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Sheldon J B Duff - One of the best experts on this subject based on the ideXlab platform.
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optimization of spent Sulfite Liquor fermentation
Enzyme and Microbial Technology, 2008Co-Authors: Steve S. Helle, Tony Lin, Sheldon J B DuffAbstract:Abstract The hemicellulose-derived sugars in spent Sulfite Liquor (SSL), a process stream of the Sulfite pulping process, may be fermented to ethanol. Low nutrient concentration, the presence of inhibitors, and a large proportion of xylose limit fermentation of the sugars in SSL. Factorial design experiments were used to find optimum conditions for hexose and xylose fermentation using a robust SSL-adapted (non-xylose fermenting) yeast strain, and Saccharomyces cerevisiae 259ST, a genetically modified strain capable of fermenting xylose. Xylose fermentation was slower than hexose fermentation, and required the detoxification of SSL and/or the addition of hexose sugars to support a large yeast concentration. Xylose fermentation and the growth of the genetically modified yeast in SSL were closely correlated. Under fermentation conditions where no growth was observed (yeast concentration ∼2 g/L and pH 5.3), an average of 5.5 g/L ethanol was obtained from xylose fermentation. SSL pretreatment and the addition of nutrients were also beneficial. For the SSL-adapted strain, ethanol yield was improved by increasing the yeast concentration, however while adding nutrients and raising the pH increased yeast growth, these changes resulted in a decrease in the ethanol yield from greater than 85% of theoretical to 70%.
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fortifying spent Sulfite pulping Liquor with hydrolyzed reject knots
Enzyme and Microbial Technology, 2007Co-Authors: Steve S. Helle, Robert A Petretta, Sheldon J B DuffAbstract:Abstract A limitation for spent Sulfite Liquor (SSL) fermentation, particularly hardwood SSL fermentation is the low sugar content. The objective of this work was to increase the sugar content of SSL through the addition and hydrolysis of reject knots from the pulp line. Factorial experiments indicated that hydrolysis yield was significantly affected by cellulase and β-glucosidase loading, concentration of SSL, knot concentration, pH and temperature. Spent Sulfite Liquor reduced the hydrolysis rate and yield, however this inhibition decreased with time. The resultant hydrolysates were readily fermented alone or in combination with 10% SSL. In 22% SSL, fermentation yields were reduced. Economic analysis indicated that knots would be an economically-viable source of supplemental sugars at a cellulase cost of up to $5 CDN per million FPU.
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xylose fermentation by genetically modified saccharomyces cerevisiae 259st in spent Sulfite Liquor
Bioresource Technology, 2004Co-Authors: Steve S. Helle, Allison Murray, Janet Lam, David R Cameron, Sheldon J B DuffAbstract:Abstract Spent Sulfite pulping Liquor (SSL) is a high-organic content byproduct of acid biSulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15–0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (
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effect of inhibitory compounds found in biomass hydrolysates on growth and xylose fermentation by a genetically engineered strain of s cerevisiae
Enzyme and Microbial Technology, 2003Co-Authors: Steve S. Helle, Janet Lam, David S Cameron, Ben White, Sheldon J B DuffAbstract:The effect of inhibitors on the recombinant xylose fermenting strain Saccharomyces cerevisiae 259ST was compared to three reference strains, including the parent strain (S. cerevisiae 259A) and an industrial strain adapted to spent Sulfite Liquor (SSL). Interaction effects between the inhibitors were verified by a fractional factorial design. S. cerevisiae 259ST was found to be as hardy as the reference strains towards acetic acid, ammonium, furfural, and osmotic effects, which are inhibitory compounds in SSL. Ammonium toxicity appeared to be due to osmotic effects. For all of the inhibitors tested, growth rate was more severely inhibited than the ethanol yield during fermentation of glucose. The ability of S. cerevisiae 259ST to ferment xylose was more severely affected than the ability to ferment glucose. At the concentrations expected in softwood SSL (pH 5), acetic acid will decrease the growth rate by 15%, while the ethanol yield on xylose would decrease by 50%, while ammonium will decrease the yeast growth rate by 20% and decrease the ethanol yield from xylose by 45%.
