FEMS MicrobiologyEcology102 (1993)225-234
© 1993 Federation of European MicrobiologicalSocieties0168-6496/93/$06.00
Published by Elsevier
225
FEMSEC 00439
Control of denitrification enzyme activity
in a streamside soil
Per Ambu8
Institute of Population Biology~ Copenhagen University, Copenhagen, Denmerk
Received 25 September 1992
Revision received 25 November 1992
Accepted 25 November 1992
Key words: NO 3 and NO~- reduction; Chloramphenicol; Kinotic parameter ( K m and Vm~x);
Temperature control; Substrate availability; Denitrification
1. SUMMARY
Progress curve analysis of NO 3 and NO~ reduction in surface soil samples from a streamside
soil gave K m values of 4.24 and 6.33 tzM, and
Vma~ values of 2.16 and 1.83 /zmol 1- I min -~,
respectively. Recoveries of reduced NO 3 and
NO~- as gaseous N averaged 82 and 108%. The
unrecovered N O 3 - N was presumably dissimilated
to NH~-N. The denitrification enzyme activity
(DEA) was examined throughout a year and
showed seasonal and spatial variabilities of only
10% to 26%, suggesting a high persistency of
denitrifying enzymes. Soil moisture and D E A
correlated significantly (r = 0,7671; P < 0.01), The
D E A in saturated subsoil also showed a relatively
little variation, with spatial variabilities of between 28 and 38%, Amendment with NO 3 rarely
enhanced the activity more than two-fold at either depth. Addition of glucose increased the
activity 2.3 and 2.5 times in the surface soil and
Correspondence to: Per Ambus, Institute of Population Biology, Copenhagen University,Universitetsparken 15, DK-2100,
Denmark.
subsoil respectively, indicating a moderate carbon
limitation of denitrification. The activation energy of D E A was found to be 64.9 kJ t o o l - t and
Qt0 values for the 2-12°C and 12-22~C temperature ranges were 2.71 and 2,53, respectivel~/, Extrapolation suggested tll0jFe would be a 4.4-fold
increase in D E A i( ~ ¢ tenaper~,t~ro was changed
from 0 to 15~C. Sttb,~t~a~ diffusion limited the
denitrifioa.tion 10 to 25 fold.
Thus, under anaerobic moist conditions it appears that changes in denitrification might primarily be due to varying diffusion of substrates
into the anaerobic soil centers. Over a year, fluctuations in DEA, temperature changes and fluctuations of electron-acceptor and -donor supply
will only have a minor effect on natural denitrification activity.
2. I N T R O D U C T I O N
R e c e n t studies have d e m o n s t r a t e d that
streamside soils have the capacity to support high
rates of biological denitrifieation [1-3] which may
be an important sink for NO 3 from agricultural
discharge, Yet, knowledge about the dynamics
226
and the control of denitrification in ecotones
between terrestrial and aquatic systems is incomplete.
Soil denitrification is primarily regulated by (a)
oxygen, (b) N O 3, and (c) available carbon [4].
The process is highly variable and the ability to
predict the extent of denitrification is limited.
The level of active denitrifiers is a combined
product of a variety of soil processes [5], and is
usually expressed as the potential denitrification
enzyme activity ( D E A ) given by Smith and Tiedje
[6]. D E A should therefore provide valuable information about the combined effect of interaction
between the various biological, chemical and
physical factors known to influence denitrification. D E A has successfully been used as a parameter for modelling denitrification spatial variability [7] and annual denitrification N-loss at the
landscape level [8], but it may [9] or may not [8,9]
be a reliable predictor of daily denitrification
activity.
The aim of this study was to identify key
factors in the regulation of denitrification activity
in a streamside soil. Investigations were made
into (a) N O 3 and N O 2 reduction kinetics ( K m
and Vma~); (b) the seasonal variation of D E A and
substrate availability; (c) the temperature effect
on DEA; and (d) the magnitude of diffusion
limited denitrification.
