Diversity Of Anammox Bacteria And Its Regulation Mechanism Of Nitrogen Metabolism In Root Zone Of Macrophytes In Wuliangsuhai Wetland

Anaerobic ammonia oxidation（Anammox）and denitrification process are important components of the wetland nitrogen cycle.The rhizosphere environment of natural wetland emergent plants provides good growth conditions for microorganisms,which plays a very important role in the biogeochemical cycle of carbon and nitrogen and reducing eutrophication.It is generally believed that the Anammox process mainly occurs under anaerobic conditions.Recent studies have found that Anammox bacteria were also detected in the rhizosphere of emergent plants in natural wetlands,whereas these studies on Anammox of emergent plants mainly focus on the functional diversity in rhizosphere and non-rhizosphere at DNA level,and the diversity of key transcriptionally active Anammox community in root-zone of emergent plants（especially plant roots）and nitrogen metabolism mechanism are still unclear.Therefore,in this study,roots and vegetated sediments of three emergent macrophytes,including Phragmites australis（PA）,Typha angustifolia（TA）and Scirpus triqueter（ST）,and unvegetated sediment were collected from Wuliangsuhai wetland,China.Based on the DNA and RNA levels,the community structure and transcriptional activity of Anammox were studied by high-throughput sequencing technology and clone library construction;in order to obtain high abundance of Anammox bacteria,Anammox bacteria were enriched of the root and rhizosphere of three emergent macrophytes and unvegetated sediment by static culture;Nitrogen metabolism Regulation mechanisms of Anammox were explored and the metabolic pathway of Anammox was reconstructed based on the results of Metagenomic assembly analysis.The main results are as follows:（1）Based on DNA levels,Anammox（hzs B）and denitrifying bacteria（nir K,nir S,nir SII and nos Z）were detected in the root and rhizosphere of three emergent macrophytes and unvegetated sediment.High-throughput sequencing results showed that the composition of Anammox bacterial community was significantly different in the root and rhizosphere of three emergent macrophytes and unvegetated sediment,Anammox bacteria depend on plant species;（2）Based on the RNA levels,active Anammox bacteria,including Candidatus Jettenia and Candidatus Brocadia,were detected only in the roots of PA;but nir K-carrying active denitrifiers,including Rhizobium,Mesorhizobium,Sinorhizobium,Bosea,Rhizobiales,Devosia,and uncultured nir K bacterium（All belong to Alphaproteobacteria Rhizobiales）,were detected in the root of TA and ST.Active root-associated ANAMMOX bacteria and nir K-type denitrifiers were influenced by plant species,and they likely contribute to nitrogen removal in eutrophic wetlands;（3）Anammox bacteria were enriched in roots and rhizosphere sediments of three emergent macrophytes and unvegetated sediment under three different substrate concentrations,including high substrate concentrations（NH₄⁺-N:200 mg/L;NO₂^--N:264 mg/L）,medium substrate concentrations（NH₄⁺-N:100 mg/L;NO₂^--N:132 mg/L）and low substrate concentrations（NH₄⁺-N:50 mg/L;NO₂^--N:65 mg/L）.The results showed that the highest increase in Anammox hzs B gene abundance was in unvegetation areas,Anammox hzs B gene abundance increased the least in plant roots;in low-substrate conditions,the abundance of Anammox hzs B gene of the root and vegetated sediment of TA and unvegetated sediment samples were significantly increased in 360 days,with a highest increase of 10⁶copies/ng DNA,the correlation analysis showed a significant effect of substrate concentration on Anammox hzs B gene abundance at different substrate concentrations;（4）Metagenomic sequencing of Anammox bacteria enriched samples showed significant differences in species composition in root and root areas after enrichment.The relative abundance of denitrifying bacteria was relatively high,mainly including Sulfurivermis（77.09%）,Thauera（68.57%）and Sulfurifustis（10.68%）;The enriched of Anammox bacteria,including Candidatus Jettenia,Candidatus Kuenenia and Candidatus Brocadia were detected in in the vegetation sediments of TA and unvegetation sediments,Candidatus Kuenenia was detected in root of TA;（5）The analysis of functional level showed significant differences in metabolic pathways of plant roots,vegetation sediments and unvegetation sediments,The key genes hzs A/hzs B and hdh involved in the Hydroxylamine oxidoreductase and Hydrazine dehydrogenase in the Anammox process were detected in vegetative sediments and unvegetative sediment.The denitrification is the main biological nitrogen nitrogen removal pathway in root of TA,and Anammox and denitrification were biological nitrogen nitrogen removal pathway in vegetative sediments of TA and unvegetative sediment.The results of redundancy analysis and co-occurrence linear network analysis show that NH₄⁺/NO₂^-and NO₂^--N significantly affects abundance of Anammox bacteria in the enrichment process.In addition,nitrogen cycle-related microorganisms have a significant impact on Anammox bacteria,mainly reflected in the competition and synergy of substrate;（6）The metagenomic assembly results showed that 110 high-quality nitrogen cycle-related MAGs were assembled,The genera,including Thauera,Sulfurivermis,Thiobacillus and Anammox Brocadiaceae families are the key functional groups involved in dissimilatory nitrate reduction,assimilation of nitrate,denitrification,nitrogen fixation and Anammox metabolic process,bring the results from average nucleotide identity（ANIb）with above published Anammox genomes and phylogenetic analysis based on whole genome together,it indicated that Anammox MAGs belonged to the two new species of Candidatus Jettenia;（7）The results of reconstructed Anammox MAGs genome metabolic pathway showed that the key genes hzs A/hzs B/hzs C and hdh were found in the Anammox MAGs genome,In addition,the nrf AH gene,involved in dissimilatory nitrate reduction process,was also detected;the analysis of the Anammox MAGs carbon and sulfur metabolism pathway revealed that Anammox MAGs underwent autotrophic carbon fixation via Wood-Ljungdahl and EMP as well as the Ru MP pathway;the sulfur metabolism pathway of Anammox MAGs were reconstructed,the conversion of H₂S from dissimilated sulfate to acetate under the action of cystine synthase（Cys OK）may participate in the carbon fixation process;and the genes SOD and ccp encoding superoxidative dismutase and cytochrome c peroxidase were found in the Anammox MAGs genome,indicating that Anammox bacteria of root zone of emergent plants in wetland likely performs oxygen detoxification with SOD and ccp genes.In this study,this paper systematically studied the active Anammox bacteria in the rhizosphere of eutrophic wetland,revealed the Anammox metabolism pathway and nitrogen cycle metabolism mechanism of the rhizosphere for the first time,and reconstructed the genome sketch of Anammox bacteria.This project provide an important theoretical support for the participation of Anammox bacteria in nitrogen cycle,and provide an important theoretical basis and technical reference for plant-microbial management in eutrophication wetland.