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Appl Environ Microbiol, July 1998, p. 2533-2538, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Kinetic Parameters of Denitrification in a
River Continuum
Roberto
García-Ruiz,1,2
Sarah
N.
Pattinson,1 and
Brian A.
Whitton1,*
Department of Biological Sciences, University
of Durham, Durham DH1 3LE, United Kingdom,1
and
Departamento de Ecología, Universidad de
Málaga, 29071, Málaga, Spain2
Received 27 October 1997/Accepted 20 April 1998
 |
ABSTRACT |
Kinetic parameters for nitrate reduction in intact sediment cores
were investigated by using the acetylene blockage method at five sites
along the Swale-Ouse river system in northeastern England, including a
highly polluted tributary, R. Wiske. The denitrification rate in
sediment containing added nitrate exhibited a Michaelis-Menten-type
curve. The concentration of nitrate for half-maximal activity
(Kmap) by denitrifying bacteria increased on
passing downstream from 13.1 to 90.4 µM in the main river, but it was
highest (640 µM) in the Wiske. The apparent maximal rate
(Vmaxap) ranged between 35.8 and 324 µmol of
N m
2 h1 in the Swale-Ouse (increasing
upstream to downstream), but it was highest in the Wiske (1,194 µmol
N m2 h1). A study of nitrous oxide
(N2O) production at the same time showed that rates ranged
from below the detection limit (0.05 µmol of N2O-N
m2 h1) at the headwater site to 27 µmol
of N2O-N m2 h1 at the
downstream site. In the Wiske the rate was up to 570 µmol of
N2O-N m2 h1, accounting for up
to 80% of total N gas production.
| |
INTRODUCTION |
The massive use of nitrogen-based
fertilizer has inevitably increased the concentrations of nitrate in
many rivers (14, 15). This has led to the acceleration of
the nitrogen cycle, especially those processes that use nitrate as the
substrate, such as denitrification, the biological reduction of
nitrogen oxides (nitrate and nitrite) to N-containing gases
(N2 and N2O) in environments with relatively
low levels of oxygen (9). Interest in denitrifying bacteria
has also been stimulated by concern over the recognition of the role of
N2O in the destruction of stratospheric ozone
(5) and in the radiative heat budget of the atmosphere (19).
Although rivers are usually the first aquatic system to receive the
excess of nitrate, fewer studies have been undertaken on them than on
soils or estuaries. Only one (2, 27) or a few sites down the
river (4) have been sampled, and it is therefore difficult
to generalize from the results. According to the river continuum
concept of Vannote et al. (28), the physical and chemical variables of water and sediments should present a gradient from the
headwaters to the estuary, which should elicit a series of responses
within the denitrifying bacterial population. For instance, in the
Swale-Ouse river system in northeastern England we have observed
(18) a consistent increase (up to 30 times) in the denitrification rate of sediment cores on passing downstream.
There have apparently been no studies to determine the apparent
affinity (Kmap) and maximal capacity
(Vmaxap) for nitrate utilization of denitrifying
bacteria down a whole river; however, this is important for clarifying
the relationship between nitrate availability in the environment and
nitrate use by denitrifying bacteria. Previous attempts to measure the
kinetics of nitrate utilization in soils and sediments have been
hampered by a number of problems. The earliest studies employed slurry
techniques (7, 12, 17), where the structure of the sediment
is destroyed and hence the in situ oxygen and nitrate gradients. The
values of Kmap and Vmaxap
obtained are only meaningful under conditions of no limitation and
cannot be incorporated into predictive models. Other researchers have
used intact sediment cores to study the kinetics of nitrate reduction
through measuring the decrease in nitrate concentration in the
overlying water (1). The long incubation period, possible
errors in measuring nitrate, and the unknown fate of the depleted
nitrate make uncertain the significance of the reported kinetic
parameters.
The aim of the present study was to investigate the effect of nitrate
concentration on the denitrification rate and N2O
production in intact sediment cores taken from a river continuum.
| |
MATERIALS AND METHODS |
Study area.
