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PNAS
2023 Vol. 120 No. 51 e2302156120
https://doi.org/10.1073/pnas.2302156120
1 of 11
Microbially induced precipitation of silica by anaerobic
methane
-
oxidizing consortia and implications for microbial
fossil preservation
Daniela Osorio
-
Rodriguez
a,1,2
, Kyle S. Metcalfe
a,1
, Shawn E. McGlynn
a,b
, Hang Yu
a
,c
, Anne E. Dekas
a
,d
, Mark Ellisman
e
, Tom Deerinck
e
,
Ludmilla Aristilde
f
, John P. Grotzinger
a
, and Victoria J. Orphan
a,2
Edited by Donald Canfield, Syddansk Universitet, Odense M., Denmark; received February 7, 2023; accepted November 6, 2023
RESEARCH ARTICLE
|
ECOLOGY
EARTH, ATMOSPHERIC, AND PLANETARY SCIENCES
Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxi-
dation of methane (AOM) in the rock record. It is not currently known whether the
microorganisms that mediate sulfate
-
coupled AOM—often occurring as multicelled
consortia of anaerobic methanotrophic archaea (ANME) and sulfate
-
reducing bacteria
(SRB)—are preserved as microfossils. Electron microscopy of ANME
-
SRB consortia in
methane seep sediments has shown that these microorganisms can be associated with
silicate minerals such as clays [Chen
et al
.,
Sci. Rep.
4
, 1–9 (2014)], but the biogenicity
of these phases, their geochemical composition, and their potential preservation in the
rock record is poorly constrained. Long
-
term laboratory AOM enrichment cultures in
sediment
-
free artificial seawater [Yu
et al
.,
Appl. Environ. Microbiol.
88
, e02109
-
21
(2022)] resulted in precipitation of amorphous silicate particles (
~
200 nm) within clus
-
ters of exopolymer
-
rich AOM consortia from media undersaturated with respect to
silica, suggestive of a microbially mediated process. The use of techniques like correlative
fluorescence in
situ hybridization (FISH), scanning electron microscopy with energy dis
-
persive X
-
ray spectroscopy (SEM
-
EDS), and nanoscale secondary ion mass spectrometry
(nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments
further revealed that they are enveloped in a silica
-
rich phase similar to the mineral phase
on ANME
-
SRB consortia in enrichment cultures. Like in cyanobacteria [Moore
et al
.,
Geology
48
, 862–866 (2020)], the Si
-
rich phases on ANME
-
SRB consortia identified
here may enhance their preservation as microfossils. The morphology of these silica
-
rich
precipitates, consistent with amorphous
-
type clay
-
like spheroids formed within organic
assemblages, provides an additional mineralogical signature that may assist in the search
for structural remnants of microbial consortia in rocks which formed in methane
-
rich
environments from Earth and other planetary bodies.
ANME
-
SRB | methane seeps | microbial biomineralization | amorphous silic
a | microfossils
The anaerobic oxidation of methane (AOM) is a microbially driven syntrophic process in
ocean sediments worldwide that modulates methane flux and mediates the production of
authigenic minerals, including carbonates and iron sulfides. AOM is frequently mediated
by multicellular consortia of anaerobic methanotrophic archaea (ANME) and sulfate
-
reducing bacteria (SRB) which couple the oxidation of methane to sulfate reduction (1,
2). The production of one mole each of bicarbonate and sulfide increases porewater alka­
linity in two total units in zones of AOM activity per mole of oxidized methane, driving
the precipitation of carbonate minerals (3, 4) following Eq.
1
:
[
1
]
Geochemical modeling (5) and isotope signatures (6, 7) in methane seep carbonates imply
rapid precipitation rates during early diagenesis, suggesting that the activity of ANME
-
SRB
consortia within these rocks and sediments stimulate carbonate precipitation (8, 9).
