Microbial Nitrate Reduction Research Articles

Microbial nitrate reduction electro-assisted by exo-electrogenic reduction of dioxygen with a Pseudomonas dominated cathodic biofilm

A nitrate reducing microbial biocathode was developed through constant polarization at -0.5 V vs SCE using two types of inoculums: a pure culture of Thiobacillus denitrificans and water collected from the artificial wetland of Rampillon (France). The results show a clear increase of the nitrate removal efficiency for the Pilots with the natural water although no catalytic nitrate reduction can be evidenced by cyclic voltammetry. Further studies show a catalytic oxygen reduction through exo-electrogenic metabolism and a correlation between the cathode polarization at -0.5 V vs SCE and the nitrate remediation. 16S rRNA gene amplicon sequencing of the biofilm bacterial DNA shows a very large predominance of Pseudomonas, a genus that includes many species able to reduce nitrate and/or reduce dioxygen (O2) using electrons from a cathode. The increased nitrate reduction performance is hypothesized to arise from an indirect bioelectro-assistance that allows the emergence of local anoxic conditions caused by microbial endo-electrogenic pathway for oxygen reduction.

High hydrostatic pressure stimulates microbial nitrate reduction in hadal trench sediments under oxic conditions

Hadal trenches are extreme environments situated over 6000 m below sea surface, where enormous hydrostatic pressure affects the biochemical cycling of elements. Recent studies have indicated that hadal trenches may represent a previously overlooked source of fixed nitrogen loss; however, the mechanisms and role of hydrostatic pressure in this process are still being debated. To this end, we investigate the effects of hydrostatic pressure (0.1 to 115 MPa) on the chemical profile, microbial community structure and functions of surface sediments from the Mariana Trench using a Deep Ocean Experimental Simulator supplied with nitrate and oxygen. We observe enhanced denitrification activity at high hydrostatic pressure under oxic conditions, while the anaerobic ammonium oxidation – a previously recognized dominant nitrogen loss pathway – is not detected. Additionally, we further confirm the simultaneous occurrence of nitrate reduction and aerobic respiration using a metatranscriptomic dataset from in situ RNA-fixed sediments in the Mariana Trench. Taken together, our findings demonstrate that hydrostatic pressure can influence microbial contributions to nitrogen cycling and that the hadal trenches are a potential nitrogen loss hotspot. Knowledge of the influence of hydrostatic pressure on anaerobic processes in oxygenated surface sediments can greatly broaden our understanding of element cycling in hadal trenches.

Spatial and seasonal pattern of microbial nitrate reduction in coastal sediments in the Vistula River plume area, Gulf of Gdańsk

Estuaries can remove and/or retain land-derived nitrogen (N) and act as filters buffering N loads to the open sea. The N coastal filter can be seasonally variable depending on water temperature and transported loads, two factors acting in synergy and strongly influenced by climate change. The capacity of sediments to mitigate riverine N loads was investigated at four sites in the Vistula River plume area (Gulf of Gdańsk, Southern Baltic Sea). Samplings were carried out in two contrasting seasons: spring and summer, characterized by different water temperatures and nitrate (NO3-) levels. Inorganic N fluxes, and rates of denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were measured in intact sediment cores by means of dark incubations and 15N-nitrate concentration-series experiments. Sampling sites were selected along a gradient of depth (5 to 24 m), that was also a gradient of sediment organic matter content. In both seasons, denitrification rates increased along with depth and from spring (6.5 ± 7.0 µmol m-2 h-1) to summer (20.4 ± 15.4 µmol m-2 h-1), despite lower NO3- concentrations in summer. In spring, at higher NO3- loading, denitrification was likely limited by low water temperature, and elevated sediment oxygen penetration. Coupled denitrification-nitrification prevailed over denitrification of water column NO3- across all sites and seasons, contributing to over 80% of the total denitrification. Notably, no anammox was detected at the sampling sites. DNRA exhibited low to undetectable rates in spring, especially at the shallowest sites. However, during summer, N recycling via DNRA increased and ranged from 0.7 to 14.9 µmol m-2 h-1. The denitrification efficiency (DE), calculated as the ratio between molecular nitrogen (N2) flux and dissolved inorganic N effluxes from sediments, ranged from 0 to 37% in spring, whereas in summer DE did not exceed 16%. Despite the dominance of denitrification over DNRA, the analyzed sediments acted as weak N buffers under in situ dark conditions. However, concentration-series experiments suggested high potential denitrification capacity, exceeding 400 µmol m-2 h-1, in response to short-term, large riverine inputs of NO3-.

