Denitri®cation rates in organic and mineral soils from riparian
sites: a comparison of N
2
¯ux and acetylene inhibition methods
Susan H. Watts*
, 1, Sybil P. Seitzinger
Rutgers, The State University of New Jersey, Institute for Marine and Coastal Sciences, P.O. Box 231, New Brunswick, NJ 08903-0231, USA
Received 22 June 1999; received in revised form 15 February 2000; accepted 23 February 2000
Abstract
Denitri®cation rates were measured in relatively dry mineral soils and water-saturated organic soils by both direct N2¯ux and acetylene inhibition methods to compare the relative sensitivity of the two methods and their applicability to dierent soil types and saturation conditions. The direct N2 ¯ux method, previously used only in submerged sediments, was modi®ed for use in terrestrial soils. Our experiments demonstrated that the N2¯ux method is adaptable for both mineral and water-logged organic soils, and that denitri®cation rates based on direct N2¯uxes can be measured after only 2 days of incubation in most cases. We used a diusive ¯ux model to separate the portion of total N2 ¯ux attributable to denitri®cation from that due to passive diusion of atmospheric N2from soil porespaces. Denitri®cation rate measurements were taken during spring, summer, and fall seasons using replicate cores from four dierent riparian sites (two with mineral soil, two with organic soil). Rates determined by N2¯ux were 35±130 and 10±245mmol N mÿ2hÿ1for mineral and organic soils, respectively. Those by acetylene inhibition, for the same sites were one, and sometimes two, orders of magnitude less with rates of 0.1±10mmol N mÿ2hÿ1for mineral soil and 0.2±3mmol N mÿ2hÿ1for organic soil. This study has demonstrated that denitri®cation measured by acetylene inhibition in riparian sites may be underestimated by at least an order of magnitude in both mineral and organic soils. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Denitri®cation; Riparian zone; Organic soil; Mineral soil; Acetylene inhibition; N2¯ux
1. Introduction
Denitri®cation is an important component of the nitrogen cycle that returns to the atmosphere inorganic nitrogen that resides in the plant and soil pools of ter-restrial and aquatic ecosystems. Under low oxygen
ten-sion, nitrate (NO3ÿ) instead of O2, is used by
denitrifying bacteria as the terminal electron acceptor for energy production, forming dinitrogen gas and
nitrous oxide (N2 and N2O) which are lost to the
at-mosphere.
Accurate measurements of denitri®cation are im-portant for many studies (e.g. assessing N loss from N-limited ecosystems or N removal from eutrophied
ecosystems), however, the determination of N2 ¯ux
is dicult to quantify against the large background of
atmospheric N2. Several methods have been developed
to cope with this diculty, but the majority involve an indirect measurement of denitri®cation rather than the
measurement of actual N2 ¯ux. The acetylene (C2H2)
inhibition method is widely used and is based on the
principle that C2H2 blocks the enzymatic reduction of
N2O to N2, so that accumulated N2O can be measured
instead of N2 (Yoshinari et al., 1977). However, it
adds an inhibitor to the system, and the inhibitor may
0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 5 6 - 0
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1
Present address. Department of Biological Sciences, University of Texas, 500 W. University Avenue, El Paso, TX 79968-0519, USA.
* Corresponding author. Tel.: 6851; fax: +1-915-747-5808.
not always be eective. Furthermore, a number of speci®c problems are associated with acetylene inhi-bition, including uneven penetration of acetylene into soil microsites (Rudolph et al., 1991) and incomplete
inhibition at low soil nitrate concentration (<10 mM)
(Knowles, 1990) or in the presence of sul®des (Adkins and Knowles, 1986; Sorensen et al., 1987). Another widely-used method is the denitrifying enzyme activity (DEA) assay that measures potential denitri®cation rates (Ambus and Lowrance, 1991; Groman and Tiedje, 1989; Schipper et al., 1993). Drawbacks of this method are that: (1) rate determinations are also based on the acetylene inhibition method, and (2) changes in denitri®cation enzyme activity have not been shown to be clearly related to actual nitrogen ¯ux (Parsons et al., 1991) due, perhaps, to the persistence of denitrify-ing enzymes in soil over long periods of time (Smith and Parsons, 1985). In the ecosystem mass balance method, no measurements are taken; any N de®cit that cannot be accounted for by other processes is assumed to be lost by denitri®cation (Peterjohn and Correll, 1984; Jacobs and Gilliam, 1985). Other esti-mates of denitri®cation have been based on the decreasing concentration of nitrate over time in ¯ood water or submerged sediment cores (e.g., Brinson et al., 1984; Gale et al., 1993). In other denitri®cation
rate measurements, labeled nitrogen (15NO3ÿ, 15NH4+,
or15N-labeled urea) has been applied to soil cores or
sediments, and then monitored for either a decrease in
the labeled compound or an accumulation of 15N2
(Buresh and Austin, 1988; Eriksen and Holtan-Hart-wig, 1993; Melin and Nommik, 1983; Nommik and Larsson, 1989; Parkin et al., 1985; Weier et al., 1993).
