Long-term variability of winter nitrate concentrations in the Northern Wadden Sea driven by freshwater discharge,decreasing riverine loads and denitrification

The hypothesis of a recent reversal in the eutrophication of the Wadden Sea and the potential of inshore waters in denitrification is explored. Salinity, temperature and nitrate concentrations in the List Tidal Basin (Northern Wadden Sea) have been measured about twice weekly since 1984. Salinity has a clear seasonal cycle with lowest salinities of about 27 in late winter and highest salinities of about 31 in summer. Mean annual deviations from the long-term mean salinity correlate significantly with riverine freshwater discharge. Winter nitrate concentrations are generally high (about 50 μM on average). The major part of the variability is related to salinity (∼35%). Temperature had a minor impact (∼1%). Superimposed on this, a long-term decrease of about 1 μM per year was found. Together, these processes account for about 45% of the nitrate variability. The long-term decrease of about 2% per year is similar to continental riverine trend in total nitrogen loads. In contrast to the List Tidal Basin, salinity explained more than 90% of nitrate variability in the off-shore German Bight. Salinity (30) normalised winter nitrate data of the German Bight also show a long-term decreasing trend. Most of the List Tidal Basin data are either on or below the nitrate–salinity relation found in the German Bight. This observation suggests that denitrification has a major impact on the winter nitrate concentrations in the Northern Wadden Sea compared to the German Bight. It is hypothesised that a large part of the unexplained variability is related to weather-dependent changes in residence time of tidal water masses in the Wadden Sea and circulation patterns within the German Bight.     

Origin and cycling of riverine inorganic carbon in the Sava River watershed (Slovenia) inferred from major solutes and stable carbon isotopes

The Sava River and its tributaries in Slovenia represent waters strongly influenced by chemical weathering of limestone and dolomite. The carbon isotopic compositions of dissolved inorganic carbon (DIC) and suspended organic carbon (POC) fractions as well as major solute concentrations yielded insights into the origin and fluxes of carbon in the upper Sava River system. The major solute composition was dominated by carbonic acid dissolution of calcite and dolomite. Waters were generally supersaturated with respect to calcite, and dissolved CO₂ was about fivefold supersaturated relative to the atmosphere. The δ¹³C of DIC ranged from −13.5 to −3.3‰. Mass balances for riverine inorganic carbon suggest that carbonate dissolution contributes up to 26%, degradation of organic matter ∼17% and exchange with atmospheric CO₂ up to 5%. The concentration and stable isotope diffusion models indicated that atmospheric exchange of CO₂ predominates in streams draining impermeable shales and clays while in the carbonate-dominated watersheds dissolution of the Mesozoic carbonates predominates.     

Removal of nitrate and phosphorus from hydroponic wastewater using a hybrid denitrification filter (HDF)

A laboratory-scale hybrid-denitrification filter (HDF) was designed by combining a plant material digester and a denitrification filter into a single unit for the removal of nitrate and phosphorus from glasshouse hydroponic wastewater. The carbon to nitrate (C:N) ratio for efficient operation of the HDF was calculated to be 1.93:1 and the COD/BOD₅ ratio was 1.2:1. When the HDF was continuously operated with the plant material replaced every 2 days and 100% internal recirculation of the effluent, a high level of nitrate removal (320–5 mg N/L, >95% removal) combined with a low effluent sBOD₅ concentration (<5 mg/L) was consistently achieved. Moreover, phosphate concentrations in the effluent were maintained below 7.5 mg P/L (>81% reduction). This study demonstrates the potential to combine a digester and a denitrification filter in a single unit to efficiently remove nitrate and phosphate from hydroponic wastewater in a single unit.     

Nitrate reductase activity and growth response of forest species to ammonium and nitrate sources of nitrogen

Summary Three tree species,Eucalyptus regnans (F. Muell.),E. obliqua (L'Herit.),Pinus radiata (D. Don) were grown in sand culture with different proportions of nitrate and ammonium. Nitrate Reductase Activity (NRA) was induced in root tissue of all species and in leaf tissue of the eucalypts. An increasing proportion of nitrate resulted in increasing NRA in all species and hence NRA alone is no indication of N-preference. The highest NRA was found withE. regnans, a result which has also been obtained in the mature forest. The growth ofE. regnans was least with NH₄ ⁺ alone, whereas that ofE. obliqua was least with NO₃ ^– alone. The soils of matureE. regnans forest have a high potential for nitrification while those ofE. obliqua forest show little nitrification. Thus the preference for particular N sources shown by seedlings in culture is supported by related properties of mature forests. It is postulated however, that the inducibility of a high level of RNA in seedlings is more likely a result of a preference for NO₃ ^– than a cause.     

