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Patel, A., Antonopoulou, I., Enman, J., Rova, U., Christakopoulos, P. & Matsakas, L. (2019). Lipids detection and quantification in oleaginous microorganisms: an overview of the current state of the art. BMC Chemical Engineering, 1, Article ID 13.
Open this publication in new window or tab >>Lipids detection and quantification in oleaginous microorganisms: an overview of the current state of the art
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2019 (English)In: BMC Chemical Engineering, ISSN 2524-4175, Vol. 1, article id 13Article in journal (Refereed) Published
Abstract [en]

Oleaginous microorganisms are among the most promising feedstocks for the production of lipids for biofuels and oleochemicals. Lipids are synthesized in intracellular compartments in the form of lipid droplets. Therefore, their qualitative and quantitative analysis requires an initial pretreatment step that allows their extraction. Lipid extraction techniques vary with the type of microorganism but, in general, the presence of an outer membrane or cell wall limits their recovery. This review discusses the various types of oleaginous microorganisms, their lipid accumulating capabilities, lipid extraction techniques, and the pretreatment of cellular biomass for enhanced lipid recovery. Conventional methods for lipid quantification include gravimetric and chromatographic approaches; whereas non-conventional methods are based on infrared, Raman, nuclear magnetic resonance, and fluorescence spectroscopic analysis. Recent advances in these methods, their limitations, and fields of application are discussed, with the aim of providing a guide for selecting the best method or combination of methods for lipid quantification.

Place, publisher, year, edition, pages
BioMed Central, 2019
Keywords
Oleaginous microorganisms, Microbial lipids, Lipid extraction, Lipid quantification, Conventional methods, High-throughput analysis
National Category
Chemical Process Engineering Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-75376 (URN)10.1186/s42480-019-0013-9 (DOI)
Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2019-08-14Bibliographically approved
Faisal, A., Holmlund, M., Ginesy, M., Holmgren, A., Enman, J., Hedlund, J. & Grahn, M. (2019). Recovery of l-Arginine from Model Solutions and Fermentation Broth Using Zeolite-Y Adsorbent. ACS Sustainable Chemistry & Engineering, 7(9), 8900-8907
Open this publication in new window or tab >>Recovery of l-Arginine from Model Solutions and Fermentation Broth Using Zeolite-Y Adsorbent
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2019 (English)In: ACS Sustainable Chemistry & Engineering, ISSN 2168-0485, Vol. 7, no 9, p. 8900-8907Article in journal (Refereed) Published
Abstract [en]

Arginine was produced via fermentation of sugars using the engineered microorganism Escherichia coli. Zeolite-Y adsorbents in the form of powder and extrudates were used to recover arginine from both a real fermentation broth and aqueous model solutions. An adsorption isotherm was determined using model solutions and zeolite-Y powder. The saturation loading was determined to be 0.2 g/g using the Sips model. Arginine adsorbed from a real fermentation broth using either zeolite-Y powder or extrudates both showed a maximum loading of 0.15 g/g at pH 11. This adsorbed loading is very close to the corresponding value obtained from the model solution showing that under the experimental conditions the presence of additional components in the broth did not have a significant effect on the adsorption of arginine. Furthermore, a breakthrough curve was determined for extrudates using a 1 wt % arginine model solution. The selectivity for arginine over ammonia and alanine from the real fermentation broth at pH 11 was 1.9 and 8.3, respectively, for powder, and 1.0, and 4.1, respectively, for extrudates. To the best of our knowledge, this is the first time recovery of arginine from real fermentation broths using any type of adsorbent has been reported.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019
Keywords
Arginine, FAU, Fermentation broth, Escherichia coli, Adsorption, Selectivity, Breakthrough
National Category
Chemical Process Engineering Bioprocess Technology
Research subject
Chemical Technology; Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73954 (URN)10.1021/acssuschemeng.9b00918 (DOI)000467351200095 ()2-s2.0-85065476739 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-05-27 (oliekm)

