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Department of Microbial Ecology, Aarhus University, Bd. 540, Ny Munkegade, DK-8000 Aarhus, Denmark
* Corresponding author (biorlm{at}biology.au.dk)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were measured. We observed an initial depletion of NO-x originally present in the soil, followed by an increase after 
1 wk caused by the activity of nitrifying bacteria. Fluxes of NO-x from the soil into the manure were calculated from NO-x profiles and compared with measurements of N2 production. Generally, NO-x fluxes and N2 production were concordant. Denitrification (NO-x consumption) was closely coupled to nitrification until Day 18, when NO-x accumulated in the anoxic zone and therefore no longer seemed to limit denitrification. Maximum denitrification rates were measured at Day 9/10 and reached 732 and 497 nmol N m-2 h-1 when measured as NO-x flux and N2 production, respectively. An experiment was set up to investigate the temporal change in NO-x profiles when nitrification was inhibited by acetylene. Profiles of NO-x were measured at 0, 2, 4, 8, 16, 24, and 48 h of incubation with 1% acetylene. Calculated NO-x fluxes were 39% of the original rates after 2 h and only 2% after 48 h. Thus, this experiment stresses the importance of very short incubation time when using the acetylene inhibition technique to measure denitrification rates.
Abbreviations: De, effective diffusion coefficient • Ds, diffusion coeeficient of the compound in a particular substrate • DW, dry weight • ESC, electrophoretic sensitivity control • NO-x, nitrate plus nitrite • PVC, polyvinyl Cl • SE, standard error
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aim of this study was to investigate the temporal variation of nitrification and denitrification associated with a manure hot-spot, using a microscale biosensor for NO-x. The biosensor makes it possible to measure the spatial distribution of NO-x in a soil with an unprecedented spatial resolution. Rates of denitrification calculated from NO-x profiles were compared with rates of N2 production. Furthermore, NO-x profiles were measured after inhibition of nitrification with acetylene to illustrate the possible underestimation of denitrification by the acetylene inhibition technique.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experimental set-up consisted of 125-mL cylindrical polyvinyl Cl (PVC) chambers (i.d. 4.47 cm) containing 10 mL of homogenized manure as an 0.3-cm thick layer in the bottom, and 11 g of soil on top of that (a layer of 0.6 cm). Unlike previous studies (Petersen et al., 1991; Petersen et al., 1992; Nielsen and Revsbech, 1994; Nielsen et al., 1996), the manure in this experiment did not contain soil and was not stabilized with silica gel. The layer of soil and manure formed a system with an oxic–anoxic interface between the aerated soil and the water-saturated manure.
Two sets of chambers were prepared: one for microsensor measurements and one for measurement of N2 production. The chambers for microsensor analysis had detachable lids so that the sample could be accessed with the sensors. The chambers were incubated at room temperature (20°C) in the dark, and during incubation the lid was closed to avoid loss of water from the soil. However, the headspace was exchanged daily with water-saturated atmospheric air to ensure a sufficient supply of O2. The chambers for measurement of N2 production were sealed with PVC lids containing two septers for injection and collection of gas samples. Measurements were performed at a 24- to 48-h interval during a period of 18 d.
Chemical Analysis
Soil samples were taken from the investigated cylinders during the experiment and analyzed for water content and the concentration of NO-3 and NO-2 to validate the biosensor measurements. The soil was collected by carefully scraping off 1 to 2 g of soil from a small area. The area of the chamber where the soil was collected was not reused for microsensor measurements. Nitrate and nitrite was extracted with demineralized water and subsequently analysed by HPLC (anion separation column LCA A14, SYKAM, Gliching, Germany) with 40 mmol L-1 NaCl as eluent. Peaks were determined at 220 nm. The water content was determined by drying the soil sample at 105°C for 24 h, and measuring the dry weight after the sampled had cooled down in a dry atmosphere.
The initial concentrations of NH+4 in the soil and in the manure were determined by extraction in 1 M KCl and subsequent analysis by the salicylate-hypochlorite method described by Bower and Hansen (1980).
The pH was determined with a pH microsensor (pH sensitive tip 50 µm in diam. and 300 µm long) constructed as described by Revsbech and Jørgensen (1986). Profiles of pH were measured at Day 1, 7, and 12.
