Measurement of nitrification rates in lake sediments: Comparison of the nitrification inhibitors nitrapyrin and allylthiourea

A method for measuring rates of nitrification in intact marine sediment cores has been modified and adapted for use in freshwater sediments. The technique involves subsampling a sediment core into minicores. Half of these cores are treated with an inhibitor of chemolithotrophic nitrification and, after incubation, differences in ammonia and nitrate concentration between inhibited and uninhibited systems are calculated. The within-treatment variability of ammonia and nitrate concentrations could be reduced by storing the cores overnight prior to subsampling. Estimates of the nitrification rate using the difference in ammonia concentrations between the inhibited and uninhibited mini-cores were always greater than the rate estimate using the difference in nitrate concentrations. Comparison between the results using the nitrification inhibitors allylthiourea (ATU) and nitrapyrin (N-Serve) indicated that the former appeared to give larger values for the nitrification rate than did the latter. Differences in the efficiency of these inhibitors in the control of nitrification under the conditions used partly explain these results. Data are also presented on the effect of N-Serve and ATU on some other nitrogen transformations affecting ammonia and nitrate concentrations.     

Effects of N-serve (2-chloro-6-(trichloromethyl)pyridine) formulations on nitrification and on loss of nitrate in sand culture experiments

Summary N-serve (2-chloro-6-(trichloromethyl)pyridine) was tested as an inhibitor of nitrification of ammonium or urea in sand cultures. Nitrification was reduced but not prevented by N-Serve present at between 5 and 20 ppm in solution or by weight of sand. In the presence of root debris and acetone, used in some experiments at 2–4 ml/l of nutrient to convey N-Serve, denitrification was stimulated under the same conditions and resulted in loss of a large proportion of nitrate, probably mainly as gaseous products and some nitrite. These losses were greater when N-serve was also present. There was also conversion of nitrate to an insoluble form in the sand. A smaller proportional loss of nitrate occurred in other treatments in the presence of root debris when N-Serve was added without acetone, either as the commercial formulation 24E or as a solid. Thus, using N-Serve to inhibit nitrification may encourage denitrifying organisms especially in the presence of carbon sources including root debris or acetone. Large decreases of nitrate reductase activity in plants produced by using N-Serve in the presence of ammonium or urea were caused as much by losses of nitrate in the presence of acetone as by prevention of nitrate formation. Other N-Serve treatments (solid or 24E) decreased enzyme induction by between 50 and 90 per cent as a result mainly of reduced nitrification.     

Nitrification on a coral reef.

We report that the algal pavement just behind the reef crest at Enewetak Atoll produces nitrate at measurable rates. In situ and in vitro incubations with N-Serve indicate that the autotrophic pathway involving two separate organisms is effective in this oxidation of ammonia to nitrate. Significant nitrification is indicated throughout the reef environment; Nitrobacter agilis has specifically been identified as at least one of the organisms responsible for the terminal oxidation of nitrite to nitrate.     

Heterotrophic nitrification and aerobic denitrification inAlcaligenes faecalis strain TUD

Heterotrophic nitrification and aerobic and anaerobic denitrification byAlcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (K_I=109 M acetate). Denitrifying or assimilatory nitrate reductases were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized byA. faecalis strain TUD, with a maximum oxygen uptake rate of 140–170nmol O₂·min^-1·mg prot^-1. Comparison of the behaviour ofA. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.     

Nitrogen leaching losses from conventional and new nitrogenous fertilizers in low-land rice culture

Summary A pot-culture experiment was conducted to assess the leaching losses of N from the conventional and new nitrogen fertilizers under low-land rice culture. Leaching losses of N were generally less than 20% of applied N with sources other than sodium nitrate and these could be reduced by blending urea with nitrification inhibitor N-Serve or coating withneem cake or by using urea super granules or slow-release N fertilizer sulphur coated urea. These new nitrogen fertilizers were more effective than urea for rice.     

