| 1. |
- Munthe, Christian, 1962
(författare)
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Precaution and Ethics: Handling risks, uncertainties and knowledge gaps in the regulation of new biotechnologies
- 2017
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Bok (övrigt vetenskapligt/konstnärligt)abstract
- This volume outlines and analyses ethical issues actualized by applying a precautionary approach to the regulation of new biotechnologies. It presents a novel way of categorizing and comparing biotechnologies from a precautionary standpoint. Based on this, it addresses underlying philosophical problems regarding the ethical assessment of decision-making under uncertainty and ignorance, and discusses how risks and possible benefits of such technologies should be balanced from an ethical standpoint. It argues on conceptual and ethical grounds for a technology neutral regulation as well as for a regulation that not only checks new technologies but also requires old, inferior ones to be phased out. It demonstrates how difficult ethical issues regarding the extent and ambition of precautionary policies need to be handled by such a regulation, and presents an overarching framework for doing so.
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| 2. |
- Ask, Magnus, 1983
(författare)
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Towards More Robust Saccharomyces cerevisiae Strains for Lignocellulosic Bioethanol Production: Lessons from process concepts and physiological investigations
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dwindling oil reserves and the negative impacts of fossil fuels on the environment call for more sustainable energy sources. First-generation bioethanol produced from sugar cane and corn has met some of these needs, but it competes with the food supply for raw materials. Lignocellulosic biomass is an abundant non-edible raw material that can be converted to ethanol using the yeast Saccharomyces cerevisiae. However, due to the inherent recalcitrance to degradation of lignocellulosic raw materials, harsh pretreatment methods must be used to liberate fermentable sugars, resulting in the release of compounds such as acetic acid, furan aldehydes and phenolics, that inhibit yeast metabolism. This thesis research aimed to identify bottlenecks in terms of inhibitory compounds related to ethanol production from two lignocellulosic raw materials, Arundo donax and spruce, and furthermore to harness the physiological responses to these inhibitors to engineer more robust yeast strains. A comparative study of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) revealed that acetic acid limits xylose utilization in pretreated Arundo donax, whereas the furan aldehydes furfural and 5-hydroxymethyl-2-furaldehyde (HMF) were hypothesized to be key inhibitors in pretreated spruce. The impacts of furfural and HMF on the redox and energy metabolism of S. cerevisiae were studied in detail in chemostat and batch cultivations. After adding the inhibitors to the feed medium of chemostat cultivations, the intracellular levels of NADH, NADPH, and ATP were found to decrease by 40, 75, and 19%, respectively, suggesting that furan aldehydes drain the cells of reducing power. A strong effect on redox metabolism was also observed after pulsing furfural and HMF in the xylose consumption phase in batch cultures. The drainage of reducing power was also observed in a genome-wide study of transcription that found that genes related to NADPH-requiring processes, such as nitrogen and sulphur assimilation, were significantly induced. The redox metabolism was engineered by overproducing the protective metabolite and antioxidant glutathione. Strains with an increased intracellular level of reduced glutathione were found to sustain ethanol production for longer duration in SSF of pretreated spruce, yielding 70% more ethanol than did the wild type strain.
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| 3. |
- 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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| 4. |
- Mayers, Joshua, 1988, et al.
(författare)
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Integrating Microalgal Production with Industrial Outputs - Reducing Process Inputs and Quantifying the Benefits
- 2016
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Ingår i: Industrial Biotechnology. - : Mary Ann Liebert Inc. - 1550-9087 .- 1931-8421. ; 12:4, s. 219-234
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Tidskriftsartikel (refereegranskat)abstract
- The cultivation and processing of microalgal biomass is resource- and energy-intensive, negatively affecting the sustainability and profitability of producing bulk commodities, limiting this platform to the manufacture of relatively small quantities of high-value compounds. A biorefinery approach where all fractions of the biomass are valorized might improve the case for producing lower-value products. However, these systems are still likely to operate very close to thresholds of profitability and energy balance, with wide-ranging environmental and societal impacts. It thus remains critically important to reduce the use of costly and impactful inputs and energy-intensive processes involved in these scenarios. Integration with industrial infrastructure can provide a number of residual streams that can be readily used during microalgal cultivation and downstream processing. This review critically considers some of the main inputs required for microalgal biorefineries - such as nutrients, water, carbon dioxide, and heat - and appraises the benefits and possibilities for industrial integration on a more quantitative basis. Recent literature and demonstration studies will also be considered to best illustrate these benefits to both producers and industrial operators. Additionally, this review will highlight some inconsistencies in the data used in assessments of microalgal production scenarios, allowing more accurate evaluation of potential future biorefineries.
