Metal limitation of cyanobacterial N2 fixation and implications for the Precambrian nitrogen cycle

Nitrogen fixation is a critical part of the global nitrogen cycle, replacing biologically available reduced nitrogen lost by denitrification. The redox‐sensitive trace metals Fe and Mo are key components of the primary nitrogenase enzyme used by cyanobacteria (and other prokaryotes) to fix atmospheric N₂ into bioessential compounds. Progressive oxygenation of the Earth's atmosphere has forced changes in the redox state of the oceans through geologic time, from anoxic Fe‐enriched waters in the Archean to partially sulfidic deep waters by the mid‐Proterozoic. This development of ocean redox chemistry during the Precambrian led to fluctuations in Fe and Mo availability that could have significantly impacted the ability of prokaryotes to fix nitrogen. It has been suggested that metal limitation of nitrogen fixation and nitrate assimilation, along with increased rates of denitrification, could have resulted in globally reduced rates of primary production and nitrogen‐starved oceans through much of the Proterozoic. To test the first part of this hypothesis, we grew N₂‐fixing cyanobacteria in cultures with metal concentrations reflecting an anoxic Archean ocean (high Fe, low Mo), a sulfidic Proterozoic ocean (low Fe, moderate Mo), and an oxic Phanerozoic ocean (low Fe, high Mo). We measured low rates of cellular N₂ fixation under [Fe] and [Mo] estimated for the Archean ocean. With decreased [Fe] and higher [Mo] representing sulfidic Proterozoic conditions, N₂ fixation, growth, and biomass C:N were similar to those observed with metal concentrations of the fully oxygenated oceans that likely developed in the Phanerozoic. Our results raise the possibility that an initial rise in atmospheric oxygen could actually have enhanced nitrogen fixation rates to near modern marine levels, providing that phosphate was available and rising O₂ levels did not markedly inhibit nitrogenase activity.     

Post-treatment of fish canning effluents by sequential nitrification and autotrophic denitrification processes

In this research study a nitrifying/autotrophic denitrifying system was used for the post-treatment of an effluent coming from an anaerobic digester treating the wastewater produced in a fish canning industry. The nitrifying reactor achieved 100% of ammonia oxidation into nitrate. The effluent from this unit was fed to the autotrophic denitrifying reactor which treated a maximum sulphide loading rate (SLR) of 200 mg S^2?/L d with removal percentages of 100% and 30% for sulphide and nitrate, respectively. The low nitrate removal efficiency is attributed to sulphide limitations.The operational costs of this system were estimated as 0.92 €/kg N_removed, lower than those for conventional nitrification/denitrification processes. For nitrogen removal the SHARON/anammox processes is the cheapest option. However the combination of nitrification and autotrophic denitrification (using elemental sulphur) processes would present a better operational stability compared to the SHARON/anammox system.     

Radiation of nitrogen‐metabolizing enzymes across the tree of life tracks environmental transitions in Earth history

Nitrogen is an essential element to life and exerts a strong control on global biological productivity. The rise and spread of nitrogen‐utilizing microbial metabolisms profoundly shaped the biosphere on the early Earth. Here, we reconciled gene and species trees to identify birth and horizontal gene transfer events for key nitrogen‐cycling genes, dated with a time‐calibrated tree of life, in order to examine the timing of the proliferation of these metabolisms across the tree of life. Our results provide new insights into the evolution of the early nitrogen cycle that expand on geochemical reconstructions. We observed widespread horizontal gene transfer of molybdenum‐based nitrogenase back to the Archean, minor horizontal transfer of genes for nitrate reduction in the Archean, and an increase in the proliferation of genes metabolizing nitrite around the time of the Mesoproterozoic (~1.5 Ga). The latter coincides with recent geochemical evidence for a mid‐Proterozoic rise in oxygen levels. Geochemical evidence of biological nitrate utilization in the Archean and early Proterozoic may reflect at least some contribution of dissimilatory nitrate reduction to ammonium (DNRA) rather than pure denitrification to N₂. Our results thus help unravel the relative dominance of two metabolic pathways that are not distinguishable with current geochemical tools. Overall, our findings thus provide novel constraints for understanding the evolution of the nitrogen cycle over time and provide insights into the bioavailability of various nitrogen sources in the early Earth with possible implications for the emergence of eukaryotic life.     