Therefore, it is surprising that silicates—not carbonates— with compositions and platy
morphologies similar to clay minerals have been found in intimate association with envi­
ronmental ANME
-
SRB consortia (10, 11) recovered from methane seep sediments. These
clay
-
like phases and Si
-
rich cements have been proposed to form on consortia through
AOM
-
initiated dissolution of sedimentary Si
-
bearing minerals (e.g., clays, quartz, or
diatom frustules) followed by reaction between dissolver porewater silicon (Si) and metal
cations adsorbed to extracellular polymeric substances (EPS) coating ANME
-
SRB con­
sortia exteriors, and possibly serving as sites for microbial surface adhesion (10). However,
as these phases have thus far only been observed on ANME
-
SRB consortia recovered from
methane seep sediments, the extent to which ANME
-
SRB consortia directly mediated
CH
4

g

+
SO
2
4

aq

HS

aq

+
HCO
3

aq

+
H
2
O.
Significance
Anaerobic methanotrophic
archaea (ANME) and sulfate
-
reducing bacteria (SRB) often
associate as multicelled consortia
in methane seep sediments and
carbonates, with a poorly
understood preservation potential
in the rock record. Here, we
provide evidence for microbially
enhanced precipitation of an
amorphous silica
-
rich phase on
the exterior of ANME
-
SRB
consortia both in methane
-
rich
sediments, carbonates, and a
methane
-
oxidizing laboratory
enrichment culture. This
consortia
-
associated mineral
phase may represent a newly
discovered microbially enhanced
biomineralization mechanism,
potentially involved in the
preservation of fossilized ANME
-
SRB consortia in ancient methane
seep carbonates. Our results
expand on the knowledge of
microorganisms and metabolisms
facilitating silica mineralization,
with important implications for
the morphological and structural
preservation of microbial fossils
in deep time on Earth and other
planetary bodies.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Copyright © 2023 the Author(s). Published by PNAS.
This article is distributed under
Creative Commons
Attribution
-
NonCommercial
-
NoDerivatives License 4.0
(CC BY
-
NC
-
ND)
.
1
D.O.
-
R. and K.S.M. contributed equally to this work.
2
To whom correspondence may be addressed. Email:
danieosro@gmail.com or vorphan@caltech.edu.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2302156120/-
/DCSupplemental
.
Published December 11, 2023.
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the precipitation of these phases, as opposed to a passive attach­
ment from the environment, remains unclear.
Understanding the biogenic vs. abiotic factors contributing to
the observed Si
-
rich phases described from uncultured environ­
mental AOM consortia from complex sediment matrices is a
challenge. Authigenic silicate precipitation can occur abiotically
in sediments through interactions between porewater Si and
clay
-
type aluminosilicates (12). Additionally, microbially mediated
sulfate
-
coupled methane oxidation increases porewater alkalinity,
which can further enhance the dissolution of silicon from quartz,
diatom frustules, and other sediment hosted silicate minerals, with
silica subsequently redepositing on the exteriors of ANME
-
SRB
consortia (10). Finally, microbial surface ligands can also favor the
nucleation of amorphous silica, as previously suggested for
ANME
-
SRB consortia (10), and for microorganisms in hot
springs and geothermal systems (13).
In this study, we leverage our sediment
-
free methane
-
oxidizing
anaerobic enrichment cultures of ANME
-
2 and SRB (14) and
laboratory
-
maintained authigenic seep carbonates with active endo
­
lithic AOM consortia (15) to examine the potential production of
these Si
-
rich phases outside of the sediment environment and under
chemically controlled laboratory conditions. ANME
-
SRB consor
­
tia in our enrichment cultures have abundant Si
-
rich nanopartic­
ulate spheres embedded in EPS, while the exteriors of many
carbonate
-
hosted endolithic AOM consortia were encrusted in
Si
-
rich rims. Notably, the de novo production of these Si
-
rich
phases on ANME
-
SRB consortia in culture occurred from solu­
tions undersaturated with respect to Si. Therefore, it is suggestive
of bona fide precipitation of a Si
-
rich phase under active
sulfate
-
coupled methane oxidation mediated by ANME
-
SRB con
­
sortia, both in cultures and in a diversity of methane seep habitats,
including carbonates (15) and sediments.
As authigenic silicates can entomb and preserve organic carbon
(16, 17), the involvement of ANME
-
SRB consortia in facilitating
silicate precipitation may enhance the preservation of methane
-
associated microbial biomass in fossil seep environments, fre­
quently occurring as
13
C depleted carbonate mounds and crusts
(18, 19). Previous work by our group and others has demonstrated
active methanotrophic ANME
-
SRB consortia living within the
interiors of modern seep carbonates (8, 9, 20), and has docu­
mented mineral precipitates with consortium
-
like size and mor­
phology (21). However, we have not yet identified living or fossil
ANME
-
SRB consortia directly in seep carbonates with high pres
­
ervation potential such as that observed in other microbial eco­
systems conferred by early silica precipitation (22). We posit that
the silica encrustation of endolithic ANME
-
SRB consortia within
seep carbonates could enhance their long
-
term preservation and
represents a key trait for identifying fossilized ANME
-
SRB con­
sortia in the rock record.