Dinitrogen Production
For N2 production measurements, chambers were incubated at room temperature. Prior to incubation, the headspace was flushed with a mixture of 80% He and 20% O2, which was humidified by conducting the gas through water. Gas samples were collected through a septum for every 24 h by injection of 500 µL He, followed by a 5 min mixing time and extraction of a 500-µL gas sample. The gas samples were analyzed for O2 and N2 on a gas chromatograph (Molsieve 5A, bead diam. 150–200 µm [80/100 mesh], column diam. 0.635 cm, column length 1.5 m) equipped with TC detector and operated at 200 mA, 25 °C, and with He carrier gas. After sampling, the headspace was flushed with the humidified He and O2 mixture. Prior to the experiment, the system was checked for errors because of N2 contamination from the atmosphere. The production of N2 was each day determined in three different cylinders and calculated as nanomoles N per squared centimeters per hour.
Microsensors
Oxygen microprofiles were measured with a Clark type O2 microsensor (Revsbech, 1989a) (tip size equals 54 µm, 90% response time = 1s and <1% stirring sensitivity). The response of the sensor was linear, and the sensor could therefore be two-point calibrated in anoxic and air saturated water.
The NO-x biosensor (tip size = 70 µm, 90% response time = 45 s, and <1% stirring sensitivity) was constructed as described by Larsen et al. (1997), and calibrated by plotting the current of the sensor against various nitrate concentrations within a relevant range. The principle of this sensor is as follows: A pure culture of denitrifying bacteria deficient of N2O reductase are immobilized in front of an electrochemical N2O sensor. The NO-x diffuses into the mass of immobilized bacteria through a semipermeable membrane in the biosensor tip and is converted to N2O by bacterial respiratory activity. The bacteria are able to reduce both nitrite and nitrate with equal efficiency. The signal of the sensor can be enhanced more than 10 times by electrophoretically mediated transport of NO-x ions into the sensor (Kjær et al., 1999). This method for increasing the sensitivity of the sensor is referred to as electrophoretic sensitivity control (ESC). By varying the electric attraction (positive tip potential) or repulsion (negative tip potential) of anions, it is possible to tailor the sensitivity and linear range of the sensor to the extremes of NO-x concentrations encountered.
The biosensor is susceptible to interference from N2O and we therefore measured profiles of N2O at Day 1, 7, and 12, but N2O could not be detected. The N2O sensor (tip size 50 µm, 90% response time of 10 s, <1% stirring sensitivity) was identical to the N2O transducer in the NO-x biosensor, except that it was equipped with an oxygen guard as previously applied in a methane sensor (Damgaard et al., 1998), but using alkaline ascorbate as a reductant. This sensor was two-point calibrated in demineralized water with no N2O and with 280 µmol L-1 N2O. The detection limit was 1 µL N2O L-1.
Profile Measurements
The experimental set-up for microsensor measurements was the same as for N2 production, except that the chambers were flushed with humidified atmospheric air instead of a He and O2 mixture. The O2 and NOx sensors were mounted on the same micromanipulator with their tips in a fixed relative position. The profiles could thereby be measured along the same vertical profile with the tip of the O2 sensor placed 0.5 mm in front of the NO-x sensor. The O2 sensor was somewhat smaller than the NO-x sensor, and it was therefore assumed that it would not cause significant physical disturbance and thereby affect the NO-x profile. The sensors were operated by a computer-controlled micromanipulator and the currents in the measuring circuits were digitized and stored in the computer. Three to four sets of profiles were measured at random sites in two different chambers at 1- to 2-d intervals. For each set of profile measurements, a soil sample was taken to determine the water content of the soil.
Acetylene Inhibition
Nitrification was inhibited in three chambers by constant flushing with 1
acetylene (Klemedtsson et al., 1988) in humidified air. Oxygen and NO-x profiles were measured after 0, 2, 4, 8, 16, 24, and 48 h of incubation. The incubation with acetylene was performed at Day 9 of the experiment, at which time denitrification rates reached maximum.
Profile Interpretation and Calculations
The flux of NO-x from the soil to the manure was calculated from the NO-x profiles using Fick's first law of diffusion (Crank, 1983):
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is the water-filled porosity, and C(x) is the concentration of the compound at depth x.
