The Isolation,Degradation Characteristics And Bacterial Community Structure Analysis Of Pb-resistant Phenanthrene Degrader Bacterial Consourtium

Polycyclic aromatic hydrocarbons(PAHs)and lead(Pb)are two common environmental pollutants in co-contaminated soil.Currently,reports are mainly focus on bacteria strain of biodegradation of PAHs,but the reports of Pb resistant phenanthrene degrader bacterial consortium are less.In this study,a heterotrophic bacterial consortium capable of degrading PAHs and resistant to Pb was isolated from Pb and PAHs co-contaminated soil in Shenyang,China.On the basis of the characteristics of consortium phenanthrene degrading,lead resistance and the structure,we can obtain the diversity and abundance of functional gene of the bacterial consortium via using the Illumina high-throughput sequencing technology.The main results are as follows:(1)The Pb-resistant and phenanthrene degrader bacterial consortium were isolated from Pb and phenanthrene selective stress,the contaminated soil samples were enriched for several times,finally a heterotrophic bacterial consortium capable of degrading PAHs and resistant to Pb were isolated.At 150 r/min,30? culture conditions,bacterial consortium can grow well in a liquid culture containing 200 mg/L phenanthrene and 120 mg/L Pb,and can removed 92.7%of phenanthrene under aerobic conditions in 48 h.(2)Using Illumina high-throughput sequencing technology to analyze the evolution of the community structure of the bacteria under different pollutant selection pressures,the results show:the diversity of bacteria was highest in the presence of Pb(S2)only,and the biomass of bacteria was the highest in the absence pollution(S1).Paracoccus,Achromobacter,Massilia,Shinella and Stenotrophomonaswere identified as the dominant bacteria.Under the pressure of phenanthrene(S3,S4),the abundance of Massilia was 18.31?30.04%,and in the absence of phenanthrene(S1,S2),the abundance of Massilia was 0.11?2.87%,which indicated that abundance of Massilia was only high in the environment containing phenanthrene.Abundance of Paracoccus and Achromobacter were stabilised at 26.94?31.82%and 14.77?17.86%under Pb stress(S2,S4).Overall,we hypothesized that Paracoccus and Achromobacter are related to Pb resistance.Massilia may act as a phenanthrene degrader.(3)Real time PCR was used to analysis the abundance of PAHs-degrading RHDa gene and detected Pb-resistant gene of bacterial consortium under different pollutant selection pressures,the results show:in the presence of phenanthrene(S3,S4),the abundance of RHD?gene was 8.18×104?2.18×105 copies/mL,in the absence of phenanthrene(S1,S2),the abundance of RHDa gene was 2.81×102?4.99×102 copies/mL,therefore,phenanthrene is the main factor determining the abundance of the RHDa gene.Simultaneously,the Pb-resistance gene was detected,but the known Pb-resistance genes weren't deteceted in the bacterial consortium.(4)We obtained two strain can grow under 60 mg/L Pb and 100 mg/L phenanthrene stress in liquid medium and named B1 and B2,respectively.Functional evalution of B1 and B2,results show both B1 and B2 don't possess the ability to degrade phenanthrene,but own characteristic of Pb-resistant.B1 was capable of resistant to Pb concentration of 100 mg/L,and B2 maximum resistance to Pb concentration of 1000 mg/L.Moreover,B2 grew faster at concentrations of 100 and 200 mg/L Pb than at concentration of 50 mg/L Pb.The isolated bacteria have certain reference value to clarify the Pb resistance of bacteria.