| 1. |
- Westman, Johan, 1983, et al.
(författare)
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Effects of encapsulation of microorganisms on product formation during microbial fermentations
- 2012
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 96:6, s. 1441-1454
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Forskningsöversikt (refereegranskat)abstract
- This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.
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| 2. |
- Franzén, Carl Johan, 1966, et al.
(författare)
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Multifeed simultaneous saccharification and fermentation enables high gravity submerged fermentation of lignocellulose.
- 2015
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Ingår i: Recent Advances in Fermentation Technology (RAFT 11), Clearwater Beach, Florida, USA, November 8-11, 2015. Oral presentation..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Today, second generation bioethanol production is becoming established in production plants across the world. In addition to its intrinsic value, the process can be viewed as a model process for biotechnological conversion of recalcitrant lignocellulosic raw materials to a range of chemicals and other products. So called High Gravity operation, i.e. fermentation at high solids loadings, represents continued development of the process towards higher product concentrations and productivities, and improved energy and water economy. We have employed a systematic, model-driven approach to the design of feeding schemes of solid substrate, active yeast adapted to the actual substrate, and enzymes to fed-batch simultaneous saccharification and co-fermentation (Multifeed SSCF) of steam-pretreated lignocellulosic materials in stirred tank reactors. With this approach, mixing problems were avoided even at water insoluble solids contents of 22%, leading to ethanol concentrations of 56 g/L within 72 hours of SSCF on wheat straw. Similar fermentation performance was verified in 10 m3 demonstration scale using wheat straw, and in lab scale on birch and spruce, using several yeast strains. The yeast was propagated in the liquid fraction obtained by press filtration of the pretreated slurry. Yet, even with such preadaptation and repeated addition of fresh cells, the viability in the SSCF dropped due to interactions between lignocellulose-derived inhibitors, the produced ethanol and the temperature. Decreasing the temperature from 35 to 30°C when the ethanol concentration reached 40-50 g/L resulted in rapid initial hydrolysis, maintained fermentation capacity, lower residual glucose and xylose and ethanol concentrations above 60 g/L.
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| 3. |
- Novy, Vera, 1984, et al.
(författare)
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Saccharomyces cerevisiae strain comparison in glucose-xylose fermentations on defined substrates and in high-gravity SSCF: convergence in strain performance despite differences in genetic and evolutionary engineering history
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The most advanced strains of xylose-fermenting Saccharomyces cerevisiae still utilize xylose far less efficiently than glucose, despite the extensive metabolic and evolutionary engineering applied in their development. Systematic comparison of strains across literature is difficult due to widely varying conditions used for determining key physiological parameters. Here, we evaluate an industrial and a laboratory S. cerevisiae strain, which has the assimilation of xylose via xylitol in common, but differ fundamentally in the history of their adaptive laboratory evolution development, and in the cofactor specificity of the xylose reductase (XR) and xylitol dehydrogenase (XDH). Results: In xylose and mixed glucose-xylose shaken bottle fermentations, with and without addition of inhibitorrich wheat straw hydrolyzate, the specific xylose uptake rate of KE6-12. A (0.27-1.08 g g(CDW)(-1) h(-1)) was 1.1 to twofold higher than that of IBB10B05 (0.10-0.82 g g(CDW)(-1) h(-1)). KE6-12. A further showed a 1.1 to ninefold higher glycerol yield (0.08-0.15 g g(-1)) than IBB10B05 (0.01-0.09 g g(-1)). However, the ethanol yield (0.30-0.40 g g(-1)), xylitol yield (0.080.26 g g(-1)), and maximum specific growth rate (0.04-0.27 h(-1)) were in close range for both strains. The robustness of flocculating variants of KE6-12. A (KE-Flow) and IBB10B05 (B-Flow) was analyzed in high-gravity simultaneous saccharification and co-fermentation. As in shaken bottles, KE-Flow showed faster xylose conversion and higher glycerol formation than B-Flow, but final ethanol titres (61 g L-1) and cell viability were again comparable for both strains. Conclusions: Individual specific traits, elicited by the engineering strategy, can affect global physiological parameters of S. cerevisiae in different and, sometimes, unpredictable ways. The industrial strain background and prolonged evolution history in KE6-12. A improved the specific xylose uptake rate more substantially than the superior XR, XDH, and xylulokinase activities were able to elicit in IBB10B05. Use of an engineered XR/XDH pathway in IBB10B05 resulted in a lower glycerol rather than a lower xylitol yield. However, the strain development programs were remarkably convergent in terms of the achieved overall strain performance. This highlights the importance of comparative strain evaluation to advance the engineering strategies for next-generation S. cerevisiae strain development.
