Doug LaRowe, Jan Amend

13 Energetic constraints on life in marine deep

sediments

13.1 Introduction

Although it is becoming clear that microorganisms are abundant in marine deep sedi-
ments [1–8], it is unclear what percentage of cells are active, how fast they are growing
or what controls their diversity and population size [9]. Addressing these issues is a
formidable task due to the relative inaccessibility of these environments, the difficulty
of cultivating representative microorganisms and the long time scales associated with
some of their lifestyles [2, 10–12]. However, quantitative limits on life in the subsur-
face can be determined by using the physiochemical data that describe their habi-
tats. In particular, the chemical composition can be used to constrain likely metabolic
strategies and rates in a given setting. This is accomplished by calculating values of
Gibbs energy available from reactions containing different combinations of the elec-
tron donors and acceptors that are found in these environments. Not only can Gibbs
energies of reaction reveal which catabolic strategies are thermodynamically possi-
ble, but they can also help determine which geochemical variables (e.g. temperature,
pressure, pH, salinity, composition) are controlling microbial activity. When reduced
to an environmentally-appropriate common factor, the energetic potential of all bio-
geochemical environments can be directly compared to assess how energy limitations
affect the amount and type of biomass in them. In the present chapter, geochemical
data obtained from sediment cores taken from the Peru Margin, South Pacific Gyre
and Juan de Fuca Ridge are used to assess the Gibbs energies of plausible catabolic
strategies including, but not limited to, the oxidation of organic matter, methane and
hydrogen by a variety of electron acceptors. In conjunction with cell-count data, the re-
sults of these calculations illustrate the importance of normalizing energy availability
to the limiting substrate and how geochemical data can be used to better understand
the distribution of life deep in marine sediments.
Perhaps one of the most enticing aspects of using Gibbs energies to characterize
the type and level of microbial activity in the deep biosphere is that the geochemi-
cal data required to do so are relatively widely available (e.g. Proceedings of the ODP
and IODP). These kinds of calculations can be used to predict microbial activity in
places where chemical data are available, but where microbiological samples have
not been taken. The calculations described below are universal in that they can il-
luminate which biogeochemical processes could be operating in any environment as
long as the prevailing temperature, pressure and composition are appropriately taken
into account.

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13.2 Previous work

The amount of energy available in an environment largely determines the metabolic

state of the resident microorganisms [13]. That is, more energy-rich settings can sup-
port larger populations, more biomass and faster growth rates than low-energy en-
vironments. Because active microorganisms ultimately derive their energy from the
catalysis of redox reactions, the Gibbs energies of these types of reactions have the
potential to reveal the metabolic state of a microbial ecosystem. This kind of ener-
getic profiling has been carried out successfully for hydrothermal systems in the deep
sea [14–20], the shallow sea [21–26] and in terrestrial systems [27–33] and, to a lesser
extent, in ocean sediments [34, 35] and basement rock [5, 36–38]. These studies have
examined more than 100 organic and inorganic redox reactions under a wide range of
environmental conditions. The calculated Gibbs energies of the thermodynamically
favored (exergonic) reactions vary between approximately 0 and −120 kJ (mol e− )−1 ,
indicating which reactions are possible catabolic strategies and how much energy
they provide.

13.3 Study site overview

The three sites examined in the current study were chosen because they represent
three significant types of marine sedimentary settings and are biogeochemically well
characterized. Sediments at the Juan de Fuca site (47°45.2󸀠 N, 127°45.8󸀠 W) are influ-
enced by hydrothermal activity resulting from the adjacent ocean spreading center.
Although these kinds of sediments do not cover a significant fraction of the ocean
floor, the water-rock interactions that typify submarine hydrothermal systems support
diverse, dense ecosystems that influence global biogeochemical cycles. Peru Margin
sediments (10°58.60󸀠 S, 77°57.46󸀠 W) lie on an active continental shelf where primary
productivity is relatively high and therefore, organic matter accumulation is signifi-
cant. Despite covering only about 7% percent of the ocean floor, nearly 58% of global
marine particular organic matter deposition occurs in such zones [39]. With its ultra-
slow sedimentation rate, great distance from continental land mass, and correspond-
ing low organic carbon sedimentation rate, the South Pacific Gyre site (23°51.04󸀠 S,
165°38.66󸀠 W), represents the majority of the world’s marine sediments.
Although most of the geochemical and microbial cell-count data for these sites
have been tabulated, several data profiles had to be extracted from figures using Plot
Digitizer (http://plotdigitizer.sourceforge.net). For all sites, the density of pore fluids
was taken to be 1.035 kg/L, the concentrations of HCO−3 were taken to be equal to the
reported dissolved inorganic carbon (DIC) and, in the absence of perfectly overlapping
data sets, several chemical concentrations and cell numbers were interpolated such
that calculations could be carried out at particular sediment depths. In order to carry
out Gibbs energy calculations, the concentrations of all reactant and product species

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 13.3 Study site overview | 281

in a given chemical reaction most be known. In the absence of such data, the con-
centrations of some compounds were estimated. These estimates, the sources of the
chemical data and other characteristics of the three study sites are summarized below
and in the Appendix.

13.3.1 Juan de Fuca (JdF)

Due to its proximity to an active rifting center and a continental margin, the sediments
penetrated by drill site U1301 on the JdF ridge are influenced by hydrothermal activ-
ity and rapid sedimentation rates (∼75 m/MY given a 3.5 MY old basaltic crust and a
262 m layer of sediments) [40]. The basaltic crust underlying these insulating sedi-
ments hosts a dynamic, heat-driven flux of fluids that are a mixture of seawater and
hydrothermal solutions that diffuse into the overlying sediments. As a result, the ther-
mal gradient in these sediments (0.228 °C/m) is steeper than that in typical marine
sediments, and the concentration profiles of various metabolically-relevant species
display complex patterns influenced by both of the sediment column boundaries (see
Appendix and 󳶳 Fig. 13.A1). Laboratory studies have shown that microorganisms from
these sediments can grow on enriched media via sulfate reduction in the presence of a
variety of organic compounds including short-chain alcohols, fatty acids and H2 , and
by fermentation of ethanol, pyruvate and betaine [41]; some isolates produced sulfide
in the presence of Fe(III) and Mn(IV), but did not show any corresponding growth.
Concentration profiles of SO2− + 2+ 2+
4 , NH4 , Mn , Fe , DIC, dissolved organic carbon (DOC)
and CH4 as well as cell numbers, pH, thermal gradient and porosity data for JdF holes
U1301C & D were taken from [40]. In the absence of concentration data for HS− and N2 ,
depth-constant concentrations of 10−5 and 10−4 mol/kg H2 O were used in the relevant
energy calculations. The water depth is 2656 m.

