| 1. |
- Munthe, Christian, 1962
(författare)
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Precaution and Ethics: Handling risks, uncertainties and knowledge gaps in the regulation of new biotechnologies
- 2017
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Bok (övrigt vetenskapligt/konstnärligt)abstract
- This volume outlines and analyses ethical issues actualized by applying a precautionary approach to the regulation of new biotechnologies. It presents a novel way of categorizing and comparing biotechnologies from a precautionary standpoint. Based on this, it addresses underlying philosophical problems regarding the ethical assessment of decision-making under uncertainty and ignorance, and discusses how risks and possible benefits of such technologies should be balanced from an ethical standpoint. It argues on conceptual and ethical grounds for a technology neutral regulation as well as for a regulation that not only checks new technologies but also requires old, inferior ones to be phased out. It demonstrates how difficult ethical issues regarding the extent and ambition of precautionary policies need to be handled by such a regulation, and presents an overarching framework for doing so.
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| 2. |
- Franzén, Carl Johan, 1966, et al.
(författare)
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Multifeed simultaneous saccharification and fermentation enables high gravity submerged fermentation of lignocellulose.
- 2015
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Ingår i: Recent Advances in Fermentation Technology (RAFT 11), Clearwater Beach, Florida, USA, November 8-11, 2015. Oral presentation..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Today, second generation bioethanol production is becoming established in production plants across the world. In addition to its intrinsic value, the process can be viewed as a model process for biotechnological conversion of recalcitrant lignocellulosic raw materials to a range of chemicals and other products. So called High Gravity operation, i.e. fermentation at high solids loadings, represents continued development of the process towards higher product concentrations and productivities, and improved energy and water economy. We have employed a systematic, model-driven approach to the design of feeding schemes of solid substrate, active yeast adapted to the actual substrate, and enzymes to fed-batch simultaneous saccharification and co-fermentation (Multifeed SSCF) of steam-pretreated lignocellulosic materials in stirred tank reactors. With this approach, mixing problems were avoided even at water insoluble solids contents of 22%, leading to ethanol concentrations of 56 g/L within 72 hours of SSCF on wheat straw. Similar fermentation performance was verified in 10 m3 demonstration scale using wheat straw, and in lab scale on birch and spruce, using several yeast strains. The yeast was propagated in the liquid fraction obtained by press filtration of the pretreated slurry. Yet, even with such preadaptation and repeated addition of fresh cells, the viability in the SSCF dropped due to interactions between lignocellulose-derived inhibitors, the produced ethanol and the temperature. Decreasing the temperature from 35 to 30°C when the ethanol concentration reached 40-50 g/L resulted in rapid initial hydrolysis, maintained fermentation capacity, lower residual glucose and xylose and ethanol concentrations above 60 g/L.
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| 3. |
- Mayers, Joshua, 1988, et al.
(författare)
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Integrating Microalgal Production with Industrial Outputs - Reducing Process Inputs and Quantifying the Benefits
- 2016
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Ingår i: Industrial Biotechnology. - : Mary Ann Liebert Inc. - 1550-9087 .- 1931-8421. ; 12:4, s. 219-234
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Tidskriftsartikel (refereegranskat)abstract
- The cultivation and processing of microalgal biomass is resource- and energy-intensive, negatively affecting the sustainability and profitability of producing bulk commodities, limiting this platform to the manufacture of relatively small quantities of high-value compounds. A biorefinery approach where all fractions of the biomass are valorized might improve the case for producing lower-value products. However, these systems are still likely to operate very close to thresholds of profitability and energy balance, with wide-ranging environmental and societal impacts. It thus remains critically important to reduce the use of costly and impactful inputs and energy-intensive processes involved in these scenarios. Integration with industrial infrastructure can provide a number of residual streams that can be readily used during microalgal cultivation and downstream processing. This review critically considers some of the main inputs required for microalgal biorefineries - such as nutrients, water, carbon dioxide, and heat - and appraises the benefits and possibilities for industrial integration on a more quantitative basis. Recent literature and demonstration studies will also be considered to best illustrate these benefits to both producers and industrial operators. Additionally, this review will highlight some inconsistencies in the data used in assessments of microalgal production scenarios, allowing more accurate evaluation of potential future biorefineries.
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| 4. |
- Wang, Ruifei, 1985, et al.
(författare)
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Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production.
