Microaerophilic metabolism of Pachysolen tannophilus at different pH values

Microaerophilic production of xylitol by Pachysolen tannophilus from detoxified hemicellulose hydrolyzate was optimal between pH values 6.0 to 7.5 when about 90% of xylose was utilized for xylitol production, the rest being fermented to ethanol. At pH values of 3.0 and 12.0, respiration became important, consuming up to 30% of available xylose. A graphic procedure suggests that histamine and cysteine are at the active site of xylose reductase in this yeast.     

Assignment of cloned genes to electrophoretically separated chromosomes of the yeast Pachysolen tannophilus

An electrophoretic karyotype of Pachysolen tannophilus has been obtained using Pulsed Field Gel Electrophoresis. Seven chromosomal bands were separated with one of the bands migrating probably as a doublet. The sizes of the chromosomes were estimated to be between 1 and 3.1 megabase pairs. Eleven loci have been assigned to chromosomal bands, including four involved in the metabolism of D-xylose.     

Reversible inactivation of d-xylose utilization by d-glucose in the pentose-fermenting yeast Pachysolen tannophilus

Abstract A major problem in fermenting pentoses using lignocellulosic substrates is the presence of d -glucose which inhibits d -xylose utilization. We previously showed that d -glucose represses the induction of xylose reductase and xylitol dehydrogenase activities, thereby inhibiting d -xylose utilization in Pachysolen tannophilus . The question arose whether d -glucose can also inactivate d -xylose fermentation. P. tannophilus cells were grown on a defined d -xylose-containing liquid medium. At about 40 h, d -glucose was added to a final concentration of 3% (w/v). This led to a rapid cessation of d -xylose utilization, which resumed after 10–12 h before d -glucose was completely consumed. This suggests that d -glucose inactivated existing d -xylose catabolic enzymes and that inactivation was reversed at low d -glucose concentrations. This reversible inactivation was distinct from d -glucose repression. Addition of cycloheximide did not block the resumption of d -xylose consumption, suggesting that reactivation was independent of protein synthesis.     

Mutants of Pachysolen tannophilus showing enhanced rates of growth and ethanol formation from d-xylose

Mutants of Pachysolen tannophilus NRRL Y-2460 have been sought that show enhanced rates of d-xylose fermentation. Mutagenesis followed by enrichment in urea-xylitol broth generally resulted in a lower frequency of good ethanol producers than enrichment in nitrate-xylitol broth. Under aerobic conditions, the best xylose-fermenting strains (which were obtained from nitrate-xylitol broth) produced ethanol from xylose twice as fast and in 32% better yield than the parent strain. Under anaerobic conditions, these strains produced ethanol from xylose 50% faster than (but in the same yield as) the parent strain. These findings show that enrichment in nitrate-xylitol broth is a promising method for obtaining mutants of Pachysolen having enhanced fermentation rates.     

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger

Abstract Resting cells of the fission yeast Schizosaccharomyces pombe , suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen iannophilus , the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.     

Fermentation of acid hydrolysates from olive-tree pruning debris by Pachysolen tannophilus

The influence of the type and concentration of acid in the hydrolysis process and its effect on the subsequent fermentation by Pachysolen tannophilus (ATCC 32691) to produce ethanol and xylitol was studied. The hydrolysis experiments were performed using hydrochloric, sulphuric and trifluoroacetic acids in concentrations ranging from 0.1 to 1.0 N, a temperature of 90 degrees C, and a time of 240 min. The fermentation experiments were conducted on a laboratory scale in a batch-culture reactor at pH 4.5 and 30 degrees C. The hydrolysis with the highest acid concentration produced the complete solubilization of hemicellulose to monosaccharides. The highest values for the specific rate of ethanol production were registered in cultures hydrolyzed with trifluoroacetic acid, and values were found to decrease as the acid concentration increased. The highest values of overall ethanol yields ( [Formula: see text] = 0.37 kg kg(-1)) were also found in the fermentation of the hydrolysates of trifluoroacetic acid.     