13.3.2 Peru Margin (PM)

The PM site, under only 78 m of water, underlies a zone of high primary productivity
that has resulted in an average sedimentation rate of 24 m/MY [42]. As a result, the
sediments here are rich in organic matter (1–12% TOC by weight) [43] and EAs such
as O2 and NO−3 disappear in the upper few centimeters of the sediment column. Sul-
fate, which is highly concentrated in seawater, initially decreases as a function of
depth, but similar to JdF sediments, it then increases again owing to diffusion from
a deep source. In particular, a Miocene brine supplies sulfate and other ions to the
sediment column from below creating complex nutrient profiles (see 󳶳 Fig. 13.A2).
Although inner-shelf environments (water depth < 150 m) only cover 5.8% of global
ocean settings, a disproportionately large amount of primary production occurs in
these waters [39]. The concentration profiles of nitrate, sulfate, DIC, ammonium,

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282 | 13 Energetic constraints on life in marine deep sediments

sulfide, methane, Mn2+ , and Fe2+ are thought to be influenced by organic matter
degradation [4]. Concentration profiles of SO2− 2+ 2+
4 , Mn , Fe , DIC, acetate, CH4 , pH
and bacteria for PM site 1229 were taken from [42]. Porosity data and a constant value
of 0.3 millimolal for [HS− ] were taken from [44].

13.3.3 South Pacific Gyre (SPG)

The slow sedimentation rate (1.1 m/M.Y.) and water depth (5695 m) at SPG are typical of
open-ocean sites that are far from land [45]. Because open-ocean sites comprise half or
more of all ocean environments [39], SPG sediments represent, by volume, a substan-
tial portion of Earth’s ecosystems. The low sedimentation rates and correspondingly
tiny amounts of organic matter delivered in these sediments results in low biomass
concentrations [46]. The near absence of electron donors at this site means that elec-
tron acceptors (EAs) such as O2 and NO−3 can penetrate deep into the sediment column
here. D’Hondt et al. [46] suggest that oxygen and nitrate are the dominant EAs and that
radiolytic hydrogen and organic matter are the most common electron donors (EDs).
Concentration profiles of SO2− − 2+ 2+
4 , NO3 , O2 , H2 , Mn , Fe , DIC, total organic carbon
(TOC), pH and cells and porosity for SPG holes U1365A & B were taken from [45]. For
sediment depths in which H2 concentration were below detection (< 2.8 nM), a nom-
inal value of 1 nM was assumed to carry out Gibbs energy calculations. In the absence
of concentration data for HS− and N2 , depth-constant concentrations of 10−7 and 10−4
mol/kg H2 O were used to calculate the catabolic potential of reactions in which they
are product species. In addition, although DOC concentrations were not reported for
this site, they were estimated from TOC data according to [DOC] = 10−8∗ (%TOC),
which produces DOC concentrations similar to those for hydrogen. Concentration data
are largely absent at 43–65 meters below the seafloor (mbsf), so concentration data
were interpolated from the values above and below these depths (see Appendix).

13.4 Overview of catabolic potential

The 18 redox reactions considered as sources of available catabolic potential are listed
in 󳶳 Table 13.1. Because not all of the concentration data that are required to evalu-
ate the energetic potential for all of these reactions have been reported for each site,
thermodynamic calculations were only carried out at all three sites for Reactions 3–
5, at two sites for Reactions 6–10 and at one site for the rest of the reactions (see
󳶳 Table 13.1). The electron donors considered include organic matter (OM), H2 , Mn2+ ,
Fe2+ , and NH+4 and electron acceptors include O2 , NO−3 , Mn(IV), Fe(III), and SO2− 4 .
Acetoclastic methanogenesis (Reaction 6) was also considered at two sites. Although
many minerals contain oxidized Fe and Mn, only goethite (FeOOH) and pyrolusite

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Table 13.1: Catabolic reactions considered in the present study.

Reaction Location
1 CH3 COO + 2O2 → H + 2HCO−
− +
3 SPG
2 5CH3 COO− + 8NO− +
3 + 3H → 4N2 + 4H2 O + 10HCO3
−
SPG
− + − 2+
3 CH3 COO + 4MnO2 + 7H → 2HCO3 + 4Mn + 4H2 O SPG, PM, JdF
4 CH3 COO− + 8FeOOH + 15H+ → 2HCO− 2+
3 + 8Fe + 12H2 O SPG, PM, JdF*
− 2− − −
5 CH3 COO + SO4 → HS + 2HCO3 SPG, PM, JdF
6 CH3 COO− + H2 O → CH4 + HCO− 3 PM, JdF
7 4HCOO− + H+ + SO2− −
4 → HS + 4HCO3
−
PM, JdF
+ 2+ −
8 CH4 + 4MnO2 + 7H → 4Mn + HCO3 + 5H2 O PM, JdF
9 CH4 + 8FeOOH + 15H+ → 8Fe2+ + HCO− 3 + 13H2 O PM, JdF
10 CH4 + SO2−
4 → H 2 O + HS−
+ HCO −
3 PM, JdF
11 H2 + 1/2O2 → H2 O SPG
12 5H2 + 2NO− +
3 + 2H → N2 + 6H2 O SPG
13 H2 + MnO2 + 2H → Mn2+ + 2H2 O
+
SPG
14 H2 + 2FeOOH + 4H+ → 2Fe2+ + 4H2 O SPG
15 4H2 + SO2− + −
4 + H → HS + 4H2 O SPG
16 2Mn + O2 + 2H2 O → 2MnO2 + 4H+
2+
SPG
17 4Fe2+ + O2 + 6H2 O → 4FeOOH + 8H+ SPG
18 3MnO2 + 4H+ + 2NH+ 2+
4 → N2 + 3Mn + 6H2 O JdF

SPG – South Pacific Gyre; PM – Peru Margin; JdF – Juan de Fuca.

