| 1. |
- Garcia Sanchez, Rosa, et al.
(författare)
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Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering
- 2010
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced. RESULTS: Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways. CONCLUSION: To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.
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| 2. |
- Sánchez I Nogué, Violeta, et al.
(författare)
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Isolation and characterization of a resident tolerant Saccharomyces cerevisiae strain from a spent sulfite liquor fermentation plant
- 2012
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 2:1, s. 68-
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Tidskriftsartikel (refereegranskat)abstract
- Spent Sulfite Liquor (SSL) from wood pulping facilities is a sugar rich effluent that can be used as feedstock for ethanol production. However, depending on the pulping process conditions, the release of monosaccharides also generates a range of compounds that negatively affect microbial fermentation. In the present study, we investigated whether endogenous yeasts in SSL-based ethanol plant could represent a source of Saccharomyces cerevisiae strains with a naturally acquired tolerance towards this inhibitory environment. Two isolation processes were performed, before and after the re-inoculation of the plant with a commercial baker’s yeast strain. The isolates were clustered by DNA fingerprinting and a recurrent Saccharomyces cerevisiae strain, different from the inoculated commercial baker’s yeast strain, was isolated. The strain, named TMB3720, flocculated heavily and presented high furaldehyde reductase activity. During fermentation of undiluted SSL, TMB3720 displayed a 4-fold higher ethanol production rate and 1.8-fold higher ethanol yield as compared to the commercial baker’s yeast. Another non-Saccharomyces cerevisiae species, identified as the pentose utilizing Pichia galeiformis, was also recovered in the last tanks of the process where the hexose to pentose sugar ratio and the inhibitory pressure are expected to be the lowest.
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| 3. |
- Almeida, Joao, et al.
(författare)
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Stress-related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae.
- 2011
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768. ; 6, s. 286-299
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Forskningsöversikt (refereegranskat)abstract
- Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.
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| 4. |
- Blomqvist, Johanna, et al.
(författare)
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Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions
- 2012
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 29:7, s. 265-274
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Tidskriftsartikel (refereegranskat)abstract
- The effect of glucose and oxygen limitation on the growth and fermentation performances of Dekkera bruxellensis was investigated in order to understand which factors favour its propagation in ethanol or wine plants. Although D. bruxellensis has been described as a facultative anaerobe, no growth was observed in mineral medium under complete anaerobiosis while growth was retarded under severe oxygen limitation. In a continuous culture with no gas inflow, glucose was not completely consumed, most probably due to oxygen limitation. When an air/nitrogen mixture (O-2-content ca. 5%) was sparged to the culture, growth became glucose-limited. In co-cultivations with Saccharomyces cerevisiae, ethanol yields/g consumed sugar were not affected by the co-cultures as compared to the pure cultures. However, different population responses were observed in both systems. In oxygen-limited cultivation, glucose was depleted within 24 h after challenging with S. cerevisiae and both yeast populations were maintained at a stable level. In contrast, the S. cerevisiae population constantly decreased to about 1% of its initial cell number in the sparged glucose-limited fermentation, whereas the D. bruxellensis population remained constant. To identify the requirements of D. bruxellensis for anaerobic growth, the yeast was cultivated in several nitrogen sources and with the addition of amino acids. Yeast extract and most of the supplied amino acids supported anaerobic growth, which points towards a higher nutrient demand for D. bruxellensis compared to S. cerevisiae in anaerobic conditions. Copyright (c) 2012 John Wiley & Sons, Ltd.
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| 5. |
- Narayanan, Venkatachalam, et al.
(författare)
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Adaptation to low pH and lignocellulosic inhibitors resulting in ethanolic fermentation and growth of Saccharomyces cerevisiae
- 2016
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Lignocellulosic bioethanol from renewable feedstocks using Saccharomyces cerevisiae is a promising alternative tofossil fuels owing to environmental challenges. S. cerevisiae is frequently challenged by bacterial contamination anda combination of lignocellulosic inhibitors formed during the pre-treatment, in terms of growth, ethanol yield andproductivity. We investigated the phenotypic robustness of a brewing yeast strain TMB3500 and its ability to adapt tolow pH thereby preventing bacterial contamination along with lignocellulosic inhibitors by short-term adaptation andadaptive lab evolution (ALE). The short-term adaptation strategy was used to investigate the inherent ability of strainTMB3500 to activate a robust phenotype involving pre-culturing yeast cells in defined medium with lignocellulosicinhibitors at pH 5.0 until late exponential phase prior to inoculating them in defined media with the same inhibitorcocktail at pH 3.7. Adapted cells were able to grow aerobically, ferment anaerobically (glucose exhaustion by 19 ± 5 hto yield 0.45 ± 0.01 g ethanol g glucose−1) and portray significant detoxification of inhibitors at pH 3.7, when comparedto non-adapted cells. ALE was performed to investigate whether a stable strain could be developed to growand ferment at low pH with lignocellulosic inhibitors in a continuous suspension culture. Though a robust populationwas obtained after 3600 h with an ability to grow and ferment at pH 3.7 with inhibitors, inhibitor robustness was notstable as indicated by the characterisation of the evolved culture possibly due to phenotypic plasticity. With furtherresearch, this short-term adaptation and low pH strategy could be successfully applied in lignocellulosic ethanolplants to prevent bacterial contamination.