While the latter method actually measures N2 ¯ux,
nutrients were added to the soil and measured rates may not accurately re¯ect in situ rates.
There are existing methods that determine
denitri®-cation rates based on direct measurement of N2 ¯ux
(N2alone or as N2:Ar ratio) (Devol, 1991; Kana et al.,
1998; Nowicki, 1994; Schole®eld et al., 1997a, b; Seit-zinger et al., 1980; SeitSeit-zinger, 1994). However, up to this time, denitri®cation determinations based on
actual N2 ¯ux have been done predominantly in
sub-merged sediments. The objective of this study was to use the method of Seitzinger (Seitzinger et al., 1980; Seitzinger, 1993) modi®ed for use in terrestrial ecosys-tems by eliminating both the overlying water phase and the week long pre-incubation period. This modi-®ed method was used with both mineral and water-logged organic soils to evaluate its applicability to dierent soil types. Denitri®cation rates determined by
direct N2 ¯ux measurements were compared to rates
determined by the widely used acetylene inhibition method.
2. Materials and methods
2.1. Study sites
Mineral soil samples (aquic fragiudults) were col-lected from riparian areas along two ®rst-order streams on the Georgia Farm Preserve located in Che-ster County, PA (USA). One watershed (17 ha) has a mature mixed hardwood riparian forest with surround-ing agricultural ®elds that have been out of production since 1992. The second watershed (13.3 ha) has a riparian zone with newly-planted trees (sugar maple (Acer saccharum), red oak (Quercus rubra), tulip
poplar (Liriodendron tulipifera), white ash (Fraxinus
americana), black walnut (Juglans nigra), and
trem-bling aspen (Populus tremuloides)), and adjacent
agri-cultural ®elds are currently cropped with corn and soybeans. Site descriptions are detailed in Newbold et al. (unpublished, ®nal report to Chesapeake Research Consortium, 1995).
Organic soil samples were collected from two acidic cedar swamps sites located within the New Jersey Pine-lands. Both wetland sites are dominated by Atlantic
white cedar (Chamaecyparis thyoides), red maple (Acer
rubrum), and Sphagnum mosses. One site (McDonalds Branch) is located in an undisturbed area of Lebanon State Forest (New Jersey). The second site (Albertsons Branch) lies in a disturbed watershed near Hammon-ton, NJ, and is sandwiched between crop orchards and a stream with treated sewage inputs. Additional infor-mation about stream chemistry and site characteristics can be found in Morgan (1987), and Morgan and Phil-lipp (1986).
2.2. Sample collection
Paired soil samples for N2 ¯ux measurements were
collected from two sites in each of the four watersheds in summer and fall of 1994 and spring 1995. Mineral soil samples were collected as soil blocks
(approxi-mately 400 cm2, 10 cm depth) and were placed into
snugly-®tting plastic bags (blocks were trimmed to ®t into incubation chambers). Organic soil samples were collected with plastic coring tubes (8 cm diameter, ap-proximately 10 cm depth). The tops of both sampling devices were left open to prevent anaerobiosis and to maintain natural diusion gradients. Air and soil tem-peratures were recorded, and the samples were stored in insulated containers during transport.
For the acetylene inhibition study, soil samples were
obtained within 3±15 days of the N2 ¯ux cores. Soil
cores were collected from ®ve sampling sites in each of the four watersheds in summer, and fall 1994 and spring 1995. At each sampling site, paired cores were
collected in PVC tubes (2.5 cm wide 15 cm deep)
sealed with rubber stoppers on the bottom, and left open on top. Air and soil temperatures were recorded, and samples were stored in insulated containers during transit.
