Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX™ pretreated corn stover

Mingjie Jin,Laura B Willis,Ragothaman Avanasi Narasimhan,Venkatesh Balan,Christa Gunawan,Benjamin D Bice,Bruce E Dale,Cory Sarks,Shane P Simonett,Trey K Sato

doi:10.1186/1754-6834-6-108

Abstract

BackgroundSimultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover, degradation products resulting from biomass pretreatment impair fermentation of sugars, especially xylose, and can synergize with high temperature stress. One approach to resolve both concerns is to utilize a strain background with innate tolerance to both elevated temperatures and degradation products.ResultsIn this study, we screened a panel of 108 wild and domesticated Saccharomyces cerevisiae strains isolated from a wide range of environmental niches. One wild strain was selected based on its growth tolerance to simultaneous elevated temperature and AFEX™ (Ammonia Fiber Expansion) degradation products. After engineering the strain with two copies of the Scheffersomyces stipitis xylose reductase, xylitol dehydrogenase and xylulokinase genes, we compared the ability of this engineered strain to the benchmark 424A(LNH-ST) strain in ethanol production and xylose fermentation in standard lab medium and AFEX pretreated corn stover (ACS) hydrolysates, as well as in SSCF of ACS at different temperatures. In SSCF of 9% (w/w) glucan loading ACS at 35°C, the engineered strain showed higher cell viabilities and produced a similar amount of ethanol (51.3 g/L) compared to the benchmark 424A(LNH-ST) strain.ConclusionThese results validate our approach in the selection of wild Saccharomyces cerevisiae strains with thermo-tolerance and degradation products tolerance properties for lignocellulosic biofuel production. The wild and domesticated yeast strains phenotyped in this work are publically available for others to use as genetic backgrounds for fermentation of their pretreated biomass at elevated temperatures.

Highlights

Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor
Phenotyping of wild S. cerevisiae strains for Ammonia fiber expansion (AFEX) and thermo-tolerance To identify S. cerevisiae strains that can tolerate AFEX pretreated corn stover (ACS) degradation products at elevated temperatures, we monitored the cell densities of 108 unique wild, domesticated or industrial isolates and laboratory control strains (Additional file 1: Table S1) cultured in 96-well plates containing Yeast extract (YEPD) medium at both 30 and 40°C or 6% and 9% glucan loading ACS hydrolysate (ACSH) at 40°C
While all or most strains doubled four to five times in cell density at 30 and 40°C in YEPD medium within 24 h, the majority of strains grew much slower in 6% and 9% glucan loading ACSH and did not reach saturation within 24 h, while doubling their cell densities one to two times

Summary

Introduction

Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Since the efficiency and rates of enzymatic hydrolysis and fermentation are often optimal at distinct temperatures, separate hydrolysis and fermentation (SHF) is commonly employed to carry out the two reactions in separate vessels, which increase capital costs and total processing time. An ideal SSCF process would occur at temperatures for optimal cellulolytic activities (e.g., 50°C for commonly-used fungal Trichoderma reesei cellulases), which is significantly above the standard culturing temperature of 30°C for Saccharomyces cerevisiae, the most commonly used organism for the production of fuel ethanol. SSCF has been conducted at lower temperatures, slowing enzymatic hydrolysis and sugar release rates and resulting in reduced fermentation rates and yields [5,6]

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