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Applied and Environmental Microbiology, August 2001, p. 3481-3487, Vol. 67, No. 8
Marine Environment Division, Environmental
Assessment Department, National Institute for Resources and
Environment, AIST, MITI, Tsukuba, Ibaraki 305-8569, Japan
Received 6 December 2000/Accepted 30 May 2001
Nitrate flux between sediment and water, nitrate concentration
profile at the sediment-water interface, and in situ sediment denitrification activity were measured seasonally at the innermost part
of Tokyo Bay, Japan. For the determination of sediment nitrate concentration, undisturbed sediment cores were sectioned into 5-mm
depth intervals and each segment was stored frozen at Nitrogen is a primary limiting
nutrient in many coastal marine environments (15, 17), and
N removal via denitrification in sediments is an important process
regulating the degree of eutrophication of shallow coastal ecosystems
(19). The availability of nitrate
(NO3 Lomstein et al. (13) demonstrated the presence of
intracellular pools of NH4+ and
NO3 In 1995, it was reported that there is a large pool of intracellular
NO3 Intracellular NO3 Furthermore, a new genus of nitrate-accumulating sulfur bacteria,
Thiomargarita, was found off the Namibian coast.
Thiomargarita also oxidizes sulfide with
NO3 Nitrate-accumulating sulfur bacteria discovered to date inhabit
sediments of upwelling areas characterized by high primary productivity, high sediment concentrations of soluble sulfide, and low
levels of dissolved oxygen in bottom waters (7, 14, 18,
25). The probable fate of the intracellular
NO3 In the present study, NO3 Study site.
The investigation was carried out at a 10-m-deep
station in the innermost part of Tokyo Bay (35.62°N, 139.98°E)
(Fig. 1). The sediment texture was that
of silty mud with a high organic-matter content (particulate organic
carbon [POC]; ~2% sediment [dry weight]), and the sediment was
poorly inhabited by benthic fauna throughout the year due to anoxia in
the bottom water during summer. Oxygen penetration depth in the surface
sediment was only 2 to 3 mm even when dissolved oxygen (O2)
in the bottom water was at air saturation (M. Sayama, unpublished
data).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3481-3487.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Presence of Nitrate-Accumulating Sulfur Bacteria
and Their Influence on Nitrogen Cycling in a Shallow Coastal
Marine Sediment
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
30°C. The
nitrate concentration was determined for the supernatants after
centrifuging the frozen and thawed sediments. Nitrate in the uppermost
sediment showed a remarkable seasonal change, and its seasonal maximum
of up to 400 µM was found in October. The directions of the diffusive
nitrate fluxes predicted from the interfacial concentration gradients
were out of the sediment throughout the year. In contrast, the
directions of the total nitrate fluxes measured by the whole-core
incubation were into the sediment at all seasons. This contradiction
between directions indicates that a large part of the nitrate pool
extracted from the frozen surface sediments is not a pore water
constituent, and preliminary examinations demonstrated that the nitrate
was contained in the intracellular vacuoles of filamentous sulfur
bacteria dwelling on or in the surface sediment. Based on the
comparison between in situ sediment denitrification activity and total
nitrate flux, it is suggested that intracellular nitrate cannot be
directly utilized by sediment denitrification, and the probable fate of
the intracellular nitrate is hypothesized to be dissimilatory reduction
to ammonium. The presence of nitrate-accumulating sulfur bacteria
therefore may lower nature's self-purification capacity
(denitrification) and exacerbate eutrophication in shallow coastal
marine environments.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) is an important factor controlling
denitrification activity in many aquatic sediments (12, 19,
20).
in deposited microalgae in the surface
sediment of a coastal bay area and found a distinct seasonal maximum
for both pools after sedimentation of a phytoplankton bloom in early
spring and a minimum in fall and winter.
