| 1. |
- 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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| 2. |
- Ask, Magnus, 1983, et al.
(författare)
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Challenges in enzymatic hydrolysis and fermentation of pretreated Arundo donax revealed by a comparison between SHF and SSF
- 2012
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; 47:10, s. 1452-1459
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Tidskriftsartikel (refereegranskat)abstract
- The perennial herbaceous crop Arundo donax is a potential feedstock for second-generation bioethanol production. In the present work, two different process options were investigated for the conversion of two differently steam-pretreated batches of A. donax. The pretreated raw material was converted to ethanol with a xylose-consuming Saccharomyces cerevisiae strain, VTT C-10880, by applying either separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). The highest overall ethanol yield and final ethanol concentration were achieved using SHF (0.27 g g(-1) and 20.6 g L-1 compared to 0.24 g g(-1) and 19.0 g L-1 when SSF was used). The performance of both SHF and SSF was improved by complementing the cellulolytic enzymes with hemicellulases. The higher amount of acetic acid in one of the batches was shown to strongly affect xylose consumption in the fermentation. Only half of the xylose was consumed when batch 1 (high acetic acid) was fermented, compared to that 94% of the xylose was consumed in fermentation of batch 2 (lower acetic acid). Furthermore, the high amount of xylooligomers present in the pretreated materials considerably inhibited the enzymatic hydrolysis. Both the formation of xylooligomers and acetic acid thus need to be considered in the pretreatment process in order to achieve efficient conversion of A. donax to ethanol.
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| 3. |
- Ask, Magnus, 1983, et al.
(författare)
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Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials
- 2013
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 12:87
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Tidskriftsartikel (refereegranskat)abstract
- Production of bioethanol from lignocellulosic biomass requires the development of robust microorganisms that can tolerate the stressful conditions prevailing in lignocellulosic hydrolysates. Several inhibitors are known to affect the redox metabolism of cells. In this study, Saccharomyces cerevisiae was engineered for increased robustness by modulating the redox state through overexpression of GSH1, CYS3 and GLR1, three genes involved in glutathione (GSH) metabolism. Overexpression constructs were stably integrated into the genome of the host strains yielding five strains overexpressing GSH1, GSH1/CYS3, GLR1, GSH1/GLR1 and GSH1/CYS3/GLR1. Overexpression of GSH1 resulted in a 42% increase in the total intracellular glutathione levels compared to the wild type. Overexpression of GSH1/CYS3, GSH/GLR1 and GSH1/CYS3/GLR1 all resulted in equal or less intracellular glutathione concentrations than overexpression of only GSH1, although higher than the wild type. GLR1 overexpression resulted in similar total glutathione levels as the wild type. Surprisingly, all recombinant strains had a lower [reduced glutathione]:[oxidized glutathione] ratio (ranging from 32--67) than the wild type strain (88), suggesting a more oxidized intracellular environment in the engineered strains. When considering the glutathione half-cell redox potential (Ehc), the difference between the strains was less pronounced. Ehc for the recombinant strains ranged from -225 to -216 mV, whereas for the wild type it was estimated to -225 mV. To test whether the recombinant strains were more robust in industrially relevant conditions, they were evaluated in simultaneous saccharification and fermentation (SSF) of pretreated spruce. All strains carrying the GSH1 overexpression construct performed better than the wild type in terms of maximum ethanol concentration, ethanol yield and furfural and HMF conversion. The strain overexpressing GSH1/GLR1 produced 14.0 g L-1 ethanol in 48 hours corresponding to an ethanol yield on hexoses of 0.17 g g-1, compared to the wild type, which produced 8.2 g L-1 ethanol in 48 hours resulting in an ethanol yield on hexoses of 0.10 g g-1. In this study, we showed that engineering of the redox state by modulating the levels of intracellular glutathione results in increased robustness of S. cerevisiae in SSF of pretreated spruce.
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| 4. |
- Ask, Magnus, 1983, et al.
