Apparent minimum free energy requirements for methanogenic Archaea and sulfate-reducing bacteria in an anoxic marine sediment (original) (raw)

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NASA Ames Research Center, MS 239-4, Moffett Field, CA 94035-1000, USA

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Department of Marine Sciences, CB# 3300, University of North Carolina, Chapel Hill, NC 27599, USA

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Department of Marine Sciences, CB# 3300, University of North Carolina, Chapel Hill, NC 27599, USA

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Department of Marine Sciences, CB# 3300, University of North Carolina, Chapel Hill, NC 27599, USA

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Revision received:

03 September 2001

Accepted:

04 September 2001

Published:

01 December 2001

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Tori M. Hoehler, Marc J. Alperin, Daniel B. Albert, Christopher S. Martens, Apparent minimum free energy requirements for methanogenic Archaea and sulfate-reducing bacteria in an anoxic marine sediment, FEMS Microbiology Ecology, Volume 38, Issue 1, December 2001, Pages 33–41, https://doi.org/10.1111/j.1574-6941.2001.tb00879.x
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Abstract

Among the most fundamental constraints governing the distribution of microorganisms in the environment is the availability of chemical energy at biologically useful levels. To assess the minimum free energy yield that can support microbial metabolism in situ, we examined the thermodynamics of H2-consuming processes in anoxic sediments from Cape Lookout Bight, NC, USA. Depth distributions of H2 partial pressure, along with a suite of relevant concentration data, were determined in sediment cores collected in November (at 14.5°C) and August (at 27°C) and used to calculate free energy yields for methanogenesis and sulfate reduction. At both times of year, and for both processes, free energy yields gradually decreased (became less negative) with depth before reaching an apparent asymptote. Sulfate-reducing bacteria exhibited an asymptote of −19.1±1.7 kJ (mol SO2−4)−1, while methanogenic Archaea were apparently supported by energy yields as small as −10.6±0.7 kJ (mol CH4)−1.

1 Introduction

Currently understood mechanisms of energy conservation require that, in order to be biologically useful, energy must be available at levels not less than one-third to one-fifth of the energy needed to phosphorylate ADP to ATP [1]. The existence of this ‘biological energy quantum’ means that a significant amount of the chemical energy available on Earth cannot be exploited by life. Likewise, the absolute magnitude of the biological energy quantum must be a critical parameter in determining the distribution of microbial life in the environment. This is particularly relevant for oligotrophic settings, such as deep-sea sediments and crustal rocks, which are now thought to potentially harbor a vast ‘deep biosphere’ [2–5].

Extensive research based on organisms in culture provides a detailed understanding of energy conservation in chemotrophic anaerobes [6–10]. These studies, combined with theoretical considerations, suggest a quantum of about −20 kJ mol−1 as the minimum energy that can be exploited by living cells [1]. A clear next step in understanding microbial energy conservation is to examine the apparent energy requirements of microorganisms in natural ecosystems, where they are often obligated to function at the energetic fringe.

This study sought to investigate in situ bioenergetic requirements by monitoring the free energy yields obtained by H2-consuming microorganisms in anoxic marine sediments from Cape Lookout Bight (CLB), NC, USA. In such sediments, H2 is produced via bacterial fermentation of complex organic matter [11,12], and is subsequently consumed by sulfate-reducing bacteria (SRB) or, when sulfate is absent, methane-producing Archaea (MPA) [13–16]:

1

2

The coupling between production and consumption of H2 in CLB is very close, resulting in very low partial pressures and very short residence times for H2[17], as is typical for sedimentary environments [18].

Previous studies have shown that in such closely coupled systems, H2 concentrations are controlled by the H2-consuming organisms in a pattern that reflects the availability of free energy. Specifically, H2-consuming organisms catalyzing more exergonic processes can generally maintain H2 partial pressures at lower levels. This effect has been demonstrated by varying the terminal electron acceptor (e.g. SO2−4 vs. CO2) [17,19,20] or the temperature [21–24], both of which affect the free energy yield of H2 consumption. In CLB sediments, pore water partial pressures of H2 are controlled quantitatively by the thermodynamics of the microbial reactions that consume it, suggesting that bulk phase H2 measurements in this environment directly reflect intracellular bioenergetics [17]. Based on this principle, we utilized measurements of H2 partial pressures in CLB sediment cores to examine the bioenergetics of methanogenesis and sulfate reduction in a functional microbial ecosystem.

