Sulfur Cycle Mediated Electron Transfers And Denitrification Pathways In Biofilm-electrode Constructed Wetlands

Excessive discharge of nutrients such as nitrogen and phosphorus from wastewater treatment plants（WWTPs）is an important source of nutrients inputs of waterbodies.In order to reduce the nutrients inputs in the WWTPs effluent,which could reduce the contribution to the eutrophication to some degree,there is an urgent need for tetiary nitrogen and phosphorus removal from WWTPs effluents.In order to solve the above problems including unsatisfied nutrients removal performance and carbon source lacking for heterotrophic denitrification,electrode biofilm-constructed wetlands were constructed for simultaneous phosphorus and nitrogen removal from WWTPs effluent.The responses of nutrients removal performance and microbial community succession to environmental factors were investigated.In addition,this paper focuses on the response,adaptation and feedback mechanism of the potential nitrogen,sulfur and carbon metabolism and multi-pathways denitrification mechanism in response to the changes of environmental factors.Moreover,PO₄^3--P removal pathways and its kinetic characteristics in the iron anode of the system were explored,and the greenhouse gas N₂O accumulation potential and its mechanism of the system were evaluated.The main conclusions of this study are as follows:The flow direction and inoculum posed significant effects on the construction of electrode biofilm-vertical flow constructed wetland system（E-VFCWs）.The NO₃^--N removal rates of upflow and downflow E-VFCWs systems were 47.9±5.8%and97.1±2.0%,respectively.The flow direction posed significant effects on the relative abundance of denitrifiers and nitrogen removal pathways,which led to the difference of nitrogen removal efficiency of the E-VFCWs.Among them,the microbial denitrification process in the upflow E-VFCWs system may only contain hydrogenotrophic denitrification,while in the downflow E-VFCWs system,and the denitrification process included multiple-pathways such as hydrogenotrophic and sulfur-dependent denitrifications.In addition,the abundance of denitrifiers in downflow E-VFCWs was higher than that in upflow E-VFCWs.The construction periods of E-VFCWs system inoculated with anaerobic sludge was shorter,and the removal efficiency of NO₃^--N and TN were higher than those when inoculated with municipal sewage sludge or without inoculation.After successful construction,the removal efficiencies of NO₃^--N and TN were 97.0±1.6%and 83.3±4.6%when inoculated with anaerobic sludge,respectively.The reason for the better nitrogen removal efficiency of E-VFCWs inoculated with anaerobic sludge may be that the copies number of bacteria after the construction of the system,and the relative abundance of functional genes related to microbial metabolic processes such as denitrification were higher than others.The electrode biofilm-constructed wetlands showed efficient and stable PO₄^3--P removal efficiency（89.7-99.4%）.HRT and current density had significant effects on PO₄^3--P removal efficiencies,while temperature or influent SO₄^2--S concentrations had little effect.The principle of phosphorus removal in the system was the adsorption by Fe OOH.When the current was lower than 0.005 A,the electroflocculation adsorption may conform to the first-order kinetics,while when the current ranged between0.025-0.050 A,the electroflocculation adsorption may conform to the second-order kinetics.Under temperature of 28℃and 12℃,the removal efficiencies of NO₃^--N were82.7-99.6%and 50.8-91.8%,respectively,and removal efficiencies of TN were 51.9-93.7%and 38.8-73.1%,respectively.Except hydrogenotrophic denitrification,the denitrification processes in E-VFCWs system also included Fe⁰ chemical NO₃^--N reduction,Fe（II）-dependent denitrification,sulfate reducing-sulfur autotrophic denitrification,fermentation or H₂/CO₂ autotrophic acetogenesis-heterotrophic denitrification,disassimilatory reduction to ammonium（DNRA）.However,the contributions of plant absorption or matrix adsorption to NO₃^--N removal within E-VFCWs was only 1.1-3.7%.Temperature was the key factor affecting diversity and functional microbial community structure related to nitrogen and sulfur transformations in E-VFCWs system.When the temperature decreased from 28℃to 12℃,the relative abundance of hydrogenotrophic denitrifiers increased,while that of the sulfate-reducing bacteria exhibited the opposite trend.The increase of relative abundance of sulfur-dependent denitrifiers at low temperature may enhance the NO₃^--N removal efficiency of E-VFCWs system.The NO₃^--N removal stability of electrode biofilm-horizontal flow constructed wetland system（E-HFCWs）with different S/N ratios in response to current or HRT changes was significantly different.Among them,the denitrification stability of E-HFCWs system to the change of current or HRT in medium-and high-sulfur groups（S/N ratio of 2.064 and 