International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 30 Characterization of Culturable Thermophilic Actinobacteria from Livingston Island, AntarcticaGousterova A., Paskaleva D. and Vasileva-Tonkova E.* Department of Applied Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 26, 1113 Sofia, BULGARIAAvailable online at: www.isca.in, www.isca.me Received 22nd October 2013, revised 21st November 2013, accepted 30th January 2014Abstract Thermophilic microorganisms in Antarctica are poorly investigated. This paper reports partial characterization and biosynthetic abilities of 26 thermophilic actinobacteria isolated from soil and penguin excrements samples from Livingston Island, Antarctica. About 15% of the tested Antarctic actinobacteria were able to tolerate up to 4% NaCl in the growth medium. We found that all strains are sensitive to eight, and 77% to ten of the 12 tested antibiotics suggesting relatively low anthropogenic impact in this Antarctic region. The Antarctic actinobacteria were tested for hydrolytic enzymes activity, antibiotic and hemolytic activity. It was found that all strains were able to hydrolyze starch, 81% - tributirin, and 65% - casein. All Antarctic actinobacteria demonstrated hemolytic activity, and about 27% - antimicrobial activityagainst some common bacterial pathogens. The results obtained revealed promising strains producers of industrially important thermostable enzymes and antibiotic compounds. Keywords: Thermophilic actinobacteria, antarctica, antibiotic susceptibility, antimicrobial activity, hemolytic activity, hydrolytic enzymes activity. IntroductionActinomycetes are one of the most widely distributed groups of Gram positive, mainly aerobic, filamentous bacteria. They are known for their metabolic versatility enabling them to survive even under extreme environmental conditions. Many actinomycetes are ecologically important and are used for production of antibiotics and enzymes1-3. Microorganisms isolated from extreme environments have been found to be promising producers of enzymes and metabolites with novel properties4-6. In recent years, researchers have shown great interest in thermophilic actinomycetes because of their economical potential in useful biological processes such as biodegradation and waste treatment7-9, and in production of antibiotics and enzymes4, 10-12. Thermophilic microorganisms are especially suitable for industrial application due to their rapid growth rate and reduced risk of contamination13. Actinomycetes have been isolated from a number of Antarctic environments14,15. In Antarctica, the presence of thermophilic microorganisms including actinomycetes has been reported16-18, which possibly due to several geothermal regions in Antarctica. Only few reports have shown the presence of thermophilic bacteria on this continent including volcanic Deception Island16-22. Previously, different ecological types of microorganisms including thermophilic actinomycetes were isolated in the Institute of Microbiology from different samples collected in the region of Livingston Island, and a culture collection was created23,24. In the present research, 26 thermophilic actinomycetes from this collection were characterized and their biosynthetic abilities investigated aiming to detect promising producers of thermostable industrially relevant enzymes and antimicrobial compounds. Material and Methods Collection of samples: Samples of soil and penguin’s excrements were collected near the Bulgarian scientific station “St. Kliment Ohridski” in Livingston Island (62°36S 60°30W), South Shetland Islands, Antarctica, during several Bulgarian Antarctic Research Expeditions since 1998 year. The