| Polycyclic aromatic hydrocarbons (PAHs) represent a major class of persistent organic pollutants (POPs), which are widespread in the environment. PAHs have attracted particular concern due to their documented acute toxicity, mutagenicity, and carcinogenicity. With the rapid urbanization and industrialization in China, increasing human activities have significantly contributed to PAHs pollution, which is characterized by high salinity, or is often co-occurred with other pollutants such as arsenic. Well-known PAHs and arsenic co-contaminated sites include wood preservation sites, coking and chemical industry sites, and mining and metallurgy industry sites. The saline PAH-contaminated sites include oil-polluted soil, marine and mangrove sediments. Microbial degradation is a major and effective alternative to physico-chemical methods in removing PAHs from contaminated sites. Numerous strains have been isolated and characterized for their abilities to degrade PAHs. However, most of them are non-halotolerant or non-arsenic resistant PAHs-degraders, making these isolates unfit candidates for remediation of hypersaline or PAHs-arsenic co-contaminated environment. Thus, the objectives of this study were to (1) obtain halophilic/halotolerant or arsenic-resistant PAHs-degraders;(2) characterize their potential in pollutant metabolism and identify the related functional genes and enzymes, and (3) propose mechanisms of pollutant metabolism. The main findings are as follows:(1) A halotolerant Martelella sp. AD-3used in the study was previously isolated by our laboratory from petroleum-contaminated soil. The PAHs biodegradability of AD-3and proposed metabolic pathway were characterized as follows:(a) The isolate AD-3could utilize anthracene, phenanthrene, or pyrene as the sole carbon source, and co-metabolize benzo[a]pyrene. After incubation of6d under the optimal conditions (salinity3%and pH9.0), the removal percentages of200mg L-1phenanthrene and25mg L-1anthracene were100%and94.6%, respectively. AD-3could remove only9.8%of10mg L-1pyrene within8d. Adding biosurfactant (rhamnolipid), PAHs intermediates (salicylate and succinate), and nutrient (yeast extract) to the medium individually greatly enhanced pyrene degradation. Co-metabolism of benzo[a]pyrene was observed in AD-3using latter three compounds as growth substrates, being highest removal efficiency with salicylate (37.9%of2mg L-1benzo[a]pyrene was removed within8d). AD-3could grow on PAHs under a range of pH6.0-10.0, being optimum at9.0, and grow on phenanthrene at salinities as high as15%(w/v). This was because isolate AD-3was screened from a highly saline and alkaline petroleum-contaminated site.(b) AD-3could degrade binary PAHs mixture, such as phenanthrene with pyrene or phenanthrene with benzo[a]pyrene. Compared to degradation of single PAH, both pyrene and benzo[a]pyrene degradation were enhanced by the presence of phenanthrene, while phenanthrene degradation was inhibited to some extent by the presence of pyrene or benzo[a]pyrene. The degree of inhibition of benzo[a]pyrene was significantly greater than that of pyrene. This may attribute to the competitive inhibition between PAHs and their co-metabolism.(c) Based on the identified metabolites, enzyme activities and the utilization profile of probable intermediates, phenanthrene degradation by isolate AD-3was proposed in two distinct routes, namely C-9,10and C-3,4routes. In C-3,4route, metabolism of1-hydroxy-2-naphthoic acid was via "naphthalene" route; Salicylic acid was converted to gentisic acid, followed by ring fission to form TCA-cycle intermediates; The transformation sequence from1-hydroxy-2-naphthoic acid to salicylic acid via the formation of1-naphthol as the intermediate,which has been seldom reported.(2) An arsenate-reducing and PAH-degrading bacterial isolate was obtained from PAHs and arsenic co-contaminated soil samples, which were taken from an aged coking plant site located in Shanghai city. The isolate, designated PAHAs-1, was identified as Pseudomonas species based on16S rRNA gene analysis. The findings are as follows:(a) MICs of As(V) and As(Ⅲ) are22mM and6mM, respectively. The isolate completely reduced1.5mM As(V) within48h and removed approximately100%and50%of60mg L-1phenanthrene and20mg L-1pyrene within60h, respectively. It could reduce As(V) when this PAHs mixture was supplied as the sole carbon source, but a slow mean reduction rate (4.62μM h-1) was observed. The presence of arsenic affected the cell growth and concurrent PAHs removal, and this depended on PAH species and arsenic concentration. Adding sodium lactate (200mg L-1) to the medium greatly enhanced the As(Ⅴ) reduction and pyrene co-metabolism.(b) The identification of a subunit of the aromatic ring-hydroxylating dioxygenase gene, arsenate reductase and arsenite transporter genes supported the dual function of this isolate. The finding of the latter two genes indicated that PAHAs-1possibly reduced As(Ⅴ) via the known detoxification mechanism. The mechanism of arsenic detoxification in PAHAs-1relies on intracellular As(Ⅴ) reduction followed by a rapid As(Ⅲ) efflux pump.(c) The effects of PAHAs-1on growth and arsenic uptake and translocation in Pteris vittata L. and phenanthrene dissipation were studied hydroponically. In an12-d experiment, PAHAs-1generally promoted plant growth, with21.0-38.7%frond biomass increase and13.5-66.3%root biomass increase. Without inoculation, arsenic concentrations in P. vittata fronds and roots were246.7-438.9and102.6-231.4mg kg-1, respectively. In the presence of PAHAs-1, elevated arsenic concentrations in P. vittata (754.1-1425.7and121.5-351.4mg kg-1As in fronds and roots, respectively) and increased arsenic transfer factors (116-315%) were observed. Given that arsenate reduction was observed only in inoculation treatments, we concluded that a PAHAs-1-mediated As(Ⅴ) reduction in the growth media was mainly responsible for the enhancement. Furthermore, inoculation with PAHAs-1in general reduced the superoxide dismutase activity and reduced glutathione level in P. vittata tissues, demonstrating the beneficial effect of PAHAs-1on alleviation of oxidative stress. The dissipation of phenanthrene from the growth media was mainly due to the degradation by PAHAs-1, while the contribution of P. vittata in phenanthrene removal only accounted for<1%.To the best of our knowledge, this work represents the first report of phenanthrene degradation by a halotolerant PAH-degrading strain via C-9,10and C-3,4routes, and an isolate showing simultaneous degradation of PAHs and reduction of arsenic. The results obtained from the study lay a foundation for the two isolates being used in the bioremediation of contaminated sites. |
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