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Christakopoulos, PaulORCID iD iconorcid.org/0000-0003-0079-5950
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Publications (10 of 253) Show all publications
Cerullo, G., Varriale, S., Bozonnet, S., Antonopoulou, I., Christakopoulos, P., Rova, U., . . . Faraco, V. (2019). Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants. New Biotechnology, 51, 14-20
Open this publication in new window or tab >>Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants
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2019 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 51, p. 14-20Article in journal (Refereed) Published
Abstract [en]

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Directed evolution, Feruloyl esterase, Fusarium oxysporum, High-throughput screening, Library
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-72926 (URN)10.1016/j.nbt.2019.01.008 (DOI)2-s2.0-85061183564 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-02-18 (svasva)

Available from: 2019-02-18 Created: 2019-02-18 Last updated: 2019-02-18Bibliographically approved
Matsakas, L., Raghavendran, V., Yakimenko, O., Persson, G., Olsson, E., Rova, U., . . . Christakopoulos, P. (2019). Lignin-first biomass fractionation using a hybrid organosolv: Steam explosion pretreatment technology improves the saccharification and fermentability of spruce biomass. Bioresource Technology, 273, 521-528
Open this publication in new window or tab >>Lignin-first biomass fractionation using a hybrid organosolv: Steam explosion pretreatment technology improves the saccharification and fermentability of spruce biomass
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2019 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 273, p. 521-528Article in journal (Refereed) Published
Abstract [en]

For a transition to a sustainable society, fuels, chemicals, and materials should be produced from renewable resources. Lignocellulosic biomass constitutes an abundant and renewable feedstock; however, its successful application in a biorefinery requires efficient fractionation into its components; cellulose, hemicellulose and lignin. Here, we demonstrate that a newly established hybrid organosolv – steam explosion pretreatment can effectively fractionate spruce biomass to yield pretreated solids with high cellulose (72% w/w) and low lignin (delignification up to 79.4% w/w) content. The cellulose-rich pretreated solids present high saccharification yields (up to 61% w/w) making them ideal for subsequent bioconversion processes. Moreover, under high-gravity conditions (22% w/w) we obtained an ethanol titer of 61.7 g/L, the highest so far reported for spruce biomass. Finally, the obtained high-purity lignin is suitable for various advanced applications. In conclusion, hybrid organosolv pretreatment could offer a closed-loop biorefinery while simultaneously adding value to all biomass components.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Softwood, Organosolv explosion, High gravity fermentation, Fractionation, Biorefinery
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-71766 (URN)10.1016/j.biortech.2018.11.055 (DOI)000453742100064 ()30471644 (PubMedID)2-s2.0-85056772752 (Scopus ID)
Funder
Swedish Energy Agency, 2015-006989
Note

Validerad;2018;Nivå 2;2018-11-27 (johcin)

Available from: 2018-11-27 Created: 2018-11-27 Last updated: 2019-01-30Bibliographically approved
Antonopoulou, I., Iancu, L., Jütten, P., Piechot, A., Rova, U. & Christakopoulos, P. (2019). Screening of novel feruloyl esterases from Talaromyces wortmannii for the development of efficient and sustainable syntheses of feruloyl derivatives. Enzyme and microbial technology, 120, 124-135
Open this publication in new window or tab >>Screening of novel feruloyl esterases from Talaromyces wortmannii for the development of efficient and sustainable syntheses of feruloyl derivatives
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2019 (English)In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 120, p. 124-135Article in journal (Refereed) Published
Abstract [en]

The feruloyl esterases Fae125, Fae7262 and Fae68 from Talaromyces wortmannii were screened in 10 different solvent: buffer systems in terms of residual hydrolytic activity and of the ability for the transesterification of vinyl ferulate with prenol or L-arabinose. Among the tested enzymes, the acetyl xylan-related Fae125 belonging to the phylogenetic subfamily 5 showed highest yield and selectivity for both products in alkane: buffer systems (n-hexane or n-octane). Response surface methodology, based on a 5-level and 6-factor central composite design, revealed that the substrate molar ratio and the water content were the most significant variables for the bioconversion yield and selectivity. The effect of agitation, the possibility of DMSO addition and the increase of donor concentration were investigated. After optimization, competitive transesterification yields were obtained for prenyl ferulate (87.5-92.6%) and L-arabinose ferulate (56.2-61.7%) at reduced reaction times (≤ 24 h) resulting in good productivities (> 1 g/L/h, >300 kg product/kg FAE). The enzyme could be recycled for six consecutive cycles retaining 66.6% of the synthetic activity and 100% of the selectivity.

