Degradation of polycyclic aromatic hydrocarbons (PAHs) by mangrove sediment fungi

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are a class of important environmental pollutants which usually occur during the uncompleted combustion process. They are widely distributed in our surroundings. Their occurrences have a major concern due to their persistent, toxic and carcinogenic properties. Microbial biodegradation is one of the most important natural processes which can influence the fate of pollutants in both terrestrial and aquatic environment. Mangroves and wetlands usually serve as sinks of different kinds of environmental pollutants, which lead to the possibility of the occurrence of a diversity of different microbes with degradation ability. The bacterial degradation of PAHs is well documented; however, little information is available of the biodegradation on PAHs by mangrove sediment fungi. The investigation reported in this dissertation aims to isolate PAHs-degrading fungal species from mangrove sediments, to explore the degradative pathways, and to further analyze the related key enzyme and its molecular characterization, by using two PAHs - anthracene (ANT) and benz[a]anthracene (BAA) as the model PAHs. Two fungi, with their respective degradative capability of ANT and BAA, were isolated from the PAHs-contaminated mangrove sediments from Ma Wan, Hong Kong, by using the mineral salt medium (MSM) with one of two PAHs as the sole carbon source. According to the morphology and molecular identification by 18S rDNA sequence, both fungi were identified as Fusarium solani, namely as MAS2 and MBS1, respectively. Results from analysis on the degradative percentage have demonstrated that removal of ANT by F. solani MAS2 and that of BAA by F. solani MBS1 reached 40% and 60% of the added amount (ANT: 50 mg l-1; BAA: 20 mg l-1) after 40 days of incubation, respectively. Investigation by solid phase microextraction (SPME) combined with gas chromatography / mass spectrometry (GC/MS) was used to detect and characterize different metabolites during the degradation. It was indicated that the fungal species can degrade the tested substrates through their respective quinone molecules to generate phthalic acid. In the lignin-degradation enzyme system (LDS), including laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), are suggested to play an important role during the biodegradation of PAHs by fungi. But during the degradation process of ANT and BAA, only laccase was detected. Further purification and characterization of laccase was carried out by incubating F. solani MAS2 in the MSM with ANT as the sole carbon source. An extracellular laccase was extracted from the aqueous portion of culture medium, and purified by ethanol precipitation, ion-exchange chromatography and gel-filtration, in sequence. Both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE have revealed that the purified laccase was a monomeric glycoprotein (72 kDa) containing approximately 12% carbohydrate. Characterization of this enzyme was carried out with 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) as the substrate. The enzyme was stable at a wide pH range between 3 and 11 and at the temperature up to 50°C, with the maximum activity being detected at pH 3.0 and at a high temperature of 70°C. Moreover, this enzyme also demonstrated a high tolerance against different heavy metal ions (20 mM), such as Pb2+, Ni2+, Cr3+. Its activity, interestingly, could be enhanced by Hg2+, which might function as an activator, similar to copper ions (Cu2+) in laccases previously reported. When incubated within the organic solvents of acetonitrile and methanol, more than 70% of laccase activity could be preserved at up to 5% of the concentration. The kinetic constants (Km and kcat) of laccase reacting with different phenolic compounds indicate that ABTS and 2,6-dimethoxyphenol (DMOP) can serve as the more specific substrates compared with catechol, guaiacol as well as syringladazine in the tested buffer of 100 mM sodium tartrate (pH 4.5). Further investigation might be carried out to understand whether the high heavy metal tolerance features of laccase is related to the high heavy metal loading in the sediments, exerting an inductive effect. Furthermore, an in vitro degradation of ANT was also carried out by directly applying purified laccase in the cell-free system. The results showed its capability on transforming the substrate, which indicates that the enzyme has the potential for practical enzymatic applications related to of environmental pollutants biodegradation, especially in the heavy metal-contaminated areas. Based on the identification of six tryptic peptides of the purified laccase from matrix-assisted laser desorption/ionization (MALDI) - Time of flight (TOF) - MS/MS, this purified laccase was confirmed having the identity of a hypothetical protein from Nectria haematococca mpVI 77-13-4, which has its asexual stage as F. solani based on the information available in Genbank. Through the analysis on the genomic DNA of that protein, the laccase-encoded gene was cloned and sequenced at both the genomic and complementary DNA (cDNA) level using the devised specific primers, and the comparisons with other laccases have exhibited the novel conformation of this laccases. In summary, this is the first study focusing on the degradation of selected PAHs (ANT and BAA) by mangrove sediment fungi. The degradative pathways were found to be similar to fungi reported in previous studies, but different from bacteria. The catalytic characterization and molecular identification of purified extracellular laccase provide the evidences that this enzyme might belong to a new member of laccase family. The less substrate-specific property confers the potential for the environmental remediation for a broad range of recalcitrant pollutants. Further studies on the exploration on expression and characteristics on the recombinant laccase in yeast strains as well as the establishment of a genetically engineered microbial producer can be undertaken for the environmental applications.