3. M A T E R I A L S A N D M E T H O D S
3.1. Study site
The study was carried out in a narrow riparian
strip bordered by a small stream on one side and
by agricultural fields on the other. The site was
located in a quaternary morainic landscape 50 km
south-west of Copenhagen. The vegetation was
Table 1
Characteristics of riparian soil
0-5 cm
5-0 cm
15-20 cm
PHH20
7.3
7.4
7.0
a Loss on ignition.
% tot-N
2.74
2.58
2.37
% tot-C
33.76
33.02
29.69
% LOI
65.3
56.7
57.5
a
dominated by Baldfngera arundindcea and Carex
sp. Soil characteristics are listed in Table 1. In the
period from October to May, water often formed
stagnant pools in inter-tussock depressions. Soil
samples were collected at 4 locations, 1, 12, 23
and 34 metres from the stream.
Kinetics of NO 7 and NO 7 reduction
Surface 0 - 1 0 cm samples were collected at the
4 locations in August 1990, composited and stored
under field-moist conditions at 4°C until analysed. Analysis took place within two weeks. Breitenbeck and Bremner [10] showed that storage of
field-moist soil samples at 4°C for up to 30 days
did not alter the denitrification capacity. The soil
was thoroughly mixed and bulky residual plant
material was removed. Soil slurries were prepared by adding 15 g field-moist soil to 120 ml
serum bottles containing 70 ml of phosphate
buffer (40 mM; p H 7.3) with 1 g 1-1 of glucose-C.
The serum bottles were sealed, evacuated and
flushed with N 2 three times, and 12 ml of the
headspace was replaced with pure C2H 2. After
15 min, 5 ml degassed solutions of K N O 3 or
N a N O 2 were added to triplicate flasks to give
final concentrations of 150 or 100 /zM, respectively. Soil slurry portions of 3.0 ml were removed
at 10-min intervals using a 13G x 4 needle permanently positioned through the stopper and
closed off with a 3 way stopcock. The 3.0 ml soil
slurry samples were immediately added to centrifuge tubes containing 3.0 ml of 100% C H 3 O H
to stop denitrification activity. The samples were
then centrifuged (30 min; 3800 x g) and the supernatant was passed through a 0.45 /zm filter
before being analyzed for N O 3 and N O ~ . Preliminary investigations showed that N O 3 reduction and denitrification in anaerobic soil slurries
with 50% C H 3 O H was repressed by 96% for at
least 2 h.
In addition, 1-ml headspace samples were
taken from the serum bottles to measure the
accumulation of N20. The gas samples were
stored in 3.5-ml Venojects® preflushed with pure
N 2•
After each sampling, 4.0 ml of 10% C 2 H 2 in
N 2 was added to the headspace to replenish the
volume sampled. The incubation was performed
227
at room temperature for 90-120 min and all
flasks were shaken by hand every 5 min.
The kinetic parameters K m and Vm~x were
estimated by fitting the NO 3 and NO 2 depletion
data to the integrate form of the MichaelisMenten equation [11] by using P R O C NLIN of
SAS [12].
Chloramphenicol interfered strongly with the
N O 3 / N O 2 analysis and could not be included
in this experiment. For that reason, the extent of
denitrifier growth was estimated from patterns of
N 2 0 evolution in the absence and presence of
growth inhibitor. Three flasks received 15 g
field-moist soil and 30 ml of a solution containing
chloramphenicol (1 g 1-1), KNO3-N (0.2 g 1-1)
and glucose-C (2 g L -1) (see (iv) below); 3 flasks
received the same but without chloramphenicol.
The flasks were flushed with N 2 and amended
with CEH 2 as described above, and the headspace
was analyzed for N 2 0 every 15 rain for 2 h.
slurries were incubated at room temperature and
shaken thoroughly by hand every 15 min; 3 1-ml
gas samples were taken at regular intervals over
30 to 60 min.
Subsamples (20 g each of field-moist soil) were
extracted with 40 ml of deionized water (rotary
shaker; 1 h). Portions (20 ml) of the water extracts were centrifuged and filtered as described
above and analyzed for N O 3 , NO~- and watersoluble carbon.
