| 1. |
- Franzén, Carl Johan, 1966, et al.
(författare)
-
Multifeed simultaneous saccharification and fermentation enables high gravity submerged fermentation of lignocellulose.
- 2015
-
Ingår i: Recent Advances in Fermentation Technology (RAFT 11), Clearwater Beach, Florida, USA, November 8-11, 2015. Oral presentation..
-
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.
|
|
| 2. |
- Franzén, D., et al.
(författare)
-
Beach-cast as biofertiliser in the Baltic Sea region-potential limitations due to cadmium-content
- 2019
-
Ingår i: Ocean and Coastal Management. - : Elsevier BV. - 0964-5691 .- 1873-524X. ; 169, s. 20-26
-
Tidskriftsartikel (refereegranskat)abstract
- Macroalgal mass blooms and accumulating beach-cast are increasing problems in many coastal areas. However, beach-cast is also a potentially valuable marine bioresource, e.g. as a biofertiliser in coastal agriculture. One limiting factor in use of beach-cast as a fertiliser is uncertainty regarding the cadmium (Cd) concentration depending on beach-cast composition and location. In this study, chemical analyses were performed on beach-cast from Burgsviken Bay off Gotland, in the Baltic Sea. The results revealed large variations in cadmium concentration depending on sampling location and beach-cast composition, with levels ranging between 0.13 and 2.2 mg Cd/kg dry matter (DM). Of 15 beach-cast samples analysed, one had a cadmium content above the Swedish statutory limit for sewage sludge biofertiliser (2 mg Cd/kg DM) and four had values above the limit suggested by the Swedish Environmental Protection Agency for 2030 (0.8 mg/kg DM). Species-specific analysis revealed that eelgrass (Zostera marina) contained significantly higher cadmium concentrations than filamentous red algae species (Ceramium and Polysiphonia spp.). Avoiding eelgrass-rich beach-cast by seasonal timing of harvesting and monitoring differences in cadmium concentrations between harvesting sites could thus facilitate use of beach-cast as biofertiliser. © 2018
|
|
| 3. |
- Gustavsson, Robert, 1987-
(författare)
-
Development of soft sensors for monitoring and control of bioprocesses
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the manufacture of bio-therapeutics the importance of a well-known process is key for a high product titer and low batch to batch variations. Soft sensors are based on the concept that online sensor signals can be used as inputs to mathematical models to derive new valuable process information. This information could then be used for better monitoring and control of the bioprocess.The aim of the present thesis has been to develop soft sensor solutions for upstream bioprocessing and demonstrate their usefulness in improving robustness and increase the batch-to-batch reproducibility in bioprocesses. The thesis reviews the potential and possibilities with soft sensors for use in production of bio-therapeutics to realize FDA´s process analytical technology (PAT) initiative. Modelling and hardware sensor alternatives which could be used in a soft sensor setup are described and critically analyzed. Different soft sensor approaches to control glucose feeding in fed-batch cultures of Escherichia coli are described. Measurements of metabolic fluxes and specific carbon dioxide production was used as control parameters to increase product yield and decrease the variability of produced recombinant proteins. Metabolic heat signals were used in uninduced cultures to estimate and control the specific growth rate at a desired level and thereby also estimate the biomass concentration online. The introduction of sequential filtering of the signal enabled this method to be used in a down-scaled system. The risk and high impact of contaminations in cell cultures are also described. An in situ microscope (ISM) was used as an online tool to estimate cell concentration and also to determine cell diameter size which enabled the detection of contaminant cells at an early stage.The work presented in this thesis supports the idea that soft sensors can be a useful tool in the strive towards robust and reliable bioprocesses, to ensure high product quality and increased economic profit.
|
|
| 4. |
- Novy, Vera, 1984, et al.
(författare)
-
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
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:1
-
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.
|
|
| 5. |
- Wang, Ruifei, 1985, et al.
(författare)
-
Kinetic modeling-based optimization of multi-feed simultaneous saccharification and co-fermentation of wheat straw for ethanol production
- 2015
-
Ingår i: 37th Symposium on Biotechnology for Fuels and Chemicals, Oral presentation.
-
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
|
|
| 6. |
- Westman, Johan, 1983, et al.
(författare)
-
A FLO1 variant which yields a NewFlo phenotype
- 2015
-
Ingår i: 32nd International Specialized Symposium on Yeasts, Perugia, Italy, September 13-17, 2015.
-
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.
|
|
| 7. |
- Westman, Johan, 1983, et al.
(författare)
-
A novel chimaeric flocculation protein enhances flocculation in Saccharomyces cerevisiae
- 2018
-
Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 6, s. 49-55
-
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.
|
|
| 8. |
- Westman, Johan, 1983, et al.
(författare)
-
Current progress in high cell density yeast bioprocesses for bioethanol production
- 2015
-
Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:8, s. 1185-1195
-
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.
|
|
| 9. |
- Westman, Johan, 1983, et al.
(författare)
-
Effects of encapsulation of microorganisms on product formation during microbial fermentations
- 2012
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 96:6, s. 1441-1454
-
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.
|
|
| 10. |
- Westman, Johan, 1983, et al.
(författare)
-
Encapsulation-Induced Stress Helps Saccharomyces cerevisiae Resist Convertible Lignocellulose Derived Inhibitors
- 2012
-
Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 13:9, s. 11881-11894
-
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.
|
|
