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Soil Science Society of America Journal 65:511-516 (2001)
© 2001 Soil Science Society of America

DIVISION S-8-NUTRIENT MANAGEMENT & SOIL & PLANT ANALYSIS

Determination of Atmospheric Volume for Direct Field Measurement of Denitrification in Soil Cores

B.P. Horgan, R.L. Mulvaney and B.E. Branham

Dep. of Natural Resources and Environmental Sciences, 1102 S. Goodwin Ave., Univ. of Illinois, Urbana, IL 61801

Corresponding author (bhorgan{at}uiuc.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
When denitrification is measured directly using 15N, the emission of labeled N2 and N2O is calculated from the volume of atmosphere confined within a closed chamber. This volume is readily estimated by measuring chamber height with a ruler; however, underestimation will occur with unsaturated soil because of air-filled porosity. A much greater complication arises when plants are present, due to the volume occupied by the aboveground biomass and the internal volume available for gas exchange. A method was developed to measure atmospheric volume from dilution of a standard addition of Ne introduced prior to circulation of air within a closed-chamber system. Ion-current measurements to determine Ne, from which the volume was obtained by regression, were performed during mass spectrometric analysis for 15N-labeled N2. Volume measurements by this method were accurate to within 3%, as compared with gravimetric measurements of air-filled porosity for bare soil varying in moisture content. When introduction of Ne into a darkened chamber was delayed to ensure stomatal closure, volume measurements for a turfgrass system were accurate to within 2.5%, based on the difference obtained before and after connecting a mason jar to provide a known volume. A more accurate method of measuring atmospheric volume will improve the accuracy achieved in direct measurement of denitrification.

Abbreviations: Db, bulk density • Dp, particle density • PVC, polyvinyl chloride


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
BECAUSE THE FINAL PRODUCT of denitrification (N2) is the major constituent of air, emission of this gas in the field can only be detected through the use of fertilizer highly enriched in 15N, in which case N isotope analyses are performed by mass or emission spectrometry (e.g., Rolston et al., 1978; Siegel et al., 1982; Craswell et al., 1985; Mosier et al., 1986; Kjeldby et al., 1987; Buresh and Austin, 1988; Mulvaney and Vanden Heuvel, 1988). The usual practice is to collect atmospheric samples using a closed chamber and to calculate the emission of labeled N2 as a proportion of the mass of ambient N2 within the chamber. The latter quantity is obtained through an ideal gas calculation from measurements of pressure, temperature, and volume. Accurate data are easily collected with a barometer and thermometer, whereas exact volume measurements are much more difficult to achieve.

In previous investigations involving direct field measurement of denitrification, the volume used in calculating emission of labeled N2 has been defined in terms of chamber dimensions. Headspace height above the soil surface is conveniently measured with a ruler, and the measurement is converted to a volume when multiplied by the cross-sectional area of the chamber. This approach will be most successful when free water seals the lower boundary of the chamber, as is the case in sampling from saturated or waterlogged soils (Lindau et al., 1988), wetlands (Xue et al., 1999), and flooded rice paddies (e.g., Buresh and Austin, 1988; Mosier et al., 1989). However, underestimation of N2 emission is almost certain to occur with unsaturated soil because air-filled porosity is not included in the headspace volume. An additional complication arises when plants are present, owing to the volume occupied by the aboveground biomass and the internal volume available for gas exchange.

Given the inherent limitations associated with the use of a ruler to estimate headspace volume for direct field measurement of denitrification, a novel technique was developed, whereby the atmospheric volume available for gas exchange is measured from dilution of a known quantity of an inert gas (Ne). The objectives of this paper are to describe this technique; to demonstrate that a high level of accuracy is achieved in measuring the atmospheric volume inside a closed chamber containing saturated or unsaturated soil, with or without turfgrass, when the bottom is sealed by an impermeable barrier; and to evaluate the effect of pumping period on volume measurements for unsaturated soil cores in the field under turfgrass.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Soil
A surface (0–20 cm) sample of Flanagan soil (fine, smectitic, mesic Aquertic Argiudoll) was collected from a site under Kentucky bluegrass (Poa pratensis L.) at the Landscape Horticulture Research Center at the University of Illinois, following removal of sod. Before use, the sample was air-dried in a forced-air oven at 60°C. Analyses of this soil as described by Mulvaney and Kurtz (1982) gave the following results: pH, 6.8; total N, 2.55 g kg-1; organic C, 30.3 g kg-1, a sand content of 125 g kg-1, a silt content of 588 g kg-1, and a clay content of 287 g kg-1. Particle density (Dp) determined by the pycnometer method (Blake and Hartge, 1996a) was 2.57 Mg m-3. The gravimetric soil moisture content, determined by oven-drying samples at 105°C, was 7 g kg-1. All analyses reported were performed in triplicate.