Results
ANME
-
SRB Consortia in Sediment
-
free Enrichment Cultures
are Associated with Nanoscale Amorphous Si
-
rich Spherical
Particles.
We investigated the hypothesis of ANME and SRB
-
mediated silicate precipitation (10) by examining anaerobic
methane
-
oxidizing ANME
-
2 and SRB consortia enriched from
seep sediments after 5 y of anoxic cultivation in the laboratory.
The inoculum for these slow
-
growing sediment
-
free cultures
was sourced from a seep sediment, and maintained under
methane headspace in artificial seawater as described in ref.
14. Using correlated fluorescence in situ hybridization (FISH),
scanning electron microscopy (SEM), and energy dispersive
X
-
ray spectroscopy (EDS) imaging (Fig. 1
A
C
) on the AOM
enrichment cultures, we identified multiple AOM consortia in an
EPS matrix that were covered with clusters of
~
200 nm diameter
spherical Si
-
rich particles (Fig. 1
D
). These were morphologically
similar to previously reported amorphous silica
-
bearing spherical
precipitates associated with cyanobacteria in hot springs (23, 24).
The SEM imaging of consortium exteriors revealed the universal
presence of Si
-
rich phases in these cultures, often embedded within
carbon
-
rich EPS material connecting clusters of multiple ANME
-
SRB consortia (
SI Appendix
, Fig. S1
). Transmission electron
microscopy (TEM) analysis corroborated these findings, showing
these Si phases were intimately integrated within the EPS matrix
within which many dozens of AOM consortia were embedded
(Fig. 1
E
and
F
). Image analysis of this TEM data estimated a
fairly uniform particle size for these silica spheres (230 ± 62 nm,
n
= 6,060), notably similar to the texture of authigenic silica
precipitated during microbial respiration of iron
-
bearing clays
(25). To compare these with environmentally acquired ANME
-
SRB consortia from seep sediments, we conducted high
-
resolution
nanoscale secondary ion mass spectrometry (nanoSIMS) analysis
of the spatial distribution of
28
Si on TEM
-
sectioned consortia
(Fig. 2
A
D
), as well as correlated FISH
-
SEM
-
EDS on a AOM
consortium after Ga
+
focused ion beam milling [Fig. 2
E
G
; (11).
These independent analyses both demonstrated that the spatial
distribution of silica
-
bearing particles associated with sediment
-
hosted ANME
-
SRB consortia from sediment was similar to that
of the Si
-
rich precipitate observed on AOM consortia grown in
sediment
-
free enrichment cultures (Fig. 1
E
). Specifically, this silica
phase is localized to the exteriors of ANME
-
SRB consortia and is
not present surrounding cells within consortia interiors.
Si
-
rich Phase on AOM Consortia Is Compositionally Distinct
from Silica Minerals in Seep Sediment.
