The immobilization of Kluyveromyces fragilis and Saccharomyces cerevisiae in polyacrylamide gel : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University

Abstract

The search for new energy sources has indicated that biomass, in the form of green plant materials and biological wastes, can provide a perpetual energy source if converted to a useful form. This study investigated the production of ethanol by the fermentation of sugars using immobilized cells. The experimental procedure involved the immobilization of two yeast species, Kluyveromyces fragilis NRRL Y 1109 and Saccharomyces cerevisiae NCYC 240, in polyacrylamide gel for the fermentation of lactose and glucose respectively. The gel methodology of two previous authors, Chibata et al. (1974) and Neuhoff (1973) was used. The former author's gel was used as a basis for batch experiments to determine the gel composition for maximum ethanol producing activity by both cell species as initial trials with this gel yielded encouraging results. Variations in monomer, BIS and cell concentration revealed that a gel containing 15% (w/v) acrylamide, 1.5% (w/v) BIS and 25% (w/v) cells in addition to 0.6% (w/v) BDMAP and 0.25% (w/v) ammonium persulfate in tris-HCl buffer pH 7.1 polymerised at 0°C produced the greatest activity in immobilized K. fragilis cells with an activity retention for immobilization of 80%. The gel composition for greatest activity in immobilized S. cerevisiae cells differed only slightly from that above containing 20% (w/v) acrylamide, 1.6% (w/v) BIS and 40% (w/v) cells and resulted in a 46% activity retention for immobilization. Further experiments at various substrate concentrations indicated that the gel imposed small or negligible limitations on the diffusion of substrate and product. Experiments to increase the cell activity retention for the immobilization of S. cerevisiae using the Neuhoff (1973) gel were unsuccessful but produced some important results. It was found that exposure to gel components, especially to the acrylamide monomer, reduced the ethanol producing ability and the viability of the cells. The general protective agents Tween 80, glycerol, gelatin and dithiothreitol proved ineffective. To minimize this damage to the cells the gels were polymerised at 0°C with rapid polymerisation being induced by high initiator and accelerant concentrations. Repeated use of the immobilized cells indicated that the simple substrate medium, of the suqar in distilled water used previously, was not sufficient to maintain stable ethanol producing activity. Although trials involving supplementation with a salt solution were unsuccessful, the incorporation 0.5% (w/v) peptone in the medium and the use of protein-containing media, such as whey, was found to stabilize activity. Experiments in continuous processing revealed that immobilized K. fragilis cells produced ethanol from deproteinised whey at an efficiency of 70 to 80% over extended periods with complete substrate utilization of full strength whey being achieved at flowrates of 0.15 SV. The half life of the activity of the immobilized cells was estimated to be at least 50 days. The experimental results suggest that this approach to fermentation may be industrially acceptable for the production of ethanol. However, a costing exercise on the production of ethanol from whey indicates that unless the product is a highly priced commodity, such as a pharmaceutical, the process is unlikely to be economically feasible due to the high cost of the immobilization support monomer