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J. Jpn. Soc. Soil Phys.

ゎࠉㄝ

土壌の物理性LecturesNo. 138, p.27 ∼ 35 (2018)

Subsurface microbial activity:
Beyond the thermodynamic ladder
Craig M. BETHKE1 and Robert A. SANFORD1

Abstract: An integration of the study of groundwaterchemistry, numerical modeling, long-term bioreactor experiments, and in-situ sampling of activity growing microbes yields a portrait of subsurface anaerobe activity thatdiffers sharply from conventional thinking. The chemical record of microbial activity on a groundwater’s redox
chemistry in a well-studied aquifer is surprisingly incomplete, casting doubt on current methods of classifying subsurface microbial activity in aquifers. We find no evidence
to support a concept by which subsurface microbial activity is arranged according to a thermodynamic ladder, nor
do we find backing for the theory of competitive exclusion. Instead, we find strong evidence that iron reducing and sulfate reducing bacteria, previously consideredlocked in competition, are in fact forming mutualistic relationships in which they tightly cooperate. Where a groupof microbes such as the methanogens is excluded from anecosystem, the explanation is more likely ecologic thanthermodynamic.
Key Words : Anaerobic microbes, microbial activity, microbial ecology, aquifer redox chemistry

1. Introduction
Subsurface microorganisms draw the energy they needto live and reproduce from the groundwater in which they
live, and in the process control many aspects of the water’s
redox chemistry. To capture energy from its environment, amicrobe catalyzes the transfer of electrons from an initiallyreduced species to an initially oxidized species, releasing
the electron donor in oxidized from, and the acceptor in
reduced form. In this way, the microbe’s activity leaves animprint on the chemistry of its environment.
In anoxic aquifers, the subject of this paper, groundwater can in many cases be divided into zones on the basisof chemical composition. By conventional thinking, thesegregation has been interpreted to reflect variation or zoning in the activity of anaerobic microbes within the aquifer
Department of Geology, University of Illinois, 1301 W. Green St. Urbana,
Illinois 61801 USA.
2018 年 1 月 16 日受稿

2018 年 3 月 1 日受理(Jones et al., 1989). On a regional scale, for example, theCretaceous Middendorf aquifer in South Carolina can bedivided into three parts, one rich in dissolved iron, a second where groundwater holds little iron, and a third where
methane accumulates in the flowing water.
The three chemical zones within the Middendorf are believed to represent the habitats, respectively, of iron reducing bacteria, sulfate reducing bacteria, and methanogens(Chapelle and Lovley, 1992). On a local scale, as a second
example, arsenic concentration in the Cretaceous Mahomet
aquifer in East Central Illinois was found to be controlled
by the activity of sulfate reducing bacteria: where sulfate
reducers are active, the arsenic concentration is invariably
small (Kirk et al., 2004).
Chemical zoning in an aquifer, in other words, signifies the differentiation of microbial activity into discretezones. The zones are commonly taken to be exclusionary,so that each is dominated by a single functional group of
microbes, according to an energetic hierarchy known as thethermodynamic ladder. In this paper, we use the results ofmore than a decade of studies within our research group— ranging from field observation to long-term laboratory
experiments to theory and quantitative modeling — to reflect on the role of thermodynamics in shaping microbial
metabolic activity in subsurface environments.

2. Concept of a thermodynamic ladder
The idea that an energetic hierarchy among functionalgroups of microbes might cause aquifers to become
zoned microbiologically arose from observations in the
Cretaceous Middendorf aquifer in South Carolina, USA(Chapelle and Lovley, 1992). An upgradient zone there
holding groundwater rich in iron is replaced along the direction of flow by a zone with low iron levels. The latterzone, in turn, gives way downgradient to a zone where dissolved methane builds up.
The three zones are believed to represent the dominancealong flow of first bacterial iron reduction, then sulfate re-

土壌の物理性

28

10

HCO3 (low δ 13C)
5

Fe2+ + H2S“electron
gap”

0
0

50

(2018)

accumulating in the flowing water, then, records the electrons donated up to that point in the flow, and the buildup
of sulfide and ferrous iron reflects how the electrons were
accepted.

