Previous Article | Next Article 
Applied and Environmental Microbiology, December 2001, p. 5530-5537, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5530-5537.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Ecology of Thioploca spp.: Nitrate and
Sulfur Storage in Relation to Chemical Microgradients and Influence of
Thioploca spp. on the Sedimentary Nitrogen Cycle
Jakob
Zopfi,1,*
Thomas
Kjær,2
Lars P.
Nielsen,2 and
Bo
Barker
Jørgensen1
Max Planck Institute for Marine Microbiology,
D-28359 Bremen, Germany,1 and Institute
of Biological Sciences, University of Aarhus, DK-8000 Aarhus C,
Denmark2
Received 27 November 2000/Accepted 25 September 2001
 |
ABSTRACT |
Microsensors, including a recently developed
NO3
biosensor, were applied to measure
O2 and NO3 profiles in marine
sediments from the upwelling area off central Chile and to investigate
the influence of Thioploca spp. on the sedimentary nitrogen
metabolism. The studies were performed in undisturbed sediment cores
incubated in a small laboratory flume to simulate the environmental
conditions of low O2, high NO3,
and bottom water current. On addition of NO3
and NO2, Thioploca spp. exhibited
positive chemotaxis and stretched out of the sediment into the flume
water. In a core densely populated with Thioploca, the
penetration depth of NO3 was only 0.5 mm and
a sharp maximum of NO3 uptake was observed
0.5 mm above the sediment surface. In sediments with only few
Thioploca spp., NO3 was
detectable down to a depth of 2 mm and the maximum consumption rates
were observed within the sediment. No chemotaxis toward nitrous oxide
(N2O) was observed, which is consistent with the observation that Thioploca does not denitrify but reduces
intracellular NO3 to
NH4+. Measurements of the intracellular
NO3 and S0 pools in
Thioploca filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO3 were found to a
depth of at least 13 cm, providing final proof for the vertical
shuttling of Thioploca spp. and nitrate transport into the sediment.
| |
INTRODUCTION |
Although the ability of
microorganisms to oxidize reduced sulfur compounds with nitrate as the
electron acceptor has been known for about one hundred years
(1) and several pure cultures have been obtained and
studied (see, e.g., references 39 and 42), only a little
is known about the ecological significance of this type of metabolism.
For instance, the first report showing a clear coupling between the
sulfur and nitrogen cycles in the marine environment was sulfide-driven
denitrification at the oxic-anoxic interface in the water column of the
Central Baltic Sea (3).
It was recently discovered that sulfide-oxidizing bacteria of the genus
Thioploca possess large nitrate-filled vacuoles
(10). Thioploca is highly abundant in the shelf
sediments along Peru and Chile (11, 12, 14, 29), and it
was therefore suspected that these organisms play a major role in
coupling the biogeochemical cycles of nitrogen and sulfur in upwelling
areas (10). The observation of benthic
Thioploca filaments in the upwelling area of the Arabian Sea
(20) and the finding of both Thioploca and the
spherical, nitrate-storing bacterium Thiomargarita off
Namibia (34) support this conclusion. Nitrate-storing
sulfide-oxidizing bacteria have also been observed at hydrothermal
vents and cold seeps and in organic-rich sediments (23, 24,
44).
Currently, none of the nitrate-storing sulfur bacteria is in pure
culture, and alternative methods have to be applied to study their
physiology and ecology. Enzyme preparations from partially purified
Beggiatoa samples showed high nitrate reductase and
ribulose-1,5-bisphosphate carboxylase-oxygenase activity and provide
the first biochemical evidence for the use of nitrate as a electron
acceptor for sulfide oxidation and chemoautotrophic growth
(23). Incubation experiments with partially purified
Thioploca filaments revealed that sulfide (H2S)
was first rapidly oxidized to [S0], which was then
further oxidized to sulfate (SO42) in a
second independent step. Intravacuolar
[NO3] served as the electron acceptor and
was reduced to ammonium (NH4+)
(25). Radiolabeled bicarbonate
(H14CO3) and
[2-14C]acetate were assimilated, indicating that
Thioploca is a facultative chemolithoautotroph capable of
mixotrophic growth (22, 25). Whole-core incubations in
small laboratory flumes have helped to unveil the chemotactic behavior
of Thioploca under changing environmental conditions.
Thioploca showed positive chemotaxis toward nitrate and low
sulfide concentrations (<100 µM) but a phobic reaction toward oxygen
and high sulfide concentrations (17). These observations
and the finding of mostly vertically oriented living filaments several
centimeters deep in the sediment led to the suggestion that
Thioploca shuttles up and down between NO3-rich bottom water and
H2S-containing sediment (10, 17).
The samples for this study were collected at stations within and off
the Bay of Concepción in central Chile, where the species composition and annual dynamics of the Thioploca population
are known from a previous study (36). On the shelf, the
community was composed mainly of T. araucae, T. chileae, and
a yet undescribed form of Thioploca with much shorter cells.
This so-called short-cell morphotype (SCM) (35, 36) is
usually found in deeper sediment layers than the two other species and
is characterized by rounded cells and a cell length/diameter ratio of
0.48. The SCM is closely related but not identical to the known
Thioploca species as revealed by partial 16S rDNA analysis
(35). Within the Bay of Concepción, large vacuolated
filaments cover the sediment surface during part of the year. They are,
apart from the lack of sheaths, phenotypically and phylogenetically
almost identical to T. araucae (36, 40).
The aim of this study was to gain information about the ecology of
Thioploca spp. and the influence of these organisms on the
nitrogen and sulfur cycles in the habitat. By the use of microsensors, we show that the nitrate uptake of the sediment is strongly influenced by Thioploca. Chemotaxis toward inorganic nitrogen compounds
was studied, and measurements of [S0] and
[NO3] concentrations were used to test the
concept of a vertical shuttling between the nitrate-rich bottom water
and the deeper sediment layers. We also demonstrate that nitrate is
transported by Thioploca down to a sediment depth of at
least 13 cm.
| |
MATERIALS AND METHODS |
Abbreviations.
Nitrate and elemental sulfur are generally
abbreviated by NO3 and S0,
respectively. For the intracellular pools
[NO3] and [S0] are used.
