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Applied and Environmental Microbiology, December 1999, p. 5314-5321, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Study of Deep-Sea Natural Microbial Populations
and Barophilic Pure Cultures Using a High-Pressure
Chemostat
Carl O.
Wirsen* and
Stephen J.
Molyneaux
Department of Biology, Woods Hole
Oceanographic Institution, Woods Hole, Massachusetts 02543
Received 9 July 1999/Accepted 9 September 1999
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ABSTRACT |
Continuous cultures in which a high-pressure chemostat was used
were employed to study the growth responses of (i) deep-sea microbial
populations with the naturally occurring carbon available in seawater
and with limiting concentrations of supplemental organic substrates and
(ii) pure cultures of copiotrophic barophilic and barotolerant deep-sea
isolates in the presence of limiting carbon concentrations at various
pressures, dilution rates, and temperatures. We found that the growth
rates of natural populations could not be measured or were extremely
low (e.g., a doubling time of 629 h), as determined from the
difference between the dilution rate and the washout rate. A low
concentration of supplemental carbon (0.33 mg/liter) resulted in
positive growth responses in the natural population, which resulted in
an increase in the number of cells and eventually a steady population
of cells. We found that the growth responses to imposed growth pressure
by barophilic and barotolerant pure-culture isolates that were
previously isolated and characterized under high-nutrient-concentration
conditions were maintained under the low-nutrient-concentration
limiting conditions (0.33 to 3.33 mg of C per liter) characteristic of the deep-sea environment. Our results indicate that deep-sea microbes can respond to small changes in substrate availability. Also, barophilic microbes that are copiotrophic as determined by their isolation in the presence of high carbon concentrations and their preference for high carbon concentrations are versatile and are able to
compete and grow as barophiles in the low-carbon-concentration oligotrophic deep-sea environment in which they normally exist.
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INTRODUCTION |
Our knowledge concerning the
physical conditions under which microbes can grow has increased
significantly in recent years. A lower temperature limit of 
7.5°C
was reported early in marine microbiology studies (4), and
the upper temperature limit was recently reported to be 113°C
(5). In most of the deep sea, microorganisms grow at 2 to
3°C and hundreds of bars of hydrostatic pressure. At nearly 11,000 m,
the Challenger Deep is the deepest known oceanic site, and the microbes
that are active there must be able to function at pressures greater
than 100 MPa. Viable microbes have been isolated from this trench and
other trenches of similar depths and studied (25, 44, 49).
While the growth temperatures of these organisms define them primarily
as psychrophiles, their pressure optima characterize them as
barotolerant, barophilic, or obligately barophilic strains. Recently,
Yayanos and coworkers isolated barophilic and obligately barophilic
strains (46, 47), and in a recent review (45)
Yayanos refers to these organisms as piezophiles and hyperpiezophiles
rather than barophiles and obligate barophiles. At this time, the
maximum growth pressure is believed to be around 1,150 × 105 Pa for an obligate barophile (11), but it
may be even higher (up to 1,400 × 105 Pa), as
reported by ZoBell (50). The ways in which genetic expression in barophilic microbes is regulated by pressure have just
recently become an active area of research (23, 28, 30). Since ZoBell's early work which showed that barophilic bacteria occur
in deep-sea samples and in most of the recent isolation and
characterization studies of barophilic isolates (11, 19, 22, 25,
26, 31, 47), the growth media employed have generally contained
very high concentrations of organic carbon (i.e., copiotrophic
conditions). This is not characteristic of the oligotrophic nutrient
conditions which these microbes generally experience in most of their
natural habitats (i.e., oligotrophic conditions).
The major genera of cultivated barophiles include the genera
Shewanella, Photobacterium, Colwellia,
and Moritella, as well as a new unidentified group
(7), and all of these organisms can be classified as
copiotrophs. True oligotrophs are often present as ultramicrobacteria
in seawater (1, 37). They are able to grow under very
nutrient-limited conditions because they have a high specific affinity
for substrates (6) and the ability to utilize a mixture of
limiting substrates (38). To date, none of these organisms
have been characterized as barophilic. The deep sea is generally
described as an oligotrophic environment that contains 0.03 to 0.2 mM
dissolved organic carbon (12, 29), and the flux of
particulate organic carbon is particularly important in determining the
residence time and turnover of carbon (35). There are
deep-sea niches, such as in animal gut tracts, in which microbes may be
periodically exposed to higher substrate concentrations (9,
42).
Limited work has been done on the growth responses of barophiles to
reduced substrate levels, and in the studies that have been done the
workers have used batch culture approaches (8, 20). If the
deep-sea barophiles that have been isolated and studied to date are
obligately copiotrophic with respect to carbon sources, then their in
situ growth activities may well vary with their level of nutrition
(44). They may not respond as barophiles at habitat
pressures in the deep sea, where growth substrates are likely limiting,
and in fact there may be an evolutionary distinction between
oligotrophic and copiotrophic barophiles (8).