- 2016
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:1, s. 88-
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Tidskriftsartikel (refereegranskat)abstract
- High content of water-insoluble solids (WIS) is required for simultaneous saccharification and co-fermentation (SSCF) operations to reach the high ethanol concentrations that meet the techno-economic requirements of industrial-scale production. The fundamental challenges of such processes are related to the high viscosity and inhibitor contents of the medium. Poor mass transfer and inhibition of the yeast lead to decreased ethanol yield, titre and productivity. In the present work, high-solid SSCF of pre-treated wheat straw was carried out by multi-feed SSCF which is a fed-batch process with additions of substrate, enzymes and cells, integrated with yeast propagation and adaptation on the pre-treatment liquor. The combined feeding strategies were systematically compared and optimized using experiments and simulations.
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| 5. |
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| 6. |
- Westman, Johan, 1983, et al.
(författare)
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Current progress in high cell density yeast bioprocesses for bioethanol production
- 2015
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:8, s. 1185-1195
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Forskningsöversikt (refereegranskat)abstract
- High capital costs and low reaction rates are major challenges for establishment of fermentation-based production systems in the bioeconomy. Using high cell density cultures is an efficient way to increase the volumetric productivity of fermentation processes, thereby enabling faster and more robust processes and use of smaller reactors. In this review, we summarize recent progress in the application of high cell density yeast bioprocesses for first and second generation bioethanol production. High biomass concentrations obtained by retention of yeast cells in the reactor enables easier cell reuse, simplified product recovery and higher dilution rates in continuous processes. High local cell density cultures, in the form of encapsulated or strongly flocculating yeast, furthermore obtain increased tolerance to convertible fermentation inhibitors and utilize glucose and other sugars simultaneously, thereby overcoming two additional hurdles for second generation bioethanol production. These effects are caused by local concentration gradients due to diffusion limitations and conversion of inhibitors and sugars by the cells, which lead to low local concentrations of inhibitors and glucose. Quorum sensing may also contribute to the increased stress tolerance. Recent developments indicate that high cell density methodology, with emphasis on high local cell density, offers significant advantages for sustainable second generation bioethanol production.
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| 7. |
- Gontia, Paul, 1984, et al.
(författare)
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Life cycle assessment of bio-based sodium polyacrylate production from pulp mill side streams: Case study of thermo-mechanical and sulfite pulp mills
- 2016
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Ingår i: Journal of Cleaner Production. - : Elsevier BV. - 0959-6526. ; 131, s. 475-484
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Tidskriftsartikel (refereegranskat)abstract
- Sodium polyacrylate (Na-PA) is a super absorbent polymer, which is commonly used in diverse hygiene products. The polymer is currently produced from fossil feedstock and its production consequently leads to adverse environmental impacts. Na-PA production from sugars present in pulp mill side streams can potentially be a successful way to achieve a more sustainable production of this polymer. In order to guide the development of a novel biochemical process for producing Na-PA, a life cycle assessment was done in which Na-PA produced from side streams of thermo-mechanical pulp (TMP) and sulfite pulp mills were compared. Furthermore, a comparison was made with Na-PA produced from fossil resources. The results show that the main determinant of the environmental impact of the bio-based Na-PA production is the free sugar content in the side streams. The lowest environmental impact is achieved by the least diluted side streams. More diluted side streams require larger amounts of energy for concentration, and, if the diluted streams are not concentrated, processes such as hydrolysis and detoxification, and fermentation are the environmental hotspots. Furthermore, the higher the yield of the fermentation process, the lower the environmental impact will be. Lastly, the production of bio-based Na-PA led to a lower global warming potential for some of the considered pulp mill side streams, but all of the other impacts considered were higher, when compared to fossil-based Na-PA production. Therefore, in parallel with efforts to develop a high-yield yeast for the fermentation process, technology developers should focus on low energy concentration processes for the side streams.
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| 8. |
- Karlsson, Emma, 1983, et al.
