Industrial challenges in the use of Saccharomyces cerevisiae for ethanolic fermentation of lignocellulosic biomass

University dissertation from Applied Microbiology, Lund University

Abstract: The sustainable production of ethanol from lignocellulosic biomass requires the combination of efficient hydrolysis and complete fermentation of all the monomeric sugars present in the raw material. The present work was aimed at tackling some of the major challenges that will be encountered in commercial-scale ethanol production using Baker’s yeast, Saccharomyces cerevisiae, the preferred microorganism for the fermentation step. During biomass pretreatment, several inhibitory compounds are released, including weak acids, furaldehydes and phenolics. The presence of these compounds in the hydrolysate reduces the ethanol yield and productivity, prolongs the lag phase, and reduces the growth rate of the yeast during fermentation. S. cerevisiae is naturally unable to utilise the pentose sugars xylose and arabinose. Evolutionary engineering was used to improve the conversion of these pentoses to ethanol in a recombinant industrial strain of S. cerevisiae expressing heterologous genes for the xylose and arabinose utilisation pathways. The evolved strain showed a higher rate of consumption of xylose and arabinose under both aerobic and anaerobic conditions, which was attributed to an increase in the transport of pentoses and the activities of xylose converting enzymes. The introduction of a short-adaptation process enabled aerobic growth at low pH in the presence of inhibitory levels of acetic acid and led to a significant reduction in the fermentation time under anaerobic conditions. In parallel, the possibility of using indigenous yeasts present in the spent sulphite liquor (SSL) ethanol plant as a source of S. cerevisiae strains with a naturally acquired tolerance to inhibitory compounds was also investigated. The isolated strain, TMB3720, exhibited a higher ethanol yield and production rate than the commercial baker’s yeast strain, regularly used as inoculum. It was hypothesised that the tolerance of this strain was related to its flocculation behaviour and its high capacity to reduce furaldehyde inhibitors. As ethanol plants are run under non-sterile conditions, S. cerevisiae must compete with other microorganisms for sugar utilisation. Therefore, competition experiments were performed with the contaminant yeast Dekkera bruxellensis and the lactic acid bacterium Lactobacillus pentosus isolated from the SSL ethanol plant. Glucose limitation, achieved by sparging a mixture of nitrogen and air (~5% oxygen) through the system, was identified as a parameter enabling D. bruxellensis to outcompete S. cerevisiae, probably due to the higher nutrient affinity of D. bruxellensis under these conditions. In parallel, reducing the pH was also found to be a possible means of reducing the levels of lactate produced by the L. pentosus strain.

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