To address the question of how natural deep-sea populations, as well as
barophilic pure cultures, respond to constant growth-limiting concentrations of organic carbon under elevated hydrostatic pressure conditions, we performed the series of experiments described below. In
this study, we used a high-pressure chemostat (21) and
continuous culture techniques to examine (i) the growth responses of
natural deep-sea populations whose growth-limiting substrate was not
known but whose growth rates could be determined from the difference between the dilution rate and the washout rate (17) and (ii) the growth responses of pure cultures of deep-sea psychrophilic barotolerant and barophilic microbes to pressure and dilution rate
changes in the presence of limiting carbon concentrations. While we
could obtain time-independent steady states in the presence of limiting
substrate concentrations with the chemostat, we had to consider the
effects of threshold substrate concentrations that affected minimum
population levels (16), the fact that steady states were
influenced by the dilution rate in the presence of various substrate
concentrations (15), and the fact that cell growth and cell
removal are not closely linked in the natural environment (i.e., deep
sea), as they are in a chemostat (18).
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MATERIALS AND METHODS |
Organisms.
Deep natural seawater (NSW) was collected from
the North Atlantic Ocean (37°23'N, 68°49'W) at depths of 4,400 to
4,500 m by using Niskin bottles rinsed with 70% alcohol and sterile
distilled water. Some of this seawater was immediately transferred to
sterile chilled bottles and stored at 105 Pa and 3°C.
Other 2-liter aliquots were filtered (pore size, 0.2 µm; sterile
Nuclepore) at 3°C. The filters with retained cells were placed in
sterile stoppered Nalgene tubes containing 10 ml of seawater obtained
from the sampling depth and pressurized to the in situ collection
pressure; these samples were stored at 3°C until they were used. When
a natural-population experiment was initiated in the laboratory, a
sterile chilled chemostat vessel was filled with cold NSW (500 ml)
obtained from the collection depth. A Nalgene tube was decompressed,
and the contents (10 ml) along with the Nuclepore filter were added to
the vessel. The contents of the chemostat were then stirred for 15 min,
the filter was removed, and samples were taken to determine zero-time
direct counts. The chemostat was then fully assembled (21),
and the experiment was initiated by starting the flow of NSW and
pressurizing the chemostat.
The following pure cultures of psychrophilic organisms were studied
with the chemostat. Barophilic isolate F1 from 4,900 m, barophilic
isolate 27AB from 5,100 m, and barotolerant isolate K-4 from 4,000 m
were originally isolated on Difco 2216 marine medium or on medium
containing 0.1% peptone and 0.1% yeast extract in chilled slush
(0.4%) agar. The growth characteristics of these organisms, including
their maximum growth rates in batch culture, have been determined
previously (19). F1 has been identified as a
Shewanella sp. (7). Slightly barotolerant strain
82 was isolated from relatively shallow water (depth, 2,600 m) on
defined sodium glutamate (0.5 g/liter) medium (41). It had a
10-h doubling time in this medium supplemented with vitamins
(3). Isolate O-96-2 was obtained from a deep-sea natural
population (depth, 4,500 m) which had been enriched in the chemostat at
450 × 105 Pa and 3°C by using 1.0 mg of yeast
extract per liter. It was isolated from a decompressed chemostat sample
on 2216 marine agar at 105 Pa and grew well in
full-strength and 10% 2216 marine broth. The doubling times at 3°C
were determined to be 6.9 h at 105 Pa and 4.0 h
at 300 × 105 Pa in batch cultures grown on medium
containing 100 mg of yeast extract per liter. Isolate O-96-12 was
obtained from a sample collected at a depth of 4,500 m which was
immediately enriched aboard a ship at 105 Pa; the strain
was isolated conventionally at 3°C by using oligotrophic AGL medium
(see below) containing 1.0 mg of C per liter. A stock culture of this
isolate was maintained on this medium before the isolate was tested in
the chemostat. While isolate O-96-12 was never exposed to more than 1 mg of C per liter in AGL medium during the enrichment and isolation
procedure, aliquots of the stock culture were found to grow quite well
on 25× AGL medium, as well as on full-strength 2216 marine agar. The
best estimate of the maximum growth rate of this organism at 3°C was
determined by using 10× and 100× AGL medium; this estimate resulted
in a doubling time of approximately 20 h at 105 Pa.
Media.
