Distribution Pattern And Ecological Function Of Comammox Nitrospira Bacteria In Estuarine Tidal Flat Wetlands

Nitrification is the key process that connects the most reduced and most oxidized inorganic nitrogen,was for over the past century considered to be a two-step nitrification process:ammonia is firstly oxidized to nitrite by ammonia-oxidizing bacteria(AOB)or archaea(AOA),and the oxidation of nitrite to nitrate is carried out by nitrite-oxidizing bacteria(NOB).Until 2015 the discovery of complete ammonia oxidizers(comammox),which alone convert ammonia to nitrate,fundamental changed our view o nitrification.Estuary tidal flat wetlands is a key transition zone of the interaction of land and sea,where the environmental processes are complex and dynamic.In recent decades,due to the intensification of upstream urbanization and impact of human activities,a large amount of anthropogenic nitrogen and phosphorus and other nutrients have entered the estuary environment,resulting in a series of ecology issues,such as eutrophication of water bodies,low-oxygen zones in the water,red tides near the estuary,etc.Estuarine tidal flat wetland is a hotpot for the nitrogen cycling and research on this newly discovered nitrifying microorganism in this region is crucial for deep understanding the biogeochemical cycle of nitrogen in estuarine ecosystems.In this study,a large-scale geographical distribution survey of nitrifying bacteria was carried out along the estuarine tidal flat wetlands in China;taking the Yangtze River estuary as a typical study area to study the distribution,diversity,community dynamics and driving environmental factors of comammox Nitrospira in estuarine tidal flat wetland ecosystem;and to identity the activity of comammox Nitrospira and its contribution to nitrification and associated nitrous oxide(N₂O)production in coastal wetland environments.The main findings are as following:(1)A large-scale survey was conducted on the distribution and diversity of comammox Nitrospira in major estuarine wetlands along the coast of China.The presence of comammox Nitrospira was found in most estuarine areas,and its gene abundances ranged from 4.15×10~5-10.66×10~6copies/g dry sediment,lower than AOA and AOB,and also lower than other ecosystems.Overall,the abundance of comammox Nitrospira showed the characteristics of the central estuarine area>the southern estuarine area>the northern estuarine area.The nitrification rate showed the same distribution trend as the gene abundance in the spatial pattern,and showed a significant positive correlation with the gene abundance of AOA,AOB and comammox Nitrospira.Clade A1 was the dominant component in the coastal estuarine wetland system,and source analysis of the obtained sequences found that they were mainly clustered with sequences from freshwater and engineered systems,indicating that upstream runoff input was the main source of estuarine tidal flat wetlands,while sequences from soil and sediment(clade A2 and clade A2 and clade B)were lower.Comammox Nitrospira showed a high positive correlation with the typical ammonia oxidizing bacteria AOA and AOB species,indicating that they are highly coexisting in the estuarine tidal flat wetland system.(2)Chongming eastern tidal flat is the largest and most well-developed tidal flat wetland at the Yangtze River Estuary.Affected by tidal action,it can be divided into high tide flat,middle tidal flat and low tide flat from land to sea,forming different environmental gradients from land to sea.The gene abundance and nitrification rate of ammonia oxidiziers in different tidal flat locations showed different distribution characteristics.The decrease of AOA and AOB showed an increasing trend,and the peak of nitrification rate in summer also appeared in the low tide flat,but the comammox Nitrospira clade A still maintained the same trend as in winter.PCo A and NMDS analysis showed that the flora of high-tidal flats clustered together,while the flora of mid-tidal flat and low-tidal flat showed a long distance in different seasons.From the perspective of the composition of different branches,the tidal flat is dominated by clade A2,which shows a downward trend with the decrease of the tidal flat.The middle and low tidal flats are dominated by clade A1,which increases with the decrease of tidal flats.Differences in tidal positions lead to differences in sediment physicochemical properties,which further affect the nitrification rate and spatial differentiation characteristics of ammonia oxidizing bacteria in sediments.(3)The temporal and spatial distribution of comammox Nitrospira and the niche characteristics of different branches in the wetland sediments of the Yangtze River Estuary were revealed by molecular biology techniques.The presence of comammox Nitrospira clade A was detected in all samples,while comammox Nitrospira clade B was detected in only 3 samples.Phylogenetic analysis showed that comammox Nitrospira clade A was further divided into clade A1 and clade A2 and clade A3.Some sequences were found in the clade A2 clade to form a unique cluster(newly named clade A2.2),and the bootstrap of the traditional clade A2(named clade A2.1)was only 37,in the source There are also differences with clade A2.1.Although clade A2.2 has only 10 OTUs,it accounts for 31.5%and 19.02%of clade A2 and the total number of sequences,respectively.Among the different branches from upstream to downstream of the estuary,clade A2.1,clade A3 and clade B were mainly distributed in the upstream area,while clade A1 and clade A2.2 were widely distributed in the downstream area.The relationship between different clades of comammox Nitrospira is more complex,indicating the coexistence of synergistic and competitive mechanisms among different clades.(4)Comammox Nitrospira showed different adaptability to different environmental variables.Salinity is an important environmental factor affecting the distribution of comammox Nitrospira in estuarine tidal flat wetlands.Clade A1 and clade A2.2 can adapt to the estuarine environment with a wide range of salinity,while clade A3 and clade B are similar to clade A2.1 and have a lower degree of adaptation to salinity.The abundance of comammox Nitrospira genes in summer was higher than that in winter,and the increase of temperature promoted the activity of ammonia oxidiziers to a certain extent.Nitrifying microorganisms all use ammonia as an energy substrate,this study found that ammonia and nitrite can promote the growth of comammox Nitrospira in a limited range.Analysis of different branches found that clade A1 and clade A2.2 were suitable for low ammonia environment,while clade A2.1,clade A3 and clade B from soil sources were more suitable for high ammonia nitrogen environment.The differences were mainly attributed to the differences in the adaptability of different branches to ammonia.In addition,comammox Nitrospira was also significantly affected by environmental factors such as p H,iron ion,TOC,particle size,etc in the sediment.(5)The contribution of comammox Nitrospira to nitrification was evaluated by screening nitrification inhibitors,combined with pure culture strain experiments and DNA-SIP experiments.It was found that the addition of chlorate completely inhibited the ammonia oxidation and nitrite oxidation processes mediated by comammox N.inopinata,but had no effect on AOA and AOB.Except for clade B,AOA,AOB and comammox Nitrospira clade A were successfully labeled by ¹³CO₂.In DNA-SIP experiments using estuarine tidal flat wetland sediment samples,the addition of chlorate significantly reduced the gene abundance of comammox Nitrospira clade A.Phylogenetic analysis after ¹³CO₂labeling found that AOA,AOB and comammox Nitrospira clade A were successfully amplified,and the addition of nitrification inhibitors changed their community structure.A certain concentration of three nitrification inhibitors PTIO,octyne and chlorate,can be used to specifically inhibit AOA,AOB and comammox Nitrospira clade A,respectively.It is speculated that the contribution of comammox Nitrospira to nitrification in estuarine tidal flat wetlands is26.89%,and its contribution to nitrous oxide is 28.47%.