| The mass production and use of antibiotics poses ecological risks.Among them,the continuous selection of antibiotic resistance genes（ARGs）is widely concerned,which has become an urgent problem in the world.At present,biological method is still the mainstream technology in practical application,especially anaerobic biotechnology due to its strong impact resistance,good ability to degrade complex organic matter and so on,widely used in antibiotic wastewater treatment.Activated sludge is not only considered as the reservoir of antibiotics and ARGs,but also the high incidence environment of ARGs transfer.However,the fate,propagation mechanisms and influencing factors of ARGs in anaerobic activated sludge are still unclear,as well as the relationship between ARGs and microbial functional metabolism remains poorly known.It is crucial to study the transfer mechanisms of ARGs for effective control and attenuation ARGs.This research explored the changes of ARGs and functional genes in sludge during anaerobic biological treatment of antibiotic wastewater,and revealed the driving mechanism of ARGs.Meanwhile,the obtained conclusions were applied to the ARGs control methods.Two reduction ways of mediating materials addition and pretreatment coupling were proposed specifically.Furthermore,their reduction mechanisms were revealed.Taking the typical antibiotics sulfamethoxazole（SMX）and sulfadiazine（SDZ）as representatives,two reactors were run in parallel to treat simulated wastewater,and explore the variations of ARGs under different antibiotics and concentrations（0.1-25mg/L）exposure.The results showed that the existence of antibiotics could promote the enrichment of ARGs.The ARGs abundance was increased by 184.1%and 103.1%with exposure to SDZ and SMX,respectively.SDZ resulted in a greater extent of ARGs enrichment,which was due to its difficult removal.The removal of the antibiotics was mainly through biodegradation,and adsorption only accounted for a small proportion.The enhanced abundance of plasmids,integrative conjugative elements and integrons among the mobile genetic factors（MGEs）is conducive to intra-and interbacterial propagation of ARGs.Combination with the abundance changes of functional genes,it could be found that antibiotic exposure caused oxidative stress in microbial communities,activated the SOS damage repair system,stimulated signal transduction to promote mutual communication and cooperation among microorganisms,as well as enhanced the bacterial type IV secretion system.These changes contributed to the transfer of ARGs.In addition,antibiotic shifted the microbial community structure to gradually enrich drug-resistant bacteria,and improved the possibility of vertical ARGs transfer.In particular,the abundance of potential hosts increased by 152.5%with SDZ exposure.To sum up,ARGs might be driven by both functional modules and bacteria community.Functional changes indirectly changed ARGs by affecting MGEs and horizontal transfer process,while microbial community effected directly on ARGs and contributed more greatly.Sludge with refractory antibiotics（SDZ）may stimulate the above two aspects to a greater extent,causing more obvious enrichment phenomenon of ARGs.The above conclusions have guiding significance for proposing the reduction strategies of ARGs:promoting the antibiotics removal,changing the functional metabolism and microbial community structure could help control the ARGs development.In this study,adding mediating materials was adopted to promote the degradation of SMX antibiotics which are easy to be degraded,and evaluated their impact on the target ARGs,including the corresponding four sulfonamides ARGs（sul1,sul2,sul3,sul4）and two non-corresponding tetracycline ARGs（tet A,tet C）,which are often detected in activated sludge and have co-enrichment phenomenon.The addition of Fe3O4,activated carbon（AC）and zero valent iron（ZVI）improved the metabolic activity of microorganisms,enhanced the interaction of bacteria and the potential direct interspecies electron transfer.The removal efficiency of SMX increased from 49.1%to 71.6-92.3%.The absolute abundance of ARGs decreased by 26.5%,62.0%and 48.5%,and the relative abundance decreased by 58.9%,65.9%and 57.7%,respectively.It could be seen that the effect of adding AC was best.The addition of all materials reduced the abundance of MGEs and the enrichment of drug-resistant bacteria,indicating that the inhibition of horizontal and vertical transfer of ARGs,which led to the decrease of ARGs spread.At the same time,they have changed the physiological features of microorganisms to varying degrees and regulated the horizontal transfer of ARGs.All of them improved the activity of antioxidant enzymes and relieved oxidative stress caused by exposure to antibiotics,but ZVI may produce additional free radicals that needs to be concerned.Further,ZVI and AC also interfered with cell communication and secretion system.In view of SDZ which is not easily biodegradable,it is obviously unwise to use the method of promoting degradation.In this study,ZVI activated persulfate was adopted to pretreat SDZ and explore its effect on target ARGs（ditto）in the subsequent activated sludge.The results showed that the concentration of SDZ decreased significantly after pretreatment,but the effect of complete mineralization was slightly worse,which led to the increase of the solution toxicity with short-time treatment.Different levels of pretreatment both effectively reduced the ARGs abundance in subsequent biological systems.The absolute and relative abundance of ARGs after 90 min treatment decreased by 81.9%and 52.9%,as well as the ARGs after 30 min treatment decreased by 59.8%and 9.1%,respectively.The effect of 90 min treatment is better than 30 min treatment.This was because the abundance of MGEs and drug-resistant bacteria both decreased after90 min pretreatment,which could effectively limit the transfer potential of ARGs.However,after 30 min treatment,the proportion of drug-resistant bacteria decreased,while the abundance of some MGEs was increased greatly,such as int I 1 and Tn916,which was not conducive to the reduction of ARGs.Based on these phenomena,multiple experiments were set up to analyze the roles of antibiotic residues（concentration and toxicity）and oxidation end product sulfate in regulation of ARGs after pretreatment.It was suggested that antibiotic residues were highly correlated with ARGs,MGEs and drug-resistant bacteria.Declined antibiotic concentration and toxicity could reduce oxidative stress,limit the occurrence of MGEs and the continuous proliferation of drug-resistant strains,which played an important role in reducing ARGs.Meanwhile,the presence of sulfate in influent supplemented microbial sulfur sources and facilitated the in-situ synthesis of antioxidant cysteine through sulfate assimilation.It limited horizontal transfer by alleviating oxidative stress and indirectly drove ARGs reduction.The increase of solution toxicity after 30 min pretreatment might enhance oxidative stress and stimulate horizontal transfer,so it is necessary to master the extent of treatment in practical use. |
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