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| 4. |
- Wang, Ruifei, 1985, et al.
(författare)
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Kinetic modeling-based optimization of multi-feed simultaneous saccharification and co-fermentation of wheat straw for ethanol production
- 2015
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Ingår i: 37th Symposium on Biotechnology for Fuels and Chemicals, Oral presentation.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Fed-batch simultaneous saccharification and co-fermentation (SSCF) enables production of lignocellulosic ethanol with high content of water insoluble solids (WIS), and therefore high cellulose loadings (the major sugar source in lignocellulose). The viscosity of the SSCF broth and the mass/heat transfer efficiency, depend on the feeding frequency of solid substrates and the hydrolytic activities of the added cellulases. An ideal feeding scheme should avoid over-feeding which leads to mixing problems, while feeding as much substrates as possible to shorten the process time and increase the final ethanol titer. A previously developed kinetic model [1] was modified to predict the performance of cellulases on steam pre-treated wheat straw, and to decide when and how much WIS to feed in the next feeding event. With this approach, mixing problems could be completely avoided up to 22.2% WIS in lab scale stirred tank reactors, and ethanol concentrations reached 56 g/L within 72 hours of SSCF. The process was tested at demonstration scale in 10 m3 reactors, and a similar fermentation performance as that in lab scale was observed. Further feeding of solid substrate (>20% WIS) did not lead to increases in the ethanol concentration, while a substantial loss of yeast viability (colony forming unit) were observed in SSCF medium at high WIS contents. This was likely due to toxic compounds retained in the pre-treated lignocellulose. We are currently investigating different xylose fermenting Saccharomyces cerevisiae strains in the SSCF process to increase the ethanol titer further. [1] Wang et al. Bioresour. Technol., 2014
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| 5. |
- Westman, Johan, 1983, et al.
(författare)
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A FLO1 variant which yields a NewFlo phenotype
- 2015
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Ingår i: 32nd International Specialized Symposium on Yeasts, Perugia, Italy, September 13-17, 2015.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Flocculation is often utilised as means of separation of yeast cells from the product in alcoholic beverage production. Brewery type strains generally start to flocculate towards the end of the fermentation process, when sugars in the wort are depleted. In Saccharomyces cerevisiae, flocculation is governed by the FLO gene family, with FLO1 generally being the main contributor to strong, Flo1 phenotype, flocculation. S. cerevisiae CCUG 53310, isolated from a spent sulphite liquor plant, has high tolerance to fermentation inhibitors typically present in lignocellulose hydrolysates (Westman et al. 2012). Furthermore, CCUG 53310 flocculates constitutively with a Flo1 phenotype that is only marginally affected by the presence of high concentrations of mannose (see figure: circles).Using primers designed for FLO1, we isolated a flocculin gene from the genome of CCUG 53310. However, constitutive expression of the gene in the otherwise non-flocculating S. cerevisiae CEN.PK 113-7D, resulted in a strain with NewFlo phenotype flocculation, being inhibited by various sugars (see figure: squares, triangles, diamonds and stars). Nonetheless, the protein was phylogenetically closely related to Flo1p and by inverse PCR we could also show that the gene is a paralog of FLO1. Homology modelling of the N-terminal part of the protein structure revealed high structural similarities to the reported structure of the Flo5p N-terminal domain. Closer examination revealed differences in certain positions that have been reported to be important for carbohydrate binding by flocculins. Not previously reported, but of special interest due to its position in a loop flanking the carbohydrate binding site, was a glutamate residue that in the corresponding position in Flo1, 5 and 9p is a glycine. We hypothesise that this glutamate residue contributes to the observed NewFlo phenotype flocculation.
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| 6. |
- Westman, Johan, 1983, et al.