2 Methods

2.1 Study site and sampling methodology

Cape Lookout Bight is a 10-m deep back barrier island lagoon located on the coast of North Carolina, USA. High rates of sedimentation [25] and organic carbon loading [26] fuel high rates of respiration, and oxygen is depleted completely within 2 mm of the sediment–water interface [27]. Below this horizon, the sediments are freely sulfidic and free of bioturbation. Sulfate reduction is the dominant terminal electron-accepting process in the upper portion of the sediment column; below the depth of sulfate depletion, which varies seasonally between 8 and 25 cm, methanogenesis is the dominant process [28].

2.2 Thermodynamic calculations

Free energy data presented in this paper are calculated using data from a previously described series of field and laboratory studies [17]. Free energy changes for H2-based sulfate reduction (Δ_G_SR) or methanogenesis (Δ_G_MP) under in situ conditions were calculated from

3

or

4

where [ ] denotes concentration; γ is an activity coefficient; P denotes partial pressure; R is the universal gas constant; T is absolute temperature; and Δ_G_0(T)-SR and Δ_G_0(T)-MP are the standard free energies of reaction for sulfate reduction and methane production, corrected to ambient temperature using the Gibbs function, Δ_G_=Δ_H_−_T_Δ_S_[29]. Standard free energies of formation of products and reactants (used to calculate Δ_G_0SR and Δ_G_0MP) were taken from [29] and [30]. Partial pressures differ from fugacities by less than 0.1% under the P_–_T conditions of our experimental systems [29], and fugacity coefficients are thus neglected in the present treatment. Activity coefficients for HSO−4 and HS− were assumed to be approximately equal and therefore to cancel one another in this treatment.

For all laboratory experiments (‘temperature experiment’ and ‘sulfate experiment’) all relevant concentrations or partial pressures were measured. For the field studies, partial pressures of H2 and CH4 were measured, along with concentrations of sulfate. Concentrations of ∑CO2 and ∑H2S were estimated by averaging published depth profiles taken at the same site, time of year, and temperature. For ∑H2S, we averaged four July/August profiles taken over 3 years and two November profiles taken over 2 years (from [31]); for ∑CO2, we used three July/August profiles taken over 3 years and two November profiles taken over 2 years (from [32] and [33]). Year-to-year variation (±1σ) in ∑CO2 was always less than ±15% at all depths in the sediment column. For ∑H2S, variation was less than ±20% at all depths, except for the upper 3 cm of the sediment column in November, where it was ±74%. Speciation in the ∑CO2, ∑H2S, and ∑H2SO4 systems was calculated based on a measured average pH of 7.2 (which remains approximately constant with depth and temperature in the sediments) and using acidity and Henry's law constants from [29,34].

Errors reported for the free energy values are calculated via propagation of errors, utilizing the standard deviation (±1σ) among replicates (for all measured quantities) and an assumed standard deviation of ±20% for ∑CO2 and ∑H2S in the down-core profiles (see Section 2.2). For the uppermost sample of the November profile, a standard deviation of ±75% for ∑H2S was assumed, per the discussion above.

3 Results and discussion

3.1 Temperature and sulfate concentration experiments

Two previously published experiments demonstrate that H2 partial pressures in CLB sediments are related quantitatively to the bioenergetics of H2-consuming microorganisms [17]. In the first, sulfate-reducing and methanogenic sediments initially at 22°C were subjected to temperature changes over the seasonal range encountered by these sediments in their natural setting (10–30°C). Relative to the 22°C starting point, the temperature increase would change Δ_G_SR or Δ_G_MP (see Eqs. 3 and 4) by about +5 kJ mol−1, and the temperature decrease would change Δ_G_SR or Δ_G_MP by as much as −12 kJ mol−1, if concentrations of all products and reactants remained the same throughout the experiment (Fig. 1). Instead, free energy yields remained essentially constant (±3%) for both treatments: Δ_G_MP=−15.8±0.5 kJ mol−1 in methane-producing sediments and Δ_G_SR=−24.7±0.6 kJ mol−1 under sulfate-reducing conditions (Fig. 1).

