Cyanobacterial Genomes from a Brackish Coastal Lagoon Reveal Potential for Novel Biogeochemical Functions and Their Evolution.

Cyanobacteria are recognised for their pivotal roles in aquatic ecosystems, serving as primary producers and major agents in diazotrophic processes. Currently, the primary focus of cyanobacterial research lies in gaining a more detailed understanding of these well-established ecosystem functions. However, their involvement and impact on other crucial biogeochemical cycles remain understudied. This knowledge gap is partially attributed to the challenges associated with culturing cyanobacteria in controlled laboratory conditions and the limited understanding of their specific growth requirements. This can be circumvented partially by the culture-independent methods which can shed light on the genomic potential of cyanobacterial species and answer more profound questions about the evolution of other key biogeochemical functions. In this study, we assembled 83 cyanobacterial genomes from metagenomic data generated from environmental DNA extracted from a brackish water lagoon (Chilika Lake, India). We taxonomically classified these metagenome-assembled genomes (MAGs) and found that about 92.77% of them are novel genomes at the species level. We then annotated these cyanobacterial MAGs for all the encoded functions using KEGG Orthology. Interestingly, we found two previously unreported functions in Cyanobacteria, namely, DNRA (Dissimilatory Nitrate Reduction to Ammonium) and DMSP (Dimethylsulfoniopropionate) synthesis in multiple MAGs using nirBD and dsyB genes as markers. We validated their presence in several publicly available cyanobacterial isolate genomes. Further, we identified incongruities between the evolutionary patterns of species and the marker genes and elucidated the underlying reasons for these discrepancies. This study expands our overall comprehension of the contribution of cyanobacteria to the biogeochemical cycling in coastal brackish ecosystems.

Comparative genome analysis of the genus Marivirga and proposal of two novel marine species: Marivirga arenosa sp. nov., and Marivirga salinae sp. nov.

BACKGROUND: The phylum Bacteroidota represents a significant proportion of heterotrophic bacteria found in marine ecosystems. Members of the phylum Bacteroidota are actively involved in the degradation of biopolymers such as polysaccharides and proteins. Bacteroidota genomes exhibit a significant enrichment of various enzymes, including carbohydrate-active enzymes (CAZymes), carboxypeptidases, esterases, isomerases, peptidases, phosphatases, and sulfatases. The genus Marivirga, a member of the family Marivirgaceae within the phylum Bacteroidota, comprises six documented species. During a microbial diversity study, three novel Marivirga strains (BKB1-2 T, ABR2-2, and BDSF4-3 T) were isolated from the West Sea, Republic of Korea. RESULTS: To explore the taxonomic status and genomic characteristics of the novel isolates, we employed a polyphasic taxonomic approach, which included phylogenetic, chemotaxonomic and comprehensive genome analysis. The three isolates were Gram-stain-negative, aerobic, rod-shaped, moderately halophilic, and had a gliding motility. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among the two isolates, BKB1-2 T and BDSF4-3 T, and the six reference strains were 70.5-76.5% for ANI and 18.1-25.7% for dDDH. Interestingly, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the strains harbor genes for a comprehensive pathway for dissimilatory nitrate reduction to ammonium (DNRA), as well as other nitrogen pathways for the reduction of nitrite, nitric oxide, and nitrous oxide. Additionally, the antiSMASH analysis indicated that the strains contained three to eight biosynthetic gene clusters (BGCs) associated with the synthesis of secondary metabolites. Furthermore, the strains carried a high number of CAZyme ranging from 53 to 152, which was also demonstrated by an in vitro analysis of degradation of the polysaccharide cellulose, chitin, laminarin, starch, and xylan. Additionally, all the strains carried genes for the metabolism of heavy metals, and exhibited tolerance to heavy metals, with minimum inhibitory concentrations (MICs) in millimoles (mM) in ranges of Co2+ (3-6), Cu2+ (0.2-0.4), Ni2+ (3-5), Zn2+ (2-4), Mn2+ (20-50), and Hg2+ (0.3). CONCLUSIONS: Based on polyphasic taxonomic approach, the three isolated strains represent two novel species names Marivirga arenosa sp. nov. (BKB1-2 T = KCTC 82989 T = InaCC B1618T), and Marivirga salinae sp. nov. (BDSF4-3 T = KCTC 82973 T = InaCC B1619T).

