Enrichment Culture Of Anaerobic Ammonium-oxidizing And Nitrite-dependent Anaerobic Methane-oxidizing Bacteria From Mangrove Sediments

Nitrite serves as the terminal electron acceptor for microorganisms that oxidize ammonium and methane under anaerobic conditions,which is a newly discovered denitification processes in mangroves wetland.N-DAMO and Anammox bacteria is assigned to NC10 and Planctomycetes phylum by reducing nitrite by oxidizing methane and ammonium under anaerobic condition,respectively.The finding of these processes greatly expands the knowledge of biogeochemistry.Due to its unique physiological and biochemical properties of Anammox and N-DAMO microorganisms,the Anammox and N-DAMO process offers an ideal option to develop new wastewater treatment technology.However,the uncultivated characteristics and slow growth rate of Anammox and N-DAMO bacteria seriously limited to get the enrichment culture.To our knowledge,the inoculums used for enrichment of N-DAMO and Anammox bacteia is mainly limited in fresh water and marine sediments,and the mangrove sediments as inoculum have not been reported.In the present work,we investigated the enrichment culture of N-DAMO and Anammox microorganisms using Zhangjiang mangrove(China)wetland sediments:1.Mangrove wetland sediment was selected as an inoculum with methane and nitrite addition about 420 days to enrich N-DAMO bacteria in sequencing batch reactor.a)The result of denitrifying activity suggested that heterotrophic denitrifying activity decrease continuously,the consumption rate of nitrite keep stably in 1.28 mg N/L/day.However,culture still do not show obvious N-DAMO activity.b)The result of qPCR and phylogenetic analyses indicated that mainly group B and group E sequences were enriched in N-DAMO culture,but not group A,the abundance of NC10 phylum 16S rRNA gene is about 3.36×105 copies/g dry soil.In the whole enrichment period,Anammox bacteria was simultaneously enriched,the copy number of hzsB gene is about 3.18×108 copies/g dry soil,the percentage of Anammox bacteria is about 10%,most of Anammox bacteria is made up of Candidatus Scalindua.The qPCR result of nosZ gene indicated that heterotrophic denitrifying microorganisms still exist in N-DAMO culture,the copy number of nosZ gene is about 6.38×1010 copies/g dry soil.2.Mangrove wetland sediment was selected as an inoculum with methane,nitrite and ammonium addition about 420 days to co-enrich Anammox and N-DAMO bacteria in sequencing batch reactor.a)The result of denitrifying activity suggested that heterotrophic denitrifying activity was highly active in earlier stage,then decrease continuously,the consumption rate of nitrite and ammonium keep stably in 16.93 mg N/L/day and 12.92 mg N/L/day,respectively.In addition,the ratio of consumption rate of nitrite-and ammonium-N was 1.2 closely to theoretical stoichiometric ratio 1.146,suggesting the denitrifying activity was dominated by Anammox process in N-DAMO and Anammox co-enrichment culture.However,culture still has not showed obvious N-DAMO activity.b)The result of qPCR and phylogenetic analyses indicated that only group B and group E sequences were enriched in N-DAMO and Anammox co-enrichment culture,the copy number of NC10 phylum 16S rRNA gene is about 4.21×105 copies/g dry soil.Anammox bacteria was simultaneously enriched,the copy number of hzsB gene is about 2.25×109 copies/g dry soil,the percentage of Anammox bacteria is about 52%,most of Anammox bacteria is made up of Candidatus Scalindua.The qPCR result of nosZ gene indicated that heterotrophic denitrifying microorganisms still exist in N-DAMO culture,the copy number of nosZ gene is about 6.50×1010 copies/g dry soil.