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Dedes, G., Karnaouri, A. C., Marianou, A. A., Kalogiannis, K. G., Michailof, C. M., Lappas, A. A. & Topakas, E. (2021). Conversion of organosolv pretreated hardwood biomass into 5-hydroxymethylfurfural (HMF) by combining enzymatic hydrolysis and isomerization with homogeneous catalysis. Biotechnology for Biofuels, 14, Article ID 172.
Open this publication in new window or tab >>Conversion of organosolv pretreated hardwood biomass into 5-hydroxymethylfurfural (HMF) by combining enzymatic hydrolysis and isomerization with homogeneous catalysis
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2021 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 14, article id 172Article in journal (Refereed) Published
Abstract [en]

Background: Over the last few years, valorization of lignocellulosic biomass has been expanded beyond the production of second-generation biofuels to the synthesis of numerous platform chemicals to be used instead of their fossil-based counterparts. One such well-researched example is 5-hydroxymethylfurfural (HMF), which is preferably produced by the dehydration of fructose. Fructose is obtained by the isomerization of glucose, which in turn is derived by the hydrolysis of cellulose. However, to avoid harsh reaction conditions with high environmental impact, an isomerization step towards fructose is necessary, as fructose can be directly dehydrated to HMF under mild conditions. This work presents an optimized process to produce fructose from beechwood biomass hydrolysate and subsequently convert it to HMF by employing homogeneous catalysis.

Results: The optimal saccharification conditions were identified at 10% wt. solids loading and 15 mg enzyme/gsolids, as determined from preliminary trials on pure cellulose (Avicel® PH-101). Furthermore, since high rate glucose isomerization to fructose requires the addition of sodium tetraborate, the optimum borate to glucose molar ratio was determined to 0.28 and was used in all experiments. Among 20 beechwood solid pulps obtained from different organosolv pretreatment conditions tested, the highest fructose production was obtained with acetone (160 °C, 120 min), reaching 56.8 g/100 g pretreated biomass. A scale-up hydrolysis in high solids (25% wt.) was then conducted. The hydrolysate was subjected to isomerization eventually leading to a high-fructose solution (104.5 g/L). Dehydration of fructose to HMF was tested with 5 different catalysts (HCl, H3PO4, formic acid, maleic acid and H-mordenite). Formic acid was found to be the best one displaying 79.9% sugars conversion with an HMF yield and selectivity of 44.6% and 55.8%, respectively.

Conclusions: Overall, this work shows the feasibility of coupling bio- and chemo-catalytic processes to produce HMF from lignocellulose in an environmentally friendly manner. Further work for the deployment of biocatalysts for the oxidation of HMF to its derivatives could pave the way for the emergence of an integrated process to effectively produce biobased monomers from lignocellulose.

Place, publisher, year, edition, pages
Springer Nature, 2021
Keywords
5-hydroxymethylfurfural, Lignocellulosic biomass, Isomerization, Homogeneous catalysis
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-86936 (URN)10.1186/s13068-021-02022-9 (DOI)000690935700002 ()34454576 (PubMedID)2-s2.0-85113600786 (Scopus ID)
Note

Validerad;2021;Nivå 2;2021-09-01 (alebob);

Forskningsfinansiär: Hellenic Foundation for Research and Innovation; General Secretariat for Research and Technology (1085)

Available from: 2021-08-31 Created: 2021-08-31 Last updated: 2021-09-06Bibliographically approved
Zerva, A., Pentari, C., Ferousi, C., Nikolaivits, E., Karnaouri, A. & Topakas, E. (2021). Recent advances on key enzymatic activities for the utilisation of lignocellulosic biomass. Bioresource Technology, 342, Article ID 126058.
Open this publication in new window or tab >>Recent advances on key enzymatic activities for the utilisation of lignocellulosic biomass
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2021 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 342, article id 126058Article, review/survey (Refereed) Published
Abstract [en]

The field of enzymatic degradation of lignocellulose is actively growing and the recent updates of the last few years indicate that there is still much to learn. The growing number of protein sequences with unknown function in microbial genomes indicates that there is still much to learn on the mechanisms of lignocellulose degradation. In this review, a summary of the progress in the field is presented, including recent discoveries on the nature of the structural polysaccharides, new technologies for the discovery and functional annotation of gene sequences including omics technologies, and the novel lignocellulose-acting enzymes described. Novel enzymatic activities and enzyme families as well as on accessory enzymes and their synergistic relationships regarding biomass breakdown are described. Moreover, it is shown that all the valuable knowledge of the enzymatic decomposition of plant biomass polymers can be employed towards the decomposition and upgrading of synthetic polymers, such as plastics.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
Lignocellulosic biomass, xylanase, carbohydrate esterase, lytic polysaccharide monooxygenase, plastics degradation
National Category
Biocatalysis and Enzyme Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-87299 (URN)10.1016/j.biortech.2021.126058 (DOI)000703676800007 ()34597805 (PubMedID)2-s2.0-85116004410 (Scopus ID)
Note