Denitrification dominates dissimilatory nitrate reduction across global natural ecosystems.

Denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) are three competing processes of microbial nitrate reduction that determine the degree of ecosystem nitrogen (N) loss versus recycling. However, the global patterns and drivers of relative contributions of these N cycling processes to soil or sediment nitrate reduction remain unknown, limiting our understanding of the global N balance and management. Here, we compiled a global dataset of 1570 observations from a wide range of terrestrial and aquatic ecosystems. We found that denitrification contributed up to 66.1% of total nitrate reduction globally, being significantly greater in estuarine and coastal ecosystems. Anammox and DNRA could account for 12.7% and 21.2% of total nitrate reduction, respectively. The contribution of denitrification to nitrate reduction increased with longitude, while the contribution of anammox and DNRA decreased. The local environmental factors controlling the relative contributions of the three N cycling processes to nitrate reduction included the concentrations of soil organic carbon, ammonium, nitrate, and ferrous iron. Our results underline the dominant role of denitrification over anammox and DNRA in ecosystem nitrate transformation, which is crucial to improving the current global soil N cycle model and achieving sustainable N management.

Nitrate reduction to ammonium: a phylogenetic, physiological, and genetic aspects in Prokaryotes and eukaryotes.

The microbe-mediated conversion of nitrate (NO3-) to ammonium (NH4+) in the nitrogen cycle has strong implications for soil health and crop productivity. The role of prokaryotes, eukaryotes and their phylogeny, physiology, and genetic regulations are essential for understanding the ecological significance of this empirical process. Several prokaryotes (bacteria and archaea), and a few eukaryotes (fungi and algae) are reported as NO3- reducers under certain conditions. This process involves enzymatic reactions which has been catalysed by nitrate reductases, nitrite reductases, and NH4+-assimilating enzymes. Earlier reports emphasised that single-cell prokaryotic or eukaryotic organisms are responsible for this process, which portrayed a prominent gap. Therefore, this study revisits the similarities and uniqueness of mechanism behind NO3- -reduction to NH4+ in both prokaryotes and eukaryotes. Moreover, phylogenetic, physiological, and genetic regulation also shed light on the evolutionary connections between two systems which could help us to better explain the NO3--reduction mechanisms over time. Reports also revealed that certain transcription factors like NtrC/NtrB and Nit2 have shown a major role in coordinating the expression of NO3- assimilation genes in response to NO3- availability. Overall, this review provides a comprehensive information about the complex fermentative and respiratory dissimilatory nitrate reduction to ammonium (DNRA) processes. Uncovering the complexity of this process across various organisms may further give insight into sustainable nitrogen management practices and might contribute to addressing global environmental challenges.

Microbial succession and denitrifying woodchip bioreactor performance at low water temperatures.

Mining activities are increasingly recognized for contributing to nitrogen (N) pollution and possibly also to emissions of the greenhouse gas nitrous oxide (N2O) due to undetonated, N-based explosives. A woodchip denitrifying bioreactor, installed to treat nitrate-rich leachate from waste rock dumps in northern Sweden, was monitored for two years to determine the spatial and temporal distribution of microbial communities, including the genetic potential for different N transformation processes, in pore water and woodchips and how this related to reactor N removal capacity. About 80 and 65 % of the nitrate was removed during the first and second operational year, respectively. There was a succession in the microbial community over time and in space along the reactor length in both pore water and woodchips, which was reflected in reactor performance. Nitrate ammonification likely had minimal impact on N removal efficiency due to the low production of ammonium and low abundance of the key gene nrfA in ammonifiers. Nitrite and N2O were formed in the bioreactor and released in the effluent water, although direct N2O emissions from the surface was low. That these unwanted reactive N species were produced at different times and locations in the reactor indicate that the denitrification pathway was temporally as well as spatially separated along the reactor length. We conclude that the succession of microbial communities in woodchip denitrifying bioreactors treating mining water develops slowly at low temperature, which impacts reactor performance.

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.

Quantifying the effect of the nitrogen biogeochemical processes on the distribution of ammonium in the riverbank filtration system.