(författare)
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A novel chimaeric flocculation protein enhances flocculation in Saccharomyces cerevisiae
- 2018
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Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 6, s. 49-55
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Tidskriftsartikel (refereegranskat)abstract
- Yeast flocculation is the reversible formation of multicellular complexes mediated by lectin-like cell wall proteins binding to neighbouring cells. Strong flocculation can improve the inhibitor tolerance and fermentation performance of yeast cells in second generation bioethanol production. The strength of flocculation increases with the size of the flocculation protein and is strain dependent. However, the large number of internal repeats in the sequence of FLO1 from Saccharomyces cerevisiae S288c makes it difficult to recombinantly express the gene to its full length. In the search for novel flocculation genes resulting in strong flocculation, we discovered a DNA sequence, FLONF, that gives NewFlo phenotype flocculation in S. cerevisiae CEN.PK 113-7D. The nucleotide sequence of the internal repeats of FLONF differed from those of FLO1. We hypothesized that a chimaeric flocculation gene made up of a FLO1 variant derived from S. cerevisiae S288c and additional repeats from FLONF from S. cerevisiae CCUG 53310 would be more stable and easier to amplify by PCR. The constructed gene, FLOw, had 22 internal repeats compared to 18 in FLO1. Expression of FLOw in otherwise non-flocculating strains led to strong flocculation. Despite the length of the gene, the cassette containing FLOw could be easily amplified and transformed into yeast strains of different genetic background, leading to strong flocculation in all cases tested. The developed gene can be used as a self-immobilization technique or to obtain rapidly sedimenting cells for application in e.g. sequential batches without need for centrifugation.
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| 7. |
- Westman, Johan, 1983, et al.
(författare)
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Current progress in high cell density yeast bioprocesses for bioethanol production
- 2015
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:8, s. 1185-1195
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Forskningsöversikt (refereegranskat)abstract
- High capital costs and low reaction rates are major challenges for establishment of fermentation-based production systems in the bioeconomy. Using high cell density cultures is an efficient way to increase the volumetric productivity of fermentation processes, thereby enabling faster and more robust processes and use of smaller reactors. In this review, we summarize recent progress in the application of high cell density yeast bioprocesses for first and second generation bioethanol production. High biomass concentrations obtained by retention of yeast cells in the reactor enables easier cell reuse, simplified product recovery and higher dilution rates in continuous processes. High local cell density cultures, in the form of encapsulated or strongly flocculating yeast, furthermore obtain increased tolerance to convertible fermentation inhibitors and utilize glucose and other sugars simultaneously, thereby overcoming two additional hurdles for second generation bioethanol production. These effects are caused by local concentration gradients due to diffusion limitations and conversion of inhibitors and sugars by the cells, which lead to low local concentrations of inhibitors and glucose. Quorum sensing may also contribute to the increased stress tolerance. Recent developments indicate that high cell density methodology, with emphasis on high local cell density, offers significant advantages for sustainable second generation bioethanol production.
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| 8. |
- Westman, Johan, 1983, et al.
(författare)
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Encapsulation-Induced Stress Helps Saccharomyces cerevisiae Resist Convertible Lignocellulose Derived Inhibitors
- 2012
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 13:9, s. 11881-11894
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Tidskriftsartikel (refereegranskat)abstract
- The ability of macroencapsulated Saccharomyces cerevisiae CBS8066 to withstand readily and not readily in situ convertible lignocellulose-derived inhibitors was investigated in anaerobic batch cultivations. It was shown that encapsulation increased the tolerance against readily convertible furan aldehyde inhibitors and to dilute acid spruce hydrolysate, but not to organic acid inhibitors that cannot be metabolized anaerobically. Gene expression analysis showed that the protective effect arising from the encapsulation is evident also on the transcriptome level, as the expression of the stress-related genes YAP1, ATR1 and FLR1 was induced upon encapsulation. The transcript levels were increased due to encapsulation already in the medium without added inhibitors, indicating that the cells sensed low stress level arising from the encapsulation itself. We present a model, where the stress response is induced by nutrient limitation, that this helps the cells to cope with the increased stress added by a toxic medium, and that superficial cells in the capsules degrade convertible inhibitors, alleviating the inhibition for the cells deeper in the capsule.
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| 9. |
- Westman, Johan, 1983, et al.