Stark Contrast in Denitrification and Anammox across the Deep Norwegian Trench in the Skagerrak

Environmental anaerobic ammonium oxidation (anammox) was demonstrated for the first time in 2002, using ¹⁵N labeling, in homogenized sediment from the Skagerrak, where it accounted for up to 67% of N₂ production. We returned to some of these original sites in 2010 to make measurements of nitrogen and carbon cycling under conditions more representative of those in situ, quantifying anammox and denitrification, together with oxygen penetration and consumption, in intact sediment cores. Overall, oxygen consumption and N₂ production decayed with water depth, as expected, but the drop in N₂ production was relatively more pronounced. Whereas we confirmed the dominance of N₂ production by anammox (72% and 77%) at the two deepest sites (∼700 m of water), anammox was conspicuously absent from two shallower sites (∼200 m and 400 m). At the shallower sites, we could measure no anammox activity with either intact or homogeneous sediment, and quantitative PCR (16S rRNA) gave a negligible abundance of anammox bacteria in the anoxic layers. Such an absence of anammox, especially at one locale where it was originally demonstrated, is hard to reconcile. Despite the dominance of anammox at the deepest sites, anammox activity could not make up for the drop in denitrification, and assuming Redfield ratios for the organic matter being mineralized, the estimated retention of fixed N actually increased to 90% to 97% of that mineralized, whereas it was 80% to 86% at the shallower sites.     

Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM⁻¹, and there was 95% inhibition at ~30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM⁻¹; 95% inhibition at ~70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM⁻¹, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ~50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.     

Seasonal oxygen,nitrogen and phosphorus benthic cycling along an impacted Baltic Sea estuary: regulation and spatial patterns

The regulatory roles of temperature, eutrophication and oxygen availability on benthic nitrogen (N) cycling and the stoichiometry of regenerated nitrogen and phosphorus (P) were explored along a Baltic Sea estuary affected by treated sewage discharge. Rates of sediment denitrification, anammox, dissimilatory nitrate reduction to ammonium (DNRA), nutrient exchange, oxygen (O₂) uptake and penetration were measured seasonally. Sediments not affected by the nutrient plume released by the sewage treatment plant (STP) showed a strong seasonality in rates of O₂ uptake and coupled nitrification–denitrification, with anammox never accounting for more than 20 % of the total dinitrogen (N₂) production. N cycling in sediments close to the STP was highly dependent on oxygen availability, which masked temperature-related effects. These sediments switched from low N loss and high ammonium (NH₄ ⁺) efflux under hypoxic conditions in the fall, to a major N loss system in the winter when the sediment surface was oxidized. In the fall DNRA outcompeted denitrification as the main nitrate (NO₃ ^?) reduction pathway, resulting in N recycling and potential spreading of eutrophication. A comparison with historical records of nutrient discharge and denitrification indicated that the total N loss in the estuary has been tightly coupled to the total amount of nutrient discharge from the STP. Changes in dissolved inorganic nitrogen (DIN) released from the STP agreed well with variations in sedimentary N₂ removal. This indicates that denitrification and anammox efficiently counterbalance N loading in the estuary across the range of historical and present-day anthropogenic nutrient discharge. Overall low N/P ratios of the regenerated nutrient fluxes impose strong N limitation for the pelagic system and generate a high potential for nuisance cyanobacterial blooms.     

Water column anammox and denitrification in a temperate permanently stratified lake (Lake Rassnitzer,Germany)

We studied microbial N₂ production via anammox and denitrification in the anoxic water column of a restored mining pit lake in Germany over an annual cycle. We obtained high-resolution hydrochemical profiles using a continuous pumping sampler. Lake Rassnitzer is permanently stratified at ca. 29 m depth, entraining anoxic water below a saline density gradient. Mixed-layer nitrate concentrations averaged ca. 200 μmol L⁻¹, but decreased to zero in the anoxic bottom waters. In contrast, ammonium was <5 μmol L⁻¹ in the mixed layer but increased in the anoxic waters to ca. 600 μmol L⁻¹ near the sediments. In January and October, ¹⁵N tracer measurements detected anammox activity (maximum 504 nmol N₂ L⁻¹ d⁻¹ in ¹⁵NH₄⁺-amended incubations), but no denitrification. In contrast, in May, N₂ production was dominated by denitrification (maximum 74 nmol N₂ L⁻¹ d⁻¹). Anammox activity in May was significantly lower than in October, as characterized by anammox rates (maximum 6 vs. 16 nmol N₂ L⁻¹ d⁻¹ in incubations with ¹⁵NO₃⁻), as well as relative and absolute anammox bacterial cell abundances (0.56% vs. 0.98% of all bacteria, and 2.7×10⁴ vs. 5.2×10⁴ anammox cells mL⁻¹, respectively) (quantified by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with anammox bacteria-specific probes). Anammox bacterial diversity was investigated with anammox bacteria-specific 16S rRNA gene clone libraries. The majority of anammox bacterial sequences were related to the widespread Candidatus Scalindua sorokinii/brodae cluster. However, we also found sequences related to Candidatus S. wagneri and Candidatus Brocadia fulgida, which suggests a high anammox bacterial diversity in this lake comparable with estuarine sediments.     