associated with filamentous sulfur
bacteria, Thioploca spp. from the continental shelf under
the oxygen-minimum zone in the upwelling region off the coast of Peru
and Chile at a water depth between 40 and 280 m (7,
26). Analysis of single filaments of Thioploca showed
that intracellular NO3 is contained in their
vacuoles at concentrations up to 500 mM (7). It was
previously assumed that Thioploca would reduce their
intracellular NO3 to dinitrogen
(N2) gas (denitrification), but the incubation experiments
of washed Thioploca sheaths with 15N
compounds showed that although conversion to dinitrogen
cannot be ruled out, dissimilatory reduction of
NO3 to ammonium
(NH4+) is the preferred pathway in
Thioploca (16). Beggiatoa spp. from
the Bay of Concepción, Chile, at a depth of 25 m, also have the capacity to concentrate NO3
intracellularly at levels ranging from 15 to 116 mM (25),
indicative of a similar nitrate-respiring metabolism.
accumulation at
concentrations of 130 to 160 mM was also reported for large, vacuolate,
filamentous sulfur bacteria, Beggiatoa spp., from a Monterey
Canyon cold seep at a depth of 900 m and from Guaymas Basin
hydrothermal vents at a depth of 2,004 m (14). A
respiratory conversion of NO3 to
NH4+ driven by oxidation of hydrogen sulfide or
endogenous stores of elemental sulfur is indicated as the metabolism
for their intracellular NO3 (D. C. Nelson, S. C. McHatton, A. A. Ahmed, and J. P. Barry, Abstr. 1999 Aquat. Sci. Meet. Am. Soc. Limnol. Oceanogr., 1999).
that is accumulated to a concentration of
100 to 800 mM in a central vacuole (18). All three genera
of nitrate-accumulating sulfur bacteria, Thioploca,
Beggiatoa and Thiomargarita, are closely related
according to 16S rRNA sequences and seem to have a similar physiology
(18).
accumulated in their vacuoles is
considered to be dissimilatory reduction to
NH4+ (11, 16; Nelson et al.,
Abstr. 1999 Aquat. Sci. Meet. Am. Soc. Limnol. Oceanogr.). The
ecological implication of their nitrate metabolism, namely, that
intracellular NO3 is dissimilatorily reduced
to NH4+ is significant, since this means that
nitrogen is conserved within the system and is recycled into pelagic
nitrogen cycling.
flux between
sediment and water, NO3 concentration profile
at the sediment-water interface, and in situ sediment denitrification
activity were measured seasonally at the innermost part of Tokyo Bay,
Japan, which is a shallow, semienclosed basin characterized by strong,
human-induced eutrophication.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Study area and sampling site ( 
) in the innermost part
of Tokyo Bay.
Sampling.
Water and sediment were sampled by scuba diving at
approximately monthly intervals. Temperature, salinity, and
concentrations of O2, nitrite
(NO2) and NO3 were
determined in samples of surface water (0.3 m below the surface) and
bottom water (20 cm above the sediment). Samples for determination of
O2 concentrations were taken in 100-ml Winkler bottles.
Samples for determination of NO2 and
NO3 concentrations were stored frozen at
30°C in 50-ml polyethylene bottles. Sediment samples were collected
in 5-cm-wide (inside diameter [i.d.]) and 25-cm-long acrylic tubes
for determination of density, water content, total
NO3 flux between sediment and water,
concentration profiles of NO3 at the
sediment-water interface, and in situ sediment denitrification activity. All sediment cores were carefully inspected, and only those
with an undisturbed sediment-water interface were used. All
measurements were initiated in the laboratory within a few hours after sampling.
Total NO3 flux.
