraksi dilakukan terhadap umbi lobak dengan etil asetat (semi polar) selama 3 hari. pengujian difusi sumur dilakukan dengan empat konsentrasi ekstrak (10, 20, 30, dan 40% (b/v) pada ketiga spesies bakteri pembusuk ikan. konsentrasi 30% ditentukan sebagai konsentrasi ekstrak terbaik penghasil zona hambat lebih dari 10 mm dengan nila minimum inhibitary concentration (mic) and minimum bactericidal concentration (mbc) berurutan sebesar 0,06% dan 0,24% (p. aeruginosa), 0,13% dan 0,50% (s. aureus), dan 0,12% dan 0,48% (b. cereus). konsentrasi terpilih digunakan pada tahap pengujian stabilitas ekstrak terhadap suhu pemanasan (80 °c dan 100 °c), waktu pemanasan (5, 10, dan 15 menit), dan nilai ph (3, 4 [kontrol], 5, 6, dan 7). perlakuan panas dan perubahan nilai ph menyebabkan ketidakstabilan ekstrak. ekstrak lobak lebih stabil pada suhu 80 °c selama 5 menit dan ph 3 menghasilkan ekstrak dengan penurunan zona hambat terkecil terhadap nilai penghambatan ekstrak kontrol. ekstrak lobak termasuk dalam kategori senyawa toksik rendah (lc = 839,52 ppm) dalam fungsinya sebagai senyawa antibakteri yang 50 mengandung senyawa antibakteri mayor (bis(2-ethylhexyl) phthalate, 1,2-benzenedicarboxylic acid, 9,12,15octadecatrienoic acid), fatty acid (n-hexadecanoic acid, butanedioic acid), carboxylic acid (isobutyric acid, malic acid, oleic acid), dan senyawa antibakteri minor (n-hydroxymethylacetamide, 2,4-bis(1,1-dimethylethyl), 2,4pentanedione,2-cyclohexen-1-one, hydrazine, cyclohexene oxide, gamma-sitosterol). kata kunci: antibakteri, ph, raphanus sativus, stabilitas, suhu, waktu microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-8128187282; email: eveline.fti@uph.edu raphanin, and essential oil which made this plant more potential for natural antimicrobial. sukhla (2011) in his research found that raphanin in radish was effective in the inhibition of pathogenic microbes such as escherichia coli, pseudomonas pyocyaneus, salmonella typhi, bacillus subtilis, staphylococcus aureus, streptococci, p. listeria, micrococcus, radish (raphanus sativus l.) is medicinal plant that commonly used as food, additive, or side dish. beside its nutritional contain radish also has bioactive compound such as tannin, flavonoids, saponin, enterococcus, lactobacillus, and pedicoccus. in -1 addition, janjua (2013) found that 100 mg ml of radish extract was effective in the inhibition of gram positive and negative bacteria. however, this antibacterial potency needs to be improved in order to expand in the food sector. in our study, we focused in antibacterial potential of radish root against three species bacteria which often causes damage to fish (pseudomonas aeruginosa, bacillus cereus, and staphylococcus aureus). related analysis such as minimum inhibitory concentration (mic), minimum bactericidal concentration (mbc), toxicity analysis using brine shrimp lethality test (bslt), and gas chromatography-mass spectrometry (gc-ms) were also assessed. based our study, we expect that this research gives more information source concerning optimal condition of radish extract which can be used in food industry. materials and methods radish extract was obtained from maceration process using ethyl acetate (semi polar) for 3 days. extract with 4 different of concentrations (10, 20, 30, and 40%) were tested using well diffusion method towards three species of bacteria test. on further, the stability test based on heating temperature was assessed by heating this extract in 80 °c and 100 °c with duration times: 5, 10, and 15 minutes and ph value (3, 4 [control], 5, 6, and 7). temperature level and heating time were determined based on syamsir (2002) research, which found that phenolic compound was stable on 121 °c for 15 minutes; and ph level was determined based on the acidity characteristic of food in daily lives, started from taste senses' acceptable acidity (3.0) to neutral condition (7.0). material. white radish (raphanus sativus l.), distilled water, k so , selenium, h so 96%, h o 2 4 2 4 2 2 35%, boric acid 4%, naoh, mix indicator, hcl, h e x a n e , b u ff e r s o l u t i o n p h 4 a n d p h 7 , phenolphthalein, alcl 2%, na, nb, dilution solution, 3 spoilage microorganism culture: staphylococcus aureus (gram positive bacteria), pseudomonas aeruaeruginosaginosa (gram negative bacteria), and bacillus cereus (spore bacteria). sample preparation phase methods. sample preparation phase was the phase used to make powder from radish root (raphanus sativus l.), that are washed, peeled, cut (3 mm), dried (oven; 60 °c; 21 hours), size reduction (dry blender), and sieved (35 mesh). radish root powder was then proximately analyzed. a growth curve was carried out on each test bacterium to determine the age of the bacteria to be used in the well diffusion test, where the number of 8 -1 bacteria used in the well diffusion test was 10 cfu ml . phase i methods. phase i research (fig 1) started by maceration process of radish root powder (1:10; 2025 °c) using ethyl acetate for 3 days. extraction was executed by constant shaking at 150 rpm. filtrate evaporation and filtration (45 °c; 35 rpm; 1 hour) was done in order to produce antibacterial compound extract. extract was then diluted to 4 level of concentration (10, 20, 30, and 40% (w/v)). antimicrobial activity test was done using well diffusion method in order to produce selected extract which was the best extract concentration. phase ii methods. in phase ii, the stability test was performed by put the extract in different temperature (80°c and 100°c) with different heating time (5, 10, and 15 minutes), and ph (3, 4 [control], 5, 6, and 7); mic and mbc test; component analysis using gc-ms; and toxicity test (atmoko 2009) were also performed done toward selected extract from phase i research. analysis. proximate analysis in phase i research was used to determine the content of water, ashes, protein, fat, carbohydrate (aoac 2005). mic and mbc in phase ii research were used to analyze the minimum value needed to inhibit and kill 90% growth of test bacteria (bloomfield 1991). in addition, to asses the cytotoxicity of extract, we used bslt method (atmoko 2009), whilst gc-ms test is used to analyze major antibacterial compound in extract. experimental design. experimental design used in phase i research was completely randomized design with one factor and four levels (10% [a ], 20% 1 [a ], 30% [a ], and 40% [a ]) and three repetition. 2 3 4 experimental design used in phase ii research for stability against heating temperature and time was completely randomized design with two factor and three repetition. temperature factor used two levels which were: 80 °c (a ) dan 100 °c (a ); while heating 1 2 time used three levels which were: 5 minutes (b ), 10 1 minutes (b ), and 15 minutes (b ). stability test against 2 3 ph value was done using completely randomized design with one factor and three repetition. ph levels were: 3 (a ), 4 [control] (a ), 5 (a ), 6 (a ), and 7 (a ).1 2 3 4 5 results phase i. based on proximate analysis on radish root extract, we found that this extracts contain volume 13, 2019 microbiol indones 91 fig 1 flowchart of radish root antibacterial activity research. source: modification from astuti (2007); janjua (2013); nuriani (2007) 18.00±0.16% water, 11.27±0.09% ashes, protein 16.72±1.25% protein, 2.50±0.30% fat, 51.51±1.23% carbohydrate. radish root powder had 79.45±1.29% yield and ph of 4.90±0.10. the bacteria used. bacteria used in test were in log phase after 6 hours of incubation period. total 8 colony used for well diffusion in this research were 10 -1 cfu ml , total colony of staphylococcus aureus, pseudomonas aeruginosaginosa, and bacillus cereus 8 -1 8 -1 used in test were 5.10x10 cfu ml , 2.61x10 cfu ml , 8 -1 and4.30x10 cfu ml . inhibition diameter based on extract concentration. according to chandra (2011), antibacterial activity could be divided into four categories based on its inhibition diameter, that were no activity (<6 mm), weak (6-7 mm), medium (7-10 mm), and strong (>10 mm). antibacterial activity test result of radish root extract using ethyl acetate was proven effectively inhibit the growth of fish spoilage bacteria (table 1). table 1 showed that extract concentration 92 eveline et al. microbiol indones significantly affecting inhibition diameter (p<0.05). the bigger the extract concentration would result in bigger inhibition diameter. according to mpila et al. (2012), bioactive compound contained in higher concentrated extract was higher than those in lower concentrated, affecting bacteria growth inhibition. table 1 also showed that 30% extract concentration was able to inhibit the bacteria with more than 10 mm inhibition diameter, it was strong inhibition according to chandra (2011); thus, the extract concentration for the next phase was 30%. phase ii. phase ii research was done in order to determine the stability against heating temperature (80 °c and 100 °c) and heating time (5, 10, and 15 minutes), and ph (3, 4 [control], 5, 6, and 7). in this step, analysis of selected extract from phase i was also done such as mic and mbc test, toxicity test, and component analysis using gc-ms. e x t r a c t s t a b i l i t y b a s e d o n h e a t i n g temperature and time. statistical test results showed that heating temperature and time interacted affected inhibition diameter of the three bacteria test (p<0.05). both factors affected the inhibition diameter so that the extract stability could be indicated. table 2 showed that heating temperature and time caused the reduction of the formed inhibition diameter, moreover at 100 °c there was no inhibition diameter formed. it could be indicated that heat treatment made the extract unstable and the presence of heat treatment caused radish extract antibacterial activity only reach medium category (7-10 mm). extract stability based on ph. the change in ph affect the inhibition diameter of the three test bacteria (p<0.05). the addition of hcl 0.1m as the acid conditioner and the addition of naoh 0.1m as base conditioner on extract gave significant difference compared to control extract with ph ~4,0 (the extract without acid or base conditioning). table 3 showed that the increase in ph from control reduce the size of inhibition diameter, while the decrement of ph value will increase the size of inhibition diameter. mic and mbc. mic and mbc value was determined based on the selected extract inhibition zone value (30% concentration). mic value was the minimum value needed to inhibit 90% growth of test bacteria, while mbc value was the minimum concentration needed to kill 90% of test bacteria (shahid et al. 2013). mic and mbc test results for s. aureus were 0.13% and 0.50%, for p. aureginosa were 0.06% and 0.24%, and for b. cereus were 0.12% and 0.48% (table 1). usually, gram negative bacteria had higher mic and mbc value compared to gram positive bacteria, but in this research the mic and mbc of gram positive bacteria (s. aureus and b. cereus) were higher. toxicity. toxicity value (lc ) indicates the safety 50 level of extract to be applied in food products. this test was done as the first step in other complex toxicity test. based on the principle of brine shrimp lethality test (bslt), the more compound needed to kill 50% of shrimp larvae, then the compound is categorized as non-toxic. juniarti (2009) divides the toxicity level of lc into three categories that are: lc >1000: non-50 50 toxic, 30<lc <1000 ppm: low toxic, dan lc <1000 50 50 ppm: toxic. test results showed that radish root extract had lc of 839.52 ppm (low toxic). 50 gc-ms. the extract contain major antibacterial compound (bis(2-ethylhexyl) phthalate, 1,2benzenedicarboxylic acid, 9,12,15-octadecatrienoic acid), fatty acid (n-hexadecanoic acid, butanedioic acid), carboxylic acid (isobutyric acid, malic acid, oleic acid), and minor antibacterial compound (nhydroxymethylacetamide, 2,4-bis(1,1-dimethylethyl), 2,4-pentanedione,2-cyclohexen-1-one, hydrazine, cyclohexene oxide, gamma-sitosterol). discussion radish root extract made using ethyl acetate with 3 days extraction time and 30% concentration effectively gave the best inhibition toward three fish spoilage bacteria (staphylococcus aureus, pseudomonas aeruginosa, and bacillus cereus). mic and mbc score against the three test bacteria respectively were: 0.13% and 0.50% (staphylococcus aureus), 0.06% and 0.26% (pseudomonas aeruginosa), and 0.12% and 0.48% (bacillus cereus). according to davidson (2009) and yunilla (2016), even though gram negative bacteria has more complex cell membrane, gram positive bacteria has membrane composed of peptidoglycan which is polar and bigger (90%) compared to peptidoglycan layer of gram negative bacteria (1050%). this would result in the phytochemical compound that are mostly non-polar getting hard to invade and destroy gram positive bacteria cell, thus more compound is needed to inhibit and kill bacteria. radish root extract was not stable against heat treatment and change in ph value; but 80 °c heat treatment for 5 min gave the closest results to control's inhibition strength. according to syamsir (2002), the reduction of inhibition rate was caused by the decrement of phenolic compound effectivity which act volume 13, 2019 microbiol indones 93 as antibacterial in the extract. phenolic compound is stable on 120 °c for 15 min, but in several phytochemical compound with low molecular weight, evaporation could easily happen when heated. this could result in the instability of extract because of the evaporation of active component. naufalin et al. (2007) added that the loss of inhibition potential of a flavonoid compound could happen because of degradation of flavonoid starting from 60 °c such as radish drying. at ph of 3, the inhibition radish root extract was increase from the inhibition strength of control extract. according to naufalin et al. (2007), there are several factors that increase the inhibition of bacteria in acid condition. the first is substitution between antibacterial compound with hcl as halogen affecting the breakdown of membrane. cl ion made bacteria cell spend more energy making it more susceptible against radish extract antibacterial compound. the second is that phenolic compound work actively in low ph condition because alkylation and hydroxylation happen easily in acid condition, improving the phenol group of bacteria whether it is in water or lipid phase. the third is that bacteria cell maintain constant ph in cell when making contact with concentration (%) inhibition diameter (mm) s. aureus p. aeruginosaginosa b. cereus 10 8.36 ± 0.19a 8.88±0.29k 8.73±0.34w 20 10.11 ± 0.34b 11.14±0.39l 9.92±0.31x 30 11.39 ± 0.21c 12.73±0.25m 12.73±0.52y 40 13.77 ± 0.30d 13.64±0.61n 13.83±0.40z heating temperature (°c) heating time (minutes) inhibition diameter (mm) s. aureus p. aeruginosaginosa b. cereus control (no heat treatment) 11.39 ± 0.20 12.73 ± 0.25 12.73 ± 0.52 80 5 7.81 ± 0.28d 9.97 ± 0.31n 10.80 ± 0.16z 10 7.05 ± 0.05c 8.72 ± 0.37m 9.78 ± 0.36y 15 6.23 ± 0.15b 7.11 ± 0.27l 8.60 ± 0.44x 100 5 0.00 ± 00a 0.00 ± 00k 0.00 ± 00w 10 0.00 ± 00a 0.00 ± 00k 0.00 ± 00w 15 0.00 ± 00a 0.00 ± 00k 0.00 ± 00w ph inhibition diameter (mm) s. aureus p. aeruginosaginosa b. cereus 3.0 11.70 ± 0.00d 13.90 ± 0.60n 14.40 ±0.38z ~4.0 (control) 11.38 ± 0.20c 12.73 ± 0.25m 12.72 ± 0.32y 5.0 5.13 ± 0.60b 6.20 ± 0.20l 6.50 ± 0.30x 6.0 0.00 ± 00a 0.00 ± 00k 0.00 ± 00w 7.0 0.00 ± 00a 0.00 ± 00k 0.00 ± 00w table 1 phase i analysis results (inhibition diameter based on extract concentration) table 2 phase ii analysis results (extract stability based on heating temperature and time) table 3 phase ii analysis results (extract stability based on ph) different notation showed that there was significant difference (p<0.05); not compared between bacteria different notation showed that there was significant difference (p<0.05); not compared between bacteria different notation showed that there was significant difference (p<0.05); not compared between bacteria 94 eveline et al. microbiol indones + acid. in low ph, proton of h ion is going to enter the cytoplasm through trans membrane proton gradient making the cytoplasm ph decrease and causing the enzyme work to return internal cell ph to normal. this would happen because the proton causing acid condition could also cause denaturation of cell component. the process to return ph to normal need a lot of energy from bacteria and could affect cell metabolism causing the cell to die. soetan and oyewole (2009) and yunilla (2016), stated that natural antinutritional compound could be formed by plant metabolism such as glycoside, tannin, lignin, lignin, triterpenoid, oxalate, dan amino acid. yunilla also said that low toxicity is also categorized as safe and is not dangerous if consumed as herbal intake, thus radish root extract was categorized as safe (not dangerous) and could be consumed at certain dose. selected radish root extract was categorized as low toxic compound (lc = 839.52 ppm) in its function as 50 antibacterial compound that contains as follows (in the order of the best antibacterial potential): major antibacterial compound (bis(2-ethylhexyl) phthalate, 1,2-benzenedicarboxylic acid, 9,12,15-octadecatrienoic acid), fatty acid (n-hexadecanoic acid, butanedioic acid), carboxylic acid (isobutyric acid, malic acid, oleic acid), and minor antibacterial compound (n-hydroxymethylacetamide, 2,4-bis(1,1dimethylethyl), 2,4-pentanedione,2-cyclohexen-1one, hydrazine, cyclohexene oxide, gamma-sitosterol). all component were categorized as essential oil that could act as antibacterial agent (hussain et al. 2011; agoramoorthy et al. 2007; pringgenies 2010; rajeswari et al. 2012; rahminiwati et al. 2010; soni et al. 2014). references agoramoorthy, chandrasekaran gm, venkatesalu v, and hsu mj. 2007. antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove from india. braz j microbiol. 38(4):739-742. doi: 10.1590/s1517-83822007000400028. th aoac. 2005. official methods of analysis 19 ed. washington: aoac inc. astuti sm. 2007. teknik mempertahankan mutu lobak (r. sativus) dengan menggunakan alat pengering vakum [technique to maintain the quality of radish (r. sativus) using a vacuum dryer]. available from http://pustaka. litbang.pertanian.go.id/publikasi/bt121079.pdf. atmoko t, amir m. 2009. uji toksisitas dan skrining fitokimia ekstrak tumbuhan sumber pakan orangutan terhadap larva artemia salina l. east kalimantan [toxicity test and phytochemical screening of plant extracts from orangutan feed on artemia salina l. larvae east kalimantan]. balai penelitian teknologi perbenihan samboja. bloomfield sf. 1991. assesing antimicrobial activity. oxford: blackwell scientific publication. chandra r, vinay d, kumar s, abhimanyu kj. 2011. detection of antimicrobial activity of oscimum sanctum (tulsi) & trigonella foenum graecum (methi) against some selected bacterial and fungal strains. res j pharm biol chem sci. 2(4):809-813. doi: 10.1046/j.13652672.1999.00780.x. d a v i d s o n m w. 2 0 0 9 . b a c t e r i a c e l l s t r u c t u r e . http://micro.magnet.fsu.edu/cells /bacteriacell.html. hussain h, badawy a, elshazly a, elsayed a, krohn k, riaz m, schulz b. 2011. chemical constituents and antimicrobial activity of salix subserrata. rec nat prod. 5(2):133-137. janjua s, maliha s, fakhir a. 2013. phytochemical analysis and in vitro antibacterial activity of root peel extract of raphanus sativus l. var niger. adv med plant res. 1(1):1-7. juniarti, delvi o, yuhernita. 2009. kandungan senyawa kimia uji toksisitas (brine shrimp lethality test) dan antioksidan (1,1-diphenyl-2-pikrilhydrazyl) dari ekstrak daun saga (abrus precatorius l.) [chemical compounds content toxicity test (brine shrimp lethality test) and antioxidant (1,1-diphenyl-2-pikrilhydrazyl) from saga leaf extract (abrus precatorius l.)]. jakarta (id): universitas yarsi. mpila da, fatimawali, weny lw. 2012. uji aktivitas antibakteri ekstrak etanol daun mayana (coleus atropurpureus benth) terhadap staphylococcus aureus, escherichia coli, dan pseudomonas aeruginosa secara in vitro [antibacterial activity test of ethanol extract of mayana leaves (coleus atropurpureus benth) against staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa in vitro]. manado (id): universitas sam ratulangi. pharmacon. 1(1):13-21. naufalin r, jenie bsl, kusnandar f. 2007. pengaruh ph, nacl, dan pemanasan terhadap stabilitas antibakteri bunga kecombrang dan aplikasinya pada daging giling [effect of ph, nacl, and heating on the antibacterial stability of kecombrang flowers and their application on ground beef]. bogor (id): institut pertanian bogor. jurnal teknologi dan industri pangan. 17(3):196-203. nuriani ad. 2007. ekstraksi komponen antibakteri dan antioksidan dari biji teratai (nymphaea pubescens willd) [extraction of the antibacterial and antioxidant components from lotus seeds (nymphaea pubescens willd)]. bogor (id): institut pertanian bogor. nurainy f, samsul r, yudiantoro. 2008. pengaruh volume 13, 2019 microbiol indones 95 96 eveline et al. microbiol indones konsentrasi kitosan terhadap aktivitas antibakteri dengan metode difusi agar (sumur) [effect of chitosan concentration on antibacterial activity with the agar diffusion method]. lampung (id): universitas lampung. jurnal teknologi & industri hasil pertanian. 13(2):117-125. doi: 10.23960/jtihp.v13i2.117%20%20125. pringgenies d. 2010. karakteristik senyawa bioaktif bakteri simbion moluska dengan gc-ms [characteristics of bioactive bacterial symbionts molluscs with gc-ms]. j ilmu dan teknologi kelautan tropis. 2(2):34-40. doi: 10.28930/jitkt.v2i2.7850. rahminiwati m, mustika aa, saadiah s, andriyanto, soeripto, unang. 2010. bioprospeksi ekstrak jahe gajah sebagai anti-crd: kajian aktivitas antibakteri terhadap mycoplasma galliseptikum dan e. coli in vitro [bioprospection of gajah ginger extract as anti-crd: study of antibacterial activity against mycoplasma galliseptic and e. coli in vitro]. jurnal ilmu pertanian indonesia. 15(1):7-13. rajeswari g, murugan m, mohan vr. 2012. gc-ms analysis of bioactive components of hugonia mystax l. (linaceae). res j pharm biol chem sci. 3(4):301-308. shahid m., shahzad a, malik a, sahai a. 2013. recent trends in biotechnology and therapeutic applications of medicinal plants. new york: springer science business media. shukla s, sanjukta c, deepak ky, geeta w. 2011. antimicrobial efficacy of r. sativus root juice. int j pharm pharm sci. 3(5):89-92. soetan ko, oyewole oe. 2009. the need adequate processing to reduce the antinutritional factors in plants used as human foods and animal feeds. afr j food sci. 3(9):223-232. soni n, mehta s, satpathy g, gupta rk. 2014. estimation of nutritional, phytochemical, antioxidant and antibacterial activity of dried fig (ficus carica). j pharmacogn phytochem. 3(2):158-165. syamsir e. 2002. mempelajari stabilitas aktivitas antimikroba ekstrak biji atung (parinarium glaberimum hassk) dengan pelarut etil asetat teknis selama penyimpanan terhadap staphylococcus aureus [study of stability of the antimicrobial activity of atung seed extract (parinarium glaberimum hassk) with technical ethyl acetate solvent during storage against staphylococcus aureus ]. bogor (id): institut pertanian bogor. yunilla i. 2016. kajian aktivitas dan stabilitas antibakteri ekstrak daun bintaro (cerbera oddolam gaertn.) [study of activity and antibacterial stability of bintaro leaf extract (cerbera oddolam gaertn.)]. tangerang (id): universitas pelita harapan. page 1 page 2 page 3 page 4 page 5 page 6 page 7 10 yogy simanjuntak_404 rev.pmd volume 3, number1, april 2009 p 47-50 issn 1978-3477 t-cell epitopes of mycobacterium tuberculosis antigen 85 complex potential for generating antibody: an immunoinformatics study yogy simanjuntak microbiology division, research center for biology, lembaga ilmu pengetahuan indonesia, jalan raya cibinong km 46, p.o box 25, cibinong 16911, indonesia phone: +62-21-8765066, fax: +62-21-8765062, email: yogy_simanjuntak@yahoo.com the present study was conducted to predict t-cell epitopes of the antigen 85 complex capable of stimulating antibody generation by using immunoinformatics approaches. by applying computational biology software, the available data of the antigen 85 complex and related-epitopes would be turned into more constructive and useful scientific informations for the development of multiepitope-based anti-tb vaccine. the identification of t-cell epitopes capable of generating antibody was done by the 3dex program, discotope analysis and pymol program. selected peptides having individual amino acids localized on the predicted antibody-binding sites were subjected to antigenic property analysis, including their hydrophilicity, flexibility and antigenic propensity. the 3dex program identified 17 peptides having at least four individual amino acids located on the antigen surface. however, after homology analysis with preselected distance of 7 å and taking into account the spatial neighborship, only seven peptides of antigen 85a, 85b and 85c (3, 3 and 1 peptide(s) respectively) had individual amino acids overlapping the predicted antibody-binding site. peptides 17838, 21780, 21275 and 36131 had an average score of antigenic propensity above 1.0. in conclusion, there are seven peptides representing t-cell epitope of antigen 85 complex that could potentially be capable of generating an antibody response. the seven peptides, p17838, p21093, p36131, p21275, p21780, p21796 and p10839, are suitable candidates for further study in order to develop a subunit-based multiepitope anti-tb vaccine. key words: mycobacterium tuberculosis, multiepitope anti-tb vaccine, t-cell epitope, antibody, immunoinformatics tuberculosis (tb) is an infectious disease caused predominantly by the pathogenic bacteria called, mycobacterium tuberculosis. tuberculosis is still a major health problem in indonesia and considered as the third main cause of death after cardiovascular and respiratory diseases. as the third country in descending order of tb case numbers after india and china, indonesia has to fight the disease seriously (http://www.who.int/gtb.html). nowadays, wide use of rifampicin containing regimens often leads to a steady increase in multidrug resistant tuberculosis (kim 2004). it is appealing to speculate that antibiotic therapy becomes a twoedged sword in tb intervention. furthermore, failure of the bcg vaccine to protect in endemic regions calls for the development of effective vaccines. instead of using whole protein preparations as vaccine candidates, researchers currently consider that t-cell epitopes may be used in the development of vaccines. some investigators are trying to identify the kind of peptide antigens that could evoke not only t-cell mediated response, but also antibody synthesis (sarhan et al. 2007). such a study is quite tricky, taking into consideration antibody designated to recognize surface epitope of the antigen, while in contrast t-cell epitopes are more hydrophobic (laver et al. 1990). the antigen 85 complex, as the most common proteins in m. tuberculosis culture fluids, would be suitable for the purpose of interest. this antigen is a strongly immunogenic, stimulating not only t-cell-mediated reaction but also humoral immune response. the representing constituents, 85a, 85b and 85c, are encoded by three genes located at different sites in the mycobacterial genome. all are a fibronectinbinding protein and strongly immunogenic both in natural and experimental studies stimulating antibody synthesis and t-cell-mediated reactions (wiker and harboe 1992). fortunately, more than a hundred t-cell epitopes of the antigen 85 complex has been reported in the immune epitope database and analysis resource (www.immuneepitope.org). however, their potency to induce antibody generation remains unclear. in view of therapeutic vaccine development that could evoke both cell-mediated and humoral immune response, an immunoinformatics study has to be done prior to experimental studies. this avoids labor-intensive, costly and time-consuming works. therefore, this study was conducted to predict t-cell epitopes of the antigen 85 complex capable of stimulating antibody generation using an immunoinformatics approaches. by applying computational biological software, the available data of the antigen 85 complex and related-epitopes can be turned into more constructive and useful scientific information for the development of multiepitope-based anti-tb vaccine. materials and methods data sources. peptide sequences representing t-cell epitopes of the antigen 85 complex were derived from many reports in the immune epitope database and analysis resource (www.immuneepitope.org), determined by various assays including elisa, cytokine bioassay, elispot, 51 chromium release and proliferation methods. the pdb files of the antigen 85 complex corresponding to known structures of antigen 85a (1sfr), 85b (1f0n) and 85c (1dqz) were retrieved from the rcsb protein data bank (www.rcsb.org). screening of t-cell epitopes. three databases consisting of 50, 57 and 25 different peptide sequences representing t-cell epitopes were analyzed for their surface structure against respective antigen 85a, 85b and 85c. the surface scan implemented in the 3d-epitope-explorer (3-dex) program was performed to screen the surface structure of each peptide. the probability of amino acids to be on surface exposure was set to equal or greater than 50% whilst joker function was activated (schreiber et al. 2005). peptide microbiol indones48 simanjuntak sequences were selected for further analysis if more than three individual amino acids were likely on the surface exposure of the respective antigen. identification of t-cell epitopes capable of generating antibody. by using the 3d-epitope-explorer program, the selected peptides were subjected to homology analysis against their respective structural pdb files. anticipating a conformational epitope, a distance of 7 å (angström) was preselected with the highest possible frame size. the previous result of surface scan was used to determine the most suitable homologous hit. antibody-binding sites of respective antigens were predicted by using discotope analysis (andersen et al. 2006). the peptide sequence and antibody binding sites could then be highlighted in the pdb structure file using the program pymol. analysis of antigenic properties. selected peptides with individual amino acids overlapping the predicted antibodybinding sites were subjected to antigenic property analysis including hydrophilicity, flexibility and antigenic propensity as described by karplus and schulz (1985); parker et al. (1986) and kolaskar and tongaonkar (1990). results three databases consisting of 50, 57 and 25 different t-cell epitopes were analyzed for their surface structure against respective antigen 85a, 85b and 85c. the 3dex software identified that 5 out of 55 peptides have more than three individual amino acids located on surface area of 3-dimensional (3d) structure-antigen 85a. as many as 11 peptides possessed more than three individual amino acids that were exposed on the surface of antigen 85b. only a single peptide representing t-cell epitope of antigen 85c was considered for further analysis (table 1). table 2 shows the homology and surface scan profile of five selected peptides against the 3d-structure antigen 85a (chain a). the 3dex software identified two peptides, p17838 and p72312, which were completely aligned within the preselected distance of 7 å against antigen 85a. however, the last contiguous peptide (p72312) in addition to peptide 21670 had only one individual amino acid located on the surface of antigen 85a (e59 and k183, respectively). although not perfectly homologous within the preselected distance, peptide p17838 and p21093 had at least 3 contiguous individual amino acids exposed on the surface of antigen 85a (f1 s2 r3 p4 g5 l6 and d46 d47 f48, respectively). interestingly, in addition to discontinuous amino acids p14 and p16, three contiguous amino acids (r3 p4 g5) of peptide p17838 overlapped the predicted antibody-binding site at the neutralizing face of antigen 85a (fig 1). four individual amino acids within the peptide p21093 were located on the surface of antigen 85a (r43 d46 d47 f48). however, only two continuous amino acids (d46 d47) overlaid the predicted antibody-binding site. in addition, two contiguous amino acids (n222 n223) within the peptide p36131 were located in the region of the predicted antibody-binding site. the 3dex software identified 5 out of 11 selected peptides (namely: smagssamil, fltselpqwl, iglsmagssamilaay, fltselpqwlsanravkp and rndptqqipkl vanntrl) that were perfect homolog within the preselected distance of 7 å against the 3d-stucture of the antigen 85b. however, most of the homologous hits did not fit with the surface scan analysis. only six selected peptides having surface structure are described in table 3. although partly aligned within the preselected distance against antigen 85b, three peptides (p21275, p21780 and p21796) had at least two individual amino acids overlapping the predicted-antibody binding site (fig 2). some amino acids within three peptides (p73294, p54977 and p20101) were also found to be part of antigen surface area. however, all of these were independent of the predicted antibody-binding site. table 4 shows homology and surface scan analysis of peptide p10839 against the 3dstructure of antigen 85c (chain b). the peptide was partly homologous within the preselected distance of 7 å against antigen 85c. the amino acids, q582 q585 s586 g588 n590, were localized on the surface exposure of the antigen. interestingly, all of those amino acids overlapped the predicted antibody-binding site (fig 3). antigenic properties of seven peptides are presented in table 5. the seven peptides had at least two individual amino acids overlapping the predicted antibody-binding site. four peptides (p17838, p21780, p21275 and p36 131) had an average score of antigenic propensity above 1.0. three peptides (p10839, p21796 and p21093) were considered the most hydrophilic compared to the others. table 2 homology and surface scan analysis of the selected peptide sequence against the 3dstructure of antigen 85a [1sfr]* peptide id p 1 7 8 3 8 p 2 1 0 9 3 p 3 6 1 3 1 p 2 1 6 7 0 p 7 2 3 1 2 distance [angströms]: 7 scan surface: threshold > 50% max. number of jokers: 2 reference atom: alpha-c atom [1sfr_85a] fsrpglpveylqvpspsmgr phe 1|ser 2|arg 3|pro 4|gly 5|leu 6|pro 7|val 8|glu 9| tyr 10|leu 11 |gln 12|val 13|pro 14|ser 15|pro 16|ser 17|met 18|gly 19| arg 20 glraqddfsgwdintpafew leu 42|arg 43|ala 44|gln 45|asp 46|asp 47|phe 48|ser 49|gly 50 | trp 51|asp 52|ile 53|asn 54|thr 55|pro 56|ala 57|phe 58|glu 59|trp60 lggnnlpakflegfvrtsni leu 219|gly 220|gly 221|asn 222|asn 223|leu 2 24|pro 225|ala 226| lys 227|phe 228|leu 229|glu 230 gpkedpawqrndpllnvgkl gly 181|pro 182|lys 183|glu 184|asp 185|pro 186|ala 187|trp 180 wdintpafewydqsglsvvm trp 51|asp 52|ile 53|asn 54|thr 55|pro 56|ala 57|phe 58| glu 59| trp 60|tyr 61|asp 62|gln 63|ser 64|gly 29|leu 66|ser 67|val 68|val 69| met 70 *underlines are homolog amino acid sequences; amino acids which are surface exposed are marked in bold table 1 screening of surface structure of t-cell epitope against respective antigen* variable antigen 85 a (n=50) antigen 85 b (n=57) antigen 85 c (n=25) none 12 16 8 1-3 4 ≤ 5 (fsrpglpveylqvpspsmgr; glraqddfsgwdintpafew;gpkedpawqrndpllnvgkl; lggnnlpakflegfvrtsni;wdintpafewydqsglsvvm) 11 (gmgpsligl; gpsliglam;smagssamil;fltselpqwl; iglsmagssamilaay; fltselpqwlsanravkp; ggykaadmwgpssdpawe; gpssdpawerndptqqip; rndptqqipklvanntrl; wyspacgkagcqtykwet; lqvpspsmgrdikvqfqsgg) 1 (dwyqpsqsngqnytykwetf) * values are expressed as the total number number of amino acid accessible on surface area 33 30 16 microbiol indones 49volume 3, 2009 discussion the only widely used vaccine against childhood tb, the bcg vaccine, is unlikely to have a significant impact on an adult tb epidemic. therefore, the domain of the anti-tb vaccine development has been explored extensively in order to find out the most efficient prophylactic vaccines. the new techniques for preparing anti-tb vaccine include development of dna vaccine, modified bcgs and multiepitope-based vaccines (sarhan 2007). the last mentioned technique becomes relatively straightforward with the availability of proteomic tools such as peptide synthesizer machine. new vaccines being developed against tb focuses on the t lymphocyte since it is the central protection against tb (sarhan 2007). interestingly, a study reported by spouge et al. (1987) revealed that strong conformational propensities enhance t-cell antigenicity. conformational epitopes are required for neutralizing antibody, thus they have to be located on the surface of a given antigen. in immune epitope database and analysis resource, more than a hundred peptides representing t-cell epitopes of the antigen 85 complex has been reported by various studies. the question now is which peptide(s) will be potentially capable to stimulate antibody response? fig 2 individual amino acids located on the surface antigen 85b 3d-structure within the six peptides (g158m159gpsligl, g5p4s2 liglam, g p182s183s184d p186 aw e r n d p t q q i p, w y83s p 8 5acg k89a90g c92 q t y k w e t, r n d p t q q ip198k199 lv anntrl and ggyk175 aa177dmwgpssdpawe). the predicted antibody-binding site of antigen 85b is shown in blue. amino acids of the respective peptides which overlap the predicted antibody-binding site are shown in magenta. individual amino acids independent of the predicted antibody-binding site are shown in red. fig 1 individual amino acids located on the surface antigen 85a 3d structure within the three peptides (f1s2r3p4g5l6pveylqv p14sp16smgr, glr43aqd46d47f48sgwdintpafew, lggn2 22n223lpakflegfvrtsni). the predicted antibody-binding site of antigen 85a (chain-a) is shown in blue. amino acids of the respective peptides overlap the predicted-antibody binding site are shown in magenta. individual amino acids independent of the predicted-antibody binding site are shown in red. *underlined are homolog amino acid sequences; amino acids which are surface exposed are marked in bold. table 3 homology and surface scan analysis of the selected peptide sequence against the 3dstructure of antigen 85b [1f0n]* peptide id p21275 p21780 p21796 p73294 p54977 p20101 distance [angströms]: 7 scan surface: threshold > 50% max. number of jokers: 2 reference atom: alpha-c atom [1f0n_85b] gmgpsligl gly 158|met 159|gly 160|pro 155|ser 156 gpsliglam gly 5|pro 4|ser 2|leu 6 gpssdpawerndptqqip gly 181|pro 182|ser 183|ser 184|asp 185|pro 186|ala 187|trp 180 wyspacgkagcqtykwet trp 180|tyr 83|ser 84|pro 85|ala 86|cys 87|gly 88|lys 89|ala 90| gly 91|cys 92|gln 93 rndptqqipklvanntrl arg 190|asn 191|asp 192|pro 193|thr 194|gln 195|gln 196|ile 197|pro 198|lys 199|leu 200|val 201|ala 202|asn 203|asn 204|thr 205|arg 206|leu 207 ggykaadmwgpssdpawe gly 88|gly 172| tyr 174|lys 175|ala 176|ala 177|asp 178|met 179|trp 180|gly 181|pro 182|ser 183|ser 184|asp 185|pro 182|ala 177|trp 180 table 4 homology and surface scan analysis of the selected peptide sequence against the 3d structure of antigen 85 [1dqz]* peptide id p 1 0 8 3 9 distance [angströms]: 7 scan surface: threshold > 50% max. number of jokers: 2 reference atom: alpha-c atom [1dqz_85c] dwyq psq sng qnytykwetf asp 579 683 |trp 580 678 |tyr 581| gln 582|pro 583|ser 584|gln 585|ser 586|asn 587|gly 588| gln 589|asn 590 *underlined are homolog amino acid sequences; amino acids which are surface exposed are marked in bold; subscript indicates an alternative amino acid. fig 3 individual amino acids located on the surface antigen 85c 3d-structure within the selected peptide (dwyq582psq585s586 ng588qn590ytykwetf). the predicted antibody-binding site of antigen 85c (chain-b) is shown in blue. amino acids of the respective peptides which overlap the predicted antibody-binding site are shown in magenta. microbiol indones50 simanjuntak in present study, the validated-3dex program only identified a few number of t-cell epitopes that potentially mimic such conformational epitopes which have individual amino acids located on surface of the antigen 85 complex. it seems the characteristic discrepancy between t-cell and b-cell epitopes is the main factor. the antibody is designated to recognize an epitope located on the surface of an antigen. generally, this kind of epitope consists of hydrophilic amino acids. in contrast, t-cell epitopes are mostly hydrophobic. it was found in the present study that only seven peptides (p17838, p21093, p36131, p21275, p21780, p21796 and p10839) have individual amino acids overlapping the predicted antibody-binding site of the respective antigen 85. this means that in addition to a cell-mediated immune response, these seven peptides could potentially stimulate antibody generation either directly or indirectly, through major histocompatibility complex (mhc) class ii. the newly-published peptide derived from m. tuberculosis rv1490 surface protein was proposed as the potential vaccine candidate taking the same item for multiepitopebased vaccine development (patarroyo et al. 2008). this comprehensive research involved a bioinformatics approach, in an in vitro and in vivo study, suggesting two peptides aeilvkyaqladkrarvyvl (11 060) and fgrveshadyhdwvcehvtp (11 073) play an important role in tb pathophysiology. the antigenic propensity of peptide 11 060, based on the finding of kolaskar et al. (1990) is 1.072, while peptide 11 073 is 1.067. interestingly, these average scores are considered lower compared with the p17838 and p21780 scores (1.088 and 1.075 respectively). this antigenic propensity serves as a gold standard in predicting antigenic determinant. antigenic propensity combines hydrophilicity, flexibility and surface accessibility scores all at the same time. ultimately, it would be very challenging to conduct a confirmation study, such as immunization of experimental animals to reveal whether these seven peptides are indeed capable of strongly evoking both the t-cell mediated immune and the humoral-immune responses. in conclusion, there are seven peptides representing t-cell epitope of the antigen 85 complex that could potentially be capable of generating an antibody response. the seven peptides, p17838, p21093, p36131, p21275, p21780, p21796 and p10839, are strong candidates proposed for further study in order to develop a subunit-based multiepitopic anti-tb vaccine. acknowledgement the author thanks to andreas schreiber (institute for biomedical research-frankfurt/main, germany) for providing a bioinformatics program, 3d-epitope-explorer. the author also thanks okta rena for editorial work. references andersen ph, nielsen m, lund o. 2006. prediction of residues in discontinuous b cell epitopes using protein 3d structures. prot sci 15:2558-67. karplus pa, schulz ge. 1985. prediction of chain flexibility in proteinsa tool for the selection of peptide antigens. naturwissenschaften 72:212-3. kim sj. 2004. second-line drug susceptibility testing: where are we and where are we going? int j tuberc lung dis 8:s6-7. kolaskar as, tongaonkar pc. 1990. a semi-empirical method for prediction of antigenic determinants on protein antigens. febs lett 276:172-4. laver wg, air gm, webster rg, smith-gill sj. 1990. epitopes on protein antigens: misconcptions and realities. cell 61:553. parker jm, guo d, hodges rs. 1986. new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites. biochem 25:5425-32. patarroyo ma, curtidor h, plaza df, ocampo m, reyes c, saboya o, barrera g, patarroyo me. 2008. peptides derived from mycobacterium tuberculosis rv1490 surface protein implicated in inhibition of epithelial cell entry: potential vaccine candidates? vaccine 26:4387-95. sarhan maa. 2007. progress in tuberculosis vaccine development. res j med med sci 2:35-41. schreiber a, humbert m, benz a, dietrich u. 2005. 3d-epitopeexplorer (3dex): localization of conformational epitopes within three dimensional structures of proteins. j comput chem 26:879-87. spouge jl, guy hr, cornette jl, margalit h, cease k, berzofsky ja, delisi c. 1987. strong conformational propensities enhance t-cell antigenicity. j immunol 138:204-12. wiker hg, harboe m. 1992. the antigen 85 complex: a major secretion product of mycobacterium tuberculosis. microbiol rev 56:6486 1 . table 5 antigenic properties of selected peptides* peptides p 1 7 8 3 8 p 2 1 0 9 3 p 3 6 1 3 1 p 2 1 2 7 5 p 2 1 7 8 0 p 2 1 7 9 6 p 1 0 8 3 9 antigen 85a 85a 85a 85b 85b 85b 85c hydrophilicity 0.746 (-1.200 to +3.271) 2.147 ( 0.286 to 4.443) 0.437 (-1.943 to 2.914) -0.438 (-0.914 to -0.200) -1.557 (-2.329 to -0.914) 3.957 ( 2.757 to 6.043) 3.571 ( 0.400 to 6.386) flexibility 0.994 (0.941 to 1.062 1.007 (0.979 to 1.030) 1.002 (0.982 to 1.052) 1.010 (0.999 to 1.021) 0.966 (0.949 to 0.983) 1.049 (1.001 to 1.103) 1.064 (0.977 to 1.148) antigenic propensity 1.088 (0.961 to 1.158) 0.960 (0.924 to 1.004) 1.017 (0.954 to 1.071) 1.028 (1.007 to 1.068) 1.075 (1.061 to 1.095) 0.946 (0.890 to 1.012) 0.969 (0.926 to 1.025) *values are expressed as average score (minimum to maximum). 05 pambudi.cdr vol.12, no.3, september 2018, p 99-105 doi: 10.5454/mi.12.3.5 the potency of aluminum hydroxide nanoparticles for dengue subunit vaccine adjuvant 1* 1 1 1 sabar pambudi , etik mardliyati , silmi rahmani , damai ria setyawati , 1 1 1 2 tika widayanti , angelina gill , asri sulfianti , and whinie lestari 1 center for pharmaceutical and medical technology, building 611-512, laptiab, bppt, serpong, indonesia, 15312; 2 litbangkes, ministry of health, jalan percetakan negara 23, jakarta, indonesia, 10560. the potency of aluminum hydroxide as an adjuvant in vaccine development is considered to depend on its particle size. in previous studies, we have successfully prepared two size particle, micro, and nano, aluminum hydroxide gel (alum) adjuvants. the potency of those particles as a candidate of adjuvant is needed to be characterized. in this study, we formulated our adjuvants with purified denv3 pre membrane envelope (prm-e) recombinant protein and evaluated the induction of nitric oxide level in mouse macrophage raw 264.7 cells. we prepared the alum adjuvant by precipitation-homogenization methods with an agitation rate at 11,000xg. secreted prm-e recombinant protein was collected from pichia pastoris x-33 fermentation which produced using bioreactor. recombinant protein purification was carried out by anion exchange chromatography followed with size exclusion chromatography. the purified prm-e recombinant protein was observed as a single band around 70 -1 kda with a concentration of 105 mg ml . complex nanoparticles alum with prm-e protein significantly (p<0.05) induced the nitric oxide level. further analysis should be conducted in order to discover the detail molecular mechanism of nanoparticle alum adjuvant, recombinant protein, and cellular immune response. key words: adjuvant, dengue, nitric oxide, prm-e potensi aluminum hidroksida (alum) sebagai adjuvan vaksin sangat bergantung dari ukuran partikelnya. pada penelitian sebelumnya, kami telah berhasil mengembangkan dua jenis ukuran partikel adjuvan dari alum, yaitu mikron dan nano. pada penelitian ini, telah dilakukan karakterisasi dari kedua jenis partikel alum yang digunakan dalam pengembangan kandidat adjuvan vaksin demam berdarah. partikel adjuvan diformulasi dengan protein rekombinan prm-e denv3 dan dievaluasi potensinya dalam menginduksi senyawa nitric oxide (no) pada galur sel mencit raw 264.7. partikel alum disiapkan dengan metode presipitasi-homogenisasi melalui proses agitasi pada kecepatan 11.000xg. protein rekombinan prm-e diproduksi dari sel ragi pichia pastoris x-33 melalui proses fermentasi menggunakan biorektor. purifikasi protein rekombinan dilakukan menggunakan metode ion exchange yang dilanjutkan dengan size exclusion chromatography. protein rekombinan yang berhasil dipurifikasi -1 tervisualisasi sebagai pita protein tunggal yang berukuran sekitar 70 kda dengan konsentrasi sekitar 105 mg ml . hasil dari penelitian pendahuluan ini menunjukkan bahwa kompleks alum adjuvan nanopartikel dengan protein rekombinan prm-e secara signifikan (p<0,05) menginduksi kadar no dibandingkan dengan kompleks alum lainnya. penelitian lanjutan perlu dilakukan untuk mendapat informasi yang lebih detil mengenai mekanisme molekular antara alum adjuvan nanopartikel, protein rekombinan dan respon imum seluler. kata kunci: adjuvan, dengue, nitric oxide, prm-e microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone/fax: 62-21-7560707; email: sabar.pambudi@bppt.go.id are often used in clinical trials and have a reputation of safety and the facilitation of long-lasting antibody responses (sun et al. 2014). alum has been widely used as human vaccine adjuvants for almost a century. it is known that their immunostimulation effect is associated with the induction of th2 responses (ulanova et al. 2001). it has also been demonstrated that alum enhances antigen uptake by the antigenpresenting cells in vitro (mannhalter et al. 1985). nitric oxide (no) has been known as one of the most important components in the immune system. it is involved in the pathogenesis and control of many infectious diseases, cancers, autoimmune and chronic degenerative diseases (bogdan 2001). initially, no vaccine adjuvant promotes the production of longlasting, efficient and specific immune responses. in the same time, the adjuvant improves the protective effect of vaccines due to a higher antibody yield and the persistence of antibodies, as well as functional t cells at high levels. freund's adjuvants are the most common adjuvant used in experimental animals. it can enhance strong antigen-specific immune responses but at the same time, it causes strong inflammation and necrosis at the injection site, which prevents its use in vaccine development. aluminum hydroxide adjuvants (alum) was described as a physiological mediator of endothelial cell relaxation plays an important role in hypotension. several cells of the innate immune system such as macrophages, neutrophils, and natural killer (nk) cells use pattern recognition receptors to recognize molecular patterns associated with pathogens. activation of macrophages inhibits pathogen replication by releasing a variety of effector molecules, including no. nitric oxide is important as a toxic defense molecule against infectious organisms. it also regulates the functional activity, growth, and death of many immune and inflammatory cell types including macrophages, t lymphocytes, antigenpresenting cells, mast cells, neutrophils and nk cells (tripathi 2007). a previous report showed that chicken spleen macrophage produced no after treated by liposomes adjuvant. the production of no by activated macrophages is an important index of immune stimulatory activity by an adjuvant (lin et al. 2011). dengue virus (denv) is a major cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions of infections each year around the globe. infections range from asymptomatic to a self-limited febrile illness, dengue fever (df), to the life-threatening dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) (guo et al. 2017). neither antiviral nor vaccine is available in the market to control dengue infection. denv vaccine development is challenging due to the existence of four serotypes of the virus (denv1–4), which a vaccine must protect against all serotypes (chokephaibulkit et al. 2013). denv is a member of the flaviviridae family and is grouped within the flavivirus genus. the viral genome consists of a positive-sense rna of ~11kb which encodes 3 structural proteins (capsid, premembrane and envelope) that form the components of the virion, and 7 non-structural proteins (ns1, ns2a/b, ns3, ns4a/b, ns5) involved in viral rna replication (perera et al. 2008). several groups have been exploited the use of pre-membrane and envelope protein as the target candidate in the development of a subunit dengue vaccine (kaufman et al. 1987; robert putnak et al. 2005). these proteins are immunogenic and confer some degree of protection against live virus challenge in animal models. the previous study showed that the formulation between these proteins with alum adjuvant could elicit virus-neutralizing antibodies in mice and rhesus macaques and confer at least partial protection against virus challenge (putnak, barvir et al. 1996; putnak, cassidy et al. 1996). in the present study, we aimed to evaluate the induction of nitric oxide level in mouse macrophage raw 264.7 cells treated with several formulations of complex alum adjuvant with denv3 recombinant prm-e protein. the results of this preliminary study will improve our understanding of the best adjuvant formulation for dengue vaccine development. materials and methods aluminum hydroxide preparation. aluminum hydroxide was prepared by the precipitationhomogenization method as described in the previous report (mardliyati 2017). an equal volume of 3.60 mg -1 ml alcl .6h o was mixed with 0.04 m sodium 3 2 hydroxide dropwise and stirred at 20xg. a small volume of 0.01 m naoh was added to adjust the ph to 6.8. after 24 hours of decantation, suspension of aluminum hydroxide microparticles was separated and washed twice with phosphate buffer ph 6.8 in order to remove sodium chloride in the suspension. aluminum hydroxide was precipitated until aqueous suspension of aluminum hydroxide gel adjuvant (alum) was formed. aluminum hydroxide nanoparticles were synthesized by downsizing (to break down particle size) it with high shear homogenization at 11,000xg for 1 hour. sodium polyphosphate 0.40% was added before downsizing to make aluminum hydroxide nanoparticles more stable. the particles size distribution was determined using a particle size analyzer (horiba la95a). mice macrophage raw 264.7 cells. the raw 264.7 mouse macrophage cell line (a kind gift from dr. peik, kribb korea) was maintained in rpmi complete media supplemented with 10% fetal bovine serum (fbs) and 1% penicillin-streptomycin in the presence of 5% co . cells were passaged every three 2 days in order to maintain the condition of the cells. production of denv3 prm-e recombinant protein. recombinant denv3 prm-e protein was constructed from denv3 clinical isolates which collected during cross-sectional study which was conducted on 2009 in jakarta (lestari et al. 2017). the prm-e gene was cloned and expressed in pichia pastoris x-33 (unpublish data). high-cell-density cultivations of pichia pastoris secreting denv3 prme recombinant protein were carried out in the bioreactor (bioflow120, eppendorf) with slight modification (gurramkonda et al. 2010). precultures for high-cell density bioreactor cultivations were o prepared in 100 ml for overnight at 30 c with agitation 100 pambudi et al. microbiol indones volume 12, 2018 microbiol indones 101 around 25xg. the overnight starting culture was transferred to the bioreactor containing 2 liters ursula rinas medium. the temperature was maintained at 30°c and ph at ph 5.5 with 12.5% (v/v) nh oh. 4 -1 aeration rate was maintained at 4 l min throughout the process. the stirrer speed was controlled between 30 to 100xg aiming at dissolved oxygen (do) concentration of 30% air saturation. in this study, we induced the secretion of prm-e protein by applying 2.5% methanol induction for 48 hours. the supernatant was collected by centrifugation followed by buffer exchanged with 20 mm tris-cl ph 8 prior purification with anion chromatography by using deae resin from ge (uk). buffer 20 mm tris-cl ph 8 with 1m nacl was used as elution buffer. subsequently, the size exclusion chromatography was performed using resin sephacryl hr200 (ge) with pbs buffer in order to purified single band of prm-e recombinant protein. all purified protein was confirmed by using sds page analysis and the concentration of the protein was determined by bca protein assay kit (thermo scientific). cell viability assay. to evaluate the cytotoxicity of the alum adjuvant to the cells, the 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide (mtt) assay was performed as described previously (mosmann 1983). the mouse macrophage 5 raw 264.7 cells were seeded on 96-well plate at 1x10 -1 cells ml . after 16-20 hours, cells were incubated in triplicate for 24 hours with a various concentration of alum ranging from 0.01% to 1.0% in 2% fbs-rpmi. after incubation, culture media with alum was removed from the cells and directly incubated with 50 l -1 of mtt solution (pbs containing 0.5 mg ml mtt) for o 4 hours at 37 c. the mtt solution was then replaced with 100 μl of 10% sds solution followed by incubation in the dark room for 22 hours. after gentle shaking for 10 min at room temperature, the absorbance at 570 nm was measured using a universal microplate reader elx800 (bio-tek instruments, inc.). the cell viability percentage for each adjuvant concentration was calculated by using graphpad prism 5 software. nitric oxide (no) assay. the production of no was determined by measuring the quantity of nitrite in the supernatant by the griess reaction (giustarini et al. 2008). the raw 264.7 cells were seeded on 96-well 5 -1 plate at 1x10 cells ml . after 2 hours, cells were incubated in triplicate for 24 hours with various concentrations of alum ranging from 0.001% to 1.0% in 2% fbs-rpmi with and without 10 μg purified prm-e recombinant protein. supernatant from the experimental cell culture was removed to another well plate in triplicate and dispense of griess solution with ratio 1:1 to all experimental samples followed by incubation in the darkroom for 10-20 minutes. the absorbance at 540 nm was measured using a universal microplate reader elx800 (bio-tek instruments, inc.) to determine the amount of no in the samples. statistical analysis. the results were analyzed using the one way anova with tukey's multiple comparison test. differences were considered significant (*) at p value < 0.05. results aluminum hydroxide particle size. the particle size of the alum adjuvant was determined by using psa as shown in table 1. the size of commercial alhydrogel in this study was around 0.913 ± 0.8316 μm. high shear homogenization at 11,000xg for 1 hour could reduce the size of alum adjuvant particle from 5.671 ± 0.3261 μm to 0.243 ± 0.0008 μm. production of recombinant denv3 prm-e protein. we performed the production of pichia pastoris expressing prm-e recombinant protein using bioreactor system in order to maximize the biomass cells and secreted protein. several major bands (200 kda, 180 kda, 100 kda, 70 kda, 10 kda) appeared from the supernatant before purification step. the collected supernatant was subjected for anion exchange and size exclusion chromatography purification steps. a single band of prm-e protein around 70 kda -1 appeared with a concentration of 105 μg ml after purification step (fig 1). cell viability of the aluminum hydroxide. the table 1 physicochemical properties of aluminum hydroxide no sample average particle size (μm) 1 ® alhydrogel 0.913 ± 0.8316 2 microparticle adjuvant 5.671 ± 0.3261 3 nanoparticle adjuvant 0.243 ± 0.0008 cell viability was evaluated by mtt assay. the viability of raw 264.7 cells was decreased in a dosedependent manner for all particle size of alum adjuvant, ® including for the commercial alhydrogel adjuvant. almost 50% of mouse macrophage raw 264.7 cells were killed after treatment with 1% of alum adjuvant. however, at a concentration of 0.5%, more than 60% of cells were survived and we used this alum concentration for further analysis (fig 2). induction of nitric oxide. no nitric oxide was induced in mouse macrophage raw 264.7 cells after treated with all alum adjuvant alone (fig 3). interestingly, recombinant prm-e protein alone could induce nitric oxide compared to negative control (media or alum only). moreover, nitric oxide was released significantly (p<0.05) from mouse macrophage raw 264.7 cells after treated with nanoparticle alum adjuvant which was formulated with prm-e recombinant protein (fig 3). discussion there is limited study reported the effect of alum particles size on the subsequent immunogenicity of the adjuvanted vaccine. in these studies, we evaluated the cytotoxicity and nitric oxide level induced by complex alum adjuvant with denv3 recombinant prm-e protein in mouse macrophages raw 264.7 cells. two fig 1 recombinant prm-e denv3 purification. (a) total secreted protein in supernatant before purification. in line 1, several bands appeared mainly at 200 kda, 180 kda, 100 kda, 70 kda and 40 kda. (b) purified prme recombinant protein after anion exchange followed by size exclusion chromatography. in line 1, prm-e recombinant protein appeared clearly with the correct size around 70 kda. 102 pambudi et al. microbiol indones 5 fig 2 raw 264.7 cell viability. mouse macrophage raw 264.7 cells were seeded on 96-well plate at 1x10 cells -1 ml . after 16-20 hours, cells were incubated in triplicate for 24 hours with various concentrations of alum ranging from 0.01% to 1.0% in 2% fbs-rpmi. at concentration 0.5%, more than 60% of cells were survived after the treatment with alum adjuvant. volume 12, 2018 microbiol indones 103 different sizes of alum particle, nano and micro, were successfully prepared in the previous study (mardliyati 2017). the toxicity of our alum particles was evaluated in this study. viability of mouse macrophages raw 264.7 cells showed slightly better when treated with our alum particles compared to the commercial ® alhydrogel adjuvant (fig 2). the previous study reported the mechanisms of action of alum of minor toxicity in some subjects by inducing cell death and inflammasome and developed persistent lumps and granulomas at the injection site (petrovsky 2015). nitric oxide has been known as one of a key player in immune response. to evaluate the possibility of our alum adjuvant to induce nitric oxide, we treated the mouse macrophage raw 264.7 cells with of micron and nano alum adjuvant together with commercial ® alhydrogel adjuvant as a control. ® similar to commercial alhydrogel adjuvant, our results showed neither micron nor nanoparticle alum alone could not induce nitric oxide (fig 3). contrary, a previous study showed that alum adjuvant alone could induce several cytokines and chemokines from macrophage cells of injected mice (mckee et al. 2009). the different immune response mechanism of alum adjuvant in vitro and in vivo system could be used to explain those phenomena. i m m u n i z a t i o n o f d e n v p r m e p r o t e i n demonstrating the immunogenicity and protective efficacy against denv infection in small animals. however, the prm-e protein that used on most denv vaccine study was formulated with adjuvant (mani et al. 2013; urakami et al. 2017). interestingly, the result from these studies showed that prm-e alone is enough to induce no level compared to control alum without prm-e protein (fig 3). development of denv vaccine by stimulating immune responses against the denv pre-membrane and envelope (prm-e) protein have been reported elsewhere (guirakhoo et al. 2000; liu et al. 2016). the envelope protein of denv is responsible for a wide range of biological activities, stimulates host immunity responses by inducing protective and neutralizing antibodies, including binding to host cell receptors, fusion and entry into host cells. therefore, the dengue prm-e protein is an important antigen for vaccine development (fahimi et al. 2018). the nanoparticle alum prm-e complexes significantly induced immune response compared to other formulation (fig 3). a previous study showed that 3 μm aluminum phosphate particles had better uptake than 17 μm aluminum hydroxide particles. alum aggregates below 10 μm are needed for efficient uptake of vaccine by apcs (morefield et al. 2005). the size of nanoparticle alum in this study is less than 1 μm (243 nm) and theoretically makes the uptake of prm-e protein by the cells more efficient compared to other formulations. alum greatly enhances priming of endogenous fig 3 induction of nitric oxide. (a) mouse macrophage raw 264.7 cells were treated with alum adjuvant only or with alum prm-e complexes. alum alone could not induce immune response however prm-e without adjuvant could induce no. nanoparticle alum complexes induced nitric oxide significantly (p<0.05) compared to other groups. cd4 and cd8 t cells independently of mast cells, macrophages and of eosinophils. however, activation of type 2 innate response orchestrated by macrophages and mast cells in vivo are not required for alum's adjuvant effects on endogenous t and b cell responses (mckee et al. 2009). in the field of drug delivery, nanoparticles have been shown to have a number of advantages over larger micron-sized particles. these advantages include increased intracellular uptake, improved bioavailability for poorly water-soluble drugs and improved pharmacokinetics, particularly in regards to tumor targeting where nanoparticles can prolong circulation of the drug, avoiding the reticuloendothelial system leading to a longer circulation time and improved efficiency of delivery to the tumor (shah et al. 2014). to the best of our knowledge, there is no study has been reported regarding the evaluation of inducible nitric oxide by nanoparticle alum adjuvant formulated with denv recombinant prm-e protein using in vitro system. further analysis should be carried out in order to discover the molecular mechanism interaction of specific particle size of alum adjuvant, recombinant protein, and cellular immune response. this kind of studies would enhance our knowledge about the detail mechanism and function of alum adjuvant in vaccine development. acknowledgment this research was supported by insinas research grant from the ministry of science, technology and higher education of the republic of indonesia year 2018. references bogdan c. 2001. nitric oxide and the immune response. nature immunol. 2: 907. doi:10.1038/ni1001-907. chokephaibulkit k, perng gc. 2013. challenges for the formulation of a universal 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doi:10.1016/j.mib.2008.06.004. petrovsky n. 2015. comparative safety of vaccine adjuvants: a summary of current evidence and future needs. drug saf. 38(11):1059-1074. doi:10.1007/s40264-015-03504. putnak r, barvir da, burrous jm, dubois dr, d'andrea vm, hoke ch, sadoff jc, eckels kh. 1996. development of a purified, inactivated, dengue-2 virus vaccine prototype in vero cells: immunogenicity and protection in mice and rhesus monkeys. j infect dis. 174(6):1176-1184. putnak r, cassidy k, conforti n, lee r, sollazzo d, truong t, ing e, dubois d, sparkuhl j, gastle w, hoke c. 1996. immunogenic and protective response in mice immunized with a purified, inactivated, dengue-2 virus vaccine prototype made in fetal rhesus lung cells. am j trop med hyg. 55(5):504-510. robert putnak j, coller ba, voss g, vaughn dw, clements d, peters i, bignami g, houng hs, chen rc, barvir da, seriwatana j, cayphas s, garcon n, gheysen d, kanesa-thasan n, mcdonell m, humphreys t, eckels kh, prieels jp, innis bl. 2005. an evaluation of dengue type-2 inactivated, recombinant subunit, and liveattenuated vaccine candidates in the rhesus macaque m o d e l . v a c c i n e . 2 3 ( 3 5 ) : 4 4 4 2 4 4 5 2 . doi:10.1016/j.vaccine.2005.03.042. shah rr, o'hagan dt, amiji mm, brito la. 2014. the impact of size on particulate vaccine adjuvants. n a n o m e d i c i n e ( l o n d ) . 9 ( 1 7 ) : 2 6 7 1 2 6 8 1 . doi:10.2217/nnm.14.193. sun x, mei m, zhang x, han f, jia b, wei x, chang z, lu h, yin j, chen q, jiang n. 2014. the extracellular matrix protein mindin as a novel adjuvant elicits stronger immune responses for rbag1, rsrs4 and rsrs9 antigens of toxoplasma gondii in balb/c mice. bmc infect dis. 14:429. doi:10.1186/1471-2334-14-429. tripathi p. 2007. nitric oxide and immune response. indian j biochem biophys. 44(5):310-319. ulanova m, tarkowski a, hahn-zoric m, hanson la. 2001. the common vaccine adjuvant aluminum hydroxide up-regulates accessory properties of human monocytes via an interleukin-4-dependent mechanism. infect immun. 69(2):1151-1159. doi:10.1128/iai.69.2.11511159.2001. urakami a, ngwe tun mm, moi ml, sakurai a, ishikawa m, kuno s, ueno r, morita k, akahata w. 2017. an envelope-modified tetravalent dengue virus-likeparticle vaccine has implications for flavivirus vaccine design. j virol. 91(23). doi:10.1128/jvi.01181-17. volume 12, 2018 microbiol indones 105 page 1 page 2 page 3 page 4 page 5 page 6 page 7 it jamilah.pmd volume 3, number 2, august 2009 p 67-71 issn 1978-3477 *corresponding author:, phone/fax: +62-251-8622833, e-mail: rusmana13@yahoo.com activity of proteolytic and amylolytic enzymes from bacillus spp. isolated from shrimp ponds it jamilah1,2, anja meryandini1, iman rusmana1*, antonius suwanto1, and nisa rachmania mubarik1 1department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 2department of biology, faculty of mathematics and natural sciences, universitas sumatera utara, jalan prof a sofyan no 3, medan 20155, indonesia accumulation of feed excess in commercial shrimp ponds due to overfeeding could decrease water quality. protein and starch are the primary components of shrimp feed. this study was conducted to characterize extracellular proteases and amylases of bacillus spp. isolated from shrimp ponds. 72 proteolytic and amylolytic bacillus spp. isolates were screened from shrimp ponds in karawang, west java. ten isolates were selected for further characterization for their growth and ability to reduce total suspended solid generated from commercial shrimp feed. bacillus sp. da 5.2.3 and l5 showed excellent activity in reducing total suspended solid, by 37 and 30% respectively. protease and α-amylase activities of bacillus sp. da 5.2.3 isolate were consistently higher than that of l5. maximum total and specific protease activity of da 5.2.3 isolate was 2.0 u ml-1 and 40.9 u mg-1 respectively, while the activities of the l5 isolate were 2.1 u ml-1 and 23.0 u mg-1 respectively. based on its 16s rrna gene sequences, bacillus sp. da 5.2.3 showed 99% similarity to bacillus cereus xhj-2-6. bacillus sp. da 5.2.3 could potentially be applied to maintain water quality by reducing total suspended solid in water columns of shrimp ponds. key words: bacillus sp. da 5.2.3, protease, α-amylase, total suspended solid overfeeding in aquaculture system can influence water quality that hampers animal growth. generally, 10% of feed remainds as waste in the water. a high load of feed in pond water can increase biological oxygen demand (bod), bacterial population, and decrease dissolved oxygen (do) content. it can also increase total suspended solid (tss) in the water. reduction of water quality has a bad influence on survival and growth of shrimps. the used of bacterial probiotics in shrimp aquacultures has been studied and reported to have great impact on shrimp growth and survival. some of these can maintain water quality by reducing the ammonia concentration in shrimp ponds, others can inhibit the growth of shrimp pathogens (rengpipat et al. 1998; vaseeharan and ramasamy 2003; decamp and moriarty 2007), stimulate the immune system of the shrimp (gullian et al. 2004) and increase digestive enzyme activity of the shrimps intestine (ziaei-nejad et al. 2006). bacillus spp. have derived great attention as a probiotic application in aquaculture in the last decade (rengpipat et al. 1998; vaseeharan and ramasamy 2003; decamp and moriarty 2007). many researchers reported their roles as biological control agents in shrimp ponds (verschuere et al. 2000; decamp and moriarty 2007). recently, ochoa-solano and olmos-soto (2006) studied the contribution of bacillus as part of the functional feed to enhance shrimp feed quality. several studies of potential protease from bacillus cereus isolated from fish gut for converting fish-wastes have been reported. however, information about the role of bacillus spp. to reduce shrimp feed wastes, was very limited. the group of bacillus bacteria is well known as a producer of a large variety of extracellular protease and amylase. therefore, this study attempted to identify potential proteolytic and amylolytic enzymes of bacillus spp. isolated from shrimp ponds for their potential application to maintain water quality in shrimp aquaculture. materials and methods sample collection and preparation. nineteen samples of four different sample sources being soil, sediment and water and shrimp intestine were collected from shrimp ponds in karawang, west java, indonesia. from samples of shrimp intestine were prepared by grinding and diluting in 0.85% (w/v) nacl. isolation and screening of bacillus. serial dilution of each sample in 0.85% nacl (w/v) was follow by heating at 80 °c for 15 min. then, 100 ml aliquots solution was spread on half strength of solid seawater complete medium (swc) (atlas 1997) added with 1% (w/v) skimmed milk. cultures were incubated at room temperature 24-48 h. proteolytic activity was shown by formation of clearing zones around colonies. amylolytic activities of bacillus spp. were screened by a similar method except swc medium was mixed with 1% (w/v) commercial cassava starch instead of glycerol was used as the carbon source. clearing zones around the colonies were detected by iodine staining solution (15 g l-1 potassium iodide and 15 g l-1 iodine in distilled water). the proteolytic index (pi) or the amylolytic index (ai) value is the ratio of hydrolysis zone (clear zone) diameter (cm) formed by a bacterium colony and its colony diameter (cm). proteolytic and amylolytic activity positive colonies were isolated on solid swc medium to give a pure culture. confirmation of amylolytic and proteolytic activity of the pure culture isolates were conducted using similar methods as to mention above. growth conditions of selected isolates. selected high pi and ai value isolates were inoculated into 50 ml swc medium in 250 ml flasks and then incubated on shaker (120 rpm) at room temperature (28 °c) to get 108 cfu ml-1 (od 620nm = 0.4). microbiol indones68 jamilah et al. table 1 isolated proteolytic and amylolytic bacillus from shrimp ponds in karawang, west java sources soil sediment water intestine number of samples 6 4 5 4 p and a 1 8 26 11 10 p a 1 0 0 3 2 0 0 3 total of isolate 2 1 26 11 14 % of isolate 2 9 36 15 19 ranges of hydrolysis zone pi±se 1.2±0.1 to 1.5±0.0 to 1.5±0.0 to 2.8±0.1 to ai±se 1.5±0.1 to 5.6±1.4 1.2±0.4 to 8.3±1.0 2.0±0.5 to 4.8±0.2 1.4±0.3 to 7.8±0.3 average of hydrolysis zone pi±se ai±se 3.7±0.3 3.4±0.4 2.6±0.2 5.8±0.4 3.0±0.3 3.0±0.3 3.6±0.2 5.3±0.6 p, proteolytic; a, amylolytic; pi, proteolytic index; ai, amylolytic index; se, standard error. then, 10 ml aliquots of cultures were inoculated into 100 ml of sf medium with 2.5% (w/v) salinity and ph 7.5. cell densities were determined at 24 and 48 h. total suspended solid (tss). fifty ml of 1.2% (w/v) shrimp feed (sf) medium (ph 7.5) was inoculated with 10 ml of bacteria with a concentration of 108 cfu ml-1. incubation was run at room temperature on a shaker (120 rpm) for 92 h. tss values were determined using filtration method. enzyme production. bacteria were grown in 1.2% (w/v) sf medium adjusted to ph of 7.5 and incubated in a shaker at 120 rpm at room temperature for 96 h. samples were taken at a 24 h time interval then centrifuged at 4500 x g for 15 for min (jouan c31) at 4 °c. the clear supernatant was used as the crude enzyme preparation. protease activity assay. the substrate used for protease activity assay was 1% (w/v) casein in 0.01 m tris-hcl buffer at ph 7.5. tyrosine 0.37 mmol l-1 was used for a standard. each mixture enzyme reaction was incubated at room temperature for 10 min and the reaction was stopped by adding of 10% (w/v) trichloro acetic acid (tca). all tests were done in replicates. protein concentration was measured using a colorimetric method (bradford 1976). bovine serum albumin was used as a standard. one unit of the enzyme activity (1u) is defined as the amount of enzyme needed to produce 1 µmol tyrosine per min, while specific activity of an enzyme is a unit of enzyme activity divided by its protein concentration. amylase activity assay. assay of α-amylase activity was measure based on the colorimetric method (benfeld 1955). soluble starch at 1% (w/v) was dissolved in 0.05 m tris-hcl buffer at ph 7.5 and used as a substrate. one unit of enzyme activity (1u) is defined as an activity of the enzyme to produce 1 µmol maltose per min. bacillus identification. identification of selected isolate was conducted based on morphology characterization. it was then confirmed with 16 s rrna gene sequencing. dna extraction was performed based on the ctab (cetyl-trimethylammonium bromide) method. the 16s rrna gene was amplified with polymerize chain reaction (pcr) machine (gene amp pcr system 2400, perkin elmer, biosystem, usa) using a specific primer for bacteria (63f (5’caggcctaacacatgcaagtc) and 1387r (5’gggcggwgtgtacaaggc) (marchesi et al. 1998) (research biolab, singapore). dna was sequenced (applied biosystem, usa) and blast analysis was conducted (ncbi: at http://www.ncbi.nlm.nih.gov). results screening of bacteria. there were 72 bacillus spp. isolated from 19 samples taken from shrimp ponds in karawang, west java. all isolates assayed positive for proteolytic and/or amylolytic activities, were gram positive and were rod shaped. colony morphology of the isolates found generally were white-cream or cream in color, concentric or circle in shapes, entire and wave or ununiform in their margin morphology. isolation results showed that sediment was the most prolific source of bacillus sp. compare to other sources, giving 36% followed by soil that gave up to 29% of the total isolates found. the lowest number of bacillus spp. found was in the water samples (15%), however it was higher than that in shrimp intestine samples. most of the isolates found (90.3%) had both proteolytic and amylolytic activities, which could be seen from clearing zone produced around colonies in half strength swc-milk and swc-starch medium (table 1). the proteolytic index (pi) and the amylolytic index (ai) of the isolates were varied. the pi value of the isolates isolated from soils were in the range of 1.2 and 6.9 while ai values were in the range of 1.5-5.6. the average of the pi and ai was 3.7 and 3.0 respectively. the l3, l5 and kat 5.1 isolates had a high value of either proteolytic or amylolytic indexes. these isolates were selected for further study. the pi and ai values of the isolates isolated from pond sediment were in the range of 1.5-7.2 and 1.2-8.3 respectively. the average of pi and ai values were 3.4 and 3.0 respectively. kpt 7.1.1 and kpt 7.1.2b isolates had a high values of either pi or ai. the average of pi and ai values of the isolates isolated from water were 2.6 and 3.6 (table 1). the isolates isolated from shrimp intestines mostly had higher values of pi and ai than isolates of other sources. the average values of pi and ai of the isolates were 5.8 (in the range of 2.7-18.8) and 5.3 (in the range of 1.4-7.8). the highest values of pi was performed by da 2.2.1 isolate, but its ai value had a lower value than other isolates. the da 5.2.3 isolate had high values of both pi and ai. this isolate was selected for further study. the dp 3.2.1 isolate had only proteolytic activity in contrast, others, such as da 2.2.3, ua 1.2.1, ua 1.2.2 isolates only assayed positive for amylolytic activity. ten isolates were selected for further characterization based on their pi and ai values (table 2). all isolates selected were gram positive, rod-shaped, catalase positive and endospore-forming bacteria. when the isolates were grown in a solid sf medium, all were able to degrade starch of shrimp feed, denoted by formation of a clear zone. however, the clearing zones did not show proteolytic activity (unpublished data). in general, the ai value of the isolates growing in swc starch medium were higher than of those grown in sf medium. growth of selected bacillus spp. all selected isolates grew well in sf medium (fig 1a and 1b). however, they had different growth rates. the bacteria grew fast for the first 24 h. the increase of the cell numbers was as high as 5 log 10 to 8 log cfu ml-1 and then the growth rate was reduced by further 6.9±2.1 7.2±0.1 3.6±0.4 18.8±0.3 microbiol indones 69volume 3, 2009 table 2 ten of 72 bacillus spp. isolated from shrimp ponds soil sediment water intestine hydrolysis index pi±se ai±se 3.7±0.4 4.7±0.6 3.3±0.2 7.2±0.1 6.6±0.9 2.3±0.1 18.8±0.3 4.2±0.6 4.0±0.3 4.7±0.3 3.5±0.5 3.5±0.3 5.2±0.6 2.5±0.0 4.0±1.4 8.3±1.0 3.6±0.4 6.0±1.6 4.8±1.4 7.8±0.3 pi, proteolytic index; ai, amylolytic index; se, standard error; ns, not selected. isolate codes l 3 l 5 kat 5.1 kpt 7.1.1 kpt 7.2.1 bkat 7.1.1 ns da 2.2.1 dp 5.1.1 dp 5.1.2 da 5.2.3 sources c e ll d e n si ty ( lo g 1 0 c fu m l -1 ) 16.0 12.0 10.0 8 . 0 6 . 0 4 . 0 2 . 0 0 . 0 0 2 4 4 8 time (h) 14.0 c e ll d e n si ty ( lo g 1 0 c fu m l -1 ) 16.0 12.0 10.0 8 . 0 6 . 0 4 . 0 2 . 0 0 . 0 14.0 0 2 4 4 8 time (h) fig 1 the growth of selected isolates of bacillus spp. as probiotic candidates over incubation time in commercial shrimp feed medium a: , isolate kat 7.1.2; , isolate dp 5.1.1; , isolate dp 5.1.2; , isolate da 2.2.1; , isolate da 5.2.3. b : , isolate l3; , isolate l5; , isolate kat 5.1; , isolate kpt 7.1.2b; , isolate kpt 7.1.1. p ro te a s e t o ta l a c t (u m l -1 ) p ro te a se s p e si fi c a c t (u m l -1 ) 2 . 5 2 . 0 1 . 5 1 . 0 0 . 5 0 . 0 2 4 4 8 7 2 9 6 60.0 50.0 40.0 30.0 20.0 10.0 0 . 0 fig 2 protease activity of bacillus sp. l5 and da 5.2.3 over time production. , da 5.2.3 total act; , da 5.2.3 specific act; , l5 total act; , l5 specific act. time (h) rate was performed by l5, dp 5.1.1 and da 5.2.3 isolates. the number of cells increased up to 7 log 10 cfu ml-1. total suspended solid (tss). reduction of tss was measured using 25 ml 1.2% sf medium in 250 ml flask, incubated on shaker at room temperature (28 °c) for 92 h. the highest reduction of tss was perfomed by l5 and da 5.2.3 isolates, which decreased tss up to 36 and 30% respectively (table 3). protease activity. bacillus sp. da 5.2.3 and l5 were characterized of their protease activities in commercial sf as medium production. the da 5.2.3 isolate showed higher proteolytic activity than the l5 isolate. the proteolytic activity of bacillus sp. da 5.2.3 was 2.0 u ml-1 higher than that of bacillus sp. l5 (1.4 u ml-1). however, specific activity graphs of both isolates had a similar pattern. the highest specific activity of da 5.2.3 and l5 isolates was 40.9 u mg-1 and 23.0 u mg-1 respectively (fig 2). amylase activity. total α-amylase activity and its specific activity for da 5.2.3 were also higher than that of the l5 isolate. the highest total α-amylase activity of the da 5.2.3 and the l5 isolate was reached in 24 h of incubation, which were 2.1 u ml-1 and 0.5 u ml-1 respectively (fig 3). the activity of this enzyme subsequently decreased with the time of incubation. specific activity of the da 5.2.3 isolate was higher than that of the l5 isolate, which were 47.3 u mg-1 and 9.1 u mg-1 respectively. identification of bacillus sp. da 5.2.3. morphology characterization of da 5.2.3 isolate showed that this isolate incubation up to 48 h. the increase of the cell was as much as 1 to 2 log 10 cfu ml-1. the data showed that a logarithmic phase was reached before 24 h of incubation, followed by a stationary phase after 48 h of incubation. the highest growth l5 da 5.2.3 dp 5.1.1 kpt 7.1.1 kat 5.1 k.pt 7.1.2b l3 kat 7.1.2 dp 5.1.2 control table 3 total suspended solid (tss) of bacillus spp. cultured in 1.2% commercial shrimp feed medium isolates tss (mg ml-1) 1640 1840 1880 1880 1920 2000 2240 2320 2400 2600 36.9 30.4 27.6 27.6 26.0 23.0 13.8 10.8 7.7 0.0 tss decreasing (%) a b microbiol indones was gram positive, rod in shape, catalase positive and an endospore-forming bacterium. pcr amplification of its 16s rdna gene showed a 1.3 kb band on electrophoresis. blast analysis of this gene sequences (ncbi) indicated that this isolate had the highest 16s rdna gene sequences similarity (99 %) with bacillus cereus xhj-2-6 (accession number: gq 19959.1). discussion higher numbers of bacillus isolated from sediment may be expected because of sediment is at the bottom site of pond, and more nutrients are concentrated there. on the contrary, bacillus isolated from shrimp digestive tract were the lowest in number. a low number bacteria was found in water samples compare to others site of ponds. bacteria in aqueous environment can reach the animal digestive tract through drinking or swallowing of water by the animals. lee et al. (2000) stated that one of factor for competency of probiotics was their ability to attach to intestine mucus. therefore, only selected bacteria could survive in the intestine. more than 90% isolates selected had both proteolytic and amylolytic activity. and most of bacillus spp. isolates have more than one extracelullar enzymes. bacillus spp. isolates taken from the shrimp digestive tract had the highest values of pi and ai. this could be due to a lot of nutrient being concentrated in intestine, so that only bacteria that have potential enzyme activity to degrade substrates could survive and could be more competitive in the intestine. denkin and nelson (1999) reported that the production of potease activity in fish digestive tract mucus of vibrio anguilarum was higher than other media. selected isolates of bacillus had the same growth pattern, and their logarithmic growth phase was reached at 24 h of incubation. santos and martins (2003) reported that logarithmic growth phase of bacillus grown on 1% soluble starch and 1% maltose was reached at between 18 and 48 h of incubation. the ability of the selected isolates to reduce total suspended solid values has potential application to maintain water quality in terms of total suspended solid reduction. total suspended solid in shrimp-pond-water is mainly consists of shrimp feed excess which mostly contains protein, starch and lipid. bacteria might degrade the feed particles into smaller ones using their extracellular enzymes. proteases hydrolyze the smaller peptides and amino acids, facilitating partial absorption. protease and amylase activities of da 5.2.3 isolate were higher than that of l5 isolate even though the pi values of the isolates were similar. this result implied that the pi value was not always correlated with enzyme activity in broth culture. in addition, the l5 isolate could decrease total suspended solid values by more than that of da 5.2.3 isolate. this result might be arise because enzyme activities were measured using different substrates. alpha-amylase of the da 5.2.3 isolate reached the maximum activities at the end of logarithmic growth phase, which was at 24 h from start of incubation. the activity was decreased sharply as the incubation time prolonged up to 48, 72 or 96 h. cordeiro et al. (2002) reported that a maximum αamylase production of a thermophilic bacillus sp. strain smia-2 was reached at 48 h or at the late stage of logarithmic growth phase in basal medium supplemented with 0.5% (w/ v) soluble starch. the activity was remained relatively constant up to 96 h. another study by santos and martin (2001) found that α-amylase of moderately thermophilic bacillus sp. was increased the production between 18-48 h of incubation in medium containing 1% (w/v) maltose as the carbon sources. the variations of exoenzyme synthesis among these three isolates are could be expected because bacteria were grown in different substrates. protease activity produced by the l5 and the da 5.2.3 isolates reached maximum activity at 48 h or at the beginning of the stationary growth phase. then, it decreased gradually as the time of incubation was extended. the length incubation times of maximum protease production of by bacillus strain was reported by some researchers, as being from 24 to 120 h from start of incubation (chu et al. 1992; mabrouk et al. 1999; beg and gupta 2003). genckal and tari (2006) stated that decreasing enzyme activity with prolonged time, could occur because of hydrolysis of the enzyme by proteases. based on all the data of this study, the da 5.2.3 isolate has potential application to maintain water qualities in shrimp ponds. based on molecular identification, this isolate was similar with bacillus cereus xhj-2-6. so far, there is no information about using this strain as probiotic in shrimp aquaculture. therefore, it will be a great challenge to explore its characteristics further for probiotic development in aquaculture industry. acknowledgement this work was supported by a phd studentship from the directorate general of higher education, department of education, indonesia to ij. 70 jamilah et al. figure 3 alpha-amylase activity of bacillus sp. l5 and da 5.2.3 over time production. , da 5.2.3 total act, , da 5.2.3 specific act; , l5 total act; , l5 specific act. bars indicate standard error. microbiol indones 71volume 3, 2009 references atlas rm. 1997. hand book of microbiological media. 2nd ed. lawrence cp, editor. boca raton: crc pr. benfeld p. 1955. amylases α and ß: method enzymol 1:149-58. beg qk, gupta r. 2003. purification and characterization of an oxidation stable, thiol-dependent serinealkaline protease from bacillus mojavensis. enzyme microb technol 32:294-304. bradford mm. 1976. a rapid and sensitive method for the quantities of protein utilizing the principle of protein-dye binding. anal biochem 72:248-54. chu im, lee c, li ts. 1992. production and degradation of alkaline protease in batch culture of bacillus subtilis atcc 14416. enzyme microb technol 14:755-61. cordeiro cam, martin mll, luciano ab. 2002. production and properties of α-amylase from thermophilic bacillus sp. braz j microbiol 33:57-61 decamp o, moriarty d. 2007. aquaculture species profit from probiotics. feed mix 15:20-3. denkin sm, nelson dr. 1999. induction of protease activity in vibrio anguillarum by gastro-intestinal mucus. appl environ microbiol 65:3555-60. genckal, tari c. 2006. alkaline protease production from alkalophilic bacillus sp. isolated from natural habitat. enzyme microb technol 30:703-10. gullian m, thompson f, rodriguez j. 2004. selection of probiotic bacteria and study of their immunostimulatory effect in penaeus vannamei. aquaculture 233:1-14. lee yk, lim cy, teng wl, ouwehand ac, tuomda em, salminen s. 2000. quantitative approach in the study of adhesion of lactic acid bacteria to intestinal cell and their competition with enterobacteria. appl environ microbiol 66:3692-7. mabrouk ss, hashem am, el-shayeb nma, ismail ms, abdel-fattah af. 1999. optimization of alkaline protease productivity by bacillus licheniformis atcc 21415. biores technol 69:155-9. marchesi jr, sato t, weighman aj, martin ta, fry jc, hiom sj, wade wg. 1998. design and evaluation of useful bacteriumspecific pcr primers that amplifygenes coding for bacterial 16s rrna. appl environ microbiol 62:2501-7. rengpipat ss, rukpratanpom s, piyatitivorakul s, menavetz p. 1998. effect of probotic bacterium on black tiger shrimp paneous monodon survival and and growth aquaculture bacillus s11. aquaculture 167:301-13. ochoa-solano jl, olmos-soto j. 2006. the functional property of bacillus for shrimp feeds. food microbiol 23:519-25. santos eo, martins mll. 2003. effect of the medium composition on formation of amylase by bacillus sp. braz arch biol technol 46:129-34. vaseeharan b, ramasamy p. 2003. control of pathogenic vibrio spp. by bacillus subtilis bt 23 a possible probiotic treatment for black tiger shrimp penaeus monodon. lett appl microbiol 36: 83-7. verschuere l, rombaut g, sorgeloos p, verstraete w. 2000. probiotic bacteria as biological control agents in aquaculture. microbiol mol biol rev 64:655-71. ziaei-nejad s, rezaei mh, takami ga, lovett dl, mirvaghefi ar, shakouri m. 2006. the effect of bacillus spp. bacteria used as probiotics on disgestive enzyme activity, survival and growth in the indian white shrimp fenneropenaeus indicus. aquaculture 252:516-24. 01. maladan.cdr vol.15, no.2, june 2021, p 37-44 doi: 10.5454/mi.15.2.1 single nucleotide polymorphism in the gene rpob mycobacterium tuberculosis from papua-indonesia and its impact on rifampicin resistance: a wholegenome sequencing analysis yustinus maladan*, tri wahyuni, hana krismawatiand center for papua health research and development, j ahmad yani no. 48, jayapura, indonesiaalan . in the antibiotic era, tuberculosis (tb) drug resistance, especially rifampicin (rif), is highly reported around the world. resistance of rif is caused by the mutation of genes associated with the rif receptor. the aims of this study are detecting the single nucleotide polymorphism of rifampicin resistant genes using whole(snp) genome sequencing (wgs) and analyzing the profile of protein changes caused by snp. twenty mycobacterium tuberculosis culture samples were passed on the wgs procedure and 19 samples were adequate to further bioinformatics analysis. single nucleotide polymorphisms analysis was performed using tbprofiler. based on tbprofiler, seventeen samples were resistant to rifampicin. the the mutations were found in gene. rpob mutations that cause the resistance are s456l, d441y, h451y, 436p, q438k. other single nucleotide polymorphisms h841r, v540m, and r230c were also found. the h841r mutants are present together with the s456l, v540m with s456l mutants, and r230c with q438k mutants. native protein for rna polymerase subunit β used was the result of separation from the crystal structure of h37rv rna mycobacterium tuberculosis polymerase (pdb: 5uhb). binding affinity rif to rna polymerase subunit using autodock vina. β calculated construction of mutant 3d structures using foldx5. from the analysis, seventeen samples were resistant to rif and two samples did not contain snp which could cause resistance to rif. key words: molecular docking, , rifampicin, genemycobacterium tuberculosis rpob di era antibiotik, resistensi obat tuberkulosis (tb), terutama rifampisin (rif), sering dilaporkan di seluruh dunia. resistensi rif disebabkan oleh mutasi gen yang berhubungan dengan reseptor rif. penelitian ini bertujuan untuk mendeteksi single nucleotide polymorphism (snp) pada gen yang berhubungan dengan resisten rifampisin menggunakan whole-genome sequencing (wgs) dan menganalisis perubahan profil protein yang disebabkan oleh snp tersebut. dua puluh sampel kultur yang telah melalui proses mycobacterium tuberculosis wgs dan 19 sampel diantaranya memenuhi untuk analisis bioinformatika lebih lanjut. analisis snp dilakukan dengan menggunakan tbprofiler. berdasarkan tbprofiler, tujuh belas sampel resisten terhadap rifampisin. mutasi ditemukan pada gen . mutasi yang menyebabkan resistensi adalah s456l, d441y, h451y, 436p, rpob q438k. snp lainnya h841r, v540m, dan r230c juga ditemukan. mutan h841r hadir bersama dengan mutan s456l, v540m dengan s456l, dan r230c dengan mutan q438k. protein native untuk rna polymerase subunit β yang digunakan merupakan hasil pemisahan dari struktur kristal rna polimerase mycobacterium tuberculosis h37rv (pdb: 5uhb). rif terhadap rna polymerase subunit β dihitung menggunakan binding affinity autodock vina. konstruksi struktur 3d mutan menggunakan foldx5. dari hasil analisis, tujuh belas sampel resisten terhadap rif dan dua sampel tidak mengandung snp yang dapat menyebabkan resistensi terhadap rif. kata kunci: gen , , , rifampisin rpob molecular docking mycobacterium tuberculosis microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ;: +62 e-mail: 8114832859 yustinus.maladan@litbang.kemkes.go.id resistance to isoniazid and (rif) with or rifampicin without resistance to other first-line tb drugs (koch et al. 2018; kemenkes ri 2019). in many studies, rif resistance was reported . the (brown 2015)et al. mechanism of action of rif is by inhibiting the transcription of dna in mycobacterium tuberculosis ( et al. floss and yu 2005; lin 2017). the analysis of tb drug resistance is performed conventionally using phenotypic drug susceptibility testing (dst). however, conventional phenotypic dst is time-consuming from more than 2 weeks until 1 month . using pcr, the time is shorter but (who 2018) lacking sensitivity and specificity, since only certain gene regions can be detected. wgs is an approach to is a bacterium that mycobacterium tuberculosis causes tuberculosis (tb). tuberculosis is a global burden disease that kills about 1,3 million people. recently, indonesia is placed third-ranked in the world of tb after india and china. based on the case detection rate (cdr), the provinces with the highest cdr are dki jakarta (122.2%), south sulawesi (84.0%), papua (78.5%) . (kemenkes ri 2019) increasing drug resistance numbers were reported around the world and become the challenge of tb elimination. mdr-tb is caused by m. tuberculosis overcome many challenges both related to conventional ph enotypes an d the limita tio ns of la ckin g comprehensive molecular tests. wgs providing detailed sequence information for whole genomes, especially genes associated with drug resistance . [810] one of the genes that can be identified by wgs is the rpob rpobgene. the gene functions to code for the formation of rna polymerase subunit β (rnap). mutations in the gene can be associated with rif resistance .(floss and yu 2005) wgs approaching possible to sequence whole part of effectively analyzes the resistance of m. tuberculosis rif through snp detection on the gene that associated with rif resistance. wgs can be used to detect minor variants as well as subpopulations with low-level drug resistance as information in treatment to anticipate the emergence of drug resistance . in (colman 2015)et al. this study, we used wgs mycobacterium tuberculosis data from papua to detect the presence of snps associated with rif resistance . (maladan 2020)et al. this study aims to detect the snp of rif resistant genes using wgs and analyzing the profile of protein amino acid substitution caused by snp. materials and methods samples. the research design was cross-sectional. a total of 20 samples were obtained from tb patients in bsl level 2 jayapura regional health laboratorypapua province. initial identification of drug resistance was carried out using genexpert. the resistant positive result sample were cultured on lowenstein-jensen's media as many as 20 samples. isolation of m. tuberculosis dna was performed using qiaamp dna mini kit (ref:51306, qiagen, german). l i b r a r y p re p a r a t i o n a n d s e q u e n c i n g . quantification of extracted dna was m. tuberculosis measured using qubit ™ 3.0 (thermo fisher scientific). ngs procedure followed nextera xt dna library prep kit . the ngs process is (illumina 2016) carried out using the miseq 600 cycle reagent kit (v3). tagging of genome was done m. tuberculosis using nextera transposome. amplification of the library was then performed using nextera pcr master mix. purification of the amplification product was performed using ampure xp beads. after purification, using nextera xt dna library preparation kits, the library was normalized. pooling dna was done on a single tube and dilution was run using the bead-based normalization method . the dna was (illumina 2017) loaded on the cartridge and wgs was run on miseq. all procedures were done in the center of papua health research and development. bioinformatics analysis. genome analysis was done using tbprofiler software on (coll 2015)et al. linux 17.10 platform. analysis of bam file reads visualization, consensus creation, and alignment were done using unipro ugene ngs 1.3.1 (okonechnikov et al. 2012). template rna polymerase subunit β of m. tuberculosis. the crystal structure of m. tuberculosis rna polymerase protein was obtained from pdb, with pdb id 5uhb. subunit rna polymerase was then β separated from the rna polymerase complex using the discovery studio software. construction of the l436p, q438k, q438k + r230c, d441y, h451y, s456l, s456l + v540m, and s456l + h841r mutants using foldx5. ligand preparation. rif is obtained by separating it from subunit rna polymerase in pdb β format. molecular docking. docking analysis was performed by using autodock vina (trott and olson 2010) (dallakyan 2015) which is integrated with pyrx application. docking results were visualized using pymol 2.0 and discovery studio 2020. result reads nucleotide sequence that was produced by the miseq v3 reagent are around 300 bp. alignment of reads was performed using h37rv m. tuberculosis ( n c _ 0 0 0 9 6 2 . 3 ) a s a r e f e r e n c e s e q u e n c e (nc_000962.3). the results of alignment read then produce a consensus which is a merging number of reads at the same location of the amplified gene. in this study, we analyze the snps in the genes rpob m. tuberculosis from papua that is associated with rif resistance. the most common mutation was thymine to cytosine mutation at position 1349 bp. other mutations are changes in thymine to cytosine at position 1.289, cytosine to adenine at position 1.294, guanine to thymine at position 1.303, and cytosine to thymine at position 1.333. mutations in these genes are characterized by the color of the mutated nucleotides (fig 1). the rpob gene was separated from the whole genome of for further analysis using m. tuberculosis the unipro ugene ngs 1.3.1 software. after separated, the genes are translated to see the changes in amino acids caused by nucleotide mutations (fig 2). changes in thymine to cytosine at position 1349 bp cause 38 maladan et al . microbiol indones volume 15, 2021 microbiol indones 39 changes in the amino acid serine to leucine (s450l). changes in thymine to cytosine at position 1289 cause changes in leucine to proline (l430p). the change in cytosine to adenine at position 1294 causes the conversion of glutamine to lysine (q432k). the change of guanine to thymine at position 1303 causes the conversion of aspartic acid to tyrosine (d435y). changes in cytosine to thymine at position 1333 cause changes in histidine to tyrosine (h445y). snps were also identified using tbprofiler to obtain mutation data in the genes. five types of mutations that cause rpob resistance to rif were found, namely s456l, l436p, q438k, d441y, and h451y (table 1). furthermore, the amino acid numbering will be adjusted according to the amino acid sequence in the h37rv m. tuberculosis rnap crystal structure by adding six to the amino acid numbering. the effect of the combination of several mutations we analyzed using the bioinformatics approach while providing an overview of the effects of mutations that have been confirmed resistant to rif. the m. tuberculosis rna polymerase molecule consists of six chains and the β rna polymerase subunit is contained in the c chain. for further analysis, the rnap is separated from the rna polymerase molecule (fig 3a). binding sites of rnap were predicted using discovery studio software (fig 3b). these binding sites were used to adjust the rif grid for docking analysis. the results of the construction of mutants l436p, q438k, q438k + r230c, d441y, h451y, s456l, s456l + v540m, and s456l + h841r show that there are differences in topology on the surface of the rnap when compared with native proteins (fig 4). mutants l436p, q438k, d441y, h451y, and s456l at the binding site, while the various combinations of r230c, v540m, and h841r are outside the binding site. the presence of mutations in the binding site area can cause loss of contact between rif and rnap and interactions that form the binding site. the surface structure of the q438k mutants and the q438k + r230c mutants did not differ much (figures 4b and 4c). likewise, the s456l and s456l v540m mutants were not much different (fig 4f and 4g), while the s456l mutants with the s456l + h841r mutants had differences in the surface around the binding site (fig 4f dan 4h). the molecular docking results showed different interactions between the native rnap m. tuberculosis proteins (fig 5). interactions occur in the form of van der waals bonds, conventional hydrogen bond, carbon hydrogen bond, pi-sigma, alkyl, pi-alkyl, pi-pi tshaped, pi-cation, unfavorable donor-donor. in the native rnap protein, interaction with rif forms hydrogen bonds with residues of s456, q438, n493, h451, f439 (fig5a). in the d441y mutant, there are only two hydrogen bonds, namely the r465 and r454 residues (fig 4e). hydrogen bonding to the r454 residue was also found in the crystal structure of the rnap . (lin . 2017)et al in the h451y mutant, hydrogen bonds are formed with the residue t451, r454, r465, q438 (fig 5f). in the s456l mutant, there are hydrogen bonds that are different from the native proteins, namely d441, h1035, q614, r613, g491 (fig 5g). the combination of the s456l and v450m mutants has almost the same hydrogen bonds as the s456l mutants but is added by the presence of the r454 hydrogen bonds (fig 5h). whereas the s456l + h841r mutant only has two hydrogen bonds, namely q438 and n443 (fig 5i). the energy required in the interaction between native rnap protein and mutants with rif is calculated in the form of binding energy (table 2). all mutants had an increase in binding energy compared to native protein rnap which was -10.1 kcal/mol. in energy stability, almost all mutants experienced a decrease in energy stability compared to native protein rnap, which was 841.36 kcal/mol. discussion wgs results showed twenty positive mdr-tb samples showed that 19 samples were of good quality and one sample of inadequate quality. merge reads on wgs results using tbprofiler. the output of tbprofiler in the form of bam format is further analyzed with unipro ugene software. mutations in genes associated with isoniazid resistance can be identified by looking at the difference in nucleotides in each read produced by the wgs process. the snps found in the gene m. tuberculosis rpob from jayapura were l436p, q438k, q438k + r230c, d441y, h451y, s456l, s456l + v540m, and s456l + h841r. in the tb284 sample, the s456 mutation was present together with the h641r mutation whereas in the tb690 sample the s456l mutation was present together with the v540m mutation (table 1). in the tb153 sample, the q438k mutation was present along with the r230c mutation. mutations l436p, q438k, d441y, h451y, s456l are mutations that have been confirmed to cause resistance to m. tuberculosis (phelan . 2016; dixit . 2019)et al et al . 40 maladan et al . microbiol indones fig 1 →mutations in the gene of of papua from wgs. the most common mutation was t c rpob m. tuberculosis position 1349 bp. other mutations are changes in t c at position 1289, c a at position 1294, g t at → → → position 1303, and c t at position 1333. some mutations that do not have the potential to cause resistance → to rif are c t at position 670, g a at position 1600, and a g at position 2504.→ → → fig 2 gene mutation that is associated with rif resistance on . according to the tbprofiler, the m. tuberculosis mutations associated with rif resistance are l430p (l436p), q432k (q438k), d435y (d441y), h445y (h451y), and l450s (l456s). fig 3 rnap and topology rnap. the rnap binding site is marked with a yellow ball. this area is the target of rif. volume 15, 2021 microbiol indones 41 fig 4 the surface structure of rnap was compared with each mutant. the circled part is the binding site. fig 5 map of native protein interactions and their respective mutants. rif is displayed in ball and stick form, while the residue is shown in ball form. 42 maladan et al . microbiol indones table 1 e. snp mutation causes mutations as well as another mutation based on the tbprofiler databas code of sample snp mutation effect mutation gene mutation position tb0065 s456l resistant tb0207 d441y resistant tb035 s456l resistant tb062 h451y resistant tb1007 h451y resistant tb1023 s456l tb1186 s456l resistant tb240 s456l tb284 s456l resistant h841r tb345 s456l resistant tb487 s456l resistant tb524 s456l tb530 s456l resistant tb618 tb674 l436p resistant tb690 s456l resistant v540m tb751 tb752 s456l resistant tb153 q438k resistant r230c table 2 rna polymerase and rif. stability energy binding energy to all mutants had an increase in binding energy compared to native protein rnap which was -10.1 kcal/mol. the combination of s456l with h841r has a greater increase of -6.8 kcal/mol. in energy stability, almost all mutants experienced a decrease in energy stability compared to native protein rnap, which was 841.36 kcal/mol. the combination of s456l with h841r had a greater reduction of 824.06 kcal/mol. receptor binding energy (kcal/mol) stability energy (kcal/mol) native protein rnap -10.1 841.36 s456l -8.3 834.7 d441y -7.2 831.1 h451y -7.8 833.68 l436p -9.9 843.88 q438k -7.2 833.75 s456l+h841r -6.8 824.06 s456l+v534m -8.3 833.34 q438k +r230c -8.1 831.34 this result is almost the same when compared to the crystal structure where hydrogen bonding occurs at the residues q438, s456, q435, r454, h451, f439 (lin . 2017)et al . the l436p mutant causes the loss of hydrogen bonds with residue s456 (fig 5b). the q438k mutant causes the loss of all key hydrogen bonds and is replaced by other hydrogen bonds such as r465, r613, and h680 (fig 5c). the presence of the r230c mutation in the q438k mutant indicates the presence of hydrogen bonding in n493 but at a further distance, namely 4.89 å (fig 4d). these results probably suggest that the r230c mutation may slightly stabilize the q438k mutant. native protein has a binding energy of -10.1 kcal/mol and stability energy of 841.36 kcal/mol. the s456l mutant and the combination of the s456l and v534m mutant have the same value, namely -8.3 kcal/mol. this value is greater than the native protein. this increase in binding energy can result in loss of contact with rif. the combination of the s456 and h841r mutants causes greater binding energy than native rnap proteins. the other mutants also volume 15, 2021 microbiol indones 43 experienced increased binding energy with rif, respectively, d441y, h451y, l436p, q438k, and q438k + r230c were -7.2, -7.8, -9.9, -7.2, -8.1 kcal/mol. changes in hydrogen bonding to the key residue and increasing energy in all mutants are indicators of resistance to rif. the results of this molecular docking analysis showed the same results as the results of the drug susceptibility test (dst) which showed that mutants l436p, q438k, d441y, h451y, s456l were resistant to rif . these (heep . 2001)et al results illustrate how resistance to rif is caused by mutations in the gene.m. tuberculosis rpob from the analysis, it was found that seventeen samples were resistant to rifampicin and two samples did not contain snp which could cause resistance to rifampicin. the snps found in the m. tuberculosis rpob gene from jayapura were l436p, q438k, q438k + r230c, d441y, h451y, s456l, s456l + v540m, and s456l + h841r. the molecular docking results showed the same results as the results of the drug susceptibility test (dst) as an illustration of how mutations in the gene could affect m. tuberculosis rpob rif. this method can be used to predict the effect of new mutations in the gene that m. tuberculosis rpob have not been confirmed by the dst method. acknowledgements thanks to the regional health laboratory of jayapura for contributing to sample collection, culture and gene expert analysis. references brown ac, bryant jm, einer-jensen k, holdstock j, houniet dt, chan jz, depledge dp, nikolayevskyy v, 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biochemistry, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia phone/fax: +62-251-8423267, e-mail: imart171@yahoo.com the yeast mitochondrial f 1 f 0 -atp synthase is a multisubunit complex that contains at least 17 different subunits. subunit 8 of yeast mitochondrial atp synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial atp8 gene. there is no homologue of subunit 8 found in bacteria. subunit 8 has three distinct domains; an n-terminal domain, a central hydrophobic domain and a c-terminal domain. subunit 8 has been shown to adopt a transmembrane topology with the central hydrophobic domain spans the inner mitochondrial membrane once. in order to elucidate the need of subunit 8 to maintain transmembrane topology for its functioning, a severely functionally defective subunit 8 variant that has been introduced with double-charged residues within the central hydrophobic domain was analysed. a gene encoding this variant was expressed in a yeast strain lacking endogenous subunit 8. the subunit 8 variant was then targeted into mitochondria. following its assembly into mitochondrial atp synthase complex, its membrane topology was determined. the results obtained showed that subunit 8 was obligatory to maintain a transmembrane topology for providing proper functioning. the transmembrane topology may be critical for subunit 8’s proposed structural roles as part of the stator stalk of the mitochondrial atp synthase complex. key words: membrane topology, atp synthase, mitochondria, yeast atp synthase, also known as f 0 f 1 -atpase, is a large complex of 600 kda that uses the proton electrochemical gradient generated by the respiratory chain to catalyse atp synthesis from adp and pi (thomas et al. 2008). the yeast mitochondrial atp synthase is a multisubunit complex composed of at least 17 subunits grouped into two sectors, a membrane-extrinsic sector (f 1 ) and a membrane-embedded sector (f 0 ). the two sectors are linked by protein stalks. the f 1 sector is comprised of five subunits a 3 , b 3 , g 1 , d 1 and e 1 , and is coupled to proton flux through the f 0 sector. the f 0 sector spans the membrane and is composed of subunits b, oscp, d, e, f, g, h, i/j, k, which are encoded by nuclear genes, and subunits 6, 8 and 9, which are encoded by mitochondrial genes. some f 0 subunits form a stator stalk anchored in the membrane that prevents futile rotation of mitochondrial atp synthase subunits relative to the rotor during coupled atp synthesis/hydrolysis (stephens et al. 2003). in the inner mitochondrial membrane the atp synthase complex can form a dimer (fronzes et al. 2006). the subunit 8 of yeast mitochondrial atp synthase is a small hydrophobic polypeptide of 48 amino acids encoded by the atp8 gene (macreadie et al. 1983). analysis of its primary structure has led to identification of three distinct domains; an n-terminal domain, a central hydrophobic domain (chd) and a c-terminal domain (devenish et al. 1992). the topology of subunit 8, which is determined by unique introduced cysteine residues, indicates that its n-terminus is located in the intermembrane space of mitochondria, whereas the c-terminus is located within the mitochondrial matrix (stephens et al. 2000). further analysis employing cysteinescanning mutagenesis showed that the first 14 and the last 13 amino acids were extrinsic to the lipid bilayer, indicating the existence of a 21 amino acid transmembrane spanning region (stephens et al. 2003). as a mitochondrially encoded protein, subunit 8 is transcribed and translated entirely within the organelle. subunit 8 is not present in prokaryotes, but is an additional subunit present in the mitochondrial atp synthase of eukaryotes. this means that bacterial atp synthase can naturally function without the presence of subunit 8 (artika 2007). the immediate question therefore is to resolve the detailed structure and roles of this subunit in the enzyme complex. several lines of evidence suggested that subunit 8 is part of the stator stalk in the yeast mitochondrial atp synthase. subunit 8 maintains close interactions with subunits b, d, f and 6, each of which has proposed roles as part of the stator stalk. no amino acid of subunit 8 directly participates in either atp synthesis/hydrolysis or proton pumping, suggesting that subunit 8 is a structural component of the mitochondrial atp synthase complex (stephens et al. 2003). in order to elucidate its detailed structure and function, an allotopic expression system for subunit 8 has been developed. allotopic expression is the deliberate relocation of organellar genes to the nucleus and delivery of the gene products from the cytoplasm to the corresponding organelle. for allotopic expression of subunit 8, a nuclear version of subunit 8 gene has been designed to be expressed in the nucleocytosolic system. to ensure that the cytoplasmically synthesized subunit 8 was imported into mitochondria, sequences encoding a mitochondrial signal peptide were fused to the n-terminus of the gene (gearing et al. 1985). the allotopic expression system has been applied to study various aspects of subunit 8 molecular biology. this system has also been successfully used to express flag taggedsubunit 8 protein (artika 2006). the allotopically expressed flag tagged-subunit 8 protein was imported into mitochondria and assembled into a functional atp-synthasecomplex. the main purpose of the flag tag addition to subunit 8 protein was to enable detection of subunit 8 protein by means of an anti-flag tag monoclonal antibody. in the present study, a flag tagged-subunit 8 variant was microbiol indones employed to analyze the need for subunit 8 to maintain transmembrane topology for function. the ability of subunit 8 to tolerate the presence of charged amino acids within its transmembrane domain is well documented. given the unfavourable thermodynamics associated with the burial of unshielded charges within a lipid bilayer, it was proposed that the chd of subunit 8 may not necessarily constitute a membrane-spanning region (stephens 2000). on the other hand, its important roles as a part of the stator stalk may necessitate subunit 8 to maintain its transmembrane topology. in addition, subunit 8 is required for the maintenance of the interaction between f 1 and f 0 as well as correct f 0 assembly (roucou et al. 1999). it is therefore important to elucidate the obligatory role of subunit 8 to maintain transmembrane topology for its function. the present study was aimed to probe the membrane topology of subunit 8 variant having double-charged residues within the chd. the hypothesis and strategy taken were as follows: upon introduction of double-charged residues within the chd, subunit 8 variant may maintain the transmembrane topology or alternatively, it may loose its transmembrane topology and effectively become a peripheral membrane protein which is still able to assemble into a functional mitochondrial atpsynthase-complex. in principal, it is possible to distinguish between a membrane peripheral protein and an integral protein as each should behave differently upon extraction with organic solvent or detergent. materials and methods materials. saccharomyces cerevisiae strain m31 (atp8, mit%, his6, ade1) used was as previously desribed (nagley et al. 1988). strain ftc2 is strain m31 but expressing flag tagged-subunit 8 gene fused with a mitochondrial signal peptide (roucou et al. 1999). strain df68 is a variant of strain m31 that expresses flag tagged-subunit 8 variant (l23d, l24d) fused with a mitochondrial signal peptide (artika 2007). isolation of mitochondria. mitochondria were prepared using the glass bead method (lang et al. 1977). the protein concentration of isolated mitochondria was determined using the bio-rad protein micro-assay procedure based on the method of bradford (1976). extraction of mitochondrial protein. mitochondrial proteins were extracted as follows. an aliquot containing 200 mg of mitochondria was centrifuged using sorvall sm-24 rotor at 10 000 rpm for 10 min at 4 °c. the pellet was then resuspended with 1 ml of appropriate extracting solution. to avoid protein degradation, protease inhibitors, phenylmethyl-sulphyl-fluoride (pmsf), para-aminobenzamidinic acid (pab) and e-amino-n-caproic acid (e-aca) were added to give final concentrations of 0.5 mm, 5 mm and 5 mm, respectively. the mixture was incubated with rotary shaking at 4 °c for 30 min followed by centrifugation using sorvall rp100-at rotor at 55 000 rpm for 30 min at 4 °c. the supernatant was carefully separated from the pellet. the protein in the pellet (subsequently called the pellet protein) was solubilised by the addition of 20 ml of 5% (w/v) sds and 20 ml of dissociation buffer (62.5 mm tris hcl, 10% (v/ v) glycerol, 2% (w/v) sds, 0.00125 (v/v) bromophenol blue, 5% (v/v) b-mercaptoethanol). unless otherwise stated, the extracted proteins in the supernatant were precipitated using the following procedures. first, 1 ml water was added and the mixture was briefly vortexed. then 4 volumes (8 ml) of methanol was added. after briefly vortexing the mixture, 2 volumes (4 ml) of chloroform was added. again the mixture was briefly vortexed followed by addition of 3 volumes (6 ml) of chloroform. the mixture was then vortexed and centrifuged using sorvall ss-34 rotor at 9 000 rpm for 2 min. after discharging the upper phase, protein was precipitated by addition of 3 volumes of methanol, vortexed and centrifuged at 9000 rpm for 2 min. after carefully removing the supernatant, the pellet was air dried. after drying, the protein (subsequently called supernatant protein) was solubilised by the addition of 20 ml 5% (w/v) sds and 20 ml dissociation buffer. both the pellet and supernatant proteins were analysed by sds-polyacrylamide-gel-electrophoresis. protein analysis. sds-polyacrylamide-gelelectrophoresis (sds-page) was performed as described by laemmli (1970) using a dual adjustable slab gel unit. following sds-page, the proteins were transferred to an immobilon-p membrane (pvdf). the membrane was then incubated overnight at 4 °c in blotting solution containing primary antibody. after washing the non-bound primary antibody, the membrane was incubated for 1 h at room temperature in blotting solution containing secondary antibody (conjugated alkaline-phosphatase). proteins were visualized using a vistra alkaline-phosphatase-conjugate-substrate kit (amersham life science, united kingdom). results detection of flag tagged-subunit 8 protein and extraction of mitochondrial protein with various solvents. a preliminary experiment was carried out to detect allotopically expressed flag tagged-subunit protein using the anti-flag monoclonal antibody following mitochondrial protein extraction using various organic solvents. mitochondria were isolated from the ftc2 yeast strain grown on ethanol medium. mitochondrial proteins were then extracted using various organic solvents (2 m urea, 4 m urea, 8 m urea and 100 mm sodium carbonate ph 11.5). both pellet and supernatant proteins were analysed by sds-page. after the transfer of protein to the pvdf membrane, the presence of subunit 8 was examined using anti-flag-tag monoclonal antibody as a probe. subunit 8 was clearly detected in all of the pellet fractions (fig 1). however, no subunit 8 was detected in any of the supernatant fractions. at this stage, the data indicated that the allotopically expressed flag tagged-subunit 8 had been successfully assembled into a functional mitochondrial atpsynthase-complex. furthermore, upon extraction with various organic solvents, the flag tagged-subunit 8 could clearly be detected using the anti-flag m2 monoclonal antibody probe. membrane topology of wildtype subunit 8. in order to analyze the membrane topology of the native subunit 8, mitochondria were isolated from strain ftc2. membrane proteins were then extracted from mitochondria to determine the extent to which wildtype subunit 8 could be extracted by 38 artika microbiol indones 39volume 3, 2009 organic solvents. both the pellet and supernatant proteins were analysed by sds-page. after the transfer of proteins to the pvdf membrane, the membrane was cut into two pieces to separate the upper part, containing proteins of a molecular weight larger than 30 kda, from the lower part, containing proteins of a molecular weight below 30 kda. the upper part of the membrane was assayed with anti-subunit-a antibody to detect the presence of subunit-a (molecular weight of 58.5 kda). the part of membrane containing proteins of molecular weight less than 14.3 kda was assayed with antiflag-tag monoclonal antibody to detect the presence of subunit 8 (molecular weight of 5.8 kda). blots were tested for subunit-a in order to determine the extent to which subunita of the membranous extrinsic f 1 sector could be recovered following extraction. as shown in fig 2, the a-subunit was generally extracted, but was not completely removed, from the membranes since it could clearly be detected in both the supernatant and pellet fractions. as expected, extraction using storage buffer (10 mm tris-maleate, 600 mm mannitol, 2 mm egta, 0.5 mm phosphate, ph 6.8) released little a-subunit into the supernatant (lane 2, a panel), and subunit-a was still mainly present in the pellet fraction (lane 1, a panel). under the same extraction conditions, no subunit 8 signal (lane 2, y8 panel) was detected in the supernatant, and subunit 8 was found to be in the pellet fraction (lane 1, y8 panel). extraction using 4 m urea released about 40% of a-subunit (lane 4 compared to lane 3, a panel). as expected, native subunit 8 was not extracted by 4 m urea. extraction using 8 m urea gave similar results to that observed using 4 m urea except that it was not clear why more a-subunit was released by extraction with urea 8 m (band in lane 6 compared to band in lane 4, a panel). however, the a-subunit was still present in the pellet fraction (lane 5, a panel). subunit 8 was not extracted by 8 m urea. membrane protein extraction was also carried out using 100 mm sodium carbonate (ph 11.5) as an extracting solvent. similarly to that observed on urea extraction, the a-subunit was extracted (lane 8, a panel) but was not completely released. as expected, the wildtype subunit 8 was not extracted by sodium carbonate (lane 8, y8 panel) and a strong subunit 8 signal was detected in the pellet fraction (lane 7, y8 panel). note that despite the amount of the starting material being the same for each extraction, a stronger subunit 8 signal was detected in the sample treated with sodium carbonate (lane 7, y8 panel). the reason for this remains unclear. finally, extraction using sds-urea, released most of the a-subunit (lane 10, a panel). surprisingly, some a-subunit was still present in the pellet fraction (lane 9, a panel). as expected, subunit 8 was extracted by sds-urea (lane 10, y8 panel) and no subunit 8 signal was detected in the pellet fraction (lane 9, y8 panel). membrane topology of subunit 8 variant. strain df68 was considered to be the most suitable strain to be used to investigate whether or not it is obligatory for subunit 8 to maintain a transmembrane topology for function. the subsequent membrane topology studies used the variant expressed in df68. membrane proteins of mitochondria isolated from strain df68 were extracted with various solvents using the same procedure as that used to extract mitochondrial membrane proteins of strain ftc2. following membrane protein extraction, samples from both the pellet and the supernatant fractions were analysed by sds-page. the presence of the a-subunit and subunit 8 was assayed in both fractions. the results are shown in fig 3. generally, the extraction pattern was similar to that obtained from the wildtype strain ftc2 expressing wildtype subunit 8. extraction using dissociation buffer released a small amount of the a-subunit (lane 2, a panel), but the vast majority of this subunit was not extracted (lane 1, a panel). the subunit 8 variant was not extracted into the supernatant (lane 2, y8 panel) and was still present in the pelleted fraction (lane 1, y8 panel). similar to what was observed with mitochondria from strain ftc2, extraction using 8 m urea released about 50% of the a-subunit (lane 4, a panel) with the remainder of the a-subunit still present in the pellet (lane 3, a panel). the subunit 8 variant was not extracted by 8 m urea (lane 4, y8 panel) and it was present in the pelleted fraction (lane 3, y8 panel) upon extraction. extraction using sodium carbonate released little a-subunit (lane 6, a panel) with the vast majority of this subunit remaining still in the pellet (lane 5, a panel). the subunit 8 variant was not extracted by sodium carbonate (lane 6, y8 panel) and all of the subunit 8 variant signal was detected in the pellet (lane 5, y8 panel). as expected, both the a-subunit and the subunit 8 variant were extracted by sds-urea (lane 8, a and y8 panels). while a small portion of a-subunit was still detected in the pelleted fraction (lane 7, a panel), little subunit 8 variant, if any, remained in the pellet (lane 7, y8 panel). discussion subunit 8 of the yeast mitochondrial-atp-synthase is unique and intriguing. while the majority of the mitochondrialatp-synthase subunits are encoded by the nuclear genome, subunit 8 is encoded by a mitochondrial gene. although it has been considered that subunit 8 plays various roles as part of the stator stalk, the bacterial enzyme naturally functions without the presence of subunit 8. the immediate questions therefore are, why subunit 8 is present in the eukaryotic system and what is the main role of this subunit in the enzyme complex. considerable progress has been made toward understanding of the molecular biology of subunit 8. however, many questions related to its function remain to be solved. the 48 amino acid protein has been found to have three distinct domains, an n-terminal domain, a c-terminal domain and a central hydrophobic domain (chd). while the c-terminal domain has been shown to be crucial for subunit 8 assembly into the complex (grasso et al. 1991), information concerning the role of the n-terminal and the chd is limited to the suggestion that the chd spans the inner mitochondrial membrane, thus indicating that subunit 8 has a transmembrane topology. detailed analyses of subunit 8 structure, topography and functionality, are still however lacking. the present study attempted to elucidate the significant interaction of the chd with the lipid bilayer of the inner mitochondrial membrane, as well as to analyze the need of subunit 8 to maintain a transmembrane topology. in principal, microbiol indones40 artika the membrane spanning domain of subunit 8 can be disrupted upon the introduction of charged amino acids, because the lipid bilayer may be unable to accommodate unshielded charged residues. in the previous study (artika 2007), six different flag tagged-subunit 8 variant genes, each having double-charged residues at a different position within the chd, were allotopically expressed in a yeast strain lacking endogenous subunit 8 (strain m31). among the functional variants, those with double-negatively-charged aspartate residues at the middle of the chd expressed in strain df68 was functionally the most defective. this was indicated by fig 1 detection of flag tagged-subunit 8 protein following extraction of mitochondrial proteins with various organic solvents and dissociation with sds plus pvdf-membrane separation. lane 1, pellet fraction from extraction with dissociation buffer; 2, supernatant fraction from extraction with dissociation buffer; 3, pellet fraction f r o m e x t r a c t i o n w i t h s o d i u m c a r b o n a t e 1 0 0 m m ( p h 1 1 . 5 ) ; 4 , supernatant fraction from extraction with sodium carbonate 100 mm (ph 11.5); 5, pellet fraction from extraction with 2 m urea; 6, supernatant fraction from extraction with 2 m urea; 7, pellet fraction from extraction with 4 m urea; 8, supernatant fraction from extraction with 4 m urea; 9, pellet fraction from extraction with 8 m urea; 10, supernatant fraction from extraction with 8 m urea. 1 2 3 4 5 6 7 8 9 10 fig 2 extracted mitochondrial protein of wildtype strain ftc2 with various solvents and dissociated with sds plus pvdf-membrane separation. lane 1, pellet fraction from extraction with dissociation buffer; 2, supernatant fraction from extraction with dissociation buffer; 3, pellet fraction from extraction with 4 m urea; 4, supernatant fraction from extraction with 4 m urea; 5, pellet fraction from extraction with 8 m urea; 6, supernatant fraction from extraction with 8 m urea; 7, pellet fraction from extraction with 100 mm sodium carbonate (ph 11.5); 8, supernatant fraction from extraction with 100 mm sodium carbonate (ph 11.5); 9, pellet fraction from extraction with sds/urea; 10, supernatant fraction from extraction with sds-urea. y8= membrane half < 14.6 kda proteins α= membrane half > 30 kda proteins 1 2 3 4 5 6 7 8 9 10 fig 3 extracted mitochondrial protein of strain df68 with various solvents and dissociated with sds plus pvdf-membrane separation. lane 1, pellet fraction from extraction with dissociation buffer; 2, supernatant fraction from extraction with dissociation buffer; 3, pellet fraction from extraction with 8 m urea; 4, supernatant fraction from extraction with 8 m urea; 5, pellet fraction from extraction with 100 mm sodium carbonate (ph 11.5); 6, supernatant fraction from extraction with 100 mm sodium carbonate (ph 11.5); 7, pellet fraction from extraction with sds/urea; 8, supernatant fraction from extraction with sds-urea. 1 2 3 4 5 6 7 8 the slowest growth rate of the strain df68 on ethanol medium. the generation time of strain df68 is much longer (11.1 ± 0.5) compared to that of strain ftc2 (6.9 ± 0.4). for this reason, strain df68 was employed in the present study to analyze the obligatory nature for subunit 8 of subunit 8 maintain its transmembrane topology. analysis of the membrane topology is based on the pattern of the protein after extraction using various solvents. as an integral protein, native subunit 8 is expected to be resistant to extraction using organic solvents. however, if the introduction of charged residues within the chd abolishes normal transmembrane topology, it may be expected that subunit 8 would then become sensitive to organic solvent extraction. to date, no other reports on the extraction of subunit 8 by the organic solvents used in the present study exist. direct comparison of data, therefore, can not be made. the results reported here suggest that the native subunit 8 is an integral protein since it is resistant to extraction by urea and sodium carbonate. this assumption is used as a basis for determining whether the subunit 8 variant adopts different membrane topology. the data presented here suggest that the subunit 8 variant of strain df68 has the same topology as the native subunit 8 protein of strain ftc2. this conclusion is based on the similarity of the protein extraction patterns of the two subunit 8 proteins. therefore, the subunit 8 variant fully retains its integral protein character. this observation further suggests that subunit 8 must maintain its transmembrane topology, because even the most functionally defective subunit 8 variant maintains the extraction pattern of the native subunit 8. the transmembrane topology may be critical for the proposed role of subunit 8 as part of the stator stalk of the mitochondrial atp-synthase-complex. both the native and variant subunit 8 proteins were resistant to extraction using organic solvents (sodium carbonate, urea), suggesting that they are integral proteins. in contrast, the peripheral f 1 a-subunit is extracted, although not completely removed, from the membrane bound-portion of the enzyme complex. as microbiol indones 41volume 3, 2009 expected, the integral subunit 8 is only released by extraction using sds-urea. the lipid bilayer is exquisitely sensitive to low concentration of amphipathic compounds, such as detergents that contain both polar and nonpolar groups (manoil and traxler 1995). in the present study, the sdsurea extraction serves as a positive control for the extraction procedures. in this case, the ability to detect subunit 8 protein from the supernatant fraction following extraction indicates that the extracted subunit 8 protein is successfully precipitated and then solubilised prior to sds-page analysis. according to manoil and traxler (1995), the introduction of a single charged residue into a membrane spanning sequence is, in most cases, unlikely to change transmembrane topology. such a substitution, however, might alter the positioning of the substituted sequence domain relative to the membrane. in df68, two charged residues are present in the spanning sequence of subunit 8, and the data suggest that those residues also do not change the subunit 8 transmembrane topology. papakonstantinou et al. (1996) suggested that a differential movement of the boundaries of the putative transmembrane domain might occur following the introduction of charged residues within the chd, such that the required hydrophobic character essential for membrane insertion, would be maintained. although data from the present studies have suggested that subunit 8 variant (df68) maintains a transmembrane topology, it is necessary to confirm this finding using a different approach. one such approach is cysteine labelling. it is possible to introduce a unique cysteine residue at either n-terminal or c-terminal end of subunit 8. the location of the cysteine residue relative to the inner mitochondrial membrane can be examined using thiol-specific reagents as probes. acknowledgements i am grateful to ausaid for sponsoring me during the course of this study. i would like to thank rodney j. devenish and phillip nagley of the department of biochemistry and molecular biology, monash university, victoria, australia, for guidance and provision of facilities. references artika im. 2006. allotopic expression of a gene encoding flag tagged-subunit 8 of yeast mitochondrial atp synthase. hayati j biosci 13:36-8. artika im. 2007. structural and functional analysis of flag taggedsubunit 8 of yeast saccharomyces cerevisiae mitochondrial atp synthase. microbiol indones 1:33-6. bradford m. 1976. a rapid sensitive method for the quantities of proteins utilising the principal of protein-dye binding. anal biochem 72:248-54. devenish rj, papakonstantinou t, galanis m, law rhp, linnane aw, nagley p. 1992. structure/function analysis of yeast mitochondrial atp synthase subunit 8. ann ny acad sci 671:403-14. fronzes r, weimann t, vaillier j, velours j, brethes d. 2006. the peripheral stalk participates in the yeast atp synthase dimerization independently of e and g subunits. biochemistry 45:6715-23. gearing dp, mcmullen gl, nagley p. 1985. chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus. biochem int 10:907-15. grasso dg, nero d, law rhp, devenish rj, nagley p. 1991. the cterminal positively charged region of subunit 8 of yeast mitochondrial atp synthase is required for efficient assembly of this subunit into the membrane f 0 sector. eur j biochem 119:2039 . laemmli uk. 1970. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227:680-5. lang bf, burger g, doxiadis i, thomas dy, bandlow w, kaudewitz f. 1977. a simple method for the large-scale preparation of mitochondria from microorganism. anal biochem 77:110-21. macreadie ig, novitski ce, maxwell rj, john u, ooi b, mcmullen g, lukins hb, linnane aw, nagley p. 1983. biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial atpase subunit 8 in saccharomyces cerevisiae. nucl acids res 11:4435-51. manoil c, traxler b. 1995. membrane protein assembly: genetic, evolutionary and medical perspectives. annu rev genet 29:1315 0 . nagley p, farrell lb, gearing dp, nero d, meltzer s, devenish rj. 1988. assembly of functional proton-translocating atpase complex in yeast mitochondria with cytoplasmically synthesised subunit 8, a polypeptide normally encoded within the organelle. proc natl acad sci usa 85:2091-95. papakonstantinou t, law rhp, nesbitt ws, nagley p, devenish rj. 1996. molecular genetic analysis of the central hydrophobic domain of subunit 8 of yeast mitochondrial atp synthase. curr genet 30:12-18. roucou x, artika, im, devenish, rj, nagley p. 1999. bioenergetic and structural consequences of allotropic expression of subunit 8 of yeast mitochondrial atp synthase. the hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex. eur j biochem 261:444-51. stephens an, khan ma, roucou x, nagley p, devenish rj. 2003. the molecular neighborhood of subunit 8 of yeast mitochondrial f 1 f 0 -atp synthase probed by cysteine scanning mutagenesis and chemical modification. j biol chem 278:17867-75. stephens an, roucou x, artika im, devenish rj, nagley p. 2000. topology and proximity relationships of yeast mitochondrial atp synthase subunit 8 determined by unique introduced cysteine residues. eur j biochem 267:6443-51. thomas d, bron p, weimann t, dautant a, giraud m, paumard p, salin b, cavalier a, velours j, brèthes d. 2008. supramolecular organization of the yeast f 1 f 0 -atp synthase. biol cell 100:5916 0 1 . cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 14, number 3, september 2020 antibiotic sensitivity pattern of bacterial isolates among diabetic outpatients with urinary tract infection in pontianak detection of liberibacter asiaticus causing citrus vein phloem degeneration from siam citrus leaves (citrus nobilis var. microcarpa) in singkawang city plantation, pontianak, west kalimantan genotypic characterization of rhizopus species from tempeh and usar: traditional inoculum of tempeh in indonesi effect of hydrocarbon-polluted seawater on the cell density of microalgae scenedesmus vacuolatus shihira & krauss detection of salmonella sp. and escherichia coli on chicken meat at tamiang layang market mardhia, mahyarudin, and abror irsan rahmawati, iliana, agus rachmat, latiffah zakaria, and mukarlina tati barus, jason wiranata sanjaya, anastasia tatik hartanti, adi yulandi, vivitri dewi prasasty, and david tandjung clara alverina santoso, noverita dian takarina, hanies ambarsari, nining betawati prihantini, and sitaresmi akhmad rizaldi and engki zelpina 89 95 101 108 117 page 1 05. sjatha.cdr vol.13, no.1, march 2019, p 36-42 doi: 10.5454/mi.13.1.5 expression of recombinant non structural 1 protein of dengue virus serotype-2 in mammalian cell line 1* 2 1 fithriyah sjatha , oktivia chandra mustika , angky budianti , and tjahjani 1 mirawati sudiro 1 department of microbiology, medical faculty, universitas indonesia, jl. pegangsaan timur no. 16, central jakarta 10320, indonesia; 2 master programme of biomedical science, faculty of medicine, universitas indonesia, indonesia. dengue infection is a global infectious disease with almost 100 million cases occurs annually in over more than 100 endemic countries. dengue virus (denv), the causative agent of dengue infection, is an 11 kbp rna positive-strand virus which encode 3 structural and 7 nonstructural proteins within its genome. non-structural 1 (ns1) protein of denv is expressed in the earlier stage of infection and having a pathogenic role in disease severity. ns1 gene of denv serotype-2 indonesian strain was amplified through pcr method using specifically designated primers. ns1 amplicon was then cloned into pumvc4.a and pcdna3.1 mammalian expression vector which confirmed through colony pcr and sequencing method. recombinant puns1 and pcns1 plasmids were transfected into cho-k1 mammalian cell line with lipid-based method. recombinant ns1 protein expression was analyzed through immunostaining using dengue patient sera and rapid ns1 detection kit. recombinant puns1 and pcns1 plasmids were successfully constructed and recombinant ns1 protein was expressed in cho-k1 mammalian cell line and shown to be reactive against dengue patient sera. our recombinant ns1 protein also tend to be released outside the transfected cho-k1 cells as detected in a rapid ns1 detection kit. recombinant dengue ns1 protein was expressed in a mammalian cell lines in both intra and extracellularly and shown to be immunogenic. key words: cho-k1 cell, dengue, ns1 infeksi dengue merupakan salah satu infeksi global dengan jumlah kasus mencapai 100 juta pert tahunnya di 100 negara endemis dengue. virus dengue (denv) selaku agen penyebab infeksi dengue merupakan virus rna untai positif dengan genom berukuran 11 kb yang mengkode 3 protein struktural dan 7 protein non-struktural. protein non-struktural 1 (ns1) virus dengue diekspresikan di fase awal infeksi dan memiliki peran penting dalam patogenisitas infeksi terkait dengan tingkat keparahan penyakit. gen ns1 dari virus dengue serotipe 2 strain indonesia diamplifikasi melalui teknik pcr menggunakan primer spesifik. gen ns1 kemudian di klona kedalam plasmid ekspresi sel mamalia, pumvc4.a dan pcdna3.1. konfimasi klona dilakukan melalui koloni pcr dan sekuensing. rekombinan puns1 dan pcns1 kemudian ditransfeksikan kedalam sel cho-k1 menggunakan penghantar lipid dan ekspresi protein ns1 rekombinan dianalisis melalui pewarnaan imunologis menggunakan serum pasien terinfeksi dengue dan kit komersial deteksi protein ns1 virus dengue. plasmid puns1 dan pcns1 rekombinan telah berhasil dikonstruksi dan protein rekombinan ns1 berhasil diekspresikan pada sistem sel mamalia cho-k1. protein rekombinan ns1 terbukti reaktif terhadap serum pasien dan dideteksi pada supernatan sel tertransfeksi yang mengindikasikan kemampuan protein rekombinan ns1 untuk dilepaskan keluar sel. protein rekombinan ns1 dari virus dengue serotipe 2 telah berhasil diekspresikan pada sel mamalia baik intra maupun ekstrasel dan terbukti imunogenik. kata kunci: dengue, ns1, sel cho-k1 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-3160491; fax: +6221-3100810; email: titi_sjatha@yahoo.com spread all over the indonesia archipelago with four viral serotypes that were found to be circulated (suwandono et al. 2006). based on the ministry of health of indonesia, dengue infection happened every year with five years outbreak periodically. during the 2007 outbreak, more than 127.687 and 1.296 deaths were reported, make it one of the largest outbreak ever happen in indonesia. until now, there is no antiviral treatment nor a globally approved vaccine available against denv infection. denv is an arthropod-borne virus which transmitted through aedes aegypti and aedes dengue fever and dengue hemorrhagic fever are infectious diseases caused by dengue virus (denv) infection which globally spread in almost 100 endemic countries all over the world (who 2011). denv is an arthropord borne virus transmitted by aedes aegypti mosquito mainly in tropical and subtropical countries. indonesia itself is one of an endemic countries with a long history of dengue infection. since it was reported in 1968 in jakarta and surabaya, dengue virus is widely volume 13, 2019 microbiol indones 37 albopictus mosquito as a viral vector. dengue virus is a member of flavivirus family with 11 kb genome size, under one open reading frame which encode three structural (c, prm, and e) proteins and seven nonstructural (ns1, 2a, 2b, 3, 4a, 4b, 5) proteins. based on the e (envelope) gene variation, dengue virus is divided into four distinct serotypes which antigenically different: denv1-4 (guzman et al. 2010 ; back ta 2013) three structural proteins (c, prm and e) were proteins that composed viral particle and having major antigenic site in the immune response. among seven non-structural proteins of denv, non-structural 1 (ns1) protein is a conserved glycoprotein with 46-55 kda in size. the immature form of this protein was found as a monomer in reticulum endoplasmic, meanwhile the mature protein, with 352 amino acids residue, can be found extracellularly soluble (sns1) or membrane bound (mns1) in infected cells (gutsche et al. 2011). ns1 protein can be detected in blood from day-1 of onset until the day-9 with concentration 0.01-1 50 μg ml in the range which make this protein a suitable marker in early diagnosis of dengue infection (alcon et al. 2002). moreover, ns1 protein has a role in viral replication and allegedly related to dengue infection pathogenesis. the ns1 protein is known can bind with heparan sulfate of epithelial cells, fibroblast, hepatocytes and endothelial cells which lead to plasma leakage through complement activation. on the other hand, an antibody against ns1 was found to be crossreactive with thrombocytes and endothelial cells. in summary, all of these phenomena can lead the increase in disease severity (amorim et al. 2014). the recombinant ns1 protein is widely developed for several purposes especially in developing diagnostic tools for dengue infection. recombinant ns1 protein expressed in e.coli or yeast expression system were found to be antigenic and immunogenic in triggering both humoral and cellular immune response in the animal model (dewi et al. 2012; tripathi and shrivastava 2018). in this study, we develop a recombinant plasmid expressing ns1 protein of denv2 originated from indonesia, puns1 and pcns1 which proved to express the recombinant ns1 protein in both intracellular and extracellularly. our recombinant ns1 protein is also recognized by dengue patient sera indicating its promising immunogenicity to be further used in developing dengue diagnostic kits. materials and methods ns1 gene, plasmids, bacterial strain, mammalian cell, and patient sera. ns1 gene of d e n v 2 w a s o r i g i n a t e d f r o m p g e x n s 9 . 1 recombinant plasmid in a previous study (dewi, 2012). original pcdna3.1 (themo) and pumvc4.a (aldevron) mammalian expression plasmid vector were propagated in e.coli dh5α (thermo) which -1 cultured in lb medium containing 100 mg ml of -1 ampicillin for pcdna3.1 or 30 mg ml of kanamycin for pumvc4.a. cho-k1 mammalian cell from kobe university, japan used for plasmid transfection, was cultured in mem media containing 10% fbs, 1% o neaa and 1% antibiotic-antimycotic at 37 c and 5% co respectively. dengue patient sera used for 2 immunostaining was derived from dengue community study with ethical approval from research ethical committee faculty of medicine, universitas indonesia no. 71/pt02.fk/etik/2009. pcr and cloning. ns1 gene was amplified through pcr method using designated primers for c l o n e d t o p c d n a 3 . 1 ; f 2 3 2 9 s b a m ( 5 ' cgcgaggatcctggataggaatgaattcacg c-3') and r ns-1-350csal (5'tccgctgtcga ctcaggctgtgaccaaggagtt-3'). meanwhile for pumvc4.a amplification, bamh1 restriction site in forward primer was changed into pst1 restriction site respectively. both primer pairs were used to amplify ns1 gene from pgexns1.9 template using primestar master mix (takara) reagent. amplified ns1 gene was then ligated into pcdna3.1 or pumvc4.a plasmid and transformed into dh5α e.coli using heat shock method. schematic insertion of ns1 gene into pcdna3.1 and pumvc4.a were depicted in figure 2. recombinant puns1 and pcns1 plasmid were confirmed for its ns1 insertion pcr method using insert primer pair in gotaq master mix (promega) reagent. insert sequence and orientation were then confirmed through the sequencing method by eijkman institute. recombinant puns1 and pcns1 transfection. recombinant puns1 and pcns1 plasmids were isolated by midi plasmid purification kit (thermo). one microgram of recombinant puns1 and pcns1 plasmids were transfected into cho-k1 cells using lipofectamine plus transfection reagent (thermo) according to manufacture instruction. native pcdna3.1 and pumvc4.a were also transfected as a negative control. briefly, 500 ng of each plasmid were transfected into 80% confluent of cho-k1 cells at m6 well plates in the presence of lipofectamine reagents for 4 hours at rt. the transfection mixture was then o replaced by cell media followed by incubation at 37 c and 5% co until further analysis. 2 immunostaining. twenty four hours after transfection, the supernatant of cho-k1 culture were discarded and cells were washed three times with excess pbs and air dried. cells were then fixated with o chilled absolute ethanol for thirty minutes at 4 c. after fixation, 1:1000 diluted dengue patient sera in pbs were applied to transfected cells and incubated for 1 h at rt. followed by 1:5000 secondary rabbit anti human igg labelled with hrp (sigma) was then added followed by another incubation for 1 h a rt. metal dab peroxidase substrate (thermo) was then applied into the cells and positively stained cell will appear in brown color under microscope observation. ns1 detection using commercial kit. seventy two hours supernatants of transfected cho-k1 cells were directly dropped into ns1 detection kit (bioline) followed by incubation at rt for 15 minutes. red band color will appear in the sample line as a positive result. results recombinant puns1 and pcns1 construction. recombinant puns1 and pcns1 were successfully achieved with the ligation-transformation rate was 80% respectively. as shown in figure 1, all single colonies of suspected recombinant puns1 and pcns1 were having ns1 gene insertion at 1000 bp size based on colony pcr analysis, thus named as puns1.1, puns1.2 and puns1.3 for pumvc4.a backbone and pcns1.20, pcns1.21 and pcns1.22 for pcdna3.1 backbone. three positive plasmids from each construct confirmed on colony pcr were further analyzed through sequencing for its nucleotide and orientation analysis as predicted in figure 2. from the sequencing result, there is no mutation in the nucleotide sequence from the ns1 gene (data not shown). immunostaining. immunostaining was performed to confirm the ability of the recombinant puns1 and pcns1 to express the recombinant ns1 protein 24 h after transfection. in immunostaining, dengue patients sera were used as primary antibody in the presence of anti-ns1 igg which confirmed by commercial dengue detection kit. as depicted in figure 3, all recombinant pcns1 and puns1 constructs (pcns1.20-22 and puns1.1-3) give red colored stained cells indicating the expression of the recombinant ns1 protein. in this experiment, pcdna3.1 and pumvc4.a backbone plasmids were also transfected into chok1 cells and show no red colored cell indicating the absence of recombinant ns1 protein and there is no non-specific binding occurs in the use of dengue patients sera. for positive transfection and staining control, a recombinant pumd2 (pumvc4.a plasmid encoding prm and e gene of denv2, (putri et al. 2015)) was used and shown similar red-colored cell indicating the transfection and staining process iscorrectly done. extracellular ns1 detection. extracellular recombinant ns1 was detected using a commercial ns1 detection kit which commonly used in dengue diagnosis (sd bioline dengue duo). briefly, supernatant from 72 hours post transfected cho-k1 cells were dropped into the detection kit and leave it at rt for 15 minutes. as depicted in figure 4, all recombinant puns1 constructs (puns1.1 – 3) and pcns1.21 gives positive red band indicating the presence of recombinant ns1 protein in the supernatant. discussion non-structural 1 (ns1) protein is a conserved glycoprotein in denv which expressed soon after viral particle infecting a cell. this glycoprotein can be expressed in soluble (sns1) and membrane-bound (mns1) form which both of these forms can induce humoral and cellular immune response through the different pathways (akey et al. 2014; rivino et al. 2013; avirutnan et al. 2007). since ns1 protein can be detected in acute phase of denv infection within various concentration level, ns1 protein is widely used as dengue diagnostic approach in detecting denv infection by using many platform. rapid test or elisa based methods are the two common methods which have been developed for this detection system (alcon et al. 2002). to develop such diagnostic method, the ns1 antigen is needed and e.coli and yeast expression system are the most common platform which has been used to obtain recombinant ns1 protein for further use in developing dengue diagnostic (huang et al. 2001; bragança et al. 2015; zhou et al. 2006). moreover, since ns1 protein is a non-structural protein which doesn't compose viral particle, ns1 protein is now also used as one approach to develop novel dengue vaccine. ns1 protein not posses the antibody-dependent enhancement (ade) activity as structural protein has as one of the advantages for using this protein which considers being safer than using structural protein (amorim et al. 2014). for our construction, we use the ns1 gene derived from denv2 of cosmopolitan genotype originated 38 sjatha et al. microbiol indones fig 1 colony pcr screening. c20-c22 : recombinant pcns1, k+ : positive control for ns1 insert cloned to pcdna3.1, m : 1 kb dna ladder, u1-3 : recombinant puns1, k+ : positive control for ns1 insert cloned to pumvc4.a. the white box showed positive ns1 target band at approximately 1 kb size. fig 2 schematic figure of recombinant puns1 and pcns1. ns1 gene of denv2 has inserted in vectors mcs regio between pst-i and sal-i restriction sites for pumvc4.a vector; and bam-hi and sal-i restriction sites for pcdna3.1. fig 3 immunostaining of transfected cho-k1 cells. cells were transfected with: 1. pcdna3.1; 2. pumvc4.a; 3. pumd2; 4 – 6. puns1.1 – 3; 7 –9. pcns1.1 – 3. 1 2 3 4 5 6 7 8 9 volume 13, 2019 microbiol indones 39 fig 4 extracellular recombinant ns1 detection. 1. puns1.1; 2. puns1.2; 3. puns1.3; 4. pcns1.20; 5. pcns1.21; 6. pcns1.22. the positive result is shown as a red line on t. 1 2 3 4 5 6 from dengue patient in indonesia. the cosmopolitan the genotype of denv2 is the common circulated genotype of denv2 in indonesia and the south east asia region (putri et al. 2015). although ns1 protein relatively a conserve glycoprotein, several antigenic properties which unique related to certain denv genotype is found so it is more suitable to use any gene or viral sources derived from circulating viral in a certain area for a better result (amorim et al. 2014; chen et al. 2018). in this study, we develop a recombinant mammalian expression system plasmid encode ns1 gene of denv2 to express recombinant ns1 protein. we use pcdna3.1 and pumvc4.a mammalian expression system with the consideration that pcdna3.1 is a high copy number the of plasmid which can amplify the number of genes. moreover, with the additional cmv enhancer in the upstream of t7 promoter tend to increase the expression level of recombinant antigen. it can be seen by a high level of recombinant ns1 expression which detected by commercial ns1 agent kit. it shown as positive band of ns1 antigen which appear from the pcns1.21 construct compare to the other pumvc4.a based construct indicating its high expression level although the initial and termination condition of transfection were similar for all recombinant constructs. another expression system that we use in this study is the pumvc4.a vector which also a mammalian cell line expression system with kanamycin resistance gene as a selection marker. a kanamycin is an antibiotic which less usable for human compare to ampicillin antibiotic which is safer to be applied in human research. from our result, recombinant puns1.1-3 were able to express the recombinant ns1 antigen but we assume the amount of the recombinant protein are less than pcns1.21 construct since there is only weak positive red band appear when testing using commercial ns1 detection kit. the negative band was obtained from pcns1.20 and pcns.22 constructs which we assume that less ns1 antigen was released outside transfected cells by these two constructs although the presence of ns1 antigen was positive at 24 h post transfection as proven by immunostaining analysis. intracellular ns1 antigen for pcns1.20 and pcns1.22 at 72 h post transfection need to be further analyzed. originally, ns1 protein can be found in two form of soluble nor membrane bound protein which in this study both form of proteins were detected as immunostaining indicate the expression of intracellular ns1 antigen (membrane-bound located will need further analysis) and ns1 in the supernatan of transfection cell. our positive immunostaining result show that our recombinant ns1 protein is recognized by dengue patients sera indicating the immunogenicity and antigenicity of our recombinant protein. among dengue non structural protein, ns1 protein is the most antigenic and immunogenic compare to the other ns protein as several studies shows that there are several b-cell epitope and t-cell epitope were located in ns1 protein which relatively conserved compare to the epitopes which located in the envelope (e) or membrane (m) protein of dengue. our ns1 recombinant protein is proven to have to conserve bcell nor t-cell epitopes when amino acid sequences were analyzed (dewi et al. 2012). immunogenicity and 40 sjatha et al. microbiol indones antigenicity of ns1 protein also one reason to use ns1 protein in developing novel dengue vaccine against denv infection, highlighting it needs to modify in order to reduce the risk of cross-reactivity of anti ns1 antibody against endothelial cell that can lead to plasma leakage. in this study, we choose mammalian cell expression system to obtain correct folding, correct protein structure or protein modification of our recombinant ns1. however, the mass production of mammalian cell line is less desirable for it highly cost production. we believe by using this expression system the antigenicity and immunogenicity of the ns1 protein can be maintained to achieve better antigenicity and immunogenicity properties. in this study, we use cho-k1 cell line derived from chinese hamster ovary wich commonly used in the recombinant protein expression studies and well known for its stability and high yield of protein production. cho-k1 cell is easy to nurture and also widely used for mass production in recombinant protein technology (dahodwala and sharfstein 2017; genzel 2015; chen th et al. 2018). from this study result, our system proved that the ns1 recombinant antigen is transported outside the transfected cells as the antigen is detected with commercial rapid ns1 antigen kit in the supernatant of transfected cells. this extracellular transport is one advantage as our recombinant ns1 protein will be much easier to be isolated and purified for further used compare with procaryotic expression systems such as e.coli which tend to make inclusion bodies to compensate the high expression pressure. the presence of inclusion bodies in procaryotic expression system is widely known as one of the obstacles in protein isolation and purification process which commonly resulting high protein loss and decreasing protein yields (wingfield et al. 2014; hoffmann et al. 2017). in conclusion, in this study we successfully obtain recombinant puns1 and pcns1 plasmid construct which able to express recombinant ns1 protein in cho-k1 mammalian cell line. and our recombinant ns1 protein is reactive against dengue patients sera, indicating it is immunogenic and antigenic to be further used in developing dengue diagnostic kit or dengue vaccines. acknowledgment this research was funded by hibah penelitian dasar unggulan perguruan tinggi by the ministry of research and higher education, republic of indonesia 2018/2019. references akey dl, brown wc, dutta s, konwerski j, jose j, jurkiw tj, delproposto j, ogata cm, skiniotis g, kuhn rj, smith jl. 2014. flavivirus ns1 structures reveal surfaces for 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insoluble (inclusion body) proteins from escherichia coli. curr protoc protein sci. 78:6.5.16.5.30. doi: 10.1002/0471140864.ps0605s78. zhou jm, tang yx, fang dy, zhou jj, liang y, guo hy, jiang lf. 2006. secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein ns1 in pichia pastoris. virus genes. 33(1):27-32. doi: 10.1007/s11262-005-0036-6. 