Available from: 2019-05-15 Created: 2019-05-15 Last updated: 2019-08-19Bibliographically approved
Ginesy, M., Enman, J., Rusanova-Naydenova, D. & Rova, U. (2019). Simultaneous Quantification of L-Arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach. Molecules, 24(4), Article ID 802.
Open this publication in new window or tab >>Simultaneous Quantification of L-Arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach
2019 (English)In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 24, no 4, article id 802Article in journal (Refereed) Published
Abstract [en]

Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
Charged aerosol detector, Fermentation, Integrated pulsed amperometric detection, Ion chromatography, L-arginine, Method validation, Monosaccharides, Reversed-phase liquid chromatography
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73081 (URN)10.3390/molecules24040802 (DOI)000460805900148 ()30813364 (PubMedID)2-s2.0-85062166570 (Scopus ID)
Funder
Bio4EnergyThe Kempe Foundations
Note

Validerad;2019;Nivå 2;2019-08-20 (johcin)

Available from: 2019-03-01 Created: 2019-03-01 Last updated: 2019-08-20Bibliographically approved
Zerva, A., Antonopoulou, I., Enman, J., Iancu, L., Rova, U. & Christakopoulos, P. (2018). Cross-Linked Enzyme Aggregates of Feruloyl Esterase Preparations from Thermothelomyces thermophila and Talaromyces wortmannii. Catalysts, 8(5), Article ID 208.
Open this publication in new window or tab >>Cross-Linked Enzyme Aggregates of Feruloyl Esterase Preparations from Thermothelomyces thermophila and Talaromyces wortmannii
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2018 (English)In: Catalysts, ISSN 2073-4344, Vol. 8, no 5, article id 208Article in journal (Refereed) Published
Abstract [en]

Cross-linked enzyme aggregates (CLEA®) technology is a well-established method in the current literature for the low-cost and effective immobilization of several enzymes. The main advantage of this particular method is the simplicity of the process, since it consists of only two steps. However, CLEA immobilization must be carefully designed for each desired enzyme, since the optimum conditions for enzymes can vary significantly, according to their physicochemical properties. In the present study, an investigation of the optimum CLEA immobilization conditions was carried out for eight feruloyl esterase preparations. Feruloyl esterases are a very important enzyme group in the valorization of lignocellulosic biomass, since they act in a synergistic way with other enzymes for the breakdown of plant biomass. Specifically, we investigated the type and concentration of precipitant and the crosslinker concentration, for retaining optimal activity. FAE68 was found to be the most promising enzyme for CLEA immobilization, since in this case, the maximum retained activity, over 98%, was observed. Subsequently, we examined the operational stability and the stability in organic solvents for the obtained CLEA preparations, as well as their structure. Overall, our results support that the maximum activity retaining and the stability properties of the final CLEAs can vary greatly in different FAE preparations. Nevertheless, some of the examined FAEs show a significant potential for further applications in harsh industrial conditions.

Place, publisher, year, edition, pages
MDPI, 2018
National Category
Chemical Engineering Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-68931 (URN)10.3390/catal8050208 (DOI)000435191500037 ()2-s2.0-85048056816 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-06-05 (rokbeg)

Available from: 2018-05-28 Created: 2018-05-28 Last updated: 2018-06-28Bibliographically approved
Zerva, A., Antonopoulou, I., Enman, J., Iancu, L., Jütten, P., Rova, U. & Christakopoulos, P. (2018). Optimization of Transesterification Reactions with CLEA-Immobilized Feruloyl Esterases from Thermothelomyces thermophila and Talaromyces wortmannii. Molecules, 23(9), Article ID 2403.
Open this publication in new window or tab >>Optimization of Transesterification Reactions with CLEA-Immobilized Feruloyl Esterases from Thermothelomyces thermophila and Talaromyces wortmannii
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2018 (English)In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 23, no 9, article id 2403Article in journal (Refereed) Published
Abstract [en]

