| 1. |
- Björlenius, Berndt, 1963-
(författare)
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Pharmaceuticals – improved removal from municipal wastewater and their occurrence in the Baltic Sea
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pharmaceutical residues are found in the environment due to extensive use in human and veterinary medicine. The active pharmaceutical ingredients (APIs) have a potential impact in non-target organisms. Municipal wastewater treatment plants (WWTPs) are not designed to remove APIs.In this thesis, two related matters are addressed 1) evaluation of advanced treatment to remove APIs from municipal wastewater and 2) the prevalence and degradation of APIs in the Baltic Sea.A stationary pilot plant with nanofiltration (NF) and a mobile pilot plant with activated carbon and ozonation were designed to study the removal of APIs at four WWTPs. By NF, removal reached 90%, but the retentate needed further treatment. A predictive model of the rejection of APIs by NF was developed based on the variables: polarizability, globularity, ratio hydrophobic to polar water accessible surface and charge. The pilot plants with granular and powdered activated carbon (GAC) and (PAC) removed more than 95% of the APIs. Screening of activated carbon products was essential, because of a broad variation in adsorption capacity. Recirculation of PAC or longer contact time, increased the removal of APIs. Ozonation with 5-7 g/m3 ozone resulted in 87-95% removal of APIs. Elevated activity and transcription of biomarkers indicated presence of xenobiotics in regular effluent. Chemical analysis of APIs, together with analysis of biomarkers, were valuable and showed that GAC-filtration and ozonation can be implemented to remove APIs in WWTPs, with decreased biomarker responses.Sampling of the Baltic Sea showed presence of APIs in 41 out of 43 locations. A developed grey box model predicted concentration and half-life of carbamazepine in the Baltic Sea to be 1.8 ng/L and 1300 d respectively.In conclusion, APIs were removed to 95% by GAC or PAC treatment. The additional treatment resulted in lower biomarker responses than today and some APIs were shown to be widespread in the aquatic environment.
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| 2. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
- 2015
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Ingår i: Frontiers in Microbiology. - : Frontiers Research Foundation. - 1664-302X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.
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| 3. |
- Guevara-Martínez, Mónica, 1989-
(författare)
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Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Microbial bio-based processes have emerged as an alternative to replace fossil-based processes for the production of fuels and chemicals. (R)-3-hydroxybutyrate (3HB) is a medium-value chemical that has gained special attention as a precursor of antibiotics and vitamins, as a monomer for the synthesis of tailor-made polyesters and as a nutritional source for eukaryotic cells. By integrating strain and bioprocess-design strategies the work of this thesis has aimed to improve microbial 3HB production by the well-studied platform organism Escherichia coli (strain AF1000) expressing a thiolase and a reductase from Halomonas boliviensis.Uncoupling growth and product formation by NH4+- or PO43-- limited fed-batch cultivations allowed for 3HB titers of 4.1 and 6.8 g L-1 (Paper I). Increasing the NADPH supply by overexpression of glucose-6-phosphate dehydrogenase (zwf) resulted in 1.7 times higher 3HB yield compared to not overexpressing zwf in NH4+ depleted conditions (Paper II). To increase 3HB production in high-cell density cultures, strain BL21 was selected as a low acetate-forming, 3HB-producing platform. BL21 grown in NH4+ limited fed-batch cultivations resulted in 2.3 times higher 3HB titer (16.3 g L-1) compared to strain AF1000 (Paper III). Overexpression of the native E. coli thioesterase “yciA”, identified as the largest contributor in 3HB-CoA hydrolysis, resulted in 2.6 times higher 3HB yield compared to AF1000 not overexpressing yciA. Overexpressing zwf and yciA in NH4+ depleted fed-batch experiments resulted in 2 times higher total 3HB yield (0.210 g g-1) compared to AF1000 only overexpressing zwf (Paper IV). Additionally, using 3HB as a model product, the bacterial artificial chromosome was presented as a simple platform for performing pathway design and optimization in E. coli (Paper V). While directly relevant for 3HB production, these findings also contribute to the knowledge on how to improve the production of a chemical for the development of robust and scalable processes.