Study area.
The continental shelf region off the
Concepción Bay (central Chile) is characterized by intense
seasonal upwelling. Between austral late spring and early fall,
southern and southwestern winds prevail and the northward-flowing
Sub-Antarctic Surface water is forced off the coast, leading to
upwelling of Equatorial Subsurface water from the Poleward Undercurrent
at 100 to 400 m (37). The Equatorial Subsurface water
is characterized by high salinity (34.4 to 34.8%), low temperature
(8.5 to 10.5°C), low oxygen concentrations (<20 µM), but high
nitrate (about 25 µM) and nutrient concentrations (37).
Upwelling off central Chile is intermittent and usually lasts between 2 and 7 days (16). Primary and secondary productivity
greatly increase when the nutrient-rich water is transported up into
the euphotic zone. For the coastal upwelling area off central Chile, a
primary production of 9.6 g of C m2
day1 has been reported (10), which is one of
the highest observed in marine environments. Due to the lack of oxygen
and sufficient amounts of alternative electron acceptors, sedimentary
organic matter is almost exclusively degraded by sulfate-reducing
bacteria (41). The sulfate reduction rates reported for
this area (170 to 4,670 nmol cm3 day1
[(8)]) are among the highest observed in coastal
margins, but free sulfide concentrations in these sediments are
surprisingly low, indicating an efficient reoxidation of sulfide
(8; J. Zopfi, M. E. Böttcher, and B. B. Jørgensen, submitted for publication).
Sampling and site description.
During January and February
1997, we repeatedly sampled sediment from three stations within and off
the Bay of Concepción (Fig. 1). The
sediment was collected from the research vessel Kay Kay of
the University of Concepción (Concepción, Chile) by means
of a small gravity corer. The cores were stored onboard at 4°C in a
refrigerator and were transported on the day of sampling to the Marine
Biological Station of the University Concepción in Dichato, where
all experiments were performed.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 1.
Sampling sites within the Bay of Concepción and on
the adjacent continental shelf off central Chile.
|
|
Station 4 (36° 38' 08" S; 073° 02' 03" W) was 24 m deep and
located within the bay (Fig.
1). The sediment at this station
was
highly sulfidic (up to 1,200 µM at 7 cm deep [Zopfi et al.,
submitted]) and was uniformly black below the brownish uppermost
3 to
4 mm. The top 4.5 to 5 cm of the sediment was a flocculent
ooze, with
mass accumulation of
Beggiatoa spp. The sediment of
Station
7 (36° 36' 05" S; 073° 00' 06" W; 32 m deep), at the mouth
of
the bay, was covered with a brown, spongy layer 1.5 cm thick,
which was
densely populated by
Thioploca spp. Below this layer
was a
0.4-cm-thick band of black iron sulfide, followed by gray-brownish
sediment. Burrows of sediment-dwelling organisms were observed.
The
sediment of Station 18 (35° 30' 08" S; 073° 07' 06" W; 88
m
deep) had a similar structure, with a spongy layer 0.5 to 1
cm thick, a
thin black layer (0.2 to 0.3 cm thick), and then gray
sediment below.
Thioploca, however, was much less abundant, and
no animal
burrows were observed. The concentration of free sulfide
was <6 µM
down to a depth of 20 cm at Stations 7 and 18. During
the sampling
period, the bottom-water concentration of O
2 was
<2 to 7 µM at all stations. The NO
3 concentrations
were about 6 µM at Station 4, and 7 to 24 µM at
Stations 7 and 18 (
36).
Microsensor measurements and flux calculations.
A Clark-type
O2 microsensor with a guard cathode was used to measure
oxygen microprofiles (28). Nitrate was measured with a
microbiosensor consisting of an electrochemical N2O
microsensor surrounded by an outer casing (19). A 100 to
200-µm-long reaction chamber was formed between the tip of the
internal N2O sensor and the ion-permeable membrane at the
tip of the outer casing. An N2O reductase-deficient culture
of Agrobacterium immobilized in the reaction chamber
transformed NO3 and
NO2 to N2O, which was detected by
the N2O microsensor. Due to this design, the microsensor
measured the combined NO3,
NO2, and N2O concentrations in a
sample. The N2O microsensor was constructed like the
O2 microsensor (28) but the cathode was plated
with silver and the electrolyte consisted of 0.5 M NaOH and 0.5 M KCl.
The N2O microsensor was polarized at 
1.2 V against an
Ag/AgCl anode immersed in the electrolyte. The current from the
microsensor was measured with a custom-made pA meter and recorded on a
strip chart recorder. The active silver surface of the N2O sensing cathode is also sensitive toward H2S. However, the
concentrations of free sulfide in the top centimeters of the Station 7 and 18 sediment was usually <1 µM, and hence no interference with
H2S was anticipated. Calibrations and measurements were
done at the same temperature. The detection limit of the sensor was
about 3 µM NO3, and the linear range was 3 to 70 µM NO3. The sensitivity for
N2O was about 1 µM, and the signal was linear up to least
1,000 µM. The response time for 90% signal intensity was about
50 s. A resting time of 1 min was used for each depth step in a
profile. The position of the sediment surface was determined for each
profile using a dissection microscope (magnification, ×10 to ×50).
Fluxes across the sediment-water interface were calculated from the
upper linear part of the microsensor profiles by Fick's
first law of
diffusion,
J =
D ×
dC/
dx, where
J is
the flux (in
micromoles per square centimeter per second),
D
is the diffusion
coefficient (in square centimeters per second), and
dC/dx is the
concentration gradient (in micromoles per cubic
centimeter per
centimeter). The activity profiles were
calculated as described
in detail elsewhere (T. Kjær, L.-H. Larsen,
and N. P. Revsbech,
unpublished data). Tabulated diffusion
coefficients were recalculated
for the
in situ temperature
and salinity (
4,
21). Since the
sediment was highly porous
in the top 1 cm, the same diffusion
coefficients of 1.8 × 10
5 and 1.41 × 10
5
cm
2 s
1 for O
2 and
NO
3 respectively, were used for the sediment
and water
phase.
Experimental setup.
