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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 1420-3049, 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)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: 2020-05-28Bibliographically 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 1422-0067, 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)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: 2020-05-28Bibliographically 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)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: 2020-04-16Bibliographically 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.

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: 2019-06-25Bibliographically approved
Zerva, A., Koutroufini, E., Kostopoulou, I., Detsi, A. & Topakas, E. (2019). A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone. New Biotechnology, 49, 10-18
Open this publication in new window or tab >>A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone
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2019 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 49, p. 10-18Article in journal (Refereed) Published
Abstract [en]

Laccase-like multicopper oxidases (LMCOs) are a heterogeneous group of oxidases, acting mainly on phenolic compounds and which are widespread among many microorganisms, including Basidiomycetes and Ascomycetes. Here, we report the cloning, heterologous expression, purification and characterization of a novel LMCO from the thermophilic fungus Thermothelomyces thermophila. The 1953 bp lmco gene sequence comprises of 3 exons interrupted by 2 introns and according to the LccED database the translated sequence belongs to superfamily 6 of multicopper oxidases. After removal of the introns, the gene was transformed into Pichia pastoris, under the control of the alcohol oxidase (AOX1) promoter. The heterologous enzyme was purified with an apparent molecular weight of 80 kDa. TtLMCO1 displayed optimum activity at pH 4 and 50 °C and appeared thermostable up to 50 °C. A variety of phenolic compounds were oxidized by TtLMCO1, including standard laccase substrates such as ABTS and 2,6 dimethoxyphenol. The UV/Vis spectrum of purified TtLMCO1 indicates that it belongs to yellow laccase-like oxidases. The enzyme was used for the bioconversion of 2′,3,4-trihydroxychalcone to 3′,4′-dihydroxy-aurone, a bioactive aurone recently shown to possess inhibitory activity against several isoforms of the histone deacetylase complex (HDAC). Overall, the thermophilic yellow LMCO TtLMCO1 presents a number of superior properties with potential use in industrial biocatalysis.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Laccase-like multicopper oxidase, Thermophile biocatalysts, Thermothelomyces thermophila, Chalcone oxidative cyclization
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-72494 (URN)10.1016/j.nbt.2018.12.001 (DOI)000454542600002 ()30529567 (PubMedID)2-s2.0-85058170054 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-01-17 (johcin)

Available from: 2019-01-17 Created: 2019-01-17 Last updated: 2019-01-17Bibliographically approved
Mandic, M., Djokic, L., Nikolaivits, E., Prodanovic, R., O’Connor, K., Jeremic, S., . . . Nikodinovic-Runic, J. (2019). Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes. Catalysts, 9(7), Article ID 629.
Open this publication in new window or tab >>Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes
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2019 (English)In: Catalysts, ISSN 2073-4344, Vol. 9, no 7, article id 629Article in journal (Refereed) Published
Abstract [en]

Laccases are multicopper-oxidases with variety of biotechnological applications. While predominantly used, fungal laccases have limitations such as narrow pH and temperature range and their production via heterologous protein expression is more complex due to posttranslational modifications. In comparison, bacterial enzymes, including laccases, usually possess higher thermal and pH stability, and are more suitable for expression and genetic manipulations in bacterial expression hosts. Therefore, the aim of this study was to identify, recombinantly express, and characterize novel laccases from Pseudomonas spp. A combination of approaches including DNA sequence analysis, N-terminal protein sequencing, and genome sequencing data analysis for laccase amplification, cloning, and overexpression have been used. Four active recombinant laccases were obtained, one each from P. putida KT2440 and P. putida CA-3, and two from P. putida F6. The new laccases exhibited broad temperature and pH range and high thermal stability, as well as the potential to degrade selection of synthetic textile dyes. The best performing laccase was CopA from P. putida F6 which degraded five out of seven tested dyes, including Amido Black 10B, Brom Cresol Purple, Evans Blue, Reactive Black 5, and Remazol Brilliant Blue. This work highlighted species of Pseudomonas genus as still being good sources of biocatalytically relevant enzymes.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
laccase, genome-mining, heterologous expression, biocatalysis, Pseudomonas
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-75724 (URN)10.3390/catal9070629 (DOI)000478652600029 ()
Note

Validerad;2019;Nivå 2;2019-08-28 (johcin)