Total
NO3 flux between sediment and water was
determined by whole-core incubation in the dark at the in situ bottom
water temperature using four intact cores in 1992 to 1993 according to
the method described by Jensen et al. (8), with some
modifications. The sediment was first adjusted in height so that the
acrylic tubes contained about 17 cm of sediment core overlaid by 8 cm of water (corresponding to about 150 ml). The water phase was then
discarded (except for a few milliliters to avoid disturbance of the
sediment-water interface) and was carefully replaced by 100 ml of fresh
bottom water that was filtered through Whatman GF/C filters
(1.2-µm-pore-size fiber glass filters) and aerated at atmospheric
saturation. Teflon-coated magnets (0.5 by 3 cm) were placed in the
water column 1 cm above the sediment surface and were gently driven by
an external, rotating magnet to ensure appropriate stirring of the
water column. The cores were transferred into a thermostatted water
bath at the in situ bottom water temperature, and total flux
measurement was started about 15 min later. The top of the core tubes
was kept open during the incubation to allow repeated sampling of the
overlying water so that the dissolved O2 concentrations in
the overlying water were always under nearly air-saturated conditions
during the total flux measurements. Two and a half milliliters of the
overlying water was taken at 0.5- to 1-h intervals, and a total of six
water samples were withdrawn during 4 h of incubation. The total
flux was calculated by measuring the changes in concentration in the
overlying water during the incubation (linear regression) and by
multiplying the obtained rate with the specific volume/surface area
ratio of the water overlying the sediment core. No correction was made
for water column activity, since filtered bottom water was used as the
overlying water.
NO3 concentrations in sediment.
NO3 concentrations in the sediments were
determined in 1992 to 1993 together with the total flux measurements.
To determine NO3 concentrations in the
sediment, three undisturbed sediment cores were sectioned into
5-mm-depth intervals, and each segment was stored frozen at 30°C
for several weeks. Pore water was separated by centrifuging the frozen
and thawed sediment samples at 2,000 × g for 10 min,
and the NO3 concentration was determined for
the supernatants.
Diffusive NO3 flux.
Molecular
diffusive flux (Fdiffusive) of
NO3 between sediment and water was calculated
from the interfacial concentration gradient using Fick's first law of
diffusion (e.g., see article by Berner [4]):
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is porosity at the sediment surface (depth, 0 to 5 mm), Ds is the molecular diffusion coefficient
in sediment (Ds = 2D0) (Ullman and Aller
[27]), D0 is the
temperature-corrected molecular diffusion coefficient in water
(5), and dC/dx is the interfacial concentration
gradient. Interfacial concentration gradients were found by linear
interpolation between the bottom water concentration at the sediment
surface and the concentration in the 0- to 5-mm-depth section assigned
to a depth of 2.5 mm.
NO3 concentration profiles before and
after freezing samples of water and sediment.
Since freezing and
thawing the samples of water and sediment seemed to have some effect on
the NO3 concentration, a change of
NO3 concentration profile before and after
freezing of the samples of water and sediment was examined in October
1993. To determine the NO3 concentration
profiles at the sediment-water interface, the overlying water in an
undisturbed sediment core was withdrawn at 5-mm intervals with a
syringe through a vertical series of silicone-filled holes (i.d., 2.5 mm) placed at 5-mm intervals along the side of the core tubes and the
sediment in the same core was then sliced into discrete depth
intervals. Each sample of water or sediment was centrifuged at
2,000 × g for 10 min immediately after sectioning (unfrozen samples) or was stored frozen at 30°C for several weeks and centrifuged immediately after thawing (frozen samples). The NO3 concentration was determined for the supernatants.
Variations of NO3 and
NH4+ concentrations in sediment caused by
different extraction conditions.
Since the
NO3 concentration found in frozen sediment of
0- to 5-mm depth was extremely high in October, variations of
NO3 and NH4+
concentrations in the sediment caused by different extraction conditions were examined in October 1993. Freshly sectioned surface (depth, 0 to 5 mm) sediment was homogeneously mixed and then treated as
follows. (i) The sediment was centrifuged immediately. (ii) Homogenized
fresh sediment (2.5 g [wet weight]) was added to a 45-ml
polypropylene centrifuge tube containing 10 ml of artificial seawater
(ASW) (27.2 g of NaCl, 0.7 g of KCl, 2.0 g of
CaCl2, and 6.0 g of MgSO4 per liter, with
an adjusted pH of 8.0 to 8.2 and 0.05% NaHCO3), and the
sediment suspension was shaken vigorously for >2 min and then
centrifuged. (iii) Procedure was the same as for ii, but deionized
water (DW; Milli-Q) was used instead of ASW. (iv) Procedure was the
same as for ii, but 1 M KCl was used instead of ASW. (v) The sediment
mixture was stored frozen at 30°C and centrifuged immediately after
thawing. Each treatment was done in triplicate, and
NO3 and NH4+
concentrations were determined for the supernatants after centrifuging at 2,000 × g for 10 min.