Feruloyl esterases (FAEs, E.C. 3.1.1.73) are biotechnologically important enzymes with several applications in ferulic acid production from biomass, but also in synthesis of hydroxycinnamic acid derivatives. The use of such biocatalysts in commercial processes can become feasible by their immobilization, providing the advantages of isolation and recycling. In this work, eight feruloyl esterases, immobilized in cross-linked enzyme aggregates (CLEAs) were tested in regard to their transesterification performance, towards the production of prenyl ferulate (PFA) and arabinose ferulate (AFA). After solvent screening, comparison with the activity of respective soluble enzymes, and operational stability tests, FAE125 was selected as the most promising biocatalyst. A central composite design revealed the optimum conditions for each transesterification product, in terms of water content, time, and substrate ratio for both products, and temperature and enzyme load additionally for prenyl ferulate. The optimum product yields obtained were 83.7% for PFA and 58.1% for AFA. FAE125 CLEAs are stable in the optimum conditions of transesterification reactions, maintaining 70% residual activity after five consecutive reactions. Overall, FAE125 CLEAs seem to be able to perform as a robust biocatalyst, offering satisfactory yields and stability, and thus showing significant potential for industrial applications.

Place, publisher, year, edition, pages
MDPI, 2018
National Category
Other Industrial Biotechnology Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70930 (URN)10.3390/molecules23092403 (DOI)30235900 (PubMedID)
Note

Validerad;2018;Nivå 2;2018-09-25 (svasva)

Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-10-10Bibliographically approved
Matsakas, L., Novak, K., Enman, J., Christakopoulos, P. & Rova, U. (2017). Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides. Bioresource Technology, 242, 287-294
Open this publication in new window or tab >>Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides
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2017 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 242, p. 287-294Article in journal (Refereed) Published
Abstract [en]

The aim of the current work was to convert an acetate-rich hemicellulose liquid fraction (LF) from hot-water extraction of Betula pendula to oils for biodiesel, with Rhodosporidium toruloides. The toxicity of acetate was circumvented by biological detoxification with an isolated alkali-tolerant and acetate-resistant Bacillus sp. strain. Removal of other lignocellulose-derived inhibitors, such as furfural and phenols, was evaluated by two strategies; an activated carbon (AC) treatment of the undiluted LF, and dilution of the LF by 25% (0.75LF) and 50%. (0.50LF). The bacterium consumed most of the acetic acid in 6-8 days in the treated or diluted media, which were subsequently used for cultivation of the yeast, for conversion of sugars to oils. The oil concentration reached 2.8 and 1.8 g/L, in the AC LF and 0.75LF medium, respectively. In comparison, the oil accumulation in the same media without prior cultivation of Bacillus sp. was 0.86 and 0.03 g/L, respectively.

Place, publisher, year, edition, pages
Elsevier, 2017
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-62873 (URN)10.1016/j.biortech.2017.04.002 (DOI)000405980700038 ()28412146 (PubMedID)2-s2.0-85017440680 (Scopus ID)
Note

Validerad; 2017; Nivå 2; 2017-08-17 (andbra)

Available from: 2017-04-04 Created: 2017-04-04 Last updated: 2018-07-10Bibliographically approved
Ginesy, M., Belotserkovsky, J., Enman, J., Isaksson, L. & Rova, U. (2015). Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis (ed.). Paper presented at . Microbial Cell Factories, 14(1), Article ID 29.
Open this publication in new window or tab >>Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis
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2015 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, no 1, article id 29Article in journal (Refereed) Published
Abstract [en]

BackgroundArginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. ResultsFirstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. ConclusionsIn this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA, ∆speC, ∆speF, ∆argR, ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.