samples were shipped to the laboratory under ice and stored frozen at –20°C in sterile containers, until processing. Isolation of thermophilic actinomycetes: Thermophilic actinomycetes used in this study were isolated previously in the Institute of Microbiology from soil (14 isolates) and penguin excrements (12 isolates) samples collected at Livingston Island, using peptone-corn steep extract agar (PCA) medium at 55°C, as described previously23,24. All isolated colonies were obtained in pure form by a series of regular subculture carried out on PCA. The identity of isolated cultures was confirmed using conventional methods of the morphological and cultural characteristics of each organism. The strains were stored at +4°C in PCA tubes and transferred after every three months onto fresh medium. The effect of temperature and NaCl: The Antarctic actinomycetes were tested for their growth in PC liquid medium at different temperatures. After inoculation, the strains were cultivated for 24 h under shaking at temperatures 30, 40, 45, 50, International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 31 55, 60, 65 and 70°C. Growth of the strains was measured by monitoring the turbidity at 570 nm.Salt tolerance of the strains was tested on PCA medium containing 3, 4, 5 and 6% (w/v) NaCl. After inoculation, the tubes were incubated at 50°C for 3 days. Antibiotic susceptibility assay: Susceptibility of the Antarctic actinomycetesto 13 antibiotics was tested using the conventional disk diffusion assay. The following antibiotics were tested: erythromycin, gentamicin, amikacin, tobramycin, lincomycin, tetracycline, chloramphenicol, vancomycin, novobiocin, ciprofloxacin, ampicillin and cefazolin. Disks impregnated with known amounts of antibiotics (BUL BIO, NCIPD, Ltd, Sofia, Bulgaria, table 1) were placed on the surface of the inoculated PCA plates. The plates were then incubated at 45°C for 24 h and the diameter of the inhibition zones was measured (including the diameter of the disks). Antimicrobial and hemolytic activity assay: The Antarctic actinomycete strains were screened for antibiotic activity using conventional well diffusion assay as described previously25. Aliquots of cell suspensions (100 µl) were added into wells (6 mm in diameter) cut in PCA plates previously seeded (1% v/v) with the indicator cultures: Bacillus subtilis ATCC 6633, Micrococcus luteus, Pseudomonas aeruginosa NBIMCC 1390 and Xanthomonas oryzae. After incubation for 24 h at 45°C, the diameter of the growth inhibition zones was measured.For extracellular hemolytic activity testing, each actinomycete culture was streaked onto blood agar plates. After 24 h incubation at 45°C, the zones of hemolysis of erythrocytes were measured. Hydrolytic enzymes activity assay: The production of extracellular hydrolytic enzymes by the Antarctic actinomycetes was determined by the agar well diffusion assay using specific substrates. After inoculation of each isolate on agar, the plates were incubated at 45°C for 24 h. Zones of clearing around the colonies on respective specific media were used as an indication of the presence of the tested enzyme activity. The production of extracellular protease was determined using 30% (v/v) skim-milk (caseinase activity). The -amylase activity was determined after growth of the strains on 1% (w/v) soluble starch and detected as a clear halo surrounding colonies after adding iodine reagent26. Lipolytic activity was detected by tributirin degradation on PCA medium supplemented with 1% (v/v) tributirin. Table-1 Growth temperature and antibiotics resistance profiles of thermophilic actinobacteria from Livingston Island, Antarctica Actinomycete code Source type