2.3. Experimental setup
2.3.1. Acetylene inhibition
The acetylene inhibition method used is detailed in Groman and Tiedje (1989) and Groman et al. (1993) and is only brie¯y summarized here. Rate measurements began within 6 h of sample collection. Cores were left in the PVC coring tubes, and each tube was sealed top and bottom with rubber stoppers; the top stopper had an attached septum. Acetylene was added to displace approximately 10% of the calculated
headspace-plus-porespace volume; headspace gases
were mixed with a 60 ml syringe, and then the cores were incubated at room temperature. At 2 h and 6 h
after C2H2addition, 3 ml headspace samples were
col-lected by syringe and placed into 3 ml vacuum tubes (Becton Dickinson Vacutainer) for analysis by gas chromatography. The denitri®cation rate was based on
the rate of N2O production between 2 and 6 h (see Eq.
(1)).
Nitrous oxide concentration was measured using a gas chromatograph (Shimadzu GC-8A with electron
capture detector (ECD); N2carrier gas; 50/80 Porapak
Q; S.S. column (2 m0.318 cm o.d.)). Mixed-gas
stan-dards in two concentrations were used for standard
curves (O2 18.7%, 20.87%; N2 3.38%, 5.87%; N2O
26.5 ppm, 70.8 ppm; CO2 3.35%, 5.98%; CH4 0.29%,
0.76%).
2.3.2. N2¯ux
The N2 ¯ux method used in this study is an
adap-tation of a method previously used in estuaries, rivers, lakes, and wetlands (Seitzinger, 1994; Nowicki, 1994; Zimmerman and Benner, 1994) and is detailed in Seit-zinger et al. (1980) and SeitSeit-zinger (1993). For the
cur-rent experiments, the chambers were modi®ed to eliminate the overlying water phase, but retained a 50±
100 cm3headspace (Fig. 1). Within 3±4 h of collection,
soil cores were cut down to approximately 8 cm
diam-eter5 cm depth and were sealed into gastight glass
chambers (O-ring and metal clamp secured bottom plate to chamber top). The chamber headspace was
then ¯ushed for 5 min using a mixture of 21% O2and
79% He (He substituted for N2 on a percent volume
basis). After headspace ¯ushing, the cores were incu-bated 2 h to allow headspace and porespace gases to equilibrate (all incubations were conducted in a dark, temperature-controlled environmental room at the soil temperature at the time of collection). Initial head-space gas samples were collected after 2 h through a
gastight port using a gastight syringe (75 ml sample)
and immediately analyzed by gas chromatography (Shimadzu GC-8A with thermal conductivity detector (TCD); He carrier gas; 45/60 Molecular Sieve 5A; S.S.
column (2.2 m0.318 cm o.d.)) to establish a baseline
concentration (Cb) for N2. After 20±24 h, a second
headspace sample (Cs) was analyzed to establish an N2
¯ux rate. The headspace gases were then re¯ushed
with O2/He, sampled for a new baseline measurement
after 2 h, incubated overnight (approximately 20 h), and then sampled again to establish a ¯ux rate for the second day. The ¯ushing/incubating/sampling cycle was repeated for 7 days.
Total ¯ux rates (mmol N mÿ2 hÿ1 ) were calculated
daily, based on the dierence between the gas
concen-trations of the baseline measurement (Cb) and the
sec-ond measurement (Cs), the volume of the headspace
and porespace (v), the surface area of the core (a),
and the number of hours between samples (h) such
that:
Flux CsÿCb v= ah 1
2.3.3. Estimating diusive ¯ux of atmospheric N2
When measuring the total ¯ux of N2from soil
pore-space into a headpore-space having a reduced concentration
of N2, one must distinguish between the two major
sources of N2: that due to degassing of atmospheric
N2from soil porespace and the portion due to
denitri-®cation. Because atmospheric N2degassing rates could
not be measured independently from N2 produced via
denitri®cation, a model was used to estimate the
amount of N2¯ux due solely to passive diusion of
at-mospheric N2 from soil porespace during each 24-h
period.
