RESEARCH ARTICLE
Weighing the deep continental biosphere
Sean McMahon & John Parnell
School of Geosciences, University of Aberdeen, Aberdeen, UK
Correspondence: Sean McMahon, School
of Geosciences, University of Aberdeen,
Aberdeen AB24 3UE, UK.
Tel.: +44 (0)1224 273433;
fax: +44 (0)1224 272785;
e-mail: [email protected]
Received 17 June 2013; revised 21 August
2013; accepted 23 August 2013.
Final version published online 19 September
2013.
DOI: 10.1111/1574-6941.12196
Abstract
There is abundant evidence for widespread microbial activity in deep continental fractures and aquifers, with important implications for biogeochemical
cycling on Earth and the habitability of other planetary bodies. Whitman et al.
(P Natl Acad Sci USA, 95, 1998, 6578) estimated a continental subsurface
biomass on the order of 1016–1017 g C. We reassess this value in the light of
more recent data including over 100 microbial population density measurements from groundwater around the world. Making conservative assumptions
about cell carbon content and the ratio of attached and free-living microorganisms, we find that the evidence continues to support a deep continental
biomass estimate of 1016–1017 g C, or 2–19% of Earth’s total biomass.
Editor: Gary King
Keywords
subsurface; biomass; groundwater; aquifer.
MICROBIOLOGY ECOLOGY
Introduction
Microbial life in continental aquifers and deep fractures
constitutes a large carbon reservoir and may play an important role in global biogeochemical cycles. However, little is
known about its total size, diversity, activity or distribution
across time and space. In a widely cited paper, Whitman
et al. (1998) estimated a continental subsurface biomass on
the order of 1016–1017 g C and a marine subsurface (i.e.
subseafloor) biomass on the order of 1017 g C. Kallmeyer
et al. (2012) found that Whitman et al. had overestimated
marine subsurface biomass by 1–2 orders of magnitude.
Hence, a reassessment of continental subsurface biomass is
timely. Here, we attempt this reassessment using recent
measurements of groundwater microbial population density, cell carbon content and the ratio of free-living to
attached microorganisms in groundwater.
Whitman et al. (1998) calculated terrestrial subsurface
prokaryotic cell numbers in the top 4 km of groundwater by
three methods based on weakly constrained estimates for
key global parameters. Using cell counts measured in a range
of marine and terrestrial unconsolidated sediments and
extrapolated below 600 m (on the assumption of a logarithmic decline with depth), they obtained a total of 2.5 9 1029
cells: a minimum value because unconsolidated sediments
represent only a small part of the continental subsurface.
Secondly, using rock porosity and cell volume as a percentFEMS Microbiol Ecol 87 (2014) 113–120
age of pore space, they obtained a total of 2.2 9 1030 cells.
Finally, using the global volume of groundwater, an average
number of unattached cells per volume of water (‘cell density’), and a ratio of grain-surface-attached to unattached
cells, they obtained 2.5 9 1030 cells. Despite the limited
scope of the minimum value calculated from unconsolidated
sediments, the cell numbers and associated biomass were
reported to range from 2.5 to 25 9 1029 cells, and 22 to
215 Pg of carbon (1 Pg = 1015 g), assuming a rather large
cell mass of 172 fg (50% C, i.e. 86 fg C). In a study of marine subsurface cell numbers, Kallmeyer et al. (2012) adopted
the arithmetic mean of these upper and lower biomass
estimates for the terrestrial subsurface, that is, 119 Pg C. Fry
et al. (2009) raised the lower estimate for the number of cells
to 6 9 1029 on the assumption that there is no decline in
cell density with depth, contrary to the inference made by
Whitman et al. (1998) from submarine trends.
From a small data set, Whitman et al. (1998) calculated
an average density of free cells of 1.54 9 105 mL1 in
groundwater and assumed that only 0.058% of cells are
free, while the rest are attached to grain surfaces, yielding
a total of 2.5 9 1030 cells in a groundwater volume of
9.5 9 1021 mL. For an assumed cellular carbon content
of 86 fg C, this represents 215 Pg carbon. Using more
recent data, we reassess the three critical parameters of
groundwater cell density, proportion of unattached cells
and cell carbon content, bearing in mind that cells in the
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
114
S. McMahon & J. Parnell
deep subsurface typically exhibit adaptation to starvation
survival (Amy et al., 1993; Kieft et al., 1997).