(författare)
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Factors affecting the viability of Saccharomyces cerevisiae in Simultaneous Saccharification and co-Fermentation of pretreated wheat straw to ethanol
- 2015
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Ingår i: 32nd International Specialized Symposium on Yeasts.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The recalcitrance of lignocellulosic materials makes economic production of second generation ethanol difficult and necessitates pretreatment prior to hydrolysis and fermentation. Dilution in these steps limits the final ethanol titre reached in the fermentation, even at high yields. A higher concentration of the raw material already in the hydrolysis step is thus required to obtain good process economy. However, this also increases the amount of toxic compounds in the fermentation.Through simultaneous saccharification and co-fermentation, SSCF, with feeding of pretreated solids, higher substrate concentrations can be reached (Wang et al 2014). Yeast cells can be adapted to the material if they are propagated in fed-batch cultivation on a medium containing the liquid fraction from the pretreatment. Yet, even with such preadaptation, the activity of the cells added to our SSCF process dropped over time. To overcome this issue, we added fresh cells to the SSCF at different time points. We observed that the viability and fermentation capacity of the cells still decreased during the process. Nutrient supplementation could not help in improving the dropping viability. However, by adding ethanol to shake flask SSCF experiments we could see that the ethanol produced in the process was likely a contributing factor to the low viability. Drop tests on agar plates containing ethanol and/or pretreatment liquor, incubated at both 30°C and 35°C, further indicated that the decreased viability was an effect of the combination of the temperature in the reactor, the inhibitors in the material, and the ethanol produced in the process.Decreasing the temperature in the reactor to 30°C when the ethanol concentration reached 40-50 g L-1 resulted in rapid initial hydrolysis and maintained fermentation capacity. The residual amount of unfermented glucose and xylose at the end of the process was reduced. With the optimized process, ethanol concentrations of more than 60 g L-1 were reached. REFERENCE: Wang R, Koppram R, Olsson L, Franzén CJ (2014) Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol. Bioresour Technol 172:303–311
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| 10. |
- Westman, Johan, 1983, et al.
(författare)
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Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production
- 2014
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 1098-5336 .- 0099-2240. ; 80:22, s. 6908-6918
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Tidskriftsartikel (refereegranskat)abstract
- Yeast has long been considered the microorganism of choice for second-generation bioethanol production due to its fermentative capacity and ethanol tolerance. However, tolerance toward inhibitors derived from lignocellulosic materials is still an issue. Flocculating yeast strains often perform relatively well in inhibitory media, but inhibitor tolerance has never been clearly linked to the actual flocculation ability per se. In this study, variants of the flocculation gene FLO1 were transformed into the genome of the nonflocculating laboratory yeast strain Saccharomyces cerevisiae CEN.PK 113-7D. Three mutants with distinct differences in flocculation properties were isolated and characterized. The degree of flocculation and hydrophobicity of the cells were correlated to the length of the gene variant. The effect of different strength of flocculation on the fermentation performance of the strains was studied in defined medium with or without fermentation inhibitors, as well as in media based on dilute acid spruce hydrolysate. Strong flocculation aided against the readily convertible inhibitor furfural but not against less convertible inhibitors such as carboxylic acids. During fermentation of dilute acid spruce hydrolysate, the most strongly flocculating mutant with dense cell flocs showed significantly faster sugar consumption. The modified strain with the weakest flocculation showed a hexose consumption profile similar to the untransformed strain. These findings may explain why flocculation has evolved as a stress response and can find application in fermentation-based biorefinery processes on lignocellulosic raw materials.
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| 11. |
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| 12. |
- Westman, Johan, 1983, et al.