*FeOOH can represent goethite and anhydrous ferrihydrite. For JdF, DOC is taken to be acetate and
for SPG, TOC was used to estimate acetate – see text. With the exceptions of MnO2 and FeOOH, all
chemical species are in the aqueous state.

(MnO2 ) were used to represent these electron acceptors. The energetic consequences
of this simplification are discussed below.
The energetic potential in the marine sediments at JdF, SPG and PM are shown as a
function of depth below the seafloor in 󳶳 Fig. 13.1 (a). In this figure, values of the Gibbs
energy of reaction, Δ𝐺𝑟 , for reactions listed in 󳶳 Table 13.1 are depicted in units of kJ per
mole of electron transferred, kJ(mol e− )−1 . The prevailing temperature, pressure and
composition of the pore fluids in these sediments were explicitly taken into account
as detailed below in Computational Methods. A prominent feature in this figure is that
the reactions separate into two groups for all three sites. The high energy group of re-
actions, which typically yield values between −70 and −105 kJ(mol e− )−1 , are those in
which O2 , NO−3 , and MnO2 are the oxidants, while the low energy group of reactions,
yielding between −20 and +5 kJ(mol e− )−1 , are methanogenesis and those in which
sulfate and FeOOH are the electron acceptors. In 󳶳 Fig. 13.1 (a), it can also be seen that
for some parts of the JdF sediment column, values of Δ𝐺𝑟 are positive for methane oxi-
dation by FeOOH and sulfate, indicating that they are not possible catabolic pathways
at these sediment depths. Also, one might expect that because the most exergonic re-
actions are computed for the oxidation of H2 and OM in SPG sediments, that this site
might have the fastest microbial metabolic rates or the largest biomass, and that the

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(a) Juan de Fuca (b) Juan de Fuca

0 0

OM + MnO
50 2 50
+
NH + MnO
4 2
2-
CH + MnO OM + SO

Depth , mbsf
4
Depth , mbsf

4 2
100 100 methanogenesis
CH + MnO
4 2

150 150 OM + FeOOH

+
NH + MnO
4 2
2-
OM + SO
4
200 methanogenesis 200 OM + MnO
2

OM + FeOOH CH + FeOOH
4
2-
CH + SO
250 4 4
250
CH + FeOOH
4

0 -20 -40 -60 -80 -7 -6 -5 -4 -3 -2 -1 0 1

Peru Margin Peru Margin

0 0
CH + MnO
OM + MnO 4 2
2
CH + FeOOH
4
CH + MnO
4 2 OM + MnO
2

50 50
Depth , mbsf

Depth , mbsf

100 100
OM + FeOOH
2-
CH + FeOOH CH + SO
4 4 4
2-
OM + SO OM + FeOOH
4
150 2- 150 2-
CH + SO OM + SO
4 4 4

methanogenesis methanogenesis

0 -20 -40 -60 -80 -100 -7 -6 -5 -4 -3 -2 -1 0 1

South Pacific Gyre South Pacific Gyre

0 0
H +O
2 2 OM + O Fe
2+
+O
2
OM + O 2
10 2 10 OM + NO
-
3 2+
H + NO
- Mn +O
2 3 OM + MnO 2
2
20 OM + NO
-
20
3
Depth , mbsf

Depth , mbsf

2+
Fe +O
30 2 30
H + MnO
2 2 H +O
40 OM + MnO 40 2 2
2
H + MnO
2 2
-
50 2+ 50 H + NO
Mn +O 2 3
2

H + FeOOH H + FeOOH
2
2
60 OM + FeOOH
60
OM + FeOOH
2-
H + SO
2- OM + SO
70 2 4 70 4
2-
OM + SO
2- H + SO
2 4
4

80 80
0 -20 -40 -60 -80 -100 -9 -8 -7 -6 -5 -4 -3 -2
3
ΔG , kJ (mol e )
- -1 log E (J/cm )
r
r

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 13.4 Overview of catabolic potential | 285

Opposite page: Fig. 13.1: Catabolic energy potential for the reactions listed in 󳶳 Table 13.1 as a func-
tion of depth in three marine sediment columns: a) Gibbs energy in units of kilojoules per mole of
electron transferred, kJ (mol e− )−1 and b) the logarithm of energy available per cubic centimeter of
sediment, log Er , (J cm-3 ). Each reaction is distinguished by the colors of the lines and their labels,
which are abbreviations including just the electron donor and acceptor. Discrete dots rather than
a continuous line represent the energy available at Juan de Fuca for methane oxidation by goethite
because this reaction is endergonic at some depths. Similarly, methane oxidation by sulfate does
not appear in the same plot because it is only exergonic for the bottom few meters in Juan de Fuca
sediments.

50
SPG
Depth , mbsf

100

150
PM

200

JdF
250
Fig. 13.2: Concentration of cells per cubic cen-
2 3 4 5 6 7 8 9 10 timeter in South Pacific Gyre (SPG), Peru Marin
3 (PM) and Juan de Fuca (JdF) sediments as a func-
log (cells / cm )
tion of depth.

