Microbial Community Structure And Enzyme Activity During Bioremediation Of Petroleum-Contaminated Soil

Petroleum contamination has become a particular problem for protection of soil ecological environment. In this work, Biolog, RFLP and Illumina high-throughput sequencing techniques were employed to explore the variation of microbial community during bioremediation of petroleum-contaminated soil and study on the catechol dioxygenase enzyme activity as well as cloning and expression of encoding genes. Firstly, microbial community structure and metabolic characteristics in petroleum-contaminated soil were studied. The results showed that bacteria were the advantage groups in polluted environment. Sixteen composite microorganisms and fifteen petroleum degrading strains were isolated from petroleum-contaminated soils, RFLP and Illumina high-throughput sequencing technology were employed to determine fingerprinting of isolated strains and species composition of microorganisms and the petroleum degradation rates were examined. Next, we investigated the effects of petroleum concentration in soil, oxidizing agent and manure on microbial community structure and metabolic characteristics. In the end, we studied characteristics of catechol dioxygen enzyme, clone and heterologous express coding genes, one strain recombinant bacteria was obtain. This study provides the theory basis for using of petroleum degradation microbio resources and bioremediation of petroleum-contaminated soil. The main research conclusions are as follows:1. Microbial community structure and metabolic characteristics in petroleumcontaminated soil in Loess plateau were studied by Biolog micro-plate technology. The results showed that the number and species composition of different soil microbial groups were various obviously under the condition of petroleum hydrocarbon pollutants stress. The number of bacteria and fungi significantly increased one order of magnitude higher than clean soil, while the number of actinomycetes significantly decreased. Bacteria were the dominant species in petroleum-contaminated soil, accounting for 99.8%-99.9%. A significant difference was observed in soil microbial community structure between uncontaminated and petroleum-contaminatd soils. The differences mostly related to the use of carbohydrates as the dominant carbon source and the carboxylic acids and amino acids. With increase concentrations of petroleum, the activity and the stability of the soil microbial community decresed. Carbon source metabolic patterns transformed sugar into polymer. Shannon index and Mc Intosh index of microbial communities were decreases, and Simpson index increased.2. Sixteen composite microorganisms and fifteen petroleum degrading strains were isolated from different petroleum-contaminated soils, RFLP, Illumina high-throughput sequencing technology and MPN method were employed to determine fingerprinting of isolated strains and species composition of microorganisms, the petroleum degradation rates and carrier of petroleum degrading composite microorganisms were examined. The results showed that the effect of temperature and petroleum concentration on of petroleum degradation rate and species composition were different due to the heterogeneity of soil. The emulsifying ability of medium temperature microorganisms were better than high temperature microorganisms, petroleum degradation rates were higher than high temperature microorganisms 7.91%-7.91%. The emulsifying ability of microorganisms obtained from low concentration is stronger, petroleum degradation rates were as 1.22-5.82 times as that of microorganisms obtained from high concentration. Achromobacter was the dominant microbial consortia is medium temperature microorganisms, Geobacillus was the dominant microbial consortia is high temperature microorganisms, no same species between the two microorganisms. There were the same species between the microorganisms obtained from different petroleum concentration, Pseudomonas was the shared species, Geobacillus and Brevibacillus were the endemic species in C4-30-20, Brucellaceae. Unclassified was the endemic species in C4-30-50. Bran was the optimum carrier, the number of petroleum degrading microbio were 1012 when solid-liquid ratio was 1:1. The petroleum degrading rates of D4109 and AD049 were 68.65% and 34.41% respectively, the phenol degrading rate of AD049 was 99%. The results of 16 S rDNA sequencing indicated that D4109 showed 96% homology with that from Brucellasuis, AD049 showed 99% homology with that from Rhodococcus pyridinivorans.3. To explore the effect of petroleum consentration on microbial community structure and metabolic characteristics during bioremediation of petroleum-contaminated soil by composting, an experiment was conducted with bacteria agent and mature chicken manure as amendment. The results showed that biodegradation of petroleum hydrocarbon followed the first-order model during composting. The half-life period increased with increase in petroleum consentration, the half-life in high consentration treatment was higher 7.07 and 6.49 days. The petroleum degradation rates were 91.45%, 91.83% and 