| 31. |
- 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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| 32. |
- 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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| 33. |
- 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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| 34. |
- Nickel, David, 1990
(författare)
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Process development for platform chemical production from agricultural and forestry residues
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As part of a bio-based economy, biorefineries are envisaged to sustainably produce platform chemicals via biochemical conversion of agricultural and forestry residues. However, supply risks, the recalcitrance of lignocellulosic biomass, and inhibitor formation during pretreatment impair the economic feasibility of such biorefineries. In this thesis, process design and assessment were developed with the aim of addressing these hurdles and improving the cost-effectiveness of lignocellulose-derived platform chemicals. To expand the feedstock base and reduce operational costs, logging residues served as underutilised and inexpensive raw material. The major impediment in converting logging residues was their high recalcitrance and low cellulose content, which resulted in low attainable ethanol titres during simultaneous saccharification and co-fermentation (SSCF). Pretreatment optimisation reduced inhibitor formation and recalcitrance, and led to enzymatic hydrolysis yields at par with those obtained for stem wood, despite the less favourable chemical composition. Upgrading logging residues with carbohydrate-rich oat hulls increased ethanol titres to >50 g/L using batch SSCF at 20% WIS loadings, demonstrating the potential to further decrease downstream processing costs. To alleviate the toxicity of inhibitors generated during pretreatment, preadaptation was applied to Saccharomyces cerevisiae . Exposure to the inhibitors in the pretreated liquid fraction improved ethanol production during subsequent fermentation. Transferring the concept of preadaptation to lactic acid production by Bacillus coagulans cut the process times by half and more than doubled the average specific lactic acid productivity, showcasing how preadaptation could decrease operational costs. To assess the performance and robustness of process designs against process input variations, a multi-scale variability analysis framework was developed. The framework included models for bioprocess, flowsheet, techno-economic, and life cycle assessment. In a case study, multi-feed processes, in which solids and cells are fed to the process using model-based predictions, were more robust against variable cellulolytic activities than batch SSCFs in a wheat straw-based ethanol biorefinery. The developed framework can be used to identify robust biorefinery process designs, which simultaneously meet technological, economic, and environmental goals.
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| 35. |
- Nickel, David, 1990, et al.
(författare)
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Uncertainty analysis as a tool to consistently evaluate lignocellulosic bioethanol processes at different system scales
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic processes are highly prone to batch-to batch variability, e.g. of raw materials and enzyme activities. This variability can be propagated throughout system scales during process development and optimization, influencing the outputs of bioreaction models, techno-economic analyses and life cycle assessments. As these outputs are the main decision variables for designing and developing lignocellulose-based processes, tools are required to evaluate the influences of process variation at different system scales. Uncertainty analysis quantifies the effects of model input variations on model outputs. It is an effective tool to consistently propagate process variation throughout scales and analyse its influence on model outputs. As an example, we use a model describing multi-feed simultaneous saccharification and co-fermentation (SSCF) of wheat straw. During the process enzymes hydrolyse the lignocellulosic material to release glucose which can be converted by microorganisms into ethanol. To investigate the impact of batch-to-batch variability in enzyme cocktails, we collected literature data on the enzymatic activity of Cellic CTec2. Retrieved data were propagated in models at bioreactor, techno-economic analysis and life cycle assessment scale. We show how uncertainty analysis can be used to guide process development by comparing different modes of operation. The method can identify economically feasible process ranges with low environmental impact while increasing the robustness of bioprocesses with high variation in raw material inputs. Furthermore, uncertainty analysis could help to identify relevant parameters to choose as response variables in experimental designs.
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| 36. |
- Pajalic, Oleg, 1964
(författare)
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BIOCHEMICALS, BIOMATERIALS, AND BIOINNOVATIONS – WHAT CAN WE GET FROM BIOMASS
- 2015
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Ingår i: 4th SCIENTIFIC SYMPOSIUM WITH INTERNATIONAL PARTICIPATION Environmental resources, sustainable development and food production OPORPH – 2015 Tuzla, Bosnia and Herzegovina November 12-13, 2015.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Environmental changes and oil price has triggered an increased interest in bio-based energy, chemicals and materials in the last decades. Thou the oil price have been dropped recently due to new findings, the transformation towards bio-based economy will continue. EU has pumped hundred millions of EURO in sustainability related research and innovation projects. Sweden has followed the pattern and has been pushed towards the green economy by establishing the vision to create and develop a bio-based economy in the first half of the twenty-first century. Sweden is rich with non-edible bio-mass i.e. forest. Traditionally the forest was used in pulp and paper industry, but digitalization boom requires paradigm shift. Green materials and green chemicals were identified as a big potential. Ambitious research was started supported by Swedish government and industry. Some of the industrial initiatives are:-The new research agenda, NRA 2020, is the collective assessment of Swedish forest-based industries regarding research, development and demonstration, which is necessary for the sector to be able to contribute to achieving. - Chemical cluster in Western Sweden started an initiative called Sustainable Chemistry 2030 with the vision that their business will be based on renewable feedstock and energy and contribute to sustainable society.
