Notes
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abundance of planktonic ciliates and microflagellates in mesohaline Chesapeake Bay waters. Estuarine Coastal Shelf. Sci. 31: 157-l 75.
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AND -.
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vertical distributions
of ciliate microzooplankton
related to anoxia in Chesapeake Bay waters. Mar.
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AND -.
199 16. A study of predacious
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benthic microfauna of an arctic tundra pond. Hydrobiologia 46: 445-464.
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the digestive cycle in Paramecium caudatum. J.
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AND B. U. SHOCKLEY. 1985. Processing of
digestive vacuoles in Tetrahymena and the effects
of dichloroisoproternol.
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1982. Ingestion rate and gut clearance in the
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Submitted: 5 November 1990
Accepted: 8 January 1991
Revised: I I February 1991
1991, 565-570
(~1199 1, by the Amencan Society of Limnology and Oceanography. Inc
Enhanced microbial methane oxidation in water from a
deep-sea hydrothermal vent field at simulated
in situ hydrostatic pressures
Abstract- Water from a hydrothermal vent field
was incubated in the presence of ‘CH, under
conditions of both atmospheric (1 atm) and simulated in situ hydrostatic pressure (-200 atm).
Methane oxidation rates measured in samples
incubated at elevated pressures were 2 l-62%
higher than those measured in replicate samples
incubated at atmospheric pressure. The magnitude of the observed effect was consistent with
that predicted to occur from changes in CH, activity with depth-dependent pressure. suggesting
that microbial CH, oxidation is a functionally
barophilic process, The data indicate that methane oxidation, as well as other microbial gas consumption processes, is likely to be affected by
moderate increases in hydrostatic pressure and
that the rates of these processes in the deep sea,
based on measurements at atmospheric pressure,
may be underestimated.
Hydrostatic pressure, which increases - 1
for every 10 m of depth, is a major
environmental
factor that can affect microbial processes in the deep sea. The effect of
intermediate hydrostatic pressures encountered in the marine environment
(corresponding to depths ~3,000 m) has been examined for several microbial processes. In
situ or simulated in situ hydrostatic pressure
atm
Acknowledgments
We thank Jody Deming for suggestions concerning
this manuscript, Eric Olson for running the CH, analysis, the crews of the RV Atlantis IZ and DSRV Alvin
for their assistance, and anonymous reviewers for comments that improved the manuscript.
This research was supported by the Office of Naval
Research.
566
Notes
has been reported to retard microbial activity in some cases and to enhance rates in
others. For example, Deming (1985) observed a pressure-enhanced response in heterotrophic bacterial growth in sediment trap
and box-core samples from a depth of 1,850
m, and Jannasch and Wirsen (1982) found
a decrease in heterotrophic uptake in undecompressed vs. decompressed samples of
seawater from a similar depth.
Wirsen et al. (1986) compared 14C0, assimilation rates in dilute hydrothermal vent
fluid in situ and onboard ship at atmospheric pressure (and the same temperature) and
found enhanced incorporation
rates at the
elevated pressures. Tuttle et al. (1983) carried out similar experiments and observed
both positive and negative pressure effects.
Cowen (1989) reported that rates of microbial Mn binding in lateral hydrothermal
plumes measured at in situ hydrostatic pressures were enhanced by a factor of up to 3.3
times relative to those measured at atmospheric pressure. The effect of elevated hydrostatic pressure on microbial CH, oxidation activity has not been reported to date.
Submarine hydrothermal
vents, which
occur at midoceanic spreading centers, are
located at depths of -2,OOO-3,000 m and
are sites of elevated microbial activity relative to ambient deep-sea water. Hydrothermal fluid venting onto the seafloor is
106-lo8 more concentrated
in methane
(CH,) than the bottom ocean water into
which it is being discharged (Lilley et al.
1983; Welhan and Craig 1979) which suggests that methane oxidation may be an important microbial process at these sites. Significant rates of aerobic CH, oxidation have
been reported in water from a deep-sea
(> 1,800-m depth) hydrothermal
effluent
plume incubated under conditions of atmospheric pressure (de Angelis 1989). This
note reports the comparison of i4CH4 uptake rates for samples of deep-sea hydrothermal vent water determined under conditions of both atmospheric and simulated
in situ hydrostatic pressure.