(författare)
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HMF and furfural stress results in drainage of redox and energy charge of Saccharomyces cerevisiae
- 2012
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Ingår i: 13th International Congress on Yeasts, Madison, WI, USA.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol produced from lignocellulosic raw materials is a promising alternative to fossil fuels and to decrease greenhouse gas emissions, but several challenges still exist. When lignocellulosic biomass is pretreated, a number of undesired degradation products are generated which may act inhibitory on microbial metabolism. Cellular damage response and repair come at an energy cost for the cell, which could be reflected by alterations in (energy) metabolism. The furaldehydes HMF and furfural have received increasing attention recently. They are formed during pretreatment from dehydration of hexoses and pentoses, respectively. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and transcriptome were investigated. Anaerobic chemostat cultivations were performed with the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 with both glucose and xylose as carbon sources. By quantifying the redox cofactors NAD(P)+ and NAD(P)H, the catabolic and anabolic reduction charges could be calculated. It was found that both reduction charges were significantly decreased in the presence of HMF and furfural, showing that HMF and furfural are draining the cells of reductive power. Furthermore, the [ATP]/[ADP] ratio of stressed cells was found to be lower than for non-stressed cells, suggesting that the energy metabolism was affected. Transcriptome analysis revealed that genes involved in xenobiotic transporter activity were significantly enriched among the up-regulated genes. The results from the present study provide valuable insights of how Saccharomyces cerevisiae deals with stress imposed by HMF and furfural, which potentially can result in strategies to improve stress tolerance.
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| 5. |
- Ask, Magnus, 1983, et al.
(författare)
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Intracellular redox state as key target for Saccharomyces cerevisiae tolerance to lignocellulosic hydrolysate inhibitors
- 2013
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Ingår i: 35th Symposium on Biotechnology for Fuels and Chemicals (April 29-May 2, 2013).
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Liberation of sugars monomers from the polysaccharides constituting lignocellulosic biomass requires pretreatment and hydrolysis. Harsh conditions during pretreatment promote the formation of a number of inhibitory compounds, among which the furaldehydes furfural and hydroxymethylfurfural (HMF) have shown to impede growth and limit ethanol productivity of the yeast Saccharomyces cerevisiae. Cellular damage response to such inhibitory molecules and repair come at an energy cost for the cell, which could be reflected by alterations in energy and redox metabolism. In this study, S. cerevisiae cultures where treated with sub-lethal concentrations of furfural and HMF, both in continuous and batch cultivations. In continuous cultures, the inhibitors concentration was as close as possible to lethal, yet allowing steady state. In batch cultivations, the chosen concentration completely inhibited growth, yet allowing growth resumption. Metabolites connected to energy and redox metabolism such as NAD(P)H, NADP+, ATP, ADP and AMP were quantified and transcriptome analysis was performed. The results, along with data from thorough physiological characterisation under the studied conditions, suggested a severe impact of furfural and HMF on energy and redox metabolism. Based on this evidence, new strain with altered redox carriers intracellular concentration were engineered. The new recombinant strains showed higher ethanol productivity in the presence of lignocellulosic hydrolysate inhibitors.
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| 6. |
- Ask, Magnus, 1983, et al.
(författare)
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Physiological studies of Saccharomyces cerevisiae for increased tolerance against furfural and HMF – two common inhibitors in lignocellulosic hydrolysate
- 2011
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Ingår i: 5th EU-Summer School Proteomic Basics, Brixen, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The use of fossil fuels in the transport sector has a significant impact on the environment through the emission of greenhouse gases such as carbon dioxide. Bioethanol is one alternative that has shown potential to at least partly replace fossil fuels. Today’s bioethanol is mainly produced from sugar cane and corn with the yeast Saccharomyces cerevisiae as production organism. Since these raw materials compete with food production, new feedstocks have to be found. A promising alternative is to make use of forest and agricultural residues, so called lignocellulosic materials. Nevertheless, there are many challenges with using lignocellulosic materials for bioethanol production. Since they are recalcitrant to decomposition, harsh conditions have to be used to break down the materials. These conditions tend to produce byproducts that can be inhibitory for the production organism, resulting in lower process productivity. Low productivity is one of the main factors affecting the feasibility of lignocellulosic ethanol production processes. Hydroxymethylfurfural (HMF) and furfural are two compounds that have received a lot of attention during the last years. By studying the effect of these inhibitory compounds on the energy metabolism of S. cerevisiae, the aim of the project is to increase the understanding of the mechanisms by which these inhibitors affect the microorganism. More specifically, the inhibitors effect is studied by quantifying intracellular key compounds such as NADH, NAD+, sugar phosphates and the adenine nucleotide pool. In addition, the biochemical data on intracellular concentrations will be integrated with transcriptomic data. In the future, this knowledge will be used to produce strains that are more tolerant to the process conditions.
