Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications

Marine fungi have been gaining interest among researchers in recent years due to their unique adaptations to high salinity and extreme pressure in the ocean, which have made them as potential bioremediators. However, studies on the degradation of heterocyclic aromatic hydrocarbons (HACs) by marine f...

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Main Author:	Mohammad Farhan Darin, Azri
Format:	Thesis
Language:	English
Published:	2022
Subjects:	QR Microbiology
Online Access:	http://ir.unimas.my/id/eprint/38367/6/Mohammad%20Farhan%20Darin_fulltext.pdf
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spelling	my-unimas-ir.383672023-03-13T03:53:38Z Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications 2022-04-22 Mohammad Farhan Darin, Azri QR Microbiology Marine fungi have been gaining interest among researchers in recent years due to their unique adaptations to high salinity and extreme pressure in the ocean, which have made them as potential bioremediators. However, studies on the degradation of heterocyclic aromatic hydrocarbons (HACs) by marine fungi in general, are still limitedly reported. This work aims to establish basic knowledge on the potential heterocyclic hydrocarbon-degrading marine fungi for bioremediation applications. Specifically, the work focused on marine fungi isolated from selected habitats in Sarawak coastal regions. A marine fungus, identified as Myrothecium cinctum, isolated from Buntal, Sarawak was found to exhibit the best performance in the degradation of carbazole, a type of HACs. Preliminary studies on the decolourisation of Remazol Brilliant Blue-R (RBBR) dye by M. cinctum showed that 80 to 100% decolourisation rate was successfully achieved when RBBR dye concentrations used was 0.01-0.05% (v/v), indicating the presence of ligninolytic enzymes in the organism. Favourable culture conditions for the degradation of carbazole by M. cinctum laccases showed that the optimal activity of M. cinctum laccases was achieved at pH 5 at an incubation temperature of 30 ºC, for a period of 12 days and in static condition. The highest activity of M. cinctum laccases recorded was 211.21 ± 2.57 U/L, representing a 1.4-fold enhancement over that produced by the control strain (Marasmius chladophyllus). Interestingly, the production of laccases in M. cinctum during the biodegradation of carbazole was found to be feasible without the addition of inducers as the presence of carbazole itself was found to mediate the production of laccases. Biotransformation of selected hydrocarbons by M. cinctum laccases showed a high percentage of degradation. Complete degradation of fluorene and dibenzofuran was achieved whilst 95.2% of degradation was observed for carbazole. Based on the capability of M. cinctum laccases to exhibit a high degree of degradation of different hydrocarbons, it can be concluded that the organism possesses low substrate specificity. In addition, a carbazole degradative pathway was proposed based on the detection of intermediates compound formed during the degradation. The presence of a single band in the SDS-PAGE analysis upon the purification of M. cinctum laccase indicates the presence of only a single isoform of laccases. The partially purified M. cinctum laccases showed the highest activity at pH 6, incubation temperature of 30 oC and carbazole concentration of 0.08% (w/v). The apparent Km and Vmax value of M. cinctum laccases were determined as 0.08 mM and 344.83 U/mg, respectively. Further analysis of the laccase gene from M. cinctum showed the enzyme has 259 bp of sequence for genomic DNA and 188 bp for cDNA isolated using generated designed primer. An intron size of 71 bp was observed spliced corresponded to the 5′GT–AG3′ rule. Copper binding region I and II were successfully sequenced, which also revealed a conserved putative cysteine supporting the presence of laccases in M. cinctum. Overall, this work has established several novel findings related to heterocyclic hydrocarbon-degrading marine fungi, which provide useful insights into their bioremediation applications. Keywords: Bioremediation, heterocyclic aromatic hydrocarbons (HACs), laccase, marine fungi, Myrothecium cinctum UNIMAS 2022-04 Thesis http://ir.unimas.my/id/eprint/38367/ http://ir.unimas.my/id/eprint/38367/6/Mohammad%20Farhan%20Darin_fulltext.pdf text en validuser phd doctoral Universiti Malaysia Sarawak Department of Molecular Biology
institution	Universiti Malaysia Sarawak
collection	UNIMAS Institutional Repository
language	English
topic	QR Microbiology