Acetone stimulation of ethanol production from d-xylose by Pachysolen tannophilus

Summary Pachysolen tannophilus, a homothallic yeast, converts xylose to ethanol at a yield of 0.3 (g/g xylose). Concomitant with ethanol production, xylitol accumulates in the culture medium at similar yields (0.3 g/g xylose). The addition of the hydrogen-accepting compound, acetone, increases the amount of ethanol produced by this organism by 50–70%. The increase in ethanol is directly correlated with a decrease in xylitol secreted. The results indicate that conversion of acetone to 2-propanol by the cells provides the NAD⁺ used as a cofactor by xylitol dehydrogenase, the enzyme responsible for converting xylitol to xylulose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.     

Characterization of Ethanol Production from Xylose and Xylitol by a Cell-Free Pachysolen tannophilus System

Whole cells and a cell extract of Pachysolen tannophilus converted xylose to xylitol, ethanol, and CO₂. The whole-cell system converted xylitol slowly to CO₂ and little ethanol was produced, whereas the cell-free system converted xylitol quantitatively to ethanol (1.64 mol of ethanol per mol of xylitol) and CO₂. The supernatant solution from high-speed centrifugation (100,000 × g) of the extract converted xylose to ethanol, but did not metabolize xylitol unless a membrane fraction and oxygen were also present. Fractionation of the crude cell extract by gel filtration resulted in an inactive fraction in which ethanol production from xylitol was fully restored by the addition of NAD⁺ and ADP. The continued conversion of xylose to xylitol in the presence of fluorocitrate, which inhibited aconitase, demonstrated that the tricarboxylic acid cycle was not the source of the electrons for the production of xylitol from xylose. Therefore, the source of the electrons is indirectly identified as an oxidative pentose-hexose cycle.     

Oxygen requirements for d-xylose fermentation to ethanol and polyols by Pachysolen tannophilus

The oxygen requirements for ethanol production from d-xylose (10 or 20 g l^?1) by Pachysolen tannophilus have been determined by controlling the availability of oxygen to shake flasks. Under anaerobic conditions no ethanol was produced whereas under aerobic conditions mainly biomass was formed. Semi-anaerobic conditions resulted in maximum ethanol production. By varying the stirring speed of a fermenter and supplying air to the liquid surface at various rates, the oxygen transfer rate (OTR) was controlled under semi-anaerobic conditions. By increasing the OTR from 0.05 to 16.04 mmol l^?1 h^?1, the ethanol yield coefficient decreased from 0.28 to 0.18 while the cell yield coefficient increased from 0.14 to 0.22. The accumulation of polyols decreased from 0.88 to 0.56 g l^?1 with increasing OTR. At OTRs between 0.09 and 1.18 mmol l^?1 h^?1, specific ethanol productivity attained a maximum value of 0.07 h^?1 and decreased with either increasing or decreasing OTR. The results indicate that the OTR must be carefully controlled for efficient ethanol production from d-xylose by P. tannophilus.     

Mutants of Pachysolen tannophilus with Improved Production of Ethanol from d-Xylose

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of d-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanol-defective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. One also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.     

外加肌醇对嗜鞣管囊酵母发酵的酒精产量及其耐酒精能力的影响

嗜鞣管囊酵母(Pachysolen tannophilus)是可以同时发酵葡萄糖和木糖为酒精的菌种,在其生长和发酵培养基中分别添加不同浓度((0～200mg/L)的肌醇以及不同起始浓度的酒精,以考察外加肌醇对嗜鞣管囊酵母生长、产酒精能力和耐酒精能力的影响.结果 表明,添加肌醇前后,嗜鞣管囊酵母的生物量及发酵的酒精产量均有所增加.外加肌醇对嗜鞣管囊酵母生长有轻微的刺激作用,酵母生长最适肌醇浓度为150mg/L;而对酵母生长的耐酒精能力却有明显的影响, 并且,菌种在YEPD培养基中的耐酒精能力高于在YEPX培养基中的耐酒精能力.经实验测定,肌醇对嗜鞣管囊酵母产酒精能力及发酵的耐酒精能力均有显著的影响.发酵培养基中未添加起始浓度的酒精时,菌种发酵的最适肌醇浓度为100mg/L,此时生成的酒精产量为45.20g/L.当分别添加起始酒精浓度为10%和12%时,随着肌醇浓度的增加,菌种发酵生成的酒精浓度均呈上升趋势;肌醇浓度为200mg/L时,两种起始酒精浓度下,酒精的净生成量均达到最大,分别为17.18g/L和16.68g/L.     

