| 1. |
- Almeida, Joao, et al.
(author)
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Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae
- 2007
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In: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575 .- 1097-4660. ; 82:4, s. 340-349
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Research review (peer-reviewed)abstract
- During hydrolysis of lignocellulosic biomass, monomeric sugars and a broad range of inhibitory compounds are formed and released. These inhibitors, which can be organized around three main groups, furans, weak acids and phenolics, reduce ethanol yield and productivity by affecting the microorganism performance during the fermentation step. Among the microorganisms that have been evaluated for lignocellulosic hydrolysate ethanol fermentation, the yeast Saccharomyces cerevisiae appears to be the least sensitive. In order to overcome the effect of inhibitors, strategies that include improvement of natural tolerance of microorganism and use of fermentation control strategies have been developed. An overview of the origin, effects and mechanisms of action of known inhibitors on S. cerevisiae is given. Fermentation control strategies as well as metabolic, genetic and evolutionary engineering strategies to obtain S. cerevisiae strains with improved tolerance are discussed.
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| 2. |
- Almeida, Joao, et al.
(author)
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NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae.
- 2008
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 78:6, s. 939-945
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Journal article (peer-reviewed)abstract
- Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.
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| 3. |
- Almeida, Joao, et al.
(author)
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Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)
- 2008
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
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Journal article (peer-reviewed)abstract
- Background: Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xyloseconsuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21. Results: In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme. Conclusion: We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.
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| 4. |
- Carrasco, Cristhian, et al.
(author)
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SO2-catalyzed steam pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse
- 2010
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In: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 46:2, s. 64-73
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Journal article (peer-reviewed)abstract
- Sugarcane bagasse is a lignocellulosic residue obtained from sugarcane milling, and a potentially interesting raw material that can be used for fuel ethanol production. In the present study, bagasse was steam pretreated at temperatures between 180 and 205 ◦C, with holding times of 5–10 min using SO2 as a catalyst to determine conditions that provide a good recovery of pentoses and a suitable material for enzymatic hydrolysis. Pretreatment conducted at 190 ◦C for 5 min gave a pentose yield of 57%, with only minor amounts of degradation compounds formed. Commercial cellulolytic enzymes were used to hydrolyze the obtained fiber fractions after pretreatment at different water-insoluble solid contents (2%, 5% and 8% WIS). The overall highest sugar yield achieved from bagasse was 87% at 2% WIS. Fermentation tests were made on both the pentose-rich hemicellulose hydrolysate obtained from the pretreatment, and the enzymatic hydrolysates obtained from the fiber fractions using the xylose-fermenting strain of Saccharomyces cerevisiae TMB3400, as well as the natural xylose-utilizing yeast Pichia stipitis CBS 6054. The pretreatment hydrolysates produced at 2% WIS as well as the enzymatic hydrolysates showed a complete glucose fermentability indicating a low toxicity to the yeasts. The best xylose conversion (more than 60%) was achieved by the strain TMB3400 at 2% WIS.
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| 5. |
- Granath, Katarina, 1971, et al.
(author)
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The YIGI (YPL201c) encoded protein is involved in regulating anaerobic glycerol metabolism in Saccharomyces cerevisiae
- 2005
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In: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 22:16, s. 1257-1268
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Journal article (peer-reviewed)abstract
- Under anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD+ from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (YpI201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two-hybrid tests with the GPP1 and GPP2 encoded glycerol 3-phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post-transcriptional levels. In agreement with such a role, a GFP-tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level. Copyright (c) 2005 John Wiley & Sons, Ltd.
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| 6. |
- Jiang, Zheshun, et al.