High Fe
Low FeElectron Equivalent (mM)

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第 138 号

100

Distance (km)Fig. 1 The “electron gap” is the discrepancy between therecord of the number of electrons donated, as reflected by
the accumulation of biogenic inorganic carbon in groundwater along the Middendorf aquifer, and evidence of thenumber of electrons acccepted, as accounted for by ferrous
iron and sulfide in solution (Park et al., 2006). Vertical line
separates zones of iron-rich and iron-poor groundwater.

duction, and finally methanogenesis. Where ferric minerals exist, at neutral to acidic pH, an iron reducer’s catabolic
reaction is favored energetically relative to those of sulfate
reducers and methanogens. Iron reduction, then, might beable to drive down electron donor concentrations (i.e., thelevels of H2 and small-molecule organic acids) to a point
low enough that insufficient energy remains in the aquiferfor sulfate reducers or methanogens to live. Similarly, sulfate reducers might employ the same strategy, known ascompetitive exclusion, to exclude methanogens.
When Park et al. (2006) looked at the redox chemistry ofgroundwater from the Middendorf aquifer, however, theyfound little evidence to support this concept. Along aflow path spanning the iron-rich and iron-poor zones, theyfound enough energy everywhere in the aquifer to drive
forward the catabolic reactions of iron-reducers, sulfate reducers, and aceticlastic methanogens. This result calls into
question the idea that thermodynamic exclusion might playa role in the aquifer’s zoning.
Park et al. furthermore attempted to balance the recordof electron donation in the aquifer with evidence of electron acceptance. As natural organic matter in the sedimentary section ferments, it liberates reduced species such asacetate that can donate electrons for microbial respiration.
In this way, four electrons are made available for each carbon that oxidizes to carbonate. The electrons may then be
accepted by sulfate reduction, which adds one sulfide tosolution per eight electrons accepted, and by iron reduction, which yields an Fe2+ for each electron. Biogenic dissolved carbonate (carbonate with isotopically light carbon)

Plotted in terms of electron equivalents (Fig. 1), thechemistry of Middendorf groundwater shows a pronounced “electron gap” across which electrons donated in
the aquifer cannot be accounted for by evidence of acceptance: there is not nearly enough iron or sulfide in the water
to balance the biogenic carbonate. We see that groundwater chemistry reveals an incomplete picture of the microbial processes occurring within an aquifer; it cannot be relied upon to characterize the microbial activity there, nor
to argue that one functional group of microbes excludes, oreven dominates other groups.

3. Thermodynamic control
on microbial kinetics
Any evaluation of the role of energetics in the originof aquifer zoning requires a thermodynamically consistentdescription of microbial kinetics. Rate laws in common
use are derived considering only the forward rate of reaction and therefore cannot account for a thermodynamic
drive or an equilibrium end point. Such laws may be applied to experiments where energy is available in abundance and the forward reaction rate overwhelms the reverse, but are not suitable for analysis of natural environments in which little chemical energy may be available.
Jin and Bethke (2007) derived a kinetic description of
microbial metabolism that rigorously honors the requirements of thermodynamics. In their analysis, electron transfer through a microbe’s transport chain is enzymaticallycoupled to the phosphorylation of ADP, adenosine diphosphate, to create ATP, adenosine triphosphate. The energy∆GU usable by the cell to drive forward its catabolic reaction is given∆GU = ∆GA − m∆GP

(1)Here, ∆GA is energy available in the environment to drive
forward the cell’s electron transfer reaction, or the negativeof the reaction’s free energy change at ambient conditions,such as −∆Gr of sulfate reduction coupled to acetate oxidation. Variable m is the ATP number, the number of ATPs
produced per turnover of the cell’s catabolic reaction; and∆GP is the free energy change of ADP phosphorylation,
about 50 kJ mol−1 .
A thermodynamic potential factor FT defines the control
of energetics on reaction rate. The factor, ranging fromzero to one, is given as from the environment’s usable energy ∆GU asBiomass (mg kg-1 )Sulﬁde20

A notable point is that in an oligotrophic environment
where available energy ∆GA is little more than the cell’sinternal energy m·∆GP , the usable energy ∆GU approaches
zero, driving FT and the reaction rate r to approach zero,
as well. The point at which available and internal energy
for a microbial catabolism are in balance, in other words,
represents the catabolism’s thermodynamic limit.

