Enrichment Of The Microorganisms Capable Of Nitrate/Nitrite-Dependentanacrobic Methane Oxidation

Nitrate/nitrite-dependent anaerobic methane oxidation(Nx-damo) is a newly demonstrated microbiological pathway for C and N cycle. This finding is significant for not only the understanding of the mechanisms of microorganisms driven cycling of biogenic elements, but also the development of sustainable wastewater treatment technology focusing on nitrogen removal and mitigation of green-house gas release.Nitrate dependent AMO(N-damo) and nitrite dependent AMO(n-damo)are more basic from the view of thermodynamics andbiochemistry.The process mediated by“Candidatus Methylomirabilis oxyfera.” M. oxyfera is affiliated with the “NC10” phylum, and effectively outcompeting ‘M. oxyfera’ in thesystem. And “Candidatus Methanoperedens nitroreducens” for the ANME-2d population and the family Candidatus‘Methanoperedenaceae’ for the ANME-2d lineage.Four SBR cultures were inoculated with mixed anaerobic sludges, to test the hypothesis that theavailable electron acceptor(nitrate or nitrite) and The temperaturewill have adirect impact on the composition of the resulting denitrifying anaerobic methane oxidation microbial community, And to explore the dynamics of the two importantmatrix of NO2-and CH4 in the process of n-damo.Finally,to analyzethe the microbial community structure of the enrichment.The main conclusions are as follows:①With mixed sludge as inoculum,NO2- and NO3- as electron acceptor, enrichment of 280 d, n-damo process achieved the maximum nitrogen removal rate of 5.25 mmol NO2--N/(L.d). A high concentration of NO2- accumulation, leading to NO2- reduction rate fell sharply, and the recovery process is relatively slow.The archaea enrichment process more slowly, enrichment of 280 d finally come to NO3-denitrification rate is much lower than with the matrix of NO2- n- damo process.Two reactors of the highest denitrification rate, respectively up to1.54 mmol NO3--N/(L.d) and 1.66 mmol NO3--N/(L.d).②Afterthe enrichmentof n-damo, through short-term batch experiments, using the Monodequation to fit the dynamiccharacteristics of NO2- reduction and CH4 oxidationin the n-damo process.Kinetics characteristicof NO2-reductionratefollows Monod mixedkinetic equation, obtained half saturation constant for 0.40 mg/L and0.30 mg/L respectivelyby two fitting.By batch experimentsphase fitting the maximum matrix degradation rate were 0.468 mg.N/( g VSS.h) and 0.949 mg.N/( g VSS.h). CH4 oxidation rate eventually reduced to first order kinetics equation, two fitting obtained CH4 oxidation rate constants were 0.096 h-1and 0.071 h-1.③ Populations of enrichment culture community structure were analyzed, PCR quantitativeresults obtained abundance of M. oxyfera bacteria in 4 reactors is 1.8870×1010 copies.g-1(dry weight)、4.4264×109 copies.g-1(dry weight)、8.7145×109 copies.g-1(dry weight)and 1.1998×1010 copies.g-1(dry weight),respectively.Only in NO3- as electron acceptor of the No. 2 and No. 3 reactor enriched M.nitroreducens Archaea.Obtainedabundance is 4.6464×10copies.g-1(dry weight) and 1.4976×1010 copies.g-1(dryweight).Phylogenetic tree of M. Oxyferabacteria16 Sr RNA and pmo A functional gene showed that in n- damo process can obtain the main enrichment culture is group A of M. oxyfera bacteria.Archaea16 Sr RNA gene diversityphylogenetic tree showed that enriched Archaea belong to the ANME class.