| 1. |
- Geijer, Cecilia, 1980, et al.
(author)
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Characterization of a novel non-GMO yeast for future lignocellulosic bioethanol production
- 2014
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In: ISSY31: 31ST International Specialised Symposium on Yeast.
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Conference paper (other academic/artistic)abstract
- CHARACTERIZATION OF A NOVEL NON-GMO YEAST FOR FUTURE LIGNOCELLULOSIC BIOETHANOL PRODUCTIONCecilia Geijer1, David Moreno1, Elia Tomas Pejo1, 2, Lisbeth Olsson11 Industrial Biotechnology , Department of Chemical and Biological Engineering Chalmers University of Technology, Gothenburg, Sweden2Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), SpainContact details: cecilia.geijer@chalmers.seConcerns about climate change and the uncertainty about future fuel supply make renewable biofuels, such as bioethanol, attractive alternatives to fossil fuels in the short/medium term. Lignocellulosic biomass (for example spruce, wheat straw and corn stover) is an abundant raw material that can be utilized to produce ethanol with the help of a fermenting microorganism. Traditionally the yeast Saccharomyces cerevisiae is used for industrial ethanol production. S. cerevisiae can be metabolically engineered to consume xylose (the second to glucose most prevalent monosaccharide in lignocellulose). However, despite many years of intensive research, it can still not ferment xylose in a satisfying way which affects the overall ethanol yield negatively. We have isolated a non-genetically modified (non-GMO) yeast species (here called C5-yeast) that has the natural ability to efficiently produce ethanol from glucose and xylose. The aim of the project is to further characterize the growth and fermentation capacities of this novel microorganism to elucidate its’ potential for lignocellulosic bioethanol production. We can show that besides glucose and xylose, the C5-yeast can also consume the pentose arabinose and the disaccharide cellobiose; both present in lignocellulosic hydrolysates. The C5-yeast rapidly converts the inhibitory sugar degradation products HMF and furfural formed during the conversion of lignocellulosic material into fermentable sugars.
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| 2. |
- Geijer, Cecilia, 1980, et al.
(author)
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Evolutionary engineered strains of Saccharomyces cerevisiae for efficient lignocellulosic bioethanol production
- 2014
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In: 36th Symposium on Biotechnology for Fuels and Chemicals.
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Conference paper (other academic/artistic)abstract
- Lignocellulosic biomass is an abundant raw material that can be utilized to produce ethanol with the help of Saccharomyces cerevisiae; a promising alternative to today’s energy sources. Conversion of lignocellulosic material (cellulose, hemicellulose and lignin) into fermentable sugars including both hexoses and pentoses results in formation of inhibitory compounds such as acetic acid, furan aldehydes and phenolics that are known to inhibit the yeasts’ metabolic processes. The aims of this study were to i) generate S. cerevisiae strains that can readily convert glucose and xylose into ethanol in the presence of inhibitory compounds, and ii) elucidate the underlying genetic changes of importance for the improved properties of the generated strains. For these purposes, a strain of S. cerevisiae containing genes for xylose reductase, xylitol dehydrogenase and xylulokinase was used. The strain was subjected to mutagenesis followed by evolutionary engineering (repetitive batch and chemostat cultivation), which resulted in populations with improved ethanol yield, improved xylose conversion rate and increased inhibitor tolerance. The complex combination of different genetic alterations in the evolved populations will now be revealed using a DNA/RNA sequencing approach. The acquired knowledge of proteins and pathways important for efficient lignocellulosic bioethanol production will then hopefully allow directed engineering for further improvement of yeast performance.
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| 3. |
- Moreno, David, 1986, et al.
(author)
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Isolation and evolution of a novel non-saccharomyces xylose-fermenting strain for lignocellulosic bioethanol production
- 2014
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In: ISSY31: 31ST International Specialised Symposium on Yeast.