Chemical analysis of the
amorphous silica
-
rich phase associated with ANME
-
SRB consortia
from our sediment
-
free enrichment cultures revealed that this
phase had a similar composition to the Si
-
rich phases analyzed
on environmental consortia directly extracted from methane seep
sediments (Fig. 3
A
), suggesting that the composition of this Si
-
rich mineral phase is common to AOM consortia despite the large
differences in conditions between the in situ seep environment
and sediment
-
free AOM enrichment cultures. Notably, the
composition of this amorphous silica
-
rich phase attached to
AOM consortia was distinct from the siliciclastic methane seep
sediment particles from which the original inoculum of ANME
-
SRB consortia was sourced. Mineral phases attached to AOM
consortia recovered from both native seep sediments and within
our sediment
-
free cultures typically had lower Si, Al, and Fe (
~
5–
20 atom % Si,
~
0–5% Al, and
~
0–3% Fe) than the sediment
particles which hosted them (
~
15–25 atom % Si,
~
5–10% Al,
and
~
3–10% Fe; Fig. 3 and
SI Appendix
, Fig. S2
). We note that
raw atom % values calculated from EDS data may underrepresent
Si, Al, and Fe in consortia given the high local C or O from
proximal biomass. Thus, we characterized the composition of
consortium
-
attached silica
-
rich precipitates by ratios of elements
typically found in octahedral sites of clays (Mg, Al, Fe) to Si and
found that AOM consortia had Al:Si ratios <0.37 and (Mg + Al
+ Fe):Si ratios <0.5, which are generally lower than those of the
sediment from which they were associated (Fig. 3). To determine
whether clay
-
like phases might precipitate with respect to the
activity of ions in our artificial seawater media (
Dataset S4
), we
prepared mineral stability diagrams with the solution composition
of our experiments (Fig. 3
B
and
SI Appendix
, Fig. S4
). The
predominant silicate minerals in equilibrium with this solution
were clays (kaolinite and illite), feldspar (albite), and chlorite
minerals (chamosite and clinochlore) (26). At our specific pH
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and dissolved Si values, predominantly aluminosilicates related
to 1:1 clays were in equilibrium with the solution composition
(Fig. 3
B
), suggesting the Si
-
rich phases associated with ANME
-
2/
SRB consortia may be associated with clays such as kaolinite
(Fig. 3
B
). Precipitation of silica (amorphous or crystalline)
would require a dissolved Si concentration at three orders of
magnitude greater than the concentration in our experiment.
Notably, the Si
-
rich phases attached to ANME
-
SRB consortia
from this work were significantly more enriched in Si than cell
-
attached silicates previously used as a model for silica precipitation
resulting from cation bridging by Fe or Al adsorbed on cell walls
(27); (
SI Appendix
, Fig. S3
). Furthermore, the Al/Si ratios in the
ANME
-
SRB
-
associated precipitates were consistently lower than
expected in clay compositions (Fig. 3
A
), supporting the biologically
mediated process in the Si enrichment in the precipitates.
Precipitated Si
-
rich Phases Attached to AOM Consortia in
Culture.
The long
-
term maintenance of our AOM enrichment
cultures and the absence of a sediment matrix provided the
opportunity to place additional constraints on the process by
which amorphous silicate particles associate with ANME
-
SRB
consortia. We formulated a simple model of ANME
-
SRB growth,
using estimates of ANME
-
SRB doubling times from the literature
(3 to 7.5 mo); (28–32), to roughly determine the maximum
number of AOM consortia in the starting inoculum that would
have resulted in a larger EPS
-
embedded aggregation of 55 or
more ANME
-
SRB consortia (e.g.,
SI Appendix
, Fig. S5
) after
5 y of anaerobic cultivation in sediment
-
free artificial seawater
supplied with methane. Our simplified model assumes that the
larger EPS embedded aggregations of AOM consortia observed in
culture were formed by consortia growth since these clusters were
not observed in the starting inoculum and in early time points
of the enrichment. The results of this model (Fig. 3
C
) indicate
that a 55
-
consortium cluster could have grown from no more
than 2 consortia, assuming a conservative doubling time of 7.5
mo. Doubling times shorter than
~
6 mo in this model imply a
55
-
consortium cluster would have grown from <1 consortium.
Similarly, clusters with less than
~
32 consortia with a doubling
time of 7.5 mo are calculated to have grown from <1 consortium,
suggesting these smaller aggregated AOM clusters formed from
consortia during the laboratory incubation that were not originally
part of the initial inoculum, or possibly represent clusters that
separated from larger aggregations during the incubation or
culture processing. Based on the observed and modeled AOM
Fig. 1.
Epifluorescence and microscopy of sediment
-
free ANME
-
2/SRB consortia in laboratory incubations. (
A
C
) Correlated FISH, SEM, and EDS imaging of
ANME
-
2/SRB consortia and associated Si
-
rich phases. Dashed ovals outline individual AOM consortia within the cluster across all three panels. (
A
) FISH image
of a cluster of ANME
-
SRB consortia, with ANME
-
2 cells stained in pink and inferred SRB cells in blue. (
B
) Paired SEM image of the same cluster of AOM consortia
in (
A
) where red arrows denote areas enriched in silica.
