Evironmental Occurrence Patterns Of DNRA And Construction And Mechanism Of Its Mediated Nitrogen Removal System

Nitrate pollution can cause ecological damage and health risks.It is imperative to control nitrate pollution.Denitrification is the most common pathway for nitrate removal in various environments,and researches on denitrification have been relatively comprehensive and mature,but less concerned about another nitrate reduction pathway,dissimilatory nitrate reduction to ammonium(DNRA).Different from denitrification,DNRA reduces nitrate to ammonium and retains it in the environment.Some studies have investigated the DNRA activity and contribution of DNRA to nitrate reduction in various natural habitats.However,little is known about the environmental occurrence patterns of DNRA microbiota and its community assembly.So,it is necessary to clarify the distribution,driving factors and assembly mechanisms of DNRA microbiota in natural habitats.Furthermore,the intermediate product nitrite and the final product ammonium of the DNRA process can be further utilized by other nitrogen cycle bacteria in a mixed bacterial community.Moreover,in the laboratory,study of DNRA processes is still at the primary stage.Therefore,it is important to clarify the synergistic relationship and microbial metabolic mechanisms between DNRA and other nitrogen cycle processes to promote the application and development of the DNRA process.In this paper,the environmental distribution pattern and community assembly mechanism of DNRA bacteria in the aquatic ecosystem were characterized.Then,in the laboratory,a novel nitrogen removal system was constructed,which the DNRA process mediated by anaerobic ammonium oxidation bacteria(AnAOB)as a key pathway.Finally,the microbial nitrogen and sulfur transformation pathways and mechanisms in the sulfur autotrophic nitrate reduction system in a high sulfur environment without the addition of organic carbon were investigated.The specific conclusions are as follows.(1)The environmental distribution patterns and community assembly mechanisms of DNRA in freshwater ecosystems were investigated from Songhua River basin as the study area.The results showed that the DNRA process was detected in six sampling sites in the Songhua River basin,with potential DNRA rates ranging from 0.25±0.23 to 4.22±0.61 μmol N/L/h,accounting for 33.07%to 98.08%of the total nitrate reduction.The nrfA gene abundance was 5.29 ×104±2.61×104-6.65×106±1.23×106 copies/g dry weight and the dominant DNRA genera were Sorangium,Corallococcus,Luteitalea,Lancunisphaera,Pseudopropionibacterium,and Actinomyces.The intra-species network of DNRA was constructed by melecular ecological network analysis.39 OTUs belonging to Module hubs and 3 OTUs belonging to Connectors were identified.Geobacteraceae is the keystone family of the DNRA intraspecific network and the whole bacterial co-occurrence network.The results of Spearman correlational analyses showed that the nrfA gene abundance,DNRA rate and the abundance of keystone OTU 6176 were significantly correlated.The results of the null model and neutral community model indicated that deterministic and stochastic processes jointly drove DNRA microbial community assembly in Songhua River sediments,where homogenous selection and drift explained 27.27%and 36.36%of DNRA microbial community assembly.(2)The environmental distribution patterns and community assembly mechanisms of DNRA in marine ecosystems were investigated in the Bohai and Yellow seas as the study area.The results showed that the DNRA process was widely distributed in the coastal marine ecosystem of northern China,especially in the Bohai Sea.The average DNRA potential rate was 8.15±6.30 μmol N/L/h,which was significantly higher than denitrification.The nrfA gene abundance ranged from 1.64×103± 4.18× 102 to 3.08× 105±2.67×104 copies/g dry weight in the Bohai and the Yellow Sea.The dominant DNRA genera were Pelobacter in Bohai and North Yellow Sea;the dominant DNRA genera were Lacunisphaera,Bythopirellula,Nibricoccus,Opitutus,and Anaeromyxobacter in the South Yellow Sea.Pelobacter and Opitutus were the Module hubs and Connectors at the genus level in the intra-species network of DNRA.The DNRA community was significantly influenced by spatial factors,which is shown by variation in DNRA community composition in different sea areas.Mantel test showed that DNRA microbial community composition in sediments was significantly correlated with sediment water content,the concentration of total nitrogen,total sulfur,NH4+-N,longitude,latitude,depth of sampling sites as well as bottom water temperature and conductivity.The null model and phylogenetic normalized stochasticity ratio indicated that stochastic processes were the main drivers of DNRA microbial community assembly.Homogenizing dispersal(16.30%)and ecological drift(72.10%)were the most important stochastic processes to control DNRA community assembly in the Bohai and the Yellow Sea.