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.

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.

Cadmium immobilization during nitrate-reducing Fe(II) oxidation by Acidovorax sp. BoFeN1: Contribution of bacterial cells and secondary minerals

Microbially nitrate-reducing Fe(II) oxidation (NRFO) is widespread in anoxic environments at circumneutral pH, in which Fe(II) oxidation is considered to be mainly catalyzed by nitrite via chemodenitrification after microbial nitrate reduction. Cadmium (Cd) is toxic to organisms, and its availability is strongly affected by iron (oxyhydr)oxides and/or bacteria. However, it remains unclear how NRFO affects the fate of Cd with environmentally relevant concentrations, and the relative contributions of microbes and secondary minerals to Cd immobilization during NRFO. Here, Acidovorax sp. BoFeN1, a typical NRFO bacterium, was used to investigate the Cd immobilization during NRFO at pH 7.0 with 0.1, 0.5, and 1.0 mg/L Cd, respectively. Higher initial Cd concentration resulted in a higher amount of Cd immobilized in precipitates, but showed stronger inhibition on nitrate reduction, acetate metabolism, and cell growth. The presence of Cd barely influenced the Fe(II) oxidation during NRFO or chemodenitrification, but retarded the mineral transformation to secondary crystalline minerals, i.e., lepidocrocite and goethite. The extracellular polymeric substances deposited in the bacteria-mineral precipitates were rich in tyrosine-like and tryptophan-like proteins with negatively charged amino and carboxyl groups. The STEM-HAADF images with EDX mapping demonstrated that Cd was strongly co-localized with Fe on the cell surface and in the periplasm. Cadmium immobilization experiments with bacterial cells or during chemodenitrification indicated that the bacterial cells and secondary minerals formed during chemodenitrification are comparably important for Cd immobilization in the Cd0.1 treatment, while the secondary minerals played a predominant role in Cd immobilization in the Cd0.5 and Cd1.0 treatments. These results suggested that Cd was likely associated with the secondary iron minerals, which precipitated with the proteins on the cell surface and in the periplasm. Our findings provide a comprehensive understanding of the roles of Cd, bacterial cells, and secondary minerals formed via chemodenitrification, as well as their interactions during the complicated NRFO process at circumneutral pH.

A long-running in situ experiment in clay: 12 years of the Bitumen–Nitrate–Clay interaction experiment at Mont Terri rock laboratory

Abstract. In various countries such as Belgium and Germany, deep clay formations are investigated as potential host rock to dispose of radioactive waste. Intermediate-level long-lived bituminised waste, produced from spent fuel reprocessing, is currently foreseen to be disposed of in such a deep repository. This type of waste typically contains large amounts of salts embedded in an organic matrix, of which NaNO3 is the most prevalent. NaNO3, along with radionuclides immobilised in the bitumen matrix, will slowly dissolve and leach out into the surrounding host rock. The transport and reactivity of nitrate in a deep clay formation, in the presence and absence of electron donors coming from bituminised waste (i.e. acetate, H2), has been studied extensively inside anoxic and water-saturated chambers in the clay in the Bitumen–Nitrate–Clay interaction (BN) experiment. This in situ experiment in Opalinus clay is located in the Mont Terri rock laboratory (Switzerland). The effect of a nitrate plume on redox-sensitive radionuclides is also studied with stable selenium in selenate form as a proxy for 79Se, an important fission product for the long-term dose to humans. The BN experiment consists of a vertical borehole containing three packed-off intervals, in contact with the surrounding clay through cylindrical sintered stainless steel filter screens. The intervals are saturated with artificial Opalinus clay pore water at a pH between 7.5 and 8, containing all major ions at concentrations specific to the location of the BN experiment but without natural organic matter. Each interval is connected to a water circulation system with a gear pump, a flow meter and water sampling containers. This ensures a chemically well-mixed solution, constant water flow and easy sampling of the interval solutions under anoxic conditions. Water samples are regularly taken to assess the chemical composition and the microbial population in the intervals. An online UV spectrophotometer and pH and redox electrodes are installed in the water circuit of the intervals, to continuously monitor the nitrate and nitrite concentrations, pH and Eh. A given solution can be injected into the intervals to follow up redox reactions affecting nitrate and selenate in situ, in the presence or absence of additional electron donors. From 2011 to 2019, several injections with nitrate were performed in the intervals to investigate the biogeochemical reactivity of nitrate in the borehole and clay. These tests showed microbial reduction of nitrate, using electron donors from Opalinus clay (e.g. dissolved organic matter or pyrite). Nitrite and nitrogenous gases were formed. Pulses of electron donors (acetate or H2) boosted the microbial activity and nitrate reduction rate. From 2019 until now, injection tests with selenate, with and without nitrate, have been performed. These tests showed clearly that the presence of nitrate, a stronger oxidiser, inhibited the microbial reduction of selenate. In the absence of nitrate, the microbial population was able to reduce selenate to selenite and more reduced Se species. The results obtained in this in situ experiment will be summarised in this work, addressing questions concerning the effect of biogeochemical perturbations of the clay on the migration of redox-sensitive radionuclides.

