Anaerobic Nitrogen Removal Flux And Its Microbial Driving Mechanism In The Sediments Of Typical Intertidal Zones In China

Nitrogen(N)is one of the necessary elements for organisms,and it's overloading causes eutrophication in the coastal ecosystems.There are close correlations between nitrogen overloading and the outbreak of red tide,formation of hypoxia and degradation of ecosystem.The intertidal zone in China stretches for thousands of miles,which is from the tropic to the cold temperate zone.In recent decades,the nitrogen inputs from terrestrial sources increased dramatically,which becomes a serious threat to the environment.The intertidal zone is an important buffer zone for the offshore environment,which is a hot area for nitrogen transformation.It is of great significance to alleviate eutrophication in offshoreareas.Denitrification and anaerobic ammonia oxidation(anammox)driven by microbes are two main ways of nitrogen removal from the intertidal zones.So,it is of great reference value to research the N-loss fluxes of denitrification and anammox,microbiological and environmental regulation mechanisms in the intertidal zone,for understanding regional N biogeochemical cycling,grasping the environmental nitrogen capacity of intertidal zone,and developing relevant measures for rational utilization and protection of intertidal zone.In this study,we collected surface sediments(0-5 cm)in 14 typical intertidal regions from Liao He Estuary to Dong zhai Port in all the four seasons.Triple samples were sampled in each intertidal region and a total of 168 samples were obtained.Environmental parameters of sediments were determined by geochemical methods;the rates of denitrification and anammox were determined using the slurry incubation and 15N isotope tracer method.The seasonal and spatial variations of abundance of denitrifiers and anammox bacteria were assessed by Real-time Quantitative PCR(Q-PCR)using nosZ gene and anammox bacterial 16S rRNA gene as molecular markers.Main results were as following:1.Generally,there was no significant seasonal difference in environmental parameters,Only NH4 concentrations showed significant difference between spring and summer(ANOVA,P<0.05),and the concentrations were higher in spring than those in summer.The regional difference of almost all environmental parameters was significant(ANOVA,P<0.05).Sediment TOC%was higher in the south region than that in the north and middle region;NH4+concentration was highest in the north region and lowest in the middle region;Heavy metal contents were the highest in the south region.2.Denitrification was the main process of natural nitrogen removal in the intertidal zone,and it's contribution ranged from 31.8%to 96.8%.Summer is the highest in season and spring is the lowest.The potential rate of anaerobic ammonia oxidation is much smaller than that of denitrification,and the contribution rate varies from 3.7%to 68.2%.Spatially,summer is the highest and spring is the lowest.3.The results of one-way and multi-factor variance analysis showed that the denitrification and total denitrification rates were mainly affected by season(P<0.05),while the anammox rates were significantly different between seasons and regions(P<0.05).In addition,the contributions of anammox were also significantly affected by the seasonal and regional interaction(P=0.00).4.Most of the functional gene abundances in intertidal sediments showed significant regional differences(P<0.03).Only AOA-amoA showed significant seasonal variation(P=0.02).Pearson correlation analysis showed that TN,TOC,heavy metals and sediment size significantly affected the abundance of most functional genes(P<0.05).5.Pearson correlation analysis showed that the denitrification and anammox rates were positively correlated with the abundance of total bacteria and archaea.In addition to AMX-16S rRNA gene,AOB-amoA and nirS,nirK,nosZI,nosZII gene abundance were positively correlated with anammox activity(P<0.05).6.We constructed multivariate stepwise regression analysis to analyze the comprehensive effects of functional gene abundance and environmental factors on the nitrogen removal activities of intertidal sediments.The results showed that environmental factors had a much larger effect on the denitrification activity than functional gene abundance.The contents of heavy metals Pb and Mn were the main factors affecting denitrification process,and the cumulative interpretation was 30%.The anammox rates were mainly affected by TN%and 8 3C,with the interpretation of 16.9%.AMX%was mainly affected by the content of Pb and Mn,and the interpretation rate is 14.6%;while ?13C,Pb and Mn are the main factors to explain the total denitrification rate,with the cumulative interpretation of 32.2%.