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ethanol production from spent Sulfite Liquor fortified by hydrolysis of pulp mill primary clarifier sludge
Applied Biochemistry and Biotechnology, 1996Co-Authors: John W Moritz, Sheldon J B DuffAbstract:Some low-yield Sulfite pulping operations ferment spent Sulfite Liquor (SSL) to remove biochemical oxygen demand associated with dissolved sugars while at the same time generating ethanol as a salable product. Simultaneous saccharification and fermentation (SSF) of primary clarifier sludge in a medium of SSL was proposed as a means of reducing the amount of sludge to be disposed of while at the same time increasing ethanol productivity. In this article, the option of fortifying existing SSL fermenting processes with the sugars produced viain situ enzymatic hydrolysis of Sulfite primary clarifier sludge (PCS) has been explored. In 100% SSL PCS hydrolysis rates as high as 3.4 g/(L·h) were observed at an initial enzyme loading of 10 filter paper units (FPU)/g PCS. To reduce the deleterious effects of glucose inhibition, single-stage SSF was carried out using cellulase enzymes andSaccharomyces cerevisiae. The production rate of ethanol in SSL was increased by as much as 25% through the SSF process.
Steve S. Helle - One of the best experts on this subject based on the ideXlab platform.
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optimization of spent Sulfite Liquor fermentation
Enzyme and Microbial Technology, 2008Co-Authors: Steve S. Helle, Tony Lin, Sheldon J B DuffAbstract:Abstract The hemicellulose-derived sugars in spent Sulfite Liquor (SSL), a process stream of the Sulfite pulping process, may be fermented to ethanol. Low nutrient concentration, the presence of inhibitors, and a large proportion of xylose limit fermentation of the sugars in SSL. Factorial design experiments were used to find optimum conditions for hexose and xylose fermentation using a robust SSL-adapted (non-xylose fermenting) yeast strain, and Saccharomyces cerevisiae 259ST, a genetically modified strain capable of fermenting xylose. Xylose fermentation was slower than hexose fermentation, and required the detoxification of SSL and/or the addition of hexose sugars to support a large yeast concentration. Xylose fermentation and the growth of the genetically modified yeast in SSL were closely correlated. Under fermentation conditions where no growth was observed (yeast concentration ∼2 g/L and pH 5.3), an average of 5.5 g/L ethanol was obtained from xylose fermentation. SSL pretreatment and the addition of nutrients were also beneficial. For the SSL-adapted strain, ethanol yield was improved by increasing the yeast concentration, however while adding nutrients and raising the pH increased yeast growth, these changes resulted in a decrease in the ethanol yield from greater than 85% of theoretical to 70%.
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fortifying spent Sulfite pulping Liquor with hydrolyzed reject knots
Enzyme and Microbial Technology, 2007Co-Authors: Steve S. Helle, Robert A Petretta, Sheldon J B DuffAbstract:Abstract A limitation for spent Sulfite Liquor (SSL) fermentation, particularly hardwood SSL fermentation is the low sugar content. The objective of this work was to increase the sugar content of SSL through the addition and hydrolysis of reject knots from the pulp line. Factorial experiments indicated that hydrolysis yield was significantly affected by cellulase and β-glucosidase loading, concentration of SSL, knot concentration, pH and temperature. Spent Sulfite Liquor reduced the hydrolysis rate and yield, however this inhibition decreased with time. The resultant hydrolysates were readily fermented alone or in combination with 10% SSL. In 22% SSL, fermentation yields were reduced. Economic analysis indicated that knots would be an economically-viable source of supplemental sugars at a cellulase cost of up to $5 CDN per million FPU.
