Applied Microbiology and Biotechnology

, Volume 92, Issue 3, pp 641–652 | Cite as

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

  • Xiongjun Shao
  • Babu Raman
  • Mingjun Zhu
  • Jonathan R. Mielenz
  • Steven D. Brown
  • Adam M. Guss
  • Lee R. LyndEmail author
Bioenergy and biofuels


Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1–2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.


Clostridium thermocellum Ethanol tolerance Genome sequencing Strain adaptation Mutations 



The authors are grateful for the support provided by funding grants from the BioEnergy Science Center (BESC), a US Department of Energy (DOE) Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science and Mascoma Corporation. The authors are also grateful for the genome sequencing support provided by the DOE Joint Genome Institute (JGI). Oak Ridge National Laboratory is managed by University of Tennessee UT-Battelle LLC for the Department of Energy under contract no. DE-AC05-00OR22725.

Supplementary material

253_2011_3492_MOESM1_ESM.pdf (175 kb)
ESM 1 (PDF 175 kb)


  1. Bahari L, Gilad Y, Borovok I, Kahel-Raifer H, Dassa B, Nataf Y, Shoham Y, Lamed R, Bayer EA (2011) Glycoside hydrolases as components of putative carbohydrate biosensor proteins in Clostridium thermocellum. J Ind Microbiol Biotechnol 38:825–832. doi: 10.1007/s10295-010-0848-9 CrossRefGoogle Scholar
  2. Brown SD, Yang SY, Guss A, Yang Z, Karpinets T, Klingeman DM, Tschaplinski TJ, Giannone RJ, Hettich RL, Engle NL, Dice L, Rodriguez Jr M, Mielenz J, Cottingham R, Hauser L, Gorin A, Davison BH, Palumbo AV, Lynd L, Keller M (2010) Analysis of the ethanol stress and tolerance mechanisms for Clostridium thermocellum through the integration of genome resequencing and systems biology studies. In: 110th ASM General Meeting, San Diego, CA, USA, May 2010Google Scholar
  3. Burdette DS, Jung S-H, Shen G-J, Hollingsworth RI, Zeikus JG (2002) Physiological function of alcohol dehydrogenases and long-chain (C30) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus. Appl Environ Microbiol 68(4):1914–1918CrossRefGoogle Scholar
  4. Ding J, Huang X, Zhang L, Zhao N, Yang D, Zhang K (2009) Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 85(2):253–263CrossRefGoogle Scholar
  5. Giovanni-Donnelly RD, Kolbye SM, Dipaolo JA (1967) The effect of carbamates on Bacillus subtilis. Mutat Res-Fund Mol M 4(5):543–551CrossRefGoogle Scholar
  6. Gowen CM, Fong SS (2010) Genome-scale metabolic model integrated with RNAseq data to identify metabolic states of Clostridium thermocellum. Biotechnol J 5(7):759–767CrossRefGoogle Scholar
  7. Herrero AA, Gomez RF (1980) Development of ethanol tolerance in Clostridium thermocellum: effect of growth temperature. Appl Environ Microbiol 40(3):571–577Google Scholar
  8. Herrero AA, Gomez RF, Roberts MF (1982) Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum. Biochim Biophys Acta 693(1):195–204CrossRefGoogle Scholar
  9. Ingram LO (1990) Ethanol tolerance in bacteria. Crit Rev Biotechnol 9(4):305–319CrossRefGoogle Scholar
  10. Isenberg HD, Schatz A, Angrist AA, Schatz V, Trelawny GS (1954) Microbial metabolism of carbamates II. Nitrification of urethan by Streptomyces nitrificans. J Bacteriol 68(1):5–9Google Scholar
  11. Jeffries TW, Jin Y-S (2000) Ethanol and thermotolerance in the bioconversion of xylose by yeasts. Adv Appl Microbiol 47:221–268CrossRefGoogle Scholar
  12. Kahel-Raifer H, Jindou S, Bahari L, Nataf Y, Shoham Y, Bayer EA, Borovok I, Lamed R (2010) The unique set of putative membrane-associated anti-σ factors in Clostridium thermocellum suggests a novel extracellular carbohydrate-sensing mechanism involved in gene regulation. FEMS Microbiol Lett 308(1):84–93CrossRefGoogle Scholar
  13. Ladisch M, Flatt J, Lynd L, Rajgarhia V, Wenger K, Hogsett DA, Wyman CE, Belcher A, van Rooyen J, Sivasubramanian MS, DiMasi D, Shao X, Draeger J, Kim Y, Ximenes E, Mosier N (2009) Development and deployment of consolidated bioprocessing for production of ethanol. In: 31st symposium on biotechnology for fuels and chemicals, “”San Francisco, CA, USA, 3–6 May 2009Google Scholar