Optimum growth temperature (°C) Antibiotic Cp G Am Tb Nb L T A C V E Cfz Inhibition zone (mm in diameter) 3A excrements 40-55 R (-) S(21) S(26) I(20) R (-) R(7) S(45) S(30) S(30) S(25) S(25) S(37) 6A excrements 40 I(18) S(21) S(35) S(23) R (-) S(22) S(55) I(20) S(27) S(45) S(25) S(45) 9A excrements 45 R(10) I(20) S(45) S(28) S(21) R(12) S(40) S(26) S(27) S(30) S(27) S(50) 10A soil 45-55 R(10) S(26) S(38) S(28) R (-) I(16) S(40) S(40) S(30) S(30) S(28) S(40) 11A soil 50 R(10) S(30) S(39) S(28) S(21) I(18) S(50) S(40) S(25) S(30) S(35) S(40) 12A soil 40-55 R (7) I(18) S(39) S(28) R(7) I(18) S(47) S(40) S(30) S(30) S(35) S(50) 13A soil 40 R (7) S(26) S(36) S(30) R (-) S(21) S(40) S(40) S(32) S(30) S(35) S(45) 14A soil 45-55 R(10) S(30) S(36) S(22) R (-) I(20) S(38) S(40) S(37) S(38) S(37) S(38) 15A excrements 40-55 R(12) S(44) S(45) S(25) R (-) I(18) S(40) S(45) S(45) S(45) S(45) S(45) 16A excrements 45-60 R(10) S(30) S(36) I(20) R (-) I(16) S(40) S(40) S(40) S(39) S(39) S(41) 17A excrements 55 R (-) S(30) S(41) S(25) R (-) S(35) S(40) S(35) S(30) S(23) S(35) S(42) 18A excrements 40-50 R(10) S(26) S(40) S(42) R (-) S(28) S(50) S(37) S(30) S(27) S(30) S(50) 20A excrements 40-55 R (-) S(29) S(35) S(22) R (-) I(16) S(35) S(30) S(40) S(22) S(25) S(40) 22A excrements 40 R (-) S(22) S(35) S(22) R (-) I(18) S(43) S(31) S(26) S(22) S(26) S(45) 23A excrements 50 I(16) S(38) S(40) S(35) R(10) S(38) S(50) S(30) S(35) S(26) S(25) S(45) 24A excrements 40 R (-) S(23) S(32) S(21) R (-) S(39) S(40) S(38) S(25) I(20) I(20) S(45) 25A excrements 40 R(8) S(38) S(40) S(25) R (-) S(25) S(50) S(40) S(35) S(26) S(32) S(50) 26A soil 40 R (-) S(30) S(40) R (-) R (-) S(28) S(45) S(40) S(32) S(30) S(45) S(50) 29A soil 45-55 R (-) S(39) S(50) S(50) R(9) R(10) S(50) S(35) S(50) S(50) S(30) S(50) 31A soil 40-45 R(8) S(30) S(28) S(32) R(10) R (-) S(52) S(36) S(35) S(26) S(40) S(50) 33A soil 40-50 R(8) S(27) S(32) R(8) R(9) R(10) S(45) S(45) S(45) S(43) S(45) S(45) 34A soil 40-45 R (-) S(30) S(46) S(24) R (-) I(18) S(45) S(34) S(29) S(34) S(27) S(43) 35A soil 40-55 R(10) S(25) S(40) I(20) R(8) S(30) S(40) S(39) S(40) S(39) S(45) S(45) 36A soil 40-60 R(10) S(32) S(58) S(25) R (-) I(20) S(45) S(40) S(40) S(30) S(30) S(45) 37A soil 40 R(11) S(26) S(36) S(22) R (-) R(11) S(40) S(32) S(32) S(25) S(30) S(50) 39A soil 45 R(10) S(30) S(31) S(21) R (-) S(25) S(50) S(50) S(50) S(50) S(40) S(55) Degree of susceptibility: S, sensitive ( 21 mm); I, intermediate (16-20 mm); R, resistant ( 15 mm). Cp, ciprofloxacin (5 µg); G, gentamicin (10 µg); Am, amikacin (30 µg); Tb, tobramycin (10 µg); Nb, novobiocin (5µg); L, lincomycin (15 µg); T, tetracycline (30 µg); A, ampicillin (10 µg); C, chloramphenicol (30 µg); V, vancomycin (30 mg); E, erythromycin (15 µg); Cfz, cefazolin (30 µg) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 32 Results and Discussion Growth temperature range and halotolerance: We found thatall Antarctic actinomycetes are thermophilic in that they grew at 55°C27. The optimum temperature for growth of most strains ranges between 40 to 55°C (table 1). The ash layers originating from the volcanic activity on the neighboring Deception Island are typical of the glaciology of Livingston Island. Thermal springs, hot soils and fumarolic emissions in Deception Island are evidence of its continuing activity, which provides excellent conditions for thermophilic bacteria21,22Therefore, it is likely that thermophilic microorganisms are transported by ash particles from Deception Island to Livingston Island. We found that all tested actinomycete strains tolerate up to 3% NaCl in the growth medium, and four strains (6A, 9A, 10A and 22A) tolerate up to 4% NaCl. It could suggest