Methods
Groundwater cell density
We compiled c. 120 individual measurements of unattached microbial cell densities (cells mL1) from 24
studies representing continental aquifers and fracture
waters at depths ranging from 10 to 3600 m (Table 1).
To determine a global average groundwater cell density as
a function of depth, the arithmetic mean cell count was
calculated for each 250-m-depth interval for each sampling site. The global arithmetic mean in each depth bin
was then calculated from these local means. Binning in
this way was intended to clarify global trends by mitigating the heavy sampling bias towards shallower depths and
Table 1. Details of groundwater cell density measurements used in this study
Host lithology
Location(s)
Cell count
method
Metabolic groups
recorded
Depth (m)
References
Carbonate
Paris, France
DAPI
800
Basso et al. (2005)
Clastic sedimentary
rocks
Shale, sandstone
Granite
Granite, sandstone,
conglomerate
Ketzin, Germany
DAPI
A, autotrophs,
heterotrophs,
CH4-gens, SR
A, SR, ?CH4-gens
647
Morozova et al. (2010)
New Mexico, USA
Mizunami, Japan
Tono, Japan
A, CH4-gens
A+, A, heterotrophs, ?NR
A+, A, heterotrophs, NR,
autotrophic SR, SX, DN
183–191
1169
104–177
Takai et al. (2003)
Fukuda et al. (2010)
Murakami et al. (2002)
Mudstone
Mudstone or
sandstone
Quartzite
Sandstone
Hokkaido, Japan
Hokkaido, Japan
AO
AO
AO, DAPI,
SYBR
Green I
AO
AO
Heterotrophs, ?CH4-gens
A+, A, SR, CH4-gens, DN
37–480
297–458
Kato et al. (2009)
Shimizu et al. (2006)
A, SR, CH4-gens
A, heterotrophs, HX,
SR, CH4-gens
A+, A, heterotrophs, SR
2830–3270
937
Moser et al. (2005)
Kimura et al. (2005)
10–101
Pedersen et al. (1996)
A+, A, autotrophs,
heterotrophs, SR, NX,
SX, CH4-trophs
A+, A, autotrophs,
heterotrophs, SR
No data
A, autotrophic and
heterotrophic acetogens
and CH4-gens, SR, FeR,
Heterotrophs
Heterotrophs, ?SR
A+, A, heterotrophs, SR, DN
Heterotrophs
Heterotrophs, CH4-gens
1300–3600
Borgonie et al. (2011)
100–1500
It€
avaara et al. (2011)
75–200
248–910
O’Connell et al. (2003)
Haveman et al. (1999)
836–1039
803–1105
240
816–1105
129–860
Pedersen & Ekendahl
Pedersen & Ekendahl
Jain et al. (1997)
Ekendahl & Pedersen
Pedersen & Ekendahl
171–978
Hallbeck & Pedersen (2008)
2825
316–1270
Lin et al. (2006)
Stevens et al. (1993)
3100
1274–1492
Kieft et al. (2005)
Olson et al. (1981)
Gauteng, South Africa
Western Queensland,
Australia
Oklo, Gabon
FC
AO
Transvaal, Free State and
Gauteng, South Africa
DAPI
Schist, ultramafics
Outokumpu, Finland
BacLight
Basalt
Gneiss, granite,
granodiorite
Southeastern Idaho, USA
Olkiluoto, Kivetty, H€astholmen
and Romuvaara, Finland
AO
AO and
DAPI
Granite
Granite
Granite
Granite
Granite/granodiorite
AO
AO
AO
AO
AO
Granite/granodiorite
Laxemar, Sweden
V€astmanland, Sweden
Manitoba, Canada
V€astmanland, Sweden
€ o
€, Avro, and Laxemar,
Asp
Sweden
€ o
€, Sweden
Asp
Metabasalt
Basalt
North West, South Africa
Washington State, USA
Syto-13
AO
Metavolcanics
Dolomitic limestone
Gauteng, South Africa
Montana, USA
FC
AO
Sandstone,
mudstone
Dolomite,
igneous rocks
AO
AO
A+, A, autotrophic and
heterotrophic acetogens and
CH4-gens, SR, FeR, MnR, NR
A, autotrophic SR, CH4-gens
A+, A, heterotrophs,
CH4-gens, SR, FeR, MnR, NR
A, SR
A, SR, CH4-gens
(1992a)
(1992b)
(1994)
(1990)
BacLight and Syto-13 are epifluorescence-based techniques. Metabolic groups overlap. Some depth limits are interval midpoints.
