Isolation,Identification,Immobilization And Degradation Performance Of The Estradiol-Degrading Bacterium(Rhodococcus Sp. JX-2)

At present,many countries in the world are facing the problem of environmental estrogen pollution,which has aroused many scholars' attention.By disrupting the transportation,secretion,synthesis and metabolism of the organism's natural hormones,environmental estrogens cause a variety of system pathological changes including reproductive system,immune system,nervous system,and result to a serious threat to the safety and health of human and animals.Sewage and livestock manure are the main sources of environmental estrogens.How to make use of the functional microorganisms to efficient degradation of sewage and livestock manure to reduce the estrogen pollution risk has become a hot topic of concern at home and abroad.The degradation activity of microorganisms which isolated in the lab is often inhibited due to microorganisms not adapt to the environment,or they are at a disadvantage with the indigenous microbial competition.How to use immobilization technology to degrade estrogens of sewage and livestock manure is worth studying,related researches at home and abroad is rarely reported.Estradiol(E2),a common natural estrogen,is the most potent one among environmental estrogens with high examination rates in sewage and livestock manure nowdays.In this work,an estradiol-degrading bacterium(strain JX-2),which utilized E2 as the sole carbon source and energy identified as Rhodococcus sp.,was isolated from activated sludge of a wastewater treatment plant.The growth and E2-degrading performance of strain JX-2 were studied;Bacteria-immobilized agents were developed;The immobilized condition of strain JX-2 was optimized;The E2 removal efficiency of bacteria-immobilized agents in sewage and cow dung was explored and discussed the degradation performance to several common antibiotics.The main results as follows:(1)Isolated bacterium JX-2 could grow well on the plates containing E2,and utilized E2 as the sole carbon and energy source.JX-2 was identified as Rhodococcus sp.based on its morphology,physiology and 16S rRNA gene sequence.The optimal growth condition of strain JX-2 in LB liquid medium was 30?,pH 8.0,the total salinity 10 g·L-1 and the liquid volume 20 mL(150 mL conical flask).Temperature 30?,pH 7.0 and inoculum size 5%(v/v)were the optimal environmental conditions for strain JX-2 to E2 degradation.Under the optimized culture condition,E2 was effectively degraded.Strain JX-2 degraded E2 more than 94%under the condition of the initial concentration of E2 30 mg·L-1,30?,at a seed of 5%and 150 r-min-"1 within 7 days.Strain JX-2 exhibited excellent environmental adaptability.It could grow well and provide efficient degradation performance in high concentration of E2(50 mg·L-1).(2)The sodium alginate embedding conditions was optimized by the orthogonal test:4%sodium alginate(m/m),bacteria:sodium alginate(1:1,v/v),5%CaCl2·2H2O(m/m)and crosslinking time(6 h)at 4?.The effects of different environmental conditions on E2 degradation of bacteria-immobilized agent were studied.The results showed that,when the environmental conditions were mild,the E2 removal rate of bacteria-immobilized agent in aqueous solution had no significant difference with free strain.But the E2 removal rate of bacteria-immobilized agent was apparently superior to that of free strain under extreme environmental conditions.The optimal environmental conditions for E2 degradation by bacteria-immobilized agents were pH 7.0,temperature 30? and inoculum size 5%(v/v).When the concentration of E2 in aqueous solution was less than 50 mg·L-1,the E2 removal rate of bacteria-immobilized agent had no significant difference with free strain.But when the concentration of E2 was greater than 50 mg·L-1,the E2 removal rate of bacteria-immobilized agent was higher than that of free strain.The concentrations of E2 in the three sewage samples of a small lake in Nanjing were detected to 55.59,39.78 and 43.35 ng·L-1.After treating with the bacteria-immobilized agents with 7 days,the E2 removal rates of the three samples were 73.5%,64.4%and 100%,respectively.The concentrations of E2 in four cow dung samples collected from cattle farm were 146.31,148.84,158.70 and 174.01 ?g·kg-1 respectively.The E2 concentration decreased after treatment with the bacteria-immobilized agents,and the degradation rate was>81%.(3)The minimum inhibitory concentration(MIC)of 12 kinds of antibiotics to strain JX-2 was measured by drug sensitive test.Among the antibiotics,strain JX-2 was extremely sensitive to penicillin and erythromycin,the drug resistance of strain JX-2 to chloramphenicol,oxytetracycline and sulfadiazine was relative higher,the drug resistance of strain JX-2 to other antibiotics was somewhere in between.The effects of low concentration(less than the MIC value)and high concentration(greater than the MIC value)of antibiotics on cell growth and E2 degradation influences were studied.In aspect of degradation performance,low concentration of antibiotics reduced E2 degradation rate of strain JX-2 compared with blank control(CK)on 1 d and 3 d.When degradation time up to 7 d,E2 degradation rate increased significantly because strain JX-2 adjusted to low concentration of antibiotics stress.The E2 degradation rate of strain JX-2 was affected obviously by high concentration of antibiotic,and the degradation rate was between 20%and 35%,significantly lower than that of CK(91.3%).In aspect of cell growth,the concentration of antibiotics that below MIC value had no effect on the growth of strain JX-2;High concentration of antibiotics could obviously.inhibit the growth of strain JX-2.Effects of mixed antibiotics on E2 degradation of strain JX-2 were studied.The mixed antibiotics had no influence on E2 degradation of strain JX-2 in aqueous solution when concentrations of antibiotics were 10.0 ?g·L-1,100.0 ?g·L-1 and 1.0 mg·L-1,respectively,and the degradation rate was more than 90%.However,when the concentration increased to 10.0 mg·L-1,the mixed antibiotics inhibited E2 degradation and the E2 degradation rate was only 48.2%in 7 days.Strain JX-2 performed well on E2 degradation in sewage when antibiotics concentrations were 10.0 ?g·L-1,100.0 ?g·L-1 and 1.0 mg·L-1,and the degradation rates were higher than 80%.