An intact core of Flanagan soil with Kentucky bluegrass sod was obtained from an adjacent site, by inserting a polyvinyl chloride (PVC) cylinder (21.5-cm o.d., 20.0-cm i.d., 30.5 cm long) to a depth of 25 cm using a tractor-mounted hydraulic press. To minimize compaction during insertion, the lower end of the cylinder was beveled to 60° on the outside surface, and an aerosol silicone lubricant was applied to the inside surface. The upper end of the cylinder was equipped with a plastic-welded PVC flange (25.4-cm o.d, 20.0-cm i.d., 1.3 cm thick) to permit the attachment of a lid for atmospheric sampling. Prior to placement in the soil, a machined brass disk (21.5-cm o.d., 1.3 cm thick) was inserted 0.65 cm into the upper end of the cylinder to protect the integrity of the flange. After removing the brass disk and verifying that infiltration rates inside and outside the cylinder did not differ, the cylinder containing the soil core was excavated and transported to a greenhouse. The turfgrass was maintained with biweekly watering and addition of Hoagland's nutrient solution. At weekly intervals, the grass sward was clipped to a height of 4.5 cm, and clippings were removed.

Experimental Technique
Research-grade Ne (MG Industries, St. Louis, MO) was introduced during atmospheric sampling via a 60-mL gas sampling tube sealed with two high-vacuum stopcocks (cat. no. 8194-03; Ace Glass, Vineland, NJ). One stopcock was connected to an interior tube for dispersion of the incoming air stream, and the other was used for exhaust. The sampling tube was flushed twice with Ne after evacuation to <0.1 mPa while connected to a four-port manifold with stopcock connections to a vacuum pump, a thermocouple gauge for vacuum measurement, and a regulated supply of Ne. Evacuation was repeated prior to filling the tube to a pressure of {approx}120 kPa. The sampling tube was then removed from the manifold, and the overpressure was relieved by momentarily opening a stopcock. Barometric pressure was measured with a Hg manometer, to permit determination of the mass of Ne inside the sampling tube from an ideal gas calculation.

Figure 1 shows the various components used to sample the atmosphere inside a PVC cylinder containing soil with or without turfgrass, following the introduction of Ne to measure atmospheric volume. The cylinder was sealed by attaching a 6.4-mm-thick brass lid and a silicone gasket to the PVC flange using four machine screws. The lid was equipped with two brass toggle valves (cat. no. B-1GS4; Whitey Co., Highland Heights, OH), one of which was fitted with a gas dispersion tube to serve as an intake port. The exhaust valve was connected via Ultra-Torr unions (Cajon Co., Macedonia, OH) and 6.4-mm-o.d. polyethylene tubing to the intake stopcock on the sampling tube previously filled with Ne. Similar connections were made between the exhaust stopcock and the intake port on a 12VDC circulating pump (model P-07530-25; Cole-Parmer Instrument Co., Chicago, IL), and between the exhaust port on the pump and the intake valve on the lid for the PVC cylinder. After completing all of the connections specified, a closed system existed for gas circulation.



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  		Fig. 1. Apparatus used to introduce Ne and collect gas samples for determination of atmospheric volume

 
Unless otherwise specified, the following procedure was employed to introduce Ne and collect an atmospheric sample after connecting all components as described above: (i) both stopcocks on the sampling tube and both toggle valves on the lid were opened; (ii) pumping (at {approx}9 L min-1) was initiated to circulate gas within the closed system; and (iii) after 20 min, pumping was terminated, the stopcocks on the sampling tube were closed, and the tube was disconnected from the circulating pump and the lid for subsequent mass spectrometric analysis for Ne.

