The Screening And Identification Of TPH-Degrading Bacteria And Their Degrading Effect

As petroleum-contaminated soil is becoming increasingly prominent in China, researchers paid more and more attention to the study of its microbial remediation.This study started from enriching and separating microorganisms from petroleum-contaminated soil samples collected in ShengLi Oil Field region. Efficient bacteria that could degrade TPH (total petroleum hydrocarbon) were screened by qualitative and quantitative methods, and they were tested for combination. Using UVS (ultraviolet spectrometry) and GC (gas chromatography) analysis the main components of petroleum degraded by consortium U and the four strains of the consortium, the degradation effect of consortium U could be fully evaluated. The results were as follows:1. 50 strains of bacteria were separated from the petroleum-contaminated soil through enrichment and domestication. The TPH degradation capacitys of the isolates were then measured using UVS respectively.And the strains PU-34, PU-15, PU-2, PU-4 and PU-1 showed higher degradation activity. These strains could make TPH decreased 58.38%, 55.55%, 55.17%, 53.09%, 52.36% respectively after they were been shaking-cultured for 4 days in the selective medium whose initial concentration of petroleum was 2500 mg ? L-1.2. Based on the results of morphological, physiological and biochemical characteristics and phylogenetic analysis, these five TPH-degrading strains were identified as Pseudoxanthomonas japonensis PU-34, Gordonia alkanivorans PU-15, Gordonia amicalis PU-2, Rhodococcus ruber PU-4, and Pseudomonas sp. PU-1, respectively.3. By comparing the biomass of the five bacteria being cultured for 48h in six kinds of medium (TSA, TSB, beef extract peptone, M79, MYG and R medium), we could draw the conclusion that the M79 medium is the optimum medium to all these five strains. So the M79 medium was chosen as seed medium for the multiplication of the strains. According to the growth curves of the strains in the M79 medium , the optimum inoculum age of each strain were 12h, 18h, 24h, 30h and 42h, respectively. Of all these five strains, PU-1 grew fastest, and PU-2 grew slowest.4. These five TPH degrading bacteria were combined randomly to form consortium and were inoculated into selective medium. After 4days'culture, the TPH degradation rate of the most consortiums was higher than a single strain. The consortium U composed by PU-34, PU-15, PU-2 and PU-4 demonstrated the highest degradation rate, reaching 80.4%.5. GC was used to analyze the saturated hydrocarbon components of petroleum after being degraded by consortium U and the four strains of it. As the results showed, strain PU-34 degraded long-chain alkanes. Strain PU-4 had effect on degrading short-and middle-chain alkanes. Strain PU-15 could degrade most kinds of alkanes. Strain PU-2 had effect not only on alkanes but also on pristane and phytane. Because the four strains which composed consortium U played different role on different substrate, the Consortium had significant degradation effect on most kinds of saturated alkanes components. The contents of straight-chain alkanes, pristane and phytane were decreased to zero by consortium U after being cultured for four days.6. The degradation effect of consortium U was assessed based on the result of UVS and GC analysis that the consortium U has ideal degradation effect and wide spectrum of substrates. After being shaking-cultured for 4 days in the selective medium whose initial concentration of petroleum was 2500 mg ? L-1, consortium U could make unsaturated hydrocarbon such as aromatic hydrocarbons decreased 80.4% by UVS measurement, and its degradation rate of alkanes, pristane and phytane were 100% by GC analysis.The study not only provides microbial resources for microbial remediation of petroleum- contaminated soil in ShengLi Oil Field region, but also lays a foundation for further developing microorganism agent. In addition, the reason for the degradation rate of consortium U could significantly increase was investigated. In this point, the study has some theoretical and practical significance.