It was assumed that the diusive ¯ux responded according to Fick's ®rst law of diusion:
F ÿDsoil dc=dz 2
whereFis the ¯ux rate, Dsoilis the diusion coecient
Fig. 1. Schematic diagram of gastight chamber used for direct N2
for soil, dc is the concentration gradient between the
soil and the overlying headspace above the core, andz
is the soil depth.
Because the rate of diusive ¯ux of gas from dry soil is much faster than from waterlogged soils,
diu-sion coecients (Dsoil) were calculated using two
dier-ent models (Ddry soil, Dsat soil). Several models predict
similar diusion coecients when air-®lled porosities are greater than 30% (Sallam et al., 1984). However, the Millington-Quirk model most accurately predicts the slower rate of diusion encountered in moist soils with air-®lled porosities as low as 5% and is, thus, ap-plicable to a wider range of soil saturation conditions than other models (Sallam et al., 1984). In the current study, the Millington-Quirk model was applied to all soil samples (mineral or organic) with air-®lled poros-ities >5% (actual range: 8±55%) (Jury et al., 1982):
Ddry soil Dair
ÿ S10=3
a =S
2
t
3
where Ddry soil is the diusion coecient of N2 in dry
soil, Dair is the diusion coecient of N2 in air (0.22
mmol N2cmÿ2sÿ1at 208C),Sais the air-®lled porosity
of the soil, andSt is the total porosity of the soil
(cal-culations forSaandStbelow).
Because the Millington-Quirk model does not accu-rately predict diusion from the wettest soils, a second model was used for saturated soils with air-®lled pore-space less than or equal to 5%. Diusion coecients (Dsat.soil) were estimated using (Rothfuss and Conrad, 1994):
Dsat:soilDdistilled water
ÿ
St2 4
whereDdistilled water (the diusion coecient of N2from
distilled water) was assumed to be 1.910ÿ5cmÿ2sÿ1
at 258C (Glinski and Stepniewski, 1985).
Total porosity (St) (the percentage of bulk volume
not occupied by solids) for both soil types was calcu-lated as (Vomocil, 1965):
St100 1ÿ Db=p 5
where Db is bulk density and p is the particle density
which was assumed to be 2.65 g cmÿ3for mineral soil
and 0.6 g cmÿ3for organic soil (Sallam et al., 1984).
Bulk density for both soil types was calculated as (Vomocil, 1965):
Db mass of air-dried soil=volume 6
Air-®lled porosity was calculated as (Vomocil, 1965):
SaStÿPv 7
where Pv is the percent volume of H2O in the soil
which was calculated as (Vomocil, 1965):
Pv100 vol H2O lost=sample vol or
PvDb wt of H2O lost=dry mass 8
After calculating the diusion coecients for each
core (range: 1.9710ÿ3 to 6.97 10ÿ2 for dry soils,
down to 3.310ÿ6 for saturated soil), the ¯ux of
at-mospheric N2from the soil in each core was calculated
daily for each ¯ux interval (2±20 h) during days 1±7. This calculation assumed that the headspace
concen-tration of N2 was zero immediately after ¯ushing and
that the initial soil porespace concentration was equal
to the calculated concentration of N2 at equilibrium
the day before. We also assumed that the rate of bio-genic denitri®cation was not great enough to aect the
equilibration rate of the atmospheric N2.
Denitri®ca-tion rates were then calculated as the dierence
between the measured total N2 ¯ux and the calculated
atmospheric N2¯ux.
3. Results
3.1. Adaptation of N2¯ux method for soils
Total ¯ux measurements for days 3±7 were generally
<600 mmol N mÿ2 hÿ1 (Figs. 2 and 3) with few days
>1000 mmol N mÿ2hÿ1 (organic soil, Figs. 2 and 3).
Fig. 2. Measured total N2 ¯ux rates for soils with >5% air-®lled
porosity (range = 8±55% ) which includes mineral soils and organic soil samples from two riparian ecosystems. Lines represent the maxi-mum amount of the total ¯ux attributable to atmospheric N2after 2
h of incubation in mineral soil (solid line) and organic soil (dashed line) (See text for details). For 95% of mineral soil samples, calcu-lations predicted no ¯ux of atmospheric N2 after 2 h incubation by
To calculate the component of the total ¯ux
attribu-table to the diusive ¯ux of atmospheric N2, we used
eitherDdry soil (cores with air-®lled porosities >5%) or
Dsat. soil(cores with air-®lled porosities <5%) (Eqs. (3)
and (4)). Based on this criteria, Ddry soil was, thus,
applied to all mineral soil cores and half of organic soil cores (air-®lled porosities ranged from 8 to 57%),
whileDsat. soilwas used for the rest of the organic soil
cores.
