Typical Carbamate Pesticide-contaminated Soil Bioremediation And Related Research

Carbamate pesticides are widely used in agriculture as insecticides, fungicides, herbicides, nematocides, or sprout inhibitors, but their residue will make environment and food severely polluted. Because this kind of pesticides has relatively high biological toxity and the residue is relatively permanent, people show more and more attention on how to remediate the polluted environment. Bioremediation of pesticides-contaminated environment, which exploits the ability of organisms to degrade and/or detoxify organic contamination, has been established as an efficient, economic, versatile, and environmentally sound treatment. Decontamination of polluted sites in our country with bioremediation has received increasing interest recently. With one kind of typical N-methyl phenyl carbamates-carbofuran as object, this thesis has studied the changes of microbial community in contaminated environment; and the characters of the isolated superior strain which capable of degrading carbofuran. The degrading activity of this strain and Degradation mechanism of this strain on target pesticides and the application of immobilization technology on bioremediation were also discussed. We also attempt to guid the practical bioremediation procedure with the research on microbial community of pollutied soils. Basical research on cloning some functional genes related to phytoremediation was also in progress. The details are as following: The soil microbial communities affected by simulated contamination of three cabamate pesticides was evaluated by using random amplified polymorphic DNA (RAPD) fingerprints. The results showed that RAPD can reflect the impacts of the three different carbamate pesticides on the microbial commnity of soil at different time in detail. As a convenient, smart and easy method, it has potential value of application on the research of risk evaluation of polluants, changes of microbial community during the process of contamination and remediation, and balance of ecology system. A superior strain, AEBL3, which was isolated from soils exposed to repeated spills of carbamate pesticides, was studied on its physical and biochemical and degrading characters. The result showed that AEBL3 is belongs to the Pseudomonas genera. By orthogonal test, it was indicated that AEBL3 could grow with best culture conditions of 32℃, pH6.0, 3 layers of gauze and 250 rpm. The strain can degrade 96.2% of 100mg/L carbofuran contained in the mineral medium by 120 hours. It also can grow with aldicarb or methomyl as sole nitrogen source. By plasmid curing test, it was indicated that the carbofuran degradase gene of AEBL3 does not locate at plasmids. The resistance of the strain to the changes of temperature and high concentration of carbofuran was improved after immobilization. A carbofuran hydrolase present in this strain was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic, anion-exchange chromatographies. The purification fold is 30.33。The optimum pH for the enzyme activity were 4.5 and 6.5. The optimum temperature is 40℃. The enzyme was stable at 30℃, but not stable at temperatures above 50℃. Its activity can be stimulated by K+ and Na+ , but was strongly inhibited by Hg2+,Cu2+,Zn2+ and Mn2+. A strain AEBL2 was isolated from selection medium in which barbon was choosen as selection pressure. The result showed that AEBL3 is belongs to the Eurwinia genera. Genomic DNA was isolated from the strain AEBL2 which can produce carotenoid and PCR was done to amplify conservative sequence of CrtI gene encoding phytoene desaturase. The product of PCR reaction is a 800bp DNA fragment. This fragment display sequence identities of 96.038% with phytoene desaturase gene of E. uredovora in Genbank. This fragment will be usefull for construction of expression vector and breeding pesticide-tolerant transgenic organism for bioremediation. PCR-DGGE (polymerase chain reaction-denatured gradient gel electrophoresis) was employed to explore the diversities and analyze the structures of the selected microbial associations under different selection pressure, such as different pesticides and pH. Jaccard correlation indicated that the different pesticides effected the isolation of different associations. However, some similar numerically-dominant bands characterized the associations. Maximum 40% difference of the microbial association structure can be produced by two different pH. AEBL3 was used as inoculant to bioremediate carbofuran-contaminating soil in bench scale. The result shows degradation rate of carbofuran in the soil was increased remarkably by the treatment. AEBL3 is mobile in the soil to a certain extent. When AEBL3 was added to the soil surface, it had a strong ability to degrade the carbofuran in 0-7cm deep soil. The effects on bioremediation of different inoculation formulations were compared. The original liquid formulation was the best. The buffed solution was the second and mixture with sand was the worst. Addition of complement will improve the effect of bioremediation in some degree, but it is not the more the better. The removal rate of carbofuran was 84% at the 8th weeks in bioaugmentation field, and the corresponding data in CK field was 23%. This result shows that the immobilized bacteria take distinct effect in bioremediation. DDGE of 16s rDNA genes revealed a dynamic change in the microbial community structure of the soil during bioremediation. AEBL3 was the relatively dominated population within the 2 weeks after inoculating, and its domination was replaced by a Bacillus population.