Ammonium (NH4+) enrichment of riverbank filtration (RBF) systems is gaining popularity. However, most previous research has concentrated on NO3- removal efficiencies, while the mechanisms of NH4+ enrichment remain unknown. A nitrogen biogeochemical process model was developed for the quantitative analysis of NH4+ enrichment in the Kaladian well field in northwest Songyuan City, NE China. Data from laboratory experiments and in-situ monitoring were used to determine initial values and calibrate the thermodynamic/kinetic parameters representing nitrogen (N) biogeochemical reactions. (1) The NO3- from river was subjected to denitrification (DNF) and dissimilatory nitrate reduction to ammonium (DNRA) within 10-14 m of the shore, whereas the NH4+ in groundwater was caused by DNRA, organic nitrogen mineralization (MIN), and mixing with laterally recharged high NH4+ groundwater. (2) DNF and DNRA were regulated by hydrodynamic processes, with the ranges of these processes being more significant in the wet season due to a higher hydraulic gradient. MIN occurred widely throughout the water flow path, with temperature primarily controlling the rates of the three reactions. (3) DNRA activity was relatively higher in the wet season when the water temperature was higher within 10-14 m of the shore. In the wet season, DNRA contributed 25%-30% to NO3- reduction, which was higher than in the dry season (5%-10%). DNRA contributed at least 40% and 15% to NH4+ enrichment in the wet and dry seasons, respectively. (4). Organic N in media gradually released NH4+ into groundwater via MIN and desorption across the entire flow path, with contributions to NH4+ enrichment reaching 75% and 85%, respectively, in the wet and dry seasons.

A nitroreductase DnrA catalyzes the biotransformation of several diphenyl ether herbicides in Bacillus sp. Za.

Diphenyl ether herbicides, typical globally used herbicides, threaten the agricultural environment and the sensitive crops. The microbial degradation pathways of diphenyl ether herbicides are well studied, but the nitroreduction of diphenyl ether herbicides by purified enzymes is still unclear. Here, the gene dnrA, encoding a nitroreductase DnrA responsible for the reduction of nitro to amino groups, was identified from the strain Bacillus sp. Za. DnrA had a broad substrate spectrum, and the Km values of DnrA for different diphenyl ether herbicides were 20.67 µM (fomesafen), 23.64 µM (bifenox), 26.19 µM (fluoroglycofen), 28.24 µM (acifluorfen), and 36.32 µM (lactofen). DnrA also mitigated the growth inhibition effect on cucumber and sorghum through nitroreduction. Molecular docking revealed the mechanisms of the compounds fomesafen, bifenox, fluoroglycofen, lactofen, and acifluorfen with DnrA. Fomesafen showed higher affinities and lower binding energy values for DnrA, and residue Arg244 affected the affinity between diphenyl ether herbicides and DnrA. This research provides new genetic resources and insights into the microbial remediation of diphenyl ether herbicide-contaminated environments. KEY POINTS: • Nitroreductase DnrA transforms the nitro group of diphenyl ether herbicides. • Nitroreductase DnrA reduces the toxicity of diphenyl ether herbicides. • The distance between Arg244 and the herbicides is related to catalytic 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.

Assessment of groundwater vulnerability to nitrates using the GIS-based DRASTIC and SI methods: a case study in Zacharo area, Greece.

A vulnerability assessment of the aquifers in the agricultural area of Zacharo in SW, Peloponnese, Greece, was conducted using the DRASTIC index and the susceptibility index (SI). Sensitivity analysis was conducted and thematic maps for each parameter were generated to analyse the impact of individual parameter on the collective groundwater vulnerability. Results derived from the DRASTIC and SI maps revealed that the extremely highly vulnerable zones are concentrated at three coastal sites in the western part of the study area. Data from these maps also indicate low vulnerability areas throughout the eastern part of the region. The distribution of nitrate concentrations in groundwater is better correlated with the DRASTIC (79.2%) compared to SI (60.2%). Neither method takes into consideration the impact of dilution and nitrate to ammonium reduction, on the nitrate content of groundwater, thus overestimating the vulnerability index. Moreover, the SI method overestimates the impact of olive groves' land use type on the susceptibility index, thus resulting to a lower correlation with the observed nitrate concentrations.

Global Patterns and Drivers of Soil Dissimilatory Nitrate Reduction to Ammonium.

