| 1. |
- 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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| 2. |
- Ferreira, Sofia, et al.
(författare)
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Metabolic engineering strategies for butanol production in Escherichia coli
- 2020
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:8, s. 2571-2587
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Forskningsöversikt (refereegranskat)abstract
- The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.
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| 3. |
- Schwarz, Hubert, et al.
(författare)
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Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies
- 2022
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290.
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability.
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| 4. |
- Zhang, Yiming, 1986, et al.
(författare)
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Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production
- 2023
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Ingår i: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: With unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis. Results: Metabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g. Conclusions: In this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories.
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| 5. |
- Trivellin, Cecilia, 1993, et al.
(författare)
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Quantification of Microbial Robustness in Yeast
- 2022
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:4, s. 1686-1691
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Tidskriftsartikel (refereegranskat)abstract
- Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.
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| 6. |
- 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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| 7. |
- Kanagarajan, Selvaraju, et al.
(författare)
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Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
- 2021
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
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Tidskriftsartikel (refereegranskat)abstract
- Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.
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| 8. |
- Harirchi, S., et al.
(författare)
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Efficacy of polyextremophilic Aeribacillus pallidus on bioprocessing of beet vinasse derived from ethanol industries
- 2020
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Ingår i: Bioresource Technology. - : Elsevier Ltd. - 0960-8524 .- 1873-2976. ; 313
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Tidskriftsartikel (refereegranskat)abstract
- This work aimed to evaluate the applicability of Aeribacillus pallidus for the aerobic treatment of the concentrated beet vinasse with high chemical oxygen demand (COD 685 g.L−1) that is defined as an environmental pollutant. This bacterium is a polyextremophilic strain and grow aerobically up to 7.5% vinasse at high temperature (50 °C). In the bioreactor and under controlled conditions, A. pallidus reduced the soluble COD content of 5% vinasse up to 27% during 48 h and utilized glucose and glycerol, completely. Furthermore, a reduction of manganese, copper, aluminum, and nickel concentrations was observed in the treated vinasse with A. pallidus. The obtained results make this strain as an appropriate alternative to be used for the aerobic bioprocessing of the vinasse. © 2020 The Author(s)
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| 9. |
- Olsson, Lisbeth, 1963, et al.
(författare)
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Microbial robustness in bioprocesses
- 2023
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Yeast is broadly exploited for industrial use, and strains are constantly improved to meet the requirements to produce the targeted product with high yield, productivity and titer. Successful strains have consistent performance also in presence of different perturbations, i.e. their performance is robust. The concept of microbial robustness will be discussed and contrasted to tolerance toward specific stresses. Furthermore, a method to quantitatively assess microbial robustness will be presented. This method allows a high throughput evaluation, in a perturbation space where different cellular function can form the basis for the evaluation. Another important tool box to examine intracellular status in face of pertubations are biosensors. Examples of applying these two methodologies towards microbial robustness will be discussed. We have used the tools to scale down bioprocesses and their perturbation, to follow adaptive laboratory evolution and to gain understanding of subpopulations.
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| 10. |
- Olsson, Lisbeth, 1963, et al.
(författare)
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Robustness: linking strain design to viable bioprocesses
- 2022
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 40:8, s. 918-931
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Forskningsöversikt (refereegranskat)abstract
- Microbial cell factories are becoming increasingly popular for the sustainable production of various chemicals. Metabolic engineering has led to the design of advanced cell factories; however, their long-term yield, titer, and productivity falter when scaled up and subjected to industrial conditions. This limitation arises from a lack of robustness – the ability to maintain a constant phenotype despite the perturbations of such processes. This review describes predictable and stochastic industrial perturbations as well as state-of-the-art technologies to counter process variability. Moreover, we distinguish robustness from tolerance and discuss the potential of single-cell studies for improving system robustness. Finally, we highlight ways of achieving consistent and comparable quantification of robustness that can guide the selection of strains for industrial bioprocesses.
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| 11. |
- Muraleedharan, Madhu Nair, et al.
(författare)
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Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases
- 2021
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier. - 0141-8130 .- 1879-0003. ; 183, s. 101-109
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Tidskriftsartikel (refereegranskat)abstract
- Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential −19.4 mV) with particles of 7–20 nm diameter, high carboxylate content and improved thermostability (To = 301 °C, Tmax = 337 °C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.