Validerad;2021;Nivå 2;2021-10-04 (beamah);

Funder: Hellenic Foundation for Research and Innovation (328)

Available from: 2021-09-30 Created: 2021-09-30 Last updated: 2021-12-13Bibliographically approved
Karnaouri, A., Zerva, A., Christakopoulos, P. & Topakas, E. (2021). Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays (2ed.). In: Nikolaos E. Labrou (Ed.), Protein Downstream Processing: Design, Development, and Application of High and Low-Resolution Methods (pp. 479-503). Springer Nature
Open this publication in new window or tab >>Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays
2021 (English)In: Protein Downstream Processing: Design, Development, and Application of High and Low-Resolution Methods / [ed] Nikolaos E. Labrou, Springer Nature, 2021, 2, p. 479-503Chapter in book (Other academic)
Abstract [en]

In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.

Place, publisher, year, edition, pages
Springer Nature, 2021 Edition: 2
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029
Keywords
Agar plate assay, Screening, Biomass-degrading enzymes, Glycoside hydrolases, Carbohydrate esterases, Oxidative enzymes, Immobilized-metal affinity chromatography
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-81306 (URN)10.1007/978-1-0716-0775-6_30 (DOI)33128767 (PubMedID)2-s2.0-85094982532 (Scopus ID)
Note

ISBN för värdpublikation: 978-1-0716-0774-9,  978-1-0716-0775-6

Available from: 2020-11-03 Created: 2020-11-03 Last updated: 2023-09-05Bibliographically approved
Zerva, A., Pentari, C. & Topakas, E. (2020). Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW). Molecules, 25(9), Article ID 2221.
Open this publication in new window or tab >>Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW)
2020 (English)In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 25, no 9, article id 2221Article in journal (Refereed) Published
Abstract [en]

The enzymatic factory of ligninolytic fungi has proven to be a powerful tool in applications regarding the degradation of various types of pollutants. The degradative potential of fungi is mainly due to the production of different types of oxidases, of which laccases is one of the most prominent enzymatic activities. In the present work, crude laccases from the supernatant of Pleurotus citrinopileatus cultures grown in olive oil mill wastewater (OOMW) were immobilized in crosslinked enzyme aggregates (CLEAs), aiming at the development of biocatalysts suitable for the enzymatic treatment of OOMW. The preparation of laccase CLEAs was optimized, resulting in a maximum of 72% residual activity. The resulting CLEAs were shown to be more stable in the presence of solvents and at elevated temperatures compared to the soluble laccase preparation. The removal of the phenolic component of OOMW catalyzed by laccase-CLEAs exceeded 35%, while they were found to retain their activity for at least three cycles of repetitive use. The described CLEAs can be applied for the pretreatment of OOMW, prior to its use for valorization processes, and thus, facilitate its complete biodegradation towards a consolidated process in the context of circular economy.

Place, publisher, year, edition, pages
MDPI, 2020
Keywords
crosslinked enzyme aggregates, Pleurotus citrinopileatus, laccase, olive oil mill wastewater
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-78918 (URN)10.3390/molecules25092221 (DOI)000535695900217 ()32397329 (PubMedID)2-s2.0-85084479755 (Scopus ID)
Note

Validerad;2020;Nivå 2;2020-05-18 (alebob)

Available from: 2020-05-18 Created: 2020-05-18 Last updated: 2023-08-28Bibliographically approved
Nikolaivits, E., Agrafiotis, A., Baira, E., Le Goff, G., Tsafantakis, N., Chavanich, S. A., . . . Topakas, E. (2020). Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates. International Journal of Molecular Sciences, 21(9), Article ID 3317.
Open this publication in new window or tab >>Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates
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2020 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 9, article id 3317Article in journal (Refereed) Published
Abstract [en]

2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.