Ethanol production from D-xylose and other sugars by the yeastPachysolen tannophilus

Summary D-Xylose was fermented to ethanol by a strain ofPachysolen tannophilus in yields greater than 0.3g ethanol per g xylose consumed. Ethanol production was influenced by xylose concentration and was at a maximum at 10%, w/v. Ethanol formation occurred at pH 2.75-2.50 but the yeast would not grow at this pH when the initial pH of the medium was less than 3.0. Ethanol was consumed by the yeast when the xylose concentration became limiting. L-Arabinose, D-glucose, D-fructose, cellobiose, D-glucuronic acid, but not sucrose,were also fermented to ethanol byPachysolen tannophilus. Kinetic studies on xylose fermentation established various parameters involved in growth, substrate utilization and ethanol formation when the yeast was fermenter grown.     

Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus

This research was designed to maximize ethanol production from a glucose-xylose sugar mixture (simulating a sugar cane bagasse hydrolysate) by co-fermentation with Zymomonas mobilis and Pachysolen tannophilus. The volumetric ethanol productivity of Z. mobilis with 50 g glucose/l was 2.87 g/l/h, giving an ethanol yield of 0.50 g/g glucose, which is 98% of the theoretical. P. tannophilus when cultured on 50 g xylose/l gave a volumetric ethanol productivity of 0.10 g/l/h with an ethanol yield of 0.15 g/g xylose, which is 29% of the theoretical. On optimization of the co-fermentation with the sugar mixture (60 g glucose/l and 40 g xylose/l) a total ethanol yield of 0.33 g/g sugar mixture, which is 65% of the theoretical yield, was obtained. The co-fermentation increased the ethanol yield from xylose to 0.17 g/g. Glucose and xylose were completely utilized and no residual sugar was detected in the medium at the end of the fermentation. The pH of the medium was found to be a good indicator of the fermentation status. The optimum conditions were a temperature of 30°C, initial inoculation with Z. mobilis and incubation with no aeration, inactivation of bacterium after the utilization of glucose, followed by inoculation with P. tannophilus and incubation with limited aeration.     

Concurrent Production and Consumption of Ethanol by Cultures of Pachysolen tannophilus Growing on d-Xylose

Growing cultures of Pachysolen tannophilus concurrently consumed and produced ethanol in the presence of substantial concentrations of d-xylose. Ethanol was also assimilated in the presence of other sugars, the amount depending on the sugar. Less ethanol assimilation occurred with d-glucose than with d-xylose. The rate of ethanol consumption decreased as the concentration of glucose was increased, but some consumption still occurred when 2% glucose was present. The rate increased with the amount of oxygen available to the culture when d-xylose or ethanol was the carbon source. In most instances, estimates of consumption were based on the extent of incorporation of C from [1-C]ethanol into trichloroacetic acid-insoluble material. The results are pertinent to the use of P. tannophilus for the production of ethanol from d-xylose.     

Multiple forms of xylose reductase in Pachysolen tannophilus CBS4044

Abstract Cell-free extracts of xylose-grown Pachysolen tannophilus exhibited xylose reductase activity with both NADPH and NADH. The ratio of the NADPH- and NADH-dependent activities varied with growth conditions. Affinity chromatography of cell-free extracts resulted in a separation of two xylose reductases. One was active with both NADPH and NADH, the other was specific for NADPH. Apart from this coenzyme specificity, the two enzymes also differed in their affinities for xylose and NADPH. The role of the two enzymes in xylose metabolism is discussed in relation to attempts to use P. tannophilus for the alcoholic fermentation of wood sugars.     

Mannitol production from glycerol by resting cells of Candida magnoliae

Production of mannitol from glycerol by resting cells of Candida magnoliae under aerobic condition was investigated. The resting cells were suspended in aqueous solution of glycerol in Erlenmeyer flasks and incubated on rotary shaker. The samples were analyzed by ion exclusion–HPLC equipped with refractive index and UV detector. The resting cells of C. magnoliae produced mannitol from fructose, sucrose and glycerol but not from glucose. Addition of yeast extract and/or potassium phosphate to the glycerol solution adversely affected its conversion to mannitol. The conversion of glycerol to mannitol was dependent on oxygen availability. Using resting cells, the yield of mannitol was as high as 45%. This is probably the first report of conversion of glycerol to mannitol by osmophilic yeast.     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.