Dissimilatory Nitrate Reduction To Ammonium Bacteria Research Articles

Unprecedented N2O production by nitrate-ammonifying Geobacteraceae with distinctive N2O isotopocule signatures.

Dissimilatory nitrate reduction to ammonium (DNRA), driven by nitrate-ammonifying bacteria, is an increasingly appreciated nitrogen-cycling pathway in terrestrial ecosystems. This process reportedly generates nitrous oxide (N2O), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how N2O is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived N2O. In this study, we characterize two novel enzymatic pathways responsible for N2O production in Geobacteraceae strains, which are predominant nitrate-ammonifying bacteria in paddy soils. The first pathway involves a membrane-bound nitrate reductase (Nar) and a hybrid cluster protein complex (Hcp-Hcr) that catalyzes the conversion of NO2- to NO and subsequently to N2O. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to N2O by Hcp-Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived N2O from other established N2O production pathways, especially through the highest 15N-site preference (SP) values (43.0‰-49.9‰) reported so far, indicating a robust means for N2O source partitioning. Our findings demonstrate two novel N2O production pathways in DNRA that can be isotopically distinguished from other pathways.IMPORTANCEStimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce N2O emission in agriculture soils. This process converts water-leachable NO3- and NO2- into soil-adsorbable NH4+, thereby alleviating nitrogen loss and N2O emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of N2O, contributing to global warming. This contribution is often masked by other N2O generation processes, leading to a limited understanding of DNRA as an N2O source. Our study reveals two widespread yet overlooked N2O production pathways in Geobacteraceae, the predominant DNRA bacteria in paddy soils, along with their distinctive isotopocule signatures. These findings offer novel insights into the role of the DNRA bacteria in N2O production and underscore the significance of N2O isotopocule signatures in microbial N2O source tracking.

Using static magnetic field to recover ammonia efficiently by DNRA process

Dissimilatory nitrate reduction to ammonium (DNRA) has garnered attention due to its ability to recover ammonia and reduce greenhouse gas emissions simultaneously. In this study, the potential of using static magnetic field (SMF) to improve DNRA process was explored from the sight of molecular biology. Functional genes, microbial community structure, and metabolism pathways were discussed. SMF of 40 mT shortened the start-up time of DNRA from 75 days to 41 days, while 80 mT SMF delayed it to 103 days. On day 80, DNRA potential rate under 40 mT SMF, reached 174 ± 11 μmol kg−1 h−1, significantly surpassing 0 mT (88 ± 6 μmol kg−1 h−1) and 80 mT SMF (52 ± 4 μmol kg−1 h−1). SMF of 40 mT also accelerated community succession and the enrichment of functional bacteria like Geobacter (from 15.71% to 32.11%). qPCR results suggested that 40 mT SMF promoted the rapid enrichment of DNRA functional gene nrfA and 80 mT SMF promoted the enrichment of nirS gene on day 40. Dynamic responses of Thauera sp. RT1901, Stutzerimonas stutzeri, Shewanella oneidensis MR-1, and Shewanella loihica PV-4 to SMF at transcriptional levels confirmed SMF could improve the nitrogen removal and electron transfer of DNRA and denitrification bacteria. Consequently, this work validated the possibility of using SMF to improve DNRA process for ammonia recovery and investigated the underlying mechanisms, which could promote the application of DNRA in full-scale.

Biodegradable microplastics boost dissimilatory nitrate reduction to ammonium (DNRA) process contributing to ammonium nitrogen retention in farmland soils

Microplastics (MPs) pollution in agriculture ecosystems profoundly affects biogeochemical cycles. However, the impacts of MPs, particularly biodegradable MPs, on dissimilatory nitrate reduction to ammonium (DNRA) process remain unclear. Herein, the alterations in denitrification and DNRA pathways and underlying mechanism by different MPs exposures (including conventional nonbiodegradable MPs (polyethylene (PE) and polypropylene (PP)) and biodegradable MPs (polylactic acid (PLA) and polybutylene adipate terephthalate (PBAT)) were explored via soil microcosm experiments accompanied with 15N isotope tracer technology and 16S rRNA high-throughput sequencing. Compared with control, PE and PP increased the rates of denitrification by 117–126% but significantly decreased DNRA rates by 39.3–63.4%, whereas DNRA activities in PLA and PBAT treatments were remarkably boosted by 163–242% and dominated soil nitrate bioreduction (6.67–8.69 μg N g−1 d −1). This differentiation was attributed to the nutrient imbalance induced by MPs and distinctly selective effects of MPs on functional microbes. Microbial interaction mechanism further suggested that limited bioavailable carbon intensified the competition between heterotrophic denitrifiers (e.g., Bacillus, Sphingomonas and Microvirga) and DNRA bacteria (e.g., Luteitalea and Streptomyces) for electron donor in nonbiodegradable MPs treatments, reducing the abundances of DNRA bacteria. However, the high bioavailable C/NOx– ratio resulting from biodegradable MPs degradation released such competition and more greatly promoted the activities of DNRA bacteria than denitrifiers, facilitating ammonium retention in soils. Collectively, this study highlights the promotion of biodegradable MPs on soil ammonium recycle via DNRA process in farmland ecosystems and can shed some light on current plastic mulch-film management toward a sustainable and cleaner agricultural industry.

