Fermentation of hexose and pentose sugars from lignocellulosic biomass using native yeast strains

To improve the utilization of xylose, simultaneous isomerization and fermentation (SIF) approach has been suggested in the literature. Here, the expectation is that continuous removal of xylulose via fermentation to ethanol will force the isomerization reaction in the direction of more xylulose formation. Unfortunately, the pH optima for the isomerization (∼ 7.5) and the fermentation (∼ 4.5) steps are vastly different. The most common approach adopted has been to run SIF at a compromised pH where both the isomerization and the fermentation take place at sub-optimal conditions. In SIF, performing under suboptimal conditions, the overall yield of ethanol and the utilization of xylose are still unsatisfactory by the limited concentrations of xylulose available to the yeast.;In an ideal situation, complete conversion of xylose to xylulose and then fermentation of all the xylulose to ethanol is expected. Towards this goal, in this thesis, we developed a novel technique capable of sustaining two different pH-microenvironments in a single vessel---one optimal for xylose isomerization and the other optimal for fermentation of xylulose. The technique involves co-immobilization of urease with xylose isomerase. These co-immobilized enzyme pellets are dispersed in a fermentation broth which contains urea in addition to the other necessary ingredients for fermentation. We showed that it is possible to sustain a significant pH gradient between the bulk liquid and the core region of the pellet because as hydrogen ions diffuse into the pellet, they are neutralized by the ammonia produced in the hydrolysis of urea by urease. The XI which is maintained at a higher pH in the inner core of the pellet then catalyzes the isomerization of the xylose to xylulose; which then becomes available for fermentation in the bulk solution at pH 4.5. Our co-immobilized enzyme strategy which provides optimal microenvironments for isomerization and fermentation is naturally benefited by the addition of borate to the fermentation broth. The advantage results from the fact that the ability of borate to bind to xylulose is pH dependent, with higher pH (6 to 7.5) favoring tighter binding. Inside the pellet, the pH is elevated, XI is active, and the ismerization equilibrium is favored by strong borate binding to xylulose. In contrast, in the low pH fermentation broth, borate has a reduced binding to xylulose, and thus produces a higher free xylulose concentration for fermentation to ethanol. This could lead to higher yields and rates of ethanol production.;In this dissertation, the isomerization of xylose to xylulose was demonstrated successfully. The results presented demonstrate the effectiveness of our co-immobilized enzyme system for isomerization under conditions optimal for fermentation by common S. cerevisiae. In our co-immobilized pellet system, we obtained xylulose conversions that are higher than those possible with the native XI pellets operating under optimal pH and borate concentration levels. In our system, tetrahydroxyborate acts to shift the equilibrium by binding to xylulose and also shuttles complexed xylulose from the pellet interior to the bulk solution (facilitated transport). We believe that this dual role of tetrahydroxyborate is responsible for the significant improvement in xylose conversion seen in our two-pH environment experiment. Hence our enzyme technology essentially eliminates the isomerization bottleneck. The fermentation of the xylulose obtained was also studied and optimal conditions and yeast strains are recommended for successful xylulose utilization by native yeasts. (Abstract shortened by UMI.)...