In situ sediment denitrification. In situ denitrification activity was measured in 1988 to 1989 separately from the flux and concentration measurements. Additional measurements were repeated to verify the reproducibility of the activity in July and November 1990. The measurements of denitrification were based upon the acetylene inhibition technique with undisturbed sediment cores (22). Acetylene, which inhibits the reduction of N2O to N2, is distributed into undisturbed sediment cores, and the rate of N2O accumulation in cores is used as a measure of in situ denitrification. I essentially used the core design and incubation procedure described by Andersen et al. (1) with slight modification.
Six sediment cores were taken for each determination of denitrification activity. In each core, the water phase was first discarded (except for a few milliliters to avoid disturbance of the sediment-water interface) and was carefully replaced with fresh bottom water to fill up the core tubes completely. The bottom water, whose dissolved O2 concentration had been kept at nearly in situ level, was used without filtration. The core was then hermetically capped with a rubber stopper, leaving ca. 8 cm (150-ml volume) of the overlying water. A small magnetic stirring bar was placed underneath the rubber stopper to mix the water phase in the same way as the total flux measurement. C2H2-saturated distilled water (600 µl) was injected into the sediment at depths from 0 to 6 cm through a vertical series of silicone-filled holes (i.d., 2.5 mm) placed at 5-mm intervals along the side of the tubes. The water phase also received C2H2-saturated water, and the final inhibitory concentrations in the pore water and in the overlying water were about 10% of saturation (vol/vol). The cores were incubated in the dark at the in situ bottom water temperature using a thermostatted water bath. Incubations of duplicate cores were terminated at 0, 1, and 2 h. After incubation, the exact height (volume) of the water in each tube was recorded. Ten milliliters of the water phase was then transferred to a closed serum bottle (70-ml volume). The bottle was shaken vigorously for >1 min to equilibrate the dissolved N2O with the gas phase. The excess pressure was then released by inserting a needle to the rubber stopper of the serum bottle, and 5 ml of the headspace gas was taken to a preevacuated glass vial of this volume (Venoject, VT-050P; Terumo Corp., Tokyo, Japan) for storage. After removal of the overlying water, the upper 6 cm of the sediment was sectioned into depth intervals of 0 to 1, 1 to 2, 2 to 4, and 4 to 6 cm. Each segment was quickly transferred to a preweighed 100-ml beaker containing 20 ml of 4% (vol/vol) neutralized formalin. The beakers were immediately closed with rubber stoppers and shaken for >1 min. After the weight of each segment had been recorded, another 5 ml of the headspace gas was stored in a preevacuated Venoject tube for analyses of N2O. In the headspace analysis, the volumetric solubility coefficients given by Weiss and Price (28) were used to calculate the concentration of N2O in the original samples of water and sediment. The cores terminated at zero time were to correct for in situ N2O content in the water and sediment. Denitrification activity at each sediment layer was calculated as the mean of the N2O accumulation rates measured in four different cores. I assumed that the N2O accumulating in the water phases of the cores was a result of diffusion from the uppermost centimeter of the sediment. This assumption seemed valid because the accumulation of N2O during the incubations with C2H2 was always highest in the uppermost centimeter, thus creating a gradient of N2O up into the water phase. Arial activity of denitrification was calculated from depth integration of the activity in the sediment at a depth from 0 to 6 cm.Chemical analysis.
