| 11. |
- 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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| 12. |
- 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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| 13. |
- 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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| 14. |
- 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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| 15. |
- 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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| 16. |
- 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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| 17. |
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| 18. |
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| 19. |
- 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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| 20. |
- 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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