Fermentation Inhibitors in the Production of Bio-ethanol: Detoxification of Lignocellulose Hydrolyzates and Physiological Effects of Furfural on Yeast

Doctoral thesis, 2004

Different strategies to overcome inhibitor problems in the production of bio-ethanol from lignocellulose hydrolyzates with the yeast Saccharomyces cerevisiae were examined. The strategies included detoxification prior to fermentation and in situ detoxification of furfural. A strain development for improved inhibitor tolerance was also initiated. Strong and weak anion-exchange resins based on different matrix materials were compared with regard to detoxification of dilute-acid hydrolyzates of spruce. Fractions collected from strong anion exchangers with styrene-based matrices showed the best fermentability and the lowest inhibitor concentrations. Detoxification at alkaline conditions using overliming (addition of calcium hydroxide) or ammonium hydroxide was investigated. Overliming was optimized in a factorial designed experiment. The effects of pH (8-12) and temperature (5-80°C) on the concentrations of fermentable sugars and inhibitors, including aliphatic acids, phenols and furan aldehydes, were determined. The model predicted that conditions with relatively low sugar degradation rate and high furan aldehyde degradation rate could be achieved. Fermentation of the treated samples showed that it was possible to find treatment conditions that resulted in an ethanol productivity that was higher than in reference fermentations on glucose. Results from experiments with detoxification by using ammonium hydroxide suggest that the improvement in fermentability was due to chemical effects during the detoxification rather than to the extra addition of nitrogen source. Inhibition effects of furfural and the kinetics of furfural uptake were studied in anaerobic and aerobic continuous cultivations of S. cerevisiae using chemostat cultures and pulse addition experiments. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. During respiratory growth furoic acid was the only product of furfural conversion. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. On the other hand, furfural was exclusively reduced to furfuryl alcohol under anaerobic conditions, where the reduction of furfural could act as an alternative redox sink, and thereby replace glycerol formation as a means of oxidizing excess NADH. Both during fermentative and respiratory growth, the maximum specific conversion rate of furfural in dynamic experiments was significantly higher than the rate obtained in the steady-state experiments. The results support the use of a fed-batch process, in which a high concentration of furan aldehydes in the culture can be avoided. A fed-batch process will be able to operate at higher specific conversion rates than a continuous process.