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| 7. |
- Ask, Magnus, 1983, et al.
(författare)
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Pulsed addition of HMF and furfural to batch-grown xylose-utilizing Saccharomyces cerevisiae results in different physiological responses in glucose and xylose consumption phase
- 2013
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:181
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Tidskriftsartikel (refereegranskat)abstract
- Pretreatment of lignocellulosic biomass generates a number of undesired degradation products that can inhibit microbial metabolism. Two of these compounds, the furan aldehydes 5-hydroxymethylfurfural (HMF) and 2-furaldehyde (furfural), have been shown to be an impediment for viable ethanol production. In the present study, HMF and furfural were pulse-added during either the glucose or the xylose consumption phase in order to dissect the effects of these inhibitors on energy state, redox metabolism, and gene expression of xylose-consuming Saccharomyces cerevisiae.Pulsed addition of 3.9?g?L-1 HMF and 1.2?g?L-1 furfural during either the glucose or the xylose consumption phase resulted in distinct physiological responses. Addition of furan aldehydes in the glucose consumption phase was followed by a decrease in the specific growth rate and the glycerol yield, whereas the acetate yield increased 7.3-fold, suggesting that NAD(P)H for furan aldehyde conversion was generated by acetate synthesis. No change in the intracellular levels of NAD(P)H was observed 1?hour after pulsing, whereas the intracellular concentration of ATP increased by 58%. An investigation of the response at transcriptional level revealed changes known to be correlated with perturbations in the specific growth rate, such as protein and nucleotide biosynthesis. Addition of furan aldehydes during the xylose consumption phase brought about an increase in the glycerol and acetate yields, whereas the xylitol yield was severely reduced. The intracellular concentrations of NADH and NADPH decreased by 58 and 85%, respectively, hence suggesting that HMF and furfural drained the cells of reducing power. The intracellular concentration of ATP was reduced by 42% 1?hour after pulsing of inhibitors, suggesting that energy-requiring repair or maintenance processes were activated. Transcriptome profiling showed that NADPH-requiring processes such as amino acid biosynthesis and sulfate and nitrogen assimilation were induced 1?hour after pulsing.The redox and energy metabolism were found to be more severely affected after pulsing of furan aldehydes during the xylose consumption phase than during glucose consumption. Conceivably, this discrepancy resulted from the low xylose utilization rate, hence suggesting that xylose metabolism is a feasible target for metabolic engineering of more robust xylose-utilizing yeast strains.
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| 8. |
- Ask, Magnus, 1983, et al.
(författare)
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TARGETING THE INTRACELLULAR REDOX STATE IN THE DEVELOPMENT OF MORE ROBUST Saccharomyces cerevisiae STRAINS FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTION
- 2014
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Ingår i: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol produced from lignocellulosic raw materials is a promising alternative to fossil fuels and to decrease greenhouse gas emissions, but several challenges still exist. When lignocellulosic biomass is pretreated, a number of undesired degradation products are generated, among which the furaldehydes furfural and hydroxymethylfurfural (HMF) have shown to impede growth and limit ethanol productivity of the yeast Saccharomyces cerevisiae. In the present study, a recombinant, xylose-utilizing S. cerevisiae strain was challenged with sub-lethal concentrations of furfural and HMF in anaerobic batch cultivations. By pulsing furaldehydes in either the glucose or the xylose consumption phase, perturbations in the intracellular NAD(P)H/NAD(P)+ ratios could be demonstrated. A genome-wide study of transcription found that genes related to NADPH-requiring processes, such as nitrogen and sulphur assimilation, were significantly induced. Moreover, the protective metabolite and antioxidant glutathione was identified as the highest scoring reporter metabolite in the transcriptome analysis. S. cerevisiae strains overproducing glutathione were constructed and the resulting strains were evaluated in simultaneous saccharification and fermentation (SSF) of pretreated spruce. The results from the present study provide valuable insights of how S. cerevisiae responds to stress imposed by HMF and furfural and how such information could be used to engineer more robust yeast strains.
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| 9. |
- 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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| 10. |
- Ask, Magnus, 1983, et al.
(författare)
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The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae
- 2013
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:22
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(+/-0.005) to 0.08(+/-0.002) and the anabolic reduction charge decreased from 0.46(+/-0.11) to 0.27(+/-0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(+/-0.002) to 0.85(+/-0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group "Cell rescue, defense and virulence" was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.
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