In situ monitoring of internal water storage reveals nitrogen first flush phenomena, intermittent denitrification, and seasonal ammonium flushing

Internal water storage (IWS) can be included in bioretention practices to increase storage capacity or promote denitrification—the microbial reduction of nitrate to nitrogen gas. IWS and nitrate dynamics are well studied in laboratory systems. However, the investigation of field environments, consideration of multiple nitrogen species, and determination between mixing versus denitrification is lacking. This study employs in situ monitoring (∼24 h duration) of water level, dissolved oxygen (DO), conductivity, nitrogen species, and dual isotopes of a field bioretention IWS system for nine storms events over a year period. Rapid peaks in IWS conductivity, DO, and total nitrogen (TN) concentrations occurred along the rising limb of the IWS water level and indicated a first flush effect. TN concentrations generally peaked during the first ∼0.33 h of sampling and the average peak IWS TN concentration (Cmax = 4.82 ± 2.46 mg-N/L) was 38% and 64% greater than the average TN along the IWS rising and falling limb, respectively. Dissolved organic nitrogen (DON) and nitrate plus nitrite (NOx) were the dominant nitrogen species of IWS samples. However, average IWS peak ammonium (NH4+) concentrations August through November (0.28 ± 0.47 mg-N/L) demonstrated statistically significant shifts compared to February through May (2.72 ± 0.95 mg-N/L). Average lysimeter conductivity measurements were more than ten times higher February through May. The sustained presence of sodium observed in lysimeters, from road salt application, contributed to NH4+ flushing from the unsaturated media layer. Dual isotope analysis showed denitrification occurred for discrete time intervals along the tail of the NOx concentration profile and the hydrologic falling limb. Longer antecedent dry periods (17 days) did not correlate to enhanced denitrification but did correspond to more leaching of soil organic nitrogen. Results from field monitoring highlight the complexities of nitrogen management in bioretention systems. First flush behavior into the IWS suggests management to prevent TN export is most critical during the onset of a storm.

Influence of carbon-based cathodes on biofilm composition and electrochemical performance in soil microbial fuel cells

Increasing energy demands and environmental pollution concerns press for sustainable and environmentally friendly technologies. Soil microbial fuel cell (SMFC) technology has great potential for carbon-neutral bioenergy generation and self-powered electrochemical bioremediation. In this study, an in-depth assessment on the effect of several carbon-based cathode materials on the electrochemical performance of SMFCs is provided for the first time. An innovative carbon nanofibers electrode doped with Fe (CNFFe) is used as cathode material in membrane-less SMFCs, and the performance of the resulting device is compared with SMFCs implementing either Pt-doped carbon cloth (PtC), carbon cloth, or graphite felt (GF) as the cathode. Electrochemical analyses are integrated with microbial analyses to assess the impact on both electrogenesis and microbial composition of the anodic and cathodic biofilm. The results show that CNFFe and PtC generate very stable performances, with a peak power density (with respect to the cathode geometric area) of 25.5 and 30.4 mW m−2, respectively. The best electrochemical performance was obtained with GF, with a peak power density of 87.3 mW m−2. Taxonomic profiling of the microbial communities revealed differences between anodic and cathodic communities. The anodes were predominantly enriched with Geobacter and Pseudomonas species, while cathodic communities were dominated by hydrogen-producing and hydrogenotrophic bacteria, indicating H2 cycling as a possible electron transfer mechanism. The presence of nitrate-reducing bacteria, combined with the results of cyclic voltammograms, suggests microbial nitrate reduction occurred on GF cathodes. The results of this study can contribute to the development of effective SMFC design strategies for field implementation.

