Hemicellulose utilization by yeast for ethanol production- adaptation, effect of inhibitors and a flux balance based analysis

Abstract

A major challenge in ethanol production using lignocellulosic feed stock is inefficient utilization of hemicellulose, which accounts for 30-40% of lignocellulosic biomass. Xylose, comprising >60% of recoverable sugars from hemicellulose is a major product of the hemicellulose hydrolysis. Utilization of this carbon source would significantly increase the ethanol yield from an estimated 60 gal/dry ton to 90 gal/dry ton. While xylose is not consumed and fermented efficiently by industrial yeast Saccharomyces cerevisiae, xylulose can be fermented by it. Isomerizing xylose to xylulose can provide an alternative to genetic modification of yeasts for xylose utilization. A method to isomerize xylose to xylulose utilizing commercially available xylose isomerase was used. A synthetic media replicating hemicellulose in composition was designed. Two yeast strains with (Schizosaccharomyces pombe) and without (Saccharomyces cerevisiae) specialized xylose transport capability were grown in the defined media. The two strains were adapted to the hemicellulose environment by growing them in chemostat for 40 days (1000hrs). The batch fermentations of the adapted and original yeast strains were compared. High pressure liquid chromatography was used to measure substrates and products of fermentation. Results indicate that the unadapted Saccharomyces cerevisiae performed better than the other three strains with respect to xylulose utilization and ethanol yield. A total of 53.1% of xylulose was utilized by the strain by the end of 120 hrs of fermentation producing 0.41 Cmoles of ethanol/ Cmole of total sugar. Furfural is an important inhibitor formed in the pretreatment process of lignocellulosic ethanol production. Furfural, mainly formed from xylose in the hemicellulose is inhibitory to the fermentation by yeasts. Effect of furfural concentration on the fermentation of hemicellulose hydrolyzate was studied in a synthetic media using the strains of Saccharomyces cerevisiae and Schizosaccharomyces pombe which were adapted to the hemicellulose like environment. Two different concentrations of furfural (0.5 and 2 g/L) were used in the medium. Also the effect of high inoculum on the fermentation of xylulose was verified by batch experiments with high initial inoculums of the original Saccharomyces cerevisiae and Schizosaccharomyces pombe strains. Performances of different strains under different conditions were compared in terms of xylulose utilization in batch growth. In Saccharomyces cerevisiae fermentations, an intermediate concentration of 0.5 g/L of furfural had highest ethanol yield of 0.375Cmoles/Cmoles of total sugar and lowest glycerol yield of 0.041Cmoles/Cmoles of total sugar. In Schizosaccharomyces pombe, absence of any furfural in the medium had highest xylulose utilization of 51.84% over 120 hrs. Higher inoculum concentrations did not improve batch xylulose utilization in both the yeast strains but the utilization rates were improved. A constraint based metabolic model of Saccharomyces cerevisiae metabolism was used to perform a flux balance based analysis of xylulose and furfural metabolism. The model outputs were comparable to the experimental production rates of products. Under the experimental conditions, the model gives an increase of 1.036 mmol/hr. g biomass of ethanol production by the consumption of xylulose. The model output rates of products suggested that, to detoxify furfural under normal metabolic functioning would reduce the specific growth rate to 0.00043 g/hr. g of biomass from 0.048 g/hr. g of biomass.