Isolation,Degradation Characteristics And Metabolic Regulation Mechanisms Of Sulfonamides-Degrading Microbial Consortium

Sulfonamide antibiotics(SAs)are the synthetic antibiotics with the highest detection frequency in China’s water environment,bringing huge potential risks to the ecological safety.The coexistence of multiple SAs in the natural water body and the wastewater of the sewage treatment plant aggravates the complexity of water environmental pollution.For the pollution of SAs,it is of great practical significance to develop efficient,low-cost and environmentally friendly biological removal methods by using biodegradation.In order to overcome the weak degradation efficiency of single SAs degrader,we aim to obtain SA degrading bacteria and synergistic metabolizing bacteria respectively through isolation and screening.Based on the screened microorganisms,we constructed a "bottom-up" composite consortium with high efficiency of SAs degradation.A comparative analysis was carried out on the biodegradation characteristics of sa.The interactions and metabolic mechanisms during the degradation of sulfonamide antibiotics were analyzed.The main research contents and results are as follows.The main contents and results are described as follows.In this study,the activated sludge was domesticated with sulfamethazine(SMZ)as a sole carbon source.We isolated and screened one strain of sulfamethoxazole-degrading and one synergistic metabolizing bacteria from the domesticated sludge subsequently.And we constructed a SMZ-degrading consortium.The results showed that strain YL1 was identified as Paenarthrobacter ureafaciens,while strain YL2 was identified as Pseudomonas koreensis.The genomes of strain YL1 and strain YL2 were sequenced and annotated by triple sequencing technology.The sequencing results showed that the genome of strain YL1 contains a gene cluster,sad ABC,which is responsible for two encoding flavoprotein monooxygenases and one encoding riboflavin reductase.Sad ABC is capable of attacking the initial sulfonamide bonds during the degradation process.The effect of the inoculum volume ratio of strain YL1 and strain YL2 on the degradation efficiency of SMZ was investigated,and the optimal ratio of YL1:YL2 = 1:1 was determined,on which the composite colonies of SAs-degrading bacteria were constructed.To clarify the biodegradation ability of SAs-degrading strain YL1,the degradation characteristics of strain YL1 for a variety of SAs were investigated under optimal degradation conditions.We established the growth kinetics of strain YL1 and the degradation kinetics of SAs to elucidate the mechanisms affecting the biodegradation of SAs by substituents.Sulfadiazine(SDZ),sulfamethoxazole(SMX),sulfamethoxazole(SMR)and SMZ were selected as the contaminants to be targeted,and the differences in the degradation efficiency of strain YL1 on SAs containing different substituents were investigated.When the strain YL1 was used to remove the mixture of various SAs,there were differences in the degradation efficacy.The results showed that the degradation rates of strain YL1 were basically the same for SDZ,SMX and SMR,while the degradation rate for SMZ was the lowest.In order to investigate the effect of the SAs’ substituents on the biodegradation rate,the growth kinetics and substrate degradation kinetics of strain YL1 were fitted with the Haldane model,respectively.The results showed that when strain YL1 was grown with four SAs substrates as a carbon source and energy source,the specific affinity of each substrate was ranked SMX>SMR>SDZ>SMZ;the maximum specific degradation rate of strain YL1 for four SAs was in the order of SMX>SDZ≈SMR>SMZ.The maximum specific degradation rate was the highest for SMX at 0.17 mg-SMX/h-mg-DW and the lowest for SMZ at 0.128 mg-SMZ/h-mg-DW.Density functional theory(DFT)was used to calculate the structural properties of the four selected SAs.Density functional theory(DFT)was used to calculate the structural properties of the four selected SAs.The effect of substituents on the biodegradation of SAs could be well explained by combining the calculated results with the characteristics of SAs such as acute and chronic toxicity.The results showed that the rate of biodegradation of those SAs with more stable structures and more toxic molecules was slower.To improve the problems of slow rate,low mineralization rate and susceptibility to environmental volatility in the process of biodegradation of SAs,a composite consortium consisting of strain YL1 and strain YL2 was constructed,and the metabolic pathways of the composite consortium for substrate degradation were investigated.The degradation efficiency between strain YL1 and the consortium with the same biomass was investigated separately using SMX with faster degradation rate as the substrate.The consortium had faster substrate degradation rate,and the mineralization rate of substrate was increased by 32.49%compared with that of strain YL1 alone.The community structure of the consortium was examined by q PCR.The results showed that the ratio of the biomass of strain YL1 and strain YL2 was largely unaffected by the initial concentration of SMX,and was able to maintain stability throughout the degradation process.The metabolic pathways of SMX degradation by the consortium were investigated by genomics and non-targeted metabolomics.A synergistic metabolic mechanism was proposed for the promotion of SAs mineralization.The results showed that strain YL2 in the consortium possesses the pathway to metabolize the key product of SMX degradation by strain YL1,4-Aminophenol(4-AP).Strain YL2 can rapidly utilize 4-AP,accelerate the mineralization of SMX,and eliminate the adverse effect caused by 4-AP;and strain YL2 can secrete riboflavin into the consortium,which enhances the mineralization of SMX.In addition,due to the addition of strain YL2,the biosynthetic pathway of various amino acids and the metabolic pathways such as ABC transporter protein in the consortium were promoted to be up-regulated,and finally promoted the biomineralization of SMX.