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

Applied Microbiology and Biotechnology volume 92, pages 641–652 (2011)Cite this article

Abstract

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.

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Acknowledgements

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.

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Author notes

  1. Babu Raman

    Present address: Bioprocess R&D, Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN, 46268, USA

Affiliations

  1. Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH, 03755, USA

    Xiongjun Shao & Lee R. Lynd

  2. Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN, 37831, USA

    Babu Raman, Jonathan R. Mielenz, Steven D. Brown & Adam M. Guss

  3. BioEnergy Science Center (BESC), Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN, 37831, USA

    Xiongjun Shao, Babu Raman, Jonathan R. Mielenz, Steven D. Brown, Adam M. Guss & Lee R. Lynd

  4. School of Bioscience & Bioengineering, South China University of Technology, University Town, Guangzhou, 510006, China

    Mingjun Zhu

  5. Mascoma Corporation, 67 Etna Road, Lebanon, NH, 03766, USA

    Lee R. Lynd

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  1. Xiongjun Shao
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Correspondence to Lee R. Lynd.

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Shao, X., Raman, B., Zhu, M. et al. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum . Appl Microbiol Biotechnol 92, 641–652 (2011). https://doi.org/10.1007/s00253-011-3492-z

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  • DOI: https://doi.org/10.1007/s00253-011-3492-z

Keywords

  • Clostridium thermocellum
  • Ethanol tolerance
  • Genome sequencing
  • Strain adaptation
  • Mutations