FEMS Microbiology Ecology 46 (2003) 63^71
www.fems-microbiology.org
Temporal variation of nitri¢cation rates in experimental freshwater
sediments enriched with ammonia or nitrite
Peter Stief
a
a;
, Andreas Schramm b , Do«rte Altmann a , Dirk de Beer
a
Max-Planck-Institute for Marine Microbiology, Microsensor Research Group, Celsiusstrasse 1, D-28359 Bremen, Germany
b
Department of Ecological Microbiology, BITOEK, University of Bayreuth, D-95440 Bayreuth, Germany
Received 17 March 2003; received in revised form 17 June 2003; accepted 7 July 2003
First published online 27 August 2003
Abstract
Two freshwater sediments (organic-poor and organic-rich) that contained their distinct natural microbial communities were incubated
3
in experimental microcosms with either NHþ
4 or NO2 in the overlying water. Microsensor measurements revealed the thin oxic surface
3
3
layer as a site of initially high rates of nitrification, i.e. O2 , NHþ
4 , and NO2 consumption, and NO3 production. Unexpectedly, during the
3
consumption
decreased
in
both
sediment
types
and
NO
consumption
decreased in the organic-rich
subsequent 4-week incubation NHþ
2
4
3
production
paralleled
these
decreases,
i.e.
the reduced NHþ
sediment. In the organic-rich sediment O2 consumption and NO3
3
4 and NO2
consumption rates were most probably due to reduced activity of nitrifiers. These microsensor data imply factors other than frequently
3
suggested competition between nitrifiers and heterotrophs for NHþ
4 , NO2 or O2 as causes for the loss of nitrification activity. We
hypothesize that experimental manipulations (e.g. removal of macrofauna, redistribution of particulate organic matter, permanent
nutrient enrichment) rendered the performance of the microbial community unstable. It is thus recommendable to restrict experiments in
such commonly used model systems to the period of highest stability.
8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Freshwater sediment; Nitrogen cycle; Nitri¢cation; Temporal variation; Sediment manipulation ; Microcosm; Microsensor
1. Introduction
Microbial degradation of organic matter in freshwater
sediments releases primarily dissolved organic carbon
(DOC), CO2 , dissolved organic nitrogen (DON), and
NHþ
4 [1,2]. These compounds are essential for both autotrophic nitri¢ers (NHþ
4 , CO2 ) and heterotrophic microorganisms (DOC, DON, NHþ
4 ). The unavoidable competiþ
tion for NH4 has long been recognized to occur in
sediments [3^6], laboratory chemostats [7,8] and bio¢lms
[9].
3
The competition for substrates such as NHþ
4 , NO2 and
O2 in these mixed communities has been thought responsible for large population shifts at the expense of nitri¢ers.
The latter was explained by lower substrate a⁄nities of
* Corresponding author. Tel. : +49 (421) 2028-843;
Fax : +49 (421) 2028-690.
E-mail address : [email protected] (P. Stief).
nitri¢ers compared to heterotrophs [3,5^8]. Sediments with
high organic contents were found least favorable for nitri¢ers [3,5,6,10,11].
Many studies on competition between sedimentary nitri¢ers and heterotrophs have been carried out using
sieved and homogenized sediments [4^6,10,12,13]. This
procedure largely removes lateral heterogeneities and
physical obstacles (e.g. pebbles and macrofauna) that
might in£uence small-scale measurements. In addition,
the validity of these model systems has been questioned
because of the massive intervention in the spatial organization of the microbial community [14]. Time for reorganization is commonly scheduled prior to the actual experiments [14^16]. Steady state is assumingly reached as soon
as the sediment^water £uxes of interest have stabilized
[14,16]. Nevertheless, sieving may change the performance
of microbial communities tremendously, and populations
with low growth rate (e.g. nitri¢ers) will recover particularly slowly from this kind of disturbance [14,17,18]. The
establishment of nitri¢cation might be further impeded by
competition for substrates with heterotrophs. From these
considerations the question arises as to whether nitri¢ca-
0168-6496 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0168-6496(03)00193-4
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P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
tion in sediment microcosms can actually establish and
then stabilize for an extended period of time. To answer
this question we investigated model sediments that were
3
continuously enriched with either NHþ
4 or NO2 via the
overlying water and could thus not be substrate-limiting
for nitri¢cation.