(author)
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Hexavalent chromium still a concern in Sweden : Evidence from a cross-sectional study within the SafeChrom project
- 2024
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In: International journal of hygiene and environmental health. - : Elsevier. - 1438-4639 .- 1618-131X. ; 256
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Journal article (peer-reviewed)abstract
- ObjectivesHexavalent chromium (Cr(VI)) is classified as a human carcinogen. Occupational Cr(VI) exposure can occur during different work processes, but the current exposure to Cr(VI) at Swedish workplaces is unknown.MethodsThis cross-sectional study (SafeChrom) recruited non-smoking men and women from 14 companies with potential Cr(VI) exposure (n = 113) and controls from 6 companies without Cr(VI) exposure (n = 72). Inhalable Cr(VI) was measured by personal air sampling (outside of respiratory protection) in exposed workers. Total Cr was measured in urine (pre- and post-shift, density-adjusted) and red blood cells (RBC) (reflecting Cr(VI)) in exposed workers and controls. The Bayesian tool Expostats was used to assess risk and evaluate occupational exposure limit (OEL) compliance.ResultsThe exposed workers performed processing of metal products, steel production, welding, plating, and various chemical processes. The geometric mean concentration of inhalable Cr(VI) in exposed workers was 0.15 μg/m3 (95% confidence interval: 0.11–0.21). Eight of the 113 exposed workers (7%) exceeded the Swedish OEL of 5 μg/m3, and the Bayesian analysis estimated the share of OEL exceedances up to 19.6% for stainless steel welders. Median post-shift urinary (0.60 μg/L, 5th-95th percentile 0.10–3.20) and RBC concentrations (0.73 μg/L, 0.51–2.33) of Cr were significantly higher in the exposed group compared with the controls (urinary 0.10 μg/L, 0.06–0.56 and RBC 0.53 μg/L, 0.42–0.72). Inhalable Cr(VI) correlated with urinary Cr (rS = 0.64) and RBC-Cr (rS = 0.53). Workers within steel production showed the highest concentrations of inhalable, urinary and RBC Cr. Workers with inferred non-acceptable local exhaustion ventilation showed significantly higher inhalable Cr(VI), urinary and RBC Cr concentrations compared with those with inferred acceptable ventilation. Furthermore, workers with inferred correct use of respiratory protection were exposed to significantly higher concentrations of Cr(VI) in air and had higher levels of Cr in urine and RBC than those assessed with incorrect or no use. Based on the Swedish job-exposure-matrix, approximately 17 900 workers were estimated to be occupationally exposed to Cr(VI) today.ConclusionsOur study demonstrates that some workers in Sweden are exposed to high levels of the non-threshold carcinogen Cr(VI). Employers and workers seem aware of Cr(VI) exposure, but more efficient exposure control strategies are required. National strategies aligned with the European strategies are needed in order to eliminate this cause of occupational cancer.
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| 7. |
- Jiang, Zheshun, et al.
(author)
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P-205 THE SAFECHROM PROJECT - EVIDENCE FROM A CROSS-SECTIONAL STUDY SHOWS THAT HEXAVALENT CHROMIUM IS STILL A CONCERN IN SWEDEN
- 2024
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In: Occupational Medicine. - 0962-7480. ; 74:Suppl 1, s. 291-292
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Conference paper (other academic/artistic)abstract
- Hexavalent chromium Cr(VI) is a human carcinogen, but the current exposure to Cr(VI) at Swedish workplaces is unknown.Recruitment of 113 workers with potential Cr(VI) exposure and 72 controls was combined with measurements of inhalable Cr(VI) (only exposed workers) and total Cr in urine and red blood cells (RBC), Bayesian analysis of occupational exposure limit (OEL) compliance was used, as well as the Swedish job-exposure-matrix.Exposed workers performed processing of metal products, steel production, welding, and plating. The geometric mean concentration of inhalable Cr(VI) in exposed workers was 0.15 μg/m3. Eight workers (7\ exceeded the Swedish OEL (5 μg/m3), and the share of OEL exceedances was estimated to be up to 19.6\ and RBC-Cr were significantly higher in exposed workers compared with controls. Workers with inferred non-acceptable local exhaustion ventilation showed significantly higher inhalable Cr(VI), urine- and RBC-Cr than those with acceptable ventilation. Workers with inferred correct use of respiratory protection had higher inhalable Cr(VI), and, paradoxically, higher urine- and RBC-Cr concentrations than workers with incorrect use. We estimate that ~17 900 Swedish workers are occupationally exposed to Cr(VI) today.Our study showed that although most air measurements were relatively low, 7\ and particularly stainless steel workers are at risk for exceeding the OEL. The existing protective measures implemented at workplaces are still inadequate and insufficient.Some workers in Sweden are exposed to high levels of the non-threshold carcinogen Cr(VI). National strategies aligned with European strategies are needed to eliminate occupational cancer.