National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-12132 (URN)10.1186/s12934-015-0211-y (DOI)000351035800001 ()25890272 (PubMedID)2-s2.0-84925218937 (Scopus ID)b351a9cd-6c4a-4042-b892-d1ba850fb659 (Local ID)b351a9cd-6c4a-4042-b892-d1ba850fb659 (Archive number)b351a9cd-6c4a-4042-b892-d1ba850fb659 (OAI)
Note
Validerad; 2015; Nivå 2; 20150323 (andbra)Available from: 2016-09-29 Created: 2016-09-29 Last updated: 2018-07-10Bibliographically approved
Enman, J., Hodge, D., Berglund, K. & Rova, U. (2012). Growth promotive conditions for enhanced eritadenine production during submerged cultivation of Lentinus edodes (ed.). Paper presented at . Journal of chemical technology and biotechnology (1986), 87(7), 903-907
Open this publication in new window or tab >>Growth promotive conditions for enhanced eritadenine production during submerged cultivation of Lentinus edodes
2012 (English)In: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 87, no 7, p. 903-907Article in journal (Refereed) Published
Abstract [en]

Background: Mycelium of the medicinal mushroom shiitake, Lentinus edodes, is a potential source for production of the blood cholesterol reducing compound eritadenine. To increase the mycelial biomass and in turn the production of eritadenine, a potential growth promoting substance in the form of a water extract of distillers dried grains with solubles (DDGS) was added to the culture media. Results: The hot water extract of DDGS was shown to considerably increase the growth of shiitake mycelia in bioreactor cultivations; the mycelial yield was 2-3 times higher than in the control, and the highest final biomass concentration obtained was 3.4 g L -1. Further, by using shake flask cultures as inoculums the bioreactor cultivation time could be reduced by 1 week for some of the experiments. The highest final titer of eritadenine in the present study was 25.1 mg L -1, which was about 2 times higher than in the control, and was also obtained when a water extract of DDGS was added to the culture medium. Conclusion: It was demonstrated that a water extract of DDGS promoted the growth of shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production

National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-8732 (URN)10.1002/jctb.3697 (DOI)000305061200005 ()2-s2.0-84862224967 (Scopus ID)7436deea-e182-48fb-b2c4-29a9f9bfb968 (Local ID)7436deea-e182-48fb-b2c4-29a9f9bfb968 (Archive number)7436deea-e182-48fb-b2c4-29a9f9bfb968 (OAI)
Note
Validerad; 2012; 20120206 (andbra)Available from: 2016-09-29 Created: 2016-09-29 Last updated: 2018-07-10Bibliographically approved
Enman, J., Patra, A., Ramser, K., Rova, U. & Berglund, K. (2011). Solid state characterization of sodium eritadenate (ed.). Paper presented at . American Journal of Analytical Chemistry, 2(2), 164-173
Open this publication in new window or tab >>Solid state characterization of sodium eritadenate
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2011 (English)In: American Journal of Analytical Chemistry, ISSN 2156-8251, E-ISSN 2156-8278, Vol. 2, no 2, p. 164-173Article in journal (Refereed) Published
Abstract [en]

Knowledge of the solid state is of great importance in the development of a new active pharmaceutical ingredient, since the solid form often dictates the properties and performance of the drug. In the present study, solid state characteristics of the sodium salt of the candidate cholesterol reducing compound eritadenine, 2(R), 3(R))-dihydroxy-4-(9-adenyl)-butanoic acid, were investigated. The compound was crystallized by slow cooling from water and various aqueous ethanol solutions, at different temperatures. Further, the compound solution was subjected to lyophilization and to high vacuum drying. The resulting solids were screened for polymorphism by micro Raman spectroscopy (λex = 830 nm) and the crystallinity was investigated by X-ray powder diffraction. Further, thermal analysis was applied to study possible occurrence of solvates or hydrates. Solids obtained from slow cooling showed crystallinity, whereas rapid cooling gave rise to more amorphous solids. Analysis of difference spectra of the Raman data for solids obtained from slow cooling of solution revealed subtle differences in the structures between crystals derived from pure water and crystals derived from aqueous ethanol solutions. Finally, from the thermal analysis it was deduced that crystals obtained from pure water were stoichiometrically dihydrates whereas crystals obtained from aqueous ethanol solutions were 2.5 hydrates; this formation of different hydrates were supported by the Raman difference analysis.