42 sjatha et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 119-124 (nyoman mantik astawan).pmd the widespread outbreaks of avian influenza (ai) in south-east asia in the recent years have led to the death of millions of domesticated birds and millions others have to be sacrificed in an effort to eradicate the disease (swayne and halvorson 2003; stegeman and bouma 2004). during this outbreak, many affected countries suffer a great deal of economic losses brought out by the collapse of their poultry industries (perkins and swayne 2002; perkins and swayne 2003; lewis 2006). more importantly, the disease also affects human causing a great concern among health authorities in the world. the availabilities of accurate, simple, safe, and fast diagnostic methods are important for an effort to prevent and to control a future outbreak of ai in both animals and man. most diagnostic methods developed in the recent years still require expensive facilities and reagents are slow to perform, lack of sensitivity and specificity, unsafe to perform, and unable to determine the virus subtype directly (gough 2004). avian influenza viruses (aivs) are a group of viruses with great genetic and antigenic diversities in nature. on the basis the antigenic characteristics of their two surface glycoproteins, haemagglitinin (ha) and neuraminidase (na), aivs are grouped into many subtypes. as many as 16 ha subtypes that can combine with 9 na subtypes have been identified (foucier et al. 2005). such antigenic diversities have often caused a great difficulty in establishing an appropriate test for an accurate detection of aiv subtypes (fouchier et al. 2005; kida dan sakoda 2006). mabs which react only with a single epitope on an antigenic structure have been widely used to detect the viral antigen in the infected hosts and also to differentiate closely related viruses (zheng et al. 2001; vareckova et al. 2002; ohnishi et al. 2005). in human influenza virus, for instance, the use of mabs microbiology indonesia, december 2007, p 119-124 volume 1, number 3 issn 1978-3477 immunological detection of avian influenza virus in infected ducks by monoclonal antibodies against aiv-h5n1 nyoman mantik astawa1*, ida bagus oka winaya1, luh putu agustini2, and nining hartaningsih2 1faculty of veterinary medicine, universitas udayana, jalan pb sudirman, denpasar 80232, indonesia 2biotechnology laboratory, disease investigation centre regional vi denpasar, jalan raya sesetan no. 266, denpasar 20223, indonesia in order to establish a detection method for avian influenza virus (aiv) infection in ducks, monoclonal antibodies (mabs) against the virus were produced. the virus used for the production of the monoclonal antibodies was aiv-h5n1 of indonesian origin. immortal mouse myeloma were fused with the lymphocytes derived from the spleen of mice immunized with the virus. the mabs were tested for their specificity by enzyme linked immunosorbent assay (elisa) and western blotting using formaldehyde inactivated virus and normal allantoic fluid as a negative control. twelve mabs which were specific against aiv were isolated and 8 of them were used for detecting of aiv antigen in duck’s tissues. aiv antigen was detected in paraffin embedded tissues of aiv-infected ducks by immunohistochemistry using mabs. aiv antigen was not detected in ducks, which were confirmed to be aiv negative. in the infected ducks, high intensity of aiv infection was detected in proventricle gland and small intestine. the aiv antigen with a lesser intensity was also detected in lungs, spleen, and bursa of fabricius, but hardly detected in muscle, brain, and several other issues. this study shows a clear evidence that mabs produced in this study are applicable for use in immunological detection of aiv in infected duck tissues. key words: avian influenza, h5n1, monoclonal antibodies, ducks, virus immunohistochemistry _____________________________________________ ________________________ * corresponding author, phone: +62-361-223791, fax: +62361-701808, e-mail: nyomanmantikastawa@yahoo.com against the ha protein of the virus is reported to have 100% sensitivity and 99.1% specificity in determining the ha subtype of the virus (vareckova et al. 2002). as in human influenza viruses, mabs against aiv is very likely to have a similar degree of sensitivity and specificity when used in detecting of aiv antigen in the infected hosts including in determining the virus subtype. among many different hosts infected by aiv, aquatic birds including ducks are suggested to play an important role in the epidemiology of ai. aquatic birds can serve as an important reservoir for low pathogenic avian influenza (lpai) viruses (alexander 2000). evidences have shown that these lpai viruses can mutate easily into highly pathogenic avian influenza (hpai) virus in water fowls, especially if they carry aiv with h5 or h7 subtype (banks and plowright 2003). in addition, there are also evidences that many outbreaks of ai in susceptible commercial flocks originate from aiv-carrier waterfowls brought into live bird markets (bulaga et al. 2003; swayne and halvorson 2003; stegeman and bouma 2004). in the carrier birds, the virus replicates mainly in the intestine of infected waterfowls, usually without showing a clear symptom. a large quantities of virus is usually excreted via feces into water, perpetuating the natural cycle of aiv infection (stegeman and bouma 2004). the detection of aiv infection in waterfowls such as ducks is therefore important both as confirmative diagnosis of clinically affected ducks and for monitoring of ducks subclinically carrying the virus. in our laboratory, several ducks with a severe clinical disease have been confirmed to be due to aiv infection (data not shown). the ducks were confirmed as ai positives by isolation of the virus in embryonated chicken eggs, identification of the virus by haemagglutination/ haemagglutination inhibition (ha/hi) test, and detection of viral nucleic acid by reverse transcriptase-polymerase chain reaction (rt-pcr). the availability of mabs against 120 astawa et al. microbiol indones of aiv-h5n1 is very likely to provide a relatively much simpler, quicker, cheaper, and safer diagnostic methods for the detection of aiv antigen in ducks. we have currently been able to produce mabs against aiv-h5n1 of indonesian isolate and the applicability of those mabs for detecting aiv antigen in duck tissues was examined. materials and methods cells. myeloma cells (p3-ns1/1-ag4.1), used for the preparation of hybridomas were obtained from murdoch university, australia. the cells were grown in dubelco’s modified essential medium with 10% newborn calf serum (nbcs) and antibiotics penicillin, 200 iu ml-1, streptomycin 200 µg ml -1. virus. formaldehyde inactivated aiv-h5n1 used in this study was an indonesian isolate. the virus was isolated in 2005 from chicken with a severe clinical disease and the virus isolate was then designated as a/ck/bali/2005. the virus was propagated in 10 days-old chicken embryonated eggs and harvested from allantoic fluids. the titer of the virus was determined by ha test (who 2002). the virus has been confirmed as h5n1 subtypes and pcr using h5 and n1 primers (data not shown). production of monoclonal antibodies. mabs against the indonesian isolate of aiv-h5n1 were produced by methods similar to those described by ohnishi et al. (2005). six to seven week-old female balb/c mice were immunized with 0.2 ml (equivalent with approximately 27 ha units) virus emulsified in freund’s complete adjuvant. fourteen and 28 days after the first immunization the mice were respectively immunized with the same antigen but emulsified in freund’s incomplete adjuvant. fourteen, 15, and 16 days after the last immunization, the mice were boosted with the same antigen but without adjuvant. the mice were then sacrificed by cervical dislocation. the spleen was removed and used for the preparation of hybridomas. as many as 2 x 107 immortal mouse myeloma cells prepared as described above were fused with 10 8 lymphocytes derived from the spleen of mice immunized with aiv-h5n1. the fusion of the two types of cells was carried out using polyethylene glycol (peg) 45% (sigma co, usa) to produce hybridomas. the hybridomas were then screened by indirect elisa (campbell 1991) for the anti-aiv antibodies using formaldehyde inactivated aiv-h5n1 as antigen and normal allantoic fluid as negative control. the hybridomas producing mabs reacted specifically with the virus were cloned by limiting dilution as described by mckearn (1980) and were then used in the production of mabs against the aiv-h5n1. titration of mabs. the titer of mabs in hybridomas’ supernatant fluid was determined by elisa according to the procedure as described by campbell (1991). elisa microtitration plate was coated overnight with formaldehyde inactivated virus diluted in carbonate-bicarbonate coating buffer (15 mm na 2 co 3, 35 mm nahco 3 ph.9.6). each plate well was coated with 100 ml antigen containing of approximately 1 ha unit of the virus. after three times washes with 0.05% tween-20 in phosphate buffered saline ph 7.2 (pbst), 100 ml blocking buffer (5% skim milk in pbst) was added and incubated for another 1 h at 37 °c. a serial two-fold dilution of mabs was prepared and 100 ml mab sample from each dilution were added to each well. the plate was incubated for 1 h at 37 °c. after three times washes as above, 100 ml anti-mouse igg-conjugated with horseradish peroxidase (hrp) (bio-rad, usa) diluted 1:2 000 in pbs-t was added to each well. the microplate was then incubated for 1 h 37 °c and washed three times as above. one hundred µl of substrate solution (1 mm 2 2’-azinodi 3ethylbenzthyazolin-6-sulfonic azide in 0.05% na citrate, 0.15% na phosphate, and 0.01% h 2 o 2 was added to each well. after incubation for 15 min at room temperature, the absorbance of the substrate solution in each well was read by multiscan spectrophotometer using 405 nm filter. titer of mabs was determined as the antilog the highest dilution giving an absorbance reading of approximately 50% of its optimal reading. determination of mab isotypes. the immunoglobulin (ig) class and subclass of the mabs were determined by indirect elisa using rabbit antimouse subtyping isotyper kits (bio-rad laboratory, usa) according to the procedures described by manufacturer. elisa microtitration plate was firstly coated overnight with formaldehyde inactivated aivh5n1 as described above. into each well, 100 µl mab diluted 1:10 in pbst were added and incubated for 1 h at 37 °c. following three times washes with pbs, rabbit anti-mouse ig isotyper from the kit was added to the wells and incubated as above. after 3 times washes, 100 ml affinity purified goat anti-rabbit igg conjugated with hrp (bio-rad, usa, diluted 1:1 000 in pbst) was added and incubated at 37 °c for 1 h. the plate was again washed as above and 100 ml substrate solution (1 mm 2.2‘-azinodi 3-ethylbenzthyazoline-6-sulfonic acid in 0.005 na citrate, 0.15 na phosphate, and 0.01% h 2 o 2 ) was added. the absorbance of the substrate solution was read in multiscan spectrophotometer with a 405 nm filter. western blotting. western blotting assay was carried out according to procedure described by zheng et al. (2001). formaldehyde inactivated aiv-h5n1 were diluted in an equal volume of sample loading buffer (1.3% sds, 5% mercaptoethanol, 0.0625 m tris-hcl ph 6.8, 10% glycerol, 0.001% bromophenol blue). the viral proteins were analysed by sodium dodecyl sulfate-polyacrylamid gel electrophoresis (sds-page) using 3% loading gel and 10% separating gel. the proteins in the gel was then transferred onto nitrocellulose membrane (bio-rad, usa). following 1 h blocking at room temperature with 3% skim milk in trisbuffered saline (tbs/100 mm tris ph 7.4 adjusted with 1 n hcl) and a brief washing with tbs, nitrocellulose membrane was then cut into 0.5 cm strips. each strip was then soaked with hybridomas’s supernatant fluid containing mabs and incubated 24 h at room temperature. following 3 times washes with tbs, anti-mouse igg coupled with biotin (bio-rad usa, diluted 1:1 000 in tbs) was the added to the membrane. after 3 times washes with tbs, streptavidin-alkaline phosphatase (promega, diluted 1:500 in tbs) was then added to the membrane. the membrane was washed 3 times as above and the aiv protein in the membrane which reacted with mabs was visualized by adding 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (bcip/nbt) substrate kit (bio-rad, usa). volume 1, 2007 microbiol indones 121 table 1 characteristics of monoclonal antibodies prepared against avian influenza virus subtype h5n1 of indonesian origin mabs isotypes elisa western blotting ihc aiv-h5n1 aiv-h5n1 nana ag8 bc12 cc5 cg1 dd9 df11 ea11 e e 8 igg1 igg1 igm igg3 igg3 igg1 igg1 igg2a 27 28 27 28 26 29 28 27 7 6 7 6 diffuse 7 6 diffuse 76/58 76/58 7 6 +++ nd nd +++ +++ nd +++ nd na: normal alantoic fluid, ihc: immunhistochemistry, -: negative, +++: strong positive, nd: not ditermined. table 2 detection of avian influenza virus antigen in infected ducks by immunostaining staining using monoclonal antibodies ducks infected duck iiinfected ducks i organs df11ag8 ag8ea11df11 e e 8 ea11 e e 8 proventricle small intestine lung spleen muscle kidney liver brain ++: moderate positive, +++: strong positive, +: weak positive, -: negative. +++ +++ ++ ++ + + + +++ +++ ++ ++ + + + +++ +++ ++ ++ + + + +++ +++ ++ ++ + + + +++ +++ ++ ++ + + + +++ +++ ++ ++ + + + detection of aiv antigen in duck tissues by immunoperoxidase staining. the ducks used in this study were cordially provided by co-assistant students in the faculty of veterinary medicine, udayana university, denpasar bali. all ducks had been tested for aiv infection by the isolation of the virus in chicken embryonated eggs and identification of the virus by haemagglutination/ haemagglutination inhibition (ha/hi) test. the results of the test were then further confirmed by reverse trancriptasepolymerase chain reaction (rt-pcr) using primers specific to h5 and n1 subtypes. two ducks that were confirmed positive to aiv-h5n1 infection and two ducks confirmed aiv negative (data not shown) were used in this study. several organs such as brain, proventricle, small intestine, liver, lung, bursa of fabricius, spleen, and kidney derived from the ducks were preserved and fixed with 10% buffered formaldehyde. paraffin embedded organs and thin sections of the organs were prepared by standard methods. immunoperoxidase staining was then carried out according to the methods similar to those described by ohnishi et al. (2005). thin sections of tissues on microscope slides were de-paraffinized twice in xylol and twice with ethanol absolute. the tissue section was washed twice with pbs and treated with 0.05% trypsin for 1 min at 37 °c. the endogenous peroxidase of the tissues was then inactivated by treatment with 3% h 2 o 2 in pbs for 20 min at room temperature. after blocking with 50% normal goat serum in pbs, mabs against aiv-h5n1 was added onto the tissue section and incubated for 18 h at room temperature. the bound mabs were detected by biotinylated goat anti-mouse igg (biodesign international) diluted 1:500 in pbs containing 10% normal goat serum and streptavidin-horse radish peroxidase (sigma co, usa) diluted 1:100 in pbs. a proper washing procedure was carried out using pbs in between each step. the aiv antigen bound with mabs was then visualized by adding diazinobenzidine (dab) substrate (sigma co, usa, 50 mg/ 50 ml pbs containing 0.07% h 2 o 2 ). result characteristic of monoclonal antibodies. as many as 12 clones of stable hybridomas secreting mabs against the aiv-h5n1 of indonesian isolate were produced. screening by elisa using formaldehyde inactivated aiv-h5n1as antigen showed that all of these 12 clones of hybridomas produced mabs against the virus, not against the normal allantoic fluid. eight mabs were further characterized and they were designated as ag8, bc12, cc5, cg1, dd9, df11, ea11, and ee8. all mabs reacted strongly in elisa test using formaldehyde inactivated aiv-h5n1. none of them reacted with normal egg allantoic fluids (negative control). the titer of mabs varied from 26 to 29 (table 1). isotyping of mabs using rabbit anti-mouse igg subtyper isotyping kit showed that 3 mabs (ag8, df11, ea11) were of igg1 subclass, 1 mab (df9) was of igm subclass, 3 mabs (cc5, cg1, dd9) were of igg3 subclass, and 1 mab (ee8) was of igg2a subclass (table 1). western blotting with mabs. in western blotting assay, a similar result was observed. all mabs reacted only with formaldehyde inactivated aiv-h5n1. no mab reacted with normal allantoic fluid. two mabs (dd9 and cc5) reacted with 2 protein bands with the molecular weight of approximately 76 and 58 kda, 5 mabs (ag8, cg1, df11, ea11, and ee8) reacted with a single protein band of 76 kda. one mab (df9) reacted with a diffuse protein band. detection of aiv antigen in duck tissues. three (cg1, ee8, ag8) produced a good and a strong result when used for the detection of aiv antigen in ducks. one mab (df11) did not react with aiv antigen in the duck tissues. aiv antigen was detected in the two infected ducks but not in uninfected ducks. aiv antigen with a high intensity was observed proventricle and in small intestine. aiv antigen at a lesser intensity was also observed in other organs such as lung, spleen, and bursa of fabricius (table 2, fig 1). aiv antigen was difficult to observe in the brain, muscle tissue, and kidney. no clear difference on the distribution of infected tissues was observed between the two infected ducks. discussion stable anti-aiv-h5n1 mabs-secreting hybridomas were successfully produced by fusion of immortal myeloma cells with lymphocytes of mice immunized with the virus. the use of formaldehyde inactivated virus for immunization of mice in the preparation of mabs appeared to be not an important factor for the production of hybridomas stably producing mabs against aiv. this is evident as all of the isolated hybrodomas consistently produced mabs against the virus and but not against the normal allantoic fluid. the use of relatively unpurified virus for immunization of mice in the preparation of mabs has been reported (wickramasinghe et al. 1993; pantophlet et al. 2001). screening method appeared to be the more important factor for successful selection of hybridomas producing 122 astawa et al. microbiol indones anti-aiv-h5n1 mabs. as in immunization, the antigen used in the elisa test for screening mabs was formaldehyde inactivated aiv-h5n1. the virus was originally propagated in the allantoic cavity of chicken embryonated eggs. the virus was then harvested from the allantoic fluid of the infected chicken embryo and was therefore expected to contain a plenty of normal allantoic fluid. it was therefore very likely that the immunization of mice with such antigen will stimulate the production of antibodies against both aiv and normal allantoic fluid. this then was confirmed when several mabs which reacted with normal ascitic fluid were detected by elisa using the unpurified aiv-h5n1 (data not shown). such mabs were excluded by further testing by elisa using normal allantoic fluid as an antigen. isotyping showed that the mabs produced in this experiment were of igm, igg1, igg2a, and igg3 subclasses. the information on the isotype of mabs is important in the selection of techniques used for the purification of mabs. in addition, the information on the mabs’ isotype is also important in the selection of techniques to be developed using mabs. the mabs with igg isotype generally produces a more specific and sensitive result and can also be used in a relatively wider range of serological tests then those of mabs of igm isotype. in some immunodetection systems, however, antibody with igm isotype is preferred as it will produce a better and a stronger reaction than mabs of igg isotype. the main problem working with igm is that it is more difficult to purify than igg. in addition, the availability of mabs of igg isotypes will also enable the purification of the mabs using protein a or g (de masi et al. 2005) which is often required for the development of a particular test such as capture elisa (ohnishi et al. 2005). in western blotting assay, all isolated mabs reacted specifically only with aiv-h5n1 antigen. none of them reacted with normal allantoic fluid which was used as an antigen for aiv negative control. the result confirmed that mabs specific to aiv-h5n1 can be produced by immunization of mice with relatively unpurified virus. the protein bands recognized by mabs were around 76 kda, 58 kda, and several other diffuse bands. the protein band with 76 kda detected by most mabs (cg1, ag8, ea11, ee8, bc12) is likely to be uncleaved haemagglutinin (ha0) of aiv-h5n1. the ha protein of aiv is a surface glycoprotein encoded by segment 4 (ha) of the viral segmented rna gemomes. the protein is initially translated as uncleaved precursor of ha0 protein with the molecular weight of around 76 kda. it is then post-translationally cleaved by host cellular proteases into two sub units, ha1 (56 kda) and ha2 (25 kda) (skehel and waterfield 1975; zhirnov et al. 2002). however, other workers on influenza a virus reported the molecular weight of ha1 varied from 50-61 kda and ha2 varied from 25-30 kda (bucher et al. 1976; boulay et al. 1987; jaspers et al. 2005). the protein contains sialic acid which plays an important role in the binding of the virus into the receptor molecules on the surface of susceptible cells (hulse et al. 2004) and such cleavage step is necessary for the infection of the virus into not react with aiv antigen in the infected ducks, suggesting that this mabs did not recognized the aiv epitope in formadehyde fixed and paraffin embedded tissues. the reason behind this is unknown. it is possible that the epitope recognized by this mab has been destroyed or hidened during the tissue proccecing. when the 4 mabs were used to immunostain tissues or organs of normal uninfected ducks, none of them produced a positive result. this showed that three of the selected mabs are applicable for use in development of specific test for the detection aiv infection in ducks. the use of mabs in the immunochemistry staining for the detection of viral antigen in the infected host has been widely reported (ohnishi et al. 2005; astawa et al. 2006). in the infected ducks, high intensity of aiv antigen was detected in organs such as preventricle and intestine villi (fig 1), suggesting that the virus replicates very efficiently in these two gastrointestinal organs. this is in accord with the finding that, in waterfowl, influenza viruses replicate preferentially in the intestinal tract, resulting in excretion of high-titer viruses in the feces (horimoto and kawaoka 2001). the combination of the availability of cells bearing the receptor for aiv and the presence of abundant proteolytic enzymes may contribute to the efficient replication of the virus in the intestine and proventricle of ducks. unlike those which originate from chicken, many aivs isolated from ducks have a strong binding activity to gangliosides with short sugar chains that were found abundant in duck gastrointestinal tissues (slemons and easterday 1978; gambaryan et al. 2003). in addition, gastrointestinal tract is rich in proteolytic enzymes (banks and plowright 2003) which are responsible for post translational cleaving of ha0 of into ha1 and ha2 (garten and klenk 1999). the cleavage of ha protein is required for the efficient replication of the virus in the two organs. the availability of mabs against aiv-h5n1 has enabled the detection of aiv antigen in duck tissues. the duck used study was previously confirmed to be infected by aiv-h5n1 but the tests used still require expensive facilities and reagents such as pcr. it is also unsafe to perform as isolation of aiv in chicken embryonated eggs and identification by ha/hi test require the use of live virus (gough 2004). the use of mabs on formaldehyde fixed and paraffin embedded tissues has made it possible to develop a relatively simpler and safer test which can be performed in laboratory with simple facilities and low biosecurity level. the immunological detection system developed in this experiment also safe to perform on daily basis as it uses formaldehyde fixed tissues which inactivates the aiv. as ducks and other aquatic birds play an important role in the transmission of aiv into susceptible hosts (matrosovich et al. 1999), the availability of test to detect ducks carrying the virus will be important in preventing the ai outbreaks brought out by this carrier water fowls. it is also important to note that most mabs produced in this experiment appeared to react with the ha protein of aiv-h5n1. this was further confirmed by hi test that mab ag8 that react with at the protein band of around 76 kda (fig 2) did exhibit inhibition of ha activity of the virus (data not shown). at this stage, however, it is still not possible to determine which mabs react specifically to h5 subtypes and which mabs cross-react aiv subtypes other than h5. if a panel of aiv isolates with several different h5 subtypes is available for study, it will be likely to be able to evaluate volume 1, 2007 microbiol indones 123 76 kda 58 kda 1 0 0 7 5 5 0 3 7 2 5 2 1 1 5 1 0 0 7 5 5 0 3 7 2 5 2 1 1 5 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8a b fig 2 reactivity of monoclonal antibodies with aiv-h5n1 and normal allantoic antigens analysed by western blotting. a: antigen: formaldehyde inactivated aiv-h5n1, and b: normal allantoic fluid. strip no. 1-8 mabs: 1: dd9, 2: cc5, 3: ag8, 4: cg1, 5: df11, 6: ea11, 7: ee8, and 8: df9, strip no. 9: standard marker. the cross-reactivity of several mabs with many different h subtypes. when mabs react specifically only with aiv of h5 subtype are available then determination of aiv-h5 subtypes can be carried out directly by immunohistrochemistry staining using mabs. this is important as aiv-h5 virus is one subype that causes most fatal infection in avian species and in mammal including human (swayne and suarez 2000). a further investigation is required in order to confirm this suggestion, especially when aiv of many different h subtypes are available for study. acknowledgement this study was funded partly by the “hibah bersaing” grand provided by directorate of higher education, department of national education, republic of indonesia. references alexander dj. 2000. a review of avian influenza in different bird 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[who]. 2002. who manual on avian influenza diagnosis and surveillance of avian influenza. geneva: who. wickramasinghe r, meanger j, enriquez ce, wilcox ge. 1993. avian reovirus protein associated with netralization of viral infectivity. virology 194:688-698. zheng l, zhang s, wood c, kapil s. wilcox ge, loughin t, minocha ac. 2001. differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of the epitope specificity. clin diag lab immunol 8:283-287. zhirnov op, ikizler mr, wright pf. 2002. cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases. j virol 76:8682-8689. 03. nandyawati.cdr vol.15, no.3, september 2021, p 91-101 doi: 10.5454/mi.15.3.3 production and characterization of thermoalkaliphilic xylanase from bacillus halodurans cm1, and its application on degumming process of ramie ( l.gaud fiber as textile raw materialboehmeria nivea ) dewi nandyawati , dea indriani astuti , niknik nurhayati *, asep riswoko , 1,2 1 2 2 and is helianti 2 1biotechnology major, graduate school, institut teknologi bandung, jalan ganesha 10 bandung, indonesia; 2center for bioindustrial technology, laboratory for the development of agroindustrial and biomedical technology, national agency for research and innovation, gedung 611, kawasan puspiptek serpong, tangerang selatan, indonesia. ramie fiber is a potential raw material to substitute imported raw materials such as cotton. due to its higher hemicellulose content, ramie fiber required hydrolysis in a process called degumming. enzymatic degumming is environmentally friendly compared to traditional process which using chemicals. alkaline thermostable xylanase has high ability in hemicellulose hydrolysis. the production of the cm1 xylanase was conducted by submerged fermentation of cm1 in a 20 l bioreactor using mamo and corncob medium under optimum bacillus halodurans conditions of temperature of 50°c, ph 9, agitation of 150 rpm, and aeration of 1 vvm. the fermentation yielded maximum cm1 xylanase activity of 641.6 u ml or 475.41 u mg after 30 h. cm1 xylanase was stable at 50°c, -1 -1 ph 9 for more than 2 h, and relatively stable against k , na , co and ca metal ions, and triton-x, sb chem + 2+ 2+ 2+ 9201 and tween-80 surfactants. degumming process was carried out by immersing ramie fibers in formulated degumming solution with rami to solution ratio of 1:20 at 50 °c, with agitation of 150 rpm for 3 h.. the highest weight loss of rami fiber of 7.72% was reached by chemical – enzymatically degumming process. the highest tensile strength of 27.51 g tex was reached by enzymatically degummed fiber that retained more than 90% of the -1 control fiber (29.62 g tex ). degumming effect on whiteness index and fiber surface was also observed in this -1 study. alkaline thermostable xylanase cm1, enzymatic degumming, ramie fiber, key words: , bacillus halodurans textile industry serat rami merupakan bahan baku potensial untuk menggantikan bahan baku impor seperti kapas. karena kandungan hemiselulosa yang tinggi, serat rami hidrolisis proses . perlu di dengan degumming degumming enzimatis lebih ramah lingkungan dibandingkan proses tradisional menggunakan bahan kimi . xilanase a alkalitermofilik memiliki kemampuan dalam hidrolisis hemiselulosa. produksi xilanase dilakukan dengan fermentasi cm1 dalam bioreaktor 20 l menggunakan media mamo dan submerged oleh bacillus halodurans tongkol jagung kondisi optimum suhu 50 °c, ph 9, 150 rpm dan 1 vvm. aktivitas spesifik optimum xilanase pada diukur dengan metode bailey pada suhu 70 °c dan ph 9 adalah 475,41 u/mg. xilanase stabil pada suhu 50 °c, ph 9 dan relatif stabil terhadap ion logam k , na , co dan ca dan surfaktan triton-x, sb chem 9201 dan tween-80. + 2+ 2+ 2+ proses degumming dilakukan dengan merendam serat rami dalam larutan degumming terformulasi dengan vlot 1:20 pada suhu 50 °c, 150 . proses degumming enzimatik dapat menggantikan atau mengurangi rpm selama 3 jam penggunaan bahan kimia karena efeknya yang signifikan terhadap kualitas serat rami. roses degumming p enzim kimia berat serat rami 7,72 % egumming enzimatis mempertahankan atis dan mengurangi sebesar . d kekuatan tarik sebesar 27,51lebih dari 90% dibandingkan perlakuan kontrol (29.62 g tex ) yaitu g tex . pengaruh -1 -1 proses degumming pada derajat putih dan permukaan serat juga diamati dalam penelitian ini. kata kunci: , enzimatis, industri tekstil, serat rami, xilanase bacillus halodurans cm1 degumming alkalitermofilik microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone/ 21-7560694;fax: +62 e-mail: niknik.nurhayati@bppt.go id. hydrolases and oxidoreductase for application in degumming, desizing and bleaching (removing impurities from cellulose fibers), finishing (removing fiber surface) and stoning (producing faded denim material). one of the important microorganism-based hydrolase enzymes in industry is xylanase. xylanase is having the ability to degrade hemicellulose containing glucose polymer of xylan into xylooligosaccharide and d-xylose (abd el aty 2018). the application of et al. the application of industrial biotechnology, known as white biotechnology, uses biocatalyst to substitute the use of chemical catalyst in industrial process (heux et al. 2015), improve end-product quality and environmentally friendly (battan 2015). et al. commonly used enzymes in textile processing are xylanase in various industrial sectors such as pulp and paper, food, textiles, biofuels, animal feed is currently increasing due to its ability to reduce the use of hazardous chemicals in the process (kumar 2017). et al. in textile industry, xylanase have been used for degumming process to remove gum or non-cellulosic impurities of natural fibers such as jute, flax and ramie. ramie is one of potential natural bast fiber which have higher fiber strength and moisture regain compared to cotton fiber. therefore, ramie is potential substitution to reduce the dependence on imported cotton (novarini and sukardan 2015). decorticated ramie fiber consists of 68-76% cellulose, 13-14% hemicellulose, 1.9-2.1% pectin and 0.6-2% lignin (lee 2019). non-et al. cellulosic impurities, called gum, should be removed from ramie fibers in order to improve spinning ability and meet the requirements of textile raw material (cheng 2020). traditional degumming process et al. commonly used alkali solution such as sodium hydroxide, sodium carbonate and others (madhu and chakraborty 2017). according to meng (2018) et al. ramie fibers consist of 74.25% cellulose, 13.8% hemicellulose, 5.16% pectin and 1.3% lignin, while ding (2014) stated that it consists of 69.83% et al. cellulose, 13.22% hemicellulose, 5.42% pectin and 1.84% lignin. xylanase is an effective enzyme for xylan depolymeration by breaking it down into small molecular weight oligomers, thereby increasing the absorption strength, whiteness and brightness of textile fibers (kaur 2016). in the previous study a et al. thermophilic cm1 has been bacillus halodurans isolated and partially characterized to have an alkaline thermostable xylanolytic activity (ulfah 2011). et al. the objectives of this study were to produce and characterize the alkaline thermostablexylanase from bacillus halodurans cm1 to be applied in degumming process of ramie fiber. analyses of ramie fiber was also conducted in order to obtain effective degumming procedure. materials and methods bacterial strains and medium the strain used in . this study was cm1 came from bacillus halodurans bppt culture collection. cm1 is bacillus halodurans a gram-positive bacteria isolated by ulfah et al (2011) from cimanggu hot springs, west java. seed culture media was a modified horikoshi media (kumar and satyanarayana, 2011) contained 1 % w/v peptone, 0.5 % w/v yeast extract, 0.1 % w/v kh po , 0.5 % w/v 2 4 na co , distilled water. production media was mamo 2 3 media with a slight modification (wibowo et al,2016) contained 5 % w/v corncob, 0.5 % w/v yeast extract, 0.1 % w/v kh po , 0.5 % w/v na co , 0.2 % w/v 2 4 2 3 nacl, 0.1 % w/v mgso , 0.01 % w/v cacl , distilled 4 2 water. chemicals and rami fiber. chemicals used in this study were 0.5 % w/v xylan beechwood, 2mg/ml xylose, 50mm ph 9 tris-cl buffer, dns reagent contained of 1 % w/v dinitrosalicylic acid, 0.2 % w/v phenol, 0.05 % w/v sodium sulfite, and 1 % w/v sodium hydroxide and 20 % w/v potassium sodium tartrate, biorad protein assay, and bovine serum albumin for enzyme and protein assay. metal ions used in this study were k , na , ca , co , cu , mn , mg , zn , ni , + + 2+ 2+ 2+ 2+ 2+ 2+ 2+ fe while surfactants used were sds, triton-x, 3+ tween-80, tween-20, edta, teepol and sb chem 9201 purchased from pt. sadya balawan, bogor, west java, indonesia. decorticated ramie fibers were purchased from cv. rabersa, wonosobo, central java, indonesia. inoculum and starter culture preparation. inoculum was prepared by inoculating one bacterial colony of cm1 into 20 ml seed culture b. halodurans media followed by incubation at 50°c, ph 9, 150 rpm until od was 0,8 – 0,9. starter culture of 600nm bacillus halodurans cm1 was produced by scaling up the volume of seed culture gradually by inoculating 10% volume of the inoculum having od nm of 0.8 -0.9 600 into seed media starting from 150 ml up to 1500 ml under the same incubation condition previously described. production of the cm1 xylanase in 20 l bioreactor. the cm1 xylanase was produced under submerged fermentation of 10 l modified mamo media inoculated by 10% volume of the bacterial starter at initial concentration of 1 x 10 cells ml in a 7 -1 20 l bioreactor (bailun bio 86-21-67867376). the fermentation was conducted at operational condition of 50°c, ph 9, 150 rpm and 1 vvm according to the protocol reported by widia (2017). samples were withdrawn every 6 h to monitor bacterial growth and enzyme production. bacterial growth was expressed as colony forming unit (cfu) obtained by counting bacterial colony from the 10 diluted fermentation -7 samples grown on horikoshi agar media. enzyme production was observed by determining volumetric and specific activity of the sample's supernatants by centrifugation of samples at 4°c, 3887 g by using gyrozen 1580 centrifuge. at the end of fermentation cm1 xylanase was recovered from the supernatant of 92 nandyawati et al . microbiol indones the culture by centrifugation at 4°c, 6000 rpm (5645 g) for 15 minutes by using tomy supreme 25 high speed refrigerated centrifuge. enzyme and protein assays. xylanase assays were conducted by using standardized internal laboratory protocol (ip.02–03/bkt/laptiab/bppt) that was based on protocol xylan hydrolysis reported by bailey (1992) followed by quantification of reducing sugar by using dinitrosalicylic acid (dns) method (miller, 1959) with some modifications as reported by haniyya (2020). one unit ofet al. xylanase activity was defined as the amount of enzyme releasing 1 mol of reducing sugar per minute under appropriate μ assayed condition. protein concentration were measured using bovine serum albumin as protein standard as reported by bradford (1976). xylanase activities were stated in volumetric activity (u ml ) -1 and specific activity (u mg ). the lat er was expressed -1 t as ratio from volumetric activity and protein concentration. effect of ph and temperature on cm1 xylanase activity. the effect of temperature and ph on the activity of cm1 xylanase were investigated in the reaction mixture containing beech wood xylan in three different buffers (50 mm): na-phosphate (ph 7 and 8), tris-hcl (ph 9 and 10), and glycine-naoh ( ph 11) at specific temperature ranging from 30 70°c. the results were presented as specific activity. enzyme stability. stability of cm1 xylanase enzyme over a temperature range of 50 to 70°c were analyzed by incubating the enzyme without xylan in the optimum ph for 120 minutes. an aliquot of the enzyme was removed and placed on ice at 15 minutes intervals. the residual xylanolytic activity of each enzyme aliquots was determined by routine assay. stability of cm1 xylanase enzyme toward metal ions and surfactants were studied by reacting the enzyme with k , na , ca , co , cu , mn , mg , zn , ni , + + 2+ 2+ 2+ 2+ 2+ 2+ 2+ fe (concentration of 1 mm and 10 mm) and with 3+ mercaptoethanol, sds, triton-x, tween-80, tween20, teepol, and sb chem 9201 (concentration of 0.1 and 1%) at room temperature for one hour. each aliquots of the enzyme was then analyzed for its residual xylanolytic activity by routine assay. the results were expressed as percentage of residual activity calculated on the basis of untreated enzyme. degumming of ramie fiber. degumming experiments of ramie fiber were conducted in three different groups of degumming solution containing enzyme (a), chemical (b), and combined enzyme and chemical (c) as presented in table 1. degumming of ramie fiber was carried on 500 ml erlenmeyer flasks containing 10 g decorticated dried ramie fiber and 200 ml degumming solution. they were incubated in a reciprocal shaking incubator at 50°c, 150 rpm for 180 minutes. aliquots of each degumming solution at the beginning and the end of the process were analyzed for xylanolytic activities by routine assay. degummed fiber samples were then rinsed by running water and oven dried at 50°c. the percentage weight loss of the dried samples was determined by comparing the fiber difference between the weight of the samples before and after degumming with that of before degumming. c h a r a c t e r i z a t i o n o f r a m i e f i b e r. characterization of ramie fiber was determined by measuring weight loss, tensile strength, whiteness index and surface morphology. tensile strength was measured by sni 08-1112-1989 using instron universal testing system 3380 series. whiteness index was measured by sni 08-0280-2004 using minolta cm-3600a spectrophotometer. surface morphology was analyzed using scanning electron microscopy. weight loss measurement was conducted in laboratory of center for bioindustrial technology, bppt while tensile strength and whiteness index were conducted at accredited laboratory of politeknik sttt bandung. surface morphology observation was conducted at cipta mikro material. results production of cm1 xylanase. the cm1 xylanase production was conducted by submerge fermentation of cm1 with ground corncob as carbon b. halodurans sources in a 20 l bioreactor. the growth of b. halodurans cm1 and production of cm1 xylanase was observed periodically for 31 hours fermentation (fig. 1). the culture underwent lag phase between 0 – 21 h of cultivation. the exponential growth phase occured between 21 30 h of cultivation and directly followed by death phase that indicated by a decreased of cell number from 2.4x10 to 1.8x10 . fig. 1a also showed 9 9 that enzyme activity increases along with the increase in the number of cells and reached maximum of 641.6 u ml at 30 h in the end of exponential growth phase. -1 the volumetric activity of cm1 xylanase at 31 h of fermentation has decreased from 641.60 u ml to 637 -1 u ml then the crude enzyme was harvested by -1 centrifugation in order to obtain the highest possible enzyme activity. extracellular protein concentration seems to increase during the exponential growth phase between 18 27 h and starting to decrease between 27 volume 15, 2021 microbiol indones 93 31 h. the specific activity of cm1 xylanase increase during the exponential growth phase and reached maximum activity of fig. 1c showed the 475.41 u mg -1 at 31 h when the enzyme was harvested as can be seen in fig. 1c. partial characterization of cm1 xylanase. effect of temperature and ph and on activity and stability of the cm1 xylanase were studied. the enzyme was active at temperature of 30°c to 90°c and ph 6 to 9. enzyme activity increased along with the increased reaction temperature and ph to reach its maximum activity of 404.37 u mg at 70°c and ph 9 as -1 can be seen in fig. 2. when incubated at its optimum ph (ph 9) cm1 xylanase retained more than 80% its activity in more than 120 min at 50°c and less than 30 min at 60°c (fig. 3). while its activity at its optimum ph and temperature (ph 9 and 70°c) seemed to be drastically dropped down to about 13% in 15 min. effect of metal ions and surfactants were studied. when various metal ion was mixed at room temperature for 1 h, k , na , ca , co , and cu + 2+ 2+ 2+ 2+ seemed able to increase cm1 xylanase activity ranging from 107 – 130%. whereas incubation with mn2+, mg2+, zn2+, ni2+ and fe3+ resulted in decreased cm1 xylanase activity ranging from 78 – 91% as presented in fig. 4. when various surfactant was added at room temperature for 1 h, triton-x, tween 80, or sb-chem 9201 at concentration of 1% seemed to increase enzyme activity ranging from 107 – 110%. whereas addition of sds, teepol, and tween 20 at concentration 1% resulted in decrease enzyme activity ranging from 90 – 97% as can be seen in fig. 5. degumming of ramie fiber. degumming effect of cm1 xylanase in various concentration as well as in combination with chemicals were studied on 10 gr decorticated dried ramie fiber in a ratio of ramie : degumming solution = 1:20. fig. 6 showed degumming effect as represented by weight loss seemed to increase along with the increased cm1 xylanase concentration with the highest weight loss of 7.23% reached by addition of 5% cm1 xylanase. chemical degumming by using naoh (0.1%), h o2 2 (1%), and their mixture showed that naoh alone was able to reduce the gum content higher (7.14%) than either h o (4.76%) or in the mixture of naoh + h o 2 2 2 2 (5.3%) as can be seen in fig. 7. effect of addition of surfactant (sb-chem 9201) in enzymatic degumming was studied. it seemed addition of sb-chem9201 resulted in slightly increased of weight loss when added with 3% cm1 xylanase as shown in fig. 8. enzymatic-chemically degumming showed the weight loss of ramie fiber seemed to increase along with the increased concentration of cm1 xylanase and reached maximum weight loss of 7.7% and 6.8% at 3% and 5% cm1 xylanase in degumming solution containing naoh and naoh + h o mixture, respectively as 2 2 presented in fig. 9. partial characterization of ramie fiber. effect of degumming on weight loss and tensile strength of ramie fiber were compared within chemically, enzymatically, and enzymatic chemically process. fig. 10 showed the highest weight loss of 7.72% and 7.67% was reached by enzymatic chemically degumming (xylanase 3% and 5%, each mixed with 0.1% naoh) followed by either chemically (naoh, 7.14%) or enzymatically (xylanase 3%, 7.15%; xylanase 5%, 7.14%) one. fig. 10 also showed enzymatically degumming (xylanase 3% and 5%) resulted in higher tensile strength of 27.5 and 26.8 g tex -1 in comparison with those of chemically (naoh, 23.7 g tex ) and enzymatic chemically (24.7 and 24.06 g tex ) -1 -1 ones. in comparison with the control of h o-treated 2 rami fiber, enzymatically degummed rami fiber retained more than 90% tensile strength whereas those of chemically and enzymatic – chemically ones retained 80 83%. degumming effect on brightness showed that in the absence of xylanase, white index of rami fiber treated by h o seemed to be higher than that of naoh 2 2 as shown in fig 11. addition of enzyme (1 – 3%) resulted in an increased white index of rami fiber treated either with naoh from 74.64% to 77.16% or h o from 2 2 76.7% to 77.06%. a morphological observation by electron microscope of those enzymatically and chemically degummed rami fiber revealed a slightly difference on those fiber's surfaces. as shown in fig. 12 the surface of enzymatically degummed ramie fiber seemed smoother with less cell wall debris in comparison with those of chemical or control one. weight loss of ramie fiber showed the reducing of non-cellulosic impurities or gum material .the result of weight loss after degumming was 4.67 – 7.72 % presented in figure 6. the highest weight loss of ramie was at enzymatic + chemical degumming (s11 = 7.72 %, s8 = 6.49 % or s9 = 7.23 %) compared to either enzymatic degumming (s2 = 7.15 % and s3 = 7.14 %) or chemical degumming (s5 = 7.14 %). this parameter should be followed by further observation such as tensile strength, whiteness index and surface morphology. the data on the tensile strength of ramie fiber was stated to be homogeneous and significantly different (p <0.05). the highest decrease in tensile strength of 94 nandyawati et al . microbiol indones fig 1 curve of bacterial growth of cm1 (●), xylanase production based on volumetric (♦) and b. halodurans specific (■) activities, and protein production (▲) in 15 l mamo medium containing ground corncob at 50˚c, ph 9, 1 vvm aeration and 150 rpm agitation. each data point represents the average of triplicate measurements. standard deviations are indicated by bars. fig 2 effect of temperature on xylanase activity in various buffer of ph 7 (○), ph 8 (◊), ph 9 (x), ph 10 (□), and ph 11 (∆). assays were conducted in triplicate. standard deviations are indicated by bars. absence of bars indicates that errors were smaller than symbols. fig 3 thermostability of xylanase activity at optimum ph 9 and specific temperature of 50˚ c (x), 60˚ c (●), and 70˚c (□) over incubation time range of 0 to 120 min. the initial activity of 416 u ml was defined as 100%. -1 assays were conducted in triplicate. standard deviations are indicated by bars. absence of bars indicates that errors were smaller than symbols. volume 15, 2021 microbiol indones 95 (a) (b) (c) fig 4 effect of metal ions on xylanase activity. the enzyme was incubated at room temperature with 1.0 mm ( ) ■ and 10.0 mm ( ) concentrations of metal ions without substrate for 1 h prior subjected to routine xylanase ■ assay. a control of untreated enzyme ( ) was 416 u ml and defined as 100%. assays were conducted in -1 ■ triplicate. fig 5 effect of surfactants on xylanase activity. the enzyme was incubated at room temperature with 0.1% ( ) and ■ 1% ( ) concentrations of surfactants without substrate for 1 h prior subjected to routine xylanase assay. a ■ control of untreated enzyme ( ) was 416 u ml and defined as 100% activity. assays were conducted in -1 ■ triplicate. standard deviations are indicated by bars. fig 6 enzymatic degumming effect on weight loss of ramie fiber with xylanase concentration range of 0 to 5% at 50°c, 150 rpm for 3 h. experiments were conducted in triplicate. standard deviations are indicated by bars. fig 7 chemical degumming effect of ramie fiber with various chemicals naoh (0.1%), h o on weight loss 2 2 (1%,) and a mixture of naoh + h o at 50°c, 150 rpm for 3 h. experiments were conducted in triplicate. 2 2 standard deviations are indicated by bars. 96 nandyawati et al . microbiol indones fig 8 enzymatic degumming effect on weight loss of ramie fiber in solution containing xylanase (concentration range of 0 to 5%) with ( ) and without ( ) addition of surfactant (sb chem 9201) at 50°c, 150 rpm for 3 h. ■ ■ experiments were conducted in triplicate. standard deviations are indicated by bars. fig 9 chemical-enzymatically degumming effect on weight loss of ramie fiber in solution containing xylanase (concentration range of 0 to 5%) with 0.1% naoh ( ), 1% h o , ( ) and a mixture of naoh + h o ( ) at 2 2 2 2■ ■ ■ 50°c, 150 rpm for 3 h. experiments were conducted in triplicate. standard deviations are indicated by bars. fig 10 effect of various degumming process on weight loss ( ) and tensil strength ( ) of ramie fiber. the process ■ ■ was conducted as control (h2o), chemically (naoh), enzymatically (xyn 3 and xyn 5), and chemicalenzymatically (xyn3+naoh and xyn5+naoh) degumming. experiments and measurements were conducted in triplicate. standard deviations are indicated by bars. fig 11 effect of various concentration of enzyme on white index in degumming solution contining naoh ( ), h o 2 2■ (■), a mixture of naoh + h o ( ), and a control h o ( ). experiments and measurements were conducted 2 2 2■ ■ in triplicate. standard deviations are indicated by bars. volume 15, 2021 microbiol indones 97 ramie fiber was on chemical degumming (s19 = 21.24 g/tex and s5 23.7 g/tex) compared to control (29.62 g/tex. the tensile strength of ramie fiber relatively maintained by enzymatic degumming (s2 = 27.51 g/tex and s3 = 26.8 g/tex) which better than chemical degumming (fig 7). the result of whiteness indexwas quite significant compared to unbleached ramie fiber. the highest whiteness index obtained by chemical degumming (s19 = 77.19 %) while enzymatic degumming also showed significant effect (s2 = 75.85 % and s3 = 76.69 %) compared to control (k = 73.70 %) (fig 8). the results of the ramie fiber morphology test using sem (scanning electron microscopy)where enzymatic degumming by using xylanase (b) showed that the gummy material of the surface was smoother than decorticated fiber used as control (a) and chemical treatment by using naoh (c) (fig 9). discussion xylanases have attracted a great deal of attention in the last decade particularly due to their eco-friendly application in various manufacture industries such as pulp and paper, biofuel, and textile. in this study cm1 xylanase production was conducted in a stirred tank 20 l bioreactor in which aeration, agitation, ph and heat transfer were able to be controlled to facilitate optimum cultivation condition. as presented above cm1 xylanase was produced by cm1 b. halodurans during the exponential phase following a relatively long lag phase of almost 20 h. it is suggested that the presence of ground corncob and mechanical agitation in the bioreactor resulted in a relatively significance change between shake flask and bioreactor environment that induced cellular stress (hallsworth 2018). thus, the bacterial cells required more extensive period of adjustment before starting to grow and in consequence lengthen the lag phase. moreover, ground corncob as carbon source could not be directly used by the bacterial cell. it explained the exponential cell growth that not started until the increased cm1 xylanase activity between 18 30 h cultivation. the enzyme obtained by using bioreactor in this study was 637.7 u ml or 475.41 u mg after 31 h cultivation. -1 -1 this result was relatively higher than that of gained by using shake flask that reached maximum activity of 522 u ml (wibowo 2016). similarly, ling ho -1 et al. (2016) reported that xylanase production by bacillus subtilis in bioreactor yielded much higher activity than that of in shake flask. the activity of cm1 xylanase towards beechwood xylan in various ph and temperature showed that the enzyme was optimally active at ph 9 and 70˚c. this optimum ph and temperature were similar with that of thermostable alkaline xylanase from anoxybacillus kamchatkensis et al reported by yadav (2018). the thermal stability of the enzyme indicated that the cm1 xylanase was fully stable after 2 h incubation at 50˚c. however, its stability that represented by residual activity, seemed to decrease down to less than 60% and 30% when the enzyme was incubated for 30 min at elevated temperature of 60 and 70˚c, respectively. it is suggested that the enzyme was denatured after 30 min incubation at 60˚c. this result was lower than that of mamo (2006) who demonstrated that the xylanase et al from s7 retained over 60% residual b. halodurans activity after 3 h incubation at 65˚c. differences in the result presumably due to differences in the origin of the bacterial isolates and the purity of the enzyme. b. halodurans cm1 was isolated from indonesian cimanggu hot spring (ulfah 2011) while that of et al. s7 strain was isolated from ethiopian soda lake (mamo 2006). in this study we used crude et al. xylanase enzyme instead of partially purified one used by mamo (2006). the activity of the cm1 et al xylanase toward metal ions indicated that the enzyme was inhibited by fe and zn and activated by na and 2+ 2+ 2+ ca . metal ions binds with enzyme catalytic site which 2+ enhance the interaction of enzyme with substrate fig ; ; . .12 surface morphology (sem) of ramie fibers. control (untreated) (a) xylanase 3% (b) naoh 0 1% (c) a cb 98 nandyawati et al . microbiol indones (anand 2013). in industrial process, metal ions et al. might be contained in water or other reagents used for its process, which will affect enzyme activity (pereira et al.2017). meanwhile the enzyme activity was slightly inhibited by almost all tested surfactants except triton x, tween 80 and sb-chem 9210. nonionic surfactants have been used as wetting agent to increase water absorption in textile processing. the use of 1.25% tween-20 enhanced the effectivity of enzymatic degumming process (singh 2019). the et al. information of enzyme activator and inhibitor was relevant in this study, in which the enzyme would be applied in degumming process of ramie fiber that was adapted to the existing method used by local small scale ramie fiber producer. the enzyme was used for degumming of ramie fiber under the designed condition considering the enzyme stability towards ph and temperature. in this study degumming effect of cm1 xylanase, alone as well as in combination with chemical (naoh), was observed and compared with that of chemical one. degumming effect was observed by determining weight loss of ramie fiber, that represented the reducing of non-cellulosic impurities or gum material. this study revealed the highest weight loss of ramie was reached by chemical enzymatically degumming proses, in which 0.1% naoh was added with either 3% or 5% cm1 xylanase. this result indicates a synergistic action of naoh in degrading gum material. addition of naoh created an alkali milieu of degumming solution by which gum material partially degrade (cheng 2020) at the same time et al. na acted as activator cm1 xylanase enhanced 2+ hemicellulose breakdown. beside metal ion of na , 2+ the addition of surfactant (sb-chem 9201) showed to enhance degradation of gum material. this is in accordance with that of singh . (2019) who et al reported the addition of 1 % surfactant, as a wetting agent, increases the absorbance capacity of the fibers so that the enzyme penetration in the fiber pores is better. in this study effect of h o on degumming was 2 2 also observed. hydrogen peroxide is usually used as bleaching agent. the result showed an opposed action of h o resulted in decreased weight loss when added 2 2 that with either naoh or cm1 xylanase in the degumming solution. degumming process, either enzymatically or chemically, reduces tensile strength of ramie fiber caused by the removal of non-cellulosic impurities by which reducing its stiffness. therefore, tensile strength analyses of ramie fiber was conducted and revealed that enzymatically degummed ramie fiber retained the higher tensile strength compared to chemically degummed. it is similar to the research of where enzymatic treatment showed tensile strength of 7.19 cn/dtex higher than alkali degumming of 6 cn/dtex. it is suggested that addition of chemicals (naoh and h o ) led to damage not only to non-cellulosic 2 2 impurities but also to cellulosic fiber. degumming effect on brightness was studied and showed that h o2 2 was a stronger bleaching agent in comparison to naoh. however, addition of cm1 xylanase in combination with naoh was able to significantly increase white index. this in accordance with bleaching boost effect of the xylanase of b. halodurans c-125 in previous study by lin (2013), who et al. demonstrated that the addition of the c-125 xylanase was able to reduce 10% chlorine as bleaching agent in wheat straw pulp bleaching. fiber surface analyses showed that enzymatically degumming ramie fiber was relatively smoother in comparison with that of the chemically one. enzymatic degumming enhances the separation of non-cellulosic with cellulosic components that makes fiber surface was smoother (rani 2019). in conclusion, cm1 xylanase either et al. alone or in combination with naoh was a better degumming solution in comparison to that of the chemical one. enzymatic degumming produced ramie fiber that relatively preserve the tensile strength and smoother surface. aiming to obtain more effective enzymatic degumming, it is necessary to conduct further study on application of pectinase in combination with cm1 xylanase. acknowledgements authors thank for the support of master program of biotechnology, institut teknologi bandung and the centre for bioindustrial technology, the agency for the assessment and application of technology. this research work has been funded by beasiswa saintek from the ministry of research and technology of the republic of indonesia and supported in part by the l p d p – r e s e a r c h g r a n t ( p r o j e c t n o . 171/e1/prn/2020) in cooperation with the ministry of research and technology of the republic of indonesia/brin. references abd el aty aa, saleh saa, eid bm, ibrahim na, mostafa fa. 2018. thermodynamics characterization and potential textile 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perrone om, boscolo m, da silva r, gomes e, martins dab. 2017. effect of metal ions, chemical agents and organic compounds on lignocellulolytic e n zy m es a c tivitie s . en z ym in hib act. do i: 10.5772/65934. rani k, ahirwar m, behera bk. 2020. comparative analysis of alkaline and enzymatic degumming process of hemp fibers. j inst eng ser e 101. doi: 10.1007/s40034-01900156-y. 100 nandyawati et al . microbiol indones ray dp, satya p, mitra s, banerjee p, ghosh rk. 2014. degumming of ramie: challenge to the queen of fibres. 1:37–41. singh a, varghese lm, battan b, patra ak, mandhan rp, mahajan r. 2019. eco-friendly scouring of ramie fibers using crude xylano-pectinolytic enzymes for textile purpose. ulfah m, helianti i, wahyuntari b, nurhayati n. 2011. characterization of a new thermoalkalophilic xylanaseproducing bacterial strain isolated from cimanggu hot spring, west java, indonesia. microbiol indones 5:139–143. doi: 10.5454/mi.5.3.7. wibowo sg, helianti i, suryani a, wahyuntari b. 2016. application of response surface method in optimization of medium composition for xylanase production by bacillus halodurans cm1 in submerged fermentation. m i c r o b i o l i n d o n e s 1 0 : 1 1 2 – 1 1 7 . d o i : 10.5454/mi.10.3.5. zhou c, xue y, ma y. 2017. characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic with potential in bacillus licheniformis ramie degumming. 54:49–58. volume 15, 2021 microbiol indones 101 02. sidauruk.cdr vol.13, no.4, december 2019, p 118-122 doi: 10.5454/mi.13.4.2 citric acid production from toba banana peel (musa acuminata colla) through submerged fermentation using aspergillus niger meva gustina e. sidauruk, surya ningsih hutauruk, merry meryam 1* martgrita, and adelina manurung bioprocess engineering study program, faculty of biotechnology, institut teknologi del, jalan sisingamangaraja, laguboti, toba samosir 22381, north sumatera, indonesia. toba banana peel waste is derived from toba banana fruit (musa acuminata colla) processing. local people utilized banana peel waste usually as livestock feed. the waste also can cause an environmental problem if it is not handled well. banana peel waste has a high content of carbohydrate that can be fermented to produce more valuable product, one of which is citric acid. citric acid is an organic acid that is consumed globally and produced in large quantities. in food and beverages industries, citric acid is used for various purposes due to its high solubility, non-toxic, and good taste characteristics. the objective of this research is to determine the optimum conditions of submerged fermentation of banana peel to produce citric acid using aspergillus niger. the treatments were various banana peel concentrations (5%, 10% and 15% w/v) added with 5% sucrose or 5% glucose (w/v). during the fermentation, ph was measured to determine ph changes indicated the production of citric acid. the results showed that the variation concentration of banana peel substrate and type of sugars affect citric acid production. the optimum condition of submerged fermentation by aspergillus niger was obtained at 15% substrate concentration by adding 5% sucrose to produce 0.651% (w/v) of citric acid. key words: aspergillus niger, citric acid, musa acuminata colla, submerged fermentation kulit pisang toba merupakan limbah buangan dari hasil pengolahan buah pisang toba (musa acuminata colla). umumnya pemanfaatan kulit pisang oleh masyarakat lokal hanya digunakan sebagai bahan pakan ternak. limbah tersebut juga dapat menimbulkan masalah lingkungan apabila tidak ditangani dengan baik. kandungan karbohidrat yang tinggi pada limbah kulit pisang toba dapat diolah melalui fermentasi untuk menghasilkan produk komersial yang lebih bernilai, salah satunya adalah asam sitrat. asam sitrat adalah asam organik penting yang dikonsumsi secara global dan diproduksi dalam jumlah besar. asam sitrat di industri pangan banyak digunakan untuk berbagai keperluan karena kelarutan asam sitrat yang tinggi, tidak beracun dan rasanya yang disukai. penelitian ini dilakukan dengan tujuan untuk mengetahui kondisi optimum submerged fermentation kulit pisang toba dalam menghasilkan asam sitrat dengan menggunakan aspergillus niger. perlakuan yang diberikan adalah variasi konsentrasi substrat kulit pisang toba (5, 10, dan 15% b/v) serta variasi jenis gula 5% sukrosa dan 5% glukosa (b/v). selama fermentasi dilakukan pengukuran ph, bertujuan untuk mengetahui perubahan ph yang mengindikasikan produksi asam sitrat. hasil penelitian menunjukkan adanya pengaruh konsentrasi substrat kulit pisang toba dan jenis gula pada produksi asam sitrat. kondisi optimum submerged fermentation kulit pisang toba oleh aspergillus niger diperoleh pada konsentrasi substrat 15% dengan penambahan 5% sukrosa yaitu menghasilkan konsentrasi asam sitrat sebesar 0,651% b/v. kata kunci: asam sitrat, aspergillus niger, musa acuminata colla, submerged fermentation microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82167244711; email: adelina.manurung@del.ac.id well. t h e b a n a n a p e e l s w a s t e c o n t a i n s h i g h carbohydrates. carbohydrates found in banana peel waste can be used to produce more valuable commercial products. along with the increase in industrial technology, the review regarded to utilizing banana peel waste is increasing. several studies have been conducted on banana peel waste as a substrate to produce useful products such as xylitol, bio methane, bioethanol, and absorbers in removing minerals in waste. this is very interesting for scientists because banana peels can be used as raw materials that are very beneficial for the community. high carbohydrate content in banana peel has the banana is a tropical fruit plant that is liked by most of the world's population. banana production continues to increase annually by 4.16% and in 2015 reached 7.3 million tons (kementerian pertanian indonesia 2016). abundant of banana production affects the increase in banana peel waste. banana peel is a waste from processing bananas. in general, the use of banana peel by local people are used as animal feedstock ingredients such as cows, goats, buffaloes and several others species. the banana peels waste also can make environmental problems if it is not handled potential to produce more valuable commercial products, one of them is citric acid. citric acid is an important organic acid that is consumed globally and produced in large quantities. citric acid in the food and beverage industry is widely used for various purposes because of the high solubility of citric acid and nontoxic. some of studies on banana peel have been done in producing citric acid through a submerged fermentation process (abbas et al. 2016; khairan, makstum and yulvizar 2019) and others through solid state fermentation (iralapati and kummari 2015; monrroy et al. 2019). in these studies, banana peel was fermented using aspergillus niger at 30°c to produce citric acid. fermentation of various species of banana peel (musa canabalisa, musa paradisiaca, musa acuminata, and musa balbisiana) obtained citric acid -1 yield average of 21,76 g kg (iralapati and kummari -1 -1 2015), 29 g kg (monrroy et al. 2019), 52 g l (abbas et al. 2016), 69.84% (khairanet al. 2019), and 87% (dhandayuthapani et al. 2008). production of citric acid from banana peel is conducted as on one of the solution to increase the quality value of banana peel and reduce environmental problems caused by banana peels. in the fermentation research conducted by varshey (2016) using mixed fruit peels, it showed a high concentration of citric acid at 30 °c fermentation conditions for 5 days, and the addition 0.25% w/v nh no and 4% methanol. based 4 3 on the previous studies, the objective of this research is to determine the optimum conditions of submerged fermentation of toba banana peel to produce citric acid using aspergillus niger. the treatments were various banana peel concentrations (5%, 10% and 15% w/v), and the addition of 5% sucrose and 5% glucose (w/v). during the fermentation, ph was measured to determine ph changes that indicates the production of citric acid. materials and methods pretreatment of substrates. toba banana peels were washed using water, chopped and dried in the oven at 60 °c for 2 days. the dried banana peel was mashed using a blender until it becomes powder. cultivation of microorganism. prepared 100 ml of potato dextrose agar (pda) media and poured into 2 sterile petri dish. then, the inoculum was grown from stock culture into a petri dish containing pda and incubated at 37 °c for 3 days. preparation of inoculum. prepared 100 ml of yeast pepton glucose (ypg) media with a composition of 1% yeast extract, 2% peptone, and 2% glucose. then, 3 inoculating loop of fungal colonies on culture media, inoculated into the media and incubated for 36 hours at 37 °c. preparation of substrate. prepared 100 ml of fermentation media with composition of 5% sugar, nh no 0.25%, kh po 0.1%, mgso 0.025%, 4 3 2 4 4 methanol 4%, and toba banana peels substrate 5%, 10%, and 15%. fermentation. the fermentation media of banana peel was inoculated by aseptically transferring 5% of starter culture to the fermentation media. the media was stirred and then incubated at 30 °c for 5 d. during the fermentation, ph measurements were conducted to determine the ph changes every 24 h using a ph meter. s e p a r a t i o n o f f e r m e n t a t i o n r e s u l t s . fermentation media that has been incubated for 5 d was cooled at room temperature. then filtered using filter paper to obtain the filtrate and saved at -4 °c. analysis of citric acid with hplc. the citric acid was analyzed using the high performance liquid chromatography (hplc). the hplc column used was zorbax eclipse plus c-18 with a mobile phase mixture of methanol and acetonitrile, ph 2.5 kh po 2 4 buffer as a solvent, and citric acid as a standard. mobile phase mixing is carried out in a ratio of 60:40. mobile phase and solvent solution was filtered using a membrane with the help of a vacuum pump. the standard citric acid curve is carried out by diluting 10% citric acid using a buffer solution. the concentrations of citric acid made are 0.05%, 0.1%, 0.5%, 1%, and 1.5%. hplc analysis results produced peaks at certain retention times of each sample and indicated citric acid compounds. the linear equations from standard solutions, used to obtain the concentration of citric acid. results the ph value of banana peel fermentation media in both additional of glucose 5% or sucrose 5% shown in figure 1. during the fermentation process there was a decrease in ph reached 3.79 for addition of glucose 5% and for addition of 5% sucrose reached 3.59, for 120 hours or five-day fermentation times. the concentration of citric acid produced in the fermentation of toba banana peel is obtained from the results of hplc analysis (table 1). from the analysis of the value of citric acid concentration, there is a significant difference in each of the concentration of the banana peel substrate 5%, 10% and 15% in the addition x sidauruk et al. microbiol indones volume 13, 2019 microbiol indones x of 5% sucrose or in the addition of 5% glucose. citric acid concentration was increase significantly with the increasing concentration of toba banana peel substrate, whereas the addition of 5% sucrose tended to increase citric acid concentration compared to the addition of 5% glucose. discussion decreased ph is one of the factors indicating the formation of organic acids. in this research, ph was decrease in the range of 3 – 4. the ph of the media changes continuously during the fermentation process as a result of the metabolic activity of microorganisms, due to the secretion of organic acids such as citric acid, gluconic acid, and oxalic acid (show et al. 2015). to accumulate citric acid, the ph of fermentation media is the most important factor for two reasons, the sporulation phase and production. in the sporulation phase, the spores need ph 5. the germinated spores absorb nitrogen derived from ammonia and release the + proton (h ), resulting in increased acidity in the media and supporting the production of citric acid. at low ph (ph ≤ 2), the formation of unexpected products such as oxalic acid and gluconic acid is inhibited, and the possibility of contamination by other microorganisms is also reduced which makes the production of citric acid higher (max et al. 2010). due to the ph range of 34, it is indicated that in the end of fermentation there are still other products in the fermentation media beside citric acid, such as gluconate acid or oxalic acid. in this study the concentration of nitrogen sources from ammonium nitrate (nh no ) was added to the 4 3 solution of 0.25% w/v equivalent to 0.124 n (rohr et al. 1981). the concentration of nitrogen sources necessary -1 for the fermentation of citric acid is 0.1-0.4 n l . this suggests that the concentration of nitrogen sources from nh no 0.25% w/v supports in terms of ph reduction 4 3 but it should still be improved. in addition to the source of nitrogen, it indicates that the ph decrease was slowly during the 120 h fermentation period, it is suspected that the ph will decrease along with the length of fermentation time. therefore, the addition of fermentation time, should be conducted to achieve fig 1 fermentation medium ph value in addition of 5% glucose (left) or 5% sucrose (right). table 1. citric acid concentration banana peel substrate % (w/v) citric acid concentration % (w/v) 5% sucrose added (w/v) 5% glucose added (w/v) 5 0.379 a (a) 0.257 a (a) 10 0.523 b (a) 0.383 b (a) 15 0.651 c (a) 0.474 c (a) note: different characters in the same column indicates a significant difference among data. the same character in parentheses in the same row does not indicate a significant difference among data. lower ph in the fermentation media. the results of citric acid concentration analysis in toba banana peels showed a significant difference in each concentration of banana peels 5%, 10% and 15%. this research showed that the increasing concentration of toba banana peel substrate will increase the production of citric acid. the highest concentration of citric acid (0.621 % w/v) was obtained in 15% of toba banana peel substrate which was added with 5% sucrose. nutrition is one of the important growth factors for fungal growth. adequate nutrients will stimulate the growth and biosynthesis of metabolite products (sukandar 2002). in the a. niger metabolic pathway in producing citric acid, phosphoenol pyruvate will be converted to oxaloacetate with the activity of phosphoenol pyruvate carboxylase enzyme. this reaction requires atp as an energy source that can be obtained from manganese, potassium, or ammonia (papagianni 2007). banana peel substrate contains manganese mineral which is one of the nutritional sources for a. niger in the incorporation of citric acid. based on research conducted by abbas et al. (2016), an increasing concentration of banana peel substrate did not always cause an increasing production of citric acid by a. niger. the highest citric acid production was in 25% concentration of banana peel substrate, whereas in 30% and 35% concentration of banana peel substrate the citric acid production was steeply decreased. this phenomenon showed that too much substrate would be a barrier for enzyme activity in citric acid cycle. in this study, fermentation with the addition of 5% sucrose resulted a higher concentration of citric acid compared to the addition of 5% glucose, due to the efficiency of a. niger to metabolize certain types of sugar to produce citric acid. in the metabolism of a. niger to produce citric acid, the metabolic pathway used is the glycolysis pathway and the entnerdoudoroff pathway which provides pyruvic acid compounds as intermediate compounds. sucrose is a disaccharide composed of glucose and fructose units. on the path of glycolysis, sucrose will be inserted into the cells through the mechanism of sucrose hydrolysis invertase to break down sucrose into glucose and fructose (boddy et al. 1993), and the two sugars are then converted to fructose 6-phosphate before being converted to acid pyruvate. fermentation of banana peel with the addition of 5% sucrose results in a higher concentration of citric acid because in the steps of glycolysis, the amount of fructose 6-phosphate is obtained from glucose and fructose, while at the addition of 5% glucose, fructose 6-phosphate will only be obtained from glucose. this results in a higher number of fructose 6-phosphate molecules that it will affect the amount of pyruvate obtained in the glycolysis process. in conclusion, although 15% of toba banana peel substrate and the addition of 5% sucrose produced a high concentration of citric acid in this research, the fermentation condition still has to be optimize to reach a higher citric acid concentration. those conditions are toba banana peel substrate concentration, sucrose concentration, ammonium nitrate concentration, and fermentation time. acknowledgements this work was supported by a grant from lppm it del. references abbas n, safdar w, ali s, choudhry s, elahi s. 2016. citric acid production from aspergillus niger using banana peel. int j sci eng res. 7(1): 1580-1583. issn 22295518. boddy lm, berges t, barreau c, vainstein mh, dobson mj, ballance dj, peberdy jf. 1993. purification and characterisation of an aspergillus niger invertase and its dna sequence. curr genetics. 24(1-2): 60-66. doi: 10.1007/bf00324666. dhandayuthapani k, thiyageswaran g, and radeep ks. 2008. production of citric acid from banana waste by aspergillus niger. int j appl bioeng. 2(1): 35-36. doi: 10.18000/ijabeg.10021. iralapati v, kummari s. 2015. production of citric acid from different fruit peels using aspergillus niger. ijser 3 (5): 129-130. issn (online): 2347-3878. kementrian pertanian indonesia. 2016. komoditas pertanian sub sektor hortikultura. agricultural commodity of horticultural subsector. [report]. jakarta: pusat data dan sistem informasi pertanian kementerian pertanian. khairan k, makstum a, yulvizar c. 2019. utilization of banana peel waste for citric acid production by aspergillus niger. iop conf. series: earth environ sci. 364 012005. doi: 10.1088/1755-1315/364/1/012005. max b, salgado jm, rodriguez n, cortes s, converti a, dominguez jm. 2010. biotechnological production of citric acid. braz j microbiol. 41(4): 862-875. doi: 10.1590/s1517-83822010000400005. monrroy m, rueda l, aparicio, al, garcia jr. 2019. fermentation of musa paradisiaca peels to produce citric acid. j chem. 2019, article id 8356712. doi: 10.1155/2019/8356712. x sidauruk et al. microbiol indones volume 13, 2019 microbiol indones x papagianni m. 2007. advances in citric acid fermentation by aspergillus niger: biochemical aspects, membrane transport and modeling. biotechnol adv. 25(3):244–263. doi: 10.1016/j.biotechadv.2007.01.002. rohr m, kubicek cp, kominek j. 1981. citric acid. in biotechnology 27: 235-239. show pl, oladele ko, siew qy, zakry faa, lan jcw, ling tc. 2015. overview of citric acid production from aspergillus niger. front life sci. 8(3): 271-283. doi: 10.1080/21553769.2015.1033653. sukandar u. 2002. proses metabolisme. metabolism processes. [book]. bandung: departemen teknik kimia, itb. varshey as. 2016. production, comparative and quantitive analysis of citric acid by aspergillus niger using food waste as a substrate. j exp food chem. 2(4): 1-6. doi: 10.4172/2472-0542.1000121. page 1 page 2 page 3 page 4 page 5 tubagus haeru.pmd volume 3, number 2, august 2009 p 56-60 issn 1978-3477 *corresponding author, phone/fax: +62-21-7890962, e-mail: haeru_tb@yahoo.com the growing economic importance of the aquaculture industry worldwide and the need to find a new approach for a probiotic method has led to increasing interest in rapid and reliable methods for detection and identification of bacteria (nilsson and strom 2002). recently, nucleic acid-based assays have been studied intensively. one of methods being developed is that of 16s ribosomal rna genes sequencing. these assays are generally considered to be more accurate, more sensitive, inexpensive and more rapid to perform than those of more conventional variety, such as morphological, biochemical/physiological and serological assays, which led to various results of species resemblance in physiology and susceptibility to environmental exchanges (macrae 2000). broad-range pcr amplification of 16s rdna with universal bacterial primers and subsequent sequence analysis of cloned products is a widely used method in molecular biology. the method can be used for identification of microorganism identity to the genus or species level (tiirola et al. 2002) and construct phylogenetic analysis which plays on important role in taxonomic positioning. it can be undertaken by comparing the gene sequences with other bacteria sequences in the genbank database using basic local alignment search tool (blast) search and continue with method of neighbor-joining to construct a phylogenetic tree (saitou and nei 1987; holmes 2003). in this paper, we identified the selected bacterial isolates from l. vannamei culture system and gut environment using the 16s ribosomal rna gene sequence and constructed and analyzed the phylogenetic trees using the program clustal x. it is important to understand that genetic diversity of selected isolates of probiotic bacteria and their taxonomic position provides basic information for developing disease control strategies in the shrimp culture industry. materials and methods sampling of bacteria and isolation. bacterial isolates were obtained from the water, sediments and shrimp’s gut using healthy wild shrimps (15 ± 1 g) collected from four districts, i.e., pandeglang, serang, tangerang and karawang. sampling stations were determined randomly with 5 to 7 spots per pond. substrates were taken from 3 substrates, i.e. pond water, mud and shrimp’ gut following the procedures of hjelm et al. (2004). water aliquots of 500 ml and mud aliquot of 50 to100 g were taken using sterile plastic bags and stored in refrigerator at 4 °c. samples from shrimp’ guts were conducted by taking living shrimps to the laboratorium using closed transportation with air and water at a ratio 3:1. sample of 5% of obtained material were taken and then isolated and enriched on tsb medium with 2% nacl and incubated for 24 h at 30 °c before streaking bacteria on marine agar. samples from mud of 50% (w/v) were diluted using sterile distilled water, whereas samples from shrimp’s guts were taken by gut-scraping. all subsequent isolates were streaked on marine agar and incubated for 24 h at 30 °c. representative colonies were selected based on phenotype, gram staining and acidity of the medium. the selected isolates were cultured on slashmarine-agar and stored at 4-10 °c after separation into three groups, i.e. original culture, stock culture and working culture. development of pure cultures of isolates. nineteen selected isolates from the slash-agar stock were streaked in identification and phylogenetic analysis of bacterial isolates from litopenaeus vannamei shrimp culture system and gut environment based on 16srrna gene sequence data tubagus haeru rahayu 1,2*, indrawati gandjar1, etty riani3, iin siti djunaidah4, and wellyzar sjamsuridzal1 1department of biology, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia; 2department of aquaculture, sekolah tinggi perikanan jakarta; jalan aup pasar minggu, jakarta 12520, indonesia; 3faculty of fisheries and marine science, institut pertanian bogor, kampus darmaga, bogor 16680, indonesia; 4agency for human resources development of marine and fisheries, departemen kelautan dan perikanan, jalan mt. haryono kav. 52-53, jakarta 12770, indonesia selected bacterial isolates from a litopenaeus vannamei shrimp culture system and gut environment were assessed using 16s rrna gene sequencing method to identify their identity and to construct their phylogenetic relationship. in a preliminary study, a total of 19 isolates were selected as probiotics. these isolates were prepared using freeze and heat-shock method to obtain the dna template. pcr amplification of 16s ribosomal rna gene of isolates was carried out using bacterial universal primers 9f and 1510r and was sequenced using an automated dna sequencer. these gene sequences were compared with other gene sequences in the genbank database (ncbi) using a blast search to find closely related sequences. alignment of these sequences with sequences available from genbank database was carried out to construct a phylogenetic tree for these bacteria. most of the isolates obtained, i.e. 17 out of the 19 isolates, belonged to different species of bacillus, sharing 95 to 99% 16s ribosomal rna identity with the respective type-strain, whereas the remaining 2 isolates belonged to micrococcus sp. and micrococcus luteus, with 97 to 99% 16s rrna homology, consecutively. key words: bacillus, micrococcus, pcr amplification of 16s rrna gene, phylogeny microbiol indones 57 duplicate on the respective media (marine agar, difco, 55.1 g.l-1) plates and incubated at 30 ºc for 48 h. the isolates were then stored in a refrigerator for short periods until further use. preliminary characterization of isolated bacteria. characterization of isolates was done following the method of akhtar et al. (2008). colonies on respective media plates were examined using the light microscope and their characteristics i.e. color, form, elevation and margin of colonies were recorded. isolates were also characterized on the basis of gram staining. dna template preparation. dna templates were prepared by using the freeze and heat-shock method (sjamsuridzal and oetari 2003). incubated isolates (48 h) were taken using sterile microtips and put into 1.5 ml microtube containing 500 µl of nuclease-free water. the cell suspension was vortexed for 1 min and then frozen for 24 h. the suspension was then boiled using a water bath (>95 ºc) for 20 min. the cells suspension was centrifuged at 13 000 rpm for 15 min. the supernatant containing the dna was stored in a refrigerator until further use. phylogenetic analysis of isolates. polymerase chain reaction amplification of 16s ribosomal rna of isolates were carried out using following primers and set of conditions: forward primer (9f; 5'-gagtttgatcctggctcag-3' and reverse primer (1510r; 5’ggctaccttgttacga-3') (nilsson and strom 2002). each vial contained 1.25 µl of each primer, 15 µl rtg suspended with nuclease free water and 7.5 µl dna template in a total 25 µl reaction volume. the reaction mixture was heated for 3 min at 95 °c and then amplification was carried out in 35 cycles. each cycle was comprised of 30 sec at 95 °c, 15 sec at 55 °c and 1 min at 72 °c. the final extension was for 5 min at 72 °c followed by storage at 25 °c. the pcr product was purified using sodium acetate/ethanol precipitation to remove excess primers and free nucleotides. the next step was cycle-sequencing reaction, in which a similar process to amplification of 16s ribosomal rna was performed. each vial contained 1 µl big dye terminator ready reaction mix v.3.1, 0.5 µl primer 9f, 7.0 µl sequence buffer, 0.5 µl nuclease free water and 1 µl dna template in a total of 10 µl reaction volume. the reaction mixture was heated for 1 min at 96 °c and then amplification was carried out over 30 cycles. each cycle was comprised of 10 sec at 96 °c, 5 sec at 55 °c and 90 sec at 60 °c. the final step was storage at 25 °c. the pcr product was re-purified using sodium acetate/ethanol precipitation to remove excess dyeterminators, followed by denaturation using 15 µl hi-di formamide and heat denaturation at 95 °c for 5 min and then immediately transferred on ice. amplified 16s ribosomal rna fragments of about 1500 bp size were obtained from each isolate and were partially sequenced (around 500 bp) using an automated dna sequencer (abi 310 prism), following the procedure given by the manufacturer. the gene sequences were compared with others in the genbank databases using the ncbi blast (www.ncbi.nlm.nih.gov). gene sequences of 16s ribosomal rna of selected organisms were obtained from genbank and aligned with gene sequence of our isolates using a multiple sequence alignment program clustal x software (thompson 1997). the aligned sequences were used to construct a distance matrix, after the generation of 100 bootstrap sets, which was subsequently used to construct a phylogenetic tree using the neighbor-joining method (holmes 2003). results colony and cell morphologies of isolates. nineteen isolates of selected putative probiotic bacteria were examined for studies on the basis of the colony color and bacterial morphology (table 1). colony pigmentation of the isolates included white, light pink, pink-orange, light yellow, yellow and pinkish red. these colonies were dominated by circular forms and only 4 isolates had circular-tending to a irregular form. colony elevation was convex in all cases. all isolates had entire margins and gave gram positive staining. pcr amplification of 16s rrna genes. electrophoretic visualization of amplified 16s rrna genes from nineteen selected probiotics using primer 9f and 1510r is shown in table 1 some characteristics of different isolates of selected putative probiotic bacteria isolates code s 18 t 1 t 17 t 28 p 18 t 9 p 43 t 21 t 26 k 48 p 10 p 16 p 11 s 9 t 23 s 23 t 18 k 52 p 7 substrate water water gut gut gut gut water gut gut sediment sediment water water gut gut sediment water water sediment color white white white white white light pink light pink pink orange pink orange pink orange pink orange pink orange pink orange light yellow yellow pinkish red pinkish red pinkish red pinkish red glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy glossy surface forms circular circular circular circular circular circular circular circular circular circular tend to irregular circular tend to irregular circular tend to irregular circular tend to irregular circular circular circular circular circular circular entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire margins elevation convex convex convex convex convex convex convex convex convex convex convex convex convex convex convex convex convex convex convex positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive gram staining macroscopic observation of colony s18 until p1 shown the isolates code of selected indigenous bacteria volume 3, 2009 microbiol indones fig 1. the characteristic bacterial 16s gene amplicon of about 1500 bp was clearly visible in all cases. identity of isolates based on 16s-rrna-sequence method. nucleotides blast search of partial sequences (500 bp) of the 16s rrna for various isolates showed that most of the isolates had variable percentage identity with bacillus (95-99%), whereas the remainder, i.e. isolate s 23 and s 9, had 97% and 99% homology to micrococcus sp. and micrococcus luteus, respectively (table 2). construction of phylogenetic tree. a phylogenetic tree was developed by aligning 16s rrna sequences of different bacteria taken from genbank, ncbi and sequences of the isolates from this study (fig 2). isolate s 9 was related to micrococcus luteus clone b14. it shared 99% similarity in its 16s ribosomal rna sequence, whereas isolate t 23 was related to micrococcus sp. mali2 and kocuria sp. with 97% similarity. based on the morphology of colony (table 1), both isolates had similarity in patters of surface, form, margin, elevation and gram staining i.e., glossy, circular, entire, convex and gram positive, consecutively. the defining feature of both isolates was that isolate s 9 had a light yellow color, whereas t 23 was yellow. meanwhile, the cluster of the bacillus group was divided into six clusters. the first cluster consisted of isolates p 18 , s 18 , t 28 , t 1 and t 17 , which were closely related to bacillus pumilus (durck14, s6-05, au39 and nuc-f) and bacillus antrachis (me-12), with similarity of 98, 98, 99, 99, 95%, consecutively, and bootstrap value 99.9%. the second cluster consisted of two isolates, i.e. t 21 and t 26 . both isolates related to bacillus sp. wrb-4, with similarity 95 and 97% consecutively with bootstrap value of 100%. the third cluster was divided into two sub-clusters. the first was isolate p 7 , which was related to bacillus sp. mali10 with similarity of both 97% while the second sub-cluster was isolates s 23 and k 52 , which were close to bacillus sp. by231ydz-fq with a similarity of 96-97%. the fourth cluster was isolate t 18 . this was close to bacillus sp. k38t with a 99% similarity. the fifth cluster was represented by t 9 and p 43 which were related to bacillus subtilis s8-04 and bacillus megaterium tk1 with a similarity of 96 and 98% consecutively. the sixth cluster was again divided into sub-clusters. the first sub-cluster consisted of isolate p 10 with a 99% similarity and related to bacillus flexus llh. the second sub-cluster was represented by isolates k 48 , p 16 and p 11 , and related to bacillus flexus gs11 and bacillus sp. bsi20565 with similarities of 95, 97, 98 and bootstrap values higher than 60%. discussion this is a preliminary study on the characteristics of nineteen isolated colonies. colonies were grown on agar media and were visible after 12 h incubation at 30 °c. this is due to the presence of sufficient nutrient, appropriateness of the agar used and appropriateness of the culture period. marine agar (difco; 55.1 g l-1) is a specific agar for heterotrophic marine bacteria. it contains 2% (w/v) nacl. such conditions were appropriated for isolates which were obtained from shrimp ponds with a salinity of about 25 ppt. thus the physiological activity from the bacteria was not disrupted. the 48 h-incubation-period was adequate enough for bacterial growth. our study showed the colonies grew table 2. nearest relative of various selected isolates of probiotic bacteria substrateisolate s 18 t 1 t 17 t 28 p 18 t 9 p 43 t 21 t 26 k 48 p 10 p 16 p 11 s 9 t 23 s 23 t 18 k 52 p 7 water water gut gut gut gut water gut gut sediment sediment water water gut gut sediment water water sediment nearest relative bacillus pumilus isolate nuc-f bacillus pumilus strain s6-05 bacillus pumilus strain au39 bacillus pumilus strain durck14 bacillus pumilus isolate egu275 bacillus subtilis strain s8-04 bacillus megaterium strain tk1 bacillus sp. wrb-4 bacillus sp. wrb-4 bacillus flexus strain gs11 bacillus flexus isolat llh bacillus flexus strain gs11 bacillus sp. bsi20565 micrococcus luteus clone b14 micrococcus sp. “mali2” bacillus sp. by231ydz-fq bacillus sp. k38t bacillus sp. ‘mali10’ bacillus sp. ‘mali10’ dq833752 e u 6 2 4 4 2 9 ef032679 a m 7 7 8 1 9 1 ef633222 e u 6 2 0 4 1 2 e u 5 8 6 0 3 4 ef636891 ef636891 dq365587 dq333292 dq365587 e u 3 3 0 3 4 1 e u 1 9 6 5 3 1 ay 2 11 0 9 6 e u 0 7 0 3 7 2 a m 9 8 3 5 2 5 ay 2 111 0 4 ay 2 111 0 4 accession number homology (%) 9 8 9 9 9 5 9 9 9 8 9 6 9 8 9 5 9 7 9 5 9 9 9 7 9 8 9 9 9 7 9 6 9 9 9 7 9 7 s18 until p1 shown the isolates code of selected indigenous bacteria fig 1 visualization of amplification of 16s rrna genes from 19 selected using primer 9f and 1510r. m, marker; bp, base pair; s18 until p1 shown the code of selected indigenous bacteria isolates. 500 bp m s18 t 1 t 17 t 28 p18 t 9 p43 t 21 t 26 k48 p10 p16 p11 s9 t 23 s23 t 18 k52 p 7 ¢ 58 rahayu et al. microbiol indones 59 fig 2 the inferred relationship based on partial 16s ribosomal rna sequence, of selected putative probiotic bacteria (boldface) to other bacteria. the tree was rooted with escherichia coli as an out-group. scale bar represents the number of inferred nucleotide substitution per site. bootstrap values (1000 replicates) are shown at the nodes. well, with the typical color of bacteria. the results were in line with akhtar et al. (2008), in that the growth of bacteria will also be determined by the period of incubation by giving enough time to reach the optimum stage. on a molecular basis, all selected isolates belonged to the gram positive group. most of the isolates (17 isolates) were bacilli (89.7%), with the remaining two isolates (10.53%) being micrococci (table 1). it indicated that bacteria belonging to bacilli group were more prevalent in healthy l. vannamei shrimp ponds. this finding was in line with those of verschuere et al. (2000), balcázar et al. (2006) and geovanny et al. (2007) in which several bacteria were identified and can be considered as probiotic bacteria in aquaculture such as: bacillus, vibrio, pseudomonas, micrococcus, bacteroides and clostridium. gram negative bacteria were not found in the 19 culture isolates, after identification based on the sequence of 16s ribosomal rna. it was probably due to the fact gram negative bacteria which were successful obtained during sampling of bacteria and isolation were not showing the characteristics of probiotic bacteria i.e. do not improve shrimps’ survival rate during challenge test (rengpipat et al. 2000). isolates related to bacilli were represented by bacillus sp. (isolates code: t 21 , t 26 , t 18 , k 52 , p 7 , p 11 and s 23 ), b. pumilus (isolates code: s 18 , t 1 , t 17 , t 28 and p 18 ), b. subtilis (isolate code: t 9 ), b. megaterium (isolate code: p 43 ), and b. flexus (isolates codes: k 48 , p 10 and p 16 ). meanwhile, micrococcus, was represented by micrococcus sp. (isolate code: t 23 ) and m. luteus (isolate code: s 9 ). as the major species found, the dominance of bacilli can be comprehended since they have a wide range of diversity in physiological ability with respect to heat, acidity and salinity tolerance. bacilli can be found in water, soil and the digestive tracts of several organisms, including shrimps (l. vannamei) (rengpipat et al. 2000). although the genus micrococcus has not a wide range of tolerance compared to bacillus, it is however considered volume 3, 2009 0.05knuc 1 0 0 0 1 0 0 0 9 7 3 9 9 9 s 9 t23 9 9 9 micrococcus luteus clone b14 (eu196531) kocuria sp g3dm 46-eu037282 micrococcus sp mali2 (ay211096)7 3 3 p 1 8 s 1 8 t1 bacillus pumilus durck14 (am778191) t28 bacillus pumilus strain au39 (ef032679) bacillus anthracis-me-12 (eu6520) bacillus pumilus isolate nuc-fdq833752 bacillus pumilus strain s6-05 (eu624429) bacillus pumilus isolate egu275 (ef633222) t17 9 9 9 5 5 4 1 0 0 0 t9 bacillus sp.wrb-4 (ef636891) t21 t26 9 5 0 9 9 7 8 8 0 5 9 2 9 9 9 p 7 bacillus sp mali10 (ay211104) bacillus sp by231ydz-fq (eu070372) s 2 3 k52 bacillus sp k38t (am983525) t18 9 8 0 9 9 1 6 2 3 7 3 8 p 4 3 bacillus subtilis (eu620412) bacillus megaterium (eu586034) 6 5 3 6 5 2 bacillus flexus llh (dq333292) p 1 0 6 4 6 bacillus sp bsi20565 (eu330341) 9 1 3 bacillus flexus strain gs11(dq365587) 6 5 4 k48 p 1 6 p11 out-group escherichia coli strain svub1(am982782) microbiol indones60 rahayu et al. as a halotolerant group. micrococcus can grow at 5% (w/v) nacl, and manage to survive in the digestive tract as well. geovany et al. (2007) reported that m. luteus was the major component of the population of the gut of rainbow trout (oncorhynchus mykiss). phylogenetic analysis indicated that nineteen strains isolated from selected local probiotic bacteria can be identified as gram positive bacteria of the bacillus and micrococcus species (fig 2). when the type of escherichia coli used for an out-group, it was clearly seen that the distribution of the nineteen of the isolates fell into two big clusters, i.e. bacillus cluster and micrococcus cluster. one was composed of seventeen bacillus genera consisted of isolate p 18 , s 18 , t 28 , t 1 , t 17 , t 21 , t 26 , p 7 , s 23 , k 52 , t 18 , t 9 , p 43 , p 10 , k 48 , p 16 and p 11 , indicated 95% in bootstrap value. the other was composed of two micrococcal strains, s 9 and t 23 , indicated 100% in bootstrap value. the higher bootstrap value (≥95%) indicated that both groups were clearly distinct (meerak et al. 2007). based on the findings above, it may be concluded that the 19 isolates of bacteria can be used as probiotic bacteria and are useful for developing shrimp culture systems. bacillus and micrococcus are frequently used in shrimp aquaculture to improve the health status toward disease outbreaks through diet or giving out into water culture (gatesoupe 1999; verschuere et al. 2000; balcázar et al. 2006). acknowledgement this research was supported partly by a local graduate scholarship, awarded by the ministry of marine affairs and fisheries, and partly by the research and development for public affairs unit of sekolah tinggi perikanan. references akhtar n, ghauri ma, iqbal a, anwar ma, akhtar k. 2008. biodiversities and phylogenetic analysis of culturable bacteria indigenous to khewra salt mine of pakistan and their industrial importance. braz j microbiol 39:143-50. balcázar jl, blas i de, zarzuela ir, cunningham d, vendrell d, múszquiz jl. 2006. the role of probiotics in aquaculture. vet microbiol 114:173-86. hjelm m, bergh ø, riaza a, nielse j, melchiorsen j, jensen s, duncan h, ahrens p, birkbeck h, gram l. 2004. selection and identification of autochthonous potential probiotic bacteria from turbot larvae (scopthalmus maximus) rearing units. syst appl microbial 27:360-71. gatesoupe fj. 1999. the use of probiotics in aquaculture. a review. aquaculture 180:147-65. geovany gr, luis bj, shen ma. 2007. probiotics as control agents in aquaculture. a review. j ocean univ china 6:76-9. holmes s. 2003. bootstrapping phylogenetic trees: theory and methods. stat sci 18:241-55. macrae a. 2000. the use of 16s rdna methods in soil microbial ecology. braz j microbiol 31:77-82. meerak jhl, watanabe y, miyashita m, sato h, nakagawa y, tahara y. 2007. phylogeny of γ-polyglutamic acid-producing bacillus strains isolated from fermented soybean foods manufactured in asian countries. j gen appl microbiol 53:315-23. nilsson wb, strom m s. 2002. detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (t-rflp) analysis of 16s rna genes. dis aqua org 48:175-85. rengpipat s, rukpratanporn s, piyatiratitivorakul s, menasaveta p. 2000. immunity enhancement in black tiger shrimp (penaeus monodon) by a probiont bacterium (bacillus s11). aquaculture 191:271-88. saitou n, nei m. 1987. the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol biol evol 4:406-25. sjamsuridzal w, oetari a. 2003. rapid preparation of fungal and bacterial genomic dna for pcr. hayati 10:122-4. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids research 24:4876-82. tiirola m, valtonen et, kinnunen pr, kulomaa ms. 2002. diagnosis of flavobacteriosis by direct amplification of rrna genes. dis aqua org 51:93-100. verschuere l, rombaut g, sorgeloos p, verstraete w. 2000. probiotic bacteria as biological control agents in aquaculture. microbiol mol biol rev 64:655-71. 9 aris tri wahyudi (81-85).pmd characterization of acid-aluminium sensitive mutants of soybean symbiont bradyrhizobium japonicum generated by transposon mutagenesis aris tri wahyudi∗, andini purnawijaya, dini nurdiani, and tedja-imas department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia acid-aluminium sensitive mutants of symbiotic bacterium bradyrhizobium japonicum bj11 (designated as aas11) and kdr15 (designated as aas15) were constructed by mini-tn5 transposon mutagenesis to study genes involved in acid-aluminium tolerance (aat) in b. japonicum. transposon delivery was carried out through conjugation between b. japonicum strains as recipients and escherichia coli s17-1 (ë pir) carrying putminitn5km1 as a donor strain. the result showed that frequency of transconjugation was in the range of 6.7 x 10-7 to 7.1 x 10-6 cell per recipients. aas11 and aas15 mutants did not grow on ayanaba media (ph 4.5) containing 50 µµµµµm aluminium. these mutants remained able to form root nodules of siratro (macroptilium arthropurpureum) plants revealing genes interrupted by transposon which were responsible for acid-al tolerance did not correlate with the nodulation genes. strains tolerant to acid-aluminium and their mutants with a wild type sensitive to acidaluminium were characterized by accumulating phosphate and aluminium absorption. compared to the wild type acid-aluminium tolerant b. japonicum, there was approximately a threeto eight-times decrease in phosphate accumulation and a fiveto seven-times increase in aluminium absorption by these mutants. these results suggest that aluminium and phosphate contents in the bacterial cells may be involved in mechanisms of acid-al tolerance of b. japonicum grown in acid-aluminium stress conditions. key words: bradyrhizobium japonicum, acid-aluminium tolerance, transposon mutagenesis, aluminium absorption, phosphate intracellular _____________________________________________ _________________ ∗corresponding author, phone/fax: +62-251-622833, e-mail: aristri2003@yahoo.com bradyrhizobium japonicum is one of the nitrogen fixing bacteria which can symbiose with either soybean or siratro plants through root nodule formation. most of the nitrogen source required by soybean plants can be provided by this symbiosis. however, the high solubility of aluminium in acid soils can poison root nodule bacteria (flis et al. 1993). this condition will influence symbiosis between root nodule bacteria and the soybean plant. soil acidity can induce nitrogen deficiency in soybeans by preventing root nodule formation. low levels of phosphorus, calcium, and molybdenum, and high levels of aluminium, iron, and manganese are important factors of soil acidity and can be toxic for plants and root nodule bacteria. soil acidity generally increases the lag time or slows the growth rate of bacteria (keyser and munns 1979). to date, there are several reports revealing that some nitrogen fixing bacteria can survive on media with a ph 4.5 containing 50 µm al, 200 µm mn, ca 50 µm, and 5 µm po4(ayanaba et al. 1983; endarini et al. 1995; wahyudi et al. 1998). reports on the acid-al tolerant bradyrhizobial or rhizobial strains exerting tolerance to acid-al are not fully understood. acid-al tolerance appears to be related with amounts of phosphate supplemented in the media or its availability in the soil (flis et al. 1993). cellular phosphate has been shown in many different bacteria as one of the mechanisms to counter the effects of metal toxicity (keasling and hupf 1996; alvarez and jerez 2004). gemell et al. (1993) and watkin et al. (1997) reported that increasing the phosphate and calcium concentration in the media at low ph will increase the growth of the root nodule bacteria, bradyrhizobial, and rhizobial strains. increasing phosphate up to 100 µm was able to encounter toxicity of aluminium (50 µm) at ph 5.5 to b. japonicum in the soil (mukherjee and asanuma 1998). a transposon is a dna fragment which can transpose from one site to another site in the genome. one of the applications of the transposon is to mutate a gene (mutagenesis) and determine the physical location of genes of interest (guilhabert et al. 2001). in this study, the transposon mini-tn5km1 (de lorenzo et al. 1990) was used to generate acid-al sensitive mutants of b. japonicum. this transposon has a relatively high frequency of transposition, low insertionally specificity, and can be expressed in most gram negative bacteria. other advantages of the transposon mini-tn5km1 is the availability of a detailed genetic and physical map. this study demonstrates the production of acidaluminium sensitive mutants of b. japonicum generated by transposon mutagenesis as defined in root nodule formation, cellular phosphate content, and aluminium absorption in relation with genes interrupted by the transposon which are involved in acid-aluminium tolerance. materials and methods bacterial strains, media, and growth conditions. two, acid-al tolerant b. japonicum strains, bj11 and kdr15 (imas 1994), were routinely grown in yeast extract mannitol agar (yma) (mannitol 10 g l-1, k 2 hpo 4 0.5 g l-1, mgso 4 ·7h 2 o 0.2 g l-1, nacl 0.2 g l-1, yeast extract 5 g l-1) supplemented with congo red (cr) 0.0025% (w/v) at room temperature. acidal sensitive b. japonicum strain (bj13) was used as a control. escherichia coli s17-1 (λ pir), which carries microbiology indonesia, august 2007, p 81-85 volume 1, number 2 issn 1978-3477 putmini-tn5km1 (de lorenzo et al. 1990), was routinely grown on luria agar (la) (tryptone 5.0 g l-1, nacl 10 g l-1, yeast extract 5.0 g l-1, agar 15 g l-1) supplemented with kanamycin (50 µg ml-1) and ampicillin (50 µg l-1) at 37 oc. the siratro plant was used for root nodulation experiments. antibiotic resistance test. acid-al tolerant b. japonicum strains bj11 and kdr15 and e. coli s17-1 (λ pir) were tested for their antibiotic resistance. four antibiotics were used in the following concentrations: kanamycin (km) (50 µg ml-1), rifampicin (rif) (50 µg ml-1), ampicilin (amp) (50 µg ml-1), and tetracyclin (tc) (50 µg ml-1). all strains were plated on suitable media supplemented with each antibiotic. this work was carried out to determine selectable markers that will be used in transposon mutagenesis experiments. transposon mutagenesis and screening of acid-al sensitive mutants. the recipients, acid-al tolerant b. japonicum bj11 and kdr15, were grown on yma + cr (0.0025% b/v) + rif (50 µg ml-1). all cultures were incubated aerobically at room temperature agitated at 140 rpm for 60-72 h. the donor, e. coli s17-1 (λ pir), was grown on lb + km (50 µg ml-1) + amp (50 µg ml-1) and incubated aerobically with agitation at 140 rpm for 18-20 h at 37 oc. the transposon mini-tn5km1 carried by e. coli s17-1 (λ pir) was transferred to recipients by diparental mating conjugation in which the ratio of donor and recipient cells was 1:1 (~ 108 cells ml-1). all matings were carried out on membrane filters (0.45 µm) placed on la modified media (la omitting nacl 1.0 g l-1) without antibiotic, and incubated for 12, 18, and 24 h at room temperature. the transconjugants were plated on yma + cr (0.0025% w/v) + km (50 µg ml-1) + rif (50 µg ml-1). screening of acid-aluminium sensitive mutants of b. japonicum was performed on ayanaba media (ph 4.5; 50 µm al) (ayanaba et al. 1983). colonies which grew on yma + cr (0.0025% w/v) + km (50 µg ml-1) + rif (50 µg ml-1) but which failed to grow on ayanaba media were choosed for further analysis. root nodulation experiment. all acid-al sensitive mutants of b. japonicum and the wild type were tested for root nodulation on siratro plants. each of these mutants were inoculated on siratro (macroptilium atropurpureum) plants at a concentration of 106 cells ml-1. the siratro was planted in 25 mm diameter x 200 mm long reaction tubes using the medium as described by speidel and wollum (1980) and grown in a green-house. root nodule formation was examined from day 7 to day 30 after inoculation. cellular phosphate content. mutants and the wild type were grown on phosphate media as described by keyser and munns (1979). all cultures were incubated at room temperature and agitated at 60 rpm. when cells reached a density until gave an od620 reading of 0.7 (~109 cell ml-1), cells were pelleted (10,000 rpm, 15 min, 4 oc) and the subsequent pellet was then incubated with 10 ml of 25% hcl for 24 h and then diluted in sterile distilled water to give a total volume of 25 ml. a 2.0 ml aliquot was then added to 2 ml hno 3 and 1 ml molybdate-vanadate solution and incubated for 20 min. total phosphate content was assayed spectrophotometrically using the molybdate-vanadate method at λ 420 nm (mukherjee and asanuma 1998). the total phosphate content on bj13 was also measured as a control. analysis of aluminium absorption. acid-al sensitive mutants of b. japonicum and the wild type were grown on phosphate media as described by keyser and munns (1979). all cultures were agitated at 60 rpm and incubated at room temperature. when cells reached a density which gave an od 620 reading of 0.7 (ca 109 cells ml-1), cells were pelleted (10,000 rpm, 15 min, 4 oc) and then resuspended in 50 ml sterilized 2-(n-morpholino) ethanesulfonic acid (mes, ph 5.4) buffer containing 50 µm al for 30 h (mukherjee and asanuma 1998). after the relevant periods, cells were pelleted (10,000 rpm, 15 min, 4 oc). aluminium was assayed using an atomic absorption spectrophotometer at λ 303 nm. the total aluminium absorption on bj13 was also measured as a control. results antibiotic resistance test. all acid-al tolerant b. japonicum strains showed resistance to ampicilin and rifampicin at 50 µg ml-1. on the other hand, e. coli showed resistance to kanamycin and ampicilin at 50 µg ml-1 (table 1). the ability of e. coli to grow on la media supplemented by kanamycin and ampicilin was due to the fact that this bacterium carried the putmini-tn5km1 component which has gene resistance to kanamycin (km1) and ampicilin (amp). therefore, kanamycin and rifampisin were choosen as a selectable markers of the b. japonicum transconjugant generated by transposon mutagenesis. transposon mutagenesis. transposon mini-tn5km1 was transferred to the recipient b. japonicum by conjugation diparental mating. this generated b. japonicum mutants which were resistant to kanamycin. the plasmid putminitn5km1 has origin of replication (ori) from pr6k. it can replicate only in a host providing the pir factor (protein initiation of replication). transfer of km1 into b. japonicum was achieved by using the mob gene of the plasmid rp4 which was driven by the products of the tra gene provided in trans on the e. coli s17-1 (λ pir) chromosome. depending on the strains, the frequency of transconjugation varied between 6.7 x 10-7 to 7.1 x 10-6 per recipient (table 2). the table 1 growth of bradyrhizobium japonicum and escherichia coli on media containing antibiotics antibiotic (µg ml-1) tc 50 rif 50 ap 50 km 50 strain bj11 kdr15 bj13 e.c + + + + + + + + + + + bj11: bradyrhizobium japonicum bj11, kdr15: bradyrhizobium japonicum kdr15, bj13: bradyrhizobium japonicum bj13, e.c: escherichia coli s17-1 (λ pir), +: grew, -: did not grow. table 2 frequency of transconjugation of transposon minitn5km1 from e. coli s17-1 (λ pir) to acid-al tolerant b. japonicum as a function of mating time frequency of transconjugationa b. japonicum x e. coli s-17-1 (λ pir) bj11 kdr15 bacterial conjugation time (h) 1 2 1 8 2 4 6.7 x 10-7 1.1 x 10-6 7.1 x 10-6 3.4 x 10-6 5.1 x 10-6 6.3 x 10-6 afrequency of transconjugation is calculated per recipient. 82 wahyudi et al. microbiol indones highest frequency of transconjugation was obtained using a mating time as long as 24 h. screening of acid-al sensitive mutants and root nodule formation. acid-al sensitive mutants are transconjugants which grow on yma + cr (0.0025% w/v) + km (50 µg ml-1) + rif (50 µg ml-1), but which failed to grow on ayanaba media (ph 4.5, al 50 mm). one acid-al sensitive mutant generated from bj11 (designated aas11) and one from kdr15 (designated aas15) were obtained. all mutants and their wild type afforded the ability to form root nodules on siratros (table 3). cellular phosphate content. total cellular phosphate declined in acid-al sensitive mutants of b. japonicum when compared to their wild type. the amounts of total cellular phosphate of aas11 were 3x lower than its wild type, and relatively lower than bj13. similarly, the total cellular phosphate of aas15 mutant was 8x lower than its wild type and 4x lower than bj13 (fig 1). aluminium absorption analysis. aluminium was found in mutants (aas11 and aas15), the wild type acid-al toleran bj11 and kdr15, and the wild type acid-al sensitive strain bj13. however, the amounts of al absorbed by aas11, aas15, and bj13 were higher than those in the cells of the acid-al tolerant strains. the amount of aluminium absorbed by aas11 was 7x higher than for its wild type, whereas the mutant aas15 absorbed al 5x higher than its wild type. the amounts of al were about two 2x in the cells of the bj13 strain than in the cells of the acid-al tolerant strains (fig 2). discussion both of the strains bj11 and kdr15 grew large watery colonies. fuhrmann (1990) divided b. japonicum into three colony types which are large watery, large mucoid, and small dry. ayanaba et al. (1983) reported that colony types appeared to influence tolerance of the acid-al stress on soybean rhizobia. strain that formed small dry, pinpoint colonies were more sensitive to acd-al than those which formed large and ‘gummy’ colonies. the transposon mini-tn5km1 from plasmid putminitn5km1 was successfully introduced into acid-al tolerant b. japonicum strains. the suicide plasmid, putmini-tn5km1, contains the transposase gene in cis configuration outside of the transposable elements. this prevents secondary transposition when the plasmid is lost from the cell (de lorenzo et al. 1990). the highest frequency of transconjugation obtained was about 7.1 x 10-6 per recipient, with a mating time as long as 24 h at 1:1 ratio between e. coli s17-1 (λ pir) and b. japonicum. this frequency is higher than b. japonicum (5.7 x 10-9) using mini-tn5km1 for mutagenesis with a 1:10 ratio between donor and recipients (wahyudi et al. 1998). these results indicate that mating time and donor:recipient ratio are affecting conjugation efficiency. the differences in frequencies between strains may have been due to the inherent properties of the bacterial cell systems. similar responses have also been reported by wahyudi et al. (1998). dna polymerase i, membrane filters, and termination transcription factor can also influence transposition events (berg 1989). one acid-al sensitive mutant generated from bj11 (aas11) and one mutant generated from kdr15 (aas15) were obtained. the inability of mutants to grow on ayanaba media is due to the transposon mini-tn5 being inserted into the b. japonicum chromosome, especially in the genes involved in acid-al tolerance. transposon mutagenesis on r. meliloti wsm419 has also been reported by goss et al. (1990). these authors observed genes that controlled acid tolerance (act). the disruption of act genes resulted in r. meliloti wsm419 not being able to maintain its internal ph when grown on acid media. riccillo et al. (2000) reported that gsh gene (gene for glutamine synthetase) was presumed to play an important role for acid tolerance on r. tropici ciat899. tucker et al. (2002) reported that acid conditions table 3 results of root nodulation of the siratro plant by strains of bradyrhizobium japonicum (wild type and mutant) no. nodule formed day number/plant position strain wild type bj 11 kdr15 mutan aas 11 aas 15 1 5 1 5 1 5 1 3 4 4 6 5 3 pr/1 sr 3 pr/1 sr 6 pr 5 pr pr: primary root, sr: secondary root. 3 0 2 5 2 0 1 5 1 0 5 0 p o 4 c e ll u la r c o n c e n tr a ti o n (p p m ) bj11 aas11 kdr15 aas15 bj13 bacterial strain fig 1 cellular phosphate content of wild type acid-al b. japonicum bj11 and its mutant (aas11), wild type b. japonicum kdr15 and its mutant (aas15) and wild type acid-al sensitive b. japonicum bj13, grown on phosphate media ph 6.4. 1 . 0 0 . 8 0 . 6 0 . 4 0 . 2 0 bj11 aas11 kdr15 aas15 bj13 a lu m in iu m a b s o rp ti o n (p p m ) bacterial strain fig 2 absorption of aluminium by wild type acid-al b. japonicum bj11 and its mutant (aas11), b. japonicum kdr15 and its mutant (aas15) and wil type acid-al sensitive b. japonicum bj13, inoculated in mes buffer ph 5.4 containing aluminium (50 µm). volume 1, 2007 microbiol indones 83 will induce genes encoding for glutamate decarboxylase in e. coli mg1655. both strains of b. japonicum wild types, bj11 and kdr15, and their mutants, aas11 and aas15, were able to perform root nodulation on siratro plant. the ability of b. japonicum mutants to form root nodules on these plants indicates that the genes for nodulation (nod) have not been affected by inactivation of the genes involved in acid-al tolerance in b. japonicum. the amounts of total cellular phosphate were larger in the acid-al tolerant strains when compared with mutants or acid-al sensitive strains. this result indicates that there was a disruption of the gene for metabolising phosphate. aluminium was able to enter the cells of tolerant, sensitive, and mutants strains, but the amount accumulated intracellularly was less in the tolerant strains. johnson and wood (1990) reported that the absorption of aluminium by cells of both acid-altolerant and -sensitive strains took place however amounts of al absorbed by the sensitive strains was 2x higher than for the tolerant strains. thus, al tolerant strains appeared to have a mechanism to protect the cells by limiting the uptake of al from the culture solution. the differences among the strains in their ability to store phosphate internally and use it under adverse conditions could be one of the determining factors to acid-al stress tolerance or sensitivity (mukherjee and asanuma 1998). it seems that available phosphate plays a major role in detoxification of al lost intraand extra-cellularly. there are reports on the role of phosphate metabolism and metal detoxification in microbial systems. alvarez and jerez (2004) reported on the role of polyphosphate on copper tolerance in acidithiobacillus ferrooxidans. they propose that one of mechanisms for heavy metal tolerance involved the hydrolysis polyphosphate and the formation of metalphosphate complexes which are then transported out of the cell. keasling and hupf (1996) demonstrated that not only formation, but also the hydrolysis of polyphosphate play a significant role in detoxification of cadmium in e. coli. they also found that two enzymes i.e. polyphosphate kinase and polyphosphatase were involved in the detoxification of cadmium (keasling and hupf 1996). the same hypothesis was proposed by remonsellez et al. (2006) who showed that the ability to accumulate and hydrolyze polyphosphate may play an important role for a copper tolerance mechanism in members of genus sulfolobus. acid conditions also stimulated an increase in intracellular polyphosphate and cellular polyphosphate kinase activity of newly isolated environmental strain candida humicola (remonsellez et al. 2006). when the cells were grown at ph 5.5, phosphate removal was found to be 4.5 fold higher than when grown at ph 7.5. this increase in phosphate removal was associated with an increase in free intracellular polyphosphate (mcgrath and quinn 2000). mukherjee and asanuma (1998) reported that in al tolerant strains, cellular absorption of al was related positively to the release of phosphate from the cells and the accumulation of intracellular phosphate (pi). from our results it is clear that the mutants, aas11 and aas15 showed a weaker ability to store phosphate internally and absorbed aluminium more strongly than for their wild type. we therefore conclude that the phosphate metabolic status of bradyrhizobial cells seems to play a major role in countering al toxicity in acid and in extracellular p-limited conditions. acknowledgement this work was supported by a competitive grant (hibah bersaing) xiii from the directorate general of higher education (dikti) of the republic of indonesia (2005) to aris tri wahyudi. we are therefore grateful for this funding and support of this research. references alvarez s, jerez ca. 2004. copper ions stimulate polyphosphate degradation and phosphate efflux in acidithiobacillus ferrooxidans. appl environ microbiol 70:5177-5182. ayanaba a, asanuma s, munns dn. 1983. an agar plate method for rapid screening of rhizobium for tolerance to acid-alumunium stress. soil sci am j 47:256-258. berg de. 1989. transposon tn5. in. berg de, howe mm (eds). mobile dna. washington: asm pr. de lorenzo v, herrero m, jakubzik u, timmis kn. 1990. tn5 transposon derivatives for insertion mutagenesis promoter probing and chromosomal insertion of cloned dna in gramnegative eubacteria. j bacteriol 172:6568-6572. endarini t, wahyudi at, imas t. 1995. screening of indigenous strains of bradyrhizobium japonicum for acid-aluminium tolerance. hayati 2:74-79. flis se, glenn ar, dilworth mj. 1993. the interaction between aluminium and the root nodule bacteria. soil biol biochem 25:403-417. furhmann j. 1990. symbiotic effectiveness of indigenous soybean bradyrhizobia as related to serological, morphological, rhizobitoxine and hydrogenase phenotypes. appl environ microbiol 56:224-229. gemell lg, roughley rj, reed ml, hartley ej. 1993. screening of rhizobium leguminosarum bv. trifolii for adaptation to acid and neutral soils using a selective agar medium. soil biol biochem 25:1463-1464. goss tj, o’hara gw, dilworth mj, glenn ar. 1990. cloning, characterization, and complementation of lesions causing acid sensitivity in tn5-induced mutants of rhizobium meliloti wsm419. j bacteriol 172:5173-5179. guilhabert mr, hoffman lm, mills da, kirkpatrick bc. 2001. transposon mutagenesis of xylella fastidiosa by electroporation of tn5 synaptic complexes. am phytopathol soc 14:701-706. imas t, wahyudi at, tjahjoleksono a, saraswati r. 1994. seleksi galur-galur bakteri bintil akar kedelai unggul pada cekaman ph rendah dan kekeringan. lembaga penelitian dan pengabdian pada masyarakat (lppm) ipb bogor: laporan penelitian. johnson ac, wood m. 1990. dna, a possible site of action of alumunium in rhizobium spp. appl environ microbiol 56:36293633. keasling jd, hupf ga. 1996. genetic manipulation of polyphosphate metabolism effects cadmium tolerance in escherichia coli. appl environ microbiol 62:743-746. keyser hh, munns dn. 1979. tolerance of rhizobia to acidity, aluminium, and phosphate. soil sci soc am j 43:519-523. mcgrath jw, ouinn jp. 2000. intracellular accumulation of polyphosphate by the yeast candida humicola g-1 in response to acid ph. appl environ microbiol 66:4068-4073. mukherjee sk, asanuma s. 1998. possible role of cellular phosphate pool and subsequent accumulation of inorganic phosphate of the aluminium tolerance in bradyrhizobium japonicum. soil biol biochem 30:1511-1516. remonsellez f, orell a, jerez ca. 2006. copper tolerance of the thermoacidophilic archaeon sulfolobus metallicus: possible role of polyphosphate metabolism. microbiology 152:59-66. riccillo pm et al. 2000. glutathione is involved in environmental stress response in rhizobium tropici, including acid tolerance. j bacteriol 182:1748-1753. 84 wahyudi et al. microbiol indones speidel kl, wollum ag. 1980. evaluating of leguminous inoculant quality: a manual. north carolina state university: department of soil science. tucker dl, tucker n, conway t. 2002. gene expression profiling of the ph response in escherichia coli. j bacteriol 184:65516558. wahyudi at, suwanto a, imas t, tjahjoleksono a. 1998. screening of acid-aluminium tolerant bradyrhizobium japonicum strains: analysis of marker genes and competition in planta. aspac j mol biol biotechnol 6:13-20. watkin elj, o’hara gw, glenn ar. 1997. calcium and acid stress interact to affect the growth of rhizobium leguminosarum bv. trifolii. soil biol biochem 29:1427-1432. volume 1, 2007 microbiol indones 85 6.mi590-endang srimurni (grayscale version) available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.3.6issn 1978-3477, eissn 2087-8575 vol 6, no 3, september 2012, p 130-134 *corresponding author; phone: +62-281-638794, fax: 62281-631700, e-mail: endang_sk@lycos.com wolbachia pipientis is common bacterial endosymbiont of arthropods and filarial nematodes; they are maternally inherited, obligate, and intracellular bacteria (mcgraw et al. 2002). interest in wolbachia stems not only from its widespread distribution in arthropods, but also from the alterations of the mode of reproduction of its hosts caused by the bacteria uch as cytoplasmic incompatibility (ci), feminization (f), parthenogenesis (p), male killing and the popcorn-effect (yu peng et al. 2008; carrington et al. 2010). the expression of cytoplasmic incompatibility, feminization, and parthenogenesis also density in many hosts of wolbachia is influenced by environmental factors such as antibiotic and temperature (mouton et al. 2007; zhukova et al. 2008; jia et al. 2009; suh et al. 2009). breeuwer and werren (1993) reported that -1 tetracycline treatment (1 mg ml in 10% sucrose) on female nasonia vitripennis infected with wolbachia induced cytoplasmic incompatibility which had a dramatic effect on bacterial densities in their eggs. on the first day after treatment, tetracycline had effectively eliminated cytoplasmic bacteria and caused reduction or absent of the microorganism in the eggs. werren and jaenike (1995) reported that in drosophila recens, wolbachia was lost with 0.2 and/or 0.25 mg tetracycline per ml within two to three generations. however, 0.1 mg tetracycline per ml apparently was not effective in curing the flies of wolbachia. kambhampati et al. (1993) showed that tetracycline treatment of aedes albopictus results a partial, but significant restoration of compatibility. s t o u t h a m e r e t a l . ( 1 9 9 0 ) i n v e s t i g a t e d trichogramma harbouring parthenogenesis-inducing wolbachia. when it was cured by antibiotics tetracycline hydrochloride, sulfamethoxazole or rifampicin (100mg -1 ml honey) the result was the appearance of male offspring because the microorganisms responsible for the expression of cytoplasmic incompatibility, parthenogenesis, and feminization in many hosts of wolbachia is influenced by environmental factors such as temperature and antibiotics. therefore, it might also affect wolbachia inducing popcorn-effect. to examine the effects of temperature and antibiotic treatment on the life span of drosophila melanogaster harbouring the popcorn-effect inducing strain of wolbachia, flies were reared in different temperature such as at 20 and 29 °c, and cultured (from egg to adult stage) on a medium -1 containing tetracycline. the tetracycline-treated wolbachia was established by placing 0.25 mg ml of tetracycline in the media. the result showed that there was no difference in the life span of d. melanogaster infected with wolbachia popcorn-effect under untreated and treated condition with tetracycline at 20 °c. therefore, there is no popcorn-effect in the d. melanogaster at low temperature (20 °c). while the life span of d. melanogaster at 29 °c, where infected flies have a shorter life span than treated flies. popcorn-effect (shorter life span) was found at 29 °c. key words: cytoplasmic incompatibility, feminisation, parthenogenesis, popcorn-effect, tetracycline ekspresi ketidakserasian sitoplasma, partenogenesis, dan feminisasi pada beberapa inang yang terinfeksi wolbachia dipengaruhi oleh faktor lingkungan seperti suhu dan antibiotik. oleh karena itu, faktor-faktor tersebut kemungkinan juga berpengaruh pada wolbachia penyebab efek-popcorn. untuk menguji pengaruh suhu dan antibiotik terhadap lama hidup drosophila melanogaster yang mengalami efek-popcorn yang disebabkan oleh infeksi wolbachia, lalat-lalat tersebut dipelihara pada suhu 20 °c dan 29 °c, kemudian dikultur dari stadium telur -1 sampai menjadi dewasa pada media baik yang mengandung tetrasiklin (0,25 mg ml ) maupun yang tidak mengandung tetrasiklin. hasil menunjukkan bahwa antara lalat terinfeksi wolbachia efek-popcorn yang diberi pengobatan maupun yang tidak diobati pada suhu 20 °c, tidak ada perbedaan dalam lama hidupnya. oleh karena itu, tidak ada pengaruh efek-popcorn terhadap d. melanogaster pada suhu 20 °c. lama hidup d. melanogaster pada suhu 29 °c yang terinfeksi wolbachia lebih pendek dibandingkan dengan yang diberikan tetrasiklin. dengan kata lain terjadi efek-popcorn pada suhu 29 °c. kata kunci: efek-popcorn, feminisasi, ketidakserasian sitoplasma, partenogenesis, tetrasiklin effects of tetracycline and temperature on drosophila melanogaster infected with wolbachia inducing the popcorn-effect endang srimurni kusmintarsih faculty of biology, universitas jenderal soedirman, jalan dr soeparno 63 grendeng, purwokerto 53122, indonesia the lytoky were killed. however, treated with gentamycin, penicillin g and erythromycin, trichogramma male offspring did not occur. when trichogramma reared at an elevated temperature >30 , the females produced male offspring as well. stouthamer et al. (1993) reported that antibiotic treatment of trichogramma deion, t. pretiosum, t. cordubensis, and mucidifurax uniraptor resulted in reversion to sexual reproduction and elimination of the microorganisms. stevens (1989) reported that when larvae of tribolium confusum were reared at 37 , the compatibility was partly restored because some larvae become cured of the infection with wolbachia. subsequently stevens and wicklow (1992) reported that tetracycline-producing streptomyces and penicillium can cure tribolium flour beetles of infection with wolbachia. fujii et al. (2001) reported feminisationinducing wolbachia in ephestia kuehniella. those cured by tetracycline produced male offspring. in original work on the wmelpop strain in drosophila melanogaster by min and benzer (1997), all infected flies held at 29 died within 14 d. however, when longevity was assessed at 25 , infected flies lived twice as long, suggesting that the virulence of wmelpop depended on temperature (carrington et al. 2009) trpis et al. (1981) reported evidence for suppression of ci with high temperature in several species. for example, in the mosquito aedes scutellaris crosses between strains that are normally incompatible became compatible when larvae were raised at a temperature of 32.5 . hoffmann et al. (1986) reported that rearing larvae at 28 the incompatibility of drosophila simulans r (riverside) males and w (watsonville) females could be suppressed. stevens (1989) reported that when larvae of t. confusum were reared at 37 , the compatibility was partly restored because some larvae become cured of the infection with wolbachia. previous experiments showed that reproduction abnormalities such as cytoplasmic incompatibility, feminization, and parthenogenesis in many hosts of wolbachia are influenced by environmental factors such as temperature and antibiotics. therefore, it might also affect wolbachia inducing popcorn-effect. to determine the possible role of the environment on the popcorn-effect inducing wolbachia, d. melanogaster infected with wolbachia popcorn-effect will be cured -1 with 0.25 mg ml tetracycline (as in the previous experiment by min and benzer, 1997) and reared at 20 and 29 . the temperature of 20 is a close approximation of the optimal physiological °c °c °c °c °c °c °c °c °c temperature of d. melanogaster and the temperature at which the originally popcorn-effect was found is 29 . materials and methods analyses of the effect of antibiotic treatment. d. melanogaster infected with wolbachia inducing popcorn-effect obtained from the lab of henk r braig, school of biological sciences, university of wales, bangor, united kingdom. to examine the effect of antibiotic treatment, flies were cultured (from egg to adult stage) on a medium containing tetracycline. the tetracycline-treated strain was established by placing -1 0.25 mg ml of tetracycline in the media. the strain was treated for three generation and tested for infection status with pcr, utilizing 81f and 691r primers, specific for the wsp gene of wolbachia (braig et al. 1998). after adults emerged the male and female flies were reared separately at a density of 15 flies per vial (2.3 cm diameter and 9.5 cm in length) with 10 replicates. to examine the effect of temperature, flies were reared in different temperature such as at 20 and 29 . statistical analysis. an effect of tetracycline and temperature in d. melanogaster infected with wolbachia popcorn-effect would be manifest by its life-span. univariate analysis of variance (spss 10.1) was used to determine the influence of temperature and tetracycline treatment. in order to apply anova, the experimental data need to show a normal distribution and an equal variability. this is not the case in these experiments. the data therefore had to be transformed before a valid statistical analysis was possible. several transformation methods were tested including using the square root of the raw data. only the transformation of the raw data to their decimal logarithm was able to satisfy the requirements of anova. results the effect of tetracycline on the wolbachia inducing popcorn-effect was analysis by pcr and the results showed the untreated sample has band that positive for wolbachia (lane 1 and 2 of fig 1, fig 2). however, treated treated samples has no visible band (lane 3, 4, 5, and 6, fig 1), which indicates that wolbachia was not exist any longer . when d. melanogaster larvae untreated and treated with tetracycline were reared at different temperature, the life-span varied. flies reared at 29 °c exhibited a shorter life-span when they were infected with wolbachia than °c °c volume 6, 2012 microbiol indones 131 °c °c where infected flies have a shorter life span than treated flies. therefore, there is no popcorn-effect in the d. melanogaster at low temperature (20 ). the analysis shows highly significant difference (< 0.001) between treatment (treated and untreated) at temperature (29 and 20 ). however, there is no significant difference between treatment and sex (male and female). at 20 untreated and treated are not significant different. however, at 29 untreated and treated are significant different. discussion when d. melanogaster larvae untreated and treated with tetracycline were reared at different temperature, the life-span varied. flies reared at 29 exhibited a shorter life-span when they were infected with wolbachia than flies treated with tetracycline (fig 3). the difference in life-span was highly significant. however, there was no significant difference in sex (male and female). tetracycline eliminated wolbachia and restored the life-span of male and female flies at 29 . this was the same temperature for which the popcorn-effect of wolbachia was originally described (min and benzer, 1997). whereas when reared at 20 (fig 4) the life span of the flies was no longer significantly different between untreated and treated, although wolbachia could still be detected when the flies were reared at 20 °c (fig 2). therefore, apparently there was no popcorn-effect at 20 . it was probably that at 20 both host and bacteria function optimally, therefore no changes in life history parameters of fitness. in other words, the host perfectly controlled the bacteria under optimal physiological condition and wolbachia did not disrupt the symbiotic relationship. werren (1997) and mcgraw et al. (2001, 2002) tried to explain the virulence of the popcorn-effect as an 'over-replication' of these bacteria. mcgraw et al. (2002) investigated the question whether the popcorn-effect might reflect the initial increased virulence commonly observed in new host-parasite associations. they transferred the popcorn-effect wolbachia into a new, naive host, d. simulans, where indeed early on reproductive fitness costs were noted caused by initial high densities of wolbachia in the ovaries that were in excess of what was required for perfect maternal transmission. these fitness costs rapidly declined in the generations after transinfection. however, the popcorn-effect was practically independent of the observed attenuation of virulence. the authors reared the transinfected d. simulans at 26 in order to observe the popcorneffect. in our hands, a temperature of 29 have been deadly already for uninfected d. simulans flies. the authors failed to make any observations at physiological temperatures. °c °c °c °c °c °c °c °c °c °c flies treated with tetracycline (fig 3). the difference in life-span was highly significant. however, there was no significant difference in sex (male and female). fig 4 shows that there is no difference in the life span of d. melanogaster between untreated and treated. unlike the life span of d. melanogaster at 29 fig 1 detection of infection of drosophila melanogaster (w1118) by wolbachia untreated and treated with tetracycline. m: one kilobase ladder; 1 and 2: d. melanogaster (w1118) untreated; 3, 4, 5, 6: d. melanogaster (w1118) treated with tetracycline. fig 2 of melanogaster (w1118) by wolbachia untreated with tetracycline at 29 and 20 °c. m: one kilobase ladder; 1: d. melanogaster infected with popcorn-effect maintained at 29 °c; 2: d. melanogaster infected with popcorneffect maintained at 20 °c; line 3: positive control (amplification of ribosomal of nucleus of d. melanogaster infected with popcorn-effect at 20 °c ). detection of infection drosophila 132 kusmintarsih microbiol indones m 1 2 43 5 6 610 bp (wsp fragment) m 1 2 3 610 bp (wsp fragment) references braig hr., guzman h, tesh rb, o'neill sl 1994. replacement of the natural wolbachia symbiont of drosophila simulans with a mosquito counterpart. nature 367(6462): 453-55. doi:10.1038/367453a0. bourtzis k, braig hr. 1999. the many faces of wolbachia. in raout d and brouqui p, editors. rickettsiae and rickettsial diseases at the turn of the third millenium. paris: elsevier. 199-219. the potential of the popcorn-effect for the manipulation of vector insects have been repeatedly stressed, e.g., bourtzis and braig (1999). the ideal would be to express the early deaths associated with the popcorn-effect, for example, in anopheles mosquitoes to prevent the transmission of malaria parasites. the finding reporter here that the popcorn-effect was in effect a laboratory artefact and was not expressed under physiological conditions put an early end to these hopes. fig 4 percentage of adult survivors of drosophila melanogaster (w1118) infected with the popcorn-effect inducing wolbachia when untreated and treated with tetracycline and maintained at 20 °c. : % of adult untreated male w1118; : % of adult untreated male w1118; : % of adult male w1118 treated with tetracycline; : % of adult female w1118 treated with tetracycline. survivor survivor survivor survivor fig 3 percentage of adult survivors of drosophila melanogaster (w1118) infected with the popcorn-effect inducing wolbachia when untreated and treated with tetracycline and maintained at 29 °c. it's shows the life span of treated d. melanogaster is longer than that of untreated. : % survivor of untreated adult male w1118; % survivor of untreated adult female w1118; % survivor of adult male w1118 treated with tetracycline; % survivor of adult female w1118 treated with tetracycline. % s u rv iv o r 0 20 40 60 80 100 120 1 7 13 19 25 31 37 43 49 55 61 67 73 days after eclosion volume 6, 2012 microbiol indones 133 days after eclosion 1 6 11 16 21 26 31 36 41 46 51 0 20 40 60 80 100 % s ur vi vo rs min kt and benzer s 1997. wolbachia, normally a symbiont of drosophila, can be virulent, causing degeneration and early death. proc natl acad sci. usa.94(20):10792-96. doi: 10.1073/pnas.94.20.10792 . mouton l, henri h, charif d, bouletreau m, vavre f. 2007. interaction between host genotype and environmental conditions affect bacterial density in wolbachia symbiosis. biol lett. 3(2): 210-213. doi : 10.1098/rsbl.2006 0590. stevens l. 1989. environmental factors affecting reproductive incompatibility in flour beetles, genus tr i b o l i u m . j i n v e r t p a t h o l . 5 3 ( 1 ) : 7 8 8 4 . stevens l, wicklow dt. 1992. multispecies interactions affect cytoplasmic incompatibility in tribolium flour beetles. am nat. 140(4): 642-53. stouthamer r, luck rf, hamilton wd. 1990. antibiotics cause parthenogenetic trichogramma to revert to sex. proc natl acad sci. usa. 87(7): 2424-27. doi: 10.1073/pnas.87.7.2424. suh e, mercer dr, fu y, dobson sl. 2009. pathogenicitty of life-shortening wolbachia in aedes albopictus after transfer from drosophila melanogaster. app environ m i c r o b i o l . 7 5 ( 2 4 ) : 7 7 8 3 7 7 8 8 . d o i : 10.1128/aem.01331-09. trpis m, perrone jb, reising, m, parker kl. 1981. control of cytoplasmic incompatibility in the aedes scutellaris c o m p l e x . j h e r e d i t y. 7 2 ( 5 ) : 3 1 3 1 7 . d o i : cgi/content/short/72/5/313. werren jh. 1997. wolbachia run amok. proc natl acad sci. usa. 94(21): 11154-55. werren jh, jaenike j. 1995. wolbachia and cytoplasmic incompatibility in mycophagous drosophila and their relatives. j heredity. 75: 320-26. doi:10.1038/hdy.1995.140. yu peng, john e, nielsen, cunningham jp, mcgraw e a. 2008. wolbachia infection alters olfactory-cued locomotion in drosophila spp. applied and environ microbiol. 74: 3943–3948. doi:10.1128/aem.02607-07. doi:10.1016/0022 2011(89)90076-1. breeuwer jaj, werren jh. 1993. cytoplasmic incompatibility and bacterial density in nasonia vitripennis. genetics 135(2): 565-74. carrington lb, hoffmann aa, weeks ar. 2010. monitoring long-term evolutionary changes following wolbachia introduction into a novel host: the wolbachia popcorn infection in drosophila simulans. proc r soc b. 277(1690: 2059–2068. doi:10.1098/rspb.2010.0166. carrington lb, leslie j, weeks ar, hoffmann aa. 2009. the popcorn wolbachia infection of drosophila melanogaster: can selection alter wolbachia longevity effects? evolution 63(10): 248-2657. doi:10.1111/j.15585646.2009.00745.x. fujii y, kageyama d, hoshizaki s, ishikawa h, sasaki t. 2001. transfection of wolbachia in lepidoptera: the feminizer of the adzuki bean borer ostrinia scapulalis causes male killing in the mediterranean flour moth ephestia kuehniella. proc r soc london b-biol sci. 268(1469): 855-59. doi: 10.1098/rspb.2001.1593. hoffmann aa, turelli m, simmons gm. 1986. unidirectional incompatibility between populations of drosophila simulans. evolution 40(4): 692-701. jia fx, yang ms, yang wj, wang jj. 2009. influence of continous high temperatur condition on wolbachia infection frequency and the fitness of lipocelus tricolor (psocoptera: liposcelididae). environt entomol. 38 (5): 1365-1372. doi :10.1603/022.0380503 kambhampati s, rai ks, burgun sj. 1993. unidirectional cytoplasmic incompatibility in the mosquito aedes a l b o p i c t u s . e v o l u t i o n . 4 7 ( 2 ) : 6 7 3 7 7 . d o i : 10.2307/2410079 mcgraw ea, merritt dj, droller jn, o'neill sl. 2002. wolbachia density and virulence attenuation after transfer into a novel host. proc nat acad sci. usa. 99(5): 2918-23. doi: 10.1073/pnas.052466499. mcgraw ea, merritt dj, droller jn, o'neill sl. 2001. wolbachia-mediated sperm modification is dependent on the host genotype in drosophila. proc r soc london bbiol sci. 268(1485): 2565-70. doi: 10.1098/rspb.2001.1839. 134 kusmintarsih microbiol indones 1: 130 2: 131 3: 132 4: 133 5: 134 05. rizaldi.cdr vol.14, no.3, september 2020, p 117-120 doi: 10.5454/mi.14.3.5 short communication detection of salmonella sp. and escherichia coli on chicken meat at tamiang layang market akhmad rizaldi¹ and engki zelpina²* ¹veterinary medical expert of the department of agriculture in east barito regency, central 2 agricultural state polytechnic of payakumbuh, jl. raya negara, km 7 tanjung pati, harau, fifty cities, 26271 salmonella sp. and escherichia coli (e. coli) are the two most important pathogens because they are indicators of food safety and sanitation indicators, because they can potentially pose a high risk of foodborne disease. this study aims to look at the prevalence of salmonella sp. and e. coli in the tamiang layang market as a supervision of food safety. a total of 6 chicken breast samples were taken at all chicken traders in the tamiang layang market. testing the presence of salmonella sp. and e. coli using mc-media pad. the existence of salmonella sp. and e. coli in chicken meat at the tamiang layang market were 66.6% and 83.3%. the need to improve hygiene and sanitation for chicken traders. key words: chicken meat, escherichia coli, market, salmonella sp. salmonella sp. dan escherichia coli (e. coli) merupakan dua patogen terpenting karena merupakan indikator keamanan pangan dan indikator sanitasi pada pangan asal hewan, karena berpotensi menimbulkan risiko tinggi penyakit bawaan makanan pada pangan asal hewan. penelitian ini bertujuan untuk melihat prevalensi salmonella sp. dan e. coli di pasar tamiang layang sebagai pengawasan keamanan pangan asal hewan. sebanyak 6 sampel dada daging ayam yang diambil pada semua pedagang ayam di pasar tamiang layang. pengujian sampel tentang keberadaan salmonella sp. dan e. coli menggunakan mc-media pad. keberadaan salmonella sp. dan e. coli pada daging ayam di pasar tamiang layang masing-masing sebesar 66,6% dan 83,3%, perlunya meningkatkan higienitas dan sanitasi para pedagang ayam. kata kunci: daging ayam, escherichia coli, pasar, salmonella sp. microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-; fax: -; email: engkizelpina03@gmail.com therefore it is necessary to identify microbial contaminants in maintaining food safety from farm to dining table (safe from farm to table). contamination of pathogenic microbes in animal foods such as chicken is a health problem that needs attention. several types of pathogenic bacteria that can contaminate chicken meat are salmonella sp. and escherichia coli (e. coli). both types of bacteria are the main cause of foodborne disease. foodborne disease is a disease that occurs due to consuming food contaminated with microbes such as bacteria with common symptoms of abdominal pain, vomiting, diarrhea, dizziness, convulsions, and fever (cdc 2018). food safety assurance or foodstuffs have become demands along with the increasing public awareness of health. tamiang layang traditional market is the main market that provides chicken meat every day for the people of east barito. at the tamiang layang traditional market, there are eight stalls selling chicken meat. chicken meat can be contaminated in the slaughterhouse, at the point of sale, and during food is the most important basic human need in maintaining a healthy body, growth, and improving people's intelligence. meat is an important food ingredient in meeting nutritional needs. chicken meat is one of the sources of high-quality animal protein that is much in demand by the community because it is easily digested, can be accepted by the majority of people and the price is relatively cheap. chicken meat is muscle tissue obtained from chicken which is commonly used for food consumption purposes. the speed at which meat breaks down depends on the number of initial microbes. the greater the number of initial microbes in the meat, the faster the damage will be. microbial contamination in foodstuffs of animal origin and its products is a problem of major concern for consumers. there are many potential critical points for the occurrence of contact and entry of microbes into food from animal origin and their processed products, processing with incomplete cooking. the possibility of salmonella sp. and e. coli in chicken meat during the sales process at the traditional tamiang layang market, it is necessary to conduct research to detect the presence or absence of salmonella sp. bacterial contamination and e. coli in chicken sold in the tamiang layang traditional market. information about the contamination of these two bacteria in chicken meat products sold at the tamiang layang traditional market will be able to increase the awareness of the tamiang layang community in particular and east barito in general in buying and consuming chicken meat sold in traditional markets in east barito. a total of 6 chicken meat samples were obtained from chicken traders at the tamiang layang market, east barito. the samples taken were put into a sterile plastic and stored in a cooler box containing an ice pack and immediately taken to the laboratory for bacteriological testing. isolation and presence of escherichia coli. a total of 50 grams of chicken thigh meat was weighed then added with 450 ml of phosphate buffered saline (fbs), after which it was homogenized using a stomacher. prepare the mc-media pad e. coli test kit by opening the aluminum cap, then opening the transparent pad cover and then dropping 1 ml of a mixture of chicken meat with homogeneous fbs earlier. enter it slowly diagonally, then close it again. mc-media pad e. coli was inserted into a petri dish, given a code, then incubated at 35 ± 1 ° c for 24 ± 2 hours. aoac oma 966.24 (cer 070901) was used as a positive control. colonies and the presence of e. coli will appear as purplish-red colonies (millipore 2017). isolation of the presence of salmonella sp. a total of 10 grams of thigh meat samples were weighed then mixed with 90 ml of phosphate buffered saline (fbs) solution, after which it was homogenized with a stomacher. prepare the mc-media pad salmonella test kit by opening the aluminum cover, then opening the transparent pad cover and then dropping 1 ml of the chicken meat mixture with homogeneous fbs earlier. insert it slowly diagonally, then close it again. mcmedia pad salmonella sp. put into a petri dish, given a code, then incubated at 35 ± 1 ° c for 24 ± 2 hours. the colony and the existence of salmonella sp. will appear as a light blue colony (millipore 2017). based on bacteriological analysis in the laboratory to determine the presence of salmonella and e. coli from chicken meat obtained from the tamiang layang market, 6 samples were examined with the following results in table 1. table 1 shows that the presence of salmonella sp. and e. coli were positive in 4 samples, i.e. sample (1,3,5,6), whereas in sample 2 it was negative salmonella sp. and positive in e. coli and in sample 4 negative for both. salmonella sp. and e. coli are food pathogenic bacteria that are transmitted by fecal or oral. the presence of these bacteria in food products of animal origin is caused by many factors of livestock/animal origin, the process of slaughtering, the process of transportation of the product to the market/retail, as well as handling at the sales location (nel et al., 2004; regalado-pineda et al., 2020) based on studies conducted on the wet market environment and small-scale processing plants in perlis and penang, malaysia, salmonella sp. not only in meat but also found in utensils and equipment used in processing, washing containers, soaking containers, tables, drains and floors around wet markets and processing plants. this suggests that this source is a potential risk for transmission of salmonella sp. on chicken meat and its table 1 presence of salmonella sp. and escherichia coli in chicken table 2 prevalence of salmonella sp. and escherichia coli in chicken sample salmonella sp. escherichia coli sample 1 + sample 2 sample 3 + sample 4 sample 5 + sample 6 + prevalence (%) (n=6) bacteria positive negative salmonella sp. 5 (83.3) 1 (16.7) 4 (66.6) 2 (33.4) escherichia coli + + + + + 118 rizaldi et al. microbiol indones environment (nidaulah et al. 2017). poor sanitation and hygiene conditions and high humidity in the slaughterhouse and chicken meat processing industry are ideal for biofilm formation by salmonella sp. bacteria. these biofilms are long-lasting and tend to protect salmonella sp. from cleaners, so it is very risky to cross-contaminate salmonella sp. in chicken and chicken meat shops (smith et al. 2007). based on table 2, the prevalence of salmonella sp. 4 (66%) and e. coli 5 (83,35). the prevalence of e. coli is higher than that of salmonella sp. high prevalence e. coli in chicken meat shows the low application of sanitation and personal hygiene in producing chicken meat at the tamiang layang market. salmonella sp. and e. coli are indicators of food safety and sanitation indicators on the food of animal origin. meat is one of the main sources of contamination from salmonella sp. and e. coli, therefore the importance of controlling and surveillance of the presence of these pathogens in food of animal origin (hedican et al. 2007; kirk et al. 2015). the condition of the tamiyang layang market, which is located in an open stall for sales of chicken meat, is carried out on the sale site until the carcass is ready for sale without refrigeration resulting in crosscontamination of the presence of salmonella sp. and e. coli. sales of chicken meat in open markets without refrigeration have a chance to be contaminated by salmonella sp. and e. coli is higher than sales in closed and refrigerated spaces (nidaulah et al. 2016). in addition, the presence of salmonella sp. and e. coli are also reported to be found in several processed chicken meat such as grilled chicken, chicken sausage and shredded chicken in chicken porridge (amiruddin et al. 2017; kartika et al. 2014; zelpina et al. 2018). poor sanitary conditions in the market for sales in tropical countries also have the potential for high risk of contamination by salmonella sp. and e. coli which can cause foodborne disease and is a zoonotic disease for the community. the presence of salmonella sp. and escherichia coli in chicken meat, not only showed poor sanitary conditions during slaughter but also showed the health status of poultry as carrier carriers against salmonella sp. and escherichia coli. the presence of salmonella sp. and escherichia coli in chicken meat sold at the tamiang layang market, stated that the implementation of sanitary and personal hygiene in every stage of the chicken meat production process has the potential to cause disease in the community because it is a foodborne disease. references amiruddin rr, darniati, ismail. 2017. isolation and identification of salmonella sp. in roasted chicken from restaurant in syiah kuala, banda aceh. jimvet. 1(3):265-274. doi: 10.21157/jim%20vet..v1i3.3152. 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10.1093/ps/86.6.1241. zelpina e, purnawarman t, lukman dw. 2018. occurance salmonella sp. in shredded chicken meat of chicken porridge sold around bogor agricultural university, dramaga, bogor. jurnal penelitian pascapanen p e r t a n i a n . 1 5 ( 2 ) : 7 3 7 9 . d o i : 10.21082/jpasca.v15n2.2018.73-79. 120 rizaldi et al. microbiol indones page 1 page 2 page 3 page 4 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 15, number 2, june 2021 single nucleotide polymorphism in the rpob gene mycobacterium tuberculosis from papua-indonesia and its impact on rifampicin resistance: a whole-genome sequencing analysis bacterial population dynamics of natural fermentation of sumbawa mare's milk using metagenomic approach molecular identification of hg-resistant bacteria and their potential in reducing mercury contamination potential zoonotic faecal bacteria from sunda porcupine (hystrix javanica) and their antimicrobial resistance profiles immunogenicity of recombinant dna vaccine encoding nonstructural protein-1 dengue virus serotype-2 in balb/c mice yustinus maladan, tri wahyuni, and hana krismawati yoga dwi jatmiko, aditya ragil suharto, irfan mustafa, and siska aditya soraya fitria nasir, ani m. hasan, aryati abdul, and yuliana sarsa nisa, rifka a. safitri, nurul inayah, achirul nditasari, susiana purwantisari, rejeki ferniah, anang achmadi, taufiq nugraha, and sugiyono saputra fithriyah sjatha, elitha sundari pulungan, and tjahjani mirawati 37 45 54 61 69 page 1 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 13, number 1, march 2019 growth characteristics of chikungunya virus isolate from indonesia in various human cell lines in vitro isolation of a functional gene encoding homologous lysophospholipase from indonesian indigenous bacillus halodurans cm1 endophytic fungi in paraserianthes falcataria: production of indole acetic acid induced defense related enzyme activities of tomato plant by indigenous endophytic bacteria and challenged by ralstonia syzigii 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endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; prof. dr-eng. eniya listiani dewi, b. eng, m.eng; president: dr. siswa setyahadi; vice president: prof. fedik a rantam, phd; general secretary: diana nurani, m.si; vice general secretary: drs. nuki b nugroho, m.si; treasurer: dr. niknik nurhayati; dr. sylva abraham; scientific and publication committee: dr. debbie s. retnoningrum; dr. is helianti; dr. iman rusmana; prof. dr. marlina, apt; prof. dra. netty widyastuti, m.si; dr. ir. nur hidayat, mp; drh. mahdi abrar; dr. supriyadi; dr. astutiati nurhasanah; tarwadi, msc.; certification committee: dr. ir. trismilah, m.si; dr. erwahyuni endah prabandari; ir. dwi kusuma indriani, mp.; dra. dini ryandini, m.si; dr. ir. maman turjaman, dea; dr. ernawati giri rahma; dra. harmastini sukiman, m.agr; dr. purwati sppd, ph.d; dr. wahyu purbowasito; organization advancement and networking: dr. puspita lisdiyanti; prof. dr. ir. endang s. rahayu, ms; sri harjati suhardi, phd; lia yulia budiarti, mkes; dr. chaidir; dr. retno indrawati, drg., m.si; alit pangestu, stp; promotion and advocacy committee: dra. mg. isworo rukmi, m.kes; jimmy hariantono, phd; yawarsa; gianina; asri sulfianti, msi debbie s retnoningrum, 2020 iman rusmana, 2020 page 1 editorial board.pdf page 1 3.mi667-wahyu irawati available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.3.3issn 1978-3477, eissn 2087-8575 vol 6, no 3, september 2012, p 107-116 *corresponding author; phone: +62-21-5460901, e-mail: w.irawati3@gmail.com the discharge of heavy metals into the environment as a result of agricultural, industrial and military operations, and the effects of this pollution on ecosystems and human health have been of concern for some years (essa et al. 2002). copper, one of the most widely used heavy metals, is mainly employed in electrical and electroplating industries and in a larger amount is extremely toxic to living organisms. the presence of copper (ii) ions cause serious toxicological concerns, as it is known to deposit in brain, skin, liver, pancreas and myocardium (davis et al. 2000). copper cannot be destroyed and tends to accumulate in soils, plants, and animals, increasing their concentrations in the superior level of food chains. this metal has been shown to be toxic to copper resistant bacteria can be isolated from environments where copper levels are abundant from mining, industrial, or agricultural activities. the aim of this work was to study the molecular and physiological characteristics of indigenous copper resistant bacteria isolated from activated sludge in an industrial wastewater treatment plant in surabaya, indonesia. the bacterial isolates were designated as strains irc1, irc2, and irc4. phylogenetic analysis based on 16s rdna sequence analysis identified isolates irc1, irc2, and irc4 as acinetobacter oleivorans (98.41% similarity), acinetobacter pitii (97.22% similarity), and cupriavidus pauculus (96.99% similarity), respectively. the addition of 5 mm of cuso in the medium affected 4 morphological appearance of all isolates to green and undulate margin might be due to the survival mechanism of bacteria by absorbing the copper. this studies indicated that copper resistance mechanism of all isolates was facilitated through the bioaccumulation of copper inside the cell, especially on the membrane fraction and inside the cytoplasm, albeit at a limited amount. it was observed that isolates irc1, irc2, and irc4 were capable of -1 accumulating 137.23 , 364.66 , and 272.07 mg l of copper, respectively from the medium containing 8 mm cuso . the capability of isolates irc1, irc2, and irc4 to accumulate copper can be exploited in bioremediation 4 process for removing copper from industrial sewage. key words : 16s rdna, accumulation, bioremediation, copper-resistant bacteria, phylogenetic analysis bakteri resisten tembaga dapat diisolasi dari lingkungan yang terkontaminasi tembaga konsentrasi tinggi akibat aktivitas pertambangan, industri, maupun pertanian. penelitian ini bertujuan untuk mempelajari karakterisasi fisiologis dan molekular bakteri resisten tembaga asli yang diisolasi dari lumpur aktif pengolahan limbah industri di surabaya, indonesia. isolat bakteri diberi nama galur irc1, irc2, dan irc4. analisis filogeni berdasarkan analisis urutan basa gen 16s rdna mengidentifikasikan masing-masing isolat irc1, irc2, and irc4 sebagai acinetobacter oleivorans (kemiripan 98,41%), acinetobacter pitii (kemiripan 97.22%), dan cupriavidus pauculus (kemiripan 96,99%). penambahan 5 mm cuso mengakibatkan perubahan morfologi 4 koloni isolat bakteri menjadi hijau dengan tepi yang bergelombang diduga berkaitan dengan mekanisme pertahanan bakteri dengan cara mengabsorbsi tembaga. hasil penelitian menunjukkan bahwa mekanisme resistensi isolat irc1, irc2, dan irc4 terhadap tembaga adalah dengan cara mengakumulasi tembaga di dalam sel khususnya di bagian membran dan membatasi jumlah tembaga di dalam sitoplasma. masing-masing isolat -1 -1 -1 bakteri dapat mengakumulasi tembaga sebesar 137,23 mg l , 364,66 mg l , dan 272,07 mg l pada medium yang mengandung 8 mm cuso . kemampuan isolat irc1, irc2, and irc4 dalam mengakumulasi tembaga dapat 4 dikembangkan dalam proses bioremediasi untuk memindahkan tembaga dari limbah industri. kata kunci : 16s rdna, akumulasi, analisis filogeni, bakteri resisten tembaga, bioremediasi molecular and physiological characterization of copper-resistant bacteria isolated from activated sludge in an industrial wastewater treatment plant in rungkut-surabaya, indonesia 1 2 2 wahyu irawati *, triwibowo yuwono , joedoro soedarsono , 3 and hari hartiko 1 department of biology, faculty of science and mathematics, universitas pelita harapan , jalan mh thamrin boulevard 1100, lippo karawaci, tangerang 15811, indonesia; 2 laboratory of microbiology, faculty of agriculture, universitas gadjah mada, sekip unit i, yogyakarta 55281, indonesia; 3 laboratory of biochemistry, faculty of biology, universitas gadjah mada, jalan teknika selatan, yogyakarta 5528, indonesia vertebrata when contaminating dietary sources, usually in the range of 100-1000 (georgopoulus et al. 2001). since heavy metals are found in microbial habitats due to natural and environmental processes, microbes have evolved several mechanisms to tolerate the presence of heavy metals (adarsh et al. 2007). metal-tolerant bacteria could survive in these habitats and could be isolated and selected for their potential application in bioremediation of contaminated sites (piotrowska-seget et al. 2005). microorganisms and microbial products have been reported to efficiently remove soluble and particulate forms of metals, especially from dilute solutions, through bioaccumulation and therefore microbebased technologies provide an alternative to the conventional techniques of metal removal/recovery. microbes are capable of accumulating toxic metal ions by two well defined processes, i.e.: (i) biosorption: an energy-independent binding of metal ions to cell walls, and (ii) bioaccumulation: energy-dependent process of metal uptake into the cells. both live and inactivated microbial mass of bacteria, fungi and algae are utilized for removing toxic metal ions (raja et al. 2006). the bioremediation of heavy metals using microorganisms has received a great deal of attention in recent years, not only as a scientific novelty but also for its potential application in industry. conventional techniques for removing dissolved heavy metals include chemical precipitation, carbon adsorption, electrolytic recovery, ion-exchange, chelation and solvent extraction or liquid membrane separationall exhibit several disadvantages, such as high cost, incomplete removal, low selectivity, high energy consumption and generation of toxic slurries that are difficult to be eliminated. therefore, much attention has been paid to the removal of metal ions by microorganisms due to its potential applications in environmental protection and recovery of toxic heavy metals (zaki and farag 2010). isolation of bacteria from metal polluted environment would represent an appropriate practice to select metal resistant strains that could be used for heavy metal removal and bioremediation purposes (malik 2004). many indigenous organisms isolated from heavy metal contaminated sites had tolerance to heavy metals toxicity (yong et al. 2008). some reports have shown that indigenous microbes tolerate high heavy metal concentrations in different ways and may play a significant role in the restoration of contaminated site (carrasco et al. 2005; ge et al. 2009). it is important to study the indigenous -1 mg l 108 irawati microbiol indones microorganisms in heavy metal polluted sites. it may provide new insight into bacterial diversity under unfavorable conditions, new isolates and probably new genetic information on heavy metal resistance, which could be exploited in future for bioremediation (fabienne et al. 2003). the aim of this work was to study the molecular and physiological characterization of indigenous copper resistant bacteria isolated from activated sludge in an industrial wastewater treatment plant in surabaya, indonesia. materials and methods bacteria and growth media. isolates irc1, irc2, and irc4 were isolated from activated sludge in an industrial wastewater treatment plant in rungkut, surabaya, indonesia. the bacterial isolates demonstrated highly copper resistance with minimum inhibitory concentration (mic) of 6 mm to 7 mm cuso . bacteria were grown in salt base solution 4 (sbs) broth containing the following (per liter): k hpo 1.5 g; kh po 0.5 g; (nh ) so 0.5 g; 2 4 2 4 4 2 4 mg so .7h o 0.2 g, supplemented with appropriate 2 4 2 concentration of copper sulfate, and in medium without copper (irawati et al. 2003). cells were incubated at 37 °c in a shaker (200 rpm). growth was monitored by measuring optical density at 600 nm. phenotypic characterization of copper resistant bacteria. phenotypic characterization was conducted to analyze bacterial cell morphology, biochemical properties, and the ability to grow at various temperature (capuccino and sherman 2005). biochemical characterization included ulitization of catalase, oxidase and citrate, h s production, and 2 hydrolysis of gelatin. tests of the bacterial ability to use different sugars as a carbon source were also conducted; these included glucose, arabinose, lactose, sucrose, galactose, d-xilose, trehalose, melibiose, dmannosa, d-manitol, d-sorbitol, inositol, and glycerol. the bacterial isolates were tested for their abblility to grow on sbs medium agar at various temperature (4 , 25, and 37 °c) . phylogenetic characterization of copper-resistant bacteria. pure culture of the target bacteria was grown overnight in sbs broth medium for the isolation of genomic dna by using the method of spooling with a glass rod as described by zyskind and bernstein (1992) and 16s rdna was amplified by using the universal bacterial 16s rdna primers. the 16s rdna region was pcr-amplified using the following primer set: 5' tggctcagaacgaacgaacgctggcggc-3' (position 20 to 43 of the escherichia coli 16s rrna genes) and 5'-taccttgttacgacttcaccccagtg-3' (position 1482 to 1507 of the e. coli 16s rrna genes). the pcr mixture (25 μl) contained 1 μl template, 2.5 μl of 10x taq dna polymerase buffer, 3.5 mm mgcl , 5 μl 2 of dntp at 1 mm, 1,25μl primers (each) at 10 μm, and 0.2 μl of 0.2 μl 5u taq polymerase. the pcr was performed in a dna engine thermal cycler (ptc-200, bio-rad, usa) with a hot start performed at 95 °c for 3 min, followed by 30 cycles of 94 °c for 1 min, 59 °c for 1 min, and 72 °c for 2 min, followed by a final extension performed at 72 °c for 3 min. pcr products were analyzed by electrophoresis on 1.5% (w/v) agarose gel in 1x tae buffer supplemented with ethidium bromide (0.5 -1 μg l ). sequencing was performed at eijkman molecular biology institute (jakarta, indonesia). the 16s rdna sequence was compared against the genbank database as described in http://eztaxon-e.ezbiocloud.net/. based on the scoring index, the most similar sequences were aligned with the sequences of other representative bacterial 16s rdna regions by using clustal x software. phylogenetic tree was constructed by using neighbor-joining tree analysis. a consensus tree was generated using mega 5.1 software (molecular evolutionary genetics analysis) (downloaded from www.megasoftware.net). c e l l u l l a r f r a c t i o n a t i o n a n d c o p p e r accumulation. for determination of the copper content of fractions of bacterial cells, copper content of bacterial cells were determined according to the method of cha and cooksey (1991) with some modifications. cells were grown in sbs broth containing 8 mm of cuso and incubated at 37 °c with 4 shaking at 200 rpm. cells were collected in an appropriate phase of bacterial growth by centrifugation at 5000x g for 20 min at 4 ̊ c and washed several times with copper-free phosphate buffer. cells were suspended with 10 ml 30 mm tris-hcl ph 8 containing 0.2 mg of dnase i and 0.2 mg of rnase, and sonicated for 20 sec at 100 w. the lysate was incubated with 1 mg of lysozyme for 30 min at room temperature and ultracentrifuged at 77 600 x g for 2.5 h to separate cytoplasmic fractions in the supernatant from the pellet of cell membrane fraction. the pellet was suspended in 10 ml of water. the pellet and the supernatant was separately disrupted with hno at 100 3 ˚c. copper content was determined by using atomic absorption spectrophotometer at 324.9 nm. copper content of the whole cell was also determined. bacterial cells from the same cultures were freezedried for determination of dry weight and total copper content. results phenotypic characterization of copper resistant bacteria. phenotypic characteristics of isolates irc1, irc2, and irc4 are given in table 1. all three isolates were gram negative and rod shape motiled bacteria. for all isolates, there was catalase activity and there was no oxidase activity. h s production and citrate utilization 2 were negative for all isolates. hidrolysis of gelatin was detected for all isolates. all isolates grew at 4, 25, and 37 °c. isolate irc1 had ability to produce acid from oxidation or fermentation of glucose, l-arabinose, lactose, melibiose, d-mannose, galactose, and d-xylose. meanwhile, isolates irc2 and irc4 were incapable of producing acid from oxidation or fermentation of glucose, arabinose, lactose, sucrose, galactose, d-xilose, trehalose, melibiose, d-mannosa, d-manitol, d-sorbitol, inositol, and glycerol. when all isolates were grown on sbs agar, colonies of the bacterial isolates were whiteopaque, light yellow-tanslucent, and white-translucent, respevtively. it was shown that the colonies of all isolates turned blue when they were grown on sbs agar containing high concentration of copper (fig 1). the presence of high copper concentration also affected the appearance of the colony margin to undulate (fig 2). phylogenetic characterization of coperresistant bacteria. phylogenetic analysis based on 16s rdna sequence analysis identified isolates irc1, irc2, and irc4 as acinetobacter oleivorans (98.41% similarity), acinetobacter pitii (97.22% similarity), and cupriavidus pauculus (96.99% similarity), respectively (table 1). comparative analysis of the sequences with the available database showed that the isolates irc1 and irc2 were close to the members of genus acinetobacter, meanwhile isolate irc4 was close to the members of genus cupriadivus (fig 3). the nucleotide sequence data of isolates irc1, irc2, and irc4 have been deposited in the ncbi nucleotide sequence database (genbank) under the accession number of jx009133, jx009134, and jx398287, respectively. copper accumulation. it was shown in this study that copper resistance mechanism of the isolates was facilitated through the accumulation of copper on the cell. the results of copper accumulation on the whole cell, membrane fraction and cytoplasm showed that copper resistance mechanism in the bacterial isolates involves accumulation of this metal especially on the membrane fraction and restricts amount of copper inside the -1 cytoplasm up to 40.37-57.6 mg l (fig 4). the highest amount of copper accumulated by isolates irc1, irc2, volume 6, 2012 microbiol indones 109 (irawati et al. 2002; irawati et al. 2003). phylogenetic analysis based on 16s rdna sequence analysis identified isolates irc1, irc2, and irc4 as a. oleivorans (98.41% similarity), a. pitii (97.22% similarity), and c. pauculus (96.99 similarity), respectively (table 1). the 16s rdna gene sequence analysis identified isolates irc1 and irc2 as strain of acinetobacter sp. meanwhile, isolate irc4 has the possibility to be a novel species of the genus -1 irc4 was 137.23, 364.66, and 272.07 mg l in the medium containing 8 mm cuso , respectively. 4 discussion isolates irc1, irc2, and irc4 were highly copperresistant bacteria isolated from activated sludge in an industrial wastewater treatment plant in rungkutsurabaya, indonesia with mic of 6-7 mm cuso 4 microbiol indones110 irawati table 2 copper-resistant bacterial strain isolated from activated sludge in an industrial wastewater treatment plant in surabaya, indonesia. bacterial strains were identified based on the 16s rdna sequencing analysis . table 1 phenotypic characteristics of copper-resistant bacteria + : positive result; : negative result bacterial isolates strain irc1 strain irc2 strain irc4 morphological colony color gram staining cell morphology motility white negative rod + light yellow negative rod + white negative rod + biochemical catalase oxidase pigmentation h2s production gelatin agar test + + + utilization of glucose (acid) glucose (gase) kcn citrate + utilization of l-arabinose lactose sucrose galactose d-xilose trehalose melibiose d-mannose d-manitol d-sorbitol inositol glicerol + + + + + + growth at : 4 °c 25 °c 30 °c + + + + + + + + + strain accession number length (bp) species most related sequence similarity (%) irc1 jx009133 1389 acinetobacter oleivorans 98.41 irc2 jx009134 1371 acinetobacter pittii 97.22 irc4 jx398287 612 cupriavidus pauculus 96.99 cupriavidus due to their 16s rdna gene sequence similarities below 97%, the threshold recognized as delineating a genospecies (tindall et al. 2010). the genus acinetobacter is known for its ability to survive a wide range of atmospheric and environmental conditions (gusten et al. 2002; simor et al. 2002; jawad et al. 2004). in comparison with other bacteria, acinetobacter is most consistently observed in the environment. acinetobacter species are important biotechnological tools, and have been utilized volume 6, 2012 microbiol indones 111 fig 1 colony morphologies of copper-resistant bacteria. (1a, 2b, 3c = isolates irc1, irc2, irc4 on medium without copper, respectively. 4a, 4b, 4c = isolates irc1, irc2, irc4 isolates on medium containing 5 mm cuso , respectively). arrows 4 showed the difference in colony color. fig 2 micrograph of colonies margin on copper-resistant bacteria. (1a, 2b, 3c = irc1, irc2, irc4 isolates on medium without copper, respectively. 4a, 4b, 4c = irc1, irc2, irc4 isolates on medium containing 5 mm cuso , respectively). colony 4 margins were observed by phase contrast microscopy at 1000 times of magnification. arrows showed the defference of colony margin. syringae accumulated up to 115 to 120 mg of copper per g dry weight of cells (cooksey and azad 1992), while bacillus sp. strain cur21 accumulated cu of only -1 0.279 mg g biomass (kunito et al. 1997). copper binding on the cytoplasm of the isolates would seem to be saturated, which might be due to the delivery of copper ions to membrane fraction (fig 4). copper in its ionic form is a required trace element for most proand eukaryotic organisms, including human. while required in a small amount, copper can easily become toxic if present in an excessive amount. this toxicity is caused mainly due to the intrinsic properties of copper, as free copper ions undergo redox cycling reactions alternating between cu(i) and cu(ii). this also results in the transfer of electrons to hydrogen peroxide and the concomitant generation of hydroxyl radicals that readily attack and damage cellular biomolecules. recently, it was found that the majority of copper stress in e. coli, as indicated by hydroxyl radical formation, occurs within the periplasm, away from the cytoplasmic dna, and is thus coppermediated oxidative stress (macomber et al. 2007). the cytoplasm might thus be better protected from coppermediated oxidative stress, and indeed cells usually prevent accumulation of significant intracellular concentrations of free copper ions (magnani et al. 2008). moreover, free copper causes a depletion of the extensively in the synthesis of enzymes and other lifesustaining macromolecules and for degradation of recalcitrant compounds (chan et al. 2011). the genus cupriavidus (formerly ralstonia) is known as bacterium that contains a high number of heavy metal resistance genes making it an interesting model organism to study microbial responses to heavy metals (mergeay et al. 2003). c. pauculus has been reported as nickel resistant bacterium isolated from the rhizosphere of rinorea bengalensis (wall.), metalpercolated ultramafic ecosystem of andaman, india (pal and paul 2010). bacteria exposed to a high level of heavy metals have adapted to this stress by employing various resistant mechanism (ahmed et al. 2005). it is shown in this study that copper resistance mechanism in isolates irc1, irc2, and irc4 involves accumulation of this metal especially on the membrane fraction and restricts amount of copper inside the cytoplasm. the highest amount of copper accumulated by isolates irc1, -1 irc2, irc4 was 137.23, 364.66, and 272.07 mg l , respectively, in the medium containing 8 mm cuso , 4 respectively. copper tolerance and bioaccumulation has been studied in bacteria-(shakoori and muneer 2002). the ability of the isolates to accumulate copper was higher than previously known copper-resistant bacteria. two highly resistant strains of pseudomonas microbiol indones112 irawati fig 3 phylogenetic tree of strains irc1, irc2, and irc4 based on 16s rdna gene sequences. genbank accession numbers are given in parenthesis. bar, 1 substitutions per 100 nucleotides. 0.01 cellular sulhydryl pool causing a pronounced decrease in cellular viability (hiniker et al. 2005). the mechanisms of injury have forced bacteria to evolve different systems to tightly control intracellular copper level in order to counter the cation toxic effect (osman and cavet 2008; waldron and robinson 2009). the volume 6, 2012 microbiol indones 113 fig 4 growth and copper accumulation of copper-resistant isolates in medium containing 8 mm cuso . (a, b, c = growth of 4 irc1, irc2, and irc4, respectively. 1, 2, 3 = copper accumulation of irc1, irc2, and irc4, respectively. bars represent standard deviations (error bars) from three independent experiments. 30 36 42 time (h) a cc u m u la ti o n o f co p p er -1 (m g l ) 400 350 300 250 200 150 100 50 0 cell membrane fraction cytoplasm 3 time (h) cell membrane fraction cytoplasm 24 30 36 a cc u m u la ti o n o f co p p er -1 (m g l ) 400 350 300 250 200 150 100 50 0 2 a cc u m u la ti o n o f co p p er -1 (m g l ) 400 350 300 250 200 150 100 50 0 42 48 60 time (h) cell membrane fraction cytoplasm 1 0 24 30 36 b 0 24 30 36 42 time (h) time (h) time (h) a c of copper resistance in p. syringae is dependent on copper sequestration and accumulation in the periplasm and outer membrane (cha dan cooksey 1991; puig et al. 2002). similar result was described in burkholderia, alcaligenes, and methylobacterium species in which the ratio of green colonies for sorbing cu increased with the increasing of cu content of the medium (kunito et al. 1997). according to rouch et al. (1985) though copper is one of the toxic heavy metals for soil bacteria, it is also an essential nutrient cation at a trace level. thus a bacteria needs a mechanism to accumulate trace cu as well as a cu-resistance system. resistance by sequestration of this ion could be a more efficient mechanism to allow further growth of the bacteria in the presence of copper (cha and cooksey 1991). the ability of microbial strain to grow in the presence of heavy metals would be beneficial in the waste water treatment where microorganisms are directly involved in the decomposition of organic matter in biological processes for waste water treatment, because often the inhibitory effect of heavy metals is a common phenomenon that occurs in the biological treatment of waste water and sewage (filali et al. 2000). isolates irc1, irc2, and irc4 developed resistance mechanism by accumulating copper and protect itself from toxic concentration of copper ions while still ensuring that these ions met their nutritional requirements. these mechanism could be utilized for detoxification and removal of heavy metals from polluted environment. such copper resistant bacterial are very useful in biotechnology for the remediation of metal contaminated environments and can also be used in the construction of biomarkers for detection of the presence of metals. acknowledgments this research was funded by university research for graduate education (urge), the searca thesis grant, and the habibie center thesis grant. references adarsh vk, mishra m, chowdhury s, sudarshan m, thakur ar, ray cs. 2007. studies on metal microbe interaction of three bacterial isolates from east calcuta wetland, onlone. j biol sci. 7(2): 80-88. doi: 10.3844/ojbsci.2007.80.88. ahmed n, nawaz a, badar u. 2005. screening of copper tolerant bacterial species and their potential to remove copper from the environment. bull environ contam toxicol. 