Characteristics And Mechanism Of Sulfamethoxazole Mineralization By Microbial Community In Activated Sludge

China has the most extensive usage and production of sulfonamides(SAs).The release of SAs into the environment has caused adverse effects on the ecological environment.The persistent toxic pressure of SAs can induce the formation of antibiotic resistance bacteria(ARB),which pose a potential threat to human and environmental health.Therefore,it is of great significance to find an efficient method for the complete mineralization of SAs.At present,most of the reported biodegradation products of SAs have not been efficiently mineralized,and could be converted back to the parent SAs by reverse pathway.In this situation,the toxicity of SAs has not been relieved.In recent years,another degradation pathway of SAs regulated by genes cluster sad ABC has contributed to the improvement of the mineralization degree toward SAs.However,the nitrogen heterocyclic products are stably accumulated as the dead-end products,which hinders the complete mineralization of SAs under aerobic conditions.In addition,considering the fact that a large number of isolated aerobic pure bacteria capable of degrading SAs do not have the ability of complete mineralization of SAs,it is unrealistic to rely on a single strain for the complete mineralization of SAs.At least there exist nitrogen heterocyclic products-degrading bacteria to be excavated.Moreover,the interactions among more than two species are needed to achieve the complete mineralization of SAs.Due to the long-term exposure to the toxic pressure of SAs,microorganisms in activated sludge can form a functional microbiome with high removal efficiency and mineralization degree toward SAs.By using activated sludge from Wenchang sewage treatment plant in Harbin as inoculation source,with sulfamethoxazole(SMX),a representative of SAs,as the sole carbon source and energy source,enrichment culture S1 and S2 with different labor-division were obtained.Both of them could completely convert 50 mg/L SMX to the equivalent 3-amino-5-methylisoxazole(3A5MI)within 13.5 h and 12 h,respectively.However,when the accumulation of3A5 MI reached the maximum,the degradation efficiency of enrichment culture S1 and S2 was significantly different.Enrichment culture S2 continued to stably accumulate 3A5 MI,while the concentration of 3A5 MI in enrichment culture S1 gradually decreased until completely removed.This phenomenon also led to a significant difference in the removal of total organic carbon(TOC).At 50 h,the TOC removal rate of enrichment culture S1 was as high as 100%,which realized the complete mineralization of SMX.However,the final TOC removal rate of enrichment culture S2 was only 63.00% ± 14%.In addition,by switching the carbon source from SMX to 3A5 MI,the enrichment culture A responsible for 3A5 MI mineralization was extracted from enrichment culture S1.On the basis of enrichment cultures with different labor division,two core degradation bacteria,Paenarthrobacter sp.P27 and Nocardioides sp.N27,were isolated.Their function is important and unique: strain P27 was the only bacterium that could realize the oxidative cleavage of C-S-N bond in SMX molecular structure(the initial and key step for SAs mineralization),and continue to degrade the phenyl part product;similarly,strain N27 was the only bacterium that could use the nitrogen heterocyclic product 3A5 MI as the sole carbon(nitrogen)source and energy source.By comparing the degradation efficiencies of co-culture pattern(P27 and N27)with enrichment culture S1,it was suggested that the indirect degradation bacteria(bacteria that were grown and reproduced by using the small molecular intermediate products formed in the SMX degradation)was necessary for the complete mineralization of SMX.The enrichment culture S1 could reach 100% mineralization rate in 50 h,but the mineralization rate under co-culture pattern was only 75.71% ±1.31% even when the sampling time was delayed to 90 h.The activated sludge microbiome that composed of core(direct)degradingbacteria(P27 and N27)and indirect degrading-bacteria was the key functional unit for the complete mineralization of SMX.The direct or indirect positive interaction between these active microorganisms directly or indirectly promoted the complete mineralization of SMX.Seven genera(Simplicispira,Sphingobium,Hydrogenophaga,Rhizobium,Achromobacter,Alicycliphilus and Acidovorax)responsible for the indirect degradation of SMX were identified by the succession analysis of microbial community structure.Another seven genera(Acidovorax,Simplicispira,Sphingobium,Alicycliphilus,Chryseobacterium,Pedobacter,Diaphorobacter)and a family Chitinophagaceae were identified as indirect 3A5 MI degraders.Pearson correlation analysis showed that Sphingobium(r = 0.5965,P = 0.0005)and Acidovorax(r =0.8453,P < 0.0001)had significant positive correlations with the acceleration of3A5 MI degradation.By the correlation verification experiment(different co-culture pattern designed using Nocardioides,Acidovorax and Sphingobium),the positive correlations were further verified.In addition,DNA stable-isotope probing(DNASIP)identified nine important OTUs that had assimilated the carbon atom in the benzene ring of SMX.Finally,based on the identified active microorganisms,the molecular ecological networks(MENs)analysis revealed the formation of positive interaction networks composed of active microorganisms under the long-term selective pressure of SAs.