Study On Biodegradation And OPH Detection Of Organophosphorus Pesticides

Organophosphorus pesticides(OPs) are a large group of pesticide widely used in agriculture. Their widespread use has caused severe environmental pollution and threated human health. So it is important to find new ways to solve these problems. Solving these problems using biological technique is thought as a promising way. The research focuses on two fields. One field is on how to elimate the pesticide pollution through biodegradation by microorganisms or enzyme. The other field is on finding a new fast and cost-efffective analytical technique to monitor the use of organophosphorus pesticides.A Triazophos-degrading strain, Klebsiella sp., was isolated by enrichment technology from soil that had been exposed long-term to Triazophos. The nutrient requirement of the isolate was very simple and it grew well at pH 7.0-8.0 with a broad temperature profile ranging from 32 ℃ to 37℃. The different degradation rates observed when Triazophos was utilized as the carbon source, or the sole nitrogen source showed that the isolate utilized Triazophos as the sole nitrogen source most effectively. The isolate degraded Triazophos through a hydrolysis mechanism. Furthermore, it can degrade the intermediate, l-phenyl-3-hydroxy-l,2,4-triazole, into inorganic compounds. This showed the great potential of the isolate in biodegradation of Triazophos pollution.It is also an effective way to biodegrade organophosphorus pesticides by enzyme. First, the separation and purification of organophosphorus hydrolase from crude enzyme was done. The process included (NH₄)₂SO₄ fractionized precipitation, desalination and gel filtration chromatography. The organophosphorus hydrolase was purified according to the result of SDS-PAGE. Second, the biodegradation of methyl-parathion by immobilized organophosphorus hydrolase was investigated. Organophosphorus hydrolase was immobilized on microfiltration membranes and then the enzyme membrane was installed into the filtration device to form an enzyme membrane bioreactor. The result of immobilization showed that hydrophile membranes were more advantageous than hydrophobe ones to act as immobilizationcarrier. Crosslinking organophosphorus hydrolase by glutaraldehyde on polyethersulfone membrane is a simple and practical immobilization method. An appropriate amount of bovine serum albumin and crosslinker is necessary to get a good result of immobilization. In the biodegradation of methyl-parathion through enzyme membrane reactor, the effect of immobilized enzyme amount, flow rate of peristaltic pump, pH of the feed, and the methyl-parathion concentration on biodegradation rate was studied. It was shown that the biodegradation rate increased with immobilized enzyme amount and substrate concentration. Biodegradation rate didn't increase when the folw rate of the peristaltic pump was larger than 7 ml/min. The degradation limit of methyl-parathion concentration was 16.65 ppm.The other way to resolve OPs is to find new analysis methods as an alternative for traditional ones. It is a simple and quick process to analyse methyl-parathion concentration using free organophosphorus hydrolase. The effect of buffer concentration and pH, enzyme amount, temperature and reaction time on detection was investigated. According to the effect of the above factors, the detection condition was decided as follows: the buffer volume was 3ml, buffer concentration 50mM, pH was 8.0, enzyme amount was 20ul, reaction time 5min, at room temperature.Biosensors immobilizing organophosphorus hydrolase are a suitable alternative for traditional organophosphorus pesticides analytical technology. In this paper, a mathematic model of biosensors immobilizing organophosphorus hydrolase and pH electrode as the transducers was set up. The solution of the differential equations describing the detection process of the biosensors summarized the main factors influencing the response characteristics of the biosensors.