50
10

Biomass
SulfateAcetate

0

0

60

ΔGA

30

ΔGU

0
0

5

10

29

question, and on which species in those reactions have concentrations in the microbe’s environment that might varyover time (see, for example, Jin and Bethke, 2002; 2003) .

100

DG (kJ (mol SO4 ) -1)

Concentra#on (mmolal)

解説：Subsurface microbial activity: Beyond the thermodynamic ladder25

Time (days)
Fig. 2 Result of a batch reactor experiment tracing bacterial sulfate reduction in pure culture.
The
Desulfobacter hydrogenophilus bacteria transform sulfate
and acetate to sulfide and carbonate, using the energy liberated to create biomass (Jin and Bethke, 2009). Symbols
show measured concentrations (top plot) and the available∆GA and usable ∆GU free energies in the experiment calculated from the measured concentrations (bottom plot), asfunctions of time elapsed. Lines show the result of a reaction model of the experiment calculated using The GeoR
chemist’s Workbench⃝
software (Bethke, 2008). Reaction in the experiment ceases when the usable energy, andhence the thermodynamic drive vanishes, even though finite amounts of sulfate and acetate remain unreacted andthe available energy is not exhausted.

Jin and Bethke (2009) set out to test the prediction ofa thermodynamic limit for the case of bacterial sulfate reduction. In a series of pure culture experiments, they foundacetotrophic sulfate reduction proceeded until available energy ∆GA dropped to ∼33 – 43 kJ (mol SO4 )−1 and thenstopped (Fig. 2), even though a finite amount of both acetate and sulfate remained unreacted. They further calculated available energy in a variety of natural environments
in which previous studies had shown sulfate reduction tohave ceased. In these cases, the available energy remaining was ∼40 – 56 kJ (mol SO4 )−1 . Energies in these ranges
map by Eq. (1) to ATP numbers m of about 2/3 – 1, inagreement with current understanding of the cellular physiology of sulfate reducers (Jin and Bethke, 2009).

4. Reactive transport modeling(3)

Bethke et al. (2008) conducted a series of numericalexperiments∗ to see how microbial communities might develop in the subsurface. In their reactive transport models, water containing a small amount of dissolved sulfate
recharges a 200-km-long aquifer. Acetate is added along
the aquifer at a slow, continuous rate, to represent the effectof natural organic matter fermenting within surrounding
fine-grained sediments. In some experiments, the aquifer
contains small amounts of goethite (ferric oxyhydroxide,
FeOOH). The aquifer in the simulations is seeded with a
few aceticlastic methanogens and a small number of sulfate
reducing and iron reducing bacteria. The initially small
populations can grow over the course of a run to colonize
the aquifer and differentiate to create a microbial community.

Here, r is the net reaction rate, the difference between theforward and reverse rates; k+ is the rate constant, which iscommonly known from experiments; [X] is biomass concentration; and FD and FA are kinetic terms related to electron acceptance and donation, the forms of which vary depending on the microbe in question, and its environment.
The forms of FD and FT depend on the natures of the electron donating and accepting reactions for the microbe in

Figure 3 shows the result of such a simulation containing no ferric iron, so only aceticlastic methanogens andacetotrophic sulfate reducers can grow, after the model has
run to a steady state at which the rates of microbial growthand decay are in balance. Dissolved sulfate decreases inconcentration as water flows away from the recharge point,
because sulfate reducers use the molecule as an electron
acceptor. Much of the acetate added to the aquifer is consumed as the electron donor for the bacteria; the remaining)(∆GU
FT = 1 − exp −
χ RT

(2)

Here χ is the average stoichiometric number, the numberof times the rate limiting step occurs per reaction turnover;
R is the gas constant; and T is absolute temperature.
The rate at which a microbe catalyzes its catabolic reaction varies with FT according tor = k+ [X] FD FA FT∗ The input files used to create the varous numerical models cited in this paper are available from repositories, as noted in original publications cited.