The 
C(x)/x at the soil–manure interface was determined as the inclination of the NO-x concentration profile at the position where O2 decreased to values significantly below atmospheric saturation. This position must represent the depth of the interface between the aerated and the water-saturated part of the system. Liquid from the manure will inevitably penetrate into the soil phase creating a narrow zone of water-saturated soil above the manure phase. The flux of NO-x observed in the upper 1 mm of the water-saturated zone is therefore assumed to take place in soil saturated with liquid from the manure. The diffusion coefficient used for calculation of the NO-x flux was therefore that of water-saturated soil (see below).
The effective diffusion coefficient, De (equal to Ds), was determined for water-saturated soil by the method described in Revsbech (1989b). The principle of this method is creation of a flux of gas (in this case N2O) through a two-layer sandwich composed of 1.5% agar and the substrate for which De is to be determined. This is done by placing the sandwich on a silicone membrane sealing a glass chamber flushed with water saturated N2O. After some hours, the agar-soil sandwich will be in equilibrium with N2O on one side and atmospheric air on the other, and there will be a constant flux of N2O through all layers. Difference in diffusive property (De) of N2O in the agar and in the water-saturated soil results in a different C(x)/x, as the flux is identical in the two substrates. By measurement of a N2O-concentration profile through the soil-agar layer, C(x)/x of agar and soil can be calculated as the slope of the profile and De is then calculated from Eq. [1].
The soil was autoclaved before the experiment to ensure that no biological activity was present. It was assumed that De for NO-3, O2, and N2O in soil were subject to the same relative change as compared with diffusion in water. The De in soil was determined to be 0.29 x De in agar. The value for De of NO-3 in agar (insignificantly different from water) at 20°C was determined to be 1.69 x 10-5 cm2 s-1 as described by Li and Gregory (1974), and the diffusion coefficient used for flux calculations was therefore 0.49 x 10-5 cm2 s-1.
Determination C(x)/x in the anoxic, water-saturated phase right above the manure results in a minor underestimation of the flux, as NO-x is consumed within the interval where the gradient is determined, and the mean inclination of the NO-x profile in that interval is therefore somewhat smaller than C(x)/x in the beginning of the interval. However, it was not possible for us to estimate Ds of ionic species in aerated soil with a reasonable accuracy. Available sensors for diffusivity that can be used in a soil environment are based on diffusion of gases (Revsbech et al., 1998), but in this case we want to measure Ds of an ion diffusing only in the water of the soil.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NH+4 concentration in the manure was determined at the beginning of the experiment to be 10 µmol NH+4 g-1 manure. This is equivalent to a concentration of 12 mmol L-1 NH+4 in the liquid fraction of the manure. The initial concentration of NO-x was 103 µmol L-1 in the liquid fraction.
Initially, pH was 6 in the soil and 5.6 in the manure. During the experiment, pH gradually rose to about 8.5 in the soil and 7.9 in the manure at Day 12. At that time, pH in the soil was almost one unit higher than in the manure.
Evaluation of the Nitrate and Nitrite Sensor Performance
This was the first time the NO-x biosensor was used in a soil system, and it was therefore necessary to perform the calibration of the sensor in soil and compare the response with that in water. The soil was washed in water (demineralized water or a 1:1 mixture of demineralized and tap water) to remove NO-x originally present in the soil. After drying the soil at 100 °C, demineralized water containing a known concentration of KNO3 was added. The sensor current was plotted against the water content of three soil samples, containing the same nitrate concentrations but with water contents of 0.1, 0.2, and 0.3 g H2O g-1 DW soil, corresponding to 60, 120, and 180% of field capacity. This was done to determine if variations in humidity of the soil during the experiment would affect the sensor calibration. We found only a 3.5% variation from the mean (data not shown). The water content of the soil during the incubation with manure increased dramatically from 0.11 to 0.34 mL g-1 DW soil (corresponding to 65 and 200% of field capacity) within the first 24 h, but hereafter it did not vary much (Fig. 1)
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The accuracy of the NO-x sensor decreased when very high NO-x concentrations were measured. From the NO-x profiles around Day 10 when the maximum NO-x concentration reached 20 mmol L-1, it appears that the NO-x concentration in the manure did not reach zero. This is considered to be caused by inaccuracy of the sensor rather than an actual accumulation of NO-x in the manure. Exposure of the sensor to such high NO-x concentrations temporarily affected the zero-reading of the sensor.
