Studies On Variations Of Microbial Communityin Soil And Characterization Of Dimethoate-Degrading Bacteria

Pesticides are a sort of biological exogenous material that has the ability to control or eliminate diseases. They can also prevent the reproduction of ruderal. In modern agricultural production, the application of pesticides, especially chemical pesticides is an important method to guarantee the stability of the crop production and help increase its production. But nowadays, the overuse of pesticides has caused ever-increasingly problem of soil pollution, along with all the associative environmental problems that can not be ignored, and because of that, technology on degradation of pesticides residue has become a hot spot of research. After pesticides are used, part of them will adhere onto the surface of plants, or maintain in the body of plants, thus polluting the plants themselves; or they will probably maintain in soil, or evaporate into the air, and come back to ground together with the rainfall, and then, flows into the rivers or lakes, thus causing pollution in water body and aquatic creatures. Meanwhile, pesticide residues in crops will enter the animals' body together with processed feed. Pesticide residues will finally enter human body after a serious of transformation and circulation, and cause diseases. Generally speaking, there are several ways of the remediation of pesticide-polluted soil including biological ways, chemical ways, and physical ways. Among the three ways listed, biological ways are acknowledged the most effective, harmless, cheap ones without a secondary pollution. A biological remediation is a process that poisonous pollutants are degraded or transferred to harmless materials through the metabolism of creatures. In the degradation of organophosphorus pesticides, the method on micro-biological remediation has a vital role to play.Efficient total sludge community DNA extraction methods were etablished. Total sludge community DNA used for PCR was extracted and the sequence amplified with actinomycetic special primer was got. Variations of bacteria gene diversity in sludge stressed by dimethoate were observed using the denaturing gradient gel electrophoresis (DGGE) technique. The result showed that there were some significant differences between bacteria communities in sludge treated with different concentrations of dimethoate and sampled at the same cultivation times. Dimethoate had obvious toxicity on bacteria community, making gene diversity decreased. Obviously, it is possible to provide a new way and foundation to illustrated bacteria gene diversity in dimethoate contaminated environments. It is feasible to study as the gene indexes for indicating the contamination degree by dimethoate.Most organophosphorus pesticides has high toxicity, and will have greatly negative effect on natural environment and creatures if they are under a great amount of use. The target organophosphorus pesticide of this thesis is Dimethoate, whose chemical formula is C5H12NO3PS2, and has a high effective on the control of insects with a medium toxicity. Its chemical name is O,O-Dimethyl phosphorodithioate, S-ester with 2-mercapto-N-methylacetamide. Dimethoate is relatively cheap, and is also quick in effect, thus having been widely used in China for a long time. At the same time, dimethoate has a long half-life and is not easy to degrade quickly in nature, thus accumulated in the environment in a large amount and caused serious pollution in water body, crops and soil. Then, it concentrates and transfers by successive stages and greatly threats human beings, especially as a potential carcinogen. As the consciousness of environmental protection increases day by day, pollution caused by dimethoate is becoming more widely concerned in the society.As far as current situation of research in this field is concerned, micro-biological ways play great role in the degradation of dimethoate. Researchers has succeeded in selecting degrading bacteria mostly from outlets of facilities for treatment of waste water containing dimethoate, or activated sludge with waste water flowing along from agricultural chemicals factory, or soil that has a high background value of dimethoate. In this thesis, soil with a low background value of dimethoate was chosen to domesticate bacteria using dimethoate as sole carbon source, which is a little different from former researches.An amount of brown soil in the grove of Shanghai Jiaotong University, which is in a low background value of dimethoate, is chosen as the target soil. The initial concentration of dimethoate was 10mg/L. Then the value was regularly and gradually increased. When the total amount of dimethoate reached 200ppm, 20mg/L untreated dimethoate started to be added weekly. During the process of domestication, water and nutrient solution has always been kept in a certain value. After three months' domestication, 1g soil was collected from it and was put in nutrient solution with a volume of 100ml and a concentration 50 mg/L of dimethoate. At a temperature of 30 centigrade degree, cultured five days in a rotary shaker (200 rpm), it was inoculated into another 100 ml of nutrient solution at an inoculation amount of 5%. This