土壌の物理性

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Conc. (μmolal)
Biomass (μg kg–1)

CH3COO–

.1
2

mAc ≥
SO4 reducers

1

Methanogens

0
1

FT

CH4(aq)10

1

Methanogens
SO4 reducers

.5

flow
0

0

(2018)The inability of methanogens to establish a stable community is instead an ecologic phenomenon. The requirement for maintaining a population is that the rate of cell
growth must meet or exceed that of decay. Bethke et al.
(2008) showed that for aceticlastic methanogens, this condition demands that the acetate concentration mAc satisfy
the inequality

100

SO42 –

第 138 号

50

100

150

200

Length (km)Fig. 3 Long-term solution to a reactive transport model inwhich sulfate reducers and aceticlastic methanogens within
an aquifer compete for acetate (Bethke et al., 2008). Sulfate
is present in the recharge water and acetate is supplied by
the fermentation of natural organic matter in surrounding
sediments. Diagrams show concentration of dissolved sulfate, acetate, and methane (top); biomass of the two functional groups of microbes (middle); and thermodynamic potential factor FT for each (bottom).

mass accumulates along the direction of flow. The aceticlastic methanogens seeded in this upgradient portion of
the aquifer, rather than growing into a population, have
died, leaving a monoculture of sulfate reducers.
A little more than 100 km along the aquifer, acetatebuilds up to a level sufficient for aceticlastic methanogens
to form a stable population. Past this point, methane accumulates in the flowing groundwater. This downgradientzone is colonized by a mixed community of methanogens
and a lesser population of sulfate reducers.
At first glance, sulfate reducers might appear to dominate the upgradient zone by competitive exclusion, but
the actual mechanism of exclusion is more interesting. As
shown in Figure 3, the thermodynamic potential factor FT
for methanogens is close to unity, whereas FT for the sulfate reducers is only about 0.7. Availability of usable energy, in other words, provides no impediment to the growthof methanogens, which are in fact thermodynamically favored over sulfate reducers. Thermodynamics, then, plays
no role in the exclusion of methanogens in this zone.

D KD·
Y k+ FT(4)

Here, Y is growth yield, KD is the aceticlasts’ half saturation constant, and k+ is the rate constant; each of thesevalues is determined experimentally. The decay constant
D appropriate for a given natural environment can be estimated from Y and the biomass concentration [X] there byassuming the population is in steady state (Bethke et al.,2008).
Methanogens in general grow slowly, have a small
growth yield Y , and with respect to acetate are associatedwith large half-saturation constants KD . As a result, theecologic criterion for maintaining a population, as given
by Eq. (4), is a relatively large acetate concentration mAc .
The ecologic minimum concentration, notably, is roughlya factor of 30 larger than the thermodynamic limit given
by Eq. (1). Simply put, aceticlastic methanogens have sufficient energy to grow everywhere in the simulation, butcannot establish a population until acetate accumulates toa level that allows cells to be replaced as quickly as theydie. Failing to replace the cells that die, the populationenters an extinction vortex and vanishes.
In a related simulation (Fig. 4), the upgradient third of
the aquifer contains goethite, allowing iron reducers togrow there. This portion of the aquifer becomes colonized
by a mixed community of a predominate population of sulfate reducers and a lesser population of iron reducers. The
community produces both sulfide and ferrous iron, so themineral mackinawite (FeS) can form according toFe2+ + HS− → FeS(s) + H+

(5)

The concentration of Fe2+ in the upgradient zone increases
along the direction of flow, because the microbial community contributes more ferrous iron to solution than sulfide.
The increment of Fe2+ in excess of sulfide cannot be taken
up by mackinawite precipitation and hence builds up in theflowing groundwater.
It is notable that ferrous iron rather than sulfide builds
up in solution, even though the iron reducing population issubordinate to the population of sulfate reducers, in termsof both biomass and reaction rate (Fig. 4). The accumulation of ferrous iron is due to the fact that iron reducers
liberate eight Fe2+ ions

解説：Subsurface microbial activity: Beyond the thermodynamic ladder
FeOOH

10

Fe2+

fluid contained sulfate but the sediment held no ferric iron,a second in which the feed lacked sulfate but the sediment
was amended with goethite, and a third that included both
sulfate in the fluid and goethite in the sediment.

