Aerobic growth at nanomolar oxygen concentrations

Aerobic growth at nanomolar oxygen concentrations
INAUGURAL ARTICLE
Aerobic growth at nanomolar oxygen concentrations
Daniel A. Stolpera,1, Niels Peter Revsbechb, and Donald E. Canfielda,2
a
Nordic Center for Earth Evolution (NordCEE) and Institute of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark; and
Department of Biological Sciences, University of Aarhus, DK-8000 Aarhus C, Denmark
b
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2007.
aerobic respiration ∣ microbial growth ∣ precambrian ∣ nanaerobe ∣
evolution
I
n discussions of the history of life, aerobic respiration is often
assumed to have been an unviable means of energy production
until oxygen levels exceeded the “Pasteur Point” (1–3). The Pasteur Point is the partial pressure of oxygen (in equilibrium with a
solution) at which facultative aerobic organisms switch to anaerobic metabolisms. It varies from organism to organism, but a
value of approximately 0.01 (1%) of the present atmospheric
oxygen level (PAL) is typical for modern prokaryotes and
single-celled eukaryotes (4). Seawater in equilibrium with this
level of atmospheric oxygen at 25 °C has a dissolved oxygen
concentration of 2.2 μM, which is comparable to the K m values
(Michaelis–Menten constants*) for oxygen utilization for many
aerobic bacteria isolated from marine environments (5). It is
known, however, that some aerobes can utilize much lower
oxygen concentrations than those corresponding to the Pasteur
Point. For example, a K m value as low as 31 nM has been measured for the facultative aerobe Aerobacter aerogenes (6). Additionally, oxygen concentrations down to 3 nM have been
measured in soybean nodules supporting aerobic respiration (7),
and the corresponding K m values for oxygen respiration of
bacteria populating these nodules are in the 5- to 8-nM range
(8). Likewise, K m values for the high-oxygen-affinity cytochrome
bd oxidase for Escherichia coli have been measured as low as
3–8 nM, in contrast to the lower affinity E. coli cytochrome bo
oxidase, which has a K m of about 200 nM (9). Despite these determinations of low-oxygen utilization by some microbes, growth
data are sparse, and we are unaware of any direct experimental
demonstrations of microbial growth based on aerobic respiration
at oxygen levels less than 230 nM (10). We emphasize the importance of growth, because to thrive in an environment an organism
must grow and reproduce. Microbes can often metabolize and
respire under environmental conditions that do not allow for
growth such as temperature extremes or limited substrate availability (11).
Understanding the oxygen limits of aerobic microbial growth is
relevant to discussions of the microbial ecology of both modern
and ancient Earth systems. On the modern Earth, low-oxygen
niches exist at all transitions from oxic to anoxic environments,
www.pnas.org/cgi/doi/10.1073/pnas.1013435107
as found, for example, in marine and freshwater sediments
(12), many soils, and in the oxygen minimum zones (OMZs)
of the global ocean (13). In the late Archean Eon (from about
2.7–2.5 billion years ago, Ga), prior to the pervasive oxygenation
of the Earth’s atmosphere at 2.3 to 2.4 Ga (14), elevated oxygen
concentrations apparently existed locally and/or for short durations (15–18). However, atmospheric oxidant models, including
those explaining the “mass-independent” fractionation of sulfur
isotopes prior to 2.3 to 2.4 Ga, restrict average oxygen levels
in the atmosphere to a maximum of ∼10−5 of the present level
(19, 20), corresponding to a concentration of about 2.2 nM in
coexisting surface waters. An unresolved question is whether such
oxygen levels were sufficient to support the growth of aerobic
communities. Using the Pasteur Point of most modern aerobic
organisms as a reference point would lead one to conclude
“no”, but biomarkers preserved in late Archean (2.67 Ga) rocks
suggest the presence of aerobic communities (21).
