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    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
    (Massey University, 1980) Dillon, Judith Ann
    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.
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    Application of alternative fermenter design in whey-ethanol production (a preliminary study) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University
    (Massey University, 1995) Tin, Calvin Siu Fan
    The performance of a crossflow-microfiltration recycle reactor for whey-ethanol production was studied. Experiments using the yeast strain Kluyveromyces marxianus Y-113, an industrial whey-ethanol strain, and reconstituted acid whey permeate powder were carried out. Unsteady state experiments (i.e. with 100% cell recycle) were conducted at 46-137 g/l feed lactose concentration and dilution rates of 0.44-1.3 hr. These experiments were used to estimate the maximum specific growth rate (μ;), biomass substrate yield coefficient (Y), product substrate yield coefficient (Y). A mathematical model for biomass, lactose and ethanol concentration prediction was also developed. The model was based on Monid kinetics incorporating the concepts of a significant biomass volume fraction and single product inhibition. Two unsteady state experiments were conducted at 53.4-55.7 g/l lactose and dilution rate of 0.88-0.95 hr to check fermentation model accuracy. Two steady state runs at 64-110 g/l lactose, dilution rates of 0.34-0.43 hr were established for comparison with the unsteady state runs and to observe the effect of operation under stable conditions with the cell concentration regulated at 10 g/l.. Productivity increases of up to 13 times over the commercial batch fermentation process using the same organism was obtained. The highest productivity obtained was 13.7 g/l.hr. when the biomass was allowed to accumulate to 29.6 g/l, but lactose utilization (46%) and ethanol concentration (10.5 g/l) were low. In general, lower values of substrate utilization and ethanol concentration were noted at high dilution rates. At high feed lactose concentrations, lower lactose utilization was obtained. It was also noted that the growth rate was not significantly affected by substrate concentration and dilution rate. The product substrate coefficient (Y) was affected by dilution rate but independent of lactose concentration. Increasing dilution rate also decreased the biomass yield coefficient (Y) and the product substrate yield coefficient (Y). Further experiments are needed to better understand the effects of these parameters on yield coefficients. Steady state runs showed close agreement to the corresponding unsteady state experiments. Major problem of the fermenter operation was insufficient membrane flux which resulted in short fermentation runs at some condition. To solve this problem, a dual membrane configuration coupled with a permeate back flushing mechanism should be introduced. The mathematical model developed was adequate, but not optimal, an uncertainties of ± 30% and ± 20% in prediction of lactose and biomass concentrations were noted. While this was acceptable in the context of preliminary economic analysis and process optimization, to further improve the model accuracy, a relationship between the various yield coefficients and operating conditions has to be determined. Better estimation of the maximum specific growth rate (μ ) and incorporating a function to describe the variation of specific growth rate (μ) with biomass and ethanol concentrations is needed. More accurate estimation of the biomass substrate yield coefficient (Y) is also necessary for further model refinement. In conclusion, the crossflow-mircofiltration recycle fermenter has demonstrated potential application in whey-ethanol production with much improved productivity over current commercial and batch systems. Further studies are needed to determine its performance as compared to other intensive fermenter designs. The mathematical model developed also provides sufficient accuracy for preliminary process economic analysis and for process optimization study.