lower energy yields at the other two sites would result in commensurately lower micro-
bial cell numbers. However, the number of cells in the respective sediment columns,
shown in 󳶳 Fig. 13.2, do not reflect this. In fact, the cell counts in JdF and PM sediments
are several orders of magnitude higher at all depths than those at SPG, the site of the
most exergonic reactions considered. This discrepancy can be readily reconciled by re-
casting the Gibbs energy calculations presented in 󳶳 Fig. 13.1 (a) to reflect the chemical
and physical characteristics at each of these three sites.
In 󳶳 Fig. 13.1 (b), the reaction energetics are plotted as energy densities in units of
Joules per cm3 of sediment. Energy densities of the 𝑟th reaction in a cm3 of sediment,
𝐸𝑟 , are calculated using:
󵄨󵄨 Δ𝐺 󵄨󵄨
󵄨 󵄨
𝐸𝑟 = 9.65 × 10−4 ⋅ 󵄨󵄨󵄨 𝑟 󵄨󵄨󵄨 [𝑖]𝜙 (1)
󵄨󵄨 𝜈𝑖 󵄨󵄨

where 𝑣𝑖 and [𝑖] stand for the stoichiometric coefficient and molal concentration, re-
spectively, of the 𝑖th limiting electron donor or acceptor and 𝜙 denotes porosity (unit-
less). The conversion factor 9.65 × 10−4 in Eq. (1) corresponds to the number of kg of
H2 O in a cm3 of seawater. Because either the electron donor or acceptor will be a lim-
iting reactant per volume of fluid, the concentration and stoichiometric coefficient of

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286 | 13 Energetic constraints on life in marine deep sediments

this limiting nutrient was used for values of 𝑣𝑖 and [𝑖] in Eq. (1) to generate the panels
in 󳶳 Fig. 13.1 (b). In order to carry out these calculations, the activities of all reactants
and products were held constant, simulating a steady state condition.
It can be seen in 󳶳 Fig. 13.1 (b) that there is far more energy available at JdF and PM
than at SPG. This is due to the fact that although the catabolic reactions considered at
SPG are more exergonic per mole of electron transferred, the concentrations of elec-
tron acceptors, and, more importantly, electron donors are far lower there than they
are at the other two sites. Within the sediment columns at each site, it can be seen that
the energy available from different reactions vary by about 6 orders of magnitude per
cm3 of sediment. For example, the energy available from Reaction (3), organic mat-
ter oxidation by MnO2 , is the most energy yielding reaction at JdF throughout most of
the sediment column, yielding 5–0.1 J cm−3 , whereas methane oxidation by FeOOH,
where it is exergonic, yields anywhere from 0.04 to < 10−6 J cm−3 . Furthermore, due
to concentration gradients in the sediments at all sites, values of 𝐸𝑟 can vary by sev-
eral orders of magnitude as a function of depth. This is most notable at JdF and PM,
but also for the reactions in SPG in which H2 is an electron donor. It should also be
pointed out that the most energetic reactions at a particular site vary with depth, sug-
gesting where physiological changes in the microbial communities in these sediment
might be observed. This is illustrated for the reactions represented by crossing lines
in 󳶳 Fig. 13.1 (b) for PM sediments and, more conspicuously, for JdF.
Acetate was used to represent organic matter in all of the Gibbs energy calcula-
tions. In the case of PM, acetate concentrations in the sediment were reported in [42],
but for JdF, the acetate concentration was taken to be equal to the concentration of
DOC. At SPG, acetate concentrations were estimated using the amount of TOC in the
sediments (see Section 13.3.2). Acetate is a common organic compound in many nat-
ural settings [47], but using a suite of organic compounds would be more reflective
of the organic milieu in marine sediments. Using acetate exclusively does have ener-
getic implications, which are shown in 󳶳 Fig. 13.3 (a). For example, values of Δ𝐺𝑟 for
sulfate reduction coupled to the oxidation of acetate (Reaction 5 in 󳶳 Table 13.1) and
formate (Reaction 7 in 󳶳 Table 13.1) are shown as a function of depth in JdF sediments.
Although these compounds are similar, the Gibbs energies of their oxidation per elec-
tron nearly span that of the known range for organic compounds [48]. Throughout the
sediment column at JdF, formate oxidation by sulfate is nearly twice as exergonic as
the analogous reaction with acetate. Furthermore, it can be seen in 󳶳 Fig. 13.3 (a) that
the form of the curve representing the energetics of formate oxidation has more pro-
nounced curvature than that for acetate. This is due to the fact that though the same
concentration of organic matter is used in both sets of calculations, the stoichiometric
coefficients for acetate and formate oxidation reactions are 1 and 4, respectively (see
Eq. (3) in Computational Methods). It should be noted that the calculations carried
out with organic carbon are an attempt to assess the energetics of readily accessible
organic matter. Taking into account the total organic matter (OM) at each site, which
includes dissolved and particulate OM, would increase the amount of energy avail-

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 13.4 Overview of catabolic potential | 287

(a) Acetate: CH COO + SO

3
- 2-
4
-
---> HS + 2HCO
-
3
(b)
+ - 2+
- + 2- - - CH COO- + 8FeOOH + 15H --> 2HCO + 8Fe + 12H O
Formate: 4HCOO + H + SO ---> HS + 4HCO 3 3 2
4 3
0 0
JdF
50 50

Depth , mbsf
Depth , mbsf

100 Goethite Anhydrous

100 Acetate
ferrihydrite

Formate 150
150

200 200

250 250 JdF

-5 -10 -15 -20 -25 -30 0 -10 -20 -30 -40 -50
- -1
ΔG , kJ (mol e )
- -1
ΔG , kJ (mol e )
r r

Fig. 13.3: Comparison of the energetic consequence of representing a) organic matter as acetate
versus formate and b) goethite versus anhydrous ferrihydrite in Gibbs energy calculations in Juan de
Fuca sediments.

able from OM degradation, but the rates at which this particulate OM is converted
into usable OM, is a complex function of numerous environmental variables [49] that
is beyond the scope of this chapter.
Similarly, the minerals goethite and pyrolusite were used in the Gibbs energy cal-
culations for reactions in which oxidized iron and manganese serve as electron accep-
tors. Although the exact mineral phases in these sediments are not known, combus-
tion analyses show the presence of iron- and manganese-bearing minerals [40, 42, 45].
As with the choice of acetate to represent OM discussed above, there is an energetic
consequence to representing Fe(III) and Mn(IV) as goethite and pyrolusite. This is il-
lustrated in 󳶳 Fig. 13.3 (b), which depicts the Gibbs energy of acetate oxidation by two
different phases of Fe(III) minerals, goethite and anhydrous ferrihydrite (FeOOH), in
JdF sediments. Despite having the same chemical formula, values of Δ𝐺𝑟 are approx-
imately three times more exergonic for the reaction with anhydrous ferrihydrite than
the analogous one with goethite, yet this is only one of many potential mineral phases
in which Fe(III) could exist. Furthermore, it should be noted that some mineral phases
may not be readily accessible if they are physically separated from the microorganisms
that can use them, or by having a crystalline structure that does not readily lend itself
to microbial utilization. Clearly, how one represents mineral phases and organic mat-
ter can have a dramatic effect on the energetics of microbially metabolized reactions.
See Computational Methods for the source of thermodynamic data for anhydrous fer-
rihydrite and pyrolusite.

