Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis

Rising crude oil prices and environmental concerns have renewed interest in renewable energy. Cellulosic ethanol promises to deliver a renewable fuel from non-food feedstocks. One technical challenge producing cellulosic ethanol economically is a robust organism to utilize the different sugars present in cellulosic biomass. Unlike starch where glucose is the only sugar present, cellulosic biomass has other sugars such as xylose and arabinose, usually called C5 sugars. This review examines the most promising naturally occurring C5 fermenting organism, Pichia stipitis. In this work, the properties that make P. stipitis unique from other organisms, its physiology and fermentation results on lignocellulosic substrates have been reviewed. P. stipitis can produce 41 g ethanol/l with a potential to cleanup some of the most concentrated toxins. These results coupled with the less stringent nutritional requirements, great resistance to contamination and its thick cell walls makes P. stipitis a viable organism for scale-up. However, P. stipitis has a slower sugar consumption rate compared to Saccharomyces cerevisiae and requires microaerophilic condition for ethanol production. Finally, future studies to enhance fermentation capabilities of this yeast have been discussed.     

Comparison of different pretreatments in ethanol fermentation using corn cob hemicellulosic hydrolysate with Pichia stipitis and Candida shehatae

A hemicellulosic hydrolysate was prepared with 0.3 M H₂SO₄ at 98 °C for 1 h. The total initial reducing sugar was maintained at 45 g l^–1 by synthetic xylose supplementation. The seven detoxification methods were employed including either the single addition of solid CaO (to pH 10 or 6) or its combinations with zeolite shaking. Over-liming gave the hydrolysate that was most completely fermented by Pichia stipitis and Candida shehatae at 30 °C, pH 6, among the tested methods.     

Ethanol tolerance of Pichia stipitis and Candida shehatae strains in fed-batch cultures at controlled low dissolved oxygen levels

Summary Fed-batch cultivations of Pichia stipitis and strains of Candida shehatae with d-xylose or d-glucose were conducted at controlled low dissolved oxygen tension (DOT) levels. There were some marked differences between the strains. In general growth was inhibited at lower ethanol concentrations than fermentation, and ethanol levels of up to 47 g·l^-1 were produced at 30°C. Ethanol production was mainly growth associated. The yeast strains formed small amounts of monocarboxylic acids and higher alcohols, which apparently did not enhance the ethanol toxicity. The maximum ethanol concentration obtained on d-xylose could not be increased by using a high cell density culture, nor by using d-glucose as substrate. The latter observation suggested that the low ethanol tolerance of these xylose-fermenting yeast strains was not a consequence of the metabolic pathway used during pentose fermentation. In contrast with the C. shehatae strains, it was apparent with P. stipitis CSIR-Y633 that when the ethanol concentration reached about 28 g·l^-1, ethanol assimilation exceeded ethanol production, despite cultivation at a low DOT of 0.2% of air saturation. Discontinuing the aeration enabled ethanol accumulation to proceed, but with concomitant xylitol production and cessation of growth.     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Segregation of altered parental properties in fusions between Saccharomyces cerevisiae and the D-xylose fermenting yeasts Candida shehatae and Pichia stipitis.

A prototrophic strain of Saccharomyces cerevisiae CSIR Y190 MATa xyl-, resistant to high levels of ethanol, was hybridized with xylose-fermenting, auxotrophic mutants of Candida shehatae and Pichia stipitis through polyethylene glycol-induced protoplast fusion in an attempt to produce ethanol-tolerant, xylose-fermenting hybrids. Mononucleate fusants were obtained, but these dissociated into a mixture of parental-type segregants. Purified Candida- and Pichia-resembling segregants failed to acquire improved ethanol tolerance but expressed other novel properties of S. cerevisiae, suggesting that karyogamy was impaired after internuclear gene transfer.     

The relation between redox potential and D-xylose fermentation by Candida shehatae and Pichia stipitis

Summary Fed-batch xylose fermentations with the yeastsCandida shehatae andPichia stipitis were conducted, using stirrer speed variation with the redox potential as control index to maintain oxygen-limited conditions. The best results were obtained withC. shehatae at 300 (±10) m V (relative to the standard hydrogen electrode), and these fermentation parameters compared favourably with those obtained previously with the dissolved oxygen tension as control variable. Redox control ofP. stipitis fermentations proved especially difficult. Cell growth during the fermentation was probably a major factor affecting redox potential.     