3.3. Temperature response
Subsamples (15 g each) of field-moist soil were
mixed with 30 ml of solution (iv) (see above),
buffered with 40 mM of phosphate (pH 7.3) and
preadjusted to 2, 6, 8, 15 or 23°C. The soil slurries were flushed with N 2 and amended with
C2H2; triplicate flasks were incubated at each of
the above temperatures. The flasks were shaken
and sampled for N 2 0 analysis at 20-min intervals
over a 160-min incubation period.
3.2. Denitrification enzyme activity (DEA)
Soil samples for examination of D E A and substrate control of the denitrification activity were
collected on 13 October in 1988 and on 16 January, 8 May and 4 September in 1989. The samples were taken with an auger from the surface 5
cm and from the top 5 cm of the saturated soil
layer. The water table was 5 cm below the surface
in October and 15 cm below in May and September. In January the whole area was flooded and
soil was consequently collected only from the
surface 5 cm. Samples from each location and
depth were treated separately and were stored as
described above, usually for less than 3 days.
After removing residual plant material, soil
slurries were made by mixing 10-20 g field-moist
samples with 20 ml of one of four solutions: (i)
chloramphenicol (1 g l-1); (ii) chloramphenicol (1
g 1-1) and KNOa-N (0.2 g 1-1); (iii) chloramphenicol (1 g L - I ) and glucose-C (2 g 1-1); and
(iv) chloramphenicol (1 g 1-1), KNO3-N (0.2 g
1-1) and glucose-C (2 g L - l ) . Solution (iv) is a
modification of the denitrification enzyme activity
assay solution used by Smith and Tiedje [6]. Triplicate incubations were performed in 120-ml
serum bottles which were flushed with N 2 and
amended with C2H 2 as described above. The
3.4. Suspension experiment
Sixteen soil cores 15 cm in length were collected in PVC tubes 30 cm long by 4.5 cm inner
diameter. The tubes were sealed at the bottom,
and brought to the laboratory for injection with
30-ml portions of deionized water containing either (i) chloramphenicol (3 g I-1); (ii) chloramphenicol (3 g 1-1) and KNO3-N (0.17 g 1-1); (iii)
chloramphenicol (3 g 1-1) and glucose-C (1.8 g
1-1); or (iv) chloramphenicol (3 g I-1), KNO3-N
(0.17 g 1-1) and glucose-C (1.8 g 1-1). The solutions were injected along the length of each of 4
replicate cores, and they were followed by 30 ml
of C2H2, which was also injectedalong the length
of each core. By the addition of the 30-ml portions the soil moisture content ended up at 104%
water filled porespace. The samples were preincubated under anaerobiosis for 16 h. The preincubation took place in a 60-L jar containing 10
kPa CEH 2 in pure N 2 and was performed to
allow dispersion of CEH 2 into the soil pores.
Following preincubation the PVC tubes were immediately capped and 1-ml headspace samples
were removed at hourly intervals over 2 h to
determine the N 2 0 accumulation at room temperature. After gas sampling, the cores were
228
Table 2
H 2 0 , N O 3 - N , and water soluble C concentrations at two
depths in a riparian soil on four occasions
H20
(kg k g - ')
NOa-N
(mg k g - l)
COD-C
(mg k g - 1)
Pet 13, 1988
0 - 5 cm
5 - 1 0 cm
3.40
2.45
ND
ND
348.3
229.2
J a n 16, 1989
0 - 5 cm
4.44
8.5
May 8, 1989
0 - 5 cm
15-20 cm
3.94
2.19
13.1
6.8
392,7
34~.6
Step 4, 1989
0-5 m
15-20 cm
3.34
2.39
12.7
5.8
517.3
323.9
flasks were sealed and 50 ml of the headspace gas
was reptit~,d with pure C 2 H 2. The denitrification
l~tes were determined from the amount of N20
evolved at 0, 1 and 2 h by the stirred slurries.