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| 12. |
- 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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| 13. |
- Helstad, Amanda, et al.
(författare)
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Protein extraction from cold-pressed hempseed press cake: From laboratory to pilot scale
- 2022
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Ingår i: Journal of Food Science. - : Wiley. - 1750-3841 .- 0022-1147. ; 87:1, s. 312-325
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: During the production of industrial hempseed oil, a press cake is formed as a byproduct, which is often used as animal feed although it contains a high amount of protein that could be used for human consumption. Extracting this valuable protein would reduce food waste and increase the availability of plant-based protein. A protein extraction process based on the pH-shift method was adapted to improve the protein extraction yield from industrial hempseed press cake (HPC). Parameters such as alkali extraction pH, time, and temperature, as well as isoelectric precipitation pH, were investigated in laboratory scale and were thereafter carried out in a pilot trial to explore the suitability for future scale up. The phytic acid content of the extracted protein isolate was also analyzed to investigate any potential inhibitory effect on mineral absorption. A final protein yield of 60.6%, with a precipitated protein content of 90.3% (dw), was obtained using a constant alkali extraction pH of 10.5 for 1 h at room temperature, followed by precipitation at pH 5.5. The pilot trial showed promising results for the future production of industrial hemp protein precipitate on a larger scale, showing a protein yield of 57.0% and protein content of 90.8% (dw). The amount of phytic acid in the protein isolate produced in the optimal laboratory experiment and in the pilot trial was 0.595 and 0.557 g phytic acid/100 g dw, respectively, which is 83%–88% less than in the HPC. This is in the range of other plant-based protein sources (tofu, kidney beans, peas, etc.). Practical Application: Industrial hempseed press cake is a byproduct in the production of industrial hempseed oil, which is mostly used as animal feed, but has the potential to become an additional source of plant-based protein for human consumption with a suitable protein extraction method. The extracted hemp protein could be used to develop new plant-based dairy or meat analog products.
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| 14. |
- Abadikhah, Marie, 1992
(författare)
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Influence of electrode material and stochastic factors on the performance and microbial community assembly in microbial electrochemical systems
- 2022
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Microbial electrolysis cells (MECs) are systems with microbial communities in the form of biofilms on electrode surfaces. The electrogenic bacteria in the anode biofilm act as catalysts for the oxidization of organic compounds, leading to release of electrons, generation of electrical current, and production of hydrogen and methane at the cathode. In addition to production of energy carriers, MECs can be used for other applications as well; for example, as biosensors to monitor biochemical oxygen demand or toxicity. The performance of MECs is determined by both deterministic and stochastic factors influencing the microbial communities on the electrode surfaces, most of which as still poorly understood. In this thesis, the effects of electrode materials on microbial community assembly and MEC performance was investigated. Two experiments were carried out. In the first, three cathode materials (carbon nanoparticles, titanium, and steel) were compared. In the second, three anode materials (carbon cloth, graphene, and nickel) were compared. The cathode materials had no significant effect on the performance of the MECs, as opposed to the anode materials where carbon cloth MECs had the highest current density and the shortest lag time during startup. The differences seen in lag time of replicate systems at the start of the experiment indicated a stochastic initial attachment of the electrogenic bacteria on the anode. Different microbial communities develop in the biofilms on the anodes and cathodes. Electrogens from the Desulfobacterota phylum dominated the anode, while various hydrogenotrophic methanogens, e.g., Methanobacterium, were found to dominate on the cathodes. Diversity and null model analysis of the electrode communities highlighted stochasticity and not electrode material as the important factor in the community assembly. Network analysis showed that the cathode communities had fewer negative interactions between taxa in comparison to the anode. Since hydrogen gas generated at the cathode surface can diffuse through the biofilm, all microorganisms on the cathode have access to the substrate, reducing the need for competition between species. In contrast, electrogens require a short distance to the anode to be able to use it as electron acceptor. Limited space on the anode and competition between electrogens shaped the anode communities and explain the higher number of negative interactions observed. Based on the findings in this thesis, it is suggested that stochastic factors have more influence than electrode material on the anode community even though there is a selective pressure for electrogenic bacteria.