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xylose fermentation by genetically modified saccharomyces cerevisiae 259st in spent Sulfite Liquor
Bioresource Technology, 2004Co-Authors: Steve S. Helle, Allison Murray, Janet Lam, David R Cameron, Sheldon J B DuffAbstract:Abstract Spent Sulfite pulping Liquor (SSL) is a high-organic content byproduct of acid biSulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15–0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (
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effect of inhibitory compounds found in biomass hydrolysates on growth and xylose fermentation by a genetically engineered strain of s cerevisiae
Enzyme and Microbial Technology, 2003Co-Authors: Steve S. Helle, Janet Lam, David S Cameron, Ben White, Sheldon J B DuffAbstract:The effect of inhibitors on the recombinant xylose fermenting strain Saccharomyces cerevisiae 259ST was compared to three reference strains, including the parent strain (S. cerevisiae 259A) and an industrial strain adapted to spent Sulfite Liquor (SSL). Interaction effects between the inhibitors were verified by a fractional factorial design. S. cerevisiae 259ST was found to be as hardy as the reference strains towards acetic acid, ammonium, furfural, and osmotic effects, which are inhibitory compounds in SSL. Ammonium toxicity appeared to be due to osmotic effects. For all of the inhibitors tested, growth rate was more severely inhibited than the ethanol yield during fermentation of glucose. The ability of S. cerevisiae 259ST to ferment xylose was more severely affected than the ability to ferment glucose. At the concentrations expected in softwood SSL (pH 5), acetic acid will decrease the growth rate by 15%, while the ethanol yield on xylose would decrease by 50%, while ammonium will decrease the yeast growth rate by 20% and decrease the ethanol yield from xylose by 45%.
Johann F Gorgens - One of the best experts on this subject based on the ideXlab platform.
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techno economics of integrating bioethanol production from spent Sulfite Liquor for reduction of greenhouse gas emissions from Sulfite pulping mills
Biotechnology for Biofuels, 2014Co-Authors: Abdul M Petersen, Kate Haigh, Johann F GorgensAbstract:Flow sheet options for integrating ethanol production from spent Sulfite Liquor (SSL) into the acid-based Sulfite pulping process at the Sappi Saiccor mill (Umkomaas, South Africa) were investigated, including options for generation of thermal and electrical energy from onsite bio-wastes, such as bark. Processes were simulated with Aspen Plus® for mass- and energy-balances, followed by an estimation of the economic viability and environmental impacts. Various concentration levels of the total dissolved solids in magnesium oxide-based SSL, which currently fuels a recovery boiler, prior to fermentation was considered, together with return of the fermentation residues (distillation bottoms) to the recovery boiler after ethanol separation. The generation of renewable thermal and electrical energy from onsite bio-wastes were also included in the energy balance of the combined pulping-ethanol process, in order to partially replace coal consumption. The bio-energy supplementations included the combustion of bark for heat and electricity generation and the bio-digestion of the calcium oxide SSL to produce methane as additional energy source. Ethanol production from SSL at the highest substrate concentration was the most economically feasible when coal was used for process energy. However this solution did not provide any savings in greenhouse gas (GHG) emissions for the concentration-fermentation-distillation process. Maximizing the use of renewable energy sources to partially replace coal consumption yielded a satisfactory economic performance, with a minimum ethanol selling price of 0.83 US$/l , and a drastic reduction in the overall greenhouse gas emissions for the entire facility. High substrate concentrations and conventional distillation should be used when considering integrating ethanol production at Sulfite pulping mills. Bio-wastes generated onsite should be utilized at their maximum potential for energy generation in order to maximize the GHG emissions reduction.
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techno economics of integrating bioethanol production from spent Sulfite Liquor for reduction of greenhouse gas emissions from Sulfite pulping mills
Biotechnology for Biofuels, 2014Co-Authors: Abdul M Petersen, Kate Haigh, Johann F GorgensAbstract:Background Flow sheet options for integrating ethanol production from spent Sulfite Liquor (SSL) into the acid-based Sulfite pulping process at the Sappi Saiccor mill (Umkomaas, South Africa) were investigated, including options for generation of thermal and electrical energy from onsite bio-wastes, such as bark. Processes were simulated with Aspen Plus® for mass- and energy-balances, followed by an estimation of the economic viability and environmental impacts. Various concentration levels of the total dissolved solids in magnesium oxide-based SSL, which currently fuels a recovery boiler, prior to fermentation was considered, together with return of the fermentation residues (distillation bottoms) to the recovery boiler after ethanol separation. The generation of renewable thermal and electrical energy from onsite bio-wastes were also included in the energy balance of the combined pulping-ethanol process, in order to partially replace coal consumption. The bio-energy supplementations included the combustion of bark for heat and electricity generation and the bio-digestion of the calcium oxide SSL to produce methane as additional energy source.
Hung Lee - One of the best experts on this subject based on the ideXlab platform.