Place, publisher, year, edition, pages
MDPI, 2020
Keywords
2, 4-dichlorophenol, marine-derived fungi, invertebrate symbionts, catechol dioxygenase, DCP metabolites
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-78914 (URN)10.3390/ijms21093317 (DOI)000535581700288 ()32392868 (PubMedID)2-s2.0-85084587186 (Scopus ID)
Note

Validerad;2020;Nivå 2;2020-05-18 (alebob)

Available from: 2020-05-18 Created: 2020-05-18 Last updated: 2022-02-10Bibliographically approved
Karnaouri, A., Asimakopoulou, G., Kalogiannis, K. G., Lappas, A. & Topakas, E. (2020). Efficient d-lactic acid production by Lactobacillus delbrueckii subsp. bulgaricus through conversion of organosolv pretreated lignocellulosic biomass. Biomass and Bioenergy, 140, Article ID 105672.
Open this publication in new window or tab >>Efficient d-lactic acid production by Lactobacillus delbrueckii subsp. bulgaricus through conversion of organosolv pretreated lignocellulosic biomass
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2020 (English)In: Biomass and Bioenergy, ISSN 0961-9534, E-ISSN 1873-2909, Vol. 140, article id 105672Article in journal (Refereed) Published
Abstract [en]

Lactic acid bioconversion processes have numerous advantages over the chemical synthesis route, not only due to the high-titer yield of the final product with great optical purity, but also due to the possibility of utilizing lignocellulosic biomass feedstocks as carbon source in an economic and environmentally friendly way. In the present study, beechwood and pine were pretreated with a novel mild oxidative organosolv process to produce cellulose-rich solid fractions, which were tested for their ability to support the growth and high lactic acid productivity of Lactobacillus delbrueckii subsp. bulgaricus (ATCC® 11842). We employed a simultaneous saccharification and fermentation (SSF) strategy in batch cultures with 9% w v−1 solids loading. The results for beechwood showed the highest production of 62 g L−1 lactic acid after 72 h of incubation, corresponding to a yield of 0.69 g g−1 of biomass (82.7% of the theoretical maximum yield) and a productivity of 0.86 g L−1 h−1. In the case of pine, the productivity was lower at 0.51 g L−1 h−1, leading to accumulation of 36.4 g L−1 lactic acid, corresponding to a yield of 0.40 g g−1 of biomass (41.4% of the theoretical maximum yield). Our study suggests that L. delbrueckii subsp. bulgaricus is an efficient lactic acid bacterial strain for the production of optically pure d-lactic acid from non-edible, organosolv pretreated hardwood and softwood biomass for the synthesis of bio-based plastics and other products.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
D-lactic acid, Lactobacillus delbrueckii subsp. bulgaricus, SSF, Lignocellulosic biomass, Organosolv pretreatment
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-80239 (URN)10.1016/j.biombioe.2020.105672 (DOI)000560059700006 ()2-s2.0-85087759469 (Scopus ID)
Note

Validerad;2020;Nivå 2;2020-07-16 (alebob)

Available from: 2020-07-16 Created: 2020-07-16 Last updated: 2020-09-03Bibliographically approved
Chalima, A., Taxeidis, G. & Topakas, E. (2020). Optimization of the production of docosahexaenoic fatty acid by the heterotrophic microalga Crypthecodinium cohnii utilizing a dark fermentation effluent. Renewable energy, 152, 102-109
Open this publication in new window or tab >>Optimization of the production of docosahexaenoic fatty acid by the heterotrophic microalga Crypthecodinium cohnii utilizing a dark fermentation effluent
2020 (English)In: Renewable energy, ISSN 0960-1481, E-ISSN 1879-0682, Vol. 152, p. 102-109Article in journal (Refereed) Published
Abstract [en]

Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen as a fuel, which however releases high amounts of polluting volatile fatty acids in the environment. In order for the process to become more competitive, the acids stream can be utilized through conversion to high added-value docosahexaenoic acid by the microalga Crypthecodinium cohnii. Docosahexaenoic acid is one of the two main omega-3 fatty acids, necessary for human nutrition. The purpose of this work was to optimize the production of omega-3 fatty acids by the cells, utilizing the organic content of a dark fermentation effluent. For that purpose, the effect of different fermentation conditions was examined, such as incubation temperature, nitrogen source and concentration, the addition of chemical modulators, as well as the feeding composition. The volatile fatty acid content of the effluent was totally depleted in a fed-batch culture of the microalga, while the cells accumulated DHA in a percentage of 35.6% of total lipids, when fed with yeast extract or 34.2% when fed with ammonium sulfate. Taking into consideration the economic feasibility of the culture conditions proposed it was concluded that the use of yeast extract could be substituted by the much economic ammonium sulfate.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Crypthecodinium cohnii, Biorefinery, Volatile fatty acids, Dark fermentation effluent, Docosahexaenoic acid
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-78552 (URN)10.1016/j.renene.2020.01.041 (DOI)000536949600010 ()2-s2.0-85078006722 (Scopus ID)
Note

Validerad;2020;Nivå 2;2020-04-16 (johcin)

