Advertisement

Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA

  • Gurdeep Rastogi
  • Geetha L. Muppidi
  • Raghu N. Gurram
  • Akash Adhikari
  • Kenneth M. Bischoff
  • Stephen R. Hughes
  • William A. Apel
  • Sookie S. Bang
  • David J. Dixon
  • Rajesh K. SaniEmail author
Original Paper

Abstract

The present study investigated the cultivable mesophilic (37°C) and thermophilic (60°C) cellulose-degrading bacterial diversity in a weathered soil-like sample collected from the deep subsurface (1.5 km depth) of the Homestake gold mine in Lead, South Dakota, USA. Chemical characterization of the sample by X-ray fluorescence spectroscopy revealed a high amount of toxic heavy metals such as Cu, Cr, Pb, Ni, and Zn. Molecular community structures were determined by phylogenetic analysis of 16S rRNA gene sequences retrieved from enrichment cultures growing in presence of microcrystalline cellulose as the sole source of carbon. All phylotypes retrieved from enrichment cultures were affiliated to Firmicutes. Cellulose-degrading mesophilic and thermophilic pure cultures belonging to the genera Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and selected cultures were studied for enzyme activities. For a mesophilic isolate (DUSELG12), the optimum pH and temperature for carboxymethyl cellulase (CMCase) were 5.5 and 55°C, while for a thermophilic isolate (DUSELR7) they were 5.0 and 75°C, respectively. Furthermore, DUSELG12 retained about 40% CMCase activity after incubation at 60°C for 8 h. Most remarkably, thermophilic isolate, DUSELR7 retained 26% CMCase activity at 60°C up to a period of 300 h. Overall, the present work revealed the presence of different cellulose-degrading bacterial lineages in the unique deep subsurface environment of the mine. The results also have strong implications for biological conversion of cellulosic agricultural and forestry wastes to commodity chemicals including sugars.

Keywords

Cellulose-degrading bacteria DUSEL Deep subsurface Thermostable enzymes Gold mine 

Notes

Acknowledgments

This research was funded by the South Dakota Governor’s program (2010) and Board of Regents grant for project title “Generating Preliminary Microbial Data on Homestake Gold Mine”. In addition, Geetha L. Muppidi acknowledges funding through the SD NASA-EPSCoR Program (NASA Grant # NCC5-588). The support of the SDSM&T’s Department of Chemical and Biological Engineering also contributed significantly to this research. We also would like to thank the anonymous reviewers whose critiques were instrumental in making our manuscript of an excellent quality.

