Construction Of High-effective Microbial Flora For Bioremediation Of Waters Polluted By Pahs And Its Degradation Characteristics

Two dominant naphthalene-degrading strains, named ZJ1H,ZJ2H, which can utilize naphthalene as sole carbon was isolated from activated sludge of a coking plant.They were identified respectively as Massilia timonae and Ralstonia mannitolilytica.The effect of initial concentration of naphthalene solutions, inoculating dosage, pH, temperature, salinity and shaking rates of rocking bed on degradation efficiencies were investigated. The degradation conditions were optimized. The results show that, the optimal conditions for degradation of naphthalene by strain ZJ1H are: initial concentration of naphthalene solutions, 20 mg/L; inoculating dosage, 15% (V/V); temperature, 30~35℃; pH, 7.0; salinity, less than 3%; shaking rates of rocking bed, 120~200rpm. The optimal conditions for degradation of naphthalene by strain ZJ2H are: initial concentration of naphthalene solutions, 40 mg/L; inoculating dosage, 10%~25% (V/V); temperature, 30~35℃; pH, 7.0; salinity, less than 3%; shaking rates of rocking bed, 120~160rpm. Broad-spectrum experiment indicated that the degradable PAHs as substrates for metabolism of strains ZJ1H and ZJ2H were diversiform. Strain ZJ1H could use chrysene, fluorene, pyrene and naphthalene as sole carbon and energy source for growth, respectively, but could't effectively use anthracene and phenanthrene. Inconsistently, strain ZJ1H could treat anthracene, chrysene, fluorene, pyrene and naphthalene these 5 PAHs as metabolic substrate, but it could not utilize phenanthrene only.At the same time, a predominant chrysene-degrading strain named CT was isolated too. The strain was initially identified as Paracoccus aminovorans by the results of morphological observation, physio-biochemical test and 16S rDNA gene sequence analysis. Under the conditions of initial concentration of chrysene 40 mg/L, inoculation amount 10% (V/V), pH 7.0 and temperature 35℃, the degradation efficiency of chrysene by the strain CT reached 85.2% within 8 days. Alkaline lysis was applied to extract plasmids from strain CT to confirm the location of degradation gene. A plasmid, greater than 15kb, was detected.The transformants obtained the ability to degrade chrysene when the plasmid of strain CT was transformed to competent cell of Escherichia coli DH10B, it can remove 43% of chrysene in the solutions with concentration of 30mg/L within 8 days. But the mutation lost the ability to degrade chrysene when its plasmid was eliminated by SDS and high temperature.This indicated that the plasmid of strain CT carried chrysene-degrading genes.Furthermore, we constructed a high-effective microbial consortium with 9 PAHs-degrading strains that separated and screened from polluted environment, result display that the degradation efficiency of microbial consortium D of both only one PAH and multiple PAHs were high.Experiments for effect of initial concentration of PAH solutions on degradation efficiencies stated clearly 80mg/L was the optimal concentration level. And experiment indicated the optimum addition quantity of surface active agent Tween-80 was 150mg/L. Experiment also showed that glucose and yeast powder were the ideal external carbon source and nitrogen source. If they existence, the degradation rate could reach 96.4% and 100%, respectively.Simulation bioremediation experiment proved that microorganism in microbial consortium D could easily incorporated into pannonibacter phragmitetus of the Yangtze River. They help each other to carry out bioremediation. The removal rate of chrysene, pyrene, fluorene reached 86.4%,79.5% and 84.8%, respectively, and the degradation rate of anthracene, phenanthrene and naphthalene were tend to 100% within 6 d.