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.     

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.     

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.     

Ethanol production from d-galactose and glycerol by Pachysolen tannophilus

Pachysolen tannophilus has recently been shown to be able to convert d-xylose, a pentose, to ethanol. Previously, d-xylose had been considered to be nonfermentable by yeasts. The present study shows that the organism can be used to obtain ethanol from other carbohydrates previously considered as nonfermentable, either by P. tannophilus in particular, d-galactose, or by yeasts in general, glycerol. Such identification for d-galactose allows P. tannophilus to be considered for fermentation of four of the five major plant monosaccharides: d-glucose, d-mannose, d-galactose and d-xylose. The ability to ferment glycerol is of potential use, in part, for the conversion of glycerol derived from algae into ethanol.     

Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.     

Factors in acid treated bagasse inhibiting ethanol production from d-xylose by Pachysolen tannophilus

The fermentation of d-xylose, the major sugar-cane bagasse hemicellulose component, to ethanol by Pachysolen tannophilus is inhibited by various factors produced or released during the acid hydrolysis of the bagasse or during the fermentation process. These include ethanol, iron, chromium, copper, nickel, acetic acid and furfural. Ethanol production by P. tannophilus is inhibited by ethanol fconcentrations >24 g l^?1. Furfural and acetic acid concentrations as low as 0.3 and 7 g l^?1, respectively, and iron, chromium, nickel and copper at concentrations of 0.07, 0.01, 0.01 and 0.004 g l^?1, respectively. Similar concentrations may be found in acid-hydrolysed bagasse. The removal of these factors by treatment with ion-exchange resin resulted in the fermentation of the sugars to ethanol. The d-glucose was used rapidly and completely whereas d-xylose utilization was slow and incomplete. An ethanol concentration of 4.1 g l^?1 was produced and an ethanol yield of 0.32 was obtained. Xylitol in significant amounts was produced.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

反相高效液相色谱在发酵制备琥珀酸中的应用

对于生物法制备琥珀酸的微生物发酵体系,利用Alltech反相Prevail C18色谱柱,以25mmol／L磷酸二氢钾（pH2.5）作为流动相,在流速1mL／min时,于210nm处紫外检测器检测,能将发酵液中琥珀酸、甲酸、乙酸和乳酸完全分离并准确定量。琥珀酸等有机酸的回收率在96％～104％之间。本方法能够快速、精确测定发酵样品中主产物琥珀酸与其它有机酸含量。     

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.     

Potential utilization of sorghum field waste for fuel ethanol production employing Pachysolen tannophilus and Saccharomyces cerevisiae

In this study, we demonstrate that the sorghum field waste, sorghum stover could be used to produce fuel grade ethanol. The alkaline treatment of 2% NaOH for 8h removed 64% of lignin from sorghum stover. Maximum of 68 and 56 g/L of ethanol yield were obtained by Saccharomyces cerevisiae (MTCC 173) and Pachysolen tannophilus (MTCC 1077) from sorghum stover under optimized condition, respectively. pH and temperature were optimized for the better growth of S. cerevisiae and P. tannophilus. A total of 51% and 48% more ethanol yield was obtained at initial sugar concentration of 200 g/L than 150 g/L by P. tannophilus and S. cerevisiae, respectively. Respiratory deficiency and ethanol tolerance of the organisms were studied. This investigation showed that sorghum field waste could be effectively used for the production of fuel ethanol to avoid conflicts between human food use and industrial use of crops.     

Effect of Nystatin on the Metabolism of Xylitol and Xylose by Pachysolen tannophilus

Ethanol production from xylitol by resting cells of Pachysolen tannophilus was increased 40-fold in the presence of nystatin, amphotericin B, and filipin, a group of antifungal agents that alter the permeability of the plasma membrane. Furthermore, these agents had little or no effect on ethanol formation from xylitol or xylose by the cell extract. During xylose metabolism, nystatin caused the intracellular xylitol to leak out into the medium at a 23-fold-faster rate but did not affect overall xylose utilization and CO₂ evolution. These observations explain the rate of xylitol utilization by cell extract being higher than that by whole cells (J. Xu and K. B. Taylor, Appl. Environ. Microbiol. 59:231-235, 1993) as well as the relative inability of P. tannophilus to utilize xylitol to support significant ethanol production and cell growth.     