The motivation for this study is to explore whether the conventional view about the limits of aerobic growth is correct and to
place quantitative limits on the lower oxygen requirements for
aerobic respiration in both the past and present. To achieve this,
we monitored growth of E. coli K-12 at low (nanomolar) oxygen
concentrations made possible by evolving oxygen sensor technology (13). We demonstrate that aerobic growth is not constrained
by the typical Pasteur Point (i.e., to > ∼ 0.01 PAL) and that
aerobic growth is viable at oxygen concentrations ≤3 nM (∼10−5
PAL), at least two orders lower than previously reported.
Results and Discussion
Growth Under Oxygen-Limiting Conditions. A typical experiment
is shown in Fig. 1A. Here, we monitored oxygen concentration
and growth for 30 h. After the initial oxygen manipulations
during the first 6 h, we established the final desired fixed pump
rate of 36 mL h−1 corresponding to an oxygen flux of 11.5
0.3 μmol L−1 h−1 (Fig. S1). After about 20 h, oxygen concentrations reached the detection limit of the sensor (3 nM), yet growth
continued to the end of the experiment 10 h later. Similarly, in a
50-h experiment with a 35-min ramp-up phase (Fig. 1B) and at a
slower pump rate of 12 mL h−1 (corresponding to an oxygen flux
of 5.5 0.3 μmol L−1 h−1 ; Fig. S1), oxygen concentrations
decreased to the detection limit of the sensor after about 12 h.
Still, growth continued for an additional 38 h (when the experiment was terminated) at oxygen concentrations at or below the
detection limit of our sensors.
Author contributions: D.A.S., N.P.R., and D.E.C. designed research; D.A.S. performed
research; N.P.R. contributed new reagents/analytic tools; D.A.S. and D.E.C. analyzed data;
and D.A.S., N.P.R., and D.E.C. wrote the paper.
The authors declare no conflict of interest .
*The Michaelis–Menten constant, K m , is the substrate concentration at which an enzyme
or a cell’s overall respiration rate operates at half its maximum rate.
1
Present address: Division of Geological and Planetary Sciences, California Institute of
Technology, MC 170-25 1200 East California Boulevard, Pasadena, CA 91125.
2
To whom correspondence should be addressed. E-mail: [email protected]
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1013435107/-/DCSupplemental.
PNAS ∣ November 2, 2010 ∣ vol. 107 ∣ no. 44 ∣ 18755–18760
ECOLOGY
Molecular oxygen (O2 ) is the second most abundant gas in the
Earth’s atmosphere, but in many natural environments, its concentration is reduced to low or even undetectable levels. Although
low-oxygen-adapted organisms define the ecology of low-oxygen
environments, their capabilities are not fully known. These capabilities also provide a framework for reconstructing a critical period in
the history of life, because low, but not negligible, atmospheric
oxygen levels could have persisted before the “Great Oxidation”
of the Earth’s surface about 2.3 to 2.4 billion years ago. Here,
we show that Escherichia coli K-12, chosen for its well-understood
biochemistry, rapid growth rate, and low-oxygen-affinity terminal
oxidase, grows at oxygen levels of ≤3 nM, two to three orders of
magnitude lower than previously observed for aerobes. Our study
expands both the environmental range and temporal history of
aerobic organisms.