A microbial-explicit model with comprehensive nitrogen processes to quantify gaseous nitrogen production from agricultural soils

Agricultural soils are a major source of anthropogenic N2O, but their N2O emission estimates are highly uncertain, mainly due to the complexity of nitrogen (N) processes. Most soil N models include only primary N processes such as nitrification and denitrification, which limits their ability to realistically simulate N transformations in soils and accurately estimate N2O emissions from soils. This study introduces a Microbial-Explicit Model incorporating Comprehensive Nitrogen processes (MEMCN) to evaluate and quantify the influences of various N processes on the production of N2O as well as NO and N2, including nitrification, denitrification, anaerobic ammonium oxidation (ANAMMOX), dissimilatory nitrate reduction to ammonium (DNRA), mineralization, and microbial assimilation (i.e., growth) and death. The MEMCN was evaluated in laboratory experiments using agricultural soils with different levels of N additions under anaerobic and aerobic conditions, and reproduced well the dynamics of NH4+, NO3−, NO2−, NO, N2O, and N2. After nitrification and denitrification, ANAMMOX and assimilation were found to be most important in controlling N transformations in agricultural soils. ANAMMOX directly increased N2 emissions by 139% at the beginning of the simulations (i.e., 48 h) under anaerobic conditions, while microbial assimilation indirectly reduced NO, N2O, and N2 emissions by 88%, 54%, and 58%, respectively, at the end of the simulations (i.e., 336 h) under aerobic conditions. Correspondingly, the biomass of ANAMMOX bacteria increased significantly at the beginning of the simulations under anaerobic conditions, while the biomass of nitrite oxidizing bacteria increased substantially under aerobic conditions. In contrast, DNRA, mineralization and microbial death had minor effect on soil N transformations, and the biomass of DNRA bacteria and heterotrophs did not change significantly during the simulations. Our study shows that it is necessary to include ANAMMOX and microbial assimilation in soil N models, while explicit simulation of microbial biomass dynamics may only be necessary if microbial biomass pools change significantly.

Fe(II) Oxidation Shaped Functional Genes and Bacteria Involved in Denitrification and Dissimilatory Nitrate Reduction to Ammonium from Different Paddy Soils.

Microbial nitrate reduction can drive Fe(II) oxidation in anoxic environments, affecting the nitrous oxide emission and ammonium availability. The nitrate-reducing Fe(II) oxidation usually causes severe cell encrustation via chemodenitrification and potentially inhibits bacterial activity due to the blocking effect of secondary minerals. However, it remains unclear how Fe(II) oxidation and subsequent cell encrustation affect the functional genes and bacteria for denitrification and dissimilatory nitrate reduction to ammonium (DNRA). Here, bacteria were enriched from different paddy soils with and without Fe(II) under nitrate-reducing conditions. Fe(II) addition decelerated nitrate reduction and increased NO2- accumulation, due to the rapid Fe(II) oxidation and cell encrustation in the periplasm and on the cell surface. The N2O accumulation was lower in the treatment with Fe(II) and nitrate than that in the treatment with nitrate only, although the proportions of N2O and NH4+ to the reduced NO3- were low (3.25% ∼ 6.51%) at the end of incubation regardless of Fe(II) addition. The dominant bacteria varied from soils under nitrate-reducing conditions, while Fe(II) addition shaped a similar microbial community, including Dechloromonas, Azospira, and Pseudomonas. Fe(II) addition increased the relative abundance of napAB, nirS, norBC, nosZ, and nirBD genes but decreased that of narG and nrfA, suggesting that Fe(II) oxidation favored denitrification in the periplasm and NO2--to-NH4+ reduction in the cytoplasm. Dechloromonas dominated the NO2--to-N2O reduction, while Thauera mediated the periplasmic nitrate reduction and cytoplasmic NO2--to-NH4+ during Fe(II) oxidation. However, Thauera showed much lower abundance than the dominant genera, resulting in slow nitrate reduction and limited NH4+ production. These findings provide new insights into the response of denitrification and DNRA bacteria to Fe(II) oxidation and cell encrustation in anoxic environments.

