| Polycyclic aromatic hydrocarbons(PAHs)are a group of persistent organic pollutants that are widely distributed in the environment.Because of their toxicity,teratogenic,carcinogenic,and mutagenic properties,PAHs pose great risk to ecosystems and human health.Microbial degradation is one of the most potential ways to remove PAHs from the environment.A comprehensive and in-depth analysis of the mechanisms of PAHs-degradation by microbes is of great significance for bioremediation of PAHs-contaminated sites.In this study,phenanthrene(PHE)and pyrene(PYR),two typical PAHs model compounds,were chosen as test pollutants.We investigated PAHs metabolic pathways and catabolic genes to provide a comprehensive understanding of the efficient PAHs-degrading bacteria,Mycobacterium sp.WY10 and Rhodococcus sp.WB9,and to elucidate their PAHs-degradation mechanisms.A highly effective PHE-degrading co-culture containing strains WY10 and WB9 was also constructed to understand their synergistic mechanism.Furthermore,the utility of PAHs-degrading bacteria for microbial bioremediation was studied to explore their mechanism in the aged PAHs-contaminated soils.The main results obtained from the study were as follows:(1)Salicylate and phthalate pathways contributed differently on phenanthrene and pyrene degradations in Mycobacterium sp.WY10A highly effective PAHs-degrading bacterium,Mycobacterium sp.WY10,almost completely degraded 100 mg L-1 PHE within 60 h and degraded 83.2%of 50 mg L-1PYR in 72 h at the optimal conditions of p H(6.5)and temperature(28°C).Strain WY10degraded PHE with initial dioxygenation mainly on C-3,4 positions,followed by meta-cleavage to produce 1-hydroxy-2-naphthoic acid that was further metabolized to tricarboxylic acid(TCA)cycle through phthalate pathway.PYR was initially oxidized at C-4,5 position to form 4,5-dihydroxypyrene,which was further metabolized to 3,4-dihydroxyphenanthrene via ortho-cleavage,entering the PHE degradation pathway.However,both phthalate and salicylate pathways played important roles in PYR degradation.Fifty-two genes located in the“major catabolic region”of strain WY10genome are related to PAHs degradation,including a complete gene set for PHE and PYR degradation towards TCA cycle via phthalate pathway.The candidate gene/ORF,BOH72?19755,encoding salicylate synthase might contribute in the salicylate pathway.(2)Accumulation of extracellular 1-hydroxy-2-naphthoic acid limited phenanthrene mineralization by Rhodococcus sp.WB9Rhodococcus sp.WB9 could not utilize PYR,but almost completely degraded 100mg L-1 PHE within 4 days at the optimal conditions of p H(7.5)and temperature(28°C).Five different hydroxyphenanthrene compounds were identified during PHE degradation,indicating multiple routes of monooxygenase attacks.Initial dioxygenation occurred at C-1,2 and 3,4 positions,which was followed by highly complex metabolic pathways.Approximately 80.7%of PHE was accumulated as extracellular 1-hydroxy-2-naphthoic acid,indicating that oxidation of 3,4-C positions,followed by meta-cleavage to form 1-hydroxy-2-naphthoic acid was a dominant pathway.131 genes in strain WB9 genome are found responsible for aromatic hydrocarbons catabolism,including the gene encoding salicylate 1-monooxygenase that catalyzes the oxidation of 1-hydroxy-2-naphthoic acid to 1,2-naphthalenediol,and the complete gene sets for salicylate and phthalate degradation to TCA cycle intermediates.Metabolic and genomic analyses revealed that strain WB9 could degrade intracellular 1-hydroxy-2-naphthoic acid towards TCA cycle,but the extracellular accumulation of 1-hydroxy-2-naphthoic acid due to not moving into cells limited PHE mineralization.(3)Metabolic cross-feeding in the co-culture of strains WY10 and WB9accelerated phenanthrene degradation and mineralizationAn efficient PHE-degrading co-culture containing Rhodococcus sp.WB9 and Mycobacterium sp.WY10 was constructed,and it almost completely degraded 100 mg L-1 PHE within 36 h at the optimal conditions of p H(6.5)and temperature(28°C).In the co-culture,strain WY10 played a major role in PHE degradation.Strain WB9degraded PHE to form 1-hydroxy-2-naphthoic acid and exported it to the culture medium through efflux transporters.Strain WB9 could not absorb extracellular 1-hydroxy-2-naphthoic acid due to the absence of 1-hydroxy-2-naphthoate transporters.However,strain WY10 could absorb and utilize extracellular 1-hydroxy-2-naphthoic acid as a growth substrate,thereby promoting PHE degradation.Strain WY10metabolized PHE and 1-hydroxy-2-naphthoic acid intracellularly to produce phthalate and protocatechuate and exported them to the culture medium through efflux transporters.Although strain WY10 had ability to degrade intracellular phthalate,it couldn not absorb extracellular phthalate due to the absence of phthalate transporters,which limited phthalate degradation by strain WY10.In the co-culture,extracellular phthalate and protocatechuate were absorbed and degraded to TCA cycle intermediates by strain WB9.Therefore,the metabolic cross-feeding of strains WY10 and WB9accelerated PHE degradation and mineralization.(4)Both strains WY10 and WB9 were effective for bioremediation of PAHs-contaminated soils,but they differed in remediation mechanismsMycobacterium sp.WY10,Rhodococcus sp.WB9 and their co-culture showed effective for bioremediation of severely PAHs-contaminated(546-1704 mg kg-1)soils near a steel plant.They simultaneously promoted the degradation of low-molecular-weight and high-molecular-weight PAHs in the soils.The PAHs-degrading bacteria degraded 17.8-45.3%of?16 PAHs in 28 d,but no significant differences in PAHs degradation were observed in different treatments of PAHs-degrading bacteria.In the treatments of WY10 and WY10+WB9,the relative abundance of the key genus Mycobacterium and the absolute content of nid A gene were significantly higher than those of other treatments.Moreover,the degradation of PAHs was positively correlated(p<0.05)with nid A gene content,showing that WY10 carrying nid A gene played a major role in PAHs degradation.In the treatment of WB9,the relative abundance of Rhodococcus and the absolute content of RHD?-GN gene were higher than those of other treatments,and the RHD?-GN gene content increased as PAHs decreased.In the period of 0-28 d,Rhodococcus had positive correlations with dozens of indigenous genera(including Mycobacterium),and it significantly promoted the proliferation of PAHs-degrading bacteria Rhodoplanes and Sphingomona(Gram-negative).The results showed that the synergistic effect of Rhodococcus sp.WB9 and Gram-negative PAHs-degrading bacteria promoted PAHs degradation.However,microorganisms in the treatment of WY10+WB9 had weaker relationships compared to microorganisms in the treatments of WY10 and WB9,and no significant increase of PAHs-degrading bacteria was found in the treatment of WY10+WB9.Therefore,the degradation of PAHs in the treatment of WY10+WB9 was not higher than that in the treatments of WY10 and WB9.Besides,the addition of strains WY10 and WB9 did not have a significant impact on bacterial community in soils,indicating that bioaugmentation of strain WY10 and WB9was an effective and environmentally friendly option for remediating PAHs-contaminated soils.To sum up,the high-efficiency degradation ability of Mycobacterium sp.WY10and Rhodococcus sp.WB9 on PHE and PYR,as well as the promotion of PHE degradation and mineralization by the metabolic cross-feeding of strains WY10 and WB9,will be worthy of further study and application in bioremediation of PAHs-contaminated sites. |
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