The mass spectrometer used was a Nuclide Model 3-60-RMS instrument equipped with double collectors, a dual-inlet system, and a microcomputer for data acquisition and control (Spectrumedix, State College, PA). A digital-to-analog converter allowed computer control of the electromagnet used for resolution of ion beams. The peak-stepping capability thereby provided was employed to automate ion-current measurements of Ne (m/z 20), so that real-time determination of atmospheric volume would be possible during direct measurement of denitrification. Prior to Ne analysis, atmospheric samples were purified to remove water vapor, CO2, and O2, following the procedure described by Mulvaney and Kurtz (1982) for measuring 15N-labeled N2. In all cases, the inlet pressure was adjusted to obtain 5 V of m/z (28 + 30) on the major collector, which eliminated the need to correct for Ar2+. The voltage at m/z 20 was converted to a volume through the equation, Y = 798.5X2 - 5463.3X + 10968.3 (R2 = 0.996), which was generated by regressing nine known volumes on their corresponding voltage measurements.

To obtain data for generating the aforementioned equation, atmospheric samples were collected as described previously, except for the use of one to three wide-mouth mason jars in place of the PVC cylinder illustrated by Fig. 1. The lids used to seal these jars were equipped with two brass toggle valves, which were attached via brass tubes (6.4-mm o.d., 4.6-mm i.d., {approx}3 cm long) that had been soldered through the lid. To promote atmospheric mixing, the intake valve was fitted with a section of Teflon tubing that extended to within 2 cm of the bottom of the jar. An exact value was obtained for the internal volume of the jars (less the volume occupied by the intake tube), and also of the circulating pump and gas sampling tube, based on the difference in weight before and after addition of deionized water. A measured density (0.9949 Mg m-3) was used in converting the weight of water to a volume. Internal volumes were calculated for the tubing and fittings from dimensional data supplied by the manufacturer. When connected to the other components used in atmospheric sampling, the mason jars provided nine known volumes that ranged from 1396 to 4057 mL.

Evaluation of Technique
To evaluate the accuracy of the Ne technique for atmospheric volume measurements with bare soil, a PVC cylinder (Fig. 1) was shortened to 12.5 cm, and the lower end was sealed by cementing to it a 1.3-cm-thick polycarbonate sheet. Prior to the addition of soil, the volume inside the sealed cylinder, plus the volume contributed by the other components used in atmospheric sampling, was determined in triplicate by the Ne technique. The volume during sampling was also obtained by measuring the difference in weight (to 0.1 g) before and after filling the cylinder with deionized water, to which was added the volume contributed by the other components (112.1 mL), determined as described in the above paragraph. After removing the water and drying the cylinder, 1976.9 g of soil was added and then compacted to achieve a Db of 1.20 Mg m-3. Triplicate measurements of atmospheric volume were performed by the Ne technique, before and after addition of sufficient water to occupy 9.8, 19.6, 48.9, or 97.9% of soil pore space (891.9 mL) determined as Vsoil(1 - Db/Dp) + MsoilGsoil, where Vsoil is the volume occupied by soil (1647.4 mL), Msoil is the mass of soil expressed as an oven-dried weight (1963.2 g), and Gsoil is the gravimetric soil moisture content (7 g kg-1). At each moisture level, atmospheric volume was determined as Vcylinder - Vsolid - Vwater + Vcomponents, where Vcylinder is the total volume inside the sealed cylinder (determined gravimetrically), Vsolid is the volume of soil solids (769.2 mL, calculated as VsoilDb/Dp), Vwater is the volume of water added, and Vcomponents is the total volume of the components used in atmospheric sampling.

A study was also conducted to evaluate the accuracy of the Ne technique for measuring atmospheric volume for turfgrass growing on a saturated soil in the greenhouse. The PVC cylinder containing an intact soil core under Kentucky bluegrass sod was placed on a 5-cm-thick layer of coarse sand in an 18.9-L bucket, and the bucket was filled with tap water to a height of 20 cm to seal the bottom of the cylinder and prevent drainage. Triplicate measurements of atmospheric volume by the Ne technique were performed before and after addition of a known volume, with or without a 160-min period of previous enclosure to effect stomatal closure. The known volumes used (262.4, 539.7, or 1059.6 mL as determined gravimetrically) were produced by connecting a mason jar (equipped as specified previously) between the PVC cylinder lid and the gas sampling tube. Following each atmospheric collection, the lid was removed from the cylinder, and a 90-min period was provided to ensure complete elimination of Ne from the turfgrass system. The difference between successive measurements was compared with the known volume of the mason jar.

