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Perez-Zabaleta, M., Guevara-Martínez, M., Gustavsson, M., Quillaguamán, J., Larsson, G. & van Maris, A. J. A. (2019). Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation. Applied Microbiology and Biotechnology, 103(14), 5627-5636
Open this publication in new window or tab >>Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
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2019 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 103, no 14, p. 5627-5636Article in journal (Refereed) Accepted
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer, 2019
Keywords
Escherichia coli, (R)-3-hydroxybutyrate, acetate, nitrogen limitation, fed batch, BL21.
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251046 (URN)10.1007/s00253-019-09876-y (DOI)000473129900012 ()2-s2.0-85066078742 (Scopus ID)
Funder
Sida - Swedish International Development Cooperation Agency, 70828
Note

QC 20190508

Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-08-15Bibliographically approved
Lindroos, M., Hörnström, D., Larsson, G., Gustavsson, M. & van Maris, A. J. A. (2019). Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor. Journal of Hazardous Materials, 365, 74-80
Open this publication in new window or tab >>Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
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2019 (English)In: Journal of Hazardous Materials, ISSN 0304-3894, E-ISSN 1873-3336, Vol. 365, p. 74-80Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Wastewater treatment, Pharmaceuticals, Membrane bioreactor, Adsorption, Surface expression
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-244080 (URN)10.1016/j.jhazmat.2018.10.081 (DOI)000456761000009 ()30412809 (PubMedID)2-s2.0-85055974597 (Scopus ID)
Note

QC 20190219

Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-02-19Bibliographically approved
Hörnström, D., Larsson, G., van Maris, A. J. A. & Gustavsson, M. (2019). Molecular optimization of autotransporter-based tyrosinase surface display. Biochimica et Biophysica Acta - Biomembranes, 1862(2), 486-494
Open this publication in new window or tab >>Molecular optimization of autotransporter-based tyrosinase surface display
2019 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1862, no 2, p. 486-494Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Autotransport, Whole-cell biocatalysis, Tyrosinase, Protein engineering, Escherichia coli
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-244107 (URN)10.1016/j.bbamem.2018.11.012 (DOI)000456764100014 ()30521785 (PubMedID)2-s2.0-85058059281 (Scopus ID)
Funder
Swedish Research Council, VR-621-2014-5293
Note

QC 20190219

Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-02-19Bibliographically approved
Guevara-Martínez, M., Perez-Zabaleta, M., Gustavsson, M., Quillaguamán, J., Larsson, G. & van Maris, A. J. A. (2019). The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli. Applied Microbiology and Biotechnology, 1-12
Open this publication in new window or tab >>The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
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2019 (English)In: Applied Microbiology and Biotechnology, p. 1-12Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer, 2019
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-249360 (URN)10.1007/s00253-019-09707-0 (DOI)000464737100008 ()2-s2.0-85062726311 (Scopus ID)
Note

QC 20190509

Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-05-14Bibliographically approved
Perez-Zabaleta, M., Sjöberg, G., Guevara-Martínez, M., Jarmander, J., Gustavsson, M., Quillaguamán, J. & Larsson, G. (2016). Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply. Microbial Cell Factories, 15(1), Article ID 91.
Open this publication in new window or tab >>Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
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2016 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, no 1, article id 91Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer, 2016
Keywords
Escherichia coli, Halomonas boliviensis, (R)-3-hydroxybutyrate, Acetoacetyl-CoA reductase, NADPH, zwf overexpression, Glutamate, Nitrogen limitation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-189084 (URN)10.1186/s12934-016-0490-y (DOI)000377167900001 ()27245326 (PubMedID)2-s2.0-84971577878 (Scopus ID)
Note

QC 20160808

Available from: 2016-08-08 Created: 2016-06-27 Last updated: 2019-06-14Bibliographically approved
Gustavsson, M. & Lee, S. Y. (2016). Prospects of microbial cell factories developed through systems metabolic engineering. Microbial Biotechnology, 9(5), 610-617
Open this publication in new window or tab >>Prospects of microbial cell factories developed through systems metabolic engineering
2016 (English)In: Microbial Biotechnology, ISSN 1751-7907, E-ISSN 1751-7915, Vol. 9, no 5, p. 610-617Article in journal (Refereed) Published
Abstract [en]

While academic-level studies on metabolic engineering of microorganisms for production of chemicals and fuels are ever growing, a significantly lower number of such production processes have reached commercial-scale. In this work, we review the challenges associated with moving from laboratory-scale demonstration of microbial chemical or fuel production to actual commercialization, focusing on key requirements on the production organism that need to be considered during the metabolic engineering process. Metabolic engineering strategies should take into account techno-economic factors such as the choice of feedstock, the product yield, productivity and titre, and the cost effectiveness of midstream and downstream processes. Also, it is important to develop an industrial strain through metabolic engineering for pathway construction and flux optimization together with increasing tolerance to products and inhibitors present in the feedstock, and ensuring genetic stability and strain robustness under actual fermentation conditions.