2. Materials and methods
2.1. Sediment origin, processing, and incubation
Two distinct study sites were chosen that both showed
high inorganic N concentrations in the water column but
di¡ered greatly in sediment properties. The ¢rst site was a
lowland stream, Rittrumer Mu«hlenbach, located near
Wildeshausen in northern Germany. Sandy, organic-poor
sediment from this stream was collected in April 2000 at a
3
3
water temperature of 12‡C. NHþ
4 , NO2 , and NO3 con31
centrations were 6, 2, and 370 Wmol l , respectively. The
second site was a man-made ditch, Millinger Landwehr, in
the £oodplain of the Lower Rhine near Rees in northwestern Germany. It was originally designed as a drainage
ditch running through intensively cultivated agricultural
land. Until 1994, it also received the e¥uents of a municipal wastewater treatment plant. Silty, organic-rich sediment from this ditch was collected in October 2000 at a
3
3
water temperature of 10.8‡C. NHþ
4 , NO2 , and NO3 con31
centrations were 22, 6, and 115 Wmol l , respectively.
Super¢cial sediment (0^10 cm depth) was sampled using
a £at shovel and ¢lled into buckets. In the laboratory the
sediments were immediately sieved through a 1-mm mesh
in order to remove pebbles, detritus and macroinvertebrates. The sediment was ¢lled into 500-ml glass beakers
up to a height of 8 cm. For each sediment sample type, 18
beakers were ¢lled and then placed into six 15-l tanks (i.e.
three beakers per tank). The sediment beakers were submersed in aerated tap water and incubated at 15‡C in the
dark. Repeated short-term NH4 Cl additions to the tanks
resulted in stable NHþ
4 disappearance rates after approximately 3 weeks [14,16]. After having reached this steady
state of sedimentary NHþ
4 consumption, three tanks for
each sediment were continuously enriched with 50 Wmol
l31 of NaNO2 , whereas the remaining three tanks were
enriched with 50 Wmol l31 of NH4 Cl. The N concentrations in the tanks were measured regularly using spectro3
photometric test kits [19]. Losses of NHþ
4 and NO2 were
corrected by adding the respective solute to the tanks
without ever replacing the water during the incubation.
The pH values in the tanks varied between 7.7 and 8.1.
2.2. Microsensor measurements
þ
Twenty-four hours after the onset of the NO3
2 or NH4
enrichment, one beaker from each tank was used for miþ
crosensor measurements (day 0). The NO3
2 and NH4 in-
cubations were continued for 4 weeks and two more measurements were performed on days 14 and 28, again
sacri¢cing one beaker from each tank for both sediment
types and enrichments. LIX-type microsensors, selective
3
3
for NHþ
4 , NO2 , and NO3 , and Clark-type O2 microsensors were constructed according to [20] and [21], respectively. Calibration of LIX-type microsensors was performed in tap water to which known amounts of
NH4 Cl, NaNO2 , and NaNO3 were added giving nominal
concentrations of 10^1000 Wmol l31 . These values were
corrected for the background concentrations of NHþ
4,
3
NO3
2 , and NO3 in pure tap water, i.e. 6 1, 6 1, and
31^45 Wmol l31 , respectively (personal communication S.
