Xylose fermentation by yeasts

Summary Xylose reductase from the xylose-fermenting yeastPichia stipitis was purified to electrophoretic homogeneity via ion-exchange, gel and affinity chromatography. At physiological pH values the thermodynamic equilibrium constant was determined to be 0.575x10¹⁰ (l·mol^-1). Product inhibiton studies are reported which clearly show that the kinetic mechanism of the xylose reductase is ordered-bi-bi with isomerisation of a stable enzyme form.     

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), baker's yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by baker's yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants.Correspondence to: M. Ciriacy     

Xylose fermentation by yeasts. 5. Use of ATP balances for modeling oxygen-limited growth and fermentation of yeast Pichia stipitis with xylose as carbon source

Kinetic studies are presented for the growth and fermentation of the yeast Pichia stipitis with xylose as the carbon source. Ethanol is produced from xylose under anaerobic as well as under oxygen-limiting conditions but only at dissolved oxygen concentrations up to 3 mumol/L Maximum yields and production rates were obtained under oxygen-limiting conditions, where the xylose metabolism may be considered to be consisted of three different components (assimilation, respiration, fermentation). The contribution of each pathway is determined by the availability of oxygen and the energy yield of each pathway. In order to describe the course of oxygen-limited fermentations, a mathematical model has been developed with the assumption that growth is coupled to the energy production. The resulting model requires only four independent parameters (Y(x/O(2) ), Y(ATP) (max), m(ATP), and P/O). These parameters were estimated on the basis of eight separate batch fermentations.     

D-xylulose fermentation in yeasts

Summary With pure D-xylulose as substrate, Schizosaccharomyces pombe produced ethanol in good yield with low quantities of polyols as by-products. Saccharomyces cerevisiae was found to be a good alcohol producer in glucose but not as good in D-xylulose. Other yeast cultures converted D-xylulose to xylitol, or D-arabitol or both, with lower ethanol yield.     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.     

A simplified routine method for determining carbohydrate fermentation by yeasts

The use of commercially available materials in a minimal medium contributes to the simplicity, reliability, and reproducibility of a method described to demonstrate carbohydrate fermentation reactions by yeasts. The medium consists of 1% yeast extract and 2% test carbohydrate in distilled water, dispensed in a modified Durham fermentation tube. Carbohydrate fermentation patterns can usually be obtained within a period of 7 days. The ability of yeasts to ferment carbohydrates is determined strictly on the basis of gas production from the substrate. The method proved reliable in reproducing established fermentation patterns for 112 different yeast strains representing 13 separate genera.

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Zusammenfassung Der Gebrauch von handelsüblichen Grundbestandteilen in einem geringen Medium trägt zu der Einfachheit, Zuverlässigkeit und Reproduzierbarkeit der beschriebenen Methode, um die Gärungsreaktionen der Karbohydrate durch Hefen zu veranschaulichen, bei. Das Medium besteht aus einem 1 prozent. Hefenauszug und aus einem 2 prozentigen Testkarbohydrat in destilliertem Wasser, die auf modifiziertes Durham Gärungsreagensglas verteilt sind. Das Karbohydrat-Gärungsmodell kann gewöhnlich innerhalb einer Periode von sieben Tagen erhalten werden. Die Fähigkeit von Hefen, Karbohydrate zu vergären, ist ausschließlich auf Grund der Gasentwicklung aus den Produkten bestimmt. Die Methode erwies sich zuverlässig in der Erzeugung festgestellter Gärungsmodelle aus 112 Hefen, die 13 verschiedene Gattungen darstellten.

     

Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha

Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose to ethanol. H. polymorpha most actively fermented sugars to ethanol at 37 degrees C, whereas the well-known xylose-fermenting yeast Pichia stipitis could not effectively ferment carbon substrates at this temperature. H. polymorpha even could ferment both glucose and xylose up to 45 degrees C. This species appeared to be more ethanol tolerant than P. stipitis but more susceptible than Saccharomyces cerevisiae. A riboflavin-deficient mutant of H. polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with riboflavin. Mutants of H. polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose, whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant.     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.     

Ethanol fermentation of HTST extruded rye grain by bacteria and yeasts

High temperature extrusion cooking of rye was used as a pretreatment for ethanol fermentation, and yeasts and bacteria were compared for their fermentation rates. Extrusion cooking caused, on average, a 7.5% increase in ethanol yield in comparison to autoclaved samples. The best results were achieved for grain with a moisture of 21-23% which was extruded at temperatures of 160-180 °C. Extrusion decreased the relative viscosity of rye grain water extracts, so it was possible to mash it without α-amylase. The efficiency of fermentation of extruded rye without Termamyl was equal to that of autoclaved and traditionally mashed rye (using α-amylase). The rate of fermentation of extruded rye grain by Zymomonas was higher during the first stage, but the final ethanol yield was similar for the bacterium and the yeast. Though both microorganisms gave good quality distillates, the concentration of compounds other than ethanol achieved from extruded rye mashes, which were fermented by Z. mobilis, was five times lower than for yeasts.     

Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts

The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis. All the cultures fermented D-glucose with the formation of ethanol. During the assimilation of D-xylose, ethanol was produced by P. stipitis and C. shehatae, whereas xylitol was produced by C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, K. fragilis, K. marxianus, P. guillermondii, and T. molishiama. The yeast P. tannophilus produced comparable amounts of both alcohols. The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.     

Protein enrichment of sweet potato residue with amylolytic yeasts by solid-state fermentation

Starchy agricultural wastes were inoculated with amylolytic yeasts for protein enrichment by solid-state fermentation. The moisture content of substrate was 65-69%, and water activity was equivalent to 0.98-0.99. The optimum conditions for protein enrichment were initial moisture content 65%, initial pH 4.5, a 1:1 mixture of ammonium sulfate and urea was incrementally added to the ferment with 1% added at zero time, 1% added at 24 h, and 0.5% added at 48 h, and incubation with amylolytic yeasts (1.0 x 10(10)/100 g substrate) at 30 degrees C for 2-3 days. The final product contained 16.11-20.82% protein.