| 1. |
- Franzén, Carl Johan, 1966, et al.
(författare)
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High gravity lignocellulose bioprocess development for ethanol and lactic acid production by multi-feed simultaneous saccharification and fermentation
- 2017
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Ingår i: Oral presentation at: Recent Advances in Fermentation Technology, RAFT12. Oct 29 - Nov 1, Bonita Springs, FL, USA..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Second generation bioethanol production is becoming established in production plants across the world. The process can also be viewed as a model biorefinery concept for biotechnological conversion of recalcitrant lignocellulosic raw materials to chemicals and other products. We have developed a Multi-Feed SSCF process: a systematic, model-driven design of fed-batch simultaneous saccharification and co-fermentation of steam-pretreated lignocellulosic materials in standard stirred tank reactors. The design includes feeding of solid substrate, enzymes, and active, robust cell factories adapted to the present substrate. The concept has been applied not only to ethanol production with S. cerevisiae, but also to lactic acid production from wheat straw by the thermophilic, cellulolytic strain Bacillus coagulans MA-13, isolated from bean processing waste. High Gravity operation, i.e. fermentation at high concentrations of water insoluble solids (WIS), pushes the process towards higher product concentrations and productivities, and improved energy and water economy. By using the multi-feed SSCF approach, the ethanol process was pushed towards final product concentrations above 60 g/L, at about 90% of the theoretical yields on consumed substrate, using 22% w/w accumulated WIS additions of acid- and steam explosion-pretreated wheat straw. Bacillus coagulans MA-13 was found to secrete cellulolytic enzymes and ferment lignocellulose-derived sugars to lactic acid; thus, it may be a potential platform for consolidated bioprocessing of lactic acid. We investigated its performance in multi-feed SSF and found that pre-adaptation of cells to the liquid fraction of the steam-pretreated lignocellulosic material improves lactate productivity and reduces the SSF time from 33 to 12 hours.
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| 2. |
- Wang, Ruifei, 1985, et al.
(författare)
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Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2 generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.
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| 3. |
- Wang, Ruifei, 1985, et al.
(författare)
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Which methods for viable yeast cell quantification can be used in lignocellulosic fermentation processes
- 2016
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Ingår i: European Symposium of Biochemical Engineering Science (ESBES) 2016, 11-14 September, Dublin, Ireland.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Cell concentration is a primary characteristic of fermentation processes. The total cell concentration in aparticle-free liquid medium can be easily assessed by cell counts, optical density or dry weight. The quantification of viable cells is not as straightforward. Viable cells can be defined as culturable, metabolically active and intact cells. Culturable cells can be assessed by colony-forming unit (CFU) assay. Metabolically active and intact cells have been quantified by e.g. qPCR, dielectric spectroscopy probes, and flow cytometry using various dyes. All these methods work well for applications in clear liquid media, but have not been validated in 2nd generation bioprocesses using lignocellulosic materials.In this study we evaluate the applicability of several methods for quantitative assessment of both total and viable cell concentrations in lignocellulosic media. In order to mimic typical conditions of lignocellulosic fermentations, we used a central composite design of experiments with known cell numbers, water insoluble solids content (WIS) and osmolality as factors. For the osmolality, we used sorbitol and NaCl to differentiate hyperosmotic conditions at different ion strengths and conductivities. The cell concentrations were determined using cell enumeration in a hemocytometer (with and without methylene blue staining), plating and enumeration of CFU, qPCR on extracted DNA and RNA, and on-line permittivity using a capacitance probe. These methods have the potential to be less affected by impurities and water insoluble solids in lignocellulosic media than e.g. dry weight and turbidity. The number and viability of cells used to create the test conditions of the experimental design were first determined from the seed culture on defined mineral medium. Considering all experimental points and some validation points within the design space, all the selected methods were used for measuring total and viable number of cells. With these data we built a quantitative model to fit all interaction effects and curvature, and to calibrate the qPCR and permittivity results to the number of total and culturable cell counts. Data of qPCR on DNA were fitted to total cell numbers, WIS level and osmolality. The permittivity measured by the dielectric probe was fitted to CFUs, WIS level, osmolality and measured conductivity. Parameter optimization resulted in statistically significant models with good predictive capacity. The results showed that cell counts and CFU were not sensitive to WIS and osmolarity levels. Therefore they can be used asreference methods in lignocellulose-based media. Furthermore, using the selected methodologies in simultaneous saccharification and fermentation (SSF) process of pre-treated wheat straw showed consistent results in total and viable cell numbers.Development of reliable and validated total and viable cell quantification methods will contribute to wellmonitored lignocellulosic fermentation processes both for research and industry in bio-based production.
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| 4. |
- 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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| 5. |
- Koppram, Rakesh, 1986, et al.