To simulate the environmental conditions
prevailing off the coast, measurements were done in a small flow
chamber where deaerated surface water from Station 7 was circulated. A
small aquarium pump was used to create flows of approximately 4 to 6 cm
s1 about 2 cm above the sediment surface. The flow
chamber consisted of two horizontal Plexiglas plates that were
separated with a spacer. Both Plexiglas plates contained a central hole
(50 cm2). A sediment core was brought into the flow through
the hole in the bottom plate, and the microsensors were introduced
through the hole in the top plate. A dissolved-O2
concentration of about 5 µM was maintained in the circulating water
by adjusting the area of air-exposed seawater at the top hole, and the
water was kept at the in situ temperature of 12°C by a thermostatted
circulating cooler. For the chemotaxis experiments,
NO3, NO2, or
N2O was added to nitrate-depleted flume water and the
behavior of Thioploca was observed from above through a
dissection microscope. The number of filaments emerging from their
sheaths was determined by setting the focus plane at about 2 mm above
the sediment surface and by counting the filaments penetrating the
plane. The length of a filament was determined with a measuring
eyepiece and by focusing down from the filament tip to the sediment surface.
Extraction and analysis of [NO3] and
[S0] in Thioploca.
Bundles of
Thioploca filaments from different sediment depths of
Station 7 were picked out and aligned in a film of seawater on a
microscope slide. Forceps and needles were used to rip the sheath apart
so that intact single filaments could be isolated.
The length (
l) and diameter (
d) of each filament
were determined, and the biovolume (
V) was calculated
(
V =
ld2/4). According to the cell
diameter and the cell-length-to-diameter
ratio, the organism was
identified as
T. araucae, T. chileae,
or SCM
(
36). With the same formula, the volume ratio between
the
cytoplasm and vacuole was determined for each species by using
the
following cell dimensions (length, diameter):
T. araucae,
14.4 µm, 15.4 µm;
T. chileae, 35.5 µm, 24.2 µm; SCM,
35.5 µm,
9.4 µm. For all three species, a mean cytoplasm thickness
of 1
µm was used (
36; H. Schulz, personal
communication).
A single filament was then picked up on the tip of a purpose-made glass
needle and left to dry in air for a few minutes, so
that the filaments
died and the cells cracked. Nitrate was extracted
from the filament by
dipping the glass needle for 5 s into a droplet
of 20 µl of
demineralized
water.
Nitrate was analyzed by the cadmium reduction method (
13),
with some adaptations to small amounts. The cadmium column typically
consisted of a 3-cm-long glass tube (inner diameter, 1.1 mm) with
a
slightly coiled, 1-mm-wide cadmium rod inside. A bent glass
capillary
with a pointed tip was mounted on the upper end of the
column, and a
straight capillary was mounted on the bottom. The
total system could
contain about 10 µl of liquid, and once activated
the column was
always kept filled with buffer solution. The column
was held almost
vertical, and when the upper tip was dipped in
liquid, gravity created
a water flow of about 30 µl min
1 through the column.
Capillary forces in the pointed tip prevented
intrusion of air when the
tip was out of water. The 20-µl sample
with the extracted nitrate was
sucked up, immediately followed
by 40 µl of buffer solution. Below
the reduction column the sample
and buffer solution was collected in a
300-µl well of a microplate.
Aliquots (20 µl) of reagents
were added by the standard procedure,
and 200 µl of demineralized
water was mixed in as well. The color
intensity was read at 670 nm, and
corrections for turbidity, if
any, were made by reading at 405 nm.
NO
3 standard solutions (20 µl each) were
processed parallel with
the filament extracts and used for calibration.
Linearity was
observed up to 250 µM. The reduction efficiency was
checked by
using NO
2 standards, and the
cadmium column was reactivated or renewed
whenever required. The
detection limit of the NO
3 assay was about 20 pmol. We analyzed filaments with biovolumes
from 0.0006 to 0.0154 mm
3; the detection limit was therefore equivalent to an
[NO
3] concentration of 33 mM for the
smallest filament and 2 mM for
the largest filament. No
NO
3 was detected when empty sheaths were
analyzed or when a filament
was dried and extracted a second time, thus
confirming that no
significant contamination or loss of nitrate
occurred in the
procedure.
After extraction of nitrate, the filament was air dried again and
immersed in 50 µl of methanol for extraction of [S
0].
The complete dissolution of sulfur globules was verified by
extraction
time series and light microscopy. Elemental sulfur
in the extract was
quantified as cyclo-octasulfur (S
8) by high-performance
liquid chromatography. Separation was done on a Zorbax ODS column
(125 by 4 mm, 5 µm; Knauer, Germany) with methanol (100%;
high-performance
liquid chromatography grade) as the eluent at a flow
rate of 1
ml min
1. S
8 eluted after 3.5 min
and was detected at 265 nm. The detection
limit was 43 pmol and was
equivalent to an S
0 concentration of 72 mM for the smallest
filament and 3 mM for
the largest filament. Repeated measurements of
[S
0] in filaments of the same species inhabiting a common
sheath
showed a variability of <10% relative standard
deviation. No loss
of S
0 was observed during the
preceding [NO
3]
extraction.
Elemental sulfur in the bulk sediment was extracted from Zn-preserved
samples with pure methanol for 16 h on a rotary shaker;
the
sediment-to-extractant ratio was about 1:20 (wet wt/vol).
S
0 in the filtered (0.45-µm-pore-size filter) extracts
was quantitated
as described above. The variability within triplicate
S
0 extractions was <14%.
Statistical treatment.
The correlation between
[NO3] and [S0] was determined
and tested for significance by the method of Spearman
(30). The
[NO3]/[S0] ratios of
filaments from different depth intervals were tested for similarity
with the H test of Kruskal and Wallis (30).
| |
RESULTS AND DISCUSSION |
Influence of Thioploca on NO3
profiles and uptake rates.