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288 | 13 Energetic constraints on life in marine deep sediments

13.5 Comparing deep biospheres

The energetics of OM degradation with three different electron acceptors are compared
in 󳶳 Fig. 13.4. The energetic comparison is made on a per mole e- basis in 󳶳 Fig. 13.4 (a)–
(c) and on a per cm3 sediment basis in 󳶳 Fig. 13.4 (d)–(f). It is immediately apparent
that the energy yields differ demonstrably at the three sites and that their relative po-
sitions change dramatically depending on the normalization procedure. For exam-
ple, OM oxidation with Mn(IV)(󳶳 Fig. 13.4 (a)) yields similar energies (per mol e− ) at
all three sites for the first 70 m of sediment. Below that, the energy yield at JdF de-
creases from −81 kJ/mol e− to −74 kJ/mol e− at ∼250 mbsf, before sharply increasing
to −80 kJ/mol e− at the sediment-water interface [42]. In comparison, the energy yields
for this reaction at PM remain fairly constant at approximately −82 kJ/mol e− over the
entire depth of the sediment column. The differences in sediment thickness at the
three sites permits only an incomplete comparison of energetics with depth.
It is striking that the energy yield for OM oxidation with Mn(IV) at JdF is consis-
tently the least exergonic on a per mol e− basis (󳶳 Fig. 13.4 (a)), but the most exergonic
on a per cm3 sediment basis (󳶳 Fig. 13.4 (d)). This same shift in relative position among
the three sites is seen in 󳶳 Fig. 13.4 (b) and (e) for OM oxidation with Fe(III): on a per
mol e− basis, SPG is the most exergonic throughout the sediment columns and JdF is
the least exergonic. However, these positions reverse on a per cm3 sediment basis with
JdF the most and SPG the least exergonic. For OM oxidation coupled to sulfate reduc-
tion (󳶳 Fig. 13.4 (c) and (f)), JdF is the most exergonic regardless of the normalization
procedure used, but SPG and PM switch relative positions.
The question of which normalization procedure should be used to present the
results of energy calculations depends on the subject matter. The amount of energy
available per cm3 sediment is arguably a far better indicator of biomass in sediments
than when energetic potential is presented per mol e− . This point is best realized by
comparing the energetic profiles in 󳶳 Fig. 13.4 to the abundance of microorganism in
each sediment column (󳶳 Fig. 13.2). Despite the similar energy yields for the catabolic
reactions considered at all three sites on a per mol e− basis (󳶳 Fig. 13.4 (a)–(c)), there
are several orders of magnitude less biomass in SPG than the other sites. Similar to
the energetics displayed in 󳶳 Fig. 13.1, this discrepancy can be resolved by present-
ing these energetic calculations in terms of the amount of energy available per cm3 .
󳶳 Figure 13.4 (d)–(f) shows the energy density (𝐸𝑟 ) as a function of depth for the same
OM oxidation reactions considered in 󳶳 Fig. 13.4 (a)–(c). For all three reactions, the

Opposite page: Fig. 13.4: Direct comparison of the energy available from organic matter oxidation in
South Pacific Gyre (SPG), Peru Margin (PM) and Juan de Fuca (JdF) sediments in units of (a)–(c) kilo-
joules per mole of electron transferred, kJ (mol e− )−1 , and (d)–(f) logarithm of the energy available
per cubic centimeter of sediment, log (J cm−3 ), for (a,b) MnO2 , (c,d) FeOOH and (e,f) SO4 2− serving
as electron acceptors.

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 13.5 Comparing deep biospheres | 289

(a) -
CH COO + 4MnO + 7H --> 2HCO + 4Mn
3 2
+
3
- 2+
+ 4H O
2
(d) 3
-
CH COO + 4MnO + 7H --> 2HCO + 4Mn
2
+
3
- 2+
+ 4H O
2

0 0

SPG
50 50 SPG
Depth , mbsf

100 100

150 150 PM

200 PM 200
JdF
JdF
250 250

-74 -76 -78 -80 -82 -84 -7 -6 -5 -4 -3 -2 -1 0 1

- -1 3
ΔG , kJ (mol e ) log E (J/cm )
r r

(b) -
CH COO + 8FeOOH + 15H --> 2HCO + 8Fe
3
+ -
3
2+
+ 12H O
2
(e) -
CH COO + 8FeOOH + 15H --> 2HCO + 8Fe
3
+
3
- 2+
+ 12H O
2

0 0

50 50 SPG
SPG
Depth , mbsf

100 100

150 150 PM
PM
200 200
JdF JdF
250 250

0 -5 -10 -15 -20 -8 -7 -6 -5 -4 -3 -2 -1 0

- -1 3
ΔG , kJ (mol e ) log E (J/cm )
r r

(c) CH COO + SO
3
-
4
2- -
---> HS + 2HCO
3
-
(f) CH COO + SO
3
- 2-
4
-
---> HS + 2HCO
3
-

0 0

50 50 SPG

SPG
Depth , mbsf

100 100

150 150 PM

200
PM 200
JdF
JdF
250 250

-2 -4 -6 -8 -10 -12 -14 -16 -10 -8 -6 -4 -2 0

3
ΔG , kJ (mol e )
- -1
log E (J/cm )
r r

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290 | 13 Energetic constraints on life in marine deep sediments

amount of energy available per cm3 spans about 7 orders of magnitude between the
sites, with SPG at the low end and JdF at the high end of the spectrum. These plots
reproduce, at least qualitatively, if not semi-quantitatively, the microbial abundances
in the sediment columns (󳶳 Fig. 13.2). It is worth noting that even in the most energy-
rich JdF sediments, the OM oxidation reaction only yields about 0.1 Joules of energy
per cubic centimeter of sediment. Note also that these are only some of the energy
yielding reactions at each site. In order to compare the total energy supply in each set-
ting, one would have to know the proportional use of each ED and EA. For example,
OM can be degraded by many EAs, providing large differences in energy availability.
Furthermore, if mineral phases such as MnO2 that are particularly energetic EAs are
not accessible to microorganisms, then other, less energetic reactions such as sulfate
reduction could be more typical catabolic strategies in these sediments.