Microbial nitrate reduction in propane- or butane-based membrane biofilm reactors under oxygen-limiting conditions

Nitrate contamination has been commonly detected in water environments and poses serious hazards to human health. Previously methane was proposed as a promising electron donor to remove nitrate from contaminated water. Compared with pure methane, natural gas, which not only contains methane but also other short chain gaseous alkanes (SCGAs), is less expensive and more widely available, representing a more attractive electron source for removing oxidized contaminants. However, it remains unknown if these SCGAs can be utilized as electron donors for nitrate reduction. Here, two lab-scale membrane biofilm reactors (MBfRs) separately supplied with propane and butane were operated under oxygen-limiting conditions to test its feasibility of microbial nitrate reduction. Long-term performance suggested nitrate could be continuously removed at a rate of ∼40–50 mg N/L/d using propane/butane as electron donors. In the absence of propane/butane, nitrate removal rates significantly decreased both in the long-term operation (∼2–10 and ∼4–9 mg N/L/d for propane- and butane-based MBfRs, respectively) and batch tests, indicating nitrate bio-reduction was driven by propane/butane. The consumption rates of nitrate and propane/butane dramatically decreased under anaerobic conditions, but recovered after resupplying limited oxygen, suggesting oxygen was an essential triggering factor for propane/butane-based nitrate reduction. High-throughput sequencing targeting 16S rRNA, bmoX and narG genes indicated Mycobacterium/Rhodococcus/Thauera were the potential microorganisms oxidizing propane/butane, while various denitrifiers (e.g. Dechloromonas, Denitratisoma, Zoogloea, Acidovorax, Variovorax, Pseudogulbenkiania and Rhodanobacter) might perform nitrate reduction in the biofilms. Our findings provide evidence to link SCGA oxidation with nitrate reduction under oxygen-limiting conditions and may ultimately facilitate the design of cost-effective techniques for ex-situ groundwater remediation using natural gas.

Nitrate-Stimulated Release of Naturally Occurring Sedimentary Uranium.

Groundwater uranium (U) concentrations have been measured above the U.S. EPA maximum contaminant level (30 μg/L) in many U.S. aquifers, including in areas not associated with anthropogenic contamination by milling or mining. In addition to carbonate, nitrate has been correlated to uranium groundwater concentrations in two major U.S. aquifers. However, to date, direct evidence that nitrate mobilizes naturally occurring U from aquifer sediments has not been presented. Here, we demonstrate that the influx of high-nitrate porewater through High Plains alluvial aquifer silt sediments bearing naturally occurring U(IV) can stimulate a nitrate-reducing microbial community capable of catalyzing the oxidation and mobilization of U into the porewater. Microbial reduction of nitrate yielded nitrite, a reactive intermediate, which was further demonstrated to abiotically mobilize U from the reduced alluvial aquifer sediments. These results indicate that microbial activity, specifically nitrate reduction to nitrite, is one mechanism driving U mobilization from aquifer sediments in addition to previously described bicarbonate-driven desorption from mineral surfaces, such as Fe(III) oxides.

PH and microbial community determine the denitrifying activity in the presence of nitrate-containing radioactive waste.