Place, publisher, year, edition, pages
Elsevier, 2019
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70723 (URN)10.1016/j.enzmictec.2018.08.007 (DOI)000452576500016 ()30396393 (PubMedID)2-s2.0-85053620494 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-11-08 (johcin)

Available from: 2018-09-03 Created: 2018-09-03 Last updated: 2019-02-01Bibliographically approved
Mu, L., Wu, J., Matsakas, L., Chen, M., Rova, U., Christakopoulos, P., . . . Shi, Y. (2019). Two important factors of selecting lignin as efficient lubricating additives in poly (ethylene glycol): Hydrogen bond and molecular weight. International Journal of Biological Macromolecules, 129, 564-570
Open this publication in new window or tab >>Two important factors of selecting lignin as efficient lubricating additives in poly (ethylene glycol): Hydrogen bond and molecular weight
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2019 (English)In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 129, p. 564-570Article in journal (Refereed) Published
Abstract [en]

Lignin, one of the most abundant natural polymers, has been successfully used as an effective lubricant additive with high value. The chemical structure of lignin is very diverse and strongly affected by both the source of lignin (i.e. plant species) and the lignin extraction process. In this work, a series of lignin from different biomass sources (hard or soft wood) and extraction process (organosolv with or without acid catalyst) has been successfully incorporated into poly(ethylene glycol) (PEG) and fortified lubricating properties were achieved. The effects of different lignin on the rheological, thermal and tribological properties of the lignin/EG lubricants were systematically investigated by different characterization techniques. Lignin in PEG significantly improves the lubricating property, where a wear reduction of 93.8% was observed. The thermal and lubrication properties of the PEG lubricants filled with different kinds of lignin are tightly related to the synergistic state of hydrogen bonding and molecular weight distribution. Lignin with broader molecular weight distribution and higher hydroxyl content shows better adhesion on metal surfaces and strengthened lubricating film, which could be used as the efficient lubricating additives. This work provides a criterion for selecting appropriate lignin as the efficient lubricant additive and accelerates the application of lignin.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Lignin, Lubrication, Poly (ethylene glycol), Hydrogen bonding, Molecular weight
National Category
Bioprocess Technology Tribology (Interacting Surfaces including Friction, Lubrication and Wear)
Research subject
Machine Elements; Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-72899 (URN)10.1016/j.ijbiomac.2019.01.175 (DOI)
Note

Validerad;2019;Nivå 2;2019-02-20 (svasva)

Available from: 2019-02-14 Created: 2019-02-14 Last updated: 2019-02-20Bibliographically approved
Raghavendran, V., Nitsos, C., Matsakas, L., Rova, U., Christakopoulos, P. & Olsson, L. (2018). A comparative study of the enzymatic hydrolysis of batch organosolv-pretreated birch and spruce biomass. AMB Express, 8(1), Article ID 114.
Open this publication in new window or tab >>A comparative study of the enzymatic hydrolysis of batch organosolv-pretreated birch and spruce biomass
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2018 (English)In: AMB Express, ISSN 2191-0855, E-ISSN 2191-0855, Vol. 8, no 1, article id 114Article in journal (Refereed) Published
Abstract [en]

A shift towards a sustainable and green society is vital to reduce the negative effects of climate change associated with increased CO2 emissions. Lignocellulosic biomass is both renewable and abundant, but is recalcitrant to deconstruction. Among the methods of pretreatment available, organosolv (OS) delignifies cellulose efficiently, significantly improving its digestibility by enzymes. We have assessed the hydrolysability of the cellulose-rich solid fractions from OS-pretreated spruce and birch at 2% w/v loading (dry matter). Almost complete saccharification of birch was possible with 80 mg enzyme preparation/gsolids (12 FPU/gsolids), while the saccharification yield for spruce was only 70%, even when applying 60 FPU/gsolids. As the cellulose content is enriched by OS, the yield of glucose was higher than in their steam-exploded counterparts. The hydrolysate was a transparent liquid due to the absence of phenolics and was also free from inhibitors. OS pretreatment holds potential for use in a large-scale, closed-loop biorefinery producing fuels from the cellulose fraction and platform chemicals from the hemicellulose and lignin fractions respectively.

Place, publisher, year, edition, pages
Springer, 2018
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70136 (URN)10.1186/s13568-018-0643-y (DOI)000438255300003 ()29992363 (PubMedID)2-s2.0-85049690419 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-07-19 (inah)

Available from: 2018-07-19 Created: 2018-07-19 Last updated: 2018-09-14Bibliographically approved
Matsakas, L., Nitsos, C., Raghavendran, V., Yakimenko, O., Persson, G., Olsson, E., . . . Christakopoulos, P. (2018). A novel hybrid organosolv: steam explosion method for the efficient fractionation and pretreatment of birch biomass. Biotechnology for Biofuels, 11(1), Article ID 160.
Open this publication in new window or tab >>A novel hybrid organosolv: steam explosion method for the efficient fractionation and pretreatment of birch biomass
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2018 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 160Article in journal (Refereed) Published
Abstract [en]

The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction.

Results

In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/wbiomass H2SO4 was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum.

Conclusions

The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive discharge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass.