References

  1. 1.
    Abdel-Fattah YR, El-Helow ER, Ghanem KM, Lotfy WA (2007) Application of factorial designs for optimization of avicelase production by a thermophilic Geobacillus isolate. Res J Microbiol 2:13–23CrossRefGoogle Scholar
  2. 2.
    Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2006) New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl Environ Microbiol 72:5734–5741. doi: 10.1128/AEM.00556-06 PubMedCrossRefGoogle Scholar
  3. 3.
    Baek SH, Im WT, Oh HW, Lee JS, Oh HM, Lee ST (2006) Brevibacillus ginsengisoli sp. nov., a denitrifying bacterium isolated from soil of a ginseng field. Int J Syst Evol Microbiol 56:2665–2669. doi: 10.1099/ijs.0.64382-0 PubMedCrossRefGoogle Scholar
  4. 4.
    Ben-Bassat A, Lamed R, Zeikus JG (1981) Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii. J Bacteriol 146:192–199PubMedGoogle Scholar
  5. 5.
    Benoit L, Cailliez C, Gehin A, Thirion J, Raval G, Petitdemange H (1995) Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11. Curr Microbiol 30:305–312. doi: 10.1007/BF00295506 PubMedCrossRefGoogle Scholar
  6. 6.
    Bischoff KM, Rooney AP, Li X, Liu S, Hughes SR (2006) Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnol Lett 28:1761–1765. doi: 10.1007/s10529-006-9153-0 PubMedCrossRefGoogle Scholar
  7. 7.
    Blumer-Schuette SE, Kataeva I, Westpheling J, Adams MW, Kelly RM (2008) Extremely thermophilic microorganisms for biomass conversion: status and prospects. Curr Opin Biotechnol 19:210–217. doi: 10.1016/j.copbio.2008.04.007 PubMedCrossRefGoogle Scholar
  8. 8.
    Deflaun MF, Fredrickson JK, Dong H, Pfiffner SM, Onstott TC, Balkwill DL, Streger SH, Stackebrandt E, Knoessen S, van Heerden E (2006) Isolation and characterization of a Geobacillus thermoleovorans strain from an ultra-deep South African gold mine. Syst Appl Microbiol 30:152–164. doi: 10.1016/j.syapm.2006.04.003 PubMedCrossRefGoogle Scholar
  9. 9.
    Dien BS, Cotta MA, Jeffries TW (2003) Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258–266. doi: 10.1007/s00253-003-1444-y PubMedCrossRefGoogle Scholar
  10. 10.
    Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson DM, Saar MO, Alexander S, Alexander EC Jr, Rohwer F (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7:57. doi: 10.1186/1471-2164-7-57 PubMedCrossRefGoogle Scholar
  11. 11.
    Felsenstein J (1989) PHYLIP-Phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  12. 12.
    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268. doi: 10.1351/pac198759020257 CrossRefGoogle Scholar
  13. 13.
    Hippe H, Andreesen JR, Gottschalk G (1992) The genus Clostridium—non-medical. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer K-H (eds) The prokaryotes, 2nd edn. Springer, New York, pp 1800–1839Google Scholar
  14. 14.
    Holt JG (1994) The aerobic endospore-forming rods and cocci. In: Holt JG (ed) Bergey’s manual of determinative bacteriology, 9th edn. Williams and Wilkins, Baltimore, pp 670–675Google Scholar
  15. 15.
    Inagaki F, Takai K, Hirayama H, Yamato Y, Nealson KH, Horikoshi K (2003) Distribution and phylogenetic diversity of the subsurface microbial community in a Japanese epithermal gold mine. Extremophiles 7:307–317. doi: 10.1007/s00792-003-0324-9 PubMedCrossRefGoogle Scholar
  16. 16.
    Ito S (1997) Alkaline cellulases from alkaliphilic Bacillus: enzymatic properties, genetics, and application to detergents. Extremophiles 1:61–66. doi: 10.1007/s007920050015 PubMedCrossRefGoogle Scholar
  17. 17.
    Kato S, Haruta S, Cui ZJ, Ishii M, Igarashi Y (2005) Stable coexistence of five bacterial strains as a cellulose-degrading community. Appl Environ Microbiol 71:7099–8106. doi: 10.1128/AEM.71.11.7099-7106.2005 PubMedCrossRefGoogle Scholar
