Degradation Characteristics And Biological Intensification Of Sulfamethoxazole By Shewanella Oneidensis MR-1

Sulfonamides are widely used in livestock and poultry breeding and human medicine,and most of them can't be completely metabolized and cause soil and water environmental residue problems.The iron-reducing microorganism Shewanella oneidensis MR-1 can use some organic acids as electron donors and gain energy.The electron shuttles can accelerate the electron transfer rate between microorganisms and extracellular electron acceptors,and enhance the degradation and conversion of pollutants in the environment.In this paper,under pure culture conditions,the degradation of sulfamethoxazole?SMX?by S.oneidensis MR-1 and the enhancement of organic acids and electron shuttles on SMX degradation by S.oneidensis MR-1 were studied.Effects of water content and organic matter content on the removal of sulfonamides in soil were studied.This study provides a theoretical basis for the microbial remediation of sulfa antibiotics in the natural environment.The main research contents and results are as follows:1.Through the pure culture of microorganisms,it was found that S.oneidensis MR-1had a degrading effect on low concentrations of SMX,and high concentrations SMX inhibited the growth of S.oneidensis MR-1.Under pure culture conditions,the SMX degradation rate was 42.2%after 5 days of culture at an 10 mg/L initial SMX concentration.When the initial concentration of SMX was 10 mg/L,there was no significant effect on the growth of S.oneidensis MR-1.As the initial concentration of SMX gradually increased,the number of S.oneidensis MR-1 gradually decreased,the growth of S.oneidensis MR-1 was almost arrested while the concentration of SMX was 70 mg/L.The initial concentration of SMX,pH,carbon source and other factors had a significant effect on the degradation of SMX by S.oneidensis MR-1.As the concentration of SMX gradually increased,the final degradation rate of SMX decreased.The optimum pH for the SMX degradation by S.oneidensis MR-1 was 7.0,and the degradative effect was better under weak alkaline conditions than weak acid condition.When 0.5 g/L glucose was used as the carbon source,the degradation rate of SMX was 66.5%after 5 days.After 0.5mmol/L sodium acetate was added as the electron donor,the SMX degradation rate was52.2%.After adding Fe-EDTA,the SMX degradation rate was 71.0%.The test results showed that:S.oneidensis MR-1 was resistant to low concentration of SMX,and the optimal pH for growth was 7.0;glucose was an appropriate carbon source for the growth of S.oneidensis MR-1,and sodium acetate was not good as an electron donor;the addition of Fe-EDTA significantly promoted the degradation of SMX by S.oneidensis MR-1.2.The effect of dissimilatory iron reduction on the SMX degradation by S.oneidensis MR-1 was studied under pure microorganism culture condition.It was found that the dissimilatory iron reduction had a significant effect on the biodegradation of SMX.The initial Fe???concentration in the system was 5 mmol/L by adding Fe?OH?₃ suspension.When the initial concentration of SMX was 10 mg/L,the final degradation rate of SMX was 67.6%,which was much higher than the control treatment.The final concentration of Fe???was 1.84 mmol/L in the control treatment.The addition of the same concentration of Fe₃O₄ suspension was not as effective as the Fe?OH?₃ suspension.The final degradation rate of SMX was only 46.1%.The final concentration of Fe???was 1.42 mmol/L.This showed that S.oneidensis MR-1 had different reducing power to different forms of iron.In the two experiments,the adsorption of Fe???on SMX was very weak.Therefore,the removal of SMX in the system was mainly due to the dissimilatory iron reduction,indicated that S.oneidensis MR-1 obtained energy from the Fe???reduction and promoted its own growth,which in turn promoted the biodegradation of SMX.3.The effect of three organic acids on the degradation of SMX and iron reduction by S.oneidensis MR-1 was studied.It was found that the addition of different concentrations of three organic acids could promote the SMX degradation,and the effect was citric acid>malic acid>oxalic acid.When the organic acid concentration was 1.0 mmol/L,the promotion effect was the most significant.The degradation of SMX was 58.5%,57.3%,and 55.4%,respectively.When 1.0 mmol/L citric acid/malic acid/oxalic acid and 5 mmol/L Fe₃O₄ suspensions were simultaneously added in the system,the SMX degradation rates were 74.6%,62.1%,and 59.4%after 5 days of incubation,respectively;when 1.0 mmol/L citric acid/malic acid/oxalic acid and 5 mmol/L Fe?OH?