| 41. |
- Tomas-Pejo, Elia, 1980, et al.
(författare)
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EVALUATION OF EVOLVED AND BARCODED XYLOSE FERMENTING STRAINS FOR BIOETHANOL PRODUCTION FROM LIGNOCELLULOSE
- 2012
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Ingår i: Science and Technology Day 2012, Chalmers University of Technology, 27th March 2012.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic raw materials for bioethanol production are today the basis for many ethanol production sites around the world. However, the utilization of engineered yeast strains for second generation ethanol production at large-scale can still be improved. Yeasts mainly use the sugars present in the lignocellulosic biomass but, toxic compounds derived from cellulose, hemicellulose and lignin degradation during pretreatment are also found in the media and inhibit yeast growth. Furthermore, wild type Saccharomyces cerevisiae is not able to ferment xylose which could constitute up to 40% of the lignocellulose material. Hence the recombinant yeast strains must be robust and ferment xylose to ethanol with high yields in the presence of inhibitors.In this study, different evolved xylose fermenting Saccharomyces cerevisiae strains have been compared in ethanol production processes from lignocellulosic hydrolysates. The differences between using single cell transformants and mixed populations will be evaluated in terms of ethanol production in large scale bioreactors.Furthermore, we have established a method to barcode the evolved yeast strains in order to be able to verify their origin. It is of outmost importance that after barcoding the original characteristics of a yeast strain are maintained. Those requirements can only be fulfilled by using a dominant selection principle. We have obtained a few hundred transformants that were shown to contain the new unique barcode DNA sequence via DNA isolation and DNA sequencing. The transformed strains must be able to grow on the lignocellulosic material and consume xylose at the same rate as before the transformation which also was tested in this study.
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| 42. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Bottlenecks in lignocellulosic ethanol production: xylose fermentation and cell propagation
- 2017
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Ingår i: European biomass conference 2017, 25th edition, June 12-15; Stockholm, Sweden..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A remaining challenge for the development of economically feasible 2nd generation bio-ethanol is low xylose consumption rate and inhibitor tolerance of the utilized Saccharomyces cerevisiae strains. Yeast starter cultures produced for ethanol production in simultaneous saccharification and co-fermentation (SSCF) processes have to meet high, seemingly conflicting requirements. A high biomass yield during propagation is required to produce the high cell concentrations required for the harsh conditions in the proceeding fermentation. Inhibitor tolerance is essential for producing a highly viable starter culture as well as favorable fermentation kinetics. Short-term adaptation of yeast cultures during propagation has been shown to have a positive effect on pentose conversion as well as inhibitor tolerance. Here we propose a model propagation strategy for evaluating physiology of yeast cultures during propagation. This model propagation strategy will be implemented in a study comparing physiology of yeast cultures with and without exposure to lignocellulosic inhibitors during propagation to assess what molecular mechanisms underlie the short-term adaptation response phenotype. For industry, a better control of yeast properties during propagation will result in an improved and consistent performance of yeast starter cultures for SSCF purposes.
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| 43. |
- Wang, Ruifei, 1985, et al.
(författare)
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Process optimization of multi-feed SSCF
- 2014
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Ingår i: 10th European Symposium on Biochemical Engineering Sciences and 6th International Forum on Industrial Bioprocesses.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Economical production of bio-ethanol from lignocellulosic materials requires an efficient and robust process which enables high-solid fermentation of pretreated lignocellulose to achieve high ethanol fermentation performance. In this work, we design and optimize a high-solid fed-batch simultaneous saccharification and co-fermentation (SSCF) process with a feed of substrate, enzyme and yeast cell for efficient production of ethanol from pretreated wheat straw in both lab and pilot scale. The yeast is prepared by pre-cultivation and adaptation in a semi-continuous cultivation in liquid hydrolysate medium in order to achieve high fermentation capacity. The feeding profiles in both pre-cultivation and SSCF steps are optimized based on a previously developed multi-feed SSCF model [1] in order to maintain high activities of both hydrolytic enzyme and yeast cell resulting in highest biomass yield during pre-cultivation and highest ethanol production efficiency during SSCF process. We also demonstrate scale up of fed-batch SSCF process in a 10 m3 pilot-scale bioreactor. The fed-batch SSCF with an optimized feed of substrate, cell and enzymes reaches high ethanol fermentation performance suggesting it to be a promising process for efficient bioconversion of lignocellulosic materials to ethanol.[1] Wang et al. Bioresour. Technol., 2014
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| 44. |
- Xiros, Charilaos, 1973, et al.