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Determinants of tolerance to inhibitors in hardwood spent Sulfite Liquor in genome shuffled Pachysolen tannophilus strains
Antonie van Leeuwenhoek, 2015Co-Authors: Nicole K. Harner, Paramjit K Bajwa, Marc B. Habash, Jack T. Trevors, Glen D. Austin, Philip A. Formusa, Chi-kin Chan, Hung LeeAbstract:Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved tolerance to inhibitors in hardwood spent Sulfite Liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to the HW SSL UV mutant (70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70–0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to the acetic acid UV mutant, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp–1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates.
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Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent Sulfite Liquor and acetic acid
Antonie van Leeuwenhoek, 2014Co-Authors: Nicole K. Harner, Paramjit K Bajwa, Marc B. Habash, Jack T. Trevors, Glen D. Austin, Hung LeeAbstract: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.
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saccharomyces cerevisiae genome shuffling through recursive population mating leads to improved tolerance to spent Sulfite Liquor
Applied and Environmental Microbiology, 2011Co-Authors: Dominic Pinel, Hung Lee, Paramjit K Bajwa, Frederic Daoust, Stephen B Del Cardayre, Vincent J J MartinAbstract:Spent Sulfite Liquor (SSL) is a waste effluent from Sulfite pulping that contains monomeric sugars which can be fermented to ethanol. However, fermentative yeasts used for the fermentation of the sugars in SSL are adversely affected by the inhibitory substances in this complex feedstock. To overcome this limitation, evolutionary engineering of Saccharomyces cerevisiae was carried out using genome-shuffling technology based on large-scale population cross mating. Populations of UV-light-induced yeast mutants more tolerant than the wild type to hardwood spent Sulfite Liquor (HWSSL) were first isolated and then recursively mated and enriched for more-tolerant populations. After five rounds of genome shuffling, three strains were isolated that were able to grow on undiluted HWSSL and to support efficient ethanol production from the sugars therein for prolonged fermentation of HWSSL. Analyses showed that greater HWSSL tolerance is associated with improved viability in the presence of salt, sorbitol, peroxide, and acetic acid. Our results showed that evolutionary engineering through genome shuffling will yield robust yeasts capable of fermenting the sugars present in HWSSL, which is a complex substrate containing multiple sources of inhibitors. These strains may not be obtainable through classical evolutionary engineering and can serve as a model for further understanding of the mechanism behind simultaneous tolerance to multiple inhibitors.
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mutants of the pentose fermenting yeast pichia stipitis with improved tolerance to inhibitors in hardwood spent Sulfite Liquor
Biotechnology and Bioengineering, 2009Co-Authors: Paramjit K Bajwa, Jack T. Trevors, Dominic Pinel, Frederic Daoust, Vincent J J Martin, Tasnina Shireen, Hung LeeAbstract:Mutants of Pichia stipitis NRRL Y-7124 able to tolerate and produce ethanol from hardwood spent Sulfite Liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars.
Barbel Hahnhagerdal - One of the best experts on this subject based on the ideXlab platform.
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activity and stability of xylose isomerase preparations from whole cells of lactobacillus brevis in spent Sulfite Liquor
Enzyme and Microbial Technology, 1993Co-Authors: Torbjorn Linden, Barbel HahnhagerdalAbstract:Three differently treated xylose isomerase preparations of Lactobacillus brevis DSM 20054 whole cells were compared with a commercial immobilized preparation, Maxazyme GI-immob., with respect to activity and stability. All isomerizations were performed in spent Sulfite Liquor. The activity (∼300 mg xylulose g−1 dry weight biocatalyst h−1) of one preparation, heat-, ethanol-, and glutaraldehyde-treated whole cells of L. brevis, was 30 to 40 times higher, at pH 5 and 5.5, respectively, than that of the commercial enzyme at pH 5.5. The commercial enzyme was not active at pH 5. During four repeated 24-h isomerizations, this L. brevis preparation retained 70% of the activity at pH 5.5 and 30% at pH 5, as compared with 66 and 0%, respectively, for the commercial preparation.
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isolation and characterization of acetic acid tolerant galactose fermenting strains of saccharomyces cerevisiae from a spent Sulfite Liquor fermentation plant
Applied and Environmental Microbiology, 1992Co-Authors: T Linden, J Peetre, Barbel HahnhagerdalAbstract:From a continuous spent Sulfite Liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent Sulfite Liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).