(3)A new nitrogen removal system in which the DNRA process mediated by AnAOB as the key pathway was constructed in the laboratory.The microbial community succession and metabolic pathways in the reactor were resolved by metagenomics analyses.The results showed that the average removal efficiency of NH4+-N and NO3--N were 60.6±9.7%and 44.4±4.3%at a COD/NO3--N ratio of 0.6,and the highest removal efficiency of NH4+-N and NO3--N were 100.0%and 68.7%at a COD/NO3--N ratio of 1.2,which indicated that the COD/NO3--N ratio was important for the AnAOB mediated DNRA process.After inhibiting AnAOB activity with methanol,heterotrophic bacteria contributed only about 20 mg/L of nitrate reduction,and the key microbiota for nitrate reduction and acetate oxidation in the reactor were AnAOB.The acetate and nitrate did not negatively affect the abundance of AnAOB,while promoting the growth of denitrification functional genes.Metagenomics analyses showed that Candidatus Brocadia,Candidatus Kuenenia and Ignavibacterium were the dominant genera in the novel nitrogen removal system,and Candidatus Brocadia sinica,Candidatus Kuenenia stuttgartiensis,Candidatus Brocadia sp.AMX2,and Candidatus Brocadia fulgida were the most dominant AnAOB in the reactor.Candidatus Brocadia sinica carrying nrfA and nrfH gene is the main bacterium performing DNRA functions in the reactor.The increased abundance of functional genes associated with the acetylCoA pathway suggested that the bacteria in the reactor metabolize acetate via the acetylCoA pathway.In conclusion,this novel nitrogen removal system may facilitate the application of AnAOB in mainstream nitrogen removal system.(4)The microbial nitrogen and sulfur transformation pathways and mechanisms in a sulfur autotrophic nitrate reduction system in a high sulfur environment without the addition of organic carbon were investigated.The results showed that the maximum transformation of NH4--N in the reactor was 51.00%and 25.10%for N/S ratios of 1 and 0.67 at the early stage of the experiment.The sulfur autotrophic DNRA and denitrification coexisted in the reactor.When N/S ratios were lower than 0.5,a significant accumulation of nitrate occurred in the reactor,which indicates that the system is mainly a short-range denitrification process.With increased incubation time,NH4+-N produced by sulfur autotrophic DNRA and SO42-S produced by sulfur autotrophic denitrification decreased simultaneously in the reactor at N/S ratios of 1 and 0.67,suggesting that NH4+-N may be oxidized to N2 with sulfate as the electron acceptor through sulfate reducing ammonium oxidation.15N isotope incubation experiments showed that the highest potential DNRA rates were achieved at N/S ratio=1 and N/S ratio=0.67,reaching 27.5±0.2 μmol N/L/h and 3.9±0.02 μmol N/L/h,respectively,which were consistent with the results of long-term experiments.The results of high-throughput sequencing showed that the fermentative bacteria Candidatus Caldatribacterium and the sulfate-reducing bacterium(Anaerolinea and Syntrophobacte)were the dominant genera in the five reactors with different N/S ratios.Soehngenia,Vulcanibacillus,and Syner-01 were only present in the reactors with N/S ratios of 1 and 0.67.In addition,the abundance of functional genes(nrfH and nrfA)for DNRA processes was significantly increased in reactors with N/S ratios of 1 and 0.67,contributing to the further transformation of ammonium from nitrate.High concentrations(>480 mg/L)of sulfide inhibited the functional genes of denitrification processes such as nirK,norB,norC,and nosZ,leading to significant nitrite accumulation in the system.The abundance of genes encoding the sulfide oxidation pathway(sqr,dsrA,dsrB,aprA,and aprB)in the reactor system was higher than that of genes encoding the Sox pathway(soxA,soxB,soxC,soxX,soxY,and soxZ),indicating that the sulfide oxidation pathway was the main pathway of the sulfur oxidation process.