Relationships between environmental factors and N-cycling microbes reveal the indirect effect of further eutrophication on denitrification and DNRA in shallow lakes

Traditional views indicate that eutrophication and subsequent algal blooms favor denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in lake ecosystems. However, lakes tend to show an increasing propensity for inorganic nitrogen (N) limitation as they become more eutrophic. Thus, the influence of further eutrophication on denitrification and DNRA in eutrophic lakes are unclear due to the uncertainty of N availability. To fill this gap, we investigated the genes abundance (AOA, AOB, nirS, nirK and nrfA) and the composition of N-cycling microbes through quantitative PCR and 16S rRNA sequencing analysis, respectively, in 15 shallow eutrophic lakes of the Yangtze-Huaihe River basin, China. The results indicated that denitrification and DNRA rates could be modulated mainly by their functional gene abundances (nirS, nirK and nrfA), followed by the environmental factors (sediment total organic carbon and nitrogen). Denitrification rates significantly increased from slightly to highly eutrophic lakes, but DNRA rates were not. An explanation is that nitrification provided ample nitrate for denitrification, and this cooperative interaction was indicated by the positive correlation of their gene abundances. In addition, Pseudomonas and Anaeromyxobacter was the dominant genus mediated denitrification and DNRA, showing the potential to perform facultative anaerobic and strict anaerobic nitrate reduction, respectively. High level of dissolved oxygen might favor the facultatively aerobic denitrifiers over the obligately anaerobic fermentative DNRA bacteria in these shallow lakes. Chlorophyll a had a weak but positive effect on the gene abundances for nitrification (AOA and AOB). Further eutrophication had an indirect effect on denitrification and DNRA rates through modulating the genes abundances of N-cycling microbes.

Synergistic interactions between anammox and dissimilatory nitrate reducing bacteria sustains reactor performance across variable nitrogen loading ratios.

Anaerobic ammonium oxidizing (anammox) bacteria are utilized for high efficiency nitrogen removal from nitrogen-laden sidestreams in wastewater treatment plants. The anammox bacteria form a variety of competitive and mutualistic interactions with heterotrophic bacteria that often employ denitrification or dissimilatory nitrate reduction to ammonium (DNRA) for energy generation. These interactions can be heavily influenced by the influent ratio of ammonium to nitrite, NH4+:NO2-, where deviations from the widely acknowledged stoichiometric ratio (1:1.32) have been demonstrated to have deleterious effects on anammox efficiency. Thus, it is important to understand how variable NH4+:NO2- ratios impact the microbial ecology of anammox reactors. We observed the response of the microbial community in a lab scale anammox membrane bioreactor (MBR) to changes in the influent NH4+:NO2- ratio using both 16S rRNA gene and shotgun metagenomic sequencing. Ammonium removal efficiency decreased from 99.77 ± 0.04% when the ratio was 1:1.32 (prior to day 89) to 90.85 ± 0.29% when the ratio was decreased to 1:1.1 (day 89-202) and 90.14 ± 0.09% when the ratio was changed to 1:1.13 (day 169-200). Over this same timespan, the overall nitrogen removal efficiency (NRE) remained relatively unchanged (85.26 ± 0.01% from day 0-89, compared to 85.49 ± 0.01% from day 89-169, and 83.04 ± 0.01% from day 169-200). When the ratio was slightly increased to 1:1.17-1:1.2 (day 202-253), the ammonium removal efficiency increased to 97.28 ± 0.45% and the NRE increased to 88.21 ± 0.01%. Analysis of 16 S rRNA gene sequences demonstrated increased relative abundance of taxa belonging to Bacteroidetes, Chloroflexi, and Ignavibacteriae over the course of the experiment. The relative abundance of Planctomycetes, the phylum to which anammox bacteria belong, decreased from 77.19% at the beginning of the experiment to 12.24% by the end of the experiment. Analysis of metagenome assembled genomes (MAGs) indicated increased abundance of bacteria with nrfAH genes used for DNRA after the introduction of lower influent NH4+:NO2- ratios. The high relative abundance of DNRA bacteria coinciding with sustained bioreactor performance indicates a mutualistic relationship between the anammox and DNRA bacteria. Understanding these interactions could support more robust bioreactor operation at variable nitrogen loading ratios.

Nitrate Bioreduction under Cr(VI) Stress: Crossroads of Denitrification and Dissimilatory Nitrate Reduction to Ammonium.

