The Effect Mechanism Of Antibiotic Mixtures On Microbial Community And Antibiotic Resistance Genes In Anaerobic Biological Treatment System

Antibiotics have become an emerging contaminant in environment due to the overuse or abuse of them.Even at low concentrations,these non-metabolized antibiotics in environment still posed a constant selection pressure on the microbial community,leading to the occurrence and dissemination of antibiotic resistant genes（ARGs）in the microbial communities.At the same time,ARGs could spread among different bacteria through horizontal transfer,resulting in serious ecological risks.Anaerobic treatment has many advantages such as wide application,low energy consumption and clean energy generation.Thus it has been used in wastewater anaerobic treatment containing antibiotics and controlling the propagation of ARGs.It is significant to comprehensively reveal the occurrence,spread and distribution of ARGs in the anaerobic treatment system,as well as the succession of microbial communities,thereby mitigating the risk of ARGs spread in the receiving environments.This dissertation chose the upflow anaerobic sludge blanket reactor as the carrier,systematically studied the effect of antibiotics mixtures on the amplification of ARGs in UASB treatment system,evaluated the influence of antibiotic mixtures on the performance of UASB anaerobic reactor,and observed the changes of dissolved organic matter in effluent and the removal of antibiotics by UASB reactors.In addition,through the study on the succession of microbial community structure and revealed its correlation among key microorganisms,antibiotics and ARGs would provide an insight into the role of functional microorganisms and the control of dissemination of ARGs.Main research contents and results are as follows:（1）The effect of continuously increasing organic load on the performance of the UASB anaerobic reactor was observed.Results showed that the COD（Chemical oxygen demand）removal rate was generally maintained at about 80%,while the organic load in influent of the reactor was rising slowly from 1.0 kg·COD/（m³·d）to4.2 kg·COD/（m³·d）.And the COD removal rate of the effluent was about 85%during the stable stage.Under the increasing concentration of influent COD,the effluent COD was gradually decreased,indicating that the anaerobic microorganisms in the reactor have good ability in digesting organic matter.In order to create suitable environment for the growth of anaerobic microorganisms in the reactor,the alkalinity of the influent was strictly controlled and the occurrence of sludge loss was prevented by measuring the effluent p H and the sludge concentration of the reactor.（2）The combined effects of three antibiotics mixtures including erythromycin（ERY）,sulfamethoxazole（SMZ）and tetracycline（TET）on the performance of UASB reactor during anaerobic digestion were investigated.The results showed that with the increasing concentration of the antibiotic mixtures in influent of the reactor,the COD removal efficiency in UASB_antibiotics showed a large fluctuation compared with that of UASB_control throughout the study.In addition,the variations of biogas composition in the anaerobic reactor were influenced by antibiotic mixtures.The removal of antibiotics by UASB_antibiotics reactor mainly included biodegradation and physical adsorption,and the degradation rate of three antibiotics were different throughout 12stages of the experiment.The dominating organic matter in effluent of two reactors was aromatic protein,but the highest protein concentration in effluent of UASB_antibioticswas 76.94 mg/L in stage 12,which was much higher than that of UASB_control（0.56 mg/L）.（3）The effect of three antibiotic mixtures（ERY,SMZ,TET）on the characteristics of low molecular weight（<580 Da）soluble microbial products（SMP）in the reactor effluent was investigated.Results showed that the formation of SMP and the number of compounds in effluent of UASB_antibiotics was significantly influenced by the gradually increasing concentration of antibiotic mixtures.The SMP of UASB_antibiotics in stage 4 was composed of more long-chain alkanes,alkenes,aromatic compounds and esters than that of UASB_control.The possible reason for this result is that the higher concentration of antibiotics inhibits the growth of anaerobic microorganisms in the reactor,leading to the slowly accumulation of these organic matters.（4）In this part,the fates of ten ARGs（ere A,erm B,erm F,sul1,sul2,sul3,tet A,tet B,tet G,tet W）and class 1 integrase（int1I）during the long-term operation of the UASB reactor were observed,as well as the correlation between ARGs and microbial communities.During anaerobic sludge digestion,the abundance and distribution of ARGs and microbial community structure were significantly influenced by the increasing concentrations of antibiotic mixtures.And the tet W was the most dominant gene in both effluent and sludge of the reactor,the maximum abundance of it in total ARGs was 93.61%in sludges.The correlation between microbial community and ARGs implied that the proliferation of ARGs in anaerobic sludge may due to the variations of the microbial hosts with ARGs.Based on redundancy analysis results,Propionibacteriaceae was significantly positively associated with sul2,sul3,and tet B,which was considered as the potential microbial host.Results also showed that most microbes were negatively correlated to ARGs.（5）The succession of microbial communities during the anaerobic digestion of wastewater containing antibiotics in UASB reactor was further studied.Results showed that the addition of antibiotic mixtures reduced the diversity of microbial community significantly in the reactor.Four dominant phyla including Firmicutes,Halobacterota,Bacteroidetes and Euryarchaeota were all existed in sludge samples of two UASB reactors throughout the experiment.In addition,Halobacterota and Euryarchaeota included most types of Archaea.RDA on the genus level evaluated the correlation between top 5 genera and three antibiotics and found that most of the genera were negatively correlated with antibiotics.Only Treponema showed the positive correlation with three antibiotics,indicating that the addition of antibiotics may inhibit the growth of microorganisms.Based on network analysis,we find that most of the genera belonged to Firmicutes in the reactor were positively correlated with three antibiotics,which have the effect of fermentation to produce acid or hydrogen to produce acetic acid.These findings showed that Firmicutes were more resistant to the changes in external environmental conditions.