3.5. Analysis
N20 was analyzed on a gas chromatograph
a Porapak-Q column and 63Ni
EC-d~tector (Shimadzu GC-8A).
F | ~ j ~l Samples of the filtrates were analyzed
for NO 3 and NO 2 on a HPLC operated with an
anion column (Waters, IC-PAK TM) and 5 mM
phosphate buffer at pH 9 as solvent. NO~- and
NO2 was detected by UV-absorption at 210 nm.
The detection limit by this method was about 0.5
~mol 1-1 of N.
The chemical oxygen demand (COD) of the
soil extracts was determined from wet-oxidation
with dichromate [13]. The COD are expressed as
glucose-C equivalents. Inorganic N and COD are
given on a dry-weight basis (105°C, 24 h). The soil
variables are listed in Table 2.
operated with
ND
ND, not determined.
transferred to 500-ml Erlenmeyer flasks and suspended in 200 ml deionized water. The slurries
were flushed for 1 min with pure N 2 (1 1 s-i), the
2.5
4. RESULTS AND DISCUSSION
• no chloramphenicol
0 with chloramphenicol
i
4.1. K m and I/ma x o f N O 3 and N O 2 reduction
Z
1.5
//'
/6
%`
z
0.5
o.o
/~
0
20
40
610
810
'
100
'
120
140
minutes
Fig. 1. N 2 0 evolution in soil slurries with and without chloramphenicol. Vertical bars indicate two standard errors, n = 3.
W h e r e no error bars are shown the S.E. was less than size of
symbol. The dashed lines shows the position of the best fit to
a sequential linear-exponential expression: Y = a - ( t ~ T ) + b,
t <T, and Y = ( a . ( t - T ) + b
) . e ( r . ( t - T ) ) , T < t < 1 2 7 . In
non-chloramphenicol flasks a, b, r, T (min), and R 2 values of
0.013, 0.672, 0.005, 52 and 0.996 were obtained. In chloramphenicol a m e n d e d flasks the N 2 0 evolution was apparently
steady for t < 127 min, and a, b, T and R 2 values of 0.014,
1.857, 127 and 0.986 were obtained.
N20 evolution from soil slurries without chloramphenicol suggested a biphasic pattern as com.
pared with the chloramphenicol amended soil
(Fig, 1). The N20 evolution pattern in non-chloramphenieol flasks is in accordance with the observations of Smith and Tiedje [6] and reflects
derepression of de novo enzyme synthesis, in the
present study, denitrification enzyme activity in
non-chloramphenicol amended soil was constant
during about one hour. The Michaelis-Menten
expression is only valid under steady-state conditions, so efforts to retard microbial growth must
be taken. Here, the soil/solution ratio in the
samples was adjusted to minimize the duration of
the experiment; the initial steady phase indicates
that the N O f - and NO 2 reduction will not have
been accelerated by an increase in biomass during the course of the experiment. The experiment
also indicated that the denitriflcation enzyme activity was not affected by the presence of chip-
229
200
Aceumulotion I of N20 from NO3 . and NO2 V
Reduction of NO:~ • and NO2 0
150' ~\
\
"6
\
o
t
0
35
~
"-'~J=c
70
c'
105
c
c
140
minutes
Fig. 2. Progress curves of N O 2- and NO 3 reduction and
accumulation of N 2 0 from N O 2 and NO~- reduction in
slurries of surface soil. Vertical bars indicate one standard
error, n = 3. Where no error bars arc shown the S.E. was less
than size of symbol.
ramphenicol which is in accordance with the finding of Smith and Tiedje [6] but opposed to the
results in a recent study by Brooks et al. [14].