This study explored the response of NO3--N bioreduction to Cr(VI) stress, including reduction efficiency and the pathways involved (denitrification and dissimilatory nitrate reduction to ammonium (DNRA)). Different response patterns of NO3--N conversion were proposed under Cr(VI) suppress (0, 0.5, 5, 15, 30, 50, and 80 mg/L) by evaluating Cr(VI) dose dependence, toxicity accumulation, bioelectron behavior, and microbial community structure. Cr(VI) concentrations of >30 mg/L rapidly inhibited NO3--N removal and immediately induced DNRA. However, denitrification completely dominated the NO3--N reduction pathway at Cr(VI) concentrations of <15 mg/L. Therefore, 30 and 80 mg/L Cr(VI) (R4 and R6) were selected to explore the selection of the different NO3--N removal pathways. The pathway of NO3--N reduction at 30 mg/L Cr(VI) exhibited continuous adaptation, wherein the coexistence of denitrification (51.7%) and DNRA (13.6%) was achieved by regulating the distribution of denitrifiers (37.6%) and DNRA bacteria (32.8%). Comparatively, DNRA gradually replaced denitrification at 80 mg/L Cr(VI). The intracellular Cr(III) accumulation in R6 was 6.60-fold greater than in R4, causing more severe oxidant injury and cell death. The activated NO3--N reduction pathway depended on the value of nitrite reductase activity/nitrate reductase activity, with 0.84-1.08 associated with DNRA activation and 1.48-1.57 with DNRA predominance. Although Cr(VI) increased microbial community richness and improved community structure stability, the inhibition or death of nitrogen-reducing microorganisms caused by Cr(VI) decreased NO3--N reduction efficiency.

Impact of seasonal change on dissimilatory nitrate reduction to ammonium (DNRA) triggering the retention of nitrogen in lake

Nitrogen (N) reduction processes including denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are critical for the eutrophication in the lake water. However, the understanding about the dominant pathways of N cycling keep limited due to the high complexity of N cycle processes in lacustrine environment. The N fractions in sediments collected from Shijiuhu Lake were measured using high-resolution (HR)-Peeper technique and chemical extraction method in varied seasons. The abundance and microbial community compositions of functional genes involved in various N-cycling processes were also obtained using high-throughput sequencing. The results showed that NH4+ concentrations in the pore water remarkably increased from the upper layer toward the deeper layer and from winter to spring. This trend suggested that higher temperature facilitated the accumulation of NH4+ in the water. Decreased NO3− concentrations were also detected at deeper sediment layers and higher temperature, indicating the intensification of N reduction on anaerobic conditions. The NH4+-N concentrations reduced in spring along with the slight change of NO3−-N in solid sediment, indicating the desorption and release of mobile NH4+ from solid phase to the solution. Remarkably decreased absolute abundances of functional genes were found in spring with DNRA bacteria nrfA gene as dominant genus and Anaeromyxobacter as the most dominant bacterium (21.67 ± 1.03%). Higher absolute abundance (146.2-788.1 × 105 Copies/g) of nrfA gene relative to other genes was mainly responsible for the increase of bio-available NH4+ in the sediments. Generally, microbial DNRA pathway predominated the N reduction and retention processes in the lake sediment at higher temperature and water depth even experiencing the suppression of DNRA bacteria abundance. These results suggested the existence of ecological risk via N retention by the action of the DNRA bacteria in the sediment on the condition of higher temperature, further provided valuable information for N management of eutrophic lakes.

Double-edged sword effects of dissimilatory nitrate reduction to ammonium (DNRA) bacteria on anammox bacteria performance in an MBR reactor

Dissimilatory nitrate reduction to ammonium (DNRA) bacteria imposing double-edged sword effects on anammox bacteria were investigated in an anammox-membrane bioreactor (MBR) experiencing an induced crash-recovery event. During the experiment, the anammox-MBR was loaded with NH4+-N:NO2−-N ratios (RatioNH4+-N: NO2−-N) of 1.20–1.60. Initially, the anammox-MBR removed over 95% of 100 mg/L NH4+-N and 132 mg/L NO2−-N (RatioNH4+-N: NO2−-N = 0.76, the well accepted stoichiometric RatioNH4+-N: NO2−-N for anammox) in the influent (Stage 0). Then, we induced a system crash-recovery event via nitrite shock loadings to better understand responses from different guilds of bacteria in anammox-MBR, loaded with 1.60 RatioNH4+-N: NO2−-N with 100 mg/L NO2−-N in the influent (Stage 1). Interestingly, the nitrogen removal by anammox bacteria was maintained for about 20 days before starting to decrease significantly. In Stage 2, we further increased influent nitrite concentration to 120 mg/L (1.33 RatioNH4+-N: NO2−-N) to simulate a high nitrite toxicity scenario for a short period of time. As expected, nitrogen removal efficiency dropped to only 16.8%. After the induced system crash, anammox-MBR performance recovered steadily to 93.2% nitrogen removal with a 1.25 RatioNH4+-N:NO2−-N and a low nitrite influent concentration of 80 mg/L NO2−-N. Metagenomics analysis revealed that a probable causality of the decreasing nitrogen removal efficiency in Stage 1 was the overgrowth of DNRA-capable bacteria. The results showed that the members within the Ignavibacteriales order (21.7%) out competed anammox bacteria (17.0%) in the anammox-MBR with elevated nitrite concentrations in the effluent. High NO2−-N loading (120 mg N/L) further caused the predominant Candidatus Kuenenia spp. were replaced by Candidatus Brocadia spp. Therefore, it was evident that DNRA bacteria posed negative effects on anammox with 1.60 RatioNH4+-N: NO2−-N. Also, when 120 mg/L NO2−-N fed to anammox-MBR (RatioNH4+-N: NO2−-N = 1.33), canonical denitrification became the primary nitrogen sink with both DNRA and anammox activities decreased. They probably fed on lysed microbial cells of anammox and DNRA. In Stage 3, a low RatioNH4+-N: NO2−-N (1.25) with 80 mg/L NO2−-N was used to rescue the system, which effectively promoted DNRA-capable bacteria growth. Although anammox bacteria's abundance was only 7.7% during this stage, they could be responsible for about 90% of the total nitrogen removal during this stage.