Place, publisher, year, edition, pages
BioMed Central, 2018
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-69455 (URN)10.1186/s13068-018-1163-3 (DOI)000434952000002 ()29930706 (PubMedID)2-s2.0-85048400180 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-06-13 (andbra)

Available from: 2018-06-13 Created: 2018-06-13 Last updated: 2018-08-08Bibliographically approved
Matsakas, L., Hruzova, K., Rova, U. & Christakopoulos, P. (2018). Biological Production of 3-Hydroxypropionic Acid: An Update on the Current Status. Fermentation, 4(1), Article ID 13.
Open this publication in new window or tab >>Biological Production of 3-Hydroxypropionic Acid: An Update on the Current Status
2018 (English)In: Fermentation, ISSN 2311-5637, Vol. 4, no 1, article id 13Article in journal (Refereed) Published
Abstract [en]

The production of high added-value chemicals from renewable resources is a necessity inour attempts to switch to a more sustainable society. 3-Hydroxypropionic acid (3HP) is a promisingmolecule that can be used for the production of an important array of high added-value chemicals,such as 1,3-propanediol, acrylic acid, acrylamide, and bioplastics. Biological production of 3HP hasbeen studied extensively, mainly from glycerol and glucose, which are both renewable resources.To enable conversion of these carbon sources to 3HP, extensive work has been performed to identifyappropriate biochemical pathways and the enzymes that are involved in them. Novel enzymeshave also been identified and expressed in host microorganisms to improve the production yieldsof 3HP. Various process configurations have also been proposed, resulting in improved conversionyields. The intense research efforts have resulted in the production of as much as 83.8 g/L 3HP fromrenewable carbon resources, and a system whereby 3-hydroxypropionitrile was converted to 3HPthrough whole-cell catalysis which resulted in 184.7 g/L 3HP. Although there are still challengesand difficulties that need to be addressed, the research results from the past four years have been animportant step towards biological production of 3HP at the industrial level.

Place, publisher, year, edition, pages
MDPI, 2018
Keywords
3-hydroxypropionic acid; metabolic engineering; building-block chemicals; glycerol; platform chemicals; Klebsiella pneumoniae; Escherichia coli; Saccharomyces cerevisiae
National Category
Industrial Biotechnology Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-67793 (URN)10.3390/fermentation4010013 (DOI)
Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2018-08-29Bibliographically approved
Antonopoulou, I., Leonov, L., Jûtten, P., Cerullo, G., Faraco, V., Papadopoulou, A., . . . Christakopoulos, P. (2018). Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions [Letter to the editor]. Applied Microbiology and Biotechnology, 102(1), 511-511
Open this publication in new window or tab >>Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions
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2018 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 1, p. 511-511Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
Springer, 2018
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-66731 (URN)10.1007/s00253-017-8631-8 (DOI)000419119100044 ()29159584 (PubMedID)
Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2018-09-14Bibliographically 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
Muraleedharan, M. N., Zouraris, D., Karantonis, A., Topakas, E., Sandgren, M., Rova, U., . . . Karnaouri, A. C. (2018). Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases. Biotechnology for Biofuels, 11(1), Article ID 296.
Open this publication in new window or tab >>Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases
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2018 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 296Article in journal (Refereed) Published
Abstract [en]

Background

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant lignocellulose in the presence of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. One of the possible systems that provide electrons to the LPMOs active site and promote the polysaccharide degradation involves the mediation of phenolic agents, such as lignin, low-molecular-weight lignin-derived compounds and other plant phenols. In the present work, the interaction of the bulk insoluble lignin fraction extracted from pretreated biomass with LPMOs and the ability to provide electrons to the active site of the enzymes is studied.

Results

The catalytic efficiency of three LPMOs, namely MtLPMO9 with C1/C4 regioselectivity, PcLPMO9D which is a C1 active LPMO and NcLPMO9C which is a C4 LPMO, was evaluated in the presence of different lignins. It was correlated with the physicochemical and structural properties of lignins, such as the molecular weight and the composition of aromatic and aliphatic hydroxyl groups. Moreover, the redox potential of lignins was determined with the use of large amplitude Fourier Transform alternating current cyclic voltammetry method and compared to the formal potential of the Cu (II) center in the active site of the LPMOs, providing more information about the lignin-LPMO interaction. The results demonstrated the existence of low-molecular weight lignin-derived compounds that are diffused in the reaction medium, which are able to reduce the enzyme active site and subsequently utilize additional electrons from the insoluble lignin fraction to promote the LPMO oxidative activity. Regarding the bulk lignin fractions, those isolated from the organosolv pretreated materials served as the best candidates in supplying electrons to the soluble compounds and, finally, to the enzymes. This difference, based on biomass pretreatment, was also demonstrated by the activity of LPMOs on natural substrates in the presence and absence of ascorbic acid as additional reducing agent.

Conclusions

Lignins can support the action of LPMOs and serve indirectly as electron donors through low-molecular-weight soluble compounds. This ability depends on their physicochemical and structural properties and is related to the biomass source and pretreatment method.

Keywords
Lytic polysaccharide monooxygenases, Lignin structural properties, Electron donor, Cyclic voltammetry, Redox potential, Forest biomass, Pretreatment
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-71479 (URN)10.1186/s13068-018-1294-6 (DOI)000448645700001 ()30386433 (PubMedID)2-s2.0-85055687969 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-11-07 (johcin) 

Available from: 2018-11-07 Created: 2018-11-07 Last updated: 2018-12-10Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-0079-5950

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