INTRODUCTION

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The importance of denitrification in microbial ecology, N₂O production, agricultural N loss and wastewater treatment has prompted a large body of research over the last 25 years. Although the basic pathway is well known (36),

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the regulation and distribution of denitrification remain poorly understood. The sensitivity of denitrification to oxygen tension is of particular interest due to (i) the recent demonstration of denitrification under aerobic conditions (40, 41), (ii) increased N₂O production under these conditions (12, 20, 28), (iii) the importance of linked nitrification-denitrification in N cycling in natural environments (11, 14), and (iv) development of "single-sludge" wastewater treatment as a low-cost alternative to traditional strategies that employ separate aerobic and anoxic reactors (26, 34, 47).

The natural-abundance ¹⁵N ratios of nitrogenous materials have been used to identify or quantify denitrification activity in low-oxygen environments, including the marine water column (24, 53, 54, 55), groundwater (15), sediments (1), and soil (25). These studies exploit the variation in the ratio of ¹⁵N to ¹⁴N in nitrogenous material that results from the isotopic discrimination of denitrification, in which ¹⁴N reacts faster than ¹⁵N. Thus, natural-abundance ¹⁵N ratios provide a small (0.366% ¹⁵N) but endogenous in situ tracer of denitrification activity, in contrast to large ¹⁵N additions (50 to 99% ¹⁵N) traditionally used to trace biological N fixation and other N transformations. However, published estimates of the extent of isotopic discrimination, or isotope effect (), of denitrification range from 13 to 40, reflecting the variety of experimental methods and denitrifying cultures used (5, 6, 10, 13, 27, 50, 52). Because the  of denitrification lies between those of other N transformations, such as N assimilation at 10 (9, 19, 30) and nitrification at 13 to 16 in situ (21) or 30 to 60 in vitro (27, 52), it is desirable that  be better constrained. In addition, possible variation in  as a function of oxygen tension has not been investigated heretofore.

We measured  of denitrification in pure cultures of Paracoccus denitrificans under dissolved-oxygen concentrations between 0 and 1.2 µM. We also measured a unique  for N₂O reduction in these cultures. For these experiments, we developed a simple, steady-state reactor which does not require a dedicated mass spectrometer or online sample preparation system, thus offering a flexible approach to investigators who do not routinely use stable-isotope techniques. The reactor was also used to measure oxygen isotope effects, which are reported elsewhere (4). This information expands the utility of stable isotope studies of denitrification in low-oxygen (<10 µM) environments. Here we describe the necessary steady-state fractionation models and the reactor configuration and performance and report the N stable-isotope effects for denitrification.

	MATERIALS AND METHODS

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Experimental design. Continuous cultures were used to control the dissolved-oxygen concentration ([dO₂]) more easily and to exploit the simplicity of steady-state fractionation models, which relate kinetic  values directly to the isotopic compositions of reactants and products. The isotopic composition of nitrogenous material is commonly expressed as a -value relative to atmospheric N₂:

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where R = ¹⁵N/¹⁴N. In a single first-order reaction, in which the substrate pool is infinitely large,  closely approximates the difference between the  values of the substrate and product (16). The isotopic dynamics of steady-state anoxic denitrification may be idealized as such a one-step process:

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where ₀ is the overall  of denitrification. Alternatively, this pseudo- may be partitioned: 1, 12, 2 ¹⁵NO₃  ¹⁵N₂O  ¹⁵N₂ (4)

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1

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(8a)

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Reactor configuration. The reactor system consisted of a medium reservoir, growth chamber, waste carboy, and connecting tubing and flow controls (Fig. 1). The 20-liter Pyrex carboy in which media were sterilized also served as the reservoir. A heavy-gauge aluminum lid and rubber gasket were secured to the reservoir with a collar and screws. The lid contained ports for gas entry, gas and medium exit to the growth chamber, and venting. Gas mixing and flow to the reservoir were controlled by a gas proportioner (Alltech, Deerfield, Ill.). Gas was conducted to the reservoir in 1/8-in. stainless steel tubing, through a 0.5-µm nominal matrix filter (Nupro, Willoughby, Ohio) and a sparging stone. Medium flow from the reservoir to the growth chamber was caused by positive pressure in the reservoir, which was in turn controlled by the sparging rate. Two needle valves (Nupro) were added to enable finer control of medium flow rate to the growth chamber. This mode of medium delivery was chosen over pumping due to the difficulty of maintaining absolutely anoxic connections between steel tubing and peristaltic pump tubing.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Reactor configuration.

