Mutation breeding of Saccharomyces cerevisiae with lower methanol content and the effects of pectinase, cellulase and glycine in sugar cane spirits (original) (raw)
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Saccharomyces cerevisiae is one of the most promising unicellular fungi on account of its vital applications in biotechnology as well as bioethanol production. Improvement of ethanol production via very high-gravity (VHG) fermentation (fermentation at high sugar levels) was successfully developed using the ethidium bromide (EtB) mutagenesis of S. cerevisiae. This study found two developed mutants of S. cerevisiae (EtB20a and EtB20b) with varied capacity for ethanol production using EtB, depending on random amplified polymorphic DNA analysis. Mutant EtB20b showed improved ethanol yield (19.5%) compared with the wild-type (18.0%), while the other mutant EtB20a exhibited retarded ethanol production (9.1%). Optimization of ethanol production by mutant EtB20b was performed under other conditions including temperature, pH, inoculum size, and incubation period. The highest production capacity of the yeasts was 20.8, 19.9, 19.5, and 19.5% at an optimum temperature of 30 °C, pH 6.0, incubati...
New approaches for improving the production of the 1st and 2nd generation ethanol by yeast
Acta biochimica Polonica, 2015
Increase in production of 1st generation ethanol from glucose is possible by reduction in the production of ethanol co-products, especially biomass. We have developed a method to reduce biomass accumulation of Saccharomyces cerevisiae by the manipulation of the intracellular ATP level due to overexpression of genes of alkaline phosphatase, apyrase or enzymes involved in futile cycles. The strains constructed accumulated up to 10% more ethanol on a cornmeal hydrolysate medium. Similar increase in ethanol accumulation was observed in the mutants resistant to the toxic inhibitors of glycolysis like 3-bromopyruvate and others. Substantial increase in fuel ethanol production will be obtained by the development of new strains of yeasts that ferment sugars of the abundant lignocellulosic feedstocks, especially xylose, a pentose sugar. We have found that xylose can be fermented under elevated temperatures by the thermotolerant yeast, Hansenula polymorpha. We combined protein engineering of ...
Applied Microbiology and Biotechnology, 1990
Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250 mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C5 sugars gave the highest triose accumulation (ca. 20 mM after 45 h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resuited in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25 mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.
Theoretical Analysis of Media Used in the Growth of Yeasts on Methanol
Microbiology, 1981
A theoretical analysis has been made of five different media which have been used for growth of the methylotrophic yeasts Hansenula polymorpha and Candida boidinii. The media compositions were found to differ significantly in their content of trace elements. Chemostat studies revealed that low growth yields, particularly at high dilution rates, were probably due to unexpected limitations in trace elements.
Biochemical principles of the use of yeast biomass and lab starter cultures in food production
Acta Alimentaria, 2009
yeast is produced as low price by-product, which could be used for further processing and application in food production. In this review we refer to the research done on yeast SCP production with special concern on strain selection/breeding to increase the biological value and on optimization of fermentation parameters to increase the yield and to improve the protein composition. The methionine-deficient provision can be taken almost as people's disease and this too, contributes to frequent incidence of various pathological processes (hepatic disease, anaemia). The daily methionine requirement for an adult is about 2.8 g and this is satisfied only to about 63%, even in case of adequate protein consumption. In monogastric animals and humans methionine is an essential amino acid, and serves not only as S-containing amino acid but it is the only methyl donor. However, supplementation with methionine alone is not efficient, as only 30-40% of the added amino acid is utilized (GEBHARDT et al., 1977; BURACZEWSKA et al., 1977). It was found that nutrients enriched with yeast proteins gave better results (TREVIS, 1979), it is believed that the need exists rather for proteins rich in limiting amino acids (like methionine) in bound form in the protein molecule than in their free forms. KOMATSU and co-workers (1974) and OKANISHI and GREGORY (1970) successfully produced yeast mutants rich in methionine (about 40% higher), however the increase in methionine was entirely the result of the rise of methionine concentration, as pool amino acid (HALÁSZ et al., 1996). Aim of the research work in CFRI was to produce yeast strains with increased protein bound methionine content. Screening of wild yeasts to produce mutant with high methionine content was the first step (HALÁSZ, 1980), as methionine content of yeast vary between wide limits (CHIAO & PETERSON, 1953). The best wild strains were treated with different mutagenic agents and protoplast fusion technique was applied to produce methionine-rich mutants. Comparing the efficiency of UV treatment, gamma irradiation, NaNO 2 treatment and protoplast fusion the results are as follows. UV treatment: out of 2080 strains tested, two mutants with auxotrophic marker were successfully produced and were stable. The frequency was about 0.1%, the stability of the auxotrophs was 14%. The highest increase in methionine content was 25% (HALÁSZ et al., 1987). Gamma irradiation and NaNO 2 treatment proved to be not suitable for this purpose (MUAYAD et al., 1983). From the protoplast fusion of the Kluyveromyces lactis hybrids two mutants showed significant increase in methionine content (Table 1). These experiences led the research group to change the mutant selection method from auxotrophic ones as it was published in the literature, to other ones: the possible increase in SO 4 requirement, sensitivity to norleucine (LAWRENCE et al., 1968) and increased methyl donor requirement. It was stated that higher SO 4 content of the medium resulted in larger colonies. However methionine anti-metabolite norleucine was not suitable to select methionine rich mutants, as the mutants showed higher sensitivity than the parent strains. This finding is in contrast to that of others' findings
Microbial Cell Factories, 2010
Background: Glycerol is the major by-product accounting for up to 5% of the carbon in Saccharomyces cerevisiae ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.
Journal of Bioscience and Bioengineering, 2013
Although many studies on the different aspects of alcoholic fermentation are available in the literature, it is still difficult to identify the possible causes of the slowing-down or stuck of fermentations, even if the change of some compositional parameters (D-glucose/D-fructose and glycerine produced/hexoses converted ratios) could be assumed as sound signals of a possible deviation from the usual Saccharomyces metabolic pathways. The reason why alcoholic yeasts preferably metabolise D-glucose rather than D-fructose was investigated by a kinetic model based on six functional parameters having a well-defined chemicalephysical meaning. The time evolution of different initial concentrations of D-glucose and D-fructose, dissolved in a model solution simulating a must (citrate buffer at pH 3.4 inoculated by a commercial strain of Saccharomyces cerevisiae), was investigated adding or not ethanol to the reaction medium. When a reduced amount of ethanol was dissolved in the reaction medium, the time evolution of the fermentation rates of these two sugars did not differ significantly, to diversify rather strongly when the alcoholic concentration increased. The hypothesised mathematical model accounts for this particular kinetic behaviour. In fact, only the sensitivity to ethanol showed by the enzymatic protein involved in the limiting steps of the fermentation process of these two sugars differed significantly, the enzymatic transformation of D-fructose being more sensitive to ethanol than D-glucose. This difference was able to justify the different kinetic behaviours shown by the two sugars when ethanol concentration in the reaction medium increased.