Nitrate has been measured in biofilms
and lake sediments by liquid ion exchanger (LIX)-based microsensors
(7, 32, 38), but similar measurements in marine
environments were not possible due to the interference of
Cl ions. In this study we used a recently developed
NO3 biosensor that allowed us to measure
microprofiles in sediments from the upwelling system off central Chile
and to study the influence of Thioploca on the
NO3 uptake. Huettel et al. (17)
suggested that filament protrusion may be a strategy to overcome the
diffusion limitation to NO3 uptake imposed by
the boundary layer and that Thioploca may thereby outcompete
NO3-consuming bacteria in the sediment. To
test this hypothesis, we incubated sediment from two different stations
in the the flume under similar conditions to those described above and
measured the oxygen and nitrate microprofiles.
At the time of sampling, the sediment of Station 7 was densely
populated by
Thioploca spp., with a total wet biomass of
44
g m
2 (
36) and the filaments
stretched out of the sediment when nitrate
was present in the flume
water.
Thioploca was much less abundant
in the second core
from Station 18, where the wet biomass was
only 9 g
m
2 (
36). Protruding filaments were only
sporadically observed
in the core studied. This difference in
Thioploca spp. abundance
was clearly reflected in the
O
2 and NO
3 profiles (Fig.
2). The profiles from Station 7 measured
in the
vicinity of
Thioploca filaments were oddly shaped,
and O
2 and
NO
3 hardly penetrated
to the sediment surface. Since the filaments
protruded into the flume
water, maximum NO
3 uptake rates occured above
the sediment surface (Fig.
3) and
the
NO
3 penetration was only 0.5 mm. Station 18 showed an O
2 microprofile
normal for marine sediments, with
a diffusive boundary layer thickness
of about 1 mm and a maximal
O
2 penetration depth of 1 mm. Nitrate
penetrated about 2 mm
into the sediment, and the linear range
of the
NO
3 gradient (Fig.
2) and maximal uptake
rates were both within the
sediment (Fig.
3). The profile structure and
penetration depth
were very similar to those found in organic-rich lake
sediments
(
19,
38).
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 2.
Laboratory flume measurements of O2 and
NO3 concentrations in sediments with a high
(Station 7) and very low (Station 18) density of Thioploca
spp. The broken line indicates the sediment-water interface.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 3.
Vertical distribution of NO3
uptake rates in sediment with a high (Station 7) and low (Station 18)
density of Thioploca filaments. The broken line indicates
the sediment-water interface.
|
|
Interestingly, the diffusive boundary layer at Station 7 was >1.5 mm,
considerably thicker than at Station 18 (Fig.
2). A
likely explanation
for this could be that
Thioploca filaments
protruding from
the sediment impede the water flow, which leads
to a thickening of the
boundary layer and thus to a lower diffusive
exchange across the
sediment-water interface. Thus, the oxygen
uptake at Station 7 was only
0.26 ± 0.11 mmol m
2 day
1
(
n = 6) compared to 0.74 ± 0.13 mmol
m
2 day
1 (
n = 6) at Station
18. The calculated diffusive NO
3 uptake at
Station 7 was 5.45 ± 1.17 mmol m
2
day
1 (
n = 4), about 55% higher than at
Station 18 (3.53 ± 0.76 mmol
m
2 day
1;
n = 5). In summary,
Thioploca spp. have a major
influence on
the sedimentary NO
3 metabolism.
They penetrate up through the diffusive boundary
layer, cause a reduced
NO
3 penetration depth, move the maximum
NO
3 uptake upward, and increase the areal
NO
3 uptake
rate.
An interesting effect of the
Thioploca community might be
that nitrate-reducing bacteria in the sediment are outcompeted for
NO
3 and that denitrification consequently
plays a minor role in sediments
densely inhabited by
Thioploca spp. The situation, however, is
probably more
complex, as indicated by denitrification measurements.
Despite the
presence of
Thioploca spp., denitrification rates
of 4.5 and
9 mmol m
2 day
1 were determined for Station
7 by adding 100 µM
15NO
3 to the
flume water (L. P. Nielsen, unpublished data). These values
are
slightly higher than usually observed in normal coastal sediments
at
similar NO
3 concentrations (references
2 and
15 and references therein)
and suggest only an
incomplete suppression of denitrification.
Nitrate for denitrification
may be supplied by leakage from
Thioploca filaments or via
advective transport of bottom water into the
sediment. Advective
transport becomes progressively more important
with increasing flow
velocities (
9) and may indeed have been
an important
process, because the top 1 to 2 cm of the sediment
had a spongy
consistency and was very porous due to the
Thioploca mat
(
26). Furthermore, the in situ flow velocity of the bottom
water near the sediment may well exceed the 5 cm s
1 that
we have used in our
experiments.
Response of Thioploca filaments to
NO2 and N2O.
The chemotactic
behavior of Thioploca spp. toward O2,
NO3, and H2S was studied by
Huettel et al. (17). However,
NO2 and N2O are also
intermediates and by-products of nitrification and nitrate reduction
processes (15, 18) and can be found in the oxygen minimum
zone of upwelling areas (5, 6). We studied whether
Thioploca spp. show chemotaxis toward
NO2 and N2O and whether they can
utilize them as electron acceptors by using cores from Station 7 and
adding NO2 or N2O to nitrate-free
flume water. On addition of 10 µM NO2, the
number and length of protruding filaments rapidly increased and reached
a maximum after 1.2 h (Fig. 4).
Single bundles were observed through the dissection microscope, and
before the NO2 addition the filament tips
were moving in and out of the sheath with frequent reversals just at
the sediment-water interface. About 30 s after the
NO2 addition, the reversals stopped and all
the filaments moved upward, protruding from the sheath. When the
NO2 concentration dropped below 2.6 µM, the
filaments began to retreat until the initial positions were
reestablished (Fig. 4). A similar sequence was observed when
NO3 was added (data not shown). It cannot
completely be ruled out that the real tactic trigger was
NO3 produced by nitrifying bacteria. However,
the fast reaction of Thioploca and the low O2
concentrations limiting nitrification in the setup do support a direct
response to NO2.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
Response of Thioploca spp. to addition of 10 µM NO2 to the flume water as indicated by
the number of filaments protruding >2 mm out of the sediment and by
the total length of all filaments exposed to flume water.
|
|
In contrast to NO
3 and
NO
2,
Thioploca filaments did not
show chemotaxis toward N
2O, suggesting that nitrous oxide
may not
be used as an electron acceptor for sulfide oxidation. Nitrous
oxide is the obligate precursor for dinitrogen formation during
denitrification and can be used by most, although not all, denitrifying
bacteria as an electron acceptor (
45). Reduction of
N
2O to ammonium
has never been reported. The absence of a
response to N
2O therefore
supports the finding of Otte et
al. (
25) that ammonium rather
than dinitrogen, as
previously assumed (
10), is the terminal
product of
nitrate
reduction.