GEOLOGY
Contributed by Donald E. Canfield, September 9, 2010 (sent for review April 4, 2010)
60
12
50
10
40
8
30
6
20
4
10
2
0
0
0
5
10
15
20
25
Cells mL-1 (x10 6)
O2 (nM)
A
30
O2 (nM)
B
60
12
50
10
40
8
30
6
20
4
10
2
0
Cells mL-1 (x10 6)
Time (h)
0
0
5
10
15
20
25
30
35
40
45
50
55
Time (h)
Fig. 1. Oxygen concentrations and cell counts for respiration experiments with E. coli at two different pump rates of oxygen through the silicone tubing in the
reactor. (A) A terminal pump rate of 36 mL h−1 was used corresponding to an oxygen input rate into the reactor of 11.5 μmol L−1 h−1 . During the first 6 h of
incubation, the pump rate was incrementally increased from zero to 36 mL h−1 in an effort to maintain oxygen concentrations at a steady value. Instead, they
fluctuated between 100 and 200 nM during this time. (B) A terminal pump rate of 12 mL h−1 was used, corresponding to an oxygen flux into the reactor of
5.5 μmol L−1 h−1 . The pump rate was brought to its terminal value after 35 min. In this particular experiment, external electrical noise produced random
fluctuations in the oxygen signal. These random peaks do not reflect actual oxygen concentrations, but rather experimental noise. Oxygen concentrations
are given by the open symbols “o,” with uncertainties given by the error bars (see SI Text for error explanation). Cell numbers are given in the closed symbols
“•.” The minimum absorbance that could be measured by the spectrophotometer used was 0.001 (corresponding to 3.47 × 105 cells mL−1 . A value of 3.47 ×
108 cells mL−1 per unit absorbance was calculated with a standard error of 8.96 × 106 cells mL−1 per unit absorbance (i.e., a standard error of 2.5%). The error
bars for the cells mL−1 are given as 5%, i.e., 2 standard errors.
In each of our experiments, the growth rate (G, in units of
Δcells mL−1 h−1 ) was constant for a given oxygen flux (or pump
rate) for periods up to 49 h, despite the fact that oxygen levels
decreased over this time period to below the detection limit of
the sensor (e.g., Fig. 1B). A constant growth rate is expected when
a limiting nutrient or metabolite is provided at a constant rate
(22), which in this case was oxygen. When comparing between
experiments, G was proportional to the oxygen flux into the reactor (R2 ¼ 0.96) (Fig. 2A), providing additional evidence for the
control of oxygen on growth. From the data in Fig. 2, we also
found a consistent growth yield between experiments of 0.51
0.06 mol of cell carbon produced per mole of O2 respired. This
value compares favorably with previous experiments of aerobic
growth of E. coli on glycerol (23) (see SI Text for calculations).
By contrast, the specific rate of growth, μðh−1 Þ, decreased during the course of an experiment and in relation to lower oxygen
concentrations (Fig. 2B). The specific growth rate is defined as
ln 2∕ðρ∕GÞ, where G is growth rate (defined above), and ρ is
the cellular density (cells mL−1 ); μ is related to cell doubling time,
tD , by tD ¼ ln 2∕μ. The lower the specific growth rate, the longer
the doubling time and the slower the growth by the organism.
Under conditions of oxygen limitation, μ should depend on
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the concentration of oxygen in the media (22). This dependence
is expected because under oxygen-limited conditions the flux of
oxygen into the cell is driven by the diffusion gradient of oxygen
into the cell. The flux of oxygen into a cell and subsequent specific
rates of growth should, therefore, decrease with decreasing oxygen
concentration. Likewise, under oxygen-limiting conditions, the
oxygen concentration in the media decreases as the population
grows. Because oxygen is removed by diffusion into cells, more
cells means more surface area to diffuse across, and lower oxygen
concentrations provide the same oxygen removal flux (balancing
the constant input rate) across the increased surface area.
The Monod equation [μ ¼ μmax ð½O2 ∕ð½O2 þ K s ] (22) expresses a common relationship between μ and substrate (in our
case oxygen) concentration. In this equation, μmax expresses the
maximum specific growth rate, and a value of 0.4 h−1 was obtained
under high-oxygen concentrations in open-air experiments (see
SI Text), whereas K s is the half saturation constant† with respect
to growth. We used the Monod equation to predict how μ would
respond to changing oxygen concentrations with variable K s
†
The K s of an organism for a particular substrate represents the concentration of that
substrate at which an organism grows at half its maximal rate.
Stolper et al.