Analysis of the progress curves (Fig. 2) gave
K_.m values of 4.24 + 4.96 and 6.33 __+1.03 /xM
(X _+S.E.), and Vm~x values of 2.16 + 0.22 and
1.83 + 0.08 /zmol 1-1 min -1 for NO~ and NO2
reduction, respectively. Recoveries of NO 3 and
NO~- as N20 averaged 82 and 108; %, respectively (Fig. 2), indicating that whilst NO2 reduction occurred entirely through denitrification,
other processes contributed to the reduction of
NO~-. In the NO3-amended flasks, the appearance of NO~- was transient (data not shown), as
expected from the differences in VmaxThe high affinities for both N O f and NO2
utilization found here are in accordance with K m
values ranging between 1.7 and 50 ~M reported
for soils [15], sediments [15-17] and pure cultures
[18-20], although a K m of 450 lzM has been
reported from a study with soil slurries [21]. A
high affinity for NO 3 utilization enables the bacteria to grow on the low concentrations of NO~usually found in habitats like mires [22] and riparian forests [23]. Even in systems receiving high
NO~- inputs, anaerobic zones may exhibit low
NO~- concentrations when the NO 3 consumption rate exceeds the supply by diffusion [17,21].
In the present soil, bulk NO 3 concentrations
rarely exceeded 200/zM (data not shown) which
makes it likely that the NO 3-reducing community
was exposed to NO 3 concentrations in the firstorder range [21]. Such conditions make it difficult
to relate the results obtained from stirred soil
slurries with homogeneous and relatively high
NO~- concentrations to the field situation. The
outcome of competition for NO~- between denitriflers and other NO 3 reducers is strongly affected by changing the NO 3 concentration, especially in the first- and mixed-order ranges which
favour denitrifiers [24]. Thus, it may well be that
denitrification in this community is responsible
for appreciably more than the apparent 80% of
the total NO3 reduction observed in the slurry
study (Fig. 2).
The low recovery of NO 3 in N20 could be
due to the presence of other NO 3 reducing processes or an incomplete blockage of the nitrous
oxide reduction [25,26], or both. But since N20
accumulated in stochiometric amounts from
NO 2, it seems most likely that the missing N
from NO 3 might be accounted for in the form of
intracellular and biomass-incorporated N or as N
dissimilated to NH~-. The chloramphenicol experiment discussed above indicated that no growth
occurred during the relatively brief incubation,
and a separate tracer study showed that NO 3
was not assimilated during anaerobiosis [27]. The
tracer study also showed that net production of
NH~- was responsible for about 6% of the dissimilatory NO 3 reduction, suggesting that the NO 3
not recovered in N20 in this experiment was
reduced to NH~-. This does not explain why NO 2
amendment did not result in N-loss since non-denitrifying soil bacteria are also capable of reducing NO 2 to NH~- by dissimilation. However,
dissimilatory NO~- reduction to NH~- takes place
apparently only following prolonged growth under NO3-free conditions when NO~- is in excess
[28]. Under natural conditions, NO£ concentrations are usually in the nM range at the present
study site and the indigenous community may not
be adapted to carrying out dissimilatory reduction
of NO 2 to NH~-.
230
20
A
7
20
0 oct
• jan
• may
I
sep
/" / t~ /
~T-- ~ ..~
15
////
10
oct
jan
may
aep
16
7
Z
I
..
0
•
•
A
\
5/
7
\
t
Z
[
12
/
/
Oe~
Z
ca
Z
E
A~
8
/
0
1,
ll2
metres from
2'3
A
4-
3'~
25
310 315 ,i0
stream
Fig. 3. Denitrification enzyme activity at 4 dates in surface soil
samples collected at different distances from the stream.
Vertical bars indicate two standard errors, n = 3. Where no
error bars are shown, the S.E. was less than size of symbol.
4.2. Variability of DEA and substrate control
T h e d e n i t r i f i c a t i o n e n z y m e activity in the surface soil varied b e t w e e n 5.2 a n d 17.9 mg N 2 0 - N
k g - t h - 1 , showing the highest a n d lowest rates in
J a n u a r y a n d S e p t e m b e r respectively (Fig. 3). T h e
seasonal variability of D E A in the surface soil
c o r r e s p o n d e d to a coefficient of variation (CV) of
26% ( n = 4) a n d the spatial variability corres p o n d e d to a C V of b e t w e e n 10 a n d 23% ( n = 4
o n the 4 occasions). O f the variables e x a m i n e d
(Table 2), it a p p e a r e d that D E A in the surface
soil was significantly r e l a t e d to the soil water
c o n t e n t (Fig. 4).
