Study On The Characteristics And Mechanism Of HMX Degradation By Rhodobacter Sphaeroides H

Octogen（HMX）is a typical explosive organic pollutant in the environment around a military factory.Microbial degradation of HMX has always been considered as the most effective way.Photosynthetic bacteria is a probiotics,and its growth,metabolism type and biodegradation function are outstanding,more and more attention has been paid to it by researchers.In this paper,using Rhodobacter sphaeroides H strain preserved in our research group as the test bacteria,the characteristics of HMX degradation by H strain were investigated,meanwhile the degradation pathway of HMX by H strain and the properties of crude enzyme solution were studied.The main results of this paper are as follows:（1）The domesticated H strain has high cell activity and can efficiently degrade HMX,meanwhile it mainly through biodegradation process.The effects of different oxygen supply light on the growth of H strain and the degradation of HMX showed that H strain could degrade HMX under all the conditions of anaerobic illumination,anaerobic darkness,aerobic illumination and aerobic darkness,and the degradation effect was the best under anaerobic illumination,up to 88.9%.Furthermore,the effects of the initial concentration of HMX,inoculation amount,pH value and temperature on the degradation of HMX by H strain were studied.The optimum degradation conditions were as follows:initial concentration of 100mg/l,pH 7.0,inoculation volume of 15%and at temperature of 30℃.The degradation rate of HMX was the highest,reaching 88.9%.When the initial concentration of HMX was 75¹50mg/l,the inoculation amount was 5%²5%,the pH value was 5.0⁹.0,and the temperature was 20⁴0℃,the degradation of HMX by H strain accords with the first-order reaction kinetic equation using the Andrews equation to simulateThe effects of different nutrient factors（including carbon source,nitrogen source and metal ion）on the degradation efficiency of HMX by H strain were studied under the optimum environmental conditions.The results showed that the optimum carbon source,nitrogen source and metal ion were malic acid,compound nitrogen source（NH₄）₂SO₂ and Ca²⁺,respectively.（2）The metabolite of HMX degradation by H strain was detected by liquid chromatography-mass spectrometry（LC-MS）.The results showed that during the degradation of HMX by H strain,three intermediates were detected,namely,mononitroso mNs-HMX of HMX and two isomers of dinitro derivatives,dNs-HMX and（MEDINA）.Of methylene dinitro amine.The first one is that HMX is degraded by reductase to mNs-HMX,and the second is that HMX is hydrolyzed to MEDINA.Because no more degradation products could be detected,the mechanism of HMX degradation by H strain needs need to be further studied.（3）The effects of different environmental factors（including initial concentration of HMX,pH value of solution,inoculum volume and temperature）on the specific activity of crude enzyme produced by H strain were studied.The results showed that the specific activity of the enzyme was enhanced at the concentration of 75,100 and 125 mg/l HMX,while the initial concentration of HMX in the culture medium reached 150 mg/l,the specific activity of the enzyme was inhibited.The specific activity of the enzyme was promoted when the pH value was 6.0⁸.0,however the specific activity of the enzyme was inhibited when the pH value was 5.0 and 9.0.The specific activity of the enzyme increased when the inoculation amount increased from 5%to 15%,the specific activity of the enzyme increasing from 15%to 25%,nevertheless the specific activity of the enzyme decreased gradually and the specific activity of the enzyme was inhibited.When the temperature increases from 20℃to 30℃,the specific activity of the enzyme increases and the specific activity of the enzyme increases.When the temperature increases from 30℃to 40℃,the specific activity of the enzyme decreases gradually and the specific activity of the enzyme is inhibited.The maximum specific activity of the crude enzyme solution was 0.405 U/mg under the initial concentration of 100 mg/l,pH 7.0,the inoculum amount 15%and at the temperature 30℃.The effect of different physical and chemical factors on the whole cell protein of crude enzyme solution produced by H strain was analyzed by polyacrylamide gel electrophoresis.The results showed that the crude enzyme protein produced by H strain was obviously promoted at the initial concentration of HMX of 75,100 and 125 mg/L.When the initial concentration of hmx is 150 mg/l,it can be inhibited..At the pH 7.0 and 8.0,the crude enzyme protein produced by H strain could be obviously promoted,When the pH value is 5.0,6.0 and 9.0,it can be inhibited.When the inoculum amount was 10%,15%and 20%,the crude enzyme protein produced by H strain had obvious promoting effect,but they could be inhibited when the inoculation amount was 5%and 25%.At 25,30 and 35℃,the strain can grow well,the crude enzyme liquid protein produced by the H strain has a significant effect,and when the temperature is 20℃and 40℃,the number of the protein bands is reduced,and the inhibition effect is exerted on the strain.