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| 4. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; , s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.
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| 5. |
- Gustavsson, Martin, 1984-, et al.
(författare)
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Biocatalysis on the surface of Escherichia coli : melanin pigmentation of the cell exterior
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.
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| 6. |
- Gustavsson, Martin, et al.
(författare)
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Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli
- 2015
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.
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| 7. |
- Hörnström, David, et al.
(författare)
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Molecular optimization of autotransporter-based tyrosinase surface display
- 2019
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : ELSEVIER SCIENCE BV. - 0005-2736 .- 1879-2642. ; 1862:2, s. 486-494
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Tidskriftsartikel (refereegranskat)abstract
- Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia colt surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.
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| 8. |
- Jarmander, Johan, et al.
(författare)
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Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 14:1, s. 51-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40 % of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose.ResultsThe Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (< 2 mg g-1 h-1) compared to carbon excess (25 mg g-1 h-1), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g-1)during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g-1 h-1) and to increase the yield of 3HB/cell dry weight to 1.38 g g-1. Nitrogen-limited fed-batch process design lead to further increased specific productivity (38 mg g-1 h-1) but also to additional cell growth (Y3HB/CDW = 0.16 g g-1). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid.ConclusionsIt was demonstrated that by using the strain E. coli PPA652ara it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB by E. coli reported here from glucose, xylose and arabinose is further comparable to the current state of the art for production of 3HB from glucose sources.
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| 9. |
- Jarmander, Johan
(författare)
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Strategies for improved Escherichia coli bioprocessing performance
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli has a proven track record for successful production of anything from small molecules like organic acids to large therapeutic proteins, and has thus important applications in both R&D and commercial production. The versatility of this organism in combination with the accumulated knowledge of its genome, metabolism and physiology, has allowed for development of specialty strains capable of performing very specific tasks, opening up opportunities within new areas. The work of this thesis has been devoted to alter membrane transport proteins and the regulation of these, in order for E. coli to find further application within two such important areas.The first area was vaccine development, where it was investigated if E. coli could be a natural vehicle for live vaccine production. The hypothesis was that the introduction and manipulation of a protein surface translocation system from pathogenic E. coli would result in stable expression levels of Salmonella subunit antigens on the surface of laboratory E. coli. While different antigen combinations were successfully expressed on the surface of E. coli, larger proteins were affected by proteolysis, which manipulation of cultivation conditions could reduce, but not eliminate completely. The surface expressed antigens were further capable of inducing proinflammatory responses in epithelial cells.The second area was biorefining. By altering the regulation of sugar assimilation, it was hypothesized that simultaneous uptake of the sugars present in lignocellulose hydrolyzates could be achieved, thereby improving the yield and productivity of important bio-based chemicals. The dual-layered catabolite repression was identified and successfully removed in the engineered E. coli, and the compound (R)-3-hydroxybutyric acid was produced from simultaneous assimilation of glucose, xylose and arabinose.
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| 10. |
- Kårelid, Victor, 1982-, et al.