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| 15. |
- Persson, Michael
(författare)
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Integrated starch and lignocellulose based biorefineries : Synergies and opportunities
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The transition from a reliance on fossil resources to the use of renewables for the production of energy, fuels and chemicals is essential for ensuring the sustainability of continued human development. Plant-based biomass is a renewable resource which can be transformed into all of these products. However, biomass is a heterogeneous material composed of several fractions with different chemical properties. Furthermore, the composition varies between species. In order to maximize the environmental and economic sustainability of biomass-based production, production systems that utilize all fractions of biomass to their fullest potential have to be developed. This is the goal of a biorefinery.The work presented in this thesis mainly revolves around biorefineries that utilize feedstocks rich in starch and lignocellulose together to produce ethanol in an integrated process. The work is focused on comparing the performance of stand-alone and integrated biorefineries by investigating the impact that feedstock blending has on parameters important for the process economy, identifying potential synergies from integration and opportunities for improved material utilization.It was found in this work, that the integration of starch- and lignocellulose-based feedstocks could result in improved ethanol productivity and yield during hydrolysis and fermentation compared to a stand-alone lignocellulose process without losing performance compared to a stand-alone starch-based process.The prospects of introducing a sequential fractionation of the lignocellulosic biomass prior to integration was investigated. It was shown that this method could be used to produce separate fractions enriched in cellulose and lignin as well as improving the hydrolyzabilty of the cellulose fraction. This kind of fractionation could facility the utilization of all biomass fractions in both feedstocks by creating new byproduct streams as well as decreasing negative impacts on existing byproduct streams.
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| 16. |
- Scheffel, Julia
(författare)
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Calcium-dependent Affinity Domains for the Purification of Antibodies and Antibody Fragments
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibodies are essential proteins in both our bodies and biotechnological research, and hold outstanding therapeutic value. The market for antibody-based therapeutics has grown exponentially during the last decades, owing to several advantages over small molecule drugs, such as fewer undesirable side effects associated with a higher target specificity. To keep up with the increasing amounts of antibodies that are on demand, emphasis has been on the optimization of upstream processes for antibody production while the advances in downstream processing and the purification of antibodies have been limited. In the downstream process, the gold standard for the primary capture step is Protein A affinity chromatography. However, elution of the antibodies from the Protein A ligand is accomplished at a low pH, which can lead to antibody aggregation and impaired biological activity. The purification procedure therefore hinders the development of new antibodies that are acid-sensitive, despite promising therapeutic potential, and may pose a threat to the increasingly popular bispecific antibodies that tend to be more aggregation prone. Further, acidic elution conditions may be an even bigger concern in the purification of antibody fragments, which also represent promising therapeutic candidates, providing several advantages over full-length antibodies in certain applications.The work in this thesis aimed to enable the purification of a more diverse group of antibodies and antibody fragments, regardless of their stability in a highly acidic environment. Efforts were also made to reduce the high antibody manufacturing costs to make these antibody therapeutics more easily accessible to patients. In order to elute the antibodies in the Protein A capture step under milder conditions, the protein ligand ZCa was developed. ZCa was isolated from a phage display library based on a Protein A domain with a grafted calcium-binding loop, and permits the calcium-dependent elution of antibodies at close to neutral pH by adding sodium chloride. The domain provides the foundation for this thesis, and was extensively optimized to achieve a high-capacity resin and an inexpensive purification strategy, yielding exceptional recoveries of pure antibody. Most importantly, ZCa was able to entirely prevent the formation of aggregates of an antibody through mild elution. Moreover, the optimized ZCa resin was applied in an integrated continuous biomanufacturing process, designed to entirely avoid the use of low pH. The implementation of the process at pilot scale for 17 days demonstrated the robustness of the novel resin along with many other promising process attributes. High productivity and yields were obtained in addition to negligible aggregate formation and low levels of residual DNA and host cell proteins, comparable to established processes.Finally, this thesis presents a combinatorial library for calcium-regulated affinity constructed from ZCa, from which numerous binders with novel target specificities were isolated. The calcium-dependent binders to single chain variable fragments (scFvs) could be used to selectively capture and elute scFv at neutral pH. Binding analysis and optimization efforts indicated potential as a platform approach for the mild and efficient purification of different scFvs.In conclusion, the purification strategies proposed in this thesis considerably improve the purification of antibodies and scFvs, and may encourage the future innovation of a wider range of antibody-based therapeutics. The continuous process supports the applicability of ZCa in a state-of-the-art commercial manufacturing process, and contributes to the more efficient manufacturing of antibodies, which can make them more affordable and accessible to the patients in need.
