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Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol

  1. Charles E. Wymana,b,c,d,2
  1. aDepartment of Bioengineering, Bourns College of Engineering, University of California, Riverside, CA 92507;
  2. bCenter for Environmental Research and Technology (CE-CERT), Bourns College of Engineering, University of California, Riverside, CA 92507;
  3. cDepartment of Chemical and Environmental Engineering, Bourns College of Engineering, University of California, Riverside, CA 92507;
  4. dBioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, TN 37831
  1. Edited by Alexis T. Bell, University of California, Berkeley, CA, and approved September 20, 2017 (received for review March 21, 2017)


Future production of renewable transportation fuels such as ethanol must rely on abundant nonfood plant sources also known as lignocellulosic biomass. However, a major historical barrier to low-cost production of ethanol from biomass is the low ethanol yields and titers that result from fermentation of biomass solids at high solids when compared with simple sugar fermentations. Here, we show that combining a cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment process with subsequent simultaneous saccharification and fermentation (SSF) can achieve similar high ethanol yields and titers that match that of separate pure glucose fermentations. We demonstrate a strategy whereby direct fermentation of biomass to ethanol is now limited by the microbe rather than by the process.


Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a long-standing limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery. Here, we illustrate how competing factors that limit ethanol yields during high-solids fermentations are overcome by integrating newly developed cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment with SSF. First, fed-batch glucose fermentations by Saccharomyces cerevisiae D5A revealed that this strain, which has been favored for SSF, can produce ethanol at titers of up to 86 g?L?1. Then, optimizing SSF of CELF-pretreated corn stover achieved unprecedented ethanol titers of 79.2, 81.3, and 85.6 g?L?1 in batch shake flask, corresponding to ethanol yields of 90.5%, 86.1%, and 80.8% at solids loadings of 20.0 wt %, 21.5 wt %, and 23.0 wt %, respectively. Ethanol yields remained at over 90% despite reducing enzyme loading to only 10 mg protein?g glucan?1 [~6.5 filter paper units (FPU)], revealing that the enduring factors limiting further ethanol production were reduced cell viability and glucose uptake by D5A and not loss of enzyme activity or mixing issues, thereby demonstrating an SSF-based process that was limited by a strain’s metabolic capabilities and tolerance to ethanol.


  • ?1T.Y.N. and C.M.C. contributed equally to this work.

  • ?2To whom correspondence should be addressed. Email: cewyman{at}engr.ucr.edu.
  • Author contributions: T.Y.N., C.M.C., and C.E.W. designed research; T.Y.N. and C.M.C. performed research; T.Y.N., C.M.C., R.K., and C.E.W. analyzed data; and T.Y.N., C.M.C., R.K., and C.E.W. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1704652114/-/DCSupplemental.

Published under the PNAS license.

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