When using Ddry soil, the diusive ¯ux model
pre-dicted that for the ``driest'' soils (air-®lled porespace
19±57%), the degassing rate of atmospheric N2 would
be <1 mmol N mÿ2 hÿ1 after 2 h of incubation on
days 3±7 (13 of 14 mineral soil samples). Thus, we
assumed that, for these, N2 ¯uxes measured after 2 h
of incubation could be attributed wholly to
denitri®ca-tion rather than degassing of atmospheric N2. For the
``wetter'' Ddry soil samples (air ®lled porosity >5% up
to 20%), the model predicted that there would still be
some ¯ux of atmospheric N2 after 2 h of incubation
due to the slower rate of diusion from soil pore-spaces. For `wet mineral' soil cores, with air-®lled por-osity as low as 20% (solid line, Fig. 2), the predicted
¯ux of atmospheric N2 after 2 h of incubation was
notable, but signi®cantly smaller than the total ¯ux, while in `wet organic' soils (air-®lled porosity as low as
7%) ¯ux of atmospheric N2was much more signi®cant
(dashed line, Fig. 2). Consequently, for these `wet'
cores, denitri®cation rates were calculated as net N2
¯ux (Fdnf) which was de®ned as the dierence between
the measured total N2¯ux (Ftot) and the predicted ¯ux
of atmospheric N2 (Fatm), such that Fdnf = FtotÿFatm
(Nowicki, 1994). In a few cases, the predicted ¯ux of
atmospheric N2 was greater than the measured total
¯ux, resulting in no net ¯ux. In such cases, denitri®ca-tion was de®ned as zero.
``Saturated'' organic soils were de®ned as those with air-®lled porosities <5% (®ve of six samples were
<1%), and ¯ux rates were calculated usingDsat.soil
dif-fusion coecients (Eq. (4)). The degassing of
atmos-pheric N2 is slower under such conditions, and the
diusive ¯ux model predicted that atmospheric N2
would be emitted continuously during the 7-day
exper-iment (Fig. 3). For net N2 ¯ux calculations, we
assumed that the predicted rate of atmospheric ¯ux at 2 h of incubation continued overnight, when it actually decreases slowly over time. Thus, we overestimated the
contribution of the atmospheric ¯ux of N2 from soil
porespaces to the total ¯ux, and underestimated the ¯ux due to denitri®cation to arrive at a conservative estimate of denitri®cation.
Because N2 ¯ux denitri®cation experiments involve
Fig. 4. Denitri®cation rates by two methods. Denitri®cation rates measured by (a) direct N2¯ux and (b) acetylene inhibition for four
sites (two with mineral soils and two with organic soil) over four sea-sons. (Error bars = S.E.). Note dierence in scale ofy-axes. Fig. 3. Measured total N2 ¯ux rates for high moisture soils. Points
represent measured total N2 ¯ux for organic soil samples with
detection of small quantities of N2 against a large
at-mospheric N2background, one must rule out
contami-nation of experimental apparatus by atmospheric N2.
Contamination could occur during sampling (when headspace samples are collected from the incubation chamber for gas chromatographic analysis) or during
incubation (due to leakage of atmospheric N2 into
chamber through gaskets or stopcocks). In order to avoid contamination during sampling, the needle of the gastight syringe and the sampling ports of both the incubation chamber and gas chromatograph were
con-tinuously ¯ushed with N2-free helium to prevent
inter-ference by atmospheric N2 (Seitzinger et al., 1980;
Seitzinger, 1993). Such ¯ushing appeared to be eec-tive in the current experiments, because replicate head-space gas samples had low variability. The coecient
of variation (c.v.) for duplicate N2 measurements
ran-ged from less than 1% (about 70% of cases) to 1± 3.5% (25% of cases). The second potential source of error is leakage of atmospheric nitrogen into the incu-bation chambers through the gasket or stopcocks. In the current experiments, the smallest detectable leak
was 20 mmol N mÿ2 hÿ1, similar to previous results
when checking the same chambers in other
exper-iments (range: 0±19210 mmol N mÿ2 hÿ1; Seitzinger
et al., 1980; Seitzinger 1987; Seitzinger et al., 1993). Although signi®cant, these rates are small compared to
most of the total N2¯uxes measured in the current
ex-periments.