NSW that was collected at a depth of 4,500 m was used
in the chemostat reservoir for natural-population studies and was
either filter sterilized twice (pore size, 0.2 µm; Nuclepore filters) or autoclaved, and it was supplemented with 1.0 g of
(NH4)2SO4 per liter, 0.015 g of
KH2PO4 per liter, and 5.0 ml of a vitamin mixture (3) per liter to ensure that growth was not limited by these nutrients and vitamins. Artificial seawater (ASW) containing the same supplements was used as the chemostat reservoir medium (21) when we studied pure cultures; this ensured that growth was limited by the organic carbon source (21).
Filter-sterilized yeast extract (1 or 10 mg/liter) or sodium glutamate
(1 or 100 mg/liter) was added to the ASW in the reservoir after
autoclaving in order to obtain the desired final concentration of
organic carbon. Yeast extract contains 33% carbon (13), and
sodium glutamate contains 35% carbon; therefore, when we used 1 mg of
yeast extract per liter and 1 mg of sodium glutamate per liter, the
limiting carbon concentrations were 0.33 and 0.35 mg/liter,
respectively. Oligotrophic AGL medium consisted of ASW to which we
added (per liter) 1 mg of glucose, 1 mg of sodium acetate, and 1 mg of
sodium lactate, equivalent to a total C concentration of 1 mg/liter. When solid AGL medium was used for enrichments cultures, Noble agar was
prewashed with 95% ethanol-acetone and then repeatedly rinsed with
distilled water to increase its purity before use.
The pure cultures used to inoculate the chemostat were pregrown at
3°C in batch cultures in ASW supplemented with either 0.1
or
0.01 g of organic substrate (e.g., yeast extract or sodium
glutamate) per liter and then added to the vessel containing ASW
during
assembly at a dilution which resulted in a carbon concentration
which
was the same as that used in the experiment. Difco 2216
marine agar was
used to determine pure-culture viable counts for
chemostat subsamples
of isolates F1, 27AB, K-4, and O-96-2, and
sodium glutamate medium
(
41) was used for barotolerant isolate
82. When the viable
O-96-12 cells were counted, AGL agar containing
a 25-fold-greater
organic compound content was used in addition
to Difco 2216 marine
agar. The pH values of all media were 7.2
to 7.3.
Chemostat operation, subsampling, and counting.
The
chemostat was sterilized, assembled, and operated at pressures ranging
from 105 to 450 × 105 Pa as previously
described (21). At time intervals subsamples were removed
from the pressurized chemostat by using a chilled sterile subsampler
apparatus. Epifluorescence direct counts (14) were obtained
in the natural-population studies. For the pure-culture studies both
viable and direct counts were obtained. Standard deviations were
determined for the pure-culture direct counts. The growth rates (µ)
of pressurized natural populations, obtained by using NSW that was not
supplemented with organic carbon, were calculated from the difference
between the dilution rate (D) and the washout rate
(A), as described by Jannasch (17):
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(1)
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(2)
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where
x0 is the number of cells per
milliliter at zero time,
x is the number of cells per
milliliter at the sampling time,
and
t is the time (in
hours).
In pure-culture studies, at least 5 retention times and generally 8 retention times were allowed after the chemostat flow
rate or pressure
was changed to allow a new steady-state population
to become
established. The subsampling instruments, as well as
the seawater used
for dilution and the growth media, were prechilled
and kept cold during
use to ensure that the psychrophilic isolates
were not exposed to high
temperatures that could injure them.
The dilution rates used for the
pure-culture studies were imposed
growth rates that were equivalent to
60 or 90% of the 10
5-Pa maximum growth rates of the
organisms. These growth rates
were determined in batch culture
experiments in which we used
the same growth substrates (e.g., 2216 marine broth, yeast extract
medium, sodium glutamate
medium).
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RESULTS |
Natural populations.
We performed the following experiments to
determine the growth responses of natural deep-sea populations at the
in situ pressure when only the naturally available dissolved organic
carbon was present, as well as how the organisms responded to limited
nutrient supplementation.
A NSW population obtained at a depth of 4,500 m was grown at a dilution
rate of 0.02 h
1 (retention time, 50 h) and a
pressure of 450 × 10
5 Pa (Fig.
1). Under these conditions, the
population grew at a
rate which was much less than the imposed growth
rate but somewhat
greater than the theoretical washout rate. This
growth rate was
determined (by using exponential curve fitting and
equation 1)
from the difference between the theoretical washout rate
(growth
rate, 0) and the actual washout rate (growth rate, >0) of
cells
for the first 160 h (Fig.
1, upper inset), which resulted in
a
generation time of 629 h (growth rate, 0.00159 h
1). Starting at 160 h, the reservoir seawater was
supplemented
with 1.0 mg of yeast extract per liter (0.33 mg of C per
liter),
and the pressurized chemostat was continued at a dilution rate
of 0.02 h
1. A positive growth response occurred over the
next 6 retention
times, and a steady population of cells containing
just more than
10
6 cells/ml became established. It was from
a subsample taken at
this point that isolate OC-96-2 was isolated. When
the population
was allowed to continue to grow as a pressurized batch
culture
from 560 to 617 h, about another doubling of cells
occurred; during
this time essentially all of the yeast extract carbon
was consumed.