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| 5. |
- 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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| 6. |
- Ylitervo, Päivi
(författare)
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Concepts for improving ethanol productivity from lignocellulosic materials : encapsulated yeast and membrane bioreactors
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic biomass is a potential feedstock for production of sugars, which can be fermented into ethanol. The work presented in this thesis proposes some solutions to overcome problems with suboptimal process performance due to elevated cultivation temperatures and inhibitors present during ethanol production from lignocellulosic materials. In particular, continuous processes operated at high dilution rates with high sugar utilisation are attractive for ethanol fermentation, as this can result in higher ethanol productivity. Both encapsulation and membrane bioreactors were studied and developed to achieve rapid fermentation at high yeast cell density. My studies showed that encapsulated yeast is more thermotolerant than suspended yeast. The encapsulated yeast could successfully ferment all glucose during five consecutive batches, 12 h each at 42 °C. In contrast, freely suspended yeast was inactivated already in the second or third batch. One problem with encapsulation is, however, the mechanical robustness of the capsule membrane. If the capsules are exposed to e.g. high shear forces, the capsule membrane may break. Therefore, a method was developed to produce more robust capsules by treating alginate-chitosan-alginate (ACA) capsules with 3-aminopropyltriethoxysilane (APTES) to get polysiloxane-ACA capsules. Of the ACA-capsules treated with 1.5% APTES, only 0–2% of the capsules broke, while 25% of the untreated capsules ruptured within 6 h in a shear test. In this thesis membrane bioreactors (MBR), using either a cross-flow or a submerged membrane, could successfully be applied to retain the yeast inside the reactor. The cross-flow membrane was operated at a dilution rate of 0.5 h-1 whereas the submerged membrane was tested at several dilution rates, from 0.2 up to 0.8 h-1. Cultivations at high cell densities demonstrated an efficient in situ detoxification of very high furfural levels of up to 17 g L-1 in the feed medium when using a MBR. The maximum yeast density achieved in the MBR was more than 200 g L-1. Additionally, ethanol fermentation of nondetoxified spruce hydrolysate was possible at a high feeding rate of 0.8 h-1 by applying a submerged membrane bioreactor, resulting in ethanol productivities of up to 8 g L-1 h-1. In conclusion, this study suggests methods for rapid continuous ethanol production even at stressful elevated cultivation temperatures or inhibitory conditions by using encapsulation or membrane bioreactors and high cell density cultivations.
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| 7. |
- Ylitervo, Päivi, et al.
(författare)
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Continuous Ethanol Production with a Membrane Bioreactor at High Acetic Acid Concentrations
- 2014
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Ingår i: Membranes. - : MDPI. - 2077-0375. ; 4:3, s. 372-387
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Tidskriftsartikel (refereegranskat)abstract
- The release of inhibitory concentrations of acetic acid from lignocellulosic raw materials during hydrolysis is one of the main concerns for 2nd generation ethanol production. The undissociated form of acetic acid can enter the cell by diffusion through the plasma membrane and trigger several toxic effects, such as uncoupling and lowered intracellular pH. The effect of acetic acid on the ethanol production was investigated in continuous cultivations by adding medium containing 2.5 to 20.0 g•L−1 acetic acid at pH 5.0, at a dilution rate of 0.5 h−1. The cultivations were performed at both high (~25 g•L−1) and very high (100–200 g•L−1) yeast concentration by retaining the yeast cells inside the reactor by a cross-flow membrane in a membrane bioreactor. The yeast was able to steadily produce ethanol from 25 g•L−1 sucrose, at volumetric rates of 5–6 g•L−1•h−1 at acetic acid concentrations up to 15.0 g•L−1. However, the yeast continued to produce ethanol also at a concentration of 20 g•L−1 acetic acid but at a declining rate. The study thereby demonstrates the great potential of the membrane bioreactor for improving the robustness of the ethanol production based on lignocellulosic raw materials.