Wahlers, swb norvia, Bremen, Germany). Calibration
curves for O2 microsensors were obtained from the reading in aerated tap water (100% air saturation) and the
lowest reading in anoxic sediment layers (0% air saturation). Sediment beakers were placed into a microsensor
setup as described previously [19]. Two to three microsenþ
3
sors (i.e. O2 +NO3
2 or O2 +NH4 +NO3 ) were mounted to a
computer-controlled micromanipulator with their horizontal distance not exceeding 10 mm. Concentration pro¢les
were measured at randomly chosen spots of the sediments
with a vertical resolution of 200^400 Wm and down to a
depth of 10 mm. During the measurements the concentraþ
þ
3
tions of NO3
2 (NO2 incubation) and NH4 (NH4 incubation) in the overlying water were held constant at 50 Wmol
l31 . Since in the NHþ
4 -enriched tanks signi¢cant amounts
of NO3
3 accumulated during the incubation, the overlying
water concentration of NO3
3 was held at approximately
31
500 Wmol l
during the microsensor measurements. In
3
contrast, in the NO3
2 -enriched tanks NO3 did not accumulate during the incubation and was thus not added
during the microsensor measurements. For both enrichments the actual overlying water concentration of NO3
3
during the microsensor measurements was determined
with a spectrophotometric test kit [19]. An extended version of Fick’s law of di¡usion was used to calculate local
volumetric conversion rates from steady-state concentration pro¢les [22]. For this purpose the second derivative of
the pro¢les was calculated. In order to reduce noise the
neighboring conversion layers were combined to obtain
conversion layers of 1 mm in thickness. Di¡usion coe⁄3
3
cients (Dw ) of NHþ
4 , NO2 , NO3 , and O2 at 15‡C in the
water phase were taken as 1.50U1035 , 1.45U1035 ,
1.44U1035 , and 1.83U1035 cm2 s31 , respectively [19].
The depth pro¢le of di¡usivity (i.e. Dw in the water column and Ds along the sediment column) was determined
for H2 in steps of 500 Wm and down to 10 mm using a
di¡usivity microsensor (Unisense A/S, Denmark). The
3
3
sediment di¡usion coe⁄cients (Ds ) of NHþ
4 , NO2 , NO3 ,
and O2 were obtained for each sediment depth by assuming the same Ds /Dw ratio as determined for H2 . Depthintegrated solute conversions within the overlap of O2
consumption and NO3
3 production, i.e. in the presumable
nitri¢cation layer, were calculated as the sum of the re-
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P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
65
Fig. 1. Concentration pro¢les (A^C) and local conversion rates (D^F) in the organic-poor sediment during a 4-week incubation with NHþ
4 . Means+S.D.
of three replicate beakers are given. S.D. bars of concentration pro¢les are omitted for clarity. Coe⁄cients of variance of replicate pro¢les were typically
1^3% in the overlying water and 10^30% in the sediment.
spective volumetric conversion rates multiplied by the
thickness of a single conversion zone.
3. Results
3.1. Microsensor measurements
2.3. Solid-phase organics
Depth distribution of organic matter was determined for
each sediment type in three randomly selected sediment
beakers at the end of the experiments. For this purpose,
sediment cores were taken using acrylic cylinders of 2.5 cm
in diameter. These cores were sectioned into 2-mm-thick
slices down to a depth of 10 mm. The slices were dried to
the constant weight at 60‡C and ¢nally combusted for 3 h
at 550‡C. The weight loss upon combustion was taken as
an estimate of the organic content of the sediment.
2.4. Statistics
Between-sediment comparison of initial areal conversion
rates was carried out using Student’s t-test with or without
correction for non-normal distribution according to Levene. Depth-integrated conversion rates within a treatment
(i.e. one sediment type exposed to one type of N enrichment) were checked for monotonous changes over time by
linear regression analysis. The obtained slopes were
checked for signi¢cant di¡erences from zero.
3.1.1. Initial status
O2 and NO3
2 typically became depleted within the top
few millimeters of both sediments types (Figs. 1A, 2A,B,
3A, and 4A,B), while NHþ
4 concentrations decreased in
the oxic and increased in the anoxic layer (Figs. 1B and
3B). In contrast, NO3
3 concentrations typically increased
in the oxic and decreased in the anoxic layer (Figs. 1^4C).
It was noticed that NO3
3 concentrations rarely reached
zero at the lower end of the pro¢les, but instead remained
stable at levels between 5 and 20 Wmol l31 . Most probably
these calculated concentrations resulted from the back3
ground signal of the NO3
3 microsensors even when NO3
was actually depleted [12,13]. No attempts were made to
correct for this inaccuracy at very low NO3
3 concentrations, since (i) nitri¢cation and not denitri¢cation was of
particular interest for this study and (ii) the sensors accurately measured the higher NO3
3 concentrations in the
nitri¢cation layer.