(författare)
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In silico and in vitro studies of the reduction of unsaturated α,β bonds of trans-2-hexenedioic acid and 6-amino-trans-2-hexenoic acid – Important steps towards biobased production of adipic acid
- 2018
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 13:2
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Tidskriftsartikel (refereegranskat)abstract
- The biobased production of adipic acid, a precursor in the production of nylon, is of great interest in order to replace the current petrochemical production route. Glucose-rich lignocel-lulosic raw materials have high potential to replace the petrochemical raw material. A number of metabolic pathways have been proposed for the microbial conversion of glucose to adipic acid, but achieved yields and titers remain to be improved before industrial applications are feasible. One proposed pathway starts with lysine, an essential metabolite industrially produced from glucose by microorganisms. However, the drawback of this pathway is that several reactions are involved where there is no known efficient enzyme. By changing the order of the enzymatic reactions, we were able to identify an alternative pathway with one unknown enzyme less compared to the original pathway. One of the reactions lacking known enzymes is the reduction of the unsaturated α,β bond of 6-amino-trans-2-hexenoic acid and trans-2hexenedioic acid. To identify the necessary enzymes, we selected N-ethylmaleimide reductase from Escherichia coli and Old Yellow Enzyme 1 from Saccharomyces pastorianus. Despite successful in silico docking studies, where both target substrates could fit in the enzyme pockets, and hydrogen bonds with catalytic residues of both enzymes were predicted, no in vitro activity was observed. We hypothesize that the lack of activity is due to a difference in electron withdrawing potential between the naturally reduced aldehyde and the carboxylate groups of our target substrates. Suggestions for protein engineering to induce the reactions are discussed, as well as the advantages and disadvantages of the two metabolic pathways from lysine. We have highlighted bottlenecks associated with the lysine pathways, and proposed ways of addressing them.
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| 9. |
- van Dijk, Marlous, 1990, et al.
(författare)
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Strain-dependent variance in short-term adaptation effects of two xylose-fermenting strains of Saccharomyces cerevisiae
- 2019
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Ingår i: Bioresource technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 292, s. 121922-
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Tidskriftsartikel (refereegranskat)abstract
- The limited tolerance of Saccharomyces cerevisiae to the inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The aim of this study was to investigate the short-term adaptation effects in yeast strains with different genetic backgrounds. Fed-batch propagation cultures were supplemented with 40% wheat straw hydrolysate during the feed phase to adapt two different pentose-fermenting strains, CR01 and KE6-12. The harvested cells were used to inoculate fermentation media containing 80% or 90% wheat straw hydrolysate. The specific ethanol productivity during fermentation was up to 3.6 times higher for CR01 and 1.6 times higher for KE6-12 following adaptation. The influence of physiological parameters such as viability, storage carbohydrate content, and metabolite yields following short-term adaptation demonstrated that short-term adaptation was strain dependent.
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| 10. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
- 2019
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
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| 11. |
- Tang, Hongting, et al.
(författare)
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Efficient yeast surface-display of novel complex synthetic cellulosomes
- 2018
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. Results: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. Conclusions: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology.
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| 12. |
- Adeboye, Peter, 1982, et al.
(författare)
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A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae
- 2016
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 212:July 2016, s. 11-19
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Tidskriftsartikel (refereegranskat)abstract
- AbstractThe conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1 mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae.
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| 13. |
- Chen, Genqiang, et al.
(författare)
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Bioconversion of waste fiber sludge to bacterial nanocellulose and use for reinforcement of CTMP paper sheets
- 2017
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Ingår i: Polymers. - : MDPI AG. - 2073-4360. ; 9:9
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Tidskriftsartikel (refereegranskat)abstract
- Utilization of bacterial nanocellulose (BNC) for large-scale applications is restricted by low productivity in static cultures and by the high cost of the medium. Fiber sludge, a waste stream from pulp and paper mills, was enzymatically hydrolyzed to sugar, which was used for the production of BNC by the submerged cultivation of Komagataeibacter xylinus. Compared with a synthetic glucose-based medium, the productivity of purified BNC from the fiber sludge hydrolysate using shake-flasks was enhanced from 0.11 to 0.17 g/(L × d), although the average viscometric degree of polymerization (DPv) decreased from 6760 to 6050. The cultivation conditions used in stirred-tank reactors (STRs), including the stirring speed, the airflow, and the pH, were also investigated. Using STRs, the BNC productivity in fiber-sludge medium was increased to 0.32 g/(L × d) and the DPv was increased to 6650. BNC produced from the fiber sludge hydrolysate was used as an additive in papermaking based on the chemithermomechanical pulp (CTMP) of birch. The introduction of BNC resulted in a significant enhancement of the mechanical strength of the paper sheets. With 10% (w/w) BNC in the CTMP/BNC mixture, the tear resistance was enhanced by 140%. SEM images showed that the BNC cross-linked and covered the surface of the CTMP fibers, resulting in enhanced mechanical strength.
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| 14. |
- Kurniawan, Tonny, et al.
