Studies On Molecular Ecotoxicological Effects Of Combined Pollution Between Butachlor And Cadmium On Soil Microbe And Bioremediation

Pesticide and heavy metals (HM) are frequently found together as contaminants in soils and have attracted much attention in recent decades. It is very important to investigate the compound pollution between pesticide and heavy metal for the management and diagnosis of soil pollution. In this paper, the combined effects of butachlor and cadmium (Cd) on soil microorganism were studied by using paddy and phaeozem soil. Moreover, the bioremediation of the combined pollution of butachlor and cadmium were also investigated. The main results are showed as follows:1. The effects of butachlor and Cd on soil urease, phosphatase and soil respiration were studied under control condition. The results showed that combined inhibition of butachlor and Cd was more significant than separate exposures on soil enzyme and soil respiration. In addition, inhibition of the combined pollutants on soil enzyme was more significant than that on soil respiration. The interaction of butachlor and Cd depend largely on incubation time and the concentration ratios of the pollutants. Joint effects of pollutants may be similar (additive) or stronger (synergistic, more than additive) or weaker (antagonistic, less than additive) than expected effects from separate exposures. In this study, the interaction of butachlor (100mg/kg) and Cd (10mg/kg) was synergistic during the whole incubation time, while the interaction of butachlor (10mg/kg or 50mg/kg) and Cd (10mg/kg) was antagonistic at the beginning of the incubation and synergistic in the end of the incubation.2. The genotoxicity of microorganism in soil contaminated with butachlor and Cd was studied by the random amplified polymorphism DNA (RAPD). The combined method with enzymatic and chemical lyses was effective for the microbial total DNA extraction. It had higher total DNA yield and purities in the products. Ten random primers were used to amplify the microbial community DNA. The results showed that combined pollution between butachlor and Cd changed the DNA sequence of soil microorganism, and caused DNA damnification. The microbial DNA damnification caused by butchlor-Cd was significantly different from their single pollution. Moreover, the number of amplified band increased with the incubation keeping on. Therefore, DNA sequence of soil microorganism was affected.3. The effects of butachlor and Cd on soil microorganism were different between in paddy and in phaeozem. The inhibition of butachlor and Cd on soil enzyme and soil respiration was more significant in paddy than that in phaeozem. Moreover, the time of inhibition in paddy occurred earlier than that in phaeozem. In addition, the number of amplified band was more in paddy than that in phaeozem according to analysis the results of RAPD. These compared results in paddy and in phaeozem showed that phaeozem had stronger ability than paddy in enduring pollutants.4. A butachlor-degrading bacterium (designated strain RE1) was isolated from the soil collected from a pesticide plant. The strain could degrade butachlor effectively. When the concentration of butachlor was 30mg/L, the degradation rate was about 90% within 3 days. Based on the general physiological and biochemical characteristics, strain RE1 was identified as Pseudomonsa putida. The degrading ability and the growth of strain RE1 was affected by incubation temperature and pH. The optimal temperature and pH for the strain's growth and butachlor degradation was 30-35℃and 6.0-8.0. In addition, strain RE1 could grow normally when the concentration of Cd was below 1 mg/L. Moreover, when the concentration of Cd was 1mg/L and the concentration of butachlor was 30 mg/L, strain RE1 could degrade butchlor and the degradation rate was over 50%. The result indicated that strain RE1 could degrade butachlor at the combined condition of butachlor and Cd.5. In this study, we tested the degradation of butachlor by the crude enzyme of ER1. In addition, we also tested butachlor and Cd as inducers of butachlor-degrading enzyme synthesis. There were significantly different in enzyme activities between the cells incubated with butachlor, butachlor + Cd and without pollutants (the controls). The activities of the enzyme protein indicated that butachlor-degrading enzyme protein was not only constitutively expressed in RE1 and was also induced by butachlor and Cd. In addition, the results of SDS-PAGE showed that the amount of some enzyme proteins increased after the strain treated with the pollutants. So it seemed that butachor and Cd induced some enzyme proteins benefit for the degrading of butachlor.6. Two-dimensional gel electrophoresis is the key in protein analysis. The sample of strain RE1 was grinded with liquid nitrogen, 10% TCA-acetone precipitation. Proteins were analyzed by immobilize dry strip pH 4-7 (linear) and 12.5% SDS-PAGE. These methods could remove the impurity that could affect the resolving capability of 2-DE. So, with these methods high resolving capability 2-DE maps were achieved. A normal SDS-PAGE replaced the Ettan DALTsix system, which could get good protein spots effectively and also save labor costs.7. Of the total bacteria proteins, eleven protein spots were found significantly changed after treated by butachor and Cd. Among these proteins, ten proteins were found to be up regulated, while one protein spot was only detected in butachlor and Cd treated cells but not in the control treated cells. To make the identification more confident and specific, the spots were excised from preparative 2-DE gels and analyzed by MALDI-TOF MS. In order to identify their functions, they were subjected to database searching against SWISS-PROT, TrEMBL and NCBI using profound software. The results showed that the identified proteins might have oxidoreductase or hydrolase activity. These proteins might be responsible for degradation of pollutants and played an essential role in cell defense against environmental stresses.