CH4(aq)2–

SO4

H2S(aq)Methanogenesis

Bethke et al. (2011) analyzed the rates of sulfate reduction, iron reduction, and methanogenesis in the experiments, as well as the usable energy available to the threefunctional groups of microorganisms. The latter experiment, in which sulfate reduction and iron reduction canoccur together, is of particular interest to us. As shown in
Figure 5, sulfate concentration in the fluid decreased gradually for about the first 100 days, and acetate decreased inabout two months to below detection, at which point thesole source of electron donor was the feed fluid.

SO4 reducers

There are several notable aspects to the results (Fig. 5).
First, despite the advantage in available energy that iron
reducers hold over sulfate reducers at neutral to acidic pH,
sulfate reduction occurs 6 to 9 times more rapidly in theexperiment than iron reduction. Second, iron reduction oc-

CH3COO–

1

15
10

SO4 reduction

5

Fe reduction

0
2

1

Methanogens

Fe reducers
flow

0

0

50

100

150

200

Length (km)Fig. 4 Results of a reactive transport similar to Fig. 3, except that goethite (FeOOH) is present in sediments within
the upgradient third of the aquifer (Bethke et al., 2008). Diagrams show dissolved concentration (top), reaction rates
on a per acetate basis (middle), and biomass of each functional group (bottom).

CH3 COO− +8 FeOOH(s) + 15 H+
2+→ 2 HCO−+ 12 H2 O
3 + 8 Fe(6)

per acetate consumed, whereas sulfate reducers+−
CH3 COO− + SO2−
4 + H → 2 HCO3 + H2 S(aq)(7)

contribute a single sulfide. A groundwater high in iron, significantly, cannot be taken as evidence that a population ofiron reducers dominates sulfate reducers in the subsurface
community.

5. Bioreactor experiments
In a series of long-term (∼300 days) experiments insemi-continuous flow bioreactors (Kirk et al., 2010), a natural consortium containing iron reducing bacteria, sulfate
reducing bacteria, and methanogens was allowed to growin the presence of natural aquifer sediments, while fluid
containing nutrients was added and reacted fluid removed.
They conducted three experiments: one in which the feed

Concentration (μmol kg –1)

Biomass (μg kg–1)

Rate (nmolal yr –1)

0.1

Rate (μmol Ac kg –1 day–1)

Conc. (μmolal)

100

31

1000

■■
■
■

■
■

800

■

●

●

600

●

400
200
0

Ac

●

Fe2+

▲
●▲▲▲▲▲ ▲▲▲▲▲▲▲▲▲▲●●●●●●●●●●●●●●●●●●●●●●●●●▲▲▲ ▲▲▲ ▲▲▲▲▲
▲▲▲
▲

50

●

Ac consumption

40●●●●●●●●●●●●●●●●●●●●●●●●
●

30
●

20
10
0

SO2–
4

■
■
■
■
■
■
■■
■■■
■■ ■
■■■
■
■■■■ ■
■

●

●
●
●

■
■
■■
■ ■■ ■■
■
■
■
■ ■
■
■
■■
■ ■
■■■
■
■

■

SO4 reduction■

FeOOH reduction■▲■▲▲▲▲▲▲▲▲▲▲ ▲ ▲▲▲▲▲▲▲▲▲▲▲▲
▲
▲
■
▲
▲▲▲▲
■

0

100

FeOOH
only

200

300

Time elapsed (days)
Fig. 5 Results of a long-term experimental study of microbial growth in a semi-continuous flow reactor conductedby Kirk et al. (2010). Sediment, along with its natural microbial community, and goethite (FeOOH) were allowed toreact over time with aliquots of water containing dissolved
sulfate and nutrients, as equal volumes of reacted water
were removed. Diagrams show concentration of sulfate, acetate (Ac), and ferrous iron in water periodically withdrawn
from the reactor (top) and rates on a per acetate basis ofsulfate reduction and iron reduction, and the rate acetate isconsumed by the microbial community (bottom). Solid line
at base of bottom diagram shows rate of iron reduction in aparallel experiment that contained goethite, but not sulfate.