Simultaneous nitrate and sulfate biotransformation driven by different substrates: comparison of carbon sources and metabolic pathways at different C/N ratios.

Nitrate (NO3-) and sulfate (SO42-) often coexist in organic wastewater. The effects of different substrates on NO3- and SO42- biotransformation pathways at various C/N ratios were investigated in this study. This study used an activated sludge process for simultaneous desulfurization and denitrification in an integrated sequencing batch bioreactor. The results revealed that the most complete removals of NO3- and SO42- were achieved at a C/N ratio of 5 in integrated simultaneous desulfurization and denitrification (ISDD). Reactor Rb (sodium succinate) displayed a higher SO42- removal efficiency (93.79%) with lower chemical oxygen demand (COD) consumption (85.72%) than reactor Ra (sodium acetate) on account of almost 100% removal of NO3- in both Ra and Rb. Ra produced more S2- (5.96 mg L-1) and H2S (25 mg L-1) than Rb, which regulated the biotransformation of NO3- from denitrification to dissimilatory nitrate reduction to ammonium (DNRA), whereas almost no H2S accumulated in Rb which can avoid secondary pollution. Sodium acetate-supported systems were found to favor the growth of DNRA bacteria (Desulfovibrio); although denitrifying bacteria (DNB) and sulfate-reducing bacteria (SRB) were found to co-exist in both systems, Rb has a greater keystone taxa diversity. Furthermore, the potential carbon metabolic pathways of the two carbon sources have been predicted. Both succinate and acetate could be generated in reactor Rb through the citrate cycle and the acetyl-CoA pathway. The high prevalence of four-carbon metabolism in Ra suggests that the carbon metabolism of sodium acetate is significantly improved at a C/N ratio of 5. This study has clarified the biotransformation mechanisms of NO3- and SO42- in the presence of different substrates and the potential carbon metabolism pathway, which is expected to provide new ideas for the simultaneous removal of NO3- and SO42- from different media.

Rapid dissimilatory nitrate reduction to ammonium conserves bioavailable nitrogen in organic deficient soils

Large amounts of nitrogen fertilized to food production are lost via denitrification and leaching. Dissimilatory nitrate reduction to ammonium (DNRA) is a bioprocess competing with denitrification, and it conserves bioavailable nitrogen in soil ecosystems. The carbon to nitrogen (C/N) ratio has long been believed essential to balance the competition between DNRA and denitrification, but increasing evidence indicates that DNRA may occur unrelated to C/N ratio via a largely unknown mechanism. Taking Geobacter as representative bacteria, we found that the kinetic of nitrite bio-reduction to ammonium was the key to enhancing DNRA and demonstrated it in pure and mixed cultures and soil ecosystems. Compared to other two strains of DNRA bacteria, Geobacter sulfurreducens was confirmed for the first time to conduct a 3-fold more rapid DNRA (NO2−→NH4+) when it grew with the kinetically matched nitrite accumulator Delftia tsuruhatensis (NO3−→NO2−). They assisted and restricted each other to bring their population ratio close to the theoretical yield ratio of 2.7:1 while performing cross-feeding with nitrite and ammonium. This rapid DNRA was further demonstrated to conserve 2–8 times more reactive nitrogen in organic deficient soils, and up to 40% of 15NO3− was reduced to ammonium. Our results found a sustainable way of nitrogen retention in soils, and have broader implications for understanding nitrogen turnover in ecosystems.