that halotolerance of microbial isolates is due to permanent impact of high salinity sea water during formation of ground microcenoses in maritime Antarctica. Antibiotic susceptibility: The Antarctic actinobacteria were tested for antibiotic resistance as a measure of human impactin this Antarctic region.All strains were found to be sensitive to most of the 12 tested antibiotics (table 1). All cultures showed high sensitivity to cefazolin, tetracycline, amikacin and chloramphenicol (inhibition zone range 37-55 mm, 38-55 mm, 26-58 mm and 25-50 mm, respectively). 96% of the strains were sensitive to ampicillin, vancomycin and erythromycin (inhibition zone range 26-50, 22-50 and 25-45 mm, respectively), 92% were sensitive to gentamicin (inhibition zone range 21-44 mm), and about 80% were sensitive to tobramycin (inhibition zone range 21-50 mm). About 92% of the cultured actinomycete strains were highly resistant only to ciprofloxacin and novobiocin, 23% - to lincomycin, and less than 8% - to tobramycin. The presence of antibiotic-resistant strains is widespread in the environment irrespective of the human use of antibiotics and may arise in response to various impacts. In some cases, antibiotic resistance can due to the presence of plasmids able to be horizontally transferred from one bacterium to another, even occasionally between phylogenetically distant bacteria, which greatly contributed to the widespread dissemination of antibiotic resistance genes in the environment28-30. Transfer of resistance to antimicrobial agents could be an essential mechanism of bacteria to adapt and survive in extreme environments31. Analysis and isolation of bacterial strains from areas with limited human activity could be a good indicator of the occurrence of anthropogenic impact on industry-free and clean natural areas, such as the Antarctic region32. Siebert et al.33 have found that some of the bacteria isolated from Antarctic sand stone in McMurdo valley were resistant to one or more antibiotics. Multiple antibiotics strains of environmental bacteria were also found in the ice core from the Greenland34, in the Arctic permafrost subsoil in Siberia35, and in King George Island, Antarctica36. We found that the majority of the tested Antarctic actinobacteria are sensitive to most antibiotics used, which suggest relatively low anthropogenic impact in this Antarctic region. Antimicrobial activity: The Antarctic actinobacteria were screened for antimicrobial activity against some common bacterial pathogens. The results showed relatively low potential of the tested strains to produce at the used conditions antimicrobial compounds (table 2). Only one strain inhibited the growth of two indicator cultures, and six strains showed antimicrobial activity against one of the target cultures. Strains 14A, 34A and 36A showed significant inhibition zones (in the range 26-30 mm) against Bacillus subtilis. Three strains (14A, 16A and 36A) exhibited antimicrobial activity against X. oryzae, and two strains (10A and 17A) inhibited the growth of P. aeruginosa. Biosynthesis of antimicrobial compounds is a strategy of many bacteria to survive in the environment and compete with other microorganisms for resources37. A large number of actinomycetes have been isolated and screened in the past decades, accounting for 70-80% of relevant secondary metabolites available commercially2,38. The resistance problem demands discovery of new antimicrobial agents effective against resistant pathogenic bacteria and fungi. Isolation of microorganisms from unusual environments such as sediments from deep sea water, hyper saline areas, hot sulfur springs, glacier regions, are expected to be potential