D E A in the subsurface soil varied b e t w e e n 1.3
a n d 6.1 mg N 2 0 - N kg -1 h -1 ( T a b l e 3). Since
s a m p l i n g d e p t h varied according to season a n d
D E A d e c r e a s e d m a r k e d l y with increasing d e p t h
(data not shown) it is n o t possible to c o m m e n t o n
seasonal variability in subsurface soil activity.
However, w h e n samples were collected at the
same depth, i.e., in May a n d S e p t e m b e r , the
D E A differed significantly only at o n e location
(Table 3). T h e spatial variability c o r r e s p o n d e d to
a CV of b e t w e e n 28 a n d 38% which is slightly
higher c o m p a r e d with the surface variability.
A d d i t i o n of 0.2 g I-1 NO~--N to surface samples caused a two-fold increase in activity u n d e r
b o t h a m b i e n t a n d h i g h - c a r b o n c o n d i t i o n s (N
,15
510 55
kg H20 kg-1
Fig. 4. Relationship between surface denitrification enzyme
activity and soil moisture (r = 0.767; P < 0.01). Horizontal
and vertical bars indicate two standard errors (n = 3). Where
no error bars are shown the S.E. was less than size of symbol.
The dashed line show the position of the geometric mean
regression line.
a d d e d / n o a d d i t i o n ratio, a n d N a n d C a d d e d / C
a d d e d ratio; T a b l e 4). It was only in J a n u a r y ,
w h e n a m b i e n t N O 3 was relatively low in the
surface soil (Table 2), that the m e a n effect of
N O 3 a d d i t i o n exceeded a t w o - f o l d increase in
activity (data n o t shown). I n the subsurface, N O 3
Table 3
Denitrification enzyme activity in saturated riparian subsurface soil at different distances from stream on three occasions
Depth and
date
Meters from stream
1
12
23
34
mg N20-N kg- 1 h - l
Oct 13, 1988
5-10 cm
4.4 b
4.6 b
3.1 c
6.1 a
3.3 aA b
3.5 aA
1.7 hA
1.8 bA
2.3 abA
1.3 cB
1.7 bcA
2.4 aA
a
May 8, 1989
15-20 cm
Sep 4, 1989
15-20 cm
Within same sampling date, means followed- by the same
lower-case letter are not significantly different (Duncan's
multiple-range test; P < 0.05).
b Within same depth and distance from stream, means followed by the same capital letter are not significantlydifferent (t-test; P < 0.05).
a
231
Table 4
Effects on denitrification in anaerobic slurries followingtreatment with different combinations of substrates
Treatments compared
Surface
Ratio of rates
between treatments
a
N added/no addition b
N and C added/C added
N and C added/N added
N and C added/no addition
1.7 + 0.4 c
2.0 + 0.6
2.3 + 0.l
4.0 + 1.0
Subsurface d
N added/no addition
N and C added/C added
N and C added/N added
N and C added/no addition
1.1 + 0.1
1.4 + 0.2
2.5 + 0.3
2.5 + 0.3
a n = 16. Four locations on 4 dates.
b N added = 0.2 g 1-1 KNO3-N;C added = 2.0 g 1-I glucoseC; N and C added=0.2 g 1-1 KNO3-N and 2.0 g 1-1
glucose-C.
e Mean + standard error of the mean.
d n = 12. Four locations on 3 dates.
addition caused increases in activity below a factor of 1.4 at both ambient and high carbon levels
(Table 4). Nitrate amended samples, from surface
and subsurface, gave an average 2.4-fold increase
in activity when 2 g I-1 of glucose-C was added
(N and C a d d e d / N added ratio; Table 4). The
carbon effect in September was the highest observed: a 2.8-fold and 3.9-fold activity increase in
surface and subsurface samples, respectively.
