The Spatial Distribution Of Anaerobic Ammonia And Anaerobic Methane-oxidizing Microbial Community In Sediments In The Water-level Fluctuation Zone Of The Tributaries Of The Yangtze River

The water-level fluctuation zone of the river is one of the essential ecosystem types related to global methane and nitrous oxide emissions.It is an important area of the nitrogen and carbon cycle in nature.The fluctuation of water level,human disturbance,and the difference in environmental factors directly or indirectly affect the soil ecosystem and microbial community structure.Anaerobic ammonia oxidation（ANAMMOX）,mediated by anaerobic ammonia-oxidizing bacteria（AAOB）,is an integral part of the biogeochemical nitrogen cycle and significantly impacts the global nitrogen cycle.The nitrate-dependent anaerobic methane oxidation（DAMO）process of anaerobic methanotrophic archaea（ANME）reveals the diversity and complexity of the biogeochemical cycle of carbon and nitrogen,which has far-reaching significance for the study of the carbon and nitrogen cycle in the natural environment.In this study,the sediments of the water-level-fluctuating zone in the Fuling section of the Yangtze River were taken as the research object.Illumina Mi Seq high-throughput sequencing and DNA-stable isotope probing（DNA-SIP）technology were used to explore the differences in the spatial distribution of ANAMMOX and DAMO and the participation of microorganisms in the sediments of different river water-level-fluctuating zones in the dry period and wet period.Combined with ecological principles,the effects of environmental factors on ANAMMOX and DAMO and the participation of microorganisms were revealed.The main results are as follows:1.The community structure characteristics of AAOB and ANME in the sediments of the water-level fluctuation zone of the tributaries.The soil collected in this section is sediment from Sanli Bay and Bazi River of the Fuling section of the Yangtze River.0～10 cm,10～20 cm,20～30 cm and 30～40 cm soil sediments were collected during dry period and wet period respectively.In the dry period,the abundance and community diversity of AAOB in the 20～30 cm soil layer were higher,and the abundance of bacteria was 2.10×10⁶～9.11×10⁶copies·g^-1dry soil.In the wet period,the abundance and diversity of AAOB in the 0～10cm soil layer were higher,and the abundance of bacteria was 6.11×10⁷～9.91×10⁷copies·g^-1dry soil.A total of 37 OTUs were detected in AAOB during the dry period,which could be attributed to Candidatus Brocadia fulgida,Candidatus Anammoxoglobus propionicus,Candidatus Brocadia anammoxidans,Candidatus Kuenenia stuttgartiensis,and Candidatus Brocadia sp.;a total of 46 OTUs of AAOB were detected in the wet period,including Candidatus Brocadia fulgida,Candidatus Anammoxoglobus propionicus,Candidatus Kuenenia stuttgartiensis,Candidatus Brocadia anammoxidans,and Candidatus Jettenia asiatica.The abundance of ANME in the 30～40cm soil layer was higher in the dry period,and the abundance was 5.18×10⁴～7.67×10⁵copies·g^-1dry soil,while that in the wet period was 5.28×10⁵～4.19×10⁶copies·g^-1dry soil.The abundance of ANME was higher in the 10～30cm soil layer during the wet period.ANME detected a total of 8 OTUs in the dry period and wet period,among which the contents of OTU8,OTU7,and OTU6 were higher.OTU8 is cultured with Uncultured Candidatus Methanperedens sp.,OTU 7 shows a very high similarity to the archaea cultured in the laboratory,and OTU6 is similar to Uncultured Methanosarcinales archaeon,an anaerobic methanosarcinales archaeon detected on the continental slopes of Mexico.In dry period,p H,soil temperature and organic matter content had a greater influence on the community structure of anammox bacteria in different river sediments,while total nitrogen content,ammonium nitrogen content,nitrate nitrogen content and nitrite nitrogen content had a lesser influence.In wet period,the environmental factors that had a great influence on the community structure of anammox bacteria in different river sediments were p H value,ammonium nitrogen content and soil temperature,followed by organic matter content and total nitrogen content.The environmental factors that had a little influence were nitrate nitrogen content and nitrite nitrogen content.In dry period,soil p H,ammonium nitrogen content and soil temperature were the most influential environmental factors on the community structure of anaerobic methane-oxidizing bacteria in different river sediments,followed by total nitrogen content and organic matter content,and nitrate nitrogen content and nitrite nitrogen content were the least influential environmental factors.In wet period,soil p H value and ammonium nitrogen content were the most important environmental factors affecting the community structure of anaerobic methane oxidizing bacteria in different river sediments,followed by total nitrogen content,nitrite nitrogen content and organic matter content,and nitrate nitrogen content and soil temperature were the least influential environmental factors.2.Differences between AAOB and ANME in sediments treated with different p HThe soils collected in this section is the sediment at a depth of 0～20cm in the Fuling section of the Yangtze River.After 56 days of anaerobic incubation by addition of ¹²CO₂and ¹³CO₂,different soil treatments and heavy ¹³C-DNA stratified by ultra-centrifugation by addition of Cs Cl were examined.The results show that the copy number of the 16S r RNA gene of AAOB in acid sediments was 2.78×10⁷～2.91×10⁷copies·g^-1dry soil,and a total of 8 OTUs were detected.The copy number of the 16S r RNA gene of AAOB in neutral soil sediments was 3.65×10⁷～3.78×10⁷copies·g^-1dry soil,and 13 OTUs were detected.The copy number of the 16S r RNA gene of AAOB in alkaline sediments was 1.77×10⁷～1.91×10⁷copies·g^-1dry soil,and 6 OTUs were detected.OTU8 is close to Candidatus Scalindua marina,OTU13 is close to Candidatus Brocadia anammoxidans,OTU1 and OTU15 are close to Candidatus Brocadia fulgida,OTU4 was assigned to Candidatus Kuenenia stuttgartiensis.The copy number of the 16S r RNA gene of AAOB in neutral soil and acid soil was significantly higher than that of AAOB in alkaline soil（P<0.05）.After 112 days of anaerobic incubation with ¹²CH₄and ¹³CH₄,the different treated soil DNA and the heavy ¹³C-DNA stratified by ultra-centrifugation by addition of Cs Cl were examined.,the copy number of the 16S r RNA gene of ANME in sediments treated with different p H values increased significantly.The 16S r RNA gene copy number of ANME in acid sediments increased to 1.27×10⁷～1.48×10⁷copies·g^-1dry soil.The 16S r RNA gene copy number of ANME in neutral sediment increased to 3.17×10⁷～3.01×10⁷copies·g^-1dry soil.The copy number of the 16S r RNA gene of ANME in alkaline sediments was7.67×10⁶～2.43×10⁷copies·g^-1dry soil.A total of 2 OTUs were detected in acidic soil,7OTUs were detected in neutral soil,and 4 OTUs were detected in alkaline soil.The copy number of the 16S r RNA gene of ANME in different p H values increased significantly,and compared with sediments treated with different p H values,ANME in neutral sediments was significantly higher than that in alkaline sediments,and ANME in alkaline sediments was significantly higher than that in acidic sediments（P<0.05）.In conclusion,AAOB and ANME have a particular spatial and temporal heterogeneity in the sediment of the fluvial water-fluctuation zone.The abundance,diversity,and community structure of AAOB and ANME were affected by p H and soil temperature.AAOB are more suitable for neutral and weakly acidic soil,and ANME are more suitable for neutral and weakly alkaline soil.