Flux (nmol l -1 h-1)
12,000
10,000
8,000
6,000
4,000
2,000
Flux ¼ 4πDrðC∞ − Ccell Þ;
0
100,000
200,000
300,000
400,000
500,000
Growth Rate (cells mL-1 h-1)
B
0.45
Specific growth rate (h -1)
0.40
0.35
0.30
0.25
0.20
0.15
Data
Ks 50 nM
Ks 100 nM
Ks 150 nM
Ks 200 nM
0.10
0.05
0.00
1
10
100
1,000
10,000
100,000 1,000,000
Oxygen (nM)
Fig. 2. (A) Growth rate of cells (cells mL−1 h−1 ) compared to the flux rate of
oxygen into the reactor. Each experiment (other than the zero point) was run
twice at a particular pump rate and the growth rates averaged. The error bar
on the y axis is the standard error of the intercept. It demonstrates that the
best-fit line passes through the origin within error. (B) Specific growth rates
(h−1 ) vs. oxygen with various fits to a Monod growth model. A series of
different K s values were explored with a constant μmax of 0.4 h−1 . See text
for details.
values, and we obtained a best-fit K s value of 121 20 nM O2
(Fig. 2B). The error represents the standard deviation around
the best-fit slope of predicted vs. measured values (Fig. 2B). To
our knowledge, this is the lowest K s ever recorded for aerobic
growth.
As described above, during aerobic respiration, oxygen is used
to fuel cell growth and also for cell maintenance requirements.
Maintenance energy refers the energy expended for all cellular
processes that do not contribute to growth including cell repair.
Typically, as μ decreases, maintenance energy becomes proportionally more important resulting in a decrease in growth yield
(23). Indeed, cell maintenance requirements could put lower
limits on rates of cell growth. Maintenance energy requirements
could have played a role in our experiments, where values of μ
were as low as 0.01 h−1 (reached at the end of the experiment
depicted in Fig. 1B). Yet despite these low values of μ, growth
yields remained constant both within (as observed by constant
growth) and among the different experiments (Fig. 2A), providing
no indication of an increasing significance of maintenance
requirements as specific rates of growth decreased.
Exploring the Lower Limits of Oxygen on Growth. Our results demonstrate aerobic growth by E. coli at oxygen concentrations of
≤3.0 nM. However, the lower oxygen limit for aerobic growth
Stolper et al.
[1]
where Flux is the flux of oxygen into the cell, D is the diffusion
coefficient for oxygen, r is the cell radius, C∞ is the oxygen concentration in the bulk media, and Ccell is the oxygen concentration
at the outer cell surface. At 37 °C (the temperature of our experiments) and in fresh water, D is 3 × 10−5 cm2 s−1 (25). We assume
that cells have the same carbon content per volume as typical
E. coli (1.85 × 10−14 mol C μm−3 ; see SI Text), that they produce
cell carbon with a growth yield of 0.5 mol C∕mol O2 respired (see
above), and that they double when they have accumulated twice
their normal cell C content. With these values and assumptions,
we can reproduce our observed specific growth rates of E. coli
at low O2 if we further assume that Ccell ¼ 1∕2C∞ (Fig. 3).
With these values, we calculate the oxygen levels that produce
doubling times of 30 and 15 d for a variety of different cell sizes
(Table 1). We chose these doubling times because in Earth–
surface environments, organisms must be able to respond to
changing temperatures, moisture, and energy supply, which are
dynamic on time scales of days to months. Such doubling times
are also typical for prokaryotes in surface marine sediments and
in the marine water column (26, 27). Model results show that
for a 1-μm cell, an oxygen concentration of 0.2 nM would be
sufficient to allow a doubling time of 30 d, and for a cell as small
as 0.5 μm [which is small for a prokaryote cell (11)], 0.050 nM
O2 would suffice (Table 1). We conclude that for organisms growing with doubling times of 30 d, 0.050 nM oxygen would appear
adequate to fuel an aerobic ecosystem. Slower growing or smaller
organisms could, in principle, grow on even lower oxygen concentrations than this. A key point is that although we have observed
aerobic growth at orders of magnitude lower oxygen concentrations than might have been expected based on the Pasteur Point
argument or has been previously observed, our results make sense
based on simple, plausible models.