An important fraction of the currently stored volume of long-lived intermediate-level radioactive waste in Belgium contains large amounts of NaNO3 homogeneously dispersed in a hard bituminous matrix. Geological disposal of this waste form in a water-saturated sedimentary formation such as Boom Clay will result in the leaching of high concentrations of NaNO3, which could cause a geochemical perturbation of the surrounding clay, possibly affecting some of the favorable characteristics of the host formation. In addition, hyper-alkaline conditions are expected for thousands of years, imposed by the cementitious materials used as backfill material. Microbial nitrate reduction is a well-known process and can result in the accumulation of nitrite or nitrogenous gases. This could lead to the oxidation of redox-active Boom Clay components, which could (locally) decrease the reducing capacity of the clay formation. Here, we compared nitrate reduction processes between two microbial communities at different pH related to a geological repository environment and in the presence of a nitrate-containing waste simulate during 1 year in batch experiments. We showed that the microbial community from in Boom Clay borehole water was able to carry out nitrate reduction in the presence of acetate at pH 10.5, although the maximum rate of 1.3 ± 0.2 mM NO3 -/day was much lower compared to that observed at pH 9 (2.9 mM NO3 -/day). However, microbial activity at pH 10.5 was likely limited by a phosphate shortage. This study further confirmed that the Harpur Hill sediment harbors a microbial community adapted to high pH conditions. It reduced twice as much nitrate at pH 10.5 compared to pH 9 and the maximum nitrate reduction rate was higher at pH 10.5 compared to that at pH 9, i.e., 3.4 ± 0.8 mM NO3 -/day versus 2.2 ± 0.4 mM NO3 -/day. Both communities were able to form biofilms on non-radioactive Eurobitum. However, for both microbial communities, pH 12.5 seems to be a limiting condition for microbial activity as no nitrate reduction nor biofilm was observed. Nevertheless, pH alone is not sufficient to eliminate microbial presence, but it can induce a significant shift in the microbial community composition and reduce its nitrate reducing activity. Furthermore, at the interface between the cementitious disposal gallery and the clay host rock, the pH will not be sufficiently high to inhibit microbial nitrate reduction.

Zero-valent iron boosts nitrate-to-ammonia bioconversion via extracellular electron donation and reduction pathway complementation

Microbial nitrate reduction to ammonia (NRA) presents a promising route to recover wastewater nitrogen resources, but its practical application is currently challenged by limited bacterial activity and reaction selectivity. Here, we propose a facile strategy to boost microbial NRA by using zero-valent iron (ZVI) as an environmentally-benign augment. Unlike the previously reported hybrid systems that rely mainly on mediated electron transfer between ZVI and bacteria for enhanced denitrification, we revealed a combined pathway of direct and mediated electron transfer from ZVI to bacteria, along with a complementation between biological and abiotic nitrogen conversion processes, to promote the NRA process. The bio-hybrid exhibited over 13-fold higher NO3− reduction activity than the individual bacteria or ZVI groups, nearly 100% NRA selectivity, and good stability for treating real wastewater. Our work provides an efficient and scalable route to combine ammonia production with wastewater valorization, which may be readily incorporated into various wastewater treatment processes to maximize resource recovery.

Underestimation about the Contribution of Nitrate Reducers to Iron Cycling Indicated by Enterobacter Strain.

Nitrate-reducing iron(II) oxidation (NRFO) has been intensively reported in various bacteria. Iron(II) oxidation is found to be involved in both enzymatic and chemical reactions in nitrate-reducing Fe(II)-oxidizing microorganisms (NRFOMs). However, little is known about the relative contribution of biotic and abiotic reactions to iron(II) oxidation for the common nitrate reducers during the NRFO process. In this study, the typical nitrate reducers, four Enterobacter strains E. hormaechei, E. tabaci, E. mori and E. asburiae, were utilized as the model microorganisms. The comparison of the kinetics of nitrate, iron(II) and nitrite and N2O production in setups with and without iron(II) indicates a mixture of enzymatic and abiotic oxidation of iron(II) in all four Enterobacter strains. It was estimated that 22−29% of total oxidized iron(II) was coupled to microbial nitrate reduction by E. hormaechei, E. tabaci, E. mori, and E. asburiae. Enterobacter strains displayed an metabolic inactivity with heavy iron(III) encrustation on the cell surface in the NRFOmedium during days of incubation. Moreover, both respiratory and periplasmic nitrate-reducing genes are encoded by genomes of Enterobacter strains, suggesting that cell encrustation may occur with periplasmic iron(III) oxide precipitation as well as the surface iron(II) mineral coating for nitrate reducers. Overall, this study clarified the potential role of nitrate reducers in the biochemical cycling of iron under anoxic conditions, in turn, re-shaping their activity during denitrification because of cell encrustation with iron(III) minerals.