AO, acridine orange; DAPI, 4′,6′-diamino-2-phenylindol; FC, flow cytometry; A+, aerobic; A, anaerobic; SR, sulphate reducers; FeR, iron reducers;
MnR, manganese reducers; NR, nitrate reducers; NX, nitrite oxidisers; HX, hydrogen oxidisers; DN, denitrifiers; SX, sulphur oxidisers; ?, inferred.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Ecol 87 (2014) 113–120
115
Weighing the deep continental biosphere
moderating the influence of large or multiple data sets
representing single sampling sites (by allowing each sampling site to count only once towards each 250-m bin).
In fact, only the shallowest six depth bins, spanning
1500 m, were sampled in four or more distinct sites
(Table 2). Mean cell densities for these bins are shown in
Fig. 1 (bars).
Groundwater volume and distribution
To convert the cell density–depth profile into total unattached cell numbers requires a known volume of groundwater and a groundwater volume–depth distribution so
that the depth-binned cell density averages can be
weighted by the proportion of groundwater present in
each depth interval. Sokolov (1977) estimated a global
groundwater volume of 2.43 9 1022 mL. Following Whitman et al. (1998), we assume that the entire volume of
groundwater represents habitable space. Whitman et al.
used a volume of 9.5 9 1021 mL groundwater in 4 km
depth. We use a volume of 1022 mL for 2 km depth (Fry,
2005), which avoids spurious precision. We assume that
the vertical distribution of this habitable space follows the
simple porosity–depth relationship (compaction curve)
given by Athy’s law (Athy, 1930):
/ðzÞ ¼ /0 ekz
where φ = porosity, φ0 = surface porosity, k = the compaction coefficient and z = depth. Because sandstone
hosts a large proportion of the world’s groundwater
(Foster & Chilton, 2003), compaction coefficients were
Table 2. Depth distribution of cell density measurements and
independent sampling sites
Depth interval (m)
Number of cell
density measurements
Number of
independent
sampling sites
0–249
250–499
500–749
750–999
1000–1249
1250–1499
1500–1749
1750–1999
2000–2249
2250–2499
2500–2749
2750–2999
3000–3249
3250–3499
3500–3749
37
21
13
14
6
4
2
0
0
0
0
3
3
1
1
11
8
9
7
4
4
2
0
0
0
0
2*
1*
1*
1*
*Sites within the Witwatersrand Basin, South Africa.
FEMS Microbiol Ecol 87 (2014) 113–120
Fig. 1. Cell density measurements from groundwater used in this
study. Each point represents one measurement (or a mean of tightly
clustered measurements from one study).
extracted from seven studies of marine and continental
sandstones to determine the required weighting (Fig. 2).
If no weighting is applied, the mean cell density is
4.1 9 105 cells mL1. Weighting by the minimum and
maximum values of k yields total average densities
4.8 9 105 and 6.1 9 105 cells mL1 respectively, assuming that the mean cell density in the 1750- to 1999-mdepth bin, for which no data were available, follows the
logarithmic trend extrapolated from the 0- to 1749-m cell
densities (R2 = 0.84). The average k value, 5.3 9
104 m1, yields an average cell density of 5.2 9 105
unattached cells mL1, an increase of c. 27% over the unweighted value. The final biomass estimate is increased
proportionally.