producers of novel secondary metabolites39. Actinomycetes that produce antibiotics have also been isolated from Antarctica40,41. The secondary metabolism of thermophilic microorganisms is poorly understood, and thermophilic actinobacteria from Antarctica are poorly explored for antagonistic activity42-44. In the present study, we detected thermoactinomycete strains promising as potential producers of antimicrobial compounds: strains 13A, 34A and 36A showing antibiotic activity against B. subtilis; 10A and 17A exhibiting antibiotic activity against P. aeruginosa. Strains 14A, 16A and 36A showing antibiotic activity against phytopathogenic strain X. oryzae are prospective for application in the agriculture for plant protection. Hemolytic activity: All tested thermophilic actinobacteria exhibited relatively high hemolytic activity on blood agar plates (table 2). Of them, six strains showed broad hemolytic zone in the range 30-35 mm, 18 strains - in the range 20-28 mm, and two strains - in the range 16-18 mm. Hemolysis followed on blood agar plates has been widely used to screen microorganisms for production of surface-active compounds45. Due to their amphiphilic character, surfactants often induce hemolysis at a given concentration46. Surface-active compounds of microbial origin are of biotechnological importance due to their potential application in different industries47. The search for different types of surface-active compounds is important since their use in International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 33 industry requires different physicochemical properties. To the best of our knowledge, there are no reports on surfactants production by thermophilic actinobacteria from Antarctica. Six Antarctic thermophilic actinobacteria, 10A, 13A, 14A, 16A, 34A and 36A, showing hemolytic activity together with antimicrobial activity can be suggested as new potential surfactant-producing strains. Hydrolytic enzymes activity: The Antarctic actinomycetes were screened for proteolytic, lipolytic and amylolytic activity on solid media. All strains showed broad zones of hydrolysis of starch; lipase activity on tributirin was detected in 81% of the strains, and caseinase activity -in 65% of the strains (table 3). Microbial extracellular enzymes play important role in degradation and utilization of a wide range of organic polymers48. Patterns of enzyme activities can be a useful tool for assessing organic matter turnover in ecosystems49. Proteases,-amylases and lipases are among the most important enzymes of great significance in present-day biotechnology, and several thermostable enzymes from thermophilic actinomycetes have been reported10,50. Thermophilic actinomycetes isolated from Antarctica have also screened for hydrolytic enzymes production51. In the present study, we detected several promising thermophilic actinobacteria that could be applied for production of industrially important thermostable proteases, -amylases and lipases. ConclusionThe results obtained from this study revealed perspective Antarctic thermophilic actinobacteria, which can be promising producers of industrially important thermostable enzymes and bioactive metabolites. Estimated low antibiotic resistance in the tested group suggests relatively low anthropogenic impact in the region of Livingston Island. Table-2 Antimicrobial and hemolytic activity of thermophylic actinobacteriafrom Livingston Island, Antarctica Actinomycete code Inhibition zone (mm in diameter) Hemolytic activity (mm) Bacillus subtilisXanthomonas oryzae Pseudomonas aeruginosa Micrococcus luteus 3A - - - - 25 6A - - - - 26 9A - - - - 25 10A w - 15 - 22 11A - - - - 30 12A - - w w 18 13A 30 - - - 