Dissimilatory nitrate reduction to ammonium (DNRA), the nearly forgotten process in the terrestrial nitrogen (N) cycle, can conserve N by converting the mobile nitrate into non-mobile ammonium avoiding nitrate losses via denitrification, leaching, and runoff. However, global patterns and controlling factors of soil DNRA are still only rudimentarily known. By a meta-analysis of 231 observations from 85 published studies across terrestrial ecosystems, we find a global mean DNRA rate of 0.31 ± 0.05 mg N kg-1 day-1, being significantly greater in paddy soils (1.30 ± 0.59) than in forests (0.24 ± 0.03), grasslands (0.52 ± 0.15), and unfertilized croplands (0.18 ± 0.04). Soil DNRA was significantly enhanced at higher altitude and lower latitude. Soil DNRA was positively correlated with precipitation, temperature, pH, soil total carbon, and soil total N. Precipitation was the main stimulator for soil DNRA. Total carbon and pH were also important factors, but their effects were ecosystem-specific as total carbon stimulates DNRA in forest soils, whereas pH stimulates DNRA in unfertilized croplands and paddy soils. Higher temperatures inhibit soil DNRA via decreasing total carbon. Moreover, nitrous oxide (N2O) emissions were negatively related to soil DNRA. Thus, future changes in climate and land-use may interact with management practices that alter soil substrate availability and/or soil pH to enhance soil DNRA with positive effects on N conservation and lower N2O emissions.

Crab bioturbation affects competition between microbial nitrogen removal and retention in estuarine and coastal wetlands.

As the important benthic animal in coastal wetlands, crab bioturbation may significantly affect the nitrogen (N) budgets by regulating microbial N transformation processes. However, the response of interaction between different microbial N processes to crab bioturbation remains poorly understood. Here, a 30-day microcosmic experiment was conducted using sediment collected from the Yangtze Estuary wetland, followed by the determination of temporal variations of physicochemical parameters, N removal (denitrification plus anammox, which is defined as N2 production) and retention rates (nitrate dissimilatory reduction to ammonium, DNRA) as well as relevant gene abundances in response to different crabs densities. The results showed that crab bioturbation simultaneously promoted the rates of N2 production and DNRA processes. These two process rates were positively associated with the intensity of crab bioturbation, which was supported by molecular analysis of relevant functional gene abundance. Crab bioturbation was more beneficial to DNRA than N2 production. Due to this disproportionate stimulation, crab bioturbation increased the importance of DNRA, indicating that N retention was becoming more significant under crab bioturbation in estuarine and coastal wetlands. The variations of sediment total organic carbon and oxygen availability driven by crab bioturbation were the critical factors mediating the changes in microbial N removal and retention. Overall, our findings highlighted that crab bioturbation can affect the N budgets in estuarine and coastal wetlands by altering the competition between microbial N removal and retention.

Molecular analysis of microbial nitrogen transformation and removal potential in the plant rhizosphere of artificial tidal wetlands across salinity gradients.

This study explored the microbial nitrogen transformation and removal potential in the plant rhizosphere of seven artificial tidal wetlands under different salinity gradients (0-30‰). Molecular biological and stable isotopic analyses revealed the existence of simultaneous anammox (anaerobic ammonium oxidation), nitrification, DNRA (dissimilatory nitrate reduction to ammonium) and denitrification processes, contributing to nitrogen loss in rhizosphere soil. The microbial abundances were 2.87 × 103-9.12 × 108 (nitrogen functional genes) and 1.24 × 108-8.43 × 109 copies/g (16S rRNA gene), and the relative abundances of dissimilatory nitrate reduction and nitrification genera ranged from 6.75% to 24.41% and from 0.77% to 1.81%, respectively. The bacterial 16S rRNA high-throughput sequencing indicated that Bacillus, Zobellella and Paracoccus had obvious effects on nitrogen removal by heterotrophic nitrifying/aerobic denitrifying process (HN-AD), and autotrophic nitrification (Nitrosomonas, Nitrospira and Nitrospina), conventional denitrification (Bradyrhizobium, Burkholderia and Flavobacterium), anammox (Candidatus Brocadia and Candidatus Scalindua) and DNRA (Clostridium, Desulfovibrio and Photobacterium) organisms co-existed with HN-AD bacteria. The potential activities of DNRA, nitrification, anammox and denitrification were 1.23-9.23, 400.03-755.91, 3.12-35.24 and 30.51-300.04 nmolN2·g-1·d-1, respectively. The denitrification process contributed to 73.59-88.65% of NOx- reduction, compared to 0.71-13.20% and 8.20-15.42% via DNRA and anammox, as 83.83-90.74% of N2 production was conducted by denitrification, with the rest through anammox. Meanwhile, the nitrification pathway accounted for 95.28-99.23% of NH4+ oxidation, with the rest completed by anammox bacteria. Collectively, these findings improved our understanding on global nitrogen cycles, and provided a new idea for the removal of contaminants in saline water treatment.