The concentration pro¢les allowed layerwise calculation
of solute conversion rates: O2 and NO3
2 were completely
consumed in the oxic layer (Figs. 1D, 2D,E, 3D, and
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P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
Fig. 2. Concentration pro¢les (A^C) and local conversion rates (D^F) in the organic-poor sediment during a 4-week incubation with NO3
2 . See Fig. 1
for more details.
4D,E), while NHþ
4 was only partly consumed in the oxic
layer and not signi¢cantly converted in the anoxic layer
(Figs. 1E and 3E). NO3
3 was typically produced in the oxic
and consumed in the anoxic layer (Figs. 1^4F). Integration
of local conversion rates across the overlap of the O2 consuming and NO3
3 -producing layer resulted in areal
þ
3
conversion rates of O2 , NO3
2 , NH4 , and NO3 in the presumable nitri¢cation layer (Tables 1 and 2): initially, the
two sediment types did not di¡er with respect to nitri¢cation activities when enriched with NHþ
4 (P s 0.05, t-test).
Di¡erences were seen though upon enrichment with NO3
2 ,
which resulted in signi¢cantly higher O2 consumption and
3
NO3
3 production rates (P 6 0.05, t-test), and lower NO2
consumption rates (P 6 0.01, t-test) in the organic-rich
versus organic-poor sediment.
3.1.2. Temporal trends
Generally, O2 consumption in the nitri¢cation layer
tended to increase in the organic-poor (NO3
2 enrichment)
3
and decrease in the organic-rich sediment (NHþ
4 and NO2
þ
enrichment). In the NH4 -enriched, organic-poor sediment
the level of O2 ¢rst increased and then decreased. These
interpretations were derived from changing O2 penetration
depths (Figs. 1^4A), local (Figs. 1^4D) and areal O2 consumption rates (Tables 1 and 2). NHþ
4 consumption decreased in both sediment types by the end of the incuba-
Table 1
Depth-integrated conversion rates (Wmol m32 h31 ) in the presumable nitri¢cation layer of the organic-poor sediment during a 4-week incubation with
3
NHþ
4 and NO2
NHþ
4 incubation
Day 0
Day 14
Day 28
NO3
2 incubation
O2
a
NHþ
4
NO3
3
DI
O2
3916 (43)
31185 (120)
3770 (133)
3162 (7)
3125 (11)
360 (31)
+375 (156)
+529 (12)
+490 (253)
2.4
2.6
3.2
3490 (67)
3832 (156)
31037 (25)
b
NO3
2
NO3
3
DI
3188 (5)
3177 (25)
3239 (12)
+90 (16)
+145 (10)
+55 (12)
2.8
2.0
3.0
Means (S.D.) of three replicate beakers are given. Positive values = production, negative values = consumption. DI : depth of rate integration [mm].
Linear regression with time as independent variable : r2 = 0.92, P 6 0.001.
b
Linear regression with time as independent variable: r2 = 0.87, P 6 0.001.
a
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P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
67
Fig. 3. Concentration pro¢les (A^C) and local conversion rates (D^F) in the organic-rich sediment during a 4-week incubation with NHþ
4 . See Fig. 1
for more details.
tion. This was indicated by less steep NHþ
4 concentration
gradients (Figs. 1B and 3B) and lower local (Figs. 1E and
3E) and areal consumption rates (Tables 1 and 2). NO3
2
consumption increased in the organic-poor and decreased
in the organic-rich sediment, likewise as NO3
2 penetration
depth decreased and increased, respectively (Figs. 2B and
4B), and local (Figs. 2E and 4E), and areal consumption
rates (Tables 1 and 2) increased and decreased, respectively. Temporal trends of the NO3
3 production varied
with the sediment type. (i) In the organic-poor sediment,
NO3
3 production ¢rst increased and then decreased inde-
3
pendently of the NHþ
4 or NO2 enrichment (Fig. 1C,F,
Table 1). (ii) In the organic-rich sediment, in contrast,
NO3
3 production decreased continuously when enriched
3
with either NHþ
4 or NO2 (Fig. 3C,F, Table 2). In the
þ
NH4 -enriched, organic-poor sediment the zone of NO3
3
production extended into the anoxic layer (Fig. 1C,F).
