Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D-xylose to D-xylulose

Summary A method to obtain the fermentative conversion by yeasts of D-xylose to ethanol is described. The method depends on a combination of two factors; (1) the ability of glucose isomerase to isomerise D-xylose to D-xylulose and (2) the ability of a number of yeasts to ferment D-xylulose.     

Direct fermentation of D-xylose to ethanol by a xylose-fermentating yeast mutant,Candida sp XF 217

Summary A mutant strain of Candida sp. XF 217, was found to produce ethanol from D-xylose aerobically as well as anaerobically. The rate of ethanol production under aerobic conditions was greater, indicating an oxygen requirement for the uptake of D-xylose in XF 217. Ethanol was also produced by XF 217 when D-glucose, D-fructose, sucrose or maltose were used as substrates. The D-xylose fermenting yeast strain is a potential organism to use for ethanol production from renewable biomass-derived hexoses and pentoses.     

D-xylose catabolizing enzymes in Neurospora crassa and their relationship to D-xylose fermentation

Summary The induction of xylose reductase (XR) and xylitol dehydrogenase (XD) activities by D-xylose under different fermentation conditions was investigated in Neurospora crassa. The induction of NADPH-linked XR preceded NADH-linked XR and the ratio of NADH to NADPH-linked XR activity displayed variation from 0.02 to 0.2 suggesting the presence of two separate enzymes. Aerobic conditions were required by N. crassa for cell growth but not for ethanol production. Maximum ethanol of 0.3 g/g of D-xylose was produced when shifted to semiaerobic condition, where high NADH-linked XR and NAD-linked XD activities were observed.     

D-xylose isomerase fromStreptomyces violaceus-ruber: Optimization of fermentation conditions

Summary Cultures ofStreptomyces violaceus-ruber produce considerable amounts of D-xylose isomerase when grown on D-xylose containing media supplemented with D-sorbitol as second carbon source. The D-xylose isomerase yield was optimized by evaluating the effects of different nutrient additions.     

Production of xylitol from D-xylulose by Mycobacterium smegmatis

Mycobacterium smegmatis transformed D-xylulose to xylitol in washed cell reactions under aerobic and anaerobic conditions. The yield of xylitol reached about 70% in anaerobic conditions (in N₂) by cells grown on media containing xylitol or D-mannitol. Cells immobilized with Ca-alginate had almost the same activity of xylitol production as washed cells.Xylitol was produced from D-xylose using commercial immobilized D-xylose isomerase from Bacillus coagulans and immobilized cells of M. smegmatis. From 10 g of D-xylose, 4 g of xylitol was produced and 5 g of D-xylose remained in the reaction mixture; no D-xylulose was detected.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Investigation of the synthesis of xylose/glucose isomerases in sixArthrobacter sp. strains

The substrate specificity of isomerases produced by six strains ofArthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A.ureafaciens B-6 strains showed low activity toward D-ribose,Arthrobacter sp. B-5 was slightly active toward L-arabinose, andA. ureafaciens B-6 andArthrobacter sp. B-2239, toward L-rhamnose. InArthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources. The synthesis of xylose/glucose isomerase induced by D-xylose inArthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol inA. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in theArthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.     

Viability of Candida shehatae in D-xylose fermentations with added ethanol

Ethanol was added at concentrations of 25 and 50 g/L to active cultures of Canida shehatae under oxygen-limited (fermentative) conditions. Added ethanol completely inhibited grwoth and fermentation of D-xylose by C. shehatae. Cultures with added ethanol rapidly declined in cell viability as measured by plate counts and methylene blue staining. The rate of decline in cell viability was dependent on the amount of added ethanol. Over the course of the fermentation, cell viability, as measured by plate counts, was significantly lower in all experiments (with or without ethanol addition) compared with the viability measurements by methylene blue staining. Thus, data from the plate counts provided a more sensitive measure of the toxic effects of added ethanol and long-term anaerobiosis on C. shehatae growth/fermentation. Mean cell volume and total cell volume declined in fermentations with added ethanol. (c) 1992 John Wiley & Sons, Inc.     