Red Inset box
: 2X magnified view of
~
200 nm amorphous silica spheres. (
C
) EDS map showing elemental
distribution, where yellow is carbon associated with ANME
-
SRB biomass, with silicon (blue) is concentrated in the extracellular matrix around individual AOM
consortia. (
D
) Higher magnification SEM image showing nanoscale silica spheres associated with AOM consortia. (
E
and
F
) TEM cross
-
section of ANME
-
SRB
consortia and closely associated Si
-
rich particles (red arrow).
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consortia growth, we reason that the Si
-
rich phases detected in
the extracellular matrix surrounding and in between individual
AOM consortia must have developed over the 5
-
y enrichment
period, as essentially all consortia associated with amorphous silica
in the enrichment cultures were predicted based on the modeled
doubling times to have grown after inoculation.
The concentration of dissolved silica in four sediment
-
free
AOM enrichments and the artificial seawater medium was
measured using ICP
-
MS calibrated standards, and these values
were compared with the [Si] equilibria of abiotic processes known
to generate amorphous silica, including silica precipitation (33)
and sorption of Si to clay minerals (34); (Table
1). Unlike in
several previous experiments examining potential microbial silicate
biomineralization, where precipitation on bacterial or archaeal cell
surfaces was only observed under conditions supersaturated with
respect to clay (27, 35, 36) or silica precipitation (37–41), the
Fig. 2.
Images of ANME
-
SRB consortia isolated from sediments. (
A
and
B
) NanoSIMS
12
C
14
N maps; (
C
and
D
) NanoSIMS
28
Si maps of the same ANME
-
SRB consortia
shown in (
A
and
B
), respectively, where silicon can be observed forming rims around the consortia. Lighter colors indicate higher counts, shown by the bars
to the left of the images. (
E
) FISH of a sediment
-
associated ANME
-
SRB consortium, with ANME in pink and SRB in green. (
F
) SEM of the consortium in (
E
) after
FIB section. (
G
) Elemental composition by EDS along a transect of the consortium in (
F
), which shows higher C and N relative abundances in the interior and an
enrichment in Si and Al at the edges of the consortia.
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silica concentrations (21 to 64
μ
M) in the artificial seawater media
and sediment
-
free enrichments (Table
1) were several orders of
magnitude below [Si] for abiotic silicate precipitation [4.3 mM
(42). Neither silica or aluminum were directly added to the arti­
ficial seawater media (14); however, it is possible that traces of
these elements may have leached from the walls of the glass serum
vials or were present as part of the original sediments where the
aggregates were retrieved from. The active formation of Si
-
rich
phases attached to ANME
-
SRB consortia in enrichment media
undersaturated with respect to Si suggests formation by a yet
unknown mechanism that is likely mediated by an aspect of the
physiology of the methanotrophic ANME
-
2 and/or associated
SRB partners, given the intimate physical association between
silica and AOM consortia in the sediment
-
free enrichment
cultures. It has been shown that monomers of silicic acid can
condense forming soluble polymeric silicic acid (PSA) particles
which can aggregate at pH <7 (43). Local low pH conditions
within ANME
-
SRB consortia, favorable for PSA formation, may
be enhanced by direct extracellular electron transfer between
ANME archaea and their SRB partner, where a build
-
up of pro­
tons from methane oxidation is predicted to accumulate resulting
in local pH gradients (44), with contributions from silica disso­
lution and carbonate precipitation (10). Organic polymers may
also enhance the condensation of monomeric silicic acid, leading
to the formation of silica nanoparticles (43). The specific mecha­
nism of formation and whether organic ligands are responsible
for the precipitation of silica on the surfaces of ANME
-
SRB con­
sortia remains an outstanding question at this time, but with
Fig. 3.