1

Variation in free energy yield with temperature in slurried CLB sediments, under sulfate-reducing (A) or methanogenic (B) conditions. Dashed lines represent the temperature-based change in Δ_G_SR or Δ_G_MP that would have occurred if the H2 partial pressure did not change from the levels measured at the initial sediment temperature of 22°C. Plot symbols represent the values of Δ_G_ calculated (per Eqs. 3 or 4) from measured concentrations or partial pressures of H2, H2S, SO2−4, CH4, and ∑CO2. Error bars represent ±1 S.D. as determined by propagation of individual measurement errors as determined on _n_=4–5 replicate sediment samples. Solid lines represent the average free energy yield that was maintained across the range of temperatures: Δ_G_SR=−24.7±0.6 kJ mol−1 and Δ_G_MP=−15.8±0.5 kJ mol−1.

In a second experiment, actively sulfate-reducing sediments were subjected to increasing sulfate concentrations ranging from 5 to 105 mM. Relative to the starting point (5 mM), the increase in sulfate would change Δ_G_SR by up to −7.5 kJ mol−1, if all other product and reactant concentrations remained the same (Fig. 2). Instead, however, Δ_G_SR was held constant (±3%) at −22.8±0.8 kJ mol−1 for all treatments (Fig. 2).

2

Variation in free energy yield with sulfate concentration in slurried CLB sediments, under sulfate-reducing conditions. The dashed line represents the sulfate-based change in Δ_G_SR that would have occurred if the H2 partial pressure did not change from the levels measured at the initial sulfate concentration of 5.9 mM. Plot symbols represent the value of Δ_G_SR calculated (per Eq. 3) from measured concentrations or partial pressures of H2, H2S, and SO2−4. Error bars represent ±1 S.D. as determined by propagation of individual measurement errors as determined on _n_=3 replicate sediment samples. The 105 mM point had only one replicate and the error propagation calculation assumes errors in H2, H2S, and SO2−4 equivalent to the average relative measurement error for the other four sulfate concentrations. The solid line represents the average free energy yield that was maintained across the range of sulfate concentrations: Δ_G_SR=−22.8±0.8 kJ mol−1.

In each case, the maintenance of constant Δ_G_SR or Δ_G_MP results because H2 partial pressures change in a way that is exactly sufficient to offset the would-be energetic changes [17], regardless of whether those changes would tend to increase or decrease Δ_G_. This can be explained if: (1) H2-consuming organisms consistently draw the H2 partial pressure down to as low a level as is physiologically possible. (2) The physiologic limitation on H2 consumers in this system is a bioenergetic one, so that the H2 partial pressure is consistently held at a level corresponding to biologically critical free energy yield.

The second point implies that H2 partial pressures measured in the bulk, extracellular pore waters of CLB sediments can be used to quantitatively assess the minimum energy requirements of methanogens and sulfate reducers in situ. In order to do so, however, it is critical to consider how the spatial distribution of H2-consuming organisms affects the partial pressure of H2 in the bulk pore fluid, for it is this pool of H2 that is sampled by the H2 measurement technique, and that is ultimately used to calculate in situ energy yields.