(författare)
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Semi-continuous reverse membrane bioreactor in two-stage anaerobic digestion of citruswaste
- 2018
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Ingår i: Materials. - : MDPI AG. - 1996-1944. ; 11:8
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Tidskriftsartikel (refereegranskat)abstract
- The presence of an antimicrobial compound called D-Limonene in citrus waste inhibits methane production from such waste in anaerobic digestion. In this work, a two-stage anaerobic digestion method is developed using reverse membrane bioreactors (rMBRs) containing cells encased in hydrophilic membranes. The purpose of encasement is to retain a high cell concentration inside the bioreactor. The effectiveness of rMBRs in reducing cell washout is evaluated. Three different system configurations, comprising rMBRs, freely suspended cells (FCs), and a combination of both (abbreviated to rMBR-FCs), are incubated at three different organic loading rates (OLRs) each, namely 0.6, 1.2, and 3.6 g COD/(L cycle). Incubation lasts for eight feeding cycles at 55 °C. Methane yield and biogas composition results show that rMBRs perform better than rMBR-FCs and FCs at all three OLRs. Volatile fatty acid profiles and H2production show that the reactors are working properly and no upset occurs. Additionally, a short digestion time of 4 days can be achieved using the rMBR configuration in this study.
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| 15. |
- Millati, Ria, 1972, et al.
(författare)
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Anaerobic digestion of citrus waste using two-stage membrane bioreactor
- 2018
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Ingår i: IOP Conference Series: Materials Science and Engineering. - 1757-8981 .- 1757-899X. ; 316:1
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Konferensbidrag (refereegranskat)abstract
- Anaerobic digestion is a promising method to treat citrus waste. However, the presence of limonene in citrus waste inhibits anaerobic digestion process. Limonene is an antimicrobial compound and could inhibit methane forming bacteria that takes a longer time to recover than the injured acid forming bacteria. Hence, volatile fatty acids will be accumulated and methane production will be decreased. One way to solve this problem is by conducting anaerobic digestion process into two stages. The first step is aimed for hydrolysis, acidogenesis, and acetogenesis reactions and the second stage is aimed for methanogenesis reaction. The separation of the system would further allow each stage in their optimum conditions making the process more stable. In this research, anaerobic digestion was carried out in batch operations using 120 ml-glass bottle bioreactors in 2 stages. The first stage was performed in free-cells bioreactor, whereas the second stage was performed in both bioreactor of free cells and membrane bioreactor. In the first stage, the reactor was set into 'anaerobic' and 'semi-aerobic' conditions to examine the effect of oxygen on facultative anaerobic bacteria in acid production. In the second stage, the protection of membrane towards the cells against limonene was tested. For the first stage, the basal medium was prepared with 1.5 g VS of inoculum and 4.5 g VS of citrus waste. The digestion process was carried out at 55°C for four days. For the second stage, the membrane bioreactor was prepared with 3 g of cells that were encased and sealed in a 3×6 cm 2 polyvinylidene fluoride membrane. The medium contained 40 ml basal medium and 10 ml liquid from the first stage. The bioreactors were incubated at 55°C for 2 days under anaerobic condition. The results from the first stage showed that the maximum total sugar under 'anaerobic' and 'semi-aerobic' conditions was 294.3 g/l and 244.7 g/l, respectively. The corresponding values for total volatile fatty acids were 3.8 g/l and 2.9 g/l, respectively. Methane production of citrus waste taken from the first stage under 'anaerobic' condition in membrane and free-cells bioreactors was 11.2 Nml and 7.2 Nml, respectively. Whereas, methane production of citrus waste taken from the first stage under 'semi-aerobic' condition in membrane and free-cells bioreactors was 8.8 Nml and 5.7 Nml, respectively. It can be seen from the results of the first stage that volatile fatty acids from 'anaerobic' condition was higher than that of 'semi-aerobic' condition. The absence of oxygen provides the optimal condition for growth and metabolism of facultative and obligatorily anaerobic bacteria in the first stage. Furthermore, polyvinylidene fluoride membrane was able to protect the cells from antimicrobial compounds.
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| 16. |
- Olsson, Joakim, 1988, et al.