Salinity was measured potentiometrically,
and a Winkler titration was performed to determine the dissolved
O2 concentration (24). Nutrient concentrations
were determined by an autoanalyzer (AutoAnalyzer II; Bran+Luebbe Inc.,
Tokyo, Japan) using the methods of Solorzano (21) for
NH4+ and Armstrong et al. (2) for
NO3 and NO2 after
thawing of the stored frozen samples. When necessary, an additional
centrifugation (2,000 × g, 10 min) was performed to clear the supernatants completely. Triplicate determinations of the
densities (grams centimeter3) and water contents (weight
loss after 24 h at 105°C) of the sediment samples served to
convert the measured concentrations and activities into appropriate dimensions.
1.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The inner part of Tokyo Bay is characterized by strong,
human-induced eutrophication, and due to the marked saline
stratification, O2 was totally depleted (anoxia) in the
bottom water during summer over a wide area (Fig.
2). As destratification was initiated in early fall, O2 concentrations in the bottom water gradually
increased and recovered to nearly air-saturated conditions during
winter and early spring. Episodic oxygen depletion in the bottom water was often observed in late spring and early summer.
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,
O2Total NO3 flux.
The directions of
the total NO3 fluxes between sediment and
water measured with undisturbed sediment cores were from the overlying water into the sediment at all seasons (Fig. 2). A maximum uptake of
0.27 µmol of N cm2 day1 was recorded in
October 1992; the uptake decreased rapidly in late fall and was
negligible in December. A dramatic increase was observed in early
spring following the sedimentation of a phytoplankton bloom, and the
uptake reached its maximum again in March to May. There was no
NO3 uptake during the marked summer
stratification when O2 and NO3
were absent in the bottom water. As destratification was initiated in
early fall, the uptake started to increase and reached its maximum
again in October 1993.
flux in July (Sayama, unpublished data).
In August, the NO3 uptake into the sediment
measured at 100% air saturation (0.07 ± 0.01 µmol of N
cm2 day1; mean ± standard error
[SE]; n = 4) was somewhat lower than that measured at
0% air saturation (0.10 ± 0.02 µmol of N cm2
day1; n = 4).
NO3 concentrations in frozen
sediment.
The NO3 concentrations
extracted from the frozen sediment samples always peaked at a depth of
0 to 5 mm throughout the year, except in July. There was a huge pool of
NO3, ca. 400 µM, in the uppermost sediment
(depth, 0 to 5 mm) in October 1992 (Fig. 2). The
NO3 pool at the sediment surface decreased
considerably in late fall and was negligible in early spring. The
NO3 pool recovered to the winter level during
early summer, but a final depletion occurred during the summer
stratification when O2 and NO3
were absent in the bottom water. As destratification was initiated in
early fall, the NO3 pool increased
dramatically and a huge pool of ca. 260 µM was found again in October
1993. The NO3 concentrations in the uppermost
sediments exceeded the concentration in the bottom water throughout the
year (Fig. 2), and the interfacial concentration gradient was the
steepest in October.
NO3 concentration profiles before and
after freezing samples of water and sediment.
The
NO3 concentration profiles changed
significantly before and after samples of water and sediment collected
in October 1993 were frozen (Fig. 3). The
NO3 pool in the surface sediment of unfrozen
samples (centrifuged immediately after sectioning) was much lower than
that of frozen samples (frozen at 30°C and centrifuged immediately
after thawing). In contrast to the concentration profile of the frozen
samples, the great peak of NO3 was never
observed in the uppermost sediment in the unfrozen samples.
NO3 concentrations of the unfrozen samples
showed a minimum in the uppermost sediment layer, followed by a small
peak at a depth of 15 to 20 mm. The most striking feature was the shift
of the interfacial concentration gradient to the inverse direction.
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Variations of NO3 and
NH4+ concentrations in sediment caused by
different extraction conditions.
