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- Albers, Eva, 1966, et al.
(author)
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Selective suppression of bacterial contaminants by process conditions during lignocellulose based yeast fermentations
- 2011
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 4
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Journal article (peer-reviewed)abstract
- BackgroundContamination of bacteria in large-scale yeast fermentations is a serious problem and a threat to the development of successful biofuel production plants. Huge research efforts have been spent in order to solve this problem, but additional ways must still be found to keep bacterial contaminants from thriving in these environments. The aim of this project was to develop process conditions that would inhibit bacterial growth while giving yeast a competitive advantage.ResultsLactic acid bacteria are usually considered to be the most common contaminants in industrial yeast fermentations. Our observations support this view but also suggest that acetic acid bacteria, although not so numerous, could be a much more problematic obstacle to overcome. Acetic acid bacteria showed a capacity to drastically reduce the viability of yeast. In addition, they consumed the previously formed ethanol. Lactic acid bacteria did not show this detrimental effect on yeast viability. It was possible to combat both types of bacteria by a combined addition of NaCl and ethanol to the wood hydrolysate medium used. As a result of NaCl + ethanol additions the amount of viable bacteria decreased and yeast viability was enhanced concomitantly with an increase in ethanol concentration. The successful result obtained via addition of NaCl and ethanol was also confirmed in a real industrial ethanol production plant with its natural inherent yeast/bacterial community.ConclusionsIt is possible to reduce the number of bacteria and offer a selective advantage to yeast by a combined addition of NaCl and ethanol when cultivated in lignocellulosic medium such as wood hydrolysate. However, for optimal results, the concentrations of NaCl + ethanol must be adjusted to suit the challenges offered by each hydrolysate.
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| 2. |
- Franzén, Carl Johan, 1966, et al.
(author)
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Multifeed simultaneous saccharification and fermentation enables high gravity submerged fermentation of lignocellulose.
- 2015
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In: Recent Advances in Fermentation Technology (RAFT 11), Clearwater Beach, Florida, USA, November 8-11, 2015. Oral presentation..
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Conference paper (other academic/artistic)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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| 4. |
- Koppram, Rakesh, 1986, et al.
(author)
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A novel process configuration of Simultaneous Saccharification and Fermentation for bioethanol production at high solid loadings
- 2012
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In: Advanced Biofuels in a Biorefinery Approach, February 28 - March 1, 2012, Copenhagen, Denmark.
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Conference paper (other academic/artistic)abstract
- Simultaneous saccharification and fermentation (SSF) is a process option for lignocellulosic bioethanol production that has proven to have several advantages compared to separate hydrolysis and fermentation. The economical viability and commercialization of cellulose-to-ethanol demands the process to work under high-solid loadings to result in high sugar yield and final ethanol titer in S. cerevisiae based SSF process. In a conventional batch SSF process practical limitations to high-solid loadings include, poor mixing and accessibility of enzymes to substrates and high inhibitors concentration that reduces the yeast viability and metabolism. In order to overcome these limitations, we propose a novel SSF process configuration involving feeding of substrate, enzyme and yeast. It is possible to overcome mixing issues associated with a batch SSF at high-solid loadings by a feed of substrate, enzyme and yeast. The feed of freshly cultivated yeast throughout the fermentation process ensures active metabolic state of yeast. In addition, the substrate feed ensures low inhibitors concentration at any given time point increasing the survival ability of yeast compared to a batch SSF. The enzyme feed ensures slow release of glucose providing an opportunity for xylose consuming yeast strain to co-consume xylose together with glucose. The aim of the current work is to understand how different combinations of feeding strategies influence the outcome of the SSF process. In the longer perspective, we aim at deducing an optimized SSF process that can handle very high-solid loadings with efficient hydrolysis and fermentation process at low enzyme and yeast loadings, respectively.
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| 7. |
- Novy, Vera, 1984, et al.
(author)
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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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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:1
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Journal article (peer-reviewed)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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| 9. |
- Wang, Ruifei, 1985, et al.
(author)
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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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In: 37th Symposium on Biotechnology for Fuels and Chemicals, Oral presentation.
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Conference paper (other academic/artistic)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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| 10. |
- Westman, Johan, 1983, et al.
(author)
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A FLO1 variant which yields a NewFlo phenotype
- 2015
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In: 32nd International Specialized Symposium on Yeasts, Perugia, Italy, September 13-17, 2015.
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Conference paper (other academic/artistic)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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