In the current experiments, average net N2 ¯uxes
ranged from 10 to 245 mmol N mÿ2hÿ1 (Fig. 4), and
the concentration of available soil N indicates that the rates are plausible. For the mineral soil cores, the
NO3ÿ pools present at the outset of the experiments
were adequate to support the observed denitri®cation rates for 2±7 days (Watts S., 1977, PhD thesis, Rutgers
University). In contrast, for the organic soils, the NO3ÿ
pools at the outset of the experiment could support the observed rates for only a few hours (Watts, loc. cit). However, nitri®cation could supply additional
NO3ÿ during incubation, and the NH4+ to support
nitri®cation could come from existing NH4+ pools or
from N-mineralization. In the mineral soils,
nitri®ca-tion of available NH4+ could support the measured
denitri®cation rates for an additional 4±25 days. In half of the organic soil cores, there was enough
avail-able NH4+ to supply NO3ÿ for 1±7 days. In addition,
N-mineralization rates were adequate to supply the required N on a daily basis (Watts, loc. cit). In aquatic sediments, which we assume are fairly similar to
satu-rated soils, nitri®cation of NH4+ released during
miner-alization of organic nitrogen has been shown to supply
up to 80% of the NO3ÿ available for denitri®cation
(Seitzinger, 1987, 1994). The N-mineralization rates in the organic soils were at least ten times greater than in
mineral soils, producing NH4
+
at rates ranging from
12.5 to 32 mmol NH4+ coreÿ1dayÿ1 (Watts, loc. cit).
This could theoretically support
nitri®cation/denitri®-cation rates of roughly 130±300mmol N mÿ2hÿ1,
ade-quate for the observed N2¯ux denitri®cation rates.
3.2. Comparison of rates by acetylene inhibition and N2
¯ux
For comparative purposes, denitri®cation rates of replicate cores were averaged for each study site by
season (days 3±7). By the N2¯ux method, average
sea-sonal rates for the organic soils ranged from 10 to 245
mmol N mÿ2hÿ1and in the mineral soils ranged from
about 35 to 130 mmol N mÿ2hÿ1(Fig. 4). The highest
mineral soil denitri®cation rates in the present
exper-iment (about 0.5 kg N haÿ1dayÿ1) are similar to `tail'
denitri®cation rates measured after peak response to
NO3ÿand glucose amendment in clay soils by a similar
method (Schole®eld et al., 1997b). By the acetylene in-hibition method, the range of seasonal averages for
denitri®cation in mineral soil (0.1±10 mmol N mÿ2
hÿ1) (Fig. 4) was one to two orders of magnitude less
than N2 ¯ux rates. In the organic soils the seasonal
range (0.2±3.0 mmol N mÿ2 hÿ1) was also one to two
orders of magnitude less than N2 ¯ux determinations
of denitri®cation. The measured ¯ux of N2O (without
acetylene addition) was <2 mmol N mÿ2 hÿ1 in
min-eral soil and <1 mmol N mÿ2hÿ1in saturated soils.
4. Discussion
4.1. Adaptation of N2¯ux method for soils
Our experiments demonstrated that the N2 ¯ux
method was adaptable for terrestrial soils, both min-eral and water-logged organic soils, and that
denitri®-cation rates based on N2 ¯uxes could be measured
after only 2 days of incubation in most cases. This method maintains physical conditions similar to in situ conditions without added nutrients or inhibitors. Using a model to estimate the rate of diusive ¯ux of
atmospheric N2 from soil porespace, we demonstrated
the ability to measure biologically-produced N2. A
drawback of the N2¯ux method is the limited number
of cores (8±12) that can be studied per experiment due to the large number of gas-chromatographic measure-ments per day. A possible modi®cation would be to study one site per week instead of two sites, and one could possibly shorten the experiment by replacing the headspace gases every 2±3 h during the ®rst day to accelerate the out-gassing of atmospheric nitrogen.