Production of N2 through Anaerobic Ammonium Oxidation Coupled to Nitrate Reduction in Marine Sediments

In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N₂, thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N₂ production in marine sediments. Incubations with ¹⁵N-labeled nitrate or ammonium demonstrated that during this process, N₂ is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N₂ production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N₂ production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N₂ production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N₂, anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.     

Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics

Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO₂ fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems.     

The marine nitrogen cycle: recent discoveries,uncertainties and the potential relevance of climate change

The ocean''s nitrogen cycle is driven by complex microbial transformations, including nitrogen fixation, assimilation, nitrification, anammox and denitrification. Dinitrogen is the most abundant form of nitrogen in sea water but only accessible by nitrogen-fixing microbes. Denitrification and nitrification are both regulated by oxygen concentrations and potentially produce nitrous oxide (N₂O), a climate-relevant atmospheric trace gas. The world''s oceans, including the coastal areas and upwelling areas, contribute about 30 per cent to the atmospheric N₂O budget and are, therefore, a major source of this gas to the atmosphere. Human activities now add more nitrogen to the environment than is naturally fixed. More than half of the nitrogen reaches the coastal ocean via river input and atmospheric deposition, of which the latter affects even remote oceanic regions. A nitrogen budget for the coastal and open ocean, where inputs and outputs match rather well, is presented. Furthermore, predicted climate change will impact the expansion of the oceans'' oxygen minimum zones, the productivity of surface waters and presumably other microbial processes, with unpredictable consequences for the cycling of nitrogen. Nitrogen cycling is closely intertwined with that of carbon, phosphorous and other biologically important elements via biological stoichiometric requirements. This linkage implies that human alterations of nitrogen cycling are likely to have major consequences for other biogeochemical processes and ecosystem functions and services.     

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C₁₂-HSL), was identified via LC-MS/MS. Furthermore, C₁₂-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C₁₂-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C₁₂-HSL in AnAOB-based applications.     

Denitrification efficiency for defining critical loads of carbon in shallow coastal ecosystems

Denitrification efficiency [DE; (N₂ − N/(DIN + N₂ − N) × 100%)] as an indicator of change associated with nutrient over-enrichment was evaluated for 22 shallow coastal ecosystems in Australia. The rate of carbon decomposition (which can be considered a proxy for carbon loading) is an important control on the efficiency with which coastal sediments in depositional mud basins with low water column nitrate concentrations recycle nitrogen as N₂. The relationship between DE and carbon loading is due to changes in carbon and nitrate (NO₃) supply associated with sediment biocomplexity. At the DE optimum (500–1,000 μmol m⁻² h⁻¹), there is an overlap of aerobic and anaerobic respiration zones (caused primarily by the existence of anaerobic micro-niches within the oxic zone, and oxidized burrow structures penetrating into the anaerobic zone), which enhances denitrification by improving both the organic carbon and nitrate supply to denitrifiers. On either side of the DE optimum zone, there is a reduction in denitrification sites as the sediment loses its three-dimensional complexity. At low organic carbon loadings, a thick oxic zone with low macrofauna biomass exists, resulting in limited anoxic sites for denitrification, and at high carbon loadings, there is a thick anoxic zone and a resultant lack of oxygen for nitrification and associated NO₃ production. We propose a trophic scheme for defining critical (sustainable) carbon loading rates and possible thresholds for shallow coastal ecosystems based on the relationship between denitrification efficiency and carbon loading for 17 of the 22 Australian coastal ecosystems. The denitrification efficiency “optimum” occurs between carbon loadings of about 50 and 100 g C m⁻² year⁻¹. Coastal managers can use this simple trophic scheme to classify the current state of their shallow coastal ecosystems and for determining what carbon loading rate is necessary to achieve any future state. Guest editors: J. H. Andersen & D. J. Conley Eutrophication in Coastal Ecosystems: Selected papers from the Second International Symposium on Research and Management of Eutrophication in Coastal Ecosystems, 20–23 June 2006, Nyborg, Denmark     

