Isolation And Identification Of Petroleum-degrading Microorganisms And Preliminary Research On Preparation Of Bacterial Agents

The bioremediation of petroleum contaminated soil and water has become one of the hot spots ofresearch all over the world. The products of microbial agents degrading petroleum have been developed inforeign countries, while the development and application of the solid petroleum degrading bacteria agent inChina are mostly in the stage of laboratory research. In this paper, efficient petroleum degrading bacteriawere isolated and identified, and furthermore, preparation technology of microbial agents and thebioremediation of petroleum contaminated soil in laboratory conditions were studied preliminarily. Themain contents are as follows:（1） Two strains of dominant bacteria degrading oil, named as KF-2and KF-4, were isolated frompetroleum contaminated soil through natural selection. The degradation rates of the two strains were38.6%and24.6%respectively8days later under experimental conditions. KF-2and KF-4were identified asAchromobacter xylosoxidans and Klebsiella pneumoniae respectively through the observation and analysisof the colony morphology, physiological and biochemical tests and16SrRNA sequence.（2） The appropriate conditions were determined through the optimization of liquid expansionculture conditions for KF-2and KF-4with the OD600value as a reference indicator. The results were asfollows: the best carbon source of the culture medium of KF-2sucrose （9g/L）,40mL medium in250mLshake flask, seed culture12h, seed liquid inoculum into the expansion culture medium14%（v/v）, expansionculture10h; the best carbon source of the culture medium of KF-4sucrose （5g/L）,30mL medium in250mLshake flask, seed culture11h, seed liquid inoculum into the expansion culture medium8%（v/v）, expansionculture4h.（3） The solid culture conditions for KF-2and KF-4were studied with the effective number of livebacteria per gram of agents as the main index. Fistly, the single-factor experiments of carrier, ratio ofmaterial to water, pH of water added into the carrier and expansion liquid inoculum into the carrier werecarried out, and then the orthogonal experiment of temperature, time and the amount of carrier during thesolid culture as well as drying temperature was designed. The optimum conditions for KF-2was as follows:carrier wheat bran, ratio of material to water1:3.6, pH of water added into the carrier9, expansion liquidinoculum into the carrier25%（v/w）, temperature25℃, culture time3d, drying temperature30℃, carrier10g; The optimum conditions for KF-4was as follows: carrier wheat bran, ratio of material to water1:3.6, pH of water added into the carrier6, expansion liquid inoculum into the carrier20%（v/w）, temperature30℃, culture time30h, drying temperature30℃, carrier10g. The final number of live bacteria of KF-2andKF-4agents reached7.3×10¹⁰CFU/g and3.16×10¹⁰CFU/g respectively.（4） The observation and study of scanning electron microscope morphology and preservationconditions for KF-2agent and KF-4agent were carried out. Results showed that wheat bran was the bestcarrier and that4℃was superior to room temperature in the aspect of preservation for the agents.（5） The simulation experiment on application of KF-2and KF-4agents to oil polluted water andsoil was carried out. The results showed that different inoculum amounts of the agents had a certain effecton the oil degradation in the simulation experiment on oil polluted water; the suitable inoculum amounts ofKF-2and KF-4agents were0.4g and0.5g respectively, and the degradation rates were88%and67%correspondingly. When the oil concentrations were1g/L,2g/L and3g/L, the oil degradation rates of KF-2agent were73.3%,61.1%,21.4%correspondingly, however, the degradation rates of KF-4agentmaintained at66%or so. Addition of glucose can promote the degradation effect of oil by KF-2agent in acertain extent. The oil degradation rate of KF-4agent was59%, which was higher compared with that ofthe other two，KF-2agent and the mixed agent. Addition of biological surfactant （LP） can promote thedegradation of oil by KF-4agent; the suitable concentration of LP was10mg/L, and the oil degradation ratereached67.6%.In the simulation test of bioremediation of petroleum contaminated soil, KF-2agent added inpetroleum contaminated soil played a certain role in the degradation of crude oil. The degradation rate were5.46%and8.29%respectively in the soil samples which contained2%and4%petroleum20days later, butthe effect was not ideal.