Attached vs. unattached cells
The ratio of attached to unattached cells in the deep continental biosphere is poorly constrained. Using data from
a single sandstone aquifer (Hazen et al., 1991), Whitman
et al. (1998) determined that 0.058% of cells in
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
116
S. McMahon & J. Parnell
supplies are high. Where nutrient levels are limited, as
expected in most of the deep biosphere, attachment
favours survival (Marshall, 1988; Griebler et al., 2002).
Indeed, the proportion of viable cells is higher in the
attached than in the unattached population (Hazen et al.,
1991). As the world’s major aquifers occupy clastic sediments and sedimentary rocks ranging in particle size from
silt to gravel (Foster & Chilton, 2003), we adopt a range
from 102 to 103 to scale up from viable groundwater cell
determinations to the whole sample; this results in an
order-of-magnitude range in the final biomass estimate.
Carbon content of cells
Fig. 2. Porosity profiles from seven studies of sandstones: (1) Mount
Simon aquifer, USA (Person et al., 2010); (2) Submarine sandstones
(Bahr et al., 2001); (3) New Jersey coastal plain, USA (Kominz &
Pekar, 2001); (4) Terrestrial oil and gas reservoirs, USA (Maxwell,
1964); (5) Nigeria delta/coastal plain (Benjamin & Nwachukwu, 2011);
(6) Mount Simon aquifer, USA (Medina et al., 2011); (7) Petroleumbearing sandstones, USA (Chapman et al., 1984). The mean (black)
closely matches Kominz & Pekar (2001).
groundwater are unattached, equivalent to an attached/
unattached ratio of 1723. Considering only viable (not
dead) cells, the same data yield a value of 0.22% cells
unattached (attached/unattached = 454), based on 21
pairs of measurements in three wells. Several data sets
have more recently become available from other aquifers.
In a pristine groundwater site, Griebler et al. (2002)
reported data equivalent to an attached/unattached ratio
of about 1050 (mean of 7 ratios over 10 months).
Alfreider et al. (1997) report data equivalent to a mean
total/unattached ratio of 1010 from four groundwater
wells, assuming 30% porosity. A simulated basalt aquifer
system has yielded 99% biomass attached to the substrate
(Lehman et al., 2001), that is, a ratio of about 100, but
using a mean particle size coarser than sand. Similarly, a
coarse (gravelly) natural aquifer yielded ratios of between
olbel-Boelke et al., 1988). For a typical fine
10 and 103 (K€
to medium sand, the particle surface area would be an
order of magnitude greater and the resultant ratio also
greater. However, still finer-grained clays and silts have
been reported to support lower abundances of microorganisms than adjacent sands, perhaps because of restrictively small pore size (Sinclair & Ghiorse, 1989;
Fredrickson et al., 1997).
Lower ratios have also been measured (ranging down
to < 1; see review in Cozzarelli & Weiss, 2007), but for
oil-bearing or contaminated aquifers where nutrient
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
The carbon content of cells varies widely, and indeed the
biomass calculation of Whitman et al. (1998) uses values
ranging from 10 to 100 fg C for different host environments, including 86 fg C for terrestrial subsurface cells. A
recent synthesis of numerous studies of carbon contents
in bacteria, not specific to the subsurface, indicates a
majority of values in the range 20–100 fg C (Romanova
& Sazhin, 2010). However, in a nutrient-limited deep biosphere, it is safer to adopt a mass typical for bacterial
cells in starvation conditions, for which a mean and consistent value of 26 fg C has been determined from 10 cultivated strains (Troussellier et al., 1997). We therefore
adopt this value, with the caveat that uncultured and
environmental species may commonly have still smaller
cell biomass.