74(2): 219-226. doi: 10.1007/s00128-004-0573-z. level of free copper within a cell must be limitted, and the transport of copper into cells and its transfer to copper-requiring enzymes needs to be highly regulated (saxena et al. 2002). copper ions within the cytoplasm cause enzyme damage (macomber and imlay 2009). free copper ions participate in redox reactions that generate hydroxyl radicals, which are highly reactive species that cause lipid peroxidation, nucleic acid cleavage, and protein damage. as such, virtually all cells have developed sophisticated homeostatic mechanism to tightly control copper uptake and its mobilization to appropriate target proteins and compartments. to ensure that sufficient copper is acquired to drive essential biochemical reactions yet prevent accumulation to levels that e n c o u r a g e h a r m f u l r e d o x c h e m i s t r y, c e l l s homeostatically control copper via dedicated proteins that facilitate copper uptake, distribution, and efflux. these homeostatic mechanisms are regulated through cellular copper sensing mechanisms 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rcular chromosomes. j b act er iol. 171(11):5850-5859. journal with doi number juniastuti, aksono eb, utsumi t, yano y, soetjipto, hayashi y, hotta h, rantam fa, kusumobroto ho, ingelusida m. 2010. analyses of precore and core promoter mutations of hepatitis b virus in patients with chronic hepatitis b in surabaya, indonesia. microbiol indones. 4(3):143-148. doi:10.5454/mi.4.3.8. journal with different language kramadibrata k, gunawan aw, aradea nn. 2005. perkembangan spo ra [t he acaul osp ora fo vea ta development of 's spore]. j mikrobiol acaulospora foveata indones. 10(2):79-80. electronic journal helianti i, nurhayati n, ulfah m, wahyuntari b, setyahadi s. 2010. constitutive high level expression of an endoxylanase gene from the newly isolated bacillus subtilis aq1 in . j biomed biotechnol. 12 p [on escherichia coli line]. doi:10.1155/2010/980567. patent carlson tl, peters em, inventors; cargill, inc (wayzata, mn), assignee. 2002 nov 5. low ph lactic acid fermentation. united state patent. us 6,475,759. conference proceeding widiastuti h. 2011. proses interaksi fungi mikoriza dengan kelapa sawit pada tanah masam [interaction process between arbuscular mycorrhizal fungi and oil palm in acid soil]. in: budi sw, turjaman m, mardatin nf, nusantara ad, triesilawati o, sitepu ir, wulandari as, riniarti m, setyaningsih i, editors. percepatan sosialisasi teknologi mikoriza untuk mendukung revitalisasi pertanian, perkebunan, dan kehutanan. 2nd indonesian congress and seminar on mycorrhizae; 2007 jul 19-20. bogor (id). bogor: ami. p 96-101 . dissertation widiyanto t. 2005. selection of nitrification and denitrification bacteria for bioremediation in shrimp farm [dissertation]. bogor (id): institut pertanian bogor. page charges permi members. page charge is idr 500 000 per printed article up to 4 pages. additional page will be charge idr 150 000 per printed page without photo(s). page with black and white photo(s) will be charged idr 500 000 and color photo(s) will be 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alamat pengiriman (address) phone/fax/email pilihan berlangganan, tidak termasuk ongkos kirim (choose of subscription, not including package and postage) ) indonesia [ ] individual: 1 yr. rp 150.000, [ ] institution: 1 yr. rp 240.000, foreign country [ ] individual: 1 yr. us$ 25.00 [ ] institution: 1 yr. us$ 45.00 pengiriman biaya (method of payment) [ ] tunai/cash [ ] wesel/bank draft rekening /transfer [ ] bank mandiri pembayaran melalui rekening (please transfer to) bank mandiri cabang menara thamrin, jakarta permi rek no 103-0002080774 kontak person: netty widyastuti; is helianti telp: +62-21-7560536 ext 7119 ; fax: +62-21-7560694 email: microbiology.indonesia@gmail.com page 1 page 2 blanko pendaftaran anggota.pdf page 1 form berlangganan 2020.pdf page 1 01. utama.cdr vol.13, no.4, december 2019, p 109-117 doi: 10.5454/mi.13.4.1 antibacterial activity test of indigenous yeast from sapodilla fruit against staphylococcus aureus and escherichia coli * gemilang lara utama , mutiara nabila, heni radiani arifin, elazmanawati lembong, and tita rialita faculty of agro-industrial technology, universitas padjadjaran, west java, indonesia. the research aimed to identify indigenous yeast antibacterial activity from sapodilla fruit against escherichia coli and staphylococcus aureus, which conducted by experimental methods and followed by descriptive analysis. this study was done by the isolation of indigenous yeast, macroscopic and microscopic identification, yeast identification using rapid yeast plus system, antibacterial test by measuring the clear zone diameter, testing of pathogenic bacteria viability against indigenous yeast and identification of organic acid produced by yeast. the results of yeast isolation obtained 1 isolate (s.cereviseae 1) from fruit and 3 isolates form sapodilla skin (s.cereviseae 2, candida famata, and pichia anomala) which had antibacterial activity against e. coli and s. aureus except c. famata isolates. isolates with the largest antibacterial activity against e. coli and s. aureus based on the clear zone diameter were s. cerevisiae (2) isolates. the results of organic acid analysis by hplc found that -1 s.cerevisiae (2) isolate produced the highest organic acid namely acetic acid as much as 2.442 mg ml . key words : antibacterial, organic acid, sapodilla fruit, yeast penelitian ini bertujuan untuk mengidentifikasi aktivitas antibakteri khamir indigenous buah dan kulit buah sawo terhadap bakteri escherichia coli dan staphylococcus aureus yang dilakukan dengan metode eksperimental dan data yang diperoleh dianalisis secara deskriptif. tahapan penelitian dilakukan dengan isolasi khamir indigenous, pengamatan khamir secara makroskopis dan mikroskopis, identifikasi khamir dengan rapid yeast plus system, pengujian aktivitas antibakteri khamir dengan pengukuran diameter zona hambat, pengujian viabilitas sel bakteri patogen terhadap khamir, dan pengujian metabolit asam organik yang dihasilkan oleh khamir. hasil isolasi khamir didapatkan 1 isolat pada bagian buah dan 3 isolat pada bagian kulit buah sawo. setelah diidentifikasi didapatkan 2 isolat saccharomyces cerevisiae, dimana isolat s.cerevisiae (1) merupakan hasil isolasi pada bagian buah sawo, 1 isolat candida famata, dan 1 isolat pichia anomala yang memiliki aktivitas antibakteri terhadap bakteri e. coli dan s. aureus kecuali isolat c. famata. s. cerevisiae (2) merupakan isolat khamir indigenous yang menghasilkan aktivitas antibakteri tertinggi terhadap bakteri e. coli dan s. aureus karena menghasilkan asam-asam organik jenis laktat, asetat, sitrat, dan malat dimana asam asetat adalah jenis asam -1 organik tertinggi dengan jumlah 2,442 mg ml . kata kunci : antibakteri, asam organik, buah sawo, khamir microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-81220272894; email: lugemilang@gmail.com pathogenic bacteria that cause diarrhea (gómez-garcía et al. 2019; utama et al. 2015). some bacteria that cause diarrhea like staphylococcus aureus and eschericia coli. these bacteria are found in food and can potentially cause infection and food intoxication so that the food consumed can cause diarrhea and even poisoning for consumers (putri et al. 2015). the results of research by mukhriani et al. (2017), sapodilla fruit extract was able to inhibit the growth of s. aureus with an optimum concentration of 1500 ppm -1 or 1.5 mg ml , while the results of arsyad and annisa (2016) the minimum inhibition concentration of sapodilla fruit extract which could inhibit total e. coli growth is 22.5% (v / v). the antibacterial properties are not only from the compounds contained in the fruit, but can also come from microorganisms such as yeast contained in the fruit. romano et al. (2019) states that yeast can be sapodilla (achras zapota l.) is one type of potential fruit plant that grows in indonesia. sapodilla fruit consumption in indonesia is growing rapidly along with the easy of planting sapodilla and sapodilla plants which can produce fruit throughout the year (ying et al. 2017). sapodilla fruit known as an herbal medicine that can cure various diseases, one of which is diarrhea. sapodilla fruit contains flavonoids, saponins and tannins, besides sapodilla fruit also contains organic acids such as citric acid and malic acid (murnisyazwani and rabeta 2019). these compounds are known to have antibacterial properties. according to jenie (1996) organic acids show antimicrobial activity against many pathogenic microorganisms, including found in a place that is rich in sugar content, for example in fruit. sapodilla fruit has a sugar content of 14% (jadhav 2018). the sugar content in sapodilla fruit can act as a substrate for yeast growth. the optimum sugar concentration for yeast growth is 14-18% (ranalli 2007). it is suspected that sapodilla fruit has yeast which can inhibit the activity of s. aureus and e. coli bacteria like antibacterial activity of sapodilla fruit extract. the antibacterial properties of indigenous yeast from sapodilla fruit are derived from the results of the yeast's own metabolism. according to raftari et al. (2009), the effect of inhibition on pathogenic bacteria by yeast is largely due to the accumulation of organic acids, where acid will cause a decrease in ph to below the ph range of bacterial growth where these acids are not dissociated and can diffuse rapidly into in pathogenic cells that cause cells to become damaged. different types of yeast can produce organic acids with different antibacterial activity. therefore, in this study we aimed to identify indigenous yeast antibacterial activity from sapodilla fruit against escherichia coli and staphylococcus aureus, which conducted by experimental methods and followed by descriptive analysis. materials and methods chemical materials. the raw materials used in this study include the skin and sapodilla fruit which are 3 months old, s. aureus, e. coli, na media (nutrient agar), nb media (nutrient broth), yma media (yeast and mold agar), yeast extract (kraft f o o d ) , a n t i b i o t i c s ( a m o x i c i l l i n 5 0 0 m g ) , chloramphenicol 500mg, aquades, physiological nacl,70% alcohol, msa (mannitol salt agar), emb (eosin methylene blue), 1% bacl2, 1% h2so4, citric acid, tartaric acid, maleic acid, oxalic acid, lactic acid, and acetic acid. the research method used is the experimental method using descriptive analysis. yeast isolation. yeast is isolated from the skin and sapodilla fruit. 1 gram of sample is diluted using physiological nacl solution of 0.85% until dilution to -3 10 100 µl of each dilution was inserted into the petri dish then poured yeast and mould agar (yma) media -1 with a composition consisting of 3 g l malt extract -1 agar and 3 g l yeast extract agar and incubated for 48 hours at room temperature. characterization of yeast isolates was carried out by observing the physical characteristics macroscopically and microscopically from yeast isolates (balia et al. 2018; ruriani et al. 2012). yeast identification. identification of yeast types is done using the rapid yeast plus system. the results of the color change data are inputted into the website and found yeast isolates (utama et al. 2016). antimcrobial activity test. yeast colony swabs in 20 ml of yeast and mold agar (yma) media aseptically and incubated for 48 hours at room temperature. swab liquid culture staphylococcus aureus and escherichia coli on nutrient agar (na) evenly. plug aseptically yeast agar plate, use a sterile forceps or needle to carefully pick up the plug and place them onto each na plates. incubate the na plates at 30°c for 48 h then diameter of the clear zones were measured at 24 and 48 hours (roostita et al. 2011). determination of antibacterial indigenous yeast activity on viability of test bacteria. fresh cultures of yeast and bacteria were inoculated as 4 -1 follows: 190 μl at a concentration of 1 × 10 cells ml of s. aureus and e. coli with 10 μl at a concentration of 6 -1 3 × 10 cells ml of each yeast, including the following controls: (i) 200 μl of bacterial culture at a 4 -1 concentration of 1 × 10 cells ml , (ii) 198 μl of 4 -1 bacterial culture at a concentration of 1 × 10 cells ml with 2 μl of amoxicilin at a concentration of 100 mg -1 ml . incubation at 30 c for 48 hours. 1 ml suspension of e. coli bacteria was platted in 20 ml of emb (eosin methylene blue agar) media while the suspension of s. aureus bacteria in msa (mannitol salt agar) media at t = 0, 12, 24, 36, and 48 hours. incubation was carried out at 37c for 24 hours and tpc calculations were carried out(acuña-fontecilla et al. 2017). identification of indigenous yeast organic acid production using hplc. yeast samples that have been dissolved in nb media added with yeast extract are filtered and inserted through the injector. the data is a chromatogram that displays retention times in the form of sums of peaks and surface area compared between standard organic acids and organic acids in the sample (kim et al. 2018). results indigenous yeasts identification. based on the results of the yeast isolation of indigenous sapodilla fruit, 1 yeast isolate from sapodilla fruit and 3 yeast isolates from the sapodilla skin were observed macroscopically and microscopically. all data from macroscopic observations have characteristics such as yeast, which is white, round (circular) with prominent elevation in the middle of the colony (ambonate), 110 utama et al. microbiol indones volume 13, 2019 microbiol indones 111 table 1 microscopic and macroscopic characteristics of sapodilla indigenous yeasts smooth or glossy texture (glistening) and full (entire) edge on s.1, s.2, and s.4, while in s.3 has a flat elevation. based on microscopic observations, the four yeast isolates have a round to oval shape with a range of 3-10 µm. yeast isolates were then purified to form pure colonies which could be used to identify the types of yeast isolates by biochemical tests using the rapid yeast plus kit. based on the results of identification, three indigenous yeast species from four isolated isolates were obtained. there are two yeast isolates of saccharomyces cerevisiae which isolated from fruit (s.1) and sapodilla skin (s.4), other isolates were candida famata (s.2), and pichia anomala (s.3) that isolated from sapodilla skin. indigenous yeasts antibacterial activities. the diameter of clear zone formed is classified into weak (d=0-3 mm), medium (d=3-6 mm), and strong (d>6 mm) (pan et al. 2009). based on the fig. 1, it known the yeast that has strong antibacterial activity against e. coli is s.1 (s. cerevisiae 1) and s.4 (s. cerevisiae 2) isolates. s.3 isolates (p. anomala) have weak antibacterial activity, whereas p.2 (c. famata) isolates do not form clear zone diameters, so it can be said that these isolates do not have antibacterial activity. the antibacterial activities towards for s. aureus has shown s. cerevisiae (1) and s. cerevisiae (2) had moderate antibacterial activity (d=3-6 mm), c. famata has no antibacterial activity because there is no clear zone diameter formation, whereas p. anomala isolates have weak antibacterial activity (d = 0-3 mm) against s. aureus. indigenous yeasts viability towards e.coli and s.aureus. based on fig. 2, it is known that almost 100% of e.coli has decreased from the 0 until 12 hour. the effectiveness of the decrease in the number of e.coli cells by s. cerevisiae (2) was 23.7% at 0 hour, while the number of viability of s. aureus decreased by 76.2% at 0 to 12 hours. organic acid production. based on the table 3, s. cerevisiae can produce acetic, citric, malic, and lactic acid compounds as indicated by an increase in the amount of organic acid. while oxalic and tartaric acid compounds experience a decrease in the amount of organic acid. discussion indigenous yeasts identification. the s. cerevisiae colonies are yellowish white, have a circular edge shape, and the surface glistening. s. 112 utama et al. microbiol indones table 2 the results of rapid yeasts plus system with eric analysis test s.1 s.2 s.3 s.4 glucose + + + + maltose + sucrose + + + + trehalose + raffinose + lipid naga + αglucoside + + + + bglucoside + + + + onpg αgalactoside bfucoside + + phs pcho urea prolyne + histidine + + leucyl-glycine + + yeast name s.cereviseae (1) c.famata p.anomala s.cereviseae (2) 1 table 3 organic acid production by potential indigenous yeast s.cereviseae (2) blank s.cereviseae metabolite oxalic 2.650 2.489 -0.161 tartaric 1.862 1.206 -0.656 lactic 3.968 4.186 0.218 acetic 1.677 4.119 2.442 citric 9.542 `0.664 1.122 malic 0.023 0.044 0.021 1 cerevisiae cells are round (spherical), sometimes ellipsoidal (oval, elongated) to cylindrical, and produce pseudomycelium. the cell size of s. cerevisiae ranges from 5-12´5-10μm (kurtzman et al. 2011). p. anomala is white, smooth with fine serrated edges. p. anomala has round, elliptical or elongated cells. the cell size of p. anomala ranges from 2-7´2-5μm (passoth et al. 2006). c. famata forms yellowish white colonies, smooth and shiny texture. c. famata is elongated round (ovoid) with cell size 2.0-3.5´3.5-5.0μm (dmytruk and sibirny 2012). based on observations, it is known that all isolates can hydrolyze substituted glycosides, namely αglu and β-glu compounds enzymatically. all isolates are known to hydrolyze glucose and sucrose types of carbohydrates, but only s.3 isolates can hydrolyze maltose and trehalose carbohydrate compounds. hist and lgy compounds which are amino acid groups can only be hydrolyzed by isolates s.1 and s.4. s. cerevisiae can hydrolyze simple sugars such as glucose and fructose, and can hydrolyze disaccharides like sucrose because it produces the enzyme sukrase (invertase) which converts sugar to be easily fermented (sainz-polo et al. 2013). p. anomala can utilize all types of sugar such as glucose, maltose, sucrose, galactose, volume 13, 2019 microbiol indones 113 fig 2 indigenous yeasts viabity towards (a) e. coli; (b) s. aureus. (a) (b) fig 1 sapodilla indigenous yeasts antibacterial activities towards (a) e. coli; (b) s. aureus. (a) (b) 114 utama et al. microbiol indones and raffinose, except lactose (tao et al. 2011). c. famata can ferment glucose, sucrose, and trehalose (gientka et al. 2016). indigenous yeasts antibacterial activities. s.cerevisiae isolates can produce antimicrobial metabolites such as organic acids, phenolic compounds, besides s. cerivisiae is known to produce several proteins that have antimicrobial properties (roostita et al. 2011). s. cerevisiae produces high concentrations of ethanol which are toxic to many microbial species and are capable of producing volatile compounds such as a r o m a t i c a l c o h o l s i n v o l v e d i n i n h i b i t i n g microorganisms (jouhten et al. 2016). the antibacterial activity of s. cerevisiae with an incubation time of two days caused by the primary metabolites produced by yeast, namely organic acid compounds. organic acids produced by s. cerevisiae such as acetic acid, malic acid, succinic acid, and lactic acid have strong antimicrobial activity '(fakruddin et al. 2017). based on research by younis et al. (2017), s. cerevisiae showed moderate antibacterial activity against e. coli bacteria and showed weak antibacterial activity against s. aureus. e. coli population decreased after exposed acetic acid, lactic acid, propionic acid, and formic acid, where the reduction in the e. coli population increased with an increase in the concentration of organic acids (raftari et al. 2009). p. anomala produces ethanol under limited oxygen conditions and produces acetic acid in aerobic conditions. p. anomala produces volatile compounds such as ethyl acetate, ethyl propanoate, phenyl ethanol, and 2-phenylethyl acetate (passoth et al. 2006). p. anomala can reduce fungal growth in several ways: production of killer poisons, secretion of β-1-3 glucanase, ethyl acetate production or by competition for nutrition (muccilli and restuccia 2015). p. anomala is known to produce acetic acid where acetic acid has an inhibitory effect on bacteria. raftari et al. (2009) stated that e. coli and s. typhimurium had a high susceptibility to lactic acid and acetic acid. p. anomala had the most influential antimicrobial activity on e. coli growth (walker 2011). the absence of a clear zone indicates no antibacterial activity in c. famata isolates. this can be caused because most of the inhibition of bacteria by c. famata is with produce the optimal killer toxin at ph 4.5 with a temperature of 20 °c. above ph 4.5 and 20 °c killer toxin activity decreases (muccilli and restuccia 2015). tests are carried out above 20 °c, so the production of killer toxin is inhibited and antibacterial activity cannot take place. the antibacterial activity of indigenous yeast to sapodilla fruit against s. aureus bacteria is lower when compared to e. coli, this can occur because test bacteria have different resistance to different types of organic acids. s. aureus test bacteria have high acid resistance when compared to e. coli, this is caused by differences in the cell wall structure of the two types of bacteria (lopez-romero et al. 2015). e. coli bacteria grow at ph 7.0-7.5 while s. aureus bacteria grows at ph 4.0-9.8 (padan et al. 2005). indigenous yeasts viability towards e. coli and s. aureus. the optimum temperature for the growth of e. coli and s. aureus is 35-37 °c, so that antibacterial testing of yeast carried out to determine the effectiveness antibacterial origin of yeast that could be use as disinfectant for e. coli and s. aureus in the environment. temperature greatly influences the growth and physiological activities of microorganisms. temperature differences can affect the speed of bacterial enzyme synthesis, enzyme inactivation, changes in metabolic processes and cell shape, and bacterial growth rate (suriani et al. 2013). the antibacterial activity of s. cerevisiae with an incubation time of two days was caused by the primary metabolites produced by yeast, namely in the form of organic acid compounds. organic acids produced by s. cerevisiae such as acetic acid, malic acid, succinic acid, and lactic acid have strong antimicrobial activity (fakruddin et al. 2017). organic acids have a bactericidal effect whose effects increase with increasing concentration (abbott et al. 2009). differences bacterial viability against organic acids can caused by the ability of strains to adapt in different acidic environments. gram-positive bacteria such as s. aureus have murein compounds that cause cell wall resistance in gram negative bacteria to be lower than gram-positive bacteria(nazzaro et al. 2013). the ability of s. aureus to survive in acidic environment is with the phase of adaptation to the acidic environment , namely by pumping protons out of the cell to maintain normal ph and also by increasing the concentration of alkaline compounds in cells to prevent cytoplasmic acidification, repair and degradation mechanisms damaged protein (bore et al. 2007). organic acid production. organic acids produced by yeast result in the accumulation of acidic end products and a decrease in ph which will inhibit the growth of both gram-positive and gram-negative bacteria (kim et al. 2018). the decrease in organic acid is caused by the use of organic acids by yeast for the fermentation process (walker and stewart 2016). volume 13, 2019 microbiol indones 115 changes in the concentration of organic acids illustrate that yeast can freely use organic acids as a source of energy and provide organic acids as intermediate compounds in cell metabolism. during fermentation s. cerevisiae can utilize acid because of the hydrolysis process by enzymes derived from yeast cells (azhar et al. 2017). s. cerevisiae cannot metabolize tartaric acid, where tartaric acid is the most commonly used acid for ph adjustment in the wine industry (jolly et al. 2014). oxalic acid hydrolysis is carried out with enzymes produced by microorganisms (pal et al. 2016). the highest type of organic acid produced by s. cerevisiae isolates is acetic acid followed by citric acid. s. cerevisiae produced acetic acid where acetic acid production increased with increasing fermentation temperature, where the optimum temperature of acetic acid production was at 30 °c (shang et al. 2016). the fermentation temperature corresponding to the optimum temperature of acetic acid production allows the highest production of acetic acid by s. cerevisiae. acetic acid formed from glucose fermentation under aerobic conditions (gomes et al. 2018). the synthesis of citric acid by s. cerevisiae is higher with dissolved oxygen in the media that is getting higher (walker and stewart 2016). yeast can produce small amounts of lactic acid. lactic acid produced at 28−30 °c. lactic acid formed by the reduction of pyruvic acid and the transformation of malic acid (vilela 2019). the best temperature for producing malic acid is 18−25 °c with optimum fermentation time for 2 to 3 days. based on observations of s. cerevisiae yeast produce small amounts of malic acid, this can be caused because most of the malic acid produced has been transformed into lactic acid or used as an energy source. several studies have reported the inhibitory effects of various organic acids on pathogenic or destructive microbes, including blom et al. (1997) which states that the use of organic acid in the form of 2.5% lactic acid and 0.25% acetic acid can extend the shelf life of roast pork for up to 5 weeks (silano et al. 2018). other studies have also been reported by castilo et al. 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fax : +6224-8505680; email : cut.nuria79@gmail.com million children under the age of 5 years. infection was usually treated by using antibiotics and the use of antibiotics could not be separated from its side effects, such as resistance, hypersensitivity, and allergic reactions (cunha 2001). in order to find alternative treatments for infection, research was conducted to find other sources mainly from natural products. one of these natural products is the purslane (portulaca oleracea l.) plant. purslane could be used for the treatment of diarrhoea, dysentery, and gastro protective a gastroenteritic infection could be caused by pathogenic bacteria namely salmonella typhimurium. salmonella is one of four key global causes of diarrhoeal diseases, which are the most common illnesses resulting from unsafe food. according to the world health organization (who) in 2018, about 550 million people falling ill each year, including 220 (kumar et al. 2022). phytoconstituents found in purslane plants were flavonoids (apigenin, kaempferol, quercetin, luteolin, myricetin, genistein, and genistin), alkaloids, coumarins, terpenoids, anthraquinone glycoside, cardiac glycoside, sterols, proteins, vitamins, and minerals (chugh et al. 2019). karlina et al. (2013) reported screening phytochemical from the purslane herb ethanol extract obtained by maceration method contained tannins, saponins and flavonoids. the purslane leaves ethanol extract from the soxhlet extraction contained secondary metabolites such as alkaloids, saponins, tannins, flavonoids, diterpenes, and proteins (wasnik and tumane 2014). different extraction methods of purslane plant could affect the chemical content in the extracts. both hot and cold extraction methods could affect the amount of chemical content extracted, because the heat in the extraction process could extract more active compounds but also affects the stability of these compounds. some compounds in natural products are likely less stable to heat. sudaryati and nusandari (2017) reported that antibacterial activity against salmonella typhimurium from the ethanol extract of macerated purslane herb occurred at concentration range of 80-100%. according to londonkar and nayaka (2011), the purslane herb ethanol extract from the soxhlet process contained many flavonoid compounds that had antibacterial activity against salmonella typhimurium. another research by nayaka et al. (2014) reported that apigenin compound that was isolated from purslane had antibacterial activity against salmonella typhimurium. the research about the antibacterial activity of the purslane herb ethanol extract with several extraction methods against salmonella typhimurium had never been reported. this study was conducted to provide information about the best extraction methods in obtaining the purslane herb ethanol extract with the greatest antibacterial activity against salmonella typhimurium. this study could also determine any significantly differences in the antibacterial activity of the purslane herb ethanol extract against salmonella typhimurium based on the extraction method variations. purslane herb were obtained from putat nganten village, grobogan regency, central java, indonesia. the plant was harvested from cultivated fields with characteristics dark green leaves, yellow flowers and reddish stem. the plant parts used are the roots, stems, leaves and flowers. the purslane herbs were sorted, then washed with water to remove impurities. fresh herbs were dried at temperature 55 °c and powdered. the extraction process. the maceration process was carried out by weighed dried purslane herb powder for 600 grams. the solvent used was 75 parts (4500 ml) of 96% ethanol and immersed for 3 days, protected from the sunlight, at room temperature with several stirring. the mixture was filtered to obtain macerate (1). the waste was soaked again with 25 parts of solvent (1500 ml) for 2 days. then mixture was stirred and filtered again to obtain macerate (2). macerate (1) and (2) were mixed and stored in a closed container and cool place, protected from the sunlight. the filtrate was stored for 24 hours. the filtrate was then filtered and concentrated by using a rotary vacuum evaporator at 55 °c until thick extract was obtained (indonesian ministry of health 2006). the percolation method was perform by measuring 900 ml 96% ethanol and then poured into 600 grams of purslane herb powder. the damp powder was stored into a percolator along with 1000 ml solvent, and then left for 2 hours. the percolates were collected together with the addition of the solvent continuously. percolation was carried out until a clear solution obtained from the percolator. percolates were concentrated by using a rotary vacuum evaporator at 55 °c until thick extract was obtained (indonesian ministry of health 2000). the reflux method was perform by weighed 600 grams of dried purslane herb powder and then stored into a round bottom flask, then 530 ml of 96% ethanol solvent was added until the dried powder was immersed in it. the reflux equipment was assembled, and the sample was extracted at 78 °c for 2 hours. the extracted filtrate was filtered with gauze and filter paper, and then stored into an erlenmeyer. this process was repeated for three times. the filtrate obtained was evaporated by using a rotary vacuum evaporator at 55 °c until a concentrated extract was obtained (list and schmidt 2000). the soxhlet method was carried out by weighed 600 grams of dried purslane herb powder then wrapped in filter paper and tied at both ends, after that it was inserted into the soxhlet extractor (zhang et al. 2018). the ethanol was poured into a round bottom flask until it filled 2/3 parts of the flask. the extraction was carried out at 78 °c. the soxhlet was assembled with a condenser and extraction was carried out until the liquid in the siphron tube was colorless (clear). the filtrate obtained was evaporated by using a rotary vacuum evaporator at 55 °c until a concentrated extract was obtained. 140 nuria et al. microbiol indones antibacterial activity test. pure culture of s. typhimurium was obtained from the laboratory of microbiology, university of muhammadiyah semarang. two point five (2.5) ml salmonella typhimurium suspension was added into 22.5 ml of warm media nutrient agar and then poured into a sterile petri dish, and let stand for the media to become solid (test media). ten (10) μl extract from various extraction methods with 30%, 35%, 40%, 45%, and 50% (b/v) concentration to organic solvent (dimetilsulfoxide/dmso) respectively, were dropped onto the surface of the paperdisk in empty sterile petri dish and were left for 10 minutes. this procedure was also carried out for 10 μl of dmso solvent as the negative control. paperdisk that already contained the test solution and dmso is affixed to the surface of the test media. the paperdisk contain 30 μg of chloramphenicol (positive control) was also attached to the test media. the petri dishes were then incubated for 24 hours at 37 °c. the test results were observed by looking at the zone of inhibition formed around the disk after the incubation period and then measured by using a caliper. the test was repeated for 3 times (banjara et al. 2012). the data were analyzed statistically with 95% level of confidence. the zone of inhibition resulted from maceration extract was 9.36-10.16 mm; percolation was 9.2310.21 mm; reflux was 8.54-9.91 mm; and soxhlet was 9.52-10.20 mm (table i.). the two way anova test showed sig <0.05 in the zone of inhibition of all extracts from the four extraction methods, and the concentration series from each extraction method also showed significant differences (p <0.05). the zone of inhibition produced by chloramphenicol was very large (27.11-28.98 mm) compared to the maceration and reflux extract (8.54-10.16 mm), therefore the antibacterial activity was significantly different. meanwhile, the difference with dmso solvent is due to the fact that the solvent did not provide any inhibition, while the extract had a zone of inhibition in a certain range. result from tukey's test for post-hoc analysis of the zone of inhibition in the percolated extract concentration series showed a significant difference between the 30-35% and 45-50% concentration. these results showed that the zone of inhibition from the 4550% concentration was relatively greater compared to zone of inhibition from the lower concentrations (3035%), and this was statistically different (p <0.05). result from tukey's test for post-hoc analysis of the zone of inhibition in the concentration series from soxhlet extract showed a significant difference between low concentrations (30%) and high concentrations (50%). meanwhile the other concentration series (35%, 40%, 45%) did not give a significant difference (p> 0.05). extraction by maceration, percolation, reflux and soxhlet method resulted in 47.7, 66.3, 61.4, and 62.8 grams viscous extract respectively. the extract organoleptic from four extraction methods showed the same characteristics, which were blackish brown color, thick consistency and distinctive odor of purslane. the antibacterial activity test of purslane herb ethanol extract (phee) against s. typhimurium was shown by the presence of a radical inhibition zone in all test concentrations. the display of phee antibacterial test results from each extraction method could be seen in figure 1. the radical inhibition zone was characterized by the presence of a clear area around the disk paper with no bacterial growth. if a radical inhibition zone was formed, an antibacterial agent could be considered to have bactericidal properties (mycek 2001). the statistic test result amongst the extraction methods showed that there were differences in the zone of inhibition produced by extract reflux method with the extracts of other methods including maceration, percolation and soxhlet. the extraction method of reflux produced the lowest zone of inhibition among the three other extraction methods, which meant that the reflux method caused the lowest phee antibacterial activity. reflux method extraction was an extraction method that used heat by boiling the dried powder with a solvent. it was possible that the heat used in the process caused some of the active compounds to break down and affect its antibacterial activity against s. typhimurium. li et al. (2017) reported the isolation of compounds from purslane herb, it had the oleraciamide a and oleraciamide b alkaloids. these compounds had 74.5-75.5 °c as its melting point which was lower than the boiling point of ethanol 96% (78 °c), so there is a possibility that these compounds was damaged due to the heat. the other three extraction methods which were the maceration, percolation and soxhlet, did not present any difference in zone of inhibition statistically (p>0.05). this meant that there was no difference in phee antibacterial activity against s. typhimurium. from the three extracts produced, it meant that the antibacterial potential is likely same among these extracts. the soxhlet extraction method used heat in the process, but the dried herb powder was not boiled along with the solvent, so the process did not involve direct volume 15, 2021 microbiol indones 141 142 nuria et al. microbiol indones table 1 antibacterial activity test results of the purslane herb ethanol extract with various extraction method against salmonella typhimurium fig 1 antibacterial activity test results view of the purslane herb ethanol extract from the extraction methods with (1a) maceration, (1b) percolation, (1c) reflux and (1d) soxhlet against salmonella typhimurium. : radical zone extraction method treatment zone of inhibition (mm) 1st attempt 2nd attempt 3rd attempt average ± sd maceration phee 3000 µg/disk phee 3500 µg/disk phee 4000 µg/disk phee 4500 µg/disk phee 5000 µg/disk chloramphenicol 30 µg/disk dmso solvent 9.96 9.97 9.98 10.00 10.55 29.03 9.09 9.71 9.73 9.91 10.30 29.03 9.03 9.33 9.38 9.39 9.65 28.99 9.36+0.52 b 9.67+0.32 b 9.69+0.30 b 9.76+0.32 b 10.16+0.46 b 28.98+0.02 percolation phee 3000 µg/disk phee 3500 µg/disk phee 4000 µg/disk phee 4500 µg/disk phee 5000 µg/ disk chloramphenicol 30 µg/disk dmso solvent 9.16 9.44 9.62 10.15 10.18 29.50 9.03 9.39 9.40 9.85 9.90 28.99 9.51 9.54 9.70 10.51 10.55 29.69 9.23+0.24 b 9.45+0.07 b 9.57+0.15 b 10.17+0.33 b 10.21+0.32 b 29.39+0.36 refluxa phee 3000 µg/disk phee 3500 µg/disk phee 4000 µg/disk phee 4500 µg/disk phee 5000 µg/disk chloramphenicol 30 µg/disk dmso solvent 8.55 9.38 9.54 9.55 9.57 29.14 8.26 9.23 9.29 9.44 10.04 26.10 8.82 9.04 9.12 9.40 10.12 26.10 8.54+0.28 b 9.21+0.17 b 9.31+0.21 b 9.46+0.07 b 9.91+0.29 b 27.11+1.75 soxhlet phee 3000 µg/disk phee 3500 µg/disk phee 4000 µg/disk phee 4500 µg/disk phee 5000 µg/disk chloramphenicol 30 µg/disk dmso solvent l 9.31 9.51 9.67 9.70 10.25 29.20 9.55 10.13 10.16 10.39 10.40 29.10 9.72 9.86 9.93 9.94 9.97 28.91 9.52+0,20 b 9,83+0,31 b 9,92+0,24 b 10,01+0,35 b 10,20+0,21 b 29,07+0,14 phee : purslane herb ethanol extract (-) : no inhibition zone was formed a ( ) significantly different (p<0.05) from other extraction methods b ( ) significantly different (p<0.05) against the positive control (chloramphenicol) and negative control (dmso solvent) group heating. this made the extract produced during the soxhlet process, still had antibacterial activity equivalent to cold extraction. the zone of inhibition profiles resulted from maceration and reflux extracts were likely the same. among all series of extract concentrations (30%, 35%, 40%, 45%, and 50%) there was no significant difference in the zone of inhibition (p> 0.05), but all series of extract concentrations had significantly different of zone of inhibition compared to chloramphenicol as positive control group. this was due to the broad spectrum with high inhibitory effect from chloramphenicol to inhibit and kill bacteria. high concentration extracts (45-50%) had greater chemical content than low concentration extracts (3035%). therefore, there was an increased inhibitory effect on the percolated extract against the growth of s. typhimurium. the antibacterial activity of the soxhlet extract at 30% concentration was the lowest compared to other concentration series. the heating process of dried purslane powder apparently very influential on its chemical content, especially compounds that were unstable to heat. the 30% concentration in the soxhlet extract also resulted in the smallest zone of inhibition compared to other concentrations (35-50%). based on the research results, with maceration, percolation and soxhlet extraction methods, extracts with the same antibacterial activity could be obtained. these three methods produced extracts with no significant difference in antibacterial activity (p> 0.05). however, for the practical purposes of research, the percolation method had the highest yield value (11.05%) compared to maceration and soxhlet (7.9510.47%). this meant that the percolation method could produce more extracts than the other two methods, and by using percolation extraction for the purslane herb, it would produce extracts that had antibacterial activity against s. typhimurium with more obtained extracts amount. from this research, it could be concluded that the purslane herb ethanol extract produced from various extraction methods (maceration, percolation, soxhlet, and reflux) had antibacterial activity against s. typhimurium. the antibacterial activity of the purslane herb ethanol extract produced by the reflux method was significantly different from other methods (maceration, percolation and soxhlet) against s. typhimurium. references banjara ra, jadhav sk, bhoite sa. 2012. antibacterial activity of di-2-ethylaniline phosphate screened by paper disc diffusion method. j. appl. pharm. sci. 2(7):230-233. doi:10.7324/japs.2012.2720. cunha ba. 2001. antibiotic side effects. med clin north a m . 8 5 ( 1 ) : 1 4 9 1 8 5 . d o i : 1 0 . 1 0 1 6 / s 0 0 2 5 7125(05)70309-6. chugh v, mishra v, dwivedi sv, sharma kd. 2019. purslane (portulaca oleracea l.): an underutilized wonder plant with potential pharmacological value. the pharma innovation journal. 8(6): 236-246. doi: 10.22271/tpi. indonesian ministry of health. 2000. parameter standar umum ekstrak tumbuhan obat [general standard parameters of medicinal plant extracts]. direktorat jenderal pengawas obat dan makanan. jakarta. indonesian ministry of health. 2006. monografi ekstrak tumbuhan obat indonesia [monographs of indonesian medicinal plant extracts]. vol.2. departemen kesehatan republik indonesia. jakarta. karlina cy, muslimin i, guntur t. 2013. aktivitas antibakteri ekstrak herba krokot (portulaca oleracea l.) terhadap staphylococcus aureus dan escherichia coli [antibacterial activity of purslane (portulaca oleracea l.) herb extract against staphylococcus aureus and escherichia coli]. lentera bio. 2(1):87–93. kumar a, sreedharan s, kashyap ak, singh p, ramchiary n. 2022. a review on bioactive phytochemicals and ethnopharmacological potential of purslane (portulaca oleracea l.). heliyon. 8(2022):e08669. doi: 10.1016/j.heliyon.2021.e08669. li c, ying z, gao m, wei w, hao d, xu l, tao x, zhang w, ying x, liu j. 2017. two new similar alkaloids from portulaca oleracea l. nat. prod. res. 31:1792-1798. doi: 10.1080/14786419.2017.1292507. list ph, schmidt pc. 2000. phytopharmaceutical technology. institute of pharmaceutical technology, university of marburg. boston. londonkar r, nayaka hb. 2011. phytochemical and antimicrobial activities of portulaca oleracea l. j. pharmacol. res. 4(10):3553-3555. mycek mj. 2001. farmakologi ulasan bergambar [pharmacological pictorial review]. widya medika. jakarta. nayaka hb, londonkar rl, umesh mk, tukappa a. 2014. antibacterial attributes of apigenin, isolated from portulaca oleracea l. int. j. bacteriol. 175851. doi:10.1155/2014/175851. sudaryati, nusandari r. 2017. karakteristik fitokimia dan aktivitas antimikroba krokot (portulaca oleracea l.) [phytochemical characteristics and antimicrobial activity of purslane (portulaca oleracea l.)]. prosiding volume 15, 2021 microbiol indones 143 seminar nasional fktp-tpi; 2017. kendari. p 318327. wasnik dd, tumane pm. 2014. preliminary phytochemical screening and evaluation of antibacterial activity of portulaca oleracea l. againts multiple drug resistance (mdr) pathogens isolated from clinical specimen. world j. pharm. res. 3(10):920-931. who. 2018. diarrhoeal disease. https://www.who.int/ news-room/fact-sheets/detail/salmonella-(nontyphoidal). accessed date 16 april 2020. zhang qw, lin lg, ye wc. 2018. techniques for extraction and isolation of natural products: a comprehensive review. chin med. 13(20): 1-26. doi: 10.1186/s13020018-0177-x. 144 nuria et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 6 okky.pmd volume 3, number 3, december 2009 p 139 145 issn 1978-3477 *corresponding author: phone: +62-251-8323848, fax: +62-251-8326851, e-mail: okky_sd@yahoo.com the quality of physic nut (jatropha curcas) seeds affected by water activity and duration of storage okky setyawati dharmaputra1,2*, rantje lilly worang1,3, rizal syarief4 and miftahudin1 1department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor16680, indonesia; 2seameo biotrop, jalan raya tajur km. 6, po box 116, bogor16134, indonesia; 3faculty of mathematics and natural sciences, universitas manado state, tondano 95615, sulawesi utara, indonesia; 4department of food science and technology, faculty of agricultural technology, institut pertanian bogor, bogor16680, indonesia the quality of physic nut (jatropha curcas) seeds should be maintained during storage, either as seeds for seedlings or oil production to be used for biodiesel. the effects of water activity and duration of storage on the quality, i.e. fungal population, lipid, fatty acid and free fatty acid contents, and viability of physic nut seeds were investigated. the results showed that the moisture content of seeds and total fungal population decreased at low water activities, and increased at high water activities (a w ). at a w 0.64, at the beginning of storage and after 20 weeks of storage, total fungal populations were 5.4 x 103 and 1.8 x 102 cfu g-1 dry basis (db), respectively. at a w 0.93, at the beginning of storage and after 20 weeks of storage, total fungal populations were 5.0 x 103 and 3.3 x 106 cfu g-1 db, respectively. at the beginning of storage fungi infecting seeds were field fungi, i.e. cladosporium sp., colletotrichum sp., fusarium semitectum, and f. verticillioides. the population of field fungi decreased with the increase of storage duration. they were replaced by postharvest fungi, i.e. aspergillus restrictus, a. penicillioides, eurotium chevalieri, e. rubrum, penicillium citrinum, p. implicatum and p. oxalicum. lipid content and viability of seeds decreased with the increase of water activities and seed moisture contents, while free fatty acid contents increased with the increase of water activities and seed moisture contents during storage. fatty acids of lipid were dominated by unsaturated fatty acids, i.e. oleic and linoleic acids. physic nut seeds could be stored at a w 0.64-0.75 up to eight weeks when the seeds will be used for seedling, or up to 16 weeks when they will be used for producing oil. key words: jatropha curcas, physic nut, water activity physic nut (jatropha curcas) seeds are good sources of biodiesel. the oil contents in the seeds and in the kernels are 2530% and 5060%, respectively. the oil contains 21% saturated fatty acids and 79% unsaturated fatty acids (gubitz et al. 1999). as the content of unsaturated fatty acids in the oil is high, it is possible that the content of free fatty acids will also increase during storage. their increase could be minimized by using good packaging and appropriate storage condition. water activity (a w ) or relative humidity and the temperature of storage were the most important physical factors affecting seed quality, because they determine the moisture content of substrate. safe appropriate content can prevent fungal infection and the hydrolysis of lipid, consequently the quality of seeds or grains could be maintained for longer period. according to sirisomboon et al. (2007), the moisture content of physic nut hulls (fruits) var. kanlueang was the highest, compared to its seeds (nuts) and kernels. wanita and hartono (2007) reported that seeds originated from physic nut fruits having yellow up to blackish yellow shell colour produce seeds with highest oil content, i.e. 2830%. physic nut seeds can also be used as seeds for seedlings. according to adikadarsih and hartono (2006), the use of physic nut seeds for seedlings should be derived from the fruits, which skin is yellow up to blackish yellow in color, because they have high percentages of viability and vigority, i.e. 89 and 81%, respectively. to obtain good quality of physic nut seeds after storage, some factors should be taken into consideration, i.e. the degree of fruit maturity, safe seed moisture content, appropriate container and storage condition, duration of storage, good viability and vigor of seeds. during storage seeds or grains could be infected by fungi which cause a decrease in viability, discolouration, loss in weight, chemical and nutritional changes, heating, caking and mycotoxin contamination. chelkowski (1991) reported that in many cases, fungi infecting seeds are seed-borne pathogens. they play an important role in the transmission of numerous pathogenic fungal species to seedlings as well as to the soil. the objective of this study was to analyze the effects of water activity and duration of seed storage on fungal population; lipid, fatty acid and free fatty acid contents, and viability of physic nut seeds. the population of each fungal species infecting physic nut seeds in various hydratation levels was also determined. materials and methods seed materials. physic nut seeds were obtained from fresh harvested fruits having yellow up to blackish yellow shell colour. the fruits were obtained from plants (lampung accession) cultivated in loyang village, cikedung subdistrict, indramayu regency, west java in may 2007. the fruit shell were peeled using a knife, then the seeds were air-dried on the selves in a shaded place up to moisture contents of about 8%. preparation of saturated salt solution. selected water activities (a w ) were 0.64, 0.75, 0.84 and 0.93 using various saturated salt solution, i.e. nano 2 , nacl, kcl and kno 3 , respectively, in sorption containers. the preparation of each salt solution was conducted by placing the salt of certain weight in a sorption container, then distilled water of certain microbiol indones140 dharmaputra et al. volume was added, consequently water activitiy selected was obtained. during storage the salt solutions were still saturated. after being checked using a thermohygrograph at every sampling, the relative humidity inside each sorption container were still the same with that at the beginning of storage. packaging and storing of physic nut seeds. as much as 100 g of physic nut seeds (moisture content ±8%) were placed in a sac made from plastic net. the sack containing the seeds was then hung in a sorption container (10 sacks per container). the seeds were stored in sorption containers with different water activities. three replicates (= 3 sorption containers) were used for each water activity. sorption containers were placed on a wooden table under room conditions. the duration of storages were 0, 1, 2, 3, 4, 6, 8, 12, 16 and 20 weeks. the ambient temperature and relative humidity of the storage room were recorded using a tinytag data logger. sampling method and to obtain working samples. samples of physic nut seeds were collected from each sack. the seeds derived from each sack in three sorption containers were collected for each storage duration. each sample derived from each sack was then mixed homogenously, manually divided into five parts to obtain working samples for the determinations of moisture content, fungal population, lipid, fatty acid and free fatty acid contents, and viability of seeds, and reserved sample. determination of seed moisture content and fungal population. moisture contents of seeds (based on wet basis) were determined based on oven method. two replicates were used for each sample. fungi from each sample were isolated and enumerated using serial dilution method followed by pour plate method on dichloran 18% glycerol agar (dg18) (hocking and pitt 1980). fungal identification was conducted based on klich and pitt (1988), pitt (1988), burgess et al. (1994), barnett and hunter (1998). determination of lipid, fatty acid and free fatty acid contents and viability. lipid, fatty acid and free fatty acid contents were determined based on soxhlet (aoac 1999), gas chromatography (aoac 1984) and titration methods, respectively. fatty acid contents were determined at the beginning of storage, subsequently after 12 and 20 weeks of storage. viability (percentage of germination) of seeds from each sample was determined by growing 25 seeds in a rectangular plastic container (36 x 27.5 x 5 cm) containing sand (5 kg/ container) seven and 10 days after planting under green house conditions.. normal seedlings were observed 7 days after planting, while normal, abnormal and death seedlings were observed on 10 days after planting. statistical analysis. split plot design with two factors was used. the first factor as the main plot was a w with four levels, i.e. 0.64, 0.75, 0.84 and 0.93. the second factor as the subplot was the duration of storage with ten levels, i.e. 0, 1, 2, 3, 4, 6, 8, 12, 16 and 20 weeks with three replications. the observed data were analyzed using anova, followed by duncan’s multiple range test at the 5% probability level. results moisture contents. seed moisture content is the most important environmental factor that influence fungal growth in stored grains. moisture content is always in equilibrium with the relative humidity of the storage. the range and the mean of ambient temperature and relative humidity of the storage is presented in table 1. at the beginning of storage the average moisture contents of physic nut seeds were between 8.17-8.36% wet basis. water activity, duration of storage and their interaction gave very significant difference on the moisture contents (table 2). at the beginning of storage the moisture contents at a w 0.64 (8.36%) was higher and significantly different from those at a w 0.75 (8.17%), but they were not significantly different from those at a w 0.84 (8.28%) and a w 0.93 (8.31%). with the increase of storage duration each a w gave different effect on the moisture contents. after one week of storage, the moisture contents at a w 0.64 (8.37%) was not significantly different with those at a w 0.75 (8.37%), but they were very significantly different from those at a w 0.84 (9.39%) and a w 0.93 (10.48%). at a w 0.64, the moisture contents decreased with the increase of storage duration after two weeks of storage, while at a w > 0.64 the moisture contents increased (table 2). fungal population. twenty one fungal species and three fungal isolates were isolated at various a w during twenty weeks of storage. they were aspergillus candidus, a. flavus, a. niger, a. penicillioides, a. restrictus, a. tamarii, aspergillus sp. b, aspergillus sp. d, cladosporium sp., c. cladosporioides, colletotrichum sp., eurotium chevalieri, e. rubrum, fusarium semitectum, f. verticillioides, lasiodiplodia sp., libertella sp., penicillium citrinum, p. implicatum, p. oxalicum, wallemia sebi, and three unidentified fungal isolates, i.e. isolates d, f, and i. cladosporium cladosporioides and p. citrinum were always isolated at various a w and duration of storage. water activity, duration of storage and their interaction gave very significant difference on total fungal population. the effect of interaction between a w and duration of storage on total fungal population is presented in table 3. from the beginning of storage until 20 weeks of storage total fungal population were not different at a w 0.64, 0.75 and 0.84, but they were significantly different from those at a w 0.93 after 16 and 20 weeks of storage. water activity of 0.64 and 0.75 are favorable to be used for storing physic nut seeds, because fungal population decreased during storage. although at a w 0.84 after 20 weeks of storage table 1 the range and mean of temperature and relative humidity of storage room during storage duration of storage (week) temperature o c) relative humidity (%) range mean range mean 0 1 25.1 28.3 26.5 61.7 80.7 72.6 1 2 24.7 27.9 26.1 63.9 82.4 75.3 2 3 24.4 27.9 26.0 65.6 82.8 75.8 3 4 25.1 27.9 26.3 60.0 80.7 71.2 4 6 25.1 28.7 26.8 58.7 80.7 71.4 6 8 25.1 28.3 26.3 48.2 78.1 67.8 8 12 25.1 29.1 26.5 55.2 78.5 69.5 12 16 25.1 28.3 26.7 46.0 80.7 68.2 16 20 25.1 28.7 26.8 55.6 82.4 72.8 ( microbiol indones 141volume 3, 2009 the total fungal population increased (4.3 x 105 cfu g-1 dry basis/db), they did not give any significant difference on the population at beginning of storage up to 16 weeks of storage. at a w 0.84 and 0.93 total fungal population was straight proportional with the increase of storage duration. at the beginning of storage the range of total fungal population was range between 5.0 x 103 – 5.4 x 103 cfu g-1 db at various a w . after 20 weeks of storage the lowest total fungal population was found at a w 0.64 (1.79 x 102 cfu g-1 db), while the highest was found at a w 0.93 (3.25 x 106 cfu g-1 db). at the beginning of storage the most often fungi isolated were categorized as field fungi, i.e. colletotrichum sp., cladosporium spp. and fusarium spp. the population of these fungi decreased with the increase of storage duration. at low a w (0.64 and 0.75) total fungal population decreased with the increase of storage duration, while at high a w (0.84 and 0.93) total fungal population increased with the increase of storage duration. lipid contents. physic nut seeds contain high lipid. at the beginning of storage and after 20 weeks of storage, the range of lipid contents were between 39.6140.85% db and 29.5932.46% db, respectively. water activity, duration of storage and their interaction gave very significant difference on lipid content. the effect of a w and duration of storage on lipid content is presented in fig 1. at the beginning of storage lipid contents of each a w were not significantly different. lipid contents of each a w decreased with the increase of storage duration. at a w 0.64, lipid contents decreased during storage, especially after 12 weeks of storage. after 20 weeks of storage lipid contents were significantly different from those at the beginning of storage up to 16 weeks of storage. at a w 0.75, lipid contents decreased during storage, especially after 16 weeks of storage. after 16 and 20 weeks of storage lipid contents were not significantly different, but they were significantly different from those at the beginning of storage up to 12 weeks of storage. at the beginning of storage, at a w 0.84, lipid contents was significantly different from those after one week of storage. lipid contents decreased continuously up to 20 weeks of storage. after 20 weeks of storage lipid contents was significantly different from those after two up to 16 weeks of storage. at a w 0.93 lipid, contents decreased significantly during storage, especially after two weeks of storage. after two weeks of storage lipid contents was significantly different from those at the beginning and after one week of storage. lipid contents decreased continuously up to 16 weeks of storage and they were significantly different from those after 20 weeks of storage. lipid contents at the four levels of a w decreased with the increase of storage duration, but they were still in the good ranges for oil production (about 30%). the highest decrease of lipid contents were found at a w 0.93 after 20 weeks of storage (29.59% db). the decrease of lipid contents was in the form of negative sigmoid, it looks like s letter upside-down. fatty acid and free fatty acid contents. lipid of physic nut seeds consist of saturated and unsaturated fatty acids. in this study fatty acid contents of physic nut seeds were dominated by unsaturated fatty acids, i.e. oleic acid (37.0541.29% wb), followed by linoleic acid (26.7329.08% wb). fig 1 effect of water activity and duration of storage on lipid content of physic nut seeds. means followed by the same letter are not significantly different according to duncan’s multiple range test at the 5% level. table 2 the effect of water activity and duration of storage on moisture content (% wet basis) of physic nut seeds duration of storage (week) water activity 0.64 0.75 0.84 0.93 0 1 2 3 4 6 8 12 16 20 8.36 rs ± 0.12 8.37 rs ± 0.09 8.26 st ± 0.03 8.19 tu ± 0.01 8.17 tu ± 0.03 8.09 u ± 0.06 8.01 vw ± 0.03 7.92 wx ± 0.05 7.84 x ± 0.06 7.64 y ± 0.07 8.27 st ± 0.09 8.37 rs ± 0.03 8.48 r ± 0.03 8.67 q ± 0.11 8.84 p ± 0.01 8.89 op ± 0.02 8.99 o ± 0.03 9.14 n ± 0.07 9.29 m ± 0.06 9.42 m ± 0.07 8.28 st ± 0.14 9.39 m ± 0.07 9.70 l ± 0.02 9.82 l ± 0.08 9.96 k ± 0.02 10.05 jk ± 0.04 10.13 ij ± 0.03 10.21 hi ± 0.02 10.29 h ± 0.03 10.44 g ± 0.11 8.31 st ± 0.06 10.48 g ± 0.25 11.11 f ± 0.07 11.27 e ± 0.06 11.38 de ± 0.02 11.42 d ± 0.01 11.49 cd ± 0.04 11.58 c ± 0.03 11.75 b ± 0.05 12.05 a ± 0.11 . , , , the composition of fatty acids of physic nut seeds stored at a w 0.64 and 0.93 at the beginning of storage, subsequently after 12 and 20 weeks of storage is presented in table 4. free fatty acid contents of physic nut seeds at the beginning of storage were between 0.13-0.27% db. at various a w free fatty acid contents increased with the increase of storage duration. water activity, duration of storage and their interaction gave very significant difference on free fatty acid contents.the effect of a w and duration of storage on free fatty acid content is presented in fig 2. at the beginning of storage free fatty acid contents at each a w were not significantly different. nevertheless, free fatty acid contents increased with the increase of storage duration. after 12 weeks of storage free fatty acid contents at a w 0.64 was not significantly different from those at a w 0.75, but they were significantly different from those at a w 0.84 and 0.93. free fatty acid contents at 1% concentration were obtained at a w 0.64 and a w 0.75 after 16 weeks of storage, at aw 0.84 after 12 weeks of storage, and at a w 0.93 after 6 weeks of storage (fig 2). the requirement of free fatty acid contents of physic nut oil to be used for biodiesel shold not be less than 1%. seed viability. at the beginning of storage the seed germination percentages were >90%. nevertheless, at each a w , the seed germination percentages decreased with the increase of storage duration. at higher a w the decrease of seed germination percentages was faster than those at lower a w . water activity, duration of storage and their interaction gave very significant difference on seed germination percentage. the effect of a w and duration of storage on seed germination percentage is presented in table 5. at the beginning of storage up to four weeks of storage at each a w the percentages of germination were not significantly different. their percentages of germination were still good (> 80%). after six and 12 weeks of storage at a w 0.64, 0.75 and 0.84 the percentages of germination were not significantly different, but they were significantly different from those at a w 0.93. after eight weeks of storage at a w 0.64 and 0.75 the percentages of germination were not significantly different, but they were significantly different from those at a w 0.84 and 0.93. discussion moisture content is the most important factor which influences fungal growth in stored seeds or grains. high moisture content is favorable for fungal growth. dirjenbun (2006) determined the safe moisture content for storing physic nut seeds was 7-9%. in this study the average moisture contents at the beginning of storage were 8.17-8.36% wet basis (table 2). water activity 0.64 was favorable a w for storing physic nut seeds up to 20 weeks, because their moisture contents were 7.648.37%. physic nut seeds were still good if stored at a w 0.75 up to eight weeks. water activity of 0.84 and 0.93 were not favorable to store physic nut seeds, because after one week of storage, their moisture contents increased to more than 9%. the moisture contents of seed or grain is in equilibrium with the relative humidity of the storage. seeds can have lost (desorption) or absorb (absorbtion) the humidity of their environment. seeds stored in a room with high relative humidity will absorb the humidity of their environment. on the other hand, seeds will have lost their humidity, if they were stored at low relative humidity. at a w 0.64 or relative humidity 64%, physic nut seeds had lost their humidity during 20 weeks of storage. their moisture contents were 8.36% (at the beginning of storage), and then they became 7.64% after 20 weeks of storage. at a w e” 0.75 during 20 weeks of storage physic nut seeds absorbed the humidity of their environment. moisture content of physic nut seeds increased at a w 0.75, 0.84 and 0.93 during 20 weeks table 4 composition of fatty acids of physic nut seeds stored at a w 0.64 and 0.93 at the beginning of storage, subsequently after 12 and 20 weeks of storage table 3 the effect of water activity and duration of storage on total fungal population (cfu g-1 db) of physic nut seeds means followed by the same letter are not significantly different according to duncan’s multiple range test at the 5% level. microbiol indones142 dharmaputra et al. duration of storage (week) water activity 0.64 0.75 0.84 0.93 0 1 2 3 4 6 8 12 16 20 5.3x103 c ± 7.0x10 4.2x103c ± 1.1x103 3.6x103 c ± 1.3x103 2.5x103 c ± 4.4x102 1.4x103c ± 7.1x102 9.9x102 c ± 3.2x102 9.5x102c ± 3.6x102 2.8x102 c ± 3.9x102 2.8x102 c ± 3.1x102 1.8x102 c ± 7.3x10 5.2x103 c ± 6.0x102 5.1x103 c ± 1.6x103 4.2x103c ± 2.4x103 3.1x103c ± 1.6x103 1.3x103 c ± 6.7x102 1.2x103 c ± 5.2x102 1.2x103 c ± 6.8 x102 6.2x102 c ± 4.1x102 2.7x102 c ± 291.96 2.7x103 c ± 1.4x102 52x103 c ± 3.1x103 5.2x103 c ± 1.5x103 5.2x103 c ± 2.2x103 5.7x103 c ± 1.7x103 6.2x103 c ± 2.8x103 2.0x104 c ± 2.2x103 2.1x104 c ± 2.5x103 2.2x104 c ± 1.1x103 2.3x105 c ± 1.1x105 4.3x105 c ± 1.2x105 5.0x103 c ± 1.5x103 6.5x103 c ± 2.3x103 8.5x103 c ± 1.6x103 1.0x104 c ± 3.8x103 3.3x104 c ± 5.5x103 1.8x105 c ± 1.2x105 3.0x105 c ± 2.4x105 308591 c ± 152907 1.2x106 b ± 2.1x105 3.3x106a ± 1.3x106 microbiol indones 143volume 3, 2009 of storage, however, a w 0.64 can maintain safe moisture content. at the beginning of storage the most often fungi isolated was field fungi, i.e. colletotrichum sp., cladosporium sp., c. cladosporioides, f. verticillioides and f. semitectum. their population decreased with the increase of storage duration, and then they were replaced by storage fungi. at a w 0.64, total fungal population decreased with the increase of storage duration (table 3). it was assumed that the decrease of total fungal population was due to the constant or the decrease of moisture contents during storage. moisture content is an important factor affecting fungal growth. at a w 0.75 total fungal population also decreased, but the decrease was not as fast as at a w 0.64. according to douglas and boyle (1996) low moisture contents inhibited fungal growth, but they did not kill the fungi. among microorganisms infecting seeds, fungi was the most tolerant to low water availability, therefore they had an important role on seed deterioration. at a w 0.84 and 0.93 total fungal population increased with the increase of storage duration due to the increase of moisture content. consequently, fungi infecting physic nut seeds grew and developed better, especially the fungi which have abundant spores such as aspergillus and penicillium.the moisture content of seeds was also affected among others by seed respiration and fungal activity. fungi absorbed the nutrition of seeds and they excreted water as the result of their metabolism. the growth of storage fungi was affected by the moisture content of the substrate, temperature, duration of storage, the level of fungal infection before storage, foreign materials, insects and mites activities. these factors were related one with anothers. seed coat was the main defense to prevent fungal penetration into the inner seed tissue. the crack of seed which occurred mechanically could give the opportunity of fungi to infect the inner part of the seeds. seed-borne fungi can cause the decrease of germination. lipid or oil of physic nut seeds which contained high unsaturated fatty acid (oleic acid) (37.05-41.29% db.), followed by linoleic acid (26.7329.08% db) (table 4) will cause hydrolyses process. this process caused the decrease of lipid and increased free fatty acid contents, either in the seeds or in the oil itself. lipid contents of physic nut seeds stored at low a w decreased with the increase of storage duration, but the decrease was not as rapid as the seeds stored at high a w (fig 1). at high a w the moisture contents of physic nut seeds were also high, so they affected the activity of lipolytic fungi to hydrolyse the lipid of the seeds, apart from lipase enzyme produced by the tissue inside of the seeds. pomeranz (1992) reported that the occurrence of fungi will accelerate the degradation of lipid during storage. the decrease of lipid content was probably due to the activity of lipolytic fungi which grew dominantly and can live for long period. this condition caused the production of free fatty acid and rancidity. lipid of seeds can be degraded by lipase table 5 the effect of water activity and duration of storage on the percentage of physic nut seed germination means followed by the same letter are not significantly different according to duncan’s multiple range test at the 5% level. fig 2 effect of water activity and duration of storage on free fatty acid content of physic nut seeds. , , , . duration of storage (week) water activity 0.64 0.75 0.84 0.93 0 1 2 3 4 6 8 12 16 20 98.67 a ± 2.31 96.00 abc ± 0 94.67 abc ± 2.31 93.33 abcd ± 2.31 92.00 abcde ± 2.31 86.67 cdef ± 2.31 84.00 defg ± 0 77.33 gh ± 4.62 62.67 ijk ± 6.11 29.33 m ± 12.22 98.67 a ± 2.31 97.33 ab ± 2.31 96.00 abc ± 0 93.33 abcd ± 2.31 90.67 abcdef ± 2.31 88.00 bcdef ± 0 88.00 bcdef ± 0 76.67 gh ± 2.31 70.67 hi ± 12.86 25.33 m ± 14.05 97.33 ab ± 2.31 94.67 abc ± 2.31 92.00 abcde ± 0 88.00 bcdef ± 0 86.67 cdef ± 2.31 82.67 efg ± 4.62 76.00 gh ± 4.00 69.33 hij ± 2.31 56.67 k ± 11.02 2.67 n ± 4.62 94.67 abc ± 4.62 90.66 abcdef ± 2.31 86.66 cdef ± 2.31 84.00 defg ± 0 81.33 fg ± 2.31 61.33 jk ± 2.31 48.00 l ± 4.00 6.67 n ± 6.11 1.33 n ± 2.31 0.00 n ± 0 into free fatty acid and glycerol, especially if the moisture content of seeds was high. free fatty acid was an index of deteriorated seeds containing lipid during storage. according to gupta and rai (2003) a. fumigatus, a. niger, alternaria sp., c. cladosporioides and p. purpuragenum isolated from soil of dairy dung, house-based waste and mill soil were the good producers of intraand extracellular lipase activities. free fatty acid contents increased with the increase of a w and storage duration. it was assumed, that the ocurrence of various fungal species in physic nut seeds can degrade lipid. according to pomeranz (1992), aspergillus, cladosporium and penicillium were capable to degrade lipid compound and have high lipolytic activity. these fungal species were often isolated from seeds containing high lipid. at the beginning fungi hydrolyze lipid enzymatically with lipase, and then they was changed into glycerol through ß-oxydation. the requirement of free fatty acid contents of physic nut oil to be used for biodiesel is 0.8% (sudradjat et al. 2007). free fatty acid contents as much as 1% at a w 0.64 and 0.75 were obtained after 16 weeks of storage (fig 2). if free acid content was high, they will block the reaction of methyl esther (biodiesel) formation, i.e. methanol, which should react with triglyceride, but methanol formation was prevented by the reaction of soap formation. in this case methanol consumption becomes high as much as 40% (more expensive), consequently the production of biodiesel decreases as much as 30%. according to sauer (1988), the decrease of seed quality was followed by the increase of free fatty acid value. the level of free fatty acid value gave an indication concerning the decrease of seed quality. the level of free fatty acid depends on fungal species infecting the seeds. in this study, free fatty acid produced by lipase of physic nut seeds was not separated from that produced by fungi. dhingra et al. (2001) reported that the viability of soybean seeds inoculated with the conidia of aspergillus ruber decreased with the increase of free fatty acid and storage duration. according to dirjenbun (2006) good seed germination was e” 80%. at the beginning of storage seed germinations were > 90%, nevertheless they decreased at various a w during storage (table 5). the decrease of germination was very rapid at a w 0.93 (< 70%) after 6 weeks of storage with the curve in the form of negative sigmoid. at a w 0.84 the seed germination percentage was good up to 6 weeks of storage, at a w 0.75 it was good up to 12 weeks of storage, while at a w 0.64 it was good up to 8 weeks of storage. decrease of seed quality was determined by the capability of germination and the vigor at the beginning of storage, the moisture content of seeds, storage condition, and fungal infection. seed germination was affected by two factors, i.e. internal and external factors. internal factor consisted of the level of seed maturity, seed size, dormancy and germination inhibitor. external factor consisted water content, temperature, oxygen, light and growth media. seeds harvested before reaching the level of physiologic maturity was attained, they do not have high viability. adikadarsih and hartono (2006) reported that the lowest percentage of physic nut germination (7%) and vigor (4.3%) were found in physic nut seeds derived from fruits which fruit skin is green in color, while the highest percentage of germination (91.67%) and vigor (84.67%) were found in seeds derived from fruits which fruit skin is yellow in color. acknowledgements the authors gratefully acknowledge theresia prawitasari for her suggestions and encoureagement. we would like also to thank the staff members of plant pathology and service laboratories, seameo biotrop, who had in one way or another contributed to this research, and for a bpps scholarship to rantje lilly worang. references adikadarsih s, hartono j. 2007. [the effect of fruit maturity on the quality of physic nut seeds. (jatropha curcas l.)] [in indonesian]. in: proceedings of workshop ii: status teknologi tanaman jarak pagar. bogor, indonesia, 29 nov 2006. p 143-8. [aoac] association of official analytical chemist. 1984. official methods of the association of agricultural analytical chemists. arlington: association of analytical chemist, inc. [aoac] association of official analytical chemist. 1999. official methods of analysis of food composition; additives; natural contaminants. 2nd volume, 16th edition. gaithersburg: aoac. barnett hl, hunter bb. 1998. illustrated genera of imperfect fungi. 4th edition. st. paul: aps press. burgess lw, summerell ba, bullock s, gott kp, backhouse d. 1994. laboratory manual for fusarium research. sydney: university of sydney. chelkowski j. 1991. fungal pathogens influencing cereal seed quality at harvest. in: chelkowski j (ed), cereal grains; mycotoxins, fungi and quality in drying and storage. amsterdam: elsevier. p 53-66. dhingra od, mizubuti esg, napoleao it, jham g. 2001. free fatty acid accumulation and quality loss of stored soybean seeds invaded by aspergillus ruber. seed sci tech 29:193 – 203. [dirjenbun] direktorat jenderal perkebunan. 2006. pedoman mutu benih jarak pagar sistem dan prosedur pembangunan sumber benih dan peredaran benih jarak pagar. jakarta: dirjenbun. douglas pl, boyle r. 1996. effect of drying control on mycotoxins production. in: proceedings of 17th asean technical seminar on grain postharvest technology; lumut, malaysia, 25-27 jul 1995. p 27-33. gubitz gm, mittelbach m, trabi m. 1999. exploitation of the tropical oil seed plant jatropha curcas l. biores technol 67:73-82. gupta p, rai v. 2003. isolation and screening of lipolytic fungi from raipur city. geobios 30:165-8. hocking ad, pitt ji. 1980. dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods. appl environ microbiol 39:488-92. klich ma, pitt ji. 1988. a laboratory guide to common aspergillus species and their teleomorphs. north ryde: commonwealth scientific and industrial research organization pitt ji. 1988. a laboratory guide to common lacey penicillium species. north ryde: commonwealth scientific and industrial research organization. pomeranz y. 1992. biological, functional, and nutritive changes of cereal grains and their product. st. paul: american of cereal chemist inc. p 55-141. sauer db. 1988. effects of fungal deterioration on grain : nutritional value, toxicity, germination. int j food microbiol 7:267–75. sirisomboon p, kitchaiya p, pholpho t, mahuttanyavanitch w. 2007 physical and mechanical properties of jatropha curcas l. fruits, nuts and kernels. biosystems eng 97:201-7. microbiol indones144 dharmaputra et al. sudradjat hr, setiawan d, widyawati y, ariatmi r, sahirman. 2007. [the problem of processing technology of biodiesel derived from physic nut oil]. [in indonesian]. in: proceedings workshop ii: status teknologi tanaman jarak pagar. bogor, indonesia, 29 nov 2006. p 195-212. wanita yp, hartono j. 2007. [the effect of fruit maturity stage on the content of physic nut oil (jatropha curcas l.)]. [in indonesian]. in: proceedings of workshop ii: status teknologi tanaman jarak pagar. bogor, indonesia, 29 nov 2006. p 177-80. microbiol indones 145volume 3, 2009 03. barus.cdr vol.14, no.3, september 2020, p 101-107 doi: 10.5454/mi.14.3.3 genotypic characterization of rhizopus species from tempeh and usar: traditional inoculum of tempeh in indonesia 1 2 2 tati barus *, jason wiranata sanjaya , david tandjung , anastasia tatik 3 2 2 hartanti , adi yulandi , and vivitri dewi prasasty 1 master of biotechnology,faculty of biotechnology, universitas katolik atma jaya, jakarta 12930, indonesia; 2 department of biology, faculty of biotechnology, universitas katolik atma jaya, jakarta 12930, indonesia; 3 food technology, faculty of biotechnology, universitas katolik atma jaya, jakarta 12930, indonesia. soybeans tempeh (tempeh) is processed by fermentation using rhizopus spp. tempeh is an important source of protein in indonesia. the traditional inoculum in tempeh fermentation locally is known as usar, which is made from the leaves of hibiscus tiliaceus. however, rhizopus information from usar is still limited. therefore, this study aims to identify and investigate the genetic diversity of rhizopus species from usar and tempeh based on the internal transcribed spacer (its) sequences and the random amplified polymorphic dna (rapd) markers. twenty-three rhizopus strains were isolated from usar and ten rhizopus strains were isolated from tempeh. based on its sequences, the isolates were similar to rhizopus microsporus (30 isolates) and rhizopus delemar (3 isolates) with 98-99% similarity. the genetics of r. microsporus and r. delemar are varied and different from the genetics of r. microsporus from tempeh. the growth temperature of r. microsporus varies from 33°c to 48°c and r. delemar can grow to a maximum at 33°c. this research needs to be continued to obtain information about the role of rhizopus from this study in determining the quality of tempeh. key words: diversity, its, rapd, rhizopus, tempeh tempe kedelai (tempe) diolah melalui fermentasi menggunakan oleh rhizopus spp. tempe adalah salah satu sumber protein penting di indonesia. inokulum tradisional dalam fermentasi tempe dikenal sebagai usar yang terbuat dari daun hibiscus tiliaceus. namun, informasi rhizopus dari usar masih terbatas. oleh karena itu, penelitian ini bertujuan untuk mengidentifikasi dan mengkaji keragaman genetik spesies rhizopus dari usar dan tempe berdasarkan urutan sekuen internal transcribed spacer (its) dan penanda random amplified polymorphic dna (rapd). dua puluh tiga strain rhizopus diisolasi dari usar dan sepuluh strain rhizopus diisolasi dari tempe. berdasarkan sekuens its maka semua strain tersebut terdiri atas rhizopus microsporus (30 isolat) dan rhizopus delemar (3 isolat) dengan kemiripan 98-99%. genetik r. microsporus dan r. delemar bervariasi dan berbeda dari genetic r. microsporus dari tempe. suhu pertumbuhan r. microsporus bervariasi dari 33°c hingga 48°c dan r. delemar dapat tumbuh hingga maksimum pada 33°c. penelitian ini perlu dilanjutkan untuk mendapatkan informasi tentang peran r. microsporus dan r. delemar dari penelitian ini dalam menentukan kualitas tempe. kata kunci: its, keragaman, rapd, rhizopus, tempe microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21; email: 5727615 tati.barus@atmajaya.ac.id tempeh is made through the fermentation of soybean, mainly by rhizopus spp. therefore, rhizopus spp. is known as an economically important mold in indonesia. many species of rhizopus spp. such as r. oligosporus, r. oryzae, r. arrhizus, and r. stolonifer were previously identified from indonesian tempeh (dwidjoseputro and wolf 1970). in the current taxonomic system, r. arrhizus is considered as a synonym of r. oryzae (abe et al. 2010), r. oligosporusas as synonym of r. microspores (dolatabadi et al. 2014), and r. oryzae as synonym of r. delemar (abe et al. 2007). at present, tempeh producers rarely use usar (traditional inoculum) (fig 1) because they generally use commercial inoculum. usar is made by growing rhizopus spp. on hibiscus tiliaceus leaves for 24 hours and then dried. after that, soybeans tempeh (tempeh) is a traditional fermented food from indonesia. it has been consumed as main source protein by indonesian for years. it contains essential compounds such as vitamin b12 (keuth and bisping 1994), isoflavon and essential fatty acids. it has also been reported that tempeh have many health benefits such as in preventing free radicals (esaki et al. 1996). tempeh can prevent diarrhea (sudigbia 1999) and anemia (astuti 1999) because of increased iron availability during fermentation. tempeh can also stimulate the formation of good bacteria populations in the intestines (stephanie et al. 2019). it is ready to be used as an inoculum in tempeh fermentation. however, information about rhizopus spp. from usar was not yet available. many molecular techniques are available to study genotypic characterization of rhizopus (abe et al. 2007). to identify of rhizopus spp. often done based on its sequences (iwen et al. 2002; lott et al. 1998). abe et al. (2007) reported the classification of three rhizopus species, i.e. r. microsporus, r. stolonifer, and r. oryzae. therefore, in this study its sequences were used to assess the genetic diversity of rhizopus isolates from usar (fig 1) and tempeh. beside its sequences, various molecular methods have been developed to get more accurate data on the genetics of an organism. one of them is random amplified polymorphic dna (rapd) marker using polymerase chain reaction (pcr). rapd markers have been successfully used in assessing differentiating fungal genetic diversity, such as the genetic diversity of colletotrichum spp. (mahmodi et al. 2014) and rhizopus stolonifera (vágvölgyi et al. 2004). therefore, this study aims to identify and investigate the genetic diversity of rhizopus species from usar and tempeh based on the internal transcribed spacer (its) sequence and the random amplified polymorphic dna (rapd) markers. materials and methods rhizopus isolation. rhizopus isolates have been isolated from usar were taken from yogyakarta, central java-indonesia. usar sampling was carried out from yogyakarta because to our knowledge that only in this area usar was still used as an inoculum in tempeh production. a total 10 tempeh is collected from yogyakarta and solo, central java-indonesia. all these tempeh are produced using commercial inoculums. a total of 23 pieces of usar that grow rhizopus has been cut off and homogenized in sterile 0.85% w/v nacl by the use of a stomacherlab-blender 400 (seward medical, london, uk) for 1 minute. all isolates were grown on potato dextrose agar (pda) and incubated at o 28 c. all suspected rhizopus isolates were stored at o 4 c for further analysis. dna extraction. genomic dnas of rhizopus isolates were extracted from four days-old mycelia grown on pda using the phytopure™ dna extraction kit (ge healthcare, uk) according to the manufacturer's protocol. dnas were visualized on 1% electrophoresis agarose gel (promega, madison, usa) then stained with ethidium bromide (sigma-aldrich, usa). amplification of its region and sequencing. amplification of its region was performed using the geneamp® pcr system 2700 (applied biosystems, carlsbad, ca, usa) and the primer pair its4 (5′–tcctccgcttattgatatgc–3′) and its5 (5′–ggaagtaaaagtcgtaacaagg–3′) (white et al. 1990). a total 50 µl of reaction mixtures were used, containing 1 µl dna template, 10 µl 5x kapa (sigma-aldrich, usa), taq extra buffer, 2.5 µl of each primer, 1.5 µl 10 mm dntpmix, 3.5 µl mgcl2, 28.5 µl nuclease-free water (nfw), and 0.5 µl kapa taq extra hotstart dna polymerase. pcr conditions were set as follow: initial denaturation at 94 °c for 2 minutes, followed by 35 cycles of denaturation at 94°c for 15 seconds, annealing at a temperature of 55°c for 30 seconds, and extension at 72°c for 1 minute. final elongation was set at 72°c for 5 minutes. pcr products were visualized on 1% agarose gel and stained with ethidium bromide. pcr products were then partially sequenced at macrogen inc., republic of korea. the dna sequencing results were compared to the genbank database using blastn (http://blast.ncbi .nlm.nih.gov/blast.cgi). phylogenetic tree was constructed using mega7. the branch support was analyzed by 1000x bootstrap analysis. growth of rhizopus isolates on various temperature. all isolates were grown on potato dextrose agar (pda). to determine the optimal growth temperature, all rhizopus isolates obtained in this study were grown on various temperature, i.e. 33°c, 102 barus et al. microbiol indones fig 1 usar: traditional inoculum of tempeh . volume 14, 2020 microbiol indones 103 42°c, 45°c, and 48°c. amplification of random amplification of polymorphic dna. amplifications of rapd markers were conducted using single six primers (table 1) through geneamp® pcr system 2700 (applied biosystems, carlsbad, ca, usa). amplification of rapd was carried out with a total of 25 μl of the reaction mixture with the same composition carried out with barus et al. (2019). amplifications of rapd markers were performed with the same condition also with barus et al. (2019). annealing conditions were done based on the melting temperature of each primer (table 1). products of amplification were separated by electrophoresis in agarose gel (1% w/v). the agarose gel was stained with ethidium bromide and uv transilluminator was used to visualize the pcr products in agarose gel. the 1 kb ladder (fermentas) was used as weight marker. clearly resolved each band was manually scored for the presence (1) or absence (0) to make binary data. dendrogram analysis among all the rhizopus was computed using roderic d.m. page software. the unweighted pair group method analysis (upgma) was used for clustering and tree view software was used for interactive visualization of the dendrogram. results a total of twenty-three rhizopus isolates had been isolated from usar (tb23-tb45) and ten rhizopus isolates had been isolated from tempeh (tb46-tb55) (fig 2). its sequences were successfully amplified and each pcr amplification showed dna fragments with single band at 700 bp (fig 2). blastn results of its sequence (± 600 nucleotides) showed that 30 isolates (tb23-tb25, tb27, tb29-tb36, tb38-tb55) were rhizopus microporus with similarity about 98-100%. only three isolates (tb26,tb28, tb37) were hizopus r delemar with similarity about 98-100%. all the its sequences have been submitted to genbank with accession numbers mf445258 mf445290. the phylogenetic tree based on the its sequences showed that the thirty three of rhizopus isolates were divided into two clusters (fig 3). the first cluster consisted of r. microsporus (30 isolates) and the second cluster consisted of r. delemar (3 isolates). all rhizopus spp. isolates were grown at 33°c, 42°c, 45°c, and 48°c. the growth temperature for all isolates r. microsporus varied . eight strains (table 2) of r. microspores could grow up to 48 , thirteen °c strains could grow up to 45 , seven strains could grow °c up to 42 , and two strains could grow up to 33 . °c °c conversely, three r. delemar isolates could only grow up to 33°c. � genomic dnas isolated from 33 rhizopus isolates were subjected to obtain rapd-pcr markers using six primers (table 1), but only 9 out of 33 rhizopus isolates produced distinc and reproducible band rapd marker using these primers. the dendogram (fig 4) describes the genetic similarity r. microsporus and r. delemar was successfully created. upgma dendrogram based on rapd – pcr separated the r. microsporus and r. delemar in two main clusters. among all r microporus, the smallest genetic similarity (gs) (35%) was found between tb34 and tb35 and the largest gs (63%) was found between tb32 and tb33. r. microporus from tempeh (tb49) is most similar to tb32 with gc 54% and most different with tb34 with gc 38%. r. delemar tb26 and r. delemar tb37 have genetic similarity 64%. discussion a study by bressa et al. (2017) showed that lifestyle enhanced health-promoting bacteria. a previous table 1 primers to amplify rapd markers of rhizopus isolates. primer sequence melting temperature sources opq6 gagcgccttg 34°c vagvolgyi et al. 2004 opa9 gggtaacgcc 34°c mahmodi et al. 2014 opj20 aagcggcctc 34°c mahmodi et al. 2014 r108 gtattgccct 30°c vagvolgyi et al. 2004 opa11 caatcgccgt 32°c mahmodi et al. 2014 opa1 caggcccttc 34°c mahmodi et al. 2014 1 104 barus et al. microbiol indones source isolate code species growth temperature (oc) 33 42 45 48 usar tb24 r. microsporus ✔ ✔ ✔ ✔ usar tb25 r. microsporus ✔ ✔ ✔ ✔ usar tb31 r. microsporus ✔ ✔ ✔ ✔ usar tb32 r. microsporus ✔ ✔ ✔ ✔ usar tb33 r. microsporus ✔ ✔ ✔ ✔ usar tb39 r. microsporus ✔ ✔ ✔ ✔ tempeh tb48 r. microsporus ✔ ✔ ✔ ✔ tempeh tb54 r. microsporus ✔ ✔ ✔ ✔ usar tb23 r. microsporus ✔ ✔ ✔ usar tb27 r. microsporus ✔ ✔ ✔ usar tb30 r. microsporus ✔ ✔ ✔ usar tb35 r. microsporus ✔ ✔ ✔ usar tb36 r. microsporus ✔ ✔ ✔ usar tb38 r. microsporus ✔ ✔ ✔ usar tb40 r. microsporus ✔ ✔ ✔ usar tb43 r. microsporus ✔ ✔ ✔ usar tb45 r. microsporus ✔ ✔ ✔ tempeh tb46 r. microsporus ✔ ✔ ✔ tempeh tb52 r. microsporus ✔ ✔ ✔ tempeh tb53 r. microsporus ✔ ✔ ✔ tempeh tb49 r. microsporus ✔ ✔ ✔ usar tb29 r. microsporus ✔ ✔ usar tb34 r. microsporus ✔ ✔ usar tb41 r. microsporus ✔ ✔ usar tb42 r. microsporus ✔ ✔ usar tb44 r. microsporus ✔ ✔ tempeh tb47 r. microsporus ✔ ✔ tempeh tb51 r. microsporus ✔ ✔ tempeh tb50 r. microsporus ✔ tempeh tb55 r. microsporus ✔ usar tb26 r. delemar ✔ usar tb28 r. delemar ✔ usar tb37 r. delemar ✔ 1 table 2 growth on various temperatures of thirty-three hizopus isolates isolated fromusar and tempeh.r reported that gut microbiota in obese subjects and/or with type-2 diabetes were different from lean and nondiabetic subjects (patterson et al. 2016). to get beneficial gut microbiota population, probiotics consumption and dietary fibers are strongly recommended. holscher (2017) reported that low fiber intake is associated with increased chronic diseases, such as obesity, cardiovascular disease, type 2 diabetes, and colon cancer. tempeh, a popular fermented food in indonesia, is one source of fiber-rich food. the main microorganism in fermentation of tempeh is rhizopus spp. at present, many molecular techniques are available for identification of rhizopus spp. however, internal transcribed spacer (its) is often used (abe et al. 2003. based on its sequence showed that 30 isolates (tb23-tb25, tb27, tb29-tb36, tb38-tb55) were rhizopus microporus with similarity about 98-100% and three isolates (tb26, tb28, tb37) were hizopus delemar with similarity r about 98-100%.the its regions have become an important molecular target for fungal taxonomy and identification (iwen et al. 2002). due to greater sequence variations, the its domains are more suitable for species identification (iwen et al. 2002; lott et al. 1998). therefore its sequences are widely used to identify and assess fungal genetic diversity. volume 14, 2020 microbiol indones 105 fig 2 results of pcr amplification sequences of internal transcribed spacer (its) sequence of rhizopus isolates. m: marker 1-kb lambda ladder. tb23-tb45: rhizopus isolates from usar. tb46-tb55: rhizopus isolates from tempeh. fig 3 phylogenetic tree generated from the internal transcribed spacer (its) sequences of 33 isolates of hizopus r spesies isolated from usar and tempeh. 106 barus et al. microbiol indones fig 4 upgma dendrogram of rhizopus strains isolated from usar and tempeh based on rapd – pcr. in the past, it was reported that various rhizopus species were used to make tempeh (dwidjoseputro and wolf 1970). in this study it was shown that only r. microspores were found in tempeh samples. this finding is similar to the report by hartanti et al. (2015), where tempeh collected from 28 locations throughout indonesia only contained r. microspores. this is caused by the use of commercial inoculums that only contain the r. microsporus. surprisingly, in this study found three isolates of r. delemar from usar. based on the rapd marker (fig 4), tb26 and tb27 are in the same cluster, but they are different types represented by different rapd markers. information on r. delemar in tempeh is still limited. therefore, the role of r. delemar in determining the quality of tempeh needs to be further investigated. the species of rhizopus may have an important contribution to the variety of tempeh flavor and nutritional value. the different species of rhizopus have different metabolic activities. moreover, it has been reported that r. delemar produced fumaric acid and malic acid (abe et al. 2007). figure 3 showed that its sequences were not sufficent to distinguish r. microsporusup to the variety level. this can be seen from the phylogenetic tree which r. microsporus var. azygosporus, r. microsporus var. chinensis, r. microsporus var. oligosporus, r. microsporus var. rhizopodiformis, and r. microsporus var. tuberosus were all grouped as one cluster (cluster 1). this indicated that the its sequences were not sufficent to distinguish r. microsporusup to the variety level. this report is in line with hartanti et al. (2015) which its sequences were not sufficent to distinguish r. microsporus spesies from tempeh up to the variety. rhizopus is the main microorganism in making tempeh. information about the growth of rhizopus isolates on various temperature is important as a basis for selecting isolates to be used as inoculums in tempeh fermentation. the growth temperature for all isolates r. microsporus (table 2) this was found strains varied . that rhizopus microsporus (tb32) can grow up to 48°c and rhizopus microsporus (tb55) can grow up to 32°c (table 2). barus et al. (2019) reported that rhizopus microsporus (tb32) produced tempeh with higher antioxidant activity compared to rhizopus microsporus (tb55). figure 4 showed that rapd markers can show genetic variation in nine rhizopus. previously it has been reported that rapd marker can be used as an important technique to investigate for the genetic variations of fungal (dwivedi et al. 2018). these reported are in line with our result, where rapd marker can also distinguish the genetic variations of nine r. microsporus and two r. delemar well. genetic of all r. microsporus isolate from usar (tb26, tb30, tb32-tb37) were different from the genetic r. microsporus from tempeh (tb49). furthermore, the genetics of r. microsporus and r. delemar derived from usar also varied. rapd markers of 24 isolates (tb 23-tb25, tb27-tb29, tb31, tb38, tb39-tb48, tb50-tb55) have not been successfully amplified using several primers (table 1) even though they have been repeated several times. this might be accessible using another primer. acknowledgements this study was funded by the competitive grant program of atma jaya catholic university of indonesia volume 14, 2020 microbiol indones 107 references abe a, asano k, sone t. 2010. a molecular phylogenybased taxonomy of the genus rhizopus. biosci biotechnol biochem 74(7): 1325-1331. doi: 10.1271/bbb.90718. abe a, oda y, asano k, sone t. 2007. rhizopus delemar is the proper name for rhizopus oryzae fumaric-malic acid producers. mycologia 99(5): 714-722. doi: 10.3852/mycologia.99.5.714. astuti m. 1999. iron availability of tempe and uses in iron deficiency anemia. the complete handbook of tempe: the unique fermented soyfood of indonesia 41-45. barus t, halim r, hartanti at, saputra pk. 2019. genetic diversity of rhizopus microsporus from traditional inoculum of tempeh in indonesia based on its sequences and rapd marker. biodiversitas journal of b i o l o g i c a l d i v e r s i t y 2 0 ( 3 ) : 8 4 7 8 5 2 . d o i : 10.13057/biodiv/d200331. bressa c, bailén-andrino m, pérez-santiago j, gonzálezsoltero r, pérez m, montalvo-lominchar mg, matémuñoz jl, domínguez r, moreno d, larrosa m. 2017. differences in gut microbiota profile between women with active lifestyle and sedentary women. plos one 12(2): 1-20. doi: 10.1371/journal.pone.0171352. dolatabadi s, walther g, van den ende ag, de hoog g. 2014. diversity and delimitation of rhizopus microsporus. fungal diversity 64(1): 145-163. doi 10.1007/s13225-013-0229-6. dwidjoseputro d, wolf ft. 1970. microbiological studies of indonesian fermented food stuffs. mycopathologia et mycologia applicata 41(3-4): 211-222. doi: 10.1007/bf02051099. dwivedi s, singh s, chauhan uk, tiwari mk. 2018. inter and intraspecific genetic diversity (rapd) among three most frequent species of macrofungi (ganoderma lucidum, leucoagricus sp. and lentinus sp.) of tropical forest of central india. journal of genetic engineering a n d b i o t e c h n o l o g y 1 6 ( 1 ) : 1 3 3 1 4 1 . doi:10.1016/j.jgeb.2017.11.008. esaki h, onozaki h, kawakishi s, osawa t. 1996. new antioxidant isolated from tempeh. journal of agricultural and food chemistry 44(3): 696-700. doi: 10.1021/jf950454t. hartanti at, rahayu g, hidayat i. 2015. rhizopus species from fresh tempeh collected from several regions in indonesia. hayati journal of biosciences 22(3):136142. doi: 10.4308/hjb.22.3.136. holscher hd. 2017. dietary fiber and prebiotics and the gastrointestinal microbiota. gut microbes 8(2):172184. doi: 10.1080/19490976.2017.1290756. iwen pc, hinrichs sh, rupp me. 2002. utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. medical mycol. 40(1): 87-109. doi: 10.1080/mmy.40.1.87.109. keuth s, bisping b. 1994. vitamin b12 production by citrobacter freundii or klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by pcr. appl environ microbiol. 60(5): 1495-1499. doi: 10.1128/aem.60.5.1495-1499.1994. lott tj, burns bm, zancope-oliveira r, elie cm, reiss e. 1998. sequence analysis of the internal transcribed spacer 2 (its2) from yeast species within the genus candida. current microbiology 36(2): 63-69. doi: 10.1007/s002849900280. mahmodi f, kadir j, puteh a, pourdad s, nasehi a, s o l e i m a n i n . 2 0 1 4 . g e n e t i c d i v e r s i t y a n d differentiation of colletotrichum spp. isolates associated with leguminosae using multigene loci, rapd and issr. the plant pathology journal 30(1): 10-24. doi:10.5423/ppj.oa.05.2013.0054. patterson e, ryan pm, cryan jf, dinan tg, ross rp, fitzgerald gf, stanton c. 2016. gut microbiota, obesity and diabetes. postgraduate medical journal 92(1087):286-300. doi: 10.1136/postgradmedj-2015133285. stephanie, kartawidjajaputra f, silo w, yogiara, suwanto, a. 2018. tempeh consumption enhanced beneficial bacteria in the human gut. food res. 3(1):57-63. doi: 10.26656/fr.2017.3(1).230. sudigbia i. 1999. tempe in the management of infant diarrhea in indonesia. the complete handbook of tempeh: the unique fermented soyfood of indonesia 23-40. vágvölgyi c, heinrich h, ács k, papp t. 2004. genetic variability in the species rhizopus stolonifer, assessed by random amplified polymorphic dna analysis. antonie van leeuwenhoek 86(2): 181-188. doi: 10.1023/b:anto.0000036123.48215.fc. page 1 page 2 page 3 page 4 page 5 page 6 page 7 04. santoso.cdr vol.14, no.3, september 2020, p 108-116 doi: 10.5454/mi.14.3.4 effect of hydrocarbon-polluted seawater on the cell density of microalgae scenedesmus vacuolatus shihira & krauss 1 1 clara alverina santoso , noverita dian takarina , hanies ambarsari *, 2 1 1 nining betawati prihantini , sitaresmi and 1 department of biology, faculty of mathematics and natural sciences, universitas indonesia, kampus ui depok 16424, indonesia; 2 center of environmental technology (ptl), agency for the assessment and application of technology (bppt), building 820 geostech, puspiptek, setu, south tangerang city, banten 15314, indonesia. study the effect of hydrocarbon-polluted seawater on the cell density of microalgae scenedesmus vacuolatus has been carried out in this study. hydrocarbon pollution derived from the oil in the sea can inhibit the photosynthesis process of microalgae. this might impact the density of microalgae cells. the purposes of this study are to determine the effect of the concentration of hydrocarbon-polluted seawater on the density of scenedesmus vacuolatus microalgae cells and to determine the optimum treatment to reduce total petroleum hydrocarbons (tph) levels. a sampling of hydrocarbon-polluted seawater was taken at kali adem port, jakarta. the treatment done in this research used a walne medium with the addition of 25% hydrocarbon-polluted seawater (a), 50% (b), 75% (c), and 100% (d). control is walne medium with sterile seawater that was not from the kali adem port. the results showed the highest average density of scenedesmus vacuolatus cells was in the control sample. this can be seen from the results of the average cell density at the peak time of 29.48 x 105 cells / ml, as well as the log phase length of scenedesmus vacuolatus. measurement of tph levels showed decreases of tph in all treatments. the optimum treatment to reduce tph levels is treatment b with a reduction percentage of 70.62%.. key words: kali adem port, scenedesmus vacuolatus, total petroleum hydrocarbon (tph), walne medium penelitian mengenai pengaruh air laut tercemar hidrokarbon terhadap kepadatan sel mikroalga scenedesmus vacuolatus telah dilakukan. pencemaran hidrokarbon yang berasal dari minyak di laut dapat menghambat proses fotosintesis mikroalga. hal tersebut dapat berdampak pada kepadatan sel mikroalga. penelitian ini bertujuan untuk mengetahui pengaruh konsentrasi air laut tercemar hidrokarbon terhadap kepadatan sel mikroalga scenedesmus vacuolatus, serta mengetahui perlakuan yang optimum untuk menurunkan kadar total petroleum hidrokarbon (tph). pengambilan sampel air laut tercemar hidrokarbon dilakukan di pelabuhan kali adem, jakarta. perlakuan dalam penelitian adalah medium walne dengan penambahan air laut tercemar hidrokarbon 25% (a), medium walne dengan penambahan air laut tercemar hidrokarbon 50% (b), medium walne dengan penambahan air laut tercemar hidrokarbon 75% (c), dan medium walne dengan penambahan air laut tercemar hidrokarbon 100% (d). kontrol yang digunakan adalah medium walne dengan air laut steril yang bukan berasal dari pelabuhan kali adem. hasil penelitian menunjukkan rata-rata kepadatan sel scenedesmus vacuolatus tertinggi yaitu pada perlakuan kontrol. hal tersebut dapat dilihat dari hasil rata-rata kepadatan sel pada masa puncak sebesar 29,48 x 105 sel/ml, serta panjang fase log dari scenedesmus vacuolatus. hasil pengukuran kadar tph menunjukkan terdapat penurunan tph pada seluruh perlakuan. perlakuan optimum untuk menurunkan kadar tph yaitu perlakuan b dengan persen penurunan sebesar 70,62%. kata kunci: medium walne, pelabuhan kali adem, scenedesmus vacuolatus, total petroleum hidrokarbon (tph) microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21 75791377; email: hanies.ambarsari@bppt.go.id impacts on the environment and living things (wibowo 2018). microalgae are photosynthetic organisms that require sunlight and co fixation to photosynthesis 2 (muchammad et al. 2013). contact between the oil layer and microalgae on the surface of the water in addition to having an impact on the photosynthesis process will also have an impact on its density. microalgae flexibility in morphology, physiology, and life cycle gives the ability to survive in polluted conditions (amirlatifi et al. 2013). response and tolerance of exposure to pollutant levels in each port is one of the places with the most human activities, especially transportation. the number of transportation activities by ships causes common pollution in the port area is oil pollution. oil pollution to the sea is the release of liquid petroleum hydrocarbon pollutants which mainly come from human activities (priyadarshani et al. 2011). petroleum hydrocarbon pollution in the sea has many negative volume 14, 2020 microbiol indones 109 type of the microalgae are different. one of microalgae genus that is adaptive in oil-polluted waters is the scenedesmus. the genus microalgae scenedesmus can adapt to oil-contaminated environments through acclimatization of physiology and genetic mutations (martinez et al. 2013). scenedesmus is a green microalga that can be found in fresh to brackish water (phinyo et al. 2017). generally, the genus scenedesmus has an ellipsoidal cell shape and forms a coenobitic with a multiple of 4 (guiry and guiry 2019). scenedesmus vacuolatus is a species of the genus scenedesmus which has a different shape. these microalgae are round in shape, do not have a prominent apex, and do not have cell-to-cell connections like the genus scenedesmus in general. s. vacuolatus is very tolerant and adaptive to the environment. lewis and flechtner in 2014, found s. vacuolatus in macrobiotic crust in deserts (lewis and flechtner 2004). in addition, these microalgae are environmentally tolerant with a wide salinity range (anand et al. 2019). hydrocarbon compounds in water can trigger eutrophication and cause an abundance of certain types of microalgae, resulting in changes in community structure. besides affecting the structure of the microalgae community, hydrocarbons also affect the size and physiology of microalgae cells. in phytoplankton exposed to hydrocarbons, the cell size is reduced in line with the increase in petroleum hydrocarbon content (nair et al. 2014). the response of each microalgae to pollutants, especially petroleum hydrocarbons is different. al obaidy and lami (2014) conducted researched on chlorophyll a levels in microcystis flos-aquae (wittr.) kircher and nostoc carneum agardh grown on a medium containing crude oil. in microcystis flos-aquae (wittr.) kircher levels of chlorophyll a decreased, whereas in nostoc carneum agardh levels of chlorophyll a tended to increase (al obaidy and lami 2014). microalgae are capable of contributing to the degradation of environmental pollutants either by directly altering pollutants, or by increasing the degradation potential of existing microbial (semple et al. 1999). several chemicals can be found in total petroleum hydrocarbons (tph) hexane, jet fuel, mineral oil, benzene, toluene, xylene, naphthalene, fluorene, and other petroleum products. one of the benzene derivatives that can be degraded by microalgae is phenol. phenol can be degraded via the meta-cleavage pathway. the meta-cleavage pathway in phenol degradation produces the final products, namely pyruvate and acetaldehyde. pyruvate can be metabolized in the krebs cycle, and acetaldehyde can be converted to acetyl-coa with the help of acetaldehyde dehydrogenase (acdh) (patel et al. 2017). the study aims to determine the effect of concentrations of hydrocarbon polluted seawater on the density of scenedesmus vacuolatus microalgae cells, and determine the optimum treatment to reduce levels of total petroleum hydrocarbons (tph). materials and methods design experiment. this study used five variations of treatment with three repetitions; control (0%), a (25%), b (50%), c (75%), and d (100%). this research used 2-litter gallons with the composition of scenedesmus vacuolatus microalgae, walne nutrient and vitamin solutions, hydrocarbon-polluted seawater samples, and also sterile seawater (not from the port) as a solvent. growth medium for microalgae. the growth medium that will be used for the growth of scenedesmus vacuolatus in the study is walne medium. the first stage of making 1 liter walne medium is to prepare distilled water as a solvent for trace metal solutions, vitamin solutions, and nutrient solutions. the next stage is the weighing of trace metal solution materials namely zncl (2.1 g), cocl .6h o (2 2 2 2 g), (nh )6mo o .4h o (0.9 g), and cuso .5h o (2 g). 4 7 24 2 4 2 the trace metal solution is dissolved into 100 ml distilled water and labeled. weigh the ingredients of the vitamin solution, namely vitamin b12 (10 mg), vitamin b1 (10 mg), and biotin (200 µg). the vitamin solution ingredients are dissolved into 100 ml distilled water and labeled. the next stage is weighing the ingredients of nutrient solutions, namely fecl .6h o (1.3 g), 3 2 mncl .4h o (0.36 g), h bo (33.6 g), edta 2na (45 2 2 3 3 g), nah po .2h o ( 20 g), nano (100 g). the nutrient 2 4 2 3 solution ingredients are dissolved into 1 l of distilled water and 1 ml of trace metal solution is added then labeled. one ml of nutrient solution has been made and then dissolved into 1 l of sea water that has been sterilized using a vacuum filter. the solution was sterilized using an autoclave at 121 °c for 15 minutes at a pressure of 0.2 mpa. after sterilization, add aseptic a 0.1 ml vitamin solution. observation of microalgae culture colors. microalgae culture colors were observed using faber castell's standard color table. observations were made every day during the study. measurement of microalgae cell density. cell size in starter culture, control culture, treatments a, b, c, and d were observed using a microscope at 1000x magnification. microalgae cell density calculations are p e r f o r m e d u s i n g a n i m p r o v e d n e u b a u e r haemacytometer then observed under a microscope. the observations are then calculated using the following formula: 4 d = 10 x q cell/ml � d: cell density 2� q: average cell count per 1 mm (andersen 2005: 249) measurement of total petroleum hydrocarbon (tph). measurement of total petroleum hydrocarbon content using gravimetric methods according to sni 6989.10: 2011 standard. the measurement of tph concentration is divided into four parts, namely oil and fat extraction, distillation, calculation of oil and fat content, and calculation of mineral oil content (total petroleum hydrocarbons). measurement of environmental parameters. o physical parameters observed were temperature ( c), salinity (‰), light intensity (lux), and culture color. retrieval of temperature data is taken using a multiparameter water quality meter [aquaread]. measurement of light intensity is done with a lux meter [uni-t]. salinity levels were measured by using a refractometer [atc brix]. chemical parameters observed were ph level, and dissolved oxygen (mg/l). sample analysis was performed aiming to determine the level of chemical content in the water sample before and after the experiment. the ph data collection was carried out using ph paper, dissolved oxygen was measured using a multiparameter water quality meter device. all of these factors were observed every day during the study then recorded. statistical methods. the data obtained were tested using the saphiro wilk normality test followed by the kruskal wallis non-parametric statistical test (α <0.05). these data were tested to determine whether or not there was a significant difference in the concentration of seawater contaminated with hydrocarbons on the density of microalgae cells scenedesmus vacuolatus. results observation of microalgae culture colors. figure 1 shows the change of microalgae cultures color during research. the color of the scenedesmus vacuolatus culture in the control, a, b, c, and d medium on day 2 to day 9 experienced the same changes. on the 12th day of observation, the treatment medium c and d experienced a darker color change, namely moss green. the macroscopic appearance of the cultures on the control, a, and b treatment mediums were the same as the 9th day, namely grass green. on the 13th day of observation, all test cultures were the same color, namely grass green. the macroscopic color change of the culture on the 14th day only occurred in the control medium, a, and b treatment mediums, which were leaf green, while the c and d treatment mediums were still the same color as the previous day (t ). on the 16th day of observation, the culture colors 13 on control medium, a, and b were still the same color as the previous day (t ), namely leaf green (leaf 14 green), while the treatment medium c and d changed to sap green. the macroscopic appearance of the culture in the control medium, a, b, c, and d treatment mediums were the same color on the 19th to the 21st day, namely sap green. overall, the color changes in the c and d cultures occurred more rapidly than in other cultures. this can be seen on the 12th and 16th days, the color of c and d cultures changed to a darker green. measurement of microalgae cell size. observation of scenedesmus vacuolatus cell size was observed through the size of cells in starter cultures, control cultures, and each treatment. microscopic observations of scenedesmus vacuolatus cells cultured in starter cultures, control cultures, treatment a, and treatment b had a cell size range between 4—6 µm. the size of s. vacuolatus cells cultured in treatment c and d ranged from 4—5 µm. overall, there is no significant difference in size for each treatment. in all treatments, the size of s. vacuolatus cells ranged from 4 6 µm. the difference in smaller cell size was only found in c and d medium. measurement of microalgae cell density. the cell density of the starter culture of scenedesmus vacuolatus entered into the test culture was 958,000 -1 cells ml in 1,800 ml of medium. the results of the mean cell density of scenedesmus vacuolatus are listed in table 2. these results are the average of three repetitions for each variation of the treatment medium. the growth curve of scenedesmus vacuolatus is shown in figure 2. the average density of scenedesmus vacuolatus cells on days 1 to 9 in the medium which was given hydrocarbon-polluted sea water (a, b, c, and d) was higher than in the control medium. the growth curve of scenedesmus vacuolatus after day 9 showed that the cell density increased significantly. after the 9th to 21st day, the average density of scenedesmus vacuolatus 110 santoso et al. microbiol indones fig 1 macroscopic color appearance of the scenedesmus vacuolatus culture from day 2 – 21. volume 14, 2020 microbiol indones 111 fig 2 microscopic observation of scenedesmus vacuolatus on starter culture (a), control medium (b), treatment medium a (c), medium b (d), medium c (e), and medium d (f). fig 3 growth curve of scenedesmus vacuolatus. a b c d e f 112 santoso et al. microbiol indones cells in the control medium was higher than the other mediums.the growth continued until it reached the peak period on the 14th day for control, a, b, and c medium, while the culture in d medium reached its peak on the 16th day. the growth rate of scenedesmus vacuolatus began to decline after day 14 in control, a, b, and c, whereas in culture d it occurred after day 16. this is indicated by a decrease in the cell density curve. kruskal wallis statistical test showed that there was no significant difference in the density of microalgae scenedesmus vacuolatus cells to variations in the concentration of sea water contaminated with hydrocarbons. the increase or decrease in s. vacuolatus cell density was not directly affected by the differences in the treatment given. measurement of total petroleum hydrocarbon (tph). the total petroleum hydrocarbon (tph) content shows the oil concentration in each treatment. the total petroleum hydrocarbon content can be determined from the residual weight of the oil after the extraction process. the tph level in treatment medium a at the beginning of the study was 719.38 mg, and at the end of the study was 266.65 mg. the decrease in tph levels in treatment medium a was 62.93%. the tph level in the b treatment medium at the beginning of the study was 1438.76 mg, and at the end of the study was 422.69 mg. the decrease in tph levels in the b treatment medium was 70.62%. the tph level in the c treatment medium at the beginning of the study was 2158.14 mg, and at the end of the study was 840.71 mg. the decrease in tph levels in the c treatment medium was 61.04%. the tph level in the d treatment medium at the beginning of the study was 2586.89 mg, and at the end of the study was 1597.91 mg. the decrease in tph levels in the d treatment medium was 38.23%. the highest decrease in total petroleum hydrocarbons was culture b, amounting to 70.62%. the results of measuring tph levels in each treatment medium at the beginning and end of the study are listed in table 3. the results of kruskal wallis's non-parametric statistical test (attachment 6) showed that there was no significant difference in the density of microalgae scenedesmus vacuolatus cells to variations in the concentration of sea water contaminated with hydrocarbons. in 1 d a y c e ll d e n sity (c e ll.m l-1) (1 05) c o n tro l (0 % ) a (2 5 % ) b (5 0 % ) c (7 5 % ) d (1 0 0 % ) 0 9 ,5 8 9 ,5 8 9 ,5 8 9 ,5 8 9 ,5 8 1 1 ,1 9 1 ,2 5 1 ,2 1 1 ,7 8 2 ,2 1 2 1 ,2 3 1 ,7 8 1 ,8 3 3 ,3 6 3 ,7 5 5 3 ,7 9 3 ,3 9 3 ,9 6 5 ,0 5 4 ,9 6 6 4 ,7 5 5 ,2 2 5 ,2 3 6 ,5 3 7 ,2 6 7 6 ,3 9 6 ,5 9 6 ,5 2 8 ,2 2 8 ,8 8 8 7 ,1 2 7 ,6 5 7 ,6 7 8 ,8 1 9 ,3 4 9 7 ,8 8 9 ,1 0 8 ,0 7 9 ,0 8 1 0 ,5 5 1 2 1 7 ,9 3 1 0 ,7 3 1 0 ,3 2 9 ,8 7 1 1 ,2 2 1 3 2 0 ,6 8 1 7 ,4 8 1 5 ,3 8 1 7 ,6 4 1 4 ,1 5 1 4 2 9 ,4 8 1 8 ,5 9 1 6 ,3 5 1 9 ,9 7 2 0 ,5 1 1 6 2 2 ,8 5 1 6 ,8 1 1 4 ,2 7 1 5 ,9 8 2 3 ,2 5 1 9 1 7 ,0 2 1 4 ,9 2 1 3 ,3 7 1 6 ,3 2 1 7 ,4 4 2 1 1 3 ,8 5 1 2 ,7 7 1 2 ,1 7 1 3 ,2 3 1 2 ,5 4 1 treatment total petroleum hydrocarbon (tph) level beginning (mg) end (mg) % residue % decreasing control (0%) 0 0 0 0 a (25%) 719,38 266,65 37,07 62,93 b (50%) 1438,76 422,69 29,38 70,62 c (75%) 2158,14 840,71 38,96 61,04 d (100%) 2586,89 1597,91 61,77 38,23 table 1 average cell density of scenedesmus vacuolatus table 2 average of total petroleum hydrocarbon (tph) level volume 14, 2020 microbiol indones 113 seawater does not directly affect the density of microalgae cells, but this study shows that s. vacuolatus can reduce the hydrocarbon levels contained in oil in sea water. measurement of environmental parameters. observation of the environmental conditions of the test culture during the study was carried out by measuring temperature, ph, salinity, dissolved oxygen in the medium, and light intensity. observation of the temperature of the test culture was carried out using a multiparameter tool. the results of the culture o temperature observations ranged from 27.3 28.3 c. observation of the salinity of the test cultures was carried out using a refractometer. the results of the salinity measurement during the study on treatment k and a were 28 ‰, while in treatment b, c, and d were 30 ‰. this difference occurs because the composition of solvent seawater is more in treatment k and a. the initial salinity measurement of solvent seawater is 28 ppt. dissolved oxygen observation in the test culture was carried out using a multiparameter device. -1 dissolved oxygen data ranged between 7,1 9,5 mg l . observation of ph of the test culture was carried out using ph paper. the result of ph measurement in the test culture during the study was 7. observation of light intensity at the place where the test culture was placed was carried out using a lux meter. the results of observations of light intensity in this study ranged from 7200 7900 lux. discussion the results of culture color observations in the study were compared to the faber castell color table. overall, the color changes in cultures c and d occur more quickly than in other cultures. this can be seen on the 12th and 16th day the colors of the c and d cultures change to a denser green. these changes can occur due to greater levels of oil pollutants. exposure to oil pollutants in high concentrations has an impact on decreased levels of carbohydrates and microalgae protein (lewis & flechtner 2004). oil pollutants in seawaters contain many fractions of compounds which can be grouped into hydrocarbon and non-hydrocarbon fractions. at higher concentrations of oil pollutants, the more non-hydrocarbon fractions on the medium. the non-hydrocarbon fraction includes several inorganic compounds such as nitrogen, sulfur, phosphorus, iron and some trace elements (anand et al. 2019). high nitrogen content can interfere with the process of formation of photosynthetic pigments in microalgae. that is because the deactivation process in photosynthetic pigment activity (valotton et al. 2008). the results of measurements of the average density of scenedesmus vacuolatus cells on days 1 to 9 in the medium given sea water contaminated with hydrocarbons (culture a, b, c, and d) are higher than in the control medium. this is caused by the ability of microalgae to accumulate and use oil (petroleum hydrocarbons) as a source of organic compounds (al obaidy & lami 2014). growth continues until it reaches its peak on the 14th day for cultures on medium k, a, b, and c, while culture on medium d reaches the peak period on the 16th day. this can occur because of the higher concentration of petroleum hydrocarbons in the medium d. provision of crude oil to microalgae culture can prolong the growth phase and produce high biomass production (semple et al. 1999). after the 9th day to the 21st day, the average density of scenedesmus vacuolatus cells in the control medium was higher than that of the entire medium with the addition of hydrocarbon polluted sea water. this can occur due to the impact of residual petroleum hydrocarbons that are unable to accumulate or are degraded by microalgae. petroleum hydrocarbons can inhibit the growth of microalgae by reducing the ability to absorb co , 2 photosynthesis, respiration, and cell division (patel et al. 2017). the growth rate of scenedesmus vacuolatus begins to decrease after the 14th day in cultures k, a, b, and c, whereas in culture d occurs after the 16th day. this is indicated by the decrease in the cell density curve. this occurs due to reduced nutrition in the medium and the effect of residual oil (petroleum hydrocarbons) which cannot be accumulated by microalgae. petroleum hydrocarbons can be toxic to microalgae when forming thick oil layers around organisms, inhibiting gas diffusion, and destruction of cell membranes due to continuous hydrocarbon uptake (el-dib et al. 2001). the highest decrease in total petroleum hydrocarbons was in culture b, by 70.62%. this shows that scenedesmus vacuolatus can reduce tph levels optimally at 50% petroleum hydrocarbon concentration. microalgae degrades hydrocarbons by accumulating and transforming the compound (phinyo et al. 2017). some chemicals that can be found in total petroleum hydrocarbons (tph) are hexane, jet fuel, mineral oil, benzene, toluene, xylene, naphthalene, fluorene, and other petroleum products (epa 2017). one of the hydrocarbons that can be degraded by microalgae is phenol which is a derivative of benzene compounds. phenols can be degraded by microalgae through the 114 santoso et al. microbiol indones meta-cleavage pathway. the degradation process produces the final product, namely acetaldehyde, pyruvate and carbon dioxide. the three end products can be used in photosynthesis and microalgae cell respiration. pyruvate can be metabolized in the krebs cycle, and acetaldehyde can be converted to acetyl-coa with the help of acetaldehyde dehydrogenase (acdh) (patel et al. 2017). the temperature range in this study is included in the optimal temperature range for the growth of the o genus scenedesmus which is 25 30 c. the maximum o growth rate of the genus scenedesmus occurs at 30 c. temperature fluctuations in culture are caused by changes in the temperature of the surrounding environment. temperature affects the chemical composition of cells, nutrient uptake and co , and 2 microalgae growth rate. salinity measurement results in studies are higher than the optimum salinity range for scenedesmus growth salinity affects the ability of scenedesmus cells to accumulate oil. in high salinity, the genus scenedesmus is able to accumulate oil up to 36% dry weight (kaewkannetra et al. 2012). according to latala's (1991) study, the genus scenedesmus can live up to 25 ‰ salinity. in this study, s. vacuolatus was grown in medium with sea water with salinity up to 30‰. this shows that s. vacuolatus is an adaptive microalgae in an environment with salinity stress. fluctuations in dissolved oxygen in the test culture are influenced by the density of scenedesmus vacuolatus cells. it is also related to the oil layer in the medium and petroleum hydrocarbons that are accumulated by microalgae (papa et al. 2009). the optimal ph range for growth of s. vacuolatus is 6.5 8.5. at ph more than 8.5 the growth rate will slow down (neuwoener & escher 2011). at ph 4.8 the growth rate of the genus scenedesmus will stop (nalewajko et al. 1997). light intensity in this study is included in the optimum range for scenedesmus growth. the genus scenedesmus is able to grow at the lowest light intensity of 2,500 lux (latiffi et al. 2017). scenedesmus vacuolatus species are able to grow optimally to a light intensity of 49,200 lux (carbone et al. 2017). authors’ contributions the main contributors were cas and ha, while the others were the supporting contributors. acknowledgements we acknowledge all the members of department of biology universitas indonesia, and center of environmental technology (ptl), agency for the assessment and application of technology (bppt) for their cooperation and assistance. this work was supported by a grant from insentif riset sistem inovasi nasional (insinas) 2020, ministry of research, technology, and higher education on behalf of ha. references al obaidy ahmj, lami mh. 2014. the toxic effects of crude oil in some freshwater cyanobacteria. journal of environmental protection. 5(5): 359-367. amirlatifi f, soltani n, saadatmand s, shokravi s, dezfulian m. 2013. crude oil-induced morphological and 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pencemaran tumpahan minyak di perairan cilacap. jurnal teknologi lingkungan. 19(2): 191-202. 116 santoso et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 03. masri.cdr vol.15, no.4, december 2021, p 135-138 doi: 10.5454/mi.15.4.3 short communication anti fungal activity of chitinolytic bacteria lysinibacillus fusiformis and brevibacillus reuszeri against the fungal pathogens rhizoctonia solani and fusarium oxysporum * mashuri masri , eka sukmawaty, and as awaliah amir department of biology, faculty of science and technology, universitas islam negeri alauddin, jalan hm yasin limpo no. 36 samata kab. gowa 90221, sulawesi selatan, indonesia. chitinolytic bacteria can produce chitinase, reported as a biocontrol agent against plants. this research aims to observe chitinolytic activity in inhibiting the growth of rhizoctonia solani and fusarium oxysporum. anti fungal testing in dual culture test by growing each of the chitinolytic bacteria, lysinibacillus fusiformis and brevibacillus reuszeri, with the pathogenic fungi, f. oxysporum and r. solani, in petri dishes containing chitin agar media facing a distance of 3 cm. the results showed that chitinolytic bacterial isolates were capable inhibit the fungus by having the activity of each index inhibition of l. fusiformis isolates (30%), b. reuszeri (77%) against f. oxysporum, and r. solani fungi isolates (100%) for each chitinolytic bacterial isolate. key words: anti fungal, chitinolytic bacteria, pathogenic fungi bakteri kitinolitik dapat menghasilkan enzim kitinase dan dilaporkan sebagai agen biokontrol terhadap tanaman. penelitian ini bertujuan untuk melihat aktivitas kitinolitik dalam menghambat pertumbuhan rhizoctonia solani dan fusarium oxysporum. pengujian anti-cendawan menggunakan teknik kultur ganda dengan menumbuhkan masing-masing bakteri kitinolitik, lysinibacillus fusiformis dan brevibacillus reuszeri, dengan jamur patogen, f. oxysporum dan r. solani, dalam cawan petri yang berisi media agar mengandung kitin secara berhadapan dengan jarak 3 cm. hasil penelitian menunjukkan bahwa isolat bakteri kitinolitik mampu menghambat cendawan dengan indeks penghambatan l. fusiformis (30%), b. reuszeri (77%) terhadap cendawan f. oxysporum dan r. solani (100%) untuk setiap isolat bakteri kitinolitik. kata kunci: anti cendawan, bakteri kitinolitik, cendawan patogen microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: : +62-411-841879; fax : +62411-8221400; email : mashuri.masri@uin-alauddin.ac.id two types of pathogens that often attack horticultural crops (liu et al. 2017; zhao et al. 2014). the main composition of fungal cell walls is chitin so that biological control strategies can be carried out by utilizing chitinolytic enzyme-producing bacteria. chitinolytic bacteria are a group of bacteria that are capable of producing the chitinase enzyme (asif et al. 2020; moon et al. 2017; wang d et al. 2018). the enzyme functions to catalyze the chitin degradation reaction by cutting the glycosidic bonds between n residues. acetylglucosamine. chitinolytic bacteria have strong antagonistic activity against fungal pathogens with hyperparasitism and antibiotic mechanisms (pliego et al. 2011).the use of chitinolic bacteria as biological control agents is expected to degrade the cell walls of pathogenic fungi so that it can inhibit the growth of pathogenic fungi in horticultural crops. two chitinolic bacteria, namely lysinibacillus fusiformis and brevibacillus reuszeri, were tested for their antifungal abilities against f. oxysporum and r. solani. horticultural crops have important potential in fulfilling nutrition and increasing farmers' income so as to support community welfare (malhotra 2016; 'kerutagi et al. 2019). however, in cultivation, there are obstacles in the form of plant diseases caused by fungal pathogens that can pose a risk of plant damage and decreased production (van bruggen et al. 2016; panth et al. 2020). this can cause economic losses in agriculture and the horticultural industry. therefore, it is necessary to take preventive measures to control these pathogens such as chemical and biological control. chemical control is a control that can be done quickly but has a bad long-term effect on the environment and can cause pathogen resistance. therefore, biological control is the main solution in preventing fungal pathogens that destroy horticultural crops. fusarium oxysporum and rhizoctonia solani are the isolates of the fungus r. solani and f. oxysporum (patimah 2018) were cultured on potato dextrose agar (pda) media and incubated for 7 days at room temperature. chitinolytic bacteria isolates, namely l. fusiformis and b. reuszeri (aditia 2016) were cultured on nutrient agar (na) media and incubated for 1x24 hours at 37 °c. the antifungal test of chitinolytic bacterial isolates was carried out using a multiple culture test. previously isolated chitinolytic bacteria, r. solani and f. oxysporum, were scratched at a distance of 2 cm from the edge of the media. after the bacteria were 24 hours old, the bacteria were tested in the dual culture test against fungi using chitin agar media. chitin agar is used to break down chitin compounds in bacteria. pathogenic fungal mycelium with a diameter of 0.5 cm was placed in a petri dish with a distance of 2 cm from the edge of the media using a needle loop. then the chitinolytic bacteria were scratched lengthwise at a distance of 3 cm from the mycelium of the pathogenic fungi, the cultures were incubated at room temperature for 7 days (anesini and perez 1993; yurnaliza et al. 2012). this test was repeated 1 time. the inhibition index used in this study was carried out by observing the resulting inhibition zone on the 7 days calculated using the formula: r1: growth radius towards the edge of the petri r2: growth radius towards antagonistic bacteria based on the results of the anti-fungal activity test, it was found that l. fusiformis produced an inhibitory index of 100% against r. solani and 30% against f. oxysporum. meanwhile, b. reuszeri produced an inhibitory index of 100% against r. solani and 77% against f. oxysporum (table 1). the inhibition index of 100% meant that there was no growth of pathogenic fungi when the antagonist was tested with chitinolytic bacteria l. fusiformis (fig 1a) and b. reuszeri (fig 1c). inhibition index <100% means that there is still growth of pathogenic fungi. this growth was indicated by the presence of hyphae formed on the surface of the growing medium when antagonist testing with chitinolytic bacteria was carried out (fig 1b and 1d). the presence of an inhibitory index showed that chitinolytic bacteria l. fusiformis and b. reuszeri were able to inhibit the growth of pathogenic fungi. this is because chitinolytic bacteria produce chitinase enzymes that are able to degrade fungal cell walls. the main component of fungal cell walls is chitin. chitin is a linear polymer composed of monomers, namely β1,4n-acetyl-glucosamine (cabib et al. 2001; roncero 2002; moon et al. 2017). in the presence of chitinase enzymes produced by chitinolytic bacteria, hydrolysis of chitin compounds occurs at -1,4-glycosidic bonds and produces oligosaccharides or n-acetylglucosamine monomers (tronsmo and harman 1993; herdyastuti et al. 2010). the magnitude of the inhibitory index produced depends on how much n-acetyl-glucosamine table 1 activity data of chitinolytic bacteria against pathogenic fungi 136 masri et al. microbiol indones chitinolytic bacteria pathogenic fungi inhibition radius inhibition index r1 r2 lysinibacillus fusiformis rhisoctonia solani 0 0 100% lysinibacillus fusiformis fusarium oxyforum 3.6 2.2 30% brevibacullus reuszeri rhisoctonia solani 0 0 100% brevibacullus reuszeri fusarium oxyforum 3.6 0.8 77% fig 1 anti-fungal activity of chitinolytic bacteria. a. lysinibacillus fusiformis against rhisoctonia solani, b. lysinibacillus fusiformis against fusarium oxysporum, c. brevibacullus reuszeri against rhisoctonia solani, d. brevibacullus reuszeri against fusarium oxysporum. monomer is produced from the chitin hydrolysis process using the chitinase enzyme. the greater the number of n monomers produced, -acetyl-glucosamine the greater the inhibitory index that will be formed. testing of the anti-fungal ability of chitinolytic bacteria was carried out simultaneously, but there were differences in the inhibitory index of each isolate of chitinolytic bacteria. this is due to the different species of chitinolytic bacteria and fungal pathogenic species used. each chitinolytic bacteria certainly produces different chitinase enzymes so that it affects the antifungal activity which is characterized by the presence of an inhibition zone. in addition, each pathogenic fungus certainly has a specific defense mechanism so that it affects self-defense against foreign compounds that interfere with its existence. based on table 1, it can be seen that the larger the inhibition zone produced, the chitinase produced by chitinolytic bacteria is able to hydrolyze large amounts of chitin so that the growth of pathogenic fungi is inhibited. the fungus f. oxysporum is more resistant to chitinase because the cell wall composition of the fungus f. oxysporum in the outer layer contains glycoprotein compounds that protect the surface of the mycelium. the glycoprotein content in the cell wall is 50-60% of the total cell wall mass (schoffelmeer et al. 1999; yurnaliza et al. 2012). chitinase is widely used as a biocontrol agent, especially for plants that are often infected with fungi, it is because chitin, which is the main component of fungal cell walls, can be degraded by the chitinase enzyme to produce an environmentally friendly product compared to chemicals (asif et al. 2020). the chitinase enzyme's role is widely used as an effective antifungal against r. solani on transgenic cotton (nicotiana tabacum l) (broglie et al. 1991), transgenic elite indica rice (datta et al. 2001), or f. oxysporum on strawberries (wang y et al. 2003), f. oxysporum f. sp. udum on pigeon pea (bapat and shah 2000), bacillus thuringiensis can inhibit the growth of several fungal pathogens and fungal cell wall degradation (hollensteiner et al. 2017). many species of bacilli are well known as plant growth-promoting bacteria (pgpb), biocontrol of pests and diseases. it was reported that species of b. brevis are effective antagonists for pathogenic fungi such as f. oxysporum f. sp. udum (bapat and shah 2000), bacillus licheniformis for aspergillus niger, magnaporthe oryzae and r. solani (cui et al. 2012), virgibacillus marismortui, bacillus subtilis, bacillus pumilus, bacillus licheniformis, terribacillus halophilus, halomonas elongata, planococcus r i f i e t o e n s i s , s t a p h y l o c o c c u s e q u o r u m a n d staphylococcus sp. for botrytis cinerea (essghaier et 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doi:10.31258/jnat.14.1.42-46. zhao x, han y, tan x qian, wang j, zhou z jiang. 2014. optimization of antifungal lipopeptide production from bacillus sp. bh072 by response surface methodology. j microbiol. 52(4):324–332. doi:10.1007/s12275-0143354-3. 138 masri et al. microbiol indones page 1 page 2 page 3 page 4 02. haniyya.cdr vol.14, no.1, march 2020, p 7-16 doi: 10.5454/mi.14.1.2 the utilization of auto-inducible plyb promoter and media optimization for cell density-dependent expression of recombinant thermoalkalophilic xylanase in bacillus subtilis db104 * haniyya, dini achnafani, maria ulfah, niknik nurhayati, and is helianti center for bioindustrial technology-agency of assessment and application of technology (bppt), building 611/614, laptiab-bppt, puspiptek area, setu, south tangerang, 15314, indonesia. strong promoters are one of the fundamental aspects to increase the level of gene expression, and one of approach to improve the recombinant enzyme productivity so that the efficiency of production cost for enzyme production in industrial scale can be reached. here we assessed the application of a cell density-dependent promoter and media optimation to promote cell growth and protein expression of bacillus subtilis without excess usage of inducers. an auto-inducible pylb promoter that is potential to provide inducer-free enzyme production was cloned and introduced into xylanase recombinant system in b. subtilis db104 by pcr cloning and protoplast transformation. a 200 bp target gene was successfully inserted in between xyncm1 orf -coding for b. halodurans cm1 xylanaseand its native promoter sequence at the upstream region. the disruption of the native promoter was intended to replace the native promoter with pylb. recombinant xylanase gene under pylb was successfully expressed in b. subtilis db104 and the enzyme was produced at stationary phase. different media with various concentrations of glucose and nitrogen were used to optimize recombinant xylanase expression. it achieved a higher level of xylanase expression compared to wild-type bacillus and recombinant xylanase with native promoter expressed in b. subtilis in media containing a 2-fold recipe of lb media thus leads to increase cell -1 density and xylanase expression (81.461 u ml ). key words: auto-inducible, bacillus subtilis db104, pylb, xylanase promotor yang kuat adalah salah satu aspek mendasar untuk meningkatkan tingkat ekspresi gen, dan salah satu pendekatan untuk meningkatkan produktivitas enzim rekombinan sehingga efisiensi biaya produksi enzim pada skala industri dapat tercapai. di dalam studi ini aplikasi promotor yang bergantung pada kepadatan sel tanpa penggunaan induser secara berlebihan dari induser dan optimasi media untuk mendorong pertumbuhan sel dan ekspresi enzim xilanase di bacillus subtilis telah dilakukan. promotor pylb yang auto inducible berpotensi untuk menyediakan produksi enzim bebas-induktor telah dikloning dan dimasukkan ke dalam sistem rekombinan xilanase di b. subtilis db104 via kloning pcr dan transformasi protoplas. gen target sepanjang 200 bp berhasil disisipkan di antara xyncm1 orf –yang mengkodekan b. halodurans cm1 xylanasedan promotor asli. gangguan promotor asli dimaksudkan untuk menggantikan promotor asli dengan promoter pylb. gen xilanase rekombinan di bawah pylb berhasil diekspresikan dalam b. subtilis db104 dan enzim diproduksi pada fase stasioner. media yang berbeda dengan berbagai konsentrasi glukosa dan nitrogen digunakan untuk mengoptimalkan ekspresi xilanase rekombinan. tingkat ekspresi xilanase yang lebih tinggi daripada b. subtilis non rekombinan ataupun b. subtilis rekombinan yang mengandung gen xilanase alkalotermofilik dengan promoter asli ditemukan di dalam media yang mengandung resep lb 2 kali lipat dari media lb sehingga -1 mengarah pada peningkatan kepadatan sel dan ekspresi xilanase (81,461 u ml ). kata kunci: auto-inducible, bacillus subtilis db104, pylb, xilanase microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-7560694; fax: +6221-7566922; email: is.helianti@bppt.go.id 2019). it is suggested that choosing the promoters that are suitable for our target genes should be done wisely to obtain the desired level of expression (schumann 2007; wenzel et al. 2011; einav and philipp 2019). there are many types of promoters used in heterologous protein expression system of recombinant host from bacillus genus, that is constitutive promoters, inducer-specific promoters, and auto-inducible promoters (schumann 2007; yu et al. 2015), among which induced-promoters are the most commonly used to date. however, these inducible promoters are not cost-efficient in the context of in gene expression, promoters are known as one of the key factors to have an important role in defining where the gene transcription starts. it is in the upstream on an open reading frame near the transcription site starts and comprises a set of sequence that has recognizable patterns. the strength of the promoters would likely affect rna polymerase binding and therefore can contribute to the enhancement of target gene expression (mekler et al. 2012; einav and philipp industrial application because of the requirement of certain chemicals and compounds to be added in the process. for example pxyl, one of the most used in industry, needs xylose as its inducers (yu et al. 2015; meyers et al. 2019). native inducible-promoter of genes encoding enzymes also needs its substrate to produce high-level protein production, such as xylan for xylanase (gupta et al. 2008; ulfah et al. 2011; helianti et al. 2018) and skim milk for protease (ulfah et al. 2011; cu et al. 2015). since the usage of constitutive promoter can generate toxic protein that can be lethal for the host, this leads us to consider the advantage of auto-inducible promoters which can ideally facilitate efficient production process at low cost with an optimum condition (yu et al. 2015; meyers et al. 2019; trung et al. 2019). several highly-efficient non-inducible promoters in bacillus subtilis had been identified by yu et al. (2015), among them was pylb which strongly promoted β-galactosidase expression and showed significant superiority from the rest of selected promoters. this promoter was not only actively expressing reporter gene bgab during stationary phase but also boosting a higher activity of β-galactosidase up to 5000 times. hence, this pylb promoter is potential to be used for overexpressing other recombinant gene product with no inducer needed (yu et al. 2015) especially one which utilizes b. subtilis as cell factory. b. subtilis db104 is a gram-positive bacterium that is generally recognized as a safe (gras) nontoxic organism (westers et al. 2004; watzlawick and altenbuchner 2011). along with members of bacillus genera, its ability to secrete protein into extracellular sphere and grow in mesophilic condition at 37 ˚c in minimum media with various carbon and nitrogen source (wenzel et al. 2011; mageshwaran et al. 2014] has attracted scientists to use them as recombinant host in large-scale production as they cover more than onethird of industrial enzyme (meissner et al. 2015; watzlawick and altenbuchner 2019]. on top of that, b. subtilis db104 has alkaline protease gene deleted (δapra3), allowing them to secrete less protease and therefore the target protein will have lower denaturation risk (kawamura and doi 1984) and it also might be more suitable to express genes from the same gram-positive bacteria than e. coli. our recent work has been focused on utilize native promoter for expression of xyncm1 gene in b. subtilis db104. whilst it could enhance the enzyme production in the host compared to the native b. subtilis db104 (haniyya et al. 2019), the productivity of recombinant clones was still almost 4 times lower than the wild-type b. halodurans cm1. the expenses of using xylan or raw corncob as inducers also rise concerns both in terms of cost-efficiency and convenience of upstream and downstream processes. the utilizing an auto-inducible promoter from b. subtilis is very much necessary to upgrade the recombinant protein production system in b. subtilis db104. hence, the current study aimed to clone and utilize pylb promoter for cell density-dependent expression of thermoalkalophic xylanase originally from b. halodurans and the strategy with optimized media. media optimization was also a highlight in our study since it was very critical for cell growth and therefore played as an important factor in determining pylb promoter efficiency. materials and methods strains, cultivation, and dna extraction. eschericia coli dh5α was used only as cloning host and b. subtilis db104 was engineered for subcloning and recombinant protein production. the wild-type strains were grown in default condition at 37 ˚c, 150 rpm for overnight in lb media (0.5% yeast extract, 1% nacl, and 1% peptone). the recombinant strains were also cultivated in stated condition using lb media with -1 respective antibiotics (100 µg ml ampicillin and 5 µg -1 ml erythromycin) unless stated otherwise. whole genomic dna of b. subtilis db104 was extracted using extraction kit from thermo fisher scientific. according to its protocol for gram-positive bacteria, an overnight culture was collected and treated with 180 µl gram-positive bacteria lysis buffer which consists of 20 mm tris-hcl ph 8.0, 2 mm edta, and -1 20 mg ml of lysozyme. after incubation at 37 °c for 30 min, 200 µl of lysis solution and 20 µl of proteinase k were added to the suspension to perform cell lysis and protein removal. the sample was incubated at 56 °c while vortexing to ensure a uniform suspension until the cells were completely lysed (about 30 min). to obtain purified genomic dna, 20 µl rnase was added to the suspension and followed with the addition of 400 µl 50% ethanol. the prepared lysate was then transferred to the filtered column and washed by wash buffer i and wash buffer ii of each contained pure ethanol. before dna elution was performed, the column was spun at maximum speed (12,000 ×g) for 3 min to dry. genomic dna was obtained after a 5-min incubation step with 200 µl elution buffer. all centrifugation was done at 8 haniyya et al. microbiol indones volume 14, 2020 microbiol indones 9 6,000—8,000 ×g except for the drying process. isolation of pylb promoter. the pylb promoter was amplified from gdna of b. subtilis db104 by a set of primers based on work of yu et al. (2015) but modified for restriction-free cloning by the addition of backbone sequences in 5' or 3' overhangs in both primer f1 and r1 (table 1). genomic dna was isolated using genejet genomic dna purification kit [#k072, thermo fisher scientific, waltham, usa]. the pylb gene was amplified by pcr in a 50 µl reaction mixture composed of 10 µl 5× hf buffer, 1 µl 10 mm dntps, 2.5 µl of 10 µm forward and reverse primers, 1.5 µl 100% dmso, 5 µl template gdna, 0.625 µl phusion hifi dna polymerase [thermo fisher scientific, waltham, usa], and 26.875 µl nuclease-free water to bring the total volume of 50 µl. after a swift initial denaturation of 98 ˚c for 30 s, amplification was performed in 30 cycles of 10 s at 98 ˚c, 15 s at 72 ˚c, and 30 s at 72 ˚c, followed by a final extension at 72 ˚c for 5 min to ensure the fragment elongation. the 256 bp fragment of pylb gene was then purified using geneaid gel/pcr dna fragments kit [df100, geneaid biotech, new taipei city, taiwan] by mixing it with 250 µl df buffer prior to sample transfer into df column by centrifugation at 14,000 ×g for 30 s. the wash step with the addition and 1-minute incubation of 600 µl wash buffer followed and the purified dna was eluted at last with 50 µl elution buffer. construction of plasmid for xylanase gene expression with mega primer approach. the recombinant plasmid was constructed through restriction-free cloning by pcr insertion of pylb gene into pske194.natprom-alkxyncm1-inlip as template plasmid. the insertion was occurred between native promoter and xyncm1 gene to replace upstream region as gene promoter. there were two megaprimers used in the experiment (fig 1), the purified pylb as the targeted gene and xyncm1 gene to assist the annealing process of targeted gene into the template (ulrich et al. 2012; mathieu et al. 2014). the assisting-gene were amplified by the method described in previous study (helianti et al. 2018). pcr was performed in a 50 µl reaction mixture composed of 10 µl 5× hf buffer, 1 µl 10 mm dntps, 1.5 µl 100% dmso, 25 ng plasmid template, 100 ng purified pylb target gene, 100 ng purified xyncm1 assisting-gene, 0.5 µl phusion hifi dna polymerase [thermo fisher scientific, waltham, usa], and 34 µl nuclease-free water to bring the total volume of 50 µl. a quick initial denaturation was performed at 98 ̊ c for 30 s and followed by 30 cycles gene amplification fig 1 the process of megaprimer-assisted cloning. 10 haniyya et al. microbiol indones table 1 list of primers used in the study primer sequence usage f1 5’gcattttactttgctacgaaaggagaatttgtgaaaagaccaacgga gcct ccg -3’ pylb cloning r1 5’ggttttctaaacagtgtaatcatacaaatctccccctttgttgtttc 3’ pylb cloning f2 5’-atgattacactgtttagaaaaccttttg -3’ xyncm1 isolation r2 5’gtatcgataattctccagtaagcaggtttc -3’ xyncm1 isolation f3 5’-ccaagcttatttcaatgagtattg -3’ recombinant ver ification r3 5’-gttgacttggctgctgtacagaag -3’ recombinant verification 1 consisted of 10 s at 98 ˚c, 30 s at 54 ˚c, and 5.5 min at 72 ˚c. at last, a final extension was wrapped at 72 ˚c for 10 min to ensure the fragment elongation. a recombinant dna was obtained and purified by geneaid gel/pcr dna fragments kit [df100, geneaid biotech, new taipei city, taiwan] with the method described previously prior to recombinant verification. the verification was again performed by pcr using f3 and r3 primers (table 1). the reaction mix in a total volume of 20 µl was simply composed by 4 µl 5× mytaq reaction buffer, 1 µl 10 µm f3 and r3 primers, 1.5 µl purified recombinant dna, 0.25 µl mytaq hs dna polymerase [bioline, london, uk], and 12.25 µl ddh o. a 30-cycles pcr was done under 2 the condition: initial denaturation at 95 ˚c for 1 min, denaturation at 95 ̊ c for 15 s, annealing at 50 ̊ c for 15 s, extension at 72 ˚c for 15 s, and final extension at 72 ˚c for 10 min. after it was verified, in-vitro ligation was proceeded by the incubation of 2 µl 10× t4 dna ligase, 16 µl pcr product, and 1 µl t4 polynucleotide kinase [promega, madison, usa] at 37 ˚c for 30 min. after the first incubation finished, 1 µl t4 dna ligase [thermo fisher, waltham, usa] was added and the incubation was prolonged at room temperature for 30 min. to eliminate non-recombinant -1 parental plasmid, 0.5 u µl dpni was added before the reaction was transformed into e. coli dh5α competent cells. verification of recombinant strains was done using both pcr (which the reaction described previously) and plasmid digestion with mfei. s u b c l o n i n g t o b . s u b t i l i s d b 1 0 4 a n d recombinant verification. plasmid transformation in bacillus was conducted using protoplasting method proposed by chang and cohen (1979). to make bacillus competent cells, the wild-type b. subtilis db104 strain was grown in lb media at 37 ˚c, 150 rpm, for overnight as a starter culture. it was inoculated into 20 ml of pennasay broth [difco, detroit, usa] until the od reached 0.4—0.6. the cell was 600 harvested using centrifugation at 3800 rpm, 4 ̊ c, for 15 min [gyrozen centrifuge, 50 ml rotor]. the protoplasts were formed after 2-h incubation at 37 ˚c in smmp solution (0.5 m sucrose, 0.02 m maleic acid, 0.02 m mgcl2.6h o, and 3.5% pennasay powder) containing 2 -1 freshly-added 2 mg ml lysozyme [sigma-aldrich, st. louis, usa]. it went under several steps of washing before a final 1.5 ml of smmp solution was gently added to resuspend cell pellets for plasmid transformation. the dna plasmid was prepared using a doublestrength smm solution (1 m sucrose, 0.04 m maleic acid, and 0.04 m mgcl .6h o) about 1:1 ratio of dna 2 2 and solution volume in a total of 50 μl. all of dna mixture was added to 0.5 ml protoplast suspension before the addition of 1.5 ml 40% peg6000 in smm solution (0.5 m sucrose, 0.02 m maleic acid, and 0.02 m mgcl .6h o). the suspension was incubated for 2 2 2 min at room temperature. about 5 ml of smmp solution was added prior to cell separation by centrifugation at 2900 rpm, 4 ˚c, for 10 min. the cell pellet was resuspended by 1 ml of smmp solution once again and incubated at 37 ˚c for 1.5 h with 100 rpm agitation. about 500 µl of cell suspension was spread on dm3 agar (0.5 m sodium succinate, 0.5% casamino acid, 0.5% yeast extract, 0.35% k hpo , 2 4 0.15% kh po , 0.5% glucose, 0.02 m mgcl .6h o, 2 4 2 2 0.01% bsa, and 1% agar) containing 5 µg/ml erythromycin and the growth of recombinant colonies could be seen after 2 days incubation at 37 ̊ c. we then selected the positive recombinant colonies by performing colony pcr and plasmid extraction for bacillus as described by voskuil and chambliss (1995). recombinant xylanase activity was checked qualitatively by measuring the ratio of clear zone diameter to bacterial colony diameter (in cm) after 24 h of incubation on lb agar ph 7 and ph 9 in the presence volume 14, 2020 microbiol indones 11 of 2% (w/v) beechwood xylan. non-recombinant b. subtilis db104 was used as negative control. plasmid extraction of recombinant b. subtilis db104. the erythromycin-resistant transformant b. subtilis db104 were selected and cultured in lb media -1 containing erythromycin (5 µg ml ) under 37 °c at 150 rpm agitation. plasmid isolation performed with set buffer (voskuil and chambliss 1993) after centrifugation at 12,000 rpm at 4 °c for 5 min to obtain cell pellet. the cell pellet resuspended in 200 µl in set buffer (25% sucrose, 0.05 m edta, 0.05 m tris-1 hcl ph 8) containing 5 mg ml lysozyme and incubated at 37 °c for 10 min. 0.2 n naoh and 1% sds was added to the suspension and flipped until clear. 5 m of cold kcooh was added and homogenized, then centrifuged. 650 µl of cold phenol:chloroform:isoamyl-alcohol (25:24:1) was added to 750 µl of supernatant and homogenized by vortexing at full speed. aqueous phase from the suspension was obtained by centrifugation. 620 µl of aqueous phase was obtained and 620 µl of chloroform:isoamyl-alcohol (24:1) was added to suspension and homogenized by vortexing at full speed. the suspension was then centrifuged and 550 µl of aqueous phase was moved to new microtube. 550 µl of cold isopropanol was added, homogenized, and centrifuged. 1 ml of alcohol 70% was added to the pellet and centrifuged at 12000 rpm at 4 °c for 5 min. the pellet dna was then resuspended in ddh2o with -1 rnase (20 µg ml ). growth curve observation and media formulation. recombinant b. subtilis db104 harboring pske194-pylb-alkxyncm1-inlip was cultured overnight in 10 ml of lb media in the -1 presence of 5 µg ml erythromycin under 37 °c at 150 rpm agitation. two percent of the culture was then used to inoculate 100 ml of fresh lb media containing the same antibiotic under 37 °c at 150 rpm agitation for 24 h. samples were taken every 2 h to observe bacterial cell density by measuring od then plotted against 600 time to obtain bacterial growth curve. media optimization strategy used to achieve highcell density with modification in yeast extract and glucose concentration. several media used were based on lb and soc media. lb media (1% tryptone, 0.5% yeast extract, 1% nacl) and 2x lb media with 2-fold 1 concentration recipe containing 5 µg ml erythromycin [sigma-aldrich, st. louis, usa] were used. soybean flour media used as an alternative source of nitrogen containing 1.89% (w/v) soybean flour and 1% nacl. for glucose optimization, soc media-based were used containing 1% and 5% (w/v) glucose for each variation. xylanase assay. xylanase activity was assayed using dinitrosalicylic acid (dns) to measure reducing sugar produced from xylan hydrolysis (bailey et al. 1992; miller 1959) in triplicate with some modifications. crude enzyme was obtained from culture supernatant after centrifugation at 12000 rpm at 4 °c for 5 min. 50 µl of supernatant was added to 450 µl xylan substrate containing 0.5% (w/v) beechwood xylan in 0.5 m tris-hcl buffer ph 9. the reaction takes place at 70 °c for 5 min at 300 rpm using a thermoshaker. after incubation, 750 µl of dns reagent (1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulfite, 1% sodium hydroxide, and 20% (w/v) potassium sodium tartrate) was added immediately. as for blank, 50 µl of sample was added after dns addition. all the samples and blanks were boiled for 5 min and let cool to room temperature. 250 µl of water was added, homogenized, and reducing sugar released was measured at 540 nm. standard curve obtained from xylose suspension ranging from 0; 0.2; 0.4; 0.6; 0.8; -1 and 1 mg ml using the same protocol. xylanase activity stated in unit (u) defined as the amount of enzyme releasing 1 µmol of reducing sugar per minute under appropriate assayed condition. results pylb cloning and verification. dna fragment of 256 bp coding for pylb promoter was integrated into pske194-natprom-alkxyncm1-inlip by pcr cloning resulting an 8757 bp recombinant plasmid (fig 2a). the insertion can occur because of the presence of 56 bp of overlapping region in both ends of pylb fragments so that they annealed into the backbone and promoting the formation of new recombinant plasmid in the next cycle of amplification. as depicted in the figure, the gene cassette was constructed of three genes with the position of pylb promoter between the native promoter and xyncm1 gene. the flanking regions of lipase locus at both 5' and 3' ends were intended for further experiments with chromosomal integration and could be used for the verification of positive recombinants (fig 2b). the positive recombinants of e. coli dh5α and b. subtilis db104 were confirmed by pcr using f3 and r3 primer set and proved to be harboring the targeted gene pylb (200 bp) within a 2061 bp gene cassette (fig 3c). this result was also supported by mfei digestion which cut the plasmid into two fragments of 7456 bp 12 haniyya et al. microbiol indones and 1301 bp after 1-hour incubation at 37 ˚c that were notably delineated from the negative one. according to the vector map, mfei digestion of recombinant plasmid generated 2 fragments of 1.3 and 7.5 kb, while control with lack of target gene formed smaller bands of 1.1 and 7.5 kb corresponding to the result (fig 3d). three out of four potential b. subtilis db104 transformants were identified as positive clones, however, only one clone (r3) was carried out for further experiments of protein expression and media formulation. qualitative assay of xylanase. qualitative assay of xylanase produced by recombinant b. subtilis performed in lb xylan media ph 7 and ph 9 to detect alkaline xylanase and compared to b. subtilis db104 wild-type. clear zone ratio 2.00 surrounding recombinant b. subtilis colony in lb media ph 9 (fig 4d) showed alkaline xylanase activity and no distinct clear zone observed in lb media ph 7 (fig 4c). as control, b. subtilis db104 wild-type grown on lb media ph 7 (fig 4a) showed distinct xylanase activity around the colony with clear zone ratio 1.40, while no clear zone detected in lb media ph 9 (fig 4b). growth curve and media optimization to achieve high cell-density. the growth curve of recombinant xylanase-producing b. subtilis was investigated to determine when the enzyme was highlyproduced. growth curve observed in lb media for 24 h with b. subtilis db104 wild-type as control bacteria (fig 5). both wild-type and recombinant b. subtilis entered the log phase after 2 h of cultivation and the recombinant b. subtilis reached the cell density peak after 9 h, while wild-type strain entered the stationary phase after 11 h. b. subtilis wild-type reached higher cell density than recombinant b. subtilis. based on the growth curve, samples were taken at 5 h (log phase), 9 h (late log phase), 14 h (stationary phase), and 24 h (late stationary phase) after inoculation to determine xylanase activity. all samples taken from recombinant and wild-type b. subtilis exhibit xylanase activity measured by dns method. both supernatant from recombinant and wildtype bacteria shows higher level of enzyme expression taken from 24-h supernatant culture (table 2). moreover, recombinant xylanase (r3) performed 2.5-1 fold higher level of enzyme expression (23.874 u ml ) fig 2 the constructed plasmid and gene cassette of pylb. (a) the whole construct of recombinant plasmid pske194-pylb-alkxyncm1-inlip (8757 bp) and its features; (b) a 2061 gene cassette containing 200 bp pylb fragment. (a) (b) pylb lip lip xyncm1 native promoter volume 14, 2020 microbiol indones 13 fig 3 the insertion and verification of pylb gene. (a) pcr amplification of 1) 256 bp pylb gene and its n) negative control from b. subtilis db104; (b) pcr amplification of 1) 1191 bp assisting-fragments xyncm1 from gdna of b. halodurans cm1; (c) colony pcr verification of positive clones harboring pske194-pylbalkxyncm1-inlip with its n) negative control of pske194-natprom-alkxyncm1-inlip (no inserted pylb gene); and (d) mfei digestion of 1) positive recombinant plasmid and its n) negative control after 1-hour incubation at 37 ̊ c. ( a ) ( b ) ( c ) ( d ) fig 4 qualitative assay of xylanase from b. subtilis db104 wild-type grown on lb in the presence of 2% (w/v) of beechwood xylan ph 7 (a); lb-xylan ph 9 (b); recombinant b. subtilis harboring pske194-plybalkxyncm1-inlip grown on lb-xylan ph 7 (c) and lb-xylan ph 9 (d). fig 5 b. subtilis db104 growth curve in lb media at 37 °c. wild-type b. subtilis (▲) showed higher peak cell density than pylb promoter-dependent recombinant xylanase (■). -1 than wild-type xylanase from b. subtilis (9.435 u ml ). it also reached almost 4 times higher than log-phase -1 recombinant xylanase (6.664 u ml ). to determine the effect of both carbon and nitrogen supplementation in media, yeast extract and glucose were used for b. subtilis growth with lb media and soc media as the base media (table 3). not only xylanase activity, cell density was also measured by spectrophotometer at 600 nm (od ). the expression 600 of recombinant xylanase produced by clone r3 harbouring pylb promoter was compared with wildtype b. subtilis and recombinant b. subtilis carrying the same xylanase gene but with the native promoter as controls (table 3). lb media with a 2-fold recipe (2x lb) used as the first step to determine the effect of higher content of yeast extract addition on the b. subtilis growth. cell density at stationary phase was increased 2-fold higher than other bacteria grown on lb media, with od 600 5.025 for recombinant b.subtilis harbouring pylb promoter. recombinant xylanase under pylb promoter control reached the highest result in enzyme activity -1 (81.461 u ml ), almost 4-fold higher than grown on lb media. it is also higher than xylanase produced by -1 wild-type b. subtilis xylanase (20.600 u ml ) and recombinant xyncm1 with native promoter (73.192 u -1 ml ). soybean flour media as an alternative source to supply nitrogen requirement in b. subtilis growth gave no better result in maintaining high activity of recombinant xylanase, even though recombinant xylanase under pylb promoter control remains the highest activity among others. s o c m e d i a w e r e u s e d w i t h d i f f e r e n t concentrations of glucose (1% and 5%) and compared to original soc media (20 mm of glucose). biomass yield from high glucose content in soc media measured by cell density (od ) revealed a decreasing 600 cell density compared to original soc media. meanwhile, wild-type and recombinant b. subtilis grown in sob media with soc media as former media starter exhibited higher cell density compared to excess glucose media, even though it did not surpass the cell density from original soc media. recombinant xylanase under pylb promoter control produced in soc media with original concentration of glucose (20 mm) showed highest xylanase activity also among other variety of soc media. discussion the pylb gene (256 bp) from b. subtilis was successfully inserted into the plasmid by the assistance of megaprimer of the same 1191 bp xylanase gene that was already incorporated in it. the cloning method this study used was slightly different from the original rf cloning in which only one megaprimer (as targeted gene) needed (mathieu et al. 2014). in our experiment we used two megaprimers, one was our pylb gene which possessed flanking regions of homology with the assisting-gene so that in the first cycle of pcr both 14 haniyya et al. microbiol indones table 2 xylanase activity measured by dns method from supernatant culture of wild-type and recombinant b. subtilis db104 time (-hour) xylanase activity (u ml -1) r3 wild-type 5 6.664 4.911 9 10.499 6.328 14 18.080 8.769 24 23.874 9.435 1 1 table 3 cell density (od ) and xylanase activity (u ml ) of recombinant b. subtilis from different media 6 0 0 compared to controls media wild-type xyncm1 with native promoter xyncm1 with pylb promoter od600 activity (u ml-1) od600 activity (u ml-1) od600 activity (u ml-1) lb 1.905 2.871 2.205 26.944 2.710 21.598 2x lb 5.050 20.600 5.680 73.192 5.025 81.461 soybean flour media 3.978 13.995 31.299 soc 7.040 29.436 6.250 22.504 5.720 60.893 soc (with 1% glucose) 3.645 15.207 2.335 10.949 2.930 19.304 soc (with 5% glucose) 3.995 61.551 3.980 28.937 4.365 47.937 sob (with starter soc) 5.965 3.294 4.525 24.508 2.200 24.766 1 volume 14, 2020 microbiol indones 15 could anneal as one joint-fragment (total size of 1447 bp). this leads to relatively easier insertion of jointfragment into the plasmid even in low annealing temperature because the assisting-gene has extended homologous region with the vector at 5' end. no additional pair of primers was used in the reaction for cloning, resulting linear amplification not exponential (ulrich et al. 2012; mathieu et al. 2014). pylb promoter is an auto-inducible promoter which promotes gene expression from late log phase to stationary phase with no requirements of inducer (yu et al. 2015). insertion of this promoter is necessary to upgrade recombinant xylanase production from recombinant b. subtilis db104. this bacteria itself (wild-type) performed xylanase activity from encoded gene in its chromosome (helianti et al. 2016) and observed in ph 7, while recombinant xylanase from b. subtilis harbouring pske194-pylb-alkxyncm1-inlip performed alkaline xylanase as the gene originated from alkalothermophilic b. halodurans cm1 (wibowo et al. 2016). this was confirmed by qualitative assay of recombinant xylanase which shown xylanase activity from recombinant bacteria in xylan plate media ph 9. in addition, recombinant xylanase were produced in increasing activity corresponding to bacterial growth curve and highly produced in stationary phase (24 h after inoculation). as stated above, gene expression under pylb promoter highly expressed along with increasing cell density (yu et al. 2015). in order to obtain a higher cell density, media composition needs to be optimized. b. subtilis biomass obtained at the end of the logarithmic phase increased following the increasing quantities of yeast extract added to the media. specific growth rate of b. subtilis supplemented with 0.8% (w/v) of yeast extract increased up to 6-fold and no further significant change above that quantity (romero-garcia et al. 2009). in this research, 2x lb media contained 1% (w/v) of yeast extract and only produced a 2-fold increase in cell density. the presence of yeast extract was confirmed to affect b. subtilis growth and increase xylanase activity if a certain amount of yeast extract were added. furthermore, recombinant xylanase under pylb promoter control constantly expressed at a higher level compared to other xylanases even if the media were switched to an alternative source of nitrogen. glucose supplementation on batch fermentation of b. subtilis obtained higher cell dry weight, with optimum glucose concentration identified as 3.07% (w/v) (zhong et al. 2014). according to the results in this experiment, excess carbon source in modified media did not enhance biomass yield and xylanase expressed from recombinant b. subtilis was not higher than in original soc media with 20 mm glucose. it turned out that excess energy source in media (usually carbon source) exhibited a high rate of carbon consumption and low energetic growth efficiency. high carbon source in the growth of microorganisms generates uncoupling of anabolism and catabolism thus lead to variety of energy spilling reaction and metabolic shifting in electron pathway to less efficient called overflow metabolism (dauner et al. 2001). in contrast, energy limitation generates catabolism coupling to anabolism so higher biomass is achieved (russel and cook. 1995). from this research, maintaining glucose concentration at certain amount while increasing nitrogen content might be able to express a higher level of recombinant xylanase with pylb promoter control in b. subtilis db104. references bailey mj, biely p, poutanen k. 1992. interlaboratory testing of methods for assay of xylanase activity. j biotechnol. 23(3):257-270. doi: 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expression system for reliable and inexpensive protein production by highcell-density fermentation. appl environ microbiol. 77(18):6419-6425. doi: 10.1128/aem.05219-11. westers l, westers h, quax wj. 2004. bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. biochim b i o p h y s a c t a . 1 6 9 4 ( 1 3 ) : 2 9 9 3 1 0 . d o i : 10.1016/j.bbamcr.2004.02.011. wibowo s, helianti i, suryani a, wahyuntari b. 2016. application of response surface method in optimization of medium composition for xylanase production by bacillus halodurans cm1 in submerged fermentation. microbiol indones. 10(3):5. doi: 10.5454/mi.10.3.5. yu x, xu j, liu x, chu x, wang p, tian j, wu n, fan y. 2015. identification of a highly efficient stationary phase promoter in bacillus subtilis. sci rep. 5:18405. doi: 10.1038/srep18405. zhong j, zhang x, ren y, yang j, tan h, zhou j. 2014. optimization of bacillus subtilis cell growth effecting jiean-peptide production in fed batch fermentation using central composite design. electron j biotechnol. 17(3):132-136. doi: 10.1016/j.ejbt.2014.04.010. 