Microprofiles of N
2O were measured during the
chemotaxis experiment, and an average profile (
n = 3)
is depicted in Fig.
5.
Since
Thioploca did not stretch out from the sediment, the
N
2O
profile exhibited a regular diffusive boundary layer of
about
0.5 mm. However, even without the contribution of
Thioploca spp.,
N
2O was rapidly (15.1 ± 1.5 mmol m
2 day
1 consumed within the first
3.5 mm of the sediment, demonstrating
the potential for sedimentary
dinitrogen formation.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 5.
Microprofile of N2O in sediment from Station
7. Nitrous oxide was added to the flume water after the depletion of
nitrate.
|
|
Internal [S0] and [NO3]
concentrations.
A concentration range of 150 to 500 nmol
mm3 has been reported for the
[NO3] content of Thioploca cells
(10), but to date nothing is known about the variability
within the different species or about the [NO3]/[S0] ratio within individual
filaments and whether it changes with depth. Additionally,
Thioploca filaments have been found down to 26 cm deep
(36), but it was not clear whether they were still alive
and contained [NO3]. Therefore,
Thioploca filaments were collected from different depths of
a core from Station 7 and the concentrations of
[NO3] and [S0] were
determined. The statistical analysis of the results from the depth
intervals 0 to 1 cm, 1 to 2 cm, 2 to 3 cm, 3 to 4 cm, 4 to 7 cm, and 7 to 10 cm did not indicate significant differences in the
[NO3]/[S0] ratios between the
different sections (P > 0.1). By the same statistical
method, it was shown that the two lowest intervals from 10 to 13 cm and
13 to 16 cm, although not different from each other, were different
from the first six intervals, with a high probability
(P < 0.001). Based on these results, the measurements of [NO3] and [S0] from 0 to
10 cm and from 10 to 16 cm deep, respectively, are grouped together
(Fig. 6A and B).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 6.
(A and B) Concentrations of
[NO3] and [S0] in single
Thioploca filaments collected at different sediment depths
from Station 7. (C) Storage concentration in sheathless sulfur bacteria
filaments from the sediment surface (0 to 2 cm) at Station 4. Open
circles, T. araucae; solid circles, T. chileae;
open triangles, SCM; dotted symbols, filaments from the deepest
sediment section (13 to 16 cm); open diamonds, sheathless filaments.
|
|
In the upper part (0 to 10 cm) of the core,
T. araucae
(57%), and
T. chileae (30%) were the dominant mat-forming
species (Fig.
6A), with the rest being SCM thioplocas. The SCM were
more abundant
(81%) in the deeper section. Similarly, Schulz et al.
(
36) found
the maximum
T. araucae and
T. chileae biomass close to the sediment-water
interface whereas the
SCM were most abundant below 7 cm deep.
The difference in the species
composition was also reflected in
the [NO
3]
and [S
0] concentrations. Whereas
[NO
3] and [S
0] varied over
similar concentration ranges (up to ~500 nmol mm
3) in
the upper section (Fig.
6A), the values were significantly
shifted
toward lower [NO
3] and higher
[S
0] concentrations in the 10- to 16-cm-deep section.
Both the SCM
and
T. chileae stored [S
0] up to
~800 nmol mm
3. However, the maximum [S
0]
concentration determined for
T. araucae was only 355 nmol
mm
3 (Fig.
6B), which nicely corresponded to a lower
volume ratio
between cytoplasm and vacuole. Whereas SCM and
T. chileae typically
have a
C/V ratio of 0.47, it is only
0.23 in
T. araucae.
Based on results from chemotaxis experiments, it was concluded that
Thioploca spp. fill their vacuoles with nitrate at the
sediment surface. Then they migrate into deeper sediment layers,
where
they oxidize sulfide to [S
0] and
SO
42 until low
[NO
3] concentrations are reached and the
upward movement is induced
again (
10,
17). Such a behavior
would imply that (i) there
exists a negative relationship between the
two storage compounds
in a filament and (ii) filaments with a high
[NO
3]/[S
0] ratio are found
predominantly at the sediment surface whereas
filaments rich in
[S
0] but depleted of [NO
3]
are found in deeper sections. This, however, seems to be valid
only for
SCM thioplocas, where a negative correlation between
[S
0]
and [NO
3] was found (
r =
0.44;
n = 24;
P < 0.05; Spearman
test) and a
decreasing
[NO
3]/[S
0] ratio correlated
with the sediment depth (
r2 = 0.71). No
significant correlations (
P > 0.05) were found in
T. araucae or in
T. chileae, which suggests that they
migrate
in a different manner. A reason for the lack of correlation was
found when the average [NO
3] and
[S
0] concentrations of all individuals of a species in a
given depth
interval were calculated (Fig.
7). Despite the variability in
the
[NO
3]/[S
0] ratios, the
average concentrations were surprisingly constant
down several
centimeters deep. This is probably best explained
by a continuous and
rapid shuttling of
Thioploca filaments relative
to the
metabolic rate. A further consequence of the continuous
shuttling is
that the filaments frequently reach the sediment
surface, where they
can recharge their [NO
3] storage. Ample
supply of nitrate, on the other hand, could allow
Thioploca
to oxidize H
2S directly to SO
42
instead of forming [S
0] first. If this is true, it also
explains why no clear correlation
between [S
0] and
[NO
3] was detected in
T. araucae
and
T. chileae, respectively. However,
final proof for a
vertical shuttling of
Thioploca spp. and nitrate
transport
into the sediment comes from the finding of living filaments
with
substantial [NO
3] concentrations (31 ± 59 nmol mm
3;
n = 9 [Fig.
6B]) in the
13- to 16-cm-deep sediment section.