Results and discussion
Biomass
Weighting the extrapolated cell density–depth distribution
(< 1749 m) by the distribution of groundwater yields an
average cell density of 5.2 9 105 cells mL1 in the top
2 km. This revises upwards the equivalent value of
1.54 9 105 determined by Whitman et al. (1998) and
represents 5.2 9 1027 unattached cells according to a
groundwater volume estimate of 1022 mL. Combining
these estimates with an attached/unattached ratio ranging
from 102 to 103 and a cellular carbon content of 26 fg C
yields a total biomass of 14–135 Pg for the top 2 km of
continental crust (Table 3). The relative insignificance of
deeper biomass can be crudely illustrated by extrapolating
the logarithmic groundwater volume–depth and cell density–depth curves to 5 km, which yields a total biomass
c. 0.2% greater than the 2-km estimate (The assumption
that cell density continues to decline logarithmically
below 2 km is discussed in the next section). Excluding
the two data points in the 1500–1749 depth bin also
increases the total biomass by < 1%.
FEMS Microbiol Ecol 87 (2014) 113–120
117
Weighing the deep continental biosphere
Table 3. Estimated key parameters determined in this study and by Whitman et al. (1998). The new average cell-density estimate is based on a
global data set weighted by the estimated vertical distribution of groundwater
This paper
Whitman et al. (1998)
Groundwater cell
density (cells mL1)
Cell biomass
(fg)
Attached/unattached
cells ratio
Groundwater
volume (mL)
Total biomass
(Pg C)
5.2 9 105
1.54 9 105
26
10–100
102–103
1723
1022
9.5 9 1021
14–135
22–215
The new biomass estimate overlaps the range suggested
by Whitman et al. (1998) for the top 4 km and represents
2–19% of Earth’s total biomass (Kallmeyer et al., 2012).
Using the marine subsurface biomass of 1.5–22 Pg estimated by Kallmeyer et al., we obtain the surprising result
that continental subsurface biomass may be equal to or
larger than the marine subsurface biomass, which
amounts to 0.2–3.5% of Earth’s total. Nevertheless, the
continental estimate should be regarded as conservative
given that:
(1) The data used in this calculation are skewed towards
aquifers (which are most commonly of sand grain-size)
and fractured crystalline rock (which are effectively
coarse-grained). Finer-grained sediments are likely to
have higher ratios of attached/unattached cells (Albrechtsen, 1994; Griebler et al., 2002), which may substantially
raise the true biomass; on the other hand, clays and silts
have been reported to support lower abundances of
microorganisms than adjacent sands (Sinclair & Ghiorse,
1989; Fredrickson et al., 1997).
(2) Measurements from coal and hydrocarbon-producing
systems, which provide exceptional carbon sources for
heterotrophic communities, were not included. Such
deposits probably do not host a large fraction of continental groundwater, but they may perhaps contribute
significantly to the total biomass (given that microbial
populations grow geometrically). Similarly, nutrient-contaminated groundwaters were not included.
(3) Most of the world’s groundwater is stored in clastic
sedimentary aquifers (Foster & Chilton, 2003), which
contribute only 30% of the available cell density database.
If the average cell density is extrapolated from these data
alone, which include the highest reported values, the estimated biomass rises about twofold. However, there are
too few data to say with confidence whether cell densities
are consistently higher in any particular lithological
context.
There are at least three other sources of major uncertainty attached to our estimate. Firstly, the wells from
which groundwater samples are taken can themselves be a
source of microbial contamination or a focus for biofilm
formation. Basso et al. (2005) found that purging and
mechanically cleaning an 800-m-deep well caused an order
of magnitude decrease in the cell density of the groundwater it sampled. Sampling techniques and precautions taken
FEMS Microbiol Ecol 87 (2014) 113–120
to avoid contamination differ widely between studies. Secondly, cell counts were measured using fluorescent stains,
which can sometimes infiltrate mineral particles, leading to
falsely high cell counts (although some studies guard
against this; Murakami et al., 2002). Thirdly, and perhaps
most importantly, the geographical and geological distributions of cell count data do not reflect the variety of the
Earth’s continental crust or groundwater. The Fennoscandian shield and the Witwatersrand Basin of South Africa
are over-represented, and most of the world’s major aquifer
systems are absent from the data set.