₃ suspensions were simultaneously added in the system,the SMX degradation rates were 90.3%,87.2%,and 78.5%,respectively.In this process,the generation of Fe???also greatly increased.Overall performance,the effect on SMX degradation and iron reduction was the most significant when citric acid and Fe?OH?₃ were added at the same time,which was mainly related to the first and second ionization constants(pK_a1,pK_a2)of citric acid.4.The microbiologically culture experiment found that both two electron shuttles had a significant effect on cell growth and SMX biodegradation.The addition of electron shuttles also promoted iron reduction.The degradation rates of SMX were 57.7%,70.7%,and 44.4%,respectively,after adding different concentrations of sodium hydrazine-2,6-disulfonate?AQDS?.After addition of different concentrations of riboflavin,the degradation rates of SMX were 79.7%,95.3%,and 50.3%,riboflavin had a stronger effect than AQDS.When electron shuttles and Fe???were simultaneously added in the system,SMX was almost completely degraded after 5 days,and iron reduction was also enhanced.The same effect of riboflavin on the reduction of iron was more pronounced.The electron transfer capacity?ETC?of the two electron shuttles was quantitatively characterized by an electrochemical method.The results showed that the electron accept capacity?EAC?of both two electron shuttles was greater than the electron donate capacity?EDC?,and riboflavin had stronger electron transfer ability than AQDS.This result explained the enhanced effect of electron shuttles on the degradation of SMX by S.oneidensis MR-1 from the perspective of chemical redox.The effect of electron shuttle on the 16S rRNA gene copy number of S.oneidensis MR-1 was determined by RT-qPCR quantitative analysis.The results showed that the copies of 16S rRNA gene copies of S.oneidensis MR-1 was significantly increased after adding electron shuttles.The relative abundance of 16S rRNA gene copy number was linearly related to AQDS and riboflavin concentration.It was demonstrated that the electron shuttle could enhance the activity of S.oneidensis MR-1 and promote the growth of the bacteria.The enhancement effect of the electron shuttle on the degradation of SMX by S.oneidensis MR-1 was explained from the biological point of view.5.In-house simulated culture experiments were conducted to study the degradation of sulfonamides in soil.The initial concentration of SMX was 10 mg/kg,the residual concentration of SMX in Jiangxi red soil was 2.20 mg/kg after 40 days of culture.Combined with the results of the control group,it was demonstrated that microbial degradation only played a part of role in SMX removal in soil.The inoculation of S.oneidensis MR-1 had little effect on the removal of SMX in soil.This might be due to the fact that the red soil used in the experiment was weakly acidic soil and was not suitable for the growth of S.oneidensis MR-1.Adding different concentrations of humic acid had little effect on the removal of single SMX in soil.This might because the humus contained in the soil could meet the test requirements,so the addition of exogenous humic acid had little effect.The initial concentrations of five sulfonamides?SAs?in soil were set as 10 mg/kg,respectively.The results showed that the SD degradation rate was the slowest among the five SAs,and the removal rate of the five SAs in the soil was lower than that of the single SMX.This might because the total antibiotic concentration was too high to inhibit the growth of microorganisms in the soil,which indicated that the type and total concentration of antibiotics in the soil affectted the final removal rate.When the test soil moisture content was 20%,50%,and 80%respectively,the final residual concentrations of the five SAs decreased significantly,and the degradation rate was SM2>SMX>SPY>SMM>SD generally.With the gradual increase of the proportion of extra sludge,the concentration of antibiotics in the soil decreased significantly,and the time to reach the stationary phase of degradation was shortened.When the sludge ratio was 8%,the final residual concentrations of SD,SPY,SM2,SMM,and SMX were 0.590,1.230,0.643,0.7311,and0.760 mg/kg,respectively.