If H2-consuming organisms are distributed randomly in relation to discrete sources of H2, they must draw their supply of H2 from the bulk pore fluid (Fig. 3A). In order to maintain mass transport into the cell, a gradient in H2 between the cell surface and the bulk pore fluid is required [35]. This would mean that H2 partial pressures measured in the bulk pool are always higher than those at the cell surface, and would consequently always overestimate the actual energy yield obtained by H2-consuming organisms. However, the quantitative relationship between intracellular thermodynamics and extracellular (bulk fluid) H2 partial pressures that is observed in CLB sediments argues against such a random distribution of H2 consumers. In the sulfate concentration experiment, for example, measured H2 partial pressures are exactly described by a rearrangement of Eq. 3 with constant Δ_G_SR[17]:

5

This equation is based on the thermodynamics of reactions occurring inside the sulfate reducer cells. If a gradient in H2 partial pressure existed between the sulfate reducer cell and the bulk pore fluid (as indicated for the random distribution model), it would have to be added to Eq. 5 to accurately predict the measured (bulk pore water) H2 partial pressure.

6

Eq. 6 ceases to be accurate in describing the bulk H2 partial pressures we measured unless Δ_P_H2 is of negligible magnitude.

3

Dependence of measured H2 partial pressure on spatial organization of H2 consumer cells. (A) Random arrangement of H2 consumer cells in relation to H2 sources. To maintain mass transport of H2, consumer cells maintain a gradient in _P_H2 between the bulk extracellular fluid and the intracellular medium. H2 measurements, which sample the bulk extracellular fluid, overestimate the intracellular partial pressure. (B) H2 consumer cells arranged in a network around individual sources of H2. An efficient network would continue to remove H2 down to the minimum biologically useful level, with the remainder escaping to the bulk extracellular fluid. H2 measurements in this fluid would thus reflect exactly the H2 partial pressure in the intracellular medium of the last ‘shell’ of H2-consuming organisms.

An alternative scheme for spatial organization of H2-consuming organisms can account for the observed relationship between intracellular bioenergetics and bulk H2 partial pressures, while still providing for H2 mass transport into the consumer cell. Both criteria are met if H2-consuming organisms are localized around discrete sources of H2 in order to intercept the outgoing flux and reduce it to the minimum biologically useful level (Fig. 3B). Conrad [36] has previously shown that such assemblages may account for a high percentage of H2 cycling in natural ecosystems. For any discrete H2 source, the impetus for continued colonization by H2 consumers would persist until the H2 efflux was reduced to a level that was no longer biologically useful. Assuming a bioenergetic limitation on H2 consumption, as suggested by the experimental data, the H2 partial pressure in the bulk pore fluid outside such a cluster would then be fixed at levels corresponding precisely to the minimum energy requirements of the H2 consumers. In this mechanism, changes in temperature or sulfate concentration would automatically adjust pore water H2 to a partial pressure exactly equivalent to the critical Δ_G_SR or Δ_G_MP, as observed for the experimental treatments.

It is important to note that in the second scenario, measured H2 partial pressures do not necessarily reflect the energetic environment experienced by all, or even most, H2-consuming organisms in the system. Instead, they must reflect the minimum energetic yields that can still drive metabolism of H2 in the outermost ‘shell’ of H2-consuming clusters – potentially only a small fraction of the H2 consuming population, but presumably having the most efficient organisms. With this in mind, we sought to use down-core H2 measurements as a means of probing the in situ minimum energy requirements of methanogens and sulfate reducers in an intact sediment microbial ecosystem.

3.2 Depth profiles of Δ_G_SR and Δ_G_MP in Cape Lookout Bight sediments

Depth distributions of H2 partial pressure in CLB sediments reported in [17], and replotted in Fig. 4, were used to calculate Δ_G_SR and Δ_G_MP for natural populations of sulfate-reducing and methanogenic bacteria. Data are presented for 2 months: August, when warmer temperatures (27.0°C) result in higher sulfate reduction rates and sulfate depletion at shallow depths (∼10 cm; Fig. 4A), and November, when relatively cool temperatures (14.5°C) allow for somewhat deeper sulfate penetration (∼16 cm; Fig. 4C). At both times of year, energy yields for sulfate-reducing bacteria increase (become less favorable) with depth before reaching an apparent asymptote. In August, an average value of −20.3±0.6 kJ mol−1 is reached by 4 cm depth and maintained until the depletion of sulfate at approximately 10 cm (Fig. 4B); under the cooler November conditions, an average of −19±1.8 kJ mol−1 is reached by 7 cm depth and maintained until the sulfate depletion depth of 16 cm (Fig. 4D). For methanogens, Δ_G_MP values were generally greater than zero when the sediments contained sulfate, consistent with H2-based competitive exclusion of methanogenesis by sulfate reducers [15,16]. Below the sulfate depletion depth (where methanogenesis becomes active [23]) Δ_G_MP values initially increase (become less favorable) with depth and then become approximately constant below 25 cm at −10.9±0.3 kJ mol−1 (August; Fig. 4B) and −10.4±0.8 kJ mol−1 (November; Fig. 4D). Interestingly, this 25 cm ‘inflection point’ marks the horizon above which the methanogenic community is seasonally disrupted by infiltration of sulfate (above 25 cm), but below which the community can adapt to permanently methanogenic conditions.