Nitrogen removal crash of denitrification in anaerobic biofilm reactor due to dissimilatory nitrate reduction to ammonium (DNRA) for tofu processing wastewater treatment: Based on microbial community and functional genes

Nitrogen removal crash of denitrification in an anaerobic biofilm reactor (ABR) occurred during tofu processing wastewater treatment. This study discovered that the crash occurred due to dissimilatory nitrate reduction to ammonium (DNRA) based on the microbial community and their functional genes. Metagenomic analysis and enzyme activity measurements were carried out to identify variations of microbial communities and their functional genes encoded for denitrification and DNRA. Total nitrogen removal efficiency of the ABR was maintained at approximately 78 ± 2 % during Stage 1 but decreased from 82 to 52 % during Stage 2 (nitrogen removal crash phase). Moreover, NO2−-N and NH4+-N increased during this crash phase with stable NO3−-N removal rate (86 ± 2 %), indicating DNRA in the ABR. Functional gene analysis found a decrease of bacteria with nitrite reductase and nitric oxide reductase, especially NirK and NorC, excluding microorganisms, containing nitrous oxide reductase, variation during the ABR operation. Most DNRA bacteria increased and replaced denitrifies as the dominant nitrate-reducing microorganisms in Stage 2. Moreover, the activities of denitrification functional enzymes, especially nitrite reductase and nitric oxide reductase, decreased from Stage 1 to Stage 2, while the DNRA nitrite reductase showed a noticeable increase. During stages 1 and 2, the tofu processing wastewater contained a high COD:TN ratio of 7.3–9.1, directly inhibiting denitrification and promoting DNRA. The COD:TN ratio reduction in Stage 3 led to denitrification recovery and DNRA inhibition. Therefore, the nitrogen removal crash in the ABR was due to DNRA, which resulted from the high COD:TN ratio.

Enrichment of DNRA bacteria: Shift of microbial community and its combination with anammox to promote TN removal

Both anammox and dissimilatory nitrate reduction to ammonium (DNRA) are important processes of nitrogen metabolism, coexisting in a variety of natural ecosystems. However, the combined nitrogen removal of DNRA bacteria and anammox bacteria has not been achieved under laboratory conditions. Here, an anammox coupled DNRA system was constructed, in which DNRA bacteria convert nitrate generated from anammox process to ammonia for anammox metabolism again. DNRA bacteria were enriched form 2.11 * 105 to 3.02 * 107 copies/ng DNA in a non-woven fabric membrane bioreactor under a high C/N ratio environment. After 137 days’ operation, ammonia production efficiency from nitrate reached 60.65%. By nrfA gene sequencing analysis, Geobacter, some strains of Desulfovibrio and Desulfobulbus and a strain of traditional denitrifying bacteria Thauera sp. MZIT exhibiting DNRA function accounted for 20% of all bacteria. Small amount of anammox bacteria were detected from the system through qPCR tests, indicating that this system can mimic the natural habitats where anammox and DNRA together contributed to enhancement of total nitrogen removal rates. This study illustrated that DNRA bacteria could be enriched at high C/N ratio and natural processes are reproducible through engineering precise control.

Insights into microbial interactive mechanism regulating dissimilatory nitrate reduction processes in riparian freshwater aquaculture sediments

Aquaculture can substantially alter the accumulation and cycling of nutrients in sediments. However, the microbial mechanisms mediating sediment dissimilatory nitrate (NO3−) reduction in freshwater aquaculture ponds are still unclear, which rule the removal and retention of N element. In the present study, three microbial NO3− reduction processes in riparian aquaculture pond sediments (i.e., crab, shrimp and fish ponds) and natural freshwater sediments (i.e., lakes and rivers) were investigated via isotopic tracing and molecular analyses. The potential rates of denitrification, anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA) significantly increased in the aquaculture ponds compared with the natural freshwaters. Denitrification contributed 90.40–94.22% to the total NO3− reduction (product as N2), followed by 2.49–5.82% of anammox (product as N2) and 2.09–5.18% of DRNA (product as NH4+). The availability of C and N substrates, rather than functional gene abundance, regulated the activities of NO3− reductions and microbiome composition. Microbial mechanism based on network analysis indicated that heterotrophic denitrifiers and DNRA bacteria (e.g., Bacillus, Micromonospora, Mycobacterium and Brachybacterium) determined the community structure and function for N conversions in aquaculture ponds, whereas the such microbial network in natural freshwater sediments was manipulated by autotrophic denitrifiers (e.g., Desulfuromonas, Polaromonas, Solitalea). Collectively, this study provides an in-depth exploration of microbial nitrogen removal in freshwater aquaculture areas and supports management strategies for N pollution caused by reclamation for aquaculture in riparian zones.

