Performance And Mechanism Of Nitrogen Removal By Coupling Denitrifying Anaerobic Methane Oxidation With Anammox

Denitrifying anaerobic methane oxidation（DAMO）process,which oxidizes methane anaerobically using nitrate/nitrite as the electron acceptor,forms an important link between the carbon and nitrogen cycles,offering a promising alternative to potentially achieve the simultaneous nitrogen removal and greenhouse gas mitigation.However,the difficulty in effective accumulation of biomass results from the extremely slow growth rate of DAMO microorganisms significantly hinders its development and practical application in wastewater treatment.Therefore,membrane granular sludge reactor（MGSR）were developed in this study,which is able to provide dissolved methane for the growth of DAMO microorganisms and to enrich DAMO microorganisms in granular sludge to achieve sufficient biomass accumulation.The aim of this study is to cultivate granular sludge coupling DAMO with Anammox,to reveal the granulation process coupling DAMO with Anammox,and to apply granular sludge coupling DAMO with Anammox to the treatment of partial nitritation effluent from sludge digestion liquor and municipal wastewater respectively,to achieve high-level nitrogen and dissolved methane removal.High-throughput sequencing,fluorescence in situ hybridization（FISH）,metagenomics and metatranscriptomics approaches,mathematical model,etc.were conducted to reveal the synergistic mechanics between DAMO and Anammox in granular sludge for nitrogen removal.Firstly,by using MGSR which was inoculated by DAMO flocs and anaerobic granular sludge,with nitrate and ammonium as nitrogen resource,granular sludge coupling DAMO with Anammox with an average diameter of 2.1 mm was cultivated.The DAMO&Anammox granules were capable of removing nitrogen at rate of 1.85kg N m^-3 d^-1 steadily in continuous operation,demonstrating excellent nitrogen removal capacity.The high-throughput sequencing analysis indicated that both Candidatus Methanoperedens and Candidatus Kuenenia dominated the granular sludge,with relative abundances of 32.65%and 6.95%respectively.The sludge granulation process of DAMO with Anammox was demonstrated from mixed inoculum including river sediment,return activated sludge and crushed anaerobic granule sludge in another MGSR.Flocculent biomass gradually turned into compact aggregates and retained as granular sludge with an average diameter of 2.2 mm after684 days’operation.Granules with density of 1.043 g m L^-1,settling velocity of 72 m h^-1 and sludge volume index of 22 m L g^-1 leaded to excellent biomass retention(42 g VSS L^-1).The abundance of methanogens decreased gradually and disappeared,while the proportion of Candidatus Methanoperedens and Anammox bacteria gradually increased and finally reached to 31.09%and 12.45%on Day 680.FISH result revealed that DAMO and Anammox microorganisms distributed homogenously throughout the granule,regardless of the differences in their abundance.Secondly,the cultivated granular sludge coupling DAMO with Anammox was applied in the treatment of anaerobic sludge digestion liquor and municipal wastewater.With influent(1000 mg TN L^-1)continuously fed to MGSR at an HRT of1.44 h,granular sludge coupling DAMO with Anammox achieved a biomass concentration of 43 g VSS L^-1 and a nitrogen removal rate of 16.53 kg N m^-3 d^-1 with efficiency of 99.21%,demonstrating outstanding nitrogen removal potential.The granular sludge coupling DAMO with Anammox presented robust capacity to withstand unstable partial nitritation effluent,achieving 98%nitrogen removal at a varied nitrite to ammonium ratio in the range of 1.15-1.45.Moreover,dissolved methane was effectively removed at a rate of 975 mg L^-1 d^-1.The cultivated granular sludge was used as inoculum in another MGSR for mainstream nitrogen removal,investigating the methane and dissolved methane removal potential by granular sludge at low temperature.A stable nitrogen removal rate of 0.94 kg N m^-3 d^-1 was achieved at 20℃with effluent concentration<3.0 mg TN L^-1.With the decreasing temperature,nitrogen removal rate dropped to 0.55 kg N m^-3 d^-1 at 10℃,while with the nitrogen removal efficiency of 98.2%and effluent concentration<1.0 mg TN L^-1.The granular sludge maintained high-level nitrogen removal steadily for more than three months under a hydraulic retention time（HRT）as low as 2.16 h at 10℃,indicating the significant potential in the treatment of mainstream wastewater.Finally,high-throughput sequencing,FISH,mtagenomic and transcriptomic approaches,and mathematical simulation were conducted to demonstrate the differences of three kinds of granular sludge coupling DAMO with Anammox under different operating conditions and to reveal the synergistic mechanism between DAMO and Anammox for niteogen removal.When treating wastewater with differernt nitrogen resources,the relative abundance of Candidatus Methanoperedens decreased from 31.4%to 25.27%and 6.4%.The Anammox relative abundance increased from 8.3%to 11.5%and 9.5%.Candidatus Methylomirabilis was enriched under mainstream condition,with a relative abundance increased to 33.3%.The high-throughput sequencing result illustrated that the nitrate in influent benefited the growth of DAMO archaea while the addition of nitrite in the influent stilulated the growth of Anammox bacteria,and the operating conditions of low temperature and low substrate concentration benefits the growth of DAMO bacteria.The Anammox community shifted from Candidatus Kuenenia to Candidatus Brocadia from sidestream condition to mainstream condition.Metagenomicit result revealed that the gene groups associated with the reverse methanogenic pathway in DAMO archaea,including genes mcr、mer、mch、frt and fmd,showed an overall increasing trend,while the genes hdh and hzs associated with hydrazine metabolism showed significant downregulation in inoculant granules.Metatranscriptomic approach revealed that the expression levels of key metabolic genes were generally positively correlated with the activity of functional microorganisms,and the expression levels were usually higher in granular sludge with higher nitrogen removal performance of functional microorganisms.Both FISH and mathematical simulation results illustrated that DAMO and Anammox microorganisms distributed evenly and located closely to each other in granules fed with nitrate and ammonium,while in granules fed with nitrite and ammonium,obvious stratification structure with Anammox bacteria on the surface and DAMO archaea in the inner layer was formed.Under differernt operation conditions including temperature,nitrogen resource and concentration,the DAMO and Anammox microorganisms reorganized their microbial spatial distribution and granular structure,adjust the relative abundance and expression level of key functional genes to adapt to differernt synergistic metabolism processes required for nitrogen removal.This study demonstrated the feasibility of integration of DAMO with Anammox in granular sludge,which achieved excellent biomass retention and high-rate nitrogen removal,investigated the nitrogen removal performance and mechanism by granular sludge coupling DAMO with Anammox,providing first insights and useful information for the design and operation of this new technology in its potential future applications.