0.25
specific growth rate (h -1)
0
data
model
0.2
0.15
0.1
0.05
0
0
10
20
30
40
50
60
70
O2 nM
Fig. 3. Specific growth rates from our experiments compared to results from
a diffusion-limited growth model. See text for details.
PNAS ∣ November 2, 2010 ∣
INAUGURAL ARTICLE
y = 0.0298x + 600.68
2
R = 0.9638
vol. 107 ∣
no. 44 ∣
18757
GEOLOGY
14,000
in E. coli, even in our experiments, is not clear given that growth
continued for up to 38 h after oxygen became undetectable using
our STOX (Switchable Trace OXygen) sensors. As E. coli appears
capable of growing at oxygen concentrations lower than the detection limit of the STOX sensor, we will explore the lower limits
for growth by modeling the fluxes of oxygen across a cell membrane. As mentioned above, when oxygen is limiting, μ is limited
by the diffusional flux of oxygen across the cell membrane, which
depends on oxygen concentration. With this in mind, we can
model how oxygen concentration limits μ for different-sized
organisms. For a spherical cell, the flux of oxygen to the cell is
given by (24):
ECOLOGY
A 16,000
Table 1. O2 concentrations required for cell doubling times of
15 and 30 d
O2 concentration, nM
Cell diameter, μM
4.0
3.0
2.0
1.5
1.2
1.0
0.8
0.6
0.5
30 d
3.2
1.7
0.79
0.44
0.28
0.20
0.13
0.07
0.05
15 d
6.4
3.4
1.6
0.88
0.56
0.40
0.26
0.14
0.10
Low-Oxygen Metabolism in Other Prokaryotes. The low-oxygen
metabolism in E. coli is made possible by the high oxygen affinity
of the cytochrome bd oxidase (9), which is expressed when oxygen
levels fall below about 10% of air saturation (28). This highaffinity oxidase is well distributed among prokaryotes (29, 30),
including cyanobacteria, many aerobes, some presumably “strict”
anaerobes (e.g., Methanosarcina barkeri, Desulfovibrio vulgaris,
and others), and also Bacteroides fragilis, which has been termed
a “nanaerobe” (29). Nanaerobes can live as strict anaerobes, cannot grow in the presence of micromolar levels of oxygen, but can
metabolize at submicromolar oxygen levels; however, the extent
to which oxygen fuels the growth of these organisms is unknown.
Another high-affinity oxidase, the cbb3-type cytochrome oxidase,
was first found in the respiratory chain of the soybean root
nodule bacterium Bradyrhizobium jaonicum (31) but is also widely
distributed among the Bacteria (30). Although our results apply
to only one organism, from the wide distribution of low-affinity
oxidase enzymes in nature, we infer that the ability to metabolize
and, in particular, to grow at nanomolar (and perhaps lower)
concentrations of oxygen is potentially common in nature.
Geobiological Implications. Low-oxygen environments are found at
the oxic-anoxic interface of aquatic sediments (11), in many soils,
in stratified lakes and marine basins (11, 32), and in OMZs of
the global ocean (33). Our results place quantitative constraints
on where in these environments aerobic metabolisms could be
supported. This issue is of particular importance in some low-oxygen environments such as OMZs, where oxygen can be reduced
to submicromolar (and perhaps even nanomolar or lower) concentrations (13). OMZs are found in upwelling regions, such
as those of the Eastern Tropical Pacific, the Benguela upwelling
current, and the Arabian Sea, and such regions are expected to
expand as a result of global warming (34). With a single exception
(13), the oxygen concentrations in these regions are not known
to concentrations better than about 1–2 μM (35), but with our
current results, the extent of possible aerobic metabolisms can
be mapped onto measurements of high-resolution oxygen distributions as made possible with the STOX sensors used here. Our
results also clearly show that aerobic metabolisms are probable in
the Bay of Bengal, another OMZ where oxygen concentrations
reach values as low as ∼2 μM (36). Such oxygen levels have typically been viewed as supporting so-called “suboxic” metabolisms such as nitrate, manganese, and iron reduction (37), with
an uncertain role for aerobic processes. Our results demonstrate
that aerobic metabolisms can operate under these conditions and
possibly overlap with some of these suboxic anaerobic metabolisms. It will be important to establish the balance between aerobic and anaerobic metabolisms in modern, natural environments
with micromolar to subnanomolar oxygen concentrations.