3.2. Solid-phase organics
The two sediments used for this experiment di¡ered
greatly in their organic matter content. At the end of the
Table 2
Depth-integrated conversion rates [Wmol m32 h31 ] in the presumable nitri¢cation layer of the organic-rich sediment during a four-week incubation with
3
NHþ
4 and NO2
NHþ
4 incubation
O2
Day 0
Day 14
Day 28
a
3976 (187)
3770 (42)
3414 (131)
Means (S.D.) of three replicate
Linear regression with time as
b
Linear regression with time as
c
Linear regression with time as
d
Linear regression with time as
e
Linear regression with time as
a
NO3
2 incubation
b
NHþ
4
c
NO3
3
DI
O2
d
NO3
2
e
NO3
3
DI
3176 (16)
3174 (30)
31 (17)
+229 (44)
+135 (54)
325 (49)
1.6
1.2
1.2
3944 (240)
3769 (67)
3683 (80)
3161 (9)
3138 (37)
393 (39)
+130 (4)
+103 (27)
+61 (19)
1.8
1.8
1.8
beakers are given. Positive values = production, negative values = consumption. DI : depth of rate integration [mm].
independent variable : r2 = 0.80, P 6 0.01.
independent variable: r2 = 0.61, P 6 0.05.
independent variable : r2 = 0.76, P 6 0.01.
independent variable: r2 = 0.53, P 6 0.05.
independent variable : r2 = 0.75, P 6 0.01.
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P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
Fig. 4. Concentration pro¢les (A^C) and local conversion rates (D^F) in the organic-rich sediment during a 4-week incubation with NO3
2 . See Fig. 1
for more details.
laboratory incubation the top layer of the organic-poor
sediment had an organic content of 2.7 T 0.3%, while
that of the organic-rich sediment was 15.4 T 4.0%
(mean T S.D., n = 3). Organic matter distribution along
the sediment column was rather uniform in the organicpoor sediment, but decreased sharply below the top layer
of the organic-rich sediment to values of around 5%.
4. Discussion
4.1. General observations
We used microsensors to determine N conversions in
two types of microbially strati¢ed model sediments, each
with its own particular characteristics. The two sediments
di¡ered greatly with respect to the organic content and,
moreover, they were sampled in di¡erent seasons. We
therefore expected to encounter two distinct microbial
communities, which under identical conditions may develop di¡erently because of their distinct initial taxonomic
composition and metabolic performance. Stable sedimentary NHþ
4 consumption rates at the end of the preincubation period were taken as indicative of having reached a
steady state [14,16]. Microsensor pro¢les recorded on day
0 of the experimental incubation revealed that the sedi-
ments were strati¢ed into (i) an oxic surface layer in which
3
3
NHþ
4 and NO2 consumption and NO3 production took
place and (ii) an anoxic layer in which NO3
3 consumption
took place. These ¢ndings agreed well with observations
on microscale N conversions in both model and ¢eld sediments [12,13,23,24]. However, in the NHþ
4 -enriched, or3
ganic-poor sediment the NO3 production zone occasionally extended into the anoxic sediment layer. This unusual
observation cannot result from a spatial mismatch of O2
and NO3
3 pro¢les, since both sensors were operated simultaneously using one and the same micromanipulator. Irreversible poisoning of the NO3
3 sensor can also be ruled out
as a source of error, because the calibrations before and
after the measurements were identical. Finally, we observed this unusual type of NO3
3 pro¢le only on days 14
and 28, but not on day 0, of the incubation. Thus, a
hypothetical compound interfering with the sensor must
have formed only during the late phase of the experimental incubation (i.e. during weeks 5 and 7). We consider this
a rather unlikely scenario and have faith in our microsensor measurements. One possible explanation for the observed phenomenon is suboxic nitri¢cation coupled to
manganese reduction as suggested by Hulth et al. [25].
The authors claim that in biogenically or physically mixed
sediments MnO2 can be buried in anoxic sediment layers
3
where it is used by microbes to oxidize NHþ
4 to NO3 .
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P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
Once produced in anoxic layers, NO3
3 tends to accumulate
when the supply of electron donors is low [25]. Physical
mixing, NHþ
4 supply, and lack of electron donors were
features of our NHþ
4 -enriched, organic-poor sediment as
well. It needs to be stressed here that for the following
considerations only the spatial overlap of O2 consumption
and NO3
3 production was treated as sedimentary nitri¢cation, whereas the suboxic NO3
3 production was ignored.