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Conference paper (other academic/artistic)abstract
- ISOLATION AND EVOLUTION OF A NOVEL NON-SACCHAROMYCES XYLOSE-FERMENTING STRAIN FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTIONAntonio D. Moreno1, Cecilia Geijer1, Elia Tomás-Pejó1,2, Lisbeth Olsson1.1Chalmers University of Technology, Department of Chemical and Biological Engineering, Industrial Biotechnology Group, Göteborg, Sweden. 2Unit of Biotechnological Processes for Energy Production, IMDEA Energy, Móstoles (Madrid), Spain.Contact e-mail: davidmo@chalmers.seThe economical success of lignocellulosic bioethanol requires the fermentation of all available sugars obtained during the process. Being the major pentose sugar in lignocellulose, the fermentation of xylose is, therefore, considered essential. The fermentative yeast Saccharomyces cerevisiae is the most promising candidate for lignocellulosic bioethanol production due to its excellent glucose fermentation capability, high ethanol tolerance and resistance to inhibitors presented in lignocellulosic streams. Nevertheless, the wild type S. cerevisae is not able to ferment xylose and all of the purpose-engineered Saccharomyces strains (genetically modified microorganisms (GMO)) are still far away from an economically viable lignocellulosic ethanol production. By chance, we have discovered a non-Saccharomyces xylose-fermenting yeast (here called C5-yeast), which shows a great potential to be used for bioethanol production from lignocellulosic streams. Unlike xylose-fermenting Saccharomyces strains, the C5-yeast is not genetically modified and its use by industries can aid in finding less legislative problems when reaching the market. In the present work, the C5-yeast was isolated from a xylose-fermenting population and evolutionary engineered to enhance its fermentation abilities and robustness. During the isolation process, three different morphologies (smooth, flat and wrinkled) of the C5-yeast were found when growing the xylose-fermenting population in plates with minimal media and xylose as a sole carbon source. Among all morphologies, flat-C5-yeast showed the highest xylose consumption rates (>90% after 72 h) and the highest ethanol conversion yields (≈50% of the theoretical considering glucose and xylose) during the fermentation of wheat straw hydrolysates. The isolated flat-C5-yeast was selected for evolutionary engineering in order to enhance its sugar conversion yields and the tolerance towards the inhibitory compounds that are present in the hydrolysate. Although further characterization is needed, an evolved C5-yeast could be considered as a suitable fermentative strain for lignocellulosic bioethanol production.
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| 4. |
- Koppram, Rakesh, 1986, et al.
(author)
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Lignocellulosic ethanol production at high-gravity: Challenges and perspectives
- 2014
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In: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 32:1, s. 46-53
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Research review (peer-reviewed)abstract
- In brewing and ethanol-based biofuel industries, high-gravity fermentation produces 10-15% (v/v) ethanol, resulting in improved overall productivity, reduced capital cost, and reduced energy input compared to processing at normal gravity. High-gravity technology ensures a successful implementation of cellulose to ethanol conversion as a cost-competitive process. Implementation of such technologies is possible if all process steps can be performed at high biomass concentrations. This review focuses on challenges and technological efforts in processing at high-gravity conditions and how these conditions influence the physiology and metabolism of fermenting microorganisms, the action of enzymes, and other process-related factors. Lignocellulosic materials add challenges compared to implemented processes due to high inhibitors content and the physical properties of these materials at high gravity. © 2013 Elsevier Ltd.
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| 5. |
- Moreno, David, 1986, et al.
(author)
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Fed-batch SSCF using steam-exploded wheat straw at high dry matter consistencies and a xylose-fermenting Saccharomyces cerevisiae strain: effect of laccase supplementation
- 2013
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:1, s. article nr. 160-
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Journal article (peer-reviewed)abstract
- Lignocellulosic bioethanol is expected to play an important role in fossil fuel replacement in the short term. Process integration, improvements in water economy, and increased ethanol titers are key considerations for cost-effective large-scale production. The use of whole steam-pretreated slurries under high dry matter (DM) conditions and conversion of all fermentable sugars offer promising alternatives to achieve these goals. Wheat straw slurry obtained from steam explosion showed high concentrations of degradation compounds, hindering the fermentation performance of the evolved xylose-recombinant Saccharomyces cerevisiae KE6-12 strain. Fermentability tests using the liquid fraction showed a higher number of colony-forming units (CFU) and higher xylose consumption rates when treating the medium with laccase. During batch simultaneous saccharification and co-fermentation (SSCF) processes, cell growth was totally inhibited at 12% DM (w/v) in untreated slurries. However, under these conditions laccase treatment prior to addition of yeast reduced the total phenolic content of the slurry and enabled the fermentation. During this process, an ethanol concentration of 19 g/L was obtained, corresponding to an ethanol yield of 39% of the theoretical yield. By changing the operation from batch mode to fed-batch mode, the concentration of inhibitors at the start of the process was reduced and 8 g/L of ethanol were obtained in untreated slurries with a final consistency of 16% DM (w/v). When fed-batch SSCF medium was supplemented with laccase 33 hours after yeast inoculation, no effect on ethanol yield or cell viability was found compared to untreated fermentations. However, if the laccase supplementation (21 hours after yeast inoculation) took place before the first addition of substrate (at 25 hours), improved cell viability and an increased ethanol titer of up to 32 g/L (51% of the theoretical) were found. Laccase treatment in SSCF processes reduces the inhibitory effect that degradation compounds have on the fermenting microorganism. Furthermore, in combination with fed-batch operational mode, laccase supplementation allows the fermentation of wheat straw slurry at high DM consistencies, improving final ethanol concentrations and yields.