土壌の物理性

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第 138 号(2018)

curs about 14 times more rapidly in this experiment thanit did in the second experiment in the series, which contained goethite but no sulfate. In other words, when theiron reducers competed with sulfate reducers for acetate,
they respired the acetate considerably more rapidly thanthey did in the absence of competition.

of the activity may in fact be recorded. Aquifer sediments
may also be retrieved and the attached microbial community analyzed by molecular methods, but the whole community does not necessarily reflect microbial activity in theaquifer, because an unknown fraction of microbes may bedormant, or nearly so.

Third, and perhaps most notably, it is clear the iron reducers and sulfate reducers enter into a tight mutualistic
relationship in which each functional group benefits theother. This point is demonstrated in Figure 6, which showsthat the community adjusts after about one month to a condition in which sulfate reduction on a per-acetate basis is
close to eight times as rapid as iron reduction. Since a sulfate reducer produces one sulfide ion per acetate, whereas
an iron reducer produces eight Fe2+ ions, this ratio of reaction rates represents the point at which sulfide and ferrous
iron are added to solution at the same rate, which by Reaction (5) is the stoichiometric ratio of mackinawite precipitation.

To address these problems, Flynn et al. (2012) undertook to measure microbial activity in the Pleistocene Mahomet aquifer in East Central Illinois, USA, using an in-

Bethke et al. (2011) suggested that iron reducers operating in isolation produce Fe2+ ions that sorb
≡ FeOH + Fe2+ → ≡ FeOFe+ + H+

(8)

onto the ferric surface, diminishing the its ability to accept
electrons. Here, ≡ FeOH and ≡ FeOFe+ represent uncomplexed and iron-complexed sites, respectively, on theferric surface. An iron reducer producing one Fe2+ foreach sulfide ion producing by a sulfate reducer, however,would likely precipitate mackinawite according to Reaction (5), thereby removing the Fe2+ from solution before it
can sorb on the ferric surface by Reaction (8). The sulfate
reducers, likewise, avoid by this relationship a buildup of
dissolved sulfide, which might lead to product inhibitionof its catabolic reaction.

6
5

rFeR

4

8 rSR 3
Excess Fe2+

2
1

HS˗

0
0

50

100

150

200

250

Time (days)

Fig. 6 Balance between iron reduction and sulfate reduction in the long-term flow-through reactor experiment conducted by Kirk et al. (2010), shown in Fig. 5, from Bethke
et al. (2011). Bethke et al. (2011) calculated the rates ofiron and sulfate reduction in the experiment from mass balance. When on a per-acetate basis the rate of iron reductionin the results equals eight times the rate of sulfate reduction,
the microbial community generates Fe2+ and dissolved sulfide in an equal molar ratio, which is the stoichiometric ratio
for mackinawite precipitation. After about one month, therates approach the 1 : 8 ratio and maintain it through the remainder of the experiment. In this way, the two groups of
microbes demonstrate mutualistic rather than competitive
behavior.

Each of the functional groups, rather than competing andattempting to exclude the other, as suggested by the concept of a thermodynamic ladder, is in fact deriving suchsignificant benefit from the other that the two catabolic reactions proceed in lockstep. The mutualistic relationshipgoes a long way toward explaining why iron reducers work
more rapidly in the presence of sulfate reducers than in
their absence.

6. In-situ sampling
Determining the nature of microbial activity in an
aquifer is most commonly an indirect process. Activity
may be interpreted on the basis of the chemical evolutionof groundwater, assuming the water maintains a record of
past microbial activity. As discussed above, however, little

Fig. 7 In-situ sampler, or “bug trap”, used by Flynn et al.
(2012) to retrieve the actively growing microbial community from wells completed in the Mahomet aquifer. Washed,autoclaved sediment from the aquifer is enclosed in a meshbag, suspended on a fishing line, and lowered to screen levelwithin the well; after a period of months, the sampler is retrieved and the biomass attached to the sediment analyzed.

