ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 20 (2015): 8-15 Reveived 5 Aug. 2014 Accepted 19 Feb. 2015 Identification and characterization of two amylase producing bacteria Cellulosimicrobium sp. and Demequina sp. isolated from marine organisms Laila S.H. Al-Naamani*1, Sergey Dobretsov 1, Jamal Al-Sabahi 1 and Bassam Soussi 1 1* Laila S.H. Al-Naamani ( ) Sultan Qaboos University, College of Agricultural and Marine Sciences, Dept. of Marine Science and Fisher- ies, Box 34, Al-Khod 123, Sultanate of Oman. email: p029787@student. squ.edu.om. Introduction The sea is a unique resource with a large diversity of natural products isolated mainly from inver-tebrates, such as sponges, tunicates, bryozoans and molluscs, as well as from bacteria (Proksch et al., 2002). Symbiotic microorganisms are often proposed as the true producers of natural products isolated from marine invertebrates. Numerous examples exist in which structurally related or identical compounds have been reported from taxonomically distinct invertebrates and cultured microorganisms (Moore, 1999; Proksch et al., 2002). Recently, research has focused on marine mi- croorganisms as sources of many metabolites that have a potential to be used as antibiotics, antimicrobials, or antitumor agents and pharmaceuticals. Marine microorganisms are a valuable source of novel enzymes with ideal characteristics because of the halophilic nature of the marine bacteria (Mohapatra et al., 1998). Halophiles are microorganisms that live, grow, and multiply in highly saline environments. Moderately halophilic bacteria are able to grow over a wide range of salt concentrations from 0.4 to 3.5M. Exoenzymes from تعريف وحتديد صفات نوعني من البكرتاي )Cellulosimicrobium sp( و )Demequina sp (املفرزة لألميليز مت عزهلما من كائنات حبرية ليلى النعماين1* وسريجي دوبريتسوف1 ومجال الصباحي1 وبسام سوسي1 Abstract. Marine sources have been known to yield novel compounds with a wide range of bioactivity with various commercial applications. In this study, the abilities of bacteria isolated from eight marine organisms to produce α-amy- lase were examined. All eight organisms were found to harbor amylase producing bacteria. Two bacterial species isolat- ed from the green alga Ulva rigida and the sponge Mycale sp. were further identified and their α-amylases were purified and characterized. The bacterial species isolated from U. rigida and Mycale sp. were identified by DNA sequencing as Cellulosimicrobium sp. and Demequina sp., respectively. Cellulosimicrobium sp. obtained maximum cell growth and amylase production at 29ºC and in the presence of lactose as a carbon source. Optimal cell growth and amylase produc- tion by Demequina sp. was observed at 35ºC. While lactose enhanced cell growth of Demequina sp., maximum amylase production was found when fructose and glycerol were the available sources of carbon. Both strains grew better in the presence of tryptone, whilst peptone stimulated amylase production. Maximal cell growth and amylase production by both of the strains was found at a medium salinity of 3% NaCl. Keywords: Natural products, enzymes, amylase, bacteria. املســتخلص:تـُْعَرف املصــادر البحريــة بتوفريهــا ملركبــات جديــدة هلــا تأثــريات حيويــة واســعة النطــاق وتطبيقــات جتاريــة متعــددة. مت فحــص القــدرة علــى إفــراز األميليــز ألفــا مــن قبــل بكرتيــات مت عزهلــا مــن مثانيــة كائنــات حبريــة. وجــد أن مجيــع الكائنــات البحريــة الثمــان حتتــوي علــى بكرتيــات منتجــة لألمليــز. كذلــك مت عــزل نوعــن مــن البكرتيــا مــن الطحلــب األخضــرUlva rigida ومــن األســفنجية Mycale sp ومت تعريفهمــا، وتنقيــة إنزمياهتمــا Cellulosimicrobium علــى أهنمــا Mycale sp ومــن Ulva rigida األميليزيــة وحتديــد صفاهتــا. مت تعريــف النوعــن البكرتيــن اللذيــن مت عزهلمــا مــن