Calculations based on the turnover
time of [NO
3] (8 to 10 days
[
25]) and a migration velocity of 5 mm h
1
(
17) showed that the internal reservoir of electron
acceptor
in
Thioploca cells is sufficient to reach such
depths.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 7.
Vertical distribution of the mean
[NO3] and [S0] concentrations
in the different Thioploca spp. from Station 7.
|
|
For comparison, [NO
3] and
[S
0] concentrations were also measured in the free
filaments living at the sediment surface of Station
4 (Fig.
6C). Their
[NO
3] content was 42 ± 27 nmol
mm
3, i.e., much lower and less variable than in the 0- to
10-cm-deep
section of Station 7 (Fig.
6A). It was also considerably
lower
than reported for
Beggiatoa spp. from Monterey Canyon
and Guaymas
Basin (
23). In general,
[NO
3] and [S
0] concentrations
of the Station 4 filaments were similar to those
of
Thioploca spp. found below 10 cm at Station 7. This was
probably
due to the high H
2S content (up to 1,200 µM) in
this sediment.
Based on the finding of a phobic response to sulfide
concentrations
of >500 µM (
17) and the high
morphological and phylogenetic
similarity to
T. araucae, it
was even suggested that these filaments
might actually be thioplocas
that had moved out of their sheaths
or did not produce them under the
prevailing environmental conditions
(
40).
Intracellular versus extracellular pools of S0 and
NO3.
In sediments of a Danish fjord and
in the Santa Barbara basin, it was observed that elemental sulfur was
associated primarily with Beggiatoa filaments (31,
43). To quantify the contribution of [S0] to the
total pool of S0 in the sediment, we manually collected all
Thioploca filaments present in a core from Station 7 and
determined the [S0] and S0 concentrations in
the remaining bulk sediment (Fig. 8). In
contrast to the two other studies, we found that [S0]
made up maximally 27% of the total S0 pool. Furthermore,
the two sulfur pools exhibited different distribution patterns. Whereas
the [S0] corresponded to the distribution of
Thioploca spp. (33) and decreased gradually
with depth, the S0 was maximal at a depth of 5 cm. Because
the turnover times of the two sulfur pools may be different, one cannot
draw quantitative conclusions about the relative significance of
Thioploca-associated and chemical sulfide oxidation, but it
clearly demonstrates that other processes contribute to sulfide
oxidation and sedimentary S0 formation. The distribution of
chemical species (H2S, Fe2+, and
S2O32) in the pore water suggests
that reducible iron oxides may also be an important oxidant for pore
water H2S (41; Zopfi et al., submitted).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 8.
Intracellular (solid symbols) and extracellular (open
symbols) pools of S0 and NO3 in
sediment of Station 7. The broken line indicates the sediment
surface.
|
|
From the known amount of [S
0] and the average
[NO
3]/[S
0]- ratio in a given depth
interval, one can calculate the amount of nitrate
being accumulated by
Thioploca cells and transported into the
sediment. If the
[NO
3] were completely released from the
cells, it would lead to pore
water concentrations of about 1 mM (Fig.
8). There is evidence
in the published literature that some
measurements of pore water
NO
3 were
influenced by [NO
3] released from
vacuolated sulfur bacteria during pore water sampling.
For example
Henrichs and Farrington (
14) noted that the
NO
3 pore water concentrations in
Thioploca-containing sediment were
higher than in the
overlying seawater. In sediments from the Peru
upwelling area,
NO
3 was found in significant concentrations
down to an unusual depth
of 10 cm and the maximum concentrations (up to
102 µM) were conspicuously
high (
11). In later
publications where unusually high nitrate
concentrations were observed,
it was already suspected that they
were affected by
NO
3 released from disrupted cells (
27,
41).
Over recent years, a variety of approaches have been applied to
estimate the contribution of
Thioploca spp. to sulfide
oxidation.
Although the reported values vary from 3 to 91% (
8,
10,
25,
41), most estimates fall in the range of 20 to 30%.
However,
even if
Thioploca spp. were not the dominant player
in sulfide
oxidation in these sediments, they are most significant for
the
sedimentary nitrogen cycling. Organic matter in the sediments
off
Concepción Bay is almost exclusively degraded via sulfate
reduction (equation
1) (
41).
|
(1)
|
|
(2)
|
If only 25% of the formed sulfide is oxidized by
Thioploca spp. according to equation
2, this would lead to
an 83% increase
of the sedimentary ammonium production. Since ammonium
is not
lost from the environment, in contrast to N
2, and
since nitrogen
tends to be the limiting factor for phytoplankton in the
marine
environment, this form of nutrient regeneration could have
considerable
consequences for the primary productivity in the
area.
| |
ACKNOWLEDGMENTS |
The staff of Dichato, the crew of R/V Kay Kay, and all
members of the Thioploca '97 expedition, V. A. Gallardo, J. G. Kuenen, S. Otte, H. Schulz, B. Strotmann, and A. Teske are thanked for their help and cooperation. A. Rusch is
acknowledged for help with the statistical analysis, and T. Ferdelman
and two anonymous reviewers are thanked for valuable comments on the manuscript.
This work was supported by the German Ministry for Education, Science,
Research and Technology (to J.Z.) and the Max-Planck Society.
| |
FOOTNOTES |
*
Corresponding Author. Present address: Institute of
Biology and Danish Center for Earth System Science, University of
Southern Denmark, Odense, Campusvej 55, DK-5230 Odense M, Denmark,
Phone: 45 6550 2745. Fax: 45 6593 0457. E-mail:
jzopfi{at}biology.sdu.dk.
| |
REFERENCES |
| 1.
|
Beijerinck, M. W.
1904.
Phénomènes de réduction produits par les microbes.
Arch. Nederl. Sci. Exactes Nat.
9:131-157.
|
| 2.
|
Binnerup, S. J.,
K. Jensen,
N. P. Revsbech,
M. H. Jensen, and J. Sørensen.
1992.
Denitrification, dissimilatory reduction of nitrate to ammonium, and nitrification in a bioturbated estuarine sediment as measured with 15N and microsensor techniques.
Appl. Environ. Microbiol.
58:303-313[Abstract/Free Full Text].
|
| 3.
|
Brettar, I., and G. Rheinheimer.
1991.
