The aim of this study is to construct petroleum degrading agent (PDA) which can effectively degrade oil. By enrichment, domestication, and separation of culture from the soil sample of Qianjiang Guanghua Oilfield, the pure culture of three petroleum degrading bacteria G-40, G-53, and G-94 was identified from the medium supplemented with oil, which served as the sole source of carbon. The species of G-40 and G-53 were preliminarily identified and classified by morphological observation, physiological and biochemical determination, and sequence analyses of 16S rDNA. The species of G-94 was preliminarily identified and classified by morphological observation, physiological and biochemical determination, and sequence analyses of ITS rDNA. The optimal inoculation proportion of these three bacteria strains and bra,n proportion in composition of PDA were determined through orthogonal test. G-40, G-53, and G-94 were isolated and identified as Brevibacillus laterosporus, Tsukamurella inchonensis, and Candida tropicalis, respectively. To construct petroleum degrading agent, the optimum inoculation proportion of the three bacteria strains was A1B3C3 (G-40:G-53:G-94 = 1:4:4); and the optimum proportion of bran was D1E1F2 (soybean meal:corn flour:bran = 1:1:2). The oil removal rate of the constructed petroleum degrading agent reached to 42.32% on day 10 under the optimal proportion of bacteria inoculation and bran composition. Petroleum degrading bacteria can effectively degrade petroleum for its own growth. This study identified three petroleum degrading bacteria strains and proposed a petroleum degrading agent by studying the optimal inoculation proportion of the three bacterial strains and the accompanying bran. Our research could provide potential microbial resources for bioremediation of petroleum-contaminated soil.
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