To investigate the applicability of the Ne technique for measuring atmospheric volume when turfgrass is grown in the field on an unsaturated soil, a study was conducted to determine the extent to which volume measurements depend on the period of pumping. To a PVC cylinder inserted as described above, five sampling tubes were connected to a modified version of the gas collection system illustrated by Fig. 1. Besides the single tube that contained Ne, four additional tubes were connected via eight three-way ball valves (Whitey model SS-43XS4) equipped with Ultra-Torr adapters, such that each of the latter tubes could be bypassed using a pair of valves to redirect the air flow. After 20, 40, 60, and 180 min, pumping was terminated, the stopcocks on one of the four additional sampling tubes were closed, the appropriate valves were switched to bypass this tube, and pumping was then resumed. A final sample was taken after 720 min, at which time pumping was terminated and the stopcocks were closed on the remaining tube. In addition to carrying out gas analyses for Ne, soil samples were collected to estimate percentage pore space from determinations of Dp and Db, the latter measurement being made by the core method described by Blake and Hartge (1996b).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The Ne technique developed in our work originated because of the need to determine atmospheric volume for direct measurement of denitrification in a turfgrass system, using the apparatus illustrated by Fig. 1. Attempts were made to estimate the volume inside the sealed cylinder from the decrease in pressure that occurred when air was removed using an evacuated bulb of known volume and pressure, based on Boyle's Law. This approach was abandoned because the return to atmospheric pressure was far too rapid to permit valid measurements with the pressure transducer employed, and the same problem vitiated subsequent measurements using positive pressure. In an effort to avoid these difficulties, a further attempt was made to estimate chamber volume by displacing the air with purified CO2, and then trapping the CO2 in NaOH as air was drawn through the chamber. Measurements by the latter technique were found to increase markedly with the concentration of NaOH, until they had almost exceeded the internal volume of the empty cylinder.

Alternatively, the volume of atmosphere in a closed system can be determined by introducing a known amount of gas, and then quantifying the dilution. This approach has been employed through the use of a low-molecular-weight hydrocarbon as an internal standard in the C2H2 reduction assay for N2 fixation (e.g., Hanson, 1977; Pedersen et al., 1978). Ideally, a noble gas would be used because of chemical and biological inertness, although other factors must be considered when atmospheric volume is to be determined in conjunction with direct measurements of denitrification, as was the case in our work. For example, mass spectrometric analyses for He were complicated by the narrow peak width at m/z 4, whereas the use of Rn was rejected because of the health hazard from ionizing radiation. Of the remaining noble gases, Ar, Kr, and Xe are at least somewhat condensible in liquid N2, which is employed for cryogenic purification prior to isotope-ratio analyses by the method of Mulvaney and Kurtz (1982). The latter problem does not apply to Ne, so this gas was used in our work. Neon is readily available at low cost, and the low water solubility of this gas (14 mL L-1) should minimize errors in measuring atmospheric volume due to incomplete recovery. The low concentration of Ne in air (18 µL L-1) improves the sensitivity of volume measurements, although a background peak occurs at m/z 20 because of Ar2+ that forms in the ion source of the mass spectrometer. No correction will be required to account for the presence of Ar if all analyses are performed at a constant inlet pressure, as was the case in our work.

For atmospheric volume to be determined accurately from dilution of a known addition of gas, the atmosphere must be of uniform composition. In our work, gaseous mixing was achieved through the use of a circulating pump in a closed-loop sampling system. There are reports that slight pressures or pressure deficits generated when air is circulated through chambers placed over the soil surface can have marked effects on gaseous emission (Denmead, 1979; Hutchinson and Mosier, 1981; Mosier et al., 1990). If desired, the chamber lid can be equipped with a low-conductance vent (e.g., 1.4-mm-i.d. tubing) to avoid pressure fluctuations. This was not done in the present project, as previous work to evaluate a similar sampling system showed that venting did not reduce short-term variability in emission of N2 or N2O (Mulvaney and Kurtz, 1984).