(författare)
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Improved inhibitor tolerance and simultaneous utilisation of hexoses and pentoses during fermentation of inhibitory lignocellulose hydrolysates by yeast at high local cell density
- 2014
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Ingår i: Lignobiotech III, Concepción, Chile, 26-29 October 2014.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Issues still creating a barrier for successful commercialization of second generation bioethanol are the inhibitory compounds present in the lignocellulose derived media and the inability of Saccharomyces cerevisiae to efficiently utilise pentoses. Both of the issues have been addressed by construction of recombinant yeast strains, often in combination with evolutionary engineering. However, hexoses and pentoses are mainly fermented sequentially by these yeasts, prolonging the total fermentation time. In our research, we have shown that encapsulation of S. cerevisiae cells in semi-permeable alginate-chitosan liquid core gel capsules increased the tolerance to lignocellulose hydrolysates and specifically furan aldehydes. The potential formation of concentration gradients of these convertible inhibitors through the cell pellet inside the capsule has been given as explanation to the increased tolerance [1]. Gradients of carbohydrates in the capsules were further hypothesised to lead to an improvement in the simultaneous utilisation of hexose and pentose sugars by the cells. To verify this hypothesis we constructed and encapsulated the xylose fermenting S. cerevisiae strain CEN.PK XXX. We found that encapsulation of the strain not only increased the inhibitor tolerance of the yeast, but also promoted simultaneous utilisation of glucose and xylose. Furthermore, during the 96 hour fermentations of a medium with glucose and xylose, the encapsulated yeast consumed at least 50% more xylose compared to the suspended cells. This led to approximately 15% higher final ethanol titres in batch fermentations. As proof of concept, an inhibitory spruce hydrolysate was fermented by suspended and encapsulated cells. The suspended cells fermented the hexoses and pentoses mainly sequentially, after a long lag phase. The encapsulated yeast, on the other hand, did not display a lag phase, and consumed glucose, mannose, galactose and xylose simultaneously from the start of the batch. However, encapsulation of yeast cells in an alginate membrane would likely not be economically permissible in an industrial setting. We therefore investigated whether keeping the cells tight together would be sufficient, even without a membrane. To this end we constructed a set of flocculating yeast strains with different flocculation strengths by expression of different variants of a flocculation gene. We found that the strongest flocculating strain, forming large dense cell flocs in the batch reactor, increased the tolerance towards furfural and increased the fermentation rate in an inhibitory spruce hydrolysate, compared to the non-flocculating strain. Overall, yeast at high local cell density comes out as a promising option for production of second generation bioethanol.References.[1] J.O. Westman et al., Int. J. Mol. Sci., 13, 11881-11894, 2012.
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| 13. |
- Westman, Johan, 1983, et al.
(författare)
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Inhibitor tolerance and flocculation of a yeast strain suitable for second generation bioethanol production
- 2012
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Ingår i: Electronic Journal of Biotechnology. - : Elsevier BV. - 0717-3458. ; 15:3
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Tidskriftsartikel (refereegranskat)abstract
- Background: Robust second generation bioethanol processes require microorganisms able to ferment inhibitory lignocellullosic hydrolysates. In this study, the inhibitor tolerance and flocculation characteristics of Saccharomyces cerevisiae CCUG53310 were evaluated in comparison with S. cerevisiae CBS8066. Results: The flocculating strain CCUG53310 could rapidly ferment all hexoses in dilute acid spruce hydrolysate, while CBS8066 was strongly inhibited in this medium. In synthetic inhibitory media, CCUG53310 was more tolerant to carboxylic acids and furan aldehydes, but more sensitive than CBS8066 to phenolic compounds. Despite the higher tolerance, the increase in expression of the YAP1, ATR1 and FLR1 genes, known to confer resistance to lignocellulose-derived inhibitors, was generally smaller in CCUG53310 than in CBS8066 in inhibitory media. The flocculation of CCUG53310 was linked to the expression of FLO8, FLO10 and one or more of FLO1, FLO5 or FLO9. Flocculation depended on cell wall proteins and Ca2+ ions, but was almost unaffected by other compounds and pH values typical for lignocellulosic media. Conclusions: S. cerevisiae CCUG53310 can be characterised as being very robust, with great potential for industrial fermentation of lignocellulosic hydrolysates relatively low in phenolic inhibitors.
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| 14. |
- Westman, Johan, 1983, et al.