Cellulosimicrobium حققــت البكرتيــا DNA بالتــوايل، وذلــك مــن خــال تقنيــة الرتتيــب التسلســلي للحمــض النــووي Demequina sp و sp sp. أقصــى منــو للخايــا و أقصــى انتــاج لألميليــز عنــد 29 درجــة مئويــة يف وجــود الاكتــوز كمصــدر كربــوين، بينمــا كان أفضــل منــو للخايــا و أفضــل إنتــاج لألميليــز لنــوع Demequina sp عنــد 35 درجــة مئويــة. ورغــم أن الاكتــوز قــد حسَّــن النمــو اخللــوي ل Demequina sp ، إال أن أقصــى إنتــاج لألميليــز يف هــذا النــوع قــد ُوِجــد عندمــا كان الفركتــوز واجليســرول مهــا املصدريــن املتاحــن للكربــون. ولقــد منــا النوعــان بشــكل أفضــل يف وجــود الرتيبتــون، .NaCl % 3 بينما حفز الببتون إنتاج األميليز. ُوِجد أيضا أن اقصى منو للخايا وإنتاج لألميليز كان يف كلي النوعن عند مســتوى امللوحة املتوســط الكلمات املفتاحية: املنتجات الطبيعية،األنزميات، األميليز 9Research Article Al-Naamani, Dobretsov, Al-Sabahi, Soussi these microorganisms with polymer degrading ability at low water activities are of interest in many harsh indus- trial processes where concentrated salt solutions would inhibit many enzymatic conversions. Furthermore, most halobacterial enzymes are known to be thermotolerant and remain stable at room temperature for a long time (Chakraborty et al., 2008(b); Mohapatra et al., 1998). Free-living bacteria and bacteria attached to marine sediments usually excrete large amounts of extracellular enzymes for the hydrolysis of intractable macromole- cules (Mohapatra et al., 2003). The enzymes secreted by the bacteria remain associated with the cell and are re- leased slowly to the environment to aid in the nutrition of their host. Until now, researchers have isolated sever- al enzymes such as acetylcholinesterase, amylase, ure- thanase, cellulase, and alginate and pectin lyases from bacteria and fungi associated with marine sponges and algae (Mohapatra et al., 2003). Although many micro- organisms produce amylase, the ones most commonly used for industrial production are Bacillus subtilis, Ba- cillus licheniformis, Bacillus amyloliquifaciens and As- pergillus niger (Vidyalakshmi, 2009). The sea around Oman is rich in marine organisms that could be a potential source of natural products that are not yet exploited. The overall objectives of this study was to isolate bacteria associated with Omani marine organisms, study their abilities to produce amylase and to investigate the optimal conditions (temperature, pH and salinity, carbon and nitrogen sources) for bacterial growth and enzyme production. Materials and methods Isolation of pure cultures from marine environ- ments Samples from eight marine organisms: the red sponge Mycale sp., the tunicate red Ascidia sp., the tunicate black Ascidia nigra, the whitesponge Chalina sp., the yellow sponge Hemiasterella sp., the sea anemone Heter- actis crispa and the green alga Ulva rigida and sea water were collected from Bandar Al Khairan (depth 1-5 m), Arabian Gulf, Sultanate of Oman. The marine organisms were transferred into plastic bags immediately after col- lection and kept in ice until transferred to the laboratory. In the laboratory, the samples were transferred to coni- cal flasks containing marine broth and placed in a rota- ry shaker at 180 rpm at 29oC for about 24 to 48 hours. After incubation, a loop full of each sample mixture was streaked onto nutrient agar and incubated for another 24 hours. The bacterial colonies were separated and sub- cultured several times until pure bacterial cultures were obtained. Screening of amylolytic enzymes production To check the amylolytic activity of the isolated bacte- rial strains, each pure bacterial culture was cultured in marine broth and incubated for about 24 hours. Subse- quently, about 20 µl of each bacterial culture was dropped on Starch agar and incubated for 24 to 48 