Experimental measurement of sediment nitrification and denitrification in Hamilton Harbour,Canada

This research examines the role of sediment nitrification and denitrification in the nitrogen cycle of Hamilton Harbour. The Harbour is subject to large ammonia and carbon loadings from a waste-water treatment plant and from steel industries. Spring ammonia concentrations rapidly decrease from 4.5 to 0.5 mg 1⁻¹, while spring nitrate concentrations increase from 1 to 2 mg l⁻¹, by mid-summer. A three-layer sediment model was developed. The first layer is aerobic; in it, oxidation of organics and nitrification occurs. The second layer is for denitrification, and the third layer is for anaerobic processes. Ammonia sources for nitrification include diffusion from the water column, sources associated with the oxidation of organics, sources from denitrification and from anaerobic processes. Diffusion of oxygen, ammonia and nitrate across the sediment-water interface occurs. Temperature effects are modelled using the Arrhenius concept. A combination of zero-order kinetics for nitrate or ammonia consumption with diffusion results in a half-order reaction, with respect to the water column loss rate to sediments. From experimental measurement, the rate of nitrification is 200 mg N 1⁻¹ sediment per day, while that of denitrification is 85 mg N 1–1 sediment per day at 20 °C. The Arrhenius activation energy is estimated as 15 000 cal/ mole-K and 17 000 cal/ mole-K for nitrification and denitrification, respectively, between 10 °C and 20 °C. Calculations of the flux of ammonia with the sediments, using the biofilm model, compare favourably with experimental observations. The ammonia flux from the water column is estimated to account for 20% of the observed decrease in water column stocks of ammonia, while the nitrate flux from the water column is estimated to account for 25% of the total nitrogen produced by the sediments.     

Some chemical and physical factors affecting the rate and dunamics of nitrification in urine-affected soil

The effects of urinary chloride and nitrogen concentration and osmotic pressure on the nitrification of ammonium in a calcareous soil treated with cow urine were examined. Urinary chloride concentrations of up to 7.4 g L^–1 had no effect on the rate of nitrification, as determined by the accumulation of soil nitrate. Osmotic stress, generated using a mixed salt solution, had an inhibitory effect on nitrification at soil osmotic pressures lower than or equal to –1.0 PMa. Nitrification was completely inhibited at a soil osmotic pressure of –2.6 MPa. Accumulation of nitrate after a lag phase of 18 days was noted in the –2.0 MPa soil osmotic pressure treatment, indicating some degree of adaptation or osmo-regulation within the nitrifying population at this stress level. High urine-N concentrations resulted in considerable nitrite accumulations and reduced nitrification activity through the effect of free ammonia. It is concluded that in most temperate grassland soils at near-neutral pH, urinary chloride and nitrogen are unlikely to reduce nitrification rates, except where urine-N concentrations exceed 16 g N L^–1. Inhibition due to osmotic stress will be directly related to soil moisture status and may be particularly severe in dry, light-textured soils.     

Nitrification and denitrification in a midwestern stream containing high nitrate: in situ assessment using tracers in dome-shaped incubation chambers

The extent to which in-stream processes alter or remove nutrient loads in agriculturally impacted streams is critically important to watershed function and the delivery of those loads to coastal waters. In this study, patch-scale rates of in-stream benthic processes were determined using large volume, open-bottom benthic incubation chambers in a nitrate-rich, first to third order stream draining an area dominated by tile-drained row-crop fields. The chambers were fitted with sampling/mixing ports, a volume compensation bladder, and porewater samplers. Incubations were conducted with added tracers (NaBr and either ¹⁵N[NO₃ ^?], ¹⁵N[NO₂ ^?], or ¹⁵N[NH₄ ⁺]) for 24–44 h intervals and reaction rates were determined from changes in concentrations and isotopic compositions of nitrate, nitrite, ammonium and nitrogen gas. Overall, nitrate loss rates (220–3,560 μmol N m^?2 h^?1) greatly exceeded corresponding denitrification rates (34–212 μmol N m^?2 h^?1) and both of these rates were correlated with nitrate concentrations (90–1,330 μM), which could be readily manipulated with addition experiments. Chamber estimates closely matched whole-stream rates of denitrification and nitrate loss using ¹⁵N. Chamber incubations with acetylene indicated that coupled nitrification/denitrification was not a major source of N₂ production at ambient nitrate concentrations (175 μM), but acetylene was not effective for assessing denitrification at higher nitrate concentrations (1,330 μM). Ammonium uptake rates greatly exceeded nitrification rates, which were relatively low even with added ammonium (3.5 μmol N m^?2 h^?1), though incubations with nitrite demonstrated that oxidation to nitrate exceeded reduction to nitrogen gas in the surface sediments by fivefold to tenfold. The chamber results confirmed earlier studies that denitrification was a substantial nitrate sink in this stream, but they also indicated that dissolved inorganic nitrogen (DIN) turnover rates greatly exceeded the rates of permanent nitrogen removal via denitrification.     

