Pathways And Mechanisms Of Microbially Mediated Nitrogen Removal From Sediments On The East China Sea Shelf

Nitrogen is often considered to be an important biogenic element of marine ecosystems and a limiting factor in primary productivity.The intensification of anthropogenic disturbances has led to the accumulation of large amounts of reactive nitrogen in the estuary and adjacent waters.Excessive nitrogen loading alters the balance of the nitrogen cycle in coastal ecosystems,resulting in a range of ecological problems such as eutrophication,harmful algal outbreaks,periodic or permanent expansion of hypoxic zones,and emissions of nitrous oxide（N₂O）.Nitrogen removal pathways in coastal marine ecosystems and their environmental influences have therefore received much attention.Anaerobic ammonia oxidation（anammox）and denitrification reactions,the primary pathways for nitrogen removal in marine ecosystems,can inhibit nitrogen accumulation by transferring reactive nitrogen from the aquatic ecosystem to the atmosphere.Although marginal seas cover only about 8%of the total ocean area,about 45%of global nitrogen removal is accomplished through denitrification and anammox of sediments in estuaries,adjacent seas and continental shelves.Therefore,exploring the sources,transport transformations,removal pathways and influencing mechanisms of nitrogen in shelf seas can help improve the understanding of nitrogen removal from sediments and the mechanistic factors.In this study,the East China Sea shelf waters were selected as the study area to analyze the relevant microbial nitrogen transformation processes and nitrogen removal pathways in the surface sediments.Moreover,the anammox activities in this area in the last century were inferred by measuring the biomarkers of sedimentary column sample,explaining the nitrogen removal pathways and mechanistic processes in the sediments from the temporal and spatial scales.In addition,anthropogenic factors were found to be an important cause of excessive nitrogen input to the East China Sea shelf waters through studies in the open sea.In order to elucidate the nitrogen removal mechanism of eutrophic seawater,a laboratory seawater simulation system was constructed,which was inoculated with marine anammox bacteria（MAB）cultivated by surface sediment enrichment in the East China Sea shelf,and the nitrogen removal mechanism of the seawater system was simulated and studied by artificially controlling the nitrogen input,providing a scheme for nitrogen removal in eutrophic seawater.This study systematically investigated the nitrogen removal pathways in the open sea and the influence of ecological and anthropogenic factors,and improved the effective nitrogen removal from eutrophic seawater through laboratory-scale incubation experiments.The following main findings were obtained:（1）Based on the physicochemical properties of surface sediments with different grain sizes and related microbial nitrogen transformation processes in the East China Sea shelf,it was found that organic carbon is a key factor in the microbial anammox and denitrification nitrogen removal pathways in marine ecosystems.The nitrogen removal from surface sediments in the East China Sea shelf was dominated by denitrification,and the nitrogen removal rate of muddy sediments was significantly higher than that of sandy sediments.Denitrification and anammox were more intense in muddy sediments rich in organic carbon compared with sandy sediments,probably because the decomposition of organic carbon produced nitrite（NO₂^–）,which was both an intermediate product of denitrification and a substrate of anammox,and denitrification and anammox produced coupling effects.The total organic carbon（TOC）content of muddy sediments in the East China Sea shelf was significantly higher than that of sandy sediments,and the mean sediment grain size was negatively correlated with TOC concentration（r=–0.78,p<0.01）,suggesting that sediment grain size is important in controlling TOC.The carbon to nitrogen ratio（C/N）（6.04 to 7.18）andδ¹³C values（–22.50‰to–20.88‰）for the study area both indicate that the organic carbon in the surface sediments is predominantly from the marine environment.The results of the most terminal meta-mixing model indicate that marine organic carbon dominates the surface sedimentary organic carbon of the East China Sea shelf,averaging about 78%.Organic carbon of terrestrial origin undergoes extensive microbial alteration after entering the marine ecosystem and is converted to marine organic carbon for reaction.Nitrogen removal in the East China Sea shelf was dominated by denitrification,with rates of nitrogen removal from muddy sediments(denitrification rate:14.39 nmol N g^-1 h^-1,anammox rate:2.73 nmol nmol N g^-1 h^-1)significantly higher than the nitrogen removal rates from sandy sediments(denitrification rate:5.55 nmol N g^-1 h^-1,anammox rate:1.57 nmol N g^-1 h^-1).This is mainly due to the abundant TOC content of the muddy sediment,which increases the copy number of functional genes associated with anammox and denitrification.The decomposition of organic carbon produces NO₂^-,which can be used as both an intermediate product of denitrification and a substrate for anammox;therefore,enhanced heterotrophic denitrification can also increase the activity of anammox.In addition,high values(24-354 ng g^-1 dw)were obtained for summed ladderane lipids in surface sediments of the East China Sea shelf,implying that anthropogenic factors have led to a large input of nutrients from the Yangtze River estuary to the East China Sea in recent years,resulting in an increase in the ongoing nitrogen loss from the shelf of East China Sea.