  14. Lamed R, Zeikus JG (1980) Ethanol production by thermophilic bacteria: relationship between fermentation product yields of and catabolic enzyme activities in Clostridium thermocellum and Thermoanaerobium brockii. J Bacteriol 144(2):569–578Google Scholar
  15. Li H, Ruan J, Durbin R (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18(11):1851–1858CrossRefGoogle Scholar
  16. Lovitt RW, Shen GJ, Zeikus JG (1988) Ethanol-production by thermophilic bacteria—biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum. J Bacteriol 170(6):2809–2815Google Scholar
  17. Lynd LR, Wyman CE, Gerngross TU (1999) Biocommodity engineering. Biotechnol Progr 15(5):777–793CrossRefGoogle Scholar
  18. Lynd LR, Baskaran S, Casten S (2001) Salt accumulation resulting from base added for pH control, and not ethanol, limits growth of Thermoanaerobacterium thermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture. Biotechnol Progr 17(1):118–125CrossRefGoogle Scholar
  19. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology (vol 66, pg 506, 2002). Microbiol Mol Biol R 66(4):739–739CrossRefGoogle Scholar
  20. Lynd LR, Jin H, Michaels JD, Wyman CE, Dale B (2003) Bioenergy: background, potential, and policy. Center for Strategic and International Studies, WashingtonGoogle Scholar
  21. Nataf Y, Bahari L, Kahel-Raifer H, Borovok I, Lamed R, Bayer EA, Sonenshein AL, Shoham Y (2010) Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors. PNAS 107:8646–18651. doi: 10.1073/pnas.1012175107 CrossRefGoogle Scholar
  22. Pei J, Zhou Q, Jiang Y, Le Y, Li H, Shao W (2010) Thermoanaerobacter spp. control ethanol pathway via transcriptional regulation and versatility of key enzymes. Metab Eng 12(5):420–428CrossRefGoogle Scholar
  23. Pei J, Zhou Q, Jing Q, Li L, Dai C, Li H, Wiegel J, Shao W (2011) The mechanism for regulating ethanol fermentation by redox levels in Thermoanaerobacter ethanolicus. Metab Eng 13(2):186–193CrossRefGoogle Scholar
  24. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738CrossRefGoogle Scholar
  25. Rydzak T, Levin DB, Cicek N, Sparling R (2009) Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405. J Biotechnol 140(3–4):169–175CrossRefGoogle Scholar
  26. Sudha Rani K, Seenayya G (1999) High ethanol tolerance of new isolates of Clostridium thermocellum strains SS21 and SS22. World J Microbiol Biotechnol 15:173–178CrossRefGoogle Scholar
  27. Tailliez P, Girard H, Longin R, Beguin P, Millet J (1989) Cellulose fermentation by an asporogenous mutant and an ethanol-tolerant mutant of Clostridium thermocellum. Appl Environ Microbiol 55(1):203–206Google Scholar
  28. Timmons MD, Knutson BL, Nokes SE, Strobel HJ, Lynn BC (2009) Analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains. Appl Microbiol Biotechnol 82:929–939CrossRefGoogle Scholar
  29. Tripathi SA, Olson DG, Argyros DA, Miller BB, Barrett TF, Murphy DM, McCool JD, Warner AK, Rajgarhia VB, Lynd LR, Hogsett DA, Caiazza NC (2010) Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant. Appl Environ Microbiol 76:6591–6599. doi: 10.1128/AEM.01484-10 CrossRefGoogle Scholar
  30. Wang DIC, Avgerinos GC, Biocic I, Wang S-D, Fang H-Y, Young FE (1983) Ethanol from cellulosic biomass. Philos Trans R Soc London Ser B 300:323–333CrossRefGoogle Scholar
  31. Williams TI, Combs JC, Lynn BC, Strobel HJ (2007) Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum. Appl Microbiol Biotechnol 74(2):422–432CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Xiongjun Shao
    • 1
    • 3
  • Babu Raman
    • 2
    • 3
    • 4
  • Mingjun Zhu
    • 5
  • Jonathan R. Mielenz
    • 2
    • 3
  • Steven D. Brown
    • 2
    • 3
  • Adam M. Guss
    • 2
    • 3
  • Lee R. Lynd
    • 1
    • 3
    • 6
    Email author
  1. 1.Thayer School of EngineeringDartmouth CollegeHanoverUSA
  2. 2.Biosciences DivisionOak Ridge National LaboratoryOak RidgeUSA
  3. 3.BioEnergy Science Center (BESC)Oak Ridge National LaboratoryOak RidgeUSA
  4. 4.Bioprocess R&DDow AgroSciencesIndianapolisUSA
  5. 5.School of Bioscience & BioengineeringSouth China University of TechnologyGuangzhouChina
  6. 6.Mascoma CorporationLebanonUSA

Personalised recommendations