16 haniyya et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 01. rahayu.cdr vol.15, no.3, september 2021, p 77-83 doi: 10.5454/mi.15.3.1 domestication and formulation of as a tempeh starterrhizopodopsis javensis gayuh rahayu *, efriwati , septina veronica 1 2 1 and 1 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia; 2 the biomedical and basic health technology research and development centre national institute of health research and development ministry of health republic of indonesia, jakarta 10560, indonesia. domestication of wild fungal strains involved in the manufacture of traditional fermented foods often occurs spontaneously. ) which taxonomically close to is a fungus type rhizopodopsis javensis (rh. javensis rhizopus naturally found in cool climates, and promisingly can be used as a tempeh starter in temperate regions. however before using it as starter, a wild strain needs to be domesticated in human-made niches. at present rh. javensis study, this species was domesticated by subculture using rice flour media at optimum growth temperature for every five days. some parameters such as spore's density, viability, and starter water content were used to determine the successfully of domestication. the result showed that grew optimally at 22 ℃. rh. javensis moreover, it has been reproducible for seven times which indicated by no changes in growing rate and colony appearance. however, compared to the commercial tempeh starter and var. wild type, r. microsporus oligosporus it has lower spore's viability with higher water content. therefore, the carrier material and other parameters such as drying processes and starter's lifespan need to be modified to increase the spore viability and starter quality. key words: food fermentation, var , spore's viability, starter quality, wild rhizopus microsporus . oligosporus strain domestikasi galur liar kapang yang terlibat dalam dalam pembuatan makanan fermentasi tradisional, sering terjadi secara spontan. ( ) merupakan salah satu galur liar kapang yang rhizopodopsis javensis rh. javensis memiliki hubungan taksonomi dekat dengan . strain liar ini ditemukan di daerah beriklim sejuk, rhizopus sehingga berpotensi untuk dikembangkan sebagai starter tempe untuk produksi di daerah beriklim sedang. untuk mendapatkan kultur yang tumbuh subur di relung ( ) buatan manusia, strain liar perlu niches rh. javensis didomestikasi terlebih dahulu. penelitian ini bertujuan untuk mendomestikasi strain liar yang rh. javensis dilanjutkan dengan memformulasikannya sebagai starter tempe. domestikasi dilakukan dengan menumbuhkan strain liar pada media tepung beras pada suhu pertumbuhan optimum dan diulangi setiap lima hari. rh. javensis kerapatan dan viabilitas spora, serta kadar air starter digunakan sebagai penilaian keberhasilan starter. hasil penelitian menemukan tumbuh optimal pada suhu 22 ℃. domestikasi dengan cara subkultur koloni rh. javensis rh. javensis rh. javensis pada media tepung beras selama 7 kali tidak mengubah kecepatan pertumbuhan dan penampakan koloni laju pertumbuhan relatif sama dengan laju pertumbuhan starter tempe . rh. javensis komersial dan var. s murni, pada suhu optimum pertumbuhan masing-masing. r. microsporus oligosporu formulasi tepung beras sebagai media pembawa starter menghasilkan kerapatan spora yang relatif rh. javensis, sama dengan starter tempe komersial, namun viabilitas sporanya rendah dan kadar airnya tinggi. starter rh. javensis belum dapat digunakan untuk membuat tempe. substrat dan proses pengeringan masih perlu dimodifikasi untuk meningkatkan viabilitas spora dan kualitas starter tempe secara keseluruhan, termasuk umur simpan starter. kata kunci: fermentasi makanan, kualitas starter, strain liar, rhizopus microsporus var. oligosporus, viabilitas spora microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 62-816-1133-725 ; : + fax: +62; gayuhrahayu@gmail.com e-mail: stolonifer were often found in indonesian traditionally tempeh-making processes (tamam 2019, putri et al. et al. r. microsporus oligosporus2021). furthermore, var. and were often used as commercial tempeh r. oryzae starters (ahnan-winarno . 2021). with the et al increasing preference of the indonesian people to use a specific commercial starter, the diversity of rhizopus species in tempeh decreases (hartanti 2015). et al. rhizopus delemar, r. which previously reported as oryzae, was no longer found in all fresh tempeh from java island (hartanti 2015). in contrast, barus et al. et tempeh is made from soybean fermented by the microbial community. although sp. is the rhizopus only microbe that is deliberately given as the tempeh starter, lactic acid bacteria (lab) and other types of bacteria (seumahu 2013, efriwati 2013), and et al. et al. yeast (efriwati 2013) were found during tempeh et al. making process. , rhizopus microsporus r. rhizopodiformis r. chinensis r. arrhizus r. , , , and al. r. delemar (2019) stated that two strains of obtained from waru ( leaves produced a better hibiscus tiliaceus) tempeh taste and more preferred than usual tempeh. therefore, it is necessary to preserve various rhizopus species from tempeh, including exploring new rhizopus s specie or their close relatives for obtainig new potential strains for tempeh production. domestication of microbial agents for food fermentation is defined as human selection to obtain cultivated strains that grow well in man-made environments, but may behave sub optimally in nature (gallone 2016)et al. . tempeh starters should include only food-grade microbes. var rhizopus microsporus . oligosporus et has not been found in nature (dolatabali al. 2013) and did not produce harmful metabolite (ahnan-winarno 2021). dolatabali (2013) et al. et al. suggseted that var might r. microsporus . oligosporus become from the domestication of . r. microsporus domestication increases functionality r. microsporus for humans. it also ensures food quality, homogeneity, and food safety (gibbons and rinker 2015). this domestication process occurs due to cultivating the mold in an isolated environment from its ancestor population to increase specific function for a human being (gallone 2020). in traditional fermented et al. food, domestication might be due to back slopping (wijaya 2015, gallone 2020). et al. et al. rhizopus oligosporus are cultivated during tempeh production, and this mold grow optimum at 30-37 ℃ (tahir 2018). tempeh production in other et al. countries with the same climate as indonesia (tropical) was easy to be done, but not in countries with a subtropical climate. within their study, ahnan-winarno et al. (2021) described tempeh production in the subtropical region was more challenging. hence, it is necessary to search for alternative molds for subtropical regions. rhizopodopsis javensis is a mucorales that is expected to be able to grow at low temperature (around 20 ℃) as that mold has been found in mountain areas (cibodas, cianjur, west java). thus, can rh. javensis probably be developed as a starter to produce tempeh in the subtropical region. however, domestication of rh. javensis as artificial selection and wild species culturing must be done prior to making it a tempeh starter. domestication is needed to obtain variants that thrive in human-made niches to meet human or industrial needs (steensels et al. 2019). according to pramudito et al. (2021), rice is usually made as a carrier material in tempeh starter. this research aimed to domesticate the wild strain of and followed by the rh. javensis formulation of domesticated strain as a tempeh starter. the domesticated strain is expected to meet the criteria for tempeh starter, which was determined based on spore density and viability, as well as starter physical characteristics such as water content. materials and methods microbial strains and media. rhizopodopsis javensis rhizopus microsporus . ipbcc 19.1478, var oligosporus ipbcc 13.1102, and commercial tempeh starter (raprima®) were used in this research. rice flour from rice var. ir 64 setra ramos was used as rh. javensis domestication media. potato dextrose agar (pda) was used as media for mold sub-culturing. working culture of and rh. javensis r. microsporus var. were obtained by rejuvenating stock oligosporus culture on pda media and incubated for 2-7 days in 1923 ℃ for and in 32 ℃ for . rh. javensis r microsporus var. . oligosporus prior to domestication, microbial domestication. rh. javensis optimum growth temperature was determined. this experiment was done by growing two-day-old culture on pda and incubating them at 20, 22, 24, 26, 28, and 30 ℃. the experiment was done in 3 replicates. the colony diameter was measured every 6 hours until the colony entirely covered the media in all the petri dishes. the growth and the sporulation rate determined the optimum growth temperature. the optimum temperature obtained was then used for rh. javensis domestication process on the tempeh starter carrier. rhizopodopsis javensis was domesticated on 50 gr sterile rice flour that was wetted by 15 ml sterilized water. a few pieces of inocula (±5 mm rh. javensis diameter) were cultured on rice flour media and incubated at optimum growth temperature rh. javensis for five days. this sub-culturing was done up to seven times. the colonies that grew from the last subculturing were regrown on pda to evaluate their domestication growth capability. the colony characteristics and the growth rate before and after domestication were then compared. the domesticated strain that showed rh. javensis comparable characters were used as materials to make starters. the inoculum, for starters, was produced in a similar procedure to that of the domestication. after five days of incubation, that culture was made into powder and dried at 30 ℃ for two days. these processes were also done to var.r. microsporus oligosporus rh. javensis and the wild strain of for 78 rahayu et al . microbiol indones volume 15, 2021 microbiol indones 79 comparison. spore density was determined by observing the suspension of diluted starter in a hemocytometer counting chamber. spore viability was determined by using the spread plate method with three replicates. the water content was determined using the oven method, according to sni 01-2891-1992. the density and viability of spore and the starter's rh. javensis water content were compared with the density and viability and water content of var. r. microsporus oligosporus and commercial starters. the kruskal-wallis test analyzes the differences within optimum growth temperature and spore viability, with 0.05 probability. colony growth data of rh. javensis r. microsporus oligosporus, var. , and rhizopus in the commercial starter, raprima®, were analyzed using the brown-forsythe test; meanwhile, data on spore density and starter water content were analyzed one-way anova with a 95% confidence level. the optimum growth temperature rh. javensis determination with distinctly different results was analyzed using the mann-whitney test at α = 0.05. all data were analyzed using spss (statistical package for the social science) software 16.00 version. result the temperature affected growth, as rh. javensis seen from the colony's diameter (fig 1 and 2). the optimum growth temperature for was rh. javensis about 22 ℃. at this temperature, grew at rh. javensis the maximum rate (fig 2b). at 28 ℃, the growth was sustained, while at 30 ℃ there was no growth (fig 1). the growth rate of domesticated was not rh. javensis significantly different from that of the wild type (fig 3). further, growth rate was not significantly rh. javensis different from var when r. microsporus . oligosporus all were grown at their optimum growth temperatures (fig 3). domestication did not change the characteristics of rh. javensis. the spore density in 1 g starter on both the wild and domesticated starters were rh. javensis relatively the same with spore concentration in commercial tempeh starter and varr. microsporus . oligosporus. this research found that all dried starters contained insignificantly different concentration of spore i.e about 10 spore/g (table 1). however, spore 6 viability in all starters was low, only 10 cfu/g or around 4 0.13-2.03 % (table 1). the water content of all starters made in this study was from 8 to 10.58 %. the water content of rh. javensis r. microsporus var. oligosporus and starter was higher but insignificantly different from that of the commercial tempeh starter (table 2). discussion in terms of growth in its optimum temperature, rh. javensis produced comparable characters to the commercial starter. grew rhizopodopsis javensis optimally and sporulated well at 22 ℃, thus classified as either facultative psychrophilic or psychrotolerant fungi. psychrotolerant microbes are mesophilic microbes that are cold-tolerant and thrive in an extensive temperature range, with the fastest growth at temperatures above 20 ℃. the growth rate of domesticated , at its optimum temperature, rh. javensis was not significantly different from its original culture (fig 3). these showed that was able to rh. javensis adapt to rice flour media. however, is rh. javensis starter type spore density number of colonies spore viability log spore/g log cfu/g (%) rh. javensis wild type 6.23±0.39 4.06±0.30 0.67a rh. javensis domesticated type 6.75±0.22 3.88±0.30 0.13a r. microsporus var. oligosporus 6.89±0.15 4.38±0.43 0.31a raprima 6.51±0.20 4.82±0.03 2.03a starter type water content (%) rh. javensis wild type 10.58±1.08a rh. javensis domesticated type 10.08±1.17a r. microsporus var. oligosporus 9.08±1.59a raprima 8.00±0.29a table 1 density and viability of spores of various molds in the starter table 2 water content during fermentation from several type of tempeh starter 80 rahayu et al . microbiol indones fig 1 growth curve at various temperatures during 48 hours incubation. vertical lines on rhizopodopsis javensis every data show the standard error. alphabets at the right show a significant difference of average colony diameter on each temperature according to the mann-whitney test at = 0.05. α fig 2 growth after 48 h incubation on pda media at (a) 20 ℃, (b) 22 ℃ (c) 24 ℃, (d) 26 ℃, rhizopodopsis javensis (e) 28 ℃, and (f) 30 ℃. fig 3 the growth rate of , var. , and in raprima on pda media rh. javensis r. microsporus oligosporus rhizopus during 60 h incubation at each optimum temperature. volume 15, 2021 microbiol indones 81 naturally found on ripe and fruit elaeagnus ficus (boedijn 1958; rahayu , unpublished). et al. further, growth was not significantly rh. javensis different from and r. microsporus var. oligosporus commercial starter (raprima®) at their rhizopus optimum growth temperature. thus, it is suggested that the starter made from could produce rh. javensis tempeh by bringing up the hyphal mass in the same compactness and times as a commercial starter. the commercial tempeh starter requires a temperature of 32 ℃ to be applied, making it challenging to use in subtropical regions (samson 2007). therefore, et al. rh. javensis that grow optimum at 22 ℃ has an opportunity to be developed as a tempeh starter for tempeh production in regions with a temperature of 22 ℃ or regions with a subtropical climate. domestication would depend on the media used. rh. javensis that was domesticated on rice flour media could grow well. colonization can be observed after three days of incubation. wijaya (2015) stated et al. that the starter is usually produced by inoculating starter powder from the previous batch to the new rice flour dough which is called as back-slopping (gallon et al. 2020). cultivation or sub-culturing that was done continuously in the human-related environment causes microbes to adapt and be more beneficial to humans. the adaptation process caused the morphological and physiological change; therefore, domesticated microbes were different from their wild type. domestication through the fermentation food process c o u l d c h a n g e o r g a n i s m s g e n e t i c a l l y a n d phenotypically (steensels 2019). however, et al. domesticated growth and colony color had rh. javensis no difference from its original culture. that rh. javensis had been sub-cultured continuously had a growth rate that was also relatively the same as its wild type culture (fig 2). besides being a medium for growth, rice flour has functioned as a carrier in starter formulation. starter carriers could also come from a mixture of rice flour and cassava flour (ahnan-winarno 2021, et al. pramudito 2021). the addition of spices such as et al. garlic or pepper traditionally used in some fermentation starter was not done in this research. the drying process in this research was not conditioned in a very sterile place. it was just prepared like a household scale fermentation. spices as a natural anti-microbe need to be considered in household-scale starter production as garlic and pepper were known as having anti-microbial and anti-fungal properties (liu et al. 2017). non-domesticated tempeh starter had rh. javensis moderate hyphae growth, mild alcohol aroma, white or white with black spot, and hyphae bound rice granules firmly (maulana 2019). the starter must contain high spores or other propagules concentration. in this research, var. and r. microsporus oligosporus rh. javensis in the starter had relatively the same spore density (table 1). however, starter had rh. javensis low spore viability. thanh and nout (2007) and putri et al. (2021) stated that spores viability may reduce by long storage. furthermore, putri (2021) stated et al. that high viability spores in starter are needed to ensure successful tempeh fermentation. low viability is presumed caused by the drying and flouring process. spore harvesting time, drying, and crushing are factors that cause of spore viability loss (thanh and nout 2002). rhizopodopsis javensis tempeh starter does not meet the quality of commercial starter. it has a lower concentration of viable spore compared to the commercial starter (raprima®). thus, the viable spore concentration does not meet the required amount of viable spore of a commercial starter in tempeh production from 1 kg of soybean. further, the water content is also still high (table 2). in a low water content starter, a fermentation agent's metabolic processes will slow down, thus maintaining cell structure and function during storage (santivarangkna et al. rhizopus 2008). some starters containing inoculum for rice wine production must have a moisture content of 4-5% or 7.8% (dung and phong 2011), while the water content of starter tempeh is about 4-5. % to 7.5% (thanh and nout 2002). the water content of the starter having was still rh. javensis high, indicating that the drying process was not optimal. the drying process at 30 ℃ for 48 hours in this study has not reached the appropriate moisture content. therefore, the drying method still needs to be modified. these characteristics indicate that the rh. javensis starter has low quality and thus cannot be used in the making tempeh trial. in conclusion, grew optimally at 22 rh. javensis ℃. the domestication using rice flour media with seven times subcultures did not change rh. javensis growth rate and its colony characteristics. in the formulation of starter using rice flour as a rh. javensis carrier resulted in its spore density relatively the same as commercial tempeh starter, but with lower spore viability and higher water content. the quality of rh. javensis starter could not be used for tempeh production in sub-tropical climate regions yet. 82 rahayu et al . microbiol indones preliminary study on tempeh production using nondomesticated . starter indicated that rh javensis rh. javensis can be used for tempeh production (maulana 2019). tempeh production using non-domesticated rh. javensis needs a longer incubation period comparing to that of commercial starter. this indicate that a robust and high concentration of viable spores is needed to s ho r te n the incu b a ti on per io d s. th e re f or e , domestication of to carrier substrate has to rh. javensis be studied further for commercialization. substrates used, adaptation method and drying process in starter production need to be modified to increase spore viability and overall starter quality, including starter lifespan. acknowledgements we are highly grateful to the department of biology ipb university and the laboratory chemicals and the biomedical and basic health technology research and development centre for support throughout the research. references ahnan-winarno ad, cordeiro l, winarno fg, gibbons j, xiao h. 2021. tempeh: a semicentennial review on its health benefits, fermentation, safety, processing, sustainability, and affordability. compr rev food sci food saf. 20:1717–1767. doi: 10.1111/15414337.12710. barus t, salim dp, hartanti at. 2019. kualitas tempe menggunakan tb 26 dan rhizopus delemar r.delemar tb 37 yang diisolasi dari inokulum tradisional tempe "daun waru". j apl teknol pangan. 8 (4): 143-148. doi:10.17728/jatp. 2019.4449. boedijn kb. 1958. notes on the mucorales of indonesia. sydowia. 12 (1-6): 321-362. dolatabadi s, walther g, gerrits van de ende ahg, de hoog gs. (2013). diversity and delimitation of rhizopus microsporus. fungal divers. doi: 10.1007/s13225-0130229-6. dung ntp, rombouts fm, nout mjr. 2005. development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine. innov food sc i e merg te c hn ol. 6 (4 ): 4294 41 . doi:10.1016/j.ifset.2005.04.007. dung ntp, phong hx. 2011. application prospects for the innovation of defined fungal starter in rice wine fermentation. j life scie. 5 (4): 255-263. doi: 10.17265/1934-7391/2011.04.002. gallone 2016. domestication and divergence of et al. saccharomyces cerevisiae beer yeasts. cell 166: 1397–1410. doi: 10.1016/j.cell.2016.08.020 gallone b, steensels j, verstrepen kj. 2020. moulded by humans: the domestication of blue-veined cheese f u n g i . m o l e c o l . 2 9 : 2 5 1 7 – 2 5 2 0 . d o i : 10.1111/mec.15525 gibbons jg, salichos l, slot jc . 2012. the et al evolutionary imprint of domestication on genome variation and function of the filamentous fungus aspergillus oryzae. curr biol. 22(15): 1403-1409. doi: 10.1016/j.cub.2012.05.033 hartanti at, rahayu g, hidayat i. 2015. species rhizopus from fresh tempeh collected from several regions in indonesia. hayati j. biosci. 22 (3): 136-142. d : oi 10.1016/j.hjb.2015.10.004 liu q, meng x, li y, zhao c, tang g, li h. 2017. antibacterial and anti-fungal activities of spices. int j mol sci. 18 (6): 1283. doi:10.3390/ijms18061283 maulana ai. 2019. pembuatan tempe kedelai menggunakan rhizopodopsis javensis sebagai starter alternatif [skripsi]. [making soya tempeh using rhizopodopsis javensis as alternative starter]. bogor (id): insitut pertanian bogor. pramudito te, putri ega, paluphi e, yogiara y. 2021. the effect of starter culture on bacterial profile in soybean tempeh. food res 5 (1): 380 – 389. doi: 10.26656/fr.2017.5(1).436 rossi m, buzzini p, cordisco l, amaretti a, sala m, raimondi s, ponzoni c, pagnoni um, matteuzzi d. 2009. growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts. fems microbiol e c ol . 6 9 ( 3) : 3 63 3 7 2 . d oi: 10. 1111 /j. 15 7 4 6941.2009.00727.x santivarangkna c, kulozik u, foerst p. 2008. inactivation mechanism of lactic acid starter cultures preserved by drying processes. j appl microbiol. 105 (1): 1-13. doi:10.1111/j.1365-2672.2008.03744.x seumahu ca, suwanto a, rusmana i and solihin dd. 2013. bacterial and fungal communities in tempeh as reveal by amplified ribosomal intergenic sequence analysis. h a y a t i j b i o s c i e . 2 0 ( 2 ) : 6 5 – 7 1 . d o i : 10.4308/hjb.20.2.65 [sni] standar nasional indonesia. 1992. cara uji makanan dan minuman (sni 01-2891-1992) [ test of food and beverage]. jakarta (id): badan standardisasi indonesia. steensels j, gallone b, voordeckers k, verstrepen kj. 2019. domestication of industrial microbes. curr biol. 29 (10): 381-393. doi:10.1016/j.cub.2019.04.025 tahir a, anwar m, mubeen h, raza s. 2018. evaluation of physicochemical and nutritional contents in soybean volume 15, 2021 microbiol indones 83 fermented food tempeh by j adv rhizopus oligosporus. biol biotechnol 17(1): 1-9. doi: 10.9734/jabb/ 2018/26770. tamam b, syah d, suhartono mt, kusuma wa, tachibana s, and lioe hn. 2019. proteomic study of bioactive peptides from tempe. j bioscie bioeng 128 (2): 241–248. doi: 10.1016/j.ijfoodmicro.2003.09.008. thanh nv, nout mjr. 2002. biomass, rhizopus oligosporus sporangiospores yield and viability as influenced by harvesting age and processing conditions. food microbiol. 19 (1): 91-96. doi:10.1006/fmic.2001.0450. thanh, nv, rombouts, fm, nout mjr. 2007. viability and physiological state transitions of rhizopus oligosporus sporangiospores in tempe starter culture. anton leeuw int jg 91(1): 35–44. doi: 10.1007/s10482-006-9093-7. wijaya ch, nurjanah s, utama qd. 2015. implementasi dan analisis keuntungan teknologi pada back-slopping pembuatan tempe" skala industri rumah tangga "quick [implementation and profit analysis using backslopping technology for “quick tempe” making at home industrial scale]. pangan, 24 (1): 49-62. http://repository.ipb.ac.id/handle/123456789/75390. i wayan.pmd volume 3, number 2, august 2009 p 72-76 issn 1978-3477 *corresponding author:, phone: +62-251-8629474, fax: +62-251-8629459, e-mail: wibawan@yahoo.com development of rapid agglutination test to detect chicken marek antibody i wayan teguh wibawan1, lia siti halimah2, titiek djannatun3, and kamaluddin zarkasie4 1laboratory of microbiology, department of infectious diseases and veterinary public health, faculty of veterinary medicine, institut pertanian bogor, jalan agatis, darmaga campus, bogor 16680, indonesia; 2 pt. biofarma persero, jalan pasteur 28, bandung 40161, indonesia; 3faculty of medicine, universitas yarsi, jalan letjen suprapto, cempaka putih, jakarta pusat 10510, indonesia; 4 pt. ipb-shigeta animal pharmaceutical bogor, jalan agatis, darmaga campus, bogor 16680, indonesia to our knowlege, there is no rapid agglutination test to detect antibodies to viruses which might be due to the small dimension of viral particles. through complex formation of staphylococcus aureus bearing protein a-rabbit igg-anti igy-igy anti marek virus, agglutination of antibodies to viruses can be achieved and visualized. to design the prototype of the test, the bacterial cells of staphylococcus aureus were coupled to a complex compound consisting of igg-igy-marek antigen. this protocol was able to detect clearly the presence of marek antibody in chicken sera, showing the rapid, clear and distinct agglutination reaction on the glass objects. no agglutination reaction was observed in the reaction of specified pathogen free chicken sera with this prototype showing the specificity of the test. this finding demonstrates a novel rapid agglutination which can be used for the detection of antibodies to various agents. key words: indirect coagglutination method, protein a, staphylococcus aureus, prototype diagnostic kit the use of specified pathogenic free (spf) chickens are absolutely necessary in the vaccine industries. the chicken must be monitored regularly for their immunological status by detecting the presence of specific antibody to certain agents. the world health organization (who) in its technical series report (trs) no. 840 (1994) stated that spf chicken used in the pharmaceutical industry must be free from and have no history of contact with adeno virus, reo virus, infectious laryngotracheitis virus, reticuloendotheliosis virus, infectious bursal disease virus, marek virus, newcastle disease virus, haemophylus gallinarum, influenza and para influenza virus, salmonella, mycoplasma, retro virus, avian encephalitis virus and pox virus. various, and sometimes complicated, serological tests are needed in the monitoring of those agents (takase et al. 2000). based on this, an indirect coagglutination test has been developed as a simple and rapid method for detecting various specific antibodies in the animal laboratory and could be used as an appropriate test in the monitoring of health and the immunological status of animal models. protein a is well known as one of the surface components of the cell wall of bacteria and is found in staphylococcus aureus (kusunoki et al. 1992; scriba et al. 2008). protein a is a polypeptide with a molecular weight of 13-45 kda, and binds covalently with the cell wall of bacteria such as s. aureus (forsgren 1970; boyle and reis 1987; kusunoki et al. 1992; takeuchi et al. 1995). protein a has an important role in the mechanism of infection, binding the crystallizable fragment (fc) of mammalian igg, but not fc of igy from chicken (boyle et al. 1985). protein a is able to bind the fc of iga and igm (langone et al. 1978). protein a binds the fc of igg, such that the epitope in antigen binding fragment (fab) is still free and has capacity to bind specific antigens (ribeiro and araújo jr 2009). coagglutination testing is a simple and rapid method in detecting antigen with a high accuracy of test results. up to now, s. aureus cowan i is mostly used and known as a rich protein a s. aureus strain. however, to date and only very limited information is available on the the possibility of using field strains of s. aureus for coagglutination tests. materials and methods bacterial isolates. in this study, 26 staphylococcus aureus isolates were used; 24 isolates were from humans and 2 isolates from bovines showing subclinical mastitis. all isolates had been identified previously as s. aureus, and existed as a collection of fakultas kedokteran hewan, institut pertanian bogor (djannatun 2002). re-identification of isolates. bacteria were inoculated onto blood agar plates for 18 h at 37 °c, and the type of colony formation and type of haemolysis were observed. the characteristics of bacteria were determined microscopically with gram staining, and the ability to utilize glucose and mannitol and the expression of catalase and coagulase activities (qian et al. 2007). detection of protein a with soft-agar and serum soft-agar. to determine s. aureus strains containing protein a, soft-agar (sa) and serum soft-agar (ssa) techniques are used. bacterial suspension (1 loop, 109 cfu) was inoculated into 10 ml of soft-agar (brain heart infussion /bhi+0.15%) (gibco europe, karlsruhe, frg) or into 10 ml of serum soft-agar (bhi+0.15% agar+100ul rabbit serum), agitated using a vortex and incubated at 37 °c for 18 h. the bacteria containing protein a will show the changes of colony formation from diffuse in sa to compact in ssa. the negative strains will remain as diffuse colonies in sa as well as in ssa. for further investigation, 3 isolates containing protein a will be selected and used as carriers (agglutinators) for the preparation of the prototypes of kits. the consistency of the binding character of the selected isolates will be confirmed in ssa using different species of mammalian sera (opdebeeck et al. 1988). detection of protein a with dot blot test. the presence of protein a on the cell surface of bacteria was confirmed with the dot blot test. the bacterial suspension (100 µl) were dropped onto a nitrocellulose membrane (bio-rad, usa), dried, submerged in skimmed milk 3% (v/v 1:10 in phosphate buffer saline/pbs), washed 2x with 5 ml pbs. nitrocellulose membranes were submerged in 5 ml pbs, incubated with 200 µl rabbit serum for 60 min, washed with 5 ml pbs (2x) and incubated with mice-anti-rabbit conjugate (25 ml conjugate + 5 ml larutan pbs) (biorad, usa) for 60 min, followed by 2x washing with 5 ml pbs. the interaction of protein a and igg was visualized by adding 5 ml of α-chloronapthol (9 ml α-chloronapthol + 3 ml metanol + 25 ml pbs) and 200 µl of h 2 o 2 (towbin et al. 1979). purification of immunoglobulin y using ion exchange chromatography. the purification of igy anti-marek’s antibodies from the chicken sera was done by ion-exchange chromatography deae-cellulose/sephadex g-25 (amersham biosciences kk tokyo, japan). using an ammonium sulphate with final concentration 65% igy (obtained from 20 ml of chicken sera) precipatate which had been resuspended with 20 ml pbs (ph 7.5-8) deae-cellulose/sephadex g-25 gel (40 ml) was added to 250 ml distillated water, washed 3x with distillated water, followed by washing with 50 ml of 1m naoh and 50 ml of 1m hcl. finally, the gel was suspended in 40 ml of phosphate buffer (ph 8) and put in a 50 cm column with a diameter of 22 cm. gel was washed with tris 10 mm until clear, 5 ml ammonium sulphate precipated chicken sera was added to the column and left for the igy to bind to deae beads. the column was washed with tris 10 mm to release the unbound proteins. the bound igy was eluted with 200 mm nacl in tris 10 mm. the eluate was collected, dialysed and stored until used (ko and ahn 2007). protein characterization in sds-page. the purified igy was measured for purity using spectrophotometer. for this, a 100 µ l aliquot of eluate was added to a cryol tube containing 100 µ l distillated water. the peak of the protein was detected at 280 nm. the specificity of purified proteins (igy anti-marek’s antibodies) was determined with agar gel precipitation test (agpt). the proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) using 3% (v/v) loading gel and 10% (v/v) separating gel and staining with coomasie brilliant blue (laemmli 1970). preparation of ig g specific to igy. rabbits were vaccinated in the first week with 0.5 ml purified igy (1 mg ml-1, intra vena/iv), the vaccination was repeated in the second week and the third week with 0.5 ml purified igy (3x, iv), respectively. one week after the last vaccination, the serum was collected and tested for the presence of specific antibody (igg) against igy using the agar-gel-precipitation test (agpt) (zhou et al. 1994). preparation of carriers-matrix. staphylococcus aureus with protein a present is used as carrier matrix coupled to the complex igg-igy-marek chicken antigen. the bacteria was preserved using formaldehyde 0.5% (v/v) for 60 min (ansorg and zarifoglu 1986). to avoid the self agglutination, box titration was done to find the appropriate composition of bacterial suspension, rabbit sera, igy and marek virus, and this complex agglutinator is known as prototype for proposed diagnostic kits. specificity of prototype diagnostic kits. to test the specificity of the prototype, the positive chicken sera, containing specific igy to marek virus and control sera from spf chickens are used. one drop (25 ul) of the prototype solution was placed on a glass object and left to react with one drop of positive serum. the negative serum was used as control. the positive reaction will appear within 1 min, as a clear and distinct agglutination reaction. results detection of protein a with soft -agar and serum soft -agar. all staphylococcus aureus isolates used in this study showed turbid growth in todd hewith broth (thb). from 24 s. aureus samples of human origin, 17 isolates showed the change of colony formation from diffuse to compact after the presence of rabbit sera in ssa. similar results were found for 2 isolates of bovine origin, and by the reference using sa cowan i strain. no comparable results were found for 7 isolates of human origin and for s. epidermidis, the colonies remained diffuse after the addition of rabbit sera in ssa. no change of colony formation was detected after the addition of chicken sera for all isolates used in this study (table 1). from 17 s. aureus isolates containing protein-a, 3 isolates were selected for preparation of carriers matrix, i.e. sa 53, sa 54 and 2pi1 based on the phenotype expression of the colonies in ssa. to detect the consistency of the binding characters, the isolates were grown in ssa using various mammalian sera. all isolates showed the change of colony formation in ssa using the respective mammalian sera, but not for chicken sera and s. epidermidis (table 2). table 1 the use of soft-agar and serum soft-agar techniques to detect the presence of protein a in staphylococcus aureus isolates isolates human isolates 12, 14, 15, 18, 29, 35, 36, 37, 38, 42, 45, 50, 53, 56, 59, 5924, 54 5, 43, 47, 48, 52, 5824, 5974 bovine isolates 3ti, 2pi staphylococcus aureus cowan 1 s. epidermidis turbid turbid turbid turbid turbid diffuse diffuse diffuse diffuse diffuse diffuse diffuse diffuse diffuse diffuse compact compact compact compact diffuse growth in tod hewitt broth (thb) colony form in soft agar colony formation using serum chicken rabbit microbiol indones 73volume 3, 2009 for further confirmation, 3 selected isolates were tested for the presence of protein a using the dot blot assay. all isolates showed the presence of protein a. these results are consistent with the results of previous experiments using ssa (table 3, fig 1). purification of immunoglobulin y. the purification of igy began with the precipitation process using ammonium sulphate which was followed by the separation step using deae-cellulose/sephadex g-25. the purified igy showing the maximum absorbance at λ 280 nm had protein concentration of 2.9-5.4 mg ml-1. this purified igy reacted specifically with marek’s antigen in agpt (data not shown). using electrophoresis and coomassie blue staining, the purified igy was found to consist of one main protein band with molecular weight of 97 kd (fig 2). igg specific to igy. one week after the last vaccination of rabbits using purified igy, the specific igg to igy was detected in the sera detected with immunodiffusion tests. the serum was collected and used for the preparation of prototype diagnostic-kits. in order to find out if the igg-anti-igy in the rabbit sera had a consistent characters to bind protein a, 100 µl of serum was used in ssa which caused the change of colony formation of sa 53, compared to the s. epidermidis. sa 53 was selected and used for the preparation of prototype reactants of the future diagnostic kits (fig 3). prototype of diagnostic kits. the prototype was designed by combining the components a and b. component a was prepared by coupling the bacterial cells of sa53 (109 cfu) with rabbit sera containing igg-anti-igy with the table 2 the interaction of selected staphylococcus aureus isolates with various mammalian sera in serum soft-agar test soft-agar serum soft-agar serum horse elephant sheep rabbit1 dog guinea pig chicken colony form of s. saureus sa 53 sa 54 sa 2pi1 diffuse compact compact compact compact compact compact compact diffuse diffuse compact compact compact compact compact compact compact diffuse diffuse compact compact compact compact compact compact compact diffuse s.epidermidis2 diffuse diffuse diffuse diffuse diffuse diffuse diffuse diffuse diffuse 1positive control; 2negative control. table 3 the presence of protein a in the selected staphylococcus aureus isolates using dot blot assay isolates sa 2pi sa 53 sa 54 s. epidermidis dot blot rabbit serum chicken serum* + + + + , s . a u r e u s c o w a n i ( d o t b l o t t + ) ; , s. epidermidis (dot blott -); *chicken serum cannot bind the protein a. a b c d fig 1 the presence of protein a in sa 53 isolate (b) and no protein a in sa 43 (c). as positive contol sa cowan i is used (a) and staphylococcus epidermidis used as a negative control (d). microbiol indones74 wibawan et al. kda 9 7 6 6 4 5 3 0 fig 2 protein bands of chicken serum after ammonium precipitation (c) and after the deae-cellulose purification (d), pure albumin was used as control (a) and markers were run in line b. a b c d fig 3 compact colonies of sa 53 in serum soft agar (ssa) using rabbit sera containing igg-anti igy, staphylococcus epidermidis colonies renmain diffuse in ssa. composition 4:1 (v/v), incubated for 60 min. component b was prepared by incubating purified specific igy with the suspension of marek virus. the relative compositions of component a and b were monitored and optimalized with box titration in order to avoid self agglutination reactions. application of the test. the specificity of the prototype reactants was undertaken by determining the presence of specific igy in chicken serum. this serum was obtained from chickens which were previously vaccinated with marek’s disease virus. for the negative control, the sera from spf chickens used was kindly provided by pt biofarma persero, bandung. the test results showed that, a specific clear and distinct agglutination reaction can be seen in positive marek serum 1 min after the addition of one drop of the kit suspension onto a glass object (fig 4). in contrast, no agglutination reaction can be detected in control sera and the suspension of kits remain homogeneous after 5 min of observing. possible. one the other hand, it is impossible to design the direct agglutination using igy of chickens because igy that cannot bind protein a. (djannatun 2002). staphylococcus aureus containing protein a is very important for this test. in this study we developed the simple method of using serum soft-agar techniques to detect the strains of s. aureus bearing protein a. this study showed that the change of colony formation of bacteria from diffuse to compact in ssa could be used an an indicator to select s. aureus isolates containing protein a. this indicator was confirmed by using the purified rabbit igg, the change of colony formation of protein a-positive-strains was also observed after the addition of purified igg in ssa (data not shown). this indicated that the interaction of igg and proteina on the bacterial cell wall is responsible for the change of colony formation. the inhibition of bacterial capsuleformation of streptococcal bacteria in ssa was caused by the presence of antibody specific to capsule antigens therefore causing the change of colony formation. these results showed that the ssa could also be used to determine the serotype of certain bacteria (wibawan and laemmler 1991; wibawan et al.1992). in summary, the prototype of the diagnostic kit was designed in complex form, using s. aureus containing protein a, rabbit sera containing igg-anti igy), purified igy anti-marek’s antibodies and marek‘s virus. this complex form is made and optimized using box titration to avoid the self agglutination reactions. this is the most crucial step in preparing the kits and the process is influenced by (i) the titre of igg in rabbit sera to igy, (ii) concentration of protein a of s. aureus, (iii) the titre of igy to marek’s virus, (iv) the titre of marek‘s antigen used, (v) incubation period and (vi) the washing step. references ansorg ra, zarifoglu fi. 1986. influence of the cultivation, devitalization and preservation of staphylococcus aureus atcc 12598 on the activity of cell-bound protein a. medical microbiol immunol 174:305-12. boyle mdp, reis jj. 1987. bacterial fc receptors. biol technol 5:697-703. boyle mdp, wallner wa, von mering go, reis kj, lawman mjp. 1985. interaction of bacterial fc receptors with goat immunoglobulins. molec immunol 22:1115-21. datta sm, janes e, simonson jg. 2008. immunomagnetic separation and coagglutination of vibrio parahaemolyticus with anti-flagellar protein monoclonal antibody. clin vacc immunol 15:1541-46. del río ml, gutiérrez cb, rodríguez fef. 2003. value of indirect hemagglutination and coagglutination tests for serotyping haemophilus parasuis. j clin microbiol 4:880-2. djannatun t. 2002. metode sederhana dan praktis pengujian keberadaan protein a staphylococcus aureus isolat asal menusia dan sapi perah serta aplikasinya dalam pembuatan perangkat diagnostik. 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and viral diseases. this coagglutination method was used mainly to detect specific antigens. however, in the present communication indirect coagglutination method is developed to detect the presence of specific antibodies in chicken serum with the marek’s disease antibody being used as a model. we endeavoured to search the scientific literatures about the use of an indirect approach in the coagglutination test, but there is only very limited information about the use of indirect coagglutination for sero-diagnosis. indirect agglutination has already been used in the development haemagglutination techniques, indirect elisa, indirect immunomagnetic separation, latex agglutination and for indirect immunofluorescence (rufli 1980; del río et al. 2003; jesudason et al. 2005; datta et al. 2008; kerremans et al. 2008). the problem in the monitoring of the serological status of laboratory animal is the use of various methods for certain agents, and the use of complicated and expensive methods. 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matuda k, sasano k. 1995. protein a in staphylococcus aureus isolates from pigs. j vet med sci 57:581-2. towbin h, staehelin t, gordon j. 1979. electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. proc natl acad sci 76:4350-4. wibawan iwt, lammler c. 1991. influence of capsul neuraminic acid on properties of streptococci of serological group b. j gen microbiol 137:2721-5. wibawan iwt, lämmler c, pasaribu fh. 1992. role of hydrophobic surface proteins in mediating adherence of group b streptococci to epithelial cells. j gen microbiol 138:1237-42. zhou em, afshar a, heckert ra, nielsen k. 1994. anti idiotypic antibodies generated by sequential immunization detect the share idiotype on antibodies to seudorabies virus antigens. j virol methods 48:301-13. microbiol indones76 wibawan et al. 04. sjatha.cdr vol.1 , no.2, june 201 , p 64-693 9 doi: 10.5454/mi.1 .2.43 levels of tnf-α in pbmc (peripheral blood mononuclear cells) induced by recombinant non structural 1 protein of dengue virus serotype-2 in itrov fithriyah sjatha oktivia chandra mustika beti ernawati dewi 1 2 1 , , , * and tjahjani mirawati sudiro 1 1 department of microbiology, faculty , universitas indonesia, indonesia; of medicine jakarta, 2 master programme of biomedical science, faculty of medicine, universitas indonesia, indonesia.jakarta, dengue infection is a global health problem with an increasing incidence every year and now endemic in more than 100 who countries. dengue infection is caused by dengue virus (denv) which is an rna virus with positive single strand, with ±11kb genome size encoding 3 structural proteins, 7 non-structural proteins, and two untranslated region (utr). ns1 protein is known to have a very important role in the development of severe denv infection, by the direct effect causing host cells damage and indirect effect by activating immune response to induce the secretion of excess cytokines. this study aims to evaluate whether recombinant pcns1 plasmids which have been proven to be able to express recombinant ns1 proteins in previous studies is able to induce cytokine secretion from peripheral blood mononuclear cells (pbmc). transfected chinese hamster ovary-k1 (cho-k1) cells with recombinant pcns1 plasmid was co-cultured with pbmc from healthy donor. after 48 hours post co-cultured, cell supernatant was collected and tnf levels and ns1 recombinant were measured by α elisa. ecombinant ns1 protein was expressed in cho-k1 mammalian cell line and the results showed that r able to induce tnf with higher levels compared to control.α key words: cho-k1 cell, dengue, ns1, tnf-α infeksi dengue adalah masalah kesehatan global dengan kejadian yang terus meningkat setiap tahun dan sekarang endemik di lebih dari 100 negara who. infeksi dengue disebabkan oleh virus dengue (denv) yang merupakan virus rna rantai tunggal positif, dengan ukuran genom ±11kb yang mengkode 3 protein struktural, 7 protein non-struktural, dan 2 daerah yang tidak ditranslasikan (utr). protein ns1 diketahui memiliki peran yang sangat penting dalam pengembangan infeksi denv yang parah, melalui efek langsung yang menyebabkan kerusakan sel inang dan efek tidak langsung yang mengaktifkan respon imun untuk menginduksi sekresi sitokin berlebih. penelitian ini bertujuan untuk menguji apakah plasmid rekombinan pcns1 yang telah terbukti mampu mengekspresikan protein ns1 rekombinan pada penelitian sebelumnya mampu menginduksi sekresi sitokin dari peripheral blood mononuclear cells chinese hamster ovary-k1(pbmc). sel (cho-k1) pasca transfeksi dengan plasmid rekombinan pcns1 di ko-kultur dengan pbmc dari orang sehat. setelah 48 jam pasca ko-kultur, supernatan sel dipanen dan kemudian kadar tnf dan ns1 rekombinan diukur menggunakan elisa.α hasil percobaan menunjukkan protein ns1 rekombinan terekspresi pada sel mamalia cho-k1 dan dapat menginduksi tnf dengan kadar yang lebih tinggi dibandingkan dengan kontrol. α : kata kunci dengue, ns1, sel cho-k1, tnf-α microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: 87865040823+62; fax: +62email: chandrasoenartono@gmail.com; cases of denv infection each year (who 2016; kementerian kesehatan ri 2017). denv is a member of flavivirus family with ±11kb genome size consists of a single strand positive rna which encoding 3 structural proteins (capsid, membrane and envelope), 7 non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5), and two untranslated region (utr). denv divided into four distinct serotypes which antigenically different: denv1-4, each serotype is divided into several genotypes based on sequential variability in the e (envelope) gene (simmonds 2017; liyun et al. et al. 2013; herman 2017). denv is an arthropod et al. borne virus that spread through the bite of infected aedes aegypti aedes albopictus and mosquitoes (aryu dengue virus infection (denv) is a major health problem in world's tropics and subtropics region, especially in southeast asia and the western pacific region. based on who (world health organization) every year around 50 million cases of dhf (dengue fever) occur worldwide, including more than 500,000 cases of severe dengue. in indonesia, denv infection was firstly reported in surabaya and jakarta in 1968, with 58 total cases where 24 of them died. nowdays, denv infection has spread to all provinces in indonesia with an increase tendency the number of candra 2010). clinical manifestations of denv infection can be asymptomatic and symptomatic. the symptomatic clinical manifestation are divided into non-typical fever, dengue fever (df), dengue hemorrhagic fever (dhf), and dengue shock syndrome (dss) (who 2011). several studies have shown an association between the severities of denv infection with the presence of high level of circulating ns1 protein (adikari et al. 2016). ns1 protein is a glycoprotein consists of 353354 amino acids encoded by 1056bp nucleotides (clyde, kyle, and harris 2006). ns1 is a key factor during denv infection which along with other viral proteins can fulfill a structural role, helping to anchor the replication complex to the membrane and induce the formation of membrane components that facilitate viral rna replication, assembly and release of viruses, immune avoidance, and various aspects of pathogenesis (glasner 2018). ns1 is found on cell et al. surface as membrane bound ns1 protein (mns1), and also secreted into the extracellular environment as soluble ns1 (sns1) hexamer and circulates in blood during acute phase which is useful as a diagnostic biomarker. membrane bound ns1 protein presented by the major histocompatibility complex (mhc) molecule is known to induce immune response, whereas sns1 can directly bind to various components of the complement that trigger or inhibit complement activity (henrique 2014; watterson 2016). et al. et al. ns1 denv also directly capable to activating endothelial cells which causes decreased integrity of endothelial cell barrier via the dependent toll-like receptors 4 (tlr4) pathway which results in endothelial cell damage and plasma leakage, activating many innate immune cells to produce any inflammatory cytokines via tlr4, and also inducing immunosuppressive cytokines from monocytes, thus potentially contributing to the pathogenesis of the disease. dosage-dependent ns1 is known to induce an increase in the production of a number of proinflammatory cytokines such as il-6, il-1β, tnf-α from pbmc cells via tlr4, which tends to contribute to cytokine storms in denv infection (modhiran et al. 2015; avirutnan 2006; young 2015; adikari et al. et al. et al. 2016). other studies also found that individual ns1 protein can induce secretion of il-6 and tnf α based on time-dependent menner (chen 2015).et al. ns1 denv has crucial roles in cases of severe denv infection and associated with excess cytokine secretion that causes severity of denv infection. hence, research is needed to determine whether the recombinant ns1 protein encoded by pcns1 from previous study is able to induce secretion of tnf-α cytokines from healthy people's pbmc in vitro that can be used as a reference for further denv research. materials and methods recombinant pcns1 plasmid and recombinant ns1 protein. recombinant pcns1 plasmid contains ns1 gene insertion and recombinant ns1 protein was expressed in cho-k1 mammalian cell line and also tend to be released outside the transfected cho-k1 cells, as confirmed by immunostaining, rapid ns1 detection kit, and ns1 elisa kit as “previously described” (sjatha 2019).et al. recombinant pcns1 transfection. cho-k1 mammalian cell used for plasmid transfection were obtained from kobe university, japan. cell propagation and recombinant pcns1 transfection procedure accordance to previous studies (sjatha et al. 2019). r e c o m b i n a n t n s 1 p r o t e i n d e t e c t i o n . recombinant ns1 protein detected from pcns1 transfected cho-k1 cells supernatant at 48 hours post co-culture with pbmc using ns1 elisa kit. the component preparation and the detection procedure in accordance with the step on product manual protocol (abbexa). the existence of recombinant ns1 protein was detected as absorbance value that higher than the negative control. pbmc (peripheral blood mononuclear cells). pbmc were isolated from a healthy patient (no fever shows for the past 1 week) and confirmed as ns1 dengue negative, igg and igm dengue negative, and prnt negative. blood used for pbmc isolation were collected with ethical approval from research ethical committee faculty of medicine, universitas indonesia no. 10901/un2.f1/etik/2018. pbmc isolation conducted by mixing 2:2 ml of blood and pbs in a falcon tube, then the mixture was put into a falcon tube containing 5 ml of ficoll-paque slowly in an oblique position 45°c. thereafter, the mixture was centrifuged without brake off with 2500 rpm of speed for 30 minutes at room temperature to collect the pbmc (shows as white ring circle). the pbmc with complete rpmi 1640 medium (10% fbs and 1% penstrep) then incubated for 2 hours in 37° c incubator with 5% co . afterward, the medium removed to 2 discard the non-adherent pbmc, then the flask was washed using rpmi 1640 medium without fbs twice with spray suction technique to collect the adherent volume 1 , 2013 9 microbiol indones 65 66 sjatha et al . microbiol indones pbmc from the bottom of the flask. hereafter, centrifuge was conducted at 1200 rpm for 5 minutes for one last time washes the pbmc, then pbmc was ready to co-culture. the number of adherent cells was counted by haemocytometer, then suspended in rpmi 1640 (sigma) with 10% fbs and 1% penstrep (gibco) to prepare the cells with a concentration of 1 x 10 6 cells/ml. pbmc co-culture. 150 μl of adherent pbmc with concentration of 10 cells/ml were added to each 6 well contains transfected cho-k1 cells at 48 hours post-transfection. 48 hours after pbmc co-culture, the supernatant was collected to measure the level of tnfα. there were three treatment groups which were done in triplicate. the groups were co-cultured pbmc with p c n s 1 t r a n s f e c t e d c h o k 1 c e l l ( c h o k1+pcns1+pbmc), co-cultured pbmc with pcdna t r a n s f e c t e d c h o k 1 c e l l ( c h o k1+pcdna+pbmc), and co-cultured pbmc with pha induced cho-k1 cell (cho-k1+pha+pbmc). tnf measurement using elisa. α tnf α levels were measured from transfected cell supernatant after co-cultured with pbmc using tnf-α human in vitro elisa kit (antibodies-online, germany). the component preparation and the measuring procedure in accordance with the step on product manual protocol (abcam). the elisa result were showed as absorbance value which then converted to levels using standard curve. results extracellular ns1 detection. extracellular recombinant ns1 protein was detected using ns1 elisa kit. the cho-k1 cell co-cultured with pbmc at 48 hours post transfection with recombinant pcns1 plasmid. at 48 hours after pbmc co-culture, the cells supernatant then collected and were used as samples in elisa assay to detect the extracellular recombinant ns1 protein. the result come out as absorbance value which was detected right after the elisa assay protocol was finished using elisa reader at 450 nm wavelength. these extracellular ns1 protein detection was conducted to reconfirm that the recombinant ns1 protein was successfully expressed in pbmc coculture. render by figure 1, the absorbance value of pcns1 transfected cell supernatant (0.239) was higher than negative control (0.176) and also higher than pcdna transfected cell supernatant (0.166) whereas pcdna transfected cell supernatant was lower than negative control. these indicated that recombinant pcns1 was able to express recombinant ns1 protein out of the cho-ki mammalian cells line. tnf levels. α tnf-α levels were measured to perceive the ability of recombinant ns1 protein in inducing the secretion of cytokine tnf-α from pbmc. as seen in figure 2, the tnf-α levels in pcns1 transfected cell supernatant after co-culture with pbmc (mean±sd: 1561.778±122.217) was higher than the tnf-α levels in pcdna transfected cell supernatant after co-culture with pbmc (mean±sd: 1524±228.351) and also higher than tnf-α levels in pbmc supernatant post pha induction (mean±sd: 818.111±626.535). significant differences in tnf-α levels (p<0.05) were shown between tnf-α levels in pcns1 transfected cell supernatants after co-culture with pbmc and the tnf-α levels in pbmc supernatant post pha induction, but not the others. these results suggested that recombinant ns1 protein expressed by cho-k1 mammalian cells line posttransfected by recombinant pcns1 plasmid was able to induce secretion of tnf from pbmc. α discussion the non-structural protein i (ns1) denv is a 46 kda glycoprotein found in infected cells, both on the cell surface and also secreted out of cells that are found circulating in the bloodstream (alayli & scholle, 2017). it has been found that denv ns1 protein with high levels is associated with severity of infection and levels >600 ng/ml in the first 72 hours of disease occurrence associated with the development of dhf (libraty 2000). in addition, it has been found that et al. denv ns1 antigens still circulate in the body of dhf patients and this may be used as a marker of the severity of denv infection (paranavitane 2014). ns1 et al. denv is known to interact with the complement system, and activate innate immune cells to produce proinflammatory cytokines such as il-6, il-1β, tnf-α from pbmc that act through tlr4, which is likely to contribute to dengue-related "cytokine storms" (modhiran 2015). modhiran 2015 found that et al. et al. doses dependent ns1 protein was able to induce the production of cytokine tnf mrnas measured from α ns1 treated maurine bmms. inflammatory cytokines are a key factor in the pathogenesis of denv (valero et al. 2014). in the other hand, ns1 can also induce immunosuppressive cytokines such as il-10 from monocytes, thus potentially contributing to the pathogenesis of the disease (adikari 2016).et al. based on the explanations above, detection of cytokine levels secreted by pbmc cells after coculture with pcns1 transfected cho-k1 cells was carried out to determine the relationship of ns1 protein to the pathogenesis of denv infection by secreting the inflammatory cytokines and to know the ability of recombinant ns1 expressed by pcns1 transfected cho-k1 cells to induce cytokine secretion, especially tnf-α from pbmc cells in vitro. detection of tnf selected in this study because α it is known that tnf and other inflammatory α cytokines play important roles during denv infection, and the low response for those cytokines by denvinfected monocytes from neonates and elderly people could important in the development of the disease (valero 2014). tnf and other inflammatory et al. α cytokines also known involved in denv hemorrhagic manifestations. in this regard, a rapid increase in the levels of cytokines, especially tnf, plays a key role α in inducing unique clinical manifestations of dhf such as plasma leakage, shock, and hemorrhagic manifestation (kurane 2007). several studies have found that denv ns1 protein is able to induce the secretion of tnf based on time α and dosage dependent, where low doses of ns1 will induce the low levels of tnf secretion and the longer α incubation time will decrease the secretion of tnf-α (modhiran 2015; chen 2015). this finding et al. et al. is consistent with what was obtained in this study. tnfα levels secreted by pbmc co-cultures with pcns1 transfected cho-k1 cells are not significantly different from tnf levels secreted by pbmc co-α cultures with pcdna transfected cho-k1 cells, possibly caused by the incubation time has passed the peak hours of tnf secretion which is 24 hours after α ns1 induction (chen 2015). to induce tnf-et al. α secretion in this study was conducted differently from fig 1 results of recombinant ns1 protein detection from pcns1 and pcdna transfected cho-k1 cells supernatant at 48 hours post co-culture with pbmc using elisa ns1 kit (antibodies-online). fig tnf-α levels from cho-k1 cells supernatant post transfected with pcns1, pcdna, and pha induction at 2 48 hours after pbmc co-culture. tnf levels was detected using tnf human in vitro elisa kit α α *p<0.05 (abcam). volume 1 , 2013 9 microbiol indones 67 others study existed. chen 2015 have been et al. measured tnf levels from pbmc supernatant after α treated with individual denv ns1 commercial protein, whereas this study used mns1 and sns1 expresed by pcns1 transfected cho-k1 cells to induce tnf secretion from pbmc. measuring the α levels of ns1 secreted by pcns1 transfected cho-k1 cells was not conducted in this study prior pbmc coculture. therefore, it is unknown yet whether the low doses of ns1 affected the low levels of tnf secreted α in this study. however, previous studies have proven that pcns1 transfected cho-k1 cells are able to secrete ns1 protein that are detected using ns1 rapid test kit and immunostaining (sjatha 2019). this et al. study also measured the ns1 at the same time as the tnf levels, and the optical density (od) value of α ns1 ​​were low. the low of od ns1 value potentialy causing the tnf levels secreted by pbmc co-α culture with pcns1 transfected cho-k1 cells were not significanly different with tnf levels secreted by α pbmc co-culture with pcdna transfected cho-k1 cells. suggested for further research to measure the ns1 levels prior pbmc co-culture to find out whether the ns1 doses affected the levels of tnf secretion. α in addition, the management of incubation time also needs to be adjusted to the peak hours of tnf α secretion to obtain the maximum levels of tnf. α in conclusion, this suggests that recombinant ns1 protein expressed by pcns1 in mamalian cho-k1 cells is able to induce secretion of 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watterson d, modhiran n, young pr. 2016. the many faces of the flavivirus ns1 protein offer a multitude of options for inhibitor design. antiviral res. 130:7-18. doi: 10.1016/j.antiviral.2016.02.014. who. 2016. dengue ontrol. retrieved march 10, 2019, from c http://www.who.int/denguecontrol/ epidemiology/en/. who regional office for south-east asia. 2011. and comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. revised and expanded edition. in searo technical. doi: 10.1017/cbo9781107415324.004 volume 1 , 2013 9 microbiol indones 69 4 happy.pmd characteristics of phosphate-solubilizing bacteria isolated from acid soil of cikopomayak, west java, indonesia happy widiastuti indonesian biotechnology research institute for estate crops, jalan taman kencana no. 1, bogor 16151, indonesia phone: +62-251-8324048, fax: +62-251-8328516, e-mail: happywidiastuti@yahoo.com phosphate-solubilizing bacteria were isolated from acid soil from cikopomayak. microbiological assay showed that the bacteria were gram negative, rod-shaped, and lacked red pigment on pikovskaya medium. isolate a synthesized red pigment on nutrient agar medium, while isolate b formed slightly red pigment on nutrient agar medium as well as on voges proskauer medium. the ratio of clearing zone to colony for isolate a and b were approximately 2.1 and 1.9, respectively. biochemical assays showed that both isolates a and b utilized glucose, lactose, sucrose, mannitol, glycerol, mannose, urea, indole, and produced catalase, but neither produced amylase and oxidase. the bacteria are identified as serratia marcescens. the ability to solubilize phosphate decreased or was even lost when subcultured on nutrient agar medium. however, the ability to solubilize phosphate was recovered gradually with the addition of phosphate. the results presented here suggest that the ability to solubilize phosphate and to synthesize the red pigment may be inversely correlated. key words: acid soil, serratia marcescens, phosphate-solubilizing bacteria, medium, pigment _____________________________________________ volume 2, number 3, december 2008 issn 1978-3477 p 115-118 the bacterium serratia sp. has been isolated from soil, water, plants, air, as well as from the gut of various insects and vertebrates. it is also found in the intestine of humans. species of serratia are gram negative, rod-shaped, ureasolubilizing and non-spore-forming (bhadra et al. 2005). these bacteria produce red pigment prodigiosin, a member of the prodiginines, which is produced by some serratia species, actinomycetes and a few other bacteria. prodigiosins have no defined role in the physiology of producing strains, but have been reported to have antifungal, anti bacterial and anti protozoal/anti malarial activities. the ability of some serratia marcescens strains to colonize a wide range of ecological niches has been linked to the production of a spectrum of extracellular products including chitinase, protease, lipase, nuclease, anti-bacterial agents, surfactants and wetting agents (matsuyama et al. 1995). gyaneshwar et al. (2002) showed that s. marcescens was an endophytic bacteria of rice that could increase the root length and dry weight. the report of dbt 2003-2004 showed that s. marcescens isolated from the tea rhizosphere was antagonistic to all plant pathogenic fungi. the assay showed that the bacteria increased plant growth, plant height, the number of shoots, the number of leaves per shoot and the phenolic chlorophyll content. in addition, a serratia species, s. phosphaticum, was reported to be able to solubilize pi as a result of the production of organic acids such as gluconic and 2-keto-gluconic acids (tilak et al. 2005). in humans, selected serratia strains cause nosocomial infection that is clinically problematic because of multidrug resistance in some species (harris et al. 2004). materials and methods bacteria were isolated using pikovskaya medium at room temperature. morphological characterization was observed by growing the bacteria on nutrient agar (na). fresh cell preparations of the bacteria were microscopically examined using a light microscope at 1000 x magnification. biochemical characterizations consisted of catalase assay, testing for growth and acid production on glucose and lactose, sucrose, mannitol, glycerol, mannose and methyl red-voges proskauer (mr-vp). in addition, testing was done for the production of amylase, urea, indole, oxidase (catalyzed oxidation of cytochrome to h 2 o 2 ) and citric acid. selected physiological characteristics were assessed, such as growth and pigment synthesis on na, pikosvkaya and vp media. the bacteria were maintained on na medium. as the bacteria were subcultured in na, the ability to solubized pi was loss. in effort to recover the lost ability to solubilize pi, the bacteria were grown on pikovskaya medium containing various levels of pi. three levels of pi were examined i. e. as in pikovskaya medium (1.6 mg pi ml-1); 50% of the pi level in pikovskaya medium (0.8 mg pi ml-1); 150% of pi level in pikovskaya medium (2.4 mg pi ml -1). results the bacteria were isolated using dilution and surface plating methods on pikovskaya media. two isolates of phosphate-solubilizing bacteria were obtained. table 1 shows that both isolate a and b produce catalase and use glucose, sucrose, mannitol, glycerol and mannose as c sources. there were no significant differences between isolate a and b except for the color of the colonies. in na medium, isolate a initially produced red pigment that was much darker compared to that of isolate b. however, there were color differences for the colonies on na and on pikovskaya media (fig 1). on pikovskaya medium, colonies were, white while on na they were red. another difference was the ability to solubilize pi. the ratio of diameter of the clearing zone to that of the colony for isolate a was 2.1 and for isolate b was 1.9. the bacteria were subcultured on na media to maintain them. after several subculture steps, the bacteria were not able to solubilize pi on pikovskaya medium but still produced red pigment (fig 2). subculturing of these two isolates in table 1 morphological and physiological traits of two isolates of serratia marcescens characteristics isolate a isolate b morphology of colony shape gram cell shape physiological traits: catalase activity glucose lactose sucrose mannitol glycerol mannose urea indole oxidase amylum (amylase activity) citrate methyl red voges proskauer colony color: nutrient agar pikovskaya voges proskauer slimy negative rod + + + + + + + + red red slimy negative rod + + + + + + + + slightly red red fig 1 color difference produced by phosphate-solubilizing bacteria isolated from cikopomayak acid soil. on pikovskaya medium the bacteria could not synthesize pigment (left) while on na the bacteria synthesized red pigment (right). fig 3 regaining of pi-solubilizing ability of isolate a induced by g r a d u a l a d d i t i o n o f p i i n p i k o v s k a y a m e d i u m . ( a ) i n i t i a l p i c o n c e n tration (50%); (b) defined pikovskaya medium (100%); (c) 25% pi addition. a b c fig 2 inability of isolate a and b to solubilize pi and synthesize pigment in pikovskaya + na (left) and pikovskaya (right) after subculturing in na. pikovskaya agar medium resulted in the loss of ability to produce red pigment. in this medium the isolates still could not solubilize pi. recovery of the pi solubilizing ability of both isolates was accomplished by gradually increasing the dose of pi in pikovskaya medium. there were differences in the dose of pi required for pi solubilization activity between two isolates. isolate b initially solubilized pi in pikovskaya medium at a 50% pi concentration (10 mm), but recovery of pi solubilizing ability for isolate a was induced with the addition of 25% pi (5 mm) (fig 3). discussion based on selected characteristics, isolate a and b were identified as s. marcescens. table 1 showed that those two isolates of phosphate-solubilizing bacteria did not utilize urea. this characteristic was in line with that of s. marcescens as reported by bhadra et al. (2005). bhadra et al. (2005) also reported that another serratia species, s. ureilytica did utilize urea. the results therefore showed that there were variations between species in serratia. in addition, the ability to solubilize pi between isolates a and b was slightly different. based on the ratio of the diameter of the clearing zone to colony, the ability of isolate a in solubilizing pi was higher than that of isolate b. the interesting characteristics of these bacteria were the differences in pigment production on selected media (table 1). it was assumed that the differences in the ability to synthesize pigment was affected by medium composition. pikovskaya medium is a simple, defined medium, while na is a complex medium. the content of both organic and inorganic components in pikovskaya is precisely measured, while in na the content of the medium substituents is uncertain because na media contains beef and yeast extracts. on pikovskaya medium, the bacteria must synthesize all of their organic compounds from glucose. however, on na, nutrients required to form organic compounds for cell growth are available. the same is true for vp medium, which is also a complex medium. on this medium the isolates formed pigment (table 1). the organic compounds present in vp medium are glucose and peptone. although glucose is the macronutrient utilized to form gluconic acid, which is needed to solubilize pi, it can also be used to form pigment. it might cause the differences in physiology especially in catabolism to produce energy, hence, the ability to synthesize pigment. the differences in pigment synthesis on pikovskaya medium and na might also be caused by the difference in the n source of the media. williams (1973) showed that the precursor of prodigiosin synthesis were serine, alanine, methionine and proline. pikovskaya medium (pigment -) contained n in inorganic form i.e. ammonium sulphate while in na (pigment +), beef extract and peptone were the source of n. peptone is also the n 2 source found in vp medium. the red pigment prodigiosin results from secondary metabolism. fineran et al. (2005) reported that prodigiosin synthesis involved quorum sensing mediated by n-acyl homoserine lactone (n-ahl). they showed that there was a transcriptional activator pigt. pigt activated transcription of biosynthestic pig a-o operon in the absence of gluconic 116 widiastuti microbiol indones acid. the addition of gluconic acid inhibited pig a-o transcription. it was also reported that p solubilization by phosphate-solubilizing gram-negative bacteria was caused by the presence of 2-keto-gluconic acid (rodriquez et al. 2001). it is possible that the lack of pigment formation in pikovskaya medium is caused by gluconic acid synthesis by phosphate-solubilizing bacteria. rodriquez et al. (2001) showed that the high pi solubilizing ability of gram negative bacteria was caused by gluconic acid and 2-keto-gluconic acid synthesized in the periplasmic space from glucose oxidation through quinoprotein-glucose-dehydrogenase (ec1.1.5.2). this result suggested that the synthesis of pigment in s. marcescens was inhibited by phosphatesolubilizing activity. however, slater et al. (2003) showed that phosphate availability regulates biosynthesis of prodigiosin in serratia via both quorum-sensing-dependent and independent pathways. they showed that in serratia 39006 pi limitation was a cause of prodigiosin operon upregulation. gluconic acid that was needed in solubilizing phosphate was synthesized from glucose in oxidation reactions (rodriguez et al. 2001). as a result, in reducing conditions the bacteria were unable to solubilize pi. it was reported that by reducing oxygen pressure, s. marcescens did not form pigment, while under the microaerobic conditions, the pigment was formed. in this situation the oxygen is used to produce co 2 . it seemed that the growth of those two isolates were similar in inducing both solubilizing pi ability and pigment synthesis whether they were incubated in pikovskaya or na. in our experiments, after several subcultures, the ability to solubilize pi on pikovskaya medium was lost when subcultured on na, but the bacteria still produced pigment. this supported the hypothesis that pigment synthesis inhibited pi solubilization. the lack of ability to synthesize red pigment in the presence of pi (initially) caused the suspicion that in pigment synthesis pi nutrient must be available. on the other hand, the inability of the isolate to form clear zone in na was caused by the sufficient availability of pi and the inhibition of gluconic acid synthesis needed in pi solubilization. the ability to form red pigment in na medium was made possible by the presence of gluconic acid that inhibited the pig-t-activator transcript as reported by fineran et al. (2005). the results suggested that in prodigiosin synthesis, pi should be available. in addition, it was suspected that the bacteria were able to solubilize pi only when pi was not available. bacterial pi solubilization might be caused by the synthesis of hydroxy acids such as oxalic acid that may chelate with ca2+ and fe2+. if this is the mechanism of pi solubilization, then it raises some questions: is there any correlation between oxalic acid and gluconic acid? is gluconic acid the oxalic acid precursor? does prodigiosin synthesis suppress the synthesis of organic acid? is there a common precursor for organic acid synthesis and prodigiosin synthesis? the observation of pi-solubilizing-ability of the bacteria at various levels of pi revealed that the ability of isolate b to solubilize pi was regained on medium containing 50% pi concentration, while on this medium isolate a could still not solubilize pi. on pikovskaya medium containing 150% pi concentration, neither isolate a nor b solubilized pi. the ability of isolate a was recovered when grown on medium with only 25% pi level of the pikovskaya medium. these results indicate that the ability of isolates a and b in solubilizing pi are different. the recovery of pi-solubilizingability of isolate a was slower compared to that of isolate b. different recovery processes between isolates a and b indicated a correlation between the ability to solubilize pi and pigment synthesis even though at the beginning of isolation the bacteria showed both the ability to solubilize pi and synthesize pigment. it was suspected that soil contained compounds that could induce these two processes. synthetic media like na medium and pikovskaya lacked these compounds, resulting in physiological changes in the bacteria. these results showed that available pi inhibited the ability of bacteria to solubilize pi. the ability of isolate a and b to solubilize pi changes according to the media composition, especially the presence of c and n compounds and the availability of pi. the recovery of solubilization activity between isolates a and b was different. phosphatesolubilizing activity of isolate a was higher compared to that of isolate b. it was in line with the recovery of the ability to solubilize pi. isolate b had less ability than isolate a in solubilizing pi as shown by ratio of clearing zone and colony that explains why it needed more pi compared to isolate a. considering these results, could the ability of bacteria to solubilize pi be increased by inhibiting pigment synthesis? likewise, could the formation of red pigment be induced by increasing the availability of free p? it was suggested that pisolubilization and pigment synthesis could be controlled by controlling the addition of pi. these results indicated that in nature, in an environment with low availability of pi, the bacteria synthesize gluconic acid that can lead to solubilization of pi, while in an environment with sufficient pi the bacteria synthesize pigment. this paper showed that there were physiological changes in the bacteria. the changes could easily be observed through the appearance of pigment and the formation of clearing zone. this paper reports the characterization of s. marcescens isolated from acid soil of cikopomayak. other characteristics such as the interactions of the bacteria with plants need to be studied further. references bhadra b, roy p, chakraborty r. 2005. serratia ureilytica sp. nov., a novel urea-utilizing species. int j syst ecol microbiol 55:21552158. fineran pc, everson l, slater h, salmond gpc. 2005. a gntr family transcriptional regulator (pigt) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in serratia. microbiology 151:3833-3845. gyaneshwar p, janes ek, mathan n, reddy pm, reinhold-hweh b, ladha jk. 2002. endophytic colonization of rice by a diazothropic strain of serratia marcescens. j bacteriol 183:26342643. harris akp, williamson nr, slater h, cox a, abbasi s, foulds i, simonsen ht, leeper fj, salmond gpc. 2004. the serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species and strain-dependent genome context variation. microbiology 150:3547-3560. volume 2, 2008 microbiol indones 117 matsuyama t, bhesin a, harshey rm. 1995. mutational analysis of flegelin-independent surface spreading of serratia marcescens 274 on a low agar medium. j bacteriol 177:987-991. rodriquez h, gonzalez t, selma g. 2001. expression of a mineral phosphate solubilizing gene from erwinia herbicida in two rhizo bacteroid strain. j biotechnol 84:155-161. slater h, crow m, everson l, salmond gp. 2003. phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in serratia via both quorum-sensing-dependent and-independent pathways. mol microbiol 47:303-320. tilak kvbr, ranganayaki n, pal kk, de r, saxena ak, nautiyal cs, mittae s, tripathi ak, jahri bn. 2005. diversity of plant growth soil health supporting bacteria. curr sci 89:134-150. williams rp. 1973. biosyinthesis of prodigiosin, a secondary metabolite of serratia marcescens. appl microbiol 25:396-402. 118 widiastuti microbiol indones 9 agus rohyadi_405.pmd volume 3, number1, april 2009 p 42-46 issn 1978-3477 neighboring plants alleviate aluminum toxicity on the external hyphae of gigaspora margarita agus rohyadi faculty of agriculture, universitas mataram, jalan majapahit 62, mataram 83125, indonesia phone: +62-370-621435, fax: +62-370-640189, email: arohyadi01@telkom.net excessive soluble aluminum (al3+) in acidic soils is toxic to the external hyphae of arbuscular mycorrhizal fungi but it can be alleviated by other soil factors. a glasshouse experiment was conducted to study the effect of increased al3+ concentration on the growth of the external hyphae of gigaspora margarita in the presence of other plants near the host plants. the experiment used compartmentalized pots to facilitate the growth of mycorrhizal-inoculated host plants, external hyphae of the fungus and not mycorhizal-inoculated neighboring plants in different compartments; and measuring the effects of al3+ and of the neighboring plants on the growth of the fungal hyphae independently. increased concentration of al3+ in soil affected the growth of external hyphae of g. margarita negatively. however, the hyphal length density of the fungus was much higher in the pots with neighboring plants than that in the other ones, despite the al toxicity. this indicates that the hyphae could be taken away from the toxic effect of al3+ by the stimulating growth from roots of the neighboring plants. key words: aluminum, cowpea, external hyphae, compartmentalized pot system, gigaspora margarita crops often suffer from adverse conditions of acid soils. aluminum (al) is considered the major stress factor because of its toxicity under acidic conditions. excessive soluble-al (al3+) in soil might create a poor plant root system by inhibiting lateral root and root hair formation, and limit the solubility and, therefore, the availability of some essential mineral nutrients, especially p (baligar et al. 1995). also, high al3+ concentration can be detrimental to soil microbes that are involved in nutrient cycling and/or forming symbiotic associations with the plant roots (wood 1995; rohyadi 2006). as a result, the plants may have limitation in taking up nutrients and water in sufficient quantity, and consequently they grow stunted. arbuscular mycorrhizas (am) are mutualistic symbioses mostly benefiting plants that have a small and/or coarse root with a lack of root hairs, and those are grown on infertile soils with particularly low available-p. hence the symbioses can be of great importance in acid soils, where poorly developed root systems and p deficiency are the main constraints for plant growth (miyasaka and habte 2001). however, the am fungi themselves are subjected to detrimental effects of mainly high al3+ concentration in acidic soils. rohyadi (2006) indicated that decreased effectiveness of an am fungus, gigaspora margarita on cowpea plant grown on acidic soils was related to the poor growth of the external fungal hyphae because of al toxicity. it is well known that the external hyphae of am fungi growing out from colonized plant roots play a crucial role. they develop to form a hyphal net-work in soils and function as an extension of root system to explore soil particularly beyond the depletion of the root zone, take up water and element nutrients there and then transport them to the roots of the host plants (smith et al. 2000). therefore, the inhibited growth of the hyphae may directly reduce the symbiotic effectiveness. only a few studies were done on the effect of al3+ on the external hyphae of the am fungi. it was reported that the growth responses of the am fungi to al toxicity are complex and much dependent on the fungal species (siqueira et al. 1985). recently, several studies in vitro showed that factors such as co 2 level, some chemical compounds and volatiles released by plant roots, and bacterial products, influence the growth of the fungal hyphae (tawaraya et al. 1996; nagahashi and douds 2000, 2003; xavier and germida 2003; scervino et al. 2005). therefore, it is believed that the presence of other plants near their hosts will alter the response of the external fungal hyphae to toxic al3+ in situ. the objective of this study was to examine if the presence of neighboring plants could modify the growth response of the external hyphae of g. margarita to the detrimental effects of al3+. materials and methods the fungus used in this study was g. margarita becker & hall (beg collection), which is tolerant to and working effectively under acidic soil conditions (rohyadi et al. 2004). the fungus was propagated in pot cultures of trifolium subterranean l. in a sand and soil (90:10, w/w) mixture for 4 months. another pot without the fungus to provide mycorrhiza-free inoculums was included. the cultivar of cowpea used was red caloona supplied by csiro tropical agriculture, brisbane, australia. the growth medium was a mixture of sand and acidic podsolic soil of grey sandy loam (90:10, w/w). the mixture was firstly fertilized with (in mg kg1 medium) 59.4 nh 4 -n, 178.2 no 3 -n, 36 p, 54 s, 214.2 k, 18.9 mg, 114.3 ca, 13.5 na, 8.1 cl, 2.7 fe, 0.45 b, 0.45 mn, 0.225 zn, 0.036 cu and 0.009 mo. then it was treated with 0, 75, 150 or 300 al 2 (so 4 ) 3 . the concentrations of al3+ (measured based on the method of close and powell 1989) in the final established media (a 0 , a 1 , a 2 or a 3 ) were 0.4, 1.1, 4.1 or 7.3 mg kg-1 soil respectively. this experiment was conducted under glasshouse conditions. it was a 2 x 3 x 2 factorial experiment consisting of two levels of mycorrhiza with (m 1 ) and without (mo) inoculation. three concentrations of al3+ established in soil were 1.1, 4.1 and 7.3 mg kg-1 soil (denoted as a 1 , a 2 and a 3 ), and the presence and absence of neighboring plants were denoted as n 1 and n 0 respectively. there were six replicates per treatment combination. microbiol indones 43volume 3, 2009 the pot system used was made of a pvc pipe (fig 1), divided into three compartments by two vertical 30 µm screen meshes that allow hyphae to pass but exclude plant roots. the first compartment, the host plant compartment (hpc), was for plants inoculated or not inoculated with mycorrhiza fungal structures, designated as the host plants. they supplied the external fungal hyphae being tested. the second compartment in the middle of the pot, the external hyphal compartment (ehc), was a root free compartment provided for the external hyphae extending from mycorrhizal-colonized roots of the host plants in the hpc. the third compartment, the neighboring plant compartment (npc), was for noninoculated plants, denoted as neighboring plants, which acted as a growth promoter for the fungal hyphae to traverse the ehc. in order to provide favorable conditions for plant roots, the hpc and npc were filled with growth medium not treated with al, a 0 (ph 5.3, containing 0.4 mg al3+ kg-1 soil and 26 ppm bray-1 p), whereas the medium in ehc was treated with al at different concentrations. therefore, the basic purpose of using the compartmentalized pot system is to allow the external fungal hyphae from mycorrhizal-colonized roots of the host plants in the hpc to extend into the ehc, interact with al and possibly traverse to the npc, and finally colonize roots of neighboring plants there. the effects of the al3+ concentrations in the ehc and the presence of neighboring plants in the npc on the growth of the external hyphae was determined by the extending of the external hyphae in the ehc and the colonization of neighboring plants in the npc by the am fungus. at the beginning of the experiment, hpc and npc were filled with 320 g of a 0 soil, but only the hpc was inoculated with 10% (w/w) of pot culture inoculums of g. margarita or was left not-inoculated to provide mycorrhizal and nonmycorrhizal colonized host plants. two pre-germinated seeds of cowpea (cv. red caloona) were transplanted into all of the hpc and a half of the npc, and then left grown for two weeks. the ehc was then filled with 260 g of a 1 , a 2 or a 3 soils in the same way. reverse osmotic (ro) water, adjusted to ph 4.6 with h 2 so 4 , was used on a weight loss basis to maintain the moisture content of the growth media at the field capacity (about 0.1 g g-1 soil) and the ph of soil in the ehc, at about 4.7 throughout the experiment. plants were harvested 6 and 10 weeks (h 1 and h 2 ) after transplanting. the dry biomass and root length of the host plants, the dry biomass of the neighboring plants, the mycorrhizal colonization on the roots of these plants, and the length density of the external fungal hyphae in the ehc were measured. plant biomass was weighed after it was dried at 70 oc for 48 hours. to measure root length and percentage of mycorrhizal colonization, root samples were cleared with 10% koh, and stained with trypan blue in lacto-glycerol and then assessed under a dissecting microscope using a gridline intersect method (giovannetti and mosse 1980). to measure length density of the external fungal hyphae in the hpc, representative samples of the growth media were taken from the compartment. the hyphae in the soil samples were then extracted following the aqueous membrane-filtration method of jakobsen et al. (1992), and assessed under microscopic observation (see detail in rohyadi 2006). the data were statistically analyzed using anova after grouping them into plant compartments and harvest times. the lsd tests at p < 0.05 were then applied to determine the significant differences among ways of treatment. results growth of host plant. dry biomass of the host plants, measured at any harvest was not affected by the al3+ concentration in the ehc or by the presence (n 1 ) and the absence (n 0 ) of neighboring plants in the npc, but was significantly affected by mycorrhizal inoculation. the dry biomass of mycorrhizal (m 1 )-plants was significantly higher than that of non-mycorrhiza (m 0 ) (table 1). similarly, mycorrhizal inoculation significantly increased the root length of the host plants in the hpc regardless of the presence or absence of neighboring plants. aluminum concentration and the presence of neighboring plants had no significant effect on the growth of roots irrespective of the mycorrhizal status of plants. mycorrhizal colonization on host plants. plants not inoculated with am fungus exhibited no evidence of am fungal colonization, while the extent of roots colonized by am fungus and the root length of host plants were stimulated by inoculation (table 1). in general, mycorrhizal colonization increased from the first to the second harvest. however, the increases were not related to the al3+ concentration in the ehc. the presence of neighboring plants reduced mycorrhizal colonization observed in the hpc at the second but not at the first harvest. the effect of al3+ concentration and neighboring plants on the length of the mycorrhizal colonized roots in the hpc was nil at the first harvest. at the second harvest, the adverse effect of al3+ on the mycorrhizal-colonized root length was dependent on the presence or absence of neighboring plants. it was found that the presence of neighboring plants was more detrimental than that the adverse effect of al3+ on the root colonization by the fungus. h o s t p la n t c o m p a rt m e n t w it h t h e f u n g a l in o c u lu m s e x te rn a l h y p h a l c o m p a rt m e n t w it h d if fe re n t a ltr e a te d s o il s n e ig h b o ri n g p la n t c o m p a rt m e n t 1 2 .5 c m 3.5 cm 2.0 cm 3.5 cm fig 1 schematic representation of the compartmentalized pot system. the compartments are separated by two vertical 30 µm nylon screen meshes ( ) that can be crossed by fungal hyphae but not by plant roots. microbiol indones44 rohyadi growth of external hyphae. the growth of external fungal hyphae in the ehc increased with harvest times; the extent was dependent on the al3+ concentration and the presence of neighboring plants in the npc. however, there was no a significant interaction between the two factors. the hyphal length density (hld) decreased with increased al3+ concentration, but the adverse effect of al3+ was nullified by the presence of neighboring plants (fig 2). mycorrhizal colonization and growth of neighboring plant. table 2 clearly shows the fungal hyphae, which traverse the ehc, were able to definitely colonize roots of plants in the npc. at h 1 , the colonization was less than 20%, but increased to more than 40% at h 2 . it also shows that different al3+ concentrations in the ehc affect the colonization rates differently. at h 1, compared to the root colonization in pots with a 1 , a significant reduction in the percentage of colonized roots only occurred in pots with a 3 ; meanwhile, at h 2 such reduction was already evident in pot with a 2 . in general, the growth of the neighboring plants increased, but was not related to soluble-al concentration in the ehc. differences in plant growth occurred between plants in pots inoculated with (m 1 ) and without mycorrhiza (m 0 ). the dry biomass of the neighboring m 1 -plants was higher than that of the neighboring m 0 -plants regardless of the al3+ concentration in the ehc, even though it was only significant at h 2 (table 2). discussion the results of this study showed that the toxicity of high al3+ concentrations in soil solution to the external hyphae of g. margarita was evident. the growth of the hyphae was depressed particularly when neighboring plants were absent. this supports and extends previous results of work in vitro indicating the inhibitory effects of al3+ on spore germination, germ tube elongation (siqueira et al. 1985), and root colonization of the fungus (rohyadi 2005). on the other hand, in pots with neighboring plants the growth of the external fungal hyphae considerably increased (fig 2), which corresponds with the decrease in percentage of the mycorrhizal colonization on roots of their host plants (table 1). this suggests the importance of the presence of neighboring plants for the growth of external hyphae of the fungus. previously, mummey et al. (2005) showed the table 1 growth and am fungal colonization on roots of host plants in the hpc in response to mycorrhizal inoculation, al3+ concentration in the ehc and the presence of neighboring plants in the npc n o t inoculated inoculated 1 . 1 4 . 1 7 . 3 1 . 1 4 . 1 7 . 3 n 0 n 1 n 0 n 1 n 0 n 1 n 0 n 1 n 0 n 1 n 0 n 1 treatments 245 a 441b 272 a 460b 260 a 411b 248 a 419b 249 a 361b 235 a 392b 311 a 829 a 327 a 815 a 341 a 792 a 345 a 814 a 361 a 797 a 325 a 745 a plant dry biomass (mg) 627 b 1202 b 706ab 1107b 682 b 1072 b 647 b 1049 b 657 b 1026 b 639 b 1035 b 876a 1420a 849 a 1488 a 872 a 1535 a 829 a 1483 a 882 a 1496 a 791 a 1458 a root length(cm) 0 0 0 0 0 0 0 0 0 0 0 0 42a 77a 46a 57b 50a 76a 42a 56b 48a 73a 42a 51b root colonization (%) mycorrhizal root length (cm) 0 0 0 0 0 0 0 0 0 0 0 0 368a b 1093 a 391a b 848 b 436 a 1167 a 349 b 830 b 423 a 1092 a 332 b 744 c neighboring plants in the npc mg al3+ kg-1 soil in the ehc mycorrhizal inoculation in the hpc ameans within a selected column followed by different superscripts are significantly different based on the lsd-test at p ≤ 0.05. hpc: host plant compartment; ehc: external hyphal compartment; npc: neighboring plant compartment.h 1 and h 2 : harvest at 6 and 10 weeks after transplanting. h 1 h 2 h 1 h 2 h 1 h 2 h 1 h 2 table 2 dry biomass and root colonization of the neighboring plants relating to mycorrhizal inoculation in the hpc and al 3+ concentrations in the ehc observed at two harvest times treatments mycorrhizal inoculation in the hpc not inoculated mg al3+ kg-1 soil in the ehc 210 a 382 b 210 a 399 b 195 a 397 b 255 a 509 a 247 a 532 a 242 a 589 a 0 0 0 0 0 0 18a 59a 17a 48b 8b 39bc root colonization (%) plant dry biomass (mg) 1 . 1 4 . 1 7 . 3 1 . 1 4 . 1 7 . 3 inoculated h 1 h 2 h 1 h 2 ameans within a selected column followed by different superscripts are significantly different based on the lsd-test at p < 0.05; hpc: host plant compartment; ehc: external hyphal compartment; h 1 and h 2 : harvest at 6 and 10 weeks after transplanting. h y p h a l le n g th ( c m g -1 s o il ) 1 2 0 0 9 0 0 6 0 0 3 0 0 0 n 0 n 1 harvest-1 harvest-2 n 0 n 1 fig 2 the influence of al3+ concentrations and neighboring plants on hyphal length density of gigaspora margarita in the external hyphal compartment (bars represent means ± sem, n=3); o, a 1 ; n, a 2 , and , a 3 are growth media with 1.1, 4.1 and 7.3 mg al3+ kg-1 soil; n 1 and n 0 are pots with and with no neighboring plants. microbiol indones 45volume 3, 2009 significance of neighboring plants on the diversity and community composition of the am fungi in the field. most studies on the interaction of am fungi and plant roots have been carried out using in vitro systems. the significant effects of the presence of plant roots on hyphal growth have been demonstrated. sbrana and giovannetti (2005) showed a chemotropism in elongation of germ tube of glomus mossea in response to the presence of plant roots. it seems that the plant roots produced influential stimuli for the growth of the am fungal hyphae. root exudates, in this case, play a key role during the pre-symbiotic phase. for instance, it was reported that hyphal growth of g. margarita was stimulated by root exudates of onion (tawaraya et al. 1996) and tomato (scervino et al. 2005). also, a component of root exudates of lotus japonicum, identified as strigolactone, induced an extensive branching of the fungal hyphae at very low concentration (akiyama et al. 2005). in general, some phenolic and flavonoid compounds, and/or volatile metabolite exuded by roots of mycotrophic plants have been recognized as chemical stimuli for hyphal elongation and branching (nagahashi and douds 2003; akiyama et al 2005), or as a chemical signal leading the hyphae to grow forward (vierheilig et al. 1998; steinkellner et al. 2007). therefore, in the present study in situ it was possible that some eliciting factors produced by roots of the neighboring plants have triggered most of the external hyphae of the fungus to grow out of the hpc, and so it left a small number of the fungal inoculums to initiate new root colonization in the plant compartment. moreover, in this study it was found that colonization of the roots of neighboring plants by the fungal hyphae traversing the ehc reached about 40% (table 2), suggesting that exposure to increasing al3+ concentrations did not really affect the function of the external hyphae of g. margarita as inoculums. the growing hyphae remained viable to initiate new colonization. this is very important for field practice since spores begin most colonization by this fungus. other results on the increased growth of neighboring plants (table 2) also indicate that the fungal hyphae did not suffer from the loss of their ability to improve the growth of cowpea plant after being exposed to high concentrations of al3+, even though there was a delay in seeing a significant contribution to the plants. to summarize, the results of this study have broadened our understanding on eco-physiological aspects of am symbiosis in acid soils particularly with problems of al toxicity. it is clear that increasing concentrations of solubleal might directly reduce the growth rate of the external hyphae of g. margarita in soil, but functions of the hyphae as inoculums and as an extension of the plant root system were not really affected. meanwhile, the presence of the roots of neighboring plants stimulated the external hyphae to grow further, irrespective of the toxic al3+ in soil. these results, in general, suggest that plants need more am symbioses to grow better particularly under adverse soil conditions, which is comparable to the requirement of the am fungi themselves to get carbon from the plants. therefore, from this point of view, developing a suitable cropping system is essential for empowering am fungi to be persistent and be able to continually develop their external hyphae. further studies however are needed to investigate deep mechanisms and other soil factors involved in triggering the growth of the fungal hyphae under regimes of different mycotrophic crop species, and of adverse soil conditions. acknowledgements i wish thank sally s smith, f andrew smith, and rob s murray (the university of adelaide, waite campus, the south australia) for their help in many ways thoughout this work. i also thank ausaid scholarship, australia for the financial support. references akiyama k, matsuzaki k, hayashi h. 2005. plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. nature 435:824-7. baligar vc, anghinoni l, pitta gve, dos santos hl, filho ec, schaffert re. 1995. aluminium effects on plant and nutrient uptake parameters of soil and solution grown sorghum genotypes. j plant nut 18:2325-38. close ea, powell hkj. 1989. rapidly extracted (0.02 m cacl 2 -soluble) ‘reactive’ aluminium as a measure of aluminium toxicity in soils. aust j soil res 27:663-72. giovannetti m, mosse b. 1980. an evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. new phytol 84:489-500. jakobsen i, abbott lk, robson ad, 1992. external hyphae of vesiculararbuscular mycorrhizal fungi associated with trifolium subterranean l. 1. spread of hyphae and phosphorus inflow into roots. new pythol 120:371-80 miyasaka sc, habte m. 2001. plant mechanisms and mycorrhizal symbioses to increase phosphorus uptake efficiency. commun soil sci plant anal 32:1101-47. mummey dl. rillig mc, holben we. 2005. neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by t-rflp analysis. plant soil 271:839 0 . nagahashi g, douds dd. 2000. partial separation of root exudate components and their effects upon the growth of germinated spores of am fungi. mycol res 104:1453-64. nagahashi g, douds dd. 2003. action spectrum for the induction of hyphal branches of an arbuscular mycorrhizal fungus: exposure sites and branching sites. mycol res 107:1075-82. rohyadi a. 2005. spore germination and colonization of gigaspora margarita as influenced by aluminium concentration. j mikrobiol indones 10:71-4. rohyadi a. 2006. elevated aluminium concentrations in soil reduce growth and function of external hyphae of gigaspora margarita in growth of cowpea plants. bionatura 8:47-59. rohyadi a, smith fa, murray rs, smith se. 2004. effect of ph on mycorrhizal colonisation and nutrient uptake in cowpea under conditions that minimise confounding effects of elevated available aluminium. plant soil 260:283-90. sbrana cm, giovannetti m. 2005. chemotropism in the arbuscular mycorrhizal fungus glomus mosseae. mycorrhiza 15:539-45. scervino jm, ponce ma, erra-bassels r, vierheilig h, ocampo ja, godeas a. 2005. arbuscular mycorrhizal colonization of tomato by gigaspora and glomus species in presence of roots flavonoids. j plant physiol 162:625-33. siqueira jo, sylvia dm, gibson j, hubbell dh. 1985. spores, germination, and germ tubes of vesicular-arbuscular mycorrhizal fungi. can j microbiol 31:965-71. smith fa, jakobsen i, smith se. 2000. spatial differences in acquisition of soil phosphate between two arbuscular mycorrhizal fungi in symbiosis with medicago truncatula. new phytol 147:357-66. steinkellner s, lendzemo v, langer i, schweiger p, khaosaad t, toussaint jp, vierheilig h. 2007. flavonoids and strigolactones in root exudates as signals in symbiotic and pathogenic plant-fungus interactions. molecules 12:1290-306. tawaraya k, watanabe s, yoshida e, wagatsuma t. 1996. effect of onion (allium cepa) root exudates on the hyphal growth of gigaspora margarita. mycorrhiza 6:57-9. vierheilig h, alt-hug m, engel-streitwolf r, mäder p, wiemken a. 1998. studies on the attractional effect of root exudates on hyphal growth of an arbuscular mycorrhizal fungus in a soil compartmentmembrane system. plant soil 203:137-44. wood m. 1995. a mechanism of aluminium toxicity to soil bacteria and possible ecological implications. plant soil 171:63-9. xavier ljc, germida jj. 2003. bacteria associated with glomus clarum spores influence mycorrhizal activity. soil biol biochem 35:471-8. microbiol indones46 rohyadi 2 aris.pmd review prospective use of 1-aminocyclopropane-1-carboxylate deaminase-producing bacteria for plant growth promotion and defense against biotic and abiotic stresses in peat-soil-agriculture edi husen1, aris tri wahyudi1*, antonius suwanto1 and rasti saraswati2 1department of biology, institut pertanian bogor, darmaga kampus, bogor 16680, indonesia; 2indonesian soil research institute, jalan ir. h. juanda 98, bogor 16123, indonesia the 1-aminocyclopropane-1-carboxylate (acc) deaminase (ec4.1.99.4) is an enzyme produced by some soil bacteria to degrade acc (the immediate precursor of ethylene) to reduce ethylene biosynthesis in higher plants. increased concentrations of ethylene in plant tissues, which are triggered by various biotic and abiotic stresses, inhibits plant growth and weakens the plant defense against the stressors. various findings on the successful use of acc deaminase producing bacteria for plant growth under unfavorable soil conditions are inspiring their use in tropical peat-soil-agriculture, which possesses bio-physical constraints. it has been proven that inoculation of plants with acc deaminase producing bacteria decreased ethylene inhibition generated by unfavorable environmental conditions, such as nutrient shortage, flooding, drought, high salts, and the presence of heavy metals and organic pollutants. understanding the mechanisms by which acc deaminase-producing bacteria act to reduce plant stress and the fitness of bacterial traits with the properties and constraints of peat-soils becomes a key to utilize these bacteria in improving crop productivity. the bacteria may ameliorate plant stress as well as promote plant growth under seasonal bio-physical changes of peat-soils that are usually encountered in the field. key words: acc deaminase, bacteria, plant growth promotion, biotic and abiotic stresses, peat-soil _____________________________________________ ________________________ * corresponding author, phone/fax: +62-251-8622833, e-mail: atriw@ipb.ac.id volume 2, number 3, december 2008 issn 1978-3477 p 107-111 peat-soils are important in the world to support human welfare, both locally and globally. they cover about 400 million ha area in the world (malmer et al. 1994; rubec 1996), 40 million ha of which are found in tropical regions and half of which are located in indonesia (driessen and subagjo 1977; widjaya-adhi et al. 1997). despite their potential use for agriculture, especially those of shallow peat-soils (widjaya-adhi et al. 1997), bio-physical constraints of these soils limit their capacity to produce optimal yields. besides high acidity (ph 3.5 to 5.0), most tropical peat-soils are deficient in macroand micro-nutrients (driessen and subagjo 1977; van breemen 1995; widjaya-adhi et al. 1997). incomplete decomposition of organic matter during peat formation also results in the accumulation of various phytotoxins, which inhibit plant growth (salampak et al. 2000; hartatik and suriadikarta 2003). water-logged conditions coupled with bio-physical constraints of peat-soils are considered both conducive and suppressive to soil-borne pathogens (hoitink and boehm 1999; hunter et al. 2006). thus, plants growing in these harsh soil conditions may be confronted with various stresses, unless a promising technology to reduce plant stress is applied. the 1-aminocyclopropane-1-carboxylate (acc) deaminase (ec4.1.99.4) is an enzyme produced by some soil borne bacteria to hydrolyze acc, the immediate precursor of ethylene in higher plants, as their source of nitrogen (jacobson et al. 1994; glick 1995). the benefits of acc deaminase-producing bacteria to diverse aspects of plant growth have been reported in various environmental conditions. these bacteria can reduce plant stress by controlling ethylene synthesis in plant tissues, which can be induced by various unfavorable environmental conditions including pathogens attack. although this plant hormone ethylene has profound effects on plant growth and physiology, increased concentration of ethylene at an early stage of plant growth inhibits root development and weakens plant defense against various stressors (glick 1995; shah et al. 1997; glick et al. 2007). a study by wang et al. (2000), using acc deaminase-producing pseudomonas sp. and enterobacter sp., demonstrated the effectiveness of these bacteria in enhancing growth and suppressing damping-off of cucumber root-rot diseases in tomato and potato. subsequent studies revealed that these bacteria helped plants cope against various environmental constraints, such as heavy metals (belimov et al. 2001), flooding (grichko and glick 2001), nutritional stress (belimov et al. 2002), drought (mayak et al. 2004), organic pollutants (reed and glick 2005) and high salts (saravanakumar and samiyappan 2007), which are present in most tropical peat-soils. many more acc deaminase-producing bacteria are still being studied to increase plant growth by using known strains or local isolates obtained from local soils. our preliminary studies on some pseudomonas sp. producing acc deaminase isolated from the rhizosphere of soybean showed significantly increased soybean root-growth upon inoculation (husen et al. 2008), so potentially this can be used as a plant helper in unfavorable soil conditions. based on the ability of acc deaminase-producing bacteria to promote plant growth and provide defense against various environmental stresses, introducing these bacteria in unfavorable tropical peat-soil-agriculture may become an attractive solution to increase crop productivity. peat-soils, characteristics and constraints peat-soils are characterized by the accumulation of organic matter from dead and partially decaying plant materials under water-saturated conditions in wetland ecosystems. they are classified as histosols and mainly divided into fibric, hemic and sapric, based on the degree of organic material decomposition, i.e. partially, intermediately and highly decomposed materials, respectively (mckenzie 1974). the thickness of peat-soils varies from shallow (50 to 100 cm) to very deep (> 300 cm). approximately 7.5 million ha of peat-soils in sumatra and kalimantan islands are suitable for agriculture (food and tree crops); especially those of < 200 cm deep (widjaya-adhi et al. 1997) peat-soils develop either from bogs which are rain-fed and nutrient-poor or fens which are fed by surface or ground water and more nutrients-rich (malmer et al. 1994). tropical peat-soils developed from ombrogenous bogs are found in shallow depressions in the natural basins between levees (banks) of rivers traversing the coastal plain (driessen and subagjo 1977). since water, organic matter and its vegetation are all interconnected, the variations of peat-soil properties are mainly determined by these three components. therefore, a change of any one of these components will fundamentally alter the nature of peat-soils (rubec 1996); such that they are considered as fragile soil ecosystems. ample reports on the constraints of peat-soils for agriculture are well documented. the extreme conditions of tropical peat-soils which prevent normal plant growth include: (i) anaerobic conditions and the presence of toxic ions in the root zone, such as fe2+, mn2+, s2(ponnamperuma 1972; driessen and subagjo 1977); (ii) water-logging or high water levels that deprive oxygen from perennial plants (van breemen 1995); (iii) spongy soil structure that weakens rooting so trees easily fall down (malmer et al. 1994); (iv) scarcity of nutrients as a result of peat accumulation (by which nutrients are fixed or chelated) and leaching by peat water (driessen and subagjo 1977; widjaya-adhi et al. 1997); (v) acidity (ph 3.5 to 5.0) caused by organic acids and cation exchange (van breemen 1995; widjaya-adhi et al. 1997); and (vi) the presence of toxic organic substances, such as phenolic acids, produced during anaerobic decomposition of organic materials (salampak et al. 2000). moreover, peatsoils are conducive to the development of soil-bornepathogen diseases, such as pythium sylvaticum-induced damping-off although some of them suppress disease (hoitink and boehm 1999; hunter et al. 2006). these conditions may inhibit plant growth and prevent the proliferation of certain beneficial microbes, unless both plants and microbes develop several strategies to survive under these unfavorable or stress conditions. feng et al. (2002) reported that some root-nodule-forming bacteria developed thickened cell walls as a mechanism for long-term survival under nutrient-limited conditions of peatsoils. involvement of certain bacteria to detoxify or transform phytotoxins generated during peat formation, which benefits both plants and microbes, has been reported (huang and kuhlman 1991; blum et al. 1999). however, since peat-soils possess various complex biophysical constraints, various levels of plant stresses under these harsh soil conditions may not be difficult to understand. most crop yields produced in peat-soils are less than those in mineral soils. the yields of food crops in peat-soils range from 2.7 to 4.1; 0.8 to 1.0; and 1.5 to 2.4 ton ha-1 for paddy field and corn; soybean and mung bean, respectively (widjaya-adhi et al. 1997). therefore, reducing plant stress would potentially increase crop yields. acc deaminase and plant-growth-promotion the acc deaminase is a cytoplasmically localed enzyme (jacobson et al. 1994) produced by soil microorganisms. it degrades cyclopropanoid amino acc (the immediate precursor of the plant growth regulator ethylene) to form ammonia and α-ketobutyrate. after the first finding of acc deaminase from pseudomonas sp. strain acp (honma and shimomura 1978), various pioneering research work conducted, such as in jacobson et al. (1994), glick (1995) and penrose et al. (2001), have successfully elaborated the biochemical properties of this enzyme and its functional roles in controlling ethylene production in higher plants. in general, the plant hormone ethylene plays important diverse roles in the growth of plants, such as root initiation and fruit ripening (burg and burg 1962; arshad and frankenberger 1991; kende 1993). as a stress hormone, ethylene is involved in various stress responses induced by biotic (pathogenic attack) and abiotic (environmental) factors. its biosynthesis starts with the s-adenosylation of methionine to s-adenosylmethionine (sam) followed by the closing of a cyclopropane ring to form acc. the acc is then oxidatively cleaved to form ethylene. in spite of its beneficial roles in root induction, increased concentrations of plant ethylene at the vegetative stage inhibits root development, nodule formation and auxin transport and promotes senescence and abscission (glick 1995; shah et al. 1997; wang et al. 1997; ma et al. 2002; glick et al. 2007). the antagonistic function of ethylene with iaa prevents the overgrowth (gigantism) of plants. iaa promotes rooting, but rooting is opposed by ethylene generated by iaa; thus, the promotion effects of iaa on root development can be outweighed by the inhibitory effect of ethylene (burg 1968; chadwick and burg 1970; arshad and frankenberger 1991). the complex role of ethylene in plant-microbe interactions elicits ideas to regulate it for various purposes; it is required by both plants for resistance to pathogenic attack and by pathogens for disease susceptibility (kunkel and brooks 2002). the importance of acc deaminase-producing bacteria on plant growth is to control ethylene biosynthesis in plant tissues which is triggered by a number of biotic and abiotic factors. inoculation of plant with acc deaminase-producing bacteria can reduce the inhibition-effects of ethylene, and so ultimately increase plant growth. evidence that acc deaminase-producing bacteria increase the growth of various agricultural crops has been reported by various researchers, including those using transformed bacterial strains receiving the acc deaminase gene (acds gene) as described by wang 108 review microbiol indones fig 1 schematic representation by which acc deaminase-producing bacteria attached to plant roots lower ethylene biosynthesis. a key enzyme acc synthase that converts adomet (s-adenosylmethionine) to acc is induced by various biotic and abiotic factors including increased iaa concentrations. bacterial uptake and hydrolysis of acc exuded by plant roots (to maintain the equilibrium between internal and external acc) prevents ethylene accumulation which inhibits plant growth. iaa, indoleacetic acid; acc, 1-aminocyclopropane-1-carboxylic acid; adomet, s-adenosylmethionine. iaa-producing microbes promotes cell proliferation and root development iaa acc synthase adomet acc oxidase ethylene acc acc acc deaminase ammonia and α-ketobutyrate wounding pathogen attacks inhibits root development, speeds aging, promotes senescence, and abscission plant root environmental (abiotic) stresses caused by: -flooding -drought -nutrient shortage -high salts -heavy metals -organic pollutants acc deaminase-producing bacteria et al. (2000). interestingly, some root-nodule-forming bacteria may produce acc-deaminase besides producing rhizobitoxine (an ethylene inhibitor) as a strategy by these bacteria to reduce the amount of ethylene synthesized in soybeans (ma et al. 2002; 2003), otherwise the nodulation process would itself be inhibited. however, since acc deaminase is a cytoplasmic enzyme in bacteria, the substrate of acc must be exuded by plant tissues and taken up by the bacteria in the rhizosphere and subsequently hydrolyzed into ammonia and α-ketobutyrate (jacobson et al. 1994; shah et al. 1997; glick et al. 1998). thus, the success of these bacteria to promote and increase plant growth will depend much on their ability to colonize the root and compete with other soil microflora. amelioration of plant stress plants grown in the tropical peat-soils may encounter various biotic and abiotic stresses as explained above. in general, biotic stresses could be due to pathogenic attack, either a biotroph which colonizes living plant tissues or necrotroph which rapidly kill plant cells to obtain nutrients. meanwhile, abiotic or environmental stresses could be due to soil acidity, water-logging (osmotic pressure), nutrient shortage and phytotoxins that are generally present in tropical peat-soils. both kinds of stressors may trigger plants to produce stress hormones as a signal to reduce the severity of the stress (wang et al. 2000; glick et al. 2007). it has been known that several plant pathogenic microbes have developed the ability to modulate signaling processes mediated by plant hormones as a strategy for manipulating plant growth or host physiology (lund et al. 1998). three kinds of plant hormones, which are important in mediating plant defenses against pathogen attack, are salicylic acid (sa), jasmonic acid (ja) and ethylene (kunkel and brooks 2002; glazebrook 2005). ethylene signaling, however, plays a pivotal role in various types of stresses including those caused by various environmental factors (glick et al. 2007). taken together, plants with a particular stress will accumulate high levels of ethylene that make them in a state of ethylene-stress and further inhibit their growth. increasing levels of ethylene eventually exacerbates the stress which weakens plant protection against the stressors. in this condition, plants may not respond well to the availability of nutrients and growth stimulators. in the presence of acc deaminase-producing bacteria residing in the root surface area, these bacteria may reduce ethylene biosynthesis in the plants by hydrolyzing acc exuded by the root and taken up by the bacteria as their source of nitrogen. thus, lowering ethylene levels in plants through the action of acc deaminase-producing bacteria will protect plants against the inhibitory effects of ethylene generated by the stressors. fig 1 shows a schematic representation developed from various sources of how acc deaminase-producing bacteria hydrolyze acc, and thus lower ethylene biosynthesis in plant roots. prospective use of acc deaminase-producing bacteria the successful use of acc deaminase-producing bacteria to assist plant growth under unfavorable soil conditions provides farmers an attractive method of volume 2, 2008 microbiol indones 109 increasing crop production. their ability to promote plant growth as well as to protect the plant against biotic and abiotic stresses may reduce the level of application of fertilizers and pesticides, which currently become more expensive and also alleviate environmental problems. the challenge that remains ahead is to obtain a good candidate of acc deaminase-producing bacteria which can live and proliferate in peat-soils and which are effective to assist plant defense against a wide range of stressors. since none of the studies on the use of acc deaminase-producing bacteria has been specifically conducted yet in tropical peat-soil conditions which posses a complex of constraints, a complete screening of 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disease-suppressive capacities. can j microbiol 46:898-907. widjaya-adhi ipg. 1997. developing tropical peatlands for agriculture. in: proceedings of international symposium on biodiversity, environmental importance and sustainability of tropical peat and peatlends. palangka raya, indonesia, sep 4-8, 1995. p 293-300. volume 2, 2008 microbiol indones 111 05. kiranasari.cdr vol.15, no.3, september 2021, p 113-117 doi: 10.5454/mi.15.3.5 comparison of n-acetyl-l-cysteine-sodium hydroxide-based and modified petroff's decontamination method for mycobacterium tuberculosis culture ariyani kiranasari muhammad rayhan 1* 2 and 1microbiology department, faculty of medicine, universitas indonesia jalan pengangsaan timur no.16, jakarta pusat, 10320, indonesia; 2 medical science, faculty of medicine, universitas indonesia. indonesia is one of 22 countries with a high incidence of tuberculosis in the world, particularly related to tbhiv and mdr-tb cases. contamination of normal flora from nasopharyngeal tract is the main problem to isolate mycobacterium tuberculosis (mtb) from sputum. therefore, a safe solution to decontaminate sputum without killing mtb bacilli is needed. n-acetyl-l-cysteine-sodium hydroxide (nalc-naoh)-based method and petroff's methods which were modified with naoh (4%) are widely used in laboratories. in the present study, we will evaluate the different of these methods. of the 110 sputum samples were collected from suspected cases of pulmonary tb, and the decontamination of sputum by these methods was performed, after acid-fast bacillus (afb) smear, then the samples were cultured in lowensteins jensen slant medium. the positive culture was validated by chromatography test for detecting the antigen of mpt-64 and pnb. based on the investigation, it has been shown that neither nalc-naoh (71%) nor modified petroff's methods (66%) had a significant effect on the positivity rate of afb smear. however, the contamination on culture was significantly higher in samples treated with nalc-naoh (21%) compared to modified petroff methods (13%) (p< 0.05). in addition, the proportion of positive culture in nalc-naoh was lower than modified petroff. in conclusion, our study proved that modified petroff methods are still more effective on sputum decontamination than nalc-naoh based on the positivity rate of mtb culture. though, not significantly different on afb microscopic examination. petroff's methods m nalc-naohkey words: decontamination, , tb, modified indonesia merupakan salah satu dari 22 negara dengan insiden tb tertinggi di dunia, khususnya yang berkaitan dengan kasus tb-hiv dan mdr-tb. kontaminasi flora normal dari saluran nasofaring merupakan masalah utama mengisolasi (mtb) dari sputum. iperlukan dalam mycobacterium tuberculosis oleh sebab itu d solusi yang aman untuk dekontaminasi sputum tanpa membunuh mtb. melakukan sampel bakteri untuk memberikan hasil yang lebih baik dekontaminan yang sering digunakan adalah metode berbasis n-asetil-lsistein-natrium hidroksida (nalc-naoh) dibandingkan dengan dari metode petroff yang dimodifikasi dengan naoh (4%) sampel sputum yang digunakan untuk pengujian berasal dari 110 kasus suspek tb paru dan . dilakukan dekontaminasi menggunakan nalc-naoh 2% atau (4% naoh) yang sebelumnya sudah dilakukan pemeriksaan bta. sputum dibiakkan media lowensteins jensen. setiap kelompok selanjutnya, pada tersebut dinilai tingkat kontaminasi dan kultur positif. kultur positif divalidasi menggunakan uji berdasarkan hasil kromatografi untuk mendeteks antigen mpt-64 dan pnb. hasil penelitian menunjukkan bahwa nalc-i adanya naoh dan metode petroff termodifikasi tidak berpengaruh nyata terhadap laju kepositifan bta, dengan nilai masing-masing sebesar 71% dan 66%. kontaminasi pada kultur secara signifikan (p=0,034) lebih tinggi pada sampel yang diberi nalc-naoh (21%) dibandingkan dengan metode modified petroff (13%). proporsi kultur positif pada sampel yang diberi perlakuan nalc-naoh lebih rendah dari modified petroff, memberikan hasil masing-masing 65% dan 70% dengan nilai p=1. kesimpulan dari penelitian ini menunjukkan bahwa dekontaminasi sputum dengan metode modified petroff masih lebih efektif dibandingkan dengan nalc-naoh untuk meningkatkan laju kepositifan kultur mtb. namun kedua metode tersebut tidak berbeda nyata untuk mendapatkan hasil positif pada pemeriksaan mikroskopis bta. kata kunci: dekontaminasi metode , m , nalc-naoh petroff's, tbmodified microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 62-21 3160491;: + e-mail: gahariyani@gmail.com (afb) is rapid, inexpensive, highly specific, and capable of identifying the most infectious cases of tb. the only disadvantage of this method is low sensitivity (varying from 50 to 80%) relative to culture (forbes et al. 2010). the gold standard for diagnosing pulmonary tb remains in culturing. decontamination of clinical specimens such as sputum is an important and critical step in the isolation of mycobacteria, to obtain better tuberculosis (tb) is one of the deadliest infectious disease which caused by .mycobacterium tuberculosis tb is a major air-borne disease in human. it remains a major worldwide health problem with global mortality ranging from 1.6 to 2.2 million lives per year (who 2016). direct smear microscopy for acid fast bacilli 114 kiranasari et al . microbiol indones results. however, bacterial and fungal contamination of the culture frequently may interfere with the interpretation data result. this condition can result the need to repeat the culture and lead to reduce the effectiveness of culture as a method of diagnosis of tuberculosis, because it will take more time for a colony to appear (bruchfeld . 2000; sjahrurachman . et al et al 2012; korean cdc 2014; gopi 2018). et al. contamination of culture and its harmful effect can be prevented by decontamination of sputum sample. decontamination and concentration of sputum samples by modified petroff's method is one of the most commonly used methods for culture. m. tuberculosis however, n-acetyl-l-cysteine-sodium hydroxide (nalc naoh) solution may recommended as a gentle but effective digesting and decontaminating agent. addition of a large volume of phosphate buffer (pb) with ph 6.8 makes strong shift in ph, washes the specimen, dilutes toxic substances and decreases the specific gravity of the specimen so that centrifugation is more effective. therefore, it is generally accepted that the nalc-naoh method (kent and kubica 1985) should be given preference over the modified petroff's methods. but, the technical and procedural factors may influence the sensitivity of each method (peres . et al 2009). tudies in different laboratory may give different s results since the result of decontamination may be influenced by various technical factors (forbes . et al 2010; peres . 2009; sjahrurachman . 2012).the et al et al aim of this study is to compare the nacl-naoh-based and modified petroff methods with 4% naoh to obtain better result on microscopic examination of afb and mtb culture . materials and methods the cross-sectional study was sputum samples. c o n d u c t e d i n t h e l a b o r a t o r yt u b e r c u l o s i s , microbiology departement, faculty of medicine, universitas indonesia from de ember 2016 jul , c to y 2017 one hundred ten . sputum samples were isolated from . suspected case of pulmonary tuberculosis the s then be decontaminated with nalc-naoh-amples based method or modified petroff's methods. mic r os c o pi c mi c ro sc opic ex am inat io n. examination was performed before and after decontamination. afb smear was conducted using ziehl-neelsen technique recommended by world health organization (who 2016; tripathi . 2014; et al burdz 2003et al. ). specimens for digestion and decontamination were mixed well using vortex and equally divided into two parts, and each treated by nalc naoh and modified petroff's method before inoculated it directly on lowenstein-jensen (lj) medium (peres et al. 2009; tripathi . 2014).et al nalc-naoh-based me thod. nalc-2% sodium hydroxide-sodium citrate solution was prepared as described by kent and kubica (tripathi et al. 2014). an equal volume of nalc-naoh citrate reagent was added into 3-5 ml of seeded sputum sample, and vortexed briefly for ec. the samples 30 s then were incubated for 15 min at room temperature and added by 0.067 m of phosphate buffer (ph 6.8) until the volume reach 50 ml. after homogenization, the sputum samples were pelleted by centrifugate it at 3,000g for 15 min. the supernatant was discarded, -20 and the pellet was resuspended with 1 ml of pb . the s smear was made and a 0.5 ml of cell suspension was inoculated on lj slopes. the culture slants were incubated at 37 c.35o modified petroff's method. in brief, 3-5 ml of sputum was homogenized for 15 min in a shaker using an equal volume of 4% naoh. after centrifugation at 3 000 for 15 min, the were neutralized with . g -20 seeds fig 1 microscopic examination of acid-fast bacilli with ziehl neelsen staining. 20 ml of sterile distilled water . or aqua pro injection the samples were again centrifuged at 3 000 for 15 . g 20 min. from the sediment, lj medium was inoculated and smear was made. the culture slants were incubated at 3 7 c (pathak . 1973; ; 5-3 o et al tripathi . 2014et al costa 2018 . all slopes were observed daily for et al. ) first week and weekly for 8 weeks. some parameters such as, growth rate, optimum temperature, colony morphology and pigmentation were also observed as growth parameters. the chromatography test was using to detect the of 4 and tests. antigen mpt-6 pnb the absence of growth at the end of 8 weeks was th regarded as negative culture. contamination, if any, was recorded separately. the number of culture failures for a certain decontamination method, included the number of specimens with negative culture as well as number of contaminated cultures. the data acquired from the study is processed using spss software version 20. bivariate analysis was done using chi-square test. result based on direct microscopy, out of samples,110 88 ( %) were smear positive and ( %) were 80 22 20 negative. the smear was carried out again after decontamination, and the result showed that 71 samples (71%) were positive by nalc-naoh and 73 samples ( %) were positive by modified petroff's 66 methods (table 1). the total number of culture failures (which includes both contamination and negative cultures) were (30%) in nalc-naoh as against ( %) in 33 38 35 modif ied petroff's methods (table 2) . the contamination rate was nalc naoh higher 23 ( %), whereas it was in modified petroff's 21 lowest methods 14 13 ( %) (table 2). a significant difference (p<0.05) in the proportion of contaminated and uncontaminated culture was shown between those treated with nalc-naoh and modified petroff. higher proportion of contaminated culture is observed from group treated with nalcnaoh in comparison to modified petroff (table 3). however, the proportions of uncontaminated culture with positive and negative results show no significant difference between group treated with nalc-naoh and modified petroff (table 4). discussion sputum culture is an important tool for tb method control programs it is more sensitive than because smear microscopy in diagnosing tb. the culture also f the but sinceacilitates drug susceptibility testing, sputum samples pass through the oropharynx tract during collection, culture contamination limits the diagnostic yield of sputum culture for tb (who 2016; forbes . 2010). et al decontamination process for removing bacteria and yeast in order to isolate in the mycobacteria sputum, unfortunately may also kill . the mycobacteria percentage of killed organisms will vary according to the method used and also the population of mycobacteria in the specimen. in this study the use of nalc-naoh for sputum digestion, as in kent and kubita method, will have final concentration about 2% naoh. such concentration will only give less destructive to compare to the used of 4% mycobacteria naoh in petroff's method (gli 2014; tripathi . et al 2014; burdz . 2003). et al in the analysis of 110 samples, the decontaminated by the nalc-naoh method provided isolation of m. tuberculosis in a percentage ( %) compared lowest 65 to modified petroff's method (7 %). this is in accordance 0 with the research of sharma . in 2012 showed that et al the proportion of positive culture results was greater in samples processed with nalc-naoh 63 7% than . samples processed with modified petroff 46 7%. . meanwhile, from the research of chatterjee . et al ( )2013 showed that the proportion of positive culture results was greater either in samples processed with nalc-naoh was 62.7% than samples processed with modified petroff 58.5% chaudhary ( 2013).et al. meanwhile, the research results of pathak sk . et al (1 )973 showed that the proportion of positive culture results was 79.4% for nalc-naoh. this figure is much greater than the proportion of positive culture results for nalc-naoh which was shown well by the study of sharma . 2012 and chatterjee . 2013et al et al . the probable reason are, 4% naoh is used s maybe for modified petroff's method as compared nalcnaoh method which uses 2% naoh while concentrating the sputum may kill or seriously injure few mycobacteria hence (korean cdc 2014). , recovery by nalc-naoh was faster and better than modified petroff's method. in our study we found smear positivity higher than the culture positivity. the reason might be that microscopy sometimes gives false positive results and in our condition, it cannot distinguish between dead and live bacteria. in such cases, the patients might be treated with antitubercular drugs and in the microscopy of these samples, the afb volume 15, 2021 microbiol indones 115 116 kiranasari et al . microbiol indones might be dead. for these reason, the dead isolates did not grow in the l-j culture media. this also reveals that afb microscopy does not always give accurate results for the diagnosis of tb sharma . 2012 ( ; gli 2014).et al the contamination rate by nalc-naoh method was % in our study which is than that reported 21 higher by other workers. sharma . (2012) 3.2%et al reported 1 while reported 4 chatterjee . (2013) .98%. several et al studies show the contamination r te by modified a petroff's was 13% in our study which is lower than that reported by other workers. sharma 2012 reported et al 23.1% while chaudary in 2013 reported 8% and et al. tripathik . in 2014 12%. our study showed, first et al the nalc-naoh method for afb smear and culture improves the sensitivity when compared to modified petroff's method. second, modified petroff method has lower contamination rate than nalc-naoh, and culture using lj medi nalc-naoh method is not um suitable for routine use. hird, t modified petroff's t he method for afb smear and culture improves the sensitivity when compared with nalc-naoh the method. in conclusion, the modified petroff is methods more effective than nalc-naoh to prevent contamination highly contaminated sputum. from however, the positivity result of microscopic examination of afb was not significantly different between this two methods. references bruchfeld j, aderaye g, palme ib, bjorvatn b, källenius g, lindquist l. 2000. sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of hiv. trans r soc trop med hyg. 94: 677-80. doi: 10.1016/s0035-9203(00)90230-x. burdz tvn, wolfe j, kabani a. 2003. evaluation of sputum decontamination methods for mycobacterium tuberculosis using viable colony counts and flow cytometry. diagn microbiol infect dis. 47: 503 9. doi: 10.1016/s0732-8893(03)00138-x. costa rr, silva sf, fochat rc, macedo rl, pereira tv, silva mr, pinto cp, leite ic. 2018. comparison between ogawa-kudoh and modified petroff techniques for mycobacteria cultivation in the diagnosis of pulmonary tuberculosis. einstein. são p a u l o . 1 6 ( 2 ) : 1 5 . d o i : 1 0 . 1 5 9 0 / s 1 6 7 9 45082018ao4214. table 1 percentage from microscopy using ziehl neelsen staining smear no. positive microscopy no. negative microscopy direct 88 (80%) 22 (20%) nalcnaoh 78 (71%) 32 (29%) modified petroff’’s 73 (66%) 37 (34%) table 2 comparison of the nalc-naoh and modified petroff's methods (in regards rate of contamination, negative cultures and culture failures) methods no of contaminated slopes negative cultures (no. of slopes with no growth up-to 8 weeks) total culture failures/ negative nalcnaoh 23 10 33 modified petroff’’s 14 24 38 table 3 chi-square test result showing a significantly higher proportion of contaminated culture in group treated with nalc-naoh acompared to the trated with modified petroff's decontamination method contamination n (%) uncontaminated n (%) p* nalcnaoh 23 (41.8%) 32 (58.2%) 0.034 modified petroff’’s 14 (25.5%) 41 (74.5%) *statistically significant for p < 0.05 table 4 chi-square test result showing no statistically significant difference in the proportion of positive and negative results in uncontaminated culture previously treated using nalc-naoh and modified petroff decontamination method negative culture n (%) positive culture n (%) p* nalcnaoh 38 (34.5%) 72 (65%) 1,000 modified petroff’’s 33 (30%) 77 (70%) *statistically significant for p < 0.05 volume 15, 2021 microbiol indones 117 chatterjee m, bhattarcharya s, karak k, dastidar sg. 2013. effects of different methods of decontamination for successful cultivation of . mycobacterium tuberculosis indian j med res. 138(4): 541 8. chaudhary sk, mishra b. 2013. comparison of hypertonic saline-sodium hydroxide method with modified petroff's method for the decontamination and concentration of sputum samples. int j infect microbiol. 2(3): 78 81. doi: 10.3126/ijim.v2i3.8664. forbes ba, sahm df, weissfeld ds. 2010. infection of lower respiratory tract: bailey and scott's diagnostic microbiology. mosby elsevier. usa. 12:805–6. gopi a, samreen f, madhulata c. 2018. a comparative study between microscopy and culture in detection of m.tb among smear negative pulmonary and extra p u l m o n a r y t u b e r cu l o s i s . i n d i a n j o u r n a l o f microbiology research, july-september, 5(3):313-317 doi:10.18231/2394-5478.2018.0066. gli, mycobacteriology laboratory manual 2014. . kent pt, kubica gp. 1985. public health microbacteriology: a guide for the level iii laboratory. centers for disease control, atlanta. korea centers for disease control and prevention. 2014. korean guidelines for tuberculosis. joint committee for the revision of korean guidelines for tuberculosis.seoul and cheongwon. : p. 2. doi: 10.4046/trd.2016.79.1.1 pathak sk, deshmukh pa, menon crn. 1973. a comparison of different culture techniques. ind j tuberc. 20: 85. peres rl, maciel el, morais cg, ribeiro fck, vinhas sa. 2009. comparison of two concentrations of nalcn a o h f o r d e co n t a m i n a t i o n o f sp ut um f o r mycobacterial culture. int j tuberc lung dis. 13(12): 1572 5. sharma m, misra rn, gandham nr, jadhav sv, angadi k, wilson v. 2012. comparison of modified petroff's and n-acetyl-l-cysteine-sodium hydroxide methods for sputum decontamination in tertiary care hospital in india. med j dr. d. y. patil university. 5(2): 97 – 100. doi: 10.4103/0975-2870.103323. sjahrurachman a, rintiswati n, gartinah t, solihin i, woro e, panjaitan r, et al. 2012. petunjuk teknis pemeriksaan biakan, identifikasi, dan uji kepekaan mycobacterium tuberculosis pada media padat. jakarta: kementrian kesehatan ri; 60 73. tripathi k, tripathi pc, nema s, shrivastava ak, dwidewi k, dhanvijay ak, et al. 2014. modified petroff's method: an excellent simplified decontamination technique in comparisoan with petroff's method. int j recent trends in science and technology. 10(3): 461 64. world health organization. global tuberculosis report. 2016. geneva: world health organization. p. 24. 5 puji rahayu-380.pmd volume 3, number1, april 2008 p 23-26 issn 1978-3477 *corresponding author:, phone: +62-21-3169503, fax: +62-21-3169510, e-mail: siswa59@yahoo.com production and purification of xylanase from indonesian isolate bacillus sp. aq-1 grown on bunch palm oil puji rahayu1, siswa setyahadi2*, and harmita1 1pharmacy department, faculty of mathematics and natural science, universitas indonesia, depok 16424, indonesia; 2center for the bioindustrial technology, badan pengkajian dan penerapan teknologi, bppt building 2, 15 floor, jalan mh. thamrin 8, jakarta 10340, indonesia xylanase (endoxylanase, ec 3.2.1.8) is a commercial enzyme that has been applied in the industrial production of fuel, food, textiles and paper. xylanase was isolated from the culture supernatant of bacillus sp. aq-1 grown on nakamura medium containing 0.5% powder bunch palm oil. the optimum ph and temperature of xylanase activity were ph 7.0 and 60 °c, respectively. the enzyme was purified by anion exchange chromatography using deae-sepharose-fast-flow column and gel filtration chromatography using sephacryl s-300 column. the results showed that purification of xylanase produced two forms of xylanase, which were identified as xylanase a and xylanase b. xylanase a can be separated from xylanase b by ultrafiltration using a 30 kda polyethersulfone membrane. the molecular weight of xylanase a and b were 15.7 and 57.7 kda, respectively. key words: xylanase, bacillus sp., purification currently, xylanolytic enzymes (endoxylanase, ec 3.2.1.8) have been the focus of much attention in industry and agriculture. these enzymes have many important applications in the saccharification of agricultural wastes (biely 1985), the improvement of bakery products (gilbert and hazlewood 1993), the food and feed industry (kulkarni et al. 1999) as well as the textile industry (querido et al. 2006). xylanase can also be converted into liquid fuel and solvents (woodward 1984; biely 1985; george et al. 2001). poutanen (1997) observed that both endogenous and added enzymes have an important effect on the quality of cereal foods. xylanase is also required for the degradation of xylan (sunna et al. 1997b). it is the second most abundant biopolymer (after cellulose) and the main component of hemicellulose, which is present in nature in large amounts and is a major by-product of the farming industry (simpson et al. 1991). the hydrolysis of xylan to xylo-oligosaccaride and xylose is catalyzed by xylanases (takahashi et al. 2000). xylanases are also applied for the bleaching of kraft pulp, increasing the brightness of the pulp in the pulp and paper industry (purkarthofer et al. 1993; viikari et al. 1994; kulkarni et al. 1999). in the process of pulp bleaching, the pulp is usually treated at high temperature at alkaline ph. therefore, thermostable xylanase would be preferred in biotechnological application, giving the advantage of running xylan digestion at high temperature. another problem in enzyme treatment in the pulp and paper industry is the availability and cost of the enzyme, due to the high cost of the substrate for enzyme production (hinnman 1994). therefore, the use of low cost substrates such as solid agricultural wastes is one of the ways to reduce production costs. bunch palm oil is one of the many solid agricultural waste materials that can be used as substrate in xylanase production. the purity of xylanase is also very important to ensure stability of enzyme in order to prevent enzyme protein denaturation. purified xylanases have possible applications in the chemical industry, nutritional industry, beverage industry, food industry and also in the production of alternative artificial sweetener that is low in calories (xylitol) (bhat 2000; goulart et al. 2005). aims of the present study were to produce and purify xylanase from bacilus sp. aq-1 using bunch palm oil as the substrate in the fermentation medium. materials and methods enzyme production. bacillus sp. aq-1 (culture collection of bioindustry laboratoy, bppt) was grown at 37 °c in luria broth medium containing 1% (w/v) bactopeptone, 0.5% (w/v) yeast extract and 0.5% (w/v) sodium chloride. the culture of bacillus sp. aq-1 was incubated at 30 °c for 24 h in the nakamura (1994) medium using powder bunch palm oil as the substrate. the substrate was prepared by sun drying for 2 days, and then was ground and sieved (35 mesh sieve). the composition of nakamura (1994) medium was as follows: 1% (w/v) bactopeptone, 0.5% (w/v) yeast extract, 0.1% (w/v) k 2 hpo 4 .3h 2 o, 0.02% (w/v) mgso 4 .7h 2 o and 0.5% (w/v) substrate (nakamura 1994). after 24 h, the cells were removed by centrifugation at 6 000 rpm using high-speed refrigerated centrifuge himac cr 21g and r10a3 rotor for 15 min at 4 °c and the supernantant from extracellular secretion was used for estimation of xylanase activity and protein content and also for xylanase purification. enzyme activity and protein concentration. xylanase activity was assayed by the dinitrosalicylic acid (dns) method, according to bailey et al. (1992). oat spelt xylan was dissolved in 50 mm sodium phosphate buffer (ph 7.0). the reaction mixture containing 50 µl of crude enzyme and 450 µl of 1% (w/v) xylan was incubated for 5 min in the thermomixer at the optimum temperature for enzyme activity. the reaction was terminated by the addition of 750 µl of dns reagent. the mixture was centrifuged at 14 000 rpm using high-speed refrigerated centrifuge himac cr 21g and r10a2 rotor for 2 min and the supernatant was boiled at 100 °c for 5 min. reducing sugars released during incubation were measured as xylose equivalents by absorbance (a) at 540 nm. one unit of xylanase activity is defined as the amount of enzyme required to liberate 1 µmol of xylose per minute at the assay condition (takahashi et al. 2000; tanaka et al. 2005). microbiol indones24 rahayu et al. protein concentration was determined by the folinphenol method, with bovine serum albumin as a standard (lowry et al. 1951). the protein content was measured at 750 nm. effect of temperature and ph on enzyme activity. xylanase activities were measured at temperatures ranging from 30-100 °c under the standard assay conditions. enzymes activities were also assayed at ph values from 4.0 to 9.0 (universal buffers) at the optimum temperature. enzyme purification. the cell-free supernantant of the culture was applied to the deae-sepharose-fast-flow column (20×200 mm). elution of the enzyme was carried out with 50 mm sodium phosphate buffer (ph 8.0) at a flow rate 1 ml min-1. each fraction was analyzed for xylanase activity and protein content. the active fractions were pooled and loaded onto sephacryl s-300 column (16×600 mm) at a flow rate 0.5 ml min-1. equilibration and elution were performed with 50 mm sodium phosphate buffer (ph 8.0). fractions were collected and analyzed for xylanase activity and protein content. the final steps of xylanase purification was carried out by ultrafiltration using a 30 kda polyethersulfone membrane. electrophoresis. the molecular weight of the purified xylanase was estimated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (sds-page). sds-page was performed in 12% (v/v) gels using low molecular weight markers (14.4-97.0 kda). a suspension of beechwood xylan at a final concentration 0.1% (w/v) was incorporated into the separating gel before addition of ammonium persulphate. proteins were visualized by silver staining with silver nitrate solution. zymogram analysis. the electrophoretic gels were washed in 2.5% (v/v) triton x-100 for 60 min and then incubated at 60 °c for 15 min in 50 mm sodium phosphate buffer (ph 7.0). the reaction was stopped by incubation at 4 °c for 15 min. the gel was then incubated in 0.1% (w/v) congo red solution for 20 min at room temperature with gentle shaking and destaining was accomplished by washing the gel with 1 m nacl until clear bands, indicating xylanase activity, became visible. the addition of 1 n hcl caused the background to turn dark blue, emphasizing the activity bands (sunna et al. 1997a). results enzyme production. microbial xylanases are usually inducible and secreted into culture medium containing xylan. the extracellular xylanase activity of bacillus sp. aq-1 grown on medium containing 0.5% (w/v) powder bunch palm oil in 24 h incubation was 65.90 u ml-1, while its protein content was 0.5 mg ml-1. xylanase activity was determined at optimum temperature and ph. effect of temperature and ph. fig 1 shows the effect of temperature and ph on xylanase activity. the optimum temperature of xylanase at ph 7.0 was observed at 60 °c (fig 1a), while the optimum ph for xylanase activity was observed at ph 7.0 (fig 1b). enzyme purification. purification was carried out at room temperature (26-28 °c). sodium phosphate buffer (50 mm, ph 8.0) was used throughout the purification procedure. the purification protocol involves two steps of column chromatography: ion exchange and gel filtration. filtrate from the culture was applied onto a deaesepharose-ff column (20×200 mm). xylanase was eluted from that column as a single protein peak that coincided with the peak of enzyme activity (fig 2). the active fractions of anion exchange chromatography (fractions 1-30) were further purified by gel-filtration-chromatography using sephacryl s-300 column (16×600 mm). fig 3 shows the xylanase activity and protein content from the gel filtration chromatogram. the active fractions from that column (fractions 185-360) were subjected to ultrafiltration. using this method, xylanase a had been separated from xylanase b. xylanase a was in the permeate solution, while xylanase b was in the retentate solution. table 1 summarizes the procedure for the purification of extracellular xylanase from bacillus sp. aq-1, indicating the degree of purification and yield for each step. electrophoresis. electrophoretic studies of crude extract and purified xylanase showed the presence of multiple bands (fig 4). a low molecular weight ranging from 14.4 to 97.0 kda was also observed. the molecular weight standards were α-lactalbumin (14.4 kda), trypsin inhibitor (20.1 kda), carbonic anhydrase (30 kda), ovalbumin (45 kda), albumin (66 kda) and phophorylase b (97 kda). zymogram analysis. the purity of xylanase was demonstrated by sds-page using 12% (v/v) gel containing 0.1% (w/v) beechwood xylan (fig 5). the permeate and retentate solution of ultrafiltration produced single activity band on each of the two gels. the molecular weight of these purified enzymes were estimated to be about 15.7 kda (xylanase a) and 57.7 kda (xylanase b) by standard curve of molecular weight. fig 1 influence of temperature (a) and ph (b) on the activity of xylanase from bacillus sp. aq-1. optimum activity temperature was measured at ph 7.0, while optimum activity ph was determined at the optimum temperature. buffers used for optimum ph determination were 50 mm sodium acetate (ph 4.0), 50 mm sodium citrate (ph 5.0), 50 mm sodium phosphate (ph 6.0-7.0) and 50 mm tris-hcl (ph 8.0-9.0) at final concentration. r e la ti v e e n z y m e a c ti v it y ( % ) 1 2 0 1 0 0 8 0 6 0 4 0 2 0 0 0 b p h 2 4 6 8 1 0 r e la ti v e e n z y m e a c ti v it y ( % ) 1 2 0 1 0 0 8 0 6 0 4 0 2 0 0 1 2 01 0 08 06 04 02 00 temperature (°c) a microbiol indones 25volume 3, 2009 fig 2 elution profile of enzyme activity (l) and protein content (�) of xylanase cell-free extract by anion exchange chromatography in deae-sepharose-ff column (20×200 mm). the column was eluted with 50 mm sodium phosphate buffer (ph 8.0). fig 3 elution profile of enzyme activity (l) and protein content (�) of pooled fractions from the first column by gel filtration chromatography in sephacryl s-300 column (16×600 mm). the column was eluted with 50 mm sodium phosphate buffer (ph 8.0). table 1 purification profile of xylanase from bacillus sp. aq-1 purification steps culture fluid deae sepharose ff sephacryl s-300 ultrafiltration permeate (xyl a) retentate (xyl b) a recoveries are expressed as percentage of initial activity. purification factors are calculated on the basis of specific activity 1 2 3 4kda 9 7 . 0 6 6 . 0 4 5 . 0 3 0 . 0 2 0 . 1 1 4 . 4 fig 4 sds-page, of crude xylanase. lane 1, protein standards; lane 2, xylanase crude extract; lane 3, permeate ultrafiltration; lane 4, retentate ultrafiltration. e n z y m e a c ti v it y ( u m l -1 ) 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 5 1 4 6 9 1 3 1 8 2 2 2 4 2 6 2 8 3 0 3 2 3 4 fractions p ro te in c o n te n t (m g m l -1 ) 0 . 5 0 0 . 4 5 0 . 4 0 0 . 3 5 0 . 3 0 0 . 2 5 0 . 2 0 0 . 1 5 0 . 1 0 0 . 0 5 0 . 0 0 -0.05 e n z y m e a c ti v it y ( u m l -1 ) 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 p ro te in c o n te n t (m g m l -1 ) 0 . 3 0 0 . 2 5 0 . 2 0 0 . 1 5 0 . 1 0 0 . 0 5 0 . 0 0 -0.05 volume (ml) 5 0 5 0 4 0 2 0 1 total activity (u) 3 2 3 8 . 1 9 2 7 3 6 . 0 6 2 2 7 0 . 2 0 2 3 0 . 0 8 6 . 1 4 protein (mg) 26.90 13.60 10.77 2 . 7 9 0 . 0 4 specific activity (u mg-1) 1 2 0 . 3 7 2 0 1 . 2 3 2 1 0 . 7 3 82.41 1 6 5 . 9 6 purification factor (fold) 1 . 0 0 1 . 6 7 1 . 7 5 0 . 6 8 1 . 3 8 recoverya (%) 1 0 0 . 0 0 84.49 70.11 7 . 1 1 0 . 1 9 fractions1 0 1 4 5 8 5 1 2 5 1 6 5 1 9 0 2 0 0 2 1 0 2 2 5 2 5 0 2 7 0 2 9 0 3 1 5 3 3 0 3 4 0 3 5 0 3 6 0 xylanase b 57.7 kda 1 2 3 kda 9 7 . 0 6 6 . 0 4 5 . 0 3 0 . 0 2 0 . 1 1 4 . 4 fig 5 sds-page of purified xylanase. lane 1, permeate solution; lane 2, retentate solution of ultrafiltration; lane 3, protein standards. microbiol indones26 rahayu et al. discussion hydrolysis of xylan requires multiple enzymes with different modes of action acting in cooperation. different microorganisms produce different kinds of xylanolytic enzymes. in this work, we have produced xylanase from bacillus sp. aq-1 at an activity level of 65.90 u ml-1 using bunch palm oil as the xylan substrate. as shown in fig 1a, the xylanolytic enzyme produced by bacillus sp. aq-1 was optimally active at 60 °c. similar results were observed for other microorganism, an optimal temperature of xylanase produced by aspergilus oryzae was at 60 °c (kitamoto et al. 1999). the enzymes reported to date are optimally active in neutral ph. the highest activity was observed at ph 7.0 when incubated at the optimum temperature (60 °c). although some microorganisms produce xylanases that have optimum activity at ph above 8, most alkaliphilic and alkali-tolerant microorganisms produce xylanases that are optimally active around neutral ph (blanco et al. 1995). using ion exchange chromatography, gel filtration chromatography and ultrafiltration, purified enzymes can be separated from other proteins. some proteins at a low concentration and lacking assayable xylanase activity, may be coeluted with the enzyme. two forms of xylanases, produced from ion exchange chromatography and gel filtration chromatography, were identified as xylanase a and xylanase b. ultrafiltration using a 30 kda polyethersulfone membrane separated both of them, one enzyme was in the permeate solution (xylanase a) and the other enzyme was in the retentate solution (xylanase b). a summary of purification steps is presented in table 1. the overall levels of recovery were 7.11 and 0.19% (xylanase a and b), with 0.68and 1.38-fold purification of xylanase a and b, and with specific activity of 82.41 and 165.96 u mg1 protein, respectively. the purification factor of xylanase a was lower than expected (less than 1). the high specific activity of xylanase b suggests that it is the major extracellular xylanase produced by bacillus sp. aq-1. the apparent purity of the enzyme was demonstrated by sdspage and zymogram analysis with gel containing 0.1% (w/v) beechwood xylan, which showed the molecular weight of xylanase a and b were 15.7 and 57.7 kda, respectively. in conclusion, we report the production of xylanase from bacillus sp. aq-1 grown on medium containing powder bunch palm oil gave high assayable enzyme activity. even though various materials can be used as a substrate in the xylanase production, bunch palm oil is a potential substrate to produce the enzyme. the results from xylanase purification also confirm that xylanase b, with an apparent molecular weight of 57.7 kda is the major xylanase secreted by bacillus sp. aq-1. in the future, purified xylanase can be applied in the chemical, beverage and functional food industry or in xylitol production. acknowledgement we thank badan pengkajian dan penerapan teknologi for encouragement, financial support and facilities to carry out this work. we also appreciate the help given by all the staff of bppt. references bailey mj, biely p, poutanen k. 1992. interlaboratory testing of methods for assay of xylanase activity. j biotechnol 23:257-70. bhat mk. 2000. cellulases and related enzymes in biotechnology. biotechnol adv 18:355-83. biely p. 1985. microbial xylanolitic system. trends biotechnol 3:289-90. blanco a, colom jf, pastor fij, vidal j. 1995. purification and properties of xylanase a from alkali-tolerant bacillus sp. strain bp-23. appl environ microbiol 61:4468-70. george sp, ahmad a, rao mb. 2001. a novel thermostable xylanase from thermomonospora sp.: influence of additives on thermostability. biores technol 78:221-24. gilbert hj, hazlewood gp. 1993. bacterial cellulose and xylanases. j gen microbiol 193:187-94. goulart aj, carmona ec, monti r. 2005. partial purification and properties of cellulase-free alkaline xylanase produced by rhizopus stolonifer in solid-state fermentation. braz arch biol tech 48:327-33. hinnman rl. 1994. the changing face of the fermentation industry. chem technol 24:45-8. kitamoto n, yoshino s, ohmiya k, tsukagoshi n. 1999. purification and characterization of overexpressed aspergillus oryzae xylanase, xynf1. biosci biotechnol biochem 63:1791-94. kulkarni n, shendye a, rao m. 1999. molecular and biotechnological aspects of xylanases. fems microbiol rev 23:411-56. lowry oh, rosebrough pj, farr al, randall rj. 1951. protein measurement with the folin phenol reagent. j biochem 193:2657 5 . nakamura s, nakai r, wakabayashi k, ishiguro y, aono r, horokoshi k. 1994. thermophilic alkaline xylanase from newly isolated alkaliphilic and thermophilic bacillus sp. strain tar-1. biosci biotechnol biochem 58:78-81. poutanen k. 1997. enzymes: an important tool in the improvement of the quality of cereal foods. trends food sci tech 8:300-06. purkarthofer h, sinner m, steiner w. 1993. cellulose-free xylanase from thermomyces lanuginosus: optimization of production in submerged and solid-stable culture. enzyme microb tech 15:6778 2 . querido ala, coelho jlc, araujo ef, chaves-alves vm. 2006. partial purification and characterization of xylanase produced by penicillium expansum. braz arch biol tech 49:475-80. simpson hd, haufler ur, daniel rm. 1991. an extremely thermostable xylanase from the thermophilic eubacterium thermatoga. biochem j 277:413-17. sunna a, prowe sg, stoffregen f, antranikian g. 1997a. characterization of the xylanases from the new isolated thermophilic xylan-degrading bacillus thermoleovorans strain k-3d and bacillus flavothermus strain lb3a. fems microbiol lett 148:209-16. sunna a, puls j, antranikian g. 1997b. characterization of the xylanolytic enzyme system of the extreme thermophilic anaerobic bacteria thermatoga martima, t. neapolitana, t. thermarum. comp biochem physiol 118a:453-61. takahashi h, nakai r, nakamura s. 2000. purification and partial characterization of a basic xylanase produced by thermoalkaliphilic bacillus sp. strain tar-1. biosci biotechnol biochem 64:887-90. tanaka h, nakamura t, hayashi s, ohta k. 2005. purification and properties of an extracellular endo-1,4-b-xylanase from penicillium citrinum and characterization of the encoding gene. j biosci bioeng 100:623-30. viikari l, kantelinen a, sundquist j, linko m. 1994. xylanases in bleaching: from an idea to the industry. fems microbiol ref 13:335-50. woodward j. 1984. xylanase: function, properties and applications. top enzyme ferment biotechnol 8:9-30. 01. rosariastuti.cdr vol.13, no.3, september 2019, p 75-82 doi: 10.5454/mi.13.3.1 the use of agrobacterium sp. i and compost as chelator combined by npk 3 fertilizer and mendong plant (fimbristylis sp.) in bioremediation of paddy soil contaminated by lead (pb) 1* 1 1 1 retno rosariastuti , abdi leonardo saragih , sudadi , supriyadi , and 2 wiwin widiastuti 1 soil science department, faculty of agriculture, universitas sebelas maret, jalan ir sutami 36a, kentingan, surakarta, 57126, indonesia; 2 regional planning research and development board, central java province, jalan pemuda 127-133, semarang 50132, indonesia. industrial waste supplies contains heavy metals such as pb which will cause pollution in paddy fields. remediation of paddy soil contaminated by pb heavy metal must be done by simple, environmental friendly, cheap and sustainable technology, that is bioremediation. the purpose of this study was to study the effectiveness of bioremediation using agrobacterium sp. i and compost as chelator combined by mendong plant and npk 3 fertilizer, and learn the ability of mendong in uptaking metal soil pb. this was field experimental research, had a factorial patern, using completly randomized block design as the base design, with three factors: (1) npk fertilizers (p0: no npk fertilizers, p1: with npk fertilizers), (2) chelator (k0: no chelator; k1: with chelator agrobacterium sp. i ; k2: with chelator compost); and (3) plant (t0: without plant; t1: with mendong plant). the 3 results showed that agrobacterium sp. i and compost were increasing pb uptake in shoot, but decreasing pb 3 uptake in root. mendong plant has highly ability in uptaking soil pb, so decreased soil pb, and effective as the phytoremediator. npk fertilizer increased plant growth so increased pb uptaken by plant. the highest pb uptake was in treatment combination of npk fertilizer + mendong plant: 80.916 µg, followed by npk fertilizer + agrobacterium sp. i + mendong plant: 76.363 µg. the highest decreased of soil pb (42.41%) was found in 3 treatment combination of compost + mendong plant. key words: agrobacterium sp. i , compost, fimbristylis sp, pb, phytoremediation3 limbah industri mengandung logam berat seperti pb, menyebabkan polusi di sawah. remediasi tanah sawah terkontaminasi pb harus dilakukan dengan teknologi sederhana, ramah lingkungan, murah dan berkelanjutan, yaitu bioremediasi. tujuan penelitian ini adalah untuk mempelajari efektivitas bioremediasi menggunakan agrobacterium sp. i dan kompos sebagai chelator yang dikombinasikan dengan tanaman mendong dan pupuk 3 npk, serta mempelajari kemampuan mendong dalam menyerap pb tanah. penelitian ini merupakan penelitian lapangan, memiliki pola faktorial, menggunakan rancangan dasar rancangan acak kelompok lengkap, dengan tiga faktor: (1) pupuk npk (p0: tanpa pupuk npk, p1: dengan pupuk npk), (2) chelator (k0 : tanpa chelator; k1: dengan chelator agrobacterium sp. i ; k2: dengan chelator kompos); dan (3) tanaman (t0: tanpa tanaman; t1: 3 dengan tanaman mendong). hasil penelitian menunjukkan bahwa agrobacterium sp. i dan kompos 3 meningkatkan serapan pb pada tajuk, tetapi menurunkan serapan pb pada akar. mendong memiliki kemampuan tinggi dalam menyerap pb tanah, sehingga dapat menurunkan pb tanah, dan efektif sebagai fitoremediator. pupuk npk meningkatkan pertumbuhan tanaman sehingga dapat meningkatkan serapan pb oleh tanaman. penyerapan pb tertinggi adalah pada kombinasi perlakuan : pupuk npk + mendong: 80,916 µg, diikuti oleh kombinasi perlakuan pupuk npk + agrobacterium sp. i + mendong: 76,363 ug. penurunan tertinggi pb tanah (42,41%) pada 3 kombinasi perlakuan : kompos + mendong. kata kunci: agrobacterium sp. i , bioremediasi, fimbristylis sp, kompos, pb3 microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82185455812; email: retnobs@staff.uns.ac.id mining waste. the metal will be accumulated by plants, consequently can affect the nutritional value and the quantity of crops in food plants, causing harm to farmers from the economic and health aspects of its consumers. ari susanti and purnawi (2013) mentioned that heavy metals are uptaken by plant roots in the form of water-soluble ions such as nutrients that come along with the water flow. sari et al. (2014) stated that plant heavy metals can inhibit metabolic processes and cell paddy fields area in kebakkramat sub-district based on the central agency of indonesia statistics (2013) is 2,258 hectares. wiyono and widodo (2004) states that 80% of paddy fields has been polluted by heavy metals in kebakkramat district, karanganyar. the source of heavy metal such as lead (pb) were industrial waste disposal, fertilizer, pesticide, and growth. its because the mechanism of reaction of heavy metals to proteins that generally attack sulphide bonds. the invaded sulphide bond always leads to its protein molecule so that it will cause structural damage. one of the ways to restore the soil environment that has been contaminated by heavy metals is by planting plants that are able to uptake the metal from the soil. this method is known as phytoremediation. in this study the plants used was mendong plants and its combination treatment with npk fertilizers, bacteria, and compost. pramono et al. (2012) described that rhizobacteria can affect the availability of soil pb. so it can cause pb easily or difficult to be uptaken by plants. in general, each plant has the ability to accumulate different heavy metals. mendong plants are known in uptake heavy metals such as pb (dewi and hindersah 2009). according to charlena (2004) the content of dangerous heavy metals such as pb and cd are available in the soil and uptaken by food crops commonly consumed by humans as a result of excessive use of fertilizers. the purpose of this study was to study the effectiveness of bioremediation using agrobacterium sp. i and 3 compost as chelator combined by mendong plant and npk fertilizers, and learn the ability of mendong in uptaking pb soil and study the most affective combination among the treatments. materials and methods the research was held on may-november 2016. the research place was located in waru village, kebakkramat sub-district, karanganyar district, central java (7030'36,4''sl and 110054'21,4 ''el, ± 108m above sea level). laboratory analysis was carried out in the chemistry, physics and biology laboratory of agriculture faculty, universitas sebelas maret, and central agricultural research institute of central java. materials. materials needed include seedlings of mendong plants, inoculum of agrobacterium sp. i3 6 -1 (10 cell.g soil), compost for mendong plant that was 5 -1 -2 t.ha (3.125 kg.m = 0.75 kg/plot), npk fertilizer for mendong plant with dose of each fertilizer that was: -1 -1 urea: 27 kg.ha (19.56 g/plot) ; sp-36: 21.6 kg.ha (25 -1 g/plot); kcl: 36 kg.ha (15 g/plot). media and chemicals needed for making inoculum of agrobacterium sp. i include: lb (luria betani: 10 g 3 pepton, 5 g nacl, 5 g yeast, 18 g agar) agar and liquid, bacterial carrier (15 kg compost, 7.5 rice bran, 750 ml em-4 , and 15 l water.) and chemicals for destruction, such as percloric acid dan nitric acid. methods. this research was conducted by field experiments. this was factorial research using completely randomized block design (rcbd) as the based design, with three factors (1) npk fertilizers (p0: withoutnpk fertilizers, p1: with npk fertilizers), (2) chelator (k0: without chelator; k1: with chelator agrobacterium sp i ; k2: with chelator compost), and 3 (3) plant (t0: without plant, t1: with mendong plant) to obtain 12 treatment combinations, each treatment combination was repeated 3 times. the observed variables were pb concentration in soil (wet destruction and detected by aas), soil parameters analyzed were cec (amonium asetat saturation), corganic (walkley and black), ph h o (electrometric), 2 and total bacterial colonies (plate count), content and uptake of pb roots and shoot of mendong plant (wet destruction detected by aas), plant height, and dry weight of biomass. preparation of bacteria carrier. materials of carrier used in this study consist of : 15 kg compost, 7.5 kg rice bran, 750 ml em-4 , and 15 l water. these materials were mixed well and then incubated for 2 months, becoming carrier of bacteria. then this carrier was sterilized using a presto pan for keeping the carrier sterile from undesirable bacteria or fungi. preparation of agrobacterium sp. i inoculum. 