| 1. |
- Nickel, David, 1990
(författare)
-
Process development for platform chemical production from agricultural and forestry residues
- 2021
-
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.
|
|
| 2. |
- Ferreira, Sofia, et al.
(författare)
-
Metabolic engineering strategies for butanol production in Escherichia coli
- 2020
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:8, s. 2571-2587
-
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.
|
|
| 3. |
- Schwarz, Hubert, et al.
(författare)
-
Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies
- 2022
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290.
-
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.
|
|
| 4. |
- Zhang, Yiming, 1986, et al.
(författare)
-
Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production
- 2023
-
Ingår i: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16:1
-
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.
|
|
| 5. |
- Trivellin, Cecilia, 1993, et al.
(författare)
-
Quantification of Microbial Robustness in Yeast
- 2022
-
Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:4, s. 1686-1691
-
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.
|
|
| 6. |
- Perruca Foncillas, Raquel
(författare)
-
Evaluation of biosensors and flow cytometry as monitoring tools in lignocellulosic bioethanol production
- 2023
-
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.
|
|
| 7. |
- Kanagarajan, Selvaraju, et al.
(författare)
-
Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
- 2021
-
Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
-
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.
|
|
| 8. |
- Harirchi, S., et al.
(författare)
-
Efficacy of polyextremophilic Aeribacillus pallidus on bioprocessing of beet vinasse derived from ethanol industries
- 2020
-
Ingår i: Bioresource Technology. - : Elsevier Ltd. - 0960-8524 .- 1873-2976. ; 313
-
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)
|
|
| 9. |
- Olsson, Lisbeth, 1963, et al.
(författare)
-
Robustness: linking strain design to viable bioprocesses
- 2022
-
Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799 .- 1879-3096. ; 40:8, s. 918-931
-
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.
|
|
| 10. |
- Muraleedharan, Madhu Nair, et al.
(författare)
-
Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases
- 2021
-
Ingår i: International Journal of Biological Macromolecules. - : Elsevier. - 0141-8130 .- 1879-0003. ; 183, s. 101-109
-
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.
|
|