解説：Subsurface microbial activity: Beyond the thermodynamic laddersitu sampling method. The authors suspended mesh bagsof autoclaved aquifer sediment (Fig. 7) at the screens of 18
wells, so that ambient groundwater flowed across the “bugtraps”. After about 14 weeks, they retrieved the initiallysterile traps and used molecular techniques to analyze themicrobial community that had grown up attached to thesediment within them. In this way, they analyzed only active microbes and hence presumably derived a more direct
characterization of microbial activity in the aquifer thanmight be possible by other techniques.
The Mahomet aquifer holds groundwater rich in dissolved iron, but poor in sulfide. As such, the aquifer falls
squarely within the iron-reducing category, as proposed by
Chapelle et al. (2009). Figure 8 shows the molecular analysis of bacteria retrieved from the bug traps, broken down
by groundwater sulfate concentration. Even though iron
reducers might be expected to dominate the community,
genera of sulfate-reducing bacteria make up a strikingly
significant fraction of the actively growing biomass sampled from the Mahomet. In wells with more than a negligible concentration of sulfate (> 0.03 mM), sulfate reducing bacteria were about equally numerous as iron reducing
bacteria. Even at wells where water is nearly devoid of sulfate, sulfate reducers make up a significant fraction of theBacterial Taxa
Bacteroidetes

HSLS

community (Fig. 8).
These results show that sulfate reducers in the Mahomet
coexist with iron reducers, even though the aquifer is nominally iron reducing. Indeed, as already discussed in thispaper, coexistence of a significant population of sulfate
reducers in the presence of high-iron groundwater is notentirely surprising, in light of the fact that iron reducers
contribute eight times as much Fe2+ to solution on a perelectron basis as sulfate reducers add sulfide.
In a broader sense, the study lends no support to the ideathat a thermodynamic ladder exerts control over the microbial community, or to the concept of competitive exclusionof sulfate reducers by iron reducers. Instead, the evidence
suggests that wherever sulfate is available in the aquifer,sulfate reducers and iron reducers live together, perhaps
in a mutualistic relationship of the sort demonstrated by
Bethke et al. (2011) and discussed in the previous sectionof this paper.

7. Discussion
We in this paper attempt to develop an understandingof the nature of subsurface microbial activity by integrating study of groundwater chemistry, numerical modeling,

NS

Functional Groups
FeIII Reducers
Geobacter
Desulfuromonas

β-Proteobacteria
Clostridia
δ-Proteobacteria
γ-Proteobacteria
Holophagae

GeothrixSO42- Reducers
Desulfobulbus
Desulfobacter

Syntrophs
Syntrophobacter
Syntrophus

Percent of Total

Other

50%

25%

Other

33

10%

Fig. 8 Relative distribution of the bacterial populations that make up a series of natural communities
sampled from the Mahomet aquifer of east-central Illinois, USA, from Flynn et al. (2012). The communities were obtained from 18 wells completed within the aquifer using the “bug traps” shown in Fig. 7.
The traps were left in place for 14 weeks before being retrieved, at which point the bacterial communitythat had grown up attached to sediment in each trap was detached and analyzed by molecular methods.
Data are grouped in the figure by groundwater sulfate concentration: column “HS” contains data fromwells where sulfate concentration is > 0.2 mM; concentration for column “LS” is between that leveland 0.03 mM; column “NS” is for data from wells with negligible sulfate, < 0.03 mM. For each taxonomic group, the diameter of the circle representing high, low, and negligible sulfate conditions shows
the group’s relative importance within the community; the radii of wedges within circles break out the
proportions of molecular sequences attributed to iron reducers (red, orange, yellow), sulfate reducers(blue), synthrophs (green), and other classes of bacteria (black). The results show that, even thoughconditions in the Mahomet aquifer are considered by conventional criteria to be strongly iron reducing,the sulfate reducing bacteria are about equally numerous as iron reducing bacteria wherever measurable
amounts of sulfate (“HS” and “LS”) are found in the groundwater. Even where sulfate concentration is
negligible (“NS”), sulfate reducers make up a significant fraction of the community.

