Biodegradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin by a ligninolytic fungus and its laccases

Research output: PhD ThesisPhD-Thesis - Research and graduation internal

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Abstract

White-rot fungi produce extracellular lignin-modifying enzymes (LME) that degrade lignin, a class of complex organic polymers, for fungal nutrition and growing. Laccase is the most well-characterized enzyme in the group of LME. Besides, because of their broad substrate specificity, laccase is also capable of degradation of many hazardous pollutants such as herbicides, pesticides, PAHs, dyes, pharmaceutical chemicals, polychlorinated biphenyls, etcetera. The working hypothesis of this study was that ligninolytic fungi and LME can degrade 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) the most toxic congener of the group of dioxins. In order to collect scientific evidence supporting the hypothesis and achieving the main aim of the thesis, which is the investigation into the 2,3,7,8-TCDD biodegradation potential of ligninolytic fungi and lignin-modifying enzymes, the experiments were conducted using white-rot fungi collected from Vietnamese forests and 2,3,7,8-TCDD as subjects and object of the research respectively. In chapter 2, the isolation and screening process for LME producing fungi were described as well as the selection of 2,3,7,8-TCDD degrading fungi. From the 76 fungal isolates collected from Vietnamese forests, 70 isolates showed LME activity based on colorimetric screening using oxidized guaiacol as an indicator. TRigidoporus sp. FMD21 (FMD21) was the only strain that showed a 2,3,7,8-TCDD degradation ability. This strain produced 238 800 U/L of laccase after ten days while MnP activity showed the highest activity of approximately 40 U/L at day 4 in the PDSRb medium consisting of potato dextrose powder, soy meal, and rice bran. In chapter 3 and chapter 4 the enzymatic degradation of 2,3,7,8-TCDD was characterized, while the degradation of 2,3,7,8-TCDD was determined using the DR-CALUX® bioassay. FMD21 ExE, which mainly consisted of laccase, degraded 77.4% of 2,3,7,8-TCDD within 36 days, while starting with an initial concentration of 0.5 pgTEQ/mL. Three pure laccase isoforms Lac_MD2, Lac_MD8 and Lac_MD9 showed 2,3,7,8-TCDD degradation activity. In this study, we found evidence for cleavage of the diaryl ether bond in the 2,3,7,8-TCDD molecule, and based on this observation we propose a new degradation mechanism of 2,3,7,8-TCDD in which 3,4-dichlorophenol is the main metabolite formed during the enzymatic degradation. The FMD21 genome consists of 38.98 Mbps with a GC content of 47.4 % and 8245 genes were predicted. Laccase and ligninolytic peroxidase genes were focused on since they are most likely involved in the degradation of 2,3,7,8-TCDD and other aromatic pollutants. The genome of FMD21 contains 12 predicted laccase genes and 13 putative ligninolytic peroxidases, which were annotated as MnP or versatile peroxidases. The diversity of FMD21 laccases were not only exhibited at the gene level, but it was also demonstrated through the complexity of the isoforms as well. In chapter 5, the purification process and the properties of FMD21 laccase isoforms are illustrated in detail. There were 13 different laccase isoforms observed in the zymogram of the FMD21 extracellular extract. Each isoform showed its distinctive properties, particularly in the catalytic pattern, where the kinetic behavior appeared to be specific and depending on the combination of isoform and substrate. These isoforms are alkaline tolerant, active at high temperatures, solvent resistant, and even enhanced by solvents such as methanol and ethanol, making them promising candidates for industrial application. Our main conclusion is that Rigidoporus sp. FMD21 appears to be an applicable fungal candidate for myco-remediation as this fungus produces LMEs with moderate levels of MnP and a high level of laccases, it grows in the simple medium and secretes LMEs and laccase isoforms that possess a capacity to effectively degrade and detoxify the most toxic dioxin congener, 2,3,7,8-TCDD. To enable the latter field applications, more studies are needed where FMD21 is applied under field conditions and are subjects of future studies.
Original languageEnglish
QualificationPhD
Awarding Institution
  • Vrije Universiteit Amsterdam
Supervisors/Advisors
  • Brouwer, A, Supervisor
  • Dang, T.C.H., Supervisor, External person
  • Roelofs, Dick, Co-supervisor
  • de Boer, Tjalf Elmer, Co-supervisor, External person
Award date28 Jan 2022
Place of Publications.l.
Publisher
Publication statusPublished - 28 Jan 2022

Keywords

  • White-rot fungi
  • ligninolytic fungi
  • lignin-modifying enzymes
  • laccases
  • dioxin
  • 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • biodegradation
  • Rigidoporus
  • isoforms

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