The NO3
concentration extracted by immediate centrifugation of unfrozen surface
(depth, 0 to 5 mm) sediment collected in October 1993 was only
0.22 ± 0.06 nmol of N cm wet sediment3 (mean ± SE; n = 3) (Fig. 4).
There was a slight increase in the NO3
concentration when ASW extraction was performed: 1.96 ± 0.22 nmol of N
cm wet sediment3. However, DW extraction and 1 M KCl
extraction resulted in a dramatic increase, with values of 46.4 ± 1.3 nmol of N cm wet sediment3 for DW extraction and
47.0 ± 2.6 nmol of N cm wet sediment3 for 1 M KCl
extraction, which were roughly comparable to the concentration
extracted from frozen sediment, 68.5 ± 1.0 nmol of N cm wet
sediment3.
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concentration (Fig. 4).
In situ sediment denitrification.
Similar to what was seen for
the NO3 pool of frozen sediment, the activity
of in situ sediment denitrification peaked at a depth of 0 to 5 mm in
all seasons (data not shown). The activity of in situ sediment
denitrification was approximately 0.02 µmol of N cm2
day1 in winter (Fig. 2). Following the spring bloom
sedimentation, a seasonal maximum activity of approximately 0.04 µmol
of N cm2 day1 was found in March to May. A
final depletion occurred during the summer stratification when
O2 and NO3 were absent in
the bottom water. As destratification was initiated in early fall, the
activity recovered to the winter level. Additional measurements in 1990 verified that there was good agreement with the activities obtained at
different years (Fig. 2). Therefore, it seems reasonable to compare
denitrification activity with the total NO3
flux measured during different years.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Intracellular nitrate pool in Tokyo Bay sediment.
In studies
of sediment nitrogen cycling in shallow coastal marine sediments,
attention has so far mainly been given to NO3
dissolved in pore water and an NO3 pool that
is not a pore water constituent has been considered unlikely to exist
in sediments.
concentrations extracted from the
frozen samples of the uppermost sediment (depth, 0 to 5 mm) always
exceeded the concentration in the bottom water (Fig. 2), and the
interfacial concentration gradient was the steepest in October. The
directions of the diffusive NO3 fluxes
predicted from the interfacial concentration gradients were out of the
sediment throughout the year. In contrast, the directions of the total
NO3 fluxes measured with the undisturbed
sediment cores were into the sediment at all seasons, and a seasonal
maximum uptake was recorded in October and in March to May (Fig. 2).
This contradiction between the directions of the diffusive flux and of
the total flux implies that a large part of the
NO3 pool extracted from the frozen surface
sediments is not a pore water constituent, particularly in October.
This hypothesis is also supported by the finding that the
NO3 pool of unfrozen surface sediments
(centrifuged immediately after sectioning) was much smaller than that
of the frozen surface sediments collected in October (Fig. 3 and 4) and
that the NO3 concentration profiles changed
significantly before and after the samples of water and sediment
collected in October were frozen (Fig. 3). In contrast to the
concentration profile of the frozen samples, the great peak of
NO3 was never observed in the uppermost
sediment in the unfrozen samples (Fig. 3). The most striking feature is
the shift of the interfacial concentration gradient to the inverse
direction. Now the direction of the diffusive
NO3 flux predicted from the interfacial
concentration gradient of the unfrozen samples is in agreement with
that of the total NO3 flux measured with the
undisturbed sediment cores.
Lomstein et al. (13) demonstrated the presence of
intracellular pools of NH4+ and
NO3 in deposited microalgae in the surface
sediment of a coastal bay area and found a distinct seasonal maximum
for both pools after sedimentation of a phytoplankton bloom in early
spring and a minimum in fall and winter. The
NO3 pool extracted from the frozen surface
sediments in Tokyo Bay is, however, inferred not to be an intracellular
pool in deposited microalgae, because the NO3
pool was small in late spring (Fig. 2) and NH4+
concentration showed only a minor change before and after the surface
sediment collected in October was frozen (Fig. 4).