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| 8. |
- Modig, Tobias, et al.
(author)
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Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress.
- 2007
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In: Applied microbiology and biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 75:2, s. 289-96
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Journal article (peer-reviewed)abstract
- Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80-90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.
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| 9. |
- Modig, Tobias
(author)
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Efficient Conversion of Lignocellulose Hydrolysates- Yeast Tolerance and Redox Metabolism
- 2007
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Doctoral thesis (other academic/artistic)abstract
- The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for ethanol production was investigated. An important problem for the successful conversion of these hydrolysates is that they often are inhibitory. The physiological effect of the furan inhibitors 5-hydroxymethyl furfural (HMF) and furfural were therefore studied, and based on the results novel strains were constructed. An additional problem for ethanol production is the by-product formation which reduces the ethanol yield. One major by-product during fermentation is glycerol, which is, furthermore, overproduced during hyperosmotic stress. Glycerol formation during anaerobic and hyperosmotic stress conditions was studied in the present work. It was shown that the enzymes alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase, all in the central carbon metabolism of S. cerevisiae, were strongly inhibited by furfural. The variability in S. cerevisiae strain response to lignocellulose hydrolysate was examined in both pulse addition and fed-batch experiments. Characterisation of the furan reduction capacity showed induced NADPH-coupled reduction for both furfural and HMF. A high constitutive NADH-coupled furfural reduction could also be seen. Based on the results of the characterisation of furan reduction, overexpression of the NADPH-dependent enzyme Adh6 and a NADH-dependent mutated Adh1 was conducted. For both enzymes, overexpression provided higher HMF conversion rate in defined medium and lignocellulose hydrolysate. Furthermore, the fermentation performance was improved in lignocellulose hydrolysate for both constructs. Glycerol formation serves to maintain the redox balance during anaerobic growth in S. cerevisiae and glycerol functions also as a osmolyte in hyperosmotic conditions. The response to combined hyperosmotic and anaerobic conditions were studied and large variations among strains could be seen. In addition, a previously uncharacterised protein (Yig1) was investigated for its possible role in regulating anaerobic glycerol formation. The overexpression of Yig1 was found to decrease the activity of glycerol-3-phosphatase.
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| 10. |
- Modig, Tobias, et al.
(author)
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Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.
- 2002
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 363:Pt 3, s. 769-776
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Journal article (peer-reviewed)abstract
- The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition.
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| 11. |
- Modig, Tobias, et al.
(author)
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Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate.
- 2008
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In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 100:3, s. 423-429
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Journal article (peer-reviewed)abstract
- The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000--previously isolated from spent sulphite liquor fermentation--was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADH-coupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.
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| 12. |
- Petersson, A, et al.
(author)
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A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance
- 2006
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In: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 23:6, s. 455-464
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Journal article (peer-reviewed)abstract
- The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity. Copyright (c) 2006 John Wiley & Sons, Ltd.
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| 13. |
- Sanchez, Gladys, et al.
(author)
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Dilute-acid hydrolysis for fermentation of the Bolivian straw material Paja Brava
- 2004
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In: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 93:3, s. 249-256
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Journal article (peer-reviewed)abstract
- Hydrolysis of the straw material Paja Brava, a sturdy grass characteristic for the high plains of Bolivia, was studied in order to find suitable conditions for hydrolysis of the hemicellulose and cellulose parts. Dried Paja Brava material was pre-steamed, impregnated with dilute sulfuric acid (0.5% or 1.0% by wt), and subsequently hydrolyzed in a reactor at temperatures between 170 and 230 degreesC for a reaction time between 3 and 10 min. The highest yield of xylose (indicating efficient hydrolysis of hemicellulose) were found at a temperature of 190 degreesC, and a reaction time of 5-10 min, whereas considerably higher temperatures (230 degreesC) were needed for hydrolysis of cellulose. Fermentability of hemicellulose hydrolyzates was tested using the xylose-fermenting yeast species Pichia stipitis, Candida shehatae and Pachysolen tannophilus. The fermentability of hydrolyzates decreased strongly for hydrolyzates produced at temperatures higher than 200 degreesC. (C) 2003 Elsevier Ltd. All rights reserved.
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