Of the sugars recovered from lignocellulose, wild type Saccharomyces cerevisiae strains can readily ferment most six-carbon sugars. However, xylose, which also forms a major portion of biomass, cannot be fermented by the same native strains of yeast. The pathway of conversion of xylose to ethanol goes via the formation of xylulose, which is a ketose isomer of xylose. There have been many attempts to genetically engineer the yeast strains with over expression of xylose isomerase genes. Our approach however is to convert the xylose to xylulose using the widely manufactured exogenous enzyme Glucose/Xylose Isomerase (XI) and then ferment the xylulose to ethanol by using different yeast strains like Saccharomyces Cerevisiae D5A and Pichia stipitis in the same reaction vessel. A major obstacle however, to this approach is that the pH at which xylose isomerase displays its optimal activity (pH of 7.5-8.0) is much different from the pH at which the fermentation is carried out. We have successfully demonstrated that the maintenance of two vastly different pH microenvironments in a single vessel was possible. This involves the use of bilayered (XI–urease)immobilized enzyme pellets. The design of the co immobilized pellets and the fermentation parameters leading to maximum ethanol production with different yeasts will be discussed.

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