(författare)
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Phenotypical and Physiological Characterization of a Flocculating Yeast Strain
- 2010
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Ingår i: Physiology of Yeast and Filamentous Fungi (PYFF4), Rotterdam, Holland, June 2010 and 2010 International Workshop on Wood Biorefinery and Tree Biotechnology, Örnsköldsvik, Sweden, June 2010.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol from lignocellulosic materials is one of the desired alternatives to meet the increased demand of renewable fuels. However, there are challenges in several steps of lignocellulose processing, including pretreatment, hydrolysis and fermentation. Using flocculating strains in the fermentation process gives a number of advantages. For example, the cells can be accumulated in the bioreactors leading to high cell concentration and rapid fermentation. They are easily separated using sedimentation and can thus be recycled to the bioreactors. Some of these strains are also better than non-flocculating strains at tolerating the possible inhibitors in the cultivation media, such as furan aldehydes, organic acids and phenolic compounds. These inhibitors make it hard for the yeast to ferment the hydrolyzate and detoxification is often necessary. A flocculating yeast strain was isolated from a Swedish ethanol plant (Domsjö Fabriker AB) fermenting sulphite liquor, and registered at Culture Collection University of Gothenburg as CCUG 53310. It has been shown that this strain can successfully ferment lignocellulosic hydrolyzates, where freely suspended reference strains failed to assimilate any sugar.In order to understand and get the possibility to improve the fermentation, a phenotypic and physiological characterization of the yeast strain has been performed. The effect of different inhibitor classes present in hydrolyzate as well as of complete hydrolyzate, on the macromolecular composition of the yeast has been investigated. Different responses can be seen from the different inhibitor classes, providing evidence for differences in metabolism between yeast cells grown in the different media.The phenotypical studies have shown that the strain, that is constitutively flocculating, belongs to the Flo1 phenotype, meaning that its flocculation is inhibited only by mannose. It has also been shown that the flocculation is dependent on cell wall proteins, and Ca2+, suggesting calcium ion dependent proteins binding to carbohydrates in neighboring cells. The flocculating cells also exhibit a significantly higher hydrophobicity than the non-flocculating reference strain; this is a factor that might also contribute to the flocculation.
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| 15. |
- Westman, Johan, 1983, et al.
(författare)
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Proteomic analysis of the increased stress tolerance of Saccharomyces cerevisiae encapsulated in liquid core alginate-chitosan capsules
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 7:11, s. e49335-
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae CBS8066 encapsulated in semi-permeable alginate or alginate-chitosan liquid core capsules have been shown to have an enhanced tolerance towards complex dilute-acid lignocellulose hydrolysates and the lignocellulose-derived inhibitor furfural, as well as towards high temperatures. The underlying molecular reasons for these effects have however not been elucidated. In this study we have investigated the response of the encapsulation on the proteome level in the yeast cells, in comparison with cells grown freely in suspension under otherwise similar conditions. The proteomic analysis was performed on whole cell protein extracts using nLC-MS/MS with TMT® labelling and 2-D DIGE. 842 and 52 proteins were identified using each method, respectively. The abundances of 213 proteins were significantly different between encapsulated and suspended cells, with good correlation between the fold change ratios obtained by the two methods for proteins identified in both. Encapsulation of the yeast caused an up-regulation of glucose-repressed proteins and of both general and starvation-specific stress responses, such as the trehalose biosynthesis pathway, and down-regulation of proteins linked to growth and protein synthesis. The encapsulation leads to a lack of nutrients for cells close to the core of the capsule due to mass transfer limitations. The triggering of the stress response may be beneficial for the cells in certain conditions, for example leading to the increased tolerance towards high temperatures and certain inhibitors.
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| 16. |
- Westman, Johan, 1983, et al.
(författare)
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Scale-up of multi feed fed-batch simultaneous saccharification and co-fermentation of pretreated wheat straw to ethanol
- 2015
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Ingår i: 37th Symposium on Biotechnology for Fuels and Chemicals.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A major remaining issue with second-generation bioethanol production is the difficulty of reaching high enough titers to facilitate an overall economical process. Utilization of approximately 20% pretreated insoluble lignocellulosic material in the process is necessary to reach an often mentioned ethanol concentration of 4-5% (w/w). The viscosity at this solids concentration becomes higher than what is easily attainable in most reactor set-ups. We have designed a fed-batch simultaneous saccharification and co-fermentation (SSCF) process for ethanol production from pretreated wheat straw up to 21% water insoluble solids in a stirred tank reactor. In addition to feeding of solids at different time points, feeding of fresh cells at different time points was found to be beneficial for the process. The fed cells were adapted to the toxic environment by pre-cultivation in the liquid fraction from the pretreatment. Enzyme addition at different time points did however not improve the process, compared to addition of the same total amount in the beginning of the fed-batch. The effectiveness of the optimized process has been proven at demonstration scale in a 10 m3 SSCF reactor, reaching ethanol concentrations of 5% (w/w). A further increase was hindered by the toxicity of the medium, lowering the cells’ fermentation capacity. We have previously shown that strong flocculation can increase the ability of yeast to ferment toxic lignocellulose hydrolysates [1]. We therefore created strongly flocculating xylose fermenting Saccharomyces cerevisiae strains and are currently investigating these in the SSCF process.[1] Westman et al. Appl Environ Microbiol, 2014.