13.6 Electron acceptor utilization

The types and relative abundance of electron acceptors encountered in marine sedi-
ments varies as a function of geographic location and depth. It is generally assumed
that the order in which they are consumed by respiring microorganisms is O2 , NO−3 ,
Mn(VI), Fe(III), SO2−
4 and CO2 (e.g. [50–52]). This explains the rapid disappearance of
O2 , then nitrate and so on as a function of depth in organic rich sediments [47]. The
generally-accepted hypothesis for this phenomenon is that the sequence of EA utiliza-
tion corresponds to the order of Gibbs energy yield of the corresponding organic mat-
ter oxidation reactions. That is, a more exergonic reaction (i.e. having a larger value
of −Δ𝐺𝑟 ) takes place before less exergonic ones. Another way of saying this is that
given a selection of electron acceptors, a microbial community will use the ones that
provide the largest amount of energy first, the second most energetic second, and so
on.
However, because the exergonicity of reactions is a function of environmental pa-
rameters such as temperature, pressure and concentrations of reactants and products,
the order of what is the most energetic electron acceptor (or donor) can deviate from
that listed above. This is clearly shown in 󳶳 Fig. 13.5 (a), which depicts the Gibbs en-
ergies of OM transformation by MnO2 , FeOOH and sulfate as well as methanogenesis
in JdF sediments. Throughout the sediment column, OM oxidation with Mn(IV) is the
most exergonic, consistently yielding approximately −80 kJ / mol e− . The other three
reactions are far less exergonic (0 to −20 kJ/ mol e− ), and their relative positions os-
cillate as a function of depth. For example, OM oxidation with Fe(III) is, at different
depths, the most and least exergonic of these three reactions.
Energetic comparisons become even more complex by examining the amount of
energy per cm3 sediment for the same set of reactions (󳶳 Fig. 13.5 (b)). Again, the re-
action with OM + Mn(IV) yields the most energy. A focus on the other three reactions
reveals that the energy yield from OM oxidation with Fe(III) is very similar to that from

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 13.6 Electron acceptor utilization | 291

(a) (b)
0 0

50 50
FeOOH + OM MnO
2
+ OM
Depth , mbsf

100 100 MnO

2
+ OM

150 150 FeOOH + OM

methanogenesis
methanogenesis

200 200 2-
SO 2-
+ OM SO + OM
4
4

250 250

0 -20 -40 -60 -80 -100 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1

3
ΔG , kJ (mol e )
- -1 log E (J/cm )
r r

Fig. 13.5: Catabolic energy potential for organic matter degradation in Juan de Fuca sediments cou-
pled to iron (FeOOH), manganese (MnO2 ) and sulfate reduction and acetoclastic methanogenesis in
units of (a) kilojoules per mole of electron transferred, kJ (mol e− )−1 , and (b) Joules per cubic cen-
timeter of sediment, J cm−3 .

methanogenesis throughout the sediment column, but that from OM coupled to sul-
fate is by far the most exergonic of the three reactions. This is noteworthy because
in the absence of accessible MnO2 (which seems to be the case since the Mn profiles
there suggest that MnO2 reduction is not especially common throughout the sediment
column – see Appendix), iron reducers, sulfate reducers and methanogens could al-
ternate as the dominant microbial communities in these sediments – considerably up-
setting the standard EA ordering listed above. In this case, methanogens and Fe(III)
and sulfate reducers would be competing as the most active organisms in JdF sedi-
ments.
By contrast, the Gibbs energies of H2 oxidation seem to follow the standard
EA trend. As an example, see the energy profiles in SPG sediments depicted in
󳶳 Fig. 13.1 (a). However, there is another story here as well. Per mole of electron,
the energy available from H2 and OM oxidation in SPG follows the canonical order
of EAs listed above with reactions with H2 being slightly more exergonic than those
with OM for the same EA. Also, per mole of electron, the oxidation of Fe2+ and Mn2+
by O2 , respectively, fall into the energy-rich and energy-poor groups of reactions con-
sidered at SPG. Once the energy availability for this site is recast into units of J per
cm3 , however, the most energy-rich reactions are, by a wide margin, Fe2+ and Mn2+
oxidation by O2 throughout the sediment column. Simply put, there are far higher
concentrations of these two electron donors than H2 or OM in SPG sediments, so, per
cm3 , there is much more energy to be had from oxidizing them.

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292 | 13 Energetic constraints on life in marine deep sediments

