Identification of Heterotrophic Nitrification in a Sierran Forest Soil

A potential for heterotrophic nitrification was identified in soil from a mature conifer forest and from a clear-cut site. Potential rates of NO₂⁻ production were determined separately from those of NO₃⁻ by using acetylene to block autotrophic NH₄⁺ oxidation and chlorate to block NO₂⁻ oxidation to NO₃⁻ in soil slurries. Rates of NO₂⁻ production were similar in soil from the forest and the clear-cut site and were strongly inhibited by acetylene. The rate of NO₃⁻ production was much greater than that of NO₂⁻ production, and NO₃⁻ production was not significantly affected by acetylene or chlorate. Nitrate production was partially inhibited by cycloheximide, but was not significantly reduced by streptomycin. Neither the addition of ammonium nor the addition of peptone stimulated NO₃⁻ production. ¹⁵N labeling of the NH₄⁺ pool demonstrated that NO₃⁻ was not coming from NH₄⁺. The potential for heterotrophic nitrification in these forest soils was greater than that for autotrophic nitrification.     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Simulated Nitrogen Deposition Reduces CH4 Uptake and Increases N2O Emission from a Subtropical Plantation Forest Soil in Southern China

To date, few studies are conducted to quantify the effects of reduced ammonium (NH₄ ⁺) and oxidized nitrate (NO₃ ⁻) on soil CH₄ uptake and N₂O emission in the subtropical forests. In this study, NH₄Cl and NaNO₃ fertilizers were applied at three rates: 0, 40 and 120 kg N ha⁻¹ yr⁻¹. Soil CH₄ and N₂O fluxes were determined twice a week using the static chamber technique and gas chromatography. Soil temperature and moisture were simultaneously measured. Soil dissolved N concentration in 0–20 cm depth was measured weekly to examine the regulation to soil CH₄ and N₂O fluxes. Our results showed that one year of N addition did not affect soil temperature, soil moisture, soil total dissolved N (TDN) and NH₄ ⁺-N concentrations, but high levels of applied NH₄Cl and NaNO₃ fertilizers significantly increased soil NO₃ ⁻-N concentration by 124% and 157%, respectively. Nitrogen addition tended to inhibit soil CH₄ uptake, but significantly promoted soil N₂O emission by 403% to 762%. Furthermore, NH₄ ⁺-N fertilizer application had a stronger inhibition to soil CH₄ uptake and a stronger promotion to soil N₂O emission than NO₃ ⁻-N application. Also, both soil CH₄ and N₂O fluxes were driven by soil temperature and moisture, but soil inorganic N availability was a key integrator of soil CH₄ uptake and N₂O emission. These results suggest that the subtropical plantation soil sensitively responses to atmospheric N deposition, and inorganic N rather than organic N is the regulator to soil CH₄ uptake and N₂O emission.     

Capacity for Denitrification and Reduction of Nitrate to Ammonia in a Coastal Marine Sediment

The capacity for dissimilatory reduction of NO₃⁻ to N₂ (N₂O) and NH₄⁺ was measured in ¹⁵NO₃⁻-amended marine sediment. Incubation with acetylene (7 × 10⁻³ atmospheres [normal]) caused accumulation of N₂O in the sediment. The rate of N₂O production equaled the rate of N₂ production in samples without acetylene. Complete inhibition of the reduction of N₂O to N₂ suggests that the “acetylene blockage technique” is applicable to assays for denitrification in marine sediments. The capacity for reduction of NO₃⁻ by denitrification decreased rapidly with depth in the sediment, whereas the capacity for reduction of NO₃⁻ to NH₄⁺ was significant also in deeper layers. The data suggested that the latter process may be equally as significant as denitrification in the turnover of NO₃⁻ in marine sediments.     