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| 6. |
- Moreno, David, 1986, et al.
(author)
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In situ laccase treatment enhances the fermentability of steam-exploded wheat straw in SSCF processes at high dry matter consistencies
- 2013
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In: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 143, s. 337-343
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Journal article (peer-reviewed)abstract
- This work evaluates the in situ detoxification of inhibitory lignocellulosic broths by laccases to facilitate their fermentation by the xylose-consuming Saccharomyces cerevisiae F12. Treatment of wheat straw slurries with laccases prior to SSCF processes decreased the total phenolic content by 50-80%, reducing the lag phase and increasing the cell viability. After laccase treatment, a negative impact on enzymatic hydrolysis was observed. This effect, together with the low enzymatic hydrolysis yields when increasing consistency, resulted in a decrease in final ethanol yields. Furthermore, when using high substrate loading (20% DM (w/v)), high concentration of inhibitors prevailed in broths and the absence of an extra nitrogen source led to a total cell growth inhibition within the first 24 h in non-treated samples. This inhibition of growth at 20% DM (w/v) was overcome by laccase treatment with no addition of nitrogen, allowing S. cerevisiae F12 to produce more than 22 g/L of ethanol.
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| 7. |
- Tomas-Pejo, Elia, 1980, et al.
(author)
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Adaption of the xylose fermenting yeast Saccharomyces cerevisiae F12 for improving ethanol production in different fed-batch SSF processes
- 2010
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In: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 37:11, s. 1211-1220
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Journal article (peer-reviewed)abstract
- An efficient fermenting microorganism for bioethanol production from lignocellulose is highly tolerant to the inhibitors released during pretreatment and is able to ferment efficiently both glucose and xylose. In this study, directed evolution was employed to improve the xylose fermenting Saccharomyces cerevisiae F12 strain for bioethanol production at high substrate loading. Adapted and parental strains were compared with respect to xylose consumption and ethanol production. Adaption led to an evolved strain more tolerant to the toxic compounds present in the medium. When using concentrated prehydrolysate from steam-pretreated wheat straw with high inhibitor concentration, an improvement of 65 and 20% in xylose consumption and final ethanol concentration, respectively, were achieved using the adapted strain. To address the need of high substrate loadings, fed-batch SSF experiments were performed and an ethanol concentration as high as 27.4 g/l (61% of the theoretical) was obtained with 11.25% (w/w) of water insoluble solids (WIS).
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| 8. |
- Tomas-Pejo, Elia, 1980, et al.
(author)
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Challenges of strain development and clone selection for bioethanol production from lignocellulose
- 2012
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In: 2nd Symposium on Biotechnology Applied on Lignocelluloses. Fukuoka, Japan. 14-17 October 2012.
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Conference paper (other academic/artistic)abstract
- Lignocellulosic biomass is one of the most promising raw materials for bioethanol production because it does not compete with food crops and is widely distributed around the world. When using lignocellulosic materials, toxic compounds derived from cellulose, hemicellulose and lignin degradation during pretreatment are also found in the media. It is well known that the most commonly used microorganism in ethanol production is Saccharomyces cerevisisae, however, wild type S. cerevisiae is not able to ferment xylose which could constitute up to 40% of the lignocellulose. Therefore, yeasts strains to be used for second-generation bioethanol production have to cope with challenging conditions that are inherent to the industrial process such as high concentration of inhibitory products, simultaneous use of different carbon sources and growth conditions that are not well controlled. Tolerance to these multiple stresses is likely to be a complex phenotype involving several cellular mechanisms and it could be difficult to perform efficient metabolic engineering. In this context, one of the most promising strategies for developing industrial strains is evolutionary engineering that includes evolution and recombination introducing genetic variability over many generations. Evolved S. cerevisiae strains engineered for xylose fermentation employed in this study have been subjected to targeted engineering for introducing a barcode in order to be able to verify their origin which also provokes random events in the population of cells. Screening after evolution or targeted engineering is challenging because of the high variability introduced during those events. Selection has to be performed carefully in order to select the best clones with best properties for a specific purpose. Furthermore, difficulties in applying novel technics such as next generation sequencing or multiomic analysis in industrial strains result from their genetic complexity such as polyploidy. In this work, mixed populations obtained by evolutionary engineering and different clones obtained after barcoding (Figure 1) are tested and evaluated in ethanol production processes from lignocellulosic hydrolysates. Differences between clones regarding xylose fermentation capability are elucidated.
