Isolation and identification of a novel thermo-alkalophilic Anoxybacillus sp. strain KB4 from Kuşburnu hot spring in Turkey

Abstract

Thermophiles are able to grow at temperatures up to 110°C and at different pH thermo-acidophiles and thermo-alkalophiles and they are an interesting source of biotechnological applications. The aim of this study was to isolate thermophilic bacterium from Kuşburnu hot water spring in the town of Diyadin in Turkey. The thermophilic strain KB4 was identified by morphological, physiological, biochemical tests and 16S rRNA gene sequencing. Strain KB4 was found to be an aerobic, Gram-positive, rod shaped, motile and yellow-pigmented. Optimal growth was obtained at pH 9.0–10.0, at 55-60 oC, and at 3% w/v NaCl. Potantial of different carbon and nitrogen sources utilization were examined. The strain KB4 was able to utilize some carbon and nitrogen sources, including glucose, galactose, sucrose, maltose, arabinose, xylose, yeast extract, peptone, tryptone and casamino acids. Phylogenetic analysis based on 16S rRNA gene sequences showed that isolate KB4 was most closely related to Anoxybacillus pushchinoensis K1 T with a 98.78% sequence similarity. In addition, this study will be a guiding study in determining the diversity of thermophilic bacteria from geothermal waters of Turkey

Keywords

• Anoxybacillus,isolation,identification,thermophilic,16S rRNA gene

Kuşburnu sıcak su kaynağından yeni termo-alkalofilik Anoxybacillus sp. KB4 şusunun izolasyonu ve tanımlanması

Abstract

Termofiller 110 oC’ye kadar sıcaklıklarda ve farklı pH’larda termo-asidofil ve termo-alkalofil büyüyebilme yeteneğine sahiptirler ve biyoteknolojik uygulamaların ilgi çeken bir kaynağıdırlar. Bu çalışmanın amacı, Türkiye’deki Diyadin ilçesi Kuşburnu sıcak su kaynağından termofilik bakteri izole etmektir. KB4 suşu, morfolojik, fizyolojik, biyokimyasal testler ve 16S rRNA gen sekanslamayla tanımlanmıştır. KB4 suşu, aerobik, Gram-pozitif, çubuk şeklinde, hareketli ve sarı pigmentli olarak bulunmuştur. Optimum üreme pH 9.0-10.0’da, 55-60 oC’de ve %3 w/v NaCl konsantrasyonunda elde edilmiştir. Farklı karbon ve azot kaynaklarının kullanımı denendi. KB4 suşu, glukoz, galaktoz, sükroz, maltoz, arabinoz, ksiloz, yeast ekstrakt, pepton, tripton ve casamino asit gibi bazı karbon ve azot kaynaklarını kullanabilmektedir. 16S rRNA gen sekansına bağlı filogenetik analizler KB4 suşunun %98.78 sekans benzerliği ile Anoxybacillus pushchinoensis K1 T ’e oldukça yakın olduğu göstermektedir. Ek olarak, bu çalışma Türkiyede’ki jeothermal sulardan termofilik bakterilerin çeşitliliğini belirlemede rehber olacaktır

Keywords

• Anoxybacillus,izolasyon,tanımlama,termofilik,16S rRNA geni

References

1. Adıguzel, A., Ozkan, H., Baris, O., Inan, K., Gulluce, M., Sahin, F. 2009. Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey. Journal of Microbiological Methods. 79.321–328.
2. Arahal, D.R., Ventosa A. 2002. Moderately Halophilic and Halotolerant Species of Bacillus and Related Genera. Applications and Systematics of Bacillus and Relatives (R. C. Berkeley, M. Heyndrickx, N. Logan, & P. De Vos, Eds.). Malden, MA: Blackwell Science.
3. Atanassova, M., Derekova, A., Mandeva, R., Sjoholm, C., Kambourova, M. 2008. Anoxybacillus bogrovensis sp. nov., a novel thermophilic bacterium isolated from a hot spring in Dolni Bogrov, Bulgaria. International Journal of Systematic and Evolutionary Microbiology. 58. 2359-2362.
4. Baltacı, M.O., Genc, B., Arslan, S., Adıguzel, G., Adıguzel, A. 2016. Isolation and Characterization of Thermophilic Bacteria from Geothermal Areas in Turkey and Preliminary Research on Biotechnologically Important Enzyme Production. Geomicrobiology Journal. 1–10.
5. Belduz, A.O., Dulger, S., Demirbag, Z. 2003. Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore-forming bacterium. International Journal of Systematic and Evolutionary Microbiology. 53. 1315-1320.
6. Bergey, D.H. 1919. Thermophilic bacteria. Journal of Bacteriology. 4/4. 301-306.
7. Bergey, D.H., Krieg, N.R., Holt, J.G. 1989. Bergey's Manual of Systematic Bacteriology. Williams & Wilkins Co, Baltimore.
8. Bradley, R.H. 2009. Isolation and characterization of Anoxybacillus from alkaline hot springs and thermal soils of Yellowstone National Park. M.S., University of Nebraska at Omaha, Omaha, ABD.

