istianto et al24042012 ISSN 1978-3477, eISSN 2087-8587 Vol 6, No 1, March 2012, p 42-47 Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.6.1.7 SHORT COMMUNICATION Application of Phenol Pretreatment for the Isolation of Rare Actinomycetes from Indonesian Soil 1 1 1 YUDHIE ISTIANTO *, RADEN SETYO ADJI KOESOEMOWIDODO , HARMOKO SAPUTRA , 2 1 1 YOSHIO WATANABE , HARDANING PRANAMUDA , AND BAMBANG MARWOTO 1 Center of Biotechnology BPPT, Kawasan PUSPIPTEK-Serpong, Tangerang Selatan 15340, Indonesia; 2 Bioresorces Laboratories, Micro Biopharm Japan, Co. Ltd.1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan Phenol treatment was applied for isolation of rare Actinomycetes using 25 soil samples collected from Pulau Seribu, Tanjung Redep, Manokwari, and Halmahera. The samples were air-dried and suspended in 1.5 % (w/v) o phenol solution at 30 C for 30 minutes, and subsequently cultured on plates of humic acid-vitamin agar (HVA) -1 -1 medium supplemented with cycloheximide (50 µg mL ) and nystatin (50 µg mL ). A total of 61 isolates were obtained and the most dominant isolates were not Streptomyces (only 24.6%), whereas other genera such as Micromonospora, Actinomadura, Microbispora and Polymorphospora were isolated with ratios of 49.2%, 13.1%, 9.8%, and 3.3%, respectively. Key words: phenol method, rare-Actinomycetes, selective isolation method, soil sample Perlakuan phenol diterapkan untuk mengisolasi Actinomycetes selain dari Streptomyces dari 25 sampel tanah yang diperoleh dari Pulau Seribu, Tanjung Redep, Manokwari, dan Halmahera. Sampel dikering anginkan o dan disuspensi dalam 1,5% larutan fenol pada suhu 30 C selama 30 menit, kemudian dikultur pada media asam -1 -1 humat-vitamin agar (HVA) yang dilengkapi dengan cycloheximide (50 µg mL ) dan nistatin (50 µg mL ). Sebanyak 61 isolat diperoleh dengan komposisi: Streptomyces hanya 24,6%, sedangkan marga lain seperti Micromonospora, Actinomadura, Microbispora dan Polymorphospora diperoleh masing-masing dengan rasio 49,2%, 13,1%, 9,8%, dan 3,3%. Kata kunci: Actinomycetes, metode isolasi selektif, perlakuan fenol, sampel tanah Actinomycetes (Actinobacteria) is a group of prokaryotic organisms belonging to the subdivision of the Gram-positive bacteria. Most of them are in the subclass Actinobacteridae and order Actinomycetales. All members of this order are characterized in part by the high G+C content (>55 mol%) in their DNA (Stackebrandt et al. 1997). Actinomycetes are a group of microbes, of which many of the members can produce antibiotics and antitumors (Chantongcome et al. 2009). A total of 7899 bioactive compounds have been identified up to 1988; 67% from Actinomycetes, 12% from bacteria, and 20% from fungi. Today, known bioactive metabolites are more than 22 000, 10 100 of which are produced by Actinomycetes. So far, 140 to 160 antibiotics have been used in human therapy and agriculture, and 100 to 120 of them are produced by Actinomycetes (Berdy 2005). New antimicrobial agents are desperately needed to combat the increasing number of antibiotic resistant pathogenic microbial strains. Natural products remain the most propitious source of novel antibiotics. It is widely accepted that Actinomycetes are prolific producers of natural bioactive compounds. The efficiency of research to discover new compounds with novel chemical structures can be increased with intensive efforts in isolating and screening rare genera of microorganisms (Takahashi, 2004 ; Kumar et al. 2010). Rare Actinomycetes are usually regarded as strains whose isolation frequency much lower than those strains isolated by conventional methods. Past and present efforts in the isolation of rare Actinomycetes have discovered some genera, such as Actinomadura, Actinoplanes, Micromonospora, Microtetraspora, that had been recovered from many soil samples (Lazzarini et al. 2001; Hayakawa et al. 2010). Although researchers have utilized numerous approaches to isolate new microbes, the number of microorganisms that have been successfully grown represents only a small portion of the total