Microsensor Profiles
Oxygen profiles showed that the soil phase was air saturated at all times. Because of soil aggregates creating metabolic or diffusional heterogeneity, there was some scattering of the datapoints of the concentration profiles in the soil. At 4- to 6-mm depth, the O2 concentration decreased dramatically and reached zero within <1 mm.
The initial NO-x concentration in the soil measured both by HPLC and by the NO-x sensor was 18 mmol L-1 (water content = 65% of field capacity). This pool of NO-x was virtually consumed within the first 2 d of the experiment, and after 4 d (Fig. 3) the maximum NO-x concentration was only about 40 µmol L-1. It should be noticed that NO-x accumulated to a very high concentration throughout in the manure after 24 h of incubation (Day 1 in Fig. 3), whereas the penetration was <1 mm from Day 4 through Day 10. A new build-up of NO-x in the soil was observed from Day 6 (Fig. 4) . The total amount of NO-x reached a maximum at Day 10, where the concentration of NO-x in the uppermost layer of the soil reached 23 mmol L-1 nitrate. Three days later, the level had decreased again to maximum concentrations of 2 mmol L-1, where it remained for the rest of the experiment. Figure 3 shows selected profiles of O2 and NO-x from the period of time when the initial pool of NO-x was consumed and the following build-up of NO-x took place. Note the different scales for NO-x concentration. To illustrate this further, Fig. 4 shows averaged NO-x profiles from each day of the period when NO-x was building up (Day 2–10) and subsequently being consumed (Day 10–15). There was a great deal of variation between individual profiles because of heterogeneity in diffusive characteristics and metabolic activity in the soil. Therefore, three to four sets of profiles were measured each day. An example of this variation, Fig. 5 shows four profiles from Day 8.
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Nitrate Plus Nitrite Fluxes and Dinitrogen Emission
Fluxes of NO-x into the manure were calculated as nanomoles of N per squared centimeters per hour from the profiles and averaged for each experimental 24-h period. These show a very low rate at Day 1 (5341 nmol N cm-2 h-1). At this time, of measuring, 24 h after setting up the experiment, most of the NO-x originally present in the soil was already consumed (maximum concentrations were 2 mmol L-1, but at the same time the soil water content had increased to more than twice the initial value). The following 3 d, the flux approached zero because of depletion of NO-x, but from Day 6 increases in NO-x concentration also led to increases in NO-x flux. Maximum rates (684–732 nmol N cm-2 h-1) were obtained at Day 9 and 10 which was also the time when the NO-x pool was at its highest level. At Day 13 the NO-x consumption had decreased to 80 nmol N cm-2 h-1, at which level it remained for the rest for the experiment.
There was generally good accordance between the rates of N2 production and the calculated flux of NO-x into the manure (Fig. 6) , except at Day 9 and 10 when the maximum rates were reached. At this time NO-x fluxes were up to 50% higher than the N2 production. However, the standard deviations of the flux measurements reveal a large variation in the flux calculations at this time of the experiment.
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In some of the profiles, there seems to be a decrease in NO-x concentration at the very top of the profile (see also Fig. 4), where the sensor makes contact with the soil surface. This apparent local NO-x minimum can presumably be ascribed to incomplete contact between the sensor and the soil pore water.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The curvature of the NO-x profiles from Day 6 and onwards indicated a wide zone of nitrification activity (Fig. 4). It is not very surprising that nitrification took place throughout the full 5- to 6-mm soil phase, as Petersen et al. (1996) measured NH+4 to penetrate up to 10 mm into the soil phase in a similar system.
Most O2 profiles show a change from atmospheric saturation to complete anoxia in just one 0.5-mm step and simultaneously measured NO-x profiles show depletion of NO-x in that same depth interval. Although denitrification has been observed in aerobically grown pure cultures of bacteria (Robertson et al., 1995), all experimental evidence indicate that very little denitrification in soils and sediments occurs under fully oxic conditions. Denitrification thus took place in a <0.5-mm depth interval.