The study was performed on the Swale-Ouse river
system. A detailed description of the geography of the area has
been provided by Jarvie et al. (8), the vegetation has been
surveyed by Holmes and Whitton (6), and biological studies
have been reviewed by Whitton and Lucas (29). The Swale
rises on the Northern Pennines, running 117 km to its confluence with
the Ure, where it becomes the Ouse (Fig.
1). The river becomes tidal (but still
freshwater) at km 145.0. (Main river distances are measured downstream
from the junction of two upland tributaries; the location of a site on
a tributary is given by a negative value indicating the
distance upstream, together with the point at which it enters the main river.)
The Swale-Ouse shows a consistent trend of changes in many physical and
chemical features on passing downstream, while the
whole river system
is subject to marked variations in discharge.
An important tributary is
the Wiske, a lowland and highly organically
polluted stream, that
receives treated sewage effluent from a
town of 10,700 inhabitants.
The oxygen concentration in water at sites on the main river was always
over 93% saturation (based on monthly data taken between
0900 to
1200 h), but this value fell to 64% in the Wiske.
Sediment and water collection.
Samples were taken from
upstream (km 
2.5 [0.0] and km 10.9), midstream (km 49.9), and
downstream (km 107.9 and km 145.0, i.e., freshwater tidal) sites (Fig.
1). In addition, sediment was collected from the Wiske (km 1.7
[86.1]). Samples were collected between 18 and 24 April 1997, with
repeat sampling at two sites, upstream (km 10.9) and the Wiske, on 18 June and 4 August 1997.
Intact sediment cores were taken in Plexiglas cylinders (3.5-cm inner
diameter, 25-cm height, with a vertical series of silicone
rubber
inserts to allow injection at different positions) from
a defined
2-m
2 area at positions where sediment was accumulating.
Each tube
was sealed with a rubber bung, and the core was removed
carefully.
The bottom of the core was sealed with an additional rubber
bung.
Special care was taken to preserve the sediment structure. The
pH
of the water was determined with a glass combination electrode
(Russell
CW76) and a Wissenschaftliche Technische Werkstätten
meter. Water
samples were taken at the same time and filtered
through 0.45-µm
(pore size) membrane filters. Nitrite and nitrate
were determined
according to the method of Stainton et al. (
26).
Depletion of in situ nitrate.
In order to establish the
required nitrate concentration during experimental studies, nitrate and
nitrite were removed from the sediment by using natural denitrification
activity, as adapted from the method of Murray et al. (12),
with river water being substituted by a medium lacking N or P. This
medium was modified from that used by Chu (3) and contained
(per liter) 4.28 mg of KCl, 54.75 mg of CaCl2 · 6H2O, 25 mg of MgSO4 · 7H2O,
15.85 mg of NaHCO3, 2.44 mg of FeCl2 · 6H2O, 3.33 mg of Na2EDTA, and 0.25 ml of AC
microelement stock (11). The medium was buffered with HEPES
(0.6 g liter1) to the natural pH. All samples were
incubated in the dark for 24 h at 15°C.
Assay for denitrification activity and N2O
production.
The acetylene (C2H2) block
method (24) was used to determine the denitrification rate.
This method is based on assessing the rate of N2O
accumulation in cores to which C2H2 has been
added to block the bacterial reduction of N2O to
N2. Net N2O production was also determined in
intact sediment cores incubated without C2H2.
After incubation to remove environmental nitrate and nitrite, the
supernatant water was drained off and replaced with the freshwater medium with the required nitrate concentration supplied as
KNO3. Control experiments with KCl showed that
K+ had no influence on the results. Nitrate concentrations
used in the assays were modified to reflect the expected
denitrification rate and ranged from 0 to 140 µM for the upstream
sites, 357 µM for downstream sites, and 11,420 µM for the Wiske.
Two triplicate sets of cores were used for each concentration assayed
at each site. The first set was incubated without
C2H2 to measure net N2O production,
while the second was incubated with C2H2 to
determine the denitrification rate (N2O plus N2
production). For the latter, C2H2 saturated
medium with the required nitrate concentration was added in the
overlying water to obtain 10% C2H2 (final
concentration). C2H2 saturated medium was
prepared by flushing for 20 to 30 min with pure
C2H2 (previously passed for 30 min through 0.1 N phosphoric acid to remove ammonium contamination). Then 1.0 ml of
this solution was injected (where required) into the sediment through
the holes in the cylinder at each centimeter depth for the top 3 cm.
