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

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.

Abstract (Swedish)

Popular Abstract in English

The production and use of bioethanol can help reducing the dependency on oil, and also represents a renewable source of energy. Bioethanol can be obtained from biomass such as agricultural and forest residues, after appropriate pretreatment and fermentation. This thesis addresses some of the challenges that must be overcome for the production of ethanol from biomass to be economically feasible.



Yeast is able to transform sugars into alcohol (ethanol) by fermentation, and this is the reason why it has been used throughout history to ferment the sugars present in cereals and grapes for the production of bread, beer and wine. In the same way, this microorganism can also be used to produce bioethanol from biomass. However, biomass consists of chains of complex sugars that must be broken down into simple sugars that can be fermented by yeast to produce ethanol. High temperature and pressure, and, sometimes, also chemicals, are usually needed to break down the complex mixture of sugars. However, severe treatment conditions cause the sugars to be degraded into aldehydes.



Biomass also contains other compounds, such as acids and phenolics, which are also released during processing. These, together with aldehydes, inhibit the metabolism of the yeast, thus reducing its ability to ferment sugars. In this work, the effect of acetic acid on yeast was investigated. An adaptation procedure that enabled yeast to be less inhibited by the presence of acetic acid was developed, resulting in a reduction of the fermentation time.



The more sugars the yeast is able to consume, the more ethanol can be produced, and the higher the process yield. However, yeast does not have the enzymes required for the consumption of xylose and arabinose, two of the simple five carbon sugars (or “pentose sugars”) that are present at significant amounts in various types of biomass. Therefore, five genes coding for the missing enzymes were introduced in an industrial yeast. The rate of consumption of these pentose sugars was later improved by gradually adapting the yeast so that it grew faster on these sugars.



Another challenge in bioethanol plants is the lack of sterile conditions during operation. As a result, other microorganisms present in the environment can contaminate the plant and compete with yeast for the available sugars. For example, some bacteria produce acids instead of ethanol. In addition, the presence of acids inhibits yeast metabolism, further reducing ethanol production. However, the lack of sterility offers the possibility of isolating yeasts that have become naturally adapted over time to the severe fermentation conditions.



In this work, a lactic acid bacterium that consumed simple sugars and produced lactic acid was isolated from an ethanol plant in Sweden. The effect of this bacterium on yeast fermentation was investigated. Possible changes in the process conditions that would reduce the level of this contaminant, and thus help maintain a high level of ethanol production by the yeast, were also studied. An indigenous yeast that dominated the fermentation process was also isolated at the same plant. This yeast systematically competed with the commercial yeast added, and eventually took over the fermentation, due to its better robustness. Such naturally adapted yeasts can provide useful information on yeast tolerance mechanisms. In addition, these yeasts could be used in other fermentation plants. Finally, competition experiments between to different yeasts were performed to investigate whether one yeast would dominate over another under conditions of limited sugar or oxygen supply.

• author: Sanchez Nogue, Violeta
• supervisor: Marie-Francoise Gorwa-Grauslund
• opponent: Dr. Chambers, Paul J., The Australian Wine Research Institute
• organization: Applied Microbiology
• publishing date: 2013
• type: Thesis
• publication status: published
• subject: Industrial Biotechnology
• keywords: Saccharomyces cerevisiae, Pentose fermentation, Evolutionary engineering, Acetic acid, pH, Short-term adaptation, Resident yeast, Spent sulphite liquor fermentation, Dekkera bruxellensis, Lactobacillus pentosus.
• pages: 172 pages
• defense location: Lecture hall C, Kemicentrum, Getingevägen 60, Lund University, Faculty of Engineering
• defense date: 2013-05-03 10:30:00
• ISBN: 978-91-7422-316-3
• language: English
• LU publication?: yes
• id: b8434df0-117f-4d3e-b595-6da0bcd6dc9c (old id 3631813)
• date added to LUP: 2016-04-04 11:01:44
• date last changed: 2018-11-21 21:02:12