Conversion of cellulose into ethanol by using fungal cellulase and calcium alginate gel containing yeast and immobilized β-glucosidase

Abstract

The ever-present shortage of food coupled with the continuing escalation of oil prices and the acceptance of the fact that fossil fuel reserves are finally running out have done much to focus attention on cellulose. It is hoped that an economical process will soon be found whereby this most abundant of organic molecules, and a renewable resource, can be converted into glucose for use as a food or for subsequent conversion into ethanol. Enzymic hydrolysis of cellulose by the thermophilic fungus Talaromyces emersonii is effected by an extracellular cellulase system consisting of endoglucanases, exoglucanases and /3-glucosidases (Folan & Coughlan, 1978, 1979; McHale & Coughlan 1980). Unfortunately, the amount of P-glucosidase produced by this organism is low, in fact less than 2% of the total extracellular protein (McHale & Coughlan, 1980). Saccharification of cellulose is less efficient as a result. Considerable quantities of cellobiose accumulate in the reaction mixture (Folan & Coughlan, 1979). The cellobiose not only inhibits cellulase action but is, in any event, much less useful than glucose as a fermentation product. The production of less than optimum amounts of P-glucosidase is not unique to Talaromyces emersonii. Indeed, in the case of Trichoderma reesei, the most powerful of cellulolytic organisms, P-glucosidase accounts for less than 0.2% of the extracellular protein (Gong & Tsao, 1979). Various ways of dealing with this problem for practical saccharification purposes have been tried. Thus Tangnu et al. (1981) have shown that P-glucosidase activities can be increased considerably by judicious choice of culture conditions. Genetic manipulation techniques have provided catabolite-repression-resistant mutants of Trichodema reesei that produce greater quantities of all of the cellulase enzymes, including a-glucosidase (Montenecourt & Eveleigh, 1977; Nevalainen et al., 1980). Other solutions to the problem have included supplementation of the cellulolytic activity of the culture filtrate in use by the addition of free or of immobilized preparations of P-glucosidase isolated from good sources such as Aspergillus phoenicis (Allen & Sternberg, 1980; Venardos et al., 1980). We are attacking the problem in two ways. With the long-term view in mind we are subjecting Talaromyces emersonii to the kind of mutagenic treatments and screening techniques that have been used by Montenecourt & Eveleigh (1977) to obtain hyper-cellulase-producing strains of Trichoderma reesei. Starting with Talaromyces emersonii CBS 8 14.70 as the parent strain, we have obtained mutants, partially resistant to catabolite repression, giving a 2-fold increase in endoglucanase, exoglucanse and P-glucosidase activities and between 1 and 2 filter-paper units/ml of culture filtrate. Thus we are now at the stage with Talaromyces emersonii that strain QM9414 of Trichoderma viride was a few years ago (Mandels et al., 1976). However, the Talaromyces system may have some practical advantages over that of Trichoderma in that the fermentation time required to achieve maximal yield of enzyme is shorter, and, being a thermophile, its enzymes are more thermostable (Folan & Coughlan, 1978; McHale & Coughlan, 1981). Simultaneously, we are investigating model systems for the conversion of cellulose into ethanol. One such system consists of cellulose, culture filtrate of Talaromyces emersonii and calcium alginate gel in which yeast cells and immobilized P-glucosidase are co-entrapped (for details of immobilization, entrapment and assay see Kierstan & Bucke, 1977; Kierstan et al., 1982). In theory the culture filtrate, which contains endoand exoglucanases as well as 8-glucosidase, would convert the cellulose into a mixture of cellobiose and glucose. These products would diffuse freely into the gel spheres, where the immobilized P-glucosidase would cleave the cellobiose to glucose, which the entrapped yeast cells would then convert into ethanol. In this way the inhibition of cellulolysis by cellobiose (Folan & Coughlan, 1979) and of P-glucosidase action by glucose (McHale &. Coughlan, 1981) would both be relieved. In practice we have shown that ethanol is produced from cellulose in such systems and that the rate of ethanol production, at least in the initial stages, is greater than in control systems lacking the supplemental immobilized P-glucosidase.