Rapid Nitrate Loss and Denitrification in a Temperate River Floodplain

Nitrogen (N) pollution is a problem in many large temperate zone rivers, and N retention in river channels is often small in these systems. To determine the potential for floodplains to act as N sinks during overbank flooding, we combined monitoring, denitrification assays, and experimental nitrate (NO₃⁻ -N) additions to determine how the amount and form of N changed during flooding and the processes responsible for these changes in the Wisconsin River floodplain (USA). Spring flooding increased N concentrations in the floodplain to levels equal to the river. As discharge declined and connectivity between the river and floodplain was disrupted, total dissolved N decreased over 75% from 1.41 mg l⁻¹, equivalent to source water in the Wisconsin River on 14 April 2001, to 0.34 mg l⁻¹ on 22 April 2001. Simultaneously NO₃⁻ -N was attenuated almost 100% from 1.09 to <0.002 mg l⁻¹. Unamended sediment denitrification rates were moderate (0–483 μg m⁻² h⁻¹) and seasonally variable, and activity was limited by the availability of NO ₃⁻ -N on all dates. Two experimental NO₃⁻ -N pulse additions to floodplain water bodies confirmed rapid NO₃⁻ -N depletion. Over 80% of the observed NO ₃⁻ -N decline was caused by hydrologic export for addition #1 but only 22% in addition #2. During the second addition, a significant fraction (>60%) of NO₃⁻ -N mass loss was not attributable to hydrologic losses or conversion to other forms of N, suggesting that denitrification was likely responsible for most of the NO₃⁻ -N disappearance. Floodplain capacity to decrease the dominant fraction of river borne N within days of inundation demonstrates that the Wisconsin River floodplain was an active N sink, that denitrification often drives N losses, and that enhancing connections between rivers and their floodplains may enhance overall retention and reduce N exports from large basins.     

Isotopic composition of nitrate-nitrogen as a marker of riparian and benthic denitrification at the scale of the whole Seine River system

Nitrogen budgets established for large river systems reveal that up to 60% of the nitrate exported from agricultural soils is eliminated, either when crossing riparian wetlands areas before even reaching surface waters, or within the rivers themselves through benthic denitrification. The study of nitrogen isotope ratios of riverine nitrates could offer an elegant means to assess the extent of denitrification and thus confirm these budgets, as it is known that denitrification results in a natural ¹⁵N enrichment of residual nitrates. The results reported here, for the Seine river system (France), demonstrate the feasibility of this isotopic approach at the scale of large watersheds. On the basis of in situ observations carried out in a large storage reservoir in the upstream Seine catchment (Der Lake), where intensive benthic denitrification occurs, as well as on the basis of laboratory experiments of denitrification under controlled conditions, it is shown that the isotopic discrimination associated with benthic denitrification is minimal ( of NO₃-N ranging from –1.5 to –3.6), probably because the rate-limiting step of the process consists of nitrate diffusion through the water-sediment interface. Riparian denitrification on the contrary, when it implies nitrate reduction during convective transfer through reducing environements, causes a much more significant isotopic enrichment of ¹⁵N of residual nitrate ( about –18). The authors report measurements of nitrogen isotopic composition of nitrate from rivers of various stream orders in the Seine river system under summer conditions. Anomalies in the data with respect to the values expected from the mixture of the various sources of nitrate are here attributed to riparian denitrification. However, the authors show that because of the patchy distribution of actively denitrifying riparian zones within the drainage network, the isotopic signature conferred to residual nitrate in river water intrinsically provides only a minimum estimate of the extent of denitrification.     

Microbial nitrate respiration--genes, enzymes and environmental distribution

Nitrate is a key node in the network of the assimilatory and respiratory nitrogen pathways. As one of the ‘fixed’ forms of nitrogen, nitrate plays an essential role in both nature and industry. For bacteria, it is both a nitrogen source and an electron acceptor. In agriculture and wastewater treatment, nitrate respiration by microorganisms is an important issue with respect to economics, greenhouse gas emission and public health. Several microbial processes compete for nitrate: denitrification, dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation. In this review we provide an up to date overview of the organisms, genes and enzymes involved in nitrate respiration. We also address the molecular detection of these processes in nature. We show that despite rapid progress in the experimental and genomic analyses of pure cultures, knowledge on the mechanism of nitrate reduction in natural ecosystems is still largely lacking.     