National Category
Bioprocess Technology Other Medical Engineering
Research subject
Biochemical Process Engineering; Medical Engineering for Healthcare
Identifiers
urn:nbn:se:ltu:diva-14595 (URN)10.4236/ajac.2011.22019 (DOI)dfe46855-6e65-47f5-b115-7b6eb6f34d7d (Local ID)dfe46855-6e65-47f5-b115-7b6eb6f34d7d (Archive number)dfe46855-6e65-47f5-b115-7b6eb6f34d7d (OAI)
Note
Validerad; 2011; 20110831 (joen)Available from: 2016-09-29 Created: 2016-09-29 Last updated: 2018-04-16Bibliographically approved
Enman, J. (2009). Fungal production and solid state chemistry of eritadenine: an integrated approach to development of an active pharmaceutical ingredient (ed.). (Doctoral dissertation). Paper presented at . Luleå: Luleå tekniska universitet
Open this publication in new window or tab >>Fungal production and solid state chemistry of eritadenine: an integrated approach to development of an active pharmaceutical ingredient
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present thesis demonstrates an integrated approach to the development of a potential active pharmaceutical ingredient, eritadenine, a cholesterol reducing compound originating from the shiitake mushroom (Lentinus edodes). The main areas covered in the thesis are a method for quantification of eritadenine, production of eritadenine by submerged cultivation of fungal mycelia and investigation of the influence of process parameters on mycelial growth and production, and finally solid state characterizations of eritadenine. The usage of the fungus as a source of eritadenine requires an analytical tool for quantification of the compound. An HPLC method was hence developed for identification and quantification of eritadenine, using chemically synthesized eritadenine as a reference. The amount of eritadenine in fruit bodies of selected strains of shiitake was determined and with the method developed in this study, eritadenine concentrations up to ten times higher than previously reported were detected. Since both fruit bodies and mycelia of shiitake have been shown to contain eritadenine submerged cultivation of shiitake mycelia was investigated as an alternative source for this compound. The mycelia were cultivated in various submerged conditions, both in shake flasks and in bioreactors. It was found that both the mycelia and the culture media contained eritadenine, of which the major part was detected in the culture media. While the biomass concentrations were higher in shake flasks, the eritadenine concentrations were considerably higher in the bioreactors, which were assigned to morphological variations. In an attempt to improve the mycelial growth and eritadenine production, a growth promotive substance in the form of a water extract of DDGS, a by-product from drygrind ethanol facilities, was added to the culture media. It was demonstrated that an amendment of the cultivation media with this extract caused a considerable growth promotive effect on shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production. If eritadenine will be used as a pharmaceutical agent, understanding about the solid state chemistry of the compound is required. Raman spectroscopy is a valuable technique for investigation of structural properties; hence, a Raman reference spectrum with line assignments for the solid state of synthetic eritadenine was established. To further investigate the solid state chemistry of eritadenine, its synthetic analogue was slowly crystallized from water and different ethanol concentrations, at different temperatures. Solids formed from slow cooling of either water or aqueous ethanol showed crystallinity. No polymorphism was detected, irrespective of solvent system or temperature. However, dissimilar thermal behaviours were observed, deducing crystals derived from water as dihydrates and crystals derived from aqueous ethanol as 2.5 hydrates.

Place, publisher, year, edition, pages
Luleå: Luleå tekniska universitet, 2009. p. 62
Series
Doctoral thesis / Luleå University of Technology 1 jan 1997 → …, ISSN 1402-1544
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-26005 (URN)c240a280-909b-11de-8da0-000ea68e967b (Local ID)978-91-86233-89-1 (ISBN)c240a280-909b-11de-8da0-000ea68e967b (Archive number)c240a280-909b-11de-8da0-000ea68e967b (OAI)
Note
Godkänd; 2009; 20090824 (joen); DISPUTATION Ämnesområde: Biokemisk och kemisk processteknik/Biochemical and Chemical Engineering Opponent: Professor Allan Myerson, Illinois Inst of Technology, USA Ordförande: Professor Kris Berglund, Luleå tekniska universitet Tid: Tisdag den 29 september 2009, kl 13.00 Plats: C 305, Luleå tekniska universitetAvailable from: 2016-09-30 Created: 2016-09-30 Last updated: 2017-11-24Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6337-6924

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