4.2. Initial status
Starting from the steady state of N conversions reached
by the end of the preincubation, we continuously enriched
3
the sediments with NHþ
4 or NO2 to exclude N limitation
of nitri¢cation. Initially, nitri¢cation in the two sediments
di¡ered with respect to NO3
2 oxidation: the depth-integrated consumption rates of O2 and NO3
3 were signi¢cantly higher in the organic-rich than the organic-poor
sediment. At the same time, however, NO3
2 consumption
rates were somewhat lower in the organic-rich than the
organic-poor sediment. Contrasting with this result the
two sediments did not di¡er with regard to NHþ
4 oxidation
irrespective of their di¡erent origin and characteristics.
Initially, areal net NO3
3 production rates were 1.3^2.3fold higher than net NHþ
4 consumption rates. Thus, the
3
stoichiometry of complete nitri¢cation (i.e. NHþ
4 :NO3 =
1:1) was not met using net rates. This can be explained by
the lowering of gross NHþ
4 consumption due to mineralization processes that liberate NHþ
4 from organic matter
3
[25,26]. Consumption of NO3
2 , in contrast, led to NO3
production rates 1.2^2.1-fold lower than expected from
the theoretical stoichiometry of complete NO3
2 oxidation
3
to NO3
3 . Hence, an additional sink for NO2 aside from
nitri¢cation, e.g. NO3
2 uptake by heterotrophs, must have
been present in the sediment. This was all the more likely
þ
3
since in the NO3
2 -enriched sediments both NH4 and NO3
as alternative N sources were in low concentration ( 6 2
and 6 45 Wmol l31 , respectively). Initial depth-integrated
rates of O2 consumption were always higher than expected
from the stoichiometry of nitri¢cation. A great proportion
3
of the O2 (i.e. 18^53% and 91^93% in the NHþ
4 - and NO2 enriched sediments, respectively) was obviously consumed
by heterotrophic metabolism or chemical reactions.
4.3. Temporal trends
In the course of the 4-week incubation the conversions
of microbial N changed in an unexpected way in the two
sediment types. Instead of a further enrichment for nitri¢cation, the N conversion rates decreased signi¢cantly in
the organic-rich sediment even though neither NHþ
4 nor
3
NO2 concentrations were limiting. By day 28 of the incubation net nitri¢cation activity was not measurable any
longer in the NHþ
4 -enriched sediment and was signi¢cantly
reduced in the NO3
2 -enriched sediment. In contrast, in the
organic-poor sediment neither an enrichment for nitri¢ca-
69
tion nor a conclusive temporal variation of nitri¢cation
activity could be observed. In previous studies a high organic loading of sediments has been shown to favor the
NHþ
4 and O2 consumption by heterotrophs rather than by
nitri¢ers [3^6]. Mostly, the lower a⁄nity of autotrophic
nitri¢ers for NHþ
4 was made responsible for their outcompetition in mixed microbial communities [3^8]. We could
demonstrate, however, that the apparent suppression of
nitrifying activity in our sediments was explicitly not due
3
to a limitation of NHþ
4 or NO2 at the sediment surface.
Alternatively, heterotrophic bacteria might have outcompeted nitri¢ers for O2 . Km values for O2 of 16 and 62 Wmol
l31 have been reported for Nitrosomonas and Nitrobacter
species, respectively [27]. The corresponding value in heterotrophic bacteria is 6 1 Wmol l31 , implying that this
group of bacteria possesses a much higher a⁄nity for O2
than nitri¢ers [27]. We do not know the species composition and the apparent Km values for O2 of the nitrifying
community in our sediments. We can state, though, that
the observed decreases of nitri¢cation activity took place
in sediment layers (0^2 mm) with O2 concentrations exceeding the above Km values considerably. It is thus unlikely that O2 limitation explains the decrease of nitri¢cation activity. Limitations of nutrients other than the ones
measured might have developed during the incubation period and caused a decrease of overall microbial metabolism. Concentrations of porewater-dissolved organic and
inorganic carbon, however, did not decrease during the
4-week incubation (data not shown). Moreover, heterotrophic O2 consumption (i.e. total O2 consumption minus O2
consumption due to nitri¢cation as calculated using the
þ
3
stoichiometry of NO3
3 production from NH4 and NO2 )
remained constant or even increased in both sediment
types (data not shown). Thus, limitations of phosphate
or iron or a general nutrient exhaustion seem rather unlikely. Porewater pH has previously been considered the
controlling factor of nitri¢cation in stream sediments, with
values lower than 7.0 signi¢cantly reducing nitri¢cation
activity [28]. In both sediment types used for our study
pH micropro¢les were measured in an independent experiment conducted at a di¡erent time of the year (unpublished data by P. Stief). The values decreased from pH
7.7 in the water column down to pH 6.5 and 5.8 in the
organic-poor and organic-rich sediments, respectively.
