Fate Of Antibiotic Resistance Genes In Bioelectrochemical Assisted Constructed Wetland System Treating Heavy Metal And Antibiotic

The coexistence of antibiotics and heavy metals in wastewater not only causes chemical pollution,but also induces the production of antibiotic resistance bacteria（ARB）and antibiotic resistance gene（ARG）in the environment.Moreover,the spread and diffusion of ARG will be intensified and endanger human health.Constructed wetland（CW）,as a widely used low-cost ecological wastewater treatment technology,also shows certain purification capacity of heavy metals,antibiotics and ARG.However,the removal efficiency of CW for target pollutants is relatively low,and CW can easily become the repository of ARG due to the characteristics of biofilm method.Therefore,further optimization of its removal efficiency for antibiotics and heavy metals is still needed to control the spread of ARG.The anaerobic and aerobic characteristics of the anode and cathode of microbial fuel cell（MFC）are consistent with the anaerobic characteristics of the bottom and aerobic characteristics of top layers in CW,respectively,which makes it possible to use MFC technology to enhance the pollutant removal.In this study,the MFC was embedded into CW to construct a new CW-MFC to enhance the removal of PPCP,antibiotics and heavy metals.Moreover,a new type manganese ore electrode CW-MFC（Mn）was developed to effectively enhance abundance of the extracellular electron transfer,COD removal,the abundance of electrochemical active bacteria（EAB）Geobacter,and to further suppress the abundance of methanogens in the system.Notably,this new system effectively solved the defects of unstable electricity generation and difficult enrichment of EAB in traditional carbon substrate CW-MFC.Furthermore,high throughput sequencing technology was used to explore the impacts of accumulation of mentioned typical pollutants and weak electric field on bacterial community composition,fate of ARGs and their corresponding host bacteria.Therefore,the mechanism of ARG abundance change was revealed during this process.Additionlly,sulfadiazine ARB（Klebsiella spp.）was isolated and purified from the effluent in a continuous running CW-MFC reactor.The changes of specific growth rate,biomass,morphology,number of viable bacteria,bacterial lethality,intracellular reactive oxygen species（ROS）,membrane permeability and expression and abundance of intracellular ARG were systematically studied under different intensities of current.Finally,a new type of three-dimensional biofilm electrode reactor（3D-BER）was constructed to preliminarily explore the response of ARG to the coexistence of antibiotics and heavy metals in electric field,aiming to provide a theoretical basis for revealing the response of ARG exposed to the electric field in CW-MFC.The main conclusions are as follows:（1）Impacts of open and closed circuit operation mode and hydraulic resisdence time（HRT）of CW-MFC on typical PPCPs and ARGs fate were studied.The results showed that the charged ions of organic molecule moved to the electrode surface under the action of the electric field to form a double electric layer in the closed circuit operation mode,and thus the adsorption capacity and metabolic activity of bacteria on the electrode were improved.Therefore,CW-MFC system has a higher PPCP removal efficiency.Moreover,closed circuit mode CW-MFC operated in high HRT contributed to increase in the diversity of microbial community.Thus,the hosts of ARGs were significantly enriched.PICRUSt function predicts results revealed that closed circuit operation exhibited positive effects on metabolic process and ARGs accumulation in the CW-MFC system.（2）To enrich EAB in CW-MFC,manganese ore constructed CW-MFC（Mn）was constructed.LEFse analysis results demonstrated that the average abundance of EAB in the manganese ore system was as high as 25.7%due to the dissimilar metal reduction reaction in the anode,while the corresponding abundance in the graphite electrode system is only 0.9%.Moreover,the CW-MFC（Mn）always maintained a higher cathode potential due to the catalysis of Mn O₂ in the cathode,so the system had a higher output voltage and power.PICRUSt prediction results confirmed that the manganese ore system significantly enriched extracellular electron transport functional genes but inhibited methanogenic functional genes.Therefore,electricity generation performance was enhanced,and higher COD removal rate was obtained.In addition,graphite substrate had higher removal efficiency of target antibiotics and ARGs due to its better adsorption capacity.Graphite substrate CW-MFC has a high removal efficiency of antibiotic despite of its low electricity generation capacity.Therefore,the removal efficiency of antibiotic does not only depend on the power generation capacity,and it can be affecred by substrate type.Redundancy analysis（RDA）showed that substrate type,antibiotic accumulation and bacterial community composition were the main factors affecting the changes of ARGs abundance.Network analysis revealed the potential hosts of ARGs and further revealed the co-occurrence pattern of ARGs and integron gene int I.