9. Canganella, F., Wiegel, J. 2014. Anaerobic Thermophiles. Life 4/1. 77–104.
10. Cihan, A.C., Ozcan, B., Cokmus, C. 2011. Anoxybacillus salavatliensis sp. nov., an α-glucosidase producing, thermophilic bacterium isolated from Salavatli, Turkey. Journal of Basic Microbiology. 51/2. 136-146.
11. Cihan, A.C. 2013. Taxonomic Classification of Anoxybacillus Isolates from Geothermal Regions in Turkey by 16S rRNA Gene Sequences and ARDRA, ITS-PCR, Rep-PCR Analyses. Polish Journal of Microbiology. 62/2. 149-163.
12. Cihan, A.C., Cokmus, C., Koc, M., Ozcan, B. 2014. Anoxybacillus calidus sp. nov., a thermophilic bacterium isolated from soil near a thermal power plant. International Journal of Systematic and Evolutionary Microbiology. 64. 211-219.
13. Claus, D., Berkeley, C.W. 1986. The genus Bacillus In: Bergey’s Manual of Systematic Bacteriology. Volume 2. Sneath pHA (Ed.) Williams & Wilkins, Baltimore, ISBN: 0-683-07893-934, pp. 1105–1139.
14. Dai, J., Liu, Y., Lei, Y., Gao, Y., Han, F., Xiao, Y., Peng, H. 2011. A new subspecies of Anoxybacillus flavithermus ssp. yunnanensis ssp. nov. with very high ethanol tolerance. FEMS Microbiology Letter. 320. 72-78.
15. Deep, K., Poddar, A., Das, S.K. 2013. Anoxybacillus suryakundensis sp. nov, a Moderately Thermophilic, Alkalitolerant Bacterium Isolated from Hot Spring at Jharkhand, India. PLoS ONE 8/12. e85493.
16. De Clerck, E., Rodriguez-Diaz, M., Vanhoutte, T., Heyrman, J., Logan, N.A., De Vos, P. 2004. Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov., isolated from contaminated gelatin batches. International Journal of Systematic and Evolutionary Microbiology. 54. 941-946.
17. Derekova, A., Sjİholm, C., Mandeva, R., Kambourova, M. 2008. Anoxybacillus rupiensis sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria). Extremophiles 11. 577-583.
18. Dulger, S., Demirbag, Z., Belduz, A.O. 2004. Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov. International Journal of Systematic and Evolutionary Microbiology. 54. 1499-1503.
19. Dussault, H.P. 1955. An improve technique for staining for halophilic bacteria. Journal of Bacteriology 70. 484-485.
20. Ercan Akkaya, S., Kıvan, M. 2008. Isolation and Identification Methods of Gr(+) Bacils Living In Hot Springs. Afyon Kocatepe University Journal of Sciences and Engineering. 2. 61-70.
21. Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package), version 3.5.1., Department of Genetics, University of Washington, Seattle, USA.
22. Filippidou, S., Jaussi, M,. Junier, T., Wunderlin, T., Roussel-Delif, L., Jeanneret, N., Vieth-Hillebrand, A., Vetter, A., Regenspurg, S., Johnson, S.L., McMurry, K., Gleasner, C.D., Lo, C.C., Li, P., Vuyisich, M., Chain, P.S., Junier, P. 2015. Genome sequence of Anoxybacillus geothermalis strain GSsed3, a novel thermophilic endospore-forming species. Genome Announce. 3/3. e00575-15.
23. Genc, B., Nadaroglu, H., Adıguzel, A., Baltacı, O. 2015. Purification and characterization of an extracellular cellulase from Anoxybacillus gonensis O9 isolated from geothermal area in Turkey. Journal of Environmental Biology. 36. 1319-1324.
24. Gül-Güven, R., Güven, K., Poli, A., Nicolaus, B. 2008. Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov., a thermophilic bacterium isolated from a hot spring in Batman. The Journal of General and Applied Microbiology. 54. 337-354.
25. Inan, K., Belduz, A.O., Canakci, S. 2011. Isolation and characterization of xylanolytic new strains of Anoxybacillus from some hot springs in Turkey. Turkish Journal of Biology. 35. 529-542.
26. Inan, K., Belduz, A.O., Canakci, S. 2013. Anoxybacillus kaynarcensissp. nov., a moderately thermophilic, xylanase producing bacterium. Journal of Basic Microbiology. 53. 410-419.
27. Kevbrin, V.V., Zengler, K., Lysenko, A.M., Wiegel, J. 2006. Anoxybacillus kamchatkensis sp. nov., a novel thermophilic facultative aerobic bacterium with a broad pH optimum from the Geyser valley, Kamchatka. Extremophiles 9. 391-398.
28. Kikani, B.A., Singh, S.P. 2012. The stability and thermodynamic parameters of a very thermostable and calcium-independent α- amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1. Process Biochemistry. 47. 1791-1798.
29. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J. 2012. Introducing EzTaxon: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. International Journal of Systematic and Evolutionary Microbiology. 62. 716-721.