Soil and fertilizer nitrogen transformations under alternate flooding and drying moisture regimes

Summary A study of changes in NH₄ ⁺ and NO₃ ^––N in Maahas clay amended with (NH₄)₂SO₄ and subjected to 4 water regimes in the presence and absence of the nitrification inhibitor N-Serve (Nitrapyrin) showed that the mineral N was well conserved in the continoous regimes of 50% and 200% (soil weight basis) but suffered heavy losses due to nitrification-denitrification under alternate drying and flooding. N-Serve was effective in minimizing these losses.Another incubation study with 3 soils showed that after 10 cycles of flooding and drying (either at 60°C or 25°C), the ammonification of soil N was enhanced. Nitrification of soil as well as fertilizer NH₄ ⁺ was completely inhibited upto 4 weeks by the treatments involving drying at high temperature. Flooding and air drying at 25°C, on the other hand, enhanced ammonification of soil N but retarded nitrification. These treatments, however, enhanced both ammonification and nitrification of the applied NH₄ ⁺ fertilizer N. Under flooded conditions rate of NH₄ ⁺ production was faster in soils that were dried at 60°C or 25°C and then flooded as compared to air dried soils.It is concluded that N losses by nitrification-denitrification and related N transformations may be considerably altered by alternating moisture regimes. Flooding and drying treatments seem to retard nitrification of soil N but conserve that of fertilizer NH₄ ⁺ applied after these treatments.     

Nitrate and nitrite in interstitial waters of eelgrass beds in relation to the rhizosphere

The distribution of nitrate and nitrite in the interstitial water of the sediment of eelgrass (Zostera marina) bed of Izembek Lagoon, Alaska, were investigated. Their concentrations were relatively high (0 to 9.8 μg-at.N·1^?1, average 4.8 for nitrate; 0 to 4.0 μ-at.N·1^?1, average 1.9 for nitrite) although the sediments were anoxic and contained hydrogen sulphide. The rates of bacterial denitrification measured by ¹⁵N tracer technique ranged from 0.49×10^?10 to 1.2 × 10^?9 g-atN·g^?1·h^?1. When a steady state is maintained, the loss of nitrate and nitrite must be balanced by their production by bacterial nitrification. Experimentally determined rate of nitrification in the sediment was of the same order. A model experiment demonstrated that oxygen is transported from leaves to rhizomes and roots of eelgrass and released into the sediment. The oxygen is used for nitrification in the rhizosphere in anoxic sediments.     

Control of nitrification of fertilizer nitrogen: Effect of inhibitors,banding and nesting

Laboratory incubation and field experiments were conducted to evaluate thiourea, ATC (4-amino-1, 2, 4 triazole hydrochloride) and N-Serve 24 E (2-chloro-6-trichloromethyl-pyridine) as inhibitors of nitrification of fertilizer N. In the incubation experiment, most of the added aqueous NH₃ or urea was nitrified at 14 days on both soils, but addition of the inhibitors to fertilizer N decreased the conversion of NH₄−N to NO₃−N markedly. There was less nitrification for ATC and thiourea but not for N-Serve 24 E when the fertilizers and the inhibitors were placed at a point as opposed to when mixed into soil. After 28 days, ATC and N-Serve 24 E were more effective in inhibiting nitrification than thiourea. ATC and N-Serve 24 E also inhibited release of mineral N (NH₄−N+NO₃−N) from native soil N. In the uncropped field experiment, which received N fertilizers in the fall, nitrification of fall-applied N placed in the 15-cm bands was almost complete by early May in the Malmo soil, but not in the Breton soil. When ATC or thiourea had been applied with urea, nitrification of fall-applied N was depressed by May and the recovery of applied N as NH₄−N was greater with increasing band spacing to 60 cm or placing N fertilizer in nests (a method of application where urea prills were placed at a point in the soil in the center of 60×60 cm area). In late June, the percentage recovery of fall-applied N in soil as NH₄−N or mineral N increased with wide band spacing, or nest placement, or by adding ATC to fertilizer N on both soils. These results indicate that placing ammonium-based N fertilizers in widely-spaced bands or in nests with low rates of inhibitors slows nitrification enough to prevent much of the losses from fall-applied N. Scientific Paper No. 552, Lacombe Research Station, Research Branch, Agric, Can.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.     

Estimates of Denitrification and Nitrification in Coastal and Estuarine Sediments

Denitrification and nitrification in sediments of Tama Estuary and Odawa Bay, Japan, were investigated by the combined use of a continuous-flow sediment-water system and a ¹⁵N tracer technique. At Odawa Bay, the nitrification rate was comparable to the nitrate reduction rate, and 70% of the N₂ evolved originated from nitrogenous oxides (nitrate and nitrite) which were produced by the action of nitrifying bacteria in the sediments. At Tama Estuary, the nitrate reduction rate was 11 to 17 times higher than the nitrification rate, and nitrogenous oxides derived from ammonium accounted for only 6 to 9% of the N₂ evolution by denitrification.     

