| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
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| 6. |
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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
- 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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