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| 17. |
- Hagman, Linda, 1991-, et al.
(författare)
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Assessment of By-product Valorisation in a Swedish Wheat-Based Biorefinery
- 2020
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Ingår i: Waste and Biomass Valorization. - : Springer Netherlands. - 1877-2641 .- 1877-265X. ; 11:7, s. 3567-3577
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Tidskriftsartikel (refereegranskat)abstract
- Biorefineries are examples of industries striving towards a circular and bio-based economy through valorising natural raw materials to a spectrum of products. This is a resource-efficient process which also decreases overall environmental impact, as the products from a biorefinery can replace fossil-based products such as plastics or fuels. To become even more resource efficient, an optimisation of the by-product use can increase the performance. This study will evaluate different scenarios for the valorisation of stillage coming from a wheat-based biorefinery. The alternatives range from the direct use of the stillage for fodder, fertiliser or incineration to three different biogas production-based scenarios. The biogas scenarios are divided into the production of fuel at a local or distant plant and the alternative of creating heat and power at the local plant. The results show how locally produced biogas for vehicle fuel and fodder usage are the better alternatives regarding greenhouse gas emissions, the finances of the biorefinery, energy balance and nutrient recycling. The results also indicate that biorefineries with several high-value products may receive lower quality by-product flows, and to these, the biogas solutions become more relevant for valorising stillage while improving value and performance for the biorefinery.
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| 18. |
- Lama, Suman, et al.
(författare)
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Production of 3-hydroxypropionic acid from acetate using metabolically-engineered and glucose-grown Escherichia coli
- 2021
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 320
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Tidskriftsartikel (refereegranskat)abstract
- Acetate can be used as carbon feedstock for the production of 3-hydroxypropionic acid (3-HP), but the production level was low due to inefficient cell growth on acetate. To better utilize acetate, a two-stage strategy, whereby glucose is used for cell growth and acetate for 3-HP formation, was attempted. Dissected malonyl-CoA reductase of Chloroflexus aurantiacus, alone or along with acetyl-CoA carboxylase and/or transhydrogenase, was overexpressed, and by-products formation pathway, glyoxylate shunt (GS) and gluconeogenesis were modified. When GS or gluconeogenesis was disrupted, cell growth on glucose was not hampered, while on acetate it was completely abolished. Consequently, 3-HP production, at production stage, were low, though 3-HP yield on acetate was increased, especially in the case of aceA deletion. In two-stage bioreactor, strain with upregulated GS produced 7.3 g/L 3-HP with yield of 0.26 mol/mol acetate. This study suggests that two-stage cultivation is a good strategy for 3-HP production from acetate.
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| 19. |
- Sjöberg, Gustav, 1991-
(författare)
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Engineering short-chain carboxylic-acid metabolism in the model microorganism Escherichia coli
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ever-increasing concern about carbon dioxide emissions has created an urgent need to develop alternative methods to cheaply and renewably produce materials, chemicals and fuels. The biorefinery is uniquely suited to deliver these products from sustainable biomass. However, cheaply and efficiently converting the dispersed, heterogenous and recalcitrant biomass to useful products requires further technical development. To address some of these challenges, the aim of this thesis was to investigate methods to improve the economic viability of the microbial biorefinery by evaluating short chain carboxylic acids as substrates (volatile fatty acids) and products ((R)-3-hydroxybutyrate, 3HB).Initially, two renewable and cheap sources of carbon were investigated as substrates for E. coli. It was determined that E. coli is a suitable microorganism for valorization of volatile fatty acids derived from food waste. Also, it was shown that lignocellulosic sugars with a composition based on a hydrolysate of wheat straw can be converted to 3HB in E. coli with similar yields and productivities as from pure glucose. To improve the yield of the model product 3HB, and thereby the potential gross profit, substrate depletion was used as a strategy throughout the thesis to control bioprocesses. Specifically, nutrient depletion was shown to decouple growth from 3HB production in nitrogen and phosphorous depleted batches, increasing the yield of 3HB. To further improve 3HB production, metabolic engineering was used to improve the availability of NADPH. Additionally, the bacterial artificial chromosome (BAC) was investigated as a robust single-copy vector for metabolic engineering in E. coli. The expression of a large operon from the BAC was shown to be comparable to chromosomal expression. Then, the specific growth rate, productivity and yield of 3HB producing strains was increased by expression of the 3HB production pathway from the BAC instead of a multi-copy plasmid. Finally, the BAC was shown to be a useful tool for the optimization of enzyme expression levels in metabolic pathways. While directly beneficial for 3HB production, the methods and strategies employed in this thesis are broadly applicable to increase the economic viability of microbial biorefineries.