Adding both N O 3 and glucose increased the
activity about two-fold more than either substrate
alone in samples from the surface (N and C
a d d e d / n o addition ratio; Table 4). In subsurface
samples the effect of adding N and C simultaneously was similar to the effect of adding glucose
alone, but two-fold higher than the N O 3 effect.
The seasonal and spatial variability of the D E A
was remarkably low compared with the variability
of natural denitrification found in this habitat [41]
and other soil ecosystems [7,29-31]. The low variability of D E A demonstrates that bacteria maintain their denitrifying enzymes under non-denitrifying conditions [5,9,32], which may make it
impossible to use D E A to predict the short-term
dynamics of naturally-occurring denitrification in
such a system. Smith and Parsons [32] suggested
that the rate of enzyme synthesis is less important
than the activation-inactivation of existing denitrification enzymes by fluctuating soil aeration
(moisture). On a long-term scale, soil moisture is
seemingly a good indicator of DEA, as also observed by Parsons et al. [33]. In January, D E A
increased with increasing distance from the
stream (Fig. 3). The reason for this is not obvious.
However, effluent soil water from the adjacent
agricultural area may expose the microbial community at the field margin to higher concentrations of substrates compared with the microbial
community at the stream margin, although this
pattern could not be derived from any of the
variables analysed.
Generally, the limitation of the activity imposed by carbon availability was greater than that
imposed by N O ; availability. At both depths the
bulk N O 3 concentration appeared to be in the
zero to mixed-order range and no profound N O 3
limitation was observed. The consistent two- to
three-fold increase in activity upon glucose addition indicated that easily-degradable carbon might
be the limiting factor in this organic soil, yet, the
carbon limitation was never very intense. Carbon
limitation has been found in other organic [34]
and mineral soils [6,23,35]. The highest level of
water-soluble carbon occurred in September (Table 2), presumably because of decaying plants.
Since this coincided with the highest glucose response, water-extractable carbon seems to be a
poor measure of available carbon. However, the
data does not rule out other possibilities. Raised
levels of water-extractable carbon might have induced an increase in the microbioal population
size which would give a higher activity upon glucose addition.
The results given here provide strong evidence
that denitrification in this type of riparian system
is a potential NO 3 sink since a high DEA, in
both surface water and shallow groundwater, is
maintained throughout the year. Yet, available
carbon is a moderate limiting factor.
4.3. Temperature response of DEA
A plot of Log e (DEA) obtained at different
temperatures versus the reciprocal absolute temperature (Fig. 5) suggests that the temperature
232
6
O
8''r-~
v
O
4-
"-
0
"-
O
-x
O
"-.O
3:
2
3,35
I
3.40
I
3,45
I
3,50
I
3.55
I
3.60
3.65
1000 * T -1
Fig. 5. Arrhenius plot o f denitrification enzyme activity. The
dashed line show the position of the regression line (r = 0.929;
P < 0.001).
response can be described by the Arrhenius
equation over the temperature range examined.
The study focused only on temperatures likely to
occur in the field and consequently no attempts
were made to determine the temperature for
maximum activity. An activation energy of 64.9 kJ
mol-~ for the denitrification enzyme activity was
calculated from the slope of the regression line,
equal to Ea/R, where E a is the activation energy
and R is the universal gas constant. By interpolation, Q10 values of 2.71 and 2.53 were obtained
for the 2 to 12°C and 12 to 22°C temperature
ranges respectively. It can also be calculated that
the activity decreases about 4.4 times when the
temperature is lowered from 15°C to 0°C.
The activation energy and the corresponding
Q10 values are within the range of temperature
coefficients reported by other workers. McKenney et al. [36] found E a values of between 50 and
55 kJ mo1-1 and a mean Q10 of 2.1(15_25) for
NO~- reduction in 2 mineral soils, and Jacobson
and Alexander [37] reported a Q10 of 1.9(15_30)
for NO 3 loss in a sandy loam. In anaerobic
slurries from an alder swamp E a and Q10 for
N 2 0 evolution varied between 70 and 76 kJ
mo1-1, and 2.8 and 3.0(0_25), respectively [38].
