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Efficient Conversion of Lignocellulose Hydrolysates- Yeast Tolerance and Redox Metabolism

Modig, Tobias LU (2007)
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.



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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. (Less)
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author
supervisor
opponent
  • Professor Kielland-Brandt, Morten, Carlsberg Laboratory, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Metabolism, Biokemi, metabolism, hyperosmotic stress, Biochemistry, S. cerevisiae, anaerobic growth, inhibitor tolerance, furfural, lignocellulose, HMF, Biotechnology, Bioteknik
publisher
Department of Chemical Engineering, Lund University
defense location
Lecture hall C, at the Centre for Chemistry and Chemical Engineering, Getingevägen 60, Lund University Faculty of Engineering.
defense date
2007-09-14 10:30
ISBN
978-91-7422-168-8
language
English
LU publication?
yes
id
7735bdfa-90b6-441e-92a0-12388cec158d (old id 548958)
date added to LUP
2007-10-09 12:49:12
date last changed
2016-09-19 08:45:05
@phdthesis{7735bdfa-90b6-441e-92a0-12388cec158d,
  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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.},
  author       = {Modig, Tobias},
  isbn         = {978-91-7422-168-8},
  keyword      = {Metabolism,Biokemi,metabolism,hyperosmotic stress,Biochemistry,S. cerevisiae,anaerobic growth,inhibitor tolerance,furfural,lignocellulose,HMF,Biotechnology,Bioteknik},
  language     = {eng},
  publisher    = {Department of Chemical Engineering, Lund University},
  school       = {Lund University},
  title        = {Efficient Conversion of Lignocellulose Hydrolysates- Yeast Tolerance and Redox Metabolism},
  year         = {2007},
}