The use of mean pool abundances to interpret 15N tracer experiments

Details are presented of a simple mathematical framework that allows ¹⁵N tracer experiments to be interpreted in terms of the main processes of the soil/plant nitrogen cycle. The calculations, all of which can be performed on a scientific calculator, yield the rates of gross mineralization and nitrification and the crop nitrogen uptake occurring as ammonium and nitrate. Two procedures are presented. One requires paired experiments with labelled ammonium and unlabelled nitrate as one treatment, and unlabelled ammonium and labelled nitrate as the other. The second procedure requires only the labelled ammonium, unlabelled nitrate treatment. Example calculations are presented using actual experimental data. The interpretative procedure uses the fact that the rate of isotopic dilution in an ammonium pool labelled with ¹⁵N is a function of the rate at which unlabelled ammonium is introduced into the pool via mineralization. Similarly, the rate of isotope dilution in an ¹⁵N labelled nitrate pool is a function of the rate at which unlabelled nitrate is introduced into the pool via nitrification.     

Quantitative traits associated with adaptation of three barley (Hordeum vulgare L) cultivars to suboptimal iron supply

A laboratory method was developed that allows determination of in situ net nitrification with high sensitivity and at high temporal resolution. Nitrate in soils is quantitatively converted into nitrous oxide under strictly anaerobic conditions in the presence of 10 kPa acetylene by the soil endogenous denitrifier population, with the N₂O detected by a gas chromatograph equipped with a ⁶³Ni electron capture detector. Thus, even low net nitrification rates, i.e. small net increases in soil nitrate concentrations can easily be detected. Comparison of results using this method with results obtained using the classical in situ incubation method (buried bag soil incubation) revealed excellent agreement. Application of the new method allowed both determination of the seasonal pattern of net nitrification as well as correlation analysis between in situ NO and N₂O flux rates and in situ net nitrification rates of the forest soils studied. Regardless of the forest site studied (spruce, spruce limed, beech), and during each year of a 3 years period (1995–1997), net nitrification varied strongly with season and was least during winter and greatest during summer. The long-term annual, mean rate of net nitrification for the untreated spruce site, the limed spruce site and the beech site were 1.54 ± 0.27 mg N kg^–1 sdw d^–1, 1.92 ± 0.23 mg N kg^–1 sdw d^–1 and 1.31 ± 0.23 mg N kg^–1 sdw d^–1, respectively. In situ rates of nitrification and NO and N₂O emission were strongly correlated for all sites suggesting that nitrification was the dominate source of NO as well as N₂O.     

Measurement of in situ rates of nitrification in sediment

A method has been developed for the measurement of nitrification rates in intact sediment cores without disturbing the concentration gradients of oxygen and ammonium. N-serve (2-chloro-6-trichloromethyl-pyridine), a specific inhibitor of the autotrophic ammonium oxidation, was injected into a 0–2 cm surface layer of the sediment (20 ppm) and added to the water column of sediment cores (5 ppm). N-serve in these concentrations was sufficient to inhibit nitrification, but did not change the rate of ammonium production or incorporation in sediment suspensions, which were incubated aerobically and anaerobically. The ammonium accumulation in cores injected with N-serve was thus equal to the amount of ammonium which was oxidized to nitrate in the control cores. Nitrification rates were in the range of 0–3 mmol N m^{–2 –1}     

Assessing Nitrification and Denitrification in the Seine River and Estuary Using Chemical and Isotopic Techniques

Downstream from metropolitan Paris (France), a large amount of ammonium is discharged into the Seine River by the effluents of the wastewater treatment plant at Achères. To assess the extent of nitrification and denitrification in the water column, concentrations and isotopic compositions of ammonium (δ¹⁵N–NH₄⁺) and nitrate (δ¹⁵N–NO₃⁻, δ¹⁸O–NO₃⁻) were measured during summer low-flow conditions along the lower Seine and its estuary. The results indicated that most of the ammonium released from the wastewater treatment plant is nitrified in the lower Seine River and its upper estuary, but there was no evidence for water-column denitrification. In the lower part of the estuary, however, concentration and isotopic data for nitrate were not consistent with simple mixing between riverine and marine nitrate. A significant departure of the nitrate isotopic composition from what would be expected from simple mixing of freshwater and marine nitrates suggested coupled nitrification and denitrification in the water, in spite of the apparent conservative behavior of nitrate. Denitrification rates of approximately 0.02 mg N/L/h were estimated for this part of the estuary.     