Recently, NO3 accumulation in an
intracellular vacuole at concentrations greater than 15 to 800 mM has
been reported for sulfur-oxidizing bacteria from offshore and deep-sea
sediments of upwelling areas (7, 14, 18, 25). Those
sulfur-oxidizing bacteria inhabit the environments characterized by
high primary productivity, high sediment concentrations of soluble
sulfide, and low levels of dissolved O2 in surrounding waters.
In hypertrophic, shallow coastal marine environments where sulfide
production in the sediment is high and O2 in the bottom water is often depleted, white mats of filamentous sulfur bacteria on
the sediment surface are frequently encountered (9, 10). In fact, there were filamentous sulfur bacteria in the surface sediment
at the site of this study in October, and massive mats of filamentous
sulfur bacteria are often formed on the sediment surface during the
period of transition from anoxic to oxic bottom water. A preliminary
microscopic inspection showed that the filamentous sulfur bacteria that
constitute the white mats covering the sediment surface in Tokyo Bay in
October 2000 are characterized by living as individual filaments,
having considerable diameters (around 9 µm), and having a large
central vacuole and intracellular globules of elemental sulfur (Fig.
5). These morphological characteristics of the bacteria are very much similar to those for a wide, vacuolate, nitrate-accumulating Beggiatoa sp. from a Monterey Canyon
cold seep and Guaymas Basin hydrothermal vents (14) and
from the Bay of Concepción, Chile (25). All
populations of large, vacuolated Beggiatoa examined to date
share the ability to concentrate NO3 at
levels ranging from 15 to 160 mM in their intracellular vacuoles. Furthermore, a preliminary examination demonstrated that the
filamentous sulfur bacteria in the surface sediment sampled at the
study site in Tokyo Bay in October 2000 accumulated
NO3 intracellularly at concentrations of
105 ± 36 mM (mean ± SE; n = 3) (Sayama and
T. Kuwae, unpublished data).
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pool extracted from the frozen surface
sediments in Tokyo Bay is contained in the intracellular vacuoles of
the filamentous sulfur bacteria, that freezing and thawing the surface
sediment result in the breakage of the bacterial cells
(14), and that intracellular NO3
is released into pore water in frozen and thawed sediments. The NO3 concentration extracted by use of ASW
from the unfrozen surface (depth, 0 to 5 mm) sediment collected in
October was negligibly low, while the NO3
concentrations extracted by use of DW and 1 M KCl were enormously high
and roughly comparable to the concentration extracted from the frozen
sediment (Fig. 4). This result can be consistently explained by the
rupture of bacterial cells due to a drastic change in osmotic pressure
and evidently supports the above conclusion.
The molecular diffusive flux (Fdiffusive) of
NO3 in October calculated using the equation
given earlier with the interfacial concentration gradient of the
unfrozen samples (Fig. 3) is 0.04 ± 0.01 µmol of N
cm2 day1 (n = 3), which is
rather small compared to the total NO3 flux
in October, 0.19 ± 0.03 µmol of N cm2
day1 (Fig. 2) (smaller by a factor of four). Therefore,
the transport process dominating the sediment-water exchange of
NO3 was not molecular diffusion at the study
site in October. This result can be also explained consistently by the
highly efficient gliding motility of Beggiatoa spp. and the
large surface area of their filaments (9).
Beggiatoa spp. may stretch their filaments up into the
overlying water, take up NO3 from the
nitrate-containing bottom water, and glide back into the
sulfide-producing sediment.
Fate of intracellular nitrate.
