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Item Investigations on the hexose-phosphorylating enzymes in the pentose-fermenting yeast, Pachysolen tannophilus : a thesis presented in partial fulfilment of the requirements for the degree in Doctor of Philosophy in Microbiology at Massey University(Massey University, 1988) Wedlock, David NeilMutants of Pachysolentannophilus, resistant to2- deoxyglucose, the toxic analogue of D-glucose, have been isolated and characterised. Their growth characteristics on hexose and pentosesugars, resistance to 2-deoxyglucose and cellular hexose-phosphorylating activities were investigated. Loss of hexose-ATP-kinase activity was found to correlate with loss of ability to grow on hexose sugars and increased resistance to 2-deoxyglucose. The growth of these mutants on D-xylose was not affected. A further series of fructose-negative and glucose-negative mutants were isolated by selecting for increased resistance To 2-deoxyglucose and by UV mutagenesis. Mutants, defective in each of the three hexose-phosphorylating enzymes found to be present in this yeast, were completely negative for growth on D-glucose,but could slowly convert this sugar to D-fructose. The conversion of D-glucose to D-fructose was hypothesised to be catalysed by the enzymes xylosereductase and xylitoldehydrogenase and experiments were conducted to investigate this possibility. Cell-free extracts from the wild type strain and several of The glucose-negative mutants were chromatographed on DEAE cellulose. The results of hexokinaseassays and anion exchange chromatography confirmed the existence of three hexose-phosphorylating enzymes in P.tannophilus. Two hexokinases which phosphorylated both D-glucose and Dfructose, exhibited F/Gratiosof1.3/1.0and3.0/1.0, while a glucokinase specific for D-glucose was also present.These enzymes were referred to ashexokinaseA and Bandglucokinase. Examination of the hexose-ATP-kinase profiles on DEAE- cellulose glucose, glucokinase of the wild type extract from cells grown on DO -xylose and glycerol indicated that the andhexokinaseB were constitutive, while hexokinaseA was inducible. Glucose repression ofxylosereductase and xylitol dehydrogenase was found to require an active hexokinaseA enzyme. This enzyme was purified from a glucokinase defective mutant by DEAE-cellulose chromatography, followed by affinity chromatography on CibacronBlueF3G-ASepharose (BlueSepharose) and examined further. The Km values for D-glucose and D-fructose were 0.36 and 2.28mM respectively. An estimated Vmaxfructose/Vmaxglucose was 1.5/1.0. When incubated with D-xylose in the presence of MgCl2 and ATP, the enzyme was inactivated. A strain of Pachysolentannophilus, defective in all three hexose-phosphorylating enzymes, was transformed with a plasmid carrying the cloned PII hexokinase gene from Saccharomycescerevisiae.The gene was expressed and the presence of the enzyme within the cells was demonstrated by DEAE-cellulose chromatography of a cell-free extract. As part of the overall plan to attempt genetic improvement in P.tannophilus, two superior ethanol producing mutants were hybridised and the segregants made available for fermentation trials at the Forest Research Institute. Hexose-negative mutants able to fermen D-xyloseinthe presence of D-glucose were selected for and subjected to fermentation trials. Several of these mutants produced promising concentrations and yields of ethanol from the fermentation of D-xylose, both as a sole carbon source and in a mixture of D-glucoseandD-xylose.Item Ethanolic fermentation of D-xylose and pine wood hydrolyzate by the yeast Pachysolen tannophilus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University(Massey University, 1984) Wong, Tze SenThis thesis reports a study of the ethanolic fermentation of D-xylose and wood hydrolyzate to ethanol by the yeast Pachysolen tannophilus with a view to developing an effective use of renewable hemi-cellulose hydrolysis products from New Zealand forest biomass residues. Initial work briefly addressed the problem of finding a suitable yeast from natural habitats suitable for the fermentation. Soon after that work commenced literature reports suggested that preliminary conversion of pentoses by enzymatic means was a possibility. Consequently, this aspect of conversion was considered and rejected. One reason for this was that literature was drawing attention to the pentose fermenting characteristics of Pachysolen tannophilus. Laboratory scale studies demonstrated the yeast Pachysolen tannophilus to be capable of fermenting the hexose and pentose sugars present in the hydrolyzate. The yeast's specific growth rate in the hydrolyzate could be improved by neutralizing the inhibitory substances with 2 g/l of anhydrous sodium sulphite. Ethanol has an inhibitory effect on growth but can also be readily assimilated by the yeast. Fermentation studied with gyration speeds of 50, 100 and 200 r.p.m. showed that oxygen was a critical parameter affecting growth and ethanol production. Batch fermentation experiments were pursued to examine this oxygen phenomenon more closely. Cell growth, substrate uptake rate and culture pH responded strongly to the supply of oxygen. However, production of ethanol accompanied cell growth only in late "exponential" phase. Fermentation characteristics were established under continuous culture at an aeration rate of 0.37 l/l.min and values obtained were as follows; maximum specific growth rate, 0.046 h-1; biomass yield, 0.04 g/g; ethanol yield, 0.17 g/g; Ks value, 13 g/l and Ki values, 0.5 g/l. A redox potential controlled chemostat study revealed that steady-state culture poised at -50 mV exhibited a 55% increased ethanol concentration and 43% decreased xylitol concentration over the value observed without redox control. With a knowledge or D-xylose fermentation as established in these batch and chemostat experiments, it was possible to consider more detailed aspects of the fermentation which would be applicable to process development. Questions addressed included which strain of Pachysolen tannophilus should be used, what quantity of inoculum was necessary, what interactions existed between fermentation variables. Statistically designed experiments were employed to answer these questions. Empirical models so developed revealed that ethanol yield has a linear relationship with initial substrate concentration. These models have given some insight into how environmental factors affect the ethanolic fermentation by this yeast and have also indicated the optimal conditions required for an effective fermentation of wood pentoses. These important fermentation process variables were established and are expected to be useful in moving the process from laboratory scale as carried out here to a pilot plant scale of operations. The values established were temperature, 28° or lower; initial medium pH for ethanol production, 5.6 to 5.8; substrate concentration used can be up to 80 g/l of pentoses; minimum inoculum density, 5.5 g/l dry weight cells and NRRL Y-2461 was recommended as the best strain to achieve the fermentation. The pre-treatment of the prehydrolyzate by 2 g/l of anhydrous sodium sulphite was highly desirable in order to enhance growth and fermentation rates. The research has shown that Pachysolen tannophilus is capable of fermenting pentose fraction of wood hydrolyzate and that the optimal conditions for this fermentation will lead to significant utilization of wood sugar. However, in the completely mixed reactor systems used in these experiments, the ethanol yields obtained were not as attractive as those observed for hexose fermentations under similar conditions. This, it is felt, points to the greater difficulty the yeast experiences in fermenting pentoses and it also suggests the need to investigate the value of other reactor formats at seme future date.