Impact of sulfamethoxazole and organic supplementation on mixotrophic denitrification process: Nitrate removal efficiency and the response of functional microbiota.

Recirculating aquaculture systems (RAS) effluent is characterized by low COD to total inorganic nitrogen ratio (C/N), excessive nitrate, and the presence of traces of antibiotics. Hence, it urgently needs to be treated before recycling or discharging. In this study, four denitrification bioreactors at increasing C/N ratios (0, 0.7, 2, and 5) were started up to treat mariculture wastewater under the sulfamethoxazole (SMX) stress, during which the bioreactors performance and the shift of mixotrophic microbial communities were explored. The result showed that during the SMX exposure, organic supplementation enhanced nitrate and thiosulfate removal, and eliminated nitrite accumulation. The denitrification rate was accelerated by increasing C/N from 0 to 2, while it declined at C/N of 5. The decline was ascribed to which SMX reduced the relative abundance of denitrifiers, but improved the capability of dissimilatory nitrogen reduction to ammonia (DNRA) and sulfide production. The direct evidence was the relative abundance of sulfidogenic populations, such as Desulfuromusa, Desulfurocapsa, and Desulfobacter increased under the SMX stress. Moreover, high SMX (1.5 mg L-1) caused the obvious accumulation of ammonia at C/N of 5 due to the high concentration of sulfide (3.54 ± 1.08 mM) and the enhanced DNRA process. This study concluded that the mixotrophic denitrification process with the C/N of 0.7 presented the best performance in nitrate and sulfur removal and indicated the maximum resistance to SMX.

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 taxonomic families (γ-proteobacteria with nrfABCDEFG and ε-proteobacteria with nrfHAIJ operon). 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.

Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.

Nitrate removal by anammox biomass with intracellular carbon source as electron donors via DNRA pathway.

In this work, a novel nitrate (NO3-) reduction pathway by anaerobic ammonium oxidation (anammox) biomass was firstly discovered with the intracellular carbon sources as the only electron donors. And the possible reaction mechanism was deduced to be intracellular dissimilatory nitrate reduction to ammonium (DNRA) pathway according to the experimental results. In batch experiments, without any external electron donors, NO3--N (about 50 mg/L) was reduced to N2 within 48 h, and a small amount of NO2--N was detected (the maximum of 2 mg/L) with the anammox biomass concentration of 4400 mg/L. Acetylene (4.46 mmol/L) addition resulted in obvious NH4+ accumulation during NO3- degradation by anammox biomass, since acetylene mainly interfered in hydrazine (N2H4) generation from NH4+ and NO. Without HCO3- addition, the NO3--N degradation rate was slower than that with HCO3- addition. Simultaneously, glycogen contents inside anammox biomass decreased to 133.22 ± 1.21 mg/g VSS and 129.79 ± 1.21 mg/g VSS with and without HCO3-, respectively, from 142.20 ± 0.61 mg/g VSS. In the long-term experiment, anammox biomass stably degraded NO3--N without external electron donors addition, and the maximum removal efficiency of NO3--N reached 55.4%. The above results indicated the anammox bacteria utilized the DNRA pathway to reduce NO3- to NO2- and further NH4+, then normal anammox metabolism would continue to convert the produced NO2- and NH4+ to N2. The intracellular stored carbon sources (e.g., glycogen) were supposed to be electron donors for NO3- degradation. This capability would enhance the viability and living space of anammox bacteria in different natural ecosystems, and make it plausible that complete nitrogen removal could be implemented only by the anammox process.

Shift of DNRA bacterial community composition in sediment cores of the Pearl River Estuary and the impact of environmental factors.