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| 8. |
- Anasontzis, George E, 1980
(författare)
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Biomass modifying enzymes: From discovery to application
- 2012
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Ingår i: Oral presentation at the Chalmers Life Science AoA conference.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- It has now been realized that the road towards the bio-based economy is a one-way street, leaving gradually the oil-based technology and driving slowly towards a more sustainable society. The current non-biodegradable hydrocarbon fuels and plastics will be replaced by new products which will derive from natural and renewable resources. The synthesis of such biofuels and biochemicals is still challenged by the difficulties to cost efficiently degrade lignocellulosic material to fermentable sugars or to isolate the intact polymers. Biomass degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in their subsequent modification, prior to further product development.Our group interests focus on all levels of applied enzyme research of biomass acting enzymes: Discovery, assay development, production and application. Relevant examples will be provided: What is our strategy for discovering novel microorganisms and enzymes from the tropical forests and grasslands of Vietnam? How do we design novel real-world assays for enzyme activity determination? Which are the bottlenecks in the enzymatic cellulose hydrolysis? How enzymes can be used to produce high added value compounds from biomass?
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| 9. |
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| 10. |
- McKee, Lauren S., et al.
(författare)
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A GH115 alpha-glucuronidase from Schizophyllum commune contributes to the synergistic enzymatic deconstruction of softwood glucuronoarabinoxylan
- 2016
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Ingår i: Biotechnology for Biofuels. - : BioMed Central. - 1754-6834. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: Lignocellulosic biomass from softwood represents a valuable resource for the production of biofuels and bio-based materials as alternatives to traditional pulp and paper products. Hemicelluloses constitute an extremely heterogeneous fraction of the plant cell wall, as their molecular structures involve multiple monosaccharide components, glycosidic linkages, and decoration patterns. The complete enzymatic hydrolysis of wood hemicelluloses into monosaccharides is therefore a complex biochemical process that requires the activities of multiple degradative enzymes with complementary activities tailored to the structural features of a particular substrate. Glucuronoarabinoxylan (GAX) is a major hemicellulose component in softwood, and its structural complexity requires more enzyme specificities to achieve complete hydrolysis compared to glucuronoxylans from hardwood and arabinoxylans from grasses. Results: We report the characterisation of a recombinant alpha-glucuronidase (Agu115) from Schizophyllum commune capable of removing (4-O-methyl)-glucuronic acid ((Me) GlcA) residues from polymeric and oligomeric xylan. The enzyme is required for the complete deconstruction of spruce glucuronoarabinoxylan (GAX) and acts synergistically with other xylan-degrading enzymes, specifically a xylanase (Xyn10C), an alpha-l-arabinofuranosidase (AbfA), and a beta-xylosidase (XynB). Each enzyme in this mixture showed varying degrees of potentiation by the other activities, likely due to increased physical access to their respective target monosaccharides. The exo-acting Agu115 and AbfA were unable to remove all of their respective target side chain decorations from GAX, but their specific activity was significantly boosted by the addition of the endo-Xyn10C xylanase. We demonstrate that the proposed enzymatic cocktail (Agu115 with AbfA, Xyn10C and XynB) achieved almost complete conversion of GAX to arabinofuranose (Araf), xylopyranose (Xylp), and MeGlcA monosaccharides. Addition of Agu115 to the enzymatic cocktail contributes specifically to 25 % of the conversion. However, traces of residual oligosaccharides resistant to this combination of enzymes were still present after deconstruction, due to steric hindrances to enzyme access to the substrate. Conclusions: Our GH115 alpha-glucuronidase is capable of finely tailoring the molecular structure of softwood GAX, and contributes to the almost complete saccharification of GAX in synergy with other exo- and endo-xylan-acting enzymes. This has great relevance for the cost-efficient production of biofuels from softwood lignocellulose.
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| 11. |
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| 12. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies
- 2013
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Ingår i: Cell Factories and Biosustainability (Hilleroed, Denmark, May 5-8 2013).
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The realization of an oil independent economy relies on the development of competitive processes for the production of fuels and chemicals from renewable resources. The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Harsh conditions during the bioconversion of lignocellulose-derived sugars to the desired products drastically hamper cell viability and therefore productivity. Microbial inhibition limits bioprocesses to an extent such that it can be said that understanding and harnessing microbial robustness is a prerequisite for the feasibility of new bioprocess and the production of renewable fuels and chemicals.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.