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| 17. |
- Westman, Johan, 1983, et al.
(författare)
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Sustaining fermentation in high-gravity ethanol production by feeding yeast to a temperature-profiled multifeed simultaneous saccharification and co-fermentation of wheat straw
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:213
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Tidskriftsartikel (refereegranskat)abstract
- Background: Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and co-fermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. Here, we investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw. Results: More than 50% cell viability was lost during the first 24 h of high-gravity SSCF. No beneficial effects of adding selected nutrients were observed in shake flask SSCF. Ethanol concentrations greater than 50 g L-1 led to significant loss of viability and prevented further fermentation in SSCF. The benefits of feeding preadapted yeast cells were marginal at later stages of SSCF. Yeast flocculation did not improve the viability but simplified cell harvest and improved the feasibility of the cell feeding strategy in demo scale. Cultivation at 30 °C instead of 35 °C increased cell survival significantly on solid media containing ethanol and inhibitors. Similarly, in multifeed SSCF, cells maintained the viability and fermentation capacity when the temperature was reduced from 35 to 30 °C during the process, but hydrolysis yields were compromised. By combining the yeast feeding and temperature change, an ethanol concentration of 65 g L-1, equivalent to 70% of the theoretical yield, was obtained in multifeed SSCF on pretreated wheat straw. In demo scale, the process with flocculating yeast and temperature profile resulted in 5% (w/w) ethanol, equivalent to 53% of the theoretical yield. Conclusions: Multifeed SSCF was further developed by means of a flocculating yeast and a temperature-reduction profile. Ethanol toxicity is intensified in the presence of lignocellulosic inhibitors at temperatures that are beneficial to hydrolysis in high-gravity SSCF. The counteracting effects of temperature on cell viability and hydrolysis call for more tolerant microorganisms, enzyme systems with lower temperature optimum, or full optimization of the multifeed strategy with temperature profile.
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| 18. |
- Westman, Johan, 1983, et al.
(författare)
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Together we are strong! Inhibitor tolerance conferred by good neighbors?
- 2010
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Ingår i: Industrial Systems Biology: Sustainable Production of Fuels and Chemicals, Gothenburg, Sweden, August 2010 and XVIII International Conference on Bioencapsulation, Porto, Portugal, October 2010.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol from lignocellulosic materials is one of the desired alternatives to meet the increased demand of renewable fuels. However, there are challenges in several steps of lignocellulose processing, including pretreatment, hydrolysis and fermentation. Using flocculating strains in the fermentation process gives a number of advantages. For example, the cells can be accumulated in the bioreactors leading to high cell concentration and rapid fermentation. They are easily separated using sedimentation and can thus be recycled to the bioreactors. Some of these strains are also better than non-flocculating strains at tolerating the possible inhibitors in the cultivation media. These inhibitors make it hard for the yeast to ferment the hydrolyzate and detoxification is often necessary.A flocculating yeast strain was isolated from a Swedish ethanol plant (Domsjö Fabriker AB) fermenting sulphite liquor, and registered at Culture Collection University of Gothenburg as CCUG 53310. It has been shown that this strain can successfully ferment lignocellulosic hydrolyzates, where the freely suspended reference strain, CBS 8066, failed to assimilate any sugar. However, upon encapsulation in Ca-alginate capsules, the strain CBS 8066 was able to successfully withstand the effect of the inhibitors and ferment lignocellulosic hydrolyzate.1There are similarities between yeast cells living in large flocs and yeast living inside a capsule, such as a high local cell concentration. It is hypothesized that this greatly enhanced local biomass concentration strongly contributes to this increased tolerance, since more cells will be able to survive. That high biomass leads to a greater number of living cells have also been known for a long time.2 Further it is thought that the cells on the outside of the floc, as well as in the outer layer inside the capsule, will convert most of the inhibitors and partly die, protecting the inner lying cells.The effect of different inhibitor classes, furan aldehydes, organic acids and phenolic compounds, present in the hydrolyzate on the flocculating strain as well as the freely suspended strain was also investigated, finding that the flocculating strain was indeed a lot better at withstanding the effect of furan aldehydes and carboxylic acids. Interestingly though, the CBS 8066 strain could tolerate the presence of phenolic compounds in the growth medium significantly better. This shows that higher biomass cannot help against all inhibitors, but there are also other underlying reasons, i.e. differences in the strains, yet to be investigated.
