Short-term adaptation of S. cerevisiae to lignocellulosic inhibitors: Underlying metabolic and physiological changes
Doctoral thesis, 2021

The limited tolerance of Saccharomyces cerevisiae (budding yeast) to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The overall aim of this thesis was to identify molecular and physiological changes during short-term adaptation.

In order to facilitate testing of S. cerevisiae physiology in lignocellulosic hydrolysate, a high-throughput methodology for the analysis of yeast strains in dark medium was developed. This methodology allows for monitoring of both aerobic and anaerobic growth of yeast in medium containing different hydrolysates at high reproducibility. The effect that individual nutrient components during propagation, rather than fermentation, has on lignocellulose fermentation performance is lacking. A high-throughput screening of certain vitamins, trace metals and nitrogen sources was performed. It was found that adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate. Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation.

Different S. cerevisiae strain backgrounds are used in the production of a suitable second-generation bioethanol host. In order to facilitate application of results obtained in laboratory experiments it is important to know whether short-term adaptation affects different strains differently. The physiology of two industrial S. cerevisiae strains were investigated while being short-term adapted. During propagation, fed with a hydrolysate containing feed, ethanol accumulation was observed for strain CR01 but not for KE6-12. Additionally, a larger increase in specific ethanol productivity for CR01 was observed than for KE6-12. Thus, short-term adaptation was found to affect S. cerevisiae physiology differently depending on strain background. To gain a more complete insight into the metabolic changes that S. cerevisiae experiences during short‑term adaptation, RNA sequencing was performed on a time-series of samples taken from propagation cultures undergoing short-term adaptation. Expression data was compared to a non‑adapted control using differential gene expression analysis. Results demonstrate, among others, an interesting role for multidrug proton antiporters YHK8 and FLR1 in the process of short-term adaptation.

lignocellulosic inhibitor tolerance

second generation bioethanol

short-term adaptation

differential gene expression

industrial yeast strains

Opponent: Professor Gunnar Lidén, Department of Chemical Engineering, Lund University, Lund, Sweden


Marlous van Dijk

Chalmers, Biology and Biological Engineering, Industrial Biotechnology

One of the great challenges facing our society today is the transition from industrial production processes that rely on petroleum to those that rely on renewable substrates. Bioethanol has the potential to replace petroleum-based transportation fuels using sugars as substrates for production by yeast. However, bioethanol produced from edible crops (first-generation) raises ethical concerns regarding land use for fuel rather than food production. Second-generation bioethanol processes utilize agricultural and forestry residues, also named lignocellulosic biomass, instead. In order to make lignocellulosic biomass suitable as a substrate, a pretreatment process is required. This process produces a hydrolysate that contains sugars, but also contains by-products which inhibit the yeast and thereby unfavorably affect process efficiency, rendering the process not economically feasible.

In my thesis I present and investigate a strategy to overcome these inhibitory effects, named short-term adaptation. Short-term adaptation is the process of growing yeast in the presence of a dilute concentration of the hydrolysate before it is used in fermentation where hydrolysate concentrations are much higher. Short-term adaptation causes the cell to experience stress without it being lethal. The experienced stress during short-term adaptation causes the yeast to become more robust during following exposure to higher levels of hydrolysate. Here, I investigated which stress response mechanisms play a role during short-term adaptation and how phenotypical characteristics of the yeast are affected by it. I also investigated the role of nutrients during yeast propagation as a tool to improve lignocellulosic hydrolysate fermentation kinetics. The results from these studies form a basis for a better mechanistic understanding of short-term adaptation and can inform future process and strain engineering strategies.

Bottlenecks in cellulosic ethanol production: xylose fermentation and cell propagation

Swedish Energy Agency (2015-006983), 2016-01-01 -- 2019-12-31.

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Subject Categories

Other Industrial Biotechnology



Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4884





Opponent: Professor Gunnar Lidén, Department of Chemical Engineering, Lund University, Lund, Sweden

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