Physio-Biochemical Characterization and 16S rRNA Sequencing of Thermophilic Geobacillus sp. stain DB2 from Dibekli Hot Spring, Turkey

Year 2017, Volume: 7 Issue: 1, 63 - 71, 31.03.2017

Fatma Matpan Bekler

Abstract

Geothermal sites are habitat for thermophilic microorganisms. In this study, a novel aerobic, rod
shaped, motile and Gram-positive thermophilic bacterium DB2 was isolated from the hot water in Dibekli in
the town of Diyadin in Turkey and it was identifed by morphological, physiological, biochemical tests and 16S
rRNA gene sequencing. Optimal cultivation was obtained at pH 6.0–6.5 and 60 oC. Strain DB2 was positive for
catalase activity, nitrate reduction, Methyl red test and starch hydrolysis, while it was negative for oxidase, urease,
gelatine and casein hydrolysis. Phylogenetic analysis based on 16S rRNA gene sequences showed that isolate DB2
was most closely related to Geobacillus subterraneus subsp. aromaticivorans Ge1(T) with a 98.97% sequence
similarity. This novel strain can be used in biotechnological application.

Keywords

16S rRNA, Geobacillus, identifcation, thermophilic

Türkiye’deki Dibekli Sıcak Su Kaynağından Termoflik Geobacillus sp. DB2 Suşunun Fizyo-Biyokimyasal Karakterizasyonu ve 16S rRNA Sekanslaması

Abstract

Jeotermal kaynaklar termofllerin habitatlarındandır. Bu çalışmada aerobik, çubuk şeklinde, hareketli
ve Gram-pozitif termoflik DB2 suşu Türkiye’deki Diyadin ilçesi Dibekli sıcak su kaynağından izole edilerek
morfolojik, fzyolojik, biyokimyasal testler ve 16S rRNA gen sekanslamayla tanımlandı. Optimal kültivasyon
pH 6.0–6.5’da, 60 oC’de elde edildi. DB2 suşunun katalaz, nitrat redükleme, Metil red testi ve nişasta hidrolizi
pozitif, oksidaz, üreaz aktivitesi, jelatin ve kazein hidrolizi negatif olarak belirlendi. 16S rRNA gen sekansına bağlı
flogenetik analizler DB2 suşunun %98.97 sekans benzerliği ile Geobacillus subterraneus subsp. aromaticivorans
Ge1(T)’e oldukça yakın olduğunu göstermektedir. Bu yeni tür, biyoteknolojik uygulamalarda kullanılabilir
özelliktedir