26 14A w 16 - - 25 15A - - w - 25 16A w 20 - - 20 17A w - 17 - 16 18A w w - - 28 20A - - w - 35 22A - - - - 27 23A - - - - 20 24A - - w w 20 25A - - - - 25 26A - - - - 26 29A - - - - 29 31A - - - - 30 33A - - - - 25 34A 30 - - - 22 35A - - - - 24 36A 26 15 - - 34 37A - - - - 30 39A - - - - 26 Symbols: -, no zone detected; w, weak zone 1 mm International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 34 Table-3Hydrolytic enzymes activity of thermophylic actinobacteria from Livingston Island, Antarctica,tested on solid media (zones, mm in diameter) Actinomycete code Protease (skim-milk) Amylase (starch) Lipase (tributirin) 3A + + + + + + + 6A + + + + + + 9A + + + + + - 10A - + + + - 11A + + + + + + + + 12A + + + + + + + 13A - + + + + + 14A + + + + + + + 15A + + + + + - 16A + + + + + + 17A - + + + + 18A - + + + 20A + + + + + + 22A - + + + + + + 23A + + + + + + + + 24A - + + + + + 25A + + + + + + + 26A + + + + + + 29A + + + + + + + 31A + + + + + + + 33A - + + + + + 34A + + + + + + + + + 35A + + + + + - 36A - + + + + 37A - + + + + 39A + + + + + - Symbols: +, 15 mm; + +, 16-29 mm; + + +, 30 mm; -, no zone detected AcknowledgementThe authors are thankful the Bulgarian Science Fund of the Ministry of Education, Youth and Science, for financial support of this work (Contract DNTS 01/1-2012). References 1.Mc Carthy A.J. and Williams S.T., Actinomycetes as agents of biodegradation in the environment – A review, Gene, 115, 189-192 (1992)2.Baltz R H., Renaissance in antibacterial discovery from actinomycetes, Curr. Opin. Pharmacol., , 557-563 (2008) 3.Su S.S., Tian L., Chen G., Li Z.Q., Xu W.F. and Pei Y.H., Two new compounds from the metabolites of a marine-derived actinomycete Streptomyces cavourensis YY01-17, J. Asian Nat. Prod. Res., 15, 265-269 (2013) 4.Yallop C.A., Edwards C. and Williams S.T., Isolation and growth physiology of novel thermoactinomycetes, J. Appl. Microbiol., 83, 685-692 (1997) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 35 5.Lee L.H., Cheah Y.K., Sidik S.M., Ab Mutalib N.S., Tang Y.L., Lin H.P. and Hong K., Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production, World J. Microbiol. Biotechnol., 28, 2125-2137 (2012)6.Mahajan G.B. and Balachandran L., Antibacterial agents from actinomycetes - a review, Front. Biosci., (Elite Ed.), , 240-253 (2012)7.Eduards C., Isolation properties and potential applications of thermophilic actinomycetes, Appl. Biochem. Biotechnol., 42, 161-179 (1993)8.Gousterova A., BraikovaD., Goshev I., Christov P., Tishinov K., Vasileva-Tonkova E., Haertlé . and Nedkov P., Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis, Lett. Appl. Microbiol., 40, 335-340 (2005)9.Gousterova A., Nustorova M., Paskaleva D., Naydenov M., Neshev G. and Vasileva-Tonkova E., Assessment of feather hydrolysate from thermophilic actinomycetes for soil amendment and biological control application, Int. J. Environ. Res., , 1065-1070 (2011)10.Kikani B.A., Shukla R.J. and Singh S P., Biocatalytic potential of thermophilic bacteria and actinomycetes, In: Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, (d., A. Mendez-Vilas), ©FORMATEX, 1000-1007 (2010)11.Waksman S.A., Schatz A. and Reynolds D.M., Production of antibiotic substances by actinomycetes. Ann. N.Y. Acad. Sci., 1213, 112-124 (2010) 12.Prakash D., Nawani N., Prakash M., Bodas M., Mandal A., Khetmalas M. and Kapadnis B., Actinomycetes: a repertory of green catalysts with a potential revenue resource., Biomed. Res. Int., 2013, article ID 264020 (2013)13.Tendler M.D. and Burkholder P.R., Studies on the thermophilic actinomycetes. I. Methods of cultivation, Appl. Microbiol., , 394-399 (1961)14.Moncheva P., Toshkov S., Dimitrova N., Chipeva