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| 9. |
- Tomas-Pejo, Elia, 1980, et al.
(author)
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Changes in the metabolism of energy reserves and gene expression during different propagation strategies of tolerant xylose fermenting yeast and its effect on the bioethanol production process
- 2013
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In: 5th Conference of Physiology of Yeast and Filamentous Fungi (PYFF5). Montpellier (France) from 4th to 7th of June 2013.
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Conference paper (other academic/artistic)abstract
- Currently, large-scale production of bioethanol is mainly based on sugar or starch-rich feedstocks. These raw materials are also employed for animal feed and human use and seem not to be sufficient to the increasing demand for biofuels. In this context, lignocellulosic raw materials are good alternatives because they do not compete with food crops and are widely distributed. However, yeast strains to be used for lignocellulosic bioethanol production have to cope with challenging conditions that are inherent to the industrial process, such as high concentration of inhibitory products produced during pretreatment of raw material, simultaneous use of different carbon sources, and growth conditions that are not well controlled. Tolerance to these multiple stresses is likely to be a complex phenotype involving several cellular mechanisms therefore it could be difficult to perform efficient metabolic engineering. The production of inhibitor tolerant xylose-fermenting Saccharomyces cerevisiae cells during the propagation of yeast biomass will have a great effect on the following fermentation process. The traits of the produced yeast will determine the fermentation performance, ethanol yield and finally the global viability of the process. In the last years, the possibility of exposing the cells to lignocellulosic hydrolysates with inhibitors during the propagation step has given good results in terms of cell viability and high ethanol concentrations in the following fermentation. It is known that one of the general stress responses in yeast is the accumulation and mobilization of energy reserves (i.e. trehalose and glycogen). Trehalose is very important for maintaining cell viability under stress conditions because it protects cells from damage, however, when inhibitors are present in the media, the trehalose synthesis and degradation can be affected. In this work we study whether different propagation strategies have an impact on the metabolism of the energy carbohydrates trehalose and glycogen. The expression of several key genes (e.g. ALD6, ADH6, ZWF1, ERG2) is also investigated using qPCR technics in order to understand the yeast response during propagation under different conditions. Differences in trehalose and glycogen concentrations and changes in gene expression during propagation of yeast could give important insights for a successful lignocellulosic bioethanol production process
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| 10. |
- Tomas-Pejo, Elia, 1980, et al.
(author)
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Effect of inhibitors present n lignocellulosic hydrolysates on evolved xylose fermenting Saccharomyces cerevisiae strains
- 2012
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In: 34th Symposium for Biofuels and Chemicals. New Orleans, USA. 30th April – 3rd May 2012.
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Conference paper (other academic/artistic)abstract
- The development of inhibitor tolerant ethanologenic yeasts is one of the important challenges for a successful bioethanol production process from lignocellulose. Furthermore, an efficient microorganism for bioethanol production has to be able to ferment xylose together with glucose since xylose represents a large fraction in the lignocellulosic biomass. Weak acids and phenolic compounds are some of the prevalent inhibitors generated during pretreatment of lignocellulose and they will be present in the fermentation broth stressing the yeast affecting the fermentation performance. Although some studies on the effect of organic acids on fermenting microorganisms have been published, there is a lack of knowledge on the effect of phenolic compounds on yeast and more concretely about the effect on the xylose fermentation performance. In this study, the effect of acetic acid and vanillin on yeast growth on glucose and xylose will be elucidated using synthetic media mimicking lignocellulosic hydrolysates. It is known that one of general stress responses in yeast is the accumulation and mobilization of energy reserves (trehalose and glycogen). Trehalose protects cells from damage, increasing cell viability, however, when inhibitors are present in the media the trehalose synthesis and degradation could be affected. Furthermore differences in gene expression of key genes involved in acetic acid and vanillin tolerance and xylose fermentation will be studied. In this work we will also compare different evolved strains and evaluate mixed populations compared to single clones, in terms of trehalose and glycogen content and inhibitor tolerance.
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