Denitrification in the central Baltic: evidence for H2S-oxidation as a motor of denitrification at the oxic anoxic interface.
Mar. Ecol. Prog. Ser.
77:157-169.
|
| 4.
|
Broecker, W. S., and T. H. Peng.
1974.
Gas exchange rates between air and sea.
Tellus
26:21-35.
|
| 5.
|
Codispoti, L. A.,
G. E. Friedrich,
T. T. Packard,
H. E. Glover,
P. J. Kelly,
R. W. Spinrad,
R. T. Barber,
J. W. Elkins,
B. B. Ward,
F. Lipschultz, and N. Lostaunau.
1986.
High nitrite levels off Northern Peru: a signal of instability in the marine denitrification rate.
Science
233:1200-1202[Abstract/Free Full Text].
|
| 6.
|
Copin-Montégut, C., and P. Raimbault.
1994.
The peruvian upwelling near 15°S in August 1986. Results of continuous measurements of physical and chemical properties between 0 and 220 m depth.
Deep-Sea Res.
41:439-467.
|
| 7.
|
de Beer, D., and J.-P. R. A. Sweerts.
1989.
Measurement of nitrate gradients with an ion-selective microelectrode.
Anal. Chim. Acta.
219:351-356[CrossRef].
|
| 8.
|
Ferdelman, T. G.,
C. Lee,
S. Pantoja,
J. Harder,
B. Bebout, and H. Fossing.
1997.
Sulfate reduction and methanogenesis in a Thioploca-dominated sediment off the coast of Chile.
Geochim. Cosmochim. Acta.
61:3065-3079[CrossRef].
|
| 9.
|
Forster, S.,
M. Huettel, and W. Ziebis.
1996.
Impact of boundary layer flow velocity on oxygen utilization in coastal sediments.
Mar. Ecol. Prog. Ser.
143:173-185.
|
| 10.
|
Fossing, H.,
V. A. Gallardo,
B. B. Jørgensen,
M. Huettel,
L. P. Nielsen,
H. Schulz,
D. Canfield,
S. Foster,
R. N. Glud,
J. K. Gundersen,
J. Kuever,
N. B. Ramsing,
A. Teske,
B. Thamdrup, and O. Ulloa.
1995.
Concentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca.
Nature
374:713-715[CrossRef].
|
| 11.
|
Froelich, P. N.,
M. A. Arthur,
W. C. Burnett,
M. Deakin,
V. Hensley,
R. Jahnke,
L. Kaul,
K.-H. Kim,
K. Roe,
A. Soutar, and C. Vathakanon.
1988.
Early diagenesis of organic matter in Peru continental margin sediments: phosphorite precipitation.
Mar. Geol.
80:309-343.
|
| 12.
|
Gallardo, V. A.
1977.
Large benthic microbial communities in sulphide biota under Peru-Chile subsurface countercurrent.
Nature
268:331-332[CrossRef].
|
| 13.
|
Grasshoff, K.,
M. Erhardt, and K. Kremling.
1983.
Methods of sea water analysis.
Verlag Chemie, Weinheim, Germany.
|
| 14.
|
Henrichs, S. M., and J. W. Farrington.
1984.
Peru upwelling region sediments near 15°S. 1. Remineralization and accumulation of organic matter.
Limnol. Oceanogr.
29:1-19.
|
| 15.
|
Herbert, R. A.
1999.
Nitrogen cycling in coastal marine ecosystems.
FEMS Microbiol. Rev.
23:563-590[CrossRef][Medline].
|
| 16.
|
Hill, A. E.,
B. M. Hickey,
F. A. Shillington,
P. T. Strub,
K. H. Brink,
E. D. Barton, and A. C. Thomas.
1998.
Eastern ocean boundaries coastal segment (E), p. 29-67.
In
A. R. Robinson, and K. H. Brink (ed.), The sea, vol. 11. John Wiley & Sons, Inc., New York, N.Y.
|
| 17.
|
Huettel, M.,
S. Forster,
S. Klöser, and H. Fossing.
1996.
Vertical migration in the sediment dwelling sulfur bacteria Thioploca spp. in overcoming diffusion limitations.
Appl. Environ. Microbiol.
62:1863-1872[Abstract].
|
| 18.
|
Knowles, R.
1982.
Denitrification.
Microbiol. Rev.
46:43-70[Free Full Text].
|
| 19.
|
Larsen, L. H.,
T. Kjær, and N. P. Revsbech.
1997.
A microscale NO3 biosensor for environmental applications.
Anal. Chem.
69:3527-3531[CrossRef].
|
| 20.
|
Levin, L.,
J. Gage,
P. Lamont,
L. Cammidge,
C. Martin,
A. Patience, and J. Crooks.
1997.
Infaunal community structure in a low-oxygen, organic rich habitat on the Oman continental slope, NW Arabian Sea, p. 223-230.
In
Proceedings of the 30th Marine Biology Symposium.
|
| 21.
|
Li, Y. H., and S. Gregory.
1974.
Diffusion of ions in sea water and in deep-sea sediments.
Geochim. Cosmochim. Acta
38:703-714.
|
| 22.
|
Maier, S., and V. A. Gallardo.
1984.
Nutritional characteristics of two marine thioplocas determined by autoradiography.
Arch. Microbiol.
139:218-220[CrossRef].
|
| 23.
|
McHatton, S. C.,
J. P. Barry,
H. W. Jannasch, and D. C. Nelson.
1996.
High nitrate concentrations in vacuolate, autotrophic marine Beggiatoa spp.
Appl. Environ. Microbiol.
62:954-958[Abstract].
|
| 24.
|
Namasaraev, B. B.,
L. E. Dulov,
G. A. Dubinina,
T. I. Zemskaya,
L. Z. Granina, and E. V. Karabanov.
1994.
Bacterial synthesis and destruction of organic matter in microbial mats of Lake Baikal.
Microbiology
63:193-197.
|
| 25.
|
Otte, S.,
J. G. Kuenen,
L. P. Nielsen,
H. W. Pearl,
J. Zopfi,
H. N. Schulz,
A. Teske,
B. Strotmann,
V. A. Gallardo, and B. B. Jørgensen.
1999.