Microphytobenthos as a source of labile organic matter for denitrifying microbes

Nitrogen loads in natural waters remain elevated in populated and agricultural areas with serious impact on estuarine and coastal ecosystems. Intertidal sediments can play a significant role in attenuating the high nitrogen levels in water via microbial nitrate reduction, in general dominated by denitrification. These heterotrophic processes are heavily mediated by both the quantity and quality of organic matter available. Benthic microalgae were experimentally investigated as organic carbon source for denitrifying microbes in intertidal mudflat sediments from the Seine Estuary (France). Dry microphytobenthos (including algae and their extracellular polymeric substances) were added to sediments and nitrate reduction rates were monitored over a two-week period using anoxic controlled flow-through reactor approach. Our results show that microphytobenthos addition resulted in significantly higher nitrate reduction (67–332% increase), highly related to the added amount of microphytobenthos. Moreover, increase of the low molecular weight carbohydrates consumption (11–39%) highlight the measurable contribution of extracellular polymeric substances to the carbon consumption during nitrate reduction. The addition of microphytobenthos increased the abundance of nitrite reductase genes, especially those encoding the nirS gene (43–152% increase) while nitrous oxide reductase genes (nosZ gene) remained constant. Microphytobenthos appeared to favor complete denitrification as suggested by an increase in nirS and a decrease in clade II nosZ gene copy numbers. This study confirms experimentally the assumption that microbes use microalgae and particularly labile extracellular polymeric substances as a carbon substrate for nitrate reduction. These results reinforce the impact played by microphytobenthos in intertidal mudflats by highlighting their role on denitrifying microbes and nitrate removal from water.

Hematite-promoted nitrate-reducing Fe(II) oxidation by Acidovorax sp. strain BoFeN1: Roles of mineral catalysis and cell encrustation.

Although nitrate-reducing Fe(II) oxidizing (NRFO) bacteria can grow sustainably in natural environments, numerous laboratory studies suggested that cell encrustation-induced metabolism limitations and cell death occurred more seriously in the absence of natural minerals. Hence, a study on how natural minerals could affect NRFO is warranted. This study examined the impact of hematite on NRFO by Acidovorax sp. BoFeN1 with different electron donors (acetate and Fe(II), acetate alone, and Fe(II) alone) and with nitrate as an electron acceptor. When acetate and Fe(II) were used as the electron donors, the amount of Fe(II) oxidation and nitrate reduction was enhanced in the presence of hematite, whereas no promotion was observed when only acetate was added as an electron donor. Under the conditions with only Fe(II) added as an electron donor, the level of Fe(II) oxidation was increased from 3.07 ± 0.06 to 3.92 ± 0.02 mM in the presence of hematite and nitrate reduction was enhanced. This suggests that hematite promotes microbial nitrate reduction by accelerating the biological oxidation of Fe(II). The main secondary minerals were goethite and lepidocrocite. After adding hematite, the assemblage of iron minerals on the cell surface decreased, and the cell crusts became thinner, indicating that hematite effectively mitigated cell encrustation. Furthermore, hematite accelerated the chemical oxidation of Fe(II) by nitrite. Hence, hematite can promote the NRFO of Acidovorax sp. BoFeN1 via two possible pathways: (i) hematite acts as nucleation sites to mitigate cell encrustation; (ii) hematite catalyzes the biological and chemical oxidation of Fe(II) through the mineral catalysis effects. This study highlights the importance of existing iron minerals on NRFO and sheds light on the survival strategy of NRFO bacteria in anoxic subsurface environments.

Distinct oxygen isotope fractionations driven by different electron donors during microbial nitrate reduction in lake sediments.

Microbial nitrate reduction can be driven by organic carbon oxidation, as well as by inorganic electron donors, such as reduced forms of sulfur and iron. An apparent inverse oxygen isotope fractionation effect was observed during nitrate reduction in sediment incubations from five sampling sites of a freshwater lake, Hongze Lake, China. Incubations with organic and inorganic electron donor additions were performed. Especially, the inverse oxygen isotope effect was intensified after glucose addition, whereas the incubations with sulfide and Fe2+ showed normal fractionation factors. Nitrate reductase encoding genes, napA and narG, were analysed with metagenomics. Higher napA/narG ratios were associated with higher oxygen fractionation factors. The most abundant clade (59%) of NapA in the incubation with glucose was affiliated with Rhodocyclales. In contrast, it only accounted for 8%-9% of NapA in the incubations with sulfide and Fe2+ . Differences in nitrate reductases might explain different oxygen isotope effects. Our findings also suggested that large variance of O-nitrate isotope fractionations might have to be considered in the interpretation of natural isotope records.

