Mechanisms Of Microbial Response And Occurrence Of Resistance Genes During The Cometabolic Degradation Of The Selected Antibiotic By Enriched AOB Sludge

Antibiotics are the core drugs in the treatment of human,animal and plant infectious diseases,and have become one of the most widely used and the most powerful drugs in the world.The widespread use of antibiotics accelerates the occurrence and proliferation of antibiotic resistance genes（ARGs）in bacteria.The continuous input of antibiotics and their low removal efficiency have made wastewater treatment plants the hot spot for the emergence,exchange,recombination and proliferation of ARGs.In order to reduce the risks of ARGs,it is necessary to enhance the removal of antibiotics and inhibit the transfer and proliferation of ARGs.Current studies have shown that ammonia oxidizing bacteria（AOB）can enhance the removal of antibiotics via cometabolism.The relationship between antibiotic removal rate and ammonia oxidation rate has been the focus of many researches,as well as product identification during the cometabolic process.However,the response mechanisms of microorganisms and the accumulation and proliferation of ARGs along the process are rarely reported.Therefore,short-term batch experiment were conducted to explore the cometabolism degradation potential of enriched AOB sludge on sulfadiazine（SDZ）and the short-term response of microorganisms.Then the long-term exposure experiment with enriched AOB sludge and conventional activated sludge（CAS）was conducted to explore the removal performance of the two kinds of sludge on conventional pollutants and SDZ and the microscopic response of microorganisms under long-term stress of SDZ exposure.The metagenomic sequencing technology was also used to analyze the functional annotation,the changes of microbial nitrogen metabolism pathway and the fate of ARGs in the enriched AOB sludge and CAS,which could provide a more comprehensive theoretical basis for the degradation of antibiotics by AOB.The main findings are summarized as follows:（1）In batch experiments,AOB’s cometabolism degradation（41.81%）was the main mechanism of SDZ removal.The ammonia oxidation activity,ATP concentration and dehydrogenase（DHA）activity of the enriched AOB sludge were negatively affected by SDZ.The abundance of amo A gene increased by 1.5 times within 24 h,which was intended to enhance substrate uptake and utilization ability and maintain stable metabolic activity.The contents of protein（PN）and polysaccharide（PS）in extracellular polymer substances（EPS）and the proportion of tryptophan protein and humic acid organic matter also increased along the process.In addition,SDZ stimulated the secretion of C4-HSL,3OC6-HSL and C8-HSL in the enriched AOB sludge,and C8-HSL may be the key signaling molecule that promotes the secretion of PS and PN.（2）In the long-term experiment,the ammonia removal efficiency（ARE）and specific ammonia oxidation rate（SAOR）in the enriched AOB sludge decreased significantly at the initial stage with 0.1mg/L SDZ addition.After adaption,ARE and SAOR recovered to 96%and17.53mg N·（g VSS·h）^-1,respectively,and the removal efficiency of SDZ increased from 83%to over 95%.When the concentration of SDZ increased to 10 mg/L,SAOR decreased to 11.05mg N·（g VSS·h）^-1,and the removal efficiency of SDZ decreased to 64%.In CAS,under 0.1 mg/L SDZ stress,the removal efficiency of TN and TP decreased significantly,and the removal efficiency of SDZ was only 26%.When the SDZ concentration increased to 2.5 mg/L,the SDZ removal efficiency increased to than 95%,which might be due to the complex microbial community in CAS.The long-term exposure to SDZ also inhibited the activity of DHA in CAS and enriched AOB sludge.The enriched AOB sludge could better adapt to the stimulation of high concentration of SDZ（10 mg/L）,and its BSI value decreased significantly.In the enriched AOB sludge,Rhodopseudomonas,Bradyrhizobium,Afipia and Nitrobacter showed a significant positive correlation with EPS,while no significant correlation was found between the microorganisms and EPS in CAS,suggesting that SDZ inhibited the enrichment of EPS secreting bacteria in CAS.In addition,C8-HSL and C6-HSL may be the key signaling molecules of PN and PS secretion by enriched AOB sludge and CAS,respectively.Nitrosomonas is the dominant bacteria in enriched AOB sludge,and SDZ promotes the enrichment of Nitrosomonas.However,Meanwhile,the bacteria conducive to SDZ degradation were also enriched in CAS,such as Planctomycetaceae,Rhodopirellula and Microbacterium.（3）In long-term experiments,COG functional annotations indicated that the main gene function in enriched AOB sludge was cell wall/membrane/envelope biogenesis,while it was mainly amino acid transport and metabolism in CAS.KEGG functional annotation indicated that the proportion of membrane transport pathway in enriched AOB sludge and the proportion of energy metabolic pathway in CAS decreased,indicating that microbial responses of the two kinds of sludge were different under SDZ exposure.The microorganisms involved in nitrogen metabolism in the two kinds of sludge were also different under the long-term exposure to SDZ.In the enriched AOB sludge,ammonia was oxidized by Nitrosomonas to hydroxylamine and nitrite,and finally to nitrate by Thiocystis,Thiorhodococcus and Rhodovulum.In CAS,ammonia was oxidized to hydroxylamine by Nitrosomonas and Rhabdaerophilum,then to nitrite by Nitrosomonas and Hyphomonas.Finally,it was oxidized to nitrate by Cyclobacteriaceae and Leptothrix.Compared with CAS,the relative abundance of signal molecule synthetic protein genes in enriched AOB sludge was higher,indicating that the stronger microbial response,more active group sensing effect,closer communication among microorganisms and more EPS secretion to resist SDZ.The enriched AOB sludge could inhibit the occurrence and proliferation of four sulfonamide ARGs under the exposure of low concentration of SDZ（0.5 mg/L）.However,when SDZ concentration increased to 10 mg/L,the relative abundance of sul1 in the enriched AOB sludge increased from 0.349%to 1.538%,and the relative abundance of other sulfonamide ARGs decreased,suggesting that high concentration of SDZ may promote the enrichment of sul1 and inhibit the occurrence of sul2,sul3 and sul4.The relative abundance of sul1 and sul2 in CAS increased with the increase of SDZ concentration,indicating that the long-term exposure to SDZ promoted the enrichment of sulfanilamide ARGs in CAS.Meanwhile,other non-sulfonamide ARGs were also detected in the enriched AOB sludge and CAS under SDZ exposure,such as mac B,tet A（58）,nov A,bcr A and ole C.In the enriched AOB sludge,the main bacteria related to these ARGs were Nitrosomonas,Comamonas and Hyphomicrobium,while they were mainly Anaerolineales,Nitrospira and Cyclobacteriaceae in CAS.These genera could be the common potential hosts of multiple antibiotic resistance genes.