D-xylose Fermentation Research Articles

Comparison of corn steep liquor with other nutrients in the fermentation of D-Xylose by Pichia stipitis CBS 6054

Pichia stipitis CBS 6054 ferments D-Xylose to ethanol in a medium containing corn steep liquor as the only source of nitrogen, amino acids, vitamins and other nutrients. The ethanol yield and fermentation rate compare favorably to those obtained with media containing more expensive sources of nitrogen, vitamins and amino acids. Corn steep liquor is a good source of nutrients that can support growth and fermentation activity of this xylose fermenting yeast.

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

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.

The effect of pH on kinetic and yield parameters during the ethanolic fermentation of D-xylose with Pachysolen tannophilus

We have studied the ethanolic fermentation of D-xylose g ethanol with Pachysolen tannophilus in batch cultures. We propose a model Y~/s g xylose to predict variations in D-xylose consumed, and biomass and g biomass ethanol produced, in which we include parameters for the specific Yx/s growth rate, for the consumption of D-xylose and production of g xylose ethanol either related or not to growth, g xylitol The ideal initial pH for ethanol production turned out to be 4.5.. fxi/$ At this pH value the net specific growth rate was 0.26 h 1, biomass g xylose yield was 0.16 g.g- 1, the cell-maintenance coefficient was 0.073 g.g-1.h-1, the parameter for ethanol production non-related to growth was 0.064 g.g- 1.h 1 and the maximum ethanol yield was 0.32 g.g-1 Greek letters

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 growth 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.

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

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.

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

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.

Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae.

The effect on D-xylose utilization and the corresponding xylitol and ethanol production by Candida shehatae (ATCC 22984) were examined with different nitrogen sources. These included organic (urea, asparagine, and peptone) and inorganic (ammonium chloride, ammonium nitrate, ammonium sulphate, and potassium nitrate) sources. Candida shehatae did not grow on potassium nitrate. Improved ethanol production (Y(p/s), yield coefficient (grams product/grams substrate), 0.34) was observed when organic nitrogen sources were used. Correspondingly, the xylitol production was also higher with organic sources. Ammonium sulphate showed the highest ethanol:xylitol ratio (11.0) among all the nitrogen sources tested. The ratio of NADH- to NADPH-linked D-xylose reductase (EC 1.1.1.21) appeared to be rate limiting during ethanologenesis of D-xylose. The levels of xylitol dehydrogenase (EC 1.1.1.9) were also elevated in the presence of organic nitrogen sources. These results may be useful in the optimization of alcohol production by C. shehatae during continuous fermentation of D-xylose.

Direct fermentation of cellulosic materials by Fusarium oxysporum 841: Acetic acid/ethanol production and tolerance.

Direct fermentation of cellulosic materials to acetic acid and ethanol was studied using a cellulolytic strain of Fusarium oxysporum DSM 841. It produces extracellular enzyme complex consisting of avicelase, CMCase, cellobiase and xylanase activities with hydrolytic potential of 80-87% of different cellulosic substrates. Acetic acid (12g/l) and ethanol (3.6g/l) were obtained with yields of 0.36g/g and 0.1 g/g from potato waste (cellulosic waste from starch industries). F. oxysporum 841 was able to ferment D-xylose as efficiently as it could D-glucose. It exhibited relatively higher product tolerance. Mechanism of inhibition of cellulase effectiveness in the presence of acetic acid/ethanol is also discussed.

Fermentation of d-xylose by Clostridium butyricum LMG 1213t 1 in chemostats

Clostridium butyricum LMG 1213t 1 was able to grow on d-xylose and l-arabinose in a minimal medium. In a chemostat in carbon limitation, the same fermentation balance (when corrected for the difference in carbon atoms) was obtained on d-xylose as on glucose in the pH range 5.5–7.0. Per mole d-xylose consumed, 1.74 mol H 2 was produced, which was equal to the H 2 production on glucose previously reported. By varying the dilution rate, the molar growth yields remained less than half these on glucose. From a critical literature survey, we propose that this was caused by an additional ATP expenditure for the active transport of d-xylose across the cell membrane. From our data we calculated that 1 mol ATP was required for the transport of d-xylose. With higher d-xylose concentrations, the butyrate production and molar growth yield Y xylose gradually decreased, but the H 2 production remained constant. On glucose-xylose mixtures (10 g l -1 total sugar), the fermentation of d-xylose was not inhibited by glucose under the conditions tested. The molar growth yields increased linearly with the weight percentage of glucose in the carbon source, while the H 2 production was not affected.

Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus.

A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.

Combined alcoholic fermentation of D-xylose and D-glucose by four selected microbial strains: Process considerations in relation to ethanol tolerance

As components of combined fermentation of both glucose and xylose to ethanol by separated or coculture processes, the effects of initial sugar concentrations on the fermentative performances ofPichia stipitis Y7124,Candida shehatae ATCC 22984,Saccharomyces cerevisiae CBS1200 andZymomonas mobilis ATCC10988 were investigated. From the characteristics of sugar and produced ethanol tolerances the most suitable microorganisms for the achievement of glucose and xylose fermentations have been selected with respect to different fermentation schemes.

