The Analysis Of Functional Microbial In Anammox Of Different Electron Acceptors

With the development of industry,serious nitrogen pollution problem has attracted more attention.Anaerobic ammonium oxidation?Anammox?provides a new understanding of the nitrogen cycle in nature.NH₄⁺ and NO₂^-,which are used as electron donor and acceptor respectively,can be converted to N2 by Anammox bacteria under anaerobic conditions,with hydroxylamine?NH₂OH?and hydrazine?N₂H₄?as intermediates.In addition to nitrite,ferric salt and bicarbonate can also be used as electron acceptors by Anammox bacteria.In order to further understand the mechanism in the Anammox reactor with different electron acceptors,the functional microorganisms were investigated in different Anammox reactor.Nitrite,ferric salt and bicarbonate with denaturing gradient gel electrophoresis?PCR-DGGE?,16 S rDNA sequencing and real time fluorescence quantitative PCR?qPCR?analysis were carried out.The results are as follows:?1?The main parameters of PCR-DGGE were optimized using universal bacteria primer of GC-F341/R518.The optimum annealing temperature,denaturant concentration,sample size,gel concentration and voltage were identified as 64?,45% to 65%,5?L to 7?L,8% and 80V/16 h,respectively.The results of fingerprints and the DNA band sequencing of PCR-DGGE showed a high abundance and a significant diversity of microorganisms in Amx?nitrite Anammox?and Fmx?Iron Feammox?reactor.?2?The results of 16 S rDNA Illumina Miseq sequencing platform showed that the most dominant species in the reactors with different electron acceptor was unclassified_Chloroflexi genera which belong to Chloroflexi.The contents of Planctomycetes in Amx,Fmx and Hmx?bicarbonate Anammox?reactors were 21.31%,16.22% and 1.65% respectively.Anammox bacterium from the genera of Candidatus Brocadia and Candidatus Kuenenia were detected.The NOB?nitrite oxidizing bacteria?only accumulated in the Fmx reactor with the proportion of 3.38%.And the AOB?ammonia oxidizing bacteria?and DB?denitrifying bacteria?showed low aboundance in three reactors.These results indicated that the dominate reaction in the Amx reactor was Anammox,while it was indicated that reaction in the Fmx reactor were Anammox and nitrite oxidation.Moreover,the Anammox reaction did not detected in the Hmx reactor.?3?The qPCR analysis showed that the Amx reactor had the highest abundance of Anammox bacteria than the other two reactors,and the Anammox bacteria appeared as the most abundant functional bacteria.In the Fmx reactor,the Anammox and NOB bacteria showed a relatively higher abundance.In contrast with the Amx reactor,the Hmx reactor gave a decreased content of Anammox bacteria and a increased content of nitrifying bacteria and denitrifying bacteria.These results indicated that the dominated bio-reaction in the Amx reactor was Anammox,while in the Fmx reactor were Anammox and nitrite oxidation.In addition,the main reactions in Hmx reactor were nitrification and denitrification rather than Anammox.?4?According to the microbial abundance analysis in the different Anammox reactor,iron salt can be used as an electron acceptor of Anammox reaction,leading to the occurence of Feammox reaction.While,bicarbonate was not be detected as the electron acceptor of Anammox reaction in Hmx reactor,in which the Nitrification and denitrification were detected.In this paper,the microbial diversity and community structure in the Anammox reactor with different electron acceptors were studied.The results provide a reference for further study on the mechanism of Anammox and the optimization of relevant processes.