hours. After incubation, amylase production was determined by the development of a colourless halo zone surrounding the bacterial colonies when Lugol’s iodine solution (1% io- dine in 2% potassium iodide w/v) was added to the plate (Fig.1). The diameters of the digestion zones were mea- sured to check the intensity of the enzyme activity of the bacteria. Two of the fastest growing bacteria with largest starch digestion zones were chosen for further identifi- cation and analysis. Bacterial identification The two bacterial strains were first identified by gram staining, API Coryne and negative staining (TEM test). Then, further identification was done by DNA sequenc- ing and fatty acid analysis profile. DNA sequencing The bacterial strains were identified by comparative analysis of their 16S rRNA gene sequences as described in Lau et al., 2002. Fragments of DNA sequences (about 700 bp) obtained from individual primers were assem- bled using the Sequencher® software package (Gene Codes, USA). The closest match to the 16S rDNA gene sequence of the respective bacteria was retrieved by comparison with data from the GenBank (www.ncbi. nlm.nih.gov). Bacterial fatty acids analysis The sample preparation and analysis for bacterial fatty acid determinations were performed as described by Sasser, 1990 using hexane mixed with methyl tert butyl ether for the extraction of the fatty acids from the bac- terial strains. Figure 1. Starch hydrolysis by amylase producing bacteria visualized by Lugol’s-iodine coloration. 10 SQU Journal of Agricultural and Marine Sciences, 2015, Volume 19, Issue 1 Identification and characterization of two amylase producing marine bacteria GC analysis One μl of each extract of bacterial fatty acids in hex- ane- methyl tert butyl ether mixture was injected into a Gas Chromatograph (Agilent 6890 N) instrument equipped with Ultra-1 (crosslinked methyl silicone gum) column, HP catalogue No. 19091A-102 (25m long x 0.2mm i.d x 0.33μl film thickness). Analytical conditions The injector and flame ionization detector (FID) tem- peratures were set at 250ºC and 260ºC, respectively. The oven temperature was programmed from 150ºC (2 min- utes) to 250ºC (5 minutes) at 4ºC/min. Helium was used as the carrier gas with a linear velocity of 20 cm/sec. The duration of the reaction was set for 40 minutes. Data analysis Retention times of the unknown bacterial fatty acids were compared to a mixture of standard bacterial fat- ty acid methyl esters (BAME) (Supelco, Catalogue No. 47080). Sensitivity of bacterial strains to antibiotics The bacterial strains were tested for their susceptibility to the following antibiotics: Ampicillin, Streptomycin, Chloramphenicol, and Penicillin G. Concentrations of 1 mg/ml and 10 mg/ml were used for each antibiotic. The test was performed using a disc diffusion bioassay. The bacterial colonies of the two strains were evenly distributed on nutrient agar using sterile swabs. Small discs (diameter = 5.5 mm and surface area = 1 cm2) con- taining each type of antibiotic were placed in the me- dia inoculated with bacteria and incubated for about 24 hours. Presence of clear zones around the discs corre- spondent to the bacterial growth inhibition was checked and their diameters were measured in millimetres with a ruler. Optimization of bacterial yield and amylase Production The effect of temperature, salinity and available carbon and nitrogen sources on bacterial growth was tested by measuring the optical density of the samples after in- cubation. Amylase production was determined by the amount of maltose produced as an end product of starch hydrolysis by amylase. Bacterial yield test Marine broth containing different NaCl concentration (0%, 3% 5%, 10% and 15% (w/v)) were prepared to test the effect of