that exists (Khana et al. 2011). Hawksworth (1997) reported that the number of known bacteria comprises not more than 10% of the estimated total number of species in the world. The isolation of unknown microbial strains thus *Corresponding author, Phone/Fax: +62-21-7560155, E-mail: yudhie_primer@yahoo.com represents an area of great potential. I n d o n e s i a i s k n o w n a s a c o u n t r y w i t h megadiversity of microorgaisms, i.e. it lies geographically on a tropical region with the highest level of biodiversity that it has high potential as a source of novel microbial bioactive metabolites. Efforts to isolate and evaluate microbial bioactive compounds from Indonesia, especially from Actinomycetes, have expanded significantly in recent years. However, most of the isolates recovered on agar plates have been identified as genus Streptomyces, which are the dominant Actinomycetes in soil. Lisdiyanti (2006) reported, a total of 982 Actinomycetes isolated from Indonesia in 2003 and 2004 using three isolation methods including sodium dodecyl sulfate yeast extract (SDS-YE), rehydration-centrifugation, and dry heating. Her study revealed that on the basis of 16S rRNA, 66.1% isolates were identified as Streptomyces and 33.8% isolates were Non-Streptomyces Groups. For the purpose of screening rare bioactive molecules, the adoption of improved methodologies for isolating the uncommon and less studied, rare Actinomycetes is required to avoid re-isolating strains that produce known bioactive metabolites and to improve the quality of the natural products screened (Takahashi and Omura 2003; Berdy, 2005; Khana et al. 2011). Over the past year, we have attempted various methods to isolate novel actinomycetes, using three isolation methods including wet method, acid treatment, dry heating on the basis of 16S rRNA, 79.5% isolates were identified as Streptomyces and 20.5% isolates are as Non-Streptomyces groups. In this paper we focused on the application of phenol pretreatment for isolating rare Actinomycetes from Indonesian soil sample. This method selectively isolated Streptomyces violaceusniger cluster (Hayakawa et al. 2004), Micromonospora (Hayakawa et al. 1991), Dactylosporangium, Microbispora (Takahashi and Omura, 2003), and Actinomadura (Hayakawa et al.1995) from Japan soil sample. A total of 25 soil samples were collected from Pulau Seribu (West Java), Tanjung Redeb (East Kalimantan), Manokwari (Papua), and Halmahera (North Maluku). Samples were passed through a 2 mm mesh sieve and air-dried at room temperature for 1 week. One gram air-dried sample were mixed with 10 mL sterilized water and vigorously stirred for 2 min. After allowing the tube to stand for 1 min, 1 mL of supernatant was transferred to 9 mL of 1.5% phenol solution. Then o the mixture was maintained at 30 C with occasional stirring. After incubation for 30 min, aliquots of mixture were spread onto humic acid-vitamin agar -1 supplemented with cycloheximide (50 µg mL ) and -1 nystatin (50 µg mL ). The experiments were done in o replicates and then all plates are incubated at 28 C for 1 to 3 weeks. A total of 61 actinomycete isolates were successfully isolated from 25 soil samples. Twenty seven isolates were isolated from Halmahera, 21 isolates from Manokwari, 8 isolates from Pulau seribu, and 5 isolates from Tanjung redep (Table 1). Colonies having characteristic features such as powdery appearance with convex, concave or flat surface and color ranging from white, gray to pinkish and yellowish were selected. Colonies observed on the first and second days were eliminated because Actinomycetes were considered as slow grower (Currie 2006). Rare Actinomycetes have usually been regarded as genera of Actinomycetes whose isolation frequency by conventional methods is much lower than that of Streptomyces. Consequently, basic knowledge on the habitat, physiology and productivity of molecules of rare Actinomycetes develops rather slowly. Due to the discovery of Actinomycetes with ecologically significant properties the screening extends into