3 propagation of agrobacterium sp. i inoculum was 3 started by preparing the lb (luria bertani) medium with the composition of 10 g tripton, 10 g nacl, 5 g yeast extract, 100 ml destilled water; 15-20 g na (nutrient agar) medium with the composition of 10 g beef extract, 10 g pepton, 5 g nacl, 1000 ml destilled -1 water, and 15 g agar l . after obtaining pure isolate of agrobacterium sp. i , purification was done in luria 3 bertani liquid in erlenmeyer and and shaked out to 10 -1 gain the density of 10 cells ml . sterile carrier was then enriched with squirted agrobacterium sp. i . the 3 comparison was 600 ml agrobacterium sp. i for 2 kg 3 of the carrier. implementation of the study. the size of the plot of land was1.5 m x 1 m (sub plot : 0.5 m x 0.5 m). seeds of mendong plant needed were six per plot (one seed per sub plot), with spacing of plant per sub plots was 50 cm x 50 cm. inoculum of agrobacterium sp. i and 3 compost were added 7 days before planting. npk fertilizer was added one day before planting. harvest was carried out 30 days after the mendong added by bacterial innoculum. field observations were plant height of one time every week and plant dry weight after harvesting. plant dry weight devided into two parts : root and shoot of mendong plant. the data were analyzed using anova test of 95% 76 rosariastuti et al. microbiol indones volume 13, 2019 microbiol indones 77 and 99% confidence level, with the continued test of dmrt 95% confidence level and correlation test. results characteristics of soil before treatment. the land used was paddy soil that has a fairly abundant water with the type of soil that is vertisol (usda 1999). the characteristics of initial soil (table 1) showed that the soil used in this study has soil ph of 7.55 (netral). -1 cec of 19.614 cmol (+) kg was classified in medium grade. the soil c-organic was high at 3.31%, which could create good environment in providing nutrients for bacteria or plants. the total population of bacterial colonies in this initial soil condition was 12.62 log 10 12 -1 cfu.g-1 (10 g ) and pb concentrations in this initial -1 soil was 12.203 μg g . based on gr (government regulation) no. 101 (2014), its value has exceeded the allowable limit. pb concentrations in soil. lead (pb) -1 concentration in initial soil was 12.203 μg g (fig 1). soil pb concentrations of all treatment including control were decreasing from soil pb concentrations of initial soil (fig 1). the results of anova showed that chelator and mendong significantly affected the decreasing concentration of pb soil. control p0k0t0 treatment has the lowest decreased of pb soil (27.18%) -1 from initial soil with value of 9.129 μg g , while p0k2t1 treatment has the highest decreased of pb soil, -1 that was 42.41% with value 7.028 μg g (fig 2). the effect of compost significantly different with treatment without chelator (fig 3). compost and mendong could decrease pb concentration in soil. mendong plant (p0k0t1 treatment) also has highly decreased in concentration of soil pb, it was 39.55% -1 from initial soil, the value was 7.376 μg g . this suggests that mendong has high ability in uptaking pb in soil. based on the correlation test of soil chemical characteristics, it is suggested that pb concentration in the soil was positively correlation with pb concentration and pb uptake in plants. pb concentration and uptake in mendong. the mean values of pb concentrations in roots and shoots of mendong have exceeded the normal limit allowed for -1 food crops ranging from 0.5 to 3 μg g (widaningrum et al. 2007). the highest pb concentration in the root -1 was in p1k0t1 treatment which was 7.527 μg.g , while the highest pb concentration in the shoot was in -1 p0k2t1 treatment which was 4.593 μg g . the average pb concentrations in roots was higher than that of shoots. based on the correlation test, pb concentration in plant was negatively correlated to height and biomass of mendong plant. the highest pb uptake in root was in p0k0t1 treatment which was 58 856 μg (fig 4). the highest pb uptake in shoot was found in p0k2t1 treatment which was 31.581 μg. generally, chelator, agrobacterium sp. i and compost, influencing the increasing of pb uptake 3 in shoots, so it could increase total pb uptaken by plant (including pb uptake in root and pb uptake in shoot), the uptake higher than plant treatment only. therefore, it is very recommended to do when the aim of bioremediation is uptake much soil metal. based on the results of anova, chelator significantly effected to pb uptaken by plant, but no interaction between fertilizer and chelator. the result of dmrt, was that mendong plant has high ability in uptaking pb in root of plant, with the highest pb uptake in root 58.856 µg (fig 5). different with plant, the influence of chelator treatment, especially compost caused the lower of pb uptake in root than that with plant treatment only which was 22.456 µg. p1k2t1 treatment tend to has low pb -1 concentration and uptaken by plant (2.720 μg g ) with an uptake value 38.707 μg. p1k0t1 treatment tend to -1 has high pb concentration in plant (5.234 μg g with uptake value of 80.916 μg (table 2). chelator tend to have low pb concentration and pb uptaken by mendong plant. height and dry biomass of mendong plant plant height. based on result of anova, fertilizer treatment very significantly effected (p <0.01) to plant height of mendong plant. but the interaction between fertilizer and chelator did not effected to plant height. the height of mendong with p0k0t1 (control) treatment, was 50 cm and the highest height of mendong found in p1k2t1 treatment that was 65.64 cm (table 3). npk fertilizer treatment, influencing the average of plant height, higher than treatment without npk fertilizer. plant dry weight (plant biomass). based on the result of anova of npk fertilizers, chelator, and its combination were not significant effect to plant biomass. the dry plant biomass of the mendong with a combination of p1k1t1 treatment (15.80 gr) tended to be higher than control (13.96 gr) and other treatments (table 3). fertilizer treatment higher effected to plant biomass, it was 15.16 g. based on the correlation test, the height of mendong plant have a positive correlation 78 rosariastuti et al. microbiol indones table 1 characteristics of initial soil table 2 total pb in mendong plant table 3 plant height and plant dry weight (plant biomass) with plant biomass. discussion characteristics of soil before treatment. the mobility of pb absorbed by plant could be influenced by soil ph (table 1). soil with low ph (acid conditions) make the metal become dissolved, and easily absorbed by root of plants. the high value of soil c-organic indicated that the soil fertility rate was high. generally the content of c-organic in land that was administered become paddy fields (wet soil) is higher (1.19-3.63%) than in dry soil (0.59-2.65%). the high c-organic in paddy field soil is superseded by the addition of organic material derived from the remnants of the roots of paddy plant also because of the slow decomposition process of soil organic matter. haryanti (2013) described that the content of soil pb was influenced by the fertility and soil organic matter content. pb in the soil was almost always strongly bounded by a precipitated organic or colloidal material. paddy field soils has higher cec than dry soil. this is due to the low washing of the soil bases (rahayu et al. 2014). soil bacterias can increase soil cec with decomposition of soil organic matter. soil microbes have an important role in immobilization of soil cations (rosariastuti et al. 2020). soil pb concentration. the pb concentration of control also decreased (fig 1). it happened maybe because of the pb leaching by rainwater or water flow in the ground. leaching is the process by which contaminants are transferred from a stabilized matrix to liquid medium, such as water or other solutions (zheng et al. 2014). this phenomenon was unwanted because where lead will accumulate in the soil was unpredicted and it could cause environment pollution including soil in the newplace of pb. high pb concentration in soil can cause the decreasing quality of crop yield, because of the absorbing pb by plant. this was due to the normal levels of pb in plants -1 ranging from 0.5 to 3 μg g (widaningrum et al. 2007). when pb entering food chain cycle, it can cause a parameters unit value grade ph 7,55 netral* c-organic % 3,31 high* source: laboratory analysis (primer) -1 cmol(+).kg 19,61 medium* information: *) according to soil research institue 2009 -1 log 10 cfu.g 12,62 high* *) government regulation no. 101 (2014) -1 mg.g 12,203 high* cation exchange capacity (cec) bacteria colonies total lead (pb) no. treatment pb concentration in plant -1 (mg.g ) pb uptaken by plant (mg) 1 p0k0t1 5,191 72,449 2 p0k1t1 4,387 63,179 source: primer p0k2t1 4,362 59,355 p1k0t1 5,234 80,916 p1k1t1 4,834 76,363 3 4 5 p1k2t1 2,720 38,7076 no. treatment plant height (cm) plant dry weight (plant biomass) (g) 1 p0k0t1 50.00 13.96 2 p0k1t1 51.42 14.40 source: primer p0k2t1 49.78 13.61 p0k0t1 53.56 15.46 p0k1t1 61.72 15.80 3 4 5 p0k2t1 65.64 14.236 volume 13, 2019 microbiol indones 79 fig 1 histogram of soil pb concentrations. fig 3 histogram of pb concentration in root and shoot of plant. fig 2 histogram of the effect of chelator to soil pb concentration. fig 4 histogram of concentration of pb uptake by mendong plant. problem to humans health. heavy metals are transferred from abiotic envieronment to living organisms, accumulated in biota at different trophic levels, and thus contaminate the food chains. trophic transfer, bioaccumulation, and biomagnification of hazardous heavy metals in food chains have important implications on wildlife and human health (ali and ezzat 2019). compost and mendong decreased pb concentration in soil (fig 2). kucasov and guvener (2009) described that compost contains humus substance (fulvic acid, humic acid, and humin) which could minimize soil heavy metals. decreasing of soil pb is not only caused by compost, but also by plant uptake. from this research, it is known that pb concentration in the soil was positively correlation with pb concentration and pb uptake in plants. schmidt (2003) explained that high concentrations of heavy metals in soil may lead to the increase of metal uptaken by plants. pb concentration and uptake in mendong. the average pb concentrations in roots was higher than shoots (fig. 3). according to indrasti et al. (2006) inhibited metal translocation from the roots to shoots, would make plant become easy to detoxify the metal. pb concentration in plant was negatively correlated to height and biomass of mendong plant, because of pb cation uptaken by the roots, would enter into the plant, and it would be an enzyme-forming inhibitor then would inhibit the metabolic process of the plant (amelia et al. 2015). generally, chelator, agrobacterium sp. i and 3 compost, influencing the increasing of pb uptake in shoots, so it could increase total pb uptaken by plant (including pb uptake in root and pb uptake in shoot), higher than plant treatment only (fig 4). it is very recommended combination when the aim of bioremediation is uptake much soil metal. compost application induced a different behavior in both species. compared to wheat and irrespective of as doses, in compost amended soils, barley plants showed an enhanced as translocation to the aerial part. the different bacterial communities structure found for each species, according to the pcr-dgge cluster analysis, suggested that specific rhizobacteria of barley may have increased as bioavability, and would therefore enhance its translocation to aerial parts (gonzales et al. 2019). mendong plant has high ability in uptaken pb in root of plant, with the highest pb uptaken in root, but agrobacterium sp. i and compost treatment decreased 3 pb concentration and pb uptaken by plant (fig5, table 2). humic acid in compost has ability in bonding either metal ion, or organic compound bonding. according to tan (2003) the negative charge in humic acid has ability to react and interact with positively charged of metal ions, thereby decreasing pb uptaken by plants, but on the other hand it could increase the metal traslocation from root to shoot of plant. height and dry biomass of mendong plant. npk fertilizer treatment, influencing the average of plant height , higher than treatment without npk fertilizer (table 3). fertilizer adduction was very effective to increase fertility and plant growth. npk fertilizer, chelator treatment and npk fertilizer+chelator interaction had a significant effect on plant biomass (rosariastuti et al. 2019). wasis and fathia (2010) explained that the use of npk fertilizers would provide substantial n supply to the soil, which would help plant growth. the average metall content in plants in all treatment combination showed the high levels of metal content in mendong plant, but the plants did not show significant damage such as chlorosis and necrosis. it suggests that mendong could be good plant for phytoremediation of pb metals. dry plant biomass of the mendong plant with a combination of p1k1t1 treatment tended to be higher than control and other treatments. fertilizer treatment higher effected to plant biomass than without fertilizer. plant biomass of the mendong were influenced by soil 80 rosariastuti et al. microbiol indones fig 5 histogram of the effect of chelator to pb uptake in root of plant. plant nutrients, so fertilizer influenced plant biomass of mendong. the height of mendong plant have a positive correlation with plant biomass. according to ekowati and nasir (2011) plant biomass was the result calculation of all plant organs. high plant which has many leaves assumed that the plant biomass would be also high. the result of correlation test also showed that higher pb levels in plant would cause the low of plant biomass. metal contamination, as a consequence of anthropogenic activities, poses threat to plants and their ecosystems. plant growing on metal contaminated soils show severe aberration in their metabolism leading to stunded plant growth and low biomass production (saboor et al. 2019). conclusion of this research are that treatment combination of compost and mendong plants (without artificial fertilizer) was the best treatment in the lowering soil pb concentration from initial soil pb -1 (7.028 μg g ) so that there was a decrease of soil pb concentration from the initial soil by 42.41%. mendong plant has high ability in uptaken pb, especially in root. chelator (agrobacterium sp. i and compost) tends to 3 increase pb uptake in root of plant, but decrease pb uptake in shoot. compost decreased total pb concentration and uptake in plant. npk fertilizer tend to increase plant dry weight (biomass), and by its combination with other treatments, the treatment combination tend to increase of pb uptaken by plant. -1 the highest pb concentration (5.234 µg.g ) and pb uptake in plant (80.916 µg) was in treatment combination of npk fertilizer and mendong plant, followed by treatment combination of npk fertilizer and agrobacterium sp. i and mendong plant which has 3 pb uptaken by plant of 76.363 µg. references ali h, ezzat k. 2019. trophic transfer, bioaccumulation, and biomagnification of non-essential hazardous heavy metals and metalloids in food chains/webs—concepts and implications for wildlife and human health. hum e c o l r i s k a s s e s s . 2 5 ( 6 ) : 1 3 5 3 1 3 7 6 . d o i : 10.1080/10807039.2018.1469398. amelia ra, rachmadiarti f, yuliani. 2015. analisis kadar logam berat pb dan pertumbuhan tanaman padi di area persawahan dusun betas, desa kapulungan, gempolpasuruan [analysis of heavy metal pb and the growth of paddy in paddy field area dusun betas, desa kapulangan, gempol-pasuruan]. lentera bio. 4(3): 187–191. ekowati d, nasir m. 2011. pertumbuhan tanaman jagung (zea mays l.) varietas bisi-2 pada pasir reject dan pasir asli di pantai trisik kulonprogo [the growth of maize (zea mays l.) variant bisi-2 in rejected and original sand in trisik beach, kulonprogo]. j manusia lingkungan. 18(3): 220-231. doi: 10.22146/jml.18445. gonzales a, pilar gg, mar gd, juan a, carmen l. 2019. compost-assisted phytoremediation of as-polluted soil. j s o i l s s e d i m e n t s . 1 9 ( 7 ) : 2 9 7 1 2 9 8 3 . d o i : 10.1007/s11368-019-02284-9. haryanti d, dedik b, salni. 2013. potensi beberapa jenis tanaman hias sebagai fitoremediasi 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[potential of several types of ornamental plants as phytoremediation of lead metal (pb) in soil]. j p e n e l i t i a n s a i n s . 1 6 ( 2 ) : 5 2 5 8 . d o i : 10.36706/jps.v16i2.72. indrasti ns, suprihatin, burhanudin, aida. 2016. penyerapan logam pb dan cd oleh eceng gondok : pengaruh konsentrasi logam dan lama waktu kontak. [pb and cd metal absorption by eceng gondok: effects of metal concentration and contact duration]. j tek ind pert.16(1): 44-50. kucasov g, gunvener z. 2009. efficiency of compost in the removal of heavy metals from the industrial wastewater. environ geol. 57: 291-296. doi: 10.1007/s00254-0081372-3. pramono a, rosariastuti r mma, ngadiman, prijambada. 2012. peran rhizobakteri dalam fitoekstraksi logam berat kromium pada tanaman jagung [the role of rhizobacteria in the heavy metal chromium extraction in c o r n p l a n t s ] . j e c o l a b . 6 ( 1 ) : 1 1 3 . d o i : 10.20886/jklh.2012.6.1.38-50. rahayu a, utami sr, rayes ml. 2014. karakterisitik dan klasifikasi tanah pada lahan kering dan lahan yang diswahkan di kecamatan perak, kabupaten jombang [characteristics and classification of soil on dry and leased land in perak district, jombang regency]. j tanah sumber daya lahan. 1(2):79-87. rosariastuti r, muhamad sf, purwanto, and suntoro. 2020. effects of agrobacterium sp. i , manure and inorganic 26 fertilizers to pb content of rice grains planted in pb polluted soil. environ nat resour j. 18(1):75-84. doi: 10.32526/ennrj. 18.1. 2020.08. rosariastuti r, selly m, sudadi sudadi, sri h, purwanto p. 2019. remediation of chromium contaminated soil by phyto-bio system (pbs) application. sains tanah j soil s c i a g r o c l i m a t o l . 1 6 ( 1 ) : 9 0 1 0 2 . d o i : 10.20961/stjssa.v16i1.24932. saboor a, ghulam m, muhammad a, muhamad a, saiiad h. 2019. seed priming and metal/metalloid stress tolerance in plants. in: priming and pretreatment of seeds and seedling. springer, singapore. 287-311. doi: 10.1007/978-981-13-8625-1_14. schmidt u. 2003. enhancing phytoextraction: the effect of volume 13, 2019 microbiol indones 81 chemical soil manipulation on mobility, plant accumulation, and leaching of heavy metals. j environ qual.32(6):1939-1954. doi: 10.2134/jeq2003.1939. tan kh. 2003. humic matter in soil and the environment. crc press: new york. wasis b, fathia n. 2010. pengaruh pupuk npk dan kompos terhadap pertumbuhan semai gmelina (gmelina arborea roxb) pada media tanah bekas tambang emas (tailing) [the effect of npk fertilizer and compost on the growth of gmelina seedlings (gmelina arborea roxb) on the land of the former gold mine (tailings)]. j ilmu pertanian indonesia. 16(2):123-129. p-issn: 0853-4217. widaningrum, miskiyah, suismono. 2007. bahaya kontaminasi logam berat dalam sayuran dan alternatif pencegahan cemarannya [danger of heavy metal contamination in vegetables and alternative prevention of contamination]. buletin teknologi pascapanen pertanian. 3:17-27. zheng sa, zheng x, chen c. 2012. leaching behavior of heavy metals and transformation of their speciation in polluted soil receiving simulated acid rain. plos one. 7(11):e49664. doi: 10.1371/journal.pone.0049664. 82 rosariastuti et al. microbiol indones page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 01. hallis.cdr vol.15, no.1, march 2021, p 1-7 doi: 10.5454/mi.15.1.1 molecular diversity of mold associated with gatotan steffanus pranoto hallis , anastasia tatik hartanti , 1 2* and agustin wydia gunawan 3 1 program of biology, faculty of biotechnology, atma jaya catholic university of indonesia. jalan jenderal sudirman 51, jakarta 12930, indonesia; 2department of food technology, faculty of biotechnology, atma jaya catholic university of indonesia, jalan raya cisauk-lapan 10, bsd city, tangerang 15345, banten, indonesia; 3 department of biology, faculty of biotechnology, atma jaya catholic university of indonesia, jalan raya cisauk-lapan 10, bsd city, tangerang 15345, banten, indonesia. gatotan gatot is a raw material to make , a fermented food made from n indonesian , especially in central java, cassava tubers. traditionally, the cassava tubers are dried for several days until the black color appears. sunhowever, natural fermentation allowed by many types of microorganisms, especially mold in this process could raise concerns about the food safety issues. previously, the identifications of molds in were based on gatotan morphological observation. here, we reported the diversity of molds associated with using molecular gatotan identification method. the molecular identification was based on ribosomal dna internal transcribed spacer (its) amplification sequences using combination of its4 and its5 primers. a total of molds were isolated ten and p ribosomal dna enceshylogenetic trees were constructed based on sequ . our results showed that the molds were classified into spp., sp., , and lasiodiplodia trichoderma aspergillus nomius, fusarium oxysporum cladosporium sphaerospermum. cassava, fungi, , internal transcribed spacerkey words: gatot makanan fermentasi indonesia, khususnya di jawa gatotan adalah bahan baku dalam pembuatan gatot, tengah yang terbuat dari umbi singkong. pembuatan gatot secara tradisional dilakukan dengan mengeringkan umbi singkong ngga muncul warna kehitaman. fermentasi alami yang langsung dengan paparan sinar matahari hi terjadi pada proses ini melibatkan pertumbuhan berbagai mikroorganisme, terutama kapang, yang tidak terkendali dan dapat menimbulkan permasalahan keamanan pangan. dentifikasi kapang yang terlibat dalam i pembuatan gatotan telah dilakukan secara morfologi. pada penelitian ini, identifikasi kapang yang berasosiasi dengan gatotan dilakukan secara molekuler. identifikasi molekuler dilakukan dengan menggunakan amplifikasi situs (its) pada dna ribosom menggunakan pasangan primer its4 dan its5. internal transcribed spacer sebanyak sepuluh kapang telah diisolasi dan pohon filogenetik disusun berdasarkan pada sekuen dna ribosom. hasil identifikasi yang diperoleh ialah lasiodiplodia trichoderma aspergillus nomius, fusariumspp., sp., oxysporum cladosporium sphaerospermum. , dan kata kunci: cendawan, gatot, , singkonginternal transcribed spacer microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ); : +62-21-80827200 (ext. 1039 fax: +62-21-5747912 e-mail: anast.hartanti@atmajaya.ac.id; cassava is usually used to make . traditionally, gatot cassava tubers are peeled off, cut, and washed until clean, then sun-dried for 3-5 days until become . gaplek gaplek is placed outdoor within approximately two weeks until the tubers colour changed to black, indicating natural fermentation to form which gatotan can be further processed into by steaming gatot (purwandari 2014a)et al. . flour had 90.33% antioxidant scavenging gatotan activity 18.92 mg × 100 g equivalent to vitamin e -1 (purwandari 2014b)et al. . development of as gatot noodle would not only increase the blood sugar level quicker but also reduce it quicker than white bread , consumption control, suggesting the low glycemic potential of . furthermore, the starch resistant of gatotan gatotan gatot (22.5%) and (21.85%) were lower than cassava (24.44%) in glucose oxidase assay fermentation of cassava ( ) has manihot esculenta been considered as one strategy to improve nutrient quality and reduce its natural cyanide content. recently, lactobacillus plantarum, a lactid acid bacteria commonly found in cassava waste, was reported decrease up to 97% of the cyanide content in cassava after 36 hours of fermentation . (hawashi 2018)et al. furthermore, together with , several l. plantarum m i c r o o r g a n i s m s c o n s o r t i u m i n c l u d i n g corynebacterium manihot saccharomyces exiguus, , and reduced the cyanide content geotrichum candidum and improved the protein and uality of fatty acid q cassava after 96 hours fermentation (samson and akomolafe 2017). in indonesia, fermentation of (puspaningtyas 2018)et al. . additionally, the dietary fiber of (14%) and (17.36%) were found gatotan gatot to be higher than cassava (8.61%), respectively suggesting a prominent as functional food for gatot people with diabetes mellitus . (sari 2018)et al. considering the benefits of as fermented food gatot product, identification of microorganisms involving in this process were reported. mold, such as aspergillus flavus rhizopus oryzae lasiodiplodia theobromae , , and were showed to be associated with from various gatotan regions in indonesia . in addition, (purwandari 2000) phenotypic identification showed predominant type of fungi, , , botryodiplodia theobromae r oligosporus. together with indigenous lactic acid bacteria l. fermentum l manihotivoransand were potential as . starter culture for controlled fermentationgatotan ( moreover, spontaneous astriani 2018)et al. . fermentation of cassava to make resulted in gatot growth of and , r. oryzae acremonium charticola proposed as indigenous fungi which had salt-tolerant properties and potential antioxidant activity (yudiarti and sugiharto 2016; sugiharto 2016)et al. . as fungal consortium in fermentation was broadly gatotan diverse, its clustering needs to be re-confirmed using molecular approaches. however, there was limited report about molecular identification regarding fungal consortium in . gatot amo ng ma ny dn a ba r c od e fo r f ung al identification, nuclear ribosomal internal transcribed spacer (its) is considered as the most accurate region to differentiate the gap between interand intraspecific variations . its region, comprised (schoch 2012)et al. of its1, its2, and small 5.8s ribosomal rna (rrna), is located between the large 18s and 28s ribosomal rna in fungal nuclear cistron. splitting process in posttranslational modification would remove its1 and its2 since they act as non-coding region (schoch et al. 2012). distinguish properties of this region influenced efficacy for systemic identification, including in diagnostic mycology area . the (ciardo 2010)et al. universal primer combination of its4 and its5 anneals to tip part of large rrna subunits, consequences in amplification of whole its region for phylogeny construction . furthermore, (white 1990)et al. improved its primer combination has been evaluated to be capable for food fungal community profiling (walters 2016)et al. . thus, its would be a promising tool as a key of mold identification in . this gatotan research aimed to identify the diversity of mold associated with using molecular approach of gatotan its region. materials and methods were made from cassava (gatotan. gatotan m. esculenta) tubers in bogor. all the water used was distilled water using gfl water distillation unit 2008 (gfl, burgwedel, germany). potato dextrose base (oxoid, hampshire, united kingdom) was mixed with 15 g l agar base for potato dextrose agar (pda), while -1 a. flavus a. parasiticus and agar (afpa), and dichloran 18% glycerol (dg18) agar were made according to the protocols (hocking and pitt 1980; pitt et al. 1983). the dna extraction reagent dna phytopure™ kit extraction was provided by ge healthcare life sciences (amersham, united k i n g d o m ) . p r i m e r p a i r s o f i t s 4 ( 5 ' tcctccgcttattgatatgc-3') (5'dan its5 gg aa gtaa a agt cgtaa caag g-3 ') w er e synthesized by integrated dna technologies (singapore). of m mold isolation. the methods old isolation were carried out using direct and dilution method . n s i direct isolation method septically, inside part of , a gatotan were placed on pda and incubated at 30 . n °c i dilution method afpa and dg18 medium were used. , first, 25 g were suspended with 250 ml of gatotan aquadest. the suspension was diluted 10 , 10 , and 10 -2 -4 -6 using physiological salt solution. a total of 1 ml diluted suspension was mixed with warm (45 °c) afpa and dg18 medium prior to solidification. solid mixtures were incubated at °c. 30 mold growth was monitored everyday for total four days. different growing colonies were placed in new pda for further examination. dna isolation, pcr mplification, and a s . equencing four days old mycelia grown on pda was ixed with distilled water ntrifuged at m , then ce 10,000 rpm for 10 pellet were mixed with glass min. beads (oxoid, hampshire, united kingdom) and vortexed to disrupt the cell wall. dna were extracted using dna phytopure™ kit extraction (ge healthcare life sciences, amersham, united kingdom) according to manufacturer's protocol. a total of 200 ng dna was amplified using its4 and its5 primer pairs to obtain its rdna sites (white et al. 1990). the pcr procedures were carried out using go taq green master mix pcr 2× (promega) with ® reaction as follows: pre-denaturation at 94 °c for 2 min, 35 cycles of denaturation at 94 °c for 15 s, annealing at 55 °c for 30 s, and elongation at 72 °c for 1 min with final elongation at 72 °c for 5 min (abe et al. 2007). the visualization was done on 1% agarose gel 2 hallis et al . microbiol indones volume 15, 2021 microbiol indones 3 electrophoresis ( ) at 60 mini-sub cell gt cell biorad ® v for 90 min followed by ethidium bromide staining and imaging using gel doc™ xr system (bio-rad, germany). pcr products were sent to first base (malaysia) for sequencing. phylogenetic analysis. the nucleotide sequence were analyzed using basic local alignment search tool ( ht tp : // www. bla s t.n c b i.n lm. nih . go v/ bla s t.c g i) (altschul 1990)et al. to acquire the mold species. the sequences were aligned using seqtrace software version 0.9.0 . the phylogenetic trees (stucky 2012) were obtained using maximum likelihood (ml) parameter on molecular evolutionary genetics analysis 6 (mega6). kimura 2 model was chosen to represent the phylogenetic tree. strength of the internal branch of phylogenetic tree was analyzed with 1,000 bootstraps (bs) . the reference and outgroup (felsenstein 1985) s e q u e n c e w e r e o b t a i n e d f r o m g e n b a n k (www.ncbi.nlm.nih.gov/genbank) (benson et al. 2005). results a total of ten mold strains were isolated and identified, including strains lasiodiplodia (4 ), trichoderma aspergillus fusarium, , (3 ), strains and cladosporium . identification (table 1) based on its sequence on showed four strains of spp. lasiodiplodia that these particular strains were found on the same clade with with low bs valuebotryosphaeria rhodina (0.47) . (fig 1) the its region sequence resolution on phylogenetic tree could not describe lasiodiplodia spp. . to species level there were two species types in one same cluster, namely with its b. rhodina anamorphic name and l. theobromae l. parva. t4 sp. strain s of was found on thetrichoderma clade trichoderma reesei, t. consisting of longibrachiatum, t. orientale, hypocrea orientalisand with bs value fig its region sequence 0.80 ( 2). resolution on phylogenetic tree based on analysis ml could not differentiate the intraspecies variations two . specimens, and were in t. reesei t. longibrachiatum , the same cluster he comparison result from . however, t the ncbi database showed 95% similarity of strain st4 with respectively (data not t. longibrachiatum, shown). one strain was identified asaspergillus a. nomius (fig 3). according to the its tree produced from ml analysis, is included in the clade with stp5 a. nomius high bs value in addition, three of0.99. fusarium strains were identified as strainsf. oxysporum (fig 4). sta2, sta4, sta5 were foundand in the f. oxysporum clade 0.63. meanwhile, with bs value s std1 train was identified as cladosporium sphaerospermum 0.86 ( 5). the with bs value fig phylogenetic tree formed specific clade of c. sphaerospermum with std1 isolate included. discussion cassava fermentation into involves several gatotan molds to produce unique and distinct taste. several previous studies showed the involvement of this particular mold genera in fermentation gatotan including a. flavus r. oryzae r. oligosporus l. , , and theobromae (astriani 2018 purwandari 2000)et al. ; . our results showed similar mold genera were identified using molecular identification (fig 1 and 3). these include lasiodiplodia a. nomius spp. and . importantly, spp. was isolated and lasiodiplodia characterized in every investigation. however, gatotan the mor pho log ic al c h ar ac te riz at io n s h owe d inconsistent identification, while purwandari (2000) identified this mold as and astriani l. theobromae (2018) as . in addition, based on b. theobromae the ref eren ce and in de x fungor um myc oba nk , table 1 bogormolecular identity of mold strains associated with fromgatotan isolation medium strain code molecular identity potato dextrose agar st2 lasiodiplodia sp. st3 lasiodiplodia sp. st4 trichoderma sp. stp5 aspergillus nomius stp6 lasiodiplodia sp. aspergillus flavus and parasiticus agar sta1 lasiodiplodia sp. sta2 fusarium oxysporum sta4 fusarium oxysporum sta5 fusarium oxysporum dichloran glycerol 18% agar base std1 cladosporium sphaerospermum 4 hallis et al . microbiol indones fig 1 phylogenetic tree and morphological characteristics of spp. was an lasiodiplodia diplodia seriata used as outgroup. phylogenetic tree was kimura2 made based on maximum likelihood analysis with model (1,000x bootstraps). fig 2 phylogenetic tree of sp. was an outgroup. phylogenetic tree wastrichoderma penicillium citrinum used as made based on maximum likelihood analysis with model kimura2 (1,000x bootstraps). fig 3 phylogenetic tree of . was an outgroup. phylogenetic tree aspergillus nomius diaporthe angelicae used as was kimura2 (1,000x bootstraps) made based on maximum likelihood analysis with model . volume 15, 2021 microbiol indones 5 lasiodiplodia is an anamorphic form from botryosphaeria and the usage of the name lasiodiplodia refers to the determination of one fungus having one name (hawksworth 2011 robert et al. et al.; 2013). importantly, since had been l. theobromae isolated in every studies, the role of this mold gatotan would be crucial in fermentation of gatotan (purwandari 2000; astriani 2018)et al. . other mold genera also reported to be associated with despite of their controversial contribution gatotan and safety for food fermentation. trichoderma sp. isolated from had been classified as unwanted gatotan mold since its potency to produce toxin ''(astriani et al. 2018). in addition, fusarium cladosporium and were isolated and identified as the major contaminants in gaplek, dried cassava tubers before fermented to gatotan . (susanti 2010) however, , t. longibrachiatum in particular, was reported to inhibit the growth of f. oxysporum penicillium oxalicum rhizoctonia solani, , , and that caused post-harvest disease sclerotium rolfsii in yam . nonetheless, (dania 2016)et al. trichoderma spp. also has an important role in solid substrate fermentation, particularly in cassava to reduce the cyanide content and improve the nutritional value (hawashi 2019)et al. . latterly, , one of generally recognized as a. niger safe microorganism, isolated from was observed gatot to produce extracellular starch degrading enzyme, αamylase . both crude and partially (angelia 2019)et al. purified α-amylase enzyme showed similar degrading activity of starch to glucose and maltose with commercial enzyme. importantly, black colonies of a. niger gatotan were also isolated in samples from various region in indonesia, suggesting their presence fig 4 phylogenetic tree of . sp. was an outgroup. phylogenetic tree wasfusarium oxysporum eurotium used as made based on maximum likelihood analysis with model kimura2 (1,000x bootstraps). fig 5 phylogenetic tree of . was an outgroup. cladosporium sphaerospermum paecilomyces variotii used as phylogenetic tree was kimura2 (1,000x made based on maximum likelihood analysis with model bootstraps). as unique characteristic of fermentation gatotan (astriani 2018 purwandari 2000)et al. ; . beside l. theobromae rhizopus, spp. was reported to have an important role in fermentation. , gatotan r. oligosporus together with , , and b. theobromae a. niger trichoderma sp., was isolated as one of indigenous fungi in fermentation . gatot (astriani 2018)et al. however, in this study, we didn't identify spp. rhizopus using molecular technique, despite of their presence during isolation. in conclusion, this study identified lasiodiplodia s p p . , s p . , , tr i c h o d e r m a f o x y s p o r u m c. . sphaerospermum, a nomius and as . molds associated with gatotan using its identification. lasiodiplodia spp. and spp., were always found in aspergillus gatotan, thus they may provide essential contribution in fermentation.gatotan acknowledgements this research was supported by faculty of biotechnology, atma jaya catholic university of indonesia through faculty research grant. references abe a, oda y, asano k, sone t. 2007. is rhizopus delemar the proper name for fumaric-malic rhizopus oryzae acid producers. mycologia. 99:714-722. altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j mol biol. 215:403-410. angelia c, sanjaya a, tanudjaja e, victor h, cahyani ad, tan tj, pinontoan r. 2019. characterization of alphaamylase from aggregate f isolated aspergillus niger from a fermented cassava gatot grown in potato peel waste medium. microbiol biotechnol lett. 47:364-371. astriani a, diniyah n, jayus j, nurhayati n. 2018. phenotypic identification of indigenous fungi and lactic acid bacteria isolated from gatotan indonesian fermented food. 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2014a. modeling and optimising the growth of lasiodiplodia theobromae during gathotan fermentation. microbiol indones. 8(3):112-120. doi: 10.5454/mi.8.3.4. purwandari u, tristiana g, hidayati d. 2014b. gluten-free noodle made from gathotan flour: antioxidant activity and effect of consumption on blood glucose level. int food res j. 21(4):1629-1634. puspaningtyas d, sari p, kusuma r. 2019. exploring the potency of gathotan and gathot as diabetes functional food: resistant starch analysis. iop conf ser: earth environ sci. 207:012042. p 1-6. doi: 10.1088/17551315/207/1/012042. robert v, vu d, amor abh, van de wiele n, brouwer c, jabas b, szoke s, dridi a, triki m, daoud sb. 2013. mycobank gearing up for new horizons. ima fungus. 4:371-379. samson so, akomolafe o. 2017. fermentation: a means of treating and improving the nutrition content of cassava ( c.) peels and reducing its cyanide manihot esculenta content. genomics appl biol. 8(3):16-24. doi: 10.5376/gab.2017.08.0003. 6 hallis et al . microbiol indones sari pm, puspaningtyas de, kusuma rj. 2018. dietary fiber and carboydrate contents of gathotan and gathot as functional food for people with diabetes mellitus. j gizi dietetik indones. 5:88-92. schoch cl, seifert ka, huhndorf s, robert v, spouge jl, levesque ca, chen w. 2012. nuclear ribosomal internal transcribed spacer (its) region as a universal dna barcode marker for . proc natl acad sci fungi usa. 109(16):6241-6246. stucky bj. 2012. seqtrace: a graphical tool for rapidly processing dna sequencing chromatograms. j biomol tech. 23:90-93. sugiharto s, yudiarti t, isroli i. 2016. assay of antioxidant potential of two filamentous fungi isolated from the indonesian fermented dried cassava. antioxidants (basel). 5(1):6-11. doi: 10.3390/antiox5010006. susanti d. 2010. identifikasi spesies-spesies kapang kontaminan dan spesies kapang dominan pada gaplek di beberapa pasar di kabupaten mojokerto [species identification of contaminant fungi and dominant fungi in gaplekfrom traditional market in mojokerto] [thesis]. malang (id): universitas negeri malang. walters w, hyde er, berg-lyons d, ackermann g, humphrey g, parada a, gilbert ja, jansson jk, caporaso jg, fuhrman ja, apprill a, knight r. 2016. improved bacterial 16s rrna gene (v4 and v4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. msystems. 1(1):e00009-15. doi: 10.1128/msystems.00009-15. white tj, bruns t, lee s, taylor j. 1990. amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis ma, gelfand dh, sninsky jj, white tj, editors. pcr protocols: a guide to methods and applications. san diego (us): academic press p 315-322. yudiarti t, sugiharto s. 2016. a novel filamentous fungus acremonium charticola isolated from gathot (an indonesian fermented dried cassava). int food res j. 23(3):1351-1354. volume 15, 2021 microbiol indones 7 04. khalila.cdr vol.14, no.1, march 2020, p 25-33 doi: 10.5454/mi.14.1.4 isolation and characterization of thermophilic bacteria as cellulolytic enzyme producer from the hot spring of ie seuum aceh besar, indonesia 1 2* 2 ruhul khalila , lenni fitri , and suhartono 1 program of biology, faculty of mathematics and natural sciences, universitas syiah kuala, jalan syech abdurrauf no.3, kopelma darussalam, syiah kuala, banda aceh 23111, aceh, indonesia; 2 department of biology, faculty of mathematics and natural sciences, universitas syiah kuala, jalan syech abdurrauf no.3, kopelma darussalam, syiah kuala, banda aceh 23111, aceh, indonesia. cellulase enzymes can be isolated from thermophilic bacteria obtained from the hot spring ie seuum, aceh besar. this research aimed to recover and characterize the isolates morphologically and biochemically followed by determination of the thermophile bacterial isolates potential as cellulolytic enzyme producers, the sampling o o o method in this research was conducted by a purposive sampling at temperature of 70 c, 60 c, and 50 c. isolation of thermophilic bacteria was carried out on nutrient agar (na) media. there were four isolates of thermophilic o o o bacteria isolated recovered at 70 c, five isolates at 60 c, and seven isolates at 50 c. of the 18 isolates obtained, 15 of them were able to produce cellulase enzymes. cellulase enzyme production can be determined by the presence of clear zones around bacterial colonies on cmc media after addition of 1% congo red drops and wash with 1 m nacl. the highest five cellulolytic index (ci) values w​​ ere obtained from isolates isb75; isb64; isb52; isb54; and isb56 that were 1.23; 2.22; 1.39; 1.59; and 1.10, respectively. biochemical tests carried out on 5 isolates with the highest cellulolytic index values showed that the bacterial isolate were suspected to be from the genera of bacillus sp. key words : cellulase enzymes, hot springs, ie seuum, thermophilic bacteri enzim selulase dapat diisolasi dari bakteri termofil yang diperoleh dari sumber mata air panas ie seuum, aceh besar. penelitian ini bertujuan untuk mendapatkan isolat, karakterisasi morfologi, menentukan isolat bakteri termofil yang mampu menghasilkan enzim selulase, dan mengetahui karakter biokimia isolat terpilih. metode o o o pengambilan sampel pada penelitian ini dilakukan dengan purposive sampling pada suhu 70 c, 60 c dan 50 c. isolasi bakteri termofil dilakukan pada media nutrient agar (na). hasil isolasi bakteri termofil yang diperoleh o o o adalah empat isolat pada suhu 70 c, lima isolat pada suhu 60 c, dan tujuh isolat pada suhu 50 c. dari 18 isolat yang didapatkan, 15 diantaranya mampu menghasilkan enzim selulase. produksi enzim selulase dapat diketahui dengan adanya zona bening di sekitar koloni bakteri pada media cmc setelah ditetesi congo red 1% dan dicuci nacl 1m. nilai indeks selulolitik (is) lima tertinggi diperoleh dari isolat isb75; isb64; isb52; isb54; dan isb56 yaitu masing-masing 1,23; 2,22; 1,39; 1,59; dan 1,10. pengujian biokimia dilakukan terhadap 5 isolat yang memiliki nilai indeks selulolitik tertinggi. hasil karakterisasi menunjukkan bahwa isolat bakteri tersebut diduga termasuk dalam genus bacillus sp. kata kunci : bakteri termofil, enzim selulase, ie seuum, sumber air panas microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-85260272003; email: lennifitri@unsyiah.ac.id plants, animals and microorganisms (said and likadja 2012). according to natsir et al. (2014), the use of enzymes originating from microorganisms in general is in great demand because it has stability at high temperatures and can be produced in large quantities. microorganisms that produce cellulase enzymes can be isolated from various sources such as bovine rumen fluid (setyoko and budi 2016), mangrove sediments (setyati and subagiyo 2012), compost (baharuddin et al. 2010) and hot springs (mukminin 2014). tuntun and huda (2014) states that hot springs are originated as a result of ground water coming out of the earth's crust after experiencing geothermal heating. one of the bacteria that can produce cellulase enzymes in hot springs is a group of thermophilic bacteria (hasanah et al. 2015). thermophile bacteria penzymes are proteins that are specifically synthesized by living cells to catalyze chemical reactions within cells (martoharsono 2006). based on the activity sites, enzymes have two types, namely intracellular enzymes, directly used in cells, and extracellular enzymes, released from cells into the environment to hydrolyze polymer molecules in the environment, such as cellulose, hemicellulose, lignin and others. one of the extracellular enzymes is cellulase (sholihati et al. 2015). according to pham et al. (2010), cellulase is a complex enzyme that is used to break down cellulose into glucose. cellulase enzymes can be produced by groups of are a group of bacteria that are able to adapt to hightemperature environmental conditions, ranging from 45‒80 °c. these thermophilic bacteria are not only tolerant of extreme environmental temperatures, but are also able to survive and reproduce at these temperature conditions (martharina 2010). one source of hot springs that can be used as a source of isolation of cellulolytic bacteria found in the aceh province is ie seuum. ie seuum is one of the hot spring bathing areas in the krueng raya valley which originates from the crater of the seulawah volcano. this hot spring area is located in ie seuum village, mesjid raya district, aceh besar regency, aceh province (wahyuni 2017). this hot spring has a temperature ranging around 45‒80 ºc and a neutral water ph of 7 so that it has the potential to obtain thermophilic bacteria that are able to produce enzymes. therefore, it is necessary to conduct a research on the ie seuum aceh besar hot spring to obtain thermophile bacterial isolates that produce cellulase enzymes. the purpose of this research was to isolate thermophilic b a c t e r i a , c h a r a c t e r i z e m o r p h o l o g i c a l l y a n d biochemically thermophilic bacteria producing cellulase enzymes from the ie seuum aceh besar hot spring. materials and methods materials. the materials used in this research were hot water samples, nutrient agar (na) media, nutrient broth (nb) media, carboxy methyl cellulose (cmc) agar media, mgso .7h o, k hpo , cacl , feso , 4 2 2 4 2 4 kno , yeast extract, glucose, agar, 1% congo red dye, 3 1m nacl, violet crystal reagent, safranin, iodine, kovac's reagent, red methyl, h o 3%, simmons 2 2 citrate agar (sca), sulfide indol motility (sim) media, triple sugar iron agar (tsia) media, methyl red-vogue proskauer (mr-vp) media, aquadest, 70% alcohol, blank disc paper, and pipette tips. water sampling. water sampling was performed using the ginting method (2009). hot water samples suspected of containing thermophilic bacteria were taken from the ie seuum aceh besar hot spring. thermophile bacterial samples were taken at three different points, namely at 70 ºc, 60 ºc and 50 ºc (fig 1). each sample point was repeated three times. water samples were collected using a long dipper which had been sterilized using 70% alcohol, then put into a hot water flask and tightly closed. sample isolation. one ml of water samples were drawn using a micropipette and dropped into a petri dish. as much as 30 ml of liquid na media was poured into a petri dish which was shaken to keep the suspension flat in the media. after freezing, the petri o dish was incubated at 50 c for 24 hours. after incubation, bacterial colonies growing on na media were re-inoculated into sterile petri dishes containing na media by means of the quadrant method, then further incubated at 50 °c for 24 hours until single colonies were visible. characterization of thermophilic bacteria. the characterization of thermophilic bacteria was conducted through macroscopic, microscopic and biochemical observations. macroscopic observation was the observation of the shape of the colony, edge, elevation, color and surface of the bacterial colony. microscopic observations were made of the shape of bacterial cells and gram staining. the isolates which had the highest cellulolytic activity index values ​​were characterized by biochemical properties consisting of catalase tests, indole and motility tests, citrate test, tsia test, methyl red (mr) test, and voges-proskauer (vp) test. screening of cellulolytic bacteria. pure isolates were grown in na media, then isolated to nb liquid media for enzymatic assay. one loop of isolate was inoculated into 5 ml nb media and incubated at 50 °c for 24 hours. a 0.5 μl isolates were transferred using micropipette and dropped on blank paper disc on 1% solid cmc media, then incubated at 50 °c for 24 hours. if the isolate had grown on disc paper, then it was flooded using 1% congo red dye for 15 minutes, then rinsed 2-3 times using 1m nacl and allowed to stand for 15 minutes to detect the clear zone indicating the cellulolytic activity (rahmadini 2012). the formed diameter of the clear zone was measured using a calipers and the cellulolytic index (ci) is calculated (sari et al. 2012) using formula as follows (kader and omar 1998): ci = cellulolytic index data analysis. data analysis was carried out descriptively and displayed in the form of tables and images. results thermophilic bacteria isolate from ie seuum hot spring, aceh besar. there were 18 thermophilic bacterial isolates recovered from different temperature comprising of five isolates were from 70 ºc, six isolates 26 k et al.halila microbiol indones ci = clear zone diameter colony diameter colony diameter volume 14, 2020 microbiol indones 27 at 60 ºc, and seven isolates at 50 ºc (fig 2). morphological characteristics of thermophile bacterial isolates. the colony characterization showed that their shapes varied ranging from a circular with spread edge, irregular, wrinkled, circular to circular with a rounded edge. the edge of the colony was branching, choppy, curved, and irregular, whereas the elevation of the colony was flat, like a button, raised and hilly. the whole isolate was creamy with the rough and smooth surface. the results of gram staining from each isolate showed that all isolates were gram positive bacteria (table 1). based on the gram staining process all isolates showed the shape of bacilli cells and were purple (maintaining a violet crystalline dye) (fig 3). screening of thermophile bacteria that produce cellulase enzymes. the screening stage aimed to determine the cellulolytic activity of the bacterial isolates. this activity is shown by the ability of bacteria to hydrolyze cmc substrate. colonies that were able to hydrolyze cmc will form a clear zone or a light orange zone around the colony after being flooded with 1% congo red (fig 4). the clear zone of 18 isolates was measured based on cellulolytic index (ci). ci value was calculated by measuring the ratio of clear zone diameter towards colony diameter(table 3). biochemical test results of cellulolytic bacteria. biochemical characterization was performed on five thermophilic bacterial isolates showing the highest cellulolytic index values, namely isolates isb75, isb64, isb52, isb54, and isb56 (table 2). biochemical testing is a way to identify pure cultures of isolated bacteria through their physiological properties. the bacteria tested showed positive result on catalase test, except for isb64 which showed negative result. the bacteria tested showed negative results onsimmons citrate test. indole test result of all isolates were negative and motile. the bacteria showed positive results on tsia media, it was shown by the color change of the media into yellow on the buttocks and slants. the test results on mr media showed positive results only on isb64 isolate and other isolates showed negative results on vp test media. isolate code macroscopis character microscopic chara cter colony shape edge of the colony colony elevation colony pigment colony surface gram shape isb71 circular with spread edge branching flat cream rough positive basil isb72 irregular irregular like a button cream smooth positive basil isb73 irregular choppy convex cream rough positive basil isb74 irregular curved convex cream smooth positive basil isb75 irregular irregular arise cream rough positive basil isb61 irregular irregular convex cream rough positive basil isb62 circular with spread edge branching raised cream rough positive basil isb63 irregular branching hilly cream rough positive basil isb64 irregular choppy raised cream rough positive basil isb65 irregular curved like a button cream smooth positive basil isb66 wrinkled irregular raised cream rough positive basil isb51 circular with a rounded edges branching raised cream rough positive basil isb52 irregular branching raised cream rough positive basil isb53 circular irregular raised cream rough positive basil isb54 circular with spread edge irregular raised cream rough positive basil isb55 irregular irregular flat cream rough positive basil isb56 circular with spread edge branching raised cream rough positive basil isb57 irregular irregular raised cream rough positive basil table 1 results of morphological (macroscopic and microscopic) characterization of thermophilic bacterial isolates from the ie seuum hot spring, aceh besar o description: isb7: ie seuum bacteria temperature of 70 c o isb6: ie seuum bacteria temperature of 60 c o isb5: ie seuum bacteria temperature of 50 c� 28 k et al.halila microbiol indones fig 1 the map of sampling location in ie seuum hot springs, aceh besar, aceh, indonesia. (a) (b) (c) (d) (e) fig 2 results of the isolation of thermophilic bacteria, (a) isb75 isolates, (b) isb64 isolates, (c) isb52 isolates, (d) isb54 isolates, and (e) isb56 isolates. fig 3 gram stain results of isb64 isolates with 1000x magnification. fig 4 screening of isb64 isolate-producing cellulase thermophilic bacteria from the ie seuum hot spring, aceh besar. (a) clear zone, (b) bacterial colony, and (c) cmc media flooded with 1% congo red. volume 14, 2020 microbiol indones 29 biochemical test isolate code isb75 isb64 isb52 isb54 isb56 catalase + + + + simmons citrate indole motility + + + + + tsia (b/s) y/y y/y y/y y/y y/y methyl red + voges proskauer identification bacillus sp.1 bacillus sp.2 bacillus sp.1 bacillus sp.3 bacillus sp.1 table 2 biochemical test results of thermophilic bacterial isolates that produce cellulase enzymes from the hot spring ie seuum, aceh besar description: + : positive reaction : negative reaction� b : butt s : slant� y: yellow table 3 average colony diameter (cm), clear zone diameter (cm), standard deviation values ​​and cellulolytic index (ci) values ​​of thermophilic bacteria producing cellulase enzymes from the ie seuum hot spring, aceh besar isolate code colony diameter (cm) clear zone diameter (cm) cellulolytic index (ci) isb71 0,96 ± 0,19 1,92 ± 0,18 1,00 isb72 0,52 ± 0,07 1,01 ± 0,12 0,94 isb73 0 0 0 isb74 1,03 ± 0,16 1,36 ± 0,14 0,32 isb75 0,65 ± 0,04 1,45 ± 0,04 1,23 isb61 0,44 ± 0,07 0,66 ± 0,07 0,50 isb62 0,75 ± 0,28 0,89 ± 0,19 0,19 isb63 0,55 ± 0,20 0,82 ± 0,14 0,49 isb64 0,60 ± 0,02 1,93 ± 0,35 2,22 isb65 0 0 0 isb66 0 0 0 isb51 0,95 ± 0,10 1,80 ± 0,26 0,89 isb52 0,61 ± 0,15 1,46 ± 0,16 1,39 isb53 0,61 ± 0,01 1,02 ± 0,02 0,67 isb54 0,57 ± 0,09 1,48 ± 0,19 1,59 isb55 0,54 ± 0,04 0,86 ± 0,05 0,59 isb56 0,79 ± 0,09 1,66 ± 0,21 1,10 isb57 0,42 ± 0,16 0,85 ± 0,04 1,02 description: o isb7: ie seuum bacteria temperature of 70 c o isb6: ie seuum bacteria temperature of 60 c o isb5: ie seuum bacteria temperature of 50 c discussion thermophilic bacteria isolate from ie seuum hot spring, aceh besar. the results obtained are different from other research, where hastuti et al. (2012), succeeded in isolating 44 thermophilic bacterial isolates from the semerup hot spring, kerinci, jambi. moreover, ardani et al. (2012), isolating four chitinolytic thermophile bacterial isolates from the ie seuum hot spring, aceh besar. lutfi (2012), succeeded isolating 6 themophilic bacteria isolates that produced cellulase from panggo hot spring, sinjai regency, south sulawesi and hasanah (2015) succeeded isolating 5 isolates of cellulase-producing thermo-alkalifilic bacteria in pariangan hot spring, west sumatra. the difference in the number of isolates successfully isolated from each point can be influenced by environmental conditions that support-bacterial life. the environmental conditions comprises biotic and abiotic factors, including the presence of dry leaves, grass and moss, as well as temperature and ph (pitri et al. 2015). environmental conditions contained in the sampling location at ie seuum were deciduous leaves, grasses, moss, inorganic waste and other organic 30 k et al.halila microbiol indones sources which were a source of energy for microorganisms. abiotic environmental conditions that occured in sampling location were temperature and ph. the temperature range at the hot water sampling location was 45‒80ºc with ph of 7. morphological characteristics of thermophile bacterial isolates. the results obtained in the study were aligned with other study (wahyuni 2017) signifying that morphological features of the thermophilic bacteria were irregular and circular colony forms with the color of the cream colony and arise and flat colony elevation, and rough and smooth shiny colonies. the result of runtuboi et al. (2018) showed that the bacteria had of regular, rhizoid, and irregular rounded colony form, the elevation of colonies were convex, like a button and flat, the colony margin were flat, bumpy, curved, and jagged, the colony pigment were white and yellow, and the texture of colony surface were shiny, rough and wrinkled. observations on the morphological characteristics of bacterial colonies need to be made in order to facilitate the process of identifying the types of bacteria. according to lay (1994), based on the morphological characteristics of bacterial colonies, the process of identification of types of microorganisms can be carried out, but to obtain perfect identification results it must proceed with biochemical tests. the results of gram staining from each isolate showed that all isolates were gram positive bacteria. based on gram staining, all isolates were bacilli and had purple color (maintaining a violet crystalline dye). this is in accordance with wahyuni (2017); mukminin (2014); and sianturi (2008) studies, the result of the studies showed that gram staining of hot water bacteria were gram positive bacilli bacteria. according to lay (1994), gram staining is used to determine the morphology of bacterial cells as well as to distinguish between gram positive and gram negative bacteria. gram-positive bacteria in gram staining will be purple while gram-negative bacteria will be red. grampositive bacteria in gram staining are purple due to the violet-iodine crystalline complex which is retained even if given a pale alcohol solution, while gram-negative bacteria are red because the complex is soluble when giving 96% alcohol solution so that it absorbs the red color from safranin. screening of thermophile bacteria that produce cellulase enzymes. the formation of clear zone around the colony on the cmc media is caused by the process of cellulose degradation by cellulolytic bacteria. hydrolyzed cellulose in the media so that if inundated by congo red 1% will produce a clear zone because congo red cannot bind to the media without β-1,4glycosidic bonds contained in cellulose polymers due to the presence of cellulase enzymes so that the binding hydrolyzed cellulose polymers (jo et al. 2011). the clear zone formed could be seen by adding congo red dye then rinsed with 1 m nacl. the addition of congo red dye was to clarify the clear zone formed so that it could be easily observed (astriani 2017). congo red is sodium salt from benzidinediazo-bis1-naphthylamine-4 sulfonic acid (c h n na o s ) so 32 22 6 2 6 2 that this dye will dissolve and be washed by other sodium salts, such as nacl (hartanti 2010). washing with nacl will fade congo red especially in the area around the colony that contains hydrolyzed cellulase derivatives such as cyclodextrin, cellobiose and glucose because congo red is not tightly bound so that a clear zone appears (seprianto 2017). the presence of clear zones caused by cellulase enzymes released into the media is colorless metabolites. bacteria produce extracellular enzymes to hydrolyze food sources that contain cellulose contained in the media. cellulose molecules cannot enter into bacterial cells because of their large size, therefore cellulose molecules are hydrolyzed in advance by cellulase enzymes to become simpler cellobiose molecules. these molecules will then be transported into bacterial cells and used as a carbon source for their growth activities (lutfi 2012). the clear zone of 18 isolates was measured based on cellulolytic index (ci) (table 3). only 15 isolates of 18 isolates obtained had the ability to produce cellulase enzymes. four isolates were recovered from 70 ºc, four isolates at 60 ºc, and at seven isolates at 50 ºc with a range of cellulolytic index of 0.19‒2.22. isolate isb75; isb64; isb52; isb54; and isb56 had the highest cellulolytic index values o​​ f 1.23; 2.22; 1.39; 1.59; and 1.10, respectively. isolate isb62 had the lowest cellulolytic index value of 0.19. according to choi et al. (2005), if the cellulolytic index value ≤ 1 is in the low category, between 1‒2 is in the medium category, and ≥ 2 is in the high category. cellulolytic index (ci) values o​​ btained in this study were moderate to high category. similar result was shown in the study by behera's (2014), the result showed that bacteria obtained from mangrove soil in mahanadi river which had cellulolytic index value of 1.18 to 2.5 cm. a different result was obtained on the study by sari et al. (2012) that succeeded isolating cellulolytic bacteria from medang river hot springs with the highest cellulolytic index value of 5.40, meanwhile a study by sonia and kusnadi volume 14, 2020 microbiol indones 31 (2015) obtained cellulolytic bacteria isolated from tengger-bromo desert that had cellulolytic index values o​​ f 1.1 to 11. a study by nababan et al. (2019) showed that cellulolytic bacteria isolated from several forest soils in bali and compost had cellulolytic index value of 4.50 to 7.98. according to pitri et al. (2015), the difference in cellulolytic index values i​​ s due to the ability of each isolate to hydrolyze cmc substrate differently so that it influences the size of the formed clear zone. according to arji et al. (2018), the size of clear zone formed depends on the bacteria ability to degrade complex molecules into simple molecules. based on the results of the enzyme testing, it can be seen that the highest number of cellulase-producing isolates were found at 50 ºc, that is, as many as seven isolates and isolates with the highest cellulolytic index o values w​​ ere found at 60 c. this is consistent with the research of sari et al. (2012) demonstrating that eleven isolates producing cellulase enzymes were recovered from 50 ºc of the medang river hot springs, but the highest cellulolytic index values w​​ ere shown by the isolates recovered from 78 ºc. ibrahim and el-diwany (2007) signified that if the selection results only get a few isolates that produce clear zones, this shows that bacteria that produce less cellulase caused by the low content of organic matter in the media that can be used as a carbon source by cellulolytic bacteria. biochemical test results of cellulolytic bacteria. bacteria isolates that showed positive catalase means that they are able to convert hydrogen peroxide into water and oxygen, allowing these bacteria classified into aerobic or facultative aerobic bacteria (sonia and kusnadi 2015). the negative result of the simmons test showed that all of these bacteria did not use citrate as their carbon source. the negative result of indole showed that the bacteria did not have the ability to break down amino acid tryptophan into indole compounds (arfah et al. 2014). bacteria that are motile indicated that the bacteria had flagellum which allowed the movement of bacteria. tsia test showed that all bacteria were able to ferment lactose and sucrose. isb64 isolate showed mr positive which means mixed acid fermentation on media was occurred. all bacteria isolates were unable to use butanadiol fermentation through vp test because it showed negative results (natsir et al. 2014). based on these data (table 2), bacterial identification can be done. identification in this study was carried out based on “cowan and steel's manual for the identification of medical bacteria third edition” by looking at the data from microscopic, macroscopic and biochemical characterization of thermophilic isolates, it was concluded that the five thermophilic bacterial isolates were thought to belong to the bacillus genera. according to corbin (2004), the colony of bacillus sp. has the general characteristics of having a whitish beige color and the shape of a round and irregular colony. bacillus sp. has a colony edge shape that is flat and uneven, the surface is rough and not slimy, and even tends to dry and powder. this is consistent with the morphological characterization results of bacterial isolates obtained. in general, the bacillus sp. has a positive stem and gram cell shape. bacillus sp. is motile, including positive gram in the form of short bacilli to single bacillus with a single arrangement, producing spores that are usually resistant to heat, aerobic, positive catalase and some species produce negative catalase. each species differs in the use of sugar as well as fermentative activity (hatmanti 2000; puspita et al. 2017). the results of this study are in accordance with huwae and aditiawati (2020), who found that cellulaseproducing bacteria obtained from sila hot spring had similarity to the genus bacillus. however, different results were obtained by arji et al. (2018), where the bacteria isolated from lainea hot spring, southeast sulawesi was likely belong to the genus listeria. references ardani f, yasmin y, fitri l. 2012. potential of chitinolytic hot springs bacteria as biological control of aedes aegypti larvae. j bio ed. 4(2): 77-81. arfah, ra, patong ar, ahmad a, djide mn. 2014. isolation and identification of amylase-producing thermophile bacteria from hot springs in lejja, south sulawesi. alkimia. 2(2): 36-46. doi: 10.24252/al-kimia.v2i2.1644. arji m, rezky w, nurhaliza wos, yanti na. 2018. isolation, characterization, and bacteria potential test of producing termostabil enzyme from lainea hot water sources, southeast sulawesi. biowallacea. 5(1): 726-734. doi: 10.33772/biowallacea.v5i1.4592. astiani m. 2017. screening for cellulolytic bacteria from banana garden (musa paradisiaca). biota journal. 3(1): 6-10. doi: 10.19109/biota.v3i1.871. baharuddin as, razak mna, hock ls, ahmad mn, aziz sa, rahman naa, shah ukm. 2010. isolation and characterization of themophilic cellulase-producing bacteria from empty fruit bunches-palm oil mill effluent compost. am j applied sci. 7(1): 56-62. doi: 10.3844/ajassp.2010.56.62. behera bc, parida s, dutta sk., thatoi hn. 2014. isolation 32 k et al.halila microbiol indones and identification of cellulose degrading bacteria from mangrove soil mahanadi river delta and their cellulose production ability. ajmr. 2(1): 41–46. doi: 10.12691/ajmr-2-1-6. choi yw, hodgkis ij, hyde kd. 2005. enzyme production by endophytes of brucea javanica. j agricultural technol. 29: 55-66. corbin bd. 2004. identification and characterization bacillus thuringiensis. j bacteriol. 186: 7736-7744. ginting y. 2009. bacterial isolation and thermophile amylase activity test from the spirit of hot springs of north sumatra mountain. 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[dissertation]. banda aceh (id): uin arraniry. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 vol.1 , no.1, march 202 , p -6 2 23 29 doi: 10.5454/mi.1 1.6. 23-29 optimization of spp. production as mycoprotein using soymilk mediarhizopus dani muliawan halim , anastasia tatik hartanti , stephanie 1 1* 2 and 1 program of food technology, faculty of biotechnology, atma jaya catholic university of indonesia. jalan raya cisauk-lapan 10, bsd city, tangerang 15345, banten, indonesia; 2 program of biology, faculty of biotechnology, atma jaya catholic university of indonesia. jalan raya cisauk-lapan 10, bsd city, tangerang 15345, banten, indonesia. mycoprotein is food with high protein content, fiber, and low in cholesterol made from fungal mycelium. in this research, mycoprotein was produced by spp. isolated from tempeh with soymilk as growth media.rhizopus this research aims to determine the best strain of spp. and optimum carbon to nitrogen ratio forrhizopus mycoprotein production two parameters were applied which were inoculum selection and carbon to nitrogen. ratio treatment in media. the best inoculum was selected from four strains of spp., ath 1, ath 24, athrhizopus 40, and ath 53. on the other hand, carbon to nitrogen ratio treatment used were as follows 20:1, 20:2, and 40:2. mycelium dry weight and protein content were measured, as well as reduction sugar, dissolved protein and total volatile base nitrogen concentration in media. the best strain for producing biomass was ath 24 with 0.6 g of0 mycelium dry weight per 50 ml of media and the protein content was 0.236 g. the best carbon to nitrogen ratio treatment was 20:1 with 0.57 gram of mycelium dry weight per 50 ml of media and the protein content was 0.2 g.0 thus, our data indicate that strain ath 24 with 20:1 of carbon to nitrogen ratio in media were highly potential for producing mycoprotein. : fermentation, mycelium, mycoprotein, , soymilkkey words rhizopus mikoprotein merupakan pangan yang tinggi protein, serat, dan rendah kolesterol yang terbuat dari miselium fungi. produksi mikoprotein pada penelitian ini menggunakan spp. yang diisolasi dari tempe denganrhizopus media pertumbuhan berupa susu kedelai. tujuan penelitian ini adalah menentukan galur spp. terbaik danrhizopus perbandingan karbon dan nitrogen optimum dalam memproduksi mikoprotein. penelitian ini terdiri dari dua tahap, yakni seleksi inokulum dan perlakuan perbandingan karbon terhadap nitrogen pada media. inokulum diseleksi dari empat galur spp. yang ath 1, ath 24, ath 40, dan ath 53, sedangkan variasi perlakuanrhizopus perbandingan karbon terhadap nitrogen terdiri dari 20:1, 20:2, dan 40:2. bobot kering dan protein biomassa ditentukan, serta kadar gula reduksi, protein terlarut, dan total nitrogen basa yang mudah menguap dalam media. galur terbaik dalam memproduksi mikoprotein adalah ath 24 dengan biomassa sebesar 0,6 gram bobot kering0 per 50 ml media dan kandungan protein sebesar 0,236 g. perlakuan perbandingan karbon dan nitrogen terbaik adalah 20:1 dengan produksi biomassa sebesar 0,57 gram bobot kering per 50 ml media dan kandungan protein sebesar 0,2 g. oleh karena itu, pilihan terbaik untuk memproduksi mikoprotein adalah menggunakan ath 240 dengan perbandingan karbon terhadap nitrogen pada media sebesar 20:1. kata kunci: fermentasi mikoprotein, miselium, , susu kedelairhizopus, microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone ;: +62-81384030344 fax: +62-; e-mail: anast.hartanti@atmajaya.ac.id azygosporus could produce chitosan by using media consist of soybean meal, peptone, glucose, and corn starch (chen . 2002). various types of media such aset al yeast nitrogen base (ynb) and sabouraud broth (sab) can be used to grow var.r. microsporus rhizopodiformis et al r. delemar(meletiadis . 2001). could produce 8 g/l biomass in glucose media (2%) and 8 g/l biomass in soybean peptone media (0.6%) (zhou et al. 2011). based on previous research chen . 2002by ( ),et al iftikhar . 2010 zhou . 2011 ,et al et al rhizopus( ), and ( ) spp. likely to grow in glucose and soybean containing media. therefore, in this research, soymilk will be used as the mycoprotein production media. soymilk, which is a natural media, has a lower cost rather than rich synthetic media, such as potato dextrose broth (pdb). indonesia has been known for its traditional food called tempeh, which is soybeans fermented by rhizopus rhizopusspp.. spp. isolated from tempeh is considered safe and potentially used for mycoprotein production. eight rhizopus spp species were identified: r. caepitosus r. delemar r. homothallicus r., , , microsporus r. oryzae r. reflexus r. schipperae,, , , and r. stolonifer et al(abe . 2010). several studies had proven that spp. can be grown on synthetic orrhizopus natural media to produce biomass. var.r. microsporus oligosporus could be grown in media consist of soybean meal and glucose, which produced yield (y ) of 0.5 g/gx/s substrate (iftikhar . 2010). var.et al r. microsporus soymilk also consists of 17.7% carbohydrate and 2.7% protein (hajirostamloo 2009). carbohydrate is the source of carbon, used as an energy source to produce cell wall polysaccharides. on the other hand, protein is the primary source of nitrogen used as an energy source to produce mycelium building blocks and shorten the adaptation phase of the cell (maulida . 2014).et al therefore, the amount of carbon and nitrogen in growth media is very important for fungal growth. it is worth noting that a high amount of carbon and nitrogen may interfere growth since optimum carbon to nitrogen ratio for fungal growth are vary (yalemtesfa . 2010).et al different strains of spp. may also haverhizopus different capabilities in producing mycoprotein. hartanti . (2015) had isolated several strains ofet al r. microsporus r. delemarand from fresh tempeh in indonesia, which will be used in this research to observe their potential as a mycoprotein source. thus, in this research, knowing the nutritional requirements for each rhizopus strain can lead to optimum biomass production, therefore, this study aims to determine the ability of spp. at different carbon to nitrogenrhizopus ratio in producing mycoprotein. materials and methods spp. strains used in thismaterials. rhizopus research consist of ath 1 (ipbcc 13.1102), ath 24 (ipbcc 13.1110), ath 40 (ipbcc 13.1120) and ath 53 (ipbcc 13.1126) obtained from atma jaya catholic university of indonesia culture collection. ath 1 is like var , ath 24r. microsporus oligosporus is like var , ath 40 isr. microsporus azygosporus r. microsporus rhizopodiformis r.like var and ath 53 is delemar et al(hartanti . 2015). all of them were maintained at 4°c on potato dextrose agar (pda) (oxoid, hampshire, gb). the fermentation media used in this research was soymilk (yeo's, singapore, sg). for the carbon to nitrogen ratio treatment, glucose (merck, darmstadt, de) and ammonium sulfate ((nh ) so ) (merck, darmstadt, de) were used.4 2 4 inoculum preparation. all spp. strainsrhizopus were grown on pda slant at 30°c for 48 hours. then, 5 ml of natrium chloride (nacl) solution with a concentration of 0.85% (w/v) was added into the slant to make a culture suspension. media preparation. the fermentation was divided into two stages, which were inoculum selection and carbon to nitrogen ratio treatment. for both stages, the growth media used was 50 ml. in inoculum selection, empty flasks were sterilized, and soymilk was poured in sterile conditions. it was similar to inoculum selection in carbon to nitrogen ratio treatment, but there were variations of carbon to nitrogen ratio of 20:1, 20:2, and 40:2. addition of glucose and (nh ) so were done using microfilter4 2 4 (axiva, faridabad, in) in sterile condition. the amount of carbon and nitrogen in soymilk was determined based on a calculation using data from literature and nutrition facts in the product (appendix 1). fermentation process. the amount of 2% culture suspension was inoculated into the media and incubated at 30°c and 125 rpm for 72 hours. yceliumm dry weight, reduction sugar, and dissolved protein were measured triplicate every 24 hours for inoculum selection. simultaneously, they were done only once for carbon to nitrogen ratio treatment at the end of fermentation with the addition of total volatile base nitrogen (tvbn) measurement. strain with the highest biomass production and protein content from inoculum selection was chosen to be used in carbon to nitrogen ratio treatment. m y c e l i u m d r y we i g h t m e a s u r e m e n t . grown mycelium from the culture was filtered using a stainless steel filter, washing was done usingthen sterile distilled water. cleaned mycelium was put into a petri dish with filter paper (sartorius stedim, göttingen, de) pads and dried using the oven at 50°c for 24 until 48 hours. dried mycelium was measured periodically until constant weight to obtain the mycelium dry weight. reduc sugar measurement.ing reducing sugar w a s m e a s u r e d b y u s i n g d n s m e t h o d spectrophotometer (thermo fisher scientific, waltham, us) at 520 nm .as reported by miller (1959) reduc sugar concentration was obtained bying calculating absorbance value into the standardglucose curve equation. dissolve protein measurement. dissolve protein was measured using spectrophotometer at 595 nm as reported by bradford (1976). dissolve protein concentration was obtained by calculating absorbance value into the standard curvebovine serum albumin equation. tnvb measurement. tnvb was measured using the conway micro diffusion method. 1 ml of boric acid (4%) was pipetted into the inner ring. for the outer ring, 1 ml of potassium carbonate (20%) and 1 ml of fermentation culture was added opposite each other. the outer ring solution was slowly homogenized by using 8 movements, and then the dish was incubated at 37°c for 2 hours. the boric acid from the inner ring was 24 halim et al. microbiol indones volume 1 2026, 2 microbiol indones 25 titrated with 0.02 n hcl and 2 drops of bcg-mr as an indicator. the titration was done when the solution's color change from green to red, and the tnvb concentration was calculated using this formula (ng and 1992):low total nitrogen measurement. total nitrogen measurement was done by sending the myceliumdried to livestock research institute in bogor, west java, indonesia. the measurement was done using the kjeldahl method, where the amount of sample was destructed using sulfuric acid. then, the sample was diluted in distilled water and distilled. the distillate was accommodated in a flask contained boric acid (4%) and was titrated using chloric acid with bcg-mr as an indicator. the titration was done when the solution's color change from green to red, and the nitrogen concentration was calculated using this formula (aoac 2005). statistical analysis. statistical analysis was performed in statistical package for the social sciences (spss) using analysis of variance (anova) with tukey's wholly significant difference (wsd) as post hoc test. all these tests were performed with p<0.05 as a significant level. results inoculum selection. based on our data, longer fermentation time resulted in higher biomass. inoculum ath 1, ath 24, and ath 40 highest mycelium dry weight were produced after 72 hours fermentation, while ath 53 was at 48 hours. ath 24 had the best capability in producing biomass. ath 24 produced 0.2 g, 0.4 g and 0.6 g of mycelium dry0 0 0 weight at 24, 48 and 72 hours, respectively, meanwhile ath 53 produced 0.34 g of mycelium dry weight at 48 hours. ath 24 biomass increased significantly from 24 hours to 48 hours and then 72 hours. it was also significantly higher than ath 1 and ath 53 (fig 1). it is indicated in our study that mycelium dry weight production was not correlated to sugar consumption. after 72 hours of fermentation, ath 24 biomass was the highest while it consumed the least amount of sugar though it was not significant to ath 1 and ath 40. the initial concentration of reducing sugar in the media was 2259 µg ml-1. reduction sugar ath 24 was kept decreasing throughout the fermentation, and the final concentration was 323 µg ml-1. while ath 1 and ath 40 reduction sugar was decreasing at 24 hours, increased at 48 hours, and decreased again at 72 hours. the final concentrations were 232 µg ml-1 and 233 µg ml-1, respectively. ath 53 had a different trend from the others because the reduction in sugar was decreasing at first 24 hours then, it increased until fermentation ended with final concentration was 1002 µg ml-1. (fig 2). unlike the reducing sugar concentration, dissolved protein concentration decreased since the fermentation started until it ended. the initial dissolved protein concentration in the media was 9725 µg ml-1. at the end of fermentation, the concentration in ath 53 was the lowest, which was 401 µg ml-1, followed by ath 1, ath 40, and ath 24 with 1138 µg ml-1, 2404 µg ml1, and 3988 µg ml-1, respectively. ath 24 consumed the least amount of dissolved protein that was significant to the others and it could produce a high amount of biomass, while ath 53 consumed the most dissolved protein but produced the lowest biomass (fig 3). based on total protein measurement, ath 53 had the highest protein content, followed by ath 24, ath 1 and ath 40 with 40.65, 38 98, 37.59 and 36.02%, . , (w/w), respectively. however, after multiplication to the mycelium dry weight, ath 24 had the highest protein content at 24, 48 and 72 hours of fermentation., at 24 hours, ath 24 had 0.079 g of protein, which was not significant to ath 40 with 0.052 g of protein, but significant to ath 53 and ath 1 with 0.047 and 0.03 g of protein, respectively. at 48 hours, ath 24 produced 0.154 g of protein, which was not significant to ath 40 and ath 53 with 0.135 and 0.138 g of protein, respectively, but significant to ath 1 with 0.084 g of protein. at 72 hours, ath 24 produced 0.236 g of protein, which was significant to ath 40, ath 1 and, ath 53 with 0.2, 0.17 and 0.102 g of protein,, respectively. therefore, ath 24 with the highest biomass and protein content was used for the carbon to nitrogen ratio treatment (fig 4). carbon to nitrogen ratio treatment. the initial carbon to nitrogen ratio in soymilk was 20:1. the addition of (nh ) so in 20:2 treatment increased the4 2 4 biomass production, protein content in mycelium dry weight, dissolved protein, and tvbn consumption compared to 20:1 treatment. while the addition of %tvbn (w/v) = (v sample titration v blank titration) x n hcl x 100 x 14.008 v sample x 1000 %n = (v sample titration v blank titration) x n hcl x 14.008 mg sample x 100% total protein (wet base) = %n x conversion factor (6.25) %protein (dry base) = % protein wb 1 water content x 100% 26 halim et al. microbiol indones fig biomass production of rhizopus spp. in soybean milk medium incubated at 30°c and 125 rpm for 72 hours.1 fig reduction sugar concentration in soybean milk media during 72 hours fermentation by rhizopus spp.2 a,b,c,d,e,f means with different superscript differ significantly (p < 0.05) experiment were done in six replicates a,b,c,d,e,f means with different superscript differ significantly (p < 0.05) experiment were done in six replicates glucose and (nh ) so in 40:2 treatment increased4 2 4 sugar and dissolved protein consumption, but decreased biomass production, protein content in mycelium dry weight and tvbn consumption compared to 20:1 treatment. the difference in biomass production and protein content in 20:2 and 40:2 treatments was not significant to 20:1 (table 1). discussion our study indicat s that ath 24 has the higheste biomass production. it could be due to the formation of azygospores (hartanti . 2015). azygospores iset al zygospores which can be formed without the mating process. zygospores are round balls that are formed when mycelia in fungi interact and fuse. after the formation, zygospores will make sporangium that contains spores for reproduction. zheng . (2007)et al stated that the formation of azygospores could be induced by lecithin in the growth media and there is lecithin in soymilk around 0.5% (van ee 2009). zheng et al r. microsporus. (2007) also stated that var rhizopodiformis was heterothallic fungi that could form zygospores. on the other hand, r. microsporus volume 1 2026, 2 microbiol indones 27 var and could neither formoligosporus r. delemar zygospores nor azygospores. homothallic fungi have male and female reproductive structures on the same mycelium, which is self-compatible. on the ,contrary heterothallic fungi need mycelium from other fungi to reproduce because they only have one type of reproductive structure in its mycelium (ulloa and hanlin 2000). therefore, ath 1, ath 40 and ath 53 could only reproduce using an asexual system, which are spores. based on this evidence, the only strain which has an advantage in reproduce was ath 24. the difference in biomass production of ath 1, ath 40 and ath 53 may be caused by the physiology characteristic and the capability to utilize nutrition in the growth media. on the other hand, protein content in mycelium dry weight was depended on the mold ability to produce amino acids (ahmed . 2017).et al therefore, ath 24 was the best strain in utilizing nutrients to produce amino acids because it has the highest protein content. rhizopus spp. can utilize nutrition in media because they can produce specific amounts of enzymes like amylase, protease, and lipase (wong 1995). there are many types of soluble carbohydrates in soybean, fig protein content in rhizopus spp. mycelium dry weight produced in soybean milk medium incubated at4 30°c and 125 rpm for 72 hours. fig 3 dissolved protein concentration in soybean milk media during 72 hours fermentation by rhizopus spp. a,b,c,d,e,f means with different superscript differ significantly (p < 0.05) experiment were done in six replicates a,b,c,d,e,f,g means with different superscript differ significantly (p < 0.05) experiment were done in six replicates 28 halim et al. microbiol indones table 1 effect of carbon to nitrogen ratio treatment to the fermentation parametres of ath 24 a,b means in same column with different superscript differ significantly (p<0.05) experiment were done in six replicates treatment (c:n) biomass (gram) rate of reduction sugar consumption (µg ml-1 h-1) rate of dissolved protein consumption (µg ml-1 h-1) protein (gram) 20:1 0.57±0.08(ab) 31.49±0.35(a) 83.94±6.13(a) 0.0009±0.0011(ab) 0.20±0.03(a) 20:2 0.61±0.08(a) 31.41±0.72(a) 86.46±8.98(a) 0.0022±0.0038(a) 0.21±0.03(a) 40:2 0.50±0.05 (b) 76.69±1.19(b) 98.66±14.09(a) -0.0021±0.0023(b) 0.17±0.02(a) ∆ [tvbn] (µg ml ) -1 such as sucrose, raffinose, stachyose, sugar alcohols, etc (obendorf and kosina 2011). at the beginning of fermentation, the molds would begin to consume simple carbohydrates in media, but complex carbohydrates would also be digested when they run out of simple carbohydrates. the increment of reduction sugar concentration in ath 1, ath 40 and ath 53 after 24 hours might be caused by their speed in hydrolyzing carbohydrates was greater than utilizing them. on the other hand, ath 24 was faster in utilizing carbohydrates rather than hydrolyzing them. the decrement of reducing sugar concentration in ath 1 and ath 40 after 48 hours was because the media was run out of complex carbohydrates. however, reducing sugar in ath 53 was increasing after 48 hours though the mold was already in the death phase. it might be caused by carbohydrase was still active in digesting the complex carbohydrates. this occasion could be because mold's enzyme was produced extracellular, which still works though the cell was dead (sinsabaugh 1994). dissolved protein concentration decreased throughout the fermentation process, indicating that the protein was consumed. tvbn measurement using conway micro diffusion method was done because bradford method can only measure protein content, where (nh ) so is4 2 4 not protein (bradford 1976). from the result, it could also be seen that the bradford method can only measure the dissolved protein content, while the conway micro diffusion method cannot measure protein content, but it can measure simple molecule with nitrogen atom-like (nh ) so . the 20:2 treatment triggered ath 24 to4 2 4 consume more dissolved protein and (nh ) so .4 2 4 however the same occasion did not happen in the 40:2 treatment because it triggered ath 24 to consume more sugar and protein. the negative result of tvbn measurement in 40:2 might be caused by the degraded protein was also measured after 72 hours of fermentation, while the original (nh ) so added into4 2 4 the media was not consumed yet. therefore, the final tvbn concentration becomes higher than the initial concentration. the 20:2 treatment produced the highest biomass and protein content in mycelium dry weight, but the difference was not significant to 20:1 (p < 0.05), while the 40:2 treatment had the lowest biomass and protein content. yalemtesfa . (2010) stated that theet al addition of (nh ) so as a nitrogen source produced a4 2 4 higher protein level in spp. andchaetomium aspergillus niger. it also applied to ath 24 in 20:2 treatment. however, it did not happen in 40:2 treatment, which might be caused by the addition of sugar disturbed the growth and ability to produced amino acids of ath 24. ahmed . (2017) stated thatet al a reasonable amount of carbon to nitrogen ratio is the key to high-quality harvesting biomass. it meant that a higher amount of carbon and nitrogen did not guarantee higher biomass and protein content production. from all those results, ath 24 with a c/n ratio of 20:2 could produce 0.61 g of mycelium dry weight with 0.21 g of protein, but difference was not significantthe (p > 0.05) compared to ath 24 with a c/n ratio of 20:1 which dproduce 0.57 g of mycelium dry weight with 0.2 g . therefore, using a 20:1 ratio will be0 of protein more economical because it is not necessary to add any other nutrient. the mycoprotein had similar protein content with beef, chicken, chicken egg, soybean and tempeh. the protein content of 0.57 g of beef, chicken, chicken egg, soybean and tempeh were 0.15, 0.15, 0.07, 0.2 , and 0.11 g, respectively (usda 2019). on0 the other hand, if it was compared to the growth media, the mycelium dry weight protein content was much higher because 0.57 ml of soymilk only had 0.01 g of protein. therefore, it was beneficial to produce mycoprotein using soymilk to increase the protein content by 20 folds. volume 1 2026, 2 microbiol indones 29 acknowledgements this research was supported by faculty of biotechnology, atma jaya catholic university of indonesia. references ahmed s, mustafa g, arshad m, rajoka mi. 2017. fungal biomass protein production from trichoderma harzianum using rice polishing. biomed res int. 2017(1): 1-9. : 10.1155/2017/6232793.doi [aoac] association of official analytical chemyst. 2005. official method of analysis of the association of official analytical of chemyst. arlington (us): aoac inc. bradford mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal biochem. 72(1-2): 248-254. : 10.1016/0003-doi 2697(76)90527-3. chen m, chan h, wu c, chuang s, hwang i, chen y, yuan g, inventor food industry research ands; development institute hsinchu( , tw), assignee. 2002 nov 26. production of chitosan and chitin. united states of america patent us 6 485 946 b1., , hajirostamloo b. 2009. comparison of nutritional and chemical parameters of soymilk and cow milk. ijmsir. 3(9): 455-457. hartanti at, rahayu g, hidayat i. 2015. speciesrhizopus from fresh tempeh collected from several regions in indonesia. hayati j biosci. 22(3): 136-142. :doi 10.1016/j.hjb.2015.10.004. iftikhar t, niaz m, zia ma, haq i. 2010. production of extracellular lipases by in arhizopus oligosporus stirred fermentor. braz j microbiol. 41(4): 1124-1132. doi: 10.1590%2fs1517-838220100004000034. kumar v, rani a, husain l. 2016. investigations of amino acids profile, fatty acids composition, isoflavones content and antioxidative properties in soy okara. asian j chem. 28(4): 903-906. : 10.14233/ajchem.doi 2016.19548. maulida r, ratnaningtyas ni, priyanto s. 2014. produksi miselium (dickson: fries) graygrifola frondosa isolate cianjur dan bobot ekstraknya pada medium mypb dengan penambahan biji bunga matahari helianthus annuus l. scripta biologica. 1(1): 26-29. meletiadis j, meis jfgm, mouton jw, verweij pe. 2001. analysis of growth characteristics of filamentous fungi in different nutrient media. j clin microbiol. 39(2): 478-484. : 10.1128/jcm.39.2.478-484.2001.doi miller gl. 1959. use of dinistrosalicylic acid reagent for determination of reducing sugar. anal chem. 31(3): 426-428. : 10.1021/ac60147a030.doi ng cs low lk, . 1992. determination of trimethylamine oxide , trimethylamine total(tmao-n) (tma-n), volatile basic nitrogen by onway's micro-(vb-n) c diffusion method (1% boric acid and 0.02n hydrochloric acid) in: miwa k, low sj, editors.. laboratory manual on analytical methods and procedures for fish and fish products. 2nd ed. singapore sg( ): southeast asian fisheries development center. p. b-3.1-b-3.7. obendorf rl, kosina sm. 2011. soluble carbohydrates in soybean. ng tb, editor. soybean: biochemistry,in: chemistry and physiology london (gb): intech.. p. 201-228. sinsabaugh rs. 1994. enzymic analysis of microbial pattern and process. biology and fertility of soils. 17(1): 6974. : 10.1007/bf00418675.doi thrane u. 2007. fungal protein for food. dijksterhuis j,in: samson ra, editor food mycology: a multifaceteds. approach to fundi and food. boca raton (us): crc press. p. 353-360. ulloa m, hanlin rt. 2000. illustrated dictionary of mycology. saint paul (us): aps press. [usda] united states department of agriculture. 2019. food data central. https://fdc.nal.usda.gov/index.html was accessed on june 22 , 2019. nd [ussec] united states soybean export council. 2010. soy n u t r i t i o n a l c o n t e n t . h t t p s : / / u s s e c . o r g / w p content/uploads/2015/10/nutrional-content-soy.pdf was accessed on march 2 , 2019. nd van ee jh. 2009. soy constituents: modes of action in lowdensity lipoprotein management. nutrition reviews. 6 7 ( 4 ) : 2 2 2 2 3 4 . : 1 0 . 1 1 1 1 / j . 1 7 5 3 -d o i 4887.2009.00192.x. wong dws. 1995. food enzymes: structure and mechanism. new york (us): chapman & hall. yalemtesfa b, alemu t, santhanam a. 2010. solid substrate fermentation and conversion of orange waste in to fungal biomass using ka-06 andaspergillus niger chaetomium spp kc-06. afr j mirobiol res. 4(12): 1275-1281. zheng r, chen g, huang h, liu x. 2007. a monograph of rhizopus. intern j mycology. 59(2): 273-372. zhou z, du g, hua z, zhou j, chen j. 2011. optimization of fumaric acid production by based onrhizopus delemar the morphology formation. bioresour technol. 102(1): 9345-9349. : 10.1016/j.biortech.2011.07. 120.doi cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 15, number 3, september 2021 domestication and formulation of as a rhizopodopsis javensis tempeh starter are and trichoderma atroviride trichoderma harzianum effective to control associated ith tomato wilt?fusarium w production and characterization of thermoalkaliphilic xylanase from cm1, and its application on bacillus halodurans degumming process of ramie ( l.gaud fiber as boehmeria nivea ) textile raw material analysis of he phytochemical characteristics of crude extract of t jasmine flower against and escherichia coli staphylococcus aureus comparison of n-acetyl-l-cysteine-sodium hydroxide-based and modifie d petroff's decontamina tion method for mycobacterium tuberculosis culture gayuh rahayu, efriwati, and septina veronica wilfridus adyatma putranto, rully adi nugroho, petrus sunu hardiyanta, and desti christian cahyaningrum dewi nandyawati, dea indriani astuti, niknik nurhayati, asep riswoko, and is helianti n a t h a s a we i s d a n i a s i h i t e , h e r l a rusmarilin, and manuntun rotua ariyani kiranasari and muhammad rayhan 77 84 91 102 113 04. sihite.cdr vol.15, no.3, september 2021, p 102-112 doi: 10.5454/mi.15.3.4 analysis of he phytochemical characteristics of jasmine flower crude extract t against and escherichia coli staphylococcus aureus nathasa weisdania sihite *, herla rusmarilin , 1 2 1 and manuntun rotua 1nutrition department, health polytechnic palembang, palembang, indonesia; 2 food technology department¸ universitas sumatera utara, medan, indonesia. indonesia is a famous country whose high diversity of plants, especially agricultural product and herbs, which has a potential of antimicrobial such as like f (l.) jasmine lower ait(jasminum sambac ). previous research discovered the presence of active compound that could inhibit bacterial growth. this study aimed to determine the phytochemical and antimicrobial activity of jasmine flower. the crude extraction was obtained by maceration, using water solvent, methanol, ethyl acetate, and n-hexane as the solvent; respectively. the inhibition assay of pathogenic microbes ( and ) was conducted using the dilution method. the escherichia coli staphylococcus aureus sample bacteria used were and this study escherichia coli (atcc 8938) staphylococcus aureus (atcc 6538). used disc diffusion method, with a complete 2-factor random design and 4 replications. data processing was conducted using anova. the results showed that the jasmine extracts with a concentration of 25%, 50%, 75% and 100% had an effect to inhibit of the growth of the bacteria. the extract with ethyl acetate solvent was the most effective in inhibiting the growth of bacteria. the results showed that jasmine flower extract contained alkaloids, flavonoids, steroids, saponins, tannins, and phenolics. minimum inhibitor concentration (mic) occurred at concentrations of 0.1% and 0.25% of jasmine extract on , staphylococcus aureus and escherichia coli respectively. jphytochemical content contained in asmine flower extracts by literature study has the potential as an antimicrobials and needs to be proven by further research. antimicrobials, jasmine, key words: escherichia coli, sthaphylococcus aureus indonesia merupakan negara yang terkenal dengan keanekaragaman hayati khususnya tanaman dan herbal yang memiliki potensi antimikroba seperti bunga melati ( (l.) ait). penelitian sebelumnya jasminum sambac menemukan adanya senyawa aktif yang dapat menghambat pertumbuhan bakteri. penelitian ini bertujuan untuk mengetahui aktivitas fitokimia dan antimikroba bunga melati. ekstraksi dilakukan dengan cara maserasi dengan menggunakan pelarut air, metanol, etil asetat, dan heksana sebagai pelarut. pengujian ekstrak dilakukan dengan metode pengenceran. sampel bakteri yang digunakan adalah dan staphylococcus escherichia coli (atcc 8938) aureus . penelitian ini menggunakan metode difusi cakram, dengan rancangan acak lengkap 2 faktor (atcc 6538) dan 4 ulangan. pengolahan data dilakukan dengan menggunakan anova. hasil penelitian menunjukkan bahwa ekstrak melati dengan konsentrasi 25%, 50%, 75% dan 100% berpengaruh dalam menghambat pertumbuhan bakteri. ekstrak bunga melati dengan pelarut etil asetat paling efektif untuk menghambat pertumbuhan bakteri. hasil penelitian menunjukkan bahwa ekstrak bunga melati mengandung alkaloid, flavonoid, steroid, saponin, tanin dan fenolik. minimum inhibitor concentration (mic) terdapat pada konsentrasi 0,1% (staphylococcus aureus escherichia coli) dan 0,25% ( ). kandungan fitokimia yang terdapat pada ekstrak bunga melati secaja kajian memiliki potensi sebagai antimikroba dan perlu dibuktikan dengan penelitian lebih lanjut lagi. kata kunci: antimikroba, bunga melati, escherichia coli, sthaphylococcus aureus microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 62-81362007718;: + e-mail: weisdania@gmail.com oil (outright), which is utilized within the cleanser, corrective, pharmaceutical, fragrance, fragrance-based treatment, and spa businesses. jasmine blossoms are too frequently utilized as an added substance in making tea . species is the (tahir 2017)et al. jasminum sambac foremost popular species in indonesia and has been named the nation's puspa flowers. . (suyanti 2003)et al. jasmine flowers have been known for a long time as a traditional medicinal ingredient including for the treatment of asthma, ulcers, and joint pain (patil and patil 2011). the flowers, leaves, and stems of jasmine contain various chemical components such as alkaloids, glycoside, saponins, terpenoids, and jasmine flower is a commodity with high economic value, its utility is not only as potted and garden ornamental plants, but also as tea fragrance, raw material perfume industry, cosmetics, traditional medicine, flower sow tomb, room decoration, and complements in traditional ceremonies (sihite et al. 2018). there are different sorts of jasmine blooms, but the one commonly found is white jasmine (jasminum sambac (l.) ait.). jasmine blossoms are utilized as a crude fabric for the fabricating preparation of jasmine flavonoids which are often used in the field of pharmacological research . the (sabharwal 2013)et al. flavonoids which function as antioxidants and have an antibacterial function in the wound healing process . the content of active (ridwan rais 2015) compounds found in jasmine flowers such as alkaloids, flavonoids, saponins and tannins can function as antiseptics and antioxidants and are believed to be used as antibacterial . the (krishnaveni and thaakur 2012) methanol extract of jasmine flowers when tested using the disc diffusion method and the dilution method had antimicrobial activity against various types of bacteria such as salmonella enteric, enterococcus faecalis, streptococcus pyogenes, bacillus cereusand (abdoullatif 2011)et al. . according to research conducted by (al-hussaini and mahasneh 2011) showed that in testing the antibacterial and antifungal activity of jasmine flower extracts produced a larger diameter of the inhibition zone when compared to jasmine flower leaves. natural preservation by utilizing plants is an alternative. some plants contain antimicrobial compounds that can be used to inhibit growth and kill pathogenic microbes, one of which is jasmine. the types of microbes studied in this study are pathogenic bacteria that can cause various diseases in humans, such as diarrheal disease by , boils, escherichia coli skin infections ulcers by staphylococcus aureus ( et al. jayalandri 2016). the use of jasmine flowers in industrial technology, especially in the food industry, needs to be followed up, therefore it is necessary to do further research on jasmine flower extract ) (j. sambac which is used as a natural antimicrobial ingredient for the food industry. it is hoped that this natural extract from jasmine flowers can be used as an alternative to preservatives and natural antimicrobials in inhibiting microbial growth. based on this background, it is necessary to research jasmine flower extract as a natural antimicrobial ingredient. the purpose of this research is to extract the antimicrobial active compound in the jasmine flower and see its effect in inhibiting the growth of microbes andescherichia coli staphylococcus aureus and see whether jasmine extract can be an alternative as natural preservatives so that it can replace the many synthetic preservatives that are synthetic preservatives circulating in society materials and methods . the material used is fresh jasmine plant material flowers with the criteria of semi-blooming florets obtained from traditional farmers in the sei-mencirim area, medan tuntung district, north sumatra. chemicals and media, namely nutrient agar (na), mueller hinton agar (mha), nutrient broth (nb), dimethyl sulfoxide (dmso), nacl 0,9%, distilled water, disc paper (oxoid), , water, methanol, ethyl acetate and n-hexane. the material used for bacterial test was plate count agar (pca). equipment used in this study were laminar air flow cabinet (astec hlf 1200l), analytical balance (mettler toledo), autoclave (express), refrigerator (toshiba), micrometer pipettes (eppendorf), oven (memmert), incubator, rotary evaporator (stuart), oxoid paper and colony counter. bacterial culture s used in the study were staphylococcus aureus escherichia coli (atcc 6538), (atcc 8938). jasmine flower extract. jasmine flower were washed and dried in the oven at 40°c for 48 h. jasmine flower powder then weighed 480 grams each and then macerated with four types of solvents (water, methanol, ethyl acetate and n-hexane) for 3 days. the dried powder jasmine was divided into 4 groups of 480 grams powder and dissolved with solvent (water, methanol, ethyl acetate and n-hexane) in a erlenmeyer then macerated for 72 h and shaking periodically. the precipitated residue was separated from the solvent by filtration and concentrated by using vacuum evaporator at 50 °c to produce a crude extract. the extract was then weighed and obtained the results of jasmine flower extract from each solvent, namely: jasmine flower extract with water solvent (19 g), jasmine flower extract with methanol (49.1 g), jasmine flower extract with ethyl acetate solvent (11.6 g), and jasmine flower extract with n-hexane solvent (6.9 g). qualitative phytochemical test were carried out on the extracts obtained to determine the presence of several chemical compounds by the method of indonesian ministry of health (indonesia ministry of health 1995) and fransworth included alkaloids, (fransworth 1996) flavonoids, steroids, saponins, tannins and phenols. antimicrobial activity. the microbial growth inhibitory potential of the crude extract was determined using disk diffusion method described by kirby-bauer . to testing the crude (kirby 1966)et al. extract used blank disc paper with a diameter of 6 mm. the disc is inserted into a sterile empty petri dish. preparation of stocks solution (100% extract) was done by dissolved 2 g of each extract into 2 ml dmso (dimethyl sulfoxide). furthermore, the stock solution was reconstituted with dmso to obtain 75%, 50% and 25% of extract concentration. the tested organisms u s e d ( at c c 8 9 3 8 ) a n d e s c h e r i c h i a c o l i volume 15, 2021 microbiol indones 103 104 sihite et al . microbiol indones staphylococcus aureus (atcc 6538) were obtained from microbiology laboratory, faculty of pharmacy, north sumatera university and were performed by agar disc-diffusion method by balouiri (balouiri et al. 2016). filter paper disc containing jasmine extracts from different solvents (25%, 50%, 75% and 100%) were placed on the agar surfaces (mueller-hinton agar). the culture was incubated at an optimum temperature of 37-38 ºc for 24 h. after the incubation period,the diameter of the inhibitory zone (clear area) around the disc is measured using a calipers. the activity of extract can be seen with the inhibition zone around the disc.the clear area around the disc paper shows a positive test for inhibition of bacterial growth (hombach 2013)et al. .the minimum inhibitory concentrations (mic) of jasmine extract was performed by a serial dilution technique (0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, and 3.0%) of the concentrates (brenes 2007)et al. . as much 10 µl of bacteria test culture were mixed with jasmine extract (the best extract of several solvent) and shaken using shaker incubator at speed 150 rpm for 24 hours. the diameters of the inhibition zones were measured in mm and the minimum extract concentration that inhibits 90% of the growth of bacteria known as mic (thaweboon and thaweboon 2018). assay method. the study was conducted in 3 stages. the first step is the phytochemical test of jasmine flower extract on various types of solvents, namely water, methanol, ethyl acetate, and hexane. p h y t o c h e m i c a l s t e s t s u c h a s a l k a l o i d s , steroids/terpenoids, saponins, tannins, flavonoids, phenolics, and glycosides was done by the method of the department of health (depkes 1995) and farnsworth (1996). the second phase of the study, namely the antimicrobial test on escherichia coli (atcc 8938 (atcc ) and staphylococcus aureus 6538), extracts using jasmine flower extract with , various concentrations, the third stage is the minimum inhibitory concentration (mic) test. the test for the antimicrobial activity of jasmine flower extracts on and staphylococcus aureus escherichia coli was determined using the agar disc diffusion method . bacteria were (balouiri 2016)et al. first incubated at 37 °c for 24 h in nutrient broth. the agar medium was spread with the inoculum discs of sterile oxoid paper of 6 mm in diameter are deposited on the plates. samples in various concentrations were injected into disc of sterile oxoid paper. after incubation at 37 °c for 24 h, the diameter of inhibition zone was measured in millimeters to the test organism. dimethyl sulfoxide (dmso) was used as negative control. processing of the data is using anova. if the results are significantly different and very real, the test is continued with the average difference test using the lsr (least significant range) test the research study . was conducted using a completely randomized design (crd) with two factorial, consisting of solvents and concentration: factor i : types of solvent (p): p1 =water p2 = methanol p3 = etyl acetate p4 = nheksane factor ii : concentration (k) jasmine extract: k1 = 25 % k2 = 50 % k3 = 75 % k4 = 100% combination treatment (pk) = 4 x 4 = 16, for the level of accuracy, this research was conducted 4 times.completely randomized design (ral) model used as below: yijk =µ + αi + βj + (αβ)ij + εijk result yield of jasmine extraction. the result of extraction with several type of solvents are shown in table 1. the highest yield of jasmine extract was found in methanol extract and followed by water, ethyl acetate and n-hexane (table 1). the highest yield in methanol is caused by the characteristic of methanol that can dissolve almost all of the components in polar, semi-polar and non-polar solvent , (pasaribu 2015)et al. is in according to research benedicta and zain (benedicta and zain 2016) that the methanol solvent can dissolve almost all organic compounds, both polar and non-polar because methanol has polar (-oh) and non-polar groups (-ch). phytochemical test results. several components of the active compounds identified in this research: alkaloids, steroids/triterpenes, saponins, tannins, flavonoids, phenolics, and glycosides. there are several bioactive compounds contained in jasmine flower extracts with water, methanol, ethyl acetate, and n-hexane as solvents (table 2). based on the qualitative phytochemical test by the method of indonesian ministry of health (indonesia ministry of health 1995) (fransworth and fransworth 1996). the test results of jasmine flower extracts in each type of solvent showed that the most optimal volume 15, 2021 microbiol indones 105 solvent to attract bioactive compounds is methanol. from the results of the phytochemical screening test, it is known that the n-hexane solvent contains compounds such as steroids/triterpenes, and glycosides. for ethyl acetate solvents, compounds such as steroids/triterpenes, flavonoids, and phenolics are obtained, whereas, in methanol solvents, almost all compounds can be withdrawn by methanol solvents except for saponin compounds. the research was conducted to study the different polarity solvent, including water, methanol, ethanol, ethyl acetate, and hexanes to phytochemical content and antioxidant activity of leaves extracts.pluchea indicia less antimicrobial activity of jasmine flower e x t r a c t s a g a i n s t a n d e s c h e r i c h i a c o l i staphylococcus aureus. the types of solvents used in jasmine flower extract had a highly significant effect (p <0.01) on the inhibition zone of growth. the e coli. relationship between the effect of the type of solvent on the zone diameter inhibition of the growth of e coli . bacteria can be seen in fig 1. each solvent has a significantly different effect. the highest inhibitory zone was obtained at ethylacetate solvent, which was 22.575 mm, while the lowest inhibition zone diameter was obtained in methanol solvent which was 9.775 mm. based on the test results obtained it can be said that jasmine flowers have the ability to inhibit the growth of bacteria e. coli (fig 1). based on the results of the study, it was found that jasmine flower extract in each concentration could inhibit the growth of bacteria at the lowest e. coli concentration of 25% to the highest concentration of 100%. from the results of variance shows that the interaction of the type of solvent and the concentration of jasmine extract gave a very significant effect (p <0.01) with the diameter of the growth inhibition zone of , so the least significant range (lsr) test was e. coli continued (fig 2). the interaction between the types of solvents and extract concentration gives a significant effect on the diameter of the zone of growth inhibition of e. coli bacteria (fig 2). the extract that could form the largest inhibitory zone in the three test bacteria was ethyl acetate extract. analysis of variance showed that the type of solvent gave a very significant effect on the diameter of the growth inhibition zone of staphylococcus aureus . the highest inhibitory zone diameter was obtained in ethyl acetate solvent, which was 23.219 mm, while the lowest inhibition zone diameter was obtained in methanol solvent which was 8.775 mm (fig 3). the ability of the ethyl acetate solvent to inhibit the growth of bacteria, the greater the staphylococcus aureus diameter of the inhibition zone is 23.219 mm, while the diameter of the inhibition zone in water solvents is 14.013 mm, in hexane solvents 13.300 mm and in methanol solvents is 8.775 mm. the results of variance table 1 the yield of jasmine extract table 2 phytochemical test results solvent yield (%) water 3,9 methanol 10,2 ethyl acetate 2,41 n-hexane 1,4 description: (+) = contains a class of compounds (-) = does not contain compounds bioactive compounds solvent jasmine flower extract water methanol ethyl acetate n-hexane alkaloids (indonesia ministry of health 1995) + + steroid/triterpenes (farnsworth, 1996) + + + + saponins (indonesia ministry of health 1995) + tannins + + flavonoids + + phenolics + + + glycosides + + 106 sihite et al . microbiol indones showed that the interaction of the type of solvent and the concentration of jasmine extract gave a very significant effect (p <0.01) with the diameter of the s. aureus growth inhibition zone so that the least significant range (lsr) test was continued. the highest increase in diameter of the inhibitory zone in the growth of was obtained in ethyl s. aureus acetate solvent which was 0.095 mm, while the lowest increase in diameter of the inhibitory zone was obtained in the methanol solvent which was 0.011 mm (fig. 4). minimum inhibitory concentration (mic) antimicrobial substrate. analysis of the mechanism antibacterial inhibition of test bacteria begins with the determination of minimum inhibitory concentration (mic) . at this stage the concentration of (yilmaz 2012) the ethyl acetate extract used in the test was 0.05%, 0.1%, 0.25 0.5%, 1%, 2%, 3%, 4%, 5%. the test bacteria used for the determination of mic antimicrobial substrates are and e coli s aureus. . . mic test results showed that the lowest concentrations that could inhibit the growth of e coli . and bacteria were 0.25% and 0.1% s aureus (e coli). . (s aureus). (table 3). discussion in the results of phytochemical screening, it was found that the extract of jasmine flowers with ethyl acetate solvent contained bioactive compounds, one of which was flavonoids. flavonoids are one of the secondary metabolites produced by a plant that can be found in the leaves, tubers, roots, wood, bark, pollen, flowers, and seeds. flavonoids in plants function to regulate growth, regulate photosynthesis, regulate antimicrobial and antiviral work, and regulate antiinsect action. this is because flavonoids have a broad spectrum of antimicrobial activity by reducing the immunity of the target organism . (davidson 2014)et al. flavonoid compounds also could coagulate protein, flavonoid compounds are also lipophilic so that they can damage the lipid layer on the bacterial cell membrane . according to krishnaveni (tuntun 2016) et al. (2011), there are several bioactive compounds contained in white jasmine flowers (jasminum sambac) such as alkaloids, flavonoids, saponins, and tannins which can function as antibacterial (krishnaveni and thaakur 2012). in this study, almost all bioactive compounds such as alkaloids, steroids/triterpenes, saponins, tannins, flavonoids, phenolics, and glycosides are contained in the extract of jasmine flowers with various solvents with different levels of polarity. from preliminary test results of antimicrobial activity, ethyl acetate extract is the most potential extract in inhibiting the growth of and e. coli s. aureus. phytochemical screening results found that jasmine flower extract with ethyl acetate solvent contains bioactive compounds, one of which is flavonoids. flavonoids in plants function to regulate g r o w t h , r e g u l a t e p h o t o s y n t h e s i s , r e g u l a t e antimicrobial and antiviral work, and regulate antifungal action. this is because flavonoids have a broad spectrum of antimicrobial activity by reducing the target organism's immunity (kuppusamy et al. 2016). the higher concentration of the extract, the greater is the number of antimicrobial compounds such as bioactive components thus the penetration of the compound into the cell becomes easier. the research was conducted with naufalin . et al (naufalin 2019) research on kecombrang that has extract bioactive components functioning as an antimicrobial, so that it can inhibit the growth of microbes including grampositive and gram-negative bacteria .it is also according to naufalin and herastuti (naufalin and herastuti 2013) kecombrang flower microcapsules table 2 minimum inhibitory concentration (mic) of jasmine extract description not inhibitory zone inhibitory zone: (-) (+) extract concentration (%) escherichia coli staphylococcus aureus 0.05 0.1 + 0.25 + + 0.5 + + 1 + + 2 + + 3 + + 4 + + 5 + + volume 15, 2021 microbiol indones 107 fig 1 effect of solvent types on diameter of inhibiting zones of bacteria growth.escherichia coli fig 2 interaction between types of solvents and the concentration of jasmine extract with growth escherichia coli inhibition zone diameter. with ethyl acetate solvent had higher antimicrobial activity than the ethanol solvent. this is probably caused by the optimum polarity of ethyl acetate, meanwhile, the methanol extract was not satisfactory in inhibiting bacteria. this is in line escherichia coli with hazimah ., who stated in their research that et al methanol is a universal solvent that can dissolve most of the components of polar compounds found in plectranthus amboinicus leaves so that the concentration of antibacterial compounds is too small or even not visible at all .(hazimah 2013)et al. the results of variance shows that the interaction of the type of solvent and the concentration of jasmine extract gave a very significant effect (p <0.01) with the diameter of the growth inhibition zone of escherichia coli. according to eveline and novita, that antimicrobial compound was categorized as active if the inhibition diameter is more than 6 mm (eveline and 108 sihite et al . microbiol indones novita 2020). antibacterial activity test result show in extract using ethyl acetate was proven to be effective in inhibiting the growth of tested bacteria. based on the results of the study it was found that dmso did not provide inhibition toward the sample bacteria. it proved that dmso does not have crucial role in inhibiting the growth of bacteria tested. in figure 2, it is known that the interaction between the types of solvents and extract concentration gives a significant effect on the diameter of the zone of growth inhibition of bacteria. the extract that could escherichia coli form the largest inhibitory zone in the three test bacteria was ethyl acetate extract. this is presumably because ethyl acetate extract has the optimum level of polarity. based on the test results obtained it can be said that jasmine flowers have the ability to inhibit the growth of bacteria. this is because the escherichia coli jasmine flower contains several active compounds that have the potential as natural antimicrobial agents such as flavonoids which have antibacterial activity, phenol fig 4 interaction between types of solvents and the concentration of jasmine extract with staphylococcus aureus growth inhibition zone diameter. fig 3 effect of solvent types on diameter inhibiting zones of bacteria growth.staphylococcus aureus volume 15, 2021 microbiol indones 109 compounds such as tannins, triterpenoid/steroid compounds that have antifungal activity (lutfiyanti et al. 2012). the compounds in the polar fraction are concidered to contain many hydroxyl groups that facilitate water-soluble antibacterial compounds which can become a habitat for bacteria. polar fraction (ethanol) is thought to have optimum polarity properties so that it diffuses more easily and can inhibit the growth of compounds that have escherichia coli. optimum polarity will have maximum antibacterial activity because hydrophilic-hydropobicbalance is needed . (wang 2019)et al. from the results, it can be seen that ethyl acetate solvent had a significant effect in inhibiting the growth of staphylococcus aureus bacteria, staphylococcus aureus is a gram-positive bacteria. generally, gram-positive bacteria are more sensitive to antibacterial compounds compared to gram-negative bacteria because the cell walls of grampositive bacteria do not have a lipopolysaccharide layer so that antimicrobial compounds that are hydrophilic or hydrophobic can pass through the walls of gram-positive bacterial cells through passive diffusion mechanisms then interact directly with peptidoglycan cells bacteria that are growing and causing cell death .(ceballos 2018)et al. ethyl acetate solvents can be used to extract semipolar compounds such as flavonoids, phenolic, glycosides and tannins meanwhile, (tanaya 2015)et al. polar solvents like water can dissolve polar compounds and nonpolar compounds because it has a large dipole moment phenolic is a compound (sulasmi 2020)et al. that contains phenols (phenol-derived compounds) that have been chemically modified to reduce their ability to irritate the skin and increase its antibacterial activity. antimicrobial activity of phenolic compounds is by damaging the lipids in the plasma membrane of microorganisms, causing the contents of the cell to come out . the results of variance (alves 2013)et al. showed that the interaction of the type of solvent and the concentration gave a very significant effect (p<0.01) on the diameter of the staphylococcus aureus growth inhibition zone. figure 3 show that the highest inhibition zone on bacteria was staphylococcus aureus found in jasmine extracted by ethyl acetate solvent with a concentration of 100%, because phenolic and flavonoid compound in extracted by ethyl acetate acts as an anti-bacterial against staphylococcus aureus (elmasri 2017) et al. . the highest inhibition zone on escherichia coli bacteria was found in jasmine extracted by ethyl acetate solvent with a concentration of 100 %. jasmine extracted by ethyl acetate contained such as steroids, phenolics and flavonoids. steroid has antimicrobial characteristics can inhibits gram-positive and gram-negative bacteria likely . the escherichia coli peroxide and vinyl bonds in steroids structures have a role as antibacterial, the mechanism of the effect of the sterols can be explained by the fact that they are similar to sterols that are normally used in the cells, and that they replace these substances in the cell membrane (dogan 2017)et al. . flavonoid compounds contained in the extract have good inhibitability of escherichia coli staphylococcus aureus bacteria and (wu et al. 2013). jasmine extract used in mic testing was extracted by ethyl acetate because ethyl acetate has been known as a good solvent for phytochemical extraction and is safe for human consumption because ethyl acetate is neither phototoxic nor photo allergenic in human tests . methanol solvent is (shukla 2019)et al. not recommended for human, because it can cause toxicity in human such as irritating to the eyes, skin and respiratory tract, acute oral and inhalation exposures, to a lesser extent, percutaneous absorption of high concentrations of methanol have resulted in central nervous system depression, blindness, coma and death (world health organization 1997). the results of the mic value of and escherichia coli staphylococcus aureus are presented in table ii, the mean values of mic jasmine extract ranged from 0.1 – 5 %. test results showed that the lowest concentrations that could inhibit the growth of and escherichia coli staphylococcus aureus bacteria were 0.25% (escherichia coli) (staphylococcus aureus). and 0.1% the results of the study explained that the height a n d d i a m e t e r z o n e s o f g r o w t h i n h i b i t i o n staphylococcus aureus escherichia coli and jasmine flower extract produced by water, methanol, ethyl acetate and n-hexane were caused by the properties of each solvent in attracting antibacterial compounds. the use of solvents in extracting foodstuffs will affect the results of testing the bioactive compounds of these foods. polar compounds are more soluble in polar solvents and non-polar compounds are more soluble in non-polar solvents. jasmine flower extract has a very significant different effect on the zone diameter of the growth inhibition of and escherichia coli staphylococcus aureus bacteria. ethyl acetate has semi-polar properties that are volatile and can dissolve semipolar compounds in cell walls such as flavonoid aglycones, non-toxic and not hygroscopic. in addition, ethyl acetate can search for compounds that provide antibacterial activity, including polyhydroxy flavonoids and other phenols (wardhani and sulistyani 110 sihite et al . microbiol indones 2012). antibacterial compounds that diffuse into the medium can inhibit the formation of thin cell walls and can lysis of the bacteria. according to suhartati et al, (2019) flavonoids can perform inhibition mechanism by interfering with the bacterial cell wall synthesis there by causing plasma leakage resulting in lysis of the bacteria . inhibition occurs also (suhartati 2019)et al. there is a process of protein synthesis, inhibition of protein synthesis occurs where there is a process of transcription and translation of genetic material so that the amino acids produced incorrectly place themselves in the peptide chain and produce proteins that do not function . flavonoid compounds (madigan 2008)et al. can damage cell walls, causing cell death (haeria and hermawati 2016). the flavonoids are group of secondary metabolites produced by plants which is a large group of polyphenols. this compound is found in all parts of the plant, including leaves, roots, wood, and bark. flavonoids can capture free radicals and inhibit lipid oxidation (zuraida 2017)et al. . overall, the results of this study were examining jasmine extract the growth of and, escherichia coli staphylococcus aureus, and which showed the growth inhibition activity (inhibitory zone). the magnitude of the microbial growth inhibition zone examined by the extract was seen as a clear area (clear zone) around the disc. the ability of extracts to inhibit microbial growth was caused by the presence of active compounds contained in jasmine extract with various solvents. in addition to solvent types, the concentration of each jasmine extract also affected the inhibition zone diameter of , and escherichia coli staphylococcus aureus .(abubakar and usman 2016) this observation is in agreement with the study of ida bagus, that the administration of different concentrations shows different effects on the resulting zone of inhibition. the amount of inhibition zone diameter also depends on the absorption/absorption of antibacterial substances into agar plates and the sensitivity of bacteria to these antibacterial agents . jasmine flower (ida bagus 2008) can be used as an alternative preservatives and pathogen control method in food material, in line with (altemimi 2019), reported a high antimicrobial et al. activity against using methanol staphylococcus aureus and ethanol extracts for beddome hopea pariviflora (altemimi 2019)et al. . the jasmine extract obtained is expected to inhibit the growth of pathogenic bacteria such as and . escherichia coli staphylococcus aureus this research is useful in providing information on the use of jasmine flowers as a natural resource to control microbial activity so that it has economic value and can be used in large and small scale food industries. it is hoped that further benefits of this jasmine extract will also be beneficial in other fields such as economics, pharmacy, and health. in this research, almost all bioactive components such as: flavonoids, glycosides, triterpenes, tannins, phenolics, and glycosides are contained in flower extracts, both in water extract, methanol, ethyl acetate, and hexane. the types of jasmine flower extract with 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acc no 103-0002080774 printed by: cv. istiqom print neung tiaamroeng, 2020 norio kurosawa, 2020 kartini kramadibrata, 2020 diana e waturangi, 2020 endang purwantini, 2020 wellyzar sjamsuridzal, 2020 yuan kun lee, 2020 yaya rukayadi, 2020 netty widyastuti sigit, 2020 general executive board of indonesian society for microbiology 2019-2023 advisory board: prof. dr. pratiwi sudarmono, phd, sp.mk; dr. mohammad dimyati; prof. dr. endang sukara; prof. dr. amin soebandrio, phd, sp.mk; prof. dr. ir. betty sri laksmi jenie, ms; prof. dr. ir. antonius suwanto, msc; dr. siswa setyahadi; president: dr. iman rusmana; vice president: prof. fedik a rantam, phd; dr. atit kanti, m.sc; general secretary: diana nurani, m.si; vice general secretary: dr. rika indri astuti, m.si ; treasurer: dr. niknik nurhayati; dr. nisa rachmania, m.si; scientific and publication committee: dr. is helianti; dr. astutiati nurhasanah; prof. dra. netty widyastuti, m.si; asri sulfianti, m.si; certification committee: dr. ir. trismilah, m.si; dra. harmastini sukiman, m.agr; dra. mg. isworo rukmi, m.kes; dra. dini ryandini, m.si; organization advancement and networking: dr. puspita lisdiyanti; lia yulia budiarti, m.kes; dr. rer. nat. catur riani; promotion and advocacy committee: jimmy hariantono, phd; gianina sebayang, m.si; ir. dwi kusuma indriani, mp; dr. tjahjani mirawati sudiro; data and information: drs. nuki b nugroho, m.si; mulyono, ph.d; jepri agung priyanto, s.si, m.si; ario fitrianto, s.si debbie s retnoningrum, 2020 iman rusmana, 2020 page 1 editorial board.pdf page 1 4.mi656-vincentia irene available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.3.4issn 1978-3477, eissn 2087-8575 vol 6, no 3, september 2012, p 117-123 *corresponding author; phone: +62-298-321212, fax: +62298-321433, e-mail: irene_meiti@yahoo.com orange ii is one of synthetic azo dyes which have been widely used for textile, food, and cosmetics. like other xenobiotic compounds, azo dyes are relatively resistant to microbial degradation under conditions normally found in wastewater treatment plants. it was reported that several microorganisms were able to transform azodyes to non-colored products or even to mineralized products (stolz 2001). bacterial azodyes degradation was usually initiated by cleavage of azo bond carried out by azoreductase of the intestinal microflora, resulting in colorless amines (chen et al. 2004; ramalho et al. 2004; kodam et al. 2005). this process is called decolorization (tan 2001; van der zee 2002). anaerobic condition is more favorable for decolorization rather than aerobic condition. it is probably related to the electron with drawing nature of azo bond and the resistance of azo dyes to oxygenases attack, or because oxygen is a more effective electron acceptor than the dyes (dos santos 2004). many researchers have reported that degradation previous work showed that enterococcus faecalis id6017 and chryseobacterium indologenes id6016 were able to decolorize the orange ii qualitatively. in that experiment, e. faecalis could decolorize orange ii more rapidly than c. indologenes. the objective of this study was to examine the decolorization of orange ii by mixed culture and the growth of both bacterial species on orange ii containing medium. the experiment was -1 done in 500 ml sterilized erlenmeyer flasks containing 285 ml growth media with 80 mg l . five different treatments were performed in this project, i.e. medium was inoculated with (i) 15 ml of sterile aquadest, (ii) 15 ml of c. indologenes, (iii) 15 ml of e. faecalis, (iv) 7.5 ml of c. indologenes and 7.5 ml of e. faecalis, and (v) 7.5 ml of e. faecalis until decolorization occured , followed by inoculation with 7.5 ml of c. indologenes. bacterial growth (total cells number), orange ii, glucose, and suphanilic acid, as intermediate product of orange ii decolorization, concentrations were measured every 2 h.the maximum decolorization of orange ii was observed in the medium inoculated with a mixed culture of e. faecalis and c. indologenes. -1 decolorization of orange ii occurred of growth and gave final concentration of sulphanilic acid of 7.06 mg l . during culture both species grow in equilibrium in terms of population. key words: chryseobacterium indologenes, decolorization, enterococcus faecalis, growth, mixed culture, orange ii penelitian sebelumnya menunjukkan bahwa enterococcus faecalis id6017 dan chryseobacterium indologenes id6016 dapat mendekolorisasi orange ii secara kualitatif. pada penelitian tersebut, e. faecalis dapat mendekolorisasi orange ii lebih cepat daripada c. indologenes. tujuan penelitian ini adalah menentukan dekolorisasi orange ii oleh kultur campur dan pertumbuhan kedua spesies bakteri pada medium yang mengandung orange ii. penelitian dilakukan dalam botol erlenmeyer 500 ml yang berisi 285 ml media dengan -1 orange ii 80 mg l . penelitian dilakukan dengan 5 perlakuan, yaitu medium diinokulasi dengan (i) 15 ml air suling, (ii) 15 ml kultur c. indologenes, (iii) 15 ml kultur e. faecalis, (iv) 7,5 ml kultur c. indologenes dan 7.5 ml kultur e. faecalis, dan (v) 7.5 ml kultur e. faecalis hingga terjadi dekolorisasi, diikuti inokulasi dengan 7.5 ml kultur c. indologenes. pertumbuhan bakteri (jumlah sel total), konsentrasi orange ii, glukose, dan asam sufanilat sebagai produk antara dekolorisasi diukur setiap 2 jam. dekolorisasi maksimum orange ii teramati pada medium yang diinokulasi dengan kultur campur e. faecalis dan c. indologenes. dekolorisasi orange ii terjadi pada 6 jam -1 pertama kultur dan memberi konsentrasi akhir asam sulfanilat sebesar 7.06 mg l . pertumbuhan kedua spesies bakteri berada dalam keseimbangan selama pertumbuhan ko-kultur. kata kunci: chryseobacterium indologenes, dekolorisasi, enterococcus faecalis, kultur campur, orange ii, pertumbuhan orange ii decolorization of orange ii by mixed culture of enterococcus faecalis id6017 and chryseobacterium indologenes id6016 1 2 vincentia irene meitiniarti *, kris herawan timotius , endang 3 4 sutariningsih soetarto , and eko sugiharto 1 faculty of biology, satya wacana christian university, jalan diponegoro 52-60, salatiga 50712, indonesia; 2 krida wacana christian university, jalan arjuna utara 6, jakarta 11510, indonesia; 3 faculty of biology, universitas gadjah mada, jalan teknika selatan, yogyakarta 55281, indonesia; 4 department of chemistry, universitas gadjah mada , sekip utara, yogyakarta 55281, indonesia of azo dyes is often carried out by mixed cultures. in the mixed cultures, a higher degree of biodegradation and mineralization can be expected when co-metabolic activities within a microbial community complement each other (pearce et al. 2003). in the anaerobic condition, orange ii can be degraded by single culture of enterococcus faecalis into sulfanilic acid as an intermediate product (meitiniarti et al. 2007). meitiniarti et al. (2005) also reported that chryseobacterium indologenes could grow in the medium containing sulfanilic acid. probably, both bacteria have synergistic relationship in orange ii degradation. for this reason, our group examined the decolorization of orange ii by mixed culture and the growth of both bacterial species on orange ii containing medium. the aim of this study was also to determine the best mixing method of both bacterial cultures on orange ii decolorization. material and methods microorganism and culture condition. c. indologenes id6016 and e. faecalis id 6017 were obtained from laboratory of microbiology, faculty of biology, satya wacana christian university, salatiga, indonesia. c. indologenes id6016 and e. faecalis id 6017 were maintained and cultivated on slant media of nutrient agar (na) and trypticase soy agar (tsa), respectively. both cultures were incubated at room temperature and sub-cultured every two weeks. growth medium. the composition of growth -1 medium (ph 6.8-7.2) for this study was (g l ): glucose, 0.90; mgso .7h o, 0.25; kh po , 2.13; k hpo , 5.55; 4 2 2 4 2 4 (nh ) so , 1.98; yeast extract, 0.25; orange ii or acid 4 2 4 orange 7, 0.08. orange ii was obtained from merck and was used for this study without any purification. pre-culture preparation. two inoculation needles of c. indologenes and e. faecalis from 24 h slant culture were inoculated aseptically into 200 ml growth medium without orange ii. pre-culture was incubated for 24-48 h in a shaker incubator with 150 rpm agitation or until optical density (λ 550 nm) of culture was 0.4 (cell 7 density of c. indologenes and e. faecalis were 26 x 10 -1 7 -1 cfu ml and 23 x 10 cfu ml ,respectively). these cultures were then used as inocula. culture condition. to determine the decolorization of orange ii by both bacterial cultures, the experiment was performed with 5 different treatments in 500 ml erlenmeyer flasks containing 285 ml growth media -1 with 80 mg l orange ii . these treatments are: (i) medium was inoculated with 15 ml of sterile aquadest, 118 meitiniarti microbiol indones (ii) medium was inoculated with 15 ml of c. indologenes, (iii) medium was inoculated with 15 ml of e. faecalis, (iv) medium was inoculated with 7.5 ml of c. indologenes and 7.5 ml of e. faecalis, and (v) medium was inoculated with 7.5 ml of e. faecalis until decolorization occured, followed by inoculating with 7.5 ml of c. indologenes. the final cell number each 7 -1 treatment at 0 h was 1.2-1.3 x 10 cfu ml . all cultures were incubated at room temperature, under static condition until decolorization occured, followed by 150 rpm agitation until 120 h. bacterial growth (total cells number), orange ii and glucose concentrations, and suphanilic acid concentration as an intermediate product of orange ii decolorization were observed every 2 h. samples were harvested and centrifugeded at 3326 g for 30 min to separate supernatant and cell mass (biomass). the supernatant was used to determine the orange ii and glucose concentrations. the ability of both bacterial strains to decolorize orange ii was determined by subtracting the initial orange ii concentration with the lowest concentration from samplings. the total cells number was determined by making a serial dilution of cell culture with sterile 5 7 aquadest and poured 0.1 ml of 10 -10 cell suspension into tsa plate media. analytical methods. samples were centrifuged at 3326 g for 30 min to separate supernatant and cell mass. the supernatant was used for determining the orange ii, glucose, and sulfanilic acid concentrations. orange ii concentration was determined at λ 482 nm (pourreza and zareian 2009). glucose concentration was determined using dns method at λ 540 nm (james 1995). the cell growth was determined using total plate count in tsa plate. in tsa plate medium, the color of c. indologenes colony was yellowish while e. faecalis was white. all absorbances were measured using shimadzu uv-vis 1201 spectrophotometer. the concentration of sulfanilic acid was determined by hplc (coughlin et al. 1999; chang et al. 2001; supaka et al. 2004). results the growth of c. indologenes as a single culture was better than its growth in the mixed culture with e. faecalis (fig 1a). on the contrary, the growth of e. faecalis together with c. indologenes inoculated as a mixed culture in the early experiment was faster than the growth of e. faecalis inoculated as a single culture (fig 1b). although, total numbers of e. faecalis cells in b 20 40 60 80 incubation time (h) 0 20 40 60 80 100 incubation time (h) 0 20 40 60 80 100 -1 4 t o ta l b ac te ri a (c f u m l ) x 1 0 1000 2000 3000 4000 5000 6000 0 -1 4 t o ta l b ac te ri a (c f u m l ) x 1 0 1000 2000 3000 4000 5000 6000 0 a the mixed culture was half of its single culture, in the end of culture its total number of e. faecalis cells was found to be the same (fig 1b). the decolorization of orange ii occurred when e. faecalis was present in the culture. the rate of orange ii decolorization by mixed culture occurred faster than by single culture (fig 2 and table 1). when inoculation of mixed culture was done early during the experiment), the rate of orange ii decolorization was found to be the -1 fastest (10.98 mg h ) (table 1). during cells growth and orange ii decolorization, glucose and sulphanilic acid concentrations significantly changed (fig 3 and 4), except when the medium was inoculated with sterile water (control) and with c. indologenes as single culture. however, sulphanilic acid was only detected in the first eight hours of culture, thereafter sulphanilic acid concentration decreased (fig 4). in the growth of c. indologenes as a single culture, aliquot of sulphanilic acid was detected until 56 h incubation. discussion the growth of e. faecalis when co-cultured with c. indologenes was faster than its growth as a single culture (fig 1b). the faster growth of e. faecalis cells caused by the presence of c. indologenes cells was which could consume sulphanilic acid and could decrease toxic effect of sulphanilic acid in this medium. meitiniarti et al. (2011) reported that c. indologenes volume 6, 2012 microbiol indones 119 fig 1 total cells of chryseobacterium indologenes (a) and enterococcus faecalis (b) in single and mixed culture in the orange ii containing medium. : medium was inoculated with 15 ml of sterile aquadest; : medium was inoculated with 15 ml of c. indologenes; :medium was inoculated with 15 ml of e. faecalis; : medium was inoculated with 7.5 ml of c. indologenes and 7.5 ml of e. faecalis ; : medium was inoculated with 7.5 ml of e. faecalis until decolorization occured, followed by inoculating with 7.5 ml of c. indologenes. inoculated after orange ii decolorized, e. faecalis grew very slowly due to the low cells density of the inoculum (5%). in low density culture, usually bacteria were less successful to colonize a natural habitat. another factor could grow on filtrate's culture of e. faecalis which contained sulphanilic acid as intermediate product of orange ii decolorization. in the fifth treatment, where c. indologenes was microbiol indones120 meitiniarti tab 1 values of growth and orange ii decolorization parameter by single culture of e. faecalis and on mixed culture with c. indologenes after 120 h incubation 0 20 40 60 80 100 -1 o ra n g e ii c o n ce n tr at io n ( m g l ) incubation time (hours) 0 2 4 6 8 10 n.d = not detected n.dt = not determined i = first control ii = early inoculation by chryseobacterium indologenes (second control) iii = early inoculation by enterococcus faecalis (third control) iv = early inoculation by both of e. faecalis and c. indologenes v = early inoculation by e. faecalis, and inoculated by c. indologenes after decolorization parameter treatments i ii iii iv v orange ii reduction (mg) (%) n.dt n.dt 65.2 0.2 90 2 65.9 0.2 89 3 69.5 0.3 91 4 time of decolorization (h) n.dt n.dt 8 6 10 decolorization rate ( -1 mg h ) n.d n.dt 8.15 0.02 10.98 0,02 6.95 0,03 glucose consumption during decolorization (mg) n.d n.dt 340 7 216 3 393 5 glucose consumption during 102 h (mg) n.d n.dt 710 + 10 673 + 6 693 + 4 sulfanilic acid concentration at decolorization (mg -1 l ) n.d n.dt 24.4 + 0.13 23.2 + 1.2 21.2 + 1.2 sulfanilic acid concentration at 128 -1 h (mg l ) n.d 1.12 18.5 + 0.5 7.06 + 0.3 10.3 + 0.7 ± ± ± ± ± ± ± ±± ±± ± fig 2 decrease of orange ii concentration in experiment with single culture of enterococcus faecalis (control) and with mixed culture (together with chryseobacterium indologenes). x: medium was inoculated with 15 ml of sterile aquadest; :medium was inoculated with 15 ml of c. indologenes; :medium was inoculated with 15 ml of e. faecalis; :medium was inoculated with 7.5 ml of c. indologenes and 7.5 ml of e. faecalis; :medium was inoculated with 7.5 ml of e. faecalis until decolorization occured, followed by inoculating with 7.5 ml of c. indologenes. bars represent erros bars from three replicates. ... ... that might affect the culture's growth could be the existence of intermediate product of orange ii decolorization that inhibits e. faecalis growth. this e. faecalis's low growth rate results in low decolorization rate. the decreased of e. faecalis growth could be due to e. faecalis sensitivity to sulphanilic acid. topaç et al. (2009) reported that the existence of sulfanilic acid > 8 -1 mg kg dry soil could decrease the number of several bacteria. on the other hand, c. indologenes were more resistant to sulphanilic acid and may be able use this compound as c, n, and energy sources. this ability could increase the cells number. athough there was sulphanilic acid in the medium (fourth and fifth treatment, table 1), the concentrations were not sufficient for c. indologenes growth. if we compare the time needed for orange ii decolorization and the rate of orange ii decolorization volume 6, 2012 microbiol indones 121 30 25 20 15 10 5 0 -1 s a c o n ce n tr at io n ( m g l ) 0 20 40 60 80 100 120 140 900 600 300 0 0 20 40 60 80 100 120 -1 g lu co se c o n ce n tr at io n ( m g l ) incubation time (hours) fig 3 change of glucose concentration in experiment with single culture of enterococcus faecalis (control) and with mixed culture (together with chryseobacterium indologenes). x: medium was inoculated with 15 ml of sterile aquadest; : medium was inoculated with 15 ml of c. indologenes; :medium was inoculated with 15 ml of e. faecalis; : medium was inoculated with 7.5 ml of c. indologenes and 7.5 ml of e. faecalis; : medium was inoculated with 7.5 ml of e. faecalis until decolorization occured, followed by inoculating with 7.5 ml of c. indologenes. bars represent erros bars from three replicates. incubation time (hours) fig 4 change of sulphanilic acid concentration in experiment with single culture of enterococcus faecalis (control) and mixed culture with chryseobacterium indologenes. x: medium was inoculated with 15 ml of sterile aquadest; :medium was inoculated with 15 ml of c. indologenes; :medium was inoculated with 15 ml of e. faecalis; :medium was inoculated with 7.5 ml of c. indologenes and 7.5 ml of e. faecalis; :medium was inoculated with 7.5 ml of e. faecalis until decolorization occured, followed by inoculating with 7.5 ml of c. indologenes. bars represent erros bars from could stimulate decolorization of azodye by bacteria. to conclude, inoculation of mixed culture of c. indologenes id6016 and e. faecalis id6017 together early during culture in orange ii containing medium gave the best orange ii decolorization. the rate of -1 orange ii decolorization was 10.98 mg h . using this treatment, the best sulphanilic acid consumption (7.06 -1 mg l ) and equilibrium growths of both species were achieved. references bras r, ferra ia, pinheiro hm, goncalves ic. 2001. batch tests for assessing decolourisation of azo dyes by methanogenic and mixed cultures. j biotech. 89(23):155-62. doi: 10.1016/s0168-1656(01)00312-1. chang js, chien c, lin yc, lin pj, ho jy. 2001. kinetic characteristics of bacterial azo dye decolorization by pseudomonas luteola. water res. 35(12): 2841-2850. doi: 10.1016/s0043-1354(00)00581-9. chang js, chen by, lin ys. 2004. stimulation of bacterial decolorization of an azodyes by extracellular metabolites from e. coli strain no . bioresour technol. 3 91(3): 243-248. doi: 10.1016/s0960-8524(03)00196-2. chen h, wang rf, cerniglia ce. 2004. molecular cloning, overexpression, purification, and characterization of an a e r o b i c f m n d e p e n d e n t a z o r e d u c t a s e f r o m enterococcus faecalis. protein expr purif. 34(2): 302310. doi: 10.1016/j.pep.2003.12.016. coughlin mf, kinkle bk, bishop pl. 1999. degradation of azo dyes containing aminonaphthol by sphingomonas sp. strain 1cx. j ind microbiol biotech. 23(4-5): 341346. dos santos ab. 2004. reductive decolourisation of dyes by thermophilic anaerobic granular sludge [disertation]. wageningen [nl]: wageningen university. james cs. 1995. analytical chemistry of foods. first ed. london: blackie academic & professional. keck a, klein j, kudlich m, stolz a, knackmuss hj, mattes r. 1997. reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of sphingomonas sp. strain bn6. appl environ microbiol. 63(9): 3684-3690. doi: 10.1128/aem.68.9.4341-4349.2002. keck a, rau j, reemtsma t, mattes r, stolz a, klein j. 2002. identification of quinoide redox mediators that are formed during the degradation of naphthalene-2-sulfonate by s. xenophaga bn6. appl environ microbiol. 68(9): 43414349. doi: 10.1128/aem.68.9.4341-4349.2002. kodam km, soojhawon i, lokhande pd, gawai kr. 2005. microbial decolorization of reactive azo dyes under aerobic conditions. world j microbiol biotech. 21(3): 367-370. doi: 10.1007/si1274-004-5957-z. meitiniarti vi, soetarto es, timotius kh, juliana c, vifian n, subarkah da. 2005. kultivasi curah c. indologenes id6016 pada media yang mengandung asam sulfanilat dan anilin. [bulk cultivation of c. indologens id6016 between single culture of e. faecalis and mixed culture of e. faecalis and c.indologenes, it seems that orange ii decolorization performed by mixed culture was better than single culture of e. faecalis (fig 2 and table 1). inoculation of e. faecalis and c. indologenes as a mixed culture at the early experiment resulted higher decolorization rate than inoculation of both bacteria in a successive way. in fourth treatment, time needed for orange ii decolorization was the shortest and the rate of -1 decolorization was the fastest (10.98 mg h ) (table 1). this could be the result of a synergistic interaction between these two bacterial species. in this condition, decolorization product of orange ii will be consumed by c. indologenes, so orange ii decolorization activity by e. faecalis will occur fast and more effective. the capability of c.indologenes to consume sulphanilic acid was demonstrated by culturing of c. indologenes id6016 using medium containing sulfanilic acid as c sole source also using (meitiniarti et al. 2005) and the fitrate's culture of e. faecalis which growing on orange ii containing medium (meitiniarti et al. 2011). the pattern of glucose consumption was similar with cell growth of e. faecalis in third and fifth treatment. although the cell number of e. faecalis inoculated in the fifth treatment was half of that shown by the third treatment, in the end the cell numbers were similar. these conditions indicated that glucose was needed by e. faecalis for growth and orange ii decolorization. mendez-paz et al. (2005) reported that the removal rate of acid orange 7 is highly favorable when glucose is added as co-substrate. bras et al. (2001) also showed that the addition of glucose or acetate ions as electron donors, apparently stimulates the reduction cleavage of azo bonds caused the oxidation of organic electron donors and/or hydrogen is coupled to the colour removal process. in the mixed culture, in addition to the presence of c. indologenes which could decrease toxic affect of sulphanilic acid for e. faecalis, probably c. indologenes could also produce intermediate products that were used as redox mediators for azoreductase produced by e. faecalis. in the degradation of 2naphthalene sulphonate (2ns), there are several mediator redoxs produced by sphingomonas sp. bn6 which could transfer equivalent redoxs to azodye (keck et al. 1997). keck et al. (2002) proposed that these redox mediators shuttle electrons from the cells to the azo dyes, which results in purely chemical, extremely nonspecific, extracellular reductive cleavage of the azo bond. chang et al. (2004) also reported that extracellular metabolite of escherichia coli strain no 3 microbiol indones122 meitiniarti on media containing sulfanilic acid and aniline]. berkala ilmiah biol. 4 (6): 373-384. meitiniarti vi, soetarto es, timotius kh, sugiharto e. 2007. products of orange ii biodegradation by e. faecalis id6017 and c. indologenes id6016. microbiol indones. 1(2): 51-54. meitiniarti vi, timotius kh, haryanto. 2011. the growth of c. indologenes on filtrate's culture of e. faecalis which continuously growing on orange ii containing medium. proceeding of international conference on biological science; 2011 sept 23-24. yogyakarta [id]. mèndez-paz d, omil f, lema jm. 2005. anaerobic treatment of azo dye acid orange 7 under batch condition. enzyme microb tech. 39(5):771-778. doi: 10.1016/j.watres.2004.11.022. pearce ci, lloyd jr, guthrie jt. 2003. the removal of colour from textile wastewater using whole bacterial cells: a review. dyes and pigments 58: 179-196. doi: 10.1016/s0143-7208(03)00064-0. pourreza n, zareian m. 2009. determination of orange ii in food samples after cloud point extraction using mixed micelles. j hazard mater. 165(1-3):1124-1127. doi: 10.1016/j.jhazmat.2008.10.132. ramalho pa, cardoso mh, cavaco-paulo a, ramalho mt. 2004. characterization of azo reduction activity in a novel ascomycete yeast strain. appl environ microbiol. 70(4): 2279-2288. doi:10.1128/aem.70.4.22792288.2004. stolz a. 2001. basic and applied aspects in the microbial degradation of azo dyes. appl microbiol biotech. 56(12): 69-80. doi: 10.1007/s002530100686. supaka n, juntongjin k, damronglerd s, delia ml, strehaiano p. 2004. microbial decolorization of reactive azo dyes in a sequential anaerobic-aerobic system. chem eng j. 99(2): 169-176. doi: 10.1016/j.cej.2003.09.010. tan ncg. 2001. integrated and sequential anaerobic/aerobic biodegradation of azo dyes [disertation]. wageningen [nl]: wageningen university. topaç fo, dindar e, uçaroğlu s, başkaya hs. 2009. effect of a sulfonated azo dye and sulfanilic acid on nitrogen transformation processes in soil. j hazard mater. 170 (23):1006-1013. doi: 10.1016/j.jhazmat.2009.05.080. van der zee fp. 2002. anaerobic azo dye reduction [disertation]. wageningen [nl]: wageningen university. volume 6, 2012 microbiol indones 123 1: 117 2: 118 3: 119 4: 120 5: 121 6: 122 7: 123 cover depan.cdr issn 1978-3477, eissn 2087-8575 volume 14, number 4, december 2020 the ethanol production activity of indigenous thermotolerant yeast 1p4pichia kudriavzevii trench construction in peat soil and the dgge analyses of nif gene and activity of dehydrogenase quality of airborne bacteria in operating theaters in several hospitals in jakarta and its surrounding areas in 2018-2019 lactic acid bacteria from tempeh and their ability to acidify soybeans in tempeh fermentation growth kinetic study of blue-green microalgae arthrospira platensis using buffalo manure as alternative media darojatul ulya, rikaindri astuti, and anja meryandini happy widiastuti, darmono taniwiryono, iman rusmana, and galuh wening permatasari conny riana tjampakasari, nabila naura, and tjahjani mirawati tati barus, gabriela giovania, and bibiana w lay brian a. sinaga, lianty simangunsong, andy trirakhmadi, monita pasaribu, and merry meryam martgrita 121 129 140 149 156 author index 163 subject index 164 3 diana.pmd analysis of pink pigmented facultative methylotroph bacteria from human environments diana elizabeth waturangi* and andreas kusuma school of biotechnology, universitas katolik indonesia atma jaya, jalan jenderal sudirman 51, jakarta 12930, indonesia the formation of pink biofilm in wet places are usually correlated with chlorine-resistant pink pigmented facultative methylotrophs (ppfm). in this study we investigated the presence of ppfm bacteria through bacterial isolation and detection of mxaf gene from wet places of human-made environments. a total of eighteen ppfm bacterial isolates were recovered from the formation of biofilm bacterial of four test places such as washstands, bathrooms, and potable water supplies. confirmation of the isolates through biochemical analysis were done using catalase, oxidase and urease tests. chlorine-resistance-activity was assayed for all of the isolates. antibiotic resistance were examined for ampicillin (25 µg), tetracycline (30 µg), kanamycin (30 µg), trimethoprim (1.25 µg), and streptomycin (10 µg) using the agar diffusion method. genomic dna was subjected to pcr analysis with primers corresponding to the 5’and 3’end conserved segments of the mxaf gene. pcr amplification followed by dna sequencing of 16s rrna gene were done for some isolates. we recovered 18 isolates of ppfm bacteria. biochemical analysis indicated that the isolates were positive for catalase, oxidase, and urease activities. chlorine-resistance-analysis showed the majority of the isolates were resistant to chlorine. antibiotic resistance assays showed all of the isolates exhibited resistance to trimethoprim but were sensitive to streptomycin, kanamycin, and tetracycline but were variably resistant to ampicilin. pcr detection using specific primers for the mxaf gene gave a positive result for all of the isolates. dna sequencing of the 16s rrna gene of two isolates showed that isolate wd10 had a 98% similarity with the mxaf gene from methylobacterium lusitanum strain mp2 and isolate wk2 had a 98% similarity to the mxaf gene from afipia felis strain rd1. the formation of pink biofilm of four wet areas in this study were correlated with the presence of chlorine-resistant ppfm bacteria and we confirmed with the presence of the mxaf gene in all of the isolates. this finding needs to be widely publicized since some ppfm bacteria were known as opportunistic pathogens. key words: pink pigmented facultative methylotroph, human environments, chlorine resistance _____________________________________________ ________________________ * corresponding author, phone: +62-21-5703306 ext. 335, fax: +62-21-5719060, e-mail: diana.waturangi@atmajaya.ac.id volume 2, number 3, december 2008 issn 1978-3477 p 112-114 pink-pigmented facultative methylotrophs (ppfm) in the genus methylobacterium is a physiologically interesting group of bacteria who preferentially utilize substrates lacking carbon-carbon bonds (e.g. methanol and methyl amine) as sources of energy and carbon, which is catalyzed by enzyme methanol dehydrogenase (hanson and hanson 1996). these organisms have been recognized as common environmental isolates from such habitats as leaf surfaces, soil-water, grasses and sewage (hiraishi et al. 1995). these bacteria also occur in wet public environments, including potable water supplies, bathrooms and washstands, where they sometimes produce pink ropy masses of growth (furuhata and matsumoto 1992). most of the methylobacterium strains isolated from these environments are highly resistant to chlorine, moreover some ppfm bacteria are known as opportunistic pathogens (hornei et al. 1999; kelley et al. 2004). this study was designed to isolate, test for resistance to antibiotics, chlorine analysis and of the methanol dehydrogenase gene (mxaf) through pcr amplification. materials and methods bacterial isolation and identification. biofilm formation of bacteria from human-made environments in atma jaya university were selected for bacterial isolation, including bathrooms, washstands and potable water supplies. samples were streaked to minimal media agar supplemented with 1% methanol (v/v) and incubated at room temperature for 7 days. identification was done through microscopic observation, morphological characteristics and biochemical analysis using oxidase and urease tests. antibiotic resistance analysis. antibiotic resistance of the isolates was determined using the agar-disk-diffusiontest with disks containing ampicillin (25 µg), tetracycline (30 µg), kanamycin (30 µg), trimethoprim (1.25 µg) and streptomycin (10 µg) (oxoid, hampshire, england). colonies of the organisms were grown on standard agar (0.25% yeast extract, 0.5% trypton, 0.1% d-glucose, 1.5% agar). performance of the susceptibility testing and evaluation of the antibiograms after incubation for 5 days at 30°c followed the clsi guidelines. chlorine resistance analysis. colonies of the organism grown on standard agar (0.25% yeast extract, 0.5% trypton, 0.1% d-glucose, 1.5% agar) at 30°c for 5 days were harvested and chlorine resistance assays were done using the method published by hiraishi et al. (1995). pcr amplification and dna sequencing of mxaf gene. genomic dna was extracted and purified using the wizard genomic dna purification kit (promega, usa). the mxaf genes were amplified from all of the dna samples in 25-µl reaction mixtures using pcr amplification (perkin elmer, usa). 2.5 u taq polymerase (new england biolabs, usa), 1x buffer, 1 µl dna template and 25 pmol forward primer f1003 (5’-gcggcaccaactggggctggt-3’) and reverse primer r1561 (5’-gggcagcatgagggctccc-3’) with 30 cycles of 92°c for 1 min, 55°c for 1min and 72°c for 1 min; followed by a final extension at 72°c for 5 min. the pcr products were separated by agarose-gel-electrophoresis and purified using a qiaquick gel extraction kit (qiagen, table 1 chlorine and antibiotics resistance test name of isolate sample source amp sp w te k chlorine washstand washstand washstand washstand washstand washstand washstand washstand washstand washstand washstand washstand washstand washstand bathroom bathroom potable water supply potable water supply wd 1 wd 2 wd 3 wd 4 wd 5 wd 6 wd 7 wd 8 wd 9 wd 10 wk 1 wk 2 wk 3 wk 4 km 1 km 2 ta 1 ta 2 i r i i s s i r r i i i i r r s s s s s s s s s s s s s s s s s s s s s r r r r r r r r r r r r r r r r r r s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s r r s r r r r s s s s s s s r r r s r: resistant, i: intermediate, s: susceptible, amp: ampicillin, s: streptomycin, w: trimethoprim, te: tetracycline, k: kanamycin. netherland). the mxaf gene of two isolates were picked randomly and dna sequencing continued using the bigdye terminator v3.1 cycle sequencing kit (applied biosystems, usa). the products were analyzed with an abi prism 377 automated dna sequencer. for comparison with known sequences, the basic local alignment search tool (blast) computer search program was used. results isolation and identification of bacteria. from the bacterial isolates we recovered 18 isolates of ppfm bacteria from 22 samples of human-made wet environments such as washstands, bathrooms and potable water supplies. all of the colonies showed pink colony, positive result for oxidase, catalase and urease activity and were identified as gram negative bacteria through gram staining. chlorine and antibiotic resistance test. chlorineresistance-analysis was performed on 9 isolates resistant to chlorine. six of these isolates were recovered from washstands, 2 from bathrooms and 1 from a potable water supply. in the antibiotic resistance test, all of the isolates were resistant to trimethoprim and 5 isolates were resistant to ampicillin. all of the isolates were sensitive to the antibiotics streptomycin, kanamycin and tetracycline (table 1). detection and dna sequencing of mxaf gene. detection of the mxaf gene using specific primers indicated that all of the isolates have positive results with the amplicon size 550 bp (fig 1). we took two isolates (wd10 and wk2) for dna sequencing of the mxaf gene. isolate wd10 showed 98% similarity to methylobacterium lusitanum strain mp2 and wk2 showed 98% similarity to methylobacterium sp. we have submitted this data to genbank with the accession number eu563216 for isolate wd10 and accession number eu563217 for isolate wk2. discussion from this study, we have shown that ppfm bacteria are widely distributed in human-made wet environments, including washstands, bathrooms and potable water supplies. there are some published reports of the presence of these bacteria in wet environments such as drinking water, tap water, washstands, bathrooms and shower curtains and most of these are resistant to chlorine and antibiotics (furuhata and matsumoto 1992; kelley et al. 2004). the presence of chlorine-resistant and antibiotic-resistant isolates in this study needs to be publicized (table 1), since methylobacterium species have also been attracting attention as opportunistic pathogens (hornei et al. 1999). antibiotic-resistance assays showed that all of the isolates were resistant to trimethoprim, sensitive to streptomycin, kanamycin and tetracycline, but variably resistant to ampicillin. chen et al. (2004) reported that methylobacterium isolated from urinary-tract-infected patients showed a high resistance to trimetophrim. trimethoprim is often used for urinary infection treatments (libecco and powell 2004). most of the isolates showed resistance to chlorine with the highest percentage of isolates from the bathroom. this might be because of the frequent use of chlorine as a desinfectant in this environment. hiraishi et al. (1995) reported that methylobacterium strains acquire the capacity for chlorine resistance by adapting to chlorinated environments. therefore, they can compete with coexisting chemoheterotrophs, and in some cases survive and exhibit massive growth in these environments (chesney et al. 1996). fig 1 shows the presence of mxaf gene in all of the isolates. our data supports the literature about the ability of ppfm bacteria to utilize substrates lacking carbon-carbon bonds (jeong et al. 2002). mcdonald and murrell (1997) reported the use of the methanol dehydrogenase structural gene mxaf as a functional gene probe for methanotrophs and methylotrophs. washstands, bathrooms and potable water supplies are all oligotrophic environments, in which carbon energy sources for the growth of chemotrophic bacteria are very scant. the presence of mxaf gene as one of the structural genes which encodes methanol dehydrogenase enzyme showed this capability. this enzyme carries out a key step in bacterial carbon-one (c1) metabolism since it catalyzes the volume 2, 2008 microbiol indones 113 fig 1 detection of mxaf genes of ppfm isolates: m 1 kb dna ladder; well 1-10, isolates (wd1-wd10); well 11-14, isolates (wk1wk4); well 15-16, isolates from potable water supply (ta1-ta2); well 17-18, isolates from bathrooms (km1-km2). m 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 production of formaldehyde, the intermediate of both assimilative and dissimilative metabolism in methylotrophs. dna sequencing of the 16srrna gene of the two isolates showed that isolate wd10 had a 98% similarity with the mxaf gene from m. lusitanum strain mp2. this was submitted to the genbank with accession number eu563216. isolate wk2 had a 98% similarity to the mxaf gene from a. felis strain rd1, with the accession number eu563217. the formation of pink biofilm in four wet places in this study were correlated with the presence of chlorine-resistant ppfm bacteria. this was confirmed with the presence of the mxaf gene in all of the isolates. this finding need to be widely distributed since some ppfm bacteria were known to be opportunistic pathogens. acknowledgement this work was supported by atma jaya research center, atma jaya catholic university of indonesia. references chen hl, ya ft, jien wl. 2004. underdiagnosis of urinary tract infection caused by methylobacterium species with current standard processing of urine culture and its clinical implications. j med microbiol 53:755-759. chesney ja, eaton jw, mahoney jr. 1996. bacterial glutathione: a sacrificial defense against chlorine compounds. j bacteriol 178:2131-2135. furuhata k, matsumoto a. 1992. some bacteriological studies on the pinkish slimy films formed on tiles using bathrooms and washstands. annu rep tokyo metrop res lab public health 43:197-204. hanson rs, hanson te. 1996. methanotrophic bacteria. microbiol rev 60:439-471. hiraishi a, furuhata k, matsumoto a, koike ka, fukuyama m, tabuchi k. 1995. phenotypic and genetic diversity of chlorineresistant methylobacterium strains isolated from various environments. appl environ microbiol 61:2099-2107. hornei b, luneberg e, schmidt-rotte h, maass m, weberk, heits f, frosch m, solbach w. 1999. systemic infection of an immunocompromised patient with methylobacterium zatmanii. j clin microbiol 37:248-250. jeong jh, kim sw, yoon sm, park jk, lee js. 2002. characterization of the conserved region of the mxaf gene that encodes the large subunit of methanol dehydrogenase from a marine methylotrophic bacterium. mol cells 13:369-376. kelley st, theisen u, angenent lt, amand as, pace nr. 2004. molecular analysis of shower curtain biofilm microbes. appl environ microbiol 70:4187-4192. libecco ja, powell kr. 2004. trimethoprim/sulfamethoxazole: clinical update. pediatrics rev 25:375-379. mcdonald ir, murrell jc. 1997. the methanol dehydrogenase structural gene mxaf and its use as a functional gene probe for methanotrophs and methylo-trophs. appl environ microbiol 63:3218-3224. 114 waturangi and kusuma microbiol indones 2 maria omega_363.pmd volume 3, number 1, april 2009 p 1-11 issn 1978-3477 *corresponding author:, phone: +61-7-38708673, fax: +61-7-33651655, e-mail: omegadvm@hotmail.com influenza a virus: phylogeny of neuraminidase primers and amplification of polymerase basic protein 2 and neuraminidase genes maria omega1*, richard lai3, hans heine4, and ross barnard2 1school of biological science, 2biotechnology department, school of chemistry and molecular biosciences, faculty of science, the university of queensland, st lucia, queensland, australia 4072; 3 biochip innovations pty. ltd., brisbane, queensland, australia, 4csiro livestock industries, australian animal health laboratory, east geelong, victoria, australia influenza a virus is a highly contagious agent that causes bird flu. to date, 16 hemagglutinin (ha) and 9 neuraminidase (na) subtypes are identified antigenically and can form any combinations or mutations with each other to confer non or low pathogenic to high pathogenic strains. mutations in viral segments that are derived from avian isolates represent a novel subtypes to which human population is infected by influenza pandemics. in this work, polymerase basic protein 2 (pb2) gene segment of 8 different avian influenza subtypes were cloned to obtain more dna samples for future work such as pb2 sequencing and to test ha primer annealing with pb2 gene. pcr amplification of na gene segment of 3 different avian influenza subtypes was the second aim of this work to test primer universal for na genes. determination of the aligned sequences between 9 na subtypes and na primer pcr products was the second aim of this work, based on blast result homology 100% and phylogenetic trees of clustal keywords: influenza a virus, phylogeny, amplification, pb2 gene, na gene avian influenza pathogenic virus is a member of the family orthomyxoviridae (lamb et al. 1996; lamb et al. 2007), divided into subtypes on the basis of two surface glycoproteins: hemagglutinin (ha) and neuraminidase (na) (hoffmann et al. 2000; wright et al. 2001). studies of pathogenicity showed that the optimal combination of ha and na enabling the cleavage of ha was important. a balance in ha and na activities is crucial; there must be enough ha activity to facilitate virus binding and enough na activity to allow release of virus progeny (mitnaul et al. 2000; hulse et al. 2004). therefore, ha and na detection of influenza a virus have been done effectively using polymerase chain reaction (pcr) strategy, ha and na universal primers to obtain information that may be related with the detection of viral infection, pathogenicity and host range of influenza a virus. in addition, phylogenetic lineages and blast homology results showed 100% between 9 na viruses and na primer pcr products which are essential to develop alignment and shared homology between subtypes. the accumulation of mutations in viral segments such as polymerase basic protein 2 (pb2), m2 and na genes can generate new chances to increase viral ability to infect species outside of natural reservoir and produce pathogenically various subtypes of virus. therefore, it is important to identify these mutations to detect this viral infection and pathogenicity. for example, the mutation ser64ala in m2 protein gives rise to amantadine resistance (barr 2005; komadina et al. 2005; russel et al. 2008). a glutamic acid to lysine substitution at position 627 in the pb2 protein (hatta et al. 2001; watanabe et al. 2008) has been associated with increased virulence of h5n1 viruses (vesudevan et al. 2005; alvarez et al. 2008). moreover, katz et al. (2000) analyzed that residues thr-223 in the na gene was unique to the h5n1 viruses of low pathogenicity and that glu-355 in pb2 was found only in the human h5n1 viruses of low pathogenicity isolated from birds in hong kong in 1997. mutations are also associated with the antiviral drug resistance, oseltamivir, such as the h5n1 strains with the h274t, r292k and n294s mutations (moscona 2005; moscona 2008; peters et al. 2008). materials and methods dna cloning of pb2 gene avian influenza a virus subtypes: preparation of escherichia coli strain dh5ααααα competent cells according to the protocol provided by nishimura et al. (1990). escherichia coli strain dh5α was used as host cells for amplification of plasmid dna. bacteria cultures were diluted 1:100 in fresh lb broth with ampicillin and then incubated further at 37 °c in a shaking incubator for overnight. 0.5 ml overnight culture was transferred into 50 ml solution a (lb, mgso 4 h 2 o, glucose) in the flask at room temperature. the cultures were then incubated at 37 °c with continuous shaking for about 3-4 h to reach a cell concentration of 8-12 x 108 cells ml-1 mid-log suspension culture indicating an od 600 of 0.8-1.2. this culture was concluded when an optical density measured at 600 nm (od 600 ) is 1.3. the culture was then transferred to microcentrifuge tubes and pelleted at 1500 g for 10 min at 4 °c. the supernatant was discarded while the pellet was resuspended in 0.5 ml solution a and 2.5 ml solution b (glycerin, peg, mgso 4 h 2 o) and aliquoted in 0.1 ml each in 1.5 ml microcentrifuge tubes. these aliquots were then stored at -80 °c. ligation. a total volume of 10 µl ligation reaction consisted of 5 µl rapid ligation buffer 2x, 1 µl taq dna polymerase (promega), and an appropriate volume of insert dna such that pgem-t essay vector: insert dna ratio was approximately 1:2. reactions were made up to final volumes using sterile distilled water and incubated at room temperature for an hour and then stored at 4 °c until transformation was carried out. heat shock transformation. for transformation, 100 µl competent e. coli strain dh5α cells were mixed with 10 µ l a-tailed ligation product (pb2 gene of h1n9 or h3n8 in p-gem-t easy vector) and chilled in ice for 10 min prior to heat shock treatment in a 42 °c water bath for 1 min. the cells microbiol indones2 omega et al. were immediately placed back in ice for another 20 min. the reaction mixture was then incubated in 1 ml prewarm lb at 37 °c for 1 h in a falcon tube. 100 µl bacterial suspension was spread per preheated 100 µg ml-1 of ampicillin selective lb agar plates that were added with 100 µl iptg and 20 µl x-gal for overnight incubation at 37 °c. the remaining bacterial suspension was also spread per lb agar plates and incubated overnight at 37 °c. screening of positive colonies. p-gem-t easy plasmid (promega) was the vector used for all cloning work. putative transformants containing this vector with the desired insert were identified based on blue/white screening. putative positive colonies were identified as white colonies and approximately 10 white colonies per plate were isolated using sterile pipette tip per colony. we did not find any white colonies that grew on agar plates because the ligation and transformation of incompetent e. coli dh5αcells were failed. consequently, we repeated the ligation and transformation with another type of competent cells such as e. coli top10 (invitrogen) cells. ligation. a total volume of 10 µl ligation reaction consisted of 5 µl rapid ligation buffer 2x, 1 µl taq dna polymerase (promega), and an appropriate volume of insert dna such that pgem-t essay vector: insert dna ratio was approximately 1:2. reactions were made up to final volumes using sterile distilled water and incubated at 4 °c for overnight and then, transformation was carried out after successful complementary annealing of vector 3’thymadine (t)overhangs with deoxyadenosine (a)-tailed pcr inserts enables ligation. rapid chemical transformation. for transformation, 50 µl competent e. coli top10 (invitrogen) cells were mixed with 10 µl each a-tailed ligation product (pb2 gene of h5n3, h6n5, h7n7, h11n6, h12n9, or h15n9 shown in table 1, in p-gem-t easy vector) and chilled in ice for 5 min. 10 µl of cells was spread on a pre-warm lb agar plate containing 100 µg ml-1 ampicillin, 100 µl iptg and 20 µl x-gal. the remaining (50 µl) of cells was also spread on another lb agar plate. these agar plates were incubated overnight at 37 °c. screening of positive colonies. p-gem-t easy plasmid (promega) was the vector used for all cloning work. putative transformants containing this vector with the desired insert were identified based on blue/white screening. putative positive colonies were identified as white colonies and approximately 10 white colonies per plate were isolated using sterile pipette tip per colony. each tip was immersed in 5 ml falcon tubes containing 2 ml lb broth supplemented with 100 µg ml-1 ampicillin. the bacterial suspension was grown overnight in a 37 °c shaking incubator. positive colonies are determined with plasmid purification followed by nanodrops spectrophotometer, restriction digestion and these clones are cryopreserved at -20 °c until use. plasmid purification. the alkaline method was used for purification of plasmid dna using the qiaprep spin miniprep kit (qiagen). briefly, putative transformants (white colonies) were cultured in 2 ml lb broth supplemented with 100 µg ml-1 ampicillin for overnight. bacterial culture grew at 37 °c in a shaking incubator plasmid extraction was carried out in accordance with the manufacturer’s recommendation. the eluted dna is stored in -20 °c freezer. determination of dna concentration. the absorbance of dna was measured at 260 nm (a 260 ) using a nanodrop untraviolet/visible spectrophotometer. dna concentrations were estimated with the assumption that 50 µg ml-1 double stranded dna has an absorbance at a 260 of 1. the nanodrop spectrophotometer was utilized in all absorbance and dna/ rna concentration determination. 