Bacteriohopanepolyols along redox gradients in the Humboldt Current System off northern Chile

Marine oxygen minimum zones (OMZs) are characterized by the presence of subsurface suboxic or anoxic waters where diverse microbial processes are responsible for the removal of fixed nitrogen. OMZs have expanded over past decades and are expected to continue expanding in response to the changing climate. The implications for marine biogeochemistry, particularly nitrogen cycling, are uncertain. Cell membrane lipids (biomarkers), such as bacterial bacteriohopanepolyols (BHPs) and their degradation products (hopanoids), have distinctive structural attributes that convey information about their biological sources. Since the discovery of fossil hopanoids in ancient sediments, the study of BHPs has been of great biogeochemical interest due to their potential to serve as proxies for bacteria in the geological record. A stereoisomer of bacteriohopanetetrol (BHT), BHT II, has been previously identified in OMZ waters and has as been unequivocally identified in culture enrichments of anammox bacteria, a key group contributing to nitrogen loss in marine OMZs. We tested BHT II as a proxy for suboxia/anoxia and anammox bacteria in suspended organic matter across OMZ waters of the Humboldt Current System off northern Chile, as well as in surface and deeply buried sediments (125–150 ky). The BHT II ratio (BHT II/total BHT) increases as oxygen content decreases through the water column, consistent with previous results from Perú, the Cariaco Basin and the Arabian Sea, and in line with microbiological evidence indicating intense anammox activity in the Chilean OMZ. Notably, BHT II is transported from the water column to surface sediments, and preserved in deeply buried sediments, where the BHT II ratio correlates with changes in δ¹⁵N sediment values during glacial–interglacial transitions. This study suggests that BHT II offers a proxy for past changes in the relative importance of anammox, and fluctuations in nitrogen cycling in response to ocean redox changes through the geological record.     

A case study for late Archean and Proterozoic biogeochemical iron‐ and sulphur cycling in a modern habitat—the Arvadi Spring

As a consequence of Earth's surface oxygenation, ocean geochemistry changed from ferruginous (iron(II)‐rich) into more complex ferro‐euxinic (iron(II)‐sulphide‐rich) conditions during the Paleoproterozoic. This transition must have had profound implications for the Proterozoic microbial community that existed within the ocean water and bottom sediment; in particular, iron‐oxidizing bacteria likely had to compete with emerging sulphur‐metabolizers. However, the nature of their coexistence and interaction remains speculative. Here, we present geochemical and microbiological data from the Arvadi Spring in the eastern Swiss Alps, a modern model habitat for ferro‐euxinic transition zones in late Archean and Proterozoic oceans during high‐oxygen intervals, which enables us to reconstruct the microbial community structure in respective settings for this geological era. The spring water is oxygen‐saturated but still contains relatively elevated concentrations of dissolved iron(II) (17.2 ± 2.8 μM) and sulphide (2.5 ± 0.2 μM) with simultaneously high concentrations of sulphate (8.3 ± 0.04 mM). Solids consisting of quartz, calcite, dolomite and iron(III) oxyhydroxide minerals as well as sulphur‐containing particles, presumably elemental S⁰, cover the spring sediment. Cultivation‐based most probable number counts revealed microaerophilic iron(II)‐oxidizers and sulphide‐oxidizers to represent the largest fraction of iron‐ and sulphur‐metabolizers in the spring, coexisting with less abundant iron(III)‐reducers, sulphate‐reducers and phototrophic and nitrate‐reducing iron(II)‐oxidizers. 16S rRNA gene 454 pyrosequencing showed sulphide‐oxidizing Thiothrix species to be the dominating genus, supporting the results from our cultivation‐based assessment. Collectively, our results suggest that anaerobic and microaerophilic iron‐ and sulphur‐metabolizers could have coexisted in oxygenated ferro‐sulphidic transition zones of late Archean and Proterozoic oceans, where they would have sustained continuous cycling of iron and sulphur compounds.     