Assembly mechanism and co-occurrence patterns of DNRA microbial communities and imprint of nitrate reduction in the Songhua River sediments of China's largest old industrial base

Dissimilatory nitrate reduction to ammonium (DNRA) reduces nitrate to ammonium nitrogen instead of nitrogen gas, which is an important internal linking process of nitrogen cycle. No literature has reported the assembly mechanism of DNRA microbial communities. Here, the deep assembly mechanisms and co-occurrence patterns of DNRA microbial communities and imprint of nitrate reduction in Songhua River Basin (an inland river located in northeastern China) were studied. The DNRA potential rates were detected at six sampling sites, 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. Spearman analysis indicated that the nrfA gene abundance was significantly positively correlated with the concentration of total nitrogen (TN) in sediments (p < 0.05), suggesting that nutrient inputs may enhance the metabolic potential of DNRA bacteria. Co-occurrence network and Spearman correlation analyses showed that the DNRA rates were significantly correlated with the abundance of keystone species, but not with the dominant genera (p < 0.05). Variance partitioning analysis (VPA) revealed that sediment physicochemical properties and spatial factors explained only 21.07% and 14.51% of the DNRA community variation. Null model and neutral community model both revealed stochastic processes play a major role in shaping DNRA microbial community structure. The drift was the most important process, explaining 36.36% of the community variation, followed by homogeneous selection and homogenizing dispersal, which accounted for 27.27% and 22.73%, respectively. Additionally, Geobacteraceae played an important role in DNRA and the entire bacterial community in Songhua River. This study explored the underestimated DNRA process and the deep community assembly mechanisms, which will contribute to understanding nitrate conversion and the impact of nitrogen pollution on microbial communities of river sediments in cold regions.

Dissimilatory nitrate reduction to ammonium (DNRA) potentially facilitates the accumulation of phosphorus in lake water from sediment.

Nitrogen (N) and phosphorus (P) are crucial nutrients for eutrophication in the lacustrine ecosystem and attract the attention worldwide. However, the interaction between them need further clarification. This study aimed to assess the influence of dissimilatory nitrate reduction to ammonia (DNRA) on the cycle of P in lacustrine sediment. Different fractions of N and P in the pore water were measured using high-resolution in-situ measurement techniques, HR-Peeper and DGT, coupling with sequential extraction for solid sediment from a shallow freshwater lake. The results showed that elevated nitrate (NO3-) reduction via DNRA rather than denitrification was verified at deeper sediment layer, suggesting the generation of inorganic ammonia (NH4+) as electron donor under anaerobic episodes. High abundance of DNRA bacteria (nrfA gene) obtained using high-throughput sequencing analysis were detected at upper layer and responsible for the accumulation of NH4+ in the sediment coupling with chemolithoautotrophic metabolism. Additionally, significant desorption of ionic ferrous iron (Fe2+) and dissolved reactive phosphate (DRP) from solid phase and the enrichment in the solution was simultaneously detected. Higher concentration of solid Fe bound P (Fe-P) at deeper layer indicated the potential re-oxidation of Fe2+ as electron donor during DNRA process and sorption of DRP toward the Fe-containing minerals. However, obvious evidence of desorption proved by DGT indicated that higher NH4+ concentrations favored the reduction of Fe(III) oxy(hydr)oxides and the desorption of DRP into the pore water and diffusion toward the overlying water. Finally, noteworthy S2- release from solid sediment was speculated to stimulate the DNRA and facilitated the accumulation of NH4+ in the solution, which further induced the enrichment of DRP in water from the solid phase. Overall, DNRA potentially facilitates the accumulation of P in lake water, and the synchronous control of N and P is important for the eutrophication management and restoration of lake eutrophication.

Homology modeling and virtual characterization of cytochrome c nitrite reductase (NrfA) in three model bacteria responsible for short-circuit pathway, DNRA in the terrestrial nitrogen cycle.

