The Enrichment Culture And Diversity Analysis Of Complete Ammonia Oxidizers From Sediments Of Yangtze Estuary

Over the past few decades,excessive input of anthropogenic nitrogen caused the perturbation of nitrogen cycle in estuary and coastal areas and trigged serious environmental problems like eutrophication and frequent occurrences of harmful algal blooms.Nitrification,a key link in biogeochemical nitrogen cycle,play an important role in nitrogen loss of eutrophicated water.Therefore,deep-going research about nitrification in estuarine and coastal ecosystems is of great significance.Nitrification,the oxidation of ammonia to nitrate via nitrite,has always been considered to be a two-step process catalysed by ammonia oxidizing microorganisms?AOM?and nitrite oxidizing bacteria?NOB?,sequentially.Until 2015,complete ammonia oxidizers?comammox?catalysing both steps of nitrification were discovered.The discovery of comammox upended the 100-year-old dogma.However,knowledge about comammox diversity,abundance and potential activity in estuarine and coastal ecosystems remain scarce.In this study,comammox were proved to be existed in tidal sediments of Yangzte Estuary based on enrichment culture.Community structure and gene expression patterns of the bioreactor biomass were analysed and retrieved gene seuqences were used for primer design,which can be used for comammox community diversity and abundance analysis in natural environments.Results of comammox diversity in this study implied the importance of comammox in nitrogen cycle.Followings are main results in this study:?1?Community structure of the reactor biomass was analysed based on metagenomics,when the reactor reached stable status and the enrichment culture showed high nitrifying activity.Four kinds of nitrifiers were detected,accounting for34.7%of all reads,which consisted of 50%NOB,48%comammox,2%?-AOB??-proteobacterial ammonia oxidizing bacteria?and 0.1%AOA?ammonia oxidizing archaea?.Results indicated the high potential of nitrite oxidizing and complete ammonia oxidizing ability of the enrichment culture and confirmed the existence of comammox in tidal sediments of Yangtze Estuary.?2?Gene expression patterns of four nitrifier groups?according to the classification in Evolutionary genealogy of genes:Non-supervised Orthologous Groups database?were analysed based on metatranscriptomics.Results showed high activity of comammox and NOB in energy production and conversion,posttranslationalmodification,proteinturnover,chaperones,andcell wall/membrane/envelope biogenesis.The expression of key nitrification genes?ammonia monooxygenase subunit A/B/C genes,amoA/B/C;hydroxylamine oxidoreductase gene,hao;NO-forming nitrite reductase K gene,nirK;nitrite oxidoreductase subunit A/B genes,nxrA/B?showed the high gene expression of comammox,followed by the gene expression of NOB.Results indicated the high metabolic activity and growth potential of comammox and NOB.?3?The phylogenetic relationships of comammox enriched in this study and other known nitrifiers were studied.Phylogeny of AmoA/B/C,Hao showed individual branches of comammox in tidal sediments,indicating that novel species may existed in this ecosystem.?4?Primers targeting the comammox amoA genes were designed,with 89%and100%coverage,based on sequences retrieved in this study and NCBI,IMG ER/MER database.Community diversity in estuary sediments were analysed using PCR and real-time QPCR,and were compared with community diversity of lake and river sediments.Comammox community structure of estuary,tidal and coastal sea sediments were similar and were different from that of lake and river sediments.The abundance of comammox in sediment environments ranged from 3.50×10⁵ to1.56×10⁶ copies g^-1 dry sediment,accounting for 0.4%-5.6%of total amoA copies?amoA copies of comammox,?-AOB and AOA?.Results indicated the important contribution of comammox in nitrification process.?5?Relationships between comammox community and environmental factors clarified that concentration of sediment NH₄⁺had influence of shaping community,while factors of salinity,pH,water content,TOC?total organic carbon?didn't have significant effect in this study.