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Ammonium Removal by the Oxygen-Limited Autotrophic Nitrification-Denitrification System

The present lab-scale research reveals the potential of implementation of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with normal nitrifying sludge as the biocatalyst for the removal of nitrogen from nitrogen-rich wastewater in one step. In a sequential batch reactor, synthetic wastewater containing 1 g of NH₄⁺-N liter⁻¹ and minerals was treated. Oxygen supply to the reactor was double-controlled with a pH controller and a timer. At a volumetric loading rate (B_v) of 0.13 g of NH₄⁺-N liter⁻¹ day⁻¹, about 22% of the fed NH₄⁺-N was converted to NO₂⁻-N or NO₃⁻-N, 38% remained as NH₄⁺-N, and the other 40% was removed mainly as N₂. The specific removal rate of nitrogen was on the order of 50 mg of N liter⁻¹ day⁻¹, corresponding to 16 mg of N g of volatile suspended solids⁻¹ day⁻¹. The microorganisms which catalyzed the OLAND process are assumed to be normal nitrifiers dominated by ammonium oxidizers. The loss of nitrogen in the OLAND system is presumed to occur via the oxidation of NH₄⁺ to N₂ with NO₂⁻ as the electron acceptor. Hydroxylamine stimulated the removal of NH₄⁺ and NO₂⁻. Hydroxylamine oxidoreductase (HAO) or an HAO-related enzyme might be responsible for the loss of nitrogen.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO₃⁻-N liter⁻¹. Traces of ¹⁵NO₃⁻ were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day⁻¹. Both assimilatory and dissimilatory reduction rates were estimated from analyses of ¹⁵N₂, ¹⁵N₂O, ¹⁵NH₄⁺, and ¹⁵N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N₂ and N₂O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO₃⁻ and NH₄⁺ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-³H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g⁻¹ day⁻¹. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N₂, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO₃⁻ was reduced to NH₄⁺. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month.     

The contribution of nitrogen transformation processes to total N2O emissions from soils used for intensive vegetable cultivation

The rapid expansion of intensively farmed vegetable fields has substantially contributed to the total N₂O emissions from croplands in China. However, to date, the mechanisms underlying this phenomenon have not been completely understood. To quantify the contributions of autotrophic nitrification, heterotrophic nitrification, and denitrification to N₂O production from the intensive vegetable fields and to identify the affecting factors, a ¹⁵N tracing experiment was conducted using five soil samples collected from adjacent fields used for rice-wheat rotation system (WF), or for consecutive vegetable cultivation (VF) for 0.5 (VF1), 6 (VF2), 8 (VF3), and 10 (VF4) years. Soil was incubated under 50% water holding capacity (WHC) at 25°C for 96 h after being labeled with ¹⁵NH₄NO₃ or NH ₄ ¹⁵ NO₃. The average N₂O emission rate was 24.2 ng N?kg^?1 h^?1 in WF soil, but it ranged from 69.6 to 507 ng N?kg^?1 h^?1 in VF soils. Autotrophic nitrification, heterotrophic nitrification and denitrification accounted for 0.3–31.4%, 25.4–54.4% and 22.5–57.7% of the N₂O emissions, respectively. When vegetable soils were moderately acidified (pH, 6.2 to ?≥?5.7), the increased N₂O emissions resulted from the increase of both the gross autotrophic and heterotrophic nitrification rates and the N₂O product ratio of autotrophic nitrification. However, once severe acidification occurred (as in VF4, pH?≤?4.3) and salt stress increased, both autotrophic and heterotrophic nitrification rates were inhibited to levels similar to those of WF soil. The enhanced N₂O product ratios of heterotrophic nitrification (4.84‰), autotrophic nitrification (0.93‰) and denitrification processes were the most important factors explaining high N₂O emission in VF4 soil. Data from this study showed that various soil conditions (e.g., soil salinity and concentration of NO ₃ ^- or NH ₄ ⁺ ) could also significantly affect the sources and rates of N₂O emission.     

Anaerobic Ammonium Oxidation Measured in Sediments along the Thames Estuary, United Kingdom

Until recently, denitrification was thought to be the only significant pathway for N₂ formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of ¹⁵N-labeled NH₄⁺, NO₃⁻, and NO₂⁻ (and ¹⁴N analogues), production of ²⁹N₂ and ³⁰N₂ was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of ²⁹N₂ in the presence of ¹⁵NH₄⁺ and either ¹⁴NO₃⁻ or ¹⁴NO₂⁻ confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO₂⁻. Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO₂⁻ or indirect reduction, following the initial reduction of NO₃⁻. Whether NO₂⁻ was directly present at 800 μM or it accumulated at 3 to 20 μM (from the reduction of NO₃⁻), the rate of ²⁹N₂ formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO₂⁻. We observed a shift in the significance of anaerobic ammonium oxidation to N₂ formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.     

Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination

The oxidation of NH₄⁺ by Nitrosomonas europaea was insensitive to 10 mM NaClO₃ (sodium chlorate) but was strongly inhibited by NaClO₂ (sodium chlorite; K_i, 2 μM). The oxidation of NO₂⁻ by Nitrobacter winogradskyi was inhibited by both ClO₃⁻ and ClO₂⁻ (K_i for ClO₂⁻, 100 μM). N. winogradskyi reduced ClO₃⁻ to ClO₂⁻ under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO₂⁻ was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH₄⁺ and NO₂⁻ was inhibited in the presence of 10 mM ClO₃⁻ after a 2-h lag period, despite the fact that, under these conditions, ClO₂⁻ was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH₄⁺ oxidation is inhibited by ClO₂⁻ produced by reduction of ClO₃⁻ by the NO₂⁻ oxidizer. The use of ClO₃⁻ inhibition of NO₂⁻ oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH₄⁺ is not affected.     

Adaptation of Denitrifying Populations to Low Soil pH

Natural denitrification rates and activities of denitrifying enzymes were measured in an agricultural soil which had a 20-year past history of low pH (pH ca. 4) due to fertilization with acid-generating ammonium salts. The soil adjacent to this site had been limed and had a pH of ca. 6.0. Natural denitrification rates of these areas were of similar magnitude: 158 ng of N g⁻¹ of soil day⁻¹ for the acid soil and 390 ng of N g⁻¹ of soil day⁻¹ at the neutral site. Estimates of in situ denitrifying enzyme activity were higher in the neutral soil, but substantial enzyme activity was also detected in the acid soil. Rates of nitrous oxide reduction were very low, even when NO₃⁻ and NO₂⁻ were undetectable, and were ca. 400 times lower than the rates of N₂O production from NO₃⁻. Denitrification rates measured in slurries of the acid and neutral soil showed distinctly different pH optima (pH 3.9 and pH 6.3) which were near the pH values of the two soils. This suggests that an acid-tolerant denitrifying population had been selected during the 20-year period of low pH.     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N2O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N₂O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg⁻¹. Without added C, peak NO emissions of 4 μg of N kg⁻¹ h⁻¹ were increased to 15 μg of N kg⁻¹ h⁻¹ by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 μg of N kg⁻¹ h⁻¹ in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 μg of N kg⁻¹ h⁻¹ after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N₂O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N₂O produced during nitrification in soil.     

Production of N2O in soil during decomposition of dead yeast cells with different spatial distributions

Production and sources of N₂O were determined in soil columns amended with autoclaved yeast cells either mixed into or added as 0.5 cm³ lumps to the soil in combination with no or 200 g NO₃ ^--N g^-1. At four occasions over a two-week study period, subsets of cores were measured for N₂O production during 4-hour incubations under atmospheres of ambient air, 10 Pa of C₂H₂, and N₂, respectively. Denitrification enzyme activity (DEA) was assessed in subsamples of cores that had been incubated continuously under air.Autoclaved yeast provided a C-source readily available for denitrifying bacteria in the soil. Nitrous oxide production was negligible in unamended columns whereas accumulated N₂O losses in the presence of yeast material were substantial, varying between 15 to 49 ng N₂O-N g^-1 h^-1. Mixing yeast into the soil caused the highest production of N₂O followed by the yeast lump and no yeast treatments. Incubation in the presence of 10 Pa C₂H₂ indicated that denitrification was the sole source of N₂O, in accordance with an increase in DEA. Nitrous oxide production and DEA peaked after 4–7 days of incubation, and both were unaffected by additional NO₃ ^-. Two-to four-fold responses to anaerobiosis and accumulation of NO₃ ^- and NH₄ ⁺ in proximity of the lumps indicated that N₂O production here was limited by relatively low C-availability. In contrast, 10- to 12-fold responses to anaerobiosis and no accumulation of inorganic N suggested a higher C-availability where yeast was mixed into the soil.     