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| 19. |
- Pages-Diaz, J., et al.
(författare)
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Semi-continuous co-digestion of solid cattle slaughterhouse wastes with other waste streams: Interactions within the mixtures and methanogenic community structure
- 2015
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Ingår i: Chemical Engineering Journal. - : Elsevier BV. - 1385-8947 .- 1873-3212. ; 273, s. 28-36
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Tidskriftsartikel (refereegranskat)abstract
- The performance of the anaerobic co-digestion process is strongly related to the characteristics of the substrates utilized. In this work, the impact of mixture interactions, i.e., synergy and antagonism, previously observed in batch operation mode were evaluated under semi-continuous co-digestion of slaughterhouse waste (SB) and its different combinations with manure (M), various crops (VC), and municipal solid waste (MSW). The effects on the process performance and the microbial community structure were investigated. The digestion of SB failed at an OLR of 0.9 gVS L-1 d(-1). However, stable performance with higher loadings was observed for mixtures that displayed synergy obtained earlier in the batch mode (i.e., SB + M, SB + VC + MSW). Bacterial and Archaeal groups increased for the SB + M and SB + VC + MSW, compared with the digestion of SB alone and that for SB + VC. The combination that showed antagonistic effects (SB + VC) resulted in unstable operation and poor representation of methanogens. It was proved that synergetic or antagonistic effects observed in batch mode due to the different mixture compositions could be correlated to process performance, as well as the development of the microbial community structure during semi-continuous operation.
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| 20. |
- Planting-Bergloo, Sara, 1978-, et al.
(författare)
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Att utveckla elevers förmåga att formulera undersökningsbara frågor i naturvetenskap : Mangling av en didaktisk modell
- 2021
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Ingår i: LUMAT. - : LUMA Centre Finland. - 2323-7112. ; 9:1, s. 774-803
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Tidskriftsartikel (refereegranskat)abstract
- En viktig målsättning för naturvetenskaplig undervisning är att utveckla förmågan att formulera undersökningsbara frågor. Syftet med den här studien är att undersöka hur undervisning som utformats med hjälp av metoden Question Formulation Technique (QFT) kan stödja utveckling av elevers förmåga att formulera naturvetenskapligt undersökningsbara frågor. QFT är en modell för att utveckla elevers förmåga att formulera och värdera sina egna frågor i allmänhet. I studien prövas QFT i en svensk skolkontext och inom ramen för naturvetenskaplig undervisning. Studien genomfördes som en interventionsstudie i gymnasieskolan och inom ramen för kursen Gymnasiearbete. I kursen ska eleverna genomföra en egen naturvetenskaplig undersökning. QFT användes för att utforma undervisning som del av introduktionen till kursen. Data består av videoinspelningar av elevsamtal från undervisning som har analyserats utifrån ett pragmatiskt ramverk med organiserande syften och praktisk epistemologisk analys. Resultaten visar vilka närliggande syften som etableras i elevernas samtal om undersökningsbara frågor i undervisningen: (A) att producera så många frågor som möjligt, (B) att bedöma vilka frågor som är mest relevanta, (C) att kategorisera frågor, (D) att hitta och specificera ett undersökningsobjekt och (E) att planera för att genomföra en undersökning. Slutsatsen är att QFT kan fungera som stöd för lärares planering av undervisning om naturvetenskapligt undersökningsbara frågor under förutsättning att läraren aktivt stödjer eleverna i att uppmärksamma centrala kvaliteter avseende undersökningsbarhet och genom att binda samman närliggande syften med det övergripande syftet.
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