(författare)
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Effects of recirculation in a three-tank pilot-scale system for pharmaceutical removal with powdered activated carbon
- 2017
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Ingår i: Journal of Environmental Management. - : Elsevier BV. - 0301-4797 .- 1095-8630. ; 193:May, s. 163-Environmental Impact Optimization of Reinforced Concrete Slab Frame Bridges
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Forskningsöversikt (refereegranskat)abstract
- The removal of pharmaceutically active compounds by powdered activated carbon (PAC) in municipal wastewater is a promising solution to the problem of polluted recipient waters. Today, an efficient design strategy is however lacking with regard to high-level overall, and specific, substance removal in the large scale. The performance of PAC-based removal of pharmaceuticals was studied in pilot-scale with respect to the critical parameters; contact time and PAC dose using one PAC product selected by screening in bench-scale. The goal was a minimum of 95% removal of the pharmaceuticals present in the evaluated municipal wastewater. A set of 21 pharmaceuticals was selected from an initial 100 due to their high occurrence in the effluent water of two selected wastewater treatment plants (WWTPs) in Sweden, whereof candidates discussed for future EU regulation directives were included. By using recirculation of PAC over a treatment system using three sequential contact tanks, a combination of the benefits of powdered and granular carbon performance was achieved. The treatment system was designed so that recirculation could be introduced to any of the three tanks to investigate the effect of recirculation on the adsorption performance. This was compared to use of the setup, but without recirculation. A higher degree of pharmaceutical removal was achieved in all recirculation setups, both overall and with respect to specific substances, as compared to without recirculation. Recirculation was tested with nominal contact times of 30, 60 and 120 min and the goal of 95% removal could be achieved already at the shortest contact times at a PAC dose of 10–15 mg/L. In particular, the overall removal could be increased even to 97% and 99%, at 60 and 120 min, respectively, when the recirculation point was the first tank. Recirculation of PAC to either the first or the second contact tank proved to be comparable, while a slightly lower performance was observed with recirculation to the third tank. With regards to individual substances, clarithromycin and diclofenac were ubiquitously removed according to the set goal and in contrast, a few substances (fluconazole, irbesartan, memantine and venlafaxine) required specific settings to reach an acceptable removal.
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| 11. |
- Kårelid, Victor, 1989-, et al.
(författare)
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Pilot-scale removal of pharmaceuticals in municipal wastewater : Comparison of granular and powdered activated carbon treatment at three wastewater treatment plants
- 2017
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Ingår i: Journal of Environmental Management. - : Academic Press. - 0301-4797 .- 1095-8630. ; 193:-1, s. 491-502
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Tidskriftsartikel (refereegranskat)abstract
- Adsorption with activated carbon is widely suggested as an option for the removal of organic micropollutants including pharmaceutically active compounds (PhACs) in wastewater. In this study adsorption with granular activated carbon (GAC) and powdered activated carbon (PAC) was analyzed and compared in parallel operation at three Swedish wastewater treatment plants with the goal to achieve a 95% PhAC removal. Initially, mapping of the prevalence of over 100 substances was performed at each plant and due to low concentrations a final 22 were selected for further evaluation. These include carbamazepine, clarithromycin and diclofenac, which currently are discussed for regulation internationally. A number of commercially available activated carbon products were initially screened using effluent wastewater. Of these, a reduced set was selected based on adsorption characteristics and cost. Experiments designed with the selected carbons in pilot-scale showed that most products could indeed remove PhACs to the target level, both on total and individual basis. In a setup using internal recirculation the PAC system achieved a 95% removal applying a fresh dose of 15–20 mg/L, while carbon usage rates for the GAC application were much broader and ranged from <28 to 230 mg/L depending on the carbon product. The performance of the PAC products generally gave better results for individual PhACs in regards to carbon availability. All carbon products showed a specific adsorption for a specific PhAC meaning that knowledge of the target pollutants must be acquired before successful design of a treatment system. In spite of different configurations and operating conditions of the different wastewater treatment plants no considerable differences regarding pharmaceutical removal were observed.
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| 12. |
- Kårelid, Victor, 1989-
(författare)
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Towards application of activated carbon treatment for pharmaceutical removal in municipal wastewater
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many pharmaceuticals are found in municipal wastewater effluents due to their persistence in the human body as well as in conventional wastewater treatment processes. This discharge to the environment can lead to adverse effects in aquatic species, such as feminization of male fish. During the past decade, these findings have spawned investigations and research into suitable treatment technologies that could severely limit the discharge. Adsorption onto activated carbon has been identified as one of the two main technologies for implementation of (future) full-scale treatment.Recent research has put a closer focus on adsorption with powdered activated carbon (PAC) than on granular activated carbon (GAC). Studies where both methods are compared in parallel operation are thus still scarce and such evaluation in pilot-scale was therefore a primary objective of this thesis. Furthermore, recirculation of PAC can be used to optimize the treatment regarding the carbon consumption. Such a setup was evaluated as a separate treatment stage to comply with Swedish wastewater convention. Additionally, variation of a set of process parameters was evaluated.During successive operation at three different wastewater treatment plants an overall pharmaceutical removal of 95% could consistently be achieved with both methods. Furthermore, treatment with GAC was sensitive to a degraded effluent quality, which severely reduced the hydraulic capacity. Both treatment methods showed efficient removal of previously highlighted substances, such as carbamazepine and diclofenac, however in general a lower adsorption capacity was observed for GAC. By varying the input of process parameters, such as the continuously added dose or the contact time, during PAC treatment, a responsive change of the pharmaceutical removal could be achieved. The work in this thesis contributes some valuable field experience towards wider application of these treatment technologies in full-scale.
