Study On Isolation And Characteristics Of Heavy Metal-tolerant Polycyclic Aromatic Hydrocarbons Degrading Bacteria

At present,with the continuous development of the level of industry and agriculture in China,serious environmental pollution has been caused,especially soil pollution.Among them,heavy metals and polycyclic aromatic hydrocarbons（PAHs）are two typical persistent compound pollutants in the environment,which have the characteristics of triple effect and are difficult to biodegrade,posing a serious threat to the ecosystem and human health.Due to the advantages of no secondary pollution,low cost and better treatment effect,bioremediation of heavy metal and polycyclic aromatic hydrocarbon composite pollution by bioremediation has become a research hotspot.In this study,pyrene（PYR）and dibenzothiophene（DBT）were selected as model compounds,and three strains of pyrene and DBT degrading bacteria resistant to heavy metals Cr（Ⅵ）,Pb²⁺,and Cu²⁺were screened and isolated from the petroleum contaminated soil of Shengli Oilfield,and they were named YH-1,YH-2 and YH-3 were identified as Pseudomonas sp,Cellulosimicrobium sp and Rhodococcus sp through 16S r RNA molecular biology technology.Further select the compound bacteria（YH-1 and YH-3）with good degradation effect to conduct single factor experiments to study the influence of compound ratio,bacterial solution inoculation amount and substrate concentration on the biodegradation effect of pyrene and DBT.The experimental results It shows that when the compound ratio is1:1,the amount of bacterial solution is 10%（v:v）,and the concentration of pyrene and DBT is 50 mg/L,the 5 d of pyrene and DBT is measured by ultrasonic extraction-UV spectrophotometry.The degradation rates were 42.36%and 86.32%,and gas chromatography-mass spectrometry（GC-MS）was used to verify the degradation ability of the compound bacteria on pyrene and DBT.The 5 d degradation rates were 46.32%and78.69%,respectively.The compound bacteria degraded pyrene while simultaneously adsorbing and transforming heavy metals Cr（Ⅵ）,Pb²⁺and Cu²⁺.The results showed The 5 d removal rates of heavy metals Cr（Ⅵ）,Pb²⁺,and Cu²⁺by the compound bacteria were 68.63%,97.66%and81.73%respectively.The functional groups such as carboxyl,hydroxyl,amino and amide groups on the cell surface of the compound bacteria participated in the heavy metal ions Cr（Ⅵ）,Pb²⁺,Adsorption of Cu²⁺.Transmission electron microscopy and TEM-EDS results show that heavy metals can pass through the cell membrane of the strain and accumulate in the cell.According to the bacterial gene scan map,the paa gene cluster of strains YH-1 and YH-3 for the degradation pathway of polycyclic aromatic hydrocarbon phenylacetic acid,the hpa gene cluster and the fad gene cluster involved in fatty acid degradation,the xyl gene cluster and cat involved in the degradation of pyrene were determined gene cluster,nah gene cluster involved in the degradation of naphthalene,in addition to heavy metal resistance genes,such as copper resistance gene pco B,chromium resistance gene chr A,mercury resistance gene cluster mer and czc,heavy metal transporter gene The credibility of cop B and KEEG annotations reached more than 99%.The existence of these functional genes confirmed the pyrene degradation ability of the compound bacteria and the resistance to heavy metals Cr（Ⅵ）,Pb²⁺,and Cu²⁺.The compound bacteria（YH-1 and YH-3）are used as the additional bacteria source and inoculated into the pyrene-heavy metal compound contaminated soil for bio-enhanced remediation.The removal rate of pyrene in the soil treated with P1,P2,P3,and P4 was67.72%,70.04%,77.94%and 66.84%,respectively,after bio-enhanced remediation for 60days.The results showed that the compound bacteria strengthened the remediation of pyrene in compound contaminated soil.High-throughput sequencing technology was used to determine the changes in the structure of the microbial flora in the soil.The results showed that after 60 days of enhanced bioremediation,Nocardioides,Bacillus,and Coccus were found in the contaminated soil.Sphaerobacter has become the dominant genus.At the same time,the BCR continuous extraction method was used to determine the different forms of heavy metals of lead,chromium,and copper in the soil.The results show that the acid extractable state of heavy metals in the soil has been reduced,while the heavy metal residue state there are different degrees of increase,indicating that the addition of compound bacteria has a certain repair effect on the heavy metals in the soil.