salinity. Solutions containing 1% of a spe- cific carbon source namely starch, glucose, fructose, lac- tose, glycerol, sucrose or maltose along with 0.1% yeast extract were prepared in sea water. Solutions containing 1% peptone or tryptone as nitrogen sources were also prepared and supplemented with 0.1% yeast extract mixed with sea water. Test tubes containing 10 ml of each solution was inoculated with the bacterial cultures except the blank and incubated at 29°C for 24h. To test the effect of temperature, test tubes containing 10ml marine broth inoculated with the bacterial cultures were incubated at different temperatures (5°C, 29°C, 35°C and 60°C) for 24h. After incubation, the blank control was used to zero the spectrophotometer and the absorbance at 600 nm was measured for all the samples. Determination of bacterial amylase production The culture solutions prepared in the bacterial yield test, as above, were centrifuged at 10,000 rpm for 5 minutes at 15ºC to separate the bacterial cells from the broth. 1 ml of cell free supernatant of each sample was removed and added to tubes containing 1 ml of 1% starch solu- tion. 1 ml of distilled water with 1 ml of 1% starch solu- tion was added to the blank tube. The tubes were incu- bated at 37oC for 12 minutes. After incubation, 1 ml of maltose colour reagent (1% 3,5-dinitrosalicylic acid, Figure 2. (a) Cellulosimicrobium sp. isolated from the green alga Ulva rigida after negative staining (TEM test) under elec- tron microscope at 25000x magnifications. (b) Demequina sp. isolated from sponge Mycale sp. after negative staining (TEM test) under electron microscope at 15000x magnification. 11Research Article Al-Naamani, Dobretsov, Al-Sabahi, Soussi 0.4 M NaOH, 1.06 M sodium potassium tartrate) was added to each tube and heated for 15 min at 100ºC. The heated mixtures were cooled immediately in ice. Then, 9 ml distilled water was added to each tube. The blank was used to zero the spectrophotometer and the absorbance at 540 nm was measured for all the samples. Results Bacterial isolation and enzyme screening All eight organisms tested were found to harbour en- zyme producing bacteria. More than 150 species of marine bacteria were isolated and more than 50 species were screened for enzyme production (Figure 1). Pure colonies have been isolated and two species among the fastest growing strains with the highest ability to hydro- lyze starch were selected from the green alga Ulva rigida (Strain I) and the red sponge Mycale sp. (Strain II). Bacterial strain identification The two strains showed characteristics of being Gram positive. According to API Coryne test, Strain I was identified from the ApiwebTM identification software as Cellulosimicrobium cellulans with 87.3% accuracy whereas Strain II was identified as Cellulomonas spp. with 99.9% accuracy. According to partial 16S RNA se- quences, the two bacterial strains were identified as fol- lows: Strain I: Cellulosimicrobium sp. with 98% accuracy (Bacteria; Actinobacteria; Actinobacteridae; Actinomy- cetales; Micrococcineae; Promicromonosporaceae) Strain II: Demequina sp. with 98% accuracy (Bacte- ria; Actinobacteria; Actinobacteridae; Actinomycetales; Micrococcineae; Cellulomonadaceae) The TEM showed the structure of the two strains under different magnifications (Figure 2). Gas chro- matographic analysis showed that the two bacterial strains had quite similar fatty acid compositions except for the absence of three types of bacterial fatty acids in Demequina sp. (Table 1). This can be explained by fact that genes Cellulosimicrobium and Demequina are high- ly similar. Previously Demequina was re-classified from Cellulomonas (Yi et al., 2007; Ue et al. 2011). This fact supports our API Coryne test data that identified strain