4

Down-core profiles of in situ concentrations and free energy yields in sediment cores from Cape Lookout Bight. (A,C) Concentrations of H2 (○) and sulfate (●) in sediment cores taken in August (27.0°C) and November (14.5°C), respectively, as redrawn from [17]. Error bars represent 1 S.D. about the mean of n_=3 replicate sediment samples. (B,D) Δ_G_MP (○) and Δ_G_SR (●) in August and November, respectively, as calculated from the concentrations in A and C, along with measured partial pressures of CH4 and estimated concentrations of ∑H2S and ∑CO2 (see Section 2). Error bars reflect the standard errors of measurement from A and C, along with estimated errors for ∑H2S and ∑CO2 (see Section 2), propagated through the Δ_G calculation. The horizontal dashed lines in each profile represent the approximate depth of transition from sulfate-reducing to methanogenic conditions.

Sulfate reducers in CLB sediments thus function with energy yields close to the biologically useful minimum of −20 kJ mol−1 that is suggested by culture-based studies [1]. Methanogens in these sediments can apparently support metabolism of H2 with energy yields as low as −10 kJ mol−1. This value is significantly lower than has been calculated for several other environments [37], but is comparable, in general, to the findings of Westermann [24]. In support of the low energy yields observed for H2-based methanogenesis, we calculate from pore water acetate concentrations [38] that acetoclastic methanogenesis in CLB sediments proceeds at very similar levels, ranging from Δ_G_MP(acetate)=−12.8 kJ mol−1 (in the summer) to −10.5 kJ mol−1 (in the winter).

3.3 The critical free energy

The very low energy yields observed for methanogens in CLB sediments, relative to organisms in culture, are likely the result of adaptation by an intact microbial community to long-term substrate limitation. In CLB sediments, the quantity of reactive organic matter (and thus, the rate of fermentative H2 production) decreases in roughly exponential fashion with depth, corresponding to age of the sediment [26]. Therefore, after colonizing the sediment to an extent that is commensurate with the initial amount of organic matter, bacteria are faced with an ever-decreasing supply of substrate. The community as a whole must adapt for years afterwards to starvation level conditions. This is reflected in each of the down-core free energy profiles (Fig. 4B,D) by initially more negative values of Δ_G_SR and Δ_G_MP that only approach asymptotic levels at depths representing many months of sediment accumulation.

A number of environmental and theoretical studies suggest that anaerobic organisms in complex natural ecosystems may be obligated to function with variable free energy yields significantly below −20 kJ mol−1 [39–41]. ATP synthesis might still be possible at free energy yields lower than −20 kJ mol−1 given sufficient variability in the parameters that ultimately determine the biological energy quantum, Δ_G_min[42]:

7

n, Δ_G_ADP→ATP, and f are defined as follows:

1. n is the stoichiometry of ions translocated by ATP synthase in chemiosmotic energy-conserving systems. n is usually taken as three ions per ATP, but may range to four or possibly five (see review in [10]). A higher value of n could reduce Δ_G_min to −15 kJ mol−1 or even lower.
2. Δ_G_ADP→ATP is the energy required for phosphorylation of ADP in vivo. This quantity depends directly on the intracellular ratio of ATP to ADP, which is about 10:1 in actively growing cells [6]. However, if the free energy available from the energy-harvesting metabolism (e.g. sulfate reduction or methanogenesis) is insufficient to catalyze ADP phosphorylation, ADP will quickly accumulate as cellular metabolism continues to hydrolyze ATP. This would lower the ATP:ADP ratio until ADP phosphorylation again becomes favorable. Every factor of 10 decrease in the ATP:ADP ratio decreases the overall energy requirement by about 5 kJ mol−1, potentially allowing organisms to capitalize on lower in situ free energy changes simply by maintaining a lower ‘energy charge’. Tran and Unden [43] demonstrated exactly such an effect for cultures of Escherichia coli. ATP:ADP ratios during stationary phase were typically much lower than during the exponential growth phase, resulting in a decrease of 5–6 kJ mol−1 in Δ_G_ADP→ATP. Under their experimental conditions, Tran and Unden found that ADP phosphorylation proceeded at energies as low as 42 kJ mol−1. Together, the values of n and Δ_G_ADP→ATP specify the minimum energy required to ‘store’ one proton for use by ATP synthase. For example, with values of _n_=4 and Δ_G_ADP→ATP=+42 kJ mol−1, proton storage could be driven by an energy of −10.5 kJ mol−1.
3. f, the thermodynamic efficiency factor, is the fraction of total available free energy that is actually conserved via proton storage. The energy-harvesting metabolism of microorganisms frequently occurs via multiple reaction steps, not all of which are coupled to energy-conserving mechanisms (e.g. ion gradient formation). Any of these non-coupled reactions which operate at a disequilibrium (as is required for a reaction to proceed in a net forward direction) have an associated free energy change, Δ_G_<0, which is lost from the system as heat. In actively growing anaerobic cultures, up to 60% of the available free energy may be lost in this way [6]. An organism would therefore have to extract about −26 kJ mol−1 from its environment in order to store a ‘10.5 kJ mol−1 proton’. However, when the availability of ‘conservable free energy’ (that which is associated with ion gradient-forming reactions) becomes limiting, ATP synthesis will become the rate-limiting step in the overall energy-harvesting metabolism. Rate limitation at this final step would cause accumulation of the products in each of the non-coupled reactions, and a corresponding decrease in forward reaction rate, thereby bringing them ever closer to equilibrium (Δ_G_=0). This approach to equilibrium would continue until the energy-coupled reactions garnered a sufficient fraction of the overall free energy (sufficiently high f) to resume ATP synthesis. For values approaching _f_=1, an organism could use essentially all of the free energy it extracts from the environment in the process of energy conservation, and the value of Δ_G_min would closely approximate the energy required for storage of one proton. A level of efficiency close to _f_=1 would be required if the apparent Δ_G_min we measure for methanogens in CLB sediments (approx. −10.6 kJ mol−1) were to be sufficient to store a proton given the parameters described above (although it is not clear that the energy required for proton storage might not be somewhat less than the value of −10.5 kJ mol−1 suggested above, since the parameters presented still relate to organisms in culture, rather than in an energy-starved ecosystem).

Finally, it is critical to mention that, in order to tap such low energy yields for energy conservation, an organism would have to direct all of the available environmental free energy into one and only one proton-storing reaction. Some organisms have multiple sites for ion gradient formation and the available free energy is split among these. This would increase the Δ_G_min to a multiple (equal to the total number of energy conserving modes) of the energy required to store a single proton. For the reasons described in (3) above, however, energy-limiting conditions might effectively turn off ion gradient formation at secondary sites, restricting it to a single active process. The specifics of how a given organism's metabolism is coupled to ion gradient formation and utilization could conceivably form a basis for significant differences in minimum energy requirements among different strains or functional groups.

The theorized biological energy quantum of −20 kJ mol−1 is based on values of _n_=3, Δ_G_ADP→ATP=+50 kJ mol−1 (ATP:ADP=10), and _f_≅0.8, as measured for cells in actively growing cultures [1]. However, the population for which we calculate an in situ Δ_G_min is almost certainly not actively growing; rather, it is likely functioning at the bare minimum required to sustain metabolic turnover of H2. As described above, the value of each parameter used in determining Δ_G_min may vary significantly, particularly for non-growing or energy-starved populations. The difference between the culture-based and in situ Δ_G_min values might thus reflect an optimization of energy-conserving efficiency by organisms in a substrate-limited natural ecosystem.