73.97% respectively. The average degradation rate was 112.08, 230.05 and 887.93 mg/(kg·d) respectively. The average well-color development(AWCD), use of carbon sources(except aromatic compounds), Shannon index and McIntosh index increased during the composting process and Microbial communities tended to be stable. The dominant microbial consortia were metabolism communities of polymers and carbohydrates in composting process. The principal component analysis results revealed that there was a significant difference in soil microbial community structure among 3 treatments and the difference was mostly related to the use of carbohydrates and carboxylic acids. Compared with the first stage of composting, the SGI in 3 treatments increased respectively by 18.26%, 20.42% and 36.41%.4. To explore the effect of oxidizing agent on microbial community structure and metabolic characteristics during bioremediation of petroleum-contaminated soil by composting, an experiment was conducted with bacteria agent and Fenton reagent as amendment. The results showed that petroleum degradation occured mainly in the middle of composting process, high Fenton treatment lagged the process. The petroleum degradation rate in middle treatment was 75.20%, as 1.24 times as control treatment. The AWCD, use of carbon sources, Shannon index, McIntosh index increased during the composting process. The dominant microbial consortia were metabolism communities of polymers and carbohydrates in composting process. The principal component analysis results revealed that there was a significant difference in soil microbial community structure among 4 treatments and the difference was mostly related to the use of carbohydrates, amino acid and carboxylic acids. Pseudomonadaceae unclassified was the dominant microbial consortia in the beginning of composting process, the average relative abundance was 76.15%. Sphingobacterium was the dominant microbial consortia in the middle and end of composting process, the average relative abundance was 44.66%. Flavobacterium was the endemic species in middle and end of composting process. Petroleum consentration and polyphenol oxidase activity, metabolism of carboxylic acids, sugars, polymer and amino acids had significant negative correlation. Azospirillum、Sphingomonas、Enterobacteriaceae. sp,Rhizobiales. sp �'� Agrobacterium were the main microbial consortia for soil enzyme and carbon metabolism. Inquilinus、Pseudomonas、Sphingobacterium �'� Steroidobacter were the main microbial consortia for degradation of petroleum.5. To explore the effect of manure on microbial community structure and metabolic characteristics during bioremediation of petroleum-contaminated soil by composting, an experiment was conducted with bacteria as amendment. The results showed that The petroleum degradation rate in low manure treatment was 71.69% and higher 20.39%, 15.48% and 9.26% than control, middle manure and high manure treatments. The AWCD, use of carbon sources, Shannon index, McIntosh index increased and the number of petroleum degrading microorganism decreased slowly, rised again and then reduced during the composting process. The dominant microbial consortia were metabolism communities of polymers in beginning, the dominant microbial consortia were metabolism communities of carbohydrate and amino acids in middle, and the dominant microbial consortia were metabolism communities of carbohydrates and polymersat the end of compsting process. The principal component analysis results revealed that there was a significant difference in soil microbial community structure among 4 treatments and the difference was mostly related to the use of carbohydrates, carboxylic acids and amino acids. The number of petroleum degrading microorganism and metabolism of polymer had significant correlation. There was a significant correlation between petroleum consentration and metabolism of polymer, carboxylic acids, carbohydrates and amines.6. AD049(Rhodococcus pyridinivorans) degraded phenol by catechol 1, 2-dioxygenase(C12O) via ortho-cleavage pathway mainly, and catechol 2, 3-dioxygenase(C23O) via meta-cleavage pathway supplementarily. C12 O synthesized in cell inducibly. Inducing effect became stronger as the increase in phenol concentrations ranged from 250 mg/L to 1500 mg/L. C12 O was belonged I, which barely degraded chlorinated aromatic compounds. Hg2+, Ag2+ and Mn2+ inhibited C12 O particularly, while Cu2+, Fe2+, Fe3+ and Zn2+ had almost no inhibition. The optimum pH of C12 O ranged from 7.0 to 8.0, and the optimum temperature ranged from 30 ℃ to 35 ℃. Calculated the Michaelis constant was 2.34×10-2 mol/L, the inhibition constant was 4.73×10-2 mol/L and the optimum substrate concentration was 3128.1 mg/L.7. The catA gene complete sequence including 944 base pairs loated in chromosome and showed 99% homology with that from Rhodococcus pyridinivorans SB3094(Query cover 99%). The cat A gene encoding C12 O successfully amplified from AD049 using specific primers, connected to p ET 28 a expression vector and transferred into E. coli BL21(DE3). The C12 O activity from recombinant strain was 1.42±0.02 μmol·min-1·mg-1, higher 10.08% than that from AD049.