  18. 18.
    Khianngam S, Tanasupawat, Lee JS, Lee KC, Akaracharanya A (2008) Paenibacillus siamensis sp. nov., Paenibacillus septentrionalis sp. nov. and Paenibacillus montaniterrae sp. nov., xylanase-producing bacteria from Thai soils. Int J Syst Evol Microbiol (in press)Google Scholar
  19. 19.
    Kublanov IV, Perevalova AA, Slobodkina GB, Lebedinsky AV, Bidzhieva SK, Kolganova TV, Kaliberda EN, Rumsh LD, Haertlé T, Bonch-Osmolovskaya EA (2008) Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera Kamchatka. Appl Environ Microbiol. doi:  10.1128/AEM.00607-08
  20. 20.
    Kumar S, Tamura K, Nei M (1993) MEGA: molecular evolutionary genetics analysis. Pennsylvania State University, University ParkGoogle Scholar
  21. 21.
    Lee YJ, Kim BK, Lee BH, Jo KI, Lee NK, Chung CH, Lee YC, Lee JW (2008) Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour Technol 99:378–386. doi: 10.1016/j.biortech.2006.12.013 PubMedCrossRefGoogle Scholar
  22. 22.
    Lu WJ, Wang HT, Yang SJ, Wang ZC, Nie YF (2005) Isolation and characterization of mesophilic cellulose-degrading bacteria from flower stalks-vegetable waste co-composting system. J Gen Appl Microbiol 51:353–360. doi: 10.2323/jgam.51.353 PubMedCrossRefGoogle Scholar
  23. 23.
    Lynd LR, Grethlein HE, Wolkin RH (1989) Fermentation of cellulosic substrates in batch and continuous culture by Clostridium thermocellum. Appl Environ Microbiol 55:3131–3139PubMedGoogle Scholar
  24. 24.
    Lynd LR, van Zyl WH, McBride JE, Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 16:577–583. doi: 10.1016/j.copbio.2005.08.009 PubMedCrossRefGoogle Scholar
  25. 25.
    Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577. doi: 10.1128/MMBR.66.3.506-577.2002 PubMedCrossRefGoogle Scholar
  26. 26.
    Madrid VM, Taylor GT, Scranton MI, Chistoserdov AY (2001) Phylogenetic diversity of bacterial and archaeal communities in the anoxic zone of the Cariaco Basin. Appl Environ Microbiol 67:1663–1674. doi: 10.1128/AEM.67.4.1663-1674.2001 PubMedCrossRefGoogle Scholar
  27. 27.
    Mahadevan SA, Wi SG, Lee DS, Bae HJ (2008) Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima). FEMS Microbiol Lett 287:205–211. doi: 10.1111/j.1574-6968.2008.01324.x PubMedCrossRefGoogle Scholar
  28. 28.
    McMullan G, Christie JM, Rahman TJ, Banat IM, Ternan NG, Marchant R (2004) Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus. Biochem Soc Trans 32:214–217. doi: 10.1042/BST0320214 PubMedCrossRefGoogle Scholar
  29. 29.
    Meintanis C, Chalkou KI, Kormas KA, Karagouni AD (2006) Biodegradation of crude oil by thermophilic bacteria isolated from a volcano island. Biodegradation 17:105–111. doi: 10.1007/s10532-005-6495-6 PubMedCrossRefGoogle Scholar
  30. 30.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. doi: 10.1021/ac60147a030 CrossRefGoogle Scholar
  31. 31.
    Onstott TC, Moser DP, Pfiffner SM, Fredrickson JK, Brockman FJ, Phelps TJ, White DC, Peacock A, Balkwill D, Hoover R, Krumholz LR, Borscik M, Kieft TL, Wilson R (2003) Indigenous and contaminant microbes in ultradeep mines. Environ Microbiol 5:1168–1191. doi: 10.1046/j.1462-2920.2003.00512.x PubMedCrossRefGoogle Scholar
  32. 32.
    Pason P, Kyu KL, Ratanakhanokchai K (2006) Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides. Appl Environ Microbiol 72:2483–2490. doi: 10.1128/AEM.72.4.2483-2490.2006 PubMedCrossRefGoogle Scholar
  33. 33.
    Rastogi G, Sani RK, Peyton BM, Moberly JG, Ginn TR (2008) Molecular studies on the microbial diversity associated with mining-impacted Coeur d’Alene River sediments. Microb Ecol. doi: 10.1007/s00248-008-9445-0