Cores were filled with the test medium, sealed without gas space, and incubated at 15°C in the dark for between 3 and 5 h. Immediately before the assay, another three cores from each site were used to
determine the initial N2O concentration. In addition,
another two sets of three cores were used to determine denitrification and N2O production by using river water from each site. For
this analysis, cores were incubated at the same time and under similar conditions to those described above but with
C2H2 saturated river water (denitrification
rate) or natural river water (N2O production).
At the end of incubation, the cores were vigorously shaken, and 5 ml of
the resulting slurry was transferred to a 12.5-ml
gas container, which
included 0.1 ml of 40% formaldehyde, and
immediately frozen until
N
2O analysis. After the gas container
was shaken for 2 min
to equilibrate gas in the headspace and sediments,
2 ml of the
headspace was removed for N
2O measurement. This was
done by
using a gas chromatograph equipped with
63Ni electron
capture detector (Perkin-Elmer 2000; detector temperature
350°C; flow
of 20 ml min
1). The N
2O concentration was
determined by using a standard curve
generated with purified
N
2O (BOC, Ltd.). The N
2O concentration
in the
headspace was used to back-calculate the amount of N
2O
in
the sediment by using Henry's law. N
2O production
(expressed
as micromoles of N
2O-N per square meter per
hour) and denitrification
rate (N
2O plus N
2,
expressed as micromoles of N per square meter
per hour) were calculated
after subtracting the initial N
2O in
the core and taking
into account the incubation period and the
area of the core.
The kinetic parameters for denitrification and N
2O
production were computed from the Lineweaver-Burk transformation
(1/
V versus
1/
S) of the Michaelis-Menten
equation, where
V is the denitrification
rate or
N
2O production, and
S is the concentration
of nitrate.
Because the denitrifying enzyme activity was assayed in a
complex
system, the kinetic parameters were termed as apparent
concentration
of NO
3 for half-maximal
activity (
Kmap) and apparent maximal activity
(
Vmaxap).
| |
RESULTS |
Environmental data.
The data for sediments obtained at the six
sites are shown in Table 1. With respect
to sediment particle size, there was an increase in the silt-plus-clay
fractions and a decrease in the sand fraction on passing downstream. In
general, total carbon and nitrogen in the sediment increased on passing
downstream.
Nitrate depletion.
The denitrification rate and
N2O production in the cores after incubation for 24 h
to remove sediment nitrate and nitrite are shown in Table
2. Denitrification was still detectable,
though rates were low: the rates were 5 µmol of N m2
h1 in the headwater site and 31.4 µmol of N
m2 h1 in the Wiske. These values represent,
respectively, 16 and 3% of the maximum rates found during assays with
the saturation level of nitrate (see below). N2O production
was very low in the Wiske (2.7 µmol of N2O
m2 h1) and lower or negative (net
consumption of N2O) at the other sites (Table 2).
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TABLE 2.
Net N2O production and denitrification rate
with no nitrate after 24 h at 15°C with nitrogen-free
freshwater medium
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|
Kinetics for denitrification and N2O production.
Nitrate addition in the overlying water resulted in an increase in
the denitrification rate (Fig. 2a). The
response of denitrification to nitrate at all sites could be fitted
successfully (minimum regression coefficient of 0.9) to a
Michaelis-Menten-type curve (Fig. 2a and Table
3). During the April survey (Table 3),
Vmaxap increased on passing down the river (from
35.8 to 324 µmol of N m2 h1), but it was
highest on the Wiske (758 µmol of N m2
h1). The value for Vmaxap in the
Wiske in August was still higher (1,194 µmol of N m2
h1). Kmap constants increased in
April on passing down the main river from 13.1 to 90.4 µM nitrate
(Table 3). The overall highest value for Kmap
(640 µM) was for the Wiske in August. Where repeat measurements were
made (km 10.9 and Wiske), values for Vmaxap and
Kmap were similar in April and June, but they
were about one-third higher in August (Table 3).
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FIG. 2.