Cell density–depth distribution
By analogy with the marine subsurface biosphere,
Whitman et al. (1998) expected groundwater cell densities to decrease logarithmically with depth below the continental surface, an inference recently challenged (Fry
et al., 2009; Breuker et al., 2011). In our data set, the six
250-m averages in the top 1499 m, each representing at
least four separate groundwater reservoirs, fit reasonably
well to a logarithmic regression (R2 = 0.78), suggestive of
a global trend over this depth interval. Only two reservoirs are sampled by the 1500- to 1749-m bin but, if
included, these measurements continue the apparent logarithmic decline (R2 = 0.84). If the very low measurement
of 2.0 9 102 cells mL1 at 1700 m (Borgonie et al.,
2011) is regarded as an outlier and excluded, R2 rises to
0.86 (increasing the final biomass estimate by < 1%).
Unfortunately, few groundwater cell density measurements are available from below this depth, and all hail
from the same geological system, the Witwatersrand Basin
of South Africa. This basin lies in a stable cratonic region
and therefore maintains very low geothermal gradients
(< 10 °C km1; Omar et al., 2003) and hence potentially
deeper habitable conditions than average continental
crust. At depths > 3 km, Kieft et al. (2005) and Borgonie
et al. (2011) recorded low cell densities (respectively
4 9 103 mL1 at 3100 m and 3.4 9 103 mL1 at
3600 m) consistent with the global logarithmic decline
suggested by the shallower data set, although Borgonie
et al. reported even lower cell densities at 1300 and
1700 m (3.0 9 103 and 2.0 9 102 mL1 respectively; the
latter not in the Witwatersrand). Inclusion of these data
improves the goodness of fit of the depth bin averages to
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
118
a logarithmic regression (R2 = 0.89). In stark contrast,
Moser et al. (2005) obtained cell densities on the order of
106 mL1 at c. 3200 and 3300 m, sharply reversing the
apparent decline with depth.
It seems plausible that a global decline in cell densities
through shallower depths might reflect the diminishing
supply of photosynthetic organic matter from above,
while deeper communities are supported independently
by geochemical carbon sources and redox reactants.
However, microbial communities in the Witwatersrand
may be atypical for continental environments at these
depths, even in a cratonic basin; local phenomena
including abiogenic hydrocarbon generation and an
anomalous radiolytic hydrogen flux may provide exceptional habitats for microorganisms (Lin et al., 2005;
Borgonie et al., 2011). Hence, more data are needed,
particularly from multi-kilometer deep settings geographically and geologically distinct from the Witwatersrand
Basin, before a ‘typical’ continental cell density–depth
profile can be constructed robustly. On balance, however, we cautiously suggest that the global data set does
support a logarithmic decline with depth in groundwater
cell densities, as postulated by Whitman et al. (1998).
This is a distinct question from the distribution of biomass per se, which is shaped by the roughly exponential
decline with depth in the availability of pore space and
groundwater.
Future prospects
To further constrain deep continental biomass, a much
wider range of depths, aquifer systems and lithological
contexts must be sampled for measurements of cell
population density and cell carbon content. The apparent
decline with depth in both habitable space and groundwater cell density suggests that biomass below 2-km
depth may contribute only marginally to the total.
However, much more extensive drilling at these depths is
necessary to clarify global trends, especially given the
unexpectedly high cell counts c. 3 km deep in the Witwatersrand Basin. Many of the world’s largest aquifer systems are missing from the present data set. The potential
for the discovery of new organisms and new community
structures is suggested by the identification of a fracture
in the Witwatersrand dominated by a single previously
unknown genus of bacterium (Chivian et al., 2008). The
most poorly constrained variable in the present study is
the ratio of attached and unattached cells; more work is
needed to understand and predict how this ratio varies
with lithology and community structure. There is also a
pressing lack of quantitative data about the global
volume of groundwater and its three-dimensional
distribution.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
S. McMahon & J. Parnell
Conclusions
A reappraisal of groundwater cell density, cell carbon
content and the ratio of attached to unattached cells predicts a total subsurface continental biomass between 1016
and 1017 g carbon. Hence, the continental subsurface biomass may be similar to or even larger than the subseafloor biomass. The data appear to indicate a global
logarithmic decline in groundwater cell density with
depth. The biomass estimate is similar to that of Whitman et al. (1998) but should be regarded as conservative
given the cautious choice of assumptions.
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