粗糙脉孢菌(Neurospora crassa)木糖发酵的研究

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌(Neurospora crassa)AS3．1602木糖发酵的影响。结果表明,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行,此时乙醇的转化率达到63．2％。木糖醇发酵适合在微好氧的条件下进行,转化率达到31．8％。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响,乙醇产生最适pH5．0,木糖醇产生最适pH4．0。在培养基pH为碱性条件时,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用,对乙醇和木糖醇的产生也有很大的影响。     

Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

Xylitol production by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

A methanol yeast, Candida boidinii no. 2201, could produce xylitol and also d-xylulose during cultivation on d-xylose medium. These fermentative products were identified by high performance liquid chromatography. A large amount of xylitol was obtained from a d-xylose medium containing ammonium acetate and yeast extract at the initial pH of 7.0. Maximum productivities of xylitol and enzymes concerned with the production were observed after 4–5 d of cultivation. A d-xylose (100 g/l) medium supplemented with 2% (v/v) methanol gave higher amounts of xylitol (48.5 g/l) and d-xylulose (3.3 g/l). Enzyme activities for d-xylose reduction, d-xylulose reduction, d-xylose isomerization, and d-xylulose phosphorylation, which could be involved in the xylitol production, were measured in cell-free extracts during cultivation and a possible pathway of xylitol production was discussed.     

Fed-batch xylitol production with recombinantXYL-1-expressingSaccharomyees cerevisiae using ethanol as a co-substrate

The bioconversion of xylose into xylitol in fed-batch fermentation with a recombinantSaccharomyces cerevisiae strain, transformed with the xylose-reductase gene ofPichia stipitis, was studied. When only xylose was fed into the fermentor, the production of xylitol continued until the ethanol that had been produced during an initial growth phase on glucose, was depleted. It was concluded that ethanol acted as a redox-balance-retaining co-substrate. The conversion of high amounts of xylose into xylitol required the addition of ethanol to the feed solution. Under O₂-limited conditions, acetic acid accumulated in the fermentation broth, causing poisoning of the yeast at low extracellular pH. Acetic acid toxicity could be avoided by either increasing the pH from 4.5 to 6.5 or by more effective aeration, leading to the further metabolism of acetic acid into cell mass. The best xylitol/ethanol yield, 2.4 gg^–1 was achieved under O₂-limited conditions. Under anaerobic conditions ethanol could not be used as a co-substrate, because the cell cannot produce ATP for maintenance requirements from ethanol anaerobically. The specific rate of xylitol production decreased with increasing aeration. The initial volumetric productivity increased when xylose was added in portions rather than by continuous feeding, due to a more complete saturation of the transport system and the xylose reductase enzyme.     

Effects of borate on isomerization and yeast fermentation of high xylulose solution and acid hydrolysate of hemicellulose

d-Xylose has been isomerized by immobilized d-glucose isomerase (EC nomenclature is now d-xylose isomerase, d-xylose ketol-isomerase, EC 5.3.1.5; EC 5.3.1.18 is a deleted EC entry). Temperature has a profound influence on the equilibrium concentration of d-xylulose. When 1 md-xylose was isomerized in the presence of various concentrations of borate, maximum conversion (80%) was observed at 0.2 m sodium tetraborate. Temperature (40–69°C) and pH (6.0–7.5) had an insignificant effect on the equilibrium when borate was present. d-Xylose (0.5 m) was isomerized by d-glucose isomerase in the presence of various concentrations of sodium tetraborate (0.0125–0.25 m). Based on the initial rate of ethanol production and the fraction of total sugar converted into ethanol after 24 h of yeast fermentation, an optimum tetraborate concentration of 0.05 m was determined for both isomerization and fermentation. At an acidic pH, the rate of fermentation was faster than at neutral pH when borate was included in the d-xylose—d-xylulose system. Acid hydrolysate of bagasse hemicellulose could not be fermented at a pH lower than 5. Therefore, a compromise condition, pH 6.0, was chosen for fermentation.     

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p,Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD⁺/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.     

Promotive action of ceramics on yeast ethanol production,and its relationship to pH,glycerol and alcohol dehydrogenase activity

Summary A yeast strain, Saccharomyces cerevisiae KPY32 isolated from pito, a traditional West-African alcoholic beverage, was immobilized in porous ceramic beads as a means of improving its ethanol production. Stationary fermentation cultures at different temperatures were made using semi-synthetic medium and fermentation parameters including ethanol production, sugar consumption, cell growth and pH were monitored. Glycerol production, and the activity of alcohol dehydrogenase (ADH) of the various systems were monitored. It was found that immobilization of the yeast resulted in improved ethanol production, at conversion rates above 93% of the theoretical value. The pH of the immobilized systems was also stabilized at around 4.0, glycerol production was higher, and the ADH activities were higher than those of free-cell systems. Ethanol production at the high temperature of 37° C was also improved by immobilization. The promotive action was found to be related to the pH, presence of glycerol and the enhancement of ADH activity.Offprint requests to: B. Demuyakor     

A strain of <Emphasis Type="Italic">Meyerozyma guilliermondii</Emphasis> isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.     