Composition of Si
-
rich phases associated with ANME
-
SRB consortia isolated from sediments and in sediment
-
free incubations. (
A
) Elemental ratios of
EDS
-
acquired compositional data of ANME
-
SRB consortium
-
attached Si
-
rich phases extracted directly from methane seep sediments (“ANME
-
SRB”) or grown under
sediment
-
free conditions (“sed. free”) compared with the range of compositions of sediments from three different seep sediment locations from Northern and
Southern California and the Costa Rican margin from which the AOM consortia were recovered (“sed.”). Sampling location details can be found in
Materials and
Methods
. Reference clay mineral compositions are also noted [arrows on right; montmorillonite (mont), illite (ill), chlorite (chl)]. Also shown is the composition of
three different Si
-
rich rings surrounding carbon
-
rich cell aggregates in a seep carbonate. (
B
) Predicted mineral stability diagram for silicate mineral formations
in the experimental solution composition with both illite, smectite, and kaolinite included as clay minerals. Feldspar refers to albite; Chlorite(1) and Chlorite(2)
refer to chamosite and clinochlore, respectively; and the red bar corresponds to ICP
-
MS measurement of [Si] in artificial seawater media from the sediment
-
free AOM enrichments. Crystalline or amorphous silica precipitation would require higher dissolved silica and lower pH values. (
C
) A general model of AOM
consortia growth showing the estimated final number of consortia (contours) within a cluster as a function of the initial consortia numbers within the enrichment,
constrained by the range of reported values for ANME
-
SRB doubling times (dashed lines).
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continued progress in cultivation for these slow
-
growing meth­
anotrophic consortia, complementary detailed analyses will likely
be possible in the future.
Amorphous Si
-
rich Minerals Occur on Endolithic ANME
-
SRB
Consortia in Seep Carbonates.
The preservation of AOM sig­
natures in paleo
-
methane seeps primarily exists within authigenic
carbonates, where mineral, isotopic, and lipid biosignatures provide
evidence for past sulfate
-
coupled methane oxidation activity
catalyzed by ANME and SRB that are analogous to those observed
in modern methane seep ecosystems worldwide (21, 48, 49). As the
greatest potential for Si
-
enhanced preservation of microfossils of
AOM consortia is likely to be found within authigenic carbonates,
we extended our investigation to active endolithic AOM consortia
within modern methane seep carbonates and the possibility that
these assemblages, like those in sediments and enrichment culture,
also showed evidence of silica precipitation. To investigate this
possibility, we examined an authigenic carbonate sample composed
primarily of calcite collected from an active deep
-
sea methane
seep site found off the coast of Costa Rica (
~
1,800 m depth).
Preliminary analyses of 16S rRNA gene sequences from subsamples
of this seep carbonate in our group showed a dominance of
ANME
-
2c and ANME
-
3 archaea, as well as members of SeepSRB1
active sulfate
-
coupled AOM activity. Correlated epifluorescence
microscopy using the general DNA stain DAPI (Fig. 4
A
) and SEM
imaging (Fig. 4
B
) of a resin
-
embedded and sectioned sample of this
seep carbonate revealed
~
10–20
μ
m diameter consortia composed
of tightly packed cocci cells. SEM
-
EDS analysis revealed these
electron
-
dense regions were more C
-
rich than the surrounding
carbonate matrix (Fig. 4
C
) and notably, were directly associated
with Si
-
rich rims (Fig. 4
D
), similar to the environmental ANME
-
SRB consortia analyzed from seep sediment. FISH hybridization
of these embedded endolithic aggregates in thin section confirmed
that they were in fact ANME
-
2/SRB (Fig. 5 and
SI Appendix
,
Fig. S6
), and correlated SEM (
SI Appendix
, Fig. S7
) and EDS
of these taxonomically defined consortia (Fig. 5) confirmed the
close association of Si with the consortia exteriors (50). FISH
hybridization of ANME
-
SRB aggregates in a carbonate matrix
was only recently reported (20), and our work advances the study of
endolithic AOM consortia in rock thin sections directly correlated
with elemental composition of these consortia and their adjacent
matrix. The morphology of the ANME
-
2/SRB aggregates in the
carbonates is consistent with FISH
-
identified ANME
-
2/SRB
consortia previously observed in sediments identified by FISH
(51). Compositional spectra from EDS (
Dataset S1
) documented
low Al, Fe, and Mg content (<0.6 % wt) in Si
-
rich rings, with
(Mg + Al + Fe):Si and Al:Si ratios similar to those of Si
-
rich
phases attached to ANME
-
SRB consortia recovered directly from
seep sediments and in our sediment
-
free laboratory enrichment
cultures (Fig. 3
A
;
Datasets S2
and
S3
). Given the compositional
and textural similarities between these Si
-
rich rings and the Si
-
rich
phases associated with ANME
-
SRB consortia, we interpreted these
Si
-
rich rings surrounding endolithic aggregates in seep carbonates
to be analogous to those found encrusting ANME
-
SRB consortia
originating from sediments.
