Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol

Patricia J. Slininger, Maureen A. Shea-Andersh, Stephanie R. Thompson, Bruce S. Dien, Cletus P. Kurtzman, Leonardo da Costa Sousa, Venkatesh Balan

Research output: Contribution to journalArticle

Abstract

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

LanguageEnglish (US)
Article numbere54227
JournalJournal of Visualized Experiments
Volume2016
Issue number116
DOIs
StatePublished - Oct 24 2016

Profile

Pentoses
Bioconversion
Yeast
Ethanol
Yeasts
Xylose
Sugars
Dilution
Enzymes
Population
Derivatives
Saccharification
Detoxification
Poisons
Glycerol
Plating
Waste disposal
Ultraviolet radiation
Biomass
Fermentation

Keywords

  • Acetic acid
  • Bioengineering
  • Corn stover
  • Diauxic lag
  • Fermentation inhibitors
  • Furfural
  • Hydrolyzate
  • Hydroxymethylfurfural
  • Issue 116
  • Pichia stipitis
  • Relative performance index
  • Scheffersomyces stipitis
  • Switch grass
  • Xylose

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Slininger, P. J., Shea-Andersh, M. A., Thompson, S. R., Dien, B. S., Kurtzman, C. P., Sousa, L. D. C., & Balan, V. (2016). Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. Journal of Visualized Experiments, 2016(116), [e54227]. DOI: 10.3791/54227

Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. / Slininger, Patricia J.; Shea-Andersh, Maureen A.; Thompson, Stephanie R.; Dien, Bruce S.; Kurtzman, Cletus P.; Sousa, Leonardo da Costa; Balan, Venkatesh.

In: Journal of Visualized Experiments, Vol. 2016, No. 116, e54227, 24.10.2016.

Research output: Contribution to journalArticle

Slininger, PJ, Shea-Andersh, MA, Thompson, SR, Dien, BS, Kurtzman, CP, Sousa, LDC & Balan, V 2016, 'Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol' Journal of Visualized Experiments, vol 2016, no. 116, e54227. DOI: 10.3791/54227
Slininger PJ, Shea-Andersh MA, Thompson SR, Dien BS, Kurtzman CP, Sousa LDC et al. Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. Journal of Visualized Experiments. 2016 Oct 24;2016(116). e54227. Available from, DOI: 10.3791/54227
Slininger, Patricia J. ; Shea-Andersh, Maureen A. ; Thompson, Stephanie R. ; Dien, Bruce S. ; Kurtzman, Cletus P. ; Sousa, Leonardo da Costa ; Balan, Venkatesh. / Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. In: Journal of Visualized Experiments. 2016 ; Vol. 2016, No. 116.
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