At this point (Fig.
1, dotted arrow), a new reservoir
containing
NSW without yeast extract was connected, and a higher
dilution
rate (0.035 h
1) (retention time, 29 h) was
imposed. At this higher dilution
rate there was no measurable
difference between the actual washout
rate and the theoretical washout
rate (Fig.
1, lower inset), which
indicated that the growth rate (or
cell division) of the existing
population on any naturally occurring
carbon had been equaled
or exceeded by the imposed dilution rate.
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FIG. 1.
Direct cell counts for a deep-sea NSW sample grown at
450 × 105 Pa and 3°C at a dilution rate of 0.02 h1 (retention time, 50 h) before and after the
introduction at 160 h (solid arrow) of 1.0 mg of yeast extract per
liter to the seawater reservoir. Growth under these conditions was
continued until 560 h, and then the flow was stopped and the
pressurized population was held as a batch culture until 617 h
(dotted arrow). This was followed by a return to a NSW flow with no
yeast extract supplement at a dilution rate of 0.035 h1
(retention time, 29 h). (Insets) Exponential curve fits for cell
numbers at actual and theoretical washout rates in NSW that was not
supplemented.
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This experiment was repeated at a pressure of 450 × 10
5 Pa with a new sample at a dilution rate (0.028 h
1) (retention time, 35 h) that was between the
dilution rates used
in the experiment described above. The actual and
theoretical
washout rates were again equivalent (dilution rate = washout rate)
for the first 160 h, indicating that there was no
measurable growth
(Fig.
2, inset) of the
population on the naturally occurring substrate
in the NSW. The size of
the population declined to 4.0 × 10
4 cells/ml, and
then the contents of the seawater reservoir were
supplemented with 10 mg of glucose per liter. Growth occurred
after this carbon source was
added (Fig.
2). The glucose concentration
in the chemostat vessel
increased asymptotically relative to the
input concentration (e.g.,
6.3, 8.6, and 9.5 mg/liter after 1,
2, and 3 retention times,
respectively). The responding population
reached a steady state soon
after 4 retention times following
glucose addition and remained at this
level from 288 to 427 h;
then the chemostat was decompressed to a
pressure of 10
5 Pa (Fig.
2, dotted arrow), and incubation
was continued at the
imposed dilution rate. There was a noticeable
decrease in the
steady-state population of cells at 10
5 Pa
compared with the population that had established at 450 ×
10
5 Pa, indicating that there was some barophilic
enrichment of the
population exhibiting better growth at the elevated
pressure,
which was lost when the chemostat was decompressed.
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FIG. 2.
Direct cell counts for a deep-sea NSW sample grown at
450 × 105 Pa and 3°C at a dilution rate of 0.028 h1 (retention time, 35 h) before and after the
introduction at 160 h (solid arrow) of 10 mg of glucose per liter
to the seawater reservoir. Growth under these conditions was continued
until 427 h (dotted arrow), and then the chemostat was
decompressed to 105 Pa with the flow continuing at the same
dilution rate. (Inset) Exponential curve fit for cell numbers at the
actual and theoretical washout rates in NSW that was not
supplemented.
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Pure cultures.
We performed a series of experiments in which
we utilized barophilic and barotolerant deep-sea isolates whose
previously determined growth characteristics identified them as
copiotrophs. The goal of these experiments was to determine if the
organisms maintained their phenotypic growth responses to pressure over
a range of growth pressures in the presence of the growth-limiting
carbon concentrations characteristic of their deep-sea habitat.
The most extensive pressure comparison at different growth rates,
temperatures, and limiting carbon concentrations was performed
with
barophilic strain F1. The data obtained at 3°C (Fig.
3) show
that at different growth rates
and substrate concentrations this
isolate maintained its overall
barophilic character; the steady-state
populations at elevated
pressures were larger than the populations
at 10
5 Pa. This
was determined by both direct counting and viable counting.
Similar
results were obtained at 8°C (Fig.
4),
which is the optimum
temperature for F1. With one exception (when
optimum growth occurred
at 450 × 10
5 Pa [Fig.
4A]),
optimum growth occurred at 300 × 10
5 Pa, which has
been determined previously to be the optimum growth
pressure for this
organism under high-substrate-concentration
batch culture conditions.
When the number of viable cells was
expressed as a percentage of the
direct count, the value was usually
higher for elevated-pressure
samples than for 10
5-Pa samples. The percentage of viable
cells was relatively low
overall compared to the direct cell count
(i.e., about 25% of
the maximum value).
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FIG. 3.