Available from: 2020-04-16 Created: 2020-04-16 Last updated: 2020-07-01Bibliographically approved
Kalogiannis, K. G., Karnaouri, A., Michailof, C., Tzika, A. M., Asimakopoulou, G., Topakas, E. & Lappas, A. A. (2020). OxiOrganosolv: A novel acid free oxidative organosolv fractionation for lignocellulose fine sugar streams. Bioresource Technology, 313, Article ID 123599.
Open this publication in new window or tab >>OxiOrganosolv: A novel acid free oxidative organosolv fractionation for lignocellulose fine sugar streams
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2020 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 313, article id 123599Article in journal (Refereed) Published
Abstract [en]

The valorization of lignocellulosic biomass towards the production of value-added products requires an efficient pretreatment/fractionation step. In this work we present a novel, acid-free, mildly oxidative organosolv delignification process -OxiOrganosolv- which employs oxygen gas to depolymerize and remove lignin. The results demonstrate that the OxiOrganosolv process achieved lignin removal as high as 97% in a single stage, with a variety of solvents; it was also efficient in delignifying both beechwood (hardwood) and pine (softwood), a task in which organosolv pretreatments have failed in the past. Minimal amounts of sugar degradation products were detected, while cellulose recovery was ~100% in the solid pulp. Enzymatic hydrolysis of pulps showed >80 wt% cellulose conversion to glucose. Overall, the OxiOrganosolv pretreatment has significant advantages, including high delignification efficiency of hardwood and softwood biomass, absence of acid homogeneous catalysis and all corresponding challenges involved, and close to zero losses of sugars to degradation products.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Biorefinery, Delignification, Enzymatic hydrolysis, Organosolv oxidation pretreatment
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-79562 (URN)10.1016/j.biortech.2020.123599 (DOI)000551494900008 ()32540692 (PubMedID)2-s2.0-85086764284 (Scopus ID)
Note

Validerad;2020;Nivå 2;2020-06-15 (alebob)

Available from: 2020-06-15 Created: 2020-06-15 Last updated: 2020-08-26Bibliographically approved
Karnaouri, A., Chalima, A., Kalogiannis, K. G., Varamogianni-Mamatsi, D., Lappas, A. & Topakas, E. (2020). Utilization of lignocellulosic biomass towards the production of omega-3 fatty acids by the heterotrophic marine microalga Crypthecodinium cohnii. Bioresource Technology, 303, Article ID 122899.
Open this publication in new window or tab >>Utilization of lignocellulosic biomass towards the production of omega-3 fatty acids by the heterotrophic marine microalga Crypthecodinium cohnii
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2020 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 303, article id 122899Article in journal (Refereed) Published
Abstract [en]

Omega-3 fatty acids have become a commodity of high nutritional and commercial value; intensive fishing and its environmental and social cost has led researchers to seeking alternative more sustainable ways of producing them. Heterotrophic microalgae such as Crypthecodinium cohnii, a marine dinoflagellate, have the ability to utilize various substrates and accumulate high amounts of docosahexaenoic acid (DHA). In this work, a mild oxidative organosolv pretreatment of beechwood pulps was employed that allowed up to 95% of lignin removal in a single stage, thus yielding a cellulose-rich solid fraction. The enzymatic hydrolysates were evaluated for their ability to support the growth and lipid accumulation of C. cohnii in batch and fed-batch cultures; the results verified the successful microalgae growth, while DHA reached up to 43.5% of the cell’s total lipids. The proposed bioprocess demonstrated the utilization of non-edible biomass towards high added value food supplements in a sustainable and efficient manner.

Place, publisher, year, edition, pages
Elsevier, 2020
Keywords
Crypthecodinium cohnii, DHA, Lipid accumulation, Lignocellulosic biomass, Oxidative organosolv pretreatment
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-77907 (URN)10.1016/j.biortech.2020.122899 (DOI)000516839600052 ()32028216 (PubMedID)2-s2.0-85078771060 (Scopus ID)
Note

Validerad;2020;Nivå 2;2020-02-28 (alebob)

Available from: 2020-02-28 Created: 2020-02-28 Last updated: 2020-03-31Bibliographically approved
Katsimpouras, C., Dedes, G., Thomaidis, N. S. & Topakas, E. (2019). A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity. Biotechnology for Biofuels, 12, Article ID 120.
Open this publication in new window or tab >>A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity
2019 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, article id 120Article in journal (Refereed) Published
Abstract [en]

Background:

The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity.

Results:

The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-d-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well.

Conclusions:

Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

Place, publisher, year, edition, pages
Springer Nature, 2019
Keywords
GH30 xylanase, Glucuronoxylan, Thermothelomyces thermophila, Xylobiohydrolase, Xylooligosaccharides
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-74533 (URN)10.1186/s13068-019-1455-2 (DOI)000467550500002 ()31110561 (PubMedID)2-s2.0-85065661255 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-06-14 (oliekm)

Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2021-03-09Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0078-5904

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