Available from: 2019-08-28 Created: 2019-08-28 Last updated: 2019-08-28Bibliographically approved
Chalima, A., Hatzidaki, A., Karnaouri, A. & Topakas, E. (2019). Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids. Applied Energy, 241, 130-138
Open this publication in new window or tab >>Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids
2019 (English)In: Applied Energy, ISSN 0306-2619, E-ISSN 1872-9118, Vol. 241, p. 130-138Article in journal (Refereed) Published
Abstract [en]

Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen- for generation of energy- that however releases high amounts of polluting volatile fatty acids, such as acetic acid, in the environment. In order for this biohydrogen production process to become more competitive, the volatile fatty acids stream can be utilized through conversion to high added-value metabolites, such as omega-3 fatty acids. The docosahexaenoic acid is one of the two most known omega-3 fatty acids and has been found to be necessary for a healthy brain and proper cardiovascular function. The main source is currently fish, which obtain the fatty acid from the primary producers, microalgae, through the food chain. Crypthecodinium cohnii, a heterotrophic marine microalga, is known for accumulating high amounts of docosahexaenoic acid, while offering the advantage of assimilating various carbon sources, such as glucose, ethanol, glycerol and acetic acid. The purpose of this work was to examine the ability of a C. cohnii strain to grow on different volatile fatty acids, as well as, on a pretreated dark fermentation effluent and accumulate omega-3. The strain was found to grow well on relatively high concentrations of acetic, butyric or propionic acid as main carbon source in a fed-batch pH-auxostat. Most importantly, C. cohnii totally depleted the organic acid content of an ultra-filtrated dark fermentation effluent after 60 h of fed-batch cultivation, therefore offering a bioprocess not only able to mitigate environmental pollutants, but also to provide a solution for a sustainable energy production process. The accumulated docosahexaenoic acid content was as high as 29.8% (w/w) of total fatty acids. 

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Biorefinery, Crypthecodinium cohnii, Dark fermentation effluent, Docosahexaenoic acid, Omega-3, Volatile fatty acids
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73234 (URN)10.1016/j.apenergy.2019.03.058 (DOI)000465509500012 ()2-s2.0-85062628275 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-03-18 (svasva)

Available from: 2019-03-18 Created: 2019-03-18 Last updated: 2019-06-18Bibliographically approved
Karnaouri, A. C., Antonopoulou, I., Zerva, A., Dimarogona, M., Topakas, E., Rova, U. & Christakopoulos, P. (2019). Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts. Bioresource Technology, 279, 362-372
Open this publication in new window or tab >>Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts
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2019 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 279, p. 362-372Article in journal (Refereed) Published
Abstract [en]

Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Thermophilic enzymes, Thermostability, Esterases, Laccases, Peroxidases, LPMOs, Biotransformation, Lignocellulose valorization
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73041 (URN)10.1016/j.biortech.2019.01.062 (DOI)000458999200043 ()30685134 (PubMedID)2-s2.0-85060269655 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-02-27 (johcin)

Available from: 2019-02-27 Created: 2019-02-27 Last updated: 2019-03-08Bibliographically approved
Katsimpouras, C., Dedes, G., Bistis, P., Kekos, D., Kalogiannis, K. G. & Topakas, E. (2018). Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides. Bioresource Technology, 270, 208-215
Open this publication in new window or tab >>Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides
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2018 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 270, p. 208-215Article in journal (Refereed) Published
Abstract [en]

Ethanol production at high-gravity promise to achieve concentrations over the threshold for an economical distillation process and concurrently reduce water consumption. However, a persisting limitation is the poor mass transfer conditions resulting in low ethanol yields and concentrations. Hereby, the combination of an acetone/water oxidation pretreatment process (AWO) with a liquefaction/saccharification step, using a free-fall mixer, before simultaneous saccharification and fermentation (SSF) can realize ethanol concentrations of up to ca. 74 g/L at a solids content of 20 wt.%. The free-fall mixer achieved a biomass slurry’s viscosity reduction by 87 % after only 2 h of enzymatic saccharification, indicating the efficiency of the mixing system. Furthermore, the direct enzymatic treatment of AWO pretreated corn stover (CS) by a GH11 recombinant xylanase, led to the production of xylooligosaccharides (XOS) with prebiotic potential and the removal of insoluble fibers of hemicellulose improved the glucose release of AWOCS by 22 %.

Place, publisher, year, edition, pages
Elsevier, 2018
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70763 (URN)10.1016/j.biortech.2018.09.018 (DOI)000447384200025 ()30218937 (PubMedID)2-s2.0-85053040053 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-09-18 (svasva)

Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2018-11-01Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-0078-5904

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