Optimization Of Di-(2-ethylhexyl)Phthalate Degrading Bacteria Culturing Conditions And Characterization Of The Enzyme

As a classical phthalate esters(PAEs),Di-(2-ethylhexyl)phthalate(DEHP),as a plasticizer,is widely used in hundreds of products,including all kinds of toys,food packaging materials,medical bags and rubber,vinyl flooring and wallpaper,detergents,lubricants,personal care products(such as nail polish,hair spray,soap and shampoo).With the application of DEHP across the world,the soil,water and air has all been polluted at by the DEHP,causing potential threat to farmland,groundwater,plants,animals and human.It is found that microbial degradation of DEHP is an effective way to remove residual DEHP organic pollutants from the environment.This study further optimized,the culturing conditions of degrading bacteria screened and separated in the early stage and studied the properties of the degrading bacteria and its degrading enzyme,all of which has laid a theoretical foundation for the optimization of the production conditions of degrading bacteria.This study is of great significance for repairing DEHP contaminated soil,promoting national food quality safety and safeguarding human health.The main contents of the study include the following aspects:(1)This study first performs a single factor experiment,with the temperature,pH,DEHP concentration,inoculation volume and shaker speed as its research objects,and the OD600 the degrading bacteria production,as its response value.On the basis of single factor experiment,the Plackett-Burman design is performed,from which three most prominent factors influcing the degrading bacteria growth are screened out from five influential factors.Based on this,the steepest climbing experiment is carried out to determine the center of the subsequent Box-Behnken response surface optimization design.Based on the results of Box-Behnken response surface optimization design's results,multiple regression analysis is performed.The best culturing conditions of the degradation bacteria is concluded:the DEHP concentration is 1000 mg·L-1,the pH is 8.0,the temperature is 30×? the inoculation amount is 3%and the rotation speed is 125 rpm·min-1.(2)Both of the growth curve and degradation curve of the degrading bacteria JQ-1 under the best culturing conditions are studyed in this research.It detected that within the early 36 hours of the experiment,the degrading bacteria grow slowly,the concentration of bacteria and the value of OD600 do not change significantly.This is the retardation period.From the 36 hour to the 6o hours,the degrading bacteria multiplies,the value of OD600 rises sharply,and the degradation of rate of the degrading bacteria reaches its peak.This period is called the logarithmic growth period.After 72 hours of cultivation,the degrading bacteria enters the decline period.After 84 hours of cultivation,the degradation rate of the degrading bacteria reachs 98%.(3)In order to determine the properties of the degrading enzymes,relative studies on the properties of the crude degrading enzyme are carried out.The intracellular enzyme and extracellular enzymes of the degrading bacteria is extracted to measure the protein level of those two kinds of enzyme,respectively.The measurement results show that the protein level of intracellular enzymes is 38 ?g/ml,while the protein level of extracellular enzymes is only 3 ?g/ml.In the DEHP enzyme reaction system,same level of intracellular enzyme and extracellular enzymes is added,respectively.After 60 hours of cultivation,the DEHP degradation rate of intracellular enzyme is detected to reached 68.72%,the degradation rate of extracellular enzyme has reached 23.23%,and the degradation rate of control group without enzyme is 20.14%.All these results show that the mainly degrading enzyme of the degrading bacteria is intracellular.(4)A further exploration on the properties of degrading enzymes is carried out to study the effect of temperature,pH,concentration of DEHP and metal ion on the DEHP degradation rate of degrading enzymes.Temperature has significant influence on the activity of degrading enzyme,and the optimal reaction temperature of degrading enzyme is 30?.When the pH is between 7.0 and 9.0,the degrading enzymes show high activity.The degradation rate of degrading enzyme has reached its peak when the concentration of DEHP is 1000 mg·L-1.The study finds that Mg2+ and Ca2+ have positive effection on DEHP degradation,Mn2+ and Cu2+ have negtive effect,and that K+and Na+ have little effect on the degradation rate of degrading enzyme.