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.