Continuous fermentation of d-xylose by immobilizedpichia stipitis comparison between cstr and cpfr

The main purpose of this work was to compare the performance of two different kinds of reactors (CSTR and CPFR) in order to enhance the ethanol productivity in the fermentation of D-xylose byPichia stipitis immobilized inx-carrageenan. Immobilization was carried out in a 4% aqueous suspension ofx-carrageenan, which was mixed with the inoculum. The bioparticles were treated with A1(NO3)3 as hardening agent. The fermenters operated during a long period of time (about 30 d). Best results were obtained in the packed-bed reactor (CPFR), which allowed operation at high final ethanol concentrations, this fact having been explained because of the observed strong product inhibition. The overall productivity reached values higher than 3.8 g/(L.h). This supposed an interesting improvement with relation to the productivities found in the literature, which as an average did not exceed 1 g/(Lh). However, the specific productivities of yeast in the continuous stirred tank reactor (CSTR) were always greater because the bioparticles were kept in close contact with the broth, whereas in the CPFR, there were at least two problems: (a) the possibility that the produced gas could prevent the intimate contact between the substrate and the particles and (b) the possible existence of preferential paths.

Acetic acid inhibition of d-xylose fermentation by Pichia stipitis

Fermentation of d-xylose by Pichia stipitis was inhibited by acetic acid and the degree of inhibition depended on the acetic acid concentration, the availability of oxygen, and the pH. A 50% inhibition of the volumetric rate of ethanol production occurred at acetic acid concentrations of 0.8 and 13.8 g l −1 at pH 5.1 and 6.5, respectively under anaerobic conditions. No acetic acid was utilized in the absence of oxygen. Under oxygen-limited conditions at pH 6.5, an acid hydrolysate of sugar cane bagasse containing (g l −1) d-xylose (40.9), d-glucose (3.1), l-arabinose (4.5), and acetic acid (9.0) was fermented to ethanol at a rate of 0.15 g(l h) −1, and an ethanol yield of 0.27 g g −1 sugar was obtained. When the hydrolysate was treated with an anion exchange resin, 84% of the acetic acid was removed and the subsequent fermentation resulted in a rate of ethanol production [0.56 g(l h) −1] and an ethanol yield (0.37 g g −1 sugar) similar to that obtained in a xylose-arabinose-glucose medium lacking acetic acid.

Temperature mediated changes of d-xylose metabolism in the yeastPachysolen tannophilus

We found that during d-xylose fermentation to ethanol by Pachysolen tannophilus the amount of xylitol produced decreased dramatically while CO2 increased when the temperature was raised from 30 to 37°C. Acetaldehyde was detected only at 37°C. However, no significant change in the amount of ethanol production was observed. A similar trend was observed when the cells were treated with 10 μM 2,4-dinitrophenol. Furthermore, the enzyme d-xylose reductase displayed a similar activation energy in cells grown either at 30 or 37°C. These data suggest that in P. tannophilus oxidative phosphorylation becomes uncoupled at higher temperature.

Temperature mediated changes of d-xylose metabolism in the yeast Pachysolen tannophilus

We found that during d-xylose fermentation to ethanol by Pachysolen tannophilus the amount of xylitol produced decreased dramatically while CO2 increased when the temperature was raised from 30 to 37°C. Acetaldehyde was detected only at 37°C. However, no significant change in the amount of ethanol production was observed. A similar trend was observed when the cells were treated with 10 μM 2,4-dinitrophenol. Furthermore, the enzyme d-xylose reductase displayed a similar activation energy in cells grown either at 30 or 37°C. These data suggest that in P. tannophilus oxidative phosphorylation becomes uncoupled at higher temperature.

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.

Glucose-negative Mutants of Pachysolen tannophilus

Mutants of the yeast Pachysolen tannophilus, exhibiting decreased ability to utilize d-glucose as the sole carbon source, were obtained by selecting for resistance to 2-deoxyglucose. Enzyme studies confirmed that these strains are defective in the hexose-phosphorylating enzymes and are unable to phosphorylate d-glucose to d-glucose 6-phosphate. The results confirmed the presence of two hexokinases, A and B, with ratios of d-glucose to d-fructose phosphorylation activity of 1·3/1·0 and 3·0/1·0, respectively, and a d-glucose-specific glucokinase. The behaviour of a hexose-negative strain, able to ferment d-xylose in the presence of d-glucose, is described.

The D-xylose fermenting capacities of immobilizedPichia stipitis andPachysolen tannophilus

The yeastsP. stipitis NRRL Y-7124 andP. tannophilus NRRL Y-2460 were entrapped in η-carrageenan beads and used for repeated batch fermentation of D-xylose, in a series of four reactors. The operating conditions finally chosen gave an oxygen coefficient (KLa) of 0.83 min−1, as measured by the sulphite method. Ethanol yields were 0.40 g/g forP. stipitis and 0.36 g/g forP. tannophilus (respectively 78.4% and 70.5% of the theoretical yields). In spite of its lower retention by the gel,P. stipitis exhibited greater fermenting capacities thanP. tannophilus.

Kinetics of ethanolic fermentation of D-xylose by Klebsiella pneumoniae and its mutants

The microbial production of ethanol from D-xylose by a new soil isolate of Klebsiella pneumoniae and the mutants K. pneumoniae MB-16 and MB-16-1048 was studied. Kinetic and physiological properties of the mutants were compared with those of the original isolate. The volumetric rates of ethanol formation by mutants MB-16-1048 and MB-16 and the original isolate were 1.58, 0.50, and 0.06 g liter-1 h-1, respectively. The cultivation times of mutants MB-16-1048 and MB-16 were 20 and 18 h, respectively, and that of the original isolate was 118 h. Both the mutants exhibited metabolic similarities with the original isolate. Ethanol was the major end product of fermentation in all three strains. Acetic acid and carbon dioxide were the other two important by-products of fermentation. Pyruvic acid was accumulated in significant proportions as an intermediate. The proportion of pyruvate in the original isolate was 54% of the total D-xylose utilized, whereas for MB-16 and MB-16-1048 the values were about 42 and 22%, respectively. The lower fractions of pyruvate in mutants MB-16 and MB-16-1048 showed up as a 41 and 82% improvement, respectively, over the original isolate in terms of the ethanol yield.