Effect Of Biochar On Nitrogen Removal Performance Of Anammox Under Different NO₂^-/NH₄⁺

Anammox is a resource-efficient and sustainable nitrogen removal wastewater biological treatment technology that can reduce organic carbon by 100%and sludge production by 90%compared to traditional nitrification and denitrification processes.However,the anammox reaction not only produces 11%of the nitrate byproducts,but also the influents NO₂^-/NH₄⁺should be strictly controlled to 1.32.Partial nitrification（PN）is often used to regulate NO₂^-/NH₄⁺,but when dissolved oxygen is too high,the excessive proliferation of nitrite oxidizing bacteria（NOB）will lead to excessive conversion of NO₂^-to NO₃^-,limiting the stable supply of matrix and causing further increase of nitrate concentration in effluent.partial denitrification（PD）coupled with Anammox can achieve the removal of accumulated nitrate in anammox effluent water,but PD process consumes organic carbon sources,and improper addition of organic matter will affect the activity of anammox bacteria（An AOB）.In recent years,studies have shown that biochar can mediate microbial electron transfer,and when NO₂^-/NH₄⁺is 1:1,the NO₃^-concentration in the effluent of anammox system can be reduced by 40.7～45.2%,and the nitrogen removal performance of the system can be enhanced.However,the long-term effect of biochar on the enhancement of Anammox under influent NO₂^-/NH₄⁺fluctuations and its mechanism are still unclear.Therefore,a biochar-anammox system was constructed in this paper.Firstly,the effects of biochar on the nitrogen removal performance of the anammox when NO₂^-/NH₄⁺was 1.32,1.0,0.8 and 0.6 were determined by comparison with the control group.Secondly,through the analysis of electron transport activity（ESTA）,characteristics of biochar,changes in extracellular polymer（EPS）concentration and composition,and changes in microbial activity,the mechanism of enhancing the nitrogen removal efficiency of Anammox by biochar at low NO₂^-/NH₄⁺was proposed.Finally,based on high-throughput and macro-factor sequencing analysis,the distribution characteristics of microbial communities in biochar surface and suspension system were determined,and the changes of microbial communities and metabolic genes under biochar mediation were revealed.The main conclusions are as follows:（1）As the influent NO₂^-/NH₄⁺decreased from 1.32 to 0.6,the contribution of biochar-induced denitrification to the total nitrogen removal rate increased from 10.01%to 18.3%.And the NRE were consistently higher than that of R_CK,the accumulation of nitrate was always lower than that of R_CK.When NO₂^-/NH₄⁺=0.6,the presence of residual ammonia nitrogen in the biochar effluent decreases NER,but the nitrite removal rate was100%,the nitrate content was half of that of the R_CK,and the NRE（85%）was still 30%higher than that of the R_CK.（2）SEM results showed that the porous structure of biochar provided adhesion sites for microorganisms,which was conducive to the enrichment of microorganisms and EPS on its surface,and greatly improved the biomass and biological activity of the reactor.In addition,MLSS and MLVSS in the R_BC increased linearly,and the growth rate of microorganisms was 3.37 times that in the R_CK.With the decrease of NO₂^-/NH₄⁺,the activity of SAA in the R_BC gradually increased,and when NO₂^-/NH₄⁺=0.6,SAA was 1.72times that of the R_CK.The nitrate reduction activity increased from 0.65 to 1.24 mg NO₃^-VSS/g/h,which was consistent with the contribution of denitrification process to the total nitrogen removal efficiency.（3）Compared with the R_CK,the electron transfer activity（ETSA）of the R_BC was increased by 18.7%,29.1%,33.6%and 41.9%at each stage,respectively,and the change of ETSA activity was significantly positively correlated with the nitrogen removal efficiency of the Anammox（R²=0.813）.The results of FTIR and XPS showed that aromatic substances on the surface of biochar may act as electron donors in the short term,and redox groups such as phenols and quinone（C--O/C-OH）may act as electron shuttles,continuously mediating electron transfer and enhancing nitrate removal.（4）The EPS analysis results showed that the total EPS of R_BC at each stage was15.76,14.16,6.9 and 1.79 mg g^-1 vss higher than that of R_CK,respectively,and the CV curve of EPS showed an irreversible reduction peak,which proved that EPS had the ability to supply electrons.In addition,the soluble microbial byproducts in EPS can be used as direct electron donors for nitrate reduction.Redox active substances such as aromatic proteins and humus may act as electron transport mediators to promote biochar induced anammox and participate in extracellular electron transfer.（5）High throughput results showed that biochar contributed to the enrichment of functional microorganisms and could promote the coexistence of An AOB and denitrifying bacteria,and when the influent NO₂^-/NH₄⁺decreased from 1.32 to 0.6,the abundance of Candidatus＿Brocadia on the surface of biochar increased from 0.79%to33.25%,and the abundance of denitrifying bacteria Dokdonella increased from 0.09%to0.96%.In addition,Macrogene analysis revealed that biochar enhanced the anaerobic ammonia oxidation reaction and nitrate reduction process by increasing the gene copy number of hydrazine dehydrogenase gene（hdh）,hydrazine synthase gene（hzs A/B/C）,and nitrate reductase gene（nap A/B/C,nar G/H）.