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| 13. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Robust S. cerevisiae strain for next generation bio-processes: concepts and case-studies
- 2013
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Ingår i: 35th Symposium on Biotechnology for Fuels and Chemicals (Portland, OR. April 29-May 2, 2013).
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The realization of an oil independent economy relies on the development of competitive processes for the production of fuels and chemicals from renewable resources. The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Sugars are released from cellulose and hemicellulose by pretreatment and hydrolysis steps. Harsh conditions result in the formation of a number of compounds, originating from sugars and lignin breakdown and acting as microorganism inhibitors. Weak organic acids, furaldehydes and phenolic compounds are sources of stress for the fermenting microorganism, as they influence cellular metabolism in a number of ways, including direct damage on cellular functions or by perturbations of the cellular energy and redox metabolism. In addition, the product of interest can act as a potent inhibitor. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose-based bioprocesses.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.
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| 14. |
- Westman, Johan
(författare)
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Ethanol production from lignocellulose using high local cell density yeast cultures. Investigations of flocculating and encapsulated Saccharomyces cerevisiae
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Efforts are made to change from 1st to 2nd generation bioethanol production, using lignocellulosics as raw materials rather than using raw materials that alternatively can be used as food sources. An issue with lignocellulosics is that a harsh pretreatment step is required in the process of converting them into fermentable sugars. In this step, inhibitory compounds such as furan aldehydes and carboxylic acids are formed, leading to suboptimal fermentation rates. Another issue is that lignocellulosics may contain a large portion of pentoses, which cannot be fermented simultaneously with glucose by Saccharomyces cerevisiae. In this thesis, high local cell density has been investigated as a means of overcoming these two issues. Encapsulation of yeast in semi-permeable alginate-chitosan capsules increased the tolerance towards furan aldehydes, but not towards carboxylic acids. The selective tolerance can be explained by differences in the concentration of compounds radially through the cell pellet inside the capsule. For inhibitors, gradients will only be formed if the compounds are readily convertible, like the furan aldehydes. Conversion of inhibitors by cells close to the membrane leads to decreased concentrations radially through the cell pellet. Thus, cells closer to the core experience subinhibitory levels of inhibitors and can ferment sugars. Carbohydrate gradients also give rise to nutrient limitations, which in turn trigger a stress response in the yeast, as was observed on mRNA and protein level. The stress response is believed to increase the robustness of the yeast and lead to improved tolerance towards additional stress. Glucose and xylose co-consumption by a recombinant strain, CEN.PK XXX, was also improved by encapsulation. Differences in affinity of the sugar transporters normally result in that glucose is taken up preferentially to xylose. However, when encapsulated, cells in different parts of the capsule experienced high and low glucose concentrations simultaneously. Xylose and glucose could thus be taken up concurrently. This improved the co-utilisation of the sugars by the system and led to 50% higher xylose consumption and 15% higher final ethanol titres. A protective effect by the capsule membrane itself could not be shown. Hence, the interest in flocculation was triggered, as a more convenient way to keep the cells together. To investigate whether flocculation increases the tolerance, like encapsulation, recombinant flocculating yeast strains were constructed and compared with the non-flocculating parental strain. Experiments showed that strong flocculation did not increase the tolerance towards carboxylic acids. However, the tolerance towards a spruce hydrolysate and especially against furfural was indeed increased. The results of this thesis show that high local cell density yeast cultures have the potential to aid against two of the major problems for 2nd generation bioethanol production: inhibitors and simultaneous hexose and pentose utilisation.
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| 15. |
- Mukesh Kumar, Awasthi, et al.
