Improving Ethanol Production from Lignocellulose Hydrolyzates: Fed-batch Fermentation, Yeast Cultivation and Strain Development for Increased Tolerance

Abstract: Ethanol production by the yeast Saccharomyces cerevisiae from dilute acid lignocellulose hydrolyzate was studied with the aim to improve fuel ethanol production. Specifically, the problem of inhibition was addressed. Lignocellulose hydrolyzate contains not only sugars, but also several compounds, such as furfural and 5-hydroxymethyl furfural (HMF), which can inhibit yeast growth and ethanol production by the yeast. It was shown that severe inhibition could be avoided during both aerobic cultivation and anaerobic fermentation in properly controlled fed-batch processes. The reason for this is that the levels of inhibitors can be maintained at low levels since the yeast has the ability to convert these substances to less toxic compounds if severe inhibition is avoided. In aerobic fed-batch cultivation aiming at high biomass yield, it is essential not only to avoid inhibiting concentrations of e.g. furfural and HMF, but also to avoid ethanol formation due to over-flow metabolism. Closed-loop feed-rate control with constant ethanol set-point, allowed a specific biomass productivity of around 0.2 g g-1 h-1 with a yield of 0.46 g biomass/g fermentable sugar. Feeding strategies were also successfully developed for anaerobic fed-batch fermentation. It was possible to increase the specific ethanol productivity several-fold in controlled fed-batches compared to batch fermentation - from 0.06 g g-1 h-1 to 0.7 g g-1 h-1. In addition to process technology, work was also made concerning inhibitor tolerance of the yeast. An alcohol dehydrogenase enzyme able to reduce HMF was identified using genome-wide analysis. The enzyme ? encoded by the gene ADH6 ? was shown to catalyze NADPH-dependant reduction of HMF and over-expression of the gene gave an increased in vivo HMF conversion ability. Furthermore, the modified yeast showed increased fermentation rate in undetoxified lignocellulose hydrolyzate.