| 1. |
- Ask, Magnus, 1983, et al.
(författare)
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The effect of pretreatment harshness on separate hydrolysis and fermentation of giant reed by a xylose-consuming Saccharomyces cerevisiae strain
- 2011
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Ingår i: World Congress on Industrial Biotechnology and Bioprocessing, May 8 - 11, 2011, Toronto, Canada.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol produced from lignocellulosic feedstocks has received increased attention during the last years. To make lignocellulosic biomass susceptible to enzymatic hydrolysis, the materials first have to be pretreated. The pretreatment is often performed under harsh conditions, which release a number of compounds that can be inhibitory for enzymatic hydrolysis and the subsequent fermentation. Xylooligomers and acetic acid are two compounds that are potential inhibitors of enzymatic hydrolysis and fermentation, respectively. The final concentration of these compounds is highly dependent on the pretreatment conditions. In this study, two different pretreatments with different harshness were performed on giant reed. The influence of the resulting material composition on enzymatic hydrolysis was then investigated. The enzymatic hydrolysis was performed at 10 % (w/w) water insoluble solid concentration (WIS) with Celluclast 1.5L and Novozyme 188 with and without the addition of HTec which is acting on hemicellulose. During the harsher pretreatment, more xylooligomers were produced which were found to have a negative effect on the enzymatic hydrolysis. One of the hydrolysates contained a substantially higher concentration of acetic acid. To investigate the effect of this, the hydrolysed giant reed was fermented with a laboratory Saccharomyces cerevisiae strain, VTT C-10880, carrying the XR/XDH pathway. It was found that the acetic acid had a significant negative effect on the xylose consumption. By supplementing the less harsh pretreated material with the same amount of acetic acid, a similar decrease in xylose consumption was observed, indicating that acetic acid is limiting xylose fermentation in this case.
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| 2. |
- Sárvári Horváth, Ilona, 1960, et al.
(författare)
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Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats
- 2003
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 69:7, s. 4076-4086
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Tidskriftsartikel (refereegranskat)abstract
- Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.
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| 3. |
- Kadić, Adnan, et al.
(författare)
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In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. Results: Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. Conclusion: Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.
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| 4. |
- Stovicek, Vratislav, et al.
(författare)
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Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate
- 2022
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Ingår i: Biotechnology for Biofuels and Bioproducts. - : Springer Science and Business Media LLC. - 2731-3654. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars—in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. Results: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. Conclusions: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.
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| 5. |
- Modig, Tobias, et al.
(författare)
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Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.
- 2002
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 363:Pt 3, s. 769-776
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Ask, Magnus, 1983, et al.
(författare)
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A comparison between simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) of spruce and giant reed using two Saccharomyces cerevisiae strains
- 2010
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Ingår i: Society for Industrial Microbiology, 60th annual meeting.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- For significant fermentative conversion of lignocellulose to ethanol, the yeast Saccharomyces cerevisiae has proved to be a robust organism, albeit inter-strain variations may have a big influence on process performance. In this study, two S. cerevisiae strains were evaluated for their ability to ferment two different lignocellulosic raw materials, giant reed and spruce at 10 % water insoluble solids (WIS). One industrial strain, Ethanol Red, and one laboratory strain carrying the XR/XDH pathway, VTT C-10880, were used. The process concept may also affect the choice of the most suitable strain. Therefore, two principal process concepts, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were evaluated.The ethanol yield on giant reed based on total soluble sugars in the SHF was higher for VTT C-10880 than for Ethanol Red. On spruce, the yield of ethanol was higher for Ethanol Red. In SSF of giant reed, VTT C-10880 performed better in terms of the ethanol yield based on total sugars in fibres and liquid. However, the ethanol yield on spruce was higher for Ethanol Red than for VTT C-10880, which only produced a minor amount of ethanol. Spruce was more inhibitory than giant reed. Ethanol Red is more robust and converted the inhibitory substances in the pretreated materials faster, and is therefore a suitable industrial strain background for fermentation of both spruce and giant reed. Interestingly, VTT C-10880 performed better in SHF than SSF, primarily due to better xylose conversion in SHF.
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| 7. |
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| 8. |
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| 9. |
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| 10. |
- García-Hidalgo, Javier, et al.
(författare)
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Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation
- 2019
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- A diversity of softwood lignin depolymerization processes yield guaiacol as the main low molecular weight product. This key aromatic compound can be utilized as a carbon source by several microbial species, most of which are Gram positive bacteria. Microbial degradation of guaiacol is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In this work we identified two proteins involved in guaiacol demethylation by the actinomycete Rhodococcus rhodochrous. Additionally, we constructed four different polycistronic operons carrying combinations of putative redox partners of this guaiacol demethylation system in an inducible expression plasmid that was introduced into the Gram negative host Pseudomonas putida EM42, and the guaiacol consumption dynamics of each resulting strain were analyzed. All the polycistronic operons, expressing a cytochrome P450 together with a putative ferredoxin reductase from R. rhodochrous and putative ferredoxins from R. rhodochrous or Amycolatopsis ATCC 39116 enabled P. putida EM42 to metabolize and grow on guaiacol as the sole carbon source.
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