Place, publisher, year, edition, pages
John Wiley & Sons, 2016
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-193009 (URN)10.1111/1751-7915.12385 (DOI)000386999300012 ()2-s2.0-84983317316 (Scopus ID)
Note

QC 20161010

Available from: 2016-09-26 Created: 2016-09-26 Last updated: 2017-11-21Bibliographically approved
Cheon, S., Kim, H. M., Gustavsson, M. & Lee, S. Y. (2016). Recent trends in metabolic engineering of microorganisms for the production of advanced biofuels. Current opinion in chemical biology, 35, 10-21
Open this publication in new window or tab >>Recent trends in metabolic engineering of microorganisms for the production of advanced biofuels
2016 (English)In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 35, p. 10-21Article in journal (Refereed) Published
Abstract [en]

As climate change has become one of the major global risks, our heavy dependence on petroleum-derived fuels has received much public attention. To solve such problems, production of sustainable fuels has been intensively studied over the past years. Thanks to recent advances in synthetic biology and metabolic engineering technologies, bio-based platforms for advanced biofuels production have been developed using various microorganisms. The strategies for production of advanced biofuels have converged upon four major metabolic routes: the 2-ketoacid pathway, the fatty acid synthesis (FAS) pathway, the isoprenoid pathway, and the reverse β-oxidation pathway. Additionally, the polyketide synthesis pathway has recently been attracting interest as a promising alternative biofuel production route. In this article, recent trends in advanced biofuels production are reviewed by categorizing them into three types of advanced biofuels: alcohols, biodiesel and jet fuel, and gasoline. Focus is given on the strategies of employing synthetic biology and metabolic engineering for the development of microbial strains producing advanced fuels. Finally, the prospects for future advances needed to achieve much more efficient bio-based production of advanced biofuels are discussed, focusing on designing advanced biofuel production pathways coupled with screening, modifying, and creating novel enzymes.

Place, publisher, year, edition, pages
Elsevier, 2016
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-193008 (URN)10.1016/j.cbpa.2016.08.003 (DOI)2-s2.0-84982306147 (Scopus ID)
Note

QC 20160926

Available from: 2016-09-26 Created: 2016-09-26 Last updated: 2017-11-21Bibliographically approved
Chung, H., Yang, J. E., Ha, J. Y., Chae, T. U., Shin, J. H., Gustavsson, M. & Lee, S. Y. (2015). Bio-based production of monomers and polymers by metabolically engineered microorganisms. Current Opinion in Biotechnology, 36, 73-84
Open this publication in new window or tab >>Bio-based production of monomers and polymers by metabolically engineered microorganisms
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2015 (English)In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 36, p. 73-84Article, review/survey (Refereed) Published
Abstract [en]

Recent metabolic engineering strategies for bio-based production of monomers and polymers are reviewed. In the case of monomers, we describe strategies for producing polyamide precursors, namely diamines (putrescine, cadaverine, 1,6-diaminohexane), dicarboxylic acids (succinic, glutaric, adipic, and sebacic acids), and ω-amino acids (γ-aminobutyric, 5-aminovaleric, and 6-aminocaproic acids). Also, strategies for producing diols (monoethylene glycol, 1,3-propanediol, and 1,4-butanediol) and hydroxy acids (3-hydroxypropionic and 4-hydroxybutyric acids) used for polyesters are reviewed. Furthermore, we review strategies for producing aromatic monomers, including styrene, p-hydroxystyrene, p-hydroxybenzoic acid, and phenol, and propose pathways to aromatic polyurethane precursors. Finally, in vivo production of polyhydroxyalkanoates and recombinant structural proteins having interesting applications are showcased.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
RECOMBINANT ESCHERICHIA-COLI, GAMMA-AMINOBUTYRATE GABA, SUCCINIC ACID PRODUCTION, MUSSEL ADHESIVE PROTEIN, CORYNEBACTERIUM-GLUTAMICUM, MICROBIAL-PRODUCTION, MANNHEIMIA-SUCCINICIPRODUCENS, PSEUDOMONAS-PUTIDA, CARBON DIAMINE, ADIPIC ACID
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-172801 (URN)10.1016/j.copbio.2015.07.003 (DOI)000367116700010 ()2-s2.0-84940063735 (Scopus ID)
Note

QC 20150831. QC 20160121

Available from: 2015-08-29 Created: 2015-08-29 Last updated: 2017-12-04Bibliographically approved
Gustavsson, M., Do, T.-H. -., Lüthje, P., Tran, N. T., Brauner, A., Samuelson, P., . . . Larsson, G. (2015). Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli. Microbial Cell Factories, 14(1), Article ID 47.
Open this publication in new window or tab >>Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli
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2015 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, article id 47Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
AIDA-I, Autotransport, Escherichia coli, Live vaccines, Salmonella enterica, Surface expression, Bacteria (microorganisms), Salmonella, Salmonella enteritidis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-167747 (URN)10.1186/s12934-015-0227-3 (DOI)000353617100001 ()2-s2.0-84928551436 (Scopus ID)
Funder
Sida - Swedish International Development Cooperation Agency
Note

QC 20150601

Available from: 2015-06-01 Created: 2015-05-22 Last updated: 2017-12-04Bibliographically approved
Jarmander, J., Janoschek, L., Lundh, S., Larsson, G. & Gustavsson, M. (2014). Process optimization for increased yield of surface-expressed protein in Escherichia coli. Bioprocess and biosystems engineering (Print), 37(8), 1685-1693
Open this publication in new window or tab >>Process optimization for increased yield of surface-expressed protein in Escherichia coli
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2014 (English)In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 37, no 8, p. 1685-1693Article in journal (Refereed) Published
Abstract [en]

The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.

Keywords
Surface expression, Autotransport, Process optimization, Proteolysis, Live vaccines
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-149969 (URN)10.1007/s00449-014-1141-5 (DOI)000339962400023 ()2-s2.0-84925884851 (Scopus ID)
Note

QC 20140904

Available from: 2014-09-04 Created: 2014-08-29 Last updated: 2017-12-05Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-3314-6060

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