13.7 Energy demand

Although the focus of this chapter is energy supply in the deep biosphere, the results
presented above also have a bearing on microbial energy demand, or at least how it
is sometimes considered. This is because the minimum quantity of energy that mi-
crobes must extract from catabolic reactions in order to remain active is often stated
in units of kJ per mole. This is alternatively referred to as a minimum Gibbs energy
(Δ𝐺min , [53]), the biological energy quantum, (Δ𝐺BQ , [54]), or a threshold Gibbs en-
ergy (Δ𝐺thr , [55]), and reported values span a very wide range from −3.8 to −49.6 kJ
per reaction turnover [54]. According to these authors, microorganisms can only cat-
alyze reactions that yield at least, e.g. −20 kJ mol−1 [56], an amount of energy related
to the mechanism of ATP synthesis. However, the energetic analysis detailed above
illustrates a potential pitfall of using this approach.
According to this commonly-used bioenergetic minimum theory, many reactions
cannot support microbial communities, because their yield is too low on a per mol
of substrate basis. However, some of these reactions – as shown above – appear to
be very energy-rich on a per cm3 of sediment basis. Based on bioenergetic minimum
theory, OM oxidation with Fe(III) and methanogenesis in JdF sediments, for example,
should not occur. However, per cm3 of sediment, these reactions yield several orders of
magnitude more energy than the knallgas reaction (O2 + H2 ) in SPG, which, according
to bioenergetic theory, yields sufficient energy for microorganisms to remain active. It
is difficult to reconcile why microorganisms that have access to nearly 1 J cm−3 from
one reaction would not use it, while those that only have 10−7 J cm−3 available from
another would. The reason that bioenergetic theory leads to this counterintuitive re-
sult is that it assumes that there is a fixed stoichiometry between catabolism and ATP
synthesis, the ultimate source of Δ𝐺min , Δ𝐺BQ , Δ𝐺thr , or whatever label is used to rep-
resent bioenergetic demand. That is, microorganisms ultimately get their energy from
catalyzing redox reactions, but ATP synthesis is a dehydration reaction; as such, they
cannot be uniquely, stoichiometrically coupled (see [57]). On a more intuitive level, in
systems with extremely low levels of H2 , as in SPG sediments, it seems unlikely that
microbes could sustain much activity powered by hydrogen oxidation. This is despite
its very negative values of Δ𝐺𝑟 (on a per mol basis). Conversely, despite their much
more modest values of Δ𝐺𝑟 , catabolic strategies such as FeOOH + OM and methano-
genesis in JdF are much more likely to fuel a microbial population due to very high
concentrations of OM present in the sediments there. Perhaps the minimum energy a
microorganism needs should be normalized like some maintenance energies are, in
units of Joules per microorganism per day (e.g [58, 59]) rather than trying to estimate
how much ATP needs to be made per mole of substrate. Although this is beyond the
scope of the present chapter, it is the focus of a study in progress [60].

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 13.9 Computational methods | 293

13.8 Concluding remarks

In a particular sediment at a particular depth, it is not only the most energy-yielding

redox reactions that are catalyzed by microorganisms. Multiple, overlapping respira-
tion and fermentation pathways can coexist [34, 44], microniches where the chemi-
cal conditions differ from the bulk sediments could support alternative pathways and
very short-lived intermediate chemical species could fuel catabolic reactions. How-
ever, at the most fundamental level, the energetics calculations presented above indi-
cate which catabolic reactions are thermodynamically favored to occur in deep marine
biosphere environments, and, in particular, at what depths in the sediment columns
these reactions are favored. By characterizing which catabolic strategies are possible
and which are not, a basic understanding of the biogeochemical dynamics of the deep
biosphere can be obtained. It is stressed that these conclusions are only possible be-
cause the environmental variables that distinguish each sediment column were taken
into account in the calculations. If only the standard state Gibbs energies would have
been considered, then all three environments would have looked identical. Further-
more, by taking into account the concentrations of the limiting electron donor or ac-
ceptor per cm3 of sediment, the variation of available energy from particular reactions
in each of these environments is revealed. By computing this energy density, a more
nuanced understanding of the deep biosphere emerges:
– The most exergonic reactions per mole of substrate may in fact provide very little
energy per cm3 sediment.
– Reactions that are not particularly exergonic per mole of substrate may provide
the most energy per cm3 sediment.
– The relative amounts of biomass can be accounted for (but only if the energetics
are quantified per volume or mass, not per mole).
– The order of EA usage by microbial communities may deviate from the canonical
order of O2 , NO−3 , MnO2 , FeOOH, SO2−
4 , CO2 and disproportionation.

Perhaps one of the greatest strengths of the approach taken in this study is that it can
be applied to any environment in order to obtain a basic understanding of bioenergetic
potential. Calculations of the Gibbs energy of reaction provide a fundamental assess-
ment of what is biologically possible in a given environment and a means to compare
how different geochemical variables govern (very) different ecosystems.

13.9 Computational methods

There is no easy way to categorize the energetic potential of particular electron donors
and acceptors without calculating the Gibbs energy of the reaction (Δ𝐺𝑟 ) at the pre-
vailing temperature, pressure and composition. In this study, values of Δ𝐺𝑟 are calcu-

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294 | 13 Energetic constraints on life in marine deep sediments

lated using
𝐾𝑟
Δ𝐺𝑟 = −𝑅𝑇 ln , (2)
𝑄𝑟
where 𝐾𝑟 and 𝑄𝑟 refer to the equilibrium constant and reaction quotient of the in-
dicated reaction, respectively, 𝑅 represents the gas constant and 𝑇 denotes temper-
ature in Kelvin. Values of 𝐾𝑟 were calculated using the revised-HKF equations of
state [61–63], the SUPCRT92 software package [64], and thermodynamic data taken
from [65–69]. Values of 𝑄𝑟 were calculated using
𝑣
𝑄𝑟 = ∏ 𝑎𝑖 𝑖 , (3)
𝑖

where 𝑎𝑖 stands for the activity of the 𝑖th species and 𝑣𝑖 corresponds to the stoichio-
metric coefficient of the 𝑖th species in the reaction of interest. Molalities of the 𝑖th
species, 𝑚𝑖 , were converted into activities using individual activity coefficients of the
𝑖th species (𝛾𝑖 ),
𝑎𝑖 = 𝑚𝑖 𝛾𝑖 . (4)
Values of 𝛾𝑖 were in turn computed as a function of temperature and ionic strength
using an extended version of the Debye–Hückel equation [70].
Complete reaction turnover refers to the situation in which the number of moles
of reactants and products processed in a chemical reaction is equal to their stoichio-
metric coefficients. For Reaction (10) in 󳶳 Table 13.1, complete reaction turnover would
indicate that 1 mole each of CH4 and SO2− −
4 are consumed and 1 mole each of HCO3 , HS
−

and H2 O are produced.