Denitrification activity and oxygen dynamics in Arctic sea ice

Denitrification and oxygen dynamics were investigated in the sea ice of Franklin Bay (70°N), Canada. These investigations were complemented with measurements of denitrification rates in sea ice from different parts of the Arctic (69°N–85°N). Potential for bacterial denitrification activity (5–194 μmol N m⁻² day⁻¹) and anammox activity (3–5 μmol N m⁻² day⁻¹) in melt water from both first-year and multi-year sea ice was found. These values correspond to 27 and 7%, respectively, of the benthic denitrification and anammox activities in Arctic sediments. Although we report only potential denitrification and anammox rates, we present several indications that active denitrification in sea ice may occur in Franklin Bay (and elsewhere): (1) despite sea ice-algal primary production in the lower sea ice layers, heterotrophic activity resulted in net oxygen consumption in the sea ice of 1–3 μmol l⁻¹ sea ice per day at in situ light conditions, suggesting that O₂ depletion may occur prior to the spring bloom. (2) The ample organic carbon (DOC) and NO₃ ⁻ present in sea ice may support an active denitrification population. (3) Measurements of O₂ conditions in melted sea ice cores showed very low bulk concentrations, and in some cases anoxic conditions prevailed. (4) Laboratory studies using planar optodes for measuring the high-resolution two-dimensional O₂ distributions in sea ice confirmed the very dynamic and heterogeneous O₂ distribution in sea ice, displaying a mosaic of microsites of high and low O₂ concentrations. Brine enclosures and channels were strongly O₂ depleted in actively melting sea ice, and anoxic conditions in parts of the brine system would favour anaerobic processes.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Denitrification of a landfill leachate with high nitrate concentration in an anoxic rotating biological contactor

The denitrification performance of a lab-scale anoxic rotating biological contactor (RBC) using landfill leachate with high nitrate concentration was evaluated. Under a carbon to nitrogen ratio (C/N) of 2, the reactor achieved N-NO₃ ⁻ removal efficiencies above 95% for concentrations up to 100 mg N-NO₃ ⁻ l⁻¹. The highest observed denitrification rate was 55 mg N-NO₃ ⁻ l⁻¹ h⁻¹ (15 g N-NO₃ ⁻ m⁻² d⁻¹) at a nitrate concentration of 560 mg N-NO₃ ⁻ l⁻¹. Although the reactor has revealed a very good performance in terms of denitrification, effluent chemical oxygen demand (COD) concentrations were still high for direct discharge. The results obtained in a subsequent experiment at constant nitrate concentration (220 mg N-NO₃ ⁻ l⁻¹) and lower C/N ratios (1.2 and 1.5) evidenced that the organic matter present in the leachate was non-biodegradable. A phosphorus concentration of 10 mg P-PO₄ ³⁻ l⁻¹ promoted autotrophic denitrification, revealing the importance of phosphorus concentration on biological denitrification processes.     

A new method of autotrophic denitrification with spent sulfidic caustic as substrate and alkalinity source

A new method based on sulfide utilizing autotrophic denitrification was adopted to remove nitrate from wastewater and to reuse spent sulfidic caustic containing high sulfide and alkalinity levels. The experiments were performed using a bench-scale upflow anoxic hybrid growth reactor (UAHGR) and an upflow anoxic suspended growth reactor (UASGR) to characterize the stoichiometric relationship between sulfur and nitrate in the process as well as the performance of the reactors. The level of nitrate removal from the UAHGR and UASGR were maintained at over 90% at a nitrate loading rate ranging from 0.15∼0.40 kgNO₃ ⁻/m³·d and no significant nitrite accumulation was observed in either reactor. Although the influent pH values were higher than the optimum range of autotrophic denitrification at 8.7∼10.1, the effluent pH was stable at 7.2∼7.9 due to the production of hydrogen ions during operation. The stoichiometric ratio of sulfate production to nitrate removal was 1.5∼2.1 mgSO₄ ²⁻/mgNO₃ ⁻ in both reactors. A comparison of the reactor performance revealed that the chemical parameters of the UAHGR operation corresponded to a plug flow like type reactor while the chemical parameters of the UASGR operation corresponded to a completely stirred tank reactor like type reactor. UAHGR did not require sludge recycling due to the packed media while UASGR required 300∼700% sludge recycling. Therefore, spent sulfidic caustic could be used in the sulfur utilizing autotrophic denitrification processes as substrate and alkalinity sources.