(författare)
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Bacterial dynamics during the anaerobic digestion of toxic citrus fruit waste and semi-continues volatile fatty acids production in membrane bioreactors
- 2022
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Ingår i: Fuel. - : Elsevier. - 0016-2361 .- 1873-7153. ; 319
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Tidskriftsartikel (refereegranskat)abstract
- Citrus wastes (CW) are normally toxic to anaerobic digestion (AD) because of flavors such as D-limonene. In this study, bacterial community was evaluated during volatile fatty acids (VFAs) production from CW inoculated by sludge in a membrane bioreactor (MBR) using semi-continuous AD with different organic loading rates (OLR). Four treatments including untreated CW filled with 4 and 8 g center dot VS center dot L(-1)d(-1) OLR (UOLR4 and UOLR8), pretreated Dlimonene-free CW filled with 4 and 8 g center dot VS center dot L(-1)d(-1) OLR (POLR4 and POLR8). The initial inoculum and the CW mixture (DAY0) was used as control for comparison. There was an obviously higher bacterial diversity in raw material (66848 sequences in DAY0), while decreased after AD and higher in POLR4 and POLR8 (65239 and 63916) than UOLR4 and UOLR8 (49158 and 51936). The key bacterial associated with VFAs production mainly affiliated to Firmicutes (37.35-84.73%), Bacteroidetes (0.48-36.87%), and Actinobacteria (0.35-29.38%), and the key genus composed of Lactobacillus, Prevotella, Bacillus, Bacteroides and Olsenella which contributed in VFA generation by degradable complex organic compounds. Noticeably, methanogen completely suppressed after MBR-AD and UOLR4 has greater acid utilizing bacteria (70.09%).
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| 16. |
- Adeboye, Peter, 1982, et al.
(författare)
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Conversion of lignin-derived phenolic compounds by Saccharomyces cerevisiae
- 2014
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Ingår i: 36th Symposium on Biotechnology for Fuels and Chemicals, April 2-May 1st, Clearwater Beach, Florids, USA.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignin breakdown during biomass pretreatment releases a wide array of phenolic compounds in lignocellulose hydrolysates. Phenolic compounds, together with organic acids and furaldehydes are known to be inhibitors of microbial fermentation, thus limiting the efficient bioconversion of lignocellulose biomass. The goal of our study is to improve S. cerevisiae tolerance to phenolic compounds from lignocellulose hydrolysates and investigate its conversion capacities. In particular, we aimed i) to establish a correlation between the phenolic compounds structure and the effect on yeast growth, and ii) to investigate the conversion/detoxification products of selected representative compounds in order to provide strain engineering strategies for enhanced phenolics conversion.First, the effect on S. cerevisiae growth of 13 different phenolic compounds commonly found in lignocellulose hydrolysates was characterized. The compounds could be grouped in three clusters, according to their effect on lag phase duration, specific growth rate and cell density. Next, coniferyl aldehyde, p-coumaric acid and ferulic acid were chosen as representative compounds and their conversion product by S. cerevisiae in aerobic culture in bioreactor were identified and followed throughout the fermentation time. Understanding the effect of different phenolics on yeast and their conversion/ detoxification pathways is the first step not only in strain engineering for enhanced robustness, but also for designing new biorefinery concepts, where the bioconversion of lignin-derived aromatics could potentially be the source of new bio-based chemicals.
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| 17. |
- Adeboye, Peter, 1982, et al.
(författare)
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DETOXIFICATION AS A STRATEGY FOR DEVELOPING TOLERANCE IN Saccharomyces cerevisiae TO PHENOLIC COMPOUNDS
- 2014
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Ingår i: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Several phenolic compounds are formed as products of lignin breakdown during pretreatment of lignocellulosic biomass. These phenolic compounds are inhibitory to cell growth and function as biocatalysts in the production of second generation biofuels from degraded lignocellulosic biomass. Our research is focused on developing a Saccharomyces cerevisiae strain with improved resistance to phenolic compounds.As part of our study, we have focused on understanding the ability of S. cerevisiae to tolerate and convert phenolic compounds. We aim to understand the conversion mechanisms of phenolic compounds and adapt the knowledge to the engineering and use of S. cerevisiae on a biotechnological platform for bioethanol production and prospective, novel bio-based chemicals.We have investigated toxicity of various phenolic compounds against S. cerevisiae. Our results showed that phenolic compounds have varied toxicity against S. cerevisiae and the toxicity may be dependent on the structure of the compound involved. Under aerobic batch cultivation conditions, we have also studied the conversion of phenolic compounds by S. cerevisiae using coniferyl aldehyde, ferulic acid and p-coumaric acid as representative phenolic compounds. We compiled a list of conversion products of the three starting compounds under investigation and we proposed a possible conversion pathway, currently being investigated.In this talk, we present the proposed conversion pathway through which S. cerevisiae converts and detoxifies coniferyl aldehyde, ferulic acid and p-coumaric acid under aerobic cultivation condition.