Effect of aerobiosis on fermentation and key enzyme levels during growth of Pichia stipitis,Candida shehatae and Candida tenuis on d-xylose

The relationship between the degree of aerobiosis, xylitol production and the initial two key enzymes of d-xylose metabolism were investigated in the yeasts Pichia stipitis, Candida shehatae and C. tenuis. Anoxic conditions severely curtailed growth and retarded ethanol productivity. This, together with the inverse relationship between xylitol accumulation and aeration level, suggested a degree of redox imbalance. The ratios of NADH- to NADPH-linked xylose reductase were similar in all three yeasts and essentially independent of the degree of aerobiosis, and thus did not correlate with their differing capacities for ethanol production, xylitol accumulation or growth under the different conditions of aerobiosis. Under anoxic conditions the enzyme activity of Pichia stipitis decreased significantly, which possibly contributed to its weaker anoxic fermentation of xylose compared to C. shehatae.     

Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis

Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose metabolism in Saccharomyces cerevisiae. We have sequenced and assembled the complete genome of P. stipitis. The sequence data have revealed unusual aspects of genome organization, numerous genes for bioconversion, a preliminary insight into regulation of central metabolic pathways and several examples of colocalized genes with related functions. The genome sequence provides insight into how P. stipitis regulates its redox balance while very efficiently fermenting xylose under microaerobic conditions.     

Xylitol production by recombinant Saccharomyces cerevisiae expressing the Pichia stipitis and Candida shehatae XYL1 genes

The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation. Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as co-substrates. Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed once the co-substrate was depleted. The recombinant strains converted xylose to xylitol with yields approaching the theoretical maxima. Xylitol production was most rapid when the co-substrate was still present. Approximately 50% of the xylose was not metabolized due to the depletion of the co-substrate. Received: 23 December 1999 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

Summary A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.     

Characterization of the genetic system of the xylose-fermenting yeast Pichia stipitis

High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads. Received: 29 February 1996 / Accepted: 29 March 1996     

Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH,temperature and substrate concentration

The effects of temperature, pH and xylose concentration on the fermentation parameters of Candida shehatae and Pichia stipitis were evaluated. The optimum pH was in the region of pH 4–5.5, with an optimum fermentation temperature of 30°C. Maximum fermentation rates were reached at 50 g l⁻¹ xylose. A maximum volumetric ethanol productivity of about 0.9 g (l h)⁻¹ was obtained with both yeast strains. The ethanol yield of C. shehatae decreased considerably when cultivated above 30°C or when the xylose concentration was increased. Xylitol accumulated concomitantly. Xylitol production by P. stipitis was observed only during cultivation at 36°C. Whereas the ethanol yield of C. shehatae was usually about 75% of the theoretical maximum, it was 85–90% with P. stipitis.     

Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate

AIMS: The objective of this study was to develop a mutant from Pichia stipitis NRRL Y-7124, tolerant of high concentrations of acetic acid and other inhibitory components present in acid hydrolysates, to improve ethanol yield and productivity. METHODS AND RESULTS: The mutant was developed through adaptation in acid hydrolysate supplemented with nutrients and minerals at 30 +/- 0.5 degrees C. When it was tested for its ability to ferment acid hydrolysate, it showed shorter fermentation time, better tolerance to acid and could ferment at lower pH. The ethanol yield (Yp/s) and productivity (Qp) were increased 1.6- and 2.1-fold, respectively. CONCLUSION: The development of a mutant and its tolerance to acetic acid present in hydrolysates is described. The selected mutant is capable of fermenting both hexoses and pentoses present in hydrolysate at lower pH in comparison with the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant could play a significant role in reducing environmental pollution by using sugars present in pulp mill effluent and, at the same time, could produce a marketable liquid fuel ethanol.     

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.     

Ethanol and sugar tolerance of Candida shehatae

Summary Candida shehatae ATCC 22984 fermented solutions of up to 260 g/L sugars derived by hydrolysis of whole barley. These solutions contained hexose: pentose 7030, the hexose being mainly glucose from the barley starch and the pentose being mainly xylose. At sugar concentrations of 180 g/L, fermentation was complete in 72 h, yielding 84 g/L ethanol, 0.47 g ethanol/g sugar. At 260 g/L, fermentation ceased when ethanol concentration reached 100 g/L, but resumed when the ethanol was removed by vacuum distillation, to yield finally 0.50 g ethanol/g sugar.     

Fermentation of cellobiose and wood sugars to ethanol byCandida shehatae andPichia stipitis

Summary Three pentose fermenting yeast strains ofCandida shehatae and three ofPichia stipitis were examined for their ability to produce ethanol from cellobiose and from sugars liberated by hydrolysis of lignocellulosic biomass. All of thePichia strains tested produced some ethanol;P. stipitis CBS 5776 gave the highest yield: 10.3 g/L on complete fermentation of 25 g/L cellobiose within 48 hours. This yeast also produced considerably more ethanol from the wood sugar mixture.