Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.

Patricia J Slininger,Bruce E Dale,Venkatesh Balan,Cletus P Kurtzman,Stephanie R Thompson,Leonardo Da Costa Sousa,Nirmal Uppugundla,Michael A Cotta,Maureen A Shea-Andersh,Bruce S Dien

doi:10.1186/s13068-015-0239-6

Abstract

BackgroundLignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment.ResultsRepetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol-challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions.ConclusionsRobust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

Highlights

Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation
Pichia stipitis is known to ferment D-xylose to ethanol more efficiently than other native yeasts previously
S. stipitis strain NRRL Y-7124 is able to produce over 70 g/L ethanol in 40 h (1.75 g/L/h) from 150 g/L sugars at a yield of 0.41 ± 0.06 g/g in high density fermentations (6 g/L cells) [7,10,11]

Summary

Introduction

Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. An estimated 1.3 billion dry tons of lignocellulosic biomass could be available annually to support ethanol production at a level that would allow the U.S to reduce its petroleum consumption by 30% [1]. S. stipitis strain NRRL Y-7124 is able to produce over 70 g/L ethanol in 40 h (1.75 g/L/h) from 150 g/L sugars at a yield of 0.41 ± 0.06 g/g in high density fermentations (6 g/L cells) [7,10,11]. Sugar uptake rate in biomass hydrolyzates needs improvement, including reducing the effects of diauxy and improving ethanol and inhibitor tolerance. To advance the science and application of S. stipitis, our objective was to apply appropriate selective pressure to guide its evolution toward an industrially robust derivative that is tolerant of diverse lignocellulosic hydrolyzates

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