NrfA is the molecular marker for dissimilatory nitrate reduction to ammonium (DNRA) activity, catalysing cytochrome c nitrite reductase enzyme. However, the limited study has been made so far to understand the structural homology modeling of NrfA protein in DNRA bacteria. Therefore, three model DNRA bacteria (Escherechia coli, Wolinella succinogenes and Shewanella oneidensis) were chosen in this study for in-silico protein modeling of NrfA which roughly consists of similar length of amino acids and molecular weight and they belong to two contrasting taxonomicfamilies (γ-proteobacteria with nrfABCDEFG and ε-proteobacteria with nrfHAIJoperon). Multiple bioinformatic tools were used to examine the primary, secondary, and tertiary structure of NrfA protein using three distinct homology modeling pipelines viz., Phyre2, Swiss model and Modeller. The results indicated that NrfA protein in E. coli, W. succinogenes and S. oneidensis was mostly periplasmic and hydrophilic. Four conserved Cys-X1-X2-Cys-His motifs, one Cys-X1-X2-Cys-Lys haem-binding motif and Ca ligand were also identified in NrfA protein irrespective of three model bacteria. Moreover, 11 identical conserved amino acids sequence was observed for the first time between serine and proline in NrfA protein. Secondary structure of NrfA revealed that α-helices were observed in 77.9%, 73.4%, and 77.4% in E. coli, W. succinogenes and S. oneidensis, respectively. Ramachandran plot showed that number of residue in favored region in E. coli, W. succinogenes and S. oneidensis was 97.03%, 97.01% and 97.25%, respectively. Our findings also revealed that among three pipelines, Modeller was considered the best in-silico tool for prediction of NrfA protein. Overall, significant findings of this study may aid in the identification of future unexplored DNRA bacteria containing cytochrome c nitrite reductase. The NrfA system, which is linked to respiratory nitrite ammonification, provides an analogous target for monitoring less studied N-retention processes, particularly in agricultural ecosystems. Furthermore, one of the challenging research tasks for the future is to determine how the NrfA protein responds to redox status in the microbial cells.

Effects of carbon load on nitrate reduction during riverbank filtration: Field monitoring and batch experiment

Riverbank filtration (RBF) is a well-established technique worldwide, and is critical for the maintenance of groundwater quality and production of clean drinking water. Evaluation of the decay of exogenous nitrate (NO3−) in river water and the enrichment of ammonium (NH4+) in groundwater during RBF is important; these two processes are mainly influenced by denitrification (DNF) and dissimilatory nitrate reduction to ammonium (DNRA) controlled by the groundwater carbon load. In this study, the effects of carbon load (organic carbon [OC]: NO3−) on the competing nitrate reduction (DNRA and DNF) were assessed during RBF using field monitoring and a laboratory batch experiment. Results show the groundwater OC: NO3− ratio did not directly affect the reaction rate of DNRA and DNF, however, it could control the competitive partitioning between the two. In the near-shore zone, the groundwater OC: NO3− ratio shows significant seasonal variations along the filtration path owing to the changing conditions of redox, OC-rich, and NO3−-limited. A greater proportion of NO3− would be available for DNRA in the wet season with higher OC: NO3− ratio (> 10), resulting in a significantly NH4+-N enrichment rate (from 1.43 × 10−3 to 9.54 × 10−4 mmol L−1 d−1) in the near-shore zone where the zone of Mn (IV) oxide reduction. However, the activity of DNRA was suppressed with lower OC: NO3− ratio (< 10) in the dry season, resulting in a stable NH4+-N enrichment rate (from 3.12 × 10−4 to 1.30 × 10−4 mmol L−1 d−1). Benefiting from seasonal variation of OC-rich and NO3−-limited conditions, DNRA bacteria outcompeted denitrifiers, which eventually led to seasonal differences in NO3− reduction in the near-shore zone. Overall, under the effect of DNRA induced by continuous high carbon load in RBF systems, nitrogen input is not permanently removed but rather retained in groundwater during RBF.

Ammonia level influences the assembly of dissimilatory nitrate reduction to ammonia bacterial community in soils under different heavy metal remediation treatments

Heavy metal remediation treatments might influence functional microbial community assembly. Dissimilatory nitrate reduction to ammonia (DNRA) contributes to the nitrogen retention processes in soil ecosystems. We assumed that remediation might reduce heavy metal toxicity and increase some available nutrients for the DNRA microbes, thus balancing the deterministic and stochastic process for DNRA community assembly. Here, we investigated the process of DNRA bacterial community assembly under different heavy metal remediation treatments (including control, biochar, limestone, rice straw, rice straw + limestone, and biochar + limestone) in an Alfisol soil. The abundance of DNRA bacteria diverged across treatments. The α-diversity of the DNRA bacterial community was correlated with pH, available phosphorus (AP), ammonium (NH4+), and extractable Fe (EFe). Metal Cd and Fe significantly affected the abundance of the nrfA gene. The β-diversity was associated with pH, NH4+, and EFe. Deterministic processes dominantly drove the assembly processes of the DNRA bacterial community. NH4+ level played an essential role in the assembly processes than the other soil physicochemical properties and metal availability. High, moderate, and low levels of NH4+ could advocate stochastic process plus selection, heterogeneous selection to stochastic process, and heterogeneous selection, respectively. Network analysis highlighted a predominant role of NH4+ in regulating DNRA bacterial community assembly. However, the relative abundance of modules and some keystone species also were influenced by pH and EFe, respectively. Therefore, the DNRA bacterial community assembly under different heavy metal remediation treatments in this study was dominantly driven by nitrogen availability. pH, phosphorus, and metal availability were auxiliary regulators on DNRA bacterial community.