II as Cellulomonas sp. Susceptibility to antibiotics Both of the strains were highly susceptible to Chloram- phenicol at concentrations of 1 mg/ml and 10 mg/ml with larger inhibition zones, specifically diameters of 30±2.8 mm and 32.5±3.5 mm for Cellulosimicrobium sp. and Demequina sp., respectively, at a concentration of 10 mg/ml. The two bacterial isolates were inhibited by 10 mg/ml Streptomycin whereas they were not affected by the same antibiotic at the lower concentration of 1 mg/ml. No inhibitory effect on the growth of both iso- lates was observed by Ampicillin and Penicillin G. The susceptibility of members of the genus Cellulo- monas to different antibiotics was studied by Funke et al. (1995). All the clinical isolates of Cellulomonas species were found to be strongly susceptible to Ciprofloxacin, Clindamycin, Erythromycin and Gentamicin. However, they were less susceptible to Penicillin G, Rifampin, Tet- racycline and Vancomycin. It was found that the bacterium Cellulosimicrobium cellulans grew well on standard synthetic media and was susceptible to a variety of antibiotics, including Pen- icillin, Macrolides, and Glycopeptides (Heym, 2005). Yoon (2007) isolated Cellulosimicrobium terreum from soil and found it to be susceptible to Streptomycin (50 μg), Chloramphenicol (100 μg) and Penicillin G (20 U). Susceptibility to Ampicillin was observed by three clin- ical strains of Cellulosimicrobium funkei (Brown et al., 2006). All these results are totally different from what we obtained from Cellulosimicrobium sp. in our study, Table 1. Results of gas chromatography mass spectrometry (GC-MS) analysis. BAME name Ret. (Min) Cm Dq 1 Methyl Dodecanoate (C12:0) 17.42 0.24 1.9 2 Methyl tridecanoate (C13:0) 20.05 0.31 0.0 3 Methyl 2 Hydroxydodeca- noate (2-OH-C12:0) 20.38 0.05 0.0 4 Methyl 3-hydroxydecanoate (3-OH-C12:0) 21.03 2.70 1.7 5 Methyl tetradecanoate (C14:0) 22.67 8.01 4.8 6 Methyl 13-methyltetradeca- noate (i-C15:0) 24.32 1.22 3.2 7 Methyl 12-methyltetradeca- noate (α-C15:0) 24.53 0.16 0.0 8 Methyl pentadecanoate (C15:0) 25.22 2.84 2.2 9 Methyl 3-hydroxytetradeca- noate (3-OH-C14:0) 26.22 4.82 3.7 10 Methyl 14-methylpentadeca- noate (i-C16:0) 26.81 1.94 0.7 11 Methyl cis-9-hexadecenoate (C16:19) 27.16 39.52 29.8 12 Methyl hexadecanoate (C16:0) 27.68 15.24 16.4 13 Methyl 15-methylhexadeca- noate (i-C17:0) 29.22 1.58 6.6 14 Methyl heptadecanoate (C17:0) 30.1 1.27 1.3 15 Methyl trans-9-octadece- noate (C18:19) 32.17 19.85 26.7 16 Methyl octadecanoate (C18:0) 32.77 0.24 0.4 12 SQU Journal of Agricultural and Marine Sciences, 2015, Volume 19, Issue 1 Identification and characterization of two amylase producing marine bacteria since it was not affected by Ampicillin and Penicillin G and was susceptible to only higher concentrations of Streptomycin. Probably this was due to isolation of our novel strain from different environment (marine). Addi- tionally, different antibiotic susceptibility could be due to antibiotic resistance of some bacterial strains (Brown et al., 2006). Bacterial yield and amylase production Effect of temperature The results showed optimal temperature for cell growth and enzyme production between 29ºC and 35ºC for Cel- lulosimicrobium sp. and Demequina sp., respectively (Fig. 3 & 4). The two strains were inactivated at 4ºC and 60ºC which indicates the mesophilic characteristics of the two bacterial strains. This property can be related to the natural habitat of the two bacterial strains in the mesophilic sea environment. This is in accordance with what was reported by Asgher et al. (2007) and Kathire- san and Manivannan (2006) that the influence of tem- perature on amylase production is related to the growth of the microorganism. Effect of salt concentration The highest bacterial growth and