2 µl each sample of eluted dna was dropped and measured at µ = 260, dna -50 using spectrophotometer program of nd1000 v3.1.2, nucleic acid. eluted dna was pure if 260/280 is 1.8-2.0. the concentration of eluted dna was also measured to obtain information in dna dilution. restriction endonuclease digestion of plasmid dna. restriction endonuclease digests were performed with a 20 µl digestion reaction mix consisting 2 µl h buffer 10x (roche) and an appropriate amount of dna such as restriction enzyme: plasmid dna ratio was approximately 1:10. reactions were made up to final volumes using sterile distilled water. the enzyme ecori was used in all restriction digests and thus, the reaction mix was carried out using the pcr cycling program (37 °c for 90 min, 65 °c for 15 min and 4 °c until end). digested products were visualized on 2% agarose gels. agarose gel electrophoresis of dna. agarose gel electrophoresis of dna was typically run on a 1.5-2% (w/v) agarose gel stained with ethidium bromide in 1x tae buffer. dna samples were combined with 6x loading dye. the size of dna fragments were determined by comparison to a standard 1 kb dna ladder mix or 2 kb dna hyperladder ii, visualized by ultra violet illumination. polymerase chain reaction amplification of na genes. pcr involves repetitive thermal cycling composed of three steps: i, melting temperature (94 °c) to denaturate dna; ii, annealing to cool the mixture (step down temperature from 56-42 °c) allowing na primers to anneal target dna sequences; and iii, extension to elongate dna with dntp by polymerase. pcr amplification of 3 subtypes of avian na genes of influenza virus subtypes (h6n5, h7n7 and h12n9) were essential as a separate step following cdna composition via rt-pcr due to limited starting volumes of rna. amplification was carried out in a 50 µl reaction consisting of 5 u µl-1 taq dna polymerase, 10x taq reaction buffer, 50 mm mgcl 2, 10 mm dntp mix, 100 µm of each forward and reverse na primer, an appropriate volume of distilled water and template dna as optimized from previously obtained rt cdna. pcr reaction was carried out using the mastercycler pcr® machine (eppendorf, qld). pcr products were stored at -20 °c (freezer) and were also visualized on 1.5% agarose gels. table 1 polimerase basic protein 2 (pb2) genes of influenza a virus subtypes subtype h1n9 h3n8 h5n3 h6n5 h7n7 h11n6 h12n9 h15n9 avian species a/shelduck/wa/1757/78 a/avian/669/wa/78 a/shearwater/aust/75 a/shearwater/aust/72 a/chicken/victoria/1/85 a/tern/aust/75 a/rnstint/wa/5754/84 a/shelduck/wa/1762/79 reference 8705-22-1100 8510-15-1215 8508-16-1600 8507-10-1200 8507-31-1430 8610-15-1330 9010-05-1330 9008-18-1000 microbiol indones 3volume 3, 2009 extraction of dna fragments from agarose gels. the minelute gel extraction kit protocol using a microcentrifuge (qiagen) was utilized for the isolation of specific dna fragments from agarose gels. experimentation was performed according the manufacturer’s instructions. generally, gel extractions were performed to elute dna from gels contained desired dna bands of pcr products with an appropriate amount of gel such that gel: buffer qg ratio was approximately 1:3 and gel: isopropanol was approximately 1:1, and added another buffer (pe) and distilled water. to elute more dna, second clean up was carried out by adding distilled water to the spin column. eluted dna was stored at -20 °c. blast searches and phylogenetic tree development and basic local alignment search tool (blast) searches. sequence data was compared to genbank dna database using blastn searches to determine the alignment (% identity) between na primers (table 2) and all virus origin (virus nucleotide database) using the national centre for biotechnology information (ncbi) blast network server available from http://www.ncbi.nlm.nih.gov/ phylogenetic tree development. phylogenetic trees were developed to test variability for regions of sequence bounded by pcr primers. 3 pairs of na primers (5f and 10r, 8f and 10r, 10f and 11r) were aligned against a selection of genbank database sequences using clustalx v 1.83 available from h t t p : / / b i p s . u ~ s t r a s b g . f r / d o c u m e n t a t i o n / c l u s t a l x . sequences based on influenza sequence databasehttp://www.flu.lanl.gov/ and na primer sequences were uploaded as fasta files and a preliminary complete alignment was done. aligned sequences were viewed, and then sequences were cropped to do alignment on sequence range defined by primers. cropped sequences were realigned and saved as a new fasta files. a region sequences defined by primers was aligned to draw bootsrapped nj trees and to open the trees using treeview. 1000 bootstrap values were carried out and can be shown at the nodes of the trees shown in fig 1, 2, and 3. mutations in na primer pcr products. mutations in the na primer products were specifically targeted for research in this project. mutations positions of the na primers have been reported to increase infection, pathogenicity and resistance to antiviral drugs. thereby, it is essential to determine any mutations that occur clinically in na primer pcr products. results amplification of pb2 gene segment dna via cloning. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length of 986 bp. lane 1 consisted of 2 kb dna hyperladder ii. lane 2 consisted of positive control, contained plasmid dna without ecori enzyme. lane 3 showed negative control, contained ecori enzyme without plasmid dna. lane 4-13 each denoted p-gem-t easy vector dna length product of 3015 bp and h5n3/a/shearwater/aust/75 pb2 full length product of 986 bp, clones 1-10. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length which was expected to be 986 bp. lane 1 consists of 2 kb dna hyperladder ii. lane 2 shows negative control, contains ecori enzyme ithout plasmid dna. lane 3-12 each denotes p-gem-t easy vector dna length product of 3015 bp and h6n5/a/shearwater/aust/72 pb2 full length product of 986 bp, clones 1-10. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length of 986 bp. lane 1 consists of 2 kb dna hyperladder ii. lane 2 shows negative control, contains ecori enzyme without plasmid dna. lane 3-12 each denotes p-gem-t easy vector dna length product of 3015 bp and h11n6/a/tern/aust/75 pb2 full length product of 986 bp, clones 1-10. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length which was expected to be 986 bp. lane 1 consists of 2 kb dna hyperladder ii. lane 2 consisted of positive control, contained plasmid dna without ecori enzyme. lane 3 showed negative control, contained ecori enzyme without plasmid dna. lane 4-13 each denoted p-gem-t easy vector dna length product of 3015 bp and h15n9/a/shelduck/wa/1762/79 pb2 full length product of 986 bp, clones 1-10. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length of 986 bp. lane 1 consisted of 2 kb dna hyperladder ii. lane 2 showed negative control, contained ecori enzyme without plasmid dna. lane 3 and 7 each consisted of positive control, contained plasmid dna without ecori enzyme. lane 4-5 each denoted p-gem-t easy vector dna length product of 3015 bp and h7n7/a/chicken/ victoria/1/85 pb2 full length product of 986 bp, clones 1-2. lane 6 showed only p-gem-t easy vector dna length product of 3015 bp, without any insert dna. lane 8-10 depicted p-gem-t easy vector dna length product of 3015 bp and h12n9/a/rnstint/wa/5754/84 pb2 full length product of 986 bp, clones 5, 9 and 10. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length which was expected to be 986 bp. lane 1 consisted of 2 kb dna hyperladder ii. lane 2 showed negative control, contained ecori enzyme without plasmid dna. lane 3 consisted of positive control, contained plasmid dna table 2 neuraminidase (na) primers id 4 0 7 7 8 8 4 0 7 7 9 3 4 0 7 7 8 5 4 0 7 7 8 7 4 0 7 7 9 2 oligo sequence 5’ – 3’ gra chc arg art c5k mrt g tgy agr gay aay tgg m55 gg cay ds5 aat gr5 acm rt5 ma5 ga cc5 5kc car ttr tcy ctr ca ccd asa rta 5cc 5ga cca rt product (bp) start end length 6 9 0 8 9 0 4 4 9 6 9 0 8 9 0 9 0 9 1 2 4 3 9 0 9 9 0 9 1 2 4 3 2 1 9 3 5 3 4 6 0 2 1 9 3 5 3 8f 10f 5f 10r 11r microbiol indones4 omega et al. gi|31339467|gb|af523409.1| gi|58429316|gb|ay862645.1| gi|50296136|gb|ay651434.1| gi|6048772|gb|af098551.1|af098 502 gi|37955308|gb|ay 207540.1| gi|30349254|gb|ay261521.1| gi|49357298|gb|ay633286.1| gi|13260576|gb|af250361.2|af25 427 444 486 gi|54037675|gb|ay664708.1| gi|30522963|gb|ay233391.1| gi|40795628|gb|ay497127.1| gi|30267602|gb|ay254106.1| gi|324495|gb|k01006.1|flaname gi|58429306|gb|ay862640.1| gi|21636452|gb|af457711.1| 607 193 368 505 523 941 gi|324537|gb|m38330.1|flanan7 gi|324419|gb|k01022.1|flanabl gi|46981860|gb|ay531030.1| 431 954 gi|324465|gb|k01039.1|flanalj gi|56425153|gb|ay684901.1| 526 gi|37955328|gb|ay207550.1| gi|37955322|gb|ay207547.1| gi|37955338|gb|ay207555.1| gi|37955300|gb|ay207536.1| 71 994 296 816 332 65 gi|37955294|gb|ay207533.1| gi|324433|gb|k01013.1|flanabmc gi|37955286|gb|ay207529.1| 811 708 gi|324515|gb|k01014.1|flanamn gi|324447|gb|k01034.1|flanala gi|47834200|gb|ay611526.1| 650 gi|56425155|gb|ay684902.1| gi|47680888|gb|ay586419.1| 5f 422 482 175 418 231 gi|49357324|gb|ay633390.1| gi|324539|gb|m37511.1|flanan9 gi|1815675|gb|u84110.1|iau8411 gi|324879|gb|m17813.1|flantern 637 809 803 10r gi|537509|gb|m24740.1|flasheau gi|324431|gb|k01019.1|flanabmb 720 492 gi|305215|gb|l06587.1|flanaita gi|49357314|gb|ay633350.1| gi|31747400|gb|ay300948.1| gi|305227|gb|l06584.1|flananew gi|305205|gb|l06585.1|flanade gi|46981876|gb|ay531038.1| 272 408 323 621 332 623 234 62 625 622 323 trichotomy 0.1 fig 1 phylogenetic tree including 5f, 10r na primers and virus subtypes. microbiol indones 5volume 3, 2009 gi|31339467|gb|af523409.1| gi|6048772|gb|af098551.1|af098 gi|50296136|gb|ay651434.1| 490 gi|58429316|gb|ay862645.1| gi|37955308|gb|ay207540.1| gi|13260576|gb|af250361.2|af25 gi|30349254|gb|ay261521.1| gi|49357298|gb|ay633286.1| 985 958 gi|37955294|gb|ay207533.1| gi|37955286|gb|ay207529.1| gi|324433|gb|k01013.1|flanabmc 10r 811 805 678 gi|305215|gb|l06587.1|flanaita gi|537509|gb|m24740.1|flasheau gi|324431|gb|k01019.1|flanabmb 992 583 gi|31747400|gb|ay300948.1| gi|49357314|gb|ay633350.1| gi|46981876|gb|ay531038.1| gi|305227|gb|l06584.1|flananew gi|305205|gb|l06585.1|flanade 498 477 574 963 964 gi|40795628|gb|ay497127.1| gi|30267602|gb|ay254106.1| gi|21636452|gb|af457711.1| 127 gi|30522963|gb|ay233391.1| gi|54037675|gb|ay664708.1| gi|324495|gb|k01006.1|flaname gi|58429306|gb|ay862640.1| 695 526 337 151 693 gi|324537|gb|m38330.1|flanan7 gi|324419|gb|k01022.1|flanabl gi|46981860|gb|ay531030.1| 549 674 gi|56425155|gb|ay684902.1| gi|47680888|gb|ay586419.1| gi|47834200|gb|ay611526.1| gi|324447|gb|k01034.1|flanala gi|324515|gb|k01014.1|flanamn 963 618 305 547 209 gi|56425153|gb|ay684901.1| gi|37955328|gb|ay207550.1| 605 gi|37955338|gb|ay207555.1| gi|37955300|gb|ay207536.1| gi|37955322|gb|ay207547.1| 949 961 514 gi|49357324|gb|ay633390.1| gi|324879|gb|m17813.1|flantern gi|324539|gb|m37511.1|flanan9 gi|324465|gb|k01039.1|flanalj gi|1815675|gb|u84110.1|iau8411 8f 728 372 125 127 226 285 200 824 767 845 569 919 268 trichotomy 0.1 fig 2 phylogenetic tree including 8f, 10r na primers and virus subtypes. gi|13260576|gb|af250361.2|af25 gi|30349254|gb|ay261521.1| gi|49357298|gb|ay633286.1| 438 gi|37955308|gb|ay207540.1| gi|50296136|gb|ay651434.1| gi|31339467|gb|af523409.1| gi|58429316|gb|ay862645.1| gi|6048772|gb|af098551.1|af098 11r 10f 946 464 202 211 240 gi|305215|gb|l06587.1|flanaita gi|537509|gb|m24740.1|flasheau gi|324431|gb|k01019.1|flanabmb 973 274 gi|31747400|gb|ay300948.1| gi|49357314|gb|ay633350.1| gi|305205|gb|l06585.1|flanade gi|305227|gb|l06584.1|flananew gi|46981876|gb|ay531038.1| 561 553 666 872 826 gi|37955294|gb|ay207533.1| gi|324433|gb|k01013.1|flanabmc gi|37955286|gb|ay207529.1| 757 994 gi|54037675|gb|ay664708.1| gi|30522963|gb|ay233391.1| gi|324495|gb|k01006.1|flaname gi|58429306|gb|ay862640.1| gi|21636452|gb|af457711.1| gi|40795628|gb|ay497127.1| gi|30267602|gb|ay254106.1| 290 218 380 701 657 973 gi|56425155|gb|ay684902.1| gi|47680888|gb|ay586419.1| 975 gi|324515|gb|k01014.1|flanamn gi|324447|gb|k01034.1|flanala gi|47834200|gb|ay611526.1| 529 914 836 gi|46981860|gb|ay531030.1| gi|324419|gb|k01022.1|flanabl gi|324537|gb|m38330.1|flanan7 406 893 gi|49357324|gb|ay633390.1| gi|324539|gb|m37511.1|flanan9 gi|1815675|gb|u84110.1|iau8411 gi|324879|gb|m17813.1|flantern 579 969 990 gi|56425153|gb|ay684901.1| gi|324465|gb|k01039.1|flanalj gi|37955328|gb|ay207550.1| 595 gi|37955322|gb|ay207547.1| gi|37955300|gb|ay207536.1| gi|37955338|gb|ay207555.1| 202 949 288 825 284 372 593 526 408 383 576 861 trichotomy 0.1 fig 3 phylogenetic tree including 10f, 11r na primers and virus subtypes. microbiol indones6 omega et al. without ecori enzyme. lane 4-13 each denoted p-gem-t easy vector dna length product of 3015 bp and h7n7/a/ chicken/victoria/1/85 pb2 full length product of 986 bp, clones 1-10. dna products generated by pcr digestion showed plasmid dna product length of 3015 bp and insert dna clone product length which was expected to be 986 bp. lane 1 consisted of 2 kb dna hyperladder ii. lane 2 showed negative control, contained ecori enzyme without plasmid dna. lane 3 consisted of positive control, contained plasmid dna without ecori enzyme. lane 4-9 each denoted only p-gem-t easy vector dna length product of 3015 bp and without any insert dna clones 1-6. determination of pb2 gene concentration. plasmid purification using qiaprep spin miniprep kit (qiagen) followed by restriction endonuclease and gel analysis (fig 49) indicated that ligations was highly efficient with most putative pb2 colonies proving to be positive. the presence of pb2 genes in plasmids of isolated clones were confirmed by dna purity and concentration via nanodrop ultraviolet (uv)/visible spectrophotometer. dna purity was estimated with the assumption that pure dna sample has an absorbance at a 260/280 of 1.8-2.0. the results showed pb2 gene h1n9 clone 7, pb2 gene h6n5 clone 3 and 6, pb2 gene h15n9 clone 1, pb2 gene h5n3 clone 5 and pb2 gene h7n7 clone 5 were less than 1.8 indicating the dna samples were not pure because of protein contaminations during plasmid dna purification procedures while the other clones were pure as expected to be between 1.8 and 2.0 (table 4). pcr amplification of na genes. to generate neuraminidase (na) gene segment samples via pcr amplification (table 5) was essential for analysis of primer annealing, mutations and phylogeny work. the genes were volume 3, 2009 microbiol indones 7 fig 7 analysis of h15n9 pb2 gene segment dna pcr product. 1 2 3 4 5 6 7 8 9 10 11 12 13 3015 bp 986 bp fig 8 analysis of h7n7 and h12n9 pb2 gene segment dna pcr product. 3015 bp 986 bp 1 2 3 4 5 6 7 8 9 10 3015 bp 986 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 fig 9 analysis of h7n7 pb2 gene segment dna pcr product. 3015 bp 986 bp 1 2 3 4 5 6 7 8 9 10 11 12 fig 6 analysis of h11n6 pb2 gene segment dna pcr product. fig 4 analysis of h5n3 pb2 gene segment dna pcr products. 3015 bp 986 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 3015 bp 986 bp fig 5 analysis of h6n5 pb2 gene segment dna pcr product. need to be isolated, amplified, analyzed then further work including sequencing. extraction of dna fragments from agarose gels. the minelute gel extraction kit protocol using a microcentrifuge (qiagen) was utilized for the isolation of specific dna fragments from varying weight of agarose gels to have eluted dna samples for dna sequencing and testing primer annealing. we had 15 µl elute dna of each sample resulted from this work and had more 8 µl elute dna from second clean up of elute dna extraction (table 6). basic local alignment search tool (blast) searches. blastn was utilized for the alignment of all sequences of na gene primers generated against a genbank consensus avian influenza virus subtypes. blast analysis formed a robust alignment with the consensus sequence indicating 100% homology between 8f, 10f, 11r primers and influenza a virus subtypes. in particular, 8f primer was labeled in the alignment to correspond with other virus origin depicted 94% homology. it may be noted that 5f primer appeared to be non-specific primer for all virus subtypes available on genbank. further confirmation of sequence was required using the outline in table 7. phylogenetic tree development. utilizing clustal-x 1.83 for multiple alignments, it was possible to test variability for regions of 44 different aiv subtype sequences divided into 9 varied na genes and bounded by na pair of primer pcr products (table 8). all trees developed underwent bootstrapping and treeview programs to distinguish phylogenetic relationships between aiv subtypes and na primers which randomly align into 0.1 distance matrices of 1000 frequency of each node or label. it was essential to analyze the bootstrap values and positions of sequences that were aligned or matched as well as mismatched or creating a gap (fig 1-3). mutations in na primer pcr products. the information shown in this table was based on scientific findings from brown et al. (1998); herlocher et al. (2003); herlocher et al. (2004); de jong et al. (2005); jackson et al. (2005); mai le et al. (2005); jackson et al. (2008); stouffer et al. (2008). as can be seen, clinical mutations occurring in 8f and 10r pair of primers at protein positions 274 (820-830 amino acid positions) and 292 (870-880 amino acid positions) and 10f and 11r pair primers at protein positions 294 (880-890 amino acid positions). discussion amplification of pb2 gene h1n9/a/shelduck/wa/ 1757/78 and h3n8/ a/avian/669/wa/78 via cloning into escherichia coli strain dh5ααααα. ligation pb2 gene into e. coli dh5α cells were failed because e. coli dh5α cells could microbiol indones8 omega et al. table 6 minelute dna from gel extraction sample h6n5 a/shearwater/aust/72 h6n5 a/shearwater/aust/72 h6n5 a/shearwater/aust/72 h6n5 a/shearwater/aust/72 h6n5 a/shearwater/aust/72 h6n5 a/shearwater/aust/72 h6n5 a/shearwater/aust/72 h7n7 a/chicken/victoria/1/85 h12n9 a/rnstint/wa/5754/84 h12n9 a/rnstint/wa/5754/84 h12n9 a/rnstint/wa/5754/84 band (bp) 3 6 0 2 0 0 2 1 9 3 0 0 3 5 3 3 5 3 1 6 9 3 5 3 2 1 9 3 5 3 4 6 0 weight of agarose gel (g) 0 . 2 0 . 3 0 . 3 0 . 3 0 . 2 0 . 4 0 . 2 0 . 3 0 . 2 0 . 2 0 . 2 table 5 expected bands for pcr products sample to run h6n5a/shearwater/aust/72 h6n5a/shearwater/aust/72 h6n5a/shearwater/aust/72 h6n5a/shearwater/aust/72 h7n7a/chicken/victoria/1/ 85(positive control) h2o(negative control) pair of primers 5f + 10r 8f + 10r 10f + 11r 8f + 9r 10f + 11r no primer product length (bp) 4 6 0 2 1 9 3 5 3 1 6 9 3 5 3 no bands results from gels (figures were unavailable) 3 bands: 200 bp, 300 bp, 400 bp 219 bp 353 bp 170 bp 353 bp no bands table 4 dna purity and concentration of pb2 gene segment clones clone (n = 80) pb2, h1n9 clones 2, 3, 4, 5, 6, 7, 9, 10 pb2, h3n8 clones 1-9 pb2, h6n5 clones 1,2,3,4,6 pb2, h6n5 clones 1-10 pb2, h11n6 clones 1-10 pb2, h15n9 clones 1-10 pb2, h5n3 clones 1-10 pb2, h7n7 clones 1-3 pb2, h7n7 clones 1-10 pb2, h12n9 clones 5,9,10 pb2, h12n9 clones 1-6 dna purity (260/280 = 1.8-2.0) clones 2, 3, 4, 5, 6, 9, 10 are good except clone 7 (1.38). h3n8 clones 1-9 are all good. h6n5 clones 1-4 are good except clone 6 (1.71). h6n5 clones 1, 2, 4-10 are good except clone 3 (1.57). h11n6 clones 1-10 are all good. h15n9 clones 2-10 are good except clone 1 (1.78). h5n3 clones 1-4, 6-10 are good except clone 5 (1.75). h7n7 clones 1, 3 are good except clone 2 (1.78). h7n7 clones 1-10 are all good. h12n9 clones 5,9,10 are all good. h12n9 clones 1-6 are all good. dna concentration (ng µl-1) clone 2 is the lowest (81.2), clone 7 is the highest (119.2) h3n8 clone 2 is the lowest (109.9), clone 9 is the highest (140.3). h6n5 clone 6 is the lowest (14.0), clone 1 is the highest (247.1). h6n5 clone 1 is the lowest (63.4), clone 10 is the highest (201.2). h11n6 clone 8 is the lowest (52.4), clone 3 is the highest (92.1). h15n9 clone 2 is the lowest (74.2), clone 9 is the highest (121.8). h5n3 clone 3 is the lowest (86.8), clone 6 is the highest (153.5). h7n7 clone 2 is the lowest (95.8), clone 1 is the highest (115.2). h7n7 clone 8 is the lowest (94.4), clone 7 is the highest (155.1). h12n9 clone 5 is the lowest (98.9), clone 10 is the highest (124.4). h12n9 clone 4 is the lowest (29.3), clone 6 is the highest (150.9). volume 3, 2009 microbiol indones 9 not be competent cells indicating mid-log suspension culture of od 600 for 3-4 hr was 1.3 (od 600 should be 0.8-1.2 means 8-12.108 cells ml-1). thereby, low efficiency competent cells (< 1 x 108 cfu µg-1 dna) showed many cells died because of lack of food source and lots of toxic metabolites. in addition, we applied improperly less incubation time (1-4 h at room temperature) for optimal ligation. as a result, the transformation efficiency (te) for e.coli dh5α cells was either low (t.e. was less than 1 x 109 cfu µg-1 supercoiled plasmid) or the cells did not grow well on agar plates. consequently, we used another type of competent cells such as e. coli top10 cells from invitrogen and the cloning experimentation was performed according to the manufacturer’s instructions. amplification of pb2 gene segment dna via cloning into top10 cells. generation of clones was required for future sequencing of the pb2 gene 6 avian influenza virus (aiv) subtypes provided (h5n3, h6n5, h7n7, h11n6, h12n9 and h15n9 subtypes shown in table 1). dna cloning procedures including ligations, transformations, plasmid purifications and restriction enzyme digestions were mostly successful with insert dna bands of the correct size resulting upon agarose electrophoresis analysis. uv analysis for other subtypes identified insert dna products of expected sizes (full length pb2 genes were detected at the large 986 bp product) shown in fig 4-9. however, no visible bands of insert dna for pb2 gene h12n9 clones 1-6 (fig 10) appearing upon gel analysis of a potential problems with dna samples following failed transformation with a lack of white bacterial colony growth on spread agar plates indicating dna absence. the transformation efficiency (te) of pb2 gene h12n9 was low (te = 8 x 106 transformants/µg plasmid dna while te of these cells should be >” 1 x 109 cfu µg-1 supercoiled plasmid). determination of pb2 gene concentration. dna concentration (table 4) was determined with the assumption tabel 7 blast result homology % between na primers and virus origin primer 5f 8f 10f 10r 11r nucleotide cay ds5 aat gr5 acm rt5 ma5 ga (192 redundancy, 48% recombination) gra chc arg art c5k mrt g (192 redundancy, 42% recombination) tgy agr gay aay tgg m55 gg (32 redundancy, 35% recombination) cc5 5kc car ttr tcy ctr ca (32 redundancy, 35% recombination) ccd asa rta 5cc 5ga cca rt (24 redundancy, 30% recombination) result homology % 100% for other viruses and bacteria, such as hiv-1, herpesvirus, bacillus, canine distemper, streptococcus so 5f primer was non-specific primer for influenza virus. 100% for h1n2, h3n2, h7n2, h4n6, h7n7, h5n7 94% for h4n6, h1n3, h3n3, h9n2, h6n6, h13n3, h3n2, h3n3, h1n2, h9n2, h7n3, h5n8, h3n6. 100% for h3n2, h1n2. 100% for h3n1, h11n1, h1n1, h9n1, h13n9, h11n9, h5n1, h2n1, h7n1, h2n9. 100% for h7n2, h6n2, h11n2, h9n2, h5n2. na subtype sequence names of 44 different subtypes table 8 sequence names for clustalx 1.83 (shown in fig 1-3) n 1 n 2 n 3 n 4 n 5 n 6 n 7 n 8 n 9 a/dk/indonesia/ms/2004(h5n1), a/duck/korea/s17/03(h6n1), a/turkey/ontario/hr2/2000(h7n1), a/aquatic bird/hong kong/m603/98 (h11n1), a/duck/shantou/2030/00(h9n1), a/duck/ohio/118c/93 (h1n1), a/mallard/alberta/47/98(h4n1), a/mallard/stralsund/41-6/81(h2n1). a/mallard/new york/6750/78 (h2n2), a/chicken/guatemala/194573/02 (h5n2), a/duck/korea/s7/03(h3n2), a/chicken/hongkong/tp38/03(h9n2), a/chicken/california/139/01(h6n2), (a/avian/ny/76247-3/00(h7n2), a/duck/nc/91347/01(h1n2). a/chicken/british columbia/04(h7n3), a/blue-winged teal/604/78 (h2n3), a/shearwater/australia/75 (h5n3), a/black-headed gull/sweden/2/99(h16n3), a/tern/astr/775/83(h3n3) a/gray teal/australia/2/79(h4n4), a/ruddy turnstone/delaware/67/98(h12n4), a/turkey/ontario/6118/68 (h8n4) a/shearwater/72 (n5), a/shearwater/australia/72 (h6n5) a/mallard/alberta/42/77(h1n6), a/duck/england/56 (h11n6), a/duck/newzealand/31/76(h4n6), a/turkey/minnesota/957/80(h6n6), a/black-headed gull/sweden/1/99(h13n6), a/pintail duck/alberta/712/80(h3n6) a/chick/n/germany/49 (h10n7), a/fpv/weybridge (h7n7), a/mallard/64650/03(h5n7) a/turkey/canada/63(h6n8), a/quail/italy/1117/65 (h10n8), a/guinea fowl/new york/4-3587/84 (h3n8), a/herring gull/de/677/88 (h2n8), a/pintail/alberta/207/99(h4n8), a/duck/ny/191255-59/02(h5n8) a/tern/australia/g70c/75 (h11n9), a/ruddy turnstone/nj/60/85 (n9), a/nws/33haa/tern/australia/g70c/75na (h1n9), a/teal/alberta/16/97(h2n9) note: h11n6 and h10n8 created gaps in na pair of primers (see fig 1-3). 3015 bp 1 2 3 4 5 6 7 8 9 fig 10 analysis of h12n9 pb2 gene segment dna pcr product. that double stranded dna sample has a calculation absorbance from 2 to 100 ng µl-1 with upper limit to detect 3700 ng µl-1. the results indicated that h6n5 clone 6 and h12n9 clone 4 had much lower concentration than other clones (the concentrations were less than 30 ng µl-1). consequently, these dna sample may not be detected by gel electrophoresis after the utilization of restriction enzyme digestions. the determination of dna concentration will be important for dna dilutions of future work such as dna sequencing, dna annealing with universal ha primer designs, and dna sampling to detect new diagnostic tools of influenza infection, virulence and pathogenesis. pcr amplification of na genes. subtypes na gene h6n5/a/shearwater/aust/72 were annealing with 8f and 10r pair of primers showing expected band of 219 bp, and 10f and 11r pair of primers indicating desired band of 353 bp. on the other hand, na gene h6n5/a/sheartwater/72 was less accurate annealing with 5f and 10r pair primers with varying bands of 200 bp, 300 bp and 400 bp (table 5). it is also important to note that 5f and 10r pair of primers was not specific primers for influenza a viruses as blast result homology 100% shown in table 7. another subtype na gene h7n7/a/chicken/victoria/1/85 used as positive control was annealing with 10f and 11r pair primers denoting expected band to be 353 bp. extraction of dna fragments from agarose gels. na gene subtype h6n5/a/sheartwater/72 was successfully extracted from isolation of expected band to be 219 bp indicating matched annealing with 8f and 10r primers and another band of 353 bp showing correct annealing with 10f and 11r primers. specific dna fragment of desired band (353 bp) was extracted from na gene h7n7/a/chicken/ victoria/1/85 showing annealing with 10f and 11r pair of primers. another subtype was na gene h12n9/a/rnstint/ wa/5754/84 denoting expected bands of each 219 bp for 8f and 10r primer annealing, 353 bp for 10f and 11r primer annealing and 460 bp for 5f and 10r primer annealing. basic local alignment search tool (blast) searches. it can be shown that 5f primer had 192 redundancies and 48% recombination and when 5f primer paired with 10r primer which had 32 redundancies and 35% recombination, the multiplicity of 5f and 10r pair of primers expected to be 6144 with 83% recombination. it may be noted that 8f primer had 192 redundancies and 42% recombination and when 8f primer paired with 10r primer which had 32 redundancies and 35% recombination, the multiplicity of 8f and 10r pair of primers expected to be 6144 with 77% recombination. of interest was 10f and 11r pair of primers that 10f primer had 32 redundancies with 35% recombination and 11r primer had 24 redundancies and 30% recombination, there by, when they paired the multiplicity became 768 with 65% recombination. the increased redundancy, multiplicity and recombination conferred problems, meaning that large amounts of primers would have to be utilized in order to anneal template dna samples. based on the analysis of homology, redundancy, multiplicity and recombination, it appeared that 10f and 11r pair of primers was better than 8f and 11r pair of primers. moreover, 8f and 11r pair of primers was much better than 5f and 10r pair of primers. for this reason, 10f and 11r pair of primers and 8f and 10r pair of primers will be important to utilize as universal primers for na gene aiv subtype detections. phylogenetic tree development. the first tree produced aligned 5f and 10r pair of primers with aiv subtypes successfully sequenced (fig 1). upon analysis, it appeared that bootstrap values of 9 na gene aiv subtypes ranged between 332 and 954 (from 33.2% to 95.4%) and there was no gap in sequence positions. as a result, n1, n2, n3, n4, n5, n6, n7, n8 and n9 were aligned or clustal within each subtype. in addition, 5f primer seemed to remain similar to n3 subtype with shortest distance of 422 (42.2%) bootstrap values. 10r primer indicated closely linked to n5 appearing shortest distance of 492 (49.2%) bootstrap value. looking at aiv aligned subtypes in fig 2, 8f and 10r pair of primers showed bootstrap values ranged between 226 and 992 (from 22.6% to 99.2%) with two gaps in n8 and n6. when analyzing tree in fig 2 consisting of two gaps, clearer pathways emerged with clear indication that n8 (h10n8) was aligned with n5 showing 583 (58.3%) bootstrap value and was also aligned with its own n8 subtype denoting 964 (96.4%) bootstrap value. moreover, n6 (h11n6) was aligned with n9 indicating 372 (37.2%) bootstrap value and was also aligned with its own n6 subtype appearing 285 (28.5%) bootstrap value. consequently, only n1, n2, n3, n4, n5, n7 and n9 were aligned within each subtype. furthermore, 8f primer appeared to remain closely related to n9 with 728 (72.8%) bootstrap value while 10r primer indicated similar to appearing n4 with 811 (81.1%) bootstrap value. fig 3 indicated the phylogenetic relationship of na gene aiv subtypes with 10f and 11r pair of primers. this tree depicted bootstrap values ranged between 825 and 994 (from 82.5% to 99.4%) with a gap in n8. when analyzing this tree consisting of a gap, accurate indication that n8 (h10n8) was aligned with n5 showing 274 (27.4%) bootstrap value and was also aligned with n8 occurring 826 (82.6%) bootstrap value. consequently, n1, n2, n3, n4, n5, n6, n7 and n9 were aligned within each subtype. furthermore, 10f and 11r pair of primer linked similarly to n1 with 464 (46.4%) bootstrap value. the resultant primers were designed in conjunction with high bootstrap value of sequences found in multiple numbers of the na gene family. that is, 10f and 11r pair of primers showed the highest bootstrap value (99.4%) followed by 8f and 10r pair of primers (99.2%), then, 5f and 10r pair of primers (95.4%). since sequence alignment was rarely perfect, the primers were degenerate to permit at sequence positions of low matches or creating gaps. once aligned, regions of high aligned sequences bounded by na primers were analyzed for the appropriateness for essential primer utilizations. for this reason, 8f and 10r pair of primers and 10f and 11r pair primers will be able to identify variable na gene families of aiv subtypes concomitantly with the detection of any mutations occurring in these primers. mutations in na primer pcr products. of interest were clinical mutations occurring in 8f and 10r pair of primers at protein positions 274 (820-830 amino acid positions) and 292 (870-880 amino acid positions) and 10f and 11r pair primers at protein positions 294 (880-890 amino acid positions). microbiol indones10 omega et al. referring to table 3, these mutations gave rise to antiviral drug resistance (oseltamivir). it appeared that 8f and 10r primers contained a tyrosine residue at position 274 (y) indicating that a possible shift from h (histidine) to y (tyrosine) is required to block neuraminidase inhibitor (oseltamivir), as well as an arginine (r)-to-lysine (k) change at position 292. it can be noted that 10f and 11r pair of primers had a serine (s) to asparagine (n) substitution at position 294 to confer oseltamivir resistance. thus, 8f and 10r pair of primers and 10f and 11r pair of primers consisted of mutations that may have a higher ability to promote virulence as a result of drug resistance pathway. acknowledgements this work was funded by biochip innovations, the ceed program and was supported by csiro. references alvarez ac, brunck meg, boyd v, lai r, virtue e, chen w, bletchly c, heine hg, barnard rt. 2008. a broad spectrum, one step reverse transcription pcr amplification of the neuraminidase gene from multiple subtypes of influenza a virus. j virol 5:77. barr ig. 2005. the evolution of h5n1 avian influenza viruses in south east asia. in: proceedings of the 3rd australian virology group meeting. cowes phillip island, australia, dec 9-12, 2005. p 39. brown ih, harris pa, mccauley jw, alexander dj. 1998. multiple genetic reassortment of avian and human influenza a viruses in european pigs, resulting in the emergence of an h1n2 virus of novel genotype. j gen virol 79:2947-55. de jong md, thanh tt, khanh th, hien vm, smith gjd, chau nv, cam bv, qui pt, ha dq, guan y, peiris jsm, 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gusdinar 1 department of pharmacy, faculty of health science, universitas muhammadiyah palangkaraya, jalan rta milono km 1,5, palangka raya, central kalimantan, indonesia; 2 department of pharmacochemistry, school of pharmacy, institut teknologi bandung, jalan ganesha no.10, bandung, indonesia. the artemisinin and its derivatives antifungal activity continue to be an interesting research object, with the potential shown to be developed as an antifungal compound. artesunate, one of the artemisinin derivatives known to have antifungal activity against various pathogenic fungi, including candida albicans. this study aims to determine the effect of artesunate on antifungal activity toward c. albicans in vitro at concentrations below 1 mg -1 ml . the method used is yeast-plate count, with a parameter of observation were the number of c. albicans colonies viable after exposure with artesunate for five days. the concentration of artesunate used was divided into -1 -2 -3 -4 -1 six groups, which were 10, 1, 10 , 10 ,10 , and 10 mg ml . compared to control, a significant decrease in colony -1 counts was only shown at the highest concentration of 10 mg ml . interestingly, at the lowest concentration of 10 4 -1 mgml , it showed an increase in a number of colonies almost twice of the blank. these results suggest that while at higher concentration of artesunate may inhibit the growth of c. albicans, a lower concentration of artesunate may stimulate their growth. key words: antifungal, artesunate, candida albicans aktivitas antifungi artemisinin dan turunannya terus menjadi objek penelitian yang menarik, dengan potensi yang ditunjukkan untuk dikembangkan sebagai senyawa antifungi. artesunat, salah satu turunan artemisinin diketahui memiliki aktivitas antifungi terhadap berbagai fungi patogen, termasuk candida albicans. penelitian ini bertujuan untuk mengetahui pengaruh artesunate terhadap aktivitas antifungi terhadap c. albicans secara in vitro -1 pada konsentrasi dibawah 1 mg ml . metode yang digunakan adalah perhitungan cawan-ragi dengan parameter pengamatan berupa jumlah koloni c. albicans yang tampak setelah terpapar dengan artesunat selama lima hari. -1 -2 -3 -4 -1 konsentrasi artesunat yang digunakan dibagi menjadi enam kelompok, yaitu 10, 1, 10 , 10 , 10 , dan 10 mg ml . dibandingkan dengan kontrol, penurunan jumlah koloni yang signifikan hanya ditunjukkan pada konsentrasi -1 -4 -1 tertinggi 10 mg ml . menariknya, pada konsentrasi terendah 10 mg ml menunjukkan peningkatan jumlah koloni hampir dua kali lipat dari blanko. hasil ini menunjukkan bahwa disaat artesunat dengan konsentrasi tinggi dapat menghambat pertumbuhan c. albicans, artesunat dengan konsentrasi yang lebih rendah justru dapat merangsang pertumbuhannya. kata kunci: antifungi, artesunat, candida albicans microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-87815093560; fax: -; email: mohammadrizkifadhilpratama@gmail.com including those caused by infection (loo et al. 2017; zuo et al. 2016). one of the artesunate properties currently being further investigated is as antifungal, where artesunate is known to have antifungal activity against various types of opportunistic pathogenic fungi such as candida albicans and cryptococcus neoformans (de cremer et al. 2015; galal et al. 2005). the exact mode of action of artesunate as antifungal itself remains elusive, but several studies have shown that artesunate and other artemisinin derivatives show antiproliferative activity on eukaryotic cells including fungi (wang et al. 2017; o'neill et al. 2010). one of the causes is that artemisinin derivatives including artesunate are known to inhibit various cyclindependent kinases (cdks) enzymes induce growth arrest of cell cycle division that are the key factors in in addition to its antimalarial activity, artemisinin and its derivatives are known to have various other activities (pan et al. 2018). artemisinin is known to have activity against various types of parasitic infections such as leishmania, schistosoma, and toxoplasma (li and zhou 2010). artemisinin and its derivatives are also known to have several other activities such as antiviral (efferth et al. 2008; pratama and gusdinar 2017), antimicrobial (appalasamy et al. 2014), and even anticancer properties (willoughby et al. 2009; li et al. 2008). artesunate, one of artemisinin derivative with potent antimalarial activity, is currently being developed for the treatment of another disease, the proliferation of fungi cells (zhang et al. 2018; tran et al. 2014; ho et al. 2014; kundu et al. 2015). however, studies conducted generally carried out in -1 doses above 1 mg ml , while testing under these doses has never been done before. thus, the antifungal activity of artesunate in this dose range is still unknown. this study aims to determine the effect of different concentration of artesunate on the antifungal activity toward c. albicans as well as proving the potency of artesunate as the antifungal compound. testing was performed with antifungal assay by counting the number of c. albicans viable colonies after administration of exposure to artesunate with multiple levels of dosage compared to controls. a decrease in the number of viable colonies showed that c. albicans cells divide at a lower rate and produce fewer colonies (kwolek-mirek and zadrag-tecza 2014). before the test, growth promotion test (gpt), and sterility test (st) are conducted to determine the ability of the medium used to grow c. albicans and its asepticity, respectively (sutton 2011). the results of this study will reveal how exactly the effect of artesunate exposure on the antifungal activity toward c. albicans. materials and methods drug and test solution. artesunate was provided by guilin pharmaceutical co. ltd, china in the form of sodium artesunate reconstitution and dissolved in sterilized sodium bicarbonate. the test compound is further diluted with physiological saline (sodium chloride 0.9%) sterile solution to obtain 6 -1 -2 -3 -4 -1 concentrations of 10, 1, 10 , 10 , 10 , and 10 mg ml , respectively. each test solution was homogenized with vortex mixer then immediately used within half an hour. fungal strains. the c. albicans strains atcc 10231 used were obtained from laboratory microbiology, school of pharmacy, institut teknologi bandung. the strain was grown in sabouraud dextrose broth (sdb) with a ph range of 5.6 + 0.2 then diluted with sdb until a number of colonies less than 100 cfu -1 ml are obtained. the inoculum suspension of c. albicans then stored in the cold room with temperature -20 °c and can be stored up to 1 week. growth promotion test and sterility test. the gpt was conducted to determine the ability of the medium used in the observation process to grow the test colony. the test medium used was sabouraud dextrose agar (sda), which has been known to grow colonies of c. albicans. as much as 1 ml of c. albicans inoculum suspension was added into sda until a mixture of 15 ml was obtained. the mixture was then homogenized with a vortex mixer for a minute, then poured into a sterile petri dish and made in triplicate. the dishes then incubated at 25°c for five days. observations were made on the fifth day by counting the number of colonies growing on each dish. all dishes should be able to grow between 30 and 300 colonies. the st was performed to ensure that the sterilization process is done successfully so that no contaminants grow in the petri dish due to non-aseptic process. as much as 1 ml of physiological saline sterile solution was added into sda until a mixture of 15 ml was obtained. the mixture was then homogenized with a vortex mixer for a minute, then poured into a sterile petri dish and made in triplicate. the dishes then incubated at 25 °c for five days. observations were made on the fifth day by counting the number of colonies growing on each dish. all dishes should not show the growth of the colony. antifungal assay. as much as 1 ml of c. albicans inoculum suspension was added to 1 ml of test solution for each concentration. the mixture was then added with sda until a final mixture of 15 ml was obtained. the mixture was then homogenized with a vortex mixer for a minute, then poured into sterile petri dishes. each series of test solution concentration was made in triplicate. the physiological saline sterile solution was used as a blank. the entire process was carried out in the laminar air flow in aseptic conditions. all petri dishes were incubated at 25 °c for five days. observations were made on the fifth day by counting the number of colonies growing on each dish then calculated the mean value of each test solution concentrations. results growth promotion and sterility. the number of colonies that grow on each petri dish was calculated entirely using a colony counter. all visible colonies were counted regardless of the size of each colony. all dishes on gpt show considerable colony growth with a colony range between 203 to 226 colonies. in contrast to gtp results, all dishes on st show no colony growth of all petri dishes (fig 1, 2). the gpt and st itself is a mandatory requirement before conducting yeast-plate count testing, where the test medium must be able to 104 pratama et al. microbiol indones volume 13, 2019 microbiol indones 105 grow the test microbes and on the other hand the working process must be guaranteed aseptic to prevent any contaminants from outside. antifungal assay. all petri dishes except on blanks covered by c. albicans colonies. the calculations of the number of colonies were performed on all parts of the petri dish. the colony growth in the antifungal assay is shown in figure 3 below. discussion surprisingly, the decrease in the number of c. albicans colonies was significantly demonstrated only by the test solutions with the highest artesunate -1 concentrations of 10 mg ml . besides the number of colonies in the concentration is still not reached half of the number of colonies on the blank, indicating that the ic of artesunate against c. albicans is in the range 50 -1 greater than 10 mg ml or equivalent to 26 µm, which is very large value. the results clearly show that the potency of artesunate as an antifungal is relatively weak, especially compared to other derivative compounds of natural products such as those derived from origanum vulgare, eucalyptus, and thymus sp like carvacrol, thymol, and α-terpineol (nazzaro et al. 2017; gucwa et al. 2018). more interesting results are at lower concentrations -1 -1 that are below 10 mg ml , artesunate actually fig 1 the result of growth promotion test. (a) the growth of c. albicans colonies in one of the petri dish; (b) number of c. albicans colonies from all petri dishes with the average of 214.3 colonies. (a) (b) fig 2 the result of sterility test. (a) no growth of colonies in one of the petri dish; (b) number of c. albicans colonies from all petri dishes. (a) (b) 106 pratama et al. microbiol indones fig 3 the result of the antifungal assay. growth of colonies on one of the petri dish from (a) negative control (blank); (b) -4 -1 -1 artesunate 10 mg ml ; (c) 10 mg ml ; (d) number of c. albicans colonies from all petri dishes from each group of -4 -1 -3 -1 -2 -1 -1 -1 -1 test solutions, with the average number for blank; 10 mg ml ; 10 mg ml ; 10 mg ml ; 10 mg ml ; 1mg ml ; and -1 10 mg ml were 67.7; 103.7; 80.7; 76.3; 65.7; 64.7; and 38.3 colonies, respectively. the dashed line indicates the average colonies number of the blank. © (d) (a) (b) triggered the growth of the number of c. albicans -4 -1 colonies. at the lowest concentration of 10 mg ml even the number of colonies that grow almost 50% more than the blank (103.7 to 67.7 colonies). the cause of the increasing number of c. albicans colonies is still unknown, but it is probably related to transcription regulator pdr1p and its target genes pdr5 and tpo1 (alenquer et al. 2006; fardeau et al. 2007). another possibility is that artesunate interacts with apoptosis protein regulators in eukaryotic cells as shown in plantaricin e and f (nurhayati et al. 2015). another interesting feature is that the increase in the number of colonies appears to occur linearly to the concentration of artesunate, where smaller artesunate concentrations lead to more c. albicans colonies growth. unfortunately, the following study is only -4 done at the lowest artesunate concentration of 10 mg -1 ml . it is interesting to observe how the change in the number of c. albicans colonies at artesunate -4 -1 concentrations smaller than 10 mg ml , whether the number of colonies is still increasing, as well as the lowest artesunate concentration which still gives an increase in the number of c. albicans. based on the author's search to date, no studies have reported this. in summary, we show for the first time an actual antifungal activity of artesunate against colonies of c. albicans, which shows that the antifungal activity of artesunate will only appear at high doses. on the contrary at low doses, artesunate actually increases the number of c. albicans colonies. the results of this study open new possibilities that the use of artesunate at low doses can actually increase the potential for candidiasis due to overgrowth of c. albicans. acknowledgement this work was supported by the internal grant from institut teknologi bandung. in addition, financial support was also provided by the institute for research and community services universitas muhammadiyah volume 13, 2019 microbiol indones 107 palangkaraya. we thank marlia singgih wibowo (institut teknologi bandung) for the help with results interpretation. references alenquer m, tenreiro s, sa-correia i. 2006. adaptive response to the antimalarial drug artesunate in yeast involves pdr1p/pdr3p-mediated transcriptional activation of the resistance determinants tpo1 and pdr5. fems yeast res. 6(8):1130-1139. doi: 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number 1, march 2021 molecular diversity of mold associated with gatotan characterization of eps7-like enterobacteria phage isolated from indonesia thermostable alkaline protease activity from aspergillus flavus ducck225 and its compatibility to local detergents identification of dermatophyte fungi causing tinea pedis and tinea unguium in malabero coastal communities, bengkulu exploration of lignocellulolytic microbes in oil palm rhizosphere on peat soils and their respiration activities steffanus pranoto hallis, anastasia tatik hartanti, and agustin wydia gunawan akhirta atikana, katsutoshi fujita, alex prima, yopi, hiroko kawasaki, ken-ichiro suzuki, and puspita lisdiyanti almalina nabila sulistyo rini, isworo rukmi, and sri pujiyanto mardhatillah sariyanti, putri maya agustria, willujeng fanny herlambang, besly sinuhaji, risky hadi wibowo, novriantika lestari, enny nugraheni, and sipriyadi happy widiastuti, siswanto, darmono taniwiryono, heru bagus pulunggono, syaiful anwar, basuki sumawinata, husni mubarok, and supiandi sabiham 1 8 15 21 27 page 1 eddy jusuf.pmd volume 3, number 2, august 2009 p 51-55 issn 1978-3477 exploration of bacillus thuringiensis ä-endotoxin derived from bacterial isolates in jabodetabek region eddy jusuf division of molecular biology, research center for biotechnology, lembaga ilmu pengetahuan indonesia, jalan raya jakarta-bogor km.46 cibinong, bogor 16911, indonesia phone: +62-21-8754587, fax: +62-21-8754588, email: eddy-jusuf@indo.net.id bacillus thuringiensis is a well-known species of bacteria producing parasporal crystalline proteins that are toxic to many insect pests, it also has strong cytocidal activity against human cancer cells. in this work we endeavoured to explore the city of jakarta and its neighbouring regencies to obtain new strains to profile its potency. the soil samples were boiled and plated on t-3 agar; the colonies which appeared were then examined under the light microscope to check for crystalline proteins. the proteins were treated with 1% sds-0.01% β-mercaptoethanol and visualized by sds-page to determine their molecular size. from 52 soil samples, 1248 putative colonies were obtained. after microscopic examination, 57 isolates showed the existence of crystalliferous protein, of those 30 indicated many sizes of ä-endotoxin protein from approximatively 150 kda to the smallest 35 kda. most of proteins examined probably showed insecticidal activity and four of those were predicted to possess cytocidal activity against human cancer cells. the geographic condition did not appear to influence the distribution of different types of äendotoxin. key words: bacillus thuringiensis, local strains, ä-endotoxin bacillus thuringiensis is a gram positive spore forming bacterium, constituting a large family of strains found in different and high specialized habitats (bernhard et al. 1997; chaufaux et al. 1997). this bacterium produces parasporal crystalline protein inclusions (ä-endotoxins), also called cry proteins, that have a natural insecticidal effect on insect pests of the lepidoptera, coleoptera, diptera, hymenoptera, homoptera, orthoptera and mallophaga orders, and against mites, platyhelminthes and nematodes. they also have germicidal activity on protozoa of medical importance such as plasmodium berghei (schnepf et al. 1998). nowadays, more than 133 types of crystal protein comprising 24 primary ranks are systematically found. molecular sizes of these proteins vary from 25 kda, found in b. thuringiensis subsp. israelensis, to 135 kda polypeptides in many others. interestingly, recent investigators have provided evidence that parasporal inclusion proteins of certain noninsecticidal b. thuringiensis isolates occasionally exhibit in vitro cytocidal activities on human cancer cells. proteins with this unique function are now categorized into a group designated parasporin (mizuki et al. 1999; xu et al. 2004). an isolate of b. thuringiensis serovar shandongiensis exhibits strong cytocidal activity against human leukaemic t cells (lee et al. 2000). lee et al. (2001) also reported that a 28 kda protein produced from this strain was a cytotoxic substance against human leukaemic t cells, and suggested that another protein cytotoxic against hela cells might also exist. until now, there are four groups of parasporin protein, ps-1 known as cry31 protein having a molecular weight of 81 kda , ps-2 known as cry 46 (37 kda) , ps-3 known as cry 41 (88 kda) and ps-4, known as cry 45 having 34 kda protein (ohba et al. 2009). until march 2002, over 359 different genes of cry protein have been identified, more than 97 different genes of cry protein have been cloned from b. thuringiensis to many other bacteria and plant species, and more than 16 thousand of strains are kept in collection all over the world (crickmore et al. 2002). among the strains listed, no one has been obtained from any region of indonesia, nor carrying a taxon name of typical of an indonesian region such as b. thuringiensis subsp. malaysiensis which was obtained from malaysia. in reality, indonesia is known as the second country having the highest diversity of biological resources after brazil. the aim of this study was to find native strains of b. thuringiensis from local natural resources. the first exploration began in jabodetabek (abbreviation of jakarta, bogor, tanggerang, depok and bekasi), a large area covering jakarta, the capital of the country, with their municipalities and regencies, and this area covers about 4563 km2. this study was limited to finding the types of ä-endotoxins distributed in this region and to predict the potencies of those proteins based on their molecular weight, in agriculture or medical fields for the next works. materials and methods sampling. the sampling was conducted in jakarta municipalities, depok, bekasi and its regency, tangerang, bogor and its regency. samples were randomly collected from environmentally diverse sources (including beach sand, forest soil, aquatic and intertidal sediments, and soils from urban, rural and agricultural areas), placed in a plastic bag and held at room temperature until processing. from central jakarta we have 7 soil samples (no 16 to 22), one sample (no 23) from southern jakarta and two samples (no 41 and 42) from eastern jakarta. from the municipality of depok (sukmajaya district), only two soil samples (no 43 and 44) were collected; from bekasi regency 8 soil samples of muara tawar coast area (no 8 to 15) and 6 samples (no 35 to 40) of bojong village were obtained. from bogor, 7 samples were obtained from cibinong district (no 1 to 7), 6 samples from bogor town (no 24 to 29) and 5 samples (no 30 to 34) were obtained from mountainous outskirts of bogor. only one sample was obtained from tanah tinggi district of tangerang town centre area (no 45). as a supplement, six samples (no 46 to 51) from the sukabumi regency (adjoining the southern bogor regency), out of jabodetabek area were added. microbiol indones52 jusuf bacterial isolation. for specifically isolating b. thuringiensis strains, the method was as described by travera et al. (1987), where 1.0 g samples were introduced into a test-tube containing 10 ml phosphate buffer 0.05 m at ph 6.8 and shaken vigorously for 15 min. the tubes were then heated at 70 °c for 30 min in a water-bath and shaken vigorously for a second time to disperse the spores. aliquots of 5, 10, 25 and 50 µl of suspension were poured on petri plates containing t-3 agar (for 1.0 l of final solution, containing 3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 m na 3 po 4 , 0.005 m mncl 2 in 12 g agar). the cultures were incubated at 28 °c for about 48 h until colonies appeared. the number of total colonies, and the colonies which resembled exactly to standard strains bts 002199a were counted. from each sample, 24 b. thuringiensis-like colonies were transferred to the fresh t-3 agar medium on the petri glass and reincubated at 28 °c for sporulation over 72 h. microscopic observation using a phase contrast microscope zeiss 003-002199 axiophate was performed to verify the presence of crystal proteins (cry protein). the crystalliferous colonies observed were harvested as the new isolates and conserved in luria-bertani slant agar for ä-endotoxin profiling. profiling of ä-endotoxins. the crystalliferous isolates being tested for the ä-endotoxin profiling were previously grown on luria-bertani agar petri for 2 x overnight before being inoculated onto t-3 agar for sporulation. the method used for this protein-profiling was as described by bel et al. (1997), where sporulated colonies growing (48 to 72 h) in t-3 agar plate were harvested and poured into chilled 1 ml 500 mm nacl in 1.5 ml microtube. the suspensions were centrifuged 13 800 x g for 10 min, and the sediment obtained was again suspended in 150 µl 1% sds-0.01% β-mercaptoethanol and heated at 110 °c for 10 min to solubilize the crystals. the suspensions were again centrifuged at 13 800 xg for 10 min. the supernatant obtained was mixed with an equal volume of sample buffer (0.15 m tris.cl ph 8.8, 3.75 mm edta, 0.75 m sucrose, 0.075% w/v bromophenolblue, 2.5 % w/v sds and 7.4 mm dithiothreitol). the mixture was heated 100 °c before visualized using electrophoresis. sds-page. to obtain the profile of ä-endotoxin from each isolate, vertical electrophoresis using polyacrylamide mixed with sodium dodecyl sulfate (laemmli 1970) was performed. electrophoretic protean ii equipment (amersham) was used with a separating gel of 10% (w/v) acrylamide. electrophoresis was performed at 75 volt using tris-glycine buffer (50 mm tris, 384 mm glycine and 0.1% (w/v) sodium dodecyl sulfate). to determine molecular weights, a combithek (boehringer mannheim cat. no. 1317-474) standard kit having the range 14.307 to 340 kd was used. gels were stained with 0.1% (w/v) comassie blue, 50% (v/v) methanol and 10% (v/v) acetic acid; and then destained by boiling the gels in water. results microscopic observation. from 51 soil samples, a total 1248 colonies were selected for microscopic observation. some samples were devoid of bacteria containing crystals, e.g. in the soil sample of southern jakarta (no 23) and of eastern jakarta (no 41), from bogor region (no 1, 2, 23, 24, 25, 26, 28, 30, 34), from depok (no 43 and 44), from tangerang (no 45), from bekasi (no 13, 14 and no 39). from sukabumi only one sample (no 50) yielded two of crystalliferous isolates, for the others (no 46, 47, 48, 49 and 51) no crystalliferous isolate was obtained. in total, from hundred colonies appeared which resembled exactly to standard strains bts 002199a in the plate of each sample, only 24 to 32 colonies selected to be observed under microsscope for the presence of crystal. the total number of crystalliferous isolates obtained after microscopic observation was 57, consisting of 20 isolates from central jakarta and 3 from eastern jakarta, 20 isolates from the whole bogor region were divided into 9 from the cibinong district, 3 from bogor town and 8 from bogor the upland plains. the 15 isolates from bekasi comprise of 8 isolates from the muara tawar coast area and 7 isolates from bojong village. there were 2 isolates from sukabumi. the number of isolates showing the presence the ä-endotoxin-protein-crystal obtained in each sample varied from one to five. the proteins obtained from the culture of those isolates were examined by sds-page for identifying the type of cry protein present. protein profiling. protein profiles obtained from cibinong (bogor) isolates are shown in fig 1a. from 9 isolates previously predicted as b. thuringiensis, there were two isolates which produced ä-endotoxin (isolate 3l from soil of housing grassland with protein weight molecular of about 130 kda, as well as isolate 6c obtained from the mud). the ä-endotoxin is thought to be cry1 or cry4. from soil under rambutan (nephelium lapaceum) trees the isolate 5k was detected having three crystal proteins of about 100 kda, 85 kda, and 35 kda each thought to be protein cry25aa1, cry2 or cry3 and cry30aa1. the isolate 3a, 3n, 4q (from soil under guava tree), 6l, 7e and 7i (from an insect cadaver near our laboratory) would be similar strains, which were previously identified as b. cereus, from which no ä-endotoxin was present. the second gel (fig 1b) shows the protein profile of all isolates obtained from muara tawar coast area of the bekasi region. isolate 8c from the soil taken under the waru tree (hibiscus tiliaceus) had one cry protein band of 130 kda, representating cry1. very dense spots of äendotoxin is found for isolate 9g obtained from the soil of post harvest rice field and found to contain 2 ä-endotoxins of about 135 kda and 120 kda. isolate 8n, 11l (from soil under mangroves in a fishering village), 12g (from mud of a milkfish (chanos chanos) pond , 15i and 15l (from soil of uncultivated land at a vapour and gas electricity generator centre) are similar, being devoid of ä-endotoxin. the ä-endotoxins present in the bacterial isolates obtained from central jakarta are shown in fig 1c and 1d. some isolates contained cry protein, among them isolate 16g (120 kda) and 16j (having two bands of 120 and 135 kda). both were obtained from garden soil under the national monument. isolates 19a and 19s (obtained from soil around the liberation monument of lapangan banteng) seems to be similar containing 3 bands of 130 kda, 85 kda and 45 kda, 20q (from soil around the istiqlal mosque) containing 85 kda; 21f (soil from the roadside of liberation monument of lapangan banteng) which shows a thick band at about 150 kda. lanes 21g, 21i, 21l and 22p seems to be similar in profile, and are considered as the same strains as b. cereus. microbiol indones 53volume 3, 2009 from bogor town center and its mountainous landscape surrounding (fig 1e and 1f), two strains, each 31a and 31t (both obtained from the soil of the camping ground of cilember), 32n (soil from the natural forest reserve of cilember) show the same profile as 21f, having a thick band of about 150 kda. isolates 27b (from soil taken from the yard of faculty of technique, universitas pakuan, bogor), 29g and 29h (soil taken from bogor botanical garden), 31e (from the soil of camping ground of cilember), 32b (soil from natural forest reserve of cilember), 33b and 33t (both the soil from pinus spp. plantation in salak mountain) and the two standard strains b. thuringiensis var. berliner hd-2, subsp. galleriae hd-29 show the same profile, having cry protein of about 70 kda, akin to cry2aa or cry2ab. the protein profile of other isolates obtained from the farm village of bojong (bekasi) in fig 1f and 1g, show the same profile as cry protein (135 kda) which is the same as the standard strain of b. thuringiensis var. aizawai hd-137 as well as var. israelensis hd-567. the isolates showing this protein type are 35r (obtained in soil taken from a house yard of the village), 37t3 (from the soil under morinda citrifolia tree attacked by insect larvae), 40a, 40g and 40i, all isolated from soil taken from a chicken barn. all isolates obtained from soil of unutilized land in a housing area of 340.0 170.0 116.4 85.2 55.6 38.2 kda 3 a 3 l 3 n 4 q 5 k 6c 6 l 7 e 7 ima 340.0 170.0 116.4 85.2 55.6 38.2 kda m 8c 8n 9g 10h 11l 12g 15i 15lb kda m 16b 16g 16j 16s 16t 17p 17q 18l 18m 18qc 340.0 170.0 116.4 85.2 55.6 38.2 340.0 170.0 116.4 85.2 55.6 38.2 kda m 19a 19s 20q 21f 21g 21i 21l 21q 22p 22qd 340.0 170.0 116.4 85.2 55.6 38.2 kda m 27b 29g 29h 31a hd2 31e 31t 31xe 1 3 5 1 3 3 1 3 0 340.0 170.0 116.4 85.2 55.6 38.2 kda mf 32b 32n 33b 33t 35r 36nh d 2 9 3 7 t 3 3 8 c h d 1 3 7 fig 1 sds-10%page of b. thuringiensis ä-endotoxin proteins representatives of the main profiles listed in table1: a, isolates from cibinong district; b, isolates from muara tawar coast area; c and d, from central jakarta; e, from central bogor and its mountainous landscape (no.31a,31e,31t and 31x); f, from bogor with surrounding mountains and bojong village of bekasi; g, from bojong village of bekasi, cipayung, eastern jakarta and sukabumi. g 40a 40g 40i 42g 42k 42t 50k 50l 1 3 4 7 8 7 2 h d 5 6 7 h d 1 3 7 microbiol indones54 jusuf cipayung (eastern jakarta), isolates 42g, 42k and 42t, and one of the two isolates from situgunung-sukabumi, (50k) show the same profile as the isolates from bojong.the total yields of ä-endotoxins obtained from this work is presented in table 1. discussion the first results in previous page reported that 17 samples were devoid of bacteria containing crystals observed under microscopic observation. it could not be said the absence of the bacteria producing parasporal crystalline proteins in the area where the samples were taken. from hundred colonies appeared which resembled exactly to standard strains in the plate of each sample, only 24 to 32 colonies selected to be observed under microsscope for the presence of crystal. it was possible that, by such random selection, the bacteria targeted were slipped away. from 57 isolates tested by protein-profiling, 40 of them showed the existence of ä-endotoxin. the uncommonly large protein crystal size (+150 kda) was found in four isolates from central jakarta and central bogor, 21f, 31a, 31t and 32n. this type protein was previously declared by schnepf et al. (1992) as cryv protein having specifically toxicity to some species of nematodes present in the strain ps17a of b. thuringiensis. this type of protein is not broadly studied because it is known that the ä-endotoxin is generally toxic to insects. it is interesting that those strains were obtained in lowland plains and upland plains. although the protein profile is similar, a serology test is necessary to distinguish those subspecies and other molecular tests to establish the precise strain. the most common large protein crystal size is the 135 kda, belonging to cry 4ba1 protein having specific toxicity to some species of mosquito. this protein type was found distributed almost in all areas studied in this work, where we get 11 isolates from the coastal area of bekasi, lowland plains of jakarta and bekasi up to upland mountainous areas of bogor and sukabumi. on the other hand, the distribution is correlated with the existence of this insect as the host for bacterial proliferation. the isolate 9c and 16j producing this protein, they produce also the 120 kda protein that is probably cry4aa being effective to kill mosquito larvae. protein of the molecular size from 130-133 kda generally belong to cry i, from cry 1aa1 to cry 1ib1 as denoted in the composed data of known cry protein gathered having specific toxicity to lepidoptera larvae. in this study we found that the isolates carrying this type of protein originated from the lowland plains area of jakarta, bogor and bekasi, and not found in mountainous areas. inversely the molecular size of protein 120-125 kda belong to cry 4aa and cry 4ab groups which are toxic towards diptera larvae, mainly mosquitoes. only one isolate from cibinong bogor (5k) showed a protein of 100 kda molecular size. such a protein is postulated as being cry 25aa1, which was previously discovered in a strain b. thuringiensis var. medellin 163-13 isolated from medellin, colombia and which are toxic to mosquito larvae. this isolate 5k has two other ä -endotoxins of 85 and 35 kda, where the 85 kda protein are also carried by isolate 20q. this is thought to be cry2ac having low toxicity against lepidoptera and diptera or possibly cry 41 (ps-3) protein of 88 kda molecular weight having low cytocidal activity againts cancer cells (ohba et al. 2009). meanwhile the ä-endotoxin of 35 kda is thought to be cry 30aa1 having toxicity against mosquito or possibly to be cry45 (ps-4) possesing 34 kda protein or cry46 (ps-2) possesing 37 kda protein, the last two were reported having high cytocidal activity againts cancer cells. isolates 19a and 19s seems to be similar containing 3 bands of 130 kda, 85 kda and 45 kda, where 130 kda protein could be cryi, 85 kda protein could be cry2ac or ps-3, and 45 kda protein could be protein mtx3 being obtained in strain b. thuringiensis subsp. galleriae. the results showed that geographic condition did not influence the different types of ä-endotoxins where all those proteins could be found somewhere. the molecular weight of the proteins found in those strains was still approximative and must be verified by other technique more sophisticated than sds-page used here. anyhow, all those strains producing ä-endotoxins will be used in the next study as table 1 ä-endotoxins distribution among b. thuringiensis isolates obtained based on the sds-page yields,mass molecular category and its type cry protein predicted category > 150 kda 135 kda 130 kda 120 kda 100 kda 85 kda 70 kda 45 kda 35 kda type of ä-endotoxins predicted cry v cry 4ba cry i or cry 4 cry 4aa1 cry 25aa1 cry 2 or cry3 cry 2aa or cry 2ab m t x 3 cry30aa1 isolate numbers carrying those protein type 21f 31a, 31t and 32n 9c 16j 35r, 37t3, 40a, 40g, 40i 42g, 42k and 42t 5 0 k 3l and 6c 8c 19a, 19s 9c 16g, 16j 5 k 5 k 19a, 19s and 20q 27b, 29g, 31e, 32b and 33b 19a and 19s 5 k origin of the isolates central jakarta central bogor bekasi coastal area central jakarta bekasi, bojong village eastern jakarta sukabumi area cibinong, bogor bekasi coastal area central jakarta bekasi coastal area central jakarta cibinong, bogor cibinong, bogor central jakarta central bogor with surrounding mountains central jakarta cibinong, bogor microbiol indones 55volume 3, 2009 of a bacillus thuringiensis serovar shandongiensis isolate exhibit a preferential cytotoxicity against human leukemic t cells. biochem biophys res comm 272:218-23. lee dw, katayama h, akao t, maeda m, tanaka r, yamashita s, saitoh h, mizuki e. 2001. a 28 kda protein of the bacillus thuringiensis serovar shandongiensis isolate 89-t-34-22 induces a human leukemic cell-specific cytotoxicity. biochim biophys acta 1547:57-63. mizuki e, ohba m, akao t, yamashita s, saitoh h, park ys. 1999. unique activity associated with non-insecticidal bacillus thuringiensis parasporal inclusions: in vitro cell-killing action on human cancer cells. j appl microbiol 86:979-84. ohba m, mizuki e, uemori a. 2009. parasporin, a new anticancer protein group from bacillus thuringiensis. anticancer res 29:4273 3 . schnepf he, schwab ge, payne jm, enarva k, foncerrada l. 1992. patent wo92/19739. schnepf he, crickmore n, rie jv, lereclus d, baum j, feitelson j, zeigler dr, dean dh. 1998. bacillus thuringiensis and its pesticidal crystal proteins. microbiol mol biol rev 62:775-806. travera ms, martin paw, reicheldelfer cf. 1987. selective process for efficient isolation of soil bacillus sp. appl environ microbiol 53:1263-6. xu z, yao b, sun m, yu z. 2004. protection of mice infected with plasmodium berghei by bacillus thuringiensis crystal proteins. parasitol res 92:53-7. biopesticide or as theuraphetic protein againts human cancer cells. the precise size of protein molecular weight of each strain is required to determine which study could be performed from which the data of the potencies of available proteins. references bel y, ganero f, alberola ta, martinez-sebastian mj, ferré j. 1997. distribution, frequency and diversity of bacillus thuringiensis in olive tree environments. spain sys appl microbiol 20:652-8. bernhard k. 1986. studies on the delta-endotoxin of bacillus thuringiensis var. tenebrionis. fems microbiol lett 33:261-5. chaufaux j, marchal m, gilois n, jehano i, buison c. 1997. research on natural strains of bacillus thuringiensis in different biotopes throughout the world. can j microbiol 43:337-43. crickmore n et al. 2002. bacillus turingiensis toxin nomenclature. the bt delta-endotoxin nomenclature committee. 6 p.[on line] http://www.biols.susx.ac.uk/home/neil_crickmore/bt/committee. html. issued mar 12, 2002. [aug 27, 2002]. laemmli uk. 1970. cleavage of structural protein during the assembly of the head of bacteriophage t4. nature 227:680-5. lee dw, akao t, yamashita s, katayama h, maeda m., saitoh h., mizuki e, ohba m. 2000. non-insecticidal parasporal proteins 05. nurtjahyani.cdr vol.14, no.2, june 2020, p 83-88 doi: 10.5454/mi.14.2.5 short communication utilization of leaves in mine reclamation land as organic fertilizer with bioactivatory of efecctive microorganism 4 (em4) and molasses supiana dian nurtjahyani 1* 2 2 3 , dwi oktafitria , sri wulan , nova maulidina , 1 4 1 imas cintamulya , eko purnomo , and ali mustofa 1 progam studi pendidikan biologi fkip; 2 progam studi biologi fmipa universitas pgri ronggolawe, east java, indonesia; 3 departemen biologi its surabaya, east java, indonesia; 4 pt. semen indonesia tbk gresik, semen indonesia group, east java, indonesia. organic fertilizer as a potentialn alternative to reduce the scarcity of chemical fertilizers, furthermore, it can improve soil fertility. pt semen gresik semen indonesia persero (tbk.) has a lot of plants in the treatment period in ex-mining land that needs fertilizer to help plant growth. in addition, in post-mining land, there is a lot of organic waste in the form of dry leaves under the stands of reclamation trees. the organic waste has the potential to be processed and used as organic fertilizer. the aims of this study was to determine the doses of molasses and em4 as bioactivators in the manufacture of organic fertilizer made from leaf litter in mine reclamation land. this study is a laboratory experimental method. the results showed the composition of solid material in the form of leaf litter, bran, cow dung and husk charcoal in a ratio of 2: 1: 1: 1. the composition of molasses and em4 with a dose of 100 ml per 100kg of solid material, respectively, dissolved in enough water (fertilizer can be clenched and expanded slowly and not broken). as conclusion composition dose molasses and em4 as bioactivators in the manufacture of organic fertilizer made from leaf litter mine reclamation land 100 ml per 100 kg (ratio 1:1) with the characteristics of the microbial composition of lactobacillus sp., streptomyces sp., yeast, rhodopseudomonas sp., actinomycetes sp. with brown em4 color indicator, sour smell, ph 3.5, and the most nutrient content is organic c. key words: em4, leaf litter, mine reclamation land, molasses, organic fertilizer pupuk organik sebagai alternatif untuk mengurangi kelangkaan pupuk kimia, selain itu dapat memperbaiki kesuburan tanah. pt semen gresik semen indonesia persero (tbk.) memiliki banyak tanaman dalam masa perawatan pada lahan bekas tambang yang membutuhkan pupuk untuk membantu pertumbuhan tanaman. disamping itu, pada lahan pasca tambang banyak sampah organik berupa dedaunan kering dibawah tegakan pohon reklamasi. sampah organik tersebut berpotensi untuk diolah dan dimanfaatkan sebagai pupuk organik. tujuan penelitian ini untuk mengetahui dosis em4 dan molase sebagai bioaktivator dalam pembuatan pupuk organik berbahan dasar serasah daun di lahan reklamasi tambang. metode penelitian yang di gunakan adalah metode eksperimen laboratoris. hasil penelitian menunjukkan komposisi bahan padat berupa serasah daun, dedak, kotoran sapi dan arang sekam dengan perbandingan 2:1:1:1; bahan berupa molase dan em4 dengan takaran masing-masing 100ml per 100kg bahan padat yang dilarutkan dalam air secukupnya (pupuk dapat dikepal dan mengembang dengan pelan-pelan serta tidak pecah). sebagai kesimpulan komposisi dosis em4 dan molase sebagai bioaktivator dalam pembuatan pupuk organik dari serasah daun di lahan reklamasi tambang masingmasing 100 ml per 100 kg (1:1) dengan karakteristik komposisi mikrobanya lactobacillus sp., streptomyces sp., yeast, rhodopseudomonas sp., actinomycetes sp. dengan indikator warna em4 coklat, baunya asam, ph 3,5, dan kandungan nutrient yang terbanyak c organik. kata kunci: em4, lahan reklamasi tambang, molase, pupuk organik, sersah daun microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-81335278182; fax:-; email: diananin39@gmail.com soil can become more loose so it is more easily penetrated by plant roots. in soils with high clay content, organic matter will be able to facilitate soil management, by improving groundwater passing and increasing water retention capacity from the soil. this will cause the soil to provide more water, especially in the dry season. organic matter can also improve soil chemical properties such as: increasing the capacity of cation exchange (exchange of nutrients or nutrients for the use of organic fertilizers can reduce dependence on chemical fertilizers whose availability is increasingly rare and expensive. it can also reduce the occurrence of environmental pollution. organic matter is known to improve the physical properties of the soil by improving soil structure, where the lumpy plants) more easily and smoothly, providing plant nutrients in the form of p, k, ca, mg, s, and elements other micro elements that are needed by plants. organic matter can improve the soil's biological condition so that the soil remains alive, durable and resistant to shocks that cause soil damage. besides organic matter can increase the efficiency of the use of chemical fertilizers, so as to reduce the cost of purchasing chemical fertilizers. thus, organic fertilizer can increase agricultural production both in quality and quantity, and improve the quality of land in a sustainable manner. the use of organic fertilizer in the long term can increase land productivity and can prevent land degradation (simanungkalit 2006). organic fertilizers have some advantages even though they also have limitations. some of the advantages of organic fertilizers are (a) organic fertilizers that contain complete nutrients, both macro nutrients and micro nutrients, (b) organic fertilizers contain organic acids, including humic acids, fulfill acids, hormones and enzymes that are not present in artificial fertilizers which are very useful both for plants and the environment and microorganisms, (c) organic fertilizers containing macro and soil microorganisms which have a very good influence on the improvement of soil physical properties and especially soil biological properties, (d) organic fertilizers can improve and maintain soil structure, (e) organic fertilizer can be a buffer of soil ph, (f) organic fertilizer can be a buffer of inorganic nutrients provided, (g) organic fertilizer can help maintain soil moisture, (h) organic fertilizer is safe to use in large quantities and even though, (i) organic fertilizer does not damage the environment. in addition to these advantages, organic fertilizer can also provide economic value to a variety of household and agricultural wastes (wahyuni 2016). reclamation and revegetation of former mines is an obligation that must be carried out minister of regulation energy and mineral resources republic indonesia 2014. decree no. 7 of 2014 the success of a reclamation is very much determined by many things, including aspects of land management, fertility of the planting media, technical planting and care of plants. pt. semen gresik semen indonesia persero (tbk.) has a lot of plants in the treatment period in ex-mining land that needs fertilizer to help plant growth. in addition, in post-mining land, there is a lot of organic waste in the form of dry leaves under the stands of reclamation trees. the organic waste has the potential to be processed and used as organic fertilizer. the purpose of this study is to utilize leaf litter in the reclaimed mine land as organic fertilizer with em 4 and molasses bioactivators. this research is a laboratory experimental study, which was carried out in the biology laboratory and in the semen indonesia tuban plant. the equipment needed in this research is pock, shovel, ziplock plastic, ph meter, thermometer, gps, camera, moisture meter, tarpaulin, and gloves. while the materials needed in this study are water used to dissolve molasses and em4, soil or media used in the process of reclamation of ex-limestone quarrying land, leaf litter taken on reclaimed land of former limestone quarrying, charcoal, fine charcoal, dirt cattle or animal manure, molasses or molasses and em4. leaf litter sample testing. leaf litter samples were taken from the reclaimed land of the former limestone quarry in 2010, 2014 and 2016, where each sample was taken twice (duplo repetition). furthermore, the quality of leaf litter was tested based on parameters namely p total (p2o5) by method olsen and bray, k total (k2o) by method spektrofotometri, organic c by method walkey and black, and nitrogen (n) by method destilasi in the laboratory. organic fertilizers. making organic leaf litter fertilizer by the bokashi method (york, 2014) begins with taking leaf litter in the reclaimed land of the former limestone quarry. the leaf litter is then put into the leaf chopper machine. the leaf chopper used is a regular chopper (or compost chopper) that has a capacity of chopping 100kg / hour with a diesel engine powered by diesel. the resulting leaf counts are weighed according to the fertilizer manufacturing plan which is 40% of 100% of the total material so that the leaf counts needed are as much as 40 kg of the total 100 kg of fertilizer. the mixture of solids is watered evenly with a bioactivator solution. bioactivators em4 and molase play a role in accelerating the fermentation process and increasing the nutrient content contained in leaf litter because with biocivators speeding up the fermentation process. the flat mixture is then put into a tarp and covered with tarps for ± 21 days. during the process the temperature is maintained less than 50ºc. organic fertilizer testing. testing the quality of organic fertilizers that have been produced is done by testing the substance content of several nutrient parameters in compost, namely nitrogen (n) by method destilasi, phosphorus (p2o5) by method olsen and bray, potassium (k2o) by method spektrofotometri and carbon (c)by method walkey and black in the laboratory. 84 et al.nurtjahyani microbiol indones volume 14, 2020 microbiol indones 85 quality of leaf litter. leaf decomposition can release a number of nutrients and materials that can support the productivity of trees and food webs in mangrove ecosystems (ashton 1999). nutritional et al. value can be defined as a c: n ratio (lower ratio means higher nitrogen concentration and higher nutritional quality) (ashton et al. 1999). the results of the analysis of the highest c: n leaf litter ratio value were obtained from the 2016 reclamation land of 19.02 and the lowest value of the 2014 reclamation land with a value of 13.11 (table 1). this shows that the reclamation land in 2014 has the highest nutrient value of leaf litter than other lands, although the percentage of n content is lower than the leaf litter on the 2016 reclamation land. this is supported by planting media used for planting teak tree seedlings in the 2014 reclamation land which comes from topsoil. planting media in the form of topsoil generally have a high nutrient content so that it allows a lot of nutrients to be absorbed by the teak tree and stored on its leaves. the results of the c: n ratio test on these leaves can be used as the basis that leaf litter found in the three reclamation lands can be utilized as a natural fertilizer for the growth of teak trees planted in the area. the low c: n ratio value of leaf litter in reclamation land in 2014 means that it has high nutritional quality and will result in a faster decomposition process. decomposer microorganisms such as bacillus and flavobacterium (alami et al. 2019, kurniawan et al. 2011) will more quickly decompose litter with high nutrient content (ashton et al. 1999, hanif et al. 2015). technical utilization of leaf litter as organic fertilizer with em4 and molasses bioactivators. the results of making organic fertilizer using the basic ingredients of leaf litter are obtained using the bokashi method (york 2014). the bokashi method is a method that utilizes em4 (effective microorganisms 4) technology. the bokashi method was chosen in this leaf litter composting technique because organic fertilizer fermented using em4 solution can be used to fertilize the soil and suppress the growth of pathogens in the soil, thereby increasing plant growth and production. in addition, the use of em4 in the fermentation process will accelerate the decay that occurs compared to the decay process without using additional microorganisms (khan et al. 2009, winduasari 2012). the results of leaf chopped that have been found are mixed with stems and twigs which cannot be chopped perfectly by a chopper machine. pieces of these stems will make the fermentation process run long and make the texture of the fertilizer coarser. therefore it is advisable to sort the leaf litter from the twigs before chopping, so that the results of leaf chopping are pure leaf litter. another solid material used in making organic fertilizer is fine bran or bran. bran or rice bran is waste from the rice milling process. bran is a good food source for decomposing bacteria in this case is em4. with high carbohydrate content and vegetable protein, bran is a complete food source for decomposing bacteria. adding bran to the production of organic fertilizer indirectly has a positive effect on the quality of the compost produced. thus it can be said that the function and benefits of bran in composting are as a food source for bacteria and as an additional ingredient for producing quality compost. bran needed is 20 kg (20% of the total 100 kg of fertilizer), temperature reaches more than 50ºc, it is feared that it will kill microorganisms in it so that microorganisms cannot live and carry out their functions. a good organic fertilizer is fertilizer that prioritizes c-organic content so that it can produce a low c / n ratio value. to achieve the c / n ratio and the content of nitrogen (n), phosphorus (p), and potassium (k) which are in accordance with the standards can be done by microbial fermentation called effective microorganism (em4) (kurniawan et al. 2011). other mixtures in making organic fertilizer are animal dung, especially cattle or manure (cow dung that has dried or has been processed). cow dung has great benefits in making organic fertilizer. the benefits of cow dung include nitrogen (n) content; phosphorus (p); potassium (k); high economic value because it can replace the use of chemical fertilizers so that production costs can be reduced; cow dung is easy to use and is applied as compost for plants; cow dung is able to provide balanced nutrients for the soil; make the soil structure more loose because cow dung will increase the number of microbes in the soil; cow dung can also improve soil ph conditions that are damaged; and by utilizing cow dung as fertilizer turned out to be able to increase crop production to 30%. with the many benefits of cow dung for soil and plants, it is highly recommended adding cow dung in making organic fertilizer. the amount of cow dung prepared is 20 kg (20% of the total 100 kg of fertilizer). cow dung used should be old cow dung and not cow dung which is still warm (new), this is because the temperature of new cow dung is still high so that it will affect other microorganisms that are added in making organic fertilizer. the last ingredient used in making organic fertilizer is husk charcoal. husk charcoal is generally used as a mixed medium in agriculture. the benefit of using the husk charcoal in making organic fertilizer is that the soil becomes loose so that it improves soil fertility and improves soil structure. in addition, the benefits of using husk charcoal are increasing soil ph thereby increasing the availability of phosphorus (p) and carbon (c). adding husk charcoal will increase the aeration system in the plant root zone. husk charcoal can also increase groundwater reserves and increase levels of potassium (k) and magnesium (mg) exchange. it is generally known that charcoal or charcoal husk has a high silicate (si) element but is low in calcium (ca) content. the silicate element itself has important benefits other than as a functional nutrient that is increasing plant immunity against pests or pathogens found in the soil, drought tolerant and against heavy metals that contaminate the soil. therefore it is recommended to use husk charcoal as an additional material in making organic fertilizer. the amount of husk charcoal prepared is 20 kg (20% of the total 100 kg of fertilizer). leaf litter, bran, cow dung and husk charcoal that have been measured according to the dose are mixed into one solid material mixture on a tarpaulin that has been opened on a flat surface. this mixing method is carried out in accordance with the bokashi mixing method by mixing all ingredients into one. mixing is done by using a shovel.the nitrogen and phosphor contents of this liquid fertilizer (92,000 ppm and 143,000 ppm) were significantly higher (p<0.05) than the em4 (0.07 and 3.22 ppm) (sastro.et al 2013). after mixing the solid material, a liquid material consisting of em4 and molasses or molasses is prepared. the em4 used is em4 which is sold freely in the market. em4 is a chocolate solution with a ph of 3.5 4.0. em4 (effective microorganisms 4) contains various fermentation microorganisms which are very numerous, approximately 80 genera and these microorganisms can work effectively in the fermentation of organic matter. the four main microorganisms in em 4 solution are photosynthetic bacteria, lactobacillus sp., saccharomyces sp., and a c t i n o m y c e t e s s p . p h o t o s y n t h e t i c b a c t e r i a (rhodopseudomonas sp.) play a role in forming substances that have benefits for plant secretions, organic matter and harmful gases by using sunlight and earth as energy sources. these substances include amino acids, nucleic acids, bioactive substances and sugars. these substances function to accelerate the growth and development of plants. besides this microorganisms can increase the growth of other microorganisms that are not pathogenic (dibia et al. 2012, fisher et al. 2000, isroi et al. 2009). lactic acid microorganisms (lactobasillus sp.) can produce lactic acid from sugar, can suppress the growth of harmful microorganisms, increase the rate of overhaul of organic materials and can destroy organic materials such as cellulose and lignin, and ferment them without causing adverse effects caused by organic materials that are not biodegradable. saccharomyces sp. or commonly called yeast / yeast functions to form anti-bacterial substances and have benefits for plant growth from amino acids and sugars released by photosynthetic bacteria. it also functions in increasing the number of active cells and root development. actinomycetes sp., aspergillus and penicilium which are included in fermented mushrooms, function in decomposing organic matter quickly to produce alcohol, esters and anti-microbial substances, eliminate odors and can prevent from harmful insects and caterpillars (aprianis et al. 2011, wahyuni 2016). em4 is not harmful to the environment because em4 culture does not contain microorganisms that are genetically modified. before use, em4 needs to be activated first because the microorganisms that are in the em4 solution are dormant (sleeping). activation of microorganisms in em4 can be done by adding water and food which can be molasses or molasses. the amount of em4 needed in making organic fertilizer with a total amount of fertilizer as much as 100 kg is 100 ml. molasses known as cane molasses is the last drop of sugar obtained from the sugar crystal separation process which is done 4 times. four times the separation process aims to find the parent leachate or the last drop of sugar. molasses is an excellent source of energy for various life forms of microorganisms. molasses is a source of carbohydrates that stimulate the growth of beneficial microorganisms. a good type of molasses for fertilizer is blackstrap molasses (unsulphered) because it has the highest concentration value of sulfur, potassium, iron and other micronutrients from original sugar cane, so it is not only the sugar content that makes molasses useful, but also the mineral content therein. molasses is an excellent chelating agent, which means that it can help convert some chemical nutrients into forms that are easily available for microorganisms. the dosage of molasses used in making organic fertilizer with a total amount of 100 kg of fertilizer is 100 ml. after em4 and molasses are measured according to size, then mixed into one with the addition of enough water. this solution is a bioactivator solution. the bioactivator solution is then poured into a solid material 86 et al.nurtjahyani microbiol indones volume 14, 2020 microbiol indones 87 table 1 result nutrient content of organic fertilizers according to indonesian national standard (sni) that has been thoroughly mixed. stirring is repeated until the water content of the fertilizer is suitable. water content in suitable fertilizers can be checked by taking a handful of fertilizers and holding it firmly (balled up), if water comes out of fertilizer and fertilizer does not expand then too much water content, but if water does not come out of fertilizer and fertilizer expands slowly slowly and not breaking then the water content is enough on the fertilizer. after all the ingredients are mixed evenly, the tarpaulin on which the fertilizer ingredients are stirred are then tightly closed so that the fermentation can proceed normally. the planned fermentation process is ± 14 days and during the fermentation process the temperature of the fertilizer is maintained not to exceed the temperature of 50ºc. if at the beginning of the fermentation. fermentation occurs anaerobically so that a closed space (tarp or gunny sack) is required. at the beginning of fermentation, fertilizer has brownish characteristics with a sweet smell (sugar), but as time goes by fermentation, fertilizer smells acidic and then smells like soil with a blackish color. this character indicates that organic fertilizer with the bokashi method can already be harvested or used. at the beginning of making organic fertilizer, leaf litter is predicted to experience maturity (ready for harvest) at ± 14 days (2 weeks) to ± 21 days (3 weeks) fermentation time. after the fermentation process is carried out for ± 21 days (3 weeks) fertilizer samples are taken to test the nutritional quality. the results of testing the elements on organic fertilizer produced showed in table 1 and table 2. but after observing when the fertilizer is aged ± 21 days (3 weeks), the fertilizer has not matured. it is possible for several things, among others: leaf litter that is used without going through the process of sorting with stems and twigs, so it is possible that the structure of stems and twigs that are classified as large will slow down the fermentation process. in addition, the condition of the leaf litter used is very dry. this is thought to be the cause of the slow fermentation process. leaf litter has a high cellulose and lignin content, especially with very dry conditions, so microorganisms will need more time in the decomposition process. therefore, to accelerate the process of fermentation and decomposition of leaf litter, leaf litter should be used in a wet or rather wet condition so that not all litter is in dry conditions. the size of leaf litter used as raw material for composting must be as small as possible to achieve aeration efficiency and to be more easily digested or broken down by microorganisms. the smaller the particles, the more the surface area of l​​ itter that is in contact with microorganisms, so that microorganisms can digest and decompose litter in a shorter time. � as conclusion, the study of the use of leaf litter in the reclaimed land of former limestone mining in the area of p​​ t. semen gresik semen indonesia persero (tbk) : leaf litter found in the reclaimed land of the former limestone quarry located in pt. semen gresik semen indonesia persero (tbk) can be utilized by processing it into organic fertilizer. the processing of chemical properties (%) permentan no. 70 th 2011 amount according to sni 19-7030 2004 organic fertilizer (i) with bioactivator em4 and molase (1:1) 2 weeks organic fertilizer (ii) with bioactivator em4 and molase(1:1) 3 weeks quality c – organik min 15 9,8 32 37,80 34,89 good n total min 4 0,40 1,64 1,80 good fosfor (p) min 4 0,10 0,47 0,42 good kalium (k) min 4 0,20 0,54 0,48 good c/n rasio 15-25 10-20 23,05 19,33 good ph 4-9 6,8 – 7,49 5,5 5 good water content 15-25 max. 50 58,82 59,15 too wet table 2 em4 data formulated with organic ingredients 88 et al.nurtjahyani microbiol indones organic waste into organic fertilizer was successfully carried out by the bokhasi method of composition of solid material in the form of leaf litter, bran, cow dung and husk charcoal with a ratio of 2: 1: 1: 1 and the search material in the form of em4 and molase with a dose of 100 ml per each 100kg (1:1) in 3 weeks of solid material dissolved in enough water (fertilizer can be clenched and expand slowly and not break) with the characteristics of the microbial composition of l a c t o b a c i l l u s s p , s t re p t o m y c e s s p , ye a s t , rhodopseudomonas sp, actinomycetes sp with brown em4 color indicator, sour smell, ph 3.5, and the most nutrient content is organic c. acknowledgements this study was suported pt semen indonesia (persero) tbk of research funding facilities, biology laboratory staff of its surabaya and biology laboratory staff of pgri ronggolawe university tuban for their cooperation in processing and analyzing samples. references aprianis y. 2011. produksi dan laju dekomposisi serasah acacia crassicarpa a. cunn. di pt. arara abadi. tekno hutan tanaman [production and decomposition rate of acacia crassicarpa a. cunn. at pt. arara abadi. plantation techno] 4(1): 41-47. alami nh, septarina wp, pratiwi t, kuswytasari nd, zulaika e, shovitri m. 2019. extracellular alkaline phosphatase from mangrove soil yeast. akta kimia indonesia. 4(1): 15-31. asthon dkk, 1999. metode penelitian [research methods] r e s p i r a t o r y u n i v e r s i t a s s u m a t r a u t a r a . http://repository.usu.ac.id/bitstream/handle/12345678 9 / 6 7 8 5 7 / c h a p t e r % 2 0 i i i v. p d f ? s e q u e n c e = 3 & isallowed=y . dibia in, dana md, trigunasih md, kusmawati y, sumarniasih mds. 2012. pembuatan kompos bokashi dari limbah pertanian dengan menggunakan aktivator em4 di desa megati tabanan [making bokashi compost from agricultural waste using activator em4 in megati tabanan village]. fakultas pertanian universitas udayana. fisher rf, binkley d. 2000. ecology and management of forest soils. john wiley & sons: new hanif, abdillah m, nursal, syafi'i w. 2015. laju dekomposisi serasah daun di kawasan hutan larangan adat rumbio kecamatan kampar kabupaten kampar sebagai pengembangan modul pembelajaran pada konsep ekosistem hutan tropis di sma kelas x [decomposition rate of leaf litter in rumbio customary forest areas, kampar district, kampar regency as the development of learning modules on the concept of tropical forest ecosystems in class x senior high school] fkip. universitas riau. isroi, yuliarti n. 2009. kompos. penerbit andi, yogyakarta. khan mai, ueno k, horimoto s, komai f, tanaka k, ono y. 2009. physicochemical, including spectroscopic, and biological analyses during composting of green tea waste and rice bran. biol fertil soils. 45:305-313. kurniawan d, kumalaningsih s, sunyoto nms. 2011. the effect of effective microorganism 4 (em4) volumes addition 1% and fermentation time on quality of bokashi fertilizer made from rabbit feces and jackfruit waste. jurnal industria. 2(1): 57-66. minister of regulation energy and mineral resources republic indonesia. 2014. decree no. 7 of 2014 about implementations of reclamation and post mine in mineral and coal mining business activities. access on 23 march 2019. available at: https://jdih.esdm.go.id/ index.php/web/result/828/detail. national standardization agency. 2004. sni 197030-2004. specifications for compost from domestic organic waste. access on 23 march 2019, available at: https:www.bsn.go.id. sastro y, bakrie b, sudolar rn. 2013. the effect of fermentation method, microbes inoculation and carbon source proportion on the quality of organic fertilizermade from liquid wastes of chicken slaughterhouse. journal indonesian tropical animal agriculture. 38(4): 257-263. doi: 10.14710/jitaa. 38.4.257-263. simanungkalit rdm, suriadikarta da, saraswati r, setyorini d, hartatik w. 2006. pupuk organik dan pupuk hayati. balai besar litbang sumberdaya lahan pertanian-badan penelitian dan pengembangan pertanian [organic fertilizer and biofertilizer. research and development center for agricultural land resources-agricultural research and development agency]. york. green calgary. 2014. bokashi composting guide. green calgary: #100-301 14th st nw, calgary, ab. t2n2a1 | www.greencalgary.org | 403.230.1443 ext. 222. wahyuni s, rianto s, muanisah u, setyanto p. 2016. the use of organic fertilizers to population bacteria and dry land paddy production. proceding biology education conference. 13(1): 752-756. windusari y, sari na, yustian i, zulkifli h. 2012. estimation of carbon biomass from the understorey and litter vegetation at tailings deposition area of pt freeport indonesia. biospecies. 5(1) 22-28. page 1 page 2 page 3 page 4 page 5 page 6 3.mi666-yenni bakhtiar available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.4.3issn 1978-3477, eissn 2087-8575 vol 6, no 4, december 2012, p 157-164 *corresponding author; phone: +62-21-7563120/7560208, e-mail: ybakhtia@hotmail.com oil palm (elaeis guinnensis jacq) is one of the highest yielding plants in oil producing crops and the most important plantation and commercial crop in indonesia. however, some fungal diseases have reduced the annual production of oil palm in south east asia (idris et al. 2003). fungal pathogen ganoderma boninense, the causal agents of basal stem rot (bsr) in oil palm, can severely attack the plant leading to the effects of mycorrhizal endosymbiotic bacteria bacillus subtilis b10 and composite of arbuscular mycorrhizal fungal spores in green house experiment were examined in order to evaluate their effectiveness and compatibility with oil palm seedlings in the presence of a fungal pathogen ganoderma boninense, the most serious pathogen in oil palm (elaeis guineensis jacq) in indonesia. a three factors experiment were conducted, with mycorrhizal inoculation (m0 and m1), bacterial b. subtilis b10 inoculation (b0 and b1), and g. boninense inoculation (g0 and g1) as the first, second, and third factors, respectively. the results showed that disease severity index, plant height, root dry-weight, and phosphorus uptake were affected by co-inoculation of mycorrhizal endosymbiotic bacteria b. subtilis b10 and composite of arbuscular mycorrhizal fungi. coinoculation of mycorrhizal endosymbiotic bacteria b. subtilis b10 and arbuscular mycorrhizal fungi did not only reduce the percentage of basal stem rot incidence, but also significantly increased plant height and phosphorus uptake by oil palm seedlings. our results suggest that in oil palm seedlings mycorrhizal endosymbiotic bacteria b. subtilis b10 worked synergistically with arbuscular mycorrhizal fungi in increasing plant adaptation toward biotic stress of pathogen g. boninese and could be promising biocontrol agents. key words: arbuscular mycorrhizal fungi, bacillus subtilis b10, biotic stress, ganoderma boninense, mycorrhizal endosymbiotic bacteria, oil palm seedlings patogen ganoderma boninense pat penyebab penyakit busuk pangkal batang merupakan patogen paling mematikan pada tanaman kelapa sawit (elaeis guineensis jacq). ko-inokulasi bakteri endosimbiotik mikoriza bacillus subtilis b10 dan komposit dari spora fungi mikoriza arbuskular pada bibit kelapa sawit telah dievaluasi efektivitas dan kompatibilitasnya dalam meningkatkan daya adaptasi bibit kelapa sawit terhadap cekaman biotik patogen g. boninense pat. percobaan tiga faktor dalam rumah kaca telah dilakukan dengan faktor pertama adalah inokulasi fungi mikoriza arbuskular yang terdiri dari tanpa inokulasi (m0) dan dengan inokulasi (m1). faktor kedua adalah inokulasi bakteri b. subtilis b10 yang terdiri dari tanpa inokulasi (b0) dan dengan inokulasi (b1), sementara faktor ketiga adalah inokulasi patogen g. boninense yang terdiri dari tanpa inokulasi (g0) dan dengan inokulasi (g1). hasil penelitian menunjukkan bahwa ko-inokulasi bakteri endosimbiotik mikoriza b. subtilis b10 dan komposit spora fungi mikoriza arbuskular mempengaruhi indeks keparahan penyakit, tinggi tanaman, bobot kering akar dan penyerapan fosfor oleh bibit kelapa sawit. ko-inokulasi bakteri endosimbiotik mikoriza b. subtilis b10 dan fungi mikoriza arbuskular tidak hanya menurunkan persentase kejadian penyakit busuk pangkal batang, akan tetapi secara signifikan juga meningkatkan pertumbuhan tinggi tanaman dan penyerapan fosfor oleh bibit kelapa sawit. hasil penelitian ini menyimpulkan bahwa bakteri endosimbiotik mikoriza b. subtilis b10 bekerja sinergis dengan fungi mikoriza arbuskular dalam meningkatkan daya adaptasi bibit kelapa sawit terhadap cekaman biotik patogen g. boninense dan berpotensi sebagai agen biokontrol. kata kunci: bacillus subtilis b10, bakteri endosimbiotik mikoriza, bibit kelapa sawit, cekaman biotik, fungi mikoriza arbuskular, ganoderma boninense, adaptation of oil palm seedlings inoculated with arbuscular mycorrhizal fungi and mycorrhizal endosymbiotic bacteria bacillus subtilis b10 towards biotic stress of pathogen ganoderma boninense pat 1,2 2 1 yenni bakhtiar *, sudirman yahya , wahono sumaryono , 3 4 meity suradji sinaga , and sri wilarso budi 1 biotech center-badan pengkajian dan penerapan teknologi, gedung 630 kawasan puspiptek, serpong, banten 15314, indonesia; 2 department of agronomy and horticulture, faculty of agriculture, institut pertanian bogor, jalan raya darmaga, bogor, west java 16680, indonesia; 3 department of plant protection, faculty of agriculture, institut pertanian bogor, jalan raya darmaga, bogor, west java 16680, indonesia; 4 department of silviculture, faculty of forestry, institut pertanian bogor, jalan raya darmaga, bogor, west java 16680, indonesia dramatic yield losses (idris et al. 2003), and could kill more than 80% of the plants (abdul razak et al. 2004). until now there is no effective treatment for ganoderma infected palms, while preventive treatments showed varying degrees of effectiveness (haniff et al. 2005). nowadays, biological control has gained much attention as a way of reducing the use of chemical products in agricultural practices. arbuscular mycorrhizal fungi (amf) live as an obligate symbiont in the roots of about 80% of land plants, including oil palm. the uses of amf to improve fertilizer efficiency and/or to protect against root diseases have been getting more attention during the last decade. their importance in controlling plant disease, nutrient cycling and soil quality are well reported (rillig and mummey 2006; smith and read 2008, lioussanne 2010). in the rhizosphere, the amf coexist with other soil organisms. several bacteria associated with am fungal spores (mycorrhizal endosymbiotic bacteria) have positive or negative effects on the amf or host plants (bharadwaj et al. 2008). bakhtiar et al. (2010) found that bacillus subtilis zj 06 had higher antagonistic activity against g. boninense in vitro and has a potential to be used as biocontrol agent against basal stem rot in oil palm. the role of amf in increasing plant resistance to pathogens is quite well known. arbuscular mycorrhizal fungi might control plant pathogens activity by direct interactions with the pathogens or mediated by mycorrhizal endosymbiotic bacteria (whipps 2004). the antagonism of bacteria inhabiting the mycorrhizal endosymbiont has also been suggested as a possible mechanism (budi et al. 1999). currently, the use of chemicals to control soil-borne pathogens is a major problem with modern crop production due to increasing concern for human health and environmental safety. the utilization of amf together with mycorrhizal endosymbiotic bacteria to control plant pathogens, combined with the positive effects on amf colonization and plant growth offer an important solution yet to be fully exploited. therefore, the aim of this study is to evaluate growth response of oil palm seedling in the green house, when inoculated with arbuscular mycorrhizal fungi and mycorrhizal endosymbiotic bacteria in the presence of root pathogen g. boninese. materials and methods preparation of arbuscular mycorrhizal fungal spores, b. subtilis b10 suspension and fungal pathogen g. boninense. the arbuscular mycorrhizal fungi (amf) used in this study was composed of amf spores isolated from rhizosphere of oil palm in previous study (bakhtiar et al. 2010). the fungi was propagated in pot cultures of pueraria javanica in zeolite sterile. the spores of amf were collected and sterilized in two steps (reimann 2005), first in 2% (w/v) of chloramine-t and tween 20 for two min and second in mixture of 20% (w/v) of streptomycin and 10% (w/v) of gentamycin (100 ml) for 10 min. all spores were stored in 50 ml of sterile tap water at 4 °c until used. isolates of b. subtilis b10 were obtained from the mycorrhizal endosymbiotic of oil palm roots colonized by amf and found to have the biggest inhibition against g. boninense in vitro (bakhtiar et al. 2010). suspension of mycorrhizal endosymbiotic bacteria b. subtilis b10 was prepared by inoculating 1 ml of bacterial suspension into 50 ml of nutrient broth. the bacterial cultures were incubated in a shaking incubator (150 rpm) at 28 °c for 12 h. inoculum of root pathogen g. boninense, grown on rubber wood block, was provided by the indonesian oil palm research institute (iopri). growth medium. the growth medium was a 1:1:1 (w/w) mixture of red yellow podzolic soil (pmk) : sand : compost in total 1 kg per polybag. the acidic pmk soil was obtained from gajrug, rangkas bitung, banten. the growth medium was sterilized using autoclave and allowed to equilibrate for 7 d. the mixture of soil media was then placed in the polybag (1 kg/polybag in the pre-nursery step and 5 kg/polybag in the main-nursery step). inoculation of amf and b. subtilis b10 into oil palm seedlings. seeds of oil palm germinated variety of dy x p dumpy type (supplied by iopri, medan, north sumatera) were grown in polybags containing 1 kg soil growth medium in the glasshouse (pre nursery step). the plant seedlings were inoculated with 200 spores of amf/seed, followed by drenching 20 ml of mycorrhizal bacterial b. subtilis b10 suspension into the soil. control polybags were prepared in a similar way but without suspension of bacteria and spores of amf. in total, five replicates per treatment were arranged and each treatment consisted of two polybags. the plant seedlings were fertilized with half strength of iopri recommended dosage. oil palm seedlings were regularly irrigated and rotated to minimize any border effects. after 12 weeks, all of the oil palm seedlings were transplanted into bigger polybags in the main-nursery step. at this step, some of the oil palm seedlings were inoculated with fungal pathogen g. boninense. the oil palm seedlings were 158 bakhtiar et al. microbiol indones then harvested after 52 weeks. the variables recorded were disease incidence of basal stem rot, height of plant and root dry weight. the phosphorus (p) use efficiency of plants was calculated and expressed as gram shoot dry weight per gram p absorbed. disease severity index (dsi). disease progression was described using the disease severity index (dsi) which depicts the severity of the disease based on the progress of the disease. the symptoms were indexed along with the following formula (abdullah 2003): 0 = for healthy plants; 1 = for appearance of three necrotic leaves; 2 = for appearance of more than three necrotic leaves; 3 = for fruiting bodies appearance at the bole; 4 = for dying/dead plant. the dsi was calculated at the end of experiment (52 weeks) based on the following formula: where: a: disease class (0, 1, 2, 3 or 4) b: number of plants showing that disease class per treatment. experimental design. the three factors experiment was arranged in a complete randomized design with four replications, resulted in a 2 x 2 x 2 treatment combinations, with mycorrhizal inoculation (m0 and m1), bacterial b. subtilis b10 inoculation (b0 and b1), and g. boninense inoculation (g0 and g1) as factors. each experiment was performed on two polybags. the quantitative data were analyzed using anova analysis and the means were separated using lsd test in sas program. results disease severity index of oil palm seedlings after inoculation with arbuscular mycorrhizal fungi and mycorrhizal endosymbiotic bacteria b. subtilis b10. in general, 52 weeks after treatments, basal stem rot disease developed slower in the seedlings inoculated with amf, whether or not the seedlings were co-inoculated with b. subtilis b10, than in the seedlings without amf (fig 1). a lower dsi would indicate disease suppression by the amf and b. subtilis b10. the seedlings treated with mixed of amf (m1) and b. subtilis b10 (b1) showed the lowest dsi (5%) (fig 1). as expected, the seedlings without amf inoculation showed the highest dsi, whether or not they were co-inoculated with b. subtilis b10. the dsi of the seedlings with and without b. subtilis b10 inoculation were 42.5% and 37.5%, respectively. inoculation with only b. subtilis b10 could not suppress the growth of pathogens g. boninense and the dsi value was very high. conversely, when the b. subtilis b10 was co-inoculated with amf, the dsi value dropped dramatically (fig 1). the leaves of infected plants were chlorotic and had white fungal mass on some parts of the plants, which then formed basidiocarp before the plants dried (fig 2). effect of amf and mycorrhizal endosymbiotic bacteria b. subtilis b10 on height of seedlings and root dry weight. the effects of mycorrhizal endosymbiotic bacteria b. subtilis b10, amf or coinoculation of them on the growth parameters and the differences in plant responses to the microbial inoculation among oil palm seedlings were observed. the presence of root pathogen g. boninense reduced the height of seedlings (fig 3). inoculation of amf, as well as b. subtilis b10, significantly increased the seedlings height compared to those without amf. interestingly, the tallest seedling (156.88 cm) was obtained when amf was co-inoculated with b. subtilis b10. it suggested that the mycorrhizal endosymbiotic bacteria b. subtilis b10 contributed to sustaining the plants' growth in the presence of pathogen g. boninense. generally, the un-inoculated seedlings (controls) challenged with g. boninense grew stunted and were the smallests (fig 3). oil palm seedlings inoculated with amf and b. subtilis b10 showed no significant difference in the root dry weight compared σ(a × b) × 100 σb × 4 disease severity index (dsi) = volume 6, 2012 microbiol indones 159 fig 1 disease severity index (dsi) at 52 weeks after planting and chalanged by fungal pathogen g. boninense. means with the same letters between treatments are not significantly different (lsd 0.05). m0: without inoculation of amf; m1: with inoculation of amf; : without inoculation of b. subtilis b10; : with inoculation of b. subtilis b10. a b a c 0 5 10 15 20 25 30 35 40 45 50 m0 m1 treatments of amf inoculation w ee k s phosphorus uptake of oil palm seedlings i n o c u l a t e d w i t h a m f a n d m y c o r r h i z a l endosymbiotic bacteria b. subtilis b10 in the presence of g. boninense. in general, the presence of fungal pathogen g. boninense reduced the uptake of phosphorus (p) by the seedlings at 52 weeks after planting, except when seedlings were co-inoculated with amf and b. subtilis b10 (fig 5). inoculation of b. subtilis b10 alone significantly increased p uptake to 27.67 g/plant when the seedlings did not treated with g. boninense. however, the p uptake decreased to 20.73 g/plant when seedlings were challenged with g. boninense. the result suggested that mycorrhizal to the control seedlings (fig 4). all seedlings had lower root dry weight when challenged with pathogenic fungus g. boninense, either inoculated with amf and or b. subtilis b10. application of mycorrhizal endosymbiotic bacteria b. subtilis b10 to the oil palm seedlings with neither amf nor g. boninense increased root dry weight over uninoculated ones. however, the root dry weight decreased when this bacteria were co-inoculated together with amf. it means that the combination of amf and the bacteria might not be compatible in increasing root dry weight of oil palm seedlings when the pathogenic fungus g. boninense was present. fig 2 comparison of healthy and infected oil palm. healthy oil palm seedlings; infected plants with the symptoms of basal stem rot caused by g. boninense. the infected plants generated chlorotic leaves, formed g. boninense fruiting bodies (yellow arrow) and the plants became dry. (a) (b) a b 160 bakhtiar et al. microbiol indones c b ab a c b b ab 100 110 120 130 140 150 160 170 b0 b1 b0 b1 m0 m1 h ei g h t o f p la n ts a t 5 2 w ee k s af te r p la n ti n g (c m ) treatments of inoculation of amf (m) and inoculation of mycorrhizal endosymbiotic bacteria (b) fig 3 height of plants at 52 weeks after planting, either challenged or not challenged by pathogen g. boninense. means with the same letters between treatments are not significantly different (lsd 0.05). m0: without inoculation of amf; m1: with inoculation of amf; : without inoculation of b. subtilis b10; : with inoculation of b. subtilis b10. boninense (m1b0g1) did not improve p uptake (14.64 g/plant). however,when the plants were treated with amf together with mycorrhizal endosymbiotic endosymbiotic bacteria b. subtilis b10 played an important role in improving p uptake. interestingly, inoculation with amf alone in the presence of g. volume 6, 2012 microbiol indones 161 fig 5 phosphorus uptake by oil palm seedlings at 52 weeks after planting, either challenged or not challenged by fungal pathogen g. boninense. means with the same letters between treatments are not significantly different (lsd 0.05). m0: without inoculation of amf; m1: with inoculation of amf; : without inoculation of b. subtilis b10; : with inoculation of b. subtilis b10. treatments of inoculation of amf (m) and inoculation of mycorrhizal endosymbiotic bacteria (b) ab a ab ab b ab b ab 0 5 10 15 20 25 30 b0 b1 b0 b1 m0 m1 p h sp h o ru s u p at ak e at 5 2 w ee k s af te r p la n ti n g 35 fig 4 root dry weight of oil palm seedlings at 52 weeks after planting, either challenged or not challenged by pathogen g. boninense. means with the same letters between treatments are not significantly different (lsd 0.05). m0: without inoculation of amf; m1: with inoculation of amf; : without inoculation of b. subtilis b10; : with inoculation of b. subtilis b10. treatments of inoculation of amf (m) and inoculation of mycorrhizal endosymbiotic bacteria (b) ab ab ab a b b b ab 0 5 10 15 20 25 b0 b1 b0 b1 m0 m1 r o o t d ry w ei g h t at 5 2 w ee k s af te r p la n ti n g ( g ) proven to be compatible with arbuscular mycorrhiza development but antagonistic towards soilborne fungal pathogens. this strain was found to produce small peptides which were responsible for the antagonistic effect against pathogens but were harmless to the symbiotic fungi. in this study, the application of amf together with mycorrhizal endosymbiotic bacteria b. subtilis b10 enhanced the height of oil palm seedlings but not root dry weight. this findings are supported by jayasinghearachchi and seneviratne (2005) that the solubilization of rock phosphate is enhanced by formation of mixed biofilms between phosphatesolubilizing saprotrophic fungi and a bradyrhizobium elkanii strain. taken together, these recent findings the effects of co-inoculation on plants depend on the particular combinations used (andrade et al. 1998; barea et al. 1998). the interactions have ranged from the very compatible (producing positive effects), the less compatible (producing neutral effects), to the incompatible (producing detrimental effects), or demonstrated better results from single inoculations rather than dual inoculations (vestberg et al. 2004). based on our study, the combination of amf and mycorrhizal endosymbiotic bacteria b. subtilis b10 produced positive effects on the height of oil palm seedlings but might be less compatible or neutral on root dry weight. it should also be noted that mycorrhizal association does not always improve plant productivity (bhromsiri and bhromsiri 2010). the competition for nutrients uptake by other microbes, may produce plant temporary nutrient starvation and result in detrimental impacts on the plant. these negative effects have occasionally been indicated, particularly when amf and pgpr were used together (raimam et al. 2007). in our study, p uptake was improved when oil palm seedlings were inoculated with amf and mycorrhizal endosymbiotic bacteria b. subtilis b10 together. the p uptake did not increase when amf were inoculated alone without mycorrhizal endosymbiotic bacteria b. subtilis b10. arbuscular mycorrhizal fungi increase host tolerance to pathogen by increasing the uptake of essential nutrients rather than phosphorus which are otherwise deficient in the non-mycorrhizal plants (gosling et al. 2006). it is often brought to mind that amf may improve p nutrition and enhance n uptake in their host plants. rhizosphere microbes, such as n fixing bacteria or p solubilizing bacteria, may interact synergistically with amf and thereby benefit plant development and growth (johansson et al. 2004; suparno 2009). bacteria b. subtilis b10 (m1b1g1), the amount of p uptake was increased (23.40 g/plant). discussion the mycorrhizal endosymbiotic bacteria b. subtilis b10 used in this study were earlier shown to have an antagonistic activity towards the fungal pathogen g. boninense (bakhtiar et al. 2010). we showed further by in vivo test of this bacteria in the presence of amf on oil palm seedlings and revealed strong antagonism against g. boninense. the possible mechanisms responsible for this biocontrol activity includes competition for nutrients or colonisation sites (johansson 2004), where siderophores play a role, niche exclusion (bharadwaj et al. 2008), production of antifungal metabolites (xiao et al. 2008) and several antibiotics (raaijmakers and desouza 2002), and induced resistance in plants (nandakumar et al. 2001). in our study, the combination of amf and b. subtilis b10 showed stronger suppression towards g. boninense compared to single application of amf or b. subtilis b10 alone. in in vitro test, single application of b. subtilis b10 indicated strong suppression against g. boninense. however, the results of in vivo test showed that this bacteria alone could not increase plant adaptation to biotic stress of g. boninense, except when this bacteria was inoculated together with amf (fig 1). it might be because the bacteria b. subtilis b10 alone could not enter the root of oil palm seedlings unless it was together with amf, which has the ability to infect the root of plants. other possible reason for this, several factors will affect the ability of bacteria in giving the benefit to the plant. some bacteria might give different results in in vitro and in vivo tests (i.e. when inoculated into plants). saharan and nehra (2011) reported that environmental factors (abiotic and biotic) may affect the growth and microbial effects of in the field. these environmental factors could be climate, weather conditions, character of soil, composition of indigenous microbes in the soil and competition among natural microbes in the soil. cruz and ishii (2012) also stated that the activity of probable endobacteria (pe) isolated from spores of amf gigaspora margarita in vitro, may not reflect their activity in the soils, especially for the bacteria living on the surface of the spore. our results suggest the possibility of integrated use of amf and their associated bacteria in biological control of soil-borne pathogens. this finding is supported by selim et al. (2005), who focused on a strain of paenibacillus isolated from the rhizosphere of sorghum 162 bakhtiar et al. microbiol indones strongly suggest that mycorrhiza-associated bacteria complement the roles of the external mycelium by mobilizing nutrients from minerals. compatible with amf in increasing oil palm height and phosphorus uptake, as well as showing an antagonistic effect against soilborne fungal pathogens the present results showed that mycorrhizal endosymbiotic bacteria b. subtilis b10, which were isolated from amf spore in the oil palm rhizosphere might be g. boninense. co-inoculation of amf and mycorrhizal endosymbiotic bacteria b. subtilis b10 did not only reduce the percentage of basal stem rot incidence, but also significantly increase the plant height and phosphorus uptake of the oil palm seedlings. our results suggest that the interaction between amf and mycorrhizal endosymbiotic bacteria b. subtilis b10 in oil palm improves phosphorus uptake and plant growth, and 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growth and inhibit the development of root pathogens. many soil bacteria have been used as pgpr, and one of them is bacillus sp. the implementation of pgpr is constrained by genotype fluctuation that makes it inactive on the rhizosphere. our previous study had characterized and revealed that 11 bacillus sp. isolated from the soybean plant rhizosphere were pgpr. to asses and compare the genetic diversity of these isolates, amplified ribosomal dna restriction analysis (ardra) and dna sequence analysis of 16s rrna were conducted. the construction of neighbor-joining trees and bootstrap analysis of 100 resamples of ardra and 16s rrna gene sequences were performed using treecon software for windows ver. 1.3b. ardra analysis was done by using four restriction enzymes (rsai, haeiii, cfri and hinfi), resulting in four phylotypes, respectively phylotype i (bacillus sp. cr24, cr33, cr64 and cr68), phylotype ii (bacillus sp. cr 31 and cr66), phylotype iii (bacillus sp. cr44 and cr71) and phylotype iv (bacillus sp. cr67, cr28 and cr69). results of blastn from 16s rrna gene sequences showed that these isolates are genetically diversed. the evolution relationship of bacillus sp. could be shown by the 16s rrna gene sequences analysis, while ardra based on the digestion sites showed their variability. key words: bacillus sp., plant-growth promoting rhizobacteria, 16s rdna, ardra, genetic diversity *corresponding author, phone/fax: +62-251-8622833, e-mail: aristri2003@yahoo.com the slow growth of plant is a major handicap that can decrease food production and ecosystem stability. synthetic fertilizers have been used to solve this problem. however, it can give negative effects on human and environment, such as pollution of the surrounding ecosystem and the death of non-target microbes. therefore, efforts to find alternative solutions, among others by using plant-growth promoting rhizobacteria (pgpr), i.e. associated soil-borne bacteria on the rizosphere that can enhance plant growth and inhibit the growth of root pathogens, such as bacillus sp. bacillus is a motile, catalase-positive, gram-positive rod, with 40-60% gc content. it forms endospore that is very resistant to extreme environmental conditions. bacillus has been known for the production of phytohormone such as indole acetic acid (glick 1995), siderophores (compant et al. 2005) and antibiotics such as zwittermicin a (silo-suh et al. 1994), bacilin, clorotetain and iturin a (phister et al. 2004). these compounds are natural and beneficial to promote plant growth. hence, it is potential to utilize those mechanisms in agriculture. its implementation, however, is constrained by the genotype fluctuation. the 16s rrna gene has been routinely used as a reliable molecular marker for phylogeny identification. it contains conserved region, a unique array of sequences that are relative among species or different species (woose 1987; moyer et al. 1994). it is the basis of molecular tools such as ribotyping, in-situ hybridization, dna sequence analysis and restriction fragment length polymorphism (rflp), which are now proposed to provide accurate genetic diversity information of microbes. based on the use of the 16s rrna, the dna sequence analysis is used in phylogenic studies (lagace et al. 2004). rflp is used to identify the difference of dna fragment length (polymorphism) by digesting with restriction enzymes. rflp analysis on 16s rrna gene or amplified rdna restriction analysis (ardra) is a useful technique for genotype identification, to infer genetic variability and similarity of microorganisms (yang et al. 2007). this study was conducted on 11 bacillus sp. isolates from the rhizosphere of soybean plant, with a focus on the analysis of their genetic diversity based on ardra and 16s rrna sequences analysis. materials and methods bacterial strains and growth condition. all plant growth promoting bacillus sp. isolates were maintained in nutrient broth medium and grown at room temperature (28 °c) for 24 h. characteristics of bacterial strains of bacillus sp. strains used in this study are listed in table 1. isolation of bacillus sp. genome. eleven isolates of bacillus sp. were cultured in 25 ml nutrient broth and incubated for 24 h at room temperature (28 °c). genomic dna isolation was carried out by using standard protocol as described by sambrook and russel (2001). amplification of 16s rrna by pcr. the amplified 16s rrna genes were obtained by pcr with forward primer 63f (5’-caggcctaacacatgcaagtc-3’) and reverse primer 1387r (5’-gggcggwgtgtacaaggc-3’) (marchesi et al. 1998), which are targeted to the conserved region of bacterial 16s rrna genes and permit the amplification of an approximately 1300-bp fragment. the pcr mixture with a total volume of 50 µl was composed of 5 µl dna template, 1 µl each primer (10 pmol) , 8 µl dntps, 25 µl taq polymerase buffer, 0.5 µl la taq polymerase, and 9.5 µl ddh 2 o (takara, japan). amplification was done in a thermal cycler 2 400 (perkin-elmer, usa). initial denaturation at 94 °c for 2 min was followed by 30 cycles of denaturation at 92 °c for 30 sec, annealing at 55 °c for 30 sec, and elongation at 75 °c for 1 min, then the final extension was carried out for 5 min at 75 °c. the presence and yield of specific pcr products were visualized by 1% agarose (w/v) gel electrophoresis for 45 min at 70 v cm-1 in tae 1x buffer. amplicons were further purified volume 3, number 1, april 2009 p 12-16 issn 1978-3477 microbiol indones 13volume 3, 2009 by gel/pcr dna fragments extraction kit according to the manufacturer’s instructions (geneaid, usa). ardra. each purified pcr products of 16s rdna were digested by four restriction enzymes, rsai, haeiii, cfri and hinfi in separated reaction. the dna digestion were performed for 3 h at 37 °c in 20 µl of reaction volumes containing 5 µl of amplicon (1.5 µg), 2 µl of buffer tango 10x, ddh 2 o and 2 units of the restriction enzyme (fermentas, usa). restriction was inactivated by heating at 65 °c for 20 min. restriction products were electrophoresed on a 1% agarose (w/v) gel electrophoresis in tae 1x buffer. the sizes of the fragments were converted into binary data and analyzed by using treecon software for windows ver. 1.3b (van de peer and de watcher 1994). sequence analysis of 16s rrna gene. the pcr products of 16s rdna were purified by gel/pcr dna fragments extraction kit (geneaid, usa), sequenced and further analyzed for bioinformatics analysis. the blastn program (www.ncbi.nlm.nih.gov) was used to find the identity and similarity of each sequences compared to the genbank database. furthermore, the clustalw program (www.ebi.ac.uk) was used in order to align those sequences. the construction of neighbor-joining tree and bootstrap analysis of 100 resamples were performed using treecon software for windows ver 1.3b (van de peer and de watcher 1994). results amplification of 16s rrna by pcr. pcr amplification of 16s rrna gene yielded dna fragments of single bands at 1300 base pairs for each bacillus sp. isolates (fig 1). these amplified dna can be used as a genetic tool to identify and classify the diversity of bacillus sp. ardra. four restriction enzymes (haeiii, rsai, cfri and hinfi) resulted in small variability of digestion profiles for each bacillus isolates. the length of dna fragment that were obtained from digestion by four restriction enzymes are shown in table 2. according to the digestion profiles, those bacillus sp. isolates were grouped into four different phylotypes as follows: phylotype i consisted of bacillus sp. cr24, cr33, cr64 and cr68; phylotype ii consisted of bacillus sp. cr31 and cr66; phylotype iii consisted of bacillus sp. cr44 and cr71; and phylotype iv consisted of bacillus sp. cr67, cr28, and cr69. most of the isolates (four isolates: cr71, cr44, cr66 and cr31) were found to be distributed within the phylotype i (fig 2), which mean their digestion sites have been evolving with the same direction. partial sequencing of 16s rrna gene and sequence analysis. there was no dominant species within 11 isolates of bacillus sp. pgpr based on bioinformatics analysis using blastn program (table 3). maximum identities for each isolate were more than 85% with e-value 0. the distributions were genetically diversed on several species of bacillus sp., such as b. subtilis, b. shandongensis, b. pumilus, b. cereus and b. thuringiensis. the phylogenetic tree based on 16s rrna gene partial sequences showed the evolutionary relationship among the isolates (fig 3). the phylogenetic tree exhibited that the 11 isolates were divided into three major groups. group i (cr66, cr33, cr24, cr 69 and cr 68); group ii (cr64 and cr71); and group iii (cr44). isolate cr44 was closely related to the reference strains. there was a significant difference between groups (phylotypes) of the isolates based on ardra compared to those obtained by phylogenetic tree analysis. discussion amplified rdna restriction analysis (ardra) is a method to analyze 16s rrna gene fragments that are produced by digestion enzymes. this method has been used for genetic analysis and diversity studies of many bacteria such as streptococcus (sasaki et al. 2004), lactobacillus (moreira et al. 2005), mycobacterium (baere et al. 2002; kurabachew et al. 2003), and type a toxin-producing clostridium (pooeshafie et al. 2005). furthermore, vaneechoutte et al. (1995) revealed that ardra can avoid dna contamination in pure culture. the restriction profiles produced by ardra can be used as a robust library for particular species (hall et al. 2001). in this study, the digestion of 16s rdna of bacillus sp. by haeiii, hinfi, rsai and cfri produced 1 to 3 bands for each treatment (table 2). genetic diversity of bacillus sp. species as revealed by ardra was low. in fact, a number of studies reported that there was no intraspecies variability and diversity (schlegel et al. 2003). the best explanation for this condition was that they had the same digestion sites in their conserved region. this has been proven by means of rsai digestion that showed homogenous pattern for all isolates with no diversity at all (table 2). meanwhile, the digestions by the other 3 restriction enzymes produced small diversity. it means, genetically, the isolates were evolving with the same direction. this study has also shown that some isolates were closely related and grouped each other as exhibited in the phylogenetic tree. isolates in the same phylotype according strain iaa production (ppm) phosphate solubilization siderophore production chitinase production cr24 cr28 cr31 cr33 cr44 cr64 cr66 cr67 cr68 cr69 cr71 15.16 12.16 5 . 4 5 3 . 2 5 3 . 7 3 7 . 5 6 3 . 0 2 0 . 8 1 0 . 8 7 4 . 3 2 9 . 6 3 + + + + + + + + + + + + + + + + + + + + + + + + + iaa, indole acetic acid table 1 the characteristics of eleven plant growth promoting of bacillus sp. isolates (unpublished data) microbiol indones14 bahri et al. table 2 the sizes of 16s rdna fragments resulted from digestion with four restriction enzymes, haeiii, rsai, hinfi and cfri isolate the size of dna fragment (bp) hae iii rsai hinfi cfri 550, 550, 200 550, 550, 200 550, 550, 200 550, 550, 200 1100, 200 1100, 200 1100, 200 1100, 200 550, 550, 200 550, 500, 200 550, 500, 200 1000, 300 1000, 300 1000, 300 1000, 300 1000, 300 1000, 300 700, 300, 300 700, 300, 300 1000, 300 1000, 300 1000, 300 650, 650 650, 650 650, 650 650, 650 650, 650 650, 650 650, 650 650, 650 650, 650 650, 650 650, 650 600, 500, 200 600, 500, 200 600, 500, 200 600, 500, 200 700, 600 700, 600 600, 500, 200 600, 500, 200 620, 480, 200 620, 480, 200 620, 480, 200 bacillus sp. cr24 bacillus sp. cr33 bacillus sp. cr64 bacillus sp. cr68 bacillus sp. cr31 bacillus sp. cr66 bacillus sp. cr44 bacillus sp. cr71 bacillus sp. cr28 bacillus sp. cr67 bacillus sp. cr69 phylotype i (bacillus sp. cr24, cr33, cr64 and cr68), phylotype ii (bacillus sp. cr31 and cr66), phylotype iii (bacillus sp. cr44 and cr71), and phylotype iv (bacillus sp. cr67, cr 28 and cr69) fig 2 dendrogram of phylogenetic and electrophoregram of bacillus sp. isolates by ardra. the dendrogram was constructed with treecon software for windows ver. 1.3b and grouped by neighbor-joining method with bootstrap analysis of 100 resamples. hae iii rsaihinfi cfri 0 . 1 9 7 6 3 5 9 9 8 9 8 filotipe iii filotipe ii filotipe i filotipe iv bacillus_cr71 bacillus_cr44 bacillus_cr66 bacillus_cr31 bacillus_cr64 bacillus_cr33 bacillus_cr24 bacillus_cr68 bacillus_cr69 bacillus_cr28 bacillus_cr67 fig 1 pcr amplification of the 16s rrna of each bacillus sp. indicated by a single band at ~1300 bp. marker (m): 100 bp ladder. m 24 28 31 33 44 64 66 67 68 69 71 1300 bp1500 bp microbiol indones 15volume 3, 2009 to the ardra profile would emerge in different groups in the phylogenetic tree. it might be due to the difference of the methods of analysis. for example, cr68 and cr69 were closely related to each other as shown in the phylogenetic tree, but these two isolates were separated from each other in the ardra profile. cr68 belongs to the phylotype i, whereas cr69 belongs to the phylotype iv. ardra profiles would much depend on the utilization of restriction enzymes. from the genotypic approach point of view we suggest the use of 16s ardra technique as tool to initially characterize bacillus sp. isolates and the use of total dna restriction profile and other molecular approaches, such as fragment selective amplification of chromosomal dna (aflp), for typing and singling out uncommon bacillus sp. strains. the diversity within species was shown by isolate cr33 and cr69. these two isolates were identified as bacillus cereus based on partial sequence analysis, although they were exhibited different ardra profile. therefore they splited into two different phylotypes and were designated as different strains. the similar characteristics between ardra and sequencing methods are actually unambiguous: a large number of characters collected from each organism and the information obtained could be used to identify the abundance and diversity of two dna sequences. the main difference between them is the asymmetry of the evolution of restriction sites vs nucleotide positions. the 4-bp recognition site for a restriction enzyme can be inactivated by any of the 12 different nucleotide substitutions. if the sequence differs from the recognition site only at a single site, then only one substitution can occur to produce that site. as a consequence, convergent losses of a restriction site are less than convergent gains and the ratio of convergent losses to table 3 similarity analysis based on 16s rrna partial gene sequences of each 11 isolates bacillus sp. by comparing to the genbank data 6 4 5 7 7 3 5 0 7 9 1 0 0 9 2 3 1 1 0 09 2 100 bcl_cr71 bcl_cr44 bcl_sub168* bcl_cr69 bcl_cr68 bcl_cr66 bcl_cr33 bcl_cr28 bcl_cr64 bcl_cr31 bcl_cr24 bcl_cr67 bcl_subnh1* 0 . 1 fig 3 dendrogram of 11 bacillus sp. isolates based on partial sequences 16s rrna gene constructed with treecon software for windows ver. 1.3b and grouped by neighbor-joining method with bootstrap analysis of 100 resampling. bcl, bacillus; *reference strains. isolate access num.maximum identityhomology bacillus sp. cr24 bacillus sp. cr28 bacillus sp. cr31 bacillus sp. cr33 bacillus sp. cr44 bacillus sp. cr64 bacillus sp. cr66 bacillus sp. cr67 bacillus sp. cr68 bacillus sp. cr69 bacillus sp. cr71 bacillus sp. 1re28 bacillus pumilus strain s2 bacillus sp. b-3(2008) bacillus cereus strain ibt016 bacillus subtilis strain cicc10166 bacillus sphaericus strain nuc-5 16 bacillus thuringiensis strain w64 bacillus pumilus bacillus sp. niot-3 bacillus cereus strain ad2 bacillus shandongensis strain sd 94% 91% 98% 95% 92% 85% 94% 94% 94% 97% 99% ef178451.1 ef439667.1 eu862293.1 ay296806.2 dq055129.1 dq833758.1 eu874887.1 ab048252.1 am981260.1 dq298080.1 eu046267.1 microbiol indones16 bahri et al. convergent gains increases as taxa become more divergent (moyer et al. 1996). the unique digestion profiles shown for each isolate can be used for reference purpose. this study has also defined the taxonomic status at the species level of all isolates following the comprehensive examination of the dna/dna similarity level of bacillus species type strains (genbank data). thus, ardra can be widely used to investigate or identify several bacillus sp. isolates inhabiting the rhizosphere possessing plant growth promoting characters. moreover, because of its simplicity and cheapness, ardra analysis will be of practical value than the more laborious sequencing analysis. acknowledgement this work was supported by incentive program for basic research from the ministry of research and technology (program insentif riset dasar, ristek) 2008 to atw. we are therefore grateful for this funding and support of this research. references baere td, mendoca r, claeys g, verschragen g, mijs w, verhelst r, rottiers s, simaey lv, ganck de c, vaneechoutte m. 2002. evaluation of amplified rdna restriction analysis (ardra) for the identification of cultured mycobacteria in diagnostic laboratory. bmc microbiol 24:1-22. compant d, duffy b, nowak j, clement c, barka ea. 2005. use of plant-growth promoting rhizobacteria for biocontrol of plant diseases: principles, mechanisms of action and future prospect. appl environ microbiol 71:4951-59. glick rb. 1995. the enhancement of plant growth promotion by free living bacteria. can j microbiol 41:109-17. hall v, taibot pr, stubbs sl, duerden bi. 2001. identification of clinical isolates of actinomyces species by amplified 16s ribosomal dna restriction analysis. j clin microbiol 39:3555-62. kurabachew m, enger o, sandra ra, lemma e, bjorvatn b. 2003. amplified ribosomal dna restriction analysis in the differentiation of related species of bacteria. j microbiol methods 55:83-90. lagace l, pitre m, jacques m, roy d. 2004. identification of the bacterial community of maple sap by using amplified ribosomal dna (rdna) restriction analysis and rdna sequencing. appl environ microbiol 70:2056-60. marchesi jr, sato t, weightman aj, martin ta, fry jc, hiom sj, wade wg. 1998. design and evaluation of useful bacterium-specific pcr primers that amplify genes coding for bacteria 16s rrna. appl environ microbiol 64:795-99. moreira jls, mota rm, horta mf, teixeira smr, neuman e, nicoli jr, nunes ac. 2005. identification to the species level of lactobacillus isolated in probiotic prospecting studies of human, animal, or food origin by 16s-23s rrna restriction analysis profiling. bmc microbiol 23:5-15. moyer cl, dobbs fc, karl dm. 1994. estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16s rrna genes from a microbial mat at an active, hydrothermal vent system, loihi seamount, hawaii. appl environ microbiol 60:871-79. moyer cl, tiedje jm, dobbs fc, karl dm. 1996. a computerstimulated restriction fragment length polymorphism analysis of bacterial small-subunit rrna genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. appl environ microbiol 62:2501-07. peer y van de, wachter r de. 1994. treecon for windows: a software package for the construction and drawing of evolutionary trees for the microsoft windows environment. comput appl biosci 10:569-70. phister tg, o’sullivan j, mckay ll. 2004. identification of bacilysin, chlorotetaine and iturin a produced by bacillus sp. strain cs93 isolated from pozol, a mexican fermented maize dough. appl environ microbiol 70:631-34. pourshafie m, vahdani p, popoff m. 2005. genotyping clostridium botulinum toxinotype a isolates from patients using amplified rdna restriction analysis. j med microbiol 54:933-36. sambrook w, russel dw. 2001. molecular cloning: a laboratory manual. 3rd ed. new york: gold spring harbor laboratory. sasaki e, osawa r, nishitani y, whiley ra. 2004. ardra and rapd analysis of human and animal isolates of streptococcus gallolyticus. j vet med sci 66:1467-70. schlegel l, grimont f, grimont pad, bouvet a. 2003 identification of major streptococcal species by rrn-amplified ribosomal dna restriction analysis. j clin microbiol 41:657-66. silo-suh la, lethbridge bj, raffel sj, he h, clardy j, handelsman j. 1994. biological activities of two fungistatic antibiotics produced by bacillus cereus uw85. appl environ microbiol 60:2023-30. vaneechoutte m, dijkshoom l, tjenberg i, elaichouni a, vos de p, claeys g, verschragen. 1995. identification of acinetobacter genomic species by amplified ribosomal dna restriction analysis. j clin microbiol 33:11-15. woose cr. 1987. bacterial evolution. microbiol rev 51:221-71. yang jh, liu hx, zhu gm, pan yl, xu ld, guo jh. 2007. diversity analysis of antagonists from rice-associated bacteria and their application in biocontrol of rice diseases. j appl microbiol 104:911 0 4 . 2.mi664-abdul munif new available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.4.2issn 1978-3477, eissn 2087-8575 vol 6, no 4, december 2012, p 148-156 *corresponding author; phone: +62-251-8629364, e-mail: abdulmunif@ipb.ac.id endophytic bacteria colonize healthy plant roots without causing apparent disease. more convey stress tolerance, induce systemic resistance, recently, endophytic bacteria have gained attention due to their interesting features related to plant growth and health stimulation. studies on endophytic bacteria have been conducted. some of the bacteria are known to increase nutrient availability, produce growth hormones, or deter plant pathogens (hallmann et al. 1997; buchenauer 1998). several reports demonstrated that endophytic bacteria are plant-associated bacteria that colonize and persist in various healthy plants, such as fruits, vegetables, stems and roots (mclnroy and kloepper 1995; sturz et al. 1997). bacterial endophytes can be isolated from surfacesterilized plant tissue or extracted from internal plant tissue. endophytic microorganisms enter plant tissue primarily through the root zone; however, aerial portions of plants, such as flowers, stems, and cotyledons, may also be used for entry (quadthallmann and kloepper 1996). specifically, the bacteria enter tissues via germinating radicles, endophytic bacteria have gained attention due to their interesting features related to plant growth and health stimulation. the objective of this research was to determine the populations and spectrum of indigenous root endophytic bacteria from tomato (lycopersicon esculentum) and the biocontrol activity of the bacteria for plant protection. the isolation procedure of these endophytic bacteria was done using surface-sterilization method using alcohol and sodium hypochlorite (naocl). general medium trypsic soy agar (tsa) was used as the growth medium for isolation. the total population density of endophytic bacteria recovered from tomato roots ranged -1 from 1.0 to 4.4 (in log scale) cfu g fresh root weight. a total of 564 strains of endophytic bacteria were isolated 10 from tomato plants grown in west java, indonesia. endophytic bacterial strains were identified based on their fatty acid profile using fame-gc-midi system. fifty species and 32 genera of endophytic bacteria were found in association with tomato root. the most abundant endophytic bacterial genera were bacillus spp and pseudomonas spp. one hundred and eighty one bacterial strains were tested for their in vitro antagonism towards rhizoctonia solani, fusarium oxysporum f.sp. radicis-lycopersici, and f. oxysporum f.sp. lycopersici. fourteen strains showed antagonism against r. solani, nine strains against f. oxysporum f.sp. radicis-lycopersici and seven strains against f. oxysporum f. sp. lycopersici. the close relationship between endophytic bacteria and their hosts make them ideal candidates for biological control and plant growth promotion. key words : bacillus spp., endophytic bacteria, pseudomonas spp., tomato bakteri endofit telah menarik perhatian karena potensinya untuk peningkatan pertumbuhan dan kesehatan tanaman. tujuan penelitian ini adalah untuk mengetahui populasi dan spektrum bakteri endofit yang berasal dari akar tanaman tomat dan potensinya sebagai agen biokontrol untuk perlindungan tanaman. prosedur isolasi bakteri endofit dilakukan dengan menggunakan metode sterilisasi permukaan dengan alkohol dan sodium hipoklorit (naocl), dan medium trypsic soy agar (tsa) sebagai media pertumbuhan untuk bakteri. strain bakteri endofit diidentifikasi berdasarkan profil asam lemaknya dengan menggunakan fame-gc-midi system. -1 kepadatan total populasi bakteri endofit yang diisolasi dari akar tomat berkisar 1.0-4.4 (pada skala log ) cfu g 10 berat akar segar. sebanyak 564 strain bakteri endofit berhasil diisolasi dari tanaman tomat dari daerah jawa barat, indonesia. hasil identifikasi menunjukkan 50 spesies dan 32 genera bakteri endofit ditemukan berasosiasi dengan akar tomat. genus bakteri endofit paling banyak ditemukan adalah bacillus spp. dan pseudomonas spp. sebanyak 181 isolat bakteri endofit diuji antagonismenya secara in vitro terhadap cendawan patogen. sebanyak 14 isolat bakteri menunjukkan reaksi antagonisme terhadap rhizoctonia solani, 9 strain terhadap fusarium oxysporum f.sp. radicis-lycopersici, dan 7 strain terhadap f. oxysporum f. sp. lycopersici. hubungan yang sangat dekat antara bakteri endofit dan tanaman inangnya menjadikan bakteri endofit sangat berpotensi sebagai agen biokontrol dan pemacu pertumbuhan tanaman. kata kunci: bacillus spp., bakteri endofit, pseudomonas spp., tomat . isolation of endophytic bacteria from tomato and their biocontrol activities against fungal diseases 1 2 2 abdul munif *, johannes hallmann , and richard a sikora 1 department of plant protection, institut pertanian bogor (ipb), kampus ipb darmaga, bogor 16680,indonesia; 2 institut fur pflanzenkrankheiten, university of bonn, nussallee 9, d 53119 bonn, germany secondary roots, stomata, or as a result of foliar damage. endophytes inside a plant may either become localized at the point of entry or spread throughout the plant (quadt-hallmann et al. 1997). variations in the populations of endophytic bacteria are attributed to plant source, plant age, tissue type, time of sampling, and environment. generally, bacterial populations are larger in roots and lower in the stems and leaves (hallmann et al. 1997). endophytic bacteria may be considered a new source of biocontrol agents (backman and sikora 2008). some reports have demonstrated that bacterial endophytes are able to improve plant growth and reduce disease symptoms caused by plant pathogens such as fusarium oxysporum subsp. vasinfectum on cotton, verticillium albo-atrum, and rhizoctonia solani on potato (hallmann et al. 1997), clavibacter michiganensis subsp. sepedonicum on potato (van buren et al. 1993), and plant-parasitic nematodes (munif et al. 2000; vertrivelkalai et al. 2010). when compared with rhizobacteria, endophytic bacteria have distinct advantages: 1) they colonize an ecological niche similar to that of vascular plant pathogens, i.e. vascular wilt pathogens and sedentary plant parasitic nematodes; 2) they face less competition with other microorganisms; 3) they have access to sufficient supply of nutrients; 4) they are less exposed to rhizosphere's abiotic and antibiotic stress factors; 5) their bioactive metabolites are better translocated within the host plant (hallmann et al. 1997; buchenauer 1998). the exploitation of the beneficial properties of endophytic bacteria requires a basic knowledge of the endophytic bacteria, such as distribution within the plant tissue, diversity, population dynamics, and characterization. the objective of this research was to determine the variations and types of indigenous endophytic bacteria from tomato plants grown in west java, indonesia, and its biological control potential against fungal disease. materials and methods isolation of endophytic bacteria. a total of 20 tomato plants were uprooted at random from tomato fields in the districts bogor, sukabumi, and bandung, west java, indonesia. the tomato plants were approximately two months old. the tomato roots were transported to the laboratory for immediate processing. tomato roots were washed under running tap water to remove adherent soil particles and then blotted dry on tissue paper. to simplify root processing, the root system was divided into three parts: top, middle, and bottom. the top section was discarded, whereas the middle and bottom parts were examined. the root was weighed and then surface sterilized with 3% and 6% sodium hypochlorite containing 0.01% tween 20 for 3 min, followed by four rinses in sterile 0.01 m potassium phosphate buffer (pb) at ph 7.0 (80 g nacl, 2 g kcl, 11.5 g na hpo , 2 g kh po ). to confirm 2 4 2 4 complete surface-sterilization (sterility check), the surface sterilized roots were imprinted on tryptic soy agar (tsa). if bacterial growth occurred within 48 h, samples were discarded. the tomato roots were then macerated with a sterile mortar and pestle in three times pb (w/v). the macerate was decanted into sterile conical flasks and shaken for 30 sec. a dilution series was made and 100 µl of each dilution was plated onto 1/10 strength tsa with a drigalski spatula (plate spreader). petri plates were incubated at 24 °c for 2-3 d and colony forming units (cfu) were calculated. three replicates were made per dilution. on each petri plate a zone containing approximately 30 bacterial colonies was marked. all bacterial colonies from this zone were transferred and purified on full strength tsa. the bacterial colonies were stored in tryptic soy broth (tsb) plus 20% glycerol at -20 °c. identification of endophytic bacteria. all bacterial strains were identified based on their fatty acid profile using fatty acid methyl ester (fame) analysis by gas-chromatography and the software microbial identification system (misi microbial id, newark, delware, usa). the extraction procedure for whole-cell fatty acid from endophytic bacteria was done following the method described by sasser (1990). harvesting. bacteria from stock cultures (-80 °c) were pre-cultivated for 24 h on tryptic soy broth agar (tsba) and then streaked on tsba for identification. each strain was streaked with a loop onto tsba in four zones with increasing dilution and incubated at 28 °c for 24 h. the bacteria from zone 2 and 3 were harvested. a loopful of bacteria (about 50 mg) was used to inoculate medium tube. saponification. lipids were saponified by adding 1.0 ml of 15% naoh in 50% methanol reagent 1 (sodium hydroxide/certified acs 45 g, methanol /reagent grade 150 ml, and deionized distilled water 150 ml) into each sample tube and the tube was sealed with a teflon-lined cap, then mixed for 5 sec. the tube was heated in a waterbath at 100 °c for 30 min, and the content was mixed again. methylation. the samples were methylated by volume 6, 2012 microbiol indones 149 adding 2 ml of 6 n hcl in 50% methanol (reagent 2: 6.0 n hydrochloride acid 325 ml, methanol (reagent grade) 275 ml), mixed for 5 sec, placed in a waterbath 80 °c for 10 min, and then rapidly cooled in water. extraction. the fames were extracted from this solution by adding 1 ml of methyl tert-butyl ester (mtbe) hexane mix (50:50, v/v) (reagent 3: hexane (hplc grade) 200 ml, methyl tert butyl ether (mtbe) 200 ml), rotated for 10 min. the top, organic phase was transferred with a pasteur pipette to test tubes. washing. the organic phase was then washed using 3.0 ml of diluted naoh (reagent 4: sodium hydroxide 10.8 g, deionized distilled water 900 ml). the organic phase was then transferred to 2 ml vials for subsequent analysis by gas chromatography using an hp 5890 (hewlett packard, mis, id, newak, delware, usa). in vitro antagonism of endophytic bacteria towards plant pathogenic fungi. a total of 181 bacterial endophytes were tested for antibiosis against fungal pathogens r. solani, f. oxysporum subsp. radicis-lycopersici, and f. oxysporum subsp. lycopersici. isolates r. solani and f. oxysporum subsp. lycopersici were obtained from the fungal collection of the institut fuer pflanzenkrankheiten, university of bonn, while isolate f. oxysporum subsp. radicislycopercisi was obtained from the fungal collection of the humboldt university of berlin. the fungi were grown on potato dextrose agar (pda) at ph = 6.4 and stored in potato dextrose broth plus 20% glycerol at -80 °c. in vitro antibiosis was tested on pda using the dual-culture technique. fungal plugs (d = 10 mm) of 6 d old cultures were placed at the center of each petri dish and the endophytic bacteria were streaked in two lines approximately 2 cm from the fungus. the bacterial strains were precultured on tryptic soy agar (tsa) for 2 d at 24 °c. plates containing fungus alone served as controls. petri dishes were maintained at 25 °c until radial growth in the control reached the border of the plate. antibiosis were scored as ' + ' in the presence of an inhibition zone or as ' ' in the absence. the experiment was replicated twice. results population of endophytic bacteria. the population densities of indigenous endophytic bacteria in tomato roots between the plants varied. the lowest -1 endophytic population was 1.8 (in log scale) cfu g 10 fresh root after surface sterilization with 3% naocl -1 and 1.0 (in log scale) cfu g fresh root after surface 10 sterilization with 6% naocl. the highest endophytic -1 populations were 4.4 (in log scale) cfu g fresh root 10 after surface sterilization with 3% naocl and 3.0 (in -1 log scale) cfu g fresh root surface sterilization with 10 6% naocl (table 1). spectrum of endophytic bacteria from tomato roots. a broad range of bacterial genera and species were recovered from tomato roots in west java. in total, 564 isolates of bacteria were identified by fame-gc and midi system. fatty acid profile is likely to become more widely used and will lead to easier identification through comparison to the literature as well as reference strains (sasser 1990). based on the similarity index (si), isolates were identified at the species level for si > 0.4, at the genus level for si 0.2-0.4, and remained unidentified for si < 0.2. fifty one bacterial species comprising 32 genera were found in tomato roots from west java, indonesia. the most abundant endophytic bacterial species were b. megaterium (14.31%) followed by p. putida (12.44%) and serratia marcescens (10.85%) (table 2). in vitro antagonism of endophytic bacteria towards fungal pathogens. a total of 181 strains of endophytic bacteria were studied for their in vitro antagonistic activity towards r. solani, f. oxysporum subsp. radicis-lycopersici, and f. oxysporum subsp. lycopersici on pda. fourteen strains (7.7 %) showed antagonism against r. solani, nine strains (5.0 %) against f. oxysporum subsp. radicis-lycpersici, and seven strains (3.9 %) against f. oxysporum subsp. lycopersici (table 3). the level of antagonism expressed as size of the inhibition zone varied from weak (<1 cm) to medium (1 to 2 cm) and strong (>2cm) (rocha et al. 2009). isolates of endophytic bacteria with pronounced antagonism towards r. solani included strains of bacillus megaterium, burkholderia cepacia, comamonas oxydovorans, enterobacter intermedius, pseudomonas chlororaphis, p. mendocina, p. putida, p. stutzeri, p. savastanoi, p. syringae, and s. marcescens. whereas, bacterial species with antagonism towards f. oxysporum subsp. radicis-lycopersici and f. oxysporum subsp. lycopersici included bacillus megaterium, burkholderia cepacia, p. chlororaphis, p. putida, and s. marcescens. in this dual culture assay, of 181 endophytic bacteria strains could inhibit r. solani, f. oxysporum subsp. radicis-lycopersici, and f. oxysporum subsp. lycopersici in varying degrees (fig 1). the inhibitory rates of the total bacterial strains were less than 10% (table 3). 150 munif et al. microbiol indones x-100 for 90 min and with 75% ethanol for 5 min, followed by 0.5% naocl in 0.05 triton x-100 for 60 min. sturz et al. (1997) isolated endophytic bacteria from red clover and potato. the root tissue of red clover and potato tuber were rinsed with 95% ethanol, then surface sterilized with 5% naocl for 3 min, followed by 2% solution of hydrogen peroxidase for 3 min, and then three washes with sterile distilled water. well defined isolation procedures for recovering discussions sodium hypochlorite is commonly used as a surface disinfectant for recovering bacterial endophytes, mainly because it is non-toxic and easy to handle. the concentration of naocl varied between 0.5 and 5% and the incubation time between 3 and 90 min. for example, misaghi and donndelinger (1990) treated cotton plants with 1.0% naocl in 0.05 triton volume 6, 2012 microbiol indones 151 table 1 population density of endophytic bacteria isolated from tomato from west java, indonesia, following surfacesterilization with sodium hypochlorite (naocl) sample origin of samples 3 % naocl 6 % naocl sterility a) check log cfu/g a) fresh root bacterial c) isolates sterility a) check log cfu bacterial t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 t13 t14 t15 t16 t17 t18 t19 t20 bogor bogor bogor bogor bogor bogor bogor bogor sukabumi sukabumi sukabumi sukabumi sukabumi sukabumi bandung bandung bandung bandung bandung bandung + + + + + + + + 3.3 3.4 2.8 3.6 3.8 3.8 3.5 4 4 4 3.6 2.9 29 30 35 31 30 30 19 32 38 32 24 21 + + + 1.6 (d) n.d 1 1.7 1.5 2.2 1.9 1.2 2.2 1.0 2.9 2.3 n.d n.d n.d 3.0 2.5 10 6 30 30 11 12 16 12 18 30 30 8 total 12 351 17 213 564 b) fresh root c) isolates a) : samples free from surface contamination, + : samples with surface contamination b) means of the population density of two replications c) : bacterial isolates were not taken, root samples were contaminated d) n.d : non detectable; population density below detection limit disinfectants. the total number of plants passing the sterility check was higher for 6% naocl (85%) than for 3% naocl (60%). however, at 6% naocl the total population density was lower than that of 3% naocl (table 1). the relatively lower number of bacterial endophytes at 6% naocl was due to the fact that surface sterilization was too strong and probably killed some endophytic bacteria within the internal root tissue. hallmann et al. (1997) reported that slight variations in the concentration and incubation time of the surface disinfestation process greatly affects the number and type of bacteria isolated. population densities of bacterial endophytes have been shown to be greatest in plant roots. in this experiment, the population densities of endophytic bacteria isolated from tomato roots were in the range -1 from 1.0 to 3.0 (in log scale) cfu g fresh weight 10 endophytic bacteria are a prerequisite for research with endophytes. due to their secluded niche inside plant tissues, endophytic bacteria are most commonly isolated from macerated plant tissue following surface sterilization (hallmann et al. 1997). disinfectants generally used for isolation of endophytic bacteria include sodium hypochlorite (naocl), ethanol, hydrogen peroxide (h o ), mercury chloride (hgcl) or 2 2 a combination of two or more of these disinfectants followed by several washes in sterile distilled water or buffer solutions (he et al. 2009). to reduce surface tension of the solvent and therefore allow the disinfectant to penetrate into micro niches on the root surface, tween 20 or tween 80 can be added (yasuda et al. 2009). in this study, 3% and 6% naocl supplemented with 0.01% tween 20 were used as surface 152 munif et al. microbiol indones table 2 species diversity of endophytic bacteria recovered from tomato roots in west java, indonesia using fame-gc (in percent of 564 isolates) species percent of total isolates 1. acidovorax avinae 2. agrobacterium radiobacter 3. alcaligenes xylosoxydans 4. alteromonas haloplanktis 5. arthrobacter citreus 6. aureobacterium barkeri 7. bacillus cereus 8. bacillus circulans 9. bacillus mycoides 10. bacillus megaterium 11. bacillus pumilus 12. bacillus sphaericus 13. bacillus thuringiensis 14. burkholderia cepacia 15. burkholderia gladioli 16. burkholderia pickettii 17. cellulomonas cellulans 18. chryseobacterium balustinum 19. chryseobacterium meningosepticum 20. chryseobacterium indologens 21. curtobacterium flaccumfaciens 22. comamonas ocidovorans 23. enterobacter tylorae 24. erwinia amylovora 25. gluconobacter oxidans 26. kluyvera cryocrescens 0.46 3.46 1.61 0.23 0.46 1.61 1.61 1.15 0.46 14.31 0.23 2.54 1.85 2.07 1.15 0.46 0.69 1.15 0.23 0.69 0.23 0.23 1.15 0.23 0.69 2.31 27. lactobacillus fermentum 28. macrococcus lutens 29. methylobacterium mesophilicum 30. ochrobactrum anthropi 31. paenibacillus pabuli 32. paracoccus denitrificans 33. peinococcus erythromixa 34. phyllobacterium myrcinacearum 35. phyllobacterium rubiacearum 36. pseudomonas aeruginosa 37. pseudomonas chlororaphis 38. pseudomonas corugata 39. pseudomonas fluorescens 40. pseudomonas flekteus 41. pseudomonas mendocina 42. pseudomonas putida 43. pseudomonas savastanoi 44. rathayibacter rathayi 45. spingobacterium multirorum 46. staphylococcus sciari 47. stenotrophomonas maltophilia 48. serratia marcescens 49. weeksella verosa 50. xanthobacter agilis 51. xanthomonas campestris 52. unidentified 1.84 0.69 0.69 2.76 0.23 0.23 0.23 0.23 0.23 0.46 2.76 0.23 3.70 0.69 2.54 12.44 0.23 0.23 0.23 0.23 1.15 10.85 0.23 1.85 0.23 13.86 percent of total isolates speciesno no indigenous bacterial endophytes found for roots and stems from cotton and sweet corn ranging between 4.0 -1 to 6.0 (in log scale) cfu g fresh weight. similar 10 results for the population densities of bacterial (when surface sterilized with 6% naocl) and from 3.0 -1 to 4.4 (in log scale) cfu g fresh weight (when 10 surface sterilized with 3% naocl). mcinroy and kloepper (1995) reported common population sizes of volume 6, 2012 microbiol indones 153 species number of strains tested number of strains with towards antibiosis activity rs 1) forl 2) fol 3) agroba cterium radiobacter 4 0 4) 0 0 aureobacterium esteroaromaticum 2 0 0 0 bacillus cereus 3 0 0 0 bacillus circulans 2 0 0 0 bacillus megaterium 28 3 3 3 bacillus sphaericus 3 0 0 0 bacillus thuringiensis 3 0 0 0 burkholderia cepacia 3 2 1 0 comamonas oxidovorans 3 1 0 0 comamonas testoteroni 2 0 0 0 chryseobacterium meningsepticum 2 0 0 0 chryseobacterium indologens 4 0 0 0 curtobacterium flaccumfaciens 2 0 0 0 chryseobacterium balustinum 6 0 0 0 enterobacter tylorae 2 0 0 0 gluconobacter oxidans 3 0 0 0 kluyvera cryocrescens 7 0 0 0 micrococcus varians 2 0 0 0 ochrobactrum anthropi 4 0 0 0 paracoccus denitrificans 2 0 0 0 phyllobacterium myrcinacearum 1 0 0 0 pseudomonas aeruginosa 3 0 0 0 pseudomonas chlororaphis 9 1 1 1 pseudomonas corrugata 2 0 0 0 pseudomonas fluorescens 8 0 0 0 pseudomonas mendocina 3 1 1 0 pseudomonas putida 27 3 1 2 pseudomonas syringae 2 1 1 0 pseudomonas stutzeri 3 1 1 0 serratia marcescens 14 2 1 1 xanthobacter agilis 4 0 0 0 unidentified 8 0 0 0 total 181 14 11 7 table 3 in vitro antagonism of endophytic bacteria from tomato roots towards rhizoctonia solani, fusarium oxysporum subsp. radicis-lycopersici, and f. oxysporum subsp. lycopersici five days after inoculation on pda media 1) rhizoctonia solani 2) fusarium oxysporum f.sp. radicis0lycopersici 3) fusarium oxysporum f.sp. lycopersici 4) 0: no antibiosis effect on pda medium endophytes were obtained from different plant roots including alfalfa, citrus twigs, sugar beet, cotton, grapevine, pine seedlings, red clover and potato of 2.0 -1 to 6.0 (in log scale) cfu g fresh root (misaghi and 10 donndelinger 1990; bell et al. 1995; hallmann et al. 1997; sturz et al. 1997). population densities of bacterial endophytes differ depending on the types of plant tissue studied and seem to be highest in the root and lower in the stem and decreases acropetally. the root is thought to be the preferred site of bacterial entrance into the plant, which would explain the high bacterial numbers in the root at early growth stages. the root system seems to provide a more buffered habitat with regards to water availability and temperature changes ( et al. 2004; he et al. 2009; sarr et al. 2010). the present experiments showed that tomato plants were found to host a diverse spectrum of endophytic bacteria. the spectrum of endophytic bacteria isolated was similar to those found for other locations and/or other host plants. mcinroy and sessitsch 154 munif et al. microbiol indones kloepper (1995) reported 46 bacterial species in 31 genera in roots and stems of sweet corn and 45 species in 32 genera in roots and stems of cotton. in grapevine 24 bacterial species in 13 genera were found (bell et al. 1995), in clover roots 31 species in 14 genera (sturz et al. 1997), in cotton roots 43 bacterial species of 28 genera and in potato tubers 24 bacterial species of 13 genera (hallmann et al. 1997). it should be mentioned that, these are only the culturable bacteria. almost nothing is known about the non-culturable bacteria inside root tissue. the diversity of endophytic bacteria reported in this study has many similarities with the endophytic spectrum detected in other crops. some bacterial genera, namely, agrobacterium, arthrobacter, bacillus, burkholderia, chryseobacterium, enterobacter, pseudomonas, and stenotrophomonas have been commonly recovered from roots of cucumber, sugar beet, corn, and lemon (mcinroy and kloepper 1995; mahaffee and kloepper 1997; hallmann et al. 1997). factors like crop rotation, organic matter, temperature, c a d b fig 1 in vitro antagonisms of 3 endophytic bacteria isolated from tomato root towards rhizoctonia solani. the figures show growth of r. solani on pda alone (a), and in the presence of endophytic bacteria showing various degrees of inhibition: strong inhibition by pseudomonas spp. mt004 (b), moderate inhibition by p. putida mt019 (c), p. savastanoi f.sp. fraxinus (d). images were taken 5 d after fungal inoculation. rainfall, soil physical, and chemical properties affect the bacterial spectrum and these factors were the source of endophytic colonizers in these tests (mahaffee and kloepper 1997; sessitsch et al. 2004). in the present study, in vitro antibiosis was tested on potato dextrose agar (pda). the choice of the growth medium can affect the degree of inhibition as demonstrated in these experiments (yang et al. 2011). strains of endophytic bacteria were screened for in vitro inhibition of r. solani, f. oxysporum f.sp. radicislycopersici, and f. oxysporum subsp. lycopersici on pda. testing in vitro antagonism of bacteria against fungal pathogens provides a rapid method to pre-select biological control candidates based on antibiosis. results showed that some bacteria inhibited the growth in vitro of r. solani (8.3%), f. oxysporum subsp. radicis-lycopersici (6.6%), and f. oxysporum subsp. lycopersici (3.9%). yang et al. (2011) reported that the inhibitory rates of endophytic bacteria in dual test assay against botrytis cinerea were varied and less than 40%. this result might be affected by medium used for antibiosis in vitro assay. fuchs and defago (1990) studied the in vitro antagonism of bacteria towards fungal disease phomopsis sclerotioides on pda, malt agar (ma), and king's b medium agar (kba). some bacteria, especially pseudomonas fluorescent, inhibited fungal pathogens on kba, but not on pda or ma and vice versa, whereas other bacteria showed antibiosis on all three media. in conclusion, endophytic bacteria can be isolated with surface-sterilized method. more than fifty one species of endophytic bacteria were recovered from tomato roots and three most abundant species, b. megaterium, p. putida, and s. marcescens, were frequently found in association with tomato roots from west java, indonesia and showed antibiosis activity against fungal diseases. the close relationship between endophytic bacteria and their hosts make them ideal candidates for biological control and plant growth promotion. references backman pa, sikora ra. 2008. endophytes: an emerging tool for biological control. biol control. 