Another limitation of the N2¯ux method is the
detec-tion limit of 20 mmol N mÿ2hÿ1which will restrict its
atmosphere method: 50 g N haÿ1 dayÿ1 or 15mmol N
mÿ2hÿ1(Schole®eld et al., 1997). Also, there is
poten-tial for enhancement of denitri®cation rates during the 7-day incubation due to both decreased competition for inorganic nitrogen from plants or from changes in N-utilization by the microbial community. Physical
disturbance has also been shown to increase N2O
pro-duction (Matthias et al., 1980). However, in the pres-ent study, a signi®cant disturbance eect was not evi-dent; there was no consistent increase or decrease in denitri®cation rates during the 7-day incubation that would indicate a sustained disturbance eect. Disturb-ance eects are not unique to denitri®cation measure-ments and should be considered when studying any microbial soil process. In situ measurements would be ideal for measurement of soil processes, however, deni-tri®cation methods are not yet available for terrestrial systems.
4.2. Comparison of rates by acetylene inhibition and N2
¯ux
Denitri®cation rates measured by the acetylene inhi-bition method were one to two orders of magnitude
less than rates measured by the N2¯ux method (Fig. 4)
in both high-moisture organic and relatively dry min-eral soils. Underestimation of denitri®cation by the acetylene inhibition method was expected in the water-logged, nutrient-poor soils for several reasons: (1) poor penetration of the inhibitor into the soil microsites (Rudolph et al., 1991), (2) inhibition of nitri®cation which may be a major source of nitrate when nitri®ca-tion and denitri®canitri®ca-tion are closely linked (as in the
current low-nutrient organic soils) (Hynes and
Knowles, 1978, 1982), and (3) incomplete inhibition
due to soil nitrate concentrations <10 mM
(corre-sponding to 75 nmol N gÿ1 dry weight) (Rudolph et
al., 1991). In the present study, extractable soil nitrate
concentrations ranged from 39 nmol N gÿ1dry weight
(spring) to 72 nmol N gÿ1 dry weight (summer)
(Watts, loc. cit).
In contrast, the denitri®cation rates determined by
N2 ¯ux and acetylene inhibition were expected to be
similar for low moisture mineral soils. Here, the pen-etration of the inhibitor should be more even. (The dif-fusive ¯ux model suggests that in drier soils, gases in the soil porespace and headspace would be in equili-brium within 2 h, from day 1.) In addition, the mineral
soils had extractable nitrate concentrations >10 mM
(Watts, loc.cit) suggesting that C2H2 inhibition should
be complete. Also, with nitrate concentrations at that level, it was not likely that nitri®cation was a
signi®-cant source of NO3ÿ, and so inhibition of nitri®cation
should not have a substantial eect. These conclusions are based on the assumption that bulk soil
measure-ments accurately re¯ect soil NO3ÿconcentrations at the
microsite level.
Other studies have demonstrated disparities between
acetylene inhibition rates and other types of N2 ¯ux
measurements. For example, ¯ux rates based on 15N2
production from 15NO3
ÿ
(measured by mass spec-troscopy) were found to be about 1.5 times greater than the rates measured by acetylene inhibition (measured by gas chromatography) in ®eld plots planted with ryegrass (Arah et al., 1993). Studies com-paring denitri®cation methods in either saturated or organic soils are generally lacking, but a comparative study in lake sediments is analogous. Denitri®cation
rates measured by acetylene inhibition were <1 mmol
N mÿ2hÿ1, while rates measured by N2¯ux and 15N±
N2 production from 15NO3ÿ were approximately 200
mmol N mÿ2 hÿ1 (Seitzinger et al., 1993). In the
or-ganic soils of the current study, N2¯ux denitri®cation
rates were one to two orders of magnitude greater than acetylene inhibition rates. Pairwise t-tests demon-strated no similarity between the two methods at any
of the four sites (p< 0.05). Correlation analysis (SAS/
STAT, SAS Institute) on pooled data indicated no statistically signi®cant relationship between the
denitri-®cation rates measured by the N2 ¯ux and acetylene
inhibition methods. Correlations were also tested sep-arately by site, soil type, and season, and again there was no statistically signi®cant relationship between the two methods.