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| 20. |
- Agnihotri, Swarnima, et al.
(författare)
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A Glimpse of the World of Volatile Fatty Acids Production and Application : A review
- 2022
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Ingår i: Bioengineered. - : Informa UK Limited. - 2165-5979 .- 2165-5987. ; 13:1, s. 1249-1275
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Forskningsöversikt (refereegranskat)abstract
- Sustainable provision of chemicals and materials is undoubtedly a defining factor in guaranteeing economic, environmental, and social stability of future societies. Among the most sought-after chemical building blocks are volatile fatty acids (VFAs). VFAs such as acetic, propionic, and butyric acids have numerous industrial applications supporting from food and pharmaceuticals industries to wastewater treatment. The fact that VFAs can be produced synthetically from petrochemical derivatives and also through biological routes, for example, anaerobic digestion of organic mixed waste highlights their provision flexibility and sustainability. In this regard, this review presents a detailed overview of the applications associated with petrochemically and biologically generated VFAs, individually or in mixture, in industrial and laboratory scale, conventional and novel applications.
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| 21. |
- Patel, Alok, Dr. 1989-, et al.
(författare)
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Microbial genetic engineering approach to replace shark livering for squalene
- 2022
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Ingår i: Trends in Biotechnology. - : Elsevier. - 0167-7799 .- 1879-3096. ; 40:10, s. 1261-1273
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Forskningsöversikt (refereegranskat)abstract
- Squalene is generally sourced from the liver oil of deep sea sharks (Squalus spp.), in which it accounts for 40–70% of liver mass. To meet the growing demand for squalene because of its beneficial effects for human health, three to six million deep sea sharks are slaughtered each year, profoundly endangering marine ecosystems. To overcome this unsustainable practice, microbial sources of squalene might offer a viable alternative to plant- or animal-based squalene, although only a few microorganisms have been found that are capable of synthesizing up to 30% squalene of dry biomass by native biosynthetic pathways. These squalene biosynthetic pathways, on the other hand, can be genetically manipulated to transform microorganisms into 'cellular factories' for squalene overproduction.
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| 22. |
- Mariam, Iqra, et al.
(författare)
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Ameliorating microalgal OMEGA production using omics platforms
- 2024
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Ingår i: Trends in Plant Science. - : Elsevier Ltd. - 1360-1385 .- 1878-4372.
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Forskningsöversikt (refereegranskat)abstract
- Over the past decade, the focus on omega (ω)-3 fatty acids from microalgae has intensified due to their diverse health benefits. Bioprocess optimization has notably increased ω-3 fatty acid yields, yet understanding of the genetic architecture and metabolic pathways of high-yielding strains remains limited. Leveraging genomics, transcriptomics, proteomics, and metabolomics tools can provide vital system-level insights into native ω-3 fatty acid-producing microalgae, further boosting production. In this review, we explore ‘omics’ studies uncovering alternative pathways for ω-3 fatty acid synthesis and genome-wide regulation in response to cultivation parameters. We also emphasize potential targets to fine-tune in order to enhance yield. Despite progress, an integrated omics platform is essential to overcome current bottlenecks in optimizing the process for ω-3 fatty acid production from microalgae, advancing this crucial field.
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| 23. |
- Ahlström, Cecilia, et al.
