Study On Whole Cell Organophosphorus Pesticides Sensor Based On Polyphosphate Accumulating Bacteria

Excessive use of organophosphorus pesticides(OP)has brought great harm to the environment and human health.The microbial degradation method is widely used in the degradation of OP because of its feature of no secondary pollution.In the previous study,we proposed a novel idea of organophosphorus pesticide sensor based on the physiological characteristics of phosphorus-accumulating organism(PAO),and demonstrated the feasibility of constructing a sensor for the analysis of hydrophilic organophosphorus pesticide residues in the environmental samples.Moreover,owing to highly hydrophobic property of most OPs a certain amount of organic solvents is indispensable to dissolve them.However,the PAO used in biological phosphorus removal treatment cannot survive in higher concentrations of organic solvents.Therefore,in the process of developing organophosphorus pesticide sensors,it is necessary to screen and cultivate a batch of organic solvent-tolerant PAOs and study their characteristics.This experiment first studied the organic solvent acclimation process of PAO and some of the characteristics of the extracellular enzymes secreted from the acclimated PAO.For the detection,a high-sensitivity dihydrogen phosphate ion-selective electrode was studied based on an ionic liquid sensitive membrane.Then we prepared a PAO whole-cell organophosphorus pesticide sensor and an organophosphorus hydrolase biosensor for OPs,which can be to determine the hydrophilic and hydrophobic organophosphorus pesticide residues in the complex environment samples.We described the acclimation research of PAO in organic solvent-water mixed solution.In this study,acetone was used as the typical organic solvent to cultivate a batch of acetone-tolerant PAO.From this batch of bacteria,we have isolated and screened out rod-shaped PAO colonies that can efficiently degrade hydrophobic OPs.The experiment explored the optimum conditions of OP degradation by acclimated PAO,and the results were compared with that of none acclimated.It was found that under the conditions of pH 7.2 none-phosphorus culturing buffer solution containing 10 ?L of acetic acid at temperature of 18?,it was most conducive to the degradation of OPs by the acclimated PAO.Under the conditions,the degradation rate of hydrophilic methamidophos was 99.4%,and the degradation rate of hydrophobic phoxim was 98.6%.We also prepared dry film-type phosphate accumulating bacteria,which is convenient for long-term preservation of the bacteria,by using the acetone acclimated PAO.Extracellular organophosphorus hydrolase(OPH)from PAOs is known to play a major role in the process of degrading OPs by PAOs.In this experiment,OPHs from the acclimated and pure PAOs were separated and purified,then their hydrolysis perfomances for OPs were studied.Through a series of ammonium sulfate precipitation,dialysis and DEAE-Sepharose FF anion exchange chromatography separation and purification process,two pure enzymes were obtained.SDS-PAGE showed that the molecular weights of these two enzymes were about 15.11 kDa(enzyme ?)and 11.98 kDa(enzyme ?),respectively.The enzyme activities of the both mixed enzymes before and after acclimation to hydrophobic phoxim were 0.105?mol/min·?g and 0.164 ?mol/min·?g,respectively.The enzymatic activity of organophosphorus hydrolase to phoxim from acclimated PAOs increased by about 1.6 times than that from none acclimated.In order to construct the detector for PAOs-based organophosphorus bacterial sensors,a dihydrogen phosphate ion selective electrode(H2PO4--ISE)was studied.In this experiment,an ionic liquid membrane electrode with high sensitivity and good stability is proposed.The indicate electrode is composed of[Hmim]PF6(1-hexyl-3-methy limidazolium hexafluorophosphate)and cyclohexanone 1:1 mixed with appropriate amount of CNT.The electrode and the reference electrode form a potentiometric detector for the sensor.The potential response time of the electrode is less than 1 s,and the minimum detection limit for dihydrogen phosphate ion could reach 1×10-10 mol/L.The H2PO4--ISE has high sensitivity,excellent selectivity with simple operation and miniaturization.On the basis of the research results above,a whole cell organophosphorus pesticide sensor based on phosphorus accumulating bacteria was prepared by immobilization of acclimated phosphorus accumulating bacteria as well as miniaturization of the H2PO4--ISE.The sensor has high sensitivity,wide linear range and good long-term performance.At the same time,based on the study of extracellular enzymes from PAOs,an enzyme electrode based potentiometric sensor was developed to realize the rapid analysis of organophosphorus pesticide residues.