Water samples for determining CH, oxidation were obtained from a hydrothermal
vent field located on the Endeavour Segment of the Juan de Fuca Ridge (47”57’N,
126’06’W) at a depth of + 2,200 m. Samples
were collected in Niskin Go-Flo bottles attached to the sampling basket of the submersible Alvin. Sample 4005 was collected
at a height of 10 m in a buoyant “black
smoker” plume. Sample 4025 was collected
outside of the plume but within 1 m of an
active black smoker. Samples were drawn
into 250-ml sterile polypropylene bottles and
stored with a headspace at 2°C for up to 3
h before incubations were begun. Samples
were shaken vigorously for a minimum of
3 min immediately after sampling and again
before incubation to remove any ambient
CH, originally present. Caps were removed
periodically to allow venting of the headspace. Previous laboratory experiments indicated that this method removed > 98% of
the ambient CH, present at these concentrations. Replicate subsamples were placed
in 7-ml septum vials (Pierce) that were sealed
with Teflon-faced
silicone septa and no
headspace. Sterile seawater that had been
previously equilibrated with i4CH4 (Amersham; sp act = 55 mCi mmol-l) was added
through the septa via a gas-tight syringe with
a sideport needle. This method ensured that
no gas phase was present in the vials.
Methane is extremely insoluble in seawater (Wiesenburg and Guinasso 1979). As
a result, formation of bubbles within the
vials may cause partitioning
of 14CH4 into
the gas phase, resulting in lower dissolved
14CH4 concentrations in the water phase.
CH, oxidation is first order with respect to
CH, (Joergensen and Degn 1983) so such
partitioning can result in lower rates of measured oxidation. To test for the effects of gas
phase formation and subsequent migration
of CH, out of solution, we ran an additional
sample set for sample 4025. Each vial in
this set was deliberately injected with a 0.25ml air bubble immediately after addition of
14CH4. In addition, vials in all sample sets
were checked for the presence of bubbles at
the end of their incubation periods. Injected
air bubbles were totally dissolved in all vials
incubated under high hydrostatic pressure.
No bubbles were observed in any of the
vials to which no air had been added.
Vial caps and septa were covered with a
double thickness of Parafilm to prevent exchange of sample and hydraulic fluid through
the needle holes in the septa. Samples were
Notes
Table 1. Experimental conditions for samples 4005
and 4025. Ambient CH, concentrations were measured
with flame ionization gas chromatography
by a modification of the method of Lilley et al. (1983) on separate samples drawn from the same Niskin bottle as
the samples for CH, oxidation determinations.
Ambient CH, in seawater samples used to determine CH,
oxidation was removed before incubation, as described
in the text. Methane concentrations in water samples
obtained throughout the vent field ranged from 32 to
9,300 nM CH,. Ambient concentrations ofCH, in ocean
bottom water from similar depths outside the vent field
were < 1 nM. Final concentrations of dissolved “CH,
in the incubation vials were calculated from the solubility tables of Wiesenburg and Guinasso (1979).
FInal
dissolved
[CH,I (nM)
[ ‘CHJ
(nM)
H&I P
Sample
(atm)
Rephcates
4005
4025
2,060
460
725
485
205
220
3
2
Amblent
incubated in the dark at 2°C for 4, 8, and
16 h. At the end of their respective incubation times, samples were killed by adding
6 N NaOH to a final pH of > 10. Controls
consisted of samples to which NaOH was
added immediately after addition of 14CH4.
Controls for both samples were < 100 dpm
(corresponding to ~8 x lop4 nmol CH,).
Experimental conditions that differed between samples 4005 and 4025 are shown in
Table 1. For each experiment,
replicate
sample sets were incubated at atmospheric
pressure (atm P) and elevated hydrostatic
pressure (high P) under otherwise identical
conditions.
All vials were incubated in
stainless-steel
pressure vessels (LECO
TernPress) in water that had been previously equilibrated to the incubation temperature. Replicate samples corresponding
to a given pressure (atm P or high P) and
incubation time (4, 8, 16 h) were placed in
a single pressure vessel. For sample 4025,
vials with and without the added bubble
were placed in the same vessel. All pressure
vessels were capped, but the valves of the
vessels in which the atm-P samples were
incubated were left open. Pressure vessels
in which high-P samples were incubated
were manually pumped to the desired pressure with an Enerpac model 1 l-400 hydraulic pump and a digital pressure readout
that could be read to - 1 atm.
Killed samples and controls were trans-
567
ferred to 40-ml vials that were sealed with
polyseal caps and stored for periods of 2-4
weeks before analysis. Any 14C0, produced
was recovered by acidifying the samples with
10 N H,SO, to a final pH of 2 and stripping
with a stream of helium (60 ml min’) for
5 min. The stripped 14C0, was trapped in
a series of four scintillation vials, each containing 0.25 ml of @-phenylethylamine (Sigma). After stripping was complete, 6 ml of
Omnifluor (New England Nuclear) scintillation cocktail was added to each vial which
was then counted on a Packard model 4430
liquid scintillation counter. Seawater blanks
were run frequently and verified that contamination did not occur between samples.