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| 18. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Yeast physiology studies and metabolic engineering for enhanced robustness
- 2014
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Ingår i: Enzitec 2014- XI Seminário Brasileiro de Tecnologia Enzimática. Barra da Tijuca-Rio de Janeiro, April 14th to 16th, 2014.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The extensive research on second-generation ethanol has paved the way to a new concept of bio-based industry, where lignocellulosic material is the primary source of sugars, to be converted to a number of fuels and chemicals. Sugars are released from cellulose and hemicellulose by pretreatment and hydrolysis steps. Harsh conditions during pretreatment promote the formation of a number of inhibitory compounds, among which weak organic acids, furaldehydes and phenolic compounds. In addition, the product of interest can act as a potent inhibitor. Regardless of the product, robust microorganisms are a prerequisite for the feasibility of lignocellulose-based bioprocesses.Current research carried out by our group focuses on the yeast Saccharomyces cerevisiae and aims at investigating the molecular bases of microbial robustness. Our efforts include the identification of the molecular targets of different classes of fermentation inhibitors aiming at understanding the complex responses of the cells to these compounds. The final goal is to engineer more robust strains. The concept of robustness will be discussed and examples of key features for S. cerevisiae robustness as well as examples of successful engineering to increase robustness will be presented.In particular, during this presentation, the following results will be discussed i) the study of redox and energy metabolism as key determinants of tolerance; ii) conversion routes of in S. cerevisiae as a way of detoxification from phenolic compounds; iii) cell membrane engineering as a strategy to achieve enhanced tolerance to weak acids.
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| 19. |
- Brink, Daniel
(författare)
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Understanding and improving microbial cell factories through Large Scale Data-approaches
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Since the advent of high-throughput genome sequencing methods in the mid-2000s, molecular biology has rapidly transitioned towards data-intensive science. Recent technological developments have increased the accessibility of omics experiments by decreasing the cost, while the concurrent design of new algorithms have improved the computational work-flow needed to analyse the large datasets generated. This has enabled the long standing idea of a systems approach to the cell, where molecular phenomena are no longer observed in isolation, but as parts of a tightly regulated cell-wide system. However, large data biology is not without its challenges, many of which are directly related to how to store, handle and analyse ome-wide datasets.The present thesis examines large data microbiology from a middle ground between metabolic engineering and in silico data management. The work was performed in the context of applied microbial lignocellulose valorisation with the end goal of generating improved cell factories for the production of value-added chemicals from renewable plant biomass. Three different challenges related to this feedstock were investigated from a large data-point of view: bacterial catabolism of lignin and its derived aromatic compounds; tolerance of baker’s yeast Saccharomyces cerevisiae to inhibitory compounds in lignocellulose hydrolysate; and the non-fermentable response to xylose in S. cerevisiae engineered for growth on this pentose sugar.The bibliome of microbial lignin catabolism is vast and consists of a long-standing cohort of fundamental microbiology, and a more recent cohort of applied lignin biovalorisation. Here, an online database was created with the long-term ambition of closing the gap between the two and make new connections that can fuel the generation of new knowledge. Whole-genome sequencing was used to investigate the genetic basis for observed phenotypes in bacterial isolates capable of growing on different kinds of lignin-derived aromatics. A whole-genome approach was also used to identify key sequence variants in the genotype of an industrial S. cerevisiae strain evolved for improved tolerance to inhibitors and high temperature. Finally, assessment of the sugar signalome of S. cerevisiae was enabled by the design and validation of a panel of in vivo fluorescent biosensors for single-cell cytometric analysis. It was found that xylose triggered a signal similar to that of low glucose in yeast cells engineered with xylose utilization pathways, and that introduction of deletions previously related to improved xylose utilization altered the signal towards that of high glucose.Taken together, the present thesis illustrates how omics-approaches can aid design of laboratory experiments to increase the knowledge and understanding of microorganisms, and demonstrates the need for a combined knowledge of molecular and computational biology in large-scale data microbiology.
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| 20. |
- Marx, Christian, 1975, et al.
