Ethanol from Lignocellulose: Physiological Effects of Inhibitors and Fermentation Strategies

Doctoral thesis, 1999

Fermentative ethanol production from dilute-acid hydrolyzates of wood using the yeast Saccharomyces cerevisiae was investigated. Of known inhibitors in hydrolyzates, acetic acid, furfural and hydroxymethyl furfural (HMF) were found in the highest concentrations (up to about 10 g/l). Physiological effects of these inhibitors were studied in synthetic media. Based on these studies, on-line control of fed-batch cultivation for in-situ detoxification of the hydrolyzates was subsequently developed. The effect of acetic acid on yeast was found to strongly depend on pH. At concentration of undissociated acetic acid higher than 5 g/l, growth stopped. However, presence of the acid in the medium at low concentration (e.g. 1 g/l) increased ethanol yield and decreased the formation of fermentation by-products. Furfural (4 g/l) severely decreases the specific growth rate of S. cerevisiae in pulse addition experiments. However, the yeast was able to convert furfural to less inhibiting products, mainly by reduction to furfuryl alcohol, with a specific conversion rate of 0.6 g/g.sigma.h. A previously unidentified metabolite was also found and was characterized by mass spectrometry. Presumably, the metabolite was formed from pyruvate and furfural. HMF is less inhibiting to yeast than furfural, but remains in the medium for about 4 times longer than furfural due to a lower conversion rate. The yeast converts HMF mainly to hydroxymethyl-furfuryl alcohol and a newly identified compound probably formed from HMF and acetaldehyde. Fed-batch fermentation was suggested as a suitable mode of operation for fermenting dilute-acid hydrolyzates from the physiological studies of the inhibitors. With a suitable feed rate, it was possible to ferment also severely inhibiting spruce and birch hydrolyzates using fed-batch operation without any pretreatment of the hydrolyzates. However, the feed rate was critical in order to obtain a successful operation. A simple feedback control strategy was therefore developed, allowing the feed rate to be determined on-line, without any other input variable than the measured carbon dioxide evolution rate.