The balance between aerobic and anaerobic metabolisms
at low-oxygen is especially important because some anaerobic
metabolisms such as denitrification can also occur in the presence
of oxygen (38). Combined with in situ studies of denitrification
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rates in oxygen gradients, our results will help provide an understanding of the cooccurrence and competition between aerobic
and anaerobic processes in nature. The nature of the coupling
between aerobic and anaerobic metabolisms in nanomolar
oxygen concentrations is virtually unknown and deserves further
research.
Low-oxygen conditions are especially important in discussions
of early life on Earth. Atmospheric oxygen levels are modeled
to have been ≤10−5 PAL (19) before about 2.3 to 2.4 Ga, which
corresponds to about 2.2 nM dissolved oxygen when saturated
with seawater at 25 °C. We have demonstrated growth to nearly
this oxygen concentration (3 nM, the detection limit of our STOX
sensor) and modeled it to much lower concentrations (equivalent
to about 2 × 10−7 PAL). As such, 10−5 PAL should support
the growth of E. coli and potentially other aerobic microbes with
similar high-affinity cytochromes. Therefore, environmental conditions may have allowed for aerobic metabolism well before the
large rise in atmospheric oxygen 2.3 to 2.4 Ga (39, 40). This is
different from many models of Earth history that require oxygen
concentrations in the atmosphere to have been at least 10−2 PAL
(i.e., based on the Pasteur Point argument) before aerobic
organisms could begin using oxygen for respiration (1–3) but is
consistent with the geologic record, where biomarkers preserved
in rocks as old as 2.67 Ga suggest the presence of eukaryotes,
cyanobacteria, and oxygen-utilizing heterotrophs and autotrophs
(21). This is fully 300 million years before the accepted widespread oxidation of the atmosphere. The calibration of geochemical proxies can be imprecise, but we suggest that an aerobic
biosphere could have been active from at least 2.67 Ga and
onward at lower levels of oxygen than inferred by occasional late
Archean “whiffs” of elevated oxygen concentrations (15, 17,
18, 41).
We have demonstrated that an active and growing aerobic
biosphere could have been maintained with oxygen concentrations at least down to about 10−5 PAL, and perhaps as low as 2 ×
10−7 PAL. Low-oxygen concentrations in the 10−7 to 10−5 PAL
range would have been rapidly depleted in metabolically active
areas, and a challenge to the aerobic biosphere may have been
access to sufficient oxygen for respiration. One could, however,
imagine aerobic communities preferentially populating a thin
veneer in soils over the land surface, being supplied by low levels
of atmospheric oxygen. Aerobic populations might also have been
found in aquatic environments in close association with oxygenproducing microbial mats and stromatolites and in the upper
photic zone where cyanobacteria were active.
One might envision that aerobic metabolisms began with the
evolution of oxygen-producing cyanobacteria, providing a source
of oxygen for aerobic respiration. If so, the combined elemental
and isotopic evidence for oxygen whiffs and eukaryotic and
cyanobacterial biomarkers (see above) place the evolution of
aerobic metabolism before about 2.7 Ga (though see ref. 42
for a different view). Independent of these geological arguments,
the history of oxidase enzymes places other constraints on the
origins of aerobiosis (30, 43, 44). Although there is active debate
on the phylogenetic history of oxidase enzymes, recent models
(30, 43, 44) support an ancient origin, even before the evolution
of oxygen-producing cyanobacteria (45). Without a cyanobacterial source, oxygen could have come from abiotic sources, such
as the rainout and disproportionation (by catalase enzymes) of
atmospherically produced H2 O2 at the Earth’s surface. This process could have supported elevated oxygen concentrations in
local environments before the evolution of cyanobacteria (46,
47). Our results do not prove that aerobic pathways predated
cyanobacterial evolution, but they allow further development
of such a hypothesis. They also provide support for an aerobic
biosphere after cyanobacterial evolution—but long before the
general oxidation of the Earth’s atmosphere some 2.3 to 2.4 Ga.