Organism and media. P. denitrificans ATCC 17741, a relatively oxygen-sensitive, classic denitrifier, was chosen for the experiments (2). Cultures were purchased from the American Type Culture Collection (Rockville, Md.) and reconstituted in nutrient broth (Difco, Detroit, Mich.) at 30°C. Subcultures were frozen in glycerol and stored at 20°C until needed. The defined medium for continuous-culture experiments contained 30 mM nitrate and 20 mM acetate, which was the sole electron donor, carbon source, and limiting substrate (39). The composition of denitrification medium was as follows (in grams per liter): KNO₃, 3.03; CH₃COONa · 3H₂O, 2.72; K₂HPO₄, 0.8; KH₂PO₄, 0.3; NH₄Cl, 0.4; MgSO₄ · 7H₂O, 0.4; trace-elements solution, 2 ml liter¹. The trace-elements solution was modified from that of Vishniac and Santer (48) and contained (in grams per liter) EDTA, 50.0; ZnSO₄, 2.2; CaCl₂, 5.5; MnCl₂ · 4H₂O, 5.06; FeSO₄ · 7H₂O, 5.0; (NH₄)₆Mo₇O₂₄ · 4H₂O, 1.1; CuSO₄ · 5H₂O, 1.57; CoCl₂ · 6H₂O, 1.61.

Reactor operation and sampling. Denitrification medium was autoclaved in 16-liter batches at 121°C and 15 lb/in² for 80 min. Beginning immediately after sterilization, the reservoir was sparged with ultra-high-purity helium or O₂ in helium (Med-Tech Gases, Medford, Mass.). The growth chamber, dO₂ probe, liquid-sampling needle, waste vessel, and tubing were autoclaved and connected while hot. The pH probe was calibrated with standard buffers (Fisher Scientific, Fair Lawn, N.J.), surface sterilized with 70% ethanol, and inserted into the growth chamber. The chamber was filled to working volume with medium, which was sampled with a syringe when cool. The culture was then inoculated with 30 ml of stationary-phase P. denitrificans and grown in batch mode at 30°C to approximately 10⁸ cells ml¹. Medium was then added at an appropriate dilution rate, and the pH was maintained at 8.0 by automatic addition of 1 M HCl. The [N₂O] of the headspace gas was monitored daily until it stabilized within a few ppm (by volume). At this point, the reactor was assumed to have reached steady state (see below).

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Cryogenic distillation. Gas samples were purified by standard cryogenic techniques (7). U-tubes were first immersed in LN₂ to freeze the N₂ or N₂O sample onto silica gel or glass beads, respectively. The large overburden of helium carrier gas was then removed with a vacuum pump. N₂ samples were further purified of CO₂ and H₂O by a LN₂-cooled trap; O₂ was removed by passing the sample over copper filings at 550°C. The purified N₂ was quantified by using a capacitance manometer (MKS Baratron). N₂O samples were further purified of H₂O by using a glass trap cooled in an ethanol-dry ice slurry. Each N₂ or N₂O sample was refrozen in a Pyrex ampoule, sealed, and stored until analysis by continuous-flow isotope ratio mass spectrometry.

Analytical methods. Nitrate [NO₃] and nitrite [NO₂] concentrations were measured by the spongy cadmium reduction method (22). The ammonia [NH₃ + NH_4⁺] concentration was determined by the colorimetric method of Strickland and Parsons (45). The ¹⁵N of (NO₃ + NO₂) was measured by the ammonia diffusion method as modified by Sigman et al. (43).