(författare)
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Pilot-Scale Protein Recovery from Cold-Pressed Rapeseed Press Cake: Influence of Solids Recirculation
- 2022
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Ingår i: Processes. - : MDPI AG. - 2227-9717. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- The agricultural sector is responsible for about 30% of greenhouse gas emissions, and thus there is a need to develop new plant-based proteins with lower climate impact. Rapeseed press cake, a by-product from rapeseed oil production, contains 30% high-quality protein. The purpose of this study was to recover protein from cold-pressed rapeseed press cakes on a pilot scale using a decanter and investigate the effect of recirculation of the spent solids fraction on protein yield. Proteins were extracted under alkaline conditions (pH 10.5) followed by precipitation at pH 3.5. Recirculating the spent solids fraction once increased the accumulated protein yield from 70% to 83%. The efficiency of the recovery process was highest in the first and second cycles. The additional yield after the third and fourth cycles was only 2%. The amino acid composition showed high levels of essential amino acids and was not reduced throughout the recovery process. The glucosinolate and phytate content was reduced in the precipitate after one cycle, although additional process steps are needed to further reduce the phytate content and limit the negative effect on mineral uptake.
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| 24. |
- Olszewska-Widdrat, Agata, et al.
(författare)
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Bioprocess optimization for lactic and succinic acid production from a pulp and paper industry side stream
- 2023
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media S.A.. - 2296-4185. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- The effective and cheap production of platform chemicals is a crucial step towards the transition to a bio-based economy. In this work, biotechnological methods using sustainable, cheap, and readily available raw materials bring bio-economy and industrial microbiology together: Microbial production of two platform chemicals is demonstrated [lactic (LA) and succinic acid (SA)] from a non-expensive side stream of pulp and paper industry (fibre sludge) proposing a sustainable way to valorize it towards economically important monomers for bioplastics formation. This work showed a promising new route for their microbial production which can pave the way for new market expectations within the circular economy principles. Fibre sludge was enzymatically hydrolysed for 72 h to generate a glucose rich hydrolysate (100 g·L−1 glucose content) to serve as fermentation medium for Bacillus coagulans A 541, A162 strains and Actinobacillus succinogenis B1, as well as Basfia succiniciproducens B2. All microorganisms were investigated in batch fermentations, showing the ability to produce either lactic or succinic acid, respectively. The highest yield and productivities for lactic production were 0.99 g·g−1 and 3.75 g·L−1·h−1 whereas the succinic acid production stabilized at 0.77 g·g−1 and 1.16 g·L−1·h−1. Copyright © 2023 Olszewska-Widdrat, Xiros, Wallenius, Schneider, Rios da Costa Pereira and Venus.
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| 25. |
- Rasool, Kashif, et al.
(författare)
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Comprehensive insights into sustainable conversion of agricultural and food waste into microbial protein for animal feed production
- 2023
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Ingår i: Reviews in Environmental Science and Biotechnology. - : Springer. - 1569-1705 .- 1572-9826. ; 22:2, s. 527-562
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Forskningsöversikt (refereegranskat)abstract
- The growing global population and higher living standards instantly demand the transition in the direction of a sustainable food system. A substantial section of means and agricultural lands are presently committed to protein-rich feed production to rear livestock for human consumption. Conversely, accelerated farming activities and the food industry have rendered a drastic increase in waste which impair the economic and environmental sustainability of the ecosystem. This situation emerges the need for developing an integrated technology for waste management and to improve sustainability footprints. Microbial protein (MP) production based on renewable electron and carbon sources has the potential as a substitute protein source. MP production for animal feed use is growing fast and is derived from bacteria, algae, and fungi including yeast. MP produced from all types of microbes is currently commercialized and in use. However, novel methods and processes are also under investigation to make MP production more economical and sustainable. Current research on MP has concentrated on the valorization of waste materials by using high protein content-containing microorganisms, which can then be used in animal feed. Using such kind of integrated approach, the agroindustry waste resources upcycling can contribute towards finding sustainable, cheaper, and environment-friendly protein sources. This review first describes the potential waste feedstock for MP production and summarizes the recent progress in the application of MP-producing microorganisms including fungus, yeast, bacteria, and phototrophic microbes. Bioprocesses, and production technology advances for MP production have been explored and discussed in detail. Finally, the MP application as animal feed, its challenges, and future perspectives in research have been evaluated.
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