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| 13. |
- Lindroos, Magnus, et al.
(författare)
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Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
- 2019
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Ingår i: Journal of Hazardous Materials. - : ELSEVIER SCIENCE BV. - 0304-3894 .- 1873-3336. ; 365, s. 74-80
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Tidskriftsartikel (refereegranskat)abstract
- Environmental release and accumulation of pharmaceuticals and personal care products is a global concern in view of increased awareness of ecotoxicological effects. Adsorbent properties make the biopolymer melanin an interesting alternative to remove micropollutants from water. Recently, tyrosinase-surface-displaying Escherichia coli was shown to be an interesting self-replicating production system for melanin-covered cells for batch-wise absorption of the model pharmaceutical chloroquine. This work explores the suitability of these melanin-covered E. coli for the continuous removal of pharmaceuticals from wastewater. A continuous-flow membrane bioreactor containing melanized E. coli cells was used for adsorption of chloroquine from the influent until saturation and subsequent regeneration. At a low loading of cells (10 g/L) and high influent concentration of chloroquine (0.1 mM), chloroquine adsorbed until saturation after 26 +/- 2 treated reactor volumes (39 +/- 3 L). The average effluent concentration during the first 20 h was 0.0018 mM, corresponding to 98.2% removal. Up to 140 +/- 6 mg chloroquine bound per gram of cells following mixed homo- and heterogeneous adsorption kinetics. In situ low pH regeneration released all chloroquine without apparent capacity loss over three consecutive cycles. This shows the potential of melanized cells for treatment of conventional wastewater or highly concentrated upstream sources such as hospitals or manufacturing sites.
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| 14. |
- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 103:14, s. 5627-5636
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Tidskriftsartikel (refereegranskat)abstract
- Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.
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| 15. |
- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
- 2016
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Ingår i: Microbial Cell Factories. - : Springer. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.
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| 16. |
- Perez-Zabaleta, Mariel, 1987-
(författare)
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Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Metabolic engineering and process engineering are two powerful disciplines to design and improve microbial processes for sustainable production of an extensive number of compounds ranging from chemicals to pharmaceuticals. The aim of this thesis was to synergistically combine these two disciplines to improve the production of a model chemical called (R)-3-hydroxybutyrate (3HB), which is a medium-value product with a stereocenter and two functional groups. These features make 3HB an interesting building block, especially for the pharmaceutical industry. Recombinant production of 3HB was achieved by expression of two enzymes from Halomonas boliviensis in the model microorganism Escherichia coli, which is a microbial cell factory with proven track record and abundant knowledge on its genome, metabolism and physiology.Investigations on cultivation strategies demonstrated that nitrogen-depleted conditions had the biggest impact on 3HB yields, while nitrogen-limited cultivations predominantly increased 3HB titers and volumetric productivities. To further increase 3HB production, metabolic engineering strategies were investigated to decrease byproduct formation, enhance NADPH availability and improve the overall 3HB-pathway activity. Overexpression of glucose-6-phosphate dehydrogenase (zwf) increased cofactor availability and together with the overexpression of acyl-CoA thioesterase YciA resulted in a 2.7-fold increase of the final 3HB concentration, 52% of the theoretical product yield and a high specific productivity (0.27 g g-1 h-1). In a parallel strategy, metabolic engineering and process design resulted in an E. coli BL21 strain with the hitherto highest reported volumetric 3HB productivity (1.52 g L-1 h-1) and concentration (16.3 g L-1) using recombinant production. The concepts developed in this thesis can be applied to industrial 3HB production processes, but also advance the knowledge base to benefit design and expansion of the product range of biorefineries.
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