Functional Gene Differences in Soil Microbial Communities from Conventional,Low-Input,and Organic Farmlands

Various agriculture management practices may have distinct influences on soil microbial communities and their ecological functions. In this study, we utilized GeoChip, a high-throughput microarray-based technique containing approximately 28,000 probes for genes involved in nitrogen (N)/carbon (C)/sulfur (S)/phosphorus (P) cycles and other processes, to evaluate the potential functions of soil microbial communities under conventional (CT), low-input (LI), and organic (ORG) management systems at an agricultural research site in Michigan. Compared to CT, a high diversity of functional genes was observed in LI. The functional gene diversity in ORG did not differ significantly from that of either CT or LI. Abundances of genes encoding enzymes involved in C/N/P/S cycles were generally lower in CT than in LI or ORG, with the exceptions of genes in pathways for lignin degradation, methane generation/oxidation, and assimilatory N reduction, which all remained unchanged. Canonical correlation analysis showed that selected soil (bulk density, pH, cation exchange capacity, total C, C/N ratio, NO₃⁻, NH₄⁺, available phosphorus content, and available potassium content) and crop (seed and whole biomass) variables could explain 69.5% of the variation of soil microbial community composition. Also, significant correlations were observed between NO₃⁻ concentration and denitrification genes, NH₄⁺ concentration and ammonification genes, and N₂O flux and denitrification genes, indicating a close linkage between soil N availability or process and associated functional genes.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.     

Distribution of nitrous oxide and regulators of its production across a tropical rainforest catena in the Luquillo Experimental Forest, Puerto Rico

Understanding of N₂O fluxes to the atmosphere is complicated by interactions between chemical and physical controls on both production and movement of the gas. To better understand how N₂O production is controlled in the soil, we measured concentrations of N₂O and of the proximal controllers on its production in soil water and soil air in a field study in the Rio Icacos basin of the Luquillo Experimental Forest, Puerto Rico. A toposequence (ridge, slope-ridge break, slope, slope-riparian break, riparian, and streambank) was used that has been previously characterized for groundwater chemistry and surface N₂O fluxes. The proximal controls on N₂O production include NO₃ ^–, NH₄ ⁺, DOC, and O₂. Nitrous oxide and O₂ were measured in soil air and NO₃ ^–, NH₄ ⁺, and DO were measured in soil water. Nitrate and DOC disappeared from soil solution at the slope-riparian interface, where soil N₂O concentrations increased dramatically. Soil N₂O concentrations continued to increase through the flood plain and the streambank. Nitrous oxide concentrations were highest in soil air probes that had intermediate O₂ concentrations. Changes in N₂O concentrations in groundwater and soil air in different environments along the catena appear to be controlled by O₂ concentrations. In general, N processing in the unsaturated and saturated zones differs within each topographic position apparently due to differences in redox status.     

Mechanisms and kinetics of nitric and nitrous oxide production during nitrification in agricultural soil

Laboratory experiments were conducted with three California agricultural soils to examine substrate and process controls over temporal variability of NO and N₂O production during nitrification, and to quantify the kinetics of HNO₂‐mediated chemical reactions. Gross NO production rates were highly correlated (r² = 0.93–0.97) with calculated concentrations of HNO₂, which were shown to originate from autotrophic microbial oxidation of NH₄ ⁺ to NO₂ ^? Production of NO was not correlated with NH₄ ⁺ or NO₃^–, or with the overall nitrification rate. Distinct periods of high NO₂^– accumulation occurred below critical pH values in each soil, apparently due to inhibition of microbial NO₂^– oxidation. Data suggest that even during periods of relatively low NO₂^– accumulation and rapid overall nitrification, HNO₂‐mediated reactions may have been the primary source of NO. Rate coefficients (k_PNO) relating NO production to HNO₂ concentrations were determined for sterile (λ‐irradiated) soils, and were similar to k_PNO values in 2 of 3 nonsterile soils undergoing nitrification. Production of N₂O was correlated with HNO₂ (r² = 0.88–0.99) in sterile soils, and with NO₂^– and NO₃^– (R² = 0.72–0.91) in nonsterile soils. Experiments using ¹⁵N confirmed that dissimilatory NO₃^– reduction contributed to N₂O production even under primarily aerobic conditions. Sterile kPNO and kPN2O values were correlated (r² = 0.90 and 0.82) with soil organic matter content. Overall, the results demonstrate that both steps of the nitrification sequence, together with abiotic reactions involving NO₂^–/HNO₂ need to be considered in developing improved models of NO and N₂O emissions from soils.