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.     

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.     

Xylitol dehydrogenase from Pachysolen tannophilus

Abstract NAD-xylitol-dehydrogenase (EC 1.1.1.9) from Pachysolen tannophilus was investigated in relation to xylitol byproduction during xylose fermentation by this yeast. For this purpose the enzyme was partially purified by a combination of affinity chromatography and fast liquid protein chromatography. The enzyme catalyzes an equilibrium reaction which at physiological pH values favours the accumulation of xylitol. The kinetics of the enzyme were shown to be Michaelis-Menten type with respect to both reaction directions. The activity of the enzyme was shown to be under the influence of the 'catabolic reduction charge' (NADH/NAD + NADH) and ATP. The apparent equilibrium constant of the enzyme may explain the considerable byproduction of xylitol during xylose fermentation by P. tannophilus .     

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes.

By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.     

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.     

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.     

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.     

Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus

The yeast Pachysolen tannophilus was found to be capable of converting D-xylose to ethanol. Batch cultures initially containing 50 g/L D-xylose yielded 0.34 g of ethanol per gram of pentose consumed. Aerobic conditions were required for cell growth but not for ethanol production. Both alcohol formation and growth were optimum when incubation temperature was 32 degrees C, when pH was near 2.5, and when D-xylose and ethanol concentrations did not exceed 50 and 20 g/L, respectively.     

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.     

Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus

Summary The yeast Pachysolen tannophilus has been identified as being able to convert an aldopentose, D-xylose, into ethanol. A feature of the conversion is that it can take place under aerobic conditions.Issued as N.R.C.C. Publication No. 19095.     

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.     

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus have been studied. When concentrations of the carbon source in the medium were lower than 100 g/l, it was found to be favorable to the mating of vegetative cells, both haploid and diploid. The addition of nitrogen and sulfur sources to the medium influenced the life phases of haploid cells and partially stabilized the vegetative growth of diploid cells. Enrichment of the nutrient medium with potassium, vitamins, and microelements was shown to be necessary for the formation and maturation of conjugated ascospores. Microelements, vitamins, and phosphorus in excessive amounts activated conjugation but did not provide for the distinct phases of formation of unconjugated asci and spores in the diploid cells. Possible reasons for the unstable diplophase in the yeast P. tannophilus are discussed.     

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.     

Draft genome sequence of the yeast Pachysolen tannophilus CBS 4044/NRRL Y-2460

A draft genome sequence of the yeast Pachysolen tannophilus CBS 4044/NRRL Y-2460 is presented. The organism has the potential to be developed as a cell factory for biorefineries due to its ability to utilize waste feedstocks. The sequenced genome size was 12,238,196 bp, consisting of 34 scaffolds. A total of 4,463 genes from 5,346 predicted open reading frames were annotated with function.     

Influence of the concentrations of d-xylose and yeast extract on ethanol production by Pachysolen tannophilus

To determine the most favorable conditions for the production of ethanol by Pachysolen tannophilus, this yeast was grown in batch cultures with various initial concentrations of two of the constituents of the culture medium: d-xylose (s_o), ranging from 1 g·l⁻¹ to 200 g·l⁻¹, and yeast extract (l_o), ranging from 0 g·l⁻¹ to 8 g·l⁻¹. The most favorable conditions proved to be initial concentrations of S_o=25 g·l⁻¹ and l_o=4 g·l⁻¹, which gave a maximum specific growth rate of 0.26 h⁻¹, biomass productivity of 0.023 g·l⁻¹·h⁻¹, overall biomass yield of 0.094 g·g⁻¹, specific xylose-uptake rate (q_s) of 0.3 g·g⁻¹·h⁻¹ (for t=50 h), specific ethanol-production rate (q_E) of 0.065 g·g⁻¹·h⁻¹ and overall ethanol yield of 0.34 g·g⁻¹; q_s values decreased after the exponential growth phase while q_E remained practically constant.     

Assignment of a PCR-amplified chitinase sequence cloned from Trichoderma hamatum to resolved chromosomes of potential biocontrol species of Trichoderma

Abstract A 1424 bp DNA sequence containing the genetic determinants of the chitinase enzyme was identified in Trichoderma hamatum by PCR amplification. High levels of similarity were observed between this sequence, named Th-ch ( T. hamatum chitinase), and the 42 kDa chitinase genes known from T. harzianum . Chromosome-sized DNAs of five potential biocontrol species of Trichoderma were separated by pulsed-field gel electrophoresis. The total number of chromosomes was six in all the species, with sizes ranging from 3.7 to 7.7 Mb; estimated genome sizes were between 30.5 and 35.8 Mb. When fractionated chromosomes of the five species were probed with radiolabelled Th-ch, strong hybridization signals developed in all cases, but the physical position of these signals varied among species indicating a polymorphic chromosomal location of the highly conserved 42 kDa chitinase gene within the genus Trichoderma .     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Application of the Melle-Boinot process to the fermentation of xylose by Pachysolen tannophilus

Pectinase production by Tubercularia vulgaris using orange-pulp pellets (OPP) or citrus pectin as carbohydrate sources was investigated. The highest levels of extracellular polygalacturonase were detected with OPP as inducing substrate. The pectinases present in the crude filtrate from T. vulgaris cultures included methyl esterase, endo-polygalacturonase and exo-polygalacturonase activities with both substrates. Moreover, in medium supplemented with OPP, the additional presence of one endo-polygalacturonase and two exo-polygalacturonases was detected.     

Peculiarities of hybrid formation and copulation activity in the yeast <Emphasis Type="Italic">Pachysolen tannophilus</Emphasis>

The copulation activity and hybrid formation efficiency have been studied in the xylose-assimilating yeast Pachysolen tannophilus. It was shown that the presence of 2% D-glucose, 0.5% yeast extract, and 2% agarose in the growth medium provided for the highest frequencies of hybrid formation. Atypical hybrid cultures similar in morphophysiological characteristics to native haploid strains of P. tannophilus were revealed in the course of hybridization. The genesis mechanism of such culture and the reasons for the restricted applicability of hybridological analysis to genetic studies of P. tannophilus are discussed.