D-xylose catabolism in fusarium oxysporum

Summary The initial steps of D-xylose catabolism inFusarium oxysporum have been studied. The presence of the oxidoreductase pathway for D-xylose catabolism was demonstrated. The enzymes involved, D-xylose reductase and xylitol dehydrogenase, were found to be inducible and relatively specific for D-xylose and xylitol. D-xylose isomerase was not detected.     

Fermentation of D-xylose, xylitol, and D-xylulose by yeasts

Fifteen yeasts which can assimilate D-xylose were examined for the ability to convert this pentose to ethanol. In six of the seven genera investigated the conversion was enhanced when air had access to the medium. Therefore, the ability to convert D-xylose to ethanol under these conditions is probably common among yeasts. Growth under the same conditions on xylitol, a putative catabolite of D-xylose, led to only traces of ethanol. The effects of growth on another putative catabolite, D-xylose, were complex, but some of the strains which were among the better producers of ethanol from D-xylose produced less from D-xylulose.     

The effect of aeration on D-xylose fermentation byPachysolen tannophilus,Pichia stipitis,Kluyveromyces marxianus andCandida shehatae

Summary The fermentation of D-xylose byPachysolen tannophilus Y2460,Pichia stipitis Y7124,Kluyveromyces marxianus Y2415 andCandida shehatae Y12878 was investigated in aerobic, anaerobic and microaerophilic batch cultures. The aeration rate greatly influenced the fermentations; growth, rate of ethanol production and oxidation of ethanol are affected. Of the strains tested,Pichia stipitis appears superior; under anaerobic conditions it converts D-xylose (20 g/l) to ethanol with a yield of 0.40 g/l and it exhibits the highest ethanol specific productivity (3.5 g of ethanol per g dry cell per day) under microaerophilic conditions.     

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.     

Metabolic transition step from ethanol consumption to sugar/ethanol

The metabolic pathway shift between only ethanol consumption to both sugar/ethanol consumption was measured by on-line analysis of respiratory quotient of a Saccharomyces cerevisiae. The experiments were carried out in a fed-batch culture under aerobic conditions. During the transition phase, respiratory quotient (RQ) profile shows that sugar can be metabolized through the fermentative pathway even to values of RQ lower than 1.Revisions requested; Revisions received 9 September 2004     

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis>

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, and incubation period) and culture medium composition (mineral salts, carbon source, and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. For optimal growth, the strain required Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g., ammonium citrate). The bacterium had no requirement for sodium acetate for either growth or production of isomerases. The production rate of enzymes was increased when metal ions were added, primarily manganese (2.5 mM). The text was submitted by the authors in English.     

The high fermentative metabolism of Kluyveromyces marxianus UFV-3 relies on the increased expression of key lactose metabolic enzymes

The aim of this work was to obtain insights about the factors that determine the lactose fermentative metabolism of Kluyveromyces marxianus UFV-3. K. marxianus UFV-3 and Kluyveromyces lactis JA6 were cultured in a minimal medium containing different lactose concentrations (ranging from 0.25 to 64 mmol l⁻¹) under aerobic and hypoxic conditions to evaluate their growth kinetics, gene expression and enzymatic activity. The increase in lactose concentration and the decrease in oxygen level favoured ethanol yield for both yeasts but in K. marxianus UFV-3 the effect was more pronounced. Under hypoxic conditions, the activities of β-galactosidase and pyruvate decarboxylase from K. marxianus UFV-3 were significantly higher than those in K. lactis JA6. The expression of the LAC4 (β-galactosidase), RAG6 (pyruvate decarboxylase), GAL7 (galactose-1-phosphate uridylyltransferase) and GAL10 (epimerase) genes in K. marxianus UFV-3 was higher under hypoxic conditions than under aerobic conditions. The high expression of genes of the Leloir pathway, LAC4 and RAG6, associated with the high activity of β-galactosidase and pyruvate decarboxylase contribute to the high fermentative flux in K. marxianus UFV-3. These data on the fermentative metabolism of K. marxianus UFV-3 will be useful for optimising the conversion of cheese whey lactose to ethanol.     