Responses of denitrification rate (a) and
N2O production (b) to nitrate additions at the sampling
sites in April 1997 ( ). For the Ivelet Bridge (km 10.9) and Wiske
sites the assays were repeated in June ( ) and August ( ) 1997. The
bars denote the standard deviations of three replicates. Note the
different scales for denitrification rate, N2O production,
and nitrate concentration.
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TABLE 3.
Kinetic parameters for denitrification rate and
N2O production estimated according to the
Lineweaver-Burk transformation of the Michaelis-Menten equation
|
|
The production of N
2O in the cores without
C
2H
2 could only be fitted to a Michaelis-Menten
curve for three sites (km 49.9,
km 145.0, and Wiske [Fig.
2b]). At
the two upstream sites (km
2.5 [0.0] and km 10.9), N
2O
production was less than 15 µmol
of N
2O-N
m
2 h
1 and without any clear trend with
increasing nitrate concentration.
The maximum values for
N
2O production were 70.2, 15.5, and 570
µmol of
N
2O-N m
2 h
1 in April for km
49.9, km 145.0, and the Wiske, respectively,
and 626 and 496 µmol of
N
2O-N m
2 h
1 in June and August
for the Wiske (Table
3). The
Kmap values
were
517, 138, and 542 µM in April for the same sites and 532
and 1,952 µM in June and August for the Wiske (Table
3). The
Kmap value for N
2O production for
these sites was higher than
the
Kmap value for
the denitrification rate, while the
Vmaxap values were lower.
The proportion of N
2O to total N gases
(N
2O + N
2) ranged between 2.4 and 38% and
between 2 and 58% at the headwater and km
10.9 sites,
respectively (Table
4), but there was no
clear trend
with increasing nitrate concentration. The values
increased on
addition of nitrate to the km 49.9 and km 107.9 samples but not
to the km 145.0 sample. In the Wiske the values
exceeded 50% at
all nitrate concentrations in April and June (Table
4).
When river water (with its natural nitrate concentration) was used
instead of medium, the denitrification rate was linearly
related
(
r2 = 0.89; slope = 0.92) to the
rate predicted from the Michaelis-Menten
curve for the nitrate
concentration in the river.
| |
DISCUSSION |
Nitrate depletion and freshwater medium.
The successful
application of kinetic studies to the sediments required removal of
nitrate and nitrite, and this was largely achieved. This suggests that
any nitrate produced via nitrification of the ammonium in the sediment
was also consumed by natural denitrification during this period. The
highest denitrification persisting after 24 h of incubation in the
absence of nitrate was at the headwater site (Ravenseat).
Influence of nitrate addition on denitrification and
N2O production.
The denitrification rate in
intact sediment cores responded strongly to nitrate addition and
followed a Michaelis-Menten-type curve, as found by other
researchers for freshwater (1) and estuarine (10,
16, 17) sediments. Repeat measurements on different dates at
particular sites showed only moderate differences.
The
Vmaxap values for denitrification rate were
mostly similar to the maximum rates reported by Pattinson et al.
(
18) for
field conditions in a 17-month survey of intact
sediment cores
from the same sites. However, comparisons with other
studies are
complicated by the fact that these have been made at a
range of
temperatures. Ideally, results would be presented for both the
field temperature and a standard laboratory temperature. In the
case of
the Swale-Ouse, the values are comparable because the
temperature at
the time of the maximum value was always within
5°C of the standard
temperature of 15°C.
Other published kinetic data apparently refer only to experiments with
slurries, so caution is needed in comparing our data
with previous
data. However, the values for
Vmaxap at the
headwater
site and at the main river (35.8 to 324 µmol of N
m
2 h
1) are well within the range of
denitrification rates measured
at near-ambient conditions for rivers (0 to 345 µmol of N m
2 h
1) and for coastal
marine sediments (0 to 888 µmol of N m
2
h
1) (
23), although denitrification was always
detectable in our
studies. The uppermost value on the Wiske (1,194 µmol of N m
2 h
1 in August) exceeded this
range. The
Kmap values may be compared
with the
lower (8 µM) and upper (344 µM) values found in previous
studies (
10,
12,
16,
17) with the slurry technique.
