(796f) Dynamic Model-Based Analysis of Furfural and HMF Detoxification By Pure and Mixed Batch Cultures of S. Cerevisiae and S. Stipitis

The conversion of lignocellulosic materials to commodity chemicals requires the efficient fermentation of sugar monomers to products. The chemical and physical processes commonly used in the pretreatment and hydrolysis of sugar polymers typically produce dehydration products such as furfural and 5-hydroxymethyl furfural (HMF) that can have a deleterious effect on fermentative microorganisms. Many microbes have the capacity to reduce these furan compounds to less toxic alcohols. Of interest in the present study is the reduction of furfural to furfuryl alcohol and the conversion of HMF to 2,5-bis-hydroxymethylfuran. Furan detoxification in yeast is achieved using a combination of enzymes from the alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase families. Depending on the specific enzyme used, furan aldehyde reduction requires the use of either NADH or NADPH. Thus, furfural and HMF induce changes to the redox balance by sequestering these cofactors in the detoxification process. Furan aldehydes can also reduce the rates of sugar uptake and ethanol synthesis by fermentative microbes.

Co-culturing different species of yeast has been shown to be an effective strategy for fermentation of sugar mixtures to ethanol. In contrast to monocultures with engineered organisms, mixed cultures allow the innate conversion strengths of two or more microbes to be combined. A variety of microbial co-cultures have been proposed for fermentation of lignocellulosic hydrolysates. For example, co-culturing a respiratory deficient mutant of the ethanologic yeast Saccharomyces cerevisiae with the xylose-fermenting yeast Scheffersomyces stipitis has been shown to produce larger amounts of ethanol from glucose and xylose mixtures than either yeast in monoculture. The use of respiratory deficient S. cerevisiae prevents the reassimilation of ethanol and allows for more precise regulation of oxygen levels needed to establish microaerobic conditions under which S. stipitis has high ethanol synthesis capabilities. Previous studies have shown that S. cerevisiae and S. stipitis respond differently to the presence of furan inhibitors in batch monoculture. HMF is reduced by S. cerevisiae at approximately a quarter of the rate that furfural is reduced. Although S. stipitis can detoxify HMF at a faster rate than S. cerevisiae, furan aldehydes have a more deleterious effect on S. stipitis growth and ethanol production.

In this paper, genome-scale reconstructions of S. cerevisiae and S. stipitis metabolism are combined with batch monoculture and co-culture experiments to develop improved understanding of furaldehyde detoxification by these yeast species. Uptake kinetics and stoichiometric equations for the intracellular reduction reactions associated with each inhibitor were added to genome-scale metabolic reconstructions of the two yeasts. Further modification of the S. cerevisiae metabolic network was necessary to satisfactorily predict the amount of acetate synthesized during HMF reduction. Inhibitory terms that captured the adverse effects of the furan aldehydes and their corresponding alcohols on cell growth and ethanol production were added to attain qualitative agreement with batch experiments. When the two yeasts were co-cultured in the presence of the furan aldehydes, inoculums that reduced the synthesis of highly toxic acetate produced by S. cerevisiae yielded the highest ethanol productivities. The model described here can be used to generate optimal cellular engineering and fermentation strategies for the simultaneous detoxification and fermentation of lignocellulosic hydrolysates by S. cerevisiae and/or S. stipitis.