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| 37. |
- Perruca Foncillas, Raquel
(författare)
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Evaluation of biosensors and flow cytometry as monitoring tools in lignocellulosic bioethanol production
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The significant environmental impact of the current fossil fuel-based industry is a major concern for society. Consequently, various initiatives are being undertaken to establish a more sustainable industrial model. One example is via the transition from conventional fossil fuel refineries to biorefineries, where renewable raw materials are utilised. Amongst these raw materials, the use of lignocellulosic biomass from agricultural residues or wood has been favoured, as it does not compete with food or land resources. In particular, extensive research has been conducted to produce biofuels such as bioethanol from lignocellulosic biomass, referred to as second-generation (2G) bioethanol.In this thesis work, the goal was to develop and apply new tools to address challenges encountered in 2G bioethanol production. Specifically, the work focused on monitoring the impact of inhibitory compounds and mixed sugars on the fermentation performance of the yeast Saccharomyces cerevisiae.Inhibitory compounds are released during the pretreatment of the lignocellulosic biomass, a crucial step necessary to break down its complex structure and to enhance sugar accessibility This thesis work specifically focused on the redox imbalance induced in cells exposed to furaldehydes such as furfural or HMF. To study this effect, a biosensor for redox imbalance, TRX2p-yEGFP, was introduced into the cells and its fluorescence signal was monitored in real-time using flow cytometry. One potential strategy for enhancing the cells' tolerance to these inhibitors is to prepare them by introducing lignocellulosic hydrolysate in the feed during cell propagation. During this pre-exposure phase, a transient induction of the TRX2p-yEGFP biosensor signal for redox imbalance was observed, which gradually diminished. This indicated that, by the time of cell collection, the cells had adapted to the inhibitor concentration within the culture. To examine whether an increased induction level of the biosensor at the time of cell collection influenced the fermentation performance, an automated control system was devised. This system utilised data from the flow cytometry analysis to control the level of inhibitors in the cultivation feed. Consequently, when the biosensor signal began to decline, higher amounts of inhibitors were added, as long as the addition did not lead to an increase in the number of damaged cells.A second biosensor was used in this thesis work to investigate the sugar signalling response of S. cerevisiae to the presence of xylose. Xylose is the second most abundant sugar in lignocellulosic biomass; however, naturally, S. cerevisiae cannot metabolise it. Genetically modified S. cerevisiae strains have been generated by introducing heterologous pathways such as the XR/XDH or XI pathways to enable xylose consumption. Nevertheless, xylose consumption rates remain lower compared to glucose. Sugar signalling emerged as a potential bottleneck in the efficient utilisation of xylose. In the present work, the response of the SUC2p-yEGFP biosensor for sugar signalling was found to vary significantly depending on the pathway employed. A higher induction for the strains carrying the XI pathway was associated with poorer growth on xylose. Lastly, the effect of introducing a xylose epimerase capable of catalysing the conversion between the two anomers, α-D-xylopyranose and β-D-xylopyranose, as a strategy to improve xylose consumption was studied. The effect was enzyme-specific and proved to be particularly beneficial in strains utilising the xylose isomerase from Lachnoclostridium phytofermentans.In conclusion, the results presented in this thesis demonstrate how biosensors can facilitate the understanding and monitoring of intracellular processes that occur within the cell under stress conditions and be a key tool for improving production processes.
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| 38. |
- Sánchez I Nogué, Violeta, et al.
(författare)
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Isolation and characterization of a resident tolerant Saccharomyces cerevisiae strain from a spent sulfite liquor fermentation plant
- 2012
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 2:1, s. 68-
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Tidskriftsartikel (refereegranskat)abstract
- Spent Sulfite Liquor (SSL) from wood pulping facilities is a sugar rich effluent that can be used as feedstock for ethanol production. However, depending on the pulping process conditions, the release of monosaccharides also generates a range of compounds that negatively affect microbial fermentation. In the present study, we investigated whether endogenous yeasts in SSL-based ethanol plant could represent a source of Saccharomyces cerevisiae strains with a naturally acquired tolerance towards this inhibitory environment. Two isolation processes were performed, before and after the re-inoculation of the plant with a commercial baker’s yeast strain. The isolates were clustered by DNA fingerprinting and a recurrent Saccharomyces cerevisiae strain, different from the inoculated commercial baker’s yeast strain, was isolated. The strain, named TMB3720, flocculated heavily and presented high furaldehyde reductase activity. During fermentation of undiluted SSL, TMB3720 displayed a 4-fold higher ethanol production rate and 1.8-fold higher ethanol yield as compared to the commercial baker’s yeast. Another non-Saccharomyces cerevisiae species, identified as the pentose utilizing Pichia galeiformis, was also recovered in the last tanks of the process where the hexose to pentose sugar ratio and the inhibitory pressure are expected to be the lowest.