Dissimilatory nitrate reduction to ammonia (DNRA) process, competing with denitrification and anaerobic ammonia oxidation (anammox) for nitrate, is an important nitrogen retention pathway in the environment. Previous studies on DNRA bacterial diversity and composition focused on the surface sediments in estuaries, but studies on the deep sediments are limited, and the linkage between DNRA community structure and complex estuarine environment remains unclear. In this study, through high-throughput sequencing of nrfA gene followed by high-resolution sample inference, we examined spatially and temporally the composition and diversity of DNRA bacteria along a salinity gradient in five sediment cores of the Pearl River Estuary (PRE). We found a higher diversity and richness of DNRA bacteria in sediments with lower organic carbon, where sea water intersects fresh water. Moreover, the DNRA bacterial communities had the specific spatially distribution coupling with their metabolic difference along the salinity gradient of the Pearl River Estuary, but no obvious difference along the sediment depth. The distribution of DNRA bacteria in the PRE was largely driven by various environmental factors, including salinity, Oxidation-Reduction Potential (ORP), ammonium, nitrate and Corg/NO3-. Furthermore, dominant DNRA bacteria were found to be the key populations of DNRA communities in the PRE sediments by network analysis. Collectively, our results showed that niche difference of DNRA bacteria indeed occurs in the Pearl River Estuary.

Mechanisms and risks of joint control of nitrogen and phosphorus through sediment capping technology in a pilot-scale study.

Purpose: Nitrogen (N) and phosphorus (P) are the key elements leading to eutrophication, and it is important to jointly control N and P release from sediments into the water column. Methods: Different mixed materials including P sorbent, natural organic carbon (C), and an oxidizing agent were applied in a 1-year pilot-scale experiment. Results: The addition of iron-rich (IR) clay and Phoslock agent promoted the formation of iron bound P (Fe(OOH)~P) and calcium bound P (CaCO3~P) in sediments, respectively. IR clay offered more advantages in immobilization of phosphorus as refractory P, and the Phoslock agent more effectively reduced the risk of P release into water, which was expressed as a low equilibrium P concentration (EPC0). Mixtures of sugarcane (SU) detritus and IR clay exhibited high carbohydrate (CHO) contents, which further fuelled both denitrification and dissimilatory nitrate reduction to ammonium (DNRA). This indicated that the SU dosage should be controlled to avoid DNRA over denitrification. Attention should be given to the fact that SU introduction significantly promoted the generation of an anaerobic state, leading to the desorption and release of Fe(OOH)~P, which could be alleviated by using Oxone. Multienzyme activity analysis showed that P and N transformation shifted from P desorption to organic P hydrolysis and from ammonification to denitrification and DNRA, respectively. Conclusion: We recommend the use of P sorbent and organic C combined with oxidizing agents as effective mixed materials for sediment remediation, which could enhance P adsorption and provide electron donors for denitrification, while also avoiding the generation of anoxia.

Coupled relationships among anammox, denitrification, and dissimilatory nitrate reduction to ammonium along salinity gradients in a Chinese estuarine wetland.

Salinization in estuarine wetlands significantly alters the balance between their nitrogen (N) removal and retention abilities but these processes have not yet been characterized effectively. In the present study, the potential rates of sediment denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) were mapped using N isotope tracing methods along salinity gradients across the Yellow River Delta wetland (YRDW) in China. The contribution of anammox to total dissimilatory N transformations in YRDW was merely 6.8%, whereas denitrification and DNRA contributed 52.3% and 40.9%, respectively. The potential rate of denitrification (5.82 µmol/kg/h) decreased significantly along salinity gradients and markedly exceeded DNRA potential rate (2.7 µmol/kg/h) in fresh wetlands, but was lower than that of DNRA in oligohaline wetlands (3.06 and 3.18 µmol/kg/h, respectively). Moreover, a significantly positive relationship between salinity and DNRA/denitrification was obeserved, indicating that increased salinity may favor DNRA over denitrification. Furthermore, total sulfur (TS) content and ratio of total organic carbon to total nitrogen (C/N) increased with the salinity gradient and showed evident positive relationships with the DNRA/denitrification ratio. In this study, we proved that increased salinization resulted in the dominance of DNRA over denitrification, possible through the addition of S and alteration of the C/N in estuarine wetlands, leading to increased N retention in estuarine wetlands during salinization, which would enhance the eutrophication potential within wetlands and in downstream ecosystems.