V., Antonova-Nikolova S. and Bogatzevska N., Characteristics of soil actinomycetes from Antartica, J. Cult. Coll.,3 -14, (2002) 15.Mavengere N.R., Isolation, identification and characterization of novel actinomycetes from Antarctic soil samples, M.Sc. Thesis, University of the Western Cape, 111 (2008)16.Nicolaus B., Marsiglia F., Esposito E., Trincone A., Lama L., Sharp R., di Prisco G. and Gambacorta A., Isolation of five strains of thermophilic eubacteria in Antarctica, Polar Biol., 11, 425-429 (1991)17.Nicolaus B., Improta R., Manca M. C., Lama L., Esposito E. and Gambacorta A., Alicyclobacilli from an unexplored geothermal soil in Antarctica: Mount Rittmann, Polar Biol., 19, 133-141 (1998)18.Logan N.A., Lebbe L., Hoste B., Goris J., Forsyth G., Heyndrick M., Murray B.L., Syme N., Wynn-Williams D.D. and De Vos P., Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich Archipelago, with the proposal of Bacillus fumarioli sp. nov., Int. J. Syst. Evol. Microbiol., 50, 1741-1753 (2000)19.Allan R.N., Lebbe L., Heyrman J., De Vos P., Buchanan C. J. and Logan N.A., Brevibacillus levickii sp. nov. and Aneurinibacillus terranovensis sp. nov., two novel thermoacidophiles isolated from geothermal soils of northern Victoria Land, Antarctica, Int. J. Syst. Evol. Microbiol., 55, 1039-1050 (2005)20.Poli A., Esposito E., Lama L., Orlando P., Nicolaus G., de Appolonia F., Gambacorta A. and Nicolaus B., Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica), Syst. Appl. Microbiol., 29, 300-307 (2006)21.Llarch A., Logan N.A., Castellvi J., Prieto M. J. and Guinea J., Isolation and characterization of thermophilic Bacillusspp. from geothermal environments on Deception island and south Shetland archipelago. Microb. Ecol., 34, 58-65 (1997) 22.Muńoz P.A., Flores P.A., Boehmwald F.A. and Blamey J.M., Thermophilic bacteria present in a sample from Fumarole Bay, Deception Island, Antarc. Sci., 23, 549-555 (2011)23.Gushterova A., Noustorova M., Tzvetkova R., Spassov G. and Chipeva V., Investigations of the microora in penguin’s excrements in the Antarctic. Bulg. Antarc. Res, Life Sci., , 1-7, Pensoft Publishers, Soa-Moscow (1999) 24.Noustorova M., Gushterova A., Tzvetkova R. and Chipeva V., Investigations of the microora in glaciers from the Antarctic, Bulg. Antarc. Res, Life Sci, , 8-12, Pensoft Publishers, Soa-Moscow (1999)25.Gesheva V. and Vasileva-Tonkova E. Production of enzymes and antimicrobial compounds by halophilic Antarctic Nocardioides sp. grown on different carbon sources, World J. Microbiol. Biotechnol., 28, 2069-2076 (2012) 26.Dhawale M.R., Wilson J.J., Khachatourians G.G. and Ingledew W.M., Improved method for detection of starch hydrolysis, Appl. Environ. Microbiol., 44, 747-750 (1982)27.Agre N.S., Taxonomy of thermophilic actinomycetes, PhD Thesis, Puschino, Moscow, 5-10 (in Russian) (1986)28.Davison J., Genetic exchange between bacteria in the environment, Plasmid42, 73-91 (1999) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 30-36, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 36 29.Dale J.W. and Park S., Molecular genetics of bacteria, 4th edition, John Wiley & Sons Inc., Chichester, UK (2004)30.Michaud L., Di Cello F., Brilli M., Fani R., Giudice A.L., and Bruni V., Biodiversity of cultivable psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea, Antarctica), FEMS Microbiol.Lett.,230, 63-71 (2004)31.Herreros M.A., Sandoval H., González L., Castro J.M., Fresno J.M. and Tornadijo M.E., Antimicrobial activity and antibiotic resistance of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese), Food Microbiol., 22, 455-459 (2005)32.Miller R.V., Gammon K. and Day M.J., Antibiotic resistance among bacteria isolated from seawater and