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| 39. |
- Spetea, Cornelia, 1968
(författare)
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Energy-efficient cultivation of marine microalgae for biomass production : Final rapport: Energimyndigheten P45907-1
- 2020
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- This project has demonstrated the principle of rotational cultivation of marine microalgae and that species adapted to cold climates can provide higher productivity during cold periods. By using marine species, and thus seawater instead of freshwater in cultivation, the environmental impact is reduced. Society faces major challenges to produce sufficient amounts of biomass for energy and material, and microalgae have a great potential to complement sources from forestry and agriculture. At Nordic latitudes year-round microalgae cultivation is debatable due to seasonal variations in productivity. Shall the same species be used throughout the year or shall seasonal-adapted species be used? The aims of the project were to identify suitable algal strains for a potential annual rotation model, where different strains are rotated during three cultivation seasons, and to further develop and optimize an energy-efficient cultivation process for the marine environment. To achieve these aims, a laboratory study was performed where two marine microalgal strains out of 167 were selected for intended cultivation at the west coast of Sweden. One strain belongs to the species Nannochloropsis granulata and the other to Skeletonema marinoi. The strains were cultivated in three simulated growth seasons: summer, winter and spring, and thereafter compared. We show that Nannochloropsis produced more biomass with more incorporated energy in lipids during summer and spring (25 MJ kg-1 compared to about 45 MJ kg-1 for diesel), whereas Skeletonema produced more biomass rich in carbohydrates and proteins during winter. Skeletonema was in general more efficient in taking up phosphate. Based on our results, biomass production as energy feedstock would be energy efficient only during the summer on the Swedish west coast. Nevertheless, species could be rotated for different purposes during the year. Biomass production could be combined with nutrient recycling of wastewater, for example, from fish industry. Our project faces a challenge in boosting the biomass produced in winter, but this could be solved, for example, by optimization of the cultivation medium and temperature increase with heat wastewater or other heat waste. The summer species Nannochloropsis proved to withstand winter by activating different lipid metabolic pathways than the cold-adapted species Skeletonema uses. Enhanced synthesis of proteins, such as enzymes, in Skeletonema during winter may compensate for their reduced activities, promoting growth and biomass production even at low temperatures. More species need to be studied to find those with higher productivity under winter conditions. In practice, the work-related consequences of a rotational cultivation should be weighed against its benefits, relative to a shorter cultivation season in each application. Potential applications mainly include cleaning of air and seawater, production of energy, biomass and biomaterials for the industry.
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| 40. |
- Tomas-Pejo, Elia, 1980, et al.
(författare)
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EVALUATION OF EVOLVED AND BARCODED XYLOSE FERMENTING STRAINS FOR BIOETHANOL PRODUCTION FROM LIGNOCELLULOSE
- 2012
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Ingår i: Science and Technology Day 2012, Chalmers University of Technology, 27th March 2012.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic raw materials for bioethanol production are today the basis for many ethanol production sites around the world. However, the utilization of engineered yeast strains for second generation ethanol production at large-scale can still be improved. Yeasts mainly use the sugars present in the lignocellulosic biomass but, toxic compounds derived from cellulose, hemicellulose and lignin degradation during pretreatment are also found in the media and inhibit yeast growth. Furthermore, wild type Saccharomyces cerevisiae is not able to ferment xylose which could constitute up to 40% of the lignocellulose material. Hence the recombinant yeast strains must be robust and ferment xylose to ethanol with high yields in the presence of inhibitors.In this study, different evolved xylose fermenting Saccharomyces cerevisiae strains have been compared in ethanol production processes from lignocellulosic hydrolysates. The differences between using single cell transformants and mixed populations will be evaluated in terms of ethanol production in large scale bioreactors.Furthermore, we have established a method to barcode the evolved yeast strains in order to be able to verify their origin. It is of outmost importance that after barcoding the original characteristics of a yeast strain are maintained. Those requirements can only be fulfilled by using a dominant selection principle. We have obtained a few hundred transformants that were shown to contain the new unique barcode DNA sequence via DNA isolation and DNA sequencing. The transformed strains must be able to grow on the lignocellulosic material and consume xylose at the same rate as before the transformation which also was tested in this study.
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