Nitrate,nitrate reduction and organic nitrogen in plants from different ecological and taxonomic groups of Central Europe

Summary 48 plant species of the families Asteraceae, Chenopodiaceae, Ericaceae, Fabaceae, Lamiaceae, Polygonaceae and Urticaceae were investigated in 14 natural habitats of Central Europe having different nitrate supplies, with respect to their nitrate content, nitrate reductase activity (NRA) and organic nitrogen content. Plants that were flowering were selected where possible for analysis. The plants were subdivided into flowers, laminae, petioles+shoot axes and below-ground organs. Each organ was analyzed separately. Differences among species were found for the three variables investigated. Apart from the Fabaceae, which had particularly high concentrations of organic N, these differences reflect mainly the ecological behaviour, i.e. high nitrate and organic N contents and NRA values per g dry weight were found in species on sites rich in nitrate, and vice versa. Nitrate content, NRA and organic N content were correlated with nitrogen figures of Central European vascular plants defined by Ellenberg (1979). By use of regression equations this correlation was tested with species from other systematic groups. Some species were attributed with calculated N figures for the first time.     

Assessing the sources and magnitude of diurnal nitrate variability in the San Joaquin River (California) with an in situ optical nitrate sensor and dual nitrate isotopes

1. We investigated diurnal nitrate (NO₃⁻) concentration variability in the San Joaquin River using an in situ optical NO₃⁻ sensor and discrete sampling during a 5‐day summer period characterized by high algal productivity. Dual NO₃⁻ isotopes (δ¹⁵N_NO3 and δ¹⁸O_NO3) and dissolved oxygen isotopes (δ¹⁸O_DO) were measured over 2 days to assess NO₃⁻ sources and biogeochemical controls over diurnal time‐scales. 2. Concerted temporal patterns of dissolved oxygen (DO) concentrations and δ¹⁸O_DO were consistent with photosynthesis, respiration and atmospheric O₂ exchange, providing evidence of diurnal biological processes independent of river discharge. 3. Surface water NO₃⁻ concentrations varied by up to 22% over a single diurnal cycle and up to 31% over the 5‐day study, but did not reveal concerted diurnal patterns at a frequency comparable to DO concentrations. The decoupling of δ¹⁵N_NO3 and δ¹⁸O_NO3 isotopes suggests that algal assimilation and denitrification are not major processes controlling diurnal NO₃⁻ variability in the San Joaquin River during the study. The lack of a clear explanation for NO₃⁻ variability likely reflects a combination of riverine biological processes and time‐varying physical transport of NO₃⁻ from upstream agricultural drains to the mainstem San Joaquin River. 4. The application of an in situ optical NO₃⁻ sensor along with discrete samples provides a view into the fine temporal structure of hydrochemical data and may allow for greater accuracy in pollution assessment.     

Risk assessment and source identification of coastal groundwater nitrate in northern China using dual nitrate isotopes combined with Bayesian mixing model

Due to the intensive and complicated human activities, the identification of nitrate pollution source of coastal aquifer is usually a challenge. This study firstly adopted stable isotope technique and stable isotope analysis in R (SIAR) model to identify the nitrate sources and contribution proportions of different sources in typical coastal groundwater of northern China. The results showed that about 91.5% of the groundwater samples illustrated significantly high nitrate concentrations exceeding the maximum WHO drinking water standard (50 mg/l), reflecting the high risk of groundwater nitrate pollution in the coastal area. A total of 57 sampling sites were classified into three groups according to hierarchical cluster analysis (HCA). The δ¹⁵N-NO₃^? and δ¹⁸O-NO₃^? values of groundwater samples from Group C (including nine samples) were much higher than those from Group A (including 40 samples) and Group B (including 8 samples). SIAR results showed that NH₄⁺ fertilizer was the dominant nitrate source for groundwater samples of Groups A and B while manure and sewage (M&;S) served as dominant source for Group C. This study provided essential information on the high risk and pollution sources of coastal groundwater nitrate of northern China.     

Detection of genes for membrane-bound nitrate reductase in nitrate-respiring bacteria and in community DNA

A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the narG gene encoding the membrane-bound nitrate reductase. This approach was used to amplify fragments of the narG gene from five Pseudomonas species previously shown to be able to express the membrane-bound nitrate reductase and from community DNA extracted from a freshwater sediment. Amino acid sequences encoded by the narG fragments were compared to one another, and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocols are specific for their intended targets. Sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.     