enzyme production for Cellulosimicrobium sp. and Demequina sp. was obtained in the presence of 3% NaCl (w/v) (Fig. 5 & 6), which is the normal salinity of sea water. These results might be related to the nature of the bacterial origin since they were isolated from marine environments. There was no amylase production in the absence of NaCl which clearly indicates the halophilic nature of the enzymes of both strains. Cellulosimicrobium sp. retained about 9% of its growth ability at 10% salt but both strains lost their enzyme productivity at this salinity. Prakash (2009b) re- ported that halophilic enzymes require high salt concen- trations for their activity due to their adaptation to high salt concentrations while they undergo denaturation at NaCl concentrations below 1M. He also suggested that halophilic bacteria can produce enzymes that can toler- ate high salt concentrations 3-15% NaCl. Effect of carbon and nitrogen sources on bacterial yield and amylase production The maximum optical density and maltose production by Cellulosimicrobium sp. in the presence of lactose was observed (Figure 7). This indicated higher cell growth and amylase production, with this carbon source. This amount of cell yield and amylase production was much higher than what was obtained in the presence of starch, which is known as the general substrate for amylase production. Hiller et al. (1997) demonstrated a similar enhanced cell growth and α-amylase production by Ba- Figure 3. Effect of temperature on cell yield of Cellulo- simicrobium sp. and Demequina sp. (λ600nm). The data are the means of two replicates ± standard deviation. Figure 4. Effect of temperature on amylase production of Cellulosimicrobium sp. and Demequina sp. (λ540nm). The data are the means of two replicates ± standard deviation. Figure 5. EEffect of salt concentration on cell yield of Cel- lulosimicrobium sp. and Demequina sp. (λ600nm). The data are the means of two replicates ± standard deviation. Figure 6. Effect of salt concentration on amylase pro- duction of Cellulosimicrobium sp. and Demequina sp. (λ540nm). The data are the means of two replicates ± stan- dard deviation. 13Research Article Al-Naamani, Dobretsov, Al-Sabahi, Soussi cillus amyloliquefaciens from lactose. The lowest bacte- rial growth and enzyme secretion was observed in the presence of fructose. Glucose was found to yield very minimum amounts of amylases from both of the bacte- rial strains regardless of its effect on cell growth. Repres- sion of amylase synthesis by glucose was also reported in thermophilic and alkaliphilic Bacillus sp. TS-23 (Lin et al., 1998), Sulfolobus solfataricus (Haseltine et al., 1996) and Bacillus sp. isolate L1711 (Bernhardsdotter et al., 2005). Haseltine et al. (1996) justified that glucose pre- vents gene expression of α-amylase and represses its se- cretion. Although maximum cell growth of Demequina sp. was obtained in the presence of lactose, the highest enzyme production was induced by fructose and glycer- ol (Figure 8). Glycerol was reported to increase enzyme production in Bacillus sp. PS-7 (Tanyildizi, 2005) and Bacillus sp. I-3 (Goyal, 2005). Higher optical density was obtained in the presence of tryptone which indicates that it enhanced the cell growth of both strains more than peptone (Figure 9 & 10). However, more maltose was produced by the addi- tion of peptone which reflected its higher stimulation of amylase production. The decrease in the amounts of maltose as the end product of starch hydrolysis in the presence of tryptone may be due to the intolerance of the bacterial strains to 0.5% tryptone. Similar results were reported previously (Chakraborty et al. 2008a). These authors found a decrease in amylase production by Saccaropolyspora species A9 at tryptone