spellingShingle	QR Microbiology Mohammad Farhan Darin, Azri Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications
description	Marine fungi have been gaining interest among researchers in recent years due to their unique adaptations to high salinity and extreme pressure in the ocean, which have made them as potential bioremediators. However, studies on the degradation of heterocyclic aromatic hydrocarbons (HACs) by marine fungi in general, are still limitedly reported. This work aims to establish basic knowledge on the potential heterocyclic hydrocarbon-degrading marine fungi for bioremediation applications. Specifically, the work focused on marine fungi isolated from selected habitats in Sarawak coastal regions. A marine fungus, identified as Myrothecium cinctum, isolated from Buntal, Sarawak was found to exhibit the best performance in the degradation of carbazole, a type of HACs. Preliminary studies on the decolourisation of Remazol Brilliant Blue-R (RBBR) dye by M. cinctum showed that 80 to 100% decolourisation rate was successfully achieved when RBBR dye concentrations used was 0.01-0.05% (v/v), indicating the presence of ligninolytic enzymes in the organism. Favourable culture conditions for the degradation of carbazole by M. cinctum laccases showed that the optimal activity of M. cinctum laccases was achieved at pH 5 at an incubation temperature of 30 ºC, for a period of 12 days and in static condition. The highest activity of M. cinctum laccases recorded was 211.21 ± 2.57 U/L, representing a 1.4-fold enhancement over that produced by the control strain (Marasmius chladophyllus). Interestingly, the production of laccases in M. cinctum during the biodegradation of carbazole was found to be feasible without the addition of inducers as the presence of carbazole itself was found to mediate the production of laccases. Biotransformation of selected hydrocarbons by M. cinctum laccases showed a high percentage of degradation. Complete degradation of fluorene and dibenzofuran was achieved whilst 95.2% of degradation was observed for carbazole. Based on the capability of M. cinctum laccases to exhibit a high degree of degradation of different hydrocarbons, it can be concluded that the organism possesses low substrate specificity. In addition, a carbazole degradative pathway was proposed based on the detection of intermediates compound formed during the degradation. The presence of a single band in the SDS-PAGE analysis upon the purification of M. cinctum laccase indicates the presence of only a single isoform of laccases. The partially purified M. cinctum laccases showed the highest activity at pH 6, incubation temperature of 30 oC and carbazole concentration of 0.08% (w/v). The apparent Km and Vmax value of M. cinctum laccases were determined as 0.08 mM and 344.83 U/mg, respectively. Further analysis of the laccase gene from M. cinctum showed the enzyme has 259 bp of sequence for genomic DNA and 188 bp for cDNA isolated using generated designed primer. An intron size of 71 bp was observed spliced corresponded to the 5′GT–AG3′ rule. Copper binding region I and II were successfully sequenced, which also revealed a conserved putative cysteine supporting the presence of laccases in M. cinctum. Overall, this work has established several novel findings related to heterocyclic hydrocarbon-degrading marine fungi, which provide useful insights into their bioremediation applications. Keywords: Bioremediation, heterocyclic aromatic hydrocarbons (HACs), laccase, marine fungi, Myrothecium cinctum
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Mohammad Farhan Darin, Azri
author_facet	Mohammad Farhan Darin, Azri
author_sort	Mohammad Farhan Darin, Azri
title	Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications
title_short	Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications
title_full	Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications
title_fullStr	Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications
title_full_unstemmed	Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications
title_sort	isolation and characterisation of heterocyclic hydrocarbon degrading marine fungi for potential bioremediation applications
granting_institution	Universiti Malaysia Sarawak
granting_department	Department of Molecular Biology
publishDate	2022
url	http://ir.unimas.my/id/eprint/38367/6/Mohammad%20Farhan%20Darin_fulltext.pdf
_version_	1783728501338144768

Isolation and Characterisation of Heterocyclic Hydrocarbon Degrading Marine Fungi for Potential Bioremediation Applications

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