Efficiency of 14C0, trapping was > 98% with
NaHi4C0,
(ICN; 55 mCi mmolll)
standards. Acidified, stripped samples were then
filtered through 0.22-pm filters (Millipore
GSWP) to test for incorporation
of label
into cell carbon. Filters were washed with
filter-sterilized
seawater, air dried, dissolved in 10 ml Omnifluor, and counted.
The rate of CH, oxidation, as measured
by conversion of 14CH4 to 14C0, and cell
14C, was at least 20% higher in samples incubated under conditions of elevated hydrostatic pressure compared to those incubated at atmospheric pressure (Fig. 1, Table
2). The high-P incubation curve for sample
4005 appeared to be nonlinear after 8 h. If
the 16-h vials are excluded, the high-P rate
for this sample is 62% higher than the atm-P
rate. Rates calculated with the portion of
the curve from 0 to 8 h and from 0 to 16 h
are included in Table 2. No significant difference was observed for the percentage of
utilized 14CH4 incorporated into cell C at
high and atmospheric pressure in either of
the samples tested.
No significant difference was observed
between rates obtained with or without the
presence of a deliberately introduced bubble
at either high P or atm P (Table 2). These
results indicate that the difference in rates
observed under incubation conditions of atmospheric and elevated hydrostatic pressure was not due to gas-phase effects.
The effect of an introduced headspace,
such as a bubble, on concentrations of dissolved CH, can be calculated with the equation of Rudd and Hamilton (1975) and the
Notes
568
2.02
_
u
a
l
I .6-
i
12
.-N
-u
.2 0.8g
*+
0.4-
01
0
4
8
Time (h)
12
16
i
O!0
1
4
I
1
I
8
1
12
1
I
16
Time (h)
Fig. I. Uptake of ‘YJH, (nM) vs. incubation time
for replicate samples of vent field water under conditions of atmospheric (+) and elevated hydrostatic pressure (0). Incubation curves are shown for (a) sample
4005 and (b) sample 4025. The amount of 14CH, oxidized was calculated as the sum of the production of
‘“CO, and cell ‘YY.
solubility coefficients for CH, in seawater
of Wiesenburg and Guinasso (1979). The
minimum bubble volume required to account for the smallest difference (2 1%) in
rates observed between high-P and atm-P
sample sets at the experimental CH, concentrations was - 100 ~1. A loo-p1 bubble
should have been easily visible in the vials.
Many smaller bubbles with a total volume
of 100 ~1 would have had the same effect
and may not have been visible. However,
if CH, does migrate out of the dissolved
phase into the gas phase on the time scale
of the incubation experiments, then similar
calculations predict that a 0.25-ml bubble
should have resulted in a 4 1% reduction of
dissolved CH, in the water phase and a proportional decrease in CH, oxidation rates.
The fact that no difference was observed
between samples with and without an introduced bubble of this size suggests that
the incubation times used in these experiments was much less than the diffusion time
of CH, into a small volume gas phase in the
atm-P vials.
The enhancement of methane oxidation
rates at positive hydrostatic pressures observed in this study may be due to the presence of barophilic methanotrophs within the
water column of the vent field or may reflect
changes in the availability
of the gaseous
substrate (CH,) under conditions of elevated hydrostatic pressure. The equilibrium
pressure, and hence activity, of gases dissolved in water increases with increasing
hydrostatic pressure (Enns et al. 1965). Using equation 3 of Enns et al. (1965) and a
partial molar volume for CH, of 35.5 ml
molk’ (Stoessel and Byrne 1982) we calculate that the activity of CH, at a hydrostatic pressure of 220 atm is 38% higher
than for the same concentration of CH, at
atmospheric pressure. This will be reflected
in a proportional increase in the CH, oxidation rate at the higher pressure, which is
consistent with the enhancement of rates
observed in this study at similar hydrostatic
pressures.
Table 2. Measured rates of CH, oxidation for samples 4005 and 4025 incubated under conditions of atmospheric and elevated hydrostatic pressure. Measured rates of CH, oxidation (i I SD) were determined from
the linear regressions of the time-series incubations (Fig. I). Percent cell C values (-t I SD) were calculated as
[cell IY?/(cell 14C + ‘“CO,)] x 100. Percentages of 14CH, incorporated into cell C were 20?4% (atm P) and 23
*5% (high P) for sample 4005, 32+4% (atm P) and 32+5% (high P) for sample 4025.