concentra- tions beyond 0.4%. Conclusions Two amylases were isolated from the two bacteri- al strains Cellulosimicrobium sp. and Demequina sp., which were correspondingly obtained from the green Figure 7. Effect of carbon and nitrogen sources on cell yield of Cellulosimicrobium sp. and Demequina sp. (λ = 600nm). The data are the means of two replicates +1 standard deviation. Figure 8. Effect of carbon and nitrogen sources on amylase production of Cellulosimicrobium sp. and Demequina sp. (λ = 540 nm). The data are the means of two replicates +1 standard deviation. 14 SQU Journal of Agricultural and Marine Sciences, 2015, Volume 19, Issue 1 Identification and characterization of two amylase producing marine bacteria alga Ulva rigida and the sponge Mycale sp. Maximum cell yield from the two bacterial strains was obtained at temperatures between 29ºC and 35ºC and 3% salinity, which indicate that the two strains have mesophilic and halophilic properties. Addition of lactose and tryptone significantly increased the growth of both of the strains. Both strains produced highest amount of amylase at temperatures between 29ºC and 35ºC, 3% salinity and in the presence of peptone. Addition of lactose increased amylase production by Cellulosimicrobium sp., whereas supplementation of the media with fructose and glycerol resulted in maximal amylase production by Demequina sp. The present study demonstrated that all eight inves- tigated marine organisms are promising sources of amy- lases producing bacteria. This finding provides the pos- sibility to isolate more enzymes-producing bacteria and search for novel bioactive compounds from the marine environment in Oman. References DeeAsgher, M., M. J. Asad, S. U. Rahman, and R. L. Legge. 2007. A thermostable amylase from Bacillus subtilis strain for starch processing. Journal of Food Engineering 79: 950-955. Bernhardsdotter, E. C. M. J., J. D. Ng, O. K. Garriott, and M. L. Pusey. 2005. Enzymic properties of an alkaline chelator-resistant amylase from an alkaliphilic Bacil- lus sp. isolate L1711. Process Biochemistry 40:2401- 2408. Brown, J. M., A. G. Steigerwalt, R. E. Morey, M. I. Dane- shvar, L. Romero, and M. M. McNeil. 2006. Charac- terization of clinical isolates previously identified as Oerskovia turbata: proposal of Cellulosimicrobium funkei sp. nov. and amended description of the genus Cellulosimicrobium. International Journal of Sys- tematic and Evolutionary Microbiology 56: 801-804. Chakraborty, S., A. Khopade, C. Kokare, K. Mahadic, and B. Chopade. 2008a. Isolation and characteri- zation of novel amylase from marine Streptomyces. Journal of Molecular Catalysis 10: 1-31. Chakraborty, S., A. Khoqade, Rinbio, X.Y. Liu, L. Zhang, K. Mahadic and C. Kokare. 2008b. Isolation, partial purification and characterization of halophilic, alkali tolerant, thermostable, surfactant and detergent sta- ble amylase enzyme from marine Saccharoplyspora species. Marine Biotechnology 11:1-35. Heym, B., P. Gehanno, V. Friocourt, M. Bougnoux, M. Le Moal, C. Husson, J. Leibowitch, and M. Nico- las-Chanoine. 2005. Molecular detection of Cellu- losimicrobium cellulans as the etiological agent of a chronic tongue ulcer in a human immunodeficiency virus-positive patient. Journal of Clinical Microbiol- ogy 43(8): 4269-4271. Funke, G., C. P. Ramos, and M. D. Collins. 1995. Identi- fication of some Clinical Strains of CDC Coryneform Group A-3 and A-4 bacteria as Cellulomonas species and proposal of Cellulomonas hominis sp. nov. for some Group A-3 Strains. Journal of Clinical Micro- biology 33(8): 2091-2097. Goyal, N., J. K. Gupta, and S. K. Soni. 2005. A novel raw starch digesting thermostable α-amylase from Bacil- Figure 9. (A) Effect of nitrogen source on cell yield of Cel- lulosimicrobium sp. measured at λ = 600 nm. (B) Effect of nitrogen source on amylase production by Cellulosimicro- bium sp. measured at λ = 540 nm. The data are the means of two replicates ± standard deviation. Figure 10. (A) Effect of nitrogen source on cell yield of De- mequina sp. measured at λ 600 nm. (B) Effect of nitrogen source on amylase production by Demequina sp. measured at λ = 540 nm. The data are the means of two replicates ± standard deviation. 