The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus

Summary The ability of a Candida shehatae and a Pachysolen tannophilus strain to ferment D-xylose to ethanol was evaluated in defined and complex media under different levels of aeration. Aeration enhanced the ethanol productivity of both yeasts considerably. C. shehatae maintained a higher fermentation rate and ethanol yield than P. tannophilus over a wide range of aeration levels. Ethanol production by C. shehatae commenced during the early stage of the fermentation, whereas with P. tannophilus there was a considerable lag between the initiation of growth and ethanol production. Both yeasts produced appreciable quantities of xylitol late in the fermentation. P. tannophilus failed to grow under anoxic conditions, producing a maximum of only 0.5 g · l^-1 ethanol. In comparison, C. shehatae exhibited limited growth in anoxic cultures, and produced ethanol much more rapidly. Under the condition of aeration where C. shehatae exhibited the highest ethanol productivity, the fermentation parameters were: maximum specific growth rate, 0.15 h^-1; maximum volumetric and specific rates of ethanol production, 0.7 g (l · h)^-1 and 0.34 g ethanol (g cells · h)^-1 respectively; ethanol yield, 0.36 g (g xylose)^-1. The best values obtained with P. tannophilus were: maximum specific growth rate, 0.14 h^-1; maximum volumetric and specific rates of ethanol production, 0.22 g (l · h)^-1 and 0.07 h^-1 respectively; ethanol yield coefficient, 0.28. Because of its higher ethanol productivity at various levels of aeration, C. shehatae has a greater potential for ethanol production from xylose than P. tannophilus.     

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.     

Characterization of hybrids obtained by protoplast fusion,between Pachysolen tannophilus and Saccharomyces cerevisiae

The genes for utilization of xylose were transferred from Pachysolen tannophilus to Saccharomyces cerevisiae. The hybrids resembled the S. cerevisiae parent morphologically and in sugar assimilation. Pulsed field gel electrophoresis showed that the chromosome banding pattern was intermediate between the two parental species. Correspondence to: H. Heluane     

Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid

A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.     

Mutants of Pachysolen tannophilus that produce enhanced amounts of acetic acid from D-xylose and other sugars

Summary Two mutants of Pachysolen tannophilus were isolated which produced considerably more acetic acid from several sugars than a wild type strain. Such mutants are of potential interest for the production of acetic acid rather than ethanol from lignocellulosic hydrolysates.Issued as NRCC No. 20810.     

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.     

Bioconversion of wheat straw cellulose/hemicellulose to ethanol by Saccharomyces uvarum and Pachysolen tannophilus

The information presented in this publication represents current research findings on the production of glucose and xylose from straw and subsequent direct fermentation of both sugars to ethanol. Agricultural straw was subjected to thermal or alkali pulping prior to enzymatic saccharification. When wheat straw (WS) was treated at 170 degrees C for 30-60 min at a water-to-solids ratio of 7:1, the yield of cellulosic pulp was 70-82%. A sodium hydroxide extration yielded a 60% cellulosic pulp and a hemicellulosic fraction available for fermentation to ethanol. The cellulosic pulps were subjected to cellulase hydrolysis at 55 degrees C for production of sugars to support a 6-C fermentation. Hemicellulose was recovered from the liquor filtrates by acid/alcohol precipitation followed by acid hydrolysis to xylose for fermentation. Subsequent experiments have involved the fermentation of cellulosic and hemicelluosic hydrolysates to ethanol. Apparently these fermentations were inhibited by substances introduced by thermal and alkali treatment of the straws, because ethanol efficiencies of only 40-60% were achieved. Xylose from hydrolysis of wheat straw pentosans supported an ethanol fermentation by Pachysolen tannophilus strain NRRL 2460. This unusual yeast is capable of producing ethanol from both glucose and xylose. Ethanol yields were not maximal due to deleterious substances in the WS hydrolysates.     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002