(författare)
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ENGINEERING GLUTATHIONE BIOSYNTHESIS TO ENHANCE REDOX ROBUSTNESS OF Saccharomyces cerevisiae
- 2014
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Ingår i: ISSY31: 31ST INTERNATIONAL SPECIALISED SYMPOSIUM ON YEAST.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The focus for biofuel production shifts to using lignocellulose biomass from forest and agricultural by-products since it does not compete with food and feed production. Polysaccharides must be pretreated to be made accessible to hydrolytic enzymes to generate monomeric sugars for the following fermentation. In this pretreatment step inhibitors of fermenting microorganisms are generated, mainly furan derivates, weak acids and phenolics. Although Saccharomyces cerevisiae is more robust than bacteria, there is demand for improvement and the development of novel yeast strains with increased inhibitor tolerance is highly desirable.Furan derivates and other inhibitors have been shown to induce the formation of reactive oxygen species. Engineering of the redox metabolism of S. cerevisiae in terms of increasing the intracellular levels of glutathione by overexpressing glutathione synthetase GSH1 resulted in increased strain robustness in a simultaneous saccharification and fermentation (SSF) process. Cell survival and final ethanol concentrations were increased in the recombinant strains compared to the wild type in industrial media [Ask et al. 2013].To show a correlation between the intracellular concentration of glutathione and the resulting effect on robustness, strains accumulating different amounts of glutathione will be created. GshF is a bi-functional enzyme found in several bacterial species, that catalyzes the formation of glutathione from its precursors without accumulation of the intermediate product γ- glutamylcysteine and without any relevant feedback inhibition. GshF will be overexpressed in a CEN.PK strain, followed by deletion of the native GSH1 and GSH2 enzymes catalyzing the two-step reaction in S. cerevisiae.
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| 21. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Bottlenecks in lignocellulosic ethanol production: xylose fermentation and cell propagation
- 2017
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Ingår i: European biomass conference 2017, 25th edition, June 12-15; Stockholm, Sweden..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A remaining challenge for the development of economically feasible 2nd generation bio-ethanol is low xylose consumption rate and inhibitor tolerance of the utilized Saccharomyces cerevisiae strains. Yeast starter cultures produced for ethanol production in simultaneous saccharification and co-fermentation (SSCF) processes have to meet high, seemingly conflicting requirements. A high biomass yield during propagation is required to produce the high cell concentrations required for the harsh conditions in the proceeding fermentation. Inhibitor tolerance is essential for producing a highly viable starter culture as well as favorable fermentation kinetics. Short-term adaptation of yeast cultures during propagation has been shown to have a positive effect on pentose conversion as well as inhibitor tolerance. Here we propose a model propagation strategy for evaluating physiology of yeast cultures during propagation. This model propagation strategy will be implemented in a study comparing physiology of yeast cultures with and without exposure to lignocellulosic inhibitors during propagation to assess what molecular mechanisms underlie the short-term adaptation response phenotype. For industry, a better control of yeast properties during propagation will result in an improved and consistent performance of yeast starter cultures for SSCF purposes.
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| 22. |
- 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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| 23. |
- Xiros, Charilaos, 1973, et al.
(författare)
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Toward a sustainable biorefinery using high-gravity technology
- 2017
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Ingår i: Biofuels, Bioproducts and Biorefining. - : Wiley. - 1932-1031 .- 1932-104X. ; 11:1, s. 15-27
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Tidskriftsartikel (refereegranskat)abstract
- The realization of process solutions for a sustainable bioeconomy depends on the efficient processing of biomass. High-gravity technology is one important alternative to realizing such solutions. The aims of this work were to expand the knowledge-base on lignocellulosic bioconversion processes at high solids content, to advance the current technologies for production of second-generation liquid biofuels, to evaluate the environmental impact of the proposed process by using life cycle assessment (LCA), and to develop and present a technically, economically, and environmentally sound process at high gravity, i.e., a process operating at the highest possible concentrations of raw material. The results and opinions presented here are the result of a Nordic collaborative study within the framework of the HG Biofuels project. Processes with bioethanol or biobutanol as target products were studied using wheat straw and spruce as interesting Nordic raw materials. During the project, the main scientific, economic, and technical challenges of such a process were identified. Integrated solutions to these challenges were proposed and tested experimentally, using wheat straw and spruce wood at a dry matter content of 30% (w/w) as model substrates. The LCA performed revealed the environmental impact of each of the process steps, highlighting the importance of the enzyme dose used for the hydrolysis of the plant biomass, as well as the importance of the fermentation yield.