13.9.1 Thermodynamic properties of anhydrous ferrihydrite and pyrolusite

The standard state thermodynamic properties of anhydrous ferrihydrite at 25 °C were

taken from [71], who measured these properties for a slightly hydrated form of this min-
eral, FeOOH*0.027H2 O, and then estimated these properties for the completely dewa-
tered version by assuming that the associated water molecules have the same thermo-
dynamic properties as liquid H2 O. In the current study, this procedure was expanded
to the standard state isobaric heat capacity (𝐶𝑜𝑝 ) data reported by [71] in order to calcu-
late the other standard state thermodynamic properties of anhydrous ferrihydrite as a
function of temperature. These values of 𝐶𝑜𝑝 were regressed as a function of tempera-
ture (not shown) using the Maier–Kelley equation [72] resulting in the following heat
capacity power function coefficients: 𝑎 = 36.387 J K−1 mol−1 , 𝑏 = 0.15358 J K−2 mol−1 ,
𝑐 = −424,514 J K mol−1 .
The standard state thermodynamic properties of pyrolusite, MnO2 , were taken
from [73] and the Maier–Kelley heat capacity coefficients were regressed as a func-
tion of temperature using the Maier–Kelley equation from heat capacity data taken

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 Appendix | 295

from the same source. The resulting values of the 𝐶𝑜𝑝 power function coefficients are
𝑎 = 12.55 J K−1 mol−1 , 𝑏 = 0.009761 J K−2 mol−1 and 𝑐 = −210,500 J K mol−1 .
A caveat of using minerals in thermodynamic calculations is that their activities
are often taken to be 1, which was also done in this study. Therefore, the resulting
Gibbs energies, or energy per cm3 of sediment, do not account for the fact that a cm3
of sediment might contain > 99% or < 1% of this mineral. If the mass of the par-
ticular minerals used in this study per cm3 were known in marine sediments, then
this quantity could be used to constrain the energy density of these reactions per cm3
sediment. Because they are not known, the limiting substrate for these reactions was
always taken to be the aqueous electron donor and never the mineral.

Appendix

The concentrations of the species appearing in the catabolic reactions considered in

this study (󳶳 Table 13.1), which are required to calculate the Gibbs energies of reac-
tion, are shown as a function of depth for the three study sites in 󳶳 Figs. 13.A1–13.A3.
The sources of these data are stated in Section 13.3. Because the concentrations for all
species have not continuously been determined as a function of depth, the concentra-
tions of some species have been estimated over the some depth ranges. In most cases,
a species concentration missing at a particular depth was assumed to be between the
concentrations measured above and below it. However, for several species, larger gaps
in the concentration record were filled in as follows. In JdF sediments, concentration
of NH+4 from 121–170 mbsf were taken to be a linear interpolation between the concen-
trations of NH+4 reported above and below this depth interval, the bounding values. In
PM sediments, the concentration of SO2− 4 from 41–66 mbsf were taken to be 10 μM be-
cause the concentrations were below detection (not stated), the concentration of CH4
between 131–156 mbsf were taken to be a mean of the bounding values and pH was
interpolated between values measured at the top and bottom of the sediment column.
In SPG sediments, at many depths, H2 concentrations were below detection (2.8 nM),
so a nominal values of 1 nM was used and between 43–63 mbsf and 74–76 mbsf and
the O2 concentrations were calculated from a 4𝑡ℎ order polynomial whose regression
coefficients were determined from O2 concentration at the other sediment depths. The
results of all of these estimates and the measured data are shown in 󳶳 Fig. 13.A1–13.A3.
Furthermore, the cell counts for SPG sediments were interpolated at many depths. In
particular, between 42 and 76 mbsf, a power-law function was applied to the rest of the
cell count data to estimate cell numbers in this interval, which is plotted in 󳶳 Fig. 13.2.

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296 | 13 Energetic constraints on life in marine deep sediments

2- 2+ 2+
DIC (mmol/kg) [SO ] (mmol/kg) [Fe ] (μmol/kg) [Mn ] (μmol/kg)
4
0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 50 100 150 0 50 100 150 200 250 300
0

50

100
Depth (mbsf)

150

200

250

+
[CH ] (log mmol/kg) DOC (mmol/kg) NH (μmol/kg) pH
4 4
-6 -5 -4 -3 -2 0 5 10 15 20 400 800 1200 1600 2000 6 6.5 7 7.5 8 8.5 9 9.5
0

50

100
Depth (mbsf)

150

200

250

Fig. 13.A1: Selected geochemical data for Juan de Fuca sediments.

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 Appendix | 297

[DIC] mmol / kg [CH ] μmol / kg [acetate] mmol / kg

4
5 10 15 20 25 0 1000 2000 3000 0 2 4 6 8 10 12 14
0

50
Depth (mbsf)

100

150

2+
[Fe ] μmol / kg 2+
[Mn ] μmol / kg
2-
[SO4 ] mmol / kg
0 3 6 9 12 15 0 2 4 6 8 10 12 0 10 20 30
0

50
Depth (mbsf)

100

150

Fig. 13.A2: Selected geochemical data for Peru Margin sediments.

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298 | 13 Energetic constraints on life in marine deep sediments

2- 2+
DIC [SO ] (mmol/kg) 2+
Fe (μmol / kg) Mn (μmol / kg) TOC, wt%
4
1.8 2 2.2 2.4 2.6 2.8 25 26 27 28 29 2 3 4 5 6 7 8 9 2 4 6 8 10 12 14 160 0.06 0.12 0.18 0.24 0.3
0

10

20
Depth (mbsf)

30

40

50

60

70

80

-
NO (μmol / kg) O (μmol/kg) [H ] nmol / kg pH
3 2 2
30 35 40 45 50 55 60 65 60 80 100120140160180200 0 5 10 15 20 25 30 35 7 7.1 7.2 7.3 7.4 7.5 7.6 7.7
0

10

20
Depth (mbsf)

30

40

50

60

70

80

Fig. 13.A3: Selected geochemical data for South Pacific Gyre sediments.

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 References | 299

Acknowledgements

Financial assistance was provided by the Center for Dark Energy Biosphere Investiga-
tions (C-DEBI), the NASA Astrobiology Institute – Life Underground (NAI-LU) and the
National Science Foundation grant OCE-1207874. This is C-DEBI contribution 169 and
NAI-LU contribution 001.

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