uncommon environments. For the purpose of screening novel bioactive molecules, several factors must be considered: pretreatment, selective medium and recognition of candidate colonies on a primary isolation plate. Production of spore mass was detected on the 21- day old cultures on Humic Vitamin Agar. Spore ornamentation was observed by Olympus BX51 microscope. The DNA was isolated using InstaGene matrix kit for DNA isolation. The pellet was lysed using a lysing matrix, combined with 100µL, and centrifugation for 3 min at 15 000 x g. Identification using the 16S rDNA was conducted by PCR using a 9F forward primer, GAGTTTGAT (C/T)(C/A)TGGCTCAG; and a 1541R reverse primer, AAGGAGGTG(A/T)TCCA(A/G)CC. The reaction mixture (50 μL total volume) contained 30 µL ddH2O, 5 µL of MgCl2, 5 µL of 10X Buffer 4 µL of deoxynucleoside triphosphates, 1 µL of each primer, 0.25 µL of Takara Taq Polymerase (Takara Bio Inc, Japan), and 5 μL of cell lysis as the template. PCR conditions were as follows: denaturation at 98 °C for 20 s, annealing at 52 °C for 45 s, and elongation at 72 °C for 2 min. A total of 30 cycles were performed, followed by a final elongation for 4 min at 72 °C. PCR products were purified with a Geneaid PCR Fragments Extraction Kit according to the manufacturer's Microbiol Indones 43Volume 6, 2012 44 ISTIANTO ET AL. Microbiol Indones instructions (Geneaid, Taiwan). Amplicon was sequenced with an automatic sequence analyzer (Applied Biosystems 3130 DNA Analyzer; Applied Biosystems, CA, USA) using the BigDye_ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Related sequences were identified by performing sequence database searches using BLAST. Sequence data for related species were retrieved from GenBank. Phylogenetic analysis was performed using CLUSTAL W software. Phylogenetic analysis by 16S rDNA sequencing (1500 bp) showed a various similarity between the strains (Fig 1). Table 1 shows that the most frequent genus found was Micromonospora sp. (30 isolates) followed by Streptomyces sp. (15 isolates), Actinomadura sp. (8 isolates), Microbispora sp. (6 isolates), and Polymorphospora sp. (2 isolates). Analysis of sequence of 16S rDNA from the 61 Fig 1 Phylogenetic trees showing the relationship between the isolates and related microorganism in the order Actinomycetales based on 16S rRNA sequences. Bar : 1% estimated sequence divergence. 0.01 Microbiol Indones 45 isolated Actynomycetes strain indicated that a total 5 strains were novel Actinomycetes strains exhibiting low similarity (<98%) with their close relatives (Table 2). To compare and confirm the identification by genetic analysis, these isolated Actynomycetes strains were identified by morphological examination. The colonies were categorized by microscope observation. From a total of 61 isolates of Actinomycetes, 15 isolates were identified as Streptomyces (spore chains with coiling, spiral and looped) (Fig 2A), 8 isolates Actinomadura (spore chains were straight and open hooked) (Fig 2B), 6 isolates Microbispora (longitudinal pair of spores on aerial mycelium) (Fig 2C), and 30 isolates Micromonospora (clusters of single conidia on substrate mycelium) (Fig 2D). When the soil samples were cultured without pretreatment, the most of the colonies recovered were bacteria other than Actinomycetes, followed by S t re p t o m y c e s , f u n g i , a n d n o n - S t re p t o m y c e s Actinomycetes. When the soil was air-dried, the number of other bacteria decreased, whereas the number of Streptomyces colonies increased. Over the past year, we applied various methods to isolate rare Actinomycetes, such as: (1) Wet Method (Streptomyces 65%, Nocardia 29%, Amycolatopsis 3.4%, Kitasatospora 1.6%, and Micromonospora 0.2%.), (2) Acid Treatment (Streptomyces 89%, Kitasatospora 5.5%, Nocardia 4.5 %, Amycolatopsis 0.6%, Actinomadura 0.4%), and (3) Dry Heating (Streptomyces 95%, Nocardia 2 %, Kitasatospora 2 % , Micromonospora 0.5 % , and Amycolatopsis 0.5 %). Pretreatment with 1.5% phenol reduced the number of non-Actinomycetes bacteria while significantly