Energy sources for aquatic animals in the Orinoco River floodplain: evidence from stable isotopes

Summary Stable carbon and nitrogen isotope ratios in autotrophs, aquatic invertebrates and fishes from the Orinoco River floodplain of Venezuela reveal that microalgae, including both phytoplankton and epiphytic (attached) forms, are predominant energy sources for many aquatic animals, even though aquatic vascular plants are much more abundant. Floating mats of the grass Paspalum repens and the water hyacinth Eichhornia spp. harbor particularly high densities of aquatic animals, but isotopic evidence indicates that few species are dependent on organic carbon originating from these plants. The stable isotopic evidence for the trophic importance of algae contradicts traditional interpretations of food webs in freshwater wetlands, which are generally thought to be based largely on detritus originating from vascular plants.     

Nitrate removal in a first-order stream: reconciling laboratory and field measurements

A combination of laboratory and field experiments were carried out to evaluate nitrate(NO ₃ ^t- ) removal during stream transport in a first-order agricultural drainage stream. Intact stream sediment cores overlain with stream and NO ₃ ^– -amended stream water indicated NO ₃ ^– losses averaging 93 — 353 mg m^–2 day^–1, with NO ₃ ^– concentration exerting a primary control on loss rate. Isotopic data indicated enrichment of NO ₃ ^– - ¹⁵N over time as NO ₃ ^– concentrations decreased, indicating a denitrification loss. Field experiments were designed to evaluate dilution of streamwater with low-NO ₃ ^– groundwater in addition to other NO ₃ ^– removal processes during transport. A series of bromide tracer and NO ₃ ^– - addition experiments were carried out in the field; groundwater dilution dominated the downstream NO ₃ ^– concentration trends, accounting for all observed decreases in NO ₃ ^– concentration. Isotopic data did not point to denitrification downstream as a major NO ₃ ^– removal process. This apparent disparity between simulated laboratory and in-situ stream removal rates appears to be a function of the hydrological processes controlling exchanges between stream bottom sediments and the overlying water. These results suggest that caution must be exercised in extrapolating potentials for NO ₃ ^– removal measured in laboratory experiments to the field, as these rates could be overestimated in some watersheds.     

Oxygen increases the steady-state level of nitrate in denitrifying cells of, Paracoccus denitrificans

Abstract The levels of nitrate in denitrifying cells of Paracoccus denitrificans were determined by centrifugation through silicone oil into phosphoric acid and ion-exchange HPLC analysis of the cell lysates, using [¹⁴C]sucrose to correct for the trapped external medium. Introduction of oxygen brought about a significant upward shift in the intracellular nitrate concentration. This result calls into question the current thinking that oxygen blocks nitrate respiration primarily due to the inhibition of nitrate transport into the cell.     

Hexadecane mineralization and denitrification in two diesel fuel-contaminated soils

The effect of nitrate, ammonium and urea on the mineralization of [(14)C]hexadecane (C(16)H(34)) and on denitrification was evaluated in two soils contaminated with diesel fuel. In soil A, addition of N fertilizers did not stimulate or inhibit background hexadecane mineralization (4.3 mg C(16)H(34) kg(-1) day(-1)). In soil B, only NaNO(3) stimulated hexadecane mineralization (0.91 mg C(16)H(34) kg(-1) day(-1)) compared to soil not supplemented with any nitrogen nutrient (0.17 mg C(16)H(34) kg(-1) day(-1)). Hexadecane mineralization was not stimulated in this soil by NH(4)NO(3) (0.13 mg C(16)H(34) kg(-1) day(-1)), but the addition of NH(4)Cl or urea suppressed hexadecane mineralization (0.015 mg C(16)H(34) kg(-1) day(-1)). Addition of 2 kPa C(2)H(2) did not inhibit the mineralization process in either soil. Denitrification occurred in both soils studied when supplemented with NaNO(3) and NH(4)NO(3), but was not detected with other N sources. Denitrification started after a longer lag in soil A (10 days) than in soil B (4 days). In soil A microcosms supplemented with NaNO(3) or NH(4)NO(3), rates of denitrification were 20.6 and 13.6 mg NO(3)(-) kg(-1) day(-1), respectively, and in soil B, they were 18.5 and 12.5 mg NO(3)(-) kg(-1) day(-1), respectively. We conclude that denitrification may lead to a substantial loss of nitrate, making it unavailable to the mineralizing bacterial population. Nitrous oxide was an important end-product accounting for 30-100% of total denitrification. These results indicate the need for preliminary treatability studies before implementing full-scale treatment processes incorporating commercial fertilizers.     