  34. 34.
    Reed DW, Fujita Y, Delwiche ME, Blackwelder DB, Sheridan PP, Uchida T, Colwell FS (2002) Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin. Appl Environ Microbiol 68:3759–3770. doi: 10.1128/AEM.68.8.3759-3770.2002 PubMedCrossRefGoogle Scholar
  35. 35.
    Rivas R, García-Fraile P, Mateos PF, Martínez-Molina E, Velázquez E (2006) Paenibacillus cellulosilyticus sp. nov., a cellulolytic and xylanolytic bacterium isolated from the bract phyllosphere of Phoenix dactylifera. Int J Syst Evol Microbiol 56:2777–2781. doi: 10.1099/ijs.0.64480-0 PubMedCrossRefGoogle Scholar
  36. 36.
    Sani RK, Geesey G, Peyton BM (2001) Assessment of lead toxicity to Desulfovibrio desulfuricans G20: influence of components of lactate C medium. Adv Environ Res 5:269–276. doi: 10.1016/S1093-0191(00)00061-7 CrossRefGoogle Scholar
  37. 37.
    Schiraldi C, De Rosa M (2002) The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol 20:515–521. doi: 10.1016/S0167-7799(02)02073-5 PubMedCrossRefGoogle Scholar
  38. 38.
    Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer grogram for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506. doi: 10.1128/AEM.71.3.1501-1506.2005 PubMedCrossRefGoogle Scholar
  39. 39.
    Somerville C (2006) The billion-ton biofuels vision. Science 312:1277. doi: 10.1126/science.1130034 PubMedCrossRefGoogle Scholar
  40. 40.
    Tai SK, Lin HP, Kuo J, Liu JK (2004) Isolation and characterization of a cellulolytic Geobacillus thermoleovorans T4 strain from sugar refinery wastewater. Extremophiles 8:345–349. doi: 10.1007/s00792-004-0395-2 PubMedCrossRefGoogle Scholar
  41. 41.
    Thamthiankul S, Suan-Ngay S, Tantimavanich S, Panbangred W (2001) Chitinase from Bacillus thuringiensis subsp. Pakistani. Appl Microbiol Biotechnol 56:395–401. doi: 10.1007/s002530100630 PubMedCrossRefGoogle Scholar
  42. 42.
    Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 6:9Google Scholar
  43. 43.
    Vargas-García MC, Suárez-Estrellaa F, Lópeza MJ, Morenoa J (2007) In vitro studies on lignocellulose degradation by microbial strains isolated from composting processes. Int Biodeterior Biodegrad 59:322–328. doi: 10.1016/j.ibiod.2006.09.008 CrossRefGoogle Scholar
  44. 44.
    Viamajala S, Peyton BM, Richards LA, Petersen JN (2006) Solubilization, solution equilibria, and biodegradation of PAH’s under thermophilic conditions. Chemosphere 66:1094–1106. doi: 10.1016/j.chemosphere.2006.06.059 PubMedCrossRefGoogle Scholar
  45. 45.
    Wang CM, Shyu CL, Ho SP, Chiou SH (2008) Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39. Lett Appl Microbiol 47:46–53. doi: 10.1111/j.1472-765X.2008.02385.x PubMedCrossRefGoogle Scholar
  46. 46.
    Wenzel M, Schönig I, Berchtold M, Kämpfer P, König H (2002) Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis. J Appl Microbiol 92:32–40. doi: 10.1046/j.1365-2672.2002.01502.x PubMedCrossRefGoogle Scholar
  47. 47.
    Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2009

Authors and Affiliations

  • Gurdeep Rastogi
    • 1
  • Geetha L. Muppidi
    • 1
  • Raghu N. Gurram
    • 1
  • Akash Adhikari
    • 1
  • Kenneth M. Bischoff
    • 2
  • Stephen R. Hughes
    • 2
  • William A. Apel
    • 3
  • Sookie S. Bang
    • 1
  • David J. Dixon
    • 1
  • Rajesh K. Sani
    • 1
    Email author
  1. 1.Department of Chemical and Biological EngineeringSouth Dakota School of Mines and TechnologyRapid CityUSA
  2. 2.Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization ResearchUS Department of AgriculturePeoriaUSA
  3. 3.Biological Systems DepartmentIdaho National LaboratoryIdaho FallsUSA

Personalised recommendations