process was repeated for another two times as a way of reproduction. After that, the enrichment was determined by GC-MS. Enrichment, in which all of the three generations of bacteria degraded more than 90% dimethoate, was regarded active. After coating the active enrichment onto plating medium adopting the method of gradient dilution, several groups of bacteria were successfully cultivated in the plating medium. Differentiate and purify the strains and cultivated them in departed nutrient solution with a concentrate 100mg/L of dimethoate, determine their ability of degradation after 2 days of cultivating at the temperature of 30 centigrade degree in rotary shaker (180 rpm). Finally, A bacterial strain, LPx, capable of utilizing dimethoate as sole source was purified and selected. Expand the amount of the strain, and then observe and study its characteristics and features on degrading dimethoate.The first step was to confirm its ability of degrading dimethoate. The conclusion was that this strain could develop in the environment that dimethoate was the sole carbon source, and had a marked ability of degradation. And the second step was to study on the physiological and biochemical properties of the bacteria. After observing the configuration of the bacteria, DNA of the bacteria was extracted as a sample, and was amplified and performed PCR for 16S rRNA gene. The primers included 5'-AGAGTTTGATCCTGGCTCAG-3' as the positive vetor primer and 5'-TACGGCTACCTTGTTA CGACTT-3' as the negative vetor primer. After sequencing the PCR product, logged in Genbank to search for the Gene, and compared it with those in the Genbank, sequenced homology analysis of the genes and based the phylogenetic tree. The result shows that the strain belonged to Pseudomonas sp., and the strain was named LPx. Pseudomonas sp. was made up by Migula in the year 1894. It is a kind of bacillus occuring in straight or slightly curved but not heliciform. It has no prosthecae or mycoclena, and does not spore. Besides, it is a gram-negative bacillus.The next step was to study on the features of LPx to degrade dimethoate. The first part was to study the effect of pH on its degradation of dimethoate. In nutrient solution with the initial concentration 100mg/L of dimethoate, and under different pH values including 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10% of LPx was inoculated. Each one had a blank sample that was not inoculated to compare with. At the temperature of 25 centigrade degree, after cultivated in rotary shaker (180 rpm) for three days, the sampled were determined on the concentration of dimethoate, and then there came the degradability. After the comparison, the result showed that the optimal pH of the bacteria for the degradation was 7.5. The second step was to study the effect of temperature on its degradation of dimethoate. In nutrient solution with the initial concentration 100mg/L of dimethoate, and under different temperatures including 15, 20, 25, 30, 35, 40 and 45 centigrade degrees, 10% of LPx was inoculated. Each one had a blank sample that was not inoculated to compare with. After cultivated in rotary shaker (180 rpm) for three days, the sampled were determined on the concentration of dimethoate, and then there came the degradability. After the comparison, the result showed that the optimal temperature of the bacteria for the degradation was about 30 centigrade degree. The third step was to study the effect of inoculation on its degradation of dimethoate. In nutrient solution with the initial concentration 100mg/L of dimethoate, and under the pH 7.5, different amount of LPx were inoculated. The amount of inoculation includes 1%, 3%, 5%, 10% and 15%. Each one had a blank sample that was not inoculated to compare with. At the temperature of 25 centigrade degree, after cultivated in rotary shaker (180 rpm) for three days, the sampled were determined on the concentration of dimethoate, and then there came the degradability. After the comparison, the result showed that the optimal inoculation of the bacteria for the degradation was 10 %. Based on the conclusions above, study on the kinetics of the degradation was followed. After confected with inoculation 10% of LPx and differenct concentration of dimethoate including 15, 30, 45, 60, 75, 90, 105mg/L, the samples were cultivated under pH 7.5 and temperature 30 centigrade degree in a rotary shaker and were determined on the concentrations of dimethoate, c1. At the same time, the concentrations of dimethoate in blank samples, c2, were also determined. Finally, the conclusion was that dimethoate with an initial concentration no more than 90mg/L could be degraded in a higher degradation rate. On optimum condition, dimethoate with an initial concentration of 100 mg/L contained in nutrient solution could be degraded to an undetectable level in 5 days. The reaction LPx degrades low concentration dimethoate follows the characteristic first-order reaction kinetics. The biodegradation process of dimethoate can be well described by enzymatic reaction of high concentration inhibition, with the maximum substrate utilization rate rmax=0.734 d^-1, Miehaelis-Menten parameter km= 21.700 mg/L, and substrate inhibition coefficient k1=259.215.