(författare)
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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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Ingår i: Advanced Biofuels in a Biorefinery Approach, February 28 - March 1, 2012, Copenhagen, Denmark.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)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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| 6. |
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| 7. |
- 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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| 8. |
- Wang, Ruifei, 1985
(författare)
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Bioprocess development for biochemical conversion of lignocellulose
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Due to its low environmental impact and high maturity of the fuel ethanol market, lignocellulosic ethanol is a promising option for reducing the carbon footprint in the transport sector. The characteristics of lignocellulosic feedstocks, such as varied sugar composition, low sugar density, low solubility, recalcitrance to enzymatic degradation, and inhibitors formed during thermochemical pretreatment, have so far limited the production process, and costs for conversion of lignocellulosic materials to ethanol are still high. In this thesis, I describe the development of a bioconversion process that pushes the limits of simultaneous saccharification and co-fermentation (SSCF) to achieve higher ethanol titre, yield and productivity on lignocellulosic feedstocks. I propose an integrated fed-batch strategy, Multi-Feed SSCF, including feeds of substrates, enzymes and adapted cells to tackle the technical challenges in operating a SSCF process at high substrate loadings. Using insights from experiments and a model-based feeding design, lignocellulose saccharification and fermentation at water insoluble solids (WIS) levels greater than 20% (w/w) was achieved. The multi-feed SSCF concept and model-aided substrate feeding design allowed rapid, reproducible, and scalable bioconversion of lignocellulose, as proven on several lignocellulosic feedstocks in both laboratory and demonstration scales. Ethanol production above 50 g/L in SSCF processes was found to be severely inhibited by the combined effects of ethanol, lignocellulose-derived inhibitors, and higher than standard cultivation temperature (35°C). Cell viability and fermentation improved significantly in a multi-feed SSCF process with a step change in temperature from 35 to 30°C, compared to operation at 35°C throughout. However, introducing the Erg3Tyr185 point mutation which has been reported to render thermotolerance in yeast, did not offer any significant improvement. Cell concentrations were determined by counting in a hemocytometer and colony forming unit assay. Their accuracy and reproducibility in lignocellulosic media, were verified by Design-of-Experiment-based calibration. Applic-ability of real time qPCR and dielectric spectroscopy as potential cell quantification methods was also investigated. With multi-feed of solid substrates, enzyme preparations, and adapted cells, the SSCF process produced > 60 g/L ethanol within 120 h, equivalent to 70% of the theoretical yield of the total sugar input, and 90% of the consumed sugar. The systematic optimisation reported in this work represents a robust and reproducible routine for developing lignocellulose-based processes. It could inspire continuous development of alternative strategies to current fossil-based chemical/fuel processes.
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| 9. |
- 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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| 10. |
- Wang, Ruifei, 1985, et al.
(författare)
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Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol
- 2014
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 172, s. 303-311
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Tidskriftsartikel (refereegranskat)abstract
- Fed-batch simultaneous saccharification and fermentation (SSF) is a feasible option for bioethanol production from lignocellulosic raw materials at high substrate concentrations. In this work, a segregated kinetic model was developed for simulation of fed-batch simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated birch, using substrate, enzymes and cell feeds. The model takes into account the dynamics of the cellulase–cellulose system and the cell population during SSCF, and the effects of pre-cultivation of yeast cells on fermentation performance. The model was cross-validated against experiments using different feed schemes. It could predict fermentation performance and explain observed differences between measured total yeast cells and dividing cells very well. The reproducibility of the experiments and the cell viability were significantly better in fed-batch than in batch SSCF at 15% and 20% total WIS contents. The model can be used for simulation of fed-batch SSCF and optimization of feed profiles.
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| 11. |
- Wang, Ruifei, 1985, et al.
(författare)
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Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production.
- 2016
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:1, s. 88-
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Tidskriftsartikel (refereegranskat)abstract
- High content of water-insoluble solids (WIS) is required for simultaneous saccharification and co-fermentation (SSCF) operations to reach the high ethanol concentrations that meet the techno-economic requirements of industrial-scale production. The fundamental challenges of such processes are related to the high viscosity and inhibitor contents of the medium. Poor mass transfer and inhibition of the yeast lead to decreased ethanol yield, titre and productivity. In the present work, high-solid SSCF of pre-treated wheat straw was carried out by multi-feed SSCF which is a fed-batch process with additions of substrate, enzymes and cells, integrated with yeast propagation and adaptation on the pre-treatment liquor. The combined feeding strategies were systematically compared and optimized using experiments and simulations.
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| 12. |
- Wang, Ruifei, 1985, et al.
(författare)
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Process optimization of multi-feed SSCF
- 2014
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Ingår i: 10th European Symposium on Biochemical Engineering Sciences and 6th International Forum on Industrial Bioprocesses.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Economical production of bio-ethanol from lignocellulosic materials requires an efficient and robust process which enables high-solid fermentation of pretreated lignocellulose to achieve high ethanol fermentation performance. In this work, we design and optimize a high-solid fed-batch simultaneous saccharification and co-fermentation (SSCF) process with a feed of substrate, enzyme and yeast cell for efficient production of ethanol from pretreated wheat straw in both lab and pilot scale. The yeast is prepared by pre-cultivation and adaptation in a semi-continuous cultivation in liquid hydrolysate medium in order to achieve high fermentation capacity. The feeding profiles in both pre-cultivation and SSCF steps are optimized based on a previously developed multi-feed SSCF model [1] in order to maintain high activities of both hydrolytic enzyme and yeast cell resulting in highest biomass yield during pre-cultivation and highest ethanol production efficiency during SSCF process. We also demonstrate scale up of fed-batch SSCF process in a 10 m3 pilot-scale bioreactor. The fed-batch SSCF with an optimized feed of substrate, cell and enzymes reaches high ethanol fermentation performance suggesting it to be a promising process for efficient bioconversion of lignocellulosic materials to ethanol.[1] Wang et al. Bioresour. Technol., 2014
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| 13. |
- 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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| 14. |
- 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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| 15. |
- 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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