Aerobic Fermentation of D-Xylose to Ethanol by Clavispora sp

Eleven strains of an undescribed species of Clavispora fermented D-xylose directly to ethanol under aerobic conditions. Strain UWO(PS)83-877-1 was grown in a medium containing 2% D-xylose and 0.5% yeast extract, and the following results were obtained: ethanol yield coefficient (ethanol/D-xylose), 0.29 g g⁻¹ (57.4% of theoretical); cell yield coefficient (dry biomass/D-xylose), 0.25 g g⁻¹; maximum ethanol concentration, 5.9 g liter⁻¹; maximum volumetric ethanol productivity, 0.11 g liter⁻¹ h⁻¹. With initial D-xylose concentrations of 40, 60, and 80 g liter⁻¹, maximum ethanol concentrations of 8.8, 10.9, and 9.8 g liter⁻¹ were obtained, respectively (57.2, 57.1, and 48.3% of theoretical). Ethanol was found to inhibit the fermentation of D-xylose (K_p = 0.58 g liter⁻¹) more than the fermentation of glucose (K_p = 6.5 g liter⁻¹). The performance of this yeast compared favorably with that reported for some other D-xylose-fermenting yeasts.     

Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis

The fermentative and respiratory metabolism of Pichia stipitis wild-type strain CBS 5774 and the derived auxotrophic transformation recipient PJH53 trp5-10 his3-1 were examined in differentially oxygenated glucose cultures in the hermetically sealed Sensomat system. There was a good agreement of the kinetics of gas metabolism, growth, ethanol formation and glucose utilisation, proving the suitability of the Sensomat system for rapid and inexpensive investigation of strains and mutants for their respiratory and fermentative metabolism. Our study revealed respiro-fermentative growth by the wild-type strain, although the cultures were not oxygen-limited. The induction of respiro-fermentative behaviour was obviously due to the decrease in oxygen tension but not falling below a threshold of oxygen tension. The responses differed depending on the velocity of the decrease in oxygen tension. At high oxygenation (slow decrease in oxygen tension), ethanol production was induced but glucose uptake was not influenced. At low oxygenation, glucose uptake and ethanol formation increased during the first hours of cultivation. The transformation recipient PJH53 most probably carries a mutation that influences the response to a slow decrease in oxygen tension, since almost no ethanol formation was found under these conditions.     

Alcoholic fermentation of glucose and xylose by Pichia stipitis,Candida shehatae,Saccharomyces cerevisiae and Zymomonas mobilis: oxygen requirement as a key factor

Summary To investigate simultaneous alcoholic fermentation of glucose and xylose derived from lignocellulosic material by separate or co-culture processes, the effect of oxygen transfer rate (OTR) on the fermentation of 50 g/l xylose by Pichia stipitis NRRL Y 7124 and Candida shehatae ATCC 22984, and the fermentation of 50 g/l glucose by Saccharomyces cerevisiae CBS 1200 and Zymomonas mobilis ATCC 10988 was carried out in batch cultures. The kinetic parameters of the xylose-fermenting yeasts were greatly dependent on the OTR. The optimum OTR values were found to be 3.9 and 1.75 mmol·1^–1·h^–1 for C. shehatae and P. stipitis, respectively. By contrast the fermentative parameters of S. cerevisiae were poorly affected by the OTR range tested (0.0–3.5 mmol·l^–1·h^–1) Under these conditions the ethanol yields ranged from 0.41 g·g^–1 to 0.45 g·g^–1 and the specific ethanol productivity was around 0.70 g·g^–1·h^–1. Z. mobilis gave the highest fermentative performance under strictly anaerobic conditions (medium continually flushed with nitrogen): under these conditions, the ethanol yield was 0.43 g·g^–1 and the average specific ethanol productivity was 2.3 g·g^–1·h^–1. Process considerations in relation to the effect of OTR on the fermentative performance of the tested strains are discussed. Offprint requests to: J. P. Delgenes