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.     

Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

Impacts of main factors on bioethanol fermentation from stalk juice of sweet sorghum by immobilized Saccharomyces cerevisiae (CICC 1308)

In order to attain a higher ethanol yield and faster ethanol fermentation rate, orthogonal experiments of ethanol fermentation with immobilized yeast from stalk juice of sweet sorghum were carried out in the shaking flasks to investigate the effect of main factors, namely, fermentation temperature, agitation rate, particles stuffing rate and pH on ethanol yield and CO(2) weight loss rate. The range analysis and analysis of variance (ANOVA) were applied for the results of orthogonal experiments. Results showed that the optimal condition for bioethanol fermentation should be A(4)B(3)C(3)D(4), namely, fermentation temperature, agitation rate, particles stuffing rate and pH were 37 degrees C, 200rpm, 25% and 5.0, respectively. The verification experiments were carried out in shaking flasks and 5L bioreactor at the corresponding parameters. The results of verification experiments in the shaking flasks showed that ethanol yield and CO(2) weight loss rate were 98.07% and 1.020gh(-1), respectively. The results of ethanol fermentation in the 5L bioreactor showed that ethanol yield and fermentation time were 93.24% and 11h, respectively. As a result, it could be concluded that the determined optimal condition A(4)B(3)C(3)D(4) was suitable and reasonable for the ethanol fermentation by immobilized Saccharomyces cerevisiae. The conclusion in the research would be beneficial for application of ethanol fermentation by immobilized S. cerevisiae from stalk juice of sweet sorghum.     

Ethanol production from pentoses by immobilized microorganisms

The capabilities of immobilized Fusarium oxysporum f. sp. lini, Mucor sp., and Saccharomyces cerevisiae in fermenting pentose to ethanol have been compared. S. cerevisiae was found to have the best fermentation rate on d-xylulose of 0.3 g l^?1 h^?1. By using a separate isomerase column for converting d-xylose to d-xylulose and a yeast column for converting d-xylulose to ethanol, an ethanol concentration of 32 g l^?1 was obtained from 10% d-xylose. The ethanol yield was calculated to be 64% of the theoretical yield.     

β-葡萄糖苷酶在酿酒酵母表面的表达

应用表面表达技术对来自Trichodermareesei的β-葡萄糖苷酶在酿酒酵母表面的表达及后期性质进行了研究。实验结果表明酵母表面表达酶有活性,该酶的最佳诱导时间为24h,最适温度是70℃,而酶活的最适pH是5．5。使异源表面表达了Bgl1的酵母在以纤维二糖为唯一碳源的培养基中生长,发酵结果表明纤维二糖被明显利用了,但在培养186h后,发酵液中仍残留一定量的纤维二糖。这种技术对纤维素发酵系统中纤维二糖酶活性低的现状有所帮助。     

Production of xylitol from D-xylose by a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis

Xylitol dehydrogenase (XDH) is one of the key enzymes in d-xylose metabolism, catalyzing the oxidation of xylitol to d-xylulose. Two copies of the XYL2 gene encoding XDH in the diploid yeast Candida tropicalis were sequentially disrupted using the Ura-blasting method. The XYL2-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing d-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter(-1) h(-1), a specific productivity of 0.76 g g(-1) h(-1), and a xylitol yield of 98%. This is the first report of gene disruption of C. tropicalis for enhancing the efficiency of xylitol production.     

Batch and membrane-assisted cell recycling in ethanol production byCandida shehatae

Summary During xylose fermentation byCandida shehatae ATCC 22984 with batch cell recycling, the volumetric ethanol fermentation rate increased two-fold, and the xylitol production rate increased three-fold as the cell density increased to ten-fold. In continuous fermentation with membrane-assisted cell recycle, the fermentation rates increased almost linearly with increasing agitation rates up to 300 rpm. The maximum continuous ethanol production rates obtained with 90 and 200 g L^–1 xylose were respectively 2.4 and 4.4 g L^–1h^–1. The cell density was 65–70 g (dry wt) L^–1. Ethanol yields ranged from 0.26 to 0.41 g g^–1.     

Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Metabolic engineering of the initial stages of xylose catabolism in yeasts for construction of efficient producers of ethanol from lignocelluloses

Plant biomass possesses a huge potential as a source for biofuel production. The main components of biomass are glucose and five-carbon sugar xylose. The yeast Saccharomyces cerevisiae that is used for industrial ethanol production from glucose is unable to xylose fermentation. Therefore a microorganism capable for efficient fermentation of both glucose and xylose has to be found in nature or constructed for economically feasible biomass conversion to ethanol. The active xylose fermentation could be performed by increasing the efficiency of initial stages of xylose metabolism. In this review the enzymes of initial stages of xylose metabolism in yeasts (xylose reductase, xylitol dehydrogenase, xylulokinase) and bacteria (xylose isomerase and xylulokinase) are characterized. The ways for construction of yeast strains capable of efficient alcoholic xylose fermentation are discussed.