Measured ‘Y’H, oxldatlon rate (nM h ‘)
Sample
atm P
4005
0.090+0.002
4025
4025 (bubble)
0. I66 to.004
0.164iO.002
high P
0.1 19iO.O05(0,
0.146iO.O04(0,
0.2OOiO.004
0.2OOiO.008
Change (%)
4, 8, 16 h)
4, 8 h)
32
62
21
22
Notes
One sample (4025) showed a smaller increase (20%) in the rate of methane oxidation when incubated under pressure than
predicted from the enhanced methane activity calculated above, while the other sample (4005) exhibited a similar (32%) or higher (62%) rate. Thus, it is not possible with
the limited number of samples in this data
set to determine whether the observed increase in CH, oxidation rates under pressure was the response of a barotolerant or
a barophilic
methanotrophic
population.
This could be tested by measuring CH, oxidation rates in replicate samples (or in a
pure culture of methanotrophs) over a wider
range of pressures and examining the deviation of the experimental response from
that predicted from changes in CH, activity
or by comparing the pressure response of
CH, oxidizers in samples collected from
surface waters vs. those collected at depth.
Restrictions imposed by doing pressure work
at sea and the limited availability
of vent
field water did not allow such tests to be
carried out.
It is clear from this data set, however, that
the process of microbial methane oxidation
itself is functionally barophilic, as has been
suggested for microbially
mediated Mn
binding and oxidation
in hydrothermal
plumes (Cowen 1989). That is. a positive
pressure effect is observed for microbial
methane oxidation.
The effect of hydrostatic pressure on this process at any depth
in the ocean will be a combination
of the
pressure response of the methanotrophs and
the pressure-dependent change of methane
activity. Using Enns et al. (1965) equation
3, we calculate that at a depth of 100 m,
CH, availability
increases by only 1.5%.
However, at 1,000 m, CH, activity is estimated to increase by 18%, while at 3,000
and 6,000 m, CH, activities increase by 77
and 167%. Although the effects of these
higher pressures on the metabolism of deepsea methanotrophs cannot be predicted, the
significant changes in CH, availability with
depth indicate that the effects of hydrostatic
pressure on methanotrophic
activity in this
environment
cannot be neglected. It also
has implications
for other microbial processes that are first order or higher with respect to any dissolved gas species.
569
This note is the first report of the effect
of hydrostatic pressure on aerobic microbial
CH, oxidation.
This study confirms the
findings of Cowen (1989) that even moderate increases in hydrostatic pressure corresponding to oceanic midwater depths can
have an appreciable effect on the rates of
microbial processes. It appears from these
experiments that microbial CH, oxidation
in natural seawater samples is enhanced by
deep-sea hydrostatic
pressures and that
measured rates of methane oxidation in the
deep sea determined under conditions of
atmospheric pressure may be underestimated. This finding can have important implications in attempts to budget losses of
CH, in hydrothermal vent and other deepsea environments.
Marie A. de Angelis’
John A. Baross
Marvin D. Lille))
School of Oceanography, WB- 10
University of Washington
Seattle 98 195
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Submitted: 27 August 1990
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Inc
Surfaces of hydrothermal vent invertebrates:
Sites of elevated microbial CH, oxidation activity
We thank A. Soeldner and E. Olson for technical
assistance, S. Jonasdottir for editorial assistance, and
M. Lilley for providing the CH, concentration
data
and for discussion. V. Tunnicliffe and K. Wilson provided assistance in species identification. We also thank
the crews of the RV .-ttlantrs II and DSRV Ahin for
their assistance.
Thts research was supported by the Offtce of Naval
Research.
Bacterial chemosynthetic processes form
the basis of primary production at deep-sea
hydrothermal vent sites and support a rich
invertebrate community associated with this
environment
(Jannasch and Mottl 1985).
Much attention has focused on sulfur oxidizers as the major microbial source of carbon and energy to the hydrothermal
vent
community because of the high concentrations of sulfide and other reduced sulfur species at these sites (Ruby et al. 198 1). Many
of these sulfur-oxidizing
and other chemosynthetic bacteria occur in endosymbiotic
association with certain vent invertebrates
(Cavanaugh 1985; Belkin et al. 1986).
Hydrothermal fluids from deep-sea vents
are also known to be enriched in reduced
gases, including CH, (Lilley et al. 1983).
However,
although
methane-oxidizing
(methanotrophic)
bacteria have been reported to have been isolated from various
vent field environments,
including animal
tissue (Jannasch and Mottl 1985) the uptake of methane has not been demonstrated.
In this note, we present the first conclusive
evidence of methanotrophic
activity associated with deep-sea hydrothermal vent animals.
The animals were obtained in September
1988 from a hydrothermal vent field located