Steady-state direct counts (mean ± standard
deviation) and viable cell counts for barophilic isolate F1 grown at
3°C at different pressures in the presence of different yeast extract
concentrations at different growth rates. (A) Culture grown in the
presence of 10 mg of yeast extract per liter at a growth rate of 0.044 h1 (60% of the maximum growth rate). (B) Culture grown
in the presence of 10 mg of yeast extract per liter at a growth rate of
0.066 h1 (90% of the maximum growth rate). (C) Culture
grown in the presence of 1 mg of yeast extract per liter at a growth
rate of 0.044 h1 (60% of the maximum growth rate). (D)
Culture grown in the presence of 1 mg of yeast extract per liter at a
growth rate of 0.066 h1 (90% of the maximum growth
rate). Stippled bars, direct counts; shaded bars, viable counts. One
bar is equal to 105 Pa.
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FIG. 4.
Steady state-direct counts (mean ± standard
deviation) and viable cell counts for barophilic isolate F1 grown at
8°C at different pressures in the presence of different yeast extract
concentrations at different growth rates. (A) Culture grown in the
presence of 10 mg of yeast extract per liter at a growth rate of 0.044 h1 (60% of the maximum growth rate). (B) Culture grown
in the presence of 10 mg of yeast extract per liter at a growth rate of
0.066 h1 (90% of the maximum growth rate). (C) Culture
grown in the presence of 1 mg of yeast extract per liter at a growth
rate of 0.044 h1 (60% of the maximum growth rate). (D)
Culture grown in the presence of 1 mg of yeast extract per liter at a
growth rate of 0.066 h1 (90% of the maximum growth
rate). Stippled bars, direct counts; shaded bars, viable counts. One
bar is equal to 105 Pa.
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Table
1 shows the percentage of the
direct count cells that were viable for each of the organisms and
culture conditions
tested. With the exception of isolate K-4, the
percentages ranged
from around 10% to almost 100%. We cannot offer an
explanation
for the variability in the percentages of viability
obtained for
strains other than (i) the possibility that pressure-grown
cells
were susceptible to decompression, (ii) the possibility that the
organisms were grown nonoptimally in the case of the cultures
grown at
10
5 Pa, and (iii) the fact that the carbon concentration in
the plating
medium was much higher than the carbon concentration in the
chemostat
growth medium.
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TABLE 1.
Numbers of viable cells, expressed as percentages of the
total direct counts obtained for barophilic and barotolerant
psychrophiles at steady states in the chemostat at different
growth pressures
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Barophilic isolate 27AB, which previously has been reported to grow
optimally at 300 × 10
5 Pa on 2216 marine broth
medium, retained this pressure optimum
when it was grown in the
chemostat at 60 and 90% of its maximum
rate growth in the presence of
10 mg of yeast extract per liter
(Fig.
5A and
B). When yeast extract was added at a
lower limiting
concentration, 1.0 mg/liter, the maximum steady-state
population
densities occurred at an even higher pressure, 450 × 10
5 Pa (Fig.
5C and D). As was the case with F1, the
steady-state
direct count for isolate 27AB decreased somewhat more than
10-fold
when the organism was grown in the presence of a 10-fold-lower
limiting substrate concentration (i.e., 1 mg/liter instead of
10 mg/liter). Again, when the number of viable cells was expressed
as a
percentage of the direct count, the value was always higher
for
elevated-pressure samples. When the number of viable cells
was
expressed as a percentage of the direct count, the value was
much
higher for isolate 27AB than for isolate F1 overall, approaching
100%
in some cases (Table
1).
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FIG. 5.
Steady-state direct counts (mean ± standard
deviation) and viable cell counts for barophilic isolate 27AB grown at
3°C at different pressures in the presence of different yeast extract
concentrations at different growth rates. (A) Culture grown in the
presence of 10 mg of yeast extract per liter at a growth rate of 0.06 h1 (60% of the maximum growth rate). (B) Culture grown
in the presence of 10 mg of yeast extract per liter at a growth rate of
0.09 h1 (90% of the maximum growth rate). (C) Culture
grown in the presence of 1 mg of yeast extract per liter at a growth
rate of 0.06 h1 (60% of the maximum growth rate). (D)
Culture grown in the presence of 1 mg of yeast extract per liter at a
growth rate of 0.09 h1 (90% of the maximum growth rate).
Stippled bars, direct counts; shaded bars, viable counts. One bar is
equal to 105 Pa.
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The next two isolates which we tested originated from seawater samples
collected at a depth of 4,400 m. Isolate O-96-2, which
was isolated
from a pressurized chemostat natural population after
enrichment with 1 mg of yeast extract per liter at 450 × 10
5 Pa (Fig.
1, at
560 h), was expected to be highly barotolerant
or perhaps even
barophilic. As shown in Fig.