increased the number of Micromonospora-like colonies (Qiu et al. 2008). Phenol treatment of soil suspension lowered the number of fungi and other bacteria, but the Actinomycetes were less affected, thus 65% of the colonies belonged to rare Actinomycetes. The phenol Sampling place Number of strain Streptomyces Micromonospora Actinomadura Microbispora Polymorphospora Pulau Seribu 8 2 5 1 0 0 Tanjun g redeb 5 0 0 1 4 0 Man okwari 21 6 11 3 0 1 Halmahera 27 7 14 3 2 1 Total 61 15 30 8 6 2 Table 1 Correlation between sampling place and isolated Actinomycetes Genus Number of strain <98% 98-99% 99-100% 100% Streptomyces 15 3 4 6 2 Micromonospora 30 0 9 19 2 Actinomadura 8 1 1 5 1 Microbispora 6 1 1 4 0 Polymorphospora 2 0 1 1 0 Total 61 5 16 35 5 Table 2 Correlation number between isolated Actinomycetes and their homologies from database Volume 6, 2012 A B C D Fig 2 Cell morphology of isolated Actinomycetes (100X). (A) Streptomyces sp.; (B) Actinomadura sp.; (C) Microbispora sp.; (D) Micromonospora sp. 46 ISTIANTO ET AL. Microbiol Indones pretreatment of the soil killed bacteria and Streptomycetes in the samples, while keeping Micromonosporae and Microbisporae a live (Hayakawa et al. 1991). Micromonospora (49.18%), Streptomyces (24.59%), Actinomadura (13.11%), Microbispora (9.83 %), and Polymorphospora (3.27 %). This effect was also suggested in previous works (Hayakawa et al.1991; Hayakawa, 2008; Kim et al. 1994). Phenols have been reported to exhibit antibacterial activities with distinguished characteristics in their reactivity with proteins related polyamides polymers (Haslam et al. 1996). The inhibition of microorganisms by phenolic compounds may be due to iron deprivation or hydrogen bonding with vital proteins such as microbial enzymes (Scalbert et al. 1991). The mechanism thought to be responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds possibly through reaction with sulfhydryl groups or though more nonspecific interaction with proteins. Phenolic compounds exert antimicrobial activity by injuring lipid-containing plasma membranes, causing leakage of cellular contents. The cell wall of rare Actinomycetes are rich in lipids, making them susceptible to the activity of phenol derivatives. The dominance of other bacteria and fungal contamination inhibited the colonization of Actinomycetes on isolation medium. When antifungal -1 agents such as nystatin (50 µg mL ) and cycloheximide -1 (50 µg mL ) were supplemented into the isolation medium, the number of fungi decreased. Thus, the isolation medium was supplemented with these antibiotics in succeeding experiments (Seong et al. 2001). Rare Actinomycetes' growth was disadvantaged when cultured on agar media together with other fast growing microorganism. Thus, the media for isolating them was designed to reduce growth of competitive microbes that it would not affect their growth. Hayakawa and Nonomura formulated Humic acid- Vitamin (HV) agar, a medium containing humic acid as the sole carbon and nitrogen source. Humic acid was extremely heterogeneous crosslinked polymers, which is resistant to biological decomposition. However, Actinomycetes can utilize it as nutrient source and also use it to supported sporulation, while it restricts the growth of non-filamentous bacteria colonies (Seong et al. 2001). Humic Acid Vitamin Agar (HVA) was used for the selective isolation of Actinomycetes. The black color of HVA made it suitable to determine the morphology of white Actinomycetes colonies. Rare Actinomycetes as well as Streptomyces grew well on HVA. Although the growth rate of Actinomycetes is low, discrimination of typical morphology of colonies was easy on HVA. The activation of spore germination by humic acid was considered as one of the causes that increases the number of diverse Actinomycetes colonies on HV agar (Hayakawa and Nanomuraea 1987). Phenol pretreatment as one of the improved selective isolation methods were applied successfully for isolating rare Actinomycetes from Indonesian soil. 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