Bioprocess development for biochemical conversion of lignocellulose
Doctoral thesis, 2017

Due to its low environmental impact and high maturity of the fuel ethanol market, lignocellulosic ethanol is a promising option for reducing the carbon footprint in the transport sector. The characteristics of lignocellulosic feedstocks, such as varied sugar composition, low sugar density, low solubility, recalcitrance to enzymatic degradation, and inhibitors formed during thermochemical pretreatment, have so far limited the production process, and costs for conversion of lignocellulosic materials to ethanol are still high. In this thesis, I describe the development of a bioconversion process that pushes the limits of simultaneous saccharification and co-fermentation (SSCF) to achieve higher ethanol titre, yield and productivity on lignocellulosic feedstocks. I propose an integrated fed-batch strategy, Multi-Feed SSCF, including feeds of substrates, enzymes and adapted cells to tackle the technical challenges in operating a SSCF process at high substrate loadings. Using insights from experiments and a model-based feeding design, lignocellulose saccharification and fermentation at water insoluble solids (WIS) levels greater than 20% (w/w) was achieved. The multi-feed SSCF concept and model-aided substrate feeding design allowed rapid, reproducible, and scalable bioconversion of lignocellulose, as proven on several lignocellulosic feedstocks in both laboratory and demonstration scales. Ethanol production above 50 g/L in SSCF processes was found to be severely inhibited by the combined effects of ethanol, lignocellulose-derived inhibitors, and higher than standard cultivation temperature (35°C). Cell viability and fermentation improved significantly in a multi-feed SSCF process with a step change in temperature from 35 to 30°C, compared to operation at 35°C throughout. However, introducing the Erg3Tyr185 point mutation which has been reported to render thermotolerance in yeast, did not offer any significant improvement. Cell concentrations were determined by counting in a hemocytometer and colony forming unit assay. Their accuracy and reproducibility in lignocellulosic media, were verified by Design-of-Experiment-based calibration. Applic-ability of real time qPCR and dielectric spectroscopy as potential cell quantification methods was also investigated. With multi-feed of solid substrates, enzyme preparations, and adapted cells, the SSCF process produced > 60 g/L ethanol within 120 h, equivalent to 70% of the theoretical yield of the total sugar input, and 90% of the consumed sugar. The systematic optimisation reported in this work represents a robust and reproducible routine for developing lignocellulose-based processes. It could inspire continuous development of alternative strategies to current fossil-based chemical/fuel processes.


strain engineering

stress tolerance

kinetic modelling

high gravity




process development

Saccharomyces cerevisiae

Opponent: Associate Prof. Anna Eliasson Lantz, Technical University of Denmark, Denmark


Ruifei Wang

Industrial biotechnology

Westman, J.O, Wang R.,, Novy, V., and Franzén C.J. Sustaining fermentation in high-gravity ethanol production by feeding yeast to a temperature-profiled simultaneous saccharification and co-fermentation of wheat straw

Wang R., Lorantfy B., Fusco S.,, Olsson L., and Franzén C.J. Comparative analysis of quantitative methods for assessing yeast cell concentrations in lignocellulosic media

Today, popular concepts such as circular economy, fossil-free and carbon neutral fuels can be readily applied to describe biofuel produced from lignocellulosic materials. The use of lignocellulosic material is essential if we are to reduce CO2 emissions.  Lignocellulosic ethanol production is becoming established worldwide. In addition to its intrinsic value, the process can be viewed as a model for the biochemical conversion of recalcitrant lignocellulosic raw materials to a range of chemicals and other products.

In this thesis, we have employed a systematic, model-driven approach to the design of feeding schemes of solid substrate, adapted yeast cells, and enzymes to fed-batch simultaneous saccharification and co-fermentation (Multi-Feed SSCF) of pretreated lignocellulosic materials. With this approach, mixing problems were avoided at high solid loadings, leading to an ethanol concentration of 50-55 g/L within 72 hours of SSCF on wheat straw. A similar fermentation performance was obtained at a 10 qubic-meter demonstration scale using several yeast strains. Key factors for an efficient lignocellulose-based bioprocess were characterised, including rapid medium liquefaction and sustained yeast viability during the SSCF process. I investigated possible strategies to circumvent the limitations to SSCF posed by limited thermotolerance of the yeast Saccharomyces cerevisiae. The concentration of active cells determines the quality of fermentation processes. I assessed the applicability of several methods for quantitative analysis of both total and viable cells in complex lignocellulosic media. In conclusion, this study established a production workflow for the conversion of pretreated lignocellulosic materials to chemicals and fuels.

Subject Categories

Industrial Biotechnology

Other Engineering and Technologies not elsewhere specified

Biological Sciences

Bioprocess Technology

Areas of Advance


Life Science Engineering (2010-2018)



Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4284




Opponent: Associate Prof. Anna Eliasson Lantz, Technical University of Denmark, Denmark

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