6, O-96-2
exhibited
barophilic properties at both 60 and 90% of its maximum
growth
rate in the presence of a limiting concentration of yeast
extract
(10 mg/liter). The optimum pressure was 300 × 10
5 Pa, and only a slight reduction in growth was observed
at 450
× 10
5 Pa. The largest steady-state cell
populations established at
a dilution rate equal to 90% (growth rate,
0.09 h
1) of the maximum growth rate compared to 60%
values; this indicated
that the maximum growth efficiency occurred at
the higher growth
rate. This organism exhibited a relatively high level
of viability,
and the maximum percentage of direct counts occurred at
300 ×
10
5 Pa at both growth rates (Table
1).
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FIG. 6.
Steady-state direct counts (mean ± standard
deviation) and viable cell counts for barophilic isolate O-96-2 grown
at 3°C in the presence of 10 mg of yeast extract per liter at
different pressures and different growth rates. (A) Culture grown at a
growth rate of 0.06 h1 (60% of the maximum growth rate).
(B) Culture grown at a growth rate of 0.09 h1 (90% of
the maximum growth rate). Stippled bars, direct counts; shaded bars,
viable counts. One bar is equal to 105 Pa.
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The second isolate from this seawater source, isolate O-96-12, was
enriched and isolated in a conventional fashion in the
presence of a
very low concentration of defined carbon substrates
(i.e., oligotrophic
conditions), as described above. Based on
a doubling time of 20 h,
this isolate was grown in the chemostat
at 90% of the maximum growth
rate. O-96-12 exhibited barophilic
growth in the presence of 1 mg of C
per liter as larger steady-state
populations became established at both
200 × 10
5 and 400 × 10
5 Pa than at
10
5 Pa (Fig.
7). When the
viable counts for samples grown at 10
5 Pa and for
decompressed subsamples were expressed as percentages
of the total
direct counts, the values were low but similar (6
to 18%) (Table
1),
whether the organisms were grown on 25× AGL
medium or on 2216 marine
agar.
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FIG. 7.
Steady-state direct counts (mean ± standard
deviation) and viable cell counts for barophilic isolate O-96-12 grown
at 3°C in the presence of 1 mg of C per liter (AGL medium) at a
growth rate of 0.031 h1 (90% of the maximum growth rate)
at different pressures. Stippled bars, direct counts; shaded bars,
viable counts on 2216 marine agar; wave pattern bars, viable counts on
25× AGL medium. One bar is equal to 105 Pa.
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Having determined that all of the previously tested strains responded
in a barophilic fashion to limiting concentrations of
substrate, we
decided to test two organisms which were known from
previous batch
culture studies to be highly or slightly barotolerant
when substrate
was not limiting. Highly barotolerant strain K-4
was grown in the
chemostat in the presence of 1.0 mg of glutamate
per liter. In batch
culture studies, this isolate has been shown
to have growth rates that
are quite similar at pressures ranging
from 10
5 to 300 × 10
5 Pa; at 400 × 10
5 Pa the growth
rate is about 20% lower (
19). As shown in Fig.
8, the steady-state populations of K-4 at
10
5 Pa were only slightly larger than the steady-state
populations
at 200 × 10
5 and 400 × 10
5 Pa. This was the case at growth rates that were 60 and
90% of
the maximum growth rate; thus, K-4 kept its highly barotolerant
growth characteristics under carbon-limiting conditions. The cell
densities established by this organism in the presence of a low
level
of available carbon indicate that anabolism was extremely
efficient.
The plate count viability of K-4 was generally very
low compared to the
total count, particularly when samples were
removed from growth under
pressure, which resulted in values less
than 1% of the direct count
(Table
1).
View larger version (45K):
[in this window]
[in a new window]
|
FIG. 8.
Steady-state direct counts (mean ± standard
deviation) and viable cell counts for barotolerant isolate K-4 grown at
3°C in the presence of 1 mg of sodium glutamate per liter at
different pressures and growth rates. (A) Culture grown at a growth
rate of 0.017 h1 (60% of the maximum growth rate). (B)
Culture grown at a growth rate of 0.028 h1 (90% of the
maximum growth rate). Stippled bars, direct counts; shaded bars, viable
counts. One bar is equal to 105 Pa.
|
|
The final pure-culture isolate tested was psychrophilic isolate 82, which previously was shown to be only somewhat barotolerant
in batch
culture experiments (
41). When isolate 82 was grown
in the
chemostat at either 60 or 90% of the maximum growth rate
in the
presence of a limiting concentration of sodium glutamate
(10 mg/liter),
the starter population washed out of the chemostat
at elevated
pressures, perhaps due to a minimum required reservoir
substrate
concentration (
16). Therefore, the sodium glutamate
concentration was increased to 100 mg/liter, and the growth rate
used
was 60% of the maximum growth rate. As shown in Fig.
