Distribution And Dynamics Of Antibiotic Resistance Genes In Oxygen-Based Biofilm Under Different Nitrogen Sources And Organic Loadings

Due to the extensive use of antibiotics in clinic,animal husbandry and agriculture,antibiotic resistance genes（ARGs）in wastewater are widely released into natural water.Because of the high fluidity of wastewater and the high contents of ARGs,ARGs can proliferate and spread in the environment through vertical gene transfer（VGT）and horizontal gene transfer（HGT）during wastewater treatment and control process,which will cause harm to the health of organisms and environment.Therefore,it is important to explore the dynamics of ARGs in wastewater treatment process and develop appropriate control technology.In this work,greywater,a kind of domestic sewage from bathroom,kitchen,laundry and other places was used as the research object.The effects of organic loading,nitrogen source and COD/TN ratio on the distribution and dynamics of ARGs were investigated during greywater treatment in an oxygen-based membrane biofilm reactor（O₂-MBfR）.The absolute quantification of ARGs was performed by real-time fluorescence quantitative polymerase chain reaction（q PCR）.The removal characteristics of pollutants were revealed by concentration and physicochemical properties analysis.The characteristics of microbial community structure in the O₂-MBfR were analyzed by high-throughput sequencing technology.By analyzing the relationship between pollutant removal and ARGs,and the influence of microbial community characteristics on the production and distribution of ARGs,the operating conditions for directional regulation of the production and distribution of ARGs in biofilms were revealed.Accordingly,the control and removal of ARGs in the process of source separation of greywater and other types of wastewater would be realized by O₂-MBfR.The main research contents and corresponding results are as follows:（1）Under different organic loading rate（OLR）,ARGs were mainly distributed in biofilms,with the highest abundance in intracellular polymeric substance（IPS）,the second in extracellular polymeric substance（EPS）,and the lowest in free liquid.The abundance of free ARGs varied greatly under different organic loadings due to the effect of bacterial uptake channel size and microbial metabolic energy consumption.Compared with other organic loadings,the low organic loading（OLR=0.192）achieved higher intI1,korB,sul1 and sul2 abunences of 10^7.63～10^7.70,10^6.28～10^6.59,10^6.20～10^6.57 and 10^6.04～10^6.28 copies/g-VSS,respectively.At high organic loading（OLR=0.512 and OLR=0.673）,the abundance of free bla _CTX-M was the highest,ranging from 10^2.76 to 10^2.87 copies/g-VSS and 10^2.61 to10^2.81 copies/g-VSS,respectively.Under the five organic loading conditions,the O₂-MBfR achieved average removal rates of LAS and TN over 97%and 75%,respectively.In addition,low organic loading could reduce the organic shock to microorganisms and increase the diversity and richness of microorganism in biofilms.On the one hand,this change reduced the stress effect on ARGs.On the other hand,it increased the possibility of ARGs migration.（2）The organic nitrogen source-CO（NH₂）₂was more conducive to the accumulation of integron intI1 and sulfonamides resistance genes（sul1 and sul2）in biofilm than the inorganic nitrogen sources（NH₄Cl and NaNO₃）,and thus organic nitrogen source was more conducive to ARGs transfer.Compared with NH₄Cl and CO（NH₂）₂,NaNO₃ significantly promoted the accumulation of housekeeping gene kor B in the effluent.In addition,nitrogen source will also impact the diversity and uniformity of bacterial groups in biofilms.Among them,the number of microbial species in the biofilm samples with NH₄Cl as the nitrogen source was the least,but the uniformity was the highest.（3）COD/TN ratio had a significant effect on ARGs distribution in the system.High COD/TN（COD/TN=30:1）was more conducive to the accumulation of ARGs（especially int I1,kor B and sul1）in biofilms than low COD/TN（COD/TN=10:1）,while low COD/TN promoted the accumulation of erm B and qnr S in the effluent.When COD/TN=10:1,the average values of Richness,Shannon and Simpson index reflecting microbial richness and diversity were 286,4.58 and 0.979,respectively,which were higher than those of COD/TN=30:1.（4）The reactor performance significantly affected the distribution and abundance of ARGs.It was found that linear alkylbenzene sulfonate（LAS）,total nitrogen（TN）and other factors had the greatest influence on the distribution characteristics of ARGs.Organic loading,nitrogen source and COD/TN ratio could affect the distribution and dynamics of ARGs by regulating the pollutant removal performance.Therefore,for inorganic nitrogen source-NaNO₃,a smaller organic loading and a larger COD/TN ratio were more conducive to the removal of TN and LAS,which in turn promoted the capture and removal of ARGs hosts and the migration of ARGs.