Denitrifying Anaerobic Methane Oxidation For Nitrogen Removal And Mechanism Of Carbon-nitrogen-sulfur Conversion

Denitrifying Anaerobic Methane Oxidation(DAMO)is a recently discovered biological process.The coupling of denitrifying and anaerobic oxidation of methane process can provide an opportunity for the design and development of a new process for biological nitrogen removal and simultaneous methane reduction.The process is catalyzed by ANME-2d archaea homologically with methanophilic archaea and bacteria belonging to phylum NC10,and can be coupled with the Anammox process to achieve the co-removal of CH₄,NO₂^-and NO₃^-.However,the nitrogen removal potential by DAMO functional microorganisms and their metabolic mechanism remains to be further revealed.This paper focuses on two key issues,namely,the construction of efficient denitrification system for DAMO process and the analysis of functional microbial metabolic pathway mechanism,in order to deepen the understanding of the key role of DAMO in biogeochemical element cycles and promote the application of DAMO process.The enrichment of DAMO coupled with Anammox and its application for high efficiency nitrogen removal were systematically studied.Under the condition of enhanced methane aeration,CH₄,NO₃^-and NH₄⁺were used as substrates to enrich and cultivate the co-enrichment culture of DAMO coupled Anammox.After 300 days of enrichment,the relative abundance of ANME-2d archaea and Anammox bacteria in the microbial population reached 56.5%and 9.7%,respectively.Considering the mass transfer rate of CH₄ gaseous substrate and the microbial retention rate of the reaction system,a Membrane Aerated Membrane Bioreactor(MAMBR)was developed,which is suitable for suspended sludge with DAMO coupled with Anammox.The total nitrogen removal rate of 5000 mg N L^-1 d^-1 can be reached within 200-day start-up when applied to wastewater denitrification.Based on the denitrification performance of the MAMBR,a numerical model was established to describe the denitrification process of the flocculent sludge DAMO process.By model calibration,compared with the biofilm system,the DAMO functional microorganisms exhibited higher growth rate and higher sludge yield rate in the suspended system;through the optimization of the model,when the SRT in MAMBR was 15?30 days,both nitrification efficiency and denitrification efficiency could be taken into account,so as to realize the process of wastewater denitrification with energy saving and high efficiency.Using the start-up strategy of“three phases based on influent”to operate the Membrane Biofilm Reactor(MBf R),the TN removal rate can be increased by 4.9 times under the premise that the start-up time can be shortened by 36%;the TN removal rate of 6656 mg N L^-1 d^-1 was achieved after 365-day operation.The nitrogen removal performance of NO₂^--AOM process and the niche differentiation and metabolic pathways of functional microorganisms were investigated.The enrichment culture with Phylum NC10 as the dominant species was successfully enriched under the conditions of CH₄ and NO₂^-.After 400-day enrichment,the relative abundance of NC10 bacteria in the microbial population reached 35.3%.The denitrification rate of MBf R inoculated with NC10 bacteria was close to 400 mg NO₂^--N L^-1 d^-1 when the influent concentration was 50 mg NO₂^--N L^-1,and the relative abundance of NC10 bacteria in the microbial population reached 65.6%at the end of the reactor operation.The 97%of its 16S r RNA gene sequences were highly similar to(>98%)those of Candidatus Methylomirabilis oxyfera species;When the influent concentration was1000 mg NO₂^--N L^-1,the 99.4%of the 16S r RNA gene sequences from NC10 bacteria were highly similar to(>99%)Candidatus Methylomirabilis sinica species;the denitrification rate of 997 mg NO₂^--N L^-1 d^-1 was achieved at the end of the operation condition,and the relative abundance of NC10 bacteria in the microbial population was44.6%.The results of substrate utilization kinetics show that Ca.M.sinica species had higher NO₂^-tolerance and lower NO₂^-affinity than Ca.M.oxyfera species.These inherent characteristics can lead to niche differentiation of different NC10 bacteria at the"species"level.Combined with nitrogen transformation and stable isotope labeling tests,it was confirmed that NC10 bacteria had both non-specific oxidation of NH₄⁺and N₂O release pathway,and the release characteristics of N₂O were related to the exposure concentration of NO₂^-and the availability of CH₄ in the environment.The nitrogen removal performance of NO₃^--AOM process and the process of DNRA catalyzed by functional microorganisms were investigated.The MBf R inoculated with co-enrichment culture for DAMO coupled Anammox process achieved a nitrate removal rate of 1013 mg NO₃^--NL^-1 d^-1 under the influent concentration of 1000 mg NO₃^--N L^-1,and the effluent TN concentration of the reactor was less than 10 mg N L^-1.During the operation of the reactor,the relative abundance of ANME-2d archaea and NC10 bacteria showed an obvious increasing trend,reaching 37.2%and 31.1%,respectively,at the end of the reactor operation.The relative abundance of Anammox bacteria decreased rapidly from 22.8%to less than 0.5%,and then gradually increased to 6.3%.The results of biofilm microelectrode analysis and functional gene transcriptional expression activity sshowed that the system for nitrogen removal was characterized by the Dissimilaory Reduction of Nitrate to Ammonium(DNRA)process,which was catalyzed by ANME-2d archaea and coupled with the anaerobic oxidation of methane.Combined with stable isotope labeling test,it was further confirmed that NO₂^-in the environment was the inducing factor for catalyzing DNRA process by ANME-2d archaea,and the produced NH₄⁺came from the reduction of NO₃^-,and NO₂^-in the environment did not participate in the nitrogen conversion process of DNRA.ANME-2d archea in biofilm can simultaneously reduce NO₃^-to NO₂^-and NH₄⁺,providing growth substrates for Anammox bacteria,which is a new mode of microbial cooperation.The nitrogen removal performance of AOM simultaneously coupled with nitrate reduction and sulfate reduction process and the key processes driving carbon-nitrogen-sulfur conversation were explored and analyzed.An MBf R was inoculated with co-enrichment culture for DAMO coupled with Anammox process coexisting NO₃^-and SO₄^2-in influent;the total nitrogen removal rates of 489 and 193 mg N L^-1 d^-1 and the dissolved methane removal rates of 353 and 144 mg CH₄ L^-1 d^-1 were achieved at 30?and 25?,respectively.ANME-2d archaea,SRB bacteria(Desulfococcus spp.),NC10 bacteria and SAD bacteria(Thiobacillus spp.and Sulfurovum spp.)were the dominant microorganisms that jointly driving the carbon-nitrogen-sulfur cycle.The concentration distribution of NO₃^-and SO₄^2-in the carbon-nitrogen-sulfur cycle affected the AOM rate.ANME-2d archaea can not only catalyze NO₃^--AOM process,but also catalyze SO₄^2--AOM process with SRB bacteria symbiosis.In the process of symbiosis between ANME-2d archaea and SRB bacteria,the transcriptional expression of gene fla B encoding flagellin and gene cyt C encoding cytochrome C in ANME-2d archaea,and gene pil A encoding the pili component protein in Desulfococcus spp.were significantly up-regulated,suggesting that ANME-2d archaea could complete SO₄^2--AOM process by transferring electrons to SRB bacteria through cytochrome C and the observed nanonet structure.The process not only expands the understanding of methane sinks in nature,but also has important potential applications in sustainable wastewater treatment processes.