The Mechanism Of Doxycycline Degraded By The Microorganism And Immobilization

Doxycycline(DC)is a second-generation tetracycline antibiotic.Compared with the first-generation tetracycline antibiotic,DC is a safer alternative.Because of its safety,high efficiency and other characteristics,DC is the most commonly used tetracycline antibiotic in China in recent years.However,due to its large dosage and low absorption,most of them were discharged in the form of parent compound,which is easy to accumulate in the environment.Residual DC in the environment seriously may affect the soil ecological environment and threaten human health.Hence,how to remove the residual antibiotics in the environment with rapid and effective ways has attracted the attention of researchers.Although some studies have reported the non-biodegradation of DC,there is almost no study reported the DC biodegradation.The process and mechanism of DC biodegradation are still unclear.Therefore,this work aims to focus on isolating DC degrading bacteria to study its biodegradation mechanism,which fills the research gap on DC biodegradation by microorganism.Furthermore,the agricultural wastes were used as the adsorption carrier to study the mechanism of DC degradation by immobilized microorganism.On the one hand,agricultural wastes are utilized as resources,the other hand,it can provide a basis for the remediation of soil and water polluted by antibiotics.In this study,we used the chicken litter to isolate the DC degrading bacteria,studied the characteristics of DC degrading bacteria,established the hydrolysis and biodegradation kinetic models under different conditions.LC-MS/MS was used to capture the products of the DC degrading bacteria and proposed the DC biodegradation pathway.In addition,the rice straw and DD1 were used to prepare immobilization.Established its kinetic models of hydrolysis,adsorption and biodegradation of DC.DC distribution and its mass transfer pathway were studied in immobilization.LC-MS/MS was used to capture the products of DC by immobilization,predicted the DC degradation pathway.The main results were as follows:(1)DD1 and DD2 with high efficiency abilities of DC biodegradation were isolated from the chicken feces,they could not use DC as the sole carbon source,but DD1 and DD2 could degrade DC with extra carbon source by co-metabolize.The strain DD1 and DD2 were identified as Brevundimonas naejangsanensis and Sphingobacterium mizutaii.Studied the growth conditions of DD1 and DD2,the results showed that:DD1 and DD2were both mesophilic bacteria,the optimum pH of DD1 and DD2 were 7 and 8,respectively.Tryptone was the optimal carbon source for DD1 and DD2.(2)The effects of temperature,pH,different carbon sources,tryptone concentration were studied on the DC degradation by DD1 and DD2.The results showed that when the initial pH was 7,the temperature was 30?,the tryptone concentration was 10 g/L,the DC degradation rate of DD1 reached 92%;the pH was 8,the temperature was 30?,when the tryptone concentration was more than 4 g/L,the DC degradation rate of DD2 reached99%.The hydrolysis and biodegradation processes of DC degradation with DD1 and DD2satisfied the first-order kinetic model.After fitting different initial DC concentrations and initial biodegradation rates,it was found it could be described well using Michaelis-Menten model.(3)The inhibition zone method was used to study the antibacterial effect of DC products,and the results showed that the inhibition of DC biodegradation and hydrolysis products on E.coli was weakened.LC-MS/MS was used to analysis and identify the DC products of the hydrolysis and biodegradation.Two hydrolyzed products(EDC,ISO-DC),four(DP-417,DP-402,DP-338,DP-323)and six(DP-431,DP-417,DP-415,DP-411,DP-403,DP-382)biodegraded products were found to be formed by DD1 and DD2,respectively.DC was biodegraded by DD1 and DD2 through decarbonylation,deamination,dehydration,and demethylation.(4)Rice straw and DD1 were used to prepare immobilization,the degradation characteristics of DC were studied.SEM results showed that DD1 cells mainly distributed on the surface of rice straw,and fewer cells attached to the porous structure to grow.The hydrolysis,adsorption and biodegradation of DC removed by immobilization at different pH,tryptone concentration,and DC concentration,all of them met the first-order kinetic model.DC adsorption process of rice straw could be fitted using the pseudo-second-order kinetic model.The adsorption process was also fitted well by the intra-particle diffusion model.The isothermal models(Langmuir,Freundlich and Temkin)were used to fit the data,the highest R~2 of the Langmuir model was 0.9933.(5)The DC distribution in the immobilization was studied by extracting DC from different positions of the immobilization.The results showed that DC was detected at the medium solution,the suspended cells,the surface and inside of the immobilization.Especially,a large amount of DC was adsorbed by the immobilization.At the same time,combined with the intra-particle diffusion model,the possible mass transfer pathway of DC was predicted.(6)The DC degradation products were analyzed and identified by LC-MS/MS,two hydrolyzed products(EDC,ISO-DC)were captured,10 products of the DC degradation were captured by immobilization,such as DP-461,DP-459,DP-443,DP-428,DP-417,DP-410,DP-402,DP-400,DP-338,DP-323.DC was degraded by the immobilization with decarbonylation,deamination,dehydration,and demethylation.