Nitrogen, carbon and sulfur metabolism in natural Thioploca samples.
Appl. Environ. Microbiol.
65:3148-3157[Abstract/Free Full Text].
|
| 26.
|
Reimers, C. E.
1982.
Organic matter in anoxic sediments off Central Peru: relations of porosity, microbial decomposition and deformation properties.
Mar. Geol.
46:175-197.
|
| 27.
|
Reimers, C. E.,
K. C. Ruttenberg,
D. E. Canfield,
M. B. Christiansen, and J. B. Martin.
1996.
Porewater pH and authigenic phases formed in the uppermost sediments of Santa Barbara Basin.
Geochim. Cosmochim. Acta
60:4037-4057.
|
| 28.
|
Revsbech, N. P.
1989.
An oxygen microelectrode with a guard cathode.
Limnol. Oceanogr.
34:472-476.
|
| 29.
|
Rosenberg, R.,
W. E. Arntz,
E. C. de Flores,
L. A. Flores,
G. Carabajal,
I. Finger, and J. Tarazona.
1983.
Benthos biomass and oxygen deficiency in the upwelling system off Peru.
J. Mar. Res.
41:263-279.
|
| 30.
|
Sachs, L.
1997.
Angewandte Statistik, 8th ed.
Springer-Verlag, Berlin, Germany.
|
| 31.
|
Schimmelmann, A., and M. Kastner.
1993.
Evolutionary changes over the last 1000 years of reduced sulfur phases and organic carbon in varved sediments of the Santa Barbara Basin, California.
Geochim. Cosmochim. Acta
57:67-78.
|
| 32.
|
Schramm, A.,
L. H. Larsen,
N. P. Revsbech,
N. B. Ramsing,
R. Amann, and K.-H. Schleifer.
1996.
Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes.
Appl. Environ. Microbiol.
62:4641-4647[Abstract].
|
| 33.
|
Schulz, H. N.,
B. B. Jørgensen,
H. A. Fossing, and N. B. Ramsing.
1996.
Community structure of filamentous, sheath-building sulfur bacteria, Thioploca spp. off the coast of Chile.
Appl. Environ. Microbiol.
62:1855-1862[Abstract].
|
| 34.
|
Schulz, H. N.,
T. Brinkhoff,
T. G. Ferdelman,
M. Hernández Mariné,
A. Teske, and B. B. Jørgensen.
1999.
Dense populations of a giant sulfur bacterium in Namibian shelf sediments.
Science
284:389-544[CrossRef].
|
| 35.
|
Schulz, H. N.
1999.
Nitrate-storing sulfur bacteria in sediments of coastal upwelling. Ph.D. thesis.
University of Bremen, Bremen, Germany.
|
| 36.
|
Schulz, H. N.,
B. Strotmann,
V. A. Gallardo, and B. B. Jørgensen.
2000.
Population study of filamentous sulfur bacteria Thioploca spp. off the Bay of Concepción, Chile.
Mar. Ecol. Prog. Ser.
200:117-126.
|
| 37.
|
Strub, P. T.,
J. M. Mesías,
V. Montecino,
J. Rutlland, and J. Salinas.
1998.
Coastal ocean circulation off western south America, p. 273-313.
In
A. R. Robinson, and K. H. Brink (ed.), The sea, vol. 11. John Wiley & Sons, Inc., New York, N.Y.
|
| 38.
| Sweerts, J. P. R. A., and D. de
Beer. Microelectrode measurements of nitrate gradients in the
littoral and profundal sediments of a meso-eutrophic lake (Lake
Vechten, The Netherlands). Appl. Environ. Microbiol.
55:754-757.
|
| 39.
|
Taylor, B. F.,
D. S. Hoare, and S. L. Hoare.
1971.
Thiobacillus denitrificans as an obligate chemolithotroph. Isolation and growth studies.
Arch. Microbiol.
78:193-204.
|
| 40.
|
Teske, A.,
M. L. Sogin,
L. P. Nielsen, and H. W. Jannasch.
1999.
Phylogenetic relationships of large marine Beggiatoa.
Syst. Appl. Microbiol.
22:39-44[Medline].
|
| 41.
|
Thamdrup, B., and D. E. Canfield.
1996.
Pathways of carbon oxidation in continental margin sediments off central Chile.
Limnol. Oceanogr.
41:1629-1650.
|
| 42.
|
Timmer ten Hoor, A.
1975.
A new type of thiosulphate oxidizing, nitrate reducing microorganism: Thiomicrospira denitrificans sp. nov.
Neth. J. Sea Res.
9:343-351.
|
| 43.
|
Troelsen, H., and B. B. Jørgensen.
1982.
Seasonal dynamics of elemental sulfur in two coastal sediments.
Esturine Coastal Shelf Sci.
15:255-266.
|
| 44.
|
Zemskaya, T. I.,
B. B. Namsaraev,
N. M. Dultseva,
T. A. Khanaeva,
L. P. Golobokova,
G. A. Dubinina,
L. E. Dulov, and E. Wada.
2001.
Ecophysiological characteristics of the mat forming bacterium Thioploca in bottom sediments of the Frolikha Bay, northern Baikal.
Microbiology
70:335-341[CrossRef].
|
| 45.
|
Zumft, W. G., and P. M. H. Kroneck.
1990.
Metabolism of nitrous oxide, p. 37-55.
In
N. P. Revsbech, and J. Sørensen (ed.), Denitrification in soil and sediments. Plenum Press, New York, N.Y.
|
Applied and Environmental Microbiology, December 2001, p. 5530-5537, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5530-5537.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Whitmire, S. L., Hamilton, S. K.
(2005). Rapid Removal of Nitrate and Sulfate in Freshwater Wetland Sediments. J. Environ. Qual.
34: 2062-2071
[Abstract]
[Full Text]
-
Kalanetra, K. M., Huston, S. L., Nelson, D. C.
(2004). Novel, Attached, Sulfur-Oxidizing Bacteria at Shallow Hydrothermal Vents Possess Vacuoles Not Involved in Respiratory Nitrate Accumulation. Appl. Environ. Microbiol.
70: 7487-7496
[Abstract]
[Full Text]
Top
Abstract
Introduction