Stolper et al.
Thus far, demonstrations of aerobic respiration at low nanomolar oxygen
concentrations have been based on the absorption spectrum associated with
variable oxygenation of root nodule leghaemoglobin (7). We used instead
the recently developed polarographic STOX sensors, which are stable and
precise down to oxygen concentrations of ≤3 nM (13) and are easily adapted
to growth experiments. We studied E. coli K-12 [strain Sø8HLT (48)] because
of its well-characterized metabolic system, rapid doubling times, and because
it possesses both high- and low-affinity cytochrome oxidases (9). E. coli is a
facultative aerobe that would normally switch to anaerobic metabolisms
at 0.005–0.02 PAL (1–4 μM O2 in solution) (49). To ensure aerobic growth
by E. coli, we used glycerol as a carbon source, which is generally considered
unfermentable (50). Although recent studies have demonstrated glycerol
fermentation by E. coli, including K-12 (51), this growth was only with the
addition of tryptone or proteinogenic amino acids (51), which were not
included in our media. Furthermore, control experiments without oxygen
addition yielded no growth of E. coli in the presence of glycerol (Fig. S2),
consistent with the absence of this metabolic pathway in our experiments.
We conducted growth experiments on E. coli K-12 at constant oxygen
fluxes into our reactor system, making continuous measurements of oxygen
concentrations down to the detection limit of the STOX sensor. We sampled
the media at intervals (generally 1 h) to measure the number of cells per unit
volume from which we determined growth rates. Our experiments were
performed in an all-glass, well-mixed, thermostated reactor sealed with a
butyl rubber stopper. Oxygen was supplied diffusively from air-saturated
water pumped at controlled rates through silicone tubing looping through
the reactor. Details of the experimental setup (Fig. S3), the media used, and
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ACKNOWLEDGMENTS. We thank Sean Crowe, Joost Hoek, Jakob MøllerJensen, Peter Søholt, Preben Sørensen, and Bo Thamdrup for helpful discussions and assistance in the lab. Tim Lyons and Dave Johnston are thanked for
insightful comments on the manuscript. D.A.S. was funded through the
generous support of a Fulbright Fellowship from the American and Danish
governments. Research was funded through the Agouron Institute and
Danmarks Grundforskningsfond.
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INAUGURAL ARTICLE
GEOLOGY
Materials and Methods
other aspects of the experimental protocols are found in SI Text). Growth was
measured by removing aliquots from the reactor, measuring the absorbance
at a wavelength of 450 nm, and converting to cell numbers based on a
working curve of cell concentrations determined by direct cell counts vs.
absorbance.
Through a series of preliminary growth experiments and negative controls, we demonstrated that growth did not occur without a flux of oxygen
into the reactor (Figs. S2 and S4). Several lines of evidence support this:
(i) when a degassed reactor inoculated with E. coli in the growth medium
was not exposed to oxygen, no growth occurred (see SI Text for details;
Fig. S2); (ii) when the flux of oxygen into the reactor was stopped, measurable growth stopped immediately (Fig. S4); and (iii) as demonstrated below,
growth rates are linearly correlated with oxygen flux and have an intercept
through the origin within error. Based on these observations, we conclude
that growth in our experiments was due to aerobic respiration, and we
rule out anaerobic respiration as a measurable source of growth. We also
explored and subsequently rejected the hypothesis that our results were
strongly influenced by biofilm growth on the silicone tubing transporting
oxygen into the reactor or by unobserved high-oxygen microniches (see
SI Text).
In a typical experiment, we inoculated E. coli into our reactor containing
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to the culture. In some experiments, we attempted to regulate oxygen to a
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less than 1 to 30 s, depending on pump rate and O2 concentration.
ECOLOGY
Conclusions
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