Keywords

16S rRNA geni, Geobacillus, tanımlama, termoflik

References

• Abd Rahman RNZR, Leow TC, Salleh AB, Basri M, 2007. Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia. BMC Microbiology, 7: 77.
• Banat IM, Marchant R, Rahman TJ, 2004. Geobacillus debilis sp. nov., a novel obligately thermophilic bacterium isolated from a cool soil environment, and reassignment of Bacillus pallidus to Geobacillus pallidus comb. nov. International Journal of Systematic and Evolutionary Microbiology, 54: 2197-2201.
• Bryanskaya AV, Rozanov AS, Slynko NM, Shekhovtsov SV, Peltek SE, 2015. Geobacillus icigianus sp. nov., a thermophilic bacterium isolated from a hot spring. International Journal of Systematic and Evolutionary Microbiology, 65: 864-869.
• Çağlayan M, Bilgin N, 2011. Cloning and Sequence Analysis of Novel DNA Polymerases from Thermophilic Geobacillus Species Isolated from Hot Springs in Turkey: Characterization of a DNA Polymerase I from Geobacillus kaue Strain NB. Applied Biochemistry and Biotechnology, 165: 1188-1200.
• Cihan AC, Ozcan B, Tekin N, Cokmus C, 2011. Geobacillus thermodenitrificans subsp. calidus, subsp. nov., a thermophilic and α-glucosidase producing bacterium isolated from Kizilcahamam, Turkey. The Journal of General and Applied Microbiology, 57(2): 83-92.
• Coorevits A, Dinsdale AE, Halket G, Lebbe L, De Vos P, Van Landschoot A, Logan NA, 2012. Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly 'thermoglucosidasius'); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov. International Journal of Systematic and Evolutionary Microbiology, 62: 1470-1485.
• Dussault HP, 1955. An improve technique for staining for halophilic bacteria. Journal of Bacteriology, 70: 484-485.
• Ercan Akkaya S, Kıvanç M, 2008. Isolation and Identification Methods of Gr(+) Bacils Living In Hot Springs. Afyon Kocatepe University Journal of Sciences and Engineering, 02: 61-70.
• Felsenstein J, 1993. PHYLIP (Phylogeny Inference Package), version 3.5.1., Department of Genetics, Unıversity of Washington, Seattle, USA.
• Fortina MG, Mora D, Schumann P, Parini C, Manachini PL, Stackebrandt E, 2001. Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov. International Journal of Systematic and Evolutionary Microbiology, 51: 2063-2071.
• Futterer O, Angelov A, Liesegang H, Gottschalk G, Schleper C, Schepers B, 2004. Genome sequence of Picrophilus torridus and its implications for life around pH 0. Proceedings of the National Academy of Sciences of the United States of America, 101:9091-6.
• Giaveno MA,MSa Urbieta, Ulloa JR, EGl Toril, Donati ER, 2013. Physiologic versatility and growth flexibility as the main characteristics of a novel thermoacidophilic Acidianus strain isolated from Copahue geothermal area in Argentina. Microbial Ecology, 65:336–46.
• Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, 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.
• Kuisiene N, Raugalas J, Chitavichius D, 2004. Geobacillus lituanicus sp. nov. International Journal of Systematic and Evolutionary Microbiology, 54: 1991-1995.
• Marmur JA, 1961. Procedure for the isolation of deoxyribonucleic acid from micro-organisms. Journal of Molecular Biology, 3(2): 208-IN201.
• Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, Lysenko AM, Petrunyaka VV, Osipov GA, Belyaev SS, Ivanov MV, 2001. Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th. International Journal of Systematic and Evolutionary Microbiology, 51: 433-446.
• Nazina TN, Lebedeva EV, Poltaraus AB, Tourova TP, Grigoryan AA, Sokolova DS, Lysenko AM, Osipov GA, 2004. Geobacillus gargensis sp. nov., a novel thermophile from a hot spring, and the reclassification of Bacillus vulcani as Geobacillus vulcani comb. nov. International Journal of Systematic and Evolutionary Microbiology, 54: 2019-2024.
• Nazina TN, Sokolova DS, Grigoryan AA, 6 other authors, 2005. Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species. Systematic and Applied Microbiology, 28: 43-53.
• Poli, A., Laezza, G., Gul-Guven, R., Orlando, P. & Nicolaus, B. 2011. Geobacillus galactosidasius sp. nov., a new thermophilic galactosidaseproducing bacterium isolated from compost. Systematic and Applied Microbiology, 34: 419–423.
• Poli A, Guven K, Romano I, Pirinccioglu H, Gul Guven R, Euzeby JPM, Matpan F, Acer O, Orlando P, Nicolaus B, 2012. Geobacillus subterraneus subsp. aromaticivorans subsp. nov., a Novel Thermophilic and Alkaliphilic Bacterium Isolated from a Hot Spring in Sirnak, Turkey. Journal of General and Applied Microbiology The Microbiology Research Foundation. 58: 437-446.
• Rainey FA, Fritze D, Stackebrandt E, 1994. The Phylogenetic diversty of thermophilic members of the genus Bacillus as revealed by 16S rRNA analysis. FEMS Microbiology Letters, 115: 205-212.
• Rastogi G, Bhalla A, Adhikari A, Bischoff KM, Hughes SR, Christopher LP, et al. 2010. Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresource Technology, 101: 8798–806.
• Schäffer C, Franck WL, Scheberl A, Kosma P, McDermott TR, Messner P, 2004. Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov. International Journal of Systematic and Evolutionary Microbiology, 54: 2361-2368. Sung MH, Kim H, Bae JW, Rhee SK, Jeon CO, Kim K, Kim JJ, Hong SP, Lee SG, Yoon JH, Park YH, Baek DH, 2002. Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from hay compost. International Journal of Systematic and Evolutionary Microbiology, 52: 2251-2255.
• Urbieta MS, Donati ER, Chan KG, Shahar S, Sin LL, Goh KM, 2015. Thermophiles in the genomic era: Biodiversity, science, and applications. Biotechnology Advances, 33: 633–647.