Microbial-Driven Nitrogen Cycling Processes And The Affecting Factors In The Yellow River Estuary

The Yellow River Estuary(YRE)locates in the confluence of Yellow River,Bohai Sea and Laizhou Bay,which is one of the three major estuaries in China.According to the report of “State of the Marine Environment of China”,nitrogen pollution has become one of the most prevalent and increasing contaminant in the YRE and its surrounding coastal areas.Excessive nitrogen may cause water eutrophication,biodiversity loss and some other serious environmental problems.Therefore,it has become a serious and urgent issue to investigate the ecological change caused by excessive nitrogen pollution in the YRE.Microbes,as drivers of biogeochemical cycles,play important roles in the elimination of nitrogen pollution in estuary habitat.The major microbial-driven nitrogen cycling processes include nitrification,denitrification,dissimilatory nitrate reduction to ammonium(DNRA)and anaerobic ammonium oxidation(anammox).Denitrification and anammox processes can directly remove nitrates from the environment.DNRA process can also achieve the conversion of nitrate in the environment.Ammonia oxidation is the rate-limiting step of nitrification and plays a key role in the cyclical conversion of ammonia nitrogen.It can be coupled with denitrification and anammox processes to achieve nitrogen remove.To data,the distribution characteristics and influencing factors of the main microbial functional groups in the nitrogen transformation process in the YRE remain unclear.By using stable isotope tracing,high-throughput sequencing,function gene clone library and qPCR methods,we aimed to clarify the abundance,community structure and influencing factors of ammonia-oxidizing microorganisms in the YRE,investigate microbial-dirven nitrate reduction rate and its influencing factors in the YRE,and explore the effect of nitrate concentration on nitrate reduction rate,bacterial abundance and community structure.The main findings are as follows:(1)Ammonia-oxidizing microorganisms can be detected in the estuary habitat of the YRE,and their abundance and community structure were different between water and sediment habitats.Ammonia oxidizing archaea(AOA)showed significant higher abundance than Ammonia oxidizing bacteria(AOB)both in sediment and water samples.AOA and AOB abundance distribution trends were consistent in sediment but distinct in water along the sampling sites.Phylogenetic analysis showed that AOA sequences were affiliated with Nitrososphaera,Nitrosopumilus and Nitrosotalea genus.Generally,Nitrososphaera was predominant in sediment,while Nitrosopumilus and Nitrosotalea dominated in water column.AOB sequences were classified into genera Nitrosospira and Nitrosomonas,and Nitrosospira dominated in both habitats.Principal coordinate analysis(PCoA)also indicated AOA community structures exhibited significant differences between the two habitats,while AOB were not.Ammonium and carbon contents were the potential key factors to influence AOMs' abundance and community structure in sediment,while no measured environmental factors were determined to have influence on AOM communities in water habitat.(2)Denitrification was the main nitrate reduction process,and nitrate might be a key factor affecting the nitrate conversion process in the YRE wetland.The results based on stable isotope labeling experiment revealed that the denitrification rate was higher than that of anammox and DNRA,indicating that denitrification was the main nitrate reduction process.The rate of anammox showed significantly higher in winter than that in summer,while the rate of denitrification and DNRA were not influenced by seasons.There was a significant positive correlation between the abundance of anammox 16 S rRNA gene and its rate,and the correlation in winter samples was significantly higher than that in summer.The abundance of DNRA nrfA gene was only significantly correlated with the rate in summer samples.The abundance of denitrification function genes showed no correlation with their rates.Correlation analysis showed that there was a significant and positive correlation between denitrification,DNRA rate and nitrate content,while anammox rate only showed a significant and negative correlation with temperature.The result indicated that the nitrate content was the key factor affected bacterial 16 S rRNA gene and DNRA nrfA gene abundance.(3)Nitrate concentration can influenced nitrate reduction rate,and affect the bacterial abundance,diversity and community composition.The denitrification rate increased with the increase of nitrate concentration,while the anaerobic ammonium oxidation and DNRA rate decreased with the increase of nitrate concentration.Diversity analysis based on high-throughput sequencing data indicated that Shannon and Evenness index were significantly lower in 400 and 600 ?M nitrate addition group than that of other groups,and 600 ?M nitrate addition group had the lowest ?-diversity.The results of ?-diversity showed that the bacterial community composition had significant difference between the group with 200 ?M or less nitrate concentrations and the group with 400 and 600 ?M nitrate concentrations.The relative abundance of Gammaproteobacteria and Actinobacteria increased with nitrate concentration increased.The nitrate concentration can be divided into high and low groups based on multiple regression trees.Co-occurrence network revealed that there was big difference in the bacterial relationship between the two groups of samples(one group with 0 and 100 ?M nitrate added,and the other group with 400 and 600 ?M nitrate added).The high nitrate concentration group has higher network complexity,revealing that the increase of nitrate concentration might be regard as a stress and leaded the microorganism to produce more complex symbiotic community relationship.Therefore,as the nitrate content increased,the more complex and tightness bacterial community may have better ability to withstand environmental stress.Linear discriminant effect analysis showed that Gammaproteobacteria and Bacilli were the indicator bacterial groups in the high nitrate concentration group.In this study,the microbial-driven nitrogen cycle processes and its influencing factors in the Yellow River estuary area were studied.The results showed that the ammonia-oxidizing archaea was the main ammonia-oxidizing microorganism in the YRE,and the abundance and community distribution of ammonia-oxidizing microorganisms were different in the water and sediment habitats.Denitrification was the main nitrate reduction in the YRE wetland.The change of nitrate concentration can affect nitrate reduction rate and bacterial community structure.The result can increase the understanding of the biogeochemical cycle drived by microorganisms in the Yellow River estuary,and provide important theoretical information for estuarine ecological protection and nitrogen pollution control.