Influences And Microbial Driving Mechanism Of Biochar Addition On N₂O Emission In Aerated Subsurface Flow Constructed Wetlands

Water pollution and global warming are two major environmental problems facing the society today.The wastewater discharge has a significant impact on the increasing of greenhouse gas concentration in the atmosphere.Constructed wetlands(CWs)has been widely used in the treatment of aquaculture wastewater,domestic sewage,and eutrophication water.However,the problem needs to be solved in traditional CWs is that the low nitrogen removal efficiencies and high nitrous oxide(N₂O)emissions,due to the uneven distribution and slow diffusion rate of dissolved oxygen(DO).In recent years,it is of rising interest in biochar as a conditioner in CWs due to the excellent structure property,large surface area,and high cation exchange capacity.Correspondingly,the microbial nitrogen transformation is complex because of the special internal environment of wetland.The effect of biochar on N₂O production pathways and the response mechanism of microorganisms are not clear.In this paper,five subsurface flow constructed wetlands were constructed on the basis of the volume ratio of biochar in common gravel with 0%,10%,20%,30%and 40%(named as CW0,BW10,BW20,BW30 and BW40,respectively),for the evaluation of pollutant removal and N₂O emission response to biochar addition.Besides,the influences of biochar addition on N₂O production pathways were studied by chemical inhibitor method and isotopomer ratio of N₂O.Furthermore,high-throughput sequencing and real-time fluorescence quantification(q-PCR)molecular biology techniques were used to explore the microbial mechanisms of nitrogen transformation under the action of biochar-added CWs.Our purposes were to define the microbial driving mechanism of N₂O emissions in CWs,providing theoretical basis and data supporting for strengthening the application of biochar in CWs.The specific conclusions of the test were as follows:(1)The dissolved DO concentration was significantly increased with bichar addition in the aeration stage.Correspondingly,the effluent DO concentration of biochar added CWs were decreased slightly,but the effect of biochar dosage on DO was not significant(p>0.05).The the effluent p H of biochar added CWs were also slightly decreased,but the effect of biochar dosage on p H was not significant(p<0.05).The conductivity(Cond)were significantly decreased with biochar addition(p<0.05).(2)The COD removal rates were improved with biochar addition,while the influence of different dosage were not significant(p>0.05).The total phosphorus(TP),ammonium(NH₄⁺-N),and total nitrogen(TN)removal rates were significantly increased.The TP removal rates increased by 2.04%,0.77%,2.78%and 4.27%;NH₄⁺-N removal rates increased by 3.49%,4.34%,9.0%,and 9.45%;TN removal rates increased by 5.22%,6.63%,10.03%and 12.10%,respectively,with 10%,20%,30%,and 40%of biochar addition in CWs.(3)The N₂O emission fluxes of all CWs under aeration stage were significantly higher than that anaerobic stage,and the N₂O emission fluxes were lower in bichar added CWs.However,there were no significant difference in the anoxic phase.Compared with contral,10%,20%,30%,and 40%of biochar addition could reduce N₂O emission by21.95%,25.12%,33.37%and 38.60%,respectively.(4)The results of chemical inhibitor method revealed that N₂O was produced via nitrification and the denitrification pathways equally in control,with the cumulative emissioms of 5.97 mg/m²,and 6.94 mg/m²,respectively.However,the relative contribution of nitrification pathways was significantly higher than that denitrification pathways in the bichar added CWs,with the cumulative emissioms of 3.72 mg/m²,and1.96 mg/m²,respectively.The addition of biochar could effectively reduce N₂O emissions both in nitrification and denitrification.The N₂O emission reduction ratios of the nitrification and denitrification were 35.75%and 71.75%,respectively,with 30%of biochar addition.(5)N₂O was mainly produced via simultaneous nitrification and denitrification,and both NH₂OH oxidation and NO₂^-reduction were important pathways of N₂O production in aeration stage.However,the N₂O emission fluxes were very low and were mainly produced by NO₂^-reduction pathway in anaerobic stage.Although AOB denitrification made a certain contribution to N₂O production in the early stage of the reaction,the NH₂OH oxidation carried out by AOB and the simultaneous denitrification carried out by heterotrophic denitrifying bacteria were the dominant pathways of N₂O emissions in the middle and later stages.The N₂O emission were reduced by reducing both the NH₂OH oxidation and NO₂^-reduction pathways.(6)The dominant microbes at phylum level in CWs were Actinobacteria,Proteobacteria,Chloroflexi,Modulibacteria,Bacteroidetes,Patescibacteria,Acidobacteria,Spirochaetes,Caldiserica,and Firmicutes.The species richness and diversity of nitrifying bacteria were increased with the addition of biochar,while the diversity of denitrifying bacteria was decreased.The structure changes of de-nitrification bacteria might improve the process of nitrogen transformation in the CWs,resulting the reduction of N₂O emissions.(7)The gene copy numbers of amo A,nir S and nosZ in the CWs were significantly increased with the increasing of biochar proportion,while the copy number of nar G were significantly decreased.The simultaneous nitrification and denitrification rates were increased with the biochar addition,which might lead to directly heterotrophic reduction of NO₂^-,showed a lower abundance of nar G.Assuredly,the NH₂OH oxidation,NO₂^-and N₂O reduction process were promoted with biochar addition,resulting in lower N₂O emission in CWs.