| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
- Aulitto, Martina, 1991, et al.
(författare)
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Bacillus coagulans MA-13: a promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:210
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Tidskriftsartikel (refereegranskat)abstract
- Background: The transition from a petroleum-based economy towards more sustainable bioprocesses for the production of fuels and chemicals (circular economy) is necessary to alleviate the impact of anthropic activities on the global ecosystem. Lignocellulosic biomass-derived sugars are suitable alternative feedstocks that can be fermented or biochemically converted to value-added products. An example is lactic acid, which is an essential chemical for the production of polylactic acid, a biodegradable bioplastic. However, lactic acid is still mainly produced by Lactobacillus species via fermentation of starch-containing materials, the use of which competes with the supply of food and feed. Results: A thermophilic and cellulolytic lactic acid producer was isolated from bean processing waste and was identified as a new strain of Bacillus coagulans, named MA-13. This bacterium fermented lignocellulose-derived sugars to lactic acid at 55 degrees C and pH 5.5. Moreover, it was found to be a robust strain able to tolerate high concentrations of hydrolysate obtained from wheat straw pre-treated by acid-catalysed (pre-) hydrolysis and steam explosion, especially when cultivated in controlled bioreactor conditions. Indeed, unlike what was observed in microscale cultivations (complete growth inhibition at hydrolysate concentrations above 50%), B. coagulans MA-13 was able to grow and ferment in 95% hydrolysate-containing bioreactor fermentations. This bacterium was also found to secrete soluble thermophilic cellulases, which could be produced at low temperature (37 degrees C), still retaining an optimal operational activity at 50 degrees C. Conclusions: The above-mentioned features make B. coagulans MA-13 an appealing starting point for future development of a consolidated bioprocess for production of lactic acid from lignocellulosic biomass, after further strain development by genetic and evolutionary engineering. Its optimal temperature and pH of growth match with the operational conditions of fungal enzymes hitherto employed for the depolymerisation of lignocellulosic biomasses to fermentable sugars. Moreover, the robustness of B. coagulans MA-13 is a desirable trait, given the presence of microbial growth inhibitors in the pre-treated biomass hydrolysate.
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