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| 24. |
- Muzamal, Muhammad, 1986
(författare)
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Steam Explosion of Wood
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rising price of petroleum and environmental concerns regarding CO2 emissions has increased interest in alternative renewable resources. Biomass can be considered as an excellent alternative raw material. A biorefinery uses biomass and produces fuel, energy and value-added chemicals. The biorefinery is an emerging field and requires much development to compete with already established petroleum-based industries. One of the greatest challenges to the biorefinery is that the raw material; biomass, has a complex chemical composition and physical structure. A pretreatment process is necessary to induce physico-chemical changes in the biomass and transform it into easily digestible material. The main factor limiting enzymatic digestion of biomass is accessibility to chemical constituents. Steam Explosion (SE) pretreatment is a promising process that has many potential benefits compared to the alternatives, e.g. it has less hazardous process chemicals and conditions, less environmental impact, fewer energy requirements and lower capital investment.Existing literature on the SE process mainly concerns products obtained after the process. Knowledge about the physical processes that take place during the SE pretreatment is limited. This licentiate thesis is based on experimental and modelling studies performed with the aim of gaining knowledge of the basic mechanisms of this process. The SE is a three-step process that involves; (i) treatment of wood with pressurized steam for a specific period of time, (ii) explosion of wood chips through the rapid release of pressure, and (iii) impact of softened wood chips with other chips and vessel walls. In the experimental part these steps have been carefully isolated and the effects of these steps on internal and external structures of single spruce wood pieces have been studied. The effect of vapour expansion and the creation of stresses during the explosion step on a single cell of spruce wood (with four layers; P, S1, S2 and S3) at high temperature and moisture content have been modelled using the Finite Element Method.The study reveals that all the steps of the SE process contribute to structural changes in the wood material and increase pore size which increases the accessibility of chemical reagents and enzymes. A wood piece disintegrates into smaller pieces during the impact step. The vapour expansion inside cells during the explosion step causes each cell to expand in all directions and creates high stress and strain fields perpendicular to the cell direction. In general, cell wall damage is more likely to occur in cells with thin walls, i.e. earlywood; damaged P, S1 and S3 layers; low MFAs; irregular cross-sections and sharp corners.
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| 25. |
- Nickel, David, 1990, et al.
(författare)
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Multi-scale uncertainty analysis – A tool to systematically consider variability in lignocellulosic bioethanol processes
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol production processes from lignocellulosic raw materials are highly prone to batch-to-batch variations. For example, raw material compositions and enzymatic activities required to release fermentable sugars from lignocellulose vary significantly between batches. To develop lignocellulosic biofuel processes and evaluate their performance regarding economics and sustainability consistently, tools are required to cope with this variability. In this presentation we will propose a multi-scale uncertainty analysis strategy to propagate input variability throughout system scales. In a first step, we use meta-data obtained from literature to define uncertainties in the process inputs. Utilizing these meta-data, uncertainty analysis is performed on a macro-kinetic model by sampling from the defined uncertain input space. The results of this uncertainty analysis are transferred to process simulations to analyze the impact of input uncertainties on the process mass- and energy balances, and on the economics of building this type of bioprocess. The generated data from process simulations (mass flows, energy integration, and economic data) are in the next step extracted and used as input to an environmental impact assessment of the process. This is done whilst keeping the simulation and systems modeling parameters constant, thus the input variability is propagated throughout the different system scales. The data generated in this integrated approach will then be compared with the variations and uncertainties observed with relevance to the estimated parameters in the process simulation and environmental impact assessment. Based on this consistent strategy, we can analyze the impact of input variability from different system perspectives, identify important bottlenecks for development, and suggest robust and sustainable process designs for different conditions and under given uncertainties. In a case study we demonstrate how integrated kinetic modeling (in Matlab), process simulation (in SuperPro Designer), and environmental impact assessment together with statistical analysis can be used for assessing how variability in enzymatic activities in bioethanol production can be propagated throughout system scales. A macro-kinetic model is used to describe the enzymatic breakdown of lignocellulose-derived polysaccharides into fermentable sugars (saccharification) and the simultaneous fermentation to bioethanol. We discuss the integration of the simulation results of the macro-kinetic model into the flowsheeting software for mass and energy balance generation, and then further on to assess environmental impacts of the process. We will evaluate different process designs regarding their robustness towards input variability. Finally, we also show how propagated uncertainties at different system scales can be integrated to design experiments at laboratory scale so that these focus on the most important parameters for developing robust kinetic models, and include the parameters that are most important for sustainable design of processes and value chains.
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