Profiles of NO-x at Day 1 showed NO-x concentrations in the manure phase of up to 1200 µmol L-1. No NO-x was initially present in the manure (as measured by HPLC). Thus, the NO-x observed in the manure at Day 1 must have diffused into the manure from the soil, where the initial concentration was 18 mmol L-1. Denitrifying bacteria are only present in very low numbers in fresh manure (Petersen et al., 1991; Nielsen et al., 1996) and the flux of NO-x into the manure apparently exceeded the NO-x-consuming capacity from Day 0 to Day 1.
At Day 18, NO-x was again present in the manure phase, and the concentration in the anoxic manure at a 2-mm depth was 50% of the maximum concentrations in the soil (Fig. 4). The deep penetration of NO-x into the manure was probably an effect of depletion of available carbon compounds and thus represents denitrification shifting from being NO-x limited to being carbon limited (Petersen et al., 1996).
The initial pool of NO-x present in the soil was depleted within the first 3 d of the experiment. The origin of the NO-x supplying denitrification during the rest of the experiment originated from nitrification of NH+4 released from the manure. The NO-x formed by nitrification could only accumulate in the 6-mm soil layer and thus not disappear from the system via diffusion or convective flow. Therefore, the two processes are inevitably coupled. Exactly how closely they are coupled depends on the physical distance between the processes, and a very tight coupling is, in this case illustrated by the finding of very small changes in the NO-x pool on two consecutive days as compared with the size of the NO-x fluxes. This means that the rates of nitrification and denitrification were virtually identical throughout the experiment (Fig. 8). Tight coupling between nitrification and denitrification also occurs, however, in systems with a deeper soil layer (Rice et al., 1988; Petersen et al., 1991; Petersen et al., 1992), and even in a system with continuous removal of NO-x behind a 1-cm thick soil layer (Nielsen and Revsbech, 1994; Nielsen et al., 1996; Nielsen and Revsbech, 1998).
Nitrifying bacteria started to produce NO-x after a lag phase of about 5 d, whereas denitrifying bacteria almost instantly (though at a low rate) started to consume the NO-x present in the system. It may be that there was only a low number of nitrifying bacteria present in the soil at the beginning of the experiment. Nitrifying bacteria are slow growing autotrophs (Brock et al., 1994), and a substantial NO-x production is therefore not to be expected until the population has built up. Once an active population of nitrifying bacteria is present, the bacteria can oxidize NH+4 at a very high rate.
The nitrifiers are dependent on the supply of NH+4 released from the manure, and the ammonification rate could also be controlling their growth and rate of NH+4 oxidation. Some studies have suggested that a long initial lag phase before nitrification starts can be because of very high (>200 mmol L-1) NH+4 concentrations inhibiting the nitrifying bacteria (Wetselaar et al., 1972; Olesen et al., 1997; Nielsen and Revsbech, 1998). In this case, however, the initial concentration of NH+4 in manure was only 12 mmol L-1 (10 mmol kg-1).
Nitrite Plus Nitrate Fluxes and Dinitrogen Emission
Both NO-x fluxes and N2 emission exhibited very low rates at Day 1 and decreased to zero after 1 or 2 d (Fig. 6) when the initial pool of NO-x was depleted. The rate of N2 emission exhibited a subsequent slow increase, whereas NO-x fluxes did not increase until Day 6. The flux was proportional to the level of NO-x concentration in the soil and peaked at Day 9 and 10. Maximum denitrification rates after 10 d were also found by Nielsen et al. (1996) and Nielsen and Revsbech (1998) by experiments with a similar two-phase system in a flow chamber. However, in their experiments the denitrification rates did not decrease as rapidly after reaching a maximum as seen in our experiments. The maximum rates obtained for NO-x fluxes and N2 emission were 732 and 497 nmol N cm-2 h-1, respectively, which is much higher than the 200 nmol N cm-2 h-1 found by Nielsen et al. (1996) and Nielsen and Revsbech (1998). The very high rates found in our experiments might be caused by the use of undiluted manure, whereas the quoted studies applied a manure-soil mix. A thorough homogenization might also have caused a very rapid mineralization.