(författare)
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Toward a sustainable biorefinery using high-gravity technology
- 2017
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Ingår i: Biofuels, Bioproducts and Biorefining. - : Wiley. - 1932-1031 .- 1932-104X. ; 11:1, s. 15-27
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Tidskriftsartikel (refereegranskat)abstract
- The realization of process solutions for a sustainable bioeconomy depends on the efficient processing of biomass. High-gravity technology is one important alternative to realizing such solutions. The aims of this work were to expand the knowledge-base on lignocellulosic bioconversion processes at high solids content, to advance the current technologies for production of second-generation liquid biofuels, to evaluate the environmental impact of the proposed process by using life cycle assessment (LCA), and to develop and present a technically, economically, and environmentally sound process at high gravity, i.e., a process operating at the highest possible concentrations of raw material. The results and opinions presented here are the result of a Nordic collaborative study within the framework of the HG Biofuels project. Processes with bioethanol or biobutanol as target products were studied using wheat straw and spruce as interesting Nordic raw materials. During the project, the main scientific, economic, and technical challenges of such a process were identified. Integrated solutions to these challenges were proposed and tested experimentally, using wheat straw and spruce wood at a dry matter content of 30% (w/w) as model substrates. The LCA performed revealed the environmental impact of each of the process steps, highlighting the importance of the enzyme dose used for the hydrolysis of the plant biomass, as well as the importance of the fermentation yield.
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| 45. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
- 2019
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
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| 46. |
- Ferreira, Sofia, et al.
(författare)
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Metabolic engineering strategies for butanol production in Escherichia coli
- 2020
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:8, s. 2571-2587
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Forskningsöversikt (refereegranskat)abstract
- The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.
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| 47. |
- Tang, Hongting, et al.
(författare)
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Efficient yeast surface-display of novel complex synthetic cellulosomes
- 2018
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. Results: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. Conclusions: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology.
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| 48. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae
- 2019
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Ingår i: Bioresource technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 292, s. 121922-
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Tidskriftsartikel (refereegranskat)abstract
- The limited tolerance of Saccharomyces cerevisiae to the inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The aim of this study was to investigate the short-term adaptation effects in yeast strains with different genetic backgrounds. Fed-batch propagation cultures were supplemented with 40% wheat straw hydrolysate during the feed phase to adapt two different pentose-fermenting strains, CR01 and KE6-12. The harvested cells were used to inoculate fermentation media containing 80% or 90% wheat straw hydrolysate. The specific ethanol productivity during fermentation was up to 3.6 times higher for CR01 and 1.6 times higher for KE6-12 following adaptation. The influence of physiological parameters such as viability, storage carbohydrate content, and metabolite yields following short-term adaptation demonstrated that short-term adaptation was strain dependent.
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| 49. |
- Zhang, Yiming, 1986, et al.
(författare)
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Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production
- 2023
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Ingår i: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: With unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis. Results: Metabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g. Conclusions: In this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories.
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| 50. |
- Muzamal, Muhammad, 1986
(författare)
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Steam Explosion of Wood
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rising price of petroleum and environmental concerns regarding CO2 emissions has increased interest in alternative renewable resources. Biomass can be considered as an excellent alternative raw material. A biorefinery uses biomass and produces fuel, energy and value-added chemicals. The biorefinery is an emerging field and requires much development to compete with already established petroleum-based industries. One of the greatest challenges to the biorefinery is that the raw material; biomass, has a complex chemical composition and physical structure. A pretreatment process is necessary to induce physico-chemical changes in the biomass and transform it into easily digestible material. The main factor limiting enzymatic digestion of biomass is accessibility to chemical constituents. Steam Explosion (SE) pretreatment is a promising process that has many potential benefits compared to the alternatives, e.g. it has less hazardous process chemicals and conditions, less environmental impact, fewer energy requirements and lower capital investment.Existing literature on the SE process mainly concerns products obtained after the process. Knowledge about the physical processes that take place during the SE pretreatment is limited. This licentiate thesis is based on experimental and modelling studies performed with the aim of gaining knowledge of the basic mechanisms of this process. The SE is a three-step process that involves; (i) treatment of wood with pressurized steam for a specific period of time, (ii) explosion of wood chips through the rapid release of pressure, and (iii) impact of softened wood chips with other chips and vessel walls. In the experimental part these steps have been carefully isolated and the effects of these steps on internal and external structures of single spruce wood pieces have been studied. The effect of vapour expansion and the creation of stresses during the explosion step on a single cell of spruce wood (with four layers; P, S1, S2 and S3) at high temperature and moisture content have been modelled using the Finite Element Method.The study reveals that all the steps of the SE process contribute to structural changes in the wood material and increase pore size which increases the accessibility of chemical reagents and enzymes. A wood piece disintegrates into smaller pieces during the impact step. The vapour expansion inside cells during the explosion step causes each cell to expand in all directions and creates high stress and strain fields perpendicular to the cell direction. In general, cell wall damage is more likely to occur in cells with thin walls, i.e. earlywood; damaged P, S1 and S3 layers; low MFAs; irregular cross-sections and sharp corners.
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