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| 6. |
- Pereira, Susana R., et al.
(författare)
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Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering
- 2015
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. Results: A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C-4) was isolated and characterized in 60% undetoxified HSSL. C-4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C-4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C-4. Conclusion: S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C-4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C-4 may represent a major advantage for the production of bioethanol using HSSL as substrate.
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| 7. |
- Sanchez Nogue, Violeta
(författare)
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Industrial challenges in the use of Saccharomyces cerevisiae for ethanolic fermentation of lignocellulosic biomass
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The sustainable production of ethanol from lignocellulosic biomass requires the combination of efficient hydrolysis and complete fermentation of all the monomeric sugars present in the raw material. The present work was aimed at tackling some of the major challenges that will be encountered in commercial-scale ethanol production using Baker’s yeast, Saccharomyces cerevisiae, the preferred microorganism for the fermentation step. During biomass pretreatment, several inhibitory compounds are released, including weak acids, furaldehydes and phenolics. The presence of these compounds in the hydrolysate reduces the ethanol yield and productivity, prolongs the lag phase, and reduces the growth rate of the yeast during fermentation. S. cerevisiae is naturally unable to utilise the pentose sugars xylose and arabinose. Evolutionary engineering was used to improve the conversion of these pentoses to ethanol in a recombinant industrial strain of S. cerevisiae expressing heterologous genes for the xylose and arabinose utilisation pathways. The evolved strain showed a higher rate of consumption of xylose and arabinose under both aerobic and anaerobic conditions, which was attributed to an increase in the transport of pentoses and the activities of xylose converting enzymes. The introduction of a short-adaptation process enabled aerobic growth at low pH in the presence of inhibitory levels of acetic acid and led to a significant reduction in the fermentation time under anaerobic conditions. In parallel, the possibility of using indigenous yeasts present in the spent sulphite liquor (SSL) ethanol plant as a source of S. cerevisiae strains with a naturally acquired tolerance to inhibitory compounds was also investigated. The isolated strain, TMB3720, exhibited a higher ethanol yield and production rate than the commercial baker’s yeast strain, regularly used as inoculum. It was hypothesised that the tolerance of this strain was related to its flocculation behaviour and its high capacity to reduce furaldehyde inhibitors. As ethanol plants are run under non-sterile conditions, S. cerevisiae must compete with other microorganisms for sugar utilisation. Therefore, competition experiments were performed with the contaminant yeast Dekkera bruxellensis and the lactic acid bacterium Lactobacillus pentosus isolated from the SSL ethanol plant. Glucose limitation, achieved by sparging a mixture of nitrogen and air (~5% oxygen) through the system, was identified as a parameter enabling D. bruxellensis to outcompete S. cerevisiae, probably due to the higher nutrient affinity of D. bruxellensis under these conditions. In parallel, reducing the pH was also found to be a possible means of reducing the levels of lactate produced by the L. pentosus strain.
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| 8. |
- Sanchez Nogue, Violeta, et al.
(författare)
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Short-term adaptation improves the fermentation performance of Saccharomyces cerevisiae in the presence of acetic acid at low pH.
- 2013
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:16, s. 7517-7525
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Tidskriftsartikel (refereegranskat)abstract
- The release of acetic acid due to deacetylation of the hemicellulose fraction during the treatment of lignocellulosic biomass contributes to the inhibitory character of the generated hydrolysates. In the present study, we identified a strain-independent adaptation protocol consisting of pre-cultivating the strain at pH 5.0 in the presence of at least 4 g L(-1) acetic acid that enabled aerobic growth and improved fermentation performance of Saccharomyces cerevisiae cells at low pH (3.7) and in the presence of inhibitory levels of acetic acid (6 g L(-1)). During anaerobic cultivation with adapted cells of strain TMB3500, the specific ethanol production rate was increased, reducing the fermentation time to 48 %.
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| 9. |
- Sanchez Nogue, Violeta, et al.
(författare)
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Xylose fermentation as a challenge for commercialization of lignocellulosic fuels and chemicals
- 2015
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Ingår i: Biotechnology Letters. - : Springer Science and Business Media LLC. - 1573-6776 .- 0141-5492. ; 37:4, s. 761-772
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Forskningsöversikt (refereegranskat)abstract
- Fuel ethanol production from lignocellulosic materials is at a level where commercial biofuel production is becoming a reality. The solubilization of the hemicellulose fraction in lignocellulosic-based feedstocks results in a large variety of sugar mixtures including xylose. However, allowing xylose fermentation in yeast that normally is used for fuel ethanol production requires genetic engineering. Moreover, the efficiency of lignocellulosic pretreatment, together with the release and generation of inhibitory compounds in this step, are some of the new challenges faced during second generation ethanol production. Successful advances in all these aspects will improve ethanol yield, productivity and titer, which will reduce the impact on capital and operating costs, leading to the consolidation of the fermentation of lignocellulosic biomass as an economically feasible option for the production of renewable fuels. Therefore the development of yeast strains capable of fermenting a wide variety of sugars in a highly inhibitory environment, while maintaining a high ethanol yield and production rate, is required. This review provides an overview of the current status in the use of xylose-engineered yeast strains and describes the remaining challenges to achieve an efficient deployment of lignocellulosic-based ethanol production.
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