15Research Article Al-Naamani, Dobretsov, Al-Sabahi, Soussi lus sp. I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme Microbiological Technology 37: 723-734. Harumi Ue, H., Y. Matsuo, H. Kasai, and A. Yokotam. 2011. Demequina globuliformis sp. nov., Demequina oxidasica sp. nov. and Demequina aurantiaca sp. nov., actinobacteria isolated from marine environ- ments, and proposal of Demequinaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology 61(6): 1322-1329. Haseltine, C., M. Rolfsmeier, and P. Blum. 1996. The glu- cose effect and regulation of α-amylase synthesis in the hyperthermophilic archaeon Sulfolobus solfatari- cus. Journal of Bacteriology 178: 945-950. Hiller, P., D. A. J. Wase, A. N. Emergy, and G. L. Solomon 1997. Instability of -amylase production and mor- phological variation in continuous culture of Bacil- lus amyloliquefaciens is associated with plasmid loss. Process Biochemistry 32: 51-59. Kathiresan, K., and S. Manivannan. 2006. Amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. African Journal of Bio- technology 5(10): 829-832. Lau, S. C. K., K. K. W. Mak, F. Chen, and P. Y. Qian. 2002. Bioactivity of bacterial strains from marine biofilms in Hong Kong waters for the induction of larval at- tachment in the marine polychaete Hydroides ele- gans. Marine Ecology Progress Series 226: 301-310. Lin, L. L., C. C. Chyau, and W. H. Hsu. 1998. Produc- tion and properties of a raw-starch-degrading amy- lase from thermophilic and alkaliphilic Bacillus sp. TS-23. Biotechnology and Applied Biochemistry 28: 61-68. Mohapatra, B. R., U. C. Banerjee, and M. Bapuji. 1998. Characterization of a fungal amylase from Mucor sp. associated with the marine sponge Spirastrella sp. Journal of Biotechnology 60: 113-117. Mohapartra, B. R., M. Bapuji, and A. Spree. 2003. Production of industrial enzymes (Amylase, Car- boxymethy and Proteanes) by bacteria isolated from marine sedentary organisms. Journal of Acta Bio- technology 23:75-84. Moore, B. S. 1999. Biosynthesis of marine natural prod- ucts: microorganisms and Macroalgae. The Royal So- ciety of Chemistry 16: 653-674. Prakash, B., M. Vidyasagar, M. S. Madhukumar, G. Mu- ralikrishna, and K. Sreeramulu. 2009b. Production, purification and characterization of two extremely halotolerant, thermostable, and alkali- stable amy- lases from Chromohalobacter sp. TVSP 101. Process Biochemistry 44: 210-215. Proksch, P., R. Edrada, and R. Ebel. 2002. Drugs from the seas - current status and microbiological impli- cations. Applied Microbiology and Biotechnology 59(2): 125-134. Sasser. 1990-revised 2001. Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. MIDI, Technical Note #101. Vidyalakshmi, R., R. Paranthaman, and J. Indhumathi. 2009. Amylase production on submerged fermenta- tion by Bacillus spp. World Journal of Chemistry 4: 89-91. Yi, H., P. Schumann, and J. Chun. 2007. Demequina aestuarii gen. nov., sp. nov., a novel actinomycete of the suborder Micrococcineae, and reclassification of Cellulomonas fermentans Bagnara et al. 1985 as Acti- notalea fermentans gen. nov., comb. nov. Internation- al Journal of Systematic and Evolutionary Microbiol- ogy 57(1): 151-156. Yoon, J., S. Kang, P. Schumann, and T. Oh. 2007. Cel- lulosimicrobium terreum sp. nov., isolated from soil. Journal of Systematic and Evolutionary Microbiology 57: 2493-2497.