Mechanisms of biodegradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria isolated from mangrove sediments
Student thesis: Doctoral Thesis
Contamination of polycyclic aromatic hydrocarbons (PAHs) in coastal sediments is of great environmental concern because of their known or suspected toxic, mutagenic and carcinogenic hazards. Recent researches have demonstrated that mangrove wetlands harbor many PAH-degrading microorganisms and are able to remedy PAHs. The present study aims to investigate the mechanisms of PAH biodegradation by the bacteria isolated from mangrove sediments, including the species diversity and activity of PAH-degrading bacteria, the genetic diversity of the initial dioxygenase, and the metabolic pathway of fluoranthene, a four-ring PAH, in the isolated bacteria. Eleven bacterial strains belonging to four genera, namely Mycobacterium (3 strains), Sphingomonas (5), Terrabacter (2) and Rhodococcus (1), were isolated from a single surface sediment sample from Ho Chung mangrove swamp. The present study for the first time isolated Terrabacter strains growing with a four-ring PAH, fluoranthene, as the sole carbon and energy source. Mycobacterium spp. were found to be responsible for the removal of four-ring PAHs, while Sphingomonas spp. were responsible for the biodegradation of three-ring PAHs. The expression of PAH-dioxygenase genes confirmed the roles of Gram-positive PAH-degrading bacteria in high molecular weight (HMW) PAH biodegradation and Gram-negative strains in the low molecular weight (LMW) PAH biodegradation. The surface mangrove sediments harbor a high diversity of PAH-degrading bacteria with different abilities to degrade PAHs of different molecular weights. A total of 32 PAH-degrading bacterial strains isolated from mangrove sediments, including strains of Mycobacterium, Sphingomonas, Terrabacter, Sphingopyxis, Sphingobium and Rhodococcus, was examined for diversity of the PAH ring-hydroxylating dioxygenase genes. Two sets of PCR primers were constructed to detect the nidA-like and nahAc-like sequences of dioxygenase α subunit, and the DNA fragments were amplified by PCR from all Gram-positive bacteria using the nidA-like primers and from all but two Gram-negative bacteria using the nahAc-like primers. Sequence analysis of the gene fragments amplified using nidA-like primers showed the presence of at least three subtypes of nidA-like genes, namely fadA1, nidA, and fadA2, in the mangrove isolates. The nahAc-like primers determined phnA1a from 8 of 16 Gram-negative isolates, and generated amplicons with 32-36% similarities to PhnA1a and 53-93% identities to p-cumate dioxygenase. These results suggested that the nidA-like and nahAc-like genes were prevalent in the PAH-degrading bacteria isolated from the mangrove sediments. A novel PAH-dioxygenase was cloned from Terrabacter sp. HH4 isolated from mangrove sediment. Five open reading frames (three complete and two partial) were identified from the 5.2 kb cloned fragment. The gene cluster shared more than 90% similarity with nidA3B3 cluster from Mycobacterium sp. PYR-1, but the fragment before the 20 bp of the start codon of fadA1 differed from that of nidA3B3 cluster and a possible transposase gene replaced the regulator protein gene nidR in nidA3B3 cluster. Recombinant expression of fadA1B1 with electron transfer chain proteins was able to transform phenanthrene, fluoranthene and pyrene to cis-dihydrodiol metabolites. The fadA1B1 was constitutively expressed in strain HH4 and responsible for the degradation of fluoranthene by Terrabacter and Mycobacterium strains in mangrove sediments. Terrabacter sp. strain HH4 was unique in that it uses fluoranthene, but not any other PAH compounds, as the sole carbon and energy source. A metabolic pathway started from 1,2-dihydroxy-, 2,3-dihydroxy- and monohydroxy-fluoranthene was determined for biodegradation of fluoranthene in strain HH4. Both dihydroxy-fluoranthene were further transformed to 9-fluorenone-1-carboxylic acid and finally converted to tricarboxylic acid cycle metabolites. This strain rapidly degraded fluoranthene without accumulation of any dead end products, as was different from Mycobacterium sp. strain HH1 that accumulated 7-methoxy-8- hydroxy-fluoranthene, suggesting that Terrabacter sp. HH4 may be useful for bioremediation of sediments contaminated with fluoranthene. The present study revealed that the prevalent Mycobacterium, Sphingomonas, and Terrabacter strains were responsible for biodegradation of PAHs in surface mangrove sediments. These strains harbored nidA, fadA1, and phnA1a as three major types of dioxygenase genes. Novel HMW PAH-degrading Terrabacter strains were isolated from the mangrove sediments and a unique metabolic pathway for fluoranthene was determined in strain HH4.
- Polycyclic aromatic hydrocarbons, Ecology, Microbiology, Biodegradation, Bioremediation, Mangrove soils, Bacteria