Competition between hydrogen peroxide and nitrate for electrons from the respiratory chains of Thiosphaera pantotropha and Rhodobacter capsulatus

Abstract Thiosphaera pantotropha and some strains of Rhodobacter capsulatus express both a periplasmic nitrate reductase and cytochrome c peroxidase when grown under aerobic conditions. Harvested cell suspensions of either species can respire nitrate in the presence of 200 μM O₂ (∼ 80% air saturation), at 70–80% of the anaerobic rate. Addition of hydrogen peroxide to such cells causes a 90% inhibition of nitrate reduction under anaerobic or aerobic conditions. The duration of the inhibition is proportional to the concentration of hydrogen peroxide added and can be ascribed to the expression of periplasmic peroxidases that compete with the nitrate reductase for electrons from the respiratory chain. The results reveal a hitherto unrecognised interaction between reactions of denitrification and the reduction of hydrogen peroxide by a periplasmic peroxidase that may have implications for the denitrification in microaerobic environments. The creation of aerobic conditions in bacterial cultures by addition of hydrogen peroxide, and relying on the generation of oxygen by endogenous catalase activity, is a commonly used technique for studying respiratory processes. The observations presented here demonstrate that results derived from such experiments should be interpreted with caution.     

Rates, controls and potential adverse effects of nitrate removal in a denitrification bed

Denitrification beds are a simple approach for removing nitrate (NO₃⁻) from a range of point sources prior to discharge into receiving waters. These beds are large containers filled with woodchips that act as an energy source for microorganisms to convert NO₃⁻ to nitrogen (N) gases (N₂O, N₂) through denitrification. This study investigated the biological mechanism of NO₃⁻ removal, its controlling factors and its adverse effects in a large denitrification bed (176 m × 5 m × 1.5 m) receiving effluent with a high NO₃⁻ concentration (>100 g N m⁻³) from a hydroponic glasshouse (Karaka, Auckland, New Zealand). Samples of woodchips and water were collected from 12 sites along the bed every two months for one year, along with measurements of gas fluxes from the bed surface. Denitrifying enzyme activity (DEA), factors limiting denitrification (availability of carbon, dissolved organic carbon (DOC), dissolved oxygen (DO), temperature, pH, and concentrations of NO₃⁻, nitrite (NO₂⁻) and sulfide (S²⁻)), greenhouse gas (GHG) production - as nitrous oxide (N₂O), methane (CH₄), carbon dioxide (CO₂) - and carbon (C) loss were determined. NO₃⁻-N concentration declined along the bed with total NO₃⁻-N removal rates of 10.1 kg N d⁻¹ for the whole bed or 7.6 g N m⁻³ d⁻¹. NO₃⁻-N removal rates increased with temperature (Q₁₀ = 2.0). In laboratory incubations, denitrification was always limited by C availability rather than by NO₃⁻. DO levels were above 0.5 mg L⁻¹ at the inlet but did not limit NO₃⁻-N removal. pH increased steadily from about 6 to 7 along the length of the bed. Dissolved inorganic carbon (C-CO₂) increased in average about 27.8 mg L⁻¹, whereas DOC decreased slightly by about 0.2 mg L⁻¹ along the length of the bed. The bed surface emitted on average 78.58 μg m⁻² min⁻¹ N₂O-N (reflecting 1% of the removed NO₃⁻-N), 0.238 μg m⁻² min⁻¹ CH₄ and 12.6 mg m⁻² min⁻¹ CO₂. Dissolved N₂O-N increased along the length of the bed and the bed released on average 362 g dissolved N₂O-N per day coupled with N₂O emission at the surface about 4.3% of the removed NO₃⁻-N as N₂O. Mechanisms to reduce the production of this GHG need to be investigated if denitrification beds are commonly used. Dissolved CH₄ concentrations showed no trends along the length of the bed, ranging from 5.28 μg L⁻¹ to 34.24 μg L⁻¹. Sulfate (SO₄²⁻) concentrations declined along the length of the bed on three of six samplings; however, declines in SO₄²⁻ did not appear to be due to SO₄²⁻ reduction because S²⁻ concentrations were generally undetectable. Ammonium (NH₄⁺) (range: <0.0007 mg L⁻¹ to 2.12 mg L⁻¹) and NO₂⁻ concentrations (range: 0.0018 mg L⁻¹ to 0.95 mg L⁻¹) were always very low suggesting that anammox was an unlikely mechanism for NO₃⁻ removal in the bed. C longevity was calculated from surface emission rates of CO₂ and release of dissolved carbon (DC) and suggested that there would be ample C available to support denitrification for up to 39 years.This study showed that denitrification beds can be an efficient tool for reducing high NO₃⁻ concentrations in effluents but did produce some GHGs. Over the course of a year NO₃⁻ removal rates were always limited by C and temperature and not by NO₃⁻ or DO concentration.