If only the acetylene block method had been used in this study for denitri®cation measurements, erroneous conclusions would have been made, not only in absol-ute rates as discussed above, but in relative rates between soil types. Based on the assumption that the physical environment of waterlogged soils would be more anaerobic than drier mineral soil, one would expect greater denitri®cation rates in waterlogged soil.
Results of the N2 ¯ux method supported this
expec-tation. The highest mean seasonal denitri®cation rates for summer and fall, 1994 did occur in waterlogged soils; in spring 1995 one mineral soil site had rates similar in one organic soil site (Fig. 4). However, acetylene inhibition showed that the highest rates occurred most often in drier mineral soils (spring 1994, fall 1994, spring 1995) (Fig. 4).
4.3. Spatial and temporal variability
In the current study, we observed that N2 ¯uxes,
and, hence denitri®cation rates, varied both temporally (day to day within a single core) and spatially (core to
core between replicates taken from a single 0.5 m2
site). Such variability in rates measured by the N2¯ux
(Smith and Parsons, 1985; Hixson et al., 1990; Merrill and Zak, 1992; Groman et al., 1992). For example, coecients of variation up to 508% for spatial varia-bility have been recorded (Folorunso and Rolston, 1984). In the current study, the coecients of variation
for N2 ¯ux were 15±200% for the mineral soils and
70±200% for the saturated organic soils.
An example of the spatial variability of denitri®ca-tion rates was demonstrated by the paired cores taken from site two of the forested buer (Table 1) where
the N ¯ux on day 5 of one core was >170 mmol N
mÿ2 hÿ1, but there was no similar large ¯ux in the
paired core on any day of the experiment. From day to day, nitrogen ¯ux from each core varied, but demonstrated no consistent pattern of increasing or decreasing N ¯ux between days 3 and 7 (only two cores over the four seasons demonstrated a signi®cant decreasing trend over time). Day-to-day variability of
<20 mmol N mÿ2 hÿ1 in daily denitri®cation rates is
not signi®cant due to the limit of analytical precision
(<20 mmol N mÿ2 hÿ1). However, in numerous cases
the day-to-day variability was much greater than 20
mmol N mÿ2 hÿ1(Table 1). For example, in one
min-eral soil core (site two, newly-planted) rates increased
from no net ¯ux on day 3 to 106mmol N mÿ2hÿ1the
next day, and then back down to no net ¯ux within 2
days. Similar variability was observed for the saturated organic soils (McDonalds Branch, site one) where the denitri®cation rate was very low for days 3 through 6
and then increased to 42 mmol N mÿ2hÿ1on day 7.
There are several explanations for the apparent tem-poral and spatial variability. One explanation is that larger ¯uxes are due to the delayed release of trapped atmospheric nitrogen. However, the diusive ¯ux cal-culations illustrated that in porous soil, gases diuse and equilibrate quickly, even when soil is relatively moist. Another explanation for short-term temporal variability of denitri®cation rates has been attributed to denitri®cation `hot spots' that are created in the soil column during the decomposition of particulate or-ganic material (Parkin, 1987; Christensen et al., 1990). As organic carbon breaks down, organic and inorganic N are released, and utilizable carbon substrates become available. As a result, respiration increases, oxygen consumption increases, thereby creating
an-aerobic conditions and enhancing denitri®cation
(Tiedje, 1988). In our experiments, the `bursts' were up to two orders of magnitude greater than any other daily rate from that core and were limited to a single day (Table 1). (The duration of `burst' activity in the current study was short-lived compared to the results of another study where the `burst' activity lasted over several days and was attributed to the decomposition of rotting plants and/or increased availability of or-ganic C from root exudates (Christensen et al., 1990)). The uneven distribution of organic C can also contrib-ute to the spatial variability of denitri®cation rates as seen when some cores in a group of replicate cores demonstrate ¯ux rates that are several times greater than those of the other cores taken from the same area (Christensen et al., 1990).
Acknowledgements
Many thanks to Mark Morgan, whose grant from the National Science Foundation, supported the work in the Pinelands and also to Denis Newbold, whose grant from the Chesapeake Research Consortium, sup-ported the work at Georgia Farm. Many thanks also
to the anonymous reviewers, whose comments
strengthened this manuscript.
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