9, isolate
82 grew best at a pressure of
10
5 Pa, and the size of the steady-state population
decreased as
the pressure was increased to 150 × 10
5
and 300 × 10
5 Pa. In contrast to the generally low
viability of the barophilic
or highly barotolerant isolates described
above (except isolate
27AB), the number of viable isolate 82 cells was
close to 100%
of the direct count in all cases (Table
1).
View larger version (60K):
[in this window]
[in a new window]
|
FIG. 9.
Steady-state direct counts (mean ± standard
deviation) and viable cell counts for barotolerant isolate 82 grown at
3°C in the presence of 100 mg of sodium glutamate per liter at a
growth rate of .037 h1 (60% of the maximum growth rate)
at different pressures. Stippled bars, direct counts; shaded bars,
viable counts. One bar is equal to 105 Pa.
|
|
| |
DISCUSSION |
Since the deep sea comprises such a large part of the earth's
biosphere, it is important to understand how the microbes in the deep
sea function under the in situ environmental conditions. Since in the
deep sea microorganisms are exposed to growth-limiting concentrations
of essential nutrients, particularly carbon (12, 29),
chemostats are well suited for metabolic studies of microorganisms grown under low-nutrient-concentration conditions (40).
Using this approach, we found that the growth rate of deep-sea
populations in the presence of natural concentrations of available
carbon could not be measured or was extremely low, but a low
concentration of supplemental carbon (0.33 mg/liter) resulted in
positive growth responses in the natural population; there was an
increase in the number of cells, and eventually a steady population of
cells became established. Our other very important finding was that the
growth responses of barophilic and barotolerant deep-sea isolates, which were isolated previously and were characterized under
copiotrophic high-nutrient-concentration conditions, were still
maintained under the low-nutrient-concentration limiting conditions
characteristic of the natural oligotrophic deep-sea environment.
An advantage of using continuous-culture approaches with a chemostat is
that relatively large, constant populations of cells growing in the
presence of low concentrations of a limiting nutrient can be
maintained; thus, the continuously changing conditions characteristic
of a batch culture are eliminated (40). It must be
emphasized, however, that in a natural system, such as the deep sea,
the supply of nutrients and cell removal are not closely linked, as
they are in a chemostat (18), and therefore, the deep sea
does not actually exhibit steady-state conditions. Chemostats, however,
have been used to obtain steady states of mixed populations obtained
from surface seawater (16, 18) in which natural enrichment occurs for the organism growing the fastest under the conditions used.
As long as the dilution rate is lower than the maximum growth rate, the
successful species outcompetes all of the other species, and a
pure-culture steady state is approached. As shown in Fig. 1 and 2, it
was evident that, based on the small amount of naturally occurring
carbon in seawater, the microbial populations present were not able to
grow at the in situ pressure at a rate equal to or even approaching the
imposed dilution rates. With respect to the available carbon, there may
be another limitation, as much of this carbon has been shown to be
relatively recalcitrant to microbial oxidation (2). However,
in the case of the lowest imposed dilution rate (Fig. 1) (0.2 h1) (retention time, 50 h), a growth rate of 0.00159 h1 (generation time, 629 h) was calculated from the
difference between the theoretical and actual washout rates for cells
in the population.
In both experiments (Fig. 1 and 2) it was possible to enrich for a
steady-state population of cells by using very low concentrations of
supplemental organic carbon (1 mg of yeast extract per liter [0.33 mg
of C per liter; 0.027 mM C] or 10 mg of glucose per liter [4 mg of C
per liter; 0.33 mM C]) at the same imposed dilution rates as those
used with unamended NSW. In both cases the populations grew, and a
steady cell number (growth rate = dilution rate) was obtained at
the in situ pressure and temperature; however, in neither case should
the population be considered a steady-state population of a single pure culture.
Members of potentially dormant (39) or extremely slowly
growing deep-sea populations may be very barotolerant or for the most
part barophilic (44, 48). As our experiments were conducted at the in situ pressure (450 × 105 Pa) measured where
the natural populations were collected, we could have expected
enrichment for pressure-tolerant or barophilic cells. This was in fact
observed with barophilic isolate O-96-2, which was enriched in the
chemostat in the presence of 1 mg of yeast extract per liter at a
dilution rate of 0.02 h1. These experiments showed that
resident deep-sea populations of microbes that exist naturally under
severely nutrient-limited conditions are able to respond to small
increases in the available carbon concentration and grow competitively
and, depending on their growth characteristics, can become the
predominant component of a population. We could speculate that deep-sea
barophilic heterotrophs are quite versatile and capable of responding
to very low levels of available carbon, as shown in this study. They
may also grow well at the high carbon concentrations which
traditionally have been used for enrichment and isolation of pure
cultures of deep-sea microbes, including the copiotrophic barophilic
isolates used in this study (e.g., isolates F1 and 27AB).