Discussion
We observed and identified Si
-
rich phases on ANME
-
SRB consor­
tium exteriors from seep sediments, authigenic carbonates, and
sediment
-
free AOM enrichment cultures that were incubated in
artificial seawater media undersaturated with respect to silica.
Measurement of [Si] in sediment
-
free incubation media via ICP
-
MS
precluded abiotic mechanisms of Si enrichment of consortium
-
attached Si
-
rich phases, as [Si] was too low to drive either amorphous
silica precipitation or Si sorption on preexisting consortium
-
attached
silicates. Additionally, most consortium
-
attached phases are enriched
in Si with respect to detrital silicates in the sediments from which
they were sourced, and also compared to known clay mineral
compositions, suggesting that these phases are not simply a product
of the attachment of clay minerals in sediment. For example, in
Si
-
rich endmember clay compositions such as montmorillonite
[(Na,Ca)
0.33
(Al,Mg)
2
(Si
4
O
10
)(OH)
2
·nH
2
O)], with a 2:1 ratio of
tetrahedral to octahedral sheets and where Si occupies all tetrahedral
sites, the octahedral cation to Si ratio is 0.5 (53); however,
consortium
-
attached Si
-
rich phases had octahedral cation: Si ratios
generally <0.5 (Fig.
3
A
). For comparison, methane seep sediment
samples from which consortia were extracted had octahedral cation
to Si ratios typically >0.5, consistent with clay minerals (Fig.
3
A
).
These results contrast with a previous report of incrustation of AOM
consortia with clay minerals (10), however, the phases reported in
that study also had cation: Si ratios generally <0.5, implying there
also existed minerals more enriched in Si compared to known clay
minerals. Our mineral stability diagrams (Fig.
3
B
and
SI Appendix
,
Fig. S4
) showed that the media solution chemistry was undersatu­
rated with respect to quartz (amorphous or crystalline), but not with
respect to clay precipitates, notably kaolinite (Fig.
3
B
). Furthermore,
the morphology of our Si
-
enriched precipitates is not consistent with
crystalline clay phases (Fig.
1 and
SI Appendix
, Fig. S1
), but rather
with the spheroid
-
like particles expected for the formation of clays
within organic assemblages (26). However, the Al/Si content is lower
than what is found in clay composition. Altogether, our findings led
us to propose that the Si
-
rich phase associated with AOM consortia
was likely a result of two types of biological processes: biominerali­
zation of Si
-
enriched precipitates from solutions undersaturated with
respect to silica and the formation of clay precipitates in intimate
associations with the organic/cellular assemblages.
Microbial precipitation of silica in undersaturated conditions
has been previously proposed to be mediated by iron (39) and
magnesium (54, 55) in solution. Experimental studies in cyano­
bacteria have shown that sulfate
-
rich EPS and an increase in pH
promoted by photosynthesis enhance the precipitation of
magnesium
-
rich silica, where magnesium likely acts as a cation
bridge between positively charged silicic acid and negative func­
tional groups in the EPS (55). Although we did document a
carbon
-
rich rim around two ANME
-
SRB consortia within the
carbonate rock (Figs.
4
B
and 5
A
), consistent with the presence of
EPS, this mechanism is not congruent with our observation that
the silicates attached to ANME
-
SRB consortia have low Al, Fe,
and Mg content (<0.6 % wt). In this study, Si
-
rich phases were
observed in association with the exteriors of ANME
-
SRB consortia,
consistent with a previously published study (10). This is surprising
since cell interiors might be more conducive to silicification in
undersaturated conditions, for example, as is observed in the intra
­
cellular Si
-
concentrating mechanisms in diatoms (56), but it is
Table 1. Silica concentrations in sediment
-
free ANME
-
SRB incubations and sample locations
Location
Si (
μ
M)
Reference
Santa Monica Basin porewater
110–300
(45)
Costa Rica cold seeps porewater
110–500
(46)
Eel River Basin porewater
700–1000
(47)
ANME
-
SRB sed
-
free enrichment 1
63.24
This study
ANME
-
SRB sed
-
free enrichment 2
21.08
This study
ANME
-
SRB sed
-
free enrichment 3
29.96
This study
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