（3）The impacts of coexistence of antibiotics and Zn²⁺on the abundance of ARGs were investigated in graphite substrate CW-MFC.The results showed that continuous Zn²⁺exposure enriched the target ARGs during the initial stage.However,excessive Zn²⁺accumulation was toxicity to bacteria and decreased antibiotic removal and the abundance of ARGs.A principal component analysis（PCA）demonstrated that ARGs and the bacterial community distribution characteristics were significantly impacted by the mass accumulation of antibiotics and Zn²⁺as well as the circuit mode.In addition,a RDA modeling,partial least squares path modeling,and Procrustes analysis revealed that the accumulation of antibiotics and Zn²⁺,the composition of the bacterial community,the circuit mode,and the abundance of int I associated with horizontal gene transfer jointly contributed to the distributions of ARGs in the electrodes and effluent.Moreover,continuous exposure to Zn²⁺decreased the bacterial diversity and changed the composition and function of the bacterial community predicted using PICRUSt tool.A variation partition analysis also showed that respective contributions of the accumulation of target pollutants and the circuit mode on the bacterial community composition and functions.The co-occurrence of ARGs,their potential hosts and bacterial functions were further revealed using a network analysis,and the result indicated they have a close and complex relationship.（4）The response of pure culture ARB to direct current was investigated.It was found that lactic dehydrogenase（LDH）increased with current in nutrition broth,indicating that the cell membrane permeability had increased.Since the adenosine triphosphate concentration increased with current,the metabolic activity enhanced in response to current stimulation.Moreover,intracellular reactive oxygen species had a positive correlation between ROS and the increased current（R=0.94）,which may be responsible for the high lethality rate of Klebsiella spp.The expression of sul I and int I significantly increased.However,the biomass,specific growth rate,the number of live bacteria decreased continuously and the proportion of dead bacteria increased continuously in simulated wastewater medium with the increase of current.Electric field reduced the activity of ARB,and the relative abundance of sul and int I.Further investigation showed that the decrease in ARGs abundance was responsible for decreased strain tolerance to SDZ in the simulated wastewater.These results showed that resistance plasmid was easy to maintain in the nutrition rich meidium,while it was easy to be lost in the nutrition minimal medium.These results are helpful to reveal the mechanism of the effect of electric field on ARB.（5）The results showed that coexposure to target antibiotics and Zn²⁺increased in the absolute and relative abundances of target ARGs in the 3D-BER reactor.Moreover,the target ARGs were enriched on the electrodes,while the abundances of ARGs were significantly reduced in the effluent.Meanwhile,RDA results revealed that the composition of bacteria carrying ARGs was greatly influenced in the cathode by the accumulation of Zn²⁺and antibiotic,which dominated the changes of ARGs abundance.Additionally,ARGs with their host bacteria revealed by network analysis were partially deposited in electrode substrates when being removed from wastewater.The results of FAPROTAX bacterial function prediction showed that the fermentation process and chemical energy heterotrophic process can be impacted by the conditions of electrical stimulation.Thus,current has impacts on the fate of pollutants,and it can reduce the risks of ARGs spread in the effluent.In conclusion,this study has important guiding significance for the research and development of efficient wastewater resource recovery and harmless treatment technology.