Research On Screening And Identification, Characteristics Of Petroleum Degrading Bacteria In Oil Polluted Wetland

Oil pollution has always been an inevitable problem in the vicinity of oil exploration areas.There are currently three main methods,namely physical,chemical and biological remediation,which are used to solve oil pollution.The microbial remediationwith low cost,high efficiency,no secondary pollution and small limitation,is becoming one of the widely using bioremediation methods.Screening out high efficiency petroleum degrading bacteria not only has important academic value,but also has important application value and practical significance for in-situ remediation of oil polluted wetland soil.In this study,oil-contaminated soils near oil wells were sampled from Daqing wetland.Taking the crude oil as the sole carbon source,we identified the 16 S rDNA sequence,the physiological and biochemical,growth and degradation characteristics of the bacteria.Simulation experienment was also carried out to verify the deoiling effect of these petroleum degrading bacteria in oil contaminated soil.We hope to screen out high efficiency petroleum degrading bacteria.(1)Through preliminary screening experiments,110 strains of petroleum degrading bacteria were found.We found that the addition of bacteria liquid is feasible for remedying oil polluted soil through carrying out simulation experiments.The remediation capacities of the top three bacteria strains for the 2 g/kg oil polluted soil were A oil 3J1-1(A3J1-1),C3 plant J1(C3J1-2)and C2 J2,and their oil removal rates up to 63.07%,39.88% and 32.22% after 7 days' experientment,respectively.The oil removal capacity of the tenth bacteria strain was 24.6%.At the same time,we found that the bacteria strain could improve the oil removal ability by cooperate with plants(yellow beans).(2)We found that the strain C3J1-2 was a bacterium belongs to the genus Exiguobacterium,the strain E1J4-1 and the strain A3J1-1 were both genus Acinetobacter by the morphology,physiological and biochemical characteristics observation and 16 S rDNA sequencing determination of the bacteria.These bacteria strains have been proven to have good degradation effects on petroleum hydrocarbons.(3)We found that all of the three petroleum degrading bacteria have the ability to produce surfactants.The strain C3J1-2 and the strain A3J1-1 have stronger surfactant-producing abilities than the strain E1J4-1 through the blood plate method.Surfactants increased the degradation rate of petroleum hydrocarbons by enhancing the hydrophobicity of the microbial surface cells.(4)We focus on studied how the growth curves(time conditions)of the three bacteria strains varied with the changes in temperatures,salinities,pH,shaking speeds and carbon sources.We aimed to understand the growth characteristics of the strains and provide partial conditional support for subsequent field restoration.(5)We further studied how the degradation abilities and characteristics of the three bacteria strains varied with the changes in the culture time,bacterial inoculum,different bacterial liquid combinations(mixed flora)and oil concentrations.We found the degradation abilities of the three bacteria strains increased continuously with the culture time.However,the increases in degradation rate were gradually slowed down with the increase of time.The degradation rate on the 20 th day's culture reached highest,up to 62.91%,when compared with other days.The bacteria inoculum of three strains directly affects the degradation rates.The optimal inoculum of strain A3J1-1 and strain C3J1-2 were both 5%,while the optimal inoculum of strain E1J4-1 was 7%.The combination of strain C3J1-2+ and strain A3J1-1 had the highest degradation rate,up to71.99%,when compared with other mixed flora or monobactera.The degradation rates of strain E1J4-1 and strain A3J1-1 first increased and then decreased with oil concentrations.The degradation ability was the strongest when the oil concentration was reached 5 000 mg/L,in which point whe degradation rates of strain E1J4-1 and strain A3J1-1 were 80.92% and 52.00%,respectively.(6)Finally,specific primers were used to identify the degradation genes of the three petroleum-degrading bacteria.We found that strain A3J1-1 have aromatic dioxygenase(Ahd-569F21,Ahd-977R21)and naphthalene dioxygenase(Nd-546F21,Nd-954R21)genes;strain C3J1-2 have naphthalene dioxygenase(Nd-546F21,Nd-954R21)gene;strain E1J4-1 have catechol dioxygenase(C23d-189F21,C23d-679R21)and naphthalene dioxygenase(Nd-546F21,Nd-954R21)genes.This study enriched the germplasm resources of petroleum hydrocarbon degrading bacteria,provided strain resources and information for microbial remediationof subsequent oil polluted wetlands,and provided partial data support for subsequent field experiments.