46(6):1-3. doi:10.1016/j.biocontrol.2008.03.009. buchenauer h. 1998. biological control of soil-borne disease by rhizobacteria. j plant dis protec. 105 (4):329-348. bell cr, dickie ga, harvey wlg, chan jwyf. 1995. endophytic bacteria in grapevine. can j microbiol. 41(1):46-53. doi: 10.1139/m95-006. fischer pj, petrini o, scott hml. 1992. the distribution of some fungal and bacterial endophytes in maize (zea mays l.) new phytol. 122(2): 299-309. fuchs j, defago g. 1990. protection of cucumber plant against black root rot caused by phomopsis sclerotioides with rhizobacteria. proc 2th international workshop on plant growth promoting rhizobacteria, interlaken, switzerland. p: 57-92. hallmann j, quadt-hallmann a, mahaffee wf, kloepper jw. 1997. bacterial endophytes in agricultural crops. can j microbiol. 43(10): 895-914. doi: 10.1139/m97131. he ri, wang gp, liu xh, zhang cl, lin fc. 2009. antagonistic bioactivity of an endophytic bacterium isolated from epimidium brevicornu maxim. afr j biotechnol. 8(2):191-195. mahaffee wf, kloepper jw. 1997. temporal changes in the bacterial communities of soil, rhizosphere, and endorhiza associated with field-grown cucumber (cucumis sativus l.). microb ecol. 34(3): 210-223. doi: 10.1007/s002489900050. mclnroy ja, kloepper jw. 1995. survey of indigenous bacterial endophytes from cotton and sweet corn. plant soil. 173(2): 337-342. munif a, hallmann j, sikora ra. 2000. evaluation of the biocontrol activity of endophytic bacteria from tomato against meloidogyne incognita. med fac landbouww univ gent. 65(2b): 471-480. misaghi i, donndelinger lr. 1990. endophytic bacteria in symptom-free catton plants. phytopathol. 80:808-811. quadt-hallmann a, kloepper jw. 1996. immunological detection and localization of the cotton endophyte enterobacter asburiae jm22 in different plant species. can j microbiol. 42:1144-1154. quadt-hallmann a, benhamou n, kloepper jw. 1997. bacterial endophytes in cotton: mechanisms of entering the plant. can j microbiol. 43(6): 577-582. rocha r, da luz de, engels c, pileggi sav, filho dsj, matiello rr, pileggi m. 2009. selection of endophytic fungi from comfrey (symphytum officinale l.) for in vitro biological control of the phytopatogen sclerotia sclerotium (lib.). brazilian j microbiol. 40(1): 73-78. doi: 10.1590/s1517-83822009000100011. sarr ps, yamakawa t, asatsuma s, fujimoto s, sakai m. 2010. investigation of endophytic and symbiotic features of rasltonia sp. tsc1 isolated from cowpea nodules. afr j microbiol res. 4(19): 1959-1963. sasser m. 1990. identification of bacteria through fatty acid analysis. in: klement z, rudolp k, sand s, edistors. methods in phytobacteriology. budapest: akademiai kiado. p 199-204. sturz av, christie br, matheson, bg, nowak j. 1997. biodiversity of endophytic bacteria potential rol which colonize red clover nodules, root stems and foliage and their influence on host growth. biol fertil soil. 25: 13-19. . sessitsch a, reiter b, berg g. 2004. endophytic bacterial communities of field-grown potato plants and their plant growth-promoting abilities. can j microbiol. 50(4): 239-249. doi: 10.1139/w03-118. volume 6, 2012 microbiol indones 155 van buren am, andre c, ishimaru ca. 1993. biological control of the ring rot pathogen by endophytic bacteria isolated from potato. phytopathol. 83:1406. vetrivelkalai p, sivakumar m, jonathan fi. 2010. biocontrol potential of endophytic bacteria on meloidogyne incognita and its effect on plant growth in bhendi. j biopest. 3(2): 452-457. yang cj, zhang xg, shi gy, zhao hy, chen l, tao k, hou tp. 2011. isolation and identification of endophytic bacterium w4 against tomato botrytis cinerea and antagonistic activity stability. afr j microbiol res. 5(2): 131-136. yasuda m, isawa t, shinozaki s, minamisawa k, nakashita h. 2009. effects of colonization of bacterial endophyte, azospirilum sp. b510, on disease resistance in rice. biosci biotechnol biochem. 73 (12): 25952599. doi: 10.1271/bbb.90402. 156 munif et al. microbiol indones 1: 148 2: 149 3: 150 4: 151 5: 152 6: 153 7: 154 8: 155 9: 156 6 desy_406(20201515).pmd volume 3, number1, april 2009 p 27-32 issn 1978-3477 *corresponding author, phone: +62-22-2502103, fax: +62-22-2504154, e-mail: dessy@chem.itb.ac.id the b’x region of yeast protein disulfide isomerase is not essential for saccharomyces cerevisiae viability at 30 °c purkan1,2, lalu rudyat telly savalas1,3, muliawati sindumarta1, and dessy natalia1* 1biochemistry research division, faculty of mathematics and natural sciences, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia, 2chemistry department, faculty of sciences and technology, universitas airlangga, jalan mulyorejo, surabaya 60115, indonesia, 3chemistry department, faculty of science education, universitas mataram, jalan majapahit 62, mataram 85125, indonesia protein disulfide isomerase (pdi) catalyzes thiol oxidation, reduction and isomerization of disulphide bond of cell surface and secreted proteins. yeast pdi1 consists of two catalytic domains (a and a’) which are separated by two non-catalytic domains (b and b’), and a x region linked the b’ and a domains. the b’ domain is important for the non-covalent binding of partially folded protein. to understand the contribution of b’ domain and x-linker of yeast pdi1 we have deleted the b’x and investigated its functional role in vitro and in vivo. yeast pdi1 without b’x region retained only 50% activity and became more sensitive toward proteinase k. interestingly, yeasts containing full length pdi1 and pdi1∆b’x showed approximately the same growth rate. however, the yeast pdi1∆b’x mutant growth impaired severely at 37 °c compared to that of the full length pdi1. our results suggested that the a-b-a’-c domains of pdi seems to be sufficient to support the growth of yeast cells in normal condition, but the b’x region might be essential in assisting refolding of highly accumulated unfolded protein at high temperature (37 °c). key words: b’domain, protein disulfide isomerase, saccharomyces cerevisiae, x-linker formation of correct disulfide bonds is essential for proper folding of the majority of cell surface and secreted proteins in eukaryotic cells. protein disulfide isomerase (pdi), which is a resident of the endoplasmic reticulum (er), catalyzes the formation, reduction and isomerization of disulfide bonds. it has also a chaperone role which mediates the folding and assembly of nascent peptides into mature proteins in the cells (wang and tsou 2003). the chaperone activity is independent of its catalytic activity as it has been demonstrated that pdi improves secretion of redox-inactive β-glucosidase (powers and robinson 2007). in addition, pdi is found to form a complex with other protein molecules, namely prolyl-4 hydroxylase and microsomal triglyceride transfer protein (pihlajaniemi et al. 1987; wetterau et al. 1991). protein disulfide isomerase is composed of four thioredoxin-like domains, a,b, b’ and a’, folowed by an acidic c-terminal extension (named as c) harboring er retention signal (freedman et al. 1994). the a and a’ domains each contain active site sequence cxxc which is required for disulphide bond formation. the b’ domain of human pdi contributes significantly to protein substrate interactions (klappa et al. 2000; denisov et al. 2009). in saccharomyces cerevisiae pdi is encoded by an essential gene pdi1 (farquhar et al. 1991). the three dimensional structure of yeast pdi1 has a twisted u-shape in which the a and a’ domain facing each other on the end of u while the b and b’ domain forming the base (tian et al. 2006). the b’ and a’ domains is connected by 17 residues which is referred as x-linker. the crystal structure shows that the b and b’ domains have hydrophobic patch which together with hydrophobic areas surrounding the active sites, form a continuous hydrophobic surfaces across the a, b’ and a’ domain (tian et al. 2006). these hydrophobic residues are involved in the interaction of pdi with its substrates. in this paper we described that yeast pdi1 without b’x region still maintains half of its insulin reductase activity, although it becomes more sensitive toward proteinase k compared to that of full length pdi1. furthermore, we demonstrated that pdi1∆b’x can support yeast viability. materials and methods materials. plasmid prs314-pdi1 (a generous gift from prof. w. j. lennarz, department of biochemistry and cell biology, state university of new york at stony brook, usa) containing pdi1 was used as a template for amplification of a dna fragment encoding for pdi1 without b’x. escherichia coli dh5 α was used for plasmid propagation and bl21 (de3) (ara, leu7967, lacx74, phoa, pvuii, phor, malf3, f’[lac, laciq), pro], trxb::kan(de3)) was used for expression host of pdi proteins. yeast s. cerevisiae strain 2736 (mata ade2-1 can1-100 ura3-1 leu2-3,112 trp1-1 his3-11,15 pdi1::his3) containing pct37-pdi1 [ura3] (obtained from prof. t. h. steven, institute of molecular biology, university of oregon, usa) and pukc639 (kindly provided by prof. m. f. tuite, school of biosciences, university of kent, united kingdom) were used for plasmid shuffling in yeast cell. plasmid pt7.7 (novagen, madison, wi) was used for expression of a pdi∆b’x mutant in e. coli. plasmid pgem®-t, vent and taq dna polymerases were obtained from promega. restriction enzymes and t4 dna ligase were obtained from new england biolabs, inc. all synthetic dna oligomers were synthesized by pro-oligo singapore. yeast cells were grown in minimal medium [0.7% (w/v) yeast nitrogen base (ynb) without amino acid, 2% (w/v) glucose or 2% (w/v) galactose] supplemented with approriate amino acids and bases, and yepd/gal yeast extract [(1% (w/v), (w/v) bacto peptone 1%, (w/v) glucose 2%, (w/v) galactose 2%)] media. escherichia coli was grown in luria-bertani (lb) (sambrook et al. 1989). construction of pdi1∆∆∆∆∆b’x mutant. a yeast pdi lacking b’x region coded by nucleotides at position 739-1149 in pdi1 gene was constructed through 3 steps of pcr (fig 1) using microbiol indones28 purkan et al. prs314-pdi1 as a template. oligonucleotide primers for pcr were designed based on published sequences of pdi1 gene (accession number 850314). primers fmp1(5’-act ttggtgaaatcgacggtaagaaccatgacgaa-3’) and rd2 (5’-gaggaggatccttacaattcatcgtgaatgg3’) were used to amplify a dna fragment corresponding to coding region 1150-1569 of pdi1. the fmp1 contained 20 nucleotides corresponding to the coding region 719-738 of pdi1, while rd2 primer had a bamh1 restriction site. the resulted dna fragment from the first round pcr as a reverse mega primer together with primer fd1 (5’-gag gac ata tga agt ttt ctg ctg gtg-3’) and a template of prs314-pdi1 were used in the second pcr. the third pcr was carried out using primers forward fd1 and reverse rd2 (5’-gaggaggatccttacaattcatcgtgaatgg-3’), and the second pcr results as a template. all pcr products were analyzed by agarose gel electrophoresis and purified by gfx purification kit. the resulted pdi1∆b’x dna fragment was ligated with pgem®-t vector and the the ligation product was transformed into e. coli dh5α using cacl 2 method (sambrook et al. 1989). the pgem®-t-pdi1∆b’x was digested with ndei and bamhi restriction enzymes and the resulted 1400 bp pdi1∆b’x fragment was isolated and then ligated with an expression vector previously digested with ndei and bamhi. the resulted pt7-7-pdi1∆b’x was confirmed by restriction analysis. the pt7-7-pdi1∆b’x was subjected to dna sequence analysis. construction of pdi1∆∆∆∆∆b’x in yeast expression plasmid. plasmid pt7.7-pdi1∆b’x as a template, and primers pdif1 (gctagcatgaagttttctgctggtgc) and pdir1 (gctagcttacaattcatcgtgaatggc) were used to amplify pdi1∆b’x. the pcr program was 94 °c for 4 min, 25 cycles of 1 min at 94 °c, 1 min at 55 °c, 2 min at 72 °c and 5 min at 72 °c. nhei sites were included in both forward and reverse primers. the pcr products were gel purified, subcloned into pgem®-t vector. a nhei fragment of pdi1∆b’x taken out from pgem®-t-pdi1∆b’x/nhei was then inserted into pukc639 previously digested with similar restriction enzyme. the resulted pukc639-pdi1∆b’x was sequenced to confirm the presence of the deletion. transformation of yeast. the resulted pukc639pdi1∆b’x was used to transform s. cerevisiae 2736 using a modified lithium acetate method (ito et al. 1983). the transformed cells were plated onto minimal medium supplemented with 0.001% (w/v) adenine and 0.002% (w/v) tryptophan and grown at 30 °c for 4-7 d. plasmid-shuffling. the yeast transformants carrying pukc639-pdi1∆b’x were grown in solid minimal medium containing 2% (w/v) galactose, 1 mg ml-1 5’fluoroorotic acid (5’foa), 0.002% (w/v) tryptophan, 0.001% (w/v) adenine and 0.001% (w/v) uracil at 30 °c for 4-7 d. the yeast cells designated as 2736d were then transferred into a minimal medium supplemented with similar amino acid and bases. protein expressions. escherichia coli strain bl21 (de3) carrying pdi1 or pdi1∆b’x genes on plasmid pt7-7 were grown in 10 ml lb media containing 100 g ml-1 ampicillin at 37 °c for 16 h. one ml of overnight culture was transferred into a fresh 50 ml lb/ampicillin media and grown until the culture od 600 reached 0.5. isopropyl-α-thiogalactopyranoside (iptg) was added to the culture to a final concentration of 0.5 mm and the culture was grown for further 2.5 h. cell cultures were precipitated by centrifugation at 2100 x g for 30 min at 4 °c. the pellet cells were resuspended in lysis buffer (50 mm tris-hcl ph 7.5, 0.2 mm nacl dan 10% glycerol) containing 1 mg ml-1 lysozyme. cells were lysed using 5 ’ 3 ’ 5 ’ 5 ’ 5 ’ 5 ’ 5 ’ 5 ’ 5 ’ 5 ’ 5 ’ 5 ’ 3 ’ 3 ’ 3 ’ 3 ’ 3 ’ 3 ’ 3 ’ 3 ’ 3 ’ a b b’ a’ c 1 1 190 1 569 rd2 pdi1 fmp1 1st pcr 719-738 megaprimer fd1 2nd pcr3 ’ fd1 rd2 3rd pcr (a) (b) (c) fig 1 strategy of deletion of dna fragment encoding b’x region of yeast pdi1 using three steps pcr. the first pcr was performed to produce a 447 bp dna fragment encoding a’c domain (a). the resulted fragment was used as a mega primer together with fd1 primer in the second pcr to produce a pdi1äb’x mutant. a low concentration of pdi1äb’x mutant (b) was obtained at this step. the third pcr was conducted to increase the concentration of pdi1äb’x mutant using fd1 and rd1 primers. this step gave a high concentration of pdi1äb’x mutant (c). microbiol indones 29volume 3, 2009 sonication with 10x 1 min burst at maximum frequency and cooled on ice for 1 min after each 1 min burst. the lysed cells was then centrifuged at 9500 x g for 30 min at 4 °c. assay of pdi protein activities. the ability of pdi1 to reduce disulphide bond was determined as described by sutter et al. (1994). the assay mixture contained 1.67 mm insulin in 100 mm potassium phosphate buffer, ph 7.0, 20 mm ethylene diamine tetraacetic acid, crude bacterial cell extract pdi, 10 mm dithiothreitol. the increase of od at 650 nm from precipitation of insulin b chain was measured. one unit activity was defined as an amount of pdi to reduce disulfide bonds of insulin in order to produce ∆α650 of 0.001 per min at experimental condition. stability test of mutant pdi1∆∆∆∆∆b’x. stability of mutant pdi toward proteinase k was conducted based on method described by klappa et al. (2000). pdi was mixed with various concentration of proteinase k (1; 3; 10 mg ml-1) in pbs buffer (nacl 140 mm, kcl 2.7 mm, na 2 hpo 4 10 mm, kh 2 po 4 1.8 mm, ph 8) for 30 min at 4 °c. the reaction was stopped with 5 mm phenyl methyl sulphonyl fluride (pmsf) for 5 min. sds-polyacrylamide gel electrophoresis and westernblotting. protein electrophoresis was performed as described by laemmli (1970). protein blotting was done by using semi-dry blotter (biorad). protein in the gel was transferred onto nitrocellulose membrane (hybond ecl). the membrane was incubated with an antibody anti pdi and subsequently with the peroxidase-conjugated anti rabbit ig antibody. the signal was developed using ecl detection reagent (amersham). results yeast pdi1∆b’x has been generated by three steps pcr. yeast pdi1∆b’x mutant was constructed by deletion of a 7391146 dna region of pdi1 corresponding to ser247-gly383 using three steps of pcr (fig 2b). the first pcr was performed to amplify the dna fragment at nucleotide position of 1150 to 1569 corresponding to the a’c domains. the resulted dna fragment with the size of 447 bp also contained 20 oligonucleotides of b domain (fig 3, lane 3). the oligonucleotide of a’c domain was used as a reverse mega primer in combination with fd1 primer to generate a low yield of 1190 bp dna fragment corresponding to aba’c domain in the second pcr (fig 3, lane 4). to increase the amount of the 1190 bp pdi1∆b’x dna fragment, a third pcr using fd1 primer and rd2 primer was performed. the resulted dna fragment (fig 3, lane 5) was first ligated with pgem®-t plasmid and then subcloned into pt7.7 expression vector. the sequence of the 1190 bp was confirmed by dideoxynucleotide sequence analysis and found to be free of mutation. a full length pdi1 generated by pcr was also subcloned into pt7-7 plasmid. mutant pdi1∆b’x produced as a soluble protein in e. coli. the full length pdi1/ pdi1∆b’x gene is placed under the control of φ10 promoter which requires t7 rna polymerase to activate the promoter. the host e. coli strain used had already been transfected with phage de3 containing the t7 rna polymerase gene. the t7 rna polymerase was under the control of of inducible lacuv5 promoter and thus addition b yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro 5 2 2 3 8 5 5 2 5 yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs .pro yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro yeast pdil sc.pro yeast pdil-db’.pro pdipacrhs.pro 6 1 6 1 7 1 1 3 0 1 3 0 1 4 2 2 0 1 2 0 1 2 0 3 2 5 6 2 4 6 2 7 4 3 2 4 2 4 6 3 3 7 3 9 4 2 5 7 4 0 6 4 6 5 3 2 8 4 7 6 a a b b’ a’ c x fig 2 primary structure of yeast pdi, pdi1äb’x and human pancreas pdi. a domain organization of pdi based on the yeast three dimensional structure (tian et al. 2006), b multiple sequences alignment of saccharomyces cerevisiae pdi1 (yeast pdi1sc.pro), pdi1äb’x (yeast pdi1 db’.pro) and human pancreas pdi (pdipancrhs.pro). microbiol indones30 purkan et al. of iptg induced the lacuv5 promoter to produce t7 rna polymerase thereby allowing transcription of the pdi1 gene. proteins were isolated from the e. coli cultures as described in material and methods. the full length pdi1 appeared as a protein with molecular weight of approximately 60 kda on sds-page while the molecular weight of pdi1∆b’x was 45 kda (fig 4). these are in agreement with the predicted molecular weight from deduced amino acids in which full length pdi1 consists of 522 amino acid residues while pdi1∆b’x contains 386 amino acid residues. mutant pdi1∆∆∆∆∆b’x has lower reductase activity and is proteinase k sensitive. to determine whether deletion of b’x region affected pdi activity, the ability of both pdi1∆b’x mutant and full length pdi1 to catalize the reductive cleavage of insulin was determined. the in vitro assay for pdi activity in the crude bacterial cell extract showed that specific activity of the pdi1∆b’x was 1.4 x 103 u mg-1 which is approximately 50% of that of the full length pdi1 (2.8 x 103 u mg-1). to know the stability of the yeast pdi lacking of b’x toward proteinase k, the pdi1∆b’x and full length pdi were treated with proteinase k. proteolysis of crude bacterial cell extracts was carried out using different concentration of proteinase k. western blot analysis using anti yeast pdi antibody showed that a significant amount of pdi1∆b’x were digested upon the increase of proteinase k concentration. there is hardly pdi1∆b’x left after treatment with 10 mg ml-1 of proteinase k (fig 5, lane 8). the full length pdi1 remains stable upon proteinase k treatment (fig 5). a lower band observed in the full length pdi could be the degradation product during protein preparation. this results suggest that pdi1∆b’x mutant is more susceptible to proteinase k degradation. mutant pdi1∆∆∆∆∆b’x can support yeast viability. since pdi1 is an essential gene, a plasmid-shuffle procedure to observe the function of mutant pdi1∆b’x on supporting yeast viability was conducted. the recombinant plasmid pukc639-gal1pdi1∆b’x containing a leu2 selectable marker was introduced into a yeast strain 2736. the strain 2736 containing pdi1::his3 null mutation carried a plasmid-borne gal1-pdi1 with a ura3 marker (tachibana and steven 1992). after transformation, the cells were grown in a medium containing 5 foa which allow cells to lose its ura based plasmid-containing full length pdi1. cells grown in the foa medium indicated that the newly introduced pdi∆b’x mutant is able to complement the chromosomal pdi1 null mutation. the growth of pdi1∆∆∆∆∆b’x yeast is almost the same as the full length pdi1 yeast. the effect of b’x deletion on the growth rate of yeast cell was investigated by growing the yeast 2736 carrying full length pdi1 and 2736d containing pdi1∆b’x in rich media containing galactose at 30 °c. the growth rate of yeast carrying full length pdi1 was 0.13 u od 600 per h, while 2736d had a growth rate of 0.12 u od 600 per h. the results show that pdi1∆b’x and full length pdi1 can support the yeast growth at nearly the same growth rate 30 °c. the pdi1∆∆∆∆∆b’x yeast impaired severely at high temperature. protein disulphide isomerase is one of the main cellular chaperones in the er. under temperature stress, the amount of unfolded proteins accumulated in the er will increase. we further investigated the effect of b’x deletion on the yeast growth at temperature stress (37 °c). as shown in fig 6 both 2736 and 2736d grew well at 30 °c. however, reduced growth were observed for both yeast strains at 37 °c in which 2736d growth impaired severely. the pdi1∆∆∆∆∆b’x yeast shows no cell wall defect. to study whether the b’x region is responsible in the folding of cell wall forming protein, the pdi1∆b’x mutant was grown in a medium containing calcofluor white. defect on the cell wall forming protein leads to calcofluor sensitive phenotype. the pdi1∆b’x mutant seems to grow in a similar manner as the full length pdi1 (fig 7) which indicates that there is no defect on the synthesis of cell wall in pdi1∆b’x. discussion protein disulphide isomerase as a key player in the formation of correct disulphide bond in protein was discovered 40 years ago by anfinsen and co workers (goldbeger et al. 1964). it is a member of thioredoxin superfamily which consists of five consecutive domains a, b, b’ a’ and c (fig 2a). there have been many studies reported on the contribution of each domain or domain combinations in pdi activities both as an enzyme or as a chaperone. we have constructed a yeast pdi without b’ domain and x-linker in which the nterminals of 19 amino acid residues of the b’ domain and 7 amino acid residues of the a’ domain were still included (fig 2b). the pdi1∆b’x was expressed as a soluble protein in e. coli and retained only 50% of its activity in the reductive cleavage of insulin b chain. this represented that b’ domain is required to pdi activity. however, other domains is also capable of interacting with insulin. other study has shown that deletion of a,b and b’a’c domains of bovine pdi resulted in a decrease of insulin reductase activity of 94% and 78%, respectively (sun et al. 2000). since pdi1∆b’x still has two a and a’ active site domains which are responsible in the reduction of disulphide bond of insulin, hence it possesess higher activity compared to the other two bovine pdi variants. several researchs have shown that multi domain fragments had enhanced catalytic activities compared with individual a or a’ domains, that the b’ domain had a particularly important role in this enhancement (darby et al. 1998; klappa et al. 1998; tian et al. 2006). to analyze whether there is a correlation between stability as defined by protease-resistance, crude bacterial cell extract containing pdi1∆b’x mutants was treated with proteinase k. it appeared that pdi1∆b’x is protease sensitive while the full length pdi1 was only slightly affected at high proteinase k concentration (fig 4). the stability of full length human pdi toward proteinase k had also been reported (klappa et al. 2000). from the crystal structure of pdi1, it was found that the two flexible catalytic a and a’ domains are attached to more rigid b’ and b domains (tian et al. 2006), hence deletion of b’x region will decrease conformational stability. furthermore, we had investigated how the function of mutant yeast pdi lacking of b’x to support yeast viability. plasmid shuffling experiment showed that pdi1∆b’x can rescue the pdi1::his3 null mutation in 2736 yeast strain. lamantia and lennarz (1993) have demonstrated that pdi1 mutant containing a, b and b’ domains can support yeast microbiol indones 31volume 3, 2009 growth, while the ab domain has lost its essential function in yeast viability. we speculate that in the pdi1∆b’x, the abac domains might adopt the modular u shape structure which presumably able to interact with polypeptide substrate to carry out its activity in vivo. crystal structure of pdi1∆b’x would be able to reveal the three dimensional structure of the mutant. protein disulfide isomerase can act both as a catalyst and also as a chaperone. deletion of the b’ domain significantly slows down the refolding rate of misfolded rnase (darby et 23 130 9 416 6 557 4 361 2 322 2 027 bp 1 2 3 4 5a fig 3 pcr product: lane 1, dna λ/hind iii; lane 2, full length pdi1 (1569 bp); lane 3, c’-a fragment (447 bp); lane 4 and 5, pdi1äb’x (1190 bp). 2 0 0 1 1 6 9 7 6 6 4 5 3 1 2 1 . 5 1 4 . 5 kda 1 2 3 4 fig 4 sds-page analysis of pdi1äb’x and full length pdi1. lane 1, protein marker; lane 2, pdi1äb’x; lane 3, full length pdi1. pdi1äb’xfull length pdi1 1 2 3 4 5 6 7 8 fig 5 western blot analysis of full length pdi1 and pdi1äb’ after treatment with proteinase k. lane 1 and 5, 0 µg ml-1; lane 2 and 6, 1 µg ml-1; lane 3 and 7, 3 µg ml-1; lane 4 and 8, 10 µg ml-1. fig 7 the effect of calcofluor on yeast growth. the parent 2736 and mutant pdiäb’x 2736d yeasts were first grown in minimal media at 30 °c for 2 d. the cultures were then diluted to obtain od 600 of 1.5. the growth rates of yeasts strains were evaluated by spotting 5 ìl of serial dilutions of yeast cells on yepgal containing calcofluor ( 5 0 ì g ml-1). the cells were incubated at 30 °c for 4 d. 2 7 3 6 2736d (pdi-1äb’x) dilution 10-1 10-2 10-3 10-4 saccharomyces cerevisiae temperature saccharomyces cerevisiae dilution 10-1 10-2 10-3 10-4 37 °c 30 °c 2 7 3 6 2736d (pdi1äb’x) 2 7 3 6 2736d (pdi1äb’x) fig 6 the effect of temperature stress on yeast growth. the parent 2736 and mutant pdi1äb’x 2736d yeasts were first grown in minimal media at 30 °c for 2 d. the cultures were then diluted to obtain od 600 of 1.5. the yeasts were spotted in serial dilutions from 10-1 to 10-4 on yepgal at 30 and 37 °c. growth was scored after 4 d incubation. al.1998; tian et al. 2006 ). furthermore, it has also been demonstrated that the b’ domain is generally important for refolding of proteins, whereas the b domain might contribute to the refolding rate in selected cases (tian et al. 2006). our data showed that the growth of pdi1∆b’x yeast strain producing the a,b,a’,c protein was reduced dramatically at non permissive temperature. at high temperature the rate of protein unfolding increseases, hence the chaperone role of pdi becomes predominant. taken together, we propose that the presence of b’x region together with b domain is required in assisting refolding of misfolded protein at non-permisive temperature. acknowledgements this research was funded partially by university research for graduate education from ministry of national education. we gratefully acknowledge p. klappa for his assistance in proteinase k sensitive experiment, references darby nj, penka e, vincentelli r. 1998. the multi-domain structure of protein disulfide isomerase is essential for high catalytic efficiency. j mol biol 276:239-47. denisov ay, maattanen p, dabrowski c, kozlov g, thomas dy, gehring k. 2009. solution structure of the bb’ domains of human protein disulfide isomerase. febs j 276:1440-9. farquhar r, honey n, murant sj, bosier p, schultz l, montgomery d, ellis rw, freedman rb, tuite mf. 1991. protein disulfide isomerase is essential for viability in s. cerevisiae. gene 108:81-9. freedman rb, hirst r, tuite mf. 1994. protein disulfide isomerase building bridges in protein folding. trends bioch sci 19:331-5. goldbeger rf, epstein cj, anfinsen cb. 1964. purification and properties of microsomal enzyme system catalyzing the reactivation of reduced ribonuclease and lysozyme. j biol chem 239:1406-10. ito h, fukuda y, murata k, kimura a. 1983. transformation of intact yeast cells treated with alkali cations. j bacteriol 153:163-8. klappa p, koivunen p, pirneskoski a, karvonen p, ruddock lw, kivirikko ki, freedman rb. 2000. mutations that destabilize the a’ domain of human protein disulfide isomerase indirectly affect peptide binding. j biol chem 18:13213-8. klappa p, ruddock lw, darby nj, freedman rb. 1998. the b’ domain provides the principles peptides-binding sites of protein disulfide microbiol indones32 purkan et al. isomerase but all domains contribute to binding of misfolding protein. embo j 17:927-35. laemli uk. 1970. cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature 227:680-5. lamantia m, lennarz wj. 1993. the essential function of yeast protein disulfide isomerase does not reside in its isomerase activity. cell 74:899-908. pihlajaniemi t, helakoski t, tasanen k, myllyla r, hntala ml, koivu j, kivirikko ki. 1987. molecular cloning of the subunit of human prolyl 4-hydroxylase. this subunit and protein disulfide isomerase are products of the same gene. embo j 6:643-9. powers sl, robinson as. 2007. pdi improves secretion of redoxinactive b-glucosidase. biotechnol prog 23:364-9. sambrook j, fritsch ef, maniatish y. 1989. molecular cloning: a laboratory manual. 2nd ed. new york: cold spring harbor pr. sun xx, yong y, liu hp, chen sm, wang cc. 2000. contributions of protein disulfide isomerase domains to its chaperone activity. biochim biophys acta 1481:45-54. sutter dk, hostens k, vandekerckhove y, fier w. 1994. production of enzymatically active rat protein disulfide isomerase in escherichia coli. gene 141:163-70. tachibana c, steven th. 1992. the yeast eug1 gene encodes an endoplasmic reticulum protein that is functionally related to protein disulfide isomerase. mol cel biol 12:4601-11. tian g, xiang s, noiva r, lennarz wj, schindelin h. 2006. the crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. cell 124:61-73. wang cc, tsou cl. 2003. protein disulfide isomerase is both an enzyme and a chaperone. faseb j 7:1515-7. wetterau jr, combs ka, mclean lr, spinner sn, aggerbeck lp. 1991. protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein. biochemistry 30:9728-35. 4 budiasih_379.pmd volume 3, number 1, april 2009 p 17-22 issn 1978-3477 *corresponding author, phone: +62-21-7560536 ext 130, fax: +62-21-7560536, e-mail: budiasih_solichin@yahoo.com effect of ph, temperature and medium composition on xylanase production by bacillus sp. aq-1 and partial characterization of the crude enzyme budiasih wahyuntari1*, nisa rachmania mubarik2, and siswa setyahadi1 1division of biocatalyst technology, badan pengkajian dan penerapan teknologi, serpong center for sciences and technology-puspiptek, tangerang 15310, indonesia; 2department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia bacillus sp. aq-1 was isolated from household aquarium sediment. the isolate produced extracellular xylanolytic enzymes on xylan containing agar medium. based on morphological, and physiological analysis, the isolate was identified as bacillus sp. aq1. the effect of temperature and ph on isolate growth and xylanase production were observed. the best condition observed for the enzyme production in luria broth supplemented with 0.5% oat spelt xylan medium was at 40 °c ph 7. the maximum enzyme production was 0.23 u ml-1 after 20 h of fermentation. two different medium compositions (a and b) were examined for xylanase production. the maximum growth of the isolate and the xylanase production was better in a medium. replacing oat spelt xylan in medium a with fruitless oil palm bunch in the medium caused the growth slightly slower than that of in the original formula. however, the xylanase production was 3 times higher in fruitless oil palm bunch medium. optimum activity of the crude enzyme was observed at 60 °c and ph 7. each ml of the crude enzyme contained 55.21 u xylanase, 8.12 u amylase and 0.50 u carboxymethylcellulase. key words: xylanase, bacillus sp. aq1, fruitless oil palm bunch xylan is a complex heteropolymer with a homopolymeric backbone chain of 1,4-linked β-d-xylopyranose units. the backbone consists of o-acetyl, α-l-arabinofuranosyl, α-1,2linked glucuronic or 4-o-methylglucuronic substituents (kulkarni et al. 1999). several hydrolytic enzymes are needed to degrade the complex structure of xylan. among these, endo 1,4-β-d-xylanase (ec 3.2.1.8), b-d-xylosidase (ec 3.2.1.37), α-l-arabinofuranosidase (ec 3.2.1.55), α-d-glucuronidase and acetyl xylan esterase (ec3.1.1.6) are the most important enzymes (rani and nand 2001). enzymatic hydrolysis of xylan produces xylooligosaccharides, xylobiose, and xylose (kulkarni et al. 1999). some bacillus spp. have been reported as xylanolytic enzymes producers (kulkarni and rao 1996; sunna et al. 1997a; archana and satyanarayana 1997; samain et al. 1997; dhillon et al. 2000a; and bocchini et al. 2002). another bacterial genus reported to produce xylanolytic enzymes was clostridium spp. (lee et al. 1987; lee and forsberg 1987; sakka et al. 1999; rani and nand 2001). since the last decade xylanolytic enzymes have been studied concerning their properties and utilization in some commodities such as in pulp and paper, feed as well as, foods and beverage industries (beg et al. 2001). in pulp and paper industry, xylanase was used as pretreatment prior to bleaching of pulp and paper to reduce application of chlorine (garg et al. 1996; kulkarni and rao 1996; chen et al. 1997; zheng et al. 2000; pala et al. 2006). in the food and beverage industry xylanolytic enzymes have been used for improving bread quality, clarification of fruit juices, wine and beer (beg et al. 2001; primo-martin et al. 2005). for industrial purposes, seeking a cheap, abundantly available substrate such as wheat straw, sugarcane baggase and rice straw for producing xylanase has been done by some researchers (jain 1995; dhillon et al. 2000a). the aims of this experiment were to study the effect of ph and temperature on isolate aq-1 growth and xylanase production, to find a better medium composition formula and to attempt using fruitless oil palm brunch as a main carbon source for xylanase production. materials and methods microorganism and growth condition. isolate aq1 used in this experiment was isolated from fresh water aquarium sediment and cultivated in luria bertani (lb) medium. morphological, physiological and biochemical properties of the isolate q-1 were observed according to case and johnson (1984). to assess the effect of temperature and ph on the cell growth and the enzyme production, the cultures were incubated at different temperatures and phs in the above medium without the addition of agar. the temperature and ph observed were 30, 40, 50 °c and ph 7.0, 8.0 and 9.0, respectively. the growth was observed by counting the cell number in a neubauer counting chamber. bacterial starter was prepared in lb medium as follows: 1 loop bacterial culture taken from slant agar and inoculated into 25 ml of medium in 125 ml erlenmeyer flask. the mixture was incubated in shaking incubator at 150 rpm for 6 h until the cell number reached about 109 cell ml-1. a sum of 5 ml of inoculums was added into 45 ml of lb + xylan medium. cell growth, xylanase activity and protein in cell free enzyme solution were observed every 4 h for 36 h incubation. cell-free enzyme solution was obtained by centrifuging the fermented broth at 4 °c, 1100 xg for 15 min. effect of medium composition on cell growth and xylanase production. after obtaining optimum temperature microbiol indones18 wahyuntari et al. and ph of xylanase production in lb medium, the isolate was cultivated in media suggested by nakamura et al. (1994) and dhillon et al. (2000b). each 100 ml of nakamura medium (medium a) contained 0.5 g polypeptone, 0.5 g yeast extract, 0.1 g k 2 hpo 4 , 0.02 g mgso 4 .7h 2 o and 0.5 g oatspelt xylan. the ph was adjusted by adding 1% na 2 co 3 . each 100 ml of dhillon medium (medium b) contained 0.1 g kh 2 po 4 , 0.1 g k 2 hpo 4 , 0.05 g mgso 4 , 0.1 g nh 4 cl, 0.3 g oat spelt xylan, 1 ml of stock vitamin solution (composition per 100 ml: biotin ± 2 mg, folic acid ± 2 mg, pyridoxine hydrochloride ± 10 mg, thiamine hydrochloride ± 5 mg, calcium dpantothenate ± 5 mg, vitamin b12 ± 0.1 mg, p-aminobenzoic acid ± 5 mg, lipoic acid ± 5 mg) and 10 ml of trace elements (composition per 100 ml of stock solution: c 6 h 9 no 6 (nitrilotriacetic acid) ± 1.5 g, mnso 4 .2h 2 o ± 1.0 g, feso 4 .7h 2 o ± 0.2 g, cocl 2 ± 0.2 g, cacl 2 .2h 2 o ± 0.2 g, znso 4 ± 0.2 g, cuso 4 .5h 2 o ± 0.02 g, al(so 4 ) 2 ± 0.2 g, h 3 bo 3 ± 0.02 g, na 2 mo 4 .2h 2 o± 0.02 g, 2 ml nitrilotriacetic acid stock solution and 1 ml of the other stocks were mixed and the volume was made to 100 ml). in the following step, the medium that showed the best bacterial growth was selected and modified. the oat spelt xylan was substituted with 0.35 g ml-1 of fruitless oil palm bunch (fpb). the fpb was prepared by chopping and screening through 35 mesh screen. the growth of bacterial cells and xylanase production was observed as previously done. assay of xylanase activity and protein content. the enzyme activity was measured according to bernfeld (1955) with modification. as much as 150 µl cell-free enzyme solution was added into 150 µl of oatspelt xylan suspension (1 g in 100 ml 0.05m phosphate buffer (ph 7 and 8) and 0.05 m trishcl buffer (ph 9)). the mixture was incubated for 15 min at the same temperature as that of the cultivation (30, 40 or 50 °c). the mixture was centrifuged at 4°c, 1100 xg for 5 min. the reaction was stopped by adding 200 µl 3,5-dinitro salicylic acid (dns) and incubated at 100 °c for 5 min. the mixture was cooled and added with 2 ml distilled water. the absorbance was measured at l540 nm against blank reagent. one unit of xylanase was defined as the amount of enzyme that released 1 µmol reducing sugar equivalent to xylose per minute at assay condition. protein content of the enzyme solution was measured according to a method suggested by bradford (1976). effect of ph and temperature on xylanase activity. xylanase activity was measured at ph ranging from 4 to 10. observation at ph 4, 5 and 6 were done using 0.05 m citratephosphate buffer, ph 7 and 8 using 0.05 m phosphate buffer, ph 9 using 0.05 m tris-hcl and ph 10 using 0.05 m glysinenaoh. the enzymatic reaction was carried out at 50 °c for 15 min. the effect of temperature on xylanase activity was observed at temperature ranging from 30 to 90 °c with interval of 10 °c at optimum ph obtained from previous experiment. activity of crude enzyme on various carbohydrate substrates. the presence of other carbohydrase was analyzed using soluble starch, carboxymethylcellulose, oatspelt and birchwood xylan. amylase, celulase and xylanase activity were measured according to bernfeld (1955), miller (1959) and bailey (1992), respectively. the enzymatic reactions were done at ph 7, 60 °c for 15 min. results identification of isolate aq1. the isolate showed xylanolytic activity on lb medium containing 2 g agar and 0.5 g oatspelt xylan per 100 ml medium. observation on morphological, physiological and biochemical properties of isolate q-1 are shown in table 1. the data showed that the isolate was a rod shaped, aerobic, gram positive, endosporeforming microorganism. the endospores were oval-shaped and located in the center of the cells. the cell diameter was less than 0.1 µm. according to bergey’s manual, these characteristics indicate that the isolate belongs to bacillus genus (claus and berkely 1986). effect of temperature and ph on bacterial growth and xylanase production. fig 1 shows bacterial growths at 30, 40 and 50 °c at various medium ph (7, 8 and 9). fig 2 shows xylanase and protein content in the fermented broth at temperature and ph observed as mentioned above. using lb medium supplemented with oatspelt xylan as xylanase inducer, the maximum cell count was observed in the culture incubated at 50 °c, ph 7 for 12 h with cell density of 1.78 x 109 (fig 1c), however, the optimum condition for xylanase production was observed at 40 °c and ph 7 for 24 h fermentation with enzyme activity of 0.24 u ml-1 and 0.26 mg ml-1 protein (fig 2b). effect of medium composition on cell growth and xylanase production. cell growth in nakamura medium was about the same as that of in dhillon medium (fig 3). the optimum growth was reached after 12 h with cell density of 4.79 x 108cfu ml-1 and 6.31 x 108 cfu ml-1 in dhillon and nakamura medium, respectively. in nakamura medium with substitution of carbon source from oatspelt xylan to fruitless oil palm bunch (fpb), the cell growth was slower compared to in original nakamura medium, where the number of cells reached at 7.94 x 108 cfu ml-1 after 18 h fermentation. protein content in the fermented broth using dhillon medium was higher (0.48 mg ml-1) and reached faster than that of in the other medium formulae observed (fig 4). the table 1 morphological, physiological and biochemical properties of isolate aq1 test aq1 xylanolytic index colony color colony shape elevation gram staining endospore growth at 50 °c growth at 7% nacl containing medium growth in anaerobic agar cell shape hydrolysis of indole methyl red voges-proskauer citrate catalase nitrate reduction carbohydrate fermentation d-glucose acid formation gas formation d-xylose l-arabinose d-mannitol 0.18 white spread flat + central + + + rod + + + + + + + + + + microbiol indones 19volume 3, 2009 maximum protein content in this medium was observed after 18 h fermentation but it did not increase significantly throughout 36 h observation, whereas the protein content in nakamura medium after 18 h fermentation was only 0.23 mg ml-1 and did not increase significantly either during 36 h of fermentation. protein in fermented broth in nakamura medium with fpb substitution was not different with that of in nakamura original formulae which was 0.24 mg ml-1 after 18 h fermentation. xylanase production in dhillon medium was slightly higher compared to in nakamura medium, which was 0.07 u ml-1 after 18 h and 0.09 u ml-1 after 30 h, respectively. however, the substitution of xylan source with fpb in nakamura medium, xylanase production was much faster and fig 3 effect of medium composition on isolate aq1 growth at 40 °c, ph 7. , oat spelt xylan (dhillon); , oat spelt xylan (nakamura), , fpb (nakamura). incubation (h) 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 c e ll n u m b e r (c e ll m l -1 ) 1.e+10 1.e+09 1.e+08 1.e+07 1.e+06 incubation (h) 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 c e ll n u m b e r (c e ll m l -1 ) 1.e+10 1.e+09 1.e+08 1.e+07 1.e+06 a incubation (h) 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 c e ll n u m b e r (c e ll m l -1 ) 1.e+10 1.e+09 1.e+08 1.e+07 1.e+06 b fig 1 effect of ph and temperature on isolate aq1 growth. a, 30 °c; b, 40 °c; c, 50 °c; ph 7, ph 8, ph 9. incubation (h) 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 c c e ll n u m b e r (c e ll m l -1 ) 1.e+10 1.e+09 1.e+08 1.e+07 1.e+06 x y la n a s e a c ti v it y ( u m l -1 ) p ro te in ( m g m l -1 ) 0 . 4 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 0 . 3 0 . 2 0 . 1 0 . 0 0 . 3 0 . 2 0 . 1 0 . 0 time (h) a x y la n a s e a c ti v it y ( u m l -1 ) 0 . 4 0 0 . 3 0 . 2 0 . 1 0 . 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 time (h) p ro te in ( m g m l -1 ) 0 . 3 0 . 2 0 . 1 0 . 0 b x y la n a s e a c ti v it y ( u m l -1 ) 0 . 4 0 . 3 0 . 1 0 . 0 p ro te in ( m g m l -1 ) 0 . 3 0 . 2 0 . 1 0 . 0 0 . 2 c 0 4 8 1 2 1 6 2 0 2 4 2 8 3 23 6 time (h) fig 2 effect of ph and temperature on xylanase production ( , , ) and protein content ( , , ). a, 30 °c; b, 40 ° c; c, 50 °c. , ph 7; , ph 8; , ph 9; , ph 7; , ph 8; , ph 9. microbiol indones20 wahyuntari et al. higher than that of in original nakamura and dhillon formula. the enzyme production was about three times higher compared to in original nakamura medium which 0.30 u ml-1 was after 30 h. effect of ph and temperature on crude xylanase activity. the ph of reaction mixture significantly affected xylanase activity as shown in fig 5, where the highest enzyme activity was observed at ph 7. the temperature higher than 60 °c decreased the enzyme activity significantly (fig 6). the optimum activity was observed at 60 °c. activity of the crude enzyme on various carbohydrate substrates. activity of the crude enzyme on some carbohydrate was showed at fig 7. the crude enzyme mainly contained xylanase as indicated by the highest activity was on oat spelt xylan (55.21 u ml-1) and birchwood xylan (43.03 u ml-1). the crude enzyme also contained amylase (8.12 u ml-1) but hardly cellulase (0.50 u ml-1). discussion based on morphological, physiological and biochemical analysis, isolate aq-1 was identified as bacillus sp. bacillus spp. have been reported as xylanolytic enzymes producers, such as bacillus sp. ncim 59 (kulkarni and rao 1996), b. thermoleovorans strain k-3d and b. flavothermus strain lb3a (sunna et al. 1997a), b. licheniformis (archana and satyanarayana 1997), bacillus sp xe (samain et al. 1997) b. circulans ab16 (dhillon et al. 2000b), b. circulans d1 (bocchini et al. 2002), b. subtilis b230 (oakley et al. 2003). growth and enzyme production condition of most b. subtilis was reported at ph 6-8 and temperature of 37-70 °c. bacillus sp. aq1 could grow and produce enzyme at the ph (7-9) and temperature (30-50 °c) observed. the data showed that even though the fastest cell growth was at 50 °c, ph 7 and 8, the growth also declined faster than that of at 30 and 40 °c. the enzyme and protein content were better at ph 7, temperature of 30 and 40 °c than that at other ph and temperature observed, therefore it can be suggested that the enzyme production was better at ph 7 and temperature close to 40 °c. previous studies reported that xylanase production by b. subtilis b230 was best at ph 8, 37 °c (oakley et al. 2003) and b. subtilis 168 at ph 7, 37 °c (st john et al. 2006). the ph and temperature conditions were similar to that condition of enzyme production by aq1 strain that was concluded as b. subtilis as well (unpublished). most xylanases were reported as inducible enzymes. some studies showed the used of different kinds of commercial extracted xylan as well as xylan containing agricultural wastes for inducing xylanolytic enzyme production. sunna et al. e n z y m e a c ti v it y ( u m l -1 ) p ro te in ( m g m l -1 ) 0 . 4 0 . 3 0 . 2 0 . 1 0 . 0 0 . 5 0 . 4 0 . 3 0 . 2 0 . 1 0 . 0 60 1 2 1 8 2 4 3 0 3 6 incubation time (h) fig 4 effect of medium composition on xylanase production ( , , ) a n d p r o t e i n c o n t e n t ( , , ) a t 4 0 ° c a n d p h 7 . , o a t s p e l t x y l a n ( d h i l l o n ) ; , o a t s p e l t x y l a n (nakamura); , fpb (nakamura); , oatspelt xylan (dhillon); , oatspelt xylan (nakamura); , fpb (nakamura). fig 5 effect of ph on xylanase activity at 50 ºc 8 9 1 0 1 1 p h 120.0 100.0 80.0 60.0 40.0 20.0 0 . 0 4 5 6 7 fig 6 effect oftemperature on xylanase activity at ph 7.0. temperature (ºc) 1 0 0 r e la ti v e a c ti v it y ( % ) 1 0 0 9 0 8 0 7 0 6 0 5 03 0 4 0 6 0 7 0 8 0 9 0 fig 7 activity of isolate aq1 crude enzyme on various carbohydrate substrates at ph 7, 60 °c. a c ti v it y ( u m l -1 ) 6 0 5 0 4 0 3 0 2 0 1 0 0 starch cmc oatspelt xylan beechwood xylan 8.12 0.50 55.21 43.03 substrates (1997a) used beechwood xylan for inducing xylanase production by b. thermoleovorans k-3d and b. flavothermus lb3a. sunna et al. (1997b) also reported that beechwood xylan was used to induce xylanolytic enzyme production by thermotoga maritima, t. neapolitana and t. thermarum. rani and nand (2000; 2001) studied substrate specificity of clostridium absonum cfr-702 using different kinds of xylans such as birchwood, larchwood, oatspelt xylan. kulkarni and rao (1996) used wheat bran as enzyme inducer for producing xylanolytic enzyme from bacillus sp. ncim 59 whereas dhillon et al. ( 2000a) used rice bran and straw, wheat and maize bran, as well as sugarcane baggase to induce xylanase production by b. circulans ab16. dhillon et al. (2000b) also used different kinds of substrates in another experiment including sugarcane bagasse, rice straw and wheat straw, as the carbon source in basal medium to produce xylanolytic enzymes by b. circulans ab 16. the experiment also reported that the present of simple sugar lowered xylanolytic enzyme production by b. circulans ab16. these studies showed that various agricultural wastes could be used as substrates for producing xylanolytic enzymes by various microorganisms. these studies also reported that an inducer for certain microorganism could be an inhibitor for others (biely 1985; paul and varma 1990; nakamura et al. 1995; oakley et al. 2003). as a consequence, it is important to choose a proper inducer for certain microorganism. in this experiment, cell growth and enzyme production in the medium containing oatspelt xylan and fpb as carbon source were observed. the results showed cell growth in medium composition according to dhillon et al. (2000b) was slightly better than that of in medium of nakamura et al. (1994), however, xylanolytic production was better in nakamura medium than that of in dhillon medium. since in dhillon medium xylanase production did not tend to increase, even decreased during fermentation time observed, therefore substitution of oatspelt xylan with fpb was done using nakamura medium. in nakamura fpb containing medium, xylanolytic enzyme production was better than that of using the original nakamura medium composition. according to irawadi (1991), fpb contained minerals as followed: 2.13% k, 0.18% ca, 0.17% mg, 0.05% mn, 0.63% na, 0.59% fe, 0.14% p 2 o 5 . the experimental results showed that enzymes production by aq-1 using fpb was three times higher than that of using oatspelt xylan therefore fpb might be advised to be used as a substrate for xylanolytic enzymes production. crude enzymes produced by bacillus sp aq1 using fpb, not only showed xylanolytic activity but also amylolytic and cellulolytic activity as well (fig 7). the presence of cellulolytic enzyme in crude xylanase is common, especially fungal xylanase (kulkarni et al. 1999). the isolate aq1 only produced very little cellulolytic enzyme (0.5 u ml-1) compared to that xylanolytic activity on oatspelt (55.21 u ml-1) and on birchwood (43.03 u ml-1). based on the available data from this experiment, the difference in crude enzyme on the different xylan substrate could not be explained yet. it is still needed more complete studies to elaborate the type of xylanolytic activities present in the crude enzyme. acknowledgement the authors thank willy agustine, alumnus of bogor agricultural institute, department of mathematics and life sciences for her help in some laboratory works. references archana a, satyanarayana t. 1997. xylanase production by thermophilic bacillus licheniformis a99 in solid state fermentation. enzyme microb technol 21:12-7. bailey mj. 1992. interlaboratory testing of methods for assay of xylanase activity. biotechnology 23:257-70. beg qk, kapoor m, mahajan l, hoondal gs. 2001. microbial xylanases and their industrial applications: a review. appl microbiol biotechnol 56:326-38. bernfeld p. 1955. amylases á dan â. in: colowick sp, kaplan no, editors. methods in enzymology. vol 1. new york: academic pr. biely p. 1985. microbial xylanolytic systems. trends biotechnol 3:288-90. bocchini da, alves-prado hf, baida lc, roberto ic, gomes e, silva r da. 2002. optimization of xylanase production by bacillus circulans d1 in submerged fermentation using response surface methodology. process biochem 38:727-31. bradford m. 1976. a rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. ann biochem 72:248-54. case cl, johnson tr. 1984. laboratory experiments on microbiology. sydney: benjamin/cummings. chen cc, adolphson r, dean jfd, erickson kel, adams mww, westpheling j. 1997. release of lignin from kraft pulp by a hyperthermophilic xylanase from thermotoga maritima. enzyme microbiol technol 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thermophilic melanocarpus abomyces iis-68. proces biochem 30:605-09. kulkarni n, rao m. 1996. application of xylanase from alkaliphilic thermophilic bacillus sp ncim 59 in biobleaching of bagasse pulp. j biotechnol 51:167-73. kulkarni n, shendye a, rao m. 1999. moleculer and biotechnological aspects of xylanases. fems microbiol rev 23:411-56. lee sf, forsberg cw. 1987. purification and characterization of an al-arabinofuranosidase from clostridium acetobutylicum atcc 824. can j microbiol 33:1011-16. lee sf, forsberg cw, rattray jb. 1987. purification and characterization of two endoxylanases from clostridium acetobutylicum atcc 824. appl environ microbiol 53:644-50. miller gl. 1959. use of dinitrosalicylic acid reagent for determination of reducing sugar. anal chem 31:426. nakamura s, ishiguro y, nakai r, wakabayashi k, aono r, horikoshi k. 1995. purification of a thermophilic alkaline xylanase from thermophilic bacillus sp strain tar-1. j mol catal b: enzymatic 1:7-15. nakamura s, nakai r, wakabayashi k, ishiguro y, aono r, hirikoshi k. 1994. thermophilic alkaline xylanase from newly isolated microbiol indones 21volume 3, 2009 alkaliphilic and thermophilic bacillus sp. strain tar-1. biosci biotechnol biochem 58:78-81. oakley aj, heinrich t, thompson ca, wilce mcj. 2003. characterization of a family 11 from bacillus subtilis b230 used for paper bleaching. acta cryst d59:627-36. pala h, mota m, gama fm. 2006. factors influencing mow deinking: laboratory scale studies. enzyme microb technol 38:81-87. paul j, varma ak. 1990. influence of sugars on endoxylanase and bxylosidase activities of bacillus strain. biotechnol lett 12:19-27. primo-martin c, wang m, lichtendonk wj, plijter jj, hamer rj. 2005. an explanation for the combined effect of xylanase-glucose oxidase in dough system. j sci food agric 85:1186-96. rani ds, nand k. 2000. production of thermostable cellulose free xylanase by clostridium absonum cfr-702. process biochem 36:355-62. rani ds, nand k. 2001. purification and characterization of xylanolytic enzymes of a cellulose free thermophilic strain of clostridium absonum cfr-702. anaerobe 7:45-53. sakka k, maeda y, takahashi n, hakamada y, shimada k. 1999. purification and some properties of xylanase from clostridium stercorarium strain hx-1. agric biol chem 55:247-48. samain e, debeire ph, touzel jp. 1997. high level production of a cellulase-free xylanase in glucose-limited fed batch cultures of a thermophilic bacillus strain. j biotechnol 58:71-8. st. john fj, rice jd, preston f. 2006. characterization of xync from bacillus subtilis subsp subtilis strain 168 and analysis of its role in depolymerization of glucoronoxylan. j bacteriol 188: 8617-26. sunna a, prowe sg, stoffregen t, antranikian g. 1997a. characterization of the xylanases from the new isolated thermophilic xylan-degrading bacillus thermoleovorans strain k-3d and bacillus flavothermus strain lb3a. fems microbiol lett 148:209-16. sunna a, puls j, antranikian g. 1997b. characterization of the xylanolytic system of the extreme thermophilic anaerobic bacteria thermotoga maritime, t neapolitana, t thermarum. comp biochem physiol 118a:453-61. wong kky, tan lul, saddler jn. 1988. multiplicity of 3-1, 4-xylanase in microorganisms. microbiol rev 52:305-17. zheng l, du y, zhang j. 2000. biobleaching effect of xylanase preparation from an alkalophilic bacillus sp on ramie fibers. biotechnol lett 22:1363-67. microbiol indones22 wahyuntari et al. 04. nisa.cdr vol.15, no.2, june 2021, p 61-68 doi: 10.5454/mi.15.2.4 potential z foonotic faecal bacteria rom sunda porcupine ( ) and hystrix javanica their antimicrobial resistance profiles sarsa a. nisa , rifka a. n. safitri , nurul inayah , achirul nditasari , susiana 1 1 2 3 purwantisari , rejeki s. ferniah , anang s. achmadi , taufiq p. nugraha , 1 1 2 2 and sugiyono saputra * 3 1 department of biology, faculty of sains and mathematics, diponegoro university, jalan prof. sudarto no.13, tembalang, semarang 50275, indonesia; 2museum zoologicum bogoriense, research center for biology, indonesian institute of sciences, jalan raya jakarta-bogor km 46, cibinong 16911, indonesia; 3 microbiology division, research center for biology, indonesian institute of sciences, jalan raya jakarta-bogor km 46, cibinong 16911, indonesia. sunda porcupine ( ) is one of the indonesian endemic species which is often sought after for hystrix javanica their meat. although it is becoming increasingly popular for extreme culinary, information regarding biological risks arising is very limited. this study aimed to assess zoonotic bacteria carried from this wildlife potential faecal by sunda porcupine and a total 22 faecal samples to investigate their antimicrobial resistance (amr) profile. were collected from captive sunda porcupine and and wo subjected to presumptive test for . tsalmonella listeria samples (9%) were regarded as positive for which indicated by black colonies salmonella was the presence of on x l dylose ysine eoxycholate (xld) agar. meanwhile, the presence for was positive in all samples (100%), listeria indicated by olour change in medium from straw to black in solation ranswab®. in total, 3 bacterial c i t 6listeria colonies were isolated a s t most and subjected for ntimicrobial usceptibility esting (ast) by disk diffusion method. importantly, multidrug-resistant bacteria and resistance to third generation cephalosporins (ceftriaxone) were not observed, indicating low risk of amr dissemination. based on gene analysis and 16s rrna phylogenetic tree construction from selected isolates, we identified several potential food-borne zoonotic pathogens, including proteus mirabilis shigella klebsiella (xh3.3, h4.2, and e1.2) (xd8.2 and g11.3), and flexneri , quasipneumoniae similipneumoniae subsp. (xf4.2) further research to confirm the pathogenicity of . those isolates is still needed but based on these results, we support the hypothesis that sunda porcupine is potential as a reservoir of pathogenic bacteria. p crucial when this reventive measures are to prevent transmission processing bushmeat. r antimicrobial resistance sunda porcupine, zoonoseskey words: , 16s rna , landak jawa ( ) merupakan salah satu spesies endemik indonesia yang sering dimanfaatkan hystrix javanica dagingnya. meski semakin populer untuk kuliner ekstrem, informasi mengenai risiko biologis yang timbul dari satwa liar ini masih sangat terbatas. penelitian ini bertujuan untuk mengkaji bakteri potensial penyebab zoonosis yang dibawa oleh landak jawa dan untuk mengetahui profil (amr) pada isolat yang antimicrobial resistance didapatkan. sebanyak 22 sampel feses diambil dari landak jawa disertakan untuk uji presumtif dan salmonella listeria salmonella . dua sampel (9%) dinyatakan positif yang ditunjukkan dengan adanya pertumbuhan koloni hitam pada (xld) agar. sementara itu, keberadaan dinyatakan positif pada xylose lysine deoxycholate listeria semua sampel (100%), ditunjukkan dengan munculnya warna hitam pada media isolation transwab®. listeria secara total, 36 koloni bakteri berhasil diisolasi dan diuji resistensinya menggunakan metode difusi cakram. bakteri multidrug-resistant dan resistensi terhadap generasi ketiga sefalosporin (ceftriaxone) tidak terdeteksi yang mengindikasikan resiko rendah penyebaran amr. berdasarkan analisis gen 16s rrna dan konstruksi pohon filogenetik isolat terpilih, kami mengidentifikasi beberapa bakteri yang potensial sebagai food-borne zoonotic pathogens flexneri, diantaranya dan , dan proteus mirabilis shigella (xh3.3, h4.2, e1.2) (xd8.2 g11.3) dan ). penelitian lebih lanjut untuk mengkonfirmasi klebsiella quasipneumoniae similipneumoniae subsp. (xf4.2 patogenisitas isolat masih diperlukan tetapi berdasarkan hasil ini, kami mendukung hipotesis bahwa landak jawa berpotensi sebagai reservoir bakteri patogen. tindakan pencegahan sangat penting dilakukan untuk mencegah penularan terutama pada saat memproses daging hewan liar ini. kata kunci: r landak jawa, zoonosis antimicrobial resistance16s rna , , microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 21-87907604; : +62fax: +6221-87907612; sugiyono.saputra@lipi.go.id e-mail: disease transmission. the existence of wild animals that should be in nature and far from human life, now carried in the form of exotic food and medicine, which can accelerate the transmission of zoonotic diseases (broad 2020). the spillover events occur due to human-animal bushmeat hunting and wildlife trade for consumption are contributing factors to zoonotic interactions during hunting, carrying, storing, raising, selling, killing, and consuming wild animals (plotkin 2020). this unsustainable wildlife hunting practices mainly operated in asia, africa and south america, leading to a significant extinction threat to several wild terrestrial mammals . (ripple 2016)et al. bushmeat from wild mammals, has been used for consumption in indonesia for decades. one of the wild mammals that is becoming more popular for consumption is sunda porcupine ( ) hystrix javanica which is an endemic indonesian wildlife that belongs to the rodentia rder . the o (lunde and aplin 2008) distribution region of these animals includes sumatra, kalimantan, java, and bali . (lunde and aplin 2008) sunda porcupine have spines as their defence system (myers . 2017)et al , nocturnal and herbivorous as in their natural habitat, they eat plant parts such as tubers, bamboo shoots, seeds, fruit, tubers, rhizomes, young shoots, and bark . sunda porcupine meat (farida 2015) have long been consumed as a source of protein for extreme culinary. they believe that consuming porcupine meat is preventing from osteoporosis and improving body vitality, while the liver and bile can cure asthma, and the thorns can be used to treat toothaches and ulcers ; (farida 2007; farida 2015 wardi et al. 2011). the international conservation status based on the iucn red list of threatened species classified sunda porcupine to least concern or not threatened. in indonesia, this wildlife is protected under the regulation of the minister of environment and forestry of the republic of indonesia number: p.106/menlhk/setjen/kum.1/12/2018. despite they have protected status, illegal hunting and trade of sunda porcupine are still rampant . (inayah 2016) additionally, biological risk information including transmission of zoonoses and antimicrobial resistance that may result from the hunting and trade this wild animal is not well recorded. in this preliminary study, we aimed to assess potential zoonotic bacteria arising from sunda porcupine faecal samples by culturedependent approach. faeces and intestinal contents are often contaminated the meat during slaughtering and processing activities. the presence of antimicrobial resistance will also be investigated by characterizing their phenotypic resistance to several antimicrobial categories. this assessment will be useful for prevention measures for tackling emerging and reemerging infectious diseases from wildlife. materials and methods sampling was conducted animals and sampling. at the small mammal research facility in research center for biology, indonesian institute of sciences. a total 22 sunda porcupine (aged 2-3 years) were used as the object of study. prior sampling, sunda porcupine had no receive any special medical treatment for the last three months with usual diet include leaves and tubers. sampling was performed using non-invasive methods without making direct contact to each individual by swabbing fresh faecal samples in the morning and transfer directly into rappaportvassiliadis enrichment broth (rv) salmonella (himedia, india) and isolation transwab® listeria (medical wire, uk) for the bacterial enrichment stage (kartika 2017)et al. . this procedure has been approved by animal ethics commission, indonesian institute of sciences (b-12220/iph/ks.02.04/iph/ix/2019). presumptive test and bacterial isolation. presumptive test for and were salmonella listeria selected in this study because these bacteria widely known as foodborne pathogens and contaminant of animal products. after inoculating faecal samples, rv salmonella enrichment broth was incubated at 42˚c for 24 hours, followed by subsequent inoculation to xylose lysine deoxycholate (xld) (himedia, india). agar xld gar is a selective medium that primarily used for a isolation and differentiation of andsalmonella shigella shigella. colonies maybe difficult to differentiate with other gram-nagative bacteria, thus presumptive test was not performed. in the other hand, typical colony growth with black spot in the center on xld gar media is easy to observe, indicating the a presence of . meanwhile,salmonella isolation listeria transwab® after is used for the detection of . listeria incubation at 37˚c for 24 hours color change from straw to black is an indication the growth of listeria spp in the sample and the results were regarded as . positive. the isolation process was continued by using columbia agar supplemented with dehydrated horse blood (5%) to detect hemolysis activity. a clear zone that appears below or around the colonies indicate beta hemolytic activity preservation of isolates were . conducted using mueller hinton broth media added with 20% glycerol and t en stored at 80˚c. h salmonella enterica enterica listeria subsp. jcm 1652 and monocytogenes jcm 7671 were used as positive controls during the isolation process. 62 nisa et al . microbiol indones volume 15, 2021 microbiol indones 63 a n t i m i c r o b i a l s u s c e p t i b i l i t y te s t i n g . antimicrobial susceptibility testing was performed using the disk diffusion method based on protocol from the eur op ea n committe e on an timicr obial susceptibility testing (eucast) . (eucast 2020) mueller hinton agar (mha) was used by inoculating bacterial inoculum with turbidity standard of 0.5 mcfarland or equivalent to 1.5×108 cfu/ml. antimicrobial disc (oxoid, usa) used in this study i n c l u d e 3 0 μ g e a c h o f a m i k a c i n ( a k , aminoglycosides , ) (amc, amoxicillin-clavulanic acid penicillin-beta lactamase inhibitors , ) (fox, cefoxitin second generation cephalosporins , ) (croceftriaxone third generation of cephalosporins). the inhibition zone diameter (izd) in agar media was indicated by the formation of a clear zone around the antibiotic disc. interpretation of izd results is species-specific and antimicrobial-specific, however, in order to describe potentially resistant isolates, we interpreted izd results (resistant, intermediate or sensitive) using breakpoints for enterobacterales according to european committee on antimicrobial susceptibility testing (eucast) guideline and zone diameter interpretive standards breakpoints for veterinary pathogens according to clinical & laboratory standards institute (clsi, 2015). we then categorised resistance cut off for amoxicillin-clavulanic acid and amikacin when with izd ≤14 cm while for cefoxitin and ceftriaxone when izd ≤17 cm. multidrugresistant bacteria are defined when bacterial isolates have resistance to one agent in three or more antimicrobial categories (clsi 2015). escherichia coli atcc 25922 and atcc 25923 staphylococcus aureus were used as control bacteria. molecular identification based on the 16s rrna. extraction of bacterial dna using chelex® 100 sodium form 10% (sigma aldrich) according to the protocol from . amplification saputra (2017)et al. of the 16s rrna gene was performed on a veriti ™ 96well fast thermal cycler (applied biosystems, usa) using bioline mytaq ™ red mix (meridian bioscience) using universal primer 27f and 1492r. pcr products were subjected to sanger dideoxy sequencing (1st base, singapore). the dna sequences obtained using primer 27f were analyzed using the mega x program and alignment of the bases sequences were performed on genbank® dna using the basic local alignment search tool (blast) menu. phylogenetic tree construction was made by neighbor joining method with 1000 bootsrap value. results presumptive test for and salmonella listeria. presumptive test for on xld gar showed salmonella a that 2 out of 22 samples tested positive (f2 and h3), which was indicated by the growth of typical black or black spots colonies. a total of 18 bacterial isolates were successfully purified and preserved. for listeria presumptive test, all samples (100%) were regarded positive which was indicated by color change in the listeria isolation transwab® from straw to black. a total of 18 isolates were successfully purified and preserved, with four isolates showing hemolytic activity (e5.1, g8.2, g11.1, and g11.3) in columbia agar supplemented with 5% dehydrated horse blood. presumptive tests and all isolates collected in this study are presented in table 1. distribution of inhibition zone diameter (izd). antimicrobial susceptibility testing only showed that one isolate that showing no inhibition zone diameter (xg10.1) and regarded as cefoxitin resistant strain. four isolates with inhibition zone diameter <10 was observed. resistance to third generation of cephalosporins (ceftriaxone) was not detected. the distribution of all isolates with the inhibition zone diameter against four can be seen in antimicrobials figure . 1 interpreted according to izd breakpoint from eucast and clsi, only two isolates that showed resistance to two antimicrobials tested (amc and fox), while five isolate showed resistance to one antimicrobials (ak or fox). resistance to all antimicrobial (multidrug-resistant bacteria) tested in this study was not observed. th of e resistance profile the seven bacterial isolates from the sunda porcupine are presented in table .1 identified bacteria and their susceptibility against antibiotics. seven bacterial isolates were selected based on colony appearance for bacterial identification using 16s rrna gene. at least ~700-800 bp of nucleotides were generated for blast analysis in genbank . percentage of identity of those isolates ® can be seen in table 2 which also include inhibition zone diameter results all s. even isolates were sensitive to amikacin, amoxicillin-clavulanic acid, cefoxitin and ceftriaxone, except for (xd8.2) which shigella flexneri was resistant to cefoxitin the phylogenetic tree . constructions of seven isolates compared with reference strains are presented in figure .2 64 nisa et al . microbiol indones discussion to the best of our knowledge, this is the first study in indonesia to assess potential zoonoses from faecal samples of sunda porcupine. this preliminary study has three main findings: 1) proportion of presumptive test for and which regarded as salmonella listeria positive were 9% (2/22) and 100% (22/22), respectively; 2) several potential zoonotic pathogens was observed, including (n=3), proteus mirabilis s h i g e l l a f l e x n e r i k l e b s i e l l a ( n = 2 ) a n d quasipneumoniae similipneumoniae subsp. (n=1); and 3) multidrug-resistant bacteria and resistance to the latest generation antimicrobial used in this study (ceftriaxone) was not detected. porcupine is one of the popular bushmeat not only in southeast asian countries (engel and ziegler 2020) but also in several african countries (bachand . et al 2012, friant . 2015). brush-tailed porcupine et al ( ) is the most preferred animal for atherurus africanus their meat in nigeria and consumed regularly up to once per week (friant . 2015). the activities et al related to this bushmeat consumption which include hunting, trade, and meat processing has led to a higher risk of zoonotic bacteria transmission (plotkin 2020). it is reported that were observed on the dorsal salmonella skin of sunda porcupine ( and prawira 2018)et al. carcass of brush-tailed porcupine (bachand . et al table 1. presumptive test results resistance 22 f samples and profile of isolated bacteria from aecal sample id presumptive salmonella presumptive listeria isolate code hemolysis activity colony appearance resistance profile d6 + xd6.2 milky white xd6.3 yellow d7 + d8 + d8.1 white xd8.2 white fox e1 + e1.2 milky white e4 + xe4.2 yellow e5 + e5.1 + transparent white, clear zone ak xe5.1 yellow e6 + e6.3 white xe6.2 yellow e7 + e7.1 transparent white e8 + e8.1 milky white ak f1 + f2 + + f2.2 white xf2.2 white f4 + f4.2 milky white xf4.2 yellowish black g4 + g4.1 transparent white amc, fox xg4.4 white xg4.5 yellowish white g5 + g8 + g8.2 + yellowish white, clear zone xg8.2 yellow g9 + xg9.3 white g10 + g10.1 transparent white ak g10.2 white ak xg10.1 yellow amc, fox g11 + g11.1 + milky white, clear zone g11.2 transparent white g11.3 + white, clear zone h1 + h1.3 white xh1.2 yellowish white h2 + xh2.3 white h3 + + h3.3 transparent white, xh3.2 black spot xh3.3 black h4 + h4.2 white xh4.1 white note: ak amikacin, amc amoxicillin-clavulanic acid, fox cefoxitin. volume 15, 2021 microbiol indones 65 fig 1 the distribution of inhibition zone diameter (izd, cm) of bacterial isolates from sunda porcupine for four antimicrobial tested. fig 2 phylogenetic tree construction from isolates xg4.4, xd8.2, xh3.3, xf4.2, h4.2, e1.2, and g11.3, was carried out using the neighbor joining method with 1000 bootsrap value. 2012) in indian porcupine , while was observedlisteria ( ) .hystrix indicus (sarangi and panda 2013) based on our study, it can be assumed that sunda porcupine has the potential as a natural reservoir for salmonella. percival and williams (2014) states that natural reservoir of is very diverse, including farm salmonella animals, domesticated animals and wild animals, including poultry, pigs, cattle, birds, dogs, rodents, turtles and cats. consumption of contaminated poultry and meat products is the most common source of transmission. meanwhile, the presumptive test listeria showed positive results for all samples, showing that listeria may presence in sunda porcupine faecal samples. and are among common salmonella listeria 66 nisa et al . microbiol indones cause of foodborne zoonotic diseases, as reported by european food safety authority (efsa 2020). three species of pathogenic bacteria were identified, including (xh3.3, h4.2, and p. mirabilis e1.2) (xd8.2 and g11.3), s. flexneri and k. quasipneumoniae subsp. similipneumoniae (xf4.2), while the status of potential pathogens of another identified isolates ( xg.4.4) pseudomonas xiamenensis was unknown (lai and shao 2008). evidence from other studies regarding zoonotic potentials of these bacteria were presented in table 2. one of the most cases of infection in humans are caused by proteus mirabilis . according to(jacobsen and shirtliff 2011) nemati (2013), is pathogenic in humans p. mirabilis because it can cause urinary tract infections. the pathogenicity of is supported by its unique p. mirabilis virulence factors, such as adhesin, flagella, toxins, quorum-sensing, enzymes, and immune invasion. together with other species such as shigella s. dysenteriae, s. boydii s. sonnei, s. flexneri and are responsible for diarrheal diseases and dysentery occurring globally and may be associated with lifethreatening complications (niyogi 2005). the spread of can occur by the faecal-oral route, where shigella infected animals excrete along with their shigella feces, then pollute the environment (said and marsidi 2017). several strains according to klebsiella simmons and gibson (2012) have been known to be pathogenic and capable of causing infection in humans, t h i s i n c l u d e k . q u a s i p n e u m o n i a e s u b s p . similipneumoniae that cause infections in human and dogs (brisse . 2014). spp. is an et al klebsiella opportunistic pathogenic bacterium from the enterobacteriaceae family that can cause respiratory infections, urinary tract infections and nosocomial infections and are commonly found as microflora in the mouth, skin, intestines of humans and animals (brisse et al. 2006, paran . 2019). et al based on antimicrobial susceptibility testing and interpretation according to eucast and clsi, resistance to ceftriaxone was not detected in all 36 isolates while resistance to amc, fox and ak was detected in seven isolates. multidrug-resistant bacteria were not observed. this result indicates that antimicrobial resistance may not disseminate in sunda porcupine, although accumulated evidence has revealed the presence of antimicrobial resistance in wildlife due to contaminated environment (allen et al. 2010). resistance to third generation cephalosporins is a major concern globally, mainly conferred by extended-spectrum b-lactamases (esbls) which commonly found in birds, mammals, livestock, and companion animals . (vittecoq 2016)et al. this study has several limitations. the method used was based on culture dependent method which only showing presumptive results for and salmonella listeria. we were also unable to recover those bacteria due to having issues with purification, preservation, and the complexity of nutrient requirements, especially for . further, detection on xld listeria salmonella agar after enrichment step may give false positive results because the growth of bacteria in the proteus table 2 antimicrobial susceptibility testing results from seven bacterial isolatesbacterial identity and isolate code species identity (%) accession inhibition zone diameter (mm) potential zoonotic pathogens status amc fox cro ak xh3.3 proteus mirabilis 99.87 nr_113344.1 24,44 24,44 31,79 18,83 yes, commonly detected in broiler chicken (jamaluddin et al. 2018) h4.2 proteus mirabilis 99.87 nr_113344.1 22,76 22,63 28,88 18,35 e1.2 proteus mirabilis 99.88 nr_113344.1 26,86 25,02 27,51 22,31 g11.3 shigella flexneri 99.98 nr_026331.1 20,73 21,31 27,04 19,11 yes, causing foodborne diseases (bintsis 2017) xd8.2 shigella flexneri 100 nr_026331.1 20,14 7,82* 29,12 20,89 xf4.2 klebsiella quasipneumoniae subsp. similipneumoniae 99.77 nr_134063.1 23,28 22,84 28,12 21,21 yes, detected in food animals with other klebsiella species (davis and price 2016) xg4.4 pseudomonas xiamenensis 99.79 nr_043533.1 30,65 24,52 35,82 31,29 unknown, regarded as denitrifying bacterium isolated from activated sludge (lai and shao 2008) note: ak amikacin, amc amoxicillin-clavulanic acid, fox cefoxitin, cro ceftriaxone. *resistance to ak, amc, fox and cro was not detected. only xd8.2 that considered as cefoxitin resistant. volume 15, 2021 microbiol indones 67 medium was resemble to colonies. salmonella additionally, not all collected isolates were identified, therefore, the diversity of culturable faecal bacteria from sunda porcupine was not fully understood. based on presumptive test results and bacterial culture, we strengthen the hypothesis that the sunda porcupine has the potential as a reservoir zoonotic of bacteria. several identified isolates reveal that they may be regarded as foodborne zoonotic pathogens, including from genus , and proteus, klebsiella shigella. due to low exposure to antimicrobials sunda porcupine in this study may not play as an important host for antimicrobial resistance. further research is needed such as identification of collected bacteria all and confirm their pathogenicity. etection ation test for d of virulence genes can be and resistance genes combined to complement the detection results through a culture dependent method so that risk factors for the spread of zoonotic diseases can be understood more comprehensively. acknowledgment this research was supported by dipa research center for biology 2018 and dipa research center for biotechnology 2019, indonesian institute of science (lipi). author contributions the contribution of each author in this article is ss as the main contributor who is responsible for the formulation and overall research objectives. san, rans contributed to laboratory investigations and ni and collected the data. san ss contributed to , an and analyzed the data. san, sp, rsf, and ss asa, tpn were responsible for original draft writing, and san and ss contributed to reviewing and editing the manuscript. references allen hk, donato j, wang hh, cloud-hansen ka, davies j and handelsman j. 2010. call of the wild: antibiotic resistance genes in natural environments. nat rev m i c r o b i o l , 8 ( 4 ) , p p . 2 5 1 – 2 5 9 . d o i : 10.1038/nrmicro2312. bachand n, ravel a, onanga r, arsenault j, gonzalez jp. 2012. public health significance of zoonotic bacterial pathogens from bushmeat sold in urban markets of gabon, central africa j wildl dis 48(3):785-9.doi: 10.7589/0090-3558-48.3.785 bintsis t. 2017. foodborne pathogens. aims microbiol. 2 0 1 7 ; 3 ( 3 ) : 5 2 9 – 5 6 3 . d o i : 10.3934/microbiol.2017.3.529 brisse s, grimont f. and grimont pad. 2006. the genus klebsiella, in. in: dworkin m., falkow s., rosenberg e., schleifer kh., stackebrandt e. 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center for biotechnology, national research and innovation agency (brin), jalan raya bogor km. 46, cibinong 16911, indonesia. ginger is a rhizomatous perennial herb that grows abundantly in tropical areas. it has been used around the world as a spice, flavoring agent, and ingredient in traditional medicine. ginger essential oils (geos) are derivatives of ginger that can be found in various products used in daily life, such as food, pharmaceutical, and cosmetics. the present study analyzed the chemical compositions, antioxidant, and antibacterial activities of three commercially available geos. the compositions of geos were identified using the gas chromatography method. the antioxidant activity was evaluated using 2,2-diphenyl-1-picryl-hydrazyl (dpph) and 2,2'-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (abts) assay methods. the antibacterial activity was determined using a disc diffusion assay based on the diameter of the inhibition zone (diz). the main compounds identified from the samples were zingiberene, α-curcumene, β-sesquiphellandrene, camphene, α-farnesene, β-bisabolene, α-pinene, -1 -1 and 3-carene. the ic values were found to be 5.3023 and 1.4504 mg ml for geo1; 0.9249 and 0.5276 mg ml 50 -1 for geo2; and 10.4463 and 3.3535 mg ml for geo3 when evaluated using dpph and abts assay methods, respectively. all samples showed antibacterial activity against staphylococcus aureus atcc 13420 and bacillus subtilis (the collection of national research and innovation agency), while only geo2 and 3 displayed inhibitory effect against escherichia coli atcc 9637. key words: antibacterial, antioxidant, chemical composition, essential oil, ginger jahe adalah tumbuhan rhizoma herba perennial yang banyak ditemukan di daerah tropis. tanaman ini banyak digunakan sebagai rempah, perisa, maupun bahan baku untuk obat tradisional. minyak jahe, merupakan salah satu produk turunan jahe, yang telah banyak digunakan dalam produk-produk yang digunakan dalam kehidupan sehari-hari seperti makanan, produk farmasi, dan kosmetik. artikel ini memberikan informasi mengenai komponen kimia, aktivitas antioksidan, dan antibakteri dari minyak jahe komersial. senyawa kimia dari minyak jahe dianalisa dengan metode kromatografi gas. aktivitas antioksidan sampel diuji dengan metode uji 2,2diphenyl1-picryl-hydrazyl (dpph) dan 2,2'-azinobis-(3-ethylbenzothiazoline-6-asam sulfonat) (abts). sedangkan aktivitas antibakteri dianalisa dengan menggunakan metode difusi cakram, dengan mengukur zona hambat yang terbentuk. senyawa kimia utama yang terdeteksi dalam sampel adalah zingiberen, α-curcumen, βsesquifellandren, camphen, α-farnesen, β-bisabolen, α-pinena, dan 3-karena. ic sampel yang diperoleh adalah 50 -1 -1 5.3023 dan 1.4504 mg ml untuk geo1; 0.9249 dan 0.5276 mg ml untuk geo2; serta 10.4463 dan 3.3535 mg -1 ml untuk sampel geo3 ketika diuji dengan metode dpph dan abts, secara berurutan. semua sampel menunjukkan aktivitas antibakteri terhadap staphylococcus aureus atcc 13420 dan bacillus subtilis (koleksi badan riset dan inovasi nasional), sedangkan hanya geo2 dan geo3 yang menunjukkan aktivitas penghambatan terhadap escherichia coli atcc 9637. kata kunci: antibakteri, antioksidan, jahe, komponen kimia, minyak atsiri microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-21-8754587, fax. +6221-8754588; e-mail: mferdina@gmail.com; linda.sukmarini @lipi.go.id antidiabetic, anticancer, antivomiting, neuroprotection, anti-inflammation, cardiovascular protector, antiobesity, antinausea, anti-emetic, protective effects against respiratory disorders, and analgesic (mao et al. 2019; abd el-hack et al. 2020; anh et al. 2020). ginger essential oil (geo) is one of ginger derivatives that can be found in various products, such as food, pharmaceutical, and cosmetics. diverse biological activities of geo have also been reported in various studies, such as antimicrobial, antioxidant, analgesic, anti-inflammatory, bronchodilator, anti-ulcer, anticancer, and immunomodulatory (mahboubi 2019). the yields of essential oil from ginger rhizomes zingiberaceae belongs to a large family of monocotyledonous perennial herbs. this plant is widely cultivated in tropical and subtropical regions (kun-hua et al. 2011), encompassing approximately 50 genera and more than 1,200 species (pintatum et al. 2020). ginger has been used widely as a spice, flavoring agent, and traditional remedy (abd el-hack et al. 2020). s t u d i e s r e v e a l e d t h a t g i n g e r h a d v a r i o u s pharmacological activities, such as antioxidant, antimicrobial, gastrointestinal modulating agent, range between 1–5% (bampidis et al. 2020). factors that affected the yields were the source, freshness, and the treatment (drying method) of rhizomes, as well as the distillation methods. the chemical constituents of geo primarily consist of monoterpenes, sesquiterpenes hydrocarbons, oxygenated sesquiterpenes, and phenylpropanoid compounds (mahboubi, 2019; pintatum et al. 2020). similar to the yields, the chemical compositions of the geos can greatly be affected by several factors i.e., the source of rhizome, its freshness or dryness, and the extraction methods used (mahboubi 2019). geo is mainly obtained from zingiber officinale, although other sources of zingiberaceae family are also available, such as z. zerumbet (koga et al. 2016; rana et al. 2017), z. cassumunar (bhuiyan et al. 2008), z. montanum (manochai et al. 2010), and z. kerrii (pintatum et al. 2020). the urgent need for antibiotics to overcome resistant bacteria underlines the importance of developing new antibiotics, one of which is by sourcing the lead compounds from natural products, including essential oils. similarly, the demands for natural food preservatives, as well as natural antioxidants for the cosmetic and food industry, are increasing. antioxidant activity of natural products is also of interest and has been investigated for its correlation to diseases, as the production of free radicals in the body correlates with various human diseases (lobo et al. 2010). as ginger has been used as traditional medicine and its medicinal properties have been widely investigated, its essential oils may be a promising source of these properties. therefore, this study aimed to elucidate the antibacterial and antioxidant activities of three different commercial geos. materials and methods procurement of geos. the essential oils of ginger were obtained through online commerce. gas chromatography-mass spectrometry analysis. the gc-ms analysis of geo was conducted using gc/ms apparatus (shimadzu qp 2010 ultra, japan) equipped with a low polarity column (30 m x 0.25 mm × 0.25 µm; restek-5ms, usa). helium as the -1 carrier gas was set at a flow rate of 0.98 ml min . the column temperature was raised to 80 °c and kept for 3 -1 min and steadily rose at 7.50 °c min to 220 °c and held for 5 min, followed by a temperature increment of -1 10 °c min rate and kept for 5 min at 270 °c. the prepared samples (10% geo in ethanol) were injected at 8 µl with a split ratio of 1:50. the peaks were identified by a combined search of retention time and comparison of ms fragments of the peak with mass spectra (nist 14 library). the percentages are calculated from the peak areas given by the gc-ms. antibacterial activity. the inhibitory effect of pure (100%) geos was tested using agar disc diffusion method against escherichia coli atcc 9637 (supelco, sigma aldrich), bacillus subtilis (collection of indonesia institute of sciences), and staphylococcus aureus atcc 13420 (supelco, sigma aldrich). the bacterial suspension was prepared using freshly grown bacteria in nutrient broth (nb; merck) that was incubated at 37 °c overnight with shaking at 180 rpm. the inoculum was diluted in liquid nutrient agar (na; merck) to achieve optical density (od) of 0.02 or 7 −1� 5×10 cfu ml at 600 nm. the inoculated na was then poured into disposable petri dishes (spl life sciences) and left to set. all samples and positive controls were applied to the blank paper discs (fioroni filters) at 10 µl and left to dry. as positive controls, streptomycin sulphate (merck) was used for b. subtilis (5 µg), whereas ampicillin (merck) was used for e. coli (10 µg) and s. aureus (3 µg). the diameter of the inhibition zone (diz) was measured after 18-24 h incubation at 37 °c. 2,2-diphenyl-1-picryl-hydrazyl (dpph) assay. the dpph assay was conducted based on a method described by yen and chen (1995) with slight modifications. briefly, stock solution of geos was prepared by diluting the samples in methanol to get -1 concentration of 100 µg ml . a total of 40 µl of a solution of 1 mm dpph in methanol was mixed with a volume of geo stock solution adjusted for a series of concentration and then added methanol to a final volume of 200 µl to get the the desired concentration. the solution was incubated for 30 min in the dark at room temperature. the absorbance of five concentration levels of the samples were measured at 515 nm using a microplate reader (tecan infinite m200 pro, switzerland). negative and positive controls were methanol and ascorbic acid (aa) (merck), respectively. the inhibition percentage of the samples was calculated according to the following formula: 2 , 2 ' a z i n o b i s ( 3 e t h y l b e n z o t h i a z o l i n e 6 sulfonic acid) (abts) assay. the abts radical scavenging assay method was adopted from lyu et al. (2020) with minor modifications. the radical mono cation of abts was prepared by mixing 7.4 mm abts with 2.6 mm potassium persulfate at 1:1 ratio, the 120 warsito et al. microbiol indones volume 15, 2021 microbiol indones 121 solution was incubated for 24 h in the dark at room temperature. the abts solution was diluted with phosphate-buffered saline (ph 7.4) to obtain an absorbance of 0.7 ± 0.02 at 734 nm. aliquots of samples (10 µl) at five concentration levels were reacted with 190 µl of the abts solution and incubated for 6 min. trolox was used as the positive control. the absorbance was measured at 734 nm using the tecan infinite m200 pro. antioxidant activities were calculated based on the equation below: the ic value shows the concentration of sample 50 that is able to inhibit the oxidation process by 50% obtained by making a linear curve between the concentration of the test solution (x-axis) and % antioxidant activity (y-axis). statistical analysis. the statistical analysis was evaluated using spss version 22 (spss inc.). the results were expressed as mean ± sd (standard deviation). pearson's correlation test was used to assess correlations between means. one-way analysis of variance was performed by anova and followed by tukey's multiple comparison tests. p < 0.05 was regarded as significant. result g c m s a n a l y s i s b a s e d c h e m i c a l compositions. fig 1 represents the total ion chromatogram (tic) of three commercial geo samples that resume intensities of all mass spectral peaks pertaining to the same scan. based on comparing retention time-ms fragments of the sample peaks with the mass spectra of a computer based-nist 14 library, the chemical compounds detected in the commercial geos tested and are listed in table 1. it is shown that the major compounds of geo1 were α-zingiberene (22.89%), camphene (16.94%), α-curcumene (11.62%), β-sesquiphellandrene (11.21%), βbisabolene (9.07%), α-pinene (5.04), and α-farnesene (4.46%). moreover, geo2 had α-pinene (25.72%) as the main component, followed by α-curcumene (11.88%), α-zingiberene (8.01%), 3-carene (7.71%), βsesquiphellandrene (6.81%), β-bisabolene (6.24%), and ar-tumerone (4.65%). while in geo3, the major identified compounds were α-zingiberene (24.88%), βsesquiphellandrene (13.67%), β-bisabolene (11.20%), α-curcumene (10.68%), α-farnesene (6.93%), camphene (5.08%), cis-sabinene (4.35%). antibacterial activity. in this study, all samples were tested for antibacterial activities against e. coli atcc 9637, b. subtilis (collection of lipi), and s. aureus atcc 13420. table 2 displays the extent of antibacterial activities of geos compared to the antibiotic controls when tested at a concentration of 100% with a volume of 10 µl. the antibacterial activities were measured based on the formation of diz and recorded in millimetres (mm). the results were categorized as follows: a diz of < 10 mm was taken as weak inhibition, a diz of 10-15 mm was taken as moderate inhibition, a diz of 16-20 mm was considered as strong inhibition, and above 25 mm was deemed as a very strong inhibition. geo 2 and geo 3 showed weak antimicrobial activity against all tested microorganisms, i.e., s. aureus, e. coli, and b. subtilis. on the other hand, geo 1 exhibited a weak activity only against the gram-positive bacteria, namely s. aureus and b. subtilis. antioxidant activity. the antioxidant activity of geos expressed as ic values is illustrated in figure 2. 50 the ic values of geo1, geo2, and geo3 when 50 evaluated using dpph assay (figure 2a) were 5.3023 ± -1 -1 0.1486 mg ml , 0.9249 ± 0.0138 mg ml , 10.4463 ± -1 0.4662 mg ml , respectively. while the ic values 50 when assessed using abts assay (figure 2b) were -1 -1 1.4504 ± 0.0551 mg ml , 0.5276 ± 0.0107 mg ml , -1 and 3.3535 ± 0.1601 mg ml for geo1, geo2, and geo 3, respectively. a similar pattern was observed with both methods as geo2 displayed the highest overall antioxidant activity, while geo3 exhibited the least activity. overall, all the geos tested had significantly lower anti-radical scavenging activity (p < 0.05) compared to the positive control used in the -1 tests, ascorbic acid (ic = 0.0059 ± 0.0002 mg ml ) in 50 the dpph assay and trolox (ic = 0.0054 ± 0.0001 mg 50 -1 ml ) in the abts assay. discussion the complex and various constituents of essential oils, including geos, possess a wide array of biological properties (miguel, 2010), including antimicrobial and antioxidant properties of geos (bellik 2014; mahboubi 2019; wang et al. 2020). the urgent need to find new sources of antimicrobial compounds and the demands for naturally-sourced preservatives are a few reasons why geo and other essential oils are often investigated for their antimicrobial and antioxidant properties. this present study aims to characterise the chemical constituents of three commercially-available geos and elaborate on 122 warsito et al. microbiol indones fig 1 total ion chromatogram of geos. (blue = geo1; black = geo2; pink = geo3). fig 2 antioxidant activity of geos based on the (a) dpph and (b) abts methods. the numbers in each bar followed by the same letter are not significantly different at the 0.05 level of tukey's multiple comparison tests. their antibacterial and antioxidant properties. it was known from the label and/or website of the ecommerce that geo1 and geo3 were produced from the root part of z. officinale r., while the geo3 source was unknown or not mentioned. previous studies revealed that the major component of z. officinale is zingiberene (kamaliroostaa et al. 2013; ribeiro et al. 2015; sharma et al. 2016; mahboubi, 2019). those were different from geo2, which contained α-pinene as the major component (25.72%). a study by pintatum et al. (2020) also identified α-pinene as the main component of z. kerrii essential oil, suggesting the presence of α-pinene can be used as the basis of identifying the source of geo within zingiberaceae family members with a high content of α-pinene. geo has been reported to have various pharmacological and biological activities, including antimicrobial and antioxidant activities (mahboubi, 2019). geo has shown antimicrobial activity against s. aureus, listeria monocytogenes, pseudomonas aeruginosa, b. subtilis, e. coli, acinetobacter baumannii, s. pyogenes, s. typhi, and multidrugresistant (mdr) a. baumannii (stoyanova et al. 2006; mesomo et al. 2013; meliani et al. 2014; wang et al. 2020). in this study, weak inhibitory effects against e. coli atcc 9637, b. subtilis, and s. aureus atcc 13420 were observed in all samples (table 2). this finding was similar to earlier studies conducted by bag and chattopadhyay (2015) and mostofa et al. (2018). on the other hand, a number of studies reported good antimicrobial activity (sivasothy et al. 2011; silva et al. 2018; mahboubi, 2019), which suggested that the volume 15, 2021 microbiol indones 123 table 1 chemical compositions of the commercial geos no. rt (min) compounds % area geo1 geo2 geo3 1 4.021 tricyclene 0.53 2 4.191 α-pinene 5.04 25.72 1.95 3 4.469 camphene 16.94 1.28 5.08 4 4.956 β-pinene 0.37 1.24 5 5.090 β-myrcene 1.57 0.41 0.51 6 5.552 3-carene 7.71 7 5.813 o-cymene 0.55 8 5.918 d-limonene 1.82 1.80 9 5.937 cis-sabinene 4.35 10 5.940 α-phellandrene 1.76 11 5.998 eucalyptol 1.31 0.83 2.60 12 7.156 α-terpinolene 0.46 0.86 0.26 13 7.317 linalool 0.51 0.32 14 8.867 endo-borneol 0.38 0.45 1.04 15 9.319 α-terpineol 0.53 0.57 16 10.343 neral 0.74 17 10.928 citral 1.03 0.23 18 11.306 bornyl acetate 0.69 19 11.502 carvacrol 1.01 20 12.658 3-allylguaiacol 1.28 21 13.114 copaene 0.58 0.39 0.82 22 13.381 β-elemene 0.58 0.41 1.34 23 13.957 caryophyllene 0.82 24 14.116 γ-elemene 0.98 25 14.402 (e)-β-famesene 0.55 26 14.655 ethyl cinnamate 1.22 27 14.956 α-curcumene 11.62 11.88 10.68 28 15.091 pentadecane 2.19 29 15.065 germacrene d 2.36 30 15.200 α-zingiberene 22.89 8.01 24.88 31 15.348 α-farnesene 4.46 2.70 6.93 32 15.435 β-bisabolene 9.07 6.24 11.20 33 15.455 β-curcumene 0.51 34 15.608 cis-muurola-4(15),5-diene 0.88 35 15.705 β-sesquiphellandrene 11.21 6.81 13.67 36 15.834 trans-γ-bisabolene 0.69 37 16.186 elemol 0.42 0.95 38 16.283 trans-nerolidol 0.56 0.59 39 17.203 zingiberenol 0.40 0.79 124 warsito et al. microbiol indones table 1 chemical compositions of the commercial geos, continuedtable 2 antibacterial activity of geos sample diameter of inhibition zone (mm)* s. aureus atcc 13420 e. coli atcc 9637 b. subtilis geo1 7.60 ± 0.24 0.00 ± 0.00 7.97 ± 0.33 geo2 7.17 ± 0.05 7.85 ± 0.31 7.42 ± 0.02 geo3 7.30 ± 0.08 7.20 ± 0.08 8.37 ± 0.33 antibiotic control# 21.52 ± 0.22 17.63 ± 0.13 14.32 ± 0.30 differences in the results may be attributed to the difference in assay methods employed in these studies. the physicochemical properties may affect the diffusion of the oil component into the agar, which interferes with the result. dilution of the geo to a surfactant may help to increase the diffusion ability and lower the density of the oil, which in turn may improve the antibacterial activity (li et al. 2015). the antimicrobial activity of ginger geos has been attributed to these chemical constituents, namely βsesquiphellandrene, caryophyllene, α-zingiberene, αfarnesene, ar-curcumene, citral, β-bisabolene, geranyl acetate, and geraniol (mahboubi 2019). these compounds were identified in the geos tested; thus, it is likely that the diffusion of the oils to the agar media was not optimal, which resulted in the lower antibacterial activity than expected. in addition, it is also likely that the lower abundance of phenolic compounds contributed to the weak antibacterial activity. this finding is in accordance with a previous study by pintatum et al. (2020) where z. kerrii extracts with fewer phenolic compounds did not display good antibacterial activity. it is also interesting to note that for geo1, s. aureus and b. subtilis were more susceptible to geo1 compared to e. coli as no diz was observed against e. coli. this finding is similar to a previous study where extracted ginger geo exhibited a better antibacterial activity against s. aureus than e. coli, potentially attributed to the structure of gram-negative bacteria that provides an additional protective barrier against antimicrobial compounds (wang et al. 2020). other studies also revealed that geo had a better antibacterial activity against s. aureus compared to e. coli (bellik 2014; mahboubi 2019; mesomo et al. 2013). the variation in the antibacterial activities of the three geos is likely to be influenced by the differences in the overall chemical compositions detected (mahboubi 2019), which may play different roles in exerting their activities against the tested bacterial strains. afterall, ginger geos can affect bacterial viability via multiple mechanisms, namely by disrupting the cell membrane permeability, interfering with the respiratory metabolism, and inhibiting dna metabolism activities (wang et al. 2020). the antioxidant activity of geos in the present study was tested using dpph and abts methods. the higher ic values obtained using dpph compared to 50 abts method (fig 2) is also similar to the results obtained in a previous study by höfer et al. (2015) where a number of methods measuring the antioxidant activity of geo were employed. geo was found to be no. rt (min) compounds % area geo1 geo2 geo3 40 17.480 β-acorenol 0.98 41 17.482 7-epi-cis-sesquisabinene hydrate 0.33 0.69 42 17.620 aromandendrene 0.55 43 17.974 β-eudesmol 0.52 44 18.044 ar-turmerone 4.65 45 18.116 tumerone 1.08 46 18.646 curlone 1.55 47 19.450 ethyl p-methoxycinnamate 1.82 volume 15, 2021 microbiol indones 125 a weak dpph reducer compared to bht (höferl et al. 2015), thus the use of other methods may elaborate further on the antioxidant activity of geos tested in this study. different plant essential oils may exhibit varying antioxidant activity when different tests are employed, as the chemical compositions of the essential oils are also different and may exert antioxidant activity by various mechanisms (miguel 2010). plants secondary metabolites that contributed to the antioxidant activities are from the phenolic groups (minatel et al. 2017), which play an important role in neutralizing the free radicals and inhibiting oxidative damage (pintatum et al. 2020). phenolic compounds are the main components that play a role in the antioxidant activity of ginger (yeh et al. 2014). as such, phenolic compounds correlate with the antioxidant activity, as exemplified in a study by pintatum et al. (2020), where a lower abundance of phenolic compounds compared to terpenoid compounds in z. kerrii extract was suggested to be associated with the weak antioxidant activity observed. the present study showed intriguing result that the antioxidant activity of geo2 was the highest, considering that the major component found was αpinene, a non-phenolic compound. despite weak antioxidant activity as tested with dpph and abts methods observed in a range of plant essential oils when low levels or absence of phenolic compounds are detected (miguel 2010), a study on essential oils from different plant parts of myrtus communis var italica showed the lack of phenolic compounds and the presence of α-pinene still translated into antioxidant activity (wannes et al. 2010). furthermore, it is also likely that the presence of some oxygenated monoterpenes such as carvacrol and α-terpineol (albeit in small amounts) may also contribute to the antioxidant activity of geo2, as such compounds have been attributed to antioxidant activity of essential oils (bayala et al. 2014). the chemical constituents of geos primarily consist of monoterpene and sesquiterpene compounds. the most abundant compound present in geos from zingiber officinale was α-zingiberene at 22.89% and 24.88% concentrations for geo1 and geo3, respectively. in comparison, geo2 constituted αpinene (25.72%) as the main component. the geos showed low antibacterial and antioxidant activities compared to the respective positive controls. the low antibacterial activity observed in this study was likely caused by the dense characteristic of the oil that caused difficulty to diffuse into the agar media. therefore, it is recommended to mix the geos with surfactant and use a dilution method to evaluate the antibacterial activity. meanwhile, the low antioxidant activity of the geos may be due to the few phenolic compounds present. as different combinations of compounds may contribute to antioxidant activity, it would be desired 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harzianum fusarium associated ith tomato wilt?w wilfridus adyatma putranto , rully adi nugroho , petrus sunu 1 1* hardiyanta desti christian cahyaningrum 2 1 , and 1 faculty of biology, satya wacana christian university, jalan diponegoro 52-60 salatiga 50711 indonesia; 2faculty of pharmacy, sanata dharma university, jalan affandi, mrican yogyakarta 55281 indonesia. the pathogenic fungi, such as in the rhizosphere of tomato ( ) negatively fusarium solanum lycopersicum affects the yield and quality of the plant. a number of biological control agents have been used for protecting tomato plants against wilt diseases including various fungal species. the objective of this study was to evaluate the antagonism effects of sp. associated t atroviride t. harzianum fusariumrichoderma and against the pathogen with tomato wilt in this study, the antagonism of these spp. against the sp. was tested . trichoderma fusarium in vitro by the dual culture technique, and the percentage inhibition of radial growth (pirg) and the antagonism reaction (scale 1-5) were evaluated. the results showed that . and . led to 70.8% pirg and t atroviride t harzianum scale 1 antagonism reaction, and 40.6% pirg and scale 3 antagonism reaction against sp. associated fusarium with tomato wilt after 7 days of incubation, respectively. these results indicate that application of . and t atroviride t harzianum fusarium. may be promising approach for biological control of wilt of tomato and may play an important role in sustainable agriculture. biological , , tomato, key words: antimicrobial activity, control wilt spp. f tusarium richoderma jamur patogen, seperti di rizosfer tomat ( ) berdampak negatif terhadap hasil fusarium solanum lycopersicum dan kualitas tanaman. sejumlah agen pengendali hayati, termasuk berbagai jenis jamur telah digunakan untuk melindungi tanaman tomat dari penyakit layu. penelitian ini bertujuan untuk mengevaluasi dampak antagonisme trichoderma atroviride t harzianum fusarium and . terhadap patogen sp. penyebab layu pada tomat. dalam penelitian ini antagonisme spp. terhadap sp. diuji secara in vitro dengan teknik kultur trichoderma fusarium ganda, dan persentase penghambatan pertumbuhan radial (pppr) dan reaksi antagonisme (skala 1-5) dievaluasi. hasil penelitian menunjukkan bahwa . dan . masing-masing menyebabkan 70,8% pppr t atroviride t harzianum dan reaksi antagonisme skala 1, dan 40,6% pppr dan reaksi antagonisme skala 3 terhadap sp. penyebab fusarium layu pada tomat setelah 7 hari inkubasi. hasil ini mengindikasikan bahwa aplikasi . dan . t atroviride t harzianum dapat menjadi pendekatan yang menjanjikan untuk pengendalian hayati layu tomat dan dapat fusarium memainkan peran penting dalam pertanian berkelanjutan. kata kunci: aktivitas antimikroba, pengendalian biologi, layu , tomat, spp.fusarium trichoderma microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 62-298-321212 ; : + fax: +62298-321433; rully.nugroho@uksw.edu e-mail: fusarium become harmful organisms for tomato plants (solanum lycopersicum) because it will cause tomato plants to be damaged and harvest failure. tomato plants that are attacked by will turn yellow on the f. oxysporum leaves, the plants will wither, growth will be stunted, the fruit will rot, then the plant will die and necrosis on one side of the stem. symptoms of necrosis begin with a change in color, then gradually dry up (wiam et al. 2019). seedlings exposed to f. sp. f. oxysporum lycopersici experience slow growth, while plants infected with cause root rot partially or fusarium completely, leaves turn yellow and then curl, plants are stunted, wilted, and may die completely (blancard 2012; hermann and lecomte 2019). can fusarium produce mycotoxins tricotisin, fumonisin, intentin, zearalenone, beauverisin, moniliformin, fusarin, fusaric acid, and fusaproliferin affect human and which can animal health (wang 2011).when ingested et al. fusarium is one of the most economically important genera of fungal plant pathogens, causing significant crop losses and contamination of grain by mycotoxins on a global basis (burgess and bryden 2012). the species most commonly involved include f. graminearum f. oxysporum et al. and (dean 2012). fusarium colonizes the xylem vessels producing mycelium and conidia. the characteristic wilt symptoms appear as a result of water stress, mainly due to vessel clogging (beckman 1987). fusarium wilt of tomato is commonly caused by the fungal pathogens, . , . or . f oxysporum f solani f equiseti (isaac . 2018; ayele . 2021). the most familiar et al et al formae species to tomato is f.sp. fusarium oxysporum lycopersici et al et al (isaac . 2018; srinivas . 2019). farmers use chemical fungicides such as benomyl, mancozeb, , and dithiocarbamate phenylamide to treat problems caused by pathogenic fungi (apriani . et al 2014; sumardiyono . 1995; et al worku and sahela 2018). however, long-term use of fungicides can affect non-target organisms such as earthworms, microbes and humans (patel 2014). biological control et al. technology plays an important role in overcoming diseases in plants that are promising and safe for humans and do not pollute the environment (kang 2019). currently there are more than 60% of , biological control species that use (abbey trichoderma et al. 2019). can be found at trichoderma species temperatures of 25-35°c (hajieghrari 2008) in et al. tropical soils, and can also be found on soils in forest, agricultural, and grassland areas (kubicek 2003). et al. as many as 35 of the 260 live trichoderma species that in roots (digamber 2017) have good economic importance because of their ability to produce enzymes, antibiotics, and secondary metabolites so that they can be used as effective biological control agents (blaszczyk 2014). is known as a et al. trichoderma biological control agent because it can survive in all kinds of conditions, has a high reproductive rate, is efficient in the absorption of nutrients from its surroundings, and shows aggressive resistance to a fungal pathogen (misra and prasad 2003 pandya ; et al. 2011). zhang (2017) stated that showed et al. trichoderma antagonistic behaviour against several phytopathogenic organisms, including bacteria, nematodes and fungi by inhibiting their growth. (jiang t asperellumrichoderma et al. t. harzianum et al. t. 2016), (ezziyyani 2007), koningiopsis et al. t. virens (delgado 2018), and (tomah 2020) have been shown to be highly et al. effective in the management of . phytophthora capsici according to jiang (2016) hyphae from isolates of et al. t. asperellum p. can penetrate hyphae and spores of capsici in the form of mycoparasitism which causes hyphal cells to degrade. mycoparasitism involves cell wall-degrading enzymes that allow mycoparasitic fungi to drill holes into other fungi and extract nutrients for their own growth (cao 2009). strains et al. trichoderma produce antibiotics or low molecular weight compounds that are useful for inhibiting the growth of plant pathogens such as 6-pentyl-pyrone (jelen et al. 2014), viridiofungin (el-hasan 2009), and et al. gliotoxin (roberts and lumsden 1990). as an interesting model for being trichoderma studied about interactions between plant hosts and its symbionts should be as aconsidered biological control alternative in the green economy era which aims to maintain human health, protect the environment, and as a promotional agent sustainable agriculture (lopezbucio 2015; guzmán 2017; sood et al. et al. et -gusmán al. 2020). therefore, this study was conducted to test whether can overcome the t. atroviride t. harzianumor pathogen tomato so that fusarium associated with wilt these two species can be used as trichoderma biocontrol agents. materials and methods isolation of ungal trains. f s the trichoderma atroviride t. harzianum and isolates were obtained from , salatiga, kptt agricultural training center indonesia and recultured in potato dextrose agar (pda merck). isolates were , spp. trichoderma incubated at 25°c for 7 days (ramteke 2019). the patogen, sp. was isolated according to a fusarium procedure developed by and (leslie summerell 2006) from the wilt infected roots of tomato plants which were collected from kptt agricultural training center . grown , salatiga, indonesia the isolates were on komada's selective medium and incubated at 30°c for 7 days, then pda and subcultured on (merck) incubated at 25°c for 5 days leslie summerell ( and 2006 . ) sp. isolate was identified on the basis fusarium of cultural and morphological characteristics which included colony color, pigmentation, surface texture, colony edge, conidiophores, septate or anseptate hyphae, presence or absence of phialid, conidia color, conidia shape, macrocoonidia or microconidia shape, presence or absence of chlamydospores (mwaniki et al. et al. 2011; sharma singh 2014; rai 2016; and redda 2018). et al. the in vitro xperiment. e effects of trichoderma spp. on were tested by the dual culture fusarium technique as described by the redda (2018). et al. trichoderma fusarium spp. isolates and sp. to be tested were cultured separately on pda for 7 days. after 7 days, 5 mm mycelial plugs (taken from the edge of fungal colonies) of each species to be tested were transferred to pda plates using cork borer. the mycelial plug of species and trichoderma fusarium sp. was placed opposite side to each other on a pda surface. pda plates inoculated with sp. was fusarium included as negative control. all plates were incubated at 25 2 c and observations were made after 7 days of + ° incubations. the percentage inhibition of radial growth (pirg) was calculated by the following formula volume 15, 2021 microbiol indones 85 86 putranto et al . microbiol indones ( ):sharfuddin and mohanka 2012 pirg = (r r )/r1 2 1×100 where radial growth of sp. in control r = 1 fusarium (cm), radial growth of sp. in dual culture r = 2 fusarium tests with spp. (cm). the antagonism trichoderma reaction of isolates were score using a trichoderma scale of 1 to 5 after 7 days of incubation, where 1 = trichoderma gr over ew the entire growth of the fusarium sp., over ew at -2 = gr least twotrichoderma third of the medium 3 = and surface, trichoderma fusarium sp. each one colonized half of the medium surface, sp. colonized at least two-third 4 = fusarium of the medium surface, sp. overgrew5 = fusarium trichoderma ( ).bell . 1982et al statistical .analysis were analysed using data spss 22 (spss inc., chicago, il). all data were calculated and analysed statistically using test (t p < 0.05) to determine differences between treatments. the table was drawn using microsoft excel 2016. result isolation and dentification of sp. i fusarium results showed that sp. had well-defined fusarium macroscopic characteristics (fig 1). fusarium sp. has pink mycelia in the center and white at the edges. fusarium sp. underwent pigmentation changes where the mycelia of sp. on the first and second day fusarium it is white and on the fifth day it changes color to a slightly brownish beige. in addition, the surface texture of sp. has a cotton-like texture and has a fusarium filamentous shape and colony edge. conidiophores fusarium sp. are dendritic conidiophores, possessing pseudohyphae, but no phialids. color of conidia fusarium sp. is a fusoidal green with thickened walls at the ends. ha chlamydiophores that are fusarium s shaped like lumps on hyphae. the effects of t fusariumrichoderma spp. on sp. using assaysin vitro . table 1 showed the pirg observed on the first until the seventh day of the test. the inhibitions of . and . are t atroviride t harzianum observed from the second and the third day of incubation, respectively. both isolates were shown to have inhibitory activity on pathogen in vitro fusarium (table 1). was significantly ( trichoderma atroviride p < 0.05) more effective as it was able to inhibit at 70.8% of sp. after 7 days of incubation, while . fusarium t harzianum demonstrated lower pirg (40.6%) after 7 days of incubation. in fig 2, a 7 day test of sp. fusarium grown with . and . clearly t atroviride t harzianum demonstrated that the mean scale for antagonism of . t atroviride t was also significantly higher than . harzianum, i.e. scale of 1 and 3, respectively. discussion the isolate obtained from tomato rhizosphere soil was confirmed as sp. the morphological fusarium characteristics are the same as mentioned by gordon (2017) and srinivas . (2019). the result of this et al study supports findings in other studies that characterized as most important fungal in fusarium tomato plantation areas in defferent countries of the world (grattidge and o'brien 1982; jones . 1991; et al steinkellner . 2005; rozlianah and sariah 2006; et al amini 2009; chehri 2016). fung that the trichoderma species are i have potential to control pathogenic fungi, has a low impact on soil balance, and has minimal impact on damage to non-target organisms (sood 2020).et al. consistent with previous findings, this study confirms the antagonism effect of .t atroviride t. harzianum and against the pathogen fusarium sp. associated with tomato wilt. the antagonism effects of both trichoderma spp. sp. showed that against fusarium both spp. affecting the development trichoderma pattern of sp. colonies. fusarium trichoderma colonies presented a faster growth in the plates of dual cultures, being capable of growing on the fusarium sp., preventing their mycelial development by nutrient and space competition as found by filizola . et al (2019). moreover, based on the antagonistic activity (soytong 1988), this study indicated that . t atroviride exhibited a very high antagonistic activity against fusarium t harzianum sp. (>75% pirg), while . table 1 the percentage inhibition of radial growth (pirg) in dual culture assay. results are expressed as mean standard deviation of pirg after 1to 7-day incubation. nd = not determined+ trichoderma strains 1-day 2-day 3-day 4-day 5-day 6-day 7-day t. atroviride nd 2.9 ± 11.94 36.5 ± 6.50 51.7 ± 3.39 60.5 ± 2.44 66.7 ± 2.34 70.8 ± 2.03 t. harzianum nd nd 5.0 ± 8.69 6.1 ± 7.08 21.3 ± 5.67 32.3 ± 4.46 40.6 ± 3.76 percentage inhibition of radial growth (pirg) (%) volume 15, 2021 microbiol indones 87 fig 1 characterization of sp. by macroscopic morphology in pda medium after 7 days. a. colony fusarium morphology; b. conidiosphores and conidia. magnification 400× . fig 2 dual culture assay after 7 days of incubation. a. / sp.; b. trichoderma virid fusarium trichoderma atro e harzianum fusarium tv trichoderma atroviride th trichoderma harzianum f fusarium/ sp.; : ; : ; : sp. showed low antagonistic activity against sp. fusarium (<50% pirg). this study shows that . t atroviride demonstrated more antagonistic activity than observed by stracquadanio . (2020). stracquadanio . et al et al (2020) found that . did not reduce the t harzianum growth of . this shows that not fusarium moniliforme all strains are able to inhibit the in vitro trichoderma growth of .fusarium previous studies have shown that has trichoderma several modes of action, including production of antibiotics and cell wall degrading enzymes, competition for key nutrients, parasitism, stimulation of plant defense mechanisms and combination of these possibilities (fravel 1988; larkin and fravel 1988; roberts and lumsden 1990 el-hasan 2009; ; john et al. et al. 2010; ; ; taghdi jelen 2014 jiang 016 et al. et al. 2 et al et . 2015). according to howel (2006) and zhang al trichoderma. 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ther etabolites from trichoderma s p n a t p ro d r e s 3 1 ( 2 3) 2 74 5 2 7 5 2 . d o i : . . : 10.1080/14786419.2017.1295235. jumsu_401.pmd volume 3, number 2, august 2009 p 56-61 issn 1978-3477 *corresponding author:, phone: +62-751-72701, fax: +62-751-72702, e-mail: ano_trisno@yahoo.co.id detection and sequence diversity of begomovirus associated with yellow leaf curl disease of pepper (capsicum annuum) in west sumatra, indonesia jumsu trisno1,2*, sri hendrastuti hidayat1, trimurti habazar2, ishak manti4, and jamsari3, 1department of plant protection, faculty of agriculture, institut pertanian bogor, darmaga campus, bogor 16680, indonesia, 2department of plant pests and diseases, 3department of agronomy, faculty of agriculture, universitas andalas, limau manis campus, padang 25162, indonesia 4balai pengkajian teknologi pertanian sumatera barat, sukarami, solok, po. box 34, padang 25001, indonesia yellow leaf curl disease of pepper has become an emerging important disease in west sumatra since early 2000. several attempts have been made, including disease survey and detection, in order to identify the causal agent of the disease. pepper (capsicum annuum) plants showing yellow leaf curl from west sumatra were analyzed for presence of begomovirus employing polymerase chain reaction (pcr) with degenerate primers pal1v 1978 and parc 715. a dna fragment of 1.6 kb was successfully amplified and subjected to direct sequencing. a stem loop region was found in the nucleotide sequence obtained, which contains the conserved nucleotide signature sequence taatattac present in begomoviruses. based on the stem loop region comparisons and phylogenetic analysis, the virus isolates from west sumatra showed the closest relationship to pepper yellow leaf curl indonesia virus (pylviv) and tomato yellow leaf curl indonesia virus (tylciv). the sequence was different from other asia begomoviruses reported earlier. these isolates were divided into three groups which were tentatively called pepper yellow leaf curl indonesia virus-west sumatra-[group 1], -[group 2] and -[group 3] {pylciv-ws-[group1], -[group2], and [group3]}. key words: begomovirus, pepper yellow leaf curl virus, sequence analysis begomovirus is a member of family geminiviridae, which is largest group of plant viruses. the geminivirus is a group of plant viruses with distinct morphological characteristies. it’s twinned isometric particles consist of circular singlestranded (ss) dna genomes (gutierrez 2000). geminiviruses are subdivided into four genera on the basis of host range, insect vector and genome organization. the genus begomovirus (sub group iii) consists of viruses that are transmitted by whiteflies to infect dicotyledonous plants with a monopartite or bipartite arrangement (fauquet and stanley 2003). begomovirus assosiated with pepper yellow leaf curl disease has become a major problem chillie pepper growing and causes crop damage in many tropical and subtropical regions wordwide (varma and malathi 2003). in indonesia, a virus infection causing yellow mosaic, upward leaf curling and stunting was observed infecting pepper throughout production area in west java, central java and jogyakarta. begomovirus-association with this disease was confirmed using polymerase chain reaction (pcr) method, and the virus was tentatively named pepper yellow leaf curl virus (pylcv) (sulandari 2004). early studies conducted by rusli et al. (1999) and sulandari (2004) showed that pylcv was not either seed or mechanically-transmitted, although the virus was easily transmitted through grafting and whitefly, bemisia tabaci genn. (hemiptera: aleyrodidae). characterization of a partial genomic sequence of the pylcv was reported later (sukamto et al. 2005; hidayat et al. 2006). the begomovirus associated with yellow leaf curl disease in pepper in west java (accession number ab246170) showed the highest sequence identity of 93 and 98% with pepper yellow leaf curl indonesia virus (pylciv) from capsicum annuum (ab189845) and pylciv from lycopersicon esculentum (ab189850), respectively. occurrence of pepper yellow leaf curl disease was reported from almost all pepper growing areas in indonesia (directorate of plant protection, personal communication), including west sumatra. a preliminary survey in several regions in west sumatra showed that disease incidence could reach 100 % (syaiful 2005). in this paper, we reported sequence analysis of the intergenic region isolated from eleven begomovirus isolates infecting pepper plants collected from different location in west sumatra. materials and methods sample collection. pepper plants showing typical symptoms of begomovirus infection (yellow mosaic, leaf curling, smaller leaflet, cupping and stunting) were collected from several pepper producing areas (18 areas, 6 district) in west sumatra (fig 1). observation was conducted directly when the plants were at the flowering stage in the field with 300-500 m2 area. samples were taken with purposive-randomsampling according to the typical begomovirus symptom. five samples were taken for each location that represent each typical symptom, thus the number of samples was 90. samples were placed in plastic bags and carried to the laboratory for dna extraction and to a screenhouse for virus isolation and propagation in the host plant. at the same time, disease incidence (%) was determined by counting the number of plants that showed the symptom from 10% of the plant population. the sample plants were determined according to systematic random sampling. however, desease severity was determined by using formula 1, with a modified scale of category according to lapidot et al. (2001). symptom microbiol indones 57trisno et al. development was evaluated according to the following scale: 0, no visible symptoms; 1, very slight yellowing of leaflet margin on apical leaf; 2, some yellowing and minor curling of leafled ends; 3, a wide range of leaf yellowing, curling and cupping, with some reduction in size, yet plants continue to develop; and 4, very severe plant stunting and yellowing, pronounced leaf cupping and curling, plant growth stopped. disease severity = %100 )( x zxn xvn ii ∑ ...........................(1) n i , sample plant in i; v i , value of symptom scale in i; z, value of highest symptom scale; n, number of observed sample plants dna extraction and pcr analysis. total dna was extracted from pepper leaves according to the method of doyle and doyle (1999) with slight modification. leaf tissue was ground in a sterile mortar in 1.0 ml of extraction buffer. the extraction buffer used for the initial homogenization contained 100 mm tris ph 8.0, 1.4 mm nacl, 20 mm edta ph 8.0 and 0.2% (v/v) β-mercaptoethanol. the extraction buffer was autoclaved and 2% (v/v) polyvinylpyrolidone (pvp) and 2% (w/v) ctab were added immediately before use. immediately after grinding, 500 µl aliquotes were transferred to a 1.5 ml microtube and incubated for 15 min at 65 °c with occasional mixing to avoid aggregation of the homogenate. to the extract was added 500 µl of chloroform:isoamylalcohol (24:1) and the mixture was vortexed thoroughly. each tube was then centrifuged for 15 min at 10 000 x g. the debris-free supernatant was then transfered to a new tube and proteins precipitated by adding 2.5 x volumes of absolute ethanol and washed twice with 70% ethanol (v/v). the pellet was dried and resuspended in 100 µl of sterilized water. this dna extract was stored at -20 °c for further use. the begomovirus genome was amplified by the pcr technique using two oligonucleotide degenerate primers for the geminivirus, palv1978 (5’-gcatctgcaggcccaca tygtcttyccngt-3’) and parc715(5’-gatttctgca gttdatrttytcrtccatcca-3’) (rojas et al. 1993). pcr reactions were prepared in 25 µl total volume, containing 10 x buffer (100 mm tris-hcl, 500 mm kcl, ph 8.3), dntp mix (4 mm), 10 mm of each primer, 1 unit of taq dna polymerase and 2 ml of the dna template. the amplification profile should be referred to rojas et al. (1993). nucleotide sequencing and phylogenetic analysis. the produc+t of the pcr amplification, approximately 1.6 kb length, was sequenced by the dideoxy-nucleotide-chain termination method with abi-prims 3100-avant genetic analyzer (applied biosystem, foster city, ca, usa) at pt. charoen pokphand indonesia, tbk, jakarta. sequence data obtained was compared with those of other geminiviruses available in genebank using the national center of biotechnology information basic local alignment search tool or ncbi blas (http://www.ncbi.nlm.nih.gov/blast). the sequences were aligned using clustal w, while phylogenetic analysis was conducted using online tool facilities available at http://www.genebee.msu.su/clustal/advaned.htm1. results of the analysis were used to construct a phylogenetic tree and the robustness of the internal branches of the tree was tested by bootstrap analysis using 100 replicates. results disease symptoms. our survey was conducted in 18 pepper-producing areas covering 6 districts in west sumatra from may to october 2007 (fig 1). it was evident that the pepper yellow leaf curl disease occurred in all the surveyed regions, with the disease incidence ranging from 37.8 to 97.0%. infection of the virus may cause various symptoms including yellowing, mosaic, curling, cupping and stunting. table 1 isolate identity, observed symptoms on collected pepper samples and disease incidence of pepper yellow leaf curl disease in west sumatra, indonesia location (district) pesisir selatan pasaman barat solok limapuluh kota tanah datar agam isolate identity peppss1-1 peppss2-3 peppbst2-3 pepso2-1 pepso3-5 p e p p y k 1 p e p t d 1 3 p e p t d 2 1 pepag1-4 pepag2-1 pepag2-4 observed symptoms on collected samples y, scr, scp m, scr y, cr, cp, st y, scr, st m, cr, cp, sl, st y, scr. cp m, sc y, cr,cp, sl, st m, scr, cp, sl m, cr, scp, st m, cr, cp, sl, st disease incidence (%) 53.0-80.0 92.5-97.0 63.3-81.7 80.0-85.0 37.8-71.7 86.7-95.0 y, yellowing; scr, severe curling; cr, curling; scp, severe cupping; cp, cupping; m, mosaic; st, stunting; sl, smaller leaf. fig 1 sampling location in west sumatra to collect pepperinfected begomovirus. l, low altitude (< 400 m: pasaman, padang, pesisir selatan); , medium altitude (400-700 m: solok, limapuluh kota); , high altitude (>700 m: tanah datar, agam). microbiol indones58 volume 3, 2009 symptoms variability was found for each region (table 1). five samples were taken from each of the 18 locations, with different and varied symptoms, so that the total samples used was. therefore, one isolate was selected for each symptom type for further detection and identification. detection of begomovirus by pcr and sequence characterization. specific dna fragments of 1600 bp were succesfully amplified from 90 leaf samples using geminivirus degenerate primers palv1978 and parc715 (fig 2). the amplified dna fragment, denoted as “top fragment”, include part of replicase gene, the full common region and part of the coat protein gene. eleven isolates from 90 samples representing six different districts with specific symptoms were subjected to direct sequencing. these isolates consisted of two samples from pesisir selatan (peppss1-1 and peppss2-3), one sample from pasaman barat (peppsbt-1), two samples from solok (pepso2-1 and pepso3-5), one sample from limapuluh kota (peppy-1), two samples from tanah datar (peptd1-3 and peptd2-1) and three samples from agam (pepag2-1, pepag2-4 and pepag1-4) (table 1). the sequence of the “top fragment” contains the nonanucleotide sequence 5’-taatattac-3’ as a part of a hairpin loop structure, and repetitive sequences known as an iteron. in his study, the hairpin loop structure was found in all sequence of begomovirus samples from west sumatra. variability in the structure and the length of hairpin loop region were observed among west sumatran samples with only three samples having similar structure, i.e. peppyk1, pepag2-1 dan pepag2-4 (fig 3). the shortest hairpin loop region was 27 bases (pepso3-5) whereas the longest was 33 bases (peptd1-3 and pepag1-4). the isolates with similar hairpin loop length, contain different structure of gc and at pairs. similar repetitive sequence 5’-ggagaca-3’ was found in all of the samples although their relative position to common region varies (table 2). phylogenetic analysis. relationship between isolates of begomovirus from west sumatra and other selected begomovirus in genebank (table 3) was evaluated based on “top fragment” sequences. eleven begomovirus isolates determined in this research could be differentiated into three groups (fig 4). the first group consisted of 6 isolates (peptd1-3, pepag1-4, pepso3-5, pepag2-4, pepag2-1 and peppss1-1). the second group consisted of 4 isolates (peppbst2-3, peptd2-1, peppss2-3 and pepso2-1), where as the third group consisted of a one isolate, i.e peppyk1. it is interesting to notice that all isolates collected from agam (pepag2-1, pepag2-4 and pepag1-4) belong to the same group 1, where as other isolates collected from same location belong to different group, for instance peptd1-3 and peptd 2-1, peppss1-1 and peppss2-3, pepso3-5 and pepso2-1. begomovirus infecting pepper in indonesia that have been reported earlier (pylciv-bgr, pylciv-to, pylciv-lbg, and pylciv-ageratum) was not belong in the same group with the begomovirus identified in the present research. however, they are closely related with group 2 of the begomovirus identified from west sumatra. in general begomovirus isolates from west sumatra were tentatively called pepper yellow leaf curl indonesia viruswest sumatera-[group1], -[group2] and -[group3] {pylciv-ws-[group1], -[group2] and -[group3]}, although sequence diversity occurred among eleven isolates from west sumatra. discussion pepper yellow leaf curl disease is considered an important new emerging disease in indonesia which causes significant damage to plants. association of begomovirus with the disease was first reported in 1999 in west java, but by early 2003 the disease ha been found in almost all pepper growing areas in java (sulandari 2004). in the same time, syaiful (2005) reported the incidence of the disease in west sumatra, and since 2005 epidemics occurred in pepper causing up to 100% disease incidence (trisno et al. 2005). the type of symptoms associated with the disease in west sumatra is different from fig 3 comparison of the hairpin loop structure of pepper begomovirus. 1, the 31 base hairpin-loop region in peppyk1, pepagam2-1, pepagam2-4, and pepylcidv; 2, the 32 base hairpin-loop region in pepsolok2-1; 3, the 27 base hairpin-loop region in pepsolok3-5; 4, the 33 base hairpin-loop region in pept.datar1-3; 5, the 29 base hairpin-loop region in pept.datar 2-1; 6, the 29 base hairpin-loop region in peppss1-1; 7, the 30 base hairpin-loop region in peppss2-3; 8, the 30 base hairpin-loop region in peppasbt 1; 9, the 33 base hairpin-loop region in pepagam1-4. the conserved nonanucleotide sequence taatatt ↓ac is in bold, and the nick site is marked by an arrow. 1 2 3 4 5 6 7 8 9 2027 bp 1353 bp 603 bp 310 bp m 1 2 3 4 5 6 7 1600 bp fig 2 agarose gel electrophoregram of dna amplified by pcr using degenerate primers pal1v 1978 dan par1c 715. m, marker (1kb ladder), lanes 1-7: begomovirus infecting pepper. microbiol indones 59trisno et al. the identity of begomovirus infecting pepper in west sumatra was determined based on their hairpin-loop structure and repetitive sequence (iteron) found in the common region. these hairpin loop structure has been found in all geminivirus sequenced so far (zhou et al. 2003) and iteron is known as the specific-binding site of the geminivirus replicationassociated proteins (ribeiro et al. 2006). both the hairpinloop structure and iteron have been found in all geminivirus sequenced to date and this might be used for analysis of their genetic diversity. it was known that differences in the common region may indicate a different begomovirus strain (hidayat et al. 1999; chatchawankanphanich and maxwell 2002; sukamto et al. 2005; ribeiro et al. 2006; santoso et al. 2008). it was found in our present research that three isolates of begomovirus infecting pepper in west sumatra (peppyk1-1, pepag2-1 and pepag2-4) have a hairpin loop table 2 comparison of iteron upstream of the c1 gene tata box in 11 begomovirus isolates from west sumatra with pepylciv-pepper yellow leaf curl indonesia virus (ab246170.1), tlciv-tomato leaf curl indonesia virus (ab100304) and tlcjav-tomato leaf curl java virus (ab162141) isolate pepylcivy tlcivy tlcjavy p e p p y k 1 pepso2-1 pepso3-5 p e p t d 1 3 p e p t d 2 1 peppss1-1 peppss2-3 peppbst2-3 pepag1-4 pepag2-1, pepag2-4 iteron (5’-3’) ggagaca ggagaca gggtctcaa ggagaca ggagaca ggagaca ggagaca ggagaca ggagaca ggagaca -z ggagaca ggagaca positionx -86 s/d -80, -93 s/d-87, -122 s/d-116 -134 s/d -128, -196 s/d -190 -102 to -94, -137 to -129 -82 s/d -76, -89 s/d-83, -118 s/d-112 -89 s/d -83, -97 s/d-91, -125 s/d-119 -84 s/d -78, -91 s/d-85, -119 s/d-113 -87 s/d -81, -94 s/d-88, -123 s/d-117 -83 s/d -77, -90 s/d-84, -117 s/d-111 -88 s/d -82, -95 s/d-89, -122 s/d-116 -85 s/d -79, -93 s/d-87, -121 s/d-115 -90 s/d -84, -97 s/d-91, -125 s/d-119 -89 s/d -83, -96 s/d-90, -124 s/d-118 xnucleotide numbering starts from the a adjacent to the nicking site within the conserved nonanucleotide (taatatt↓ac), ydatabase genebank, znona data. table 3 list of geminivirus used for phylogenetic analysis (database genebank) genebank accession number ab246170.1 ab267838 ab189845 d q 0 8 3 7 6 4 nc_008267 af189018 nc 004005 nc_005347.1 eu585781.1 e u 4 8 7 0 4 0 ab162141 af327436.1 dq116884.1 dq641697.1 nc_000869.1 eu249457.1 aj495813.1 ab306314.1 ef544600 ab189913.1 x 7 4 5 1 6 . 1 organism pepper yellow leaf curl indonesia virus pepper yellow leaf curl indonesia virus pepper yellow leaf curl indonesia virus pepper yellow leaf curl indonesia virus-bogor tomato yellow leaf curl indonesia virus-lembang tomato yellow leaf curl indonesia virus-lembang tomato yellow leaf curl virus pepper yellow vein mali virus pepper leaf curl yunnan virus tomato leaf curl philippines virus tomato lea curl java virus tomato leaf curl malaysia virus tomato leaf curl pakistan virus tomato leaf curl virus vietnam tomato yellow leaf curl china virus tomato yellow leaf curl thailan virus ageratum yellow vein china virus ageratum yellow vein virus-ishigaki ageratum yellow vein taiwan virus ageratum yellow vein virus-indonesia ageratum yellow vein virus nukleotide’s length (bp) 2 7 6 0 2 7 6 0 1 5 6 3 2 7 4 3 2 7 6 2 2 7 6 2 2 7 8 1 2 7 8 6 2 7 4 7 2 7 5 5 1 5 6 2 2 7 5 4 2 7 5 9 2 7 4 5 2 7 3 4 2 7 4 3 2 7 6 8 2 7 5 3 2 7 5 3 1 5 5 7 2 7 4 1 indonesia, bogor indonesia, bogor indonesia, bandung, lembang indonesia, bogor indonesia, bandung, lembang indonesia, bandung, lembang almeria, spain mali china philipines, batanga indonesia, magelang malaysia, klang pakistan vietnam, hanoi china thailan china, hainan japan, okinawa china fujian indonesia, bandung, lembang china geography origin host plant c. annuum ageratum conyzoydes l. esculentum capsicum annuum l. esculentum l. esculentum l. esculentum c. annuum l. esculentum l. esculentum l. esculentum l. esculentum l. esculentum l. esculentum c. annuum ageratum conizoydes l. esculentum ageratum conizoydes ageratum conizoydes ageratum conizoydes acronim pylcvi-co pylcvi-ag pylcvi-t2 tylcvi-bgr tylcvi-lbg tylcvi-lba tylcv pylvmiv plcyuv tlcphiv tlcjav tlcvmal tlcpav tlcvit tylcvci tylctaiv ayvciv aylcvishi ayvtaiv aylcindv ayvv those found in java, which commonly caused bright yellow colouring (sulandari 2004). the differences in symptoms type of the disease between west sumatra and java may be caused by the difference of: (i) the type of cultivar used, where peper cultivars that are generally planted in west sumatera is the local cultivar “lado keriting darek” (from direct interview with farmers). meanwhile, the cultivar that is generally planted in java are an introduction cultivar, such as tm 999, tm 888 and jatilaba (sulandari 2004), (ii) the strain of geminivirus; this research showed the different strains of geminivirus excisted between west sumatera and java (fig 4). idris and brown (1998) stated that there are different virus strains that cause plant desease with different symptoms. since high genetic diversity of begomoviruses has been previously reported, it is important to determine the identity of begomovirus infecting pepper crops in west sumatra. microbiol indones60 volume 3, 2009 group 1 group 2 group 3 fig 4 phylogenetic tree based on the alignments of the common region sequences of pepper-infecting begomovirus from west sumatra with other selected begomovirus (genebank database). squares (ÿ ) show position of the samples. structure similar to that of pylciv-bogor (hidayat et al. 2006), whereas the isolates from the rest of west sumatra have different structures. repetitive sequences for most isolates of begomovirus infecting pepper in west sumatra (ggagaca) were similar with those of tolciv (sukamto et al. 2005) and pylciv-bgr (hidayat et al. 2006), but different from those of tolcjav (sukamto et al. 2005). differences were also observed with geminiviruses reported from other countries, for instance bean golden mosaic virus-guatemala (ikegami et al. 1988), tomato leaf curl joydebpur virus-mild (tolcjv-mild), and tomato leaf curl new delhi virus-severe from jessore (tolcndv-svr[jes])(maruthi et al. 2005), each with repetitive sequence of tgcgagtgtctccaa, ggtgt, ggagt, respectively. variability in hairpin-loop structure and iteron sequences indicates the possibility of genetic diversity among begomoviruses from west sumatra. therefore, further analysis of genome sequences is necessary. phylogenetic analysis based on the common-region sequences revealed that the west sumatra isolates of begomovirus infecting pepper can to differentiated into three groups. sequence similarity ranged from 63 to 91% with selected begomoviruses from asia (table 3). padidam et al. (1995) proposed that virus isolates displaying more than 90% sequence identity sh+ould be considered as isolates or strains rather than different viruses. in this study, strain grouping didnot represent different altitude for sample location, where the group 1 comes from locations sampled at low, medium and high altitudes, i.e. peppss1-1, pepso3-5, pepag1-4, and peptd1-3, respectively. we could not make any conclusion yet regarding the identity of begomovirus from west sumatra based on analysis of he common-region sequences. availability of the full genome sequence will provide comprehensive and complete analysis enable such a conclusion. however, there is an indication that genetic variability of begomovirus isolates infecting pepper occurred in west sumatra and that the viruses are related to begomovirus infecting pepper in java. this fact should be considered in developing strateies to control pepper yellow leaf curl disease, especially in the development of resistant varieties. acknowledgements this research was supported in part by research grant from department of agriculture (kerjasama kemitraan penelitian pertanian dengan perguruan tinggi /kkp3t) and directorate genderal of higher education, department of national education, indonesia (hibah bersaing). references chatchawankanpanhich o, maxwell dp. 2002. tomato leaf curl karnata virus from bangalore, india, appears to be a recombinant begomovirus. j phytopathology 92:637-45. doyle jj, doyle jl. 1999. isolation of plant dna from fresh tissue. j focus 12:13-5. fauquet cm, stanley j. 2003. geminivirus classification and nomenclature: progress and problems. ann appl biol 142:1658 9 . gutierrez c. 2000. strategies for geminivirus dna replication and celecycle interference. j physiol mol plant pathol 60:219-30. hidayat sh, chatchawankanpanich o, rusli e, aidawati n. 2006. begomovirus associated with pepper yellow leaf curl disease in west java, indonesia. j microbiol indones 11:87-90. idris am. brown jk. 1998. sinaola tomato leaf curl geminivirus: biological and molecular evidence for a new subgroup iii virus. j phytopatology 88:648-57. ikegami m, morinaga t, miura k. 1988. potential gene products of bean golden mosaic virus have higher sequence homologies to those of tomato golden mosaic virus than those of cassava laten virus. j virus genes 1:191-203. lapidot m, friedmen m, pilowsky m, ben-joseph r, cohen r. 2001. effect of host plant resistance to tomato yellow leaf curl virus (tylcv) on virus acquisition and transmission by its whitefly vector. j phytopathology 91:1209-13. maruthi mn, alam sn, kader ka, rekha ar, colvin j. 2005. nucleotide sequencing, whitefly transmission, and screening tomato for resistance against two newly described begomoviruses in banglades. j phytopathology 95:1472-81. padidam m, beachy rn, fauquet cm. 1995. clacification and identification of geminivirus using sequence comparation. j gen virol 76:249-63. ribeiro sg, martin dp, lacoste c, simoes is, orlandini drs, inouenagata ak. 2006. molecular and biological characterization of tomato chlorotic mottle virus suggests that recomendation underlies the evolution and diversity of brazilian tomato begomoviruses. j phytopathology 96:702-11. rojas mr, gilbertson rl, russel dr, maxwell dp. 1993. use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. j plant dis 77:340-7. rusli es, hidayat sh, suseno r, tjahjono, b. 1999. geminivirus on pepper: symptom variation and transmission study. bul hpt 11:126-31. santoso tj, hidayat sh, duriat as, herman m, sudarsono. 2008. indentity and sequence diversity of begomovirus assciated with yellow leaf curl diseases of tomato in indonesia. j microbiol indones 2:1-7. sukamto, kon t, hidayat sh, hase s, takahashi h, ikegami. 2005. begomoviruse associated with leaf curl disease of tomato in java, indonesia. j phytopathology 95:562-6. sulandari s. 2004. karakterisasi biologi, serologi dan analisis sidik jari dna virus penyebab penyakit daun keriting kuning cabai. [dissertation]. bogor: institut pertanian bogor. trisno j, charnita r, hanafiah a. 2005. karakteristik gejala dan deteksi virus kuning tanaman cabai di sumatera barat. j manggaro 6:21-9. varma a, malathi vg. 2003. emerging geminivirus problems: a serious threat to crop production. ann appl biol 142:145-64. zhou x, xie y, tao x, zhang z, li z, fauquet cm. 2003. characterization of dna-b assocated with begomoviruses in china and evidence for co-evolution with their cognate viral dna-a. j gen virol 84:237-247. trisno et al. microbiol indones 61 02. yuliana.cdr vol.14, no.2, june 2020, p 52-65 doi: 10.5454/mi.14.2.2 in silico study on testing antidiabetic compounds candidate from azaphilone monascus sp. 1 1 1 anna yuliana , hilman fitriaji s p , khofi siti mukhaufillah , and lina 2 rahmawati rizkuloh 1 department of pharmacy, stikes bakti tunas husada, jl.cilolohan no.36,tasikmalaya 46184, indonesia; 2 department of pharmacy, universitas perjuangan, jl. pembela tanah air (peta) no. 177, tasikmalaya 46115, indonesia. monascus sp. can be used as an ingredient in rice fermentation to produce red rice, called angkak. in asia, angkak is used as traditional medicine and food containing bioactive compounds, one of which is monakolin that has the potential to be a nutraceutical. monascus sp. produces five main pigments: red (monascorubramin, rubropunktamin), orange (monascorubrin, rubropunktatin), and yellow (monaskin, ankaflavin) which have biological activity. in subsequent developments, many new pigments were found which derivatives of the main pigments monascus sp. are, but information regarding their biological effects is still very limited. the purpose of this study was to determine the pigment derivative compounds of monascus sp. as a candidate compound for antidiabetic drugs. this study used 57 pigment derivative compounds monascus sp. which is done in silico. 2gpa protein (glycogen phosphorylase) is used as an antidiabetic receptor. the software used in this research includes chemdraw, marvin sketch, molegro molecular viewer, biovia discovery studio, and autodock. the adme study was conducted using preadmet web-based software. the results of the drug scan test on the mps4 isolate compound have a value that meets the requirements in all parameters such as molecular weight, proton donor, proton acceptor, log p and molar refractory. the adme test results on the mps4 isolate compound have a value that meets the requirements in all parameters of caco2, hia (human intestinal absorption), and in ppb (protein plasma binding). the results of the docking, the isolate mps4 compound are the best compounds and meet the requirements because they have smaller binding affinity than natural ligands and comparison ligands (glibenclamide). the results of this study, mps4 isolate can be used as a candidate for new antidiabetic drugs, but still requires further research, including in vitro and in vivo tests. key words: antidiabetic, azaphilone, docking, in silico, monascus sp. monascus sp. dapat digunakan sebagai bahan fermentasi beras sehingga menghasilkan beras berwarna merah yang disebut angkak. di asia, angkak digunakan sebagai pengobatan tradisional dan juga makanan yang mengandung senyawa bioaktif, salah satunya adalah monakolin yang berpotensi sebagai nutraceutical. monascus sp.menghasilkan pigmen utama yaitu merah (monaskorubramin, rubropunktamin,), orange (monaskorubrin, rubropunktatin), dan kuning (monaskin, ankaflavin) yang memiliki aktivitas biologis. pada perkembangan berikutnya, ditemukan banyak pigmen baru yang merupakan turunan dari pigmen utama monascus sp., tetapi informasi mengenai efek biologisnya masih sangat terbatas. tujuan penelitian ini adalah untuk mengetahui senyawa turunan pigmen monascus sp. sebagai senyawa kandidat antidiabetes. penelitian ini menggunakan 57 senyawa turunan pigmen monascus sp. secara in silico. sebagai reseptor antidiabetes digunakan protein 2gpa(glikogen fosforilase). perangkat lunak yang digunakan dalam penelitian ini meliputi chemdraw, marvin sketch, molegro molecular viewer, biovia discovery studio, danautodock. studi adme dilakukan dengan software berbasis web preadmet. hasil uji drug scanmenunjukkan senyawa isolate mps4 memiliki nilai yang memenuhi syarat dalam semua parameter seperti berat molekul, donor proton, akseptor proton, log p dan refractory molar. hasil uji adme pada senyawa isolate mps4 memiliki nilai yang memenuhi syarat dalam semua parameter caco2, hia (human intestinal absorption), maupun pada ppb (protein plasma binding). hasil uji docking menunjukkan senyawa isolate mps4 menjadi senyawa yang terbaik dan memenuhi syarat karena memiliki binding affinity lebih kecil daripada ligan alami dan ligan pembanding (glibenklamid). hasil dari penelitian ini, isolate mps4 dapat dijadikan salah satu kandidat obat baru antidiabetes, namun masih memerlukan penelitian lebih lanjut diantaranya uji in vitro dan in vivo. kata kunci: bakteri pelarut fosfat, fosfor, kultur murni dan campuran, pertanian, tanah microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-265-334740; fax: +62265-334745; email: anna_yuliana@stikes-bth.ac.id hyperglycemia, protein, and fat related to a lack of insulin secretion. polyuria, polydipsia, weight loss, tingling, polyphagia are symptoms that are felt by people with diabetes mellitus (fatimah 2015). the mechanism of dm type 2 is a decrease in diabetes mellitus is a disease that characterized by disruption of carbohydrate metabolism and insulin sensitivity (insulin resistance) and failure of pancreatic beta cell function which results in decreased production of insulin that cause hyperglycemia (perkeni 2015). monascus sp.can be used as rice fermentation material to make red rice, called angkak. in asia, angkak is used as traditional medicine and food, contains bioactive compounds. one of them is a monacolin compound, which has potential as a nutraceutical (nguyen et al. 2017). research on monascus sp. pigments is growing rapidly, including the discovery of new pigments, which are derived from the main pigments: yellow, orange and red. the research data obtained is still a separate data from any research journals, so that complete data are needed, particularly information regarding biological activity. from the six major pigments, 57 derivatives have been found until now. (yuliana et al. 2017). as seen from table 1, some pigments derived from monascus sp. include red (monascorubramin, r u b r o p u n k t a m i n ) , o r a n g e ( m o n a s c o r u b r i n , rubropunctatus), and yellow (monaskin, ankaflavin) which have biological activity. monascus sp. pigments widely used as a food coloring and flavoring in the food industry, especially for fish such as fish paste, surimi and meat products such as sausages and ham. in addition, this pigment can be used not only in the food sector, but also in the cosmetic and pharmaceutical industries (seyedin et al. 2015). pigments that have the potential to reduce blood table 1 the main pigments structure of monascus sp. (yuliana et al. 2017) volume 14, 2020 microbiol indones 53 54 yuliana et al. microbiol indones glucose levels are rubropunctamine (red) and rubropunctatine (orange) pigments (sulistyaning 2013). the purpose of this study was to determine the pigment derivative compounds of monascus sp. as a candidate compound for antidiabetic drugswith in silico test. materials and methods the tools used in this study are personal computers with processor specifications: intel core i7, 4.0 gb ram; 64 bit operating system, operating system: windows 10 pro. the software used is marvin sketch version 5.2.5.1, molegro molecular viewer (mmv) version 2.5,chemdrawultra, biovia discovery studio 2017, autodock, and web programs such as preadmet, prodrg2, pharm mapper and pdbsum. all software is open source. the materials in this study include the receptors that have been identified, downloaded from protein data bank (pdb) and 57 dyestuff compounds isolated from azaphilone derivatives from monascus sp. ligand preparation. ligands were drawn using chemdraw software and then optimized by protonation at 7.4 ph at marvin sketch (ruswanto et al. 2014). the procedure is performed on azaphilonederived ligands and then stored in the mol2 format. optimization of the structure aims to see the conformation of molecules and see the low potential energy that has been adjusted based on ph conditions in the body. drug scan. drug observation was carried out on dyestuff compounds derived from monascus sp.by considering lipinski's rule of five as well as oral and ligand bioavailability. the parameters used were<500 g / mol molecular weight, <5hydrogen bond donors, <10 hydrogen bond acceptors, and 40-130 molar refractory.these parameters can be determined with the help of marvin sketch software (ruswanto 2015). receptor preparation. there are 5 enzyme structures (receptors) for antidiabetic: 1c8l (glicogen phosporilase a), 1h5u (glicogen phosporilase b), 2fw3 (karnitin palmitoyltranspherase 2), 2qmj (maltase glucoamilase), and 2gpa (glicogen phosporilase). pdb format is created after the codes 1c8l, 1h5u, 2fw3, 2qmj, 2gpa are downloaded from pdb (protein data bank). then it is prepared to separate from its natural ligand, adding hydrogen and removing solvent molecules (ruswanto et al. 2018). admet study. admet compounds profiling is done by applying the admet descriptor algorithm and the admestar database that freely available at (http://admetexp.org) (qidwai 2017). docking method validation. before the compound docking process is carried out with protein, the docking method is validated using autodock 4.2.6. to see the root mean square deviation (rmsd)as docking validityparameters (lelita et al. 2017). validation was done by using natural ligand redocking on each receptor were used. re-docking is the process of separating the natural ligand crystal structure docking receptor then carried back to the receptor. the docking parameter is valid if the rmsd value from the redocking results is ≤ 2.0 å (sherman et al. 2006). the value of rmsd indicate acceptable accuracy, whereby if the value of rmsd <2 å means indicates that the smaller the error of the docking results, so it can be said to be valid ( ).lelita et al. 2017 molecular tethering. test compounds and proteins downloaded from pdb were prepared with autodock 4.2.6, before the tethering process, the parameters, dimensions, and the coordinates of the grid box were adjusted. the dimensions of the grid box are adjusted to the size of each ligand, while the coordinates of the grid box are adjusted based on the coordinates of the center of its natural ligand. before that, the location of the gridboxwas determined using the autodock program tools. grid box is the location of the ligand mooring space which will be docking and have settingsincludes center_x, center_y, and center_z. to determine the size of a grid box is setby using spacing (armstrong). the placement of the gridboxis based on the location of the test ligand and the active side of the protein.the grid box used is x = 31,864, y = 21,182, and z = 26,477.the parameter used is the calculation of 100 times runs ga (genetic algorithm). compounds for testing and prepared proteins are put together in one folder for further gridding and docking (yuliana et al. 2019). results drug scan. on the results of the drugscan test on azaphilone derivatives in table 2, shows that not all azaphilone derivatives fulfill the lipinski's rule of five parameters requirements. such as glycyl rubropuntatin, isolate mps3, isolate mps2, isolate m p s 1 , n g l u t a r y l m o n a s c u r o b a m i n e , n table 2 drug scan test result no name of compound drug scan m olecular weight proton donor proton acceptor log p refractory m olar < 500 g/mol < 5 < 10 < 5 40-130 1 glycyl rubropunctatin 413.470 1 11 3,33 114,09 2 isolate mps4 439.552 1 9 4,32 126,87 3 isolate mps3 439.508 1 11 4,08 123,54 4 isolate mps2 538.645 5 14 3,74 161,46 5 isolate mps1 510.591 5 14 2,85 152,26 6 n-glutaryl monascurobamine 511.571 2 15 4,30 138,82 7 n-glutaryl rubropunctamine 483.517 2 15 3,41 129,62 8 pp-v 412.461 3 10 3,26 125,22 9 n-glycosyl rubropunctamine 557.640 4 17 2,65 149,92 10 n-glycosyl monascurobamine 585.694 4 17 3,54 159,12 11 compound r3 374.433 1 10 2,05 101,41 12 red derivat 1 453.535 1 11 4,65 128,03 13 red derivat 2 425.481 1 11 3,76 118,83 14 red derivat 3 497.544 2 15 4,01 134,07 15 red derivat 4 469.490 2 15 3,12 124,87 16 red derivat 5 453.535 1 11 4,65 128,03 17 red derivat 6 425.481 1 11 3,76 118,83 18 red derivat 7 497.544 2 15 4,01 134,07 19 red derivat 8 469.490 2 15 3,12 124,87 20 un named 375.465 3 9 1,16 103,91 21 monascopyridine a 355.434 0 7 4,23 98,81 22 monascopyridine b 383.488 0 7 5,12 108,01 23 monascopyridine c 357.450 1 9 3,90 99,88 24 monascopyridine d 343.467 1 7 4,61 100,56 25 new red pigmen 375.465 3 9 1,16 103,91 26 monascuskaodione a 356.418 0 8 3,36 100,67 27 monascuskaodione b 384.472 0 8 4,27 109,87 28 red shandong 1 303.402 4 7 0,54 91,77 29 red shandong 2 331.456 4 7 1,43 100,98 30 monankarin a-b 358.3851 2 8 2.38 98,10 31 monankarin c-d 372.4117 2 7 2.90 103.14 32 monankarin e 358.3851 2 7 2.53 98.67 33 monankarin f 356.4123 2 6 2.99 103.21 34 monascusone a 254.2790 3 6 -0.99 67.08 35 monascune b 302.3218 0 7 1.64 82.07 36 fk 17-p2b2 236.2637 2 5 0.35 66.34 37 xantomonascin a 388.4111 2 8 4.00 102.20 38 xantomonascin b 414.4914 2 8 3.76 126.81 39 y3 448.571 6 8 0.34 115.88 40 monapurones a 330.4180 1 6 2.98 97.87 41 monapurones b 344.4446 0 5 3.93 101.83 42 monapurones c 344.4446 0 5 3.93 101.83 43 monaphilones a 374.5137 1 6 4.61 111.50 44 monaphilones b 332.4339 1 6 3.27 97.70 45 monaphilones c 336.4657 1 7 4.14 95.95 46 monashexenone 320.4232 1 7 3.70 92.33 47 rubropuctin 358.4712 1 6 4.02 107.15 48 monarubrin (y,bf) 330.4180 1 6 3.13 97.95 49 yellow ii 372.4547 1 7 4.31 116.32 50 purpureus one 390.5131 0 8 5.43 107.95 51 monascuspiloin 360.4440 1 6 3.11 101.32 52 monaphilol a 384.4654 1 6 3.62 111.10 53 monaphilol b 356.4123 1 6 2.73 101.89 54 monaphilol c 440.5287 1 8 3.59 125.32 55 monaphilol d 412.4755 1 8 2.70 116.12 56 monasfluor a 354.4394 0 5 3.98 104.30 57 monasfluor b 384.4654 0 7 4.27 109.87 volume 14, 2020 microbiol indones 55 g l u t a r y l r u b r o p u n c t a m i n e , n g l y c o s y l r u b r o punctamine, n-glycosylmonascurobamine, red derivat 1, red derivat 2, red derivat 3, red derivat 4, red derivat 5, red derivat 6, red derivat 7, red derivat 8, monascopyridine b, y3, purpureus one, while the azaphilone derivative compounds that fulfill lipinski's rules are isolate mps4, pp-v, compound r3, un named, monascopyridine a, monascopyridine c, m o n a s c o p y r i d i n e d , n e w r e d p i g m e n t , monascuskaodione a, monascuskaodione b, red shandong 1, red shandong 2, monankarin c-d, monankarin a-b, monankarin e, monankarin f, fk 17-p2b2, monascusone a, monascusone b, monasfluor a, monasfluor b, xantomonascin a, xantomonascin b, monapurones a, monapurones b, monapurones c, monaphilol d, monaphilol a, monaphilol b, monaphilol c, monaphilones b, monaphilones a, monaphilones c, monashexenone, rubropuctin, monarubrin (y,bf), yellow ii, monascuspiloin, so that it can be used as a candidate for drug compounds for further testing. receptor preparation. there are 5 enzyme structures (receptors) for antidiabetic, which is 1c8l, 1h5u, 2fw3, 2qmj, 2gpa. to separate them from the original ligand, the addition of hydrogen and the removal of solvent moleculesare needed. then the enzymes / receptors that have been downloaded and stored in the form of pdb were analyzed using pdbsum. figure 1 showed that the 2gpa receptor shows a stable structure because the percentage of residue in the most favored region is 89.7% and in the disallowed region is 0.1%. the quality of the protein structure is considered good if the residue in the disallow region is less than 15% and the amino acid residue in the most favored region is greater than 50%. the greater percentage of amino acid residues in the most favored and the lower percentage of residues in the disallowed region, the better the quality of the structure. so it can be said that the protein structure of 2gpa receptor has good quality and can be used for further analysis. admet study. based on adme test results using web-based preadmet software in table 2, azaphilone derivative compounds have medium permeability values, which are in the range of 4-70%. the process of absorption in the human intestine is in a good range that is in the range of 70-100%. then for the binding of proteins in the blood compound ; mps4 isolate, mps3 isolate, red derivate 1, red derivate 5, monascopyridine b, monascopyridine d, monascuskaodione b, xantomonascin a, xantomonascin b, monaphilones a, rubropuctin, m o n a s c o p y r i d i n e b , m o n a s c o p y r i d i n e d , m o n a s c u s k a o d i o n e b , x a n t o m o n a s c i n a , xantomonascin b, monaphilones a, rubropuctin, monarubrin (y, bf) yellow, monaphilol a, monaphilol b, monasfluor a, monasfluor b have a high value of> 90% showing a strong bond with plasma proteins in the body. docking method validation. validation is done by autodock 4.2.6. based on the data from table 4, the result shows that the 2gpa pdb is valid where the rmsd value has fulfil the ≤2 requirements, which is have value of 0.77. meanwhile the 1h5u, 2fw3, 1c8l pdb are not valid because the rmsd result shows that the values​​exceed the specified ≤ 2 range. the rmsd results of those three gdp codes are 2.85, 2.03, and 3.41. in the other hand, the 2qmj pdb still fulfills the requirements, it have value of 1.28, but when the rmsd value of the 2gpa pdb compared with the 2qmj pdb, it can be confirmed that the rmsd of the 2gpa pdb is smaller, because if the rmsd value is getting smaller, it shows that the position of the ligand is better because it getting near to the original conformation. molecular tethering. as can be seen from figure 2, the receptor used with the docking ligand obtained an rmsd value of <2.0 å, it shows that it has fulfilled the validity requirements. discussion a protein structure is declared good if the most favored regions are ≥ 90% and disallowed regions less ≤ 1%. another parameter used in the selection of receptors is that the rmsd value must be ≤ 2, because if the rmsd value is getting closer to zero then the original ligand with a copy of the ligand is increasingly similar. from these parameters, the enzyme / receptor with the code 2gpa (glycogen phosphorylase) fulfills the requirements because it has an rmsd value of 0.77. caco2 cells are the distribution of drugs through intestinal epithelium that derived from human colon adenocarcinomas that have multiple transport routes. hia is the result of absorption and bioavailability processes that are evaluated from the amount of expenditure through bile, urine, and feces. ppb (protein plasma binding) is a part of the drug that is available in a free form for circulation to all tissues in the body (nursamsiar et al. 2016). validation of docking is done with water and without water, it is intended to determine the effect of 56 yuliana et al. microbiol indones no name of compund caco2 (nm/sec) hia (human intestinal absorption) % ppb (protein plasma binding) % 1 glycyl rubropuntatin 20.9325 medium 96.702970 good 87.663371 weakly bonded 2 isolate mps4 25.6923 medium 99.303546 good 92.314035 strongly bonded 3 isolate mps3 21.5677 medium 98.309963 good 90.416630 strongly bonded 4 isolate mps2 8.92909 medium 78.193552 good 78.061242 weakly bonded 5 isolate mps1 9.25845 medium 74.407933 good 64.976138 weakly bonded 6 n-glutaryl monascurobamine 19.8389 medium 92.538271 good 90.486415 strongly bonded 7 n-glutaryl rubropuctamine 19.7598 medium 90.311224 good 88.136616 weakly bonded 8 pp-v 4.01366 medium 92.923736 good 86.114618 weakly bonded 9 n-glucosyl rubropuctamine 12.4451 medium 85.412276 good 65.185992 weakly bonded 10 n-glucosyl monascurobamine 12.5306 medium 87.690078 good 79.078783 weakly bonded 11 compound r3 17.4163 medium 95.677133 good 73.231309 weakly bonded 12 red derivat 1 22.1952 medium 98.668455 good 90.932647 strongly bonded 13 red derivat 2 21.2765 medium 97.891289 good 88.641765 weakly bonded 14 red derivat 3 20.5128 medium 91.491229 good 89.736964 weakly bonded 15 red derivat 4 20.4423 medium 88.981951 good 86.838945 weakly bonded 16 red derivat 5 22.1952 medium 98.668455 good 90.932647 strongly bonded 17 red derivat 6 21.2765 medium 97.891289 good 88.641765 weakly bonded 18 red derivat 7 20.5128 medium 91.491229 good 89.736964 weakly bonded 19 red derivat 8 20.4423 medium 88.981951 good 86.838945 weakly bonded 20 un named 17.9896 medium 89.666473 good 66.572743 weakly bonded 21 monascopyridine a 26.367 medium 98.750840 good 91.953695 strongly bonded 22 monascopyridine b 32.7272 medium 98.912525 good 93.500167 strongly bonded 23 monascopyridine c 22.7729 medium 96.648798 good 89.685299 weakly bonded 24 monascopyridine d 22.9611 medium 96.270412 good 97.268350 strongly bonded 25 new red pigmen 17.9896 medium 89.666473 good 66.572743 weakly bonded 26 monascuskaodione a 28.3725 medium 98.770329 good 88.897997 weakly bonded 27 monascuskaodione b 36.0846 medium 98.771155 good 92.381422 strongly bonded 28 red shandong 1 13.6843 medium 85.719545 good 71.398196 weakly bonded 29 red shandong 2 14.2523 medium 87.122330 good 87.278357 weakly bonded 30 monankarin a-b 21.4435 medium 93.56731 good 85.58307 weakly bonded 31 monankarin c-d 21.9971 medium 93.909198 good 87.093425 weakly bonded 32 monankarin e 21.4031 medium 93.567883 good 84.456494 weakly bonded 33 monankarin f 35.5122 medium 93.789307 good 88.543275 weakly bonded 34 monascusone a 19.3778 medium 78.683369 good 34.939799 weakly bonded 35 monascune b 22.9891 medium 97.536574 good 61.438550 weakly bonded table 3 test results of adme azaphilone derivatives volume 14, 2020 microbiol indones 57 table 3 test results of adme azaphilone derivatives -continuedno name of compund caco2 (nm/sec) hia (human intestinal absorption) % ppb (protein plasma binding) % 36 fk 17-p2b2 0.993 rendah 90.43201 good 56.07652 weakly bonded 37 xantomonascin a 19.6124 medium 88.865206 good 96.842726 strongly bonded 38 xantomonascin b 26.5245 medium 94.538788 good 94.764093 strongly bonded 39 y3 19.3732 medium 50.125685 medium 67.649190 weakly bonded 40 monapurones a 26.9554 medium 95.760530 good 86.135746 weakly bonded 41 monapurones b 44.272 medium 97.697949 good 88.300451 weakly bonded 42 monapurones c 44.272 medium 97.697949 good 88.300451 weakly bonded 43 monaphilones a 48.8546 medium 96.052979 good 94.089855 strongly bonded 44 monaphilones b 40.8229 medium 96.054536 good 90.112573 strongly bonded 45 monaphilones c 26.9554 medium 95.760530 good 86.135746 weakly bonded 46 monashexenone 22.609 medium 95.857284 good 88.158433 weakly bonded 47 rubropuctin 46.2928 medium 96.050080 good 95.896405 strongly bonded 48 monarubrin (y,bf) 40.5147 medium 96.071613 good 92.263384 strongly bonded 49 yellow ii 34.2219 medium 96.423561 medium 91.738989 strongly bonded 50 purpureus one 31.1835 medium 98.247814 good 90.721848 strongly bonded 51 monascuspiloin 30.4892 medium 96.468903 good 90.306522 strongly bonded 52 monaphilol a 36.7103 medium 96.501044 good 95.427557 strongly bonded 53 monaphilol b 29.408 medium 96.568439 good 91.926288 strongly bonded 54 monaphilol c 34.038 medium 97.366571 good 89.549401 weakly bonded 55 monaphilol d 27.7997 medium 97.311031 good 83.639250 weakly bonded 56 monasfluor a 49.808 medium 97.649968 good 92.794086 strongly bonded 57 monasfluor b 36.0846 medium 98.771155 good 92.381422 strongly bonded information�: caco-2� : low <4 ; medium 4-70 ; high > 70 hia � : bad 0-20 % ; medium 20-70 % ; good 70-100 % ppb � : strongly bonded > 90 % ; weakly bonded < 90 % table 4 docking validation results receptors grid box rmsd x y z 2gpa 31.864 21.182 26.477 0.77 1h5u 28.47 21.132 31.662 2.85 2qmj -29.911 7.647 -17.894 1.28 2fw3 23.57 4.509 34.021 2.03 1c8l 27.833 20.552 31.813 3.66 water on the docking process. in validation the presence of water shows physiological conditions in the body, because water will affect the ligand bond to the receptor and also the formation of hydrogen bonds with the receptor, besides this validation can also show the comparison of the position of the original ligand with the comparative ligand against the receptor when they are docked. the rmsd value is a parameter that can be used, because if the rmsd value obtained from the validation results is ≤2, that is mean the result is good. molecular tethering of pigments from monascus sp. was carried out on 57 pigment compounds of monascus sp. with for the results of interaction 2 gpa receptor. and molecular tethering showed in table 5. there are only 2 pigments that have smaller binding energy valueswhen compared with antidiabetic drugs 58 yuliana et al. microbiol indones fig 1 visualization results of ramachandran plot and procheck statistics. fig 2 visualization of docking result. docking ligand (yellow) natural ligand (green). fig 3 visualization of 2d and 3d docking results for isolate mps4 compounds 2 gpa receptor. with on the market (glibenclamide), the value of free energy bonds on isolate mps4 refers to the smallest value, because the value is -11.58 kcal / mole which indicates it has the most stable position. ligands from glibenclamide produce asn284, gly675 hydrogen bonds. this interaction is also found in isolate mps4 that shows the affinity of isolate mps4 is better than the comparative ligand (glibenclamide). the visualization of 2d and 3d docking results for isolate mps4 compounds 2 gpa receptor with can be seen from figure 3. the next lowest free energy bond value is the red derivate 6. red derivate 6 compound has a smaller binding energy when compared to original ligands and comparative ligands (glibenclamide) so that they have a stable conformation. the bonds that occur in red derivatives 6 are hydrogen bonds, namely asn284 and gly675 which are also found in original hydrogen ligand and comparative ligand (glibenclamide) bonds. therefore, red derivate 6 has the potential to become volume 14, 2020 microbiol indones 59 no compounds run binding affinity kkal/mol hydrogenbond amino acid (residue contact) 1 glibenclamide (comparison) 55 -10.48 asn284 gly675 asn282, ala383, phe286, phe285, his341, asp339, thr378, ala673, his377, asn484, ser674, leu139, gly675, thr676, glu672,gly135, gly677, val567, arg569, tyr648, gly134, lys574, asn133, leu136, lys568, asp284, glu88, asn284 2 original ligand 32 -6.08 leu136 asn284 glu672 ser674 gly675 asn484 leu136, asn284,glu672, ser674, gly675, asn484, lys574, tyr573, his377, ala673, thr676, val455, leu139, gly135 3 glycyl-rubropunctatine 28 -10.11 thr676 asn484 his377 thr676, asn484, his377, arg569, tyr648, lys568, lys574, asn678, gly677, glu672, gln665 gly675, ala673, ser674, val455, leu139, glu88, asn133, gly137, asp283, asn284, leu136, gly135, gly134 4 isolate mps4 21 -11.58 gly677 thr676 his377 asn284 gly677, thr676, his377 asn284, arg569, tyr648, lys574, lys568, gln665, gly675, asn678, glu672, asn484, ala673, ser674, val455, leu136, gly137, glu88, asn133, asp283, his341, asn282, asp339, thr378, gly134, gly135, tyr573 5 isolate mps3 40 -9.78 tyr537 glu672 ala673 his377 thr676 gly677 tyr537, glu672, ala673 his377, thr676, gly677, asp339, asn284, gly135, leu136, thr378, glu88, asn133, asp283, gly134, arg569, tyr648, lys568, lys574, asn678, gly675, asn484, ser674, val455 6 isolate mps2 47 -7.45 his377 glu672 gly675 lys680 his377, glu672, gly675, lys680, thr378, asp339, ala383, phe285, asp283, asn282, asn284, leu136, arg569, his341, asn133, val567, gly677, gly134, tyr648, arg138, thr676, gly135, asn678, gln665, ser674, tyr573, ala673, val455 7 isolate mps1 83 -9.53 gln665 ser674 gly677 thr676 gly675 his377 gln665, ser674, gly677, thr676, gly675, his377, lys568, glu672, tyr573, ala673, val455, his341, thr378, asp339, leu136, asn284, asp283, leu139, gly135, gly134, arg569, lys574, tyr648, asn678, gly694, ala695, ser667, asn696, val567 8 n-glutaryl monascurobamine 52 -9.30 leu136 asp283 leu136, asp283, lys574, lys568, val567, gly677, thr676, lys680, gly135, glu672, arg138, gly675, ser674, ala673, tyr573, his377, gly137, asn133, asp339, asn284, gly134, phe285, his341, ala383, asn282, arg569, tyr648 9 n-glutaryl rubropuctamine 18 -9.66 asp283 leu136 thr676 asp283, leu136, thr676, gly137, asn133, asn282, glu88, phe285, ala383, asn284, gly134, arg569, his341, lys574, tyr648, lys568, val567, gly677, lys680, arg138, gly675, glu672, ser674, gly135, ala673, tyr573,his377 10 pp-v 89 -9.99 leu136 asp283 thr676 leu136, asp283, thr676, phe285, phe286, asn133, gly137, his341, gly134, gly135, lys574, lys568, gln665, gly675, gly677, glu672, ser674, ala673, val455, tyr573, his377, asn284, thr378, glu88, asp339, asn282, ala383 11 n-glycosyl rubropuctamine 15 +7.08 asn284 his377 asn484 gly135 asn284, his377, asn484, gly135, his341, phe285, ala383, leu384, asp339, thr378, ala673, glu672, val455, ser674, gly675, lys568, lys574, val567, gly677, thr676, arg138, leu139, asp283, asn133, glu88, asn282, leu136 table 5 the results of docking pigment tests from monascus sp. with 2 gpa (glycogen phosphorylase) receptor 560 yuliana et al. microbiol indones table 5 the results of docking pigment tests from monascus sp. with 2 gpa (glycogen phosphorylase) receptor -continuedno compounds run binding affinity kkal/mol hydrogenbond amino acid (residue contact) 12 n-glycosyl monascurobamine 90 +7.83 asn484 ser674 his377 asn284 asn484, ser674, his377, asn284, gly677, lys568, lys574, gly123, leu139, gly134, thr676, arg569, asp283, leu136, asn133, glu88, asn282, his341, phe285, leu384, thr378, ala383, asp339, ala673, val455, glu6782, gly675 13 compound r3 72 -9.90 asp283 leu136 his377 asn484 gly675 gly677 thr676 asp283, leu136, his377, asn484, gly675, gly677, thr676, glu672, gln665, asn678, lys574, gly135, asn284, asn282, asn133, glu88, gly137, gly134, leu139, val455, ala673, ser674, lys568 14 red derivat 1 19 -8.80 gly675 glu672 his377 gly675, glu672, his377, ser674, tyr573,ala673,val455, leu139, leu136, thr378, asn284, his341, asp339, ala383, phe285, gly135, arg569, tyr648, gly134, gly677, thr676, val567, lys568, lys574 15 red derivat 2 18 -8.86 asn484 leu136 gly137 asp283 gly135 thr676 asn484, leu136, gly137, asp283, gly135, thr676, gly675, ala673, ser674, val455, tyr573, glu672, asn284, his377, asp339, his341, ala383, thr378, asn282, glu88, asn133, gly134, arg569, lys574, gly677, leu139 16 red derivat 3 78 -8.09 thr676 gly677 glu672 his377 thr676, gly677, glu672, his377, lys568, gly675, lys574, tyr573, ser674, ala673, val455, leu139, thr378, his341, phe285, ala383, asp339, leu136,asn284, gly135, asp283, gly134, tyr648, arg569, asn133 17 red derivat 4 86 -8.51 gly677 thr676 glu672 his377 gly677, thr676, glu672, his377, gly675, ser674, tyr573, ala673, val455, thr378, leu136, leu139, asn284,asp283, gly134, asn133, arg569, tyr648, lys568, lys574, asn678, gly135, gln665 18 red derivat 5 57 -10.30 gly677 thr676 his377 gly677, thr676, his377, val455, leu136, thr378, gly137, asn133, glu88, asn282, asp283, asn284, gly134, gly135, tyr573, tyr648, arg569, lys680, asn678, lys568, lys574, gly675, glu672, asn484, ser674, ala673 19 red derivat 6 100 -10.63 gly677 thr676 asn484 his377 gly677, thr676, asn484, his377, lys680, asn678, gly675, lys568, lys574, ser674, glu672, val455, ala673, leu139, leu136, asp283, gly137, asn284, gly135, glu88, asn133, gly134, tyr648, arg569 20 red derivat 7 2 -9.86 gly677 asn484 his377 asn284 gly677, asn484, his377, asn284, lys568, tyr648, lys574, lys680, thr676, gly675, glu672, ser674, ala573, val455, thr378, leu136, phe285, glu88, asn282, asn133, his341, asp283, gly135, tyr573, arg569, gly134, asn678, gln665 21 red derivat 8 21 -9.50 gly677 thr676 his377 gly677, thr676, his377, arg569, asn678, lys568, lys574, tyr648, lys680, gly675, arg138, asn484, ser674, glu672, ala673, val455, leu136, thr378, asp339, ala383, his341, phe285, asn284, gly135, gly134 22 un named 32 -9.11 gly135 asp283 gly675 asn484 his377 glu672 gly135, asp283, gly675, asn484, his377, glu672, gly134, leu136, thr676, leu139, val455, ser674, ala673, thr671, thr378, val379, asn284, leu380, tyr573, lys574, lys568, tyr648, arg569 volume 14, 2020 microbiol indones 61 table 5 the results of docking pigment tests from monascus sp. with 2 gpa (glycogen phosphorylase) receptor -continuedno compounds run binding affinity kkal/mol hydrogenbond amino acid (residue contact) 23 monascopyridine a 49 -9.81 leu136 asp283 gly135 glu672 leu136, asp283, gly135, glu672, phe286, ala383, phe285, sn284, asn133, gly137, gly134, lys574, thr676, gly675, ser674, his377, ala673, thr378, thr671, tyr573, val379, leu380, asp339, his341 24 monascopyridine b 35 -9.21 his377 ala673 leu136 asp283 his377, ala673, leu136, asp283, leu139, tyr573, gly135, gly137, gly134, asn133, asn284, asp339, phe285, phe286, ala383, val567, his341, gln665, lys568, gly677, asn678, thr676, gly675, thr378, glu672, lys574, ser674 25 monascopyridine c 23 -8.59 ser674 gly675 glu672 gly135 leu136 asp283 asn284 ser674, gly675, glu672, gly135, leu136, asp283, asn284, thr378, his341, ala383, phe285, asp339, leu384, leu139, val455, asn484, ala673, thr676, his377, tyr573, lys574, gly134 26 monascopyridine d 59 -9.03 leu136 asp283 asn284 glu672 leu136, asp283, asn284, glu672, his341, asp339, ala383, phe285, thr378, ser674, gly675, asn484, thr676, leu139, val455, lys574, ala673, tyr573, gly135, his377, gly134 27 new red pigment 52 -9.59 asp283 leu136 his377 asn484 gly675 thr676 gly677 asp283, leu136, his377, asn484, gly675, thr676, gly677, val567, tyr648, lys574, lys568, gly135, asn284, asn133, glu88, gly137, gly134, val455, leu139, ser674, ala673, glu672 28 monascuskaodione a 76 -9.48 his377 asn484 gly675 thr676 his377, asn484, gly675, thr676, gly677, lys574, asn678, lys568, gln665, glu672, asn284, asp238, glu88, asn282, asn133, leu136, val455, leu139, ala673, ser674, gly135 29 monascuskaodione b 86 -9.46 his377 asn484 gly675 thr676 his377, asn484, gly675, thr676, val455, leu139, ala673, ser674, glu672, gly135, gly677, lys574, gly134, lys568, arg569, tyr648, asp283, leu136, asn284, asn133, glu88, gly137 30 red shandong 1 60 -8.36 his377 asn284 glu672 his377, asn284, glu672, ala673, val455, tyr573, lys574, leu139, asn484, gly675, gly135, ser674, yhr676, leu136, asp283, gly134, gly137, glu88, asn133, his341, asp339 31 red shandong 2 37 -8.15 gly135 asp283 lys574 tyr573 gly135, asp283, lys574, tyr573, his377, asn133, asn284, thr378, his341, asp339, phe285, ala383, leu136, gly675, glu672, thr676, gly677, asn678, gln665, lys568, val567, arg569, gly134, his571 32 fk 17-p2b2 55 -7.74 his377, asn484, gly675, glu672, asn284 leu136, his377, asn484, gly675, glu672, asn284, asn133, glu88, asp283, tyr573, gly135, lys574, ser674, thr676, ala673, val455, leu139. 