New barophilic and obligately barophilic bacteria have been isolated
and described recently (25, 33, 34, 36), and the
phylogenetic diversity of these new bacteria is quite broad (7). While the presence of psychrophilic and barophilic
bacteria is well established, it should be pointed out that many
isolates are classified as barophilic on the basis of growth
experiments performed at 10 to 15°C (24, 26, 33, 34). When
many of the same isolates are grown at 4°C, a temperature
characteristic of their natural habitat, or even at temperatures up to
10°C, they do not respond as barophiles but exhibit only barotolerant properties. This implies that many deep-sea bacteria do not exist in
situ under conditions which allow them to act as barophiles, and it
follows that deep-sea bacteria that actually grow as barophiles (44) under natural conditions may not be ubiquitous. With
respect to the present study, a question posed by Yayanos
(44), whether the pressure tolerance of these microbes
varies with nutrition, is important. This question is important not
only with respect to the mode of nutrition (e.g., autotrophy,
heterotrophy, and oligotrophy) but also with respect to the
concentration of the growth-limiting substrate.
While the copiotrophic barophiles that have been described do appear to
be adapted for life in their extreme environment (25, 32,
43), it remains to be determined whether this phenotypic pressure
adaptation is retained under carbon-limiting conditions. These
conditions may dramatically shift the metabolism/incorporation ratio,
as previously shown for nonbarophilic psychrophiles, and may make them
less efficient under oligotrophic conditions (41). The
numerical predominance of oligotrophic marine bacteria over more
copiotrophic heterotrophs has been shown to occur when carbohydrates are supplied at concentrations less than 0.5 mg/liter (1). However, invariably, isolated obligately oligotrophic bacteria have
been found to adapt and become facultative oligotrophs capable of
growth in the presence of a higher concentration of substrate (37). Conversely, decreasing the substrate concentration (to the limits that can be tested in batch cultures) has been shown to
induce a more efficient barophilic response in certain copiotrophic deep-sea psychrophiles (8, 20).
The deep sea contains a very limited supply of utilizable organic
carbon, and the functioning of barophilic microbes is linked to the
saturation constant (Ks) and to the efficiency
of substrate uptake, as well as to the ability to grow at high
pressures and at low temperatures. In the present study we obtained
evidence showing that there are not two distinct classes of barophilic microbes with respect to the carbon concentration requirement (i.e.,
microbes that are oligotrophic and microbes that are copiotrophic taking advantage of different growth niches in the deep sea and not
being able to adapt to growth in characteristically different nutrient
niches). As previously shown, barophilic bacteria can exist as actively
metabolizing cells in association with gut tracts of abyssal animals
(9) or with nutrient-rich particulates (10). However, in most of the deep-sea biosphere the free-living cells in the
water and in the surface sediments exist under severe, carbon-limiting
conditions. Our data show that the moderately barophilic, highly
barotolerant, and weakly barotolerant organisms which were isolated and
characterized in batch cultures under high-nutrient-concentration
(copiotrophic) conditions can adapt to and grow in the presence of a
wide range of substrate concentrations, including oligotrophic levels.
This conclusion is supported by the fact that the
Ks values for a number of copiotrophic marine bacteria are the same order of magnitude as the
Ks value for the upper limit for oligotrophic
growth (27), which makes the copiotrophs competitive with
the oligotrophs under nutrient conditions characteristic of the deep
sea. In the present study, each of the isolates maintained its pressure
optimum (barophilic or barotolerant) under nutrient-limiting conditions
in the chemostat; thus, these organisms are quite versatile competitors
in their low-temperature, high-pressure, nutrient-limited, deep-sea
milieu. This conclusion may be extended in reverse based on the data
obtained for oligotrophic isolate O-96-12, which exhibited a barophilic
growth pattern under carbon-limiting conditions but also grew well in
batch cultures in the presence of high concentrations of carbon, such
as the concentration in 2216 marine broth. Such versatility is critical
for deep-sea bacteria, which must maintain metabolic activity as they
encounter changing substrate concentrations.
| |
ACKNOWLEDGMENTS |
This work was supported by grant OCE-9415371 from the National
Science Foundation.
We acknowledge the expert engineering contribution of Kenneth Doherty,
who designed the high-pressure chemostat.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biology
Department, Woods Hole Oceanographic Institution, MS # 33, Woods Hole,
MA 02543. Phone: (508) 289-2307. Fax: (508) 457-2134. E-mail:
cwirsen{at}whoi.edu.
Contribution no. 10019 of the Woods Hole Oceanographic Institution.
This paper is dedicated to Holger W. Jannasch, who was our longtime
mentor, coworker, and friend.
| |
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Top
Abstract
Introduction