Fluxes of NO-x and N2 production did not differ significantly during most of the experiment. Free NO-x can be removed from the soil in three ways: It can be immobilized by assimilation, reduced to NH+4, or denitrified to N2O or N2 gas. The flux of NO-x from the soil to the manure phase thus does not necessarily equal the denitrification rate. We measured microprofiles of N2O, which could not be detected, and the fairly good agreement between NO-x fluxes and N2 production throughout most of the experiment indicates that most NO-x was consumed by denitrification and gave rise to N2 production. There might have been some consumption of NO-x by other processes, but the rate of this consumption was too small and the variation in the measurements too large for it to cause a significant difference between NO-x consumption and N2 production.
It is important to consider the inaccuracies involved in the calculation of NO-x fluxes. The inclination of the concentration profile at the manure interface varied considerably among individual profiles, which gave rise to the high standard deviations (Fig. 6). This is most conspicuous around Day 9 and 10 when the rates were highest. The inclination was measured at the depth where the O2 concentration dropped considerably below atmospheric saturation. The profiles presented here were measured with 500-µm step size, and that gives rise to some inaccuracy in the determination of the exact depth of the interface between the aerated and the water-saturated soil. When the O2 concentration drops from atmospheric concentration to zero within one 500-µm step, we only know that the position of the interface is somewhere within that interval. It is thus likely that the inclination of the NO-x profile was measured in an interval that was not entirely within the anoxic, water-saturated soil. The gradient may thus have been measured in an interval characterized by a very large change in diffusive properties, and this would severely affect the calculated flux values. Furthermore, the consumption of NO-x within the same depth interval where the gradient is determined inevitably leads to underestimation of the real NO-x flux.
Comparison of the NO-x fluxes to the NO-x consumption calculated from the change in total NO-x pool during incubation with acetylene showed no significant difference (Fig. 7). This supports the use of the diffusion coefficient of water-saturated soil for flux calculations, and it indicates that fluxes we have calculated are representative of the real fluxes.
Acetylene Inhibition
Nitrous oxide production following inhibition of the N2O reductase of denitrifying bacteria by addition of acetylene is an often-used procedure for the estimation of denitrification rates. This procedure may, however, lead to underestimation of the denitrification rate when nitrification and denitrification are closely coupled (Nielsen et al., 1996), as also nitrification is inhibited by acetylene (Berg et al., 1982). In soil with low rates of denitrification and only few hot-spots there is little coupling between nitrification and denitrification, and Parkin et al. (1984) found no effect on denitrification rates in that type of soil after incubation with acetylene for 48 h. Several studies, including this, have shown that nitrification and denitrification associated with hot-spots in soil is closely coupled (Rice et al., 1988; Nielsen and Revsbech, 1994; Nielsen et al., 1996; Nielsen & Revsbech, 1998). The use of the acetylene inhibition technique for estimation of denitrification in such systems will therefore give severely underestimated rates.
Profiles of NO-x (Fig. 9) show the gradual depletion of the pool of NO-x after the addition of acetylene. The decreasing NO-x concentrations were reflected by decreasing fluxes of NO-x to the denitrification zone in the manure phase (Fig. 10), and after 2 h the mean flux had decreased to 38% of the original rate. Nielsen et al. (1996) made simulations of NO-x profiles following acetylene addition in their diffusion chamber system. They assumed that all nitrification activity was taking place within a distance of 0.7 to 1 mm from the manure surface. The 10-mm thick soil layer was in touch with a water column and NO-x could thus diffuse both into the water and into the manure. Their model estimated that denitrification coupled to nitrification would be reduced to 46 and 25% of its original rate after 1 and 3 h of incubation, respectively. In our experiment, the manure was the only NO-x sink, and nitrification took place throughout the 5- to 6-mm soil phase. A substantial (62%) decrease in NO-x fluxes was observed after just 2 h, so nitrification and denitrification rates were indeed closely coupled. This shows that even with short incubation times, e.g., of 2.5 h as used in the experiment by Petersen et al. (1996), acetylene may severely affect denitrification rates.
This study illustrates the steep O2 and NO-x gradients associated with manure hot-spots in soil and also provides an example of the dynamics of processes and pool sizes and of the nitrification and denitrification processes. The success with the application of a NO-x biosensor in soil furthermore opens for studies of other processes involving nitrate and nitrite, such as root uptake.
Received for publication January 15, 2001.
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