Isolation, Identification And Growth Condition Optimization Of Degrading Bacteria Of Oil Contaminated Soil

Bioremediation (in particular microbial) technology to eliminate petroleum-contaminated soil with its low-cost method, no secondary pollution is widespread concern. At present, microbial remediation of oil pollution has been carried out under laboratory conditions for bioremediation applications in practice, provides reference and guidance. The oil from the Daqing oil field has long been contaminated soil collected soil samples to screen crude oil as sole carbon source selected by direct separation advantage of a group of petroleum hydrocarbon degrading bacteria; select one of ten pure strains as the research object,16SrDNA sequence analysis combined with morphological and physiological and biochemical tests of efficient oil degrading strains and their preliminary identification of the best growth conditions were explored (pH, salinity, nutrients) to study the different factors on microbial growth and the optimal level combination; Finally, under laboratory conditions, the oil contaminated soil on soil enzyme activity awakened determined. The results are as follows:1 Through the 7 samples of petroleum-contaminated soil were enrichment culture, we selected 10 strains of bacteria are growing well. Named 15,18,7,9,11,21, L, H, X,4.2 By morphological, physiological and biochemical analysis and sequence analysis of the combination of 16SrDNA, initially identified 11 strain identified as Bacillus pumilus,15 strain was Rhizobium sp,18 strain of bacteria was Licrobacterium oxydans, H was Arthrobacter sp, 7,9,4,21, L and X strain were all Bacillus sp.3 (1) The single factor method that changed the initial pH, salt concentration, N source and P source of the initial in order to search for the optimal growth conditions of each strain, then found that:L strain conditions for optimum growth pH=7, salt 1%, nitrogen and phosphorus were NH4NO3 and NaH2PO4. The optimum growth conditions of strain H:pH=7, 3% of salt, nitrogen and phosphorus sources were KNO3 and K2HPO4:KH2PO4=1:2 (two P sources.) The optimum pH is 8 of 18 strain,3% salt concentration, nitrogen and phosphorus sources were NH4NO3 and K2HPO4.15 strain of the optimal pH of 7,3% salt concentration, nitrogen and phosphorus sources were NH4Cl and two phosphorus source. The optimum pH is 7 X isolates,3% salt concentration, nitrogen and phosphorus sources were NH4Cl and two P sources.4 strain optimum pH of 8,3% salt concentration, nitrogen and phosphorus sources were NH4NO3 and KH2PO4. The optimum pH was 7 of 11 strain,3% salt concentration, nitrogen and phosphorus sources were NH4Cl and KH2PO4.7 strain of the optimal pH of 6,1% salt concentration, nitrogen and phosphorus sources were KNO3 and two phosphorus source.9 strain optimum pH of 7,3% salt concentration, nitrogen and phosphorus sources were NH4Cl and two P sources. The optimum pH was 7 of 21 strain,1% salt concentration, nitrogen and phosphorus sources were NH4Cl and K2HPO4.(2) The orthogonal test was to explore a variety of factors influence the growth of strain to find the bset conditions for growth of strain combinations. The results showed that the best conditions for each combination of strain, respectively:L strain was 1B2A3B4D, H strain was 1B2A3B4D,18 strain was 1B4B3A2C,15 strain was 2B1B4C3A,4 strain was 2B3A1B4A, X strain was 1A3D2B4D,7 strain was 1B2C3D4A,9 strain was 1A2A3A4A,21 strain was 1B3B2B4B/C,11 strain was 1A2A3A4A.4 The measurement results of enzymatic activity in oil contaminated soil:(1) Polyphenol oxidase activity(mg/g), respectively:61D was 35.78851,61M was 25.28901,61S was 22.87682,97D was 49.79176,97S was 32.4608,97M was 32.84066, oil soil was 30.55726.(2) Urease activity(mg/g), respectively:61M was 0.321074,97D was 0.276984,61S was 0.904971,61D was 0.053245,97S was 0.320985,97M was 0.296232, oil soil was 0.247786.(3) Invertase activity in soil were (mg/g):61M was 0.425707,97D was 0.397302,61S was 0.876511,61M was deep 0.128437,97S was 0.556353,97M was 0.395122, oil soil was 0.432914. (*D is deep,M is middle,S is surface.)(4) The correlation between soil enzyme analysis showed that:Invertase and urease very significant positive correlation. Invertase and polyphenol oxidase showed a significant negative correlation. Polyphenol oxidase and urease negative correlation between the non-significant...