Assessment of Nitrification Potential in Ground Water Using Short Term, Single-Well Injection Experiments

Nitrification was measured within a sand and gravel aquifer on Cape Cod, MA, using a series of single-well injection tests. The aquifer contained a wastewater-derived contaminant plume, the core of which was anoxic and contained ammonium. The study was conducted near the downgradient end of the ammonium zone, which was characterized by inversely trending vertical gradients of oxygen (270 to 0 μM) and ammonium (19 to 625 μM) and appeared to be a potentially active zone for nitrification. The tests were conducted by injecting a tracer solution (ambient ground water + added constituents) into selected locations within the gradients using multilevel samplers. After injection, the tracers moved by natural ground water flow and were sampled with time from the injection port. Rates of nitrification were determined from changes in nitrate and nitrite concentration relative to bromide. Initial tests were conducted with ¹⁵N-enriched ammonium; subsequent tests examined the effect of adding ammonium, nitrite, or oxygen above background concentrations and of adding difluoromethane, a nitrification inhibitor. In situ net nitrate production exceeded net nitrite production by 3- to 6- fold and production rates of both decreased in the presence of difluoromethane. Nitrification rates were 0.02–0.28 μmol (L aquifer)⁻¹ h⁻¹ with in situ oxygen concentrations and up to 0.81 μmol (L aquifer)⁻¹ h⁻¹ with non-limiting substrate concentrations. Geochemical considerations indicate that the rates derived from single-well injection tests yielded overestimates of in situ rates, possibly because the injections promoted small-scale mixing within a transport-limited reaction zone. Nonetheless, these tests were useful for characterizing ground water nitrification in situ and for comparing potential rates of activity when the tracer cloud included non-limiting ammonium and oxygen concentrations.     

Patterns of hydrological exchange and nutrient transformation in the hyporheic zone of a gravel-bottom stream: examining terrestrial—aquatic linkages

• 1 The terrestrial-aquatic interface beneath a riparian corridor was investigated as a region of hydrological and biological control of nutrient flux. Subsurface flow paths were defined from the channel toward the riparian zone and also from the riparian zone toward the channel using tracer-injection studies. Solute transport had a rapid channel component (m min^?1) and a slow hyporheic flow component (mh^?1, m day^?1). Subsurface flow beneath the riparian zone approximated a straight path entering at meanders but could also cross beneath the stream, possibly using relic channels.
• 2 Dissolved oxygen (DO) concentration in the hyporheic zone ranged from <1.0 to 9.5mgl^?1 due to permeability variations in bankside sediments. DO concentration was related to the proportion of stream water in the lateral hyporheic zone, indicating that the channel water was the DO source.
• 3 The magnitude and riming of lateral water exchange was linked to previously published studies of nitrification and denitrificarion. Both nitrification potential and channel exchange decreased with distance from the channel and were absent at sites lacking effective exchange, due to low DO. Field amendment of ammonium to an aerobic flow path indicated nitrification potential under natural hydrological conditions. Denitrification potential was inversely related to channel exchange and was insignificant in channel sediments. Field amendment of acetylene plus nitrate to a flow path with low DO and minimal channel exchange indicated denitrificarion of amended nitrate.
• 4 Comparison of hydraulic head to distribution of the biologically important solutes DO, ammonium, and nitrate was useful for interpreting previous findings and conceptualizing the riparian zone as a functioning ecotone between terrestrial and aquatic systems.

     

The fate of labelled15N urea and ammonium nitrate applied to a winter wheat crop

Labelled urea or ammonium nitrate was applied to winter wheat growing on a loamy soil in Northern France. Two applications of fertilizer were given: 50 kg N ha^–1 at tillering (early March) and 110 kg N ha^–1 at the beginning of stem elongation (mid-April). The kinetics of urea hydrolysis, nitrification of ammonium and the disappearance of inorganic nitrogen were followed at frequent intervals. Inorganic nitrogen soon disappeared, mainly immobilized by soil microflora and absorbed by the crop. Net immobilization of fertilizer N occured at a very similar rate for urea and ammonium nitrate. Maximum immobilization (16 kg N ha¹) was found at harvest for the first dressing and at anthesis for the second dressing (23 kg N ha¹). During the nitrification period, the labelled ammonium pool was immobilized two to three times faster than the labelled nitrate pool. No significant net¹⁵N remineralization was found during the growth cycle.The actual denitrification and volatilization losses were probably more important than indicated from calculations made by extrapolation of fluxes measured over short intervals. However microbial immobilization was the most important of the processes which compete with plant uptake for nitrogen.