It is important to point out that ‘optimization’, in the sense it is employed above, implies a system in which the energy-conserving (ATP-producing) metabolism is approaching equilibrium with respect to the catabolic (ATP-utilizing) processes. In some respects, this can be considered an inevitable consequence of chemistry. If the free energy available in the environment is less than the amount required in ATP-driven catabolic reactions, every reaction involved in ATP formation will inescapably relax any associated disequilibrium until it reaches Δ_G_=0. At this point, of course, the organisms would cease to function in any net sense. At the other end of the spectrum, under conditions of active growth (rapid catabolism and ATP expenditure), organisms sacrifice (lose to heat) large quantities of free energy in order to ensure rapid forward progress in metabolic processes. Somewhere in the middle lie cells that are simply maintaining themselves with a bare minimum of ATP expenditure. Does this allow them to become more efficient with respect to energy conservation, and does the rate of ATP cycling in the cell scale to the degree of thermodynamic efficiency that is possible (or vice versa)? How efficient can an organism be in energy conservation before its rate of ATP cycling drops below the critical minimum required for cell maintenance? Given appropriately high rate constants for the relevant energy-conserving reactions, it is quite possible to maintain a significant net production of ATP with an energy-conserving apparatus that is operating very close to thermodynamic equilibrium (i.e. with a very high level of efficiency). Such critical issues may well lie at the heart of understanding the nature of microbial processes in oligotrophic settings.

The possible existence of a gap between the minimum energy required for growth and that required simply for sustained metabolic activity carries critical significant implications for understanding the distribution and function of microorganisms in the natural world. Clearly, estimates of growth energy requirements are needed to address the potential for microbial colonization of oligotrophic environments. Once established, however, microbial populations might persist and sustain metabolism for long periods of time with much smaller energy yields. Integrated over the lifetime of a given ecosystem, the chemistry catalyzed during sustained metabolism likely makes a much greater contribution to global biogeochemistry than the chemistry catalyzed during the brief initial growth phase of the population. Similarly, environmental microbiology studies in natural ecosystems may frequently encounter populations in ‘maintenance mode’, rather than growing ones. In both cases, an understanding of energy metabolism at maintenance levels is critical.

This point is illustrated by anaerobic methane oxidation (AMO). This process is widespread in the marine environment and significant in global geochemistry [44], but the responsible microbial agents have not been successfully cultured. Most recent evidence implicates a two-member microbial consortium consisting of an archaeal (methanogenic?) and a sulfate-reducing partner [23,45–50]. The total energy available from AMO under most environmental conditions would generally be insufficient to support two organisms with a −20 kJ mol−1 growth energy requirement [1]. However, biological catalysis of AMO by an established consortium would carry a significantly lower energy requirement. For example, AMO actively occurs in CLB sediments at H2 partial pressures that translate to energy yields of −13.5±1.0 kJ mol−1 for the methanogenic partner [23] and −18.8±1.2 kJ mol−1 for the sulfate-reducing partner – meeting the apparent in situ free energy requirements of both organisms.

How many other environmentally relevant processes might function in the region between the energy quantum observed in actively growing cultures and the apparent critical free energies required for intact communities in situ? The answer to this question gains added significance when considering that the function of >99% of the microorganisms on Earth remains uncharacterized from the standpoint of culture-based studies [51]. Establishing the magnitude of the gap, if any, between critical growth energies and critical survival energies may be an important step in understanding the distribution and nature of microbial life in oligotrophic environments.

Acknowledgements

The authors are grateful to B. Schink, D. Des Marais, B. Bebout, and members of the NASA-Ames EMERG group for comments and discussion on an early draft of this manuscript. The work was supported by NSF grants OCE 92-17570 and OCE 96-33465. T.M.H. was supported by NDSEG, Royster, and NRC fellowships.

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