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Budget calculations. Dissimilatory (denitrification) and assimilatory N budgets were calculated for the reactor system. Each term in the dissimilatory budget was expressed as a percentage of the NO₃ supplied in the medium:

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The NO₃ and NO₂ terms were their respective steady-state concentrations (denoted by the subscript "ss") expressed as percentages of the initial NO₃ concentration. The N₂O term (%N₂O_ss) was the N₂O-N production rate expressed as a percentage of the NO₃ supply rate:

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where X_N2O is the mole fraction of N₂O in growth chamber headspace gas. The analogous N₂ term, %N_2ss, is shown below. The first factor in this equation was the amount of N contained in a 34-ml grab sample, which was measured manometrically during cryogenic distillation: (12)

	RESULTS

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Reactor performance. Dissimilatory N recovery from the reactor system was near 100% over a range of dilution rates (Fig. 2). Residual [NO₃] increased with increasing dilution rate, but the cultures were not in danger of washout at the rates tested (39). Dissimilatory N recovery varied as a function of the sparging rate (Fig. 3), with particularly high N₂ recovery at the lowest sparging rate, as discussed below. Assimilatory N recovery was 80 to 100% over the same range of dilution and sparging rates (data not shown). Dilution and sparging rates of 0.7 day¹ and 100 ml min¹, respectively, were held constant in further experiments, in which variable [dO₂] was the sole experimental treatment.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Dissimilatory N budget as a function of the dilution rate. Symbols are means and SE (n = 3). NO2 and N2O made up less than 1% of total N.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Dissimilatory N budget as a function of the sparge rate. Symbols are means and SE (n = 3).

Experimental results. Under anoxic steady-state conditions, the reactor system yielded ¹⁵NO₃, ¹⁵N₂O, and ¹⁵N₂ of 15.5 ± 0.3, 0.08 ± 3.1, and 11.0 ± 1.2, respectively (mean ± standard error [SE], n = 6). According to these data and equations, 5a and 5b, ₁ = 26.5 ± 1.2 and ₂ = 11.1 ± 3.1 under anoxic conditions. The independent estimates of ₁ calculated from equation 8b and ¹⁵NO₃ and ¹⁵N₂O from multiple steady states at different [dO₂] are similar although more variable (26.9 ± 2.6 and 24.3 ± 4.0, respectively). However, ₁ calculated from ¹⁵N₂ from multiple steady states was 14 (Fig. 4). This discrepancy and the high N₂ recovery at the low sparging rate (Fig. 3) suggested that significant isotopic signal from atmospheric N₂ had biased the ¹⁵N₂ values. To quantify this bias, the response of measured ¹⁵N₂ to systematic air contamination, or a "handling blank," was simulated (Fig. 5). The expected ¹⁵N₂ was first calculated by subtracting the slope of the NO₃ regression in Fig. 4 (i.e., ₁) from points on that regression. The resulting straight line represents the ¹⁵N₂ expected from constant fractionation and no air contamination. The expected ¹⁵N₂ for the highest [dO₂] treatment (the least biogenic N₂) was compared to the measured value, and the amount of atmospheric N₂ necessary to create the difference was calculated by mass balance. Simulation of constant fractionation with constant air contamination was then made by adding the isotopic contribution of this amount (0.5 µmol) of atmospheric N₂ to the constant-fractionation values. The resulting curve fits very closely with the measured ¹⁵N₂, suggesting that both ₁ and the amount of air contamination during sampling were constant for all [dO₂] treatments.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   15N as a function of NO3 consumption (f). Different fractions of f were caused by dO2 treatments. Symbols are means and SE (n = 3). The slope and SE of the slope of regressions of 15N versus f are 14.5 and 2.0 (N2), 24.3 and 4.0 (N2O), and 26.9 and 2.6 (NO3). The asterisk represents the 15N of the NO3 supplied (3.6).

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Comparison between measured 15N2 from dO2 experiments (solid triangles) and dilution rate experiments (open triangles), the expected 15N2 given constant fractionation (straight line), and the expected value with a constant amount of air contamination (curved line). See the text for details.

View this table: [in this window] [in a new window]   TABLE 1.   Isotope effects for different dO2 treatments, calculated from transformed 15N2

	DISCUSSION

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The value of ₁ reported here falls well within the range in the literature (Table 2). All estimates of biological fractionation are well below 90, the maximum theoretical fractionation of N---O bond rupture (50). The precision of ₁ as measured in our steady-state system compares favorably with that measured by batch culture experiments (5, 13), in which ₁ was calculated by using the classic Rayleigh equation, which is sensitive to error in f (46). The value and precision of ₁ are similar to those reported by Mariotti et al. (27), who measured f by the acetylene block technique.