An important consideration in determining atmospheric volume by the technique described is the period of pumping required to ensure a uniform concentration of Ne. Preliminary work showed mixing to be complete within 5 min, based on the finding that no difference was detected in Ne concentration when duplicate samples were collected by connecting sampling tubes to the intake and exhaust valves on the lid attached to a PVC cylinder (one end of which had been sealed) containing an unsaturated bare soil. Yet this period of pumping proved to be inadequate, as a considerably larger value was obtained when atmospheric volume was determined gravimetrically, which can be attributed to incomplete transport of Ne through soil pore space. Better agreement was achieved by pumping for 10 min, and the difference did not exceed 3% when pumping was performed for 20 min, regardless of soil moisture content (Table 1). Perfect agreement was not expected because error could have occurred in gravimetric measurement of atmospheric volume, particularly since Dp was determined on a small sample of soil (10 g), and then extrapolated to the entire core (2.0 kg). That such error did occur is confirmed by the finding that the difference between the two methods was not reduced when the pumping period was increased to 30 min.


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  		Table 1. Comparison of gravimetric and Ne measurements of atmospheric volume in a closed system with or without soil at varying moisture contents

 
The Ne technique also proved accurate in measuring atmospheric volume for turfgrass when the lower boundary of the PVC cylinder was sealed with water, provided the introduction of Ne followed a period of enclosure to limit gas exchange via stomates. This is illustrated by Table 2, which shows the results obtained when measurements were performed before and after addition of a known volume, either with or without a previous period of enclosure. When the introduction of Ne was delayed for 160 min after attaching the lid, the error did not exceed 2.5%; otherwise, the addition of known volume was overestimated by 9 to 15%. The latter error can be attributed to entrapment of Ne within the turfgrass, which reduced the concentration of this gas in the external atmosphere and thereby increased the volume determined. This was confirmed by checking for the liberation of Ne from excised plant tissue that had been transferred to a sealed mason jar. Neon was only detected when no period of previous enclosure had been provided.


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  		Table 2. Accuracy of Ne technique for measuring known atmospheric volumes added to a turfgrass system with or without a previous period of enclosure to promote stomatal closure{dagger}

 
The Ne technique was developed to measure atmospheric volume in the field even if the soil is unsaturated. Under such conditions, the measured volume should increase with the pumping period employed, owing to the diffusive transport of an inert gas through soil pore space (Rolston et al., 1991). This is confirmed by Fig. 2 , which shows that atmospheric volume had increased by almost 80% in 12 h, from 3 to 5.5 L. The latter volume exceeds the upper limit of the regression equation used in our work and is therefore invalid. The remaining volumes indicate Ne penetration to a depth of 14 to 22 cm, as estimated from measurements of soil water content (173 mL kg-1), Db (1.3 Mg m-3), and Dp (2.57 Mg m-3) after taking into account the 5.5-cm height that existed above the soil surface inside the sealed cylinder. An optimal period of pumping cannot be specified exactly because of the effects of soil moisture content and porosity, but prolonged pumping should be avoided to preclude the possibility that atmospheric volume will exceed the soil pore space contained within the cylinder as the soil dries.



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  		Fig. 2. Effect of pumping period on atmospheric volume measured by the Ne technique for turfgrass growing on unsaturated soil in the field. Standard deviations for triplicate determinations ranged from 10 to 23 mL

 
Although developed to improve the accuracy obtainable when denitrification is measured directly using 15N, the Ne technique described may be useful for other measurements that depend on atmospheric volume. This technique could, for example, be useful when C2H2 is employed to inhibit the reduction of N2O to N2 in measuring denitrification. Other applications might arise in regard to determining air-filled porosity for soil or the internal volume within a plant that is available for gas exchange. Instead of mass spectrometry, atmospheric analyses could be performed by gas chromatography, which provides the option of substituting another inert gas for Ne, such as He or Ar.


   ACKNOWLEDGMENTS
 
Partial support was provided by a grant from the United States Golf Association.

Received for publication June 21, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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