34

土壌の物理性bioreactor experiments, and the in-situ sampling of actively growing microbes from an aquifer. Taken together,
these studies portray microbial communities and their relationship to ecosystem chemistry in a manner distinct fromconvention interpretations.
The nature of subsurface microbial activity has mostcommonly been inferred by comparing the redox chemistry of groundwater from place to place. The variation
might be observed on a sub millimeter scale in soil, orover a range of hundreds of kilometers in a regional flow
regime. A spot where groundwater is rich in iron, for example, would have been taken to reveal a community ofiron reducing bacteria inhabiting an aquifer.
The Middendorf aquifer of South Carolina, USA, however, shows a large electron gap: groundwater in theaquifer records just a small fraction of the respiration occurring there (Fig. 1). In a reactive transport model (Fig. 4)
and a bioreactor experiment (Fig. 5), furthermore, groundwater becomes enriched in iron, even though iron reduction in both the model and the experiment is subordinate tosulfate reduction. Clearly, the presence of dissolved ironcannot be taken as evidence of a subsurface community
dominated by iron reducers.
As well, conventional thinking has held that the distribution of microbial populations in the subsurface falls
along a thermodynamic ladder in which iron reducers holdan advantage over sulfate reducers, which in turn dominate methanogens. By the theory of competitive exclusion,
wherever an advantaged population can live, it displaces
other groups of microbes from the habitat by denying themthe energy they need to live.
We see no evidence that subsurface communities behavein this manner. In the Middendorf, sufficient energy exists
everywhere for iron reducers, sulfate reducers, and aceticlastic methanogens to live. In a reactive transport experiment, methanogens are excluded from part of an aquifer,
but not by the lack of energy. Instead, these methanogens
enter an extinction vortex because they fail to reproduce
quickly enough to replace cell decay; their exclusion fromthe community is an ecologic rather than thermodynamic
phenomenon.
In contrast to the conventional perspective, we find thatmicrobial communities commonly exist as mixed populations, shaped as much by mutualism as competition. In
the Middendorf aquifer, the electron gap (Fig. 1) might be
explained by a community of coexisting iron reducers andsulfate reducers. If the Fe2+ and sulfide these groups add
to solution were to precipitate as mackinawite, the groundwater would lose part of the record of electron acceptance.
The microbial communities that grow up in the reactive
transport modeling, furthermore, are as commonly mixed

第 138 号(2018)

communities than monocultures.
Perhaps most striking is the evidence that iron reducers
and sulfate reducers can establish a close mutualistic relationship. In a long-term bioreactor experiment (Fig. 6),
the relationship between the two groups becomes so tightly
balanced that the community produces Fe2+ and sulfide ina one-to-one ratio. Just as notably, the rate of iron reduction in the experiment was roughly 15 times faster than ina parallel experiment lacking sulfate. When the iron reducers had to compete with sulfate reducers for acetate, they
actually worked more quickly than in the absence of competition.
In-situ sampling of actively growing microbes from the
Mahomet aquifer, which is taken by conventional criteria
to be strongly iron reducing, seems to confirm results of thebioreactor experiment. Despite its classification, the Mahomet wherever sampled is seen to have a significant population of active sulfate reducers. Given a small amount of
sulfate in the groundwater, the sulfate reducers are roughly
equivalent in population size to the iron reducers.
We argue that classifying microbial activity on the basis of an ecosystem’s redox chemistry is obsolete, as is
the concept that communities reflect an energetic hierarchy
among functional groups of microbes. Instead, microbial
populations distribute themselves according to ecologic
phenomena that are of necessity subject to the constraintsof thermodynamics, but do not directly reflect them.

Acknowledgments
This paper is possible in light of the considerable efforts
and insights of our former graduate students Qusheng Jin,
Matthew Kirk, Ted Flynn, Dong Ding, and Jungho Park.
We thank them all.

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