4.183,respectively）were much higher than those in low-sulfur group（S/N ratio of 0.271）.In addition,a significantly linear positive correlation relationship between the improvement rate of NO₃^--N removal efficiencies within the medium-or high-sulfur groups and SO₄^2--S accumulation in E-HFCWs system in E-HFCWs system indicated that sulfur cycle may play an important role in improving the anti-shock load capacity of nitrogen removal efficiency of E-HFCWs system under the condition of low current or HRT.Based on the assumption that sulfur cycle enhanced denitrification efficiency,this experiment verified that adding pyrite to enhance sulfur autotrophic denitrification rate was an effective emergency measure to deal with the deterioration of denitrification efficiency of E-HFCWs system,especially for medium-and high-sulfur groups.In this study,microbial community succession and processes concerning nitrogen,sulfur and carbon metabolisms（including denitrification,sulfur cycle,carbon fixation,hydrogen oxidation,acetic acid oxidation and methane oxidation）in E-HFCWs system were studied by metagenomic sequencing.When the current was 0.1 A and the HRT was 24 h,the potential denitrifiers in the E-HFCWs system of the low-sulfur group mainly included Thauera,Azoarcus,unclassified＿Comamonadaceae,Meiothermus,and the possible electron donors mainly included H₂ or acetic acid.At this stage,the composition and structure of potential denitrifying bacteria in E-HFCWs system of high-sulfur group was similar to that of E-HFCWs system of low-sulfur group.The possible electron donors included H₂,acetic acid,CH₄,and sulfur.When HRT decreased to 6 h,the composition and structure of hydrogenotrophic denitrifying bacteria in E-HFCWs system of low-sulfur group changed significantly,and the relative abundance of potential denitrifying bacteria was significantly lower.When the HRT was 6 h,compared with the HFCWs system of low-sulfur group,the relative abundance of denitrifying bacteria in high-sulfur group HFCWs system was higher,the denitrification type was more abundant,and the function related to nitrogen,sulfur and carbon metabolism and the number and degree of nodes（species）in the species co-occurrence network were（degree）higher.Therefore,the denitrification efficiency of high-sulfur group E-HFCWs was more stable.The N₂O accumulation rate of E-VFCWs system at low temperature（20.9-34.2%）is much higher than that at room temperature（<0.86%）.The main reason is that the inhibitory effect of low temperature on N₂O reduction functional gene is stronger than that of N₂O producing functional genes.In addition,the N₂O accumulation rate of the system increases with the decrease of current or HRT,and under the condition of low current or HRT,the E-HFCWs accumulation rate of medium-and high-sulfur group is significantly lower than that of low-sulfur group,indicating that the electron storage and reuse mode of sulfur cycle in medium-and high-sulfur group improves the utilization rate of electrons in the whole operation process.In addition,the addition of pyrite could quickly alleviate the N₂O accumulation in the electrolytic biofilm-constructed wetlands system,especially for medium-and high-sulfur groups E-HFCWs.In this study,the model of electron transfer（electron storage in the stage of electron reuse）in the process of sulfur cycle in electrode biofilm-constructed wetlands system,and its regulation on denitrification efficiency and pathways were proposed for the first time.When HRT,current density and temperature are higher,more effective electrons per unit of water can be obtained.At this time,the electrons provided by electrolysis are not only used for NO₃^--N reduction in the biofilm-constructed wetlands system with medium-or high-sulfur group,but also deposited in the system in the form of solid-state reduced sulfur.At this stage,the main role of sulfur cycle in the biofilm-constructed wetlands system with medium-and high-sulfur groups electrodes is electronic storage period.However,when HRT,current density or temperature are lower,the number of available electrons per unit of water is insufficient,and the number of electrons provided by electrolysis is not enough to meet a satisfied NO₃^--N removal effect.The solid-state reduced sulfur deposited in the system in the electron storage period will provide additional electrons for NO₃^--N reduction through sulfur-dependent denitrification.At this stage,the main role of sulfur cycle in the biofilm-constructed wetland system with middle-high sulfur groups electrode is electron resupply,which could play important role in strengthening the ability of the system to cope with NO₃^--N load fluctuation or low temperature stress.The research perfects the theory of nitrogen transformation mechanism in autotrophic system,and provides new perspective for deep phosphorus and nitrogen removal from WWTPs effluents.The research has important theoretical value and practical significance.