Oligotrophic Growth of Nitrate-Dependent Fe2+-Oxidising Microorganisms Under Simulated Early Martian Conditions.

Nitrate-dependent Fe2+ oxidation (NDFO) is a microbially mediated process observed in many anaerobic, low-nutrient (oligotrophic) neutral–alkaline environments on Earth, which describes oxidation of Fe2+ to Fe3+ in tandem with microbial nitrate reduction. Evidence suggests that similar environments existed on Mars during the Noachian epoch (4.1–3.7 Ga) and in periodic, localised environments more recently, indicating that NDFO metabolism could have played a role in a potential early martian biosphere. In this paper, three NDFO microorganisms, Acidovorax sp. strain BoFeN1, Pseudogulbenkiania sp. strain 2002 and Paracoccus sp. strain KS1, were assessed for their ability to grow oligotrophically in simulated martian brines and in a minimal medium with olivine as a solid Fe2+ source. These simulant-derived media were developed from modelled fluids based on the geochemistry of Mars sample locations at Rocknest (contemporary Mars soil), Paso Robles (sulphur-rich soil), Haematite Slope (haematite-rich soil) and a Shergottite meteorite (common basalt). The Shergottite medium was able to support growth of all three organisms, while the contemporary Mars medium supported growth of Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002; however, growth was not accompanied by significant Fe2+ oxidation. Each of the strains was also able to grow in oligotrophic minimal media with olivine as the sole Fe2+ source. Biomineralised cells of Pseudogulbenkiania sp. strain 2002 were identified on the surface of the olivine, representing a potential biosignature for NDFO microorganisms in martian samples. The results suggest that NDFO microorganisms could have thrived in early martian groundwaters under oligotrophic conditions, depending on the local lithology. This can guide missions in identifying palaeoenvironments of interest for biosignature detection. Indeed, biomineralised cells identified on the olivine surface provide a previously unexplored mechanism for the preservation of morphological biosignatures in the martian geological record.

Enhanced microbial nitrate reduction using natural manganese oxide ore as an electron donor

Nitrate contamination of groundwater is a global problem. Enhanced biological nitrate reduction by liquid organics combined with low-cost natural materials (as electron donors) can cost-effectively remove nitrate from groundwater. Dissolved Mn(II) as an electron donor has been thoroughly investigated to support microbial nitrate reduction. However, most Mn in soil and sediments is in solid form, and the ability of solid-phase natural manganese oxide ore (NMO) as electron donor and for supporting microbial nitrate reduction is unknown. Therefore, a microcosm experiment was conducted to bridge this gap in knowledge. The results demonstrated that microbial nitrate reduction (mainly converted to nitrite) was enhanced by NMO (rich in cryptomelane). The electrochemical and X-ray photoelectron spectroscopy analyses suggested that NMO may be oxidized by microbial metabolism. Illumina Miseq sequencing results indicated that Acidovorax spp. played a crucial role in NMO-supported nitrate reduction. Further Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analyses indicated that bacterial extracellular electron transfer may be one of the mechanisms for the microbial NMO oxidation. The results of our study highlight the potential importance of NMO in nitrate reduction in the natural environment and may pave the way for NMO-assisted technology for nitrate removal from groundwater with less usage of organic electron donors.

Performances and mechanisms of microbial nitrate removal coupling sediment-based biochar and nanoscale zero-valent iron

Immobilized microorganism technology has attracted increasing attention for high concentration of microbes, low cell loss and high resistance to impact of environment. The microbial reduction of nitrate in the presence of sediment-based biochar (SBC) and nanoscale zero-valent iron (nZVI) was investigated in four different free systems. NZVI-SBC/bacteria system realized the best nitrate removal of 97.61% within 3 days through the synergistic effect of SBC and nZVI on denitrifying bacteria. Accumulation of nitrite and ammonium in nZVI-SBC/bacteria system also decreased. High-throughput sequencing results showed that the proportion of denitrifying bacteria in microbial community structure increased after adding nZVI-SBC. The performance of nitrate removal was then studied through PVA/SA-immobilization. Immobilized active pellets performed better nitrate removal (98.89%) and stronger tolerance under different conditions than the free bacterial cells. Overall, this study provided a promising approach by utilizing SBC and nZVI for the bio-remediation of nitrate-contaminated water in practical application.