33 monankarin a-b 42 -8.39 glu162, glu273 arg277, ile275, val278, gln295, arg277, asn274, ile275, ala246, ser245, ile159, arg160, glu162, glu273 34 monankarin c-d 38 -8.17 val567, lys568, glu672, gly675, his377, arg569 lys574, leu136, gln665, gly677, asn678, thr676, tyr573, ser674, ala673, val455, asn284, gly135, asp283, gly134, tyr648, val567, lys568, glu672, gly675, his377, arg569 62 yuliana et al. microbiol indones table 5 the results of docking pigment tests from monascus sp. with 2 gpa (glycogen phosphorylase) receptor -continuedno compounds run binding affinity kkal/mol hydrogenbond amino acid (residue contact) 35 monankarin e 70 -9.14 glu672, lys568, gly135, asp283, asn284 lys574, leu136, his377, gly134, arg569, tyr648, val567, gly677, gln665, asn678, thr676, gly675, val455, ser674, ala673, glu672, lys568, gly135, asp283, asn284 36 monankarin f 3 -8.71 gly135, his377, asn484, glu672, lys568, val567 lys574, tyr573, ala673, tyr648, arg569, gly134, leu136, asp283, asn284, thr378, ser674, val455, leu139, gly675, gln665, thr676, gly677, asn678, gly135, his377, asn484, glu672, lys568, val567 37 monaphilones a 100 -8.46 thr676, glu672, tyr573, his377 lys568, arg569, tyr648, lys680, gly134, gly677, gly135, asn133, lys574, asn284, leu136, asp283, glu88, asn282, his341, val455, ala673, ser674, gly675, tyr90, thr676, glu672, tyr573, his377 38 monaphilones b 73 -8.35 lys568, gly677, thr676, glu672, asn284 tyr648, arg569, val567, gly134, asn133, tyr90, arg649, lys608, ser674, val455, asn484, gly135, his377, leu136, gly675, tyr573, lys574, lys568, gly677, thr676, glu672, asn284 39 monaphilones c 69 -8.82 glu672, gly675, asn484, his377 tyr573, leu380, lys568, arg569, tyr648, gly134, lys574, thr378, thr671, val379, asn284, ala673, val455, ser674, leu139, thr676, leu136, gly135, gly677, glu672, gly675, asn484, his377 40 monapurones a 81 -9.55 tyr573, asn284, thr676, gly675, asn484, his377, glu672, lys568, ala673, leu380, val379, thr671, thr378, val455, leu139, leu136, ser674, gly135, gly677, asn678, gln655, val567, lys574, tyr573, asn284, thr676, gly675, asn484, his377 41 monapurones b 27 -8.35 asn484, his377 lys568, leu136, ala383, val567, lys574, gly675, tyr573, asn284, asp339, thr378, phe285, his341, glu672, gly677, thr676, gly135, leu139, ala673, val455, ser674, asp283, asn484, his377 42 monapurones c 94 -8.17 asn484, lys574 gly675, lys568, leu136, ser674, leu139, gly135, thr676, gly677, val567, gln665, tyr573, asn133, glu672, asn282, glu88, his341, asp283, asn284, gly137, thr378, his377, gly134, val455, ala673 43 monarubrin (y,bf) 15 -8.57 asp283, asn284, glu672, his377 his341, gln665, gly675, leu136, lys568, asn678, gly677, thr676, gly134, lys574, arg569, gly135, tyr573, ala673, thr378, asp339, phe285, asp283, asn284, glu672, his377 44 monascusone a 42 -8-08 asp283, leu136, ser674, glu672 val455, ala673, his377, thr378, asn284, phe285, his341, asn282, asn133, glu88, gly137, gly134, gly135. asn484, gly675, tyr573, asp283, leu136, ser674, glu672 45 monascusone b 23 -9.19 asn484, his377 leu136, lys568, his341, ala673, val455, leu139, gly135, ser674, thr676, val567, gly677, glu672, lys574, gly675, tyr573, asn284, thr378, glu88, asn133, asn484, his377 46 monascuspiloin 74 -8.32 lys574, thr676, gly675, glu672 tyr648, lys568, arg569, gly134, asn133, tyr90, leu136, ala383, asp339, thr378, his377, asn284, ala673, ser674, tyr573, gly135, gly677, asn678, lys574, thr676, gly675, glu672 47 monashexenoone 31 -9.50 glu162, glu273, arg277, ile275, tyr161, val278, gln295, arg277, asn274, ile275, ala246, ser245, ile159, arg160, glu162, glu273 volume 14, 2020 microbiol indones 63 64 yuliana et al. microbiol indones table 5 the results of docking pigment tests from monascus sp. with 2 gpa (glycogen phosphorylase) receptor -continuedno compounds run binding affinity kkal/mol hydrogenbond amino acid (residue contact) 48 purpureus one 10 -8.20 leu136, asp283, asn484, gly675, ser674, gly677 arg569, lys574, gly135, asn284, gly134, tyr90, asn133, tyr573, his377, val455, tht676, thr378, lys680, glu672, val567, tyr648, lys568, ala673, leu136, asp283, asn484, gly675, ser674, gly677 49 robropuctin 4 -9.22 gly672, asn284, his377, gly135, asp283 tyr648, arg569, lys568, his341, tyr90, asn133, tyr573, ala673, thr378, asp339, phe285, leu136, lys574, ala383, gly675, gly134, gly672, asn284, his377, gly135, asp283 50 xantomonascin a 89 -9.12 his377, ser674, lys574, leu136, asp283 his341, tyr573, ala673, thr671, thr378, val455, asn284, asp339, phe285, asn282, glu88, asn133, gly134, arg569, gly135, lys568, thr676, glu672, gly675, his377, ser674, lys574, leu136, asp283 51 xantomonascin b 9 -10.13 asp283, leu136, thr676, his377, ser674 lys574, ala383, tyr573, his341, asp339, asn284, thr378, thr671, ala673, glu672, val455, gly675, gly677, gly135, arg569, lys568, gly134, asn133, glu88, asn282, phe285, asp283, leu136, thr676, his377, ser674 52 y3 87 -9.29 asp283, gly677, thr676 lys574, lys568, gln665, asn678, gly675, ala673, ser674, val455, asn484, leu139, his377, thr378, leu136, gly135, asn133, gly137, asn284, gly134, arg569, tyr573, glu672, asp283, gly677, thr676 53 yellow ii 64 -8.80 his377, leu136, asp283 ala383, his341, asp339, thr378, ala673, ser674, glu672, gly675, thr676, lys568, gly677, lys574, gly135, gly134, gly137, asn133, asn282, asn284, glu88, phe285, phe286, his377, leu136, asp283 54 monaphilol a 63 -9.13 asp283, glu672, asn284, his377 tyr648, arg569, lys568, leu136, his341, phe285, asp339, thr378, gly675, gly135, tyr573, lys574, gly134, asn133, his571, asn282, asp283, glu672, asn284, his377 55 monaphilol b 15 -8.93 asp283, asn284, glu672 tyr648, arg569, lys568, leu136, his341, asn282, phe285, asp339, his377, thr378, tyr573, gly675, gly135, lys574, gly134, asp283, asn284, glu672 56 monaphilol c 42 -9.96 asp283, leu136, thr676, glu672, gly675, asn484, his377, lys574, lys568, ala383, his341, ala673, val455, ser674, tyr573, thr378, asn284, phe285, asp339, asn133,glu88, gly134, gly137, val576, gln665, gly677, ala673, asp283, leu136, thr676, glu672, gly675, asn484, his377 57 monaphilol d 48 -9.94 his377, asn484, thr676, leu136, asp283 his341, lys568, glu672, val455, ser674, leu139, ala673, tyr573, asn284, thr378, phe285, ala383, asp339, gly134, gly137, gly135, lys574, asn678, gln665, val567, gly677, gly675, his377, asn484, thr676, leu136, asp283 58 monasfluor a 87 -8.92 his377, asn484, gly675, thr676, leu136, val455, leu139, ser674, ala673, gly677, lys568, asn678, gln665, lys574, glu672, gly135, tyr573, asn284, asp283, asn282, glu88, asn133, his377, asn484, gly675, thr676 59 monasfluor b 16 -9.47 his377, asn484, thr676, his377, asn484, thr676, leu136, asn133, gly134, gly137, glu88, asp283, asn284, ser667, gln665, asn696, lys568,gly677, gly675, lys574, asn678, gly135, ala673, leu139, ser674, val455. an antidiabetic drug that works on the glycogen phosphorylase receptor. as conclusion, from drug scan test results on the isolate mps4 compound have the values t​​ hat fulfill all parameters' requirements such as molecular weight, proton donors, proton acceptors, log p and molar refractory. adme test results on isolate mps4 compounds have the values t​​ hat fulfill all parameters' requirements of caco2, hia (human intestinal absorption), as well as on ppb (plasma protein binding). the docking test results on the isolate mps4 compound were the best and qualified because it have smaller binding affinity than original ligands and comparative ligands (glibenclamide). therefore, mps4 isolate can be used as a candidate for new antidiabetic drugs, but still requires further research, including in vitro and in vivo tests. references fatimah rn. 2015. diabetes melitustipe 2 [type 2diabetes mellitus]. artikel review medical faculty lampung university. 4(5):94. lelita r, gunawan r, astuti w. 2017. studi docking molekular senyawa kuersetin, kalkon dan turunannya sebagai inhibitor selkankerpayudara mc-7 (michigan cancer foundation-7) [study of molecular docking compounds of quercetin, chalcone and their derivatives as mc-7 (michigan cancer foundation-7) breast cancer cell inhibitor. j atomik. 2(2):190-196. nguyen t, karl m, santini a. 2017. red yeast rice. foods. 6(3):19. doi:/10.3390/foods6030019. nursamsiar n, toding at, awaluddin a. 2016. studi in silico senyawa turunan analog kalkon dan pirimidin sebagai antiinflamasi: prediksi absorpsi, distribusi, dantoksisitas [in-silico study chalcone and pyrimidine analog derivatives as anti-inflammmatory: prediction of absorption, distribution, and toxicity]. pharmacy: p h a r m j o f i n d o n e s . 1 3 ( 1 ) : 9 2 1 0 0 . d o i : 10.30595/pji.v13i1.891. omran z, rauch c. 2014. acid-mediated lipinski's second rule: application to drug design and targeting in cancer. eurbiophys j 43(1):199–206. doi: 10.1007/s00249-0140953-1. qidwai t. 2017. qsar modeling, docking and admet studies for exploration of potential anti-malarial compounds against plasmodium falciparum. in silico pharm. 5(1):6. rudjianto a, yuwono a, shahab a, manaf a, pramono b, lindarto d, purnamasari d, sanusi h, zufry h, novida h, suastika k, sucipto kw,sasiarini l, dwipayana mp, saraswati mr, soetedjo nn, soewondo p, soelistijo sa, sugiarto, langi ya. 2015. konsensus pengelolaan dan pencegahan diabetes melitus tipe2 di indonesia [consensus of management and prevention of type 2 diabetes mellitus in indonesia]. pengurus besar perkumpulan endokrinologi indonesia (pb perkeni), jakarta. ruswanto r, nofianti t, mardianingrum r, and lestari t. 2018. desain dan studi in silico senyawa turunan kuwanon-h sebagai kandidat obat anti-hiv [design and in silico study of kuwanon-h as anti-hiv drug candidate]. valensi. 4(1):57-66. ruswanto r. 2015. desain dan studi interaksi senyawa n'-(3, 5 d i n i t r o b e n z o y l ) i s o n i c o t i n o h y d r a z i d e p a d a mycobacterium tuberculosis enoyl-acyl carrier protein reductase (inha) [design and interaction study of the c o m p o u n d n ' ( 3 , 5 d i n i t r o b e n z o y l ) i s o n i c o t i n o h y d r a z i d e o n m y c o b a c t e r i u m tuberculosisenoyl-acyl carrier protein reductase (inha)]. jurnal kesehatan bakti tunas husada. 12(1):192-201. ruswanto r. 2015. molecular docking empat turunan isonicotinohydrazide pada mycobacterium tuberculosis enoyl-acyl carrier protein reductase (inha) [molecular docking of four isonicotinohydrazide derivatives in mycobacterium tuberculosis enoyl-acyl carrier protein reductase (inha)]. jurnal kesehatan bakti tunas husada, 13(1). seyedin a, hatamian-zarmia, rasekh b, mirderikvand m. 2 0 1 5 . n a t u r a l p i g m e n t p r o d u c t i o n b y monascuspurpureus: bioreactor yield improvement through statistical analysis. appl food biotechnol. 2(2):23-30. sherman w, beard hs, farid r. 2006. use of an induced fit receptor structure in virtual screening. chem biol drug. 67(1):83-84. singgih m, permana b, maulidya sai, yuliana, a. 2019. studi in silico metabolit sekunder kapangmonascus sp. sebagai kandidat obat antikolesterol dan antikanker [in silico study of monascus sp. as candidates for anticholesterol and anticancer drugs]. alchemy. 15(1):104-123. sulistyaning ar, wirawanni y. 2013. pengaruh pemberian angkak (red yeast rice) terhadap kadar glukosa darah puasa pada wanita prediabetes [the effect of red yeast rice on fasting blood glucose levels in prediabetic women] (doctoral dissertation, diponegoro university). yuliana a, singgih m,julianti e, blanc p. 2017. derivates of azaphilone monascus pigments. biocatalysis and agricultural biotechnology. 9(1):183-194. volume 14, 2020 microbiol indones 65 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 7.mi676-ratna wahyuni available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.3.7issn 1978-3477, eissn 2087-8575 vol 6, no 3, september 2012, p 135-138 *corresponding author; phone: +62-31-5965304/5, e-mail: ratna_wahyuni@yahoo.com mycobacterium leprae is the causal agent of leprosy, a chronic infectious disease that still becomes major problem especially in indonesia. since mdt) for leprosy has been introduced in 1982, the prevalence of leprosy worldwide significantly decreased (lockwood and suneetha 2005). indonesia has reached elimination program in year 2000 with prevalence rate less than multidrug therapy ( 1/10.000 inhabitant (departemen kesehatan ri 2008), but new cases still remain high (world health organization (who) 2011). there are some endemic pocket areas in indonesia with high prevalence and high number of patient (who 2010). as an effective treatment for leprosy, mdt regiment has effectively reduced prevalence of the disease, but the drug resistance to the mdt component still emerged. drug resistance cases have been reported from many areas (dela cruz et al. 1996; kai et al. 1999; cambau et al. 2002; matsuoka et al. 2007), and profile of mutation on drug resistance mycobacterium leprae isolates in indonesia collected during 2003-2011 1 1 1 2 ratna wahyuni *, dinar adriaty , iswahyudi , cita rosita sigit prakoeswa , 2 1 indropo agusni , and shinzo izumi 1 leprosy study group, institute of tropical disease, universitas airlangga, jalan mulyorejo kampus c unair, surabaya 60115, indonesia; 2 departement of dermatology and venereology , faculty of medicine, universitas airlangga; jalan prof dr moestopo 47, surabaya 60131, indonesia short communication multidrug therapy (mdt) regiment has been used for leprosy all over the world for more than 20 years. drug resistances of mycobacterium have been reported from many areas. the resistance mostly occurred due to mutation on the gene coding protein targeted by anti-leprosy drugs. two hundreds and seventy m. isolates from some area in indonesia were examined for studying the profile of mutation among isolates collected during 2003-2011. drug resistance determining region of the folp1 gene and the rpob gene was sequenced. the results showed 5 isolates of m. leprae harboured mutation only in the folp1 gene and another isolate harbored mutation in both the folp1 and rpob gene. the point mutation in the folp1 gene that was found in 2 isolates occurred in codon 53 (acc gcc; thr ala). double point mutations on codon 53 that was found in two isolates were acc aga (thr arg) and acc agg (thr arg). the point mutation in the folp1 gene occurred in codon 55 were found in two isolates were ccc ctc (pro leu) and ccc cgc (pro arg). whereas mutation in the rpob gene in one isolate occurred in codon 410 was gat tat (asp tyr). these mutations that altered the amino acids of the protein revealed that isolates of m. leprae were resistant to drug with variable profiles. key words: dapsone, drug resistance, mutation, mycobacterium leprae, rifampicin terapi kombinasi (multidrug therapy) telah digunakan untuk pengobatan penyakit kusta di seluruh dunia selama lebih dari 20 tahun dan kasus-kasus resistensi mycobacterium leprae terhadap obat anti kusta telah dilaporkan dari beberapa daerah. resistensi terjadi karena adanya mutasi pada gen yang mengkode protein target dari obat-obat anti kusta. dua ratus tujuh puluh isolat m. leprae dari beberapa daerah di indonesia diperiksa untuk mengetahui profil mutasi isolat-isolat yang dikoleksi selama tahun 2003-2011. daerah gen yang bertanggung jawab pada resistensi terhadap obat yakni gen folp1 dan rpob telah disekuensing. hasil penelitian menunjukkan 5 isolat m. leprae mengalami mutasi hanya pada gen folp1, sementara satu isolat mengalami mutasi pada gen folp1 dan rpob. mutasi titik pada gen folp1 yang ditemukan pada dua isolat terjadi di kodon 53, yaitu acc gcc (thr ala). mutasi titik ganda di kodon 53 ditemukan juga pada dua isolat, yaitu acc aga (thr arg) dan acc agg (thr arg). mutasi titik pada gen folp1 yang terjadi di kodon 55 ditemukan pada dua isolat, yaitu ccc ctc (pro leu) dan ccc cgc (pro arg). sementara mutasi pada gen rpob ditemukan hanya pada satu isolat, yakni pada kodon 410 (gat tat; asp tyr). mutasi-mutasi yang ditemukan mengubah asam amino dari protein, menunjukkan bahwa isolat-isolat m. leprae yang mengalami mutasi tersebut resisten terhadap obat dengan profil mutasi yang bervariasi. kata kunci: dapson, mutasi, mycobacterium leprae, resisten obat, rifampisin leprae leprae . 136 wahyuni microbiol indones water, 20 μl 2x premix g, 0.4 l taq polymerase, 1.6 µl primer each (stock solution 5m), dna template was added for 4 μl, total volume was 40 μl. pcr kit tm (premix g, taq polymerase) was from failsafe pcr system (epicentre biotechnologies). pcr product tm was purified using gfx pcr dna and gel band purification kit (ge healthcare). tm dual cydye terminator sequencing kits (ge healthcare) was used in the preparation of sequencing reaction. the reaction was performed according to the manufacture's manual. the sequencing reaction was done in biorad i-cycler machine under the following condition: 95 °c for 20 sec, tm of sense primer +3 °c for 15 sec, 70 °c for 1 min. the condition was done for 35 cycles. the sequencing product was then purified by ethanol precipitation and dried followed by dissolving in 2 μl of loading dye and was loaded into prepared tm acrylamide gel in long-read tower system (amersham biosciences). sequence analysis was done tm using long-read tower system (amersham biosciences) with the temperature was set on 60 °c as described as in the protocol. study showed that all isolates for dapsone and rifampicin resistances were positive in pcr examination. sequencing result showed, there were six out of two hundred and seventy m. leprae isolates (2.22%) harbour mutation in the folp1 gene. four isolates had mutation on codon 53 and two isolates on codon 55. different five profiles of mutation in the folp1 gene have been found. only one out of two hundreds and seventy m. leprae isolates (0.37%) had mutation in the rpob gene on codon 410 (table 1). the partial dna sequence of the m. leprae isolates showed that all profiles of mutation found in this study were varied (fig 1) and were same to the types of mutation that have been previously reported (matsuoka et al. 2008). the first profile of mutation in the folp1 gene was point mutation occurred in codon 53 from acc (threonine) to gcc (alanine). it was occurred in two isolates. the second profile was double point mutation on codon 53 from acc (threonine) to aga (arginine). the third was also double point mutation on codon 53 which was changed to agg (arginine). the fourth was point mutation occurred on codon 55 which was from ccc (proline) changed to ctc (leucine). the last profile was point mutation on codon 55 which was changed to cgc (arginine). all these profiles of mutation that changed amino acid of the protein indicated that isolates were resistance to dapsone. only one profile of mutation found in the rpob gene on μ+some are multidrug resistance (cambau et al. 1997; matsuoka et al. 2000; maeda et al. 2001; you et al. 2005). mdt prescribed by who, containing dapsone, rifampicin, and clofazimine, is the major regiment for leprosy treatment, and it is important to detect the resistant cases for evaluating efficacy of mdt and supporting leprosy elimination program. the resistance mostly occurred due to mutation on the gene coding protein targeted by anti-leprosy drugs (kai et al. 1999; cambau et al. 1997; matsuoka et al. 2000; maeda et al. 2001). clofazimine resistance has not been described, and its mechanism and molecular methods to detect this, are unknown who regional office for south-east asia. 2009; matsuoka 2010). some m. leprae isolates from some areas in indonesia were examined for studying the profile of mutation in the folp1 and rpob genes among isolates collected during 2003-2011. the study was established for exploring mutation on m. leprae isolates as information of drug resistance cases in indonesia. two hundreds and seventy m. leprae isolates were obtained from multibacillary (mb) leprosy patients by taking skin slit specimens from the lesion or ear lobe. samples were taken from jawa timur, nusa tenggara barat, nusa tenggara timur, south sulawesi, maluku, and papua barat. one sample from kalimantan were taken in surabaya. samples were taken from 144 under mdt treatment patients, 63 release from treatment (rft) patients, 59 new case patients, 3 rok (rifampicin, ofloxacin, clarithromycin) treatment patients, and 1 single (dapsone) treatment patient. specimens were put in 1.5 ml eppendorf tube contain phosphate buffered saline (pbs) solution. dna isolation from the specimens was done by using qiaprep spin miniprep kit (qiagen) with procedures as described in manual book. amplification of dna by pcr was done in biorad i-cycler machine using folp1-folpr2 primers (folp1 5'gcttctcgtgccgaagcgctc-3'; folpr2 5'gcgcgtagtatcgatacttac-3'; pcr product length 305 bp) and rpobf-rpobr primers (rpobf 5'caggacgtcgaggcgatcac-3'; rpobr 5'cagcggtcaagtattcgatc-3'; pcr product length 374 bp) to obtain dna sequence target of the folp1 and rpob genes. the condition of both pcr was as follows : 1) 98 °c for 2 min; 2) 98 °c for 30 sec, 63 °c -59°c down 1 degree percycle for 30 sec, 72 °c for 30 sec; for 5 cycles; 3) 98 °c for 30 sec, 58 °c for 30 sec, 72 °c for 30 sec, for 40 cycles; 4) 72 °c for 5 min. the mixture for all pcr condition was 12.4 µl of distilled the drug-resistant strains (robert 1976) unfortunately there is no data on medication compliance among isolates available in this study, so it could not be described further. here, we are describing a method for detecting the mutation that responsible for drug resistances by pcr followed by direct sequencing which is simpler and faster than mouse footpads culture method. it is also very convenience to conduct but the implementation in developing country such as indonesia is still not easy. it is also not suitable and expensive for high number of sample. development of simple detection methods as a leprosy drug susceptibility-dna microarray (ldsda) and a reverse hybridization dna strip test will be very much helpful for maintaining drug resistance study in developing country where the prevalence of leprosy is still high (matsuoka 2010; matsuoka et al. 2008; cambau et al. 2012). this study supports and volume 6, 2012 microbiol indones 137 no isolate treatment status drug gene no. wild type mutant 1 . surabaya1 under mdt treatment dapsone folp1 53 acc (thr) gcc (ala) 2 . surabaya2 under mdt treatment 53 acc (thr) gcc (ala) 3 . surabaya3 new case 53 acc (thr) aga (arg) 4 . makassar1 under mdt treatment 53 acc (thr) agg (arg) with dapsone monotherapy before 5 . kalimantan1 new case 55 ccc (pro) ctc (leu) 6 . pasuruan1 relapse case 55 ccc (pro) cgc (arg) rifampicin rpob 410 gat (asp) tat (tyr) table 1 mutation profile of six drug resistance case-mycobacterium leprae isolates in indonesia fig 1 the partial dna sequence of the mycobacterium leprae isolates; a. wild type of folp1 gene on codon 53 and 55, b-d. mutant of folp1 gene on codon 53, e-f. mutant of folp1 gene on codon 55, g. wild type of rpob gene on codon 410, h. mutant of rpob gene on codon 410 (blue square represent no mutation, red square represent mutation). codon 410 that was alteration from gat (aspartic acid) to tat (tyrosine) and again indicated that the isolate was resistant to rifampicin. while treatment status of six drug resistance casem. leprae isolates are known, it can be concluded that two isolates were primary resistant to dapsone, since both were new cases. other isolates were regarded to be secondary resistant to dapsone. one isolate (pasuruan1) harboured mutation on both gene (folp1 and rpob). the isolate was regarded to be secondary resistant to dapsone and rifampicin as the isolate was taken from relapse case assumed to be resistant. treatment with inappropriate regiments is one main reason of drug-resistant strains development and the occurrence of relapses with a resistant strain (matsuoka 2010). it was found in one isolate (makassar1) that had dapsone monotherapy. beside that inconsistent prolonged treatment can also raises kai m, matsuoka m, nakata n, maeda s, gidoh m, maeda y, hashimoto k, kobayashi k, kashiwabara y. 1999. diaminodiphenylsulfone resistance of mycobacterium leprae due to mutation in the dihydropteroate synthase gene. fems microbiol lett. 177(2):231-235. doi: 10.1111/j.1574-6968.1999.tb13737.x. lockwood dn, suneetha s. 2005. leprosy: too complex a disease for a simple elimination paradigm. bulletin of world health organiztion. 83(3): 230-235. maeda s, matsuoka m, nakata n, kai m, maeda y, hashimoto k, kimura h, kobayashi k, kashiwabara y. 2001. multidrug resistant mycobacterium leprae from patients with leprosy. antimicrob agents chemother. 45(12):3635-3639. doi:10.1128/aac.45.12.36353639.2001. matsuoka m. 2010. drug resistance in leprosy. jpn j infect dis. 63(1): 1-7. matsuoka m, aye ks, kyaw k, tan ev, balagon mv, saunderson p, gelber r, makino m, nakajima c, suzuki y. 2008. a novel method for simple detection of mutations conferring drug resistance in mycobacterium leprae, based on dna microarray, and its applicability in developing countries. j med microbiol. 57(10):12131219. doi: 10.1099/jmm.0.2008/002600-0. matsuoka m, budiawan t, aye ks, kyaw k, tan ev, cruz ed, gelber r, saunderson p, balagon v, pannikar v. 2007. the frequency of drug resistance mutations in mycobacterium leprae isolates in untreated and relapsed leprosy patients from myanmar, indonesia, and the philippines. lepr rev. 78(4):343-352. matsuoka m, kashiwabara y, namisato m. 2000. a mycobacterium leprae isolate resistant to dapsone, rifampin, ofloxacin and sparfloxacin. jacobsen print j lepr. 68(4): 452-455. world health organization (who). 2010. weekly epidemiological report. 85: 249-264. world health organization (who). 2011. weekly epidemiological report. 86: 389-400. world health organization regional office for south-east asia. 2009. guidelines for global surveillance of drug resistance in leprosy. world health house, india. you ey, kang tj, kim sk, lee sb, chae gt. 2005. mutations in genes related to drug resistance in mycobacterium leprae isolates from leprosy patients in korea. j infect. 50(1): 6-11. doi:10.1016/j.jinf.2004.03.012. microbiol indones138 wahyuni complements on the information about mutation profile of drug resistance m. leprae isolates in indonesia. acknowledgments this work was supported by jica, leprosy ngo from japan and phki (program hibah kompetisi berbasis institusi/institution-based grant program competition) from indonesia government. thank you for masanori matsuoka from leprosy research center tokyo japan for supervising technique of molecular detection of drug resistant m. leprae. references cambau e, bonnafous p, perani e, sougakoff w. ji, and v. jarlier. 2002. molecular detection of rifampin and ofloxacin resistance for patients who experience relapse of multibacillary leprosy. clin infect dis. 34(1): 39-45. doi: 10.1086/324623. cambau e, perani e, guillemin i. 1997. multidrugresistance t o d a p s o n e , r 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j, cole st. 1993. a simple and rapid technique for the detection of rifampin resistance in mycobacterium leprae. int j lepr. 61(4): 600-604. 1: 135 2: 136 3: 137 4: 138 5 endang.pmd volume 3, number 3, december 2009 p 121 125 issn 1978-3477 horizontal transfer of the “popcorn-effect” strain of wolbachia from drosophila melanogaster to stomoxys calcitrans endang srimurni kusmintarsih faculty of biology, universitas jenderal soedirman, jalan dr soeparno no 63 grendeng, purwokerto 53122 phone: +62-0281-638794, fax: 62-0281-631700, e-mail: endang_sk@lycos.com the wolbachia containing haemolymph from wolbachia infected drosophila third/fourth instar larvae was transferred through microinjection into 5-days-old pupae of the blood sucking stable fly, stomoxys calcitrans. there was no previous record of wolbachia being present in s. calcitrans. from a total of 682 emerging adults, 236 were females and of these, seven females were tested positive (approximately 3%) for wolbachia infection. using electron microscopy, it was shown that wolbachia were present in the muscle tissues of s. calcitrans. key words: drosophila melanogaster, stomoxys calcitrans, ‘popcorn-effect’, haemolymph, microinjection wolbachia infection was first discovered in mosquitoes (hertig 1936). since then, these intracellular bacteria have been found in a variety of insect species, mites and isopods (rousset et al. 1992; breeuwer and jacobs 1996; werren 1997; grenier et al. 1998). wolbachia can induce different effects in the reproductive organs of its hosts. these include cytoplasmic incompatibility (ci) in isopods, insects and mites (breeuwer and jacobs 1996), parthenogenesis/thelytoky (t) in parasitoid insects (stouthamer et al. 1990), feminisation (f) in isopods (juchault et al. 1994), male-linked lethality in drosophila bifasciata and the butterfly acraea encedon (hurst et al. 1999 and 2000), and the ‘popcorn-effect’ in d. melanogaster in which an early death of the insect occurs (min and benzer 1997). a sudden massive degeneration of the fly’s cells occurs in response to bacterial multiplication. the bacteria are present in low numbers during development through the embryonic, larval and pupal stages. as soon as the adult flies emerge, the bacteria start to multiply rapidly, causing sudden death of their host. it might be possible to make use of the ‘popcorn-effect’ induced by wolbachia to control insect vectors of important human diseases in which the pathogen or parasite is transmitted at the end of the insect’s life span after it has taken three to four blood meals. in the future, this may play a vital role in the prevention of arthropod-borne diseases; disease transmission depends on the older insect and wolbachia infection has been found to shorten the life span of insects. wolbachia is maternally inherited. however, there is strong evidence for horizontal transfer between species that are distantly related, based on non-congruence of host and bacterial phylogenies (o’neill et al. 1992; rousset et al. 1992; werren et al. 1995). comparative molecular phylogenies of 20 parthenogenetic trichogramma populations and their symbiotic wolbachia suggest the occurrence of occasional horizontal transmission (schilthuizen and stouthamer 1997). this is a good indication that individual wolbachia species or strains are not restricted to a single host. the ‘popcorneffect’-inducing wolbachia causes a reduction in insect life span, so if this strain could be transferred to insects that are disease vectors, it might be an effective vector control. for example, adult anopheles gambiae, which transmits plasmodium spp. (causing malaria), has a life expectancy of 15 days (garrett-jones and shidrawi 1969) whilst plasmodium has an incubation period of 11 days (baker 1966). similarly, adult aedes trivittatus has a life expectancy of about 25 days in the laboratorium (christensen 1978) whilst the parasite it transmits (dirofilaria immitis) has an incubation period of 16 days at 22.5oc (christensen and hollander 1978). they will only be infectious and pathogens transmitted after the host has taken at least three to four blood meals. if the insect was infected with the ‘popcorn-effect’-inducing wolbachia, the insect will experience early and sudden death in its adult stage perhaps preventing disease transmission to humans. experimental transfers have been performed, in which wolbachia has been transferred by microinjection (within and between species, between families, and between orders (boyle et al. 1993; braig et al. 1994; chang and wade 1994; juchault et al. 1994; karr 1994; rousset and stordeur 1994; rigaud and juchault 1995; sinkins et al. 1995; clancy and hoffmann 1997; dunn and rigaud 1998; grenier et al. 1998; poinsot et al. 1998; van meer and stouthamer 1999; fujii et al. 2001). the interfamily transfer of wolbachia was demonstrated by braig et al. (1994). they found that bacteria from the mosquito aedes albopictus could be transferred by microinjection into uninfected embryos of d. simulans, conferring complete ci on the adults. recently, embryonic microinjection protocol for wolbachia transfection have been developed for the mosquitoes a. albopictus (xi et al. 2006) and a. aegypti (xi et al. 2005), as well as wolbachia transfer protocols based on injection of symbionts directly into adult drosophila and aedes mosquitoes (frydman et al. 2006; ruang-areerate and kittayapong 2006). grenier et al. (1998) successfully transferred the symbiote within the trichogrammatidae. however, there is no study to date on horizontal transfer of the ‘popcorn-effect’. to investigate whether the ‘popcorn-effect’ phenotype is specific to drosophila or whether it can be transmitted to another species, the popcorn-effect inducing wolbachia was transferred from d. melanogaster by microinjection into uninfected s. calcitrans pupae. there is no previous record of wolbachia being present in s. calcitrans. to verify the presence of wolbachia in newly infected individuals of s. calcitrans, the tissues from s. calcitrans which tested positive for wolbachia by pcr were then microbiol indones122 kusmintarsih examined by electron microscopy. this is to confirm that the positive pcr amplification represented the identification of real wolbachia, and not a fortuitous pcr amplification of genomic dna. materials and methods microinjections. stomoxys calcitrans was obtained from prof lehane’s and d. melanogaster larvae harbouring wolbachia ‘popcorn-effect’ was obtained from dr. braig’s laboratory school of biological sciences, university of wales, bangor, united kingdom. microinjection experiments using cell micro-injector pm 1000, a d. melanogaster fourth instar larva, derived from a previously established colony, was placed in front of the microcapillary needle and punctured. subsequently, the haemolymph was drawn into the needle via capillary action of the filament. a 5-day-old s. calcitrans pupa was placed in front of the needle and punctured with the use of the macroand microcontrols. the d. melanogaster haemolymph in the needle flowed into the pupa when the control pedal was pressed. the microcapillary needle was then removed. if the needle broke or if backflow into the needle occurred, then the pupa was discarded. a new needle was used for each transfer. in order to ensure that all pupae used in this experiment were of the same age (5-day-old pupae), all stock used was 17 days old from egg collection. the pupae were kept on moist cotton wool (checked daily, water being added if necessary). rearing, collection, and killing of flies. after microinjection the pupae were put in a plastic cup (10.5 cm diameter, 4 cm height) containing moist cotton wool. humidity was maintained by regular addition of water. the plastic cups were housed in net cages (26 cm length, 26 cm width and 26 cm height) at 25oc. the number of pupae in every plastic cup container depended on the number of pupae that were injected in the same day (187, 69, 173, 48, 30, 184, 199, 119, 87, 169, 109, 109, 109, 116 and 37). the cages were placed on paper in order to facilitate egg collection (next generation). adults emerged after several days and were allowed to mate. adult flies were fed daily with pig’s blood (by placing cotton wool, which was soaked in the pig’s blood, on top of the net cage). eggs were transferred from the paper into a plastic cup container (10 cm diameter and 4 cm height) containing larval feeding medium after 28 days from emergence, adult flies were killed and the females tested for wolbachia infection. the period of 28 days after emergence, is the longest possible time before risking loosing any flies due to natural mortality. the flies were not killed sooner in order to allow the bacteria to grow in the new host. the period of 28 days gave the female flies the possibility to lay eggs so that the line would continue to the next generation. as wolbachia are maternally inherited, the bacteria would be transmitted to the progeny just through the females, not the males. dna extraction, electrophoresis, and electron microscopy. the procedure of dna extraction was using the dneasy tissue kit (qiagen). the extracted dna from the adult stomoxys calcitrans female was analysed using the polymerase chain reaction (pcr) to determine evidence of wolbachia infection. the procedure was according to the manufacture instructions (promega). one master mix was used to detect wolbachia (using wolbachia surface protein wsp gene). forward (w81f: 5′-tgg tcc aat aag tga tga aga aac-3′) and the reverse primer (w691r: 5′-aaa aat taa acg cta ctc ca-3) were designed to amplify a fragment of 610 bp (braig et al. 1998); as a control to detect mitochondria, forward primer 12sbif (5′-aag agc gac ggg cga tgt gt-3′) and reverse primer 12sair (5′aaa cta gga tta gat acc cta tta t-3′ ) were used. the microcentrifuge tubes were then placed in a thermal cycler (technogene). an initial denaturation at 95°c for 5 minutes was followed by 35 cycles of 95°c for 1 minute, 55°c for 1 minute and 72°c for 2 minutes: followed by 72°c for 10 minutes and finally 4°c. to verify the presence of wolbachia in individuals of s. calcitrans, which were infected by microinjection of haemolymph from d. melanogaster infected with the popcorn strain of wolbachia, the tissues from s. calcitrans tested positive for wolbachia by pcr were further examined by electron microscopy. this is to confirm that the positive pcr amplification represented the identification of real wolbachia, not fortuitous pcr amplification of genomic dna. the tissues (muscle, testes, ovaries, brain and eyes) were taken from flies dissected in pbs (phosphate buffer saline) and put in karnofsky’s fixative (glauert and lewis 1998) consisting of 0.4% paraformaldehyde and 0.16% glutaraldehyde in sodium cacodylate, 10% w/v sucrose, ph 7.2 for 1 h, then washed twice using pbs. the tissues are then put in 1% osmium tetroxide plus cacodylate buffer (1:1) for one hour, washed twice with water and put in 2% uranyl acetate and kept in the fridge (4oc) overnight. after fixation the material was dehydrated using serial ethanol changes, and embedded in the resin. a glass knife was used to shape and cut the embedded sample in the piramitome (lkb leica) and ultratome (lkb leica). the sections were collected on grids (pioloform coated nickel 200 mesh grids) and then stained with lead citrate for 6 minutes. sections were observed using a philips em 60 kv. results from a total of 682 emerging adults, 236 were females and of these, seven females (approximately 3%) tested positive for wolbachia infection. fig 1 and 2 show gel electrophoreses of injected female s. calcitrans testing positive for wolbachia infection. the first lane was a one kilobase ladder, second lane was wolbachia positive control from d. melanogaster infected with ‘popcorn-effect’. therefore, the next lanes, which had the same size of band as the d. melanogaster positive control, indicated that s. calcitrans were wolbachia positive lane 4, 6, 8, 10, 18 and 20 in fig 1 and lane 4 in fig 2. mitochondrial primers (12sbif and 12sair) were used for checking whether the conditions for the pcr were appropriate in every sample. using electron microscopy it was shown that wolbachia were present in the muscle tissues of s. calcitrans (fig 3 and 4). to provide evidence that s. calcitrans did not contain bacteria before injection with wolbachia, the muscle tissue was examined by pcr and electron microscope. fig 5 shows gel electrophoresis of s. calcitrans testing negative for microbiol indones 123volume 3, 2009 fig 1 gel electrophoresis of stomoxys calcitrans testing positive for wolbachia infection. lane 1, 1 kb ladder; lane 2, positive control for wolbachia from drosophila melanogaster; lane 3, positive control for mitochondria from d. melanogaster; lane 4, 6, 8, 10, 18 and 20. wolbachia positive results from s. calcitrans; lane 5, 7, 9, 11, 13, 15, 17, 19 and 21, mitochondria from s. calcitrans; lane 12, 14, 16. wolbachia negative results from s. calcitrans. fig 2 gel electrophoresis of stomoxys calcitans testing positive for wolbachia infection. lane 1, 1 kb ladder; lane 2, positive control for wolbachia from d. melanogaster; lane 3, positive control for mitochondria from d. melanogaster; lane 4, wolbachia positive result from s. calcitrans; lane 5, 7, 9, and 11. mitochondria from s. calcitrans; lane 6, 8, and 10. wolbachia negative from s. calcitrans. fig 3 stomoxys calcitrans muscle tissue (51,000 x). using electron microscopy it was shown that wolbachia were present in the muscle tissues of s. calcitrans. m, mitochondria and f, fibril of muscle. arrow: wolbachia. fig 4 stomoxys calcitrans muscle tissue (19,000 x). m, mitochondria and f, fibril of muscle. arrow: wolbachia. fig 5 gel electrophoresis of stomoxys calcitrans testing negative for wolbachia infection before injecting. lane 1, 1 kb ladder; lane 2. positive control for wolbachia; lane 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 mitochondria primer of s. calcitrans; lane 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 wolbachia primer of s. calcitrans there were no bands indicating negative of wolbachia. figs 6 muscle of uninfected stomoxys calcitrans before injection (20 000 x): a, a horizontal section and b, a transversal section. m, mitochondria and f, fibril of muscle. there is no wolbachia in the tissue. a b microbiol indones wolbachia infection before injecting and fig 6 show muscle tissues of s. calcitrans, where no wolbachia or any other bacterial structures were detected. this indicated that uninjected (normal slide) s. calcitrans were negative for wolbachia. discussion from 236 females, only seven females (approximately 3%) tested positive for wolbachia infection. there are several possible reasons for why such a low number of positive results were obtained. firstly, the wolbachia density might have been too low to produce an infectious dose in the pupae, resulting in elimination of the inoculum. secondly, that the interaction between wolbachia symbiont from d. melanogaster and the new host genome of s. calcitrans is unfavourable (kondo et al. 2005). therefore, the success of transmission for the bacteria are influenced by several factors, including bacterial density and host genotype (breeuwer and werren 1993; bourtzis et al. 1996; grenier et al. 1998; kondo et al. 2005; duron, et al. 2006; mouton et al. 2006). thirdly, the pupae might have been too young. young pupae have a very up-regulated immune system whereas older pupae (which are protected by an additional exoskeleton) exhibit a highly down-regulated immune system (braig hr 2009, personal communication). if the pupae are too young at the time of infection, the inoculum might be destroyed by the immune system regardless of its dose. however, if the pupae are much too old, the barrier between haemolymph and germ line tissue might become unpenetrable for wolbachia. strong somatic infections might result but permanent, inheritable infections might not be established. even a germ line infection requires a certain threshold density of bacteria before a productive infection in the next generation can be secured. reaching this threshold for an endosymbiont that has been adapted to a different host for thousands and thousands of generations might constitute an immense barrier to be overcome. although pcr is far from quantitative, some of the wolbachia signals from females in lanes 4, 6, 8, 10, 18 and 20 in fig 1 and lane 4 in fig 2 were similar the intensity of the wolbachia signal of the infected donor, being the ‘popcorneffect’ of d. melanogaster. the strong signal of females (aged 28 days) suggests a productive infection. the pcr assay is not sensitive enough to pick up the inoculum. lane 4 is presumably an example of a weak infection. unfortunately, even the six strongest infections did not lead to any inheritance. similar results have been obtained in flour beetles (tribolium confusum) in which the infection has been lost in the second generation as well (chang and wade 1994). van meer and stouthamer (1999) transferred wolbachia from muscidifurax uniraptor (hymenoptera), which harbours parthenogenesis-inducing wolbachia to d. simulans (diptera) without establishing a stable infection. for s. calcitrans that were positive for wolbachia after transferring these bacteria by microinjection, no specific effects on the host were detected and the bacteria were not maintained stably. however, investigation by electron microscopy showed that the bacteria were present in the tissues of infected s. calcitrans such as the thoracic muscle. this indicates that wolbachia develops in the tissues of the new host. it was assumed that the bacteria did not reach the reproductive organs when injected, thereby causing no maternal transmission to the next generation. furthermore, host-symbiont interactions are important for success in the establishment of an infection. another example of a failed transfer of wolbachia has been obtained for the isopods chaetophiloscia elongata and armadillidium vulgare, which are distantly related species. however, wolbachia could be transferred between closely related species of isopods (a. nasatum to a. vulgare) (juchault et al. 1994). on the other hand, phylogenetic work on wolbachia (werren et al. 1995) provides evidence that, at least in some cases, this bacterium has successfully transferred over large phylogenetic distances in its interspecific movement. it remains unknown what further permits particular inter-family transfers to occur successfully, other than time. using electron microscopy it was shown that wolbachia were present in the muscle tissues of s. calcitrans. the most common form observed was an elongated cylinder with convex ends typical of bacterial rods; it is the most common shape of bacteria, after all, the word bacterium means rod or staff. the second form of wolbachia that had been encountered was a barrel shape, oval like an egg and showed a maximum diameter along its length. the presence of wolbachia in s. calcitrans was reported for the first time in this study. it was confirmed by em following a positive pcr result. however, although there were positive results in s. calcitrans, no permanent establishment of wolbachia from d. melanogaster occurred in s. calcitrans and the wolbachia could not be detected in the next generation. a reason for the infection not being stable is that wolbachia may not be physiologically adapted to its new host. the wolbachia found in somatic muscle tissues of s. calcitrans, were morphologically similar to the wolbachia present in d. melanogaster acknowledgement this work was supported by due batch ii, universitas jenderal soedirman, purwokerto, indonesia. i would like thank to mj lehane and henk r braig, school of biological sciences, university of north wales, bangor, united kingdom for guidance and providing facilities, and thank to alison bell for the help with electron microscopy. references baker jr. 1966. the fine structure of the exoerythrocytic stages of plasmodium fallax. trans r soc trop med hyg 28:355–73. bourtzis k, nirgianaki a, markakis g, savakis c. 1996. wolbachia infection and cytoplasmic incompatibility in drosophila species. genetics 144:1063-73. boyle l, o’neill sl, robertson hm, karr tm. 1993. interspecific and intraspecific horizontal transfer of wolbachia in 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population. science 310:326–8. xi z, khoo cc, dobson sl. 2006. interspecific transfer of wolbachia into the mosquito disease vector aedes albopictus. proc biol sci 273:1317–22. 129-134 (andi utama).pmd recombination is integral to the evolution of enteroviruses, including poliovirus (pv). the first evidence of recombination of pv was detected in vaccine-related isolates with chimeric sequences excreted by children exposed to the trivalent oral poliovirus vaccine (opv) (king 1988; cammack et al. 1988). similar isolates were later detected in a number of patients with vapp (driesel et al. 1995; li et al. 1996; martin et al. 2002) as well as in healthy opv recipients (cammack et al. 1988; tatem et al. 1991; blomqvist et al. 2003). the heterologous sequences of most vaccinerelated isolates were derived from the other sabin opv strains, with recombinants frequently found among vaccinerelated isolates of all serotypes (cammack et al. 1988; lipskaya et al. 1991). a small proportion of vaccine-related isolates have capsid sequences derived from the opv strains and noncapsid sequences derived from other, nonvaccine viruses. recombination among the opv strains is readily detectable because the sequences of the parental vaccine strains are well defined (toyoda et al. 1984). recombination also occurs during the circulation of wildtype polioviruses and other enteroviruses. recombination of opv and circulating wild-type pv was reported in china (liu et al. 2000; liu et al. 2003), and recombination of opv and circulating non-polio enteroviruses were reported in the dominican republic and haiti (kew et al. 2002), in madagascar (rousset et al. 2003), in egypt (yang et al. 2003), and in the philippines (shimizu et al. 2004). sequence analysis showed that the above circulating vaccine-derived polioviruses (cvdpvs) were recombinants between pv and unidentified enterovirus that underwent recombination in the nonstructural protein-coding regions of the genome. microbiology indonesia, december 2007, p 129-134 volume 1, number 3 issn 1978-3477 in vitro recombination of poliovirus with coxsackie a virus serotype 18 at downstream nonstructural protein-coding regions andi utama1* and hiroyuki shimizu2 1research center for biotechnology, lembaga ilmu pengetahuan indonesia, jalan raya bogor km 46, cibinong 16911, indonesia, 2department of virology ii, national institute of infectious diseases, 4-7-1 gakuen, musashimurayama, tokyo 208-0011 many genetic recombinations of poliovirus (pv) are to be found in excreted viruses, including viruses from vaccineassociated paralytic poliomyelitis (vapp) as well as healthy vaccine recipients. most recombinations were among different serotypes of pvs. however, recombination can also occur between pv and other enteroviruses. it was predicted that the hot spot of the recombination is in the nonstructural protein-coding regions, but the exact site is may be different in each recombination. we have demonstrated that the construct recombinant virus between pv and coxsackie a virus serotype 11 (cav-11), or with cav-17 with recombination site in the n-term of 2c-coding region, were viable. however, the recombination of pv with cav-18 at this site was not viable. to determine if the recombination between pv and cav-18 can occur at other sites, eight chimeric cdnas (between pv [isolate pj156] and cav-18 [pj156/cav-18]), all having different recombination sites (2c-8, 2c-133, 2c-235, 2c-268, 2c-287, 2c-327, 3a-67, 3c-60) were constructed using the long-pcr method. the cdna was then transcribed in vitro and then transfected into the hep-2 cell-line. as expected, the recombinant virus pj156/ cav-18, with recombination sites 2c-327, 3a-67, and 3c-60 were viable, while all the others were not. the recombinant viruses displayed a slightly smaller plaque size, but demonstrated quite similar growth as compared to the parental control pj156. since analysis for similarity has shown that the homology between pv and cav-18 was high around these regions, these results supported the copy-choice mechanism of enterovirus recombination. key words: poliovirus, cav-18, recombination _____________________________________________ ________________________ * corresponding author, phone: +62-21-8754587, fax: +62-21-8754588, e-mail: andiutama2002@yahoo.com although the significance and the mechanism of natural genetic recombination are still not understood, it can be suggested that it has a biological role in genetic recombination in pv evolution, especially for the prolonged circulation of opv-related pv. details of the mechanism of pv recombination are not well understood. however, based on current knowledge, the recombination is believed to occur by the ‘copy-choice’ mechanism with homologous genome templates (wimmer et al. 1993). generally, for single-stranded rna viruses such as pv, the mechanism is probably copy choice (template switching during rna replication) rather than trough true recombination i.e. the mechanism is analogous to gene conversion (kirkegaard and baltimore 1986). however, it may be different for the different virus recombinations. furthermore, the hot spot of recombination in the genome of pv is not well defined. we previously demonstrated that chimeric cdna constructed between pv and cav-11 or cav-17, with the crossover site in the n-term. part of 2c-coding region, resulted in viability of the virus (utama and shimizu 2005; utama and shimizu 2006). however, the virus was not viable when an rna transcript derived from chimeric cdna between pv and cav-18, with the same crossover site, was transfected into the cell-line. in this study, chimeric cdnas between pv and cav-18, with various crossover sites, were artificially constructed. the rna transcripts were then transfected into hep-2 cell-line. the viability of the virus was analyzed and the viable recombinant viruses were characterized. materials and methods viral rna extraction and construction of chimeric cdna. pj156 isolated from an acute flaccid paralysis case in 130 utama et al. microbiol indones fig 1 strategy for construction of chimeric virus pj156/cav-18. the detail procedure in materials and methods. pj156 p 1 p 2 p 3 p3a (with t7 promoter sequence) a b cav-18 p 4 rt-pcr (25x) rt-pcr (25x) p 3 a pcr 5x pcr 10x in vitro transcription transfection to hep-2 cells p 4 the philippines in 2001 (shimizu et al. 2004), and cav-18 stored in our laboratory, were used as the parental viruses. viral rnas were extracted from freeze-thawed lysates of infected cell culture-supernatants using a high-pure-viralrna kit (roche, germany). chimeric cdna constructs between pj156 and cav-18 were produced using the longpcr method, similarly as previously described (fig 1) (utama and shimizu 2005; utama and shimizu 2006). briefly, the 5’ and 3’ end of the genome of both viruses were separately amplified by the rt-pcr reaction using the titan one tube rt-pcr system (roche). twenty-five cycles of the pcr reaction were performed after 40 min of the rt reaction. amplified cdna fragments were purified with wizard sv gel and pcr clean-up system (promega, madison, usa). these fragments were then fused to each other in a pcr reaction using the expandtm long template pcr system (roche, germany) to obtain a target of chimeric full-length cdna. rna transcription and transfection. full-length chimeric cdnas were produced using in vitro transcription by using the ribomaxtm large-scale rna-production system (promega). five ml aliquots from 20 ml rna transcript solution were mixed with 100 ml of 0.5 mg·ml-1 deae dextran in 1 × hebss buffer (5 g·l-1 of hepes, 8 g·l-1 of nacl, 0.36 g·l-1 of kcl, 0.125 g·l-1 of na 2 hpo 4 2h 2 o, 10 g·l-1 of dextrose), as previously described (utama and shimizu 2005; utama and shimizu 2006). the transcript-deae-dextran mixtures were spread over hep-2 cells in 24-well plates. the cells were then rocked on a shaker for 30 min at room temperature. the fluids were aspirated off and the cells then incubated at 35 °c in dulbecco’s modified eagle’s medium supplemented with 2% bovine-calf-serum (maintenance medium). cytophatic effects were observed up to 7 days. hep-2 cells were subsequently infected with recombinant viruses which showed cpe to confirm the infectivity of the viruses. if cpe was not observed, a 3x blind passage was performed to confirm that no virus had been recovered from the rna transfection. plaque purification of the recombinant virus. all recovered viruses were cloned by plaque-assay on an hep-2 cell monolayer (utama and shimizu 2005; utama and shimizu 2006). a ten-fold serial dilution of viruses, prepared in the maintenance medium, were inoculated in hep-2 cells using 6-well plates, and incubated at 35 °c for 30 min. the cells were covered with 2 ml of 0.5% (w/v) agarose-me in dmem with 5% (v/v) bovine calf serum. after incubation at 35 °c for 3 days, plates were stained with 2 ml of 0.5% (v/v) neutral-red in maintenance medium containing 0.5% (w/v) agarose-me. plaque size was measured, and plaque numbers were calculated after incubation at 35 °c for a further day. one-step growth-curve and temperature sensitivity analyses. one-step growth-curve experiments were conducted by infecting a monolayer of hep-2 cells with viruses at a multiplicity of infection of 10 ccid 50 per cell (utama and shimizu 2005; utama and shimizu 2006). at different times after infection, the cells and supernatant were collected, frozen and thawed three times, and then centrifuged (10 000 x g, 5 min) to remove cell debris. virus titers in the supernatants were determined by the end-point dilution method in hep-2 monolayer-cultures in 96-well plates at 35 °c. to test temperature sensitivity, one-step growth experiments were carried out at 35 °c and at 39.5 ºc, respectively. results construction chimeric virus pj156/cav-18. cdna chimers between pj156 and cav-18, with a crossover site in the n-term. part of 2c-coding region (amino acid no. 8 of 2c), (pj156/cav-18(2c-8)) were firstly constructed (fig 2). after rna transfection into hep-2 cells and incubation at 35 °c, no cpe was observed, which implied that the virus was not viable. these results suggested that recombination might be not occurring between pj156 and cav-18 at the 2c-8 site. to find out whether recombination can occur at other sites, various chimeric cdnas between pj156 and cav-18 with different crossover sites (2c-8, 2c-133, 2c-235, 2c-268, 2c-287, 2c-327, 3a-67, 3c-60) were constructed using appropriate sets of primers (table 1). after rna transfection into hep-2 cells following by incubation at 35 °c, only the volume 1, 2007 microbiol indones 131 fig 2 viability of recombinant viruses derived from chimeric pj156/cav-18 cdna. the number in parentheses describes the number of amino acid in each protein. 3c(60) o p 1 p 2 p 3 crossover site 2c(8) 2c(133) 2c(235) 2c(268) 2c(287) 2c(327) 3a(67) x x x x x o o viability table 1 primers used for construction of chimeric cdnas recombinant crossover site* sequence a/b pj156/cav-18 (2c-8) pj156/cav-18 (2c-133) pj156/cav-18 (2c-235) pj156/cav-18 (2c-268) pj156/cav-18 (2c-287) pj156/cav-18 (2c-327) pj156/cav-18 (3a-67) pj156/cav-18 (3c-60) 2c (8) 2c (133) 2c (235) 2c (268) 2c (287) 2c (327) 3a (67) 3c (60) 5’-ggagatagctggctcaagaagttcacagaggcgtgcaatgc-3’ 5’-gcattgcacgcctctgtgaacttcttgagccagctatctcc-3’ 5’-ggtgcacggcagcccagggacaggaaaatctgttgccaccagc-3’ 5’-gctggtggcaacagattttcctgtccctgggctgccgtgcacc-3’ 5’-cacccccaactgtggctcacagtgatgccctaagccgg-3’ 5’-ccggcttagggcatcactgtgagccacagttgggggtg-3’ 5’-ggcgatggccactgaaatgtgcaaggaatgccctcaaccagc-3’ 5’-gctggttgagggcattccttgcacatttcagtggccatcgcc-3’ 5’-cccttggtgtgtggcaaggccatccaattaatggac-3’ 5’-gtccattaattggatggccttgccacacaccaaggg-3’ 5’-gggaactgtatggaagctctgttccaaggaccaatcagc-3’ 5’-gctgattggtccttggaacagagcttccatacagttccc-3’ 5’-cctccaggctgtcactacctttgcagctgtggctgg-3’ 5’-ccagccacagctgcaaaggtagtgacagcctggagg-3’ 5’-ggaggttgaggtgttagacgccaaggcccttgaagaccaggcaggg-3’ 5’-ccctgcctggtcttcaagggccttggcgtctaacacctcaacctcc-3’ 5’-catgctaatacgactcactataggttaaaacagctctggg-3’ ** 5 ’ t t t t t t t t t t t t t t t t t t t t t t c t c c g 3 ’ a b a b a b a b a b a b a b a b *( ) described amino acid no. in each protein, ** t7 promoter sequence is underlined. p 3 a p 4 *( ) described amino acid no. in each protein. table 2 plaque phenotype and amino acid substitution in determined region of each recombinant virus virus crossover site* plaque size (mm) titer (pfu/ml) pj156 pj156/cav-18 (2c-327) pj156/cav-18 (3a-67) pj156/cav-18 (3c-60) 2c (327) 3a (67) 3c (60) 2 6 1 2 1 4 1 4 6.6 x 108 3.5 x 108 3.4 x 108 2.6 x 108 recombinant pj156 and cav-18 with crossover sites at 2c-327, 3a-67, and 3c-60 (each virus designated as pj156/ cav-18(2c-327), pj156/cav-18(3a-67), and pj156/cav-18 (3c-60)) were viable, whereas recombinant viruses with crossover sites at (2c-8, 2c-133, 2c-235, 2c-268, 2c-287 were not viable (fig 2). we also attempted to recover non-viable viruses by transfecting the cells with the rna derived from their chimeric cdnas and incubating at 30 °c. however, no cpe was observed, even though the process was repeated for another two times. these results suggested that recombination could only occur between pj156 and cav-18 at downstream of 2c-coding regions, different with recombination of pv and cav-11 and cav-17 as previously reported (utama and shimizu 2005; utama and shimizu 2006). plaque assay of recombinant viruses. plaque assay of recovered recombinant viruses, along with their parental pj156 isolate, was performed to analyze the effect of recombination on viral phenotype. the results showed that pj156/cav-18 (2c-327) expressed small plaques (1-2 mm in diameter), as compared with pj156 (2-6 mm in diameter) (table 2, fig 3). two other recombinant viruses, pj156/cav-18 (3a67) and pj156/cav-18 (3c-60), showed intermediate-sized plaques (1-4 mm in diameter), but still smaller compared to the parental pj156. the titer of all recombinant viruses was also varied, namely 3.5 x 108 pfu·ml-1, 3.4 x 108 pfu·ml-1, and 2.6 x 108 pfu·ml-1, respectively for pj156/cav-18 (2c-327), pj156/cav-18 (3a-67), and pj156/cav-18 (3c-60), and was close to half that of the parental pj156 (6.6 x 108 pfu·ml-1) (table 2). these findings suggest that recombination between pj156 and cav-18 at different crossover sites resulted in only slight or no differences in phenotypes as defined by plaque size and virus titer. 132 utama et al. microbiol indones fig 3 plaque size of the parental and recombinant viruses. the positions of crossover site are described in parentheses. pj156/cav-18 (2c-327) pj156/cav-18 (3a-67) pj156/cav-18 (3c-60)pj156 fig 4 one-step growth of viruses at 35 °c (a) and 39.5 °c (b). sabin 1, mahoney pj156, pj156, 156/ca 18(2c-327), 156/ca 18(3a-67), 156/ca 18(3c-60). t it e r (t c id 5 0 /5 0 µ l) 10 9 10 8 10 7 10 5 10 4 10 3 10 6 10 2 10 1 0 2 4 6 8 1 0 time of post infection (h) b time of post infection (h) t it e r (t c id 5 0 /5 0 µ l) a 0 2 4 6 8 1 0 1010 10 9 10 8 10 7 10 5 10 4 10 3 10 2 10 1 10 6 fig 5 similarity analysis between pv and some coxsackei a viruses based on the entire amino acids using simplot software version 3.5.1. pv-1, poliovirus serotype 1; pv-2, poliovirus serotype 2; pv-3, poliovirus serotypy 3; cav-1, coxsackie a virus serotype 1; cav-11, coxsackie a virus serotype 11; cav-13, coxsackie a virus serotype 13; cav-17, coxsackie a virus serotype 17; cav-18, coxsackie a virus serotype 18; cav-21, coxsackie a virus serotype 21. s im il a ri ty ( % ) 1 0 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 0 4 0 0 6 0 0 8 0 0 1 000 1 200 1 400 1 600 1 800 2 000 position (aa) one-step growth of recombinant viruses. a one-step growth experiment of the recombinant viruses, along with their parental viruses at 35 and 39.5 °c, was conducted to analyze the effect of recombination on viral growth and temperature sensitivity. pj156/cav-18 (2c-327), pj156/cav18 (3a-67) and pj156/cav-18 (3c-60), pj156/cav-17 showed a similar growth pattern compared to the parental pj156 at 35 °c (fig 4a). other reference viruses such as sabin 1 and mahoney strains showed a similar pattern at this temperature. all the pj156/cav-18 recombinant viruses also demonstrated a similar growth to the pj156 and mahoney at 39.5 °c (fig 4b), which means that all the viruses were equally temperature sensitive. moreover, the sabin 1 virus was sensitive to this temperature. these results imply that recombinant between volume 1, 2007 microbiol indones 133 pj156 and cav-18 at different crossover site in nonstructural protein-coding region result in recombinant viruses which have similar temperature-sensitivity characteristics. discussion recombination has been shown to occur in pv, either among vaccine strains (cammack et al. 1988; lipskaya et al. 1991) or between vaccine strains and wild-type pv (liu et al. 2000; liu et al. 2003) and other unidentified non-polio enteroviruses (rousset et al. 2003; yang et al. 2003; shimizu et al. 2004). we have shown that pv could recombine with cav-11 or cav-17 at the n-term. part of the 2c-coding region (utama and shimizu 2005; utama and shimizu 2006). however, the cpe was not apparent when rna derived from chimeric cdna of pj156/cav-18, with the crossover site at n-term. part of 2c-coding region, was transfected into the hep-2 cell-line. similarity analysis has demonstrated that homology was high between pv and cav-18 at the 3 region (represents 3a, 3b, 3c, and 3d regions) (utama and shimizu 2006) (fig 5). therefore, it is predicted that pv can recombine with cav-18 in these regions. however, there is no direct evidence to prove this hypothesis. by using the long-pcr method, eight chimeric cdnas between pj156 and cav-18, each with different crossover sites, were constructed. the chimeric cdnas were respectively designated pj156/cav-18 (2c-8), pj156/cav-18 (2c-133), pj156/cav-18 (2c-235), pj156/cav18 (2c-268), pj156/cav-18 (2c-287), pj156/cav-18 (2c-327), pj156/cav-18 (3a-67), and pj156/cav-18 (3c-60). after in vitro transcription, each transcript was transfected into hep-2 cell-line. as expected, pj156/cav-18 (2c-327), pj156/ cav-18 (3a-67), and pj156/cav-18 (3c-60) showed cpe in the cell-line, while others did not (fig 2). these results imply that recombination can occur at a site in which the homology is high between both viruses. based on this evidence, it is suggested that the recombination between pv and cav-18 occur by a ‘copy-choice’ mechanism. pj156/cav-18 (2c-327) showed smaller plaque size (1-2 mm) compared to pj156/cav-18 (3a-67) and pj156/cav18 (3c-60) (1-4 mm), and compared with the parental pj156 (2-6 mm). the titer of the recombinant viruses also varied from 2.6 x 108 to 3.5 x 108 pfu·ml-1, and was close to half that of the parental pj156 strain (6.6 x 108 pfu·ml-1) (table 2). these findings suggest that the recombination between pj156 and cav-18 at different crossover sites resulted in only very slight or no differences in phenotypes for plaque sizes and virus titer, but different phenotype compared to the parental pj156. also all of the pj156/cav-18 recombinant viruses demonstrated a similar growth pattern both at 35 °c (fig 4a) and at 39.5 °c (fig 4b). thus, all the recombinant viruses were similarly temperature resistant (fig 4b). this is the same as for the parental pj156 and mahoney strains, which are highly pathogenic strains (fig 4b), suggesting that recombination between pv and cav-18 may result in viruses which have characteristics similar with highly pathogenic viruses. to elucidate to pathogenic characteristic of the recombinant viruses, however, the neurovirulence test using pv receptor-transgenic mice has to be performed. references blomqvist s, bruu al, stenvik m, hovi t. 2003. characterization of a recombinant type 3/type 2 poliovirus isolated from a healthy vaccinee and containing a chimeric capsid protein vp1. j gen virol 84:573-580. cammack n, phillips a, dunn g, patel v, minor pd. 1988. intertypic genomic rearrangements of poliovirus strains in vaccinees. virology 167:507-514. driesel g, diedrich s, kunkel u, schreier e. 1995. vaccine-associated cases of poliomyelitis over a 30 year period in east germany. eur j epidemiol 11:647-654. kew om, morris-glasgow v, landaverde m, burns c, shaw j, garib z, andre j, blackman e, freeman cj, jorba j, sutter r, tambini g, venczel l, pedreira c, laender f, shimizu h, yoneyama t, miyamura t, van der avoort h, oberste ms, kilpatrick d, cochi s, pallansch m, de quadros c. 2002. outbreak of poliomyelitis in hispaniola associated with circulating type 1 vaccine-derived poliovirus. science 296:356-359. king amq. 1988. preferred sites of recombination in poliovirus rna: an analysis of 40 intertypic cross-over sequences. nucleic acids res 16:11705-11723. kirkegaard k, baltimore d. 1986. the mechamism of rna recombination in poliovirus. cell 47:433-443. li j, zhang lb, yoneyama t, yoshida h, shimizu h, yoshii k, hara m, nomura t, yoshikura h, miyamura t, hagiwara a. 1996. genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients. arch virol 141:10471054. lipskaya gy, muzychenko ar, kutitova ok, maslova sv, equestre m, drozdov sg, perez-bercoff r, agol vi. 1991. frequent isolation of intertypic poliovirus recombinants with serotype 2 specificity from vaccine-associated polio cases. j med virol 35:290-296. liu hm, zheng dp, zhang lb, oberste ms, kew om, pallansch ma. 2003. serial recombination during circulation of type 1 wildvaccine recombinant polioviruses in china. j virol 77:109941 1 0 0 5 . liu hm, zheng dp, zhang lb, oberste ms, pallansch ma, kew om. 2000. molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in china. j virol 74:11153-11161. martin j, samoilovich s, dunn g, lackenby a, feldman e, heath a, svirchevskaya e, cooper g, yermalovich m, minor pd. 2002. isolation of an intertypic poliovirus capsid recombinant from a child with vaccine-associated paralytic poliomyelitis. j virol 76:10921-10928. rousset d, rakoto-andrianarivelo m, razafindratsimandresy r, randriamanalina b, guillot s, balanant j, mauclère p, delpeyroux f. 2003. recombinant vaccine–derived poliovirus in madagascar. j infect dis 9:885-887. shimizu h, thorley b, paladin fj, brussen ka, stambos v, yuen l, utama a, tano y, arita m, yoshida h, yoneyama t, benegas a, roesel s, pallansch m, kew om, miyamura t. 2004. circulation of type 1 vaccine-derived poliovirus in the philippines in 2001. j virol 78:13512-13521. tatem j, weeks-levy c, mento sj, di michele sj, georgiu a, waterfield wf, sheip b, costalas c, davies t, ritchey mb, cano fr. 1991. oral poliovirus vaccine in the united states: molecular characterization of sabin type 3 after replication in the gut of vaccinees. j med virol 35:101-109. toyoda h, kohara mm, kataoka y, suganuma t, omata t, imura n, nomoto a. 1984. complete nucleotide sequences of all three poliovirus serotype genomes: implication for genetic relationship, gene function and antigenic determinants. j mol biol 174:561-585. 134 utama et al. microbiol indones utama a, shimizu h. 2005. construction of a recombinant virus between poliovirus and coxsackie a virus 11. ann bogorienses 10:19-26. utama a, shimizu h. 2006. construction and characterization of chimeric virus between poliovirus and coxsackie a virus serotype 17. j mikrobiol indones 11:77-81. wimmer e, hellen cut, cao x. 1993. genetics of poliovirus. annu rev genet 27:353-436. yang cf, naguib t, yang sj, nasr e, jorba j, ahmed n, campagnoli r, van der avoort h, shimizu h, yoneyama t, miyamura t, pallansch m, kew om. 2003. circulation of endemic type 2 vaccine-derived poliovirus in egypt from 1983 to 1993. j virol 77:8366-8377. 02. atikana.cdr vol.15, no.1, march 2021, p 8-14 doi: 10.5454/mi.15.1.2 characterization of eps7-like enterobacteria phage isolated from indonesia akhirta atikana , katsutoshi fujita , alex prima , yopi , 1,* 2 1 1 hiroko kawasaki , ken-ichiro suzuki , puspita lisdiyanti 2 2 1* and 1 research center for biotechnology, the indonesian institute of sciences, jl raya bogor km. 46, cibinong 16911, indonesia; 2resource collection division, biological resource center (nbrc), national institute of technology and evaluation (nite), kisarazu-shi, chiba, japan bacteriophages are the most abundant entities in earth. the order is the largest and most caudovirales widespread group among bacterial viruses. the purpose of this study was to characterize bacteriophages from indonesian waters. during this experiment, we collected sample from kuningan (west java) and buleleng (bali), indonesia. we used an overlay agar method with three strains of as a host (nbrc 13965, nbrc 12713 and e. coli nbrc 13168) combined with digestion profiling using three restriction enzymes (pvuii, ecorv and hincii) and transmission electron microscope (tem) to characterize the morphology of the phage from indonesia. our results showed that phage lipi13-bp006 is in a group of and highly similar to enterobacteria phage eps7. caudovirales bacteriophage, bali, enterobacteria phage eps7, environment, key words: e. coli merupakan organisme yang keberadaannya paling melimpah di muka bumi. orde bacteriophages caudovirales bacteriophages merupakan orde yang paling besar dan paling luas diantara orde yang lainnya. penelitian ini bertujuan untuk karakterisasi dari perairan indonesia. pada penelitian ini, koleksi sampel faga dilakukan di kuningan, jawa barat dan buleleng, bali. karakterisasi pada penelitian ini bacteriophages dilakukan dengan menggunakan: metoda menggunakan tiga strain sebagai host (nbrc overlay agar e. coli 13965, nbrc 12713 and nbrc 13168) yang dikombinasikan dengan menggunakan tiga digestion profiling enzim restriksi (pvuii, ecorv and hincii) dan (tem). hasil penelitian transmission electron microscope menunjukkan bahwa phage lipi13-bp006 termasuk dalam grup dan memiliki kemiripan yang caudovirales tinggi dengan . enterobacteria phage eps7 kata kunci: , bali, bacteriophages enterobacteria phage eps7, e. coli microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 81228766056; : +62fax: + 6 2 e m a i l : a; a k h i r t a . a t i k a n a @ g m a i l . c o m , puspita.lisdiyanti@bioteknologi.lipi.go.id vaccines, for the detection of pathogenic bacterial strain, for genetic screening vectors and for therapeutics application. phages can also be used as bio control agents in agriculture and petroleum industry (clark and march 2006, mc. auliffe . 2007, ul haq . et al et al 2012). the current state of bacteriophage genomics showed that the genetic diversity of the population is very high, actively evolving with active engagement of horizontal genetic exchange, and that their genomes are consequently pervasively mosaic in their architectures. early indications from genomic sequencing and metagenomic analysis indicate that natural phage communities are reservoirs of the uncharacterized genetic diversity on earth (canchaya . 2007). et al indonesia is well known as mega biodiversity country, included the diversity of microorganism. ho we ver, i nfo rm at io n o n t he div er si ty of bacteriophages in indonesia is limited to research on a lytic pradovirus-like ralstonia phage (addy . et al 2018), a bacillus-phage (handoko 2019), and the et al. bacteriophage family of myoviridae (sujonoputri . et al 2020, wardani . 2020). therefore, the purpose of et al bacteriophages are viruses that infect bacteria and the most abundant life forms on earth. the first bacteriophages attack documented by frederick twort (1915) and felix d'herelle (1917). an estimated viral population of bacteriophages is approximately 10 million per cubic centimeter of any environmental niche (mc. auliffe . 2007). according to the et al international committee on taxonomy of viruses (ictv), there are 17 families of phages, where their hosts are archaea and bacteria. the classification of bacteriophage is based on the morphology and the nucleic acids: from an enveloped head to a nonenveloped head, from a linear to a circular nucleic acid (carstens 2012). research on bacteriophages developed from the study of dna and the discovery of messenger rna, until the basic molecular interactions and genetic regulation. bacteriophage is valuable in the modern biotechnology industry, as diagnostic tools, vehicles for this research is to characterize bacteriophages from indonesia. this paper will elaborate detection, isolation and identification of bacteriophages from indonesian environment, especially from kuningan and bali. we hope to find new type of bacteriophages isolated from indonesia to enrich the database of indonesian bacteriophages. materials and methods during the year 2012-2013, samples collection. water samples were collected from waduk dharma, kuningan (west java) and lovina beach, buleleng (bali island). the no. 802 broth media (polypeptone 1%, yeast extract 0.2%, mgso4-7h2o 0.1%) were added to the sample for bacteriophage enrichment. the supernatant for further isolation process were collected by centrifugation. bacteriophages isolation, host specificity and titer test. the isolation methods were done using agar overlay methods, using the no. 802-broth medium, the no. 802-agar medium (no. 802 broth + 1.5 % agar) and the no. 802 agar top medium (no. 802 broth + 0.8 % agar). three different strains of were used for e. coli host specificity test ( b (nbrc 13168), e. coli e. coli k12 f (nbrc 13965), and k12 (nbrc + e. coli 12713). the serial dilution was used to determine the viral titer (pfu/ml). purification of phage was performed by single plaque selection and filter centrifugation. afterwards, the pure isolated phage was preserved in 7% dmso and stored in -80ºc. phage characterization. restriction digestion analysis, dna sequencing and electron microscope were done for the characterization of the phage. the morphology of phage was observed using tem where the phage was negatively stained by uranyl acetate. for enzyme restriction analysis, the dna phage was extracted using the phage dna isolation kit (norgen biotek corp. canada). the dna was digested using 3 restriction enzymes ( ii, ii, and rv). the pvu hinc eco restriction mixture was used to build a phage-cloning library. the dna fragment was ligated in the ii hinc site of plasmid puc118 and grown in jm109. e. coli the colony containing the insert was selected using the blue-white selection method. colony direct pcr was performed using universal primer m13-m4 primer (5'gttttcccagtcacgac-3') and m13-rv primer (5'-cagga aacagctatgac-3'). the pcr amplicon was used for dna sequencing and the nucleic acid sequences were analyzed using the dna data bank of japan (ddbj). results during the year of 2012-2013, we collected samples from west java and bali. preliminary research showed that at least we have 8 candidates of bacteriophages isolated from indonesian environments (table 1). three different strains of : nbrc 13168, e. coli nbrc 13965 and nbrc 12713 were used as the host. our result showed that the sample s2012-bp003 and s2013-bp005 formed plaque in every host, meanwhile the sample s2012-bp002 formed plaque in only two of the host (nbrc 13168 and nbrc 13965). the viral titers were determined using serial dilutions. table 2 showed the result of viral titer test among samples. the result showed that the titers were varies in range from 10 pfu/ml until 10 pfu/ml. these imply that the 5 10 amount of the phages among samples is quite high to be used for further characterization. during phage purification, three different of single plaques (a, b and c) were chosen for specificity test against the three host (nbrc 13168, nbrc e. coli 13965 and nbrc 12713). however, no plaque observed in sample lipi13-bp006c and lipi13bp008a, therefore in total we only have 66 sample phages. table 3 showed the result of specificity test among three host (nbrc 13168, nbrc 13965 e. coli and nbrc 12713). our specificity result (table 3) did not show the possibility of having specific host e. coli among phages, and only phages from bali (lipi13bp006a, lipi13-bp006b, lipi13-bp008c and lipi13-bp008c) that was not formed plaques while using host strain nbrc 13168. e. coli however, the host specificity test (table 3) shown different profile compared to our pre-eliminary research (table 1). table 1 showed that sample s2012bp003 and s2013-bp005 formed plaque in all of the three of host (nbrc 13168, nbrc 13965 and e. coli nbrc 12713), meanwhile sample s2012-bp002 formed plaque in only two of the host (nbrc 13168 and nbrc 13965). meanwhile the specificity test (table 3) showed that sample from kuningan (s2012bp002 and s2012-bp003) formed plaque in all three e. coli hosts. also, the specificity test of sample from bali (s2013-bp005) indicate that phage lipi13-bp007 formed plaque in all of the host, but phage e. coli lipi13-bp006 and phage lipi13-bp008 did not formed plaque in host nbrc 13168. this result e. coli indicate that at least we have two candidate of phages isolated from kuningan and three candidate of phages isolated from bali. also, from this specificity profile volume 15, 2021 microbiol indones 9 10 atikana et al . microbiol indones we can predict that samples from kuningan might contain similar types of phages; meanwhile sample from bali might have different types of phages. furthermore, the phage genomic dna extracted from pure phage lysate and it was used for phage characterization. the phage genomic dna digested with 3 restriction enzymes ( rv, ii and ii). eco hinc pvu the digestion profile (table 4) shown that the enzyme pvuii did not digested the dna of the phages but lipi13-bp006a, lipi13-bp006b, and lipi13bp007a. the enzyme hincii digested all of the phages, meanwhile the enzyme ecorv digested all the phages but phage lipi13-bp007a and lipi13-bp007b. from the digestion profile, it showed that phage lipi13bp007 has three different restrictions profile. therefore, this result indicates that the phage lipi13bp007 might contain three types of bacteriophages. the enzyme hincii digested the dna of all the sample phages; therefore it was used for further analysis (phage cloning library). the clone library was conducted using vector plasmid puc118 and grown in e. coli jm109. colonies selected from blue-white selection and used for sequence analysis. during this experiment, fifteen colonies of the phage lipi13bp006 were used for sequencing. the result of sequence analysis (table 5) revealed that every colony with inserted dna fragment of the phage lipi13bp006 is highly similar with enterobacteria phage eps7. however, the nucleotide sequences analysis of the other seven phages did not show any similarity in the dna databank. in our opinion, these other seven phages might be potential candidates for new bacteriophages from indonesia. therefore, further characterization using whole genome analysis can be used to gain more complete information among phages. the morphology of phage lipi13-bp006 was observed using the electron microscope. figure 1 confirms that phage lipi13-bp006 is in the group of order caudovirales, identify with icosahedral head and long tail. this figure is in line with the result of our sequencing analysis (table 5), which shows that the p ha g e l i p i 13 b p 0 0 6 i s h i g h l y s im i l a r t o enterobacteria phage eps7, the group of order caudovirales. discussion the enterobacteria phage eps7 first isolated from sewage sample in korea. it is a t5-like group phage, grouped in the family of , order of siphoviridae caudovirales (tailed phages). the genome size of enterobacteria phage eps7 is 111,382 bp. it is a linier dna phage with icosahedral head (diameter 65 nm) and non-contractile tail (185 nm). the original hosts of enterobacteria phage eps7 are , escherichia coli salmonella typhimurium salmonella enteritidis, and (hong 2008). the caudovirales (tailed phages) et al. are the oldest virus and known as typical phages that are infected both eubacteria and archaea. the phages can be virulent (lytic) or temperate (ackermann, 2005). virulent (lytic) phages proceed with immediate replication after infecting the host cell. new viruses released in large numbers by lysis of the host cell after infection. meanwhile, temperate (lysogenic) phages do not necessarily start replicating immediately. the phages may integrate their nucleic acid (genome) into the host cell until it induced to become autonomous again. afterwards, it starts to replicate and lyse the host cell (grabow 2001). phages are associated with almost all bacterial genera and grouped on the basis of a few characteristics: range, morphology, nucleic acid, strategies of infection, morphogenesis, phylogeny, serology, sensitivity to physical and chemical agents, and dependence on properties of hosts and the environment. a host is required to evaluate the existence of phages and various host strains have been used for phages detection. most of these host strains detect groups of phages, in particular to somatic coliphages. the abundance and the distribution of phages are based on the existence of their host organisms. the host-specificity of phages can be a useful tool to classify certain bacteria. phages may not only be specific for species of bacteria, but also for strains of bacteria, allowing typing beyond the level of species. however, no single method was established to detect phages in a specific host (grabow 2001, clokie et al. 2011). phages can be recovered and detected by many techniques and approaches. major reasons for any inconsistencies are the host bacteria that are used for the detection of various groups of phages (grabow 2001). traditional and molecular approaches can be combined to have the overall picture of the viral community. the epifluorescent microscopy or the flow cytometry can be used to determine the number of phages that infect all hosts. meanwhile, the morphological diversity can be investigated using transmission electron microscopy (tem). appropriate hosts can be isolated specifically from the environment of interest, or a model permissive host can be used. table 1 bacteriophages isolated from kuningan and bali sampling area sample number acc. number bacteria host e. coli kuningan, west java s2012-bp002 lipi13-bp001 b (nbrc 13168) lipi13-bp002 k12 f+ (nbrc 13965) kuningan, west java s2012-bp003 lipi13-bp003 k12 (nbrc 12713) lipi13-bp004 b (nbrc 13168) lipi13-bp005 k12 f+ (nbrc 13965) buleleng, bali s2013-bp005 lipi13-bp006 k12 (nbrc 12713) lipi13-bp007 b (nbrc 13168) lipi13-bp008 k12 f+ (nbrc 13965) table 2 viral titer tests no. acc. number e. coli host plaque forming unit (pfu) 10 µl 100 µl 1 lipi13-bp001 nbrc 13168 15 × 106 1 × 106 2 lipi13-bp002 nbrc 13965 12 × 108 38 × 107 3 lipi13-bp003 nbrc 12713 5 × 107 1 × 107 4 lipi13-bp004 nbrc 13168 4 × 106 45 × 105 5 lipi13-bp005 nbrc 13965 5 × 106 15 × 105 6 lipi13-bp006 nbrc 12713 2 × 1010 18 × 109 7 lipi13-bp007 nbrc 13168 25 × 108 1 × 109 8 lipi13-bp008 nbrc 13965 4 × 1010 2 × 1010 table 3 host specificity test no. acc. number nbrc 13168 nbrc 13965 nbrc 12713 sample number plaque plaque plaque a b c a b c a b c 1 s2012bp002 lipi13-bp001          2 lipi13-bp002          3 s2012bp003 lipi13-bp003          4 lipi13-bp004          5 lipi13-bp005          6 s2013bp005 lipi13-bp006 × × n/a   n/a   n/a 7 lipi13-bp007          8 lipi13-bp008 n/a × × n/a   n/a   note: (n/a) no samples, (√) plaques formed, (x) no plaques formed volume 15, 2021 microbiol indones 11 table 4 digestion profile no. acc. number pvuii hincii ecorv plaque plaque plaque a b c a b c a b c 1 lipi13-bp001 × × ×       2 lipi13-bp002 × × ×       3 lipi13-bp003 × × ×       4 lipi13-bp004 × × ×       5 lipi13-bp005 × × ×       6 lipi13-bp006   n/a   n/a   n/a 7 lipi13-bp007  × ×    × ×  8 lipi13-bp008 n/a × × n/a   n/a   table 5 nucleotides blast analysis of phage lipi13-bp006 clone library no source sequences id number of nucleotides blast analysis percentages 1 bali > jsat13-2bp006a_01 114 letters cp000917.1 enterobacteria phage eps7 99% 2 bali > jsat13-2bp006a_02 634 letters cp000917.1 enterobacteria phage eps7, 93% 3 bali > jsat13-2bp006a_03 769 letters cp000917.1 enterobacteria phage eps7 96% 4 bali > jsat13-2bp006a_04 114 letters cp000917.1 enterobacteria phage eps7 99% 5 bali > jsat13-2bp006a_05 791 letters cp000917.1 enterobacteria phage eps7 95% 6 bali > jsat13-2bp006a_07 775 letters cp000917.1 enterobacteria phage eps7 95% 7 bali > jsat13-2bp006a_08 114 letters cp000917.1 enterobacteria phage eps7 98% 8 bali > jsat13-2bp006a_09 285 letters cp000917.1 enterobacteria phage eps7 100% 9 bali > jsat13-2bp006a_12 114 letters cp000917.1 enterobacteria phage eps7 99% 10 bali > jsat13-2bp006a_15 264 letters cp000917.1 enterobacteria phage eps7 96% 11 bali > jsat13-2bp006b_09 105 letters cp000917.1 enterobacteria phage eps7 99% 12 bali > jsat13-2bp006b_10 264 letters cp000917.1 enterobacteria phage eps7 96% 13 bali > jsat13-2bp006b_13 428 letters cp000917.1 enterobacteria phage eps7 95% 14 bali > jsat13-2bp006b_15 114 letters cp000917.1 enterobacteria phage eps7 99% 15 bali > jsat13-2bp006b_16 264 letters cp000917.1 enterobacteria phage eps7 96% note: (n/a) no samples, (√) digested, (x) not digested 12 atikana et al . microbiol indones furthermore, no universal molecular marker can be used to determine the phages, because no gene is suitably conserved within all phages. but restriction fragment length polymorphisms (rflp) can also be used to assess the bacteriophage diversity (clokie . et al 2011). studies of phages in environments have been reported from most parts of the world. during this research we characterize bacteriophages isolated from indonesian environment using three model host e. coli (nbrc 13168, nbrc 13965 and nbrc 12713) combined with digestion profiling using three restriction enzymes rv, ii and ii) and (eco hinc pvu transmission electron microscope (tem). however, these approaches only identify phages that infect the specific strains that were being used as a host e. coli and did not show the actual proportion of the phage. bacteriophage is recently used in modern biotechnology as well as for the detection of pathogenic bacterial strain. a phage can be used individually to treat a bacterial infection by lysing the bacterial cell as it is having the lytic potential (ul haq et al. 2012). some phages have a global distribution while others may be endemic to particular environments. phages and bacteria (and archaea) are probably co-existed and evolved together. therefore it makes sense that the symbiotic relationship between bacteria and phage is advantageous because it may boost the bacterial ability to survive by encoding toxins and other useful genes (clokie 2011). et al. during the experiment, we identify the phage lipi13-bp006 isolated from bali as enterobacteria phage eps7. eight candidates of bacteriophage were isolated from indonesian environment, where five candidates isolated from kuningan, west java and three candidates isolated from buleleng, bali. the bacteriophage candidate isolated from bali (lipi13bp006) is highly similar to enterobacteria phage eps7. however, the other phages have no similarity with any phages in the databank. these unidentified phages are potential candidates as new bacteriophages isolated from indonesian environment. little information is known about bacteriophages from indonesia. studies on the indonesian bacteriophages were focusing on a lytic pradoviruslike ralstonia phage (addy . 2018), a bacillus-et al phage (handoko 2019), and the bacteriophage et al. family of myoviridae (sujonoputri . 2020, et al wardani . 2020). the pradovirus-like ralstonia et al phage was isolated from soil, infecting ralstonia solanacearum, an aerobic non spore gram-negative bacterium (addy 2018). the bacillus-phage was et al isolated from soil samples, infecting sp., a bacillus gram-positive bacteria (handoko . 2019). while. et al while the myoviridae family were isolated from food and soil samples (sujonoputri 2020), as well as et al liquid waste, cow and chicken intestines, chicken skin (wardani . 2020). the present study isolated et al e n t e r o b a c t e r i a p h a g e e p s 7 a n d u n k n o w n bacteriophages from water samples, infecting escherichia coli, a gram-negative bacteria. therefore, we think that the information in this paper is valuable to support the research of bacteriophages in indonesia. acknowledgements this work was supported by science and technology research partnership for sustainable development (satreps) program on development of internationally standardized microbial resource center to promote life science research and biotechnology and government research funding fy 2012-2013 from research center for biology, the indonesian institute of sciences and research center for biotechnology, the indonesian institute of sciences. we would like to thank to ir. ahmad jauhar fig 1 the morphology of enterobacteria phage eps7 from bali, indonesia (bar: 100nm, blue arrow: head, white arrow: tail). volume 15, 2021 microbiol indones 13 arif, m. sc as project leader and manager of indonesian culture collection (inacc) and dr. yantyati widyastuti as the head of laboratory of applied microbiology, research center for biotechnology, indonesian institute of sciences. references addy, h.s., farid, m.m., ahmad, a.a. et al. host range and molecular characterization of a lytic pradovirus-like ralstonia phage rsop1idn isolated from indonesia. a r c h v i r o l 1 6 3 , 3 4 0 9 – 3 4 1 4 ( 2 0 1 8 ) . https://doi.org/10.1007/s00705-018-4033-1. ackermann hw. 2005. bacteriophage classification. in 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(ed) bacteriophage: genetics and molecular biology. uk. caister academic press. p. 43-59. carlson k. 2005. working with bacteriophages: common techniques and methodological approaches. in kutter, e. and a. sulakvelidze (ed) bacteriophages biology and applications. new york. crc press. p. 439-490. carsten eb. 2012. in king amq, adams mj, lefkowitz ej, carstens eb. (ed) virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses. p. 1-7. clark jr, march jb. 2006. bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. trends in biotechnology. p. 212-218. clokie mrj, andrew dm, andrey vl, shaun h. 2011. review: bacteriophages in nature. bacteriophage 1:1, p. 31-45. handoko ya, wardani ak, sutrisno a, widjanarko sb, thurgood tl, thompson dw, sharma r, grose jh. 2019. genome sequences of two bacillus phages isolated from indonesia. microbiol resour announc 8:e01058-19. https://doi.org/10.1128/mra.01058-19. sujonoputri fr, ketty, nd, marcello, n, waturangi, de. 2020. isolation, characterization, and application of bacteriophages against several pathogenic and food spoilage bacteria. international journal of infectious d ise a ses . 10 1 (s 1) . https://d oi. org /10. 1016 / j.ijid.2020.09.116. grabow, wok. 2001. bacteriophages: update on application as models for viruses in water. water sa 27: 2. p. 251-268. mc auliffe o, ross rp, fitzgerald gf. 2007. the new phage biology: from genomics to applications. in mc grath s, van sinderen d (ed) bacteriophage: genetics and molecular biology. uk. caister academic press. p. 141. moineau s, tremblay d, labrie s. 2002. phages of lactic acid bacteria: from genomics to industrial applications. asm news. p. 388-393. ul haq i, chaudhry wn, akhtar mn, andleeb s, qadri i. 2012. bacteriophages and their implications on future biotechnology: a review. virology journal 9: 9. doi:10.1186/1743-422x-9-9. van twest r, kropinski am. 2009. bacteriophages enrichment from water and soil. in clokie mrj, kropinski am (ed) methods in molecular biology, bacteriophages methods and protocols, volume 1: isolation, characterization and interactions. humana press, new york. p. 15-21. wardani, ak, nurbayu, ir, and qodriyah, nl. 2020. isolation of lytic bacteriophages and their potential to control cronobacter spp. opportunistic food-borne pathogens. iop conf. ser.: earth environ. sci. 475 012086. 14 atikana et al . microbiol indones 01. ulya.cdr vol.14, no.4, december 2020, p 121-128 doi: 10.5454/mi.14.4.1 the ethanol production activity of indigenous thermotolerant yeast pichia kudriavzevii 1p4 darojatul ulya, rikaindri astuti*, and anja meryandini department of biology, faculty of mathematics and natural science, ipb university, ipb dramaga campus, dramaga 16680, bogor, west java, indonesia. pichia kudriavzevii 1p4 is a thermotolerant-ethanologenic yeast potential for application in ethanol industry. in this study we evaluated the stress tolerance phenotype of p. kudriavzevii 1p4 in dealing with fermentation related-stresses, including high temperature stress, high sugar content, ethanol content and the fermentation capacity of the particular isolate. based on spot assay, 1p4 showed stress tolerance phenotype against high sugar concentration for up to 30% sugar content and 10% ethanol stress. in addition, 1p4 was capable to show o temperature-stress tolerance phenotype for up to 42 c, suggesting that 1p4 belong to thermotolerant yeast isolate. fermentative activity was measured by using glucose consumption and ethanol production assay. we evaluated o o o the fermentative and growth rate of 1p4 at various temperature condition which were 27 c, 37 c and 42 c using ypd media (at initial glucose of 2%, 10% and 20%). interestingly, 1p4 consumed the highest glucose in 20% of o concentration at 37 c (15.29%), simultaneously with the highest concentration of ethanol (32.05 g/l ethanol and 0.67 g/l/h ethanol productivity). cell growth analysis showed that growth of 1p4 isolate increased with higher initial glucose condition yet decreased as temperature during fermentation was raised. the growth rate of 1p4 was o found high in 20% initial glucose at 37 c than 2% and 10% at same temperature. in addition, 1p4 exhibited short lag phase at high-temperature fermentation. our data indicate that 1p4 can potentially be applied as fermentation agent especially in high-temperature ethanol fermentation. key words: bioethanol production, fermentation related-stress, thermotolerant yeast pichia kudriavzevii 1p4 merupakan khamir etanologenik-termotoleran yang berpotensi diaplikasikan dalam industri etanol. dalam penelitian ini kami mengevaluasi karakter ketahanan cekaman isolat 1p4 terhadap cekaman fermentasi, meliputi ketahan terhadap suhu tinggi, kadar gula tinggi, kadar etanol tinggi dan aktivitas fermentasi isolat tersebut. berdasarkan spot tes analisis, 1p4 menunjukkan fenotipe ketahanan terhadap cekaman gula tinggi hingga 30% dan etanol 10%. lebih lanjut, 1p4 mampu menunjukkan ketahanan terhadap suhu hingga o 42 c, yang mengindikasikan bahwa 1p4 termasuk dalam isolat khamir termotoleran. aktivitas fermentasi diukur menggunakan metode konsumsi glukosa dan produksi etanol. kami mengevaluasi laju fermentasi dan o o o pertumbuhan 1p4 pada berbagai kondisi media yaitu, 27 c, 37 c dan 42 c, menggunakan media ypd (dengan kadar gula awal 2%, 10% dan 20%). menariknya, 1p4 mengkonsumsi konsentrasi gula tertinggi pada media o dengan kadar gula 20% pada suhu 37 c (15.29%), simultan dengan produksi konsentrasi etanol tertinggi pada -1 -1 kondisi tersebut (32.05 gl etanol dan 0.67 g l h produktivitas volumetrik etanol). analisis pertumbuhan sel menunjukkan bahwa pertumbuhan isolat 1p4 meningkat dengan naiknya konsentrasi glukosa namun menurun seiring meningkatnya suhu fermentasi. laju pertumbuhan 1p4 tinggi pada media dengan kadar gula awal 20% dibandingkan media 2% dan 10% pada suhu yang sama. 1p4 juga menunjukkan fase lag yang pendek pada fermentasi dengan suhu tinggi. hasil penelitian kami mengindikasikan bahwa 1p4 potensial diaplikasikan sebagai agen fermentasi terutama pada fermentasi etanol dengan suhu tinggi. kata kunci: cekaman fermentasi, khamir termotoleran, pichia kudriavzevii microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone: +62-82113538835; email: rikaindriastuti@apps.ipb.ac.id stress (saini et al. 2018). highly concentrated medium containing high sugar concentration may lead to higher ethanol product at the end of fermentation, yet high sugar and ethanol content during fermentation could influence the growth, viability and fermentative activity of ethanologenic yeast (d'amore 1992). in addition, bioethanol fermentation generated heat in the process due to exothermic reaction and thereafter raised the temperature of fermentation broth (kumar et al. 2013). elevated temperature during fermentation was significantly inhibited growth of yeast in due time affects bioethanol production (divine et al. 2016). the improvement of consumption fossil fuel and environmental pollution led to the finding of environmental sustainable energy sources (azhar et al. 2017). bioethanol is a promising alternate for fossil fuel that can be produced through yeast fermentation (branco et al. 2019). however during bioethanol fermentation, yeast cells are confronted with many different fermentation related-stresses including high temperature stress, high osmotic stress and ethanol thus, in order to meet the requirement of ethanologenic yeast for high-temperature ethanol fermentation (htef), we need yeast isolates with high tolerance against fermentation related-stresses while capable to produce higher ethanol yields in high temperature and sugar concentration conditions. using thermotolerant yeast for bioethanol production in high temperature fermentation could gain some advantages such as higher saccharification and fermentation rates, energy saving through reduction cooling cost and minimalize risk of contamination (arora et al. 2015). saccharomyces cerevisiae is industrial ethanologenic yeast which is most commonly used for bioethanol production worldwide. saccharomyces cerevisiae had known as a good ethanol producer with high ethanol tolerance phenotype (chi et al. 2010). however as mesophilic yeast, most of s. cerevisiae strains could not grow and produce ethanol in high temperature fermentation (kitagawa et al. 2010). moreover, stressful condition like an increase in ethanol concentration, temperature, osmotic stress and bacterial contamination are the reason why the yeast cannot survive during fermentation (basso et al. 2008). for these reasons, exploration and investigation of nonconventional yeast for bioethanol production in high temperature still gained significant interest worldwide. pichia kudriavzevii had received major interest as an ethanol fermentation agent for more than a decade due to its capability to grow at high temperature and high sugar concentration (isono et al. 2012, kaewkrajay et al. 2014, diaz-nava et al. 2017, martha et al. 2020). previous study reported many strains of p. kudriavzevii had good ability to grow and produce o ethanol at high concentration temperature of over 40 c (pongcharoen et al. 2018). then, it is important to investigate characteristic of newly isolated p. kudriavzevii strain as potential fermentation agent for htef. yeast strain 1p4 is p. kuriavzevii isolated from o cacao fermentation that able to grow at 40 c which is potential as an ethanol fermentation agent at htef (inderiani 2017). in this study, we evaluated the stress tolerance phenotype of p. kudriavzevii 1p4 in dealing with fermentation related-stresses, including high temperature stress, high sugar content and ethanol content. furthermore, we performed fermentative activity analysis using glucose consumption and ethanol production assay at different initial glucose and temperatures. these data are key determinant to evaluate yeast fitness as a fermentation agent for htef. materials and methods yeast strains and morphology characterization. yeast strain had been isolated and identified previously as p.kudriavzevii 1p4 at microbiology lab ipb university (inderiani 2017). for this study, this strain was regrown from freeze-dried isolate into ypd liquid media (1% yeast extract, 2% peptone, 2% glucose) for o 24 h at room temperature (27 c). the culture from ypd liquid media were aseptically streaked into ypd solid media. pure colonies of p. kudriavzevii 1p4 were used as working culture for all experiments. for morphology characterization, 1p4 isolate was grown at ypd media for 24 h at room temperature then observed using light microscope to identify cell shape, cell size and budding cell type. for stress tolerance experiment, saccharomyces cerevisiae by4741 was used as a comparison isolate (riles and fay 2019). stress tolerance assay. stress tolerance of p. kudriavzevii 1p4 at ypd media with high sugar content, high ethanol content at high temperatures was evaluated by spot assay method (astuti et al. 2018). in this regards, yeast s. cerevisiae was applied as control. yeasts were cultured in ypd media for 18-24 h as the main culture. serial dilution was performed with starting od of 0.5 od . about 2 µl of each dilution 600nm was spotted at ypd solid media with 2% initial glucose o o o and incubated at temperatures 27 c, 37 c, 40 c and o 42 c for evaluating temperatures tolerance. ypd solid media with 2%, 10%, 20% and 30% glucose concentration were used as spot media for sugar tolerance assay while ypd without ethanol, with 5%, 10% and 15% ethanol were used as spot media for ethanol tolerance assay. the experiment was carried out in triplicate for 72 h observation. glucose consumption. yeast 1p4 was cultured in ypd liquid media with optical density 0.9-1 at 600 nm as the main culture. about 5% of an active inoculum was transferred into fresh liquid ypd media with initial o glucose of 2%, 10% and 20% then incubated at 27 c, o o 37 c and 42 c with shaking 120 rpm. the experiment was carried out in 15 ml reaction tubes with a working volume of 10 ml ypd liquid media and conducted in triplicate. the sugar concentration was determined per 8 h during 48 h observation by dns method (miller 1959). growth cell. the procedures of preparation to evaluate growth cell p. kudriavzevii 1p4 were same as measuring glucose consumption as described earlier. yeast growth was monitored by measuring optical density of the culture at ypd media with different 122 ulya et al. microbiol indones volume 14, 2020 microbiol indones 123 initial glucose of 2%, 10% and 20% and temperatures o o o (27 c, 37 c and 42 c) at 600 nm (od ) per 3 h 600nm during 48 h of fermentation. ethanol production. the fermentation culture of 1p4 isolate was prepared in ypd media with different o o initial glucose of 2%, 10% and 20% at 27 c, 37 c and o 42 c using the same procedures as evaluating glucose consumption and growth cell. ethanol concentration was measured at 48 h of fermentation using gas chromatography. experiment was conducted in duplicate for each condition. results morphological identification. morphological characteristic of 1p4 isolate was identified using light microscope after 24 h incubation at ypd media. as macroscopic identification, colony of p. kudriavzevii 1p4 was found round and had white cream color (figure 1a). based on microscopic observation, single cell 1p4 was found ovoid with size 4 – 10 µm and monopolar budding cell type (figure 1b). stress tolerance assay. yeast p. kudriavzevii 1p4 was more tolerant to fermentation-related stresses than s. cerevisieae by4741 (figure 2). growth of yeast p. kudriavzevii 1p4 was unchanged following high temperatures stress conditions (figure 2a-d) while s. o cerevisiae could not grow at temperatures of 40 c and o 42 c (figure 2c-d). interestingly, both of isolates of p. kudriavzevii 1p4 and s. cerevisiae by4741 exhibited high sugar stress tolerance up to 30% initial glucose (figure 2e-h). the difference was yeast 1p4 had wider and thicker colony isolate than comparison isolate by4741. 1p4 isolate showed good growth at control condition and spot media with 5% ethanol (figure 2i-j). however, high ethanol concentration affected the growth of 1p4 isolate at ethanol stress media. growth of 1p4 isolate was slightly decreased in media containing 10% ethanol compared to control condition while growth of by4741 was severe at those stress media (figure 2k). both isolates exhibited incapability to grow against 15% ethanol stress conditions (figure 2l). glucose consumption. glucose utilization of p. kudriavzevii 1p4 was measured in different initial glucose (2%, 10% and 20%) and temperature levels o o o (27 c, 37 c and 42 c) with 2% of initial glucose at o 27 c as a control condition (figure 3). the reducing sugar was rapidly consumed by 1p4 isolate during the first 24 h at media with 2% initial glucose at all o o o temperatures (27 c, 37 c and 42 c). 1p4 isolate utilized glucose almost completely at this temperature, with only 0.15%-0.08% (w/v) glucose remaining at 48 h. the similar pattern in glucose consumption was o o showed at condition of 10% at 37 c and 42 c. however, the reducing sugar rate consumption was o o faster at 37 c than at 42 c. 1p4 assimilated reducing sugar almost completely at 36 h, respectively 4 h faster o than at condition of 42 c. indeed, 1p4 isolate showed higher glucose consumption rate of all initial glucose o concentration at 37 c compared other conditions. 1p4 consumed the most glucose content of 20% initial o glucose at 37 c (15.29%) than 2% initial glucose (1.8%) and 10% initial glucose (9.9%), respectively. cell growth of yeast 1p4. cell growth of yeast p. kudriavzevii 1p4 at ypd media with initial glucose 2%, 10% and 20% were grouped in three different o o o temperatures (27 c, 37 c and 42 c), with condition of o 2% sugar at 27 c was designated as control experiment. our data shows that od of yeast 1p4 600nm increased with higher initial glucose condition yet decreased as temperature during fermentation was raised (figure 4). 1p4 had higher final od value at o o o 27 c than both of 37 c and 42 c conditions. in addition, the final od value was higher at media of o 20 c initial glucose than of 2% and 10% initial glucose o at 37 c though extremely low at 2% initial glucose at o 42 c. as found in this study, the growth pattern of 1p4 isolate differs depending on availability of glucose on the media and levels of temperatures. based on od 600nm data, yeast 1p4 showed lag phase up to 6 hours and followed by exponential growth for up to 24 h at control condition. it is worth nothing that 1p4 isolate also showed diauxic phenomenon after 24 h. however, o growth pattern of 10% and 20% initial glucose at 27 c o and all conditions at 37 c showed lag phase then followed by exponential phase until 48 h. in contrast, cell growth of 10% and 20% initial glucose showed progressive increase of turbidity value for up to 24 hours as exponential growth. the duration of lag phase of 1p4 isolate was affected by initial glucose and temperature. the higher initial glucose at media prolonged the lag phase although raising the temperature shortened the duration. interestingly, 1p4 isolate grown at media o with 10% initial glucose at 27 c, all initial glucose at o o 37 c and 20% initial glucose at 42 c had lag phase as same duration as control condition. however, lag phase became longer than control at 20% initial glucose at o 27 c hence shorter than control at 2% and 10% initial o glucose at 42 c. ethanol production. the kinetic parameters of ethanol production 1p4 in media ypd with different 124 ulya et al. microbiol indones fig 3 glucose consumption of 1p4 isolate at media ypd of 2%, 10% and 20% initial glucose at various o o o temperatures (27 c, 37 c and 42 c) during 48 h fermentation. glucose concentration was measured using o dns method. condition of 2% initial glucose at 27 c was used as control experiment. ypd media with 2% initial glucose. ypd media with 10% initial glucose. ypd media with 20% initial glucose. a b fig 1 morphological characterization of p. kudriavzevii 1p4 grown at ypd solid media after 24 h incubation. (a) colony of 1p4 isolate. (b) cell of 1p4 isolate under light microscope (1000x). fig 2 effect of temperature, sugar and ethanol stresses on the cell growth of p. kudriavzevii 1p4 after 72 hours o incubation. s. cerevisiae by4741 was used as control isolate. temperature stress: a. 27 c (control), b. o o o 37 c, c. 40 c, d. 42 c. sugar stress: e. 2% (control), f. 10%, g. 20%, h. 30%. ethanol stress: i. 0% (control), j. 5%, k. 10%, l. 15%. by4741: isolate s. cerevisiae by4741, 1p4: isolate p. kudriavzevii 1p4. volume 14, 2020 microbiol indones 125 o fig 4 growth curve of p. kudriavzevii 1p4 in different initial glucose (2%, 10% and 20%) and temperatures (27 c, o o 37 c and 42 c) during 48 h fermentation. cell growth was measured using spectrophotometer at od 600nm o absorbance. condition of 2% initial glucose at 27 c was used as control experiment. ypd media with 2% initial glucose. ypd media with 10% initial glucose. ypd media with 20% initial glucose initial glucose and temperatures was shown at table 1. the ethanol concentration of 1p4 isolate increased as temperature of fermentation was raised. as found in this study, 1p4 isolate produced significantly higher o o ethanol concentrations at 37 c and 42 c than those at control condition. moreover, 1p4 isolate yielded o higher ethanol concentration at 37 c (32.05 g/l) than o 42 c (14.80 g/l), respectively. on the other hand, the availability of glucose at fermentation media affected the ethanol production of 1p4 isolate. ethanol concentration was found increasing as more glucose was added at fermentation media. the ethanol production of 1p4 was found high in 20% initial o glucose (14.80 g/l) at 42 c than 2% (4.15 g/l) and 10% (9.55 g/l) at same temperature, respectively. the highest ethanol concentration of 1p4 was showed of o 20% initial glucose at 37 c (32.05 g/l). this suggests that 1p4 isolate is potential for good candidate of fermentation agent at high-temperature ethanol fermentation. 1p4 isolate showed the highest ethanol yield, ethanol productivity and fermentation efficiency at o condition of 37 c (table 1). furthermore, the highest ethanol yield and fermentation efficiency were o exhibited of 2% initial sugar at 37 c (0.44 g/g and 87.62%, respectively) whereas the highest ethanol productivity was found of 20% initial glucose at same temperature (0.67 g/l/h). as found in this study, the ethanol yield of 1p4 and fermentation efficiency was decreased at higher initial glucose available at fermentation media. the ethanol yield and fermentation efficiency of 1p4 of 2% initial glucose at o 42 c were 0.22 g/g and 42.85%, respectively, compared to the condition 20% initial glucose (0.12 g/g and 12.10%, respectively). in contrast, ethanol productivity of 1p4 was shown increasing at higher initial glucose. the ethanol productivity of 1p4 of 2% o initial glucose at 37 c was 0.17 g/l/h compared to the condition of 20% initial glucose (0.67 g/l/h). discussion competent thermotolerant yeast as a fermentation agent is one of important key for successful producing ethanol in htef (yuangsaard et al. 2013). as found in this study, 1p4 showed high tolerance phenotype against o 42 c and 30% initial glucose. however, severe growth of 1p4 at 10% ethanol concentration indicated that ethanol tolerance mechanism was independent pathway from temperature and sugar tolerance at 1p4 isolate. temperature has been reported causing protein damage and yeast responding to this condition by inducing of hsps and trehalose (techaparin et al. 2017, chamnipa et al. 2018). hsps played role as molecular chaperones to protecting protein cells from thermally damage (saini et al. 2018). previous studies reported that trehalose is an important marker for potential stress resistance in yeast, since yeast with high trehalose accumulation showed temperature and ethanol tolerance (lahiri et al. 2014, gibson et al. 2007). however, it was reported that the phenotype of high tolerance against fermentation related-stresses depends on several factors including yeast strain, composition of fermentation medium, intracellular ethanol accumulation, incubation temperature and osmotic pressure (banat et al. 1998). the important characteristics of yeast in order to be applied for successful fermentation agent are capable to grow and produce high ethanol yield during htef. according to the growth curve of 1p4, cell density of 1p4 changed due to different glucose concentration and temperature. high cell density of 1p4 was presented in control condition, although the final od at all initial o glucose was almost similar in 27 c. as expected, the cell density was gradually decreased as temperature of fermentation was raised, although higher cell density was found at higher initial glucose. this might be related to temperature stress that inhibited the cell growth of 1p4. previous study reported that elevated temperature suppressed several proteins that involve in various metabolism pathways and affected protein transport and vesicle organization (choudary et al. 2016). interestingly, control condition showed diauxic phenomenon which is related to availability of glucose concentration in the media. in this study, we also found that concentration of glucose and temperatures affected duration of lag phase. lag phase of 1p4 became shorter at htef although higher initial glucose extended the duration. this characteristic might be an advantage for 1p4 as a fermentation agent for htef, considering that 1p4 isolate can adapt more quickly at high temperature. duration of lag phase is obviously an important of yeast phenotype in industrial fermentation, since short lag time allows yeast cells to grow more quickly in substrate fermentation (varelas et al. 2017). fermentation was conducted in nine conditions of initial glucose and temperatures. from our study, we found that 1p4 showed markedly better fermentation activity at htef than control. amongst all condition, 1p4 consumed the most substrate of 20% initial glucose o at 37 c simultaneously with the highest ethanol production at same condition. the ethanol production of 1p4 was higher compared to s. cerevisiae rl-11 that produced 11.7 g/l ethanol concentration and scheffersomyces stipitis cbs 6054 that obtained 8.2 g/l ethanol concentration (mussatto et al. 2012, scordia et al. 2012). moreover, the ethanol production of 1p4 o decline at 42 c. this pattern was similar to ethanol production of p. kudriavzevii strains that showed its ethanol production decreased gradually as the temperature was raised (techaparin et al. 2017, ndubuisi et al. 2018). it is important to note that 1p4 showed highest fermentation rate at media with high o glucose at 37 c, suggesting that this temperature is optimum condition for 1p4 to produce ethanol. this finding is considered high than that of s. cerevisiae o strains which had 30 c of optimum temperature for ethanol production (choi et al. 2010), and of p. 126 ulya et al. microbiol indones table 1 kinetic parameters of ethanol production by p. kudriavzevii 1p4 with different initial glucose and temperatures conditions p: ethanol concentration (g/l), yp/s: ethanol yield (g/g), qp: ethanol productivity (g/l/h), ey: fermentation efficiency (%). temperatures initial sugar p (g/l) yp/s (g/g) qp (g/l/h) ey (%) 2% 5.05±3.32 0.26±0.17 0.11±0.07 51.46±0.34 27oc 10% 12.90±1.84 0.21±0.03 0.27±0.04 41.11±0.06 20% 12.30±2.40 0.14±0.03 0.26±0.05 27.20±0.05 2% 8.25±0.63 0.44±0.03 0.17±0.01 87.62±0.07 37oc 10% 23.10±2.26 0.23±0.02 0.48±0.05 45.49 ±0.04 20% 32.05±7.42 0.20±0.49 0.67±0.15 40.96±0.09 2% 4.15±2.19 0.22±0.12 0.09±0.05 42.85±0.22 42oc 10% 9.55±2.80 0.10±0.03 0.20±0.06 18.83±0.05 20% 14.80±8.77 0.12±0.07 0.31±0.18 24.10±0.14 volume 14, 2020 microbiol indones 127 o kudriavzevii strain reported previously which had 25 c of optimum condition (agrawal et al. 2019). in conclusion, p. kudriavzevii 1p4 performed higher fermentative activity at htef. 1p4 consumed the o highest concentration of glucose of 20% at 37 c (15.29%), simultaneously with the highest concentration of ethanol (32.05 g/l ethanol and 0.67 g/l/h ethanol productivity). 1p4 isolate also showed high tolerance against sugar stress up to 30% glucose o and temperature stress up to 42 c. interestingly, the cell growth analysis exhibited short lag phase of 1p4 as temperature of fermentation was raised. this indicates that p. kudriavzevii is potentially applied as ethanol fermentation agent for high temperature ethanol fermentation. thus further study in large scale fermentor is required. acknowledgements the authors thank the ministry of research, technology and higher education of republic of indonesia for funding through scheme penelitian tesis magister to ria (no. 27/e1/kpt/2020). references agrawal t, quraishi a, jadhav sk. 2019. bioethanol production from madhuca latifolia l. flowers by newly isolated strain of pichia kudriavzevii. energy environ. 30(8): . doi:10.1177/0958305x19852475. 1477-1490 astuti ri, 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p. 2017. hightemperature ethanol production using thermotolerant yeast newly isolated from greater mekong subregion. b r a z j m i c r o b i o l . 4 8 ( 3 ) : 4 6 1 4 7 5 . d o i : 10.1016/j.bjm.2017.01.006. varelas v, sotiropoulou e, karambini x, liouni m, nerantzis et. 2017. impact of glucose concentration and nacl osmotic yeast on yeast cell wall β-d-glucan f o r m a t i o n d u r i n g a n a e r o b i c f e r m e n t a t i o n . f e r m e n t a t i o n . 3 ( 4 4 ) : 1 2 0 . doi:10.3390/fermentation3030044. yuangsaard n, yongmanitchai w, yamada m, limtong s. 2013. selection and characterization of a newly isolated thermotolerant pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate. anton leeuw int j g. 103(3):577-88. doi: 10.1007/s10482-012-9842-8. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 5.mi688-mochamad amin available online at http://jurnal.permi.or.id/index.php/mionline doi: 10.5454/mi.6.4.5issn 1978-3477, eissn 2087-8575 vol 6, no 4, december 2012, p 173-179 *corresponding author; phone: 31-5992445, e-mail: ingelusida@yahoo.com +62-31-5992446. fax: +62the hepatitis c virus (hcv) has been proven to be a major etiologic agent of parentally transmitted virus. hcv has become a major cause of liver cancer and is one of the commonest indications for liver transplantation (francois et al. 1993; pawlotsky et al. 2003; ghany et al. 2009). who estimated that approximately 170 million people were chronically infected with hcv, which is around 3% of the world population (http://www.who.int/emc). in general, approximately 20% of chronically hcv infected the prevalence of hepatitis c virus (hcv) infection has been as high as approximately 80% in patients with maintenance hemodialysis in public hospitals in indonesia. however, the prevalence in private hospitals has not been examined yet. the aim of this study was to investigate the prevalence of anti-hcv antibody and the subtype distribution in patients with hemodialysis in a private hospital in surabaya, indonesia. sera were obtained from 41 hemodialysis patients in a private hospital in surabaya. the positive prevalence of anti-hcv antibody was carried out by the enzyme-linked immunosorbent assay (elisa). anti-hcv-positive sera were subjected to reverse transcription-pcr (rt-pcr) to detect hcv rna and then direct sequencing. the hcv subtype was examined by phylogenetic analysis. twenty five patients (61%) out of 41 were positive for anti-hcv antibody, and hcv-rna was detected in 19 patients. the positive prevalence of anti-hcv antibody was 7.7% (one out of 13 patients) among patients who had undergone hemodialysis for less than one year, whereas it was 85.7% (24 out of 28 patients) among patients who had undergone hemodialysis for over one year. phylogenetic analysis revealed hcv-1a (52.6%) was the most common subtype, followed by 1b (15.8%), 1c (15.8%), 2a (5.3%), and 3k (5.3%). in conclusion, the prevalence of hcv infection among hemodialysis patients in a private hospital was as high as that in general hospitals. the predominant subtype was hcv-1a, which is in accordance with the previous studies in general hospitals in surabaya, indonesia. key words: anti-hcv, hepatitis c virus, hemodialysis, subtypes, surabaya prevalensi infeksi virus hepatitis c (hcv) diperkirakan 80% pada pasien dengan perawatan hemodialisis di rumah sakit umum di indonesia. akan tetapi, prevalensi di rumah sakit swasta belum diketahui. tujuan dari penelitian ini adalah untuk mengetahui prevalensi antibodi anti-hcv dan distribusi subtipenya pada pasien dengan hemodialisis di sebuah rumah sakit swasta di surabaya, indonesia. serum diperoleh dari 41 orang pasien hemodialisis di sebuah rumah sakit swasta di surabaya. deteksi prevalensi antibodi anti-hcv dilakukan dengan metode elisa. reverse-transcription-pcr (rt-pcr) dilakukan dengan menggunakan serum anti-hcvpositif untuk mendeteksi hcv rna, lalu disekuensing. subtipe hcv dianalisa dengan menggunakan pohon kekerabatan. dua puluh lima pasien (61%) dari 41 diketahui positif memiliki antibodi anti-hcv, dan hcv-rna terdeteksi pada 19 pasien. prevalensi positif antibodi anti-hcv adalah 7,7% (satu dari 13 pasien) di antara pasien yang telah menjalani hemodialisis selama kurang dari satu tahun, sedangkan itu 85,7% (24 dari 28 pasien) di antara pasien yang telah menjalani hemodialisis selama lebih dari satu tahun. analisa pohon kekerabatan mengungkapkan hcv-1a adalah subtipe yang paling umum sebanyak 52,6%, diikuti oleh 1b (15,8%), 1c (15,8%), 2a (5,3%), dan 3k (5,3%). sebagai kesimpulan, prevalensi infeksi hcv di antara pasien hemodialisis di rumah sakit swasta adalah sama tinggi dengan yang di rumah sakit umum. subtipe dominan adalah hcv-1a, yang juga sesuai dengan studi sebelumnya di rumah sakit umum di surabaya, indonesia. kata kunci: anti-hvc, hemodialisis, hepatitis c virus, subtipe, surabaya the prevalence and subtype distribution of hepatitis c virus infection among hemodialysis patients in a private hospital in surabaya, indonesia 1,6 1,2,6 6,7 7 mochamad amin , juniastuti , takako utsumi , yoshihiko yano , 1,4 1,5 1,2,6 mochamad yusuf , mochammad thaha , priyo budi purwono , 1,3 1,3,6 7 retno handajani , soetjipto , hak hotta , 7 1,2,6 yoshitake hayashi , and maria inge lusida * 1 institute of tropical disease, universitas airlangga, jalan mulyoreijo, surabaya 60115, indonesia; 2 3 4 5 department of microbiology; department of biochemistry; department of cardiology; department of internal medicine, faculty of medicine, universitas airlangga, jalan mayjen prof dr moestopo 47, surabaya 60131, indonesia; 6 indonesia-japan collaborative research center for emerging and re-emerging infectious diseases, universitas airlangga, jalan mulyoreijo, surabaya 60115, indonesia; 7 center for infectious diseases, kobe university graduate school of medicine, hyogo 650-0017, japan patients progress to liver cirrhosis over a period of 20 to 30 years, with hepatocellular carcinoma (hcc) arising from cirrhosis at an estimated rate of 1% to 4% per year (lusida et al. 2000; lavanchy et al. 2009). it was reported from surabaya that the prevalence of hcv infection was 2.3% of healthy blood donors, 64.7% of hepatocellular carcinoma and 76.3% of hemodialysis patients (soetjipto et al. 1996). transmission through medical procedures, such as blood transfusions and hemodialysis, is highly prevalent in developing countries. the prevalence of hcv among hemodialysis patients was variable, from 1.9 to 84.6% in different countries in recent years (fissell et al. 2004; alavian et al. 2010; mohammad et al. 2010). the prevalence of hcv infection is much higher in developing countries as compared to developed countries. previous studies from developing countries reported that the transmission of hcv among hemodialysis patients was generally nosocomial, and the possible risk factors were as follows: the failure to disinfect equipment between patients, the sharing of single-use vials for infusions, poor sterilization techniques, poor cleaning of dialysis machines, and inadequate distance between chairs (zampieron et al. 2004; theodore et al. 2006). several studies on hemodialysis patients showed that the prevalence of anti-hcv antibody positive was high in general hospitals in surabaya, but the prevalence in private hospitals, which were generally considered to have a better standard of operation, has not been reported yet (soetjipto et al. 1996; lusida et al. 2003; santoso et al. 2010). the aims of this study were to determine the seropositive prevalence of anti-hcv antibody and the distribution of hcv subtypes in hemodialysis patients in a private hospital in surabaya. materials and methods hemodialysis unit, sample collections and serological analysis for anti-hcv antibody. sera were collected from 41 hemodialysis patients (mean age 43.9 ± 11.8 years, 28 male, 13 female) in a private hospital in surabaya from january to june 2011. collected samples were stored at -80 °c in a freezer in the hepatitis laboratory, institute of tropical disease, airlangga university, surabaya, indonesia. the antihcv antibody was serologically examined using enzyme-linked immunosorbent assay (elisa) kit (hepalisa, indec, jakarta, indonesia). limited data was available regarding the risk factors associated with 174 amin et al. microbiol indones hcv infection. separate rooms were not available for patients seropositive for anti-hcv. polymerase chain reaction and direct sequencing for hcv-rna. following this, hcvrna was examined by reverse transcriptionpolymerase chain reaction (rt-pcr) among anti-hcv antibody positive samples, as described previously (doi et al. 1996; soetjipto et al. 1996; murphy et al. 2007). at first, rt-pcr was carried out using different sets of primers in ns5b region. the amplification in 5'utr region was then carried out by rt-pcr in case of negative for ns5b region (hotta et al. 1994; doi et al. 1996; soetjipto et al. 1996; murphy et al. 2007). these methods have been shown to be highly sensitive for the detection of hcv rna in anti-hcvpositive sera from patients with chronic liver disease in japan and indonesia (soetjipto et al. 1996). the pcr products were visualized on 2% agarose gel with ethidium bromide. isolation and purification of the pcr product used qiaquick pcr purification kit (qiagen gmbh, hilden, germany). nucleotide sequences of the amplified fragments were determined with the bigdye v1.1 terminator cycle sequencing kit and abi prism 310 genetic analyzer (applied biosystems, usa). determination of hcv subtype. the genotype and subtype of hcv nucleotide sequences of the specimens were compared with those of the international dna data bank (ddbj/embl/genebank) to determine hcv subtypes by following the criteria (simmonds et al. 2005). a phylogenetic tree was reconstructed by the unweighted pair group method using arithmetic averages (upgma) method using clustering in genetyx for windows version 9.0. results seropositive prevalence for anti-hcv antibody. among 41 hemodialysis patients, 25 patients (61%) (18 men and 7 women, aged 21 to 64 years) were positive for anti-hcv antibody. the positive prevalence of anti-hcv antibody was 7.7% (one out of 13 patients) among patients who underwent hemodialysis for less than one year, whereas it was 85.7% (24 out of 28 patients) among patients who underwent hemodialysis for more than one year. detection of hcv-rna and determination of genotype and subtype. hcv-rna in ns5b region was detected in 18 out of 25 hemodialysis patients with anti-hcv antibody positive. phylogenetic analysis revealed hcv-1a (n=10) was the most common subtype, followed by 1b (n=3), 1c (n=3), 2a (n=1), and 3k (n=1) (fig 1). among hcv-1a isolates, the homology was more than 95% of 294 bp (data were not shown). hcv-rna in 5'utr region was also examined and confirmed the positive prevalence of hcv-rna. only one sample (hd50) detected hcvrna based on 5'utr region among those negative samples based on ns5b region. in the multiple alignments, this amplified fragment of 5'utr showed similarities of nucleotide sequences with genotype 1, both in motif1 and motif2 (fig 2). in total, 19 (76%) out of 25 hemodialysis patients with anti-hcv antibody positive were positive for hcv-rna. the number of genotypes and subtypes are summarized in fig 3. discussion it was reported that the anti-hcv antibody seropositive prevalence among hemodialysis patients in the general hospital was 73.5% in 2003, from a study in west java, indonesia and 81% in 2013, from a study in central java, indonesia (saketi 2003 and rinonce 2013, respectively). the high prevalence of hcv infection is associated with the combination of volume 6, 2012 microbiol indones 175 fig 1 phylogenetic analysis based on ns5b sequences (294bp). collected samples in present study (printed in bold) were aligned with strains from the dna data bank (ddbj). the genotypes were written in the roof of the branch and subtypes were indicated in the right side. bar, 10 substitution per 100 nucleotides. genotype 7 were found in central africa (murphy et al. 2007). the present study revealed that several subtypes, including subtype 1a, 1b, 1c, 2a, and 3k, were found in hemodialysis patients. a previous study revealed that subtype 2a (52%) and 1b (15%) were prevalent among healthy blood donors from surabaya, indonesia (soetjipto et al. 1996). recent studies have revealed that subtypes 1b (47%) and 1c (19%) were prevalent among hcv-related liver diseases in jakarta, indonesia, and subtypes 1a (52%) and 3k (16%) were prevalent among hcv and human immunodeficiency virus (hiv) co-infected patients from yogyakarta, indonesia (utama et al. 2010; anggorowati et al. 2012). the genetic diversity differs depending on the geographic and clinical distributions. previous studies from surabaya and yogyakarta revealed that 1a was dominant among hemodialysis patients in the general hospitals (hadiwandowo et al. 1994; soetjipto et al. 1996; rinonce et al. 2013). present study also found that subtype 1a was the most prevalent among hemodialysis patients. it has still not been determined why 1a was predominant in hemodialysis patients in indonesia, while subtype 1b was the most prevalent among hcv-related liver diseases in jakarta and surabaya (hotta et al. 1994; soetjipto et al. 1996; utama et al. 2010). on the other hand, frequent exposure to hcv-contaminated blood may favor the frequent blood transfusions and the lack of adherence to universal infection precautions (santoso et al. 2010). present studies revealed that the hcv prevalence among hemodialysis patients in this private hospital was 61% and which was in accordance with our previous study in the general hospital (soetjipto et al. 1996; lusida et al. 2003). these results suggested that hcv infection among hemodialysis patients was still highly prevalent in surabaya, east java. hcv has been classified as the sole member of a distinct genus called hepacivirus in the family flaviviridae. the hcv genome carries a single long open reading frame (orf) encoding a polyprotein that is proteolytically cleaved into a set of distinct products. analysis of the hcv genome has revealed the high heterogeneity of the virus. in humans, at least seven different genotypes have been classified based on the relatively well-conserved regions of the genome (5'utr, c, e1, ns5b or complete genome) (doi et al. 1996; bartenschlager et al. 2000; simmond et al. 2005; utama et al. 2009). epidemiological studies also show a link of a certain hcv genotype with the mode of transmission and geographic distribution (simonds et al. 2005). genotypes 1 to 3 were detected globally, while genotype 4 was common in the middle east and africa (bukh et al. 1993; abdel-hamid et al. 2007), genotypes 5 and 6 were found in south africa (smuts et al. 1995) and southeast asia (lu et al. 2007) and microbiol indones176 amin et al. fig 2 determination of genotype based on 5'utr region. hcv-rna in one sample (hd50) was negative for hcv-rna by ns5b region but positive for 5'utr region. the genotyping was determined based on motif 1 and 2 in the 5'utr region. fig 3 summary of genotype and subtype in hemodialysis patients in this study. subtypes (n=18) were determined by ns5b region and genotypes were determined by ns5b and 5'utr region. subtype genotype genotype 1 2 3 subtype 1a 1b 1c 2a 3k subtype undetermined 1n=10 n=3 n=3 1 1 1 hd 50 t a ca c cgg aa t tg cca gga cga ccgggt c ct t t c _ _ t t gga t c _ a a cc cgc t caa tg cc tggaga t t t gggcg tgc c cc c m62321-genotype 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ _ . . . . . . . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ab047639-genotype 2 . . . . . . . . . . . . . . . g . . . a . . . t . . . . . . . . . . _ _ . . . . . . . _ . . . . . a . . . t . . . . . c . . c c . . . . . . . . . . . . . . . . d63821-genotype 3 . . . . . . . . . . . c . . . g . . t t . . . . . . . . . . . . . . _ _ . . . . . . a _ t . . . . . . . . . . . . . . c . . . a . . . . . . . . . . . . . . . . y11604-genotype 4 . . . . . . . . . . . c . . . g . . . t . . . . . . . . . . . . . . _ _ . . . . . a . _ . . . . . . . . . . . . . . . c . . . a . . . . . . . . . . . . . . . . y13184-genotype 5 . . . . . . . . . . . . . . . g . . . t . . . . . . . . . . . . . . _ _ . . . . . . t _ . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . y12083-genotype 6 . . . . . . . . . . . . . . . . . . . t . . . . . . . . . . . . . . c a . . . . . . a a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . motif 1 motif 2 subtype of hcv infection in hemodialysis patients in a private hospital was similar as that in general hospitals in surabaya. further investigation in other private hospitals with different levels of quality will be necessary to evaluate the hcv infection among hemodialysis patients in indonesia. both routine precautions and education of the unit staff will be necessary to reduce the viral transmission in the hemodialysis unit. acknowledgment we wish to thank nasronudin of the institute of tropical disease (itd)-universitas airlangga, surabaya, indonesia for his cooperation and m. soetomo of the al irsyad hospital, surabaya, indonesia, for the collection of samples from the hemodialysis department of al irsyad hospital. this study was supported in part by a grant-in-aid from the japan initiative for global research network on infectious diseases (j-grid) program from ministry of education, culture, sports, science, and technology, japan. references abdel-hamid m, el-daly m, molnegren v, el-kafrawy s, abdel-latif s, esmat g, strickland gt, loffredo c, albert j, widell a. 2007. genetic diversity in hepatitis c virus in egypt and possible association with hepatocellular carcinoma. j gen virol. 88(5): 15261531. doi: 10.1099/vir.0.82626-0. agarwal sk, dash sc, gupta s, pandey rm. 2009. hepatitis c virus infection in haemodialysis: the 'noisolation' policy should not be generalized. nephron clin pract. 111:c133-140. doi: 10.1159/000191208. alavian sm. 2010. hepatitis c in hemodialysis patients needs more attention for control and review the risk factors. saudi j kidney dis transpl. 21(2): 357-358. anggorowati n, yano y, heriyanto ds, rinonce ht, utsumi t, deshinta pm, subronto yw, hayashi y. 2012. clinical and virological characteristics of hepatitis b or c virus co-infection with hiv in indonesian patients. j med virol. 84(6): 857-865. doi: 10.1002/jmv.23293. bartenschlager r, lohmann v. 2000. replication of hepatitis c virus. review article. j gen virol. 81:1631-1648. bukh j, purcell rh, miller rh. 1993. at least 12 genotypes of hepatitis c virus predicted by sequence analysis of the putative e1 gene of isolates collected worldwide. proc natl acad sci usa. 90(17): 8234-8238. doi: 10.1073/pnas.90.17.8234. doi h, apichartpiyakul c, ohba ki, mizokami m, hotta h. 1996. hepatitis c virus (hcv) subtype prevalence in chiang mai, thailand, and identification of novel subtypes of hcv major type 6. j clin microbiol. volume 6, 2012 microbiol indones 177 emergence of mixed genotype infection. subtype 1a might be easily transmitted among hemodialysis patients through the adaption of impaired immunity. when mixed-genotype infection involving hcv/1a occurs, this subtype tends to prevail and persists as the only subtype during the course of the disease (qian et al. 2000). the possible sources of the high prevalence of hcv infection are blood transfusion and nosocomial infection in the hemodialysis unit. a recent study from yogyakarta also revealed the possibility of nosocomial infection based on the molecular analysis of hcv genome among patients using the same hemodialysis unit (rinonce et al. 2013). the present study also revealed that hcv genome of 5'utr and ns5b regions among patients of the same hcv subtype mostly showed high homology with each other and this was suggested to be nosocomial infection. the sequence homology among hcv isolates of the same subtype was quite high (more than 95% for hcv-1a), even though it was not 100%, because of the nature of quasispecies of hcv. the most important factor of hcv transmission in the hemodialysis unit is considered to be a crosscontamination from supplies and unit staff (fabrizi et al. 2008). it was reported from yogyakarta that hcv infection was independently associated with hemodialysis frequency and the number of blood transfusions (rinonce et al. 2013). according to the current concept, dialyzer reuse was not identified as a risk factor for hcv acquisition in multicenter databases and no randomized controlled trials existed on the impact of isolation on the risk of transmission of hcv to hemodialysis patients (fabrizi et al.2008). compared to our previous study in 1996 (soetjipto et al. 1996), this study also shows that there is no improvement in 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virus in south africa. j clin microbiol. 33(6): 16791681. soetjipto, handajani r, lusida mi, darmadi s, adi p, soemarto, ishido s, katayama y, hotta h. 1996. differential prevalence of hepatitis c virus subtypes in healthy blood donors, patients on maintenance hemodialysis, and patients with hepatocelluler carcinoma in surabaya, indonesia. j clin microbiol. 34(12): 2875-2880. theodore sy, jamal m. 2006. epidemiology of hepatitis c virus (hcv) infection. int j med sci. 3(2): 41-46. utama a, budiarto br, monasari d, octavia ti, chandra is, gani ra, hasan i, sanityoso a, miskad ua, yusuf i, lesmana la, sulaiman a; tai s. 2008. hepatitis c virus genotype in blood donors and associated liver 34(3): 569-574. fabrizi f, messa p, martin p. 2008. transmission of hepatitis c virus infection in hemodialysis: current concepts. int j artif organs. 31(12): 1004-1016. francois m, dubois f, brand d, bacq y, guerois c, mouchet c, tichet j, goudeau a, barin f. 1993. prevalence and significance of hepatitis c virus (hcv) viremia in hcv antibody-positive subjects from various populations. j clin microbiol. 31(5): 1189-1193. fissell rb, bragg-gresham jl, woods jd, jadoul m, gillespie b, hedderwick sa, rayner hc, greenwood rn, akibat t, young ew. 2004. patterns of hepatitis c prevalence and seroconversion in hemodialysis units from three continents: the dopps. kidney international 65:2335-2342. doi:10.1111/j.15231755.2004.00649.x. ghany mg, strader db, thomas dl, seeff lb. 2009. diagnosis, management, and treatment of hepatitis c: an update. hepatology 49(4):1335-1374. doi: 10.1002/hep.22759. hadiwandowo s, tsuda f, okamoto h, tokita h, wang y, tanaka t, miyakawa y, mayumi m. 1994. hepatitis b virus subtypes and hepatitis c virus genotypes in patients with chronic liver disease or on maintenance hemodialysis in indonesia. j med virol. 43(2):182-186. doi: 10.1002/jmv.1890430216. hotta h, handajani r, lusida mi, soemarto w, doi h, miyajima h, homma m. 1994. subtype analysis of hepatitis c virus in indonesia on the basis of ns5b region sequences. j clin microbiol. 32(12): 3049-3051. lavanchy d. 2009. the global burden of hepatitis c. liver international 29(s1):74-81. doi:10.1111/j.14783231.2008.01934.x. lu l, li c, fu y, thaikruea l, thongswat s, maneekarn n, apichartpiyakul c, hotta h, okamoto h, netski d, pybus og, murphy d, hagedorn ch, nelson e. 2007. complete genome for hepatitis c virus subtypes 6f, 6i, 6j and 6m: viral genetic diversity among thai blood donors and infected spouses. j gen virol. 88(5): 15051518. doi: 10.1099/vir.0.82604-0. lusida mi, soetjipto, handajani r, nidom ca, soemarto, darmadi s, adi p, fujii mn, fujita t, ishido s, hotta h. 2000. viral load in indonesian patients with chronic liver disease and in blood donors infected with different subtypes of hepatitis c virus. jpn j infect dis. 53(2): 67-69. lusida mi, surayah, sakugawa h, nagano-fujii m, soetjipto, mulyanto, handajani r, boediwarsono, setiawan pb, nidom ca, ohgimoto s, hotta h. 2003. genotype and subtype analyses of hepatitis b virus (hbv) and possible co-infection of hbv and hepatitis c virus (hcv) or hepatitis d virus (hdv) in blood donors, patients with chronic liver disease and patients on hemodialysis in surabaya, indonesia. microbiol immunol. 47(12): 969-975. mohammad wz. 2010. prevalence of hepatitis c virus in hemodialysis patients: five year experience from a single center. saudi j kidney dis transpl. 21: 548-554. murphy dg, willems b, deschênes m, hilzenrat n, disease in indonesia. intervirology 51(6): 410-416. doi: 10.1159/000205515. zampieron a, jayasekera h, elseviers m, lindley e, de vos jy, visser r, harrington m. 2004. european study on volume 6, 2012 microbiol indones 179 epidemiology and the management of hcv in the haemodialysis population, part 1: centre policy. edtna erca j. 30(2): 84-90. doi: 10.1111/j.17556686.2004.tb00341.x. 05. sjatha.cdr vol.15, no.2, june 2021, p 69-75 doi: 10.5454/mi.15.2.5 immunogenicity of recombinant dna vaccine encoding non-structural protein-1 dengue virus serotype-2 in balb/c mice fithriyah sjatha tjahjani mirawati 1 2* , elitha sundari pulungan , and sudiro 1 1 department of microbiology, medical faculty, universitas indonesia, indonesia; 2master programme of biomedical science, faculty of medicine, universitas indonesia, indonesia. dengue hemorrhagic fever (dhf) is an infectious disease caused by the dengue virus (denv) which spread widely in tropical and subtropical regions of the world. denv is a single-positive strand rna virus with a genome size of ± 11kb which encodes three structural proteins, seven non-structural proteins, and two untranslated regions (utr). the non-structural protein-1 (ns1) of denv is known to have important role in dengue pathogenesis also promising to be developed as dengue vaccine. lately, novel approach by dna vaccine immunization have given new perspective for a safe, stable, and immunogenic vaccine platform. previously, we had successfully constructed dna vaccine encoding ns1 protein of denv2 (puns1) which expressed recombinant ns1 protein in mammalian cells line. thus, in this current study the ability of puns1 to induce humoral immune response will be further analyzed by in mice immunization. sixteen balb/c mice aged of 4 weeks were immunized 3 times with puns1 or pumvc4a on 2 weeks interval. blood sampling was 100 µg of carried out . antibodies titer just before immunization and termination was done 2 weeks after last immunization from individual mice sera against denv-2 were measured with in-house elisa. anti-dengue ns1 igg titer from mice group immunized with showed elisa absorbances five times higherrecombinant puns1 than pumvc4a group. this result the ability of puns1 to induce humoral immune response against ns1 denv-2 in-suggested vivo. recombinant puns1 can induce humoral immune response in mice. dengue virus serotype-2 (denv-2), humoral immune response, ns1, recombinant dnakey words: demam berdarah dengue (dbd) adalah infeksi yang disebabkan oleh virus dengue (denv) yang tersebar luas di wilayah tropis dan subtropis di dunia. denv merupakan virus rna rantai tunggal dengan ukuran genom ± 11kb yang mengkode tiga protein struktural, tujuh protein non-struktural, dan dua daerah yang tidak ditranslasikan (utr). protein non-struktural ( ) denv diketahui memiliki peran yang sangat penting dalam ns1 patogenesis infeksi denv dan sebagai pengembangan vaksin dengue yang menjanjikan. saat ini, pengembangan vaksin baru dengan dna yang diimunisasikan memberikan perspektif baru karena aman, stabil, dan imunogenik. pada penelitian sebelumnya, kami telah berhasil mengonstruksi vaksin rekombinan dna yang mengkode protein nsi dari denv-2 (puns1) dan diekspresikan pada sel mamalia. oleh karena itu, pada penelitian ini dilakukan analisis lebih lanjut untuk melihat kemampuan puns1 dalam menginduksi respon imun humoral dengan imunisasi pada mencit. sebanyak 16 mencit balb/c yang berumur 4 minggu diimunisasi sebanyak 3 kali dengan 100 µg puns1 atau pumvc4a dalam interval waktu 2 minggu. pengambilan sampel darah mencit dilakukan sebelum imunisasi dan dilakukan terminasi 2 minggu setelah imunisasi terakhir. titer antibodi dari serum masingmasing mencit diukur dengan elisa in-house. titer igg anti protein ns1 dari denv2 dari kelompok mencit yang diimunisasi dengan rekombinan puns1 menunjukkan nilai absorbansi yang tinggi, 5 kali lebih tinggi dari kelompok pumvc4a. hal ini membuktikan kemampuan puns1 dalam menginduksi respon imun humoral terhadap ns1 denv-2 secara in-vivo. rekombinan puns1 dapat menginduksi respon imun humoral mencit. kata kunci: dengue virus serotipe 2 (denv-2), dna rekombinan, ns1, respon imun humoral microbiology indonesia available online at http://jurnal.permi.or.id/index.php/mionline issn 1978-3477, eissn 2087-8575 *corresponding author: phone 82218723138;: +62 e-mail: elithasundaripulungan gmail@ .com denv belongs to the family flaviviridae, of the genus . dengue virion morphology is flavivirus spherical with a diameter of ± 50 nm. the outer part of the virion is covered by a sheath in the form of a membrane lipid with a sheath thickness of ± 10 nm with nucleocapsid inside about ± 30 nm in diameter. denv genetic material in the form of positive rna single strand with a length of ± 11 kb. the genome of this virus encodes a large polyprotein which is divided into three structural proteins (capsid (c); membrane precusor (prm); and envelope (e)), seven non-structural (ns) dengue hemorrhagic fever (dhf) is an infectious disease caused by the dengue virus (denv). dengue virus infection is a global health problem that occurs in tropical and subtropical regions of the world. based on data from the who (world health organization) this disease has progressed to cause nearly 390 million people to be infected each year, including more than 960,000 cases of severe dengue (who 2016). proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5), and two regions that are not translated or called the untranslated region (utr) (putnak 2003; lin et al. et al. 2012). base on genetically, dengue virus has four different serotypes are denv-1, denv-2, denv-3, and denv-4. each denv serotype has a protein e amino acid sequence homology of about 70% (lin et al. 2012). ns1 protein is a glycoprotein weighing 42-50 kda from 353-354 amino acids encoded by 1056bp of genes (clyde 2006) this protein with other viral proteins et al. . can fulfill a structural role, helping to anchor the replication complex to the membrane and induce the formation of membrane components that facilitate viral rna replication, assembly and release of viruses, immune avoidance, and various aspects of pathogenesis (glasner 2018). there are two form of ns1 which et al. expressed by infected host cell, cell surface on (mns1 protein) and secreted into the extracellular environment (sns1 protein). in the process of infection, mns1 will be presented by major histocompatibility complex (mhc) molecules and can induce the immune response. meanwhile, sns1 can directly bind to various components of the complement pathway which either trigger or inhibit the activity of complement also can be used as a marker because the presence of high levels of ns1 have shown an association between the severity of dengue virus infection (henrique 2014; watterson et al. et al. 2016). the ns1 protein is able to activate endothelial cells directly and decrease the integrity of the endothelial cell barrier through dependent tlr4 pathway (henrique 2014). when denv infects, et al. the body can activate the cellular immune system and the humoral immune system in response to fight. as a result of the humoral immune system, dengue virus infection can lead to the formation of short-term protective antibodies in the form of immunoglobulin m (igm) and long-term protective antibodies in the form of immunoglobulin g (igg) against the virus (shu p.y. et al. 2000). in contrast to viral structural proteins that induce neutralizing antibodies, ns1 protein can induce antins1 antibodies. so, it's not inducing ade (antibody dependent enhancement) activity which raised from m or e protein based vaccine (hertz, 2017). et al. recombinant dna plasmid can be delivered to the host in several ways and methods so that it may mimic some aspects of the natural infection of the host cells. compared to recombinant protein and recombinant virus vaccines, dna vaccines are relatively inexpensive, have low production costs, easy to manufacture and use, and safe because it is not pathogenic but still immunogenicity. this is based on the possibility of the dna vaccine can induce cd8 and + cd4 t cells so that stimulate the immune response + through the mhc-i and mhc-ii pathways (leitner et al. 2007). in addition, a recombinant dna plasmid is another potential strategy for the development of an ns1-based vaccine (liu, y. 2016). et al. several denv vaccine approach based on ns1 protein are widely developed. in we our previous study, successfully constructed and proven that our recombinant puns1 (recombinant pumvc4.a encoding ns1 protein of denv-2) able to express its protein in mammalian cell line . (sjatha 2019)et al. furthermore, this current study will be analyzed the ability of our recombinant puns1 to induce humoral immune response in mice as initial evaluation of puns1 antigenicity as dna vaccine. materials and methods all protocols within ethics statement and mice. this experiment have been ethically approved by health research ethics committee faculty of medicine universitas indonesia-rscm number: ket1350/un2.f1/etik/ppm.00.02/2019. animal experiments were carried out using 16 balb/c mice in total, which were divided into two groups: (1) eight mice were immunized with pumvc4.a control plasmid and (2) eight mice were immunized with puns1. experiment in mice was carried out by experienced and certified animal welfare staff and under the supervision of attending veterinary. the animal experiment was carried out in the animal care facility of the department of microbiology, faculty of medicine universitas indonesia with a closed cage system and ad libitum feed-drink. viruses. the virus used as antigen for in-house elisa was dengue virus serotype-2 (denv-2) ncg (new guinea c) strain harvested at 6 d.p.i titer of 5.02 x 10 ffu/ml. 7 propagation of harboring escherichia coli plasmid. dh5α competent cells escherichia coli that has carried the recombinant puns1 and pumv4.a plasmid propagated in liquid luria bertani (lb) were medium. to produce starter cultures, bacterial clones containing recombinant plasmids were streaked on solid lb medium containing 50 μg/μl kanamycin and incubated at 37°c for 24 hours, then one loop of bacterial colony was inoculated in 5 ml liquid lb 70 sjatha et al . microbiol indones volume 15, 2021 microbiol indones 71 medium containing 50 μg/μl kanamycin and incubated at 37°c, 200 rpm. after 18-20 hours, 2 ml of liquid culture was moved in to 200 ml of fresh liquid lb medium containing 50 µg/µl kanamycin. then, reincubated at 37°c, 200 rpm for 18-20 hours. when the cell density reach od equal to 3-4 x 10 cells/ml, the 9 cells were harvested. the bacterial culture was centrifuged at 4000 rpm for 10 minutes at 17°c to get bacterial cell pellet. plasmid isolation. plasmid isolation from bacterial pellet was carried out following the hispeed® plasmid midi kit (qiagen, catalog no: 12643) protocol. pure dna plasmids were confirmed by gel electrophoresis and stored at -30°c. plasmid c o n c e n t r a t i o n s w e r e m e a s u r e d u s i n g spectophotometry method. confirmation of puns1 by pcr. pcr reaction was performed using (invitrogen, catalog no: 10342053). composition of pcr reaction as much: 2.5 μl of pcr master mix; 15.25 μl of dw; 2.5 μl of dntp 2mm; 1 μl of mgcl 50 mm; 0.8 μl of reverse primer 2 350 (10 mm); 0.8 μl of primary forward 2329 xbai sbam taq dna polymerase (10 mm); 0.15 μl of enzyme (invitrogen, catalog no: 10342-053).; and 2 l μ of plasmid template. the pcr cycles used were: 95°c for 5 minutes; followed by 40 cycles of 95°c for 30 seconds; 55°c for 30 seconds; 72°c for 90 seconds; and ended was 72°c for 5 minutes. marker used was λ dna iii (tiangen, catalog no: md202). hind sixteen mice were mice immunization. balb/c immunized with 100 μg puns1 and pumvc4.a plasmid using nfi (needle free injector, shimajet, japan) apparatus. held within 3 immunizations were doses in 2 weeks interval and retro-orbital blood were taken prior to immunization under the sedation of ketamine-xylasine treatment. two weeks after third immunization, mice were terminated through cervical dislocation and blood were drawn by cardiac puncture. mice blood were then centrifuged at 3000 rpm for 5 minutes and collected sera were stored at -70 c until o further examination. anti-ns1 igg of denv2 analysis with in-house elisa. termination individual mice sera from both groups were tested for igg recognizing ns1 of denv2 with in-house elisa. briefly, flat bottom 96well elisa plate (nunc, catalog no: 442404) were coated with 10 ffu/ml denv2 in bicarbonate buffer 4 ph 9.6 overnight at 4 c. plate were washed third o with pbs containing 0.05% tween 20 negative control . antigen was pbs 1x, negative control sera were preimmunized sera, and positive control were mice sera immunized with denv-2. elisa plate wells were coated with denv-2 antigen with the same titer in each well. the coating process is carried out overnight at 4°c. the plate washed 3 times with pbs containing 0.05% tween 20, followed by 5% skim milk blocking at 37°c for 1-hour incubation. then, elisa plate was washed followed by addition of 100 μl of diluted (1:200) serum samples and incubation at 37°c for 1 hour. after incubation, plate was washed and diluted horse anti mouse-igg hrp labeled (1:5000; vector laboratories, catalog no: pi-2000) were added followed by incubation at 37°c for 1 hour. after incubation, plate was washed and 80 l of tmb μ substrate (1-step, catalog no: 34028) was added followed by incubation in dark with gentle agitation for 20 minutes at rt. finally, 30 l of stop solution was μ added and absorbances were measured at 450 nm. numerical data obtained statistical analysis. from elisa were analyzed using spss program, tested for its normality by shapiro wilk test, followed by t-test. for significances at p value <0.05 and confidence level 95%. results recombinant plasmid puns1 confirmation. pcr was performed to confirm the ns1 gene in puns1. the pumvc4.a plasmid was used as negative control. as seen in figure 1, the presence of ns1 could be detected in the recombinant plasmid puns1. this is indicated by the presence of a band in the puns1 lane. recombinant puns1 had ns1 gene insertion of 1056 bp size based on colony pcr analysis. evaluation of anti denv2 igg titer. pre and post-immunization serum of individual mice sera from both puns1 and pumvc4.a groups were used to measure igg antibody titers induced by recombinant puns1 against denv2. as seen in figure 2, preimmunization serum from both puns1 (mean ± sd: 0.137 ± 0.008) (mean ± sd: 0.138 ± and pumvc4.a 0.009) group showed similar absorbances value. however, post-immunization serum mice's with puns1 (mean ± sd: 1.508 ± 0.039) had anti denv2 igg value higher than mice immunized with pumvc4a (mean ± sd: 0.294 ± 0.034). this difference is said to be significant with a confidence level of 95% (p < 0.05). these results indicate that the puns1 recombinant was able to induce humoral immune response in mice, with 5 times fold higher than pumvc4.a control group in recognizing denv-2. 72 sjatha et al . microbiol indones discussion secreted and membrane-associated of denv ns1 are highly immunogenic because it can induce immune system correspond to the production of antibody against ns1 which also detected in infected patients ( ). it has been found that chuang, y-c, 2013et al. denv ns1 protein with high levels is associated with severity of infection and levels >600 ng/ml in the first 72 hours of disease occurrence associated with the development of dhf (libraty 2000). thus, ns1 et al. is also developed as denv vaccine approach since its advantages for not inducing ade (antibody dependent enhancement) activity which raised from m or e protein based vaccine (hertz, 2017). et al. although ns1 can induce non-neutralizing antibodies, there have been reported problems. antins1 antibodies can cross-react with coagulationrelated cells or molecules, such as human plasminogen, thrombin, platelets, and endothelial cells (chuang, yc, 2013). these anti-ns1 autoantibodies can et al. cause thrombocytopenia and apoptosis mediated by nitric oxidation in endothelial cells in vitro (lin, c-f, et al. 2002). because of the homology of the sequences between the ns1 denv protein and proteins in platelets and endothelial cells, it is possible that these autoantibodies are induced by ns1 through molecular mimicry (lin, y-s, 2011). although in vitro has et al. shown the potential of cross-reaction between antins1 antibodies and endothelial cells and platelets, this finding has not been supported or proven in vivo (sun, d-s, 2007).et al. fig 1 ns1 insert confirmation by pcr. m: marker, 1: recombinant plasmid puns1, 2: plasmid pumvc4.a, k-: negative control pcr. fig 2 antibody titer from mice group immunized with puns1 and pumvc4a using in-house elisa (*p < 0.05). volume 15, 2021 microbiol indones 73 alternative vaccine delivery strategy an can be performed to get better vaccine outcome. in this study, we use nfi (needle free injector) as apparatus to introduce dna vaccine in balb/c mice which proven to be more effective in delivering dna in to target cell compare to syringe-based immunization. in the present needle-free immunization strategy, the vaccine plasmid could directly enter a relatively large number of muscle cells in the thigh of host animals (imoto, j-i, 2005). et al. after immunization, dna vaccine can be uptake by the muscle cells and neighboring antigen presenting cells. the injected dna vaccine will express the recombinant protein as endogenous antigen and presented by mhc class i. on the other hand, antigen can be released through extra cellular and further taken up by circulated apcs which lately presenting recombinant protein by mhc class ii. the antigenexpressing apcs then migrate to lymph nodes where they activate the t and b lymphocytes to induce cellular and humoral immune responses (leitner et al. 2007). although antigen presentation by both mhc class i and ii, dna vaccine have other advantages. dna vaccine are safer, more stable for storage and shipping, easy to handle, induce cellular immune responses, licensed veterinary vaccine, relatively generic construction and production, and potent prime in animal studies (khan 2013; liu 2010). one of disadvantages of dna vaccine is inducing antibody production against dna (khan 2013). in our result, igg response of control pumvc4.a group is also detected in low level of absorbances. this result may occur because of factors influencing immune responses against the vector such as route of administration, dose of vector, host-related factors, and promoters (bessis 2004). et al. antivector immunity was found in mice injected with the plasmid intraperitoneally, intravenously, or subcutaneosly. besides producing antibodies against the inserted gene, specific cytotoxic t cells were also found in the plasmids used. however, in mice injected with plasmids intramuscularly, the humoral immune response was obtained but with a lower cytotoxic t cell response (brockstedt , 1999). a study of plasmid-et al. induced endothelial cells demonstrated the productivity of the cytokines tnf-α, il-1β, and il-4 which increased promoter activity (ritter 2000). et al. sjatha 2019, showed that levels of the cytokine et al. tnf-α in cho-k1 mammalian cell line which transfected by pcns1 was higher compared control. n our result showed igg recognizing denv-also i 2 obtaining from puns1 group has shown 5 times higher absorbances level compare to control pumvc4.a group, indicating high production of recombinant ns1 expression and its immunogenicity in inducing humoral igg response. kim 2010, et al. showed dna vaccines may have a relatively poor immunogenicity. this can be overcome by increasing the potential mechanisms of dna vaccine such as plasmid alterations, increasing the stability of the dna by formulations and encapsulation, delivery, and augmentation of immunity (liu 2010). to evaluate our recombinant puns1 as dna vaccine candidate, induced antibody may further analyzed for its ability to overcome denv infection in-vitro or in-vivo. and also to evaluate the possibility of cross-reaction with other denv serotype or other flavivirus, cross-recognizing of antibody against ns1 protein to endothelial cell which lead to plasma leakage and also its ability to trigger complement cascade as vaccine safety concern. in conclusion, we successfully prove the ability of our puns1 to induce humoral immune response in mice. further analysis for its antibody protection against denv can be performed as one parameter in 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