（2）Based on the physicochemical characteristics of sediment columns and anammox-specific biomarkers in the East China Sea shelf,it was found that the summed ladderane lipids content was increasing in the last 60 years and the anammox was gradually enhanced.Summed ladderane lipid content was low until the 1960s,but increased significantly since 1960,corresponding to the time of rapid economic development in China.It may be that anthropogenic discharge of large amounts of nutrient and organic carbon into the East China Sea has led to a large depletion of dissolved oxygen in the water column while the eutrophication of seawater has become more serious,which in turn has caused serious hypoxia in seawater and provided a good living environment for anammox bacteria.The results of the phylogenetic tree established by the anammox 16S rRNA gene retrieved sequences mainly from Candidatus Scalindua,closely related to sequences from environmental sources such as the surface sediments of the Yangtze River estuary（KU218333.1,JX243219.1）,marine sediments of the Zhoushan Islands（KF029964.1）and surface sediments of the South China Sea（HQ665746.1）.Similar trends were observed between the concentrations of the component ladderanes and with the summed ladderanes content,all showing an increasing trend with time,indicating that the anammox activity in the East China Sea shelf has intensified in recent years.The higher levels of ladderanes in suspended particulate matter were mainly found in bottom water layer,a phenomenon that suggests that ladderanes in suspended particulate matters from the East China Sea shelf is generated in the water column rather than from the terrestrial environment or sediment resuspension.Summed ladderane lipids content fluctuated at relatively low levels(16.36-47.11 ng g^-1 dw)until the 1960s and then began to increase significantly(45.31-233.60 ng g^-1 dw)after the 1960s.The period of enhanced anammox corresponds to the rapid economic development of China after1960,which accelerated the growth of anammox bacteria due to the discharge of large amounts of nutrients into the coastal shelf of the East China Sea,which in turn led to eutrophication and anoxia near the mouth of the Yangtze River.After 1960,there was a significant positive correlation between TOC content and summed ladderane lipids content in DH5-0 sedimentary column samples（r=0.366,p=0.031）,indicating that eutrophication in the study area is closely related to anammox reactions.The transport of suspended particulate matter in the Yangtze estuary and the input of large amounts of organic carbon due to anthropogenic factors are the main reasons for the increase in sedimentary organic carbon.The branched GDGTs showed a significant positive correlation with the summed ladderane lipids,which further suggests that this region has enhanced anammox activity due to hypoxia.（3）Studies on the temporal and spatial scales of sediments in the East China Sea shelf have identified anthropogenic influences as an important cause of excessive regional nitrogen inputs.The nitrogen removal mechanism of eutrophic seawater was obtained based on the determination of ladderane lipids analysis and¹⁵N isotope tracing technique in a seawater simulation system.Anammox is the main nitrogen removal process in the seawater simulation system because the unique physiological properties of the inoculated MAB make it extremely tolerant in high salinity environments and enable it to enhance nitrogen removal from eutrophic seawater.The genus Candidatus Scalindua,a marine anammox bacterium,was dominant in the simulated system,and its abundance increased with increasing anthropogenic control of nitrogen input（10.10%to 81.71%）,and the environment in the system was suitable for the growth of this genus.A seawater simulation system was constructed in the laboratory and inoculated with MAB enriched and cultured from surface sediments of the East China Sea shelf,and the correlation between nitrogen removal from eutrophic seawater and bacterial communities was investigated by artificially controlling the nitrogen input.The results show that the average effluent removal efficiencies of NH₄-N（83.57%for Strength1,87.19%for Strength2,and 90.14%for Strength3）,NO₂-N（75.20%for Strength1,76.99%for Strength2,and 83.57%for Strength3）and TN（74.86%for Strength1,78.92%for Strength2,and 83.52%for Strength3）were significantly higher as the intensity of the influent nitrogen input increased.The optimal nitrogen removal was obtained by the system Strength3 with the highest influent nitrogen input.This is because increasing influent strength reduces the dissolved oxygen level in the water of the seawater system,increasing the space and activity of the associated anaerobic microorganisms,thus promoting the growth of anammox bacteria and improving the removal efficiency of nitrogen.The increase in nitrogen loading strength enhanced the nitrogen removal capacity of the MAB-inoculated seawater system,and the ratio of anammox rate to total nitrogen removal rate（ra）was all above 95%,indicating that anammox was the main nitrogen removal pathway of the system.In addition,the results of high-throughput sequencing showed that Candidatus Scalindua was dominant within the constructed seawater system（10.10%in Strength1,25.89%in Strength2,and 81.71%in Strength3）,indicating that the environment within the system was suitable for the growth and survival of MAB.nos Z and anammox 16S rRNA genes abundance increased with increasing nitrogen input strength,corresponding to the effect of nitrogen removal in the system.This study improved the nitrogen removal efficiency of eutrophic seawater by inoculating MAB and adjusting the strength of nitrogen input,allowing it to be applied to a wide range of saline raw wastewater treatments.