Another problem in assessing results is the fact that denitrification
in the sediment has been related to the nitrate concentration
in the
overlying water. Although transport of nitrate into the
sediment is
likely to be rapid, the lower and variable nitrate
concentration in the
sediment means that the
Kmap value has
inevitably
been overestimated. However, we advocate the use of cores to
obtain
realistic measurements of kinetic parameters to be incorporated
into integrating models.
Net N
2O production occurred at all sites, but only at some
sites was there an increase with increasing nitrate concentration.
Compared to other studies, the proportions of N
2O related
to total
N gases (N
2 + N
2O) for the highest
nitrate concentrations (Table
4) were high, reaching 80% in the Wiske
in June. The published
values for N
2O in aquatic sediments
have been obtained from pore
water profiles (
25) and direct
N
2O flux measurements from cores
(
13,
21); net
N
2O flux is generally less than 2 µmol of N
2O
m
2 h
1 and less than 5% of N
2
production (
23). As N
2O production is
an
intermediate product in at least three processes in the nitrogen
cycle,
each influenced by a range of environmental factors, it
is difficult to
explain these high values. However, eutrophication
is one factor
reported to lead to increased N
2O production, as
shown in
Narragansett Bay (
20), where N
2O fluxes
increased around
10-fold from the relatively unpolluted lower- and
mid-bay sediments
to the eutrophic upper-bay sediments. A study
(
22) on a marine
mesocosm showed that N
2O flux
from sediment and the N
2O/N
2 ratio
increased
markedly in relation to nitrate input.
Ecological significance.
The Vmaxap
value showed a marked increase coincident with increasing nitrate
concentration on passing downriver, and it seems almost certain that
this is a key factor. However, other factors must be considered when
assessing the results shown in Fig. 2. These factors include the number
of denitrifying bacteria, their genetic and physiological properties,
and the optimal conditions of the process (e.g., the concentrations of
suitable carbon substrates, phosphate, and oxygen). Downstream, the
sediment particles were finer, presumably decreasing oxygen
penetration, and the carbon content of the upper 2 cm of sediment was
higher (Table 1), all features likely to favor denitrification.
Although the changes in oxygen concentration were not investigated, the
high O2 saturation usually present in the river water and
the relatively low incubation period minimized the disturbance of
sediment in our experimental setup.
The
Kmap values increased on passing downstream
and were maximum in the Wiske. This trend agrees with the observed
increase
in nitrate concentration on passing downstream. In
environments
with high affinity (low
Kmap) and
high nitrate concentration or
in those with low affinity (high
Kmap) and low nitrate concentration,
nitrate
reduction by denitrifying bacteria would be inefficient.
The
Kmap values calculated in this study for each
site were compared
with the nitrate data available for 1995 to 1996 from other sources
(Environment Agency and the LOIS data base). For the
upland sites,
the
Kmap values were higher than
the nitrate concentration in
the river, and therefore the response of
denitrification to the
nitrate concentration is likely to be linear for
most of the year.
However, the
Kmap values for
all sites on the main river and the
Wiske were lower than the nitrate
concentration for most of the
year, and therefore nitrate reduction by
denitrifying bacteria
is probably nitrate saturated, especially during
winter and spring.
This suggests that other variables, such as
temperature, organic
carbon, and oxygen, must be considered when
studying the seasonal
changes in denitrification (
18). As
denitrifying bacteria are
taxonomically heterogeneous
(
10), it is likely that these factors
also exert a selective
pressure on groups that are dominant at
particular times of the year.
| |
ACKNOWLEDGMENTS |
This work was performed as part of the Land Ocean Interaction
Study programme (NERC grant GST/01/A770), which included a studentship for S.N.P. R.G. acknowledges a postdoctoral grant from the
Ministerio de Educación y Cultura, Subprograma General en el
Extranjero.
We are most grateful to R. V. Smith, Department of Agricultural
and Environmental Science, the Queens University of Belfast, Belfast,
Northern Ireland, for helpful discussion.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Durham, South Road, DH1 3LE, Durham, United Kingdom. Phone: 44 (0) 191 374 2419. Fax: 44 (0) 191 386 0619. E-mail: B.A.Whitton{at}durham.ac.uk.
This is Land Ocean Interaction Study programme publication no.
414.
| |
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0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
