Study On The Remediation Characteristics Of Petroleum-contaminated Soil By Bioaugmentation Of Inoculating Petroleum-dagrading Bacteria

Biosugmentation is based on inoculating exogenous degrading bcteria into the soil to degrade petroluem and It is a targeted,effcient and fast degradable bioremediation approach.Petroleum-degrading bacteria consortium which were screened from petroleum-contaminated soil around a certain oil well site in Yanchang,Shaanxi and isolated by enrichment culture were inoculated into the different polluted-time petroleum-contaminated soils?freshed and aged petroleum-contaminated soil?to investigate the effects of moisture content?5.1%and 15.0%?and different inoculation amount(10³,10⁴,10⁵,10⁷,10⁸ cfu·g^-1)on petroleum hydrocarbon degradation.The growth of inocula,the changes of indigenous bacterial community,and the effect of bioaugmentation on the gene expression of polycyclic aromatic hydrocarbon functional gene estradiol dioxygenase?EDO?.were determined using high throughput sequencing and qPCR technology.Results showed that the composition of petroleum-degrading bacterial consortium are mainly Proteobacteria phylum?99.75%?,Gamma-proteobacteria class?99.49%?,Pseudomonadales order?99.36%?,Moraxellaceae family?87.33%?,Acinetobacter Sp.?87.32%?,and Pseudomonadaceae family?12.04%?,Pseudomonas Sp.?12.00%?.The removal effect of petroleum hydrocarbons is not obvious under natural soil moisture for bioaugmentation of freshed petroleum-contaminated soil.Repairing for 60days,the removal rates of inoculating MC are from 2.09%to 6.93%.Keeping the soil about 15%moisture is benefit for bioremediation efficiency than in the 5.1%moisture.When soil moisture was 15.0%,the inoculation amounts of degrading strains were 10⁸cfu·g^-1,the best removal efficiencies of total petroleum hydrocarbon achieved 14.25%in freshed petroleum-contaminated soil after 60 days.The concentration of petroleum hydrocarbons decreased from 14111 mg·kg^-11 to 12100 mg·kg^-1.The concentration of alkanes decreased from 11567 mg·kg^-1to 10367 mg·kg^-1,and the concentration of polycyclic aromatic hydrocarbons decreased from 1433 mg·kg^-11 to 1225 mg·kg^-1respectively.The inoculated degrading bacteria played a certain role in removing alkanes in the soil,but had no obvious effect on the removal of polycyclic aromatic hydrocarbons.After 60 days of inoculation,the Shannon index and ACE index of soil decreased from 7.26 and 12853.04 to 1.77 and 6947.85,respectively.the relative abundance of Proteobacteria phylum increased from 27.72%to 89.86%;Acinetobacter genus increased from 0.05%to 77.87%;Pseudomonas genus increased from 0.14%to5.38%.It shows that the degrading bacteria can survive and grow in freshed contaminated soil with 15%soil moisture.When soil moisture was 15.0%,the inoculation amounts of degrading strains were10⁸ cfu·g^-1,the best removal efficiencies of total petroleum hydrocarbon achieved18.67%in aged petroleum-contaminated soil after 60 days of bioremediation.The concentration of petroleum hydrocarbons decreased from 15233 mg·kg^-11 to 12389mg·kg^-1.The concentration of alkanes decreased from 12000 mg·kg^-11 to 10392 mg·kg^-1,and the concentration of polycyclic aromatic hydrocarbons decreased from 3200mg·kg^-11 to 1967 mg·kg^-1.The inoculation of MC degrading bacteria promoted the removal of polycyclic aromatic hydrocarbons.After 60 days of inoculation,the Shannon index and ACE index of soil decreased from 7.29 and 11841.83 to 5.77 and11234.11,respectively.the relative abundance of Proteobacteria phylum increased from28.22%to 50.22%;Acinetobacter genus increased from 0.04%to 20.70%;Pseudomonas genus increased from 0.26%to 2.41%.It shows that the inoculated degrading bacteria can grow and survive in the aged contaminated soil,but its growth and survival is worse than that in the freshed contaminated soil.Inoculation of exogenous degrading bacteria in the two different polluted-time soils changed the compositions of indigenous bacterial community and decreased alpha diversity significantly.The soil microbial diversity and richness both showed a decreasing trend.After inoculation with MC 10⁷ cfu·g^-11 into freshed and aged polluted soil for 7 days in both 5.1%and 15.0%soil moisture,the relative abundance of Proteobacteria phylum increased from 27.72%to 89.01%?92.99%;Acinetobacter genus increased from 0.05%to 65.09%?85.85%and Pseudomonas genus increased from 0.14%to 3.50%?15.12%in the freshed petroleum-contaminated soil.The relative abundance of Proteobacteria phylum increased from 28.22%to 57.98%?66.35%;Acinetobacter genus increased from 0.04%to 25.86%?30.25%and Pseudomonas genus increased from 0.26%to 5.03%?30.87%in the aged petroleum-contaminated soil.increased the abundance of Proteobacteria in the soil.The research Indicated that the inocula could grow rapidly and keep dominant in the petroleum-contaminated soil during 60 days of bioaugmentation treatment.After 7 days of inoculation of petroleum hydrocarbon degrading bacteria into contaminated soil,the EDO gene copy number was reduced in different polluted-time polluted soils compared with the control.This indicates that the bioemediation of oil-contaminated soil inhibits the copy of microbial EDO genes.In the condition of5.1%and 15%soil moisture,the EDO copy number of aged contaminated soil was 8.1and 17.8 times than that of the freshed contaminated soil,respectively.It shows that the contaminated soil under long-term pollution has more polycyclic aromatic hydrocarbon functional gene estradiol dioxygenase?EDO?gene.And especially in 15%soil moisture.