| Polycyclic aromatic hydrocarbons(PAHs) are ubiquitous refractory organic pollutants in the environment, which have carcinogenic, teratogenic, mutagenic properties. The problem of environmental pollution caused by PAHs has received more and more attention. A preponderant pyrene-degrading strain was isolated and purified from activated sludge collected from aeration basin of a coking plant to provide microorganism resource and scientific basis for bioremediation of the environment polluted by polycyclic aromatic hydrocarbons. We call it B-1. Strain B-1 was identified by the results of morphological observation, physio-biochemical test and 16S rDNA sequence analysis. The characteristic of B-1's degrading pyrene and the effect of incubation amount, the initial pyrene concentration of liquid medium, pH value and salinity on pyrene degradation efficiency were inspected. Studied the different effects of adding exotic carbon sources, which were glucose, vitamin C, salicylic acid, phthalic acid, naphthalene andα-ketoglutaric, on pyrene degradation. We choosed the appropriate vector to immobilize strains and used orthogonal experiment to determine the optimum conditions for immobilization. Comparing the immobilized cells with free cells, we observed the change of the pyrene tolerance and optimum range of pH value.The results showed strain B-1 was initially identified as Bacillus with concentration up to about 130mg/L. Under the conditions of incubation amount 15% (V/V), initial pyrene concentration 20~100 mg/L and temperature 30℃, strain B-1 could preserve high degradation activities of pyrene in the range of the pH 4.0-11.0 and the salinity less than 1%. With the initial pyrene concentration of 80 mg/L, incubation amount of 13% (V/V), pyrene degradation percentage by strain B-1 was 92.4% after 10 days.Adding six different kinds of exotic carbon sources separately, there are different effects of promoting the degradation of pyrene. When the glucose concentration in the reaction reached 100mg/L, pyrene degradation percentage by strain B-1 was 94%, increased by 17.5% compared with the control. However, pyrene degradation rate declined after enhancing the concentration of vitamin C from 50 mg/ L to 100 mg/L. 50 mg/L was the optimum dosage, with the pyrene degradation rate reaching 89%. When the concentration of phthalic acid in the medium was 100 mg / L, the removal rate of pyrene reached 97.8%. 50 mg/L was the most suitable concentration of salicylic acid for pyrene degradation, increasing by 21% compared with the control. Adding 1~20 mg/Lα-ketoglutarate to the culture system enhanced pyrene degradation efficiency obviously. The most suitable concentration was 20mg/L, with pyrene degradation rate reaching 89%. Adding some naphthalene promoted the degradation efficiency of pyrene, When the naphthalene concentration was 50 mg/L, pyrene degradation rate by strain B-1 reached 92% after 13 days, increasing by 16% compared with the control.B-1 was immobilized using alginate and PVA as the vector and adding certain amount of kaolin. Orthogonal experiment determined the optimum operation conditions: 8%polyvinyl alcohol, 5%kaolin, 0.2%sodium alginate and 15ml embedded cell. After the strain immobilized, the capability of degrading pyrene greatly improved, 80 mg/L of pyrene in the medium was almost completely removed after 13 days. Compared with free cells, immobilized cells had a wider range of PH value for B-1 growth. In the range of pH 6~11, the removal rate of pyrene reached more than 84%. Immobilized B-1 had the tolerance against higher concentrations of pyrene. When the pyrene concentration in the reaction was 160 mg/L, pyrene degradation rate could reach 59%. The removal of pyrene by immobilized B-1, on the one hand, was because of contribution of the biological degradation by strain B-1, the other hand, related to the fixed materials'absorption of pyrene. |
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