Hence, it seems as if denitrifiers from different
communities respond differently to temperature
changes and that the highest activation energy
may apply to denitrifiers in organic soils. Different responses to temperature may also be due to
adaption of denitrifying populations to different
local temperatures [39]. Yet, the alder swamp [38]
was situated less than 70 km from the field site
used here. According to Focht and Verstraete
[40] it is most likely that disparate temperature
effects can be explained by differences in the
amount and quality of organic substrates.
The present data show, that the temperature
effect is more important than the effects caused
by NO 3 and carbon additions (Table 4), at least
when a change from 0 to 15°C (representing the
transition from prevalent winter to prevalent
summer temperatures) is considered. Under in
situ conditions, however, it might often be impossible to differentiate between the direct effect of
temperature changes on denitrification enzyme
activity and indirect effects caused by the impact
of temperature changes on for example respiration, nitrification and 0 2 solubility, all of which
are important factors in the regulation of denitrification. Therefore, the relative importance of
temperature compared with substrate supply
might change depending on the conditions.
4.4. Effect of suspending soil cores under suboptimum and optimum substrate conditions
Denitrification increased markedly when both
untreated and substrate-amended cores were
made into slurries (Table 5), indicating an intense
limitation of denitrification in intact soil due to
substrate diffusion. The suspension effect in untreated samples and in samples where only one
substrate has been added was not significantly
Table 5
Ratio between denitrification in anaerObic slurries and cores
unde r suboptimum and optimum substrate conditions
Substrate
treatment
Slurry/core
ratio
ambient
+ 5.1 mg N O 3 - N s a m p l e - 1
+ 54 mg glucose-C sample - 1
+ NO~-, glucose
24 + 8 a
21 + 3
215:2
10 + 0.2
a Mean 5: standard error, n = 4.
233
differetit. However, t h e r e was a t e n d e n c y towards
a lower degree of diffusional l i m i t a t i o n w h e n o n e
substrate was a d d e d c o m p a r e d with u n t r e a t e d
samples. W h e n N O 3 a n d glucose were a d d e d
s i m u l t a n e o u s l y the s u s p e n s i o n effect was two-fold
lower t h a n with u n t r e a t e d samples or with a
single additive.
Overall, these results d e m o n s t r a t e that the
detlitrifiers in this c o m m u n i t y possess a high substrate affinity a n d are limited slightly by the
a m o u n t of available substrate. T h e d e n i t r i f i c a t i o n
p o t e n t i a l is r e l a t e d to soil m o i s t u r e a n d varies
rolatively little t h r o u g h o u t the year, It is importarif tO n o t e that d e n i t r i f i c a t i o n c a n p r o c e e d even
at t e m p e r a t u r e s close to 0°C, which is the prevalent t e m p e r a t u r e at times w h e n r u n o f f from adjacent agricultural areas c o m m o n l y takes place.
Thus, w h e n N O f e n t e r s the soil pores d u r i n g the
winter r u n o f f period it is likely to b e r e d u c e d , at
least 80% t h r o u g h denitrification. However, the
efficiency of the r i p a r i a n z o n e as a N O 3 filter is
severely c o n s t r a i n e d by the limiting effect of substrate diffusion o n the rate of denitrification.
ACKNOWLEDGEMENTS
T h e technical assistance of L o n e Schou is
greatly appreciated, a n d the v a l u a b l e criticism of
the m a n u s c r i p t by S ~ r e n C h r i s t e n s e n a n d H e l e n
Clayton is gratefully acknowledged. T h e work was
s u p p o r t e d by the D a n i s h N a t u r a l Science Research Council a n d by the N a t i o n a l A g e n c y of
E n v i r o n m e n t a l P r o t e c t i o n in D e n m a r k .
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