(författare)
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Ensiling of Saccharina latissima and Laminaria digitata with organic acid additives
- 2016
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Ingår i: Nordic Seaweed Conference, 12-13 October, Grenå.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Seaweeds are a promising source of biomass to provide food, fuels and chemicals in a sustainable future. However, some issues for a biorefinery are that its composition as well as availability varies seasonally and a fresh source cannot be provided all year around. To secure a steady biomass supply the harvest has to be preserved and today drying (especially sun-drying) is commonly used for seaweeds in the carrageenan industry. In colder climates, however, relying on sun-drying could be more problematic and energy intensive drying utilizing hot air is the alternative.An alternative method is ensiling, which is common in agriculture for preserving animal feed. Ensiling is not well researched for seaweeds, though it is receiving more and more attention. It offers a low energy alternative for preservation that relies on acidification by lactic acid bacteria in an anaerobic environment efficiently hampering growth of unwanted microbes. To ensure that the microbial community is beneficial for a good ensiling process additives can be used e.g. inoculum, organic acids, enzymes and sugars. In this study, six different organic acids have been tested at three different concentrations to investigate them as potential additives for ensiling of Saccharina latissima and Laminaria digitata. The content of protein and the monosaccharide profile in the biomass has been analysed before and after 90 days of ensiling to elucidate how the preservation process affects the biomass composition. Such knowledge is important for a biorefinery of seaweeds.
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| 17. |
- Satari, Behzad, et al.
(författare)
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Co-Production of Fungal Biomass Derived Constituents and Ethanol from Citrus Wastes Free Sugars without Auxiliary Nutrients in Airlift Bioreactor
- 2016
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 17:3
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Tidskriftsartikel (refereegranskat)abstract
- The potential of two zygomycetes fungi, Mucor indicus and Rhizopus oryzae, in assimilating citrus waste free sugars (CWFS) and producing fungal chitosan, oil, and protein as well as ethanol was investigated. Extraction of free sugars from citrus waste can reduce its environmental impact by decreasing the possibility of wild microorganisms growth and formation of bad odors, a typical problem facing the citrus industries. A total sugar concentration of 25.1 g/L was obtained by water extraction of citrus waste at room temperature, used for fungal cultivation in shake flasks and airlift bioreactor with no additional nutrients. In shake flasks cultivations, the fungi were only able to assimilate glucose, while fructose remained almost intact. In contrast, the cultivation of M. indicus and R. oryzae in the four-liter airlift bioreactor resulted in the consumption of almost all sugars and production of 250 and 280 g fungal biomass per kg of consumed sugar, respectively. These biomasses correspondingly contained 40% and 51% protein and 9.8% and 4.4% oil. Furthermore, the fungal cell walls, obtained after removing the alkali soluble fraction of the fungi, contained 0.61 and 0.69 g chitin and chitosan per g of cell wall for M. indicus and R. oryzae, respectively. Moreover, the maximum ethanol yield of 36% and 18% was obtained from M. indicus and R. oryzae, respectively. Furthermore, that M. indicus grew as clump mycelia in the airlift bioreactor, while R. oryzae formed spherical suspended pellets, is a promising feature towards industrialization of the process.[on SciFinder (R)]
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| 18. |
- Wang, Guokun, 1988, et al.
(författare)
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RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production in Saccharomyces cerevisiae
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:19, s. 9324-9332
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Tidskriftsartikel (refereegranskat)abstract
- The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.
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| 19. |
- Antonopoulou, Io, 1989-
(författare)
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Development of biocatalytic processes for selective antioxidant production
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.
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| 20. |
- Borja, Gheorghe M., et al.
(författare)
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Metabolic engineering and transcriptomic analysis of Saccharomyces cerevisiae producing p-coumaric acid from xylose
- 2019
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Aromatic amino acids and their derivatives are valuable chemicals and are precursors for different industrially compounds. p-Coumaric acid is the main building block for complex secondary metabolites in commercial demand, such as flavonoids and polyphenols. Industrial scale production of this compound from yeast however remains challenging. Results: Using metabolic engineering and a systems biology approach, we developed a Saccharomyces cerevisiae platform strain able to produce 242 mg/L of p-coumaric acid from xylose. The same strain produced only 5.35 mg/L when cultivated with glucose as carbon source. To characterise this platform strain further, transcriptomic analysis was performed, comparing this strain's growth on xylose and glucose, revealing a strong up-regulation of the glyoxylate pathway alongside increased cell wall biosynthesis and unexpectedly a decrease in aromatic amino acid gene expression when xylose was used as carbon source. Conclusions: The resulting S. cerevisiae strain represents a promising platform host for future production of p-coumaric using xylose as a carbon source.
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| 21. |
- Huang, Mingtao, 1984, et al.
(författare)
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Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:47, s. E11025-E11032
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Tidskriftsartikel (refereegranskat)abstract
- Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.
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| 22. |
- Jers, C., et al.