The probable fate of the
intracellular NO3 accumulated in the
nitrate-accumulating sulfur bacteria discovered to date is considered to be dissimilatory reduction to NH4+
(11, 16; Nelson et al., Abstr. 1999 Aquat. Sci. Meet. Am. Soc. Limnol. Oceanogr.). The ecological implication of their nitrate metabolism, that intracellular NO3 is
dissimilatorily reduced to NH4+, is
significant, since this means that nitrogen is conserved within the
system and is recycled into pelagic nitrogen cycling.
uptake by the sediment in almost all
seasons, especially in March to May and in October (Fig. 2). The
acetylene inhibition technique is subject to potential problems
primarily because nitrification, and hence coupled denitrification, is
inhibited by acetylene (3) and because inhibition of
denitrification by acetylene can sometimes be incomplete, particularly
in the presence of sulfide (6, 23), the concentration of
which is generally very high in eutrophied coastal marine sediments.
However, the difference between the NO3
uptake by the sediment and the in situ sediment denitrification activity was so large that it cannot be explained as an underestimation caused by the technical problems of the acetylene inhibition.
In addition, there was a significant correlation between
NO3 and NO2
concentrations in the bottom water and the in situ sediment
denitrification activity (r2 = 0.339; P < 0.05;
n = 13). This suggests that the major source of
NO3 for the sediment denitrification was
coming from the overlying water at the study site.
From these results, it seems reasonable to hypothesize that the
intracellular NO3 pool found in Tokyo Bay
sediment cannot be directly utilized by the sediment denitrification
and that the probable fate of it is also dissimilatory reduction to
NH4+.
Influence of nitrate-accumulating sulfur bacteria on nitrogen
cycling in sediment.
Judging from the difference between the
NO3 concentrations in the bottom water and in
the frozen and thawed surface sediments, there seems to be a
significant amount of intracellular NO3 in
almost all seasons (Fig. 2). The nitrate-accumulating sulfur bacteria
therefore may play an important role in annual nitrogen cycling in
shallow coastal marine sediment. In the innermost part of Tokyo Bay,
the annual NO3 uptake into the sediment was
45.8 µmol of N cm2 year1 (Fig. 2), but
the annual denitrification in the sediment was only 9.5 µmol of N
cm2 year1 (Fig. 2), and the rest of
NO3 taken up by the sediment (36.3 µmol of
N cm2 year1) is hypothesized to be reduced
dissimilatorily to NH4+. The ecological
consequence of the huge pool of intracellular NO3 and its dissimilatory reduction to
NH4+ is increased efflux of
NH4+ to the water column (internal loading).
Indeed, the annual NH4+ efflux was 207 µmol
of N cm2 year1 at the site of this study
(Sayama, unpublished data), and about 18% of the
NH4+ released from the sediment is estimated to
be coming from dissimilatory reduction of
NO3.
is transported into the intracellular
NO3 pool in the surface sediment directly
from the overlying bottom water by vertical migration of
nitrate-accumulating sulfur bacteria. The accumulated intracellular
NO3 is not directly available for sediment
denitrification, and the probable fate of the intracellular
NO3 is hypothesized as dissimilatory
reduction to NH4+ and being recycled into
pelagic nitrogen cycling together with the NH4+
coming from the mineralization of particulate organic nitrogen. Therefore, the presence of filamentous sulfur bacteria that accumulate a huge amount of NO3 in their intracellular
vacuoles and reduce the intracellular NO3
dissimilatorily to NH4+ lowers nature's
self-purification capacity (denitrification) and stimulates pelagic
primary production or exacerbates eutrophication in shallow coastal
marine environments.
|
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ACKNOWLEDGMENTS |
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I am very grateful to Peter Bondo Christensen (National Environmental Research Institute, Silkeborg, Denmark) for critical review of the manuscript and to S. Shimamura for technical assistance.
This study was supported by a grant from the Environmental Agency, Tokyo, Japan.
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FOOTNOTES |
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* Mailing address: Marine Environment Division, Environmental Assessment Department, National Institute for Resources and Environment, AIST, MITI, 16-3 Onogawa, Tsukuba, Ibaraki 305-8569, Japan. Phone: 81-298-61-8375. Fax: 81-298-61-8357. E-mail: m.sayama{at}aist.go.jp.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, August 2001, p. 3481-3487, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3481-3487.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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