However, in the layer of highest nitri¢cation activity (i.e.
the 0^1-mm layer) pH was never lower than 6.9^7.1. Given that in the study presented here the pH gradients were
of similar shape, there was a small chance that porewater
pH had a negative e¡ect on nitri¢cation activity. However,
it remains inconclusive why the drop in pH reduced nitri¢cation only in the organic-rich, but not in the organicpoor sediment. To overcome these uncertainties, in future
studies pH microsensors should be included in the set of
routinely used microsensors.
Factors related to the processing of the sediments might
deliver alternative explanations for our results. (i) Sedi-
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70
P. Stief et al. / FEMS Microbiology Ecology 46 (2003) 63^71
ment sieving removed macrofauna, which may have controlled the abundance of lower trophic levels, such as
meio- and microfauna [29]. The absence of a top-down
control of bacterivorous meio- and microfauna may in
return have reduced the abundance of bacteria to the
point where their activities were reduced [30,31]. Slowly
growing populations, such as nitri¢ers, may be unable to
compensate for these grazing losses [14,17,18]. On the other hand, there are also reports indicating that increased
bacterivory may keep bacteria in the exponential growth
phase and thus stimulate their activity [32,33]. (ii) During
the ¢lling of the sediment beakers organic particles, including attached heterotrophic bacteria, became enriched
in the top layer of the sediments. The release of allelopathic compounds by these heterotrophs may have directly
inhibited the nitri¢ers growing in the same sediment layer
[5,7]. (iii) High virus-to-bacteria ratios have been found in
several sediment types [34,35], suggesting a great potential
for viral infection of benthic bacteria. Since metabolically
active bacteria are the preferred hosts of viruses (‘viruses
kill the winner’) [36], also the non-substrate-limited nitri¢ers of our sediments might have represented vulnerable
targets of viral infection. Again, the growth rates of nitri¢ers might have been too low to compensate for losses due
to cell lysis.
5. Summary and conclusions
Microsensor measurements in model freshwater sediments demonstrated that the unexpected decrease of nitri3
¢cation rates was not due to limitation by NHþ
4 , NO2 , or
O2 . Three alternative hypotheses were presented for the
suppression of nitri¢cation: (i) meio- and microfaunal
grazing, (ii) allelopathic compounds excreted by heterotrophic bacteria, or (iii) viral infection. These modes of suppression might have been promoted by processing the sediment and by the experimental incubation : (i) sieving
eliminated members of the benthic food chain, (ii) ¢lling
the sediment beakers led to an accumulation of organic
particles and heterotrophic bacteria at the sediment surface, and (iii) nutrient enrichment might have rendered the
nitrifying subpopulation vulnerable to viral infection.
Whatever the exact mechanism is, it is apparent that sediment manipulation puts the benthic nitrifying community
at risk of being non-persistent. Care must be taken that
experimental sediment incubations do not exceed the transient period of stability.
Acknowledgements
Dr. Jost Borcherding and Detlef Henning are acknowledged for their support during sediment collection in the
¢eld. Anja Eggers, Gaby Eickert, and Ines Schro«der kindly
provided us with O2 microsensors. Our thanks are also
due to Dr. Armin Gieseke for helpful comments on the
manuscript. This study was supported by the Max Planck
Society and a grant by the German Research Foundation
(STI202/1-2).
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