penguin fecal samples collected near Palmer Station, Antarctica, Can. J. Microbiol., 55, 37-45 (2009)33.Siebert J., Hirsch P., Hoffmann B., Gliesche C.G., Peissl K. and Jendrach M., Cryptoendolithic microorganisms from Antarctic sandstone of Linnaeus Terrace Asgard Range: diversity, properties and interactions, Biodivers. Conserv., , 1337-1363 (1996)34.Miteva V.I., Sheridan P.P. and Brenchley J.E., Phylogenetic and physiological diversity of microorganisms isolated from a deep Greenland glacier ice core, Appl. Environ. Microbiol., 70, 202-213 (2004)35.Mindlin S., Soina V., Petrova M. and Gorlenko Z., Isolation of antibiotic resistance bacterial strains from Eastern Siberia permafrost sediments, Russian J. Genetics, 44, 27-34 (2008)36.WongC., TamH., AliasS., GonzálezM., González-RochaG. and Domínguez-YévenesM., Pseudomonas and Pedobacter isolates from King George Island inhibited the growth of foodborne pathogens, Polish Polar Res., 32, 3-14 (2011)37.O’Brien A., Sharp R., Russell N. and Roller S., Antarctic bacteria inhibit growth of food-borne microorganisms at low temperatures, FEMS Microbiol. Ecol., 48, 157-167 (2004)38.Kumar N., Singh R.K., Mishra S.K., Singh A.K. and Pachouri U.C., Isolation and screening of soil actinomycetes as source of antibiotics active against bacteria, Int. J. Microbiol. Res., , 12-16 (2010)39.Tiwari K. and Gupta R.K., Rare actinomycetes: a potential storehouse for novel antibiotics, Crit. Rev. Biotechnol., 32, 108-132 (2012)40.Nedialkova D. and Naidenova M., Screening the antimicrobial activity of actinomycete strains isolated from Antarctica, J. Cult. Coll., , 29-35 (2004)41.Gesheva V. and Vasileva-Tonkova E., Production of enzymes and antimicrobial compounds by halophilic Antarctic Nocardioides sp. grown on different carbon sources, World J. Microbiol. Biotechnol., 28, 2069-2076 (2012)42.Schuurmans D.M., Olson B.H. and San Clemente C.L., Production and isolation of thermoviridin, an antibiotic produced by Thermoactinomyces viridis n. sp., Appl. Microbiol., , 61-66 (1956)43.Moppett C.E., Don T.D. T., Johnson F. and Coronelli C., Structure of thermorubin A, the major orange-red antibiotic of Thermoactinomyces antiobioticus J. Am. Chem. Soc., 94, 3269-3272 (1972)44.Kanoh K., Matsuo Y., Adachi K., Imagawa H., Nishizawa M. and Shizuri Y., Mechercharmycins A and B, cytotoxic substances from marine-derived Thermoactinomyces sp. YM3-251, J. Antib., 58, 289-292 (2005)45.Moran A.C., Martnez M.A. and Sińeriz F., Quantification of surfactin in culture supernatants by hemolytic activity, Biotechnol. Lett., 24, 177-180 (2002)46.Vinardell M.P. and Infante M.R., The relationship between the chain length of non-ionic surfactants and their hemolytic action on human erythrocytes, Comp. Biochem. Physiol., Part C, 124, 117-120 (1999)47.Banat I.M., Franzetti A., Gandolfi I., Bestetti G., Martinotti M.G., Fracchia L., Smyth T.J. and Marchant R., Microbial biosurfactants production, applications and future potential, Appl. Microbiol. Biotechnol., 87, 427-444 (2010)48.Munster U. and De Haan H., The role of microbial extracellular enzymes in the transformation of dissolved organic matter in humic waters, Ecol. Studies, 133, 199-257 (1998)49.Ferrer M., Golyshina O., Beloqui A. and Golyshin P.N., Mining enzymes from extreme environments, Curr. Opin. Microbiol., 10, 207-214 (2007)50.Gomes J. and Steiner W., Extremophiles and Extremozymes, Food Technol. Biotechnol. 42, 223-235 (2004)51.Hristova I., Nedelcheva P., Gushterova A., Paskaleva D. and Krastanov A., Isolation of thermophilic actinomycetes producers of thermostable proteases, In: (Ed. A. Méndez-Vilas), Microbes in Applied Research: Current Advances and Challenges, 423-426 (2012)