(författare)
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Production of 3-hydroxypropanoic acid from glycerol by metabolically engineered bacteria
- 2019
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 7:MAY
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Forskningsöversikt (refereegranskat)abstract
- 3-hydroxypropanoic acid (3-HP) is a valuable platform chemical with a high demand in the global market. 3-HP can be produced from various renewable resources. It is used as a precursor in industrial production of a number of chemicals, such as acrylic acid and its many derivatives. In its polymerized form, 3-HP can be used in bioplastic production. Several microbes naturally possess the biosynthetic pathways for production of 3-HP, and a number of these pathways have been introduced in some widely used cell factories, such as Escherichia coli and Saccharomyces cerevisiae. Latest advances in the field of metabolic engineering and synthetic biology have led to more efficient methods for bio-production of 3-HP. These include new approaches for introducing heterologous pathways, precise control of gene expression, rational enzyme engineering, redirecting the carbon flux based on in silico predictions using genome scale metabolic models, as well as optimizing fermentation conditions. Despite the fact that the production of 3-HP has been extensively explored in established industrially relevant cell factories, the current production processes have not yet reached the levels required for industrial exploitation. In this review, we explore the state of the art in 3-HP bio-production, comparing the yields and titers achieved in different microbial cell factories and we discuss possible methodologies that could make the final step toward industrially relevant cell factories.
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| 23. |
- Lorantfy, Bettina, 1986, et al.
(författare)
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Characterization of the respiratory physiology of Lactococcus lactis for starter culture production with improved acidification capacity
- 2015
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Ingår i: Microbial Stress: From Molecules to Systems.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Commercial freeze-dried starter cultures for cheese making are produced mainly via anaerobic batch processes. A recent discovery has shown that some lactic acid bacteria (LAB) are able to sustain respiration under aerobic conditions when hemin is added to the growth medium, since it completes the electron transport chain for respiration, which is otherwise defective [1]. Respiration is energetically beneficial: compared to fermentation, under respiratory conditions the biomass yield is higher and a different by-product pattern is observed. However, it is also important to consider whether the different metabolism can affect the performance of the starter culture. Thus, this project investigates LAB respiratory physiology, aiming to clarify the molecular reasons behind the milk acidification capacity of the respiratory culture. Our bioreactor results demonstrate that with hemin addition, cells switch from the fermentation to respiration only in the late exponential phase of growth. Although presence of oxygen is an additional stress for LAB, in the presence of hemin under aerobic conditions cells have surprisingly better fermentation behaviour, i.e. higher lactate yield before the respiratory switch. Therefore, we hypothesize that with improved fermentation a certain energy threshold is achieved for the respiratory switch. This energy requirement might be related to the intake of the hemin, however this aspect needs further investigation, as hemin transport into the cells has not been characterized yet.Reference: [1] Lechardeur D et al Curr Opin Biotechnol 2011,22(2):143
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| 24. |
- Lorantfy, Bettina, 1986, et al.
(författare)
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What induces respiration in lactic acid bacteria? Characterization of respiratory metabolism of Lactococcus lactis in bioreactors for production of starter cultures with improved acidification capacity
- 2015
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Ingår i: RAFT 11 Recent Advances in Fermentation Technology.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Commercial freeze-dried lactic acid bacteria starter cultures for cheese making are produced mainly via anaerobic batch fermentations. Recently, it has been shown that some Lactococcus lactis species are able to sustain respiration under aerobic conditions when hemin is added to the growth medium, since it completes the electron transport chain for respiration, which is otherwise defective. Respiration is energetically beneficial and under respiratory conditions, higher biomass yield is obtained together with a changed by-product pattern, compared to fermentation [1]. So far it has not been studied how the different culture conditions and thereby different metabolism affect the starter culture performance. In this project, we investigate respiratory culture conditions, and the effect on the milk acidification capacity of the culture. Since Lactococcus lactis is a fastidious microorganism, a rich chemically defined medium was developed to support the nutrient requirements, and was applied for bioreactor cultivations with quantitative approaches. The product profile and on-line gas analysis revealed that with hemin addition at the start of the process, cells switch to respiratory metabolism only in the second phase of growth, after an initial mixed-acid fermentative phase. To characterize the observed respiratory switch, a multivariate study was performed: a set of bioreactor batch experiments were carried out with different initial sugar concentrations under anaerobic, aerobic, and respiratory conditions. The results indicate that hemin addition together with some yet not defined threshold must be met in order to induce the respiratory metabolic state of the culture.[1] Lechardeur D et al Curr Opin Biotechnol 2011,22(2):143‐149
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| 25. |
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