30. Logan, N.A. 2002. Modern Methods for Identification. Applications and Systematics of Bacillus and Relatives (R. C. Berkeley, M. Heyndrickx, N. Logan, & P. De Vos, Eds.). Malden, MA: Blackwell Science.
31. Marmur, J.A. 1961. Procedure for the isolation of deoxyribonucleic acid from micro-organisms. Journal of Molecular Biology. 3/2. 208-IN201.
32. Meintanis, C., Chalkou, K.I., Kormas, K.A. (2008). Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the diff erentiation of species within the genus Geobacillus. Letters in Applied Microbiology. 46. 395-401.
33. Namsaraev, Z.B., Babasanova, O.B., Dunaevsky, Y.E., Akimov, V.N., Barkhutova, D.D., Gorlenko, V.M., Namsaraev, B.B. 2011. Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, Central Mongolia. Microbiology. 79. 491-499.
34. Patel, J.B. 2001. 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. The Journal of Molecular Diagnostics. 6. 313-321.
35. Pikuta, E., Lysenko, A., Chuvilskaya, N., Mendrock, U., Hippe, H., Suzina, N., Nikitin, D., Osipov, G., Laurinavichius, K. 2000. Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov. International Journal of Systematic and Evolutionary Microbiology. 50. 2109-2117.
36. Pikuta, E., Cleland, D., Tang, J. 2003. Aerobic growth of Anoxybacillus pushchinoensis K1T: emended descriptions of A. pushchinoensis and the genus Anoxybacillus. International Journal of Systematic and Evolutionary Microbiology. 53. 1561- 1562.
37. Poli, A., Esposito, E., Lama, L., Orlando, P., Nicolaus, G., Appolonia, F., Gambacorta, A., Nicolaus, B. 2006. Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica). Systematic and Applied Microbiology. 29. 300-307.
38. Poli, A., Romano, I., Cordella, P., Orlando, P., Nicolaus, B., Ceschi Berrini, C. 2011. Anoxybacillus thermarum sp. nov., a novel thermophilic bacterium isolated from thermal mud in Euganean hot springs, Abano Terme, Italy. Extremophiles. 13. 867–874.
39. Rainey, F.A., Fritze, D., Stackebrandt, E. 1994. The Phylogenetic diversty of thermophilic members of the genus Bacillus as revealed by 16S rRNA analysis. FEMS Microbiol Letter. 115. 205-212.
40. Rothschild, L.J., Mancinelli, R.L. 2001. Life in extreme environments. Nature. 409. 1092-1101.
41. Rosselló-Mora, R. 2005. Updating prokaryotic taxonomy. J Bacteriol 187. 6255-6257. Rothschild LJ, Mancinelli RL (2001) Life in Extreme Environments. Nature. 409. 1092-1101.
42. Sayeh, R., Roussel, Gannoun, Birrien, Hamdi, and Prieur, R.L. 2007. Anoxybacillus tunisiense sp. nov, a new thermophilic facultative http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=480415. bacterium isolated from a geothermal hot spring in Tunisia.
43. Woese, C.R., Winker, S., Gutell, R.R. 1990. Architecture of ribosomal RNA: constraints on the sequence of ‘tetraloops’. Proceedings of the National Academy of Sciences of the United States of America. 87. 8467-8471.
44. Yavuz, E., Gunes, H., Harsa, S., Yenidunya, A.F. 2004. Identification of extracellular enzyme producing thermophilic bacilli from Balcova (Agamemnon) geothermal site by ITS rDNA RFLP. Journal of Applied Microbiology. 97. 810-817.
45. Yumoto, I., Kikue, H., Kawahara, T., Yoshinobu, N., Hidetoshi, O., Hidetoshi, M., Yuji, Y., Kenji, N., Tamotsu, H. 2004. Anoxybacillus voinovskiensis sp. nov., a moderately thermophilic bacterium from a hot spring in Kamchatka. International Journal of Systematic and Evolutionary Microbiology. 54. 1239-1242.
46. Yücel Şengün, İ., Kılıç, G. 2016. Isolation, identification and current taxonomy of acetic acid bacteria. Biological Diversity and Conservation. 9/1. 154-162
47. Zhang, C.M., Huang, X.W., Pan, W.Z., Zhang, J., Wei, K.B., Klenk, H.P., Tang, S.K., Li, W.J., Zhang, K. 2011. Anoxybacillus tengchongensis sp. nov. and Anoxybacillus eryuanensis sp. nov., facultatively anaerobic, alkalitolerant bacteria from hot springs. International Journal of Systematic and Evolutionary Microbiology. 61. 118-122.
48. Zhang, X.Q., Zhang, Z.L., Wu, N., Zhu, X.F., Wu, M. 2013. Anoxybacillus vitaminiphilus sp. nov., a strictly aerobic and moderately thermophilic bacterium isolated from a hot spring. International Journal of Systematic and Evolutionary Microbiology. 63. 4064-4071.