The results reported here indicate that ₁ in P. denitrificans is constant over a range of dO₂ concentrations. This constancy supports the validity of using ₁ to quantify, identify, or rule out denitrification fluxes in environments containing [dO₂] gradients. However, other work indicates that biological kinetic fractionation can vary with environmental conditions. For sulfate reduction, Rees (38) hypothesized that the greater fractionation often measured in situ is due to slower growth in the field than in pure culture, but our dilution rate experiments indicated that the growth rate per se did not cause ₁ to vary (Fig. 5). Bryan et al. (8) showed that the overall  of denitrification does vary with the denitrification rate in whole cells and cell extracts of Pseudomonas stutzeri limited by [NO₂], increasing to a maximum value of 25 ± 3.2 at initial [NO₂] > 2.5 mM. The electron acceptor concentrations in our experiments were well within the asymptotic range reported by Bryan et al. (above 2.5 mM). These authors also found a negative correlation between  and denitrification rate when the rate was increased by higher electron donor concentrations.

The isotopic composition of N₂O in our experiments was quite distinct from both ¹⁵N₂ and ¹⁵NO₃. The combined effects of ₁ and ₂ resulted in ¹⁵N₂O being about 13 heavier and 15 lighter than ¹⁵N₂ and ¹⁵NO₃, respectively, at steady state. To our knowledge, this is the first report of an isotope effect for nitrous oxide reduction in a denitrifying system. Yoshida et al. (53) cited unpublished data which yielded a value of 27 for ₂ in P. denitrificans, but they did not specify whether the bacteria were supplied with N₂O, NO₂, or NO₃. Yamazaki et al. (51) reported a maximum  of 39 for N₂O reduction by the N₂ fixer Azotobacter vinelandii, but nitrogenase, not nitrous oxide reductase, appeared to be responsible for the observed activity.

The expression of isotopic fractionation by P. denitrificans was strongly influenced by [dO₂]. Within the narrow range between 0 and slightly more than 1.2 µM dO₂, the ¹⁵N of NO₃ and N₂O varied up to 26 and ¹⁵N₂ probably varied to an equal extent (Fig. 4). However, the usefulness of [dO₂] as a predictor of ¹⁵N in denitrifying environments may be limited to the extent to which it controls NO₃ consumption. The expression of  in natural and applied systems will also depend on the distribution of denitrifiers. P. denitrificans is very sensitive to dO₂ in comparison to some denitrifiers, such as Comamonas sp. (35) and Thiosphaera pantotropha (39), which under aerobic conditions maintain 40 and 25% of their anaerobic denitrifying activity, respectively. However, Pseudomonas fluorescens, which frequently dominates denitrifying environments, denitrifies over approximately the same range of [dO₂] as P. denitrificans (28). Chemostat studies of P. halodenitrificans revealed only slightly higher tolerance to dO₂, to ~2 µM (20).

It is hoped that ₁ and ₂ may be used to help distinguish between denitrification and nitrification as sources of N₂O and may serve as in situ tracers of both processes. For example, if the ¹⁵N of the substrate pools for both processes, NO₃ and NH₄⁺, respectively, are assumed to be zero, then denitrification- and nitrification-produced ¹⁵N₂O in open systems at steady state would be ca. 15 and 65, respectively (52). However, the isotopic composition of substrate pools in real systems could obscure this distinction. The ¹⁵N of NO₃ and NH₄⁺ vary from 23 to +43 and 20 to +50, respectively, depending upon the source and the combined fractionation effects of redox reactions in the environment (49). Given these ranges, it is possible that denitrification- and nitrification-produced ¹⁵N₂O would be indistinguishable. Linked nitrification-denitrification may also confuse ¹⁵N₂O signatures by increasing the range of potential substrate molecules, which may in turn may have variable ¹⁵N (56). In environments such as sediments and biofilms, spatial linkage between nitrification and denitrification is on the order of 1 mm or less, and in single microorganisms such as Thiosphaera pantotropha, which both denitrifies and nitrifies in aerobic environments, N₂O may be produced from NH₄⁺, NO₃, and NO₂ (3). However, in many environments (9, 19, 29, 31), the ¹⁵N of substrate pools can be constrained within a range of 10 (see, e.g., references 1, 32, and 42), and the isotopic composition of N₂O could provide a simple index to the relative contribution of the two processes producing N₂O.